nanosystems interface

CHAPTER

3

Understanding and utilizing the biomolecule/ nanosystems interface

SUBCHAPTER

Biomolecule immobilization and delivery strategies for controlling immune response

Subchapter3.1 Esther Y. Chen, Wendy F. Liu

The Edwards Lifesciences Center for Advanced Cardiovascular Technology, University of California, Irvine, CA, United States

1 INTRODUCTION Materials are a cornerstone of modern medicine, used in a wide range of applications including implanted and extracorporeal devices, vehicles for drug delivery, and scaffolds for tissue engineering or regenerative medicine.1 These materials interface directly with proteins, cells, and tissues, and as a foreign body, often trigger a host response that aims to eliminate or isolate the material from the body.2 The physical and chemical properties of the biomaterial play an important role in governing the adsorbed protein species, including serum components involved in the complement and coagulation cascades, as well as their conformation (Fig. 3.1.1A).3 These events in turn influence the downstream interactions with blood clotting factors and inflammatory cells, including platelets, neutrophils, macrophages, and dendritic cells (Fig. 3.1.1B).4 These immune cells are important for mediating the overall host response, and also help orchestrate the wound healing and tissue repair processes (Fig. 3.1.1C).5 This final phase involves resident tissue cells, including fibroblasts that lay down new matrix to form scar tissue (Fig. 3.1.1D). Overcoming this foreign body response is essential for reducing fibrosis, which ultimately prevents the functional integration of the device with surrounding host tissue. Many recent advances in biomaterial engineering have focused on designing surfaces to actively modulate local immune cells to control the foreign body response.9

Nanotechnologies in Preventive and Regenerative Medicine. http://dx.doi.org/10.1016/B978-0-323-48063-5.00003-4 Copyright © 2018 Elsevier Inc. All rights reserved.

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FIGURE 3.1.1  Schematic Depicting Parallels Between the Foreign Body Response After Implantation of Biomaterials (Right) and the Wound Healing Process After Injury (Left) Following implantation, proteins adsorb to the surface of the biomaterial (A). The adsorbed proteins, along with injury to the tissue, lead to recruitment and activation of immune cells (B), followed macrophages, and dendritic cells (C). Macrophages fuse to form giant cells, and fibroblasts proliferate and become activated forming a fibrous connective tissue (D). Eventually, the fibrous capsule of the implant isolates the implant from the local tissue. During homeostasis, host cells express a variety of self-markers that prevent immune cells from adhesion and activation, including CD200,6 CD47,7 and heparin sulfate proteoglycan8 (E).

While altering the chemical and physical composition can impact the host protein and cellular interactions,10,11 immobilizing immunomodulatory molecules onto materials has been used as a strategy to directly target immune cells and control their activity.12 These biomolecules include proteins thought to mitigate the inflammatory response, such as complement inhibitors, cytokines, extracellular matrix components, or self proteins (Fig. 3.1.1E). A major challenge in engineering biomolecule–synthetic-material hybrids has been the development of methods to effectively tether biomolecules to materials and control their delivery. As biomolecules often require aqueous environments to maintain their conformation and activity, this becomes a major design criteria that limits the available material fabrication methods. Therefore, depending

2 Noncovalent immobilization of biomolecules

on the biological, chemical, and physical properties of the materials, as well as the desired release profile, molecules may be encapsulated within or immobilized onto biomaterials through noncovalent interactions or covalent conjugation methods. In this subchapter, we will review different strategies to engineer delivery of immunomodulatory molecules from materials, highlighting the successes achieved and challenges remaining in this emerging field.

2  NONCOVALENT IMMOBILIZATION OF BIOMOLECULES Noncovalent attachment of biomolecules to materials relies on hydrophilic/ hydrophobic or electrostatic interactions, and is advantageous because of the ease of fabrication. The method of immobilization largely depends on the hydrophilicity of the material. For example, for hydrophilic materials, proteins may be dispersed within the material during the fabrication process, which is typically carried out in an aqueous environment. However, for hydrophobic materials that often require the use of organic solvents, proteins need to be encapsulated within a separate aqueous phase, or adsorbed to the surface of the material postfabrication. The following section provides several examples of combining biomolecules with materials using noncovalent strategies.

2.1  ENCAPSULATION OF BIOMOLECULES Encapsulation of biomolecules within materials can be easily performed for hydrophilic polymers, including natural biopolymers, such as alginate, agarose, or extracellular matrix–based materials, as well as synthetic polymers, such as poly(ethylene glycol) (PEG). These polymers typically form hydrogels, which are characterized by a network of crosslinked molecules that contain large amounts of water because of the hydrophilic nature of the polymer. Biomolecules are introduced by directly mixing into the aqueous polymer solutions during fabrication. Retention of biomolecules within these polymers and their subsequent release into the local environment is dependent on many factors, including interactions with the polymer itself and the pore sizes created by the polymeric mesh. Delivery of cytokines or chemokines, small molecules that mediate recruitment and activation of immune cells, via encapsulation within biomaterials has been explored for immunomodulation in tissue engineering. For example, biomaterialbased nerve guidance channels provide a scaffold for directed nerve growth during regeneration; however, the overall performance has been limited due to acute and chronic inflammatory response following surgery and biomaterial implantation. To mitigate inflammation, Mokarram et al. filled polysulfone nerve guidance channels with agarose gel that was physically blended with recombinant interleukin-4 (IL4)13 (Fig. 3.1.2A), a cytokine released during wound healing. Local delivery of IL-4 from the agarose-filled channels promoted polarization of recruited macrophages toward a prohealing (also known as M2) phenotype. M2 macrophage polarization

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FIGURE 3.1.2  Noncovalent Conjugation Methods (A) Biomolecules physically blended into hydrogels during fabrication.13 (B) Biomolecules encapsulated in hydrophobic biomaterials using a double emulsion technique.14 (C) Direct adsorption of biomolecules on the surface of biomaterials.15,16 (D) Ionic immobilization or electrostatic binding of biomolecule to a biomaterial.17 IL, Interleukin; PLGA, poly(lactic-co-glycolic acid); RGD, tripeptide arginine-glycine-aspartic acid.

resulted in enhanced recruitment of Schwann cells and faster axonal growth, suggesting that encapsulation and delivery of IL-4 not only modulate the host immune response, but may also have significant potential in improving the overall regenerative process. For hydrophobic polymers, encapsulation of proteins is challenging as the materials are typically fabricated in organic solvents that are detrimental to the function of most proteins. To maintain the stability of proteins during encapsulation within hydrophobic polymers, a double-emulsion (water-in-oil-in-water or W/O/W) technique is used, where the protein is encapsulated within a second aqueous phase.18 Poly(lactic-co-glycolic acid) (PLGA) is a hydrophobic polymeric biomaterial that has been widely explored in tissue engineering applications and drug delivery systems because of its tunable degradation and biocompatible degradation products. A few studies have incorporated pharmacological agents into PLGA using double emulsion. These include the therapeutic hormone estradiol,19 as well as tetanus toxoid for vaccine delivery.20 In addition, proteins including insulin,21 proteinase inhibitors,22 and vascular endothelial growth factor (VEGF) have all been encapsulated within PLGA.23 In an ischemia-reperfusion model, VEGF delivery using PLGA microparticles (MP) increased angiogenesis 1 month after treatment. However, implantation of PLGA still elicits an immune response that leads to scar deposition.24 Since protein encapsulation within hydrophobic polymers, such as PLGA remains difficult, researchers have alternatively encapsulated genes, which are more stable in the fabrication environment. Nucleic acid encoding the antiinflammatory cytokine,

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IL-10, was encapsulated in PLGA using a similar W/O/W emulsion technique,14 and delivered by intramuscular injection in mice for therapy to treat type 1 diabetes (Fig. 3.1.2B). IL-10 gene delivery led to higher serum levels of IL-10 and lower IFN-γ blood glucose levels for up to 6 weeks after implantation. Moreover, reduced infiltration of lymphocytes and neutrophils into the pancreatic islet was observed, suggesting the utility of immunomodulatory gene therapy in treatment of autoimmune diseases.

2.2  DIRECT ADSORPTION OF BIOMOLECULES To prevent exposure of biomolecules to the harsh conditions of biomaterial fabrication, biomolecules may be simply adsorbed on the surface of biomaterials after fabrication. In this case, biomolecules interact with the material surface through hydrophobic/hydrophilic and electrostatic interactions, or may be covalently attached [described in more detail in Section 3 (Subchapter 3.1)]. Following implantation, a layer of host proteins immediately adsorbs to the surface. The type of adsorbed proteins plays an important role in the recruitment and activation of immune cells. For instance, a polyethylene terephthalate (PET) surface coated with fibrinogen promote inflammation, and cause increased phagocyte accumulation on the implant surface.25 Surface adsorbed proteins clearly play a role in biomaterial interactions with immune cells (Fig. 3.1.1A and E), but adsorption under physiological conditions is nonspecific and difficult to control. To better understand how different species of adsorbed proteins regulate local immune activation, Zaveri et al. examined the response to polystyrene coated with various ECM proteins15 (Fig. 3.1.2C). ECM proteins were physically adsorbed on MP by incubation with either human plasma–derived fibronectin, vitronectin, bovine plasma fibrinogen, as well as fetal bovine serum or bovine serum albumin (BSA). They found that fibrinogen-coated MP elicited higher macrophage phagocytosis than other ECM coating. This response was mediated by macrophage integrin Mac-1, which plays an important role in MP uptake and proinflammatory cytokine secretion. Interestingly, macrophages from Mac-1 knockout mice or macrophages blocked with tripeptide arginine-glycineaspartic acid (RGD)–binding peptides present significantly lower phagocytosis of fibrinogen-opsonized MP. In addition, subcutaneous implantation of PET disks encapsulating RGD-containing peptide reduced fibrous capsulation around implantation, further suggesting that RGD-binding integrins are involved. More recently, different ECM proteins were immobilized on ultrahigh-molecular-weight polyethylene (UHMWPE), a polymer used in orthopedic implants, and inflammatory effects were examined using an established mouse calvarial model.26 Again, the uptake of opsonized ultrahigh molecular weight polyethylene MP (BSA, fibrinogen, or fibronectin) was suppressed by Mac-1 knock out, while cytokine secretion was inhibited under RGD blocking condition. Together, these results suggest that modifying biomaterial surface with antiintegrin molecules may be used as a strategy to mitigate the inflammatory response.

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In another example, a decellularized trabecular bone scaffold was coated with cytokines to modulate the immune response.16 The goal of this study was to provide dynamic regulation of macrophages, promoting inflammatory (also known as M1) activation followed by M2 activation, to promote initiation and propagation of new blood vessels, respectively.27,28 To accomplish this, IFN-γ, an inflammatory cytokine was physically adsorbed on the surface through hydrophobic interaction, whereas IL-4, the prohealing cytokine, was conjugated using biotin–streptavidin (method described in more detail in Section 4.1 of Subchapter 3.1). By providing a short release of IFN-γ, followed by sustained release of IL-4, the scaffold promoted sequential polarization of M1 and M2, as suggested by gene expression and cytokine secretion of macrophages. Cells receiving the sequential cytokine expressed higher level of proangiogenic gene VEGF followed by platelet-derived growth factor. This study applied different conjugation strategies to control the release of different biomolecules to initiate angiogenesis and promote vascularization sequentially. While nonpolar molecules are typically immobilized to materials through hydrophobic interaction, polar or charged molecules can be effectively bound through ionic interactions. Ionic bonding involves electrostatic interactions between oppositely charged ions, and can significantly enhance the binding strength of biomolecules immobilized to materials. These interactions can be leveraged for the adsorption of highly charged molecules, for example, heparin, an anticoagulant that mimics the function of heparin sulfate proteoglycans expressed by endothelial cells, and is highly negatively charged.29 Coating of heparin and soluble administration of heparin are both clinically used in cardiovascular devices and procedures, although it is thought that immobilization of heparin, as opposed to soluble delivery, provides better antithrombotic properties by inhibiting the activation of the coagulation cascade initiated by the surface of the biomaterial.30 As a result, several recent studies have immobilized heparin on the surface of biomaterials using a variety of methods,31 including ionic bonding. Initially, colloidal graphite was thought to provide an antithrombotic surface itself. However, it was later discovered that the antithrombotic property was due to the adsorption of a clinical disinfectant, benzalkonium chloride (positively charged), which becomes firmly bound to the surface of graphite and in turn assists heparin immobilization.17 Benzalkonium chloride provides a electrostatic bond for negatively charged heparin32,33 (Fig. 3.1.2D). The heparin-modified stents successfully demonstrated an anticoagulant ability. Other positively charged biomaterials, such as polyurethanes have also been heparinized through ionic bonding to enhance thromboresistance.34–36

2.3 CHALLENGES Noncovalent immobilization of biomolecules within or on biomaterials is advantageous because of ease of fabrication, and immunomodulation is likely effective over the short term. However, the primary challenge is maintaining the stability of molecules and controlling their release profile. The therapeutic activity of biomolecules, such as proteins, is highly dependent on the conformational structure. During the

3 Covalent conjugation of biomolecules

biomaterial fabrication process, proteins are potentially exposed to extreme conditions, such as different hydrophilicity of solvent, temperature, or pH. These external factors denature proteins or vary their conformation and aggregation, affecting their functionality. In addition, adsorption of proteins onto a surface likely affects their conformation and activity, even upon desorption. Indeed, surfaces presenting different functional groups modulate the conformation of adsorbed fibronectin, and thus integrin binding and cell adhesion.37 Moreover, the release profile of these biomolecules is another major challenge. As the period of inflammation and wound healing can vary from days to months, controlled release of biomolecules is essential to manipulate immune cell activities over this time course. Thus, researchers have been exploring controlled release of biomolecules by tuning material degradation. For example, the molecular weight or the lactic to glycolic acid ratio of PLGA influences it degradation dramatically, and can be altered to achieve a desired degradation profile.38,39 Another challenge for noncovalent biomolecule immobilization is the longevity and stability of the attachment. Under physiological conditions, adsorbed biomolecules likely are eventually replaced by other proteins present in the local environment, and long-term effects of these biomaterials may be difficult to predict. While it is indeed possible that early control of the immune response will have longterm benefits, improved methods to tether molecules to biomaterial surface that offer lasting stability will provide better control of immunomodulation.

3  COVALENT CONJUGATION OF BIOMOLECULES To overcome the instability associated with encapsulation or adsorption of biomolecules to materials, covalent conjugation provides an irreversible bond between the biomolecule and material. These methods rely on available functional groups on the biomolecule and material, including amine (NH2), sulfhydryl (SH), or carboxylic acid (COOH) groups. Recent advances in bioconjugation techniques and the development of specific crosslinkers have significantly expanded the range of biomolecule–material combinations that can be created. The following section details several examples of conjugation chemistries that can be used to tether biomolecules to materials, categorized by the crosslinking methods used.

3.1  CARBODIIMIDE CROSSLINKERS One of the most commonly used crosslinking agents are carbodiimides, which are used to bind NH2 to COOH groups. Given that biomolecules, such as proteins contain abundant primary NH2 groups available on the surface, carbodiimide chemistries have been one of the most popular and versatile methods for crosslinking NH2 to COOHs of biomaterials. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is used in combination with N-hydroxysuccinimide (NHS) to modify the carboxyl biomaterial surface and create an NHS ester, or NH2-reactive intermediate, allows conjugation of biomolecules containing primary NH2.

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FIGURE 3.1.3  Covalent Conjugation Methods Covalent conjugation of biomolecules relies on the existing functional groups on the molecules, such as amine (NH2) or sulfhydryl (SH) group. (A) Carboxyl on the surface of biomaterials can be modified with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to become amine reactive. Biomolecules containing primary amines groups are immobilized on the surface through carbodiimide bond.40–42 (B) SH-reactive crosslinkers can be used for conjugation to a thiol or maleimide surface. Biomolecules containing primary amine groups are converted into thiol-reactive compounds using succinimidyl 3-(2-pyridyldithio) propionate (SPDP). Biomolecules may also be treated with both SPDP and dithiothreitol (DTT) to react with maleimide surfaces.43–47

To reduce the thrombotic response to PLGA, heparin was immobilized to PLGA through carbodiimide crosslinking40 (Fig. 3.1.3A). In this study, acid-terminated PLGA was first treated with EDC/NHS to allow conjugation of chitosan, which contains numerous NH2 groups. Heparin, which contains COOH groups, was further bound to chitosan through carbodiimide crosslinking to create a heparin–chitosan– PLGA surface. Heparin–chitosan–PLGA exhibited reduced platelet adhesion, while maintaining cell compatibility as suggested by a hepatocyte proliferation assay. To mitigate thrombosis to polyethylene, Larm et al. treated the surface with polyethylenimine (PEI) and covalently conjugate heparin on the surface. Specifically, they introduced carboxylic and sulfate groups on the surface by treating polyethylene with sulfuric acid.41 The surface was further treated with PEI to present NH2 groups to allow covalent heparin conjugation through amide bond. Since PEI is highly negatively charged, heparin can also be immobilized through ionic bonding. Interestingly, in comparing immunomodulatory effect of ionic and covalent binding of heparin, they found that covalent conjugation of heparin on PEI-activated PE tubing exhibited approximately threefold less platelet adhesion than ionic conjugation.

3.2  SULFHYDRYL-REACTIVE CROSSLINKERS In addition to NH2-reactive chemistries, SH-reactive compounds are another commonly used crosslinking chemistry. SHs, also referred to as thiols, are found in the

3 Covalent conjugation of biomolecules

side chain of the amino acid cysteine. Many proteins have SHs but these functional groups are less abundant than NH2, thus making crosslinking via SHs more selective. Furthermore, cysteines can be introduced recombinantly to provide a specific site for conjugation. For biomolecules or biomaterials that contain NH2 groups can be converted to SHs via succinimidyl 3-(2-pyridyldithio) propionate (SPDP) followed by DTT treatment. SPDP is a compound that contains both NH2- and SH-reactive functional groups. SH-reactive chemical groups can be used in bioconjugation include maleimides and pyridyl disulfides. For example, proteins containing amino groups can be transferred into pyridyldithiol-activated protein, and later form disulfide bonds with a SH-activated surface. In this section, we discuss few examples that use SH-reactive crosslinkers to covalently conjugate biomolecules. Conjugation of a naturally occurring complement inhibitor, factor H, on the surface of polystyrene has been explored as a method to limit complement activation in response to materials.43 Primary NH2 were introduced to polystyrene by treating polystyrene with polyallyamine and SPDP crosslinkers, and then treated with DDT to create a SH-containing surface (Fig. 3.1.3B). Factor H was treated with SPDP to become reactive to SHs, and then covalently conjugated to the polystyrene surface. After incubating the material with serum, plasma or whole blood from healthy blood donors, immobilization of BSA (control) showed more C3 fragments deposited on the polystyrene compared to factor H-immobilized polystyrene. Immobilization of factor H prevented not only C3 fragment binding but also soluble C3a and C5b-9 generation in vitro. Their results demonstrated the effectiveness of factor H immobilization on biomaterials for preventing complement activation. In addition to SPDP, maleimide is another thiol-reactive compound that has been used in bioconjugation. Nilsson et al. applied thiol–maleimide conjugation strategy to immobilize the factor-H capture peptide, 5C6, on surfaces of biomaterials.44,45 Glass surfaces were chemically modified to contain maleimide groups, and then conjugated with a cysteine-modified 5C6 peptide through a SH–maleimide reaction. Covalent immobilization of 5C6 and an ATP hydrolysis catalyst, apyrase, was found to reduce platelet consumption and complement activation (C3a and sC5b-9) significantly after incubating with whole human bloods for an hour. Surfaces conjugated with 5C6 peptide have been shown to capture factor H from circulation without interfering with its activity, thus inhibiting biomaterial-induced complement activation.44

3.3  ACRYLATE-BASED CROSSLINKING Acrylates are light-sensitive functional groups that can be introduced to polymers or proteins and peptides, and have been largely used in functionalizing and polymerizing PEG hydrogels. PEG molecules are functionalized at each end with an acrylate to form homobifunctional monomers, polyethylene glycol diacrylate (PEGDA), that crosslink upon addition of a photoinitiator and ultraviolet light. To modify PEG hydrogels with a biomolecule, acrylate-modified proteins or peptides can be introduced to the polymerization mixture, and will tether to one end of the PEG molecule, while allowing the other end to be integrated within the gel.

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For immunomodulation, the adhesive RGD has been chemically tethered it with PEGDA hydrogel.48,49 RGD-modified PEG hydrogels attenuated the foreign body response in comparison to unmodified PEG hydrogel at 4 weeks after subcutaneous implantation, as indicated by a significant difference of thickness of the fibrous capsule around the implanted hydrogel.49–51 Additionally, PEGDA hydrogel can potentially be chemically functionalized with NH2, thiol, maleimide, and biotin for bioconjugation, making PEG a good candidate for delivery of biomolecules for immunomodulation. The reactivity between acrylate and thiol groups also provides a robust reaction for polymerization of peptide that contains cysteine into PEGDA hydrogels,52 and has been applied in immunomodulatory decoration. Hume et al. thiolated TGF-β1 and IL-10 via Traut’s reagent and fabricated immunomodulatory PEG hydrogels.53 Dendritic cells seeded on hydrogels functioned with immunosuppressive protein secreted lower amounts of IL-12 and expressed lower MHCII after 3-day culture, compared to stimulated controls. Using the thiol-acrylate polymerization, proteins were likely present throughout the hydrogel network for considerably long time period until the hydrogels were fully degraded.

3.4 CHALLENGES While primary NH2 and thiol groups present on proteins provide a direct means of conjugation to biomaterials, these conjugation methods still pose significant challenges. The accessibility of available NH2 and thiol groups on proteins can sometimes depend on protein conformational structure, and as a result, the conjugation efficiency can be unpredictable. In addition, the abundance of NH2 groups on proteins causes conjugation using carbodiimide crosslinkers to result in a number of different orientations, which can also influence the function of the protein. The therapeutic activity of some biomolecules, for example, membrane-bound proteins, can be highly dependent on structural orientation. Therefore, site-specific conjugation is required for more predictable conjugation and effective immunomodulation.

4  SITE-SPECIFIC IMMOBILIZATION OF BIOMOLECULES 4.1  NONCOVALENT SITE-SPECIFIC IMMOBILIZATION Site-specific conjugation, or the tethering of a protein at a precise molecular location, relies on recombinant DNA technology and the use of nucleic acid tags. The protein of interest is recombinantly cloned to contain a conjugation tag, usually at either C or N-terminus. AviTag is a popular fusion tag that encodes for 15 amino acids that allow biotinylation of protein for binding to streptavidin. Streptavidin, a protein from bacterium Streptomyces avidinii, has four high-affinity binding sites for biotin. Several studies have used biotin–streptavidin interaction to site specifically immobilize biomolecules on biomaterials.42,54 This method allows proteins to be tethered in an oriented manner, which can be critical for their function, particularly membrane-bound

4 Site-specific immobilization of biomolecules

FIGURE 3.1.4  Site-Specific Immobilization of Biomolecules (A) Biotin–avidin interaction used for noncovalent site-specific conjugation. Proteins of interest are cloned with AviTag sequence for biotinylation, and then immobilized onto avidin-coated surfaces.42,55 (B) Sortase-mediated conjugation used for covalent and sitespecific immobilization. Proteins are fused with LPETG, a sortase recognition peptide, which is site specifically modified with a functional group, such as azide for conjugation with biomaterials.56–58 TM, Thrombomodulin.

receptors that engage to ligands in a particular orientation. Several membrane bound immunomodulatory molecules are found on host cells, and serve as a mechanism for self/nonself discrimination. These include CD200 (OX-2) and CD47, which are recognized by immune cells through inhibitory receptors, such as CD200 receptor (CD200R) and signal regulatory protein-α (SIRP-α), respectively.6,7 Site-specific immobilization of immunomodulatory molecules on biomaterials can potentially promote self recognition to local immune cells, and thus prevent immune cell activation. To create materials that mimic CD200 expressed by host cells, the extracellular domain of CD200 with AviTag sequence was recombinantly produced42 (Fig. 3.1.4A). Streptavidin–biotin conjugation was used to tether CD200 to the material at the C-terminus by first biotinylating CD200-AviTag, and attaching it to a carboxyl-terminated polystyrene surface that had been treated with NHS/EDC and conjugated with streptavidin. Examining the effect on macrophage activation in vitro suggested that site-specific conjugation of CD200 was necessary to effectively suppress inflammatory activation. Furthermore, CD200-coated particles decreased macrophage infiltration in response to subcutaneous implantation in vivo. Biotin–avidin interaction has also been used to tether the self molecule, CD47, to particles for prolonged circulation. Phagocytes clear particles and foreign cells that do not exhibit markers of self. CD47 is highly expressed on red blood cells and interacts with SIRPα, an inhibitory transmembrane receptor present on myeloid cells, to prevent uptake.7 Cells lacking CD47 are readily phagocytosed, and activate T-cells.59 Moreover, apoptotic cells downregulate CD47 as a mechanism to promote clearance.60,61 Biotinylated recombinant CD47 was tethered to streptavidin-coated nanoparticles to prolong nanoparticle circulation by delaying phagocytosis by macrophage.55

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The immunomodulatory effect was further improved by synthetic CD47 peptide, which stabilized the conjugation and promoted drug delivery to tumors. Various strategies have been used to site-specifically bind CD47 to a biomaterial surface. Stachelek et al. treated polyvinyl chloride (PVC) to render a thiolated surface, and then further conjugated with avidin via disulfide bonds using SPDP.46 Biotinylated CD47 was immobilized on the avidin-coated surface. They found that CD47 modification significantly reduced attachment of human neutrophils and human monocyte-derived macrophages after 4 h and 3 days, respectively. The biotin– avidin conjugation exhibited a retention of CD47-immobilized subdermal implantation for 10 weeks. In another approach, Finley et al. produced recombinant CD47 with polylysine tail and treated with SPDP to create a thiol-reactive CD47, which was then conjugated on thiol-activated PVC surface. After 3 h incubation with whole blood, unmodified PVC that exhibited high-platelet adhesion and increased activation of both platelets and neutrophils.47 Biotin–avidin interactions have provided a versatile method for site-specific conjugation of proteins to biomaterial surfaces. However, this interaction is noncovalent and does not provide long-term stability in physiological conditions. In fact, the half life of the biotin–avidin interaction is thought to be as little as 35 h,62 which is a major drawback for modification of implant surfaces with immunomodulatory molecules. Therefore, a covalent conjugation method is needed for many clinical applications that require long-term stability.

4.2  COVALENT SITE-SPECIFIC IMMOBILIZATION To site specific modify protein for covalent ligation, the biosynthetic machinery of an Escherichia coli methionine auxotroph can be used to introduce an azide functional group into proteins, which can be further used for conjugation.63 Specifically, a methionine surrogate, azidohomoalanine, which is activated by E. coli synthetase, replaces methionine in during translation. Recombinantly produced protein containing azidohomoalanine are further selectively modified by Staudinger ligation using triarylphosphine (TPP). Using this reaction, Qu et al. immobilized recombinant thrombomodulin (TM) onto the luminal surface of PU-coated poly(tetrafluoroethylene) vascular graft.64 TM is a membrane protein highly expressed on myeloid and endothelial cells that inactivates thrombin and therefore reduces blood coagulation65. In this study, expanded PTFE grafts were coated with PU, which was first activated with hexamethylene diisocyanate to bind NH2–PEG–TPP. TM expressing a single C-terminal azide moiety (TM-N3) was then ligated onto TPP by Staudinger ligation. The TM-modified grafts exhibited reduced platelet activation in a chronic arteriovenous shunt model after a 60 min perfusion period, suggesting that TM actively limits biomaterial-induced thrombosis. However, this approach has limited yield of azidemodified proteins, and scalability is still a major challenge for its clinical translation. Another strategy for site-specific conjugation utilizes sortases as conjugation enzymes. Sortases are a class of bacterial transpeptidase enzymes that decorate the surface of bacteria with a diverse array of proteins and enable microbes to interact with

4 Site-specific immobilization of biomolecules

the microenvironment. Sortases can be categorized into different families based on their primary sequences and functions. SrtA from Staphylococcus aureus recognizes a C-terminal LPXTG motif of a protein, cleaves the peptide between threonine and glycine residues, and covalently attaches the protein to the NH2 group of a pentaglycine crossbridge forming an amide bond.66,67 Ton-That et al. used SrtA to link the carboxyl group of threonine to the NH2 group of NH2-Gly3.68 Several studies have further evaluated the suitability of sortase in protein–peptide or protein–protein ligation.69–71 In addition, LPXTG may be genetically fused to the proteins and sortase may be subsequently used for specific labeling of surface proteins with fluorophores or other molecules of interest.72,73 Recombinant TM containing a LPETG sequence for SrtA recognition was produced and then used to generate TM-N3 using SrtA as a catalyst56 (Fig. 3.1.4B). PU-coated expanded PTFE graft was activated with hexamethylene diisocyanate and further conjugated with DIBO–PEG–NH2 (DIBO, dibenzocyclooctyne). TM was then immobilized on DIBO-modified graft through azide and DIBO ligation, a highly efficient copper-free “click” reaction, a term commonly used to describe fast and high-yielding chemical reactions.74 The TM-immobilized grafts showed promising suppression of platelet adhesion in the baboon arteriovenous shunt model after 1 h exposure. The same research group also applied the sortase-mediated bioconjugation to attach TM and/or a single-chain antibody (scFv) through a C-terminal LPETG motif to protein micelles that expressed GGG oligopeptide for site-specific targeting of activated platelets and delivery of an antithrombotic agent (TM).57 In a model of laser-injury–induced thrombosis, TM/scFV-micelle administration inhibited thrombus formation within 180 s. Using sortase-mediated bioconjugation, they observed no decrease in surface-bound TM over 4 weeks in vitro. However, degradation and denaturation under physiological conditions limited the performance of immunomodulatory coating. More recently, a regenerative TM coating was created using sortase transpeptidation to enable multiple cycles of TM binding and cleavage.58 A pentaglycine-modified biomaterial surface was charged with TM-LPETG under SrtA catalysis, and then stripped by additional delivery of triglycine and SrtA. Impressively, the regenerative coating was cycled over 28 days in vitro without loss in efficiency of SrtA. Implantation of pentaglycine-modified catheters in mice confirmed immobilization and removal of TM over the course of one hour. Taken together, sortase-mediated bioconjugation may provide a means of permanent covalent conjugation with the capacity to regenerate, and can be applied on a number of potential biomolecules for finite stability and activity as immunomodulatory probes.

4.3 CHALLENGES While covalent conjugation methods are becoming more widely used to immobilize biomolecules to biomaterials, these techniques continue to evolve and become adapted for different molecules and materials. Many challenges still lie in maintaining protein activity and longevity of conjugation. In addition, the overall process often contains many steps and would benefit from simplification and/or optimization.

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For example, a fusion protein consisting of an N-terminal 6xHis, SrtA, and LPXTG followed by the protein of interest can undergo self-cleavage for one-step purification.69 Moreover, purification and site-specific conjugation can be combined into a single step using Sortase-tag expressed protein ligation.75 Here, a single protein construct containing LPXTG, SrtA, and 6xHis is tagged to the C-terminal end, and upon purification using a nickel column. In the future, these strategies may be applied to immunomodulatory proteins for site-specific “click” chemistry conjugation to biomaterials. Another covalent site-specific immobilization strategy that has been used for biological imaging, and can potentially be applied to create immunomodulatory biomaterials is ligation through the enzyme lipoic acid ligase (LplA). This system is similar to the Avitag-based biotin–avidin system described in Section 4.1 of Subchapter 3.1, insofar as it also attaches a substrate to a peptide tag that is recombinantly introduced to a protein. Fernandez-Suarez et al. redirected a microbial LplA to specifically attach a chemical probe to an engineered LplA acceptor peptide, a 13–amino acid peptide fused to the end of the protein.76 The LplA acceptor peptide fusion protein was constructed and ligated with azide using LplA as a catalyst, and then used for protein labeling inside living cells. In the future, the use of LplA can be applied to site-specific conjugation of immunomodulatory proteins to biomaterial surfaces.

5  CONCLUSIONS AND FUTURE DIRECTIONS To overcome the foreign body response to biomaterial implantation, immunomodulatory biomolecules have been incorporated in materials using a variety of methods. Strategies including noncovalent immobilization offer fast release of biomolecules, whereas covalent conjugation methods provide a longer lasting modification for long-term stability. Biomolecules can be dispersed within hydrophilic polymers, but encapsulation of biomolecules within hydrophobic polymers usually requires double-emulsion or direct adsorption to the surface of the biomaterial postfabrication via hydrophobic interaction or ionic immobilization. However, ultimately noncovalent immobilization methods usually result in short-term release of biomolecules. In contrast, covalent conjugation provides a more stable bond for biomolecule tethering. NH2 groups present in abundance on the surface of proteins are often used to covalently immobilized proteins onto biomaterials through carbodiimide crosslinkers (EDC/NHS). Sulfhydryl groups are less abundant, allowing more selective conjugation through SH-reactive crosslinking with maleimides or pyridyl disulfides. SHs can also be recombinantly introduced to biomolecules or be converted from NH2 by using the SPDP reagent. Finally, acrylate-based crosslinking may be used to form biomolecule–polymer hybrids. Together, these covalent conjugation methods provide more stability than noncovalent methods, but typically do not tether biomolecules at a particular site or in specific orientations.

References

For biomolecules that depend highly on structural orientation for functionality, site-specific conjugation is desirable. Proteins can be recombinantly fused with peptide tags for biotinylation or functionalization. Biotin–avidin is a noncovalent reaction that has been employed for site-specific conjugation on biomaterials, but to improve the stability of site-specific conjugation, other peptide sequence that can be recognized by specific enzymes, including sortase A and lipoic acid ligase have also been explored. Modified biomolecules can further be chemically immobilized onto biomaterials through “click” chemistry. Selecting the best immobilization strategy among the various available conjugation methods depends on the biomaterial used and the desired biomolecule release profile. Noncovalent methods provide a quick approach to deliver biomolecules in the short term, whereas covalent conjugation results in improved stability. Given the dynamic nature of the host immune response, a combination of different conjugation strategies may potentially be used for temporal control of immune cell function, and thus improve the regenerative response.

REFERENCES 1. Hubbell JA. Biomaterials in tissue engineering. Biotechnology 1995;13(6):565–76 (Nature Publishing Company). 2. Anderson JM, Rodriguez A, Chang DT. Foreign body reaction to biomaterials. Semin Immunol 2008;20(2):86–100. 3. Wilson CJ, Clegg RE, Leavesley DI, Pearcy MJ. Mediation of biomaterial-cell interactions by adsorbed proteins: a review. Tissue Eng 2005;11(1–2):1–18. 4. Luttikhuizen DT, Harmsen MC, Luyn MJV. Cellular and molecular dynamics in the foreign body reaction. Tissue Eng 2006;12(7):1955–70. 5. DiPietro LA. Wound healing: the role of the macrophage and other immune cells. Shock 1995;4(4):233–40. 6. Hoek RM, Ruuls SR, Murphy CA, Wright GJ, Goddard R, Zurawski SM, et al. Downregulation of the macrophage lineage through interaction with OX2 (CD200). Science 2000;290(5497):1768–71. 7. Oldenborg P-A, Zheleznyak A, Fang Y-F, Lagenaur CF, Gresham HD, Lindberg FP. Role of CD47 as a marker of self on red blood cells. Science 2000;288(5473):2051–4. 8. Sarrazin S, Lamanna WC, Esko JD. Heparan sulfate proteoglycans. Cold Spring Harbor Perspect Biol 2011;3(7):a004952. 9. Morais JM, Papadimitrakopoulos F, Burgess DJ. Biomaterials/tissue interactions: possible solutions to overcome foreign body response. AAPS J 2010;12(2):188–96. 10. Wang D, Phan N, Isely C, Bruene L, Bratlie KM. Effect of surface modification and macrophage phenotype on particle internalization. Biomacromolecules 2014;15(11):4102–10. 11. Ward WK, Slobodzian EP, Tiekotter KL, Wood MD. The effect of microgeometry, implant thickness and polyurethane chemistry on the foreign body response to subcutaneous implants. Biomaterials 2002;23(21):4185–92. 12. Kim YK, Chen EY, Liu WF. Biomolecular strategies to modulate the macrophage response to implanted materials. J. Mater. Chem. B 2016;4(9):1600–9. 13. Mokarram N, Merchant A, Mukhatyar V, Patel G, Bellamkonda RV. Effect of modulating macrophage phenotype on peripheral nerve repair. Biomaterials 2012;33(34):8793–801.

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14. Basarkar A, Singh J. Poly (lactide-co-glycolide)-polymethacrylate nanoparticles for intramuscular delivery of plasmid encoding interleukin-10 to prevent autoimmune diabetes in mice. Pharm Res 2009;26(1):72–81. 15. Zaveri TD, Lewis JS, Dolgova NV, Clare-Salzler MJ, Keselowsky BG. Integrin-directed modulation of macrophage responses to biomaterials. Biomaterials 2014;35(11):3504–15. 16. Spiller KL, Nassiri S, Witherel CE, Anfang RR, Ng J, Nakazawa KR, et al. Sequential delivery of immunomodulatory cytokines to facilitate the M1-to-M2 transition of macrophages and enhance vascularization of bone scaffolds. Biomaterials 2015;37:194–207. 17. Gott VL, Whiffen JD, Dutton RC. Heparin bonding on colloidal graphite surfaces. Science 1963;142(3597):1297–8. 18. Cohen S, Yoshioka T, Lucarelli M, Hwang LH, Langer R. Controlled delivery systems for proteins based on poly(lactic/glycolic acid) microspheres. Pharm Res 1991;8(6):713–20. 19. Mittal G, Sahana DK, Bhardwaj V, Ravi Kumar MNV. Estradiol loaded PLGA nanoparticles for oral administration: effect of polymer molecular weight and copolymer composition on release behavior in vitro and in vivo. J Control Release 2007;119(1):77–85. 20. Alonso MJ, Gupta RK, Min C, Siber GR, Langer R. Biodegradable microspheres as controlled-release tetanus toxoid delivery systems. Vaccine 1994;12(4):299–306. 21. Soriano I, Evora C, Llabrés M. Preparation and evaluation of insulin-loaded poly (dl-lactide) microspheres using an experimental design. Int J Pharm 1996;142(2):135–42. 22. Esposito E, Cortesi R, Bortolotti F, Menegatti E, Nastruzzi C. Production and characterization of biodegradable microparticles for the controlled delivery of proteinase inhibitors. Int J Pharm 1996;129(1):263–73. 23. Formiga FR, Pelacho B, Garbayo E, Abizanda G, Gavira JJ, Simon-Yarza T, et al. Sustained release of VEGF through PLGA microparticles improves vasculogenesis and tissue remodeling in an acute myocardial ischemia-reperfusion model. J Control Release 2010;147(1):30–7. 24. Anderson JM, Shive MS. Biodegradation and biocompatibility of PLA and PLGA microspheres. Adv Drug Deliv Rev 2012;64:72–82. 25. Hu W-J, Eaton JW, Ugarova TP, Tang L. Molecular basis of biomaterial-mediated foreign body reactions. Blood 2001;98(4):1231–8. 26. Zaveri TD, Dolgova NV, Lewis JS, Hamaker K, Clare-Salzler MJ, Keselowsky BG. Macrophage integrins modulate response to ultra-high molecular weight polyethylene particles and direct particle-induced osteolysis. Biomaterials 2017;115:128–40. 27. Mountziaris PM, Mikos AG. Modulation of the inflammatory response for enhanced bone tissue regeneration. Tissue Eng B 2008;14(2):179–86. 28. Spiller KL, Anfang RR, Spiller KJ, Ng J, Nakazawa KR, Daulton JW, et al. The role of macrophage phenotype in vascularization of tissue engineering scaffolds. Biomaterials 2014;35(15):4477–88. 29. Greinacher A, Michels I, Liebenhoff U, Presek P, Mueller-Eckhardt C. Heparin-associated thrombocytopenia: immune complexes are attached to the platelet membrane by the negative charge of highly sulphated oligosaccharides. Br J Haematol 1993;84(4):711–6. 30. Sanchez J, Elgue G, Riesenfeld J, Olsson P. Studies of adsorption, activation, and inhibition of factor XII on immobilized heparin. Thromb Res 1998;89(1):41–50. 31. Linhardt RJ, Murugesan S, Xie J. Immobilization of heparin: approaches and applications. Curr Top Med Chem 2008;8(2):80–100. 32. Gott VL, Whiffen JD, Koepke DE, Daggett RL, Boake WC, Young WP. Techniques of applying a graphite-benzalkonium-heparin coating to various plastics and metals. ASAIO J 1964;10(1):213–7.

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51. Lynn AD, Kyriakides TR, Bryant SJ. Characterization of the in vitro macrophage response and in vivo host response to poly(ethylene glycol)-based hydrogels. J Biomed Mater Res A 2010;93(3):941–53. 52. Salinas CN, Anseth KS. Mixed mode thiol−acrylate photopolymerizations for the synthesis of peg−peptide hydrogels. Macromolecules 2008;41(16):6019–26. 53. Hume PS, He J, Haskins K, Anseth KS. Strategies to reduce dendritic cell activation through functional biomaterial design. Biomaterials 2012;33(14):3615–25. 54. Tsai RK, Discher DE. Inhibition of “self” engulfment through deactivation of myosin-II at the phagocytic synapse between human cells. J Cell Biol 2008;180(5):989–1003. 55. Rodriguez PL, Harada T, Christian DA, Pantano DA, Tsai RK, Discher DE. Minimal “Self” peptides that inhibit phagocytic clearance and enhance delivery of nanoparticles. Science 2013;339(6122):971–5. 56. Qu Z, Krishnamurthy V, Haller CA, Dorr BM, Marzec UM, Hurst S, et al. Immobilization of actively thromboresistant assemblies on sterile blood-contacting surfaces. Adv Healthc Mater 2014;3(1):30–5. 57. Kim W, Haller C, Dai E, Wang X, Hagemeyer CE, Liu DR, et al. Targeted antithrombotic protein micelles. Angew Chem 2015;127(5):1481–5. 58. Ham HO, Qu Z, Haller CA, Dorr BM, Dai E, Kim W, et al. In situ regeneration of bioactive coatings enabled by an evolved Staphylococcus aureus sortase A. Nat Commun 2016;7:11140. 59. Oldenborg PA, Zheleznyak A, Fang YF, Lagenaur CF, Gresham HD, Lindberg FP. Role of CD47 as a marker of self on red blood cells. Science 2000;288(5473):2051–4. 60. Gardai SJ, McPhillips KA, Frasch SC, Janssen WJ, Starefeldt A, Murphy-Ullrich JE, et al. Cell-surface calreticulin initiates clearance of viable or apoptotic cells through transactivation of LRP on the phagocyte. Cell 2005;123(2):321–34. 61. Azuma Y, Nakagawa H, Dote K, Higai K, Matsumoto K. Decreases in CD31 and CD47 levels on the cell surface during etoposide-induced Jurkat cell apoptosis. Biol Pharm Bull 2011;34(12):1828–34. 62. Chilkoti A, Stayton PS. Molecular origins of the slow streptavidin-biotin dissociation kinetics. J Am Chem Soc 1995;117(43):10622–8. 63. Kiick KL, Saxon E, Tirrell DA, Bertozzi CR. Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation. Proc Natl Acad Sci 2002;99(1):19–24. 64. Qu Z, Muthukrishnan S, Urlam MK, Haller CA, Jordan SW, Kumar VA, et al. A biologically active surface enzyme assembly that attenuates thrombus formation. Adv Funct Mater 2011;21(24):4736–43. 65. Conway EM. Thrombomodulin and its role in inflammation. Semin Immunopathol 2012;34:107–25. 66. Mazmanian SK, Liu G, Ton-That H, Schneewind O. Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science 1999;285(5428):760–3. 67. Ton-That H, Liu G, Mazmanian SK, Faull KF, Schneewind O. Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif. Proc Natl Acad Sci 1999;96(22):12424–9. 68. Ton-That H, Mazmanian SK, Faull KF, Schneewind O. Anchoring of surface proteins to the cell wall of Staphylococcus aureus. Sortase catalyzed in vitro transpeptidation reaction using LPXTG peptide and NH2-Gly3 substrates. J Biol Chem 2000;275(13):9876–81. 69. Mao H, Hart SA, Schink A, Pollok BA. Sortase-mediated protein ligation: a new method for protein engineering. J Am Chem Soc 2004;126(9):2670–1.

1 Introduction

70. Pritz S, Wolf Y, Kraetke O, Klose J, Bienert M, Beyermann M. Synthesis of biologically active peptide nucleic acid-peptide conjugates by sortase-mediated ligation. J Org Chem 2007;72(10):3909–12. 71. Proft T. Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilisation. Biotechnol Lett 2010;32(1):1–10. 72. Popp MW, Antos JM, Grotenbreg GM, Spooner E, Ploegh HL. Sortagging: a versatile method for protein labeling. Nat Chem Biol 2007;3(11):707–8. 73. Tanaka T, Yamamoto T, Tsukiji S, Nagamune T. Site-specific protein modification on living cells catalyzed by sortase. ChemBioChem 2008;9(5):802–7. 74. Kolb HC, Finn M, Sharpless KB. Click chemistry: diverse chemical function from a few good reactions. Angew Chem Int Ed 2001;40(11):2004–21. 75. Warden-Rothman R, Caturegli I, Popik V, Tsourkas A. Sortase-tag expressed protein ligation: combining protein purification and site-specific bioconjugation into a single step. Anal Chem 2013;85(22):11090–7. 76. Fernandez-Suarez M, Baruah H, Martinez-Hernandez L, Xie KT, Baskin JM, Bertozzi CR, et al. Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes. Nat Biotechnol 2007;25(12):1483–7.

SUBCHAPTER

Nanoproteomics approaches in biomarker discovery: The critical role of protein corona on nanoparticles as drug carriers

Subchapter3.2

Loreto Megido, Paula Díez, Manuel Fuentes Cancer Research Centre (IBMCC/CSIC/USAL/IBSAL), Salamanca, Spain

1 INTRODUCTION The study of nanoscience has spread out1 during the last century. It has experienced an increasing development, especially since the 1990s. Nanotechnology is currently involved in our daily life as a part of cosmetics, food packaging, drug delivery systems, therapeutics, or biosensors among others,2 and it has been estimated that the production of nanomaterials will increase in 2020 by 25 times what it is today.3 There is a wide range of applications for these nanomaterials in numerous fields, from commercial products—such as electronic components, cosmetics, household appliances, semiconductor devices, energy sources, food color additives or surface coatings—to medical products like biological sensors, drug carriers, biological probes, implants, and medical imaging.4 In fact, biomedicine is one of the most

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promising fields of nanoscience, and “nanomedicine” is recognized as a highly interdisciplinary field to provide personalized medicine for many pathologies. There are several properties that make nanomaterials [such as nanoparticles (NPs)] interesting as medical tools useful in diagnostics, in vivo imaging, and disease treatment, such as their small size, their large surface area and the kinetics of adsorption, among others.1 Despite the future dependence of our society on nanomaterials, their safe incorporation in our lives has been poorly assessed.5 The risk–benefit balance for these materials together with their toxicological profile, and the potential adverse pathogenic reactions derived from exposure should be tested to define their influence in the clinical outcomes and applications.1 In this sense, several risks attributed to manufactured nanomaterials have been reported6 concerning sources of NPs and ways to enter the human body: skin penetration, injection, or inhalation.7 Environmental epidemiology holds that exposure to nanomaterials could cause significant public health drawbacks, such as pulmonary or cardiovascular diseases as a consequence of a long-term exposition to the nanomaterial.8 Once NPs are inside the body, the direct interaction with cells and organs is possible, allowing in this interphase several biomedical applications, such as targeted drug delivery,9 cellular therapy,10 tissue repair,11 hyperthermia,12 magnetic resonance imaging (MRI),13 magnetic resonance spectroscopy,14 magnetic separation,15 and sensors for metabolites and other biomolecules.16 In addition, the small size and electromagnetic properties of magnetic NPs make them useful for biomolecule labeling in bioassays and as MRI contrast agents.17 Carbon nanotubes can be also used as biosensors, probes, actuators, nanoelectronic devices, drug delivery systems, and tissue-repair scaffolds due to their physical, mechanical, and electronic properties.18 Targeted drug delivery involves most of the applications of nanomedicine that are currently in research stage.19 The main prominent disease focus for it has been in cancer. Some NP formulations for cancer treatment have already been approved by regulatory agencies and are routinely used in several clinical therapies, showing reduced adverse events compared to traditional treatments.20 The principal purpose of targeted drug delivery is to locally treat the disease by improving the drug–target interaction, especially for drugs that present difficulties in delivery because of their poor water solubility. NPs used in drug delivery include virus-based NPs, lipid-based polymers, and dendrimers. They improve the drug uptake by altering the exposure time and also the specific targeting, which allows a targeted-drug release reducing side effects.1 Nanodelivery systems can easily achieve favorable drug release profiles, but toxicity, stability, and degradation properties of the nanomaterial are yet to be well evaluated.20 The process requires that the particles traverse the cell membranes and interact with specific cells.7 Poor penetration and difficulties in drug release have been reported as the main problems for this strategy. A powerful propulsion and enhanced navigation capabilities are needed for an efficient delivery to the specific target, to overcome the limited tissue penetration, the autonomous release of the drug, and the swimming against blood low.1 Moreover, toxicity testing of NPs is not easy, as in vitro models do not allow the fully characterization of the variabilities in how the different tissues and organs interact with NPs.20 Thereby, the success of the

2 Structure and composition of the protein corona

interaction lies on the biocompatibility of NPs with the biological medium,7 which is termed as “protein corona effect.” This phenomenon is so far poorly understood, causing the major deficiency for nanomaterials to be commercialized as biomedical tools. The protein corona includes the proteins and other biomolecules that cover the surface of the NPs forming layers inside the biological medium. Since the surface of the NP changes due to the protein coatings, the actual perception of the NP for a biological entity becomes totally different from the expected one. Protein corona also involves the creation of a new interface between the nanomaterials and the biological medium that is called bio–nano interface, causing dissociation between the NP properties and the resulting biological response. This issue establishes the need to assess the potential risk of the physicochemical peculiarities of nanomaterial interaction with the human body. That is the reason why the characterization of the protein corona is one of the main objectives in nanomedicine. Dynamic interactions, kinetics, and thermodynamic exchanges between the surface of the NPs and the biological components should be examined.1 Complete characterization includes measurements of size, curvature, porosity, chemistry of the used material, composition, state of dispersion, surface area, chemistry and functionalization, crystallinity, heterogeneity, roughness, and hydrophobicity or hydrophilicity.21,22

2  STRUCTURE AND COMPOSITION OF THE PROTEIN CORONA As said before, protein corona modifies the size and the surface composition of the NPs, giving them a new identity that determines the full physiological response, which is unique to each nanomaterial and depends on the recently cited parameters.1 Blood is the first physiological environment that the NP “meets” after entering the human body. Blood plasma contains about 1000 different proteins and lipids in various concentration ranges.23 These components adsorb to the surface of NPs in function of their protein–NP affinity (the so-called “Vroman’s effect”24) and protein–protein interaction. According to this effect, high-affinity proteins in low concentration could replace highly concentrated proteins with low affinity for the NPs. The structure and composition of the protein corona depends on the features of the chosen nanomaterial (size, shape, composition, surface charges, etc.), the biological environment (blood, interstitial fluid, cell cytoplasm, etc.), the conditions, and the time of exposure. A scheme of the protein corona surrounding a NP and its possible interaction paths with cells inside the human body is shown in Fig. 3.2.1. The composition of the protein corona is dynamic and highly dependent on the initial biological environment, which indicates the possibility of exposure memory.25 High-affinity proteins form the “hard” corona (tightly-bounded proteins that do not desorb), while low-affinity proteins conform the “soft” corona (loosely bounded proteins). Composition of the corona changes over time, corresponding the larger exchange times to the hard corona,23 which defines the biological and physiological identity of the particle.26 A recent model proposed by Simberg et al.27 suggests that the hard-corona proteins directly interact with the NP surface and the soft-corona proteins interact

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FIGURE 3.2.1  Scheme of Protein Corona Different layers of biomolecules are coating the nanoparticle (NP) surface, which depends directly on the component of the biological fluids. The protein corona has strong influence on the biological effect of the targeted drug delivery, which can be carried out by (A) direct interaction with a membrane receptor of the cell. (B) Direct penetration of the NP through the cell membrane and release of the drug in the cytoplasm. (C) Penetration of the cell membrane and interaction with a cytoplasmic receptor. Each of the processes triggers a response to the drug inside the cell.

with the hard corona via protein–protein interactions.28 A summary of the proteins than mainly conform protein coronas around NPs are collected in Table 3.2.1. Moreover, secondary binders would alter the activity and/or conformation of the primary binders or mask them, avoiding the direct interaction with the surrounding environment. Thereby, there are several protein layers in each NP, and the thickness of the protein corona depends on several factors, most of them related with the composition and characteristics of the environment, for example, protein concentration, particle size, and surface properties. Walkey and Chan recently identified 125 plasma proteins that were screened in a protein corona involving at least one nanomaterial.28 In the last 25 years, mostly of the reported results show that a “typical” protein corona consists of about 2–6 high abundance tightly adsorbed proteins, and many others with lower abundances. During adsorption, proteins may experience conformational and thermodynamically favorable changes that are typically irreversible. Protein corona is affected by the material of the NP, the size, and the surface morphology in combination with the chemical functionalization, hydrophilicity, hydrophobicity, and the biological environment.1 There are other issues whose effects have

2 Structure and composition of the protein corona

Table 3.2.1  Mostly Described Proteins Integrating Protein Coronas Main Protein Corona Composition Serum albumin Actin Myosin Hemoglobin Vitamin-binding proteins Tubulin Fibrinogen Transferrins Immunoglobulins Complement proteins Ig k chain C region Ig λ-2 chain C region Ig γ-3 chain C region Ig γ-1 chain C region Lipoproteins Apolipoprotein A-I Apolipoprotein E Apolipoprotein A-IV Glycoproteins α-2-HS-glycoprotein β-2-glycoprotein 1 Hemopexin Adapted from Corbo C, Molinaro R, Taraballi F, Toledano Furman NE, Hartman KA, Sherman MB, et al. Unveiling the in Vivo Protein Corona of Circulating Leukocyte-like Carriers. ACS Nano 2017;11:3262–7329; Pelaz B, Dullin C, Parak WJ, Pelaz B, Alexiou C, Puebla RAA, et al. Diverse applications of nanomedicine. ACS Nano 2017;11:2313–8120; Pisani C, Gaillard JC, Dorandeu C, Charnay C, Guari Y, Chopineau J, et al. Experimental separation steps influence the protein content of corona around mesoporous silica nanoparticles. Nanoscale 2017;9:5769–72.30

yet to be studied, like temperature and gradient plasma. Conformational changes in proteins mainly focus on secondary structure of hard corona proteins, which conform the closest layer to the nanomaterial.31 Some of the reported modifications include changes from a closed to open conformation of proteins,32 a state maintained after the removal of NPs, destabilization and structural loss of the bound proteins,33 or protein function alteration by induced protein-folding changes.34 However, conformational changes in proteins remains being a wide area yet that has to be deeply studied and further characterization is still required The effect of protein corona inside the human body has been so far characterized mainly by in vitro experiments, which have been shown to minimally reproduce in vivo phenomena. A recent study characterized the in vivo protein corona on biomimetic liposomes (called leukosomes).29 This experiment revealed a lower accumulation

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of NPs in mononuclear phagocyte system organs and a prolonged circulation time in blood when comparing to an in vitro control, so it is demonstrated that the in vivo protein corona differently modifies the behavior of NPs in the biological fluid, and subsequently the interaction with the cells. The initial stages of plasma membrane receptor recognition of the NP–protein coronas are crucial to understand the biodistribution and some signaling responses initiated on the membrane.35 Otherwise, many biological responses to the environment are initiated intracellularly, so in larger timescales, when biomolecules have been fully degraded, these processes will mediate in the NP–cell interaction. For intermediate times, intracellular recognition may mediate in the processing, degradation, and lysosomal export of NPs (as in case of the immune system). Endogenous proteins seem to remain a longer time carried on the NP surface, which could induce subtle biological effects that must be yet identified.

3  METHODS FOR IDENTIFICATION AND EVALUATION OF THE PROTEIN CORONA The studies for determining the interaction of proteins with NPs in biological media are complex, since more than 3700 proteins in different concentrations coexist and compete for the surface of NPs.36 Li et al.37 and Mahmoudi et al.32 recently reviewed characterization methods to study these interactions. Some of these techniques can be only employed to study the interaction of a single protein with NPs, while others can simultaneously evaluate the interaction with multiple proteins. In Fig. 3.2.2, a scheme of the techniques mostly employed of evaluation of the protein corona is shown. Conventional approaches to assess the protein corona involve the incubation of NPs with mixtures of proteins obtained from different biological environments (such as plasma, serum, interstitial fluid, etc.) for variant periods of time (between 10 min and a few hours), and the elimination of unbounded proteins using ultracentrifugation,38 column chromatography,39 or density gradient purification.40 After 3–4 centrifugation washing steps, the wasted proteins are mostly eliminated, although the risk of losing weakly bounded proteins has to be assumed. Thickness, density, protein identity, protein quantity, protein affinity, protein arrangement, and protein conformation are the main parameters that identify the protein corona. In this sense, Aggarwal et al.41 collected recent studies focused on the techniques for protein isolation (e.g., centrifugation, gel filtration, magnetic separation for magnetic NPs, affinity chromatography), and separation and identification [electrophoresis (2D-PAGE, SDS-PAGE) followed by tandem mass spectrometry (MS/MS)]. Next, the main employed techniques are summarized and discussed.

3.1 CENTRIFUGATION Most of the experiments for the evaluation of protein corona require incubation time of NPs with the biological medium or proximal fluid (such as blood plasma, interstitial fluid, cell cultures, etc.). Centrifugation is the conventional method for isolating the protein–NP complex from the medium and the nonbounded proteins. It is

3 Methods for identification and evaluation of the protein corona

FIGURE 3.2.2  Summary of Methodological Approaches Useful for the Characterization of the Protein Corona

necessary to consider the washing steps, gradient, and solution volume, because it could affect the protein corona. To obtain a high-yield isolation of the proteins of interest, centrifugation can be employed combined with other methods like gel filtration, size-exclusion chromatography, magnetic separation, and microfiltration.1 Walczyk et al.42 studied the stability and structure of protein–NP complexes when they are incubated in normal human plasma. For many nanomaterials, protein corona is about 10 nm thick, so differential centrifugation sedimentation would allow to semiquantitatively measure the size distribution of the complexes in a protein–NP mixture. Thanks to differential centrifugation sedimentation, it has been determined that the increase or dilution of NPs in plasma solution changes the thickness of hard corona.1 In fact, NPs could aggregate or change the size distribution depending on each particular biological environment. Thus, the NP tracking and analysis techniques allow measuring NPs in suspension particle-by-particle, offering a higher resolution and a better understanding of aggregation than classical light-scattering techniques. Montes-Burgos et al.43 applied the NP tracking and analysis technique to a multiparameter analysis for the characterization of the NP size. This multiparameter measurement allows resolving, in situ and in real time, NP subpopulations with different features in a complex mixture.

3.2  CIRCULAR DICHROISM Secondary structures of proteins have their own circular dichroism (CD) spectra in the ultraviolet (UV) region. Thereby, this method has been applied for monitoring conformational changes of the proteins because of the interaction with NPs surface.44

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NPs do not interfere in the CD spectra because they are not chiral. This approach gives an average of the entire molecular population, so it can be determined which percentage of the protein structure is α-helix or β-sheet. The CD spectrum in the nearUV (250–350 nm) can also be sensitive to certain aspects of tertiary structure thanks to the chromophores that produce the CD signals at that wavelength. The spectra can be sensitive to small changes in the tertiary structure produced by the protein–protein interactions and/or by the solvent. CD cannot be applied on complex mixtures, but it provides useful information on protein structure changes adsorbed on the NPs’ surface. A recent research developed by the teams of Joint Research Center, Institute for Health and Consumer Protection (JCR-IHCP) in collaboration with scientists at the diamond synchrotron radiation resource in the UK showed that it is possible to measure the alterations generated in proteins attached to NPs using synchrotron radiationbased circular dichroism spectroscopy, displaying sensitivity levels that have not been previously described.45 The technique allows the measurement of critical parameters related to protein–NP interaction, and provides data on the relative stability of biological proteins. It also helps understanding and predicting the potential toxicology of nanomaterials, so it may contribute to the design of the next generation NPs that could be nontoxic and then ideal for being employed as drug delivery systems.

3.3  ISOTHERMAL TITRATION CALORIMETRY Isothermal titration calorimetry can quantitatively determine thermodynamic parameters in solution, such as binding affinity, binding stoichiometry, and binding enthalpy change. Temperature variation is measured after titration of protein to the NP solution, and these variations are fitted to isothermal functions. An example of application for this technique is the human serum albumin, which in association with NPs produce an exothermic reaction. The protein–material interaction increases proportional to its hydrophobic capacity, which leads to a realistic identification and quantification of the protein.46

3.4  SDS-PAGE AND WESTERN BLOTTING Electrophoresis is a largely known method for the separation and analysis of protein mixtures. Charged molecules are dispersed in a solution and they migrate under an electric field. Several variants of the method have been described along the years, like capillary electrophoresis and gel electrophoresis (1D or 2D), which are conventionally employed for analyzing protein–NP complexes.23,47 SDS-PAGE and Western blotting are qualitative methods based on comparison, so it is quite complicated to obtain quantitative data out of them. In general, they can detect between 1 and 50 ng of a single protein band.41

3.4.1  Capillary electrophoresis

Separation of proteins can be registered using a UV or fluorescence detector.47 The detection sensitivity is not very high, but this method was used to study the adsorption

3 Methods for identification and evaluation of the protein corona

of albumin onto poly-(methoxypolyethyleneglycolcyanoacrylate-co-geadecylcyanoacrylate) NPs allowing direct quantification without a desorption procedure.

3.4.2  1D gel electrophoresis The proteins are separated during migration on a gel of acrylamide and bisacrylamide by different size and electrophoretic mobility.48 Proteins can be analyzed in their native form or after denaturalization. The negatively charged proteins are obtained by boiling them with reducing agents (like DTT or mercaptoethanol) and anionic detergents. The treatment with SDS causes ionic repulsion and so the proteins detach from the NP surface. Proteins in gel can be stained (i.e., Coomassie, silver stain) and a routine densitometry analysis is enough to quantify their abundance.23 SDS-PAGE is a quick and cheap low-cost method; however, there are difficulties on protein separations when the protein sample is too complex.

3.4.3  2D gel electrophoresis In the early years of proteomics area, it was one of the powerful methods that allows separating more than 5000 proteins in two dimensions within the same gel.47 In the first dimension, they are separated according to their charge by isoelectric focusing. In the second dimension, they are separated according to their molecular weight by SDS-PAGE. This technique has been optimized for the analysis of adsorbed plasma proteins on polystyrene particles, solid lipid NPs and magnetite NPs.49 After separation, a staining method is required to visualize the protein spots in the gel and then they are transformed in a digital image. It should be sensitive to low-abundance proteins and allow a linear dynamic range for all the spots in the gel. Ammoniac silver staining and fluorescent approaches ensure high sensitivity and detect less than 1 ng of protein. Difference in gel electrophoresis (DIGE) is another interesting approach that requires the use of cyanine fluorophore dyes (Cy dyes). This compound covalently binds proteins in a mixture before the isoelectric focusing and 2D-PAGE. DIGE requires the label of each sample with one Cy dye, but it allows a fluorescent scanner after the protein migration that shows different images from the same gel, using emission and excitation settings for each dye. If two sample conditions are run in the same gel, this technique allows identifying spot changes in the same gel, reducing possible errors generated by running samples in different gels. After image capture, same spots across the gels must be matched, which can be quite challenging. Significantly different gel spots can be analyzed by mass spectrometry (MS) assessing the protein identification.1 Although 2D techniques are relatively quick and provide a reproducible method for identifying proteins, obtaining high-gel quality can be time consuming, especially when dealing with fluorescent staining. 2D-PAGE method has been widely applied to characterize protein corona.50,51 The obtained data is compared with a master map of human proteins, normally a master map of the human plasma proteome (it is the most studied biological medium). This map is versatile and depends on the donor or the anticoagulant used for the collection process. To increase accuracy, N-terminal sequencing on individual spots have been employed, to better identify the proteins in the 2D-PAGE pattern. Identification

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of proteins in each spot can be performed by MS followed by immunoblotting or peptide sequencing and Western blotting.41 Besides 2D-PAGE, gel filtration like size-exclusion chromatography or affinity chromatography can also be employed for protein separation. Collected proteins from chromatography do not usually need more separation and can be identified by MS and peptide or N-terminal sequencing approaches.41

3.5  UV–VISIBLE SPECTROSCOPY UV–visible spectroscopy is based on the measurement of the ratio of the passed light with respect to the incident light in the wavelength range from the UV to the visible. The adsorption of proteins on the surface of the NPs induces changes on the absorption spectrum, which makes this method a fast and simple strategy that does not require sophisticated sample preparations or equipment. Nonetheless, it is not a very accurate method for a quantitative analysis, since the absorbance depends on multiple parameters (aggregation, temperature, pH, etc.). In fact, this approach is useful to provide macroscopic information about the nature of the protein corona for metallic NPs, as plasmonic peaks can be monitored during the protein adsorption, a process that is highly sensitive to the conditions of the NPs. In some studies, using gold NPs, plasmonic peaks showed a redshift due to the formation of a dense dielectric layer onto the surface of the mentioned nanomaterial.52,53

3.6  FLUORESCENCE SPECTROSCOPY This technique measures the fluorescence emitted by a chemical compound that is excited by a determined wavelength. Some amino acid groups like tyrosine, tryptophan, and phenylalanine have fluorescent properties and so are called fluorophores. Either the NP or the protein, or even both, can be fluorescent, and if they are not, a fluorophore can be added to the system; however, it is necessary to mention that this addition could modify the conformation and the affinity of proteins for the NPs. Fluorescence spectroscopy is sensitive to protein dynamics because of the time scale of the excitement process (nanoseconds), which is the same time scale of many important biological processes like conformational changes or molecular binding.45 The protein–NP binding can be monitored by steady-state54 or time-resolved fluorescence spectroscopy, fluorescence resonance energy transfer, or stepwise single-molecule photobleaching. A derivative of poly (p-phenylene ethynylene) has been used as fluorescent indicator to study the adsorption on the surface of gold NPs.55 The attachment results in the transfer of excited electrons from the fluorophore to the metallic NP, so that fluorescence is quenched. If proteins adsorb to the surface, the poly (p-phenylene ethynylene) detaches, and fluorescence intensity increases. Röcker et al.56 analyzed the adsorption of human serum albumin onto polymercoated FePt and CdSe/ZnS by fluorescence correlation spectroscopy, which formed a 3.3 nm monolayer protein corona. Fluorescence correlation spectroscopy provided

3 Methods for identification and evaluation of the protein corona

quantitative data on the dynamism of the protein corona in a particular biological environment. This application could be useful for other nanoscience uses.

3.7  MASS SPECTROMETRY Mass spectrometry is an analytical essential technique in proteomics studies to identify protein identities. It ionizes samples after digesting and fragmenting them in small peptides providing qualitative and quantitative information of the protein mixture. It has been widely applied to identify protein coronas after a previous sample separation on SDS-PAGE. The band of interest on the electrophoresis gel is cut and digested in gel using a peptidase (e.g., trypsin, Lys-C) to extract the peptides that would be analyzed by MS. Conversely, this digestion can be performed in solution. Moreover, protein denaturation is needed, and the denaturing agent should be carefully chosen to avoid reduction of trypsin efficacy. Peptides can be separated by 2D HPLC chromatography (ion exchange followed by reverse-phase chromatography before MS/MS studies) to ease the process. This combination has resolved thousands of plasma proteins over 104 dynamic concentration ranges without depleting abundant proteins. It has been applied to analyze plasma proteins that bind to dextrancoated iron oxide NPs.27 Tenzer et al.57 recently studied long-lived blood plasma-derived corona on monodispersed amorphous silica NPs of different sizes (20, 30, and 100 nm). They used liquid chromatography, MS, 1D and 2D gel electrophoresis, and immunoblotting to analyze the protein corona composition. They identified 125 proteins and observed an enrichment of specific lipoproteins and coagulation proteins. Immunoglobulins displayed a lower affinity for the particles. They demonstrated that electrostatic effects by themselves are not the main driving force in regulation of the protein–NP interactions. MS has experienced great developments when referring to protein quantification, to improve the accuracy. One of these improvements is the “shotgun MS/MS,” which is a proteomic method that combines an LC system connected online to a tandem mass spectrometer operated in electrospray ionization mode (LC–MS/MS). In LC, the peptide components present in the sample are separated by reversed phase liquid chromatography and analyzed by MS in the full-scan and MS/MS modes. The method is uniquely suited for protein components identification, including their posttranslational modifications. If it is used with stable isotope-based labeling, it allows not only protein qualification, but also quantification. The system works acquiring continuous series of fragment-ion spectra of selected peptide ions after proteolytic digest of the samples. These spectra, combined with information on the precursor ions, are analyzed to determine the amino acid sequence of the fragmented peptides and to infer the proteins from which the peptides originated. The method is almost exclusively applied for discovery experiments, aiming to identify large sets of proteins in complex samples, such as the ones bonded to a NP.58 Another MS/MS improvement is the selected reaction monitoring. It is a nonscanning technique generally performed on triple-quadrupole instruments (QQQ) in

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which fragmentation is used to increase selectivity and sensitivity. Two mass analyzers are used as static mass filters to monitor a particular fragment ion of a selected precursor ion. The selectivity resulting from the two filtering stages combined with the high-duty cycle results in quantitative analyses with unmatched sensitivity. The specific pair of m/z ratio values associated with the precursor and fragment ions selected is referred to as a “transition”.59 Multiple reaction monitoring is the application of selected reaction monitoring to multiple product ions from one or more precursor ions.60 This technique is widely applied for targeted qualitative proteomics. If it is combined with isotopic labeling, multiple reaction monitoring can be used to construct a calibration curve that provides the absolute quantification of the native peptide, and, therefore, its parent protein.59 Among qualitative characterization by MS of the protein corona, it is possible to provide quantitative information of the proteins which are forming the different layers of the protein corona of a specific nanomaterial in a proximal fluid of interest. Then, several MS-based strategies are quite useful and here they are described.

3.7.1 SILAC Stable isotope labeling with amino acids in cell culture (SILAC) and its derivatives, such as N-labeling61 and NeuCode (neutron encoded) SILAC,62 are technologies in which cells or even organisms are grown in media supplemented with arginine or lysine residues containing combinations of 13C and 15N stable isotopes, generating proteins with altered masses.63 The principle of SILAC is based on metabolically incorporating these isotopes into the entire proteome during protein metabolism. Two populations of cells grow in different culture media, the “light” medium containing the natural isotope in the amino acids, and the “heavy” medium containing stable isotope-labeled aminoacids. After at least five cycles of cell divisions, proteins of the cells in heavy medium theoretically contain amino acids in the heavy state. When cultures are digested and analyzed by LC–MS, each digested peptide contains at least one of these labeled amino acids, inducing small shifts in the detected m/z ratio and enabling the relative quantification of isotopelabeled peptides compared to unlabeled peptides.64 SILAC-based approaches have been applied to quantitatively identify protein coronas, such as carbon nanotube protein corona.65 A HeLa-labeled-cells lysate was incubated with the NPs, and after the protein–NP interaction happened, agglomerates were collected and analyzed by SDS-PAGE. The isotope labeling allowed identifying the proteins that bind to the NPs. Established SILAC reagents have only allowed, until recently, the analysis of three samples simultaneously (the so-called light, medium and heavy labeling), but this number has been increased by new developments in the field. NeuCode SILAC applies a collection of isotopologues of lysine with subtle mass differences achieved by the incorporation of deuterium into its side chain, and can be used to analyze up to 18 different samples at the same time.62

3 Methods for identification and evaluation of the protein corona

3.7.2  iTRAQ Isobaric tags for relative and absolute quantitation (iTRAQ) is an isobaric labeling method in MS/MS applied to determine the proteins coming from different sources in a single experiment. Peptides are covalently labeled with stable isotope molecules with tags of varying mass introduced from iTRAQ specific reagents in the N-terminus and side chain amines of peptides after proteolysis.66 iTRAQ reagents are isobaricmass designed, so differentially labeled peptides appear as single peaks in MS scans, reducing the probability of pear overlapping. When iTRAQ-tagged peptides are subjected to MS/MS analysis, the mass balancing carbonyl moiety is released as a neutral fragment, and so it liberates isotope-encoded reporter ions that provide relative quantitative information on proteins. Four different reagents are available, therefore comparison of two to four samples within a single MS run is feasible. Enzymatic digestion of proteins prior to labeling artificially increases sample complexity, but it has been shown as a requirement for quantitative evaluation of protein dynamics.67 iTRAQ approach is proposed to be best applicable to samples of moderate to low complexity that can be separated by cation-exchange chromatography followed by HPLC. iTRAQ strategy has shown a great propensity to identify proteins of high abundance, which reflects the limited effectiveness of peptide separation and, therefore, a lower probability to identify low-abundance proteins in complex biological samples.68 iTRAQ is useful for protein corona quantification to identify the hard-corona proteins after unbinding proteins from NPs and digesting them for an improved resolution of results. This technique is robust and presents more sensitivity and better accuracy of quantification than 2D-DIGE.69

3.7.3  Label-free quantification Label-free quantification is a method in MS that determines the relative amount of proteins in two or more biological samples, but unlike other quantitative methods, is does not use a stable isotope that chemically binds and labels the protein.70 Typically, peptide signals are detected at the MS1 level, and their isotopic pattern allows distinguishing them from chemical noise. Patterns are then tracked across the retention time dimension and used to reconstruct a chromatographic elution profile of the monoisotopic peptide mass. The total ion of the peptide signal is then integrated and used as a quantitative measurement of the original peptide concentration. For each detected peptide, all isotopic peaks are first found and the charge state is then assigned. Label-free quantification is based on precursor signal intensity and presents issues when identifying the peptide precursor ion that is being measured, which might be a different peptide with a similar m/z ratio and the same time elution, or it could overlap with other peptides. Spectral counting has problems since the peptides are identified, thus making it necessary to run an additional MS/MS scan which takes time reduces the resolution of the experiment.71 In contrast to differential labeling, every sample must be measured separately in a label-free experiment. The extracted peptide signals are then mapped across LC–MS measurements using their coordinates on the m/z ratio and retention-time

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dimensions. Differential processing of biological samples reveals the need of having a standard that can be used to adjust the results. Peptides that are not expected to change in their expression levels in different biological samples can be used for this purpose. Normalization methods are used to remove systematic background noise in the peptide intensity values between LC–MS measurements.70 Then, discriminatory peptides are identified by selecting the peptides whose normalized intensities are different among the samples.72 Several label-free quantification methods have been used to study proteins adsorbed to NPs simultaneously to labeling approaches.73

3.8  FOURIER TRANSFORM INFRARED AND RAMAN SPECTROSCOPIES Raman and Fourier transform infrared (FTIR) spectroscopies give information about the surface properties of protein–NP complexes and allow detecting the protein binding onto the surface. They also allow confirming the protein attachment to NPs through the appearance of additional characteristic bands.1 Vibrational spectroscopy has spectral crowding as associated experimental problem. Raman spectroscopy allows measuring the protein–NP complexes in aqueous solution and its spectrum is simpler than the one obtained from IR because the localized vibrations of doubleor triple-bonded or electron-rich groups produce intense Raman bands than vibrations of single-bonded or electron-poor groups. FTIR has been applied to monitor the structure of protein–NP complexes, estimating the protein secondary structures thanks to the absorption of amine bonds.44

3.9 NMR Nuclear magnetic resonance (NMR) has been used to identify lipophilic molecules like cholesterol, triglycerides, or phospholipids from human plasma that bind copolymer NPs after size-exclusion chromatography.74 The binding patterns agree with the composition of high-density lipoprotein. Apolipoproteins bind to many other NPs, suggesting that lipid and lipoproteins could generally associate to NPs under physiological conditions. Solid-state NMR has also been applied to the study of in situ secondary-structure determination of statherin peptides on hydroxyapatite surfaces.75 Thanks to this method, it was possible to design biomimetic fusion peptides that use nature’s crystalrecognition mechanism to display accessible and dynamic bioactive sequences from the hydroxyapatite surface.

3.10 X-RAY X-ray crystallography is also a routine technique to determine how a drug interacts with its target and what modifications could improve the interaction. Prakasham et al.76 have investigated diastase enzyme immobilized on nickel-impregnated silica paramagnetic NPs and they characterized them by FTIR and X-ray crystallography.

4 Conclusions and perspectives

Immobilized enzyme showed more affinity to substrate than the free one, and it was further characterized for its biocatalytic activity. Thus, the enzyme–NP binding profile varied by changing the physiological conditions (i.e., pH of the reaction mixture).

4  CONCLUSIONS AND PERSPECTIVES NPs have a great relevance and potential applications in biomedical sciences from diagnostic tools to targeted drug delivery systems. Prior to its daily and normalized use, their effect on human body and health should be concretely determined. The interactions of NPs with biological media induce the formation of a layer of biomolecules, mostly proteins, coating the NP. This layer is called protein corona and it may modify the NP identity inside the human body, acquiring new characteristics and capabilities. Protein corona needs to be deeply identified, quantified and characterized because it has a critical role in toxicity and biocompatibility of nanomaterials within the human fluids and limits and modifies the recognition of NPs by cells, tissues, etc. The behavior of nanomaterials is also conditioned by the protein–NP interactions. Specifically focusing on targeted drug delivery, protein corona could affect the process so that the drug does not reach the target, and is degraded or even delivered to the wrong target. Bearing this in mind, it is required to identify biomarkers useful to understand the protein corona and its effects on the mechanism for an efficient targeted drug delivery. Wide proteomic methods have been reported to identify and characterize the compounds of the protein corona. The process can be very complex because of the complexity of protein coronas, so further studies are needed to simplify the quantification and improve the speed and quality on protein corona identification. Protein corona could be exploited as a great resource to improve the vectorization of NPs inside the body, but since their composition is barely known and it is complicated to predict their composition and the potential biological implications, it is necessary to keep studying the behavior of protein corona in vivo. Experimentation on this field is still ongoing to acquire better knowledge of toxicity, biocompatibility and behavior of NPs when contacting biological media. An optimal understanding of protein–NP interactions will allow applying nanomaterials in many complex biomedical processes, making them easier and faster, and constituting a challenge to be achieved by nanoscience and nanomedicine.

ACKNOWLEDGMENTS We gratefully acknowledge financial support from the Spanish Health Institute Carlos III (ISCIII) for the grant: FIS PI114/01538. We also acknowledge Fondos FEDER (EU), Junta Castilla-León (grant BIO/SA07/15), and Fundación Solórzano (FS23/2015). The Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001, of the PE I + D + I 2013–2016, funded by ISCIII and FEDER. P.D. and C.D. are supported by a JCYLEDU/346/2013 PhD scholarship.

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40. Moore A, Weissleder R, Bogdanov A. Uptake of dextran-coated monocrystalline iron oxides in tumor cells and macrophages. J Magn Reson Imaging 1997;7:1140–5. 41. Aggarwal P, Hall JB, McLeland CB, Dobrovolskaia MA, McNeil SE. Nanoparticle interaction with plasma proteins as it relates to particle biodistribution, biocompatibility and therapeutic efficacy. Adv Drug Deliv Rev 2009;61:428–37. 42. Walczyk D, Bombelli FB, Monopoli MP, Lynch I, Dawson KA. What the cell “sees” in bionanoscience. J Am Chem Soc 2010;132:5761–8. 43. Montes-Burgos I, Walczyk D, Hole P, Smith J, Lynch I, Dawson K. Characterisation of nanoparticle size and state prior to nanotoxicological studies. J Nanopart Res 2010;12:47–53. 44. Xiao Q, Huang S, Qi Z-D, Zhou B, He Z-K, Liu Y. Conformation, thermodynamics and stoichiometry of HSA adsorbed to colloidal CdSe/ZnS quantum dots. Biochim Biophys Acta 2008;1784:1020–7. 45. Mu Q, Liu W, Xing Y, Zhou H, Li Z, Zhang Y, et al. Protein binding by functionalized multiwalled carbon nanotubes is governed by the surface chemistry of both parties and the nanotube diameter. J Phys Chem C 2008;112:3300–7. 46. Lindman S, Lynch I, Thulin E, Nilsson H, Dawson KA, Linse S. Systematic investigation of the thermodynamics of HSA adsorption to N-iso-propylacrylamide/N-tertbutylacrylamide copolymer nanoparticles. Effects of particle size and hydrophobicity. Nano Lett 2007;7:914–20. 47. Kim HR, Andrieux K, Delomenie C, Chacun H, Appel M, Desmaële D, et al. Analysis of plasma protein adsorption onto PEGylated nanoparticles by complementary methods: 2-DE, CE and Protein Lab-on-chip® system. Electrophoresis 2007;28:2252–61. 48. Cedervall T, Lynch I, Lindman S, Berggard T, Thulin E, Nilsson H, et al. Understanding the nanoparticle-protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles. Proc Natl Acad Sci 2007;104:2050–5. 49. Kai T, Martin L, Wolfhard S, Rainer H, Müller MK. Determination of plasma protein adsorption on magnetic iron oxides: sample preparation. Pharm Res 1997;14:905–10. 50. Gessner A, Waicz R, Lieske A, Paulke BR, Mäder K, Müller RH. Nanoparticles with decreasing surface hydrophobicities: influence on plasma protein adsorption. Int J Pharm 2000;196:245–9. 51. Göppert TM, Müller RH. Polysorbate-stabilized solid lipid nanoparticles as colloidal carriers for intravenous targeting of drugs to the brain: comparison of plasma protein adsorption patterns. J Drug Target 2005;13:179–87. 52. Tessier PM, Jinkoji J, Cheng YC, Prentice JL, Lenhoff AM. Self-interaction nanoparticle spectroscopy: a nanoparticle-based protein interaction assay. J Am Chem Soc 2008;130:3106–12. 53. Casals E, Pfaller T, Duschl A, Oostingh GJ, Puntes V. Time evolution of the nanoparticle protein corona. ACS Nano 2010;4:3623–32. 54. Shang L, Wang Y, Jiang J, Dong S. PH-dependent protein conformational changes in albumin: gold nanoparticle bioconjugates: a spectroscopic study. Langmuir 2007;23:2714–21. 55. You C-C, Miranda OR, Gider B, Ghosh PS, Kim I-B, Erdogan B, et al. Detection and identification of proteins using nanoparticle-fluorescent polymer “chemical nose” sensors. Nat Nanotechnol 2007;2:318–23. 56. Röcker C, Pötzl M, Zhang F, Parak WJ, Nienhaus GU. A quantitative fluorescence study of protein monolayer formation on colloidal nanoparticles. Nat Nanotechnol 2009;4:577–80.

References

57. Tenzer S, Docter D, Rosfa S, Wlodarski A, Kuharev J, Rekik A, et al. Nanoparticle size is a critical physicochemical determinant of the human blood plasma corona: a comprehensive quantitative proteomic analysis. ACS Nano 2011;5:7155–67. 58. Domon B, Aebersold R. Options and considerations when selecting a quantitative proteomics strategy. Nat Biotechnol 2010;28:710–21. 59. Lange V, Picotti P, Domon B, Aebersold R. Selected reaction monitoring for quantitative proteomics: a tutorial. Mol Syst Biol 2008;4:222–35. 60. Murray KK, Boyd RK, Eberlin MN, Langley GJ, Li L, Naito Y. Definitions of terms relating to mass spectrometry (IUPAC Recommendations 2013)*. Pure Appl Chem 2013;85:1515–609. 61. Ippel JH, Pouvreau L, Kroef T, Gruppen H, Versteeg G, Van Den Putten P, et al. In vivo uniform 15N-isotope labelling of plants: using the greenhouse for structural proteomics. Proteomics 2004;4:226–34. 62. Hebert AS, Merrill AE, Bailey DJ, Still AJ, Westphall MS, Strieter ER, et al. Neutronencoded mass signatures for multiplexed proteome quantification. Nat Methods 2013;10:332–4. 63. Carneiro DG, Clarke T, Davies CC, Bailey D. Identifying novel protein interactions: proteomic methods, optimisation approaches and data analysis pipelines. Methods 2016;95:46–54. 64. Chen X, Wei S, Ji Y, Guo X, Yang F. Quantitative proteomics using SILAC: principles, applications, and developments. Proteomics 2015;15:3175–92. 65. Cai X, Ramalingam R, Wong HS, Cheng J, Ajuh P, Cheng SH, et al. Characterization of carbon nanotube protein corona by using quantitative proteomics. Nanomed Nanotechnol Biol Med 2013;9:583–93. 66. Ow SY, Salim M, Noirel J, Evans C, Rehman I, Wright PC. iTRAQ underestimation in simple and complex mixtures: “the good, the bad and the ugly”. J Proteome Res 2009;8:5347–55. 67. Wiese S, Reidegeld KA, Meyer HE, Warscheid B. Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research. Proteomics 2007;7:340–50. 68. DeSouza L, Diehl G, Rodrigues MJ, Guo J, Romaschin AD, Colgan TJ, et al. Search for cancer markers from endometrial tissues using differentially labeled tags iTRAQ and cICAT with multidimensional liquid chromatography and tandem mass spectrometry. J Proteome Res 2005;4:377–86. 69. Wu WW, Wang G, Baek SJ, Shen RF. Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel- or LC-MALDI TOF/TOF. J Proteome Res 2006;5:651–8. 70. Bantscheff M, Schirle M, Sweetman G, Rick J, Kuster B. Quantitative mass spectrometry in proteomics: a critical review. Anal Bioanal Chem 2007;389:1017–31. 71. Asara JM, Christofk HR, Freimark LM, Cantley LC. A label-free quantification method by MS/MS TIC compared to SILAC and spectral counting in a proteomics screen. Proteomics 2008;8:994–9. 72. Müller LN, Brusniak M-Y, Mani DR, Aebersold R. An assessment of software solutions for the analysis of mass spectrometry based quantitative proteomics data. J Proteome Res 2008;7:51–61. 73. Zhang H, Burnum KE, Luna ML, Petritis BO, Kim J-S, Qian W-J, et al. Quantitative proteomics analysis of adsorbed plasma proteins classifies nanoparticles with different surface properties and size. Proteomics 2011;11:4569–77.

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74. Hellstrand E, Lynch I, Andersson A, Drakenberg T, Dahlbäck B, Dawson KA, et al. Complete high-density lipoproteins in nanoparticle corona. FEBS J 2009;276:3372–81. 75. Stayton PS, Drobny GP, Shaw WJ, Long JR, Gilbert M. Molecular recognition at the protein-hydroxyapatite interface. Crit Rev Oral Biol Med 2003;14:370–6. 76. Prakasham RS, Devi GS, Rao CS, Sivakumar VSS, Sathish T, Sarma PN. Nickel-impregnated silica nanoparticle synthesis and their evaluation for biocatalyst immobilization. Appl Biochem Biotechnol 2010;160:1888–95.

SUBCHAPTER

Soft materials and coatings for controlled drug release

Subchapter3.3 Cecilia Fager, Eva Olsson

Chalmers University of Technology, Gothenburg, Sweden

1 INTRODUCTION Controlled drug release coatings have been around for more than 50 years and their performance has increased significantly since the beginning.1 The development and improvement of the coatings are partly due to more controlled chemistry during their fabrication. The aim of a pharmaceutical product is to obtain an optimal therapeutic and benign treatment.2 It consists of a combination of an effective active pharmaceutical ingredient and a tailored controlled release (CR) rate. It also involves a minimization of the amount of drug that is needed and a reduction of plausible side effects.3–5 Different aspects need to be addressed to obtain the specific drug release behaviour that is intended. Very often, concerning oral CR formulations, the gastrointestinal (GI) tract is of great importance to consider since it is the location of the drug release and from which the drug will be further transported to the target location. The upper part of the GI tract contains, for example, the stomach while the lower part contains the colon.6 The pH-value varies throughout the GI tract and does also vary depending on fed or fasting condition. The pH value varies between 1 and 5.5 in the upper part while it is between 5.5 and 7 in the lower part.7,8 The different pH values of the different parts need to be taken into account when, for example, selecting the soft material to be designed to dissolve in the upper GI tract. Immediate release (IR) formulations, also denoted as conventional formulations, give a rapid release of the drug from the formulation. IR can be provided by several formulation systems, such as tablets, capsules, and pellets, through adding a coating that surrounds the drug. The IR formulations are desired for occasions, such as acute drug delivery targeting diseases in the GI tract or infections that need a fast delivery

1 Introduction

of the drug.9,10 Another application of IR formulations is to provide a barrier to prevent oxygen degradation. Some drugs are sensitive to the presence of oxygen. They can oxidize and thus degrade, and in such cases an IR coating can act as an oxygen barrier.11 Studies performed on free polymer films indicate a reduction in penetration rate of oxygen through the free polymer films. This effect can be used to increase the durability of the product.12 However, if controlled drug release over a longer period of time is desired, then the choice of material is CR formulations. The fundamental constituents of a CR formulation are either a drug core coated with a film (also denoted as membrane) or a matrix in which the drug is dispersed.1 A common factor for both IR and CR are that they are very frequently soft materials consisting of a combination of polymers for a tailored release. Both natural and synthetic polymers are used. Examples of natural polymers are various types of modified cellulose: cellulose derivatives. They are frequently used due to their susceptibility to chemical modification offering possibilities for material design.13,14 Even though two cellulose derivatives have the same backbone, substitution groups can be added, resulting in one cellulose derivative becoming hydrophobic while another can become hydrophilic. Since the purpose of the formulations is to increase the pharmaceutical efficiency for the patient, all involved materials need to fulfill the requirements for being human-body friendly. Hence, free from toxicity and not being harmful either before or after degradation. Polymers used in CR formulations will be further discussed in Section 3 (Subchapter 3.3). The major difference between IR and CR is thus the drug release rate. The intake of IR formulations usually occurs as a repeated administration. When the drug has done its impact, the administration of a new IR formulation occurs, while a CR formulation lasts for a longer period of time. Fig. 3.3.1 shows the different drug plasma

FIGURE 3.3.1  Illustration of the Different Drug Plasma Concentrations For immediate release (IR) formulations, broken line, and controlled release (CR) formulations, solid line. Two administrations of the IR formulations are marked with arrows and denoted as first and second dose. The different drug plasma concentrations are marked with thinner solid lines, which are toxic range, therapeutic range, and ineffective range. Inspired by Langer R, Peppas N. Chemical and physical structure of polymers as carriers for controlled release of bioactive agents: a review. Macromol Sci C 1983;23(1):61–126.15

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concentrations corresponding to IR and CR formulations, where two administrations of IR formulations and one administration of CR formulation occur during the illustrated time period. The focus of this subchapter is from now and onwards directed to CR formulations. There are several factors to be considered when designing a CR formulation. A major factor is the mechanisms of the drug release. Different release mechanisms can occur depending on how the formulation is manufactured and its resulting material nanostructure. There are two main systems of CR formulations. They are defined as reservoir CR system and matrix CR system.15,16 The reservoir CR system is based on a coating that encapsulates the drug, while the matrix-controlled drug release system is based on a drug that is dispersed throughout a matrix. The major release mechanisms for CR formulations are diffusion, dissolution, osmotic pumping, and swelling.13,15,17 These release mechanisms are described in more detail later. The two most frequently encountered release rates for oral CR drug formulations are zero order and first order.15 The zero-order release rate provides a constant drug release rate in the therapeutic range while the first order provides a decreasing rate.

2  CONTROLLED DRUG RELEASE SYSTEMS The CR formulations are divided into two main systems: reservoir-controlled release system and matrix-controlled release system. Even though there exists a huge number of different oral CR formulations, they are often based upon one or a combination of the following release mechanisms: diffusion, dissolution, osmotic pumping, and swelling.15,17 One important factor regarding the diffusion-based release mechanism is the thickness of the coating or matrix, that is, the diffusion path. One way to control the drug release with diffusion is, therefore, to alter the diffusion path in a controlled manner. An example is to utilize a pellet with a coating consisting of two polymers that possess different water solubility. They phase-separate during fabrication. When the pellet reaches the GI tract, the water-soluble polymer will leach out, creating a transport path for the drug along the remaining porous network of the water insoluble polymer. The diffusion path can be controlled by varying the polymer volume ratios.18–20 Characterization of the transport path will be discussed in Section 3.4 of Subchapter 3.3. Regarding dissolution, the importance of the solubility of the material within the surrounding media plays an important role. The release rate depends partly on how easily the coating or matrix is dissolved within the surrounding media. For the case of osmotic pumping, a semipermeable membrane gives rise to a pressure difference which will release the drug from the CR formulation. The last release mechanism that is covered within this subchapter is swelling. One important factor regarding swelling is that there is no dissolution taking place in the material as in the case of the dissolution mechanism. Instead, the release of the drug occurs only after the material has swollen and allows diffusion through the swollen CR formulation.15 These different release mechanisms are explained in the following subchapters.

2 Controlled drug release systems

2.1  RESERVOIR-CONTROLLED RELEASE SYSTEM The reservoir-controlled release system is based upon a coating that surrounds a drug core and provides the CR function. To deliver the drug with a CR rate, some examples of important criteria that should be fulfilled are as follow. The film should be without cracks and uniform in terms of thickness and density. The film quality is important as its purpose is to provide a stable storage of the final formulation, as well as a light protection for the drug that increase the lifetime of the final product.13 Additionally, the film coating ought to be well adherent to the drug core. It is common that the coating is categorized as being either hydrophobic or hydrophilic and nonporous or porous. Depending on the selected coating material, different release mechanisms or a combination of several are involved. Three possible release mechanisms regarding reservoir CR formulations are presented below. They are diffusion, dissolution, and osmotic pumping. For the case of diffusion as the release mechanism for a reservoir CR system, the coating can be made out of water insoluble polymer and thus possess hydrophobic properties. The coating will thus remain compact in a water environment. The release of the drug arises due to a drug concentration gradient between the inside of the coating and its outside. A constant release, with a zero-order release rate profile, follows since the thickness of the hydrophobic coating and the concentration gradient is not changed for the ideal case.15 The diffusion through a hydrophobic nonporous coating is illustrated in Fig. 3.3.2. Hence, in this case the drug diffuses across the coating. It is also possible to use two materials, for example, two polymers with different solubility within the GI tract. When the CR formulation reaches the GI tract, one polymer

FIGURE 3.3.2  Cross-Section of a Spherical Reservoir CR System The release mechanism is diffusion. The figure shows a nonporous coating where diffusion across the coating has occurred after time t.

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FIGURE 3.3.3  Cross-Section of a Spherical Reservoir CR System The release mechanism is diffusion. (A) Coating consisting of two phase-separated polymers, black represents the water insoluble polymer and gray the water soluble polymer. (B) The coating after contact with water during time t. The water-soluble polymer has been leached leaving a porous transport path through which the drug diffuses.

is leached out.13 This results in creation of porous network within the remaining insoluble polymer. The drug will then diffuse through this porous nanostructure that provides a transport path for the drug. A reservoir CR formulation using a porous coating is shown in Fig. 3.3.3. The release mechanism of dissolution, also denoted erosion, for reservoir CR systems is based on the dissolution of the coating material within the environment that it is surrounded by and thus the drug is released. As for the case of diffusion, one way of controlling the drug release rate for a dissolution system is to control the thickness of the coating. Hence, the solubility properties of the coating material in the surrounding environment play an important role. If the coating has high solubility in the surrounding environment, the dissolution of the coating material occurs at a high rate and the drug will be released faster compared to a coating with a lower solubility. As mentioned in the beginning of the introduction, large pH variations within the GI tract are a fact. Hence, the dissolution of the coating can also be designed to occur depending on the pH value of the surrounding environment, and using this approach, the formulation can be targeted to release the drug at a specific position within the GI tract.13 The release mechanism of osmotic pumping is obtained from a coating, which is semipermeable, with an orifice (Fig. 3.3.4). Provided that the surrounding environment is water and the coating material is water permeable, only water can diffuse through the coating into the drug core. Note that the coating is not expanded or dissolved upon contact with water.13 To utilize the osmotic pumping release mechanism, the drug needs to be water soluble. The solute, drug dissolved in water, will then be released by transportation through the orifice in the same rate as water crosses the coating.

2 Controlled drug release systems

FIGURE 3.3.4  Cross-Section of a Spherical Reservoir CR System The release mechanism is osmotic pumping. The coating is semipermeable, where it is permeable only for water but not for the drug. The figure to left shows the situation at time 0 and the figure to the right shows at time t, where the drug is transported through the orifice.

2.2  MATRIX-CONTROLLED RELEASE SYSTEM The matrix CR systems take advantage of the fact that the drug is either dissolved or dispersed within the matrix material. This type of release system is easier to fabricate in comparison to the reservoir CR systems, but it is harder to achieve a zero order release rate as the distance that the drug is transported across varies with time.13,15 As for the case of reservoir-controlled drug release systems, one or several drug release mechanisms can be involved. Three possible release mechanisms for matrix controlled release systems are presented in the following section. For the case of diffusion as release mechanism for matrix CR system, an insoluble matrix material toward its surrounding environment is selected. The drug particles are dispersed within the matrix. The drug closest to the surface in the matrix will be released initially while the drug further inside the matrix will have to diffuse a continuously increasing diffusion path to be released. This results in a drug-depleted matrix. The depleted matrix volume increases with time as the drug is continuously released, see Fig. 3.3.5. As a consequence, zero-order drug release rates are rare cases for solid matrix CR systems. Contrary to the nondissolved matrix material for diffusion, the matrix material for dissolution is intended to be dissolved within the surrounding environment. The drug particles are distributed throughout the soluble matrix material. With time, the matrix will dissolve and thus the drug will be released in a controlled manner, see Fig. 3.3.6.

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FIGURE 3.3.5  Cross-Section of a Spherical Matrix CR System The release mechanism is diffusion. The drug is dispersed within the matrix. With time, drug is released and the matrix is drug depleted. The diffusion path for the drug increases with time.

FIGURE 3.3.6  Cross-Section of a Spherical Matrix CR System The release mechanism is dissolution. The drug is dispersed within the matrix. With time, the matrix material will dissolve and drug will be released after time t.

3 Soft materials for controlled drug release

FIGURE 3.3.7  Cross-Section of a Spherical Matrix CR System The release mechanism is swelling. The drug is dispersed within a nonswollen polymer matrix. After time t, the polymer swell due to the surrounding environment and the drug release occurs.

The drug release occurs according to zero order rate as the drug release decreases with dissolved matrix material. Soft materials used for the release mechanism of swelling have properties in between the materials for diffusion and dissolution release mechanisms.15 The drug particles are distributed throughout the matrix material and when this material comes in contact with the surrounding environment in, for example, the GI tract, the material swells. As an example, if the matrix material is a hydrophilic polymer, the polymer chains will relax upon contact with water, which would result in swelling. The drug transport properties are different for the nonswollen and swollen states, which thus can be used to control the drug release. Fig. 3.3.7 shows a nonswollen polymer matrix CR system to the left and to the right a swollen polymer matrix.

3  SOFT MATERIALS FOR CONTROLLED DRUG RELEASE The materials for either reservoir or matrix CR systems are frequently made from polymers. Therefore, this paragraph gives an overview of polymers in CR formulations and presents various types of polymeric materials. The polymers are divided into the groups of natural polymers and synthetic polymers and this division is based upon which source is used to produce the polymers.14 Some advantages of natural polymers are that they have high accessibility due to their source, that is, plants, they are hydrophilic and have high affinity for biological tissues. Synthetic polymers, on the other hand, can be synthesized in a controlled manner, resulting in a more controlled drug release rate as the degradation of the polymer can be controlled better.

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Very pure polymers can be produced, which minimizes the risk for contaminations. They can also fairly easily be modified by adding functional groups, which improve the CR properties even further. Some major drawbacks with natural polymers are that they do contain contaminations and need to be purified before they can be used in any pharmaceutical formulations, which is not the case for synthetic polymers; on the other hand, synthetic polymers need to be synthesized. Excipients are additives that can be added to CR formulations. They are added to improve the performance of the formulations. If the aim is to increase the flexibility and thus reduce the brittleness and the risk for cracks within a coating, a so-called plasticizer can be added.13 Other excipients that can improve or affect the release mechanisms are excipients that create pores within the CR formulations; these are denoted as porogens. It is also important to be able to store the final CR formulations in safety, hence no agglomeration of the formulations due to stickiness should occur. This is why excipients denoted antiadherents are added to avoid potential storage problem. Sometimes, the market requests attractive final CR formulations, which can involve coloring of the coatings. For this purpose, excipients can be pigments to provide an attractive color of the formulations.

3.1  NATURAL POLYMERS Natural polymers, also denoted as biopolymers, consist of four groups, which are polysaccharides, polypeptides, lipids, and polynucleotides. However, the biopolymer that is mostly used for pharmaceutical purposes is polysaccharides. Polysaccharides are synthesized by nature, more specific, by plants, wood, bacteria, fungi, and algae.21,22 Polysaccharides possess some very important properties that contribute as good soft materials for the pharmaceutical formulations. Polysaccharides are stable within a certain pH range and they are nontoxic. The stability in different pH environments is important for delivery to the GI tract, as described earlier. Since the CR formulations are introduced to the body, the nontoxicity of the polysaccharides and their degradation products are highly prioritized properties. Some polysaccharides also hold the possibility of acting antibacterially, which is an excellent property in, for example, tissue applications and reduces the risk that the body repels the inserted object. Natural polymers are commonly water soluble and to create pharmaceutical CR formulations, the coatings need to provide the possibility of water insolubility sometimes, which can be achieved by cross linking the polysaccharides. The cross linking is possible as the polysaccharides possess the ability to be chemically modified.21–23 These outstanding properties of the polysaccharides are unfortunately outweighed by their flaws: contaminations. Another factor that is crucial for natural polymers is that the molecular weight of natural polymers often varies that can cause problems considering the reproducibility.24 Research has been performed during the last decades to handle these major factors, make the natural polymers more pure and obtain more uniform molecular weights. Therefore, some polysaccharides, such as cellulose, starch, and alginates have become approved and also standards within pharmaceutical applications. Further on, research to implement the polysaccharide,

3 Soft materials for controlled drug release

FIGURE 3.3.8  Chemical Molecular Structures for Four Common Polysaccharides Used in CRelease Pharmaceutical Formulations The molecular structures correspond to (A) cellulose, (B) chitosan, (C) alginates (alginic acid), and (D) starch (amylose).

chitosan, in CR formulations is ongoing.25 The molecular structures for these four polysaccharides are shown in Fig. 3.3.8.22 The diversity of the applications for cellulose is very broad and reaches from household to pharmaceutical products. Cellulose has a high crystallinity and it is water insoluble in its natural state. However, chemical modification of cellulose is possible, and thus the cellulose can be modified to be water soluble, denoted as cellulose derivatives.26 This can be applied for the CR formulations. For the case of reservoir CR systems, coatings can be produced containing two chemically modified cellulose derivatives.13,18,27 Thus, polymers with opposite water solubility properties, one water-soluble polymer and one water-insoluble polymer can be combined. When the coating, composed of a binary polymer system, is brought in contact with water, the water-soluble polymer will leach out. This creates a porous nanostructure within the water insoluble polymer, which provides a transport path for drug. By varying the ratios of the polymers in a binary polymer system, different release rate profiles are observed.18 Starch, derived from potato, corn, wheat, and rice is the most commonly used starch in pharmaceutical formulations. As for the case of cellulose, starch can be chemically modified and thus the release properties accommodate the desired properties. Amylose is one frequently used starch derivative.22 Starch commonly appears as excipients in different formulations. It can be used to dilute the formulations, such as capsules or tablets. It can also act as binders in, tablets. Alginates are polysaccharides that are used in pharmaceutical products but are still under development to be more explored. Alginates are commonly encountered as excipients as they possess the property of forming gels that stabilize systems. Aliginic acid is an alginate that is of high interest due to its highly viscous properties and the ability to be hydrated at low pH. As mentioned earlier, one drawback

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with natural polymers is that they contain contaminations, which in turn gives rise to their extraordinarily good properties, but for the case of alginic acid, research has led to development of a very pure form. This pure alginic acid is used for implants. As a fourth and last example of possible natural polymers that can be used for controlled drug release formulations, such as a film or as a gel is chitosan, that originates from chitin. Chitin can be found in fungi. Chitosan does possess important possibilities that can give rise to interesting future CR products.28 Chitosan is a positively charged hydrophilic polysaccharide with pKa around 6.5–7, thus a cationic polymer.25,28 As mentioned previously, a formulation that can either dissolve or be kept solid throughout different parts of the GI tract is of great importance. Chitosan holds the capability to form films, and it is not dissolved in the upper GI tract as the enzymes present in this area are not capable of decomposing chitosan. However, the enzymes within the colon can decompose chitosan, which thus can be used to prevent the drug from being released within the upper GI tract and instead allowing to be released later in the colon. Since chitosan possesses hydrophilic properties, it forms a gel when it is hydrated. This matrix can thus be applied for the dissolution CR matrix system, where the drug is released in a controlled manner as the chitosan matrix is dissolved with time.

3.2  SYNTHETIC POLYMERS Synthetic polymers, in contrast to natural polymers are polymers that are chemically synthesized. The synthesized polymers exhibit a high degree of purity since they are produced in a controlled manner. This purity is an important factor for polymers in order for them to be used in pharmaceutical formulations.17 Synthetic polymers occur frequently in CR formulations as they possess good properties, making them suitable for coatings, such as films. They can possess a controlled drug release rate as they can be synthesized in a way that the polymer degradation is more controlled. The polymers can be synthesized with a high purity that minimizes the risk for contaminations. They can also fairly easily be modified by adding functional groups, which improve the CR properties even further. Another important factor for the applicability of the material for CR formulations is that the material can degrade without toxic by-products. Since these polymers are synthesized in a controlled manner, the degradation rate can be modified depending on how the polymers are synthesized. This is thus one of the reasons behind the CR rate of the drug from formulations produced from synthetic polymers. The most common synthetic polymer applied for CR formulations is polyester. However, polyethers, polyamides, and polyacrylates also occur as potential material.29 The molecular structures of some examples of the frequently encountered synthetic polymers, polyesters can be seen in Fig. 3.3.9. The PLA (poly lactic acid) and PGA (poly glycolic acid) are examples of homopolymers, which means that they consist of repeating units of one monomer.30,31 PLGA (poly d,l lactide-co-glycolide) is an example of a copolymer that consists of more than one type of monomer.32

3 Soft materials for controlled drug release

FIGURE 3.3.9  Molecular Structures for Three Common Polyesters Which Are Frequently Used as Synthetic Polymers for CR Formulation (A) Poly lactic acid (PLA), (B) poly glycolic acid (PGA), and (C) poly d,l lactide-co-glycolide (PLGA).

3.3  POLYMERIC MATERIALS FOR CONTROLLED RELEASE FORMULATIONS An important factor determining which material should be used for the CR formulation is where the drug is aimed to be released to achieve the most efficient therapeutic treatment. As described throughout this subchapter, either reservoir CR system or matrix CR systems can be used and there are almost the same polymers used in both. However, depending on where the drug release is intended to occur, different polymers are selected to be the main part of the CR formulation. Therefore, the following described polymers will be divided into three groups, that is, polymers that are soluble in the GI tract or not and polymers that are soluble at a certain pH.13 Polymers that are soluble within the GI tract are usually water soluble, that is, hydrophilic polymers, such as cellulose derivatives; hydroxypropyl cellulose (HPC), hydroxypropyl ethyl cellulose, hydroxylpropyl methylcellulose, and methyl cellulose.14 Other examples of polymers that are soluble in the GI tract are alginates, chitosan, gelatin, and starch. Several derivatives from vinyl are also included within this group, such as polyvinyl alcohol, polyvinyl acetate phthalate, and polyvinyl pyrrolidone. When the aim is to release the drug in a specific pH environment within the GI tract, possible polymers that fulfill this purpose are cellulose acetate phthalate, and hydroxypropyl methylcellulose phthalate. The last group of polymers, which involves polymers that are not soluble in the GI tract, are usually not soluble in water either. This group involves cellulose derivatives, vinyl derivatives, as well as some other polymers. For the case of the cellulose derivatives, EC is one polymer that is very often selected. Polyethylene, polypropylene, and polyvinyl chloride are some other examples of these water-insoluble polymers and silicon-based polymers are also included within this group. A combination of polymers that are soluble within the GI tract and polymers that are not soluble can be used to tailor the release profiles for a certain CR formulation. An example here is a reservoir CR system where a phase-separate polymer film is applied. Two immiscible polymers, one polymer that dissolves in the GI tract, HPC, and one that do not dissolve in the GI tract, EC, will be immiscible. When the coated CR formulation is inserted to the body by oral intake, the GI tract–soluble polymer

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will leach out from the GI tract–insoluble polymer and create a porous film. This porous nanostructure provides the transport path for the drug. A combination of release mechanisms occurs and the drug is released in a controlled manner.18,27

3.4  CHARACTERIZATION OF CONTROLLED RELEASE FORMULATIONS To be able to control the drug release from a formulation with high accuracy, the understanding of the correlation between material nanostructure and the mass transport properties is of great importance. For this purpose, a novel in situ environmental electron microscopy method has been developed.20 The environmental electron microscope (ESEM) provides high-spatial resolution and uses electrons for imaging and spectroscopy. The advantage of an ESEM compared to regular scanning electron microscopes (SEMs) is that organic samples can be investigated without the need of being coated, and thus no surface modification is needed to study the samples that remains in their original state. The main components of a conventional SEM are an electron source, electron column, and sample chamber, all operated in vacuum. As the vacuum is a requirement, the samples need to be electrically conductive to avoid accumulation of electrons at the surface of the sample. The samples can either be electrically conductive or be coated with, for example, gold, if insulating. The drawback with coated samples is that the morphology of the surface may be distorted, therefore in the case of imaging most organic samples, it is convenient to utilize an ESEM. The ESEM enables imaging of insulating organic samples as it allows water vapor inside the sample chamber.33 The additional components, compared to a conventional SEM, that allow the water vapor inside the ESEM specimen chamber are the pressure limiting apertures within the electron column. The electrons of the electron beam may collide with the water gas molecules and ionize them, which results in positive ions and negative electrons. The positive ions neutralize the negatively charged surface and prevent accumulation of the electrons. In addition, the electrons generated from the gas molecules amplify the signal to the detector. Thus, imaging of organic samples without a deposited electrically conductive coating can be performed in an ESEM. A novel method for controlled wetting experiments in ESEM involves an in situ sample stage, which utilizes a nanomanipulator to bring the sample into contact with a water reservoir.34,35 This method makes it possible to study the water transport and the nanostructure of the soft material at the same time. The set up for the wetting experiments performed with this novel in situ method is illustrated in Fig. 3.3.10. This novel in situ sample stage for studying water transport makes it easier to understand the relationship between the water transport properties and the nanostructure of the soft material. More specific, water transport through free-standing phase-separated polymer consisting of EC and HPC were studied.18 When the films are brought upon water contact, the HPC leaches out and creates a nanostructure within the EC, which is the transport path for the drug. The different stages of the leaching process were studied using this novel in situ method in ESEM. The studies showed the effect of different volume ratios of the polymers on the nanostructure of

3 Soft materials for controlled drug release

FIGURE 3.3.10  Set Up of the Novel In Situ Sample Stage Designed for Performing Wetting Experiments in ESEM Using Nanomanipulator In (A) is the set up viewed from the side while in (B) the set up is viewed from above.

the coatings for controlled drug release and also the effect on the interaction mechanisms between the film and the liquid. A new initial stage preceding the steady-stage of liquid transport through the coating was revealed. In addition to the ESEM studies, focused ion beam combined with a SEM (FIBSEM) can be used to extract information about the internal nanostructure. A schematic cross-section of a coated pellet with a phase-separated polymer film is shown in Fig. 3.3.11A), where the difference between the pellets are unleached (left pellet) and leached (right pellet) conditions. The FIB-SEM uses the ion beam to remove material with a very high accuracy while simultaneously imaging with the electron beam for high-spatial resolution. An SEM image of an internal nanostructure, the poros transport path for the drug, can be seen in Fig. 3.3.11B).

FIGURE 3.3.11 (A) Schematic cross-section of a coated pellet with a phase-separated polymer film. The pellet to the left is unleached, hence the phase-separated polymer film contains both polymers. The pellet to the right is leached (one of the polymers have been removed), thus the porous transport path for the drug have been created. (B) SEM image visualizing the porous transport path for the drug.

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4  CURRENT STATUS AND OUTLOOK This subchapter has presented an overview of oral-controlled drug release formulations and provided information regarding possible soft materials that can be applied for this purpose. The research activity within this field is continuously increasing to seek improved and higher efficiency of the CR formulations and thus improve the drug therapy.14 The research aimed to find and improve the soft materials that are used for CR formulations is intense since combined with the optimized drugs for specific therapeutic treatment, it will provide an improvement of the treatment of diseases. Research is ongoing on the so called “smart polymers,” which, for example, involves chitosan as mention in previous subchapter. These types of smart polymers are examples of stimuli-responsive polymers.36 This can involve an external stimulation of the polymer by either chemicals or electromagnetism to release the drug and also target the drug delivery to locations where it is to be released. It can also be smart polymers that are further developed and very sensitive to a certain pH and thus provide another way to release the drug in a very specific site.37 The smart polymers can also be trigged by either light or temperature if they are designed to be light or temperature sensitive.38 Common for all systems are that the material nanostructure determines the release mechanisms and rates. It is therefore crucial to identify the relationships among fabrication parameters, material nanostructure, and drug-release properties.

REFERENCES 1. Rhodes CT, Porter SC. Coatings for controlled-release drug delivery systems. Drug Dev Ind Pharm 1998;24(12):1139–54. 2. Klein E. The role of extended-release benzodiazepines in the treatment of anxiety: a risk-benefit evaluation with a focus on extended-release alprazolam. J Clin Psychiatry 2002;63(14):27–33. 3. Hutton JT, Morris JL. Long-acting carbidopa-levodopa in the management of moderate and advanced Parkinson’s disease. Neurology 1992;42(Suppl 1):S51–6. 4. Edsbäcker S, Bengtsson B, Larsson P, Lundin P, Nilsson Å, Ulmius J, et al. A pharmacoscintigraphic evaluation of oral budesonidegiven as controlled-release (Entocort) capsules. Alim Pharmacol Ther 2003;17(4):525–36. 5. Anderson RU, Mobley D, Lank B, Saltzstein D, Susset J, Brown JS. Once daily controlled versus immediate release oxybutynin chloride for urge urinary incontinence. J Urol 1999;161(6):1809–12. 6. Gruber P, Longer MA, Robinson JR. Some biological issues in oral, controlled drug delivery. Adv Drug Deliv Rev 1987;1(1):1–18. 7. Rawlings JM, Lucas ML. Plastic pH electrodes for the measurement of gastrointestinal pH. Gut 1985;26:203–7. 8. Davenport HW. Physiology of the digestive tract. Chicago, IL: Year Book Medical Publishers; 1982. 9. Divoll M, Greenblatt DJ, Ochs HR, Shader RI. Absolute bioavailability of oral and intramuscular diazepam: effects of age and sex. Anasth Analg 1983;62(1):1–8.

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10. Salzman C, Shader RI, Greenblatt DJ, Harmatz JS. Long vs short half-life benzodiazepines in the elderly kinetics and clinical effect of diazepam and oaxzepam. Arch Gen Psychiatry 1983;40(3):293–7. 11. Felton LA. Characterization of coating systems. AAPS PharmSciTech 2007;8(4):E112. 12. Felton LA, Timmins GS. A nondestructive technique to determine the rate of oxygen permeation into solid dosage forms. Pharm Dev Technol 2006;11:141–7. 13. Sakellariou P, Rowe RC. Interactions in cellulose derivative films for controlled drug delivery. Prog Polym Sci 1995;20(5):889–942. 14. Langer R, Peppas N. Advances in biomaterials, drug delivery, and bionanotechnology. Am Inst Chem Eng J 2003;49(12):2990–3006. 15. Langer R, Peppas N. Chemical and physical structure of polymers as carriers for controlled release of bioactive agents: a review. Macromol Sci C 1983;23(1):61–126. 16. Sershen S, West J. Implantable, polymeric systems for modulated drug delivery. Adv Drug Deliv Rev 2002;54:1225–35. 17. Uhrich KE, Cannizzaro SM, Langer RS, Shakesheff KM. Polymeric systems for controlled drug release. Chem Rev 1999;99:3181–98. 18. Marucci M, Hjärtsam J, Ragnarsson G, Iselau F, Axelsson A. Coated formulations: new insights into the release mechanism and changes in the film properties with a novel release cell. J Control Release 2009;136(3):206–12. 19. Siepmann J, Peppas NA. Modeling of drug release from delivery systems based on hydroxypropyl methylcellulose (HPMC). Adv Drug Deliv Rev 2001;48:139–57. 20. Jansson A, Boissier C, Marucci M, Nicholas M, Gustafsson S, Hermansson A-M, et al. Novel method for visualizing water transport through phase-separated polymer films. Microsc Microanal 2014;20(02):394–406. 21. Malafaya PB, Silva GA, Reis RL. Natural–origin polymers as carriers and scaffolds for biomolecules and cell delivery in tissue engineering applications. Adv Drug Deliv Rev 2007;59:207–33. 22. Rinaudo M. Main properties and current applications of some polysaccharides as biomaterials. Polym Int 2008;57:397–430. 23. Klemm D, Heublein B, Fink H-P, Bohn A. Cellulose: fascinating biopolymer and sustainable raw material. Angew Chem Int Ed 2005;44:3358–93. 24. Andersson H, Hjärtstam J, Stadig M, Corswant von C, Larsson A. Effects of molecular weight on permeability and microstructure of mixed ethyl-hydroxypropyl-cellulose films. Eur J Pharm Sci 2013;48(1–2):240–8. 25. Jennings JA, Wells CM, McGraw GS, Pulgarin DAV, Whitaker MD, Pruitt RL, et al. Chitosan coatings to control release and target tissues for therapeutic delivery. Ther Deliv 2015;6(7):855–71. 26. Doelker E. Cellulose derivatives. Adv Polym Sci 1993;107:199–265. 27. Lecomte F, Sepmann J, Walther M, MacRae RJ, Bodmeier R. Blends of enteric and GITinsoluble polymers used for film coating: physicochemical characterization and drug release patterns. J Control Release 2003;89(3):457–71. 28. Hsu S-H, Whu SW, Hsieh S-C, Tsai C-L, Chen DC, Tan T-S. Evaluation of chitosanalginate-hyaluronate complexes modified by an RGD-containing protein as tissueengineering scaffolds for cartilage regeneration. Artif Organs 2004;28(8):693–703. 29. Nair LS, Laurencin CT. Biodegradable polymers as biomaterials. Prog Polym Sci 2007;32:762–92. 30. Gliding DK, Reed AM. Biodegradable polymers for use in surgery polyglycolic/poly(actic acid) homo- and copolymers: 1. Polymer 1979;20:1459–64.

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31. Braunecker J, Baba M, Milroy GE, Cameron RE. The effect of molecular weight and porosity on the degradation and drug release from polyglycolide. Int J Pharm 2004;282:19–31. 32. Gunatillake PA, Adhikari R. Biodegradable synthetic polymers for tissue engineering. Eur Cells Mater 2003;5:1–16. 33. Stokes D. Royal microscopical society: principles and practice of variable pressure : environmental scanning electron microscopy (VP-ESEM). West Sussex, UK: John Wiley & Sons Ltd; 2008. 34. Jansson A, Nafari A, Sanz-Velasco A, Svensson K, Gustafsson S, Hermansson A-M, et al. Novel method for controlled wetting of materials in the environmental scanning electron microscope. Microsc Microanal 2013;19:30–7. 35. Svensson K, Jompol Y, Olin H, Olsson E. Compact design of a transmission electron microscope-scanning tunneling microscope holder with three-dimensional coarse motion. Rev Sci Instrum 2003;74(11):4945–7. 36. Kumar A, Srivastava A, Galaev IY, Mattiasson B. Smart polymers: physical forms and bioengineering applications. Prog Polym Sci 2007;32:1205–37. 37. Ding H-M, Ma Y-Q. Controlling cellular uptake of nanoparticles with pH-sensitive polymers. Sci Rep 2013;3:2804. 38. Alvarez-Lorenzo C, Bromberg L, Concheiro A. Light-sensitive intelligent drug delivery systems. Photochem Photobiol 2009;85:848–60.

SUBCHAPTER

Dual function nanoconjugates for biomedical imaging and targeted drug delivery

Subchapter3.4

Isabel Gessner, Sanjay Mathur Institute of Inorganic Chemistry, University of Cologne, Cologne, Germany

1 INTRODUCTION Engineered nanostructures with bioactive surface ligands and/or functional groups that can chemically attach to biomolecules or therapeutic drugs have emerged as versatile carrier systems for biomedical imaging and therapy. The experimental capabilities to synthesize nanoparticles in different sizes and morphologies together with the availabilities of diverse surface chemistry protocols have expanded the potential of nanoparticles as probes for molecular imaging and early diagnosis of diseases, such as cancer.1–3 In most cases, the interaction of nanoparticles with tumor tissues and cells is attributed to an enhanced permeation and retention of macromolecules and nanoparticles at the tumorous sites the so-called enhanced permeability and retention (EPR)

1 Introduction

effect; however, with the aid of exogenous targeting ligands conjugated to nanoparticulate carriers, the EPR phenomenon can be used for more effective vectorization of drugs and imaging functionalities. A number of recent studies have shown that upon successful coupling of nanoparticles and tumor targeting ligands, such as peptides, antibodies, hormones, etc., high specificity in nanobio interactions can be achieved.4–6 The current research thrust on the conjugation of biomolecules on nanoparticles is triggered by the need of unifying imaging and drug-delivery units to achieve both tumor detection and treatment in a single event. The enormous application potential of nanoparticle based theranostic systems in tracking and targeting of tumors has been demonstrated in several in vitro studies; however, their efficacy in quantifying the amount of carrier (nanoparticle) and biologically active cytotoxic drug payload (anticancer agent) required is currently limited and pose an existing barrier in their clinical applications due to limited reproducibility in cellular uptake and poor receptor specificity. A number of functional biomolecules, such as proteins, peptides, DNA, si-RNA, vitamins, plasmids, and polysaccharides, as well as fatty acids have been attached to a variety of nanoparticles and used for cell tracking and imaging,7–9 which suggest the pressing need to develop more refined methods for covalent immobilization and characterization of surface grafted units especially to quantitatively estimate the surface units. In contrast to the nonspecific chemical conjugation of biologically active molecules to nanoparticles that is mostly driven by ionic or chemisorptive interactions, covalent modes for the controlled attachment and detachment of targeting units are more promising because in adsorptive bioconjugates (1) very high concentration of active biomolecules is required against the amount of carrier nanoparticles, (2) specific bio-response is not guaranteed due to random attachment of biomolecules, (3) the stability of nanobio conjugates is rather limited, so that active drug molecules or targeting units can be replaced by other molecules present in the biological environment, and (4) nonspecific interactions make the nanobio conjugates more sensitive to local changes in pH and temperature. The field of nanomedicine involving a broad range of bioimaging techniques and therapies has grown tremendously to involve an enormous variety of materials, including inorganic, polymeric, and liposomal structures.10 Optimization of synthetic methods and reaction parameters enabled engineering of desired materials and properties, which possess a precisely controlled size, shape, phase, and functional surface groups required for any specific application. Tuning of chemical characteristics which are determined by particle composition, size, morphology, and surface chemistry plays a decisive role in the cell–nanoprobe recognition events. However, considering their employment for medicinal purposes thus their administration into the human body, the surface functionalization with (bio)molecules becomes necessary.9 As nanoparticles often suffer from fast macrophage clearance and thus low-blood circulation times, grafting with biocompatible polymers, such as poly(ethylene)glycol (PEG) is commonly performed to enhance their bioavailability that additionally provides anchor points for the subsequent covalent attachment of biomolecules.11 Conjugation of specific biomolecules on nanoparticles not only leads to an enhanced biocompatibility but also adds a specific functionality to the nanoparticle making them carriers of bioreceptivity specific to cellular uptake mechanism pointing out their potential in biomedicine. For instance, therapeutic or diagnostic (macro)molecules can be transported with high payloads thus

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FIGURE 3.4.1  Only Few Nanoparticles Have the Ability to Cross the Blood–Brain Barrier (BBB)

even offering significant advances over the administration of free molecules. Nevertheless, the activity of nanoparticulate systems is highly dependent on their ability to cross physiological barriers and thus reach the intended site of action. In this context, complex structures, such as the blood–brain barrier (BBB) are a prominent example and still remain an almost insurmountable obstacle inside the body (Fig. 3.4.1). The transport of drugs or contrast agents to the brain is particularly relevant for the treatment and imaging of central nervous system related diseases, therefore, nanocarriers have been designed to penetrate the BBB either through active or passive targeting mechanisms.12 Although very small nanoparticles are able to passively cross biological membranes through the EPR effect, active transport through, for example, receptor or carrier mediated uptake are favored as they offer more control over biodistribution and uptake kinetics.13 Therefore, the identification of cell-specific receptors on cell membranes has enabled directing nanoprobes to the site of interest by attaching targeting units, such as antibodies, peptides, or hormones, which would trigger endocytosis. Recently, the integration of multiple functionalities on one carrier has gained increasing popularity to obtain theranostic nanoprobes (Fig. 3.4.2) as the bifunctional nature of such beads additionally provides simultaneous tracking and targeting of drug carriers. While the term biofunctionalization is very broad-ranging, generally referring to the surface decoration of materials for biocompatibility reasons or the immobilization of biomolecules, bioconjugation is defined as covalent derivatization of biomolecules.14 Recent reports state the disadvantages of simple physisorbed biomolecules on nanostructured surfaces, which often leads to low stability in vivo and thus the simple replacement with endogenous proteins and fast degradation by enzymes.9 To realize the widely acclaimed advantages of integrating diagnostic imaging, drug delivery, and therapeutic monitoring in a single nanotheranostic probe,

2 Typical classes of nanoparticles for biomedical applications

FIGURE 3.4.2  Synthesis of Theranostic Nanoprobes Through Bioconjugation Strategies

the clinical utilization of engineered nanoprobes demands deeper understanding of chemical interface between the nanoparticle surface and interacting units of the biomolecules. For instance, chemisorption of PEG molecules on nanoparticles render them hydrophilic thus enable the formation of hydrogen bonds to water molecules that evidently reduces nonspecific adsorption of serum proteins thereby enabling longer circulation times.15 Similarly, positively charged nanoparticles can enhance endocytosis or phagocytosis.16 Therefore, in this subchapter, we will mainly focus on defined chemical interactions between nanoparticle surfaces and biomolecules, although few examples of physisorption are included for comparison sake, and in terms of drug and gene transport, will be mentioned. For reasons of clarity and due to the enormous variety of available biomolecules, we will only highlight recent advances that have been made in the attachment of popular natural or biopharmaceutical molecules.

2  TYPICAL CLASSES OF NANOPARTICLES FOR BIOMEDICAL APPLICATIONS The range of employed nanoparticle materials that have been developed for medicinal purposes is very broad and involves inorganic, such as metals or metal oxides, as well as organic, for example, polymeric or liposomal structures comprising both advantages, as well as disadvantages pertaining to their use in biomedical applications.10 While inorganic nanoparticles compared to their polymeric counterparts provide higher mechanical strength, chemical stability, and protection against disintegration by biological fluids,17 organic nanoparticles are more often biodegradable hence demonstrating excellent safety in vivo so that several of them have already been clinically approved.6 Due to the immense variety of materials, this subchapter will only highlight some of the most commonly used material types.

2.1  INORGANIC NANOPARTICLES 2.1.1  Iron oxide nanoparticles (IONPs) IONPs have been widely used for biomedical applications, including magnetic resonance imaging (MRI) contrast enhancement, bioseparation, drug delivery, hyperthermia, and tissue repair.18 In addition to hematite (α-Fe2O3) particles, magnetite (Fe3O4),

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and maghemite (γ-Fe2O3) are most commonly used iron oxide phases due to their excellent magnetic properties. Below a critical size, IONPs exhibit superparamagnetic behavior and can thus be magnetized in the presence of an external magnetic field but feature zero-field demagnetization upon its removal. These outstanding properties are often exploited for biomedical applications making them very suitable constructs for, for example, magnetic targeting processes whereby an external magnetic field guides the sample to the place of interest. For instance, magnetic nanoparticles conjugated to plasmid DNA expressing enhanced green fluorescent protein (GFP) have been directed to the heart and kidney upon intravenous injection into mice by means of external magnets of 25 Gauss or 2 kA/m.19 The magnetic nature of IONPs has additionally been exploited for the separation of proteins,20,21 nucleic acids,22 and cancer cells,23 as well as for waste water treatments.24 Furthermore, in contrast to other metal oxides, iron oxides are considered as biocompatible since iron ions are found naturally in human cells, being essential for cell metabolism and respiration. Nevertheless, polymeric or silica coatings are commonly performed on IONPs to avoid excess metal ion release and to control pharmacokinetics of the particles. Additionally, polymeric shells have been used as anchor points for the subsequent biofunctionalization with targeting ligands to enable a receptor-specific cell uptake.25,26

2.1.2  Gold nanoparticles (Au NPs) Owing to their exceptional optical properties (plasmonic) and chemically inert nature, Au NPs are among one of the most investigated inorganic materials in terms of biomedical applications. Due to the size and shape dependent surface plasmon resonance, not only spherical Au NPs in various sizes but also nanorods with tunable aspect ratios, as well as nanoshells have been developed.27–29 Besides their use in diagnostic X-ray based imaging techniques,30 gold nanostructures have been employed for photothermal therapy.31 The simultaneous use of both, imaging and therapy, was demonstrated by Huang et al. by successfully attaching a cancer cell–specific antibody to the surface of gold nanorods (GNRs) to guide the nanoprobes to the malignant tissue.32 Subsequent heat radiation using an 800 nm laser resulted in two times faster photothermal destruction of cancer cells in which GNRs have been accumulated compared to nonmalignant cells. Comparable results were obtained by Loo et al., who used anti-HER2 antibody conjugated gold nanoshells for the selective destruction of targeted cancer tissues (Fig. 3.4.3).33 Aside from antibodies, a variety of different biomolecules has been attached to the surface of Au NPs including DNA,34 biotin,35 proteins, and peptides.36

2.1.3  Silicon dioxide nanoparticles (SiO2 NPs)

The high biocompatibility combined with the easy surface functionalization due to the abundant presence of surface hydroxyl (silanol) groups has resulted in the intense research activity related to the use of silica-based nanomaterials for biomedical applications. Moreover, compared to other nanostructures, porosity of silica nanoparticles can easily be tuned during particle synthesis. Due to the development of molecular sieves in the 1990s, the employment of porous silica materials as drug delivery vehicles has increased tremendously37 as they offer a protected cavity for

2 Typical classes of nanoparticles for biomedical applications

FIGURE 3.4.3  Combined Imaging and Therapy of SKBr3 Breast Cancer Cells Using HER2Targeted Nanoshells Scatter-based darkfield imaging of HER2 expression (top row), cell viability assessed via calcein staining (middle row), and silver stain assessment of nanoshell binding (bottom row). Cytotoxicity was observed in cells treated with a NIR-emitting laser following exposure and imaging of cells targeted with anti-HER2 nanoshells only. Note increased contrast (top row, right column) and cytotoxicity (dark spot) in cells treated with a NIR-emitting laser following nanoshell exposure (middle row, right column) compared to controls (left and middle columns). Reprinted with permission from Loo C, Lowery A, Halas N, West J, Drezek R. Immunotargeted Nanoshells for Integrated Cancer Imaging and Therapy. Nano Lett 2005;5:709–11.33 Copyright 2005 The American Chemical Society.

small molecules, such as drugs or dyes38 but also larger molecules, including genes.39 Besides the encapsulation of compounds into their porous structure, the large surface area can be modified with targeting ligands, therapeutic biomolecules and imaging agents to achieve highly efficient theranostic agents.40 Furthermore, due to their bioceramic nature, bisphosphonate-loaded mesoporous silica particle have been employed as implantable drug delivery systems for osteointegration.41

2.1.4  Lanthanide-based nanoparticles One major group of optically active nanoparticles are upconversion nanoparticles (UCNPs), which usually consist of a fluoride-based matrix, such as NaGdF4 or NaYF4 to incorporate lanthanide ions as optical centers. The main advantage of these particles are their outstanding optical properties, as they are able to convert low-energy NIR photons into a higher energy visible light photon. This provides low autofluorescence, as well as low photodamage to biological tissues. As UCNPs are commonly produced

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in high-boiling organic solvents, a subsequent ligand exchange or alternative surface modification is often necessary to obtain water-dispersible particles. While an additional silica coating is often performed to obtain a hydrophilic surface which can easily be functionalized with polymers or targeting units, such as folic acid,42 polymers can alternatively directly be attached electrostatically during a ligand-exchange reaction to form stable aqueous dispersions. These nanoprobes have been further vectorized with biomolecules, such as antibodies, proteins, or oligonucleotides.43 Additionally, UCNPs have recently been exploited as drug delivery vehicles, whereby the drug can either be conjugated covalently or noncovalently44 to the particle surface.

2.1.5  Composite structures Multicomponent nanoparticle architectures that comprise more than one material composition represent composite structures, such as hybrids and core–shell structures (Fig. 3.4.4). Composite structures demonstrate enhanced dispersibility, biocompatibility, and easier functionalization compared to single material particles and have gained increasing popularity for biomedical applications. Besides a broad variety of combinations, encapsulations of inorganic structures within silica and polymers prevail as they assure reduced metal ion leakage and offer high-colloidal stability.45 The surface anchor points obtained after coatings with silica provide a basis for the covalent attachment of biomolecules and has been frequently performed for metal oxides, as well as quantum dots and rare-earth doped nanoparticles. Moreover, core–shell structures can be easily converted to hollow or rattle-type structures by removing (parts of) the core material.46 As-obtained hollow cavities exhibit excellent properties for drug delivery applications, being able to incorporate high payloads of drugs and simultaneously offering control over the release kinetics while the surface can be biofunctionalized.47 Composite structures whereby inorganic nanoparticles are embedded inside a polymeric matrix enable the integration of several particle specific functionalities as it has been demonstrated by Song et al., who incorporated quantum dots and maghemite nanoparticles in a polymer nanostructure. As-prepared magnetic-fluorescent nanoprobes were then functionalized with monoclonal antibodies (mAb) to isolate cancer cells out of a cell mixture through fluorescent microscopy and magnetic separation48 (Fig. 3.4.5).

FIGURE 3.4.4  Schematic Drawing of Composite, Core–Shell, Hollow, and Hybrid Structures While the mixed materials can clearly be distinguished in terms of composite and core– shell particles, no clear interfaces can be observed in terms of hybrid materials. Hollow structures can easily be obtained via removal of the core of core–shell structures.

2 Typical classes of nanoparticles for biomedical applications

FIGURE 3.4.5  Schematic Drawing of the Recognition of Specific Cancer Cells by Nanobioprobes (A) Two types of nanobioprobes coated with anti-CD3 or anti-PSMA monoclonal antibody (mAb) recognize Jurkat T cells or LNCaP cells, respectively. (B) Magnetic isolation of cancer cells bound by nanobioprobes. (C) Fluorescent imaging of target cancer cells under a fluorescence microscope. When a mixture of the two types of cancer cells in A was bound by their respective nanobioprobes, they can be distinguished under UV due to different colors of the attached nanobioprobes. Reprinted with permission from Song EQQ, Hu J, Wen CYY, Tian ZQQ, Yu X, Zhang ZLL, Shi YBB, Pang DWW, Fluorescent-magnetic-biotargeting multifunctional nanobioprobes for detecting and isolating multiple types of tumor cells. ACS Nano 2011;5:761–70.48 Copyright 2011 American Chemical Society.

2.2  ORGANIC NANOPARTICLES 2.2.1  Polymer nanoparticles Among all polymeric particles that have been designed as diagnostic or therapeutic tools, two main classes can be identified: naturally occurring polymers, such as gelatins and polysaccharides, which also include the prominent group of chitosan (aminopolysaccharides) and synthetic polymers, such as poly(lactic-co-glycolic acid) (PLGA) and PEG. Especially the biodegradability makes these biopolymers a very attractive material for the administration to humans. For instance, degradation of PLGA particles results in the formation of the metabolites lactic acid and glycolic acid, which can easily be reincorporated into the natural cell cycle.49,50 To enable the targeted transport of these drug carriers, a vast variety of mainly receptor specific ligands have been attached to the surface of polymeric nanoparticles.6

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2.2.2 Liposomes

Liposomes have first been described in 1965 as model for cellular membranes.51,52 Compared to polymeric particles, they are structures that are build-up of a bilayer phospholipid membrane prepared through self-assembly in aqueous media and thus comprise a hydrophilic cavity and a hydrophobic shell. Therefore, nonpolar, as well as polar substances can be encapsulated inside the particles while the surface can additionally be modified, for example, with polymers, such as PEG to enhance blood circulation times or with specific receptors to improve the cellular uptake efficiency (Fig. 3.4.6).53 The high biocompatibility together with the easy surface functionalization and the high versatility of possible cargo molecules has led to the clinical approval of so far 12 liposomal agents.10

FIGURE 3.4.6  Evolution of Liposomes (A) Early traditional phospholipids “plain” liposomes with water soluble drug (a) entrapped into the aqueous liposome interior, and water-insoluble drug (b) incorporated into the liposomal membrane (these designations are not repeated in other figures). (B) Antibody-targeted immunoliposome with antibody covalently coupled (c) to the reactive phospholipids in the membrane, or hydrophobically anchored (d) into the liposomal membrane after preliminary modification with a hydrophobic moiety. (C) Long-circulating liposome grafted with a protective polymer (e), such as poly(ethylene)glycol (PEG), which shields the liposome surface from the interaction with opsonizing proteins (f). (D) Long-circulating immunoliposome simultaneously bearing both protective polymer and antibody, which can be attached to the liposome surface (g) or, preferably, to the distal end of the grafted polymeric chain (h). (E) New-generation liposome, the surface of which can be modified (separately or simultaneously) by different ways. Among these modifications are the attachment of protective polymer (i) or protective polymer and targeting ligand, such as antibody (j); the attachment/incorporation of the diagnostic label (k); the incorporation of positively charged lipids (l) allowing for the complexation with DNA (m); the incorporation of stimuli-sensitive lipids (n); the attachment of stimuli-sensitive polymer (o); the attachment of cell-penetrating peptide (p); the incorporation of viral components (q). In addition to a drug, liposome can loaded with magnetic particles (r) for magnetic targeting and/or with colloidal gold or silver particles (s) for electron microscopy. Reprinted with permission by Torchilin VP. Recent advances with liposomes as pharmaceutical carriers. Nat Rev Drug Discov 2005;4:145–60.53 Copyright 2005 Nature Publishing Group.

3 Functionalization of nanoprobes with common biomolecules

3  FUNCTIONALIZATION OF NANOPROBES WITH COMMON BIOMOLECULES The surface decoration of nanoconjugates with biomolecules has found increasing attention during the last years as it allows to tune their biodistribution thereby affecting their blood circulation half-life, their ability to cross biological barriers, cell uptake efficiency, and kinetics, as well as clearance and accumulation in specific organs.54,55 A vast variety of biomolecules has been synthetically conjugated with nanomaterials to impart new surface functionalities for targeting, therapy, or imaging of cells (Fig. 3.4.7).

3.1  TARGET-SPECIFIC BIOMOLECULES While antibacterial or antiviral diseases can nowadays efficiently be treated by administering highly specific drugs, the therapy of cancer is still associated with surgery and chemotherapy, which implies strong and painful adverse effects. This can mainly be explained by the low specificity of anticancer agents, which do not only affect malignant but also healthy cells.67 Therefore, the administration of high doses is necessary to ensure the uptake of sufficient concentrations, which in turn leads to high systemic toxicities. However, during the last years research has elucidated several cell surface targets on tumorous cells that can be used for the selective delivery of anticancer agents. The design of targeted nanocarriers that can carry high payloads of drugs has enabled a reduction of the minimum effective doses of drugs but also

FIGURE 3.4.7  Bioconjugated Nanobeads With Versatile Core Materials and Biomolecules

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significantly reduced the systemic toxicity. Although nanoparticles in general tend to passively accumulate in tumor cells due to the EPR effect,68 the uptake efficiency can significantly be enhanced by attaching a specific targeting moiety to the particle surface (Fig. 3.4.8). A variety of targeting units have been identified that mainly trigger a receptormediated endocytosis of nanoparticles.4 In this subchapter, we will highlight some of the most commonly used target-specific biomolecule classes.

FIGURE 3.4.8  Passive Versus Active Targeting (B) Particles tend to passively extravasate through the leaky vasculature, which is characteristic of solid tumors and inflamed tissue, and preferentially accumulate through the enhanced permeability and retention (EPR) effect. In this case, the drug may be released in the extracellular matrix and diffuse throughout the tissue for bioactivity. (A) Once particles have extravasated in the target tissue, the presence of ligands on the particle surface can result in active targeting of particles to receptors that are present on target cell or tissue resulting in enhanced accumulation and cell uptake through receptormediated endocytosis. This process, referred to as “active targeting,” can enhance the therapeutic efficacy of drugs, especially those that do not readily permeate the cell membrane and require an intracellular site of action for bioactivity. Adapted with permission from Farokhzad OC, Langer R. Impact of nanotechnology on drug delivery. ACS Nano 2009;3:16–20.69 Copyright 2009 American Chemical Society.

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3.1.1  Antibodies and other proteins Among all types of protein-based targeting ligands, antibodies are the most intensively studied group owing to their outstanding targeting efficiencies. This can mainly be explained by their high specificity and affinity, for example, for cell surface receptors.6 Especially considering cancer cell targeting, a variety of cell surface receptors has been identified which can be specifically targeted with antibodies, several of them being even clinically approved.70 A prominent example is the family of epidermal growth factor (EGF) receptors that are targeted by a variety of mAbs, including trastuzumab (Herceptin), as well as cetuximab (Erbitux)71. For instance, Eghtedari et al. reported the covalent attachment of Herceptin to GNRs via carbodiimide coupling chemistry. Enhanced uptake of antibody-linked nanorods was oberserved for HER2 overexpressing cells compared to particles, which were not functionalized with specific antibodies (Fig. 3.4.9). These results were further supported through in vivo experiments, whereby intravenous injection of antibody-linked nanoparticles into breast cancer tumor bearing mice resulted in the efficient accumulation in tumor tissues.72 Similar results could be obtained for Herceptin-conjugated mesoporous silica nanoparticles,73 as well as polymeric nanoparticles.74

FIGURE 3.4.9 Left: Schematics of Her-PEG gold nanorods (GNRs). The particles are attached covalently to Herceptin through Nanothinks Acid. PEG-thiol molecules are attached to GNR through their thiol functional group. Right: Bright field microscopic images of SK-BR3 cells that were incubated with PEG GNRs (A) or Her-PEG GNRs (B), Her GNRs (C), or no GNRs (D) in vitro. Samples were silver-stained to reveal GNR as dark spots. The total number of dark spots that indicate GNRs is higher when Her-PEG GNRs or Her GNRs were used as compared to the groups where PEG GNRs or no GNRs were used. Adapted with permission from Eghtedari M, Liopo AV, Copland JA, Oraevsky AA, Motamed M. Engineering of hetero-functional gold nanorods for the in vivo molecular targeting of breast cancer cells. Nano Lett 2009;9:287–91.72 Copyright 2009 American Chemical Society.

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Similar to antibodies, target-specific proteins, such as transferrin, a glycoprotein that is mainly responsible for the iron transport in mammalian cells, can sensitively bind to transferrin receptors that are upregulated on the surface of many cancer cells.75 A variety of transferrin-nanoconjugates have been reported in the literature ranging from polymeric nanoparticles76 and liposomes77 to Au NPs.78

3.1.2  Small biomolecules: folic acid and biotin Folic acid, also known as vitamin B9, is a high-affinity ligand for the folate receptor which is overexpressed by many tumor cells, including ovarian, endometrial, and breast cancers.79 The linkage of folic acid to nanoparticles of different materials has thus been widely employed to guide them to folate overexpressing tumor tissues.80 Due to the expression of folate receptors on malignant cells is patient dependent, their visualization is of particular importance in terms of disease diagnosis. In this context, biolabeling of folate receptors was achieved using traceable nanocarriers, such as UCNPs81 or IONPs,82 which additionally proved their successful uptake via receptor-mediated endocytosis. Another vitamin that is commonly used in terms of cancer cell targeting is vitamin B7, better known as biotin. Biotin-related transporters on cell surfaces have been identified, which can be found in higher concentrations on tumor cells compared to healthy cells.83 Besides the high affinity of biotin for these surface receptors enabling the targeted transport of biotinylated nanoparticles,84 biotin shows strong affinity to the proteins streptavidin and avidin (see Section 4 of Subchapter 3.4), which has been exploited for biosensing85 and bioseparation processes.20

3.1.3 Peptides

Integrins, such as αvβ3 are cell adhesion molecules, which are hardly detected in healthy adult epithelia but are overexpressed in many cancer types.86 As the peptide arginine-glycine-aspartic acid (RGD) interacts with high affinity with the active binding site of αvβ3,87 it has been demonstrated that RGD functionalized nanoparticles can be used to sensitively image the cell surface receptors. Especially, IONPs26,88 and UCNPs89 have found application owing to their MRI contrast enhancement and near-infrared imaging capabilities, respectively. For instance, intravenous injection of nanophosphors to which cRGD was covalently bound via carbodiimide coupling strategy resulted in the accumulation in integrin αvβ3 overexpressing U87MG tumor cells, while no luminescence was observed in MCF-7 tumors (Fig. 3.4.10).89 Moreover, targeting αvβ3 integrin by RGD has enabled the transport and release of drugs in integrin overexpressing cells via nanoprobes. In this context, mesoporous silica nanoparticles that have been loaded with the anticancer drug doxorubicin and surface modified via click chemistry with RGD peptides have been developed. By incorporating a glutathione sensitive disulfide linker between the particle surface and RGD, the peptides form a nanovalve that triggers drug release upon endocytosis.90

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FIGURE 3.4.10 Time-dependent in vivo upconversion luminescence imaging of subcutaneous U87MG tumor (left hind leg, indicated by short arrows) and MCF-7 tumor (right hind leg, indicated by long arrows) borne by athymic nude mice after intravenous injection of UCNP-RGD over a 24 h period (A–C: 1 h, D–F: 4 h, G–I: 24 h). All images were acquired under the same instrumental conditions (power ≈ 80 mW/cm2 and temperature ≈21.5°C on the surface of the mouse). (H) The in vivo signal-to-noise ratio (SNR) calculation. Region of interest (ROI) 1, specific uptake; ROI 2, nonspecific uptake; ROI 3, background. SNR) [(mean fluorescence intensity of the specific uptake)−(mean fluorescence intensity of background)]/[(mean fluorescence intensity of the nonspecific uptake)−(mean fluorescence intensity of background)]. UCNP, Upconversion nanoparticles. Reprinted with permission of Xiong L, Chen Z, Tian Q, Cao T, Xu C, Li F, Targeted imaging in vivo using peptide-labeled nanophosphors, Anal Chem 2009;81:8687–94.89 Copyright 2009 American Chemical Society.

Although not directly related to the field of target-specific biomolecules, cellpenetrating peptides (CPPs) can likewise enhance the uptake of nanocarriers into cells. In contrast to earlier mentioned targeting units, the mechanism of cell uptake is not yet completely understood in terms of many CPPs.91 However, some recent studies demonstrated the strong ability of CPPs to transport even big cargos, such as inorganic nanoparticles92 or liposomes93 into cells.

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3.2  THERAPY-SPECIFIC BIOMOLECULES Patient-specific delivery of therapeutic molecules has aroused as a crucial factor for disease treatments. In this context, drug delivery is one of the most employed applications of nanoparticles. Drugs can thereby not only be incorporated inside hollow structures but can also be attached onto the carrier surface incorporating sensitive linkers that enable a triggered release.94,95 Besides the broad range of synthetic drugs, some biomolecules have demonstrated therapeutic properties and have found applications in the treatment of a variety of diseases. Especially the two main classes of biopharmaceuticals and nucleic acids have gained increasing attention in terms of novel therapeutic approaches and will thus be presented in this subchapter.

3.2.1  Biopharmaceuticals and biological drugs Among a broad variety of therapeutic biomolecules, insulin is by far the most prominent example of a biopharmaceutical agent. Discovered during the early 1920s, the pancreatic protein was since then further engineered so that to date biotechnological approaches can be used to prepare large quantities of human insulin analogues.96 While insulin is usually administered intravenously, current research focuses on alternative routes of administrations with the preferred one being oral administration. However, due to the low-oral bioavailability of insulin and the high-degradation rate in the stomach, a variety of different nanocarriers have been developed to overcome current limitations. Besides polymeric and polymer-coated inorganic nanoparticles, solid lipid particles have emerged as promising candidates for the efficient transport and release of high payloads of insulin and furthermore have demonstrated an improved gastrointestinal absorption.97–99 By covalently attaching insulin to GNRs via carbodiimide chemistry, the blood sugar level in diabetic mice could be significantly decreased for time periods, which were three times longer compared to the free protein (Fig. 3.4.11). Due to the strong bond to the nanocarrier, degradation by enzymes could be decelerated thus leading to a prolonged bioactivity.100 In addition to their ability to target-specific cells in our body, antibodies can also be useful in terms of novel therapeutic treatments of diseases. In fact, killing of malignant cells can be induced through, for example, receptor blockade, which can thus hinder tumor cell signaling.101 Common examples are monoclonal anti-EGFR antibodies which were successfully attached to COOH-PEG decorated IONPs via carbodiimide coupling chemistry. Obtained nanoconjugates could not only be traced via MRI but also demonstrated a significant reduction in cell viability in EGFR expressing cells.25 Moreover, hormones, such as the estrogen estradiol have been administered to reduce postmenopausal symptoms. Huge payloads of the therapeutic molecule could be encapsulated in polymeric102 nanostructures and were loaded onto PEGylated UCNPs.103 Release studies of the hormone from estradiol loaded UCNP system revealed a rapid release during the first 12 h, which decelerates afterwards so that <60% of the drug is released after 60 h (Fig. 3.4.12).

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FIGURE 3.4.11 Left: Synthesis and characterization of INS-GNPS: (A) schematic diagram of the synthesis of 20 nm GNPs and functionalization with insulin. (B–D) TEM images of 20, 50, and 70 nm GNPs, respectively (scale bar 100 nm). Right: Profiles of blood glucose levels following subcutaneous administration of free insulin and INS-GNPs in diabetic mice. 50 nm INS-GNPs (30 mg/mL, 200 µL) or free insulin (0.12 IU) were subcutaneously injected into diabetic mice. Blood glucose levels were monitored every 0.5 h, up to 6 h postinjection. The results are presented as percentage of blood glucose levels, mean ± S.D. *P < 0.05 INS-GNPs versus free insulin, U-test value = 0.000457. Adapted with permission from Shilo M, Berenstein P, Dreifuss T, Nash Y, Goldsmith G, Kazimirsky G, et al. Insulin-coated gold nanoparticles as a new concept for personalized and adjustable glucose regulation. Nanoscale 2015;7:20489–96.100 Copyright 2015 Royal Society of Chemistry.

3.2.2 Oligonucleotides The emerging field of gene therapy is based on the successful transport of therapeutic nucleic acids into cells where they can selectively promote gene expression or silencing. However, due to the strong negative charge of nucleic acids, penetration of biological membranes is very poor. Therefore, carrier systems have been designed to enable the efficient incorporation into cells based on liposomes, polymeric particles, and a vast variety of inorganic nanoparticles.104,105 For instance, Yang et al. proved the successful transport of small interfering RNA (siRNA) using silica-coated UCNPs.106 Oligonucleotide attachment was performed electrostatically by employing positively charged photocaged linkers, which also enabled the NIR light triggered release of siRNA in vitro. The electrostatic interaction between positively charged surface ligands and negatively charged nucleic acids is also regularly employed in terms of organic particles, such as polymeric- and liposomal-based structures. Besides the commonly used polyethyleneimine, a variety of cationic polymers with high-DNA binding affinity and thus DNA transfection potential have been identified.107 However, nucleic acids that are only adsorbed to particle surfaces are easily degraded by nucleases.108 Therefore, silica based particles which often exhibit a porous nature offer a protected cavity for oligonucleotides and are thus of great interest in terms of nucleic acid transport. Nevertheless, considering the large size of nucleic acids, Kim et al. have demonstrated that the pore size of mesoporous silica nanoparticles has a significant influence on transfection rates.39

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FIGURE 3.4.12 Left: Nanosystem was synthesized by coating an amphiphilic block-polymer C18PMH-PEG (pPEG) on core−shell nanocrystal NaY- F4:Yb, Er@NaLuF4 (UCNPs), and then loading 17β-estradiol (E2) into the surface hydrophobic layer. Right: Release behavior of E2 from E2-UCNP@pPEG in PBS solution (pH 7.4). Adapted with permission from Hu Y, Li J, Zhu X, Li Y, Zhang S, Chen X, et al. 17β-estradiol-loaded peglyated upconversion nanoparticles as a bone-targeted drug nanocarrier, ACS Appl Mater Interfaces 2015;7:15803– 11.103 Copyright 2015 American Chemical Society.

As an alternative, the covalent attachment of oligonucleotides to nanoparticles has gained increasing popularity as it offers high stability of the nanobioconjugate. This was recently demonstrated by Zhang et al., who prepared inorganic nanoparticle–DNA conjugates using a three-step functionalization method. First, carboxylic acid groups were grafted onto the surface of nanoparticles followed by the attachment of streptavidin via carbodiimide chemistry and the final conjugation with biotinylated DNA. The variability of the core material was proven by employing plasmonic (gold), magnetic (Fe2O3), catalytic (palladium), and luminescent (CdSe/Te@ZnS and CdSe@ZnS) materials. Self-assembly of heterogenous DNA-functionalized particles

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could be achieved by using complementary bases on nanoparticles or employing a complementary linker molecule.109 Furthermore, Chen et al. presented the covalent attachment of DNA strains to silica nanoparticles via copper-catalyzed click chemistry. A stimuli responsive system was created by anchoring DNA to the pores of the particles which formed a cap and thereby hindered release of incorporated drug molecules. Upon heat treatment and/or addition of DNA-degrading enyzmes, DNA caps were removed giving way for the cargo.110

3.3  IMAGING TECHNIQUES AND BIOFUNCTIONALIZATION Visualization of anatomical structures and cellular processes inside the human body has triggered enhanced diagnostic capabilities and has thus contributed to a faster and more efficient treatment of diseases. Recent advances in real time imaging of nanodimensional biological processes have been of particular relevance to understand the complex cellular mechanisms in organisms. During the last years, a variety of bioimaging methods [e.g., MRI, positron emission tomography (PET), single photon emission computed tomography (SPECT), ultrasonography (US), computed tomography (CT), optical imaging (OI)], which differ significantly in sensitivity and resolution has been developed, among which some commonly used types will be presented in the following section.

3.3.1  Magnetic resonance imaging MRI is one of the most commonly used imaging techniques in clinics and has enabled a noninvasive diagnosis of diseases by creating images with excellent anatomic details. The basic principle relies on the substantial number of protons present in biological tissues, mainly in the form of water. Although the entire process itself is very complex, it can be summarized in the following steps: By applying an external magnetic field, a longitudinal magnetization occurs whereby hydrogen nuclear spins align parallel to the external field. Radiofrequency pulses that are applied perpendicular to the external magnetic field then cause a transverse magnetization. After removal of the radiofrequency pulse, relaxation of hydrogen nuclear spins occurs in two different forms: as realignment along the longitudinal external magnetic field (T1) and as transverse or spin–spin relaxation (T2). As these relaxation times differ significantly depending on the hydrogen atom surrounding tissue, anatomical images can be obtained with soft tissue contrast.111 Both relaxation times, T1 and T2, can be shortened by employing adequate magnetic contrast agents. Paramagnetic gadolinium-based contrast agents are currently most commonly used due to the high-magnetic moment of Gd (III) ions. However, superparamagnetic IONPs have found increasing attention during the last years and have even made their way through clinics and are nowadays commercially available.112 Compared to gadolinium-based agents, IONPs offer an enhanced uptake efficiency in tumor cells and a prolonged blood-circulating half life.113 To obtain a safe contrast agent, the biocompatibility of administered nanoparticles is of crucial importance. Currently, commercially available IONP contrast agents namely Feridex and

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FIGURE 3.4.13  Nanoparticle Uptake Analyzed by Means of Magnetic Resonance Imaging (MRI) (A) T2-weighted images of CT26 and J774A.1 cells in gelatin (2 × 106 cells/0.3 mL) incubated with increasing concentrations of green tea (GT)-, the oxidized form of epicatechin (ECq)-, and the oxidized form of epigallocatechin gallate (EGCGq)-coated iron oxide nanoparticles (IONPs) for 3 h. The increasing negative contrast in T2-weighted images clearly demonstrate the particle uptake. (B) Metabolic activity of cells upon exposure to increasing concentrations of IONPs, as assessed by MTT assay. Cells were incubated with nanoparticles or remained untreated (control) for 3 h. The lines represent mean ± standard deviation (n = 3). Adapted with permission from Xiao L, Mertens M, Wortmann L, Kremer S, Valldor M, Lammers T, et al. Enhanced in vitro and in vivo cellular imaging with green tea coated water-soluble iron oxide nanocrystals. ACS Appl Mater Interfaces 2015;7:6530–40.115 Copyright 2015 American Chemical Society.

Resovist are based on dextran or carboxydextran coated iron oxide nanocrystals.114 As an alternative, Xiao et al. recently presented a green and facile synthesis of IONP-based MRI contrast agents using natural products, such as vitamin C and green tea.115,116 Both biomolecules functioned not only as reducing agent during the nanoparticle synthesis but also simultaneously formed a capping layer of the oxidized moieties on the nanoparticle surface imparting them higher water dispersibility that enhanced the cellular uptake. Consequently, a strong T2* contrast was obtained in vitro (Fig. 3.4.13) and in vivo thus demonstrating their great potential as MRI contrast agents.

3.3.2 Radiolabeling During the last years, PET has evolved as one of the most powerful imaging techniques particularly regarding to cancer diagnosis as it offers, in contrast to MRI,

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imaging of biomolecular processes in vivo.117 During the radioactive decay, a positron is emitted, which then travels through the surrounding tissue until it recombines with an electron. This annihilation results in the generation of two photons which possess an energy equivalent to the mass of an electron and a positron. As these photons travel in opposite directions (180 degree), detectors on opposing sides of the sample can register annihilation events in a predefined time-window. Clinical PET scanners usually consist of a circular-shaped detector so that precise information about the location and quantity of radioactive decays can be determined to provide detailed data on biochemical processes. The common radioisotopes 11C, 13N, 15 O, and 18F can easily be incorporated into organic molecules to obtain efficient and diagnostic specific PET tracers.117,118 The most prominent PET tracer that has been widely used for clinical imaging of oncology, cardiac, and neuronal diseases is 2-[18F]-fluoro-2-deoxy-d-glucose (FDG). Accumulation of FDG in specific cells thus provides detailed information on the glucose metabolism that in turn delivers relevant conclusions for the diagnosis of tumorous tissues.119 Aside from molecular tracers, several research groups are currently focusing on the development of radioactively labeled nanoparticles which are commonly prepared by attaching chelating ligands, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) in which radionuclides can be incorporated.120 The effect of chemical structure and size was found to enhance the blood circulation times as observed in DOTA-labeled PEG conjugates, which were nanosized when compared to short-chained molecules.121 Nowadays, several bioconjugated nanoparticles have been employed for PET imaging.122 In this context, the modification of nanoparticles with radiolabeled biomolecules has found increasing attention, as it offers tracing of bioconjugated nanobeads. For instance, the attachment of 124I radiolabeled antibodies that specifically target the carbohydrate antigen 19-9 (CA19-9) to the surface of polymercoated liposomes has demonstrated their PET imaging capabilities for pancreatic cancer in mice (Fig. 3.4.14).123 As an alternative, the attachment of radioactively labeled RGD peptides on the surfaces of nanoparticles might open up new possibilities in terms of a simultaneous αvβ3 integrin targeting and PET imaging.124,125 Besides PET, SPECT (Single Photon Emission Computed Tomography) offers nuclear medicine imaging based on the detection of γ-rays.126 In this context, biodistribution and accumulation of 111In-labeled mAbs that have been linked to the surface of dextrane-coated IONPs could be observed via SPECT in breast cancer tumor bearing mice. An additional therapeutic effect could be obtained by applying an alternating magnetic field upon injection of theranostic bioprobes to achieve effective thermal ablation of tumor cells.127

3.3.3  Optical imaging In direct comparison to MRI or PET, OI offers an advantageous cost effectiveness and a simple procedure for the visualization of cells and tissues. Nevertheless, OI techniques, such as fluorescence and bioluminescence are often associated with lowspatial resolutions and limited penetration depths into tissues and have thus mainly been used for small animal and cell-culture imaging.128 Still, the development of

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FIGURE 3.4.14  In Vivo Micro-PET and Micro-CT Images of Pancreatic Cancer (BxPC3) Xenografts at 4 and 20 h With Those Associated Tumor-To-Blood Ratios and Positive-toNegative Tumor Ratios Obtained After the 20-h-Image CT, Computed tomography; PET, positron emission tomography. Adapted with permission from Girgis MD, Federman N, Rochefort MM, McCabe KE, Wu AM, Nagy JO, et al. An engineered anti-CA19-9 cys-diabody for positron emission tomography imaging of pancreatic cancer and targeting of polymerized liposomal nanoparticles. J Surg Res 2013;185:45–55.123 Copyright 2013 Elsevier.

super-resolution microscopes, which was awarded with the Nobel Prize in chemistry in 2014 points toward an encouraging future of fluorescence imaging in the nanodimension.129 As an alternative to the vast variety of molecular fluorophores, fluorescent nanoparticles have been developed which often feature improved brightness and lower unspecific binding by cellular proteins.130 Besides optically active nanoparticles, such as UCNPs, Au NPs or quantum dots, the incorporation of fluorescent labels and dyes into nonfluorescent nanocarriers has enabled their tracking in cells via OI techniques. Due to the small size of dyes, they have commonly been incorporated into hollow structures as present on mesoporous silica particles but have also been attached to the surface of nanoparticles.38 Prominent examples of biomolecular dyes are the family of coumarins and curcumin, which additionally exhibit beneficial antiinflammatory, antioxidant, and anticancer properties.131,132 Curcumin, the yellow-orange dye that is commonly associated with curry powder, naturally derives from an Indian plant called Curcuma longa and has its main absorbance at 549 nm if measured in ethanol.133 However, owing to its hydrophobic nature, the fluorescent drug is generally encapsulated inside polymeric or liposomal structures and has also been incorporated into inorganic/organic nanocomposites.134 Recent studies have demonstrated that curcumin additionally leads to enhanced anticancer

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FIGURE 3.4.15 MCF7 cellular (upper) and MCF7 multicellular tumor spheroids (lower) uptake of different forms of curcumin after 24 h of treatment: (A) control MCF7 cells, (B) pure curcumintreated MCF7 cells, (C) curcumin (Cur)-poly (lactide)-tocopheryl polyethylene glycol succinate (PLA-TPGS) NPs-treated cells, (D) [Cur + paclitaxel (PTX)]-PLA-TPGS NPstreated cells (hoechst—blue light, curcumin—green light). Adapted from Nguyen HN, Ha PT, Nguyen AS, Nguyen DT, Do HD, Thi QN, et al. Curcumin as fluorescent probe for directly monitoring in vitro uptake of curcumin combined paclitaxel loaded PLA-TPGS nanoparticles. Adv Nat Sci Nanosci Nanotechnol 2016;7:1–6.136

properties compared to the free drug by simultaneously controlling the release kinetics.135 Tracking of curcumin-loaded nanoparticles is easily possible via confocal microscopy and can additionally be exploited to determine the loading capacity of nanoparticles via UV–vis spectroscopy. Recently, Nguyen et al. have proven the suitability of curcumin-loaded polymeric micelles for cancer theranostics, whereby the strong fluorescence was still detectable even at the presence of paclitaxel, an additional anticancer drug. Moreover, uptake studies using MCF-7 cells and tumor spheroids demonstrated increased uptake efficiencies compared to free curcumin, as evidenced in Fig. 3.4.15.136 Due to its ability to exhibit a bright green fluorescence upon UV light irradiation, the GFP is to date one of the most studied proteins in biochemistry. It has thus found application as marker for several proteins, receptors, and for gene expressions.137 Moreover, the reversible attachment of GFP onto cationic Au NP surfaces via electrostatic interactions has recently been employed for biomedical sensing applications in which healthy and cancerous cells could be optically differentiated. The successful interaction between the particle and the protein could be observed, as GFP fluorescence was quenched upon binding to Au NPs.138 Alternatively, GFP derivatives have also been incorporated into the particle material to obtain fluorescent nanoparticles that can be optically traced.139

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3.3.4  Multimodal imaging To overcome imaging specific limitations in resolution, sensitivity, and penetration depth, the combination of multiple imaging modalities has gained increasing popularity. Since the employment of clinical dual PET/CT scanners around two centuries ago, a variety of tandem scanners have been developed. The design of multimodal contrast agents is hence a major issue of medicinal research as it offers reduced stress for patients by performing a single administration for multiple measurements.140 Especially, bioconjugated nanostructures have found increasing attention as they provide besides their biocompatibility a large surface area compared to their small volume for the attachment of different moieties. Integrated dual MRI/PET and MRI/SPECT systems combine beneficial high-anatomical spatial resolutions and soft tissue contrast with noninvasive molecular imaging.141 Dual MRI/SPECT contrast agents based on IONPs that were surface modified with 125I-radiolabeled cRGD have demonstrated great potential for breast cancer imaging in vivo. Long-circulation half-lives, as well as high-tumor uptake could be obtained leading to sensitive tumor detections with high resolutions.142 Trimodal imaging agents that combine MRI, PET, and OI have recently been developed based on human serum albumin coated IONPs. As-prepared bioconjugated platforms were dually labeled with fluorophores (Cy5.5), as well as 64CuDOTA and subsequently injected into glioblastoma bearing mice.143 Arifin et al. recently presented the synthesis of Au NPs functionalized with gadolinium chelates, which have been coencapsulated with human pancreatic islet cells in alginate microcapsules. As prepared systems exhibited strong contrast for MRI, CT, and ultrasonography and simultaneously demonstrated efficient therapeutic treatments of diabetes.144

4  TYPICAL SURFACE GRAFTING TECHNIQUES Attachment of suitable chemical groups on nanoparticle surfaces can affect the toxicity profile, accumulation in organs, and influence their pharmacokinetics and -dynamics. While physisorption-based methods are still commonly performed due to their simple handling and variability, they usually suffer from low stability in vivo, their pH sensitivity and the resulting random orientation of attached molecules on the particle surface.145 Chemisorption in contrast provides stronger bonds, which are more stable under physiological conditions and control over the reaction product.146 A large number of advanced strategies have been demonstrated to chemically functionalize nanoparticles by attaching surface groups that render the surface neutral or charged (positive or negative). In addition to the common chemisorptive mechanisms, a variety of covalent conjugation strategies have been developed during the last years for the efficient linkage of biomolecules to nanoparticle surfaces, with the most common ones being carbodiimide and click reactions, as well as biotin– streptavidin interactions (Fig. 3.4.16).

4 Typical surface grafting techniques

FIGURE 3.4.16  Typical Surface Modification Mechanisms for the Bioconjugation of Nanoparticles

The reaction requirements, as well as the selectivity of the functional groups are the crucial factors that should be considered when choosing the adequate coupling strategy for each system.

4.1  CARBODIIMIDE COUPLING Carbodiimides which are unsaturated compounds based on an allene structure are considered as one of the most important molecular classes in organic chemistry and have found particular interest in the preparation of nucleotides and peptides.147 The formation of an amide by linking a free amine and a carboxylic acid group through carbodiimide crosslinkers can easily be performed in organic solvents using DCC (N,N-dicyclohexylmethandiimin) or in aqueous media using EDC (1-ethyl-3-(3′dimethylaminopropyl)carbodiimide), whereby the latter plays the more important role in terms of bioconjugation. The pH plays a crucial role during carbodiimide couplings as EDC loses its activity at pH lower 4.5 forming urea derivatives, while amide bond formation can be enhanced at neutral and higher pH ranges under very mild conditions.148 Moreover, Singh et al. recently demonstrated that the attachment of G-protein subunits to Au NPs via a covalent carbodiimide chemistry and a noncovalent interaction significantly alters G-protein activity. While a covalent conjugation leads to a ∼fivefold acceleration of protein activity, kinetics were retarded in terms of a simple electrostatic interaction.149 EDC coupling strategies

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were also achieved for the covalent attachment of a vast variety of biomolecules including mAbs,127 transferrin,77 trypsin,150 cRGD,26 and BSA.151 However, while on one hand the presence of high quantities of amine and carboxyl-groups in many biomolecules provides high versatility for carbodiimide chemistry, it often lacks a site-specific conjugation, which can be overcome by employing more specific grafting techniques.

4.2  CLICK CHEMISTRY Click reactions were first defined in 2001 by Kolb et al. as coupling strategies, which are based on mild-reaction conditions, a high selectivity, and stereospecificity, high yields and the easy removal of undesired by-products.152 The family of click reactions spans from Diels–Alder, Michael addition, and thiol-ene reactions to Huisgen cycloadditions of which the copper catalyzed alkyne/azide (CuAAC) reaction is the most prominent and extensively used type of a click reaction. Formation of the stable triazole ring thereby represents the driving force for this reaction. While the noncatalyzed Huisgen reaction produces a mixture of 1,4 and 1,5-disubstitution products, the CuAAC delivers selectively 1,4-disubstituted 1,2,3-triazoles. The catalyst source is very variable ranging from Cu(II) salts to Cu(0) nanoparticles, provided the reactive Cu(I) species is formed.153 The high selectivity, the high yield even at room temperature syntheses and the solubility of all components in water offers optimal conditions for bioconjugation strategies.154 A vast variety of reported protocols can be found for the covalent attachment of RGD peptides,90 enzymatic lipases,155 biotin,20 folic acid,156 proteins,157 and nucleic acids110 via CuAAC. However, a demand for copper-free click reactions has increased due to the potential severe damage of remaining copper ions to biomolecules and cells158 and the potential conflicts with Cu sensitive materials.159 Thus the employment of a strain-promoted azide−alkyne cycloaddition, which usually describes the reaction between an azide and a cyclooctyne, has become very popular for the bioconjugation of nanoparticles and has even been transferred to the intracellular labeling of biomolecules.160 In this context, an attempt toward in vivo click chemistry has been presented by Koo et al., who demonstrated the labeling of cancer cells with azides using naturally occurring monosaccharides which could then be selectively targeted by cycloocyne functionalized liposomes.161 Besides alkyne-azide cycloadditions, thiol-yne and thiol-ene reactions have found increasing attention as surface modification technique. For instance, Zhang et al. have recently demonstrated the potential of thiol-ene click reaction to prepare boronic acid modified magnetic nanoparticles, which can be used for the selective capture of glycoproteins out of egg white samples.162 The higher efficiency of click reactions compared to carbodiimide couplings was recently proven by Bolley et al., who prepared fluorescent superparamagnetic nanoparticles targeting αvβ3 integrins using both linkage strategies. In fact, due to the increased number of cRGD peptides grafted on nanoparticle surface via click chemistry (CuACC and thiol–yne), enhanced targeting efficiencies could be observed

4 Typical surface grafting techniques

compared to the carbodiimide counterpart.163 Comparable results have been previously reported by Thorek et al., who investigated the coupling efficiencies to prepare nanoparticle–antibody conjugates. In this case, IgG and anti-CD20 antibodies were attached using the above-mentioned two functionalization techniques to the surface of IONPs.164 Furthermore, since many years, the linkages between malemides and thiols are considered as very efficient Michael-type addition click reactions that have been used ubiquitously in organic chemistry and have nowadays found application for bioconjugation strategies.165 Bioconjugated IONPs have been prepared by covalently attaching the enzyme collagenase exploiting the high affinity between malemide and sulfhydryl groups.151 Moreover, drug and dye loaded mesoporous silica particles could be directed toward αvβ3 integrin overexpressing cells by linking cRGD via the same conjugation chemistry, leading to highly efficient theranostic probes.166

4.3  BIOTIN–STREPTAVIDIN INTERACTION Even though they officially do not belong to the group of covalent coupling strategies, biotin–streptavidin, as well as biotin–avidin interactions have become very popular for the surface decoration of nanoparticles due to their unusual high binding affinity (dissociation constant Kd = 10−15 M). The tetrameric structure of both, streptavidin and the homologous protein avidin, which originally derive from the actinobacterium Streptomyces avidinii and ovarian egg yolk, respectively, enables the binding of four biotin molecules to each protein. The formation of multiple hydrogen bonds and van der Waals interactions, as well as the conformational changes upon biotin binding are the critical parameters for their high affinity, making it one of the strongest known noncovalent interactions in nature.167 These remarkable properties have commonly been exploited for biosensing applications whereby a variety of biological molecules could be detected with high sensitivities.168–170 Moreover, the surface decoration of nanoparticles with avidin allows the selective attachment of biotinylated antibodies as recently demonstrated by Song et al.48 The coupling of proteins to Au NPs surfaces was tested by Chirra et al., who compared the efficiency of a covalent carbodiimide reaction and the biotin streptavidin interaction. The particle surface was thereby functionalized with carboxylic acid groups and biotin molecules, respectively. Both methods resulted in the formation of bioconjugated Au NPs that was proven by a detectable shift in absorbance (Fig. 3.4.17). In direct comparison between these two methods, higher amounts of catalase were attached via carbodiimide chemistry although the reaction method also resulted in decreased enzyme activities.171 In this context, the order of functionalization steps seems to play a decisive role as it was recently indicated that higher amounts of biomolecules can be grafted onto the surface of nanoparticles by first performing a streptavidin coating on the particles surface, which then offers four binding sites per streptavidin molecule for biotinylated bits.109

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FIGURE 3.4.17 Top: Schematic representation of the two approaches involved in the coupling of catalase to gold nanoparticles (Au NPs). Bottom: Kinetic study showing the crosslinking of biotinylated GNP in the presence of low streptavidin concentration (9 nM) confirming biotin–streptavidin interaction. Also shown as a dotted line is the noncrosslinked high-concentration streptavidin (100 µM) coated GNP profile. Inset shows the initial noncrosslinked dispersed GNPs in the right and the final crosslinked settled GNPs in the left. Also shown in black dotted line is the streptavidin-coated biotinylated GNPs (using high-streptavidin concentration). Reprinted with permission from Chirra HD, Sexton T, Biswal D, Hersh LB, Hilt JZ, Catalase-coupled gold nanoparticles: comparison between the carbodiimide and biotin-streptavidin methods. Acta Biomater 2011;7:2865–72.171 Copyright 2011 Elsevier.

5 Current challenges and future prospectives of bioconjugated nanobeads

5  CURRENT CHALLENGES AND FUTURE PROSPECTIVES OF BIOCONJUGATED NANOBEADS The emerging field of nanotechnology is thought to revolutionize future diagnosis and therapy of diseases.1,172 Nanomedicine represent a versatile platform for integrating the multifunctional properties of nanoparticles with various synthetic coupling chemistries to conjugate specific biomolecules on the surface of nanoparticles used as carriers. The unique opportunity of imparting therapeutic, diagnostic, or tunneling properties to nanoparticles by anchoring adequate drug payloads offers the potential for increasing the binding affinity of a particle toward a target tissue and cells. Among the nanoparticle platforms, inorganic materials show specific advantages of long-term stability, defined surface chemistry and possibility to combine multivalent targeting through a single nanoparticle. In this context, iron oxide nanocarriers have been shown to be suitable for use as theranostic agents due to their intrinsic magnetic properties required for MRI applications and surface-conjugation with targeting (antibodies, peptides, small molecules, etc.) or therapeutic units (genes, drugs, or radioactive nuclides) to provide multimodal solutions. Still, the tremendous expansion of nanostructured materials during the last years has enlarged toxicity issues,173 thus the employment of biocompatible nanoarchitectures that also involves the use of biomolecules has become very important. Despite the promising potential of nanobioconjugates in theranostic applications, the translational effort of employing the in vitro protocols for in vivo systems is currently challenged by the missing validation of design parameters. Nanoparticles are intrinsically beneficial due to their size, surface charge and established methods of surface functionalization that ensure their interaction with cellular systems; however, using them for targeted delivery to specific tissues is still an overarching challenge mainly due to their small sizes and nonspecific internalization by various cells. As the functionality of nanoparticles becomes more complex, such as the addition of targeting ligands, there is a need to precisely engineer optimally designed nanoparticles with the physicochemical and biological properties to achieve each of the desired functions with selective identification and quantitation. Technologies, such as isothermal titration calorimetry have been proven successful for the elucidation of the nonspecific binding between proteins and nanoparticles, for example, the formation of a protein corona.174 Kinetics, binding affinities, and quantification of surface biomolecules can be performed in simple experimental setups using a mixture of selected proteins and model nanoparticles. Nevertheless, new biophysical or biochemical methods are clearly needed to continue investigations in complex biological media combined with a variability in particle material. For the possible application of nanoparticles in clinical experiments, better and more reliable characterization strategies including pharmacokinetics and long-term toxicity studies are desired. It is essential that engineered nanostructures can be reproduced in terms of their size, surface consistency, and composition with high figure of merit as these factors also influence their pharmacokinetic and pharmacodynamics in biological settings. Therefore, by considering the transfer of a nanoparticle-based

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system in medicine from the bench to the clinic, the changes from their “chemical identity” in lab toward their “biological identity” (in vitro) to their “physiological identity” (in vivo) have to be taken into account. Numerous nanoparticle platforms are currently under various stages of preclinical and clinical development, including various liposomes, polymeric micelles, quantum dots, and Au NPs. Nevertheless, considering the enormous number of researchers working on nanomedicine, the yield of clinically approved agents on the market is surprisingly small.175 The lack of clear guidelines for nanomedicine approval, as well as the low support of big pharmaceutical companies for complex nanocarriers whose production is hence very time- and cost-consuming are two of several reasons for this. Still, the future points toward a strong increase of clinical trials in the field of nanomedicine implying that bioconjugated nanocarriers might play a crucial role in future disease diagnosis and therapy.176

ACKNOWLEDGMENTS Authors kindly acknowledge the financial and infrastructural support provided by the University of Cologne, Germany. We gratefully acknowledge Dr. Thomas Fischer, Dr. Shaista Ilyas, Dr. Laura Wortmann, and Dr. Shifaa Siribbal for fruitful discussions during the preparation of this subchapter.

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