(bio-) conjugation of inorganic nanoparticles for targeted delivery

ADR-12430; No of Pages 12 Advanced Drug Delivery Reviews xxx (2013) xxx–xxx

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Controlled antibody/(bio-) conjugation of inorganic nanoparticles for targeted delivery☆ Jose-Maria Montenegro a, Valeria Grazu b, Alyona Sukhanova c, d, Seema Agarwal e, Jesus M. de la Fuente b, Igor Nabiev c, d, Andreas Greiner e, Wolfgang J. Parak a,⁎ a

Fachbereich Physik and WZMW, Philipps Universität Marburg, Marburg, Germany Instituto de Nanociencia de Aragon, University of Zaragoza, Spain Dept. EA3798 “Detection and Nanotechnological Therapeutical Approaches to Biological Mechanisms of Defence”, University of Reims Champagne-Ardenne, Reims, France d Laboratory of Nano-Bioengineering, Moscow Engineering Physics Institute, 115409 Moscow, Russian Federation e Lehrstuhl für Makromolekulare Chemie II, Universität Bayreuth, Germany b c

a r t i c l e

i n f o

Article history: Received 13 December 2011 Accepted 21 December 2012 Available online xxxx Keywords: Colloidal nanoparticles Quantum dots Gold nanoparticles Magnetic nanoparticles Antibodies Drug delivery Bioconjugation Molecular recognition Controlled targeting

a b s t r a c t Arguably targeting is one of the biggest problems for controlled drug delivery. In the case that drugs can be directed with high efficiency to the target tissue, side effects of medication are drastically reduced. Colloidal inorganic nanoparticles (NPs) have been proposed and described in the last 10 years as new platforms for in vivo delivery. However, though NPs can introduce plentiful functional properties (such as controlled destruction of tissue by local heating or local generation of free radicals), targeting remains an issue of intense research efforts. While passive targeting of NPs has been reported (the so-called enhanced permeation and retention, EPR effect), still improved active targeting would be highly desirable. One classical approach for active targeting is mediated by molecular recognition via capture molecules, i.e. antibodies (Abs) specific for the target. In order to apply this strategy for NPs, they need to be conjugated with Abs against specific biomarkers. Though many approaches have been reported in this direction, the controlled bioconjugation of NPs is still a challenge. In this article the strategies of controlled bioconjugation of NPs will be reviewed giving particular emphasis to the following questions: 1) how can the number of capture molecules per NP be precisely adjusted, and 2) how can the Abs be attached to NP surfaces in an oriented way. Solution of both questions is a cornerstone in controlled targeting of the inorganic NPs bioconjugates. © 2012 Elsevier B.V. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Nanoparticle synthesis with a controlled number of functional ligands . 3. Functionalization of nanoparticles with antibodies . . . . . . . . . . 4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Due to their innate high specificity and large diversity, antibodies (Abs) are one of the most used biomolecules to provide specificity and ☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Inorganic nanoparticle platforms”. ⁎ Corresponding author. E-mail address: [email protected] (W.J. Parak).

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bioactivity to nanoparticles (NPs). Nowadays, thanks to Abs production and screening innovations, it is feasible to obtain or engineer specific Abs against virtually any substance in high amounts. Thus it is possible to find Abs that recognize an extraordinarily large number of antigens ranging from large pathogens (viruses, bacteria, etc.) to small molecules (drugs, hormones, bacterial toxins, allergenic peptides, etc.) [1,2]. Moreover, Abs have shown high binding affinities with amazing specificity for target molecules even in complex sample matrices (heterogeneous food mixtures, cell lysates, etc.) and with low target concentrations

0169-409X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.addr.2012.12.003

Please cite this article as: J.-M. Montenegro, et al., Controlled antibody/(bio-) conjugation of inorganic nanoparticles for targeted delivery, Adv. Drug Deliv. Rev. (2013), http://dx.doi.org/10.1016/j.addr.2012.12.003

J.-M. Montenegro et al. / Advanced Drug Delivery Reviews xxx (2013) xxx–xxx

b) +

+

Binding Coating

Affinity Chromatography

c) + + + + +

+ + +

+ + +

Adsorption to Beads

+

+ + -+

+ + +

+ + +

+ ++

+ NP Binding Desorption from Beads and Bead Removal

-

The synthesis of NPs such as fluorescent semiconductor NPs (Quantum Dots, QDs), noble metal NPs, or superparamagnetic NPs, and their surface chemistry has been described in many recent reviews [20–26]. After synthesis the surface of the NPs in general is coated with a ligand shell, which provides colloidal stability [27]. In particular in the case of Au, and with some limitations also in the case of CdSe/ZnS core/shell NPs, (biological) molecules can be directly attached via thiols to the NP surface by partially replacing the original ligand shell. Alternatively, (biological) molecules can be also linked to the original ligand shell. One classical way for such subsequent (bio-) conjugation is the use of crosslinkers [28]. These link functional groups such as \COOH, \NH2, \OH, \SH as provided by the original ligands on the NP surface with functional anchor groups of the (biological) molecules. Prominent examples are the use of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) chemistry [29] and sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) [30] to link molecules bearing \NH2 and \SH groups with \COOH, \NH2 and \OH groups on the NP surface. In order to ensure attachment of a very limited number of capture molecules per NP, one may use simultaneously two types of polyethylenglycol (PEG)based polymers for NPs solubilisation or encapsulation: a very small and defined quantity (b5%) of polymer containing valences available for bioconjugation (e.g. SH–PEG–NH2 for sulfo-SMCC-based conjugation protocol) supplemented with saturating quantity (>95%) of polymer of the same size with polar groups ensuring NP solubility, but not available for bioconjugation through the same protocol (e.g. SH-PEG-OH polymer). By varying the ratio of these polymers on the surface of NPs and by decreasing the number of valences available for NP bioconjugation one may tag a very limited and controlled number of functional ligand molecules per NP [30]. However, as motivated above for many applications, controlled bioconjugation involving an even more precise control of the number of molecules attached per NP is desirable. This is a nontrivial task. Assuming 100% linking efficiency, incubation with n ligands per NP will statistically lead also to NPs with n − 1, n + 1, n − 2, etc. ligands per NP. Thus, by simply adjusting the ratio between ligands

a)

+ + -+

+ + +

+ + +

+ ++

-

2. Nanoparticle synthesis with a controlled number of functional ligands

and NPs during the linking reaction it is not possible to make NPs with a precisely defined number of ligands per NP. In the following, different strategies are discussed on how NPs with precisely n ligands per NP can be obtained. Arguably the easiest conjugates are NPs with exactly one ligand per NP. This is for example possible by using huge ligands, so that for geometrical reasons only one ligand fits per each NP. This can be done for example by using polymers modified with the functional compound of interest as ligand for coating the NPs (cf. Fig. 1a). One good example is the work of Wilson et al. where, using a polymer chain with one functional biotin moiety for wrapping around a NP,

-

[3–10]. Therefore, the interest of Ab-NP formulations focuses on many biotechnological and biomedical applications. This is reflected in the number of articles, as indexed by ISIWeb of Knowledge® (Topic = antibodies and nanoparticles), showing their potential applications, which increased almost exponentially during the last years. Cell sorting, bioseparation, and purification are currently the main biotechnological applications for Ab-conjugated NPs (Ab-NPs). Indeed, a significant number of enterprises are commercializing them for this purpose (Invitrogen-Life Sciences, BBInternational, etc.). Another field of application is the developing of nanomaterial-based biosensors for disease diagnosis, food safety examination, pathogenic detection and environment monitoring. The use of Ab-NPs for diagnosis has allowed not only for improving well-established detection techniques (Enzyme Linked ImmunoSorbent Assay, ELISA; Polymerase Chain Reaction, PCR, etc.), but also for developing new strategies of detection which are more simple and sensitive [11–14]. This will enable in a near future the construction of point-of-care immunosensor devices which are portable, rapid, robust, and user-friendly, with enhanced sensitivity and an integrated format while lowering their costs. Moreover, attachment of Abs onto NPs can be useful for a large variety of biomedical applications especially in the field of in vivo diagnosis and even in human therapy. The bulky list of biomedical application of Ab-NPs includes targeted drug delivery, gene therapy, cell labeling/tracking, magnetic or optical hyperthermia treatments, molecular imaging, etc. [15–19] However, it is important to note that the controlled conjugation of the Abs to the surface of the NPs is a key issue to finally achieve success in any of these applications.

-

2

d) Binding +

Fractionation

Fig. 1. a) Only one ligand fits per NP. b) A low number of ligands added per NP makes sure that at maximum one ligand is attached per NP. Subsequent affinity chromatography collects the NPs with exactly one ligand per NP. c) Schematic drawing of place exchange reactions of ligands. Ligands bind first with their charged functional group to the surface of oppositely charged beads. NPs can bind to the other terminal of the ligands via specific attachment. The resulting ligand-NP conjugates are then desorbed from the beads. d) NPs can be fractionated according to the number of ligands attached per NP. Legend: the (inorganic) particle core is drawn in deep blue, the surface capping of the NP in light blue. The ligand (e.g. a biological molecule or a polymer) which is attached to the NP is drawn in red. The functional group of the ligand is drawn in green, and a group on the ligand which is used for specific attachment to the NP is drawn in yellow.

Please cite this article as: J.-M. Montenegro, et al., Controlled antibody/(bio-) conjugation of inorganic nanoparticles for targeted delivery, Adv. Drug Deliv. Rev. (2013), http://dx.doi.org/10.1016/j.addr.2012.12.003

J.-M. Montenegro et al. / Advanced Drug Delivery Reviews xxx (2013) xxx–xxx

it is assumed that in the average each NP is coordinated by only one polymer chain [31]. Here, aminodextran was functionalized by pyridyldithio propionate and a controlled number of biotin moieties. This functionalized dextran wrapped around Au NPs exposing the biotin moieties as functional groups. If an excess of ligands is added (i.e.> > 1 ligand per NP), still due to its large size only one ligand is bound per each NP and the probability for NPs with no ligand is low. In the opposite direction, by adding only a very low fraction of (small) ligands (i.e. b b 1 ligand per NP) during the conjugation most NPs will have no ligand, whereas some NPs will have exactly one ligand attached per NP. The probability of getting NPs with more than 1 ligand per NP is very low in this case (cf. Fig. 1b). In order to isolate the NPs with 1 ligand per NP from the NPs without ligand, affinity chromatography has been used [32]. Levy et al. have demonstrated this approach by using metal ion affinity chromatography (IMAC) to purify peptide-capped NPs. These peptide-capped NPs are modified with a His-tag sequence, binding to an immobilized nickel–nitriloacetic (Ni–NTA) functionalized beads. Controlled starting NPs and peptides concentrations lead to particles with statistically 1 peptide per NP. The methodology of gel electrophoretic fractionation was used by Howarth et al. to purify streptavidin–QDs conjugates. The replacement of wild streptavidin coating QDs for monovalent streptavidin containing a low amount of biotin binding site, permits to obtain monovalent QDs–Streptavidin conjugates [33]. Following another strategy, place-exchange reactions of alkanethiols on NPs can be used for the precise functionalization of NPs [34]. The use of solid-state immobilized alkanethiols has led to NPs with a discrete number of functionalities [35–41]. Following this approach of place exchange reactions on solid supports, slightly cross-linked polymer beads, e.g. Wang resin, is functionalized with e.g. mercaptohexanoic acid by covalent bonding. Due to the low density of mercaptohexanoic acid on the beads surface, alkanethiol-coated Au NP conjugates undergo place exchange reactions with only one molecule of mercaptohexanoic acid, yielding monocarboxy-functionalized Au NPs (cf. Fig. 1c). Recently, it has been shown that amino-functionalized silica gel can be used for non-covalent bonding with mercapto hexanoic acid, following the same concept [38]. In extension of place exchange reactions, the higher reactivity of Au NPs on their poles was used for bi-functionalization of NPs. These bi-functionalized NPs were copolymerized to yield copolymers composed of organic moieties and NPs [42]. A new method for the preparation of monofunctionalized Au NPs has been introduced by using polymerizable thiol ligands for Au NPs [43]. The initiation of free radical polymerization of ligands on the surface of Au NPs with a carboxy-functionalized radical initiator, led to Au NPs with one carboxylic group per NP. Besides monofunctionalized NPs, also NPs with a controlled number of multiple ligands can be synthesized. As mentioned, a typical approach in which NPs are incubated with a defined number of ligands per NP ends up with a statistical distribution of them in the particle surface, whereby the main species can be controlled by the number of added ligands. Fractionation of the ligand-NP conjugates according to the number of ligands per NP leads to samples with conjugates with an exactly defined number of ligands per NP. Such fractionation has been demonstrated by using gel electrophoresis [44] and chromatography [45] (cf. Fig. 1d). For example, Sperling et al. designed a protocol where it was possible to separate NPs functionalized with a controlled number of functional groups attached on the surface [46]. This methodology is based on the use of a long polyethylene glycol (PEG) chain (>5 kDa), attached to the carboxylic groups on the surface of polymer coated NPs [47]. This PEG functionalization introduces a change in the NPs size that leads to the fractionation of NPs with a discrete number of PEG attached (0,1,2…) via gel electrophoresis. The chemical modification of the PEG terminal end with the molecule of interest leads to NPs modified with an exactly defined number of substituents. NPs with a discrete number of DNA [44,48–51], PEG [46,47,52], and streptavidin [47,53] have been produced with this method. Also, NPs with exactly one Ab per NP have been demonstrated based on NPs with exactly

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one streptavidin per NP and biotinylated Abs [53]. HPLC techniques have been used for ligand-NP fractionation as well. Mullen et al. have examined amide coupling methods for quantitative assessment of dendrimer distributions. A skewed Poisson distribution was observed and quantified using HPLC. The prepared samples had an average number of ligands per dendrimer ranging from 0.4 to 13, in agreement with the mean ligand/dendrimer ratio, measured by 1H NMR, gel permeation chromatography (GPC) and potentiometric titration[54]. These results provide insight into the heterogeneity of distributions that are obtained for many classes of nanomaterials to which ligands are conjugated. A general problem of all the above-mentioned reports is the analytical proof for the functionalization of NPs with exactly 1, 2, etc. functional ligand molecules per NP. The most common method for this purpose is the formation of controlled NP groupings which can be imaged by transmission electron microscopy (TEM). Assuming for example the case that one NP has exactly one functional group, and another NP has exactly one other functional group which binds to the functional group of the first NP, then both NPs should form a dimer, which could be observed by TEM. However, such NP architectures could also be generated by e.g. electrostatic forces as previously reported [55]. Therefore TEM analysis always requires statistical evaluation and single images of just one NP grouping are not sufficient proof. Controlled NP groupings and thus proof of discrete functionalization have been very successfully demonstrated in the case of DNA as biological ligand [35,48,49,56]. Progress in proving the functionalization of Au NPs and their covalent attachment to macromolecular chains was achieved recently by gel permeation chromatography (GPC) of Au NPs with controlled functionality [57]. Here, free radical polymerization of 4-vinylthiophenol was initiated on the surface of Au NPs by a functionalized macroinitiator resulting in Au NP-coumarin conjugates with one coumarin moiety per Au NP linked by a polystyrene spacer. Clear evidence was given by GPC that the Au NP, PS spacer, and coumarin moiety are chemically linked together. Another elegant proof of discrete functionalization has been given by Carstairs et al. [58]. They have synthesized QDs with a defined number of DNA chains by conjugation of streptavidin-coated QDs to biotinylated DNA duplexes. Controlled DNA-QD ratios followed by purification through ion exchange sepharose-packed spin columns using increasing NaCl concentrations, led to the purification of QD-DNA conjugates with a defined number of DNA chains. Fluorophores eventually photobleach upon excitation. A generally accepted proof for single molecules is discrete photobleaching [59]. This means that the fluorescence intensity of a small grouping of fluorophores goes down stepwise by the subsequent photobleaching of one fluorophore after the other. Bumb et al. demonstrated that NPs with n fluorescent ligands undergo exactly n photobleaching steps, which demonstrates that the NPs are functionalized exactly with n ligands per NP [60]. The above-mentioned concepts demonstrate that in fact, conjugation of NPs can be done in a way that the number of attached functional ligand molecules is precisely controlled. However, not all of these concepts can be carried out with any type of NP. Several methods involve direct coupling of the (biological) molecules to the NP surface. In the case of Au NPs this is easily achieved via terminal thiol groups on the molecules, which for other NP materials is not possible. Methods in which the (biological) molecules are linked to the original ligand coating of the NPs are more general in this direction, as they do not depend of the NP material. Still this field is in its infancy and has been further developed to become routine. The biggest bottlenecks are that several methods only work for certain NP materials, and that so far functionalization has been demonstrated only with few different molecules. In this direction a universal protocol for monofunctionalization of NPs with Abs is still missing. In the particular case of antibodies, multivalency is not desirable for many envisioned applications. For example Ab monovalency is highly desirable when studying biological process that could be affected by protein clustering. This is the case of in vivo single-molecule imaging studies in cells as multivalency could generate cross-linking of surface

Please cite this article as: J.-M. Montenegro, et al., Controlled antibody/(bio-) conjugation of inorganic nanoparticles for targeted delivery, Adv. Drug Deliv. Rev. (2013), http://dx.doi.org/10.1016/j.addr.2012.12.003

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proteins and activate unwanted signaling pathways which could dramatically alter cell behavior [33]. Moreover, NP functionalized with only one antibody are also desirable in biodetection to improve the limit of detection (LOD) of nanobiosensors that use NPs as labels (fluorescence or electrochemical labeling, mass or gold/silver colorimetric enhancement, etc)[61,62]. Though as mentioned to our knowledge no general protocol for synthesizing monofunctional Ab-NP conjugates the methods as described here may offer a starting point in this direction. To give an example, with gel-electrophoresis NPs with a precisely determined number of PEGs per NP can be obtained [46]. By using PEG with a group on the free end which is reactive towards antibodies, as for example the O6-Alkylguanine-DNA Transferase (SNAP) technology by Johnsson et al. [63], one can imagine first making NPs with one sticky end, which can bind only at one Ab [64]. The concept of the SNAP technology will be further introduced below.

attached to the NP surface. Bioactivity, avidity or targeting efficiency of the functionalized NPs will depend on that. However, attachment of Abs to the surface of NPs can impair this function, for example when the antigen binding sites are chemically modified or sterically blocked upon conjugation. For this reason, oriented attachment of Abs is desirable. In this case conjugation will be made at a non-active region of the Ab and the antigen binding sites point towards solution. In order to understand the different strategies that could be used to achieve oriented immobilization, it is important to review first the 3D structure of the Abs. There are five classes (isotypes) of immunoglobulins or Abs (IgG, IgA, IgM, IgD and IgE). Hereby immunoglobulin G (IgG) is the most abundant in normal serum and the most extensively used for biofunctionalization of NPs. The basic structure of an IgG molecule comprises two identical light chains and two identical heavy chains, which are linked together by disulfide bonds (cf. Fig. 2) with a molecular weight of about 150 kDa and an average size of 14.5× 8.5 × 4 nm3 [65]. The light chains contain one variable (VL) domain and one constant (CL) domain, whereas the heavy chains have one variable (VH) and three constant (VL–VH) domains. The 4 protein chains are assembled with a specific “Y-shaped” geometry. IgG molecules are bifunctional, and the two identical antigen-binding sites are localized at the end of

3. Functionalization of nanoparticles with antibodies Besides that for certain applications it is essential to control the Ab valency of functionalized NPs as explained above, another task is to keep the Ab capability of recognizing its corresponding antigen once

a)

Antigen-binding sites (BS)

N-terminus

b)

N-terminus

Disulfide bonds

Hinge region

Carbohydrate

C-terminus

c) Reduction Agent (RA) RA RA

Functional BS

Non-functional BS

Light chain fragment (25 kDa)

Disulfide bonds

Partially-cleaved heavy-light chain fragment (75 kDa)

Heavy chain fragment (50 kDa)

d) Antigen-binding sites

Hinge region

Single Variable Domaine (VhH) 13 kDa

6 His + Cys Engineered VhH

Fig. 2. Schematic cartoon showing: a) the Y-shaped structure of an Ab (which is the ligand to be attached to the NP, and thus is drawn in red), the two light chains (variable regions) and the heavy chains (constant regions) are colored in violet and red respectively. The specific functional sites with which the Ab can bind antigens are drawn in green. Groups which can be used for attachement to NPs are drawn in yellow. b) Three-dimensional model of an Ab from studies by X-ray crystallography. The Ab structure was taken from the Protein Data Bank (PDB) and visualized using PyMol v0.99. The PDB entry 1IGY was selected for this 3D representation. Light chains are colored in orange and cyan. Heavy chains are colored in yellow and green. Carbohydrate residues are colored in purple. c) Fragmentation of an Ab into function Ab fragments. d) Fragmentation of the llama heavy chain Ab (HcAb) into the single variable domain (VhH) Ab fragment.

Please cite this article as: J.-M. Montenegro, et al., Controlled antibody/(bio-) conjugation of inorganic nanoparticles for targeted delivery, Adv. Drug Deliv. Rev. (2013), http://dx.doi.org/10.1016/j.addr.2012.12.003

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the arms of the Y. These two antigen-binding ends (or amino-terminal ends) of the Ab molecule are called the Fab fragments (for antigen binding fragment). The stem of the Y (or carboxyl-terminal end) is the Fc fragment (for crystallizable fragment). This stem region of an Ab molecule ensures that each Ab generates an appropriate immune response for a given antigen by triggering effector functions. It links the Ab to other actors in the immune response by directing different physiological effects such as complement-mediated lysis, enhanced phagocytosis, or allergy (in some cases) [66–72]. However, full-size Abs have a large size, which impedes intratumoral distribution due to interstitial tumor pressure and limits their intracellular and intratissue penetration especially in solid tumors. Furthermore, the conditions used for Abs conjugation often provoke IgG unfolding [73]. For this reason Abs can be fragmented and only the antigen-binding fragments are used (cf. Fig. 2c). Small Ab fragments conjugated with NPs in a highly oriented manner can be considered as an attractive possibility for the generation of smallest targeted nanoprobes. Unfortunately, the physico-chemical properties of Fab and scFv (single chain variable fragment), including their stability, tendency to agglomeration and production cost, do not qualify them as good candidate for routine preparation and application as targeting reagents [74]. Additionally, the disadvantage of using Ab fragments such as Fab or scFv is that they have reduced Ab avidity. The strategy to overcome this problem includes engineering of even smaller functional Ab fragments and delivering the desired number of such fragments through a common carrier, e.g., NPs. Multivalent nanoprobes can also be engineered wherein the smallest functionally active fragments of different Abs against different antigens may be tagged with the same NP. This way more than one cellular target can be identified by a single multivalent Ab fragment conjugate [75]. Going in this way, Sukhanova et al. engineered ultra-small nanoprobes through oriented conjugation of semiconductor QDs with 13-kDa single-domain antibodies (sdAbs) derived from llama IgG (cf. Fig. 2d) [30]. Monomeric sdAbs are 12-times smaller than monoclonal Abs and demonstrate excellent capacity to refolding. sdAbs were tagged with NPs through an additional cysteine residue integrated within the C-terminal of the sdAb. Careful variation of sdAb/NP ratios in their specific conjugation reaction was controlled by the Bradford assay [76] and allowed for developing ultrasmall sdAb-NP nanoprobes comprising, in average, four copies of sdAbs coupled per NP in a highly oriented manner [77]. sdAb-NP conjugates against carcinoembryonic antigen (CEA), a well-known cancer biomarker [78], demonstrated excellent specificity of flow cytometry quantitative discrimination of CEA-positive and negative tumor cells. Moreover, the immunohistochemical labeling of biopsy samples was found to be comparable or even superior to the quality obtained with gold standard protocols of anatomo-pathology practice [30]. The use of scFv Ab fractions has also been reported. Colombo et al. have used scFv Ab recombinant fractions for targeting specific cancer cells. The linkage to NPs was carried out exploiting the SNAP irreversible reaction with modified guanine functionalized NPs. As mentioned above, gel electrophoresis allows for monofunctional guanin-PEG-NP conjugates, as it can fractionate NPs with a different number of attached PEGs per NP [46]. This approach yielded scFv-magnetic NPs that were effectively used for targeting specific cancer cells. Although in this example, magnetic nanoparticles were used, the method is potentially universal [64]. It also has the potential to yield monofunctional Ab-NP conjugated. The methods for linking Abs to NPs as described below are in general also valid for linking Ab fragments to NPs, though oriented attachment of the Ab fragments is favored in some cases due to their geometry or due to the kind of methodology used. It is true that for therapeutic applications, the faster speed of penetration in solid tumors of antibody fragments over intact antibodies is a remarkably advantage. In the late 80's it was already established that an intact molecule of IgG took 54 h, while a Fab fragment only 16 h to penetrate 1 mm into a solid tumor [79]. However, the functionalization of NPs with intact antibodies for targeted drug and

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gene delivery together with magnetic or optical hyperthermia increased exponentially during the last years as indexed by ISIWeb of Knowledge® (Topic = antibodies and nanoparticles and therapy). Moreover, for in vitro diagnosis purposes there is a wide interest in the functionalization of inorganic NPs with intact IgG Abs due to their higher avidity and stability. As (full-size) Abs are asymmetric molecules they could adopt four possible spatial orientations upon conjugation to the surface of a NP (cf. Fig. 3) [80,81], whereby the actual orientation may be a combination of these. Both, in the case of “head-on” and “sideways-on” orientations, the efficiency of antigen recognition of the functionalized NPs is hampered due to steric hindrance. Therefore, a good option for oriented bioconjugation is through the Fc fragment. This naturally involves that the owned biological activity of the Fc fragment may be compromised or sacrificed. Several conjugation strategies guarantee oriented attachment of an Ab through such an “end-on” orientation [28,82–90]. This ensures free Fab regions far away from the NP surface. While ensuring an oriented binding, all the strategies developed for site-specific conjugation of Abs through their Fc region have the added advantage of their generality of use among different IgG Abs. This is possible as amino acid sequencing studies have shown that only small differences can be found in the protein sequence of different Abs. These differences are mostly localized in the tip of the arms of the Abs (antigen recognition site). Hence the Fc region is identical in all Abs of the same isotype and species, and well-conserved among different species. However, it needs to be pointed out that recently also “flat-on” orientations have been demonstrated which do not alter the antigen-binding activity [91–93]. For the development of Ab coupling strategies onto NPs, one may take advantage of existing experience in the development of immunoafinity chromatography columns and immunosensing platforms using microstructured materials such as agarose beads, glass/silicon or gold surfaces, etc. However, Ab functionalization of NPs has some limitations when compared to surfaces and microbeads. The biofunctionalization of NPs is not trivial as it involves several stages in which it is essential that they remain colloidally stable. This is a problematic task as many NPs tend to agglomerate upon small changes of pH and/ or ionic strength. This is because the colloidal stability of NPs is governed by a delicate balance among attractive (van der Waals and/ or magnetic) and repulsion forces (electrostatic and/or steric). Since there is a wide variety of NPs (magnetic NPs, plasmonic NPs, quantum dots, etc.) which are very different in terms of size, surface area, organic shell composition, density of reactive groups, colloidal stability, etc., there are no standardized functionalization protocols, which make it necessary to optimize them for each particular case. Therefore, the choice of the optimal coupling method will not only depend on the functional groups which are present on the surface linking layer of the NPs, but also on the density per nm2 of these groups (cf. the first part of this review), and on the colloidal stability of the NPs at different pH and ionic strength values. In the following we will describe 4 different concepts for linking Abs to NPs, which are based on: a) physical adsorption, b) direct covalent linkage between the surface of the NP and the Ab, c) using adapter molecules, or d) combining ionic adsorption and covalent binding (cf. Fig. 4; Table 1).

a)

End-on

b)

c) Head-on

Sideways-on

d)

Flat-on

Fig. 3. Schematic representation of the four different orientations of antibodies on surfaces. “End-on” (Fc attached to the support) (a) and "Flat-on" (all three fragments attached to the support) (d) orientations of the antibodies, permits to keep its functionality. However, this reactivity is compromised with the “Head-on” (Fabs attached to the support) (b) and “Sideways-on” (one Fc and one Fab attached to the support) (c) orientation.

Please cite this article as: J.-M. Montenegro, et al., Controlled antibody/(bio-) conjugation of inorganic nanoparticles for targeted delivery, Adv. Drug Deliv. Rev. (2013), http://dx.doi.org/10.1016/j.addr.2012.12.003

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J.-M. Montenegro et al. / Advanced Drug Delivery Reviews xxx (2013) xxx–xxx

b)

a) +++ + - - - + - -

c) CONH

d) Protein G

e) Biotin Streptavidin

+++ + - - - + - -

CONH

Fig. 4. Schematic representation of the different strategies used to functionalize NPs with Abs: a) electrostatic adsorption; b) covalent binding via amine groups on the Ab; c) covalent binding via carbohydrate groups on the Ab; d) use of adapter biomolecules (streptavidin–biotin, Protein G); e) ionic adsorption plus covalent binding.

Physical adsorption is generally based on hydrophobic, electrostatic, hydrogen binding, and van der Waals attractive forces between the Ab and the surface of the NP. It provides the simplest and most straightforward immobilization process. Its facility and versatility is based on the fact that it is not necessary to chemically modify the Ab nor to activate the functional groups of the NP. Hydrophobic interaction is one of the methodologies most used, but often suffers from denaturation of immobilized Abs, yielding poor reproducibility. Extensive denaturation occurs because the hydrophobic interaction with the NP surface and the Ab, thermodynamically forces a change in the native three-dimensional structure of the Ab. As a consequence, there is a time dependent and irreversible loss of the biological activity of the attached Abs [94–96]. Electrostatic attraction is governed by a charge interaction between oppositely charged NPs surfaces and Abs molecules (cf. Fig. 4a). It is important to note that different Abs can have large differences among their isoelectric points (pIs), i.e. the pH at which they are neutral. Therefore, it is important to know the pI of the Ab of interest in order to work at a pH that promotes its ionic adsorption, i.e. at a pH lower (Ab is positively charged) or higher (Ab is negatively charged) than the pI. Recently, some studies showed that this simple coupling methodology can be also used to orient Abs [97,98]. Ionic adsorption is a multipunctual process, and because of this, its rate depends mainly on the number of charged groups present on the Ab surface. Therefore, it may be expected that an Ab may be immobilized on the support by its surface region where the highest possibilities of getting this multipoint adsorption exists. Since the Ab molecule is asymmetric, the region with the greatest number of charges is the plane involving the four Ab subunits during the adsorption process. This means that the Ab will adopt a “flat-on” molecular orientation in which the antigen recognition sites will remain close to the NP surface though they will not be involved in immobilization. In this way, antigen binding capacity is preserved. The main disadvantage of this method is that the attachment is weak and pH dependent. The adsorbed Abs molecules may be removed by changes of pH and/or ionic strength of the buffer used for performing assays. Furthermore, as not all the charged groups of the NP are used for Ab binding, nonspecific adsorption of matrix proteins cannot be avoided. In this sense, in vivo studies recently showed that adsorbed Abs can suffer competitive displacement by serum proteins [99]. Direct covalent binding of Abs is not as straightforward as physical adsorption. It requires several steps including the introduction of functional groups on the NP surface (cf. the first part of this review), the use of chemical linkers, and/or the chemical modification of the Ab. Depending on the coupling chemistry selected, the binding can occur with or without chemical modification of the Ab. Certain coupling chemistries allow the direct reaction with primary amines, one of the most available groups on the Ab surface. Others, however,

need a previous chemical modification of the Ab such as oxidation of its sugar moieties, reduction of disulfide bonds, etc. At any rate, despite being a more complex process, immobilization via covalent coupling provides a number of distinct advantages over physical adsorption such as higher stability of the bioconjugate, and better reproducibility. Binding through primary amines of the Ab is straightforward and the amine groups of Abs are probably the most used functional moieties to covalently immobilize them (cf. Fig. 4b). Their widespread popularity is due to the following factors: i) they are residues abundant in most proteins, ii) they are usually located on the surface of the Ab, and iii) they are very reactive without any previous activation with a wide variety of reactive groups on the NP surface. Thus, amine groups can react directly with NPs containing reactive groups such as aldehydes, epoxides or cyanogen bromide [100,101]. In the case of NPs having carboxylic (\COOH) or amine groups, their previous activation with carbodiimide/N-hydroxysuccinimide or glutaraldehyde allows for covalently linking them to the primary amines of the Ab [28,102]. However, Ab immobilization using its amine groups is not the most appropriate coupling methodology as it results in randomly oriented Abs on the NP surface. The high reactivity of all the aminebinding linkers makes them very unstable at alkaline pH values. As a consequence, the coupling reaction must be carried out under mild pH values and occurs therefore mainly via the most reactive and exposed amine groups. There are two types of amine groups on the Ab surface, the ε-amino of Lys and the terminal amino group. The most reactive amino group under mild pH conditions is the terminal amino group (pKa around 7–8 versus 10.5–10.7 for ε-amino of Lys). When studying the distribution of amino residues in the IgG Ab, it is possible to observe that the four terminal amino groups of the Ab are near to the antigen recognition place. Consequently, most of the Ab molecules immobilized through their most reactive amine groups would adopt “head-on” and “sideways-on” spatial orientations. This causes the loss of antigen binding capacity due to direct binding of the antigen-binding site or steric hindrance by the NP surface. For this reason often binding through thiol groups of the Ab is used [103–106]. In Abs the sulfhydryls are oxidized as disulfides, and they are important contributors to the Ab function as they: i) contribute to the tertiary structure of each protein subunit, ii) covalently connect heavy and light chains, and iii) join the two Ab halves at the hinge region. As only free sulfhydryls can be conjugated directly with thiol-reactive groups of NPs, the native disulfide bonds of the Abs must be selectively cleaved with reducing agents such as 2-mercapthoethylamine, mercaptoethanol, dithiotreitol, thiopropylagarose, etc. [107–109]. As the disulfides in the hinge region are the most susceptible to reduction, it is possible to selectively cleave only these disulfides and thus to split the Ab into two monovalent halves without altering their 3D structure and antigen-binding efficiency. By the introduction of thiol reactive groups (malemide, iodoacetyl, 2-piridyl disulfide, etc.) on the NP surface the Ab fragments can be selectively immobilized. Although this strategy has the drawback that a previous chemical modification of the Ab is necessary, it has the advantage that it ensures an oriented immobilization (“end-on” orientation). This chemical modification can also be combined with fragmentation of the IgG Ab “under” the hinge region by the use of proteolytic enzymes (pepsine, ficin…). In this way it is also possible to conjugate small Ab fragments such as F(ab')2 and Fab' [84]. The scFv (single chain variable fragment) Ab fragment, consisting of the IgG heavy- and light-chain variable domains, which are connected with a flexible peptide linker (cf. Fig. 2c), maintains a high binding affinity and specificity. These functionally active Ab fragments were used for preparation of nanoprobes shown to be efficient in specific in vivo and in-situ tumor targeting and imaging [75]. Thus high antigen binding capacity can also be achieved with Ab fragments, which allows for increasing the number of antigenbinding sites per nm2 of the NP surface. In the special case of Au NPs the reduced monovalent halves can be immobilized directly

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Table 1 Comparison of the immobilization protocols of Abs onto NPs. Strategy

Functional groups needed on the NP surface

Physical adsorption

Hydrophobic interaction

Hydrophobicity

Electrostatic adsorption

ionizable groups (positive or negative charges)

Covalent binding Through primary amines

Through thiols

Through sugar moieties

Reactive moieties -epoxides -aldehydes -cyanogen bromide Activable moieties -carboxylic groups -amine groups -Gold surface or -thiol reactive groups: -piridyl disulfide -maleimide -hydrazide groups -amine groups

Use of adapter biomolecules Via biotin binding proteins -Indifferent

Advantages

Disadvantages

-Facility -versatility -not necessary to modify the Ab

-Reversible attachment

-Ab denaturation -poor reproducibility -Oriented binding -A minimum density of charged groups is needed -binding depends on pH/ionic strength -limited by NP stability with pH -Better reproducibility -Activation of the NP and/or the Ab is -stable bond needed -Not necessary to modify -Random binding the Ab -low biological antigen-binding capacity -instability of most of the reactive moieties with time and pH

-Oriented binding

-Necessary to reduce the Ab

-necessary to oxidize the Ab. -some Abs do not contains sugars -Usually oriented binding -Reversible attachment -NP functionalization with (strept)avidin -Oriented binding (⁎) -binding resists to harsh -site-specific Ab biotinilization conditions -difficult to control NP valency -(strept)avidin is expensive

-Working pH greater or smaller than the pI of Ab

-Possibility to combine with protease fragmentation

-oriented binding

Via secondary Ab

-Indifferent

-Oriented binding -not necessary to modify the Ab -easier to control NP valency

Via Fc-binding proteins

−Indifferent

−Oriented binding −not necessary to modify the Ab

Via nucleic acid -Indifferent hybridization Ionic adsorption + covalent Ionizable groups + reactive attachment groups

Observations

-Oriented binding (⁎) -multiplexing capacity -Easy -oriented binding -not necessary to modify the Ab -strong binding

onto gold surface through Au–S linkage. This strategy could also be used in the case of recombinant scFv (single chain variable fragment). An engineered C-terminal cysteine residue at the light chain constant domain of a scFv fragment could be successfully applied to attach scFv onto a gold surface [110]. This direct immobilization strategy has been extensively used as the chemisorption of the SH group to Au is very selective and strong (≈ 30 kcal/mol) [111–114]. However, it is usually difficult to control the valency of the NPs as there is a minimum amount of Ab molecules necessary to bind to the Au NPs in order to guarantee their colloidal stability. Moreover, recent studies showed that this direct chemisorption induces inactivation of the antigen binding capacity of the Ab fragments. This inactivation takes place gradually, and can be diminished if the Ab fragments are coimmobilized with polyethylene glycol (PEG)[115]. Reduced Abs can not only be immobilized onto gold NPs. By the introduction of thiol-reactive groups (malemide, iodoacetyl, 2-piridyl disulfide,…), they can be selectively immobilized on virtually any kind of NP [116–118]. The third covalent attachment strategy involves binding through sugar moieties of the Ab. Also this coupling strategy guarantees an oriented Ab immobilization (“end-on” orientation)

-Reversible binding -site directed immobilization of the secondary Ab -secondary Ab must be monoclonal -not compatible with sandwich-assay formats in biosensing −Reversible binding −difficult to control NP valency −not compatible with sandwich-assay formats in biosensing -Protein A, G or A/G are expensive -Site-directed modification of the Ab -A minimum density of charged groups is needed -limited by NP stability with pH

(⁎) Only via a site-directed biotinilation of the Ab -valency can be controlled by the use of monovalent forms of (strept)avidin. -The host species of the primary Ab must match with the specificity of the secondary Ab

-IgG affinity: Protein G > Protein A -IgG binding activity: Protein G > Protein A

(⁎) Only via a site-directed labeling of the Ab -During immobilization the working pH must be greater or smaller than the pI of Ab

2 doas it takes advantage of the carbohydrate chains attached to the CH main within the Fc region (cf. Fig. 4c) [119,120]. This method needs a first step in which the carbohydrates chains are mildly oxidated by the use of sodium periodate to create reactive aldehydes (–CHO) for coupling [82,101,85]. Then, aldehyde-activated (oxidized) sugars can be reacted directly to NPs containing primary amines through reductive amination or to NPs that have been activated with hydrazide groups. Although this strategy needs the chemical modification of the Abs, the optimization of mild oxidation conditions often allows for obtaining modified Abs with high retention of their antigen binding capacity. In the special case of Au NPs, also the modification of the oxidized Ab with a low molecular weight heterobifunctional linker such as dithiol-PEG-hydrazide has been described, whereby the dithiol group is conjugated directly to the gold surface [92]. Another strategy that can be used and avoids the oxidation of the Ab implies the use of NPs functionalized with boronic acid (BA) [84]. A major limitation of this methodology is the necessity for the Ab molecule to be glycosilated. Polyclonal Abs (from antisera) are usually glycosylated, but other Ab preparations may not have carbohydrates such as recombinant Abs

Please cite this article as: J.-M. Montenegro, et al., Controlled antibody/(bio-) conjugation of inorganic nanoparticles for targeted delivery, Adv. Drug Deliv. Rev. (2013), http://dx.doi.org/10.1016/j.addr.2012.12.003

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fermented in bacteria, or some monoclonal Abs which may not suffer post-translational glycosilation. Oriented immobilization can also be accomplished by using adapter biomolecules which are themselves directly coupled to the NP surface (cf. Fig. 4d). There are several options described in the literature involving i) biotin binding proteins (avidin, streptavidin), ii) Abs that specifically recognize the Fc portion of IgG of a certain specie (secondary Abs), iii) proteins that specifically bind to the Fc region (Protein A, G, A/G), iv) nucleic acid mediated hybridization, and v) genetically engineered Abs with terminal linker-peptide residues. Each of these alternatives has its own advantages and disadvantages. The binding is much stronger than the one achieved by physical adsorption. The strategy of Ab binding via biotin binding proteins makes use of the natural strong binding of avidin or streptavidin to the small molecule biotin (vitamin H) [121–124]. Hereby the Abs need to be modified with biotin, and the surface of the NPs needs to be functionalized with (strept)avidin. The (strept)avidin–biotin interaction is similar to Ab-antigen or receptor-ligand recognition, but with higher affinity constants (Kd around 10−14 M and 10−15 M for streptavidin and avidin, respectively). It is among the most rapid and strongest non-covalent interactions known. This makes the (strept)avidin–biotin complex very resistant to a wide range of pH values, elevated temperatures, and harsh chemical conditions (up to 8 M urea or 3 M guanidine, organic solvents) [84,125–127]. However, it has been recently shown that incubation in non-ionic aqueous solvents above 70 °C can break the biotin–avidin bonding. Avidin has the disadvantage of being a glycoprotein with high pI (≈ 10). This limits its use due to its tendency for agglomeration and to bind unspecifically other components than biotin. Streptavidin on the other hand is purified from the bacteria Streptomyces avidinii and thus it is not a glycoprotein. In addition it has a much lower pI than avidin (around 5–6). This reduces the non specific binding due to ionic interaction with other molecules, and moreover there is no potential for binding to carbohydrate receptors. Also neutravidin (pI ≈ 6.3), a deglycosilated form of avidin, can be used to overcome avidin drawbacks. Since each (strept)avidin molecule contains a maximum of four biotin binding sites, any immobilization protocol can be used to anchor the (strept)avidin onto the NP surface, as it is impossible to provoke steric hindrance of all of them. It is possible to find articles where ionic adsorption or covalent binding via its carboxylic or amine groups were used for linking of (strept)avidin to NPs. However, the stability of the biotin–streptavidin complex can be altered depending on the immobilization methodology selected [128,129]. The multivalency is also an advantage in some applications where amplification of the immunoassay signal is of interest. However, the tetrameric nature of (strept)avidin becomes a problem when control of the Ab stochiometry is needed. To overcome this problem it is possible to use recombinant monomerics forms of (strept)avidin, but taking into account that their affinity for biotin is much lower (Kd around 10−7 M) [130]. Naturally, for the use of this Ab binding strategy it is not only necessary to functionalize the NP with (strept)avidin, but it is additionally required to functionalize the Ab with the binding partner (biotin). In order to avoid a random immobilization it is compulsory that the biotinilation of the Ab is site specific, which can be within its Fc region via its carbohydrates moieties or via thiols obtained after reduction of the disulfides located in the hinge region [131,132]. The second concept is based on attaching Abs via secondary Abs. In this strategy primary–secondary Ab binding is used for the oriented immobilization of Abs to NPs. A secondary Ab refers to Abs specific for other Abs. In contrast, the primary Abs are specific for the non-Ab target of interest. The most popular and customizable assay format of ELISA platforms (sandwich format) is based on this specific recognition between primary and secondary Abs. To obtain NPs functionalized with the primary Ab of interest it is necessary to first immobilize a secondary Ab onto the NP surface that specifically recognizes the Fc region of the primary one. Then these functionalized NPs are incubated with the primary Ab of interest. This facilitates the single-step functionalization of NPs and rapid multiplexing, as chemical modification of the primary Ab is not needed. However certain considerations must to be taken into account: i) the secondary Ab has

to be immobilized in an oriented manner to maximize the antigen binding efficiency of the NPs; ii) the primary Ab to be used has to be generated in host species recognized by the secondary Ab; and iii) the secondary Ab must be monoclonal as the use of polyclonal ones may results in the agglomeration of the NPs after primary Ab addition. An important advantage of this approach is that the valency of NPs is easily controlled by varying the concentration of the incubated primary Abs. This is much easier than trying to control the valency at the chemical conjugation step as it is usually done when using a direct conjugation strategy. A potential drawback of the strategy is that Abs are not attached to the NP surface as strongly as if they were covalently immobilized. A third possibiliy involves Ab binding via Fc-binding proteins, in which first NPs are modified with Fc-binding proteins, which then in turn act as attachment point for the Fc region of immunoglobulin (Ig). There are several cell wall bacteria proteins (Protein A, G) that bind specifically to the Fc region of Ig. Abs can be immobilized via these binding proteins in an oriented “end-on” configuration onto the NP surface [91,133–136]. All these proteins bind almost exclusively with the IgG class of Abs, but their binding properties differ among species and subclasses of IgG. Protein A is generally preferred for rabbit, pig, dog and cat IgG. However, in general, the affinity of Protein G for IgGs is higher than Protein A. Moreover, Protein G shows a broader binding activity to Abs of different species as well as to different isotypes [84]. Nowadays, researchers use recombinant forms of these proteins which lack the albumin and cell surface binding domains that are present in their native forms. This minimizes nonspecific interactions with serum/ cell proteins. Also a recombinant protein known as Protein A/G is now commercially available [137–139]. This is a recombinant fusion protein that includes the IgG-binding domains of both Protein A and Protein G. Therefore, Protein A/G is ideal for binding the broadest range of IgG subclasses from rabbit, mouse, human and other mammalian samples. As in the case of strept(avidin), the random covalent binding of these Fc-binding proteins does not represent a problem, as all these proteins present several IgG binding sites per molecule. It is clear that Ab immobilization using these Fc-binding proteins is much easier than via the streptavidin–biotin linkage, as it does not require any Ab modification (as biotinylation in the case of (strept)avidin linkage). However, for biosensing applications, the use of Ab-functionalized NPs with this strategy is rather limited to only competitive-assays in which only a single antibody is used unless a monomeric form of this proteins is used [140,141]. Forth, Ab binding can also be achieved via nucleic acid hybridization. Ab immobilization can be also achieved taking advantage of specific Watson–Crick base pairing of two complementary nucleic acid chains. This strategy requires a two step processes: i) conjugation of the Ab with a DNA sequence, and ii) conjugation of the NP with another DNA sequence complementary to the one attached to the Ab [142,143]. Conjugation to the NP is easily obtained since hybridization of complementary DNA strands is highly specific and spontaneous below their melting temperature. Another format that was recently reported takes advantage of this specific base pairing to site-specifically tagged Protein G to NPs. In this case, protein-DNA conjugate was used to label functionalized NPs with IgG in an oriented way. This methodology is interesting due to its versatility and especially when there is the need of binding several different Abs onto the same NP [91]. Furthermore, this method offers also the possibility to attached a controlled number of Abs per NP, as NPs with a controlled number of DNA molecules have already been reported, as described in the first part of the review [44,48–51]. Recently approaches with genetically engineered Abs with terminal linker-peptide residues have been reported [71]. Since most recombinant Ab fragments are expressed with an affinity tag (additional amino acids at one of their termini) in order to facilitate purification, this tag could also be used for their oriented attachment on the NP surface [144,145]. These linker-peptides selectively bind to small molecular counterparts which have to be immobilized to the NP surface. Examples are the snap-tag and his-tag. There are two main advantages. First, as the Abs are linked via their peptide-modified terminus ensuring their oriented attachment. Second, it is easier to bind the small molecular counterparts to the surface of NPs than directly binding a whole Ab. The

Please cite this article as: J.-M. Montenegro, et al., Controlled antibody/(bio-) conjugation of inorganic nanoparticles for targeted delivery, Adv. Drug Deliv. Rev. (2013), http://dx.doi.org/10.1016/j.addr.2012.12.003

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disadvantage of this method is that Abs have to be genetically expressed with the additional peptide residues. Ionic adsorption plus covalent binding: oriented binding in two steps. As it was mentioned above, the promotion of ionic adsorption of Abs is a strategy to achieve oriented immobilization (cf. Fig. 4e). The advantages of this strategy are its simplicity and the fact that there is no need of Ab modification steps or the use of expensive adapter proteins. However, the reversibility of ionic interactions limits its further applicability. A few years ago a smart approach that combines ionic adsorption with covalent binding was developed for agarose and epoxiacrylic based supports. The strategy is based on the existing kinetic differences among ionic adsorption processes and covalent reactions. Usually covalent reactions are much slower than physical adsorption processes, and thus it is possible to promote a two step binding method which promotes first the rapid ionic adsorption of the Ab followed by a further covalent attachment step [146]. In this way the ionic adsorption step guarantees a “flat on” orientation of the Ab. Next, further site-specific covalent binding occurs only with the amine-groups which are facing the NP surface, which assures a strong binding [147]. Recently this approach has been extended to polystyrene NPs [148] and magnetic NPs [101]. To achieve this it was necessary to prepare bifunctional NPs containing: i) ionizable groups (that allow attachment of Abs via ionic adsorption) and ii) reactive groups (for further covalent attachment of the Ab). The obtained covalently functionalized NPs with a “flat on” orientation of the Ab had the same biological efficiency than the ones in which the Ab was bound in an “end-on” orientation via its carbohydrate moieties [101]. This methodology is easily applicable to any kind of Ab as long as the pH of incubation is adjusted depending on the pI of the Ab. Moreover, it can be also easily extended to any kind of NP as long as the NP maintains its colloidal stability at the pH of incubation, and has a high enough density of charged groups to promote ionic adsorption of the Ab. It is clear that the oriented binding of Abs is a key feature to obtain highly bioactive NPs. However, another parameter that is also important to take into account is to optimize the surface packing density of the Ab molecules. The efficiency of the functionalized NPs tends to drop drastically if the antigen binding sites are sterically hindered by adjacently immobilized Abs. Hence, along with a proper orientation, an optimization of their surface density is necessary. This again goes back to the first part of the review, in which methods for making NPs with a controlled number of functional ligands and thus anchor points for Abs have been discussed. However, combination of controlling as well the number as the orientation of Abs on the surface of NPs is nowadays still a technical challenge and needs future development. We nevertheless predict that future bioconjugation strategies will focus upon a combination of these two important features, as they will allow for maximum control of the antigen-binding properties of Ab-NP conjugates. Finally, another milestone towards biological applications of Abfunctionalized NPs is to ensure a correct passivation of the NP's surface once the Ab was bound. A high amount of the functional groups on the surface of the NPs are not used to anchor the antibody. These groups must be masked (passivation of the NP surface) to prevent nonspecific adsorption of proteins. Moreover, an inadequate passivation/inertization process can affect the sensitivity limits achieved in nanodiagnosis. Moreover if the Ab modified NPs are going to be used in therapy applications unspecific binding of plasma proteins to nanoparticles can influence their biodistribution and therapeutic efficacy [149]. Once nanomedicines are in the blood, if these non-used functional groups and/or hydrophobicity of the NPs surface are not correctly masked, protein binding to their surface occurs. This gives rise to what it is known as the formation of the “protein corona” [150]. Certain components of this corona, called opsonins, induce NP uptake by the RES and consequently their removal from the bloodstream before they reached the therapeutic target for which were designed [151]. It is known that hydrophilic and neutral carriers will adsorb fewer proteins. Different strategies to diminish RES

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recognition and increase nanomedicines blood half-lives have been extensively reviewed [152–154]. Although detailing these strategies is outside the scope of this review, it is important to highlight the importance of achieving a good inertization of the NPs surface after the Ab was bound which may add additional constraint on the choice of the most adequate bio-conjugation scheme. 4. Conclusions Current methodologies concerning the functionalization of inorganic-core NPs with Abs have been presented. The control of the number of functional groups attached per NP, as well as the characterization and purification techniques available have been treated in detail. The particular characteristics of the Abs, concerning their shape, function and limited stability, makes their attachment to NPs a non-trivial task where orientation and how they are linked (electrostatic, covalent, indirect, etc.) is capital to keep the Ab activity intact for the desired purpose. Strategies of oriented linkage via covalent binding, the use of other biomolecules directly coupled to the NP surface or adsorption approaches through physical interactions, maintaining the Ab activity, have been summarized. Moreover, the application of the immobilization techniques can be also applied to Abs fragments containing the original Ab specificity such as sdAbs or scFv fractions. The reduced specificity compared to full Abs in some of these cases of Ab fragments can be overcome by their properties as lower molecular mass and higher stability, being useful for applications requiring nanoprobes of controlled size. Acknowledgments This study was supported in part by the European Commission through the FP7 Cooperation Program (grant nos. NMP-20094.0-3-246479 NAMDIATREAM to IN and WJP, and NANOGNOSTICS to WJP), ERC-Starting Grant NANOPUZZLE and ARAID to JMF and by the MEGA-grant of the Ministry of High Education and Science of the Russian Federation (grant no. 11.G34.31.0050 to IN). This article is dedicated to the memory of Professor Dr. Rafael Suau Suarez, Ph.D. director of JMM, an eminent scientist and a good friend, who passed away in November 2010. References [1] L. Holmquist, O. Vesterberg, Direct on air sampling filter quantification of cat allergen, J. Biochem. Biophys. Methods 51 (2002) 17–25. [2] W. Wels, M. Biburger, T. Muller, B. Dalken, U. Giesubel, T. Tonn, C. Uherek, Recombinant immunotoxins and retargeted killer cells: employing engineered antibody fragments for tumor-specific targeting of cytotoxic effectors, Cancer Immunol. Immunother. 53 (2004) 217–226. [3] E. Garber, K. Venkateswaran, T. O'Brien, Simultaneous multiplex detection and confirmation of the proteinaceous toxins abrin, ricin, botulinum toxins, and Staphylococcus enterotoxins A, B, and C in food, J. Agric. Food Chem. 58 (2010) 6600–6607. [4] A. Williams, G. Galfre, C. Milstein, Analysis of cell-surfaces by xenogeneic myeloma-hybrid antibodies — Differentiation antigens of rat lymphocytes, Cell 12 (1977) 663–673. [5] M. DeLisa, Z. Zhang, M. Shiloach, S. Pilevar, C. Davis, J. Sirkis, W. Bentley, Evanescent wave long period fiber Bragg grating as an immobilized antibody biosensor, Anal. Chem. 72 (2000) 2895–2900. [6] V. Popii, G. Baumann, Laboratory measurement of growth hormone, Clin. Chim. Acta 350 (2004) 1–16. [7] W. Yin, J. Liu, T. Zhang, W. Li, W. Liu, M. Meng, F. He, Y. Wan, C. Feng, S. Wang, X. Lu, R. Xi, Preparation of monoclonal antibody for melamine and development of an indirect competitive ELISA for melamine detection in raw milk, milk powder, and animal feeds, J. Agric. Food Chem. 58 (2010) 8152–8157. [8] X. Ai, B. Butts, K. Vora, W. Li, C. Tache-Talmadge, A. Fridman, H. Mehmet, Generation and characterization of antibodies specific for caspase-cleaved neo-epitopes: a novel approach, Cell Death Dis. 2 (2011). [9] A. Singh, D. Senapati, S. Wang, J. Griffin, A. Neely, P. Candice, K. Naylor, B. Varisli, J. Kalluri, P. Ray, Gold nanorod based selective identification of Escherichia coli bacteria using two-photon Rayleigh scattering spectroscopy, ACS Nano 3 (2009) 1906–1912. [10] M. Pavao, A. Traish, Estrogen receptor antibodies: specificity and utility in detection, localization and analyses of estrogen receptor alpha and beta, Steroids 66 (2001) 1–16.

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