- Email: [email protected]
Nanotransporters for drug delivery
Available online at www.sciencedirect.com
ScienceDirect Nanotransporters for drug delivery Tessa Lu¨hmann and Lorenz Meinel Soluble nanotransporters for drugs can be profiled for targeted delivery particularly to maximize the efficacy of highly potent drugs while minimizing off target effects. This article outlines on the use of biological carrier molecules with a focus on albumin, various drug linkers for site specific release of the drug payload from the nanotransporter and strategies to combine these in various ways to meet different drug delivery demands particularly the optimization of the payload per nanotransporter. Address Institute for Pharmacy and Food Chemistry, University of Wu¨rzburg, Am Hubland, DE-97074 Wu¨rzburg, Germany Corresponding author: Meinel, Lorenz ([email protected])
Current Opinion in Biotechnology 2016, 39:35–40 This review comes from a themed issue on Nanobiotechnology Edited by Michael Nash and Oded Shoseyov
polymer), genetic engineering and modern, bioorthogonal chemistries can help to overcome this obstacle thereby reducing the risk of development [3]. Stability challenges of the nanotransporters are also related to the inherently instable drug linker, supposed to readily disintegrate at the site in need with premature cleavage during circulation potentially driving off target effects [4]. Lastly, validated targets for nanotransporter homing are few in number and extrapolated from existing therapeutic antibodies and their life cycle extension through cytotoxic drug decoration. However, the common denominator for valuable targets are a high target expression in the diseased tissue and low expression elsewhere. These building blocks — carrier, drug-linker, antigen selection — are addressed in the following sections (Figure 1). Future perspectives are delineated by outlining recent developments within each block while outlining the potential when combining these driven by the medical need.
Carrier systems http://dx.doi.org/10.1016/j.copbio.2015.12.013 0958-1669/# 2016 Elsevier Ltd. All rights reserved.
Introduction Paul Ehrlich’s vision of selectively delivering highly potent drugs primarily to the seat(s) of a disease has become reality. Particularly indications with limited alternative treatments (e.g. oncology, some rare diseases) benefit from the combination of a targeting moiety to cell surface antigens and concomitant delivery of a covalently connected cytotoxic drug providing that the resulting nanotransporters have adequate pharmacokinetic (PK) properties [2]. These drug nanotransporters expand the armentarium in fighting challenging diseases to very toxic drugs thereby inappropriate for untargeted, systemic use. Some of these nanotransporters integrate responsive linker technologies, such that the targeting moiety is separated from the drug in response to an extra-cellular or intracellular stimulus or in response to applied triggers. Learnings from the development of antibody–drug conjugates (ADC) have been extremely helpful in defining the necessary building blocks for nanotransporters and increased the understanding when tuning their properties. For example, heterogeneity in product quality outcome and clinical performance is a direct result of the availability of various potential conjugation sites on a carrier (i.e. the antibody or another macromolecule or www.sciencedirect.com
From a PK perspective, carriers derived from natural sources can be separated in those with intrinsic binding to the neonatal Fc Receptor (FcRn; such as albumin [5] and antibodies [6]) and those which do not. FcRn binding controls the fate of bound proteins in circulation with diminution of renal clearance [7] and by rescue from intracellular degradation [8]. FcRn binds antibodies and albumin — a 66.5 kDa protein — at acidic pH as found in endosomes, or the extracellular milieus in neoplastic or generally inflamed tissues [8]. Albumin concentrations in human plasma are typically around 0.6 mM and among other functions such as regulating the osmotic pressure, the protein evolved as multifunctional transporter for various drugs, nutrients, waste products, ions, bilirubin, or fatty acids among others. The binding site of albumin for the FcRn is known and serves as an insight to modulate the PK of the carrier as a function of albumin’s decoration site [9]. Interference with the albumin’s FcRn binding site through targeted decoration will likely impact the distribution of the carrier and its catabolism. The precise characterization of albumin detailed ridges and valleys through known binding affinities to various small molecules [10]. These insights might be particularly interesting for site specific decoration with linkers of known propensities to cleavage in plasma when exposed to certain micro-environmental conditions on the carrier surface, as has been reported for ADC [3]. Albumin requires decoration for targeting of epitopes, whereas antibodies possess both properties, targeting and stabilization. An optimization of the number of bound molecules/targeting moieties is essential, such that the physical stability (aggregation) of the conjugates Current Opinion in Biotechnology 2016, 39:35–40
36 Nanobiotechnology
Figure 1
(a)
(b) Drug
Drug
Drug
Drug
[n]
Carrier
Drug Drug
Targeting
Carrier
Targeting
Drug
Drug
Drug
Drug
[n]
= Cleavable Linker Current Opinion in Biotechnology
Composition of the nanotransporter. (a) Individual drug-linker molecules are conjugated to the carrier. (b) The payload comprises series of connected drug linker conjugates.
is controlled [3,11,12]. Alternative options include the use of synthetic polymers which has been elegantly reviewed before [13–15] and intriguing bioinspired polymers, emerging complementation to more controllable platforms [16], promising advanced compatibility with established bioconjugation chemistries.
Cleavable linkers Different types of linkers prone to cleavage by enzymes or responsive to variations in pH, redox potential or
hydrogen peroxide levels are available for conjugation to attain (bio)-responsiveness of the nanotransporter in the target tissue or cellular compartments (Figure 2).
Enzyme triggered cleavage Bio-responsible drug delivery systems devoted to enzymes with activity in intracellular compartments or present in the extracellular space can be modularly assembled dependant on the desired site of action of the target drug. Lysosomal delivery of, for example, ADCs is
Figure 2
Enzyme triggered
Chemically triggered
Protease
pH O
NH2
H N
HO O
N H
O
OH O
SH
Redox system
Intracellular
Extra-cellular
Hydrogen peroxide Current Opinion in Biotechnology
Endogenous stimuli for bioresponsive drug delivery. Current Opinion in Biotechnology 2016, 39:35–40
www.sciencedirect.com
Nanotransporters for drug delivery Lu¨hmann and Meinel 37
by endocytosis of the antibody upon binding to its epitope on the cell and entry into the lysosomal pathway. As a result of the acid milieu in endosomal and lysosomal compartments, the drug is liberated from the ADC due to cleavage of the linker between the drug and the antibody by the catalytic activity of lysosomal enzymes, such as cathepsins, which recognize dipeptides composed of basic amino acids (Lys, Arg, Cit) connected to a hydrophobic amino acid (Phe, Val). Using this approach, cathepsin sensitive linkers have been developed comprising both a p-aminobenzyloxycarbonyl (PABC) spacer and the dipeptide valine-citrulline (Val-Cit) (Figure 3a). After enzymatic cleavage in the lysosome the chemical unmodified parent drug is released within the cell due to spontaneous elimination from the PABC spacer [17–19]. This elegant principle has been intensively used to conjugate potent chemotherapeutic drugs such as auristatins and doxorubicin to albumin or antibody carriers [20,21].
recognized by extracellular matrix metalloproteinases (MMP), which degrade extracellular matrix (ECM) components and promote bioactive processing of growth factors [22,23]. There is more and more evidence that in pathophysiological conditions MMP levels are temporally upregulated [24,25]. It is this MMP dysregulation in local microenvironments, which can be exploited as stimuli for the release of target drugs from the carrier. ECM derived and synthetic peptide sequences have been well characterized as substrates for MMPs, which share a common consensus sequence pattern (Figure 3b) [26]. FRET (fluorescence resonance energy transfer) based peptide sensors detecting tumor associated MMP2/9 activity in vivo have been recently developed and proved successful in tumor tissue detection [27]. Moreover, MMP2-sensitive PEG–drug conjugates were developed for delivery of paclitaxel to tumor cells [28], indicating the high potential of MMP based targeting in cancer therapy. Although MMPs hydrolyze peptide bonds of their substrates with a high level of specificity, targeting of individual MMPs has proven to be difficult
Responsiveness of drug delivery systems can be further achieved by integration of short peptide sequences,
Figure 3
(a)
(b) Val
P4 P3 P2 P1 P1’ P2’ P3’ P4’
PABC
Cit
O O
H N
N H
R1
Peptide sequence
R2
O
O
N H
N H
O
(c) O R1
OH
N H
NH
B O
O
R2
NH2
N H
OH
O
Reactive oxygen species cleavable linker
Cathepsin B cleavable linker
(d) O N
N
R
R
Imine
N H
Hydrazone
O
N R
N H
O
Carboxylic hydrazone
Vinyl ether
O
H N
O
HO
O
O Carboxy dimethylmaleic anhydride
O
Orthoester
O O Ester Current Opinion in Biotechnology
Cleavable linker systems used in bioresponsive drug delivery. Red color highlights the cleavable bond and orange color indicates further cleavage by fragmentation. (a,b) Enzyme triggered cleavage, (c) ROS mediated cleavage and (d) acid cleavable linkers.Source: Modified from [1]. www.sciencedirect.com
Current Opinion in Biotechnology 2016, 39:35–40
38 Nanobiotechnology
due to their high structural homology and enzymatic promiscuity [29]. Besides MMPs, enzymes of the endogenous blood coagulation cascade such as thrombin and tumor associated prostata specific antigen were successfully used as bioresponsive stimuli for local drug delivery [21,30].
Chemical triggered cleavage Exploiting variations in pH in diseased and healthy tissue is another strategy for bioresponsive drug delivery. Acid labile chemical groups are depicted in Figure 3d and can be integrated as building blocks for conjugates targeting either extra-cellular or intracellular environments. Proton sensitive linker systems have been extensively studied in cancer therapy due to the acidic conditions with pH values down to 6.5 within the solid tumor microenvironment or in the acidic extracellular milieu in inflammatory tissue [31,32]. A recent study deployed a boronic acid cleavable linker, reacting with endogenous hydrogen peroxide as sensor of oxidative stress produced at sites of inflammation (Figure 3c) [33]. Similar linker systems, targeting reactive oxygen species (ROS) at inflammatory sites, might be instrumental for bioresponsive drug delivery applications in the future as they enable the release of the parent drug after fragmentation. Bio-reducible linkers, which comprise disulfide bonds, can be reduced to thiols due to different concentrations of glutathione in the extracellular and intracellular space and found in tumor tissues compared to healthy ones [34]. Although numerous studies have used disulfide linked carrier systems, stability problems of the conjugate derive from the fact that disulfide bonds lack stability and are exposed to disulfide exchange in plasma over time. For successful drug delivery, maintenance of the linkage between the attenuate drug and the carrier in circulation is fundamental. Hydrazone and disulfide linked ADCs showed shorter half-lives in vivo than similar ADC linked by VC-PABC [35]. Moreover, the situation becomes complex as the in vivo stability of the conjugate does not only rely on the chemical nature of the linker itself but also on the selected conjugation site on the carrier [3,4]. Similarly, the amount of payload charged on the carrier and resulting hydrophobicity of the formed ADC was recently studied and the impact of loading on PK properties was detailed with hydrophilic linkers promoting in vivo stability [36]. The bottleneck of low drug number per nanotransporter has to be overcome in the future to convey a sufficient amount of the (toxic) drug to the target site. Therefore, the nanotransporter must retain its (PK) stability combined with an increased drug to nanotransporter ratio. Potential strategies might deploy modern biorthogonal conjugation strategies to assemble series of Current Opinion in Biotechnology 2016, 39:35–40
connected cleavable drug linker conjugates to be sitespecifically conjugated to a carrier in the future (Figure 1b).
Conclusion and future perspectives The essential building blocks for nanotransporters for drugs — carrier, drug-linker, antigen selection — can be freely combined. Thereby, tailor made, soluble systems can be provided, allowing the shuttle of quite toxic (but efficacious) drugs due to targeting, a bioresponsive release of the (parent) drug showering the tissue(s) in need, or generally dormant systems when circulating which are activated by the flare of a disease. In spite of having this fascinating vision in reach, deploying these systems has to date been limited to ADCs and at least in part fueled by pharmas’ need for life cycle management of therapeutic antibodies facing or in patent expiry and the arguably lower development risk when existing building blocks and drugs are newly combined in contrast to the development of entirely novel molecules. We would argue that systematically generating similar knowledge levels — as existing for a therapeutically used antibody which is to be profiled as ADC during life cycle management — for each building block is essential to build a short-term or medium-term interest. For example, the dependency of the selected carrier site on the stability of the drug linker (vide supra) is a caveat which can be addressed through site specific decoration chemistries [12]. Similarly, the reported challenge of drug ‘overcharged’ antibodies may be reduced more elaborate selection strategies of the coupling sites on the carrier [37]. Lastly, the desired dynamics of cleavage of the nanotransporters may be species dependent and extrapolation of results from, for example, rodents to humans may be misleading. Understanding the carrier in detail may help the selection of preferential sites for decoration providing particular stability across species [4]. Current nanotransporters — such as the ADCs — are typically charged with small (toxic) molecules or peptides/ peptidomimetics, pharmacologically activated upon release from the system. Extrapolating these approaches to (anabolic) growth factors and immune stimulating cytokines may help to address neoplastic safety concerns associated with some of these. Shielding the receptor binding sites of macromolecular drugs when bound to the nanotransporters and uncovering these through cleavage may positively skew their risk–benefit profile such that therapeutic use may be envisonaged or extended to more benign indications beyond the current. What are alternatives to meet the major drawback of the low molecular weight ratio of payload to the carrier. Obviously, the carrier is essential for targeting and the PK properties but it is for the large carrier that this ratio is negatively impacted. However, in analogy to previous studies [38] one could design the payload charged part of www.sciencedirect.com
Nanotransporters for drug delivery Lu¨hmann and Meinel 39
the nanotransporter with a maleimide functionality. Upon administration and when in contact with the albumin (and the Cys-34 on albumin), the maleimide functionalized element would need to readily bind to endogenous albumin in the patient, deploying the large endogenous albumin pool for conjugation. As attractive as this may be, off site binding to other thiol groups than the free one on albumin and incomplete binding need to be carefully assessed to understand the limit of this approach.
functional consequences. Biochim Biophys Acta 2013, 1830:5405-5417. 9.
Andersen JT, Dalhus B, Cameron J, Daba MB, Plumridge A, Evans L, Brennan SO, Gunnarsen KS, Bjoras M, Sleep D et al.: Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor. Nat Commun 2012, 3:610.
10. Goncharova I, Orlov S, Urbanova M: The location of the highand low-affinity bilirubin-binding sites on serum albumin: ligand-competition analysis investigated by circular dichroism. Biophys Chem 2013, 180–181:55-65. 11. Perez HL, Cardarelli PM, Deshpande S, Gangwar S, Schroeder GM, Vite GD, Borzilleri RM: Antibody–drug conjugates: current status and future directions. Drug Discov Today 2014, 19:869-881.
In summary, the exciting variability leading to tailored nanotransporters through combination of (available or future) building blocks is substantially improving our armamentarium for smart therapeutic systems of the future. Providing the necessary details on appropriate conjugation sites on the carrier, reliable linkers across species, finding discriminating targeting moieties for yet unexplored diseases, and optimizing the payload/nanotransporter ratio are major, exciting research areas which when sensitively combined will lead our way to the vanishing point of revolutionary medicines of the future.
15. Jeong B, Bae YH, Lee DS, Kim SW: Biodegradable block copolymers as injectable drug-delivery systems. Nature 1997, 388:860-862.
Acknowledgments
16. Sun J, Zuckermann RN: Peptoid polymers: a highly designable bioinspired material. Acs Nano 2013, 7:4715-4732.
The financial support by the BMBF (Federal Ministry of Education and Science, 13N13454) and by the FET Open FP7 European project MANAQA (Magnetic Nano Actuators for Quantitative Analysis, 296679) is gratefully acknowledged.
17. Toki BE, Cerveny CG, Wahl AF, Senter PD: Protease-mediated fragmentation of p-amidobenzyl ethers: a new strategy for the activation of anticancer prodrugs. J Org Chem 2002, 67:18661872.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
2.
Luhmann T, Germershaus O, Groll J, Meinel L: Bone targeting for the treatment of osteoporosis. J Control Release 2012, 161:198213. Senter PD, Sievers EL: The discovery and development of brentuximab vedotin for use in relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Nat Biotechnol 2012, 30:631-637.
3.
Shen BQ, Xu K, Liu L, Raab H, Bhakta S, Kenrick M, ParsonsReponte KL, Tien J, Yu SF, Mai E et al.: Conjugation site modulates the in vivo stability and therapeutic activity of antibody–drug conjugates. Nat Biotechnol 2012, 30:184-189. First study reporting the impact of the conjugation site on antibody drug conjugate stability in vivo.
4.
5.
Dorywalska M, Strop P, Melton-Witt JA, Hasa-Moreno A, Farias SE, Galindo Casas M, Delaria K, Lui V, Poulsen K, Loo C et al.: Effect of attachment site on stability of cleavable antibody drug conjugates. Bioconjug Chem 2015, 26:650-659. Chaudhury C, Mehnaz S, Robinson JM, Hayton WL, Pearl DK, Roopenian DC, Anderson CL: The major histocompatibility complex-related Fc receptor for IgG (FcRn) binds albumin and prolongs its lifespan. J Exp Med 2003, 197:315-322.
6.
Brambell FWR, Hemmings WA, Henderson M, Kekwick RA: Electrophoretic studies of serum proteins of foetal rabbits. Proc R Soc Lond B: Biol Sci 1953, 141:300-314.
7.
Montoyo HP, Vaccaro C, Hafner M, Ober RJ, Mueller W, Ward ES: Conditional deletion of the MHC class I-related receptor FcRn reveals the sites of IgG homeostasis in mice. Proc Natl Acad Sci U S A 2009, 106:2788-2793.
8.
Kragh-Hansen U, Minchiotti L, Galliano M, Peters T Jr: Human serum albumin isoforms: genetic and molecular aspects and
www.sciencedirect.com
12. Strop P, Liu SH, Dorywalska M, Delaria K, Dushin RG, Tran TT, Ho WH, Farias S, Casas MG, Abdiche Y et al.: Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates. Chem Biol 2013, 20:161-167. 13. Langer R, Tirrell DA: Designing materials for biology and medicine. Nature 2004, 428:487-492. 14. Duncan R: The dawning era of polymer therapeutics. Nat Rev Drug Discov 2003, 2:347-360.
18. Dubowchik GM, Firestone RA: Cathepsin B-sensitive dipeptide prodrugs. 1. A model study of structural requirements for efficient release of doxorubicin. Bioorg Med Chem Lett 1998, 8:3341-3346. 19. Dubowchik GM, Mosure K, Knipe JO, Firestone RA: Cathepsin Bsensitive dipeptide prodrugs. 2. Models of anticancer drugs paclitaxel (Ttaxol (R)), mitomycin C and doxorubicin. Bioorg Med Chem Lett 1998, 8:3347-3352. 20. Doronina SO, Bovee TD, Meyer DW, Miyamoto JB, Anderson ME, Morris-Tilden CA, Senter PD: Novel peptide linkers for highly potent antibody-auristatin conjugate. Bioconj Chem 2008, 19:1960-1963. 21. Elsadek B, Graeser R, Warnecke A, Unger C, Saleem T, ElMelegy N, Madkor H, Kratz F: Optimization of an albuminbinding prodrug of doxorubicin that is cleaved by prostatespecific antigen. ACS Med Chem Lett 2010, 1:234-238. 22. Uebersax L, Merkle HP, Meinel L: Biopolymer-based growth factor delivery for tissue repair: from natural concepts to engineered systems. Tissue Eng Part B Rev 2009, 15:263-289. 23. Page-McCaw A, Ewald AJ, Werb Z: Matrix metalloproteinases and the regulation of tissue remodelling. Nat Rev Mol Cell Biol 2007, 8:221-233. 24. Malemud CJ: Matrix metalloproteinases (MMPs) in health and disease: an overview. Front Biosci J Virtual Library 2006, 11:1696-1701. 25. Vartak DG, Gemeinhart RA: Matrix metalloproteases: underutilized targets for drug delivery. J Drug Target 2007, 15:1-20. 26. Nagase H, Fields GB: Human matrix metalloproteinase specificity studies using collagen sequence-based synthetic peptides. Biopolymers 1996, 40:399-416. 27. Savariar EN, Felsen CN, Nashi N, Jiang T, Ellies LG, Steinbach P, Tsien RY, Nguyen QT: Real-time in vivo molecular detection of primary tumors and metastases with ratiometric activatable cell-penetrating peptides. Cancer Res 2013, 73:855-864. Seminal work showing the detection of primary tumor tissue by matrix metalloprotease cleavable peptide sequences. Current Opinion in Biotechnology 2016, 39:35–40
40 Nanobiotechnology
28. Zhu L, Wang T, Perche F, Taigind A, Torchilin VP: Enhanced anticancer activity of nanopreparation containing an MMP2sensitive PEG–drug conjugate and cell-penetrating moiety. Proc Natl Acad Sci U S A 2013, 110:17047-17052. 29. Overall CM: Molecular determinants of metalloproteinase substrate specificity — matrix metalloproteinase substrate binding domains, modules, and exosites. Mol Biotechnol 2002, 22:51-86. 30. Tanihara M, Suzuki Y, Nishimura Y, Suzuki K, Kakimaru Y, Fukunishi Y: A novel microbial infection-responsive drug release system. J Pharm Sci 1999, 88:510-514.
34. Brulisauer L, Gauthier MA, Leroux JC: Disulfide-containing parenteral delivery systems and their redox-biological fate. J Control Release 2014, 195:147-154. An excellent review article about current applications and limitations of redox sensitive linker systems in drug delivery. 35. Sanderson RJ, Hering MA, James SF, Sun MM, Doronina SO, Siadak AW, Senter PD, Wahl AF: In vivo drug-linker stability of an anti-CD30 dipeptide-linked auristatin immunoconjugate. Clin Cancer Res 2005, 11:843-852.
31. Ye WL, Zhao YP, Na R, Li F, Mei QB, Zhao MG, Zhou SY: Actively targeted delivery of doxorubicin to bone metastases by a pH-sensitive conjugation. J Pharm Sci 2015, 104:2293-2303.
36. Lyon RP, Bovee TD, Doronina SO, Burke PJ, Hunter JH, Neff LaFord HD, Jonas M, Anderson ME, Setter JR, Senter PD: Reducing hydrophobicity of homogeneous antibody–drug conjugates improves pharmacokinetics and therapeutic index. Nat Biotechnol 2015, 33:733-735. A straightforward study illuminating design rules for antibody drug conjugates.
32. Lv SX, Tang ZH, Zhang DW, Song WT, Li MQ, Lin J, Liu HY, Chen XS: Well-defined polymer-drug conjugate engineered with redox and pH-sensitive release mechanism for efficient delivery of paclitaxel. J Control Release 2014, 194:220-227.
37. Hamblett KJ, Senter PD, Chace DF, Sun MMC, Lenox J, Cerveny CG, Kissler KM, Bernhardt SX, Kopcha AK, Zabinski RF et al.: Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res 2004, 10:7063-7070.
33. Weinstain R, Sayariar EN, Felsen CN, Tsien RY: In vivo targeting of hydrogen peroxide by activatable cell-penetrating peptides. J Am Chem Soc 2014, 136:874-877. This study highlights the application of hydrogen peroxide cleavable linkers as sensor for the detection of inflammation sites in the lung.
38. Fiehn C, Kratz F, Sass G, Mueller-Ladner U, Neumann E: Targeted drug delivery by in vivo coupling to endogenous albumin: an albumin-binding prodrug of methotrexate (MTX) is better than MTX in the treatment of murine collagen-induced arthritis. Annals Rheumatic Dis 2008, 67:1188-1191.
Current Opinion in Biotechnology 2016, 39:35–40
www.sciencedirect.com
ScienceDirect Nanotransporters for drug delivery Tessa Lu¨hmann and Lorenz Meinel Soluble nanotransporters for drugs can be profiled for targeted delivery particularly to maximize the efficacy of highly potent drugs while minimizing off target effects. This article outlines on the use of biological carrier molecules with a focus on albumin, various drug linkers for site specific release of the drug payload from the nanotransporter and strategies to combine these in various ways to meet different drug delivery demands particularly the optimization of the payload per nanotransporter. Address Institute for Pharmacy and Food Chemistry, University of Wu¨rzburg, Am Hubland, DE-97074 Wu¨rzburg, Germany Corresponding author: Meinel, Lorenz ([email protected])
Current Opinion in Biotechnology 2016, 39:35–40 This review comes from a themed issue on Nanobiotechnology Edited by Michael Nash and Oded Shoseyov
polymer), genetic engineering and modern, bioorthogonal chemistries can help to overcome this obstacle thereby reducing the risk of development [3]. Stability challenges of the nanotransporters are also related to the inherently instable drug linker, supposed to readily disintegrate at the site in need with premature cleavage during circulation potentially driving off target effects [4]. Lastly, validated targets for nanotransporter homing are few in number and extrapolated from existing therapeutic antibodies and their life cycle extension through cytotoxic drug decoration. However, the common denominator for valuable targets are a high target expression in the diseased tissue and low expression elsewhere. These building blocks — carrier, drug-linker, antigen selection — are addressed in the following sections (Figure 1). Future perspectives are delineated by outlining recent developments within each block while outlining the potential when combining these driven by the medical need.
Carrier systems http://dx.doi.org/10.1016/j.copbio.2015.12.013 0958-1669/# 2016 Elsevier Ltd. All rights reserved.
Introduction Paul Ehrlich’s vision of selectively delivering highly potent drugs primarily to the seat(s) of a disease has become reality. Particularly indications with limited alternative treatments (e.g. oncology, some rare diseases) benefit from the combination of a targeting moiety to cell surface antigens and concomitant delivery of a covalently connected cytotoxic drug providing that the resulting nanotransporters have adequate pharmacokinetic (PK) properties [2]. These drug nanotransporters expand the armentarium in fighting challenging diseases to very toxic drugs thereby inappropriate for untargeted, systemic use. Some of these nanotransporters integrate responsive linker technologies, such that the targeting moiety is separated from the drug in response to an extra-cellular or intracellular stimulus or in response to applied triggers. Learnings from the development of antibody–drug conjugates (ADC) have been extremely helpful in defining the necessary building blocks for nanotransporters and increased the understanding when tuning their properties. For example, heterogeneity in product quality outcome and clinical performance is a direct result of the availability of various potential conjugation sites on a carrier (i.e. the antibody or another macromolecule or www.sciencedirect.com
From a PK perspective, carriers derived from natural sources can be separated in those with intrinsic binding to the neonatal Fc Receptor (FcRn; such as albumin [5] and antibodies [6]) and those which do not. FcRn binding controls the fate of bound proteins in circulation with diminution of renal clearance [7] and by rescue from intracellular degradation [8]. FcRn binds antibodies and albumin — a 66.5 kDa protein — at acidic pH as found in endosomes, or the extracellular milieus in neoplastic or generally inflamed tissues [8]. Albumin concentrations in human plasma are typically around 0.6 mM and among other functions such as regulating the osmotic pressure, the protein evolved as multifunctional transporter for various drugs, nutrients, waste products, ions, bilirubin, or fatty acids among others. The binding site of albumin for the FcRn is known and serves as an insight to modulate the PK of the carrier as a function of albumin’s decoration site [9]. Interference with the albumin’s FcRn binding site through targeted decoration will likely impact the distribution of the carrier and its catabolism. The precise characterization of albumin detailed ridges and valleys through known binding affinities to various small molecules [10]. These insights might be particularly interesting for site specific decoration with linkers of known propensities to cleavage in plasma when exposed to certain micro-environmental conditions on the carrier surface, as has been reported for ADC [3]. Albumin requires decoration for targeting of epitopes, whereas antibodies possess both properties, targeting and stabilization. An optimization of the number of bound molecules/targeting moieties is essential, such that the physical stability (aggregation) of the conjugates Current Opinion in Biotechnology 2016, 39:35–40
36 Nanobiotechnology
Figure 1
(a)
(b) Drug
Drug
Drug
Drug
[n]
Carrier
Drug Drug
Targeting
Carrier
Targeting
Drug
Drug
Drug
Drug
[n]
= Cleavable Linker Current Opinion in Biotechnology
Composition of the nanotransporter. (a) Individual drug-linker molecules are conjugated to the carrier. (b) The payload comprises series of connected drug linker conjugates.
is controlled [3,11,12]. Alternative options include the use of synthetic polymers which has been elegantly reviewed before [13–15] and intriguing bioinspired polymers, emerging complementation to more controllable platforms [16], promising advanced compatibility with established bioconjugation chemistries.
Cleavable linkers Different types of linkers prone to cleavage by enzymes or responsive to variations in pH, redox potential or
hydrogen peroxide levels are available for conjugation to attain (bio)-responsiveness of the nanotransporter in the target tissue or cellular compartments (Figure 2).
Enzyme triggered cleavage Bio-responsible drug delivery systems devoted to enzymes with activity in intracellular compartments or present in the extracellular space can be modularly assembled dependant on the desired site of action of the target drug. Lysosomal delivery of, for example, ADCs is
Figure 2
Enzyme triggered
Chemically triggered
Protease
pH O
NH2
H N
HO O
N H
O
OH O
SH
Redox system
Intracellular
Extra-cellular
Hydrogen peroxide Current Opinion in Biotechnology
Endogenous stimuli for bioresponsive drug delivery. Current Opinion in Biotechnology 2016, 39:35–40
www.sciencedirect.com
Nanotransporters for drug delivery Lu¨hmann and Meinel 37
by endocytosis of the antibody upon binding to its epitope on the cell and entry into the lysosomal pathway. As a result of the acid milieu in endosomal and lysosomal compartments, the drug is liberated from the ADC due to cleavage of the linker between the drug and the antibody by the catalytic activity of lysosomal enzymes, such as cathepsins, which recognize dipeptides composed of basic amino acids (Lys, Arg, Cit) connected to a hydrophobic amino acid (Phe, Val). Using this approach, cathepsin sensitive linkers have been developed comprising both a p-aminobenzyloxycarbonyl (PABC) spacer and the dipeptide valine-citrulline (Val-Cit) (Figure 3a). After enzymatic cleavage in the lysosome the chemical unmodified parent drug is released within the cell due to spontaneous elimination from the PABC spacer [17–19]. This elegant principle has been intensively used to conjugate potent chemotherapeutic drugs such as auristatins and doxorubicin to albumin or antibody carriers [20,21].
recognized by extracellular matrix metalloproteinases (MMP), which degrade extracellular matrix (ECM) components and promote bioactive processing of growth factors [22,23]. There is more and more evidence that in pathophysiological conditions MMP levels are temporally upregulated [24,25]. It is this MMP dysregulation in local microenvironments, which can be exploited as stimuli for the release of target drugs from the carrier. ECM derived and synthetic peptide sequences have been well characterized as substrates for MMPs, which share a common consensus sequence pattern (Figure 3b) [26]. FRET (fluorescence resonance energy transfer) based peptide sensors detecting tumor associated MMP2/9 activity in vivo have been recently developed and proved successful in tumor tissue detection [27]. Moreover, MMP2-sensitive PEG–drug conjugates were developed for delivery of paclitaxel to tumor cells [28], indicating the high potential of MMP based targeting in cancer therapy. Although MMPs hydrolyze peptide bonds of their substrates with a high level of specificity, targeting of individual MMPs has proven to be difficult
Responsiveness of drug delivery systems can be further achieved by integration of short peptide sequences,
Figure 3
(a)
(b) Val
P4 P3 P2 P1 P1’ P2’ P3’ P4’
PABC
Cit
O O
H N
N H
R1
Peptide sequence
R2
O
O
N H
N H
O
(c) O R1
OH
N H
NH
B O
O
R2
NH2
N H
OH
O
Reactive oxygen species cleavable linker
Cathepsin B cleavable linker
(d) O N
N
R
R
Imine
N H
Hydrazone
O
N R
N H
O
Carboxylic hydrazone
Vinyl ether
O
H N
O
HO
O
O Carboxy dimethylmaleic anhydride
O
Orthoester
O O Ester Current Opinion in Biotechnology
Cleavable linker systems used in bioresponsive drug delivery. Red color highlights the cleavable bond and orange color indicates further cleavage by fragmentation. (a,b) Enzyme triggered cleavage, (c) ROS mediated cleavage and (d) acid cleavable linkers.Source: Modified from [1]. www.sciencedirect.com
Current Opinion in Biotechnology 2016, 39:35–40
38 Nanobiotechnology
due to their high structural homology and enzymatic promiscuity [29]. Besides MMPs, enzymes of the endogenous blood coagulation cascade such as thrombin and tumor associated prostata specific antigen were successfully used as bioresponsive stimuli for local drug delivery [21,30].
Chemical triggered cleavage Exploiting variations in pH in diseased and healthy tissue is another strategy for bioresponsive drug delivery. Acid labile chemical groups are depicted in Figure 3d and can be integrated as building blocks for conjugates targeting either extra-cellular or intracellular environments. Proton sensitive linker systems have been extensively studied in cancer therapy due to the acidic conditions with pH values down to 6.5 within the solid tumor microenvironment or in the acidic extracellular milieu in inflammatory tissue [31,32]. A recent study deployed a boronic acid cleavable linker, reacting with endogenous hydrogen peroxide as sensor of oxidative stress produced at sites of inflammation (Figure 3c) [33]. Similar linker systems, targeting reactive oxygen species (ROS) at inflammatory sites, might be instrumental for bioresponsive drug delivery applications in the future as they enable the release of the parent drug after fragmentation. Bio-reducible linkers, which comprise disulfide bonds, can be reduced to thiols due to different concentrations of glutathione in the extracellular and intracellular space and found in tumor tissues compared to healthy ones [34]. Although numerous studies have used disulfide linked carrier systems, stability problems of the conjugate derive from the fact that disulfide bonds lack stability and are exposed to disulfide exchange in plasma over time. For successful drug delivery, maintenance of the linkage between the attenuate drug and the carrier in circulation is fundamental. Hydrazone and disulfide linked ADCs showed shorter half-lives in vivo than similar ADC linked by VC-PABC [35]. Moreover, the situation becomes complex as the in vivo stability of the conjugate does not only rely on the chemical nature of the linker itself but also on the selected conjugation site on the carrier [3,4]. Similarly, the amount of payload charged on the carrier and resulting hydrophobicity of the formed ADC was recently studied and the impact of loading on PK properties was detailed with hydrophilic linkers promoting in vivo stability [36]. The bottleneck of low drug number per nanotransporter has to be overcome in the future to convey a sufficient amount of the (toxic) drug to the target site. Therefore, the nanotransporter must retain its (PK) stability combined with an increased drug to nanotransporter ratio. Potential strategies might deploy modern biorthogonal conjugation strategies to assemble series of Current Opinion in Biotechnology 2016, 39:35–40
connected cleavable drug linker conjugates to be sitespecifically conjugated to a carrier in the future (Figure 1b).
Conclusion and future perspectives The essential building blocks for nanotransporters for drugs — carrier, drug-linker, antigen selection — can be freely combined. Thereby, tailor made, soluble systems can be provided, allowing the shuttle of quite toxic (but efficacious) drugs due to targeting, a bioresponsive release of the (parent) drug showering the tissue(s) in need, or generally dormant systems when circulating which are activated by the flare of a disease. In spite of having this fascinating vision in reach, deploying these systems has to date been limited to ADCs and at least in part fueled by pharmas’ need for life cycle management of therapeutic antibodies facing or in patent expiry and the arguably lower development risk when existing building blocks and drugs are newly combined in contrast to the development of entirely novel molecules. We would argue that systematically generating similar knowledge levels — as existing for a therapeutically used antibody which is to be profiled as ADC during life cycle management — for each building block is essential to build a short-term or medium-term interest. For example, the dependency of the selected carrier site on the stability of the drug linker (vide supra) is a caveat which can be addressed through site specific decoration chemistries [12]. Similarly, the reported challenge of drug ‘overcharged’ antibodies may be reduced more elaborate selection strategies of the coupling sites on the carrier [37]. Lastly, the desired dynamics of cleavage of the nanotransporters may be species dependent and extrapolation of results from, for example, rodents to humans may be misleading. Understanding the carrier in detail may help the selection of preferential sites for decoration providing particular stability across species [4]. Current nanotransporters — such as the ADCs — are typically charged with small (toxic) molecules or peptides/ peptidomimetics, pharmacologically activated upon release from the system. Extrapolating these approaches to (anabolic) growth factors and immune stimulating cytokines may help to address neoplastic safety concerns associated with some of these. Shielding the receptor binding sites of macromolecular drugs when bound to the nanotransporters and uncovering these through cleavage may positively skew their risk–benefit profile such that therapeutic use may be envisonaged or extended to more benign indications beyond the current. What are alternatives to meet the major drawback of the low molecular weight ratio of payload to the carrier. Obviously, the carrier is essential for targeting and the PK properties but it is for the large carrier that this ratio is negatively impacted. However, in analogy to previous studies [38] one could design the payload charged part of www.sciencedirect.com
Nanotransporters for drug delivery Lu¨hmann and Meinel 39
the nanotransporter with a maleimide functionality. Upon administration and when in contact with the albumin (and the Cys-34 on albumin), the maleimide functionalized element would need to readily bind to endogenous albumin in the patient, deploying the large endogenous albumin pool for conjugation. As attractive as this may be, off site binding to other thiol groups than the free one on albumin and incomplete binding need to be carefully assessed to understand the limit of this approach.
functional consequences. Biochim Biophys Acta 2013, 1830:5405-5417. 9.
Andersen JT, Dalhus B, Cameron J, Daba MB, Plumridge A, Evans L, Brennan SO, Gunnarsen KS, Bjoras M, Sleep D et al.: Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor. Nat Commun 2012, 3:610.
10. Goncharova I, Orlov S, Urbanova M: The location of the highand low-affinity bilirubin-binding sites on serum albumin: ligand-competition analysis investigated by circular dichroism. Biophys Chem 2013, 180–181:55-65. 11. Perez HL, Cardarelli PM, Deshpande S, Gangwar S, Schroeder GM, Vite GD, Borzilleri RM: Antibody–drug conjugates: current status and future directions. Drug Discov Today 2014, 19:869-881.
In summary, the exciting variability leading to tailored nanotransporters through combination of (available or future) building blocks is substantially improving our armamentarium for smart therapeutic systems of the future. Providing the necessary details on appropriate conjugation sites on the carrier, reliable linkers across species, finding discriminating targeting moieties for yet unexplored diseases, and optimizing the payload/nanotransporter ratio are major, exciting research areas which when sensitively combined will lead our way to the vanishing point of revolutionary medicines of the future.
15. Jeong B, Bae YH, Lee DS, Kim SW: Biodegradable block copolymers as injectable drug-delivery systems. Nature 1997, 388:860-862.
Acknowledgments
16. Sun J, Zuckermann RN: Peptoid polymers: a highly designable bioinspired material. Acs Nano 2013, 7:4715-4732.
The financial support by the BMBF (Federal Ministry of Education and Science, 13N13454) and by the FET Open FP7 European project MANAQA (Magnetic Nano Actuators for Quantitative Analysis, 296679) is gratefully acknowledged.
17. Toki BE, Cerveny CG, Wahl AF, Senter PD: Protease-mediated fragmentation of p-amidobenzyl ethers: a new strategy for the activation of anticancer prodrugs. J Org Chem 2002, 67:18661872.
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Current Opinion in Biotechnology 2016, 39:35–40
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