Polypeptides and Engineered Proteins - American Chemical Society

Chapter 6

Polypeptides and Engineered Proteins

Downloaded by UNIV OF FLORIDA on October 26, 2017 | http://pubs.acs.org Publication Date (Web): October 25, 2017 | doi: 10.1021/bk-2017-1252.ch006

Xinyu Liu, Jin Hu, Zhuoran Wang, Zhikun Xu, and Weiping Gao* Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084, China *E-mail: [email protected]

Polypeptides and engineered proteins are a class of bioinspired and biomedical materials. Polypeptides like elastin-like polypeptides, collagen polypeptides and silk polypeptides are a kind of biopolymers that are biocompatible and biodegradable, which are widely used in biomedicine ranging from drug delivery to tissue engineering. These polypeptides together with chemically synthesized polypeptides are introduced in this chapter. Engineered proteins like genetically engineered antibodies, antibody conjugates, protein-polymer conjugates and protein nanoparticles are a class of biologically active biomaterials that are of great use for molecular diagnosis and imaging, and therapy of diseases. The advances in the development of engineered proteins are described in this chapter. The prospects of polypeptides and engineered proteins are discussed at the end of this chapter.

Introduction Polypeptides and engineered proteins are a class of bioinspired and biomedical materials that have been extensively studied for biomedical applications ranging from drug delivery to tissue engineering. Both proteins and polypeptides are biopolymers composed of amino acids, which can be biologically degraded into amino acids as essential nutrition for the body. Proteins are a class of polypeptides in three-dimensional folded structures and have been identified as the major structural and functional components of tissues and organs. Native proteins are usually modified to form engineered proteins for specific biomedical applications. Due to the biological nature, polypeptides and engineered proteins have been recognized as preferred biomaterials for drug delivery, molecular diagnosis and © 2017 American Chemical Society Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

imaging, and tissue engineering. The purpose of this chapter is to highlight recent advances in preparation and biomedical applications of polypeptides and engineered proteins. In this chapter, biologically synthesized elastin-like polypeptides, collagen polypeptides and silk polypeptides, and chemically synthesized polypeptides are emphasized in the polypeptide part. Engineered antibodies, protein-polymer conjugates and protein nanoparticles are the focus of the engineered protein part. Future perspectives for polypeptides and engineered proteins are discussed at the end of this chapter.


Polypeptides Polypeptides have attracted much attention and been widely used in drug delivery (1), tissue engineering (2), bioadhesion (3) and other biomedical fields (4), due to their great biocompatibility and biodegradability. In this section, polypeptides are classified into two categories of biologically synthesized polypeptides and chemically synthesized polypeptides in light of their sources.

Biologically Synthesized Polypeptides Elastin-Like Polypeptides (ELPs) Elastin is a protein rich in proline, glycine, alanine, and valine, which is found in most of animals and has a critical function in numerous organs (5). As the major component of over 90% elastic fibers, elastin possesses important biological functions, including providing integrity of macroscale structure, interacting with the receptor to control cell functions, and playing a role in regulating cell proliferation, differentiation, chemotaxis and migration (6). There have been many elastin-based copolymers that combine elastin with other molecules together. For example, elastin-collagen scaffolds can support vascular cells to grow, which is in particular relevance to engineering of vascular tissue (7). However, as a biomaterial, elastin has shortcomings, such as insolubility, troublesome purification, and ease to be calcified in implantation (8). In order to overcome these challenges, recombinant techniques have been utilized to construct elastin-like polypeptides (ELPs) that can retain the main properties of elastin (9). ELPs are synthetic polypeptides that retain physical properties of elastin. They have several unique benefits compared to other engineered biopolymers. First, ELPs are derived from human tropoelastin and composed of Val-Pro-Gly-Xaa-Gly repeat, in which the Xaa could be any amino acid except proline. Second, the molecular weight and gene composition of ELPs can be precisely encoded, which makes ELPs uniform in structure. Third, they are biodegradable (10), non-immunogenic and non-toxic (11), and have good pharmacokinetic properties (12). Fourth, ELPs can be expressed in E. Coli at high yield, and they have a reversible characteristic phase transition temperature(Tt), so they can be rapidly purified by inverse transition cycling (ITC) (Figure 1) (13). Due to these benefits, ELPs have extensively been used for drug delivery in different ways such as drug nanocarriers, thermally coacervated depots, and 94 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


cross-linked or self-assembled nanoparticles (14). Moreover, ELPs have also been used in the form of fibres or gels as injectable scaffolds for soft tissue replacement and cartilage tissue repair (15).

Figure 1. Schematic illustration of ELP purification by ITC. An ELP conjugated with hydrophobic drug doxorubicin (DOX) could selfassemble into nanoparticles due to the amphiphilicity of the conjugate (16). By attaching to the Cys residues, DOX was sequestered to form the core of the micelle and ELP formed the micelle corona (17). The drug could be targeted to tumor by the enhanced permeation and retention (EPR) effect (Figure 2), which limited the toxicity to healthy tissues. After 24 h, the nanoparticles concentration showed a 3.5-fold increase over free drug in tumor (18). Based on these nanoparticles, a recombinant ELP was further fused with a targeting peptide to improve drug delivery into tumor (19).

Figure 2. ELP–drug micelles accumulate in the tumor by EPR effect. 95 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


In addition, ELPs have also been utilized to improve the circulating time of therapeutic proteins. For example, a C-terminal interferon alpha-ELP (IFNELP) fusion protein was genetically constructed by Gao et al. for the first time (Figure 3), it was long acting and accumulated dozens of times more in tumor than IFN, thus highly potent for cancer therapy. These results provide a simple yet efficient approach to precisely design protein-polymer conjugates for the treatment of cancer and viral diseases (20).

Figure 3. The synthesis and model of IFN-ELP (generated from PDB code 1lTF) (20). Reproduced with permission from reference (20). Copyright 2015 John Wiley & Sons.

Thermally responsive ELPs are another promising kind of recombinant biopolymers and have been used for drug delivery into solid tumors (Figure 4). In a recent study, a 125I-labeled ELP was used for local treatment, in which the ELP depots prolonged the residence time of radionuclide in mouse tumors (21). In another application, an ELP unimer with a Tt of 40 °C could aggregate upon local hyperthermia and thus preferentially accumulated in tumors. Tumors treated with hyperthermia showed 2-fold higher temperature-sensitive ELP accumulation than tumors without hyperthermia treatment (22).

Figure 4. Thermal targeting of ELP–drug conjugates to a heated tumor by the phase transition behaviour. 96 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Collagen Polypeptides Collagens make up nearly 30% of total proteins in the body. Collagens are characteristic of protein polymers mainly composed of glycine-proline-oxyproline repeating unit that can fold into a triple-helical structure. The family of collagens includes 28 members in which vertebrates contain several triple-helical domain ones numbered from I to XXVIII. Collagen molecules have different rod length ranging from about 75 nm to 425 nm. By supramolecular assemblies, collagens can be divided into several subfamilies, including fibrils, networks, beaded filaments and anchoring fibrils (Figure 5). The main function of collagens is to maintain the integrity of the extracellular matrix structure in the body (23). Besides, collagens also directly regulate cell growth, differentiation and adhesion, participate in tissue repair, platelet aggregation and wound healing (24), and thus interacts with the extracellular environment.

Figure 5. Supramolecular families of collagens (25). Because of the enzymatic degradability, biocompatibility, and cross-linking properties (26), collagen is widely used in medical applications. For example, collagen-based explants can be used as biomaterials such as porcine heart valves, sutures, and blood vessels. In clinical application, there are many FDA-approved collagens such as Cosmoderm and Zyplast used in cosmetic procedures as filler materials. Besides, collagen scaffolds have been successfully applied for burn wounds and dermal burn dressings, such as Apligraf and Integra because they can improve cellular proliferation and adhesion. In addition, collagen-based materials have been widely used in tissue engineering, such as bone graft substitute and collagen scaffolds to support the vascular structures (27). Collagens also have potential in gene therapy. In many tissues, it seems to be essential to remain the structural integrity by correct expression of collagen genes. Probably more than 30 collagen gene mutations can lead to produce different disease phenotypes. 97 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Therefore, in order to test the potential of gene therapy, experiments have been conducted to rescue the mutate genes produced phenotypes. However, relative research is still at early stage (28).


Silk Polypeptides Silk is a kind of protein polymer whose structure, property, and composition are dependent highly on their source. For natural silk, the most common silk source is Bombyx silkworm, widely used for biomedical applications (29). Meanwhile, some other animals and insects such as honeybees, ants, wasps, spiders and mussels can produce silk besides silkworm. Silk is degradable in vivo over several months, which is primarily regulated by foreign body response. Silk proteins are also able to be degraded by enzymes including actinase, carboxylase, and chymotrypsin (30). But many other factors, such as the mechanical properties of the implantation site, type of the physical properties of the silk scaffold, and the processing conditions of the silk at that site, can influence the exact time for degradation. Silk has an ultimate tensile strength ranging from about 500 to 900 MPa, which tends to form crystals or β-sheets structures and is highly dependent on the structure of hydrophobic regions (31). Silk polypeptides are considered as attractive biomaterials for tissue engineering. This is because of their excellent permeability for water and oxygen, lower immunogenicity, higher tensile strength, slow degradation, and good ability to sustain cell growth and adhesion. In order to improve the bioactivity of the materials, the silk surfaces can be modified with grow factors or some peptides such as RGD. As an example, silk coated with RGD improved the mineralized matrix level produced by osteoblasts (32). For another example, silk proteins can form fibrous silk matrices by several approaches (29). Fibrous silk matrices can not only promote mesenchymal stem cells to differentiate into ligament-specific cells by the enhancement of mechanical bioreactor (31), but also have potential in ligament tissue engineering. Moreover, since spider silk and silkworm silk have a vital role in supporting the growth of chondrocyte, recombinant spider silk and silkworm silk were successfully transformed into hydrogels, which showed potential in tissue engineering of cartilage. Besides, in rabbit models, silkworm silk hydrogels showed the ability to repair the defection of cancellous bone (33).

Chemically Synthesized Polypeptides In previous several sections, we introduced some biologically inspired polypeptides. Typically, these polypeptides are biologically synthesized. Alternatively, chemistry is another powerful tool for the synthesis of polypeptides. Generally speaking, the chemical methods for the synthesis of polypeptides can be divided into two kinds, solid-phase polypeptide synthesis and liquid-phase polypeptide synthesis. Solid-phase polypeptide synthesis was first invented by Merrifield (34) in 1963 and was widely applied in the synthesis of small polypeptides with less than 50 residues. However, because of the tedious operation, high expense, and most importantly the limitation of polypeptide 98 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


length, the solid-phase polypeptide synthesis method is not suitable for the preparation of large polypeptides with more than 100 residues. In contrast, the liquid-phase polypeptide synthesis method is more efficient, in which the most economical and simple process is the ring-opening polymerization (ROP) of α-amino acid-N-carboxyanhydrides (NCAs). NCAs were first synthesized by Leuchs in 1906 (35), and its ROP can be initiated by different kinds of nucleophiles and bases, and among them, the most usual are primary amines and alkoxide anions. The efficiency of initiation is affected by several factors, including the ratio of monomer to initiator, the existence of impurities like water and the degree of polymerization (36). The initiation efficiency of primary amines is good due to their nucleophilicity, and the polymerization process of fast initiation and slow propagation make the degree of polymerization less than 200. Compared with primary amines, the tertiary amines, alkoxides, and other initiators can be used to prepare high molecular weight polypeptides, because their basicity is stronger than nucleophilicity, which leads to the slow initiation and fast propagation (37). The mechanisms of ring-opening polymerization of NCAs are thought to be the combination of “Normal Amine Mechanism” (NAM) and “Activated Monomer Mechanism” (AMM) (38). The former one is that the amines attack electrophilic carbonyl of NCA monomers so that the nucleophiles induce the ring opening and polymerization. The latter one is that the amines firstly deprotonate the NCA monomers, which makes NCA nucleophilic to initiate the ROP (Figure 6). These two mechanisms are side reactions for each other. Because of the existence of side reactions, chain length cannot be predicted and chain-end functionality cannot be controlled, so that the polymerization process cannot be considered as a living polymerization process. In 1997, Deming reported the first living ROP of NCAs (40). The strategy they employed is to utilize transition metal complexes as the initiator of the ring opening reaction and meanwhile as the living group in the propagating chain end to control the next addition of NCA monomer. In order to reduce the possibility of chain transfer reactions, they tried different transition metal complexes with alkyl, alcohol, amine and carbamate, and they found that the metal alkoxides (i.e., CpTiCl2OR and (CuOtBu)4) showed inert compared with alkoxide anions. The early transition metal complexes (i.e., CpTiCl2NR2) could not act as feasible initiators, while the late transition metal complexes (i.e., (bpy)NiMe2 and (bpy)Ni(Me)N(R)Ar, where bpy = 2, 20-bipyridine) could. Anyway, the living polymerization needs to be controlled by introducing a special group, such as the transition metal complexes, to the propagating chain end. Given that the oxidative addition reaction can happen between metals and cyclic anhydrides, Deming (41) chose zerovalent cobalt and nickel complexes (i.e., (PMe3)4Co, and bpyNi(COD), where COD = 1,5-cyclooctadiene) as initiators, and after the oxidative addition with anhydrides to obtain the intermediate, a second NCA monomer will react with intermediate to get six-membered amido-alkyl metallacycles, which can further react with other NCA monomer to get five-member amido-amidate metallacyles. Next, the migration of amide proton frees the living chain-end from the metal (42), and getting the amido-amidate complexes, which would repeatedly initiate the ROP of other 99 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


NCAs and meanwhile prevent the side reactions (Figure 7). The dispersity of the polypeptides gotten via this method are less than 1.20 and the molecular weight can range from 500 to 5 × 105. In 2007, Cheng et al. developed a new reagent, hexamethyldisilazane (HMDS) (43), as an initiator to realize the living ROP of NCAs, and the polypeptides obtained showed less than 1.3 in dispersity, well-controlled molecular weight and high yield. The mechanism they proposed exhibited that HMDS would firstly deprotonate the NCA monomer like AMM mechanism to form TMS amine, leaving the other TMS group linked with NCA monomer, and then the TMS amine induced the ring-opening of NCA monomer to get the propagating species, which would repeatedly initiate the ROP of other NCAs (Figure 8). And in 2008, they further reported that N-TMS amines could be directly served as initiators (44), which made it easier to construct a large number of C-terminally functionalized polypeptides because of the diversity of N-TMS amines. However, this method also has some drawbacks, for example, it could not be used to polymerize N-substituted NCAs and the reaction rate of polymerization is slower than transition metal initiators.

Figure 6. Two mechanisms of ROP of NCAs, (a) NAM; (b) AMM (39). Reproduced with permission from reference (39). Copyright 2011 Springer.

100 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 7. Mechanism of ROP of NCAs initiated by transition metal complexes (39). Reproduced with permission from reference (39). Copyright 2011 Springer.

Figure 8. Mechanism of polypeptides synthesis initiated by HDMS (39). Reproduced with permission from reference (39). Copyright 2011 Springer.



In addition to transition metal complexes and HDMS system, there are many other strategies developed to control NCAs polymerization. For instance, Schlaad et al. employed primary amine hydrochlorides salts to initiate the ROP (45), which could efficiently prevent the deprotonation of NCA monomers; Hadjichristidis et al. reported that high vacuum techniques could be used to control the ROP of NCAs well (46); and Vayaboury et al. found that low temperature could also be useful to eliminate the side reaction during the ROP and get a well-controlled polymerization process (47). With the development of living ROP of NCAs, many kinds of polypeptides and polypeptide-based copolymers can be synthesized as what people designed, and they show a variety of potential applications. For example, Deming et al. reported that the polymeric vesicles assembled by polyarginine and polyleucine could be used to deliver water-soluble species into cells and showed potential application in drug delivery (Figure 9) (48); Hammond et al. developed a series of efficient antimicrobial polypeptides by the ROP of γ-propargyl-L-glutamate NCAs, which exhibited broad-spectrum antimicrobial activity yet very low hemolytic activity (Figure 10) (49).

Figure 9. Schematic diagram of proposed self-assembly of amphiphilic polypeptide vesicles, which can be used in intracellular delivery (48). Reproduced with permission from reference (48). Copyright 2007 Nature Publishing Group. 102 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 10. The structures and activities of antimicrobial polypeptides (49). Reproduced with permission from reference (49). Copyright 2011 American Chemical Society.

Engineered Proteins In previous section, we introduced several kinds of polypeptides synthesized by both biological and chemical methods, and their applications in biomedical fields. Generally speaking, the polypeptide-based materials show very limited biological functions but more than the advantages of their biocompatibility and biodegradability as functional materials. Unlike these polypeptides, proteins are a special class of polypeptides, which have three-dimensional folded structures and specific biological functions. Thus, to develop the protein-based biomaterials is of great importance and has attracted much interest in recent year. With the development of protein engineering, native proteins are able to be engineered into the specific forms for the specific applications. In this section, we will highlight the main advances in three emerging fields of engineered proteins, which are engineered antibodies, protein-polymer conjugates and protein nanoparticles.

Engineered Antibodies Antibody (Ab), which is also called immunoglobulin (Ig), is a kind of large, Y-shaped protein (just for monomer, such as IgD, IgG, IgE). It is excreted mainly by B cells and shows high specificity, which can be used to identify the exogenous pathogens and then neutralize them. Since the hybridoma technology was developed by Kohler and Milstein in 1975 (50), people have been able to obtain various monoclonal antibodies (mAbs) targeting to the antigens they want, and from then on, the monoclonal antibodies have showed great potential in in vivo therapeutics, in vitro diagnostics and basic biological research fields. However, there are still some shortcomings, for example, the ineluctable immunogenicity will appear when using murine mAbs produced by hybridoma technology in human directly, which may cause fatal allergy (51). With the development of recombinant DNA technology, more and more engineered recombinant proteins have been made, including the chimeric, humanized Abs (53, 54). As shown in Figure 11, the chimeric mAbs are constructed by combining human constant regions (about 75%) and murine variable regions, and because of the replacement of constant regions which take 103 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


the main responsibility for the immunogenicity (55), the chimeric mAbs are much less immunogenic than murine mAbs. To further reduce the influence of murine regions, the humanized mAbs have been developed. They contain about 85~90% human part and remain only the murine complementarity determining regions (CDRs). Many humanized mAbs have been approved by FDA; however, their binding affinity is usually lower than murine one, which can be partially solved by mutations in CDRs (56).

Figure 11. Cartoon schematics of mouse, chimeric, humanized and fully human antibodies (52). Reproduced with permission from reference (52). Copyright 2014 John Wiley and Sons. Even so, there are still murine components in chimeric and humanized mAbs. To develop fully human mAbs and abolish the immunogenicity completely, the transgenic mice technology (57) and phase display technology (58) have been built. The transgenic mice are such strains whose Ig gene segments have been replaced by human’s, which leads to the production of fully human mAbs when they are injected with antigens. It is worth noting that the glycosylation patterns of the mAbs produced by transgenic mice are still murine patterns (59). Ipilimumab, an anti-CTLA-4 antibody, one of approved antibodies of the emerging cancer immunotherapy, is the fully human mAb produced by transgenic mice (60). The phage display technology can fuse the human antibody’s gene with the phage gene that encodes its coat protein, so the expressed fusion protein of antibody-coat protein will be displayed on the surface of phase. This technology can be used to build an antibody library for the screening of more antigens. Fully human mAbs have solved the immunogenicity of murine mAbs, while there are other problems existing, such as poor tissue penetration, relatively 104 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


low binding affinity, high expense of production (62). Recently, many novel formats of mAbs have been created to solve these problems. Antibody fragments (Figure 12), such as Fab, single-chain fragment variable (scFv), single-domain antibodies (nanobodies), diabodies, and minibodies are all smaller than the intact antibodies, which means improved tumor penetration rate. What’s more, these antibody fragments like scFv can be produced by cheap and efficient prokaryotic expression system rather than eukaryotic expression system. However, their smaller sizes also make it quicker to be cleared from blood and thus cause poor pharmacokinetics (63). This problem can be solved by further biological or chemical modifications to get scFv-Fc (64) or PEG-scFv (65).

Figure 12. Cartoon schematics of different antibody formats and their molecular weights (61). Reproduced with permission from reference (61). Copyright 2005 Nature Publishing Group.

Another innovative strategy to engineer antibodies aims to turn the normal monovalency into multivalency. By decreasing the off-rates and increasing the retention time on the target, the multivalency antibody formats will exhibit better affinity than monovalency. Both chemical and biological approaches can be applied to link the monovalent scFv into diabodies, triabodies and tetrabodies. Furthermore, the cross-linking will improve the serum half-life by enlarging the size of antibodies (66). The other example of multivalency is bispecific antibodies (BsAbs). BsAbs are designed to be able to target two distinct antigens at the same time for they are composed of two different variable regions (67). Blinatumomab developed by Amgen to treat Philadelphia chromosome-negative relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL) (68), one of the most expensive anti-tumor drugs on market, is the first BsAbs approved by FDA. It targets CD3 of T cells and CD19 of tumor cells simultaneously and thus engages cytotoxic T cells to attack tumor cells. Recently, with the development of bioconjugate chemistry, more and more antibody modification methods have been developed, which leads to the successful construction of antibody conjugates. So far, many kinds of antibody conjugates have been developed by linking different payloads, such as drugs (69, 70), radionuclides (71), proteins (72), RNAs (73), antibiotics (74), fluorophores (75), to the antibodies that serve as the targeting groups. Among these antibody conjugates, antibody-drug conjugates (ADCs) is a very promising 105 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


topic in antitumor therapeutics (76). The concept of ADCs can go back to the “Magic Bullets” invented by Paul Ehrlich (77). ADCs consist of three parts: antibody, drug and linker, and they can deliver the highly potent drugs to the target tumor cells. ADCs combine the superiorities of both antibodies (high specificity) and drugs (high potency), meanwhile eliminate the shortcomings of both antibodies (low efficacy) and drugs (severe side effects). There are two ADCs products on market, brentuximab vedotin for Hodgkin’s lymphoma (HL) (78) and Ado-trastuzumab emtansine for HER2-positive breast cancer (79). Many advanced technologies are being developed to improve current antibody conjugates. For example, Sutro Biopharma inserted the non-natural amino acids into the antibody sequence to precisely control the linking site and number of payloads (81); Gao et al. developed fluorescent polymer-antibody conjugates by site-selective in situ growth method, which could be used to amplify the signals detected from antigens (Figure 13) (80). The method developed can be further applied to create a variety of antibody-polymer-dye/drug conjugates for in vitro diagnosis and in vivo imaging and therapy.

Figure 13. Site-selective in situ growth of antibody-polymer-dye conjugates and their applications in signal amplification (80). Reproduced with permission from reference (80). Copyright 2015 Elsevier.

Protein-Polymer Conjugates There has been a dramatic increase in the number of proteins that are used as therapeutic agents due to their high biological activity and specificity. Nevertheless, the delivery of therapeutic proteins in their native forms has several drawbacks that include short circulating half-life, high immunogenicity, low solubility and poor stability. One effective approach to solve the problems is to 106 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


attach a synthetic polymer to the proteins to yield protein-polymer conjugates with more favorable properties. The synthesis of protein-polymer conjugates can be simply divided into two approaches- grafting to and grafting from (Figure 14). The most successful and widely used strategy of grafting to approach is PEGylation, the typical representative of post-polymerization. This strategy is to covalently attach non-ionic, hydrophilic poly(ethylene glycol) (PEG) to a protein. Grafting from is to directly grow polymers from proteins attached with initiators via controlled/living polymerization. This section focuses on PEGylation as well as the newly developed methods to prepare protein-polymer conjugates.

Figure 14. General strategies for the synthesis of protein-polymer conjugates.

PEGylation The preparation of PEGylated proteins is to attach PEG with reactive targets on the proteins via a variety of coupling reactions. The targets may include side-chains of reactive amino acid residues such as lysine and cysteine (82, 83). The first PEGylated proteins were reported in 1977, where monomethoxy-PEG (mPEG) was conjugated to albumin and catalase at the sites of lysine side-chains, which brought substantial changes in properties and particularly increased in vivo circulation half-life relative to native proteins (84, 85). Since then, the technique of PEGylation developed rapidly and various PEGylated proteins were used in clinic as therapeutics. One route of PEGylation is to conjugate mPEG to the lysine residues of proteins. The hydroxyl group of mPEG is activated into functional groups in advance, followed by the coupling reaction with lysine residues on the proteins (Figure 15). The activated mPEG derivatives include PEG dichlorotriazine(PEG-DCT) (86), tresylate(PEG-TS) (87), succinimidyl carbonate (PEG-SC) (88, 89), benzotriazole carbonate (PEG-BTC) (90), p-nitrophenyl carbonate (PEG-pNPC) (91), trichlorophenyl carbonate (PEG-TCP) (91) and succinimidyl succinate (PEG-SS) (92). These PEGylation methods can be regarded as the first-generation PEGylation. However, these coupling methods have many shortcomings, such as weak linkage between protein and PEG, difunctional PEG contamination formed from diol PEG, restriction to mPEG of small size, complexity of synthetic steps, inefficiency and non-specificity of conjugation (93–95). 107 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 15. Reactive mPEG derivatives.

Figure 16. PEGylation using oxime “click” chemistry (99, 100). Reproduced with permission from reference (99). Copyright 1996 American Chemical Society. Another route of PEGylation was to attach mPEG to proteins via imine bonds or amide bonds (Figure 15). There are two strategies: 1a) mPEG is transformed to mPEG-aldehyde (96) or mPEG-acetal derivatives (97), and the latter can be in situ transformed to the former, followed by reacting with the amino groups, in most cases, the N-terminal amino groups of the proteins to form Schiff bases that can be further reduced into stable amine bonds; 1b) mPEG is transformed to activated mPEG-carboxylic derivatives, particularly active esters (98), followed by reacting with the amino groups of the proteins to form amide bonds; 2) the N-terminal amine of proteins is oxidated into carbonyl groups, followed by reacting with the aminooxy activated mPEG (mPEG-O-NH2) to form oximes (Figure 16) (99, 100). These strategies can be regarded as the second-generation PEGylation as they can 108 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


avoid the effect of diol PEG, and the conjugates can well retain the activity of the proteins. But they still have some shortcomings such as low selectivity of the N-terminal amine oxidation, heterogeneous products, and multi-steps reactions. Along with the above two routes, there is another kind of PEGylation targeting the free surface thiols of cysteine residues on the proteins (Figure 15). In order to react with the cysteine residues, mPEG is transformed into activated derivatives such as PEG maleimide (PEG-MI) (101, 102), vinylsulfone (PEG-VS) (103), iodoacetamide (PEG-IA) (102), or disulfide (PEG-DS) (104, 105). After reaction, the PEG is conjugated to the proteins via alkyl sulfide or disulfide bond. These methods can also be classified as the second-generation PEGylation. PEGylation targeting the cysteine residues is usually regarded as a site-specific modification way because the free surface thiols of cysteines are usually rare. In the cases of proteins without cysteine residues, a free cysteine could be added by genetic engineering to create a reaction site. The conjugates via site-specific PEGylation usually have retained bioactivity and decreased immunogenicity. The shortcoming of this PEGylation strategy is that the disulfide bond is not stable in reducing environments, and the PEG-IA can react with amino groups, which results in heterogeneity in products. PEGylation has been developed for nearly 40 years and is regarded as safe, biocompatible and “stealthy” from the clearance, which can significantly improve the pharmacological properties of proteins. Several PEGylated proteins have been approved by FDA and used as therapeutic drugs (Table 1) (106, 107), meanwhile an increasing number of PEG conjugates such as PEG-interleukin-2, PEG-hirudin, PEG-bovine hemoglobin etc., are being studied in clinical trials. Grafting From Growing polymers directly from proteins attached with initiators provides the opportunity to avoid all postpolymerization modification and multiple protein-polymer coupling reactions. This grafting from method can not only simplify purification process and enhance conjugation yield but also prepare well-defined protein-polymer conjugates. Particularly, controlled/living radical polymerization (CRP), including atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain transfer polymerization (RAFT)), has been used to grow polymers directly from proteins at defined initiation sites (82, 83, 93), which is a straightforward route to prepare protein-polymer conjugates. ATRP and RAFT are ideal for the grafting from method, in which ATRP initiators or chain transfer agents (CTAs) are attached to proteins to form protein-based macro-ATRP initiators or macro-CTAs for in situ ATRP or RAFT (Figure 17). The main process of ATRP is a transition metal catalyzed redox equilibrium between the active radical intermediate species and the halogen atom capped dormant species. The main process of RAFT includes two initiation procedures called initiation and re-initiation, respectively, each initiation procedure consists of a chain-transfer and chain-propagation equilibrium. ATRP and RAFT-based grafting from strategies can minimize steric hindrance and perform in various reaction conditions with a wide range of monomers. Additionally, the purification steps can be greatly simplified since only unreacted 109 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

monomers, without free polymer chains, are remained in the products, and the molar-mass dispersity (ĐM) of the conjugate is relatively narrow.


Table 1. The FDA-approved protein–polymer conjugates (106, 107)a PEG conjugate

Size and type of PEG

Sites of modification

Indications

Adagen (PEGadenosine deaminase)

5 kDa (11-17 chains); linear

Random; Lys; Ser; Tyr; His

Severe combined immunodeficiency

Oncospar (PEG-Lasparaginase)

5 kDa; linear

Random; Lys; Ser; Tyr; His

Acute lymphoblastic leukemia

PEG-Intron (PEG-interferon α-2b)

12 kDa; linear

Random; His 34 (major); Ser; His; Lys

Chronic hepatitis; cancer; multiple sclerosis; HIV/AIDS

PEGASYS (PEG-interferon α-2a)

40 kDa; branched

Random; Lys 31, 121, 131, or 134

Chronic hepatitis

Neulasta (PEG-GCSF)

20 kDa; linear

N-Terminal methionine

Febrile neutropenia

Somavert (PEGGH receptor antagonist)

5 kDa (4-6 chains); linear

Random; Lys 38, 41, 70, 115, 120, 140, 145, 158, and phenylalanine 1

Acromegaly

Mircera (PEGCREA)

30 kDa; linear

Lysine 52 or 46

Chronic kidney disease

Cimzia (Anti-antiTNFα)

20 kDa (2 chains); linear

C-terminal Cys

Crohns disease; rheumatoid arthritis

Krystexxa (PEG-uricase)

10 kDa; linear

Random; lysines

Gout

Omontys (PEGinesatide)

20 kDa (2 chains); linear

The single lysine

Anemia in chronic kidney disease

Plegridy (PEGinterferon β-1a)

20 kDa; linear

The α-amino group of N-terminal

Relapsing multiple sclerosis

Adynovate (PEG-antihemophilic factor VIII)

20 kDa (2 chains); branched

Mainly ε-amino groups of lysines

Hemophilia A

a

G-CSF, granulocyte colony-stimulating factor; GH: growth hormone; CREA: continuous erythropoietin receptor activator; TNFα: tumor necrosis factor alpha.



Figure 17. Structures of ATRP initiator, RAFT CTA, ATRP polymer and RAFT polymer. The grafting from strategy contains two parts: (i) attachment of functionalized ATRP initiators to the desired sites on a protein, (ii) followed by CRP to form protein-polymer conjugates. The chemistry of initiator attachment is similar to that for PEGylation. The amine side-chains of lysine have been targeted to synthesize protein-polymer conjugates via the grafting from method. Russell et al. modified chymotrypsin at lysine residues with 2-bromoisobutyramide groups and then in situ polymerized mPEG-methacrylate from the protein initiators (108). Wang et al. and Haddleton et al. also used the same modification method to in situ grow polymers from horse spleen apoferritin (109), lysozyme (LYS) and bovine serum albumin (BSA) macroinitiator (83), respectively. Unfortunately, in most cases, proteins have multiple lysines on their surfaces. This modification occurs at random and multiple sites on the protein, resulting in ill-defined bioconjugates with reduced bioactivity. Random modification results in heterogeneous products with greatly reduced biological activity. Therefore, the development of site-specific grafting from (SGF) is of vital significance. The precise number and placement of polymer chains is mainly determined by the defined initiators on the proteins. Typically, site-specific modification can simply be divided into three parts: Nterminal modification; C-terminal modification and cysteine modification. Since the pKa values of α-amino group of the N-terminal amino acid residue is 7.6-8.0 (110), while that of the ε-amino groups of the Lys residues distributed on the proteins is 10.0-10.2 (111), the pKa difference makes the selectivity of N-terminal modification come true. Chilkoti et al successfully prepared well-defined myoglobin-polymer conjugates with high yield, low dispersity and excellent retention of bioactivity for targeted delivery of proteins (112). The polymerization includes three steps: first, the N-terminus was transformed to an aldehyde through a pyridoxol-5′-phosphate (PLP)-mediated transamination reaction; second, a hydroxylamine-functionalized ATRP initiator (ABM) was conjugated to the N-terminus to form a macroinitiator (Mb-Br); third, the PEG-like polymer poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) was in situ grown from myoglobin (Mb) by ATRP (112) (Figure 18). Although this approach may be applicable to a large subset of proteins, it is not devoid of problems: 1) the transamination reaction is usually carried out at 37 °C 111 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


overnight, which might not be so mild for unstable proteins; 2) the attachment of initiator to proteins includes two steps, which might complicate the synthesis process; 3) this method is not suitable for proteins with active sites near their N-termini.

Figure 18. In situ growth of a stoichiometric PEG-like conjugate at the N-terminus of myoglobin (112).

Figure 19. a) The mechanism of intein-mediated protein ligation (116). Reproduced with permission from reference (116). Copyright 1994 Oxford University Press. b) In situ growth of a stoichiometric PEG-like conjugate at the C-terminal of GFP (113). 112 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Although the C-terminal is the conservative site of a protein, it cannot be chemoselectively modified as N-terminus, since the pKa of C-terminal carboxylic acid group (~3.0) is very close to that of the carboxylic acid group on the side chains of Asp (pKa ~3.7) and Glu (pKa ~4.0) residues on the protein scaffold (113, 114). Instead, the genetic engineering technology can effectively solve the problem. Intein-mediated protein ligation (IPL) can be used for the C-terminal modification of proteins. Intein is a protein sequence embedded in-frame in a protein precursor that can be cleaved and spliced itself out to rearrange protein intramolecularly during maturation (Figure 19a) (115, 116). Inteins can be mutated and designed for precise cleavage of peptide bonds at either N- or C-terminus of itself, so that the cleaved proteins possess N-terminal cysteine or C-terminal thioester moiety for site-specific modification (117, 118). Chilkoti et al. adopted IPL to attach an ATRP initiator at the C-terminal of green fluorescent protein (GFP) and produced well defined GFP-POEGMA conjugates via ATRP (Figure 19b). The conjugates exhibited increased blood exposure and tumor accumulation after intravenous administration (113). The IPL-based C-terminal modification has two potential problems: 1) disulfide bonds in the folded protein might not be tolerated as thiol chemicals are needed during reaction; 2) the protein can be premature and automatically cleaved during expression, thus reducing the final yield. Sortase-mediated protein ligation (SPL) has attracted much attention for the preparation of bioconjugates. Sortase A (SrtA) is a transpeptidase that recognizes the LPXTG motif of protein and catalyzes the cleavage of the Thr-Gly amide bond with an active-site cysteine, generating a covalent acyl-enzyme intermediate. The thioester intermediate then undergoes nucleophilic attack on the carboxyl group of Thr by the N-terminal amino group of oligoglycine (Figure 20a) (119). Chilkoti et al. exploited this method and in situ grew POEGMA from the C-terminal of GFP (120). More recently, Gao et al. employed the same modification method to in situ polymerize OEGMA from the C-terminal of Interferon (IFN) (Figure 20b) (121). The IFN-POEGMA conjugate possessed highly retained biological activity, improved pharmacokinetics and enhanced tumor accumulation. Above all, in a murine cancer model, IFN-POEGMA showed better anticancer efficacy than PEGASYS, a gold standard for IFN delivery in clinic. Cysteine residue is also selected as the site for site-specific modification as it distributes rarely on the protein, and its number and placement are defined. As mentioned above, cysteine can also be incorporated via genetic engineering technology. The thiol group on the side chain of cysteine can react with ATRP initiators and CTA functionalized with maleimide, pyridyl disulfide and activated ester. Maynard et al. modified cysteine residues of BSA and a mutant T4 LYS (V131C) with ATRP initiators functionalized with pyridyl disulfide or maleimide and in situ polymerized N-isopropylacrylamide (NIPAAM) from BSA and LYS (Figure 21a) (122). The bioactivity of the BSA-/LYS-PNIPAAM conjugates synthesized via ATRP was found to be completely retained after initiator attachment and polymerization. Velonia et al. also employed the same BSA macroinitiator to prepare giant amphiphiles (123). Gao et al. introduced a free cysteine residue for the installation of maleimide-functionalized initiator near the C-terminal and synthesized a POEGMA conjugate from a cyclized GFP (124), and they also employed the same maleimide-functionalized initiator to modify the 113 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


free cysteine residue of HSA, to further preparing HSA-PHPMA nanovesicles for enhanced intracellular protein delivery (125). Bulmus and Davis et al. employed RAFT polymerization for in situ formation of disulfide-linked BSA-POEGMA and BSA-PNIPAAM conjugates in aqueous medium in the presence of a BSA macro-CTA (126, 127). The pyridyl disulfide functionalized CTA was attached to the cysteine on BSA through its “Z-group”. Most recently, Sumerlin et al. attached the R-group of a maleimide-functionalized CTA to the free cysteine residue of BSA in order to more reasonably design a protein macro-CTA (Figure 21b) (128). However, this approach is not well site-specific for proteins with multiple cysteine residues distributed on their surfaces and restricted to the proteins with cysteine residues far away from the active sites.

Figure 20. a) The mechanism of sortase-mediated protein ligation (119). Reproduced with permission from reference (119). Copyright 2009 John Wiley and Sons. b) In situ growth of POEGMA at the C-terminal of IFN (121). Reproduced with permission from reference (121). Copyright 2016 Elsevier.



Figure 21. a) Site specific attachment of CTA to the cysteine on BSA and LYS (122). Reproduced with permission from reference (122). Copyright 2005 American Chemical Society. b) In situ polymerization of oligo(ethylene glycol) acrylate (PEG-A) from the BSA macro-CTA (128). Reproduced with permission from reference (128). Copyright 2008 American Chemical Society.



Protein Nanoparticles Proteins are natural biological molecules indispensable for living organisms. They have unique functionalities and potential applications both in biological and material fields. With the development of nanotechnology and nanomedicine, proteins are also able to be designed as a class of nanomaterials for medical use, which are known as protein nanoparticles. The combination of protein and nanotechnology has many advantages in medical application, including their large surface-to-volume ratio, specific delivery of therapeutics, low antigenicity, biodegradability, and small size for overcoming biological barriers in our body. Protein size ranges from few to hundreds nanometers, depending on their molecular mass. By using genetic engineering, it is easy to modify their structure and surface charge, to allow heterologous ligand display, and to improve stability. More importantly, proteins can be designed to form multimeric structures with the ability to self-assemble in a similar way as viral capsid proteins do. These properties open up a novel concept for imaging and therapy in nanomedicine by using the repetitive nature of protein nanoparticles to conjugate multiple drug or dyes molecules on their surfaces. These properties enable protein particles to be widely used in targeted delivery of therapeutic drugs, vaccine designing, diagnosis, and gene therapy. Moreover, some protein particles themselves are therapeutic agents. Here, we select three types of representative protein nanoparticles to illustrate their advantages and applications.

Albumin Based Nanoparticles Albumin is the most abundant protein in plasma with a molecular weight of 66.5 kDa, and plays an important role in maintaining blood colloidal osmotic pressure, nutrition and immunity. Albumin is water soluble, stable in a wide range of pH (4-9) and temperature (up to 60 °C) (129), and easily obtained from various sources. Albumin and albumin based nanoparticles are ideal carriers for drug delivery because albumin is nontoxic and non-immunogenic, and particularly has two binding sites for attaching both hydrophilic and hydrophobic drugs (130) to increase drug’s solubility and half-time (131). Albumin and albumin based nanoparticles are also found accumulated in cancer tissue through enhanced permeation and retention (EPR) effect and receptor mediated transporting. Based on these properties, they are widely used as drug carriers in cancer therapy (132, 133). In 1990s, albumin was first used to study tumor uptake by radioactive labeling or conjugation with dyes. Those studies showed that 3% to 25% of albumin could be detected in the tumor tissue rather than in other tissues (129). For instance, after administering [111In]-DTPA labeled albumin, more than 20% of protein accumulated in tumor after a single dose of injection (134). Afterwards, instead of labeling albumin with radiopharmaceuticals, researchers started to conjugate therapeutic drugs to albumin for clinical use. The most famous one was albumin-based nanoparticles conjugated with paclitaxel. Paclitaxel (PTX) was obtained from Taxus brerifolia in 1960s, which could inhibit tumor growth 116 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


through suppressing the synthesis of cytoskeletal microtubule (135). PTX drug (Taxol) that has been approved by FDA has restricted clinical use because of its poor aqueous solubility (< 0.03 mg/ml) (136, 137) and side effects. To solve these problems, PTX-albumin nanoparticle (Abraxane) with a size of 130 nm was generated for the purpose of treating breast cancer and was approved by FDA in 2005 (138) (Figure 22). Compared with Taxol, Abraxane had higher maximum tolerated dose (300 mg/m2 for Abraxane vs 175 mg/m2 for Taxol every 3 weeks), less infusion time (30 min for Abraxane vs 3–24 h for Taxol), higher tumor response rates (33% for Abraxane vs 19% for Taxol), and longer tumor retention (23 weeks for Abraxane compared with 16.9 weeks for Taxol) (139). In addition, another anti-cancer drug Doxorubicin (DOX) was used to generate protein nanoparticle with albumin for cancer therapy. DOX could inhibit cancer growth by interacting with DNA and disrupting cell membranes (140). However, similar to PTX, DOX also has the problems of short circulation time and strong side effects (141). To solve these problems, like Abraxane, DOX as a hydrophilic drug was adsorbed or incorporated to albumin to form DOX-albumin nanoparticles (142) (Figure 22). DOX-albumin nanoparticles ranged from 150 nm to 500 nm in size, and showed higher maximum tolerated dose (10 mg/kg for DOX vs 30 mg/kg for DOX-albumin). Particularly, the cardiotoxicity of DOX was significantly reduced (143).

Figure 22. Albumin-drug nanoparticles

Virus-Like Nanoparticles Virus-like particles (VLPs) is a kind of protein nanoparticles composed of virus structure proteins such as envelope and capsid. These proteins are able to self-assemble into nanoparticles, but do not contain viral genetic materials (144). VLPs are considered as a kind of vaccines with high efficiency and safety because 117 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


VLPs display antigenic epitopes on their surfaces and thus mimic native virions. VLPs are safer than those prophylactic vaccines because they do not contain viral genomes (145). VLPs exhibited high-immune response by stimulating B cell activation and thus high antibody production, due to the high density of antigenic epitopes displayed on the VLP surface (146, 147). Moreover, VLPs could be taken up by T cells due to their nanometer-size, leading to the activation of T cells (148). Based on this mechanism, the first HBsAg (hepatitis B surface antigen) was produced using recombinant DNA technology in yeast in 1981 and was licensed as the first recombinant human vaccine (149). Since then, various VLPs have been developed. Some of them were commercialized, such as Engerix®and Recombivax® that are hepatits B virus vaccine (150, 151), and Cervarix® and Gardasil® that are papillomavirus vaccine (152, 153). Additionally, many other VLPs like influenza, parvovirus, Norwalk and various chimeric VLPs are in clinical trials or in preclinical evaluation (154–156). VLPs are also considered as a kind of new nanomaterials for gene delivery since some of them have the capacity of DNA binding like native virus (Figure 23). In 1980s, polyoma (JC) based VLP was proven to be able to bind viral DNA and deliver it into cells in vitro, and later studies showed that the polyoma VLPs could bind oligonucleotides up to ~9 kbp. These results indicate that JC VLP was an ideal carrier for gene therapy (157). Since then, VLPs have been used to as carriers for the transportation of therapeutic nucleic acids, chemical polymers and proteins into cells or animals (158–160). Besides, the abundant amino acids such as cysteine, lysine, tyrosine, aspartic, and glutamic displayed on VLPs provide targets for chemical coupling (158).

Figure 23. VLP structure

Other Self-Assembled Protein Nanoparticles Like VLPs, there are many other proteins that can self-assemble into nanoparticles, such as ferritin, protein vaults and peptide-based assemblies. Ferritin plays an important role in iron sequestration. It is composed of 24 subunits and each unit has a molecular weight of 20kDa (161). Those units can self-assemble into octahedral symmetry with a size around 12 nm (Figure 24A) (162). Ferritin subunits contain many regions that allow genetic insertion of 118 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


small proteins or peptides for the purpose of therapeutic delivery, meanwhile their ability of self-assembly is not disturbed. For instance, antigenic peptides of ovalbumin (OVA) were inserted to ferritin to form a ferritin-OVA nanoparticle. This nanoparticle could be internalized inside dendritic cells, which led to the proliferation of CD4+ and CD8+ T cells both in vitro and vivo (163). Antibodies, such as trastuzumab, a human anti-HER2 antibody that was used to treat breast cancer, could also be conjugated to ferritin nanoparticle using genetic method (164).

Figure 24. Schematics of ferritin (A) (162) and vault (B) (170). Reproduced with permission from reference (170). Copyright 2012 Elsevier.

Vault particle is a kind of ribonucleoprotein particle in eukaryotic cells, with a size of 70 nm × 40 nm × 40 nm and a mass of 13 mDa (165). Vault’s function is still unclear, but its structure has been well studied. It contains 79 major vault proteins (MVP), and 38 MVPs compose half of the vault (Figure 24B) (166). Vault opening and closing can be regulated by pH. There are some positive charges inside vault, allowing DNA and RNA to bind inside for gene delivery (167). Moreover, vault can deliver other proteins or peptides through a protein named mINT that can bind to the N-terminal of MVP (168). Base on this rationale, CCL21 (a chemoattractant for T cell)-mINT fusion protein was encapsulated inside vault, which could be used to inhibit lung tumor growth in a mouse tumor model (169). Recently, several cationic and amphipathic peptides have been used for the production of protein nanoparticles (Figure 25). For example, the mixture of a peptide coming from influenza hemagglutinin and cationic polylysine (K16) could form spherical nanoparticles with a size ranging from 120 nm to 800 nm. After fused with a pro-apoptotic peptide (PVKRRLFG), these nanoparticles were delivered inside cells and showed apoptosis activity (171). Cationic peptides such as R9, T22, CXCL12, vCCL2, and V1 were fused with green fluorescence protein (GFP) containing His tag. Due to the interaction between cationic peptide and His tag, these proteins could self-assemble into nanoparticles with a size around 20 nm (172). Among them, T22-GFP-His nanoparticles could specifically bind to CXCL4 receptor over-expressed in many types of cancer, which led to the accumulation of GFP in tumor tissue in vivo. This suggests that other therapeutic proteins could be delivered through this method (173). 119 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 25. Schematic of peptide-driven protein nanoparticles. (CP = cationic peptide; L = linker; GFP = green fluorescent protein; 6His = six-histidine tag)

Future Perspectives A major challenge for materials science, drug delivery, molecular diagnosis and imaging, and tissue engineering is to design functional biomaterials that are well-defined in structure and function, and stimuli-responsive to environmental changes in pH or temperature or to cell-mediated processes. In this regard, protein-based biomaterials, such as polypeptides and engineered proteins, play unique roles and receive increasing interest as a class of bioinspired and biomedical materials for advanced biomedical applications. Although many significant advances in synthesis and application of polypeptides and engineered proteins have been achieved in the past decades, precision synthesis of well-defined functional polypeptides and engineered proteins in a cost-effective way remains a considerable challenge for future biomedical applications. For instance, it is challenging to synthesize site-specific protein-polymer conjugates with high efficiency for protein delivery. It is particularly problematic to efficiently synthesize site-specific antibody-drug/dye conjugates with high payload for molecular imaging and diagnosis, and drug delivery. With the development in genetic engineering, protein engineering, chemical biology and polymer chemistry, these problems would be solved. Therefore, it is highly promising to develop novel functional polypeptides and engineered proteins for biomedical applications.


References 1. 2. 3. 4.


5. 6. 7.

8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

22. 23. 24. 25.

McDaniel, J. R.; Callahan, D. J.; Chilkoti, A. Adv. Drug Delivery Rev. 2010, 62, 1456–1467. Ma, P. X. Adv. Drug Delivery Rev. 2008, 60, 184–198. Wang, J.; Liu, C.; Lu, X.; Yin, M. Biomaterials 2007, 28, 3456–3468. Liwo, A.; Khalili, M.; Scheraga, H. A. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 2362–2367. Vrhovski, B.; Weiss, A. S. Eur. J. Biochem. 1998, 258, 1–18. Rodgers, U. R.; Weiss, A. S. Biochimie 2004, 86, 173–178. Monzack, E. L.; Rodriguez, K. J.; McCoy, C. M.; Gu, X.; Masters, K. S. In Biomaterials for tissue engineering applications; Burdick, J. A., Mauck, R. L., Eds.; Natural materials in tissue engineering applications, 8; Springer: 2011; vol. 8, pp 225−226. Daamen, W. F.; Nillesen, S. T.; Hafmans, T.; Veerkamp, J. H.; van Luyn, M. J.; van Kuppevelt, T. H. Biomaterials 2005, 26, 81–92. Mithieux, S. M.; Rasko, J. E.; Weiss, A. S. Biomaterials 2004, 25, 4921–4927. Shamji, M. F.; Betre, H.; Kraus, V. B.; Chen, J.; Chilkoti, A.; Pichika, R.; Masuda, K.; Setton, L. A. Arthritis Rheum. 2007, 56, 3650–3661. Urry, D. W.; Parker, T. M.; Reid, M. C.; Gowda, D. C. J. Bioact. Compat. Polym. 1991, 6, 263–282. Liu, W. E.; Dreher, M. R.; Furgeson, D. Y.; Peixoto, K. V.; Yuan, H.; Zalutsky, M. R.; Chilkoti, A. J. Controlled Release 2006, 116, 170–178. Trabbic-Carlson, K.; Liu, L.; Kim, B.; Chilkoti, A. Protein Sci. 2004, 13, 3274–3284. MacEwan, S. R.; Chilkoti, A. J. Controlled Release 2014, 190, 314–330. Kyle, S.; Aggeli, A.; Ingham, E.; McPherson, M. J. Trends Biotechnol. 2009, 27, 423–433. MacKay, J. A.; Chen, M.; McDaniel, J. R.; Liu, W.; Simnick, A. J.; Chikoti, A. Nat. Mater. 2009, 8, 993–999. McDaniel, J. R.; Callahan, D. J.; Chilkoti, A. Adv. Drug Delivery Rev. 2010, 62, 1456–1467. Elzoghby, A. O.; Samy, W. M.; Elgindy, N. A. J. Controlled Release 2012, 161, 38–49. Hu, J.; Xie, L.; Zhao, W.; Sun, M.; Liu, X.; Gao, W. Chem. Commun. 2015, 51, 11405–11408. Hu, J.; Wang, G.; Liu, X.; Gao, W. P. Adv. Mater. 2015, 27, 7320–7324. Liu, W.; MacKay, J. A.; Dreher, M. R.; Chen, M.; McDaniel, J. R.; Simnick, A. J.; Callahan, D. J.; Zalutsky, M. R.; Chikoti, A. J. Controlled Release 2010, 144, 2–9. Meyer, D. E.; Shin, B. C.; Kong, G. A.; Dewhirst, M. W.; Chilkoti, A. J. Controlled Release 2001, 74, 213–224. Chung, H. J.; Park, T. G. Adv. Drug Delivery Rev. 2007, 59, 249–262. Brown, R. A.; Phillips, J. B. Int. Rev. Cytol. 2007, 262, 75–150. Ricard-Blum, S. Cold Spring Harbor Perspect. Biol. 2011, 3, a004978. 121 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


26. Nair, L. S.; Laurencin, C. T. Adv. Biochem. Eng. Biotechnol. 2006, 102, 47–90. 27. Cen, L.; Liu, W.; Cui, L.; Zhang, W.; Cao, Y. Pediatr. Res. 2008, 63, 492–496. 28. Darwin, J. P. Annu. Rev. Biochem. 1995, 64, 403–434. 29. Kluge, J. A.; Rabotyagova, O.; Leisk, G. G.; Kaplan, D. L. Trends Biotechnol. 2008, 26, 244–251. 30. Cao, Y.; Wang, B. Int. J. Mol. Sci. 2009, 10, 1514–1524. 31. Wang, Y.; Kim, H. J.; Vunjak-Novakovic, G.; Kaplan, D. L. Biomaterials 2006, 27, 6064–6082. 32. Sofia, S.; McCarthy, M. B.; Gronowicz, G.; Kaplan, D. L. J. Biomed. Mater. Res. 2001, 54, 139–148. 33. Fini, M.; Motta, A.; Torricelli, P.; Giavaresi, G.; Nicoli Aldini, N.; Tschon, M.; Giardino, R.; Migliaresi, C. Biomaterials 2005, 26, 3527–3536. 34. Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149–2154. 35. Leuchs, H. Ber. Dtsch. Chem. Ges. 1906, 39, 857–861. 36. Sekiguchi, H. Pure Appl. Chem. 1981, 53, 1689–1714. 37. Wang, M.; Du, J. Acta Polym. Sin. 2014, 9, 1183–1194. 38. Hadjichristidis, N.; Iatrou, H.; Pitsikalis, M.; Sakerllariou, G. Chem. Rev. 2009, 109, 5528–5578. 39. Cheng, J.; Deming, T. J. In Peptide-based materials; Deming, T. J., Ed.; Synthesis of polypeptides by ring-opening polymerization of α-amino acid N-carboxyanhydrides, 1; Springer: Berlin, 2011; Vol. 310, pp 1−26. 40. Deming, T. J. Nature 1997, 390, 386–389. 41. Deming, T. J. Macromolecules 1999, 32, 4500–4502. 42. Deming, T. J.; Curtin, S. A. J. Am. Chem. Soc. 2000, 122, 5710–5717. 43. Lu, H.; Cheng, J. J. Am. Chem. Soc. 2007, 129, 14114–14115. 44. Lu, H.; Cheng, J. J. Am. Chem. Soc. 2008, 130, 12562–12563. 45. Dimitrov, I.; Schlaad, H. Chem. Commun. 2003, 23, 2944–2945. 46. Aliferis, T.; Iatrou, H.; Hadjichristidis, N. Biomacromolecules 2004, 5, 1653–1656. 47. Vayaboury, W.; Giani, O.; Cottet, H.; Deratani, A.; Schué, F. Macromol. Rapid Commun. 2004, 25, 1221–1224. 48. Holowka, E. P.; Sun, V. Z.; Kamei, D. T.; Deming, T. J. Nat. Mater. 2007, 6, 52–57. 49. Engler, A. C.; Shukla, A.; Puranam, S.; Buss, H. G.; Jreige, N.; Hammond, P. T. Biomacromolecules 2011, 12, 1666–1674. 50. Köhler, G.; Milstein, C. Nature 1975, 256, 495–497. 51. Stern, M.; Herrmann, R. Crit. Rev. Oncol. Hemat. 2005, 54, 11–29. 52. Chari, R. V. J.; Miller, M. L.; Widdison, W. C. Angew. Chem., Int. Ed. 2014, 53, 3796–3827. 53. Jones, P. T.; Dear, P. H.; Foote, J.; Neuberger, M. S.; Winter, G. Nature 1985, 321, 522–525. 54. Morrison, S. L.; Johnson, M. J.; Herzenberg, L. A.; Oi, V. T. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 6851–6855. 55. Chames, P.; Van Regenmortel, M.; Weiss, E.; Baty, D. Br. J. Pharmacol. 2009, 157, 220–233. 122 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


56. Chothia, C.; Lesk, A. M.; Tramontano, A.; Levitt, M.; Smith-Gill, S. J.; Air, G.; Sheriff, S.; Padlan, E. A.; Davies, D.; Tulip, W. R.; Colman, P. M.; Spinelli, S.; Alzari, P. M.; Poljak, R. J. Nature 1989, 342, 877–883. 57. Jakobovits, A. Curr. Opin. Biotechnol. 1995, 6, 561–566. 58. Smith, G. P. Science 1985, 228, 1315–1317. 59. Kim, S. J.; Park, Y.; Hong, H. J. Mol. Cells 2005, 20, 17–29. 60. Lipson, E. J.; Drake, C. G. Clin. Cancer Res. 2011, 17, 6958–6962. 61. Holliger, P.; Hudson, P. J. Nat. Biotechnol. 2005, 23, 1126–1136. 62. Zarnani, A. H.; Bozorgmehr, M.; Shabani, M.; Barzegar-Yarmohammadi, L.; Ghaemimanesh, F.; Jeddi-Tehrani, M. In Cancer Immunology; Rezaei, N., Ed.; Monoclonal Antibodies for Cancer Immunotherapy, 16; Springer Science & Business Media: Berlin, 2015; pp 293−328. 63. Pavlinkova, G.; Beresford, G. W.; Booth, B. J.; Batra, S. K.; Colcher, D. J. Nucl. Med. 1999, 40, 1536–1546. 64. Slavin-Chiorini, D. C.; Kashmiri, S. V. S.; Schlom, J.; Calvo, B.; Shu, L. M.; Schott, M. E.; Milenic, D. E.; Snoy, P.; Carrasquillo, J.; Anderson, K.; Hand, P. H. Cancer Res. 1995, 55, 5957s–5967s. 65. Yang, K.; Basu, A.; Wang, M.; Chintala, R.; Hsieh, M. C.; Liu, S.; Hua, J.; Zhang, Z.; Zhou, J.; Li, M.; Phyu, H.; Petti, G.; Mendez, M.; Janjua, H.; Peng, P.; Longley, C.; Borowski, V.; Mehlig, M.; Filpula, D. Protein Eng. 2003, 16, 761–770. 66. Kortt, A. A.; Dolezal, O.; Power, B. E.; Hudson, P. J. Biomol. Eng. 2001, 18, 95–108. 67. Kontermann, R. mAbs 2012, 4, 182–197. 68. Przepiorka, D.; Ko, C.-W.; Deisseroth, A.; Yancey, C. L.; CandauChacon, R.; Chiu, H.-J.; Gehrke, B. J.; Gomez-Broughton, C.; Kane, R. C.; Kirshner, S. Clin. Cancer Res. 2015, 21, 4035–4039. 69. Panowski, S.; Bhakta, S.; Raab, H.; Polakis, P.; Junutula, J. R. mAbs 2014, 6, 34–45. 70. Zolot, R. S.; Basu, S.; Million, R. P. Nat. Rev. Drug Discovery 2013, 12, 259–260. 71. Steiner, M.; Neri, D. Clin. Cancer Res. 2011, 17, 6406–6416. 72. Ronca, R.; Sozzani, S.; Presta, M.; Alessi, P. Immunobiology 2009, 214, 800–810. 73. Pardridge, W. M. Adv. Drug Delivery Rev. 2007, 59, 141–152. 74. Franzman, M. R.; Burnell, K. K.; Dehkordi-Vakil, F. H.; Guthmiller, J. M.; Dawson, D. V.; Brogden, K. A. Int. J. Antimicrob. Agents 2009, 33, 14–20. 75. Kaushal, S.; McElroy, M. K.; Luiken, G. A.; Talamini, M. A.; Moossa, A. R.; Hoffman, R. M.; Bouvet, M. J. Gastrointest. Surg. 2008, 12, 1938–1950. 76. Liu, X.; Gao, W. In Biomedical Nanomaterials; Zhao, Y., Shen, Y., Eds.; Antibody-Drug Conjugates, 6; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, DE, 2016; Vol. 6, pp 149−176. 77. Ehrlich, P. The collected papers of Paul Ehrlich 1956, 1, 596–618. 78. Senter, P. D.; Sievers, E. L. Nat. Biotechnol. 2012, 30, 631–637. 79. Amiri-Kordestani, L.; Blumenthal, G. M.; Xu, Q. C.; Zhang, L.; Tang, S. W.; Ha, L.; Weinberg, W. C.; Chi, B.; Candau-Chacon, R.; Hughes, P. Clin. Cancer Res. 2014, 20, 4436–4441. 123 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


80. Zhang, L.; Zhao, W.; Liu, X.; Wang, G.; Wang, Y.; Li, D.; Xie, L.; Gao, Y.; Deng, H.; Gao, W. Biomaterials 2015, 64, 2–9. 81. Hallam, T. J.; Wold, E.; Wahl, A.; Smider, V. V. Mol. Pharm. 2015, 12, 1848–1862. 82. Gauthier, M. A.; Klok, H. A. Chem. Commun. 2008, 39, 2591–2611. 83. Nicolas, J.; San, M. V.; Mantovani, G.; Haddleton, D. M. Chem. Commun. 2006, 45, 4697–4699. 84. Abuchowski, A.; Van Es, T.; Palczuk, N. C.; Davis, F. F. J. Biol. Chem. 1977, 252, 3578–3581. 85. Abuchowski, A.; Mccoy, J. R.; Palczuk, N. C.; Van, E. T.; Davis, F. F. J. Biol. Chem. 1977, 252, 3582–3586. 86. Zalipsky, S.; Lee, C. In Poly(Ethylene Glycol) Chemistry; Harris, J. M., Ed.; Use of Functionalized Poly(Ethylene Glycol)s for Modification of Polypeptides, 21; Springer Science & Business Media: Berlin, 1992; pp 1−15. 87. Francis, G. E.; Fisher, D.; Delgado, C.; Malik, F.; Gardiner, A.; Neale, D. Int. J. Hematol. 1998, 68, 1–18. 88. Zalipsky, S.; Seltzer, R.; Menon-Rudolph, S. Biotechnol. Appl. Biochem. 1992, 15, 100–114. 89. Miron, T.; Wilchek, M. Bioconjugate Chem. 1993, 4, 568–569. 90. Dolence, E. K.; Hu, C. Z.; Tsang, R.; Sanders, C. G.; Osaki, S. Electrophilic polyethylene oxides for the modification of polysaccharides, polypeptides (proteins) and surfaces. U.S. Patent 5,650,234, 22 July 1997. 91. Veronese, F. M.; Largajolli, R.; Boccù, E.; Benassi, C. A.; Schiavon, O. Appl. Biochem. Biotechnol. 1985, 11, 141–152. 92. Abuchowski, A.; Kazo, G. M.; Verhoest, C. R., Jr.; Van Es, T.; Kafkewitz, D.; Nucci, M. L.; Viau, A. T.; Davis, F. F. Cancer Biochem. Biophys. 1984, 7, 175–186. 93. Roberts, M. J.; Bentley, M. D.; Harris, J. M. Adv. Drug Delivery Rev. 2002, 54, 116–127. 94. Pelegrio’Day, E. M.; Lin, E. W.; Maynard, H. D. J. Am. Chem. Soc. 2014, 136, 14323–14332. 95. Kozlowski, A.; Harris, J. M. J. Controlled Release 2001, 72, 217–224. 96. Molineux, G. Curr. Pharm. Design 2004, 10, 1235–1244. 97. Bentley, M. D.; Harris, M. J. Poly(ethylene glycol) aldehyde hydrates and related polymers and applications in modifying amines. U.S. Patent 5,990,237, 23 Nov. 1999. 98. Zalipsky, S.; Barany, G. J. Bioact. Compat. Polym. 1990, 5, 227–231. 99. Gaertner, H. F.; Offord, R. E. Bioconjugate Chem. 1996, 7, 38–44. 100. Gilmore, J. M.; Scheck, R. A.; Esser‐Kahn, A. P.; Joshi, N. S.; Francis, M. B. Angew. Chem. 2006, 45, 5307–5311. 101. Goodson, R. J.; Katre, N. V. Nat. Biotechnol. 1990, 8, 343–346. 102. Kogan, T. P. Synthetic Commun. 1992, 22, 2417–2424. 103. Morpurgo, M.; Veronese, F. M.; Kachensky, D.; Harris, J. M. Bioconjugate Chem. 1996, 7, 363–368. 104. Woghiren, C.; Sharma, B.; Stein, S. Bioconjugate Chem. 1993, 4, 314–318. 124 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


105. Karpusas, M.; Nolte, M.; Benton, C. B.; Meier, W.; Lipscomb, W. N.; Goelz, S. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 11813–11818. 106. Veronese, F. M.; Pasut, G. Drug Discovery Today 2005, 10, 1451–1458. 107. Alconcel, S. N. S. Polym. Chem. 2011, 2, 1442–1448. 108. Bhalchandra, S. L.; Hironobu, M.; Matyjaszewski, K.; Russell, A. J. Biomacromolecules 2005, 6, 3380–3387. 109. Zeng, Q.; Li, T.; Cash, B.; Li, S.; Xie, F.; Wang, Q. Chem. Commun. 2007, 14, 1453–1455. 110. Kinstler, O.; Molineux, G.; Treuheit, M.; Ladd, D.; Gegg, C. Adv. Drug Delivery Rev. 2002, 54, 477–485. 111. Wong, S. S. Chemistry of Protein Conjugation and Crosslinking; Reactive groups of proteins and their modifying agents, 2; CRC Press: Boston, MA, 1991; pp 27−29. 112. Gao, W.; Liu, W.; Mackay, J. A.; Zalutsky, M. R.; Toone, E. J.; Chilkoti, A. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 15231–15236. 113. Gao, W.; Liu, W.; Christensen, T.; Zalutsky, M. R.; Chilkoti, A. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 16432–16437. 114. Zhao, W.; Liu, F.; Chen, Y.; Bai, J.; Gao, W. Polymer 2015, 66, A1–A10. 115. Hirata, R.; Ohsumk, Y.; Nakano, A.; Kawasaki, H.; Suzuki, K.; Anraku, Y. J. Biol. Chem. 1990, 265, 6726–6733. 116. Perler, F. B.; Davis, E. O.; Dean, G. E.; Gimble, F. S.; Jack, W. E.; Neff, N.; Noren, C. J.; Thorner, J.; Belfort, M. Nucleic Acids Res. 1994, 22, 1125–7. 117. Muralidharan, V.; Muir, T. W. Nat. Methods 2006, 3, 429–438. 118. Esser-Kahn, A. P.; Francis, M. B. Angew. Chem. 2008, 47, 3751–3754. 119. Tsukiji, S.; Nagamune, T. ChemBioChem 2009, 10, 787–798. 120. Qi, Y.; Amiram, M.; Gao, W.; Mccafferty, D. G.; Chilkoti, A. Macromol. Rapid Commun. 2013, 34, 1256–1260. 121. Jin, H.; Wang, G.; Zhao, W.; Liu, X.; Zhang, L.; Gao, W. Biomaterials 2016, 96, 84–92. 122. Heredia, K. L.; Bontempo, D.; Ly, T.; Byers, J. T.; Sven Halstenberg, A.; Maynard, H. D. J. Am. Chem. Soc. 2005, 127, 16955–16960. 123. Le, D. B.; Velonia, K. Angew. Chem., Int. Ed. 2008, 47, 6263–6266. 124. Hu, J.; Zhao, W.; Gao, Y.; Sun, M.; Wei, Y.; Deng, H.; Gao, W. Biomaterials 2015, 47, 13–19. 125. Liu, X.; Gao, W. ACS Appl. Mater. Interfaces 2017, 9, 2023–2028. 126. Liu, J.; Bulmus, V.; Herlambang, D. L.; Barner-Kowollik, C.; Stenzel, M. H.; Davis, T. P. Angew. Chem. 2007, 46, 3099–3103. 127. Boyer, C.; Bulmus, V.; Liu, J.; Davis, T. P.; Stenzel, M. H.; BarnerKowollik, C. J. Am. Chem. Soc. 2007, 129, 7145–7154. 128. De, P.; Li, M.; Gondi, S. R.; Sumerlin, B. S. J. Am. Chem. Soc. 2008, 130, 11288–11289. 129. Kratz, F.; Beyer, U. Drug Delivery 1998, 5, 281–299. 130. Desai, N.; Trieu, V.; Yao, Z.; Louie, L.; Ci, S.; Yang, A.; Tao, C.; De, T.; Beals, B.; Dykes, D.; Noker, P.; Yao, R.; Labao, E.; Hawkins, M.; SoonShiong, P. Clin. Cancer Res. 2006, 12, 1317–1324. 131. Venditto, V. J.; Szoka, F. C. Adv. Drug Delivery Rev. 2013, 65, 80–88. 125 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


132. John, T. A.; Vogel, S. M.; Tiruppathi, C.; Malik, A. B.; Minshall, R. D. Am. J. Physiol. Lung Cell Mol. Physiol. 2003, 284, L187–L196. 133. Gibbs, J. B. Science 2000, 287, 1969–1973. 134. Stehle, G.; Sinn, H.; Wunder, A.; Schrenk, H. H.; Stewart, J. C. M.; Hartung, G.; Maier-Borst, W.; Heene, D. L. Crit. Rev. Oncol. Hematol. 1997, 26, 77–100. 135. Wang, T. H.; Wang, H. S.; Soong, Y. K. Cancer 2000, 88, 2619–2628. 136. Zuylen, L. V.; Verweij, J.; Sparreboom, A. Invest. New Drugs 2001, 19, 125–141. 137. Gradishar, W. J.; Tjulandin, S.; Davidson, N.; Shaw, H.; Desai, N.; Bhar, P.; Hawkins, M.; O’Shaughnessy, J. J. Clin. Oncol. 2005, 23, 7794–7803. 138. Nabholtz, J. M.; Gelmon, K.; Bontenbal, M.; Spielmann, M.; Catimel, G.; Conte, P.; Klaassen, U.; Namer, M.; Bonneterre, J.; Fumoleau, P. J. Clin. Oncol. 1996, 14, 1858–1867. 139. Thorn, C. F.; Oshiro, C.; Marsh, S.; Hernandezboussard, T.; Mcleod, H.; Klein, T. E.; Altman, R. B. Pharmacogenet. Genom. 2011, 21, 440–446. 140. Danesi, D. R.; Fogli, S.; Gennari, A.; Conte, P.; Tacca, M. D. Clin. Pharmacokinet. 2002, 41, 431–444. 141. Dreis, S.; Rothweiler, F.; Michaelis, M.; Cinatl, J. C., Jr.; Kreuter, J.; Langer, K. Int. J. Pharm. 2007, 341, 207–214. 142. Yuan, A.; Wu, J.; Song, C.; Tang, X.; Qiao, Q.; Zhao, L.; Gong, G.; Hu, Y. J. Pharm. Sci. 2013, 102, 1626–1635. 143. Pumpens, P.; Grens, E. In Artificial DNA: Methods and Applications; Khudyakov, Y. E., Fields, H. A., Eds.; Artificial Genes for Chimeric Virus-Like Particles, 8; CRC Press LLC: Boca Raton, FL, 2002; Vol. 8, pp 249−327. 144. Zhao, Q.; Li, S.; Yu, H.; Xia, N.; Modis, Y. Trends Biotechnol. 2013, 31, 654–663. 145. Bachmann, M. F.; Zinkernagel, R. M. Annu. Rev. Immunol. 1997, 15, 235–270. 146. Brun, A.; Bárcena, J.; Blanco, E.; Borrego, B.; Dory, D.; Escribano, J. M.; Gall-Reculé, G. L.; Ortego, J.; Dixon, L. K. Virus Res. 2011, 157, 1–12. 147. Bachmann, M. F.; Dyer, M. R. Nat. Rev. Drug Discovery 2004, 3, 81–88. 148. Michel, M. L.; Tiollais, P. Pathol. Biol. 2010, 58, 288–295. 149. Keating, G. M.; Noble, S. Drugs 2012, 63, 1021–1051. 150. Venters, C.; Graham, W.; Cassidy, W. Exp. Rev. Vaccines 2014, 3, 119–129. 151. Elliott, W. T.; Chan, J. Internal Medicine Alert 2009, 31, 190–191. 152. Mcintyre, J. A.; Leeson, P. A. Gardasil. Drugs Future 2006, 31, 97–100. 153. Quan, F. S.; Huang, C.; Compans, R. W.; Kang, S. M. J. Virol. 2007, 81, 3514–3524. 154. Maranga, L.; Rueda, P.; Antonis, A.; Vela, C.; Langeveld, J.; Casal, J.; Carrondo, M. Appl. Microbiol. Biotechnol. 2002, 59, 45–50. 155. Tacket, C. O.; Sztein, M. B.; Losonsky, G. A.; Wasserman, S. S.; Estes, M. K. Clin. Immunol. 2003, 108, 241–247. 156. Fang, C. Y.; Lin, P. Y.; Ou, W. C.; Chen, P. L.; Shen, C. H.; Chang, D.; Wang, M. J. Virol. Methods 2012, 182, 87–92. 157. Lee, L. A.; Niu, Z.; Wang, Q. Nano Res. 2010, 2, 349–364. 126 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


158. Ng, B. C.; Chan, S. T.; Lin, J.; Tolbert, S. H. ACS Nano 2011, 5, 7730–7738. 159. Newman, M.; Chua, P. K.; Tang, F. M.; Su, P. Y.; Shih, C. J. Virol. 2009, 83, 10616–10626. 160. Andrews, S. C.; Harrison, P. M.; Yewdall, S. J.; Arosio, P.; Levi, S.; Bottke, W.; Darl, M. V.; Briat, J. F.; Laulhère, J. P.; Lobreaux, S. J. Inorg. Biochem. 1992, 47, 161–174. 161. Lawson, D. M.; Artymiuk, P. J.; Yewdall, S. J.; Smith, J. M. A.; Livingstone, J. C.; Treffry, A.; Luzzago, A.; Levi, S.; Arosio, P.; Cesareni, G. Nature 1991, 349, 541–544. 162. Zhang, Y.; Orner, B. P. Int. J. Mol. Sci. 2011, 12, 5406–5421. 163. Kang, H. J.; Kang, Y. J.; Lee, Y. M.; Shin, H. H.; Sang, J. C.; Kang, S. Biomaterials 2012, 33, 5423–5430. 164. Suprenant, K. A. Biochemistry 2002, 41, 14447–14454. 165. Tanaka, H.; Kato, K.; Yamashita, E.; Sumizawa, T.; Zhou, Y.; Yao, M.; Iwasaki, K.; Yoshimura, M.; Tsukihara, T. Science 2009, 323, 384–388. 166. Querol-Audí, J.; Casañas, A.; Usón, I.; Luque, D.; Castón, J. R.; Fita, I.; Verdaguer, N. EMBO J. 2009, 28, 3450–3457. 167. Kickhoefer, V. A.; Garcia, Y.; Mikyas, Y.; Johansson, E.; Zhou, J. C.; RavalFernandes, S.; Minoofar, P.; Zink, J. I.; Dunn, B.; Stewart, P. L. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 4348–4352. 168. Lai, C. Y.; Wiethoff, C. M.; Kickhoefer, V. A.; Rome, L. H.; Nemerow, G. R. ACS Nano 2009, 3, 691–699. 169. Collins, L.; Parker, A. L.; Gehman, J. D.; Eckley, L.; Perugini, M. A.; Separovic, F.; Fabre, J. W. ACS Nano 2010, 4, 2856–2864. 170. Casañas, A.; Guerra, P.; Fita, I.; Verdaguer, N. Curr. Opin. Biotechnol. 2012, 23, 972–977. 171. Vazquez, E.; Roldán, M.; Diezgil, C.; Unzueta, U.; Domingoespín, J.; Cedano, J.; Conchillo, O.; Ratera, I.; Veciana, J.; Daura, X. Nanomedicine 2015, 5, 259–268. 172. Céspedes, M. V.; Unzueta, U.; Tatkiewicz, W.; Sánchezchardi, A.; Conchillosolé, O.; P, Á.; Xu, Z.; Casanova, I.; Corchero, J. L.; Pesarrodona, M. ACS Nano 2014, 8, 4166–4176. 173. Ugutz, U.; Virtudes, C. M.; Neus, F.-M.; Isolda, C.; Juan, C.; Luis, C. J.; Joan, D.-E.; Antonio, V.; Ramón, M.; Esther, V. Int. J. Nanomed. 2012, 7, 4533–4544.


Polypeptides and Engineered Proteins - American Chemical Society

Recommend Documents