Single chain protein-polymer<unk> s Attaching a single<unk>  chain to a specific site away from the active center of the protein has less impact on protein activity compared with random attachments. In practice, attaching a single<unk>  chain can be used to adjust chemical properties of the therapeutic protein. For example, conjugation of a single chain of the hydrophilic polyethylene glycol (PEG) can increase the hydrodynamic radius of the protein conjugate by 5-10 fold. Attachment to PEG was mainly achieved by covalent conjugation via the grafting to strategy, targeting chemo-selective anchor groups. Other<unk> s, such as oligosaccharides and polypeptides, offer different properties to the enzymes attached to them. Stimuli responsive<unk> s Heat Researchers conjugated the thermo-responsive<unk>  poly(N-isopropylacrylamide) (pNIPAm) with the biotin-recognizing protein streptavidin close to its recognition site. At temperatures above the lower critical solution temperature (LCST), the<unk>  collapses and blocks the binding site, thus reversibly preventing biotin from binding to streptavidin. By copolymerization with two different thermosensitive<unk> s poly(sulfobetaine methacrylamide) (pSBAm) and pNIPAm together, researchers can control enzyme activity in a small temperature window. Light ((N,N′-dimethylacrylamide)-co-4-phenylazophenyl acrylate) at the active site of endoglycanase creates a photoswitchable protein<unk> . The resulting<unk>  catalyzes the hydrolysis of glycoside when irradiated by 350 nm UV light, but turns inactive under 420 nm visible light depending on the conformation of the conjugated<unk> .<unk>  shell protein core A<unk>  shell is formed by conjugation of multiple molecules of<unk> s onto the protein core. The<unk>  shell can either protect the protein core from unwanted degradation or create desired interactive sites for guest molecules. The first generation of<unk>  shell protein core structures mainly used of Polyethylene glycol (PEG) chains to increase the hydrodynamic radius and reduce immune response to proteins. However, the PEG shell can reduce protein activity in the inner core. More advanced designs use biodegradable linkers to achieve programmed release of the protein core in specific tissues. Several therapeutic designs with biodegradable PEG shells are already being developed in vivo. Direct conjugation of<unk> s (“grafting to” strategy) can efficiently construct a<unk>  shell with diverse<unk>  types, however, it suffers from low<unk>  density, especially with large<unk> s. In contrast, “grafting from” strategy allows the formation of a dense and uniform<unk>  shell. The protein core can also function as a carrier for other therapeutic molecules, such as plasmid DNA. Dendrite<unk>  shells have a high volume to molecular weight ratio compared with traditional<unk>  shells. Using branched carbohydrates can give unique biological properties while maintaining molecular definition. Non-covalent conjugation Although covalent conjugation has been the dominant strategy for constructing<unk> s, noncovalent chemistry can add another level of complexity and provides the opportunity to create higher-ordered structures. Specifically, self-assembly by non-covalent interactions is progressing rapidly. Supramolecular self-assembly can create nanoparticles, vesicles/micelles, protein cages, etc. Metal-binding interactions, host-guest, and boronic acid-based chemistries are widely studied as non-covalent conjugation methods to create<unk> s.<unk> -Streptavidin system Streptavidin is a protein purified from the bacterium Streptomyces avidinii, which has a high affinity for biotin. By covalently linking streptavidin and<unk> s, well defined supramolecular constructs can be created due to the high specificity of Streptavidin for both biotin and its analogues. Building upon the covalent core shell strategy, several<unk> –streptavidin systems have been developed for affinity separation, bio-sensors and diagnostic applications due to the robust binding conditions and stability of the protein. Streptavidin can be used as a macro-initiator for in situ ATRP, through grafting from strategy, a stoichiometrically well defined<unk> -protein conjugate can be synthesized.<unk>  streptavidin systems can also be empowered to cross the cellular membrane by conjugating with cell penetrating molecules such as peptides and membrane disturbing<unk> s.<unk>  streptavidin systems can also be modulated to respond to certain environmental changes such as pH. By incorporating pH responsive poly(propylacrylic acid) (PPAAc) into the system, tumor cell suppressor p53 and cytochrome C can be delivered into cancer cells efficiently. For biomolecules that are not hampered by the biotin-streptavidin interaction, iminobiotin, an analogue of biotin, has been applied as a pH-sensitive linker that allows the controlled and reversible assembly and intracellular release of cargo molecules in acidic intracellular compartments. Protein-polymer<unk>  supramolecular structures<unk> -protein conjugates can also form a higher ordered supramolecular structure via self-assembly of amphiphilic<unk> s into micelles and microcapsules, which is one of the most promising strategies to generate drug delivery systems. Such systems have the innate advantage of rapid preparation, a high drug loading capacity, ease of surface decoration, and the potential to be stimuli responsive. Micelles Micelles refers to a type of supramolecular structure consisting of amphiphilic molecules self-assemblies, usually hollow centered. Researchers successfully conjugated a diblock copolymer site specifically onto GFP, the resulting amphiphilic<unk> -protein conjugate is capable of reversible self-assembly into micelles. In addition to retaining the native globular shape of proteins, the polypeptide backbone of denatured proteins can also be utilized to be conjugated with hydrophilic<unk>  chains to generate higher ordered structure through hydrophobic interactions. For example, nanoconjugates of poly-ethylene glycol(PEG) and denatured bovine serum albumin(BSA) will spontaneously self-assemble into a micellar structure, whose protein core can adsorb high numbers of hydrophobic drugs. Nanoparticles An efficient way to synthesize protein-polymer<unk>  nanoparticles is to take advantage of photoinitiated reversible addition−fragmentation chain transfer (RAFT)<unk> ization-induced self-assembly(PISA) by using multi-RAFT modified bovine serum albumin (BSA) as a macromolecular chain transfer agent. RAFT mediated growth of the PHPMA chains will graft from the BSA-RAFT, and increase the hydrophobicity of the star BSA−PHPMA conjugates. At the critical aggregation concentration, they form nanoparticles due to the hydrophobic interactions. The resulting nanoparticles show excellent encapsulation capability for both hydrophobic and hydrophilic molecules, such as cancer drugs and DNA. A rather easy method to prepare protein-polymer<unk>  nanoparticles is nanoprecipitation. Spherical nanoparticles composed of BSA-PMMA with diameters of around 100 nm were obtained and the water insoluble chemotherapeutic drug camptothecin was encapsulated within the hydrophobic core consisting of PMMA. Such protein-polymer<unk>  nanoparticles possess tunable sizes and surface charges, have attractive bio-compatibilities and allow efficient cell uptake. Camptothecin-encapsulated BSA-PMMA nanoparticles revealed enhanced anti-tumor activity both in vitro and in animals. Beyond the nanoscale, protein-polymer conjugate could also be used as building blocks for constructing more complicated structures such as microcapsules through hydrophobic interactions. By performing pickering emulsion technique to process BSA–pNIPAm nanoconjugates into hollowed microcapsules consisting of a closely packed monolayer of conjugated protein–polymer building blocks (named proteinosomes). These proteinosomes exhibit protocellular properties such as guest molecule encapsulation, selective permeability, controllable mobilization, gene-directed protein synthesis and membrane-gated internalized enzyme catalysis. Based on the above mentioned method, a multi responsive microcapsule has been synthesized by incorporating photoswitchable spiropyran units and the thermoresponsive monomer N-isopropylacrylamide into the membrane. Stimuli responsive membrane exhibited advantages in the capture and release of different-molecular-weight products by opening and closing the photoresponsive spiropyran ligands, under body temperature, room temperature, UV, redox. Another effective way to modulate the permeability of microcapsules was based on a ‘‘self-sacrificing’’ strategy. By selectively using lysozyme and BSA as building blocks as well as ‘‘self-sacrificing’’ components, the corresponding pores could be generated in the membrane, and then the permeability of the generated microcapsules could be increased from10 kDa to 22 kDa and then to 71 kDa. By loading FITC-Lys (14 kDa), RBITCdextran (70 kDa) and DNA (90 kDa) into the microcapsules, a programmed release of the encapsulants from low molecular weight to high molecular weight was realized. Using similar strategy, pH-sensitive protein-polymer microcapsules were developed. Both doxycycline (DOX) and folic acid were incorporated onto the surface of protein covalently. The very low toxicity of<unk> -protein nanoconjugates effectively avoided the high toxicity of DOX, which is expected to not only reduce toxic side effects, but also improve anticancer efficiency in vitro examinations. Protein Nanocages Protein nanocages are natural nanocarriers composed of protein subunits with a porous structure. They benefit from monodispersity, intrinsic high stability for protection of internalized drugs from enzymatic degradation and controllable assembly for cargo loading and release. However, their application might be blocked by immunogenicity, broad biodistribution and significant function and property variations. The incorporation of<unk>  chains by performing in situ ATRP on the outer surface of or inside the protein nanocages can be an effective way to mitigate those drawbacks. For example, increased loading density of cargo molecules and enhanced stability of the cage assembly can be obtained via internal ATRP inside the cavity of the virus capsid. Beyond virus type particles, large multimeric proteins such as the iron storage protein ferritin have emerged as attractive tools to be used as well-defined nano-containers. Using a grafting from strategy,<unk> s can be introduced to ferritin in a highly regular fashion for precise spatial control. These<unk> –ferritin constructs exhibited protease resistance, enabling longer retention time within the bloodstream while reducing possible antibody interactions.
Polymer-protein hybrid
Bioactive compound
Immunoconjugate
Polysaccharide peptide
Mutagen
Tetrameric protein
Glycol nucleic acid
Azo compound