Protein-polymer Bioconjugates Research Articles

A Protein-Polymer Bioconjugate-Coated Upconversion Nanosystem for Simultaneous Tumor Cell Imaging, Photodynamic Therapy, and Chemotherapy.

Combined cancer therapy possesses many advantages including improved tumoricidal efficacy, reduced side effects, and retarded drug resistance. Herein, a protein-polymer bioconjugate-coated multifunctional upconversion nanosystem, consisting of upconversion nanoparticles (UCNs) core, tailored amphiphilic protein-polymer bioconjugate shell, and photosensitizer zinc phthalocyanine (ZnPc) and antitumor drug doxorubicin coloaded inside, was elaborately developed for combined photodynamic therapy (PDT) and chemotherapy. In this system, UCNs core could convert deep penetrating near-infrared light to visible light for simultaneous cell fluorescence imaging and photodynamic therapy by activating ZnPc to generate cytotoxic ROS, while the protective shell of bovine serum albumin-poly(ε-caprolactone) (BSA-PCL) offered excellent water solubility, good stability, and low cytotoxicity. The ROS production test showed that this nanosystem could successfully generate singlet oxygen under NIR irradiation. A cellular uptake study demonstrated that intense fluorescence emission of the UCNs could be observed in HeLa cells, indicating their outstanding real-time imaging capability. More importantly, compared with single PDT or chemotherapy systems, the constructed combined therapy UCNs system demonstrated significantly enhanced tumor cell killing efficiency. On the basis of our findings, this multifunctional UCNs nanosystem could be a promising versatile theranostic nanoplatform for image-guided combined cancer therapy.

Synthesis of Protein–Polymer–Protein Bioconjugates by the Combination of Atom Transfer Radical Polymerization and Click Reaction

Novel protein–polymer–protein bioconjugates were synthesized by the successive reaction between both chain ends of a polymer and two different types of proteins. A poly(2‐(2‐methoxyethoxy)ethyl methacrylate) (OH‐PMEO2MA‐Br) was synthesized by atom transfer radical polymerization (ATRP) from a hydroxyl‐terminated initiator. The Br‐terminated chain ends of the resulting polymer in one end were transformed to azido groups (OH‐PMEO2MA‐N3 ) and subsequently the OH‐terminated chain ends in the other end were transformed to 4‐nitrophenyl formate‐derivated group (NPC‐PMEO2MA‐N3 ). A model protein, cationized bovine serum albumin (cBSA) was reacted with 4‐nitrophenyl formate groups of NPC‐PMEO2MA‐N3 to form cBSA‐PMEO2MA‐N3 . The click reaction between azido‐terminated cBSA‐PMEO2MA‐N3 and alkyne‐derived bovine serum albumin (BSAalkyne ) yielded novel cBSA‐PMEO2MA‐BSA bioconjugates with two proteins at both polymer ends.

Kinetic Effects on Self-Assembly and Function of Protein-Polymer Bioconjugates in Thin Films Prepared by Flow Coating.

The self-assembly of nanostructured globular protein arrays in thin films is demonstrated using protein-polymer block copolymers based on a model protein mCherry and the polymer poly(oligoethylene glycol acrylate) (POEGA). Conjugates are flow coated into thin films on a poly(ethylene oxide) grafted Si surface, forming self-assembled cylindrical nanostructures with POEGA domains selectively segregating to the air-film interface. Long-range order and preferential arrangement of parallel cylinders templated by selective surfaces are demonstrated by controlling relative humidity. Long-range order increases with coating speed when the film thicknesses are kept constant, due to reduced nucleation per unit area of drying film. Fluorescence emission spectra of mCherry in films prepared at <25% relative humidity shows a small shift suggesting that proteins are more perturbed at low humidity than high humidity or the solution state.

Facile Construction of Near Infrared Fluorescence Nanoprobe with Amphiphilic Protein-Polymer Bioconjugate for Targeted Cell Imaging.

A simple, straightforward, and reproducible strategy for the construction of a near-infrared (NIR) fluorescence nanoprobe was developed by coating CuInS2/ZnS quantum dots (CIS/ZnS QDs) with a novel amphiphilic bioconjugate. The amphiphilic bioconjugate with a tailor-designed structure of bovine serum albumin (BSA) as the hydrophilic segment and poly(ε-caprolactone) (PCL) as the hydrophobic part was fabricated by chemical coupling the hydrophobic polymer chain to BSA via the maleimide-sulfhydryl reaction. By incorporating CIS/ZnS QDs into the hydrophobic cores of the self-assembly of BSA-PCL conjugate, the constructed NIR fluorescence nanoprobe exhibited excellent fluorescent properties over a wide pH range (pH 3-10) and a good colloidal stability in PBS buffer (pH = 7.4) with or without 10% fetal bovine serum. The presence of the outer BSA shell effectively reduced the nonspecific cellular binding and imparted high biocompatibility and low-toxicity to the probe. Moreover, the NIR fluorescence nanoprobe could be functionalized by conjugating cyclic Arg-Gly-Asp (cRGD) peptide, and the decorated nanoprobe was shown to be highly selective for targeted integrin αvβ3-overexpressed tumor cell imaging. The feasibility of the constructed NIR fluorescence probe in vivo application was further investigated and the results demonstrated its great potential for in vivo imaging. This developed protocol for phase transfer of the CIS/ZnS QDs was universal and applicable to other nanoparticles stabilized with hydrophobic ligands.

Self-Assembly of Temperature-Responsive Protein-Polymer Bioconjugates.

We report a simple temperature-responsive bioconjugate system comprising superfolder green fluorescent protein (sfGFP) decorated with poly[(oligo ethylene glycol) methyl ether methacrylate] (PEGMA) polymers. We used amber suppression to site-specifically incorporate the non-canonical azide-functional amino acid p-azidophenylalanine (pAzF) into sfGFP at different positions. The azide moiety on modified sfGFP was then coupled using copper-catalyzed “click” chemistry with the alkyne terminus of a PEGMA synthesized by reversible addition–fragmentation chain transfer (RAFT) polymerization. The protein in the resulting bioconjugate was found to remain functionally active (i.e., fluorescent) after conjugation. Turbidity measurements revealed that the point of attachment of the polymer onto the protein scaffold has an impact on the thermoresponsive behavior of the resultant bioconjugate. Furthermore, small-angle X-ray scattering analysis showed the wrapping of the polymer around the protein in a temperature-dependent fashion. Our work demonstrates that standard genetic manipulation combined with an expanded genetic code provides an easy way to construct functional hybrid biomaterials where the location of the conjugation site on the protein plays an important role in determining material properties. We anticipate that our approach could be generalized for the synthesis of complex functional materials with precisely defined domain orientation, connectivity, and composition.

Polymersomes prepared from thermoresponsive fluorescent protein-polymer bioconjugates: capture of and report on drug and protein payloads.

Polymersomes provide a good platform for targeted drug delivery and the creation of complex (bio)catalytically active systems for research in synthetic biology. To realize these applications requires both spatial control over the encapsulation components in these polymersomes and a means to report where the components are in the polymersomes. To address these twin challenges, we synthesized the protein-polymer bioconjugate PNIPAM-b-amilFP497 composed of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and a green-fluorescent protein variant (amilFP497). Above 37 °C, this bioconjugate forms polymersomes that can (co-)encapsulate the fluorescent drug doxorubicin and the fluorescent light-harvesting protein phycoerythrin 545 (PE545). Using fluorescence lifetime imaging microscopy and Förster resonance energy transfer (FLIM-FRET), we can distinguish the co-encapsulated PE545 protein inside the polymersome membrane while doxorubicin is found both in the polymersome core and membrane.

Polymersomes Prepared from Thermoresponsive Fluorescent Protein–Polymer Bioconjugates: Capture of and Report on Drug and Protein Payloads

AbstractPolymersomes provide a good platform for targeted drug delivery and the creation of complex (bio)catalytically active systems for research in synthetic biology. To realize these applications requires both spatial control over the encapsulation components in these polymersomes and a means to report where the components are in the polymersomes. To address these twin challenges, we synthesized the protein–polymer bioconjugate PNIPAM‐b‐amilFP497 composed of thermoresponsive poly(N‐isopropylacrylamide) (PNIPAM) and a green‐fluorescent protein variant (amilFP497). Above 37 °C, this bioconjugate forms polymersomes that can (co‐)encapsulate the fluorescent drug doxorubicin and the fluorescent light‐harvesting protein phycoerythrin 545 (PE545). Using fluorescence lifetime imaging microscopy and Förster resonance energy transfer (FLIM‐FRET), we can distinguish the co‐encapsulated PE545 protein inside the polymersome membrane while doxorubicin is found both in the polymersome core and membrane.

Aqueous photoinduced living/controlled polymerization: tailoring for bioconjugation

We report a photoinduced living polymerization technique able to polymerize a large range of monomers, including methacrylates, acrylates and acrylamides, in water and biological media as well as organic solvents. This polymerization technique employs ultra-low concentrations of a ruthenium-based photoredox catalyst (typically 1 ppm to monomers) and enables low energy visible LED light to afford well-defined polymers with narrow polydispersities (Mw/Mn < 1.3). In this paper, different parameters, including photocatalyst concentrations and solvent effects, were thoroughly investigated. In addition, successful polymerizations in biological media have been reported with good control of the molecular weights and molecular weight distributions (Mw/Mn < 1.4). Finally, protein–polymer bioconjugates using a “grafting from” approach were demonstrated using bovine serum albumin as a model biomacromolecule. The enzymatic bioactivity of the protein was demonstrated to be maintained using a standard assay.

Well-Defined Aminooxy Terminated N-(2-Hydroxypropyl) Methacrylamide Macromers for Site Specific Bioconjugation of Glycoproteins

Syntheses and characterization of aminooxy terminated polymers of N-(2-hydroxyproyl) methacrylamide (HPMA) of controlled molecular weight and narrow molecular weight distribution are presented here. Design of a chain transfer agent (CTA) containing N-tert-butoxycarbonyl (t-Boc) protected aminooxy group enabled us to use reversible addition-fragmentation (RAFT) polymerization technique to polymerize the HPMA monomer. An amide bond was utilized to link the aminooxy group and the CTA through a triethylene glycol spacer. As a result, the aminooxy group is linked to the poly(HPMA) backbone through a hydrolytically stable amide bond. By varying the monomer to initiator ratios, polymers with targeted molecular weights were obtained. The molecular weights of the polymers were determined by gel permeation chromatography (GPC) and mass spectrometry (ESI and MALDI-TOF). The t-Boc protecting group was quantitatively removed to generate aminooxy terminated poly(HPMA) macromers. These macromers were converted to rhodamine B terminated poly(HPMA) by reacting N-hydroxysuccinimide (NHS) ester of the dye with the terminal aminooxy group to form a stable alkoxyamide bond. Utility of these dye-labeled polymers as molecular probes was evaluated by fluorescence microscopy by studying their intracellular uptake by renal epithelial cells. These aminooxy terminated poly(HPMA) were also tested as biocompatible carriers to prepare chemoselective bioconjugates of proteins using transferrin (Tf) as the protein. Oxidation of the sialic acid side chains of Tf generated aldehyde functionalized protein that was reacted with aminooxy terminated poly(HPMA), which resulted in protein-polymer bioconjugates carrying oxime linkages. These bioconjugates were characterized by gel electrophoresis and MALDI-TOF mass spectrometry.

Advances in polymer and polymeric nanostructures for protein conjugation

Linear polymers have been considered the best molecular structures for the formation of efficient protein conjugates due to their biological advantages, synthetic convenience and ease of functionalization. In recent years, much attention has been dedicated to develop synthetic strategies that produce the most control over protein conjugation utilizing linear polymers as scaffolds. As a result, different conjugate models, such as semitelechelic, homotelechelic, heterotelechelic and branched or star polymer conjugates, have been obtained that take advantage of these well-controlled synthetic strategies. Development of protein conjugates using nanostructures and the formation of said nanostructures from protein–polymer bioconjugates are other areas in the protein bioconjugation field. Although several polymer–protein technologies have been developed from these discoveries, few review articles have focused on the design and function of these polymers and nanostructures. This review will highlight some recent advances in protein-linear polymer technologies that employ protein covalent conjugation and successful protein-nanostructure bioconjugates (covalent conjugation as well) that have shown great potential for biological applications.

Recent developments in polymer–block–polypeptide and protein–polymer bioconjugate hybrid materials

In the last few years, polymer bioconjugates have been shown to be useful in many emerging areas of materials science. Consequently, the synthesis of polymer bioconjugates has suddenly become a central topic in polymer chemistry. The versatility and robust nature of modern synthetic methods such as controlled radical polymerisation (CLRP),1Abbreviations: AGET, activators generated by electron transfer; AM, amine mechanism; AMM, activated monomer mechanism; ATRP, atom-transfer radical polymerisation; BLG, β-benzyl-L-glutamate; Boc, butylcarbonate; BSA, bovine serum albumin; CLRP, controlled radical polymerisation; CTAs, chain-transfer agents; DCC, dicyclohexylcarbodiimide; DLS, dynamic light scattering; DMAP, dimethylaminopyridine; DSC, differential scanning calorimetry; DMAEMA, dimethylaminoethyl methacrylate; FT-IR, Fourier-transform infrared; GFP, green fluorescent protein; GPC, gel permeation chromatography; HMA, hostasol methacrylate; His, histidine; HO−EbiB, 2-hydroxyethyl 2-bromoisobutyrate; HO−POEOMA, hydroxy-functionalised poly(oligo(ethylene oxide) monomethyl ether methacrylate); HRP, horseradish peroxidase; LCST, lower critical solution temperature; MePEG α-SPA, monomethoxy poly(ethylene glycol)-succinimidyl propionate; MW, molecular weight; n-BuA, butyl acrylate; NAM, N-acryloylmorpholine; NCAs, N-carboxyanhydrides; NHS, N-hydroxysuccinimidyl; NMP, nitroxide-mediated radical polymerisation; NMR, nuclear magnetic resonance; N-TMS, N-trimethylsilyl; PAGE, polyacrylamide gel electrophoresis; PBLA, poly(β-benzyl-L-aspartate); PBLG, poly(γ-benzyl-L-glutamate); PBS, phosphate-buffered saline; PDI, polydispersity index; PDS, pyridyl disulphide; PEG, poly(ethylene glycol); PEGA, poly(ethylene glycol) methyl ether acrylate; PEGMA, poly(ethylene glycol) methylether methacrylate; p(HEMA), poly(2-hydroxyethyl methacrylate); pHPMA, poly(N-(2-hydroxypropyl)methacrylamide); PMA, poly(methyl acrylate); pNIPAM, poly(N-isopropylacrylamide); PS, polystyrene; PyBOP, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; PZLL, poly(ε-carbobenzyloxy-L-lysine); RAFT, reversible addition-fragmentation chain-transfer polymerisation; rh–GH, recombinant human growth hormone; RMA, rhodamine methacrylate; ROP, ring-opening polymerisation; sCT, salmon calcitonin; SDS-PAGE, sodium dodecyl sulphate PAGE; SPPS, solid-phase supported synthesis; t-BA, tert-butyl acrylate; TCEP, tris(2-carboxyethyl)phosphine; THF, tetrahydrofuran; TMB, 3,3′,5,5′-tetramethylbenzidine; VLP, virus-like particle.1 ring-opening polymerisation (ROP), and ‘click’ chemistry make them excellent tools for the preparation of tailor-made polymer bioconjugates. CLRP in combination with other techniques has been shown to be a mature technology for building tailor-made block copolymers and protein–polymer conjugates with a wide range of applications, especially in biomedical domains. This review describes the recent advances and progress in the rapidly expanding field of bioconjugation, outlining the work performed up to 2012.

Fluorescent nanohybrids: quantum dots coupled to polymer recombinant protein conjugates for the recognition of biological hazards

The present research introduces the concept of developing a novel nanohybrid system based on fluorescent quantum dots coupled to polymer–protein bioconjugates for the detection of potential biological hazards. The organic–inorganic hybrids were constructed by the chemical conjugation of carboxylic functionalized poly(vinyl alcohol) with the designed synthetic recombinant protein specific to bovine herpesvirus (rHBoV5), followed by coupling to II–VI semiconductor quantum dots (QDs). These nanostructures were characterized by UV-visible spectroscopy, Fourier transform infrared (FTIR) spectroscopy, photoluminescence (PL) spectroscopy, and transmission electron microscopy (TEM). The results clearly showed that the polymer–protein bioconjugates (PVA–COOH/rHBoV5) were synthesized via the proposed zero-length linker route. Moreover, these bioconjugates proved to be successful capping agents for producing CdS and CdSe quantum dots using aqueous colloidal chemistry. The TEM images associated with the optical absorption results indicated the formation of nanocrystals with estimated diameters in the range of 4.0–5.0 nm. The “blue-shift”, in the visible absorption spectra, and the PL values present strong evidence that the CdS and CdSe QDs behaved as fluorophores in the quantum-size confinement regime. Finally, the hybrid system was validated by immunochemical assay of bovine herpesvirus utilized as the model for detecting biological hazards. This research opens a window of opportunity of using a new class of hybrid nanomaterials for the rapid detection of potentially threatening biological species.

Construction of Polymer–Protein Bioconjugates with Varying Chain Topologies: Polymer Molecular Weight and Steric Hindrance Effects

We report on the fabrication of well-defined polymer-protein bioconjugates with varying chain architectures, including star polymers, star block copolymers, and heteroarm star copolymers through the specific noncovalent interaction between avidin and biotinylated synthetic polymer precursors. Homopolymer and diblock precursors site-specifically labeled with a single biotin moiety at the chain terminal, chain middle, or diblock junction point were synthesized by a combination of atom-transfer radical polymerization (ATRP) and click reactions. By taking advantage of molecular recognition between avidin and biotin moieties, supramolecular star polymers, star block copolymers, and heteroarm star copolymers were successfully fabricated. This specific binding process was also assessed by using the diffraction optic technology (DOT) technique. We further investigated the effects of polymer molecular weights, location of biotin functionality within the polymer chain, and polymer chain conformations, that is, steric hindrance effects, on the binding numbers of biotinylated polymer chains per avidin within the polymer-protein bioconjugates, which were determined by the standard avidin/2-(4-hydroxyazobenzene)benzoic acid (HABA) assay. The binding numbers vary in the range of 1.9-3.3, depending on the molecular weights, locations of biotin functionality within synthetic polymer precursors, and polymer chain conformations.

Fabrication of a Thermoresponsive Biohybrid Double Hydrophilic Block Copolymer by a Cofactor Reconstitution Approach

The fabrication of a thermoresponsive biohybrid double hydrophilic block copolymer (DHBC) by a cofactor reconstitution approach is reported. Poly(N-isopropylacrylamide) (PNIPAM) bearing a porphyrin moiety at the chain terminal, PPIXZn-PNIPAM, is synthesized by the combination of ATRP and a click reaction. The subsequent cofactor reconstitution process between apomyoglobin and PPIXZn-PNIPAM affords well-defined myoglobin-b-PNIPAM protein-polymer bioconjugates. Behaving as typical responsive DHBCs, the obtained myoglobin-b-PNIPAM biohybrid diblock copolymer exhibits thermo-induced aggregation behavior in aqueous solution as a result of the presence of the thermoresponsive PNIPAM block, as revealed by temperature-dependent transmittance, dynamic laser light scattering measurements, transmission electron microscopy, and scanning electron microscopy. This work represents the first report of the preparation of responsive biohybrid DHBCs by the cofactor reconstitution process.