Polymer Brushes and Surface Nanostructures: Molecular Design, Precise Synthesis, and Self-Assembly.

For over two decades, polymer brushes have found wide applications in industry and scientific research. Now, polymer brush research has been a significant research focus in the community of polymer science. In this review paper, we give an introduction to the synthesis, self-assembly, and applications of one-dimensional (1D) polymer brushes on polymer backbones, two-dimensional (2D) polymer brushes on flat surfaces, and three-dimensional (3D) polymer brushes on spherical particles. Examples of the synthesis of polymer brushes on different substrates are provided. Studies on the formation of the surface nanostructures on solid surfaces are also reviewed in this article. Multicomponent polymer brushes on solid surfaces are able to self-assemble into surface micelles (s-micelles). If the s-micelles are linked to the substrates through cleavable linkages, the s-micelles can be cleaved from the substrates, and the cleaved s-micelles are able to self-assemble into hierarchical structures. The formation of the surface nanostructures by coassembly of polymer brushes and "free" polymer chains (coassembly approach) or polymerization-induced surface self-assembly approach, is discussed. The applications of the polymer brushes in colloid and biomedical science are summarized. Finally, perspectives on the development of polymer brushes are offered in this article.

Synergistic Effects of the Superhydrophilic and Superhydrophobic Components on the Anti-freezing Performances of Latex Particles and Anti-icing Properties of Latex Films.

The development of new materials for anti-freezing and anti-icing applications is a big challenge in industry and academic area. In this research, inspired by the antifreeze proteins, latex particles with superhydrophilic zwitterionic shells and superhydrophobic cores are synthesized by RAFT emulsion polymerization, and the applications of the latex particles in anti-freezing and anti-icing applications are investigated. In anti-freezing study, the critical aggregate temperature (CAT) of the latex particles decreases, and the separation of the melting and freezing temperature of ice increases with the particle concentration. Enzyme molecules can be cryopreserved in the presence of the latex particles; and their bioactivities are well maintained. Latex particles are casted into latex films with dynamic surfaces. Anti-icing performances including anti-frosting properties, freezing delay time and ice adhesion strengths, are studied; and the water-treated latex films present stronger anti-icing properties than other films, due to the synergistic effects of the superhydrophilic and superhydrophobic components. In addition, latex particles with zwitterionic shells and poly(n-butyl methacrylate) cores, and latex particles with small molecular surfactant on the surfaces are synthesized, respectively. The anti-freezing performances of the different latex particles and anti-icing properties of the latex films are compared. This research provides a facile and effective method for the synthesis of anti-freezing and anti-icing materials. This article is protected by copyright. All rights reserved.

Polymerization-Induced Proteinosome Formation Initiated by Artificial Cells.

Cellular communication is essential for living cells to coordinate the individual cellular responses and make collective behaviors. In the past decade, the communications between artificial cells have aroused great interest due to the potential applications of the structures in bioscience and biotechnology. To mimic the cellular communication, artificial cell assisted synthesis of proteinosomes was studied in this research. Multienzyme proteinosomes with glucose oxidase (GOx) and horseradish peroxidase (HRP) decorated on the membranes were synthesized by the thermally triggered self-assembly approach. Free radicals produced in a cascade reaction taking place on the surfaces of the multienzyme proteinosomes initiated reversible addition-fragmentation chain transfer (RAFT) polymerization of NIPAM at a temperature above LCST of PNIPAM in the presence of bovine serum albumin (BSA) or alcohol dehydrogenase (ADH)/acetaldehyde dehydrogenase (ALDH), and daughter proteinosomes with BSA or ADH/ALDH on the surfaces were fabricated. The structures of the GOx/HRP initiator proteinosomes, and the synthesized daughter proteinosomes were characterized with transmission electron microscopy, atomic force microscopy, fluorescence microscopy, dynamic light scattering, and micro-DSC. Enzyme activity assays demonstrate the high bioactivities of the enzymes on the surfaces of the initiator and the synthesized daughter proteinosomes.

Polymer-Protein Nanovaccine Synthesized via Reactive Self-Assembly with Potential Application in Cancer Immunotherapy: Physicochemical and Biological Characterization In Vitro and In Vivo.

Nanovaccines composed of polymeric nanocarriers and protein-based antigens have attracted much attention in recent years because of their enormous potential in the prevention and treatment of diseases such as viral infections and cancer. While surface-conjugated protein antigens are known to be more immunoactive than encapsulated antigens, current surface conjugation methods often result in low and insufficient protein loading. Herein, reactive self-assembly is used to prepare nanovaccine from poly(ε-caprolactone) (PCL) and ovalbumin (OVA)-a model antigen. A rapid thiol-disulfide exchange reaction between PCL with pendant pyridyl disulfide groups and thiolated OVA results in the formation of nanoparticles with narrow size distribution. High OVA loading (≈70-80 wt%) is achieved, and the native secondary structure of OVA is preserved. Compared to free OVA, the nanovaccine is much superior in enhancing antigen uptake by bone marrow-derived dendritic cells (BMDCs), promoting BMDC maturation and antigen presentation via the MHC I pathway, persisting at the injection site and draining lymph nodes, activating both Th1 and Th2 T cell immunity, and ultimately, resisting tumor challenge in mice. This is the first demonstration of reactive self-assembly for the construction of a polymer-protein nanovaccine with clear potential in advancing cancer immunotherapy.

Synthesis of Cleavable Polymers via Oxidation of Thioether Moieties and Thiol Click Reactions: A Click-Declick Strategy.

In the past decades click chemistries including thiol chemistries have found wide applications in the synthesis of well-defined polymers. In this research, a click-declick strategy based on the oxidation of heteroaromatic thioethers and the substitution reactions between the oxidized groups and thiols, is proposed for the synthesis of the cleavable polymers. In proof-of-concept experiments, block copolymers (BCPs) and star-like polymers are synthesized by thiol-phenylsulfone substitution reactions, and heteroaromatic thioethers are produced at the junction points of the BCP chains or on the crosslinking sites of the star-like polymer. The thioethers can be oxidized to heteroaromatic sulfoxides or sulfones, depending on the oxidization condition. It is demonstrated that both sulfoxides or sulfones can have base catalyzed nucleophilic substitution reactions with thiols, leading to the cleavage of the polymers.

Ca2+-Chelation-Induced Fabrication of Multistimuli-Responsive Charged Nanogels from Phospholipid-Polymer Conjugates and Use for Drug/Protein Loading.

Thermoresponsive phospholipid-poly(N-isopropylacrylamide) (PL-PNIPAM) conjugates were synthesized via reversible addition fragmentation chain transfer polymerization mediated by a phospholipid-modified trithiocarbonate. Temperature triggered the micellization of the PL-PNIPAM conjugate to form phosphate group-decorated micelles in the aqueous solution. Driven by the chelation of phospholipids and Ca2+, the PL-PNIPAM conjugate and Ca2+ ions formed size-tunable nanoclusters at a temperature beyond the lower critical solution temperature. To fabricate cross-linked nanogels, NIPAM was copolymerized with N-succinimidyl acrylate (NSA) to obtain the PL-P(NIPAM-co-NSA) conjugate bearing pendent cross-linkable functionalities. Subsequently, the size-controllable nanogels containing disulfide linkages were generated at 37 °C by cross-linking the PL-P(NIPAM-co-NSA)/Ca2+ nanoclusters with cystamine through modulation of Ca2+ concentrations. These negatively charged nanogels demonstrate temperature/pH/reduction triple responsiveness. The nanogels can be efficiently loaded with doxorubicin (DOX) and proteins with various isoelectric points. The DOX-loaded nanogels exhibited a temperature/pH/reduction triple-responsive release profile. The immobilized RNase A, BSA, and GOx retained the protein bioactivity. The release of RNase A-loaded nanogels possesses a temperature-responsive profile. The immobilization of Lys and cytochrome C in nanogels inhibited protein bioactivity. However, the addition of NaCl triggered the recovery of bioactivity. These multistimuli-responsive nanogels can provide a versatile platform applicable in biotechnology and drug/protein delivery.

Surface Coassembly of Binary Mixed Polymer Brushes and Linear Block Copolymer Chains.

Binary mixed polymer brushes (BMPBs) are two different homopolymer chains that are covalently anchored to the solid surfaces at high grafting densities. One feature of the BMPBs is the unique ability to make surface phase separation under external stimuli. In this research, we demonstrate that different surface nanostructures can be fabricated by surface coassembly of BMPBs and free block copolymer (BCP) chains. Polystyrene/poly(2-(dimethylamino)ethyl methacrylate) (PS/PDMAEMA) BMPBs on silica particles (PS-PDMAEMA-SiO2) are synthesized by a two-step "grafting to" approach. PDMAEMA-b-PS block copolymer (BCP) chains and PS-PDMAEMA-SiO2 make surface self-assembly and a variety of surface nanostructures are formed in methanol. The grafting densities of PS and PDMAEMA brushes, solvent, and the BCP structures all exert significant influences on the surface morphology. With an increase in PDMAEMA grafting density, the surface structures change from perforated layers, to rods, and to spherical surface micelles (s-micelles). The PS grafting density also exerts an effect on the formation of the surface nanostructures. At low PS grafting density, sparsely distributed s-micelles are produced, and at high density, densely distributed s-micelles are observed. Based on transmission electron microscopy and scanning electron microscopy results, a surface phase diagram is constructed, which provides a guide to the surface morphology control.

Polymer brush-based nanostructures: from surface self-assembly to surface co-assembly.

Surface structures play an important role in the practical applications of materials. The synthesis of polymer brushes on a solid surface has emerged as an effective tool for tuning surface properties. The fabrication of polymer brush-based surface nanostructures has greatly facilitated the development of materials with unique surface properties. In this review article, synthetic methods used in the synthesis of polymer brushes, and self-assembly approaches applied in the fabrication of surface nanostructures including self-assembly of polymer brushes, co-assembly of polymer brushes and "free" block copolymer chains, and polymerization induced surface self-assembly, are reviewed. It is demonstrated that polymer brush-based surface nanostructures, including spherical surface micelles, wormlike surface structures, layered structures and surface vesicles, can be fabricated. Meanwhile, the challenges in the synthesis and applications of the surface nanostructures are discussed. This review is expected to be helpful for understanding the principles, methods and applications of polymer brush-based surface nanostructures.

Janus Surface Micelles on Silica Particles: Synthesis and Application in Enzyme Immobilization.

In these years, synthesis and applications of Janus structures have aroused great interest for large-scale applications in chemistry and materials science. Up to now, Janus particles with different morphologies and different functionalities have been synthesized in solutions, but the synthesis of Janus particles on solid surfaces has not been touched. In this research, Janus surface micelles (JSMs) are fabricated on the surfaces of silica particles by polymerization induced surface self-assembly (PISSA) approach, and the JSMs are used for enzyme immobilization. Usually, enzyme immobilization should be able to optimize the performance of the immobilized enzymes, and an ideal immobilization system must offer protection to the immobilized enzyme with retained bioactivity. Herein, it is demonstrated that JSMs on silica particles can be used as an ideal platform for the immobilization of enzymes. To prepare JSMs, poly(2-(dimethylamino) ethyl methacrylate) macro chain transfer agent (PDMAEMA-CTA) brushes on silica particles and poly(di(ethylene glycol) methyl ether methacrylate) macro CTA (PDEGMA-CTA) are employed in reversible addition-fragmentation chain transfer dispersion polymerization of styrene. After polymerization, JSMs with polystyrene cores and PDMAEMA/PDEGMA patches on the surfaces are prepared on silica particles. After quaternization reaction, the quaternized PDMAEMA patches are used for the immobilization of enzymes. Experimental results turn out that enhanced bioactivities of the immobilized enzymes are achieved and the enzyme molecules are well protected by surface Janus structures.

Biosurfaces Fabricated by Polymerization-Induced Surface Self-Assembly.

Surface biofunctionalization provides an approach to the fabrication of surfaces with improved biological and clinical performances. Biosurfaces have found increasing applications in many areas such as sensing, cell growth, and disease detection. Efficient synthesis of biosurfaces without damages to the structures and functionalities of biomolecules is a great challenge. Polymerization-induced surface self-assembly (PISSA) provides an effective approach to the synthesis of surface nanostructures with different compositions, morphologies, and properties. In this research, application of PISSA in the fabrication of biosurfaces is investigated. Two different reversible addition-fragmentation chain transfer (RAFT) agents, RAFT chain transfer agent (CTA) on silica particles (SiO2-CTA) and CTA on bovine serum albumin (BSA-CTA), were employed in RAFT dispersion polymerization of N-isopropylacrylamide (NIPAM) in water at a temperature above the lower critical solution temperature (LCST) of poly-(isopropylacrylamide) (PNIPAM). After polymerization, PNIPAM layers with BSA on the top surfaces are fabricated on the surfaces of silica particles. Transmission electron microscopy results show that the average PNIPAM layer thickness increases with monomer conversion. Kinetics study indicates that there is a turn point on a plot of ln([M]0/[M]t) versus polymerization time. After the critical point, surface coassembly of PNIPAM brushes and BSA-PNIPAM bioconjugates is performed on the silica particles. The secondary structure and the activity of BSA immobilized on top of the PNIPAM layers are basically kept unchanged in the PISSA process. To prepare permanently immobilized protein surfaces, PNIPAM layers on silica particles are cross-linked. BSA on the top surfaces presents a reversible "on-off" switching property. At a temperature below the LCST of PNIPAM, the activity of the immobilized BSA is retained; however, the BSA activity decreases significantly at a temperature above the LCST because of the hydrophobic interaction between PNIPAM and BSA. Based on this approach, many different biosurfaces can be fabricated and the materials will find applications in many fields, such as enzyme immobilization, drug delivery, and tissue engineering.

Methionine-Based pH and Oxidation Dual-Responsive Block Copolymer: Synthesis and Fabrication of Protein Nanogels.

In this paper, we synthesized a block copolymer containing pendent thioether functionalities by reversible addition-fragmentation chain transfer polymerization of a tert-butyloxycarbonyl (Boc)-l-methionine-(2-methacryloylethyl)ester (Boc-METMA) monomer using a poly(ethylene glycol) (PEG)-based chain transfer agent. The deprotection of Boc groups resulted in an oxidation and pH dual-responsive cationic block copolymer PEG-b-P(METMA). The block copolymer PEG-b-P(METMA) possessing protonable amine groups was water-soluble at pH < 6.0 and self-assembled to form spherical micelles at pH > 6.0. In the presence of H2O2, the micelles first became highly swollen with time and completely disassembled at last, demonstrating the H2O2-responsive feature because of the oxidation of hydrophobic thioether to hydrophilic sulfoxide. The anticancer drug curcumin (Cur) was entrapped in the polymeric micelles and the Cur-loaded micelles displayed a H2O2-triggered release profile as well as a pH-dependent release behavior, making PEG-b-P(METMA) micelles promising nanocarriers for reactive oxygen species-responsive drug delivery. Taking advantage of the protonated amine groups, the cationic polyelectrolyte PEG-b-P(METMA) formed polyion complex micelles with glucose oxidase (GOx) through electrostatic interactions at pH 5.8. By cross-linking the cores of PIC micelles with glutaraldehyde, the PIC micelles were fixed to generate stable GOx nanogels under physiological conditions. The GOx nanogels were glucose-responsive and exhibited glucose-dependent H2O2-generation activity in vitro and improved storage and thermal stability of GOx. Cur can be encapsulated in the GOx nanogels, and the Cur-loaded GOx nanogels demonstrate the glucose-responsive release profile. The GOx nanogels displayed high cytotoxicity to 4T1 cells and were effectively internalized by the cells. Therefore, these GOx nanogels have potential applications in the areas of cancer starvation and oxidation therapy.

Biomimetic Mineralization of Protein Nanogels for Enzyme Protection.

Protein nanogels have found a wide variety of applications, ranging from biocatalysis to drug/protein delivery. However, in practical applications, proteins in nanogels may suffer from enzymic hydrolysis and denaturation. Inspired by the structure and functionalities of the fowl eggshells, biomimetic mineralization of protein nanogels was studied in this research. Protein nanogels with embedded porcine pancreas lipase (PPL) in the cross-linked nanostructures were synthesized through the thiol-disulfide reaction between thiol-functionalized PPL and poly(N-isopropylacrylamide) with pendant pyridyl disulfide groups. The nanogels were further reacted with reduced bovine serum albumin (BSA) and BSA molecules were coated on the nanogels. Mineralization of BSA leads to the synthesis of biomineralized shells on the nanogels. With the growth of CaCO3 on the shells, the nanogels aggregate into suprastructures. Thermogravimetric analysis, XRD, dynamic light scattering, and TEM were employed to study the mechanism of the biomineralization process and analyze the structures of the mineralized nanogels. The biomineralized shells can effectively protect the PPL molecules from hydrolysis by trypsin; meanwhile, the nanosized channels on the mineralized shells allow the transport of small-molecule substrates across the shells. Bioactivity measurements indicate that PPL in the nanogels maintains more than 80 % bioactivity after biomineralization.

Hydrophobic Interaction-Induced Coassembly of Homopolymers and Proteins.

Studies on the fabrication of polymer-protein hybrid self-assemblies have aroused great interest over the past years because of a broad range of applications of the materials in drug/protein delivery, biosensors, and enhancement of protein stability. The hybrid assemblies are usually fabricated from polymer-protein bioconjugates, which may suffer from the damages to the protein structures and the loss of functionalities in the synthesis. Herein, we report a simple and efficient approach to the fabrication of vesicle-like structures based on coassembly of homopolymer chains and protein molecules. At room temperature, poly(N-isopropylacrylamide) (PNIPAM) and bovine serum albumin (BSA) are able to form complexes through hydrophobic interactions in aqueous solution. Upon heating to a temperature above the cloud point of PNIPAM, vesicle-like structures with collapsed PNIPAM in the walls and BSA at the surfaces are formed. The size and membrane thickness of the assemblies can be tuned by the molar ratio of PNIPAM to BSA. The hydrophobic interaction between PNIPAM and BSA plays a key role in the complex formation and self-assembly process. The complexes and assembled structures are analyzed by using micro differential scanning calorimetry, light scattering, and transmission electron microscopy. BSA in the assemblies retains over 90% of its activity, and the protein stability is enhanced because of the hydrophobic interaction between proteins and polymers. This approach allows us to prepare polymer-protein assemblies without bioconjugate synthesis. Meanwhile, possible damages to the protein structures and the loss of bioactivities of proteins can be avoided.

Surface Reconstruction by a Coassembly Approach.

Materials with switchable surfaces, capable of changing surface properties under external stimuli, are playing a pivotal role in many applications, such as tissue engineering, biosensors, and drug/protein delivery. In this research silica particles with patterned and switchable surfaces are fabricated. Surface micelles on silica particles are formed by coassembly of polymer brushes and "free" block copolymer chains in a selective solvent. The cores of the surface micelles are crosslinked by anthracene photodimerization. After quaternization of the coronae, amphiphilic surface micelles are prepared. The surface micelles are able to rearrange in different media. After treatment with an organic solvent, the surfaces of silica particles are occupied by hydrophobic polymer components; in aqueous solution, the positively charged polymer chains are on the surfaces. The switching of the surface micelles results in changes in surface composition and wetting behaviors.

Amphiphilic Janus Twin Single-Chain Nanoparticles.

In these years, synthesis and self-assembly of nanoparticles with various sizes, geometries and compositions have aroused great interest due to their scientific significance and potential applications. The nanoparticles with asymmetric structures are able to self-assemble into hierarchical structures for constructing functional materials. To investigate the self-assembly behaviors of the nano-sized asymmetric polymer particles, synthesis and self-assembly of twin single-chain nanoparticles (TSCNPs), a type of covalently bonded SCNPs, are reported in this research. A block copolymer with pendant anthracene groups and bromines on two blocks were synthesized by two-step reversible addition-fragmentation chain transfer polymerization. After two-step intramolecular cross-linking reactions, anthracene photodimerization and atom transfer radical coupling, TSCNPs were prepared. The amphiphilic TSCNPs possess surfactant properties. Upon addition of the TSCNPs, the surface tension of water is reduced. In aqueous solution, the TSCNPs self-assemble into vesicles with the hydrophobic particles in the inner walls and the hydrophilic particles on the surfaces.

Bioassemblies Fabricated by Coassembly of Protein Molecules and Monotethered Single-Chain Polymeric Nanoparticles.

Molecular nanoparticles have been used as building blocks in the synthesis of functional materials. The grand challenges in the synthesis of the functional materials are precise control of the structures and functionalities of the materials by using nanoparticles with different architectures and properties. Monotethered single-chain polymeric nanoparticles (SCPN) are a type of nanosized asymmetric particles formed by intramolecular cross-linking of linear diblock copolymer chains. Monotethered SCPNs can be used as elemental building blocks for the fabrication of well-defined advanced structures. In this research, synthesis of biohybrid materials based on coassembly of bovine serum albumin (BSA) molecules and monotethered SCPNs is investigated. Due to the asymmetric structure of the SCPNs, positively charged SCPNs and negatively charged protein molecules coassemble into biohybrid vesicles with SCPNs on the layers and protein molecules in the walls. The self-assembled structures were analyzed by using dynamic light scattering, transmission electron microscopy, cryo-transmission electron microscopy, and atomic force microscopy. The average size of the biohybrid vesicles can be controlled by the molar ratio of SCPNs to BSA. The protein molecules in the biohybrid vesicles maintain most of the activities. This research paves a new way for the synthesis of functional biohybrid structures, and the materials can be used as protein carriers.

Surface Coassembly of Polymer Brushes and Polymer-Protein Bioconjugates: An Efficient Approach to the Purification of Bioconjugates under Mild Conditions.

Well-defined polymer-protein bioconjugates are widely used in therapeutics and biocatalysis. One of the challenges in the synthesis of bioconjugates is the efficient separation of the target conjugate molecules from reaction systems. In this research, surface coassembly of polymer brushes and polymer-protein bioconjugates is investigated, and it is demonstrated that the coassembly approach can be applied in the purification of polymer-protein bioconjugates. Bovine serum albumin-poly( N-isopropylacrylamide) (BSA-PNIPAM) bioconjugates were synthesized by the "grafting from" approach, and PNIPAM brushes on silica particles were prepared by the "grafting to" approach. PNIPAM brushes on silica particles are able to coassemble with BSA-PNIPAM at a temperature above the lower critical solution temperature of PNIPAM. Two-layer surface structures with collapsed PNIPAM in the inner layers and BSA in the outer layers are formed on the silica particles. The size of the silica particles and molecular weight of PNIPAM on the bioconjugates exert influences on the coassembly. The coassembly approach can be used in the purification of bioconjugates. After repeated coassembly centrifugation-release cycles, all the BSA-PNIPAM bioconjugates can be removed from the reaction solutions, and the purified bioconjugates are obtained.

Fabrication of Polymer-Protein Hybrids.

Rapid developments in organic chemistry and polymer chemistry promote the synthesis of polymer-protein hybrids with different structures and biofunctionalities. In this feature article, recent progress achieved in the synthesis of polymer-protein conjugates, protein-nanoparticle core-shell structures, and polymer-protein nanogels/hydrogels is briefly reviewed. The polymer-protein conjugates can be synthesized by the "grafting-to" or the "grafting-from" approach. In this article, different coupling reactions and polymerization methods used in the synthesis of bioconjugates are reviewed. Protein molecules can be immobilized on the surfaces of nanoparticles by covalent or noncovalent linkages. The specific interactions and chemical reactions employed in the synthesis of core-shell structures are discussed. Finally, a general introduction to the synthesis of environmentally responsive polymer-protein nanogels/hydrogels by chemical cross-linking reactions or molecular recognition is provided.

Nanoscale Proteinosomes Fabricated by Self-Assembly of a Supramolecular Protein-Polymer Conjugate.

Proteinosomes are a type of protein-based spherical capsules, which have potential applications in drug delivery, cell imaging, gene expression, and biocatalysis. In this research, a novel approach to the fabrication of proteinosomes entirely composed of protein molecules based on self-assembly of a supramolecular protein-polymer conjugate is proposed. A supramolecular protein-polymer conjugate was prepared by mixing ßCD-modified bovine serum albumin (BSA) and adamantane-terminated poly(N-isopropylamide) (Ad-PNIPAM) in aqueous solution. The BSA-PNIPAM bioconjugate self-assembled into micelles with PNIPAM cores and BSA coronae at a temperature above the lower critical solution temperature (LCST) of PNIPAM. After cross-linking of BSA in the coronae, and followed by addition of excess ßCD, PNIPAM chains were cleaved from the micellar structures, and nanoscale proteinosomes were prepared. The dual-responsive proteinosomes dissociated in the presence of trypsin or glutathione.

Covalently Connected Polymer-Protein Nanostructures Fabricated by a Reactive Self-Assembly Approach.

The synthesis of polymer-protein nanostructures opens up a new avenue for the development of new biomaterials. In this research, covalently connected polymer-protein nanostructures were fabricated through a reactive self-assembly approach. Poly(tert-butyl methacrylate-co-pyridyl disulfide methacrylamide) (PtBMA-co-PPDSMA) was synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization. Covalently connected nanostructures (CCNs) with hydrophobic polymer cores and hydrophilic protein coronae were prepared by adding solutions of PtBMA-co-PPDSMA/DMF to aqueous solutions of bovine serum albumin (BSA). The thiol-disulfide exchange reaction between pyridyl disulfide groups on the polymer chains and thiol groups on the protein molecules plays a key role in the fabrication of CCNs. The self-assembly process was investigated by dynamic light scattering (DLS) and stopped-flow techniques. DLS results indicated that the sizes of the CCNs were determined by the initial polymer concentration, the BSA concentration, and the average number of thiol groups on BSA molecules. TEM and sodium dodecyl sulfate polyacrylamide gel electrophoresis were used to analyze the nanostructures. Far-UV circular dichroism results demonstrated that the original folded conformations of BSA molecules were basically maintained in the reactive self-assembly process. Compared with native BSA, the secondary structure and conformation change of coronal BSA induced by urea or thermal treatment were remarkably suppressed. The cytotoxicity assays demonstrated that the CCNs were essentially nontoxic to Hela and COS-7 cells.