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.

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.

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.

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.

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.

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.

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.

Co-assembly of Patchy Polymeric Micelles and Protein Molecules.

The development in the synthesis and self-assembly of patchy nanoparticles has resulted in the creation of complex hierarchical structures. Co-assembly of polymeric nanoparticles and protein molecules combines the advantages of polymeric materials and biomolecules, and will produce new functional materials. Co-assembly of positively charged patchy micelles and negatively charged bovine serum albumin (BSA) molecules is investigated. The patchy micelles, which were synthesized using block copolymer brushes as templates, leads to co-assembly with protein molecules into vesicular structures. The average size of the assembled structures can be controlled by the molar ratio of BSA to patchy micelles. The assembled structures are dissociated in the presence of trypsin. The protein-polymer hybrid vesicles could find potential applications in medicine.

Surfactant Behavior of Amphiphilic Polymer-Tethered Nanoparticles.

In recent years, an emerging research area has been the surfactant behavior of polymer-tethered nanoparticles. In this feature article, we have provided a general introduction to the synthesis, self-assembly, and interfacial activity of polymer-tethered inorganic nanoparticles, polymer-tethered organic nanoparticles, and polymer-tethered natural nanoparticles. In addition, applications of the polymer-tethered nanoparticles in colloidal and materials science are briefly reviewed. All research demonstrates that amphiphilic polymer-tethered nanoparticles exhibit surfactant behavior and can be used as elemental building blocks for the fabrication of advanced structures by the self-assembly approach. The polymer-tethered nanoparticles provide new opportunities to engineer materials and biomaterials possessing specific functionality and physical properties.

Preparation of Janus Graphene Oxide (GO) Nanosheets Based on Electrostatic Assembly of GO Nanosheets and Polystyrene Microspheres.

A facile and versatile method for the synthesis of Janus graphene oxide (GO) nanosheets with different structures is reported. Based on electrostatic assembly, Janus GO nanosheets can be easily functionalized with a template polymer or be defunctionalized by altering the ionic strength. By using this approach, Janus GO nanosheets are prepared successfully with hydrophobic polystyrene chains on one side and hydrophilic poly(2-(dimethylamino)ethyl methacrylate) chains on the other side.

Pore-blocking steric mass-action model for adsorption of bioparticles.

The steric mass-action (SMA) model has been widely reported to describe the adsorption of proteins in different types of chromatographic adsorbents. Here in the present work, a pore-blocking steric mass-action model (PB-SMA) was developed for the adsorption of large-size bioparticles, which usually exhibit the unique pore-blocking characteristic on the adsorbent and thus lead to a fraction of ligands in the deep channels physically inaccessible to bioparticles adsorption, instead of being shielded due to steric hindrance by adsorbed bioparticles. This unique phenomenon was taken into account by introducing an additional parameter, Lin, which is defined as the inaccessible ligand densities in the physically blocked pore area, into the PB-SMA model. This fraction of ligand densities (Lin) will be deducted from the total ligand (Lt) for model development, thus the steric factor (σ) in the proposed PB-SMA will reflect the steric shielding effect on binding sites by adsorbed bioparticles more accurately than the conventional SMA model, which assumes that all ligands on the adsorbent have the same accessibility to the bioparticles. Based on a series of model assumptions, a PB-SMA model was firstly developed for inactivated foot-and-mouth disease virus (iFMDV) adsorption on immobilized metal affinity chromatography (IMAC) adsorbents. Model parameters for static adsorption including equilibrium constant (K), characteristic number of binding sites (n), and steric factor (σ) were determined. Compared with those derived from the conventional SMA model, the σ values derived from the PB-SMA model were dozens of times smaller and much closer to the theoretical maximum number of ligands shielded by a single adsorbed iFMDV, indicating the modified model was more accurate for bioparticles adsorption. The applicability of the PB-SMA model was further validated by the adsorption of hepatitis B surface antigen virus-like particles (HBsAg VLPs) on an ion exchange adsorbent with reasonably improved accuracy. Thus, it is considered that the PB-SMA model would be more accurate in describing the adsorption of bioparticles on different types of chromatographic adsorbents.

Interface-directed self-assembly of gold nanoparticles and fabrication of hybrid hollow capsules by interfacial cross-linking polymerization.

Amphiphilic gold nanoparticles (AuNPs) were produced at liquid-liquid interface via ligand exchange between hydrophilic AuNPs and disulfide-containing polymer chains. By using oil droplets as templates, hybrid hollow capsules with AuNPs on the surfaces were obtained after interfacial cross-linking polymerization. The volume ratio of toluene to water exerts an important effect on the size of capsules. The average size of the capsules increases with the volume ratio. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM) were used to characterize the hollow structures. In this research, not only one-component but also multicomponent hollow capsules were prepared by copolymerization of acrylamide and hybrid AuNPs at liquid-liquid interface. Because of the improvement in hydrophilicity of the hollow capsules, the average size of multicomponent capsules is bigger than one-component ones in aqueous solution.

Self-assembly of gold nanoparticles and polystyrene: a highly versatile approach to the preparation of colloidal particles with polystyrene cores and gold nanoparticle coronae.

Colloidal particles with polystyrene (PS) cores and gold nanoparticle (AuNP) coronae were prepared on the basis of the self-assembly of AuNP's and PS. Citrate-stabilized AuNP's were dispersed in aqueous solution, and PS with thiol terminal groups (PS-SH) was dissolved in toluene. A stable emulsion was obtained by mixing the two solutions. Optical microscope images indicate that after grafting of PS-SH to the citrate-stabilized AuNP's at liquid-liquid interface, the interfacial tension is reduced and the average size of toluene droplets in the emulsion decreases. Transmission electron microscope (TEM) results also prove the grafting of PS-SH to AuNP's and the location of the hybrid nanoparticles at the liquid-liquid interface. Colloidal particles with PS cores and AuNP coronae were prepared by adding the emulsion to excess methanol. The weight ratio of PS-SH to AuNP exerts a significant effect on the size of colloidal particles. TEM and dynamic light scattering results both indicate that the size of colloidal particles increases with the weight ratio. The application of the core-shell-structured colloidal particles to protein separation was also investigated in this research. Colloidal particles with PS-coated magnetic nanoparticles in the cores were also prepared by this strategy.

Copolymers of styrene and gold nanoparticles.

The use of stoichiometrically functionalized gold nanoparticles (AuNPs) as building units in polymerization reaction is described; the obtained copolymers, comprised of AuNPs and polystyrene, behave differently in various solvents, micellar structures with PS cores and AuNPs corona are obtained in water.

Silica particles with immobilized protein molecules and polymer brushes.

In this research thermo-responsive polymer brushes and protein molecules are immobilized on the surfaces of silica particles by covalent bonds. Pyridyl disulfide functionalized silica particles are prepared by surface chemical reactions, and thiol-terminated poly(oligo(ethylene glycol) monomethyl ether methacrylate) (POEGMA) and bovine serum albumin (BSA) molecules are grafted to the silica particles by thiol-disulfide exchange reactions. X-ray photoelectron spectroscopy, thermogravimetric analysis, dynamic light scattering, confocal laser scanning microscopy, far-UV circular dichroism and transmission electron microscopy are employed to characterize the polymer/protein mixed layers on silica particles. The POEGMA brushes not only protect the protein molecules but also improve the dispersibility of the hybrid particles in aqueous solution. The activity of the immobilized BSA protein can be controlled by the thermo-responsive POEGMA brushes. At a temperature below the lower critical solution temperature (LCST) of POEGMA, BSA activity is not affected by polymer brushes; however, BSA activity decreases significantly at a temperature above the LCST of POEGMA. STATEMENT OF SIGNIFICANCE: In this research, both protein molecules and polymer brushes were anchored to the silica particles by highly efficient thiol-disulfide exchange reaction, and their grafting density can easily be determined by UV-vis. Owing to the temperature-sensitive nature of the grafted polymer brushes, the protein molecules can be protected by the collapsed polymer brushes above the LCST, and their catalytic activity can be controlled. Moreover, the protein molecules on silica particles can be easily separated from the solution and can be reused.

Enzyme-polymer hybrid nanogels fabricated by thiol-disulfide exchange reaction.

In this paper a novel method for the fabrication of hybrid nanogels based on thiol-disulfide exchange reaction is reported. Poly(oligo(ethylene glycol) monomethyl ether methacrylate-co-di(ethylene glycol) methyl ether methacrylate-co-2-(2-pyridyldisulfide) ethyl methacrylate) (POEGMA-co-PDEGMA-co-PDSMA) was synthesized by reversible addition-fragmentation chain transfer polymerization. Pyridyl disulfide functionalized porcine pancreatic lipase (PPL-S-S-Py) was prepared by treatment of PPL with Traut's reagent (2-iminothiolane) and 2,2'-dithiodipyridine. Upon addition of meso-2,3-dimercaptosuccinic acid into aqueous solutions of PPL-S-S-Py and POEGMA-co-PDEGMA-co-PDSMA, enzyme-polymer hybrid nanogels were prepared. The hybrid nanogels show thermal responsiveness. With an increase in the content of PPL in the nanogels, the lower critical solution temperature (LCST) shifts to the higher temperature. At a temperature below LCST, PPL molecules are in the shells of the nanogels, and at a temperature above LCST, PPL molecules are embedded inside the nanosized structures. The immobilized PPL show enhanced heat resistance and good reusability.

Thermoresponsive nanohydrogels cross-linked by gold nanoparticles.

Thermoresponsive nanohydrogels cross-linked by gold nanoparticles (AuNPs) were prepared by 1,3-dipolar cycloaddition reactions and in situ reversible addition-fragmentation chain-transfer (RAFT) polymerization. In order to synthesize thermoresponsive nanohydrogels, AuNPs decorated with azide groups (AuNPs-N(3)) were prepared through ligand exchange. Click reactions between AuNPs-N(3) and dialkynetrithiocarbonate yielded cross-linked AuNP aggregates. The size and cross-linking density of AuNP aggregates increased with the molar ratio of acetylene groups to azide groups. After click reactions, the absorption maximum of the plasmon band of AuNPs red-shifted to a long wavelength. Thermoresponsive nanohydrogels were prepared by in situ RAFT polymerization of N-isopropylacrylamide (NIPAM) using trithiocarbonate in the cross-linked AuNP aggregates as chain-transfer agents. The thermoresponsive nanohydrogels presented a low critical solution temperature at around 32 degrees C due to the "coil-to-globule" transition of connecting PNIPAM chains in the nanohydrogels. The size of the thermoresponsive nanohydrogels was determined by the molar ratio of acetylene groups to azide groups.

Bioconjugation of biotin to the interfaces of polymeric micelles via in situ click chemistry.

Azido-containing amphiphilic triblock copolymer poly(ethylene glycol)-b-poly(azidoethyl methacrylate)-b-poly(methyl methacrylate) (PEG-b-PAzEMA-b-PMMA) was prepared by postpolymerization functionalization of poly(ethylene glycol)-b-poly(hydroxyethyl methacrylate)-b-poly(methyl methacrylate) (PEG-b-PHEMA-b-PMMA). In aqueous media, PEG-b-PAzEMA-b-PMMA self-assembled into spherical micelles with the azide groups at the hydrophobic/hydrophilic interface due to the molecular architecture. Biotin was conjugated to the micelles by in situ click chemistry between azide groups and alkynated biotin, resulting in the formation of a functional interface between the hydrophilic shell and the hydrophobic core. The bioavailability of biotin to avidin was demonstrated by an avidin/4'-hydroxyazobenzene-2-carboxylic acid (avidin/HABA) assay, transmission electron microscopy, and dynamic light scattering investigations.