Cooperative Macromolecular Self-Assembly toward Polymeric Assemblies with Multiple and Bioactive Functions

ConspectusIn the past decades, polymer based nanoscale polymeric assemblies have attracted continuous interest due to their potential applications in many fields, such as nanomedicine. Many efforts have been dedicated to tailoring the three-dimensional architecture and the placement of functional groups at well-defined positions within the polymeric assemblies, aiming to augment their function. To achieve such goals, in one way, novel polymeric building blocks can be designed by controlled living polymerization methodology and advanced chemical modifications. In contrast, by focusing on the end function, others and we have been practicing strategies of cooperative self-assembly of multiple polymeric building blocks chosen from the vast library of conventional block polymers which are easily available. The advantages of such strategies lie in the simplicity of the preparation process and versatile choice of the constituent polymers in terms of their chemical structure and functionality as well as the fact that cooperative self-assembly based on supramolecular interactions offers elegant and energy-efficient bottom-up strategies. Combination of these principles has been exploited to optimize the architecture of polymeric assemblies with improved function, to impart new functionality into micelles and to realize polymeric nanocomplexes exhibiting functional integration, similar to some natural systems like artificial viruses, molecular chaperones, multiple enzyme systems, and so forth.In this Account, we shall first summarize several straightforward designing principles with which cooperative assembly of multiple polymeric building blocks can be implemented, aiming to construct polymeric nanoassemblies with hierarchal structure and enhanced functionalities. Next, examples will be discussed to demonstrate the possibility to create multifunctional nanoparticles by combination of the designing principles and judiciously choosing of the building blocks. We focus on multifunctional nanoparticles which can partially address challenges widely existing in nanomedicine such as long blood circulation, efficient cellular uptake, and controllable release of payloads. Finally, bioactive polymeric assemblies, which have certain functions closely mimicking those of some natural systems, will be used to conceive the concept of functional integration

Stabilization of Multimeric Enzymes against Heat Inactivation by Chitosan-<i>graft</i>-poly(<i>N</i>‑isopropylacrylamide) in Confined Spaces

The inactivation of multimeric enzymes is a more complicated process compared with that of monomeric enzymes. Stabilization of multimeric enzymes is regarded as a challenge with practical values in enzyme technology. Temperature-sensitive copolymer chitosan-<i>graft</i>- poly­(<i>N</i>-isopropylacrylamide) was synthesized and encapsulated with multimeric enzymes in the confined spaces constructed by the W/O microemulsion. In this way, the quaternary structures of multimeric enzymes are stabilized and the thermal stabilities of them are enhanced. The whole process was studied and discussed. This method, which works well for both glucose oxidase and catalase, can be developed as a general protection strategy for multimeric enzymes

Artificial Peroxidase/Oxidase Multiple Enzyme System Based on Supramolecular Hydrogel and Its Application as a Biocatalyst for Cascade Reactions

Inspired by delicate structures and multiple functions of natural multiple enzyme architectures such as peroxisomes, we constructed an artificial multiple enzyme system by coencapsulation of glucose oxidases (GOx) and artificial peroxidases in a supramolecular hydrogel. The artificial peroxidase was a functional complex micelle, which was prepared by the self-assembly of diblock copolymer and hemin. Compared with catalase or horseradish peroxidase (HRP), the functional micelle exhibited comparable activity and better stability, which provided more advantages in constructing a multienzyme with a proper oxidase. The hydrogel containing the two catalytic centers was further used as a catalyst for green oxidation of glucose, which was a typical cascade reaction. Glucose was oxidized by oxygen (O₂) via the GOx-mediated reaction, producing toxic intermediate hydrogen peroxide (H₂O₂). The produced H₂O₂ further oxidized peroxidase substrates catalyzed by hemin-micelles. By regulating the diffusion modes of the enzymes and substrates, the artificial multienzyme based on hydrogel could successfully activate the cascade reaction, which the soluble enzyme mixture could not achieve. The hydrogel, just like a protective covering, protected oxidases and micelles from inactivation via toxic intermediates and environmental changes. The artificial multienzyme could efficiently achieve the oxidation task along with effectively eliminating the toxic intermediates. In this way, this system possesses great potentials for glucose detection and green oxidation of a series of substrates related to biological processes

Pure Anisotropic Hydrogel with an Inherent Chiral Internal Structure Based on the Chiral Nematic Liquid Crystal Phase of Rodlike Viruses

Imparting ordered structures into otherwise amorphous hydrogels is expected to endow these popular materials with novel multiple-stimuli responsiveness that promises many applications. The current contribution reports a method to fabricate pure polymeric hydrogels with an inherent chiral internal structure by templating on the chiral nematic liquid crystal phase of a rodlike virus. A method was developed to form macroscopically homogeneous chiral templates by confinement induced self-assembly in the presence of monomers, cross-linkers and initiators. Polymerization induced gelation was performed without perturbing the elegant 3D chiral organization of the rodlike virus bearing double bonds. Furthermore, a suitable method was found to remove the organic virus template while keeping the desired polymeric replica intact, resulting in a pure polymeric hydrogel with a unique internal chiral feature that originates from the 3D chiral ordering of the cylindrical pores left by the virus. Multiple-stimuli responsiveness has been demonstrated and can be quantified by the change of the pitch of the chiral feature. The chiral structure endows the otherwise featureless hydrogel with a unique material property that might be used as a readout signal for sensing and acts as the basis for responsive, biomimetic nanostructured materials

Pure Anisotropic Hydrogel with an Inherent Chiral Internal Structure Based on the Chiral Nematic Liquid Crystal Phase of Rodlike Viruses

Imparting ordered structures into otherwise amorphous hydrogels is expected to endow these popular materials with novel multiple-stimuli responsiveness that promises many applications. The current contribution reports a method to fabricate pure polymeric hydrogels with an inherent chiral internal structure by templating on the chiral nematic liquid crystal phase of a rodlike virus. A method was developed to form macroscopically homogeneous chiral templates by confinement induced self-assembly in the presence of monomers, cross-linkers and initiators. Polymerization induced gelation was performed without perturbing the elegant 3D chiral organization of the rodlike virus bearing double bonds. Furthermore, a suitable method was found to remove the organic virus template while keeping the desired polymeric replica intact, resulting in a pure polymeric hydrogel with a unique internal chiral feature that originates from the 3D chiral ordering of the cylindrical pores left by the virus. Multiple-stimuli responsiveness has been demonstrated and can be quantified by the change of the pitch of the chiral feature. The chiral structure endows the otherwise featureless hydrogel with a unique material property that might be used as a readout signal for sensing and acts as the basis for responsive, biomimetic nanostructured materials

Reversible Interactions of Proteins with Mixed Shell Polymeric Micelles: Tuning the Surface Hydrophobic/Hydrophilic Balance toward Efficient Artificial Chaperones

Molecular chaperones can elegantly fine-tune its hydrophobic/hydrophilic balance to assist a broad spectrum of nascent polypeptide chains to fold properly. Such precious property is difficult to be achieved by chaperone mimicking materials due to limited control of their surface characteristics that dictate interactions with unfolded protein intermediates. Mixed shell polymeric micelles (MSPMs), which consist of two kinds of dissimilar polymeric chains in the micellar shell, offer a convenient way to fine-tune surface properties of polymeric nanoparticles. In the current work, we have fabricated ca. 30 kinds of MSPMs with finely tunable hydrophilic/hydrophobic surface properties. We investigated the respective roles of thermosensitive and hydrophilic polymeric chains in the thermodenaturation protection of proteins down to the molecular structure. Although the three kinds of thermosensitive polymers investigated herein can form collapsed hydrophobic domains on the micellar surface, we found distinct capability to capture and release unfolded protein intermediates, due to their respective affinity for proteins. Meanwhile, in terms of the hydrophilic polymeric chains in the micellar shell, poly­(ethylene glycol) (PEG) excels in assisting unfolded protein intermediates to refold properly via interacting with the refolding intermediates, resulting in enhanced chaperone efficiency. However, another hydrophilic polymer-poly­(2-methacryloyloxyethyl phosphorylcholine) (PMPC) severely deteriorates the chaperone efficiency of MSPMs, due to its protein-resistant properties. Judicious combination of thermosensitive and hydrophilic chains in the micellar shell lead to MSPM-based artificial chaperones with optimal efficacy

Aggregation Behavior of the Template-Removed 5,10,15,20-Tetrakis(4-sulfonatophenyl)porphyrin Chiral Array Directed by Poly(ethylene glycol)-<i>block</i>-poly(l‑lysine)

Complexation between 5,10,15,20-tetrakis­(4-sulfonatophenyl)­porphyrin (TPPS) and poly­(ethylene glycol)-<i>block-</i>poly­(l-lysine) (PEG-<i>b</i>-PLL) was performed via electrostatic interaction. Two kinds of primary arrays of TPPS with different supramolecular chirality induced by PLL were obtained in the resultant complex by inverting the mixing procedure of the two components. These arrays could be displaced by poly­(sodium-<i>p</i>-styrenesulfonate) (PSS) from the chiral PLL template through competitive electrostatic complexation, and then PSS formed a polyion complex micelle with PEG-<i>b</i>-PLL. The template-removed TPPS arrays preserved their induced chirality and served as primary subunits for the secondary aggregation of TPPS. The morphology of the secondary aggregates was strongly dependent upon the asymmetric primary supramolecular arrangement of TPPS. The rodlike nanostructure that was ∼200 nm in length was composed of the primary arrays that showed opposite exciton chirality between the J- and H-bands. In contrast, the micrometer-sized fibrils observed were composed of the arrays with the same exciton chirality at the J- and H-bands

Controlled Release of Ionic Drugs from Complex Micelles with Charged Channels

Oral administration of ionic drugs generally encounters with significant fluctuation in plasma concentration due to the large variation of pH value in the gastrointestinal tract and the pH-dependent solubility of ionic drugs. Polymeric complex micelles with charged channels on the surface provided us with an effective way to reduce the difference in the drug release rate upon change in pH value. The complex micelles were prepared by self-assembly of PCL-<i>b</i>-PAsp and PCL-<i>b</i>-PNIPAM in water at room temperature with PCL as the core and PAsp/PNIPAM as the mixed shell. With an increase in temperature, PNIPAM collapsed and enclosed the PCL core, while PAsp penetrated through the PNIPAM shell, leading to the formation of negatively charged PAsp channels on the micelle surface. Release behavior of ionic drugs from the complex micelles was remarkably different from that of usual core–shell micelles where diffusion and solubility of drugs played a key role. Specifically, it was mainly dependent on the conformation of the PAsp chains and the electrostatic interaction between PAsp and drugs, which could partially counteract the influence of pH-dependent diffusion and solubility of drugs. As a result, the variation of drug release rate with pH value was suppressed, which was favorable for acquiring relatively steady plasma drug concentration

Synthetic Nanochaperones Facilitate Refolding of Denatured Proteins

The folding process of a protein is inherently error-prone, owing to the large number of possible conformations that a protein chain can adopt. Partially folded or misfolded proteins typically expose hydrophobic surfaces and tend to form dysfunctional protein aggregates. Therefore, materials that can stabilize unfolded proteins and then efficiently assist them refolding to its bioactive form are of significant interest. Inspired by natural chaperonins, we have synthesized a series of polymeric nanochaperones that can facilitate the refolding of denatured proteins with a high recovery efficiency (up to 97%). Such nanochaperones possess phase-separated structure with hydrophobic microdomains on the surface. This structure allows nanochaperones to stabilize denatured proteins by binding them to the hydrophobic microdomains. We have also investigated the mechanism by which nanochaperones assist the protein refolding and established the design principles of nanochaperones in order to achieve effective recovery of a certain protein from their denatured forms. With a carefully designed composition of the microdomains according to the surface properties of the client proteins, the binding affinity between the hydrophobic microdomain and the denatured protein molecules can be tuned precisely, which enables the self-sorting of the polypeptides and the refolding of the proteins into their bioactive states. This work provides a feasible and effective strategy to recover inclusion bodies to their bioactive forms, which has potential to reduce the cost of the manufacture of recombinant proteins significantly

Effect of the Surface Charge of Artificial Chaperones on the Refolding of Thermally Denatured Lysozymes

Artificial chaperones are of great interest in fighting protein misfolding and aggregation for the protection of protein bioactivity. A comprehensive understanding of the interaction between artificial chaperones and proteins is critical for the effective utilization of these materials in biomedicine. In this work, we fabricated three kinds of artificial chaperones with different surface charges based on mixed-shell polymeric micelles (MSPMs), and investigated their protective effect for lysozymes under thermal stress. It was found that MSPMs with different surface charges showed distinct chaperone-like behavior, and the neutral MSPM with PEG shell and PMEO₂MA hydrophobic domain at high temperature is superior to the negatively and positively charged one, because of the excessive electrostatic interactions between the protein and charged MSPMs. The results may benefit to optimize this kind of artificial chaperone with enhanced properties and expand their application in the future