Hypoxia Reversion by Low-Immunogenic Ultra-Acid-Sensitive Comicelles of Protein-Polymer Conjugates Sensitizes Tumors to Photodynamic Therapy.

Hypoxia is characteristic of the tumor microenvironment, which is correlated with resistance to photodynamic therapy (PDT), radiotherapy, chemotherapy, and immunotherapy. Catalase is potentially useful to catalyze the conversion of endogenous H2O2 to O2 for hypoxia reversion. However, the efficient delivery of catalase into the hypoxia regions of tumors is a huge challenge. Here, we report the self-assembly of ultra-acid-sensitive polymer conjugates of catalase and albumin into nanomicelles that are responsive to the acidic tumor microenvironment. The immunogenicity of catalase is mitigated by the presence of albumin, which reduces the cross-linking of catalase with B cell receptors, resulting in improved pharmacokinetics. The ultra acid sensitivity of the nanomicelles makes it possible to efficiently escape the lysosomal degradation after endocytosis and permeate into the interior of tumors to reverse hypoxia in vitro and in vivo. In mice bearing triple-negative breast cancer, the nanomicelles loaded with a photosensitizer effectively accumulate and penetrate into the whole tumors to generate a sufficient amount of O2 to reverse hypoxia, leading to enhanced efficacy of PDT without detectable side effects. These findings provide a general strategy of self-assembly to design low-immunogenic ultra-acid-sensitive comicelles of protein-polymer conjugates to reverse tumor hypoxia, which sensitizes tumors to PDT.

Hypoxia-Triggered Bioreduction of Poly(N-oxide)-Drug Conjugates Enhances Tumor Penetration and Antitumor Efficacy.

PEGylation prolongs the blood circulation time of drugs; however, it simultaneously reduces the tumor penetration of drugs due to the nonfouling function and bulky hydrodynamic volume of PEG, leading to unsatisfactory outcomes in the treatment of solid tumors. Herein, we report the in situ growth of a bioreducible polymer of poly(N-oxide) from an important protein drug of interferon alpha (IFN) to generate site-specific IFN-poly(N-oxide) conjugates with higher bioactivity than a clinically used PEGylated IFN of PEGASYS. An IFN-poly(N-oxide) conjugate is screened out to have a circulating half-life as long as 51 h, which is similar to that of PEGASYS but 96-fold greater than that of IFN. However, the conjugate greatly outperforms PEGASYS and IFN in tumor penetration and antitumor efficacy in mice bearing melanoma. This enhanced tumor penetration is ascribed to the adsorption-mediated transcytosis of the conjugate whose poly(N-oxide) is biologically reduced into poly(tertiary amine), under hypoxia, which can be further protonated in the acidic tumor microenvironment. These novel findings demonstrate that poly(N-oxide)s are not only long-circulating but also bioreducible under hypoxia and are of great promise as next-generation carriers to deliver drugs into the interior of solid tumors to enhance their antitumor efficacy.

N-Terminal Lysozyme Conjugation to a Cationic Polymer Enhances Antimicrobial Activity and Overcomes Antimicrobial Resistance.

Microbial resistance to antibiotics is one of the greatest global healthcare challenges. There is an urgent need to develop effective strategies to overcome antimicrobial resistance. We, herein, report photoinduced in situ growth of a cationic polymer from the N-terminus of lysozyme. The attachment of the cationic polymer improves the proteolytic and thermal stability of lysozyme. Notably, the conjugate can efficiently overcome lysozyme resistance in Gram-positive bacteria and antibiotics-resistance in Gram-negative bacteria, which may be ascribed to the synergistic interactions of lysozyme and the cationic polymer with the bacteria to disrupt their cell membranes. In a rat periodontitis model, the lysozyme-polymer conjugate not only greatly outperforms lysozyme in therapeutic efficacy but also is superior to minocycline hydrochloride, which is the gold standard for periodontitis therapy. These findings may provide an efficient strategy to dramatically enhance the antimicrobial activities of lysozyme and pave a way to overcome antimicrobial resistance.

Thermosensitive Polymer Conjugated Prodrug-Activating Enzyme with Enhanced Tumor Retention and Antitumor Efficacy.

Enzyme-activated prodrug therapy has emerged as an effective strategy for cancer therapy. However, the inefficient delivery of prodrug-activating enzymes into tumor tissues leads to unsatisfactory antitumor efficacy and undesirable toxicity to normal tissues. Herein, we report in situ growth of a thermosensitive polymer of poly(diethylene glycol) methyl ether methacrylate (PDEGMA) from horseradish peroxidase (HRP) to yield a HRP-PDEGMA conjugate with well-retained activity as compared to HRP. The conjugate shows a sharp phase transition behavior with a lower critical solution temperature of 23 °C. The conjugate catalyzes the conversion of non-cytotoxic indole-3-acetic acid (IAA) into cytotoxic species for killing tumor cells. Notably, the PDEGMA conjugation not only increases the stability and cellular uptake of HRP but also prolongs the tumor retention time of HRP upon intratumoral injection. As a result, in mice bearing melanoma, the conjugate inhibits the growth of melanoma much more efficiently than HRP. These results demonstrate that the thermosensitive polymer conjugation of an enzyme is an effective strategy that can enhance the antitumor efficacy of an enzyme-activated prodrug.

Polymerization-Induced Coassembly of Enzyme-Polymer Conjugates into Comicelles with Tunable and Enhanced Cascade Activity.

Living organisms utilize spatially organized enzyme complexes to carry out signal transduction and metabolic pathways in an efficient and specific way. Herein, inspired by natural enzyme complexes, we report the polymerization-induced coassembly (PICA) of enzyme-polymer conjugates into comicelles with tunable and enhanced cascade activity by using the cascade reaction implemented by glucose oxidase (GOX) and horseradish peroxidase (HRP) as a model system. Notably, the cascade activity of GOX/HRP-polymer comicelles monotonically increases with the GOX/HRP ratio. The cascade activity of GOX/HRP-polymer comicelles is up to 4.9 times higher than that of free GOX and HRP mixtures at the same GOX/HRP ratio. We further demonstrate that our system can quickly detect glucose in contrast with a commercially available glucose assay kit. These findings provide a new and general method of PICA for the controlled construction of artificial enzyme complexes with tunable and enhanced activity in enzyme cascades for advanced biomedical applications.

Precision Conjugation: An Emerging Tool for Generating Protein-Polymer Conjugates.

Protein-polymer conjugates are increasingly being applied in biomedicine because of the unique combination of the biological activity of the proteins and the multifunctionality and flexibility of the polymers. However, traditional protein-polymer conjugation techniques suffer from some unavoidable drawbacks, including nonspecificity and low efficiency. In this Minireview, we discuss a new approach based on "precision conjugation" for the construction of the next-generation protein-polymer conjugates in a more controlled, more efficient, and tailorable fashion for a broad range of advanced applications. In illustrating the concept, we highlight two general methods: site-specific in situ growth and intrinsically disordered polypeptide fusion, with a focus on the in situ, efficient, and controllable formation of protein-polymer conjugates. At the end, the challenges associated with this emerging concept are further discussed.

Polymerization Induced Self-Assembly of a Site-Specific Interferon α-Block Copolymer Conjugate into Micelles with Remarkably Enhanced Pharmacology.

Conjugating a hydrophilic and protein-resistant polymer to a protein is a widely used strategy to extend the in vivo half-life of the protein; however, the benefit of the half-life extension is usually limited by the bioactivity decrease. Herein we report a supramolecular self-assembly strategy of site-specific in situ polymerization induced self-assembly (SI-PISA) to address the dilemma. An amphiphilic block copolymer (POEGMA-PHPMA) was directly grown from the C-terminus of an important therapeutic protein interferon-α (IFN) to in situ form IFN-POEGMA-PHPMA conjugate micelles. Notably, the in vitro bioactivity of the micelles was 21.5-fold higher than that of the FDA-approved PEGylated interferon-α PEGASYS. Particularly, the in vivo half-life of the micelles (83.8 h) was 1.7- and 100-fold longer than those of PEGASYS (49.5 h) and IFN (0.8 h), respectively. In a tumor-bearing mouse model, the micelles completely suppressed tumor growth with 100% animal survival, whereas at the same dose, PEGASYS and IFN were much less effective. These findings suggest that SI-PISA is promising as a next-generation technology to remarkably enhance the pharmacological performance of therapeutic proteins with short circulation half-lives.

Site-Selective in Situ Growth-Induced Self-Assembly of Protein-Polymer Conjugates into pH-Responsive Micelles for Tumor Microenvironment Triggered Fluorescence Imaging.

Self-assembly of site-selective protein-polymer conjugates into stimuli-responsive micelles is interesting owing to their potential biomedical applications, ranging from molecular imaging to drug delivery, but remains a significant challenge. Herein we report a method of site-selective in situ growth-induced self-assembly (SIGS) to synthesize site-specific human serum albumin-poly(2-(diisopropylamino)ethyl methacrylate) (HSA-PDPA) conjugates that can in situ self-assemble into pH-responsive micelles with tunable morphologies. Indocyanine green (ICG) was selectively loaded into the core of sphere-like HSA-PDPA micelles to form pH-responsive fluorescence nanoprobes. The nanoprobes rapidly dissociated into protonated individual unimers at a transition pH of around 6.5, that is the extracellular pH of tumors, which resulted in a sharp fluorescence increase and markedly enhanced cellular uptake. In a tumor-bearing mouse model, they exhibited greatly enhanced tumor fluorescence imaging as compared to ICG alone and pH-nonresponsive nanoprobes. These findings suggest that pH-responsive and site-selective protein-polymer conjugate micelles synthesized by SIGS are promising as a new class of tumor microenvironment-responsive nanocarriers for enhanced tumor imaging and therapy.

Sortase-catalyzed initiator attachment enables high yield growth of a stealth polymer from the C terminus of a protein.

Conventional methods for synthesizing protein/peptide-polymer conjugates, as a means to improve the pharmacological properties of therapeutic biomolecules, typically have drawbacks including low yield, non-trivial separation of conjugates from reactants, and lack of site- specificity, which results in heterogeneous products with significantly compromised bioactivity. To address these limitations, the use of sortase A from Staphylococcus aureus is demonstrated to site-specifically attach an initiator solely at the C-terminus of green fluorescent protein (GFP), followed by in situ growth of a stealth polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) by atom transfer radical polymerization (ATRP). Sortase-catalyzed initiator attachment proceeds with high specificity and near-complete (≈95%) product conversion. Subsequent in situ ATRP in aqueous buffer produces 1:1 stoichiometric conjugates with >90% yield, low dispersity, and no denaturation of the protein. This approach introduces a simple and useful method for high yield synthesis of protein/peptide-polymer conjugates.

In situ growth of a PEG-like polymer from the C terminus of an intein fusion protein improves pharmacokinetics and tumor accumulation.

This paper reports a general in situ method to grow a polymer conjugate solely from the C terminus of a recombinant protein. GFP was fused at its C terminus with an intein; cleavage of the intein provided a unique thioester moiety at the C terminus of GFP that was used to install an atom transfer radical polymerization (ATRP) initiator. Subsequent in situ ATRP of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) yielded a site-specific (C-terminal) and stoichiometric conjugate with high yield and good retention of protein activity. A GFP-C-poly(OEGMA) conjugate (hydrodynamic radius (R(h)): 21 nm) showed a 15-fold increase in its blood exposure compared to the protein (R(h): 3.0 nm) after intravenous administration to mice. This conjugate also showed a 50-fold increase in tumor accumulation, 24 h after intravenous administration to tumor-bearing mice, compared to the unmodified protein. This approach for in situ C-terminal polymer modification of a recombinant protein is applicable to a large subset of recombinant protein and peptide drugs and provides a general methodology for improvement of their pharmacological profiles.

Thermo-pH-Sensitive Polymer Conjugated Glucose Oxidase for Tumor-Selective Starvation-Oxidation-Immune Therapy.

Protein drugs are increasingly used as therapeutics for the treatment of cancer. However, their inherent drawbacks, such as poor stability, low cell membrane and tissue permeability, lack of tumor selectivity, and severe side effects, limit their wide applications in cancer therapy. Herein, screening of a thermo-pH-sensitive polymer-glucose oxidase conjugate that can controllably self-assemble into nanoparticles with improved stability is reported. The size, surface charge, and bioactivity of the conjugate can be tuned by adjustment of the solution temperature and pH. The cellular uptake, intracellular hydrogen peroxide generation, and tumor cell spheroid penetration of the conjugate are greatly enhanced under the acidic tumor microenvironment, leading to increased cytotoxicity to tumor cells. Upon a single intratumoural injection, the conjugate penetrates into the whole tumor tissue but hardly diffuses into the normal tissues, resulting in the eradication of the tumors in mice without perceivable side effects. Simultaneously, the conjugate induces a robust antitumor immunity to efficiently inhibit the growth of distant tumors, especially in combination with an immune checkpoint inhibitor. These findings provide a novel and general strategy to make multifunctional protein-polymer conjugates with responsiveness to the acidic tumor microenvironment for selective tumor therapy.

Active-targeting long-acting protein-glycopolymer conjugates for selective cancer therapy.

Non-fouling polymers are effective in improving the pharmacokinetics of therapeutic proteins, but short of biological functions for tumor targeting. In contrast, glycopolymers are biologically active, but usually have poor pharmacokinetics. To address this dilemma, herein we report in situ growth of glucose- and oligo(ethylene glycol)-containing copolymers at the C-terminal site of interferon alpha, an antitumor and antivirus biological drug, to generate C-terminal interferon alpha-glycopolymer conjugates with tunable glucose contents. The in vitro activity and in vivo circulatory half-life of these conjugates were found to decrease with the increase of glucose content, which can be ascribed to complement activation by the glycopolymers. Additionally, the cancer cell endocytosis of the conjugates was observed to maximize at a critical glucose content due to the tradeoff between complement activation and glucose transporter recognition by the glycopolymers. As a result, in mice bearing ovarian cancers with overexpressed glucose transporter 1, the conjugates with optimized glucose contents were identified to possess improved cancer-targeting ability, enhanced anticancer immunity and efficacy, and increased animal survival rate. These findings provided a promising strategy for screening protein-glycopolymer conjugates with optimized glucose contents for selective cancer therapy.

In situ growth of a stoichiometric PEG-like conjugate at a protein's N-terminus with significantly improved pharmacokinetics.

The challenge in the synthesis of protein-polymer conjugates for biological applications is to synthesize a stoichiometric (typically 1:1) conjugate of the protein with a monodisperse polymer, with good retention of protein activity, significantly improved pharmacokinetics and increased bioavailability, and hence improved in vivo efficacy. Here we demonstrate, using myoglobin as an example, a general route to grow a PEG-like polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) [poly(OEGMA)], with low polydispersity and high yield, solely from the N-terminus of the protein by in situ atom transfer radical polymerization (ATRP) under aqueous conditions, to yield a site-specific (N-terminal) and stoichiometric conjugate (1:1). Notably, the myoglobin-poly(OEGMA) conjugate [hydrodynamic radius (R(h)): 13 nm] showed a 41-fold increase in its blood exposure compared to the protein (R(h): 1.7 nm) after IV administration to mice, thereby demonstrating that comb polymers that present short oligo(ethylene glycol) side chains are a class of PEG-like polymers that can significantly improve the pharmacological properties of proteins. We believe that this approach to the synthesis of N-terminal protein conjugates of poly(OEGMA) may be applicable to a large subset of protein and peptide drugs, and thereby provide a general methodology for improvement of their pharmacological profiles.

Cytokine conjugates to elastin-like polypeptides.

Cytokines are a group of pleiotropic proteins which are crucial for various biological processes and useful as therapeutics. However, they usually suffer from the poor stability, extreme short circulation half-life, difficulty in high-yield and large-scale production and side effects, which greatly restricts their applications. Over the past decades, conjugation of cytokines with elastin-like polypeptides (ELPs), a type of promising biomaterials, have showed great potential in solving these challenges due to ELP's thermal responsiveness, excellent biocompatibility and biodegradability, non-immunogenicity, and ease of design and control at the genetic level. This review presents recent progress in the design and production of a variety of ELP conjugated cytokines for extended circulation, enhanced stability, increased soluble protein expression, simplified purification, improved drug delivery, and controlled release. Notably, the unique thermoresponsive properties of cytokine-ELP conjugates make it possible to self-assemble into micelles with drastically extended circulatory half-life for targeted delivery or to in situ form drug depots for topical administration and controlled release. The challenges and issues in the emerging field are further discussed and the future directions are pointed out at the end of this review.

Pyridine-2,6-dicarboxaldehyde-Enabled N-Terminal In Situ Growth of Polymer-Interferon α Conjugates with Significantly Improved Pharmacokinetics and In Vivo Bioactivity.

Polymer-protein conjugates are a class of biohybrids with unique properties that are highly useful in biomedicine ranging from protein therapeutics to biomedical imaging; however, it remains a considerable challenge to conjugate polymers to proteins in a site-specific, mild, and efficient way to form polymer-protein conjugates with uniform structures and properties and optimal functions. Herein we report pyridine-2,6-dicarboxaldehyde (PDA)-enabled N-terminal modification of proteins with polymerization initiators for in situ growth of poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA) conjugates uniquely at the N-termini of a range of natural and recombinant proteins in a mild and efficient fashion. The formed POEGMA-protein conjugates showed highly retained in vitro bioactivity as compared with free proteins. Notably, the in vitro bioactivity of a POEGMA-interferon α (IFN) conjugate synthesized by this new chemistry is 8.1-fold higher than that of PEGASYS that is a commercially available and Food and Drug Administration (FDA) approved PEGylated IFN. The circulation half-life of the conjugate is similar to that of PEGASYS but is 46.2 times longer than that of free IFN. Consequently, the conjugate exhibits considerably improved antiviral bioactivity over free IFN and even PEGASYS in a mouse model. These results indicate that the PDA-enabled N-terminal grafting-from method is applicable to a number of proteins whose active sites are far away from the N-terminus for the synthesis of N-terminal polymer-protein conjugates with high yield, well-retained activity, and considerably improved pharmacology for biomedical applications.

Introducing Temperature-Controlled Phase Transition Elastin-like Polypeptides to Transient Electronics: Realization of Proactive Biotriggered Electronics with Local Transience.

Transient electronics have dramatically changed inner-body therapy in health care. They stand out because of their harmless dissolution in the human body with no lingering electronic trash. However, high-precision biomedical implants require programmable and serial remedy operations, and controlling the whole-device destruction is not proactive and precise. Thus, a novel biotriggered and temperature-controlled transient electronics fabrication method using elastin-like polypeptides (ELPs) as triggers is proposed. Biocompatible ELPs simply mixed with trace silver nanowire (AgNW) can serve as the "switch" for the electronics to respond to local temperature changes in deionized water, exhibiting an agile response time. A ratio gradient experiment of the ELPs and AgNW shows that more programmable and precise transience properties (initial resistance, ready time, response time, and stable resistance) can be achieved by using a designated proportion. Further, we validated that the 3D-printing-based ELP-triggering transient electronics fabrication method is very simple yet effective for preparing transient wireless charging LEDs. Transient devices comprising ELPs-AgNW and PLGA-Ag respond within 160 s below 10 °C and degrade within a certain period.

Site-selective protein modification with polymers for advanced biomedical applications.

Protein modification with polymers has led to intriguing and new types of bioconjugates. They combine the tunable physicochemical properties of the polymers with the specific biological activity of the proteins. These unique attributes of protein-polymer conjugates render them interesting and useful in biomedicine. However, the application potential of protein-polymer conjugates is limited by the mostly non-selective protein modification with polymers due to the lack of site-selective protein modification technology. Recent advances in site-selective protein modification and controlled polymerization have made it possible to modify proteins with polymers in a site-selective and controlled manner. In this review, recent advances in site-selective protein modification with polymers are depicted in five parts as follows: site-selective protein modification; site-selective polymer modification; site-selective in situ growth of polymers from proteins; biosafety of polymers; and biomedical applications. Site-selective protein-polymer conjugates are superior to non-selective ones in precise control of structures and functions, which makes them more interesting for advanced biomedical applications ranging from protein delivery to diagnostics. Particularly, important examples in this regard are highlighted in this review. Additionally, major challenges and future directions in this emerging research field are also discussed in the perspective section of this review.

In Situ Growth of Self-Assembled Protein-Polymer Nanovesicles for Enhanced Intracellular Protein Delivery.

We report a new and general method, in situ growth, for designing self-assembled protein-polymer nanovesicles for intracellular protein delivery.In situ polymerization of a water-soluble monomer from a protein attached with a polymerization initiator yields amphiphilic protein conjugates of a water-insoluble polymer. These conjugates can in situ self-assemble into nanostructures with tunable morphologies from spheres to vesicles. Interestingly, an exogenous protein can be in situ encapsulated inside protein-polymer nanovesicles for enhanced intracellular protein delivery. The in situ growth method may open up new opportunities for designing a variety of self-assembled protein-polymer nanostructures tailored to specific applications.

In situ growth of a C-terminal interferon-alpha conjugate of a phospholipid polymer that outperforms PEGASYS in cancer therapy.

Conjugating therapeutic proteins and peptides to poly(ethylene glycol) (PEG) can improve their pharmacokinetics and therapeutic potential. However, PEGylation suffers from non-specific conjugation, low yield and immunogenicity. Herein we report a new and general methodology to synthesize a protein-polymer conjugate with site-specificity, high yield and activity, long circulation half-life and excellent therapeutic efficacy. A phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), was grown solely from the C-terminus of interferon-alpha to form a site-specific (C-terminal) and stoichiometric (1:1) PMPC conjugate of interferon-alpha in high yield. Notably, the PMPC conjugate showed 194- and 158-fold increases in systemic exposure and tumor uptake as compared with interferon-alpha, respectively. The in vitro antiproliferative bioactivity of the PMPC conjugate was 8.7-fold higher than that of PEGylated interferon-alpha (PEGASYS). In a murine cancer model, the PMPC conjugate completely inhibited tumor growth and cured 75% mice, whereas at the same dose, no mice treated with interferon-alpha or PEGASYS survived. We believe that this new approach to synthesize C-terminal protein conjugates of PMPC may be applicable to a large subset of protein and peptide drugs, thereby providing a general platform for the development of next-generation protein therapeutics.

Site-specific in situ growth of an interferon-polymer conjugate that outperforms PEGASYS in cancer therapy.

Conjugating poly(ethylene glycol) (PEG), PEGylation, to therapeutic proteins is widely used as a means to improve their pharmacokinetics and therapeutic potential. One prime example is PEGylated interferon-alpha (PEGASYS). However, PEGylation usually leads to a heterogeneous mixture of positional isomers with reduced bioactivity and low yield. Herein, we report site-specific in situ growth (SIG) of a PEG-like polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), from the C-terminus of interferon-alpha to form a site-specific (C-terminal) and stoichiometric (1:1) POEGMA conjugate of interferon-alpha in high yield. The POEGMA conjugate showed significantly improved pharmacokinetics, tumor accumulation and anticancer efficacy as compared to interferon-alpha. Notably, the POEGMA conjugate possessed a 7.2-fold higher in vitro antiproliferative bioactivity than PEGASYS. More importantly, in a murine cancer model, the POEGMA conjugate completely inhibited tumor growth and eradicated tumors of 75% mice without appreciable systemic toxicity, whereas at the same dose, no mice treated with PEGASYS survived for over 58 days. The outperformance of a site-specific POEGMA conjugate prepared by SIG over PEGASYS that is the current gold standard for interferon-alpha delivery suggests that SIG is of interest for the development of next-generation protein therapeutics.