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Rational design of efficient cytosolic protein delivery carriers holds enormous promise for biotherapeutics development. Several delivery systems have been developed during the past decades, while tailoring the balance between extracellular protein binding and intracellular cargo release is still challenging. In this study, we synthesized a series of oxygen-sensitive reactive polymers, rich in boron, by radical polymerization and post-modification for cytosolic protein delivery in vitro and in vivo. The introduction of boronate building blocks into the polymer scaffold significantly enhanced its protein binding affinity, and the polymer/protein complexes with high stability were obtained by tailoring the molecular ratios between the boronate ligands and the amine groups. The lead material screened from the polymer library exhibited efficient protein delivery efficacy that can release cargo proteins in cytosol in a reactive oxygen species responsive manner, which enables intracellular delivery of proteins with maintained bioactivity. In addition, the polymer-based nanoformulations efficiently delivered saporin, a toxin protein, into osteosarcoma cells and tumor tissues, and exhibited high therapeutic efficacy in an osteosarcoma mouse model. The synthesized polymer in this study can be developed as a promising nanocarrier for cytosolic delivery of protein therapeutics to treat a variety of diseases., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Polymers are one of the most promising protein delivery carriers; however, their applications are hindered by low delivery efficacy owing to their undesirable performance in protein binding, cellular uptake and endosomal escape. Here, we designed a series of histidine-based coordinative polymers for efficient intracellular protein delivery. Coordination of metal ions such as Ni 2+ , Zn 2+ , and Cu 2+ with histidine residues on a polymer greatly improved its performance in protein binding, complex stability, cellular uptake and endosomal escape, therefore achieving highly improved protein delivery efficacy. Among the coordinative polymers, the Zn 2+ -coordinated one exhibited the highest cellular uptake, while the Cu 2+ -coordinated one exhibited the highest endosomal escape. The Ni 2+ -coordinated polymer formed large-sized aggregates with cargo proteins and showed insufficient protein release after endocytosis. The results obtained in this study provided new insight into the development of coordinative polymer-based protein delivery systems.

Polymer-mediated intracellular protein delivery systems are important for the development of protein-based biotechnologies and therapeutics. However, intracellular delivery of cargo proteins in the presence of serum remains challenging due to competitive binding of serum proteins with the polymers. Here, we reported a dendrimer engineered with a high density of 4-diethylaminophenyl groups on the surface to address this issue. The dendrimer showed a pH-responsive phase-transition behavior and could assemble with cargo proteins into stable nanoparticles in serum solutions. It efficiently delivered cargo proteins into living cells, and exhibited a pH-responsive disassembly behavior after cell internalization. As a result, various cargo proteins were delivered into the cytosol of living cells with maintained bioactivity. This study provided a convenient and efficient strategy to design polymers with high serum-tolerance in cytosolic protein delivery.

Oxidative stress is a hallmark of the pathophysiogenesis of retinal ischemia. The direct delivery of antioxidant enzymes such as superoxide dismutase (SOD) into retinal cells provides a promising option for the down-regulation of oxidative stress in retinal ischemia, however, efficient intracellular protein delivery remains a major challenge for this application. Here, a boronic acid-rich polymer was used for the intracellular delivery of SOD both in vitro and in vivo. The polymer assembled with SOD into uniform nanoparticles with high binding affinity, and transported the cargo protein into several cell lines with maintained bioactivity and low cytotoxicity. We investigated the intraocular biodistribution, therapeutic efficacy and safety of the SOD nanoformulation in a retinal ischemia/reperfusion (I/R) injury model. After intravitreal injection, the nanoparticles rapidly diffused through the vitreous and penetrated into retinal ganglion cells (RGCs). Compared to free SOD, the nanoformulation exhibited much enhanced therapeutic efficacy with reduced RGC apoptosis and protected retinal function. Enzymatic results confirmed that the SOD nanoformulation reduced malondialdehyde expression and increased glutathione level in the ocular tissues, and thereby down-regulated oxidative stress and prevented RGC loss. Overall, this work offers a new therapeutic option for the treatment of retinal ischemic disorders by direct delivery of antioxidant proteins., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cytosolic protein delivery is important for the development of protein therapeutics towards intracellular targets. Guanidyl polymers exhibit high binding affinity with cargo proteins, and thus were designed as carriers for intracellular protein delivery. However, the structure-activity relationship and mechanism of these polymers in cytosolic protein delivery remained to be investigated. In this study, we synthesized a total number of eighteen guanidyl-rich polymers by grafting various guanidyl containing compounds onto a polyethylenimine scaffold. The investigated guanidyl analogues were consisted of a guanidyl group and a hydrophobic component including cyclohexane, benzene, and alkanes with various chain lengths. It is surprising that only the polymers with both benzene and guanidyl possessed high efficiency in cytosolic protein delivery. Further results showed that all the synthesized polymers have efficient protein binding in water and high cellular uptake, but these polymers except the benzene-guanidyl based one enter the cytosol of cells without carrying their cargo proteins, suggesting poor stability of the polymer/protein complexes in culture medium. Paired guanidinium-π interactions between the ligands on benzene-guanidyl polymers are critical to the stabilization of polymer/protein complexes. In addition, a lead polymer in the library exhibited robust delivery efficacy to various cargo proteins, while maintaining their bioactivity after cell internalization. The results suggest that complex stability is a critical factor in polymer-mediated intracellular protein delivery systems, and provide new insights to guide design of polymeric protein vehicles., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cytosolic protein delivery offers opportunities to develop protein-based therapeutics specifically modulate intracellular processes, especially those linked to 'undruggable' targets. Since protein molecules are generally membrane-impermeable due to their macromolecular nature and hydrophilic property, carriers are needed to facilitate protein transport through cell membranes. Current delivery techniques usually require chemical modification or genetically engineering on cargo proteins to strengthen the binding affinity between proteins and the carriers. However, these approaches are usually accompanied with complicated syntheses, and the bioactivity of proteins might be irreversibly changed after modification. In this review, we discussed recent advances in the rational design of polymers for cytosolic delivery of native proteins with distinct isoelectric points and sizes. Functional ligands were grafted onto cationic polymers to strengthen the binding affinity between polymers and proteins, and/or to reduce the charge repulsion between cationic polymers during the formation of polymer/protein complexes. The principles of developed polymers in cytosolic protein delivery were intensively discussed. Furthermore, the possibility of developed polymers in the delivery of therapeutic proteins to treat diseases in vivo was evaluated. We hope to provide theoretical and technical supports to the development of polymers for cytosolic protein delivery., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cytosolic protein delivery is critical for the development of protein-based therapeutics. However, an efficient and robust carrier that can deliver native proteins without biological or chemical modifications into cells is highly desired. Here, we developed a guanidinium-rich polymer consisting of a cationic polymer scaffold modified with both phenyl and biguanide moieties. The polymer showed much higher protein binding affinity and endocytosis and reduced cytotoxicity compared to a control polymer by replacing the biguanide with monoguanide moieties. The guanidinium-rich polymer is capable of transporting proteins with various molecular weights and charge properties into the cytosol of living cells efficiently, while maintaining their bioactivities. This study permits the development of cationic polymers modified with phenylbiguanide moieties for efficient intracellular protein delivery.

The design of efficient and universal carriers to transport proteins into cells is highly desirable in the development of biotherapeutics. However, intracellular protein delivery remains a major challenge owing to the multiple barriers in protein delivery process, such as protein encapsulation, endocytosis, endosomal escape and intracellular protein release. Recently, it was reported that fluorination on polymers is beneficial for their self-assembly and intracellular trafficking. We hypothesize the fluoropolymers can be used to encapsulate proteins and developed as a new class of carriers to break down the barriers during intracellular protein delivery. In this study, we successfully discovered an efficient and non-toxic polymer for protein delivery in a library of fluoropolymers. The lead material A6-2 in the library efficiently transported various proteins including bovine serum albumin, β-galactosidase, saporin and a cyclic hendecapeptide into the cytosol of living cells with minimal cytotoxicity. More importantly, activities of delivered proteins and peptides were maintained after delivery. The A6-2/saporin complex was further coated with a hyaluronic acid shell, and the yielding nanoparticle efficiently suppressed tumor growth in a breast cancer model by targeted protein delivery. This study provides a new insight into the design of efficient and biocompatible polymers for protein delivery., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The efficient delivery of biopharmaceutical drugs such as proteins and peptides into the cytosol of target cells poses substantial challenges owing to their large size and susceptibility to degradation. Current protein delivery vehicles have limitations such as the need for protein modification, insufficient delivery of large-size proteins or small peptides, and loss of protein function after the delivery. Here, we adopted a rational approach to design a polymer with robust efficacy for intracellular protein and peptide delivery. The polymer is composed of a dendrimer scaffold, a hydrophobic membrane-disruptive region, and a multivalent protein binding surface. It allows efficient protein/peptide binding, endocytosis, and endosomal disruption and is capable of efficiently delivering various biomacromolecules including bovine serum albumin, R-phycoerythrin, p53, saporin, β-galactosidase, and peptides into the cytosol of living cells. Transduction of apoptotic proteins and peptides successfully induces apoptosis in cancer cells, suggesting that the activities of proteins and peptides are maintained during the delivery. This technology represents an efficient and useful tool for intracellular protein and peptide delivery and has broad applicability for basic research and clinical applications.

Developing protein delivery systems holds tremendous promise for the next-generation of protein-related biotechniques and biotherapeutics. Polycatechols inspired from nature polyphenols such as epigallocatechin gallate represent a class of polymers with highly efficacy in cytosolic protein delivery. Besides catechol, there are other benzenediol structures with two hydroxyls on an aromatic ring such as resorcinol and hydroquinone, and their behaviors in protein binding and intracellular delivery are yet unknown. Here, we firstly reported a type of polyresorcinols by grafting resorcinol moieties on cationic dendrimers for efficient cytosolic protein delivery. The materials exhibited comparable protein delivery efficacy with polycatechols but much higher stability towards oxidation in air. The resorcinol modification greatly improved the protein binding, cellular internalization, and endosomal disruption of the cationic polymer and reduced its cytotoxicity as well. The obtained polyresorcinols delivered various native proteins into the cytoplasm with maintained protein bioactivities, and successfully delivered a model antigen ovalbumin into dendritic cells to induce antigen cross-presentation for the treatment of B16-OVA melanoma in vivo. This study gives new insights into the physicochemical behaviors of phenolic polymers and developed a class of polyresorcinols for efficient cytosolic protein delivery. [Display omitted] • The resorcinol modification significantly enhanced the protein delivery efficacy of the cationic polymer in multiple ways. • Polyresorcinols exhibited higher stability towards oxidation compare to polycatechols and polyhydroquinone. • Polyresorcinols delivered a model antigen ovalbumin into dendritic cells to induce antigen cross-presentation for the treatment of B16-OVA melanoma in vivo. [ABSTRACT FROM AUTHOR]

RNA interference with the ability to specifically silence target genes has shown great potential to treat various diseases. The primary challenge for RNA interference is to effectively and selectively deliver genetic materials such as small interfering RNA (siRNA) to targeted tissues and cells in vivo. Numerous efforts have been made to overcome the extracellular and intracellular barriers during siRNA delivery. In this review, we focused on most recent advances in the design of functional polymers to achieve efficient siRNA delivery via addressing the various parameters, including siRNA binding, serum stability, specific targeting, tissue penetration, cellular internalization, endosomal escape, and intracellular siRNA release. The beneficial roles of assembled polymer nanostructures and polymer‐based hybrid nanocomposites in siRNA delivery were also reviewed. Finally, the challenges and perspectives in the future development of polymer‐based siRNA delivery systems will be discussed. [ABSTRACT FROM AUTHOR]

During pond culture of Eriocheir sinensis, a high limb-impairment rate restricts the industry development and quality. Therefore, research on limb autotomy and regeneration has important practical significance for the industrial development and basic biology of E. sinensis. This study evaluated the changes in bud morphology, growth-related gene expression and nutritional status during cheliped regeneration in E. sinensis. The study found that the new cheliped was pre-formed in the bud and then regenerated with the completion of molting of E. sinensis. The new cheliped was similar in morphology to the normal cheliped after the first molting but smaller in size. The qRT-PCR results of growth-related genes showed that the expression levels of EcR-mRNA (ecdysteroid receptor) and Chi-mRNA (chitinase) were significantly up-regulated, whereas the expression of MIH-mRNA (molt-inhibiting hormone) was significantly down-regulated (P < 0.05). The nutritional status during the regeneration process showed that the hepatopancreas total lipid content decreased significantly within 28 days and was significantly lower in the autotomy group than in the control group at 14 d and 21 d (P < 0.05). The hepatopancreas fatty acid composition results showed that saturated fatty acids (SFA), highly unsaturated fatty acids (HUFA) and n-3/n-6 were significantly higher in the autotomy group than in the control group at 21 d (P < 0.05), whereas the ∑ n-6 PUFA and ∑ n-3 PUFA at 1 d and 7 d, and the monounsaturated fatty acid (MUFA) at 28 d in the autotomy group were significantly lower than in the control group (P < 0.05). Moreover, the levels of eicosatetraenoic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) showed that DHA was significantly lower at 7 d and significantly higher at 21 d in the autotomy group than in the control group (P < 0.05), whereas ARA and EPA were not significantly different between the two groups. Muscle L-tryptophan content was significantly lower at 1 d and significantly higher at 7 d in the autotomy group than in the control group (P < 0.05). These results indicate that during the cheliped regeneration process, crabs could accelerate molting and regeneration by regulating growth-related gene expression (e.g., EcR-mRNA and MIH-mRNA) and nutrient metabolism (e.g., lipid metabolism). [ABSTRACT FROM AUTHOR]

Innenrücktitelbild: A Coordinative Dendrimer Achieves Excellent Efficiency in Cytosolic Protein and Peptide Delivery (Angew. B Ein koordinatives Dendrimer b transportiert 30 verschiedene Proteine und Peptide in das Zytosol lebender Zellen, wobei ihre Bioaktivität nach der intrazellulären Freisetzung erhalten bleibt. Zytosolische Freisetzung, Dendrimere, Enzyme, Proteinfreisetzung, Peptidfreisetzung. [Extracted from the article]