Dual-responsive nanocarriers for efficient cytosolic protein delivery and CRISPR-Cas9 gene therapy of inflammatory skin disorders.

Developing protein drugs that can target intracellular sites remains a challenge due to their inadequate membrane permeability. Efficient carriers for cytosolic protein delivery are required for protein-based drugs, cancer vaccines, and CRISPR-Cas9 gene therapies. Here, we report a screening process to identify highly efficient materials for cytosolic protein delivery from a library of dual-functionalized polymers bearing both boronate and lipoic acid moieties. Both ligands were found to be crucial for protein binding, endosomal escape, and intracellular protein release. Polymers with higher grafting ratios exhibit remarkable efficacies in cytosolic protein delivery including enzymes, monoclonal antibodies, and Cas9 ribonucleoprotein while preserving their activity. Optimal polymer successfully delivered Cas9 ribonucleoprotein targeting NLRP3 to disrupt NLRP3 inflammasomes in vivo and ameliorate inflammation in a mouse model of psoriasis. Our study presents a promising option for the discovery of highly efficient materials tailored for cytosolic delivery of specific proteins and complexes such as Cas9 ribonucleoprotein.

Dual-responsive bioconjugates bearing a bifunctional adaptor for robust cytosolic peptide delivery.

Peptide drugs have been successfully used for the treatment of various diseases. However, it is still challenging to develop therapeutic peptides working on intracellular targets due to their poor membrane permeability. Here, we proposed a type of dual-responsive bioconjugates bearing a heterobifunctional adaptor containing both aldehyde and catechol moieties for efficient cytosolic peptide delivery. Hydrazine-terminated cargo peptides were tagged to a boronated dendrimer with the help of the adaptor via dynamic acylhydrazone and catechol­boronate linkages. The bioconjugates efficiently delivered peptides with distinct physicochemical properties into various cells, and could release the cargo peptides triggered by intracellular reactive oxygen species and endolysosomal acidity, restoring the biofunctions of delivered peptides. In addition, the designed complexes efficiently delivered a pro-apoptotic peptide into osteosarcoma cancer cells and successfully inhibited the tumor growth both in vitro and in vivo. This study provides a universal and efficient platform for cytosolic therapeutic peptide delivery to intracellular targets for treating various diseases.

Robust Reversible Cross-Linking Strategy for Intracellular Protein Delivery with Excellent Serum Tolerance.

Intracellular protein delivery has attracted increasing attentions in biomedical applications. However, current delivery systems usually have poor serum stability due to the competitive binding of serum proteins to the polymers during delivery. Here, we report a reversible cross-linking strategy to improve the serum stability of polymers for robust intracellular protein delivery. In the proposed delivery system, nanoparticles are assembled by cargo proteins and cationic polymers and further stabilized by a glutathione-cleavable and traceless cross-linker. The cross-linked nanoparticles show high stability and efficient cell internalization in serum containing medium and can release the cargo proteins in response to intracellular glutathione and acidic pH in a traceless manner. The generality and versatility of the proposed strategy were demonstrated on different types of cationic polymers, cargo proteins, as well as cell lines. The study provides a facile and efficient method for improving the serum tolerance of cationic polymers in intracellular protein delivery.

Polycatechols with Robust Efficiency in Cytosolic Peptide Delivery via Catechol-Boronate Chemistry.

Cytosolic delivery of peptides remains a challenging task because of the limited binding sites on peptides and the existence of multiple intracellular barriers. Here, we proposed the use of polycatechols with a high cell permeability to deliver peptides of different physicochemical properties using the catechol-boronate chemistry. Peptides were decorated with boronate moieties via three strategies, and the introduced boronate groups greatly increased the binding affinity of cargo peptides with polycatechols. The loading peptides could be released under the endolysosomal acidity. When the cargo peptide was modified with boronate moiety via a p-hydroxybenzylcarbamate self-immolative spacer, it could be loaded by polycatechols and released in a traceless manner triggered by reactive oxygen species. The proposed strategies greatly promote the cytosolic delivery efficiency of different peptides into various cell lines and restored their biofunctions after intracellular delivery and release. This study provides a general and robust platform for the intracellular delivery of membrane-impermeable peptides.

Tailoring guanidyl-rich polymers for efficient cytosolic protein delivery.

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.

A Guanidinium-Rich Polymer for Efficient Cytosolic Delivery of Native Proteins.

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.

Statistical versus block fluoropolymers in gene delivery.

Fluoropolymers have shown great promise in non-viral gene delivery. The current fluoropolymers developed for gene delivery are synthesized by grafting fluoroalkyls or fluoroaromatics onto cationic polymers. To expand the family of fluoropolymers for the transduction of nucleic acids, new strategies to synthesize fluoropolymers are required. In this study, we synthesized both statistical and block copolymers of poly(2-dimethylaminoethyl methacrylate) (pDMAEMA) and poly(heptafluorobutyl methacrylate) (pHFMA) via reversible addition-fragmentation chain transfer polymerization, and the transfection efficiencies of the fluorocopolymers were evaluated. The statistical fluorocopolymer exhibited dramatically higher performance in gene delivery than the block one, which is attributed to more efficient and sustained DNA uptake by the transfected cells. Moreover, the statistical copolymer of DMAEMA and HFMA showed a fluorine effect in gene delivery, and its efficiency was much superior to non-fluorinated polymers. The results revealed the structure and activity relationships of fluoropolymers consisting of DMAEMA and HFMA, and provided a new insight to guide the design of fluoropolymers for efficient gene delivery.