Cytosolic protein delivery is of central importance for the development of protein-based biotechnologies and therapeutics; however, efficient intracellular delivery of native proteins remains a challenge. Here, we reported a boronic acid–rich dendrimer with unprecedented efficiency for cytosolic delivery of native proteins. The dendrimer could bind with both negatively and positively charged proteins and efficiently delivered 13 cargo proteins into the cytosol of living cells. All the delivered proteins kept their bioactivities after cytosolic delivery. The dendrimer ensures efficient intracellular delivery of Cas9 protein into various cell lines and showed high efficiency in CRISPR-Cas9 genome editing. The rationally designed boronic acid–rich dendrimer permits the development of an efficient platform with high generality for the delivery of native proteins.

Cytosolic protein delivery is of great importance for basic cell biology and the discovery of novel protein-based biotherapeutics. It remains a challenging task because of the limited binding sites on proteins and their relatively large size. As a result, most current approaches for cytosolic protein delivery need covalent modification on native proteins, which is usually involved with complicated synthesis, reduced protein bioactivity, and unexpected safety concerns. In this study, we proposed a novel strategy to deliver proteins of different molecular sizes and isoelectric points by specific recognitions between natural polyphenols and boronic acid-containing polymers. Protein molecules were decorated with polyphenols via noncovalent hydrogen-bond/hydrophobic interactions or reversible dynamic covalent bonds. The natural polyphenols increase the binding affinity between proteins and boronic acid-containing polymers, allow the release of bound proteins in acidic environments because of pH-sensitive property of catechol–boronate esters, and thus greatly promote the cytosolic delivery efficiency. This strategy showed robust efficiency in the delivery of various proteins such as bovine serum albumin, phycoerythrin, and ribonuclease A and maintained the protein bioactivity after intracellular release. The reported strategy permits the development of a polyphenol-involved polymer platform for cytosolic protein delivery.

Based on the high frequency of concurrent adenomatous polyposis coli (APC) and KRAS mutations and their strong cooperative interaction in human colorectal cancer (CRC) promotion, we herein develop a CRISPR-Cas9-based genome-editing nanomedicine to target both APC and KRAS mutations for the treatment of CRC. To this end, a hyaluronic acid (HA)-decorated phenylboronic dendrimer (HAPD) was designed for the targeted delivery of Cas9 ribonucleoprotein (RNP), by which both APC and KRAS genetic mutations harboring in CRC cells can be synergistically disrupted. Systemic administration of Cas9 RNP targeting APC and KRAS enabled by HAPD significantly inhibits tumor growth on xenografted and orthotopic CRC mouse models and also greatly prevents CRC-induced liver metastasis and lung metastasis. Thus, this duplex genome-editing system provides a promising gene therapy strategy for the treatment of human CRC and can be extended to other types of cancers with activated Wnt/β-catenin and RAS/extracellular signal-regulated kinase (ERK) pathways.

The transfection efficiency of siRNA mediated by cationic polymers is limited due to the instability of polymers/siRNA complexes in the presence of serum. Poly(ethylene glycol) (PEG) is usually applied to modify cationic polymers, so as to reduce protein and cell adsorption and then to improve siRNA transfection efficiency. However, the polymers’ modification with PEG mostly consumes the free amino of the polymers, which can, in turn, reduce the charge density and limit their siRNA transfection efficacy. Here, a new PEG modification strategy that need not consume the surface aminos of polymers is proposed. Catechol–PEG polymers are coated on the surface of phenylboronic acid (PBA)‐modified Generation 5 (G5) poly(amidoamine) dendrimers (G5PBA) via reversible boronate esters to establish PEG‐modified dendrimer/siRNA nanoassemblies for efficient siRNA delivery. The PEG/G5PBA/siRNA nanoassemblies have positive charge and show excellent gene silencing efficacy in the absence of serum in vitro. More importantly, the PEG/G5PBA/siRNA nanoassemblies also exhibit excellent serum resistance and gene silencing efficacy in serum‐containing medium. Furthermore, the effective antiserum and gene silencing efficacy elicited by these nanoassemblies lead to excellent antitumor effects in vivo. This proposed strategy constitutes an important approach to reach an excellent gene silencing efficacy in the presence of serum.

Polymers are widely used as non-viral carriers for siRNA delivery, but concern has also arisen in their limited efficacy and inherent toxicity. Whilst many of previous efforts have been documented towards improving the performance of polymers via chemical modifications, the structure-activity relationships (SAR) of these ligand-modified polymers are not well understood. To address this issue, we systemically prepared a library of surface-engineered dendrimers (>300) as the screening pool to discover efficient siRNA carriers. The modified ligands include alkyls and fluoroalkyls, amino acids, benzene derivatives and heterocyclic compounds. Gene silencing results showed that the lead material shows excellent efficacy even in hard-to-transfect cells such as mesenchymal stem cells. The SAR studies revealed that ligands containing appropriate hydrophobicity, or ligands with both hydrophobic and functional atoms/groups are essential for polymers to achive efficient knockdown efficacy. A second-generation library designed based on the above principles further confirms the proposed design criteria. The results enable the future rational design of potent siRNA carriers.