Combined Hydrophobization of Polyethylenimine with Cholesterol and Perfluorobutyrate Improves siRNA Delivery.

Polyethylenimine (PEI) is a promising delivery vector of nucleic acids, but cytotoxicity and only moderate transfection efficacy with small RNAs limit its applications. Here we hypothesized that hydrophobization of PEI by combined modification with perfluorinated moieties (F) and cholesterol (Ch) will help in addressing both the cytotoxicity and siRNA delivery efficacy. To test the hypothesis, we synthesized a series of copolymers (F-PEI-Ch) by modifying PEI by reaction with heptafluorobutyric anhydride and cholesteryl chloroformate. We investigated and compared the effect of the modifications on siRNA delivery in vitro and in vivo. We found that the F-PEI-Ch copolymers assembled into micellar structures and that the copolymer with the highest Ch content exhibited the best siRNA delivery performance, including lower cytotoxicity, enhanced cell uptake, improved endosomal escape, and the best siRNA silencing efficacy in vitro and in vivo when compared with control PEI, F-PEI, and PEI-Ch. Overall, hydrophobization of PEI with a combination of cholesterol and superhydrophobic perfluorinated moieties represents a promising approach to the design of siRNA delivery vectors with decreased toxicity and enhanced transfection efficacy.

Reversible Covalent Cross-Linked Polycations with Enhanced Stability and ATP-Responsive Behavior for Improved siRNA Delivery.

Cationic polyplex as commonly used nucleic acid carriers faced several shortcomings, such as high cytotoxicity, low serum stability, and slow cargo release at the target site. The traditional solution is covering a negative charged layer (e.g., hyaluronic acid, HA) via electrostatic interaction. However, it was far from satisfactory for the deshielding by physiological anions in circulation (e.g., serum proteins, phosphate). In this study, we proposed a new strategy of reversible covalent cross-linking to enhance stability in circulation and enable stimuli-disassembly of polyplexes in tumor cells. Here, 25k polyethylenimine (PEI) was chosen as model polycations for veriying the hypothesis. HA-PEI conjugation was formed by the cross-linking of adenosine triphosphate grafted HA (HA-ATP) with phenylboronic acid grafted PEI (PEI-PBA) via the chemical reaction between PBA and ATP. Compared with noncovalent polyplex by electrostatic interaction (HA/PEI), HA-PEI exhibited much better colloidal stability and serum stability. The covered HA-ATP layer on PEI-PBA could maintain stable in the absence of physiological anions, while HA layer on PEI in HA/PEI group showed obvious detachment after anion's competition. More importantly, the covalent cross-linking polyplex could selectively release siRNA in the ATP rich environment of cytosol and significantly improve siRNA silence. Besides, the covalent cross-linking with HA-ATP could effectively reduce the cytotoxicity of cationic polyplex, improve the uptake by B61F10 cells and promote the endosomal escape. Consequently, this strategy of HA-PEI conjugation significantly enhanced the siRNA transfection in the absence or presence of FBS (fetal bovine serum) on B16F10 cells and CHO cells. Taken together, the reversible covalent cross-linking approach shows obvious superiority compared with the noncovalent absorption strategy. It held great potential to be developed to polish up the performance of cationic polyplex on reducing the toxicity, enhancing the serum tolerance and achieving controlled release of siRNA at target site.

GSH depletion liposome adjuvant for augmenting the photothermal immunotherapy of breast cancer.

The high redox level of tumor microenvironment inhibits the oxidation treatment and the immune response. Here, we innovatively develop maleimide liposome (ML) adjuvants to promote immunogenic cell death (ICD) induction and dendritic cells (DCs) maturation by glutathione (GSH) depletion for augmenting the photothermal immunotherapy of breast cancer. The ML effectively depletes the intracellular GSH and up-regulates reactive oxygen species (ROS) in both tumor cells and DCs. In tumor cells, the ROS boosted the ABTS·+ production to activate photothermal-induced ICD. In DCs, it relieved the immunosuppression, promoting DC maturation (57%) and antigen presenting. As a result of the ML assistant, the therapeutic systems improved the infiltration of CD8+ T cells to 53% in tumor tissues, eliciting strong abscopal effect and antimetastasis effect. The MLs were believed to be a superior candidate of adjuvants for enhancing immune response and cancer therapeutic efficacy.

pH-Switchable Coordinative Micelles for Enhancing Cellular Transfection of Biocompatible Polycations.

Inefficient transfection of biocompatible low-molecular-weight (LMW) polycations, such as 1.8k polyethylenimine (PEI), is a major challenge for successful nucleic acid delivery. Current strategies to improve transfection efficiency are bottlenecked by maintaining the balance between efficient gene encapsulation and on-demand cargo release. Here, we developed a new class of Zn(II)-coordinated micelles, which showed tight small interfering RNA (siRNA) binding and pH-switchable release. The dipicolylamine-modified PEI 1.8k (PD) and dopamine-conjugated cholesterol (Chol-Dopa) assemble into coordinative micelles (Zn-PD/Chol-Dopa) via the coordination of 2,2'-dipicolylamine (DPA) and Dopa through Zn(II) as a bridge. The high phosphate-binding affinity of Zn-DPA enhanced the siRNA packaging and the interaction between cholesterol and cell membranes enhanced cellular uptake. Moreover, the coordination effect weakened in the acidic environment of lyso/endosome, triggering the disassembly of micelles and siRNA release. These properties of the micelles resulted in strong siRNA transfection efficiencies in various cell lines. Our strategy of constructing coordinative micelles improves the transfection efficiency of LMW PEI and holds tremendous potential to develop the endogenous responsive gene delivery systems.