Gang Chen

Combining Fluorination and Bioreducibility for Improved siRNA Polyplex Delivery

Polycations are promising vectors for the delivery of siRNA therapeutics but they often suffer from toxicity and low in vivo delivery efficacy. This study tests the hypothesis that combining fluorination and bioreducibility of polycations will overcome problems with both the toxicity and delivery efficacy. To test the hypothesis, we synthesized bioreducible (RHB) and nonreducible (NHB) poly(amido amine)s. The RHB were additionally fluorinated using reaction with heptafluorobutyric anhydride to obtain F-RHB. We found that both RHB and F-RHB showed significantly reduced cytotoxicity compared with NHB, which allowed their safe use in a wider range of doses than NHB. All three synthesized polycations formed polyplexes with siRNA. F-RHB achieved the best siRNA silencing efficacy in multiple cell lines in vitro, which was at least in part because of fluorination-induced enhancement of cellular uptake and improved endosomal escape. Lastly, F-RHB showed greatly improved Luc silencing efficacy in tumors in vivo when compared with polyplexes based on RHB, NHB, as well as control poly(ethylenimine) (PEI). This study suggests that combining fluorination with bioreducibility of polycations is a promising strategy to the design of siRNA delivery vectors with improved toxicity and in vivo activity profiles.

Oral Nanostructured Lipid Carriers Loaded with Near-Infrared Dye for Image-Guided Photothermal Therapy

Photothermal therapy exerts its anticancer effect by converting laser radiation energy into hyperthermia using a suitable photosensitizer. This study reports development of nanostructured lipid carriers (NLCs) suitable for noninvasive oral delivery of a near-infrared photosensitizer dye IR780. The carrier encapsulating the dye (IR780@NLCs) was stable in simulated gastric and intestinal conditions and showed greatly enhanced oral absorption of IR780 when compared with the free dye. As a result of increased oral bioavailability, enhanced accumulation of the dye in subcutaneous mouse colon tumors (CT-26 cells) was observed following oral gavage of IR780@NLCs. Photothermal antitumor activity of orally administered IR780@NLCs was evaluated following local laser irradiation of the CT-26 tumors. We observed significant effect of the photothermal IR780@NLCs treatment on the rate of the tumor growth and no toxicity associated with the oral administration of IR780@NLCs. Overall, orally administered IR780@NLCs represents a safe and noninvasive method to achieve systemic tumor delivery of a photosensitizing dye for applications in photothermal anticancer therapies.

Cyclam-Modified PEI for Combined VEGF siRNA Silencing and CXCR4 Inhibition To Treat Metastatic Breast Cancer

Chemokine receptor CXCR4 plays an important role in cancer cell invasion and metastasis. Recent findings suggest that anti-VEGF therapies upregulate CXCR4 expression, which contributes to resistance to antiangiogenic therapies. Here, we report the development of novel derivatives of polyethylenimine (PEI) that effectively inhibit CXCR4 while delivering anti-VEGF siRNA. PEI was alkylated with different amounts of a CXCR4-binding cyclam derivative to prepare PEI-C. Modification with the cyclam derivatives resulted in a considerable decrease in cytotoxicity when compared with unmodified PEI. All the PEI-C showed significant CXCR4 antagonism and the ability to inhibit cancer cell invasion. Polyplexes of PEI-C prepared with siVEGF showed effective silencing of the VEGF expression in vitro. In vivo testing in a syngeneic breast cancer model showed promising antitumor and antimetastatic activity of the PEI-C/siVEGF polyplexes. Our data demonstrate the feasibility of using PEI-C as a carrier for simultaneous VEGF silencing and CXCR4 inhibition for enhanced antiangiogenic cancer therapies.

Self-assembled hemoglobin nanoparticles for improved oral photosensitizer delivery and oral photothermal therapy in vivo

AIM The aim of the present study was to use hemoglobin (Hb) nanoparticles (NPs) to improve oral bioavailability of a near-infrared dye IR780 for in vivo antitumor application in photothermal therapy. METHODS One-step acid-denaturing method was used to encapsulate IR780 into self-assembled Hb NPs (IR780@Hb NPs). Pharmacokinetics, biodistribution and antitumor effect were studied in vivo. RESULTS The Hb NPs showed high stability in enzymatic and acidic conditions similar to the gastric environment, and enhanced absorption of IR780 into the blood. In vivo imaging revealed that IR780 could accumulate at the tumor sites and effectively caused photothermal effect, which resulted in tumor ablation after oral administration in tumor-bearing mice. CONCLUSION Hb NPs represent a promising delivery system for improving oral absorption of photosensitizer dyes, which could open new treatment modalities in cancer.

Development of fluorinated polyplex nanoemulsions for improved small interfering RNA delivery and cancer therapy

We report the development of a small interfering RNA (siRNA) delivery vector based on cationic perfluorocarbon nanoemulsions. We have prepared perfluorodecalin (PFD) emulsions with a positive surface charge provided by a fluorinated poly(ethylenimine) (F-PEI). The fluorinated emulsion (F-PEI@PFD) reduced cytotoxicity of F-PEI and demonstrated effective binding with siRNAs to form nanosized emulsion polyplexes. The prepared emulsion polyplexes enhanced cellular uptake and improved endosomal escape of the siRNA. In addition to increased reporter gene silencing in multiple cancer cell lines, when compared with control F-PEI and PEI polyplexes, the siRNA emulsion polyplexes showed an excellent resistance to serum deactivation and maintained high activity, even in high-serum conditions. The F-PEI@PFD emulsion polyplexes carrying an siRNA to silence the expression of Bcl2 gene induced apoptosis and inhibited tumor growth in a melanoma mouse model in vivo and showed potential for in vivo ultrasound imaging. This study demonstrates the potential of F-PEI@PFD emulsions as a multifunctional theranostic nanoplatform for safe siRNA delivery, with integrated ultrasound imaging functionality.

Fluorination Enhances Serum Stability of Bioreducible Poly(amido amine) Polyplexes and Enables Efficient Intravenous siRNA Delivery

The use of small interfering RNA (siRNA) in cancer treatment has been limited by the lack of effective systemic delivery methods. Although synthetic polycations have been widely explored in siRNA delivery, polycation/siRNA polyplexes often suffer from insufficient stability in vivo. Here, rationally designed siRNA delivery systems that meet the requirements for systemic siRNA delivery to distant tumors are reported. The hypothesis that modular design of delivery systems based on poly(amido amine)s that combine fluorination for systemic stability with bioreducibility for easy intracellular siRNA release, and PEGylation for improved safety and colloidal stability will overcome problems with contradicting siRNA delivery demands is tested. PEGylated, fluorinated, and bioreducible copolymers (PEG-PCD-F) with different degree of fluorination are thus synthesized. The fluorinated copolymers readily formed polyplexes with siRNA and achieved greatly improved gene silencing efficacy in multiple cell lines in vitro when compared with nonfluorinated controls. The results show fluorination-induced enhancement of stability, cellular uptake, and endosomal escape of the polyplexes, while exhibiting efficient siRNA release in reducing intracellular environment. PEG-PCD-F polyplexes with siRNA against Bcl2 inhibit breast tumor growth following systemic intravenous administration. The results provide strong evidence of successful combination of bioreducibility with fluorination and PEGylation to achieve systemic siRNA polyplex delivery.

ATP-activated decrosslinking and charge-reversal vectors for siRNA delivery and cancer therapy

Stimuli-responsive polycations have been developed for improved nucleic acid transfection and enhanced therapeutic efficacy. The most reported mechanisms for controlled release of siRNA are based on polyelectrolyte exchange reactions in the cytoplasm and the degradation of polycations initiated by specific triggers. However, the degradation strategy has not always been sufficient due to unsatisfactory kinetics and binding of cationic fragments to siRNA, which limits the gene silencing effect. In this study, a new strategy that combines degradation and charge reversal is proposed. Methods: We prepared a polycation (CrossPPA) by crosslinking of phenylboronic acid (PBA)-grafted 1.8k PEI with alginate. It was compared with 25k PEI, 1.8k PEI and 1.8k PEI-PBA on siRNA encapsulation, ATP-responsive behavior and mechanism, cytotoxicity, cell uptake, siRNA transfection, in vivo biodistribution and in vivo anti-tumor efficacy. The in vitro and in vivo experiments were performed on 4T1 murine breast cancer cells and 4T1 tumor model separately. Results: The crosslinking strategy obviously improve the siRNA loading ability of 1.8k PEI. We validated that intracellular levels of ATP could trigger CrossPPA disassembly and charge reversal, which resulted in efficient and rapid siRNA release due to electrostatic repulsion. Besides, CrossPPA/siRNA showed strong cell uptake in 4T1 cells compared with 1.8k PEI/siRNA. Notably, the cytotoxicity of CrossPPA was pretty low, which was owing to its biodegradability. Furthermore, the crosslinked polyplexes significantly enhanced siRNA transfection and improved tumor accumulation. The high gene silencing ability of CrossPPA polyplex led to strong anti-tumor efficacy when using Bcl2-targeted siRNA. Conclusion: These results indicated that the ATP-triggered disassembly and charge reversal strategy provided a new way for developing stimuli-responsive siRNA carriers and showed potential for nucleic acid delivery in the treatment of cancer.

Pulmonary delivery of polyplexes for combined PAI-1 gene silencing and CXCR4 inhibition to treat lung fibrosis

This report describes the development of polyplexes based on CXCR4-inhibiting poly(ethylenimine) derivative (PEI-C) for pulmonary delivery of siRNA to silence plasminogen activator inhibitor-1 (siPAI-1) as a new combination treatment of pulmonary fibrosis (PF). Safety and delivery efficacy of the PEI-C/siPAI-1 polyplexes was investigated in vitro in primary lung fibroblasts isolated from mice with bleomycin-induced PF. Biodistribution analysis following intratracheal administration of fluorescently labeled polyplexes showed prolonged retention in the lungs. Treatment of mice with bleomycin-induced PF using the PEI-C/siPAI-1 polyplexes resulted in a significant down-regulation of the PAI-1 expression and decreased collagen deposition in the lung. The results of this study provide first evidence of the potential benefits of combined inhibition of CXCR4 and PAI-1 in the pulmonary treatment of PF.

Reversibly Stabilized Polycation Nanoparticles for Combination Treatment of Early- and Late-Stage Metastatic Breast Cancer

Metastatic breast cancer is a major cause of cancer-related female mortality worldwide. The signal transducer and activator of transcription 3 (STAT3) and the chemokine receptor CXCR4 are involved in the metastatic spread of breast cancer. The goal of this study was to develop nanomedicine treatment based on combined inhibition of STAT3 and CXCR4. We synthesized a library of CXCR4-inhibiting polymers with a combination of beneficial features that included PEGylation, fluorination, and bioreducibility to achieve systemic delivery of siRNA to silence STAT3 expression in the tumors. An in vivo structure-activity relationship study in an experimental lung metastasis model revealed superior antimetastatic activity of bioreducible fluorinated polyplexes when compared with nonreducible controls despite similar CXCR4 antagonism and the ability to inhibit in vitro cancer cell invasion. When compared with nonreducible and nonfluorinated polyplexes, improved siRNA delivery was observed with the bioreducible fluorinated polyplexes. The improvement was ascribed to a combination of enhanced physical stability, decreased serum destabilization, and improved intracellular trafficking. Pharmacokinetic analysis showed that fluorination decreased the rate of renal clearance of the polyplexes and contributed to enhanced accumulation in the tumors. Therapeutic efficacy of the polyplexes with STAT3 siRNA was assessed in early stage breast cancer and late-stage metastatic breast cancer with primary tumor resection. Strong inhibition of the primary tumor growth and pronounced antimetastatic effects were observed in both models of metastatic breast cancer. Mechanistic studies revealed multifaceted mechanism of action of the combined STAT3 and CXCR4 inhibition by the developed polyplexes relying both on local and systemic effects.