Co-delivery Of Sorafenib And VEGF-siRNA Via PH-sensitive Liposomes For The Synergistic Treatment Of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a malignant tumor that threat to human health. The complexity of HCC occurrence and development depends on several genetic or epigenetic alterations. The expansion of knowledge on molecular biology promotes tumor early diagnosis and treatment as well as innovation of therapeutic agents and strategy. However, a single agent and schema may not be able to achieve satisfactory effects. Thereby, combination therapy based on modulation of different genetic mechanisms provides another choice to improve therapeutic effect and to reduce undesied toxicity, which has become a hot topic of tumor treatment. Recently, combination of anti-tumor drug with small interfering RNA (siRNA) has been employed as a promising therapeutic modality. siRNA is able to inhibit the progression of cell cycle, proliferation, angiogenesis or other cellular pathways through exerting the RNA-induced silencing complex (RISC) in cytoplasm and inducing sequence-specific gene silencing. Correspondingly, siRNA shows great potential in treating specific tumor types and several research have demonstrated the efficiency with drug/siRNA combination therapy on down-regulating cancer-related genes to enhance chemotherapeutic effect at the tumor site. Non-viral nanocarrier affords a platform for drug and siRNA combination, the focus of which is to load drug and siRNA into a single carrier, allowing for co-delivery and a synergistic effect at tumor site.Since the antitumor drug and siRNA own distinct physicochemical property respectively, it is a big challenge to construct a co-delivery carrier with high entrapment efficiency. The carrier should keep stable in systemic circulation and achieve tumor site specific delivery to produce their antitumor effect. Liposomes represent one of the most successful nanocarriers, having been proven in several clinical trials. Liposomes can be prepared easily with the hydrophilic and hydrophobic drug co-loading, own good biocompatibility and scale-up ability. Therefore, this study used cationic liposome (CL) to encapsulate sorafenib (Sf), which is a small molecular multitarget receptor kinase inhibitor, producing Sf loaded cationic liposomes (Sf-CL). Then Sf-CL adsorbed siRNA to obtain Sf and siRNA co-loaded cationic complex SiSf-CL. To realize Sf and siRNA tumor-site released, pH sensitive materials, O-carboxymethyl-chitosan (CMCS) was coated on the surface of SiSf-CL via electrostatic interaction to obtain pH sensitive Sf and siRNA co-loaded cationic liposome (CMCS-SiSf-CL). In our study, VEGF-siRNA was chose as model gene to test the co-delivery ability of this pH sensitive liposome, providing a new idea for HCC therapy. The main contents and results are as follows:1. Preparation and characterization of sorafenib cationic liposomesThe methodology of determinating Sf by HPLC was developed. The results showed that Sf had a good linear relation within determined concentration. Precision and recovery met the requirements. Dissolve-ultracentrifugation method was used to separate liposomes and free Sf. Sf content and encapsulation efficiency was determined by HPLC. Sorafenib cationic liposomes (Sf-CL) were prepared by the thin film dispersion method. The effects of factors on encapsulation efficiency by single-factor test, including Sf to DOPE molar ratio, cholesterol to DOPE molar ratio and DOTAP to DOPE molar ratio. Under the optimum formulation: the molar ratio of Sf and DOPE of 1:6.5, the molar ratio of cholesterol and DOPE of 1:2, the molar ratio of DOTAP and DOPE of 1:3, the mean encapsulation efficiency of three batchs was 90.36%±0.63%, loading efficiency was 5.19%±0.035%, particle size was 147.0±2.2 nm, PDI was 0.135±0.027, zeta potential was 33.5±1.7 mV. Blank cationic liposomes (CL) were prepared by the same method. Particle size of CL was 122.5±2.6 nm, PDI was 0.104±0.006, zeta potential was 38.0±2.1 mV. Both Sf-CL and CL was spherical and the dispersity was good.2. Preparation and characterization of sorafenib and siRNA co-loadedcationic liposomesSf-CL were incubated with Cy3-siRNA according to different mass ratio of CL and siRNA to developed Sf and siRNA co-loaded cationic liposomes SiSf-CL Agarose gel electrophoresis and laser particle analyzer were used to screen the optimized ratio. The results showed that siRNA could be condensed completely at the CL to siRNA mass ratio of 20:1. Particle size and zeta potential of SiSf-CL was 164.5±3.1 nm and -10.6±1.0 mV, respectively, which was suitable for futher modification. SiSf-CL were incubated with CMCS according to different mass ratio of CMCS and siRNA to develop CMCS modified Sf and siRNA co-loaded cationic liposome, CMCS-SiSf-CL, the optimized radio of which was CMCS to siRNA mass ratio of 3.8:1. Particle size and zeta potential of CMCS-SiSf-CL was 200.1±7.9 nm and -10.6±1.0 mV, respectively. CMCS-SiSf-CL was able to protect siRNA against degradation within determined time. The particle size changed barely after incubation for 120 min in 37℃,10% plasma or storage for 28 days at 4℃. The results demonstrated that CMCS-SiSf-CL have some biological stability and physical stability. The the pH sensitivity of CMCS was determined by acid-base titration, the titration curve became flat after pH 6.24. In vitro release of CMCS-SiSf-CL and CMCS-Sf-CL in different pH condition was tested by dynamic membrane dialysis method. The results showed Sf was sustainably released from CMCS-SiSf-CL and CMCS-Sf-CL with 26.08%±3.19% and 31.75%±2.09% respectively in pH 7.4 PBS. The release of Sf was over 34.52%±2.16% and 40.78% ±4.07% from CMCS-SfSi-CL and CMCS-Sf-CL in pH 6.5 PBS, respectively, faster than that in pH 7.4, which indicated that CMCS modified carrier had pH sensitivity (P<0.05).3. In vitro and in vivo siRNA distribution and sorafenib antitumor efficacy of pH sensitive sorafenib and siRNA co-loaded liposomes.In vitro cytotoxicity and cellular uptake of the carriers were tested on HepG2 cell model. Flow cytometry and fluorescence microscope was used to investigate Cy3-siRNA cellular uptake in HepG2 cells in different pH condition. The flow cytometry analysis showed that celluar uptake efficiency of CMCS-SiSf-CL in pH 6.5 condition was twice as much that in pH 7.4,58,18%±12.83% and 27.69%±4.10% (P<0.05), respectively. In addition, the celluar uptake efficiency was similar as that of SiSf-CL and commercial Lipofectamine-2000/siRNA complex,55.49%±8.47% and 66.93%±2.52%, respectively (P>0.05). The results demonstrated that CMCS could fall off from the surface of the carrier owing to charge reversal in tumor microenvironment mimicking condition. The exposed SiSf-CL could enter into cells efficiently, while free siRNA could not be uptaken by cells, which was corresponding to the results of fuorescent microscope. MTT assay was used to test Sf cytotoxicity on HepG2 cells at 24 h. The results showed that the cytotoxicity of Sf, Sf-CL, CMCS-Sf-CL, CMCS-SiSf-CL was dose dependent, the IC50 was 12.16±1.81μM, 7.80±1.48 μM,11.82±0.38 μM,11.44±0.83 μM, respectively. Blank carrier, CMCS-CL, did not diplay obvious cytotoxicity. Tumor accumulation of the carriers was investigated by the fluorescence intensity of Cy5-siRNA. The live image and tumor section images showed that CMCS-SiSf-CL could deliver siRNA to tumor region efficiently, while free siRNA could not. The in vivo tumor growth inhibition of Sf carriers was investigated by Kunming mice bearing H22 tumor model. The mice were treated tail iv injection once every 3 days of Sf (4.5 mg/kg), Sf-CL,(4.5 mg/kg), CMCS-Sf-CL (4.5 mg/kg), CL or CMCS-CL for 19 days (total for six doses). The results showed that Sf-CL and CMCS-Sf-CL could inhibit tumor growth efficiently compared with saline group. HE staining images indicated that no significantly toxic pathological changes were observed in main organs of the mice after injection, revealing that this carrier could deliver Sf to tumor to generate antitumor effect and did not injure normal organs.4. In vitro and in vivo co-delivery of pH sensitive sorafenib and siRNA co-loaded liposomesLaser scanning confocal microscope was used to investigate the distribution of CMCS modified Cy3-siRNA/Coumarin 6 co-loaded liposomes in two-dimension cultured HepG2 cells. Cy3-siRNA and Coumarin 6 was representive of siRNA and Sf respectively. The results showed that CMCS modified co-loaded liposomes were able to co-deliver Cy3-siRNA and Coumarin 6 into cells, and co-delivery effect was stronger in pH 6.5 condition than that in pH 7.4 condition. Lysotracker was used to stain the lysosomes in cells. The results showed that Cy3-siRNA was able to escape from lysosomes after 3 hours. Three dimension cultured HepG2 tumor spheroids were developed and the penetration of co-loaded liposomes into tumor spheroids was captured by laser scanning confocal microscope. The results showed that after 5 hours incubation CMCS modified co-loaded liposomes penetrated into tumor spheroids up to 107.8 mm in pH 6.5 condition and 84 mm in pH 7.4 condition. The merged fluorescence intensity was obviously higher in pH 6.5 condition than that in pH 7.4 condition under the same interval of Z-stack scan, which demonstrated the penetratio of CMCS modified Cy3-siRNA/Coumarin 6 co-loaded liposomes into tumor spheroids under the acid condition. siRNA was replaced with Cy5-siRNA in vivo co-delivery study. The frozen sections of mice tumor tissues showed that CMCS-modified co-loaded liposomes could co-deliver Cy5-siRNA and Coumarin 6 into tumor regions.5. In vitro inhibiting hepatocellular carcinoma study of pH sensitive sorafenib and siRNA co-delivery liposomesVEGF-siRNA was selected as model gene to study the inhibition of the carrier on VEGF high expressed HepG2. The transfection dose of VEGF-siRNA was tested by Realtime PCR and was set at 100 nM. The VEGF gene silencing efficiency of different single-delivery and co-delivery carriers was investigated on HepG2 cells at 48 h with the Sf final concentration of 2.8 μM and siRNA final concentration of 100 nM. The results showed that VEGF mRNA relative expression of free Sf and free siRNA treated group was 78.31%±18.18% and 和 108.84%±33.04%, respectively. Treatment with CMCS-Sf-CL, CMCS-Si-CL, SiSf-CL and CMCS-SiSf-CL resulted in a downregulation of VEGF mRNA of 73.12%± 14.99%,68.93%±3.91%. 35.29%±12.77% and 66.31%±5.56%, respectively, which means that compared with free siRNA and single loaded carrier, co-delivery carrier exhibited gene silencing effect (P<0.05). In pH 6.5 condition that mimics tumor microenvironment, treatment with CMCS-SiSf-CL resulted in a downregulation of VEGF mRNA (38.41%±2.72%) compared with that in pH 7.4 condition (P<0.05), further demonstrating that CMCS modified liposomes were pH sensitive. CMCS could drop off in tumor acid microenvironment, followed by the exposure of cationic SiSf-CL cellular uptake and co-delivery of siRNA/Sf into the cytosol, which produced gene silencing. VEGF protein expression after 48 h transfection was measured by Western Blot and the results were in accordance with that of Realtime PCR. MTT assay was used to test the cell viability of HepG2 after treating with various formulations. The results showed that Sf could inhibit cell viability of HepG2, while VEGF-siRNA had less effect on cell viability during the transfection progress. The results of cell apoptosis test showed that various formulations could induce cell early apoptosis expect free siRNA and CMCS-NCsi-CL. The apoptosis rate of cells treated with SiSf-CL and CMCS-SiSf-CL in pH 6.5 condition was higher than other groups (38.59% and 36.16%, respectively). The results of cell cycle test showed the combination of Sf and VEGF-siRNA did not affect HepG2 cell cycle.Above all, CMCS modified liposomes could load Sf and siRNA efficiently and release Sf and siRNA at tumor region not only via enhanced permeability and retention effect but also the pH sensitivity, co-delivering Sf and siRNA to tumor region, improving the cellular uptake and antitumor ability of Sf and siRNA. The preparation of the carrier is simple and reproductive, laying the foundation for the co-delivery systems development and providing new area for HCC therapy.