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Abstract A cationic liposomal gene delivery system comprising DOTAP, DOPE, and cholesterol was prepared and optimized. The results showed that the liposome/DNA (LP/DNA) system had spherical morphology, with a particle size of around 150 nm and zeta potential of approximately 30 mV. Cytotoxicity experiments showed that cells treated with all of the liposome carriers- with the exception of LP1-had more than 80% viability even at a weight ratio of 30. The in vitro transfection efficiency was measured using a Promega™ Luciferase Assay System. Of the tested lipoplexes, LP2/DNA showed the highest cell transfection efficiency (at a weight ratio of 10)-which was similar to or slightly lower than that of Lipofectamine® 2000 in HeLa, A549, and SPC-A1 cell lines. After freeze-drying, the cell transfection efficiency decreased slightly (P>0.05). The cell uptake mechanism study showed that LP/DNA lipoplexes mainly entered cells via clathrin-mediated and caveolin-mediated endocytic pathways. The results confirmed that LP2 has potential for use as an effective gene carrier, and provides experimental evidence to support its further development as a safe and effective gene delivery system.
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@#Nowadays, there is still no mature gene delivery system for safe and effective transfection on primary dendritic cells (DC). Herein, we constructed a liposome-based gene delivery system for primary DCs and optimized the preparation method to improve the transfection efficiency of siRNA on primary DCs. In this study, different methods, including co-incubation method, ethanol injection method, and protamine compound method, were used to prepare liposome/siRNA complexes based on different cationic lipids. Moreover, particle size, zeta potential, siRNA loading capacity, safety, stability, uptake efficiency and gene silencing efficiency of various liposome/siRNA complexes were detected to screen the optimal cationic lipid as well as its preparation method. We demonstrated that the OA2/siRNA delivery system prepared by the co-incubation method exhibited the best safety, uptake efficiency and gene silencing effect, compared to other siRNA delivery systems including the commercial Lipo2000. In summary, we provide a safe and effective gene delivery vector for primary DC cells through simple preparation method, which could also offer a gene delivery platform for other immune cells.
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OBJECTIVE: To prepare Wheat germ agglutinin (WGA) modified vinorelbine (VRB) cationic liposomes (WGA-VRB cationic liposomes), and to optimize the formulation and conduct cytotoxicity test.METHODS: Thin-film diffusion and ammonium sulfate gradient method were used to prepare WGA-VRB cationic liposomes using phospholipid and cholesterol as excipient, 3β-[N-(N' -N' -dimethyl aminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) as cationic material, distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) as long cycle chain. Using encapsulation rate as index, central composite design-response surface methodology was used to optimize the amount of DC-Chol, cholesterol and VRB. The contents of VRB in VRB liposomes and WGA-VRB cationic liposomes were determined. The effects of them and blank cationic liposomes on survival rates of human breast cancer cell MCF-7 and human non-small cell lung cancer cells A549 were compared. RESULTS: The optimal formulation of 5 mL WGA-VRB cationic liposomes was as follows as phospholipid 22 mg, cholesterol 12 mg, DC-Chol 8 mg, VRB 0. 5 mg. Encapsulation rate of the liposomes was (92. 24 ± 1. 21)％ (n=3), relative error of which to predicted value was 5. 3％. The contents of VRB in VRB liposomes and WGA-VRB cationic liposomes were (96. 01 ± 3. 26), (93. 39 ± 1. 59) μg/mL(n=3). Compared with blank cationic liposomes and VRB liposomes, WGA-VRB cationic liposomes could significantly reduce survival rate of MCF-7 and A549. CONCLUSIONS: WGA-VRB cationic liposomes are prepared successfully. Inhibitory effect of WGA-VRB cationic liposomes on MCF-7 and A549 cell survival is stronger than that of VRB liposomes.
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OBJECTIVE:To optimize the formulation of Asiaticoside cationic liposomes,and to investigate the characteristics of drug release in vitro. METHODS:The thin film dispersion method was used to prepare liposome;using encapsulation efficiency and drug-loading amount as index,the formulation of Asiaticoside liposomes was optimized by central composite design-response surface method with the ratio of drug to lipid(X1),the ratio of cholesterol to lipid(X2)and the concentration of D-mannose(X3) as factors. Using sodium lauryl sulfate as medium,in vitro release characteristics of cationic liposomes prepared with 1%octadecyl-amine was investigated by bag filter method,and compared with those of Asiaticoside solution and common liposome. RESULTS:The optimal formulation was X1 0.07,X2 0.17 and X3 0.03 g/ml. The encapsulation efficiency was (75.529 ± 1.071)%,and drug-loading amount was(2.539±0.029)%(n=3);the deviation from the predicted values were -0.217% and 0.205%;1% oc-tadecylamine was add into formulation to obtain cationic liposomes,and the Zeta potential had changed from -5.6 mV to 20.8 mV. in vitro accumulative release rates of Asiaticoside solution,common liposomes and cationic liposomes were 89.13%(12 h), 87.58%(72 h) and 94.46%(72 h),and the latter was in line with Weibull model. CONCLUSIONS:Asiaticoside cationic lipo-somes have high encapsulation efficiency,and can releases for 72 h.
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OBJECTIVE:To study the anti-tumor effects of Vinblastine (VLB) hydrophilic group modified cationic liposomes in tumor-bearing mice. METHODS:Tumor-bearing model were induced by inoculating yellow ascites of S180 ascites tumor mice. Tumor-bearing mice were randomly divided into model group,VLB sulfate injection group,VLB liposomes group,VLB hydrophil-ic group modified liposomes group,VLB cationic liposomes group and VLB hydrophilic group modified cationic liposomes group, i.e. group A,B,C,D,E and F,with 18 mice in each group. Group A was given normal saline intravenously via mice tail,other groups were given VLB 1.5 mg/kg every 2 days for consecutive 5 times. The anti-tumor effects of different VLB preparations were compared,using living conditions,survival time,tumor volume and weight,and tissue pathological section as indexes. RE-SULTS:Compared with group A,B,C,D and E,the mice of group F were more active,and had longer survival time,smaller tumor volume and lighter tumor weight,with statistical significance(P<0.05). The tissue pathological section of mice in group F indicated that coagulation necrosis,disintegration,and dissolution of tumor cell nucleus. CONCLUSIONS:VLB hydrophilic group modified cationic liposomes have obvious anti-tumor effect,which are better than other VLB preparations.
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OBJECTIVE: To explore the feasibility of the reversal of multidrug resistance in breast cancer using vector-based small interference RNA(siRNA) and to solve the problems of siRNA transient expression and siRNA delivery in vivo. METHODS: Based on the siRNA sequence which was screened in previous studies that could effectively inhibit the expression of MDR1 gene, the expression plasmid was constructed. The siRNA expression plasmid was then encapsulated in new nano-sized stealth cationic liposomes. Pharmacodynamic studies of the liposomes were carried out in vitro and in vivo. RESULTS: The results showed that the cationic liposomes loaded with MDR1 siRNA expression plasmid could effectively inhibit the expression of MDR1 gene both in vitro and in vivo. CONCLUSION: Multi-drug resistance of breast cancer cells is reversed to a great extent by this siRNA-containing cationic liposomes. Nano-sized cationic liposomes are ideal delivery vehicle of siRNA, which could protect the siRNA from degradation and deliver siRNA into the tumor region where it could exert functions.
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AlM:To construct recombination eukaryotic expression plasmid of human thyrotropin receptor extracellular domain encapsulated with cationic liposomes. METHODS:We amplified the target gene of shuttle vector PHMCMVTSHR289, conjugated the target gene and eukaryotic expression plasmid pcDNA3. 1 +, and accredited whether pcDNA3. 1+/TSHR289 was connected or not by enzymatic digestion and sequencing. Cationic liposomes encapsulated the recombination plasmid pcDNA3. 1+/TSHR289. RESULTS: Recombination plasmid pcDNA3. 1+/TSHR289 digested with enzyme Hindlll and the fragment through 0. 8% gel electrophoresis showed 512bp strip. Recombination plasmid pcDNA3. 1+/TSHR289 were found synonymous mutation through forward ( AAC to AAT ) and reverse sequencing ( GCG to GCT) . The volume ratio of cationic liposomes and recombinant plasmid was 3:1. CONCLUSlON: lt is successful to construct the recombination plasmid pcDNA3. 1+/TSHR289 by accredit it through enzymatic digestion and sequencing.
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Objective To investigate the inhibitory effects of hepatitis B virus (HBV) S gene-specific antisense locked nucleic acid (LNA) on HBV replication and expression in HepG_22.2.15 cells,and to screen the effective short sequence of LNA.Methods Four different lengths of short sequence of antisense locked nucleic acid which were complementary to the initiator of HBV S gene were designed,synthesized and transfected by cationic liposomes into HepG_22.2.15 cells.The HBsAg and HBV DNA of supematant was tested by enzyme linked immunoadsorbent assay(ELISA) and real-time fluorescent quantitative PCR(FQ-PCR) at 24,48 and 72 hours after treatment.LNA's cyto-toxicity on cell was evaluated by MTT method.Results Four different lengths of short sequence of LNA inhibi-ted the expression of HBsAg and the replication of HBV DNA with the inhibition rates of 46.58%,54.38%,72.43% ,69.92% and 27.09% ,28.77% ,34.71% ,32.68% respectively after 72 hours.There's no obvious tox-icity on cell.Conclusion Antisense LNA that targeting at HBV S gene has strong inhibition on HBV in vitro,and the optimal length of LNA sequence might be in the range of 15 base to 25 base.It has a therapeutic potential in the treatment of patients infected with HBV.
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Objective To evaluate the inhibitory effect of cationic Liposomes complexed to plasmids encoding endostatin and/or angiostatin on the growth and metastasis of Lewis lung cancer in mice model. Methods C57BL/6j mice were established as mice model. Cationic liposome complexed plasmids encoding angiostatin and endostatin were administrated intratumorally to inhibit the growth and metastasis of the implanted tumor. The size change of the tumor; metastasis in lung, the activity, nourishment, survival period of the mice were observed to evaluate the function of cationic liposome complexed plasmids. Results The treatment group could inhibit the growth and metastasis of the implanted tumor. Comparing with control group, they showed significance in tumor size, metastasis in lung and survival period of the mice (P
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Objective To explore the influence of polyamine-cholesterol cationic liposome (PCL)-mediated CpGODN aerosol on eosinophiles in the lung tissue of mouse asthma model. Methods Mouse asthma model was replicated by challenging with 1% ovalbumin aerosol. Mice were categonied into four groups, namely normal control, asthma control, CpGODN/PCL treatment group and CpGODN treatment group (6 each). The left lungs of mice were harvested, serially sectioned, hematoxylin and eosin (HE)-stained, and the infiltration of eosinophiles (EOS) was examined under microscope. Meanwhile, the bronchoalveolar lavage fluid (BALF) was collected for total and eosinophil cells count. Results An ovalbumin challenged mouse asthma model was successfully replicated. Pathological observation of the lung of asthma control showed increase in mucous secretion in alveolar space and peribronchial infiltration of large amount of inflammatory cells, primarily EOS and lymphocytes. The total cell number, EOS number and the ratio in BALF were significantly higher in asthma control group compared with that in both normal control group and CpGODN treatment group (P