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The extraordinary advantages associated with mRNA vaccines, including their high efficiency, relatively low severity of side effects, and ease of manufacture, have enabled them to be a promising immunotherapy approach against various infectious diseases and cancers. Nevertheless, most mRNA delivery carriers have many disadvantages, such as high toxicity, poor biocompatibility, and low efficiency in vivo, which have hindered the widespread use of mRNA vaccines. To further characterize and solve these problems and develop a new type of safe and efficient mRNA delivery carrier, a negatively charged SA@DOTAP-mRNA nanovaccine was prepared in this study by coating DOTAP-mRNA with the natural anionic polymer sodium alginate (SA). Intriguingly, the transfection efficiency of SA@DOTAP-mRNA was significantly higher than that of DOTAP-mRNA, which was not due to the increase in cellular uptake but was associated with changes in the endocytosis pathway and the strong lysosome escape ability of SA@DOTAP-mRNA. In addition, we found that SA significantly increased the expression of LUC-mRNA in mice and achieved certain spleen targeting. Finally, we confirmed that SA@DOTAP-mRNA had a stronger antigen-presenting ability in E. G7-OVA tumor-bearing mice, dramatically inducing the proliferation of OVA-specific CLTs and ameliorating the antitumor effect. Therefore, we firmly believe that the coating strategy applied to cationic liposome/mRNA complexes is of potential research value in the field of mRNA delivery and has promising clinical application prospects.
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A combination of cationic liposome/green fluorescence protein (GFP)-p53 complexes and a chemotherapeutic drug,cisplatin, was evaluated for therapeutic activity in human carcinoma cells. Cationic liposome/GFP-p53 complexes andcationic liposome/PEI (polyethylenimine) 0.8k/GFP-p53 complexes were synthesized and evaluated in HeLa and in A549cells for uptake and cytotoxicity, alone and in combination with cisplatin. The particle size of cationic liposome/GFP-p53complexes and cationic liposome/PEI 0.8K/GFP-p53 complexes was 318 ± 18 to 754 ± 108 nm, and zeta potentials were-15.7±2.8–+27±08 mV. The GFP expression on the delivery by cationic liposome/pEGFP (enhanced green fluorescenceprotein) complexes and cationic liposome/PEI 0.8K/pEGFP complexes was demonstrated. Treatment of the cells with eithercationic liposome/GFP-p53 complexes or cationic liposome/PEI 0.8K/GFP-p53 complexes inhibited the cell growth. Onpost treatment with cisplatin, the growth inhibition of the complexes was further increased in HeLa cells and significantlyincreased in A549 cells on the lipid-to-DNA ratio. This study concluded that the sensitivity to the cancer cells to cisplatinwas dependent on the cell line and the ratio of cationic liposome and GFP-p53. GFP-p53 expression delivered by cationicliposome/GFP-p53 complexes would be useful to increase the effect of cisplatin on the treatment of cancer cells.
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@#Liposome injection is one of the most successful special injections that use nanotechnology to enhance drug efficacy and reduce accompanied toxicity. New liposomes with special structures and functions have emerged since the first liposome injection containing doxorubicin was marketed. This review summarized the principles and research progress of Stealth liposome technology and cationic liposome technology, analyzed the structural and functional characteristics and clinical application advantages of liposome products that have been marketed from the perspective of pharmacology, introduced current research hotspots of new liposomes, and analyzed the current regulatory status of liposome injection at home and abroad, thereby providing theoretical reference for the research and development（R&D）, clinical translation and supervision of liposome injection.
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To target neovasculature and tumor cells, a novel cationic liposome with verteporfin (BPD) active-loaded in lumen (CLL) was designed and its basic in vitro and in vivo behaviors were evaluated in this study. Calcium acetate gradient loading method was applied to encapsulate BPD actively and cationic lipid (2,3-dioleoy-loxy-propyl)-trimethylammonium (DOTAP) was added by post-insertion for the positive charge of CLL. Results of characterization showed that the diameter and zeta-potential of CLL were around 100 nm and 28 mV, respectively. Compared with passive loading liposomes, CLL significantly enhanced the stability of BPD loading. What's more, the loaded BPD in lumen could switch off the fluorescence and photosensitization during blood circulation by homo-fluorescence resonance energy transfer (homo-FRET) effect, leading to the diminished phototoxicity to normal tissues. In vitro cellular uptake and cytotoxicity assay exhibited that positive charge dramatically enhanced the uptake of CLL both in vascular endothelial cells and tumor cells leading to superior therapeutic efficacy. In vivo study further showed that CLL reduced the clearance rate and increased tumor accumulation compared with passive loading group. Quantitative results of exvivo organ indicated that negligible CLL distributed in normal organs contributing to low phototoxicity. Animal experiments were conducted according to the Guidelines of the Experimental Animal Ethics Committee of Peking University Health Science Center and International Animal Experiments. In conclusion, we successfully designed a novel cationic targeting liposome that overcame the limitations of passive loading and significantly enhanced the efficacy of photodynamic therapy.
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@#The combination of diagnosis and treatment through nanotechnology is conducive to the development of cancer treatment. Fluorescent imaging in the second near-infrared window(NIR-II)developed rapidly in recent years due to its imaging advantages. In this paper, we prepared a novel nano drug system, DOX-IR1061-cationic liposome, in which NIR-II fluorescent probe IR1061 was loaded as imaging agents and doxorubicin was loaded as therapeutic agents. It also explores enhanced cellular uptake and cancer cell inhibition rate through octadecylamine. Our NIR-II performance test on liposomes showed that DOX-IR1061-cationic liposome has NIR-II imaging ability. Analysis of liposome cell uptake behavior and cancer cell inhibition experiments demonstrated that octadecylamine can promote liposome uptake by cells and synergize with DOX to enhance anticancer effects. This suggests that the DOX-IR1061-cationic liposome can be used to achieve imaging and therapy effect with further research value.
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OBJECTIVE: To synthesize a novel cationic lipid,N,N-dimethyl-[N',N'-di-(stearoyl-1-ethyl)] 1,3-diaminopropane (DMSP),and evaluate its feasibility as methotrexate(MTX) carrier. METHODS: DMSP and phosphatidylcholine were employed to prepare liposomes by reverse phase evaporation method,and then MTX was entrapped by physical mixing. The entrapment efficiency was determined by ultracentrifugation,and its release ratio was evaluated by dialysis. The morphology of liposomes was observed under transmission electron microscope. The average diameter and Zeta potential were determined by laser particle size analyzer. MTT test was used to evaluate the cytotoxicity of liposomes as drug carrier and the inhibition of cancer cells growth. RESULTS: The obtained liposomes showed regular shape and uniform size,with a mean Zeta potential of +(36.26±4.77)mV and average diameter of 120 nm. The liposomes had low hemolytic activity and cytotoxicity. With the help of DMSP the cationic liposomes achieved a very high entrapment efficiency for the hydrophilic drug MTX (91.50±1.02)%. The inhibition of the MTX liposomes on cancer cells growth was much higher than that of MTX solution. CONCLUSION: DMSP is a novel cationic lipid with low cytotoxicity and high entrapment efficiency,which has a great application potential in drug delivery system.
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OBJECTIVE: To develop a polyethylene glycol (PEG) modified cationic liposomes for the co-delivery of siRNA and honokiol to improve tumor therapy. METHODS: The PEG-modified cationic liposomes were prepared by thin film hydration method. Honokiol was loaded in the liposomes with hydrophobic interaction and siRNA was loaded with electrostatic interaction. The optimal formulation was screened according to size, Zeta potential, entrapment efficiency and serum stability. The liposomes were characterized with cellular uptake and intracellular localization of siRNA. The pharmacodynamic effect of honokiol-loaded liposomes (LH) and hono-kiol-siRNA-loaded liposomes (LH-siRNA) were verified by inhibition of cancer cell growth. RESULTS: All the honokiol-loaded liposomes had an average particel size of about 100 nm with high drug entrapment efficiency. The optimal liposome formulation (N/P ratio of 5) exhibited the best cellular uptake. The results of pH-dependent hemolysis and intracellular localization suggested that the LH-siRNA had good ability of endosomal escape. In vitro cell growth experiments showed that both LH and LH-siRNA had good inhibition action on tumor cell growth based on the effects of honokiol and siRNA. CONCLUSION: The PEG-modified cationic liposomes can be a potential carrier for co-delivery of siRNA and honokiol to tumors.
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OBJECTIVE: To synthesize a novel cationic cholesterol derivative, N-(N',N'-dimethyl) propyl succinic mono-cholesteryl mono-amide (DMAPA-CHEMS), and evaluate its feasibility as a none-viral gene vehicle. METHODS: DMAPA-CHEMS and phospholipid were employed to produce liposomes by thin membrane dispersion method, and the morphology of liposomes was observed under transmission electron microscope. The average diameter and Zeta potential were determined by laser particle size analyzer. The combination to and protection for DNA were investigated via agarose gel electrophoresis. The uptake of FITC-labelled oligonucleotides into HepG2 cells was determined by flow cytometry, while the transfection promotion of liposomes for GFP-plasmid DNA was observed by inverted fluorescence microscope. RESULTS: The obtained liposomes showed regular shape and uniform size, with a mean diameter of 94.0 nm and Zeta potential of 24 mV. The liposomes could combine DNA completely and protect DNA from the degradation of DNase I when the charge ratio of cationic cholesterol to DNA was more than 4. The cationic liposomes had low cytotoxicity. Although the uptake efficiency of liposomes made of DMAPA-CHEMS was lower than that of DC-Chol, the gene expression efficiency was higher. CONCLUSION: DMAPA-CHEMS is a novel gene carrier with high transfection efficiency and low cytotoxicity, which has a great potential for application.
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The efficacy of chemotherapeutic drug in cancer treatment is often hampered by drug resistance of tumor cells, which is usually caused by abnormal gene expression. RNA interference mediated by siRNA and miRNA can selectively knock down the carcinogenic genes by targeting specific mRNAs. Therefore, combining chemotherapeutic drugs with gene agents could be a promising strategy for cancer therapy. Due to poor stability and solubility associated with gene agents and drugs, suitable protective carriers are needed and have been widely researched for the co-delivery. In this review, we summarize the most commonly used nanocarriers for co-delivery of chemotherapeutic drugs and gene agents, as well as the advances in co-delivery systems.
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Objective To evaluate the influence of preconditioning with and anti-myosinmonoclonal antibody (mAb2G4)-nuclear factor-kappa B decoy oligodeoxynucleotide (ODN)-lipofectamine (lip) on hypoxia-reoxygenation (H/R) injury in H9c2 cardiomyocytes.Methods H9c2 cardiomyocytes were seeded in 6-well plate at the density of 1×105/ml (2 ml/well),and were divided into 3 groups (n=9 each) using a random number table:control group (group C),H/R group and mAb2G4-ODN-lip group (group MOL).The cells underwent 2 h of hypoxia in an air-tight bag,followed by 1 h reoxygenation.In MOL group,the cells were treated with mAb2G4-ODN-lip (2 μg ODN) for 4 h and then cultured in the common culture medium for 8 h before hypoxia.At the end of reoxygenation,proliferation of cells was measured using MTT assay,and the cells and supernatant of the culture medium were collected to determine the activity of lactate dehydrogenas (LDH),content of malondialdehyde (MDA),concentrations of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) (by ELISA).The rate of proliferation inhibition was calculated.Results Compared with group C,the rate of proliferation inhibition,LDH activity,MDA content,and concentrations of TNF-α and IL-6 were significantly increased in the other two groups.Compared with group H/R,the rate of proliferation inhibition,LDH activity,MDA content,and concentrations of TNF-α and IL-6 were significantly decreased in MOL group.Conclusion mAb2G4-ODN-lip can mitigate H/R injury in H9c2 cardiomyocytes.
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OBJECTIVE: To investigate the preparation of novel cationic liposomes encapsulating siRNA, and evaluate their silence efficiency on target mRNA in LNCap cells. METHODS: Reverse evaporation technique was used to prepare the cationic liposomes. Protamine and calf thymus DNA were added to increase the encapsulation efficiency. The physicochemical property of the liposomes was evaluated. Human prostate cancer cells, LNCap, were used as the cell model. The silence efficiency on target gene in LNCap cells was evaluated with lipofectamine 2000 as the positive control. RESULTS: The optimum liposomes were obtained when the ratio of DOTAP and cholesterol was 3:1 and the ratio of liposomes and siRNA was 300:1. The size distribution and Zeta potential of the liposomes were (117 ± 4.3) nm and (43 ± 3.6) mV, respectively. The liposomes significantly improved siRNA accumulation in cells. CONCLUSION: The novel cationic liposome showed high transfection efficiency and distinct silence efficiency for target gene, which are expected to become a highly effective drug delivery system for anti-cancer drugs.
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Objective To investigate whether ultrasound-mediated microbubble destruction(UMD) could enhance cationic liposome (CL) induced plasmid DNA delivery or not,and optimize the transfection conditions.Methods Multiple parameters were explored to obtain the optimal transgene efficiency by means of with or without serum in culture medium,various CL or nano-liposomal bubble(NB) concentrations,different time point of ultrasonic irradiation.The transfection efficiency was assessed by fluorescence microscopy and flow cytometer,and cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assay.Results The serum could protect the cells but show little impact on transfection efficiency induced by CL.CL and plasmid DNA at a weight ratio of 4 ∶ 1 exhibited high transfection efficiency of (17.71-± 0.79)％ and high cell viability of (91.28 ± 0.76) ％.CL combining with ultrasonic irradiation at the time point of 1 hour could increase the transfection efficiency to (24.85 ± 0.78)％ (P ＜0.01).Higher transfection rate (32.47 ± 4.01) ％ was obtained by adding NB at the concentration of 10 ％ (P ＜0.05).Conclusions UMD accompanied with CL could enhance gene delivery effectively,which would provide a new method for gene therapy.
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OBJECTIVE:To investigate the effect of liposome formulation in promoting hepatocytes' uptake of tenofovir and cytotoxic effect. METHODS: The tenofovir cationic liposomes were prepared by tert-butyl lyophilization, and its encapsulation efficiency and physico-chemical property were determined. Using human hepatic carcinoma cells SMMC-7721 as a model to investigate the effect of liposome formulation in promoting hepatocytes' uptake of tenofovir. MTT assays were used to examine the toxicity of tenofovir cationic liposomes in different conditions. RESULTS: The prepared tenofovir cationic liposomes had an encapsulation efficiency of (88.3?1.6)%, a size distribution of (278.4?67.6)nm and a Zeta potential of (31?5)mV. The results showed that the liposome formulations containing galactose and PEG modifying lipids resulted in significantly higher and prolonged drug accumulation inside cells as compared with free drugs. The cell survival rates were above 80% when the tenofovir liposome was at a concentration of 7.5?g?mL-1 and lipids at a concentration of 30?g?mL-1, and little toxicity was noted. CONCLUSION: The prepared cationic liposome can greatly enhance hepatocytes' uptake and protection of tenofovir, which is expected to become a highly effective drug delivery system of antiviral drugs such as tenofovir etc.
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OBJECTIVE:To prepare and characterize lansoprazole(LAP) cationic liposomes. METHODS:Liposomes were prepared by ethanol injection technique.An orthogonal test was utilized to optimize the formulation and preparation of LAP liposomes.The encapsulation efficiency was determined by ultrafiltration.The morphological examination of LAP liposomes was performed using transmission electron microscopy.The particle size and Zeta potential of the liposomes were measured.The release rate of LAP from liposomes was tested. RESULTS:The liposomes with spherical or ellipsoidal shape and better stability featured the encapsulation efficiency of(80?1.23)%,the mean partical size of (184?21)nm,and Zeta potential of (36.1?5)mV.The release kinetics in vitro obeyed first-order equation.The stability of LAP was better. CONCULSION:The selected formulation and preparation technic of lansoprazole liposomes were rational and stable and liposomes featured a sustained release in vitro.
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@#ObjectiveTo evaluate the osteoinductivity of cationic liposome mediated recombined human bone morphogenetic protein-2 gene (rhBMP-2) transfer in skeletal muscle stem cells (SMSCs) of rabbits. MethodsSMSCs were transfered by pAGFP rhBMP-2 liposome complex. Gene transferred SMSCs were evaluated with immunohistochemistry.ResultsThe transfection rate of SMSCs transferred by cationic liposome mediated pAGFP-rhBMP-2 was 14.18%. Positive stain of rhBMP-2, alkaline phosphatase(ALP),collegenⅠhad been found in the transferred SMSCs.ConclusionQuantity of SMSCs is enough to be target cells of gene therapy. Cationic liposome can mediated bone morphogenetic protein-2 gene transfected SMSCs in vitro. Compared with AdrhBMP-2,the transfection rate of liposome mediated bone morphogenetic protein-2 gene transfected SMSCs is lower.
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Summary: The efficiency and safe range of LipofectamineTM2000 (LF2000)/bcl-xl applied in human keratocytes, the optimal ratio of LF2000/bcl-xl and the bcl-xl gene expression in human keratocytes were investiaged. By using trypan-blue staining, the effects of LF2000 and bcl-xl on the survival rate of the cultured human keratocytes were measured respectively. By using semi-quantitative RT-PCR, the efficiency and the expression of LF2000-mediated bcl-xl transfection into keratocytes were examined. The results showed that the survival rate of human keratocytes had no signficant change in the presence of LF2000 (20 μg/ml) or bcl-xl (10 μg/ml) for 24 h. LF2000 could effectively mediate the transfection of exogenous gene bcl-x1 into human keratocytes. The best transfection efficiency could be obtained when the ratio of bcl-xl/LF2000 was 1:8. One day after transfection, the positive cells for bcl-x1 could be detectable, and the positive rate reached the peak on the post-transfection day 3 (48.3 %), then gradually decreased. Fifteen days after transfection, there were few positive cells. It was suggested that LF2000 could effectively transfer the exogenous gene bcl-xl into human keratocytes without obvious toxicity during a concentration range. LF2000/bcl-xl may be likely to play an important role in gene therapy of human keratocytes.
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OBJECTIVE: To overcome the limitations of the single gene transfer, the authors present the results of wild-type p16 and p53 combined genes transfer in vitro to the U251MG and U373MG cell lines using cationic liposome as a vector. METHODS: To compare the therapeutic effect of the combined p16 and p53 genes transfer with the single p16 and p53 gene transfer, full length of wild-type human p16 and p53 gene, and combined p16-p53 genes were transferred in vitro to the U251MG and U373MG cell lines using cationic liposome as a vector. As the U251MG and U373MG cell lines are devoid of p16 and p53 genes, the therapeutic effect of the three groups of gene transfer could be evaluated by the growth suppression or percentage of the viable cells. Reverse transcription polymerase chain reaction(RT-PCR), flow cytometry, and electron microscopy(EM) were used for evaluation of the growth suppression or apoptosis of the tumor cells. RESULTS: p16 gene, p53 gene and the combined p16-p53 genes were effectively transferred to the cell lines using cationic liposome as a vector resulting in dramatic decrease of the viable tumor cells in comparison to the control group(p=0.004). The cytotoxic effect of the gene transfer in the U251MG cell line was the most significant in the combined p16-p53 group. However, in the U373MG cell line p53 single gene transfer group showed more significant effect than the combined gene transfer group. Apoptosis was confirmed by EM in the combined p16-p53 genes group. The G1 phase arrest effect, confirmed by the flow cytometry was more prevalent in the p16 gene transfer group than the other groups. CONCLUSION: Cationic liposome-mediated transfer of combined p16-p53 genes to the human glioblastoma cell lines is proven effective. However, the therapeutic effect of the combined p16-p53 genes transfer was not consistently superior to the single p16 or p53 gene transfer.
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Humans , Apoptosis , Cell Line , Flow Cytometry , G1 Phase , Genes, p16 , Genes, p53 , Glioblastoma , Liposomes , Reverse TranscriptionABSTRACT
Objective:To prepare recombinant pCDNA3.1+/Ag85A DNA vaccine encoding tuberculosis Ag85A gene mediated with LipofectamineTM 2000 and to study the effect on T cell subpopulation via oral vaccination.Methods:Recombinant plasmid containing Ag85A using liposome as a vector was constructed and administered to C57BL/6 mice via oral route.Determination of the contents of IFN-r,IL-4 in serum of C57BL/6 mice was performed by double antibody sandwich ELISA.Furthermore,the ability of splenocytes to secret IFN-? and IL-4 was tested by ELISPOT method.When the mice were vaccinated with recombinant eukaryotic expressing vector 5 weeks later,titers of serum antibody against Ag85A were detected by ELISA.The percentage of the CD4+ and CD8+ T cell subsets in the splenocytes was determined by flow cytometry.Results:Lymphocytes obtained from the spleen of liposomal pcDNA3.1+/Ag85A vaccine-immunized mice exhibited lower IFN-? production and higher IL-4 production than those of pcDNA3.1+ vector immunized mice.The number of spleen MNC secreting IFN-? stimulated by Ag85A protein in vitro was significantly lower than that of plasmid vector group.Liposomal pcDNA3.1+/Ag85A vaccine immunized mice elicited higher Ag85A-specific antibodcy titres.The percentage of the CD4+ and CD8+ T cell subsets in the splenocytes was decreased.The subset both were shown the profile of Th2 responses.Conclusion:The oral recombinant plasmid pCDNA3.1+/Ag85A mediated with LipofectamineTM 2000 It shows down-regulation effect on the subsets of CD4+ T cells and CD8+ T cells.The regimen has good immunogenecity and could induce Th2 type humoral response in C57BL/6 mice.The immunization suppresses secretion of IFN-?.It can greatly enhance the titres of Ag85A-specific antibodies.LipofectamineTM 2000 can act as an adjuvant through comparation with negative control group.
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An eukaryotic expression plasmid Rc/CMV GHcDNA containing CMV late promoter and hGH cDNA was constructed and introduced into mouse skeletal muscle by means of direct injection with or without the cationic liposome (Lipofectin and Lipofectamine reagent) intramuscularly. The expression of the gene was detected through both RT-PCR and IRMA (Immunoradiometric assay) at the level of transcription and protein respectively. The gene expression can be detected even after 90 days of single plasmid injection. Compared with the mice injected with the plasmid itself, the expression levels of mice injected with the plasmid-cationic liposome complex seem to be higher, and the expression period longer. Furthermore, the effect of Lipofectamine seems to be even better than that of Lipofectin. The results indicate that direct intramuscular injection with recombinant expression plasmid or plasmid-cationic liposome complex is a simple, efficient and economical way for foreign gene transfer and expression in experimental mice in vivo.
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Objective To preparation a new cationic liposome of which the positive component is a new cytofectin as a gene delivery vehicle for laboratory research of gene transfection and gene therapy in lung diseases. Methods The new cationic liposome was prepared by film. Electron microscopy and gel electrophoresis were employed to define the condition for transfection. The prepared cationic liposome was used in the transfection of a fluorescence plasmid to lung cancer cell line SPC and the rate of the transfection was evaluated. Results The prepared new cationic liposome was global microcapsule in size of 100 nm-1 ?m. The highest transfection rate of 60% was attained when the liposome was in a ratio of (6-8)∶1 with plasmid. Conclusion The new cationic liposome is prepared with cytofectin N 4-spermine cholesteryl carbamate as its positive component, and sound transfection activity is observed under a certain circumstance. It provides a basis for gene transfection and gene therapy.