Liposome Complexes Research Articles

Co-delivery of oxygen and erlotinib by aptamer-modified liposomal complexes to reverse hypoxia-induced drug resistance in lung cancer.

Tumor hypoxia is a common feature of the tumor microenvironment and has been regarded as one of the key factors in driving the emergence of drug resistance in solid tumors. To surmount the hypoxia-associated drug resistance, we fabricated the novel multifunctional liposomal complexes (ACLEP) that could co-deliver oxygen and molecular targeted drug to overcome the hypoxia-induced drug resistance in lung cancer. The ACLEP were fabricated with liposomes anchored with anti-EGFR aptamer-conjugated chitosan to co-administrate erlotinib and PFOB to EGFR-overexpressing non-small-cell lung cancer. Our results showed that the ACLEP possessed desired physicochemistry, good biostability and controlled drug release. The entrapped PFOB in nanoparticle facilitated the uptake of ACLEP in either normoxia or hypoxic condition. Comparing to those nanoparticles loading erlotinib alone, our innovative oxygen/therapeutic co-delivery system showed a promising outcome in fighting against hypoxia-evoked erotinib resistance both invitro and invivo. Hence, this work presents a potent drug delivery platform to overcome hypoxia-induced chemotherapy resistance.

Enhanced efficacy of anti-miR-191 delivery through stearylamine liposome formulation for the treatment of breast cancer cells

MicroRNAs are gaining rapid attention as promising targets for cancer treatment; however, efficient delivery of therapeutic miRNA or anti-miRNA into cancer cells remains a major challenge. Our previous work identified miR-191 as an oncogenic miRNA overexpressed in breast cancer that assists in progression of malignant transformation. Thus, inhibition of miR-191 using antisense miR-191 (anti-miR-191) has immense therapeutic potential. Here, we have developed a stearylamine (SA) based cationic liposome for delivery of miR-191 inhibitor (anti-miR-191), and studied its efficacy in breast cancer cells (MCF-7 and ZR-75-1) in culture. SA liposomes alone inhibited cancer cell growth with lesser IC50s (50% inhibitory concentration) values as compared to normal mouse fibroblast cells (L929). The efficient delivery of anti-miR-191 in SA liposome complex was found to be highly effective in killing the cancer cells than a comparable dose of SA free anti-miR-191 liposome complex. The formulation also showed negligible cytotoxicity in human erythrocytes. Combined treatment of SA liposome with anti-miR-191 markedly enhanced apoptotic cell death and suppressed the migration of cancer cells in vitro. Notably, anti-miR-191 loaded SA liposome complex increased chemosensitivity of breast cancer cells to currently used anti-cancer drugs (doxorubicin or cisplatin) in free form. Our work demonstrates that anti-miR-191 loaded in SA liposome complex has promising clinical application for breast cancer therapy.

Enediol-Ligands-Encapsulated Liposomes Enables Sensitive Immunoassay: A Proof-of-Concept for General Liposomes-Based Photoelectrochemical Bioanalysis.

Sensitive photoelectrochemical (PEC) bioanalysis usually relies on enzyme-assisted signal amplification. This work describes the first proof-of-concept study for liposome-based PEC bioanalysis. Specifically, unilamellar liposomes were prepared and then utilized to carry the enediol-ligands and antibodies within their internal cavities and upon their external surfaces, respectively. On the other hand, the 96-well plate was used for accommodating the sandwich immunocomplexing, and then the confined liposomes were directed to release the encapsulated enediol-ligands into an individual well. The subsequent in situ sensitization of the TiO2 nanoparticles (NPs) electrode was then used to transduce the recognition events. This facile strategy allows for sensitive immunoassay without the involvement of laborious electrode fabrication and enzymatic amplification. Importantly, the protocol can be extended as a general PEC method for numerous other targets of interest. We believe this work could offer a new perspective for the rational implementation of various liposome complexes for novel PEC bioanalysis.

Endocytosis and intracellular traffic of cholesterol-PDMAEMA liposome complexes in human epithelial-like cells

Liposomes are generally used as delivery systems, as they are capable of encapsulating a wide variety of molecules (i.e. plasmids, recombinant proteins, therapeutic drugs). However, liposomal drug delivery have to fulfill different requirements, such as the effective internalization by the target cells and avoidance of the degradative activity of the intracellular compartments. The use of polymer lipid complexes (PLCs), by including different polymers in the liposome formulation, could improve internalization and intracellular release of drugs. The aim of the present work is to study the mechanisms of cellular uptaking and the intracellular trafficking of PLCs formed with cholesterol-poly(2-(dimethylamino)ethyl methacrylate) CHO-PDMAEMA and lecithin (LC CHO-PD). Calcein-loaded liposomes were used to determine cellular uptake and intracellular localization by flow cytometry and confocal microscopy. Incorporation of CHO-PDMAEMA to lecithin liposomes enhanced the internalization capacity of PLCs. Internalization of PLCs by human epithelial-like cells (HEK-293) diminished at 4°C, suggesting uptake by endocytosis. PLCs showed no co-localization with acidic compartments after internalization. Experiments with endocytosis inhibitors and co-localization of liposomes and albumin, suggested the caveolae endocytic pathway as the most probable route for intracellular trafficking of PLCs.In this work, we demonstrated an efficient uptake of LC CHO-PDs by human epithelial-like cells (HEK-293) through the non-degradative caveolae endocytic pathway. The mode of internalization and the intracellular fate of liposomes under study, suggest a promising use of LC CHO-PDs as drug delivery systems.

Cationic liposome-mediated delivery of reovirus enhances the tumor cell-killing efficiencies of reovirus in reovirus-resistant tumor cells

Reovirus induces tumor cell death efficiently and specifically, and thus is currently undergoing clinical testing as an anticancer agent. In the intracellular trafficking of reovirus, proteolytic disassembly of reovirus capsid-proteins and subsequent penetration of viral particles into the cytosol are crucial steps. Cathepsins B and L are largely responsible for the proteolytic disassembly of reovirus. Reovirus efficiently lyses tumor cells exhibiting relatively high activities of cathepsins B and L, while tumor cells with low activities of cathepsins B and L are often refractory to reovirus, probably due to inefficient endo/lysosomal escape. In this study, in order to enhance the tumor cell-killing efficiencies of reovirus by promoting endo/lysosomal escape, especially in reovirus-resistant tumor cells, reovirus was complexed with a cationic liposome transfection reagent. Reovirus alone and reovirus-cationic liposome complex (reoplex) exhibited similar levels of tumor cell-killing efficiencies in reovirus-susceptible tumor cells, while reoplex mediated more than 30% higher levels of tumor cell-killing activities in reovirus-resistant tumor cells than reovirus alone. Reoplex-mediated tumor cell death was efficiently induced in the tumor cells pretreated with cathepsin inhibitors. The mRNA levels of interferon (IFN)-β and apoptotic genes were significantly elevated following reoplex treatment. These results suggest that cationic liposomes efficiently promoted delivery of reovirus to the cytosol, leading to induction of apoptosis.

Prevention of liver tumor formation in woodchucks with established hepatocellular carcinoma by treatment with cationic liposome-DNA complexes

BackgroundApproximately 250 million people worldwide are chronically infected with hepatitis B virus (HBV) and more than half of the hepatocellular carcinoma (HCC) cases are attributed to this infection. As HCC has a high mortality rate, and current treatment options are remarkably limited, the development of new therapeutic treatment strategies is warranted.MethodsIn this study, woodchucks infected with woodchuck hepatitis virus (WHV), and with pre-existing liver tumors, were used as a model to investigate if complexes of cationic liposomes and non-coding DNA (JVRS-100) were effective in treatment of HCC.ResultsIt was observed that the high serum viral load that is present in a typical chronic WHV infection (i.e., approximately 100-fold higher than human viral loads) results in immune suppression and resistance to treatment with JVRS-100. Treatment of woodchucks with lower serum viral load that more closely matched with the viral load usually seen in human HBV infection appears a better model for immunotherapeutic development based on the responsiveness to JVRS-100 treatment. In the latter case, marked declines in WHV DNA and WHV surface antigen were determined over the 12-week treatment period and WHV markers stayed suppressed during most time points of the 12-week follow-up period. Even more remarkably, the formation of new liver tumors was not observed in woodchucks treated with a well-tolerated dose of JVRS-100, as compared to several new tumors that developed in vehicle-treated control animals.ConclusionsAlthough there was little decrease in the volumes of the liver tumors existing at the time of treatment, it is generally accepted that preventing the spread and metastasis of almost always fatal cancers such as HCC and thus, reducing it to a chronic and treatable disease can also be a successful therapeutic approach. The results in woodchucks warrant the investigation of JVRS-100 as an intervention to prevent liver cancer in patients chronically infected with HBV and at high risk for HCC development.

Multifunctional quantum dots and liposome complexes in drug delivery.

Incorporating both diagnostic and therapeutic functions into a single nanoscale system is an effective modern drug delivery strategy. Combining liposomes with semiconductor quantum dots (QDs) has great potential to achieve such dual functions, referred to in this review as a liposomal QD hybrid system (L-QD). Here we review the recent literature dealing with the design and application of L-QD for advances in bio-imaging and drug delivery. After a summary of L-QD synthesis processes and evaluation of their properties, we will focus on their multifunctional applications, ranging from in vitro cell imaging to theranostic drug delivery approaches.

Aptamer-Conjugated Chitosan-Anchored Liposomal Complexes for Targeted Delivery of Erlotinib to EGFR-Mutated Lung Cancer Cells.

Lung cancer is the leading cancer and has the highest death rate. The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib has had a promising response in lung cancer therapy. Unfortunately, individuals with TKI-resistant EGFR mutations often develop acquired resistance against erlotinib. To overcome this resistance, in the present study, we developed liposomes anchored with anti-EGFR aptamer (Apt)-conjugated chitosan (Apt-Cs) as stable carriers to deliver erlotinib to the target. We loaded erlotinib into Apt-Cs-anchored liposomal complexes (Apt-CL-E) and characterized the physicochemistry of Apt-CL-E. The nanoparticles showed good biostability and a binding specificity for EGFR-mutated cancer cells guided by the Apt. The specific binding facilitated the uptake of Apt-CL-E into EGFR-mutated cancer cells. A cytotoxicity study showed an advantage of Apt-CL-E over their nontargeted liposomal counterparts in delivering erlotinib to EGFR-mutated cancer cells, resulting in cell cycle arrest and apoptosis. These results provide a good platform for future in vivo animal studies with Apt-CL-E.

Payload release by liposome burst: Thermal collapse of microgels induces satellite destruction

We present a smart liposome carrier system for stimulated release, consisting of cationic, thermo-responsive microgels. At low temperature, the swollen microgels adsorb about 200 anionic liposomes, 50 nm in diameter, per microgel. When heated from 39 °C to 41 °C, the microgel–liposome complex particles collapse from approx. 370 nm down to approx. 270 nm. Upon the thermo-induced collapse, the adsorbed liposome satellite layer is squeezed until the initially spherical liposomes explode and release their payload (antitumor drug doxorubicin) into the surrounding. This burst release mechanism, taking place over a narrow temperature range, is newly reported and of possible biomedical importance.

Transfection Studies with Colloidal Systems Containing Highly Purified Bipolar Tetraether Lipids from Sulfolobus acidocaldarius.

Lipid vectors are commonly used to facilitate the transfer of nucleic acids into mammalian cells. In this study, two fractions of tetraether lipids from the archaea Sulfolobus acidocaldarius were extracted and purified using different methods. The purified lipid fractions polar lipid fraction E (PLFE) and hydrolysed glycerol-dialkyl-nonitol tetraether (hGDNT) differ in their structures, charge, size, and miscibility from conventional lipids. Liposomes were prepared by mixing tetraether lipids with cholesterol (CH) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) resulting in stable vectors for gene delivery. Lipoplexes were prepared by complexation of liposomes with a luciferase expressing plasmid (pCMV-luc) at certain nitrogen-to-phosphorus (N/P) ratios and optimised for the transient transfection of ovarian adenocarcinoma cells (SK-OV-3). Complexation efficacy was investigated by gel-red fluorescence assay. Biophysical properties, like size, surface charge, and morphology, were investigated by differential light scattering (DLS), atomic force microscopy (AFM), and scanning electron microscopy (Cryo-SEM), respectively, revealing structural differences between liposomes and lipoplexes. A range of stable transfecting agents containing tetraether lipids were obtained by incorporating 5 mol% of tetraether lipids. Lipoplexes showed a decrease in free gel-red with increasing N/P ratios indicating efficient incorporation of plasmid DNA (pDNA) and remarkable stability. Transfection experiments of the lipoplexes revealed successful and superior transfection of SK-OV-3 cell line compared to the commercially available DOTAP and branched polyethyleneimine (25 kDa bPEI).

Physicochemical properties of lipopeptide-based liposomes and their complexes with siRNA

siRNA/cationic liposome complexes are efficient systems for transmembrane delivery. The aim of this study was to prepare a novel complex consisted of lipotripeptide OrnOrnGlu(C16H33)2 and siRNA molecule and examined their physicochemical properties. Electron microscopy study has shown that the siRNA/liposome complex (m/m 1/10) tends to form sandwich-like structures that may protect nucleic acid from nuclease degradation. Photon correlation spectroscopy data indicate that the particle size increased after siRNA adding, but did not exceed 300 nm in diameter, while z-potential of lipoplexes decreased from 22 mV to 14 mV, compared to the empty liposomes thus indicating positive charge neutralization by negatively charged siRNA. These data allow to hypothesize that such size and total positive charge could provide efficient cellular uptake by endocytosis. That may have good prospects for gene silencing therapy.

A comparison between conventional liposome and drug-cyclodextrin complex in liposome system

A drug-cyclodextrin complex in liposome system was prepared in order to make a comparison with conventional risperidone-loaded liposome. Thin film hydration, reverse phase evaporation and ethanol injection methods were taken as preparation means to obtain the two types of liposome. Differential thermal analysis (DTA) and transmission electron microscopy (TEM) were used to investigate the thermal characters of inclusion complexes and morphology of liposome, respectively. Particle size, zeta potential and encapsulation efficiency were studied by light scattering analysis and ultrafiltration. In vitro release was carried out in the pH 7.4 phosphate buffer solution and samples were collected at the certain time. As a result, drug-cyclodextrin complex in liposome prepared by various methods displayed lower encapsulation efficiency than conventional liposome. However, size was larger and its stability was better than the latter. The second release phase of novel delivery system was slightly slower after initial burst release at the first phase, while the conventional liposome displayed a more regular trait. Thus, the novel liposome have potential to be developed as co-administration formulation with long-acting injection.

Effect of a monoclonal antibody against human relaxin-2 on cancer cell growth inhibition

Relaxin-2 has gained much attention because of its importance in cancer cell proliferation, invasion, and angiogenesis. However, the potential of relaxin-2 as a target for immunotherapy has never been directly validated before. In the present study, we produced a human relaxin-2-specific antibody by immunizing mice using a specific liposome complex containing relaxin-2 epitope peptide and CpG-DNA. We isolated a hybridoma clone, 1B11F12, producing the anti-relaxin-2 monoclonal antibody. We confirmed with immunoprecipitation analysis that the antibody specifically recognizes native human relaxin-2. Treatment with the antibody suppressed the growth of some cancer cell lines. Therefore, we suggest that relaxin-2 could be a reasonable target of cancer therapeutics, and the monoclonal antibody could be used to treat cancer in humans.

Inhibitory effect of RNA interference targeting GFI-1 on the proliferation of atypical chronic myelogenous leukemia NT1 cells

To investigate the inhibitory effects of RNA interference targeting GFI-1 on growth and proliferation of atypical chronic myelogenous leukemia (aCML) NT1 cells. NT1 cells were transfected with PBS and liposome complex (vehicle group), scrambled siRNA and liposome complex (negative control, NC group), and GFI-1 siRNA and liposome complex (GFI-1 siRNA group), respectively. Real-time quantitative RT-PCR (qRT-PCR) and Western blot were performed to examine the expression levels of GFI-1 mRNA and protein, respectively. The proliferation abilities of NT1 cells of the three groups were evaluated by MTT assay. The cell cycle in cells of the three groups was analyzed by flow cytometry. Moreover, nude mouse xenograft model was used to detect the tumor formation ability in the three group cells. Quantitative real-time PCR data showed that the expression level of GFI-1 mRNA in GFI-1 siRNA group was significantly lower than those of NC group and vehicle group [(0.367±0.017) vs. (0.918±0.006) and (1.010±0.005), respectively, (P<0.05)]. Western blot results showed that the GFI-1 protein expression level in the GFI-1 siRNA group was also significantly reduced, compared with those of the NC group and vehicle group (P<0.05 for both). From MTT assay data, the absorbance value of NT1 cells in the GFI-1 siRNA group (0.667±0.059) was significantly lower than those of the NC group (1.096±0.049) and vehicle group (1.193±0.064, P=0.023). Flow cytometry data showed that sub-G1 and G0/G1 phase proportions of the GFI-1 siRNA group were significantly higher than those of the NC and vehicle groups [sub-G1: (8.2±2.5)% vs. (1.9±1.3)% and (2.0±3.6)%, respectively, (P<0.05); G0/G1: (66.7±3.8)% vs. (53.3±4.5)% and (48.6±3.2)%, respectively, (P<0.05)]. Furthermore, the tumor weight in the GFI-1 siRNA group [(0.37±0.02) g] was significantly lower than those in the NC group [(0.83±0.06) g] and vehicle group [(0.92±0.04) g] (P<0.05). RNA interference targeting GFI-1 inhibits the growth and proliferation of NT1 cells, which may provide a new therapeutic target for atypical chronic myelogenous leukemia.

A Phase l Study of a Tumor-targeted Systemic Nanodelivery System, SGT-94, in Genitourinary Cancers

Gene therapy development has been limited by our inability to target multifocal cancer with systemic delivery. We developed a systemically administered, tumor-targeted liposomal nanodelivery complex (SGT-94) carrying a plasmid encoding RB94, a truncated form of the RB gene. In preclinical studies, RB94 showed marked cytotoxicity against tumor but not normal cells. SGT-94 was administered intravenously in a first-in-man study in metastatic genitourinary cancer. Minimal side effects were observed; dose-limiting toxicity (DLT) has not been reached in 11 evaluable patients. There was evidence of clinical activity at the 2.4 mg dose with one complete remission (CR) and one partial remission (PR). The patient in CR was retreated upon progression and had a second PR. Furthermore, there was tumor-specific targeting of the SGT-94 complex. One patient had wedge resections of two lung metastases which demonstrated RB94 expression at the DNA level by polymerase chain reaction (PCR) and at the protein level by Western blotting, with no RB94 present in normal contiguous lung. In conclusion, systemically delivered SGT-94 showed evidence of selective tumor targeting and was well tolerated with evidence of clinical activity. Additional studies are warranted to explore the activity of this drug as a single agent and in combination therapy.

A randomised, double-blind, placebo-controlled trial of repeated nebulisation of non-viral cystic fibrosis transmembrane conductance regulator (CFTR) gene therapy in patients with cystic fibrosis

BackgroundCystic fibrosis (CF) is a chronic, life-limiting disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene leading to abnormal airway surface ion transport, chronic lung infections, inflammation and eventual respiratory failure. With the exception of the small-molecule potentiator, ivacaftor (Kalydeco®, Vertex Pharmaceuticals, Boston, MA, USA), which is suitable for a small proportion of patients, there are no licensed therapies targeting the basic defect. The UK Cystic Fibrosis Gene Therapy Consortium has taken a cationic lipid-mediatedCFTRgene therapy formulation through preclinical and clinical development.ObjectiveTo determine clinical efficacy of the formulation delivered to the airways over a period of 1 year in patients with CF.DesignThis was a randomised, double-blind, placebo-controlled Phase IIb trial of theCFTRgene–liposome complex pGM169/GL67A. Randomisation was performed via InForm™ version 4.6 (Phase Forward Incorporated, Oracle, CA, USA) and was 1 : 1, except for patients in the mechanistic subgroups (2 : 1). Allocation was blinded by masking nebuliser chambers.SettingsData were collected in the clinical and scientific sites and entered onto a trial-specific InForm, version 4.6 database.ParticipantsPatients with CF aged ≥ 12 years with forced expiratory volume in the first second (FEV1) between 50% and 90% predicted and any combination ofCFTRmutations. The per-protocol group (≥ 9 doses) consisted of 54 patients receiving placebo (62 randomised) and 62 patients receiving gene therapy (78 randomised).InterventionsSubjects received 5 ml of nebulised pGM169/G67A (active) or 0.9% saline (placebo) at 28 (±5)-day intervals over 1 year.Main outcome measuresThe primary end point was the relative change in percentage predicted FEV1over the 12-month period. A number of secondary clinical outcomes were assessed alongside safety measures: other spirometric values; lung clearance index (LCI) assessed by multibreath washout; structural disease on computed tomography (CT) scan; the Cystic Fibrosis Questionnaire – Revised (CFQ-R), a validated quality-of-life questionnaire; exercise capacity and monitoring; systemic and sputum inflammatory markers; and adverse events (AEs). A mechanistic study was performed in a subgroup in whom transgene deoxyribonucleic acid (DNA) and messenger ribonucleic acid (mRNA) was measured alongside nasal and lower airway potential difference.ResultsThere was a significant (p = 0.046) treatment effect (TE) of 3.7% [95% confidence interval (CI) 0.1% to 7.3%] in the primary end point at 12 months and in secondary end points, including forced vital capacity (FVC) (p = 0.031) and CT gas trapping (p = 0.048). Other outcomes, although not reaching statistical significance, favoured active treatment. Effects were noted by 1 month and were irrespective of sex, age orCFTRmutation class. Subjects with a more severe baseline FEV1had a FEV1TE of 6.4% (95% CI 0.8% to 12.1%) and greater changes in many other secondary outcomes. However, the more mildly affected group also demonstrated benefits, particularly in small airway disease markers such as LCI. The active group showed a significantly (p = 0.032) greater bronchial chloride secretory response. No difference in treatment-attributable AEs was seen between the placebo and active groups.ConclusionsMonthly application of the pGM169/GL67A gene therapy formulation was associated with an improvement in lung function, other clinically relevant parameters and bronchial CFTR function, compared with placebo.LimitationsAlthough encouraging, the improvement in FEV1was modest and was not accompanied by detectable improvement in patients’ quality of life.Future workFuture work will focus on attempts to increase efficacy by increasing dose or frequency, the coadministration of a CFTR potentiator, or the use of modified viral vectors capable of repeated administration.Trial registrationClinicalTrials.gov NCT01621867.FundingThis project was funded by the Efficacy and Mechanism Evaluation (EME) programme, a Medical Research Council and National Institute for Health Research partnership.

Effect of glycol chitosan on functional and structural properties of anionic liposomes

The possibility of using glycol chitosan to obtain stabilized liposome containers for doxorubicin delivery is demonstrated. The dissociation constants of the liposome complexes with glycol chitosan (3.4 × 10–4 and 1.10 × 10–5 depending on the aggregation state of the liposomes) are determined. It is shown that the formation of liposome complexes with glycol chitosan has a significant prolongation effect on the release of doxorubicin from the liposomes at pH 7.4.

Optimization of a cationic liposome-based gene delivery system for the application of miR-145 in anticancer therapeutics.

In order to improve the delivery efficiency of microRNA (miRNA or miR)-145, the present study examined several factors which may affect cationic liposome (CL)-based transfection, including the hydration medium used for the preparation of liposomes, the quantity of the plasmid, the molar ratio of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP)/cholesterol (chol), or DOTAP/chol, and the weight ratio of DOTAP/DNA. In order to enhance the transfection efficiency, protamine was selected as a DNA-condensing agent to form liposome‑protamine‑DNA (LPD) ternary complexes. An agarose gel retardation assay was used to examine the DNA binding affinity of the CLs. Following transfection, GFP fluorescence images were captured and flow cytometry was performed to determine the transfection efficiency. Furthermore, an MTT assay was performed to determine the cytotoxicity of the liposome complexes. The final optimal conditions were as follows: 5% glucose as the hydration medium, a molar ratio of DOTAP/chol at 3:1 for the preparation of CLs, a weight ratio of DOTAP/protamine/DNA of 3:0.5:1, with 8 µg plasmid added for the preparation of the LPD complexes. In vitro, the LPD complexes exhibited an enhanced transfection efficiency and low cytotoxicity, which indicated that the presented LPD vector enhanced the transfection efficiency of the CLs. The HepG2 cells were found to have the lowest expression levels of miR‑145 out of the cell lines tested (A549, BGC-823, HepG2, HeLa, LoVo and MCF-7). Following the transient transfection of the HepG2 cells with miR‑145, the results revealed that the overexpression of miR‑145 inhibited the proliferation of the HepG2 cells and downregulated the expression of cyclin-dependent kinase 6 (CDK6), cyclinD1, c-myc, and Sp1 transcription factor (Sp1). In conclusion, in this study, we optimized a liposome‑based delivery system for the efficient delivery of miR‑145 into cancer cells. This may provide a foundation for further research into the use of miR‑145 in anticancer therapeutics.

Self-assembled Micelle Interfering RNA for Effective and Safe Targeting of Dysregulated Genes in Pulmonary Fibrosis

The siRNA silencing approach has long been used as a method to regulate the expression of specific target genes in vitro and in vivo. However, the effectiveness of delivery and the nonspecific immune-stimulatory function of siRNA are the limiting factors for therapeutic applications of siRNAs. To overcome these limitations, we developed self-assembled micelle inhibitory RNA (SAMiRNA) nanoparticles made of individually biconjugated siRNAs with a hydrophilic polymer and lipid on their ends and characterized their stability, immune-stimulatory function, and in vivo silencing efficacy. SAMiRNAs form very stable nanoparticles with no significant degradation in size distribution and polydispersity index over 1 year. Overnight incubation of SAMiRNAs (3 μm) on murine peripheral blood mononuclear cells did not cause any significant elaboration of innate immune cytokines such as TNF-α, IL-12, or IL-6, whereas unmodified siRNAs or liposomes or liposome complexes significantly stimulated the expression of these cytokines. Last, the in vivo silencing efficacy of SAMiRNAs was evaluated by targeting amphiregulin and connective tissue growth factor in bleomycin or TGF-β transgenic animal models of pulmonary fibrosis. Intratracheal or intravenous delivery two or three times of amphiregulin or connective tissue growth factor SAMiRNAs significantly reduced the bleomycin- or TGF-β-stimulated collagen accumulation in the lung and substantially restored the lung function of TGF-β transgenic mice. This study demonstrates that SAMiRNA nanoparticle is a less toxic, stable siRNA silencing platform for efficient in vivo targeting of genes implicated in the pathogenesis of pulmonary fibrosis.

A COMPARISON OF UNILAMELLAR AND MULTILAMELLAR LIPOPLEXES IN TERMS OF TRANSFECTION EFFICIENCY OF HUMAN EMBRYONIC KIDNEY CELLS

Non-viral gene delivery systems have attracted many attentions in the field of gene therapy. Liposome nanoparticles are regarded as one of the most effective non-viral gene delivery vectors. With attention to the fact that one important feature of these carries is their transfection efficiency, in this study we compared the efficiency of unilamellar and multilamellar liposomes in transfecting human embryonic kidney cells by LacZ gene. A liposomal formulation made of DOTAP/DOPE/Cholesterol was fabricated through modified lipid film hydration method. In order to obtain unilamellar lipoplexes extrusion was performed. Lipoplexes were prepared by complexation of liposomes with LacZ gene. Zeta potential, size and polydispersity of lipoplexes and liposomes were measured. In order to determine transfection ability of them, LacZ expression was measured using beta-galactosidase assay. The results show that multilamellar liposomes in spite of their larger sizes have higher transfection compared to unilamellar ones. This is indicative of better performance of multilamellar lipoplexes in gene therapy applications.