1-2-dioleoyloxy-3-(trimethylammonium)propane and Lung-Neoplasms

Tumor suppressor gene TUSC2/FUS1 (TUSC2) is frequently inactivated early in lung cancer development. TUSC2 mediates apoptosis in cancer cells but not normal cells by upregulation of the intrinsic apoptotic pathway. No drug strategies currently exist targeting loss-of-function genetic abnormalities. We report the first in-human systemic gene therapy clinical trial of tumor suppressor gene TUSC2.. Patients with recurrent and/or metastatic lung cancer previously treated with platinum-based chemotherapy were treated with escalating doses of intravenous N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP):cholesterol nanoparticles encapsulating a TUSC2 expression plasmid (DOTAP:chol-TUSC2) every 3 weeks.. Thirty-one patients were treated at 6 dose levels (range 0.01 to 0.09 milligrams per kilogram). The MTD was determined to be 0.06 mg/kg. Five patients achieved stable disease (2.6-10.8 months, including 2 minor responses). One patient had a metabolic response on positron emission tomography (PET) imaging. RT-PCR analysis detected TUSC2 plasmid expression in 7 of 8 post-treatment tumor specimens but not in pretreatment specimens and peripheral blood lymphocyte controls. Proximity ligation assay, performed on paired biopsies from 3 patients, demonstrated low background TUSC2 protein staining in pretreatment tissues compared with intense (10-25 fold increase) TUSC2 protein staining in post-treatment tissues. RT-PCR gene expression profiling analysis of apoptotic pathway genes in two patients with high post-treatment levels of TUSC2 mRNA and protein showed significant post-treatment changes in the intrinsic apoptotic pathway. Twenty-nine genes of the 82 tested in the apoptosis array were identified by Igenuity Pathway Analysis to be significantly altered post-treatment in both patients (Pearson correlation coefficient 0.519; p<0.01).. DOTAP:chol-TUSC2 can be safely administered intravenously in lung cancer patients and results in uptake of the gene by human primary and metastatic tumors, transgene and gene product expression, specific alterations in TUSC2-regulated pathways, and anti-tumor effects (to our knowledge for the first time for systemic DOTAP:cholesterol nanoparticle gene therapy).. ClinicalTrials.gov NCT00059605.

Topics: Apoptosis; Dose-Response Relationship, Drug; Fatty Acids, Monounsaturated; Gene Expression Profiling; Gene Transfer Techniques; Genetic Therapy; Humans; Lung Neoplasms; Maximum Tolerated Dose; Nanoparticles; Plasmids; Positron-Emission Tomography; Quaternary Ammonium Compounds; Reverse Transcriptase Polymerase Chain Reaction; Tumor Suppressor Proteins

The molecular targeted drug ATRA demands a suitable carrier that delivers to the cancer site due to its poor bioavailability and drug resistance. ATRA, being a lipid with carboxylic acid, has been nano-formulated as a cationic lipo-ATRA with DOTAP:cholesterol:ATRA (5:4:1) and its pH-responsive release, intracellular drug accumulation, and anticancer effect on human lung cancer (A549) cell line analysed. The analysis of the physicochemical characteristics of the developed lipo-ATRA (0.8 µmol) revealed that the size of 231 ± 2.35 d.nm had a zeta potential of 6.4 ± 1.19 and an encapsulation efficiency of 93.7 ± 3.6%. The ATRA release from lipo-ATRA in vitro was significantly (p ≤ 0.05) higher at acidic pH 6 compared to pH 7.5. The intracellular uptake of ATRA into lipo-ATRA-treated A549 cells was seven-fold higher (0.007 ± 0.001 mg/ml) while only three-fold uptake was observed in free ATRA treatment (0.003 ± 0.002 mg/ml). The lipo-ATRA treatment caused a highly significant (p ≤ 0.001) decrease in percent cell viability at 48 h when compared with the free ATRA treatment. Overall, the results proved that the developed lipo-ATRA has suitable physicochemical properties with enhanced ATRA release at acidic pH, while maintaining stability at physiologic pH and temperature. This resulted in an increased ATRA uptake by lung cancer cells with enhanced treatment efficiency. Hence, it is concluded that DOTAP lipo-ATRA is a suitable carrier for ATRA delivery to solid cancer cells.

Topics: Cholesterol; Fatty Acids, Monounsaturated; Humans; Hydrogen-Ion Concentration; Liposomes; Lung Neoplasms; Quaternary Ammonium Compounds; Tretinoin

Immunization with synthetic mRNA encoding tumor-associated antigens is an emerging vaccine strategy for the treatment of cancer. In order to prevent mRNA degradation, promote antigen-presenting cells antigen presentation, and induce an anti-tumor immune response, we investigated the nasal administration of mRNA vaccines with positively charged protamine to concentrate mRNA, form a stable polycation-mRNA complex, and encapsulate the complex with DOTAP/Chol/DSPE-PEG cationic liposomes. Cationic liposome/protamine complex (LPC) showed significantly greater efficiency in uptake of vaccine particles in vitro and stronger capacities to stimulate dendritic cell maturation, which further induced a potent anti-tumor immune response. Intranasal immunization of mice with cationic LPC containing mRNA encoding cytokeratin 19 provoked a strong cellular immune response and slowed tumor growth in an aggressive Lewis lung cancer model. The results of this study provide evidence that cationic LPC can be used as a safe and effective adjuvant and this mRNA formulation provides a basis for anti-cancer vaccination of humans.

Topics: Administration, Intranasal; Animals; Cancer Vaccines; Carcinoma, Lewis Lung; Cell Differentiation; Dendritic Cells; Fatty Acids, Monounsaturated; Female; Humans; Immunotherapy; Keratin-19; Liposomes; Lung Neoplasms; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Protamines; Quaternary Ammonium Compounds; RNA, Messenger; Tumor Burden

Targeting the specific molecular proteins or genes which are responsible for the suppression of cancer growth is currently an emerging molecular method to treat cancer. ATRA treatment is now considered as a molecular targeted therapy for many cancers. As ATRA exhibits its therapeutic effect through its receptors, this study was focused to investigate the effect and action of liposomal-ATRA treatment on the expression of RAR-β protein which is also a tumor suppressor. The liposomal-ATRA was developed with cationic DOTAP and cholesterol by thin-film formation method. The benzo(a)pyrene(50 mg/kg b.wt)-induced mice were treated with free and liposomal-ATRA(0.60 mg/kg b.wt). The RAR-β protein expression in lung and liver tissue samples were analyzed by immunohistochemistry (IHC) and western blotting (WB) on the 30th and 120th days. Almost nil expression of RAR-β protein was observed in B(a)P cancer control and liposome alone-treated groups. A comparatively elevated expression was seen in the free ATRA-treated group (IHC score-2+ in lung on the 120th day with band density of 14.46 ± 1.24% in WB). Interestingly, the liposomal-ATRA treatment demonstrated a significantly (p ≤ 0.01) higher RAR-β expression in lung (35.20 ± 3.398% band intensity and score 4+ in the 120th day) than that of in ATRA alone treatment. This study results indicate that the RAR-β protein expression was suppressed by B(a)P during cancer induction even on the 30th day itself. The treatment could reactivate the suppression and the lipo-ATRA treatment could show significantly higher RAR-β expression on the 120th day, which implies that it accumulated more ATRA in target site and sustained it for enhanced action.

Topics: Animals; Antineoplastic Agents; Benzo(a)pyrene; Cholesterol; Disease Models, Animal; Fatty Acids, Monounsaturated; Liposomes; Liver; Lung; Lung Neoplasms; Male; Mice; Quaternary Ammonium Compounds; Receptors, Retinoic Acid; Tretinoin

The purpose of this study was to assess whether cationic nanoliposomes could address tumor vaccines to dendritic cells in the lungs in vivo. Nanoliposomes were prepared using a cationic lipid, dimethylaminoethanecarbamoyl-cholesterol (DC-cholesterol) or dioleoyltrimethylammoniumpropane (DOTAP), and dipalmitoylphosphatidylcholine (DPPC), the most abundant phospholipid in lung surfactant. The liposomes presented a size below 175 nm and they effectively entrapped tumor antigens, an oligodeoxynucletotide containing CpG motifs (CpG) and the fluorescent dye calcein used as a tracer. Although the liposomes could permanently entrap a large fraction of the actives, they could not sustain their release in vitro. Liposomes made of DOTAP were safe to respiratory cells in vitro, while liposomes composed of DC-cholesterol were cytotoxic. DOTAP nanoliposomes were mainly taken up by alveolar macrophages following delivery to the lungs in mice. Few dendritic cells took up the liposomes, and interstitial macrophages did not take up liposomal calcein more than they took up soluble calcein. Stimulation of the innate immune system using liposomal CpG strongly enhanced uptake of calcein liposomes by all phagocytes in the lungs. Although a small percentage of dendritic cells took up the nanoliposomes, alveolar macrophages represented a major barrier to dendritic cell access in the lungs.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Adjuvants, Immunologic; Animals; Cancer Vaccines; Cell Line, Tumor; Cell Survival; Cholesterol; CpG Islands; Dendritic Cells; Drug Delivery Systems; Fatty Acids, Monounsaturated; Female; Fluoresceins; Fluorescent Dyes; gp100 Melanoma Antigen; Lipopeptides; Liposomes; Lung; Lung Neoplasms; Macrophages, Alveolar; MART-1 Antigen; Mice; Nanoparticles; Quaternary Ammonium Compounds; Tissue Distribution

Molecular targeted therapy for specific genes is an emerging research. Retinoic Acid Receptor (RAR-β) is a key tumor suppressor which is found to be lost drastically during much cancer progression. We hence, analyzed the expression level of RAR-β gene during B(a)P induced lung cancer development in mice and studied the lung cancer targeted action of All Trans Retinoic Acid (ATRA) in DOTAP liposomal formulation. The effect of its treatment on lung cancer was determined by histopathological analysis. RAR-β gene expression was assessed by RT-PCR and qPCR. A distinct band for RAR-β gene (density - 0.5123 for lung and 0.5160 for liver) was observed in normal mice, whereas no visible band was observed in cancer induced group, indicating loss of RAR-β gene expression. Both ATRA and lipo-ATRA treated groups showed detectable RAR-β expression with relatively lesser density than the normal group. The expression was more intense in lipo-ATRA treatment (density-0.2973) compared with free ATRA treatment (density-0.1549) in lung tissues. The qPCR results also have highlighted a highly significant (p ≤ 0.01) variation RQ values between lipo-ATRA group (15.46 ± 1.54) and free ATRA group (7.58 ± 1.30) in lung tissue sample on 30th day. The mean RQ value for normal lung on 30th day was 20.86 ± 2.58 against the cancer control. The 120th day mice also showed the similar RAR-β expression pattern with further declined expression levels as there was no treatment given after 30 days. Interestingly, the lipo-ATRA treatment could show a highly significant (p ≤ 0.001) expression (12.00 ± 2.31) when compared with free ATRA treatment (3.31 ± 0.58) which implies that the lipo-ATRA formulation could result in sustained delivery of ATRA in target site. Histopathology of lung and liver on 120th day also revealed an effective therapeutic indication in lipo-ATRA treatment compared to free ATRA treatment due to lipo-ATRA's stealth property and it efficiently inhibited the metastasis to liver. These results revealed that the lipo-ATRA treatment has efficiently delivered ATRA into target site than free ATRA and in-turn it might have induced the expression of RAR-β gene or prevented loss of RAR-β gene in cancer animals.

Topics: Animals; Antineoplastic Agents; Benzo(a)pyrene; Fatty Acids, Monounsaturated; Gene Expression; Liposomes; Liver; Lung; Lung Neoplasms; Mice; Quaternary Ammonium Compounds; Receptors, Retinoic Acid; Tretinoin; Up-Regulation

DESI2 is a novel pro-apoptotic gene. We previously reported that DESI2 overexpression induces S phase arrest and apoptosis by activating checkpoint kinases. This work was to test whether the combination of endostatin, an endogenous antiangiogenic inhibitor, with DESI2 could improve the therapy efficacy in vitro and in vivo. The recombinant plasmid co-expressing DESI2 and endostatin was encapsulated with DOTAP/Cholesterol cationic liposome. Mice bearing CT26 colon carcinoma and LL2 lung cancer were treated with the DNA-liposome complex. We found that, in vitro, the combination of DESI2 and endostatin more efficiently inhibited proliferation of CT26, LL2, HCT116 and A549 cancer cells via apoptosis, as assessed by MTT assay, colony-formation assays, flow cytometric analysis, hoechst staining and activation of caspase-3, respectively. In addition, DESI2 overexpression caused up-regulation of RPS7, a substrate of DESI2 deubiquitination. Furthermore, siRNA targeting RPS7 partially abrogated, whereas RPS7 overexpression enhanced DESI2-induced inhibition of cell proliferation. Importantly, the combination also caused DNA lesions accumulation, which further promotes apoptosis. Mechanistic rationale suggested that endostatin first inhibits DNA-PKcs kinase, and partly abrogated DESI2-induced phosphorylation of DNA-PKcs, leading to increase of DNA damage, then contributes to DESI2-induced apoptosis. In vivo, the combined gene therapy more significantly inhibited tumor growth and efficiently prolonged the survival of tumor bearing mice than mono therapy. The improved antitumor effect was associated with inhibition of cell proliferation via apoptosis, as analyzed by TUNEL assay and PCNA immunostaining. The combination also inhibited angiogenesis, as assessed by alginate-encapsulated tumor cell assay and CD31 staining. Our data suggest that the combined gene therapy of DESI2 and endostatin can significantly enhance the antitumor activity as a DNA lesions accumulator, apoptosis inducer and angiogenesis inhibitor. The present study may provide a novel method for the treatment of cancer.

Topics: A549 Cells; Angiogenesis Inhibitors; Animals; Apoptosis; Carbon-Nitrogen Lyases; Caspase 3; Cell Proliferation; Cholesterol; Colonic Neoplasms; DNA Fragmentation; Endostatins; Fatty Acids, Monounsaturated; Female; Gene Expression Regulation, Neoplastic; Genetic Therapy; HCT116 Cells; Humans; Liposomes; Lung Neoplasms; Mice; Mice, Inbred C57BL; Plasmids; Quaternary Ammonium Compounds; Ribosomal Proteins; RNA, Small Interfering; Signal Transduction; Survival Analysis; Xenograft Model Antitumor Assays

To investigate the cytotoxicity of the homemade peptide cationic liposome CDO14 and its efficacy of RNA interference (RNAi). MTT method was used to determine the cytotoxicity of the liposome to a human lung cancer cell line Luc-A549 that can express luciferase stably. Luciferase siRNA (Luc-siRNA) was transfected into Luc-A549 cells by CDO14. Contents of luciferase in the transfected cells were detected by luminous instrument and contents of total protein in these cells were detected by BCA method. Nude mice were inoculated with Luc-A549 cells in axilla to establish xenograft tumor model. Complexes of Luc-siRNA and the cationic liposomes were injected into the modeling mice via tail vein. Contents of luciferase in the transfected mice were detected by the whole body imaging system. The cytotoxicity of the homemade cationic liposome was similar to that of commercial liposome DOTAP, and lower than that of Lipo2000. The siRNA transfection efficacy mediated by CDO14 was higher than that mediated by DOTAP. The homemade peptide cationic liposome CDO14 is expected to serve as delivery vector in gene therapy because of its low cytotoxicity and high transfection efficiency.

Topics: Animals; Cations; Cell Line, Tumor; Fatty Acids, Monounsaturated; Genetic Therapy; Genetic Vectors; Humans; Liposomes; Luciferases; Lung Neoplasms; Mice; Mice, Nude; Peptides; Quaternary Ammonium Compounds; RNA Interference; RNA, Small Interfering; Transfection

Modifying the viral genome to express potent and cancer-selective therapeutic genes has enhanced the role of adenoviruses (Ads) in cancer molecular therapeutics. However, the efficacy of Ad systemic delivery in vivo is limited by neutralizing antibodies, short blood circulation time, and high levels of nonspecific liver uptake resulting in hepatotoxicity. We therefore investigated the systemic delivery of tumor necrosis factor-related apoptosis-inducing ligand-expressing oncolytic Ad genome DNA (pmT-d19/stTR) via lipid envelopment as an alternative approach for cancer virotherapy in an orthotopic lung cancer model. Cationic liposomes (DOTAP/DOPE) were complexed with pmT-d19/stTR to generate pmT-d19/stTR+DOTAP/DOPE with the average diameter of which was 143.3 ± 5.7 nm at the optimal DNA:lipid ratio (1:6). Systemic administration of pmT-d19/stTR+DOTAP/DOPE elicited highly effective antitumor responses in vivo, with tumor volumes decreasing 94.5%, 90.5%, and 92.4% compared to phosphate buffered saline-, naked Ad (mT-d19/stTR)-, or pmT-d19/stTR-treated groups, respectively. Additionally, innate immune responses and Ad-specific neutralizing antibodies were significantly decreased in pmT-d19/stTR+DOTAP/DOPE-treated mice compared to those in the mT-d19/stTR-treated group. The biodistribution profile analyzed by quantitative-PCR and immunohistochemical analysis demonstrated that viral replication occurred preferentially in tumor tissues. Moreover, the viral genome tumor-to-liver ratio was significantly elevated in pmT-d19/stTR+DOTAP/DOPE-treated mice, which was 934- and 27-fold greater than the mT-d19/stTR- and pmT-d19/stTR-treated mice, respectively. These results demonstrate that systemic delivery of oncolytic viral genome DNA with liposomes is a powerful alternative to naked Ad, overcoming the limited clinical applicability of conventional Ads and enabling effective treatment of disseminated metastatic tumors.

Topics: Adenoviridae; Adenovirus E1A Proteins; Animals; Apoptosis; Cell Survival; Cytokines; Cytopathogenic Effect, Viral; Drug Carriers; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Genetic Vectors; Genome, Viral; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Oncolytic Virotherapy; Oncolytic Viruses; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Real-Time Polymerase Chain Reaction; TNF-Related Apoptosis-Inducing Ligand; Virus Replication; Xenograft Model Antitumor Assays

In the present study, we have used plasmid-based RNA interference (RNAi) strategy to downregulate the expression of epidermal growth factor receptor (EGFR) in EGFR wild-type (H292) and mutant (H1975) lung tumor models. The targeted knockdown of EGFR by small hairpin RNA not only inhibited growth of H292 xenograft but also inhibited H1975 lung cancer cell and xenograft, which bore L858R/T790M EGFR and was resistant to EGFR tyrosine kinase inhibitors. These data demonstrated that small hairpin RNA was an effective therapy against mutant EGFR-expressing cancer cells and thus considered to be a promising strategy in the treatment of lung cancers.

Topics: Animals; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; ErbB Receptors; Fatty Acids, Monounsaturated; Female; Humans; Liposomes; Lung Neoplasms; Mice; Mutation; Neoplasm Invasiveness; Quaternary Ammonium Compounds; RNA, Small Interfering

DOTAP/cholesterol-based lipoplexes are successfully used for delivery of plasmid DNA in vivo especially to the lungs, although low systemic stability and circulation have been reported. To achieve the aim of discovering the best method for systemic delivery of DNA to disseminated tumors we evaluated the potential of formulating DOTAP/cholesterol lipoplexes with a polyethylene glycol (PEG)-modified lipid, giving the benefit of the shielding and stabilizing properties of PEG in the bloodstream.. A direct comparison of properties in vitro and in vivo of 4 different DOTAP/cholesterol-based lipoplexes containing 0%, 2%, 4%, and 10% PEG was performed using reporter gene activity and radioactive tracer lipid markers to monitor biodistribution.. We found that 10% PEGylation of lipoplexes caused reduced retention in lung and heart tissues of nude mice compared to nonPEGylated lipoplexes, however no significant delivery to xenograft flank tumors was observed. Although PEGylated and nonPEGylated lipoplexes were delivered to cells the ability to mediate successful transfection is hampered upon PEGylation, presumably due to a changed uptake mechanism and intracellular processing.. The eminent in vivo transfection potency of DOTAP/cholesterol-based lipoplexes is well established for expression in lung tumors, but it is unsuitable for expression in non first pass organs such as xenograft flank tumors in mice even after addition of a PEG-lipid in the formulation.

Topics: Animals; Carcinoma, Small Cell; Cell Line, Tumor; Cholesterol; Drug Delivery Systems; Fatty Acids, Monounsaturated; Gene Expression; Genes, Reporter; Genetic Therapy; Humans; In Vitro Techniques; Liposomes; Lung Neoplasms; Male; Mice; Mice, Nude; Nanomedicine; Polyethylene Glycols; Quaternary Ammonium Compounds; Transfection; Transplantation, Heterologous

The antisense oligonucleotide 2'-O-methyl-RNA is a selective telomerase inhibitor targeting the telomerase RNA component and represents a potential candidate for anticancer therapy. The poor cellular uptake of 2'-O-methyl-RNA is a limiting factor that may contribute to the lack of functional efficacy. To improve delivery of 2'-O-methyl-RNA and consequently antitumoral efficiency in human lung cancer cells, we have investigated several transfection reagents. The transfection reagents DOTAP, MegaFectin 60, SuperFect, FuGENE 6 and MATra-A were tested for intracellular delivery. A FAM-labeled 2'-O-methyl-RNA was used to assess the intracellular distribution by confocal laser scanning microscopy in A549 human non-small cell lung cancer cells. Telomerase activity was measured using the telomeric repeat amplification protocol. Cell viability after transfection was quantified by the MTT assay. All transfection reagents enhanced 2'-O-methyl-RNA uptake in A549 cells but the cationic lipid reagents DOTAP and MegaFectin 60 were most efficient in the delivery of 2'-O-methyl-RNA resulting in telomerase inhibition. Among both DOTAP exhibited the lowest cytotoxicity. Our experiments show that DOTAP is the most suitable transfection reagent for the delivery of 2'-O-methyl-RNA in human lung cancer cells according to its relatively low cytotoxicity and its ability to promote efficient uptake leading to the inhibition of telomerase.

Topics: Antineoplastic Agents; Capillary Electrochromatography; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Cholesterol; Drug Delivery Systems; Enzyme Inhibitors; Excipients; Fatty Acids, Monounsaturated; Humans; Indicators and Reagents; Liposomes; Lung Neoplasms; Microscopy, Confocal; Oligonucleotides; Oligonucleotides, Antisense; Quaternary Ammonium Compounds; RNA, Antisense; Telomerase; Tetrazolium Salts; Thiazoles; Transfection

To compare systemic intravenous and local intratracheal delivery of doxorubicin (DOX), antisense oligonucleotides (ASO) and small interfering RNA (siRNA).. "Neutral" and cationic liposomes were used to deliver DOX, ASO, and siRNA. Liposomes were characterized by dynamic light scattering, zeta-potential, and atomic force microscopy. Cellular internalization of DOX, ASO and siRNA was studied by confocal microscopy on human lung carcinoma cells. In vivo experiments were carried out on nude mice with an orthotopic model of human lung cancer.. Liposomes provided for an efficient intracellular delivery of DOX, ASO, and siRNA in vitro. Intratracheal delivery of both types of liposomes in vivo led to higher peak concentrations and much longer retention of liposomes, DOX, ASO and siRNA in the lungs when compared with systemic administration. It was found that local intratracheal treatment of lung cancer with liposomal DOX was more efficient when compared with free and liposomal DOX delivered intravenously.. The present study outlined the clear advantages of local intratracheal delivery of liposomal drugs for the treatment of lung cancer when compared with systemic administration of the same drug.

Topics: Administration, Inhalation; Animals; Antibiotics, Antineoplastic; Cell Line, Tumor; Cell Proliferation; Doxorubicin; Drug Carriers; Fatty Acids, Monounsaturated; Genetic Therapy; Humans; Injections, Intravenous; Light; Liposomes; Lung Neoplasms; Mice; Mice, Nude; Microscopy, Atomic Force; Microscopy, Confocal; Oligonucleotides, Antisense; Particle Size; Quaternary Ammonium Compounds; RNA Interference; RNA, Small Interfering; Scattering, Radiation; Surface Properties; Tissue Distribution; Xenograft Model Antitumor Assays

Cationic hyaluronic acid (HA)-modified DOTAP/DOPE liposomes were designed for the targeted delivery of anti-telomerase siRNA to CD44 receptor-expressing lung cancer cells. DOTAP/DOPE liposomes modified with 1%-20% (w/w) HA-DOPE conjugate were obtained by the ethanol injection method. Their size was below 170 nm and they exhibited zeta potentials higher than +50 mV. Lipoplexes prepared at different +/-ratios with siRNA were in the range of 200 nm and below and their zeta potentials were strongly dependent on the degree of modification and the +/-charge ratio. The presence of HA did not compromise binding, protection of siRNA from degradation, and complex stabilities in serum but rather resulted in an improvement of these properties. Liposome cytotoxicity, investigated by the MTT assay and LDH release after treatment of CD44(+) A549 cells and CD44(-) Calu-3, was demonstrated only at high concentrations. However, the addition of siRNA to HA-modified liposomes prevented cytotoxic effects compared to all other formulations. As shown by flow cytometry, transfection of siRNA into A549 cells was markedly improved with HA-modified liposomes, but not into Calu-3 cells. Using a qPCR-TRAP assay to test telomerase activity, no difference was demonstrated in the efficiency between HA-modified and nonmodified preparations. Moreover, some reduction in telomerase activity was observed with liposomes alone, lipoplexes prepared with nonsense siRNA and lipofectamine, indicative for some direct inhibitory effect of the lipids and siRNA on the expression of this enzyme. HA-modified DOTAP/DOPE liposomes represent a suitable carrier system for siRNA since properties like binding or protection of siRNA are not altered. They display an improved stability in cell culture medium and a reduced cytotoxicity. Furthermore, these novel lipoplexes could successfully be targeted to CD44-expressing A549 cells opening interesting perspectives for the treatment of lung cancer.

Topics: Cell Line, Tumor; Drug Delivery Systems; Fatty Acids, Monounsaturated; Humans; Hyaluronan Receptors; Hyaluronic Acid; Liposomes; Lung Neoplasms; Phosphatidylethanolamines; Quaternary Ammonium Compounds; RNA, Small Interfering; Telomerase

All-trans retinoic acid (ATRA) has been shown to exert anti-cancer activities in a number of types of cancer cells. However, it has been reported that many NSCLC exhibited resistance to ATRA treatment. In the present study, we hypothesized that intracellular delivery of ATRA would overcome the ATRA resistance in A549 cells. Here, we investigated the induction of apoptosis by ATRA incorporated in cationic liposomes composed of DOTAP/cholesterol in A549 human lung cancer cells, which are insensitive (resistant) to the growth inhibitory effects of ATRA. The zeta potentials of DOTAP/cholesterol liposomes and DSPC/cholesterol liposomes were about +50 and -3 mV. In A549 cells, [(3)H]ATRA incorporated in DOTAP liposomes showed increased cellular association compared with [(3)H]ATRA or [(3)H]ATRA incorporated in DSPC/cholesterol liposomes. ATRA incorporated in DOTAP/cholesterol liposomes showed much higher cytotoxic effects and apoptosis-inducing activity compared with ATRA or ATRA incorporated in DSPC/cholesterol liposomes. The enhanced expression of TIG3 mRNA tumor suppressor gene by ATRA incorporation into DOTAP/cholesterol liposomes might partly explain the mechanism of enhanced cytotoxicity and/or apoptosis. These observations provide valuable information to help in the design of differentiation therapy by ATRA in non-small cell lung carcinoma.

Topics: Apoptosis; Cholesterol; Fatty Acids, Monounsaturated; Humans; Liposomes; Lung Neoplasms; Quaternary Ammonium Compounds; Tretinoin; Tumor Cells, Cultured

Delivering phosphodiester ONs (PO-ONs) remains an attractive but challenging goal in antisense therapy. Both in the literature and in our experiments, most cationic liposomes fail in generating an antisense effect with PO-ONs, while they succeed with chemically modified ONs such as phosphothioate ONs (PS-ONs). This work aims to explain the biological activity of PO- and PS-ONs delivered by DOTAP/DOPE liposomes based on a detailed understanding of their cell biological behavior by means of fluorescence correlation spectroscopy and confocal laser scanning microscopy. We conclude that DOTAP/DOPE liposomes are not suited to deliver PO-ONs due to the release of naked PO-ONs in the cytosol at the time of the endosomal escape of the liposomes and the subsequent rapid degradation of the naked PO-ONs. Carriers that would not release the PO-ONs upon endosomal escape but would continue to carry the PO-ONs until they arrive at the target mRNA could therefore be better suited to delivering PO-ONs. In the case of PS-ONs, the ONs are not degraded upon release at the time of the endosomal escape of the liposomes, creating a pool of intact, biologically active PS-ONs and thus making DOTAP/DOPE liposomes mainly suitable for delivering nuclease resistant ONs. However, the cells seemed to display an export pathway for removing intact PS-ONs from the cells, limiting the presence of naked PS-ONs in the nucleus to approximately 8 h following the delivery.

Topics: Drug Delivery Systems; Fatty Acids, Monounsaturated; Fluorescence Resonance Energy Transfer; Humans; Liposomes; Lung Neoplasms; Microscopy, Confocal; Oligonucleotides; Oligonucleotides, Antisense; Phosphatidylethanolamines; Quaternary Ammonium Compounds; RNA, Messenger; Time Factors; Tumor Cells, Cultured

NPRL2 is one of the novel candidate tumor suppressor genes identified in the human chromosome 3p21.3 region. The NPRL2 has shown potent tumor suppression activity in vitro and in vivo and has been suggested to be involved in DNA mismatch repair, cell cycle checkpoint signaling, and regulation of the apoptotic pathway. In this study, we analyzed the endogenous expression of the NPRL2 protein and the cellular response to cisplatin in 40 non-small-cell lung cancer cell lines and found that expression of NPRL2 was significantly and reciprocally correlated to cisplatin sensitivity, with a Spearman correlation coefficient of -0.677 (P < 0.00001). Exogenously introduced expression of NPRL2 by N-[1-(2,3-dioleoyloxyl)propyl]-NNN-trimethylammoniummethyl sulfate:cholesterol nanoparticle-mediated gene transfer significantly resensitized the response to cisplatin, yielding a 40% greater inhibition of tumor cell viability and resulting in a 2- to 3-fold increase in induction of apoptosis by activation of multiple caspases in NPRL2-transfected cells compared with untransfected cells at an equal dose of cisplatin. Furthermore, a systemic treatment with a combination of NPRL2 nanoparticles and cisplatin in a human H322 lung cancer orthotopic mouse model significantly enhanced the therapeutic efficacy of cisplatin and overcame cisplatin-induced resistance (P < 0.005). These findings implicate the potential of NPRL2 as a biomarker for predicting cisplatin response in lung cancer patients and as a molecular therapeutic agent for enhancing response and resensitizing nonresponders to cisplatin treatment.

Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cholesterol; Cisplatin; Drug Resistance, Neoplasm; Fatty Acids, Monounsaturated; Female; Gene Transfer Techniques; Genetic Vectors; Humans; Lung Neoplasms; Mice; Mice, Nude; Nanoparticles; Neoplasm Proteins; Quaternary Ammonium Compounds; Random Allocation; Recombinant Fusion Proteins; Tumor Stem Cell Assay; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

The purpose of this study was to investigate whether all-trans retinoic acid (ATRA), an active metabolite of retinal, incorporated in cationic liposomes composed of 1,2 dioleoyl-3-trimethylammonium propane (DOTAP)/cholesterol could inhibit established metastatic lung tumors by delivery to the pulmonary tumor site after intravenous injection. After intravenous injection in mice, the highest lung accumulation of [(3)H]ATRA was observed by the DOTAP/cholesterol liposomes formulation, while other formulations including [(3)H]ATRA dissolved in serum or [(3)H]ATRA incorporated in distearoyl-l-phosphatidylcholine (DSPC)/cholesterol liposomes produced little accumulation in the lung. In mice used as a model of lung cancer metastasis, ATRA incorporated in DOTAP/cholesterol liposomes, injected intravenously, reduced the number of tumor nodules compared with free ATRA or ATRA incorporated in DSPC/cholesterol liposomes. These results suggest that ATRA incorporated in cationic liposomes would be an effective strategy for differentiation therapy of lung cancer metastasis.

Topics: Animals; Antineoplastic Agents; Cations; Cell Line, Tumor; Cell Transplantation; Chemical and Drug Induced Liver Injury; Chemical Phenomena; Chemistry, Physical; Colonic Neoplasms; Drug Carriers; Fatty Acids, Monounsaturated; Injections, Intravenous; Liposomes; Lung; Lung Neoplasms; Male; Mice; Neoplasm Transplantation; Quaternary Ammonium Compounds; Rats; Rats, Inbred F344; Solubility; Tissue Distribution; Tretinoin

Delivery of genes to airway mucosa would be a very valuable method for gene therapy and vaccination. However, there have been few reports on suitable gene delivery systems for administration. In this study, we use a cationic emulsion system, which is physically stable and facilitates the transfer of genes in the presence of up to 90% serum, as a mucosal gene carrier.. Cationic lipid emulsion was formulated with squalene and 1,2-dioleoyl-sn-glycero-3-trimethylammoniumpropane (DOTAP) as major components. Emulsions formed stable complexes with DNA and protected and transferred DNA to target cells against DNase I digestion in the presence of mucosal destabilizers such as heparin sulfate (a polysaccharide of the glycosaminoglycan family in mucosa) and Newfectan (a natural lung extract of bovine) in an in vitro system. In contrast, commercial liposomes and counter liposomes, made with an identical lipid composition of emulsions, failed. After in vivo intranasal instillation, the cationic emulsion showed at least 200 times better transfection activity than the liposomal carriers in both nasal tissue and lung.. These findings show that cationic emulsions can mediate gene transfection into airway epithelium, making it a good choice for transferring therapeutic genes and for genetic vaccination against an pathogenic infection via an airway route.

Topics: Carcinoma, Large Cell; Carcinoma, Squamous Cell; Cations; Cell Culture Techniques; DNA; Emulsions; Epithelial Cells; Fatty Acids, Monounsaturated; Gene Expression; Gene Transfer Techniques; Genes, Reporter; Humans; Lipids; Liposomes; Luciferases; Lung Neoplasms; Nasal Mucosa; Phosphatidylethanolamines; Plasmids; Polysorbates; Quaternary Ammonium Compounds; Squalene; Transfection; Tumor Cells, Cultured; X-Ray Diffraction

Studies conducted in non-tumor-bearing, immunocompetent mice have shown that intravenous administration of liposome-DNA complex elicits an inflammatory response that results in a failure to sustain adequate transgene expression. In the present study, however, we investigated the effects of a cationic liposomal DOTAP:cholesterol (DOTAP:Chol)-DNA complex on cytokine production and transgene expression in both experimental lung tumor-bearing (TB) mice and non-tumor-bearing (NTB) syngeneic mice and nude mice. Intravenous injection of DOTAP:Chol-luciferase (luc) DNA complex resulted in tumor necrosis factor-alpha levels that were 50% lower and interleukin-10 levels that were 50-60% higher in TB mice than in NTB mice. Furthermore, a significant increase in luc expression (P = 0.001) that persisted for 7 days was observed in TB mice. In contrast, luc expression decreased significantly from day 1 to day 2 in NTB mice. Also, luc expression was two- to threefold higher in TB mice that were given multiple injections of DOTAP:Chol-luc complex than in mice who received a single injection. In contrast, luc expression was significantly suppressed following multiple injections in NTB mice (P = 0.01). Further analysis revealed IL-10 protein expression by the tumor cells in TB mice. Injection of anti-IL-10 antibody in TB mice resulted in a significant decrease in luc expression (P = 0.01) compared with that in mice injected with a control antibody. Based on these findings, we conclude that transgene expression persists in TB mice and is partly mediated by IL-10. Additionally, multiple injections of liposome-DNA complex can increase transgene expression in TB mice. These findings have clinical applications in the treatment of cancer.

Topics: Animals; Cholesterol; Cytokines; DNA; Enzyme-Linked Immunosorbent Assay; Fatty Acids, Monounsaturated; Fibrosarcoma; Gene Expression Regulation, Neoplastic; Immunohistochemistry; Inflammation; Interleukin-10; Liposomes; Luciferases; Lung; Lung Neoplasms; Macrophages, Alveolar; Mice; Mice, Inbred C3H; Mice, Nude; Models, Biological; Neoplasms; Plasmids; Quaternary Ammonium Compounds; Time Factors; Transgenes

We have investigated the effects of an improved liposomal formulation (extruded DOTAP:cholesterol (DOTAP:Chol)-DNA complex) on transgene expression in tumor cells and normal cells of murine and human origin both in vitro and in vivo. In vitro, transgene expression was significantly increased (P = 0.01) in human tumor cells compared to normal human cells. The increased transgene expression was due to increased uptake of the liposome-DNA complex by tumor cell phagocytosis. Furthermore, immunohistochemical analysis demonstrated a greater transgene expression in lung tumors than in surrounding normal tissues. Increased transgene expression due to enhanced uptake of the liposome-DNA complexes by tumor cells in vivo was also demonstrated using fluorescently labeled DOTAP:Chol liposomes. Finally, evaluation of lung tissue explants obtained from patients undergoing pulmonary resection demonstrated significantly higher (P = 0.001) transgene expression in tumor cells than in normal cells. Thus, we demonstrated that intravenous injection of DOTAP:Chol-DNA complex results in increased transgene expression in tumor and is due to increased phagocytosis of the complexes by tumor cells.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Survival; Cholesterol; DNA; Fatty Acids, Monounsaturated; Genetic Therapy; Humans; Immunoenzyme Techniques; Injections, Intravenous; Liposomes; Luciferases; Lung Neoplasms; Mice; Mice, Inbred C3H; Phagocytosis; Pinocytosis; Quaternary Ammonium Compounds; Transgenes; Tumor Cells, Cultured

Topics: Animals; Breast Neoplasms; Dextrans; DNA; Drug Carriers; Drug Delivery Systems; Fatty Acids, Monounsaturated; Fluorescent Dyes; Gene Products, tat; HIV-1; Humans; Immunoglobulin G; Intracellular Fluid; Liposomes; Lung Neoplasms; Mice; Peptides; Phosphatidylethanolamines; Polyethylene Glycols; Quaternary Ammonium Compounds; Rats; tat Gene Products, Human Immunodeficiency Virus

Delivery of therapeutic genes to disseminated tumor sites has been a major challenge in the field of cancer gene therapy due to lack of an efficient vector delivery system. Among the various vectors currently available, liposomes have shown promise for the systemic delivery of genes to distant sites with minimal toxicity. In this report, we describe an improved extruded DOTAP:cholesterol (DOTAP:Chol) cationic liposome that efficiently delivers therapeutic tumor suppressor genes p53 and FHIT, which are frequently altered in lung cancer, to localized human primary lung cancers and to experimental disseminated metastases. Transgene expression was observed in 25% of tumor cells per tumor in primary tumors and 10% in disseminated tumors. When treated with DOTAP:Chol-p53 and -FHIT complex, significant suppression was observed in both primary (P < 0.02) and metastatic lung tumor growth (P < 0.007). Furthermore, repeated multiple treatments revealed a 2.5-fold increase in gene expression and increased therapeutic efficacy compared to single treatment. Finally, animal survival experiments revealed prolonged survival (median survival time: 76 days, P < 0.001 for H1299; and 96 days, P = 0.04 for A549) when treated with liposome-p53 DNA complex. Our findings may be of importance in the development of treatments for primary and disseminated human lung cancers.

Topics: Acid Anhydride Hydrolases; Animals; Fatty Acids, Monounsaturated; Female; Gene Expression; Genes, p53; Genes, Tumor Suppressor; Genetic Therapy; Humans; Liposomes; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Proteins; Proteins; Quaternary Ammonium Compounds; Time Factors; Transgenes; Transplantation, Heterologous; Tumor Cells, Cultured

Intravenous (i.v.) administration of a lipopolyplex consisting of a ternary complex of DOTAP:cholesterol cationic liposomes, protamine sulfate, and noncoding plasmid DNA (LPD-pDNA) is capable of stimulating a potent Th-1 cytokine response and inhibiting the growth of established tumors in mice. Both activities are mainly elicited by unmethylated CpG motifs in the plasmid DNA (pDNA) component, which are bacterial in origin. Since oligodeoxynucleotides (ODN) that possess a consensus immunostimulatory CpG motif of RRCpGYY (R is purine and Y is pyrimidine) can mimic the immunostimulatory actions of bacterial DNA, we hypothesized that i.v. administration of LPD prepared with GpG-ODN would mimic the ability of LPD-pDNA to stimulate Th-1 cytokines and antitumor activity and provide an improved vector for probing the immune mechanisms underlying the observed antitumor effects. These hypotheses were tested for the treatment of established 24JK experimental pulmonary metastases that are syngeneic in C57BL/6 mice. Mice treated with LPD containing 25 microg of the prototypical phosphodiester (PO) CpG-ODN 1668 (tccatGACGTTcctgatgct, motif capitalized) demonstrated a dramatic reduction in lung tumor burden (>80% inhibition, P<0.01) compared to dextrose-treated controls. The antitumor effect was dependent on the CpG dinulceotide and correlated with the ability to stimulate serum Th-1 cytokines (TNF-alpha, IL-12, and IFN-gamma). Both activities required assembly of CpG-ODN in a cationic liposome/DNA complex (lipoplex) or the LPD lipopolyplex. LPD delivery of both PO-1668 and phosphorothioated (PS)-1668 stimulated a greater cytokine response compared to delivery of free ODN. Furthermore, within the LPD complex, both PO- and PS-1668 had similar ability to stimulate Th-1 cytokines with respect to potency and duration of response, thus eliminating the need for the PS modification. In tumor cell lysis assays, LPD-CpG DNA stimulated development of an acquired, tumor-specific CD8+ cytotoxic T-lymphocyte (CTL) activity that was dependent on CpG DNA. LPD was also capable of stimulating NK activity; however, this was not dependent on CpG DNA. Only formulations that concomitantly stimulated NK activity and CpG-specific, Th-1 cytokine were capable of stimulating the development of tumor-specific CTL activity and significant inhibition of tumor growth. Thus, we propose a model where CpG DNA in complex with cationic liposome-based lipoplexes or lipopolyplexes stimulates antitumor NK a

Topics: Animals; Cholesterol; CpG Islands; Cytotoxicity, Immunologic; Fatty Acids, Monounsaturated; Liposomes; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Plasmids; Quaternary Ammonium Compounds; Th1 Cells

ras mutations represent one of the most common oncogenetic lesions in human non-small cell lung cancer (NSCLC) and adversely affect the survival of patients afflicted with this disease. ras-directed gene therapy in the past employed primarily antisense oligonucleotides (AS-ODN) or expression vectors (such as a viral vector construct) that deliver the antisense sequence to inactivate the mutant oncogene message. These approaches produced minimal toxicity, and yet were limited in efficacy. Ribozymes present a viable alternative in antisense therapy by virtue of their renewable catalytic capability for site-specific RNA cleavage. We recently produced an adenoviral vector with a hammerhead ribozyme transgene (KRbz) that is specific for the K-ras codon 12 mutant sequence GUU, given the considerations that (a) in the United States, approx 30% of human NSCLCs express K-ras oncogene mutations, nearly all of which reside in codon 12; (b) anti-K-ras, anti-H, as well as anti-N-ras hammerhead ribozymes are potent growth inhibitors in various human cancers tested; and (c) in vitro and animal model studies suggest that ribozymes directed at oncogene (K- and H-ras C-fos, BCR-ABL) or human immunodeficiency viral gene messages are more effective than their antisense counterpart. This article describes the techniques involved in the production of the KRbz-adenoviral vector that is specific for the K-ras mutation GTT, and summarizes its in vivo antitumor effect against NSCLC xenografts expressing the relevant K-ras mutation in athymic mice.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line; DNA Mutational Analysis; Fatty Acids, Monounsaturated; Genes, ras; Genetic Techniques; Genetic Vectors; Humans; Liposomes; Lung Neoplasms; Mutation; Plasmids; Quaternary Ammonium Compounds; Recombination, Genetic; RNA, Catalytic; Sequence Analysis, DNA; Transfection

Cationic liposomes spontaneously interact with negatively charged plasmid DNA to form a transfection competent complex capable of promoting the expression of a therapeutic gene. This work aims to improve the understanding of the poorly defined mechanisms and structural rearrangements associated with the lipid-DNA interaction. Specifically, dimethyl dioctadecylammonium bromide (DDAB):dioleoyl phosphatidylethanolamine (DOPE) and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) liposomes were mixed with a reporter plasmid (pADbeta or pCMVbeta) to form lipid-DNA complexes. The size and charge characteristics of the complexes as determined by photon correlation spectroscopy and microelectrophoresis were found to be dependent on the lipid:DNA ratio, with both DDAB:DOPE-DNA and DOTAP-DNA complexes aggregating at around neutral zeta potential. Negative stain transmission electron microscopy demonstrated at least three distinct complex structures being formed at the same DOTAP:DNA ratio. We postulate that two of these aggregates are structural moieties involved in the formation of the efficient transfection particle. Gel electrophoresis was used to determine the efficiency and extent of lipid-DNA complex formation. Results showed that only DOTAP liposomes were capable of preventing ethidium bromide intercalation with DNA and protecting the enclosed plasmid from nuclease digestion. When a range of lipid-DNA complexes were transfected into in vitro cell lines, the efficiency of reporter gene (beta-galactosidase) expression was found to depend on the type of liposome used in the complex, the ratio of lipid:DNA and the transfected cell line. Our results challenge the requirement for DOPE to be included in the formulation of cationic lipid vectors, especially in the case of DOTAP containing liposomes.

Topics: Animals; Cell Line; Drug Delivery Systems; Drug Interactions; Electrophoresis; Electrophoresis, Agar Gel; Fatty Acids, Monounsaturated; Fibroblasts; Fluorescent Dyes; Haplorhini; Humans; Kidney; Lipids; Liposomes; Lung Neoplasms; Microscopy, Electron; Particle Size; Plasmids; Quaternary Ammonium Compounds; Spectrophotometry; Static Electricity; Transfection; Tumor Cells, Cultured

Our laboratory has recently developed a lipopolyplex consisting of DOTAP:cholesterol liposomes, protamine sulfate, and plasmid DNA (LPD) that provides improved systemic gene delivery compared with lipoplex following tail vein injection in mice. Because endothelial cells are the primary cells transfected in the lung, it was hypothesized that LPD might be an effective vector for gene therapy of pulmonary metastases. This hypothesis was examined by testing the efficacy of cytokine (IL-12) and tumor suppressor (p53) strategies for treatment of an experimental model of pulmonary metastasis in C57Bl/6 mice. Surprisingly, all LPD complexes including those containing an 'empty' plasmid provided a potent (>50% inhibition) and dose-dependent antitumor effect, compared with dextrose-treated controls. In addition, i.v. injections of LPD containing 'empty' plasmid also inhibited tumor growth in a subcutaneous model of C3 fibrosarcomma. The antitumor effect correlated well with a strong and rapid proinflammatory cytokine (TNF-alpha, IL-12 and IFN-gamma) response. Naked plasmid DNA did not elicit a cytokine response and the response required assembly of DNA into a lipoplex or the LPD lipopolyplex. Except for the heart, elevated levels of cytokine were observed in all organs (lung, liver, kidney and spleen) where LPD is known to have gene transfer activity. Methylation of immune-stimulatory CpG motifs in the plasmid component of LPD inhibited the proinflammatory cytokine response as well as the antitumor effect of LPD in both tumor systems. This suggests that i. v. administration of LPD elicits a systemic proinflammatory cytokine response that mediates the antitumor activity of the lipopolyplex. In addition, the antitumor activity was not observed in SCID mice suggesting a possible role for B or T lymphocytes in the antitumor response initiated by LPD. This represents the first demonstration that an intravenously administered cationic liposome-based nonviral vector can promote a systemic, Th1-like innate immune response. The immune adjuvant properties of LPD might prove to be suitable for delivering tumor-specific antigens in the context of DNA vaccination.

Topics: Animals; Cell Division; Cholesterol; Cytokines; Drug Combinations; Fatty Acids, Monounsaturated; Fibrosarcoma; Injections, Intravenous; Liposomes; Lung Neoplasms; Methylation; Mice; Mice, Inbred C57BL; Mice, SCID; Plasmids; Protamines; Quaternary Ammonium Compounds; Vaccines, DNA

Antisense oligonucleotides complementary to specific mRNA sequences are widely used inhibitors of gene expression in vitro and in vivo . In vitro cationic lipids have been demonstrated to increase the pharmacological activity of antisense oligonucleotides by increasing cellular uptake and facilitating nuclear accumulation. We have investigated the intracellular fate of oligonucleotide/cationic lipid complexes using fluorescently labeled lipids and oligonucleotides targeted to protein kinase C-alpha. After addition to cells the lipids initially co-localized with the oligonucleotide on the cell surface and in fine punctate structures within the cytoplasm. At later times the oligonucleotide began to accumulate in the nucleus as well as the cytoplasm. In contrast, the cationic lipid remained localized to the cell surface and the cytoplasm and was never found in the nucleus. Expression of protein kinase C-alpha mRNA did not begin to decline until after oligonucleotide was seen in the nucleus. This was also coincident with the transient appearance of a smaller mRNA transcript believed to result from RNase H cleavage of protein kinase C-alpha mRNA. These data suggest that although cationic lipids facilitate uptake of oligonucleotides, the complex must disassociate before the oligonucleotide can gain access to the nucleus and induce degradation of targeted mRNA.

Topics: Boron Compounds; Cell Nucleus; Drug Carriers; Fatty Acids, Monounsaturated; Fluorescent Dyes; Humans; Isoenzymes; Kinetics; Lung Neoplasms; Molecular Structure; Oligonucleotides, Antisense; Protein Kinase C; Protein Kinase C-alpha; Quaternary Ammonium Compounds; RNA, Messenger; Thionucleotides; Transcription, Genetic; Tumor Cells, Cultured

Antisense oligodeoxy-nucleoside phosphorothioates (OPTs) of L-myc were encapsulated into reconstituted influenza-virus-A envelopes (virosomes). The envelopes of the virosomes consisted of a single positively charged (cationic) lipid bilayer. Binding of cationic virosomes to cellular receptors that are membrane glycoproteins or glycolipids containing terminal sialic acid is mediated by the hemagglutinin glycoprotein (HA) of the influenza virus. After internalization through receptor-mediated endocytosis, cationic virosomes fuse efficiently with the membranes of the endosomal-cell compartment, and as a consequence the encapsulated OPT are delivered to the cell cytoplasma. Examination by fluorescence microscopy of the cellular uptake of cationic virosomes containing fluorescein-labeled OPT showed rapid and efficient incorporation of virosomes. Addition of cationic virosomes (75-150 microl) containing antisense L-myc OPT in the picomolar range to small-cell-lung-cancer (SCLC) cell cultures that expressed highly the L-myc oncogene led to strong inhibition of thymidine incorporation in a concentration-dependent manner. Virosome-entrapped sense L-myc OPT and random-order OPT had only minimal effects on the thymidine uptake. Cells of SCLC cell line NCI-H82 expressing a very low level of L-myc were not affected by antisense-L-myc virosomes. In Western-blot analysis, expression of L-myc protein was suppressed in the antisense-virosome-treated NCI-H209 cells but not in untreated control NCI-H209 cells. These results suggest that cationic virosomes may have great potential as an efficient delivery system for antisense oligonucleotides in cancer therapy.

Topics: Antibodies, Monoclonal; Carcinoma, Small Cell; Cell Division; Endocytosis; Fatty Acids, Monounsaturated; Fluorescent Dyes; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Influenza A virus; Liposomes; Lung Neoplasms; Oligonucleotides, Antisense; Proto-Oncogene Proteins c-myc; Quaternary Ammonium Compounds; Transfection; Tumor Cells, Cultured