1-2-dioleoyloxy-3-(trimethylammonium)propane and Disease-Models--Animal

Topics: Animals; Cations; Cell Line, Tumor; Colorectal Neoplasms; Disease Models, Animal; Fatty Acids, Monounsaturated; Female; Genetic Therapy; HEK293 Cells; Humans; Immunotherapy; Injections, Intralesional; Injections, Intraperitoneal; Lipids; Mice; Micelles; Nanoparticle Drug Delivery System; Polyesters; Polyethylene Glycols; Quaternary Ammonium Compounds; Receptors, Interleukin; RNA, Messenger; Tissue Distribution

Acanthamoeba keratitis is an ophthalmic disease with no specific treatment that specially affects contact lens users. The silencing of serine phosphatase (SP) and glycogen phosphorylase (GP) proteins produced by Acanthamoeba has been shown to significantly reduce the cytopathic effect, although no vehicle was proposed yet to deliver the siRNA sequences to the trophozoites. In this study, PEGylated cationic liposomes were proposed and optimized using Box-Behnken design. The influence of DOTAP:DOPE ratio, DSPE-PEG concentration, and siRNA/DOTAP charge ratio were evaluated over both biological response and physicochemical properties of liposomes. The ratio of DOTAP:DOPE had an effect in the trophozoite activity whereas the charge ratio influenced both size and protease activity. The predicted values were very close to the observed values, yielding a formulation with good activity and toxicity profile, which was used in the following experiments. A murine model of ocular keratitis was treated with siGP + siSP-loaded liposomes, as well as their respective controls, and combined treatment of liposomes and chlorhexidine. After 15 days of eight daily administrations, the liposomal complex combined with chlorhexidine was the only treatment able to reverse the more severe lesions associated with keratitis. There was 60% complete regression in corneal damage, with histological sections demonstrating the presence of an integral epithelium, without lymphocytic infiltrate. The set of results demonstrate the efficacy of a combined therapy based on siRNA with classical drugs for a better prognosis of keratitis caused by Acanthamoeba.

Topics: Acanthamoeba; Acanthamoeba Keratitis; Animals; Chlorhexidine; Cornea; Disease Models, Animal; Drug Administration Schedule; Drug Compounding; Drug Delivery Systems; Drug Therapy, Combination; Factor Analysis, Statistical; Fatty Acids, Monounsaturated; Gene Expression Regulation; Glycogen Phosphorylase; Humans; Liposomes; Phosphatidylethanolamines; Phosphoric Monoester Hydrolases; Polyethylene Glycols; Protozoan Proteins; Quaternary Ammonium Compounds; Rats; Rats, Wistar; RNA, Small Interfering; Trophozoites

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

For the past few years, gene-therapy has recently shown considerable clinical benefit in cancer therapy, and the applications of gene therapies in cancer treatments continue to increase perennially. EZH2, an ideal candidate for tumor gene therapy, plays an important role in the tumorigenesis.. In this study, we developed a novel gene delivery system with a self-assembly method by Methoxy polyethylene glycol-polycaprolactone (MPEG-PCL) and DOTAP(DMC). And EZH2si-DMC was used to research anti-glioma both in vitro and in vivo.. DMC with zeta-potential value of 36.7 mV and size of 35.6 nm showed good performance in the delivery siRNA to glioma cell in vitro with high 98% transfection efficiency. EZH2si-DMC showed good anti-glioma effect in vitro through inducing cell apoptosis and inhibiting cell growth. What's more, treatment of tumor-bearing mice with DMC-EZH2si complex had significantly inhibited tumor growth at the subcutaneous model in vivo by inhibiting EZH2 protein expression, promoting apoptosis and reducing proliferation.. The EZH2 siRNA and DMC complex may be used to treat the glioma in clinical as a new drug.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Enhancer of Zeste Homolog 2 Protein; Fatty Acids, Monounsaturated; Female; Glioma; Humans; Mice, Inbred BALB C; Mice, Nude; Micelles; Molecular Targeted Therapy; Nanoparticles; Polyesters; Polyethylene Glycols; Quaternary Ammonium Compounds; RNA, Small Interfering

Certain types of cationic lipids have shown promise in cancer immunotherapy, but their mechanism of action is poorly understood. In this study, we describe the properties of an immunotherapeutic consisting of the pure cationic lipid enantiomer R-1,2-dioleoyl-3-trimethyl-ammonium-propane (R-DOTAP) formulated with modified viral or self-peptide Ags. R-DOTAP formulations with peptide Ags stimulate strong cross-presentation and potent CD8 T cell responses associated with a high frequency of polyfunctional CD8 T cells. In a human papillomavirus tumor model system, a single s.c. injection of tumor-bearing mice with R-DOTAP plus human papillomavirus Ags induces complete regression of large tumors associated with an influx of Ag-specific CD8 T cells and a reduction of the ratio of regulatory/Ag-specific CD8 T cells. R-DOTAP also synergizes with an anti-PD1 checkpoint inhibitor, resulting in a significant inhibition of B16 melanoma tumor growth. We found that R-DOTAP stimulates type I IFN production by dendritic cells in vivo and in vitro. s.c. injection of R-DOTAP results in an IFN-dependent increase in draining lymph node size and a concomitant increase in CD69 expression. Using knockout mice, we show that type I IFN is required for the induction of CD8 T cell activity following administration of R-DOTAP plus Ag. This response requires Myd88 but not TRIF or STING. We also show that R-DOTAP stimulates both TLR7 and 9. Collectively, these studies reveal that R-DOTAP stimulates endosomal TLRs, resulting in a Myd88-dependent production of type I IFN. When administered with Ag, this results in potent Ag-specific CD8 T cell responses and antitumor activity.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Fatty Acids, Monounsaturated; Humans; Immunotherapy, Adoptive; Interferon Type I; Lymphocyte Activation; Melanoma; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, SCID; Mice, Transgenic; Myeloid Differentiation Factor 88; Nanoparticles; Papillomaviridae; Papillomavirus Infections; Quaternary Ammonium Compounds; Skin Neoplasms; T-Lymphocytes, Cytotoxic

Oral drug delivery with nanoparticles has demonstrated great potential for drugs with poor bioavailability. Efficient delivery is possible by overcoming both the mucus and epithelial barrier of the gastrointestinal tract (GIT). Cationic lipid-assisted nanoparticles (CLANs), which are composed of amphiphilic block copolymers and cationic lipids, have been well studied and have been proved beneficial for drug delivery. In this study, CLANs prepared by poly(ethylene glycol)-block-poly(lactic acid) (PEG-b-PLA) and 1,2-dioleoyl-3-trimethylammonium-propanechloride (DOTAP) or N,N-bis(2-hydroxyethyl)-N-methyl-N-(2-cholesteryloxycarbonyl aminoethyl)ammoniumbromide (BHEM-Chol) were used for oral delivery of tacrolimus (FK506) for ulcerative colitis treatment. The average size of these nanoparticles is around 110 nm and the zeta-potential is 35 mV. These nanoparticles maintained their size in buffer solutions of pH 1.2 and 6.8, and slowly release the encapsulated drug. CLANs can be accumulated in the colon and transported through the epithelium in the colitis model by dextran sulfate sodium salt (DSS), leading to attenuation of DSS-induced colitis.

Topics: Administration, Oral; Animals; Anti-Inflammatory Agents; Cholesterol Esters; Colitis, Ulcerative; Disease Models, Animal; Drug Carriers; Drug Compounding; Drug Liberation; Fatty Acids, Monounsaturated; Female; Hydrogen-Ion Concentration; Kinetics; Lactates; Mice; Mice, Inbred C57BL; Nanoparticles; Particle Size; Polyethylene Glycols; Quaternary Ammonium Compounds; Sodium Dodecyl Sulfate; Tacrolimus

Infectious diseases are the second leading cause of death worldwide, suggesting that there is still a need for the development of new and improved strategies for combating pathogens effectively. Streptococcus pneumoniae is the most virulent bacteria causing pneumonia with high mortality, especially in children and the elderly. Because of the emergence of antibiotic resistance in S. pneumoniae, employing a serotype-independent mucosal vaccine would be the best approach to prevent and treat the diseases caused by S. pneumoniae. In this study, we have developed a pneumococcal nasal vaccine, consisting of pneumococcal surface protein A (PspA) and cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and cholesteryl 3β-N-(dimethylaminoethyl)-carbamate (DC-chol) (DOTAP/DC-chol liposome). The efficiency of this cationic liposome-based PspA nasal vaccine was examined in a murine model of S. pneumoniae infection. Intranasal vaccination with PspA and DOTAP/DC-chol liposomes conferred protective immunity against lethal inhalation of S. pneumoniae, improving the survival rate of infected mice. Moreover, intranasal immunization with PspA and DOTAP/DC-chol liposomes not only induced the production of PspA-specific IgA and IgG by both mucosal and systemic compartments but also elicited PspA-specific Th17 responses, which play a pivotal role in controlling S. pneumoniae infection by host innate immune response. We further demonstrated that DOTAP/DC-chol liposomes enhanced PspA uptake by nasal dendritic cells (DCs), which might be a mechanism for the induction of protective immune responses to S. pneumoniae infection. These results show that DOTAP/DC-chol liposome would be an efficient mucosal vaccine system for a serotype-independent universal nasal vaccine against pneumococcal infection.

Topics: Administration, Intranasal; Animals; Antibodies, Bacterial; Antigens, Bacterial; Bacterial Proteins; Cholesterol; Disease Models, Animal; Fatty Acids, Monounsaturated; Female; Humans; Immunity; Immunoglobulin A; Liposomes; Mice; Mice, Inbred BALB C; Pneumococcal Infections; Quaternary Ammonium Compounds; Streptococcal Vaccines; Streptococcus pneumoniae; Th17 Cells; Vaccination

This study was designed to prepare and characterize nanoliposomal vaccine formulation encapsulating AE36 HER2/neu-derived peptide with or without CpG and evaluate the immunologic and therapeutic responses of that in BALB/c mice model of Her2 overexpressing breast cancer. AE36 was encapsulated in liposomes composed of DOTAP, DOPE and Cholesterol (DDC) or DD with. The formulations could induce both CD8+ and CD4+ responses and stimulate production of cytokines which was detected by Enzyme-linked immunospot assay (ELISpot) kits, cytotoxicity test and intracellular cytokine assay by flow cytometry. The formulation showed both therapeutic and prophylactic effects in BALB/c mice bearing Her2

Topics: Animals; Breast Neoplasms; Cancer Vaccines; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cholesterol; Cytokines; Cytotoxicity, Immunologic; Disease Models, Animal; Fatty Acids, Monounsaturated; Female; Humans; Immunotherapy; Liposomes; Mice; Mice, Inbred BALB C; Nanostructures; Oligodeoxyribonucleotides; Peptides; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Receptor, ErbB-2

Although there have been several attempts to develop a vaccine against leishmaniasis, no vaccine in human has been developed yet. Liposomes consisting of 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) encapsulating soluble Leishmania antigens (SLA) enhance protective immunity of SLA against Leishmania major infection in mice. However, they immobilized at the injection site because of their positive charge. To overcome the problem, shielding the surface charge with polyethylene glycol (PEGylation) was chosen in this study. Liposomal SLA consisting different concentrations of PEG (1.9%-15% mol) were prepared. BALB/c mice were immunized three times in 3 weeks intervals with different formulations. Lesion development and parasite burden in footpad and spleen were evaluated to specify the type of generated immune response and extent of protection. Th1/Th2 cytokine profiles and IgG isotypes were also analysed. The maximum protection was observed in mice immunized with Lip-SLA or pLip-SLA (1.9%) due to smaller footpad swelling, reduction in parasite load, an increase in IgG2a and IFN-γ production. Our results showed that immunization of mice with a high level of PEG (>7.5%) did not improve protective immunity of liposomal SLA. The presence of PEG, particularly more than 3.75%, is not recommended for protection against leishmaniasis.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Protozoan; Antigens, Protozoan; Cytokines; Disease Models, Animal; Fatty Acids, Monounsaturated; Female; Immunoglobulin G; Interferon-gamma; Leishmania major; Leishmaniasis; Leishmaniasis Vaccines; Liposomes; Mice; Mice, Inbred BALB C; Parasite Load; Polyethylene Glycols; Quaternary Ammonium Compounds; Spleen; Th1 Cells; Th2 Cells; Vaccination

Mucopolysaccharidosis type I (MPS I) is an autosomal disease caused by alpha-L-iduronidase deficiency. This study proposed the use of cationic nanoemulsions as non-viral vectors for a plasmid (pIDUA) containing the gene that codes for alpha-L-iduronidase. Nanoemulsions composed of medium chain triglycerides (MCT)/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)/1,2-dioleoyl-sn-glycero-3-trimethylammonium propane (DOTAP)/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) were prepared by high pressure homogenization. Formulations were prepared by the adsorption or encapsulation of preformed pIDUA-DOTAP complexes into the oil core of nanoemulsions at different charge ratios. pIDUA complexed was protected from enzymatic degradation by DNase I. The physicochemical characteristics of complexes in protein-containing medium were mainly influenced by the presence of DSPE-PEG. Bragg reflections corresponding to a lamellar organization were identified for blank formulations by energy dispersive X-ray diffraction, which could not be detected after pIDUA complexation. The intravenous injection of these formulations in MPS I knockout mice led to a significant increase in IDUA activity (fluorescence assay) and expression (RT-qPCR) in different organs, especially the lungs and liver. These findings were more significant for formulations prepared at higher charge ratios (+4/-), suggesting a correlation between charge ratio and transfection efficiency. The present preclinical results demonstrated that these nanocomplexes represent a potential therapeutic option for the treatment of MPS I.

Topics: Animals; Disease Models, Animal; Emulsions; Fatty Acids, Monounsaturated; Gene Expression; Genetic Therapy; Humans; Iduronidase; Kidney; Liver; Lung; Male; Mice, Inbred C57BL; Mice, Knockout; Mucopolysaccharidosis I; Nanostructures; Phosphatidylethanolamines; Plasmids; Polyethylene Glycols; Quaternary Ammonium Compounds; Spleen; Transfection; Triglycerides

Cationic immune stimulating complexes (PLUSCOMs) are particulate antigen delivery systems. PLUSCOMs consist of cationic immunostimulatory complexes (ISCOMs) derivatives and are able to elicit in vivo T cell responses against an antigen.. To evaluate the effects of PLUSCOMs containing Leishmania major antigens (SLA) on the type of immune response generated in the murine model of leishmaniasis.. PLUSCOMs consisting of 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used as antigen delivery system/immunoadjuvants for soluble SLA. BALB/c mice were immunized subcutaneously, three times in 2-week intervals. Footpads swellings at the site of challenge and parasite loads were assessed as a measure of protection. The immune responses were also evaluated by determination of IgG subclasses and the level of IFN-γ and IL-4 in cultured splenocytes.. There was no significant difference (p<0.05) between the sizes of lesions in mice immunized with different formulations. Also, there was no significant difference in the number of parasites in the footpad or spleen of all groups compared with the control group. The highest level of IFN-γ secretion was observed in the splenocytes of mice immunized with PLUSCOM/SLA (p<0.001) and lower amounts of IL-4 was observed in PLUSCOM group (p<0.001) as compared to negative control.. Our results indicated that SLA in different formulations generated an immune response with mixed Th1/Th2 response that was not protective enough despite the activation of CD4+ T cells with secreting IFN-γ in groups which received PLUSCOM with antigen.

Topics: Adjuvants, Immunologic; Animals; Antigens, Protozoan; CD4-Positive T-Lymphocytes; Cells, Cultured; Disease Models, Animal; Drug Delivery Systems; Fatty Acids, Monounsaturated; Female; Immunization; Immunoglobulin G; Immunotherapy; Interferon-gamma; Interleukin-4; Leishmania; Leishmaniasis; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Multiprotein Complexes; Parasite Load; Quaternary Ammonium Compounds

Cationic liposomes have been used as efficient antigen delivery systems for cancer vaccination. The current study has investigated whether the incorporation of the helper-fusogenic lipid dioleoylphosphatidylethanolamine (DOPE) in cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)-cholesterol enhances the cytosolic delivery of p5 HER-2/neu derived peptide (p5) and promotes cytotoxic T lymphocytes (CTL) response. The p5, which is a very hydrophobic peptide, was encapsulated into liposomes by using three different methods and characterized for their colloidal properties. A chaotropic loading method using 7 M urea provided the highest encapsulation yields. Mice were first immunized with encapsulated p5 in liposomes composed of either DOTAP-cholesterol or DOTAP-cholesterol-DOPE, alone or co-administered with CpG-ODN, as an immunoadjuvant, then, inoculated with a subcutaneous injection of TUBO tumor cells. Results obtained from enzyme-linked immunospot, cytotoxicity and intracellular cytokine assays as well as tumor sizes and animal survival analysis demonstrated that p5 encapsulated in DOTAP-cholesterol-DOPE liposomes co-administered with CpG-ODN greatly enhanced the cytotoxic T lymphocytes response and highly inhibited the tumor progression. The outperformance of DOTAP-cholesterol-DOPE liposomes+CpG-ODN was found to be attributed to its capability in induction of both CD8+ and CD4+ responses. This formulation could be a suitable vaccine candidate against Her2 positive cancers and merits further investigations.

Topics: Adjuvants, Immunologic; Animals; Cancer Vaccines; Cell Line, Tumor; Cytokines; Cytotoxicity, Immunologic; Disease Models, Animal; Fatty Acids, Monounsaturated; Female; Humans; Immunization; Liposomes; Mice; Neoplasms; Oligodeoxyribonucleotides; Peptide Fragments; Quaternary Ammonium Compounds; Receptor, ErbB-2; Spleen; T-Lymphocyte Subsets; Tumor Burden

A suitable adjuvant and delivery system are needed to develop an effective vaccine against leishmaniasis. To induce a Th1 type of response and protection in BALB/c mice against Leishmania major infection, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) nanoliposomes bearing an intrinsic adjuvanticity, were used as an antigen delivery system and immunoadjuvant for soluble Leishmania antigens (SLA). DOTAP liposomes containing different concentrations of SLA were prepared by using lipid film method followed by sonication. The prepared vesicles showed a diameter of about 100nm, a positive zeta potential and approximately 70% encapsulation efficiency of SLA. BALB/c mice were immunized subcutaneously (SC), three times in a 3-week interval with different concentrations of liposomal SLA (12.5, 25, and 50μg of SLA/50μl/mice), free SLA and as well as free liposome. The group of mice received 50μg of SLA in DOTAP-nanoliposomes showed a significantly (p<0.001) smaller footpad swelling and the lowest spleen and footpad parasite burden after the challenge. This group also showed the highest IFN-γ production compared to the other groups, lower IL-4 level and higher IgG2a antibody titer. Taken together, the results indicated that simple DOTAP nanoliposome containing 1μg/μl SLA are appropriate delivery systems to induce a Th1 type of immune response and protection against L. major infection in BALB/c mice.

Topics: Adjuvants, Immunologic; Animals; Antigens, Protozoan; Disease Models, Animal; Fatty Acids, Monounsaturated; Female; Foot; Injections, Subcutaneous; Leishmania major; Leishmaniasis; Leishmaniasis Vaccines; Liposomes; Mice; Mice, Inbred BALB C; Nanoparticles; Parasite Load; Quaternary Ammonium Compounds; Vaccination

Short interfering RNA (siRNA) is a potent activator of the mammalian innate immune system. When considering possible clinical applications of siRNA for humans, the adverse immunostimulatory effects must also be taken into account. Here, we show that atelocollagen-mediated systemic delivery of siRNA without chemical modifications did not cause any immunostimulation in both animals and human peripheral blood mononuclear cells (PBMCs), even if the siRNA harbored an interferon (IFN)-inducible sequence. In contrast, systemic delivery of immunostimulatory RNA (isRNA)-mediated by a cationic lipid (such as Invivofectamine) induced potent type-I IFNs and inflammatory cytokines. Regarding the mechanism by which the isRNA/atelocollagen complex avoided adverse effects on immunostimulation, we revealed that this complex was not incorporated into PBMCs. On the other hand, Invivofectamine delivered isRNA into PBMCs. The use of either atelocollagen or Invivofectamine as a vehicle elicited significant and undistinguishable therapeutic effects in a contact hypersensitivity (CHS) inflammatory model mouse, when we intravenously injected the siRNA targeting monocyte chemoattractant protein-1 as the complex. For the goal of realizing siRNA-based medicines for humans, atelocollagen is an excellent and promising delivery vehicle, and it has the useful advantage of evading detection by the "radar" of innate immunity.

Topics: Adjuvants, Immunologic; Animals; Cells, Cultured; Chemokine CCL2; Collagen; Dermatitis, Contact; Disease Models, Animal; Fatty Acids, Monounsaturated; Female; Gene Transfer Techniques; Humans; Inflammation Mediators; Interferon Type I; Kidney Function Tests; Leukocytes, Mononuclear; Liver; Macrophages; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred ICR; Quaternary Ammonium Compounds; Receptors, Cell Surface; RNA, Small Interfering

The efficacy of an antisense oligonucleotide (ODN17) cationic nanoemulsion directed at VEGF-R2 to reduce neovascularization was evaluated using rat corneal neovascularization and retinopathy of prematurity (ROP) mouse models. Application of saline solution or scrambled ODN17 solution on eyes of rats led to the highest extent of corneal neovascularization. The groups treated with blank nanoemulsion or scrambled ODN17 nanoemulsion showed moderate inhibition in corneal neovascularization with no significant difference with the saline and scrambled ODN17 control solution groups, while the groups treated with ODN17 solution or Avastin® (positive ODN17 control) clearly elicited marked significant inhibition in corneal neovascularization confirming the results reported in the literature. The highest significant corneal neovascularization inhibition efficiency was noted in the groups treated with ODN17 nanoemulsion (topical and subconjunctivally). However, in the ROP mouse model, the ODN17 in PBS induced a 34% inhibition of retinal neovascularization when compared to the aqueous-vehicle-injected eyes. A significantly higher inhibition of vitreal neovascularization (64%) was observed in the group of eyes treated with ODN17 nanoemulsion. No difference in extent of neovascularization was observed between blank nanoemulsion, scrambled ODN17 nanoemulsion, vehicle or non-treated eyes. The overall results indicate that cationic nanoemulsion can be considered a promising potential ocular delivery system and an effective therapeutic tool of high clinical significance in the prevention and forthcoming treatment of ocular neovascular diseases.

Topics: Angiogenesis Inhibitors; Animals; Cations; Corneal Neovascularization; Disease Models, Animal; Drug Carriers; Emulsions; Fatty Acids, Monounsaturated; Humans; Infant, Newborn; Male; Mice; Mice, Inbred C57BL; Nanostructures; Oligonucleotides, Antisense; Quaternary Ammonium Compounds; Rats; Rats, Sprague-Dawley; Retinopathy of Prematurity; Vascular Endothelial Growth Factor Receptor-2; Vitreous Body

Previously we have shown cationic lipid (R)-DOTAP as the immunologically active enantiomer of the DOTAP racemic mixture, initiating complete tumor regression in an exogenous antigen model (murine cervical cancer model). Here, we investigate the use of (R)-DOTAP as an efficacious adjuvant delivering an endogenous antigen in an aggressive murine solid tumor melanoma model. (R)-DOTAP/Trp2 peptide complexes showed decreasing size and charge with increasing peptide concentration, taking a rod shape at highest concentrations. The particles were stable for 2 weeks at 4 °C. A dose of 75 nmol of Trp2 (formulated in (R)-DOTAP) was able to show statistically significant tumor growth delay compared to lower doses of 5 and 25 nmol, which were no different than untreated tumors. (R)-DOTAP/Trp2 (75 nmol) treated mice also showed increased T cell IFN-γ secretion after restimulation with Trp2, as well as CTL activity in vivo. This vaccination group also showed the highest population of functionally active tumor-infiltrating lymphocytes, indicated by IFN-γ secretion after restimulation with Trp2. Thus, (R)-DOTAP has shown the ability to break tolerance as an adjuvant. Its activity to enhance immunogenicity of other tumor associated antigens should be studied further.

Topics: Adjuvants, Immunologic; Animals; Antibody Formation; Cancer Vaccines; Cell Line, Tumor; Disease Models, Animal; Fatty Acids, Monounsaturated; Female; Immunotherapy; Interferon-gamma; Lymphocytes, Tumor-Infiltrating; Melanoma, Experimental; Membrane Proteins; Mice; Mice, Inbred C57BL; Microscopy, Electron, Transmission; Neoplasm Transplantation; Particle Size; Peptide Fragments; Quaternary Ammonium Compounds; Stereoisomerism; T-Lymphocytes, Cytotoxic; Tumor Microenvironment; Vaccines, Subunit

Single-stranded RNA stimulates immune cells and induces the secretion of pro-inflammatory cytokines and type I IFN. As adjuvant RNA can induce a T(h)2 type of humoral response, however, its potency in the induction of cytotoxic T cells in vivo has not been analyzed. Here we show that immunization with the antigen ovalbumin (OVA) and the adjuvant phosphodiester RNA complexed to the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) induced a Toll-like receptor-7-dependent cytotoxic T cell and humoral response. Staining with SIINFEKL-K(b) tetramers demonstrated the induction of antigen-specific T cells that were functional in in vivo cytotoxic T cell assays against SIINFEKL-loaded target cells. In infection experiments with OVA-secreting Listeria monocytogenes, the cytotoxic T cell response strongly reduced the bacterial load in liver and spleen. The RNA-driven humoral response was characterized by OVA-specific antibodies of the IgG1 isotype whereas CpG-DNA induced antigen-specific antibodies of the IgG2a (BALB/c) or IgG2c (C57BL/6) isotype. Furthermore, stimulation with RNA did not induce splenomegaly, a common feature of CpG-DNA-driven immune activation in mice. Taken together, our data confirm that RNA can be used as a safe adjuvant and induces a strong antibody response of the IgG1 isotype. Additionally, we demonstrate that RNA induces an antigen-specific immunity characterized by a potent cytotoxic T cell response to infection.

Topics: Adjuvants, Immunologic; Animals; Antibody Formation; Antigens; Bacterial Vaccines; Cells, Cultured; Disease Models, Animal; Fatty Acids, Monounsaturated; Female; Immunization; Immunoglobulin G; Listeria monocytogenes; Listeriosis; Lymphocyte Activation; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Oligodeoxyribonucleotides; Oligonucleotides; Ovalbumin; Quaternary Ammonium Compounds; RNA; T-Lymphocytes, Cytotoxic; Th2 Cells; Toll-Like Receptor 7

The present study aimed to establish an efficient targeted nonviral strategy for IL-12 gene transfer in colon carcinoma in vivo employing transferrin (Tf)-lipoplexes. Complexes for in vitro experiments were prepared at a 5/1(+/ - ) (lipid/DNA) charge ratio, with the ligand Tf (32 (microg/(microg DNA). Complexes for in vivo experiments contained 144 mM of total lipid (DOTAP/Chol), 60 (microg of pCMVLuc or pCMVIL-12 and 32 (microg of Tf-lipoplexes per microgram of plasmid. For intratumoral studies, CT26 (5 x 105 cells) in 50 microl of PBS were inoculated subcutaneously into the back of the mouse. Treatments began when tumor sizes reached 5-6 mm in diameter. Complexes were injected by a single intratumoral injection in a volume of 50 microl. Our in vitro results indicate that Tf-lipoplexes always mediate higher gene expression in colon (CT26) tumor cells, compared to plain-lipoplexes (without ligand) or naked plasmid. At the same time, CT26 tumor-bearing animals treated with Tf-lipoplexes containing the therapeutic gene IL-12, showed tumor growth inhibition, leading to a complete tumor regression in 75% of the treated mice (p < 0.001), without signs of recurrence. High levels of IL-12 and IFN-gamma were detected in the sera of treated mice. Mice survival also improved considerably by treatment with this system, with a survival rate of 88%, at 23 days post-administration. In summary, in this study we have developed an efficient, targeted cationic lipid-based system for the treatment of colon tumors. The vector has the advantages of ease of preparation and economy, in comparison with commercial transfection reagents, as well as, the possibility of a large scale production.

Topics: Animals; Cations; Cells, Cultured; Cholesterol; Colonic Neoplasms; Disease Models, Animal; Fatty Acids, Monounsaturated; Female; Gene Expression Regulation; Gene Transfer Techniques; Genetic Therapy; Interferon-gamma; Interleukin-12; Mice; Mice, Inbred BALB C; Plasmids; Quaternary Ammonium Compounds; Survival Rate; Transferrin