RESUMO
Clinical trials of cholesterol ester transfer protein (CETP) peptide vaccine were stopped after disappointing results in humans due to the inadequacy of adjuvant aluminum hydroxide in stimulating the immune response against the self-antigen of CETP. To increase the efficacy of the CETP vaccine, we developed a novel liposomal form of tetanus toxoid-CETP (TT-CETP) peptide (Lip CETP) with well-characterized properties and high encapsulation efficiency. The vaccine efficacy against atherosclerosis was evaluated in rabbits challenged with a high cholesterol diet. Rabbits were immunized with Lip-CETP or liposome containing CETP with CpG ODN (Lip CETP/CpG). Control groups received empty liposomes or buffer. Anti-TT-CETP specific antibodies in serum were determined and gene expression of cytokine IFN-γ and IL-4 were measured in blood peripheral mononuclear cells. Therapeutic response was evaluated by titration of plasma lipoproteins during the study and pathologic analysis of aorta atherosclerotic lesions at the end. Lip-CETP/CpG elicited strong anti-TT-CETP antibodies and a higher IFN-γ level than the buffer. IL-4 was lower than the buffer in all vaccinated groups. Plasma lipoproteins showed no significant difference in the studied groups. Atherosclerosis thickness grade of the aorta was lower than the buffer group (p < 0.001) in rabbits vaccinated with Lip-CETP but not with Lip-CETP/CpG. In conclusion, Lip-CETP showed a strong atheroprotective effect.
Assuntos
Aterosclerose/prevenção & controle , Proteínas de Transferência de Ésteres de Colesterol/administração & dosagem , Vacinas/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/uso terapêutico , Animais , Aorta/patologia , Aterosclerose/patologia , Proteínas de Transferência de Ésteres de Colesterol/uso terapêutico , Lipossomos/química , Masculino , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/uso terapêutico , Coelhos , Vacinas/uso terapêuticoRESUMO
The size of nanoliposome-encapsulated drugs significantly affects their therapeutic efficacy, biodistribution, targeting ability, and toxicity profile for the cancer treatment. In the present study, the biodistribution and anti-tumoral activity of PEGylated liposomal Doxorubicin (PLD) formulations with different sizes were investigated. First, 100, 200, and 400 nm PLDs were prepared by remote loading procedure and characterised for their size, zeta potential, encapsulation efficacy, and release properties. Then, in vitro cellular uptake and cytotoxicity were studied by flow cytometry and MTT assay, and compared with commercially available PLD Caelyx® . In vivo studies were applied on BALB/c mice bearing C26 colon carcinoma. The cytotoxicity and cellular uptake tests did not demonstrate any statistically significant differences between PLDs. The biodistribution results showed that Caelyx® and 100 nm liposomal formulations had the most doxorubicin (Dox) accumulation in the tumour tissue and, as a result, considerably suppressed tumour growth compared with 200 and 400 nm PLDs. In contrast, larger nanoparticles (200 and 400 nm formulations) had more accumulation in the liver and spleen. This study revealed that 90 nm Caelyx® biodistribution profile led to the stronger anti-tumour activity of the drug and hence significant survival extension, and showed the importance of vesicle size in the targeting of nanoparticles to the tumour microenvironment for the treatment of cancer.
Assuntos
Neoplasias do Colo , Doxorrubicina , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Doxorrubicina/análogos & derivados , Lipossomos , Camundongos , Camundongos Endogâmicos BALB C , Polietilenoglicóis , Distribuição Tecidual , Microambiente TumoralRESUMO
Leishmaniasis is a serious health problem that needs a suitable vaccine delivery system to control the disease. Cationic lipids such as 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) have been widely used in nanoliposomes' formulation to deliver antigen and adjuvant at the same time to induce protection against Leishmaniasis. Therefore, it is necessary to accurately quantify DOTAP concentration in the formulation and biological materials. Due to the poor UV absorbance of DOTAP, the use of the conventional HPLC-UV method was impossible. Currently, an evaporative light scattering detector (ELSD) or MS/MS detector in conjunction with HPLC is used to quantify DOTAP. These methods have several disadvantages, including time- consuming during extraction procedure and decrease or/and even remove some components of the formulation. According to the advantages of the quantitative 1H Nuclear Magnetic Resonance (1H-NMR) spectroscopic method, a free extraction approach was developed to the assay of DOTAP in nanoliposomes containing Leishmania antigens. This method was carried out based on the relative ratio of signal integration of DOTAP [CH2 (CH2-CH = CH-CH2)] in δ 2 ppm to a definite amount of an internal standard called dimethyl sulfone (DMSO2). The q1H-NMR method showed good precision (intra-day RSD = 1.8 % and inter-day RSD = 2.5 %), linearity (in the ranges of 1.3-7.8 mg. mL-1 with correlation coefficients at 1), repeatability (RSD ≤ 2.39 %), and stability (RSD ≤ 2.32 %) for the quantification of the DOTAP without any extraction method. Considering all the experiments conducted in this study, NMR can be a feasible alternative to other traditional techniques for the simultaneous quantification of lipids in liposome formulations.
Assuntos
Leishmania , Ácidos Graxos Monoinsaturados , Lipídeos , Lipossomos , Propano , Espectroscopia de Prótons por Ressonância Magnética , Compostos de Amônio Quaternário , Espectrometria de Massas em TandemRESUMO
To overcome the lack of selectivity and nonspecific biodistribution of drugs in the body, targeted delivery of chemotherapeutic agents with aptamers is a very effective method. In this strategy, aptamers could be specifically identified and attach to targeted molecules on the cancerous cells and deliver the chemotherapeutic agents to desired tissue with minimal or no damage to the normal cells. In this study, we designed anti-epithelial cell adhesion molecule (EpCAM) RNA aptamer conjugated PEGylated liposomal doxorubicin (ER-lip) to investigate its in vitro and in vivo anticancer abilities. Data showed that EpCAM aptamer was able to enhance cell uptake and cytotoxic effects of Dox in C26 cell line. The biodistribution study indicated that ER-lip enhanced the tumor accumulation of Dox compared to Caelyx. Also, double staining of isolated tumor cells with anti-CD44-PE-cy5 and anti-EpCAM Cy-7 antibodies indicated that tumor cells expressed a high level of EpCAM+ CD44+ cells (p ≤ .001) compared to cultured C26 cell line. in vivo results showed that ER-lip promoted survival and reduced tumor growth rate in animal model compared to Caelyx. In conclusion, these results suggested that the ER-lip could be served as promising formulation for the treatment of cancers with the high expression of EpCAM.
Assuntos
Antineoplásicos , Aptâmeros de Nucleotídeos , Doxorrubicina/análogos & derivados , Sistemas de Liberação de Medicamentos/métodos , Molécula de Adesão da Célula Epitelial/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Aptâmeros de Nucleotídeos/farmacocinética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/química , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Molécula de Adesão da Célula Epitelial/química , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Polietilenoglicóis/farmacologia , Distribuição TecidualRESUMO
AIM: This study aimed to investigate the existence of phospholipase-A (PLA) activity in Soluble L. major Antigens (SLA) because of no reports for it so far. Liposomes were used as sensors to evaluate PLA activity. OBJECTIVES: Liposomal SLA consisting of Egg Phosphatidylcholine (EPC) or Sphingomyelin (SM) were prepared by two different methods in different pH or temperatures and characterized by Dynamic Light Scattering (DLS) and Thin Layer Chromatography (TLC). METHODS: Lipid hydrolysis led to the disruption of EPC liposomal SLA in both methods but the Film Method (FM) produced more stable liposomes than the Detergent Removal Method (DRM). RESULT: The preparation of EPC liposomal SLA at pH 6 via FM protected liposomes from hydrolysis to some extent for a short time. EPC liposomes but not SM liposomes were disrupted in the presence of SLA. CONCLUSION: Therefore, a phospholipid without ester bond such as SM should be utilized in liposome formulations containing PLA as an encapsulating protein.
Assuntos
Leishmania major/enzimologia , Vacinas contra Leishmaniose/química , Leishmaniose Cutânea/prevenção & controle , Fosfolipases A/metabolismo , Proteínas de Protozoários/química , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/metabolismo , Antígenos de Protozoários/administração & dosagem , Antígenos de Protozoários/química , Antígenos de Protozoários/metabolismo , Composição de Medicamentos/métodos , Estabilidade de Medicamentos , Ensaios Enzimáticos , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Leishmania major/imunologia , Vacinas contra Leishmaniose/administração & dosagem , Vacinas contra Leishmaniose/metabolismo , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Lipossomos/química , Lipossomos/metabolismo , Fosfatidilcolinas/administração & dosagem , Fosfatidilcolinas/metabolismo , Fosfolipases A/isolamento & purificação , Proteínas de Protozoários/administração & dosagem , Proteínas de Protozoários/metabolismo , Esfingomielinas/administração & dosagem , Esfingomielinas/metabolismoRESUMO
HER2/neu is an immunogenic protein inducing both humoral and cell-mediated immune responses. The antigen-specific cytotoxic T lymphocytes (CTLs) are the main effector immune cells in the anti-tumor immunity. To induce an effective CTL specific response against P5+435 single peptide derived from rat HER2/neu oncogene, we used a liposome delivery vehicle. In vivo enhancement of liposome stability and intracytoplasmic delivery of peptides are the main strategies which elevate the liposome-mediated drug delivery. Liposomes containing high transition temperature phospholipids, such as DSPC, are stable with prolonged in vivo circulation and more accessibility to the immune system. Incorporation of DOPE phospholipid results in the effective delivery of peptide into the cytoplasm via the endocytotic pathway. To this end, the P5+435 peptide was linked to Maleimide-PEG2000-DSPE and coupled on the surface of nanoliposomes containing DSPC: DSPG: Cholesterol with/without DOPE. We observed that mice vaccinated with Lip-DOPE-P5+435 formulation had the highest number of IFN-γ- producing CTLs with the highest cytotoxic activity that consequently led to significantly smallest tumor size and prolonged survival rate in the TUBO mice model. In conclusion, our study indicated that the liposomal form of P5+435 peptide containing DOPE can be regarded as a promising prophylactic anti-cancer vaccine to generate potent antigen-specific immunity.