RESUMEN
In the present study, a Poloxamer 407-based amphotericin B (AmpB)-containing polymeric micelles system (AmpB/M) was employed in the treatment of Leishmania amazonensis-infected BALB/c mice. Initially, the in vitro antileishmanial activity (IC50 value) of AmpB/M and B-AmpB/M (empty micelles) against stationary promastigotes and amastigotes-like forms of the parasites was determined, and results were of 1.83 ± 0.4 and 22.1 ± 0.7 µM, respectively, for the promastigotes, and of 2.27 ± 0.5 and 33.98 ± 2.6 µM, respectively, for the amastigotes-like. The cytotoxic concentration (CC50) values of these products were also evaluated, and we found the results of 119.5 ± 9.6 and 134.7 ± 10.3 µM, respectively. With these values, the selectivity index (SI) was calculated and results were of 65.3 and 5.4, respectively, for the promastigotes, and of 59.3 and 3.96, respectively, for the amastigotes-like of the parasites. Free AmpB showed IC50 values of 1.2 ± 0.3 and 2.5 ± 0.5 µM for the promastigotes and amastigotes-like, respectively, whereas the CC50 value was of 9.5 ± 0.4 µM. The SI values of this drug were of 7.9 and 3.8, respectively, for the promastigote and amastigote-like stages of the parasites. After, animals were infected and received saline or were treated subcutaneously with free AmpB, AmpB/M or B-AmpB/M. In the results, free AmpB-treated and infected mice showed reductions in their body weight, which were associated with hepatic and renal damage; however, no organic alteration was observed in the AmpB/M-treated animals. In addition, these animals showed significant reductions in their lesion average size and in the parasite burden in all evaluated infected tissue and organs, when compared to the other groups; as well as significantly higher levels of antileishmanial IFN-γ, IL-12, GM-CSF and nitrite, which were associated with low production of IL-4, IL-10 and IgG1 isotype antibodies. In conclusion, this AmpB/M system could be considered as an alternative for future studies in the treatment of tegumentary leishmaniasis.
Asunto(s)
Anfotericina B/administración & dosificación , Antiprotozoarios/administración & dosificación , Excipientes , Leishmania mexicana/efectos de los fármacos , Leishmaniasis Cutánea/tratamiento farmacológico , Poloxámero , Anfotericina B/farmacología , Anfotericina B/uso terapéutico , Anfotericina B/toxicidad , Animales , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Antiprotozoarios/toxicidad , Supervivencia Celular/efectos de los fármacos , Citocinas/biosíntesis , Relación Dosis-Respuesta a Droga , Femenino , Inmunoglobulina G/biosíntesis , Concentración 50 Inhibidora , Leishmania mexicana/crecimiento & desarrollo , Leishmania mexicana/inmunología , Leishmaniasis Cutánea/inmunología , Leishmaniasis Cutánea/parasitología , Leishmaniasis Cutánea/patología , Hígado/parasitología , Ganglios Linfáticos/parasitología , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Micelas , Polímeros , Ratas , Bazo/citología , Bazo/inmunología , Bazo/parasitologíaRESUMEN
The current treatment of leishmaniasis has been hampered due to the high toxicity of the available drugs and long duration protocols, which often lead to its abandonment. In the present study, a poloxamer 407-based delivery system was developed, and a molecule, 8-hydroxyquinoline (8-HQN), was incorporated with it, leading to an 8-HQN/micelle (8-HQN/M) composition. Assays were performed to evaluate the in vitro antileishmanial activity of 8-HQN/M against Leishmania amazonensis stationary promastigotes. The cytotoxicity in murine macrophages and in human red cells, as well as the efficacy of the treatment in macrophages infected by parasites, was also assessed. This product was also evaluated for the treatment of murine tegumentary leishmaniasis, using L. amazonensis-infected BALB/c mice. To evaluate the in vivo efficacy of the treatment, the average lesion diameter (area) in the infected tissue, as well as the parasite load at the site of infection (skin), spleen, liver and draining lymph nodes were examined. Non-incorporated micelle (B-8-HQN/M) and the free molecule (8-HQN) were used as controls, besides animals that received only saline. The parasite burden was evaluated by limiting dilution and quantitative real-time PCR (qPCR) techniques, and immunological parameters associated with the treatments were also investigated. In the results, the 8-HQN/M group, when compared to the others, presented more significant reductions in the average lesion diameter and in the parasite burden in the skin and all evaluated organs. These animals also showed significantly higher levels of parasite-specific IFN-γ, IL-12, and GM-CSF, associated with low levels of IL-4 and IL-10, when compared to the saline, 8-HQN/M, and B-8-HQN groups. A predominant IL-12-driven IFN-γ production, against parasite proteins, mainly produced by CD4+ T cells, was observed in the treated animals, post-infection. In conclusion, 8-HQN/M was highly effective in treating L. amazonensis-infected BALB/c mice and can be considered alone, or combined with other drugs, as an alternative treatment for tegumentary leishmaniasis. Graphical Abstract Therapeutic scheme and immunological and parasitological parameters developed in the present study.
Asunto(s)
Leishmania mexicana/efectos de los fármacos , Leishmaniasis Cutánea/tratamiento farmacológico , Oxiquinolina/uso terapéutico , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Eritrocitos/parasitología , Femenino , Humanos , Leishmaniasis Cutánea/parasitología , Hígado/parasitología , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Micelas , Oxiquinolina/administración & dosificación , Carga de Parásitos , Polímeros , Bazo/parasitología , Linfocitos T/inmunologíaRESUMEN
The present study was carried out to evaluate the intravaginal vaccine potential against bovine alphaherpesvirus type 5 (BoHV-5). Sixty three cows were divided into seven groups (n: 9) and inoculated intravaginally (VA) or intramuscularly (IM) with inactivated BoHV-5, associated with the recombinant B subunit of the heat-labile enterotoxin of E. coli (rLTB), 2-hydroxyethylcellulose (Drug Delivery System A - DDS-A) or Poloxamer 407 (Drug Delivery System B - DDS-B) as follows: G1 (DDS-A + BoHV-5 + rLTB), G2 (DDS-A + BoHV-5), G3 (DDS-B + BoHV-5 + rLTB), G4 (DDS-B + BoHV-5), G5 (BoHV-5 + rLTB), G6 (Negative control) e G7 (Positive control). The local and systemic humoral responses were measured by indirect ELISA (IgA and IgG) and serum neutralization tests, and the cellular response was measured by a quantitative direct ELISA (IL-2 and IFN-Gamma). The results showed the group inoculated by the IM route, G5, demonstrated the highest levels of IgG in the vaginal mucosa among the experimental groups (p < 0.05). In the groups tested with polymers (G1 and G3) in the vaginal mucosa, even higher levels of IgG were seen in comparison to the positive control (G7; p < 0.01). Higher levels of IgA were also noted in relation to the other groups (p < 0.05) on days 30, 60 and 90 post-inoculations. The groups G1 and G3 also provided higher titers of neutralizing antibodies (Log2) in relation to other treatments (p < 0.01) 90 days after inoculation. In the nasal mucosa, there was an increase in the levels of IgA and IgG with the use of vaccines from groups G1 and G3, in relation to the positive control, G7 (p < 0.05) at 60 and 90 days after the first inoculation. Moreover, neutralizing antibodies titers were detected at 60 and 90 days by serum neutralization. The inclusion of the evaluated polymers resulted in a superior response (p < 0.05) of immunoglobulins and IL-2 and IFN-Gamma in relation to the treatment using only rLTB (G5). This data demonstrates the capabilities of a vaccine with an intravaginal application in cattle to stimulate a local and systemic immune response.
Asunto(s)
Escherichia coli , Vacunas Virales , Animales , Femenino , Bovinos , Vacunas de Productos Inactivados , Interleucina-2 , Anticuerpos Neutralizantes , Inmunoglobulina G , Inmunoglobulina A , Polímeros , Anticuerpos AntiviralesRESUMEN
New therapeutic strategies against leishmaniasis are desirable, since the treatment against disease presents problems, such as the toxicity, high cost and/or parasite resistance. As consequence, new antileishmanial compounds are necessary to be identified, as presenting high activity against Leishmania parasites, but low toxicity in mammalian hosts. Flau-A is a naphthoquinone derivative recently showed to presents an in vitro effective action against Leishmania amazonensis and L. infantum species. In the present work, the in vivo efficacy of Flau-A, which was incorporated into a Poloxamer 407-based micelle system, was evaluated in a murine model against L. amazonensis infection. Amphotericin B (AmB) and Ambisome® were used as controls. The animals were infected and later treated with the compounds. Thirty days after the treatment, parasitological and immunological parameters were evaluated. Results showed that AmB, Ambisome®, Flau-A or Flau-A/M-treated animals presented significantly lower average lesion diameter and parasite burden in tissue and organs evaluated, when compared to the control (saline and micelle) groups. Flau-A or Flau-A/M-treated mice were those presenting the most significant reductions in the parasite burden, when compared to the others. These animals developed also a more polarized antileishmanial Th1 immune response, which was based on significantly higher levels of IFN-γ, IL-12, TNF-α, GM-CSF, and parasite-specific IgG2a isotype; associated with low levels of IL-4, IL-10, and IgG1 antibody. The absence of toxicity was found in these animals, although mice receiving AmB have showed high levels of renal and hepatic damage markers. In conclusion, results suggested that the Flau-A/M compound may be considered as a possible therapeutic target to be evaluated against human leishmaniasis.
Asunto(s)
Antiprotozoarios/uso terapéutico , Leishmania/efectos de los fármacos , Leishmaniasis/tratamiento farmacológico , Micelas , Naftoquinonas/uso terapéutico , Poloxámero/uso terapéutico , Animales , Antiprotozoarios/química , Antiprotozoarios/farmacocinética , Excipientes/química , Excipientes/farmacocinética , Excipientes/uso terapéutico , Femenino , Leishmania/metabolismo , Leishmaniasis/metabolismo , Ratones , Ratones Endogámicos BALB C , Naftoquinonas/química , Naftoquinonas/farmacocinética , Poloxámero/química , Poloxámero/farmacocinética , Resultado del TratamientoRESUMEN
Clioquinol (5-chloro-7-iodoquinolin-8-ol or ICHQ) was recently showed to presents an in vitro effective antileishmanial action, causing changes in membrane permeability, mitochondrial functionality, and parasite morphology. In the present study, ICHQ was incorporated into a Poloxamer 407-based polymeric micelles system (ICHQ/M), and its antileishmanial activity was in vivo evaluated in L. amazonensis-infected BALB/c mice. Amphotericin B (AmpB) and its liposomal formulation (Ambisome®) were used as controls. Parasitological and immunological evaluations were performed 30â¯days after the treatment. Results indicated more significant reductions in the average lesion diameter and parasite burden in ICHQ or ICHQ/M-treated mice, which were associated with the development of a polarized Th1 immune response, based on production of high levels of IFN-γ, IL-12, TNF-α, GM-CSF, and antileishmanial IgG2a antibody. Control groups´ mice produced high levels of IL-4, IL-10, and IgG1 isotype antibody. No organic toxicity was found by using ICHQ or ICHQ/M to treat the animals, although those receiving AmpB and Ambisome® have presented higher levels of renal and hepatic damage markers. In conclusion, results suggested that the ICHQ/M composition can be considered as an antileishmanial candidate to be tested against human leishmaniasis.
Asunto(s)
Antiprotozoarios/inmunología , Antiprotozoarios/uso terapéutico , Clioquinol/inmunología , Clioquinol/uso terapéutico , Leishmania mexicana/efectos de los fármacos , Leishmaniasis Visceral/tratamiento farmacológico , Poloxámero/administración & dosificación , Anfotericina B/administración & dosificación , Anfotericina B/uso terapéutico , Anfotericina B/toxicidad , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/administración & dosificación , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/uso terapéutico , Antiprotozoarios/administración & dosificación , Antiprotozoarios/toxicidad , Clioquinol/administración & dosificación , Citocinas/biosíntesis , Citocinas/inmunología , Sistemas de Liberación de Medicamentos/métodos , Humanos , Inmunoglobulina G/sangre , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Leishmania mexicana/crecimiento & desarrollo , Leishmaniasis Visceral/inmunología , Ratones , Ratones Endogámicos BALB C , Micelas , Carga de Parásitos , Poloxámero/química , Células TH1RESUMEN
Metronomic chemotherapy is a novel approach to the control of advanced cancer, as it appears to preferentially inhibit endothelial cell activity in the growing vasculature of tumors. Doxorubicin-containing sterically stabilized liposomes (DXR-SL) accumulate in large amounts in tumor tissue, resulting in enhanced antitumor effects of the encapsulated DXR. In the present study, it was hypothesized that metronomic chemotherapy may further augment the accumulation of DXR-SL, improving its therapeutic efficacy. This study tests the antitumor efficacy for the combination of a metronomic cyclophosphamide (CPA)-dosing schedule with sequential intravenous injections of DXR-SL in the treatment of lung metastatic B16BL6 melanoma-bearing mice. Three dosing schedules for the combination of metronomic CPA injections (s.c. 170 mg/kg every 6 days) plus either a low or a high dose of DXR-SL (i.v. 1 or 5 mg/kg every 6 days) were set: Schedule I, DXR-SL was given 3 days before the first CPA treatment; Schedule II, DXR-SL and CPA were given simultaneously; and, Schedule III, DXR-SL was given 3 days after the first CPA treatment. Lung weight and median survival time (MST) were evaluated. As expected, both the dosing schedule as well as the dose of DXR-SL improved therapeutic efficacy. Schedule I with the low DXR dose and Schedule II with the low or high DXR dose significantly increased MST, compared with regular metronomic CPA therapy. Under the dosing schedules (Schedule I with the low DXR dose and Schedule II with the high DXR), there was a strong relationship between increased MST and decreased lung weight. However, Schedule I with high DXR dose resulted in significantly lower lung weights, but did not increase MST, suggesting that chemotherapy may result in increased toxicity in some conditions. Although treatment regimens require optimization, the results of the present study may prove useful in further explorations of combining metronomic chemotherapy with liposomal anticancer drugs in the treatment of solid tumors.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Ciclofosfamida/administración & dosificación , Doxorrubicina/análogos & derivados , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Polietilenglicoles/administración & dosificación , Animales , Línea Celular Tumoral , Doxorrubicina/administración & dosificación , Esquema de Medicación , Masculino , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BLRESUMEN
In this report, we investigated the combined effect of drug liposomalization and addition of glycerol on the transdermal delivery of isosorbide 5-nitrate (ISN) in rat abdominal skin in vitro. Occlusive application of both liposomal and aqueous ISN solution, with and without addition of 5% glycerol, showed that drug liposomalization and addition of glycerol has far-reaching implications for ISN permeation and accumulation in 4 and 8 weeks old rat abdominal skin. Using 8 weeks old rat abdominal skin, the optimal concentration of glycerol to be added to liposomal ISN was found to be 5%. The ISN mean values permeated through and accumulated in stripped 8 weeks old rat abdominal skin from those formulations described above were not significant different, which might indicate the combined effect of glycerol and liposomal ISN resides solely in the stratum corneum (SC). Based on previous reports, the enhancement effect of glycerol might be due to an increase in the SC hydration, and perhaps due to subtle changes in the lipid organization caused by penetration of liposomal lipids within the SC intercellular spaces. These data might provide evidence that glycerol action on SC is useful to facilitate skin permeation and accumulation of drugs formulated in liposome.
Asunto(s)
Glicerol/farmacología , Dinitrato de Isosorbide/análogos & derivados , Liposomas/química , Vasodilatadores/administración & dosificación , Administración Cutánea , Animales , Química Farmacéutica , Portadores de Fármacos , Técnicas In Vitro , Dinitrato de Isosorbide/administración & dosificación , Dinitrato de Isosorbide/farmacocinética , Masculino , Ratas , Ratas Wistar , Absorción Cutánea , Vasodilatadores/farmacocinéticaRESUMEN
Efficient delivery is a key issue in translating interference RNA technology into a feasible therapy. The efficiency of carrier systems used for this technology is commonly tested by co-transfection, i.e. simultaneous transfection with an exogenous gene and with the siRNA. Two approaches can be distinguished: (1) with the two transfectants in the same carrier complex (siRNA/pDNA/carrier) and (2) with the two transfectants in different carrier complexes (pDNA/carrier and siRNA/carrier). The process to prepare the nucleic acid(s)-carrier complexes and the transfection procedure may affect the effectiveness of the gene-silencing process. In this study, two preparation methods were compared, namely the co-preparation of an siRNA/pDNA/liposome lipoplex (Method I) and the separate preparation of an siRNA/liposome lipoplex and a pDNA/liposome lipoplex (Method II). siRNA in the lipoplex produced by Method I showed a stronger gene-silencing effect than that in the lipoplexes prepared by Method II. There was no significant difference between the two methods in the amount of siRNA delivered to cells. Cellular entry and intracellular trafficking of siRNA/pDNA/liposome lipoplex is likely to differ from those of the separate lipoplexes. When in Method II non-transcriptional pDNA was included in the complex with siRNA, the gene-silencing effect was significantly enhanced. If and to what extent the experimental design is suitable to quantify RNA interference remains to be demonstrated.
Asunto(s)
Lípidos/química , Liposomas , Plásmidos/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transfección/métodos , Animales , Cationes , Línea Celular Tumoral , Colesterol/química , Citoplasma/metabolismo , Etanolaminas/química , Genes Reporteros , Luciferasas de Luciérnaga , Luciferasas de Renilla , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Ratones , Miristatos/química , Tamaño de la Partícula , Fosfatidiletanolaminas/química , ARN Interferente Pequeño/genéticaRESUMEN
In order to investigate the effect of liposomal drugs on skin delivery, it was postulated that the process of liposomalization might lead the drug to an overpredicted solubility state which has far-reaching implications for drug skin permeation and accumulation. In this regard, conventional (CL) and flexible liposomes (FL) were prepared by the lipid film hydration method and the particles were downsized by sonication using hydrocortisone (HC) as a poorly water soluble model drug. The solutions derived from the whole CL and FL suspensions eluted on a Sephadex G-50 column (SG-50) demonstrated that most part of HC not only resides solely in the water phase but also it might exist in an improved solubility state. The results of the in vitro study using rat abdominal skin and occlusive application indicated that HC penetrated and accumulated much better solely than when associated with CL or FL. In regard to the penetration of the non-entrapped HC associated to liposomes bilayer fragments, a very small amount of phospholipids in the non-liposomal part eluted on SG-50 was found that could not justify by itself the penetration of HC associated to liposome bilayer fragments. It was proposed that all the steps of the liposomes preparation process might contribute for the increased HC solubility state, but definitively the presence of phospholipids played a crucial role on improving the HC solubility in the absence of sodium cholate. In comparison with commercially available ointments, the non-entrapped HC solution derived from the whole CL suspension eluted on SG-50 showed a higher concentration of HC accumulated and more uniformly distributed as well in the epidermis and dermis compartments. In addition, the thermodynamic activity of the non-entrapped HC solutions maintaining a driving force of the drug across the skin barrier pointed out that the level of HC solubility achieved during liposome preparation has far-reaching implication for drug skin permeation and accumulation in the experimental conditions used. The findings also indicated that the non-entrapped drug solutions obtained on the process of liposomalization could be useful on transdermal drug delivery systems, particularly for improving the permeation and accumulation capacity of poorly soluble drugs.
Asunto(s)
Antiinflamatorios/administración & dosificación , Hidrocortisona/administración & dosificación , Administración Cutánea , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacocinética , Portadores de Fármacos , Hidrocortisona/química , Hidrocortisona/farmacocinética , Técnicas In Vitro , Liposomas , Masculino , Microscopía Electrónica de Rastreo , Pomadas , Tamaño de la Partícula , Fosfolípidos/análisis , Fosfolípidos/química , Ratas , Ratas Wistar , Piel/química , Absorción Cutánea/efectos de los fármacos , SuspensionesRESUMEN
We recently demonstrated that agitation during lipoplex formation (vorLTsiR) improves the gene knockdown effect of siRNA because the resultant decrease in lipoplex size leads to an enhanced uptake by cells. In furthering this line of research, the present study was focused on the interaction of siRNA to cationic liposomes during lipoplex preparation. A fluorescence resonance energy transfer (FRET) study indicated that the application of agitation in the presence of siRNA effectively reorganized positively charged lipids (DC-6-14 and DOPE) in an order that effectively promoted further electrostatic interaction between the negatively charged phosphate backbone of siRNA and the positively charged lipids in the cationic liposome membrane. A circular dichroism (CD) study indicated that the agitation did not bring about a change in the A-form helix of siRNA, therefore the interactions between the lateral anionic groups of siRNA - responsible for the characteristic bands of the A-form helix - and cationic liposomes were effectively promoted. Factorial design coupled with response surface methodology was used to statistically analyze the influence of vortex speed and time and siRNA dose on the in vitro gene knockdown effects of siRNA-lipoplex that were spontaneously formulated (spoLTsiR) along with that formulated under agitation (vorLTsiR). The analysis indicated that vortex speed plays the most important role in enhancing the gene knockdown effect of siRNA among the three variables, although all three are important. It was concluded that the high energy transmitted by applying agitation during lipoplex formation harmonized the interaction of siRNA to positively charged lipids (DC-6-14 and DOPE) in cationic liposomes, resulting in a superior gene knockdown efficacy of vorLTsiR compared to spoLTsiR. Our study suggests that the preparation procedure is one of the critical factors in producing the enhanced gene knockdown effect of siRNA.
Asunto(s)
Etanolaminas/química , Técnicas de Silenciamiento del Gen , Miristatos/química , Fosfatidiletanolaminas/química , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transfección/métodos , Cationes , Dicroismo Circular , Análisis Factorial , Transferencia Resonante de Energía de Fluorescencia , Células HeLa , Humanos , Liposomas , Luciferasas/biosíntesis , Luciferasas/genética , Movimiento (Física) , Conformación de Ácido Nucleico , ARN Interferente Pequeño/química , Factores de TiempoRESUMEN
The successful delivery of therapeutic siRNA to the designated target cells and their availability at the intracellular site of action are crucial requirements for successful RNAi therapy. In the present study, we focused on the siRNA-lipoplex preparation procedure and its effect on the gene-knockdown efficiency of siRNA in vitro. Agitation (vortex-mixing) during siRNA-lipoplex (vor-LTsiR) preparation and its effect on the gene-knockdown efficiency of stably expressed cell GFP was investigated, and their efficiency was compared with that of spontaneously formed lipoplex (spo-LTsiR). A dramatic difference in size between lipoplexes was observed at the N/P ratio of 7.62 (siRNA dose of 30 nM), even though both lipoplexes were positively charged. With the siRNA dose of 30 nM, vor-LTsiR accomplished a 50% gene-knockdown, while spo-LTsiR managed a similar knockdown effect at the 120 nM level, suggesting that the preparation procedure remarkably affects the gene-knockdown efficacy of siRNA. The uptake of vor-LTsiR was mainly via clathrin-mediated endocytosis, whereas that of spo-LTsiR was via membrane fusion. In addition, by inhibiting clathrin-mediated endocytosis, the gene-knockdown efficiency was significantly lowered. The size of the lipoplex, promoted by the preparation procedure, is likely to define the entry pathway, resulting in an increased amount of siRNA internalized in cells and an enhanced gene-knockdown efficacy. The results of the present study definitively show that a proper siRNA-lipoplex preparation procedure makes a significant contribution to the efficiency of cellular uptake, and thereby, to the gene-knockdown efficiency of siRNA.
Asunto(s)
Técnicas de Silenciamiento del Gen/métodos , Silenciador del Gen , Lípidos/química , ARN Interferente Pequeño/administración & dosificación , Cationes , Línea Celular Tumoral , Clatrina/metabolismo , Endocitosis , Humanos , LiposomasRESUMEN
In the last two decades, cationic liposomes have been investigated as vehicles for nucleic acids [plasmid DNA (pDNA) and small interfering RNA (siRNA)] delivery in vitro and in vivo. The formation of cationic liposomes-nucleic acids complexes, termed lipoplexes, depends on a number of experimental variables. The quality of the nucleic acid and the cationic liposome as well as the selection of diluents for diluting the concentrated stocks strongly affect the resulting lipoplexes and their efficiency of gene-expression or gene-silencing effect following transfection. In addition, the molar ratio of cationic lipid nitrogen (N) to siRNA or pDNA phosphate (P) (N/P ratio) influences the final characteristics of the lipoplexes, such as size, surface zeta potential, and reproducibility, thereby reflecting their efficiency following transfection. The methods presented in this chapter could be helpful to obtain reliable and reproducible lipoplexes and experimental results.
Asunto(s)
ADN/administración & dosificación , Liposomas/química , ARN Interferente Pequeño/administración & dosificación , Transfección , Cationes/química , ADN/química , Silenciador del Gen , Células HeLa , Humanos , Liposomas/clasificación , Plásmidos/administración & dosificación , ARN Interferente Pequeño/químicaRESUMEN
PURPOSE: The purpose of this study is to determine if the treatment with siRNA-lipoplexes significantly influences on global gene expression in the treated cells. METHODS: We investigated global gene expression in a HT1080 cell line by a cDNA microarray. We also evaluated the effect of lipofection on global gene expression by determining the change of the expression of an exogenous gene, green fluorescence protein (GFP), and also determined treatment-related cytotoxicity. RESULTS: Treatment of the cells with either siRNA-lipoplexes or cationic liposomes altered the expression of approximately 2,500 genes. When lipoplexes containing non-specific siRNAs were used, GFP expression was enhanced. In this case the effect was independent on the dose and type of siRNA in the formulation. By contrast, when lipoplexes containing a specific siRNA against GFP was used, GFP expression was markedly diminished. These results clearly indicate that an efficient reduction of a targeted gene expression by a specific siRNA is accompanied by a significant alteration of the expression of numerous non-targeted genes. In addition, treatment-related cytotoxicity increased with siRNA- and cationic lipid-doses, but was not dependent on siRNA type. CONCLUSION: Non-specific effects of siRNA-lipoplexes may either enhance, attenuate or even fully mask the desired outcomes of siRNA-based biochemical studies and therapies.
Asunto(s)
Perfilación de la Expresión Génica , Liposomas , ARN Interferente Pequeño/administración & dosificación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Interferente Pequeño/farmacologíaRESUMEN
Cationic liposomes (CL) are one of the most widely studied non-viral vectors for gene delivery. It is well-known that CL induces cytotoxicity following lipofection. However, little is known regarding the mechanism involved in the cytotoxicity. In this study, the in vitro cytotoxicity of CL and its complex with pDNA (lipoplex) was investigated, and a part of the mechanism of induction as well. While free pDNA did not show any cytotoxicity, pDNA increased the cytotoxicity of CL via the formation of lipoplex. In addition, the lipoplex-induced cytotoxicity increased in a lipoplex dose-dependent manner, irrespective of the type of pDNA, cell line and the absence or presence of serum. An assay showed that apoptosis was largely induced by treatment with the lipoplex (lipofection), but not with CL alone, in the tested range of concentration of CL and pDNA. Furthermore, following treatment with lipoplexes, the cells exhibited the morphological features of apoptosis and DNA fragmentation. A cDNA microarray study showed that the lipofection up-regulated 45 genes related to apoptosis, transcription regulation and immune response. These results clearly indicate that pDNA in the lipoplex increases the cytotoxicity of CL as a result of inducing apoptosis. The fundamental principle for gene therapy is to deliver gene-based therapeutics to target cells for specific gene targeting with minimal cytotoxicity. Our results suggest the possibility that cytotoxicity induced by lipofection, accompanied by gene changes, could intrinsically exacerbate, attenuate or even mask the desired effects of gene-based therapy.