RESUMEN
Intradermal delivery of antigens for vaccination is a very attractive approach since the skin provides a rich network of antigen presenting cells, which aid in stimulating an immune response. Numerous intradermal techniques have been developed to enhance penetration across the skin. However, these methods are invasive and/or affect the skin integrity. Hence, our group has devised zinc oxide (ZnO) nano-rods for non-destructive drug delivery. Chemical vapour deposition was used to fabricate aligned nano-rods on ZnO pre-coated silicon chips. The nano-rods' length and diameter were found to depend on the temperature, time, quality of sputtered silicon chips, etc. Vertically aligned ZnO nano-rods with lengths of 30-35 µm and diameters of 200-300 nm were selected for in vitro human skin permeation studies using Franz cells with Albumin-fluorescein isothiocyanate (FITC) absorbed on the nano-rods. Fluorescence and confocal studies on the skin samples showed FITC penetration through the skin along the channels formed by the nano-rods. Bradford protein assay on the collected fluid samples indicated a significant quantity of Albumin-FITC in the first 12 h. Low antibody titres were observed with immunisation on Balb/c mice with ovalbumin (OVA) antigen coated on the nano-rod chips. Nonetheless, due to the reduced dimensions of the nano-rods, our device offers the additional advantage of excluding the simultaneous entrance of microbial pathogens. Taken together, these results showed that ZnO nano-rods hold the potential for a safe, non-invasive, and painless intradermal drug delivery.
RESUMEN
Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate-release (IR) solid oral dosage forms containing stavudine (d4T) are reviewed. According to Biopharmaceutics Classification System (BCS), d4T can be assigned to BCS class I. No problems with BE of IR d4T formulations containing different excipients and produced by different manufacturing methods have been reported and, hence, the risk of bioinequivalence caused by these factors appears to be low. Furthermore, d4T has a wide therapeutic index. It is concluded that a biowaiver is appropriate for IR solid oral dosage forms containing d4T as the single active pharmaceutical ingredient (API) provided that (a) the test product contains only excipients present in the IR d4T drug products that have been approved in a number of countries for the same dosage form, and (b) both test product and its comparator are either "very rapidly dissolving" or "rapidly dissolving" with similarity of dissolution profiles demonstrated at pH 1.2, 4.5, and 6.8.
Asunto(s)
Estavudina/química , Estavudina/farmacocinética , Administración Oral , Animales , Disponibilidad Biológica , Biofarmacia , Química Farmacéutica , Formas de Dosificación , Excipientes/química , Humanos , Permeabilidad , Solubilidad , Equivalencia TerapéuticaRESUMEN
Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing metronidazole are reviewed. Metronidazole can be assigned to Biopharmaceutics Classification System Class I. Most BE studies that were identified reported the investigated formulations to be bioequivalent, indicating the risk of bioinequivalence to be low. Formulations showing differences in bioavailability showed dissimilarities in in vitro dissolution profiles. Furthermore, metronidazole has a wide therapeutic index. It is concluded that a biowaiver for solid IR formulations is justified, provided: (a) the test product and its comparator are both rapidly dissolving; (b) meet similarity of the dissolution profiles at pH 1.2, 4.5, and 6.8; (c) the test product contains only excipients present in IR drug products approved in International Conference on Harmonisation (ICH) or associated countries in the same dosage form; and (d) if the test product contains sorbitol, sodium laurilsulfate, or propylene glycol, the test product needs to be qualitatively and quantitatively identical to its comparator with respect to these excipients [corrected]..
Asunto(s)
Antiinfecciosos/administración & dosificación , Antiinfecciosos/farmacocinética , Metronidazol/administración & dosificación , Metronidazol/farmacocinética , Administración Oral , Animales , Antiinfecciosos/química , Control de Medicamentos y Narcóticos , Humanos , Metronidazol/química , Solubilidad , Comprimidos , Equivalencia TerapéuticaRESUMEN
Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing levofloxacin as the only active pharmaceutical ingredient (API) are reviewed. According to the current Biopharmaceutics Classification System, levofloxacin can be assigned to Class I. No problems with BE of IR levofloxacin formulations containing different excipients and produced by different manufacturing methods have been reported and hence the risk of bioinequivalence caused by these factors appears to be low. In addition, levofloxacin has a wide therapeutic index. On the basis of this evidence, a biowaiver is recommended for IR solid oral dosage forms containing levofloxacin as the single API provided that (a) the test product contains only excipients present in IR levofloxacin drug products that have been approved in International Conference on Harmonization (ICH) or associated countries and which have the same dosage form; (b) both the test and comparator dosage form are "very rapidly dissolving" or "rapidly dissolving" with similarity of the dissolution profiles demonstrated at pH 1.2, 4.5, and 6.8; and (c) if the test product contains polysorbates, it should be both qualitatively and quantitatively identical to its comparator in terms of polysorbate content.
Asunto(s)
Antibacterianos/administración & dosificación , Levofloxacino , Ofloxacino/administración & dosificación , Administración Oral , Animales , Antibacterianos/química , Antibacterianos/farmacocinética , Antibacterianos/uso terapéutico , Control de Medicamentos y Narcóticos , Excipientes , Humanos , Ofloxacino/química , Ofloxacino/farmacocinética , Ofloxacino/uso terapéutico , Solubilidad , ComprimidosRESUMEN
The aim of this study was to evaluate the ability of TMC, with different degrees of quaternization, to increase insulin absorption in vivo following nasal and rectal administration in rats. Two batches of TMC with different degrees of quaternization (TMC-L, 12.3% quaternized and TMC-H, 61.2% quaternized) and chitosan hydrochloride were administered intranasally (0.25 and 0.5% w/v) and rectally (0.5% w/v) with insulin (4 IU/kg body weight), at a pH of 4.40 and 7.40, in rats. Blood samples were taken over a period of 2 h for measurement of blood glucose levels and plasma insulin levels. Local toxicity evaluation was done by histological examination of the nasal and rectal epithelia. At pH 4.40 all these polymers were able to increase nasal and rectal insulin absorption, compared to the control groups. However, at a pH of 7.40, only TMC-H was able to increase the nasal and rectal absorption of insulin. These results relate to the insolubility of chitosan hydrochloride at neutral pH values, while the charge density of TMC-L is still too low for any significant interaction at pH 7.40. Histological evaluation of the nasal and rectal eptihelia shows no changes in the morphology of the cells after exposure to these polymers. Only slight congestion of the nasal submucosa was observed and all these polymers led to a mild increase in mucus secretion at pH 4.40. Highly quaternized TMC proves to be a potent absorption enhancer in vivo, especially at neutral pH values where chitosan salts are ineffective.
Asunto(s)
Quitosano/química , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/farmacocinética , Insulina/administración & dosificación , Insulina/farmacocinética , Absorción , Administración Intranasal , Administración Rectal , Aminación , Animales , Glucemia/análisis , Quitosano/efectos adversos , Portadores de Fármacos/efectos adversos , Portadores de Fármacos/farmacología , Concentración de Iones de Hidrógeno , Insulina/efectos adversos , Insulina/farmacología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Secreciones Intestinales/efectos de los fármacos , Masculino , Moco/metabolismo , Mucosa Nasal/efectos de los fármacos , Mucosa Nasal/metabolismo , Mucosa Nasal/patología , Compuestos de Amonio Cuaternario/química , Ratas , Ratas Sprague-Dawley , Recto/efectos de los fármacos , Recto/metabolismo , Recto/patología , SolubilidadRESUMEN
It has long been known that protection against pathogens invading the organism via mucosal surfaces correlates better with the presence of specific antibodies in local secretions than with serum antibodies. The most effective way to induce mucosal immunity is to administer antigens directly to the mucosal surface. The development of vaccines for mucosal application requires antigen delivery systems and immunopotentiators that efficiently facilitate the presentation of the antigen to the mucosal immune system. This review provides an overview of the events within mucosal tissues that lead to protective mucosal immune responses. The understanding of those biological mechanisms, together with knowledge of the technology of vaccines and adjuvants, provides guidance on important technical aspects of mucosal vaccine design. Not being exhaustive, this review also provides information related to modern adjuvants, including polymeric delivery systems and immunopotentiators.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Sistemas de Liberación de Medicamentos/tendencias , Diseño de Fármacos , Inmunidad Mucosa/inmunología , Vacunas/administración & dosificación , Animales , Sistemas de Liberación de Medicamentos/normas , Humanos , Membrana Mucosa/química , Membrana Mucosa/inmunología , Guías de Práctica Clínica como Asunto/normas , Vacunas/síntesis química , Vacunas/inmunologíaRESUMEN
During persistent infection and hypoxic-stress, Mycobacterium tuberculosis (Mtb) expresses a series of Mtb latency antigens. The aim of this study was to evaluate the immunogenicity of a DNA vaccine encoding the Mtb latency antigen Rv1733c and to explore the effect of pulmonary delivery and co-formulation with poly (d,l-lactide-co-glycolide) (PLGA)-polyethyleneimine (PEI) nanoparticles (np) on host immunity. Characterization studies indicated that PLGA-PEI np kept their nanometer size after concentration and were positively charged. The np were able to mature human dendritic cells and stimulated them to secrete IL-12 and TNF-alpha comparable to levels observed after lipopolysaccharide (LPS) stimulation. Mtb latency antigen Rv1733c DNA prime combined with Rv1733c protein boost enhanced T cell proliferation and IFN-gamma secretion in mice in response to Rv1733c and Mtb hypoxic lysate. Rv1733c DNA adsorbed to PLGA-PEI np and applied to the lungs increased T cell proliferation and IFN-gamma production more potently compared to the same vaccinations given intramuscularly. The strongest immunogenicity was obtained by pulmonary priming with np-adsorbed Rv1733c DNA followed by boosting with Rv1733c protein. These results confirm that PLGA-PEI np are an efficient DNA vaccine delivery system to enhance T cell responses through pulmonary delivery in a DNA prime/protein boost vaccine regimen.
Asunto(s)
Antígenos Bacterianos/genética , Mycobacterium tuberculosis/genética , Linfocitos T/inmunología , Vacunas contra la Tuberculosis/administración & dosificación , Vacunas contra la Tuberculosis/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/farmacología , Administración por Inhalación , Animales , Antígenos Bacterianos/inmunología , Células Cultivadas , Células Dendríticas/inmunología , Femenino , Humanos , Inmunización Secundaria , Interferón gamma/metabolismo , Interleucina-12/metabolismo , Ácido Láctico/administración & dosificación , Ácido Láctico/farmacología , Ratones , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/inmunología , Nanopartículas/administración & dosificación , Polietileneimina/administración & dosificación , Polietileneimina/farmacología , Ácido Poliglicólico/administración & dosificación , Ácido Poliglicólico/farmacología , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Vacunas contra la Tuberculosis/genética , Factor de Necrosis Tumoral alfa/metabolismo , Vacunas de ADN/genética , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunologíaRESUMEN
We recently described a delivery system that is composed of a chitosan core to which the hepatitis B surface antigen (HBsAg) was adsorbed and subsequently coated with sodium alginate. In this present work, alginate coated chitosan nanoparticles were evaluated as a subcutaneous adjuvant for HBsAg. HBsAg loaded, alginate coated or uncoated chitosan nanoparticles, associated or not with CpGODN were subcutaneously administered to mice and several immunological parameters were evaluated. A high anti-HBsAg IgG titer (2271+/-120 mIU/ml), with the majority of antibodies being of Th2 type, was observed within group I, vaccinated with HBsAg loaded onto coated nanoparticles. However, regarding cellular immune response, no significant differences were observed for antigen-specific splenocyte proliferation or for the secretion of IFN-gamma and IL-4, when compared to the control group. The co-delivery of antigen-loaded nanoparticles in the presence of the immunopotentiator, CpG ODN 1826, resulted in an increase of anti-HBsAg IgG titers that was not statistically different from the first group; however, an increase of the IgG2a/IgG1 ratio from 0.1 to 1.0 and an increase (p<0.01) of the IFN-gamma production by the splenocytes stimulated with the HBV antigen was observed. The enhancement of the immune response observed with the antigen-loaded nanoparticles demonstrated that chitosan is a promising platform for parenteral HBsAg delivery and, when co-administered with the CpG ODN, resulted in a mixed Th1/Th2 type immune response.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Quitosano/administración & dosificación , Sistemas de Liberación de Medicamentos , Antígenos de Superficie de la Hepatitis B/administración & dosificación , Nanopartículas/administración & dosificación , Vacunas contra Hepatitis Viral/administración & dosificación , Adyuvantes Inmunológicos/química , Alginatos/química , Animales , Anticuerpos Antivirales/sangre , Materiales Biocompatibles/administración & dosificación , Células Cultivadas , Quitosano/química , Quitosano/inmunología , ADN/inmunología , Femenino , Ácido Glucurónico/química , Antígenos de Superficie de la Hepatitis B/inmunología , Ácidos Hexurónicos/química , Inmunidad Celular/efectos de los fármacos , Inmunidad Celular/inmunología , Inmunidad Mucosa/efectos de los fármacos , Inmunidad Mucosa/inmunología , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Interleucina-10/biosíntesis , Interleucina-10/inmunología , Interleucina-4/biosíntesis , Interleucina-4/inmunología , Ratones , Ratones Endogámicos BALB C , Mitógenos/farmacología , Nanopartículas/química , Oligodesoxirribonucleótidos , Bazo/inmunología , Bazo/metabolismo , Vacunas contra Hepatitis Viral/inmunologíaRESUMEN
Alginate coated chitosan nanoparticles were previously developed with the aim of protecting the antigen, adsorbed on the surface of those chitosan nanoparticles, from enzymatic degradation at mucosal surfaces. In this work, this new delivery system was loaded with the recombinant hepatitis B surface antigen (HBsAg) and applied to mice by the intranasal route. Adjuvant effect of the delivery system was studied by measuring anti-HBsAg IgG in serum, anti-HBsAg sIgA in faeces extracts or nasal and vaginal secretions and interferon-gamma production in supernatants of the spleen cells. The mice were primed with 10 microg of the vaccine associated or not with nanoparticles and associated or not with 10 microg CpG oligodeoxynucleotide (ODN) followed by two sequential boosts at three week intervals. The association of HBsAg with the alginate coated chitosan nanoparticles, administered intranasally to the mice, gave rise to the humoral mucosal immune response. Humoral systemic immune response was not induced by the HBsAg loaded nanoparticles alone. The generation of Th1-biased antigen-specific systemic antibodies, however, was observed when HBsAg loaded nanoparticles were applied together with a second adjuvant, the immunopotentiator, CpG ODN. Moreover, all intranasally vaccinated groups showed higher interferon-gamma production when compared to naïve mice.
Asunto(s)
Islas de CpG , Anticuerpos contra la Hepatitis B/biosíntesis , Antígenos de Superficie de la Hepatitis B/administración & dosificación , Antígenos de Superficie de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/administración & dosificación , Vacunas contra Hepatitis B/inmunología , Oligonucleótidos/administración & dosificación , Administración Intranasal , Alginatos , Animales , Formación de Anticuerpos/inmunología , Proliferación Celular/efectos de los fármacos , Quitosano , Portadores de Fármacos , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunidad Mucosa/inmunología , Indicadores y Reactivos , Interferón gamma/biosíntesis , Ratones , Ratones Endogámicos BALB C , Nanopartículas , Polímeros , Bazo/citología , Bazo/inmunología , Bazo/metabolismoRESUMEN
Insulin (INS), like other peptides, has low therapeutic activity when administered orally due to degradation by proteolytic enzymes. Polymeric nanoparticles have been introduced as a useful carrier for peptide oral delivery, because they can protect these compounds from degradation. The objective of the present study is to develop an INS nanoparticulate system by using chitosan (CS), triethylchitosan (TEC), and dimethyl-ethylchitosan (DMEC, a new quaternized derivative of CS). INS-polymer nanoparticles were prepared by the polyelectrolyte complexation method. The physicochemical properties of the nanoparticles including particle size distribution, zeta potential, and polydispersity index were determined by using dynamic light scattering technique. Transmission electron microscopy was also used to observe the morphology of the nanoparticles. The amount of INS loaded into the nanoparticles was determined by measuring the association efficiency and also the content of INS in the nanoparticles. In vitro release studies showed a relatively small burst effect at the beginning and then a sustained release characteristic for 5 hours.
Asunto(s)
Quitosano/química , Preparaciones de Acción Retardada/química , Composición de Medicamentos/métodos , Insulina/química , Nanopartículas/química , Administración Oral , Preparaciones de Acción Retardada/administración & dosificación , Difusión , Insulina/administración & dosificación , Ensayo de Materiales , Nanopartículas/ultraestructuraRESUMEN
The aim of the present study was to develop insulin nanoparticulate systems by using chitosan (CS), triethylchitosan (TEC) and dimethyl-ethylchitosan (DMEC, a new quaternized derivative of chitosan) for colon delivery. The nanoparticles were prepared by the polyelectrolyte complexation (PEC) method. Particle size distribution, zeta potential and polydispersity index of the nanoparticles were determined using dynamic light scattering technique. Transmission electron microscopy (TEM) was also used to observe the morphology of the nanoparticles. It was found that the nanoparticles carried positive charges and showed a size distribution in the range of 170-270 nm with spherical morphology and smooth surface structure. The amount of insulin loaded into the nanoparticles was determined by measuring the association efficiency and also the content of insulin in the nanoparticles. Insulin loading was found to be more than 80% for all of the nanoparticles. In vitro release studies showed a small burst effect at the beginning and then a sustained release characteristic for 5h. Ex vivo investigations revealed better insulin transport across the colon membrane of rats for nanoparticles made with quaternized derivatives than those made of chitosan. In vivo studies in rats have showed enhanced colon absorption of insulin by using these nanoparticles compared to free insulin in diabetic rats. The insulin absorption from the rat's colon was evaluated by its hypoglycemic effect.
Asunto(s)
Quitosano/química , Sistemas de Liberación de Medicamentos , Hipoglucemiantes/administración & dosificación , Insulina/administración & dosificación , Animales , Quitosano/análogos & derivados , Colon/metabolismo , Preparaciones de Acción Retardada , Diabetes Mellitus Experimental/tratamiento farmacológico , Electrólitos/química , Hipoglucemiantes/farmacocinética , Insulina/farmacocinética , Luz , Masculino , Microscopía Electrónica de Transmisión , Nanopartículas , Tamaño de la Partícula , Ratas , Ratas Wistar , Dispersión de RadiaciónRESUMEN
The purpose of this work was to assess the ability of recombinant hepatitis B vaccine, encapsulated in alginate-coated chitosan nanoparticles, to induce local and systemic immune responses following oral vaccination. The antigen was administered either alone or in combination with the immunopotentiator, synthetic oligodeoxynucleotide containing immunostimulatory CpG motif (CpG ODN) as adjuvant, and associated or not with the alginate-coated chitosan nanoparticles. After two immunizations the group I (HBsAg associated with nanoparticles) and the group VI (HBsAg and CpG, both associated with nanoparticles) showed enhanced immune responses. Both groups showed significant higher values of the CD69 expression in CD4+ and CD8+ T-lymphocytes and lower values of this marker in B lymphocytes. Moreover, a strongest proliferative response of the splenocytes, ex vivo stimulated with concanavalin A, was observed in the same groups. Although with a presence of non-responder mice within the groups, only mice of the groups I and VI elicited the generation of anti-HBsAg antibodies detected in serum (IgG) and in the intestinal washings (sIgA). The results demonstrated that coated chitosan nanoparticles might have potential for being used as a deliver system for oral vaccination with the recombinant hepatitis B surface antigen.
Asunto(s)
Alginatos/administración & dosificación , Quitosano/administración & dosificación , Vacunas contra Hepatitis B/administración & dosificación , Vacunas contra Hepatitis B/inmunología , Nanopartículas/administración & dosificación , Administración Oral , Alginatos/química , Animales , Antígenos CD/biosíntesis , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos de Diferenciación de Linfocitos T/inmunología , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Quitosano/química , Quitosano/inmunología , Composición de Medicamentos , Femenino , Esquemas de Inmunización , Lectinas Tipo C , Ratones , Ratones Endogámicos BALB C , Nanopartículas/química , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/inmunología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , VacunaciónRESUMEN
Chitosan of different molecular weights (Chi-P, MW=2.7x10(5) g/mol and Chi-A, MW=5.0x10(5) g/mol) and trimethyl chitosan chloride (TMC) of various degree of quaternization (DQ) including TMC-20, TMC-40 and TMC-60 were evaluated as adjuvants for inducing of immune responses to ovalbumin (OVA). OVA in solution and in alum were used as controls. Groups of BALB/c mice were immunized on days 0, 7 and 14. The IgG and IgA titers were examined on days 0, 13 and 21. It was found that for both days 13 and 21, Chi-A could elicit higher IgG responses to OVA than Chi-P. On day 13, OVA in TMC-40 induced IgG responses significantly higher than that in solution, Chi-P and TMC-60. Moreover, OVA in TMC-40 could induce IgG responses higher than OVA in alum. Although a significant difference was not observed at day 21, OVA in TMC-40 was shown to induce higher IgG responses than that in TMC-20, TMC-60 and solution. The IgA responses were the most pronounced on day 21. Again, Chi-A could elicite higher IgA responses than Chi-P and TMC-40 induced the highest IgA responses. In conclusion, these findings demonstrate that both MW of chitosan and DQ of TMC influence the level of immune induction. TMC-40 shows to be the most potent adjuvant for intranasal administration.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Quitosano/administración & dosificación , Inmunoglobulina A/biosíntesis , Inmunoglobulina G/biosíntesis , Ovalbúmina/inmunología , Administración Intranasal , Animales , Quitosano/inmunología , Femenino , Ratones , Ratones Endogámicos BALB CRESUMEN
CD69 is a very early cell activation antigen expressed on the surface of activated immune cells. It can appear within 1-2h of activation and exhibits maximal expression levels between 18 and 24h after stimulation. In this work, the expression profile of CD69 in mice splenocytes was evaluated following exposure to the biopolymers, chitosan or alginate and the immunostimulatory factors, CpG ODN 1826 or concanavalin A. We have shown that both polymers are able to upregulate expression of CD69 on B cells and CD4+ T-lymphocytes, with alginate as the least potent stimulus. Moreover, the expression of the CD69 molecule on CD8+ T-lymphocytes was observed only in splenocytes cultured with chitosan. However, activation of lymphocytes did not result in cell proliferation. On the other hand, CpG ODN proved to be a potent B cell stimulator, as evidenced by the upregulation of CD69, but had less effect on T-cells. These results, together with previous discoveries reported in scientific literature, may contribute to the clarification of the adjuvant effect, which has been attributed to chitosan and alginate formulations or to the biopolymers itself.
Asunto(s)
Alginatos/farmacología , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Materiales Biocompatibles/farmacología , Quitosano/farmacología , Portadores de Fármacos , Activación de Linfocitos/efectos de los fármacos , Subgrupos Linfocitarios/efectos de los fármacos , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Concanavalina A/farmacología , ADN/inmunología , Ácido Glucurónico/farmacología , Ácidos Hexurónicos/farmacología , Lectinas Tipo C , Subgrupos Linfocitarios/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Mitógenos/farmacología , Oligodesoxirribonucleótidos , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunología , Regulación hacia ArribaRESUMEN
In this study, the potential of N-trimethyl chitosan (TMC) nanoparticles as a carrier system for the nasal delivery of a monovalent influenza subunit vaccine was investigated. The antigen-loaded nanoparticles were prepared by mixing a solution containing TMC and monovalent influenza A subunit H3N2 with a tripolyphosphate (TPP) solution, at ambient temperature and pH 7.4 while stirring. The nanoparticles had an average size of about 800 nm with a narrow size distribution and a positive surface charge. The nanoparticles showed a loading efficiency of 78% and a loading capacity of 13% (w/w). It was shown that more than 75% of the protein remained associated with the TMC nanoparticles upon incubation of the particles in PBS for 3h. The molecular weight and antigenicity of the entrapped hemagglutinin was maintained as shown by polyacrylamide gel electrophoresis and Western blotting, respectively. Single i.n. or i.m. immunization with antigen-loaded TMC nanoparticles resulted in strong hemagglutination inhibition and total IgG responses. These responses were significantly higher than those achieved after i.m. administration of the subunit antigen, whereas the IgG1/IgG2a profile did not change substantially. The i.n. administered antigen-TMC nanoparticles induced higher immune responses compared to the other i.n. antigen formulations, and these responses were enhanced by i.n. booster vaccinations. Moreover, among the tested formulations only i.n. administered antigen-containing TMC nanoparticles induced significant IgA levels in nasal washes of all mice. In conclusion, these findings demonstrate that TMC nanoparticles are a potent new delivery system for i.n. administered influenza antigens.
Asunto(s)
Administración Intranasal , Quitosano/administración & dosificación , Sistemas de Liberación de Medicamentos , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H3N2 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza , Nanopartículas/administración & dosificación , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/administración & dosificación , Antígenos Virales/química , Antígenos Virales/inmunología , Quitosano/química , Modelos Animales de Enfermedad , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/administración & dosificación , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Humanos , Inmunoglobulina A Secretora/análisis , Inmunoglobulina G/sangre , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/química , Vacunas contra la Influenza/inmunología , Ratones , Ratones Endogámicos C57BL , Nanopartículas/química , Vacunación/métodosRESUMEN
The design of particulate vaccine delivery systems, particularly for mucosal surfaces, has been a focus of interest in recent years. In this context, we have previously described the development and the characterization of a new nanosized delivery system, consisting of a model antigen adsorbed to chitosan particles and coated with sodium alginate. In the present work the ovalbumin release profiles from these coated nanoparticles in different pH buffers were investigated and compared to those of the uncoated particles. Cytotoxicity of the polymers and nanoparticles was assessed using the MTT assay. Finally, particle uptake studies in rat Peyer's patches were performed. It was demonstrated that the coating of the nanoparticles with sodium alginate not only avoided a burst release observed with uncoated particles but also increased the stability of the particles at pH 6.8 and 7.4 at 37 degrees C. At neutral pH, the release was lower than 5% after 3.5 h incubation in a low ionic strength buffer. For both, chitosan and alginate polymers, and for the nanoparticles, comparable cell viability data close to 100%, were obtained. Additionally, based on confocal laser scanning microscopy observations, it was shown that alginate coated nanoparticles were able to be taken up by rat Peyer's patches, rendering them suitable carriers for intestinal mucosal vaccination.
Asunto(s)
Alginatos/administración & dosificación , Quitosano/administración & dosificación , Sistemas de Liberación de Medicamentos , Nanoestructuras , Ovalbúmina/administración & dosificación , Ganglios Linfáticos Agregados/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Femenino , Ácido Glucurónico/administración & dosificación , Ácidos Hexurónicos/administración & dosificación , Concentración de Iones de Hidrógeno , Masculino , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/química , Ovalbúmina/inmunología , Ratas , Ratas Wistar , Solubilidad , Bazo/citología , Bazo/efectos de los fármacos , VacunaciónRESUMEN
In this study, the potential of N-trimethyl chitosan (TMC) nanoparticles as a carrier system for the nasal delivery of proteins was investigated. TMC nanoparticles were prepared by ionic crosslinking of TMC solution (with or without ovalbumin) with tripolyphosphate, at ambient temperature while stirring. The size, zeta-potential and morphology of the nanoparticles were investigated as a function of the preparation conditions. Protein loading, protein integrity and protein release were studied. The toxicity of the TMC nanoparticles was tested by ciliary beat frequency measurements of chicken embryo trachea and in vitro cytotoxicity assays. The in vivo uptake of FITC-albumin-loaded TMC nanoparticles by nasal epithelia tissue in rats was studied by confocal laser scanning microscopy. The nanoparticles had an average size of about 350 nm and a positive zeta-potential. They showed a loading efficiency up to 95% and a loading capacity up to 50% (w/w). The integrity of the entrapped ovalbumin was preserved. Release studies showed that more than 70% of the protein remained associated with the TMC nanoparticles for at least 3 h on incubation in PBS (pH 7.4) at 37 degrees C. Cytotoxicity tests with Calu-3 cells showed no toxic effects of the nanoparticles, whereas a partially reversible cilio-inhibiting effect on the ciliary beat frequency of chicken trachea was observed. In vivo uptake studies indicated the transport of FITC-albumin-associated TMC nanoparticles across the nasal mucosa. In conclusion, TMC nanoparticles are a potential new delivery system for transport of proteins through the nasal mucosa.
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Quitosano/química , Sistemas de Liberación de Medicamentos/métodos , Nanoestructuras/química , Proteínas/administración & dosificación , Animales , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Embrión de Pollo , Cilios/efectos de los fármacos , Cilios/fisiología , Electroforesis en Gel de Poliacrilamida , Geles , Humanos , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Masculino , Microscopía Confocal , Microscopía Electrónica de Rastreo , Mucosa Nasal/citología , Mucosa Nasal/efectos de los fármacos , Mucosa Nasal/inmunología , Ovalbúmina/administración & dosificación , Ovalbúmina/química , Ovalbúmina/farmacología , Proteínas/química , Proteínas/farmacología , Ratas , Ratas Wistar , SolubilidadRESUMEN
Chitosan is a biocompatible polysaccharide of natural origin that can act as a permeation enhancer. In this study, we used an integral in vitro/in vivo correlation approach to: a) investigate polysaccharide-mediated absorption kinetics of the peptide drug octreotide across mammalian airway epithelium, b) assess formulation toxicity, c) correlate the mechanism of permeation enhancement. The 20% and 60% N-trimethylated chitosan derivatives (TMC20 and TMC60) were synthesized by alkaline methylation using chitosan as starting material. Octreotide was administered in control buffers or in 1.5% (w/v) gel-phase formulations of pH 5.5 for chitosan and pH 7.4 for TMCs. In vitro, reconstituted Calu-3 cell monolayers were used for trans-epithelial electrical resistance (TEER), transport and cytotoxicity assays. Intratracheal instillation in rats was used to determine octreotide kinetics and formulation toxicity in vivo. Chitosan, TMC20 and TMC60 decreased TEER significantly and enhanced octreotide permeation in vitro by 21-, 16- and 30-fold. In vivo, sustained release properties of the formulations were observed and the bio-availability was enhanced by 2.4-, 2.5- and 3.9-fold, respectively. Interestingly, we found a linear in vitro/in vivo correlation between calculated absorption rates (R2=0.93), suggesting that the permeation enhancement by polysaccharides, both in vitro and in vivo, proceeds via an analogous mechanism. Cell viability and histology studies showed that the TMCs are safer than chitosan and that Calu-3 cell monolayers are a valuable model for predicting paracellular transport kinetics in airway epithelia. Additionally, cationic polysaccharides are promising enhancers for peptide drug absorption with prospect for sustained-release formulations.