Plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B

This work investigates the preparation and in vivo efficacy of plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B. Chitosan pDNA nanoparticles were prepared using a complex coacervation process. Prepared nanoparticles were characterized for size, shape, surface charge, plasmid loading and ability of nanoparticles to protect DNA against nuclease digestion and for their transfection efficacy. Nasal administration of nanoparticles resulted in serum anti-HBsAg titre that was less compared to that elicited by naked DNA and alum adsorbed HBsAg, but the mice were seroprotective within 2 weeks and the immunoglobulin level was above the clinically protective level. However, intramuscular administration of naked DNA and alum adsorbed HBsAg did not elicit sIgA titre in mucosal secretions that was induced by nasal immunization with chitosan nanoparticles. Similarly, cellular responses (cytokine levels) were poor in case of alum adsorbed HBsAg. Chitosan nanoparticles thus produced humoral (both systemic and mucosal) and cellular immune responses upon nasal administration. The study signifies the potential of chitosan nanoparticles as DNA vaccine carrier and adjuvant for effective immunization through non-invasive nasal route.     

Evaluation of the immune response following a short oral vaccination schedule with hepatitis B antigen encapsulated into alginate-coated chitosan nanoparticles

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.     

Improved stability and immunological potential of tetanus toxoid containing surface engineered bilosomes following oral administration

The present study was designed with the objective to investigate the stability and potential of glucomannan-modified bilosomes (GM-bilosomes) in eliciting immune response following oral administration. GM-bilosomes exhibited desired quality attributes simultaneously maintaining the chemical and conformation stability of the tetanus toxoid (TT) entrapped in to freeze dried formulations. The GM-bilosomes exhibited excellent stability in different simulated biological fluids and sustained release profile up to 24 h. GM-bilosomes elicited significantly higher (P < 0.05) systemic immune response (serum IgG level) as compared to bilosomes, niosomes and alum adsorbed TT administered through oral route. More importantly, GM-bilosomes were found capable of inducing mucosal immune response, i.e. sIgA titre in salivary and intestinal secretions as well as cell mediated immune response (IL-2 and IFN-γ levels in spleen homogenate) which was not induced by i.m. TT, the conventional route of immunization. Conclusively, GM-bilosomes could be considered as a promising carrier and adjuvant system for oral mucosal immunization.From the Clinical EditorThis team reports on the development and effects of a glucomannan-modified bilosome as an oral vaccine vector, using tetanus toxoid in the experiments. These GM-bilosomes not only elicited significantly higher systemic immune response as compared to bilosomes, niosomes and alum adsorbed orally administered TT, but also demonstrated mucosal immune response induction as well as cell mediated immune responses, which were not induced by the conventional route of immunization.     

Nasal immunization studies using liposomes loaded with tetanus toxoid and CpG-ODN.

To increase the systemic and mucosal immune responses against the nasally administered tetanus toxoid, liposomes as a drug delivery system and CpG-ODN as an adjuvant were evaluated. Rabbits were nasally immunized with entrapped tetanus toxoid (TT) and CpG-ODN in neutral liposomes and systemic and mucosal immune responses were determined. Liposomes containing TT and CpG-ODN were prepared by dehydration-rehydration method. The volume mean diameter of liposomes was 2.3+/-0.6 microm. Encapsulation efficiency of TT and CpG-ODN was determined as 54.0+/-8.8 and 60.1+/-7.4, respectively. The leakage of the encapsulated TT from liposomes reached 7.38% after 3 months. Encapsulated TT kept its intact structure, and its immunoreactivity was also completely preserved, as shown by SDS-PAGE and ELISA methods. The highest serum IgG and antitoxin titers were observed in groups immunized with solution formulations (P < 0.001). However the highest mucosal sIgA titers were achieved by liposomes encapsulated with TT. CpG-ODN as an adjuvant was able to increase the serum IgG and antitoxin titers when co-administered with TT solution (P < 0.05) or co-encapsulated with TT in liposomes (P < 0.01), but failed to increase the sIgA titers in nasal lavages. No hemolysis occurred on incubation of liposomes and human RBCs. Also after nasal administration of plain liposomes to human volunteers, no local irritation was seen. Intranasal administration of liposomes encapsulated with vaccines showed to be an effective way for inducing the mucosal immune responses.     

Cholera toxin B subunit conjugated bile salt stabilized vesicles (bilosomes) for oral immunization

Bile salt stabilized vesicles, bilosomes appear to be a promising and potential carrier system for oral delivery of peptides and proteins. Bilosomes containing bovine serum albumin (BSA), a model antigen, were prepared and conjugated with cholera toxin B subunit (CTB) in order to enhance their affinity towards M cells of Peyer's patches. Stability studies were undertaken to ascertain the effect of simulated gastric fluid (SGF, pH 1.2), simulated intestinal fluid (SIF, pH 7.5) and different concentrations of bile salts. Intactness and biological activity of CTB were checked by hemagglutination test. A single oral dose of CTB-conjugated bilosomes produced almost equivalent response compared to parenteral administration of antigen with Freund's complete adjuvant (FCA). However, in contrast to FCA, oral administration of bilosomes is convenient and devoid of any adverse effects that are observed with parenteral administration of FCA. Serum IgG titers after single administration were significantly better (P < 0.05) than oral administration of antigen with other systems for 3 consecutive days, suggesting an effective stimulation of systemic immune response. Mucosal IgA titers obtained advocated a possible application of CTB-conjugated bilosomes as oral vaccine delivery system.     

Development and characterization of chitosan succinate microspheres for the improved oral bioavailability of insulin

The present study describes the fabrication of insulin loaded chitosan succinate microspheres to improve the efficacy of orally administered insulin. Chitosan succinate polymer was synthesized and its microspheres were prepared by emulsion phase separation technique. The microspheres were characterized by FT-IR spectroscopy, scanning electron microscopy, particle size, X-ray diffraction, and swelling index. Insulin was loaded into the microspheres by passive absorption technique. The ability of microspheres to protect insulin from gastric enzymatic degradation was investigated. Stability of insulin in the microspheres was determined by gel electrophoresis and circular dichroism (CD). In vitro release studies were performed under simulated gastric and intestinal pH conditions (pH 2.0 and pH 7.4). The pharmacokinetic parameters were monitored after oral administration of insulin loaded chitosan succinate microspheres, chitosan succinate-insulin solution, as well as after subcutaneous injection of insulin to diabetic rats. The degree of succinate substitution in the synthesized polymer was 16%. The prepared microspheres were spherical with an average diameter of 49 +/- 2 microm. The insulin-loading capacity was 62%. Chitosan succinate microspheres were found to protect the degradation of insulin from gastric enzymes. The encapsulated insulin was quickly released in simulated intestinal fluid (SIF, pH 7.4), whereas a small fraction of insulin was released in simulated gastric fluid (pH 2.0). The relative pharmacological efficacy for chitosan succinate microspheres (16 +/- 4%) was almost fourfold higher than the efficacy of the chitosan succinate-insulin solution administration (4 +/- 1.5%). The results suggest that chitosan succinate microspheres could be used as a potential carrier for oral insulin delivery.     

Induction of high antitoxin titers against tetanus toxoid in rabbits by intranasal immunization with dextran microspheres

Poor absorption of protein antigens through the mucosal membranes necessitates the use of mucoadhesive delivery systems. Regarding the advantages of mucosal immunization and also the penetration enhancement potential of dextran microspheres, in this study the adjuvant potential of these microspheres was compared with CpG-ODN. Cross-linked dextran microspheres (CDMs) were loaded with tetanus toxoid (TT). In vitro release studies were performed in a model, simulating the nasal cavity. The immunoreactivity of encapsulated TT was assayed by ELISA. Membrane toxicity and local irritating potential of CDM was examined by erythrocyte hemolysis and nasal administration to human nose, respectively. The various formulations were nasally administered to rabbits (n=4). Alum-adsorbed TT (AATT) was injected as the positive control. The serum IgG and nasal lavage sIgA titers were determined by ELISA method. Serum antitoxin titers were determined by toxin neutralization (TN) bioassay method. Mean diameter of CDM was 128.1+/-25.8 microm. Mean encapsulation efficiency was 20.3+/-3.2% (n=3). Antigenicity of encapsulated TT was 90.5+/-1.8% (n=3) that of original TT. Hemolysis studies showed no membrane disruption by CDM and none of the human subjects reported nasal irritation. Among the nasally immunized animals, the highest antitoxin titers was seen in the group immunized with CDM+TT (P<0.0001). The serum IgG titers of the CDM+TT group was higher than the TT solution group (P<0.05). The adjuvant potentials of CDM and CpG-ODN in inducing IgG titers was not significantly different (P>0.05). The lowest sIgA titers in the bronchial lavage were seen in the group of animals received AATT parenterally. Considering the proper release characteristics, desirable preservation of the antigen activity of TT, good mucoadhesion properties and also safety of CDM+TT, these microspheres could be regarded as an efficient mucosal adjuvant and antigen delivery system. These microspheres could induce very high antitoxin titers following nasal administration, while the CpG-ODN could not induce such titers. The antitoxin titers induced by CDM+TT was 175 times higher than the protective levels.     

Adjuvanted,antigen loaded N-trimethyl chitosan nanoparticles for nasal and intradermal vaccination: Adjuvant- and site-dependent immunogenicity in mice

N-trimethyl chitosan (TMC) nanoparticles have been shown to increase the immunogenicity of subunit antigens after nasal and intradermal administration. This work describes a second generation of TMC nanoparticles containing ovalbumin as a model antigen (TMC/OVA nanoparticles) and an immunopotentiator (TMC/OVA/immunopotentiator nanoparticles). The selection of immunopotentiators included Toll-like receptor (TLR) ligands lipopolysaccharide (LPS), PAM₃CSK₄ (PAM), CpG DNA, the NOD-like receptor 2 ligand muramyl dipeptide (MDP) and the GM1 ganglioside receptor ligand, cholera toxin B subunit (CTB). The TMC/OVA/immunopotentiator nanoparticles were characterised physico-chemically and their immunogenicity was assessed by determining the serum IgG, IgG1, IgG2a titres and secretory IgA levels in nasal washes after intradermal and nasal vaccination in mice.After nasal vaccination, TMC/OVA nanoparticles containing LPS or MDP elicited higher IgG, IgG1 and sIgA levels than non-adjuvanted TMC/OVA particles, whereas nanoparticles containing CTB, PAM or CpG did not. After intradermal vaccination, the TMC/OVA/CpG and TMC/OVA/LPS nanoparticles provoked higher IgG titres than plain TMC/OVA particles.Altogether, our results show that co-encapsulation of an additional immunopotentiator with the antigen into TMC nanoparticles can further improve the immunogenicity of the vaccine. However, the strength and quality of the response depends on the immunopotentiator as well as the route of administration.     

Biodegradable microparticles for the mucosal delivery of antibacterial and dietary antigens

Mucosal administration of antigen is known to be appropriate for vaccine purposes as well as tolerance induction. Biodegradable poly(DL-lactide-co-glycolide) (PLGA) microparticles were used to deliver both antibacterial phosphorylcholine (PC) and dietary antigen beta lactoglobulin (BLG) by mucosal route. In a first study, the protective immunity elicited by intragastric vaccination with PC encapsulated in microparticles was evaluated in a mouse model against intestinal infection by Salmonella typhimurium and pulmonary infection by Streptococcus pneumoniae. A significant rise in anti-PC immunoglobulin A (IgA) titers, as measured by an enzyme-linked immunosorbent assay, was observed in the intestinal secretions after oral immunization with PC-loaded microparticles compared with the titers of mice immunized with free PC-thyr or blank microparticles. This antibody response correlated with a highly significant resistance to oral challenge by S. typhimurium. IgA in pulmonary secretion were not able to protect against S. pneumoniae infection. BALB/c mice were, therefore, immunized intranasally (i.n.). Immunization was followed by a rise in anti-PC IgA and IgG titers in serum and in pulmonary secretions by both free and encapsulated PC-Thyr. The survival rates were 91 and 76% in the two groups of mice, respectively. In a second study and in order to prevent allergy against milk by inducing oral tolerance, one of the major allergenic milk protein, BLG was entrapped into microparticles. Oral administration of microparticles containing BLG reduced significantly (by 10000) the amount of protein necessary to decrease both specific anti BLG IgE and DTH response. These studies demonstrate the ability of microparticles to induce both mucosal immunity and oral tolerance.     

Oral hepatitis B vaccine: chitosan or glucan based delivery systems for efficient HBsAg immunization following subcutaneous priming

The World Health Organization encourages “the development of oral formulations to simplify their transport, storage and administration in poor countries”, and to “facilitate an effective immunization program to prevent sexually transmitted hepatitis B”. Thus, two distinct and promising delivery systems were developed: recombinant hepatitis B surface antigen (HBsAg) encapsulated into alginate-coated chitosan particles (AlgChiPs) and into glucan particles (GPs) mainly composed of β-1,3-d-glucan. In vitro preliminary studies showed that both could be internalized by peripheral blood mononuclear cells and murine Peyer’s patches, an imperative aspect regarding oral immunization. Chitosan particles (ChiPs) have shown interesting immunostimulating properties as mast cells activators. Vaccination studies reveal that three oral immunizations induced serum anti-HBsAg Immunoglobulin G (IgG) in 60 % of the animals and anti-HBsAg secretory IgA in faeces for both formulations. When subcutaneous (SC) priming was done, followed by two oral boosts, all mice were responder and much higher serum anti-HBsAg IgG titers were observed, besides mucosal protective immunity.     

Immune response by nasal delivery of hepatitis B surface antigen and codelivery of a CpG ODN in alginate coated chitosan nanoparticles

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 na?ve mice.     

Oral delivery of microparticles containing plasmid DNA encoding hepatitis-B surface antigen

The role of albumin-based chitosan microparticles on enhancing immune response of plasmid DNA (pDNA) to hepatitis-B surface antigen (HBsAg) vaccine after oral administration was investigated in mice. The pDNA encoding HBsAg was entrapped in albumin microparticles using a one-step spray drying technique optimized in our laboratory. The encapsulated particles were also characterized in vitro for their shape, size, encapsulation efficiency, content, and stability. Albumin microparticles could protect the DNA from nuclease degradation as confirmed in our agarose gel study. Further immune modulating effect was studied in our formulation by measuring IgG antibodies in serum as well as IgA antibodies in fecal extracts. The mice were immunized with a prime dose of 100 μg of pDNA in microparticle formulations with and without interleukins biweekly until week 7 followed by a booster dose of equivalent strength on week 33 to compare the response with the subcutaneous group. The oral immunization with the pDNA to HBsAg microparticles gave significantly higher titer level of both sIgA and IgG at week 9 and 34, respectively, in oral vaccine with interleukins group when compared with the subcutaneous group. Thus, we observed an augmentation of both humoral and cellular immune responses for prolonged periods after immunization.     

Oral delivery of microparticles containing plasmid DNA encoding hepatitis-B surface antigen

The role of albumin-based chitosan microparticles on enhancing immune response of plasmid DNA (pDNA) to hepatitis-B surface antigen (HBsAg) vaccine after oral administration was investigated in mice. The pDNA encoding HBsAg was entrapped in albumin microparticles using a one-step spray drying technique optimized in our laboratory. The encapsulated particles were also characterized in vitro for their shape, size, encapsulation efficiency, content, and stability. Albumin microparticles could protect the DNA from nuclease degradation as confirmed in our agarose gel study. Further immune modulating effect was studied in our formulation by measuring IgG antibodies in serum as well as IgA antibodies in fecal extracts. The mice were immunized with a prime dose of 100 μg of pDNA in microparticle formulations with and without interleukins biweekly until week 7 followed by a booster dose of equivalent strength on week 33 to compare the response with the subcutaneous group. The oral immunization with the pDNA to HBsAg microparticles gave significantly higher titer level of both sIgA and IgG at week 9 and 34, respectively, in oral vaccine with interleukins group when compared with the subcutaneous group. Thus, we observed an augmentation of both humoral and cellular immune responses for prolonged periods after immunization.     

Nasal immunization studies by cationic, fusogenic and cationic-fusogenic liposomes encapsulated with tetanus toxoid

Particulate antigens are more effective than soluble antigens in induction of systemic and mucosal immunity; possibly because they are more efficiently endocytosed by mucosal-associated lymphoid tissue (MALT) M cells. In this study, we determined the systemic and mucosal immune responses in rabbits following intranasal immunization with tetanus toxoid (TT) entrapped in cationic, fusogenic and cationic-fusogenic liposomes. Liposomes containing TT were prepared by dehydration-rehydration method. The volume mean diameter of cationic, fusogenic and cationic-fusogenic liposomes were 3.4 +/- 0.6, 4.3 +/- 2.3 and 3.4 +/- 1.5 microm, respectively. Encapsulation efficiency of TT in cationic, fusogenic and cationic-fusogenic liposomes was respectively determined as 49.1 +/- 8.4%, 48.5 +/- 2.1% and 50.8 +/- 4.9%. After 3 months, the leaking of encapsulated TT from liposomes ranged between 2.02 - 5.46%. Immunoreactivities of encapsulated TT in all kinds of liposomes were completely preserved, as studied by Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Enzyme-Linked Immunosorbent Assay (ELISA). The highest serum immunoglobulin G (IgG) and antitoxin titers were observed in groups immunized with solution formulation (P< 0.001). However, the highest mucosal secretory IgA (sIgA) titers were achieved by fusogenic liposomes (five times more titers compared with TT solution, and 15 times more titers compared with i.m. vaccine), followed by cationic-fusogenic liposomes. No hemolysis was occurred on incubation of liposomes and human erythrocytes. Also after nasal administration of plain liposomes to human volunteers, no local irritation was seen. This study suggests that intranasal administration of fusogenic and cationic-fusogenic liposomes encapsulated with vaccines could be an effective way for inducing mucosal immune responses.     

Dendritic cell targeted chitosan nanoparticles for nasal DNA immunization against SARS CoV nucleocapsid protein

This work investigates the formulation and in vivo efficacy of dendritic cell (DC) targeted plasmid DNA loaded biotinylated chitosan nanoparticles for nasal immunization against nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) as antigen. The induction of antigen-specific mucosal and systemic immune response at the site of virus entry is a major challenge for vaccine design. Here, we designed a strategy for noninvasive receptor mediated gene delivery to nasal resident DCs. The pDNA loaded biotinylated chitosan nanoparticles were prepared using a complex coacervation process and characterized for size, shape, surface charge, plasmid DNA loading and protection against nuclease digestion. The pDNA loaded biotinylated chitosan nanoparticles were targeted with bifunctional fusion protein (bfFp) vector for achieving DC selective targeting. The bfFp is a recombinant fusion protein consisting of truncated core-streptavidin fused with anti-DEC-205 single chain antibody (scFv). The core-streptavidin arm of fusion protein binds with biotinylated nanoparticles, while anti-DEC-205 scFv imparts targeting specificity to DC DEC-205 receptor. We demonstrate that intranasal administration of bfFp targeted formulations along with anti-CD40 DC maturation stimuli enhanced magnitude of mucosal IgA as well as systemic IgG against N protein. The strategy led to the detection of augmented levels of N protein specific systemic IgG and nasal IgA antibodies. However, following intranasal delivery of naked pDNA no mucosal and systemic immune responses were detected. A parallel comparison of targeted formulations using intramuscular and intranasal routes showed that the intramuscular route is superior for induction of systemic IgG responses compared with the intranasal route. Our results suggest that targeted pDNA delivery through a noninvasive intranasal route can be a strategy for designing low-dose vaccines.     

Immunoadjuvant potential of cross-linked dextran microspheres mixed with chitosan nanospheres encapsulated with tetanus toxoid

Context: Nasal mucosa is a desirable route for mucosal vaccine delivery. Mucosal co-administration of chitosan nanoparticles with absorption enhancers such as cross-linked dextran microspheres (CDM, Sephadex^®) is a promising antigen delivery system.

Objective: In the current study, the chitosan nanospheres loaded with tetanus toxoid (CHT:TT NPs) was prepared and characterized. The immune responses against tetanus toxoid after nasal administration of CHT:TT NPs alone or mixed with CDM were also determined.

Materials and methods: Chitosan nanospheres were prepared by ionic gelation method. Particle size, releasing profile and antigen stability were evaluated by dynamic light scattering, diffusion chamber and SDS-PAGE methods, respectively. Rabbits were nasally immunized with different formulations loaded with 40 Lf TT. After three times immunizations with 2 weeks intervals, sera IgG titres and nasal lavage sIgA titres were determined.

Results: Mean size of CHT NPs and CHT:TT NPs were 205?±?42?nm and 432?±?85?nm, respectively. The release profile showed that 42.4?±?10.5% of TT was released after 30?min and reached to a steady state after 1.5?h. Stability of encapsulated TT in nanospheres was confirmed by SDS-PAGE. The antibody titres showed that CHT:TT NPs-induced antibody titres were higher than TT solution. CHT NPs mixed with CDM induced the systemic IgG and nasal lavage sIgA titres higher than intranasal administration of TT solution (p?Discussion and conclusion: As the results indicated, these CHT:TT NPs when co-administered with CDM were able to induce more immune responses and have the potential to be used in mucosal immunization.     

Dextran microspheres could enhance immune responses against PLGA nanospheres encapsulated with tetanus toxoid and Quillaja saponins after nasal immunization in rabbit

Potent immunoadjuvants are needed to elicit responses following mucosal delivery. PLGA (poly[D,L-lactic-co-glycolic acid]) nanospheres, Quillaja saponin (QS) and cross-linked dextran microspheres (CDM) as drug delivery and absorption enhancer adjuvants were evaluated. PLGA nanospheres were prepared by solvent evaporation method. Particulate characteristics of nanospheres were studied by optical and scanning electron microscopes and dynamic light scattering technique. The mean diameter of nanospheres encapsulated with TT and TT?+?QS determined as 425 and 390?nm. Loadings of TT and QS were 30?±?1.9% and 23?±?2.8%. Nanospheres encapsulated with TT or QS were intranasally administered to rabbits, three times in two-week intervals and the serum IgG and nasal lavage IgA titers were determined by ELISA. The serum IgG titer induced with (TT)_PLGA nanospheres was higher than TT solution (P?<?0.001). IgG titers induced with (TT?+?QS)_PLGA was higher than (TT)_PLGA (P?<?0.0001). When (TT)_PLGA and (TT?+?QS)_PLGA nanospheres were mixed with CDM, higher IgG titers were induced (P?<?0.001). The highest mucosal sIgA titers were seen in animals immunized with (TT?+?QS)_PLGA?+?CDM. Co-encapsulation of QS and TT in PLGA nanospheres increased sIgA titers. In conclusion, the highest immune responses were observed by concomitant use of three adjuvants.     

Induction of systemic and mucosal immune responses by intranasal administration of alginate microspheres encapsulated with tetanus toxoid and CpG-ODN

In the induction of systemic and mucosal immunity, particulate antigens are more effective than soluble antigens; possibly because they are more efficiently endocytosed by mucosal-associated lymphoid tissue (MALT) M cells. In this study, we determined the systemic and mucosal immune responses in rabbits following intranasal immunization with encapsulated tetanus toxoid (TT) and CpG-ODN in alginate microspheres. The microspheres were less than 4 microm in diameter. Encapsulation efficiency of TT and CpG-ODN was determined as 47.7+/-6.6 and 34.2+/-7.4, respectively. Release of TT and CpG-ODN in a simulated model with nasal cavity was 14.2+/-3.06 and 36.7+/-2.4% after 4 h. Encapsulated TT preserved its intact structure, but its immunoreactivity was decreased to about 91+/-5%. The highest serum IgG and antitoxin, and nasal lavage IgA titers were observed in groups immunized with microsphere formulations. CpG-ODN as an adjuvant could increase the serum IgG and antitoxin titers when co-administered with TT solution, but its co-encapsulation with TT in alginate microspheres failed to potentiate the systemic immune response while induced high IgA titers in nasal lavages. No hemolysis was occurred on incubation of alginate microspheres and human RBCs. Also after nasal administration of plain microspheres to human volunteers, no local irritation was observed. Intranasal administration of microspheres encapsulated with vaccines showed to be an effective way for inducing a variety of immune responses and that a strong systemic IgG and mucosal IgA responses can be induced in rabbits with intranasal administration of alginate microspheres encapsulated with TT.     

Chitosan microparticles containing plasmid DNA as potential oral gene delivery system.

The potential of chitosan as a polycationic gene carrier for oral administration has been explored since 1990s. Chitosan has been shown to effectively bind DNA in saline or acetic acid solution and protect DNA from nuclease degradation. In this study, pDNA (plasmid DNA) was encapsulated in chitosan microparticles. Chitosan-DNA microparticles were prepared using a complex coacervation process and stability of plasmid DNA was investigated in this complex. The chitosan-DNA microparticles could protect the encapsulated plasmid DNA from nuclease degradation. Release of pDNA from microparticles was studied in simulated gastric, simulated intestinal medium and acidic PBS (phosphate buffer saline) (pH 4.5) buffer at 37 degrees C, and released pDNA was assayed spectrophotometrically. In vitro release of pDNA from chitosan microparticles was dependent on pH, as the pH of the release medium increased release profile decreased. In in vivo-animal studies blue color was observed with X-gal (4-chloro-5-bromo-3-indolyl-beta-galactosidase) staining of histological stomach and small intestine sections after oral administration of pDNA-chitosan microparticles as an indicator of exogeneous gene expression.     

Enhanced mucosal and systemic immune responses obtained by porous silica nanoparticles used as an oral vaccine adjuvant: Effect of silica architecture on immunological properties

Three different kinds of silica (S2, S1 and SBA-15) with different particle sizes (130, 430nm and 1-2μm) and different pore characteristics (i.e. pore size and shape) were developed as oral vaccine immunological adjuvants and the relationship between the silica architecture and immunological properties was investigated. The silica particles were characterized using SEM, TEM and nitrogen adsorption. Model antigen bovine serum albumin (BSA) was successfully entrapped into the silica pores to produce a sustained release vaccine delivery system. Compared with the responsiveness induced by parenteral administration of BSA emulsified in Freund's complete adjuvant (FCA), oral immunization with the silica/BSA formulation produced a stimulated humoral and mucosal (sIgA) response. The IgG and IgA titers induced by loading BSA was as follows: S1>S2>SBA-15. The highest IgG and IgA titers of S1 were attributed to its large honeycombed pores and the optimal particle diameter of 430nm. The corresponding IgG1 and IgG2a titers were also investigated to confirm that BSA loaded in nanoparticles by oral immunization can induce both T-helper 1- and T-helper 2- (Th1 or Th2) mediated responses. We believe that the results of our research will open up new avenues for the formulation of oral vaccines.