Cationic immune stimulating complexes containing soluble leishmania antigens: preparation, characterization and in vivo immune response evaluation

Background: Cationic immune stimulating complexes [PLUSCOMs] are particulate antigen delivery systems. PLUSCOMs consist of cationic immunostimulatory complexes [ISCOMs] derivatives and are able to elicit in vivo T cell responses against an antigen

Objective: To evaluate the effects of PLUSCOMs containing Leishmania major antigens [SLA] on the type of immune response generated in the murine model of leishmaniasis

Methods: PLUSCOMs consisting of 1, 2-dioleoyl-3-trimethylammonium-propane [DOTAP] were used as antigen delivery system/immunoadjuvants for soluble SLA. BALB/c mice were immunized subcutaneously, three times in 2-week intervals. Footpads swellings at the site of challenge and parasite loads were assessed as a measure of protection. The immune responses were also evaluated by determination of IgG subclasses and the level of IFN- gamma and IL-4 in cultured splenocytes

Results: There was no significant difference [p<0.05] between the sizes of lesions in mice immunized with different formulations. Also, there was no significant difference in the number of parasites in the footpad or spleen of all groups compared with the control group. The highest level of IFN- gamma secretion was observed in the splenocytes of mice immunized with PLUSCOM/SLA [p<0.001] and lower amounts of IL-4 was observed in PLUSCOM group [p<0.001] as compared to negative control

Conclusion: Our results indicated that SLA in different formulations generated an immune response with mixed Th1/Th2 response that was not protective enough despite the activation of CD4+ T cells with secreting IFN- gamma in groups which received PLUSCOM with antigen

In vivo evaluation of mucoadhesive properties of nanoliposomal formulations upon coating with trimethylchitosan polymer

Drug delivery via mucosal routes has been confirmed to be effective in inducing strong immune responses. Liposomes could enhance immune responses and mucoadhesive potentials, make them useful mucosal drug delivery systems. Coating of liposomes by mucoadhesive polymers succeeded in enhancing immune responses. Our studies aim at preparation and characterization of trimethylchitosan-coated nanoliposomes for nasal delivery of a model antigen, tetanus toxoid [TT]. Anionic liposomes were prepared by dehydration-rehydration method with an average size of 100 nm and were coated with 0.01% [w/v] solution of trimethyulchitosan [TMC] with 50 +/- 10% of quaternization. Surface properties and zeta potential were evaluated by DLS. Antigen stability and integrity were studied by SDS-PAGE electrophoresis. Nasal clearance rate and mucoadhesive properties of liposomes were studied by gamma scintigraphy method using 99mTc-labelled liposomes. The zeta potential of non-coated and TMC-coated liposomes was -40 and +38.8, respectively. Encapsulation rate of tetanus toxoid was 77 +/- 5.5%. SDS-PAGE revealed that the antigens remained intact during formulation procedure. Gamma scintigraphy confirmed that both types of liposomes could remain in nasal cavity up to ten folds over the normal residence time for conventional nasal formulations. TMC-coated nanoliposomes have several positive potentials including good mucoadhesive properties, preserved integrity of loaded antigen and presence of TMC as a mucoadhesive polymer with innate immunoadjuvant potential which make them suitable for efficient adjuvant/delivery system

Nano-adjuvanted polio vaccine: preparation and characterization of chitosan and trimethylchitosan [TMC] nanoparticles loaded with inactivated polio virus and coated with sodium alginate

It is proposed that particulate antigens could better interact with the antigen presenting cells [APCs]. A fast, simple and scalable process for preparation of polymeric nanoparticles [NPs] is coating of charged antigenic particles, like viruses, with oppositely charged polymers. A second coating with a charged polymer could increase the stability and modify the immunomodulatory potentials of NPs. Negatively charged inactivated polio virus [IPV] was coated with cationic polymers, chitosan [CHT] and trimethylchitosan [TMC] by a simple incubation method. CHT: IPV and TMC: IPV NPs were coated by anionic polymer, sodium alginate [ALG]. Physical characteristics and stability of NPs were studied. Cytocompatibility of NPs was checked with MTT assay. DC maturation study was used for evaluation of the NPs potential in interaction with DCs. Among the various polymer to antigen ratios tested, the least size and PDI and the highest ZP was seen in TMC: IPV [2:1], CHT: IPV [2:1], ALG: TMC: IPV [2:2:1] and ALG: CHT: IPV [4:2:1]. The physical stability of TMC: IPV and CHT: IPV was preserved until 15 days. After an early de-association of some part of coated alginate, ALG: CHT: IPV and ALG: TMC: IPC NPs were stable until the end of study [25[th] day]. No one of the NPs formulations had a negative effect on cell viability. Compared with plain IPV, nanoparticulate IPV formulations failed to increase the expression of CD40 and CD86 markers of DCs. NPs prepared with simple and scalable method, had reasonable physical characteristics, stability and cytocompatibility and could be tested in vivo for their immunoadjuvant potential

Mucosal adjuvant potential of quillaja saponins and cross-linked dextran microspheres, co-administered with liposomes encapsulated with tetanus toxoid

Intranasal vaccination is particularly a striking route for mucosal immunization, due to the ease of administration and the induction of both mucosal and humoral immunity. However, soluble antigens [Ag] are not sufficiently taken up after the nasal administration and need to be co-administered with adjuvants, penetration enhancers or encapsulated in particles. So, in this study, tetanus toxoid [TT] as a model Ag was entrapped in nonionic liposomes. The effect of the co-administration of Quillaja saponin [QS] as an adjuvant and cross-linked dextran microspheres [CDM] as penetration enhancer on immune responses was also studied. TT or TT + QS loaded liposomes were prepared by dehydration-rehydration method [DRV], followed by the extrusion through 400 nm filters. Some formulations were mixed with CDM. Liposomes were first characterized for their size range, mean diameter and morphology using particle size analyzer, optical and transmission electron microscopes. The volume mean diameter of liposomes was determined as 3836 +/- 179 and 624 +/- 114 nm before and after the extrusion, respectively. Structural efficiency of TT extracted from liposomes was confirmed by SDS-PAGE method. Encapsulation efficiencies of TT and QS were 44 +/- 8.50% and 60 +/- 6.02%, respectively. Rabbits were nasally immunized with various formulations and serum IgG titers and nasal lavage slgA titers were determined by an ELIS A method. TT + QS liposomes induced higher slgA levels in comparison with TT liposomes [p < 0.05], but the difference in serum [gG levels was not significant. Results indicated that neutral liposomes administered nasally, have a good potential for induction of mucosal immunity and co-encapsulation of QS and TT in liposomes improved the systemic and mucosal immune responses

PLGA nanospheres loaded with autoclaved leishmania major [ALM] and CPG-ODN: preparation and in vitro characterization

Several antigens, adjuvants and delivery systems have been evaluated for induction of protective immune responses against Leishmaniasis, but most of them have been inefficient. In this study, PLGA nanospheres as antigen delivery system CpG-ODN as an immunoadjuvant for increasing the immune responses against Autoclaved Leishmania major [ALM] were prepared and characterized. PLGA nanospheres prepared by a double-emulsion [W/O/W] technique. The internal aqueous phase contained ALM and CpG-ODN, while the oily phase contained the solution of PLGA in dichloromethan and the external aqueous phase was PVA 7.5% [WIV] solution. Particulate characteristics were studied by scanning electron microscopy and particle size analysis. The encapsulation efficiency was determined by Lowry method for ALM and UV spectroscopy at 260 nm for CpG-ODN. The release profiles of antigen and CpG-ODN from nanospheres evaluated for one week. Nanospheres were spherical in shape, having smooth surfaces. Mean diameters for blank and ALM + CpGODN loaded nanospheres recorded as 302 +/- 129 and 333 +/- 128 nm respectively. Also, the encapsulation efficiencies of ALM and CpG-ODN were 71.6 +/- 8.8 and 49.1 +/- 2.4%, respectively. Evaluation of the release profiles of ALM and CpG-ODN from nanospheres showed that 44.8 +/- 0.8% of ALM and 29.5 +/- 0.2% of CpGODN released from nanospheres in one week. The prepared nanospheres with desirable size, encapsulation efficiency, and slow rate of release, had acceptable features for future in vivo studies

Preparation and characterization of PLGA nanospheres encapsulated with autoclaved leishmania major [ALM] and quillaja saponin

Several antigens, adjuvants and delivery systems have been evaluated for induction of protective immune responses against leishmaniasis, but have mostly been inefficient. In this study, poly [d,l-lactide-co-glycolide] [PLGA] nanospheres as antigen delivery system and Quillaja saponins [QS] as an immunoadjuvant have been used to increase the immune responses against Autoclaved Lieshmania major [ALM]. PLGA nanospheres were prepared using a double emulsion [W/O/W] technique. The internal aqueous phase contained ALM and saponin, while the oily phase contained the solution of PLGA in dichloromethane and the external aqueous phase was polyvinylalcohol [PVA] 7.5% [W/V] solution. Particulate characteristics were studied by scanning electron microscope and particle size analyzer. The encapsulation efficiency was determined by Lowry method and the release profile of antigen and saponin from nanospheres was evaluated for one week. Nanospheres were spherical in shape having smooth surfaces. Mean diameters for nanospheres loaded with ALM and ALM+QS were 300 +/- 123 nm and 294 +/- 106 nm respectively. Encapsulation efficiencies for ALM and QS were found 71 +/- 14.8% and 55.8 +/- 23.1%, respectively. Evaluation of the release profiles of ALM and QS from nanospheres in one week showed that 44.8 +/- 0.8% of ALM and 29.5 +/- 0.21% of QS had been released from nanospheres. In conclusion, the prepared nanospheres with desirable size, encapsulation efficiency, and slow rate of release, had acceptable features for future in vivo studies

Evaluation of the clearance characteristics of liposomes in the human nose by gamma-scintigraphy

The nasal cavity possesses many advantages as a site for drug delivery, such as, ease of administration, applicability for long term treatments and a large surface area for absorption. One important limiting factor for nasal drug delivery is the limited time available for absorption within the nasal cavity due to mucociliary clearance. Several drug delivery systems including different kinds of microspheres and liposomes have been tried for encapsulation of drugs and increasing the residence time in nasal cavity. In this study the clearance rate of three kinds of liposomes: neutral [phosphatidylcholin [PC] and cholesterol [Chol]], cationic [PC, Chol and stearylamine] and fusogenic [PC, Chol, dioleoyl phosphatidylethanol amine] was determined by gamma scintigraphy with lactose powder being used as negative control. Liposomes were prepared by dehydration-rehydration method. 99mTc labeled liposomes were prepared using technetium pertechnetate in the presence of a potent reducing agent, stannus chloride. The labeling procedure was set in a manner that each 150 ml of liposome suspensions contained 2 MBq of radioactivity. Labeling efficiency was calculated by paper chromatography using acetone as mobile phase. Each delivery system containing 2 MBq of activity was sprayed into right nostril of four healthy volunteers and one-minute static views were repeated each half hour until 4 hours. Clearance rates were compared using two Regions of Interest [ROIs]; the initial site of deposition of particles, and all of nasopharynx region. The clearance rate of each one of liposomes was calculated after applying the physical decay corrections. The mean labeling efficiencies for neutral, cationic and fusogenic liposomes were calculated as 91%, 20% and 69%, respectively. The cleared percent of preparations from nasopharynx region after 4 hours was determined as follows: neutral liposomes 18 +/- 2.9%; fusogenic liposomes 53.5 +/- 1.2%; cationic liposomes 69.7 +/- 4.2%; lactose powder 74.5 +/- 4.9%. Neutral liposomes showed the lowest clearance rate compared to lactose powder [P<0.0001], followed by fusogenic liposomes [P<0.01] and cationic liposomes [P<0.05]. The clearance profiles of formulations from deposition ROI and nasopharynx ROI were identical. This study shows the neutral liposomes have the highest mucoadhesion properties and are suitable nasal delivery systems. Furthermore, this study proves that limiting step for the nasal clearance of nasally administered particulate systems is their dislocation from the initial site of deposition, and their following interactions with mucus layer in the rest of nasal passage does not significantly affect the clearance time