Development of Novel Octanoyl Chitosan Nanoparticles for Improved Rifampicin Pulmonary Delivery: Optimization by Factorial Design.

A novel hydrophobic chitosan derivative, octanoyl chitosan (OC) with improved organic solubility was synthesized, characterized, and employed for the preparation of rifampicin (Rif) encapsulated nanoparticle formulations for pulmonary delivery. OC was characterized to confirm acyl group substitution and cytotoxicity in A549 epithelial lung cells. OC nanoparticles were produced by the double emulsion solvent evaporation technique without cross-linking and characterized for particle size distribution, morphology, crystallinity, thermal stability, aerosol delivery, and drug release rate. OC was successfully synthesized with substitution degree of 44.05 ± 1.75%, and solubility in a range of organic solvents. Preliminary cytotoxicity studies of OC showed no effect on cell viability over a period of 24 h on A549 cell lines. OC nanoparticles were optimized using a 32 full factorial design. An optimized batch of OC nanoparticles, smooth and spherical in morphology, had mean hydrodynamic diameter of 253 ± 19.06 nm (PDI 0.323 ± 0.059) and entrapment efficiency of 64.86 ± 7.73% for rifampicin. Pulmonary deposition studies in a two-stage impinger following aerosolization of nanoparticles from a jet nebulizer gave a fine particle fraction of 43.27 ± 4.24%. In vitro release studies indicated sustained release (73.14 ± 3.17%) of rifampicin from OC nanoparticles over 72 h, with particles demonstrating physical stability over 2 months. In summary, the results confirmed the suitability of the developed systems for pulmonary delivery of drugs with excellent aerosolization properties and sustained-release characteristics.

Mono-N-carboxymethyl chitosan (MCC) and N-trimethyl chitosan (TMC) nanoparticles for non-invasive vaccine delivery.

Mucosal application of a vaccine can effectively induce both systemic and mucosal immune responses. In general, mucosal applications of antigens result in poor immune responses. Therefore, adjuvant/delivery systems are required to enhance the immune response. Chitosan is a cationic biopolymer which exerts advantages as a vaccine carrier due to its immune stimulating activity and bioadhesive properties that enhance cellular uptake and permeation as well as antigen protection. Similar effects are also shown by chitosan derivatives. In this study, the nanoparticulate systems were prepared by using differently charged chitosan derivatives, N-trimethyl chitosan (TMC, polycationic), and mono-N-carboxymethyl chitosan (MCC, polyampholytic) for mucosal immunisation. The derivatives were synthesised and characterised in-house. The aqueous dispersions of the derivatives were also prepared for comparison. The cytotoxicity studies (MTT assay) on Chinese hamster ovary (CHO-K1) cell lines showed that cell viability was in the order of MCC, chitosan and TMC. Nanoparticles were prepared using ionic gelation method and loaded with tetanus toxoid (TT). Nanoparticles with high loading efficacy (>90% m/m), particle size within the range of 40-400nm, with a negative surface charge for MCC and positive surface charge for TMC and chitosan were obtained. The structural integrity of the TT in the formulations was confirmed by SDS-PAGE electrophoresis analysis. The effective uptake of the FITC-BSA loaded nanoparticles into the cells was demonstrated by cellular uptake studies using J774A.1 cells. Immune responses induced by the formulations loaded with tetanus toxoid were studied in vivo in Balb/c mice. Enhanced immune responses were obtained with intranasal (i.n.) application of nanoparticle formulations. Chitosan and TMC nanoparticles which have positively charged surfaces induced higher serum IgG titres when compared to those prepared with MCC which are negatively charged and smaller in size. Nanoparticle formulations developed in this study can be used as promising adjuvant/delivery systems for mucosal immunisation.

Effect of vitamin E TPGS on immune response to nasally delivered diphtheria toxoid loaded poly(caprolactone) microparticles.

The nasal mucosa has many advantages as a potential site for drug and vaccine delivery. The present study has sought to exploit this route of delivery using microparticles composed of D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) as a matrix material blended with poly(caprolactone) for nasal immunisation with diphtheria toxoid. Particles were prepared by a double emulsion method, followed by spray drying and the effect of TPGS on size, zeta potential, loading and release of antigen was assessed. Particles composed of TPGS-PCL blends were spherical, smooth and monodisperse, displaying increasing yields after spray drying with increasing concentrations of TPGS. The immune response to diphtheria toxoid loaded PCL-TPGS microspheres after nasal administration was shown to be higher than that achieved using PCL microspheres alone. We conclude that TPGS shows significant potential as a novel adjuvant either alone or in combination with an appropriate delivery system.

Immobilisation of vaccines onto micro-crystals for enhanced thermal stability.

The thermal instability of many vaccines leads to the wastage of half of all supplied vaccines. In this note, we report the application of a novel technology: protein-coated micro-crystals (PCMC) to improve the thermostability of a model vaccine (diphtheria toxoid, DT). The latter was immobilised onto the surface of a crystalline material (L-glutamine) via a rapid dehydration method, resulting in the production of a fine free-flowing powder. The PCMC consisted of thin, flat crystals with an antigen loading of 3.95% (w/w). The DT-coated glutamine crystals and free DT (the controls) were incubated at different temperatures for a defined time period (4 degrees C, RT and 37 degrees C for 2 weeks and 45 degrees C for 2 days), after which the crystals were suspended in buffer and intramuscularly administered to mice. Incubation of DT (free and crystal-coated) at room temperature and at 37 degrees C for 2 weeks did not result in any change in the antibody response compared to DT that had always been stored properly (i.e. in the refrigerator). In contrast, incubation of free DT at 45 degrees C resulted in a reduced IgG response, indicating thermal instability of free DT at that temperature. The antibody response was not reduced, however, with the crystal-coated DT. These preliminary studies show that PCMC is a promising technology for the thermal stabilisation of vaccines.

Biodegradable mucoadhesive particulates for nasal and pulmonary antigen and DNA delivery.

Biodegradable polymer and particulate carriers have been shown to be of considerable potential for the delivery of peptides, proteins and DNA in animal models. In the context of vaccine delivery to the upper and lower respiratory tracts, the use of mucoadhesive agents offers a strategy for the facilitation of increased residence time and increased vaccine efficacy. Additional concerns addressed here include the potential of uptake of vaccine formulations by the primary olfactory nerves in the nasal cavity, effective delivery to the lung, strategies to maximise the immunopotentiation of candidate vaccine formulations, as well as the evaluation of animal models and interpretation of engendered immune responses in terms of antigen-specific antibody production. Experimental data are presented that demonstrate the potential of muco- and bioadhesive agents in combination with liposomes for intranasal (i.n.) delivery of tetanus toxoid in mice. A delivery system utilising chitosan for the formulation of microspheres by the spray-drying method is described and assessed for intranasal vaccine delivery, and porous particles with potential for pulmonary administration are also outlined.

Adjuvant synergy: the effects of nasal coadministration of adjuvants.

Modern peptide and protein subunit vaccines suffer from poor immunogenicity and require the use of adjuvants. However, none of the currently licensed adjuvants can elicit cell-mediated immunity or are suitable for mucosal immunization. In this study we explored the immunological effect of nasal co-administration of adjuvants with distinct functions: cholera toxin subunit B, a potent mucosal adjuvant that induces strong humoral responses, muramy di-peptide (MDP), an adjuvant known to elicit cell mediated immunity but rarely used nasally, and chitosan, an adjuvant that achieves specific physiological effects on mucosal membranes that improve antigen uptake. Groups of five female BALB/c mice received on days 1 and 56 nasal instillations of the recombinant Helicobacter pylori antigen urease admixed to single or multiple adjuvant combinations. Serum IgG kinetics were followed over 24 weeks. At the conclusion of the experiment, local antibody responses were determined and antigen-specific recall responses in splenocyte cultures were assayed for proliferation and cytokine production. The combination of adjuvants was shown to further contribute to the increased antigenicity of recombinant H. pylori urease. The data presented here outline and support facilitation of increased immunomodulation by an adjuvant previously defined as an effective mucosal adjuvant (chitosan) for another adjuvant (MDP) that is not normally effective via this route.

Immunisation against plague by transcutaneous and intradermal application of subunit antigens.

We have investigated immunological responses in BALB/c mice following transcutaneous (TC) delivery of fraction 1 (F1) and V subunits from Yersinia pestis in conjunction with an enterotoxin-derived adjuvant (cholera toxin, CT). It was found that two or more TC applications of F1 and V subunits (admixed with cholera toxin) served to elicit significant levels of anti-F1 and V antibodies in the serum of immunised mice. IL-6 secretion from cultured splenocytes derived from immunised mice indicated that a single TC application of F1 and V subunits (admixed with cholera toxin) conferred a cell-mediated response. As compared with intranasal or direct intradermal injection of F1 and V, the numbers of F1/V-specific antibody-forming cells in the spleens of animals immunised by TC application of F1 and V (admixed with CT) was relatively low. It was noted that TC application of F1 and V admixed with CT was very effective for priming responses that were boosted by intranasal or intradermal routes. Similarly, it was found that TC application of F1 and V admixed with CT could be used to efficiently boost pre-existing responses engendered by intradermal injection or intranasal instillation of F1 and V. In order to assess if TC application of F1 and V admixed with CT could protect experimental animals from plague, immunised mice were injected with a virulent strain of Y. pestis. It was found that two TC applications of F1 and V admixed with CT conferred only limited protection against 10(2) MLDs. However, three TC applications of F1 and V admixed with CT conferred solid protection against 10(2) MLDs. Hence we have shown, for the first time, that TC application of F1 and V admixed with CT can protect animals against challenge with a virulent strain of plague causing bacteria. These data suggest that transcutaneous immunisation may be a simple and non-invasive method for immunising individuals against plague.

Adjuvant action of melittin following intranasal immunisation with tetanus and diphtheria toxoids.

Melittin, a 26-amino acid peptide and the major active component of the venom of the honey bee--Apis mellifera--has recently been shown to have absorption enhancing properties in Caco-2 cells at levels well below the level required for the generation of cytotoxicity. Given the potential of absorption enhancing agents to act as adjuvants when administered nasally [Alpar, H.O., Eyles, J.E., Williamson, E.D. and Somavarapu, S. (2001) "Intranasal vaccination against plague, tetanus and diphtheria", Adv. Drug Delivery Rev. 51, 173-201] we hypothesized that melittin may have potential as a mucosal adjuvant. Following our initial studies reported here, it was found that the co-administration of 4 microg of melittin in conjunction with tetanus toxoid in BALB/c mice was effective in eliciting markedly enhanced antibody titres in comparison to control groups and groups receiving free antigen administered intranasally. Lower concentrations of melittin were less effective and mice receiving 4 microg of melittin plus antigen exhibited antibody titres significantly higher (i.e. P<0.05) than any of the other groups tested. The observed IgG2a titres were shown to be dependent upon the dose of melittin co-administered with the immunising antigen in a similar fashion to the observed total IgG responses. In summary, melittin has been shown here to have potential as a novel mucosal adjuvant for antigens administered via the nasal route.

Oral plasmid DNA delivery systems for genetic immunisation.

The use and optimisation of plasmid DNA delivery systems for the purposes of eliciting transgene specific immune responses to orally administered DNA encoded antigen represents a significant challenge. Here, we have outlined a multicomponent polymer modified liposomal delivery system that offers potential for oral administration of plasmid DNA. It is shown that the polymer/liposome formulated DNA is able to elicit markedly enhanced transgene specific cytokine production following in vitro restimulation of splenocytes with recombinant antigen. This is discussed with reference to recent publications and the potential of plasmid DNA delivery systems for the purposes of genetic immunisation, as reported in selected literature, is assessed.

Intranasal vaccination against plague, tetanus and diphtheria.

Plague is an extremely virulent and potentially lethal infection caused by the bacterium Y. pestis. The current vaccine used to immunise against plague often fails to engender solid (100%) protection against inhalational infection with Y. pestis. Similarly, logistical factors favour the development of non-parenteral immunisation protocols to counter plague. Recently an improved parenteral vaccination strategy for plague, based on the recombinant subunit approach, has entered clinical trails. The Yersinia pestis subunit antigens (F1 and V) have been successfully incorporated into novel vaccine delivery systems such as biodegradable microspheres composed of poly-L-(lactide) (PLLA). Intranasal and intratracheal administration of PLLA microencapsulated F1 and V serves to protect experimental animals from inhalational and subcutaneous challenge with virulent Y. pestis bacilli. Liposomes have also been used to improve the immunogenicity of intranasally administered Y. pestis antigens, and the effectiveness of this approach to plague immunisation has been evaluated. Tetanus and diphtheria still cause many deaths worldwide. The maintenance of protective immunity to diphtheria and tetanus requires booster injections of the currently licensed toxoid vaccines. Consequently, many people remain unprotected. Improved coverage may well result from the development of effective non-invasive vaccines that could be readily distributed and potentially self-administered. To this end, the intranasal and inhalational routes of administration have been extensively investigated. Tetanus and diphtheria toxoids have been delivered intranasally to experimental animals using a wide variety of adjuvants (enterotoxin derivatives), penetration enhancers (cyclodextrins, bile salts, surfactants, cationic polymers) and delivery systems (microspheres and liposomes). As compared with parenteral vaccination, nasal immunisation has been shown favourably effective in small animal models, and a limited number of early phase clinical trails. As a caveat to this, adjuvantisation of toxoid/subunit molecules appears to be a requisite for elicitation of appreciable immunological responses, following nasal administration of acellular immunogens. Testing in larger animal models and humans is needed to ascertain if the promising results obtained in rodents can be reciprocated without compromising safety.