By manufacturing a single-particle system in two particulate forms (i.e., micrometer size and nanometer size), we have designed a bacterial vaccine form that exhibits improved efficacy of immunization. Microstructural properties are adapted to alter dispersive and aerosol properties independently. Dried ''nanomicroparticle'' vaccines possess two axes of nanoscale dimensions and a third axis of micrometer dimension; the last one permits effective micrometer-like physical dispersion, and the former provides alignment of the principal nanodimension particle axes with the direction of airflow. Particles formed with this combination of nano-and micrometer-scale dimensions possess a greater ability to aerosolize than particles of standard spherical isotropic shape and of similar geometric diameter. Here, we demonstrate effective application of this biomaterial by using the live attenuated tuberculosis vaccine bacille Calmette-Gué rin (BCG). Prepared as a spray-dried nanomicroparticle aerosol, BCG vaccine exhibited high-efficiency delivery and peripheral lung targeting capacity from a low-cost and technically simple delivery system. Aerosol delivery of the BCG nanomicroparticle to normal guinea pigs subsequently challenged with virulent Mycobacterium tuberculosis significantly reduced bacterial burden and lung pathology both relative to untreated animals and to control animals immunized with the standard parenteral BCG.bacillus Calmette-Gué rin ͉ inhaled particles ͉ live-attenuated vaccines ͉ spray drying ͉ tuberculosis A erosol vaccination via the lungs targets an epithelium critical to host defense against inhaled pathogens (1), potentially avoids needle injection, and provides an exciting opportunity in the development of a newer and more effective tuberculosis (TB) vaccine. Published studies of inhaled vaccines for measles, influenza, and TB (2) have mostly involved nebulized solutions, a process that necessitates large volumes of water with long administration times; dried forms of vaccines, as formed traditionally by freeze drying and recently by spray drying (15), present an alternative to nebulization but pose risks to the integrity of dried bacteria and produce powders with suboptimal dispersive characteristics. Here, we show that by designing bacterial vaccines via rapid drying we can exploit the natural tendency of dried bacteria to assume elongated shapes and achieve remarkably effective airborne properties that combine the positive attributes of their submicrometer radial axes and micrometer-scale longitudinal axes. We demonstrate vaccine delivery from an inhaler designed for delivery to newborns. Furthermore, we report better TB protection with an aerosol form of bacillus Calmette-Guérin (BCG) than is achievable by standard s.c. and intradermal injection using a low-inoculum, aerosol infection and challenge in the sensitive guinea pig model. ResultsIn initial experiments, powders of dry bacteria were prepared by spray drying with a model nonpathogenic Mycobacterium, Mycobacterium smegmatis. We added a sec...
One third of the world population is infected with tuberculosis (TB), and new infections occur at a rate of one per second. The recent increase in the emergence of drug-resistant strains of Mycobacterium tuberculosis and the dearth of anti-TB drugs is threatening the future containment of TB. New drugs or delivery systems that will stop the spread of TB and slow down or prevent the development of drug-resistant strains are urgently required. One of the reasons for the emergence of drug-resistant strains is the exposure of mycobacteria to sub-therapeutic levels of one or more antibiotics. Lung lesions containing large numbers of bacteria are poorly vascularized and are fortified with thick fibrous tissue; conventional therapy by the oral and parenteral routes may provide sub-therapeutic levels of anti-TB drugs to these highly sequestered organisms. Administering drugs by the pulmonary route to the lungs allows higher drug concentrations in the vicinity of these lesions. Supplementing conventional therapy with inhaled anti-TB therapy may allow therapeutic concentrations of drug to penetrate effectively into lung lesions and treat the resident mycobacteria.
Microparticle phagocytosis induces responses in infected murine macrophages that are indicative of activation of innate bactericidal mechanisms, and are inimical to bacterial survival. It is likely that such responses augment straightforward drug action on the bacterium and contribute to the unexpectedly high efficacy of microparticles in experimental tuberculosis.
Three-dimensional encapsulation of cells within nanostructured silica gels or matrices enables applications as diverse as biosensors, microbial fuel cells, artificial organs, and vaccines; it also allows the study of individual cell behaviors. Recent progress has improved the performance and flexibility of cellular encapsulation, yet there remains a need for robust scalable processes. Here, we report a spray-drying process enabling the large-scale production of functional nano-biocomposites (NBCs) containing living cells within ordered 3D lipid-silica nanostructures. The spray-drying process is demonstrated to work with multiple cell types and results in dry powders exhibiting a unique combination of properties including highly ordered 3D nanostructure, extended lipid fluidity, tunable macromorphologies and aerodynamic diameters, and unexpectedly high physical strength. Nanoindentation of the encasing nanostructure revealed a Young's modulus and hardness of 13 and 1.4 GPa, respectively. We hypothesized this high strength would prevent cell growth and force bacteria into viable but not culturable (VBNC) states. In concordance with the VBNC state, cellular ATP levels remained elevated even over eight months. However, their ability to undergo resuscitation and enter growth phase greatly decreased with time in the VBNC state. A quantitative method of determining resuscitation frequencies was developed and showed that, after 36 weeks in a NBC-induced VBNC, less than 1 in 10,000 cells underwent resuscitation. The NBC platform production of large quantities of VBNC cells is of interest for research in bacterial persistence and screening of drugs targeting such cells. NBCs may also enable long-term preservation of living cells for applications in cell-based sensing and the packaging and delivery of live-cell vaccines.
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