Intracellular Bacteria and Protozoa - Intracellular Delivery: Fundamentals and Applications - page 773

surfaces. The majority of N1 were localized in the cytoplasm, whereas N2
colocalized with the endosomes/lysosomes. Colocalization was not observed
between nanostructures and intracellular Salmonella bacteria. However, significant
in vitro reductions in bacterial counts (0.44 log 10 ) were observed after incubation
with N1, suggesting that the surface property of the nanostructure influences intra-
cellular bacterial clearance. In view of these results, GEN loaded in the same core-
shell nanostructures (nonionic amphiphilic shells N1 and ionic cores) and employed
for therapy against intracellular pathogens, including Salmonella (Ranjan et al.
2010 ). The intracellular pathway of GEN-N1 nanostructures was determined by
flow cytometry and confocal microscopy, finding that their uptake into J774A.1
macrophages proceeded mainly by fluid-phase endocytosis and clathrin-mediated
pathways. The nanostructures were nontoxic in vitro at doses of 50-250 mg/ml, and
they significantly reduced the amounts of intracellular Salmonella (0.53 log).
Finally, GEN was uniformly loaded in a porous (1.7-3.3 mm pore diameter) bio-
degradable silica xerogel matrix for the prolonged release for treatment of Salmonella
infection in a mouse model. Three ip doses administered to mice of porous silica
loaded with GEN reduced the CFU of S. thyphimurium in livers of infected mice by
0.48 log compared to 0.13 log with free drug (Munusamy et al. 2009 ).

4 Francisella

F. tularensis is a highly virulent and contagious, facultative intracellular bacterium. It
causes the zoonotic disease tularemia in a large number of mammals and can be a
potentially fatal human disease if untreated. F. tularensis is a potential biological war-
fare/bioterrorism agent, and medical and public health management of the infectious
disease caused by this bacterial agent is important (Dennis et al. 2001 ). The bacteria
are spread via multiple transmission routes including arthropod bites, physical contact
with infected animal tissues, contaminated water and inhalation of aerosolized organ-
isms. Inhalation of as few as 10 CFU are sufficient to initiate lung colonization and the
subsequent development of pulmonary tularemia, which is the most lethal form of the
disease exhibiting mortality rates as high as 60% (Fuller et al. 2009 ).
Infection by F. tularensis involves the RES system, dendritic and alveolar epithe-
lial cells and leads to bacterial growth within the lungs, liver and spleen (Fortier et al.
1991 ). Following uptake or invasion of a host cell wild type, F. tularensis cells escape
the phagosome and replicate within the cytoplasm of infected cells (Clemens et al.
2004 ; Fortier et al. 1995 ; Golovliov et al. 2003 ; Santic et al. 2005 ). Following a period
of multiplication where relatively high numbers of bacteria are produced when com-
pared to other pathogens such as Salmonella , intracellular F. tularensis induces apop-
tosis leading to death of the infected macrophage and release of the bacteria into the
extracellular environment (Lai et al. 2001 ). Extracellular bacteria are subsequently
thought to infect naive macrophages and bacterial multiplication begins again.
Initially, bacteria colocalized with the late endosomal/lysosomal markers
LAMPs, but not with cathepsin D. Moreover, the phagosomes containing