Targeted delivery of antibiotics to intracellular chlamydial infections using PLGA nanoparticles
Introduction
Human infections by the intracellular bacterial pathogens Chlamydia trachomatis and Chlamydia pneumoniae present enormous health care problems. C trachomatis is the most prevalent sexually transmitted bacterium, and is associated also with blinding trachoma; C pneumoniae is a respiratory pathogen responsible for a significant proportion of community-acquired pneumonia. In addition, both C trachomatis and C pneumoniae have been shown to cause, or are strongly associated with, diverse chronic diseases [1], [2]. Both C trachomatis and C pneumoniae are obligate intracellular bacteria and undergo a biphasic, intracellular developmental cycle. The developmental cycle is initiated when elementary bodies (EB), the infectious extracellular form of the organism, attach to the target host cell. Once bound, EB enter host cells and reside in a host cell membrane-bound cytoplasmic inclusion within which they spend their intracellular tenure. In the inclusion, EB develop into reticulate bodies (RB), the metabolically active growth form. Each RB undergoes 7-8 rounds of cell division, after which most dedifferentiate back to EB. Newly-formed EB are released by host cell lysis or exocytosis (see [3] for review). Many studies have demonstrated that both C trachomatis and C pneumoniae often disseminate widely from their sites of primary infection [4], [5]. At sites of their disseminated residence, both organisms enter an unusual biological state referred to as ‘persistence’ [5], [6]. In this state, a block in gene expression obviates the full completion of the normal developmental cycle, and the organisms display several unusual morphological, transcriptional and other properties [7], [8]. Importantly, persistently infecting Chlamydiae can elicit powerful immunopathogenic responses that can contribute to chronic diseases associated with the organism and thus present important therapeutic targets. However, persistent infections by both organisms have proved to be refractory to antibiotic therapy. The lack of therapeutic efficacy results from the attenuated metabolic rate of persistently infecting Chlamydiae in combination with the modest intracellular drug concentrations achievable by normal delivery of antibiotics to the inclusions within which Chlamydiae reside in the host cell cytoplasm. Thus, strategies that improve the penetration of antibiotics into the inclusions could potentially improve the treatment efficacy.
Recent studies show that Chlamydiae hijack the host cell machinery to obtain lipid, protein and other nutrients from extracellular sources. Specifically, it was shown that following chlamydial infection, cellular lipid nanoparticles, which are about 100 nm in size, are actively rerouted to the inclusions [9], [10], [11]. Previous studies showed that nanoparticles formulated using the biodegradable poly(d-l-lactide-co-glycolide) (PLGA) polymer and in the 100–300 nm size range can enter mammalian endothelial, epithelial, and smooth muscle cells efficiently and deliver encapsulated drug intracellularly at a sustained rate [12], [13], [14]. Based on this, we hypothesized that PLGA nanoparticles can be used as Trojan horses to enhance the delivery of antibiotics to the chlamydial inclusion complexes. We initially studied the trafficking of PLGA nanoparticles in C trachomatis-infected cells. We then evaluated nanoparticles for the delivery of antibiotics to the inclusions. Our studies show that nanoparticles target intracellular chlamydial inclusions and organism, and enhance the effectiveness of antibiotics in reducing microbial burden.
Section snippets
Materials
PLGA was purchased from Absorbable Polymers (Pelham, AL). Glacial acetic acid (LC-MS grade) was procured from Alfa Aesar (Ward Hill, MA). Ammonium acetate crystals and sodium hydroxide was purchased from Mallinckrodt (Phillipsburg, NJ). Azithromycin was purchased from Astatech Inc (Bristol, PA). Rifampin, N-acetyl-l-cysteine (NAC), polyvinyl alcohol (PVA) (average Mw 30,000–70,000 Da), 6-coumarin and carboxy methyl cellulose sodium salt (CMC) (average Mw 90000 Da) were purchased from
Nanoparticle formulation and characterization
PLGA nanoparticles labeled with a green fluorescent marker, 6-coumarin, were formulated with an average particle size of ∼260 nm, which was in the range previously reported for PLGA nanoparticles [12], [15]. Nanoparticles loaded with the two antibiotics, rifampin and azithromycin, were also in similar size range (Table 1). While the extent of loading achieved here was less than optimal, especially for nanoparticles loaded with both the antibiotics, we proceeded to use these formulations,
Conclusions
Intracellular trafficking studies show that PLGA nanoparticles efficiently concentrate in inclusions in both acute and persistent C trachomatis infections. Encapsulation of antibiotics, azithromycin and rifampin, either singly or in combination, improved their effectiveness in reducing the infectious load of chlamydia based on inclusion size and numbers. Our studies suggest that enhanced intracellular and intra-inclusion accumulation, in combination with sustained drug release, contributes to
Acknowledgments
Funding from NIH (R01AI080928).
References (36)
- et al.
Prevention of pelvic inflammatory disease by the control of C. trachomatis infection
Int J Gynaecol Obstet
(2002) - et al.
Lower genital tract infection and endometritis: insight into subclinical pelvic inflammatory disease
Obstet Gynecol
(2002) - et al.
Fluorescence and electron microscopy probes for cellular and tissue uptake of poly(D, L-lactide-co-glycolide) nanoparticles
Int J Pharm
(2003) - et al.
Characterization of nanoparticle uptake by endothelial cells
Int J Pharm
(2002) - et al.
UEA I-bearing nanoparticles for brain delivery following intranasal administration
Int J Pharm
(2007) - et al.
Preparation of hemoglobin-loaded nano-sized particles with porous structure as oxygen carriers
Biomaterials
(2007) - et al.
Single-step surface functionalization of polymeric nanoparticles for targeted drug delivery
Biomaterials
(2009) - et al.
Preparation of stock solutions of macrolide and ketolide compounds for antimicrobial susceptibility tests
Clin Microbiol Infect
(2004) - et al.
Lipid rafts, caveolae, caveolin-1, and entry by Chlamydiae into host cells
Exp Cell Res
(2003)
Evidence of systemic dissemination of Chlamydia pneumoniae via macrophages in the mouse
J Infect Dis
Persistent Chlamydiae and chronic arthritis
Arthritis Res
Chlamydial persistence: beyond the biphasic paradigm
Infect Immun
Viability and gene expression in Chlamydia trachomatis during persistent infection of cultured human monocytes
Med Microbiol Immunol (Berl)
Expression of Chlamydia trachomatis genes encoding products required for DNA synthesis and cell division during active versus persistent infection
Mol Microbiol
Trafficking from CD63-positive late endocytic multivesicular bodies is essential for intracellular development of Chlamydia trachomatis
J Cell Sci
Cytoplasmic lipid droplets are translocated into the lumen of the Chlamydia trachomatis parasitophorous vacuole
Proc Natl Acad Sci USA
New insights into Chlamydia intracellular survival mechanisms
Cell Microbiol
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