U.S. patent application number 14/146537 was filed with the patent office on 2014-05-01 for nanoparticles for imaging and treating chlamydial infection.
This patent application is currently assigned to Wayne State University. The applicant listed for this patent is Wayne State University. Invention is credited to Alan P. Hudson, Jayanth Panyam, Judith A. Whittum-Hudson.
Application Number | 20140120038 14/146537 |
Document ID | / |
Family ID | 40282132 |
Filed Date | 2014-05-01 |
United States Patent
Application |
20140120038 |
Kind Code |
A1 |
Panyam; Jayanth ; et
al. |
May 1, 2014 |
NANOPARTICLES FOR IMAGING AND TREATING CHLAMYDIAL INFECTION
Abstract
Compositions of nanoparticles and targeting moieties for imaging
and treating Chlamydial infection are provided, including
nanoparticles conjugated to folic acid and comprising at least one
antibiotic effective against Chlamydia.
Inventors: |
Panyam; Jayanth; (Plymouth,
MN) ; Whittum-Hudson; Judith A.; (Novi, MI) ;
Hudson; Alan P.; (Novi, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Wayne State University |
Detroit |
MI |
US |
|
|
Assignee: |
Wayne State University
Detroit
MI
|
Family ID: |
40282132 |
Appl. No.: |
14/146537 |
Filed: |
January 2, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12670191 |
Jan 22, 2010 |
8647673 |
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PCT/US2008/070901 |
Jul 23, 2008 |
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14146537 |
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60951643 |
Jul 24, 2007 |
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Current U.S.
Class: |
424/9.6 ;
514/254.11; 514/29 |
Current CPC
Class: |
A61K 31/49 20130101;
A61K 47/6935 20170801; A61K 31/519 20130101; A61K 47/22 20130101;
A61K 31/519 20130101; A61K 31/49 20130101; A61K 31/7052 20130101;
A61K 31/496 20130101; A61K 47/6921 20170801; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 47/6937 20170801; A61K 47/551
20170801 |
Class at
Publication: |
424/9.6 ; 514/29;
514/254.11 |
International
Class: |
A61K 47/22 20060101
A61K047/22; A61K 47/48 20060101 A61K047/48; A61K 49/00 20060101
A61K049/00; A61K 31/7052 20060101 A61K031/7052; A61K 31/496
20060101 A61K031/496 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under grants
AR 42541, Al 44493 and AR 48331 awarded by the National Institutes
of Health. The government has certain rights in the invention.
Support was also provided by the Wilson Foundation and
Nanotechnology Enhancement funding from OVPR.
Claims
1-5. (canceled)
6. A composition comprising nanoparticles conjugated with folic
acid, wherein said particles comprise a first antibiotic in a form
and in a dosage suitable for treatment of a persistent
intracellular Chlamydia infection wherein Chlamydia of said
infection lack ftsK transcription or lack ftsK and ftsW
transcription at the time the composition is administered.
7. The composition of claim 6 comprising nanoparticles conjugated
with folic acid, wherein said particles further comprise one or
more other antibiotic(s) active against persistent intracellular
Chlamydia infection.
8. The composition of claim 6, wherein said first antibiotic is
selected from the group consisting of azithromycin, amoxicillin,
rifampicin, erythromycin, erythromycin ethylsuccinate, ofloxacin,
levofloxacin doxycycline, and tetracycline.
9. The composition of claim 7, wherein said one or more other
antibiotics is selected from the group consisting of azithromycin,
amoxicillin, rifampicin, erythromycin, erythromycin ethylsuccinate,
ofloxacin, levofloxacin doxycycline, and tetracycline, and said one
or more other antibiotics is different from said first
antibiotic.
10-12. (canceled)
13. A composition for imaging persistent intracellular Chlamydia
infection in a cell, comprising nanoparticles conjugated with folic
acid, wherein said nanoparticles further comprise at least one
detectably labeled moiety and wherein Chlamydia of said infection
lack ftsK transcription or lack ftsK and ftsW transcription at the
time of imaging.
14. The composition of claim 13, wherein said nanoparticles are
fluorescently labeled.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a 371 national phase of International
Application No. PCT/US2008/070901 filed on Jul. 23, 2008, which is
a non-provisional of U.S. Provisional Application No. 60/951,643
filed on Jul. 24, 2007, which applications are incorporated by
reference in their entirety herein.
TECHNICAL FIELD
[0003] The present invention is directed to compositions of
nanoparticles and targeting moieties for imaging and treating
Chlamydial infection.
BACKGROUND OF THE INVENTION
[0004] All chlamydial species are obligate intracellular bacterial
parasites of eukaryotic cells, and all are pathogenic to their
various hosts. In addition to their known etiologic roles in
elicitation of various acute diseases, the human pathogens C.
trachomatis (CT) and C. pneumoniae (CP) have been shown to cause,
or are strongly associated with, diverse chronic clinical entities.
Such conditions are often caused by persistent infection.
[0005] Many studies indicate, however, that existing antimicrobial
drugs are ineffective against such persistent chlamydial
infections, and neither C. trachomatis nor C. pneumoniae requires
an animal reservoir for maintenance. Because persistent chlamydial
infections are highly prevalent and can have severe chronic disease
sequelae, treatments to eradicate such persistent infections are
urgently needed.
BRIEF SUMMARY OF THE INVENTION
[0006] A method of treating a Chlamydia bacterial infection in a
patient in need of such treatment is provided, comprising
administering to the patient a therapeutically effective amount of
nanoparticles comprising at least one antibiotic agent, wherein the
antibiotic agent is effective against Chlamydia. The antibiotic may
be selected from the group consisting of azithromycin, amoxicillin,
rifampicin, erythromycin, erythromycin ethylsuccinate, ofloxacin,
levofloxacin doxycycline, and tetracycline, and the nanoparticles
comprise folic acid, such as in a form conjugated to the
nanoparticle surface. The Chlamydia may be selected from the group
consisting of C. trachomatis and C. pneumoniae and other species
such as C psittaci, C pecorum, C caviae or C suis.
[0007] Also provided is a composition comprising nanoparticles
conjugated with folic acid, wherein the particles comprise a first
antibiotic in a form and in a dosage suitable for treatment of a
Chlamydia infection. The nanoparticles conjugated with folic acid
or other targeting moieties may further comprise one or more other
antibiotic(s) active against Chlamydia. The first antibiotic may be
selected from the group consisting of azithromycin, amoxicillin,
rifampicin, erythromycin, erythromycin ethylsuccinate, ofloxacin,
levofloxacin doxycycline, and tetracycline. The one or more other
antibiotics may be selected from the group consisting of
azithromycin, amoxicillin, rifampicin, erythromycin, erythromycin
ethylsuccinate, ofloxacin, levofloxacin doxycycline, and
tetracycline, wherein the one or more other antibiotics is
different from the first antibiotic.
[0008] Further provided is a method for inhibiting the growth of C.
pneumoniae or C. trachomatis comprising contacting C. pneumoniae or
C. trachomatis, or a cell infected by C. pneumoniae or C.
trachomatis, with a composition comprising folic acid-conjugated
nanoparticles and one or more antibiotics. The antibiotics may be
selected from the group consisting of azithromycin, amoxicillin,
rifampicin, erythromycin, erythromycin ethylsuccinate, ofloxacin,
levofloxacin doxycycline, and tetracycline, or newer generations of
one or more of these antibiotics.
[0009] Also provided is a method of imaging Chlamydia infection in
a mammal, comprising the steps of administering to the mammal a
composition comprising nanoparticles comprising folic acid and at
least one imaging agent, and detecting the nanoparticles in the
mammal, wherein the particles target folic acid receptor-expressing
cells infected with Chlamydia. The nanoparticles may be
fluorescently labeled.
[0010] Also provided is a composition for imaging Chlamydia
infection in a cell, comprising nanoparticles conjugated with folic
acid, wherein the nanoparticles further comprise at least one
detectably labeled moiety, such as a fluorescent label.
BRIEF DESCRIPTION OF THE FIGURES
[0011] Exemplary embodiments are illustrated in referenced figures.
It is intended that the embodiments and figures disclosed herein
are to be considered illustrative rather than restrictive.
[0012] FIG. 1 shows RT-PCR analysis of relative transcript levels
encoding the three folic acid receptor (FR) subtypes in
chlamydia-infected and uninfected cells. Results are presented as
fold-increase in receptor expression in infected cells compared to
uninfected cells for each subtype.
[0013] FIG. 2 shows quantitative analysis of nanoparticle uptake in
chlamydia-infected and uninfected cells. Cells were treated with
nanoparticles with or without folic acid (FANP, NP, respectively)
and nanoparticle uptake quantitated by HPLC. Uptake normalized to
total cell protein. *P<0.05
[0014] FIGS. 3A and B shows images demonstrating the homing of
labeled nanoparticles to infected host cells, using the RTM-3
microscope. Panel A, visible light image of a 24 hr-infected HEp-2
cell pulsed with particle. Panel B, the same cells viewed under
epifluorescence. The inclusion is indicated by arrows.
[0015] FIG. 4A-C shows images demonstrating delivery of NP
containing DNA to cultured HEP-2 cells. Green indicates the
presence of NP; red/yellow red indicates DNA released from NP
inside inclusions (arrows). FIG. 4A shows 72 hours post-infection,
and FIG. 4B shows 48 hours post-infection. FIG. 4C shows two Z cuts
of the same field.
[0016] FIGS. 5A and 5B. McCoy cells infected with 10.sup.4 IFU/well
C. trachomatis (serovar K), and treated immediately following
infection with serial two-fold dilutions of azithromycin or
rifampin to a final concentration range of 31-2000 ng/ml and 1-64
ng/ml, respectively, and incubated for 24 or 48 hrs. Both
azithromycin and rifampin, showed no significant change in
MIC.sub.50 with length of treatment.
[0017] FIGS. 6A-C. McCoy cells with 10.sup.4 IFU/well C.
trachomatis (serovar K) and treated immediately following infection
with serial two-fold dilutions of azithromycin or rifampin
encapsulated nanoparticles to a final concentration range of
31-2000 ng/ml and 1-64 ng/ml respectively and incubated for 24 or
48 hrs post infection. The mean inclusion number was plotted as a
function of concentration. Both azithromycin and rifampin
encapsulated nanoparticles are as effective as individual free
drug.
[0018] FIG. 7A-C. McCoy cells (2.times.10.sup.5/well) infected with
10.sup.4 IFU/well C. trachomatis (serovar K), then treated at 0, 24
or 48 hours post infection with serial two-fold dilutions of
rifampin. The cells were incubated for 24 hours in the presence of
drug and then fixed. The final concentration range was 2.5-10 ng/ml
for rifampin. Statistical significance based on pair-wise rank sum
test is shown.
[0019] FIG. 8A-C. McCoy cells (2.times.10.sup.5/well) infected with
10.sup.4 IFU/well C. trachomatis (serovar K), then treated at 0, 24
or 48 hours post infection with serial two-fold dilutions of
azithromycin. The cells were incubated for 24 hours in the presence
of drug and then fixed. The final concentration range was 25-100 ng
for azithromycin. Statistical significance based on pair-wise rank
sum test is shown.
[0020] FIGS. 9A and 9B. McCoy cells (2.times.10.sup.5/well) were
infected with 10.sup.4 IFU/well C. trachomatis (serovar K), then
treated at 0 or 24 hours post infection with serial two-fold
dilutions of rifampin. The cells were incubated for 24 hours in the
presence of drug and then fixed. The final concentration range for
rifampin was 2.5-80 ng/ml and for azithromycin was 25-800 ng/ml.
Improved sensitivity was observed at 40 and 80 ng/m. A 50%
reduction in inclusion number was observed for rifampin
nanoparticles compared to free drug at 40 ng/ml.
[0021] FIGS. 10A and 10B. McCoy cells (2.times.10.sup.5/well) were
infected with 10.sup.4 IFU/well C. trachomatis (serovar K), then
treated at 0 or 24 hours post infection with serial two-fold
dilutions of azithromycin. The cells were incubated for 24 hours in
the presence of drug and then fixed. The final concentration range
for azithromycin was 25-800 ng/ml. Inclusion number expressed as
percent of control was plotted as a function of concentration.
There was no improvement in response to drug using drug-loaded
nanoparticles compared to free drug when treatment was added at 24
hpi (hours post-infection).
[0022] FIGS. 11A and 11B. McCoy cells (2.times.10.sup.5/well) were
seeded onto 96-well microtiter plates and then infected the next
day with 10.sup.4 IFU/well C. trachomatis (serovar K). The cells
were treated at 24 hours post infection with serial two-fold
dilutions of rifampin. The cells were incubated for 24 hours or 48
hours in the presence of drug and then fixed. When drug was added
at 24 hpi and left on for 24 hours, there was no difference in
sensitivity between cells treated with nonFA or FA targeted
nanoparticles. Both showed an MIC.sub.50 between 40 and 80 ng/ml.
When drug was added at 24 hpi and left on for an additional 48
hours, there was an enhanced sensitivity compared with 24 hour drug
exposure. The MIC50 shifted to 20-40 ng/ml. A significant change in
slope at 10 ng/ml suggests that FA targeted nanoparticles improved
sensitivity over drug encapsulated nanoparticles alone.
[0023] FIGS. 12A and 12B. McCoy cells (2.times.10.sup.5/well) were
seeded onto 96-well microtiter plates and then infected the next
day with 10.sup.4 IFU/well C. trachomatis (serovar K). The cells
were treated at 24 hours post infection with serial two-fold
dilutions of azithromycin. The cells were incubated for 24 hours or
48 hours in the presence of drug and then fixed. When drug was
added at 24 hpi and then left on for 24 hours there was a minimal
response with non FA nanoparticles, however, the MIC.sub.50 was
still greater than 800 ng/ml. In contrast when drug was added at 24
hpi and left on for 48 hours both targeted and non targeted
nanoparticles showed enhanced sensitivity with an MIC.sub.50 near
to 800 ng/ml.
[0024] FIG. 13 shows .sup.1H NMR spectrum of PEG-FA conjugated PLGA
nanoparticles (PEG: CH.sub.2 at 3.59 ppm; PLA: CH at 1.25 ppm and
CH.sub.3 at 5.22 ppm; and folic acid: aromatic protons at 6.62 and
7.61 ppm and aliphatic amide proton at 8.1 ppm).
[0025] FIG. 14. SPR analysis of folic acid (FA) conjugated
nanoparticles (solid line) on anti-folic acid monoclonal antibody
coated surface. The anti-folate monoclonal antibody (Chemicon) was
amine coupled to the sensor chip and nanoparticle formulations were
injected at a concentration of 10 mg/ml. Nanoparticles without
folic acid (dashed line) as control.
[0026] FIGS. 15A-C. Shown in FIGS. 15A-C are high resolution images
of a McCoy cell from cultures infected and grown in the presence of
PenG. Cells are unstained and captured under real time microscopy
using the RTM-3 microscope with Richardson contrast (A), DIC-like
mode (B), and fluorescence mode (C). IFN.gamma. treatment induced
smaller inclusions filled with atypical large RB at 48 hr p.i.;
untreated, infected cultures had large inclusions primarily filled
with EB (not shown). 100.times. original mag., RTM3 microscope.
[0027] FIG. 16. Representative DnaA and FtsW PCR results for two
mice originally positive for 16S rRNA, plasmid and/or omp1 as
described above (upper vs lower lanes) are shown in this agarose
gel: Lanes contain: 1, 100 bp Std; 2, +ctrl DNA; PCR for dnaA; 3,
-ctrl, water with dnaA; 4, +ctrl, DNA, PCR for ftsW; 5, -ctrl for
ftsW; 6, Knee A, cDNA, RT-PCR for dnaA; 7, Knee A, cDNA, RT-PCR for
ftsW; 8, Knee B, cDNA, dnaA; 9, Knee B, cDNA, ftsW. These results
clearly demonstrate that dnaA remains positive while ftsW has been
shut off. Positive controls (acutely infected McCoy cells) for the
two products were run in the same experiments. .beta.-actin was
used for normalization
[0028] FIG. 17. Images demonstrating targeting of folic
acid-conjugated nanoparticles to infected host cells, using the
Kodak imaging system. Panel A, fluorescence image of an infected
mouse injected intravenously with 6 coumarin-folic acid-conjugated
particles. Panel B, the same mouse viewed under X-ray. The joint
tissue showing accumulation of nanoparticles is indicated by
arrows.
[0029] FIG. 18. Accumulation of folic acid-conjugated nanoparticles
in infected host tissue. Nanoparticles with folic acid on the
surface (FANP) or without (NP) were injected intravenously in an
infected mouse. NP concentration in the different tissues was
determined by HPLC. *P<0.05
[0030] FIGS. 19A-E. Antibiotic delivery in nanoparticles
effectively reduces Chlamydial viability. McCoy cells were infected
with C trachomatis serovar K at MOI=1. After adsorption, at the
time of overlay medium addition, different concentrations of
rifampin (R), azithromycin (Z) or combination of both drugs (Z+R)
were also added. Drugs were administered either encapsulated in
nanoparticles (top panel) or dissolved in growth medium (bottom
panel). Chlamydial viability was determined by counting the number
of inclusions/well. Infected cells without either drug yielded a
mean of .about.550 IFU (top) and 632 IFU/well (bottom).
Concentrations for Z+R=Z (loading was 5 .mu.g/ml with 6 .mu.g/ml R)
(top panels). Free drugs in combination are compared to single drug
(open symbols, bottom panels).
[0031] FIGS. 20A and 20B. FIG. 20 shows free and
nanoparticle-encapsulated antibiotics added at either t0 (time of
infection) or after 24 hr of infection (t24), and included for
controls blank nanoparticles.
[0032] FIG. 21. Rifampin release from nanoparticles in phosphate
buffered saline (pH 7.4) containing 0.1% Tween 80 and 0.1% N-acetyl
cysteine to maintain sink conditions and stability, respectively.
Released drug was quantitated by HPLC. Data are expressed as
mean.+-.S. D.
DESCRIPTION OF THE INVENTION
[0033] The urgent need for improved methods and compositions for
localizing (imaging) and treating Chlamydia infection and its
serious sequelae, including arthritis, blindness, COPD, and many
others, is addressed herein using nanoparticles conjugated with
folic acid, wherein the nanoparticles target Chlamydia-infected
cells. The need is due in part to the persistence of Chlamydia in
the body and the previous inability to specifically locate and
treat such persistent infections. Both C. trachomatis and C.
pneumoniae can disseminate widely from their sites of primary
infection and cause significant long-term disease consequences. For
the most part, those consequences result from a powerful
immunopathogenic response to the organisms, as well as from the
terminal host cell lysis/scarring at the end of the developmental
cycle.
[0034] As with all chlamydiae, C. trachomatis and C. pneumoniae
undergo an unusual biphasic developmental cycle. The cycle is
initiated when elementary bodies (EB), the infectious extracellular
form of the organism, attach to the target host cell. Once bound,
EB are brought into a membrane-bound cytoplasmic inclusion within
which they spend their intracellular tenure. In the inclusion EB
develop into reticulate bodies (RB), the growth form. Each RB
undergoes seven or eight rounds of cell division, after which most
dedifferentiate back to EB. Newly-formed EB are released by host
cell lysis or exocytosis (Hatch, T. P., 1999, In:
Chlamydia--Intracellular Biology, Pathogenesis, and Immunity,
Stephens, R. S., Ed. ASM Press, Washington D.C., pp. 29-67).
[0035] Many studies have demonstrated that both C. trachomatis and
C. pneumoniae often disseminate widely from their sites of primary
infection; when they do so, these organisms can take up long-term
residence at distant anatomic locations (Moazed T. C., et al.,
1998, J. Infect. Dis. 177:1322-1325; Villareal C., et al., 2002,
Arthritis Res. 4:5-9). At sites of their disseminated residence,
both organisms enter an unusual biological state referred to as
`persistence` (Villareal C., et al., 2002, Arthritis Res. 4:5-9;
Hogan, R. J., et al., 2004, Infect. Immun. 72:1843-1855). 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 (Byrne,
G. I., et al., 2001, Infect. Immun. 69:5423-5429; Gerard, H. C., et
al., 2001, Mol. Microbiol. 41:731-741). The means by which
persistently infecting chlamydiae engender pathology is not well
understood, but it is clear that they can elicit a powerful
immunopathogenic response that can contribute to chronic diseases
discussed herein.
[0036] Although trachoma largely disappeared in developed nations
during the twentieth century, mainly because of improved public
sanitation and availability of clean water, trachoma remains a
significant disease in rural regions of many parts of the world,
including the Middle East, Africa, southeast Asia, and the Indian
subcontinent.
[0037] Chlamydia psittaci (also referred to as Chlamydophila
psittaci) is a bacterium that can be transmitted from birds, such
as pet birds, to humans, causing psittacosis. Beginning with a
flu-like set of symptoms, the disease may progress to severe
pneumonia and other non-respiratory problems. Persons at risk of
contracting the disease include veterinarians, zoo workers, and
poultry industry employees, such as turkey handlers at risk for
pneumonia and systemic disease. (Fenga, C. et al., 2007, Ann Agric.
Environ. Med. 14:93-96.) Suitable treatments and preventive
measures include administering compositions of the invention to
birds and to humans.
[0038] In the developing world, trachoma is still the primary cause
of treatable/preventable blindness (Mabey, D. C., et al., 2003.
Lancet, 362, 223-229; West, S. K., 2004. Prog. Ret. Eye Res., 23,
381-401). Estimates of the prevalence of the disease world-wide
suggest that nearly six million individuals currently are blind
from trachoma, and perhaps twice that number are at significant
risk for blindness (West, S. K., 2004. Prog. Ret. Eye Res., 23,
381-401); as many as 150 million people world-wide are affected in
some way by the disease (Kumaresan, J. A., et al., 2003. Am. J.
Trop. Med. Hyg., 69(5 suppl.), 24-28). One estimate of the overall
economic impact of trachoma gives a figure of $2.9 billion annual
loss overall in productivity due to this disease (Kumaresan, J. A.,
et al, 2003. Am. J. Trop. Med. Hyg., 69(5 suppl.), 24-28). Although
now confined primarily to the developing world, trachoma remains a
disease of major importance, and it currently is a focus of
intensive eradication efforts.
[0039] C. pneumoniae is responsible for a large proportion of cases
of community-acquired pneumonia (Grayston, J. T., et al., 1992.
Annu. Rev. Med., 43, 317-323). The incidence of seroconversion is
low in children, but rises steeply after childhood. In the US,
about 50% of 20 year-olds show evidence of prior infection (CDC
website); by the seventh decade, seropositivity rates approach
80-90% (Leinonen, M., et al., 1993. Eur. Heart J., 14(suppl.K),
57-61).
[0040] Importantly, C. pneumoniae has been associated with several
more severe pulmonary diseases, including sarcoidosis, chronic
obstructive pulmonary disease (COPD), arthritis, late-onset
Alzheimer's disease, multiple sclerosis, giant cell arteritis, and
others (for example, Balin, B. J., et al., 1998; Sriram, S., et
al., 1999 Ann. Neurol., 46, 6-14; Swanborg, R. H., et al., 2003. J.
Neuroimmunol., 136, 1-8). Given the high levels of adult
seroposivity to C. pneumoniae observed in all studies, the costs
associated with the disease must be substantial.
[0041] Cardiovascular disease is the leading cause of death in
essentially all developed nations. In the late 1980's, a group in
Finland published a landmark study indicating an association
between C. pneumoniae infection and heart disease (Saikku, P., et
al., 1988. Lancet, 2, 983-986). This association is now reasonably
well-accepted in the medical and scientific communities, and it is
particularly focused on atherosclerosis, a critical contributing
factor to cardiovascular disease (reviewed in Belland, R. J., et
al., 2004. Cell. Microbiol., 6, 117-127; and Campbell, L. A., et
al., 2004. Nat. Rev. Microbiol., 2, 23-32). The organism has been
identified in atherosclerotic plaques by several standard screening
techniques and by many independent laboratories (Ciervo, A., et
al., 2003. Mol. Cell. Probes., 17, 107-111). A biochemical
mechanism by which C. pneumoniae may contribute to plaque formation
has been defined (Kalayoglu, M. V., et al., Byrne G. I., 1998b.
Infect. Immun., 66, 5067-5072).
[0042] As a leading cause of death and incapacity in developed
nations, cardiovascular disease imposes an enormous burden on the
health care systems of all those countries. For just one example, a
recent study from the Netherlands indicated that the cost of
treatment for a single affected individual can be a high as
C=380000 (Groot, et al., 2004. Health Econ., 13, 850-872). This
does not include the costs of productivity losses attributable to
affected individuals. Thus, it is feasible that C. pneumoniae
infection alone may be responsible for an exceptionally large
proportion of health care costs in developed nations.
[0043] Sexually-transmitted infections are among the most common
diseases world-wide, and C. trachomatis is almost certainly the
most common sexually-transmitted bacterial infection among them.
One report estimated that in 1995, 89 million people between 15 and
45 years of age globally had a chlamydial infection of the
urogenital tract (Gerbase, A. C., et al., 1998. Sex. Transm.
Infect., 74(suppl. 1), S12-S16). In the United States, genital
infections by C. trachomatis must be reported to the Centers for
Disease Control (CDC) by each of the fifty states and the District
of Columbia. In 2003, almost 900,000 new cases of C. trachomatis
infection were reported by the states and the District together
(Centers for Disease Control and Prevention, 2004). Estimates from
various sources range as high as 4 million active cases of genital
C. trachomatis at any one time in the U.S. Genital infections by C.
trachomatis are of particular concern for women due to the
potential long-term reproductive sequelae that they can
engender.
[0044] Estimates of the prevalence of genital chlamydial infection
in Europe appear to be equivalent, or even somewhat higher, than
those for the U.S. For example, a recent study showed a prevalence
of 5.9% for genital C. trachomatis infection among men 17-35 years
of age in Ireland (Powell, J., et al., 2004. Sex. Transm. Infect.,
80, 349-353); a similar prevalence was identified among young women
screened in three cities in Scotland. In England, where a National
Chlamydia Screen Program was initiated in 2002 for men and women
under 25 years of age, the first year's data indicated a
10.1.degree.)/0 prevalence among women tested and a 13.3%
prevalence among men (LaMontagne, D. S., et al., 2004. Sex. Transm.
Infect., 80, 335-341). In Eastern Europe, estimates of genital
chlamydial infection among young women give values of 4.5% or
higher (Masata, J., et al., 2004. Deak, J. (Ed.) Proceedings of the
Fifth Meeting of the European Society for Chlamydia Research,
Pauker Nyomdaipari Kft, Budapest, Hungary).
[0045] The standard treatment for active primary urogenital
infection with C. trachomatis is a course of doxycycline or
erythromycin or a single dose of azithromycin, and these have
proved to be effective in eradicating organisms (Tobin, J. M., et
al., 2004, Intl J. STD AIDS 15:737-739); treatment with
azithromycin has also proved effective for active pulmonary C.
pneumoniae infection. Unlike treatment of active chlamydial
infections, antimicrobial therapy of persistent infections by
either organism is problematic. For example, either species
infecting joint tissues in arthritis patients respond poorly or not
at all to standard treatment with antibiotic because they reside in
those tissues in the persistent state (Whittum-Hudson J. A., et
al., 2006, In: Chlamydia--Genomics and Pathogenesis, Bavoil P.,
Wyrick P., Eds., pp. 475-504 (Horizon Bioscience Press (Norfolk, U.
K.)).
[0046] The most important sequelae to genital C. trachomatis
infection include PID, salpingitis, occlusion of the fallopian
tubules, and other fertility-abolishing or -diminishing problems in
women; epididymitis can follow genital infection in men. Recent
estimates indicate that about 8% of women with lower genital tract
infection with C. trachomatis progress to PID; this number appears
to account for half of all PID cases (Honey, E., et al., 2002.
Intl. J. Gynecol. Obstet., 78, 257-261).
[0047] As importantly, nearly two thirds of women with infertility
due to occluded fallopian tubes show antibodies against this
organism; C. trachomatis may be the causative agent in up to 40% of
ectopic pregnancies (Honey, E., et al., 2002. Intl. J. Gynecol,
Obstet., 78, 257-261). Other reports indicate that genital C.
trachomatis is the most common cause of acute salpingitis, and that
perhaps 25% of women with the acute disease become infertile
(Guaschino, S., et al., 2000. Ann. N. Y. Acad. Sci., 900, 293-300).
The incidence of new cases of PID and other reproductive problems
due to genital chlamydial infection is high among urban young women
in the U.S. (Kelly, A. M. et al., 2004. J. Pediatr. Adolesc.
Gynecol., 17, 383-388) and in Europe (Grio, R., et al., 2004.
Minerva Ginecol., 56, 141-147).
[0048] Genital infection with C. trachomatis also can elicit a
painful inflammatory arthritis in both men and women, similar to
the arthritis that can follow C. pneumoniae infection
(Whittum-Hudson, J. A., et al. 2006. Pathogenesis of
Chlamydia-associated arthritis, in Bavoil, P., Wyrick, P. (Eds.),
(supra); Whittum-Hudson, J. A., et al., 2005. Chlamydia pneumoniae
and inflammatory arthritis, in Yamamoto, Y., Friedman, H.,
Bendinelli, M. (Eds.), Chlamydia pneumoniae Infection and Diseases.
NY, Kluwer/Academic Press, 227-238). The prevalence of acute C.
trachomatis-induced arthritis has not been firmly established, but
conservative estimates from the United States and elsewhere suggest
it to be in the range of 5-10% of individuals with a documented
prior genital infection with the organism (Rich, E., et al. 1996.
Arthritis Rheum., 39, 1172-1177).
[0049] Nanoparticles provide a powerful new means for finding and
eradicating the sites of persistent Chlamydia infection that can
lead to the broad range of chronic health issues discussed above.
The term "nanoparticle" has been used to refer to nanometer-size
devices consisting of a matrix of dense polymeric network (also
known as nanospheres) and those formed by a thin polymeric envelope
surrounding a drug-filled cavity (nanocapsules) (Garcia-Garcia E,
(2005). Int J Pharm 298:274-92). Nanoparticles can penetrate into
small capillaries, allowing enhanced accumulation of the
encapsulated drug at target sites (Calvo P, et al. (2001). Pharm.
Res. 18:1157-66). Nanoparticles can passively target tumor tissue
through enhanced permeation and retention effect (Monsky W L, et
al. (1999). Cancer. Res. 59:4129-35; Stroh M, et al. (2005). Nat.
Med. 11:678-82).
[0050] Nanoparticles can be delivered to distant target sites
either by localized catheter-based infusion (Panyam J, (2002). J.
Drug Target. 10:515-523) or by attaching a ligand to nanoparticle
surface that has affinity for a specific tissue (Shenoy, D.,
(2005). Pharm Res 22:2107-14). Because of sustained release
properties, nanoparticles can prolong the availability of the
encapsulated drug at the target site, resulting in greater and
sustained therapeutic effect (Panyam J and Labhasetwar V (2003).
Adv Drug Deliv Rev 55:329-47).
[0051] "PEGylation" refers to the decoration of particle surface by
covalently grafting or adsorbing of PEG chains. The purpose of PEG
chains is to create a barrier to the adhesion of opsonins present
in the blood, so that delivery systems can remain longer in
circulation, invisible to phagocytic cells (Kommareddy S, (2005).
Technol Cancer Res Treat 4:615-26).
[0052] This disclosure is based in part on the important new
discovery herein that Chlamydia-infected cells overexpress folic
acid receptors. Thus, folic acid conjugates can be used to
specifically target Chlamydia-infected cells. Although the reduced
folate carrier is present in virtually all cells, folate-conjugates
are not substrates and are taken up only by cells expressing
functional folate receptors (Hilgenbrink A R et al., (2005a). J.
Pharm. Sci. 94:2135-46). Folic acid conjugation allows endocytic
uptake of the conjugated carrier via the folate receptor, resulting
in higher cellular uptake of an encapsulated drug or targeting
moiety. The high affinity of folic acid to its receptor (binding
constant .about.1 nm) and folate's small size make it ideal for
specific cell targeting. Furthermore, the ability of folic acid to
bind its receptor is not altered by covalent conjugation to
delivery systems (Lee, R. J. et al., (1994). J. Biol. Chem.
269:3198).
[0053] Effective treatments for active urogenital and ocular
infections by C. trachomatis are available in the form of various
antimicrobials. The standard treatment for urogenital infection
with C. trachomatis is a course of doxycycline or erythromycin, or
a single dose of azithromycin, and these have proved to be
effective in eradicating actively-growing organisms (Tobin, J. M.,
2004, Intl. J. STD ADIS, 15, 737-739 2004). Treatment with
azithromycin has also proved reasonably effective for active
pulmonary C. pneumoniae infection. Many studies indicate, however,
that existing antimicrobial drugs are ineffective against
persistent chlamydial infections, and neither C. trachomatis nor C.
pneumoniae requires an animal reservoir for maintenance. Thus, the
distribution of active and persistent chlamydial infections in any
given human population must be assessed before large-scale
treatment programs can be designed appropriately.
[0054] Unlike treatment of active C. trachomatis or C. pneumoniae
infections, antimicrobial treatment of persistent infections by
either organism await improved diagnostic and therapeutic agents
and methods, which are now provided by the present invention. For
example, Chlamydia of either species infecting joint tissues in
arthritis patients respond poorly or not at all to standard
treatment with a single antibiotic because they reside in those
tissues in the persistent, rather than the actively-growing state
(Whittum-Hudson, J. A., et al., 2006. Pathogenesis of
Chlamydia-associated arthritis, in Bavoil, P. Wyrick, P. (Eds.),
Chlamydia--Genomics and Pathogenesis, Horizon Bioscience Press,
Norfolk, England, 2006, Chapter 21.); (Whittum-Hudson, J. A., et
al., 2005. Chlamydia pneumoniae and inflammatory arthritis, in
Yamamoto, Y., Friedman, H., Bendinelli, M. (Eds.), Chlamydia
pneumoniae Infection and Diseases, NY, Kluwer/Academic Press,
227-238). In the case of atherosclerosis, it is not clear whether
C. pneumoniae resides in vessel tissues in the persistent or
actively-growing form or some mixture of each, but large-scale
antibiotic trials have failed to show improvement in patients with
aortic or other plaques (Grayston, J. T., et al., 2005, N. Engl. J.
Med., 352, 1637-1645). Using the imaging compositions and methods
disclosed herein, the location of C. pneumoniae can now be
investigated and the resulting atherosclerosis treated.
[0055] As shown in FIG. 1, folic acid receptor subtypes were
expressed at a higher level in Chlamydia-infected human or mouse
cells. By showing that Chlamydia-infected cells overexpress folic
acid receptors, this disclosure provides a new mechanism for
finding, imaging, and treating previously unidentifiable persistent
infections.
[0056] FIG. 2 shows that nanoparticles derivatized with folic acid
accumulated more in Chlamydia-infected HEp-2 cells than in
non-infected cells. Nanoparticles also targeted inclusions within
Chlamydia-infected cells. As described in detail in the Examples,
and as shown in FIG. 3, HEp-2 cells infected with Chlamydia were
pulsed with labeled nanoparticles and examined under
epifluorescence using a RTM-3 microscope. This discovery was then
used to deliver plasmid DNA into Chlamydial inclusions (FIG. 3).
HEp-2 cells were infected with Chlamydia (CT) and then pulsed with
nanoparticles containing plasmid DNA (FIG. 4). Delivery to
inclusions was imaged using separate fluorescent markers on the DNA
and the nanoparticles. The results are discussed in more detail in
the Examples.
[0057] The relevance of these in vitro studies to imaging and
treating true Chlamydia infection was demonstrated in mouse models
of infection. As described in the Examples, folic acid-derivatized
nanoparticles targeted Chlamydial infected sites in mice at day 8
and day 11 of infection. Infection was undetectable using
nanoparticles without folic acid. Sites of infection imaged were
the genital tract (ex vivo) and joints (knees and paws/ankles);
spleen, kidney and the liver were imaged ex vivo as well.
[0058] Nanoparticles formulated using a FDA-approved, biodegradable
polymer PLGA were used in the disclosed studies. Applicants'
previous studies demonstrated that PLGA nanoparticles are non-toxic
and biocompatible (J. Panyam, et al. Int J Pharm 262: 1-11 (2003),
and are suitable for in vivo drug delivery (J. Panyam, et al. J
Drug Target 10: 515-23 (2002). Applicants previously showed that
nanoparticles can efficiently encapsulate and sustain the release
of hydrophobic drugs like dexamethasone (J. Panyam, et al. J Pharm
Sci 93: 1804-14 (2004) and paclitaxel, and nucleic acids (S.
Prabha, et al. Int J Pharm 244: 105-15 (2002). An important
advantage of PLGA nanoparticles is that the rate of drug/nucleic
acid release from nanoparticles, and therefore, the therapeutic
efficacy, can be controlled by varying the polymer properties such
as molecular weight, lactide-glycolide ratio and end-group
chemistry (J. Panyam, et al. Mol Pharm 1:77-84 (2004); (S. Prabha,
et al. Pharm Res 21: 354-64 (2004).
[0059] In summary, the data disclosed herein demonstrate that
nanoparticles can be targeted to cells infected with Chlamydia
using folic acid. The invention provides for new methods of
detection and treatment using, for example, antibiotics, small
molecules, antibodies, and polynucleotides.
[0060] Suitable antibiotics include those previously demonstrated
to be effective against one or more form of Chlamydia infection,
such as azithromycin, amoxicillin, rifampicin, erythromycin,
erythromycin ethylsuccinate, ofloxacin, levofloxacin doxycycline,
and tetracycline. A polynucleotide such as DNA can be incorporated
in the nanoparticles to affect the life cycle of the organism, for
example as an antisense treatment. Although it has not been
developed in the present context, all chlamydial species are
difficult to work with in in vitro systems because no genetic
modification system is currently available for these organisms.
That is, while the genomes of many, perhaps most, bacterial
pathogens can be manipulated by various means so as to assess the
function of particular genes or sets of genes of interest, no such
system currently exists for chlamydiae. Development of a system for
genetic manipulation of CT or CP or both would allow elucidation of
the function(s) of the many genes in the genome of each organism
for which no function is known. Such elucidation would almost
certainly lead to additional means to eradicate active human
chlamydial infections, and it would probably provide new strategies
for obviation of persistent infections by these organisms. The
general means of genetic manipulation involves delivering
oligonucleotides or other constructs to the living organism of
interest, the result of which is most often intended to be
attenuation of expression of the gene(s) of interest. In the
present context, we will load the nanoparticles with
oligonucleotides or other constructs to affect expression of
chlamydial genes that we suspect will disallow completion of the
intracellular developmental cycle and/or obviate production of
chlamydial molecules involved intimately in the pathogenesis
process.
[0061] Guidance for doses and dosing regimens are well-known to the
practitioner, and will be adapted for use in the context of the
nanoparticle administration methods herein. For example,
azithromycin can be given at a dose of 1 g orally as a single dose.
Doxycycline can be given 100 mg orally twice a day for 7 days.
Erythromycin can be given 500 mg orally four times a day for 7
days; erythromycin ethylsuccinate can be given 800 mg orally four
times a day for 7 days; oflaxicin can be given 300 mg orally twice
a day for 7 days; and levofloxacin can be given 500 mg orally for 7
days.
[0062] Several imaging modalities can be used to image chlamydial
infection. As described in the Examples, fluorescently-labeled
folate conjugates can be used for optical imaging of chlamydial
infections. Radiolabeled folate conjugates can be used in
conjunction with CT scanning to image chlamydial infection (Muller
C, et al, J Nucl Med. 2006 December; 47(12):2057-64). Folic acid
can also be conjugated to agents like gadolinium or
superparamagnetic nanoparticles for magnetic resonance imaging
(MRI) of chlamydial infections (Sun C et al, J Biomed Mater Res A.
2006 Sep. 1; 78(3):550-7).
[0063] Human infections by the intracellular bacterial pathogens
Chlamydia trachomatis and C pneumoniae present an enormous health
care problem. Infections by these pathogens have been associated
with engendering and/or exacerbating several chronic diseases, and
some of these Chlamydial infections have proved to be refractory to
antibiotic therapy. The lack of therapeutic efficacy results from
the attenuated metabolic rate of infecting chlamydiae under some
circumstances, in combination with the modest intracellular
concentrations achievable by normal delivery of such drugs to the
inclusions within which chlamydiae reside in the host cell
cytoplasm.
[0064] The major therapeutic goal of the disclosure herein provides
a means by which antibiotics or other therapeutic agents can be
delivered in a targeted manner to the intracellular Chlamydial
inclusion at effective concentrations, without toxicity to the host
cell or infected tissue. Chlamydial infection elicits increased
expression of host cell folic acid receptors (FAR), and that folic
acid-conjugated nanoparticles provide a novel and highly effective
means of intracellular delivery of therapeutic agents to
Chlamydia-infected cells. According to the present disclosure, host
cells infected with either C trachomatis or C pneumoniae can be
cleared of actively- or persistently-infecting organisms via
nanoparticle-mediated targeted delivery of effective concentrations
of antibiotics known to work against active Chlamydial
infections.
[0065] Human infections by the intracellular bacterial pathogens
Chlamydia trachomatis and C pneumoniae present an enormous burden
to the US health care system. The former is the most prevalent
sexually transmitted bacterium in developed nations. In the US,
recent data indicate that there are an estimated 2-4 million new
STD cases annually caused by C trachomatis. C pneumoniae is a
respiratory pathogen responsible for a significant proportion of
community-acquired pneumonia.
[0066] Both C trachomatis and C pneumoniae can and often do
disseminate widely from their sites of primary infection. At
anatomic locations distant from those primary infection sites, both
organisms may enter an unusual biologic state designated
"persistence", and it is in this form that both have been strongly
associated with engendering chronic diseases, including
inflammatory arthritis, tubal occlusion leading to ectopic
pregnancy, and cervical cancer (C trachomatis); C pneumoniae has
been compellingly associated with atherosclerosis, inflammatory
arthritis, and temporal arteritis, among several other
diseases.
[0067] Primary infections with C trachomatis and C pneumoniae can
usually be treated effectively with antibiotics. However, for
reasons that remain to be fully elucidated, persistent infections
by both organisms have proved to be refractory to such treatments.
In large part, the lack of therapeutic efficacy results from the
attenuated metabolic rate of persistently infecting Chlamydiae in
combination with the modest intracellular concentrations achievable
by normal delivery of such drugs to the inclusions within which
Chlamydiae reside in the host cell cytoplasm. The major therapeutic
goal, and the long-term goal of the disclosure herein, is to
provide means by which antibiotics or other therapeutic agents can
be delivered in a targeted manner to the intracellular Chlamydial
inclusion at effective concentrations, with minimal toxicity to the
host cell or infected tissue.
[0068] Chlamydial infection elicits increased expression of host
cell folic acid receptors (FAR), and folic acid-conjugated
nanoparticles provide a novel and highly effective means of
targeted intracellular delivery of therapeutic agents to
Chlamydia-infected cells. Without being limited to a specific
mechanism, the present disclosure is based in the finding that host
cells persistently infected with either C trachomatis can be
cleared of persistently-infecting organisms via
nanoparticle-mediated targeted delivery of effective concentrations
of antibiotics known to work against active Chlamydial infections.
Nanoparticle-facilitated delivery requires reduced amounts of
therapeutic materials for both acute and persistent infections,
thus engendering significant health care cost reductions.
[0069] All Chlamydial species are obligate intracellular bacterial
parasites of eukaryotic cells, and all are pathogenic to their
various hosts [1]. In addition to their known etiologic roles in
elicitation of various acute diseases, the human pathogens C
trachomatis and C pneumoniae have been shown to cause, or are
strongly associated with, diverse chronic clinical entities.
Trachoma is caused by repeated ocular infection with C
trachomatis.
[0070] As the leading cause of infectious blindness in humans
[1,2], half a billion people suffer from trachoma, and up to
one-fourth of those infected will become blind. Although trachoma
has largely disappeared from North America and Europe, extraocular
Chlamydial infections remain of great importance in the latter
areas; ie, 2-4 million new Chlamydial sexually transmitted
infections (STI) are reported each year in the US alone, with an
annual cost exceeding $1 billion. Chlamydial genital tract
infection is over 5 times more common than gonorrhea [3] and has
been correlated with increased risk of HIV infection and other STI
pathogens [4].
[0071] C trachomatis is the leading cause of tubal infertility and
pelvic inflammatory disease, eg [5,6]. Chlamydial genital infection
occurs in 5-15% of pregnant women, and 50% of their babies will
develop inclusion conjunctivitis or respiratory infections [7],
making C trachomatis the most common ocular pathogen in infants
[8]. Genital infections also predispose to development of a
significant proportion of reactive arthritis cases [9], in which
viable, metabolically active organism is present in synovial
tissue, primarily within monocyte/macrophages [10]. C pneumoniae,
identified as a cause of community-acquired pneumonia in adults,
also has been associated with atherogenesis [11,12];
seroepidemiologic studies suggest that the majority of adults have
been exposed to C pneumoniae.
[0072] Although controversial, C pneumoniae has been associated
with other chronic inflammatory diseases, including late onset
Alzheimer's disease [13,14], and one or more forms of multiple
sclerosis [15]. C psittaci infects avian species and can have major
economic impact on poultry production, as well as placing poultry
handlers at risk for transmission [16]; recent data implicates C
psittaci as well as other Chlamydial species in temporomandibular
joint disease ([17]). The application also provides a method to
treat, cure or prevent chlamydia-associated reactive arthritis, as
well as treating, curing or preventing disease at other sites where
Chlamydiae disseminate and cause inflammation, including atheromas,
sites in the lungs, and sites in the brain.
[0073] While cause-effect relationships have not been proven in
several of these diseases, and several negative reports have been
published, data confirming a presence of C pneumoniae in CSF from
additional MS patients, and in brains of independent samples from
AD brains have been presented at national/international meetings by
respected researchers [14,18]. Thus, the public health significance
of Chlamydial infection is enormous, and a drug-targeting approach
capable of selective delivery of antibiotics to the infected cells
has industrial applicability.
[0074] As with all Chlamydiae, C trachomatis and C pneumoniae
undergo an unusual biphasic developmental cycle. The cycle is
initiated when elementary bodies (EB), the infectious extracellular
form of the organism, attach to the target host cell. Once bound,
EB are brought into a membrane-bound cytoplasmic inclusion within
which they spend their intracellular tenure (stage 1). In the
inclusion, EB develop into reticulate bodies (RB; stage 2), the
growth form. Each RB undergoes 7-8 rounds of cell division (stage
3), after which most dedifferentiate back to EB. Newly-formed EB
are released by host cell lysis or exocytosis ([20]).
[0075] Many studies have demonstrated that both C trachomatis and C
pneumoniae often disseminate widely from their sites of primary
infection; when they do so, these organisms can take up long-term
residence at distant anatomic locations [21,22]. At sites of their
disseminated residence, both organisms enter an unusual biological
state referred to as `persistence` [22,23] (stage 5). 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 (eg,
[24,25]).
[0076] The means by which persistently infecting Chlamydiae
engender pathology is not well understood, but it is clear that
they can elicit a powerful immunopathogenic response that can
contribute to genesis/exacerbation of the chronic diseases
mentioned above. Various cell stresses can induce transition into
the `persistent` state in vitro and presumably in vivo: starvation,
heat shock, penicillin G, interferon-.gamma. (IFN.gamma.), iron
deprivation. Morphologically, persistent forms are distinct,
aberrantly large RB (stage 5); transcriptionally, not all models of
persistence are entirely consistent. A monocyte model of
persistence has been described, which has features in common with
persistence seen in human and murine reactive arthritis, eg.
[25,26].
[0077] The inventors compared the transcript profiles of C.
pneumoniae in the persistent state as elicited by IFN.gamma.
treatment, penicillin G treatment, and iron deprivation, all
compared to the transcript profile of the same strain (CWL029)
persistently infecting normal human monocytes. In these in vitro
experiments, transcript profile was not entirely consistent among
all these persistence models, nor was it fully consistent with
that/those of C. trachomatis whose persistent state is elicited by
parallel means. However, there are some features held in common for
all models of persistence. For example, ftsK is always off, whereas
ftsW is not. Thus, a number of molecular genetic and other details
regarding `persistent` Chlamydial infections remain to be
defined.
[0078] A recent study in ReA patients has shown that combination
antibiotic therapy may be more effective in vivo for acute and
persistent synovial infection [28]; this is supported by in vitro
data from several studies. Because of growing concerns about
antibiotic resistance, improved combined therapies for Chlamydial
infections would be highly desirable. A challenge to treatment of
Chlamydial infections is the complex intracellular developmental
cycle in which drugs and other therapeutics must cross several
membranes, not simply the bacterial membrane. According to the
present disclosure, the size and rapid entry of nanoparticles into
infected cells enables encapsulated antibiotics to more
efficiently/successfully reach the bacterial targets. Persistent
Chlamydial infections are highly prevalent and can have severe
chronic disease (inflammatory) sequelae. Improved treatments to
eradicate such persistent infections are urgently needed and have
industrial applicability.
[0079] Folate receptors are overexpressed following infection.
Human and murine cells encode several receptor isoforms for folic
acid, of which .alpha., .beta., and .gamma. are the most prominent
[29]. These receptors are differentially expressed as a function of
cell type, growth conditions, and the general health of the cell
[30]. Studies by others suggest that folic acid and folate
conjugates have equal affinity for the different isoforms [31].
Recent studies indicate that folic acid receptors (FAR) are
overexpressed on activated macrophages in the synovium of arthritic
joints [32]. Chlamydial infection causes activation of monocytes
and macrophages and their localization to the joint (and other
infected tissues). Studies by the inventors showed an increase in
the mRNA encoding FAR in cultured human macrophages and human
epithelial cells 24 hrs post-infection with C trachomatis.
Increased expression of FAR on Chlamydia-infected cells indicates
that such cells can be targeted using folic acid as targeting
ligand.
[0080] Nanoparticles for Targeted Antibiotic Delivery.
[0081] Nanoparticles refer to nanometer-size devices with a matrix
core of dense polymeric network, with antimicrobials, vaccines, or
other therapeutic molecules encapsulated in the polymer core. The
inventors' previous research has demonstrated that PLGA
nanoparticles are non-toxic and biocompatible [38], and they are
suitable for in vivo drug delivery [39]. The inventors also have
shown that nanoparticles can efficiently encapsulate and sustain
the release of hydrophobic drugs such as dexamethasone [40] and
paclitaxel and nucleic acids [41]. important advantage of PLGA
nanoparticles is that the rate of drug release from nanoparticles,
and therefore, the therapeutic efficacy, can be controlled by
varying the polymer properties such as molecular weight,
lactide-glycolide ratio and end-group chemistry [42,43].
[0082] PLGA nanoparticles are rapidly taken up into cells by
endocytosis, resulting in higher cellular uptake of the entrapped
drug compared to that following conventional drug treatment [44].
The therapeutic efficacy of nanoparticles is further enhanced by
their ability to protect the drugs from degradation by lysosomal
enzymes [45]. Nanoparticles, because of their colloidal nature and
serum stability [44], can be easily dispersed in saline and
injected intravenously. Because of their small size, such particles
can penetrate small capillaries, allowing enhanced accumulation of
encapsulated drug or other therapeutic molecule at target sites.
Nanoparticles can be delivered to distant target sites by attaching
a ligand such as folic acid to the particle surface which has
affinity for a specific tissue. Nanoparticles can be employed to
deliver either single or combination antibiotics to targeted sites,
and they may be useful for specific delivery of vaccines or other
therapeutic modalities. Folic acid-conjugated nanoparticles rapidly
target Chlamydia-infected cells in both in vitro and in vivo
systems of such infections.
[0083] Animal models of disseminated Chlamydial infection. Animal
models have provided important information regarding Chlamydiae and
the immune/inflammatory responses these bacteria induce in vivo,
eg, [46-54]. These studies have added insights into pathogenic
processes such as the association of repeated infection with
increased clinical and histopathologic disease, and immunization
studies testing several logical vaccine candidate antigens
including MOMP and hsp60 [51,55,56]. Until recently, most mouse
models have utilized the mouse pneumonitis strain (MoPn) of C
trachomatis to study the pathogenesis and immune responses during
pneumonia and genital infection Eg, [57-63]. Human biovars of C
trachomatis have also been used to establish murine genital
infection models [47,48,64-66]. While some features of each of the
models have direct relevance to human disease, many differences
between the diseases in models and human disease have been
noted.
[0084] Few animal studies have investigated Chlamydia-associated
reactive arthritis (ReA), a good disease model for studying
persistent infections. In earlier studies, we observed that ocular
infection of mouse conjunctivae (an ocular mucosal tissue) resulted
in Chlamydial dissemination to synovium [67]. More recently, we
have focused primarily on a genital infection model to induce
murine ReA since this infection route is more representative of
human Chlamydia-associated ReA. The latter model allowed us to
document dissemination of C trachomatis to synovial tissues and
associated knee pathology. An overview of the synovial inflammation
induced in the co-PI's murine ocular and genital infection models
has been published [54,67].
[0085] Chlamydial dissemination occurs in other animal models: C
pneumoniae was shown by Moazed et al to disseminate to distant
sites after intranasal challenge of mice, but synovium was not
assayed [21]. Studies by Rank et al with a mouse pneumonitis strain
of C trachomatis (MoPn)-induced genital infection resulted in an
acute arthritis [68], but these studies preceded knowledge of
persistent infection in ReA and availability of molecular screening
to demonstrate presence of viable organism. The latter studies
utilized either presensitization or intra-articular Chlamydial
challenge, making it less physiologic than natural dissemination
from genital infection. Rank and colleagues have also shown in
guinea pigs dissemination of GPIC from genital tract to joint
[69].
[0086] The inventors' model for C trachomatis-associated ReA offers
distinct, demonstrated advantages for the proposed studies because
of its noninvasive mode of disease generation and reproducible
inflammation and infection. In addition, we show the feasibility of
imaging nanoparticle localization in addition to targeting sites of
Chlamydial infection with drug-loaded nanoparticles. Thus, means
are used to monitor nanoparticle trafficking, success of drug
delivery, and subsequent proof of cure by imaging, histology and
molecular analyses, in addition to quantification of nanoparticles
in tissues.
[0087] The data disclosed herein provide a new and unique
combination of basic and applied science-oriented approaches to
precise targeting of therapeutic agents to target
Chlamydia-infected cells and tissues. Overexpression of folate
receptors following Chlamydial infection and the use of folic acid
conjugated delivery system to target (Chlamydia) infected cells
have not been reported before. This approach can have a significant
impact on treatment of several severe chronic diseases associated
with persistent infection by C trachomatis and C pneumoniae.
Targeted antibiotic delivery using nanoparticles is expected to
reduce the amount of drug required, which would have a substantial
impact on drug costs for Chlamydial infection treatment/cure.
Additionally, all nine species comprising the Order Chlamydiales
are pathogenic to their various hosts, and the seven animal
pathogen species have enormous economic impact on domestic and
other animals. Importantly, this approach to development of a
targeting system for therapeutic agents can provide a model for
development of congruent systems to eradicate other intracellular
pathogens, including Mycobacterium tuberculosis, Listeria
monocytogenes, and others. The constitution of nanoparticles can be
designed for either rapid or long-term release of the encapsulated
agent(s) [40]. The disclosure is applicable to developing systems
providing sustained delivery of antimicrobial or other therapies to
infected tissues, transformed cells, etc. Thus, the data are
expected to have a significant impact on the field of infectious
diseases.
[0088] The following examples are intended to illustrate but are
not to be construed as limiting of the specification and claims in
any way.
EXAMPLES
Example 1
Chlamydia-Infected Cells Overexpress Folic Acid Receptors
[0089] Mammalian cells encode multiple folic acid receptors,
designated .alpha., .beta., and .gamma. (Ross, J. F. et al. (1992)
Cancer 73:2432-2443). The present Example was performed to examine
the expression of those receptors in Chlamydia infected cells.
Nearly confluent monolayers of cycloheximide-treated cells were
infected in vitro at MOI 5:1 with K serovar C. trachomatis. The
cell lines employed in these experiments were RAW 264.7 and U937
(murine and human macrophage lines, respectively), and HEp-2 (human
epithelial cell line).
[0090] At 24 hr post-infection, infected and uninfected control
cultures were harvested, and RNA/cDNA was prepared for real time
RT-PCR analysis to determine relative levels of mRNA encoding each
of the three receptor subtypes in infected versus uninfected cells.
As shown in FIG. 1, C. trachomatis infection resulted in increased
expression of the .beta., and .gamma. receptors in human cells. The
over-expressed isoform was dependent on the cell type. That is, a
murine cell line showed the largest increase in the folic acid
receptor alpha(.alpha.) isoform, unlike human lines, which
upregulated the .beta. and .gamma. receptor isoforms most when
infected with Chlamydia. These results indicate that chlamydial
infection results in upregulation of folic acid receptors in the
cell lines tested.
Example 2
Preparation of Nanoparticles with Folic Acid on the Surface
[0091] This example uses techniques developed by applicants to
anchor PEG and PEG-folate conjugate on the surface of
nanoparticles, and described in Provisional Application Ser. No.
60/871,404, filed Dec. 12, 2006, incorporated herein by reference.
The technique relies on the interfacial activity of PEG-X block
copolymer conjugate, where X is any hydrophobic polymer (example,
polylactide, polypropylene oxide, etc). Most nanoparticle
formulations involve an emulsion step in the preparation. Following
the formation of the emulsion, a solution of PEG-containing block
copolymer (for example PLA-PEG (1000/5000 Da), with or without
conjugated ligand (folic acid, for example) in an organic solvent
(methanol, chloroform, etc), is added to the emulsion. PLA-PEG is a
surface active block copolymer, composed of hydrophobic PLA chains
and hydrophilic PEG chains.
[0092] Addition of the block copolymer to the emulsion results in
the hydrophobic polylactide chain inserting itself into the oil
phase and the hydrophilic PEG (or PEG-folate) chain remaining in
the outer-most aqueous phase. This results in nanoparticles that
contain PEG (or folate-PEG) chains on the surface. Because this
method relies only on the interfacial activity of the copolymer,
the technique is independent of the polymer used for nanoparticle
formulation or the targeting ligand that is being used, in this
case, folic acid.
[0093] Folic acid conjugation allows endocytic uptake of the
conjugated carrier via the folate receptor, resulting in higher
cellular uptake of the encapsulated drug. The high affinity of
folic acid to its receptor (binding constant .about.1 nm) and
folate's small size allow its use for specific cell targeting. The
ability of folic acid to bind its receptor is not altered by
covalent conjugation to delivery systems.
[0094] As described in the following Examples, introduction of
folic acid on the nanoparticle surface enhanced Chlamydia-infected
cell-specific accumulation of both PLGA nanoparticles.
Nanoparticles used in the Examples herein were prepared as
follows:
[0095] An aqueous solution of bovine serum albumin was emulsified
in an organic solution of a biodegradable polymer such as
poly(D,L-lactide-co-glycolide) and a fluorescent probe such as
6-coumarin. This simple emulsion was further emulsified into an
aqueous solution of polyvinyl alcohol to form a
water-in-oil-in-water type emulsion. Following this, a solution of
PEG-containing block copolymer (for example PLA-PEG (1000/5000 Da),
with or without conjugated ligand (folic acid, for example) in an
organic solvent (methanol, chloroform, etc), was added to the
emulsion. The emulsion was then stirred for .about.18 hours.
Nanoparticles formed were recovered and washed by repeated
ultracentrifugation steps (140,000 g for 1 hour, 3.times.), and
then lyophilized. The dry nanoparticle preparation was dispersed in
appropriate physiological medium as required at the time of an
experiment. Bovine serum albumin used in the formulation helps in
stabilizing the nanoparticles formulation, and can be replaced with
serum albumin from other species, if necessary.
Example 3
Folic Acid-Derivatized Nanoparticles Accumulate More in Infected
Cells
[0096] This example was performed to study the targeting of
fluorescently-labeled nanoparticles to chlamydial inclusions within
HEp-2 host cells infected with C. trachomatis serovar K. Nearly
confluent monolayers of cycloheximide-treated cells were infected
at MOI 5:1, and at 24 hr post-infection, infected cells were pulsed
with folic acid-conjugated poly(D,L-lactide-co-glycolide) (PLGA)
nanoparticles labeled with 6-coumarin. Quantitative studies
indicated that infected cells accumulate significantly more folic
acid-conjugated nanoparticles than uninfected cells (FIG. 2), and
correlates well with increased expression of folic acid receptors
in infected cells (FIG. 1).
[0097] This discovery was then used to deliver plasmid DNA into
Chlamydial inclusions (FIG. 4). HEp-2 cells were infected with
Chlamydia (CT) and then pulsed with nanoparticles containing
plasmid DNA (FIG. 4). Delivery to inclusions was imaged using
separate fluorescent markers on the DNA and the nanoparticles. FIG.
4A shows confocal images detecting TOTO3-labeled (red) DNA within
inclusions (white arrow) vs 6 coumarin-labeled nanoparticles
(green) in cells which were infected 72 hrs previously with
Chlamydia trachomatis. Co-localization of red released DNA and
nanoparticles within the inclusion are seen. 4B shows an image
collected at 48 hr after infection also pulsed with the
nanoparticles containing DNA. A cell with a large inclusion (arrow)
clearly has evidence of released DNA (red) with nanoparticles
(green) within the inclusion. FIG. 4C shows two Z cuts of the same
field showing localization of DNA within the inclusion and perhaps
within reticulate bodies (arrow); clearly the nanoparticles are
also associated with chlamydial membranes within the inclusion and
the inclusion membrane.
[0098] Nanoparticle uptake in infected cells was also imaged on a
RTM-3 microscope. Nanoparticles homed quite rapidly to the
inclusion (FIGS. 3A, 3B); fluorescence was focused at the inclusion
membrane and at the membranes of RB resident at the internal side
of that membrane. Fluorescence was dimmer on elementary bodies
(center of inclusion), consistent with their smaller size. This
Example shows that nanoparticles constructed as described in
Example 2 can be utilized to target chlamydial inclusions, thereby
delivering to the inclusion the contents of the particle.
Example 4
Folic Acid-Derivatized Nanoparticles Target Chlamydia Infection In
Vitro
[0099] This example was performed to investigate targeting
fluorescently-labeled nanoparticles to chlamydial infections in a
mouse model of Chlamydia-induced arthritis. Balb/c mice were
infected with K serovar C. trachomatis as described (G. A.
Altenberg. PNAS 91: 4654-4657 (1994)), and then injected
intravenously with fluorescently labeled, folic acid conjugated
nanoparticles 10-14 days post-infection. Animals were then imaged
on Kodak animal imager. Previous studies have shown that in this
model, infected macrophages reside in the synovium and genital
tract (G. A. Altenberg. PNAS 91: 4654-4657 (1994)). Folic
acid-conjugated nanoparticles targeted the synovial tissue in
infected mice. Image analysis of excised genital tract from the
treated mice showed nanoparticle accumulation in the upper genital
tract. This tissue-specific targeting was absent in controls
(infected mice injected with nanoparticles without folic acid,
uninfected mice injected with either nanoparticle formulation).
This Example shows that folic acid-directed targeting of
nanoparticles to Chlamydia-infected cells operates not only in
vitro, but also in vivo.
Example 5
Targeting of Folic Acid-Conjugated Nanoparticles to Infected Cells
In Vivo
[0100] Following infection at the epithelial surfaces of the
cervix, C. trachomatis can ascend to the upper reproductive tract.
In the case of urogenital or ocular (conjunctival) infections,
organisms can disseminate to distant anatomic locations such as the
joint, to engender chronic sequelae. The results above in Example 4
indicate that folic acid-conjugated nanoparticles will target
infected tissues in the murine model of Chlamydia-induced
arthritis. Whole animal imaging indicated that appropriately
labeled folic acid-conjugated nanoparticles would be useful for
imaging infection sites to monitor the progression or eradication
of infection, in addition to their use in drug delivery.
[0101] Experiments are performed to determine whether persistent
joint infections in vivo can be attenuated or eliminated using
antibiotics delivered by nanoparticles to infected tissues. BALB/c
mice are infected genitally with 10.sup.7 K serovar C. trachomatis
EB, and at different time intervals (1 to 21d) post-infection,
upregulation of the folate receptor subtypes is assessed in
synovial and genital tissues harvested from infected mice;
assessment is done by real time RT-PCR as above, and by western
analysis for folic acid receptors.
[0102] The relative load of C. trachomatis is monitored in DNA
preparations from each synovial sample at each time point, again by
real time PCR analysis as described (M. R. Lugo and F. J. Sharom,
et al. Biochemistry 44: 643-655 (2005). Some infected mice are
subjected to IHC to determine the morphology/identity of infected
cells. Chlamydial infections are identified by IHC as described
above. Once these studies are completed, groups of mice are
infected as above, then injected intravenously with fluorescently
labeled, folic acid conjugated nanoparticles at 1, 3, 7, 10, 14,
and 21 d post-infection. Animals are euthanized 30-360 min after
injection and target synovial and genital tissues are harvested.
Tissues are homogenized, lyophilized and extracted with
methanol.
[0103] Nanoparticle concentration in the tissues are quantified
using HPLC, and the data are normalized to the wet weight of the
tissue. Some mice that are similarly infected and injected with
nanoparticle formulation are used for imaging purposes using a
Kodak whole animal imager, as above. X-rays with imaging will
define the anatomic localization of nanoparticles. Data are
correlated among time course of infection, bacterial load, and
nanoparticle biodistribution. Distribution of folic acid conjugated
nanoparticles is expected to co-localize with infected cells in
synovium. Eradication of organism from infected sites is performed
using folate conjugated nanoparticles containing azithromycin or
other antibiotics, as described above; assessment of efficacy is
done by real time PCR targeting chlamydial chromosome number in
treated as compared to untreated animals and normalized to host 18S
rDNA.
Example 6
Folic Acid Conjugated Particles for Delivery of Antibiotics
[0104] The data in Example 3 indicated that folic acid-conjugated
PLGA particles labeled with 6-coumarin homed to C.
trachomatis-containing inclusions at 24 hr post-infection in
actively infected HEp-2 cells. The present Example is performed to
measure several parameters: homing at all times post-infection,
homing in cells harboring persistent chlamydiae, the efficacy of
particle delivery at one or more specific times post-infection, and
the efficacy of particle delivery at times post-infection in
epithelial cells (active) or monocytic cells (persistent) harboring
C. pneumoniae.
[0105] To perform this Example, 6-coumarin-labeled, folic acid
conjugated (empty) PLGA particles are prepared as described above,
and targeting to cultured epithelial cells (HEp-2) and monocytic
cells (U937) is assessed by fluorescence microscopy using the RTM-3
microscope. Targeting is assessed at 6, 24, 48, and 72 hr
post-infection of each cell type, using cells infected at
MOI.about.1:1 with C. trachomatis serovar K or C pneumoniae strain
AR-39; assessment of the relative level of fluorescence within each
infected cell is quantified by computer analysis in each time
course experiment with each chlamydial species and by HPLC on
extracted 6-coumarin. Those data are correlated with data regarding
the relative level of upregulation of each folate receptor cell
type in both HEp-2 and U937 cells.
[0106] Particles are prepared containing the antibiotic
azithromycin, which is known to be bactericidal for
actively-growing chlamydiae of both species. Drug-containing
particles are administered to HEp-2 and U937 cells at various times
post-infection, allowed to home to the inclusions for 24 hr, then
cultures are harvested for determination of viable chlamydiae by i)
real time PCR targeting the relative level of chlamydial chromosome
relative to that of control cells (identically infected but given
empty particles or no particles), and recovery of IFU. Chlamydiae
of either species infecting U937 cells do not fully enter the
persistent state. If delivery of the antibiotic is effectively
bactericidal in U937 experiments, analysis is extended to infection
(both species) of normal human monocytes at 72 hr post-infection;
at 72 hr post-infection, both C. trachomatis and C. pneumoniae are
fully and completely in the persistent infection state.
Example 7
Antibiotic Loaded Nanoparticles
[0107] Cells and growth of Chlamydia. McCoy cells were seeded onto
96-well microtitre plates at a concentration of 2.times.10.sup.5
cells per well and allowed to adhere overnight at 37.degree. C. and
5% CO.sub.2 in Eagles MEM supplemented with 10% FBS and 200 mM
Glutamine. The following day, the cells were inoculated with C.
trachomatis (UW-31, serovar K) at 10.sup.4 IFU/well and centrifuged
at 2500 rpm for 1 hr at room temperature, followed by incubation at
37.degree. C. for 1 hr. The inoculum was then aspirated and
replaced with the appropriate two fold dilution of free drug or
encapsulated nanoparticles prepared in antibiotic-free growth media
containing 0.06% glucose and 1 .mu.g/ml cyclohexamide. The cells
were incubated in the presence of drug for 24 or 48 hrs post
infection and then fixed with 100% MeOH. Chlamydial inclusions were
detected by immunofluorescence using BioRad Pathfinder.RTM.
according to the manufacturer's instructions and the plates
subsequently stored protected from light at 4.degree. C.
[0108] Microsusceptibility of C. trachomatis to azithromycin and
rifampin. McCoy cells were seeded onto 96-well microtiter plates
and then infected the next day with 10.sup.4 IFU/well C.
trachomatis (serovar K). The cells were treated immediately
following infection with serial two-fold dilutions of azithromycin
or rifampin to a final concentration range of 31-2000 ng/ml and
1-64 ng/ml respectively and incubated for 24 or 48 hrs. FIGS. 5A
and 5B show the results; the mean inclusion number was plotted as a
function of concentration. The MIC.sub.50 was defined as the
minimum drug concentration needed to reduce the total number of
inclusions by 50% and was determined to be .about.40 ng/ml for
azithromycin and .about.5 ng/ml for rifampin. These data are
consistent with previous sensitivity levels reported for both drugs
(Suchland et al., 2003; Kutlin et al., 2005). In addition, both
azithromycin and rifampin, showed no significant change in
MIC.sub.50 with length of treatment.
Example 8
Comparison of Free Drug Vs Encapsulated Nanoparticles
[0109] McCoy cells were seeded onto 96-well microtiter plates and
then infected the next day with 10.sup.4 IFU/well C. trachomatis
(serovar K). The cells were treated immediately following infection
with serial two-fold dilutions of azithromycin or rifampin
encapsulated nanoparticles to a final concentration range of
31-2000 ng/ml and 1-64 ng/ml respectively and incubated for 24 or
48 hrs post infection. The mean inclusion number was plotted as a
function of concentration.
[0110] To prepare free antimicrobials and drug encapsulated
nanoparticles, stock solutions of azithromycin (Asta, N-66072) and
rifampin (Sigma, R-5777) were prepared at a concentration of 25
mg/ml in sterile water by adding glacial acetic acid dropwise until
the compounds were completely dissolved as described by Barry et
al., 2004. The stocks were stored at -80.degree. C. in single use
aliquots. Subsequent dilutions were prepared immediately prior to
use in defined growth media without antibiotics. Azithromycin and
rifampin were also encapsulated into biodegradable, poly
(D,L-lactide-co-glycolide) (PLGA) nanoparticles.
[0111] The results are shown in FIG. 6 A-C. When used individually,
both azithromycin and rifampin nanoparticles were as effective as
free drug exhibiting similar MIC.sub.50 of 40 ng/ml and 5 ng/ml
respectively. Combination nanoparticles containing approximately
equal amounts of azithromycin and rifampin showed an enhanced
effect compared to azithromycin alone with an MIC.sub.50 of 20
ng/ml. This is consistent with other studies that have shown that
combined treatment with both azithromycin and rifampin are more
effective at treating chlamydial infections than individual free
drugs (Wolf and Malinverni, 1999). There was no significant shift
in MIC.sub.50 for individual or combined nanoparticle therapy with
length of treatment. These results show that both azithromycin and
rifampin encapsulated nanoparticles are as effective as individual
free drug.
Example 9
Effectiveness of Free Drug Vs Nanoparticles with Treatment at 0, 24
or 48 Hours Post Infection
[0112] McCoy cells (2.times.10.sup.5/well) were seeded onto 96-well
microtitre plates and then infected the next day with 10.sup.4
IFU/well C. trachomatis (serovar K). The cells were treated at 0,
24 or 48 hours post infection with serial two-fold dilutions of
azithromycin or rifampin. The cells were incubated for 24 hours in
the presence of drug and then fixed. Based on previous experiments
we refined the concentrations ranges for each of the two free drugs
and encapsulated nanoparticles. The final concentration range was
2.5-10 ng/ml for rifampin and 25-100 ng for azithromycin.
[0113] When free drug or encapsulated nanoparticles were added
immediately following infection, the response was similar to that
observed previously with an MIC.sub.50 of 5 ng/ml for rifampin and
40 ng/ml for azithromycin. In contrast, when drug was added 24 or
48 hours post infection a marked insensitivity was observed,
reflected in a minimal decrease in inclusion number with increasing
dose. (FIG. 7 A-C, FIG. 8, A-C.) There was some indication that the
cells treated with drug encapsulated nanoparticles were more
sensitive than those treated with free drug, albeit the MIC.sub.50
was shifted out of the range of this experiment. Statistical
significance based on pair-wise rank sum test is written on the
figures.
[0114] Secondary inclusions were observed at 72 hpi. These
inclusions showed sensitivity similar to infected cells treated
immediately post infection. This suggests that sensitivity to
azithromycin and rifampin is related to a specific window in the
Chlamydial life cycle.
Example 10
Responsiveness of Infected Cells to Drug Encapsulated Nanoparticles
Vs Free Drug when Treatment is Delayed for 24 Hpi
[0115] McCoy cells (2.times.10.sup.5/well) were seeded onto 96-well
microtitre plates and then infected the next day with 10.sup.4
IFU/well C. trachomatis (serovar K). The cells were treated at 0 or
24 hours post infection with serial two-fold dilutions of
azithromycin or rifampin. The cells were incubated for 24 hours in
the presence of drug and then fixed. Previous data indicated that
there is a dramatic shift in MIC.sub.50 when drug treatment is
delayed. We, therefore, extended the concentration ranges for the
two drugs. The final concentration range for rifampin was 2.5-80
ng/ml and for azithromycin was 25-800 ng/ml.
[0116] The results are shown in FIG. 9A-B and indicate that
sensitivity to drug may be enhanced when rifampin is delivered in
nanoparticles relative to free drug. Inclusion number expressed as
percent of control was plotted as a function of concentration. In
the case of rifampin improved sensitivity was observed at 40 and 80
ng/ml. A 50% reduction in inclusion number was observed for
rifampin nanoparticles compared to free drug at 40 ng/ml. In the
case of azithromycin, there was no improvement in response to drug
using nanoparticles compared to free drug when treatment was added
at 24 hpi (FIG. 10A-B).
[0117] Consistent with previous data, for both rifampin and
azithromycin, nanoparticle delivered antimicrobials were as
efficient as free drug when treatment was added immediately post
infection. This is also supportive that it is EB to RB transition
and early stages of inclusion formation that seem to be the window
of maximum sensitivity.
[0118] The next experiment was performed to determine whether
extending the length of exposure to drug would enhance the shift of
MIC.sub.50 observed for nanoparticles vs free drug, and whether
there is a difference in targeted nanoparticles vs drug
encapsulated alone. McCoy cells (2.times.10.sup.5/well) were seeded
onto 96-well microtitre plates and then infected the next day with
10.sup.4 IFU/well C. trachomatis (serovar K). The cells were
treated at 24 hours post infection with serial two-fold dilutions
of azithromycin or rifampin. The cells were incubated for 24 hours
or 48 hours in the presence of drug and then fixed.
[0119] When drug was added at 24 hpi and left on for 24 hours,
there was no difference in sensitivity between cells treated with
nonFA or FA targeted nanoparticles. Both show an MIC.sub.50 between
40 and 80 ng/ml. (FIG. 11.) This is consistent with previous
results and show an advantage over free drug alone where MIC.sub.50
is greater than 80 ng/ml. When drug is added at 24 hpi and left on
for 48 hours, there is an enhanced sensitivity compared with 24
hour drug exposure. The MIC50 shifts to 20-40 ng/ml. A significant
change in slope at 10 ng/ml suggests that FA targeted nanoparticles
improve sensitivity over drug encapsulated nanoparticles alone.
[0120] For azithromycin, when drug was added at 24 hpi and then
left on for 24 hours there was a minimal response with non FA
nanoparticles, however, the MIC.sub.50 was still greater than 800
ng/ml. In contrast when drug was added at 24 hpi and left on for 48
hours both targeted and non targeted nanoparticles showed enhanced
sensitivity with an MIC.sub.50 near to 800 ng/ml. (FIG. 12.)
Example 11
Folic Acid Conjugated Nanoparticles Target Chlamydia Infected
Cells
[0121] This example was performed to investigate the targeting of
fluorescently-labeled nanoparticles to Chlamydial inclusions in an
in vitro cell culture model. Folic acid conjugated-nanoparticles
were formulated using a simple, interfacial activity-assisted
method of nanoparticle surface functionalization (manuscript
submitted). This method utilizes the fact that when an ampiphilic
diblock copolymer is introduced into a biphasic (oil/water) system,
the copolymer adsorbs at the interface. The hydrophobic block of
the copolymer tends to partition into the oil phase while the
hydrophilic block tends to remain in the aqueous phase. In the
proposed approach, we introduce a diblock copolymer such as
polylactide-polyethylene glycol (PLA-PEG) with folic acid
conjugated to the PEG chain (PLA-PEG-folic acid). This results in
partitioning of PLA block into the polymer containing oil phase and
PEG-ligand block into the aqueous phase. Removal of the organic
solvent results in the formation of nanoparticles with PEG-folic
acid on the nanoparticle surface. Micelles formed due to the
self-assembly of the PLA-PEG block copolymer are removed by
extensive dilution and washing of the system. We call this method
Interfacial Activity Assisted Surface Functionalization (IAASF).
Using this approach, we fabricated nanoparticles from a
biodegradable polymer PLGA and surface functionalized with PEG and
folic acid as targeting ligand. Incorporation of PLA-PEG segments
along with the folic acid in nanoparticles was confirmed by proton
NMR (FIG. 13). Presence of PEG and folic acid on the surface was
confirmed by contact angle measurement, and surface plasmon
resonance (FIG. 14).
[0122] Decrease in the contact angle of water from 49.+-.5 to
33.+-.3 for unconjugated and PEG-conjugated nanoparticles
(P<0.05) suggests that incorporation of PEG significantly
increased the hydrophilicity of the nanoparticle surface. This was
expected, since PEG is more hydrophilic than PLGA. The decreased
hydrophilicity of PEGylated nanoparticles is expected to contribute
to the decreased biorecognition and increased circulation time of
particles. Surface plasmon resonance studies indicated that not
only was folic acid present on the surface of the particles but was
also available for binding. A significant difference of about 200
RU in binding to the anti-folate antibody coated surface was
observed for nanoparticles with and without folic acid on the
surface (FIG. 14).
Example 12
Development of Persistence Phenotype with K Serovar
[0123] Two approaches were used with in vitro infected McCoy cells
to test whether aberrant inclusions developed. Cells were exposed
to penicillin G (PenG, 100 U/ml added at 24 hr p.i.) [70,71]. Pen G
induced the typical large aberrant RB shown by others (FIG. 15). In
these inclusions, only the small number of EB had motion, and the
aberrant forms were stationary in the inclusion. Treatment of
infected cells with recombinant IFN.gamma. for 24 hr induced small
inclusions that appeared to contain exclusively RB (not shown).
With primary cultured human monocytes, no treatment was necessary
to elicit persistence by day 3 post-infection.
[0124] This was supported by additional experiments with acutely
Chlamydia-infected and uninfected cells: semi-confluent monolayers
of HEp-2 cells were infected with C trachomatis serovar K at an MOI
5 and overlaid with cycloheximide-containing medium. At 24 hr p.i.,
infected and uninfected cells were pulsed for 5-15 min. with folic
acid-conjugated nanoparticles labeled with 6-coumarin. Quantitative
studies indicate that infected cells accumulate significantly more
folic acid-conjugated nanoparticles than uninfected cells (FIG. 2),
and correlates well with increased expression of folic acid
receptors in infected cells (FIG. 1).
[0125] Nanoparticle uptake in live, infected cells was also imaged
on the RTM-3 microscope. Remarkably, nanoparticles appeared to have
homed quite rapidly to the inclusion within 15 min of addition
(FIG. 3 A, B); indeed fluorescence is focused at the inclusion
membrane and at the membranes of RB resident at the internal side
of that membrane and differentiated EB deeper in the inclusion.
Thus, initial studies suggest that particles constructed in the
manner given can be utilized to target Chlamydial inclusions,
thereby delivering to the inclusion the contents of the
particle.
Example 13
Nanoparticles Deliver their Payload into Chlamydial Inclusions
[0126] This example was performed to determine whether
nanoparticles can deliver their contents into the inclusion
complexes. PLGA nanoparticles containing TOTO3-labeled plasmid DNA
were prepared using previously published methods. TOTO-labeled DNA
was chosen as the payload because: (1) TOTO-labeled DNA is red
fluorescent and enables the use of epi-fluorescence microscopy to
track intracellular delivery; and (2) high binding affinity of TOTO
for DNA and the lack of fluorescence in the absence of DNA binding
ensure low false positives. For these studies, nearly confluent
monolayers of HEp-2 cells were infected with C trachomatis as
above. At 24 hr p.i. cells were pulsed with DNA-loaded
nanoparticles that were also labeled with 6-coumarin. At 48 hr
p.i., nanoparticles (green fluorescence) were found within the
inclusions. Importantly, nanoparticles were found to release their
DNA payload (red fluorescence) within the inclusion complexes (not
shown). This example indicates that PLGA nanoparticles can be
utilized to deliver their contents into Chlamydial inclusion
complexes.
Example 14
Molecular Evidence for Persistent Infection in Mouse Knees
Following Genital Infection
[0127] Persistently infected cells may be a potent source of
reactivated infection, increased inflammation at the tissue sites
including upper genital tract, synovial tissue, and disseminated
sites associated with C trachomatis and C pneumoniae. In two
studies of acutely infected HEp-2 cells and persistently infected
human monocytes [25,26], it was found that transcription of genes
important in Chlamydial cell division and energy
metabolism/transfer were markedly different under the two infection
states and congruent with decreased Chlamydial cell division and
altered metabolic activity during persistence. The key genes for
comparison which are relevant to this example are dnaA (Chlamydial
DNA replication; turned on in cells with acute or persistent
infection), ftsK and ftsW (Chlamydial cell division; turned off in
persistent infection in monocytes; supported by evidence from
Belland et al [72] to be downregulated in IFN.gamma.-treated
persistently infected cells) and others. Human synovial samples
from ReA patients yielded data for dnaA/ftsW and other genes
supporting persistent infection in those tissues. Total nucleic
acids were obtained from 17 mouse synovial samples days 7, 10 or 14
p.i., and screened by RT-PCR for Chlamydial 16SrRNA or primary
transcripts [75], MOMP [76], and plasmid [77]. Ten of 17 samples
were positive by at least one assay, 7 were positive by all three
assays. Positive samples were then tested for presence of
transcripts from dnaA and ftsW [25,78] by RT-PCR (see Detailed
Methods for details). In the RT-PCR, 2 samples (d.10 p.i.) were
positive for both dnaA and ftsW transcripts, 7/17 were negative for
both messengers, and 7 of 17 were positive for dnaA transcripts but
negative for ftsW and reflective of persistent infection. A gel
showing results for two different mice is given in FIG. 16. These
data show that a molecular phenotype for persistent infection
develops in knees of the mouse genital infection model, and support
the use of targeted nanoparticle therapy on active vs persistent
infection in synovial tissues.
Example 15
Folic Acid Conjugated Nanoparticles Target Chlamydia Infected
Tissue
[0128] BALB/c mice were infected with C trachomatis (UW-31/K) as in
previous studies [22], and then injected I. V. with 6
coumarin-labeled, folic acid-conjugated nanoparticles 10-14 days
p.i. Animals were then imaged in a Kodak animal imager. Previous
studies have shown that in this model, infected mononuclear
cells/macrophage reside in the synovium and genital tract [22]. As
shown in FIG. 17, folic acid conjugated nanoparticles appear to
target the synovial tissue in infected mice. This tissue-specific
targeting was absent in controls (infected mice injected with
nanoparticles without folic acid, uninfected mice injected with
either nanoparticle formulation; not shown) Image analysis of
excised genital tract from the treated mice showed particle
accumulation in the upper genital tract (not shown).
[0129] In addition to imaging, nanoparticle levels in various
tissues from the same mice were also quantified. In agreement with
the imaging experiment, folic acid conjugated particles were found
to accumulate in significantly higher quantities in knee joints,
liver and genital tract than nanoparticles without folic acid (FIG.
18, P<0.05; n=3-4). Thus, folic acid-directed targeting of
nanoparticles to Chlamydia-infected cells operates not only in
vitro, but also in vivo.
Example 16
Effect of Combination Antibiotic Treatment on Chlamydial
Viability
[0130] This example describes the effect of combining azithromycin
and rifampin and their encapsulation in nanoparticles on the IC50
of the individual drugs. Nanoparticles used in this example were
not conjugated to folic acid. As can be seen from FIG. 19,
combination therapy was significantly more effective than
individual drugs, both free and encapsulated in nanoparticles. When
treated individually, MIC50s for both free and
nanoparticle-encapsulated drugs were 0.008 .mu.g/ml and 0.032
.mu.g/ml, respectively. However, in the presence of 0.004 .mu.g/ml
rifampin, the MIC50 of azithromycin dropped to 0.016 .mu.g/ml,
while in the presence of 0.016 .mu.g/ml azithromycin, the MIC50 of
rifampin reduced to 0.002 .mu.g/ml. It was interesting to note that
despite releasing only a fraction of the drug(s), nanoparticle
antibiotics were at least as effective as free drugs. The
inventors' previous studies have shown that, in general, less than
50% of encapsulated drugs are released in 24-48 hrs [40].
Example 17
Nanoparticles to Treat Chylamydia Infection
[0131] Data disclosed herein indicate that chlamydiae-infected
cells up-regulate expression of genes encoding folic acid receptors
(FAR), suggesting that the association of folic acid with the
therapeutic modality will target infected cells in a reasonably
specific fashion. Assessment of FAR expression patterns will be
followed by production of non-derivatized and folate-derivatized
nanoparticles and testing the ability of those particles to target
infected cells, using well-developed and characterized in vitro
systems. An in vivo murine model of chlamydial infection is
used.
[0132] Overexpression of FAR as a function of chlamydial infection,
and the use of a folic acid conjugated delivery system to target
cells infected with chlamydiae have not reported previously.
Molecular genetic analytical systems will determine the
relationship between progression of infection over time and
differential folate receptor expression at both cellular and tissue
levels. This information is used to determine the optimal time for
targeting the infected cells/tissue and whether such therapy will
work later during chronic/persistent infection.
[0133] Examples described above indicated that chlamydial infection
induces differential up-regulation of FAR in cultured human
epithelial cells and macrophages, and murine macrophages in vitro
at 24 hrs pi. Translation of the up-regulated mRNA encoding the
receptors should follow, and be reasonably congruent with, the
transcript level increase. Initial results suggest that the
up-regulated receptors are present on the cell membrane of the host
cell, and possibly on the inclusion membrane as well since the
latter is derived from host cell membrane/components. In Aim One,
expression of mRNA encoding each of the FAR subtypes will be
defined following infection with C trachomatis serovars (see below)
using HEp-2 and McCoy cells as hosts; HEp-2 cells are of primary
interest since human epithelial cells represent the target host
cell type in primary infection.
[0134] Monocytic cells also must be analyzed since they have been
shown to be the host cell type for dissemination and persistent
infection [22,79]. Relative levels of folic acid receptor mRNA/cDNA
will be determined quantitatively for each of the three dominant
receptor types (.alpha., .beta., .gamma.) by real time RT-PCR at
t0, 6, 12, 24, 36, 48, and 72 hrs post-infection of each of the two
host cell types during normal active infection. Cells will be grown
and infected at MOI .about.1-5 as in our earlier publications [eg,
[25,26]. Because we have used K serovar for most previous studies,
we will use it for initial experiments. However, ocular and genital
strains of C trachomatis have been shown to differ at the genetic
level [80], and we thus will assess FAR transcript levels in the
same host cell types infected with C (ocular) serovar.
[0135] Primers targeting each of the three folate receptor subtypes
(human and mouse) have been designed and tested in real time RT-PCR
assays. Relative transcript levels will be expressed as a function
of time post-infection relative to their levels in uninfected host
cell types of the same line. Concomitant assessment of the relative
level of translation product from each receptor subtype gene will
be done using image analysis software on western analyses for each
subtype. The mAb to be used in these westerns are commercially
available (Santa Cruz and Abcam) and differentially recognize the
three versions of the human and mouse receptor.
[0136] In addition to quantitation of transcription and translation
product levels from the three receptor genes, we will use the same
FAR-targeted mAb in standard immunistochemical (IHC) analyses to
assess the localization and overall level of each receptor on
chlamydiae-infected vs uninfected host cells of each type; receptor
density and distribution will be determined in each host cell type
for infection by K serovar and for infection by C serovar. For the
Western and IHC experiments, cells will be grown and infected and
the cell lysates obtained at different time points will be analyzed
by Western blotting. IHC will be performed. In these IHC
experiments, we will determine whether the three receptor subtypes
are present differentially or otherwise over time on the inclusion
membrane compared to cell membrane, and if so what their density
and overall distribution is. Image analysis software will assist in
these analyses.
[0137] Chronic disease sequelae from chlamydial infections involve
primarily organisms in the persistent infection state, as in
Chlamydia-induced inflammatory arthritis [25,73,81]. In order for
NP-based therapy to be generally useful for treating chlamydial
infections, it will preferably be effective again both normal
active (primary) infections and persistent infections. We will
assess expression from the .alpha., .beta., and .gamma. FAR in
standard in vitro models of chlamydial persistence, including the
normal human monocyte model utilized by inventors, and treatment of
infected HEp-2 cells with low levels of IFN-.gamma. and
(separately) penicillin G. We also will assess FAR gene expression
in the murine macrophage cell line RAW 264.7. Blood samples will be
procured from volunteer donors, normal monocytes prepared, and
infected at MOI of 1-5 [25,73]. In the normal monocyte model of
infection, persistence is fully in force by 3 d post-infection.
[0138] Samples will be analyzed for transcripts from the
FAR-encoding genes at 6, 24, 48, 72, and 96 hr post-infection in
that model. Translation products will be assessed at the same time
points by western analyses and IHC, as above, to insure that
protein FAR protein levels are increased in some reasonable
proportion to those of the encoding transcripts. As in studies
above for normal active infection of human and mouse cells, FAR
up-regulation will be examined in the monocyte model of persistence
using K, E and C serovars.
[0139] Another model of persistence that has been well studied in
many laboratories is that elicited by treatment of
chlamydiae-infected HEp-2 cells with low levels of recombinant
IFN-.gamma.[82,83]. Persistence elicited by penicillin G has been
somewhat less extensively studied, but recent results from APH's
laboratory indicate that the transcript pattern of chlamydial cells
in which persistence has been elicited by IFN-.gamma. treatment vs
penicillin G treatment are not entirely congruent [APH, manuscript
in prep.; see also [72,84]]. In experiments parallel to those given
just above for the normal monocyte model, we will elicit
persistence in K, E and C serovar-infected HEp-2 cells and McCoy
cells as extensively described by others, eg, [85]; we will assess
FAR transcripts and translation products as a function of time
post-infection. Time points to be examined include 4, 8, 14, 24,
and 48 hr post-infection.
[0140] Extension to use of serovar E is reasonable because this is
a very common STD serovar in the US, and has been used extensively
by the inventors and others. Results for mRNA and protein analyses
of FAR expression will be compared between the IFN-.gamma. and
penicillin G treated cultures, and both will be compared to results
from the normal monocyte model of persistence.
[0141] Further experiments will extend the culture-based results to
the mouse model of genital chlamydial infection. Female BALB/c mice
will be infected genitally with K and E serovar in separate
experiments, as extensively described by us [86,87]. In additional
experiments, BALB/c will be ocularly infected with C serovar [54].
Tissues will be procured at 1, 3, 7, 10 and 12 d post-infection for
analysis to determine relative levels of mRNA from the three
FAR-encoding genes, using real time RT-PCR as above. Western
analyses also will be performed to assess translation product
levels from those genes. Tissues to be so analyzed include the
synovia, genital tracts, conjunctivae, and tissues in which
nanoparticles initially accumulate such as liver, spleen or
kidneys. Results from these analyses will be compared with those
from the various in vitro model systems above.
[0142] The results will provide molecular background, and will
provide a significant amount of new data regarding responses of the
various host cell types to chlamydial infection. The results may
also further demonstrate differences in host responses to ocular vs
genital serovars of C trachomatis. These studies will distinguish
between targeting inflamed, infected tissues and expected clearance
via other tissues.
Example 18
Targeting of Chlamydia-Infected Cells by Folic Acid-Derivatized
Nanoparticles Containing Antibiotics
[0143] Data disclosed in the examples above strongly suggest that
folic acid-conjugated PLGA particles labeled with 6-coumarin home
to C trachomatis-containing inclusions at 24 hr and later
post-infection in actively infected HEp-2 cells. It remains to be
demonstrated, however, whether that homing will obtain at
significant level at all times post-infection, whether homing will
occur in cells harboring persistent chlamydiae, whether the
efficacy of particle delivery is higher at one or more specific
times post-infection, and whether it will occur at all/some times
post-infection in epithelial cells (active) or monocytic cells
(persistent) harboring C trachomatis.
[0144] To address these issues, fluorescently-labeled, folic acid
conjugated PLGA particles will be added to cultured epithelial
cells (HEp-2) and normal human monocytic cells and uptake will be
assessed by fluorescence microscopy. In addition, using
fluorescence microscopy, we will study the intracellular
trafficking of nanoparticles in infected and uninfected cells to
understand the mechanism of nanoparticle targeting to inclusions.
Those data will be correlated with data from Example 17 regarding
the relative level of upregulation of each folate receptor cell
type in the various host cell types. If targeting is equivalent at
all times examined for both serovars in all cell types examined, as
we expect, we will prepare particles containing the antibiotics
azithromycin and rifampin and determine the bactericidal and
bacteriostatic activity in cell culture.
[0145] To be able effectively to target chlamydial inclusions, it
is important to determine the times post-infection when the uptake
of folate-conjugated nanoparticles is the highest. Further, it is
also essential to demonstrate that host cells harboring persistent
organisms take up targeted nanoparticles. HEp-2 cells will be
infected with C trachomatis serovars K, E, or C in separate
experiments, and nanoparticles will be added to the infected cells
at 0 hr, 2 hrs, 6 hrs, 12 hrs, 18 hrs, 24 hrs, 36 hrs and 48 hrs
post-infection; fluorescently-labeled nanoparticles will be used
for the studies. Nanoparticles with folic acid, nanoparticles with
just PEG and no folic acid, nanoparticles without PEG and folic
acid will be used as treatments with the latter two nanoparticle
types serving as controls. Cells will be imaged over a period of 60
min following nanoparticle addition, and uptake of the various
particles into infected vs uninfected cells (and inclusions, see
just below) will be correlated with temporal FAR expression during
chlamydial infection from examples above.
[0146] In order to further confirm the effect of microbe's
developmental stage on nanoparticle targeting to inclusions, we
will quantify particle levels in the inclusion using HPLC. HEp-2
cells will be grown in 24 well plates and infected as before.
Fluorescently-labeled nanoparticles will be added to the infected
cells at 0 hr, 2 hrs, 6 hrs, 12 hrs, 18 hrs, 24 hrs, 36 hrs and 48
hrs post-infection. Cells will then be lysed and protein content of
lysates determined using the Pierce protein assay kit.
[0147] The developmental stage of the microbe will be confirmed by
determining the expression of specific genes known to be expressed
at different stages of the developmental cycle. These will include
but not be limited to early genes (eg, omp1, others), middle period
genes (eg, pyk, dnaA, others), and late genes (eg, omcB, others)
[see [88]]. Cell lysates will then be lyophilized and the
fluorescent label will be extracted using methanol.
[0148] Nanoparticle concentration in the inclusions will be
determined by HPLC. Increased nanoparticles uptake into cells may
not reflect increased uptake into the inclusions. However, we
expect that any increase in cellular uptake of nanoparticles
following infection is due to FAR overexpression and a general
increase in cellular uptake of nanoparticles will translate into
increase in nanoparticle accumulation in inclusions. We will
confirm the results of this study by quantitating the
nanoparticles-associated fluorescence in inclusions using confocal
microscopy.
[0149] Examples above indicated that folate-conjugated
nanoparticles rapidly (<10 min) target chlamydial inclusions
when added to infected host cells in culture (FIG. 12). However,
the mechanism of this targeting is not known. We will investigate
the intracellular trafficking of nanoparticles with and without
conjugated folic acid in chlamydiae-infected cells. It is well
established that chlamydial inclusions are neither acidified nor
fusogenic with lysosomes. It has been shown that the inclusion,
rather than interacting with the endocytic pathway, is fusogenic
with exocytic vesicles containing sphingomyelin and cholesterol on
their way from the Golgi apparatus to the plasma membrane
[89-91].
[0150] While the question of whether there is any direct folic acid
receptor-mediated endocytosis at the inclusion membrane is not yet
answered, studies [29] suggest that there could be some overlap
between the host folate receptor-mediated endocytosis and
cholesterol transport. Folate receptors (.alpha. and .beta.
isoforms) belong to a special class of membrane proteins, namely
glycosylphosphatidyliositol (GPI)-anchored proteins.
[0151] GPI-anchored proteins are trafficked differently from
transmembrane-anchored proteins such as transferrin receptors. It
has been shown that the trafficking of GPI-anchored proteins is
regulated by cellular levels of cholesterol and sphingolipids.
Further, GPI-anchored proteins are thought to be constituents of
lateral nonhomogeneities in the exoplasmic leaflet of the plasma
membrane termed as rafts.
[0152] While there is some disagreement in the literature on the
role of the rafts in chlamydial entry into host cells (for example,
[92]) Stuart et al showed that C pneumoniae, C psittaci, and C
trachomatis serovars E and F (but not serovars A, 36B, and C, LGV,
L2 and MoPn) enter host cells via cholesterol-rich lipid raft
microdomains [93]. Because the inclusions (organism) may use the
rafts to derive cholesterol and sphingolipids [89,93,94], we expect
nanoparticles to also use the same pathway to traffic to
inclusions. To test this hypothesis, we will determine the effect
of inhibition of cholesterol transport on nanoparticle trafficking
to inclusions.
[0153] Fluorescently-labeled nanoparticles with and without folic
acid will be administered to HEp-2 and normal human monocytes at
various times post-infection and then visualized using the RTM-3
microscope under fluorescence mode and Richardson contrast mode to
define the localization of nanoparticles inside the cells with
reference to other organelles. The fluorescent label (6-coumarin)
stays attached to nanoparticles and does not leach out in the time
frame of the studies proposed here but control experiments will
deliver 6 coumarin alone to infected cells to confirm this in the
chlamydia infection models [38,44,95]. Images will be obtained at
5, 10, 15, 30 and 60 min post-nanoparticle addition. Initially,
localization of nanoparticles within the endocytic pathway will be
determined using markers for early endosomes and lysosomes.
[0154] Texas red-conjugated transferrin will be used as a marker
for early endosomes while Lysotracker Blue will be used as a marker
for acidic lysosomes. Presence of nanoparticles in these
compartments will be determined by the colocalization of
nanoparticle-associated green fluorescence with marker-associated
red or blue fluorescence.
[0155] Following this, the ability of the LDL pathway to contribute
to nanoparticle trafficking will be investigated. Cells will be
incubated with nanoparticle suspension prepared in lipid-deficient
serum in the presence of ammonium chloride to prevent the
acidification of lysosomes [96]. If the LDL pathway contributes to
trafficking of nanoparticles to inclusions, ammonium chloride is
expected to reduce or prevent this trafficking. Finally, we will
study the effect of Brefeldin A and nocodazole on nanoparticle
trafficking to inclusions. Brefeldin A is a Golgi inhibitor while
nocodazole inhibits microtubules. Previous studies [89] showed that
both these inhibitors reduce cholesterol and sphingolipid
accumulation in inclusions through the exocytic pathway. If
nanoparticles use the same pathway to accumulate in inclusions,
these inhibitors should reduce nanoparticle trafficking to
inclusions if the inhibitors are added prior to incubation with
nanoparticles.
[0156] The effect of above inhibitors on nanoparticle accumulation
in the inclusions will be determined using fluorescence microscopy
with image analysis as above. We will confirm the results of the
microscopic studies by quantitation of nanoparticle levels in the
inclusions following different treatments. EBs will be purified
from treated cells by density gradient centrifugation and then
extracted with methanol. The methanolic extract will be analyzed
for 6-coumarin concentration by HPLC and the results will be
expressed as the amount of nanoparticles per mg chlamydial protein.
(see Methods); controls will be infected cells pulsed with
nanoparticles and extracted directly for HPLC analysis. In an
independent set of experiments, we will determine the effect of the
above inhibitors (without nanoparticle treatment) on inclusion
counts to confirm that these treatments do not affect chlamydial
viability.
[0157] As discussed above, monotherapies with antibiotics such as
azithromycin, doxycycline or rifampin are effective in eliminating
acute (active) infections but are less effective against persistent
forms. A few previous studies have shown that the combination of
azithromycin and rifampin is effective against both C trachomatis
and C pneumoniae [27,28]. In this example, we will determine the
efficacy of azithromycin and rifampin combination administered in
folic acid conjugated nanoparticles against persistent infection.
We have already optimized the formulation conditions for loading
the combination of azithromycin and rifampin in nanoparticles.
Studies by others show that a combination of 0.5 .mu.g/ml of
azithromycin and 0.015 .mu.g/ml of rifampin was effective in
reducing C trachomatis serovar K infection in HEp-2 cells [97]
while a combination of 0.1 .mu.g/ml of azithromycin and 0.075
.mu.g/ml of rifampin was effective against C pneumoniae in vitro
[98]. Based on our initial studies above, we will use concentration
ranges of 0.01 to 0.5 .mu.g/ml of azithromycin and 0.001 to 0.1
.mu.g/ml of rifampin in our studies.
[0158] Infected cells will be treated with nanoparticles loaded
with both antibiotics, nanoparticles loaded with either antibiotic,
empty nanoparticles, antibiotics in combination or separately
dissolved in the medium or medium alone. Treatments will be tested
on HEp-2 and normal monocytes for human models; for mouse cell
models, we will use McCoy because they are good for drug titrations
and RAW 264.7 cells. Loaded nanoparticles will be given at various
times post-infection as determined above, allowed to home to the
inclusions for 24-48 hr, then cultures will be harvested for
determination of viable chlamydiae by i) real time PCR (APH lab)
targeting the relative level of chlamydial chromosome relative to
that of control cells (identically infected but given empty
particles or no treatment), and recovery of IFU by titration of
lysates on fresh indicator cells (JAW-H lab).
Example 19
Therapeutic Efficacy of Antibiotic-Loaded Nanoparticles In Vivo
[0159] Following infection at the epithelial surfaces of the
urogenital system, C trachomatis can ascend to the upper
reproductive tract, and then disseminate to distant anatomic
locations such as the joint, to engender chronic inflammatory
sequelae. Initial experiments suggested that folic-acid conjugated
nanoparticles home to infected synovial tissues in a mouse model of
C trachomatis-induced arthritis. Whole animal imaging study
indicated that appropriately labeled folic acid-conjugated
nanoparticles might be useful for imaging infection sites to
monitor the progression or eradication of infection in addition to
their use in drug delivery.
[0160] By a combination of in vivo imaging, molecular genetic, and
other methods, we will extend these results to determine if
delivery of antibiotic-loaded nanoparticles to Chlamydia-infected
mice clears persistent synovial infection in vivo. We will define
the host and pathogen responses to delivery of antibiotic loaded
nanoparticles that reduce synovial chlamydial load after mucosal
infection. We will determine if reduction in synovial chlamydial
load correlates with localization of nanoparticles and reduced
synovial inflammation, and whether nanoparticle-delivered therapy
alters the articular/synovial persistent and/or acute
infection.
[0161] We have obtained key molecular data that persistently
infected cells are present in mouse knees after genital infection
of mice with serovar K-EB. Because it is a more prevalent genital
serovar than K, we will test E serovar in BALB/c and possibly C3H
mice to confirm the observation. Next, antibiotic-treated mice will
be compared to untreated controls (Table 1 below shows the
different treatment groups). Treatments will be administered at a
time point corresponding to maximal tissue folic acid expression
observed above in Example 17.
[0162] Following treatment, animals will be euthanized at 48 h and
later time points (a time course will be performed) and different
tissues including the synovium, genital tract, and liver will be
collected. Nucleic acids will be extracted from synovium of hind
knees and other tissues from at least five mice per group. PCR will
confirm that total chlamydial DNA (16S rRNA) is reduced after
antibiotic treatment. cDNA will be prepared from RNA isolated from
these groups using standard techniques and quantitative real time
RT-PCR will be performed targeting genes whose
expression/non-expression is characteristic of acute vs persistent
chlamydial infection.
[0163] Initially we will use the genes for chlamydial cell division
(eg, dnaA vs ftsW), but additional genes such as the differentially
expressed Hsp60 genes will be targeted as additional
genes/functions associated with persistence in human patients/cells
are identified. The mouse arthritis model, based on adequate
quantities of DNA and RNA can be extracted to perform the proposed
studies. Opposite hind knees and paws will be used for
histopathology in some experiments.
TABLE-US-00001 TABLE 1 Treatment groups Group # Treatment Purpose 1
Azithromycin + Rifampin To determine efficacy of NP - Targeted
targeted combination therapy 2 Azithromycin NP - Targeted To
determine efficacy of targeted mono therapy 3 Rifampin NP -
Targeted To determine efficacy of targeted mono therapy 4 Blank NP-
Targeted Control for groups 1 to 3 5 Azithromycin + Rifampin
Non-targeted control for group 1 NP - Non-targeted 6 Azithromycin +
Rifampin Solution control for group 1 free in solution 7
Azithromycin free in solution Solution control for group 2 8
Rifampin free in solution Solution control for group 3 9 Vehicle
Untreated control
[0164] We will determine whether reduced joint inflammation as well
as chlamydial load are results of reduced acute and/or persistent
genital tract infection vs reduced dissemination to joint vs active
anti-chlamydial responses occurring within the joints. We will test
whether there are fewer organisms in genital tract/conjunctivae
(site of primary infection) following targeted antibiotic therapy
based on chlamydial DNA/RNA. If reduced chlamydiae are detected in
joints and genital tracts/conjunctivae of treated compared to
non-treated mice, this will support reduced dissemination from
genital tract.
[0165] If there are fewer acutely or persistently infected
monocytes/DC in GT, joint protection would be attributed to an
antibiotic therapy-induced reduction in total load and thereby
reduction in persistently infected cells either at the level of the
genital tract or during trafficking from genital tract. If there is
no detectable change in GT load based on treatment but reduced knee
pathology/load, we will conclude that local, intra-articular
antimicrobial effects probably contribute to reduced arthritis most
significantly (see below). Several time points will be tested
initially (d. 4, 6, 8, 10 pi) using only the targeted combination
therapy nanoparticles (Group 1; Table). Using the data obtained
from the initial studies, we will narrow the window for testing of
other groups as indicated in the Table.
[0166] Reduction of chlamydial load in synovium will be determined
by screening PCR followed by real time PCR/RT-PCR on positive
samples. We will establish a standard curve for chlamydial
chromosome number and transcripts relative to IFU input from
purified EB stock. Our hot phenol extraction methods allow for both
DNA and RNA isolation. Given the smaller amount of total nucleic
acids we can obtain from mouse synovium (average, 10 .mu.g) we will
limit our tests for the acute/persistence infection to assays for
genes expected to be positive and negative as described earlier. If
inter-mouse variability is low, we may pool either 2 knees/mouse or
2-4 knees/2-4 mice to allow a larger panel of genes to be tested by
qPCR.
[0167] Synovial tissue, while not as complex as the genital tract,
has some `black box` features. For instance, in human reactive
arthritis (ReA), it is not known whether the inflammation begins at
the level of the synovial lining cells or at vessels from which
activated, infected cells egress. Synovial macrophages have been
shown by EM/IEM to harbor EB/RB-like particles by the JWH/APH
collaborator HR Schumacher [10] and more recently HRS/APH showed
IEM of aberrant RB in human synovium [99]. There is only one very
early report of culture-positive samples from synovial fluid. It is
now appreciated by most in the field that persistent chlamydial
infection of reactive arthritis joints is a result of localization
of cells/monocytes harboring viable, metabolically active C
trachomatis beneath the synovial lining cells [79].
[0168] We have shown previously that Th1 and Th2 cytokine
expression is upregulated in synovial tissues from ReA mice, as was
shown for human ReA synovium (JW-H, unpublished). Our preliminary
studies with mouse synovium showed that genital infection resulted
in upregulation of transcripts for IL-4, IL-10, IL-12 and
TNF.alpha., with lesser increases in IL-12 at the time point
assessed (d 21 p.i.). We expect to detect downregulation of
pro-inflammatory responses and clearing of infected cells in
successfully treated mice.
[0169] The most direct method to test this possibility initially
will be to use real time PCR/RT-PCR to assess key
cytokine/chemokine transcript levels with respect to chlamydial DNA
load with and without nanoparticle-mediated therapy. Knees from the
treated mouse populations (treatments as in the Table) will be
removed and either snap frozen for DNA/RNA extraction or, for some
mice, synovium will be dissected from under the patella using a
high-power dissecting microscope. The latter samples will be
processed immediately for molecular and culture experiments, or
snap-frozen until processing for NP extraction. Knees/paws from
additional mice will be fixed for histologic studies.
[0170] An important strength of the model is that we have assessed
the genital tract/joint axis in several mouse strains and currently
we are expanding the chlamydial serovars tested. The window of
biological relevance for events involving nanoparticle delivery to
both genital tract and distant tissue can be narrowed quite simply.
We will target knees at 10-21 days post-injection initially;
earlier time points will be used if necessary. Such temporal
studies are simply not possible in studies of patients with
chlamydial STD.+-.ReA, and studies of the dissemination stage via
peripheral blood are limited in both animals and humans. Our
proposed studies in the murine model offer novel ways to
investigate effects of antibiotic therapy locally, in transit and
at peripheral sites.
[0171] Further, we will determine if inflammatory response
parameters are altered in the synovium of treated mice. We expect
this to be the case, but such responses are unlikely to be
detectable until after dissemination of chlamydia to joints. We
will perform
[0172] RT-PCR (conventional semi-quantitative PCR for selected
cytokine/chemokines and then real time RT-PCR to maximize our
quantitative information on these small samples). The combination
of tests of host responses vs pathogen gene expression will provide
important information regarding the relation of host responses to
the replicative state of the bacteria within synovium and the
effect of targeted antibiotic therapy on the host responses. This
information has implications for future drug development for human
ReA caused by chlamydia initially infecting mucosal tissues.
[0173] Nanoparticle Preparation.
[0174] Antibiotic loaded nanoparticles will be formulated using a
modification of our previously published emulsion solvent
evaporation technique [95]. In a typical procedure, antibiotics
azithromycin and rifampin (16 mg each) along with the polymer PLGA
(30 mg) are dissolved in 1 ml chloroform. This solution is added to
12 ml of aqueous 2% w/v polyvinyl alcohol solution and is sonicated
using a probe sonicator (Misonix) to form water-in-oil emulsion.
Precaution is taken to maintain the temperature of the emulsion
around 4.degree. C. during sonication in order to maintain the
stability of antibiotics. The emulsion is stirred overnight to
evaporate chloroform. Nanoparticles formed are recovered by
ultracentrifugation (140,000.times.g), washed two times with
distilled water to remove unentrapped antibiotics, and then
lyophilized for 48 hrs.
[0175] Nanoparticles with Folic Acid on the Surface.
[0176] Following the preparation of emulsion in polyvinyl alcohol
(see above), a methanol solution (100 .mu.l) of polylactide
(PLA)-PEG-folic acid conjugate is added to the emulsion. This
results in the anchoring of the PLA segments into nanoparticles,
with PEG-folic acid chains on the surface. Following this, the
emulsion is stirred to evaporate organic solvents and nanoparticles
are processed as described above. This procedure was used in the
Preliminary Studies to obtain nanoparticles containing PEG-folic
acid conjugate on the surface (FIGS. 3-5).
[0177] Nanoparticle Characterization.
[0178] To quantitate antibiotic loading, nanoparticles will be
incubated with methanol for 8 hrs, and the concentration of
azithromycin and rifampin in methanol extract will be determined by
HPLC. For azithromycin, a Beckman HPLC system consisting of C-18
column (Beckman) heated at 50.degree. C. and UV detection (210 nm)
will be used for drug quantification. Two mobile phases (Mobile
phase A: Phosphate buffer pH 8.5:Methanol (90:10) and Mobile Phase
B: Acetonitrile:Methanol (90:10)) in the ratio of 25:75 will be
used at a flow rate of 1 ml/min. For rifampin, a similar HPLC setup
and column conditions will be used. Mobile phase consisting of
phosphate buffer (pH 6.8) and acetonitrile (50:50) and UV detection
(238 nm) will be used. To determine the release of antibiotics,
nanoparticles (1 mg/ml) will be suspended in PBS (pH 7.4; 0.15 M)
containing 0.1% Tween 80 (to maintain sink conditions), and kept at
37.degree. C. and 100 rpm. Antibiotic concentration in the release
buffer will be determined by HPLC.
[0179] Nanoparticles that release different doses of antibiotics
will be formulated by varying the dose-ratios of antibiotics in the
formulation and by using polymers of different molecular weights
and hydrophobicity. PLGA polymers of different molecular weights
and composition are available commercially (Birmingham Polymers).
Particle size and size distribution of nanoparticles will be
determined by dynamic light scattering (DLS) and atomic force
microscopy (AFM). For DLS studies, a suspension of nanoparticles (1
mg/ml) will be subjected to particle size analysis in 90Plus
particle size analyzer (Brookhaven). For AFM studies, a suspension
of nanoparticles (1 mg/ml) will be added to silicon substrate that
is pre-coated with polyethyleneimine, and air-dried. AFM images
will be obtained with an E scanner (Nanoscope III, VEECO).
[0180] Determination of Nanoparticle Concentration in Cells and
Organelles.
[0181] Nanoparticle concentration in cell or organelle lysates will
be determined using our previously published HPLC assay procedures
(REF). Lysates will be initially lyophilized to maximize the
extraction efficiency. Following this, 1 ml methanol will be added
to the lyophilized samples and incubated for 6 hr. Concentration of
the fluorescent label, 6 coumarin, will be determined using HPLC. A
C-18 column (4.6 mm.times.25 cm) with 5 um (fix micron) packing
(Beckman) will be used. Separations will be achieved using
acetonitrile:water:1-heptane sulfonic acid sodium salt
(65:35:0.005M) as the mobile phase. 6-coumarin will be quantified
using a fluorescence detector (Jasco; .lamda.(Ex) 450
nm/.lamda.(Em) 490 nm). Concentration of nanoparticles will be
determined from a standard curve of peak area versus nanoparticle
concentration, and the results expressed as amount of nanoparticles
normalized to cell protein.
[0182] Infection of Cell Lines and Primary Monocytes Cultures.
[0183] Various cell lines will be used. In most cases, McCoy
(mouse) or HEp2 (human) cells will be infected. The former will be
used for titrations of chlamydial stocks and for drug dose-response
assays. Cells will be infected with an MOI .about.1 in 96 well
plates. Plates will be centrifuged at RT for 1 hr (1200.times.g),
incubated at 37 C for 1 hr, then overlay medium containing 0.5-1.0
ug/ml cycloheximide applied; for antibiotic titrations, antibiotics
will be added to overlay medium.
[0184] Genital or Conjunctival Chlamydial Infection of Mice
[54,67,100].
[0185] Mice are DepoProvera treated prior to challenge with human
biovars of C trachomatis. Serovars K and E are currently in use as
genital challenge inocula; C serovar will be used for ocular
(conjunctival) infection. These and other chlamydial stocks are
grown and Percoll purified in the JWH laboratory. Challenge doses
usually are 5.times.10.sup.6-10.sup.7 IFU delivered topically
intravaginally in 30 .mu.l SPG; for ocular challenge, 5000 IFU
(.about.5.times.10.sup.6 IFU) are topically delivered in 5 .mu.l to
each eye. High-titered crude stocks are used in some experiments.
Vaginal or conjunctival swabs are collected for culture and DFA at
weekly intervals until the end of each experiment.
[0186] Uninfected mice kept in separate filter-top cages will be
examined and swabbed on the same schedule to control for
nonspecific effects of procedures. After swab sample collections,
samples are randomized and then coded; the code is only broken by
the PI after results are obtained.
[0187] Specimen Collection.
[0188] Genital tracts (GT and Conjunctivae) are dissected out using
sterile technique and new instruments for each tissue per mouse.
For histopathology combined with culture or molecular analyses GT
is divided into ovary/oviduct/upper one-third of uterine horn
(R1/L1); mid-uterine horn (R2/L2); lower horn with cervix/vagina
(R3/L3). The cervix/vagina is divided at the bifurcation of the
horns (one half for molecular study, the other embedded for
histology) for snap freezing (molecular analysis) or OCT embedding.
For confirmation of NP targeting, half of the GT or one
conjunctival sample will be frozen for extraction and HPLC analysis
of 6-coumarain. Tracts are photographed in situ as well as ex vivo
for documentation of vaccine effects to correlate with histology,
with mouse number included in each image. Signs of inflammation are
scored at the time of sacrifice based on edema, vascularity, overt
inflammation (color change). DepoProvera treated uninfected mice
are included as controls in all experiments. Conjunctivae are
obtained as we published [54]; hormone treatment is not needed.
[0189] Knees/Synovium.
[0190] After sacrifice by exsanguinations, hind legs are removed
aseptically after removing skin from hind legs. One knee is
generally snap frozen for sterile harvesting of synovium, and the
other hind leg is formalin fixed. In some experiments, synovium
will be dissected on fresh tissues under a binocular microscope.
This tissue would be snap embedded in OCT for histology, or snap
frozen for biochemical and/or molecular analyses. Since samples are
so small, synovium from both hind legs would be collected and
pooled for this purpose. Paws/ankles will be processed
separately--these are often the first joints inflamed in other
rodent arthritis models. These methods are published [67,100].
Selected tissues will be collected to determine loads of NP (based
on extraction of 6-coumarin), homing of NP to inflamed tissues vs
to expected routes of clearance; tissues may include spleen, liver,
lungs, kidneys, genital tracts/conjunctivae and joints.
[0191] Microbiologic Assays.
[0192] Culture/DFA will be performed from genital or conjunctival
swabs by standard methods. Inclusions are graded on a 0-4+ scale on
a fluorescent microscope; mean inclusion counts/ml for each mouse
are calculated from first passage samples in duplicate 96 wells.
DFA smears are processed as for monkeys or humans except a minimum
of 200 cells will be used for conjunctival smears and total EB
scored (0-4+ scale) for each smear. Antibiotic dose-response curves
will be generated with infected McCoy or other susceptible cells
for each preparation of drug-loaded NP to ensure expected loading
prior to delivery in vitro or in vivo. Molecular screening for
chlamydial DNA will be used regularly as proposed under the
specific aims and in our published studies. Chlamydia-specific
antibodies are commercially available [54,87,100,101].
[0193] Molecular and Cellular Assays.
[0194] Extraction and purification of DNA and RNA will be by
standard methods using hot phenol or Trizol [102,103]. We routinely
prepare RNA from small samples of murine tissues and cells,
including mouse synovium. Particular care is taken to avoid
cross-contamination of samples with chlamydial DNA/RNA; animal
dissections, nucleic acid extractions, and molecular assays are
each performed in different rooms. The hoods used for RT reactions
are not used for assays involving intact or live chlamydia; indeed,
those hoods are in separate buildings. Standard negative controls
are included in assays to document integrity of samples and absence
of chlamydial/other DNA.
[0195] RT-PCR will be used in some experiments to detect cytokine
mRNA in mouse genital tissues, conjunctivae and LN under different
experimental conditions; the co-PI has published the methodology to
be used [67,104-107] Quantitative real time RT-PCR (qPCR) is in use
in the co-PI's and co-I's labs for both chlamydial and host gene
expression [25,73,74,108,109] (see Biosketches); cDNA obtained by
RT reactions with random hexamer primers are used for q-PCR
reactions on either the Applied Biosystems 7700 or Roche Light
Cycler using SybrGreen for product detection. There is also a new
Applied Biosystems 7500 instrument in the co-I's laboratory. All
primers for qPCR for chlamydial genes have been (re)designed for
use with mouse tissues/cells guided by the primers used in our
human studies; preliminary tests have determined that no products
are seen when chlamydia-specific primers are used for uninfected
mouse cells.
[0196] Immunohistochemical Staining.
[0197] ABC immunoperoxidase or fluorescent antibody staining is
used for detection of cellular and chlamydial antigens and
cytokines. Antibodies to molecules of interest are available
commercially or through ATCC. Immunohistochemical analyses of
surface antigens and receptors, and cytokines in tissue sections
and single cell preparations are published [105,110]. Molecular
Probe bioprobes will be used for dual staining under wavelengths
using living cells on the RTM-3 microscope. Additional experiments
will use a Nikon E600 epifluorescence microscope with appropriate
filters to analyze co-expression; results will be documented by
digital photography and analyzed with ImagePro software in the PI's
lab.
[0198] Real time microscopy will also be performed with the RTM3
microscope to test how infected target cells respond to various
antibiotic delivery approaches. These images will be digitally
captured on DVC tape and in Volocity (Improvision Inc) software
(Mac G4 platform) customized for the RTM-3; heated perfusion
chambers will allow longer term experiments and time lapse
photography documentation.
[0199] EM/IEM.
[0200] EM/IEM analyses are used to extend our observations in
synovium, and to identify cells containing organism, and the state
of the organism (classical or aberrant forms).
[0201] Statistical Analyses.
[0202] Relative transcript levels will be compared by treatment
.+-.infection after chlamydial and host values are normalized to
16S rRNA and 18S rRNA, respectively. ANOVA will be used to compare
results from different treatment groups. Student's t-test will be
used to compare differences between treatment groups (chlamydial
titers). Gene expression differences between treatment groups will
be analyzed by Kruskal-Wallis one-way ANOVA on ranks, with multiple
comparisons of groups done by Student-Newman-Keuls method. Repeated
measures ANOVA will be used for in vivo time-course
experiments.
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Example 20
Comparison of Free and Nanoparticle-Encapsulated Antibiotics
[0313] Free and nanoparticle-encapsulated antibiotics added at
either t0 (time of infection) or after 24 hr of infection (t24),
were compared along with blank nanoparticles as controls. As shown
in FIG. 20, blank particles at the same particle concentrations
used for each antibiotic had no effect on inclusion numbers even at
the higher concentrations of nanoparticles. In this same
experiment, rifampin-loaded nanoparticles significantly reduced
inclusion counts near the MIC50 to the highest drug concentration.
Similar results were obtained for Azithromycin added at t0, but
results for the latter in nanoparticles did not differ from free
drug with t24 hr addition.
[0314] Rifampin is highly unstable in solution compared to
Azithromycin (the t1/2 of Rifampin is hours compared to days for
Azithromycin). The data herein suggest that encapsulation of
Rifampin increases the effective t1/2 of this antibiotic. In vitro
release experiments under simulated physiologic conditions showed
that nanoparticles release about 60% of the encapsulated rifampin
in 24 hrs and the remainder is released over 7 days (FIG. 21).
Increased t1/2 by delivery of antibiotics or other drugs
encapsulated in nanoparticles has important implications for in
vivo therapeutic applications.
[0315] All publications herein are incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference.
[0316] One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. Indeed, the
present invention is in no way limited to the methods and materials
described.
* * * * *