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Enhanced intraphagocytic killing of Brucella abortus in bovine mononuclear cells by liposomes-containing gentamicin
Veterinary Immunology and Immunopathology , 8 (:1985) 171--182 Elsevier Science Publishers B.V., A m s t e r d a m -- Printed in The Netherlands
171
ENHANCED INTRAPHAGOCYTIC KILLING OF BRUCELLAABORTUSIN BOVINE MONONUCLEAR CELLS BY LIPOSOMES-CONTAINING GENTAMICIN C. DEES*, M.W. FOUNTAIN, 3.R. TAYLOR, AND R.D. SCHULTZ* Animal Health Research and Department of Microbiology, School of Veterinary Medicine and Agriculture Experiment Station, Auburn University, AL 36849{U.S.A. {Accepted 29 June 1984)
ABSTRACT Dees, C., Fountain, M.W., Taylor, J.R., and Schultz R.D., 1985. Enhanced Intraphagocytlc k i l l i n g of Brucella abortus in bovine mononuclear cells by llposomes-contalnlng gentamlcln.
Vet. Immunol. Immunopathol., 8: 171-182.
In v i t r o Intraphagocytlc k i l l i n g of Brucella abortus in bovine mononuclear leukocytes was enhanced by cationic, anionic, and neutral multllamellar llposomes-contalnlng gentamlcln.
Free gentamlcln not entrapped in llposomes
and llposomes without a n t i b i o t i c dld not enhance intraphagocytlc k i l l i n g of B__~. abortus in bovine phagocytes. In vlvo k i l l l n g of B. abortus in guinea pigs was also enhanced by llposomes-contalnlng gentamlcln when compared to free gentamlcln.
Llposomes-contalnlng alpha tocopherol acetate failed to enhance
In vlvo k i l l i n g of B. abortus.
INTRODUCTION Few bacteria are able to survive for long periods inside phagocytic cells. B__~.abortus, however, is not only able to survive for long periods of time within leukocytes but can also replicate within phagocytic cells (Phllllpon e t . a l , ]g77), Schaffer e t . a l . , 1953). The a b i l i t y of B. abortus to survive and replicate in phagocytic ceils is an important factor when one considers the nature of the disease produced by thls bacterium.
The a b i l i t y of
B. abortus to survive I n t r a c e l l u l a r l y can result in long term chronic disease (Schaffer e t . a l . , 1953). Relapses following antlmlcroblal therapy can also be explained by the I n t r a c e l l u l a r location of B. abortus since I n t r a c e l l u l a r Brucella are protected from treatment by most antibiotics (Schaffer e t . a l . , 1953) *Authors to whom reprint ruquests should be addressed: Departmentof Pathoblologlcal Sciences, School of Veterinary Medicine, University of Wlsconsln-Madlson 53706. Current Addresses; M.W. Fountain, The Liposome Co., Princeton, N,J. 08540 J.R. Taylor, USDARegional Parasite Lab, Auburn, AL 36849. 0165-2427/85/$03.30
© 1985 Elsevier Science Publishers B.V.
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Therefore, new treatment regimens and delivery vehicles for antlmlcroblal agents need to be developed to enhance penetration of antibiotics into phagocytic cells to k i l l i n t r a c e l l u l a r B. abortus. Recently, liposomes (multilamellar vesicles) were reported to be effective delivery vehicles for a number of chemotherapeutic agents including dlgoxln (Ryman et. a l . , 1978), insulin (Fountain et. a l . , 1980), and antibiotics (Bonventre and Gregorladias, 1978; Morgan and Williams, Ig80).
Delivery of
antibiotics via liposomes has several potential advantages: l ) prolonged serum levels of a n t i b i o t i c ; 2) antibiotics may be targeted to sites not reached by free a n t i b i o t i c ; 3) antibiotics may be delivered into phagocytic cells resulting in enhanced I n t r a c e l l u l a r k i l l i n g of bacteria.
(Bonventreand
Gregoriadias, 1978; Fountain et. a l . , 1982; Morgan and Williams, 1980). Bonventre and Gregorladlas (19BO) reported that Intraphagocytlc k i l l i n g of Staphylococcus aureus is enhanced by llposomes-contalnlng dlhydrostreptomycln.
I t has also been reported that llposomes-contalning
streptomycin enhance I n t r a c e l l u l a r k i l l i n g in vivo of Mycobacterlum tuberculosis (Vladimirsky and Hadlgina, 1982). Dees et. a l . , (1983) has also reported that llposomal delivery of gentamicln can enhance the penetration of gentamlcln into bovine phagocytic cells when compared to free drug.
I t was also suggested that gentamlcln
inserted into phagocytic cells had a long Intracellular survival time (Dees, et. a l . , 1983). Liposomal delivery of gentamlcln might be useful in enhancing the Intraphagocytlc k i l l i n g of B. abortus.
Secondly, since llposoma delivered
gentamlcln has a long survival time in bovine phagocytes, i t may be possible to insert gentamicln into a phagocytic cell before i t ingests a pathogenic bacterium llke B. abortus.
Thus, a phagocytic c e l l ' s a b i l i t y to k i l l B.
abortus or other bacteria might be prophylactically enhanced by llposome delivery of gentamicln prior to phagocytosls of the bacteria preventing any p o s s i b i l i t y of long term intraphagocytlc survival. Th~s report presents the results of in v i t r o and in vivo studies to determine the effects of llposomes-contalnlng gentamlcln on the Intracellular k i l l i n g of B. abortus in bovine phagocytic cells. Hopefully, llposomal delivery of antibiotics w i l l prove useful in enhancing intraphagocytlc penetration of a n t i b i o t i c , targeting of antibiotic to organs of the retlculoendothellnal system, and prophylactically protecting phagocytic cells from colonization wlth B. abortus.
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MATERIALS AND METHODS Liposome preparation Charged and neutral llposomes (multilamellar) were prepared as previously described (Fountain et. a l . , 1980; Magee, 1978; Fountain et. a l . , 1982). Lipid Films of a ?:2 molar r a t i o of phosphatldylcholine (PC) (Avantl Polar Lipids, Birmingham, A]) and dlcetylphosphate (DCP), 7.2 molar r a t i o of PC and stearylamlne (ST), and PC alone were drled under nitrogen.
The t o t a l
phosphollpld concentration for each llposome preparation was lO mg/ml. Gentamlcln sulfate was supplied as a lyophillzed preservative free powder (Scherlng Veterinary, Kenilworth, N.Y.) s t e r i l e normal saline.
Gentamlclnpowder was rehydrated with
A one ml solution of rehydrated gentamlcln sulfate at
a concentration of 50 mg/ml was added to the dried l i p i d films. Liposomes were also prepared from a 7:I:0.5 ratio of PC, ST and Vitamin E (a tocopherol acetate) and PC, cholesterol (Chol), ST (?:2:1) that used for In vlvo studies. The individual llposome preparations were agitated and then passed over a Sephadex G-50 column with Hank's Balanced Salt Solution as the elutlng buffer.
The llposomal entrapped gentamlcln concentrations for neutral,
anionic, and cationic llposomes was determined by biological assay using S. aureus ATCC strain 25923 similar to gentamlcln determinations described previously (Bennet et. a l . , 1966; Morgan and Williams, 1980). Intrallposomal gentamlcln concentrations were also confirmed by radloenzymatlc assay as described (Fountain et. a l . , 1982). In v i t r o k i l l i n g assays Mononuclear phagocytic cells were obtained from heparinlzed bovine peripheral blood by Ficoll-Hypaque (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) density gradient centrlfugation similar to procedures described previously ( T e r r i t o et. a l . , 1976). Mononuclear cells at the plasma-Flcoll interface were removed and placed In RPMI-1640 medium (Gibco Inc., Grand Island, N.Y.).
Mononuclear cells were counted with electronic
cell counter and placed in tissue culture flasks.
The mononuclear cells were
incubated for one hour at 39°C. The non-adherent cells ( p r i m a r i l y lymphocytes) were removed by decanting the RPMI-1640 medium. The adherent cells ( p r i m a r i l y monocytes) were used for incubation with B. abortus and
174
llposomes.
The number of adherent cells was determined by calculating the
difference between the i n i t i a l
c e l l counts and the c e l l s (not adhering)
removed In the RPMI-1640. Mononuclearphagocytic cells at a concentration of approximately l.O x lO ? cells/ml were used In a l l studies. Bovine phagocytic cells were incubated wlth B. abortus strain 19 (Colorado Serum Co., Denver, CO) or s t r a i n 2308 at concentrations approximately equal to t h a t of the phagocytic c e l l s for a period of 24 hours at 39°C in RPMI-1640 medium. B o t h strains of B. abortus used in the in v i t r o k i l l i n g assays were found to be sensitive to gentamlcln and the minimum i n h i b i t o r y concentration of gentamlcln for both b a c t e r i a l strains was determined to be less than O.5~g/ml.
Pooled bovine sera from B. abortus infected c a t t l e , heat
i n a c t l v a t e d , and d i l u t e d l:lO0 was used to opslnlze the B. abortus that were incubated wlth the phagocytes.
After 24 hours incubation the unphagocytosed
bacteria were removed by washing the c e l l cultures wlth RPMI-1640 medium. The f i n a l wash was cultured to assure complete removal of free B. abortus.
The
mononuclear c e l l s a f t e r washing were covered wlth a three ml volume of medium and lO0 ~l of the c a t i o n i c , anionic, or neutral llposomes-contalnlng gentamlcln.
Control cultures were incubated wlth appropriate amounts of
charged or neutral llposomes not containing a n t i b i o t i c or wlth free gentamlcln (25 ~g/ml) equal to or greater than gentamlcln In the llposomes.
Phagocytic
c e l l s wlth or without llposomes were incubated for 5 hours 39°C. The unincorporated llposomes were removed by washing wlth RPMI-1640 medium. The l a s t wash was examined by b i o l o g i c a l and radloenzymatlc assays to assure complete removal of gentamlcln.
The number of surviving cells of B. abortus
was determined by freeze-thawlng to lyse the cells and then determining the number of bacterial colonies s i m i l a r to procedures previously described (g). Quantltatlon of viable Intraphagocytlc B. abortus was performed by the method of Miles and Mlsra as cited (Alton et. a l . , 1975). The e f f e c t of incubating bovine mononuclear cells wlth llposomes-contalnlng gentamlcln before challenge wlth B. abortus was also examined.
Cell cultures
were incubated wlth neutral or charged llposomes alone, free gentamlcln (25 ~g/ml), or wlth llposomes-contalnlng gentamlcln f o r 24 hours at 39°C. The phagocytic c e l l s were then washed as described above to remove llposomes or gentamlcln not associated wlth the c e l l s .
The l a s t wash was examined for
gentamlcln by b i o l o g i c a l and radloenzymatlc assay.
The cells that had been
p r o p h y l a c t i c a l l y treated wlth llposomes-contalnlng gentamlcln and the control
175
cultures were incubated for 48 hours in RPMI-1640 medium. B. abortus was added to the mononuclear cells and incubated for 24 hours at 3g°c.
The
surviving B. abortus in the phagocytic cells was determined as described above. In vlvo k i l l i n g of B. abortus by 11posomes containing gentamicln Twenty five guinea pigs were divided into 5 separate groups. pigs were caged as treatment groups.
All guinea
Guinea plgs were inoculated
Intraperltoneally with 0.5 ml of B. abortus strain 2308 at a concentration of 8.8 x lO9 bacteria per ml.
Treatmentof groups was started 48 hours after
bacterial inoculation wlth virulent B. abortus.
A control group of guinea
pigs received no treatment except bacterial inoculation.
The five guinea pigs
in the second group were treated with appropriate concentrations of llposomes alone (PC:ST:Vit E., 7 : I : 0 . 5 ) .
The t h i r d treatment group received free
gentamlcln at a concentration of 2 mg/kg body weight.
The fourth group
received liposomes with entrapped gentamlcln and free gentamlcln at a total concentration equal to 2 mg/kg, (the concentration given to the free gentamlcin treatment group), the last group received llposomes with no entrapped gentamicln plus free gentamlcln (2 mg/kg body weight). were given every three days for a t o t a l of three treatments. were given as Intraperltoneal injections.
Treatments
At1 treatments
After the third injection, no
treatment was given for 5 days. After 5 days, a l l guinea pigs were k i l l e d and t h e i r spleens removed and homogenized. The spleen homogenatewas sampled and the surviving B. abortus per gram of spleen was determined as described previously (Woodard et. a l . , 1980). Liposomes-contalnlng gentamlcln With a composition of PC: Chol:ST (7:2:1) were also used to examine k i l l i n g of B. abortus in vIvo.
Procedures were
similar to those described above for llposomes-contalnlng Vitamin E.
Guinea
pigs were treated daily for 5 days with entrapped or free gentamlcln (5 mg/kg body weight).
After the last i n j e c t i o n , no treatment was given for 5 days
similar to procedures described above. Spleen weights for the treatment groups after treatment were also determined slmilar to procedures described (Woodard et. a l . , IgSO). S t a t i s t i c a l Methods Data obtained by the in vlvo studies were subjected to log transformation
176
to obtain homoscedastic treatment groups variances ( B a r t l e t t and Kenda]l, 1946).
After log transformation, variances from the treatment groups were
examined for unlformity using B a r t l e t t ' s Test ( B a r t l e t t , 1937). All treatment groups means were examlned by an analysis of varlance ( B a r t l e t t and Kendall, 1946).
Significant differences in the treatment groups from the control
groups mean were determined by Dunnett's Test (Dunnett, 1946). Significant differences between two individual group means were determined by using student's T-Test (Fisher, 1925). Data for the in v i t r o studies were also subjected to log transformation when appropriate.
Significance of the difference between means were
determined by the same methods described above. RESULTS Liposomal Entrapment All PC l~posomes (no cholesterol or stearylamlne) prepared with the 50 mg/ml solution of gentamlcln sulfate were found to contain gentamlcln by biological assay and radloenzymatlc assay.
However,charged llposomes
contained the highest intrallposomal levels of gentamlcin.
Anionic llposomes
contained approximately 220 ~g/ml of gentamlcln, cationic llposomes contained 190 ~g/ml of gentamlcln, and neutral llposomes contained 30 ~g/ml of gentamlcln.
Cationic llposomes used In vlvo studies composed of
PC:ST:VIt E contained 8.52 mg of gentamlcin per lOB mg of t o t a l phosphollpld. Llposomes composed of cholesterol (PC:ChoI:ST) contained 8.76 mg of gentamlcln per lOB mg phosphollpld. In v i t r o
killln~ of B. abortus
Liposomes-contalning gentamlcln s i g n i f i c a n t l y (p
Liposomescontaining gentamlcin were also able to enhance the
%ntracellular k i l l i n g of B. abortus strain 2308 (not shown) similar to the i n t r a c e l l u l a r k i l l i n g of B__~.abortus strain 19 as shown in Table l . Free gentamicin was unable to enhance the %ntraphagocytic k i l l i n g of B__~. abortus.
Table l shows that the surviving %ntraphagocytic bacteria in bovine
phagocytic cells a f t e r treatment with free gentamlcln was not s i g n i f i c a n t l y d l f f e r e n t from control cultures that had not been treated wlth a n t i b i o t i c .
177
Cationic, anionic, and neutral llposomes alone were also unable to enhance Intraphagocytlc k i l l i n g of B. abortus. Table l also shows that the pre-treatment of bovine phagocytic cells wlth llposomes-contalnlng gentamlcln caused a prophylactic enhancement of Intraphagocytlc k i l l i n g of B. abortus.
The surviving number of bacteria in
bovine phagocytic cells treated with llposomes-contalnlng gentamlcln was s i g n i f i c a n t l y lower than the surviving number of bacteria in cells treated with Free gentamlcln (p
Bacteria in cells treated wlth anionic
llposomes were s i g n i f i c a n t l y lower compared to cells treated with cationic or anlon~c llposomes-contalning gentamlcln (p
The surviving bacteria in
cells treated with cationic or anionic llposomes-contalnlng gentamlcln was not s l g n I f l c a n t l y d i f f e r e n t when these two groups were compared. The number of bacteria in cells pre-treated with free gentamlcln or wlth charged or neutral llposomes alone was not s i g n i f i c a n t l y d i f f e r e n t from the control group mean that had no pre-treatment. In vlvo enhancement of bacteria k i l l i n g Guinea pigs treated with liposomes (PC:ST:VIt E) containing low doses of gentamlcln (2 mg/kg body weight) had s i g n i f i c a n t l y reduced numbers of B_.~. abortus per gram of spleen when compared to control treatment groups that received no amlnoglycoslde (Table 2) (p
However, llposomes without entrapped gentamicln
plus free gentamlcin when compared at a p r o b a b i l i t y level of p
178 1ABLE l
In v i t r o k i l l i n g of B. abortus s t r a i n 19 in Bovine Phagocytes Treated Bacterla/ml
_
Mean log
Prgphwlactlc
Bacterla/ml
Mean log
Control Cells
3.8 x lO 5
5.3596 a
7.6 x lO 7
?.8837 a
Cells + Free Gentam%cln (25~g/ml)
3.3 x lO 5
5.4923 b
6.3 x IOT
7.8777 b
Cells + Anionic Liposomes
2.4 x 105
5.0791 b
4.3 x 107
7.2077 b
Cells + Anionic Liposomes-containing Gentamlcln (22~g/ml gentamicln) Cells + Cationlc Liposomes Cells + Cationic Liposomes-contalning Gentamicin (I9~g/ml gentamlcin) Cells + Neutral Liposomes Cells + Neutral Liposomes-contalning
0
0c,d
0
6.6 x lO ?
0c,d
5.3 x lO 5
5.6461 b
7.8777b
2.3 x lO l
1.0099c, d
5.3 x I05
5.6253b
7.0 x lOT
7.8450b
l.O x I03
3.0004c,d
3.0 x I02
2.6334c,d
0
oc,d
Gentamicin (3~g/ml gentamlcln) a b c d
N=3 f o r a l l treatment groups. A l l mean log values are ± 0.157 standard e r r o r of the mean f o r treated samples and ± 0.0074 f o r prophylactic studies. Means t h a t are not s i g n i f i c a n t l y d i f f e r e n t from the control mean. Meanst h a t are s i g n i f i c a n t l y d i f f e r e n t from the control mean (p
Liposomes alone or free gentamicin did not significantly reduce the number of B. abortus in guinea pig spleens, compared to untreated animals.
The
animals treated with empty liposomes and free drug also did not have a
179
Table 2.
Enhanced in vivo killing of B. abortus in Guinea Pigs by Liposomes-containing Gentamicin (PC:ST:VIt E) B. abortus/gram spleen
Mean Log
Control (no gentamlcln)
5.9 x 105
5.4088a
Free gen~amicln (2 mg/kg body weight)
4.6 x lO5
5.1190b
Liposomes alone
1.6 x lO5
5.16g0b
Liposomes with Entrapped Gentamlcln and Free Gentamlcln (2 mg/kg weight t o t a l )
9.5 x I03
3.7764b,c
Liposomes and Free gentamicln (none entrapped. Free drug at 2 mg/kg weight)
1.4 x I05
4.9789b,c
N=5 f o r a l l treatment groups. e rr o r of the mean.
Mean logs reported are ± 0.159 standard
S t a t i s t i c a l analysis using Ounnett's Test shows that free gentamicin and liposomes alone are not slgn%flcantly d i f f e r e n t from the control mean. Liposomes with entrapped gentamlcln and llposomes plus free gentamicin are s i g n i f i c a n t l y lower than the control mean (p
slgniflcant reductlon of Intrasplenic B. abortus.
(Table 2).
Spleen Weights No s i g n i f i c a n t differences In spleen weights were found in any treatment group compared to spleen weight of untreated animals. DISCUSSION This study demonstrates that llposomes-contalnlng gentamicin enhance the lntraphagocytic k l l l t n g of B. abortus an organism that is not readtly k i l l e d by phagocytic c e l l s alone.
Furthermore, l n t r a c e l l u l a r k1111ng of B. abortus
was enhanced p r o p h y l a c t i c a l l y In v i t r o by i n s e r t i o n of ltposomesmcontalning gentamicin Into phagocytic c e l l s .
Charged (anionic and c a t i o n i c ) llposomes
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Table 3
In vivo k i l l i n g of B. abortus In Guinea Plgs Administered PC:ChoI:ST [-~posomes-contalnlng Gentamlcln
Treatment Groups
Bacteria per gram spleen
Log transform mean
Control
3.17 x 105
5.44 a
Llposomes alone
3.62 x lO5
5.233NS
Llposome and Free Gentamlcln
3.38 x lO5
5.218NS
3.02 x 104
4.461"*
Liposome Entrapped Gentamlcln a
N=5 f o r a l l groups.
All means are ± 0.295 mean square error.
b
All anlmals treated with a n t i b i o t i c s received 5 mg per kg of body weight Intraperltoneally for 5 days.
NS
Not s i g n l f l c a n t l y d i f f e r e n t from the control group mean by ANOVA and Dunnet's Test.
**
Significantly lower than control group mean by ANOVA and Dunnet's Test (p
s i g n i f i c a n t l y enhanced Intraphagocytlc k i l l i n g of B. abortus when compared to k i l l i n g of B. abortus caused by anionic liposomes-contalning gentamlcln.
Thls
finding indicates that k i l l i n g of I n t r a c e l l u l a r B. abortus Is d i r e c t l y related to the amount of Intrallposomal gentamicin since neutral liposomes were shown to contain lower amounts of entrapped amlnoglycoslde. The mechanism of enhanced Intraphagocytlc k i l l i n g of B. abortus shown here remains to be elucidated.
Previous studies ( R l t t e r et. a l . , 1981) have
suggested that phosphatldylchollne llposomes similar to those used in this study can affect the permeability of cell membranes resulting In entry of free drug Into the cells without llposome entrapment.
However, llposomes with the
highest Intrallposomal levels of gentamlcln in thls study gave the highest enhancement of I n t r a c e l l u l a r k i l l i n g .
Further, studies in this laboratory
(Dees et. a l . , 1983) have suggested that llposomes with entrapped gentamlcln when incubated with bovine phagocytic cells insert higher amounts of gentamicin into cells than llposomes plus free gentamlcin (un-entrapped) and free drug Is unable to enter bovine phagocytic c e l l s .
Studies are In progress
to c l a r i f y the mechanism of the enhanced i n t r a c e l l u l a r k i l l i n g reported here and to determine i f llposomes containing gentamlcln adversely affect phagocyte function.
181
Enhanced _ i _~nvlvo k i l l l n g of B. abortus by cationic llposome containing gentamicin is also demonstrated in thls study.
Liposomes-contalnlng
a n t i b i o t i c stabilized wlth vitamin E and cholesterol e f f e c t l y reduced the number of surviving B. abortus in guinea plg spleens compared to free antlblotlc.
However, longer treatment schedules, correct dosage and treatment
routes need to be investigated to improve the k i l l i n g of B. abortus.
The
exact mechanism of enhanced I n t r a c e l l u l a r k i l l i n g of B. abortus In vlvo is not known. Enhanced I n t r a c e l l u l a r kl111ng of B. abortus during in v i t r o studies appears to be the result of enhanced I n t r a c e l l u l a r delivery of a n t i b i o t i c into phagocytic c e l l s .
Enhanceddevllery of a n t i b i o t i c Into phagocytic cells by
l%posomes in vlvo may account For the reduced numbers of Intrasplenlc B. abortus compared to results obtained when Free drug Is used. However, llposomal delivery of therapeutic agents, l l k e a n t i b i o t i c s , increases the b l o a v a l l a b i l i t y of the llposomally encapsulated drug and prolongs serum drug levels (Morgan and Wllllams, 1980). Liposomaldellvery of a n t i b i o t i c s also may target specific tissues p r e f e r e n t i a l l y .
For example, l i v e r and spleen
levels of a n t i b i o t i c are increased when compared to free drug (Morgan and Williams, 1980). A n t i b i o t i c targeting to spleen, prolonged serum a n t i b i o t i c levels, and enhanced delivery of a n t i b i o t i c into phagocytic cells by llposomes contalnlng gentamlcln are probably a l l important in enhancing the I n t r a c e l l u l a r k i l l i n g of B. abortus in vlvo. Of interest, free drug and empty llposomes formed with PC and cholesterol dld not s i g n i f l c a n t l y reduce Intrasplenlc B. abortus.
T h l s finding is in
contrast to in vlvo results wlth a similar treatment group using llposomes stabillzed wlth vitamin E.
The present in v i t r o
studies using llposomes
without cholesterol or vitamin E suggest that empty llposomes can enhance penetration of free drug are In agreement wlth previous studies ( R i t t e r et. a l . , IgSl).
Ln vlvo studies using llposomes wlth vitamin E are in agreement
with in v l t r o studies presented here and agree with data presented by Rltter (IgSl).
Inclusion of cholesterol Into phosphatldylchollne liposome may
adversely affect permeability of free drug Into c e l l s . necessary to investigate thls phenomena.
Further studies are
Llposomes stabilized with vitamin E
and with no entrapped gentamlcln also did not appear to enhance clearance of B. abortus in vlvo. The present study demonstrates that liposomes-containing gentamicin are able to enhance in vitro and in vivo killing of B__~.abortus. Liposomes-containlng amlnoglycoside may provide an improved treatment method for brucellosis in animals and humans by increasing the Intraphagocytlc killing of bacteria, targeting of the antibiotic to infected areas of the reticuloendothelial system including spleen and lymph nodes (Morgan and
182
Williams, 1980) and prophylactically protecting uninfected cells by Insert%on of a n t i b i o t i c prior to infection. Acknowledgements This project was p a r t i a l l y funded by U.S.D.A. special research grant #59-2011-I-2-01B0 and and Eastern A r t i f i c i a l Insemination Cooperative, Ithaca, N.Y. References Alton, G.G., Jones, L.M., and Pietz, D.E., 1975. Laboratory Techniques in Brucellosis, p. 60, World Health Organization, New York. Bartlett, M.S. and Kendall D.G., 1946. Bartlett M.S., 1937.
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Bennet, J.V., Brodle, J.L., Brenner, E.J., and Kirby, W.R., 1966. Appl. Microb%ol., 14:170-177. Bonventre, P.F., And Gregoriadias, G, 1978. Antlmicroblal. Agents Chemother., 13:1049-I051. Dees, C. and R.D. Schultz, 1983. Evidencefor long term survival of gentamlcln in phagocytic cells after insertion by llposomes. Fed. Proc., 42:802. Dunnett, C.W., ]946.
J. Amer. Stat. Assoc., 50:I096-I121.
Fisher, R.A., 1925. Metron, 5:90-I04. Fountain, M.W., Dees, C., and Schultz, R.D., 1982. Curr. Microblol., 6:6-66. Fountain, M.W., Pedersoll, W.D., GanJam, V.K., and Ravls, W.R., 1980. Curr. Ther. Res., 28:558-561. Magee, W.E., 1978. Ann N.Y. Acad. Scl., 308:308-324. Morgan, J.R., and Williams, K.E., 1980. Antimicroblal. Agents. Chemother., I?:544-548. Phillppon, A.M., Plommet, M.G., Kazmlerczak, A., Marly, J.L., and Nevot, P.A., 1977.
J. Infect. Dis., 136:482-488.
Pomales. A., Lebron, A., Stlnebrlng, W.R., 1957. Proc. Soc. Exp. B i o l . , 94:?8-83. Ritter, C., Lyewgar, C.W., and Ruttman, R.J., 1981. Can. Res., 41:2366-2371. Ryman, B.E., Jewkes, R.F., Jeyaslngh, K., Osborn, M.P., Patel H.M., Richardson, V.J., Tattersall, M.H.N., and Tyrell, D.A., 1978. N.Y. Acad. Sci., 308: 281-305. Shaffer, J.M.M Jucera, C.J., and Splnk, W.W., 1953. Terrlto, M.C., Golde, D.W., and Cllne, M.J., 1976.
J. Exp. Med., 9?:??-89. in N.R. Rosse and
H. Freldman (ed.) Manual of Clinical Immunoloqy. p. 142, American Society for Microblology, Washington, D.C.
171
ENHANCED INTRAPHAGOCYTIC KILLING OF BRUCELLAABORTUSIN BOVINE MONONUCLEAR CELLS BY LIPOSOMES-CONTAINING GENTAMICIN C. DEES*, M.W. FOUNTAIN, 3.R. TAYLOR, AND R.D. SCHULTZ* Animal Health Research and Department of Microbiology, School of Veterinary Medicine and Agriculture Experiment Station, Auburn University, AL 36849{U.S.A. {Accepted 29 June 1984)
ABSTRACT Dees, C., Fountain, M.W., Taylor, J.R., and Schultz R.D., 1985. Enhanced Intraphagocytlc k i l l i n g of Brucella abortus in bovine mononuclear cells by llposomes-contalnlng gentamlcln.
Vet. Immunol. Immunopathol., 8: 171-182.
In v i t r o Intraphagocytlc k i l l i n g of Brucella abortus in bovine mononuclear leukocytes was enhanced by cationic, anionic, and neutral multllamellar llposomes-contalnlng gentamlcln.
Free gentamlcln not entrapped in llposomes
and llposomes without a n t i b i o t i c dld not enhance intraphagocytlc k i l l i n g of B__~. abortus in bovine phagocytes. In vlvo k i l l l n g of B. abortus in guinea pigs was also enhanced by llposomes-contalnlng gentamlcln when compared to free gentamlcln.
Llposomes-contalnlng alpha tocopherol acetate failed to enhance
In vlvo k i l l i n g of B. abortus.
INTRODUCTION Few bacteria are able to survive for long periods inside phagocytic cells. B__~.abortus, however, is not only able to survive for long periods of time within leukocytes but can also replicate within phagocytic cells (Phllllpon e t . a l , ]g77), Schaffer e t . a l . , 1953). The a b i l i t y of B. abortus to survive and replicate in phagocytic ceils is an important factor when one considers the nature of the disease produced by thls bacterium.
The a b i l i t y of
B. abortus to survive I n t r a c e l l u l a r l y can result in long term chronic disease (Schaffer e t . a l . , 1953). Relapses following antlmlcroblal therapy can also be explained by the I n t r a c e l l u l a r location of B. abortus since I n t r a c e l l u l a r Brucella are protected from treatment by most antibiotics (Schaffer e t . a l . , 1953) *Authors to whom reprint ruquests should be addressed: Departmentof Pathoblologlcal Sciences, School of Veterinary Medicine, University of Wlsconsln-Madlson 53706. Current Addresses; M.W. Fountain, The Liposome Co., Princeton, N,J. 08540 J.R. Taylor, USDARegional Parasite Lab, Auburn, AL 36849. 0165-2427/85/$03.30
© 1985 Elsevier Science Publishers B.V.
172
Therefore, new treatment regimens and delivery vehicles for antlmlcroblal agents need to be developed to enhance penetration of antibiotics into phagocytic cells to k i l l i n t r a c e l l u l a r B. abortus. Recently, liposomes (multilamellar vesicles) were reported to be effective delivery vehicles for a number of chemotherapeutic agents including dlgoxln (Ryman et. a l . , 1978), insulin (Fountain et. a l . , 1980), and antibiotics (Bonventre and Gregorladias, 1978; Morgan and Williams, Ig80).
Delivery of
antibiotics via liposomes has several potential advantages: l ) prolonged serum levels of a n t i b i o t i c ; 2) antibiotics may be targeted to sites not reached by free a n t i b i o t i c ; 3) antibiotics may be delivered into phagocytic cells resulting in enhanced I n t r a c e l l u l a r k i l l i n g of bacteria.
(Bonventreand
Gregoriadias, 1978; Fountain et. a l . , 1982; Morgan and Williams, 1980). Bonventre and Gregorladlas (19BO) reported that Intraphagocytlc k i l l i n g of Staphylococcus aureus is enhanced by llposomes-contalnlng dlhydrostreptomycln.
I t has also been reported that llposomes-contalning
streptomycin enhance I n t r a c e l l u l a r k i l l i n g in vivo of Mycobacterlum tuberculosis (Vladimirsky and Hadlgina, 1982). Dees et. a l . , (1983) has also reported that llposomal delivery of gentamicln can enhance the penetration of gentamlcln into bovine phagocytic cells when compared to free drug.
I t was also suggested that gentamlcln
inserted into phagocytic cells had a long Intracellular survival time (Dees, et. a l . , 1983). Liposomal delivery of gentamlcln might be useful in enhancing the Intraphagocytlc k i l l i n g of B. abortus.
Secondly, since llposoma delivered
gentamlcln has a long survival time in bovine phagocytes, i t may be possible to insert gentamicln into a phagocytic cell before i t ingests a pathogenic bacterium llke B. abortus.
Thus, a phagocytic c e l l ' s a b i l i t y to k i l l B.
abortus or other bacteria might be prophylactically enhanced by llposome delivery of gentamicln prior to phagocytosls of the bacteria preventing any p o s s i b i l i t y of long term intraphagocytlc survival. Th~s report presents the results of in v i t r o and in vivo studies to determine the effects of llposomes-contalnlng gentamlcln on the Intracellular k i l l i n g of B. abortus in bovine phagocytic cells. Hopefully, llposomal delivery of antibiotics w i l l prove useful in enhancing intraphagocytlc penetration of a n t i b i o t i c , targeting of antibiotic to organs of the retlculoendothellnal system, and prophylactically protecting phagocytic cells from colonization wlth B. abortus.
173
MATERIALS AND METHODS Liposome preparation Charged and neutral llposomes (multilamellar) were prepared as previously described (Fountain et. a l . , 1980; Magee, 1978; Fountain et. a l . , 1982). Lipid Films of a ?:2 molar r a t i o of phosphatldylcholine (PC) (Avantl Polar Lipids, Birmingham, A]) and dlcetylphosphate (DCP), 7.2 molar r a t i o of PC and stearylamlne (ST), and PC alone were drled under nitrogen.
The t o t a l
phosphollpld concentration for each llposome preparation was lO mg/ml. Gentamlcln sulfate was supplied as a lyophillzed preservative free powder (Scherlng Veterinary, Kenilworth, N.Y.) s t e r i l e normal saline.
Gentamlclnpowder was rehydrated with
A one ml solution of rehydrated gentamlcln sulfate at
a concentration of 50 mg/ml was added to the dried l i p i d films. Liposomes were also prepared from a 7:I:0.5 ratio of PC, ST and Vitamin E (a tocopherol acetate) and PC, cholesterol (Chol), ST (?:2:1) that used for In vlvo studies. The individual llposome preparations were agitated and then passed over a Sephadex G-50 column with Hank's Balanced Salt Solution as the elutlng buffer.
The llposomal entrapped gentamlcln concentrations for neutral,
anionic, and cationic llposomes was determined by biological assay using S. aureus ATCC strain 25923 similar to gentamlcln determinations described previously (Bennet et. a l . , 1966; Morgan and Williams, 1980). Intrallposomal gentamlcln concentrations were also confirmed by radloenzymatlc assay as described (Fountain et. a l . , 1982). In v i t r o k i l l i n g assays Mononuclear phagocytic cells were obtained from heparinlzed bovine peripheral blood by Ficoll-Hypaque (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) density gradient centrlfugation similar to procedures described previously ( T e r r i t o et. a l . , 1976). Mononuclear cells at the plasma-Flcoll interface were removed and placed In RPMI-1640 medium (Gibco Inc., Grand Island, N.Y.).
Mononuclear cells were counted with electronic
cell counter and placed in tissue culture flasks.
The mononuclear cells were
incubated for one hour at 39°C. The non-adherent cells ( p r i m a r i l y lymphocytes) were removed by decanting the RPMI-1640 medium. The adherent cells ( p r i m a r i l y monocytes) were used for incubation with B. abortus and
174
llposomes.
The number of adherent cells was determined by calculating the
difference between the i n i t i a l
c e l l counts and the c e l l s (not adhering)
removed In the RPMI-1640. Mononuclearphagocytic cells at a concentration of approximately l.O x lO ? cells/ml were used In a l l studies. Bovine phagocytic cells were incubated wlth B. abortus strain 19 (Colorado Serum Co., Denver, CO) or s t r a i n 2308 at concentrations approximately equal to t h a t of the phagocytic c e l l s for a period of 24 hours at 39°C in RPMI-1640 medium. B o t h strains of B. abortus used in the in v i t r o k i l l i n g assays were found to be sensitive to gentamlcln and the minimum i n h i b i t o r y concentration of gentamlcln for both b a c t e r i a l strains was determined to be less than O.5~g/ml.
Pooled bovine sera from B. abortus infected c a t t l e , heat
i n a c t l v a t e d , and d i l u t e d l:lO0 was used to opslnlze the B. abortus that were incubated wlth the phagocytes.
After 24 hours incubation the unphagocytosed
bacteria were removed by washing the c e l l cultures wlth RPMI-1640 medium. The f i n a l wash was cultured to assure complete removal of free B. abortus.
The
mononuclear c e l l s a f t e r washing were covered wlth a three ml volume of medium and lO0 ~l of the c a t i o n i c , anionic, or neutral llposomes-contalnlng gentamlcln.
Control cultures were incubated wlth appropriate amounts of
charged or neutral llposomes not containing a n t i b i o t i c or wlth free gentamlcln (25 ~g/ml) equal to or greater than gentamlcln In the llposomes.
Phagocytic
c e l l s wlth or without llposomes were incubated for 5 hours 39°C. The unincorporated llposomes were removed by washing wlth RPMI-1640 medium. The l a s t wash was examined by b i o l o g i c a l and radloenzymatlc assays to assure complete removal of gentamlcln.
The number of surviving cells of B. abortus
was determined by freeze-thawlng to lyse the cells and then determining the number of bacterial colonies s i m i l a r to procedures previously described (g). Quantltatlon of viable Intraphagocytlc B. abortus was performed by the method of Miles and Mlsra as cited (Alton et. a l . , 1975). The e f f e c t of incubating bovine mononuclear cells wlth llposomes-contalnlng gentamlcln before challenge wlth B. abortus was also examined.
Cell cultures
were incubated wlth neutral or charged llposomes alone, free gentamlcln (25 ~g/ml), or wlth llposomes-contalnlng gentamlcln f o r 24 hours at 39°C. The phagocytic c e l l s were then washed as described above to remove llposomes or gentamlcln not associated wlth the c e l l s .
The l a s t wash was examined for
gentamlcln by b i o l o g i c a l and radloenzymatlc assay.
The cells that had been
p r o p h y l a c t i c a l l y treated wlth llposomes-contalnlng gentamlcln and the control
175
cultures were incubated for 48 hours in RPMI-1640 medium. B. abortus was added to the mononuclear cells and incubated for 24 hours at 3g°c.
The
surviving B. abortus in the phagocytic cells was determined as described above. In vlvo k i l l i n g of B. abortus by 11posomes containing gentamicln Twenty five guinea pigs were divided into 5 separate groups. pigs were caged as treatment groups.
All guinea
Guinea plgs were inoculated
Intraperltoneally with 0.5 ml of B. abortus strain 2308 at a concentration of 8.8 x lO9 bacteria per ml.
Treatmentof groups was started 48 hours after
bacterial inoculation wlth virulent B. abortus.
A control group of guinea
pigs received no treatment except bacterial inoculation.
The five guinea pigs
in the second group were treated with appropriate concentrations of llposomes alone (PC:ST:Vit E., 7 : I : 0 . 5 ) .
The t h i r d treatment group received free
gentamlcln at a concentration of 2 mg/kg body weight.
The fourth group
received liposomes with entrapped gentamlcln and free gentamlcln at a total concentration equal to 2 mg/kg, (the concentration given to the free gentamlcin treatment group), the last group received llposomes with no entrapped gentamicln plus free gentamlcln (2 mg/kg body weight). were given every three days for a t o t a l of three treatments. were given as Intraperltoneal injections.
Treatments
At1 treatments
After the third injection, no
treatment was given for 5 days. After 5 days, a l l guinea pigs were k i l l e d and t h e i r spleens removed and homogenized. The spleen homogenatewas sampled and the surviving B. abortus per gram of spleen was determined as described previously (Woodard et. a l . , 1980). Liposomes-contalnlng gentamlcln With a composition of PC: Chol:ST (7:2:1) were also used to examine k i l l i n g of B. abortus in vIvo.
Procedures were
similar to those described above for llposomes-contalnlng Vitamin E.
Guinea
pigs were treated daily for 5 days with entrapped or free gentamlcln (5 mg/kg body weight).
After the last i n j e c t i o n , no treatment was given for 5 days
similar to procedures described above. Spleen weights for the treatment groups after treatment were also determined slmilar to procedures described (Woodard et. a l . , IgSO). S t a t i s t i c a l Methods Data obtained by the in vlvo studies were subjected to log transformation
176
to obtain homoscedastic treatment groups variances ( B a r t l e t t and Kenda]l, 1946).
After log transformation, variances from the treatment groups were
examined for unlformity using B a r t l e t t ' s Test ( B a r t l e t t , 1937). All treatment groups means were examlned by an analysis of varlance ( B a r t l e t t and Kendall, 1946).
Significant differences in the treatment groups from the control
groups mean were determined by Dunnett's Test (Dunnett, 1946). Significant differences between two individual group means were determined by using student's T-Test (Fisher, 1925). Data for the in v i t r o studies were also subjected to log transformation when appropriate.
Significance of the difference between means were
determined by the same methods described above. RESULTS Liposomal Entrapment All PC l~posomes (no cholesterol or stearylamlne) prepared with the 50 mg/ml solution of gentamlcln sulfate were found to contain gentamlcln by biological assay and radloenzymatlc assay.
However,charged llposomes
contained the highest intrallposomal levels of gentamlcin.
Anionic llposomes
contained approximately 220 ~g/ml of gentamlcln, cationic llposomes contained 190 ~g/ml of gentamlcln, and neutral llposomes contained 30 ~g/ml of gentamlcln.
Cationic llposomes used In vlvo studies composed of
PC:ST:VIt E contained 8.52 mg of gentamlcin per lOB mg of t o t a l phosphollpld. Llposomes composed of cholesterol (PC:ChoI:ST) contained 8.76 mg of gentamlcln per lOB mg phosphollpld. In v i t r o
killln~ of B. abortus
Liposomes-contalning gentamlcln s i g n i f i c a n t l y (p
Liposomescontaining gentamlcin were also able to enhance the
%ntracellular k i l l i n g of B. abortus strain 2308 (not shown) similar to the i n t r a c e l l u l a r k i l l i n g of B__~.abortus strain 19 as shown in Table l . Free gentamicin was unable to enhance the %ntraphagocytic k i l l i n g of B__~. abortus.
Table l shows that the surviving %ntraphagocytic bacteria in bovine
phagocytic cells a f t e r treatment with free gentamlcln was not s i g n i f i c a n t l y d l f f e r e n t from control cultures that had not been treated wlth a n t i b i o t i c .
177
Cationic, anionic, and neutral llposomes alone were also unable to enhance Intraphagocytlc k i l l i n g of B. abortus. Table l also shows that the pre-treatment of bovine phagocytic cells wlth llposomes-contalnlng gentamlcln caused a prophylactic enhancement of Intraphagocytlc k i l l i n g of B. abortus.
The surviving number of bacteria in
bovine phagocytic cells treated with llposomes-contalnlng gentamlcln was s i g n i f i c a n t l y lower than the surviving number of bacteria in cells treated with Free gentamlcln (p
Bacteria in cells treated wlth anionic
llposomes were s i g n i f i c a n t l y lower compared to cells treated with cationic or anlon~c llposomes-contalning gentamlcln (p
The surviving bacteria in
cells treated with cationic or anionic llposomes-contalnlng gentamlcln was not s l g n I f l c a n t l y d i f f e r e n t when these two groups were compared. The number of bacteria in cells pre-treated with free gentamlcln or wlth charged or neutral llposomes alone was not s i g n i f i c a n t l y d i f f e r e n t from the control group mean that had no pre-treatment. In vlvo enhancement of bacteria k i l l i n g Guinea pigs treated with liposomes (PC:ST:VIt E) containing low doses of gentamlcln (2 mg/kg body weight) had s i g n i f i c a n t l y reduced numbers of B_.~. abortus per gram of spleen when compared to control treatment groups that received no amlnoglycoslde (Table 2) (p
However, llposomes without entrapped gentamicln
plus free gentamlcin when compared at a p r o b a b i l i t y level of p
178 1ABLE l
In v i t r o k i l l i n g of B. abortus s t r a i n 19 in Bovine Phagocytes Treated Bacterla/ml
_
Mean log
Prgphwlactlc
Bacterla/ml
Mean log
Control Cells
3.8 x lO 5
5.3596 a
7.6 x lO 7
?.8837 a
Cells + Free Gentam%cln (25~g/ml)
3.3 x lO 5
5.4923 b
6.3 x IOT
7.8777 b
Cells + Anionic Liposomes
2.4 x 105
5.0791 b
4.3 x 107
7.2077 b
Cells + Anionic Liposomes-containing Gentamlcln (22~g/ml gentamicln) Cells + Cationlc Liposomes Cells + Cationic Liposomes-contalning Gentamicin (I9~g/ml gentamlcin) Cells + Neutral Liposomes Cells + Neutral Liposomes-contalning
0
0c,d
0
6.6 x lO ?
0c,d
5.3 x lO 5
5.6461 b
7.8777b
2.3 x lO l
1.0099c, d
5.3 x I05
5.6253b
7.0 x lOT
7.8450b
l.O x I03
3.0004c,d
3.0 x I02
2.6334c,d
0
oc,d
Gentamicin (3~g/ml gentamlcln) a b c d
N=3 f o r a l l treatment groups. A l l mean log values are ± 0.157 standard e r r o r of the mean f o r treated samples and ± 0.0074 f o r prophylactic studies. Means t h a t are not s i g n i f i c a n t l y d i f f e r e n t from the control mean. Meanst h a t are s i g n i f i c a n t l y d i f f e r e n t from the control mean (p
Liposomes alone or free gentamicin did not significantly reduce the number of B. abortus in guinea pig spleens, compared to untreated animals.
The
animals treated with empty liposomes and free drug also did not have a
179
Table 2.
Enhanced in vivo killing of B. abortus in Guinea Pigs by Liposomes-containing Gentamicin (PC:ST:VIt E) B. abortus/gram spleen
Mean Log
Control (no gentamlcln)
5.9 x 105
5.4088a
Free gen~amicln (2 mg/kg body weight)
4.6 x lO5
5.1190b
Liposomes alone
1.6 x lO5
5.16g0b
Liposomes with Entrapped Gentamlcln and Free Gentamlcln (2 mg/kg weight t o t a l )
9.5 x I03
3.7764b,c
Liposomes and Free gentamicln (none entrapped. Free drug at 2 mg/kg weight)
1.4 x I05
4.9789b,c
N=5 f o r a l l treatment groups. e rr o r of the mean.
Mean logs reported are ± 0.159 standard
S t a t i s t i c a l analysis using Ounnett's Test shows that free gentamicin and liposomes alone are not slgn%flcantly d i f f e r e n t from the control mean. Liposomes with entrapped gentamlcln and llposomes plus free gentamicin are s i g n i f i c a n t l y lower than the control mean (p
slgniflcant reductlon of Intrasplenic B. abortus.
(Table 2).
Spleen Weights No s i g n i f i c a n t differences In spleen weights were found in any treatment group compared to spleen weight of untreated animals. DISCUSSION This study demonstrates that llposomes-contalnlng gentamicin enhance the lntraphagocytic k l l l t n g of B. abortus an organism that is not readtly k i l l e d by phagocytic c e l l s alone.
Furthermore, l n t r a c e l l u l a r k1111ng of B. abortus
was enhanced p r o p h y l a c t i c a l l y In v i t r o by i n s e r t i o n of ltposomesmcontalning gentamicin Into phagocytic c e l l s .
Charged (anionic and c a t i o n i c ) llposomes
180
Table 3
In vivo k i l l i n g of B. abortus In Guinea Plgs Administered PC:ChoI:ST [-~posomes-contalnlng Gentamlcln
Treatment Groups
Bacteria per gram spleen
Log transform mean
Control
3.17 x 105
5.44 a
Llposomes alone
3.62 x lO5
5.233NS
Llposome and Free Gentamlcln
3.38 x lO5
5.218NS
3.02 x 104
4.461"*
Liposome Entrapped Gentamlcln a
N=5 f o r a l l groups.
All means are ± 0.295 mean square error.
b
All anlmals treated with a n t i b i o t i c s received 5 mg per kg of body weight Intraperltoneally for 5 days.
NS
Not s i g n l f l c a n t l y d i f f e r e n t from the control group mean by ANOVA and Dunnet's Test.
**
Significantly lower than control group mean by ANOVA and Dunnet's Test (p
s i g n i f i c a n t l y enhanced Intraphagocytlc k i l l i n g of B. abortus when compared to k i l l i n g of B. abortus caused by anionic liposomes-contalning gentamlcln.
Thls
finding indicates that k i l l i n g of I n t r a c e l l u l a r B. abortus Is d i r e c t l y related to the amount of Intrallposomal gentamicin since neutral liposomes were shown to contain lower amounts of entrapped amlnoglycoslde. The mechanism of enhanced Intraphagocytlc k i l l i n g of B. abortus shown here remains to be elucidated.
Previous studies ( R l t t e r et. a l . , 1981) have
suggested that phosphatldylchollne llposomes similar to those used in this study can affect the permeability of cell membranes resulting In entry of free drug Into the cells without llposome entrapment.
However, llposomes with the
highest Intrallposomal levels of gentamlcln in thls study gave the highest enhancement of I n t r a c e l l u l a r k i l l i n g .
Further, studies in this laboratory
(Dees et. a l . , 1983) have suggested that llposomes with entrapped gentamlcln when incubated with bovine phagocytic cells insert higher amounts of gentamicin into cells than llposomes plus free gentamlcin (un-entrapped) and free drug Is unable to enter bovine phagocytic c e l l s .
Studies are In progress
to c l a r i f y the mechanism of the enhanced i n t r a c e l l u l a r k i l l i n g reported here and to determine i f llposomes containing gentamlcln adversely affect phagocyte function.
181
Enhanced _ i _~nvlvo k i l l l n g of B. abortus by cationic llposome containing gentamicin is also demonstrated in thls study.
Liposomes-contalnlng
a n t i b i o t i c stabilized wlth vitamin E and cholesterol e f f e c t l y reduced the number of surviving B. abortus in guinea plg spleens compared to free antlblotlc.
However, longer treatment schedules, correct dosage and treatment
routes need to be investigated to improve the k i l l i n g of B. abortus.
The
exact mechanism of enhanced I n t r a c e l l u l a r k i l l i n g of B. abortus In vlvo is not known. Enhanced I n t r a c e l l u l a r kl111ng of B. abortus during in v i t r o studies appears to be the result of enhanced I n t r a c e l l u l a r delivery of a n t i b i o t i c into phagocytic c e l l s .
Enhanceddevllery of a n t i b i o t i c Into phagocytic cells by
l%posomes in vlvo may account For the reduced numbers of Intrasplenlc B. abortus compared to results obtained when Free drug Is used. However, llposomal delivery of therapeutic agents, l l k e a n t i b i o t i c s , increases the b l o a v a l l a b i l i t y of the llposomally encapsulated drug and prolongs serum drug levels (Morgan and Wllllams, 1980). Liposomaldellvery of a n t i b i o t i c s also may target specific tissues p r e f e r e n t i a l l y .
For example, l i v e r and spleen
levels of a n t i b i o t i c are increased when compared to free drug (Morgan and Williams, 1980). A n t i b i o t i c targeting to spleen, prolonged serum a n t i b i o t i c levels, and enhanced delivery of a n t i b i o t i c into phagocytic cells by llposomes contalnlng gentamlcln are probably a l l important in enhancing the I n t r a c e l l u l a r k i l l i n g of B. abortus in vlvo. Of interest, free drug and empty llposomes formed with PC and cholesterol dld not s i g n i f l c a n t l y reduce Intrasplenlc B. abortus.
T h l s finding is in
contrast to in vlvo results wlth a similar treatment group using llposomes stabillzed wlth vitamin E.
The present in v i t r o
studies using llposomes
without cholesterol or vitamin E suggest that empty llposomes can enhance penetration of free drug are In agreement wlth previous studies ( R i t t e r et. a l . , IgSl).
Ln vlvo studies using llposomes wlth vitamin E are in agreement
with in v l t r o studies presented here and agree with data presented by Rltter (IgSl).
Inclusion of cholesterol Into phosphatldylchollne liposome may
adversely affect permeability of free drug Into c e l l s . necessary to investigate thls phenomena.
Further studies are
Llposomes stabilized with vitamin E
and with no entrapped gentamlcln also did not appear to enhance clearance of B. abortus in vlvo. The present study demonstrates that liposomes-containing gentamicin are able to enhance in vitro and in vivo killing of B__~.abortus. Liposomes-containlng amlnoglycoside may provide an improved treatment method for brucellosis in animals and humans by increasing the Intraphagocytlc killing of bacteria, targeting of the antibiotic to infected areas of the reticuloendothelial system including spleen and lymph nodes (Morgan and
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Williams, 1980) and prophylactically protecting uninfected cells by Insert%on of a n t i b i o t i c prior to infection. Acknowledgements This project was p a r t i a l l y funded by U.S.D.A. special research grant #59-2011-I-2-01B0 and and Eastern A r t i f i c i a l Insemination Cooperative, Ithaca, N.Y. References Alton, G.G., Jones, L.M., and Pietz, D.E., 1975. Laboratory Techniques in Brucellosis, p. 60, World Health Organization, New York. Bartlett, M.S. and Kendall D.G., 1946. Bartlett M.S., 1937.
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