J. MICROENCAPSULATION,
1991, VOL. 8, NO. 1, 29-36
Ampicillin-loaded liposomes and nanoparticles: comparison of drug loading, drug release and in vitro antimicrobial activity
Journal of Microencapsulation Downloaded from informahealthcare.com by Nyu Medical Center on 12/05/14 For personal use only.
E. FATTAL, J. ROJAS, L. R O B L O T - T R E U P E L , A. ANDREMONT and P. COUVREUR Laboratoire de Pharmacie GalCnique, URA CNRS 1218, FacultC de Pharmacie, Chatenay-Malabry, France, Laboratoire de Microbiologie, FacultC de Pharmacie, Chatenay-MalFbry, France and Laboratoire d'Ecologie Microbienne, Institut Gustave Roussy, Villejuif, France (Received 6 March 1990; accepted 28 March 1990)
In this paper, we report the physico-chemical properties of negatively charged liposomes and of polyisohexylcyanoacrylate nanoparticles loaded with ampicillin. Although the carriers were of the same size (200nm), drug-loading capacity was 20 times higher for nanoparticles than for liposomes. After freeze-drying or storage at 4"C, no drug escaped from polymeric nanoparticles. On the other hand, in the same conditions, ampicillin leaked rapidly from liposomes. Drug release in foetal calf serum was gradual (of zero order) with nanoparticles, whereas it was rapid with liposomes. Finally, the antimicrobial activity of ampicillin-entrapped liposomes or nanoparticles was studied in vitro.
+
Introduction Liposomes and nanoparticles have aroused interest as potent drug carriers that are capable of enhancing the intracellular delivery of antibiotics (Alving 1988). Resistance of intracellular infections to chemotherapy was shown to be related to the low intracellular uptake of commonly used antibiotics or to their reduced activity at the acid p H of lysosomes. Acidic antibiotics (b-lactams) d o not diffuse through the lysosomal membrane because of their ionic character at neutral extralysosomal p H (Renard et al. 1987). Recently, we have shown in experimentally infected mice that linkage of ampicillin to polyisohexylcyanoacrylate (PIHCA) nanoparticles increased the efficiency of the drug in experimental murine salmonellosis (Fattal et al. 1989). We have also shown an increased efficacy of this preparation in chronic listeriosis in athymic nude mice (Youssef et al. 1988). Surprisingly, Bakker-Woundenberg et al. (1985), did not observe, on the same model, a similar effect with liposome-entrapped ampicillin. However, the dose of ampicillin bound to lipsomes in this study was poor and only 23 per cent of that was bound to nanoparticles. A straightforward comparison of the two treatments was thus not possible. Because drug targeted by aid of liposomes or nanoparticles may result in a different intracellular distribution profile (Guise et al. 1987), the efficiency against intralysosomal infections can differ. I n addition, owing to the specific behaviour of each carrier with seric components (single opsonization for polymeric nanoparticles Address for correspondence: Prof. Patrick Couvreur, Laboratoire de Pharmacie GalCnique, URA CNRS 1218, 5 Rue J. B. CICment, 92296 Chatenay-Malabry, France. 0265-2048191 $3.00 0 1991 Taylor & Francis Ltd
30
E . Fattal et al.
Journal of Microencapsulation Downloaded from informahealthcare.com by Nyu Medical Center on 12/05/14 For personal use only.
or liposomal destabilization by H D L ) (Juliano 1988), drug release profile may also vary. Thus, the need to design a carrier with optimized efficacy against intracellular infections requires comparison of the activity of antibiotics targeted by either liposomes or nanoparticles with comparable drug loading. Therefore, we report here the comparison of the major physicochemical properties of liposomes and nanoparticles loaded with the same amount of ampicillin.
Materials and method Materials Soybean phosphatidylcholine was obtained from Lecithos (Saint Maur, France). Cholesterol, phosphatidylglycerol, esterase were purchased from Sigma (Saint Louis, USA). Sodium ampicillin was obtained from Bristol (Paris, France) and ampicillin trihydrate from Negma (Buc, France). Isohexylcyanoacrylate was synthetized by Sopar (Brussels, Belgium). Spores of Bacillus subtilis (ATCC 6663) were used as inoculum for classical microbiological assay. Assay medium no. 2 was obtained from Difco Laboratory (Detroit, USA). Foetal calf serum was obtained from Gibco (New York, USA).
Preparation of liposome-entrapped ampicillin Liposomes were prepared from a lipid mixture of 168pmol of soybean phosphatidylcholine, 210 pmol of cholesterol and 42 pmol of phosphatidylglycerol. The dried lipid film, obtained on rotary evaporator, was redissolved in 14ml of diethylether. A 50 mg/ml solution of sodium ampicillin in buffer ( T R I S 0.1 M, NaCl 0.15 M, p H = 7-4) was added. After 3 min of bath sonication, the diethylether was removed under vacuum. Liposomes were extruded through a 0.2 pm polycarbonate membrane (Nucleopore, Pleasontown, USA). Unentrapped ampicillin was then separated by Sephadex G50 (Pharmacia, Upssala, Sweden) filtration. Liposomes fractions were pooled and concentrated by ultracentrifugation (300 000 g for 30 min). The amount of ampicillin encapsulated in liposomes was determined using a reverse phase liquid chromatography assay (Margosis 1982). T h e size of the liposomes was determined using a laser light scattering method (Nanosizer coulter, Coultronics, Margency, France). Drug-free liposomes were prepared by the same way. Preparation of nanoparticle-entrapped ampicillin Isohexylcyanoacrylate monomer (100pl) was added under mechanical stirring to lOml of an aqueous polymerization medium (dextran 70 1 per cent in 10-3 N HCI) containing 2 mg/ml of ampicillin trihydrate. After 6 h of polymerization, a milky suspension was obtained, neutralized with 1 N NaOH and brought to isotonicity by glucose 5 per cent. Measurements of ampicillin entrapped into nanoparticles were carried out on the supernatant after ultracentrifugation of the samples at 110OOOgfor 1 h 30 min. Analytical determinations were made using an HPLC method (Margosis 1982). Freeze-drying Ampicillin entrapped into liposomes or nanoparticles was freeze dried at - 40°C using a freeze-dryer (Christ alpha 1.5, Bioblock, Vanves, France) for 24 h under vacuum. Glucose 5 per cent was used as cryoprotector. Resuspension of nanoparticle
Liposomal and nanoparticulate ampicillin
31
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liposomal pellet was carried out by a single addition of distilled water (10 ml) to each vial. T h e size and drug content of the carriers were determined before and after freeze-drying. Stability a t +4"C Stability of ampicillin entrapped into liposomes or nanoparticles was studied after storage during 21 days at +4"C. This experiment was carried out at pH4.9 which corresponded to the optimum of stability for ampicillin (Savello and Shangraw 1971). Size and drug content were determined at different time intervals (days 1, 7, 15 and 21). T h e stability of free ampicillin was tested in the same experimental conditions.
Drug release in foetal calf serum Eight millilitres of the suspension of liposome-entrapped ampicillin was incubated with 32 ml of foetal calf serum (FCS) at 37°C. T h e final concentration of ampicillin was of 0*46mg/ml. This preparation was taken off at different time intervals (0,30,60 and 120min). Released ampicillin was separated by Sephadex Gel filtration. Then, liposomal fractions were pooled and ampicillin content measured after precipitation of serum proteins with 1.2 M perchloric acid. Eight millitres of the suspension of nanoparticle-entrapped ampicillin was incubated with 32ml of fetal calf serum (FCS) at 37°C. T h e concentration of ampicillin was 0.40 mg/ml. Samples (10 ml) were taken off at different time intervals ( 0 , 30, 60 and 120min). Released ampicillin was measured in the supernatant after ultracentrifugation at 110 OOOg for 1h. Antimicrobial activity in vitro Antimicrobial activity of ampicillin entrapped within liposomes or nanoparticles was compared with free ampicillin using spores of Bacillus subtilis as inoculum (Sabath and Anhalt 1980). Experiments were carried out with Triton X100-treated liposomes or esterases treated nanoparticles in order to induce the release of ampicillin. Practically, the assay plates were prepared by adding 0.2 ml of B . subtilis spore suspension to 300 ml of molten assay medium at 48"C, pouring 50 ml of the uniformly seeded agar into plastic petri dishes (120 m m x 120 mm). Small 5 mm holes were punched in the plates and 50p1 samples of different concentrations of ampicillin were then placed in them (10,5,2.5 and 1.25 pg/ml). T h e diameter of each zone of inhibition was measured after incubation of the seeded agar at 37°C for 18 h. Results are expressed in millimetres.
Results Size and entrapment eficiency Both nanoparticles and liposomes were of the same dimension, with a diameter around 200 nm (table 1). Nevertheless, the entrapment efficiency of ampicillin in PIHCA nanoparticles (90 per cent) was about 20 times higher than that of liposomes (46 per cent) (table 1). Consequently, to entrap a similar amount of ampicillin in both drug carriers, larger quantities of lipids were needed for liposomes than PIHCA polymer for nanoparticles. Therefore, the ratio of the drug to the solid phase was higher for nanoparticles (0.18) than for liposomes (0.08).
E. Fattal et al.
32
Size and entrapment efficiency of liposomes (PC/Ch/P,G:4/51 1) and nanoparticles (PI HCA).
Table 1.
Liposomes
Nanoparticles
208 70 46 008/1
187f13 90 0.18/1
~
Journal of Microencapsulation Downloaded from informahealthcare.com by Nyu Medical Center on 12/05/14 For personal use only.
Size (nm) Ampicillin entrapped (per cent) Ampicillin/mg of lipids or polymer (me)
Table 2.
Size and entrapment efficiency before and after freeze-drying of liposomes (PC/Ch/PG: 4/5/1) and nanoparticles (PIHCA). Ampicillin entrapped (per cent)
Size (nm)
~~~
Nanoparticles
Liposomes
Nanoparticles
Liposomes
205 & 33 224f 16
185f23 199&34
89 86
100 17
~
Before freeze-drying After freeze-drying
Freeze-drying Freeze-drying was not found to modify significantly the size of both drug carriers (table 2). However, a very large amount of ampicillin was released from liposomes after lyophilization whereas no leakage of the drug was observed from nanoparticles. Stability at +4"C T h e size of nanoparticles was not altered during the 21 days of storage (figure 1). A very slight increase in the size of the liposomes (from 228nm to 271 nm) was observed at day 21. Concerning drug entrapment, ampicillin was found firmly
0
5
10
15
20
25
Days Figure 1.
Effect of storage (+4"C) on the size of liposomes (PC/Ch/PG: 4/5/1) nanoparticles (PIHCA) (0).
(m)and
Liposomal and nanoparticulate ampicillin
33
100 A
8
Y
80
U
2Ql 2
c C
t
undegraded free amplcillln ( O L )
60
9)
t
Journal of Microencapsulation Downloaded from informahealthcare.com by Nyu Medical Center on 12/05/14 For personal use only.
. d
3
.-0
40
E
a
20
5
0
10
15
20
25
Days Figure 2. Effect of storage (+4"C) on the amount of ampicillin entrapped into liposomes (PC/Ch/PG: 4/5/1(W ) and into nanoparticles (PIHCA) ( 0 ) .x : free ampicillin still intact as a control.
1001
Figure 3 .
Release of ampicillin from liposomes (PC/Ch/PG:4/5/1) ( W ) and from nanoparin foetal calf serum at 37°C. ticles (PIHCA) (0)
34
E. Fattal et al.
bound to the nanoparticles during the whole storage time (figure 2). On the other hand, ampicillin was quickly released from liposomes. On day 21, only 33 per cent of ampicillin initially associated with liposomes was still entrapped (figure 2).
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Drug release in foetal calf serum After incubation in foetal calf serum, ampicillin was released very slowly from nanoparticles, following zero-order kinetics (figure 3). On the other hand, ampicillin was released rapidly from liposomes: 60 per cent of the entrapped drug had already leaked after 30 min incubation (figure 3). Antimicrobial activity in vitro Tables 3 and 4 show the antimicrobial activity of four progressive dilutions of ampicillin-loaded nanoparticles and liposomes, respectively. Entrapment of ampicillin into nanoparticles was found to decrease of about 50 per cent the in vitro antimicrobial activity of the drug. This was observed even with esterases predegraded nanoparticles (table 3). With liposomes, the zones of inhibition were obviously equivalent to those obtained with free ampicillin at the same concentrations (table 4).Absence of inhibition was noted with both nanoparticles and liposomes free of drug.
Table 3. Ampicillin-loaded PIHCA nanoparticles: antimicrobial activity in witro (PIHCA). Zone of inhibition (mm) for ampicillin concentration (pglml) of
Free ampicillin Ampicillin entrapped into nanoparticles Ampicillin entrapped into nanoparticles (pretreated with esterases: 1.5 mg/ml) Free-ampicillin nanoparticles
10
5
2.5
1.25
25
15
21 12 12
19 9 8
16 0 0
0
0
0
0
14
Table 4. Ampicillin encapsulated into liposomes (PC/Ch/PG:4/5/1):antimicrobial activity in witro. Zone of inhibition (mm) for ampicillin concentration (pglrnl) of
Free ampicillin Ampicillin entrapped into lipsomes Ampicillin entrapped into liposomes (pretreated with Triton x 100) Free-ampicillin liposomes
10
5
2.5
1-25
25 25
21 22
20 20
16 17
25 0
22
20 0
17 0
0
Liposomal and nanoparticulate ampicillin
35
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Discussion and conclusion Physico-chemical properties of ampicillin-loaded nanoparticles were compared with liposomes made by the REV method and composed of PC/Ch/PG: 4/5/1. These REV negatively charged liposomal vesicles were chosen because they were known to be recognized by the reticuloendothelial system (Schroit 1986). I n addition, they were found to possess the better encapsulating efficiency of ampicillin as compared with other liposomes (data not shown) (Fattal and Rojas, personal communication 1989). T h e data obtained have shown that ampicillin was more efficiently entrapped into polymeric nanoparticles than within liposomes. Furthermore, although freezedrying did not modify the size of both carriers, this process induced dramatic leakage of ampicillin from liposomes. No drug release from nanoparticles was observed after freeze-drying. Likewise, at 4"C, nanoparticles were more stable than liposomes in terms of drug retention. After incubation with serum, drug release from nanoparticles was slow and regular with a zero-order profile. Liberation of ampicillin from nanoparticles was probably the consequence of the polymer's bioerosion due to esterases (Lenaerts et al. 1984). More surprising is the fact that no burst effect was observed as described by Henry-Michelland et al. (1987) in phosphate buffer free of serum. This suggests that opsonization of the polymeric particles by seric proteins could play the role of a barrier against the quick desorption of the superficially adsorbed drug molecules. When incubated in presence of serum, liposomes quickly lost their drug content, due probably to the destabilization of the vesiclues by serum components as suggested by Scherphof et al. (1980). Finally, antimicrobial activity of ampicillin was not altered after encapsulation into liposomes. On the other hand, drug activity was reduced by 50 per cent after entrapment into nanoparticles. This could be due to the fact that even in the presence of esterases, the polymer was probably not entirely degraded. Indeed, entrapment of ampicillin into nanoparticles was found to improve dramatically rather to reduce the therapeutic efficiency of the drug in vivo (Youssef et at. 1988, Fattal et al. 1989). These results allow a better characterization of ampicillin-loaded nanoparticles or liposomes. They should be useful for choosing the optimal nanocarrier to target antibiotics intracellularly.
+
Acknowledgements This study was supported by Laboratoires Negma, (Buc, France), by the Conseil scientifique de la Faculti: de Pharmacie, (University of Paris XI), and by the Ministery of National Education, (Reseau Vectorisation, France). We thank Anne Sophie Dumont for excellent technical assistance.
References ALVING,C. R., 1988, Macrophages as targets for delivery of liposome-encapsulated antimicrobial agents. Advances in Drug Delivery Review, 2, 107-1 28. BAKKER-WOUNDENBERG, I. A. J. M., LOKERSE, A. F., ROERDINK, F. H., REGTS,D., and MICHEL, M. F., 1985, Free versus liposome-entrapped ampicillin in treatment of infection due to Listeria monocytogenes in normal and athymic (nude) mice. Journal of Infectious Diseases, 151 (9,917-924. FATTAL, E., YOUSSEF, M., COUVREUR, P., and ANDREMONT, A., 1989, Treatment of experimental salmonellosis in mice with ampicillin-bound nanoparticles. Antimicrobial Agents in Chemotherapy, 33 (9), 154C1543.
Journal of Microencapsulation Downloaded from informahealthcare.com by Nyu Medical Center on 12/05/14 For personal use only.
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Liposomal and nanoparticulate ampicillin
GUISE,V,, JAFFRAY, P., DELATTRE, J . , PUISIEUX, F., ADOLPHE, M., and COUVREUR, P., 1987, Comparative cell uptake of propidium iodide associated with liposomes or nanoparticles. Cellular and Molecular Biology, 33, (3), 397-405. HENRY-MICHELLAND, S., ALONSO,M. J., ANDREMONT, A,, MAINCENT, P., SAUZIERES, J., and COUVREUR, P., 1987, Attachment of antibiotics to nanoparticles: preparation, drugrelease and antimicrobial activity in vitro. International Journal of Pharmaceutics, 35, 121-127. JULIANO, R. L., 1988, Factors affecting the clearance kinetics and tissue distribution of liposomes, microspheres and emulsion. Advances in Drug Delivery Review, 2, 31-54. LENAERTS, V., COUVREUR, P., CHRISTIAENS-LEYH, D., JOIRIS,E., ROLAND, M., ROLLMAN, B., and SPEISER, P., 1984, Degradation of poly(isobutylcyanoacry1ate) nanoparticles. Biomaterials, 5, 65-68. MARGOSIS, M., 1982, Quantitative reversed-phase high-performance liquid chromatographic analysis of ampicillin. Journal of Chromatography, 236, 469-480. RENARD, C., VANDERHAEGHE, H. J., CLAES,P. J., ZENEBERGH, A., and TULKENS, P. M., 1987, Influence of conversion of penicillin G into a basic derivative on its accumulation and subcellulat localization in cultured macrophages. Antimicrobial Agents in Chemotherapy, 31 (3), 410-416. SABATH, L. D., and ANHALT,J. P., 1980, Assay of antimicrobics: Laboratory test in chemotherapy. Manual of Clinical Microbiology, edited by E. H. Lenette (American Society of Microbiology Publishers), pp. 4 8 5 4 9 0 . SAVELLO, D. R., and SHANGRAW, R. F., 1971, Stability of sodium ampicillin solutions in the frozen and liquid states. American Journal of Hospital Pharmaceutics, 28, 754-759. SCHERPHOF, G., ROERDINK, D., HOEKSTRA, D., ZBOROWSKI, J., and WISSE,E., 1980, Stability of liposomes in blood constituents: consequences for uptake of liposomal lipid and entrapped compounds by rat liver cells. Liposomes in Biological Systems, edited by G. Gregoriadis and A. C. Allisson (John Wiley & Sons), pp. 179-209. SCHROIT, A. J . , MADSEN, J., and NAYAR,R., 1986, Liposome-cell interactions: in vitro discrimination of uptake mechanism and in vivo targeting strategies to mononuclear phagocytes. Chemistry and Physics of Lipids, 40,373-393. YOUSSEF, M., FATTAL, E., ALONSO, M. J., ROBLOT-TREUPEL, L., SAUZIERES, J., TANCREDE, C., OMNES,A., COUVREUR, P., and ANDREMONT, A., 1988, Effectiveness of nanoparticlebound ampicillin in the treatment of Listeria monocytogenes infection in athymic nude mice. Antimicrobial Agents in Chemotherapy, 8, 1204-1 207.
1991, VOL. 8, NO. 1, 29-36
Ampicillin-loaded liposomes and nanoparticles: comparison of drug loading, drug release and in vitro antimicrobial activity
Journal of Microencapsulation Downloaded from informahealthcare.com by Nyu Medical Center on 12/05/14 For personal use only.
E. FATTAL, J. ROJAS, L. R O B L O T - T R E U P E L , A. ANDREMONT and P. COUVREUR Laboratoire de Pharmacie GalCnique, URA CNRS 1218, FacultC de Pharmacie, Chatenay-Malabry, France, Laboratoire de Microbiologie, FacultC de Pharmacie, Chatenay-MalFbry, France and Laboratoire d'Ecologie Microbienne, Institut Gustave Roussy, Villejuif, France (Received 6 March 1990; accepted 28 March 1990)
In this paper, we report the physico-chemical properties of negatively charged liposomes and of polyisohexylcyanoacrylate nanoparticles loaded with ampicillin. Although the carriers were of the same size (200nm), drug-loading capacity was 20 times higher for nanoparticles than for liposomes. After freeze-drying or storage at 4"C, no drug escaped from polymeric nanoparticles. On the other hand, in the same conditions, ampicillin leaked rapidly from liposomes. Drug release in foetal calf serum was gradual (of zero order) with nanoparticles, whereas it was rapid with liposomes. Finally, the antimicrobial activity of ampicillin-entrapped liposomes or nanoparticles was studied in vitro.
+
Introduction Liposomes and nanoparticles have aroused interest as potent drug carriers that are capable of enhancing the intracellular delivery of antibiotics (Alving 1988). Resistance of intracellular infections to chemotherapy was shown to be related to the low intracellular uptake of commonly used antibiotics or to their reduced activity at the acid p H of lysosomes. Acidic antibiotics (b-lactams) d o not diffuse through the lysosomal membrane because of their ionic character at neutral extralysosomal p H (Renard et al. 1987). Recently, we have shown in experimentally infected mice that linkage of ampicillin to polyisohexylcyanoacrylate (PIHCA) nanoparticles increased the efficiency of the drug in experimental murine salmonellosis (Fattal et al. 1989). We have also shown an increased efficacy of this preparation in chronic listeriosis in athymic nude mice (Youssef et al. 1988). Surprisingly, Bakker-Woundenberg et al. (1985), did not observe, on the same model, a similar effect with liposome-entrapped ampicillin. However, the dose of ampicillin bound to lipsomes in this study was poor and only 23 per cent of that was bound to nanoparticles. A straightforward comparison of the two treatments was thus not possible. Because drug targeted by aid of liposomes or nanoparticles may result in a different intracellular distribution profile (Guise et al. 1987), the efficiency against intralysosomal infections can differ. I n addition, owing to the specific behaviour of each carrier with seric components (single opsonization for polymeric nanoparticles Address for correspondence: Prof. Patrick Couvreur, Laboratoire de Pharmacie GalCnique, URA CNRS 1218, 5 Rue J. B. CICment, 92296 Chatenay-Malabry, France. 0265-2048191 $3.00 0 1991 Taylor & Francis Ltd
30
E . Fattal et al.
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or liposomal destabilization by H D L ) (Juliano 1988), drug release profile may also vary. Thus, the need to design a carrier with optimized efficacy against intracellular infections requires comparison of the activity of antibiotics targeted by either liposomes or nanoparticles with comparable drug loading. Therefore, we report here the comparison of the major physicochemical properties of liposomes and nanoparticles loaded with the same amount of ampicillin.
Materials and method Materials Soybean phosphatidylcholine was obtained from Lecithos (Saint Maur, France). Cholesterol, phosphatidylglycerol, esterase were purchased from Sigma (Saint Louis, USA). Sodium ampicillin was obtained from Bristol (Paris, France) and ampicillin trihydrate from Negma (Buc, France). Isohexylcyanoacrylate was synthetized by Sopar (Brussels, Belgium). Spores of Bacillus subtilis (ATCC 6663) were used as inoculum for classical microbiological assay. Assay medium no. 2 was obtained from Difco Laboratory (Detroit, USA). Foetal calf serum was obtained from Gibco (New York, USA).
Preparation of liposome-entrapped ampicillin Liposomes were prepared from a lipid mixture of 168pmol of soybean phosphatidylcholine, 210 pmol of cholesterol and 42 pmol of phosphatidylglycerol. The dried lipid film, obtained on rotary evaporator, was redissolved in 14ml of diethylether. A 50 mg/ml solution of sodium ampicillin in buffer ( T R I S 0.1 M, NaCl 0.15 M, p H = 7-4) was added. After 3 min of bath sonication, the diethylether was removed under vacuum. Liposomes were extruded through a 0.2 pm polycarbonate membrane (Nucleopore, Pleasontown, USA). Unentrapped ampicillin was then separated by Sephadex G50 (Pharmacia, Upssala, Sweden) filtration. Liposomes fractions were pooled and concentrated by ultracentrifugation (300 000 g for 30 min). The amount of ampicillin encapsulated in liposomes was determined using a reverse phase liquid chromatography assay (Margosis 1982). T h e size of the liposomes was determined using a laser light scattering method (Nanosizer coulter, Coultronics, Margency, France). Drug-free liposomes were prepared by the same way. Preparation of nanoparticle-entrapped ampicillin Isohexylcyanoacrylate monomer (100pl) was added under mechanical stirring to lOml of an aqueous polymerization medium (dextran 70 1 per cent in 10-3 N HCI) containing 2 mg/ml of ampicillin trihydrate. After 6 h of polymerization, a milky suspension was obtained, neutralized with 1 N NaOH and brought to isotonicity by glucose 5 per cent. Measurements of ampicillin entrapped into nanoparticles were carried out on the supernatant after ultracentrifugation of the samples at 110OOOgfor 1 h 30 min. Analytical determinations were made using an HPLC method (Margosis 1982). Freeze-drying Ampicillin entrapped into liposomes or nanoparticles was freeze dried at - 40°C using a freeze-dryer (Christ alpha 1.5, Bioblock, Vanves, France) for 24 h under vacuum. Glucose 5 per cent was used as cryoprotector. Resuspension of nanoparticle
Liposomal and nanoparticulate ampicillin
31
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liposomal pellet was carried out by a single addition of distilled water (10 ml) to each vial. T h e size and drug content of the carriers were determined before and after freeze-drying. Stability a t +4"C Stability of ampicillin entrapped into liposomes or nanoparticles was studied after storage during 21 days at +4"C. This experiment was carried out at pH4.9 which corresponded to the optimum of stability for ampicillin (Savello and Shangraw 1971). Size and drug content were determined at different time intervals (days 1, 7, 15 and 21). T h e stability of free ampicillin was tested in the same experimental conditions.
Drug release in foetal calf serum Eight millilitres of the suspension of liposome-entrapped ampicillin was incubated with 32 ml of foetal calf serum (FCS) at 37°C. T h e final concentration of ampicillin was of 0*46mg/ml. This preparation was taken off at different time intervals (0,30,60 and 120min). Released ampicillin was separated by Sephadex Gel filtration. Then, liposomal fractions were pooled and ampicillin content measured after precipitation of serum proteins with 1.2 M perchloric acid. Eight millitres of the suspension of nanoparticle-entrapped ampicillin was incubated with 32ml of fetal calf serum (FCS) at 37°C. T h e concentration of ampicillin was 0.40 mg/ml. Samples (10 ml) were taken off at different time intervals ( 0 , 30, 60 and 120min). Released ampicillin was measured in the supernatant after ultracentrifugation at 110 OOOg for 1h. Antimicrobial activity in vitro Antimicrobial activity of ampicillin entrapped within liposomes or nanoparticles was compared with free ampicillin using spores of Bacillus subtilis as inoculum (Sabath and Anhalt 1980). Experiments were carried out with Triton X100-treated liposomes or esterases treated nanoparticles in order to induce the release of ampicillin. Practically, the assay plates were prepared by adding 0.2 ml of B . subtilis spore suspension to 300 ml of molten assay medium at 48"C, pouring 50 ml of the uniformly seeded agar into plastic petri dishes (120 m m x 120 mm). Small 5 mm holes were punched in the plates and 50p1 samples of different concentrations of ampicillin were then placed in them (10,5,2.5 and 1.25 pg/ml). T h e diameter of each zone of inhibition was measured after incubation of the seeded agar at 37°C for 18 h. Results are expressed in millimetres.
Results Size and entrapment eficiency Both nanoparticles and liposomes were of the same dimension, with a diameter around 200 nm (table 1). Nevertheless, the entrapment efficiency of ampicillin in PIHCA nanoparticles (90 per cent) was about 20 times higher than that of liposomes (46 per cent) (table 1). Consequently, to entrap a similar amount of ampicillin in both drug carriers, larger quantities of lipids were needed for liposomes than PIHCA polymer for nanoparticles. Therefore, the ratio of the drug to the solid phase was higher for nanoparticles (0.18) than for liposomes (0.08).
E. Fattal et al.
32
Size and entrapment efficiency of liposomes (PC/Ch/P,G:4/51 1) and nanoparticles (PI HCA).
Table 1.
Liposomes
Nanoparticles
208 70 46 008/1
187f13 90 0.18/1
~
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Size (nm) Ampicillin entrapped (per cent) Ampicillin/mg of lipids or polymer (me)
Table 2.
Size and entrapment efficiency before and after freeze-drying of liposomes (PC/Ch/PG: 4/5/1) and nanoparticles (PIHCA). Ampicillin entrapped (per cent)
Size (nm)
~~~
Nanoparticles
Liposomes
Nanoparticles
Liposomes
205 & 33 224f 16
185f23 199&34
89 86
100 17
~
Before freeze-drying After freeze-drying
Freeze-drying Freeze-drying was not found to modify significantly the size of both drug carriers (table 2). However, a very large amount of ampicillin was released from liposomes after lyophilization whereas no leakage of the drug was observed from nanoparticles. Stability at +4"C T h e size of nanoparticles was not altered during the 21 days of storage (figure 1). A very slight increase in the size of the liposomes (from 228nm to 271 nm) was observed at day 21. Concerning drug entrapment, ampicillin was found firmly
0
5
10
15
20
25
Days Figure 1.
Effect of storage (+4"C) on the size of liposomes (PC/Ch/PG: 4/5/1) nanoparticles (PIHCA) (0).
(m)and
Liposomal and nanoparticulate ampicillin
33
100 A
8
Y
80
U
2Ql 2
c C
t
undegraded free amplcillln ( O L )
60
9)
t
Journal of Microencapsulation Downloaded from informahealthcare.com by Nyu Medical Center on 12/05/14 For personal use only.
. d
3
.-0
40
E
a
20
5
0
10
15
20
25
Days Figure 2. Effect of storage (+4"C) on the amount of ampicillin entrapped into liposomes (PC/Ch/PG: 4/5/1(W ) and into nanoparticles (PIHCA) ( 0 ) .x : free ampicillin still intact as a control.
1001
Figure 3 .
Release of ampicillin from liposomes (PC/Ch/PG:4/5/1) ( W ) and from nanoparin foetal calf serum at 37°C. ticles (PIHCA) (0)
34
E. Fattal et al.
bound to the nanoparticles during the whole storage time (figure 2). On the other hand, ampicillin was quickly released from liposomes. On day 21, only 33 per cent of ampicillin initially associated with liposomes was still entrapped (figure 2).
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Drug release in foetal calf serum After incubation in foetal calf serum, ampicillin was released very slowly from nanoparticles, following zero-order kinetics (figure 3). On the other hand, ampicillin was released rapidly from liposomes: 60 per cent of the entrapped drug had already leaked after 30 min incubation (figure 3). Antimicrobial activity in vitro Tables 3 and 4 show the antimicrobial activity of four progressive dilutions of ampicillin-loaded nanoparticles and liposomes, respectively. Entrapment of ampicillin into nanoparticles was found to decrease of about 50 per cent the in vitro antimicrobial activity of the drug. This was observed even with esterases predegraded nanoparticles (table 3). With liposomes, the zones of inhibition were obviously equivalent to those obtained with free ampicillin at the same concentrations (table 4).Absence of inhibition was noted with both nanoparticles and liposomes free of drug.
Table 3. Ampicillin-loaded PIHCA nanoparticles: antimicrobial activity in witro (PIHCA). Zone of inhibition (mm) for ampicillin concentration (pglml) of
Free ampicillin Ampicillin entrapped into nanoparticles Ampicillin entrapped into nanoparticles (pretreated with esterases: 1.5 mg/ml) Free-ampicillin nanoparticles
10
5
2.5
1.25
25
15
21 12 12
19 9 8
16 0 0
0
0
0
0
14
Table 4. Ampicillin encapsulated into liposomes (PC/Ch/PG:4/5/1):antimicrobial activity in witro. Zone of inhibition (mm) for ampicillin concentration (pglrnl) of
Free ampicillin Ampicillin entrapped into lipsomes Ampicillin entrapped into liposomes (pretreated with Triton x 100) Free-ampicillin liposomes
10
5
2.5
1-25
25 25
21 22
20 20
16 17
25 0
22
20 0
17 0
0
Liposomal and nanoparticulate ampicillin
35
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Discussion and conclusion Physico-chemical properties of ampicillin-loaded nanoparticles were compared with liposomes made by the REV method and composed of PC/Ch/PG: 4/5/1. These REV negatively charged liposomal vesicles were chosen because they were known to be recognized by the reticuloendothelial system (Schroit 1986). I n addition, they were found to possess the better encapsulating efficiency of ampicillin as compared with other liposomes (data not shown) (Fattal and Rojas, personal communication 1989). T h e data obtained have shown that ampicillin was more efficiently entrapped into polymeric nanoparticles than within liposomes. Furthermore, although freezedrying did not modify the size of both carriers, this process induced dramatic leakage of ampicillin from liposomes. No drug release from nanoparticles was observed after freeze-drying. Likewise, at 4"C, nanoparticles were more stable than liposomes in terms of drug retention. After incubation with serum, drug release from nanoparticles was slow and regular with a zero-order profile. Liberation of ampicillin from nanoparticles was probably the consequence of the polymer's bioerosion due to esterases (Lenaerts et al. 1984). More surprising is the fact that no burst effect was observed as described by Henry-Michelland et al. (1987) in phosphate buffer free of serum. This suggests that opsonization of the polymeric particles by seric proteins could play the role of a barrier against the quick desorption of the superficially adsorbed drug molecules. When incubated in presence of serum, liposomes quickly lost their drug content, due probably to the destabilization of the vesiclues by serum components as suggested by Scherphof et al. (1980). Finally, antimicrobial activity of ampicillin was not altered after encapsulation into liposomes. On the other hand, drug activity was reduced by 50 per cent after entrapment into nanoparticles. This could be due to the fact that even in the presence of esterases, the polymer was probably not entirely degraded. Indeed, entrapment of ampicillin into nanoparticles was found to improve dramatically rather to reduce the therapeutic efficiency of the drug in vivo (Youssef et at. 1988, Fattal et al. 1989). These results allow a better characterization of ampicillin-loaded nanoparticles or liposomes. They should be useful for choosing the optimal nanocarrier to target antibiotics intracellularly.
+
Acknowledgements This study was supported by Laboratoires Negma, (Buc, France), by the Conseil scientifique de la Faculti: de Pharmacie, (University of Paris XI), and by the Ministery of National Education, (Reseau Vectorisation, France). We thank Anne Sophie Dumont for excellent technical assistance.
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Liposomal and nanoparticulate ampicillin
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