98 PAUL ET AL.

© 1998 Wiley-Liss, Inc.

DRUG DEVELOPMENT RESEARCH 43:98–104 (1998)

Research Article

Pentamidine-Loaded Poly(D,L-lactide) Nanoparticles:Adsorption and Drug Release

Muriel Paul,1* Abdelkader Laatiris,1 Hatem Fessi,2 Barbara Dufeu,1 Rémy Durand,3

Michèle Deniau,3 and Alain Astier1

1Laboratoire de Pharmacotechnie, Service Pharmacie, Créteil, France2Faculté de Pharmacie Claude Bernard, Lyon, France

3Laboratoire de Parasitologie, Faculté de Médecine de Créteil, Créteil, France

ABSTRACT This work describes the loading capacity of poly(D,L-lactide) nanoparticles, the factors in-fluencing pentamidine release, and the cytotoxicity of nanoparticles. The nanoprecipitation method wasused to prepare pentamidine-loaded poly(D,L-lactide) nanoparticles. Various concentrations of pentami-dine base and polymer were tested. The influence of the dilution, temperature, and ionic strength wasevaluated. The cytotoxicity on J 774 cells of unloaded nanoparticles, pentamidine-loaded nanoparticles,and pentamidine isethionate were tested. The percentage of binding decreased significantly with drug load.A nonlinear increase in drug uptake per unit mass of polymer with the equilibrium pentamidine concentra-tion was found. A Langmuir-type sorption was suggested (r = 0.998). 390 µg/ml was found to be thehighest level of drug incorporation. The increase of polymer concentration did not improve the pentami-dine fixation yield. The increase in temperature or buffer molarity induced a significant release of pentami-dine. The increase in dilution also induced an increase in release of pentamidine. The cytotoxicity ofpentamidine-loaded nanoparticles and unloaded nanoparticles was superimposable. After 24 h of incuba-tion, pentamidine-loaded nanoparticles presented an IC50 value significantly lower than that of free drug(0.39 vs. 6.5 g/ml). Drug Dev. Res. 43:98–104, 1998. © 1998 Wiley-Liss, Inc.

Key words: pentamidine; poly(lactide) nanoparticles; adsorption; release

Contract grant sponsor: Baxter Dubernard Hospital Foundation.

*Correspondence to: Dr. Muriel Paul, 51 avenue du Maréchalde Lattre de Tassigny, 94 010 Créteil, France. E-mail: alastier@micronet.fr

Received 25 July 1997; Accepted 26 February 1998

INTRODUCTION

Drug targeting, using colloidal systems, has led bothto a reduction in toxicity and an increase in efficacy ofantileishmanial drugs [Alving, 1986]. Colloid carriers arepreferentially taken up by cells of the mononuclear ph-agocyte system and thus target the drug directly to theparasitized host cells [Youssef et al., 1988; Puisieux et al.,1994]. Many drugs, such as amphotericin B, pentavalentantimonials, and primaquine, have been evaluated on dif-ferent colloidal systems [Modabber, 1992; Gaspar et al.,1992; Rodrigues et al., 1995; Paul et al., 1997a].

Pentamidine, a major antileishmanial drug whichhas long been a second-line treatment of leishmaniasesafter antimony failure or intolerance [Davidson and Croft,1993], has been loaded on several carriers (human red

cell ghosts, liposomes) [Berman et al., 1988; Banerjee etal., 1996]. We previously loaded pentamidine onpolymethacrylate nanoparticles and studied their physi-cochemical properties and the in vitro delivery of the drugfrom this carrier [Paul et al., 1997a]. It was found thatpentamidine was bound to nanoparticles by ionic inter-action. In vitro and in vivo experiments were alsoperformed in order to evaluate the action of pentamidine-

Strategy, Management and Health Policy

Venture CapitalEnablingTechnology

PreclinicalResearch

Preclinical DevelopmentToxicology, FormulationDrug Delivery,Pharmacokinetics

Clinical DevelopmentPhases I-IIIRegulatory, Quality,Manufacturing

PostmarketingPhase IV

PENTAMIDINE PLA NANOPARTICLES 99

bound nanoparticles against intracellular Leishmania[Deniau et al., 1993; Fusaï et al., 1994]. Pentamidineloaded on polymethacrylate was sixfold more active thanfree drug in Leishmania infantum-infected mice [Durandet al., 1997a]. These studies showed that targeted penta-midine should be of major interest in the treatment ofvisceral leishmaniasis. Despite a very slight cytotoxicityassociated with good animal tolerance, the main prob-lem with these nanoparticles was their low biodegrad-ability [Rolland, 1987].

Conversely, polyester polymers have become veryinteresting for sustained drug delivery since they are knownto be completely biodegradable and well tolerated by tis-sues [Kulkarni et al., 1971]. More recently, the physicochemi-cal properties and the stability of pentamidine-loadedpoly(D,L-lactide) (PLA) nanoparticles were described [Paulet al., 1997b]. The in vivo study demonstrated that penta-midine-loaded PLA nanoparticles were less active than pen-tamidine-loaded methacrylate nanoparticles (efficiencymultiplied by 3) [Durand et al., 1997b].

As physicochemical characterization of nano-particles remains an important prerequisite for the suc-cess of nanoparticle development, it seemed importantto identify the interaction between drug and polymer. Inthe present study, we characterized the loading capacityof PLA nanoparticles and studied the factors that influ-ence the release of pentamidine.

MATERIALS AND METHODS

Materials

Pentamidine isethionate was purchased from Bellon(Neuilly sur Seine, France). The phospholipid mixture(PL) (Lipoid S 75) was kindly supplied by Lipoid GmbH(Ludwigshafen, Germany). Poloxamer (Symperonic PE/F-68®) was purchased from ICI (Clamart, France). Poly-mer poly(D,L-lactide) (PLA, mol. wt. (GPC) 200,000) wassupplied by Boehringer Ingelheim (Ingelheim, Ger-many). Other reagents were analytical grade.

Production of Pentamidine Free Base

Pentamidine base was obtained by precipitation ofpentamidine isethionate solution in alkaline medium(25% ammonium hydroxide) at 4°C under magnetic stir-ring. The precipitate was filtered, washed twice with coldammoniacal water, and dried under vacuum. Pentami-dine base was characterized by UV (Jasco 7800, Prolabo,Paris, France) and infrared spectroscopy (Perkin Elmer2000 FT-IR, St Quentin en Yvelines, France).

Preparation of PLA Nanoparticles

PLA nanoparticles were prepared according to themethod reported by Fessi et al. [1992]. Pentamidine basein ethanolic solution (1mg/ml), phospholipids (125 mg), and

PLA were dissolved in acetone (25 ml) under magnetic stir-ring at 60°C. This solution was mixed under magnetic stir-ring with an alkaline aqueous solution (1 ml of 25%ammonium hydroxide) containing Poloxamer (250 mg). Af-ter 15 min of stirring, the acetone and some of the waterwere evaporated on a rotary evaporator under reduced pres-sure (0.8 bar) at 60°C. The final volume was 10 ml.

Physicochemical Characterization of NanoparticlesDetermination of drug loading

Total pentamidine concentration was determinedafter dissolution of nanoparticle suspension (100 µl) in amixture composed of acetonitrile (80%) and water (20%)(dilution 1/100). Free pentamidine was determined afterseparation of the loaded nanoparticles from the aqueousmedium by a combined ultrafiltration–centrifugation tech-nique (Ultrafree MC®, Millipore, Bedford, MA).Ultrafiltrate was then diluted in the same solvent mixture(1/50). The bound percentage (BP) was calculated as:

BP = [ (total pentamidine - free pentamidine) /total pentamidine ] × 100.

Pentamidine were assessed by high-performance liq-uid chromatography (HPLC). The chromatographic systemconsisted of a solvent delivery pump (880 PU, Jasco, Tokyo,Japan), an autosampler equipped with a 100 µl loop (Spec-tra AS 100 XR, Thermo Separation Product, CA), and a UVscanning detector (UV 975, Jasco, Tokyo, Japan). Eachsample was injected onto a reverse-phase C18 column(Hypersil, 5 µm, 250 mm × 4.6 mm, I.D., Shandon, France).The mobile phase was composed of 700 ml of methanol and300 ml of heptane-sulfonic acid (0.05 M) and diethylamine(0.014M) aqueous solution. The pH of the aqueous solutionwas adjusted to 3.00 using phosphoric acid. The flow ratewas 0.9 ml/min. Detection of pentamidine was performedby ultraviolet absorption at 280 nm. Data were acquiredand processed with the Winilab Chromatography manager(Perichrom, Saulx-les-Chartreux, France).

Photon correlation spectroscopyParticle size distribution, average size, and poly-

dispersity index were measured by laser scattering usinga monochromatic laser ray diffusion counter (NanosizerN4, Coultronics, Margency, France).

Determination of pHThe pH of nanoparticle suspensions was deter-

mined at 20°C using a digital pH meter 646 (Prolabo,Paris, France).

Determination of nanoparticle suspensionabsorbance

The nanoparticle suspension was diluted in water(1/20) and the optical density was measured at 510 nm.

These assays were performed in triplicate.

100 PAUL ET AL.

Loading Capacity of PLA

Various concentrations of pentamidine ranging from0.1 to 0.5 mg/ml were tested. Unloaded nanoparticleswere prepared according to the same formulation, omit-ting pentamidine. Various concentrations of polymerranging from 5 to 15 mg/ml were tested with a 0.5 mg/mlpentamidine concentration.

Factors Influencing ReleaseInfluence of dilution

In order to investigate the release of pentamidinefrom nanoparticles, pentamidine-loaded PLA nano-particles (0.2 mg/ml) were diluted (1/10 to 1/500) withphosphate-buffered saline (PBS) (0.1M, pH 7.4) at 37°C.Immediately after dilution, aliquots were filtered by cen-trifugal ultrafiltration process. Ultrafiltrates were assayedfor free pentamidine by HPLC.

Influence of temperature

Pentamidine-loaded PLA nanoparticles (0.2 mg/ml)were diluted (1/20) in PBS (0.1 M, pH 7.4) at five tem-peratures (4, 22, 37, 60, and 80°C). Immediately afterdilution, aliquots of nanoparticle suspensions (0.5 ml)were collected and filtered by centrifugal ultrafiltrationprocess. Ultrafiltrates were assayed for free pentamidineby HPLC.

Influence of molarity

Pentamidine-loaded PLA nanoparticles (0.2 mg/ml)were diluted (1/20) in PBS at 4°C. Different molaritiesranging from 10–5 M to 1 M were tested. The same pro-cess was applied to determine pentamidine release.

These assays were performed in duplicate.

Cytotoxicity Assay

The macrophage-like cell line, J774, derived froma BALB/c mouse, was used to evaluate the cytotoxicityof pentamidine-loaded PLA nanoparticles, unloadednanoparticles, and pentamidine isethionate. Cells wereseeded in 24-well plates at a cellular density of 5 105 cells/ml and were cultivated in RPMI-1640 medium with20 % (v/v) heat-inactivated fetal calf serum supplemented

with L-glutamine and sodium bicarbonate. In each well,0.5 ml of cell suspension was introduced. Cells were in-cubated at 37°C in a humidified atmosphere containing5% CO2 for 4, 24, and 48 h with 100 µl of various drugdilutions. For unloaded nanoparticles, polymeric concen-trations ranged from 3.125 10–3 to 0.5 mg/ml. For penta-midine-loaded nanoparticles, concentrations ranged from0.025 to 4 µg/ml and for pentamidine isethionate from0.1 to 8 µg/ml. Cytotoxicity was determined by measur-ing the inhibition of cell growth using the tetrazoliumdye (MTT) assay.

Statistical Analysis

Results were expressed as mean ±SD. A one-wayanalysis of variance or a U-test was performed to com-pare the influence of the various parameters. A P-valueless than 0.05 was considered a significant difference.

RESULTS AND DISCUSSION

Our results showed that pentamidine was essen-tially bound to PLA nanoparticles by adsorption. Actu-ally, the drug uptake per unit mass of polymer particlesdid not increase linearly with the equilibrium concen-tration of pentamidine (Fig. 1). For concentrations un-

Fig. 1. Langmuir sorption of pentamidine base on poly(D,L-lactide)nanoparticles at pH 8 at 20°C.

TABLE 1. Physicochemical Characteristics of Pentamidine-Loaded PLA Nanoparticles at Various Concentrations

Pentamidine (mg/ml) 0.1 0.2 0.3 0.4 0.5

Percentage of binding 100 ± 0ab 100 ± 0ab 95.2 ± 2.1b 89.0 ± 1.5b 75.8 ± 1.8Size (nm) 148 ± 25 124 ± 20 129 ± 18 139 ± 35 131 ± 15.6pH 8.25 ± 0.10 8.15 ± 0.07 8.12 ± 0.05 8.05 ± 0.10 8.0 ± 0.05

Results are expressed as means ± standard errors.aP < 0.01:0.1 mg/ml vs. 0.4 mg/ml, 0.2 mg/ml vs. 0.4 mg/ml, 0.3 mg/ml vs. 0.5 mg/ml, and 0.4 mg/ml vs. 0.5 mg/ml.bP < 0.001:0.1 mg/ml vs. 0.5 mg/ml

PENTAMIDINE PLA NANOPARTICLES 101

der 200 µg/ml, the percentage of binding was maximal.For higher concentrations, the percentage decreasedand reached 75.8 ± 1.8% for a 0.5 mg/ml pentamidineconcentration. Under our experimental conditions, aconcentration of 390 µg/ml was found to be the highestlevel of drug incorporation achieved within nanoparticle(Table 1).

A plot of these values suggested a Langmuir-typesorption isotherm (correlation coefficient = 0.998). TheLangmuir equation [Zografi, 1995] is:

X/m = a × bc / 1 + bcwhere X/m is the ratio of adsorbed drug to adsorbate, cis the equilibrium concentration of drug, and a and bare constants related to the surface area of adsorbentand to the enthalpy of adsorption, respectively.

Plotting the values of c/(x/m) vs. c gave a linearrelation with a slope of 1/a and a y-axis intercept of 1/ab.The values for Langmuir sorption a and b were alsocalculated and found to be 87.6 mg/g and 0.00115 l/mgfor a and b, respectively. The high value of a (constantrelated to the surface area of polymer) associated to avery low value of b (constant related to the enthalpy ofadsorption) showed that PLA polymer had a high ca-pacity of adsorption and that the standard enthalpy ofsorption was probably low, excluding a chemiosorptionmechanism. The values of a and b are similar to thoseobtained with verapamil hydrochloride-loaded poly-butylcyanoacrylate nanoparticles [El Egakey and Speiser,1982]. The adsorption of many drugs, such as dauno-rubicin, dactinomycin on polymethyl, and polyethyl-cyanoacrylate, has been described [Couvreur et al., 1979;Illum et al., 1986]. On the contrary, the loading of manydrugs on poly(lactide) polymers was studied but the ad-sorption mechanism has never been evaluated [Guterreset al., 1995; Rodrigues et al., 1995].

Pentamidine loading did not induce modificationof mean diameters and pH of nanoparticle suspensions(Table 1).

Polymer concentration had no effect on the bind-ing of pentamidine, suggesting that the surface area wasnot modified by higher polymer concentrations (P =0.624). The mean diameter of nanoparticles was not in-

fluenced by the polymer concentration. The maximalpercentage of fixation was 80.6 ± 0.6%. The pH of thenanoparticle suspensions was not significantly modifiedwith the increase in polymer concentration (P = 0.562).On the contrary, a significant increase of the absorbanceat 510 nm was observed with the increase in polymerconcentration (Table 2). An exponential relation betweenthe absorbance and the polymer concentration was evi-denced (r = 0.98) (Fig. 2).

As the sorption is a reversible process, the adsorbeddrug could be completely desorbed. Actually, our in vitrostudies showed that the release can reach 70 to 80%.Every parameter able to increase the solubility of penta-midine can induce its release in the medium.

Increasing the temperature enhanced a significantpentamidine release from PLA polymer (P = 0.0013).Nevertheless, a temperature of 60°C should be reachedto induce a significant release compared with a tem-perature of 4°C. The results are summarized in Table3. This could be due to a weakening of the attractiveforces between drug molecules and the polymer. Inaddition, the increase in temperature induced an in-crease in pentamidine solubility and, thus, decreasedits affinity for the polymer.

TABLE 2. Influence of Polymer Concentration on Physicochemical Characteristics of Pentamidine-Loaded PLA Nanoparticles (0.5 mg/ml)

Polymer concentration(mg/ml) 5 7.5 10 12.5 15

Percentage of binding 75.1 ± 2.9 75.5 ± 4.2 75.9 ± 2.5 78.1 ± 2.3 80.6 ± 0.6pH 7.79 ± 0.07 8.00 ± 0.21 7.85 ± 0.08 8.00 ± 0.03 7.86 ± 0.04Do (510 nm) 0.092 ± 0.001abc 0.113 ± 0.004ac 0.148 ± 0.004bc 0.28 ± 0.03c 0.339 ± 0.02

Results are expressed as means ± standard errors.aP < 0.05: 5 mg/ml vs. 10 mg/ml, 7.5 mg/ml vs. 10 mg/ml.bP < 0.01: 5 mg/ml vs. 10 mg/ml and 10 mg/ml vs. 12.5 mg/ml.cP < 0.001: 5 mg/ml vs. 12.5 mg/ml and 15 mg/ml, 7.5 mg/ml vs. 12.5 mg/ml and 15 mg/ml, 10 mg/ml vs. 15 mg/ml, 12.5 mg/ml vs. 15 mg/ml.

Fig. 2. Influence of polymeric concentration on the absorbance of di-luted nanoparticle suspensions (1/20) at 510 nm.

102 PAUL ET AL.

The increase in the ionic strength of the mediumalso induced an important and significant release of pen-tamidine, involving a better solubility of pentamidine (P< 0.0001). The maximal release (81.4%) was obtainedwith the highest buffer molarity (1 M). Nevertheless, indiluted buffer (10-5 M), no release of pentamidine in themedium was observed. A hyperbolic relation was foundbetween the molarity of buffer and the release of penta-midine in the medium (r = 0.996) (Fig. 3).

In vitro release studies associating the effects oftemperature, dilution, and ionic strength showed an im-mediate release of pentamidine in the medium, reaching70% for the highest dilution (1/500). A hyperbolic rela-tion was found between the dilution factor and the per-centage of release (r = 0.98) (Fig. 4). Similar results werefound with primaquine-loaded PLA nanoparticles (69.6%)[Rodrigues et al., 1995].

Under each of these conditions, pentamidine solu-bility could be predicted by its ionization status. Duringnanoparticle preparation, pentamidine was introducedin its base form to improve the fixation yield. But afterpreparation, the pH of nanoparticle suspensions was 8.At this pH, pentamidine was essentially ionized (pKa =11.4). Pentamidine ionization induces a modification of

the UV spectra. The λmax for the base was 250 nm and theλmax for the salt 269 nm. A bathoachromic shift (16 nm)was observed with pentamidine-loaded nanoparticles. Anintermediate λmax (266 nm) was found. Therefore, it waspossible to calculate the percentage of base and salt ofpentamidine in the pentamidine-loaded PLA nano-particles. A low percentage of base (17%) was associatedwith a high percentage of salt (83%), which confirmedthe ionized status of pentamidine in nanoparticles. Thisphenomenon was also observed with other drugs, suchas rose bengal [Illum et al., 1986].

Pentamidine-loaded PLA nanoparticles were rela-tively toxic to the J774 cells after 24 or 48 h of incubation(IC50 = 0.39 and 0.18 µg/ml, respectively) (Table 4). Asshown in Figure 5, the cytotoxicity of pentamidine-loadednanoparticles and unloaded nanoparticles was super-imposable. A Michaelis-Menten relation was found be-tween drug concentration and the percentage of mortality(r = 0.99). After 48 h of incubation, a similar relation wasfound. The inhibitory concentrations were lower than thoseobtained after 24 h of incubation. The cytotoxic effect wasrelated to the nature of the polymer: after hydrolysis, thePLA polymer induces a production of lactic acid, toxic forcells. Nevertheless, studies with other drugs have shown

TABLE 3. Influence of Temperature on Pentamidine Release

Temperature(°C) 4 22 37 60 80

Percentage of release 45.8 ± 2.0b 47.3 ± 3.0a 51.7 ± 1.5a 60.3 ± 4.0a 82.1 3.0

Nanoparticles were diluted in PBS (1/20).Results are expressed as means ± standard errors.U-test was used for statistical analysis.aP < 0.05: 22°C, 37°C, and 60°C vs. 80°C.bP < 0.01: 4°C vs. 80°C.

Fig. 3. Plot of pentamidine release percentage with dilution factor.Nanoparticles were diluted in PBS (0.1 M, pH 7.4) at 37°C. Data werefitted to a Michaelis-Menten model (r = 0.98).

Fig. 4. Semilogarithmic plot of pentamidine release percentage with buffermolarity. Nanoparticles were diluted (1/20) in PBS (0.00001 M to 1 M, pH7.4) at 4°C. Data were fitted to a Michaelis-Menten model (r = 0.996).

PENTAMIDINE PLA NANOPARTICLES 103

that PLA polymer was relatively well tolerated by cells[Vernier-Julienne et al., 1995; Nemati et al., 1994]. Com-parison between these studies is very difficult, as the na-ture of the cells was different from one assay to another.

CONCLUSION

We have demonstrated that pentamidine was boundto PLA polymer by adsorption, explaining the high andrapid release induced by dilution, the increase in tem-perature, and ionic strength. The definition of the natureof the interaction between pentamidine and polymer isimportant, as it can influence the in vivo drug distribu-tion. In vivo, all parameters are optimal for a rapiddefixation (37°C, high dilution in blood and ionic strengthrelatively high). We studied these nanoparticles in Leish-

Fig. 5. Semilogarithmic plot of mortality percentage with drug concen-tration: 2: unloaded nanoparticles (polymeric concentration ranging from0.003 to 0.5 mg/ml); 7: pentamidine loaded PLA nanoparticles (drugconcentration ranging from 0.05 to 4 mg/ml); Z: pentamidine isethionate(drug concentration ranging from 0.5 to 8 mg/ml). Drugs were incubated24 h and cytotoxicity assessed by MTT method. Data were fitted to aMichaelis-Menten model (runloaded nanoparticles = 0.995, rpentamidine loaded

nanoparticles = 0.981, and rpentamidine isethionate = 0.998).

TABLE 4. Cytotoxic Effect of Unloaded Nanoparticles, Pentamidine-Loaded PLA Nanoparticles, and Pentamidine Isethionate on J774 Cells

H4 H24 H48

IC25a IC50 IC90 IC25 IC50 IC90 IC25 IC50 IC90

Unloaded nanoparticles — — — 0.019 ± 0.004 0.059 ± 0.01 0.67 ± 0.06 0.01 ± 0.003 0.03 ± 0.004 0.14 ± 0.02(mg/ml)

Pentamidine-loaded 1.44 ± 0.02 — — 0.17 ± 0.002d 0.39 ± 0.14b 6.5 ± 1.7b 0.06 ± 0.007c 0.18 ± 0.015c 0.98 ± 0.07b

nanoparticles (µg/ml)Pentamidine — — — 2.5 ± 0.1 6.5 ± 1.7 31 ± 10 0.37 ± 0.04 1.01 ± 0.12 4.65 ± 0.65

isethionate (µ/ml)aIC = Inhibitory concentration.U-test was used for statistical analysis.bP < 0.05.cP < 0.01.dP < 0.0001 (pentamidine-loaded nanoparticles vs. pentamidine isethionate).

mania-infected BALB/c mice in comparison with freedrug [Durand et al., 1997b]. The efficacy was multipliedby three (based on the inhibitory concentration). How-ever, when pentamidine is bound to another polymer(methacrylate) by ionic interaction (not influenced bydilution, temperature, etc.), the efficiency is multipliedby 6.5 [Durand et al., 1997a]. Therefore, it is very impor-tant to understand the nature of binding of drug to poly-mer to predict its efficacy in vivo.

ACKNOWLEDGMENTS

We thank Mrs. Christine Fernandez for her linguis-tic assistance. This investigation received financial sup-port from Baxter Dubernard Hospital Foundation.

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