In vitro evaluation of bisnaphthalimidopropyl derivatives loaded into pegylated nanoparticles against Leishmania infantum protozoa

International Journal of Antimicrobial Agents 39 (2012) 424–430

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In vitro evaluation of bisnaphthalimidopropyl derivatives loaded into pegylated nanoparticles against Leishmania infantum protozoa Sofia Costa Lima a,∗,1 , Vasco Rodrigues a,1 , Jorge Garrido b,c , Fernanda Borges b , Paul Kong Thoo Lin d , Anabela Cordeiro da Silva a,e a

Parasite Disease Group, IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal CIQUP, Departamento de Química e Bioquímica, Faculdade de Ciências, 4169-007 Porto, Portugal c Departamento de Engenharia Química, Instituto Superior de Engenharia do Porto, Rua Dr António Bernardino de Almeida 431, 4200-072 Porto, Portugal d School of Pharmacy and Life Sciences, Robert Gordon University, St Andrew Street, Aberdeen AB25 1HG, UK e Faculdade de Farmácia da Universidade do Porto, Departamento de Ciências Biológicas, Rua Aníbal Cunha 164, 4050-047 Porto, Portugal b

a r t i c l e

i n f o

Article history: Received 29 June 2011 Accepted 4 January 2012 Keywords: Bisnaphthalimidopropyl derivatives PEG–PLA nanoparticles Leishmania infantum Murine J774 macrophages Human THP-1 macrophages

a b s t r a c t Bisnaphthalimidopropyl (BNIP) derivatives have recently been shown to have potential as antileishmanial agents. However, these compounds have some drawbacks, including their low aqueous solubility and some toxic effects. In this study, we designed a drug delivery system for enhanced delivery of BNIP derivative compounds whilst reducing adverse toxic effects, and hence increasing their biological efficacy. A coated drug delivery system based on polymeric nanoparticles of pegylated poly(lactic acid) (PLA), a biodegradable polymer, was successfully achieved. The pegylated PLA nanoformulations loaded with BNIP derivatives were evaluated in an in vitro model of intracellular amastigotes in murine J774 and human THP-1 cells for visceral leishmaniasis using luciferase-expressing Leishmania infantum parasites. Pegylation of PLA nanoparticles significantly reduced the capacity of empty nanoparticles in inhibiting intracellular parasite growth. The BNIP derivatives BNIPDadec and BNIPDaoct exhibited EC50 values (concentration of compound necessary to decrease cell viability to 50% of the untreated control) of ca. 4.5 ␮M for THP-1 and J774 cells and ca. 9.0 ␮M for mouse bone marrow-derived macrophages. Nanoparticle encapsulation of the BNIP derivative compounds decreased their toxicity towards macrophages by ≥10-fold as evaluated by the MTT assay. The antileishmanial activity of free BNIPDadec was 1.02 ± 0.41 ␮M and 0.73 ± 0.06 ␮M for THP-1 and J774 macrophages, respectively. Pegylation of PLA nanoparticles loaded with BNIPDadec resulted in EC50 values of 1.43 ± 0.63 ␮M and 1.79 ± 0.77 ␮M for THP-1 and J774 macrophages, respectively. A similar trend was observed for free BNIPDaoct and pegylated BNIPDaoct PLA nanoparticles (2.43 ± 0.19 ␮M and 1.23 ± 0.40 ␮M for THP-1 macrophages and 1.36 ± 0.17 ␮M and 1.52 ± 0.57 ␮M for J774 macrophages). The nanoformulations were more efficient in reducing parasitic growth inside human macrophages than in murine cells, suggesting host cell-dependent metabolism. © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

1. Introduction The leishmaniases are a group of diseases caused by protozoan parasites of the genus Leishmania, characterised by the ability of these parasites to survive and replicate inside host phagocytes. This property poses a major problem in the development of effective anti-Leishmania therapeutic agents, since the intracellular localisation of the parasite is able to protect it from exposure to drugs that do not readily diffuse into cells. Therefore, use of drug carriers capable of delivering anti-Leishmania compounds to infected cells

∗ Corresponding author. Tel.: +351 22 607 4923; fax: +351 22 609 8539. E-mail address: [email protected] (S. Costa Lima). 1 These two authors contributed equally to this work.

should improve the therapeutic efficiency of those drugs and at the same time reduce their toxicity by modifying the pharmacokinetic and biodistribution profiles of the drug. In fact, polymeric nanoparticles have already been tested as nanocarriers for the delivery of antileishmanial compounds [1]. In general, these systems have led to an increase in drug efficacy and a decrease in toxicity, resulting in an improved therapeutic ratio [2]. Previous reports have shown empty polymeric nanoparticles to exhibit an antileishmanial effect and this may be due to activation of the macrophage effector mechanisms following internalisation of the nanoparticles. For example, empty poly(alkyl cyanoacrylate) nanoparticles exhibited anti-Leishmania activity owing to activation of the macrophage with consequent production of hydrogen peroxide [3]. Furthermore, others reported on the anti-Leishmania activity of empty poly(lactic-co-glycolic acid) nanoparticles in an

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S. Costa Lima et al. / International Journal of Antimicrobial Agents 39 (2012) 424–430

in vitro model of Leishmania infantum infection [4]. Since the absence of immunogenic properties is one of the requirements for a nanocarrier, the non-specific activation of macrophages by polymeric nanocarriers is not a desirable event. The pegylation reaction is often applied since it lowers toxicity and prevents activation of the complement system [5] or the coagulation cascade [6]. One of the synthetic polymers generally used in nanoparticle drug delivery systems is poly(lactic acid) (PLA), a linear aliphatic polyester derived from lactic acid monomers [7,8]. PLA is therefore considered a safe polymer since it has been used in several clinical trials that have been approved by the US Food and Drug Administration (FDA) as well as European regulatory authorities [9]. The presence of ester linkages in the PLA backbone allows hydrolytic degradation of the polymer in an aqueous environment. The degradation rate was shown to be much faster in vivo compared with in vitro tests in buffer solutions [10,11]. Recently, bisnaphthalimidopropyl (BNIP) polyamine derivative compounds were identified and characterised as novel antileishmanial agents [12,13]. However, these compounds have some drawbacks, including low aqueous solubility and some toxic effects. It is therefore of great importance to design a system for the effective delivery of BNIP compounds in order to improve targeting and reduce adverse cellular toxicity. Here we report the synthesis of copolymer poly(lactic acid)–polyethylene glycol (PLA–PEG), followed by its use for the first time in the preparation of polymeric nanoparticles to encapsulate two BNIP derivative compounds (BNIPDaoct and BNIPDadec) (Fig. 1). The physical properties of the nanoparticles (e.g. size, zeta potential, stability and morphology) were studied. Furthermore, the effects of drug-loaded and unloaded nanoparticles were assessed on human and murine macrophage cell viability. Nontoxic concentrations of the nanoformulations determined for the mammalian cells were evaluated in an in vitro model of leishmaniasis infection based on the intracellular stage of the parasite. 2. Materials and methods 2.1. Materials The bisnaphthalimidopropyldiamino derivative compounds used were BNIPDaoct and BNIPDadec as previously described [12] (Fig. 1). Poly(d,l-lactide) [molecular weight (MW) 75 000–120 000 Da] and poly(vinyl alcohol) (PVA) (87–89% hydrolysed, MW 13 000–23 000 Da). Polyethylene glycol methyl ether (MePEG) (average MW 750 Da) was dried under high vacuum at room temperature before use. d,l-Lactide was purified by recrystallisation (twice) from dried ethyl acetate and dried in vacuo over P2 O5 . The polymerisation catalyst stannous octoate was prepared as a 0.3% (w/v) solution in anhydrous toluene. All reagents were analytical grade from Sigma-Aldrich (Sintra, Portugal) and were used as received.

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2.2. Spectral characterisation Proton nuclear magnetic resonance (1 H NMR) spectra were recorded on an AVANCETM III 400 MHz spectrometer (Bruker, Madrid, Spain) in CDCl3 at 25 ◦ C. Tetramethylsilane signal was used as an internal standard. 2.3. Synthesis of polyethylene glycol methyl ether–poly(lactic acid) copolymer The MePEG–PLA diblock copolymer was synthesised by ringopening polymerisation of d,l-lactide in the presence of MePEG and stannous octoate, according to a previously reported method with minor modifications [14]. Briefly, d,l-lactide and MePEG were dissolved in freshly distilled anhydrous toluene and heated under a nitrogen atmosphere. Stannous octoate was then added and polymerisation was carried out under reflux at 120 ◦ C under a nitrogen atmosphere for 5 h. The reaction was cooled to room temperature and poured in cold water with stirring to hydrolyse unreacted d,llactide monomers, followed by extraction with dichloromethane. The synthesised PEG–PLA diblock polymers were purified by precipitation in cold diethyl ether. The resulting solid was filtered and dried at room temperature in vacuo (yield 80%). 1 H NMR (CDCl ): 5.19 (m, –CH, PLA repeating unit), 3.65 (s, –CH , 3 2 PEG end group; and –CH2 , PEG repeating unit), 3.41 (s, –CH3 O, end group of PEG), 1.59–1.55 (m, –CH3 , PLA end group; and –CH3 , PLA repeating unit). 2.4. Preparation of poly(lactic acid) nanoparticles loaded with bisnaphthalimidopropyl derivative compounds PLA nanoparticles were prepared by the nanoprecipitation method [15] in the presence of varying amounts of each BNIP derivative compound (% drug loading). PLA (ca. 15 mg) was carefully weighed (ABS Analytical Balance; Kern & Sohn, Balingen, Germany) and was dissolved in a defined volume of acetone in a glass tube to achieve a concentration of ca. 7 mg of polymer per millilitre of acetone. A volume of stock solution at 10 mg/mL of BNIPDadec or BNIPDaoct in dimethyl sulphoxide (DMSO) was then added to obtain the desired % drug loading (mass of compound per mass of polymer used, w/w). The content of the glass tube was injected in 10 mL of an aqueous solution of PVA 1% (w/v) using a 19G syringe. The emulsion was left stirring for 10 min, followed by evaporation of the organic solvent using a rotator evaporator for 5 h. Removal of the organic solvent yielded the nanoparticles, which were washed by centrifugation. An initial centrifugation at 5000 × g for 5 min at 20 ◦ C (Sigma 3K18 Laboratory Centrifuge; Philip Harris Scientific, Lutterworth, UK) was performed to remove larger particle aggregates. The resulting supernatant was recovered and centrifuged at 20 000 × g for 20 min at 20 ◦ C and was designated S1. The nanoparticle sediment was washed twice with 0.45 ␮m-filtered distilled water by centrifugation at 20 000 × g for 20 min at 20 ◦ C. The sediment was then re-suspended in 2.0 mL of phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2 HPO4 and 2.0 mM KH2 PO4 ; pH 7.4). The supernatants obtained from the washing steps (S1, L1 and L2) were stored at 4 ◦ C for quantification of drug encapsulation. Unloaded PLA nanoparticles were prepared using the same procedure without the addition of compound. Nanoparticle suspensions were sterilised by exposure to ultraviolet light (20 min) and stored at −20 ◦ C until further use. 2.5. Preparation of polyethylene glycol-coated polymeric nanoparticles

Fig. 1. Chemical structure of the bisnaphthalimidopropyl (BNIP) derivative compounds (A) BNIPDaoct and (B) BNIPDadec.

PLA (15 mg) and PLA–PEG diblock copolymer (4 mg) were dissolved in acetone in a glass tube to give a concentration of ca. 7 mg

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of total polymer per millilitre of acetone. A determined volume of stock solution of BNIPDadec or BNIPDaoct in DMSO (10 mg/mL) was then added and its content was injected in 10 mL of a solution of PVA 1% (m/v) whilst stirring. Solvent evaporation, recovery, washing and storage of PEG-coated PLA nanoparticles were performed in the same way as non-coated particles. Empty particles were prepared by excluding the addition of compound. Incorporation of PEG in the nanoparticle structures was confirmed by NMR analysis. 2.6. Nanoparticle characterisation 2.6.1. Size, polydispersity index and zeta potential Size, polydispersity index and zeta potential determinations were performed by dynamic light-scattering methods using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) with a detection angle of 173◦ . Measurements were made in triplicate at 25 ◦ C. The sample for dynamic light-scattering analysis was prepared by diluting the nanoparticle suspension in PBS to a final concentration of 1 mg/mL in polymer. 2.6.2. Encapsulation efficiency The encapsulation efficiency of the BNIPDadec and BNIPDaoct compounds in uncoated and PEG-coated PLA nanoparticles was determined indirectly by quantification of the non-encapsulated compound that was retained in the aqueous phase following particle recovery. Therefore, the amount of encapsulated compound was obtained by the difference between the total amount initially employed in the preparation of particles and the non-encapsulated amount. The BNIP compounds have intrinsic fluorescent properties exhibiting two peaks in the emission spectra, at 390 nm and 490 nm, after excitation at 345 nm (Supplementary Fig. S1). Non-encapsulated drug present in the supernatants obtained after recovery and washing of the nanoparticles (S1, L1 and L2) was quantified by measuring the fluorescence intensity at 490 nm in a spectrofluorometer (L550B; PerkinElmer, Waltham, MA). Encapsulation efficiency was determined indirectly as the amount of compound non-encapsulated/total amount of compound added initially. The non-encapsulated compound was determined in three fractions from the washing steps (S1, L1 and L2), as illustrated in Supplementary Fig. S1. Supplementary material related to this article can be found, in the online version, at doi:10.1016/j.ijantimicag.2012.01.003. 2.7. Parasite and cell culture 2.7.1. Parasites A cloned line of L. infantum (MOM/MA671TMAP263) promastigotes, stably expressing the luciferase (luc) gene, was grown in RPMI 1640 medium supplemented with 10% heat-inactivated foetal bovine serum (FBS), 2 mM l-glutamine, 20 mM HEPES, 100 U/mL penicillin and 100 ␮g/mL streptomycin (all from BioWhittaker, Verviers, Belgium). Selection of luc-positive parasites was done by adding geneticin® sulphate (Sigma-Aldrich, St Louis, MO) to the culture media at a final concentration of 5 ␮g/mL. Parasites were maintained in culture at 26 ◦ C by subpassage (106 parasites/mL) every 5 days. Leishmania infantum axenic amastigotes stably expressing the luc gene were derived from promastigotes by culturing them in MAA (Medium for Axenic Amastigotes). MAA consisted of modified medium 199 with Hank’s balanced salt solution (Gibco-Invitrogen, Barcelona, Spain) supplemented with 0.5% trypto–casein–soy broth (Bio-Rad, Bath, UK), 15 mM d-glucose (Panreac, Barcelona, Spain) and 4 mM NaHCO3 (Sigma-Aldrich, St Louis, MO). The pH was adjusted to 5.8 and the media was 0.2 ␮m-sterilised by filtration and further supplemented with 0.023 mM bovine hemin (Fluka, St Louis, MO), 5 mM l-glutamine and 25% heat-inactivated FBS.

Amastigotes were maintained in culture at 37 ◦ C in an atmosphere containing 5% CO2 by subpassage (105 parasites/mL) every 5 days. 2.7.2. Cell lines The human leukaemia monocytic cell line THP-1 and the murine macrophage cell line J774 were cultured as a monolayer at 37 ◦ C in a humidified atmosphere containing 5% CO2 . Cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mM l-glutamine, 100 U/mL penicillin and 100 ␮g/mL streptomycin and were maintained in culture by subpassage every 3 days. 2.8. Toxicity of bisnaphthalimidopropyl derivative compounds and nanoformulations to macrophages For macrophage differentiation, 105 human THP-1 cells were incubated in the presence of 20 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, Sintra, Portugal) for 18 h at 37 ◦ C and were left for another 24 h with fresh medium containing no PMA. Murine macrophage cells were incubated in 96-well plates at a cell density of 105 cells/well and were allowed to adhere to the bottom of the wells for a period of 2 h at 37 ◦ C in 5% CO2 . Serial dilutions of BNIPDadec, BNIPDaoct or the nanoformulations in culture media were added to the wells, in quadruplicate, and were incubated for 72 h at 37 ◦ C in 5% CO2 . Cell viability was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay as previously described [16]. The EC50 value, i.e. the concentration of BNIP compound necessary to decrease cell viability to 50% of the untreated control, was determined by linear regression analysis. 2.9. Growth inhibition assays against intracellular Leishmania infantum amastigotes Murine J774 cells were incubated in 96-well plates (105 cells/well) and were allowed to adhere for 2 h. Subsequently, differentiated THP-1 macrophages and J774 cells were infected for a period of 4 h with the stationary phase LUC-expressing L. infantum axenic amastigotes at a parasite:cell ratio of 5:1. After the infection period, cells were washed with culture medium to remove non-internalised parasites. Serial dilutions of BNIP compounds and empty or BNIP-loaded PLA nanoparticles in culture medium were added to the wells, in quadruplicate, and were incubated for 72 h at 37 ◦ C in 5% CO2 . After the incubation period, the luciferase activity of intracellular amastigotes was determined accordingly to the manufacturer’s instructions. The percentage of growth inhibition was calculated as: % growth inhibition =



1−



relative luminescence units of treated cells × 100 relative luminescence units of untreated cells

The BNIP concentration, either free or in PLA nanoparticles, necessary to decrease parasite viability to 50% (EC50 ), was determined by linear regression analysis. 3. Results 3.1. Synthesis and characterisation of polyethylene glycol–poly(lactic acid) copolymers Block copolymers of PLA and PEG were prepared by ringopening polymerisation of d,l-lactide with PEG as a macroinitiator and stannous octoate as a catalyst. NMR spectra clearly indicated the production of the copolymer with typical resonance signals at 1.58 ppm assigned to methyl groups of the lactic acid units,

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Table 1 Physicochemical characteristics of poly(lactic acid) (PLA) and polyethylene glycol (PEG)-coated PLA nanoparticles loaded with bisnaphthalimidopropyl (BNIP) derivative compoundsa . Nanoparticle

Size (nm)

PLA PEG–PLA BNIPDaoct PLA BNIPDaoct PEG–PLA BNIPDadec PLA BNIPDadec PEG–PLA

176.2 175.4 231.0 223.9 235.2 231.5

± ± ± ± ± ±

3.1 6.5 12.9* 11.4* 9.1* 6.5*

Polydispersity index 0.048 0.095 0.083 0.052 0.112 0.076

± ± ± ± ± ±

0.005 0.009 0.019 0.010 0.021 0.005

Zeta potential (mV)

Encapsulation efficiency (%)

−7.05 −5.16 −3.01 −1.64 −3.91 −1.90

N/A N/A 77.9 ± 3.7 80.4 ± 6.5 83.1 ± 7.3 78.8 ± 6.1

± ± ± ± ± ±

1.99 0.36 0.97 0.79* 0.77 0.25*

N/A, not applicable. a Values are presented as mean ± standard deviation of at least four independent experiments. * P < 0.001 compared with empty PEG–PLA nanoparticles.

and singlet at 3.65 ppm owing to the methylene groups of PEG (–O CH2 CH2 O) and at 5.19 ppm owing to the methine groups of the lactic acid residues (–COCHCH3 ). 3.2. Characterisation of bisnaphthalimidopropyl-loaded poly(lactic acid) nanoparticles The BNIP derivative compounds BNIPDaoct and BNIPDadec were successfully incorporated into uncoated and PEG-coated PLA nanoparticles by the nanoprecipitation method [15,17]. Characterisation of the nanoformulations was carried out by determining the following parameters: size; polydispersity index; zeta potential; encapsulation efficiency; and morphology. Table 1 summarises the physicochemical characteristics of the developed nanoformulations. The morphology of the PLA nanoparticles, either uncoated or coated with PEG, was observed by scanning electron microscopy (Fig. 2). From the two images, only the formulation with PEG (Fig. 2B) exhibited a heterogeneous distribution of the nanoparticle size, with a large polydispersity index value (0.09), as confirmed by dynamic light scattering.

amastigotes, nanoparticles of uncoated and PEG-coated PLA were prepared with a drug loading of 5% (w/w) and concentrations in polymer of 0.25 mg/mL for empty PLA nanoparticles and 0.5 mg/mL for PEG-coated PLA nanoparticles. 3.4.1. Toxicity of the nanoformulations to macrophages The toxicity of the free compound as well as uncoated and PEG-coated PLA nanoparticles was evaluated in human THP-1 differentiated macrophages, murine J774 macrophages and murine bone marrow-derived macrophages by the MTT assay (Table 2).

3.3. In vitro evaluation of unloaded nanoparticles 3.3.1. Toxicity of poly(lactic acid) and polyethylene glycol–poly(lactic acid) nanoparticles to macrophages Before evaluating the antileishmanial properties of both types of empty nanoparticles, their toxicity to the host cell used in the infection assays (J774 and THP-1 differentiated macrophages) was assessed by the MTT assay, as illustrated in Fig. 3. Both cell types exhibited 80% viability after 3 days in the presence of 2.0 mg/mL of both types of polymer. 3.3.2. Growth inhibition of unloaded nanoparticles against Leishmania infantum intracellular amastigotes Empty PLA and PEG–PLA nanoparticles were evaluated for their ability to inhibit the growth of intracellular amastigotes. The main purpose of this assay was to determine the maximum polymer concentration for both types of nanoparticles that did not significantly inhibit the growth of L. infantum intracellular amastigotes. Results in Fig. 4 showed that empty PLA nanoparticles are able to inhibit the growth of L. infantum intracellular amastigotes in a dose-dependent manner, achieving a growth inhibition >60% at a concentration of 2.0 mg/mL. In contrast, PEG-coated nanoparticles did not exhibit this behaviour but instead showed a maximum growth inhibition of 20% at 2 mg/mL. 3.4. Evaluation of the antileishmanial effect of the bisnaphthalimidopropyl compound nanoformulations To evaluate the antileishmanial activity of the nanoformulations without the effect of the polymer itself on intracellular

Fig. 2. Scanning electron microscope images of (A) uncoated and (B) polyethylene glycol (PEG)-coated poly(lactic acid) (PLA) nanoparticles.

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Fig. 3. Viability of murine J774 macrophages and human THP-1 differentiated macrophages following treatment with increasing concentrations of uncoated poly(lactic acid) (PLA) nanoparticles () and copolymer poly(lactic acid)–polyethylene glycol (PLA–PEG) nanoparticles ().

Table 2 Cytotoxicity of bisnaphthalimidopropyl (BNIP) derivative compounds and nanoformulations to murine J774 macrophages, murine bone marrow-derived macrophages (BMM␾) and human THP-1 differentiated human macrophages. Formulation

Free BNIPDadec BNIPDadec PLA BNIPDadec PEG–PLA Free BNIPDaoct BNIPDaoct PLA BNIPDaoct PEG–PLA

EC50 (␮M)a THP-1

J774

BMM␾

4.63 ± 0.63 >50 >50 4.43 ± 0.35 >50 >50

4.54 ± 0.56 >50 >50 4.47 ± 0.40 >50 >50

9.31 ± 0.57 >50 >50 8.59 ± 0.79 >50 >50

EC50 , concentration of compound necessary to decrease cell viability to 50% of the untreated control; PLA, poly(lactic acid); PEG, polyethylene glycol. a Results are shown as the mean ± standard deviation of three independent experiments.

Incorporation of BNIP compounds into PLA or PEG–PLA nanoparticles reduced its toxicity to all types of macrophage studied, as revealed by EC50 values, by ≥10-fold (Table 2). 3.4.2. Growth inhibition of intracellular Leishmania infantum amastigotes by the bisnaphthalimidopropyl compound formulations The antileishmanial activity of the nanoformulations was evaluated using an in vitro model of infection. EC50 values obtained from the inhibition assays are given in Table 3.

Table 3 Antileishmanial activity of free bisnaphthalimidopropyl (BNIP) derivative compounds and nanoformulations against Leishmania infantum intracellular amastigotes in murine J774 macrophages and human THP-1 differentiated macrophages. Formulation

EC50 (␮M)a THP-1

Free BNIPDadec BNIPDadec PLA BNIPDadec PEG–PLA Free BNIPDaoct BNIPDaoct PLA BNIPDaoct PEG–PLA

1.02 0.95 1.43 2.43 0.98 1.23

± ± ± ± ± ±

J774 0.41 0.12 0.63 0.19 0.22** 0.40*

0.73 5.29 1.79 1.36 6.19 1.52

± ± ± ± ± ±

0.06 0.31 0.77 0.17 0.29 0.57

EC50 , concentration of compound necessary to decrease intracellular parasite viability to 50% of the untreated control; PLA, poly(lactic acid); PEG, polyethylene glycol. a Results are shown as the mean ± standard deviation of four independent experiments. * P < 0.05 compared with the free form of the compound. ** P < 0.001 compared with the free form of the compound.

Incorporation of BNIP compounds in uncoated and PEG-coated PLA nanoparticles revealed that pegylation retained (in murine macrophages) or increased (in human macrophages) the efficacy in inhibiting intracellular amastigote growth (Table 3). However, BNIP-loaded PLA nanoparticles exhibited a decreased ability to inhibit the growth of L. infantum intracellular amastigotes in murine J774 macrophages, as revealed by the higher EC50 values (Table 3). 4. Discussion Here, pegylation of nanoparticles based on PLA was performed to investigate the influence of PEG on the antileishmanial activity of BNIP-loaded nanoparticles. In the strategy used, polymer, copolymer and BNIP compound were dissolved in an organic solvent (acetone) followed by their addition to an aqueous phase. Removal of the organic solvent resulted in an aqueous dispersion of polymeric nanoparticles with PEG chains at the surface of the nanoparticles. Size is the most important characteristic of a nanoparticulate system since it is a major determinant of the uptake mechanism and intracellular route by which nanoparticles are internalised in cells [18]. All prepared nanoformulations exhibited a mean diameter in the range 175–235 nm, with polydispersity index values lower than or around 0.1 (Table 1). No differences in size were observed between empty uncoated and PEG-coated PLA nanoparticles. A ca. 30% increase in the mean size was observed when PLA nanoparticles were loaded with 5% (w/w) amounts of both BNIP compounds (Table 1). The volume of stock solution of BNIP compounds added to the organic phase was kept constant when preparing nanoparticles with increasing amounts of compound by increasing the concentration of BNIP compounds in the stock solution. Thus, the observed increase in particle size cannot be attributed to an effect of the DMSO solvent used to prepare the stock drug solution, indicating that the increase in size was due to the compounds in the PLA particle structure, hence causing an expansion of the particle matrix [18]. Zeta potential is often used as a measure of a particle surface charge [15]. BNIP-loaded PLA nanoparticles exhibited negative zeta potential values in the range of −3.01 mV to −3.91 mV (Table 1). No differences in zeta potential were found with the different polymers used to prepare nanoparticles, which is in accordance with previous findings [19]. Incorporation of BNIP compounds, at 5% (w/w) drug loading, resulted in a non-statistically significant decrease in zeta potential of PLA nanoparticles, from −7 mV for empty PLA nanoparticles to −3 mV for BNIP-loaded PLA nanoparticles. This may be due to a masking effect of the superficial carboxylic groups by the drug adsorbed on the particle

S. Costa Lima et al. / International Journal of Antimicrobial Agents 39 (2012) 424–430

surface, since residual amounts of PVA remain associated with nanoparticles even after repeated washings [19]. Inclusion of PEG chains on the surface of the PLA nanoparticles usually results in a less negative zeta potential because, like PVA, it is able to cover the surface-charged carboxylic groups of PLA [17]. A decrease in the zeta potential values was observed for empty PLA nanoparticles after PEG-coating (from −7 mV to −5 mV for PLA nanoparticles) (Table 1). These results suggest that at least some of the PEG is located in the nanoparticles’ surface. However, it is not possible to discriminate whether the observed decreased zeta potential values for pegylated nanoparticles are due to the presence of PEG chains on the particle’s surface and/or to the presence of residual PVA. BNIP encapsulation in nanoparticles was in the range 79–83% of BNIP compounds in all prepared nanoformulations as a result of the hydrophobic nature of BNIP compounds. This is in accordance with previous reports in the literature that indicate high encapsulation efficiencies for hydrophobic compounds in nanoparticles prepared by nanoprecipitation owing to strong interactions between the drug and polymers, which prevents their diffusion to the aqueous phase during particle formation [2]. Moreover, the efficiency of encapsulation did not decrease with increasing amount of BNIP compound used in nanoparticle preparation [up to 5% (w/w) drug loading as amount tested]. Both the uncoated and PEG-coated PLA nanoparticles do not exert cytotoxicity on murine J774 and THP-1 differentiated macrophages up to 2 mg/mL. As shown in Fig. 4, pegylation of the empty nanoformulations was efficient in reducing their antileishmanial activity, most probably by decreasing non-specific activation of the macrophage [3,4]. To reduce this antiparasitic effect, only 0.25 mg/mL of uncoated PLA nanoparticles should be used, whilst for PEG-coated PLA nanoparticles 2.5-fold more polymer led to the same outcome. Free BNIPDadec and BNIPDaoct exhibited toxicities to THP-1 differentiated macrophages and murine J774 and bone marrowderived macrophages between 4.43 ± 0.35 ␮M and 9.31 ± 0.57 ␮M (Table 2). These were rather elevated toxicities and may be explained by the anticancer properties exhibited by BNIP compounds, facilitated by their ability to intercalate DNA [12,20]. Encapsulation of the BNIPDadec and BNIPDaoct on polymeric nanoparticles significantly reduced their toxicity towards the macrophages studied. In fact, these results were in accordance with other in vitro and in vivo drug studies in which encapsulation of a drug into a nanocarrier was proven to be effective in reducing the toxicity of the free drug [21].

Fig. 4. Growth inhibition of uncoated () and polyethylene glycol (PEG)-coated () poly(lactic acid) (PLA) nanoparticles against Leishmania infantum intracellular amastigotes in murine J774 macrophages. Results are given as mean ± standard deviation of three independent experiments. *P < 0.05; **P < 0.01.

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For murine J774 macrophages, a decrease in the antileishmanial activity of BNIP compounds, particularly BNIPDadec, was observed when encapsulated in PLA and PLA–PEG nanoparticles compared with the free compound (Table 3). BNIP compounds are hydrophobic in nature and therefore are easily able to cross the plasma membrane, diffuse through the cytoplasm and reach the parasitised phagolysosomes without the need of any specialised carrier; however, this may also account for their elevated toxicity to cells. In addition, the release of BNIP compounds from nanoparticles in culture should be slow and thus results in a decreased capacity to inhibit the growth of intracellular amastigotes of L. infantum. Therefore, an increase in the incubation period might be beneficial for enhanced antileishmanial activity of the BNIP nanoformulations, since a higher amount of compound would be released from the nanoparticles. The pegylated nanoparticles exhibit a similar effect to that of the free compounds. It is possible that PEG-coated nanoparticles release the encapsulated BNIP compound more rapidly to the culture medium compared with PLA nanoparticles and this in turn results in a higher efficacy in the growth inhibition of L. infantum intracellular amastigotes. The nanoformulations were more efficient in reducing parasite growth inside human THP-1 differentiated macrophages. In fact, the inhibitory effect of BNIPDaoct was significantly more evident when encapsulated in PLA nanoparticles, by an order of 2.5-fold increase. The observed variations are probably due to differences in host cell metabolism, the internalisation pathway and/or response to infection. To summarise, encapsulation of BNIP compounds in nanoparticles considerably reduced their toxicity towards mammalian cells, which allows the delivery of higher concentrations of compounds in order to eliminate the parasite effectively. The effect of a PEG coat in the performance of BNIP-loaded nanoparticles is host cell-dependent and should become clearer on future in vivo studies. Funding: The authors acknowledge funding from Calouste Gulbenkian Foundation (project P-105348) and Fundac¸ão para a Ciência e a Tecnologia (FCT) (project SAU-ENB/113151/2009). SCL thanks FCT and FSE (Fundo Social Europeu, III quadro comunitário) for grant SFRH/BPD/37880/2007. Competing interests: None declared. Ethical approval: Not required.

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