Paromomycin-loaded albumin microspheres:Efficacy and stability studiesWahid Khan,a Rajendra Kumar,b Sukhvinder Singh,b

Sunil Kumar Arorab and Neeraj Kumara*

In theQ4 present work, paromomycin-loaded albumin microspheres (PM-MS) have been formulated for passive targeting ofparomomycin (PM) to macrophages, for the treatment of visceral leishmaniasis (VL). PM-MS were prepared by spray-dryingmethod with a mean particle size of� 3mm. Thermal and chemical cross-linking methods were used for controlling drugrelease from the prepared microspheres (MS). PM-MS were then tested for efficacy and stability studies. In efficacy study,in vitro promastigote assay was carried out to assess the susceptibility of promastigote to PM in the concentration range of5.0–150mg/ml; cytotoxicity assay was performed to determine possible toxicity of PM for the host cells (peritoneal macro-phages) and intracellular amastigote assay was carried out to determine the efficacy of free PM (PM solution) and encapsu-lated PM (PM-MS). Results obtained indicated a significant increase in efficacy of PM-MS in comparison to PM solution atequivalent concentration. Subsequently, stability studies of prepared formulation was carried out at various temperatureand humidity conditions, these studies provided stability of formulation at all tested conditions including accelerated condi-tions. Thus, it can be concluded that present work provides an optimized formulation with stability and enhanced efficacy.Copyright © 2011 John Wiley & Sons, Ltd.

Keywords: leishmaniasis; paromomycin; microspheres; amastigote; promastigote

Introduction

Visceral leishmaniasis (VL) is a communicable disease that isendemic and causes high morbidity and mortality worldwide.[1]

Treatment for leishmaniasis has remained unsatisfactory due tovarious unresolved issues such as poverty, malnutrition, inade-quate vector control, insensitive diagnostic tools, and unaffordabil-ity of treatment. Leishmania parasite is an obligate intracellularparasite that replicates and cause infection exclusively within thephagolysosomes of the host macrophages.[2] Biopharmaceuticalissues get more complicated in VL where the response of anti-parasitic drugs is mainly governed by the drug concentrationachieved in microenvironment of intracellular location of parasiterather than the availability in host systemic circulation. As no vac-cine is available to date for leishmaniasis, the management ofleishmania relies primarily on chemotherapy.[3] Major frontlinedrugs available for the treatment of leishmaniasis are eithertoxic or ineffective against the parasite due to emergence ofresistance.[4] Thus, there is a need for a better delivery system todeliver this drug. Among the available delivery options, the besttreatment available today for VL is AmBisome (liposomal productof amphotericin B) which is a good trade-off between activityand toxicity, but is very expensive and unaffordable.[5]

In 2006, paromomycin (PM) intramuscular injection was ap-proved in India for treatment of VL and in 2007, it was includedin 15th edition of the WHO Model List of Essential Medicines fortreatment of VL.[6,7] PM is an efficacious, but one of the leastexploited molecules, due to its low permeability which resultsin poor bioavailability and dose-dependent adverse effects in-cluding local toxicity at the site of injection, ototoxicity, nephro-toxicity, and tetany.[8] To address these problems, biodegradablealbumin microspheres have been designed as a specific carrier

system for passive targeting of PM to the macrophages wherethe leishmania parasites reside. This delivery system would resultin increased efficacy of PM by reducing the dose required fortreatment and subsequently reducing associated toxicities. Thepre-formulation,[9] formulation development and targeting,[10] andpharmacokinetics and toxicity[11] studies of prepared formulationhave already been reported elsewhere. In the present work, PM-MS were evaluated mainly for efficacy and stability of the preparedformulation. Stability studies were carried out to optimize theformulation, so that it can be stored at room temperature and canbe used in the remote areas where VL is most prevalent.

Experimental

Reagents and materials

Paromomycin sulfate (labelled potency 675mg/mg) was a giftsample by Gland Pharma Ltd (Hyderabad, India). Bovine serumalbumin (Bovine Fraction V) and Trypsin (2000 units/mg) wasobtained from sd fine-chem. Ltd. (Mumbai, India). RPMI-1640medium and foetal calf serum (FCS) was purchased from Sigma(St Louis, MO, USA). Penicillin and streptomycin was purchasedfrom HiMedia Laboratories Pvt. Ltd (Mumbai, India). Ultrapure

* Correspondence to: Neeraj Kumar, Department of Pharmaceutics, National Instituteof Pharmaceutical Education&Research (NIPER), Sector 67, S.A.S. Nagar-160062, India.E-mail: neeraj@niper.ac.in

a National Institute of Pharmaceutical Education & Research (NIPER), Nagar, India

b Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh,India

Drug Test. Analysis (2011) Copyright © 2011 John Wiley & Sons, Ltd.

Research ArticleDrug Testing

and Analysis

Received: 15 September 2011 Revised: 20 October 2011 Accepted: 21 October 2011 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/dta.389

1Journal Code Article ID Dispatch: 12.11.11 CE:

D T A 3 8 9 No. of Pages: 6 ME:

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water was obtained using the Elgastat water purification system(Bucks, UK). Other chemicals and reagents used in the experi-ments were purchased from local Indian suppliers.

Parasites

The experiment was performed on Leishmania parasites namelyLeishmania donovani (MHOM/IN/80/Dd8). L. donovani promasti-gotes were grown at 25 �C and cultured in RPMI-1640 mediumsupplemented with 10% (v/v) heat inactivated fetal calf serum,100 IU/ml penicillin and 100mg/ml streptomycin. Metacyclic-stage parasites were isolated from 6-day-old parasite cultures,centrifuged, washed twice with 0.02M phosphate buffered saline(PBS) pH 7.2 and resuspended in RPMI-medium. The strain wasmaintained in Syrian golden hamster (Mesocricetus auratus).

Preparation of PM-loaded albumin microspheres

A spray-drying method was used for preparation of PM-MS.Briefly, an aqueous solution of PM and albumin was sprayed ina spray dryer to form MS. Particles of desired size (≤5 mm) witha good yield were obtained by selecting low feed flow rate(2ml/min), 5% total solid content of sprayed solution, inlet tem-perature 120 �C, and by selecting other optimized instrumentparameters.[10] MS obtained were kept at 60 �C for 3 h to get adried product. Thereafter, particles were cross-linked by chemicaland thermal methods.

Thermal/chemical cross-linking of microspheres

MS prepared by spray drying need to be cross-linked for obtainingthe optimum release profile and for extended shelf life of theproduct. Two methods of cross-linking – (1) chemical cross-linkingusing glutaraldehyde, and (2) thermal cross-linking using heattreatment at high temperatures – were used in present experi-ments.[12–14] Thermal cross-linking was carried out at 120 º C fordifferent time periods (12 and 24h).[10] Whereas, in chemicalcross-linking, four sets of 100mg of MS were suspended in 5mlof 1-butanolat room temperature and MS were stabilized by add-ing glutaraldehyde (25% glutaraldehyde solution) to attain 1%,2%, 3%, and 4% glutaraldehyde concentration. This suspensionwas then stirred at 700 rpm for 90min to allow the cross-linkingof MS. Thereafter, cross-linked MS were filtered under vacuumusing 0.2mm nylon filter and washed with 1-butanol. Finally, MSwere vacuum dried overnight at room temperature.

In vitro drug release studies

In vitro drug release studies were carried for PM-MS, both ther-mally cross-linked (heated at 120 �C, for 12 and 24 h) and chem-ically cross-linked (with 1, 2, 3, and 4% glutaraldehyde). PMreleased from these MS was studied over a period of 9 h usingdialysis bag method. Briefly, 10mg MS (equivalent to approxi-mately 1mg of PM) were transferred to dialysis tubing (M.W.cut off 10 000 Da) containing 3ml of 0.2% trypsin in PBS (pH7.4), and bag was immersed in a vial containing 17ml of PBS(pH 7.4). Vial was placed on a reciprocal shaker water bath main-tained at 37 �C.[10] One ml aliquot was withdrawn at a presettime point and was simultaneously replenished with the samevolume of release media. Samples were assayed for PM releaseusing developed high performance liquid chromatography(HPLC) method.[9]

In vitro antileishmanial activity of formulation

In vitro antileishmanial activity of both thermally and chemicallycross-linked PM-MS formulations was investigated and comparedwith the PM solution. In these experiments, promastigote assay(to determine IC50 value for parasites) was initially performed fol-lowed by cell cytotoxicity assay (to determine drug concentrationrange for intracellular amastigote assay) and lastly intracellularamastigote assay was performed (to determine activity of devel-oped formulation against intracellular amastigotes).

Promastigote assay

The susceptibility of promastigote to PM was assessed using milte-fosine (MF) as a standard drug. Promastigotes (105 parasites/well) intheir logarithmic growth phase were cultured in 96 well plates.[15]

Various dilutions of the drugs were added in each well (n= 3) tomake final concentration of MF (2.5, 5, 10, and 20mg/ml) and PM(5, 10, 50, 100, and 150mg/ml) and incubated at 26 �C for 48 h. Afterthis incubation, 20ml of Alamar-Blue reagent was added and keptfor 5 h. Thereafter, absorbance was measured at 570 and600nm.[16] The concentration that produced 50% reduction ingrowth (IC50) of promastigotes as compared to control (untreated)was determined for both PM and MF.

Cytotoxicity assay for the peritoneal macrophages

Peritoneal macrophages were harvested from Balb/C mice andincubated overnight in a 96 well plate (104 cells/well) in RPMI-1640 medium at 37 �C in 5% CO2. Thereafter, macrophages wereincubated with different concentrations (0.5, 5, 50, 100, and150 mg/ml) of PM solution, PM-MS (both thermally and chemicallycross-linked) and equivalent amount of blank MS (n = 3), threewells were left as untreated control wells. PM solution and MSformulations were removed after 6 h, and then macrophageswere washed and placed in medium for an additional 42 h. Afterthis time period, medium was aspirated and fresh medium sup-plemented with Alamar-Blue reagent was added, incubated forfurther 5 h and absorbance at 570nm and 600nm were deter-mined. Results were expressed as percentagemacrophage viability,compared with untreated control wells.[16–18]

Intracellular amastigote assay

In this experiment, in vitro antileishmanial activity of PM-MSformulation (thermally and chemically cross-linked) was investi-gated by intracellular amastigote assay. Peritoneal macrophageswere harvested from Balb/C mice and 5 x 105 macrophagecells/well were cultured in 6-well plates. On subsequent Q3days,these macrophages were exposed to promastigotes (L. donovani,MHOM/IN/80/Dd8) at multiplicity of infection of 10:1 (parasites:macrophage) and incubated at 37 �C for 12 h. Non-internalizedpromastigotes were removed, whereas internalized promasti-gotes were allowed to convert into amastigotes. These cells werethen incubated with 1ml of different concentrations (0.5, 5, 50,and 100mg/ml) of PM solution and with the same concentrationof PM-MS; two wells of untreated infected macrophages wereused as control. These formulations (PM solution and PM-MS)were removed after 6 h by washing and then macrophages werecultured in complete culture media for an additional 42 h. Afterthat, cells were fixed with methanol and stained with Giemsastain. The intracellular amastigotes were counted to determinenumber of amastigotes per macrophages.[19]

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Stability studies of formulation

To perform stability study of PM-MS (thermally cross-linked)samples were divided into three sets and were stored at 4 �C(refrigerator), ambient temperature/humidity and at 40 �C/75%RH (stability chamber) for 1 and 3months.[20] Samples in triplicatewere kept for specified condition and for each time point. At1month and 3months time period, samples were withdrawn andanalyzed for physical change, drug content, and release profileusing developed HPLC method.[9]

Statistical analysis

Statistical analysis for the efficacy of PM in solution and PM-MSwas performed. Comparison of mean values between variousgroups was performed by one-way ANOVA, followed by pair wisemultiple comparison by Bonferroni t-test.

Result and discussion

Preparation of PM loaded albumin microspheres

PM-MS of desired size (≤5mm) were prepared by a spray-dryingmethod. Various factors affecting size and product yield wereoptimized in step wise manner by varying one parameter at atime while keeping other parameters constant and vice versa.

PM-MS with a mean particle size of� 3 mm were obtained witha reasonable good product yield (45%).

Cross-linking of microspheres and in vitro drug releasestudies

Cross-linking of these prepared MS was carried out to change thesurface property of MS from hydrophilic to hydrophobic, therebyaltering drug release profile. The higher the cross-linking, thelower would be the solubility of albumin hence the slower wouldbe the drug release from MS. Both chemical cross-linking usingglutaraldehyde and thermal cross-linking using heat treatmentwere used in the present experiment. Glutaraldehyde has a widespectrum of industrial and biomedical applications as a cross-linking agent. The aldehyde group of glutaraldehyde reacts witha primary amine of albumin to form a Schiff base, altering watersolubility of albumin. In thermal cross-linking (at temperatureabove 80 �C) albumin undergoes intermolecular aggregationand irreversible structural changes i.e. denaturation. The aggre-gation is due to the formation of intermolecular S-S bonds,whereas, denaturation depends on the temperature, exposuretime, pH, and salt concentration. Typical denaturation tempera-tures range from 80 to 160 �C used to stabilize albumin MS withdifferent exposure time according to thermal stability of thedrug.[21] PM was found to be stable at 120 �C for 24 h.[9] Therefore,120 �C was selected as thermal cross-linking temperature.

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Figure 1. Drug release behaviour of PM-loaded microspheres. Drug release studies were conducted for two types of microspheres (A) thermally cross-linked at 120 �C for 12 h and 24 h and (B) chemically cross-linked with different concentration of glutaraldehyde for 90min. Release study was carried inPBS (pH 7.4) with 0.2% trypsin at 37 �C (n = 3).

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Drug Testing

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In vitro drug release profiles of PM loaded albumin MS stabilizedat 120 �C for different time periods are shown in FigureF1 1A. MSexhibited a biphasic release pattern, where an initial burst releasewas followed by a slower release. These release results for ther-mally cross-linked MS were previously reported,[10] but given againin this manuscript to obtain better comparison with chemicalcross-linking method. MS cross-linked by chemical method isshown in Figure 1B. PM release from albumin MS cross-linked withdifferent glutaraldehyde concentrations (1, 2, 3, and 4%) showedthe dependence of drug release on the amount of cross-linkingagent. Higher the amount of cross-linking agent, lower would bethe amount and rate of PM release obtained from PM-MS. Theseresults indicate that time taken during thermal cross-linking andconcentration of cross-linking agent in chemical cross-linkingsignificantly affect the release profile of drug from albumin MS.The objective of this formulation is to deliver the drug intracellu-

lar to macrophages where leishmania parasites reside. Dependingon the size and surface properties of MS, in vivo uptake of MS inmacrophages occurs maximum within minutes of intravenousinjection.[22]Therefore, MS stabilized at 120 �C for 24 h (thermallycross-linked) and with 3% glutaraldehyde (chemically cross-linked)were used for further experiments. It is also pertinent to mention

that these MS were able to hold the higher amount of drug forlonger period and could release the drug in controlled mannerthereby more drug could be targeted to the macrophages.

In vitro antileishmanial activity of formulation

Promastigote assay

In vitro antileishmanial activity assay was performed on the extra-cellular promastigote form of L. donovani, Dd8 strain. In thisassay, activity of different concentrations of PM (5–150mg/ml)against parasites was determined. Miltefosine in different con-centrations (2.5–20mg/ml) was used as a standard drug. As ob-served from Figure F22, when drug concentration increased, it ledto reduced numbers of viable promastigotes. The concentrationthat produced 50% reduction in viability of promastigotes(IC50) was determined and found to be 119mg/ml for PM and11.4 mg/ml for MF.

Cytotoxicity assay for the peritoneal macrophages

This experiment was carried out to determine possible toxicity ofPM for the host cells (peritoneal macrophages) to determine theeffect of used doses on intracellular amastigotes on the survivalof macrophage cell itself. There is only one report availabledescribing that there was no cytotoxicity observed with PM upto27 mg/ml of PM.[23] In this experiment, macrophages were incu-bated with different concentrations (up to 150mg/ml) of PMsolution, PM-MS (both chemically and thermally cross-linked) andblank MS. It was found that both PM solution and encapsulated

Figure 3. Cytotoxicity behaviour of PM solution and different PM-MSformulations. PM neither in solution form nor as PM-MS produced anycytotoxicity in the given concentration range.

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Figure 4.Q1 Effect of PM solution and PM-MS (thermally cross-linked) atequivalent drug concentration on intracellular amastigotes of leishmaniaparasites. Results are expressed as number of amastigotes/macrophage.#P value <0.05, Vs. control for all samples, *P value <0.05 Vs. PM solutionof respective group. Experimental values are expressed as mean� SD.Comparison of mean values between various groups was performed byone way ANOVA, followed by pair wise multiple comparison by Bonferronit-test.

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Figure 5. Effect of PM solution and PM-MS (chemically cross-linked byglutaraldehyde) at equivalent drug concentration on intracellular amasti-gotes of leishmania parasites. Results are expressed as number of amasti-gotes/macrophage. #P value <0.05, Vs. control for all samples, *P value<0.05 Vs. PM solution of respective group. Experimental values areexpressed as mean� SD. Comparison of mean values between variousgroups was performed by one way ANOVA, followed by pair wise multiplecomparison by Bonferroni t-test.

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Table 1. Observations of stability studies on % drug content in dif-ferent storage conditions.

Storage conditions % Drug content (�SD)

1month 3months

Refrigerator 99.73 (4.11) 98.40 (2.19)

Ambient T/H 100.40 (3.65) 98.73(0.12)

Stability chamber 98.70 (0.91) 99.17 (0.35)

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MS (0.5–150mg/ml) did not produced any toxicity to macrophages(FigureF3 3). Therefore, it can be concluded that tested concentrationrange 0.5–150mg/ml can be used for in vitro amastigote assaywithout affecting cell viability.

Intracellular amastigote assay

In vitro antileishmanial activity of new PM-MS formulations wasinvestigated on intracellular amastigotes of leishmania. Since, onlyone report describing in vitro activity of PM in different strains ofleishmania parasites was available in literature,[24] therefore drugconcentrations were selected on the basis of promastigote assay,keeping cell cytotoxicity data in consideration. The activity at dif-ferent concentrations of PM in various formulations against intra-cellular L. donovani (Dd8 strain) was assessed in macrophages. Itwas observed that equivalent amount of PM encapsulated in albu-min MS gave a significantly higher efficacy of PM in comparison to

PM solution. PM solution showed 5, 29, 35, and 41% reduction inintracellular parasite number at the doses of 0.5, 5, 50, and100mg/ml, respectively. Whereas, PM-MS formulations showedbetter efficacy as compared to PM solution with thermally cross-linked formulation causing 33, 41, 66, and 75% parasite reductionat 0.5, 5, 50 and 100mg/ml, respectively (Figure F44) and chemicallycross-linked formulation was equally efficacious showing 41, 46,and 67% parasite inhibition at similar doses (Figure F55). Therefore,it can be concluded that PM-MS have better efficacy of PM inLeishmania amastigote clearance.

Stability studies of formulation

Stability of any formulation is an important parameter for prod-uct development. It is necessary that the product should not losethe specification during storage and thus a stability study of PM-MS (thermally cross-linked) samples was carried out. Samples

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Figure 6. Release profile of stability samples kept in different conditions for (A) 1month and (B) 3months.

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Figure 7. SEM images of MS after keeping samples for 3months in different storage conditions.

Paromomycin loaded albumin microspheres: Efficacy and stability studies

Drug Testing

and Analysis

Drug Test. Analysis (2011) Copyright © 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/dta

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were stored at 4 �C (refrigerator), ambient temperature/humidityand at 40 �C/75% RH (stability chamber). At predetermined timepoints, samples were withdrawn and analyzed for drug content,physical change, and for release profile. Drug content of allformulations (TableT1 1) as determined by the developed HPLCmethod was found to be within acceptable limit of 100(�5%).In vitro release studies revealed that all samples exhibits samerelease pattern comparable to control (FigureF6 6). In all samplesafter 1 and 3months, no physical changes were observed bySEM (FigureF7 7). Therefore, it can be concluded that developedformulation was stable in the tested storage conditions.

Conclusions

PM-loaded albumin MS for parenteral (intravenous) administra-tion were prepared by spray-drying method. Thermal and chem-ical cross-linking method was successfully used for cross-linkingof MS and to achieve the desired in vitro drug release pattern.In vitro efficacy studies carried out in macrophage cell showedsignificantly increased efficacy of PM-MS, when compared withPM solution. Stability studies also showed the stability of formu-lation in accelerated conditions. Present experiment providespassive targeting of PM to macrophages with increased efficacycompared to free PM.

Acknowledgements

WK is thankful to NIPER for the PhD fellowship. Financial assis-tance from NIPER grant (C1111-NJK) to carry out this researchwork is duly acknowledged.

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[21] A.G.A. Coombes, V. Breeze, W. Lin, T. Gray, K.G. Parker, T. Parker.Lactic acid-stabilised albumin for microsphere formulation andbiomedical coatings. Biomaterials 2001, 22, 1.

[22] F. Chellat, Y. Merhi, A. Moreau, L. Yahia. Therapeutic potential ofnanoparticulate systems for macrophage targeting. Biomaterials2005, 26, 7260.

[23] R.A. Neal, S.L. Croft. An in vitro system for determining the activity ofcompounds against the intracellular amastigote form of Leishmaniadonovani. J. Antimicrob. Chemother. 1984, 14, 463.

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W. Khan et al.

Drug Testing

and Analysis

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