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Tuberculosis

Nanoparticle encapsulated antitubercular drugsas a potential oral drug delivery system againstmurine tuberculosis

Rajesh Pandey, A. Zahoor, Sadhna Sharma, G.K. Khuller*

Department of Biochemistry, Postgraduate Institute of Medical Education and Research,Chandigarh 160 012, India

Summary Patient non-compliance is the major drawback associated with the long-duration chemotherapy of tuberculosis (TB); hence, reduction in dosing frequencyforms an important therapeutic strategy. The present study reports the formulation ofthree frontline antitubercular drugs (ATD), i.e. rifampicin (RIF), isoniazid (INH) andpyrazinamide (PZA) encapsulated in poly (DL-lactide-co-glycolide) (PLG) nanoparti-cles. Drug encapsulation efficiencies were 56.972.7% for RIF, 66.375.8% for INH and6875.6% for PZA. Following a single oral administration of these preparations tomice, the drugs could be detected in the circulation for 6 days (RIF) and 9 days (INH/PZA), whereas therapeutic concentrations in the tissues were maintained for 9–11days. Further, on oral administration of drug-loaded nanoparticles to Mycobacteriumtuberculosis-infected mice at every 10th day, no tubercle bacilli could be detected inthe tissues after 5 oral doses of treatment. Therefore, nanoparticle-based ATDtherapy forms a sound basis for reduction in dosing frequency for better managementof TB.& 2003 Elsevier Ltd. All rights reserved.

Introduction

Tuberculosis (TB) is a chronic communicable dis-ease caused by the bacterium Mycobacteriumtuberculosis that infects over 1.8 billion peopleworldwide and is responsible for 1.5 million deathsannually.1 Moreover, TB has emerged as an occupa-tional disease in the health care set-up.2 Althoughan effective therapeutic regimen is available,patient non-compliance [because of the need oftaking antitubercular drugs (ATD) daily or several

times a week] results in treatment failure as well asthe emergence of drug resistance. Patient-compli-ance can be improved by the use of ATD-formula-tions, which reduce the dosing frequency of thedrugs. For this purpose, ATD encapsulated inliposomes3 and polymers such as poly (DL-lactide-co-glycolide) (PLG)4,5 have been successfully usedas injectable formulations in experimental TBmodels.

In the last decade, nanoparticle-based drugdelivery systems have been developed with thegoal of better management of diverse clinicalconditions.6–9 Because PLG polymers are biode-gradable and biocompatible,10 they have beenthe most commonly used drug carriers. Thepresent communication demonstrates the use of

KEYWORDS

Tuberculosis;

Antitubercular drugs;

Nanoparticles

*Corresponding author. Tel.: þ 91-172-747-585; fax: þ 91-172-747-401.E-mail address: gkkhuller@yahoo.co.in, medinst@pgi.chd.

nic.in (G.K. Khuller).

1472-9792/$ - see front matter & 2003 Elsevier Ltd. All rights reserved.doi:10.1016/j.tube.2003.07.001

Tuberculosis (2003) 83, 373–378

PLG-nanoparticles (PLG-NP) as oral carriers forthree frontline ATD, i.e. rifampicin (RIF), isoniazid(INH) and pyrazinamide (PZA), and their chemo-therapeutic potential against murine TB.

Materials and methods

Chemicals and drugs

PLG (50:50) resomer RG 506, Mr 2500, waspurchased from Boehringer Ingelheim (Germany),polyvinyl alcohol (PVA, 87–89% hydrolysed, averageMr 13,000–23,000), RIF, INH and PZA were obtainedfrom Sigma (St. Louis, MO, USA). All other reagentswere obtained from standard companies.

Animals

Mice (Laca) of either sex (4–5 weeks old, weighing18–20 g), obtained from the Central Animal House,PGIMER, Chandigarh were used in the study. Theanimals were fed standard pellet diet and water adlibitum. The study was approved by the Institute’sEthical Committee.

Culture

The culture of M. tuberculosis H37Rv was originallyobtained from the National Collection of TypeCultures (NCTC), London, UK, and was maintainedon Youman’s modified medium.

Preparation of PLG-nanoparticles (PLG-NP)

PLG-NPs were prepared by the multiple emulsiontechnique described by Lamprecht et al.11 withslight modifications. Briefly, 1ml of an aqueousdrug solution was first emulsified in 10ml ofdichloromethane (DCM) containing the polymer(drug:polymer::1:1 w/w) by sonication for 1min.In case of RIF, the drug was directly added to DCMprior to sonication. The primary emulsion waspoured into 8ml of 1% aqueous PVA solution andsonicated for 3min to form the second water-in-oil-in-water emulsion. The latter was allowed to stircontinuously overnight for complete removal ofDCM. The PLG-NP were recovered by centrifugation(8000–10,000 rpm, 15min), washed thrice withdistilled water and vacuum-dried. Drug-free NPwere prepared by substituting normal saline for theATD. The PLG-NP were resuspended in normalsaline prior to each experiment. It should be notedthat all the three drugs were encapsulated simul-taneously in PLG-NP.

Nanoparticles characterization

The drug-loaded PLG-NP were analysed for theirsize and polydispersity index (PI) on a Zetasizer1000 HS, Malvern Instruments, based on photoncorrelation spectroscopy. The amount of drugsentrapped within PLG-NP was determined bymeasuring the amount of un-encapsulated drug inthe aqueous solution recovered after centrifuga-tion and washing of the particles. Drugs wereanalysed using standard protocols-RIF by microbio-logical assay (sensitivity 0.25 mg/ml),12 INH byspectrofluorimetry (sensitivity 0.01 mg/ml)13 andPZA by spectrophotometric method (sensitivity5 mg/ml).14 Results were also confirmed by LC-Massspectrometry (LCMSMS, API 3000). The LC-MS assaysensitivities were RIF 25 ng/ml, INH 100 ng/ml andPZA 1000 ng/ml, with an accuracy of 95–97%for each drug. The range of standards were RIF0.1–4.0 mg/ml, INH 0.1–10 mg/ml and PZA 1–100 mg/ml whereas the quality control range were RIF0.1–4 mg/ml, INH 0.3–4.8 mg/ml and PZA 3–48 mg/ml. The drug encapsulation efficiency was ex-pressed as the percentage of drug entrapped withrespect to the theoretical value, and the drugloading was expressed as the amount of drugentrapped per gram of polymer. For in vitrodrug release profile of PLG-NP in phosphatebuffered saline (PBS, pH 7.2–7.4), 25mg of vacuumdried drug-loaded PLG-NP were suspended in 2mlof PBS and kept in an incubator (without agitation)at 371C. At each time point (day 1, 2, 3, 4, 7, 14,21, 28, 35 and 42), the samples were centrifuged(8000 rpm, 15min), supernatant collected anddrugs were assayed. The results were expressedas percentage of drug released over time. Thepellet was resuspended in 2ml of fresh PBS forsubsequent studies.

In vivo drug disposition studies

Mice were divided into the following groups (6–8mice per group)FGroup 1. Drug-loaded PLG-NP,oral; Group 2. Free drugs, oral; Group 3. Free drugsmixed with drug-free PLG-NP, oral (to exclude thesurface binding of drugs to nanoparticles); Group 4.Drug-free or empty PLG-NP, oral (a positive controlgroup to exclude the influence of empty nanopar-ticles, if any, on drug estimations). In each case,drugs were administered once in therapeutic dosecombinations, i.e. RIF 12mg/kg, INH 10mg/kg, PZA25mg/kg b.wt. Mice were bled via the retro-orbitalplexus at different time points. In addition, animalswere sacrificed at various intervals. In order toexclude the possibility of drug accumulation

374 R. Pandey et al.

following repeated dosing, PLG-NP were alsoadministered at day 0, 10, 20, 30 and the micewere sacrificed on day 11, 21, 31 and 41. Drugconcentrations were determined in plasma and 20%organ homogenates (whole organ or 100mg tissuein 500 ml PBS) using standard protocols. Plasma druglevels were also confirmed by LC-MS. Each experi-ment was repeated 4–5 times.

Hepatotoxicity studies

Mice were divided into four groups (5–6 mice pergroup). Group 1 served as controls, group 2 wasadministered oral-free drugs in combination daily,group 3 was administered oral drug-loaded PLG-NPevery 10 days (3 doses) and group 4 was adminis-tered oral-empty PLG-NP every 10 days (3 doses).The drug doses were the same as in the in vivostudies. At day 26, mice were bled and the sera wasanalysed for total bilirubin, alanine transaminase(ALT) and alkaline phosphatase (ALP) using stan-dard kits.

Experimental infection and chemotherapy

Mice were inoculated via the lateral tail vein with1.5� 105 viable bacilli of M. tuberculosis H37Rv in0.1ml of 0.9% sterile saline. Fifteen days afterinoculation, infection was confirmed by ZiehlNeelsen staining of tissue smears of lungs andspleen after killing two animals. The animals weredivided into three groups with 7–9 animals pergroup. Group 1 received free drugs orally, Group 2received drug-loaded PLG-NP orally and Group 3received PBS orally. In each case, the drug dosagewas the same as stated earlier. Free drugs weregiven daily, whereas PLG-NP was administeredonce every 10 days and the animals were sacrificedafter 46 days of chemotherapy (46 doses of freedrugs and 5 doses of drug-loaded PLG-NP). Lung andspleen homogenates (whole organs) were preparedin 3ml sterile PBS. 50 ml of undiluted, 1:10 dilutedand 1:100 diluted homogenates in duplicates wereinoculated on Lowenstein-Jensen medium in milkbottles or plates for enumeration of colony formingunits (cfu).

Results

Physicochemical characterization ofdrug-loaded PLG-NP

The majority (480%) of the nanoparticles were inthe size range of 186–290 nm with a polydispersity

index of 0.3870.04 (Table 1). Drug encapsulationranged from 57% to 68%, whereas drug loadingvaried from 570 to 680mg drug per gram ofpolymer. In vitro drug release profile in PBS showedan initial (up to 48 h) burst release followed by anegligible release of either drug up to 6 weeks.

In vivo drug disposition studies

PlasmaFollowing the oral administration of drug-loadedPLG-NP, the drugs were detectable in the plasmafrom 6h onwards. In the case of RIF, minimuminhibitory concentration 90 (MIC) levels wereobserved until day 4, whereas INH and PZA werepresent at MIC up to day 9. On the other hand, nodrug was detected in the plasma beyond 12–24 h,following the oral administration of free drugs, orfree drugs mixed with empty PLG-NP. A similar drugrelease profile was observed after single doseadministration of PLG-NP encapsulated drugs toanimals infected with M. tuberculosis.

OrgansAll three drugs were present at MIC levels in thetissues at day 7 and day 9, following the single oraladministration of drug-loaded PLG-NP (Fig. 1). Atday 11, INH was present at therapeutic concentra-tion in all the organs. However, RIF levels wereo0.25 mg/ml, whereas PZA was not detected in thelungs. In case of free drugs (alone and mixed with

Table 1 Physicochemical characterization of drug-loaded PLG-NP.

Size range 186–290 nm (480%particles)

Polydispersity index 0.3870.04

Drug encapsulationefficiency (%)

RIF 56.9972.72

INH 66.3175.83PZA 68.0275.58

Drug loading (mg/gpolymer)

RIF 570727

INH 663758PZA 680756

In vitro release (% ofencapsulated drug) inphosphate bufferedsaline (pH 7.2–7.4)

Burst release (3–7%) inthe first 48 h followedby minimal (o1%)release up to 42 days

Results are mean7SD of 5 nanoparticles preparations.

Nanoparticle encapsulated antitubercular drugs against murine tuberculosis 375

drug-free PLG-NP), no drug was detectable in thetissues after 48 h. The results of drug accumulationstudies, i.e., tissue drug levels following repeatedoral dosing with drug-loaded PLG-NP on every 10thday are shown in Table 2. In case of RIF, drug levelswere o0.25 mg/ml in the lungs on day 11, 21, 31and 41, whereas in liver/spleen, drug levels werep0.4þ 0 mg/ml. Similarly, in case of INH and PZA,drug levels in either organ did not show anyprogressive increase, thereby excluding the possi-bility of drug accumulation on repeated dosing.

Hepatotoxicity studies

The results of oral administration of free drugs,drug-loaded PLG-NP and empty PLG-NP for 3weeks, on serum bilirubin, ALT and ALP, showedthat: (i) total serum bilirubin was in the range0.32–0.48, 0.32–0.53 and 0.38–0.63mg/100ml incase of free drugs, drug-loaded PLG-NP and emptyPLG-NP respectively (normal range up to 1.0mg/100ml in mice); (ii) serum ALT was in the range4.1–11.0, 6.3–19.1 and 5.1–10.8 U/l in case of freedrugs, drug-loaded PLG-NP and empty PLG-NPrespectively (normal range up to 70U/l in mice);and (iii) serum ALP ranged from 1.4–3.1, 0.8–2.6and 1.4–2.6 U/l in case of free drugs, drug-loadedPLG-NP and empty PLG-NP, respectively (normalrange up to 70U/l in mice). Thus, there was noevidence of biochemical hepatotoxicity in eithergroup.

Chemotherapeutic efficacy

Following the inoculation of tissue homogenates(50 ml) on Lowenstein-Jensen media, no visiblegrowth of M. tuberculosis occurred till day 25 inmice receiving free drugs daily or drug-loaded PLG-NP every 10 days, with undiluted, 1:10 diluted or1:100 diluted homogenates (Table 3). However,control (untreated) animals revealed a bacterialcount of 4.7270.05 and 4.7370.03 cfu/ml in lungand spleen homogenates, respectively.

Discussion

Patient non-compliance has been a major obstaclein the successful management of TB, and hascompelled researchers to develop sustained-release drug formulations so that the dosingfrequency may be reduced.3–5 The present studyevaluates the therapeutic potential of PLG-NP

Table 2 Tissue drug levels following the repeated oral administration of drug-loaded PLG-NP to mice on every10th day.

RIF (mg/ml homogenate) INH (mg/ml homogenate) PZA (mg/ml homogenate)

Lung Liver Spleen Lung Liver Spleen Lung Liver Spleen

Day 11 o0.25 0.470 0.470 0.0570.01 0.1770.04 0.2070.01 Not detected 40.075.0 34.075.0Day 21 o0.25 0.470 o0.25 0.0470.01 0.1870.04 0.2070.02 Not detected 43.072.0 32.073.0Day 31 o0.25 o0.25 0.470 0.1070.02 0.2070.03 0.2170.04 Not detected 38.074.0 34.073.0Day 41 o0.25 o0.25 o0.25 0.0570.01 0.1570.03 0.1770.02 Not detected 30.073.0 28.073.0

Results are mean7SD, n¼ 5–6.0.25 mg/ml was the sensitivity of analytical method for RIF.PZA was below the detection limit in the lungs.

Figure 1 Tissue drug levels following the single oraladministration of PLG-NP encapsulated drugs to mice.(a) RIF, (b) INH and (c) PZA. Results are mean7SD,n¼ 6–8.

376 R. Pandey et al.

based ATD-delivery system when administeredorally.

The double-emulsion and solvent-evaporationprocess is useful for the encapsulation of water-soluble drugs and involves: (i) the formation ofdroplets in the primary emulsion followed by theremoval of solvent from the droplets of the secondemulsion and (ii) precipitation of the polymer andsolidification of the core of the PLG-NP. In ourstudy, vigorous mixing during the preparationprocess, the use of a sonicator and the emulsionstability obtained with PVA as a surfactant, resultedin particles with a size range 186–290 nm (Table 1).Only 12–18% particles were in the higher size rangeas indicated by a polydispersity index of 0.3870.04(the index is a measure of dispersion homogeneity,values closer to zero indicate a homogeneousdispersion). The large surface area of the PLG-NPas well as the water solubility of the drugs are thetwo key factors which accelerate drug loss into theaqueous phase during nanoparticle preparation.15

However, drug encapsulation/loading was found tobe satisfactory and infact better than our previousexperience with microparticles.5 Further, we ob-served that when drug-loaded PLG-NP were sus-pended in PBS, there was a negligible (initial 3–7%followed by o1%) in vitro drug release for 6 weeks.Although, hydration-mediated degradation of PLGis well reported,16 the residual PVA associated withthe nanoparticles was probably responsible for theminimal release, by forming a diffusional barrier.

Our previous studies17 in mice have demon-strated that following the oral administration ofATD encapsulated in PLG microparticles, therapeu-tic plasma concentrations are maintained for 72 h(RIF and INH) and 108 h (PZA) respectively. Thepresent study shows that an enhanced sustainedrelease of the drugs (without altering the drugdosage) is possible with PLG-NP. Following the oraladministration of drug-loaded PLG-NP, the drugswere present at or above MIC in the plasma for 4days in case of RIF, and 9 days in case of INH and

PZA. This is in contrast to free drugs (alone ormixed with drug-free PLG-NP), which were clearedfrom the plasma within 12–24 h of oral administra-tion. Further, the drugs were possibly encapsulatedin the polymers rather than being surface adsorbedbecause the administration of free drugs mixedwith empty (drug-free) PLG-NP failed to improvethe duration of drug stay in plasma/organs.

It is known that PLG-polymers have a bioadhesiveproperty, which enables them to stick to theintestinal mucosa.18 PLG microparticles, becauseof their comparatively large size, may fail to crossthe intestinal mucosa. However, nanoparticles canbe absorbed directly via different routes (intracel-lular/paracellular) through the intestinal muco-sa.6,7 Drugs are released from the PLG-NP slowlyand not all at once, a phenomenon that holds truefor all controlled release systems in general. Thesefactors explain the enhanced sustained drug re-lease in the plasma in case of PLG-NP as comparedto PLG microparticles.

All the three drugs were present at above MIC inthe organs up to day 9 (Fig. 1). At day 11, INH waspresent at therapeutic concentration in all theorgans. However, RIF levels were below the limitsof detection (o0.25 mg/ml) in the lungs, whereasPZA was undetectable. This formed the basis ofadministering drug-loaded PLG-NP orally to miceonce in every 10 days for evaluating the che-motherapeutic efficacy. Further, results of the drugaccumulation studies showed that there was nosteady increase in the tissue drug levels followingrepeated oral dosing, thereby indicating that therewas no cumulative or carry-over effect of eitherdrug (Table 2). The chances of drug related toxiceffects were drastically reduced because the samedrug amount was administered every 10 days(encapsulated in PLG-NP) rather than daily (freedrugs). Hepatotoxicity studies further substan-tiated these findings.

Oral administration of free drugs daily or drug-loaded PLG-NP every 10 days to M. tuberculosis

Table 3 Chemotherapeutic efficacy of free drugs administered daily and drug-loaded PLG-NP administered every10 days orally to M. tuberculosis infected mice, following 46 days of treatment.

ControlLog cfu/ml(mean7SD)

Oral free drugs, daily Oral drug-loaded PLG-NP,every 10 days

Lung homogenates 4.7270.05 No tubercle bacilli detectedn No tubercle bacilli detectedn

Spleen homogenates 4.7370.03 No tubercle bacilli detectedn No tubercle bacilli detectedn

Lung/spleen homogenates were prepared by homogenizing whole organs in 3ml sterile PBS (pH 7.2–7.4) and 50 ml of preparedhomogenates were used for inoculation.Results are based on visible growth of M. tuberculosis on Lowenstein-Jensen media at day 25 post inoculation, n¼ 5–6.nNo growth of tubercle bacilli occurred following the inoculation of undiluted, 1:10 diluted and 1:100 diluted homogenates.

Nanoparticle encapsulated antitubercular drugs against murine tuberculosis 377

H37Rv infected mice for 46 days, resulted incomplete bacterial clearance from the organs ascompared to untreated controls (Table 3). This isbetter than our previous reports,5,19 which showedthat the subcutaneous administration of drug-loaded PLG-microparticles significantly reducedthe bacilli count in infected mice, but not to zerolevels. This could be attributed to the use of threedrugs (instead of two, i.e. RIF and INH in theprevious reports) as well as an enhanced sustainedrelease profile of drug-loaded PLG-NP. Although inthe present study, free drugs and drug-loaded PLG-NP showed equal efficacy, it should be emphasizedthat 46 doses (free drugs daily) have been reducedto 5 doses (PLG-NP every 10 days). These resultsbear important implications for TB therapy becausereduction in dosing frequency would certainlyenhance the patient compliance and, hence, bettermanagement of the disease.

Literature pertaining to the encapsulation of ATDin nanoparticles has been scanty. Dalencon et al.20

reported the loading of rifabutin in nanocapsules;however, the preparation was evaluated in experi-mental toxoplasmosis. The encapsulation of ethio-namide, a second-line ATD, was reported by Lopeset al.21 but the authors did not carry out any in vivostudies. Therefore, the present study communi-cates, for the first time, the preparation, char-acterization, biodistribution and chemotherapeuticefficacy of three frontline ATD (RIF, INH and PZA)encapsulated in nanoparticles. The preparationsare suitable for oral administration at every 10 daysand bear significant therapeutic potential for themanagement of TB.

Acknowledgements

Authors are thankful to M/s Panacea Biotec Ltd.Lalru, Punjab, for carrying out drug analysis byLC-MS and particle sizing.

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