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Tuberculosis (2005) 85, 415–420

Tuberculosis

KEYWORDSolid lipidnanoparticDrug delivAntitubercDrugs;TuberculosChemothe

1472-9792/$ - sdoi:10.1016/j.t

�Correspondifax: +91 172 27

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http://intl.elsevierhealth.com/journals/tube

Oral solid lipid nanoparticle-based antitubercularchemotherapy

Rajesh Pandey, Sadhna Sharma, G.K. Khuller�

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

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ng author. Tel.: +91 17244 401.ess: gkkhuller@yahoo.c

Summary The present study was planned to evaluate the chemotherapeuticpotential of oral solid lipid nanoparticles (SLNs) incorporating rifampicin, isoniazidand pyrazinamide against experimental tuberculosis. The SLNs were prepared by the‘‘emulsion solvent diffusion’’ technique with an encapsulation efficiency of 5175%for rifampicin, 4574% for isoniazid and 4174% for pyrazinamide. Following a singleoral administration to mice, therapeutic drug concentrations were maintained in theplasma for 8 days and in the organs (lungs, liver and spleen) for 10 days whereas freedrugs were cleared by 1–2 days. In M. tubercuIosis H37Rv infected mice, no tuberclebacilli could be detected in the lungs/spleen after 5 oral doses of drug loaded SLNsadministered at every 10th day whereas 46 daily doses of oral free drugs wererequired to obtain an equivalent therapeutic benefit. Thus, SLN based antituberculardrug therapy forms a sound basis for reducing dosing frequency and improvingpatient compliance for better management of tuberculosis.& 2005 Published by Elsevier Ltd.

Introduction

Nanoparticles are known to hold promise astherapeutic drug carriers and solid lipid nanoparti-cles (SLNs, defined as nanocrystalline suspensionsin water prepared from lipids which are solid atroom temperature)1 are a new form of particulatecarriers besides the more conventional ones such asliposomes, lipid emulsions and polymeric nanopar-ticles.2 The SLNs combine the virtues of traditional

Published by Elsevier Ltd.

2755 175;

o.in (G.K. Khuller).

nanoparticles while eliminating some of theirdemerits. Hence, it is not surprising that SLNs havebeen employed in formulation development, 3,4 toincorporate drugs thereby improving their bioavail-ability,5 as well as for targeted drug delivery.6

Though liposomes7 and polymeric nanoparticles8

proved to be successful antitubercular drug (ATD)carriers in experimental tuberculosis (TB), it wasonly recently that SLNs were explored as aninhalable ATD carrier.9 However, the fact that theoral route supersedes all other routes for thepurpose of drug administration need not be over-emphasized. Therefore, the present study wasplanned to co-incorporate three frontline ATDs

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(rifampicin, isoniazid and pyrazinamide) in SLNsand to evaluate its chemotherapeutic potential viathe oral route in experimental murine TB.

Materials and methods

Chemicals and drugs

Stearic acid (Mr 284.5), rifampicin, isoniazid,pyrazinamide and polyvinyl alcohol (PVA, Mr

13 000–23 000, 85% hydrolyzed) were obtained fromSigma Chemical Co. (St. Louis, MO, USA). All otherreagents were obtained from standard companies.

Animals

Laca mice (20–25 g body weight) were obtainedfrom the Central Animal House, Post GraduateInstitute of Medical Education and Research,Chandigarh (India). Animals were housed in biosaf-ety cabinets (Nuaire Instruments, NU 605-600E,Series 6) and provided with pellet diet/water adlibitum. The study was approved by the Institute’sEthical Committee.

Culture

The culture of M. tuberculosis H37Rv originallyobtained from the National Collection of TypeCultures (NCTC, London) was maintained on You-man’s modified medium.

Preparation of SLNs

The preparation of SLNs was based on the principleof ‘emulsion solvent diffusion method in water,’10

with slight modifications.9 Briefly, 10mg of eachdrug (rifampicin, isoniazid and pyrazinamide) and30mg of stearic acid were put into a mixture ofacetone/ethanol (12ml each) and heated to60–70 1C in a water bath. The total drug: lipid ratiowas maintained at 1:1W/W. The resulting solutionwas poured into 25ml of 1% w/v aqueous PVA at4–8 1C under mechanical stirring. The SLNs formedinstantaneously which were recovered by centrifu-gation at 35,000g for 30min at 4–8 1C. The pelletwas washed thrice with distilled water and vacuumdried. It should be noted that the three drugs wereco-incorporated in the SLNs. Drug-free/emptynanoparticles were prepared by substituting so-dium chloride in place of the ATDs.

Characterization of SLNs

Five mg of SLNs were put into 5ml of ethanol andheated to 50 1C to lyse/dissolve the particles andrelease the drugs. The percentage drug incorpora-tion efficiency with respect to initial amount ofdrug taken was calculated by the formula: (amountof drug released from the lysed SLNs/amount ofdrug initially taken to prepare the SLNs) x 100.Rifampicin was analyzed by a microbiological assaythat was specific for the drug, using Bacillus subtilis(MTCC 441) as the indicator strain (sensitivity0.25 mg/ml, linearity 0.25–10 mg/ml).11 Isoniazidwas estimated by a spectroflourimetric methodbased on the principle that in the presence ofsalicylaldehyde, the drug formed a hydrazonewhich was soluble in isobutanol and a highlyfluorescent compound which could be measuredat an excitation wavelength of 392 nm and emissionof 478 nm (sensitivity 0.1 mg/ml, linearity0.1–10 mg/ml).12 Pyrazinamide was analyzed spec-trophotometrically, based on the formation of acoloured complex between the drug and sodiumnitroprusside at alkaline pH, which could bemeasured at 495 nm (sensitivity 5.0 mg/ml, linearity5–70 mg/ml).13 Residual PVA was analyzed color-imetrically based on the reaction between twoadjacent hydroxyl groups of PVA and an iodinemolecule to form a coloured complex which couldbe measured at 695 nm (sensitivity 25 mg/ml,linearity 25–550 mg/ml).14 Residual acetone/etha-nol was analyzed by headspace GC.

In vitro drug release studies

Five mg of drug-loaded SLNs were put into 50ml ofsimulated gastric fluid (SIF, 0.1MHCl, pH 1.2) orsimulated intestinal fluid (SIF, phosphate buffer, pH6.8), both without enzymes, prepared according tothe USP.15 At various time points (30min, 1, 2, 3, 4,6, 12, 24, 48, 72 h), 1ml aliquots were drawn fordrug analysis and replaced with an equal volume ofbuffer. The results were expressed as the percentdrug released with respect to the theoretical value.

Preparation of SLNs for in vivo studies

The drug doses used throughout the study wererifampicin 12mg/kg+isoniazid 10mg/kg+pyrazina-mide 25mg/kg body weight, as per the standardhuman doses used in our previous reports.8,9 Sincethe doses were different for the three drugs, theinitial amount of each drug required for SLNpreparation was calculated by the formula9:(amount of drug required per animal/mean drug

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incorporation efficiency) x 100. Once the total drugquantities required were known, equivalentamount of stearic acid was used in the preparation(to maintain drug/lipid ratio at unity). The basicprocedure for SLN preparation remained the sameas discussed above. Five milligrams of the SLNformulation (containing 0.24mg rifampicin+0.20mg isoniazid+0.50mg pyrazinamide) com-prised a therapeutic dose combination, which wassuspended in 100 ml distilled water just before oraldosing in each experiment. Similarly, free drugswere also freshly dissolved in distilled water/methanol(5:1 v/v) immediately before dosing.

In vivo drug disposition studies

Mice were divided into 2 groups of 10 animalseach—Group 1, oral drug-loaded SLNs; and Group2, oral free drugs. Following drug administration,the animals were bled at different time points,plasma was separated and analyzed for the drugs bythe methods mentioned above. In addition, theanimals were sacrificed at various time points toanalyze the drug content in 20% organ homogenates(100mg tissue in 500 ml isotonic saline) of lungs,liver and spleen. The results are expressed as mgdrug per ml plasma/g organ weight.

Pharmacokinetic analysis

The plasma drug concentration versus time datawas used to determine the area under theconcentration—time curve (AUCo–t) on a SigmaPlot software (version 8.0). The terminal AUCt–N

was obtained by dividing the last measurableplasma drug concentration by the elimination rateconstant (obtained by regression analysis). The sumof AUC0–t and AUCt–N yielded the total AUC0–N

while the area under moment curve/area undercurve gave the mean residence time (MRT). Therelative bioavailability of SLN incorporated drugswas computed by the formula: (AUC0–N of oral SLNincorporated drugs/AUC0–N of oral free drugs) x(Dose of oral free drugs/dose of oral SLN incorpo-rated drugs).

Experimental infection and chemotherapy

Mice were infected intravenously with 1� 105

viable bacilli of M. tuberculosis H37Rv in 0.1ml ofsterile isotonic saline (standardized procedure inour laboratory in lieu of the absence of aerosolinfection facilities).8 Fifteen days later, 6 animalswere sacrificed, the lungs and spleen were removedaseptically and homogenized in sterile isotonic

saline. The establishment of infection was con-firmed by Ziehl–Neelsen staining of the homoge-nates. In addition, the homogenates were plated onMiddlebrook 7H10 agar base (supplemented withOADC) to obtain the basal infection load. Theremaining animals were divided into the followinggroups of 6 animals each—Group 1, untreatedcontrol; Group 2, oral drug-free SLNs every 10 days(5 doses); Group 3, oral free drugs daily (46 doses);and Group 4, oral drug-loaded SLNs every 10 days (5doses). The drug doses were the same as mentionedabove. At day 46 following the initiation ofchemotherapy, the animals were killed and lung/spleen homogenates were prepared in sterileisotonic saline. The homogenates (50 and 100 ml ofundiluted and 1:10 diluted) were inoculated onMiddlebrook medium for enumeration of colonyforming units (cfu). The colonies were counted onday 21 post inoculation and the cfu data wereanalyzed by one-way analysis of variance (ANOVA)followed by Student’s unpaired t-test.

RESULTS

Characterization of SLNs

The drug incorporation efficiency was 5175% forrifampicin, 4574% for isoniazid and 4174% forpyrazinamide. The amount of residual PVA was10.5–12.5%w/w of vacuum dried particles. Noresidual acetone/ethanol could be detected.

In vitro drug release

In case of isoniazid/pyrazinamide, the drug re-leased in SGF was o15% in the first 6 h and 12–15%during 6–72 h. Rifampicin was released to a lesserextent, i.e. 9% in the first 6 h and 11% during 6–72 h.The drug released in SIF was no more than 20% upto6 h and 11% from 6 to 72 h, in the case of isoniazid/pyrazinamide while rifampicin released was in therange of 8–12% during the entire study period.

Drug distribution profile in plasma/organsand pharmacokinetics

Following a single oral administration of ATD-loaded SLNs to mice, all the drugs were detectablein the plasma from 3h onwards up to day 8 (Fig. 1).At each time point, the drug concentrations wereat or above the minimum inhibitory concentration(MIC90) already established in our laboratory(rifampicin 0.2 mg/ml, isoniazid 0.3 mg/ml, pyrazi-namide 8 mg/ml).16 On the other hand, free drugs

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were cleared from the circulation within 12 h oforal administration. All the three drugs could bedetected in the lungs, liver and spleen of theanimals up to day 10 following the oral adminis-tration of ATD-loaded SLNs (Fig. 2) whereas freedrugs were cleared by 24–48 h from the tissues.Furthermore, in the case of oral drug-loaded SLNs,the MRT, AUC and hence the relative bioavailabilitywas increased 10–29 fold as compared with freedrugs (Table 1).

Table 1 Salient pharmacokinetic parameters following tcompared to oral free drugs.

Mean residence time (MRT, h) Area un

RifampicinOral free drugs 6.2071.00 8.37Oral SLNs 91.3076.30 92.00IsoniazidOral free drugs 5.5070.70 10.62Oral SLNs 97.60711.20 314.00PyrizinamideOral free drugs 6.6071.00 188.00Oral SLNs 100.6079.30 2505.00

Values are mean7SD, n ¼ 5:

Figure 1 Plasma drug levels following a single oraladministration of drug-loaded SLNs to mice.

Chemotherapy

The basal cfu load at the start of treatment (15days post-infection) was 4.2070.04 log in the lungsand 4.3470.04 log in spleens (results are mean7SD of 6 animals). Five doses of drug-loaded SLNsresulted in undetectable cfu in the organs of M.tuberculosis H37Rv infected mice whereas 46conventional doses were required to achieve thesame therapeutic benefit. Untreated control and

he oral administration of ATD-loaded SLNs to mice, as

der curve (AUC0–N, mg h/ml) Relative bioavailability

71.10 1.0077.20 10.99

72.10 1.00729.00 29.57

712.00 1.007113.00 13.32

Figure 2 Tissue drug levels following a single oraladministration of drug-loaded SLNs to mice.

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Table 2 Assessment of chemotherapeutic efficacy of oral SLNs in M. tuberculosis H37Rv infected mice.

Log cfu

Lung Spleen

Untreated controls 5.1870.31 5.2170.26Oral empty SLNs, every 10 days (5 doses) 5.1070.27ns 5.2470.18ns

Oral drug-loaded SLNs, every 10 days (5 doses) o1.0� o1.0�

Oral free drugs, daily (46 doses) o1.0� o1.0�

Values are mean7SD ðn ¼ 6Þ.ns, non-significant ðp40:05Þ compared to untreated controls.�no cfu detected with undiluted/1:10 diluted homogenates after 21 days of plating on Middle brook medium.

Oral solid lipid nanoparticle-based antitubercular chemotherapy 419

animals receiving empty SLNs showed comparableðp40:05Þ bacterial load (Table 2).

Discussion

The reduction in the dosing frequency of ATDsremains a therapeutic challenge and hence thepresent study was designed to evaluate thepotential of oral SLN- based ATD delivery in amurine TB model. The SLNs prepared according tothe ‘‘emulsion solvent diffusion’’ method 10 showeda drug incorporation efficiency of 5175%, 4574%and 4174% for rifampicin, isoniazid and pyrazina-mide, respectively. Rifampicin showed the highestincorporation owing to the lipid-based nature ofthe formulation. The stability of the particles wasindicated by the slow drug release observed in SGF/SIF which could be attributed to the residual PVA(10.5–12.5%w/w) associated with SLNs. Particlestability is known to be enhanced by PVA whichforms a barrier to the diffusional release ofincorporated compounds.17 The SLNs being a lipid-based formulation, it is expected that lipophilicdrugs would remain incorporated for a longer timeperiod whereas hydrophilic drugs would be releasedmore. Hence, rifampicin (a hydrophobic drug) wasreleased to a lesser extent in SGF/SIF as comparedwith the hydrophilic drugs, isoniazid/pyrazinamide.

Following a single oral administration of the SLNformulation, the drugs could be detected in theplasma from 3 h onwards up to 192 h (Fig. 1). Onthe other hand, free drugs could not be detected inthe plasma beyond 12 h of oral administration.Further, in the case of ATD-loaded SLNs, sustaineddrug levels were maintained in the organs till day10 (Fig. 2), whereas, free drugs were cleared by24–48 h from the tissues. Because of the slow andsustained release of drugs from the SLNs, the MRTwas increased by several fold as compared to free

drugs (Table 1). This resulted in an enhancedAUC0–N and relative bioavailability of the 3 ATDs.Other workers have demonstrated the improve-ment in bioavailability for tobramycin18 and idar-ubicin 5 when administered in SLNs.

Considering the fact that the drugs were presentin the tissues till day 10 in the case of SLNs (Fig. 2),the chemotherapy schedule was designed accord-ingly in which oral drug-loaded SLNs were adminis-tered every 10 days for 6 weeks whereas the parentdrugs were administered orally daily (conventionaltherapy) (Table 2). No cfu were detected in theorgan homogenates in the case of oral drug-loadedSLNs or conventional chemotherapy. Although theresults were similar, it should be emphasized that46 conventional doses could be reduced to 5 oraldoses. The results bear important implicationsbecause reduction in dosing frequency wouldcertainly enhance patient compliance and hence,improve management of TB. Further, no alterationsin serum bilirubin, alanine aminotransferase (ALT)or alkaline phosphatase (ALP) were observed in theTB-infected animals receiving drug loaded or emptySLNs which indicated the safety of the formulationas far as biochemical hepatotoxicity is concerned(Khuller et al., unpublished results).

Although polymeric nanoparticles8 and lipo-somes7 are efficient ATD carriers, the advantagewith SLNs is that unlike liposomes, their long-termstability as well as drug incorporation efficiency isbetter whereas in contrast to polymeric formula-tions, the risk of residual organic solvents isminimum.19 Lipids such as stearic acid (used forpreparing SLNs in the present study) are a part ofthe regular diet and their intermittent use as a drugcarrier is not likely to pose any health hazard. Thisis in contrast to the use of synthetic polymerswhose safety by the oral route must be welldocumented. Another limiting factor towards theuse of synthetic drug carriers is their high cost.However, SLNs were not yet explored for the oral

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delivery of ATDs and our experiments are the firstendeavour in this direction. Our findings suggestthat SLNs offer an economical and patient friendlyapproach for the administration of anti-TB drugs,bearing a high chemotherapeutic potential.

Acknowledgements

This research was partially funded by grant fromDepartment of Science and Technology, Govt. ofIndia, New Delhi.

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