Nanostructured drug delivery for better management of tuberculosis

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Journal of Controlled Release xxx (2014) xxx–xxx

COREL-07115; No of Pages 15

Contents lists available at ScienceDirect

Journal of Controlled Release

j ourna l homepage: www.e lsev ie r .com/ locate / jconre l

Review

Nanostructured drug delivery for better management of tuberculosis

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Indu Pal Kaur ⁎, Harinder SinghUniversity Institute of Pharmaceutical Sciences, UGC-Centre of Advanced Study Panjab University, Chandigarh 160014, India

⁎ Corresponding author. Tel.: +91 172 2534191.E-mail addresses: [email protected] (I.P. Kaur),

http://dx.doi.org/10.1016/j.jconrel.2014.04.0090168-3659/© 2014 Published by Elsevier B.V.

Please cite this article as: I.P. Kaur, H. Singhhttp://dx.doi.org/10.1016/j.jconrel.2014.04.0

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Article history:Received 17 January 2014Accepted 3 April 2014Available online xxxx

Keywords:Nanostructured drug deliveryTuberculosisAntitubercular drugs (ATDs)Sustained releasePolymersReduced side effectsDose reduction

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ROWith almost 30% of the world population suffering from tuberculosis (TB) including its resurgence in the devel-

oped world, better management of this global threat is highly desired. The emergence of multidrug-resistant TB(MDR-TB) against first-line drugs and extensively drug resistant TB (XDR-TB) due tomisuse of second-line antitu-bercular drugs (ATDs) is a further concern. Recommended treatment involves long termandmultipledrug therapywith severe side effects. In this context, nanostructured systems efficiently encapsulating considerable amounts ofATDs, eliciting controlled, sustained and more profound effect to overcome the need to administer ATDs at highand frequent doses, would assist in improving patient compliance and circumvent hepatotoxicity and/ornephrotoxicity/ocular toxicity/ototoxicity associated with the prevalent first-line chemotherapy. Nanostructureddelivery systems constitute a wide range of systems varying from liposomes, micelles, micro- and nanoemulsions,to polymeric nanoparticles (PNPs) and solid lipid nanoparticles (SLNs). Improved bioavailability, solubility, stabil-ity and biocompatibility make them an ideal choice for delivery of ATDs. Present review comprehensively coversresearch carried out on first-line antitubercular drug therapy using these nanostructured systems.

© 2014 Published by Elsevier B.V.
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Contents

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1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02. Tuberculosis: some key facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.1. Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.2. Nature of causative agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.3. Emergence of MDR and XDR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.4. Secondary infections in HIV patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3. Antitubercular drug (ATD) therapy: issues and concerns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.1. WHO treatment guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.2. Major issues related to therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

4. Need for novel and sustained delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.1. Initial efforts with implants and microparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

5. Nanodelivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05.1. Nanoemulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05.2. Nanosuspensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05.3. Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05.4. Niosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05.5. Polymeric nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05.6. Solid lipid nanoparticles (SLNs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05.7. Micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05.8. Dendrimers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

6. Nanomedicine as an answer to cerebral, drug-resistant and latent TB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 07. Patented systems/status of ATD nanodelivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 08. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

[email protected] (H. Singh).

, Nanostructured drug delivery for better management of tuberculosis, J. Control. Release (2014),09

http://dx.doi.org/10.1016/j.jconrel.2014.04.009

mailto:[email protected]

mailto:[email protected]


http://www.sciencedirect.com/science/journal/01683659


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2 I.P. Kaur, H. Singh / Journal of Controlled Release xxx (2014) xxx–xxx

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1. Introduction

Tuberculosis (TB), a persistent and lethal infectious disease [1], isone of the major challenges in modern day community health [2]. Re-gardless of potentially remedial pharmacotherapies being available forover 50 years, TB remains themost important cause of avertable deaths.Although TB appears as a chronic disease with comparatively slow de-velopment, multidrug-resistant strains can kill immunocompromisedpatients in very short periods of time [3].

Nanotechnologywith its sophistication and advanced techniques hasprovided us a new tool to understand the scientific developments at ananoscale [4] and treat dreadful diseases such as tuberculosis and AIDSwith a greater ease using nanoparticles as drug delivery systems.Confronted with frequent therapeutic failures and emergence of multi-drug resistance strains, researchers have developed novel ways to defydrug resistance, to restrict the treatment duration andmore importantlyto lessen side effects, toxicity and drug interactions. The present reviewdiscusses hurdles in the effective treatment of TB and use of nanostruc-tured delivery systems to counter these problems.

2. Tuberculosis: some key facts

2.1. Epidemiology

After HIV/AIDS, TB is the most commonly occurring and fatal infec-tious disease [5]. Roughly 2 billion people at present are infected world-wide with Mycobacterium tuberculosis, representing about 30% of thetotal population. In 2012, 8.6 million people fell illwith TB and1.3 milliondied from TB. Though prevalent in budding countries where elevatedmortality indexes have been reported [6], howsoever, infection has alsoresurged significantly in the urbanized countries. A WHO self studyestimated that every second a newperson is infectedwith TB [7]. Thoughbillions of dollars are spent each year and the governments all over theworld stand committed to the eradication of TB, however the diseasestill remains out of bound, infecting millions and killing thousands ofinfected population.

2.2. Nature of causative agent

M. tuberculosis, is an acid fast bacteria, which forms acid-stable com-plexes with arylmethane dyes [8]. The Mycobacteria are plentiful in soiland water, butM. tuberculosis is mainly identified as a pathogen whichlives in the host and several species of theM. tuberculosis complex havespecifically adapted their genetic structure to infect human populations.

2.3. Emergence of MDR and XDR

Mismanagement of first-line drugs results in the emergence ofmultidrug-resistant TB (MDR-TB), cure for which takes even longer. It re-quires use of second-line drugs, which are costlier and showmore exten-sive and severe undesirable effects. Globally, only 48% ofMDR-TB patientsin the 2010 cohort of detected cases were successfully treated, and highmortality rates and poor follow-upwas reflected. Globally in 2012, an es-timate of 450 000 people, spread over virtually all the surveyed countriesdeveloped MDR-TB and there were an estimated 170,000 deaths fromMDR-TB.

When the second-line ATDs aremisused (including use of quinolonesfor normal non-mycobacterial infections), extensively drug resistant TB(XDR-TB) which is resistant to both first and second line anti-TB drugs[9–11]. XDR-TB strains have been reported from South Africa and otherparts of the world, with its high occurrence in HIV-positive individuals.

2.4. Secondary infections in HIV patients

It is estimated that 1/3 of the 36 million people on earth with HIV/AIDS are co-infected with M. tuberculosis [12]. This co-infection has

Please cite this article as: I.P. Kaur, H. Singh, Nanostructured drug delivehttp://dx.doi.org/10.1016/j.jconrel.2014.04.009

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been named the “cursed duet,” requiring approximately 30% of theyearly income of an infected household in direct and indirect costs,thus becoming a socio-economic calamity for these families [13].

Countries with themaximumTB/HIV co-infection rates exist in Sub-Saharan Africa. Worldwide, the biggest increase in co-infection with TBandHIV has taken place in the 25–44 year old population [14]. Since thisage group generally involves the active workforce of any country, hencethe consequent economic impact is huge.

3. Antitubercular drug (ATD) therapy: issues and concerns

3.1. WHO treatment guidelines

WHO recommends the use of first line ATDs (Table 1) in TB patientsat the onset of the disease. The most effective pharmacotherapy iscomposed of a multi-drug regimen of isoniazid (INH), pyrazinamide(PZA) and rifampicin (RIF). Thefirst 2 months involve intensive therapywith these three agents together with ethambutol (EMB) [15,16]. Forthe remaining 4 months, RIF and INH are administered. These fourdrugs collectively with streptomycin (parenteral aminoglycoside)represent the so-called first-line treatment.

To simplify dosing schedules and to minimize mono-therapy-associated resistance to RIF, the WHO and the International UnionAgainst Tuberculosis and LungDisease (IUATLD) suggests administrationof fixed dose combination (FDC) of RIF and INH plus PZA or PZA withEMB [17,18]; FDC's combine at least 2 first-line ATDs drugs in one singleformulation.

All the ATDs (except RIF) are hydrophilic in nature (Table 1), andinvariably manifest severe side effects especially upon long term use.Any carrier system which can help improve the permeability of theseagents (low or negative log P as shown in Table 1, indicate poor perme-ability of these ATDs; BCS class III) will result in improved effectivenessat lower dosewith lesser incidence of side effects. Later will in turn leadto improved compliance with lowered health costs. It is in this contextthat encapsulation technology (micro- or nano-encapsulation) mayplay an important role for formulating ATDs into sustained-releasesystems.

Treatment of MDR-TB comprises the administration of PZA concur-rently with second-line drugs such as ethionamide, prothionamide,clycloserine, capreomycin, p-aminosalicylic acid or fluoroquinolones[27]. The second-line drugs exhibit more toxicity, are more expensiveand are less potent than the first-line agents.

The present review is majorly focused on the use and developmentof first-line ATDs.

3.2. Major issues related to therapy

To assure therapeutic effectiveness, extended treatments (9–12 months) are usually recommended [28]. In this regard, low patientcompliance and obedience to the dosage regimens turn into criticaldrawbacks of the pharmacotherapy. Variable bioavailability of theseATDs further creates an additional crucial limitation [29].

Amongst HIV/TB co-infected patients, RIF and EMB show a decline inintestinal absorption [28]. Generally variable bioavailability of RIF isotherwise also a major clinical problem [30]. It also displays a strongpH-dependent solubility (1 part in 5, 10, 250, and 360 parts of waterat pH-values of 1.5, 2, 5.3, and 7.5, respectively, at 25 °C) [3,31]. RIFdisplays low aqueous solubility and moderately good absorption inthe stomach and was earlier classified as Class II drug, according to theBCS [32] but has later been reclassified as BCS class IV drug [32]. Absorp-tion of RIF from any FDC which also incorporates INH, is significantlycompromised due to its reaction with INH, in the gastric medium, toform its insoluble hydrazone [33–35]. RIF, in combination products,with other ATDs, is also known to undergo degradation under acidicconditions [36]. Shishoo et al. [37] have indicated in an in vitro studythat in the presence of INH, RIF tends to undergo faster and greater

ry for better management of tuberculosis, J. Control. Release (2014),


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t1:1 Table 1t1:2 First line antitubercular drugs, their physicochemical properties and side effects.

Drugs Partition coefficient (log P) Solubility mg/mL (in water) Mol. wt. Side effects Referencet1:3

Isoniazid (INH) −0.639 140 137.14 Hepatotoxicity, neuropathy [19,20]t1:4

Rifampicin (RIF) 3.719 1.4 822.94 Hepatotoxicity [21–23]t1:5

Ethambutol (EMB) −0.14 10 204.31 Hepatotoxicity, ocular toxicity [1,24]t1:6

Streptomycin (STR) −6.400 – 581.57 Ototoxicity, nephrotoxicity [25,26]t1:7

Pyrazinamide (PZA) −1.884 15 123.11 Hepatotoxicity [17,26]t1:8

3I.P. Kaur, H. Singh / Journal of Controlled Release xxx (2014) xxx–xxx

decomposition at acidic pH (in stomach), as compared to when presentalone. The decomposition of RIF under these conditions varies from 8.5to 50% in the time range corresponding to the gastric residence time inhumans (15 min to 3 h) [35].

RIF-INH interaction was also confirmed when the extent of loss ofdrug under in vitro fasting pH condition (pH 2.0) [38] equaled itsin vivo drop in bioavailability [29,37]. An almost 30% fall in bioavailabil-ity of RIFwas observed in these studies. The results emphasize a need todevelop a suitable delivery system which can prevent its degradation.

EMB is also unstable in the presence of the INH, PZA or RIF, and par-ticularly in the presence of INH. Thus, it is indicated that EMB must notbe administered as an FDCwith other ATDs. Its bioavailability is reducedto half when co-encapsulated with the other three ATDs.

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Novel Antitubercular TherapyPatient compliancePhysician friendly

Drug Development and NCEsExtensive researchCostlyTime dependentPotency?Side effects?

Nanostructured ATD MedicineExperimental evidence of successReduced dosing frequencyReduced side effectsVariety of delivery systemsCost effectiveTackle MDR-TBChoice of route of administration

Need to address issues likeScale upStabilityClinical trialsToxic solvent residues & nanotoxicology

Answer

Substitute?

Status?

Conventional TB TreatmentMultiple drugsFrequent dosingSide effectsPoor patient complianceDrug resistance

Will result in

Fig. 1. Options for effective antitubercular therapy.

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4. Need for novel and sustained delivery systems

Tuberculosis (TB) has been a leading killer disease globally and amultiple of first-line ATDs must be administered regularly for at least6 months for relief of the disease.

Howsoever, control of tuberculosis with antimycobacterial chemo-therapy is a tricky and arduous taskmainly because (i) therapy involveslong duration of treatment, (ii) competency of the antimycobacterialdrugs to reach targets and induce antimycobacterial effect are pretty in-adequate (poor permeability and stability), (iii) all antimycobacterialdrugs are highly toxic, and (iv) significant patient noncompliance toprescribed medicines is observed especially due to the length of thetherapy and also due to the therapy associated severe side effects. Acommon and dangerous side effect of these ATDs is hepatotoxicity,and the effect is dose related. Further to this, INH causes neurotoxicity,EMB causes ocular toxicity and STR (though not hepatotoxic) causesnephrotoxicity and irreversible ototoxicity.

Most of these factors are attributable to the inadequate modes ofdrug administration and thus present a great challenge for drug deliverytechnology and for the scientific community including formulationscientists [6].

Nanotechnology science has been a boon to current pharmacologyand biopharmaceutical enhancement of drug performance. It is possibleto design drug delivery systems capable of targeting phagocytic cellswhich are infected by intracellular pathogens, such asmycobacteria. De-livery systems based on nanotechnology offer wide opportunities forimproving the therapy for a range of diseases including TB (Fig. 1).

Keeping in mind the previously discussed facts in terms of increasedfrequencyofMDR strains as an outcome of erratic and poorlymaintaineddosage regimen and the limited capacity to correctly and efficiently diag-noseMDR and XDR-TB, it seems appropriate and worthwhile to developnovel ways of improved bioavailability and site specific targeting of firstline ATDs therapeutics to avoid development of drug resistance [39]. Fur-ther to it these systemsmay result in reduced toxicity due to low concen-tration of free drug in plasma at any given time, assuming that the carriersystem with entrapped drug acts as a reservoir continuously supplyingthe required concentration of drug in plasma.

To extort amaximum therapeutic benefit, a drugmust be formulatedcarefully and this forms the fundamental concept behind a drug deliverysystem. Four “D's” are assigned to a drug delivery system i.e. drug,destination, disease and delivery, out of which the last one is the onlychangeable factor [40]. When the rate or place (or both) of drug release


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of a formulation is altered, the formulation is known asmodified-releasesystem. It is usually attained by using encapsulation techniques. Encap-sulation technology has widespread application in the pharmaceuticalindustry for the controlled release of drugs. Amongst the handiest poly-mers used for the purpose are poly(lactide-co-glycolide) (PLG), alginicacid, and chitosan, although, nonpolymeric drug carriers like lipids inthe form of solid lipid nanoparticles (SLNs) and liposomes are alsogaining popularity. Irrespective of the carrier system, the crucial aim indrug delivery is to improve the drug's bioavailability by bypassing oneor several of the probable factors identified to influence it. Diverse



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nanoparticulate approaches have been developed and reported toexclusively deliver chemotherapeutic agents to target sites. This helpsimprove the therapeutic index of the drug by stringently localizingits pharmacological action to the target site or organ, and limiting itsnon-target and unwanted side effects. Thus it may be said thatnanoparticulate drug delivery systems help improve tolerance for toxicchemotherapeutics in addition to influencing their bioavailability [41].It may be noted that use of nanocarrier systems for ATDs is anticipatedin result to several benefits viz. smaller dose complemented withoverwhelming of first pass metabolism, dodging of enzymatic or pH de-pendent degradation and circumventing of any efflux systems prevalentin gastrointestinal tract.

4.1. Initial efforts with implants and microparticles

One of the earlier efforts reported to develop a sustained-release ATDformulation was the incorporation of INH in three different polymers:poly(methyl methacrylate), poly(vinyl chloride), and carbomer [42].The main intent of the study was to attain persistent plasma INH levelsin fast acetylators of the drug. Utilizing another polymer, Eudragit RS100, the encapsulation and systematic release kinetics of INH werestudied in the early 1990s [43]. Spherical microcapsules were developedusing various polymers and it was understood that aliphatic poly(esters)such as PLA, PGA, and PLG have exceptional biocompatibility, biodegrad-ability, and mechanical strength [44] making them easy to formulateand use for delivery of numerous drug molecules. These polymers areapproved by the US Food and Drug Administration for drug delivery.Consequently, with the use of PLG as a carrier for INH, the era of aliphaticpoly(esters) as ATD carriers began [45]. Themethod included drymixingof INH and PLG to generate a film under pressure and implanting thisfilm under the skin of mice resulted in maintenance of sustained plasmaINH levels for 6 weeks. The levels attained with the formulation in agroup of animals were equivalent to the levels in the daily dosing of con-ventional free (unencapsulated) drug. Considerable antimycobacterialactivity was achieved in the liver and lung homogenates of animals eu-thanized 6 weeks post-implantation. The method was also applied toother drugs (e.g., clofazimine, a second-line ATD)with significant results.In beige mice infected withMycobacterium avium–M. intracellulare com-plex, equivalent reductions in colony-forming units (CFU)were achievedin the PLG-clofazimine (single implant) and daily free-clofaziminegroups [46]. These PLG films were then modified to prepare rods; laterwas easy to recover and remove the formulation once all the drugswere released from the system [47]. PLG implants encapsulating PZAwere also reported with equally encouraging results [48]. Three timesthe daily dose of PZA when administered as a single PLGA polymerimplant exhibited sustained levels up to 54 days. When explored forchemotherapeutic efficacy, single implant was comparable to standardoral treatment with PZA given daily for 8 days. On the other hand, thedisadvantage of the systemwasmainly the invasive method of adminis-tration, because implantation necessitates surgery including partial an-esthesia. The toxicity of N-methylpyrrolidone used in manufacturingthe implants was also a major concern. Moreover, no study with ATDsis absolute unless RIF is included and evaluated for efficacy. RIF, owingto its high hydrophobicity and proneness to loss of activity due to irre-versible acid degradation in g.i.t., is at times difficult to encapsulatewith significant efficiency. These tribulations encouraged scientists toopt for an injectable sustained-release system that might work equallywell for INH and RIF. Using emulsification and solvent evaporationtechniques, three kinds of PLG microparticles incorporating RIF wereprepared. Amongst the porous, nonporous, or hardened particles, thehardened particles proved to be the most excellent formulation interms of drug release, since a single subcutaneous dose of hardenedPLG microparticles exhibited sustained release of RIF in the organs(lungs, liver, and spleen) of mice up to 6 weeks [49]. The reason behindthismay possibly be the strong hydrophobic interactions of RIFwith PLG,thus resulting in slow but sustained release of RIF. Additionally, the high


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PVA concentrations (20%w/v) lead to the formation of a stable emulsionproducing a depot, at the site of injection, from which the drug releasedslowly. To see the possibility of applying this system for encapsulatinghydrophilic molecules, INHwas chosen as the model drug. After a singlesubcutaneous injection of PLG-encapsulated INH to mice, therapeuticdrug levels were maintained in the organs for 49 days as against 1 dayin case of free INH [50]. Another technique i.e. multiple-emulsion wasemployed using the natural polymer cellulose for the encapsulation ofINH, and the formulation was assessed by in vitro means [51]. Further,a combination of RIF and INH encapsulated separately in PLG andmixed before dosing was evaluated. The results showed that the combi-nation administered as a single subcutaneous dose to mice maintainedtherapeutic drug levels for 6 weeks. Moreover, in M. tuberculosis-infected mice, subcutaneous PLG microparticles encapsulating RIF orINH (or both) significantly reduced the CFU counts in the infected organs[50]. The therapeutic potential of subcutaneously injected drug-loadedPLG microparticles is also reported by other researchers [52,53]. Theoperational principle of parenteral PLG microspheres encapsulatingATDs, including PZA was also found to hold promise for oral drug deliv-ery. Each drug was formulated separately and the formulations werecombined before oral dosing. A single oral dose tomicemaintained ther-apeutic drug levels in plasma for 3 to 5 days as compared to 1 day for freedrugs respectively. Noticeably, the ATDs were also present in the tissuesfor up to 5 to 7 days in the PLG group, while free drugs were clearedwithin 2 days. Along with improvement in bioavailability, there was en-hancement in most of the reported pharmacokinetic parameters whenPLG was used as a vehicle for oral delivery of ATDs [54]. This formedthe basis for a subsequent chemotherapeutic study in which drug-loaded PLG microparticles were administered weekly for 6 weeks toM. tuberculosis-infected mice resulting in a significant decline, compara-ble to that achieved by daily free-drug therapy, in bacilli counts ofvarious infected organs. The study thus established oral PLG microparti-cles as a suitable system for efficient reduction in dosing frequency [55].Achieving such promising results with the microparticulate systems,motivated researchers to look for alternative and more potential systemin terms of: (i) higher drug encapsulation efficiency, (ii) higher drugloading, (iii) enhanced drug bioavailability, and, (iv) reduction in dosingfrequency.

Majority of these issues were suitably addressed by the use ofnanoparticulate systems for suitable development of these ATDs [4].

5. Nanodelivery systems

In the recent times, nanotechnology has emerged as a highlysophisticated and advanced technology, referring to the nanoscale sizerange of atoms, molecules and macromolecules with exclusive orsignificantly better (with respect to the free drug) physicochemicalproperties [4]. With the advent of nanotechnology, it may becomepossible to more effectively treat dreadful diseases such as tuberculosisand AIDS. Frequent therapeutic failures and emergence of multidrugresistance strains, a need to curtail the treatment duration andmore im-portantly to reduce drug interactions are the major factors whichsuggest the need for developing nanocarrier systems for drug agentsused for these diseases. Regardless of having a successful chemothera-peutic regimen for tuberculosis, the prevailing treatment scheduleis burdensome and presents considerable problems such as patientnon-compliance, severe and often fatal hepatotoxicity, and need of co-administering multiple antitubercular drugs. The pharmaceutical tech-nologists of today are aiming at improving the efficiency and reducingassociated toxicity of ATDs by purposely targeting the site of infections.The potential to develop more effective and compliant therapy withexisting molecules seems to lie with nanotechnology. This is especiallyrelevant since not even a single new ATD was launched/approved inthe last four decades until a recent FDA approved agent ‘Sirturo’,which is chemically known as bedaquiline. It is thefirstmedicine specif-ically designed for treating MDR TB tuberculosis. Though effective, the



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agent shows serious side effect like induction of potentially fatal heartrhythms [56], hence the doors are still open for novel and safetherapeutics.

Polymer-based ATD loaded nanodelivery systems, reported thus farin literature, indicate significant advantages with respect to bioavail-ability, decreased dosing frequency, and drug targeting. Further to thisa variety of other nano-based delivery systems such as nanoemulsions,nanosuspensions, and solid lipid nanoparticles are also being exploredas explained in the following passages. Table 2 summarizes variousnanodrug delivery systems of ATDs developed by different researchersfor the treatment of TB.

5.1. Nanoemulsions

Spontaneously formed oil-in-water dispersions of small size be-tween 10 and 100 nm, which can be produced in large amounts havebeen in use, since long, for the delivery and improved uptake ofentrapped drugs by the cells of the phagocytic system [1,90] and lipo-protein receptors in the liver subsequent to oral administration [91].Nanoemulsions constitute a popular drug delivery system as they arethermodynamically stable and can be sterilized by filtration [92].

A stable oral nanoemulsion (mean particle size of 80.9 nm, polydis-persity index of 0.271, in vitro drug release of 95%) of ramipril, with a rel-ative bioavailability of 229.62% as compared to conventional capsule and539.49% as compared to that of drug suspension is reported. The studysignified the use of ramipril nanoemulsion for pediatric and geriatric pa-tients [93]. In another study, the authors evaluated the effect of labrasolon self-nanoemulsification efficiency of ramipril nanoemulsion [94].Different o/w nanoemulsions of RIF for IV administration by means ofpharmaceutically acceptable excipients: Sefsol® 218 as the oil phase,Tween® 80, Tween® 85 and saline water as the surfactant, the cosurfac-tant and the aqueous phase, respectively [95] and with a mean droplet

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Nano drug delivery systems proposed and evaluated for the treatment of tuberculosis.

Delivery system (key component) Drug Advant

Nanoparticles (PLGA) RIF Strongdrug le

Nanoparticles (poly(butyl-2-cyanoacrylate)) Moxifloxacin EnhancNanoparticles (wheat germ agglutinin and PLGA) RIF IncreasNanoparticles (PLGA) RIF, INH, PZA Total cAerosolized liposomes (egg-PC and cholesterol) RIF High coNebulized solid lipid nanoparticles (nanocrystalline lipid) RIF, INH, PYR BetterNanoparticles (stearic acid) RIF, INH EffectivSpherical micelles (PLA-modified chitosan oligomers) RIF SustainMicelles (poly(ethylene glycol)–poly (aspartic acid) conjugate) INH, PZA, RIF SustainDendrimeric nanocarriers RIF Improv

increasMesoporous silica nanoparticle (polyethyleneimine) RIF, INH IntraceNanoparticles (gelatin) RIF SustainNanoemulsion (Sefsol®) RIF High eMultiple emulsion INHNanosuspension (nanocrystals) Clofazimine ReductMicroparticles 4 ATDs SustainLiposomes (phosphatidylcholine-cholesterol) PZA, rifabutin ReduceLiposomes (phosphatidylcholine-cholesterol) ATDs 40% drLiposomes (phosphatidylcholine-cholesterol) INH, RIF ReduceNiosomes (spans) RIF SustainNiosomes (spans) RIF SustainNanoparticles (PLG) RIF, INH, PZA Sustain

adminiNanoparticles (PBCA) RIF, INH, STR High inSLNs (stearic acid) RIF, INH, PZA SustainSLNs (Compritol® 888 ATO) RIF, INH, STR ImprovMicelles (PEO-PPO) RIF 5–7 timMicellar gel (caprolactone/coglycolide) RIF In vitroMicelles (PLA-chitosan) RIF In vitroConjugates (PEG-PAA) INH 5–6 folNanoparticles (PEG, PAA) INH, RIF, PZA StrongDendrimers (polypropylimine) RIF Increas

intrace


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size of 47–115 nm are also reported. The encapsulation efficiency ofmore than 99% and the homogeneity was exceptional for all thenanoemulsions. An initial burst effect was noticed in in vitro drug releasestudies ranging from40 to 70% after 2 h, followed by a restrained release.Stability assays over a 3 month period indicated a small but insignificantincrease in the droplet size and viscosity both at 4 °C and 25 °C. Stabilitystudies concluded that the optimized formulation was stable andsuitable for IV delivery of RIF.

Mehta et al. carried out physicochemical analysis of INH micro-emulsions and established that the release of drug frommicroemulsionwas non-Fickian [73]. Studywas extended to observe changes in themi-crostructure of Tween 80-basedmicroemulsion in the presence of ATDsviz. INH, PZA, and RIF. The particle size ranged from 210 nm to 320 nmfor these ATD nanoemulsions. The formulations showed controlledrelease with maximum drug release in 2 h found to be 40, 35, and 10%for INH, PZA, and RIF, respectively [96].

5.2. Nanosuspensions

These are sub-micron colloidal dispersions of pure drugs stabilizedby means of surfactants [97]. Nanonization (reducing the mean size ofsolid drug particles to the nano-scale generally by top milling orgrinding) is a helpful methodology to improve the solubility of drugs il-lustrating strong solute–solute interactions and highmelting pointswitha poor water and lipid solubility [98]. Clofazimine, a riminophenazinecompound, is an agent helpful for treating patients with M. aviuminfection. However, the use of this drug was constrained because of itspoor solubility. Clofazimine was then formulated as a nanosuspension(particle size 385 nm) and administered to mice by intravenous route.This lead to significant reduction of bacterial loads in the liver, spleen,and lungs of mice infected with M. avium [74]. The pharmacokineticdata indicated that the drug concentrations in the studied organs

ages References

therapeutic efficacy, high encapsulation efficiency, prolonged plasmavels, greater dispersibility

[57]

ed in vitro and in vivo therapeutic efficacy [58,59]ed bioavailability, prolonged plasma drug levels [57,60]learance of TB bacilli after 5 oral treatments [60]ncentration of the drug in the target organ (e.g., lung) [61]therapeutic efficacy in Guinea pigs [62]e and sustained drug release [63]ed release of the drug (in vitro) [64]ed drug release and 6-fold increase in anti-TB activity [65–67]ed selective uptake of drug-loaded nanocarriers by macrophages,ed drug entrapment

[68,69]

llular delivery [70]ed release, high concentrations in various organs [71]ntrapment efficiency of 99% [72]

[73]ion in bacterial load in lungs [74]ed release up to several days [75,76]d bacterial load, higher antimicrobial activity [77]ug in lungs [78]d CFU in tissues [79]ed release, high concentration in various organs [80,81]ed release, high concentrations, 145 times higher accumulation in lungs [82]ed release, complete bacterial clearance after five doses—single dosestered every 10 days

[83]

tracellular concentration, INH 4.5 times, RIF 22 times, STR 7 times [84]ed release up to 5 days, sterilizing effect [63]ed bioavailability, protection against degradation [85–87]es increase in solubility [88]sustained release up to 32 days [89]sustained release 5 days [66]d increase in antitubercular activity [66]antitubercular activity [65]ed solubility, in vitro sustained release effect up to 120 h, increasedllular concentrations

[68,69]



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reached high levels, well in surplus of MIC for mostM. avium strains. Theeffects of clofazimine nanocrystalswere analogous to those of the liposo-mal formulation taken as a control. Reverchon et al. developed RIF submicronic particles (400 nm to 3 μm) using supercritical CO2 assistedatomization suitable for parenteral and aerosolizable drug delivery sys-tems. Latter is an approach convenient for TB pharmacotherapy adminis-tering drugs locally to the lungs. The authors studied the effect of varioussolvents on particle size and drug degradation [75,76]. Out of the varioussolvents tried to solubilize the drug, DMSO was found to be the mostappropriate for nanonization.

5.3. Liposomes

Liposomes are vesicles in a nano- to micro-range and comprise ofphospholipid bilayer surrounding an aqueous core encapsulating thedesired drug. For prolonged sustainability and circulation time, lipo-somes are at times PEGylated. It was reported that intravenous admin-istration of liposome encapsulated gentamicin resulted in a significantreduction in the mycobacterial count in the liver and spleen of amouse model of disseminated M. avium complex infection [97]. Simi-larly, liposome encapsulated second-line antibiotics presented analo-gous results [99,100]. Lung-specific stealth liposomes composed ofphosphatidylcholine, dicetyl-phosphate, O-steroyl amylopectin, choles-terol, and monosialogangliosides–distearylphophatidylethanolamine–poly(ethylene glycol) 2000 were utilized for the targeted deliveryof anti-TB drugs (INH and RIF; entrapment 8–10% and 44–49% respec-tively) to the lungs. Significant accumulation of the nanocarriers inthe lungs proved their prospective use in targeted drug delivery [101].The liposomal drugs have also been shown to considerably decreasethe bacterial load when compared to the free drug [77], improving theantimycobacterial efficacy and decreasing the toxicity of the encapsu-lated drug.

In vivo biodistribution following intravenous administration inhealthy and tuberculosis infected mice showed marked increase in ac-cumulation in lungs from 5.1% for conventional liposomes to 31% forPEGylated liposomal systems after 30 min. The rate of accumulationwas invariably attributed to the composition of the liposomal vesicles.Drug uptake levels in the lungs increased to approximately 40% for thePEGylated nanocarriers, within 30 min of administration in infectedanimals, while a 30–50% drop in uptake and accumulation in the reticu-loendothelial organs viz. the liver and spleen was observed [102]. Drug-loaded nanocarriers showed a considerable decrease in the toxic effectswhen assessed for cytotoxicity in peritonealmacrophages, in contrast tofree drugs. A 12 mg/kg dose of liposomal and free INH decreased thenumber of CFU in the lungs to about 3.9 and 4.5 log units, whereas at10 mg/kg, the corresponding reduction was by 3.8 and 4.3 log units, re-spectively. Decrease in CFU was observed in a parallel pattern for theliver and spleen. The therapeutic activity of free and encapsulated INHand RIF was estimated at both therapeutic and sub-therapeutic doses.Administration of sub-therapeutic doses (4 and 3 mg/kg for INH andRIF respectively) also showed a sharp decrease in CFU for encapsulateddrugs in comparison to the free drugs [101]. These observations have astrong implication for expected decrease in toxic side effects by usinglower but effective doses.

With the aim of improving the anti-TB activity, reducing the toxicityand facilitating the parenteral administration of highly lipophilicclofazimine, drug-loaded liposomes (entrapment efficiency 95–100%)were produced [103] andpreclinically assessed in acute and chronicmu-rine infections [104]. Drug encapsulation led to a decrease in the in vitroand in vivo toxicity of the drug and enhanced the anti-TB activity in bothacute and chronic models. In addition, chronically infected mice treatedwith the encapsulated drug exhibited total clearance of bacilli from theliver and spleen with no signs of recurrence 2 months post-treatment.In the lungs, a gradual reduction in CFUwas observed, however a relapsewas observed 2 months post-treatment. Pyrazinamide- [105] andrifabutin-containing liposomes [106] are other examples representing


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potential and resourcefulness of liposomal nanocarriers. Liposomal PZA(entrapment efficiency 11.2%) at two-fifths the dose of daily free PZAwas found to be superior to free drug in the management of TB infectionin experimental animals. Rifabutin liposomes prepared with dipalmitoylphosphatidylcholine:dipalmitoyl phosphatidylglycerol (DPPC:DPPG)exhibited lower bacterial loads in the spleen (5.53 log10 vs. 5.18 log10)and liver (5.79 log10 vs. 5.41 log10) as compared to free rifabutin suggest-ing that liposomal RFB is a promising approach for the treatment ofextrapulmonary TB in human immunodeficiency virus co-infectedpatients.

5.4. Niosomes

Charged phospholipids (stearyl amine and dicetylphosphate) andnonionic surfactants (monoalkyl or dialkyl polyoxyethylene ether)formed as a result of cholesterol hydration lead to the development ofliposome like vesicles called niosomes [107]. Niosomes have severaladvantages over liposomes, being more stable, showing less difficultyin scaling up, and having low-cost of production. Niosomes can host hy-drophilic drugs inside the core and they tend to entrap lipophilic drugsin hydrophobic provinces. Niosomes are visualized as alternative deliv-ery systems that can overcome the drawbacks associated with steriliza-tion, high production costs, scale-up difficulties and the instability of thephospholipidic components of liposomes upon light exposure even atroom temperature. Various experiments were performed using arange of surfactants (viz. Spans 20, 40, 60, 80, 85) and different sizedniosomes for successful delivery of RIF to the lungs after loading intoniosomes [80,81]. A slow drug release, majorly attributed to the lipo-philic nature of the surfactant, but significantly higher drug concentra-tions were achieved in the target organs via intra-peritoneal route ofadministration in contrast to intra-venous route. A significant increasein accumulation of the RIF-loaded niosomes was observed in the lungsafter intra-tracheal administration.

In another study, Jain and Vyas prepared microsized (8–15 μm)RIF-loaded niosomes comprising Span® 85 as the surfactant (90% re-lease in N48 h) [108]. In vivo studies indicated that by regulating thesize of the carrier, up to 65% localization of drug can be achieved in thelungs. Only 15% of the administered drug was found in the lungs whenan equivalent amount of free drug was administered. Remaining drugwas found to be distributed in the liver (20%), spleen (17%), kidney(12%), and blood (36%). In a different investigation, the same group ofresearchers extended the findings and studied the biodistribution ofniosomes with smaller sizes (1–2 μm) consisting of different sorbitanesters (Span® 20, 40, 60, 80 and 85) and cholesterol in a 50:50% molefraction ratio [109]. The encapsulation gradually increased with the in-crease in hydrophobicity of the surfactant and ranged between 20 and35%. In vitro release showed 80% maximal and 52% minimal levels in120 h for Span-20- and Span-85-based systems, respectively; slowerdrug release was observed for the more lipophilic surfactants in theaqueous medium. Niosomal formulations reached substantially higherRIF concentrations i.e. 46.2% of the administered dose, in thoracic lymphnodes when administered via the i.p. route as compared to 13.1%for the free drug. These observations recommended that the drugcompartmentalised into the lymphoid tissuewhen loaded into niosomes.On the contrary, after intravenous administration of the drug-loadedcarriers, only 7.3% of the drug was detected in the thorax, with the accu-mulation extent being lower than the 11.5% obtained for free RIF.Mullaicharam and Murthy, investigated the organ biodistribution of RIFniosomes (5 mg/mL) after intravenous and intratracheal administrationin comparison to that of the free drug in albino rats [82]. Generally, a sig-nificant increase in the total drug concentration in the lungs, kidneys,liver and blood serum was obvious for rifampicin-loaded niosomes.After intravenous administration, niosomes preferentially accumulatedin the lung, liver and kidney with the organ to serum AUC ratios being117,060 for the lungs/serum, 67 for the liver/serum and 3068 for thekidney/serum, while administration of free RIF resulted in a less selective



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delivery (558.3 for the lung/serum, 16.1 for the liver/serum and 332.6 forthe kidney/serum). After intratracheal administration, the lung/plasmaratios were 128,585 and 885 for niosomes and free drug, respectively,showing a 145-fold increase in the accumulation capacity of RIF-loadedniosomes in the lungs as compared to the free drug.

In another study, RIF loaded niosomes (particle size 1.13 μm, poly-dispersity index 0.14, entrapment efficiency 65%) were prepared by re-verse phase evaporation method and a negative charge was inducedusing dicetyl phosphate [110]. Attempts were also made to incorporateINH in niosomes (particle size 2.3 μm, entrapment efficiency 80%,in vitro release 90% in N48 h) [111]. Karki et al. [111] proved that thedrug accumulation by niosomal drug delivery in visceral organs (lung,kidney, liver, spleen) was lower than free drug indicating less inci-dences of toxicity of niosomal drug delivery system than free drug.

5.5. Polymeric nanoparticles

These are widely used delivery carriers for drug solubilization, sta-bility, and specific targeting [112–114]. Excellent stability, simplicity ofadministration by various routes, and possibility of loading hydrophilicand hydrophobic drugs have made their reputation as one of the mostadmired techniques for drug encapsulation [115].

Based on the technique used for their production, two categories ofsystems can be formed, namely nanocapsules and nanospheres. In theprevious, the drug is solubilized in aqueous or oily solvents and isenclosed within a polymeric membrane. On the contrary, the latter iscomposed of solid matrices of variable porosity in which the activemolecules are homogeneously distributed throughout the particle anddispersed at the molecular level. A wide range of biomaterials exist forthe production of polymeric nanoparticles (PNPs) [115]which are elim-inated from the system by opsonization and phagocytosis [116]. Toavoid detection by the host immune system and to extend circulationtimes in the blood stream, the alteration of the exterior with extremelyhydrophilic chains (e.g., PEG) has been widely investigated. The tech-nique is one of themost expansively studiedwith regard to antitubercu-lar drug delivery systems.

Semete et al. projected that PLGA nanocarriers (200 and 350 nm,polydispersity index 0.1, zeta potential −10–18 mV, specific surfacearea 3–10 m2/g) could be a safe way to deliver ATDs at the target. Amean 40.04% of the particles were localized in the liver, 25.97% in thekidney, and 12.86% in the brain [117].

Pandey et al. [83] reported sustained release of RIF, INH and PZAfrom orally administered poly(lactideco-glycolide) (PLG) nanoparticles.Drug encapsulation efficiencieswere 56.9±2.7% for RIF, 66.3±5.8% forINH and 68± 5.6% for PYA with particle size range 186–290 nm (N80%particles) and polydispersity index of 0.1–0.2. The drugs could be de-tected in the mice of plasma for up to 4 days for RIF and 9 days forINH and PZA; while therapeutic concentrations in the tissues were ob-served till 9 to 11 days following single oral dose of nanoparticles. Incontrast free drugs were cleared from the plasma within 12 to 24 h.Complete bacterial clearance from the organs of tubercle bacillus infect-ed mice was achieved after five oral doses of nanoparticles every 10thday, while free drugs took 46 doses to obtain identical results. Nanopar-ticles exhibited comparable results when tested in guinea pigs. A singleoral administration of the formulation resulted in sustained drug levelsin the plasma for 7–12 days and in the organs for 11–14 dayswith a sig-nificant improvement in mean residence time as well as drug bioavail-ability [118]. In a study, microparticles were tested and found to beless effective in comparison to nanoparticles [50,54]. For improving effi-ciency of nanoparticles, the surface of particles wasmodified employingwheat germ agglutinin. Lectins grafted PLGA nanoparticles showed asubstantially extended plasma life of up to 6–14 days in mice ascompared to uncoated ones (4–9 days) after administration by oral/inhalation route. Furthermore, absolute bacterial clearance was ob-served in various organs (lungs, liver and spleen) only after 3 doseseach administered at 14 days interval [119]. The presence of lectins on


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the surface of these nanoparticles (particle size 350–400 nm, encapsu-lation efficiency of 54%–66%) resulted in (i) enhanced mucoadhesion(ii) faster biorecognition by glycosylated structures in the intestineand (iii) increased serum half life [120]. After oral administration inmice, these nanoparticles demonstrated prolonged serum levels for6–7 days for RIF, 13–14 days for INH and PZA as compared to uncoatednanoparticles which were observed for 4–6 days for RIF and 8–9 daysfor INH and PZA loaded unmodified nanoparticles. Surface modifiednanoparticles illustrated detectable drug levels in the lungs, liver, andspleen for up to 15 days. Three oral doses of coated nanoparticles admin-istered every 14 days (versus 45 daily doses of free drugs) achievedcomplete bacterial clearance.

Pandey and Khuller also studied ATDs encapsulated in nanoparticlesadministered via pulmonary route [63]. The drug incorporation efficien-cy was 51± 5% for RIF, 45 ± 4% for INH and 41 ± 4% for PZA. The drugreleased in simulated intestinal fluid (SIF) was not more than 20% up at6 h and 11% for a period from 6 to 72 h, in the case of INH/PZAwhile RIFreleasewas in the range of 8–12% during the entire study. In guinea pigs,a single nebulization of RIF, INH, and PZA encapsulated nanoparticlesproduced same effect as that elicited by oral administration, i.e.sustained therapeutic drug levels in the plasma and in lungs for up to6 to 8 days for RIF and 11 days for INH and PZA, respectively. Followingpulmonary administration, complete sterilization of the lungs wasachieved only after administration of five doses at every 10th daywhereas to produce same effect orally 46 daily doses were necessary.While agglutinin surface modified nanoparticles needed only threedoses administered every 14 days for 45 days. Passive targeting of che-motherapeutic compounds to alveolar macrophages has been studiedusing pulmonary administration of nanoparticles for the treatment oftuberculosis infections [121,122]. Versatility of the nanoparticle-basedformulations was further established by effective subcutaneous injec-tions tomice infectedwithM. tuberculosis [123]. The drug encapsulationefficiency was 56.9± 2.7% for INH, 66.3 ± 5.8% for INH and 68.0 ± 5.6%for PZA. A single subcutaneous dose of RIF, INH, and PZA loaded poly-(DL-lactide-co-glycolide) PLG nanoparticles showed sustained thera-peutic drug levels in plasma for 32 and 36 days in the lungs/spleenand resulted in absolute clearance of bacteria as compared with freedrug which required daily treatment (35 oral doses).

Econazole and moxifloxacin loaded nanoparticles were prepared bythe solvent evaporation and multiple emulsion technique and wereassessed individually and in combination in murine mice model so as todevelop an effective orally administered regimen for TB. The drug encap-sulation efficiency of PLG nanoparticles for econazole and moxifloxacinwas found to be 52.27 ± 3.80% and 33.69 ± 3.88%, respectively. The av-erage size of the PLG nanoparticles was 217 nm, with a polydispersityindex of 0.38. Econazole and moxifloxacin loaded PLG nanoparticlesdemonstrated prolonged plasma levels up to 5 and 4 days, respectively,maintaining drug levels in the lungs, liver and spleen up to 6 days in com-parison to free drugs being cleared within 12–24 h. Only 8 doses of PNPsof each were sufficient to suppress bacterial clearance in M. tuberculosisinfected mice, while 56 daily doses of free moxifloxacin and 112 dosestwice a day of free econazole were required to elicit a similar effect.Combination of twodrugswas significantly efficacious in contrast to indi-vidual drugs. Moreover, when a third drug RIF was added to this combi-nation, it resulted in a complete bacterial clearancewithin 8 weeks. Studythus signifies the use of these nanoparticulate carriers for intermittenttubercular chemotherapy [72]. Anisimova et al. reported RIF, INH andstreptomycin loaded PBCA (poly(n-butylcyanoacrylate)) and PIBCA(poly(isobutylcyanoacrylate)) nanoparticles and studied their in-vitrocellular uptake in humanbloodmonocytes to develop drug depot system.There was a great enhancement in intracellular to the extracellular con-centrations of encapsulated INH (4–8 fold), streptomycin (7 fold) andRIF (22–25 fold)when compared to free drug INH(similar), streptomycin(undetectable) and RIF (5 times) [84].

In another study, moxifloxacin-loaded PBCA nanoparticles (418 nm)were prepared by anionic polymerization of poly(butyl-2-cyanoacrylate)



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[58,59]. Drug encapsulation efficiency ranged between 41.0% and 47.6%and the average size was 418 nm. Un-encapsulated drug (∼55%) wasnot removed from the formulation. In vitro drug release showed initialburst release followed by sustained drug release with 65% drug beingreleased at end of 48 h; but the developed NPs were found to be moretoxic than free drug in a cytotoxicity assay. When infected cells wereexposed to the drug in the free and the encapsulated form, a pronouncedincrease (2–3 fold) in the intracellular concentration from 125 to 175 to375 μg/mL was illustrated. In addition, encapsulated moxifloxacin wasmore effective than the free form to kill intracellular bacilli. Evaluationof the anti-TB activity in mice infected withM. tuberculosis showed a sig-nificant decrease in the mycobacterial count in the lungs after IV admin-istration [58,59]. PLGA (poly(lactic-co-glycolic acid)) nanoparticle-basedimplants were injected subcutaneously in a murine model and a singlesubcutaneous dose was sufficient to sustain drug levels in plasma, lungsand spleen for b1 month with untraceable bacterial counts in theseorgans [40]. Zahoor et al. prepared alginate nanoparticles of first-lineanti-TB drugs using ionotropic gelation method. The average size of algi-nate nanoparticleswas found to be 235 nmwith a polydispersity index of0.44; drug encapsulation was 70–90% for INH and PZA, 80–90% for RIFand 88–95% for EMB. After oral administration to mice, free drugswere undetectable in blood after 12 to 24 h but were traceable in tissues(e.g. spleen, liver and lung) till next day, whereas PNPs were detected inplasma up to 7 days for EMB, 9 days for RIF, 11 days for INH and PZA andin various organs (liver, lungs, spleen) till the 15th day [124]. du Toit et al.prepared emulsion-based INH-loaded polymeric nanoformulations(particle size 77–414 nm, zeta potential of −24 mV) employingnanoprecipitation technique. They could accustom the size of the nano-particles by varying the polymer concentration; lower polymer concen-trations led to smaller particles. In vitro release studies showed initialburst release till 2 h depending upon the technique used for preparationof nanoparticles [125]. Pandey and Khullar prepared PLGA nanoparticlesloaded with RIF, INH, PZA and EMB with respective encapsulation effi-ciency of 55.91% for RIF, 67.34% for INH, 68.32% for PZA and 43.11% forEMB. On concurrent oral administration of these nanoparticles to mice,therapeutic levels were maintained for 5–8 and 9 days in blood andplasma, respectively [126]; one administration every 10th day (5 doses)eliminated the bacteria in the meninges.

Clemens et al. employed mesoporous silica nanoparticles (MSNP;particle size 100 nm) coated with polyethyleneimine (PEI) loadedwith RIF or with cyclodextrin-based pH-operated valves which openonly at acidic pH to release INH into M. tuberculosis-infected macro-phages. The MSNP are engulfed/phagocytized efficiently by humanmacrophages, trafficked to acidified endosomes, and discharge highconcentrations of ATDs intracellularly. PEI-coated MSNP expressedmuch greater loading of RIF than uncoated MSNP and a great deal of ef-ficacy against M. tuberculosis-infected macrophages. MSNP showed nocytotoxicity at the doses employed for drug delivery. In the same way,the INH delivered by MSNP comprising pH-operated nanovalves killedM. tuberculosis within macrophages more effectively as compared toan equivalent amount of free drug. Hence MSNP provides a resourcefulstage that can be utilized to optimize the intracellular release andloading of particular drugs for the treatment of tuberculosis [70].

Saraogi et al. reported gelatin based nanostructured carriers aspotential vectors for efficient management of tuberculosis [71]. Theauthors developed RIF loaded gelatin nanoparticulate (GP) delivery sys-tem using two-step desolvationmethod. The drug loaded GPs exhibitedparticle size of 264 nm, with polydispersity index 0.24, zeta potential15.32 mV and encapsulation efficiency of 59.5%. The drug releaseexhibited a biphasic pattern i.e. initial burst which was followed by asustained release pattern (81.4% release in 72h). The cytotoxicity proto-cols confirmed the safety of nanoparticles in comparison to free drug. Invivo biodistribution study indicated higher localization of drug loadedGPs in various organs, in comparison to free RIF solution in PBS(pH 7.4). In comparison to free drug, the nanoparticles not only main-tained the plasma levels but also improved the AUC andmean residence


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time (MRT), suggestive of superior pharmacokinetics of drug. RIF GPs inaddition showed significant reduction in the bacterial counts in thelungs and spleen of TB-infected mice representing promising prospectsfor increasing drug targetability vis a vis decreasing dosing frequencywith the interception of side effects, for improved management oftuberculosis.

Hulda Swai's (CSIR's Centre for Polymer Technology, South Africa)team have developed nanoparticles (200 nm) of four frontline anti-TBdrugs aspiring to shorter treatment regimen with single dose drug ap-plication that will last for several days. They established that the nano-particles release the drugs into the bloodstream at a slower rate andfor a more prolonged period as compared to the conservative therapy[127].

5.6. Solid lipid nanoparticles (SLNs)

SLNs are aqueous nanocrystalline suspensions, made from lipidswhich are solid at room temperature [128]. The SLNs are an innovativeform of nanoparticulate carriers besides the more conventional onessuch as liposomes, lipid emulsions, and PNPs. SLNs have good tolerabil-ity (due to their origin from physiological lipids), scaling-up feasibility,the capacity to incorporate hydrophobic or hydrophilic drugs, and animproved stability of encapsulated drugs. Therefore, SLNs are inimitablein the sense that they mingle the virtues of traditional NPs while eradi-cating some of their problems [129]. It is remarkable that the SLNs ex-hibit important advantages, such as the composition (physiologiccompounds) and the possibility of large-scale production favored bythe feasibility to avoid organic solvents in the manufacturing process[130].

SLNs loaded with ATDs prepared by the emulsion solvent diffusionmethod to entrap RIF, INH, and PZA together is reported. The drugincorporation efficiency was 51 ± 5% for rifampicin, 45 ± 4% for INHand 41 ± 4% for PZA. The drug released in SIF was no more than 20%up to 6 h and 11% from6 to 72 h, in the case of INH/PZAwhile RIF releasewas in the range of 8–12% during the entire study period. The chemo-therapeutic potential of the formulation was estimated via the respira-tory route in guinea pigs. A sustained drug release was achieved for5 days in plasma and for 7 days in the organs. The pharmacokineticswas unaffected in healthy as well as TB-infected guinea pigs. Sevenweekly doses of the formulation led to undetectable bacilli in the organsof TB-infected guinea pigs, replacing 46 conventional doses [63]. A sin-gle oral administration of SLN formulations to mice, maintained thera-peutic drug concentrations in plasma till 8 days and in the organs richin mononuclear phagocyte system (MPS) i.e. the lungs, liver and spleenfor 10 days in comparison to free drugs which were cleared within1–2 days. Moreover, plasma concentrations were equal to or abovethe MIC at all time points of measurement. InM. tuberculosis H37Rv in-fected mice, 5 oral doses of drug loaded SLNs, every 10th day, were suf-ficient to completely suppress bacterial load in the lungs/spleenwhereas 46 daily oral doses of free drug were required to get a same ef-fect. Initial CFU count, 15 days after the infection with M. tuberculosisH37Rv was 4.20 and 4.34 log in lungs and spleen, respectively [63].

In our lab, we have developed SLNs of first line anti-TB drugs viz.INH, PZA, RIF, EMB and STR.We could achieve high drug loading and en-capsulation efficiency for these ATDs [86]. INH loaded SLNs (particle size48.4 nm, polydispersity index 0.266, entrapment efficiency 69%, zetapotential −0.101 mV) showed a significant improvement in relativebioavailability in the plasma (6 times) and brain (4 times) after admin-istration of INH-SLNs with respect to the free drug solution at the samedose [86]. RIF loaded SLNs (particle size 141.2 nm, polydispersity index0.320, entrapment efficiency 65.3%, zeta potential −3.5 mV) with drugloading of up to 50% w/w were prepared by a novel patented method[33,85]. SLNs provided protection to RIF against INH induced degrada-tion in acidic media. The extent of degradation was reduced to morethan half when RIF was incorporated in SLNs and further reduced toone fourth when INH was also present as SLNs [33]. RIF SLNs when



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administered in rats enhanced bioavailability by 8 times as compared tofree drug [85]. RIF SLNs also provided significant protection against INHinduced degradation, in vivo, when administered concomitantly withfree INH to rats (unpublished data). Another highly hydrophilic mole-cule, STR was encapsulated in SLNs with entrapment efficiency ofmore than 60% [87]. Furthermore, EMB loaded SLNs prepared with anencapsulation efficiency of more than 60% were administered to ratsshowing an enhancement of plasma bioavailability by 5 times ascompared to free drug solution (unpublished data).

5.7. Micelles

As a result of self assembly, amphiphilic polymers give rise to thepolymeric micelles in water. Micellar shell is formed due to the contactof hydrophilic blockswith the aqueousmedium, assisting the solubiliza-tion of amphiphile in water and stabilizing the aggregate. Conversely,hydrophobic blocks make the inner micellar core which facilitates thesolubilization of poorly water-soluble drugs [131] shielding them fromchemical and biological degradation. These micelles can be mademore lipophilic to improve, the penetration of incorporated drug intothe pathogen, and its antibacterial activity against Mycobacterium.

Commercially available and FDA-approved poly(ethylene oxide)–poly(propylene oxide) (PEO–PPO) block copolymers (linear poloxamersand branched poloxamines) are amongst the majority of importantmicelle-forming materials [132]. Preliminary studies that explored thesolubilization of RIF within polymeric micelles of a range of linear andbranched PEO–PPO with a broad spectrum of compositions illustrateda minimal solubilization effect (∼2-fold) [88]. These observations pro-pose that the size of themicellar core strongly restricts the encapsulationof the very bulky RIFmolecule. Other amphiphilic block copolymers syn-thesized by linking mono and bifunctional PEG precursors of differentmolecular weights with poly(ε-caprolactone) (PCL) enabled the finetuning of the HLB and the amplification of the micellar core, improvingthe solubilization extent by 5- to 7-fold [88]. Jiang and co-workerssynthesized thermo-responsive poly(ε-caprolactone-coglycolide)–poly(ethylene glycol)-poly(ε-caprolactone-co-glycolide) (P(CL-GA)–PEG-P(CL-GA)) smart block copolymers with micelle-forming and gela-tion properties [81]. The sol–gel transition temperature was fine tunedby changing theGA/CL ratio and the length of the hydrophobic segments.RIF-loaded (2 mg/mL) 25% gels were used to characterize the in vitrorelease profile of the matrices over time; the release was sustainedover 32 days. Although, these micellar systems did not show a substan-tial enhancement in the solubility of the drug, they could find applicationin the development of a drug depot system for the sustained release ofthe drug. To prolong the delivery of RIF, drug-loaded stereocomplexmicelles were formed by the particular assembly of enantiomeric poly(ethylene glycol)–poly(L-lactide) (MPEG-PLLA) and poly(ethyleneglycol)-poly(D-lactide) (MPEG-PDLA) block copolymers in a 1:1 ratio ofL-PLA- and D-PLA-containing block copolymers [133]. The RIF loading ca-pacity and encapsulation efficiency of the stereocomplexes were higherthan in the enantionerically pure micelles. Drug delivery experimentsin vitro illustrated a fast initial release (50% after 4–8 h) and amoremod-erated one (100% after 48 h) at later times. Moreover, the in vitro drugrelease could be regulated by the molecular weight of the polymer. Wuet al. developed PLA-modified chitosan oligomers competent of aggre-gating in water to form spherical micelles having sizes between 154and 181 nm [134]. Entrapment of 10% RIF into the nanocarriers resultedin core expansion to sizes in the range of 163–210 nm. In vitro release ex-periments illustrated a burst effect (35% within 10 h) followed bysustained release until day 5. To achieve higher effectiveness and longeranti-TB activity while limiting the toxic effects, Silva et al. synthesizedINH-poly(ethylene glycol)–poly(aspartic acid) conjugates that sustainthe release of the drug over time [66]. The micelle-forming prodrugshowed a 5.6-fold increase in antitubercular activity againstM. tuberculosis in vitro in comparison to the free drug. The mechanismproposed principally involves micelle uptake and intracellular release


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of the drug following the hydrolysis of the linkage. The identical synthet-ic pathwaywas pursued in order to encapsulate PZA and RIF [65,67]. Dueto relatively low CMC values (5 × 10−4–5 × 10−5 mg/L) micelles werestable in vitro. The size and distribution of these micellar nanoparticleswere found to be 78 nm, 84 nm and 99 nm, respectively, for PZA, INHand RIF conjugates and level of the conjugated drug was in the range of65.0–85.7% [65]. Size of themicelleswould avoid renal filtration, increas-ing the residence times in the blood stream. Furthermore, a strongerantimycobacterial activity was apparent. To conquer resistance, Jin andcollaborators designed INH lipid derivatives [135]. The new amphiphilicmolecules formedmonolayers at the air/water interface. The aggregationbehavior was closely related to the character of the hydrophobic tail.Flexible medium-long tails formed nano-sized vesicles. On the contrary,short lipid tail-derivatives displayed weak hydrophobic interactions andthey did not self-assemble. Molecules with very long tails led to the for-mation of crystal-like structures. The promising antibacterial activity ofthese micelles was demonstrated against Mycobacterium due to a morelipophilic structure that improved the penetration of the drug into thepathogen.

5.8. Dendrimers

Dendrimers are macromolecules exhibiting well defined, regularlyhyperbranched and three-dimensional design, with relatively lowmolecularweight and polydispersity, and, high and adjustable function-ality. The concept was proposed in the early 1980swith the synthesis ofpolyamidoamines (PAMAM), the first described dendrimers [136].These functional molecules are capable of drug encapsulation by virtueof the dendrimeric core, complexation and conjugation on the surface[137]. Regardless of the limited usefulness of pristine PAMAMs, the in-tangible approach did motivate the design of surface-modified deriva-tives with improved in vivo performance [138]. Due to their uniquestructure, dendrimers come across as striking candidates for the entrap-ment and delivery of anti-TB agents for a variety of administrationroutes. However, only a few researchers have yet explored theirpotential for this application. With an intent to target the drug to mac-rophages, Kumar et al. prepared RIF-loaded mannosylated 5th genera-tion (5G) polypropylenimine (PPI) dendrimeric nanocarriers [69].Surface modification with sugar molecules like mannose recognizableby lectin receptors situated on the surface of phagocytic cells enhancedthe selective uptake of the drug-loaded nanocarriers by cells of the im-mune system. Encapsulation efficiency for RIF was approximately 37%with hydrophobic interactions and hydrogen bonding contributing tothe physical binding of the drug to the core. The solubility of RIF withinunchanged dendrimers was ~52 mg/mL, while the superficial mannosemolecules sterically hindered the complexation and encapsulation ofthe drug and the solubilization of RIF was considerably less efficient atabout 5 mg/mL (2-fold as compared to the aqueous solubility of RIF).Mannosylation however significantly reduced the hemolytic toxicityassociated with dendrimers with amine terminals, from 15.6 to 2.8%.RIF-dendrimerswhen assayed indicated a beneficial effect of the carrier,decreasing the intrinsic hemolytic effect of free RIF from 9.8 to 6.5%. Asimilar trend was noticed when the viability of a kidney epithelial cellline was tested; encapsulation enhanced the survival of the cells from∼ 50% for free RIF to ∼85% for encapsulated RIF. Drug release studiesconducted in vitro showed that the modified dendrimers sustained therelease for about 120 h, as opposed to the fast delivery (10 h) foundwith regular dendrimers. The phagocytic uptake of RIF and RIF-loadeddendrimers was explored with alveolar macrophages harvested fromrat lungs. A clear increase in the intracellular concentration of the anti-biotic was apparent. Using analogous approach, the suitability of RIF-containing 4th and 5th generation PEGylated-PPI dendrimers to sustainthe delivery of RIF was investigated [68]. PEGylation resulted in a con-siderable increase in the percentage of drug encapsulation from 28and 39% to 47 and 61% for G4 and G5 derivatives, respectively. In addi-tion, the surfacemodification led to a better controlled release; i.e., 97.3



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and 46.3% cumulative release values were respectively found for 4G PPIand PEG-PPI after 36 h. Finally, PEG-grafted dendrimers exhibited aminimal hemolytic activity (1–3%) as opposed to the NH2-terminatedones (14–20%).

6. Nanomedicine as an answer to cerebral, drug-resistant andlatent TB

Across the globe, cerebral TB is perhaps the most horrible form ofextrapulmonary TB because of the associated high mortality [139]. Nu-merous reports support the feasibility of NP (loaded with other drugs)localization to the brain in experimental models [140–143], a processthat is predisposed by the targeting procedure [144]. Neutral particles(e.g., PVA stabilized PLG; SLNs) are reported to show a better of cerebraluptake and a lesser risk of toxicity to the blood–brain barrier [145]. Asingle oral dose of ATD PLG-NPs was found to result in sustained druglevels in the brain for 9 days. In a murine TB model, five oral doses ofthe formulation administered every 10th day resulted in undetectablebacilli in themeninges, as evaluated on the basis of CFU andhistopathol-ogy [126]. These results definitely merit evaluation in a higher animalmodel. We have also observed significant concentration of INH inbrain when administered as SLNs [145].

Poor patient compliance remains themain reason behind treatmentfailure and emergence of drug resistance including MDR-TB [77]. Inview of the rising incidence of MDR-TB and its deadly alliance withHIV [146,147], it was contemplated that the concept of nanomedicineshould be extrapolated to encapsulate second-line ATDs as well.Lopes et al. reported the nanoencapsulation of ethionamide [148].Ethionamide-loaded PLG NPs were also developed and evaluated by adifferent research group [149]. A single oral dose of the formulationproduced a sustained release of drug for 6 days in plasma, whereasthe free drug was cleared in 6 h in mice. Ethionamide was detected inthe organs for 5–7 days, suggestive of a weekly therapeutic regimenin MDR-TB.

Another exhilarating development has been the nanoencapsulationof azole antifungals and fluoroquinolones [58,150]. Azole antifungals(clotrimazole, econazole) have shown potent antimycobacterial activityagainst drug-sensitive and drug-resistant strains of M. tuberculosis aswell as the latent bacilli [151–153]. Fluoroquinolones, especiallymoxifloxacin, also possess strong antimycobacterial activity and achieveprolonged, high concentrations in alveolarmacrophages predominantlyfollowing encapsulation. These significant findings were reported byKisich et al. who established that moxifloxacin encapsulated inpoly(butyl cyanoacrylate) NPs accumulated in alveolar macrophagesthree times more efficiently than free moxifloxacin [58]. Moreover,the encapsulated drug was detected intracellularly for six times longerperiods than free drug, even at similar extracellular levels. An intracellu-lar concentration of 0.1 μg/mL with encapsulated moxifloxacin wascomparable to 1.0 μg/mL of free drug in terms of inhibiting mycobacte-rial growth [58]. InM. tuberculosis-infected mice, eight weekly doses ofthe PLG NP-encapsulated triple-drug combination (moxifloxacin +econazole + RIF) showed complete bacterial clearance from theorgans [72]. In addition, it has been recognized that the addition ofmoxifloxacin has the potential to shorten the duration of treatment[154]. Studies with econazole entrapped in alginate NPs confirmedthat the system has an edge over the PLG NPs, in terms of both pharma-cokinetics and pharmacodynamics, reducing the dosing frequency by15-fold [72,151]. The system is worth investigating for intermittenttherapy against MDRTB and latent TB.

Antibiotic STR has become less admired over the time because of theneed to administer it parenterally and its potential for nephrotoxicity.Though, it is one of the most cost-effective ATDs and is recommendedin certain special cases including relapse or treatment failure, with-drawal of INH and RIF, TB meningitis, co-treatment with HIV proteaseinhibitors, and certain cases of MDR-TB [14]. STR has been formulatedinto anoral dosage formbynanoencapsulation [155] and the STR loaded


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PLGNPs could not only be given orally tomice but also displayed an en-hanced bioavailability, therapeutic efficacy, and showed absence ofnephrotoxicity in contrast to free drug. We have recently reported onan enhanced availability of STR loaded SLNs in the brain with a reduceddistribution to the kidneys (STR is nephrotoxic) upon intranasal admin-istration. Reports of nanosystemspreparedwith newanti-TB drugs (e.g.,isoxyl) [156] or modified old drugs (e.g., fullerene-INH conjugate) [157]have also appeared.

7. Patented systems/status of ATD nanodelivery systems

US patent 20100310662A1 [158] shows the preparation of an oralnanoparticulate drug delivery system for the treatment of TB. Systemcomprises of poly DL-poly lactide co-glycolide (PLG) nanoparticlesencapsulating at least one azole, moxifloxacin and RIF. The inventorsprepared NP of individual drugs, encapsulating them for improved bio-availability and retention time. The invention disclosed the multipleemulsion and solvent evaporation technique for preparation of nano-particles. The average particle size of the nanoparticles was between217nmand250 nm. In vivodrug disposition studies revealed that singleoral dose of PLG nanoparticles maintained therapeutic drug concentra-tion in plasma for several days in comparison to free drug. The inventorsclaimed it as the most potent regimen that yielded total bacterial clear-ance in mice within eight weeks of the therapy and the results wereequivalent/comparable to the conventional treatment with four firstline ATDs. All the free ATDs (INH, RIF, PZA and EMB)were administeredonce daily while the encapsulated econazole, moxifloxacin and RIF(combination) were administered every 10th day with the exceptionof ethambutol which was administered weekly. This observation wasexplained on the basis of the fact that all three drugs of this combinationare active against activelymultiplying aswell as nondividing bacilli. Thecombination was proposed more beneficial than conventional regimenon the basis that econazole andmoxifloxacin are highly active drugs, ef-fective against multidrug resistant bacilli or its latent and persistentform. All the three drugs exhibit sterilizing effect owing to their activityagainst non-replicating bacilli as against conventional regimen whereonly RIF bears this activity. This regimen is also expected to reduce thetotal duration of tuberculosis chemotherapy. The main advantage ofthe invention was its ability to be administered orally, circumventingthe pain and drawbacks of subcutaneous or intravenous routes.

US patent 6054133 [159] revealed methods and composition fortargeting intracellular pathogens like M. tuberculosis. The ATDs wereconjugated with transferrin or other ligands to form conjugates thatare selectively taken up by the infected phagosomes. The inventionstates that similar to transferrin, low density lipoproteins are also suit-able vehicles for drug delivery which like transferrin traffic to earlyendosomes. They prepared three types of formulations viz. PLG NPencapsulating RIF + INH + PZA, PLG nanoparticles encapsulatingRIF + INH and PLGNP encapsulating EMB. The findings of this inventiondisclose thatMIC is not achievedwhen EMB is part of the four active sub-stances encapsulated together however separate encapsulation of EMBprovided the required MIC. The inventors have taken special care to en-sure that drug interactions areminimized and the desiredMIC is attainedfor each agent. The inventionwas proposed to show critical advantage ofdelivering antibiotics directly and selectively to the pathogens thusmax-imizing their efficacy. US patent 07018657 [160] discloses a particulatecomposition for ATDs, inwhich nanoparticles are prepared from a colloi-dal system comprising a continuous phase and micelles; the micellescomprising the surfactant material. In this process, the dispersed phaseis quenched to a solid state and the continuous phase and solvent arethen removed to produce NPs. The NPs can be incorporated in an aerosolcomposition suitable for deep lung delivery by means of a metered doseinhaler.

WO 2009002227 A1 [161] describes an invention which relates to anovel generation of controlled-activity antimicrobial preparationswhich are based on rifabutin and contain nanoparticles. The inventive



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method for producing said agent consists in heating the rifabutin,milk and glycol acid copolymer, D-mannitol, polysorbate 80 anddimethylsulphoxide mixture at a determined ratio to a temperature of50–60 °C, in stirring said mixture until a homogeneous transparent liq-uid is formed and in cooling said liquid to the room temperature. Theproduced suspension, with nano sized particles of 200–400 nm, afterhaving been diluted with water, can be put to good use. Said medicinalagent exhibits broad-spectrum antimicrobial activity and is non-toxic.

WO 2006/109317 [126] reveals a process for the preparation of polyDL-lactide-coglycolide nanoparticles encapsulating ATDs. A single oraladministration of PLG nanoparticles to mice could achieve the MIC forEMB (1.5 μg/mL) in the plasma, only when it was encapsulated and ad-ministered separately. This is important from the point of view of treat-ment of TB because if the drug levels are below MIC, the treatmentbecomes ineffective and may result in generating resistant strains. Infact, when PLG-NP co-encapsulating EMB along with other 3 drugswere administered to mice, EMB levels in the blood were below MICthroughout the study period. Furthermore, with reference to free EMBwhose bioavailability is considered to be 1, the bioavailability of PLG-NP-encapsulated EMB (alone) was 10.6, whereas, the bioavailability ofPLG-NP-encapsulated EMB (along with other 3 drugs) was 5.1. SinceEMB is unstable in the presence of the INH, PZA or RIF, and particularlyin the presence of INH, thus, it should not be co-encapsulated with INH.The encapsulation of EMB was not known before these inventions.

U.S. Patent 20060222716A1 [162] reported the preparation of SLNs ofSTR (131nm), RIF andmoxifloxacin (100–450nm)by adding the10–20%of water phase at 70–80 °C to molten lipid-surfactant phase containingdrug. After formation of homogenous mixture, remaining amount of thewater phase was added and stirred using rotor–stator mixer. U.S. Patent5,858,410 [163] reported pharmaceutical nanosuspensions of RIF andEMB,whichwere grounded in an air jet, in an aqueous surfactant solutionhomogenized in a piston-gap homogenizerwhichwas further reduced insize to make a nanosuspension of 800 nm. The advantage of this inven-tion was that organic solvents were avoided during preparation of thecarrier system.

U.S. Patent 6994862 [164] described liquid composition of RIF con-taining particles of 321.3 nm prepared by dissolving monoglycerideand emulsifier in organic solvent and evaporating organic solventunder nitrogen to form viscous liquid i.e. solubilizing composition towhich a solvent like ethanol, propylene glycol, polyethylene glycolwas added to prepare a homogenous mixed liquid formulation. Liquidformulationwas lyophilized using a cryoprotectant. The liquid formula-tion according to the present invention exhibited a sustained releasepattern while 80% drug was released in 5 h in case of the free drug.

WO 2013098841 A1 [165] reports a hybrid lipid-polymer nanoparti-cles of RIF and STR prepared by emulsification process. Lipid-polymer-drug dissolved in organic solvent was added to aqueous tween 80solution, sonicated for 60 s and homogenized using high-pressure ho-mogenizer. Organic solvent was evaporated under reduced pressureand suspension was concentrated. The minimum particle size attainedwas 185 nm and 223 nm in case of RIF and STR nanoparticles.

PCT Application No. PCT/IN2012/000154 [86] from our lab describedsolid lipid nanoparticles entrapping INHwith high entrapment efficien-cy of up to 69% and a process for preparing the same. A 6 times higherbioavailability was observed with INH-SLNs probably due to the bypassof its first passmetabolism. RES avoidance of INH-SLNs due to small par-ticle size (d90≤ 48.4 nm) is especially expected to reduce its eliminationfrom the body. Small size complemented with the presence of hydro-philic Tween® 80 in the prepared INH-SLNs is further expected to ap-prehend RES removal. Indian Patent Application 3356/DEL/2012 [87]from our group reported preparation of SLNs of RIF its to improve bio-availability and limit its drug interaction with INH. Latter reduced itsdegradation to ∼ 9% (from 26.50%) when present alone and to ∼ 20%(from 48.81%) when INHwas also present. While, when INHwas incor-porated into SLNs, thepercent degradation of RIFwas further reduced to12.35%. Furthermore, the degradation of INH in combination with RIF


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also reduced significantly from 13.2% to 2.7% when both the drugswere encapsulated individually within SLNs.

Indian Patent Application 3093/DEL/2012 [87] from our lab de-scribes STR loaded SLNs with entrapment efficiency of 60% and particlesize of 140.2 nm. It is claimedbyus that the nanocolloidal dispersion canbe useful in achieving successful and effective oral, intranasal or topicaldelivery of STR. The carrier system resulted in an increased bioavailabil-ity of STR in the blood and brain.

8. Conclusions

Besides quinolones and the long-acting rifamycins, no importantadditions have been made to the ATD armamentarium, since long. Al-though a number of novel anti-TB candidates are in the pipeline [154],however, the major disadvantage linked with new drug developmentincludes: immense research efforts, cost, and time; difficulty in dealingwith MDR and latent bacilli; and doubt with respect to toxicity and re-sistance [166]. By utilizing nanotechnology, synthetic and naturalpolymer-based controlled-release ATD nanomedicine formulationshave been developed, encapsulating key first-line as well as second-line ATDs. They are amore convenient alternative to “NewDrugDiscov-ery” which is time consuming (launch of any new molecule takes al-most 20–30 years), and costly (about 100 billion dollars/molecule).Packaging or remodeling of existing ATDs demonstrating significantantimycobacterial potential with low MIC, using a suitable nanostruc-tured carrier systems tends to address multiple issues like solubility,stability, permeability, drug degradation or interaction, and severe ad-verse effects. In addition, the resulting product is protectable andcommercializable because of the “newness” assigned to it. Out of theavailable choices PLG NPs present the flexibility of selecting differentroutes of administration and exhibit a sustained drug release to anextent that it is feasible to replace daily usual free-drug treatmentwith intermittent doses of NP-based drugs. The improved drug bioavail-ability and therapeutic efficacy were witnessed even at subtherapeuticdoses. In addition to a lower effective dose, the period of chemotherapycan also be shortened by use of these products. These factors are deci-sive in substantially curtailing the cost of treatment, reducing interac-tions with anti-HIV drugs, and better management of MDR-TB andlatent TB. Elaborate studies with alginate NPs have shown its advan-tages over PLG NPs in terms of simplicity of production, cost of polymer,higher drug encapsulation and loading, and better sustained release ofencapsulated drugs. However, aside from the issue of the choice ofpolymer and some key milestones yet to be achieved, nanomedicinemay be the long-sought solution for improving patient compliance inTB chemotherapy.

Further to above, lipidic nanoparticles may be even more suitabledelivery system for these ATDs. Lipidic nanoparticles would have aspecific advantage for treatment of mycobacterium infections becausethe latter have a special affinity for lipidic substrates [28]. Furthermore,mycobacteriumpossesses a special range of lipases to act on lipidic sub-strates [28]. In view of these facts, it may be assumed that lipidmatrix ofSLNs may attract mycobacterium and action of their constituent lipaseswould work to their disadvantage by the release of encapsulated ATDsin close vicinity of the organism producing amuch faster and higher an-timicrobial effect. Similar observations have beenmade for anti-fungalslike amphotericin B, where use of lipidic carriers improved its efficacyand reduced its toxicity. Furthermore, these SLNs being nano in sizemay also intercalate or enter more freely with and across the macro-phage wall so that high local concentration of drug would be presentat the seat of high occupancy of the mycobacterium.

A major breakthrough expected out of these nanoantibiotic systemsentrapping ATDs within nanocarriers is their capacity to enter into andact on the mycobacterium by a variety of mechanisms. The latterinclude endocytosis including pinocytosis encompassing their activeuptake; passive diffusion and para- and transcellular uptake intomacro-phages as well as the mycobacterium. It is thus proposed that since a



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series of transport sites are involved in the uptake and subsequent ac-tion of the nanoantibiotic therapy, hence the pathogenicmicroorganism(mycobacterium) may not be equipped to mutate at so many sites tobring about its resistance to the action of nanoantibiotics [167]. Reasonsassigned for induction of resistance of mycobacterium to these ATDs in-variably involve its decreased affinity for the latter e.g. in case of RIF[168]; or poor cytoplasmic permeability across mycobacterial cell walle.g. in case of STR [169]; and a general reduction in binding sites. Allthese factors will be promisingly addressed by use of nanocarrierswhich tend to improve the physicochemical attributes (several ofwhich seem to be responsible for their poor activity and inducedMDR) rather than the intrinsic nature of the entrapped drug. Further-more, an exposure to inadequate therapy at sublethal concentrationsfor inappropriate time usually results in development of resistantstrains, however, the cited literature studies clearly demonstrateachievement of significant concentrations (above MIC) for a prolongedperiod in the plasma and also within macrophages in some cases,when nanocarriers were used for these ATDs. Thus it may be concludedthat these nanosystems will be able to harness the antimicrobial actionof entrapped ATDs to overcome drug resistance.

It is thus opportune to conduct lab studies to evaluate the potentialof ATD loaded nanodelivery systems to penetrate into and kill the resis-tant strains. This should be followed by conduct of preclinical acute andrepeated dose toxicity studies followed by phase I and II clinical trials.

However, the nanodelivery systems require stringent evaluation ofparameters like particle size, drug leakiness and stability to aggregationor agglomeration upon storage. The scientific community is nowawaiting regulatory guidelines for their evaluation, especially in termsof stability and toxicity; former are yet missing for monitoring of suchproducts. However absence of such guidelines should not deter the in-vestors and manufacturers from filing approvals once they are able toelicit specific advantages associated with these systems. Furthermore,most of the approvals may be sought within the framework of existingguidelines.

In the past decade, the role of all-trans retinoic acid (ATRA) andcholecalciferol (vitamin D3) is being explored in TB. Administration ofthese vitamins has been considered beneficial for the treatment of TB[170]. ATRA increases the resistance of cultured human macrophagesto infection by virulent strains ofM. tuberculosis [171] while tuberculo-sis patients are reported to be deficient in vitamin D3 [172,173].

ATRA acts synergistically with vitamin D3 to inhibit Mycobacteriumentry as well as survival within macrophages, possibly through the res-cue of phagosomematuration arrest [174]. As Mycobacterium is highlyresistant and can also shift strains, treatment with antitubercular drugsmay fail at times, while, the host mediated macrophage-directed path-way, observed with ATRA and vitamin D3 can be a gunshot treatmentfor tuberculosis.

In our lab, ATRA and vitamin D3 were incorporated into SLNs to en-hance their bioavailability by presenting them as an aqueous dispersionwith high permeability and long circulation times in addition toprotecting them against photodegradation and oxidative degradation.Improved stability, including photostability, and pharmacokinetic per-formance of these vitamin loaded SLNs was confirmed by us [175,176].

The successful translation nanocarrier systems encapsulating strate-gic ATD therapy, to themarket as viable products (in terms of therapeuticbenefits, safety and commercialization), is being eyed with enthusiasmand trepidation both by the researchers and the pharma industry. Morethan that the governments of the developed and the developing coun-tries and social agencies like WHO also look at these products with ahope and anticipation.

Acknowledgments

We are thankful to the Department of Biotechnology, Governmentof India for providing financial help to carry out the research workreported from our lab. We are also grateful to Ms Parneet Kaur and


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Ms Simarjot Kaur of the University Institute of Pharmaceutical Sciences,Panjab University, Chandigarh for their valuable help in the preparationand editing of this review.

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Nanostructured drug delivery for better management of tuberculosis

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