Advances in nanotechnology-based carrier systems for targeted delivery of bioactive drug molecules with special emphasis on immunotherapy in drug resistant tuberculosis – a critical review

Abstract

From the early sixteenth and seventeenth centuries to the present day of life, tuberculosis (TB) still is a global health threat with some new emergence of resistance. This type of emergence poses a vital challenge to control TB cases across the world. Mortality and morbidity rates are high due to this new face of TB. The newer nanotechnology-based drug-delivery approaches involving micro-metric and nano-metric carriers are much needed at this stage. These delivery systems would provide more advantages over conventional systems of treatment by producing enhanced therapeutic efficacy, uniform distribution of drug molecule to the target site, sustained and controlled release of drug molecules and lesser side effects. The main aim to develop these novel drug-delivery systems is to improve the patient compliance and reduce therapy time. This article reviews and elaborates the new concepts and drug-delivery approaches for the treatment of TB involving solid-lipid particulate drug-delivery systems (solid-lipid micro- and nanoparticles, nanostructured lipid carriers), vesicular drug-delivery systems (liposomes, niosomes and liposphere), emulsion-based drug-delivery systems (micro and nanoemulsion) and some other novel drug-delivery systems for the effective treatment of tuberculosis and role of immunomodulators as an adjuvant therapy for management of MDR-TB and XDR-TB.

• Carbon nanotubes
• immunomodulators
• nanostructured lipid carriers
• quantum dots
• tuberculosis

Introduction

Tuberculosis (TB) is a major cause for billions of active TB cases and deaths across world. However, there are effective and cheap treatments are available by the government and non-Government health care authorities (Meena, 2010). Over five decades of various TB treatment programs, still the potentially effective drugs for TB have failed to reduce the prevalence of the disease. TB is mainly caused by rod shaped microorganism Mycobacterium tuberculosis. TB is an airborne infection which is a major cause of mortality and morbidity. Disease mainly affects the peoples in both developing as well as in developed countries (Zumla et al., 2013). Mostly, patients having active pulmonary TB may have mild/excessive coughing, fever, fatigue, loss of appetite, weight loss, night sweats, bloody sputum (Jeong & Lee, 2008). It is estimated that more than 200 million peoples will be affected by this disease and that expected 35 million peoples will die due to disease in this decade if effective control and preventive measures are not taken by the concerns authorities (Meena, 2010). Further the emergence of human immunodeficiency virus (HIV) and multidrug resistant (MDR)/extremely drug resistant (XDR) TB poses a major challenge to the control it across the world (Sarkar & Suresh, 2011). There are several modes of transmission of causative agents from infected persons to un-infected persons. In general, infection spreads by forming droplets by an infected person during coughing, sneezing, speaking may transmit to the air. TB is generally classified as latent TB or active TB. The latent phase of TB in which bacterium is present inside human body as inactive form without showing any kind of symptoms even shows negative result on a Mantoux tuberculin test. In the active phase, the host’s immune system becomes weakens and the bacterium becomes active and cause some secondary complications (Gupta et al., 2015).

Pathogenesis of tuberculosis

Mycobacterium tuberculosis is anaerobic, non-spore-forming, non-motile rod shaped bacteria which is highly resistant to alcohol and acid. Its mode of transmission is mainly from one infected person to another person via droplet nuclei in air due to coughing. When this mycobacterial agent reaches to the alveolar region in lungs, it invades and replicates within alveolar macrophages. Alveolar macrophages immediately take them, where they undergo phagocytosis. Further mycobacteria interacts with T lymphocytes which results in the formation of differentiated macrophages into the histocytes and epithelioid (Chaudhary & Garg, 2015). These epithelioid histocytes and lymphocytes aggregates into different small clusters and form granulomas. In the granulomas, CD4 cells T Lymphocytes which is also called as effector T cell, secretes different kinds of cytokines such as interferon-γ which further activates the macrophages in order to destroy the bacteria with which they are infected previously. CD8 T-Lymphocytes (cytotoxic T cells) also directly kills the infected cells (Jeong & Lee, 2008). Pathogenesis of tuberculosis infection is shown in Figure 1.

Figure 1. Pathogenesis of tuberculosis.

Drugs for treatment of tuberculosis

Treatment of active TB is very complex due to the emergence of multidrug-resistant (MDR-TB) and extremely drug-resistant (XDR-TB). There are only 10 drugs approved by the US Food and Drug Administration for TB treatment. In India, available TB treatment is directly observed therapy (DOTS) which comprises the administration of four oral antibiotics for the period of 6 months or more. TB chemotherapy initially started with the discovery of streptomycin in 1944 only a very small fraction of anti-tubercular drugs reaches the alveoli which are the desired target of drug action. Some other drugs used in TB treatment include clofazimine (having direct anti-tubercular and immune-suppressive properties), amoxicillin (which inhibits the synthesis of the cell wall), rifabutin (which inhibits the synthesis of bacterial RNA) and long-acting rifamycins and inhaled interferon-gamma, clarithromycin (which inhibits the synthesis of proteins). Major drugs used for TB treatment is listed in Table 1.

Table 1. Classification of anti-TB drugs and their mechanism of action.

Drug	Mechanism of action	Adverse or side effects	Role	BCS class
Isoniazid (INH) (1951)	It inhibits the synthesis of mycolic acid (an essential cell wall component) thus interferes with cell wall synthesis in mycobacterium.	Hepatitis, burning sensation in extremities, drug interaction, fatigue, fever etc. (Sensi, 1983; Kochi et al., 1993)	As a pro-drug conversion and as a drug target product (Zhang & Yew, 2009)	BCS-III class (Hari et al., 2010)
Rifampicin (RIF) (1966)	Inhibits the synthesis of bacterial RNA polymerase thus inhibits the nucleic acid synthesis.	Stomach upset, flu-like symptoms, bleeding (Sensi, 1983). Rashes and other hypersensitivity reactions, hepatitis-1 may also appear (Wade & Zhang, 2004).	As a drug target (Zhang & Yew, 2009)	BCS-II class (Hari et al., 2010)
Ethambutol (ETB) (1961)	Inhibits bacterium arabinogalactan synthesis and finally cell wall synthesis get inhibited.	Chances of visual disturbances. (Mikusová et al., 1995)	As a drug target (Zhang & Yew, 2009)	BCS-III class. (Hari et al., 2010)
Pyrazinamide (PYZ) (1952)	Depletes and inhibit the membrane energy.	Hepatitis, joint aches rashes and other hypersensitivity reactions. (Zhang et al., 2003)	As a drug target and as a pro-drug conversion (Zhang & Yew, 2009)	BCS-III class. (Hari et al., 2010)
Aminoglycosides Streptomycin (1944), kanamycin (1957), capreomycin (1960), amikamycin (1957)	Inhibition of protein synthesis irreversibly	Nephrotoxicity, ototoxicity, electrolyte abnormalities like hypokalemia etc. (Allen et al., 1983)	As a drug target (Zhang & Yew, 2009)	Amikacin-III BCS class. Kanamycin-Na (Hari et al., 2010)
Ethionamide (1956)	Inhibition of mycolic acid synthesis	Gastrointestinal upset, hepatotoxicity, neurotoxicity and anorexia etc. (Morlock et al., 2003)	As a pro drug conversion and drug target (Zhang & Yew, 2009)	BCS-II class. (Hari et al., 2010)
Amino salicylic acid (1946)	Inhibits the incorporation of PABA into folic acid and iron metabolism.	Gastrointestinal distress, hypothyroidism and other hypersensitivity reactions (Wade & Zhang, 2004).	Drug activation (Zhang & Yew, 2009)	Na (Hari et al., 2010)
Cycloserine	Inhibits the incorporation of d-alanine into peptidoglycan	CNS toxicity, Stevens–Johnson syndrome etc. (Caceres et al., 1997)	Drug target (Zhang & Yew, 2009)	BCS-IV/II class (Hari et al., 2010)

Nanotechnology-based drug-delivery systems in tuberculosis

Nanotechnology-based drug-delivery systems are mainly consist of different formulations, each of them contains different functional and structural properties (Garg et al., 2015b). They are able to modify easily by varying or changing the composition of drug, polymers, excipients and other additives used in their preparation in order to reduce the problems associated with conventional drug-delivery systems (Garg et al., 2015c) which are shown in Figure 2.

Figure 2. Problems with conventional drug delivery systems.

As the use of nanosized drug-delivery systems is widely accepted and commercially applicable (Garg et al., 2015e). In general, most of the drug-delivery systems are used as drug carrier systems due to their high stability, high carrier-loading capacity and ability to incorporate both lipophilic/hydrophilic drug substances and due to feasibility of administration through different kinds of routes like oral, topical, parenteral and pulmonary routes (Garg et al., 2015a; Kaur et al., 2015a). The advantages of nanotechnology-based system are shown in Figure 3. They can also be designed for modified drug release from different carriers systems (Malik et al., 2015). Nanotechnology-based drug-delivery systems are broadly classified into four groups including the solid-lipid particulate dosage forms, emulsion-based systems, solid-lipid tablets and vesicular systems (Salome & Ikechukwu, 2013).

Figure 3. Advantages of nanotechnology-based drug delivery systems.

These formulations provide increased drug solubilization for water-insoluble drugs (Chaudhary et al., 2015b). Further the drug absorption is observed to be better than other conventional systems of drug delivery which might be due to the property of wetting of the hydrophobic drug particles in the presence of polymer matrix and presence of surfactants (Chaudhary et al., 2015a). Therefore, these drug-delivery systems represent diverse group of formulations comprising several classes of excipients for example triglyceride oils, mixed glycerides, surfactants like lipophilic surfactants, hydrophilic surfactants and water-soluble co-solvents with different kinds of properties (Salome & Ikechukwu, 2013). The nanotechnology-based drug-delivery systems are shown in Figure 4.

Figure 4. Classification of nanotechnology-based drug delivery systems.

Solid–lipid particulate dosage forms

Solid–lipid micro and nanoparticles

In general, the colloidal particles in 10–1000 nm size range are commonly referred as nanoparticles (Momoh et al., 2013; Garg et al., 2014b). The successful use of nanoparticles in different drug-delivery systems depends on their different physicochemical properties and their ability to cross through several anatomical barriers inside body (Rohilla et al., 2014b; Pandey et al., 2015). They are effective in sustained drug release of entrapped or encapsulated drug contents (Pardeshi et al., 2013; Chaudhary et al., 2014). Solid-lipid micro/nanoparticles often shows interesting features concerning about therapeutic purposes (Jawahar et al., 2012; Morie et al., 2014). Solid-lipid nanoparticles were introduced in the early 1990s as an alternative drug-delivery system to other available traditional colloidal carrier systems such as liposomes, niosomes, emulsions and polymeric nanoparticulates (Sharma et al., 2014a; Weber et al., 2014). The hopeful strategy to overcome various problems with conventional drug-delivery system is to develop suitable drug carrier system like solid-lipid nanoparticles (Pallerla & Prabhakar, 2013). Solid-lipid micro/nanoparticles are aqueous colloidal dispersions comprises of melted biodegradable lipids in water or in aqueous solution of surfactant and size ranging between 50 and 1000 nm (Jeong & Lee, 2008; Singh et al., 2014a). Solid-lipid nanoparticles is an emerging field of nanotechnology with potential applications in various fields such as in effective drug delivery (Singh et al., 2014b), clinical medicines (Singh et al., 2012), cosmeceuticals (Garg et al., 2012b) and as well as in research (Wang & Wu, 2006; Garud et al., 2012). Solid-lipid nanoparticles containing lipid concentrations up to 2.5% do not show any cytotoxic effects in vitro (Jawahar et al., 2012). Commonly used lipids are triglycerides, fatty acids, partial glycerides, fats and waxes (Guo et al., 2012). The biggest advantages of solid-lipid nanoparticles is the use of lipids in the preparation are generally physiological lipids which have the less chances of acute/chronic toxicities (Weber et al., 2014). Lipophilic drugs are better suited for solid-lipid nanoparticles (Garud et al., 2012). Further, the use of solid-lipid instead of liquid-lipid in their preparation is more beneficial in order to control the release kinetics of encapsulated drug molecules and to improve the stability of active ingredients (Garg et al., 2013; Weber et al., 2014). SLNs mainly possess a central solid–lipid core matrix that can solubilize lipophilic molecules stabilized by use of different surfactants (Acevedo-Morantes et al., 2013). The melting point of lipids used must exceed body temperature (Alam et al., 2013). Biological membrane lipids used in solid-lipid micro/nanoparticles are lecithin or soya lecithin, biocompatible non-Ionics lipids such as ethylene oxide or propylene oxide copolymers (Swathi et al., 2012; Goyal et al., 2013).

Various advantages of solid-lipid nanoparticles are that they are effective in drug-targeting purposes (Seyfoddin et al., 2010). It have high drug stability (Johal et al., 2014), high drug-loading efficiency or high percentage encapsulation efficiency (Joshi et al., 2014a), ability to encapsulate both lipophilic and hydrophilic drug substances (Kaur et al., 2014c), less or no use of organic solvents (Liu et al., 2009), increased bioavailability of entrapped bioactive compounds (Goyal et al., 2014a), less biotoxicity of carrier system and better protection of incorporated labile compounds (Dong et al., 2011). Pandey et al. (2005) developed SLNs, which was prepared with an encapsulation efficiency for rifampicin (51%), pyrazinamide (41%) and (45%) for isoniazid, after a single oral administration to mice. Therapeutic drug concentrations were maintained in the plasma for 8 days and in the different organs lungs, liver and spleen for 10 days where as the free drug solution were found cleared in 1–2 days (Pandey et al., 2005). Different examples of solid-lipid micro/nanoparticles in tuberculosis treatment are shown in Table 2.

Table 2. Examples of solid-lipid micro and nanoparticles in tuberculosis treatment.

Polymer used	Anti-TB drug used	Results obtained	References
Stearic acid and sodium taurocholate	Rifampicin	Good release kinetics was found and good flow properties of dry powder of rifampicin SLNs.	Maretti et al. (2014)
Compritol ATO 888, Tween 80	Rifampicin	The percentage degradation in combination of rifampicin solid-lipid nanoparticles and isoniazid solid-lipid nanoparticles was found decreased up to 12.35%	Singh et al. (2013)
Soya lecithin, compritol ATO 888, stearic acid, Tween 80	Isoniazid	In this study, the prepared SLNs give high entrapment efficiency (68%) and small size (48 nm) for isoniazid loaded SLNs. The marked improvement in bioavailability in both in plasma and brain was observed after giving isoniazid loaded SLNs and free/plain drug solution at the same dose.	Bhandari & Kaur (2013)
Cetyl palmitate, Tween 80, poloxamers 188	Rifampin	Rifampicin loaded SLNs were found spherical in shape with diameter up to 100 nm, low and negative zeta potential and encapsulation efficiency up to 82% and the anti-mycobacterial activity was found improved against Mycobacterium fortuitum. MIC of drug loaded SLNs were found to be eight times fewer than free rifampin solution administered.	Aboutaleb et al. (2012)
Phospholipon R 80 H, tristearin	Isoniazid	The prepared SLNs were of spherical in shape with diameter of 164 nm. Further the use of polysorbate80 and sonication time was found to be effective for entrapment efficiency and for reduction in size of particles.	Nair et al. (2011)
Mannose	Rifabutin	In this particular study, the cellular uptake studies was done which shows due to mannose coating, SLNs formulations for alveolar macrophages are better suited when compared to un-coated formulation (without mannose).	Nimje et al. (2009)

Nanostructured lipid carrier

The colloidal drug carrier system not only offers many advantages in the case of targeted drug delivery (Luan et al., 2013) but also increase bioavailability of some poorly soluble drugs and protection for sensitive active compounds (Wang & Xia, 2014). As nanostructured lipid carriers (NLCs) were developed in the midlines of the 1990s as an alternative carrier system to the existing traditional carriers (Kamble et al., 2012). NLCs are the second generation of SLNs (Wang et al., 2013). In order to obtain the blends of particles, matrix mainly solid lipids are mixed with melted lipids in a ratio of 70:30 (Jia et al., 2010). They can also incorporate both lipophilic and hydrophilic drugs under the optimized conditions (Zheng et al., 2012). NLCs can be produced by high-pressure homogenization or any other modified techniques to yield more lipid particulate dispersions with solid contents 30–80% (Patidar et al., 2010). They can be administered via different routes such as oral, ocular, pulmonary and intravenous route (Parhi & Suresh, 2012). NLCs mainly accommodates drug due to their highly unordered lipid structures (Kamble et al., 2012). Song et al. (2015) developed nanostructured lipid carrier to encapsulate rifampicin. The particle size was found around 160 nm. The rifampicin was found to be entrapped above 90% within the system and high uptake was observed in NR8383 cells and alveolar macrophages (Song et al., 2015).

Emulsion-based drug-delivery systems

Microemulsion

Microemulsions are mainly clear, transparent, thermodynamically stable, isotropic dispersions composed of an aqueous component and oily component (Nikumbh et al., 2015). Microemulsion also contains emulsifying agents, surfactant or co-surfactant mixtures (Kaur et al., 2014b). Microemulsion improves drug solubilization, absorption and permeability (Jain et al., 2009; Mostafa et al., 2014). It also provides protection against enzymatic hydrolysis (Hu et al., 2011). The selection of suitable surfactant combinations is a critical parameter in order to make thermodynamically stable emulsion (Wang et al., 2009). Establishment of a phase diagram is also necessary for the selection of multiple components in microemulsions (Narang et al., 2007). Agatonovic-Kustrin et al. (2003) formulated a colloidal dosage form for oral delivery of isoniazid and rifampicin using artificial neural network (ANN) data modeling, which helps to understand the basic process of microemulsion formation and their stability within pseudo-ternary and ternary colloidal systems. The solubility behavior of two combined drugs and stability can be predicted based on the separation of two drugs into oil and water phases depending upon their solubility (Agatonovic-Kustrin et al., 2003).

Nanoemulsion

As emulsion is a biphasic dosage form with wider range of applications (Mahajan et al., 2014). In nanoemulsion, one phase is usually dispersed in another phase in which it is usually immiscible (Devarajan & Ravichandran, 2011). Nanoemulsion can be prepared by different methods, but the homogenization method is most commonly adopted (Kotta et al., 2015). In general, the term “nanoemulsion” is a system which is thermodynamically stable and isotropically clear dispersion of two immiscible liquids such as water and oil which are usually stabilized by use of surfactant molecules in different proportion (Thakur et al., 2013). In the last few decades, these o/w nanoemulsions found enormous application in various fields like healthcare, cosmetics, foods, agrochemicals, pharmaceuticals and biotechnology (Setya et al., 2013). Nanoemulsions can be defined as emulsion with mean droplet diameters size ranging from 50 to 1000 nm (Bhatt & Madhav, 2011). Nanoemulsions contains oil phase, aqueous phase, emulsifying agent and active pharmaceutical ingredients such as drugs or diagnostic agents (Abolmaali et al., 2011). The surfactants and co-surfactants helps to form a thermodynamically as well as kinetically stable nanoemulsion (Kumar & Singh, 2012). Nanoemulsion can be prepared readily and spontaneously without high-energy inputs. In general, emulsions are cloudy but the nanoemulsions have translucent and clear appearance. Nikonenko et al. (2014) developed phospholipid-based nanoemulsion system of SQ-641-NE which was found to be active against Mycobacterium tuberculosis. Different examples of anti-tubercular drug loaded emulsion systems are given in Table 3.

Table 3. Examples of anti-tubercular drug loaded emulsion systems.

Polymer used	Anti-TB drug used	Results obtained	References
Brij 96, nile red, ruthenium dichloride	Rifampicin, Isoniazid, Pyrazinamide	The micro emulsion has been tested using different dye solutions. It was found that rifampicin has strong interaction with Nile red dye and pyrazinamide/isoniazid has interaction with ruthenium dichloride.	Kaur & Mehta (2014).
Oleic acid, ethanol, Tween 80	Rifampicin	An optically clear/stable microemulsion was obtained without phase separation. The phase changing between w/o microemulsion to o/w microemulsion and controlled/sustained release of the drug was achieved.	Mehta et al. (2007)
Oleic acid, ethanol, Tween 80	Rifampicin	In this method, the microemulsion-based system composed of oleic acid, Tween 80, ethanol and phosphate buffer was developed. A stable microemulsion was found after incorporating rifampicin in terms phase separation. The microemulsion get changes into o/w emulsion at infinite dilution conditions as depicted by particle size.	Mehta et al. (2007)
Sefsol, Tween 80	Rifampicin	In this study, different oil-in-water (o/w) nanoemulsions were prepared. The optimized formulation was also subjected further to stability studies as per the ICH guidelines. The formulation was found stable more than period of 19 months.	Ahmed et al. (2008)
Oleic acid, ethanol, Tween 80	Isoniazid	In this study, the microemulsion was found stable after the incorporation of isoniazid in terms of optical texture, phase separation and pH, as far as dissolution studies are concerns the sustained/controlled release of drug from o/w emulsion droplet was found.	Mehta et al. (2008)
Oleic acid, ethanol, Tween 80	Rifampicin, Isoniazid, Pyrazinamide	In this study, the degradation of rifampicin in the presence of isoniazid was seen. The results depicted that anti-tubercular drugs in single and mixed drug formulations was followed non-Fickian release behavior except for rifampicin in pH 7.4 release medium.	Mehta et al. (2010)

Vesicular drug-delivery systems

Liposome

Liposomes are the concentric bilayered vesicle in which an aqueous volume is enclosed by lipid bilayer membrane (Mansoori et al., 2012). Liposomes were discovered in an accidental discovery by Dr Alec. D. Banghamam, a British hematologist in 1961. When he dispersed phosphatidyl choline in water, it forms a closed bilayer-type structure which contains an aqueous phase inside which was entrapped by outer lipid bilayer (Thulasiramaraju et al., 2012). The name liposome is derived from two Greek words Lipos meaning fat and Soma meaning body (Mansoori et al., 2012). Liposome can be defined as “simple microscopic vesicles in which an aqueous volume is entirely enclosed by a membrane composed of lipid molecule” (Anwekar et al., 2011). They are able to encapsulate both hydrophilic and lipophilic drug substances (Utreja et al., 2011). It can also be used as a non-toxic vehicle to encapsulate insoluble drugs also (Patel & Panda, 2012). The size range is usually in 0.05–5.0 μm in diameter (Mansoori et al., 2012). They can be formulated and processed in different size, composition, charge and lamellarity (Patel & Panda, 2012). The wide range of applicability of liposomes makes them more advantageous (Garg, 2014). Liposome act as a vehicle to deliver the active therapeutic substances like proteins, peptide, antifungal, immunomodulators, diagnostics, enzymes, vaccines, cancer, ophthalmic, genetic material etc. (El-Badry et al., 2014). Liposomes are also effective in loading of water-soluble drugs in central aqueous compartment and lipid soluble agents in outer membrane (Paliwal et al., 2015). Due to the advancements in the methods of preparing and formulating liposomes, high-entrapment efficiencies can be made possible to incorporate different types of drugs into them (Kulkarni et al., 2011). Based on the structure, they are classified as unilamellar vesicles (ULVs) and multi-lamellar vesicles (MLVs). There are various advantages of liposomal system as a drug-delivery system such as they are biocompatible, biodegradable, non-toxic, flexible and non-immunogenic for systemic and non-systemic administrations, acts as a reservoir of drug and therapeutic index of drug may increase and modulates the distribution of drugs (Nasr et al., 2013). Basic component of liposomes are phospholipids which include natural phospholipids like phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, synthetic phospholipids such as di-palmitoyl-phosphotidyl-choline, di-stearoylphosphotidylcholine, di-palmitoylphosphotidylglycerol, di-palmitoylphosphotidylserine, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) are mainly used in liposomes. Di-oleaylphosphotidylcholine is also used as unsaturated lipidic phase (Rahman et al., 2012). Sphingolipids like Sphingomyelin and glycosphingolipids like gangliosides can also be employed as lipidic phase. Liposomes are mainly taken up by the phagocytic cells following the mechanisms like surface adsorption upon cell membrane, vesicular internalization, lysosomal fusion with endocytic vesicles and enzymatic degradation by lysosomal enzymes. After degradation, they release the encapsulated drug within phagocytic cells (Rojanarat et al., 2012). Liposomes are effective against intracellular pathogens as they can reach deep in lungs. Uptake and recycling of liposomal phospholipids occurs through the alveolar type II cells. Liposomes avoid decomposition of the entrapped drug molecules and release the entrapped drug molecules at designated targets in a controlled manner (Akbarzadeh et al., 2013). Liposomes are already developed for controlled and sustained delivery of various anti-TB drugs, proteins and peptides. Different examples of liposomal-based system in tuberculosis are given in Table 4.

Table 4. Examples of liposomal-based preparations for tuberculosis.

Lipid used	Anti-TB drug used	Results obtained	References
Di-palmitoyl phosphatidyl choline (DPPC)	Isoniazid, Rifampicin, Pyrazinamide, Ethambutol	In this study, the liposomal formulation shows high entrapment efficiency, effective sustained/prolonged drug release, improved recovery/uptake of drugs from affected/target site.	Bhardwaj et al. (2013).
Soybean phosphatidylcholine, porous mannitol, cholesterol	Pyrazinamide	Pyrazinamide loaded pro-liposomes were developed and 4.26–4.39 µm aerodynamic diameter and up to 45% encapsulation efficiency was observed.	Rojanarat et al. (2012)
Di-palmitoyl phosphatidyl choline (DPPC)	Isoniazid	Controlled/sustained release of isoniazid from liposomes was observed over period of 1 day.	Chimote & Banerjee (2010)
DNA-hsp65 vaccine	DNA-hsp65 vaccine	The study shows that vaccine can be entrapped or complexed within liposomal system and can be used to immunize the animal (mice) by intra-nasal or intra-muscular routes.	Rosada et al. (2008).
Dipalmitoyl phosphatidyl choline, Dipalmitoyl phosphatidyl glycerol	Rifabutin	Significant decline in CFUs count was measured/observed in organs like lungs, liver etc. when compare to free/plain rifabutin in infected mice.	Gaspar et al. (2008)
Di-palmitoyl phosphatidyl choline, cholesterol	Pyrazinamide	High therapeutic efficacy was observed in infected mice when pyrazinamide loaded liposomes were injected twice weekly.	El-Ridy et al. (2007)
Phosphatidyl choline, cholesterol, dicetyl phosphate	Isoniazid, Rifampicin	The loading capacity of rifampicin was found better as compared to the loading of isoniazid in liposomal system.	Pandey et al. (2004)
Di-palmitoyl phosphatidylcholine (DPPC), hydrogenated phosphatidylcholine (HPC) and distearoyl phosphatidyl choline (DSPC)	Capreomycin	Capreomycin loaded liposomes were found suitable for inhalable formulations as with diameter of 200 nm or less. The liposome was found to provide the high capreomycin sulfate content.	Ricci et al. (2006)
Di-stearoyl phosphatidyl choline and cholesterol	Kanamycin	In this study, the encapsulation efficiency up to 63% was obtained for kanamycin. The lipidic vesicles were found with mean diameter of at least 132 nm.	Justo & Moraes (2005)
Di-stearoyl phosphatidyl choline and cholesterol	Isoniazid, Rifampicin, Ethionamide, Pyrazinamide, Streptomycin	In this study, mean vesicle diameters were found varied from 286 to 329 nm. No drug leakage or mean vesicle diameter changes were observed for 3 weeks.	Justo & Moraes (2003)
Phosphatidyl choline, cholesterol, dicetyl phosphate	Isoniazid, Rifampicin	In this study, the liposomal formulation encapsulating rifampicin and isoniazid for at least 6 weeks was found to reduce the mycobacterial load in lungs and other organs of infected mice when compared with untreated animals after administered once in a week.	Labana et al. (2002)
Phosphatidyl choline and phosphatidyl serine	Rifabutin	In this study, the rifabutin was incorporated in liposomal system which results in significant enhancement of its anti-tubercular activity against M. avium infection when compared to rifabutin in free form or plain solution.	Gaspar et al. (2000)
Bristol	Amikacin	In this particular study, the liposomal entrapment of amikacin was found high. The minimum inhibitory concentration (MICs) for M. avium was found for at least 28 days.	Leitzke et al. (1998)
Egg phosphatidyl choline, cholesterol, dicetyl phosphate, O-SAP and monosialogangliosides, di-stearyl phosphatidyl ethanolamine-poly(ethylene glycol)	Isoniazid, Rifampin	In this study, biodistribution studies shows that liposomes containing O-SAP were 15% accumulated in lungs when compared to conventional liposome which was 5.1% accumulated in lungs preparation after 30 min of injection.	Deol & Khuller (1997)
Phosphatidyl glycerol, phosphatidyl choline, cholesterol	Sparfloxacin	In this method, treatment with free or sparfloxacin loaded liposome for 24 h was studied in animals, which shows reduction in the growth index up to 25% for untreated group and 30% for control groups. Free sparfloxacin reduced the growth index up to 6% of the untreated control group, while liposome-encapsulated sparfloxacin was found to reduce it up to 8% to that of the control group when cultures were treated for 4 days.	Düzgüneş et al. (1996)
Poly ethylene glycol, distearoyl phosphatidyl ethanolamine, distearoyl phosphatidyl choline, cholesterol, phosphatidylinositol, phosphatidyl glycerol, phosphatidyl choline	Streptomycin	In this study, liposomal system was prepared which encapsulates streptomycin. They were found to reduce the level of MAC (Mycobacterium avium complex) infection in high level when compared to MAC infection in un-treated (Control groups) and they were also found bacteriostatic in lungs.	Gangadharam et al. (1995)

Niosome

Among all types of carrier systems liposome and niosomes are very well documented in controlled drug-delivery system (Mujoriya et al., 2011). Niosomes have potential as a carrier system for the delivery of different types of drugs, antigens and hormones (Gopalkrishnan & Chenthilnathan, 2012). The first report or idea on the use of certain non-ionic surfactants vesicles was from the cosmeceuticals prepared by L’oreal (Chandu et al., 2012). They are usually bi-layered structures which are able to encapsulate both hydrophilic and lipophilic drug molecules either in aqueous layer or in lipidic vesicular membrane (Abdelkader et al., 2014). In niosomes, non-ionic surfactant such as span-60, span-80 can be used because they can be stabilized by cholesterol and some other lipid addition (Madhav & Saini, 2011). They behave as liposomes as they help to prolong the blood circulation of entrapped drug moieties/molecules (Mullaicharam & Murthy, 2004). The materials used to prepare niosomes are cheaper and make them more stable, thus niosomes have more advantages over the liposomes (Diljyot, 2012). Usually, the niosomes are microscopic in size (Gandhi et al., 2014). Niosomes also provide therapeutic activity in a well-controlled manner for a definite period of time (PravinaGurjar & Chouksey, 2014). They are also chosen over liposomes due to high-stability and more economical (Madhav & Saini, 2011) characteristics. Niosomes are now favorably used for controlled and targeted drug delivery, as the “Paul Elrich” in 1909, gives the concept of Drug-targeting and nanotechnology (Sankhyan & Pawar, 2012). Niosomes also helps to overcome the problems associated with liposomal system such as susceptibility to oxidation, high cost and difficulty in procuring high quality/purity levels. In noisome, hydrophobic drug molecules can be encapsulated inside surfactant bilayer and hydrophilic drug molecules inside the lipidic vesicle (PravinaGurjar & Chouksey, 2014). Therefore, niosomes are a promising carrier system for drug delivery being non-ionic, less toxic and it also improves the therapeutic index of drug molecules by restricting its action only towards target cells and tissues (Mullaicharam & Murthy, 2004). The advantages of niosomes as a drug-delivery system are like the surfactants used in niosomes are generally biodegradable, biocompatible and non-immunogenic, osmotically more active and stable, increase the oral bioavailability of drugs, improves the therapeutic performance of the drug molecules by delayed its clearance from the circulation or organs, no special storage conditions required, can be administered by oral, parenteral as well as topical routes and they are cost effective as compared to liposomes (Sharma et al., 2012). Basic components of niosomes are same as used in liposomes except the use of non-ionic surfactants. Different niosomal systems for incorporating anti-tubercular drugs are listed in Table 5.

Table 5. Examples of niosomes preparations in tuberculosis.

Polymers used	Anti-TB Drug used	Results obtained	References
Cholesterol, dicetyl phosphate, stearyl amine	Ethambutol	The drug-loading efficiency of ethambutol was found increased from 12.20% to 25.81% and the zeta potential was found up to neutral values. Drug targeting towards lungs was also got increased by niosomal system.	El-Ridy et al. (2015)
Sorbitan monostearate, cholesterol, dicetyl phosphate diethyl ether	Isoniazid	In this study, drug entrapment efficiency gets increased up to an optimum level by enhancing the cholesterol content and there after decrease in encapsulation efficiency was found. Study also proves that isoniazid loaded niosomes are capable of providing reduced drug dose as well as dose frequency. The reduced toxicity was observed when the formulation was given through parenteral route or via any other suitable routes	Singh et al. (2011)
Cholesterol, dicetyl phosphate, stearyl amine, span 60 and span 85	Pyrazinamide	In this study, the highest percentage of pyrazinamide-loading efficiency was found using surfactant span-60.	El-Ridy et al. (2011)
Triton X 100, polyethylene glycol 2000, span 80	Rifampicin, Isoniazid	In this method, the prepared niosomal particles was found in range of 200–300 nm size and high drug entrapment efficacy was found for rifampicin and isoniazid.	Mehta et al. (2011)
Cholesterol, triton-X-100, diethyl ether, span-80 and span-85	Rifampicin	This study depicts that effective targeting of the rifampicin mainly happens in the lymphatic regions. Rifampicin was entrapped in niosomes successfully and was found useful for treatment/management of disease conditions.	Jain et al. (2006)

Lipospheres

Lipospheres formulation is a aqueous microdispersion of water-insoluble solid spherical particles with size range between 0.01 and 100 μm in diameter (Elgart et al., 2012). The lipospheres or lipidic microspheres are generally made up of solid hydrophobic triglycerides with outer monolayer of phospholipids embedded on the surface (Islan & Castro, 2014). Liposphere formulation is better suited for oral, parenteral and topical drug-delivery system (Barakat & Yassin, 2006; Rawat et al., 2008). The solid core in liposphere is generally used to dissolve or dispersed the drug molecules in a solid fat matrix. Several techniques such as solvent evaporation, hot and cold homogenization and high-pressure homogenization have been used for the production of liposphere (Elgart et al., 2012). Takenaga et al. (2008) developed lipid microsphere to encapsulate rifampicin. The particle size was found around 247 nm and their size remained stable on different temperature (storage conditions) such as at 4 or 25 °C for at least 4 weeks period (Takenaga et al., 2008).

Miscellaneous lipid-based drug-delivery systems

Dendrimer

Dendrimers are emerging class of polymeric materials which are highly branched, mono-disperse macromolecules. The dendrimer systems have been found impactive on the physical and chemical properties of drug substances (Jana et al., 2012). Due to the multivalent and mono-disperse nature of dendrimers, they are emerging field of chemistry and biology especially in drug delivery, gene therapy and chemotherapy (Doshi, 2011). Dendrimer chemistry was first introduced in by Fritz Vogtle in 1978 (Jana et al., 2012). The word dendrimer derived from Greek word Dendron means tree and meros meaning part, so they are highly branched polymeric system. In general, dendrimers are synthetic nanomaterials that lie between 2 and 10 nm in diameter with defined molecular weights. Large number of functional groups can be attached on the external surface of dendrimers (Doshi, 2011). Dendrimers are known to have well-established entrapment properties. The dendrimer surface contains many different sites to which drug substances, ligands can be attached as well as materials such as polyethylene glycol (PEG) etc. (Mutalik et al., 2014). These kinds of attachments are used to modify the structure of dendrimers so that it can interact with the target receptors (Kaur et al., 2015b). Dendrimers generally have large interior void space which may be used to encapsulate or incorporate small drug molecules which help to reduce their toxicity and facilitate its controlled or targeted drug release (Thomas et al., 2008). Dendrimer solution has lower viscosity than the linear polymers (Kumar et al., 2015). The presence of many chain-ends or functional groups in dendrimers is responsible for high solubility, reactivity and miscibility (Sharma et al., 2015). The solubility of dendrimers can also influenced by the nature of groups attached on surface (Jana et al., 2012).

Dendrimers are significant as they have unique structural features (Garg et al., 2015d). Large numbers of functional groups are available at the surface of dendrimers for drug substance and ligand attachment which makes them more specific in targeting (Kaur et al., 2015). As the generations of dendrimer increases, the more functional groups can be attached at the branching sites (Thomas et al., 2008). Table 6 shows the use of dendrimer-based anti-tubercular preparations.

Table 6. Dendrimer-based anti-tuberculosis preparations.

Polymer used	Anti-TB drug used	Results obtained	References
Poly ethylene glycol, poly propyleneimine	Rifampicin	In this study, it was found that dendrimer-based system helps to increase the drug encapsulation capacity and reduce the drug release rate etc. The systems were found more suitable for sustained/controlled delivery of rifampicin in tuberculosis treatment.	Vijayaraj Kumar et al. (2007)
Poly ethylene glycol, poly propyleneimine	Rifampicin	In this study, rifampicin loaded dendrimers were found to reduce release rate of drug in 7.4 pH. The cytotoxicity/hemolytic toxicity were also found reduced. The dendrimer-based system was found effective in delivery of rifampicin in tuberculosis treatment.	Kumar et al. (2006)

Nanoparticles/microparticles/polymeric nanoparticles/microspheres

Polymeric nanoparticles are well known to have good biocompatible/biodegradable features (Gagandeep et al., 2014; Kaur et al., 2014e). They are more sustainable and suitable candidates as drug-delivery carriers (Li et al., 2009; Banyal et al., 2013). Generally, the polymeric nanoparticles can be made structurally more stable by selecting different kinds of polymers, different surfactants and organic solvents (Cekié et al., 2007; Ohashi et al., 2009; Kaur et al., 2014f). They can be easily synthesized with various desirable properties like drug release, particle size/shape and zeta potential (Verma et al., 2008; Garg et al., 2014c). Polymeric nanoparticles usually have different active/functional groups depending upon polymers used and they can easily transform themselves according to either structural moiety of drugs or ligands attached (Shi et al., 2015). Therefore, they are more suitable as drug-targeting candidate (Banyal et al., 2013). Further, the lectin or guar gum, PLGA etc. conjugated nanoparticles are able to improve mucoadhesion and biorecognition on bacterial cell wall (Kaur et al., 2014c). They also help to induce prolonged half-life (Singh & Pai, 2014). Due to the size versatility of the nanoparticles, it has advantages over conventional techniques in the case of effective drug delivery (Smith, 2011). There are wide varieties of polymers available which can be used in tuberculosis chemotherapy via micro or nanoparticles through different routes of administration (Parikh et al., 2014). Microspheres are small spherical particles, with diameters in the micrometer range (typically 1–1000 μm) (Joshi et al., 2014b). Microspheres are characteristically free flowing powders consisting of proteins or synthetic polymers which are biodegradable in nature (Cao et al., 2011). Microspheres are defined as “Monolithic sphere or therapeutic agent distributed throughout the matrix either as a molecular dispersion of particles” can be defined as structure made up of continuous phase of one or more miscible polymers in which drug particles are dispersed at the molecular or macroscopic level (Comoglu et al., 2008; Modgill et al., 2014). Microspheres can be manufactured from various natural and synthetic materials (Kalia et al., 2014). Glass microspheres, polymer microspheres and ceramic microspheres are commercially available (Barrow et al., 2007; Rohilla et al., 2014a). There are several studies that show nanoparticle delivery of anti-tubercular drugs in both blood plasma as well as organ tissues, some studies are listed in Table 7.

Table 7. Examples of microparticles/nanoparticles/polymeric nanoparticle-based anti-TB preparations.

Delivery system	Anti-TB drug used	Results obtained	References
Gelatin nanoparticles (mannosylated)	Isoniazid	In this particular study, the nanoparticles (both plain and mannosylated gelatin nanoparticles) was prepared and the particles was found to be in size ranging from 261 to 380 nm. The drug loading was found up to 55%. The nanoparticles were found effective in targeting isoniazid to alveolar regions as the considerable drop in bacterial counts within the infected mice after IV administration was observed. The decline in the hepatic toxicity was also observed.	Saraogi et al. (2011)
Chitosan nanoparticles	Isoniazid	In this method, isoniazid loaded chitosan/tripolyphosphate nanoparticles was found to decrease the MIC against Mycobacterium avium infection. The nanoparticles were finally spray dried. Reduction in size of particles was observed as the concentration of leucine was increased.	Pourshahab et al. (2011)
PLGA nanoparticles	Ethionamide	In this study, ethionamide loaded nanoparticles was prepared. The size was found 287 nm. The drug entrapment efficiency was found 35% and a nanoparticle also found to generate controlled drug release for at least 6 days within plasma.	Kumar et al. (2011)
PLGA microspheres	Rifampicin	Rifampicin loaded PLGA microspheres were prepared and spherical shaped particles was observed with a diameter of 1.9 μm. It was found that they were effectively taken up by the NR-8383 cells were found localize in phago-lysosomes.	Onoshita et al. (2010), Feng et al. (2014)
PLGA oral nanoparticles	Ethionamide	The oral nanoparticles were found 286 ± 26 nm in size; with zeta-potential was −13 ± 2.5 mV. The drug encapsulation efficiency and loading capacity were found 35.2 ± 3.1% and 38.6 ± 2.3%.	Kumar et al. (2011)
PLGA microspheres	Rifampicin	In this case, rifampicin loaded microspheres was found 1.72 ± 0.16 μm in diameter.41% release of rifampicin over 4–5 days was also observed.	Doan & Olivier (2009)
PLGA microparticles	Rifampicin	In this study, it was found that the uptake of rifampicin from the PLGA microspheres was larger (about 4%) at 1 h and 9.3% at 4 h. An in vivo imaging study was done which depicts that the micron sized particles were rapidly excreted from the lungs but nanosized particles were found retained within lungs.	Ohashi et al. (2009)
Porous nanoparticle-aggregates particles (PNAPs) of PLGA	Rifampicin	The porous nanoparticle aggregates was found effectively deposited within the lungs. Initial rapid release of rifampicin was observed, when in vitro release study was employed. Rifampicin concentrations were found to be measurable in tissue and cells (Lungs) for 8 h.	Sung et al. (2009)
Inhalable microparticles	Isoniazid, Rifabutin	The inhalable microparticles incorporating first line anti-tubercular drugs were found to maintain the intra-cellular concentration of isoniazid for 24 h and rifabutin for 96 h period of time.	Verma et al. (2008)
Inhalable microparticles of poly(lactic acid) (l-PLA)	Isoniazid, Rifabutin	In this study, the prepared inhalable microparticles of isoniazid/rifabutin were found to have drug content up to 50%. Particle size was found up to 5 μm. Aerodynamic diameter was found up to 3.58 μm. About 70% of the drug load was found released in 10 days. Macrophage targeting was found effectively.	Muttil et al. (2007)
Oral nanoparticles of poly-lactide-co-glycolide (PLG)	Isoniazid, Rifampicin, Pyrazinamide, Ethambutol	It was found that after administration of the formulation to animal (mice) via oral route it maintains controlled drug levels for at least 5–8 days within plasma. Significant improvement in the pharmacokinetic parameters was found as compared with drug solution (Plain or free).	Pandey & Khuller (2006a)
PLG nanoparticles	Streptomycin	The prepared nanoparticles were found to be 153 nm in size. Drug encapsulation was found 32.12% and drug loading was found to be 14.28%. Streptomycin levels were found to be maintained for 4 days in the plasma and for 7 days in the different organs after single oral administration of streptomycin loaded nanoparticles.	Pandey & Khuller (2006b)
Alginate nanoparticles	Rifampicin, Ethambutol, Isoniazid, Pyrazinamide	In this study, high drug entrapment efficiency was achieved from 70% to 90%. It was found that after a single oral dose administration, therapeutic drug concentrations were found to be retained in the plasma for 7–11 days and in the organs like lungs etc. for at least 15 days when compared to free drug solutions.	Ahmad et al. (2006)
Alginate nanoparticles	Isoniazid, Rifampicin, Pyrazinamide	In this study, the alginate nanoparticles were prepared. The particles were found 235 nm in size. The drug entrapment efficiencies were found upto70–90% for pyrazinamide and isoniazid, respectively, and 80–90% for Rifampicin. The majority of particles were found in the respirable range. The aerodynamic diameter was found around 1.1 μm.	Zahoor et al. (2005)
Inhalable microparticles of poly(d,l-lactic acid)	Isoniazid, Rifampicin	This study depicted that large amount of particles can be delivered to the lungs by 2 min exposure to fluidized particles.	Sharma et al. (2001)
PLGA microspheres	Rifampicin	In this study, animals were treated with PLGA microspheres encapsulating rifampicin which shows significantly reduction in numbers of bacteria, inflammation and lung damage.	Suarez et al. (2001)
PLG microparticles	Isoniazid, Rifampicin	In this study, the PLG microparticles was found to be effective to clear bacteria more from lungs when administered to an experimental murine model of tuberculosis.	Dutt & Khuller (2001)
PLGA microspheres	Rifampicin	In this study the rifampicin loaded PLGA-microparticles was prepared. Finally, spray drying was done and the particles were found 3.45 μm in size.	O'Hara & Hickey (2000)
PLGA microspheres	Rifabutin	Rifabutin loaded microspheres were prepared and in vivo studies were conducted in J774 murine and Mono Mac 6 (MM6) human monocytic cell lines. The rifabutin caused significant reduction of intra cellular replicating Mycobacterium avium complex (MAC).	Barrow et al. (2007)

Carbon nanotubes

Carbon nanotube is an emerging field of nano-biotechnology which has wide potential applications in drug delivery. They are known to have several micrometers in length with cross-sectional diameter in the range of 1–100 nm (Petersen et al., 2011; Hussain et al., 2014). They are mainly of two types, single-walled and multi-walled (Kaur et al., 2014a). The biggest advantage of carbon nanotubes is that they can be easily functionalized with a number of different chemical/functional groups and drug moieties to their surface according to the desired range of properties (Banyal et al., 2013). Carbon nanotubes can behave in both metallic and non-metallic manner (Kaur et al., 2014h). By increasing their biochemical utility manifolds and different functional groups such as proteins/peptides and nucleic acids can be coupled to their surface to makes them easily available to cells, organs and various tissues for targeting purpose (Yang & Han, 2000; Pabreja et al., 2014).

Quantum dots

Quantum dots are semi-conductor particles which generally have size ranging from 1 to 10 nm. That can be made to fluorescent in different colors depending on their size (Sharma et al., 2014b). As they are very good fluorophore, considering their wide emission spectra, they may be utilized for the detection and estimation of TB-infected cells easily and faster with high efficacy in different targeting organs (Banyal et al., 2013).

Nanosuspension

Nanosuspensions are generally micron-sized, colloidal dispersions (Rana & Murthy, 2013). Nanosuspension usually stabilized with use of different surfactants (Wang et al., 2015). Nanonization is a also useful methodology to improve the solubility of profile of drugs (Patel et al., 2011). A nanosuspension is a “very fine solid particles which are dispersed in a suitable aqueous vehicle” intended to be used through different routes like oral, topical, parenteral etc. (Kumar & Kumar, 2011; Kaur et al., 2014d). The size of particles in nanosuspension generally should be between 200 and 600 nm (Feng et al., 2011; Goyal et al., 2014b). Peters et al. (2000) developed a clofazimine-loaded nanocrystalline suspension in order to reduce their toxicity and the low solubility (0.3 μg/mL) of the drug. The bacterial counts were found less in all organs. The results show that the clofazimine-loaded nanosuspension is suitable for IV injection. The drug concentration in liver, spleen and lungs were found to be high, so nanosuspension was found effective against Mycobacterium avium strains (Peters et al., 2000). Reverchon et al. (2002) developed supercritical carbon-di-oxide assisted atomization process to produce rifampicin-loaded nanosuspension (sub-micron sized particles) (Reverchon et al., 2002).

Drug resistance in tuberculosis

Multidrug resistant (MDR) tuberculosis and XDR tuberculosis are the emerging threats in management and treatment of TB. MDR-TB is the TB strains in which the resistance mainly produced to first line drugs such as isoniazid and rifampicin (Thakare et al., 2012). Whereas the extensively drug-resistant tuberculosis (XDR-TB) is a new TB strain which is mainly resistant to fluoro-quinolone agents and one or more second line injectable anti-tubercular agents such as amikacin and kanamycin capreomycin (Denholm et al., 2012). In general, the drug resistance is a man-made problem which results from misuse of available anti-tubercular drugs and due to the poor management of disease course or therapy (Yashodhara et al., 2010). Drug resistant strains are now accounts about 10% deaths of all TB cases. Now a days, India and China are those countries having more cases of patients with multi-drug-resistant TB. In India, “Revised National Tuberculosis Control Program” (RNTCP) has been introduced DOTS therapy which is effective and safe (Thakare et al., 2012). Most of the second line drugs used in MDR-TB is costly and toxic. Whole therapy lasts up to 2 years resulted patient stops taking medication which leads to more severity in TB. Major factors responsible for drug resistance in tuberculosis are shown in Figure 5.

Figure 5. Factors responsible for drug resistance in tuberculosis treatment.

Current treatments available for drug-resistant tuberculosis are inadequate and outcomes are less effective than for drug-susceptible TB patients previously treated with second-line drugs (Zumla et al., 2013). As per the WHO report among 7063 patients from 13 different countries who started second-line treatment for MDR-TB in 2007 only 37% cases were successfully treated and 20% cases either died or undergoes failure of treatment (Thakare et al., 2012). As per WHO report 2014, anti-TB drug resistances were evaluated in 144 countries which account 95% of the World’s population. In 2013, six countries out of the 36 with high MDR-TB cases completed drug resistance surveys these countries includes Azerbaijan, Thailand and Vietnam, Myanmar, Pakistan, Philippines. In mid-2014, other surveys were conducted in five of these countries which include India, South Africa, China, Kenya and Ukraine. Extensively drug-resistant TB (XDR-TB) has been reported by 100 countries on average 9.0% of people with MDR-TB have XDR-TB. Treatment options for patients with MDR-TB and XDR-TB are extremely limited. Further, the poor efficacy, tolerability, treatment discontinuation in many of cases leads to resistance which in turn increases the risk of high mortality rate (Zhang & Yew, 2009). Table 8 shows the first line drugs and their resistance.

Table 8. First line drugs and their resistance.

Drugs	Gene involved in resistance	Gene function	Mechanism of drug resistance
Isoniazid	katG inhA	Catalase-peroxidase Enoyl ACP reductase	In this type of drug resistance, the Kat GS315T mutation is the most common mutation in most of the strains. This resistance was seen 94% of all isoniazid resistant clinical isolates that were reported. Further mutations in the mabA or inhA (promotor region) can also induce resistance (Zhang & Yew, 2009).
Rifampicin	rpoB	β subunit of RNA polymerase	Generally in this case of drug resistance, the mutation occurs in the 81 base pair region of the rpoB. It was found in about 96% of rifampicin resistant M. tuberculosis isolates reported. Mutations in rpoB generally results in high level resistance and cross-resistance (Palomino & Martin, 2014)
Ethambutol	embB	Arabinosyl transferase	This particular kind of resistance occurs due to the mutations in the embCAB operon particular in emb band and occasionally in embC but the embB codon 306 mutation is most frequent in clinical isolates resistant to ethambutol as it was found in 68% of the resistance strains (Zhang & Yew, 2009).
Pyrazinamide	pncA	Nicotinamidase, pyrazinamidase	Most of pyrazinamide resistant M. tuberculosis strains have mutations in pncA region as it accounts for 72–97% of the resistance cases reported. However, in some resistant, strains do not have pncA type mutation (Zhang & Yew, 2009).

Implications of nanotechnology in MDR-TB and XDR-TB

As there are no first line drugs or treatment available for drug resistance in the case of TB. However, nanotechnology-based approaches can also be utilized to incorporate several first and second line anti-tubercular drugs. The lack of nanotechnological-based studies in resistance profiles in drug resistant tuberculosis is that the resistance vary greatly on individual treatment basis (Jassal & Bishai, 2009). There is no treatment options are available against the TB and drug resistance TB. However, our current strategy in various TB programs is that, drug resistance programs, research, and drug development is based on the hypothesis/believes that we will develop a suitable carrier system against rising drug resistance TB epidemics in future. The development of novel TB drugs is on priority basis to fight with TB-resistance.

Novel carrier system in MDR-TB and XDR-TB

Nanodispersions

Nanodispersions are submicron dispersions which are generally colloidal in nature of pure drugs which is stabilized via use of different surfactants (Newa et al., 2008; Malik et al., 2014). They also improve the solubility profile drugs (Chokshi et al., 2007). They are thermodynamically stable with the size of droplet mostly ranges between 10 and 100 nm (Varshosaz et al., 2006). They can host hydrophilic drugs inside core and lipophilic drugs within hydrophobic domains respectively (Jores, 2004; Kaur et al., 2014g).

Polymeric micelles

These are nanocarrier systems which are generated by the self-assembly or self-arrangement of a polymers in water above its critical micellar concentration (CMC) (Lu et al., 2010; Garg et al., 2014a). They are also effective in high drug-loading and drug release, specifically in the case of anti-tubercular drugs (Halperin & Alexander, 1989). They are also effective in drug targeting. Moretton et al. (2010) developed polymeric micelles for rifampicin. It was found during study that large micelles were found by increasing the poly-ethylene glycol concentration. Rifampicin was found to be entrapped within the system appropriately (Moretton et al., 2010).

Future prospective in the treatment of MDR-TB and XDR-TB

Some newer drug candidates in tuberculosis treatment must be needed in order to reduce the burden of different kinds of resistance in tuberculosis and some other severe complications. Some of new drug candidates are listed in Table 9.

Table 9. Newer drug candidates in tuberculosis treatment (Mohan et al., 2013).

Drugs under pre-clinical development stages	Drugs under clinical development
Nitromidazole (TBA-354)	Phase-I	Phase-II	Phase-III
Capuramucin (SQ-641)	Oxazolidinone AZD5847	Rifamycin: (Rifapentine)	Fluoroquinolones: (Gatifloxacin) (Moxifloxacin)
Benzothiazone (BTZ-043)		Oxazolidinone: (Sutezolid (PNU-100480) (Linezolid)	4-Thioureido-iminomethylpyridinium: (Perchlorate) (Perchlozone)
Imidazopyridine (Q201)		Diarylquinoline: (Bedaquiline TMC207)	Nitro-dihydro-imidazooxazole: (Delamanid -OPC67683)

Further, the management of treatment for MDR-TB and XDR-TB should be in a proper way in order to reduce the burden of these resistances and secondary complications in disease. Some of second line anti-tubercular drugs used in the treatment of MDR-TB and XDR-TB are listed in Table 10.

Table 10. Second line anti-tubercular drugs used in the treatment of MDR-TB and XDR-TB (Prasad, 2007).

	Daily dosage (mg)
Anti-TB drugs	Minimum dosage (mg)	Maximum dosage (mg)	Type of anti-TB activity	Duration of treatment
Fluoroquinolone Ciprofloxacin Levefloxacin Ofloxacin Gatifloxacin	1500 mg 500 mg 600 mg 400 mg	1500 mg 750 mg 800 mg 400 mg	Weak bactericidal action	Minimum duration: 6 months. Maximum duration: 12–18 months.
Polypeptides: Capreomycin	500–750 mg	1000 mg	Bactericidal action	Minimum duration: 6 months. Maximum duration: 12–18 months.
Analog of d-alanine Cycloserine	500 mg	750 mg	Bacteriostatic action	Minimum duration: 6 months. Maximum duration: 12–18 months.
Thioamides Ethionamide	500 mg	750–1000 mg	Bacteriostatic action	Minimum duration: 6 months. Maximum duration: 12–18 months.
Amino glycosides Kanamycin Amikacin	500–750 mg 500 mg	1000 mg 750–1000 mg	Bactericidal Bactericidal against actively multiplying organisms.	Minimum duration: 6 months. Maximum duration: 12–18 months.

Role of immunomodulators in treatment of MDR-TB and XDR-TB

Immunomodulation is the emerging and promising therapeutic alternate, specifically in the tuberculosis management and treatment (Thakare et al., 2012). The weak immune response of host is accountable for the relapse of infection. Immunomodulators may be effective for this purpose such as immune stimulatory can be used for stimulation of weak immune response. Some of immunostimulant are listed in Table 11.

Table 11. Examples of immunostimulant (Patil et al., 2012).

Immunostimulant	Mechanism of action
Interferon’s: alpha, beta, gamma	They are able to induce certain enzymatic reactions, inhibition of cell proliferation and enhancement of immune activities like increased phagocytosis by macrophages and augmentation of specific cytotoxicity by T lymphocytes.
Interleukins: aldesleukin, des-alanyl-1, serine-125 human IL-2	They mainly help to activate cellular immunity profoundly with lymphocytosis, eosinophilia and thrombocytopenia and by release of multiple cytokines (e.g. TNF, IL-1 and interferon-g).
Levamisole, immuvac, reimun	Others

Similarly, the immune suppressors are also useful in decreasing an elevated immune response in individual to get beneficial results. Some of examples of immune suppressants are given in Table 12.

Table 12. Examples of immunosupressants (Patil et al., 2012).

Immunosupressant drugs	Mechanism of action
Cyclosporine, tacrolimus sirolimus, everolimus	Inhibitors of lymphocyte signaling (a) Calcineurin inhibitors (b) mTOR inhibitors
Glucocorticoids	Inhibitors of lymphocyte gene expression
Anakinra, daclizumab, basiliximab, etanercept, infliximab, adalimumab	Cytokine inhibitors
Efalizumab (LFA-1 inhibitor)	Inhibitors of immune cell adhesion
Antithymocyte globulin (ATG), alemutuzmab (antiCD-52 antibodies), muromunab (anti CD-3 antibodies, OKT-3)	Antibodies against specific immune cell molecules
Rho (D) immune globulin	Others

Therefore, immunomodulation is an approach based on the fact or principle that a particular microorganism causes disease conditions in a host organism due to the host’s susceptibility rather than its capability or characteristics alone. This principle may be utilized to obtain marked improved results in management and treatment of tuberculosis (Wallis & Johnson, 2009). Therefore, impairment of immune response of the host may thus effective. For this, combination of an immune potentiating agent along with chemotherapy in tuberculosis would be beneficial to prevent such severe disease conditions (Patil et al., 2012). The immunomodulators therapy is useful in tuberculosis due to their efficacy in:

• preventing infections to proceed further in disease conditions and secondary complications (Garg & Goyal, 2012);

• preventing the re-occurrence of later stage infections (Garg & Goyal, 2014b,c);

• helps to achieve early controls and measures of infections when given in conjunction with specific chemotherapeutic agents in tuberculosis (Garg & Goyal, 2014a);

• they also help to achieve early clinical response like weight gain etc.

Current therapy of tuberculosis consists of use of ordinary first line anti-tubercular drugs and specific antibiotics either for a particular period of time or for long period (Garg et al., 2012a).

This increases the chances for generation of resistance and MDR strains. All the countries in the world besides their social or economic status are suffering from MDR-TB and XDR-TB. The continuous development of MDR-TB and XDR-TB is considerably reducing the outcome of anti-tubercular drugs which in turn causes therapy failure (Bhattacharya et al., 2013). Furthermore, there is a chance of TB re-activation and re-infection which also suggests that these chemotherapeutics and antibiotics cause impairment in immune response of host (Bhattacharya et al., 2013). Therefore, an approach for TB treatment reduces chances of immune impairment, able to reduce therapy time of treatment and able to diminish toxicities.

Immunomodulation using immune therapeutic vaccines for tuberculosis

Immune therapeutic vaccines are one of the first immune therapeutic applications of vaccines to show some promise approach in a clinical trial was the heat-killed Mycobacterium vaccae. Its mode of action has been proposed to be an enhancement of Th1 and down-regulation of Th2 cytokine expression (Reljic & Ivanyi, 2012). Table 13 shows the vaccines used in tuberculosis immunotherapy.

Table 13. Vaccines used in immunotherapy in tuberculosis.

Vaccines used	Results obtained	References
Ag 85 (A) DNA vaccine	In this study, the effect of Ag85 (A) DNA vaccine alone and in combination with rifampin and pyrazinamide was observed in mice. Ag85A DNA vaccine was found to be effective in production of IFN-gamma but it lowered the production of interleukins-4. Ag85 (A) DNA vaccine alone and in combination with rifampin and pyrazinamide was also found to lower the pulmonary and spleen bacterial loads by 1.03–1.38 logs.	Liang et al. (2011)
RUTI (which is a vaccine)	This study was based on principle of correlation between ESAT-6-specific interferon (IFN)-γ secretion and pulmonary lesions development. In this study animals were infected using Mycobacterium caprae. The animals were taken in three different groups. Group 1 is Untreated group (CT). Group 2 was treated with Isoniazid (INH). Group 3 was treated with Isoniazid (INH) and RUTI. RUTI vaccine was found effective to enhance the interferon-γ production.	Domingo et al. (2009)

Immunomodulation using cytokines in TB-immunotherapy

Cytokines are highly pleiotropic proteins which are effective to promote host immune defense mechanisms. For the effective treatment of mycobacterial infections, the administered cytokines must first reach their target cells after binding to the specific receptors and finally to activate an intact signal transduction pathway to produce cellular response in host cell. The dose and route of administration of cytokines must be carefully chosen in order to avoid the risk of toxicity/unwanted pharmacological responses or effects. Several cytokines have been considered for treatment of mycobacterial infections, including IFN-gamma, IL-2, IL-12, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF) (Reljic & Ivanyi, 2012).

Immunomodulation using antibodies in TB-immunotherapy

Antibodies could have played a vital role in immunotherapy since normal human sera contain high antibody titers for mycobacterial heat shock proteins. Some of examples regarding the use of antibodies in TB-immunotherapy are given in Table 14. Table 15 shows the role of immunomodulators as adjuvant to chemotherapy in treatment of tuberculosis.

Table 14. Antibodies in tuberculosis immunotherapy.

Antibody used	Results obtained	References
Polyclonal antibodies	In this study, SCID mouse model was taken for aerosol infection by M. tuberculosis, which was followed by 3–8 weeks of treatment of chemotherapy with isoniazid and rifampicin (INH-25 mg/kg + RIF-10 mg/kg). The animals (mice) were divided in two groups. Both the groups were treated with intra-peritoneal passive immunization using hyper-immune serum (HS) for 10 weeks. The infected mice were treated with chemotherapy (INH + RIF) for 8 weeks and inoculated with RUTI (HS group) or with normal serum (CT group). Significant differences were found between HS and CT groups in terms of number of bacilli in the lungs.	Guirado et al. (2006)
Heparin-binding hemagglutinin adhesin (HBHA)	In this study, the heparin binding hemagglutinin adhesin (HBHA) was identified as an antigen. Lymphocytes from 60% of healthy infected individuals (n = 25) produced interferon (IFN)-γ after stimulation with HBHA, compared with only 4% of patients with active tuberculosis (n = 24) was found. Most of the individual (82%) in study was found to produce anti-HBHA antibodies as compared with 36% of the infected healthy subjects. These observations indicate that HBHA is recognized differently by the immune systems of patients with tuberculosis and by healthy individuals.	Masungi et al. (2002)

Table 15. Studies showing role of immunomodulators as adjuvant to chemotherapy in the treatment of tuberculosis.

Type of study	Description and results obtained	References
To determine effect of Dzherelo on HIV and TB co-infected patients.	In this study, the clinical trial (open label) was conducted in 40 patients. The main aim of the trial was to determine the effect of oral immune modulator i.e. Dzherelo on the immune and other different viral parameters. The patients under the study were taken in two equal groups. In the group A (control group) patients was given first line anti-tubercular drugs, i.e. isoniazid, rifampicin, pyrazinamide, streptomycin and ethambutol and in the group B patients received Dzherelo as an immunomodulator twice in a day along with first line HRZSE regimens. After two months of the treatment in 40 patients, it was found that the total lymphocytes (CD3+) were increased upto728–920 cells/μl in group B (Dzherelo receivers) but in the group A the lymphocytes were found to reduce from 650 to 585 cells/μl.	Nikolaeva et al. (2008b)
Therapy for interferon gamma for pulmonary tuberculosis treatment.	In this trial study, total nine trials were conducted with interferon-γ. In 1 trial: patients were aerosolized or subcutaneously administration, In 5 trial: patients were only aerosolized, In 3 trials: patients were intramuscularly administration In these trials, patients were aerosolized by IFN-γ which was found effective in sputum negative conversion in patients.	Gao et al. (2011)
Adjuvant immune-therapy for XDR-TB management and treatment	In this study, the different trials were conducted on seven patients having XDR-TB. In this treatment, the patients received Immunoxel (Dzherelo) during trials. An increase in hemoglobin up to 104.1–118 g L^−1 was found. The time for mycobacterium clearance in infected patients was found to 25–28 days which is effective result.	Prihoda et al. (2009)
Role of recombinant interferon-α2b in MDR-tuberculosis treatment	In this trial study, five patients were taken to study the effect of recombinant-interferon-α2b. The patients under the study were given of recombinant-interferon-α2b. The subcutaneous administration was done in every week to the patients under the study till 12 weeks period. The patients under the trial study were observed for clinical examination, radiological examination etc. before treatment and after treatment also. Two patients out of total five patients were found to be negative sputum smear after recombinant-IFN-α2b therapy. One patient was found to show clinical improvement and negative smear after therapy after examination. The other two patients showed no response in this trial study.	Palmero et al. (1999)
Immuno-modulation therapy with recombinant interferon-γ1b in management and treatment of pulmonary tuberculosis	In this study, it was found that in the immune-adjunctive therapy with nebulized r-interferon-γ1b along with DOTS treatment can significantly reduce various lung inflammatory cytokines in 16 weeks. The immune modulation-based treatment was done on5 MDR-patients. In all five patients, the sputum was found negative with improved symptoms.	Dawson et al. (2009)
Adjunct immune therapy for relapsed TB, first-diagnosed TB, treatment-failed TB and multidrug-resistant TB and TB and HIV co-infected patients.	This study trial was done to check the effectiveness of the (V-5) immunomodulator in different TB infected cases when Immunitor, an immunomodulator which is also known as V-5 was given as an adjunct immunotherapy along with DOTS regimens, i.e. HRSZE. The trial study was conducted after dividing patients into two groups. One group (group A) in which 62 patients was taken in trial and was given above immunomodulator once in a day. The control group (group B) which contains 61 patients who received adjunct treatment for at least 30 days along with DOTS therapy regimens. After therapy period about 1 month with the treatment with immunomodulator, i.e. V-5 the following results were obtained. Fifty-five patients from group A become negative sputum smear testing and this result accounts for (88.7%) of the trial study results. Whereas in the case of the (group B) 9 patients out of 61 patients was found converted as it accounts for (14.8%). Thirty-three TB infected patients in (group B) gained 0.8 kg weights on average basis which is less effective result. Fifty-seven patients in (group A) gained at least 2.8 kg weight during trial study which accounts for (91.9%). No adverse or side effects were found. The chance of TB reactivation was observed. Therapy period up to 1 month with improved patient compliance.	Dawson et al. (2009)
Immunotherapy using IL-2 and GM-CSF in potential management and treatment for MDR-tuberculosis	This study mainly investigated the therapeutic effects of interleukin (IL)-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF) in co-administration with anti-tubercular agents, i.e. isoniazid and rifampin to treat animal model (mice) of tuberculosis. The TB strain, i.e. H37Rv was used to cause tubercular infection in the mice. To check the effectiveness of IL-2 and GM-CSF. The animals (mice) who was receiving immunotherapy in trial study, a very few lungs lesions was developed as compared with animals (mice) who were receiving anti-bacterial drug therapy or immune therapy and anti-bacterial drug therapy both as adjunct. Survival rate up to 30% was found in animals (mice) was found under the trial study who received immune-therapy and less CFUs in the lungs was found, i.e. was 1.02 log CFUs. In the case of spleen 1.34 log CFUs was found. Therefore, from the study, it is clear that both interleukin-2 and GM-CSF may be effective to treat tuberculosis cases and some other secondary complications too.	Zhang et al. (2012)
To study the effect of adjuvant Immunoxel and Anemin immunomodulators in HIV and Pulmonary tuberculosis co-infected patients.	In this study, when Immunoxel and Anemin as an immunomodulators in combination with anti-tubercular therapy (ATT) were given to patients under trials. Total 60 patients were taken in the study which was taken into three groups. Group 1: ATT. Group2: ATT + ImmunoxelGroup3: ATT + Immunoxel + Anemin both as immunomodulators. Blood samples from the patients were taken and were measured in order to check plasma levels of tumor necrotic factor-α, interferon-γ, interleukins-2, inter leukins-6 and interferon-α. After the therapy time, i.e. 6 months period it was found that group 2 (ATT + Immunoxel) produce 61% and group 3 (Immunoxel + Anemin combinations) developed 44.4% higher levels of interleukin-2. Whereas in group 1 (ATT) it was declined or 33.1%. The levels of interleukin-6 were found increased by 17% in group 1 (ATT group) The interleukins-6 levels were found to be reduce up to 26.2% in group 2 (ATT + Immunoxel). The levels of interleukins-6 were to reduced up to 21.3% in group 3 (ATT + Immunoxel + Anemin) Tumor necrotic factor-α was suppressed in two immunotherapy groups by 19.1 and 76.3% in groups 2 and 3, respectively, but had risen by 14% in group 1 patients.	Nikolaeva et al. (2008a)

Conclusion

As the pulmonary tuberculosis is a second leading cause of death after AIDS since time immemorial and it continues to cause immense human misery today up to greater extent. The emergence of MDR-TB and XDR-TB has been threatening and imposing various problems to destabilize TB control globally in developing as well as developed countries. TB has remained a neglected disease, but there is an urgent need for new anti-TB drugs that should be more effective and should have less side effects and toxicity. There is also a need for newer and innovative anti-TB drug-delivery systems in the better management of tuberculosis.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. The author Dr. Amit K. Goyal (AMR/43/2011-ECD-1 dated 1 July 2013) is thankful to the Indian Council of Medical Research (ICMR), New Delhi, India, for providing financial assistance to carry out research work.