Approaches to treating tuberculosis by encapsulating metal ions and anti-mycobacterial drugs utilizing nano- and microparticle technologies

Tuberculosis (TB) is caused by a bacterial infection that affects a number of human organs, primarily the lungs, but also the liver, spleen, and spine, causing key symptoms of fever, fatigue, and persistent cough, and if not treated properly, can be fatal. Every year, 10 million individuals become ill with active TB resulting with a mortality approximating 1.5 million. Current treatment guidelines recommend oral administration of a combination of first-line anti-TB drugs for at least 6 months. While efficacious under optimum conditions, ‘Directly Observed Therapy Short-course’ (DOTS) is not without problems. The long treatment time and poor pharmacokinetics, alongside drug side effects lead to poor patient compliance and has accelerated the emergence of multi-drug resistant (MDR) organisms. All this, combined with the limited number of newly discovered TB drugs to treat MDR-TB and shorten standard therapy time, has highlighted the need for new targeted drug delivery systems. In this respect, there has been recent focus on micro- and nano-particle technologies to prepare organic or/and metal particles loaded with TB drugs to enhance their efficacy by targeted delivery via the inhaled route. In this review, we provide a brief overview of the current epidemiology of TB, and risk factors for progression of latent stage tuberculosis (LTBI) to the active TB. We identify current TB treatment regimens, newly discovered TB drugs, and identify studies that have used micro- or nano-particles technologies to design a reliable inhalation drug delivery system to treat TB more effectively.


Introduction
TB, an airborne infectious disease caused by Mycobacterium tuberculosis (M.tb), is a major public health issue associated with a high rate of morbidity and mortality [1]. Pulmonary infection with M. tb. occurs when droplets (droplet nuclei, 1-5 microns) containing M.tb are released into the air by infected individuals and are inhaled and reach the deep lung including the respiratory alveolar units; the pathogen infects immune cells (alveolar macrophages) which surround the bacteria to contain the infection, leading to the formation of granulomas within the pulmonary tissues. Long-term immunemediated containment of infection in such structures does not eradicate infection but can lead to latent TB where bacteria switch metabolism and can survive for years or decades in this state with no overt clinical signs (sub-clinical infection). Latent tuberculosis can reactivate and lead to active TB progression of LTBI to TB was reported to increase by 20-fold in those with HIV due to the depletion of CD4T-lymphocytes and other immune cells that control and contain LTBI [4]. The increased incidence of both diseases involves a decline in immunological responses which can lead to death if left untreated. The chance of people living with HIV (PLHIV) to get TB infection is 19 times higher than that for healthy people without HIV. In 2018, Africa had the highest-burden, 862 000patients, who were infected with both HIV and TB.
The chance of active TB development is 3-fold higher in diabetic patients and the death risk is 1.89 times greater compared with TB patients without diabetes [18]. The rate of diabetes (type 2 diabetes) is steadily increasing in poorer countries where TB is also prevalent, such as India, central and south America, and a few countries in Africa. Diabetes damages the adaptive and innate immune responses, again leading to a substantial increase in the survival and proliferation of M.tb. A recent study showed a remarkable number of M.tb organisms in diabetic mice with decreased production of IFN-γ and other cytokine mediators which resulted in weakened T-cell immunity. Furthermore, the recruitment of neutrophils was decreased in diabetic patients, enhancing the likelihood of progression to active TB [19]. Conversely, glucose intolerance and deterioration in glycaemic control are induced during TB which makes controlling diabetes more difficult [20].
Many studies have shown a strong link between tobacco smoking and active TB due to the negative effect of cigarette smoke on mucosal secretion defenses and the protective role of alveolar macrophages [21]. Nicotine markedly reduces the immune response; for example, the number of M.tb bacteria was increased in a model of mice exposed to tobacco smoke [22]. A similar effect was shown by consuming a large volume of alcohol, which also subdues the immune system and affects cytokine mediator production [23]. Similarly, air pollution substantially elevated the risk of active TB progression, and this effect was mainly associated with inhaling particles with a diameter less than 10 mm (PM10) and increasing levels of pollutant gases, nitrogen dioxide (NO2), nitrogen oxides (NOX), carbon monoxide (CO) and sulfur dioxide (SO2) [24]. The mechanism by which these pollutants increase the risk of TB was found to be multifactorial. For instance, diesel exhaust is found to affect macrophage function leading to reduced levels of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) which play important roles in controlling the activation of LTBI. Likewise, the interleukin-10 (IL-10) level was increased for mice exposed to CO particles resulting in a higher risk of progression to active TB [25]. Furthermore, the risk of developing active TB was shown to increase substantially, between 6-10-fold, with malnutrition [26]. This is exacerbated in those with TB who exhibit reduced appetite, malabsorption of nutrients, and wasting [27].
In addition to these risk factors, genetic variation in both humans and the different strains of the M.tb pathogens can impact on the prevalence, morbidity, and mortality of TB. Genetic variations in humans also affect the progression of LTBI into active TB [28]. This is a complex area and regardless of the underlying predispositions to acquire TB and progress to active TB, effective targeted treatment that will rapidly kill the organisms and reduce transmission, and prevent the development of MDR-TB is a desirable outcome.

Current TB treatment guideline and vaccination
Thus, the main aims of TB-therapy are to cure patients effectively, reduce transmission of infection to other people, and prevent the occurrence of MDR-TB [29]. The WHO recommended 'Directly Observed Therapy Short-course' (DOTS) treatment for drug-sensitive TB consists of a 2-month intensive phase with 4 drugs (Rifampin (RIF), Isoniazid (INH), Pyrazinamide (PZA), and Ethambutol (EMB) taken daily, followed by a continuation phase of RIF plus INH daily for 4 months. Patients must be observed taking their medication to ensure compliance and under these circumstances, cure rates can exceed 85%. There are a number of variations of DOTS depending on factors such as co-infection with HIV or other co-morbidities, and MDR-TB requires longer treatment with more toxic drugs [30,31].
Bacillus Calmette-Guérin (BCG) is the only licensed vaccine available for TB and is most effective when given to neonates in high burden countries and provides protection against disseminated forms of tuberculosis. It works less well in older children, especially those in high burden countries, for reasons that are not fully understood [32].

Promising new TB drugs and vaccines
The lengthy, complex drug treatment regimen, and toxic side effectswhich lead to patient non-compliance are major obstacles to successful TB therapy and contribute to the development of MDR-TB. Thus, new drugs and targeted drug therapy formulations are urgently required to overcome the current challenge of eradicating TB as a global health problem. This means that new treatments need to be shorter, simpler, safer, and affordable, with fewer adverse drug interactions [33]. There are over 20 promising anti-mycobacterial agents in various stages of clinical development, or recently licensed [11,34]. These are either new chemical entities or derivatives of existing compounds, and include drugs with novel targets, such as Bedaquiline, which inhibits the mycobacterial ATP synthase, and drugs with known targets such as Sutezolid and Delpazolid which both inhibit protein synthesis. However, these agents fall into around only 10 functional classes, targeting, for example, translation, lipid transport, lipid biosynthesis and catabolism, ATP synthase, meaning that their longterm value may be undermined by the potential for cross-resistance. Moreover, many drugs that have been approved for other conditions are being tested in combination with current multi-drug therapy, including higher doses of more potent, longer-lasting Rifamycins, and fluoroquinolones such as moxifloxacin.
Similarly, ∼20 novel vaccines are in clinical trials at various stages and include modified versions of the current BCG vaccine, protein subunit vaccines delivered with adjuvant, recombinant viral vectors expressing one or more mycobacterial antigens, and live attenuated M.tb [35]. In the one hundred years since the original BCG was developed, nothing better has emerged. The continued quest for new vaccines in the face of uncontrolled TB reflects our lack of insight to the exact immunological mechanisms involved hence the inability to effectively contain, or protect people from, the disease.

Micro-and nano-technology for treatment of TB
The availability of new anti-TB drugs may not be sufficient to combat MDR-TB and eradicate TB at the rate required to meet the WHO END-TB plan. Finding alternative approaches to overcome the poor pharmacokinetics and pharmacodynamics of currently used oral TB drugs could advance the treatment of this disease [36]. First-line TB treatments have limited solubility in water which reduces their absorption from the gastrointestinal tract leading to low bioavailability [37], even when high doses are taken. They also have short half-lives and are eliminated rapidly from the body [38]. Consequently, oral or even intravenous administration of these drugs may deliver insufficient therapeutic dose to the affected organ and particularly the lungs [39]. The interest in utilizing unique nanotechnology approaches for the treatment of TB has increased substantially over recent years, particularly for targeted inhalation therapy, due to the potential advantages this has over conventional treatments. This relates to the ability of the inhalation system to deliver the drug to the site of infection, accelerate the onset of treatment using potentially less dose of drugs and mimicking the way that M.tb harnesses the host defence and other mechanisms to infect lung through M.tb-contaminated aerosol. However, a challenge for inhalation therapy is to produce an optimal biopharmaceutical formulation that is physically and chemically stable and efficiently and reproducibly delivered to the lungs at the requisite doses needed to achieve clinical efficacy, with favourable pharmacokinetic profiles.

Nano-and micro-particles for targeted therapy of TB
Several studies have investigated the use of nano-or microparticles (NP and MPs, respectively) loaded with traditional TB drugs as inhalation therapy for TB. Formulations include the use of different nano-or microcarriers, including nanoemulsions, liposomes, nanosuspensions, and polymers, to encapsulate and deliver antitubercular drug(s) to the infection site. Nanoemulsions are thermodynamically stable oil-in-water dispersions with droplet size ranging between 10 and 100 nm. Particle dispersion can be stabilized by various surfactants and cosurfactants, producing large interfacial areas, and a stable dispersion and stability for inhalation therapy. For example, a nanoemulsion encapsulating Rifampicin showed an extended drug-release profile of up to 2 h, with a drug entrapment efficiency of 100% and osmotic pressure that compares with blood and is suitable for IV delivery [40]. Liposomes are made up of spherical vesicles composed of a phospholipid bilayer; their unique structure gives them the ability to deliver hydrophobic, amphipathic, and hydrophilic medicines. Nanosuspensions are biphasic systems consisting of pure drug particles in aqueous vehicles. The vehicles are generally composed of either surfactants or polymers that stabilize the nanosized particles. These nanosuspensions are used to increase the solubility and bioavailability of drugs with strong intermolecular interactions that cause low solubility in aqueous and oily media. In a nanosuspension form, Rifampicin's solubility was increased 50 times compared with the free drug and the cytotoxicity on epithelial lung cells was reduced. In one study, Rifampicin nanoparticles were prepared using a microemulsion-based technique, cetyl palmitate, and Tween 80 [41]. This resulted in spherical Rifampicin nanoparticles with a size of 100 nm, low negative zeta potential, and encapsulation efficiency of 82% were produced. Rifampicin was released with a sustained drug release profile for 72 h showing an improved antibacterial effect against M. fortuitum, being effective at one eighth the concentration of Rifampicin alone. Table 1 lists other recent approaches to utilizing these micro-and nanotechnologies to design efficient antitubercular lipid-based therapies. The results given in the table show that encapsulating a single TB drug within nano-or micro-particles enhanced the release profile, improved the antitubercular efficacy, and produced reliable drug delivery systems that can be administered by several routes, such as oral, i.v. or inhalation. Importantly, dual loading of different TB drugs within nanoparticles showed better antitubercular results compared with the mixture of free drugs. These results are promising for future research aimed at designing nanoor micro-particles containing multiple TB drugs. Interestingly, nanotechnology could also play an important role in vaccination, or enhance the effects of vaccines used to treat or prevent TB. For example, vaccination using a plasmid DNA vector encoding the antigen 85A (Ag85A) of M.tb was facilitated when adsorbed to poly (D,L-lactide-co-glycolide) (PLGA) microparticles and delivered intramuscularly. Thus, similar protection to that seen with BCG was observed with the PLGA-adsorbed DNA encoding Ag85A, which in turn was 100 times more effective than naked DNA-encoding Ag85A, when assessed in an aerosol challenged mouse model of M.tb [42,43].

Metal nanoparticles for targeted treatment of TB
The resistance of M.tb to the antitubercular drugs along with a lack of new drugs for the treatment of bacterial infections suggest the need to utilize novel antibacterial agents. In this respect, metal nanoparticles such as silver and zinc have been studied extensively as a potential treatment for many medical conditions [44]. Silver nanoparticles (AgNPs) are antibacterial and have been utilized in several therapeutic [45,46], and diagnostic applications, as well as in optoelectronics [47], and in water disinfection [48]. Studies have reported the antimicrobial effect of AgNPs on resistant strains of bacteria through several mechanisms [49]. These include disturbance of bacterial membranes and cell walls leading to cell leakage by increasing membrane permeability [50], initiating lipid peroxidation and reduction in the levels of the antioxidant, glutathione, depolarization of mitochondria, and oxidative damage of DNA with apoptotic cell death [51], damaging of bacterial cell DNA by binding to its sulfur and phosphorus groups [52], and by releasing Ag ions which play an important antibacterial role by interacting with bacterial cell membranes [53]. Silver nanoparticles in colloidal form and suspension of silver ions with silver nanoparticles in an aqueous medium show superior antibacterial effects by serving as a catalyst which disrupts essential enzymes that microbes need for oxygen metabolism [54]. Similarly, zinc nanoparticles show efficient antibacterial and UV-blocking properties. These attributes are why they are widely used in personal care products, such as cosmetics and sunscreen [55]. Zinc oxide nanoparticles are delivered in a spray form to relieve redness and itching sensations triggered by skin conditions such as lichen planus, eczema, seborrheic dermatitis, psoriasis, and increased skin dryness [56]. Currently, a combination of silver and zinc oxide nanoparticle sprays are prescribed as an antibacterial spray for acute relief of conjunctivitis, skin inflammation, sinusitis, and earache [57]. Many recent studies have been exploring the use of metal nanoparticles for treating TB and MDR-TB as shown in Table 2. Table 2 shows the antitubercular effect of metal/metal oxide NPs, and TB drugs loaded with metal/metal oxide NPs, against different strains of M.tb bacteria. Importantly, all metal-containing agents, such as silver, gold, titanium, zinc oxide, and gallium, showed a bactericidal effect when applied to M.tb [58]. Similarly, a synergistic effect was reported for first-line TB drugs such as Rifampicin when formulated within a metal/metal oxide NP system leading to an increase in the antitubercular effect of these drugs and reducing their minimal inhibitory concentration [59]. Interestingly, silver and zinc oxide NPs showed only a bacteriostatic effect when used against resistant strains of M.tb [60]. These metal/metal oxide NPs showed low toxicity when incubated with eukaryotic THP-1 and Vero cells, suggesting the possibility of using an advanced therapy containing a cocktail of metal/metal oxide NPs along with TB drugs to improve the efficacy of TB treatment and shorten the duration of therapy. Similarly, metal NPs showed a significant improvement in the diagnostic approach to TB. Employing gold NPs in the detection of TB DNA for diagnostic purposes, using a paper-based analytical platform, resulted in a highly sensitive detection limit of 1.95 × 10 -2 ng/ml for TB DNA [61].   Spherical NPs with a mean size of 19.9 nm with a negative zeta potential of −13.8 mV. The antibacterial effect was observed against all strains and didn't show any toxic effects when exposed to L929 fibroblasts.

[90]
Assess the efficacy of silver NPs on TB bacteria N/A Silver NP NPs were prepared using a single dispersion and stabilized by polyvinylpyrrolidone. Characterization: Size, in vitro antitubercular effects against H37Rv strain, and in vivo efficacy using mice.
NP with size 43.6 ± 10.7 nm were prepared to enhance the suppression of TB bacteria by 2 fold and decrease the count of TB bacteria in the spleen and lungs by 2 x in mice.

[91]
Design a green synthesis of silver NPs N/A Silver Nano composi te NPs were prepared by a one-step reaction using Chitosan. Characterization: Size, antitubercular effect, and cytotoxicity.
Spherical nanocomposites with a size 11-17.5 nm and IC50 against normal lung cells was 357.2 μg/ ml. M. tuberculosis was inhibited by an MIC of 1.95 μg/ml.

[92]
Prepare silver NP loaded with antibacterial drugs

Vancom ycin
Silver NP NPs were prepared by a citrate reduction process. Characterization: Size, morphology, antibacterial effect against M. smegmatis.
Spherical NP with a size 30 ± 3 nm were prepared successfully. The internalization of the drug inside the bacteria was enhanced through formulation with NPs.
11 [93] Investigate the antimycobacterial activity of silver N/A Silver NP Silver NP were prepared by a chemical reduction method. Characterization: Size, morphology, MIC against M. tuberculosis H37Rv.
Spherical and tetrahedral silver NP with an average size of 59 nm were prepared. The system showed an antibacterial effect on this TB strain with an MIC of 1 mg/ml.

[63]
Fabricate a nanoscale multi-drug delivery system RIF, PZA Silver NP The biodegradable polymer chitosan-grafted-(cetyl alcoholmaleic anhydride-pyrazinamide) was made by multiple reactions. Silver NP and RIF were incorporated. Characterization: Size, morphology, drug release, in vitro cytotoxicity on Vero and THP-1 cells, and antitubercular activity.
FTIR confirmed the successful synthesis of silver, RIF, and polymer containing PZA. PZA and RIF were in an amorphous phase with a size of 140 nm and their full release was performed within 12 h. The combination therapy showed better antitubercular effect than their single administration.
13 [94] Design and characterize biodegradable silver NPs  18 [99] Compare the efficacy of silver and gold NPs N/A Silver, Gold NP Biological synthesis of silver and gold NPs was done using environmental bacterium. Chemical synthesis was performed using reduction reactions. Characterization: Size, Morphology, antitubercular effects in vitro and ex vivo using infected THP-1 cells and cytotoxicity assays.
Ag and AuNPs were spherical and Polyhedral in morphology, respectively. AgNPs showed better antitubercular efficacy than the gold NPs.
19 [98] To  Spherical NP with diameter of 16 nm showed size and concentration-dependent antitubercular effects with a 3-4 times decrease in TB metabolic activity with very minimal toxicity when the maximum dose was applied.

Conclusions
TB remains a significant global disease that causes high morbidity and mortality for which current therapeutic strategies are often inadequate; consequently, it continues to be a major health concern. New strategies are being examined, broadening the scope of the research, with innovative novel approaches to improve treatment, and reduce mortality. Currently, the number of newly formulated TB drugs is low. Different technologies such as micro-and nano-technologies are being used as carriers for targeted M.tb treatment by encapsulating single and combinations of traditional antibiotics, as well as combining antibiotics with antibacterial metals; the metal nanoparticles have an additive/synergistic effect allowing use of lower doses of drug(s) with reduced side effects, whilst exhibiting high efficacy. Promising results report increasing the antitubercular efficiency of many first and second-line TB drugs when formulated within nano-and microparticles which facilitates drug uptake by M.tb. infected macrophages [62]. Similarly, utilizing metal/metal oxide NPs together with TB drug(s) increased their efficacy [63]. Importantly, there is a need to establish reliable in vivo models to examine the impact of the metal NP-drug formulations in animal models as the number of in vivo studies on these drug formulations is very limited. Metal-based formulations have an element of risk related to the potential toxicity of the metal, the dose of which would need to be carefully monitored and adjusted for human use.
Organic and metal micro-and nano-carriers offer great potential for more effective delivery of TB drugs to the affected site, alone and in combination, to increase their potency, particularly in the presence of antibacterial metal nanoparticles. Introduction of potent, novel, and repurposed drugs will increase the impact of such systems and hopefully reduce the impact of M.tb. on human health. Uniformly distributed ZnO NPs with a diameter of 11 nm and zeta potential of +19.1 mV. The MIC for the ZnO NP was 256 mg/ml but 32 mg/ml of ZnO NPs were able to reduce the MIC of RIF from 64 mg/ ml to 16 mg/ml which confirms a synergistic effects between both antitubercular agents.