Multi-functionalized nanocarriers targeting bacterial reservoirs to overcome challenges of multi drug-resistance

Abstract

Introduction

Infectious diseases associated with intracellular bacteria such as Staphylococcus aureus, Salmonella typhimurium and Mycobacterium tuberculosis are important public health concern. Emergence of multi and extensively drug-resistant bacterial strains have made it even more obstinate to offset such infections. Bacteria residing within intracellular compartments provide additional barriers to effective treatment.

Method

Information provided in this review has been collected by accessing various electronic databases including Google scholar, Web of science, Scopus, and Nature index. Search was performed using keywords nanoparticles, intracellular targeting, multidrug resistance, Staphylococcus aureus; Salmonella typhimurium; Mycobacterium tuberculosis. Information gathered was categorized into three major sections as ‘Intracellular targeting of Staphylococcus aureus, Intracellular targeting of Salmonella typhimurium and Intracellular targeting of Mycobacterium tuberculosis’ using variety of nanocarrier systems.

Results

Conventional management for infectious diseases typically comprises of long-term treatment with a combination of antibiotics, which may lead to side effects and decreased patient compliance. A wide range of multi-functionalized nanocarrier systems have been studied for delivery of drugs within cellular compartments where bacteria including Staphylococcus aureus, Salmonella typhimurium and Mycobacterium tuberculosis reside. Such carrier systems along with targeted delivery have been utilized for sustained and controlled delivery of drugs. These strategies have been found useful in overcoming the drawbacks of conventional treatments including multi-drug resistance.

Conclusion

Development of multi-functional nanocargoes encapsulating antibiotics that are proficient in targeting and releasing drug into infected reservoirs seems to be a promising strategy to circumvent the challenge of multidrug resistance.

Graphical abstract

Introduction

Infectious diseases continue to be a distressing burden on global economies. Lack of surveillance, poor control, and prevention strategies have lead antibiotic treatment to chaos where most of the infectious organisms have developed resistance towards conventional therapeutic agents [1]. Moreover, research for the development of newer chemotherapeutic agents is lagging far behind the pace of emergence of drug resistance by such pathogens [2]. Multidrug-resistant bacteria have evolved to become less susceptible to more than one chemotherapeutic agent. Mechanisms involved behind such resistance may include modifications in the protein of interest, inactivation of targeting agent by bacterial enzymes and preventing the drug to reach its target [3].

Staphylococcus aureus being one of the most prevalent multi drug-resistant bacteria presents a major threat towards community-acquired infection-associated morbidity and mortality [4]. Likewise, Mycobacterium tuberculosis and Salmonella species are a major challenge for researchers to fight the ever-increasing incidence of antibiotic resistance [5]. An important concern associated with these pathogens is their intracellular persistence. These microorganisms are known to invade macrophages and other polymorphonulcear cells, where they get hold of degradative mechanisms adopted by phagocytic cells. This allows the pathogen to reside inside cellular compartments where they persist, replicate and disseminate infection on finding suitable circumstances. Thus, targeting intracellular pools of microorganisms can be a useful strategy for complete eradication of drug at lesser doses with reduced toxicity [6].

Despite the research in progress, the potential of bacteria to develop resistance against newer antibiotics can never be ignored. This scenario requires modifications in current strategies exploited to counter the mechanisms adopted by bacteria to develop resistance against new and conventional drug candidates [7]. Ongoing research intensively suggests the use of nanomedicines to overcome the barriers of antibiotic therapy. High flexibility in surface modifications offers a wide range of possibilities that can be exploited to target a variety of extra or intracellular mediators of drug resistance. Montanari and his coworkers have investigated such modifications to reach intracellular targets [8]. Nanomedicines have also been successfully investigated as cargoes to deliver a drug to its destination, protecting it from undesired modifications while roaming before reaching the target [9]. Various nanocarriers that have been investigated for targeting of intracellular pathogens include different polymeric nanoparticles, metal-based nanoformulations, and lipid-based carrier systems. These carrier systems have been discussed in this review for targeting of intracellular Staphylococcus aureus, Salmonella typhimurium, and Mycobacterium tuberculosis [10].

Mechanism for intracellular uptake of nanocarriers

Eradication of intracellular microorganisms has always been challenging for scientists, which seems even more difficult in the case of multidrug-resistant bacteria. Drug must reach in sufficient concentration within cell where pathogen has colonized for their complete removal. Intracellular infection caused by S. aureus has shown decreased response towards conventional treatment strategies. These shortcomings have been combated using nanocarrier systems [11, 12]. Some commonly employed mechanisms for cellular uptake of drug loaded functionalized nanocarriers have been presented in Fig. 1. Drug-carrier complex may enter target cells either by receptor-dependent or receptor-independent endocytosis, such as clathrin/ caveolin mediated internalization or micropinocytosis, respectively [13]. Drug carrier conjugate may also enter the cell by surface electrostatic interactions between the carrier and cell membrane, that may distort membrane integrity, allowing passage of drug-carrier complex across the cell membrane [14]. At present, a large share of research has been focused on intracellular delivery of therapeutic agents for such infectious pathogens that exploit intracellular milieu for their survival [15]. In this context, nanocarriers have been widely explored for delivery of antibiotics to cells with high microbial burden, thus, helping complete eradication of invading microorganisms with reduced chances of relapse and an expected decrease in the emergence of resistance against therapeutic agents [16]. Armstead and Li have reviewed the use of nanomedicines for targeting intracellular pathogens including Mycobacterium tuberculosis, Hepatitis and human immunodeficiency virus [17]. Some major sites for intracellular targeting have been depicted in Fig. 2.

Fig. 1.

Common mechanisms for cellular uptake of drug loaded functionalized nanocarriers, I; uptake by formation of pore in cell membrane, II, carrier mediated uptake of ligand-drug carrier complex (clathrin or caveolin dependent endocytosis), III; uptake of drug-carrier conjugate through disoriented cell membrane, IV, uptake of liposomal formulation across cell membrane, V; uptake of drug-carrier conjugate by micropinocytosis

Fig. 2.

Major sites for intracellular targeting, I; promoting acidification of phagosomes, II; targeting of phagosomes harboring pathogen, III; stimulating phagosome-lysosome fusion

Staphylococcus aureus

Staphylococcus aureus (S. aureus) is a significant member of our microbial flora and exists as one of the most prevalent multi-drug resistant pathogens [18]. The first strain of S. aureus that developed resistance against methicillin emerged in 1961 and now strains resistant against vancomycin have also been surfaced [19]. Among all resistant strains of S. aureus, the most dominant cause of resistance appears to be the interspecies transmission of genes [20]. S. aureus colonizes various anatomic regions of the human host with most prominent sites including anterior nares, respiratory passage, gastrointestinal region and skin [21]. Once the natural host defenses are breached, S. aureus may enter adjacent tissues and blood. Bacteremia and endocarditis are the most prominent invasive diseases associated with S. aureus. Endothelial cells are supposed to be a major site for the progression of infection. In the case of tissue damage, S. aureus has been known to interact with glycoproteins (fibronectin and laminin) on surface connective tissues for adhesion. In the absence of tissue injury, a case of bacteremia or endocarditis, the initial interaction observed is with the surface of endothelial cell [22, 23].

Intracellular subsistence of S. aureus

One of the key health concerns associated with S. aureus infection is its relapse, which may ultimately lead towards the development of resistance against antibiotics. This problem has been thought to be a result of inability of antibiotics to reach the sites where bacteria persists. Usually, such regions comprise the intracellular constituencies where bacteria seem to colonize. Phagocytic invasion of S. aureus was reported by Rous and Jones in guinea pigs in 1916 [24–26]. S. aureus has been observed to reside for a long duration within endothelial cells [27]. Likewise, S. aureus also invades immune cells where they proliferate and escape the natural defenses [28]. Pathogenic strains of S. aureus have been reported to escape the cytoplasm of polymorphonuclear leukocytes, following lysis of cell [29]. Studies have confirmed that following uptake of S. aureus, pathogen survived within host mononuclear cells. It was also observed that antibiotics showing poor uptake into S. aureus infected cells were not successful in the complete elimination of pathogen [30]. Abu Humaidan and coworkers have discussed the intracellular survival of S. aureus in epidermal keratinocytes, which lead to complement activation [31]. Moriwaki and associates have demonstrated the persistence of S. aureus atopic dermatitis accumulates within the lysosomal compartment of keratinocytes [32]. In another study, Lacoma and colleagues have suggested the persistence and replication of S. aureus in murine alveolar macrophages [33]. Fraunholz and Sinha have reviewed various intracellular fates of S. aureus [34]. Zhou and coworkers have discussed use of nanocarriers for effective intracellular eradication of methicillin resistant S. aureus [35]. Labruère and associates have also reviewed fabrication and development of various nanoparticulate systems for treatment of S. aureus associated infections [36]. Hibbitts and O’Leary have also discussed various nanocarrier systems for targeting intracellular S. aureus [37].

Polymeric nanoparticles for intracellular targeting of S. aureus

Maya and coworkers have described the efficiency of O-carboxymethyl chitosan nanoparticles that were encapsulating tetracycline against intracellularly invading S. aureus. The study indicated a six-fold increase in efficiency of this delivery system against intracellular S. aureus (HEK-293 and differentiated THP1) in contrast to tetracycline alone [11]. In another study, a cationic antimicrobial peptide (plectasin) was loaded into PLGA (poly-(lactic-co-glycolic acid) nanoparticles that allowed the release of peptide over a period of 24 h. S. aureus infected Calu-3 cell lines were used to investigated antimicrobial efficiency of formulation while its distribution was studied in Calu-3, THP-1, and A549 cell lines. It was established that nanoparticles containing plectasin were successfully internalized by Calu-3 and THP-1 cells but not significantly by A549 cells. Antimicrobial efficacy of formulation was found to be improved as compared to free peptide [38]. Smitha and associates have developed chitin nanoparticles encapsulating rifampicin for its delivery to polymorphonuclear leukocytes. Nanoformulation exhibited sustained release of drug and an improved delivery (5–6 folds) into S. aureus infected polymorphonuclear cells [39]. Montanari and coworkers have investigated levofloxacin loaded cholesterol conjugated hyaluronan based nanohydrogels. This nanocarrier system showed preferential accumulation in lysosomal compartments colonizing S. aureus [40]. Sémiramoth and colleagues have investigated pH sensitive and pH insensitive penicillin G bioconjugates with squalene for targeting intracellular infection. Squalene conjugated penicillin was found to have increased activity against intracellular S. aureus in comparison to unconjugated penicillin [41]. Qui and coworkers have studied hyaluronic acid conjugated streptomycin for intracellular eradication of S. aureus and Listeria monocytogenes. This conjugate was more efficient in reduction of intracellular bacterial burden with significantly reduced nephrotoxicity when compared to free streptomycin [42]. Mu and colleagues have also investigated gentamycin gold nanoparticles conjugated with phosphatidylcholine against intracellular S. aureus [43].

Liposomes for intracellular targeting of S. aureus

Liposomes have been established as a promising system for intracellular delivery of antimicrobials. These systems are known to exhibit significant uptake by cells and thus, can be employed for the delivery of a variety of drugs against intracellular parasitic and bacterial infections [44]. In a study, pegylated and non-pegylated liposomal formulations of vancomycin were investigated for intracellular targeting of methicillin-resistant S. aureus. The non-pegylated liposomal formulation was successful in achieving an adequate amount of vancomycin inside macrophages to exert its bactericidal action. In contrast, the pegylated liposomal formulation was not much successful in achieving effective concentration within target cells because of delay in the process of phagocytosis offered by stealth effect of pegylation [45]. Onyeji and his colleagues have also investigated the use of liposomes in enhancing the cellular uptake of vancomycin and teicoplanin. Liposomal encapsulation of both drugs enhanced the killing of S. aureus inside human macrophages and a significant increase in efficacy was observed in comparison to free drugs [46]. Dihydrostreptomycin encapsulated in liposomal carrier system has also been investigated for targeting of intracellular S. aureus. The results indicated an augmented killing of bacteria invading phagocytic vacuoles [47]. Fountain and coworkers have studied entrapment and surface charge properties of aminoglycosides (amikacin, gentamicin, kanamycin, and tobramycin) with cationic, anionic and neutral liposomes. They have also investigated the role of these aminoglycoside entrapped liposomes in canine monocyte associated intracellular killing of S. aureus. Results showed an increase in canine monocyte associated intracellular eradication of S. aureus when liposomal formulations were introduced into cell cultures [48]. Table 1 summarizes various nanoformulations investigated for intracellular eradication of S. aureus.

Table 1.

Summary of nanoformulations investigated for intracellular targeting of S. aureus

Polymer	Carrier System	Therapeutic agent	Year published	Reference
Hyaluronic acid/ cholesterol	Nanohydrogels	Levofloxacin	2018	[40]
Hyaluronic acid/	Nanoparticles	Streptomycin	2017	[42]
Phosphatidylcholine/gold	Gold nanoparticles	Gentamycin	2016	[43]
Poly-(lactic-co-glycolic acid)	Nanoparticles	Plectasin	2015	[38]
Chitin	Nanoparticles	Rifampicin	2015	[39]
O-carboxymethyl chitosan	Nanoparticles	Tetracycline	2012	[11]
Squalene	Nanoparticles	Penicillin G	2012	[41]
1,2-distearoyl-sn-glycero-3-phosphocholine/methylpolyethyleneglycol–1,2-distearoyl-phosphatidyl ethanolamine conjugate	Liposomes	Vancomycin	2010	[45]
Egg phosphatidylcholine/ diacetylphosphate/ cholesterol	Liposomes	Vancomycin/ teicoplanin	1994	[46]
Dimyristoylphosphatidylcholine/ dicetylphosphate/ stearylamine	Liposomes	Amikacin, gentamicin, kanamycin, tobramycin	1981	[48]
Egg phosphatidylcholine/ cholesterol/ phosphatidic acid	Liposomes	Dihydro streptomycin	1978	[47]

Salmonella typhimurium

Salmonella typhimurium (S. typhimurium), a commensal microbe is known to be one of the major causes of gastroenteritis in humans. Salmonella typhimurium has been recognized to alter microbiota and develop mechanisms to overcome natural defenses to establish infection [49]. Infection disseminates following the invasion of intestinal epithelium and adopts various modes of virulence [50]. Various mediators that allow a bacterium to endure harsh intracellular environment involve adhesins for anchoring the epithelial surface, Salmonella pathogenicity islands (SPI-1 & SPI-2) for its translocation into the cell; allowing engulfment along with triggering inflammation. Once colonizing Salmonella containing vacuoles (SCV), S. typhimurium may persist within vacuole or it may escape into the cytoplasm for replication [51].

S. typhimurium hijacking host macrophages

Being an intracellular pathogen, S. typhimurium has been found to infect macrophage, a substantial participant of natural immune response [52]. Macrophages along with neutrophils seem to get galvanized in response to S. typhimurium invasion of intestinal epithelial cells along with mononuclear cells, triggering various mediators of the complement cascade. This reaction is responsible for the release of peptides (α-defensin and cathelicidins) that are antimicrobial in nature and prove fatal for the pathogen [53]. Macrophages and other mononuclear cells activated in response to this infection, phagocytose bacteria and kill them via discharge of reactive oxygen species. However, studies have demonstrated certain genetic expressions (sigma factors) to help S. typhimurium in combating stresses offered by phagocytic cells that were originally employed to control replication and dissemination of pathogens [54]. Other factors that help pathogen survive the host environment involve pSLT plasmid, biofilm associated proteins, adhesins, and flagella [53].Studies have also demonstrated that S. typhimurium utilizes BES system (base pair excision system) to maintain the integrity of bacterial DNA [55, 56].

Such defensive mechanisms adopted by pathogen necessitates that a drug must reach inside the cell sheltering bacteria for its complete annihilation. Intracellular targeting of S. typhimurium has also been proposed to be substantial for overcoming the problem of emerging multidrug resistance, as antibiotic is delivered in effective concentrations at the site of bacterial growth and replication, that otherwise evades natural host defenses and contributes towards the failure of conventional antibiotic therapy [57]. Jajere has reviewed intracellular persistence of S. typhimurium and its ability to adapt to host environment [58]. Ibarra and Steele-Mortimer has also reviewed in detail the mechanism for intracellular survival of S. typhimurium and key virulence factors that help it in accessing intracellular niche [59]. Ranjan and associates have reviewed mechanisms adopted for intracellular survival of S. typhimurium in host along with reasons for failure of conventional therapy and role of nanoparticles for removal of intracellular pathogens [60].

Polymeric nanoparticles for targeting intracellular S. typhimurium

Nanoparticles have also been successfully investigated against intracellular S. typhimurium. Nanocarriers have been effectively manipulated to improve the physicochemical characteristics of conventional agents employed for the eradication of S. typhimurium [61]. Chitosan nanoparticles have been investigated to enhance the penetration of ceftriaxone sodium inside the cell (caco-2 and macrophage-J774.2 cell lines). Results indicated increased uptake of drug into cells with a significant decline in the cellular burden of S. typhimurium [61]. In another study, polyisohexylcyanoacrylate nanoparticles were developed and loaded with ampicillin. This formulation was investigated against S. typhimurium infected murine macrophages (J774 and peritoneal cell cultures). Nanoparticles significantly increased the delivery of drug into macrophages and bacterial killing inside the cells was observed. Labeling of nanoparticles with tritium confirmed entry of formulation inside the cytoplasm and infected vacuoles [62]. Pinto-Alphandary and colleagues have also studied targeting of intracellular S. typhimurium using ampicillin loaded polyisohexylcyanoacrylate nanoparticles. Nanoparticles were found both isolated and in association with S. typhimurium entrapped within phagosomes/phagolysosomes. Thus, penetration of ampicillin was found to be enhanced via delivery using nanoparticles, as it can reach intracellular bacterial reservoirs more efficiently [63]. Fattal and coworkers have also established the effectiveness of polyisohexylcyanoacrylate nanoparticles loaded with ampicillin for its targeted delivery in S. typhimurium infected mice (C57BL/6) model. It was observed, that formulation delayed mortality, at a dose of 0.8 mg compared to free ampicillin that demonstrated similar effect but at a much higher dose (32 mg). Such result was attributed to enhanced accumulation of drug-carrier system in liver and spleen suggesting intracellular targeting an effective mean to restrain S. typhimurium infection [64]. Mudakavi and associates have developed protamine and pectin coated mesoporous silica nanoparticles that were further decorated with arginine for intravacuolar targeting of ciprofloxacin. These drug-loaded nanocarriers exhibited improved antibacterial activity against S. typhimurium because of localized delivery of drug [65]. In another study, chitosan nanoparticles and fucoidan coated chitosan nanoparticles were prepared for intracellular delivery of ciprofloxacin. Fucoidan coated nanoparticles exhibited superior antibacterial activity against S. paratyphi as compared to chitosan nanoparticles. Fucoidan coated nanocarriers showed better uptake by macrophages in contrast to chitosan nanoparticles [66].

Lipid based formulations for eradication of intracellular S. typhimurium

Scientists have also investigated solid-lipid nanoparticles (SLNs) for the delivery of antibiotics against intracellular S. typhimurium. Xie and coworkers have explored docosanoic acid SLNs for efficient intracellular delivery of enrofloxacin against S. typhimurium in RAW 264.7 cell lines. SLNs exhibited enhanced (27–38 folds) intracellular accumulation of the drug and strong inhibitory effect against intracellular S. typhimurium. The study also indicated slower removal of drug from inside of cells compared to free drug following extracellular drug elimination [67]. Desiderio and associates have developed liposomal formulation comprising cholesterol, phosphatidylcholine and phosphatidyl serine for intracellular delivery of cephalothin. The drug-liposome complex was successfully taken up by pre-infected murine peritoneal macrophages of S. typhimurium and significantly reduced intracellular bacterial burden as compared to free cephalothin [68]. Lutwyche and associates have also studied intracellular delivery of gentamycin using a liposomal carrier system. Gentamycin was incorporated in non pH-responsive dipalmitoylphosphatidylcholine and pH-responsive dioleoylphosphatidylethanolamine based liposomes and formulations were examined for delivery of drug into murine J744A.1 cell line that were pre-infected by wild-type S. typhimurium and recombinant-hemolysin expressing S. typhimurium strain. These formulations showed enhance uptake of gentamycin and successfully eliminated both strains that were colonizing inside macrophage cell lines [69]. In another study, gentamycin was encapsulated in pH-sensitive liposomal carrier to target intracellular S. typhimurium. Results showed a significant increase in intracellular accumulation of drug in infected liver and spleen due to liposomal encapsulation that led to a corresponding rise in anti-bacterial effect [70].

Metal based nanoparticles as potential systems for intracellular targeting of S. typhimurium

The growing interest in metal nanoparticles have directed researchers towards potential intracellular drug targeting. Yeom and coworkers have investigated gold nanoparticles for the delivery of antimicrobial peptides against intracellular S. typhimurium in preinfected HeLa cell line. Results showed improved cellular viability due to significant reduction in intracellular S. typhimurium. It was also observed, that antimicrobial peptides conjugated with gold nanoparticles, when introduced in S. typhimurium mice model, led to complete eradication of pathogens from colonized organs. Thus, gold nanoparticles were proposed to be a promising option for the eradication of intracellular pathogens in mammals [71]. In another study, gold nanoparticles have been studied for their potential antibacterial effect against S. typhimurium via apoptosis, intracellularly [72]. A brief summary of nanocarrier systems explored against intracellular S. typhimurium has been presented in Table 2.

Table 2.

Summary of nanoformulations investigated for intracellular targeting of S. typhimurium

Polymer	Carrier System	Therapeutic Agent	Year published	References
Gold	Nanoparticles	Gold	2019	[72]
Protamine/ pectin/ arginine	Mesoporous Nanoparticles	Ciprofloxacin	2017	[65]
Fucoidan/ Chitosan	Nanoparticles	Ciprofloxacin	2017	[66]
Docosanoic acid	Solid lipid nanoparticles	Enrofloxacin	2017	[67]
Gold conjugated with DNA aptamer	Metal nanoparticles	Histamine tagged DNA aptamer	2016	[71]
Chitosan	Nanoparticles	Ceftriazone	2012	[61]
Phosphatidylethanolamine/ N-succinyldioleoyl- phosphatidylethanolamine	pH Sensitive Liposomes	Gentamicin	2000	[70]
Dipalmitoylphosphatidylcholine/ dioleoylphosphatidylethanolamine conjugates/ polyethylene glycol-ceramide	pH sensitive liposomes	Gentamicin	1998	[69]
Polyisohexylcyanoacrylate	Nanoparticles	Ampicillin	1994	[63]
Polyisohexylcyanoacrylate	Nanoparticles	Ampicillin	1989	[64]
Cholesterol, phosphatidyl choline and phosphatidyl serine	Liposomes	Cephalothin	1983	[68]

Mycobacterium tuberculosis

Despite the availability of vaccine and a range of antibiotics, tuberculosis remains a top-notch killer among infectious diseases [73]. Although the primary site of infection is lungs, Mycobacterium tuberculosis (Mtb) may disseminate to other regions causing secondary extra-pulmonary infections in bones, joints, and CNS etc. Mtb has known to develop several mechanisms that protect it against the aggressive environment of host and allows it to breach natural defenses [74]. The presence of lipid rich cell wall containing mycolic acid, appears to be a key virulence feature. Mtb utilizes various cell surface proteins (integrin complement receptors and mannose receptors) to acquire access inside macrophages where they can persist and replicate, waiting for favorable circumstances for the dissemination of infection [75]. Once inside macrophages, Mtb buds out of fused phagolysosomes into vacuoles that are not capable to coalesce with lysosomes, and are recognized as a primary site for killing bacteria that gain entry into the host [76]. Mtb entrapped in phagosomes have been observed to be less acidic that is responsible for the termination of pathogen. This inhibition of phagosomes-lysosome fusion allows prolonged subsistence of Mtb within macrophages [77, 78]. Mtb has also been observed to arrest the maturation of phagosomes because of the continual presentation of Rab5 and enhanced survival of bacilli inside the macrophage [79]. Another intracellular survival strategy adopted by Mtb involves recruitment and retention of TACO (host protein) inside phagocytes invaded by bacilli averting transport of pathogen to lysosomes [80]. Various researchers have reviewed the survival strategies adopted by Mtb along with mechanisms for intracellular targeting. Kiran and coworkers have discussed various mechanisms involved in Tb granuloma development and resolution along with host targeted treatment strategies [81]. Donnellan and Giardiello have discussed several forms of nanomedicines for intracellular targeting of Mtb [82]. Nasiruddin and colleagues have reviewed nanopartricles as a convincing strategy for intracellular targeting of Mtb [83].

Multidrug resistant Mycobacterium tuberculosis

Regardless of the massive exploration of newer therapeutic choices, Mtb associated multidrug resistance is yet an untangled phenomenon [84]. Emerging resistance is demanding enormous contests in the implementation of Mtb control and prevention strategies. Multidrug resistant Mtb, thus, contributes towards the racing incidence of mortality. Moreover, late detection of a multidrug resistant-strain of Mtb delays the commencement of accurate therapy and further belittles the efforts to confine damages [85]. The emergence of extensively resistant strains of Mtb have also been identified [86]. Researchers have ascribed many different mechanisms to portray multidrug-resistance. Among these, genetic mutations hold a decent share that modifies drug targets or may cause a decrease in sufficient concentrations of drug at the site of infection, that can eradicate pathogen by inhibiting its activation [87]. One notable constraint, that makes treatment more challenging is hiding of Mtb inside mononuclear cells. This renders it difficult for drugs to reach the site of Mtb growth and replication at adequate concentrations, sponsoring the incidence of multidrug resistance [88]. Thus, it is essential to study the molecular mechanisms that are contributing towards the development of multidrug resistance [89].

Polymeric nanoparticles targeting intracellular Mycobacterium tuberculosis

Intracellular hiding of Mtb and its ability to escape natural defenses as well as conventional therapeutic strategies has forced the researchers to investigate mechanisms that can allow the drug to reach within Mtb reservoir at effective concentrations. This can be particularly helpful to overcome the lackings in conventional strategies, that have not been successful in controlling Mtb infection up to this far [90]. A study has demonstrated the use of modified mesoporous silica nanoparticles for intracellular delivery of rifampin and isoniazid. Mesoporous silica nanoparticles showed better uptake and release of drug intracellularly. Polyethyleneimine coating of rifampin loaded mesoporous silica nanoparticles exhibited better loading and efficacy of drug against Mtb infected macrophages as compared to uncoated nanoparticles. Mesoporous silica nanoparticles were also equipped with pH-dependent valves, comprised of cyclodextrin that exhibited release of entrapped isoniazid only in response to an acidic stimulus [91].

Anisimova and coworkers have described intracellular delivery of three drugs (isoniazid, rifampin, and streptomycin), using poly-n-butylcyanoacrylate and poly-iso-butylcyanoacrylate nanoparticles as carrier systems. Results demonstrated an enhanced intracellular accumulation of all three drugs [92]. In another study, poly-n-butylcyanoacrylte based nanoparticles have been employed for the delivery of moxifloxacin against intracellular Mtb. Drug loaded nanoparticles were observed to be distributed into the cytoplasm of macrophages and occasionally in close association with bacteria inside cells. As compared to free moxifloxacin, encapsulated drug exhibited increased intracellular accumulation, prolonged intracellular residence and retarded growth of Mtb at lower concentrations [93].

de Faria and associates have investigated citral derived, very lipophilic analog of anti-tubercular drug isoniazid that was observed to appreciably enhance nanoencapsulation and increase intracellular delivery of drug. Results indicated that nanoparticles interact with Mtb residing inside macrophages as well as extracellular bacteria [94]. Sharma and colleagues have developed porous-nanoparticle aggregate particles (micron-sized inhalable platform) for delivery of magainin I peptide in the lungs with improved stability. These nano complexes showed a prolonged antibacterial effect. This nanocarrier system was also observed to prevent the fusion of phagosome-lysosome in macrophages invaded by Mtb and promoted apoptosis in these cells [95]. In another study, poly(ε-caprolactone)-b-poly(ethylene-glycol)-b-poly(epsilon-caprolactone) based micelles were prepared, followed by coating with chitosan or hydrolyzed galactomannan and investigated for macrophage targeting of rifampicin. Formulations showed enhanced encapsulation of drug. Significant internalization of the drug into murine RAW 264.7 macrophages was observed for micelles coated with galactomannan, however, chitosan coating hindered cellular uptake of encapsulated drug [96].

Horvati and coworkers have investigated isoniazid loaded lipopeptide carrier system directed against macrophage-specific molecule tuftsin. Conjugate exhibited targeted delivery of drug to Mtb infected macrophages in the rat model (H₃₇RV) as compared to free drug. Bioavailability of drug was further enhanced by encapsulating the conjugate into poly(lactide-co-glycolide) based nanoparticles [97]. Lemmer and colleagues have developed isoniazid loaded poly d-lactic-co-glycolic acid-based nanoparticles that were modified with mycolic acid as a ligand. This carrier system was investigated for targeting of isoniazid in Mtb infected macrophages. Results suggested that mycolic acid increased the uptake of nanoparticles inside infected macrophages, while phagosomes containing these carriers were readily processed to phagolysosomes [98]. Edagwa and coworkers have suggested a longer acting nanocarrier system for intracellular targeting of rifampicin and pentenyl-isoniazid. Formulation exhibited increased internalization and longer retention by macrophages while both drugs were found to be localized in late and recycling endosomes [99]. Another study has proposed the loading of isoniazid into mannosylated gelatin-based nanoparticles, for its targeted delivery into alveolar macrophages. Results indicated efficient uptake of nanoformulation by alveolar macrophages with a significant increase in antibacterial action [100]. Choi and associates have investigated six nanoformulations of gallium-III and rifampin for their intracellular delivery. The study suggested use of folate or mannose conjugated block copolymer for the entrapment of gallium III that resulted in sustained release of encapsulated molecule along with appreciable inhibition of Mtb within macrophages. Dendrimer based nanoformulations for intracellular delivery of both gallium III and rifampin were also investigated that showed the promising inhibitory effect on intracellular Mtb [101]. In another study, gallium loaded nanoparticles were investigated for intracellular uptake by macrophages infected with a virulent strain of mtb. These nanoparticles showed significant uptake by infected macrophages along with sustained drug release for 15 days [102]. Tenland and coworkers have also investigated peptide-loaded mesoporous silica nanoparticles for intracellular targeting. The system was readily taken up by macrophages and showed an increased killing of Mtb in comparison to free peptide [103].

Abdelghany and associates have investigated alginate coated PLGA nanoparticles and alginate loaded PLGA nanoparticles for intra-macrophage delivery of two second-line anti-mycobacterial agents, amikacin and moxifloxacin. Both formulations showed rapid uptake by infected macrophages and an improved anti-mycobacterial activity [104]. Sharma and coworkers have demonstrated anti-mycobacterial activity of free motif (Pep-H) and its chitosan and gold-based nanoparticles. Mycobacterial burden was significantly reduced with both free motif Pep-H and its nanoparticle formulations, however, later systems exhibited higher activity at much lower concentrations [105]. Cotta and colleagues have investigated norfloxacin coated iron oxide nanoparticles for targeting macrophages infected with wild type and resistant strains of Mycobacterium smegmetis. Drug coated iron oxide nanoparticles exhibited 4-fold higher uptake by infected macrophages [106]. Grotz and associates have prepared rifampicin encapsulated polymeric micelles using poly (vinyl caprolactam)-poly (vinyl acetate)-poly (ethylene glycol) graft copolymer for pulmonary administration. These inhalable drug-loaded nanocarriers showed enhanced macrophage uptake and anti-mycobacterial activity [107]. Machelart and associates have explored the intrinsic anti-mycobacterial activity of poly β-cyclodextrin nanoparticles. These nanocarriers decreased colonization of Mtb inside macrophages by intervening with lipid rafts [108]. Rossi and coworkers have investigated inhalable powder for targeting alveolar macrophages that were successful in reducing mycobacterial burden. Sodium hyaluronate nanocomposite based respirable microparticles were used as a carrier for delivery of rifampicin, isoniazid, and verapamil [109]. Tripod and colleagues have reported vitamin E functionalized inulin micelles for delivery of rifampicin and were evaluated for macrophage uptake [110]. In another study, chitosan and tween 80 coated sodium alginate nanoparticles have been explored for coadministration of rifampicin and ascorbic acid via inhalation [111].

Liposomes for eradication of intracellular Mycobacterium tuberculosis

Dicetylphosphate modified phosphatidylcholine and cholesterol-based aerosolized liposomal formulation encapsulating rifampicin have been investigated for targeting of alveolar macrophages invaded by Mycobacterium smegmatis. Second strategy employed for intracellular targeting involved coating of phosphatidylcholine and cholesterol-based liposomes, with ligands specific for alveolar macrophages. Both formulations exhibited an increased concentration of rifampicin in the lungs. Ligand anchored liposomes showed increased delivery of drug to alveolar macrophages [112]. Mannosylated ciprofloxacin loaded liposomes were developed for pulmonary administration of drug to alveolar macrophages in rat. Results showed that mannosylated liposomes exhibited better targeting of drug with enhanced antibacterial effect in alveolar macrophages infected with Mtb as compared to unmodified liposomes [113]. Khademi and coworkers have investigated lipid-modified PLGA nanoparticles containing HspX/EsxS fusion protein as a delivery system against Mtb [114]. Table 3 presents a summary of nanoformulations that have been investigated for intracellular targeting of Mtb.

Table 3.

Summary of nanoformulations investigated for intracellular targeting of M. tuberculosis

Polymer	Carrier System	Therapeutic Agent	Year Published	References
Gallium	Nanoparticles	Gallium	2019	[102]
Silica	Mesoporous Nanoparticles	Peptide NZX	2019	[103]
Alginate/ Poly(lactide-co-glycolide)	Nanoparticles	Amikacin/ moxifloxacin	2019	[104]
Chitosan/ gold	Nanoparticles	Motif (Pep-H)	2019	[105]
Iron oxide	Nanoparticles	Norfloxacin	2019	[106]
Poly (vinyl caprolactam)-poly (vinyl acetate)-poly (ethylene glycol)	Micelles	Rifampicin	2019	[107]
Poly β-cyclodextrin	Nanoparticles	Poly β-cyclodextrin	2019	[108]
Sodium hyaluronate	Nanocomposite based microparticles	Rifampicin, Isoniazid, Verapamil	2019	[109]
Vitamin E/Inulin	Nanoparticles	Rifampicin	2019	[110]
Chitosan/ tween 80/sodium alginate	Nanoparticles	Rifampicin/ ascorbic acid	2019	[111]
Poly(lactide-co-glycolide)	Nanoparticles	Magainin I peptide	2018	[95]
Mannose or folate anchored poloxamer F127/ dendrimers	Nanoparticles	Ga(III)/ rifampicin	2017	[101]
Mycolic acid conjugated poly(lactide-co-glycolide)	Nanoparticles	Isoniazid	2015	[98]
Poly(lactide-co-glycolide)/ tuftsin	Lipopeptide nanocarrier	Isoniazid	2014	[97]
Poly(lactide-co-glycolide)	Nanoparticles	Rifampicin/ pentenyl isoniazid	2014	[99]
Poly(ε-caprolactone)/ poly(ethylene-glycol)/ chitosan/ hydrolyzed Galactomannan	Micelles	Rifampicin	2013	[96]
Poly ethyleneimine/ silica	Mesoporous silica nanoparticles	Rifampicin	2012	[91]
Poly(lactide-co-glycolide)	Nanoparticles	Citral derived isoniazid	2012	[94]
Mannosylated gelatin	Nanoparticles	Isoniazid	2011	[100]
4-aminophenyl-a-d-mannopyranoside	Liposomes	Ciprofloxacin	2008	[113]
Poly-n-butylcyanoacrylte	Nanoparticles	Moxifloxacin	2007	[93]
Egg phosphatidylcholine/ cholesterol / dicetylphosphate,/ maleylated bovine serum albumin/ o-steroyl amylopectin	Aerosolized liposomes	Rifampicin	2004	[112]
Poly-n-butylcyanoacrylate/ poly-iso-butylcyanoacrylate	Nanoparticles	Isoniazid/ rifampicin/ streptomycin	2000	[92]

Correlation of functionalized nanocarriers with in vivo animal models

Nanoparticles have gained wide acceptance in different therapeutic fields including drug delivery, prompt detection, sensing and catalysis [115, 116]. Translation of drug or formulation from preliminary research to sound clinical application involve both in vitro cell evaluation and in vivo studies. Although in vitro analysis of nano-formulation is of great importance, the microenvironment of in vitro cell and tissue cultures has been observed to be markedly different from diseased state [117]. Therefore, infections where pathogens reside and proliferate inside cells, such nanocarrier systems are designed that specifically deliver the drug to target reservoirs. In such cases, it is necessary to evaluate the uptake of drug-loaded functionalized nanocarriers in infected in vivo animal models along with in vitro cellular uptake studies [118]. In vivo animal models have also been widely explored to investigate potential toxicities associated with nanocarriers [119].

Conclusion

Intracellular invasion of pathogens including Staphylococcus aureus, Salmonella typhimurium, and Mycobacterium tuberculosis have been regarded as one of the major contributing factors towards emerging multidrug resistance. Such microorganisms have developed various survival mechanisms to escape the natural host defenses. Targeting these mechanisms through functionalized nanocarriers may be helpful in the utilization of already available compounds and decreasing the incidence of multidrug resistance. Scientists have thus, been trying to target intracellular reservoirs of pathogens, that will allow larger concentrations of drug to come in contact with bacteria, residing inside the cellular compartments. An appreciable reduction in bacterial burden can be observed at much lesser drug concentrations, along with decreased toxicity via transporting drug into bacterial reservoirs. In this regard, various nanocarrier systems (polymeric nanoparticles, metal-based nanoparticles, liposomes, and solid-lipid nanoparticles, etc.) have been investigated to deliver a drug into infected cells. These systems have shown great promise for the uptake of drug by target cells via attaching cell-specific ligands (mannose, mycolic acid and tuftsin, etc.) and exploiting other phagocytic mechanisms. Carriers bearing molecules isolated from or closely resembling bacterial components have also shown promising uptake into intracellular compartments. Nanocarriers have also demonstrated to prolong the release and residence of a drug inside target macrophages, thus, considerably decreasing intracellular bacterial burden. Based on these assessments, nanocarriers can play an important role in intracellular drug targeting in future. Exploration of novel cell specific surface molecules, intracellular markers and bacterial surface antigens may open new avenues for innovative targeting strategies and reducing the incidence of multi drug resistance.

Authors’ contribution

Maria Hassan Kiani and Muhammad Imran contributed in research and data acquisition. Abida Raza revised the manuscript. Basic concept and idea were conceived by Gul Shahnaz.

Compliance with ethical standards

Study does not include research on animal or human participants for clinical trials. Article has not been submitted to any other journal. Authors have studied COPE guidelines and tried their best to conform to ethical standards requested by journal.

Conflict of interest

The authors declare no conflict of interest.