Impact of nanoparticles on amyloid β-induced Alzheimer’s disease, tuberculosis, leprosy and cancer: a systematic review

Abstract Nanotechnology is an interdisciplinary domain of science, technology and engineering that deals with nano-sized materials/particles. Usually, the size of nanoparticles lies between 1 and 100 nm. Due to their small size and large surface area-to-volume ratio, nanoparticles exhibit high reactivity, greater stability and adsorption capacity. These important physicochemical properties attract scientific community to utilize them in biomedical field. Various types of nanoparticles (inorganic and organic) have broad applications in medical field ranging from imaging to gene therapy. These are also effective drug carriers. In recent times, nanoparticles are utilized to circumvent different treatment limitations. For example, the ability of nanoparticles to cross the blood−brain barrier and having a certain degree of specificity towards amyloid deposits makes themselves important candidates for the treatment of Alzheimer’s disease. Furthermore, nanotechnology has been used extensively to overcome several pertinent issues like drug-resistance phenomenon, side effects of conventional drugs and targeted drug delivery issue in leprosy, tuberculosis and cancer. Thus, in this review, the application of different nanoparticles for the treatment of these four important diseases (Alzheimer’s disease, tuberculosis, leprosy and cancer) as well as for the effective delivery of drugs used in these diseases has been presented systematically. Although nanoformulations have many advantages over traditional therapeutics for treating these diseases, nanotoxicity is a major concern that has been discussed subsequently. Lastly, we have presented the promising future prospective of nanoparticles as alternative therapeutics. In that section, we have discussed about the futuristic approach(es) that could provide promising candidate(s) for the treatment of these four diseases.


Introduction
Nanoparticles are particulate dispersions of solid particles ranging in size from 10 to 100 nm [1]. Nanoparticles have widespread applications in several disciplines including molecular biology, physics, organic and inorganic chemistry, medicine and material science due to their unique chemical, optical, electrical and magnetic characteristics compared with their bulk counterparts [2]. It has been found that reducing bulk materials to nano-size alters their physicochemical properties, which can be used in a variety of biomedical applications [3]. The large surface area-to-volume ratio of nanoparticles generally makes them highly reactive and confers a high adsorption capacity which in turn allows them to transport or interact with other molecules such as proteins, drugs, chemical compounds, etc [3]. Moreover, these interactions modulate the molecular or cellular activities, which make them a promising candidate for a variety of biological applications [3]. behaviour are extensively used due to their biodegradable and biocompatible nature for diagnosis and treatment including magnetic hyperthermia treatments. Magnetic NPs enable magnetic delivery of drugs ideal for directed delivery to affected tissue under the action of a magnetic field. However, potential neurotoxicity associated with different metallic/inorganic nanoparticles such as oxidative stress, free radical formation, immune response, lysosomal dysfunction and cell necrosis is a matter of concern for many researchers [50].
However, several inorganic and metallic nanoparticles provide good platform for the efficient inhibition of fibrillation process either in bare form or in conjugation with different fibrillation inhibitors. The most widely used metallic nanoparticle owing to few unique features like inert behaviour, local surface plasmon resonance absorption, tuneable structural and chemical properties is gold nanoparticle (AuNP) [51][52][53][54][55][56][57][58][59]. Bare AuNPs and AuNPs modified with peptides or other therapeutic molecules are majorly used for the treatment of AD and has shown anti-fibrillation property [54]. Bastus et al. showed that a combination of heat and gold nanoparticles can dissolve the amyloid deposits locally and remotely [55]. Moore et al. studied the effect of electric charge and surface chemistry of gold nanospheres on the modulation of Aβ fibrillation. They used gold nanoparticles coated with citrate, CTAB (cetyltrimethylammonium bromide), PAA [poly(acrylic acid)] or PAH [polyelectrolytes poly(allylamine)hydrochloride] and showed that surface chemistry as well as size determines inhibition ability to a great extent and electric charge determines the morphological features of aggregates. Among all the coatings, PAA-coated nanoparticles (with a diameter of 18 nm and less) are non-toxic in nature and showed the highest inhibition of Aβ 1-40 aggregation/fibrillation via a dynamic exchange between bulk solution and NP-localized peptide at a lower stoichiometric ratio of 1:2,000,000 with the peptide [56].
NIR absorbing Au nanocages developed by Shi et al., entraps a chelator that chelates metal ions like Cu 2+ that induce amyloid fibrillation and oxidative stress. In this novel methodology, Au nanocages with human IgG as a pore blocker bound via redox as well as thermal-sensitive arylboronic ester bond to generate phenylboronic acid functionalized Au nanocages (AuNC-PBA) that entraps clioquinol and causes its oxidative stimuli-response controlled release to act at the target site. AuNC-PBA possesses an edge length of about 50 nm and a negative charge over the surface that confer the ability to cross the BBB. When Aβ aggregation increase the level of H 2 O 2 , the arylboronic ester interaction breaks which is also accompanied by NIR light induced local heating that release clioquinol and helps in the chelation of Cu 2+ to dissolve amyloid-β plaques and inhibition of H 2 O 2 production. By this mechanism, this nanoformulation also helped in increasing cell viability of pheochromocytoma cells (PC12) to approximately 70% in contrast to cells induced with 5 μM Aβ-Cu 2+ toxicity that reduced the cell viability to 41% [57].
The resonating capability of gold nanoparticles upon the exposure to the light of specific energies, producing heat that can be used for photothermal therapy (PTT) to disintegrate amyloid deposits. In a study, penetratin peptide-modified poly(ethylene glycol)-stabilized gold nanostars (AuNS) further modified with ruthenium complex as luminescent probes has been synthesized. The conjugation of penetratin peptide with the AuNP enhances the permeability across the BBB. Additionally, the irregular morphology of the nanostars resulted a larger surface area and a high NIR absorption-scattering ratio. Aβ aggregation by the BCA protein assay in absence and presence of nanostars (10 and 20 μg/ml), showed an increase in soluble content of Aβ from 26% to 73% and 86%, respectively. Additionally, photothermal experiment (Ru@Pen@ PEG-AuNS−Aβ aggregates irradiated by 808 nm laser for 3 min) was performed by exploiting the NIR absorption property of AuNS. ThT fluorescence signal decreased more when Ru@Pen@ PEG-AuNS−Aβ aggregates were exposed to NIR than of non-irradiated Ru@Pen@ PEG-AuNS−Aβ aggregates. Possibly, the AuNS produce local heat upon laser irradiation that dissociate Aβ fibrils and destroy their amyloidogenic potential [58].
An excellent hybrid nanoparticle combining two peptide inhibitors/β-sheet breaker peptides (VVIA and LPFFD) has been designed by Xiong et al. First, a molecular hybrid has been developed by combining these two peptide inhibitors into a single sequence. Thereafter cysteine residues have been incorporated to the end parts of these short peptides/ β-sheet breaker peptides (VVIA and LPFFD) which allows their conjugation onto gold nanoparticle surface via Au-S chemistry. This hybrid AuNPs significantly inhibited Aβ 1-42 fibrillation process. Such inhibition mainly occurs due to hindrance in oligomerization process and reduction of β-structures and conversion to a higher proportion of random coils. The synergistic inhibition of these nanoparticles was greater as compared to single inhibitor sequence. Additionally, an increase in cell viability from 48% to 82% was achieved at a lower dosage of hybrid AuNPs (0.1 nmol/L of nanoparticles containing 40 nmol/L of inhibitor peptides) [59].
Similar to gold nanoparticles, silver nanoparticles can cause rapid dissolution of the fibrils. In a study, poly(vinyl) pyrrolidone (PVP)-stabilized negatively charged triangular silver nanoplates (AgTNPs) were found to be more effective than the PVP-stabilized silver nanospheres. The workers showed that when Aβ fibrils were treated with these AgTNPs under near infrared illumination, only 1 h was needed to dissolve the fibrils as compared with nanospheres that took roughly 70 h. Most effective NPs i.e. AgTNPs selectively bind to the positively charged residues present within the amino acid sequence of the peptide disrupted the fibril structure which led to dissolution of Aβ fibrils (as revealed from TEM and AFM studies) and thereby reduces cytotoxicity (as revealed from cell viability studies using SH-SY5Y and BE-(2)-C cells) [60].
Similarly, selenium nanoparticles have been demonstrated to be a useful therapeutic approach [46,61,62]. In a study, selenium nanoparticles were conjugated with two targeting peptides (LPFFD and TGN) and further conjugated with chitosan which produce 'dual function' selenium nanoparticles [46]. Those are L1T2-SeNPs, L1T1-SeNPs and L2T1-SeNPs where selenium nanoparticles having a 1:2, 1:1 and 2:1 concentration ratio of LPFFD to TGN, respectively. These SeNPs can serve two major functions; inhibition of Aβ aggregation and facilitates penetration through BBB. Among these three selenium nanoparticles, L1T1-SeNPs was most effective for inhibiting the aggregation process and reducing associated cytotoxicity in PC12 cells [46]. Synergistic effect of peptide and nanoparticle block the active site of fibril formation and reduce free monomeric peptide concentration, thus causing inhibition of Aβ 1-40 aggregation via hydrophobic and electrostatic connection. Also these nanoparticles facilitates penetration through BBB, and reduced Aβ-induced apoptosis by preventing ROS generation [46].
Leblanc and coworkers studied the anti-fibrillation potency of dihydrolipoic acid (DHLA) capped CdSe/ ZnS quantum dots of size approximately 2.5 nm. When these nanoparticles were mixed with amyloid β, they form conjugates with peptide leading to reduction in the fibrillation process which was studied using a fluorescence marker (Thioflavin T). Studies performed by using different analytical tools like TEM and AFM demonstrated that there is a noticeable change in morphology of fibrils conjugated to the quantum dots. Both the length and width of the fibrils were significantly altered [62].
Inorganic NPs having magnetic properties have also been utilized for the targeted delivery of different drug molecules and phytochemicals to the affected tissue. For example, a nano formulation has been made by a group of researchers that involves magnetic core-shell mesoporous silica nanomaterials as a carrier for the site directed delivery of quercetin, a polyphenolic phytochemical with known anti-fibrillation and anti-oxidant activity. This novel nanomaterial having a surface-modified monodispersed magnetite core, prepared using sol-gel process by taking tetraethoxysilane as a precursor. Subsequent evaluation of the biological activity of the nanoparticles revealed (i) relatively high entrapment efficiency and loading capacity of quercetin (70.35% and 14.51%, respectively), (ii) a high releasing percentage of 50.28% and (iii) increase in cellular viability of primary hippocampal neuronal cells up to 85.2%. When these NPs encapsulated quercetin, the stability and bioavailability of this phytochemical were greatly enhanced. The magnetic behaviour of these NPs enabled magnetic force guided on site release of quercetin which effectively inhibits nanoparticles administered to PC12 cells equivalent to 10 μM of resveratrol, better cytoprotective effect was observed as compared with free resveratrol by neutralizing the toxic effect of the amyloid beta peptide [70].
Few researchers have tagged polymeric nanoparticles to certain phytochemicals along with a carrier protein ApoE (apolipoprotein E). A study reported that ApoE3 mediated stable poly(butylcyanoacrylate)(PBCA) nanoparticles containing curcumin (ApoE3-CPBCA) has been formulated to enhance cellular uptake and anti-amyloidogenic activity of the tagged compounds [71]. Cytotoxicity studies on SH-SY5Y neuroblastoma cells and anti-apoptotic activity studies suggested the higher efficacy and increased uptake of curcumin by ApoE3-C-PBCA as compared with curcumin not conjugated with any nanoparticles. These nanoparticles imparted excellent neutralization effect against Aβ-induced cytotoxicity due to the synergistic effect of ApoE3 and curcumin [33].
Similar to phytochemicals, several peptide molecules have the potential to inhibit the Aβ induced toxicity effectively. However, their hydrophilic nature restricts them to cross the BBB [33]. In this regard, nanotechnological intervention specifically polymeric nanoparticles based approach provides a useful targeting strategy to overcome the obstacle. For example, Agyare et al. developed such type of nanoparticles that enclosed the sub-fragments of Aβ peptide in a tripolyphosphate gelation modified polymeric chitosan. It was further coated with polyamine modified F(ab ) portion of antibody IgG4.1 which is an anti-amyloid antibody. This engineered nanoparticles were able to cross the BBB and targeted amyloid formed by the aggregation of the causative peptide [72]. Thus, polymeric nanoparticles are excellent tools for the delivery of therapeutic molecules that otherwise would not have gained entry through the BBB. Several polymeric substances have been described to increase the circulation time of tagged therapeutic molecules substantially and protect nanoparticles from opsonization process by acting as a steric barrier. The muco-adhesive features and highly functionalized structure of polymeric substances helps in the site directed sustainable release of the drug molecules to the affected site of brain. The biodegradability nature of this class of nanomaterials is the major attraction for many researchers. Upon degradation, they produce metabolites that could be easily processed through biochemical pathways occurring inside the body. However, one disadvantage of polymeric nanoparticles is they sometimes release the tagged therapeutic molecules in sites other than the targeted area as a result of which only empty polymeric vector is delivered to the affected site. In contrast, various metallic/inorganic nanoparticles themselves possess the anti-aggregation properties and effectively inhibit aggregation/fibrillation process. Like polymeric nanoparticles metallic nanoparticles can also be tagged with different chemical groups such as ligands, antibodies, peptides etc. Their unique structural features help them to get combined with other treatment methods for fibril dissolution such as magnetic hyperthermia treatment, photothermal therapies etc. Interestingly, the mode of action(s) exerted by metallic/inorganic nanoparticles towards the inhibition of in vitro and in vivo Aβ fibrillation process is/are almost similar to that exerted by different polymeric nanoparticles (Figure 1). While inorganic nanoparticles are excellent in preventing amyloid fibrillation, polymeric nanoparticles are more efficient in dissolving formed fibrils. Despite the advantages, several in vitro and in vivo studies have reported excessive production of ROS upon exposure to metallic nanoparticles. Production of ROS increases the oxidative stress, inflammation and thereby causing damage to several vital structures of the body such as DNA, proteins and cell membranes [73]. Hence, right selection of nanoparticles is the determining factor for the effectiveness of the nanomaterial-based therapeutics.

Self-assembled nanoparticles
Various other nanoparticles are also studied for their therapeutic activity against amyloidal aggregation such as self-assembled nanoparticles. Taylor et al. prepared click-curcumin liposomes by attaching the curcumin derivative [N-propargyl 2-(3 ,5 -di(4-hydroxy-3-metoxystyryl)-1H-pyrazol-1 -yl)-acetamide] on preformed liposomes by click chemistry in PBS (pH 6.5). The mean diameter of these self-assembled nanoparticles varies from 52.8 to 218 nm. When tested for the fibrillation inhibition potential by ThT fluorescence assay and immunoassay, a substantial inhibition was observed in both oligomer and fibril formation. This is may be due to two possible factors. Firstly, due to the high affinity binding involving multivalent interactions between Aβ and click-curcumin liposomes. Secondly, because of the protrusion of curcumin-derivative molecule from the liposome surface, they are more accessible for the interaction with the Aβ species [74].
In another study, the amphiphilic nature of the N-terminal peptide fragment pN1-22 of ovalbumin is exploited and nanoparticles are synthesized by the self-assembly process in PBS at a temperature of 65 • C and pH 2.2. Additionally, these nanoparticles are noncytotoxic as evident from cell viability assay performed on PC12 cells that showed 100% viability at a concentration of 46 μM. The resulting nanoparticles possessed a diameter of around 30 nm and ζ-potential value of 35 mV. ThT fluorescence assays and morphological studies by TEM imaging showed the concentration dependent inhibition of Aβ 1-42 fibrillation in presence of pN1-22 nanoparticles. The half time (t 1/2 ) of Aβ fibrillation was increased with increasing concentration of the NPs that indicates the pN1-22 nanoparticles prevented Aβ fibrillation by binding to Aβ monomer as well as oligomers majorly through hydrophobic interaction and hydrogen bonding [49].
Li et al., reported self-assembled nanoparticles based novel two-in-one strategy of real time assessing as well as inhibition of Aβ peptide. Initially, the group self-assembled Aβ 15-20 peptide (KLVFF) and polyoxometalate (POM) which yielded hybrid colloidal nanospheres having the diameter ranging from 70 to 100 nm. Subsequently, they incorporated Congo red (a clinically used amyloid β fibril specific staining dye) into this hybrid colloidal nanosphere so as to monitor the fibrillation inhibition process via change in Congo red fluorescence. The synthesized nanomaterials possess combined anti-aggregation property of the β-sheet breaker peptide and POMs in a single system that resulted in higher inhibitory potential. At a 2:1 concentration ratio of Aβ 1-40 and the self-assembled POM-peptide nanospheres, the fibril bound ThT fluorescence was reduced greatly (65%). Whereas the same was reduced by 45% and 35% in presence of identical amount of peptide (KLVFF) and POM, respectively. In addition to this, the nanoparticles conferred targeted inhibition of Aβ aggregation in mice cerebrospinal fluid and reduced Aβ induced cytotoxicity [75].
Recent advances in the field of nanoquencher based biosensing has guided many researchers to formulate novel biosensing based inhibitors. Xia and co-workers prepared a nanosystem where self-assembled polydopamine nanospheres (PDANS), a nanoquencher, were conjugated with a carboxyfluorescein (FAM)-labelled DNA aptamer fluorophore. This nanosystem is useful for selective detection of Aβ oligomers via 'fluorescence-signal on' strategy. The mechanism behind this detection strategy involves the specific interaction of Aβ oligomers with FAM-DNA aptamers causing the fluorophore to change its conformation into a hairpin structure and is subsequently released from the surface of the nanoquencher PDANS that leads to the increase in the FAM-DNA fluorescence signal. A detection limit of 12.5 nM was achieved by this strategy. Further, the fibrillation inhibition potential of PDANS was also observed because of its interaction with Aβ monomeric species via hydrogen bonding [76]. This nanosystem could be an efficient diagnostic tool for detection of Aβ oligomers associated amyloid beta induced Alzheimer's disease.
At present, the implementation of nanotechnology-based therapeutics has gained tremendous consideration because of their ability to penetrate through the BBB and being able to safeguard the native structure of the inhibitor molecules. Several metallic/inorganic, polymeric as well as self-assembled nanoparticles conjugated with well-established inhibitors (phytochemicals, enzymes) have been designed as dual functional nanoparticles that confer the ability to get penetrated through BBB and have efficient anti-Aβ activities (summarized in Table 1). Additionally, many metallic nanoparticles are also being employed to treat several infectious diseases like tuberculosis and leprosy.

Nanotechnology based strategies to treat tuberculosis Tuberculosis and drug resistance
Tuberculosis (TB) is referred as an immemorial human malady and the discovered etiological agent was Mycobacterium tuberculosis [77]. According to the WHO report 2021, approximately 4.8 million people were diagnosed with pulmonary TB in 2020 globally. For efficacious treatment of tuberculosis, a six-month course of drug is administered to the TB diseased patients. The four first-line drugs: isoniazid (INH), rifampicin (RF), ethambutol (EMB) and pyrazinamide (PYZ) are basically used [78]. In spite of development of various effective anti-TB drugs, TB is still considered as a dreaded disease which affects the mankind with highest mortality and morbidity worldwide [79]. The drug resistance phenomenon is considered as the major obstruction in the proper treatment of TB. Consequently, in patients who developed rifampicin-resistant TB (RR-TB) and multidrug-resistant TB (MDR-TB, where the resistance is developed towards isoniazid and rifampicin, the two most effective anti-TB drugs), the treatment is prolonged, expensive with several side effects as well [78]. Apart from this, pre-XDR TB and XDR TB are of major concern. Pre-XDR TB is a diseased condition where the patient is resistant to rifampicin and any fluoroquinolone, a class of second-line anti-TB drug and XDR TB is a type of diseased condition where the patient is resistant not only to rifampicin and any fluoroquinolone, but also to at least one of the drugs i.e., bedaquiline and linezolid. The universally used vaccine for TB is bacille Calmette-Guerin (BCG) vaccine. This vaccine consists of live attenuated strain of Mycobacterium bovis. It has achieved success in prevention of TB in children but is inefficient to prevent the disease in adult individuals [80]. To overcome the problem of drug resistance, lack of effective vaccine and to achieve the End TB Strategy targets for 2030 and 2035, there is an urgent need of adequate research and innovation in this regard [78]. Highest inhibition in a lower stoichiometric ratio of 1:2,000,000 These nanoparticles are able to cross BBB and easily undergo clearance. 18 nm non-toxic PAA-coated NPs showed complete inhibition of Aβ 1-40 aggregation via a dynamic exchange between bulk solution and NP-localized peptide.
In vitro and cell culture study [56] NIR absorbing gold nanocages In vitro and cell culture study [59] poly(vinyl) pyrrolidone (PVP)-stabilized negatively charged silver nanoparticles PVP-stabilized negatively charged triangular silver nanoplates (AgTNPs) shown to be more effective than the silver nanospheres and dissolve fibrils in only 1 h upon near infrared irradiation. Nanospheres Edge length of nanoplates: 70 nm and diameter of nanospheres: 20 nm Increase in cell viability: 65% 30 nM administered into SH-SY5Y and BE-(2)-C cells AgTNPs selectively binds to the positively charged residues present within the amino acid sequence of the peptide disrupt the fibril structure and led to dissolution. The larger surface area of nanoplates adsorbs greater number of Aβ monomers.
In vitro and cell culture study [60] Selenium nanoparticles Selenium NPs conjugated with two targeting peptides (LPFFD and TGN), further conjugated to chitosan act as "dual function" selenium nanoparticles; prepared in presence of ascorbic acid; peptides taken in ratios of 1:2, 1:1 and 2:1, and the resultant NPs termed L1T2-SeNPs, L1T1-SeNPs and L2T1-SeNPs; L1T1-SeNPs shows highest aggregation inhibition and reduced cytotoxicity in PC12 cell. Size: around 100 nm 0.5 mg/kg Synergistic effect of peptide and nanoparticle block the active site of fibril formation and reduce free monomeric peptide concentration thus, causing inhibition of Aβ 1-40 aggregation via hydrophobic and electrostatic connection. Also facilitates penetration through BBB; reduced Aβ induced apoptosis by preventing ROS generation.
In vitro cell culture and animal studies [46] Continued over Nanoparticles interact with amyloid β, to form conjugates leading to change in morphology of fibrils and reduced fibrillation.
In vitro [62] Novel hybrid systems of magnetic core-shell mesoporous silica NP and quercetin.
Synthesized by sol-gel process in the presence of a precursor tetraethoxysilane and modification with PEG3K for reduced cytotoxicity; relatively high entrapment efficiency and loading capacity of quercetin, increase in cellular viability. In vitro/ in vivo [72] Continued over In vitro [74] The use of nanoparticles for the treatment of tuberculosis Mycobacteria are blessed to have a critical system of survival, which consequently help them to exist in the hostile environment of the host. Furthermore, the patient incompliance and the drug resistance phenomenon (both multidrug-resistance, MDR/ extensively drug-resistance, XDR) adds a bit more complexity for the treatment of tuberculosis. The current approach of nanotechnology involves greater prospect to be utilized for the treatment of TB, by preparing different nanoformulations which is yet to be explored [81]. The primacy of this therapeutic approach have several features: (i) efficient carrying capacity, (ii) more time span activity, (iii) facile route of administrations, (iv) loading of various drugs or easy encapsulations and (v) lower toxicity towards the host cells [82]. Myriad of metallic nanoparticles have been employed as anti-mycobacterial agents for several years [83]. Among them, silver and gold nanoparticles (AgNPs and AuNPs) are considered as important and effective because of their distinctive physical, chemical and biological characteristics [84]. In former times, silver was considered as an essential antimicrobial agent in treatment of various infectious entities [85]. So, both of these AgNPs and AuNPs may exhibit antimycobacterial effect alone or by being conjugated with various stabilizing agents/antibiotics/peptides/polymers to show a combinatorial effect which will help in the treatment of MDR/XDR TB [84]. It has been shown that these two nanoparticles can inhibit the growth of bacillus Calmette-Guerin (BCG), which is used as a surrogate for TB. Liang and co-workers used tdTomato, an important fluorescent protein expressed by mycobacteria in culture, so as to determine the number of viable mycobacterial cells by examining the fluorescence intensity. It was observed that the BCG fluorescence had decreased maximally upon the treatment with lower concentrations of citrate capped AuNPs (0.1 and 1 μg/ml) and higher concentrations of polyallylamine hydrochloride (PAH) capped AuNPs (10 and 20 μg/ml). On the other hand, the extent of inhibition of BCG fluorescence was almost similar in presence of 1-20 μg/ml AgNPs. The alterations in the number of colony forming units (CFUs) observed after the treatment with these nanoparticles were in agreement with the results of fluorescence experiment [86]. An independent study by Ignatov and coworkers revealed that the growth of M. tuberculosis H37Rv was inhibited in the in vitro treatment of 0.1-50 μg/ml polyvinylpyrrolidone (PVP) stabilized single dispersed silver nanoparticles (SNPs-PVP) [85]. The maximum inhibition (by two times) was observed in presence of 50 μg/ml SNPs-PVP. Similar results had also obtained in in vivo studies with experimental mouse model. The mycobacterial load in lungs and spleen of the mice had decreased by two times due to the SNPs-PVP treatment (0.1 mg/kg). Additionally, the effect of the treatment was also examined for inflammatory phenomenon in the lungs and the survival rate of the mice. Inflammation in lungs were decreased substantially and the survival rate had risen up to 60%. The interleukin-4 level (IL-4; an anti-inflammatory cytokine), in the broncho-pulmonary lavage fluid (BPL) after the SNPs-PVP treatment was reduced by two fold which possibly led to the decrease in inflammatory phenomenon in the lungs of experimental animals. SNPs-PVP treatment additionally reduced the level of different immunological markers such as interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α) etc. in body fluids of experimental TB mice. By considering all the results obtained from both the in vitro and in vivo studies, it can be concluded that SNPs-PVP can be considered as effective therapeutic agents against TB [85].
Attempts have also been made to study the inhibitory effect of silver nanoparticles towards drug resistant (both MDR and XDR) M. tuberculosis strains. Chen et al. explored the anti-mycobacterial activity of synthesized AgNPs [where alginate (ALG) was utilized as a capping agent] against numerous pathogenic mycobacterial strains which includes both drug-sensitive and drug-resistant strains (MDR and XDR strains) [81]. It was concluded from this study that alginate capped silver nanoparticles (ALG-AgNPs) inhibited the growth of above mentioned mycobacterial strains, without conferring any toxicity to the THP1 cells. In another approach of this study, when latency was induced in vitro, it had been observed that the growth of M. tuberculosis H37Rv was getting reduced appreciably in a dose-dependent manner of ALG-AgNPs, which implied that these nanoparticles can also be used to treat latent TB by subduing the dormant M. tuberculosis. Fluorescence-based cell permeability experiment revealed that ALG-AgNPs treatment increases the cell permeability which may be the possible mechanism behind its anti-mycobacterial activity. The therapeutic effect of ALG-AgNPs was further assessed by executing the in vivo studies on zebrafish and mouse TB model which unveiled that the bacterial load was reduced upon the treatment of these nanoparticles without causing any cytotoxicity in the host cell [81]. Even, silver containing mesoporous silica-based nanosystems (MSNs-AgBrNPs and Ag@MSNs) have growth inhibitory effects [87]. Interestingly, MSNs-AgBrNPs had shown efficient antimycobacterial effect in comparison with Ag@MSNs. The possible mechanism of better efficiency of MSNs-AgBrNPs over Ag@MSNs could be due to the distribution of AgBrNPs throughout the silica network and mesoporous channels; thus more exposed to the media, in comparison with the Ag core present in Ag@MSNs. The morphological studies (carried out by cryo-EM) further revealed that MSNs-AgBrNPs exerted efficient anti-mycobacterial activity by damaging the cellular envelope of M. tuberculosis H37Rv. Furthermore, in this paper, authors hypothesized that various drug candidates can also be loaded with these mesoporous nanosystems which can be considered as a better therapeutic approach for the treatment against drug resistant (both MDR and XDR) M. tuberculosis strains [87].
The anti-mycobacterial activities of biosynthesized AuNPs, AgNPs, and Au-AgNPs (synthesized from leaf extract of B. prionitis, root extract of P. zeylanica and bark extract of S. cumini) have also been explored. A study by Singh et al. depicted the growth inhibitory effect of these nanoparticles against M. tuberculosis in latent and active disease conditions [88]. The bimetallic nanoparticles i.e. Au-AgNPs are much more efficient in context of anti-mycobacterial activities than the monometallic ones (AuNPs and AgNPs). This study also revealed that Au-AgNPs synthesized from the bark extract of S. cumini plant, exhibit higher specificity and selectivity to the pathogen M. tuberculosis. Therefore, it can be concluded that the bimetallic nanoparticles i.e. Au-AgNPs could be more promising drug candidate than monometallic nanoparticles (AuNPs and AgNPs) for the treatment of tuberculosis [88].
Silver nanoparticles were found to enhance the cell membrane permeability by generating nano-sized pores on it. This consequently might be helpful for the entry of antibiotics [89]. It had also been examined that by encapsulating silver to a biocompatible polymer (e.g. poly (D,L-lactide-co-glycolide); PLGA), effective antibiotics can be delivered directly to the infected area of alveolar macrophages [83].
Interestingly, it has been observed that the anti-mycobacterial activity of gold nanosystems [gold nanorods (AuNRs) and gold nanospheres (AuNSs)] can be improved upon their administration with different conventional TB drugs. In a study by Ali et al., a drug carrier system had been prepared where AuNRs and AuNSs were conjugated with rifampicin (RF) which was meant to be delivered to the macrophage cells after the engulfment, and has shown anti-TB effect. It can be concluded from this study that these gold nanosystems could be considered as highly efficient drug carrier system in the therapeutic approach towards tuberculosis [90]. Other investigators also examined whether silver nanoparticles are able to carry drugs which are usually used for the TB treatment. Sun et al. revealed that the AgNP-Vancomycin (AgNP-VAM) conjugates which were tested on Mycobacterium smegmatis, showed significant anti-mycobacterial effects [91]. It has been found that by conjugating with AgNPs, the uptake of VAM was significantly enhanced, resulting in proper delivery of the antibiotic into the bacterium. By considering these results, it can be concluded that the current approach can be utilized for a better delivery of drug for the treatment of TB [91]. Another independent study by Golikov and coworkers, it was revealed that the inhibition of growth of mycobacteria was maximum with the usage of silver nanoparticles along with different drugs (isoniazid, rifampicin, ethionamide, levofloxacin and ofloxacin) and minimum when AgNPs were used along with kanamycin [92]. Similar results had also been observed in case of in vivo studies performed by this group. The survival rate of mice treated with silver nanoparticles with isoniazid were maximum i.e. ∼95%. While, mice treated only with the silver nanoparticles, the survival rate was found to be ∼35%. From these studies, it can be concluded that silver nanoparticles in combination with different antibiotics could be used as a promising therapy for the treatment of TB [92]. The results obtained have paved the way for the possibilities of use of these drug delivery systems to target infected macrophages in order to treat drug resistant TB (both MDR and XDR) [90]. All the studies mentioned in this section clearly depicted that bare/capped monometallic (AgNPs, AuNPs, citrate capped AuNPs, PAH capped AuNPs, ALG capped AgNPs, PVP capped AgNPs etc.) and bimetallic nanoparticles (Ag-AuNPs) can easily penetrate through the mycolic acid cell wall of M. tuberculosis and are the potential candidates for the treatment of tuberculosis (Figure 2 and Table 2).

Limitations of conventional leprosy therapy
Leprosy is a highly debilitating disease that has affected the mankind since the prehistoric times. Leprosy is treated by the Multidrug therapy (MDT) regimen introduced by the WHO in the 1980s. Despite the success of MDT in the treatment of leprosy, poor patient-drug compliance has been observed over the years due to the adverse side effects of these MDT drugs, namely rifampicin, dapsone and clofazimine [93]. These drugs, though highly effective against leprosy, are classified as Class II drugs according to the Biopharmaceutics Classification System as they have low water solubility and oral drug bioavailability [94]. Clofazimine tends to re-crystallize in cells and intestinal lumen due to  In vitro [87] Biosynthesized AgNPs, AuNPs, and bimetallic Au-AgNPs

Au-AgNPs dosage was 3 μg/ml
The bimetallic nanoparticles i.e. Au-AgNPs are much more efficient in context of anti-mycobacterial activities than the monometallic ones (AuNPs, AgNPs  The inhibition of growth of mycobacteria and mice survival rate were maximum with the usage of silver nanoparticles along with isoniazid In vitro and in vivo (mouse model) [92] the high dosage [95]. There are also incidences of antibiotic resistance cropping up due to such issues. Brazil, India and other parts of south-eastern Asia heavily contribute towards the global leprosy burden. Such problem demands the emergence of new therapeutic tools against leprosy. To counter the issues of poor patient-drug compliance and adverse side effects of these drugs towards patients, several research groups are working towards developing various nano-systems that would help in targeted delivery of these drugs. These nano-systems may potentially reduce the dosage of these drugs as well as promote sustained drug release on targeted site and also increases the bioavailability of these drugs. This approach also decreases the side effects associated with these drugs.

Usage of nanoparticles/nanoformulations for the effective delivery of anti-leprotic drugs and treatment of leprosy
Dapsone (Dap) loaded pH sensitive nanoparticles were prepared by Chaves et al. by employing a copolymer Eudragit L100 (EL100), which is soluble at a pH above 6.0. Optimized using the Plackett Burman Design and Box-Behnken Design, the nanoparticles were of the size ∼198 nm. These nanoparticles retained the drug in simulated stomach environment at pH 1.2 and most of the drug was released upon reaching pH 6.8. This formulation was non-cytotoxic towards various cell lines (upto 400 μg/ml) and it also had a sustained drug release pattern with better permeability compared to free drug [96]. The same group also developed poly (D,L-lactide-co-glycolide); PLGA copolymer based clofazimine (Clz) nano-delivery system using Plackett Burman Design (particle size: ∼211 nm), which gave similar advantages over free clofazimine. These advantages could be attributed to the properties of copolymer PLGA. Degradation of PLGA matrix consequently helped sustained drug release [97]. They also used the combination of these two nano-systems (Clz-PLGA and Dap-EL100) and found efficient intestinal permeability. Such combination would also circumvent the free drug associated toxicity to an extent, avoiding intestinal damage, hence improving patient compliance [98]. Mesoporous silica nanoparticles loaded with high amounts of clofazimine by using acetophenone as a co-solvent were developed by Wei Chen et al. These nanoparticles released clofazimine in high amounts which enabled the killing of M. tuberculosis bacteria without affecting the macrophages [99]. Many solid-lipid nanoparticles (SLNs) have also been used for the effective delivery of different anti-leprotic drugs. Reis and coworkers utilized Box Behnken design for the optimization of mannosylated SLNs having particle size of ∼333.2 nm [100]. The release rate of drug from mannosylated SLNs coated dapsone depends on pH i.e., released faster at acidic pH as compared with neutral pH. This also targeted the intestinal microfold cells (M-cells) which transports antigens from intestinal lumen to the immune system and mannosylation helps in this uptake. These nanoparticles were stable for 8 weeks [100]. Another SLNs i.e., Lactonic sophorolipids stabilized with various non-ionic polymeric surfactants (Pluronic F68 and F127) were utilized by Kanwar et al., to prepare dapsone and rifampicin loaded SLNs whose drug release profile was modelled kinetically. It was observed that drug transport mechanism for dapsone and rifampicin are Fickian driven process and Non-Fickian process, respectively. It had high entrapment efficiency and loading capacity as well.
Despite the high release rate of the drugs from the SLNs, the drug concentrations were well within therapeutic limits indicating controlled drug release [101,102]. Nanoemulsions derived from micellar solutions are efficient delivery systems which can increase drug absorption by protecting the drugs from hydrolysis and degradation [102][103][104]. Dapsone containing oral nanoemulsion and topical dapsone nanoemulsion was developed by Monteiro et al. [103] and Borges et al. [105], respectively. The dapsone oral nanoemulsion was developed using propylene glycol as a co-solvent and was a water in oil (w/o) system which was inverted in vivo to an oil in water (o/w) system. Such inversion increased the drug permeability as well as the drug dissolution compared with powdered dapsone [103]. Topical administration of drugs resulted in dose reduction In vitro [108] that limited the adverse effects caused due to higher dosage of the drugs [105]. A topical dapsone nanoemulsion was developed by using isopropyl myristate and n-methyl pyrrolidone, where isopropyl myristate containing nanoemulsion showed higher in vitro epidermal permeation whereas n-methyl pyrrolidone had higher dapsone solubilization and in vitro release rate. These formulations were also stable for a period of 3 months [105]. The mode of action, anti-leprotic drug release along with other important details of all these above mentioned nanoparticles have been summarized in Table 3. Meanwhile, Taufikuromah et al. used a combination of gold and silver nanoparticles to topically treat epidermal wounds due to leprosy. They found that such a combination was effective in reducing the wound width by 80% in patients under the MDT regimen while the wound width of the control group had decreased only by 50% (Table  3) [106]. However, the mode of action of these two nanoparticles towards the inhibition of growth and survival of Mycobacterium leprae (leprosy causing pathogen) is mostly unclear. The non-culturable nature of this pathogen in artificial medium hinted towards this understanding. Our group has taken an indirect approach to understand the possible mode of action of these two nanoparticles. The effect of ∼20 nm citrate capped silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) on HSP18, an 18 kDa immunodominant small heat shock protein belonging to M. leprae was tested. This small heat shock protein is also responsible for the growth and survival of this pathogen in host macrophages by virtue of its chaperone function [93,107]. Fluorometric characterization of binding of these two nanoparticles with HSP18 revealed the nature/stoichiometry of binding i.e., one silver nanoparticle/gold nanoparticle binding per subunit of HSP18 with binding affinity in the submicromolar range [108]. It was further found that the interaction of these two nanoparticles with HSP18 affects the structure, chaperone function and protective efficacy of HSP18 in a distinctly opposite manner. HSP18-AgNPs interaction led to oligomeric dissociation, decreased surface hydrophobicity, thermal stability, protective efficacy and reduced chaperone function of HSP18, while all of these were enhanced upon interaction of HSP18 with AuNPs ( Figure 3 and Table 3) [108]. This particular study evokes the possibility of nanoparticles for the effective treatment of leprosy by targeting different overexpressed antigenic proteins within the pathogen during infection [108]. In future, nanoformulations that have the ability to target HSP18, an important mycobacterial small heat shock protein, could be employed for treating leprosy patients. It is noteworthy to mention here that heat shock proteins especially large HSPs are the important drug targets in cancer and nanotechnology has been extensively used to carry their inhibitors to the cancer cells.

Nanotechnology-based approaches for the treatment of cancer by targeting large heat shock proteins HSP90: an important drug target in cancer
Cancer is a generic term for a group of diseases that possess serious threat to public health globally. It is characterized by the development of abnormal cells formed by the uncontrolled cell division which eventually leads to tissue destruction. Considering its diverse forms, high lethality and widespread occurrence in population, designing of specific diagnosis as well as effective treatment is a huge challenge to combat this disease. Currently, various approaches have been implemented for the specific diagnosis and therapeutic development for cancer. However, conventional diagnostic and therapeutic approaches for cancer are severely threatened by the typical features of malignant tumours, including their infinite capability for replication, metastasizing ability, immune evasion and heterogeneity [109,110]. Presently, numerous genes, active enzymes and proteins in connection to different stages of cancer manifestation are being targeted for the proper diagnosis and effective treatment of cancer [111,112]. One such target is a large heat shock protein namely HSP90 which has been widely investigated and different HSP90 targeting molecules have reached clinical trials. HSP90 plays an essential role in many physiological activities [113,114]. HSP90 family proteins are a group of evolutionarily conserved protein found in eukaryotic cells, having monomeric molecular weight of ∼90 kDa. HSP90 acts as molecular chaperones by aiding in the proper folding of newly synthesized polypeptide chains and preventing the aggregation of stressed client proteins [113,114]. Members of mammalian HSP90 family are located in various cellular compartments-HSP90 α and HSP90 β in the cytoplasm, glucose-regulated protein 94 (GRP94) in the endoplasmic reticulum (ER) and tumour necrosis factor receptor-associated protein 1 (TRAP1) in the mitochondrial matrix [113,115]. These proteins are involved in important cellular processes and biochemical pathways like apoptosis, cell cycle control, cell survival, protein folding and degradation and cell signalling [113][114][115]. It is well known that HSP90 plays a vital role in protein homeostasis, cell differentiation and development [113][114][115]. However, overexpression of HSP90 is related to many diseases, including viral infections, inflammation, neurodegenerative diseases and several types of cancers [113][114][115].
It has been reported that HSP90 performs critical roles in maintaining the activity, proper folding, stability, functioning and proteolysis of several oncoproteins in various types of cancers [116]. It is usually overexpressed in cancer cells which lead to increase in tumour growth, adhesion, invasion, metastasis and angiogenesis [117]. Moreover, it has been shown that cancer cell epithelial-mesenchymal transition (EMT), invasion, migration and tumour metastasis are all facilitated by HSP90 expression, in accordance with the activation of hypoxia-inducible factor 1α (HIF-1α) and nuclear factor kB (NF-kB) [115]. Furthermore, HSP90 was shown to regulate the transcription and expression of vascular endothelial growth factor receptor (VEGFR), which is a key molecule involved in the process of angiogenesis. Hence, the overexpression of HSP90 could potentially increase tumorigenesis, migration, penetration and angiogenesis [118]. It is also known that the expression level of HSP90 can serve as a possible biomarker for a number of different cancers including, lung cancer, esophageal carcinoma, bladder cancer, melanoma, leukemia, non-small cell lung cancer (NSCLC) and breast cancer [119][120][121][122].

Different HSP90 inhibitors and their transportation in different cancer cells using nanosystems
As HSP90 is an important drug target, investigators have developed several HSP90 inhibitors. These inhibitors are broadly classified into two categories: (i) N-terminal HSP90 inhibitors; examples are geldanamycin (GA), tanespimycin or 17-allylaminogeldanamycin (17-AAG), radicicol and 17-(2-dimethylaminoethyl)amino-17-demethoxygeldanamycin (17-DMAG) [123][124][125][126]. (ii) C-terminal HSP90 inhibitors such as novobiocin (nvb), 6-bromo-3-[4-methoxyphenylcarboxamide]-quinolein-2-one (BrCaQ), cyclic peptide analogs (SM122, SM145 and SM253) [127,128]. The most commonly used FDA approved HSP90 inhibitor is 17-AAG. Despite its selective mechanism of action on cancer cells, 17-AAG faces challenging issues due to its poor aqueous solubility, short biological half-life and hepatotoxicity which hindered its progression into clinical trials. Nanotechnology based approach has been employed to overcome these problems and to deliver 17-AAG and other HSP90 inhibitors in different cancer cells. Xioang et al. have reported the use of degradable amphiphilic diblock polymers of poly(ethylene oxide)-block-poly(D,L-lactide) [PEO-b-PDLLA] as nano-carriers which increased the aqueous solubility of 17-AAG by 150-fold as compared with the solubility of 17-AAG in absence of these nano-carriers [129]. Use of nano-carriers has increased the half-life of 17-AAG in serum and blood along with an increased volume of distribution in rat model [129]. Chandran et al., have formulated PEG-DSPE micellar nano-carriers (prepared using dry film method) for the delivery of 17-AAG without the inclusion of any organic solvent [130]. They have found that by modulating the PEG-DSPE concentration and incorporation of D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) in the micelle composition, has decreased the release of 17-AAG significantly from the micelles [130]. In an independent study, Hussain and co-workers synthesized polylactide-co-glycolide-polyethylene glycol-folic acid (PLGA-PEG-FA) using nanoprecepitation method. Then, this nanoparticles containing 17-AAG were characterized and studied their cellular uptake and cytotoxicity in MCF-7 breast cancer cell lines. They observed a superior cellular uptake and increased cytotoxicity of folate targeted nanoparticles in comparison to free 17-AAG [131]. The same group also synthesized Pluronic ® P-123 and F-127 mixed micelles loaded with 17-AAG using thin film hydration method which can internalize inside the human brain tumour glioblastoma cell lines [132]. These 17-AAG loaded mixed micelles showed increased cytotoxicity to the glioblastoma cells as compared with free drug. Thus, this particular study hinted towards usage of 17-AAG loaded Pluronic ® P-123 and F-127 mixed micelles for the treatment of the most common and aggressive malignant primary brain tumour in human glioblastoma multiforme (GBM) [132]. Polymeric micelles carrying 17-AAG were synthesized by Larson et al., using poly(styrene-co-maleic acid) [SMA] copolymers that exhibited potent in vitro activity against DU145 human prostate cancer cells [133]. This nanoformulation was also well tolerated and exhibited potent anti-cancer activity in mouse model harbouring prostate cancer tumour xenograft. Von Hoff and co-workers had commenced a Phase I trial of a nanoemulsion of 17-AAG, namely CNF1010, but due to the toxicity issue, it was dropped from clinical trials [134]. In another Phase II clinical trial carried out by Tao et al., the drug 17-AAG was successfully converted to albumin-bound nanoparticles (nab-17-AAG) but the trails were withdrawn later [135]. Similarly, a study carried out by Won et al. described a self-assembled biodegradable recombinant human gelatin (rHG) modified with α-tocopheryl succinate (TOS) nanoparticles via EDC/NHS reaction that was efficiently encapsulated with 17-AAG [136]. This 17-AAG-loaded nanoparticles were non-immunogenic and more efficient than free 17-AAG. Along with this, from studies executed in tumour mouse model, the enhanced permeability and retention effect of nanoconjugated 17-AAG was evident [136]. Rubinstein and co-workers used co-precipitation/reconstitution method to solubilize 17-AAG into extendable (PEGylated), biocompatible and biodegradable sterically stabilized phospholipid nano-micelles that increased intracellular uptake of 17-AAG-loaded phospholipid nanomicelles thus, amplifying the drug potency and its cytotoxicity to MCF-7 human breast cancer cells [137].
Additionally, co-administration of polymer-based nanoshells which consists of multiple drugs along with 17-AAG was initiated. Kwon and his co-workers designed a 3-in-1 polymeric micelle nano-container for delivering three drugs that includes, 17-AAG (HSP90 inhibitor), rapamycin and paclitaxel [138]. The polymeric nano-carrier poly(ethylene glycol)-block-poly(d,l-lactic acid) [PEG-b-PLA] micelles were capable to solubilize the drugs. According to the findings of this study, micelles composed of 3-in-1 PEG-b-PLA exerted a powerful synergistic effect in breast cancer cell lines (MCF-7 and 4T1) [138]. Notably, intravenous (IV) injection of 17-AAG, paclitaxel and rapamycin using micelles composed of 3-in-1 PEG-b-PLA was well tolerated by FVB albino mice [138]. In an independent study, Kozak and co-workers formulated a multidrug-loaded nano-micelle, triolimus, containing paclitaxel, rapamycin and 17-AAG [139]. The combination of these three drugs in nano-micelle exhibited potent cytotoxic synergy against lung cancer cell line (A549) and breast cancer cell line (MDA-MB-231). This nano-drug conjugate had an inhibitory effect on the Ras/Raf/mitogen-activated protein kinase pathway as well as the PI3K/Akt/mTOR pathway [139]. In a tumour xenograft mouse model, using this nano-micelle triolimus, a delay in tumour growth as well as a 50% reduction in tumour cell proliferation was observed as compared to paclitaxel-containing micelles. Similar kind of study was performed using different combinations of drugs in nano-carrier were found to be effective against a wide range of cancers. Some of the examples are: 17-AAG/Paclitaxel combination in polyethylene glycol-poly(D, L-lactic acid) [PEG-PLA] and PEG-distearoylphosphatidylethanolamine/tocopheryl polyethylene glycol 1000 (PEG-DSPE/TPGS) nano-micelles against human ovarian cancer [140], 17-AAG/Docetaxel drugs in hyaluronic acid (HA)-decorated poly(lactic-co-glycolic acid) (PLGA) nanoparticles [HA-PLGA NPs] against squamous cell carcinoma [141], 17-AAG/Doxorubicin combination in polypeptide based nanogels against ErbB2-driven breast cancer [142].
Beside 17-AAG, another N-terminal HSP90 inhibitor is geldanamycin (GA). Attempt has been taken for delivery of this anti HSP90 inhibitor utilizing nanoformulations. Forrest and coworkers have formulated a lipophilic GA prodrug, 17'GAC (16)Br encapsulated in acid-catalyzed ring opening polymerization mediated methoxy-capped poly(ethylene glycol)-block-poly(epsilon-caprolactone) [mPEG-b-PCL] micelles [143]. This micellar nanoformulation exhibited significant accumulation and lower systemic toxicity in rat models. Semi-synthetic derivative of geldanamycin and 17-DMAG (another important N-terminal HSP90 inhibitor) were also delivered into cancer cells by utilizing nano-carriers. It has been reported that mRNA expression of HSP90 gene in lung cancer and breast cancer cell lines was inhibited by 17-DMAG, which was physically encapsulated in PLGA-PEG (poly (DL-lactic-co-glycolic acid)-polyethyleneglycol) nanoparticles. PLGA-PEG was synthesized using double emulsion method [144,145]. This nanoencapsulated complex exhibited higher inhibitory effect on lung cancer A549 cell line and T47D breast cancer cell line as compared to 17-DMAG alone [144,145]. SNX-2112 is another N-terminal HSP90 inhibitor and a promising anticancer agent but has poor solubility. To enhance its solubility, Wu and co-workers developed a nanocrystal formulation for SNX-2112 and found that, due to their cosolvent-like pharmacokinetic behaviour, the nanocrystals rapidly released the SNX-2112 drug molecules in in vivo rat-model [146]. The same group further formulated glucose-based mesoporous carbon nanospheres (MCNs) using hydrothermal reaction to efficiently load SNX-2112 which significantly enhanced the cellular uptake, biodistribution and anti-tumour effect in xenograft of breast cancer of mice model [147].
Scientists have also utilized various nano-carrier to deliver different C-terminal HSP90 inhibitors into cancer cells. Sansalvamide A derivatives, a novel series of C-terminal HSP90 inhibitors were synthesized by Kim et al. and conjugated them to the star polymer core to generate nanoparticles [148]. These polymer conjugated HSP90 inhibitors induced apoptosis by a caspase 3-dependent pathway and also imparted cytotoxicity to a colon cancer cell line [148]. Sauvage et al. have loaded another C-terminal HSP90 inhibitor, 6BrCaQ [N-(6-bromo-1-methyl-2-oxoquinolin-3-yl)-4-methoxybenzamide] into nanometre-scaled liposomes and examined their suitability for drug delivery to solid tumours. Administration of liposomal 6BrCaQ showed decrease in HSP90 expression and significant anticancer activity towards prostate cancer cell lines [149].
Interestingly, one novel gene therapy strategy was taken to deliver micro-RNA (miRNA) of HSP90 using nanotechnology approach. Banerjee and co-workers designed an artificial anti-HSP90 miRNA plasmid vector which was delivered through a glucocorticoid receptor (GR) targeted liposome complex for targeting and down-regulation of HSP90 in cancer cells [150]. GR-mediated delivery of miRNA-HSP90 plasmid in tumour-bearing mice enforced apoptosis in angiogenic vessels and in tumour mass as well as significantly shrunk tumour-volume [150]. As a whole, this unique strategy showed significantly higher anti-cancer activity against tumour models of melanoma and lung cancer.

Utilization of inhibitors of HSP90 and HSP70 in cancer thermotherapy
The delivery of these anti-HSP90 nanoformulations also has usage in the thermotherapy of cancer. Thermotherapy involves heating the tumour to cause local hyperthermia. One of the major concerns impeding the advancement of this therapy is the damage of adjacent healthy tissues due to heating. Hyperthermia also causes a rise in the expression of heat shock proteins, notably HSP90 and HSP70, which counteracts the induction of apoptosis and makes the cell resistant to thermal injury. To overcome this limitation, nanotechnology-based approaches have been adopted for precise and selective 'intracellular' heating along with diminishing the protective functions of HSP90 and/or HSP70. For example, Fraifeld and co-workers have described that human ovarian cancer cells (SKOV-3) exhibited a greater sensitivity to hyperthermia when treated with geldanamycin (GA) and novobiocin (nvb) [151]. Furgeson's group reported that poly(K) 8 -poly(VPGXG)60 block copolymers [K 8 -ELP(1-60)] conjugated with geldanamycin can be used in conjunction with heat to produce an effective chemotherapy regimen [152]. In addition to these, HSP90 inhibitors in conjugation with various nano-encapsulation were co-delivered with photothermal agents to reduce the thermo-resistance of cancer cells [153][154][155][156].
Attempts have also been made to produce hyperthermia utilizing nanoformulations containing HSP90 inhibitor. Liposomes loaded with HSP90 inhibitor and iron oxide nanoparticles (also known as magnetoliposomes) generated heat when exposed to a magnetic field. This form of liposome was employed to encapsulate 17-AAG in a recent study, and it was coated with an antibody targeting CD90 to improve uptake by tumour stem cells [157]. Furthermore, in vitro and in vivo experiments using liver cancer stem like cells (LCSCs) were carried out so as to assess hyperthermia inducing potency of this magnetic nanoformulation. Over-expression of HSP90 was predicted to be observed in LC-SCs after exposure to magnetic hyperthermia. Magnetoliposomes targeted to CD90 that included 17-AAG showed greater anti-tumour effects upon thermotherapy compared to magnetoliposomes without the encapsulated drug. Recently, the effectiveness of 17-AAG in poly (lactic-co-glycolic acid) nanoparticles loaded with Fe 3 O 4 for the treatment of pancreatic cancer was evaluated by Rochani et al. [158]. The pancreas cancer cell line has been shown to lose 75% of its viability after being subjected to magnetic field induced hyperthermia for 3 h. This finding demonstrates the effectiveness of combining heat and heat shock protein suppression in cancer treatment [158].
Apart from HSP90, another important large heat shock protein which has been targeted in nanoparticles based thermotherapy of cancer is HSP70. Gold nanoparticles (AuNPs) are used as drug (some well-known HSP70 inhibitors) carriers to treat various types of cancer. These nanoparticles, due to their EPR effect, can easily accumulate in tumours. More importantly, the ability of gold nanoparticles to convert light energy into heat, makes them important candidates for tumour-selective photothermal therapy. To find out the effect of VER-155008, a well known HSP70 inhibitor, on the sensitivity of tumour cells to heat, Tang et al. conjugated VER-155008 with methoxy-polyethylene-glycol-coated-gold-nanorods (MPEG-AuNR) [159]. It has been found that VER-155008 micelles down-regulated the expression of HSP70, thus attenuated the heat-resistance of tumour cells and enhanced the therapeutic outcome of mild-temperature photothermal therapy against colon cancer [159]. In an independent study, where Wang and co-workers employed core-shell structure Au@SiO 2 nanomaterials with high photothermal performance conjugated with an HSP70 inhibitor 2-phenylethynesulfonamide (PES) into breast cancer tumour cells and exposed them to near-infrared light (NIR), they found that this irradiation generated mild heat induced apoptosis or necrosis at relatively low temperature [160]. All evidences presented in this section clearly depicted the immense potential of HSP90 inhibitor/HSP70 inhibitor based nanoformulations towards the treatment of different types of cancer ( Figure 4 and Table 4).

Toxicity of nanoparticles
Irrespective of the several added advantages of nanoparticles over traditional therapeutic agents, the success rate of nanoparticles to be utilized clinically is majorly halted due to their toxicity. Nanotoxicity is of great concern particularly in case of nanoparticles mediated drug delivery process. It has been reported that cationic nanoparticles impart more toxicity compared to that of anionic nanoparticles [1]. Interestingly, some of the inherent characteristics of nanoparticles like higher surface reactivity, higher surface-to-volume ratio etc., contribute towards their toxicity [161]. When nanoparticles interact with different biomolecules especially with protein, 'protein-corona' forms, such formation sometimes alters the pharmacological behaviour of nanoparticles in such a manner which could induce toxicity [162]. Basically, variable interaction or affinity of different nanoparticles with this protein corona can generate reactive oxygen species (ROS) which consequently causes cellular damage. Metal based nanoparticles can also produce ROS through surface modulation and through Fenton, Fenton-like and Haber-Weiss reactions [163]. Several attempts have been made to assess the toxic potential of nanoparticles of different blood cell components [164][165][166][167][168][169][170]. Huang and co-workers revealed the toxic effect of silver nanoparticles to red blood cells (RBCs) [164]. This group convincingly showed that the silver nanoparticles of 15 nm diameter induce more haemolysis and membrane damage than silver nanoparticles having larger size (50 and 100 nm). Some other inorganic nanoparticles (TiO 2 and ZnO nanoparticles) induced toxicity also and increase haemolysis as well as the monocyte-derived dendritic cell (MDDC) death [165]. The size of nanoparticles also contributes towards their toxicity. Battal et al. demonstrated that the size of SiO 2 nanoparticles greatly influence its genotoxic/mutagenic and cytotoxic effects [166]. These two effects of nanoparticles also depend on surface coating. Magdolenova et al. demonstrated that iron oxide coated with oleate are more cytotoxic and genotoxic than uncoated iron oxide nanoparticles [167]. When Delogu and co-workers subjected various pre-coated nanocapsules (Pluronic coated, Chitosan coated and Polyethylene glycol coated) to human primary immune cells, they observed that these nanocapsules differentially affect immune cells [168]. Yu and co-workers studied the effect of multiwall carbon nanotube on the DNA damage and cytotoxicity in male human peripheral blood lymphocytes and they found that these nanoparticles were able to induce considerable DNA damage  17-AAG and PEO-b-PDLLA (PEO and PDLLA) was dissolved in dimethylacetamide.

Breast cancer
In vitro and in vivo [131] Pluronic ® P-123 and F-127 mixed micelles Prepared using thin film hydration method and was dissolved in ethanol along with 17-AAG.

Human brain tumour glioblastoma
In vitro and in vivo [132] poly(styrene-co-maleic acid) [SMA] copolymer micelles Tanespimycin (dissolved in minimal DMSO) was added to SMA solution dropwise.

Human prostate cancer
Phase I clinical trial (withdrawn) [133] human gelatin (rHG) modified with alpha-tocopheryl succinate (TOS) nanoparticles rHG-TOS was formed by EDC/NHS reaction. DMSO was used to dissolve it and 17-AAG.

Lung and breast cancer
In vivo and ex vivo [139] polyethylene glycol-poly(D, L-lactic acid) [PEG-PLA] nano-micelles Prepared using solvent evaporation method. Paclitaxel, 17-AAG and co-polymer were dissolved in chloroform.

Squamous cell carcinoma
In vivo and ex vivo [141] polypeptide based nanogels Nanogels were prepared using PEG-b-PPGA/Ca 2+ block ionomer complex.

ErbB2-driven breast cancer
In vivo and ex vivo [142] magnetoliposomes Synthesized using co-precipitation method. Encapsulation of drug in PLGA was preformed using solvent evaporation and self-assembly technique.

Colon cancer
In vivo and ex vivo [143] ELP K 8 -poly(VPGXG)60 Prepared using recursive directional ligation method and its conjugates were dissolved in DMSO.

Lung cancer and breast cancer
In vivo [144,145] SNX-2112 glucose-based mesoporous carbon nanospheres (MCNs) Prepared using hydrothermal reaction and glucose was used as carbon source.

Breast cancer
In vivo, ex vivo [146,147] Sansalvamide A derivatives star polymer Synthesized using reversible addition-fragmentation chain transfer polymerization technique.

Colon cancer
In vivo [148] Continued over

Colon cancer
In vivo and ex vivo [159] PES core-shell structure Au@SiO 2 nanomaterials Gold rods were coated with mesoporous silica to form Au@SiO 2 nanocomposites.

Breast cancer tumour cells
In vitro and in vivo [160] and cytotoxicity [169]. In a separate study, Sun and co-workers reported that silicon nanoparticles can induce cytotoxicity and inflammation in human umbilical vein endothelial cells (HUVECs) by activating potassium channels [170]. Since, nanoparticles can induce toxicity when they come in contact with our blood cells, optimization of the biocompatibility of the nanoparticles must be looked upon very carefully. More importantly, the degradation process of nanoparticles inside body should be investigated carefully to overcome the toxic effects of nanoparticles.

Conclusions and promising future prospective
The introduction of nanotechnology and nanomaterials had led to revolutionary changes in various domains of medical science. This review has presented an overview of the current advances in nanotechnology for the treatment of four important diseases (Alzheimer's disease, tuberculosis, leprosy and cancer). The studies presented here clearly suggest that the ability of penetrating the blood-brain barrier makes nanoparticles excellent candidates to be applied in AD treatment, especially by inhibiting the fibrillation process and toxic effects of amyloid beta peptide. However, in future, a systematic study in this research field by exploring more efficient biodegradable nanomaterials that can be tagged with several disease modifying agents to successfully treat this disorder is highly demanding. Furthermore, it can be concluded that metallic nanoparticles are more useful towards the treatment of tuberculosis and leprosy possibly due to their ability to enhance the permeability of mycolic acid cell walls. Since, multimetallic nanoparticles exert better antimycobacterial activity than the monometallic nanoparticles, attempts should be made to screen the antimycobacterial potency of various other multimetallic nanoparticles. This futuristic approach could provide promising candidate(s) for the treatment of these two infectious diseases. This report also concludes that inorganic and/or organic based nanoparticles can be efficiently utilized for the delivery of various drugs used in tuberculosis, leprosy and cancer. Additionally, considering the toxic effects of nanoparticles, the evaluation of their biodegradability and cytotoxicity is extremely required. Such evaluation will certainly help us to assess their safe usage in drug delivery. Lastly, in future, more emphasis should be given to prepare non-toxic and biocompatible nanoparticles with improved pharmacokinetic behaviour, for their safer biomedical applications.