Biomedicine & Pharmacotherapy 141 (2021) 111920

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Current status of nanoscale drug delivery and the future of nano-vaccine

development for leishmaniasis – A review
Pragya Prasanna a, 1, Prakash Kumar a, 1, Saurabh Kumar a, Vinod Kumar Rajana a, Vishnu Kant a,
Surendra Rajit Prasad a, Utpal Mohan b, V. Ravichandiran a, b, Debabrata Mandal a, *
a
Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Hajipur 844102, India
b
National Institute of Pharmaceutical Education and Research, Kolkata 700054, India

A B S T R A C T

The study of tropical diseases like leishmaniasis, a parasitic disease, has not received much attention even though it is the second-largest infectious disease after
malaria. As per the WHO report, a total of 0.7–1.0 million new leishmaniasis cases, which are spread by 23 Leishmania species in more than 98 countries, are
estimated with an alarming 26,000–65,000 death toll every year. Lack of potential vaccines along with the cost and toxicity of amphotericin B (AmB), the most
common drug for the treatment of leishmaniasis, has raised the interest significantly for new formulations and drug delivery systems including nanoparticle-based
delivery as anti-leishmanial agents. The size, shape, and high surface area to volume ratio of different NPs make them ideal for many biological applications. The
delivery of drugs through liposome, polymeric, and solid-lipid NPs provides the advantage of high biocomatibilty of the carrier with reduced toxicity. Importantly,
NP-based delivery has shown improved efficacy due to targeted delivery of the payload and synergistic action of NP and payload on the target. This review analyses
the advantage of NP-based delivery over standard chemotherapy and natural product-based delivery system. The role of different physicochemical properties of a
nanoscale delivery system is discussed. Further, different ways of nanoformulation delivery ranging from liposome, niosomes, polymeric, metallic, solid-lipid NPs
were updated along with the possible mechanisms of action against the parasite. The status of current nano-vaccines and the future potential of NP-based vaccine are
elaborated here.

1. Introduction
Nanotechnology has gained importance in a wide range of applica­
tions because it can be tuned to attain desirable attributes by manipu­
lating physicochemical properties like size, shape, charge, etc. [1]. The
NPs are considered as the fundamental element of nanotechnology
whose size varies in the wide range of 1–1000 nm and beyond with
multiple applications in biology and medicine [2]. Nowadays a trend of
synthesizing NPs using biomaterials such as plants and microorganisms
is regarded as a reliable approach where both gold and silver NPs of
varied shapes and sizes have been synthesised using plants extracts.
These NPs are advantageous in terms of greater antimicrobial activity
and reduced toxicity compared to chemically synthesized NPs [3].
Further, antimicrobial textiles have also been developed by attaching
NPs in the fibers to make self-decontaminating antimicrobial textiles
[4]. For the safety and toxicity issues, the study of NPs- protein in­
teractions are also required. There are multiple proteomic studies that
have studied the cytotoxicity of NPs by studying NP- protein corona
level (protein adsorption layers located on the surface of colloidal NPs
level [5]. Advances in nanotechnology have provided numerous drug
delivery options to reduce dosage, enable improved release promoting
high drug concentrations at the target sites. For instance, specific and
targeted macrophage delivery will be highly beneficial in case of
intracellular infections like leishmaniasis [6,7].
Leishmaniasis is a spectrum of diseases primarily prevalent in poor
and underdeveloped economies with an estimated 7,00,000–1 million
cases reported annually (WHO) [8]. The causative agent of leishmani­
asis, Leishmania is transmitted by the bite of infected female phleboto­
mine sandflies and it completes its life cycle in two different hosts: sand
flies and vertebrates [9]. Therapeutic agents like antimonials, Ampho­
tericin B (AmB), paromomycin, sitamaquine, and miltefosine became
insufficient due to side effects like toxicity, increased drug resistance,
and in some cases due to cost-associated with its formulation. The heavy
metal, antimony, in the pentavalent form (SbV) as sodium antimony

* Correspondence to: Department of Biotechnology, NIPER-Hajipur, Export Promotions Industrial Park (EPIP)-Hajipur, District: Vaishali, Pin 844102 Bihar, India.
E-mail addresses: pragyaprasanna36@gmail.com (P. Prasanna), bbhaskar.kumar73@gmail.com (P. Kumar), sahilsaurabh25@gmail.com (S. Kumar),
vinoddhniper@gmail.com (V.K. Rajana), vishnukant378@gmail.com (V. Kant), surendra.science@gmail.com (S.R. Prasad), mohan.utpal@gmail.com (U. Mohan),
directorniperkolkata@gmail.com (V. Ravichandiran), debabrataman@gmail.com (D. Mandal).
1
Contributed equally in this manuscript.

https://doi.org/10.1016/j.biopha.2021.111920
Received 5 May 2021; Received in revised form 9 July 2021; Accepted 12 July 2021
Available online 20 July 2021
0753-3322/© 2021 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
P. Prasanna et al.
gluconate (SAG) has been the basis of drug treatment for visceral
leishmaniasis (VL) since the 1940s. However, clinical resistance to SAG
has been a problem in India since the late 1990s and a cure rate of 60%
was reported [10]. Later, the region endemic for VL in North Bihar,
India, got the unique distinction of being the only region in the world
where widespread primary failure to SAG has been reported [11]
causing complete discontinuation of SAG for the treatment of VL.
AmBisome, a liposomal formulation of AmB, is the most widely accepted
treatment to date for the disease VL which is caused by the parasite
Leishmania donovani [12] although it is come with a huge financial
burden due to costly lipid-based ingredients [13]. The safety and effi­
cacy of miltefosine, the only orally administered drug for VL, is also
compromised due to the high cost of production and teratogenicity [14].
Besides, no registered vaccines are available to date [15]. The challenges
such as limited therapeutic options, suboptimal diagnostics, poor com­
munity awareness, drug resistance, toxicity, asymptomatic carriers, and,
most importantly, absence of vaccines encourage more investigation
including nanoscale drug delivery for more effective treatment of
leishmaniasis. Most important step towards effective treatment of
leishmaniasis, at present, includes integrating national control strategies
with simple, reliable, and affordable field serological test along with the
creation of cost-effective and affordable diagnostic kits, drugs or vac­
cines considering the socio-economic problem associated with the dis­
ease [16,17].
The application of nanoformulation using antileishmanial drugs for
improved delivery started in 1992 [7]. Since then, many NFs both in the
form of drugs and vaccines have been prepared and tested at preclinical
levels for leishmaniasis [18,19]. However, low success rates of nano­
scale delivery system in vivo and at clinical studies so far, encouraged us
to analyze the possible reasons for the failure of NP-based delivery
system and a possible future for the same.
2. Importance of nanoscale delivery systems
NPs are the most fundamental component in the fabrication of a
nanostructure, which behaves differently from its bulk counterparts
[20]. Initially, the idea of nano-based drug delivery arose to address the
limitations and harmful effects of formulation excipients caused due to
fast and random distribution as well as toxic consequences by acting as
vectors for drugs that have improved pharmacokinetic parameters e.g.
systemic bioavailability. Nowadays, the branches of nanotechnology are
being developed especially to overcome biological barriers such as
gastrointestinal pH, blood-brain barrier, etc. [21,22]. NP-based drug
delivery systems have advantages such as high stability and loading with
the cargo, ease of incorporation of both hydrophilic and hydrophobic
substances, feasibility of variable routes of administration, including
oral and inhalation routes which makes better treatment options for
many diseases including leishmaniasis [23]. Furthermore, they provide
the luxury of improvement of drug bioavailability and other pharma­
cokinetic parameters conveniently without any loss of drug efficacy.
NP-based drug delivery act as a vehicle to alter the physio-chemical
properties of a drug easily. For instance, to improve the aqueous solu­
bility of poorly water-soluble drugs, a water-soluble polymer or metallic
NP-based delivery system can be easily linked with the drug molecule
covalently or non-covalently without any significant loss in its biological
activity [24]. Besides, these delivery systems would, most often, also
protect drugs from quick inactivation/degradation in circulation.
Increasing the aqueous solubility of drug moiety through chemical
modification is a daunting task and has a risk of loss of its bioactivity.
The same process with the help of nanotechnology-based modifications
of drug molecules is easier and restores the inherent drug properties.
Since the delivery vehicle is a separate entity that carries the drugs as
cargo, it can be conjugated with receptors. ligands and signaling mole­
cules destined for a targeted tissue/organ delivery subsequently
reducing the dosage by promoting increased drug accumulations at the
target sites [25,26]. Engineering of these drug delivery systems also
2
Biomedicine & Pharmacotherapy 141 (2021) 111920
facilitates the improved and sustained release of cargo at the specific
organ or tissues where it is destined. Encapsulation of drugs into
nanocarriers, also, provides enhanced protection against intracellular
and extracellular degradation compared to naked drugs [27].
Nanoformulation-based targeted drug delivery has shown promise to
overcome drug resistance associated with leishmaniasis [28]. Multiple
drug delivery strategies targeting drug resistance strains have been
formulated with considerable success in vitro and in vivo using animal
models [23]. Different liposomes and hybrid NPs loaded with anti­
leishmanial agents have shown promising results against drug-resistant
strains of the parasite. In most cases, these drug delivery systems
generally target the drug efflux pumps as the emergence of drug resis­
tance is very often associated with over-expression of these pumps e.g.,
ABC transporter, P-glycoprotein containing efflux pumps like MDR1 and
MRP1 [28]. High drug efflux causes a decrease in the intracellular
concentrations of the internalized drug subsequently decreasing the
therapeutic effect. Simultaneously drug efflux causes localized toxicity
of deposited drugs to the healthy tissues which can be evaded using
nanoformulation-based delivery [19]. Another beneficial aspect of
nanoscale delivery is the pH adaptability of the drug moieties in mi­
croenvironments. The majority of antileishmanial agents are adminis­
tered through parenteral mode. The paucity of orally administered
antileishmanial demands alternatives with high aqueous solubility and
stability at gastrointestinal pH. There are several lipid-based delivery
systems like palmitoyl homocysteine [29,30], hyaluronic hydrogels,
stealth polyethylene glycol (PEG) layers, and pH-sensitive linkers which
can be used to prepare pH-sensitive delivery vehicles favoring drug
release in a wide pH range [31]. This is important since the parasite
resides in the macrophages of the liver and spleen of the infected host
where the pH is low from its bulk counterpart.
3. Important physiochemical advantages of nanoscale drug
delivery
3.1. Shape and size of particles
NPs can be synthesized in different shapes and sizes [20]. These
variations in turn determine the toxicity and efficacy of NPs. Several
studies have highlighted that size, shape, charge, and surface coating of
NPs are major determinants of uptake and toxicity that could be
manipulated solely to make them non-toxic NPs. Spherical NPs are
comparatively less toxic than other dimensions whereas platelet-shaped
AgNPs are detrimental to epithelial cells [32]. Spherical GNPs and both,
spherical and cubic ZnO NPs, had shown lower cytotoxicity than other
structural forms [33]. The size of the NP is important for cellular uptake,
its interactions with the immune system, and its clearance from the body
[34]. In VL the parasite primarily infects the macrophages of the liver
and spleen. Therefore, uptake studies of macrophage cell lines using
different NPs could provide a strategy for improved drug delivery for VL
[35]. Using three different types of NPs (stars, rods, and triangles) of the
same size it was observed that uptake was much higher for triangular
NPs than the others in murine macrophages [36]. The clathrin-mediated
endocytosis pathway was found to be important in the uptake of gold
NPs. The anionic surface coated triangular GNPs were compared with
spherical GNPs for their uptake in mouse macrophage cell line RAW
264.7 and Hela cell lines. It was found that triangular GNPs were
internalized more with increasing size whereas spherical GNPs are
internalized more with decreasing size [37]. The synthesized GNPs of
sub-10 nm size (2,4 and 6 nm) show internalization based on size and
nature of surface charge [38]. For zwitterionic and anionic GNPs, uptake
decreased with increasing size, whereas for cationic NPs uptake de­
creases with reducing size. In another study, macrophage uptake was
found to be more for PEG-coated gold nanospheres than nanorods [39].
These studies show the importance of NP geometry and surface prop­
erties on transport across biological barriers. These studies also estab­
lished that uptake of NPs in macrophages are not only dependent on size
P. Prasanna et al.
but also on the geometry of NPs and associated surface charge.
3.2. Surface charge of NPs
Surface charge is important for uptake and biodistribution of NPs in
vivo [40]. NPs are routinely tested for use in medical products, like in
imaging, gene, and drug delivery. For these applications, cellular uptake
is usually important and is governed by surface characteristics such as
hydrophobicity and charge along with size. Cationic NPs cause more
damage to plasma-membrane integrity, stronger mitochondrial and
lysosomal damage, and a higher number of autophagosomes than
anionic NPs. It was observed that nonphagocytic cells uptake cationic
NPs to a higher extent, but charge density and hydrophobicity are
equally important. Phagocytic cells have a preference to take up anionic
NPs [41]. Indeed, negatively charged phosphatidylserine (PS) in the
outer leaflet of the Leishmania spp. the plasma membrane was found to
be important for its attachment to macrophages [42,43]. Cells do not use
different uptake mechanisms for cationic and anionic NPs, but high
uptake rates are always associated with increased biological effects.
Phagocytic THP-1 cells or non-phagocytic A549 cells when incubated
with fluorophore-conjugated polystyrene NPs (F-PLNPs) show differ­
ential uptake which correlated well with zeta potential [44]. This result
shows that surface charge is a critical parameter in determining cellular
uptake efficiency, although other factors such as aggregation/agglom­
eration, protein corona formation which, in turn, depend on surface
charge can also influence the cellular uptake partly or indirectly. Neutral
NPs have been found to have a slower opsonization rate than charged
NPs [45]. Zwitterionic NPs have been found to have prolonged circu­
lation. The charge is important for mucoadhesion or diffusion, cellular
uptake, bioavailability, and toxicity [38]. By changes in surface charge
and functionality, the biocompatibility and uptake efficiency can be
changed for NPs [46]. Surface modifications using hydro­
philic/hydrophobic components having different ionizable groups
facilitate drug release mechanisms after penetration through the
Fig. 1. It represents different physiochemical properties of NPs. (A) Shape (B) Size & its correlation with different biomaterials in nature and (C) Surface charge
associated with functionalization of NPs.
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Biomedicine & Pharmacotherapy 141 (2021) 111920
biological barrier. Commonly used surface modifiers of metal NPs are
disulfide, amine, thiolate, dithioline, carboxylate, and phosphine
groups. Modification by PEG reduces the cytotoxicity whereas attaching
carboxyl groups (-COOH) increase hydrophilic properties thereby
altering the internalization of drugs [39]. The polymeric NPs and lipo­
somes interact with the macrophages depending on their surface charge
[45]. Liposomes displaying a negatively charged surface, exhibit a much
higher rate of phagocytosis by macrophages as compared to neutral
vesicles [47]. A schematic presentation was given in Fig. 1 regarding the
role of different physicochemical properties including shape, size, and
surface charge in NP-based delivery.
3.3. pH-dependent targeting of NPs
Leishmania species after infection live in an acidic phagolysosomal
compartment where the pH is low. A drug moiety capable of reaching a
target should resist the variable pH seen across different tissues or
subcellular compartments and, therefore, pH-dependent drug release
will be very important for the site-specific accumulation and action of
drugs. pH-responsive NP platform with polymer as the inner core,
amphiphilic lipid-poly(ethylene glycol) (lipid-PEG) as the outer shell,
and a peptide encoded surface for efficient internalization and sharp
endosomal pH response has been reported [48]. NPs consisting of a
drug-loaded polylactic-co-glycolic acid (PLGA) core and wrapped with
acid-triggered membrane peptide have shown facilitated internalization
into cells within the acidic microenvironment where intracellular con­
ditions degrade the NPs, thereby releasing the chemotherapeutic cargo
[49]. The single-walled carbon nanotubes (SWNTs) can selectively
deliver tyrosine phosphatase inhibitor 1 (TPI) as a drug intracellularly to
macrophages in a pH-Dependent manner [50]. Here it was shown that at
pH> 7 the drug is stably bound to SWNTs but under acidic conditions at
pH < 6.0 the drug is released inside the macrophages. So, this kind of pH
specific delivery can be advantageous for the treatment of VL which are
associated with macrophages of the liver, spleen, lymph nodes, etc.
P. Prasanna et al.
Except for miltefosine, delivery in leishmaniasis lacks drugs that
could be orally administered. For a drug to be administered orally and
exert its effect, it should be able to survive the pH of the gastrointestinal
tract that varies throughout fasted and fed state [51] with a consistent
acidic environment in the stomach (<2) and alkaline in the small in­
testine (>6) [52]. When pH-sensitive lipids like palmitoyl homocysteine
(PHC) were incorporated into liposomes composed of dipalmitoyl,
stearoyl, and diheptadecanoyl L- α phosphatidylcholine (DPPC, DSPC,
and DHPC respectively), the greatest pH-differential of drug release was
obtained with serum at an ambient temperature of pH range 6–7.4 [53].
Here, the thiolactone form of PHC was found to be more stable at acidic
pH. For an NP to be effective in leishmaniasis it is very important to
protect it from degradation by the acidic environment and release its
payload to the target site to exert potential antiparasitic activities. This
takes into account factors like charge, hydrophobicity, nature of func­
tionalized ligands affecting cellular uptake, distribution, interaction
with immune cells, plasma proteins, and clearance from the body [54].
Adsorption of plasma proteins on hydrophobic surfaces is higher [55]
which in turn has an impact on the uptake, distribution, and clearance.
Organic NPs get degraded in a highly acidic lysosomal environment
whereas GNPs have been found to shift the pH towards the alkaline side
and reduces the lysosomal activity [56]. This indicates NP-based de­
livery can make the drug more pH tolerant. A chitosan and chondroitin
sulfate-based NP delivery system with AmB at pH 7.4 shows its effec­
tiveness in treating tegumentary leishmaniasis than standard AmB due
to improved stability and less toxicity at that pH [57]. Biopolymer of
polycaprolactone loaded with AmB has shown better efficacy than
AmBisome for cutaneous leishmaniasis (CL) at a physiological pH of 7.4
as well as a skin-relevant pH of 5.5. It is to be noted that pH dependent
delivery is directly correlated with surface charge of NPs since the
change in pH micro-environment affects ionization of coated functional
groups attached with NPs. Till date; no exhaustive study was performed
to check the efficacy of NP-based delivery on leishmania model of
macrophage infection at different pH. However, it is obvious that the
microenvironment of the phagocytes, including extreme pH, enzymatic
or ROS-mediated degradation, and permeability of the cell membrane is
critical in determining the fate of internalized NPs.
4. Different NP-based drug delivery approaches in leishmaniasis
4.1. Natural and synthetic compounds/derivatives with nanoformulation
Through advancement in high-throughput screening of compounds,
it is now possible to screen new compounds against the sensitive and
resistant strains of parasite compared to known antileishmanial agents.
The drug resistance in leishmania strains is a result of increased mem­
brane fluidity, decreased intracellular drug accumulation, and high
expression of ATP-binding cassette (ABC) transporters (MDR1 and
MRPA) [58]. In search of new antileishmanial agents quinoline, indolyl
quinoline analogs, naphthoquinones, artemisinin, coumarins, synthetic
derivatives of AmB, Miltefosine, anti-cancer drug doxorubicin, etc have
been reported with in vitro antileishmanial activity [59–61]. Natural
and plant-derived compounds, namely andrographolide (AG), curcu­
min, bisnaphthalimidopropyl (BNIP), Oleanolic acid (OA), etc. have
shown strong antileishmanial activity [62,63]. The majority of these
compounds show enhanced efficacy in nanoformulations than their
native counterpart. The chemotherapeutic applications of AG, a diter­
penoid lactone, are seriously hampered because of poor bioavailability,
short plasma half-life, and unwanted tissue localization. AG nano­
particles loaded in 50:50 PLGA reduced the dose by almost 1/4th
compared to standard AG in vitro against the parasite [62]. Similarly,
OA which has low aqueous solubility and low permeability, when
administered with polymeric NPs reduced ~ 98% amastigote burden in
the spleen of BALB/c mice, significantly higher than OA alone [63].
Encapsulation of BNIP diaaminooctane (BNIPDaoct) loaded PLGA NPs
allow the drug to be delivered orally and also reduced the parasitic
4
Biomedicine & Pharmacotherapy 141 (2021) 111920
burden with ~80% efficiency than the active alone [64].
Artemisinin-PLGA NPs displayed increased leishmanicidal activity with
minimal macrophage toxicity compared to free artemisinin [65].
Moreover, PLGA NPs of curcumin, a bioactive compound with reported
leishmanicidal activity were found to be efficacious in combination with
miltefosine at a subcurative dose with negligible nephrotoxicity [66].
Compound 8-hydroxyquinoline (8-HQN) [67,68] in combination with a
polymeric micelle system showed better efficacy than free 8-HQN or
AmB [68]. Additionally, phytochemicals (i.e., alkaloids, flavonoids,
terpenoids, and sterols) based nanomedicines with improved PK/PD
profiles are also being developed to prepare cost-effective drug alter­
natives with fewer side effects [66]. All these results suggest nano-based
delivery is more effective than standard drug delivery even for natural
compounds or newly developed antileishmanial agents. This is possible
because the solubility and bioavailability of all these compounds are
generally increased by nanoscale delivery system. A schematic presen­
tation of different NPs that is used for leishmaniasis delivery is shown in
Fig. 2.
4.2. Liposomes
The liposome encapsulates part of the aqueous medium with
dispersed hydrophilic substances whereas it accommodates hydropho­
bic molecules in the bilayer and behaves as a semipermeable membrane,
relative to the material encapsulated in the aqueous core of the vesicles.
Liposomal delivery promotes superior tissue absorption, favoring drug
penetration into the macrophages and retarding its clearance from the
site of action with increased bioavailability. The liposome can carry both
water-soluble compounds and lipids and, therefore, considered drug-
delivery systems of wide application [69]. Liposomes can deliver
drugs, proteins, enzymes, and genetic materials to living cells [70,71].
Liposomes are more often studied for the treatment of leishmaniasis
compared to any other parasitic disease, mainly because Leishmania
colonizes spleen and liver macrophages, which are also responsible for
liposome clearance in vivo. Consequently, liposome-based delivery has
minimal toxicity compared to other nano-based delivery systems. In
fact, in its early applications, liposome and its physicochemical prop­
erties were studied for macrophages, in the treatment of leishmania
infection [72]. The efficacy of various antimonial drugs such as
meglumine antimoniate (AME) or sodium stibogluconate (SSG) when
encapsulated in liposomes has always shown better efficacy than the free
drug [73]. Due to drug resistance associated with free antimonials,
miltefosine [74] and AmB, delivery systems with liposomal form were
preferred for the treatment of various types of leishmaniasis [75,76].
Papagiannaros and coworkers (2005) demonstrated the advantages of
using liposomal miltefosine over free miltefosine with improved efficacy
against miltefosine-resistant Leishmania promastigotes. Liposomal
amphotericin B (L-AmB) was > 350-fold more active than AME, and
2–5 times more active than non-liposomal AmB, when tested against
experimental VL [77]. L-AmB eliminated 99% of the amastigotes in the
liver with a concomitant improvement in the AmB therapeutic index
when tested in L. donovani-infected squirrel monkeys. Other lipid-based
AmB products such as AmB colloidal dispersion (Amphocil®) and the
AmB lipid complex Amphotec® have shown less efficacy against VL than
the L-AmB formulation [78] although they are less toxic and more
efficacious than fungizone, an AmB-deoxycholate (D-AmB) formulation.
Ergosterol-rich liposomal AmB formulation (Kalsome™10) tested in
murine models was found to induce no hepato- or nephrotoxicity,
leading to a significant reduction in parasite burden from liver and
spleen [79]. Liposomes coated with sugars like galactose, mannose, and
fucose were designed to target more specifically macrophages as these
cells have receptors to recognize those glycoside residues [80,81].
Mannose-coated liposomal systems were more effective in vivo [82] and
also the overall toxicity was reduced compared to conventional lipo­
somes and the free drugs [26,83].
Negatively charged lipids as liposomal form were found to favor the
P. Prasanna et al.
Fig. 2. A representative figure of different NPs (liposomal, niosomal, SLN, Polymeric, metallic etc.) that is used for leishmaniasis delivery.
drug-encapsulation efficiency [84], whereas cationic lipid-containing
positively charged liposomes had shown increased drug uptake in
macrophages [85]. Using PS-loaded L-AME, a high in vivo efficacy with
> 130 times reduction in the dose of total antimony was observed in
parasite-infected hamsters [86]. The association of SAG with phospha­
tidylcholine- stearylamine (PC-SA) liposomes had shown a significant
reduction in antileishmanial activity in vivo [87]. Similarly, cationic
liposomal formulation of SSG was found to be very effective for the
treatment of infection caused by SSG-sensitive and -resistant L. donovani
[88]. Due to preferential binding of peptides on macrophage receptors,
different peptide-modified liposomes have also shown improved dug
delivery for SAG [89] and primaquine, a known antimalarial drug under
the 8-aminoquinoline group, against leishmania parasites [90]. Due to
the higher success of liposomes, immunoliposomes containing lipo­
somes coated or bound with antibodies were also tested since macro­
phages have receptors to bind to the Fc portion of antibodies.
Immunoglobulin G (IgG)-coupled liposomes [91] and liposome-bound
antibody formulation containing doxorubicin [92] are more effective
in killing leishmania parasites than their liposome-free counterpart. In
short, the liposomal delivery system for leishmaniasis has huge potential
considering the early success of AmBisome and its extensive use to date.
Liposomal formulation of drugs like SAG/SSG, which are stopped due to
high resistance cases, may provide a new opportunity for future appli­
cations. However, the liposomal formulations are generally of short
half-life, high-cost [93] and there is a chance of leakage or fusion of
encapsulated drugs leading to decreased stability and toxicity in the
micro-environment [94,95]. Cationic liposomes have been so far known
to be highly stable which if loaded with antigens acts as potent adjuvants
to induce long-term protection against L. donovani and represents an
alternative to DNA vaccination [96]. Stabilty of L-AmB can be increased
by loading with tufstin, an immunomodulator of tetrapeptide sequence
originated from IgG antibody. In vitro stability studies revealed that
tuftsin-bearing AmB liposomes released ~2 fold less AmB in the serum
of BALB/c mice, compared to tuftsin-free AmB liposomes [97]. Both
tufstin-bearing and fufstin-free L-AmB shows ~ 5 fold reduced toxicity
5
Biomedicine & Pharmacotherapy 141 (2021) 111920
than D-AmB formulation as measured by hemolysis assay. Toxicity
measured by serum creatinine and urea level was also significantly
lower for both tufstin-bearing and fufstin-free L-AmB than D-AmB.
These studies indicate that although liposomal formulation as are fairly
stable and non-toxic there is a possibility of improving their delivery by
other additional carriers. The details of liposome-based delivery of
nanoformulation against leishmaniasis are listed in Table 1.
4.3. Niosomes
The niosomes are lipid vesicles that contain a significant fraction of
non-ionic surfactants in their composition [98]. Niosomes were found to
have a higher retention capability than liposomes in vivo when the lipid
moiety was absent and the size is smaller. They are structurally similar
to liposomes in their bilayer; however, the prepared niosomes are more
stable than liposomes. It can entrap both hydrophilic and lipophilic
drugs, either in an aqueous layer or in a vesicular membrane made of
lipid material. As a result, the therapeutic effectiveness of such anti­
leishmanial compounds was found to be superior to that of liposomal
antileishmanial compounds. For a long time, niosomes were considered
a viable alternative to liposomes as drug vehicles because they were less
expensive, least toxic, biodegradable, and non-immunogenic [98].
Itraconazole niosomes were effective against L. tropica with an IC50
value of 0.24 µg/ml than 0.43 µg/ml for itraconazole alone [99]. Nio­
somal SSG was found to be better than liposomal vesicular formulations
or free drugs, against experimental murine VL by increasing drug levels
in the infected reticuloendothelial system [100]. Their data showed that
antimony level in the liposomal form is removed much faster than nio­
somal form in the circulation as tested in L. donovani–infected BALB/c
mice models. The non-ionic surfactant vesicles with AmB have shown
higher drug uptake in the lungs and skin than AmB alone thus signifi­
cantly reducing parasite burdens in the liver [101]. These niosomes
were also used as a nebulizer indicating a different way of delivery.
Niosomes composed of either AmB, glucantine or both were prepared as
large spherical multi-layer vesicles having a size range of 10–18 µM
P. Prasanna et al. Biomedicine & Pharmacotherapy 141 (2021) 111920

Table 1
Liposome-based NP delivery against leishmaniasis.
Chemotherapeutic agents/ Model of Study Mechanism of action and Advantage in using the nano-formulation Refs.
Drugs

L. donovani
Meglumine antimoniate Mice Greater drug penetration through skin [73]
Miltefosine In vivo Enhanced hepatic accumulation [74]
AmB Human Improved pharmacokinetic profile of AmB; Improved patient tolerance and reduced systemictoxicity [75,
76]
AmB PEM; Mice Improved activity due to enhanced drug uptake [78]
Ergosterol Mice No hepatotoxicity, Significant reduction in parasite burden from liver and spleen [79]
Sugars Ex vivo Macrophage specific targeting [80,
81]
Antimony Hamsters Enhanced efficacy in comparison to antimony alone [86]
SAG Ex vivo Improved drug delivery to macrophages [89]
Primaquine In vitro and in vivo Macrophage specific targeting [90]
Doxorubicin Human Enhanced killing efficacy due to targeted delivery to macrophage [91,
92]
SSG BALB/c mice Complete suppression of disease-promoting IL-10 and TGF-β, upregulation of Th1 cytokines. Increased [179]
retention of SSG within parasites.
Amphotericin-B Human Improved pharmacokinetic profile of AmB. Improved patient tolerance and reduced systemictoxicity [180]
Tuftsin decorated Hamster Increased uptake; enhanced drug tolerance [181]
IFN-g and doxorubicin- BALB/c mice Reduced levels of IL-4 but increased levels of IL-12 and iNOS.Synergistic effect of drug and liposomes. [182]
mannosylated
Artemisinin BALB/c Th1-biased immune response. Improved efficacyand no toxicity [183]
AmB Swiss mice Hamsters Up-regulation of Th-1 cytokines, iNOS, down regulation of Th-2 cytokines.Drug is morestable and less [184]
toxic
Andrographolide Hamster High macrophage delivery, effective against drug-resistant strains.Reduced parasitic burden, hepatic and [185]
renal toxicity
Cholesterol BALB/c mice Down regulation of virulence factor and Macrophage activation. Increased drug uptake [186]
L. infantum
Meglumine Antimonate Mongrel dogs and BALB/ Enhanced circulationtime. Reduction in systemic toxicity. [25]
cmice

Abbreviations: SSG, Sodium Stibogluconate; iNOS, Inducible Nitric oxide synthase; TGFβ, Transforming growth factor beta.

[102]. These glucantine and gulacntine-AmB niosomes reduce the
splenic parasite burden in L. major-infected BALB/c mice ~84% when
applied topically twice daily with 12 h intervals for 30 days. Niosomes
have been also evaluated as vaccine carriers for CL using purified gp63
glycoprotein as antigens [103]. These gp63 proteins were encapsulated
into niosomes and used to vaccinate C57BL/10 mice which were inoc­
ulated subcutaneously twice at two weeks intervals (4 mg of pro­
tein/mouse). When vaccinated mice were exposed to a homologous
challenge (i.e. mice vaccinated with L. mexicana gp63 and infected with
the same parasite), significant disease resistance with ulcerated lesions
was observed that began to heal. Niosome formulated with inactivated
whole L. major parasites were evaluated as an encapsulation model for
vaccine candidates[98]. Although niosome-based delivery has shown
some success against leishmaniasis in animal models, till date no nio­
somal formulation was evaluated as a replacement of L-AmB formula­
tion. But, in general, the preparation of niosomes are much cheaper than
liposomes since niosomes use surfactants like Tween 60, Tween 80 etc
which are less costly than bilayered phospholipids used in liposomes
[104]. These surfactants are physically and chemically more stable than
phospholipids making the niosomes more stable and non-toxic than li­
posomes. Therefore, niosome-based delivery system may provide a
suitable replacement for liposomal delivery system in the future.
4.4. Solid lipid nanoparticles
Solid lipid Nanoparticles (SLNs) are nanospheres that consist of solid
lipids of ~ 50–1000 nm in size with a solid lipid matrix stabilized by
physiologically compatible emulsifiers [105]. Although the size of SLNs
is bigger in comparison to the conventional NPs, they have been proved
to be a very efficient drug-delivery system.Chitosan-coated SLNs loaded
with AmB had shown better internalization in J774.1 cells and less
cytotoxic effect than marketed formulations [27]. To improve AmB
bioavailability and prepare a formulation for oral delivery of AmB, a
vitamin B12-stearic acid (VBS) conjugate coated and SLNs encapsulated
with AmB were fabricated. It showed an enhanced efficacy over
conventional AmB without showing any toxic effects on macrophage
cells [106]. To prepare an oral formulation for VL, AmB and paromo­
mycin (PM) were together loaded in 2-hydroxypropyl-β-cyclodextrin
(HPCD) modified SLNs [107]. The dual drug-loaded SLNs (DDSLNs) had
shown complete cellular internalization of SLNs within 24 h of incuba­
tion, low cytotoxic effect without any hepatic/renal toxicity in Swiss
albino mice. Further, the DDSLNs had shown better in vivo efficacy than
miltefosine-treated control. PM-loaded in SLNs was studied against
L. major-infected BALB/c mice where it showed improved effectiveness
than PM alone in killing the parasite by switching towards Th1 immune
response [108]. The SLNs coated with macrophage specific ligand
O-palmitoyl mannan (OPM) and entrapped with AmB, showed better
parasite killing than D-AmB formulation with equivalent dose [109].
The organ distribution of the AmB bearing SLNs (both AmB-SLN and
OPM-AmB-SLN) was compared with the D-AmB in albino rats. Here
OPM-coated SLNs were found to accumulate more AmB in the liver,
spleen, and lungs, the organs which are rich in macrophages. SLNs can
improve the drug delivery by improving the solubility of poorly
water-soluble molecules like 17-N-allylamino-17-demethoxygeldana­
mycin (17-AAG, tanespimycin) which is a known inhibitor of heat
shock protein 90 (Hsp90). The entrapment of 17-AAG by ~80% in SLN
along with high macrophage uptake within 2 h indicated the advantage
of drug delivery with SLNs. The biggest advantage of SLN-based delivery
is its biocompatibility since the lipids used in formulation are 100%
biodegradable [110]. SLNS can be synthesized without organic solvents
and they can be sterilized if required. These provide more stability and
longer storage capacity for the SLNs. The major disadvantages of SLNs
are the initial burst release and low drug loading capacity or expulsion
during the crystallization process.
4.5. Polymer-based nanoparticles
The polymeric systems are popular because they are more biocom­
patible and biodegradable [111]. Several polymers such as poly-lactic
acid (PLA), poly-glycolic acid (PGA), poly-lactide-co-glycolide (PLGA),

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P. Prasanna et al. Biomedicine & Pharmacotherapy 141 (2021) 111920

poly-caprolactone (PCL), and poly-cyanoacrylate (PCA) and natural
protein polymers, such as albumin and gelatin, and polysaccharides are
used for the preparation of polymeric drug delivery systems [112]. The
PLA, PGA, PLGA, PCL, and PCA polymers are approved by FDA for
human applications due to their high biocompatibility. AmB entrapped
in PLGA NPs and stabilized by TPGS (a known p-GP efflux inhibitor) had
shown higher oral bioavailability and reduced nephrotoxicity [113,114]
in rats than standard fungizone. Miltefosine encapsulated PLGA–PEG
NPs of a size range of 10–15 nm are more effective antileishmanial
agents[115] in the hamster model compared to standard miltefosine and
AmB. AmB encapsulated cationic stearylamine lipid-polymer hybrid NPs
(LPNPs) of ~ 200 nm, which had characteristics of both polymeric NPs
and liposomes [116,117], had shown increased efficacy with higher
macrophage uptake and rapid plasma clearance. AmB encapsulated
sodium alginate-glycol chitosan stearate NPs (AmB-SA-GCS-NP). which
has been prepared by strong electrostatic interaction between oppositely
charged polymer and copolymer, had shown higher macrophagic uptake
in J774.1 macrophages and faster in vivo localization in tissues of liver,
spleen, lung, and kidney than standard AmB [118]. PS–coated gelatin
NPs with encapsulated AmB had shown higher in vivo organ distribution
in Wistar rats and also causing a more reduced parasite burden (85% vs.
50%) compared to standard AmB [119]. Polymeric NPs have also
become useful devices for the oral administration of water-insoluble
molecules with improved bioavailability and reduced toxicity due to
the ability of polymers to cross the mucosal barrier with specialized
uptake mechanisms [120,121]. AmB and PLGA polymeric NPs, which
are made using E-TPGS (D-α-Tocopherol polyethylene glycol 1000 suc­
cinate) as a stabilizer, have improved the drug’s oral bioavailability and
minimize its side effects[113]. Moreover, PLGA NPs and
nano-suspensions containing AmB were also coined as an alternative of
cost-effective delivery compared to Fungizone and AmBisome [122]. For
application against CL, AmB nanoencapsulation in PLGA/dimercapto­
succinic acid (DMSA) NPs (Nano-DMS-AmB) was developed and tested
against C57BL/6 mice infected with L. amazonensis. The nano-
DMS-AmB showed a greater reduction in the number of parasites than
standard D-AmB thereby reducing the effective dose of the nano­
formulation[123]. The toxicity of AmB-loaded PLGA-polyethylene gly­
col (PEG) NPs was lower compared to that of the free drug when tested
in the macrophage model [124]. Mannose-anchored and
AmB-encapsulated PLGA NPs had shown specific targeting on macro­
phage receptors thereby improving the efficacy of the drug [125].
Mannosylation of thiolated chitosan (TC) NPs (MTC) was found to be a
useful approach for improving AmB-intramacrophage localization as
sustained drug release for 10 days was obtained for mannosylated NPs
[126]. Therefore, conventional antileishmanial agents like AmB and
miltefosine can be delivered very efficiently using polymeric NPs both in

Table 2
Polymeric NPs-based delivery against leishmaniasis.
Chemotherapeutic agents/ drugs Model of study Mechanism of action and Advantage in using the nano-formulation Refs.

L. amazonensis
Pentamidine In vitro Controlled release; Reduced nephrotoxicity [187]
AmB In vitro Reduced IC50 against L. amazonensis; Highmacrophage uptake [188]
L. infantum
AmB (BMM-ϕ) Balb/c mice Rapid phagocytosis by macrophages and dendritic cells; Increased cytotoxic T cell [189]
response
AmB BALB/c mice Preferential accumulation in the visceral organs and increased CD-8T cell response [190]
AmB Hamsters Increased accumulation of AmB in the spleen and liver, reduced dose [191]
L. donovani
Doxorubicin Hamsters Cell cycle arrest at G1 and S phase inducing apoptosis; Increased Th1 and decreased Th2 [192]
response
Wistar rats Controlled delivery; Low cost [193]
AmB Swiss albino mice Selective macrophage targeting [125]
Rifampicin Albino rat; macrophages Greater macrophage uptake using mannose receptors [194]
AmB J774A.1; BALB/c mice Enhanced drug uptake and effective against drug-resistant strains. [126]
AmB J774A.1 Swiss albino mice Enhanced antileishmanial activity; Non- toxic [195]
AmB BMDM Equally effective but oral delivery instead of intravenous [196]
AmB Hamsters BALB/c mice Non-invasive nebulisation method; Both pulmonary and hepatic delivery [101]
Sprague–Dawley rats
Miltefosine Hamster 50% decreased dose and CD14 mediated Selective targeting [115]
Betulin J774A.1 11-fold decrease in IC50 Controlled release [197]
Curcumin and Miltefosine Hamster Synergistic effect of drugs. increased lymphocyte proliferation. [66]
Oleanolic acid BALB/c Increased solubility and bio-availability of drug; Effective against drug-resistant strains. [198]
Andrographolide – 4-fold decrease in IC50 and faster macrophage uptake [199]
Polymethyl methacrylate of Human RBC/THP-1 cells 3-fold decreased IC50 with no toxicity on human RBCs [200]
octylgallate
Polyalkylcyanoacrylate BALB/c 2-fold decreased ED50 with reduced cytotoxicity [24]
Primaquine-loaded nanoparticles J774A1 Synergistic effect of NP and primaquinone, 21-fold increase in efficacy [201]
PEG-PLA - bisnaphthalimidopropyl THP-1, J774 cells Reduced toxicity and increased liver uptake [64]
derivatives
Miltefosine In vitro and In vivo No hemolytic and cytotoxic properties [202]
Andrographolide BALB/c Sustained drug release, decrease in dosage and effective against resistant strains [203]
Curcumin J774.1 Increased solubility of curcumin and better macrophage uptake [37]
AmB BALB/c mice Rapid clearance from plasma and low distribution in kidneys with reduced toxicity [204]
Resiquimod RAW 264.7BALB/c mice 100% drug release; Increased immune response in macrophages [205]
AmB Wistar Rats, Hamster High AmB accumulation in liver and spleen with enhanced anti-leishmanial activity [119]
Artemisinin BALB/c Escalated IgG2a levels, enhanced pro-inflammatory cytokines (IFN-γ and IL-2) and [173]
suppression of Th2 cytokines (IL-10 and IL-4)
AmB Human 100-fold increased efficacy of AmB against AmB-resistant strain with poloxamer 188 [75,
206]
L. chagasi
Pentavalent antimony BALB/c mice, Hamsters Selective and improved uptake by macrophage scavenger receptors; Reduced toxicity [207]
N-(2-hydroxypropyl) THP-1 cells/ (PEMs)/ (BMMs) 19-fold increase in efficacy in vitro. [208,
methacrylamide– AmB. 209]

Abbreviations: AmB, Amphotericin B; PEM, Peritoneal macrophages; BMM, Bone marrow derived macrophages.

7
P. Prasanna et al.
vitro and in vivo. The main advantages of the polymeric NPs are their
easier & faster preparation method and high drug loading with mini­
mum leakage due to the stability of polymeric NPs. More information on
Polymeric NP-based drug delivery was summarized in Table 2.
4.6. Metallic nanoparticles
Sizes, shape, high aspect ratio, and optical features of metal NPs are
different from their macro-scaled counterparts thus, making them ideal
to be used for drug-delivery and biosensor-based applications. In recent
years, biological methods have been discovered to be the most suitable
for the synthesis of metal NPs, as chemical and physical methods have
drawbacks [127]. Active chemical ingredients which are present in
medicinal plants and plant extracts are very routinely used for the green
synthesis of metallic NPs. Since these plant products are already known
as antileishmanial agents, the synthesized NPs show high efficacy
against the parasite-infected animal model studies [128–130] but with
significantly reduced cytotoxicity than chemically synthesized metallic
NPs. Compared to AmB, the gold nanoparticles (GNP) conjugated AmB
(GL-AmB) had shown 2.5- and a 5-fold reduction in IC50 against pro­
mastigote and amastigote forms of the parasite, respectively [131].
These GL-AmB NPs have reduced cytotoxicity on macrophages and were
less hemolytic than standard AmB. Quercetin-functionalized GNPs were
found to be highly effective against wild and resistant strains of
L. donovani parasites, with low toxicity against macrophages [128,132].
In a recent study, we have shown that 7,8-dihydroxyflavone (DHF)
synthesized GNPs (DHF-GNP) are more antileishmanial in the amasti­
gote model than the promastigote model [133]. The synthesized
DHF-GNPs of ~35 nm size kills the sensitive- and drug- (AmB and SAG)
resistant parasites with equal efficacy. A diagram showing the synthesis
of NPs from the herbal extract and its application in macrophage de­
livery, uptake, and parasite killing is shown in Fig. 3. Adil et al. showed
silver nanoparticles (AgNPs) having enhanced antileishmanial activity
against L. tropica by inhibiting proliferation and metabolic activity of
promastigote by 1.5–3 fold respectively in dark conditions and
2–6.5 fold under UV light irradiation [134]. Phytochemicals from the
aqueous extract of Isatis tinctoria were used as reducing and capping
Fig. 3. A diagram showing the synthesis of NPs from herbal extract and its application in macrophage delivery, uptake and parasite killing.
(a) Giemsa staining and parasite killking data were reproduced from [255]. (b) TEM imags of macrophage NP uptake was reproduced from [256].
8
Biomedicine & Pharmacotherapy 141 (2021) 111920
agents to prepare AmB where AmB was successfully adsorbed on the
surface of biogenic AgNPs. These NPs show antileishmanial activity with
> 96% percent killing under visible light, indicating a better window of
using photodynamic therapy (PDT) for treating CL with skin lesions
[135]. Akhtar et al. showed that PEGylated silver doped zinc oxide (ZnO
NPs) were highly effective against leishmaniasis by developing reactive
oxygen species (ROS) at low concentrations, making them ideal for PDT
[136]. The biogenic AgNPs (35 nm) obtained from Anethum graveolens
leaf extract enhances the activity of miltefosine on L. donovani pro­
mastigotes and have increased biocompatibility than normal AgNPs on
macrophages [137]. The biogenic GNPs (30 nm) obtained using May­
tenus royleanus have been shown to inhibit L. tropica promastigote’s
growth in a time-dependent manner where parasite survival is directly
co-related with internalized GNP concentration [138]. Green synthe­
sized titanium oxide (TiO2) NPs, synthesized using aqueous leaf extract
of Euphorbia prostrate, showed comparatively weak leishmanicidal ac­
tivity after 24 h exposure when compared with AgNPs [139]. Novel
Au-Ag alloy NPs, which were synthesized by Dioscorea bulbifera tuber
extract, had shown a MIC value of 32 µg/ml against L. donovani pro­
mastigotes along with anti-biofilm activity against bacteria indicating a
multipurpose application[140]. Iron oxide (Fe3O4) NPs of ~4 nm of size
synthesized by Rosmarinus officinalis leaf extract had shown an anti­
leishmanial effect on L. Major with an IC50 value of 350 µg/ml [141].
ZnO NPs synthesized from Verbena officinalis and Verbena tenuisecta
plant leaf extracts had shown anti-leishmanial efficacy which is corre­
lated with the phenolic compounds present in those extracts [142].
Therefore, green synthesized metallic NPs synthesized have a significant
prospect in anti-leishmanial drug delivery. Among different metallic
NPs, SiO2 and ZnO, are generally considered non-toxic since the bulk
material inherently is less toxic [143]. However, there was always a
concern over the toxicity related to GNPs and AgNPs. Production of
reactive oxygen species (ROS) leading to damage of cell membrane,
DNA, and protein oxidation was considered to be a general outcome of
toxicity associated with GNP and other metallic NPs [144]. The GNPs
which are administered in BALB/c mice at a dose of 8 mg/kg/week
caused severe sickness and death of mice in 21 days. These phenomena
is observed only with 8–37 nm size of GNPs which are chemically
P. Prasanna et al. Biomedicine & Pharmacotherapy 141 (2021) 111920

Table 3
Metallic and miscellaneous NPs for drug-delivery against leishmaniasis.
Type of NP Chemotherapeutic Model of Study Tested Mechanism of action and Advantage in using the nano-formulation Reference
agents/ Drugs parasite

Carbon- AmB BALB/c mice L. donovani Greater solubility,increased stability at gastric pH andlow toxicity [210]
based NPs J774A.1
AmB J774A.1 No hepatic/renal toxicity of NP [211]
Hamster
Doxorubicin Hamsters High drug accumulation in parasite [212]
AmB Hamster Oral intake possible, Increase in CC50 [213]
AmB J774A.1, Wistar Increased protective proinflammatory mediators’ expression and spleen [214]
rats uptake.
Doxorubicin J774.2 Stability at lysosomal pH, high cellular uptake [215]
hamsters
Protein NPs AmB BALB/c mice L. amazonensis Reduced lesion size at infected footpad, AmB related toxicity reduced as [216]
measured by histopathological studies
Meglumine Antimonate In vitro L. major Highest activity was observed with glutaraldehyde (10 µl/ ml) [217]
Metal NPs AmB J774A.1,Wistar L. donovani Superior efficacy, desired stability and reliable safety of cost-effective [214]
rats
AmB J774A.1, Reduced cytotoxicity Increased uptake and activity [218]
Hamster
Natural compounds In vitro, in vivo Increased efficacy [127]
AgNPs In vitro Amplified anti-leishmanial effect [137]
TiO2 NPs In vitro Significant increase in G0/G1 phase of the cell cycle with a subsequent [139]
decrease in S and G2/M phases leading to growth-inhibition
AmpB In vitro, ex vivo Low cytotoxicity to macrophage cells and reduced AmpB cytotoxicity [131]
Quercetin Ex vivo Equally effective in drug-resistant strains [128]
Rhazya stricta decne-AuNPs In vitro L. tropica Effective delivery with low cytotoxicity to macrophage cells [130]

AgNPs Ex vivo Greater antimicrobial efficacy [134]

AmpB In vitro Enhanced antimicrobial activity [135]
ZnONPs In vitro Photodynamic therapy against leishmaniasis [136]
AgNPs In vivo L. major Enhanced drug-delivery [129]
Fe3O4 NPs In vitro Low toxicity [141]

synthesized (citrate or sodium borohydride) but not with 3–5 nm or
> 50 nm sized GNPs [145]. However, GNPs synthesized using plant
extracts/natural products does not cause toxicity and mostly well
tolerated in vitro and in vivo irrespective of the size of GNPs. This clearly
indicates that various methods of surface modification of GNPs is related
to there toxicity and not the bulk component of the synthesized GNP.
Further, recent studies have shown that GNPs are gradually degraded by
NADPH oxidase in human fibroblast cells for 2–6 months [146]. This
study supports the possible safety of GNP-based delivery since degraded
particles of size < 3 nm can be easily cleared by circulation without
causing any long-term deposition in different tissues and cells like
macrophages. Table 3 summarizes metallic and other NP-based delivery
methods against leishmaniasis.
5. Nano-based vaccine delivery for leishmaniasis
The dream of an effective human vaccine against leishmaniasis could
not be realized due to challenges such as failure in conferring the im­
munity, lack of suitable adjuvants, differential virulence dynamics of
Leishmania species, and lack of experimental human models. Addition­
ally, the majority of the population suffering from the disease belongs to
poor sections of the society; therefore, low profitable recovery from the
market discourages robust research initiatives to invest in vaccine-based
research for leishmaniasis. Several NP-based delivery as prophylactic
nanovaccines for the treatment of leishmaniasis was studied [147]. A
scheme showing development of different nano-vaccine and its delivery
against animal and human models in Fig. 4. Traditional vaccines against
leishmaniasis were based on live or attenuated parasites or their sub­
units. However, to date, only a few vaccines are under different phases
of the clinical trial. A summary of vaccines available for leishmania to
date has been presented in Table 4. All the vaccine candidates for
leishmaniasis are categorized into three types: (i) Live leishmania; (ii)
Killed leishmania or parasite fractions with or without adjuvants (first
generation) and (iii) recognized leishmania molecules including re­
combinant proteins or DNA (second generation) [148]. DNA vaccines,
known as third-generation vaccines, are of particular interest because
they can effectively induce both CD8 + and CD4 + T cells, produce
long-lived antigens and properly folded polypeptides, etc.[149]. How­
ever, DNA vaccines are still in earlier phases of clinical trials.
First-generation vaccine, leishmanization against CL suffers standardi­
zation and safety setbacks whereas second-generation vaccines e.g. re­
combinant proteins or DNA molecules (leish-111f) suffer from a lack of
an appropriate adjuvant [150]. The second generation of vaccines
against Leishmaniain clinical trials is of relatively low efficacy due to
limited CD8 + T-cell response emerging from cross-presentation [151].
Three types of leishmanial vaccines, Leish-F1, F2, and F3, designed at
the Infectious Disease Research Institute (IDRI), USA were in clinical
trials [152]. LEISH-F1 which is a fusion protein made up of three
polypeptides in tandem and formulated with monophosphoryl lipid
A-stable emulsion (MPL-SE), showed encouraging results in phase 1 with
a shorter time to cure. The clinical trial was organized in the USA,
Colombia, Brazil, Peru, and India. The observed safety and immunoge­
nicity in endemic and non-endemic populations have inspired a phase II
clinical trial with LEISH-F2 where the histidine tag of recombinant
protein was removed. IDRI is going for phase I trial of LEISH-F3 where
the fused peptide is composed of nucleoside hydrolase from L. donovani
and sterol 24-c-methyltransferase from L. infantum. SLNs can serve as an
efficient tool to synthesize and carry a leishmanial vaccine [105]. In
2005, a group of researchers prepared a nanovaccine delivery method in
mice by loading recombinant Leishmania superoxide dismutase
(SODB1) in a chitosan NPusing the ionotropic gelation method. NPs
loaded with SODB1 showed a higher cell-mediated immune response
and higher IgG2a levels. This showed the potential of nanovaccine for
the treatment of leishmaniasis. Nanoliposomes used as the nanocarriers
for soluble Leishmania antigens (SLA) showed parasite clearance in the
footpad and spleen of a mouse model injected with this formulation
[153]. Liposomal formulations of ChimeraT (a combination of 3 leish­
manial proteins) when delivered subcutenously protected mice against
L. infantum infection, by reducing the parasite load in spleen, liver, bone
marrow, and lymph node. The cure was associated with a stimulated

9
P. Prasanna et al. Biomedicine & Pharmacotherapy 141 (2021) 111920

Fig. 4. Development of nano-vaccine and its delivery in vivo against animal and human models.

effective adjuvant is one of the reasons for the limited efficacy. There­
Table 4
fore, a delivery system carrying both antigen and immune-stimulatory
List of vaccines developed for the treatment of leishmaniasis.
adjuvants to the antigen-presenting cells (APCs) is expected to play a
Generation Vaccine Antigen Adjuvants Refs. very significant role in enhancing efficacies. A list of nano-vaccine de­
I Leishvaccine IFLA/BR/1967/ BCG [219] livery systems prepared as potential antileishmanial vaccines is listed in
PH8 Table 5.
ALMϼ ALM Alum + BCG [220]
Nano based delivery systems (NDS) could potentially deliver target
Autoclaved L. BCG [221]
major vaccines to the site of action within the host’s body and enhance im­
Leishmune LiESP FML [222] mune responses by facilitating its absorption and uptake of antigens by
+ saponin APCs [156], prevent degradation of antigens such as peptides, proteins,
CaniLeish LiESP – [223] or oligonucleotides [157], promoting the controlled release of antigen
Attenuated Attenuated L. [224]
[158] and modulate the type of immune responses induced by the an­
–
vaccine donovani
Attenuated L. gentamycin [225] tigens [159]. NDS may improve the solubility of hydrophobic com­
mexicana and L. pounds in an aqueous medium to render them suitable for parenteral
major administration. Delivering antigens with the help of NPs to APCs pre­
II LEISH‑F1 LmSTI1 +LeIF MPL‑SE [](150,
sents an attractive way of activating the host immune system [148]. It
LEISH‑F2 – MPL‑SE 152)
LEISH‑F3 – GLA‑SE [152], could be used in delivering antigens and adjuvants either to enhance
[226] their uptake by APCs, [160] or generate Th1 type immune response
Leish‑Tec A2 Saponin [227] [161]. The use of nano-based vaccines could offer a stronger immuno­
SMTγ + NHμ SMT+NH GLA‑SE [228] logical response due to the delivery to the same APC [162]. Studies
KMP-11 CpG-ODN [229]
suggest that polymeric NPs when used as adjuvants could develop more
pSP IL-12 [230]
Leishmanolysin GP63 BCG, (MPL- [231] potent immunogenicity against Leishmania attributed to its physico­
TDM) chemical properties.
III ChAd63–KH ChAd63 – [232] In the case of mucosal immunization, NDS may help in the protection
Abbreviations: ALM, Autoclaved-killed L. major; SMT, sterol 24-c-methyltran­ of antigen against the gastrointestinal environment (acid and proteolytic
ferase; MPL-SE, monophosphoryl lipid A in structure stimulating Toll-like re­ enzymes), enhancement of antigen translocation to the mucosa-
ceptor; GLA, glucopyranosyl lipid A. associated lymphoid tissue, and ease of delivery via the oral or intra­
nasal route [163]. NDS-mediated delivery of antigens ensures the de­
Th1-type response in vivo by producing higher levels of IFN-γ, IL-12, and livery of both antigen and adjuvants into the same APCs which
GM-CSF cytokines [154,155]. Fig. 5. efficiently enhances activation of APCs. Additionally, encapsulated
Some encouraging efforts of developing first-generation (killed par­ formulations of immunostimulatory adjuvant into the delivery system
asites) vaccines have failed to provide convincing results in phase III restrict the systemic circulation of the adjuvant thereby preventing
trials due to standardization and safety issues whereas others are still in adverse effects [164].
earlier phases. In this regard, researchers admit that the lack of an A cationic solid–lipid nanoparticle (cSLN) formulation withA2 anti­
gen of L. donovani, and cysteine proteinases of type I (CPA) and II (CPB)

10
P. Prasanna et al.
Fig. 5. A graphical abstract showing delivery of different NPs against leishmania-infected macrophages involving different mechanisms of killing.
of L. infantum without its unusual C-terminal extension (CTE) showed an
efficient vaccine delivery system against VL [165]. Administration of the
pcDNA-A2-CPA-CPB(-CTE) delivered by either electroporation or cSLN
formulation protects BALB/c mice against L. infantum challenge asso­
ciated with high levels of IFN-γ and lower levels of IL-10 production,
leading to aelevatedTh1 and reduced Th2 immune response. The ratio of
IFN-γ: IL-10 induced upon re-stimulation was always significantly
higher in vaccinated animals. Nitric oxide production, parasite burden,
and histopathological analysis also showed a better cures for
vaccine-treated animal. In a murine model, cationic liposomes formu­
lated with a novel whole Leishmania lysate (WLL) from
detergent-solubilized L. major promastigotes in a liposomal form, were
used as a vaccine for leishmaniasis [166]. The parasite burden, footpad
swelling, and Th1/Th2 response were analyzed in the mice model where
good efficacy was observed for vaccinated animals. In another study, a
murine model of leishmaniasis was used to evaluate the role of
liposome-polycation-DNA (LPD)-NPs containing recombinant major
surface glycoprotein of Leishmania (rgp63) in defense against leish­
maniasis [167]. Reduced parasites in the spleen, the increased levels of
IgG2a, minimal footpad swelling and 5-fold increased IFN-γ secretion
compared to standard SLA treatment indicated that proper adjuvants
and strong antigen-delivery systems are important for vaccine delivery
against leishmaniasis. By using a single emulsion solvent evaporation
process, two hypothetical proteins from L. panamensis were encapsu­
lated into PLGA particles and immune response was studied in BALB/c
11
Biomedicine & Pharmacotherapy 141 (2021) 111920
mice infected with L. panamensis [168]. The high encapsulation efficacy
provides a tool for better antigenicity for vaccine delivery by this
method. Vaccination strategies involving live, killed, or attenuated
(physically or genetically) leishmania parasites have produced limited
success. The delivery of peptide-, protein-, or DNA-based vaccines
although showed some promise in animal models but failed in human
targets. For a suitable vaccine candidate, a strong host protective T
response is important [169]. However, L. donovani infection decreases
the expression of both MHC-I and MHC-II, which are responsible for
effective T cell response, in macrophages and dendritic cells [170]. This
provides a possible explanation of the failure in vaccination strategy in
leishmaniasis. In parallel, lack of a potential adjuvant was also reasoned
as a major problem in vaccine delivery against leishmania. It is believed
that nano-based vaccine delivery can replace, at least, the role of a
suitable adjuvant since it provides improved stability of antigen, a role
reminiscent of most adjuvants.
6. Possible mechanism of action of Nano-based delivery in
leishmaniasis
Polyamine-dependent trypanothione reductase (TR) redox system
and ergosterol biosynthesis pathway are two unique pathways that are
not present in the mammalian system. Therefore, any drug delivery
methods which can target these pathways may be highly selective in
killing the parasite without causing any defect to its human host. The
P. Prasanna et al. Biomedicine & Pharmacotherapy 141 (2021) 111920

Table 5
Nano-based vaccine delivery approaches for leishmaniasis.
Type of Antigen Model Mechanism of action References
Nanoformulation as
vaccines

Liposomes Leishmania lysate Mice Low parasite burden, footpad swelling, and Th1/Th2 response [166]
rgp63 Mice Reduced parasites in the spleen, the increased levels of IgG2a, minimal [167]
footpad swelling and 5-fold increased IFN-γ secretion
Imiquimod + SLA BALB/c mice Enhance CD-4 + and CD-8 + T cells response [233]
L. donovani antigens BALB/c mice Induction of IFN-γ and IgG2a [234]
rGP63 BALB/c mice High IgG2a/IgG1 ratio and IFN-γ production and thelowest IL-4 level [235]
Soluble leishmanial antigens BALB/c mice Strong Th1 response [236]
Recombinant L. major stress- BALB/c mice Low spleen parasite burden, high IgG2a antibody; Lower IL-4 level [237]
inducible protein 1
Recombinant LiHyR protein BALB/c mice The increased concentration of IgG2aantibody wasobserved with [238]
significantparasitic burden reduction
Recombinant glycoprotein (rgp63) BALB/c mice Induce valuable immune responseevidenced by asignificant uptickin Th-1 [167]
cells mediated immuneresponse and level of IgG2a antibody
SLA BALB/c mice Inadequate protection against Leishmaniasis [239]
Recombinant Leishmania major BALB/c mice Lowering in footpad lesion thicknessamong immunized subjects [240]
class Inuclease (rLmaCIN) withsignificantly higher levels of IgG2aand INF-γ
Freeze thawed promastigotes BALB/c mice Reduction in parasitic burden due toenhanced Th-1 cells response [241]
andassociated cytokines
Recombinant cysteine protease Syrian golden Improved anti-leishmanial responsewith increased survival time [242]
mixture Hamsters andcytokines level however immuneresponse was not adequately
evaluated
Thiolated antibody (IgG-SH) + SLA BALB/c mice Improved Cytotoxic T-cells response [243]
Virosomes Qβ-virus like α-Gal trisaccharide C57BL/6 mice The successful killing of parasites anderadication of infection [244]
particles evaluatedthrough the RT-PCR technique
Polymeric NPs PLGA Hypothetical proteins BALB/c mice High encapsulation efficiency [168]
(L. panamensis)
SLA + TLR agonists L. major Results have clearly shown improvedmacrophage activation [245]
promastigotes accompaniedby the significant reduction inpro-inflammatory cytokines

SLA + Lipophosphoglycan BALB/c mice Elevated NO, INF-γ, and IL-12 levelsdisplay 80% disease remission [246]
ininfected BALB/c
Recombinant proteins BALB/c mice Immune mediated parameters are not evaluated [168]
(rLpanUA.22; rLpanUA.27)
Recombinant proteinsChimeric BALB/c mice Improved NO levels and cell-mediated immunityfollowed by [247]
peptides (CPA and CPB) immunization
CPA Transgenic mice Enhance CD-4 + and CD-8 + T cellsresponse; Increased production of IL- [248]
12
SLA with Toll like receptor BALB/c mice Pronounced activation of cellularimmunity due to enhanced Th-1cells [248]
mediated immune responseThe levels of interleukins observed tobe
minimized(IL-6 and IL-10)
Chitosan Leishmanial SLA BALB/c mice Poor immune protection was observed [249]
PMMA pcDNA3 L. major infected BALB/c mice Improve immuneprotection wasobserved [250]
clearly evaluated throughuprise in IgG2a levels
Niosomes Autoclaved L. major BALB/c mice Amplified immune response [98]
Gp63 entrapped C57BL/10 mice Stable and controlled release [103]
AmB BALB/c Decreases in lesion size and parasite burden [251]
Solid Lipid Nanoparticles Cysteine proteinase COS-7 cells Deliver immunogenic CP genes [105]
A2 antigen BALB/c Compare electroporation and cationic solid-lipid nanoparticle (cSLN) [165]
mediated delivery to administer a DNA vaccine
rCPB C57BL/c Maximize entrapment efficiency with reduction in the particle size and its [252]
distribution
Chitosan nanoparticles Superoxide dismutase BALB/c Increased antigenicity [253]
Nanoemulsion Glucopyranosyl Lipid A (LEISH- Mice Macrophage activation [226]
F3 +GLA-SE)
Escheriosomes L. donovani promastigote soluble Hamster, BALB/c Elicit strong CD8 + cytotoxic T lymphocyte (CTL) response [254]
antigens

Abbreviations: SLA, Soluble Leishmania antigen; IgG, Immunoglobulin; IL, Interleukin; CTL, Cytotoxic T lymphocytes

crystal structure of L. infantum TR in complex with NADPH and Ag(0)
was solved at 3.3 Å resolution [171]. Ag binds with high affinity to the
catalytic triad composed of Cys52, Cys57, and His461 residues of
L. infantum TR thereby causing parasite death. Further in vitro assay
with Ag showed that Ag can inhibit TR activity in nanomolar concen­
tration and it can be more effective than antimony. Green synthesized
AgNPs and TiO2 NPs kills the parasite by cell cycle arrest at G0/G1 phase
thereby reducing the promastigotes in the S and G2/M phases of the cell
cycle [139]. Further, increased ROS production leading to increased
apoptosis and necrosis was observed. GNP conjugated with AmB
(GL-AmB) kills the parasite by decreasing the ergosterol content ~2
times more than the same equivalent of free AmB. In parallel,
trypanathione reductase (TR) activity and subsequently reduced thiol
content was found to be decreased under similar conditions. This in­
dicates that conjugate GL-AmB acts on both targets involving ergosterol
biosynthesis and the TR redox pathway. Although, GL-AmB causes
increased ROS necrosis, instead of apoptosis, was observed as evidenced
by DNA fragmentation, FACS, and lactate dehydrogenase (LDH) leakage
assay. The drugs which generate ROS are preferred leishmanicidal
agents, as Leishmania has to survive under the high oxidative stress of
macrophages where they primarily infect and reside [136]. Metallic NPs
which produce ROS are always effective against many pathogenic mi­
crobes albeit with a problem related to cytotoxicity [172]. The NPs
entrapped with natural compounds, phytochemicals or flavonoids,

12
P. Prasanna et al.
therefore, showed increased uptake and less cytotoxicity in
macrophage-infected parasites although generally, they do not show
enhanced effect in vitro against promastigotes than their naked coun­
terpart [66,68,173]. Green synthesized GNPs were found to deposit in­
side the macrophages within hours of treatment thereby conferring their
effectiveness against drug-resistant strains of L. donovani [130,174,175].
GNP functionalized with DHF showed that inhibition of arginase activity
of polyamine biosynthesis pathway of the parasite is the major cause of
death [133]. DHF-GNP was found to be a more potent inhibitor of
arginase than DHF alone but DHF-GNP was more biocompatible than
citrate-reduced GNPs. Interestingly, both DHF and DHF-GNP showed
inhibition of drug transporter, MDR1, thereby making it effective
against drug-resistant strains of the parasite. This study further confirms
that the toxicity of metallic NPs can be reduced by molecules used in
surface functionalization. The liposome or polymeric NP-based delivery
of AmB has always reduced cytotoxicity than free AmB, thereby,
allowing increased dosage for application in animal models [77–79]. In
the macrophage model, NP-based delivery with reduced parasite count
always comes with a common observation which is concomitant with
increased Th1 and decreased Th2 response [27,79,88,131].
Up-regulation of proinflammatory cytokines such as IL-12 and IFN-γ
(Th1 markers) and limiting the expression of IL-10 (Th2 markers) along
with Th1 cytokines-mediated increased expression of inducible nitric
oxide synthase (iNOS) was found to be the most common mechanism for
clearing parasites in vivo in leishmaniasis [176]. In nano-based vaccine
delivery system increased stability of antigen [158]. The higher ratio of
Th1/Th2 cytokine response [166].and increased IgG2a/IgG1 ratio for
polysaccharide encapsulated NPs [167] was observed for BALB/c mice
which are vaccinated.
7. Conclusion
Macrophages (~2000 nm) composed of DNA (~2.5 nm) and pro­
teins (~1–20 nm) can be considered very efficient targets in leishman­
iasis for NP-based drug delivery due to their different sizes in the nm
range. However, very little progress was made for efficient use of NP-
based drugs in leishmaniasis, except the liposomal formulation of
AmB which was FDA approved in the year 1997. The toxicity of most
chemotherapeutic drugs, drug resistance, high cost of liposomal for­
mulations, and consequent lack of interest of private firms due to low
benefit in treating a poorer class of people in 3rd world countries had
created a big hole in eradicating the disease by 2020 as proposed by
WHO [8]. Post Kala-Azar dermal leishmaniasis (PKDL) which is usually
a sequel of VL and the emergence of HIV-VL co-infections generates a
new sociological problem related to leishmaniasis treatment. Since
1995, more than 50 nano pharmaceuticals have received FDA approval
and are currently available for clinical use for various diseases including
different forms of cancer, multiple sclerosis, SCID, and even for diseases
related to iron deficiency [177]. However, no progress was made so far
for VL and CL. These nanodrugs are typically administered orally,
intravenously, and less frequently transdermally and in different forms
(Polymeric, liposomal, metallic, nanocrystals, etc.) very successfully.
Currently, there are also many formulations being developed for other
indications, including metabolic disorders, autoimmune conditions,
anesthesia, ophthalmic conditions, neurological and psychiatric dis­
eases, and others [178]. It indicates the possible safety and advantage of
targeted drug delivery of nanoformulation which is a major problem in
conventional delivery. Most of the nanodrugs approved to date have
shown reduced toxicity rather than improved efficacy compared to
conventional formulations. The most obvious advantage of NP-based
delivery is the customization of the delivery system with different
sizes, shapes, and cargo thereby providing targeted and sustained drug
release at the desired tissues and organs. Therefore, nanoscale- drug
delivery may become a suitable method for treatments of diseases like
leishmaniasis where the mechanism of action and efficacy in reducing
the parasite burden, in vivo, by different nano-formulations was already
13
Biomedicine & Pharmacotherapy 141 (2021) 111920
shown by various studies.
CRediT authorship contribution statement
PP, PK, and DM designed the concept; PP, PK, SK, VKR, VK, SRP and
DM collected information and data. PP, PK,SK,VKR, and DM written the
manuscript; PP, PK, UM, VR and DM analyzed the manuscript. All au­
thors read and approved the final manuscript.
Funding
The funding for this research work was provided by the Ministry of
Chemicals and Fertilizers, Government of India.
Competing Interests
The authors report no conflicts of interest in this work.
Acknowledgment
We acknowledge the financial assistance in research funding to Dr.
Debabrata Mandal and Ph.D.fellowship to Pragya Prasanna, Vinod
Kumar Rajana, Saurabh Kumar, Vishnu Kant and Surendra Rajit Prasad
from the Ministry of Chemicals and Fertilizers, Government of India. Dr.
Prakash Kumar was assisted by a Post doctoral research fellowship from
the Ministry of Chemicals and Fertilizers, Government of India.
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