seminar

1. NANOTECHNOLOGICAL STRATEGIES FOR
TREATMENT OF LEISHMANIASIS
PREPARED BY:
SHAHID ANSARI
ROLL NO :2 UNDER THE GUIDENCE OF:
M.Pharm (1st Year) Dr. G.J.KHAN
(M.Pharm, Ph.D)
Principal
ALI - ALLANA COLLEGE OF PHARMACY
AKKALKUWA DIST: NANDURBAR, M.H.(425415)
1

2. Contents
2
HEADS
PAGE
NO.
HEADS
PAGE
NO.
INTRODUCTION 3 SOLID LIPID NANOPARTICLE 16
TYPES 5 MICROEMULSION 18
LIFE-CYCLE 6
METALLIC NANOPARTICLES &
CARBON BASED MATERIALS
19
HISTORY & CURRENT STRATEGIES 7 NANOIMMUNIZATION 20
CURRENT DRUG & LIMITATIONS 8 ABSORPTION & DISTRIBUTION 21
NOVEL NANOTECHNOLOGY 9
FUTURE OF ANTILEISHMANIAL
AGENTS
23
LIPOSOMES 11 CONCLUSION 24
POLYMERIC NANOPARTICLES 14 BIBLIOGRAPHY 25
LIPID NANOCAPSULE 15

3. • The World Health Organization (WHO) considers leishmaniasis to be one of the main neglected
diseases in the world, affecting primarily the poor population of underdeveloped and developing
countries. The etiologic agents are several different species, all belonging to the genus Leishmania
which maintain their life cycle through transmission between an insect, a sandfly, and a mammalian
host.
• Leishmania is a genus of trypanosomes that are responsible for the disease leishmaniasis.
Trypanosomatida is a group of kinetoplastid. Kinetoplastida is a group of flagellated protists
A protist is any eukaryotic organism that is not an animal, plant or fungus distinguished by having
only a single flagellum.
• Leishmania organisms are dimorphic protists alternating between the promastigotes (in the insect
vector) and the amastigotes (in vertebrate hosts).
• Leishmaniasis is endemic in 98 countries, with 350 million people at risk, in Asia, Africa, Southern
Europe and South and Central America. 3

4. • Leishmaniasis is caused by different species that belong to the genus Leishmania, including following
The different species are morphologically indistinguishable, but they can be differentiated by
monoclonal antibodies.
L. donovani L. infantum L. mexicana
L. amazonensis L. venezuelensis L. tropica
L. aethiopica L. braziliensis L. guyanensis
L.panamensis L. peruviana.
STRUCTURE
Depending on the stage of their lifecycle
• Amastigote form -found in the mononuclear phagocytes
and circulatory systems of humans. It is an intracellular
and nonmotile form, being devoid of external flagella.
The short flagellum is embedded at the anterior end
without projecting out. It is oval in shape, and
measures 3–6 µm in length and 1–3 µm in breadth. 4

5. • Promastigote Form- It is found in the alimentary tract of sandflies. It is an extracellular and motile form.
It is considerably larger and highly elongated, measuring 15-30 µm in length and 5 µm in width. It is
spindle-shaped, tapering at both ends. A long flagellum is projected externally at the anterior end. The
nucleus lies at the centre.
TYPES OF LEISHMANIASIS
• Cutaneous Leishmaniasis-is the most common form, which causes an open sore
at the bite sites, which heals in a few months to a year and half, leaving an
unpleasant looking scar. Diffuse cutaneous leishmaniasis produces widespread
skin lesions which resemble leprosy, and may not heal on its own.
Cutaneous leishmaniasis
• Visceral leishmaniasis- or kala-azar ('black fever') is the most serious form, and is potentially fatal if
untreated. Other consequences, which can occur a few months to years after infection, include fever,
damage to the spleen and liver, and anaemia. It is characterized by irregular bouts of fever, weight loss,
enlargement of the spleen and liver, and anaemia.
5

6. • Mucocutaneous leishmaniasis causes both skin and mucosal ulcers with damage primarily of the
nose and mouth. It leads to partial or total destruction of mucous membranes of the nose, mouth and
throat. It accounts Over 90% of leishmaniasis.
LIFE CYCLE-
Mucocutaneous leishmaniasis
Life cycle of protozoa Leishmania
6

7. HISTORY & CURRENT STRATEGIES ON LEISHMANIASIS TREATMENT
• The standard drugs for 70 years, remain being used as first-line drugs in several parts of the world as
sodium stibogluconate, Amphotericin B (AmB) deoxycholate has been used since the early 1960s as
second-line treatment for leishmaniasis Alternatively, pentamidine isethionate, miltefosine and
paromomycin are available, but their use is limited due to toxicity or high cost of treatment.
• The current treatment for leishmaniasis is based on chemotherapy and poses limitations such as
toxicity, difficult route of administration and lack of efficacy.
• These drugs are known to have severe side effects, including nausea, abdominal colic, diarrhoea, skin
rashes, hepatotoxicity, cardiotoxicity, nephrotoxicity and pancreatitis. Nowadays, there are two
approaches to develop new therapies: one is the search for new drugs and the other is the optimization
of actual drug formulation. A possibility to improve leishmaniasis treatment would be the application
of drug delivery systems which can enhance the therapeutic potency of existing drugs by optimizing
their adsorption, distribution, metabolism and excretion (ADME) and reducing toxicity.
7

8. CURRENT ANTILEISHMANIAL DRUGS ASSOCIATED LIMITATION AND MECHANISMS OF ACTION
DRUGS ADMINISTRATION ASSOCIATED PROBLEMS MECHANISM OF ACTION
SODIUM-
STIBOGLUCONATE
Parenteral
Hepatotoxicity Nephrotoxicity
Cardiotoxicity
Inhibition of glycolysis and
fatty acid oxidation
AMPHOTERICIN B Intravenous
Nephrotoxicity
Hypokalemia Interferes with DNA synthesis
PENTAMIDINE Intramuscular Pain, nausea ,vomiting hypertension Interferes with DNA synthesis
MILTEFOSINE Oral
Contra indicated in pregnancy
Hepatotoxicity Nephrotoxicity
Cardiotoxicity
Associated with phospholipid
biosynthesis
SITAMAQUINE Oral
Vomiting methemoglobinemia(for
individuals with glucose-6-phosphate
dehydrogenase deficiency)
Inhibit translocation of
ribosomal subunits
8

9. NOVEL NANOTECHNOLOGY-BASED APPROACHES FOR ANTI-LEISHMANIAL DRUG
DELIVERY
• Nanomedicine is the field of science involved in the design and development of nanotechnology based
therapeutics and diagnostics with dimensions in the range of 1 nm to 1000 nm. The design of
nanometersized delivery systems for drugs represents one of the most promising developments for
antimicrobial therapies because such systems may lead to treatments with higher target delivery effect,
resulting in higher therapeutic efficacy and lower toxicity.
• Moreover, such systems can improve bioavailability and protect the incorporated drugs from being
metabolized, causing prolonged drug residence in the human body, and therefore allowing to increase the
time between administrations. Several nanotechnological strategies, such as lipid-based colloid systems,
nanostructured layered films, polymeric nanoparticles and silver nanoparticles have been explored for
precise drug delivery in leishmaniasis treatment, increasing effectiveness and safety of drug therapy.
• Many drugs are poorly soluble in aqueous phase which compromise their distribution into and within the
bloodstream. Solubilisation of hydrophobic drugs in colloidal particles is a possible approach to overcome
the problem of bringing a hydrophobic substance into the aqueous blood compartment.
9

10. • Nanoparticles can be classified either by the way in which they carry substances or by the
characteristics of the matrix. According to the classification based on the type of material from which
the matrix is made one can distinguish organic nanoparticles and inorganic nanoparticles.
• Nanotechnology-drug delivery systems include organic NPs, liposomes, solid lipid nanoparticles
(SLNs), nanostructured lipid carriers (NLCs), polymeric nanoparticles and microemulsions. The
inorganic nanoparticles involve the use of different inorganic oxides and have different sizes, shapes,
solubility, and long-term stability. They are usually synthesized by chemical reduction of the metallic
salt with a reducing agent.
10

11. LIPOSOMES-
• Liposomes are closed vesicles forms when dry phospholipid is fully hydrated composed of lipids that occurs
naturally in the cell membrane such as Phosphatidylcholine & cholesterol making formulation biodegradable
made of one or more bilayer composed basically composed of phospholipids & cholesterol.
• Their application to the treatment of leishmaniasis has evolved together with advances in knowledge on
relationships between liposomal structure via administration pharmacokinetics and biodistribution. They are
useful in targeting by passive delivery using EPR.
• At the end of the 1970s, the groups of Ward and Hanson were the first to understand the strategic utility of the
intravenous administration of liposomes for selective delivery to the liver and spleen macrophages infected
with visceral leishmaniasis. These liposomes also had antileishmanial activity against experimental cutaneous
leishmaniasis but only if they were administered intravenously. The main advantage of liposomes is their
ability to provide sustained drug release, leaving the drug available in the blood circulation for a long period
of time, which increases the prophylactic effect and reduces the drug dosage additionally, these structures are
biocompatible with biological fluids, decreasing the toxicity of drugs and limiting any local inflammatory
reactions.
11

12. • There are 3 different preparations of liposomal Amphotericin B namely Ambiosomes, Amphotericine B and
Amphotec. Ambiosomes is one of the few liposomal formulation to have been registered by USFDA & other
national drug authorities & proven to be successful against leishmaniasis in 1981.
• For antileishmanial therapy, drug targeting can be achieved by liposomal encapsulated drugs allowing them to reach
the intracellular Leishmania amastigotes the drug containing liposomes naturally enter in the macrophages by
phagocytosis and hence deliver the drugs passively to the phagolysosome where they then can act directly on the
parasites. Severe acute and chronic side effects have limited the use of AmB deoxycholate as antileishmanial agent.
• The commercial liposomal AmB, Ambisome, produced by Gilead Sciences, is the only drug approved in 1997 by
the FDA for VL treatment. Ambisome presented low toxicity and high cure rate (above 90%) when compared to
AmB deoxycholate and is recommended by the WHO as first-line therapy for treatment of VL caused by L.
donovani in India, Bangladesh, Bhutan and Nepal or L. infantum in the Mediterranean Basin, Middle East, Central
Asia and South America. A liposomal formulation for CL not only enhances the therapeutic effect of current
chemotherapeutics as administration of hydrophilic drugs such as Glucantime apart from this Paramomycin can also
be loaded. Blood circulation time liposomes can be improve by addition of polyethylene glycol to the surface of
liposome and improves half life.
12

13. Advantages: Increased drug stability, Biocompatible, Completely biodegradable, Increased efficacy and
therapeutic index of drug, Can be used preferentially for parenteral or cutaneous route.
PHARMACOLOGY & PHARMACOKINETICS -The drug binds to parasite ergosterol precursors,
causing disruption of the parasite membrane. The specialized formulation has characteristics that increase
efficacy while minimizing toxicity effective penetration and sustained levels in tissue, especially liver and
spleen; high transition temperature leading to stability in blood, macrophages, and tissues; presence of
cholesterol in the liposome, which minimizes drug interaction with mammalian cell membranes and
decreases toxicity and high affinity for ergosterol and its precursors ensuring antimicrobial efficacy.
13

14. POLYMERIC NANOPARTICLES-
• This are colloidal structures size ranging from 10nm-1µm composed of synthetic & natural polymers its having
advanced physicochemical property that improves BA, biodegradability, enhance cellular dynamics & useful in
targeting drugs. Its valuable in the therapy of infectious diseases such as leishmaniasis as having small size &
proficient passage through biological barrier & enhanced cellular uptake & delivery of drug in targeted tissues.
They can be used as Nanocapsule & Nanosphere depending of nature of drugs can either attached to surface or
incorporated in to matrix.
• Liposomes are unique potential carriers but it is having certain limitations as low encapsulated efficiency, rapid
leakage of water soluble drugs polymeric nanoparticles having specific advantages over it.
• Primaquine loaded nanoparticles reported to be 21 times more effective than the free primaquine in eradicating
the leishmania parasites. Gasper & coworker were the first to evaluate the polymeric nanoparticles against the
L. donovani infected macrophage. In 2013 costa lima developed PLGA based nanoparticles containing AmB
with suitable antiparasitic activity for VL. As acidic end group of PLGA is responsible for drug incorporation &
that increased chain length may increase the drug- polymer interaction leading to improve drug incorporation
efficiency.
14

15. LIPID NANOCAPSULES-
• This are the biomimetic composed of an oily core of triglycerides of capric & caprylic acid surrounded by shell
of lecithin & solutol. Solutol is the mixture of free PEG 660 & PEG 660 hydroxystearate they mimic the
lipoprotein & size ranging from 20-100 nm hybrid between liposomes & polymeric nanocapsule.
• They are attracted great interest because of their protective coating which is usually easily oxidized main
advantages are increment in the drug selectivity & effectiveness.
• Targetting the lipid nanocapsules to specialized phagocytes via Phosphatidylserine (PS) specific ligand bearing
doxorubicin acting against leishmania mexicana and having a 1.75 fold higher uptake if PS is on surface
compared to non- PS containing lipid nanocapsule –DOX.
15

16. SOLID LIPID NANOPARTICLES AND NANOSTRUCTURED LIPID CARRIERS –
• To overcome the limitations of polymeric nanoparticles as polymer having slow precipitation due to slow rate of
solvent removal allowed more time for drug molecule to partition into aqueous phase leading to low %
encapsulated efficiency lipids used as carrier this lipid nanoparticles called as SLNs. Basically they are made of
solid lipid core with a monolayer phospholipid shell the solid state of the nanoparticle matrix provides
protection to chemically labile drugs.
• This are submicron colloidal carriers ranging from 50- 1000nm containing lipid disperse in water or may be in
the aqueous solution of surfactant.
• Gupta & coworkers developed AmB containing SLN as well as AmB loaded SLN coated by macrophage
specific ligand, O- palmitoyl mannose shows higher efficiency of AmB modified SLNs over the other. Chitosan
coated SLNs loaded with AmB have been developed for Immuno adjuvants chemotherapy of leishmania
infection, Invitro activity revealed that this are more potent than commercial formulation.
• SLNs loaded by cysteine, proteinase gene showed a promising DNA vaccine- delivery system for leishmaniasis.
16

17. • For the treatment of CL, drugs topically applied must be able to cross the skin and reach the Leishmania
amastigotes within the phagolysosomes of infected macrophages localized in the deep dermal layer of the skin.
Particularly for CL, SLNs could improve the interaction of the drug with the stratum corneum and other layers
of the skin and thus provide the possibility of topical administration with controlled release, reduced drug
toxicity and so to optimize its treatment.
• To overcome the limitation of SLNs nanostructured lipid carriers have been introduced with the aim to increase
drug loading & stability there by reducing drug expulsion.
17

18. MICROEMULSIONS-
• This are thermodynamically stable, isotropically clear dispersion of two immiscible liquids such as oil &
water which is stabilized by interfacial film of surfactant, size ranging from 10- 200nm.
• The Invitro biological performance of fluconazole loaded microemulsion for the topical treatment of
cutaneous leishmaniasis has been seen as fluconazole favours the topical treatment of CL as having high
affinity for keratin & prolonging its retention in the skin. Chalcone and derivatives have been used as
potential drug candidates for leishmaniasis treatment shows promising activity against both the promastigote
& amastigote forms of leishmania.
Advantages:
• Solubilize hydrophilic and lipophilic drugs.
• Easy preparation.
• Long shelf-life.
• Thermodynamically stable.
• Small size droplets and can be sterilized by filtration.
18

19. METALLIC NANOPARTICLES AND CARBON-BASED MATERIALS-
• Nanoparticles are based on small well-defined aggregates of the noble metals in the zero valent state. The
size and the shape of the nanoparticles can be controlled by the reducing agent, the capping agent, and the
reaction conditions used in the preparation. Inherent properties of the noble metals, enhanced due to the
greater surface area of the nanoparticulate form, could be of interest for the treatment of Leishmaniasis.
• The production of reactive oxygen species (ROS) by silver NP is an antibacterial well-known effect and
Leishmania parasites seem to be very sensitive to it as well. An antileishmanial effect using Ag NPs at
concentrations ranging from 25 to 200 μg/mL.
• The effects of Ag-NPs on biological parameters of L. tropica promastigotes such as morphology, metabolic
activity, proliferation, infectivity, and survival in host cells in vitro. When exposed to Ag-NPs in the dark, L.
tropica promastigotes lost their shape and their internal organelles were no longer distinguishable. Similarly,
when exposed to Ag-NPs in UV light, the promastigotes membranes were disrupted and had an atypical
appearance. Furthermore, Ag-NPs showed a significant antileishmanial effect by inhibiting the proliferation
and metabolic activity of promastigotes.
19

20. NANOIMMUNIZATION-
• Nanoparticles have not only been used as drug vehicles but also as promising vaccine carriers. Vaccines are
composed of appropriate antigens responsible for induction of a protective immune response against a specific
pathogen, boosted by adjuvants. Despite many antigens are immunogenic to the host, the lack of universal
protection may result to the inability of the vaccine to elicit desirable immune response.
• The use of nanotechnology in delivering antigens and adjuvants have different aims:
(i) To enhance their uptake by APCs
(ii) To generate Th1 type immune response,
(iii) To induce a stronger immunological effect due to a simultaneous delivery to the same APC, compared to the
free antigen and adjuvant.
• Dendritic cells (DCs) are a class of specialized APCs that coordinate both innate and acquired immunity.
They sense the presence of microorganisms and recognize conserved pathogen associated molecular patterns.
Mature DCs migrate from the infection site to the closer draining lymph node for antigen presentation to naive
T-cells. The physicochemical properties of polymer nanoparticles when used as adjuvants in a vaccine could
mean a more effective immune response against Leishmania.
20

21. ABSORPTION AND DISTRIBUTION OF NANOPARTICLES-
• The absorption and distribution profile of a nanoparticle is greatly determined by physicochemical properties
such as size, charge, hydrophobicity, and targeting molecules, and these properties are dependent on the type
of the delivery system. The size of the nanoparticle is important for its entry into cells, its interactions with the
immune system, and its clearance. Cell uptake mechanisms are partially dependent on size.
• Endocytosis is the process through which nanoparticles or small molecules enter cells; the specific type of
endocytosis through which the nanoparticle enters the cell determines the translocation of the entrapped
molecule inside the endosome. Hydrophobicity affects cellular uptake, distribution, interaction with immune
cells and plasma proteins, and clearance from the body.
• Charge is important for mucoadhesion or diffusion, cellular uptake, and toxicity. Targeting affects
biodistribution and immune responses. These physicochemical characteristics of the nanoparticle surface also
play a decisive role in uptake. According to the physicochemical characteristics of the nanocarrier and the
nature of the target cells, two main internalization pathways may occur either phagocytosis or other endocytic
pathways (macropinocytosis or endocytosis) Distribution and uptake can also be driven by active vectoring.
21

22. • This involves the addition of specific compounds (ligands) to the nanoparticle surface. Such ligands favor
interactions with the cell membrane, thus enhancing the recognition of nanoparticles by cells. The ligand is
chosen dependent on its stability and selectivity with regard to the target cells. Other factors such as
availability and interactions with immunologic cells or with membranes are also taken into account. It is
noteworthy that polymeric nanoparticles and liposomes, whose structure and chemical composition strongly
differ, still show similar interactions with macrophages, based on their surface electric charge.
• Liposomes displaying a negatively charged surface, generally containing the negatively charged
phospholipids phosphatidylserine (PS) and phosphatidylglycerol (PG), exhibit a much higher binding to and
phagocytosis by macrophages as compared to neutral vesicles. Indeed, up-regulation of the level of negatively
charged PS in the outer leaflet of the Leishmania spp. amastigote plasma membrane was recently
demonstrated. This seems to be highly relevant for their attachment to macrophages, as negatively charged
phospholipids expressed in the outer membrane leaflet of cells are recognized by macrophages (via scavenger
receptors) as apoptotic cells.
22

23. FUTURE OF ANTILEISHMANIALAGENTS-
• The potential of some colloidal drug carriers such as emulsions, liposomes, and nanoparticles is of great
interest, mainly due to their versatile nature and attractive advantages in the context of parasitic diseases. The
potential of medicine delivery systems based on the controlled release of drugs, especially nanoparticles
(NPs), is attracting increased attention.
• Biodegradable nanoparticles are frequently used as drug delivery vehicles because of their therapeutic value
in reducing the risks of toxicity of some medicinal drugs. They can increase the bioavailability, solubility, and
retention time of many potent drugs that are difficult to deliver orally Nanoparticles can be useful in treating
some macrophage mediated diseases. Mononuclear phagocyte cells engulf Leishmania parasites, but also
remove drug particles from the body circulation.
• Liposomes and nanoparticles have shown great potential for improving the efficacy and tolerability of
antileishmanial drugs such as liposomal amphotericin Solid lipid nanoparticles and nanostructured lipid
carriers represent a second generation of colloidal carriers, mainly because of their better stability and ease of
commercialization.
23

24. CONCLUSION-
Targeting of antileishmanial drugs to infected macrophages via novel drug delivery systems presents a
promising approach to overcome the limitations associated with the current treatment protocols. The
unmodified and surface engineered drug carriers result resulted in the reduced toxicity and increased effect of
drug on the Leishmania parasites. The development of drug delivery systems will allow to overcome not only
toxicity and drug effectiveness but also contribute to lower costs which will not only benefit the treatment of
patients with leishmaniasis The ability to successfully treat Leishmania depends on not only identifying an
effective drug, but also requires production of a formulation that delivers the drug at parasiticidal levels into
infected macrophages, which can reside at multiple sites within the body.
24

25. Bibliography-
• Menezes J, Guedes C, Petersen A, Fraga D, Veras P. Advances in Development of New Treatment for
Leishmaniasis. BioMed Research International 2015; 1-10.
• Bruni N, Stella B, Giraudo L, Della Pepa C, Gastaldi D, Dosio F. Nanostructured delivery systems with
improved leishmanicidal activity: a critical review. International Journal of Nanomedicine 2017; 12: 5289–
5311.
• Balasegaram M, Ritmeijer K, Lima M, Burza S, Genovese G, Milani B, Gaspani S, Potet J, Chappuis F.
Liposomal amphotericin B as a treatment for human leishmaniasis. Expert Opin. Emerging Drugs 2012;
17(4):493-510.
• Gutierrez V, Seabra A, Reguera R, Khandare J, Calderon R. New approaches from nanomedicine for treating
Leishmaniasis. Chemical Society Reviews 2015; 1-28.
• Tiuman T, Santos A, Nakamura T, Filho A, Nakamura C. Recent advances in leishmaniasis treatment.
International Journal of Infectious Diseases 2011; 525–532.
• Norouzi R. A review on Most Nanoparticles Applied against Parasitic Infections. Journal of Biology and
Today's World 2017; 6 (10): 196-203.
• Almeida L, Terumi A, Lucia M, Bruno F, Braga K, Chorilli M, Marcia A. Nanotechnological Strategies for
Treatment of Leishmaniasis. Journal of Biomedical Nanotechnology 2017; 13:117–133.
• Daboul M. Is the Amastigote Form of Leishmania the Only Form Found in Humans Infected With Cutaneous
Leishmaniasis. Laboratory Medicine 2008; 39:38- 41.
25

26. •Dawit G, Girma Z, Simenew K. A Review on Biology, Epidemiology and Public Health Significance of
Leishmaniasis. Journal of Bacteriology and Parasitology 2013; 4:2-7.
•Tamiru A, Tigabu B, Yifru S, Diro E, Hailu A. Safety and efficacy of liposomal amphotericin B for treatment of
complicated visceral leishmaniasis in patients without HIV, North-West Ethiopia. BMC Infectious Diseases
2016; 16(548) :1-7.
• Gohil C, Kher J. Review on Leishmaniasis. Biomedical Journal of Scientific & Technical Research 2017; 1(5):
1-3.
• Berman J. Human Leishmaniasis: Clinical, Diagnostic, and Chemotherapeutic Developments in the Last 10
Years. Clinical Infectious Diseases 1997; 24:684-703.
• Roy P, Das S, Auddy R, Mukherjee A. Biological targeting and drug delivery in control of Leishmaniasis.
Journal of Cell and Animal Biology 2012; 6(6): 73-87.
• Croft S, Seifert K, Yardley V. Current scenario of drug development for leishmaniasis. Indian J Med Res
2006; 123: 399-410.
• Haldar A, Sen P, Roy S. Use of Antimony in the Treatment of Leishmaniasis: Current Status and Future
Directions. Molecular Biology International 2011; 1-23.
26

27. 27