Nanoparticles as multifunctional devices for the topical treatment of cutaneous leishmaniasis

1. Introduction

2. Factors affecting the efficacy

of CL topical chemotherapies:

benefits of NPs

3. AMPs and their symbiosis with

NPs

4. NO: a waste of therapeutic

potential without release

systems

5. Silver nanoparticles: new dress

with old fabric

6. PDT: selective parasite

destruction with NPs

7. NPs as immunomodulators

8. Conclusion

9. Expert opinion

Review

Nanoparticles as multifunctionaldevices for the topical treatmentof cutaneous leishmaniasisEsther Moreno, Juana Schwartz, Celia Fernandez, Carmen Sanmartın,Paul Nguewa, Juan Manuel Irache & Socorro Espuelas†

†University of Navarra, Tropical Health Institute & Pharmacy and Pharmaceutical Technology

Department, Pamplona, Spain

Introduction: Cutaneous and mucocutaneous leishmaniasis are major tropical

skin diseases. Topical treatment is currently limited to the least severe forms of

cutaneous leishmaniasis (CL) without risk of dissemination. It is also recom-

mended in combination with systemic therapy for more severe forms.

Progresses in this modality of treatment are hindered by the heterogeneity

of the disease and shortcomings in the clinical trials.

Areas covered: This review overlooks three major modalities of topical thera-

pies in use or under investigation against CL: chemotherapy, photodynamic

therapy and immunotherapy; either with older compounds such as paramo-

mycin or more recent nitric oxide donors, antimicrobial peptides or silver

derivatives. The advantages and limitations of their administration with

newer formulation strategies such as nanoparticles (NPs) are discussed.

Expert opinion: The efficacy of a topical treatment against CL depends not

only on the intrinsic antileishmanial activity of the drug but also on the

amount of drug available in the dermis. NPs as sustained release systems

and permeation enhancers could favour the creation of a drug reservoir in

the dermis. Additionally, certain NPs have immunomodulatory properties or

wound healing capabilities of benefit in CL treatment. Pending task is the

selective delivery of active compounds to intracellular amastigotes, because

even small NPs are unable to penetrate deeply into the skin to encounter

infected macrophages (except in ulcerative lesions).

Keywords: cutaneous leishmaniasis, dermal retention, immunostimulation, nanoparticles,

nitric oxide, photodynamic therapy, silver nanoparticles, topical treatment

Expert Opin. Drug Deliv. (2014) 11(4):579-597

1. Introduction

The term ‘leishmaniasis’ describes a range of diseases caused by different species ofprotozoa of the genus Leishmania that are transmitted by phlebotomine sandflies.Leishmania parasites are injected into the vertebrate host as a promastigote (theelongated form with an external flagellum), which is phagocytosed by differentphagocytic cells in the host. Within cells of the mononuclear phagocyte system(its habitat), promastigotes differentiate into amastigotes (the round form withoutan external flagellum) and then proliferate, establishing the infection [1].

Leishmaniasis encompasses visceral and tegumentary forms [2]. Visceral leishman-iasis (VL) is the most severe form, in which the parasites have migrated to vitalorgans. It is fatal if untreated. Tegumentary leishmaniasis is one of the major trop-ical dermatosis of immense public health significance. It is prevalent in 88 countriesand it is estimated that 350 million people worldwide are at risk of infection, withan estimated prevalence of 12 million cases and an annual incidence of be 1.5million cases per year. Most (90%) of the cases are reported in Africa (mainly in

10.1517/17425247.2014.885500 © 2014 Informa UK, Ltd. ISSN 1742-5247, e-ISSN 1744-7593 579All rights reserved: reproduction in whole or in part not permitted

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http://informahealthcare.com/journal/EDD

Morocco, Ethiopia and Tunisia), the Middle East (mainly inAfghanistan, Pakistan, Iran, Iraq, Syria and Saudi Arabia) andLatin America (mainly in Brazil, Bolivia, Colombia, Ecuador,Peru and Venezuela) [3,4].Tegumentary leishmaniasis [3] is divided into three major

clinical phenotypes: localised cutaneous leishmaniasis (LCL),diffuse cutaneous leishmaniasis and mucocutaneous leishman-iasis (MCL), whose manifestations range from small cutaneousnodules to gross mucosal tissue destruction. A fourth form ofCL is the sequel of VL, known as post kala-azar dermal leish-maniasis. This clinical diversity is a consequence of the numer-ous Leishmania species responsible for the disease pathogenesisas well as the immune response and the genetic susceptibility ofthe host to infection. In general terms, species prevalent in theOld World (Africa, Asia and Southern Europe) (OWCL)produce limited clinical manifestations compared with NewWorld species (Latin America) (NWCL).The treatment of CL (even self-curing form) is recom-

mended to expedite healing, reduce the risk of scarring, pre-vent parasite dissemination and reduce chance of relapse[5,6]. The therapy is decided according to the clinical lesionsand the etiological species and its ability to develop intomucosal leishmaniasis. Parenteral treatment is given for severecutaneous disease, for the treatment of Leishmania specieswith the potential to disseminate, and for establishedMCL [7]. The first-line treatment is based on antimonial

derivatives given by parenteral route, daily for at least 3 weeks.However, systemic antimonials are associated with consider-able toxicity, and there are reports of emerging leishmanialresistance. Amphotericin B (AmB), pentamidine or paramo-mycin (PM) can be used as first-line agents because theyhave been found as effective as the antimonials, butthese are also limited by toxicity and parenteral route ofadministration.

For uncomplicated LCL, local chemotherapy [8], topical orintralesional, or physical therapies have been assayed inclinical practice: intralesional antimonials, topical PM with12% methylbenzethonium chloride (PM-MBCL), cryother-apy or thermotherapy. More recently, photodynamic therapy(PDT) with 5-aminolevulonic acid (5-ALA) or immunother-apy with imiquimod (IMQ) in a cream have also been evalu-ated. However, there are several difficulties to determinewhich ones of these local therapies were really effective, firstof all because the heterogeneity in the clinical features of CLlesions. Moreover, clinical trials usually failed to define com-mon clinical end points, follow-up periods and rarely includeplacebo controls, which are important given the potential ofCL in particular to resolve spontaneously, making it difficultto assess their relative merits [9,10].

In general, most studies have found the highest clinicalefficacy and lowest recurrence rates with intralesional antimo-nials [11,12]. Thermotherapy was more than or as effective asparenteral antimonials for the treatment of Leishmania major(L. major) and NWCL infections, respectively. PDT wassuperior to topical PM-MBCL in L. major infections.

As far as topical PM-MBCL is concerned, a meta-analysisrecently published that overviewed 14 randomised controlledtrials (RCTs) involving 1221 patients concluded that its activ-ity was similar than intralesional antimonials in L. majorinfections. Its efficacy was inferior in treating NWCL [13].Up to now, there are no sufficient clinical trials to supportthe use of topical immunotherapy with 5% IMQ, althoughthe treatment had significant cure rates than placebo and acure rate of 90% given in combination with systemic antimo-nial at the beginning of the therapy. Besides, clinical trialsaddressed with topical AmB did not provide sufficientevidence for their use in OWCL or NWCL [11,12].

This review analyses the different local therapies assayed inthe treatment of CL either in clinical or preclinical settings.They are classified in three major groups: chemotherapy, phys-ical therapies and immunotherapy. Within the chemotherapy,we approach not only the use of the most common drugs, asPM and others (splendidly reviewed in [14,15]), but also othercompounds such as antimicrobial peptides (AMPs), nitricoxide (NO) donors or silver derivatives (summarisedin Table 1). The utility of their administration by means ofnanoparticles (NPs) instead of conventional dosage forms(like creams, ointments) is discussed for each therapeuticmodality. Among physical therapies, we only analysed thePDT because it was the sole for which application of anyformulation was required.

Article highlights.

. Current status of cutaneous leishmaniasis (CL) topicaltherapies appeals to investigate in the performance ofnewer tools, as nanotechnologies.

. Drug-loaded nanoparticles (NPs) as sustained releasesystems and permeation enhancers can increase theamount of drug arriving to the dermis (where infectedmacrophages reside) and modulate the permeation rate,enhancing the efficacy of the treatment with commonantileishmanial drugs. Preclinical studies have confirmedthe performance of liposomes to enhance the efficacyof topical paramomycin.

. The use of NPs is mandatory to take advantage ofantimicrobial properties of nitric oxide (NO) andNO donors.

. Silver NPs have emerged as a safer and more effectiveform of administering old silver derivatives. They couldbe useful for the treatment of CL in combination withother actives.

. Major handicap of current photodynamic therapy with5-aminolevulonic acid or methylene blue is theunspecific destruction of all the treated tissue. NPs couldallow a more selective destruction of the parasite andreduce the risk of scarring.

. Besides loading active compounds, NPs themselves,because of their composition or particulate dimensions,have immunomodulatory properties or wound healingcapabilities (e.g., cationic NPs) of great interest in CLtopical therapy.

This box summarises key points contained in the article.

E. Moreno et al.

580 Expert Opin. Drug Deliv. (2014) 11(4)

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Table 1. Examples of topical formulations assayed against CL in preclinical and/or clinical settings.

Agent Formulation Animal/human studies

ChemotherapyPM PM (15%) + MBCL ointment Animals: 100% curation in Leishmania major-infected

BALB/c mice given twice daily for 6 days starting 30 daysafter infection [22]

Humans: efficacy similar to intralesional SbV in OWCL andinferior in NWCL [13]

PM (15%) in WR base with gentamicin(0.5%)

Humans: around 80% cure rate with or without 0.5%gentamicin [30]

Liposomes Animals: absence of parasite burden in the spleen andcomplete wound healing 8 weeks after infection in BALB/cmice infected with L. major treated twice a day for4 weeks with 5 -- 7.5 mg PM/mice. No comparison withointment [150]

Transferosomes Animals: more effective than the cream inL. major-infected BALB/c mice after treatment twice a dayfor 4 weeks with absence of parasites in spleen andreduction of lesion size [40]

HEC hydrogel (10%) Animals: similar efficacy (evolution of lesion size) thanantimonials after topical application twice a day for20 days in hamster infected with L. braziliensis andL. amazonensis [38]

AmB Fungizone�: micellar solubilisation with DOCEthanolic solution of liposomes

Animals: efficacy of Amphocil� and Albecet� but notFungizone in L. major-infected BALB/c mice following21 days administration [151]

Humans: no evidences of efficacy in OWCL [12]

Humans: 1 trial with 17 patients in Israel treated withAmphocil disperser in 5% ethanol. The lesions healedfaster than placebo [50]

Sitamaquine Anhydrous gelEmulsionsW/S emulsions

Animals: no reduction of lesion size or parasite burden inL. major infected BALB/c after treatment with 50 mgformulation per day beginning 10 -- 11 days after infectionuntil day 25 -- 28 [32]

BPQ Hydrous gelW/O emulsionAnhydrous gel

Animals: reduction of lesion size and parasite burden afterdaily administration for 13 days starting 23 days post-infection [44]

MIL Miltex�

Mixture with other lipidsAnimals: 1.5 mg daily applied for 5 or 2 weeks reduceparasite load and lesion healing in BALB/c and C57BL/6 mice infected with L. major or L. mexicana, althoughrelapse occurred [152]

Silver Nanosilver Animals: no differences with control group inL. major-infected BALB/c mice [109]

NO donors GSNO in PBS Animals: reduction in size lesions, parasite burden andwound healing of BALB/c mice infected with L. major andIFN-g-KO C57BL/6 mice infected with L. braziliensis [90]

SNAPCream (200 µmol/l)(cetyl alcohol, quaternary ammonium, water)

Humans: healing of ulcers after 30 days of application in16 patients [89]

NO patch Humans: 37.1 cure rate versus 94.8% glucantime inpatients infected with L. panamensis [93]

PDTMB Aqueous solution

CreamAnimals: reduction of lesion size and parasite burden inlymph nodes in hamster infected with L. amazonensis10 min before LED irradiation for 1 h 3 times a week for3 months in solution or cream [153]

Humans: faster wound healing of a patient infected withL. amazonensis as compared with parenteral SbValone [117]

5-ALA: 5-Aminolevulonic acid; AmB: Amphotericin B; BPQ: Buparvaquone; CL: Cutaneous leishmaniasis; DOC: Sodium deoxycholate; GSNO: S-nitrosoglutatione;

HEC: Hydroxyethylcellulose; IMMA: Intramuscular meglumine antimoniate; IMQ: Imiquimod; IPMA: Intarperitoneal meglumine antimoniate; LED: Light-emitting

diode; MB: Methylene blue; MBCL: Methylbenzethonium chloride; NO: Nitric oxide; NWCL: New World CL; OWCL: Old world CL; PDT: Photodynamic therapy;

PM: Paromomycin; SbV: Pentavalent antimonial compounds; SNAP: S-nitroso-N-acetylpenicillamine; W/O: Water-in-oil; W/S: Water-in-silicone.

Nanoparticles as multifunctional devices for the topical treatment of cutaneous leishmaniasis

Expert Opin. Drug Deliv. (2014) 11(4) 581

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As there is not international consensus about what a NP is,we point out that in this review we have considered a NP asany microscopic particle whose size is measured in nano-metres. According to their structure and composition, thereare many types of NPs such as liposomes, solid lipid NPs orpolymeric NPs. Liposomes are the nanocarriers most widelyinvestigated in the topical treatment of CL [14].

2. Factors affecting the efficacy of CL topicalchemotherapies: benefits of NPs

The efficacy of any therapy is determined by two factors:i) the intrinsic activity of the active molecule and ii) the deliv-ery of this molecule to its site of action at a relevant concentra-tion for a sufficient period of time to do its action. Thesecriteria, applied to CL, are summarised in Figure 1. The targetin CL is the infected macrophages, localised deeply in theskin. Local delivery of actives at this level is particularly prob-lematic because it has to contend, first, with the stratum cor-neum (SC) barrier and/or, second, with a rapid clearance tosystemic circulation. In normal conditions, the SC barrieronly permits small molecule diffusion [16]. Therefore, thedrug penetration level is governed by the mechanism of pas-sive diffusion from the upper layers and convective blood,lymphatic and interstitial flow from the bottom. The rapidclearance to systemic circulation or the transport to papillarydermis (lower dermis) and underlying tissues will depend onthe drug binding with skin compounds and plasma proteins.Drugs with high protein binding have more chance to

accumulate deeply in the skin [17]. Moreover, this process iscontrolled by multiple factors related to physicochemicalproperties of the drug (such as molecular weight (MW) andlogP), the skin status at the site of application, formulation/vehicle type and the complex relationships among them [16],as explained below.

2.1 Physicochemical properties of drugsUsually, optimal candidates for CL topical therapies havebeen mismatched with compounds with the ideal physico-chemical properties and flux (J) parameters for percutaneousabsorption through intact skin. These include low MW,logP 1 -- 3, solubility parameter 9 -- 10 and few functionalgroups capable of hydrogen binding [18]. Therefore, formula-tions for these compounds providing high permeability coef-ficient (Kp) and J values (estimated according with Fick’slaw of diffusion) [19] in ex vivo studies with Franz diffusioncells were considered as the most promising ones.

As summarised in Table 2, PM, the only drug currentlyaccepted for topical CL therapy, does not fit the ‘optimal’physiochemical properties for percutaneous absorptionthrough skin. It has a MW > 500, it is freely soluble in water(logP ~ -8.3), has > 11 functional groups capable of hydrogenbonding and it is positively charged at physiological pH.Accordingly, the Kp calculated using the Potts--Guy equa-tion [20] is one of the lowest among common drugs andthus, theoretically, PM has poor chance to penetrate intothe skin. Furthermore, this drug has rather modest intrinsicactivities against Leishmania spp. (Table 2). On the contrary,

Table 1. Examples of topical formulations assayed against CL in preclinical and/or clinical settings (continued).

Agent Formulation Animal/human studies

5-ALA 5-ALA w/o cream or 20% solution Animals: eradication of parasite burden in BALB/c infectedmice in the lesion after 50 J/cm2 4 h irradiation 1 weeklater [125]Humans: evidences of the higher efficacy of PDT weeklyfor 4 weeks than PM + MBCL twice daily for 28 days inL. major infections [12]

ImmunotherapyIMQ Aldara� (5% IMQ ointment) Animals: no improvement of topical PM in L. major

infected mice after 10 days treatment twice a day [137]

Animals: improvement of glucantime therapy alone inL. major-infected BALB/c mice (100 mg/kg/day IPMA) for12 days and the dream once every 3 days for2 weeks [138]

Humans: 90% cure in 12 patients that no responded toIMMA alone after daily treatment for 20 days with250 mg containing 5% IMQ [136]

Humans: no evidences of synergistic effect combined withIMMA in L. tropica and NWCL [11,12]

5-ALA: 5-Aminolevulonic acid; AmB: Amphotericin B; BPQ: Buparvaquone; CL: Cutaneous leishmaniasis; DOC: Sodium deoxycholate; GSNO: S-nitrosoglutatione;

HEC: Hydroxyethylcellulose; IMMA: Intramuscular meglumine antimoniate; IMQ: Imiquimod; IPMA: Intarperitoneal meglumine antimoniate; LED: Light-emitting

diode; MB: Methylene blue; MBCL: Methylbenzethonium chloride; NO: Nitric oxide; NWCL: New World CL; OWCL: Old world CL; PDT: Photodynamic therapy;

PM: Paromomycin; SbV: Pentavalent antimonial compounds; SNAP: S-nitroso-N-acetylpenicillamine; W/O: Water-in-oil; W/S: Water-in-silicone.

E. Moreno et al.


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compounds ‘apparently’ more suitable were not effective atall; they showed contradictory results in mice and humansamong different clinical trials. For example, miltefosine(MIL) reduced lesion size and healed established lesions inL. mexicana- and L. major-infected BALB/c and C57BL/6mice; however, MIL failed to cure nodular CL lesions inhumans [21]. According to Potts--Guy equation, MIL has the-oretically the highest Kp among compounds enumeratedin Table 2.

The first study evaluating the efficacy of topical PM wasaddressed by El-On et al. 30 years ago [22]. They showedthat a 15% PM plus 12% MBCL ointment produced a com-plete cure of L. major lesions in BALB/c mice. In the sameseries of experiments, PM in combination with other quater-nary ammonium disinfectants or dimethyl sulfoxide(DMSO) (a known penetration enhancer), gave significantlevels of activity. By contrast, AmB, pentamidine, antimonialsor allopurinol showed lower activity.

Indeed, neither AmB nor antimonials are good topical can-didates because of predictable poor capacity to permeate theskin. However, both of them and, specially, AmB has EC50

much lower than PM. Pentamidine and allopurinol havenot only higher Kp than PM, but also EC50 in general lowerthan the aminoglucoside (Table 2).

The higher efficacy of PM in this set of experiments can beexplained taking into consideration the skin permeabilityalteration in CL lesions and the relationships within thetrinity vehicle-drug-skin.

2.2 Skin alterations in CL lesions

The skin barrier is seriously damaged in CL lesions. It is well-

known that the skin alteration enhances the skin penetration

of drugs [23], most significantly for those compounds that

have low penetration rates through human skin and, espe-

cially, hydrophilic ones [24,25]. Moreover, because of its hydro-

phobic nature and packing structure, the hydrophobic SC is a

major obstacle to skin compounds penetration, especially, for

hydrophilic molecules. On the contrary, skin damage does

not affect the penetration and permeation of extremely lipo-

philic compounds. This type of compound usually remains

retained in the SC because the increasing hydrophilicity

from SC to dermis hampers the diffusion towards deeper

layers in the skin [26,27].Due to the alterations produced in the skin with CL

lesions, the optimal attributes of drugs capable to permeate

will be different to those described for intact skin [18] and

move towards more hydrophilic compounds that have more

chance to arrive at the dermis in damaged skin. Accordingly,

we could predict a higher increase in PM skin permeation

for damaged skin in comparison with the effect produced,

for example, in the absorption of AmB, a more hydrophobic

compound. In fact, it was determined that PM permeation

increases between 2 and 52 times when different formulations

of the drug were evaluated with intact or stripped hairless skin

(skin without SC) of mice, respectively [28]. A recent work

determined a 6 -- 20% of dose absorbed in patients [29] after

• SC damage • MW

• logP

• Skin metablism

• Binding to skin

• Sustained release

• Permeation enhancement

Skin status1.1. Drug release fromthe formulation

1. Tissue level: formation of a dermis reservoir

UlcersNodules

E

D

D

1.3. Clearance by dermalblood supply

1.2. Permeationthrough the skin:partitionbetween skinlayers

Drug properties

Vehicle

2. Cellular level: intrinsic activity of drugagainst Leishmania spp. amastigotes

• Inflammation

Figure 1. Factors affecting the efficacy of CL topical therapy. At tissue level, it depends on the formation of an active drug

reservoir in the dermis. At cellular level, it is governed by the intrinsic antileishmanial activity of the drug. The status of the

skin in CL lesions and the relationships within the trinity vehicle--drug--skin determine the amount of drug available in the

dermis, which stress the importance of formulation design in the topical therapy of CL.CL: Cutaneous leishmaniasis.



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20 days of daily treatment with a cream containing 15% of

PM and 0.5% of gentamicin in a complex base called

WR279,396 [30].Another factor related to skin status that can be important

is the inflammation that present CL lesions also accompaniedby the efflux of plasma proteins [31]. Little attention has beengiven to the effect that drug--skin compounds or drug--plasmaprotein interactions has in their skin retention as well as intheir activity against Leishmania spp. Garnier et al. observedsitamaquine accumulation in the skin, whereas several typesof topical formulations (gel, water-in-oil and oil-in-wateremulsions) loaded with the drug did not reduced the parasiteburden in L. major-infected BALB/c mice [32]. The authorssuggest that the interaction of sitamaquine with melanin orother proteins decreased the amount of the compound thatcan be internalised by infected dermal macrophages anddestroy the parasite. In this context, we emphasise the lowprotein binding of PM, the only drug with confirmed efficacy(Table 2). Obviously, protein binding is only one of themultiple factors that determine the topical efficacy of aformulation. Fluconazole, a hydrophilic azole with lowprotein binding, administered at a 10% concentration in acream was not effective in mice infected by L. major andL. amazonensis [33].

Other aspects that complicate CL treatment and canexplain the variability in the obtained results [3] are the hetero-geneity of lesions depending on Leishmania species, thepatient, the anatomical localisation of lesions or the courseof the infection. Erythematous nodules, indurated plaques,scaly plaques or ulcers show variable structural and functionalalterations of the skin layers (as well as parasite load) thatdifferently affect the drug absorption [27,34]. For example,the efficacy of PM ointment ranged from 31 to 74% inpatients infected by L. major [3].

Moreover, there are also important differences in theskin characteristics between the animal models andhumans. Experiments of transdermal penetration can becarried out in vitro with pig ear skin because of the bigsimilarity between pig and human skin [15]. Next, treat-ment efficacy should be tested in vivo in Leishmania-infected mice or hamster before evaluation in patients.The contradictory results obtained for mice and humansand among the different preclinical and clinical trials thatcomplicate the drug and formulation algorithm selectionduring the screening of new treatments should not besurprising [3].

2.3 Formulation/vehicles issues: NP attributesConcerning the characteristics of the vehicles, it is well knownthat the interactions between drug--skin, vehicle--drug andvehicle--skin are determinant in topical administration [35].El-On et al. used the same cream (prepared by adding waterto cetomacrogol ointment B.P) for all the drugs, withdifferent properties [22].T

able

2.Propertiesofdrugswithantileishmanialactivityofinterest

intopicaldelivery.

Drugs

LogP

Melting

point(ºC)

MW

Solubility

(mg/m

l)

Hydrogendonor/

acceptorco

unt

Kp*

Plasm

aprotein

binding

Invitro

activity

Leishmania

spp.

amastigotes(µM)

Leishmania

spp.

AmphotericinB

0.8

170

924.1

0.75

29

1.70E-06

90%

0.1

L.amazonensis[154]

Miltefosine

2.25

232--234

407

25--100

63.72E-06

96%

1.72--6.25

L.donovani[155]

Paromomycin

-8.31

220

617

50

32

7.89E-09

Low

9.2

µM--75mM

L.donovani[156]

Pentamidine

4186

340.4

Very

soluble

10

10.61

60%

2.89

L.donovani[157]

SitamaquineHCl

4.16

179--180

416.1

Very

soluble

571.08

n.d.

2.24

L.donovani[157]

Buparvaquone

4.74

178--184

326.4

Insoluble

n.d.

638.12

n.d.

1.50

L.infantum

chagasi

[158]

Gentamicin

-3.14

105

477.6

100

20

2.33E-04

Low

n.d.

n.d.

Ketoconazole

4.35

146

531.4

Insoluble

614.77

n.d.

1E-7

L.m.mexicana[159]

Fluconazole

0.4

138--140

306.3

86

0.77

11.5

300

L.donovani[155]

Itraconazole

5.66

166.2

705.6

Insoluble

9195.35

n.d.

1E-7

L.mexicana[159]

Allopurinol

-0.55

350

136.2

0.57

74.67

3%

>250

L.amazonensis[154]

Sodium

stibogluconate

-3.41

98

912

Very

soluble

22

3.37E-07

n.d.

57.3

L.donovani[160]

*KpcalculatedwithPotts--G

uyequation

[22].

MIL:Miltefosine;MW:Molecularweight;n.d.:Nodata

found.

E. Moreno et al.


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According to Fick’s law of diffusion commonly applied toskin permeation studies of drugs [18], formulations should bedesigned to obtain the maximal thermodynamic activity ofthe compound [16,36]. That means a maximum of solubility(and drug concentration) in the formulation as well as moreaffinity for the skin than for the formulation. Comparedwith other antileishmanials, PM is characterised by its highlyaqueous solubility that allows the easy incorporation of highamount of solubilised drug in the aqueous phase of creamsor other hydrophilic vehicles. The higher the drug gradientconcentration is, the higher the driving force for diffusionacross the skin will be. On the contrary, AmB needs to besolubilised in DMSO for its incorporation in the cetomacro-gol ointment B.P [22]. The formation of a micellar suspensionafter mixture of all the components during the preparation ofthe cream is highly likely to occur.

However, it is necessary not only a concentration gradientbut also a certain tendency of the compound to escape fromthe vehicle to the skin, both aspects within the concept ofthermodynamic activity. Comparing two vehicles containingthe same concentration of a drug, the thermodynamic activityand the penetration into the skin will be higher in the vehiclewith the lower solubility except when interactions betweenvehicle and skin occur. Otherwise, the drug will remain inthe formulation. The best formulation will be made withthe vehicle solubilising the maximal amount of drug andwith a solubility parameter similar to skin as expressed bythe well-known paradigm ‘apply wet on wet and dry ondry’ [16]. In general, vehicles with high affinity by the skin usu-ally promote drug partitioning (K) by enabling the compoundto diffuse inside the skin in a concentration equivalent to thatin the vehicle [35]. Hydrophilic formulations will haveenhanced interactions with dermal membranes or skin withremoved SC that result in changes in partition and diffusionproperties of the drug and thus, modified permeation.Accordingly, 10% PM applied in hydrophilic gels haveslightly greater efficacy than the reference formulation (15%PM in MBCL ointment) in BALB/c mice infected byL. major [37]. The activity of this formulation was higherthan that observed for parenteral antimony treatment inhamster infected by L. amazonenis and L. braziliensis [38].

Thus, following the paradigm of topical treatment, thevehicle should be fitted according to the type of lesions tobe treated that in CL ranges between ulcers and the oppositedry lesions, complicating their selection and optimisation.Finally, the clearance rate of drugs arriving at the dermis inCL therapies has not specifically been determined. Instead,several works have confirmed the importance of skin retentiontime of drug in the efficacy of CL topical therapy. Theenhanced activity of several PM liposomal formulations hasbeen associated with greater skin retention and not with skinpenetration. The PM cream has better skin penetration andwas less active than liposomal formulation [39-41]. ConcerningAmB, Amphocil� dispersed in a hydroalcoholic solution wasthe only AmB formulation that showed a certain activity after

topical application in agreement with higher retention indermal membranes as compared with Fungizone� [42].

Other studies addressed with buparvaquone (BPQ) alsoconfirm the retention of the drug in skin and especially in der-mal membranes as a good predictor of efficacy [43]. A hydrogelloaded with BPQ was found as effective as an anhydrous gelloaded with a phosphate prodrug of BPQ (3-POM-BQP),with four-fold lower drug-loading concentration. Theretention of the drug in the skin from this formulation washigher [43,44].

Thus, formulations for the topical treatment of CL shouldbe designed to promote the partitioning of the drug into theskin (K) from the formulation, rather than changing thedrug’s diffusion coefficient (D). In fact, it seems that quickdiffusion through the skin was detrimental to PM activity [40].

In this context, we highlight the major strategies commonlyaddressed to improve the dermal accumulation, namely occlu-sion, permeation enhancers and vehicles or delivery systems.

The use of an occlusive dressing has an adjuvant effect byseveral mechanisms [45]. First, the dressing prevents theremoval of the ointment from the lesion by protecting theskin from scratching, rubbing and scraping. Second, occlusionon burns or wounds favours the epidermal regeneration.Finally, water retention by occlusive dressing results in hydra-tion of the application zone and likely improves the skin pen-etration and diffusion of compounds [46]. However, thisapproach is not really easy to accomplish in preclinical studiesand it has not been standardised in patients. Lesions are cov-ered with a sterile gauze secured with tape at the most [30] andit cannot be considered as a real occlusive dressing [10].

Permeation enhancers usually tend to increase D and thepermeation rate [47,48]. They allow many drugs to penetrateto deeper skin layers. However, they also tend to acceleratethe permeation rate through the skin. The time of contactwith the microbes seems to be critical in antimicrobialchemotherapy.

Vehicles (i.e., liposomes) with strong interactions with skinusually promote drug partitioning (K) by enabling the com-pound to diffuse inside the skin in a concentration equivalentto that in the vehicle [35]. NPs, as vehicles in skin delivery, canenable a sustained release of drug by virtue of their controlledrelease properties [49]. The delivery systems should be able toprovide controlled release in enough amounts to be active.In some cases, the rate of release could be too long and eventhe NPs can be removed from the skin before the drug hasbeen released.

As indicated before, the performance of NPs for the topicaltreatment of CL was confirmed by the enhanced efficacy ofPM liposomes [39-41]. Similarly, compared with Fungizone,the three lipidic formulations of AmB had significant effectwhen topically applied to L. major lesions, in presence ofethanol as permeation enhancer [50].

Although in vitro experiments with Leishmania amastigoteshave clearly established the performance of NPs to improvethe efficacy of drugs such as PM or AmB, the probability of



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NPs to penetrate in the skin deep enough to directly encoun-ter with infected macrophages in the dermis is really low. Yetmatter of debate, current evidence is that NPs preferentiallyaccumulate in the hair follicle openings and remain in theupper layers of the SC of intact skin [51]. It is also clear thatNPs are able to penetrate deeper in damaged skin and thatpenetration depend on the particle size [52,53]. Furthermore,it also appears that organic and inorganic particles penetratethe skin quite differently. According to their composition,their interaction with the skin can provoke alterations thatfacilitate the passage of the loaded drugs [54]. The incorpo-ration of permeation enhancers such as single chain surfac-tant, ethanol or terpenes to lipid vesicles has generated novelfamilies of lipid vesicles called, respectively, transferosomes,ethosomes and invasomes [55]. Either due to the direct effectof the permeation enhancers in the skin or the modulationin the flexibility of the vesicles produced by their incorpo-ration in the vesicle composition, these types of NPs seemto have the capability to penetrate deeper in the skin than con-ventional vesicles: they have been visualised deeper in the SCand viable epidermis and more amounts of the loaded drugspermeated through the skin. A work confirmed the superiorefficacy of transferosomes containing PM [40] versus PMcream in L. major-infected BALB/c mice, estimated as evolu-tion in the lesion size and parasite burden in the spleen,although conventional liposomes were not compared in thesame experimental work.In brief, formulations that produce a high and sustained

retention of a compound in ex vivo studies with Franz diffusioncells and dermal membranes could have probably more chanceto be effective, at least in CL ulcerative lesions. Aiming a topicaltreatment, this determination is as important as the intrinsicactivity of the active against Leishmania spp. The experimentalestimation of both parameters should be addressed duringthe screening of compounds and formulations in CL topicaltherapy.

3. AMPs and their symbiosis with NPs

Besides conventional organic molecules, several AMPs haveshown activity against Leishmania spp. [56]. AMPs belong toseveral families such as temporins, bombinins, magainins,histatins, cathelidins, indolicins and melittins [57]. These pep-tides lack any specific consensus amino-acid sequences, butmost of them maintain common features such as containingpositive charge and relatively hydrophobic and amphipathicstructure. The charge and amphipathic features allow themto interact with plasma membranes resulting in pore forma-tion and release of cytosol components. This is their principalmechanism of action, although some of them also interactwith intracellular targets and produce biochemical changesresembling apoptosis [57].Their selective action on protozoans is based on their higher

concentration of anionic phospholipids in their cell membranecompared with mammalian cells. Their immunomodulatory

properties [58] and ability to recruit inflammatory cells to siteof parasite infection also affect parasite replication and viabil-ity [59,60]. Their use has several important limitations and infact, although many AMPs have been evaluated in vitro againstseveral species of Leishmania, only two peptides have beenevaluated in vivo in a model of VL [61]. First, their intrinsicactivity is usually in the range of µM versus often nM-activecommon drugs [56]. Moreover, they have a high inherent sus-ceptibility to chemical and enzymatic degradation [62]. Theirbig size, in the range of kDa (whereas optimal skin candidatesshould be < 500 Da), and positive charge besides their poorintrinsic activity predict insufficient penetration through theskin to be effective. However, this supposition should be con-firmed in view of the lack of correlation between compounds,physicochemical properties and CL topical efficacy previouslydescribed. Thus, AMPs could merit a place in the topical treat-ment of CL.Moreover, AMPs are abundantly present in mam-malian skin (cathelicidins and b-defensins), where theyprovide a rapid first line of immune defence against patho-gens [63]. Poor attention has been paid to theirimmunomodulating properties [64] and their role in the infec-tion by Leishmania that can be a valuable strategy ofimmunotherapy.

In another context, a group of peptides collectively calledcell-penetrating peptides (CPPs) have been evaluated forthe ability to transport diverse cargoes into cells and tissues[65] and proposed as transdermal drug delivery systems [66].Therefore, some CPPs such as Tat, Pep-1, penetratin orMAP have showed antimicrobial activities [67,68]. Further-more, it could be interesting to study the antileishmanialactivity of CPPs or vice versa to analyse the CPPs propertiesof leishmanicidal AMPs. In fact, a magainin pore-formingpeptide was able to improve the transdermal penetration offluorescein [69] and certain peptides belonging to magaininsfamily have antileishmanial activity. Similarly, gramicidinincreased the percutaneous permeation of benzoic acid, amodel drug [70].

The ability of CPPs to improve skin penetration of com-pounds has not been compared with other permeationenhancers. However, they can be complexed or conjugated toother leishmanicidal compounds, producing an improvementin their skin penetration and also a synergistic leishmanicidaleffect.

NPs have been proposed as a strategy to enhance theactivity of AMPs due to the protection exerted by the car-riers against degradation and more selective delivery tothe infective agents [71]. These benefits, as previously indi-cated, do not entirely apply on skin delivery because ofthe poor chance of NPs to approach infected macro-phages in the dermis. However, CPPs can take advantageof NPs skin delivery: the surface modification of NPswith CPPs such as polyarginine [72] or TAT peptide hasbeen described as an option to translocate NPs with theirpayloads into deeper skin layers [73], nearer to Leishmaniaparasites.

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4. NO: a waste of therapeutic potentialwithout release systems

Endogenous NO is considered one of the most versatile

endogenous molecules, which is involved in many physiolog-

ical processes such as neurotransmission, blood pressure regu-

lation, immune response, cell differentiation and apoptosis.

Oxidation of L-arginine to L-citrulline by a family of nitric

oxide synthase enzymes leads to NO production. NO has a

huge therapeutic potential that may range from antimicrobial

activity [74] to vasodilatation, pro- or anti-inflammatory,

wound-healing or immunomodulatory effects [75].The role of nitrogen species in killing of Leishmania para-

sites is clearly established. IFN-g produced by T-helper 1(Th1) cells induces the expression of inducible nitric oxidesynthase enzyme by macrophages. This enzyme catalysesNO production, which kills the parasite [76].

Unfortunately, the use of NO as therapeutic agent presentsmany hurdles due to the small size, the abrupt half-life andthe instability of the molecule. In order to harness the poten-tial benefits, this evanescent gas needs to be delivered neartarget cells at precise concentrations and long enough periodsto exert its biological action [77].

After topical application, the D of NO is enough to pene-trate the epidermis, to reach the microcirculation of the upperhorizontal plexus and the upper layer of the dermis [78,79]. Thesignificant transcutaneous penetration of NO (up to 3 mm indepth in the human dermis) has motivated the pursuit of top-ical administration of NO and NO donors for the treatmentof skin diseases [80-83]. The delivery strategies evaluatedinclude gaseous NO from a tank, organic nitrates, acidifiednitrite, NO donated from a pro-drug (NO donors) andrelease and generation from NPs [74,77,84-86].

NO donors more currently evaluated belong to two major

families such as diazeniumdiolates and S-nitrosothiols[74,77,87,88]. Although the NO release from diazeniumdioalates

(NONOates) can be controlled depending on the formulation,some concerns about their toxicity and the possibility for form-ing carcinogenic secondary nitrosamines have limited their

clinical utility. S-Nitrosothiols represent an important and amore desirable class of NO donors because they aregenerated endogenously. However, the topical application of

these NO donors is limited by the low stability of the S-NObond. In 1998, Lopez-Jaramillo et al. reported the therapeutic

effect of S-nitroso-N-acetylpenicillamine (SNAP), a S-nitroso-thiol, in CL. Sixteen Ecuadorean patients infected by L. brazil-iensis were treated with a SNAP cream administered topically

every 4 h (except during sleep) for a total of 10 days. After5 days, improvement of the ulcers were observed and by

30 days all lesions were healed and new skin appeared [89].More recently, S-nitrosoglutathione was found to be cytotoxicto intracellular amastigotes and promoted the healing of

topically treated L. major and L. braziliensis skin lesions,respectively, in BALB/c and IFN-g-KO C57BL/6 mice [90].

Another cost-effective alternative assayed in Leishmania isthe topical application of NO-generating creams with acidifiednitrite [91]. NO is generated non-enzymatically by the acidifica-tion of nitrite (KNO2) by organic acids such as ascorbic acid(ASC) or salicylic acid (SAL). Experiments in vitro showedthat the combination of KNO2 with ASC or SAL killedpromastigotes and amastigotes of L. major in a dose-andtime-dependent manner. However, the combinations showedmodest efficacy in healing L. major CL lesions of BALB/cmice. Forty patients with L. tropicaCL were treated for 4 weekswith KNO2 in aqueous cream combined with ASC or SAL.Only 11 (28%) patients showed improvement and only5 were cured at 2 months. Moreover, the cream provokedskin irritation due to the acid pH of the formulation [92].

The utility of NO donors is limited by their payloads, toorapid release and lack of targeted NO delivery [84]. NO releasesystems provide an excellent platform to deliver precise NOdoses over an extended period of time directly to specific tis-sues. In topical application, the sustained release is the majoradvantage of NPs over other NO donors or NO-generatingcreams that produce large or low amounts of NO with a rapidreturn to baseline. A wide range of organic and inorganic(silica) NPs have been developed as carriers of NO donors [84],but only one type of NO-release system, more specifically apatch, has been assayed in the topical treatment of CL. Inpatients infected with L. panamensis, the cure rates after3 months were 94.8% for the control group that receivedGlucantime compared with 37.1% in the group treated withthe patch [93].

More studies about NO doses and release rates from deliverysystems that are required to eliminate the parasite are necessaryto fully exploit the therapeutic potential of NO-release NPs inthe topical treatment of CL. Moreover, it could be interestingto combine NO administration with other drugs implicated inthe redox process of the parasite, such as T(SH)2 pathways.The combination could amplify the leishmanicidal activity ofboth NO and drug at lower concentrations.

5. Silver nanoparticles: new dress withold fabric

For centuries, silver compounds have been extensively usedfor healing purposes due to their strong bactericidal effectsas well as a broad spectrum of antimicrobial activity [94]. Silverfell into disuse because of the advent of antibiotics. Today, itsuse has re-emerged as a clinical treatment or prevention ofinfections in wounds, ulcers and burns [95]. The differentsilver compounds that are used as antimicrobial include topi-cal creams, ointments and solutions with silver nitrate, silversulfadiazine or silver oxide.

The antimicrobial activity of silver resides in its ionisationand high reactivity of Ag+. Ag+ adheres to the negativelycharged cell wall, changing cell wall permeability. This action,coupled with protein denaturation and generation of reactiveoxygen species (ROS), induces mitochondrial damage, cell



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lysis and, finally, cell death. The antibacterial activity of Ag+ isalso related to its ability to modify the DNA replicationmechanisms as well as to cause abnormalities in size, cyto-plasmic contents and outer cell layers of sensitive cells [95].Topically applied, silver compounds can also be toxic to fibro-blasts and epithelial cells by a similar mechanism of action,although higher concentrations are required. Moreover, silverions can permeate through the skin and potentially lead tointernal organ injury [96].More recently, metallic silver in the form of silver NPs

(AgNPs) has emerged as a safer and more effective form ofadministering silver compounds [97-99]. However, there is stillsome controversy about its mechanism of action [94]. Somestudies associate the action of AgNPs with its gradual releaseof silver ions from their surface [100,101]. They showed betteractivity than non-particulate silver because they can releaseAg+ ions at a greater rate than bulk silver, due to their largesurface area. Moreover, after internalisation, the NPs them-selves and the Ag ion-reactive released induce generation ofROS, mitochondrial damage and, ultimately, the death ofthe organism [101]. The stimulatory effect of AgNPs on macro-phages can also contribute to their antimicrobial activity.Because of their particulate dimensions, after being phagocy-tosed, macrophages released NO, ROS and inflammatorycytokines such as TNF-a, macrophage inflammatory proteinsand G-CSF [102], which help to eliminate the microorgan-ism [103]. Although they are less toxic than other forms ofsilver administration [98], there are still some concerns aboutthe toxicity of AgNPs as was recently demonstrated in asubchronic study in guinea pigs [104].Any effect produced by AgNPs in the cells, such as toxicity,

inflammation, stimulation or antimicrobial efficacy, weregreatly influenced by the particle size. The smaller the par-ticles are, the higher the surface exposed to media able to gen-erate Ag+ is [100]. On the other hand, smaller particles entercells more easily than larger ones [105,106].As far as Leishmania is concerned, AgNPs showed activity

against Leishmania promastigotes and infected macrophagesamastigotes [107,108]. However, neither the intralesional nortopical administrations of AgNPs were able to control theevolution of lesions produced by L. major in BALB/c mice,although the secondary infections were eradicated [109].A low penetration of AgNPs through skin could explain theirinefficacy against Leishmania in vivo. In fact, it has been pre-viously reported that the penetration profile of metallic NPsthrough skin is greatly affected by the particle size and skinstate. At most, small AgNPs (around 10 nm) could bedetected in the deepest layers of the SC and the uppermoststrata of the viable epidermis of abraded human skin sam-ples [110]. In human samples of burned skin treated withAgNPs dressing, AgNPs were found in the upper part of der-mis of unhealed and healed lesions [111]. Because of the local-isation of infected macrophages in CL, we can assume thatonly silver ions released from AgNPs from the skin surfacecould diffuse through the skin deep enough to eliminate the

parasites. AgNPs were also administered intralesionally with-out success in spite of the injection avoiding the skin barrier.The low amount of AgNPs (3 -- 4 µg Ag per each injection)given could explain the poor efficacy in this case. Thein vitro studies reported a 10 µg/ml dose required to observea 50% reduction in the infection index. Therefore, althoughAgNPs have showed antileishmanial activity per se, their useas carriers or their combination with other drugs would berecommended to decrease the required dosage, reduce the tox-icity of both agents and also enhance the leishmanicidal prop-erties [112]. Synergistic effect of AgNPs with antibiotics such asamoxicillin, ampicillin, gentamicin, vancomycin or kanamy-cin against several bacteria (Staphylococcus aureus, Escherichiacoli and Pseudomonas aeruginosa) has been previouslyreported [113]. Because of that, the combination of AgNPswith effective compounds against Leishmania could be a novelapproach in the treatment of CL.

6. PDT: selective parasite destructionwith NPs

PDT is a therapeutic modality, whereby diseased cells and tis-sues are destroyed by a combination of special drugs, calledphotosensititisers (PSs), and light of the correct wavelengthwhen it is absorbed by the PS, in the presence of oxygen.The excited PS transfers energy to molecular oxygen andproduces ROS such as single oxygen and hydroxyl radical,killing the cells and tissues where the PS is localised prior tophotoactivation.

To date, the clinical application of PDT has been mainlylimited to areas of the body easily accessible to irradiationfrom laser or incoherent light sources. Consequently, PDThas been widely investigated as a treatment for topical dis-eases [114], such as cancer, infections [115] and other patholo-gies of the skin, bladder, mouth and female reproductivetract, for example psoriasis or acne.

Due to the clinical efficacy of PDT in localised cutaneousdiseases and its antimicrobial activity, PDT was tested to treatCL [116]. According to the Cochrane database [11,12], a meta-analysis of 49 trials involving different strategies of interven-tion confirmed the efficacy of a weekly session of PDT with5-ALA or its derivative methyl-ALA (MAL) for 4 weeksagainst L. major infections. In a RCT from Iran that com-pared PDT with topical treatment of PM daily for 28 days,cures rates were significantly higher in the PDT group andthe prevention of scarring was similar between them. Thesestudies also showed the absence of recurrences and the goodcosmetic results of PDT. A recent study has corroboratedthe efficacy of another inexpensive PDT based on methyleneblue (MB) as a PS and a non-coherent light source to treatCL caused by L. amazonensis [117].

Among the PSs evaluated in CL, 5-ALA, its derivativeMAL [118,119] and MB [120] are the most common PSs usedin the clinical practice against several skin diseases. In deep,ALA is a metabolic precursor in the biosynthesis of the natural

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PS protoporphyrin IX and MB is a phenothiazine derivative.Both ALA and MB have been administered by oral or topicalroute with a good security profile. However, 5-ALA and MBlack some properties of ideal PSs. They have low capacity togenerate ROS. Ideal PSs should have long-lived excited tripletstate and increased capacity of ROS generation. Another cur-rent limitation of PSs is their poor permeability through theskin. Despite this, 5-ALA and MB are small and hydrophilicmolecules (167.8 and 319.85 Da, respectively) and, althoughtheir hydrophilicity can compromise their penetrationthrough intact skin, their low octanol:water partition coeffi-cient could favour their penetration in situations of disorderedSC and disrupted epithelial barrier, which occurs in CLlesions. In fact, it has been demonstrated that MB and5-ALA were able to achieve depths of respectively 400 [121]

and 150 µm [119], down to several mm in tissues without SC.New generation of PSs, such as chlorines or phthalocya-

nines derivatives are more effective in the generation of singletoxygen after light exposure, compared with traditional pheno-thiazine derivatives and are activated at longer wavelengths.The ideal PS should be photo-activated mainly in the redand far-red. PSs absorbing light at longer wavelengths maybe photo-activated in deeper tissue layers and promote amore effective photo-activation in these regions [122]. This isespecially important in CL because parasites reside deeply inthe dermis. However, they have high MWs. Either they arehighly hydrophobic molecules, tending to form inactiveaggregated complexes, or they are highly hydrophilic mole-cules. Anyway, new generation of PSs have more problemsto move across the skin and to interact with biologicalmembranes.

NPs have been extensively investigated for improving theperformance of PDT [123] in skin cancer treatment. One ofthe advantages of delivering PSs by means of NPs is, as previ-ously described for chemotherapeutic drugs, the enhancementof their penetration through the skin. Additionally, NPs canavoid the tendency to aggregation and subsequent inactiva-tion of many PSs. In fact, better 5-ALA penetration andskin production and accumulation of protoporphyrin IX hasbeen demonstrated with several types of NPs [124].

Another handicap of current PDT against CL is the indi-rect destruction of the parasite, mediated either by immuneactivation of macrophages or killing them [125,126]. It is clearlyestablished that parasites cannot transform the precursor5-ALA to protoporphyrin IX. Accordingly, in vitro studiesshowed that 5-ALA-based PDT induced no phototoxicresponse in L. major. However, a systemic immune response,with strongly increased levels of monocyte chemoattractantprotein-1 and IL-6, was found. These results support theidea that the mechanism of action of 5-ALA could bemediated by immune responses.

Thus, the most significant advance in the PDT of CLshould be to achieve the direct action of PSs against the para-site instead of through the host cell death. It would benefit thehealing process of ulcer and scarring [127]. Several PSs with

confirmed in vitro activity against Leishmania spp. promasti-gotes and amastigotes have been previously reported [124,128],as well as the improvement in the targeting by means ofNPs. In an in vitro study conducted by Montanari et al. [129]using ultradeformable liposomes (UDL) to treat CL due toL. braziliensis, a clear difference was observed between thePS (zinc phthalocyanine, ZnPc) and the PS loaded in UDL(UDL-ZnPc). The activity increased from 20% after lightirradiation for the PS alone until 100% anti-promastigoteand 80% anti-amastigote activity at the same light doseapplied in NPs. Moreover, penetration studies confirmedthat UDL-ZnPc reached deeper skin layers carrying seven-folds higher amount of ZnPc. In vivo studies using NPs inPDT of CL are still lacking [129,130].

Among different vehicles, metallic NPs are particularlyappealing in PDT. By the effect of surface-localised plasmonresonance, they increased the PS capacity to produceROS [131] and could enhance the PDT efficacy.

7. NPs as immunomodulators

The use of immunomodulators for the treatment of leishman-iasis, especially in combination with drugs, is well justified bythe crucial influence of the immunological status of the hostin the clinical pattern of the disease and due to the efficacyof the chemotherapy [132,133]. High failure of the parenteraltreatment with antimonials in immunocompromised patientshas been described. In fact, most antileishmanial drugs haveimmunomodulatory effects that contribute to their effectagainst the parasite [134,135]. Studies have demonstrated thatantimonials increase phagocytosis, production of proinflam-matory cytokines (IL-6, IL-1b and TNF-a) and productionof oxidants (ROS and NO) in Leishmania-infected phago-cytes, human monocytes and neutrophils; the underlyingmechanism may be related to ERK1/2 and p38 MAP kinasephosphorylation. MIL stimulates T cells and macrophages,increases secretion of proinflammatory cytokines, includingIFN-g , and enhances the production of oxidants. An increasein IFN-g has been observed in vivo after MIL therapy. Immu-nomodulation, as a treatment option, could also counteractthe mechanisms of immune evasion and immunosuppressiondeveloped by Leishmania parasites, such as abrogation of theoxidative burst, suppression of MHC II-mediated antigenpresentation and inhibition of Th1 immune response.

Successful immunotherapy of leishmaniasis has beenachieved through activating antigen-presenting cells withToll-like receptors (TLR) agonists, as IMQ.

IMQ, a TLR7 agonist, originally approved for the topicaltreatment of external anogenital warts, has been evaluatedwith success in combination with pentavalent antimonialsfor the treatment of NWCL (L. braziliensis) awaiting furtherstudies for robust clinical evidence of the synergistic combina-tion [136]. The benefit of IMQ was less clear in OWCL infec-tions (L. tropica), although the compound was active in vitroand in vivo against L. major-infected mice [137,138]. Differences



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in the permeability of skin to IMQ, higher in ulcers ofpatients infected with L. braziliensis than through nodularlesions of L. tropica could explain the differences of efficacyamong species [14].The use of immunotherapy or chemoimmunotherapy is

expected to become a more important therapeutic optionwith the increase of the prevalence of leishmaniasis due toresistant species and the increasing number of immunocom-promised patients. However, this modality of treatmentpresents the risk of generating an overdrive proinflammatoryscenario that can not only reduce the infection but also aggra-vate tissue destruction and scar formation [139]. Ulceration andtissue destruction in CL seems to be mainly a consequence ofimmune activation evoked by the infection rather than adirect effect of the parasitic infectious burden, although defi-nite proof of this hypothesis has not been shown to date [23].Therefore, ulcerative lesions of Leishmania parasites are char-acterised by low infection burden and strong T-cell-mediatedresponse, whereas nodular disease shows abundant parasiticinfiltration and antigen-specific T-cell unresponsiveness [23].Thus, immunotherapy should be able to modulate thedelicate balance between protection and overinflammationand the immune response to Leishmania, so that the antileish-manial effects are maximised but tissue destruction mini-mised. It should be possible to target more restricted T-cellpopulations, macrophages and cells producing specific cyto-kines [140]. IMQ and other TLR agonists affect immune cellsso they should be carefully used because of risk of tissuedestruction exacerbation. In this context, tissue injury pro-duced by the TNF-a is well-known and oral pentoxyfylline,a TNF-a inhibitor, has been successfully used as adjuvant to

intramuscular meglumine antimoniate in the treatment ofOWCL and NWCL [141]. More recently, the role of Fasligand and TNF-related apoptosis-inducing ligand expressionin ulcer formation and inhibition leading to ulceration reduc-tion without increasing infective loads has been described[142,143].

Besides IMQ, cytokines like GM-CSF have been alsoassayed by topical administration. In patients infected withL. braziliensis, the combination of topical GM-CSF withGlutamine� supposed a market improvement as comparedwith the administration of glucantime alone (100 vs 57%cure) [144].

Several pharmaceutical excipients could also be useful inthe context of immunomodulation, cooperating with drugsfor parasite elimination or facilitating wound healing. Chito-san is well-known for its regenerating properties and theincorporation of this polymer in wound dressings is com-mon [145]. NPs and, especially, cationic ones activate severalintracellular signalling pathways [146,147] such as NF-kb orinflammasome. NF-kb activation is crucial for the immunedefence against Leishmania parasites [148]. Inflammasome-mediated particles activation seems to play a protective rolein the protection against Leishmania [149] as well as againsttissue injury.

8. Conclusion

Local treatment is a therapeutic option with applicationrestricted to the least severe forms of CL without risk of dis-semination. WHO recommends the use of PM ointments,thermotherapy, cryotherapy or intralesional antimonials for

Sustained release:prolong exposition of parasite to drug(limited by removal from the skin surface)

Immunomodulatoryproperties

(NF-κβ/inflammasome activation)(i.e., cationic NP)

Permeation enhancers:increase amount of drug in dermis(i.e., lipid nanocarriers, ethosomes,

transferosomes)

Wound-repaireffect

(i.e., chitosan NP)

Multifunctional

NP

Figure 2. Attributes of NPs of benefit in CL topical therapy. As sustained release systems and permeation enhancers, NPs can

increase the amount of the loaded drug arriving at the dermis and modulate the permeation rate. Besides targeting more

amount of drug near the parasite, certain NPs themselves have immunomodulatory properties or wound healing capabilities

that could maximise the efficacy of the topical therapy.CL: Cutaneous leishmaniasis; NPs: Nanoparticles.

E. Moreno et al.


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the treatment of patients with OWCL that do not fulfil thecriteria for which only local wound care is considered enough.The experience with local therapy for NWCL is limited and,because self-healing is rare and the evolution of the diseaseis potentially severe, systemic treatment is usually morepreferred.

The efficacy of a topical treatment against CL depends notonly on the intrinsic antileishmanial activity of the drug, butalso on the amount of drug available in the dermis (Figure 1).NPs as sustained release systems and permeation enhancers(Figure 2) could favour the creation of a drug reservoir inthe dermis. Their use seems to be mandatory for profitingthe antileishmanial activity of NO, a small molecule that oth-erwise diffuses freely through the skin. Additionally, certainNPs have immunomodulatory properties or wound-healingcapabilities to benefit in CL treatment (Figure 2). CationicNPs (e.g., chitosan NPs) have been demonstrated to activatethe NF-kb system, which is crucial against Leishmania. More-over, chitosan has been used for the preparation of occlusivedressings because it promotes the collagen synthesis and fibro-blast proliferation necessary for the healing of CL woundswithout scarring. These activities could enhance or even syn-ergise with the loaded antileishmanial compound, decreasingthe amount required to destroy the parasite and the develop-ment of resistances.

Pending task is the selective delivery of active compoundsto intracellular amastigotes because even small NPs are unableto penetrate deeply in the skin to encounter infected macro-phages (except in ulcerative lesions). It would be particularlyuseful for improving the performance of current PDT thatindirectly destroys the parasite by elimination of all thesurrounding tissue.

9. Expert opinion

The idea of healing CL lesions with a topical treatment hasalways been particularly appealing. However, there are impor-tant handicaps that could limit this therapeutic option. Theheterogeneity of the disease and also the subsequent variabilityof efficacy can lead physicians to ignore this alternative. More-over, its use, currently restricted to LCL as monotherapy,could be extended to any form of tegumentary leishmaniasisin combination with parenteral drugs, on increasing its bene-fits. Local therapies’ target should not only be able to elimi-nate the parasite and prevent the risk of dissemination, butalso be able to reduce the scar formation and disfigurement.As disadvantage, more requirements complicate the conductand analysis of CL clinical trials and the attainment ofevidence base for recommendations in the treatment.

Physical therapies such as cryotherapy or thermotherapyand the intralesional administration of antimonials have thecommon feature of overcoming the skin barrier that couldexplain their high general efficacy regardless of the type ofCL lesion. However, they are far to be satisfactory for thepatient because they are painful and destroy the skin tissue(not only the parasite), worsening the scarring.

In our opinion, future local therapy will be topical andaddressed to heal CL lesions without scarring, probably incombination with systemic drugs to mitigate partially theinherent variability of this type of therapy.

The treatment choice should be mainly dictated by rationaltherapeutic indications instead of being determined on eco-nomic considerations. In fact, economical issues are the majorproblem for the implementation of NPs as a powerful tool toenhance current scenario of leishmaniasis therapy. The hand-icap of the cost is still higher if the benefits of the treatmentare not clear, limited to improve the cosmetic appearance ofthe lesions or addressed only to accelerate the healing of theless severe forms of the disease.

As far as research is concerned, alterations in skin perme-ability and the high spectrum of CL lesions complicate theestablishment of criteria for choosing or discarding drug andformulation candidates. The design of algorithms to move adrug candidate through the pipeline to get new and betterCL therapies becomes a hard task. In this context, a criticalmilestone will be the determination of the parameter/s relatedto drug and formulation (such as MW, logP, J, Kp, retentionin dermis, interaction with skin compounds) that correlate themost with CL efficacy. Mathematical model-based predic-tions and in vivo methods for the assessment of topical drugavailability are challenging topics of intensive research thatcould provide valuable information for achieving thispurpose.

Acknowledgments

E Moreno and J Schwartz have contributed equally to thiswork.

Declaration of interest

We would like to thank the ADA Foundation (University ofNavarra) for the grant that was awarded to J Schwartz andthe Tropical Health Institute-Universidad de Navarra andFIMA (Fundacion para la Investigacion Medica Aplicada)for supporting the research group of “Pharmacotherapy ofleishmaniasis”. The authors state no conflict of interest andhave received no payment in preparation of this manuscript.



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AffiliationEsther Moreno1, Juana Schwartz1,2,

Celia Fernandez1, Carmen Sanmartın1,4,

Paul Nguewa1,3, Juan Manuel Irache2 &

Socorro Espuelas†1,2

†Author for correspondence1University of Navarra, Tropical Health Institute,

Irunlarrea, 1 E-31008 Pamplona, Spain

Tel: +34948425600;

Fax: +34948425619;

E-mail: [email protected] of Navarra, Pharmacy &

Pharmaceutical Technology Department,

Irunlarrea, 1 E-31008 Pamplona, Spain3University of Navarra, Microbiology &

Parasitology Department, Irunlarrea,

1 E-31008 Pamplona, Spain4University of Navarra, Organic &

Pharmaceutical Chemistry Department,

Irunlarrea, 1 E-31008 Pamplona, Spain



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mailto:[email protected]


Nanoparticles as multifunctional devices for the topical treatment of cutaneous leishmaniasis

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