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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Activity of Chitosan and Its Derivatives against <named-content content-type="genus-species">Leishmania major</named-content>
and <named-content content-type="genus-species">Leishmania mexicana</named-content>
<italic>In Vitro</italic>
</title>
<author><name sortKey="Riezk, Alaa" sort="Riezk, Alaa" uniqKey="Riezk A" first="Alaa" last="Riezk">Alaa Riezk</name>
<affiliation><nlm:aff id="aff1"><addr-line>Department of Infection Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Raynes, John G" sort="Raynes, John G" uniqKey="Raynes J" first="John G." last="Raynes">John G. Raynes</name>
<affiliation><nlm:aff id="aff1"><addr-line>Department of Infection Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Yardley, Vanessa" sort="Yardley, Vanessa" uniqKey="Yardley V" first="Vanessa" last="Yardley">Vanessa Yardley</name>
<affiliation><nlm:aff id="aff1"><addr-line>Department of Infection Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Murdan, Sudaxshina" sort="Murdan, Sudaxshina" uniqKey="Murdan S" first="Sudaxshina" last="Murdan">Sudaxshina Murdan</name>
<affiliation><nlm:aff id="aff2"><addr-line>Department of Pharmaceutics, UCL School of Pharmacy, University College London, London, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Croft, Simon L" sort="Croft, Simon L" uniqKey="Croft S" first="Simon L." last="Croft">Simon L. Croft</name>
<affiliation><nlm:aff id="aff1"><addr-line>Department of Infection Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">PMC</idno>
<idno type="pmid">31871082</idno>
<idno type="pmc">7038302</idno>
<idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038302</idno>
<idno type="RBID">PMC:7038302</idno>
<idno type="doi">10.1128/AAC.01772-19</idno>
<date when="2020">2020</date>
<idno type="wicri:Area/Pmc/Corpus">000408</idno>
<idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000408</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Activity of Chitosan and Its Derivatives against <named-content content-type="genus-species">Leishmania major</named-content>
and <named-content content-type="genus-species">Leishmania mexicana</named-content>
<italic>In Vitro</italic>
</title>
<author><name sortKey="Riezk, Alaa" sort="Riezk, Alaa" uniqKey="Riezk A" first="Alaa" last="Riezk">Alaa Riezk</name>
<affiliation><nlm:aff id="aff1"><addr-line>Department of Infection Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Raynes, John G" sort="Raynes, John G" uniqKey="Raynes J" first="John G." last="Raynes">John G. Raynes</name>
<affiliation><nlm:aff id="aff1"><addr-line>Department of Infection Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Yardley, Vanessa" sort="Yardley, Vanessa" uniqKey="Yardley V" first="Vanessa" last="Yardley">Vanessa Yardley</name>
<affiliation><nlm:aff id="aff1"><addr-line>Department of Infection Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Murdan, Sudaxshina" sort="Murdan, Sudaxshina" uniqKey="Murdan S" first="Sudaxshina" last="Murdan">Sudaxshina Murdan</name>
<affiliation><nlm:aff id="aff2"><addr-line>Department of Pharmaceutics, UCL School of Pharmacy, University College London, London, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Croft, Simon L" sort="Croft, Simon L" uniqKey="Croft S" first="Simon L." last="Croft">Simon L. Croft</name>
<affiliation><nlm:aff id="aff1"><addr-line>Department of Infection Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
</author>
</analytic>
<series><title level="j">Antimicrobial Agents and Chemotherapy</title>
<idno type="ISSN">0066-4804</idno>
<idno type="eISSN">1098-6596</idno>
<imprint><date when="2020">2020</date>
</imprint>
</series>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc><textClass></textClass>
</profileDesc>
</teiHeader>
<front><div type="abstract" xml:lang="en"><p>There is an urgent need for safe, efficacious, affordable, and field-adapted drugs for the treatment of cutaneous leishmaniasis, which newly affects around 1.5 million people worldwide annually. Chitosan, a biodegradable cationic polysaccharide, has previously been reported to have antimicrobial, antileishmanial, and immunostimulatory activities.</p>
</div>
</front>
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<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Antimicrob Agents Chemother</journal-id>
<journal-id journal-id-type="iso-abbrev">Antimicrob. Agents Chemother</journal-id>
<journal-id journal-id-type="hwp">aac</journal-id>
<journal-id journal-id-type="pmc">aac</journal-id>
<journal-id journal-id-type="publisher-id">AAC</journal-id>
<journal-title-group><journal-title>Antimicrobial Agents and Chemotherapy</journal-title>
</journal-title-group>
<issn pub-type="ppub">0066-4804</issn>
<issn pub-type="epub">1098-6596</issn>
<publisher><publisher-name>American Society for Microbiology</publisher-name>
<publisher-loc>1752 N St., N.W., Washington, DC</publisher-loc>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">31871082</article-id>
<article-id pub-id-type="pmc">7038302</article-id>
<article-id pub-id-type="publisher-id">01772-19</article-id>
<article-id pub-id-type="doi">10.1128/AAC.01772-19</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Experimental Therapeutics</subject>
</subj-group>
</article-categories>
<title-group><article-title>Activity of Chitosan and Its Derivatives against <named-content content-type="genus-species">Leishmania major</named-content>
and <named-content content-type="genus-species">Leishmania mexicana</named-content>
<italic>In Vitro</italic>
</article-title>
<alt-title alt-title-type="running-head">Antileishmanial Activity of Chitosan</alt-title>
<alt-title alt-title-type="short-authors">Riezk et al.</alt-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Riezk</surname>
<given-names>Alaa</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>a</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Raynes</surname>
<given-names>John G.</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>a</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Yardley</surname>
<given-names>Vanessa</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>a</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Murdan</surname>
<given-names>Sudaxshina</given-names>
</name>
<xref ref-type="aff" rid="aff2"><sup>b</sup>
</xref>
</contrib>
<contrib contrib-type="author" corresp="yes"><name><surname>Croft</surname>
<given-names>Simon L.</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>a</sup>
</xref>
</contrib>
<aff id="aff1"><label>a</label>
<addr-line>Department of Infection Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom</addr-line>
</aff>
<aff id="aff2"><label>b</label>
<addr-line>Department of Pharmaceutics, UCL School of Pharmacy, University College London, London, United Kingdom</addr-line>
</aff>
</contrib-group>
<author-notes><corresp id="cor1">Address correspondence to Simon L. Croft, <email>simon.croft@lshtm.ac.uk</email>
.</corresp>
<fn fn-type="other"><p><bold>Citation</bold>
Riezk A, Raynes JG, Yardley V, Murdan S, Croft SL. 2020. Activity of chitosan and its derivatives against <italic>Leishmania major</italic>
and <italic>Leishmania mexicana in vitro</italic>
. Antimicrob Agents Chemother 64:e01772-19. <ext-link ext-link-type="uri" xlink:href="https://doi.org/10.1128/AAC.01772-19">https://doi.org/10.1128/AAC.01772-19</ext-link>
.</p>
</fn>
</author-notes>
<pub-date pub-type="epreprint"><day>23</day>
<month>12</month>
<year>2019</year>
</pub-date>
<pub-date pub-type="epub"><day>21</day>
<month>2</month>
<year>2020</year>
</pub-date>
<pub-date pub-type="collection"><month>3</month>
<year>2020</year>
</pub-date>
<pub-date pub-type="pmc-release"><day>21</day>
<month>2</month>
<year>2020</year>
</pub-date>
<pmc-comment> PMC Release delay is 0 months and 0 days and was based on the . </pmc-comment>
<volume>64</volume>
<issue>3</issue>
<elocation-id>e01772-19</elocation-id>
<history><date date-type="received"><day>3</day>
<month>9</month>
<year>2019</year>
</date>
<date date-type="rev-request"><day>10</day>
<month>10</month>
<year>2019</year>
</date>
<date date-type="accepted"><day>8</day>
<month>12</month>
<year>2019</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2020 Riezk et al.</copyright-statement>
<copyright-year>2020</copyright-year>
<copyright-holder>Riezk et al.</copyright-holder>
<license license-type="open-access" xlink:href="https://creativecommons.org/licenses/by/4.0/"><license-p>This is an open-access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="https://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution 4.0 International license</ext-link>
.</license-p>
</license>
</permissions>
<self-uri content-type="pdf" xlink:href="AAC.01772-19.pdf"></self-uri>
<abstract abstract-type="precis"><p>There is an urgent need for safe, efficacious, affordable, and field-adapted drugs for the treatment of cutaneous leishmaniasis, which newly affects around 1.5 million people worldwide annually. Chitosan, a biodegradable cationic polysaccharide, has previously been reported to have antimicrobial, antileishmanial, and immunostimulatory activities.</p>
</abstract>
<abstract><title>ABSTRACT</title>
<p>There is an urgent need for safe, efficacious, affordable, and field-adapted drugs for the treatment of cutaneous leishmaniasis, which newly affects around 1.5 million people worldwide annually. Chitosan, a biodegradable cationic polysaccharide, has previously been reported to have antimicrobial, antileishmanial, and immunostimulatory activities. We investigated the <italic>in vitro</italic>
activity of chitosan and several of its derivatives and showed that the pH of the culture medium plays a critical role in antileishmanial activity of chitosan against both extracellular promastigotes and intracellular amastigotes of <named-content content-type="genus-species">Leishmania major</named-content>
and <named-content content-type="genus-species">Leishmania mexicana</named-content>
. Chitosan and its derivatives were approximately 7 to 20 times more active at pH 6.5 than at pH 7.5, with high-molecular-weight chitosan being the most potent. High-molecular-weight chitosan stimulated the production of nitric oxide and reactive oxygen species by uninfected and <italic>Leishmania</italic>
-infected macrophages in a time- and dose-dependent manner at pH 6.5. Despite the <italic>in vitro</italic>
activation of bone marrow macrophages by chitosan to produce nitric oxide and reactive oxygen species, we showed that the antileishmanial activity of chitosan was not mediated by these metabolites. Finally, we showed that rhodamine-labeled chitosan is taken up by pinocytosis and accumulates in the parasitophorous vacuole of <italic>Leishmania</italic>
-infected macrophages.</p>
</abstract>
<kwd-group><title>KEYWORDS</title>
<kwd>cutaneous leishmaniasis</kwd>
<kwd><italic>Leishmania major</italic>
</kwd>
<kwd><italic>Leishmania mexicana</italic>
</kwd>
<kwd>chitosan</kwd>
<kwd>macrophage uptake</kwd>
</kwd-group>
<funding-group><award-group id="award1"><funding-source><institution-wrap><institution>Council for At-Risk Academics (CARA) UK</institution>
</institution-wrap>
</funding-source>
<principal-award-recipient><name><surname>Riezk</surname>
<given-names>Alaa</given-names>
</name>
</principal-award-recipient>
</award-group>
<award-group id="award2"><funding-source><institution-wrap><institution>London School of Hygiene and Tropical Medicine (LSHTM)</institution>
<institution-id>https://doi.org/10.13039/100009660</institution-id>
</institution-wrap>
</funding-source>
<principal-award-recipient><name><surname>Riezk</surname>
<given-names>Alaa</given-names>
</name>
</principal-award-recipient>
</award-group>
</funding-group>
<counts><count count="1" count-type="supplementary-material"></count>
<fig-count count="8"></fig-count>
<table-count count="4"></table-count>
<equation-count count="0"></equation-count>
<ref-count count="74"></ref-count>
<page-count count="17"></page-count>
<word-count count="11797"></word-count>
</counts>
<custom-meta-group><custom-meta><meta-name>cover-date</meta-name>
<meta-value>March 2020</meta-value>
</custom-meta>
</custom-meta-group>
</article-meta>
</front>
<body><sec sec-type="intro" id="s1"><title>INTRODUCTION</title>
<p>Leishmaniasis is an infectious disease caused by protozoan parasites belonging to the genus <italic>Leishmania</italic>
. The parasite is transmitted between humans and mammalian reservoirs (e.g., dogs and rodents) through the bite of a female phlebotomine sand fly (<xref rid="B1" ref-type="bibr">1</xref>
). There are two main clinical forms, cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL), with CL being the most common (<xref rid="B2" ref-type="bibr">2</xref>
). In addition to “simple” CL, there are other complex cutaneous manifestations, including mucocutaneous leishmaniasis (MCL), diffuse cutaneous leishmaniasis (DCL), recidivans leishmaniasis (RL), and post-kala-azar dermal leishmaniasis (PKDL) (<xref rid="B3" ref-type="bibr">3</xref>
, <xref rid="B4" ref-type="bibr">4</xref>
).</p>
<p>CL is caused mainly by <named-content content-type="genus-species">Leishmania tropica</named-content>
, <named-content content-type="genus-species">Leishmania major</named-content>
, and <named-content content-type="genus-species">Leishmania aethiopica</named-content>
in the Old World and by <named-content content-type="genus-species">Leishmania braziliensis</named-content>
, <named-content content-type="genus-species">Leishmania guyanensis</named-content>
, <named-content content-type="genus-species">Leishmania mexicana</named-content>
, and <named-content content-type="genus-species">Leishmania amazonensis</named-content>
in the New World (<xref rid="B5" ref-type="bibr">5</xref>
). Of the 88 countries where CL occurs, 90% of the cases are in Afghanistan, Brazil, Iran, Peru, Saudi Arabia, and Syria (<xref rid="B1" ref-type="bibr">1</xref>
). In the mammalian host, the parasite survives and multiplies within macrophages. The cellular immune responses in CL play a critical role in the control and progress of the disease and include two main mechanisms of macrophage activation: (i) the classical pathway (M1 macrophages), in which Th1 and NK cells produce cytokines (such as gamma interferon [IFN-γ]) that stimulate the production of nitric oxide (NO) and reactive oxygen species (ROS) and the activation of other lysosomal antimicrobial activities that are responsible for killing the <italic>Leishmania</italic>
parasites, and (ii) the alternative pathway, mediated by Th2 cytokines like interleukin-4 (IL-4) and IL-13 in the early stages of infection, forming a favorable environment for <italic>Leishmania</italic>
proliferation (<xref rid="B6" ref-type="bibr">6</xref>
, <xref rid="B7" ref-type="bibr">7</xref>
).</p>
<p>The pentavalent antimonial compounds sodium stibogluconate (Pentostam), and meglumine antimoniate (Glucantime) have been the standard treatment for CL for the past 70 years (<xref rid="B8" ref-type="bibr">8</xref>
). These drugs have several limitations, including difficulty of administration, toxicity of the drug, and varying sensitivities among <italic>Leishmania</italic>
species (<xref rid="B9" ref-type="bibr">9</xref>
). Second-line treatments include the polyene antifungal amphotericin B, which also suffers from toxicity, the oral phospholipid miltefosine, the use of which is limited by teratogenicity, and the aminoglycoside antibiotic paromomycin (PM), which has low cure rates for certain <italic>Leishmania</italic>
species (<xref rid="B10" ref-type="bibr">10</xref>
<xref ref-type="bibr" rid="B11">–</xref>
<xref rid="B12" ref-type="bibr">12</xref>
). Treatment with intravenous liposomal amphotericin B (AmBisome) is safe and has achieved clinical success against CL at a dose of 3 mg/kg of body weight daily for 7 days (<xref rid="B13" ref-type="bibr">13</xref>
, <xref rid="B14" ref-type="bibr">14</xref>
), but the high cost of this formulation limits its use (<xref rid="B15" ref-type="bibr">15</xref>
). Two Cochrane analyses have clearly shown clinical deficiencies of most drugs (<xref rid="B16" ref-type="bibr">16</xref>
, <xref rid="B17" ref-type="bibr">17</xref>
). There is an urgent need for new treatments which can eliminate the parasites and improve the healing process and are safe, reliable, and also field adaptable for use in diverse health care systems.</p>
<p>Chitosan is a biodegradable, biocompatible, positively charged nontoxic mucoadhesive biopolymer produced by the deacetylation of chitin. Chitosan has a pK<sub>a</sub>
of approximately 6.3 and is insoluble at alkaline pH but soluble in weak acidic solvents like acetic acid, where the amino groups become protonated. Many reports have described the antimicrobial activity of chitosan, but the actual mechanism of action has not been fully elucidated (<xref rid="B18" ref-type="bibr">18</xref>
), although three direct mechanisms have been suggested. The first is the interaction between the protonated NH<sub>3</sub>
<sup>+</sup>
groups of chitosan and the negatively charged cell membrane of microbes. This interaction changes the permeability of the microbial cell membrane, causing osmotic imbalances and consequently killing the microbe (<xref rid="B18" ref-type="bibr">18</xref>
, <xref rid="B19" ref-type="bibr">19</xref>
). The second suggested mechanism is that chitosan binds to microbial DNA and inhibits DNA transcription, assuming that chitosan penetrates the microbial cell membrane and reaches the DNA (<xref rid="B19" ref-type="bibr">19</xref>
, <xref rid="B20" ref-type="bibr">20</xref>
). The third mechanism is via chitosan’s chelation of metals and binding of basic nutrients essential for microbial growth (<xref rid="B19" ref-type="bibr">19</xref>
). An indirect mechanism of action may be related to the known proinflammatory effect of chitosan on macrophages. This involves stimulation of tumor necrosis factor alpha (TNF-α), IL-6, NO, ROS, and IFN-γ, which play critical roles in the proinflammatory response against intracellular microbes by enhancing the production of microbicidal reactive nitrogen species (<xref rid="B21" ref-type="bibr">21</xref>
<xref ref-type="bibr" rid="B22">–</xref>
<xref rid="B25" ref-type="bibr">25</xref>
). Chitosan activates polymorphonuclear leukocytes, macrophages, and fibroblasts, and these properties promote wound healing (<xref rid="B18" ref-type="bibr">18</xref>
, <xref rid="B26" ref-type="bibr">26</xref>
).</p>
<p>The poor solubility of chitosan and the loss of the cationic charge in neutral and alkaline environments are two of the major obstacles to the consideration of chitosan as a useful antimicrobial. Recently, the chemical modification of chitosan to produce various derivatives to improve its solubility and widen its application has gained attention (<xref rid="B27" ref-type="bibr">27</xref>
, <xref rid="B28" ref-type="bibr">28</xref>
). Chitosan and its derivatives have been shown to have <italic>in vitro</italic>
antileishmanial activity with 50% effective concentrations (EC<sub>50</sub>
s) ranging from 70 to 240 μg/ml against <named-content content-type="genus-species">L. infantum</named-content>
, <named-content content-type="genus-species">L. amazonensis</named-content>
, and <named-content content-type="genus-species">Leishmania chagasi</named-content>
promastigotes and amastigotes (<xref rid="B29" ref-type="bibr">29</xref>
<xref ref-type="bibr" rid="B30">–</xref>
<xref rid="B34" ref-type="bibr">34</xref>
). All this makes chitosan an appropriate candidate for further studies to evaluate its suitability for the treatment of CL.</p>
<p>The aim of our work was to (i) determine the <italic>in vitro</italic>
antileishmanial activity of chitosan and its derivatives against <named-content content-type="genus-species">L. major</named-content>
and <named-content content-type="genus-species">L. mexicana</named-content>
promastigotes and intracellular amastigotes at two different pHs (the culture medium pH of 7.5 and a lower pH of 6.5, which are both suitable for macrophage and parasite growth) (<xref rid="B35" ref-type="bibr">35</xref>
<xref ref-type="bibr" rid="B36">–</xref>
<xref rid="B37" ref-type="bibr">37</xref>
), (ii) evaluate the <italic>in vitro</italic>
role of chitosan in the activation of the macrophage M1 proinflammatory phenotype via the measurement of NO, ROS, and TNF-α production by host cells and by measuring parasite survival, and (iii) investigate chitosan uptake by macrophages to explain its activity against intracellular amastigotes.</p>
</sec>
<sec sec-type="results" id="s2"><title>RESULTS</title>
<sec id="s2.1"><title><italic>In vitro</italic>
activities of chitosan and derivatives against <named-content content-type="genus-species">L. major</named-content>
and <named-content content-type="genus-species">L. mexicana</named-content>
.</title>
<p>The antileishmanial activities (against promastigotes and amastigotes) of high-, medium-, and low-molecular-weight (HMW, MMW, and LMW, respectively) chitosan and its derivatives (a total of 11 compounds) were tested. Dose-dependent activity (Fig. S1 and S2 in the supplemental material) against <italic>Leishmania</italic>
promastigotes and amastigotes was observed for chitosan and its derivatives, except for carboxymethyl chitosan, which showed no activity against either parasite stage within the experimental parameters tested (pH 7.5 or 6.5 and concentrations up to 400 μg/ml). In the 72-h assays, chitosan and its derivatives (except for carboxymethyl chitosan) were 7 to 20 times more active against <named-content content-type="genus-species">L. major</named-content>
and <named-content content-type="genus-species">L. mexicana</named-content>
promastigotes and intracellular amastigotes (infecting peritoneal mouse macrophages [PEMs]) in culture medium at pH 6.5 than at pH 7.5 (<italic>P</italic>
< 0.05 by <italic>t</italic>
test) (<xref rid="T1" ref-type="table">Tables 1</xref>
and <xref rid="T2" ref-type="table">2</xref>
). HMW, MMW, and LMW chitosan, from both crustacean and fungal sources, exhibited significantly higher activities than chitosan derivatives against promastigotes and intracellular amastigotes (EC<sub>50</sub>
s of ≈6 μg/ml against <named-content content-type="genus-species">L. major</named-content>
promastigotes and 10 μg/ml against <named-content content-type="genus-species">L. mexicana</named-content>
promastigotes, and EC<sub>50</sub>
s of ≈12 μg/ml against <named-content content-type="genus-species">L. major</named-content>
amastigotes and 16 μg/ml against <named-content content-type="genus-species">L. mexicana</named-content>
amastigotes) at pH 6.5 (<italic>P</italic>
< 0.05 by an extra-sum-of-squares F test) (<xref rid="T1" ref-type="table">Tables 1</xref>
and <xref rid="T2" ref-type="table">2</xref>
). Additionally, <named-content content-type="genus-species">L. major</named-content>
promastigotes and amastigotes were significantly more sensitive to chitosan and its derivatives than <named-content content-type="genus-species">L. mexicana</named-content>
promastigotes and amastigotes (approximately 1.5 to 2 times more sensitive [<italic>P</italic>
< 0.05 by the extra-sum-of-squares F test]).</p>
<table-wrap id="T1" orientation="portrait" position="float"><label>TABLE 1</label>
<caption><p><italic>In vitro</italic>
activities of chitosan and its derivatives against promastigotes at two pHs</p>
</caption>
<alternatives><table frame="hsides" rules="groups"><colgroup span="1"><col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
</colgroup>
<thead><tr><th rowspan="4" colspan="1">Compound</th>
<th colspan="8" rowspan="1">Mean value ± SD (μg/ml) at<xref ref-type="table-fn" rid="T1F1"><sup><italic>a</italic>
</sup>
</xref>
:<hr></hr>
</th>
</tr>
<tr><th colspan="4" rowspan="1">pH 7.5<hr></hr>
</th>
<th colspan="4" rowspan="1">pH 6.5<xref ref-type="table-fn" rid="T1F2"><sup><italic>b</italic>
</sup>
</xref>
<hr></hr>
</th>
</tr>
<tr><th colspan="2" rowspan="1"><named-content content-type="genus-species">L. major</named-content>
<hr></hr>
</th>
<th colspan="2" rowspan="1"><named-content content-type="genus-species">L. mexicana</named-content>
<hr></hr>
</th>
<th colspan="2" rowspan="1"><named-content content-type="genus-species">L. major</named-content>
<hr></hr>
</th>
<th colspan="2" rowspan="1"><named-content content-type="genus-species">L. mexicana</named-content>
<hr></hr>
</th>
</tr>
<tr><th rowspan="1" colspan="1">EC<sub>50</sub>
</th>
<th rowspan="1" colspan="1">EC<sub>90</sub>
</th>
<th rowspan="1" colspan="1">EC<sub>50</sub>
</th>
<th rowspan="1" colspan="1">EC<sub>90</sub>
</th>
<th rowspan="1" colspan="1">EC<sub>50</sub>
</th>
<th rowspan="1" colspan="1">EC<sub>90</sub>
</th>
<th rowspan="1" colspan="1">EC<sub>50</sub>
</th>
<th rowspan="1" colspan="1">EC<sub>90</sub>
</th>
</tr>
</thead>
<tbody><tr><td rowspan="1" colspan="1">Amphotericin B</td>
<td rowspan="1" colspan="1">0.05 ± 0.01</td>
<td rowspan="1" colspan="1">0.2 ± 0.02</td>
<td rowspan="1" colspan="1">0.14 ± 0.01</td>
<td rowspan="1" colspan="1">0.3 ± 0.03</td>
<td rowspan="1" colspan="1">0.07 ± 0.02</td>
<td rowspan="1" colspan="1">0.3 ± 0.1</td>
<td rowspan="1" colspan="1">0.13 ± 0.07</td>
<td rowspan="1" colspan="1">0.3 ± 0.02</td>
</tr>
<tr><td rowspan="1" colspan="1">HMW chitosan</td>
<td rowspan="1" colspan="1">105 ± 12</td>
<td rowspan="1" colspan="1">1,549 ± 525</td>
<td rowspan="1" colspan="1">140 ± 12</td>
<td rowspan="1" colspan="1">2,187 ± 928</td>
<td rowspan="1" colspan="1">5.9 ± 0.5</td>
<td rowspan="1" colspan="1">37 ± 9</td>
<td rowspan="1" colspan="1">10.4 ± 1.6</td>
<td rowspan="1" colspan="1">98 ± 33</td>
</tr>
<tr><td rowspan="1" colspan="1">MMW chitosan</td>
<td rowspan="1" colspan="1">113 ± 9</td>
<td rowspan="1" colspan="1">1,277 ± 580</td>
<td rowspan="1" colspan="1">150 ± 12</td>
<td rowspan="1" colspan="1">2,223 ± 681</td>
<td rowspan="1" colspan="1">6.2 ± 0.3</td>
<td rowspan="1" colspan="1">43 ± 8</td>
<td rowspan="1" colspan="1">10.9 ± 1.4</td>
<td rowspan="1" colspan="1">96 ± 27</td>
</tr>
<tr><td rowspan="1" colspan="1">LMW chitosan</td>
<td rowspan="1" colspan="1">118 ± 11</td>
<td rowspan="1" colspan="1">1,238 ± 582</td>
<td rowspan="1" colspan="1">157 ± 13</td>
<td rowspan="1" colspan="1">2,225 ± 723</td>
<td rowspan="1" colspan="1">6.7 ± 0.3</td>
<td rowspan="1" colspan="1">40 ± 8</td>
<td rowspan="1" colspan="1">10.2 ± 1.5</td>
<td rowspan="1" colspan="1">84 ± 28</td>
</tr>
<tr><td rowspan="1" colspan="1">Fungal chitosan</td>
<td rowspan="1" colspan="1">118 ± 11</td>
<td rowspan="1" colspan="1">1,228 ± 560</td>
<td rowspan="1" colspan="1">150 ± 13</td>
<td rowspan="1" colspan="1">1,991 ± 580</td>
<td rowspan="1" colspan="1">6.2 ± 0.3</td>
<td rowspan="1" colspan="1">42 ± 6</td>
<td rowspan="1" colspan="1">10.5 ± 1.3</td>
<td rowspan="1" colspan="1">61 ± 17</td>
</tr>
<tr><td rowspan="1" colspan="1">Chitosan oligosaccharide</td>
<td rowspan="1" colspan="1">153 ± 15</td>
<td rowspan="1" colspan="1">1,680 ± 506</td>
<td rowspan="1" colspan="1">190 ± 20</td>
<td rowspan="1" colspan="1">2,366 ± 461</td>
<td rowspan="1" colspan="1">62.5 ± 4</td>
<td rowspan="1" colspan="1">446 ± 92</td>
<td rowspan="1" colspan="1">77 ± 2.7</td>
<td rowspan="1" colspan="1">452 ± 36</td>
</tr>
<tr><td rowspan="1" colspan="1">Chitosan oligosaccharide-lactate</td>
<td rowspan="1" colspan="1">98 ± 9</td>
<td rowspan="1" colspan="1">1,226 ± 130</td>
<td rowspan="1" colspan="1">125 ± 14</td>
<td rowspan="1" colspan="1">765 ± 83</td>
<td rowspan="1" colspan="1">14 ± 0.1</td>
<td rowspan="1" colspan="1">135 ± 2</td>
<td rowspan="1" colspan="1">23 ± 1.4</td>
<td rowspan="1" colspan="1">311 ± 25</td>
</tr>
<tr><td rowspan="1" colspan="1">Chitosan HCl</td>
<td rowspan="1" colspan="1">96 ± 7</td>
<td rowspan="1" colspan="1">1,189 ± 211</td>
<td rowspan="1" colspan="1">110 ± 24</td>
<td rowspan="1" colspan="1">746 ± 169</td>
<td rowspan="1" colspan="1">13.2 ± 1</td>
<td rowspan="1" colspan="1">118 ± 34</td>
<td rowspan="1" colspan="1">20.8 ± 2.4</td>
<td rowspan="1" colspan="1">264 ± 61</td>
</tr>
<tr><td rowspan="1" colspan="1">PC1-chitosan<xref ref-type="table-fn" rid="T1F3"><sup><italic>c</italic>
</sup>
</xref>
</td>
<td rowspan="1" colspan="1">111 ± 20</td>
<td rowspan="1" colspan="1">1,875 ± 230</td>
<td rowspan="1" colspan="1">176 ± 14</td>
<td rowspan="1" colspan="1">2,832 ± 412</td>
<td rowspan="1" colspan="1">19.9 ± 2.8</td>
<td rowspan="1" colspan="1">187 ± 90</td>
<td rowspan="1" colspan="1">32 ± 2.2</td>
<td rowspan="1" colspan="1">328 ± 48</td>
</tr>
<tr><td rowspan="1" colspan="1">PC2-chitosan<xref ref-type="table-fn" rid="T1F3"><sup><italic>c</italic>
</sup>
</xref>
</td>
<td rowspan="1" colspan="1">104 ± 6</td>
<td rowspan="1" colspan="1">1,485 ± 259</td>
<td rowspan="1" colspan="1">170 ± 8</td>
<td rowspan="1" colspan="1">2,744 ± 377</td>
<td rowspan="1" colspan="1">16.5 ± 2.7</td>
<td rowspan="1" colspan="1">138 ± 49</td>
<td rowspan="1" colspan="1">28 ± 2.4</td>
<td rowspan="1" colspan="1">296 ± 53</td>
</tr>
<tr><td rowspan="1" colspan="1">PC3-chitosan<xref ref-type="table-fn" rid="T1F3"><sup><italic>c</italic>
</sup>
</xref>
</td>
<td rowspan="1" colspan="1">119 ± 19</td>
<td rowspan="1" colspan="1">1,860 ± 365</td>
<td rowspan="1" colspan="1">187 ± 16</td>
<td rowspan="1" colspan="1">3,175 ± 580</td>
<td rowspan="1" colspan="1">23.3 ± 2.5</td>
<td rowspan="1" colspan="1">218 ± 44</td>
<td rowspan="1" colspan="1">37 ± 2.5</td>
<td rowspan="1" colspan="1">442 ± 65</td>
</tr>
<tr><td rowspan="1" colspan="1">Carboxymethyl chitosan</td>
<td rowspan="1" colspan="1">—<xref ref-type="table-fn" rid="T1F4"><sup><italic>d</italic>
</sup>
</xref>
</td>
<td rowspan="1" colspan="1">—</td>
<td rowspan="1" colspan="1">—</td>
<td rowspan="1" colspan="1">—</td>
<td rowspan="1" colspan="1">—</td>
<td rowspan="1" colspan="1">—</td>
<td rowspan="1" colspan="1">—</td>
<td rowspan="1" colspan="1">—</td>
</tr>
</tbody>
</table>
<graphic xlink:href="AAC.01772-19-t0001"></graphic>
</alternatives>
<table-wrap-foot><fn fn-type="other" id="T1F1"><label>a</label>
<p>Experiments were conducted in triplicate cultures. Experiments were reproduced a further two times with confirmed similar data (not shown). Amphotericin B deoxycholate was used as a positive control. Both RPMI alone at pH 6.5 and chitosan solvent did not show any activity against promastigotes. Statistically significant differences were found for the EC<sub>50</sub>
s of chitosan and its derivatives at pH 6.5 and pH 7.5 (<italic>P</italic>
< 0.05 by using <italic>t</italic>
test).</p>
</fn>
<fn fn-type="other" id="T1F2"><label>b</label>
<p><named-content content-type="genus-species">L. major</named-content>
promastigotes were significantly more susceptible to chitosan and derivatives than <named-content content-type="genus-species">L. mexicana</named-content>
at pH 6.5 (<italic>P</italic>
< 0.05 by the extra-sum-of-squares F test).</p>
</fn>
<fn fn-type="other" id="T1F3"><label>c</label>
<p>Phosphorylcholine-substituted chitosan (see <xref rid="T4" ref-type="table">Table 4</xref>
).</p>
</fn>
<fn fn-type="other" id="T1F4"><label>d</label>
<p>—, no activity up to 400 μg/ml.</p>
</fn>
</table-wrap-foot>
</table-wrap>
<table-wrap id="T2" orientation="portrait" position="float"><label>TABLE 2</label>
<caption><p><italic>In vitro</italic>
activities of chitosan and its derivatives against amastigotes infecting PEMs, as well as cytotoxicities toward KB cells</p>
</caption>
<alternatives><table frame="hsides" rules="groups"><colgroup span="1"><col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
</colgroup>
<thead><tr><th rowspan="5" colspan="1">Compound</th>
<th colspan="10" rowspan="1">Mean value ± SD (μg/ml)<xref ref-type="table-fn" rid="T2F1"><sup><italic>a</italic>
</sup>
</xref>
<hr></hr>
</th>
</tr>
<tr><th colspan="8" rowspan="1">Activity against amastigotes at<xref ref-type="table-fn" rid="T2F2"><sup><italic>b</italic>
</sup>
</xref>
:<hr></hr>
</th>
<th colspan="2" rowspan="3">Cytotoxicity toward KB cells at pH 6.5<xref ref-type="table-fn" rid="T2F3"><sup><italic>c</italic>
</sup>
</xref>
<hr></hr>
</th>
</tr>
<tr><th colspan="4" rowspan="1">pH 7.5<hr></hr>
</th>
<th colspan="4" rowspan="1">pH 6.5<hr></hr>
</th>
</tr>
<tr><th colspan="2" rowspan="1"><named-content content-type="genus-species">L. major</named-content>
<hr></hr>
</th>
<th colspan="2" rowspan="1"><named-content content-type="genus-species">L. mexicana</named-content>
<hr></hr>
</th>
<th colspan="2" rowspan="1"><named-content content-type="genus-species">L. major</named-content>
<hr></hr>
</th>
<th colspan="2" rowspan="1"><named-content content-type="genus-species">L. mexicana</named-content>
<hr></hr>
</th>
</tr>
<tr><th rowspan="1" colspan="1">EC<sub>50</sub>
</th>
<th rowspan="1" colspan="1">EC<sub>90</sub>
</th>
<th rowspan="1" colspan="1">EC<sub>50</sub>
</th>
<th rowspan="1" colspan="1">EC<sub>90</sub>
</th>
<th rowspan="1" colspan="1">EC<sub>50</sub>
</th>
<th rowspan="1" colspan="1">EC<sub>90</sub>
</th>
<th rowspan="1" colspan="1">EC<sub>50</sub>
</th>
<th rowspan="1" colspan="1">EC<sub>90</sub>
</th>
<th rowspan="1" colspan="1">LD<sub>50</sub>
</th>
<th rowspan="1" colspan="1">LD<sub>90</sub>
</th>
</tr>
</thead>
<tbody><tr><td rowspan="1" colspan="1">Amphotericin B</td>
<td rowspan="1" colspan="1">0.07 ± 0.01</td>
<td rowspan="1" colspan="1">0.13 ± 0.05</td>
<td rowspan="1" colspan="1">0.19 ± 0.05</td>
<td rowspan="1" colspan="1">1.5 ± 0.2</td>
<td rowspan="1" colspan="1">0.06 ± 0.01</td>
<td rowspan="1" colspan="1">0.11 ± 0.06</td>
<td rowspan="1" colspan="1">0.18 ± 0.06</td>
<td rowspan="1" colspan="1">1.7 ± 0.3</td>
<td rowspan="1" colspan="1">58 ± 8</td>
<td rowspan="1" colspan="1">190 ± 9</td>
</tr>
<tr><td rowspan="1" colspan="1">HMW chitosan</td>
<td rowspan="1" colspan="1">98 ± 6</td>
<td rowspan="1" colspan="1">1,635 ± 245</td>
<td rowspan="1" colspan="1">119 ± 9</td>
<td rowspan="1" colspan="1">1,804 ± 304</td>
<td rowspan="1" colspan="1">11.4 ± 1</td>
<td rowspan="1" colspan="1">69 ± 18</td>
<td rowspan="1" colspan="1">15.4 ± 2</td>
<td rowspan="1" colspan="1">103 ± 28</td>
<td rowspan="1" colspan="1">752 ± 90</td>
<td rowspan="1" colspan="1">3,022 ± 366</td>
</tr>
<tr><td rowspan="1" colspan="1">MMW chitosan</td>
<td rowspan="1" colspan="1">103 ± 8</td>
<td rowspan="1" colspan="1">1,652 ± 287</td>
<td rowspan="1" colspan="1">125 ± 10</td>
<td rowspan="1" colspan="1">1,793 ± 323</td>
<td rowspan="1" colspan="1">12.9 ± 1</td>
<td rowspan="1" colspan="1">81 ± 18</td>
<td rowspan="1" colspan="1">16.3 ± 2</td>
<td rowspan="1" colspan="1">122 ± 34</td>
<td rowspan="1" colspan="1">758 ± 89</td>
<td rowspan="1" colspan="1">3,019 ± 400</td>
</tr>
<tr><td rowspan="1" colspan="1">LMW chitosan</td>
<td rowspan="1" colspan="1">102 ± 7</td>
<td rowspan="1" colspan="1">1,651 ± 282</td>
<td rowspan="1" colspan="1">125 ± 10</td>
<td rowspan="1" colspan="1">1,795 ± 320</td>
<td rowspan="1" colspan="1">12.1 ± 1</td>
<td rowspan="1" colspan="1">74 ± 14</td>
<td rowspan="1" colspan="1">16.1 ± 2</td>
<td rowspan="1" colspan="1">116.6 ± 33</td>
<td rowspan="1" colspan="1">803 ± 90</td>
<td rowspan="1" colspan="1">3,088 ± 420</td>
</tr>
<tr><td rowspan="1" colspan="1">Fungal chitosan</td>
<td rowspan="1" colspan="1">102 ± 7</td>
<td rowspan="1" colspan="1">1,650 ± 276</td>
<td rowspan="1" colspan="1">124 ± 9</td>
<td rowspan="1" colspan="1">1,796 ± 316</td>
<td rowspan="1" colspan="1">12.6 ± 3</td>
<td rowspan="1" colspan="1">92 ± 27</td>
<td rowspan="1" colspan="1">16.9 ± 2</td>
<td rowspan="1" colspan="1">144 ± 44</td>
<td rowspan="1" colspan="1">759 ± 91</td>
<td rowspan="1" colspan="1">3,134 ± 380</td>
</tr>
<tr><td rowspan="1" colspan="1">Chitosan oligosaccharide</td>
<td rowspan="1" colspan="1">145 ± 12</td>
<td rowspan="1" colspan="1">2,473 ± 500</td>
<td rowspan="1" colspan="1">175 ± 14</td>
<td rowspan="1" colspan="1">2,543 ± 505</td>
<td rowspan="1" colspan="1">73 ± 4</td>
<td rowspan="1" colspan="1">260 ± 32</td>
<td rowspan="1" colspan="1">86.2 ± 6</td>
<td rowspan="1" colspan="1">288 ± 39</td>
<td rowspan="1" colspan="1">765 ± 93</td>
<td rowspan="1" colspan="1">3,232 ± 400</td>
</tr>
<tr><td rowspan="1" colspan="1">Chitosan oligosaccharide-lactate</td>
<td rowspan="1" colspan="1">93 ± 7</td>
<td rowspan="1" colspan="1">1,957 ± 174</td>
<td rowspan="1" colspan="1">120 ± 9</td>
<td rowspan="1" colspan="1">2,365 ± 239</td>
<td rowspan="1" colspan="1">39 ± 1</td>
<td rowspan="1" colspan="1">201 ± 16</td>
<td rowspan="1" colspan="1">47 ± 2</td>
<td rowspan="1" colspan="1">245 ± 23</td>
<td rowspan="1" colspan="1">754 ± 92</td>
<td rowspan="1" colspan="1">3,058 ± 390</td>
</tr>
<tr><td rowspan="1" colspan="1">Chitosan HCl</td>
<td rowspan="1" colspan="1">97 ± 11</td>
<td rowspan="1" colspan="1">2,080 ± 516</td>
<td rowspan="1" colspan="1">121 ± 15</td>
<td rowspan="1" colspan="1">2,402 ± 667</td>
<td rowspan="1" colspan="1">40 ± 2</td>
<td rowspan="1" colspan="1">210 ± 23</td>
<td rowspan="1" colspan="1">47.9 ± 3</td>
<td rowspan="1" colspan="1">243 ± 33</td>
<td rowspan="1" colspan="1">781 ± 92</td>
<td rowspan="1" colspan="1">3,589 ± 405</td>
</tr>
<tr><td rowspan="1" colspan="1">PC1-chitosan<xref ref-type="table-fn" rid="T2F4"><sup><italic>d</italic>
</sup>
</xref>
</td>
<td rowspan="1" colspan="1">144 ± 10</td>
<td rowspan="1" colspan="1">1,292 ± 217</td>
<td rowspan="1" colspan="1">169 ± 12</td>
<td rowspan="1" colspan="1">1,365 ± 212</td>
<td rowspan="1" colspan="1">68 ± 3</td>
<td rowspan="1" colspan="1">246 ± 26</td>
<td rowspan="1" colspan="1">81.7 ± 6</td>
<td rowspan="1" colspan="1">274 ± 38</td>
<td rowspan="1" colspan="1">756 ± 93</td>
<td rowspan="1" colspan="1">3,364 ± 398</td>
</tr>
<tr><td rowspan="1" colspan="1">PC2-chitosan<xref ref-type="table-fn" rid="T2F4"><sup><italic>d</italic>
</sup>
</xref>
</td>
<td rowspan="1" colspan="1">133 ± 6</td>
<td rowspan="1" colspan="1">1,005 ± 194</td>
<td rowspan="1" colspan="1">159 ± 6</td>
<td rowspan="1" colspan="1">1,705 ± 170</td>
<td rowspan="1" colspan="1">60 ± 3</td>
<td rowspan="1" colspan="1">202 ± 22</td>
<td rowspan="1" colspan="1">71.9 ± 5</td>
<td rowspan="1" colspan="1">237 ± 36</td>
<td rowspan="1" colspan="1">800 ± 92</td>
<td rowspan="1" colspan="1">3,709 ± 410</td>
</tr>
<tr><td rowspan="1" colspan="1">PC3-chitosan<xref ref-type="table-fn" rid="T2F4"><sup><italic>d</italic>
</sup>
</xref>
</td>
<td rowspan="1" colspan="1">163 ± 11</td>
<td rowspan="1" colspan="1">1,052 ± 144</td>
<td rowspan="1" colspan="1">187 ± 10</td>
<td rowspan="1" colspan="1">1,107 ± 142</td>
<td rowspan="1" colspan="1">71 ± 4</td>
<td rowspan="1" colspan="1">251 ± 30</td>
<td rowspan="1" colspan="1">83.5 ± 6</td>
<td rowspan="1" colspan="1">286 ± 41</td>
<td rowspan="1" colspan="1">786 ± 93</td>
<td rowspan="1" colspan="1">3,719 ± 378</td>
</tr>
<tr><td rowspan="1" colspan="1">Carboxymethyl chitosan</td>
<td rowspan="1" colspan="1">—<xref ref-type="table-fn" rid="T2F5"><sup><italic>e</italic>
</sup>
</xref>
</td>
<td rowspan="1" colspan="1">—</td>
<td rowspan="1" colspan="1">—</td>
<td rowspan="1" colspan="1">—</td>
<td rowspan="1" colspan="1">—</td>
<td rowspan="1" colspan="1">—</td>
<td rowspan="1" colspan="1">—</td>
<td rowspan="1" colspan="1">—</td>
<td rowspan="1" colspan="1">1,184 ± 99</td>
<td rowspan="1" colspan="1">3,999 ± 500</td>
</tr>
</tbody>
</table>
<graphic xlink:href="AAC.01772-19-t0002"></graphic>
</alternatives>
<table-wrap-foot><fn fn-type="other" id="T2F1"><label>a</label>
<p>Experiments were conducted in quadruplicate cultures. Experiments were reproduced a further two times with similar results (data not shown).</p>
</fn>
<fn fn-type="other" id="T2F2"><label>b</label>
<p>Both RPMI alone at pH 6.5 and chitosan solvent did not show any activity against amastigotes. Statistically significant differences were found between the EC<sub>50</sub>
s of chitosan and its derivatives at pH 6.5 and pH 7.5 (<italic>P</italic>
< 0.05 by using <italic>t</italic>
test).</p>
</fn>
<fn fn-type="other" id="T2F3"><label>c</label>
<p>Chitosan and its derivatives had low cytotoxicities toward KB cells at both pH 6.5 and 7.5, and there was no significant difference in the cytotoxicities at these two pHs (<italic>P</italic>
< 0.05 by <italic>t</italic>
test). No statistically significant difference was found between LD<sub>50</sub>
s (50% lethal dose) of the three types of chitosan and other derivatives against KB cells (except for carboxymethyl chitosan, which was the least toxic) (<italic>P</italic>
> 0.05 by the extra-sum-of-squares F test).</p>
</fn>
<fn fn-type="other" id="T2F4"><label>d</label>
<p>Phosphorylcholine-substituted chitosan (see <xref rid="T4" ref-type="table">Table 4</xref>
).</p>
</fn>
<fn fn-type="other" id="T2F5"><label>e</label>
<p>—, no activity up to 400 μg/ml.</p>
</fn>
</table-wrap-foot>
</table-wrap>
<p>To allow like-for-like comparison, EC<sub>50</sub>
s were recalculated in terms of molarity, using estimated molecular weights (HMW chitosan, 342.5 kDa; MMW chitosan, 250 kDa; LMW chitosan, 120 kDa, and fungal chitosan, 130 kDa) at pH 6.5. Based on molarity (Tables S4 and S5), HMW chitosan was significantly more active against <named-content content-type="genus-species">L. major</named-content>
and <named-content content-type="genus-species">L. mexicana</named-content>
promastigotes and amastigotes and, hence, was used in all subsequent studies.</p>
</sec>
<sec id="s2.2"><title>Host cell dependence of the antileishmanial activity of HMW chitosan at pH 6.5.</title>
<p>We aimed to assess the host cell dependence of the antileishmanial activity of HMW chitosan and amphotericin B (Fungizone) by evaluating the <italic>in vitro</italic>
activity against <named-content content-type="genus-species">L. major</named-content>
amastigotes in three different macrophage types; the EC<sub>50</sub>
s and EC<sub>90</sub>
s in the three different macrophage populations are summarized in <xref rid="T3" ref-type="table">Table 3</xref>
. There was a significant difference in the activity of HMW chitosan depending on the type of macrophage (PEMs, bone marrow-derived macrophages [BMMs], or human leukemic monocyte-like-derived cells [THP-1 cell line]) (<italic>P</italic>
< 0.05 by the extra-sum-of-squares F test). HMW chitosan was significantly more active against intracellular amastigotes in PEMs and BMMs than in differentiated THP-1 cells.</p>
<table-wrap id="T3" orientation="portrait" position="float"><label>TABLE 3</label>
<caption><p>HMW chitosan activities against <named-content content-type="genus-species">L. major</named-content>
amastigotes in three different macrophage cultures</p>
</caption>
<alternatives><table frame="hsides" rules="groups"><colgroup span="1"><col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
</colgroup>
<thead><tr><th rowspan="3" colspan="1">Host cells</th>
<th colspan="4" rowspan="1">Mean value ± SD<xref ref-type="table-fn" rid="T3F1"><sup><italic>a</italic>
</sup>
</xref>
<hr></hr>
</th>
</tr>
<tr><th colspan="2" rowspan="1">HMW chitosan (μg/ml)<hr></hr>
</th>
<th colspan="2" rowspan="1">Amphotericin B (μM)<hr></hr>
</th>
</tr>
<tr><th rowspan="1" colspan="1">EC<sub>50</sub>
</th>
<th rowspan="1" colspan="1">EC<sub>90</sub>
</th>
<th rowspan="1" colspan="1">EC<sub>50</sub>
</th>
<th rowspan="1" colspan="1">EC<sub>90</sub>
</th>
</tr>
</thead>
<tbody><tr><td rowspan="1" colspan="1">PEMs</td>
<td rowspan="1" colspan="1">10.31 ± 1.22</td>
<td rowspan="1" colspan="1">89.07 ± 20.46</td>
<td rowspan="1" colspan="1">0.02 ± 0.004</td>
<td rowspan="1" colspan="1">0.27 ± 0.07</td>
</tr>
<tr><td rowspan="1" colspan="1">BMMs</td>
<td rowspan="1" colspan="1">14.60 ± 1.79</td>
<td rowspan="1" colspan="1">145.7 ± 36.2</td>
<td rowspan="1" colspan="1">0.04 ± 0.005</td>
<td rowspan="1" colspan="1">0.43 ± 0.1</td>
</tr>
<tr><td rowspan="1" colspan="1">THP-1</td>
<td rowspan="1" colspan="1">24.28 ± 2.87</td>
<td rowspan="1" colspan="1">200.1 ± 48.8</td>
<td rowspan="1" colspan="1">0.08 ± 0.006</td>
<td rowspan="1" colspan="1">1.15 ± 0.37</td>
</tr>
</tbody>
</table>
<graphic xlink:href="AAC.01772-19-t0003"></graphic>
</alternatives>
<table-wrap-foot><fn fn-type="other" id="T3F1"><label>a</label>
<p>More than 80% of macrophages were infected at 24 h for all cell lines. Cultures were grown at pH 6.5 and analyzed after 72 h. Experiment was conducted in quadruplicate cultures. Experiment was reproduced a further two times with similar results (data not shown). There were statistically significant differences in EC<sub>50</sub>
s between the three types of cells (<italic>P</italic>
< 0.05 by the extra-sum-of-squares F test); chitosan and amphotericin B were significantly more active in PEMs and BMMs than in THP-1 cells. RPMI and DMEM alone at pH 6.5 and chitosan solvent did not show any activity against amastigotes.</p>
</fn>
</table-wrap-foot>
</table-wrap>
</sec>
<sec id="s2.3"><title>Effects of HMW chitosan on the production of TNF-α by uninfected or <named-content content-type="genus-species">L. major</named-content>
-infected BMMs at pH 6.5.</title>
<p>The activation of M1 macrophages by Th1 lymphocytes plays an important role in the control of CL (<xref rid="B6" ref-type="bibr">6</xref>
, <xref rid="B38" ref-type="bibr">38</xref>
, <xref rid="B39" ref-type="bibr">39</xref>
). Therefore, we measured TNF-α production by BMMs stimulated by HMW chitosan. Following exposure to HMW chitosan, the TNF-α production by BMMs was found to be dose dependent, in a bell-shaped manner, in both <italic>Leishmania</italic>
-infected and uninfected cells, as shown in <xref ref-type="fig" rid="F1">Fig. 1</xref>
. After 24 h, the levels of TNF-α in the culture fluid of BMMs exposed to HMW chitosan at concentrations of 14.8, 44.4, and 133.3 μg/ml were significantly higher than the levels in culture fluid of BMMs (infected and uninfected) that had not been exposed to chitosan, with TNF-α being the highest at 44.4 μg/ml chitosan. Meanwhile, at other concentrations (1.64, 4.9, and 400 μg/ml), HMW chitosan did not stimulate BMMs to produce TNF-α (<italic>P</italic>
< 0.05 by <italic>t</italic>
test).</p>
<fig id="F1" orientation="portrait" position="float"><label>FIG 1</label>
<caption><p>TNF-α production in uninfected and <named-content content-type="genus-species">L. major</named-content>
-infected BMMs after 24 h of exposure to 1.64, 4.9,14.8, 44.4, 133.3, or 400 μg/ml of chitosan at pH 6.5. The dose response in both uninfected and <named-content content-type="genus-species">L. major</named-content>
-infected BMMs was bell shaped. TNF-α production was significantly decreased (<italic>P</italic>
< 0.05 by <italic>t</italic>
test) by infecting the cells with <named-content content-type="genus-species">L. major</named-content>
. Experiment was conducted in quadruplicate, and data are expressed as mean values ± SD. Experiment was reproduced a further two times with similar results (data not shown). Positive control was BMMs treated with LPS at 10 μg/ml. Negative control was BMMs not exposed to chitosan. Initial macrophage infection rate was >80% after 24 h. Chitosan solvent did not cause any TNF-α production.</p>
</caption>
<graphic xlink:href="AAC.01772-19-f0001"></graphic>
</fig>
<p>HMW chitosan at concentrations of 14.8, 44.4, and 133.3 μg/ml stimulated BMMs to produce TNF-α at concentrations of 87 ± 4.5 (mean ± standard deviation [SD]), 712 ± 9, and 48 ± 3 pg/ml, respectively, in uninfected BMMs and 56 ± 3.5, 464 ± 10, and 32 ± 4 pg/ml, respectively, in <named-content content-type="genus-species">L. major</named-content>
-infected BMMs. Less TNF-α was generated when the chitosan concentration was increased to 133.3 μg/ml and above. Lipopolysaccharides (LPS) from <named-content content-type="genus-species">Escherichia coli</named-content>
O26:B6 (positive control) stimulated TNF-α production in both uninfected and infected BMMs after a 24-h incubation period at a significantly higher level than did chitosan (<italic>P</italic>
< 0.05 by <italic>t</italic>
test). Our results indicated that HMW chitosan activated M1 macrophages.</p>
</sec>
<sec id="s2.4"><title>Effects of HMW chitosan on the production of ROS by BMMs at pH 6.5.</title>
<p>ROS plays an important role in the killing of intracellular amastigotes (<xref rid="B6" ref-type="bibr">6</xref>
, <xref rid="B38" ref-type="bibr">38</xref>
, <xref rid="B39" ref-type="bibr">39</xref>
), and therefore, we measured ROS production by BMMs stimulated by HMW chitosan. HMW chitosan (at concentrations of 14.8, 44.4, and 133.3 μg/ml) increased the production of ROS (indicated by H<sub>2</sub>
DCFDA [2′,7′-dichlorodihydrofluorescein diacetate] fluorescence) after 4 h of incubation but did not stimulate ROS after 8 h of incubation (Table S1). Other concentrations of HMW chitosan (1.64, 4.9, and 400 μg/ml) did not stimulate BMMs to produce ROS after 4 h or 8 h of incubation.</p>
<p>The ROS dose response in both uninfected and infected BMMs was bell shaped, similar to that seen with TNF-α. Increasing the chitosan concentration from 14.8 to 44.4 μg/ml increased ROS production, after which further increases in concentration reduced ROS production. In addition, ROS production by BMMs was significantly decreased (<italic>P</italic>
< 0.05 by <italic>t</italic>
test) by infecting the cells with <named-content content-type="genus-species">L. major</named-content>
, as shown by the results in <xref ref-type="fig" rid="F2">Fig. 2</xref>
.</p>
<fig id="F2" orientation="portrait" position="float"><label>FIG 2</label>
<caption><p>ROS production in uninfected and <named-content content-type="genus-species">L. major</named-content>
-infected BMMs after 4 h of exposure to 1.64, 4.9,14.8, 44.4, 133.3, or 400 μg/ml of HMW chitosan at pH 6.5. High levels of ROS were induced by both uninfected and <named-content content-type="genus-species">L. major</named-content>
-infected BMMs exposed to HMW chitosan compared to the levels in BMMs that were not exposed to chitosan (<italic>P</italic>
< 0.05 by <italic>t</italic>
test). Maximum production of ROS occurred at 44.4 μg/ml of chitosan. ROS production by <named-content content-type="genus-species">L. major</named-content>
-infected BMMs was significantly lower than in uninfected cells (<italic>P</italic>
< 0.05 by <italic>t</italic>
test). Experiment was conducted in quadruplicate, and data are expressed as mean values ± SD. Experiment was reproduced a further two times with similar results (data not shown). Positive control was BMMs treated with 25 mM H<sub>2</sub>
O<sub>2</sub>
(a known ROS inducer). Negative control was BMMs not exposed to chitosan. Initial macrophage infection rate was >80% after 24 h. Chitosan solvent alone did not cause any ROS production.</p>
</caption>
<graphic xlink:href="AAC.01772-19-f0002"></graphic>
</fig>
<p>We found that <italic>in vitro</italic>
, HMW chitosan had a stimulatory effect on BMM ROS production after 4 h of incubation. We therefore investigated whether this ROS plays any role in the activity of HMW chitosan against intracellular amastigotes. For these experiments, the 4-h-posttreatment time point was taken because ROS peaked at this point in BMMs in response to chitosan treatment, at a time when chitosan does not induce NO in BMMs (Table S3). Scavenging of ROS by the ROS scavenger 5 mM <italic>N</italic>
-acetyl-<sc>l</sc>
-cysteine (NAC) had no significant impact on the activity of chitosan against intracellular amastigotes (<italic>P</italic>
> 0.05 by <italic>t</italic>
test) (<xref ref-type="fig" rid="F3">Fig. 3</xref>
). The ROS scavenger caused complete scavenging of ROS production after 4 h (Table S2) and had no cytotoxicity against human squamous carcinoma cells (KB cells) or leishmanicidal activity against <named-content content-type="genus-species">L. major</named-content>
amastigotes (data not shown). Even though chitosan stimulated ROS production, ROS did not play a role in the antileishmanial activity of chitosan.</p>
<fig id="F3" orientation="portrait" position="float"><label>FIG 3</label>
<caption><p>Activity of HMW chitosan against <named-content content-type="genus-species">L. major</named-content>
amastigotes in BMMs after 4 h, with and without ROS scavenger at pH 6.5. Infected macrophages were preincubated with 5 mM NAC for 2 h, after which HMW chitosan at a concentration of 1.64, 4.9,14.8, 44.4, 133.3, or 400 μg/ml was added and the cells were incubated for a further 4 h. Chitosan activity against intracellular amastigotes was evaluated as described in Materials and Methods. Values are expressed as percentages of inhibition of infection relative to the results for untreated controls. After 4 h, there was no significant difference in the antileishmanial activities of chitosan after scavenging of ROS (<italic>P</italic>
> 0.05 by <italic>t</italic>
test). Experiment was conducted in quadruplicate, and data are expressed as mean values ± SD. Experiment was reproduced a further two times with similar results (data not shown). Initial macrophage infection rate was >80% after 24 h.</p>
</caption>
<graphic xlink:href="AAC.01772-19-f0003"></graphic>
</fig>
</sec>
<sec id="s2.5"><title>Effects of HMW chitosan on the production of NO by BMMs at pH 6.5.</title>
<p>NO plays an important role in the killing of intracellular amastigotes (<xref rid="B6" ref-type="bibr">6</xref>
, <xref rid="B38" ref-type="bibr">38</xref>
, <xref rid="B39" ref-type="bibr">39</xref>
), and therefore, we measured NO production by BMMs stimulated by HMW chitosan. We showed that chitosan did not have a stimulatory effect on BMM NO production after 4 h of incubation (Table S3). However, after 24 h of incubation, HMW chitosan had a stimulatory effect on BMM NO production at pH 6.5 in a clearly bell-shaped, dose-dependent manner (<xref ref-type="fig" rid="F4">Fig. 4</xref>
). HMW chitosan at concentrations of 14.8, 44.4, and 133.3 μg/ml induced both uninfected and infected BMMs to produce NO (at 14.9 ± 0.3, 34 ± 1.2, and 11 ± 1 μM, respectively, in uninfected BMMs and 11 ± 1, 26 ± 2.5 and 8 ± 1.2 μM, respectively, in infected BMMs), NO being highest at 44.4 μg/ml. Meanwhile, other concentrations of HMW chitosan (1.64, 4.9, and 400 μg/ml) did not stimulate BMMs to produce NO after 24 h of incubation.</p>
<fig id="F4" orientation="portrait" position="float"><label>FIG 4</label>
<caption><p>NO production in uninfected and <named-content content-type="genus-species">L. major</named-content>
-infected BMMs after 24 h of exposure to 1.64, 4.9,14.8, 44.4, 133.3, or 400 μg/ml of chitosan at pH 6.5. The response in both uninfected and infected BMMS was bell shaped and related to chitosan concentration. Maximal production of NO was stimulated by 44.4 μg/ml of chitosan. NO production was significantly decreased (<italic>P</italic>
< 0.05 by <italic>t</italic>
test) when the cells had been infected with <named-content content-type="genus-species">L. major</named-content>
. Experiment was conducted in quadruplicate cultures, and data are expressed as mean values ± SD. Experiment was reproduced a further two times with similar results (data not shown). Positive control was BMMs treated with 10 μg/ml LPS. Negative control was BMMs not exposed to chitosan. Initial macrophage infection rate was >80% after 24 h. Chitosan solvent alone did not cause any NO production.</p>
</caption>
<graphic xlink:href="AAC.01772-19-f0004"></graphic>
</fig>
<p>LPS caused significantly higher NO production than did HMW chitosan (<italic>P</italic>
< 0.05 by <italic>t</italic>
test) in both uninfected and infected BMMs. The levels of NO produced by <named-content content-type="genus-species">L. major</named-content>
-infected BMMs exposed to LPS (positive control) or HMW chitosan were significantly lower than the levels produced by uninfected BMMs (<italic>P</italic>
< 0.05 by <italic>t</italic>
test) (<xref ref-type="fig" rid="F4">Fig. 4</xref>
).</p>
<p>As HMW chitosan had an <italic>in vitro</italic>
stimulatory effect on BMM NO production after 24 h of incubation, we investigated further whether NO has any role in the activity of HMW chitosan against intracellular amastigotes. Inhibition of NO production by the NO inhibitor N<sup>G</sup>
-methyl-<sc>l</sc>
-arginine acetate salt (L-NMMA) at 0.4 mM had no significant influence on the activity of chitosan against intracellular amastigotes (<italic>P</italic>
> 0.05 by <italic>t</italic>
test) (<xref ref-type="fig" rid="F5">Fig. 5</xref>
), although the NO inhibitor did cause a complete inhibition of NO production (Table S2) after 24 h and had no cytotoxic effects against KB cells and no leishmanicidal activity against intracellular <named-content content-type="genus-species">L. major</named-content>
amastigotes (data not shown). Even though chitosan stimulated NO production, it did not play a role in the antileishmanial activity of chitosan.</p>
<fig id="F5" orientation="portrait" position="float"><label>FIG 5</label>
<caption><p>Activity of HMW chitosan against <named-content content-type="genus-species">L. major</named-content>
-infected BMMs after 24 h in the presence or absence of an NO inhibitor at pH 6.5. Infected macrophages were preincubated with the NO inhibitor L-NMMA (0.4 mM) for 2 h, following which HMW chitosan at a concentration of 1.64, 4.9,14.8, 44.4, 133.3, or 400 μg/ml was added and the cells were incubated for a further 24 h. Chitosan activity against intracellular amastigotes was evaluated as described in Materials and Methods. Values are expressed as percentages of inhibition of infection relative to the results for untreated controls. After 24 h, there was no significant difference in the activity of chitosan after inhibition of NO (<italic>P</italic>
> 0.05 by <italic>t</italic>
test). Experiment was conducted in quadruplicate cultures, and data are expressed as mean values ± SD. Experiment was reproduced a further two times and the results were confirmed (data not shown). Initial macrophage infection rate was >80% after 24 h.</p>
</caption>
<graphic xlink:href="AAC.01772-19-f0005"></graphic>
</fig>
</sec>
<sec id="s2.6"><title>Cellular uptake of HMW chitosan and inhibition of endocytosis.</title>
<p>We found that the activation of M1 macrophages by HMW chitosan did not play a role in its activity against intracellular amastigotes. Therefore, we investigated whether the antileishmanial effects of HMW chitosan against intracellular amastigotes after 4 h and 24 h of exposure were dependent on the direct activity of chitosan following its entry into the macrophages at pH 6.5. No significant difference was observed in the activity of chitosan against intracellular amastigotes when it was added after prior phagocytosis inhibition with cytochalasin D (<italic>P</italic>
> 0.05 by <italic>t</italic>
test) (<xref ref-type="fig" rid="F6">Fig. 6</xref>
). In contrast, dynasore, an inhibitor of pinocytosis (clathrin-mediated endocytosis [CME]), did significantly affect chitosan-mediated parasite killing at pH 6.5 (<italic>P</italic>
< 0.05 by <italic>t</italic>
test) (<xref ref-type="fig" rid="F6">Fig. 6</xref>
). The same activity was seen at pH 7.5 (<xref ref-type="fig" rid="F6">Fig. 6C</xref>
). The two inhibitors had no cytotoxicity against KB cells or activity against intracellular <named-content content-type="genus-species">L. major</named-content>
amastigotes at the concentrations used. Pinocytosis (CME) played a critical role in the efficacy of HMW chitosan against intracellular amastigotes.</p>
<fig id="F6" orientation="portrait" position="float"><label>FIG 6</label>
<caption><p>Activities of HMW chitosan against <named-content content-type="genus-species">L. major</named-content>
-infected BMMs after 4 h at pH 6.5 (A), 24 h at pH 6.5 (B), or 24 h at pH 7.5 (C), with or without phagocytosis inhibitor or pinocytosis (CME) inhibitor. We found that chitosan requires pinocytosis (CME) and not phagocytosis by BMMs for killing of <named-content content-type="genus-species">L. major</named-content>
amastigotes at pH 6.5 and 7.5. BMMs were infected with stationary-phase promastigotes. Some of the infected macrophages were preincubated with cytochalasin D (phagocytosis inhibitor) or dynasore (pinocytosis [CME] inhibitor) and exposed to various concentrations (1.64, 4.9,14.8, 44.4, 133.3, or 400 μg/ml) of chitosan for 4 h and 24 h, followed by microscopic counting of the number of infected macrophages. There was no significant difference in the activities of HMW chitosan after inhibition of phagocytosis (<italic>P</italic>
> 0.05 by <italic>t</italic>
test). In contrast, significant inhibition of chitosan-mediated parasite killing occurred in the presence of dynasore at the two pH values (<italic>P</italic>
< 0.05 by <italic>t</italic>
test). Values are expressed as percentages of inhibition of infection relative to the results for untreated controls. Experiments were conducted in quadruplicate cultures, and data are expressed as mean values ± SD. Experiments were reproduced a further two times and the results confirmed (data not shown). Initial macrophage infection rate was >80% after 24 h.</p>
</caption>
<graphic xlink:href="AAC.01772-19-f0006"></graphic>
</fig>
</sec>
<sec id="s2.7"><title>Fluorescence microscopy of the uptake of chitosan by macrophages.</title>
<p>Rhodamine-labeled chitosan was used to track the delivery of chitosan to the parasitophorous vacuoles (PVs) of <italic>Leishmania</italic>
-infected macrophages. <xref ref-type="fig" rid="F7">Fig. 7</xref>
illustrates the cellular uptake of chitosan by BMMs infected with green fluorescent protein (GFP)-labeled <named-content content-type="genus-species">L. major</named-content>
(<named-content content-type="genus-species">L. major</named-content>
-GFP) or <named-content content-type="genus-species">L. mexicana</named-content>
-GFP after 4 h and 24 h of exposure to rhodamine-labeled chitosan. There was colocalization of chitosan and intracellular amastigotes after 4 h and 24 h, with normalized mean deviation product (nMDP) color indexes of 0.7 and 1, respectively (see “Microscopic imaging of the cellular uptake of rhodamine-labeled chitosan” in Materials and Methods). The uptake of chitosan increased in a time-dependent manner. The results in <xref ref-type="fig" rid="F7">Fig. 7XA</xref>
and <xref ref-type="fig" rid="F7">XB</xref>
, panels D and E, show this uptake after 4 h and 24 h, respectively, as well as the accumulation of chitosan in PVs (<xref ref-type="fig" rid="F7">Fig. 7</xref>
, colocalization of rhodamine and GFP shown by yellow color). The results in <xref ref-type="fig" rid="F7">Fig. 7XA</xref>
and <xref ref-type="fig" rid="F7">XB</xref>
, panels F, also show that the inhibition of pinocytosis (CME) with dynasore prevented the uptake of chitosan, with a negative nMDP color index that represents no colocalization of chitosan and amastigotes. This is also supporting evidence for the uptake by pinocytosis as seen by the results in <xref ref-type="fig" rid="F6">Fig. 6</xref>
.</p>
<fig id="F7" orientation="portrait" position="float"><label>FIG 7</label>
<caption><p>Fluorescence microscopy images of the cellular uptake of rhodamine-labeled chitosan at 4 h and 24 h at pH 6.5 by BMMs infected with <named-content content-type="genus-species">L. major</named-content>
-GFP (XA) or with <named-content content-type="genus-species">L. mexicana</named-content>
-GFP (XB). Blue represents the nuclei of BMMs, green represents intracellular amastigotes, red represents labeled chitosan, and yellow represents merged red chitosan and green <italic>Leishmania</italic>
parasites. (A to F) Infected BMMs unexposed to chitosan after 4 h (A) or 24 h (B), exposed to chitosan after 4 h (D) or 24 h (E), unexposed to chitosan after 24 h (C), and exposed to chitosan and pinocytosis inhibitor (dynasore) after 24 h (F).</p>
</caption>
<graphic xlink:href="AAC.01772-19-f0007"></graphic>
</fig>
</sec>
</sec>
<sec sec-type="discussion" id="s3"><title>DISCUSSION</title>
<p>The literature on the antileishmanial activity of chitosan and its derivatives is limited, especially pertaining to its mechanism(s) of action (<xref rid="B19" ref-type="bibr">19</xref>
, <xref rid="B40" ref-type="bibr">40</xref>
, <xref rid="B41" ref-type="bibr">41</xref>
). In this study, we assessed the antileishmanial activity of various forms of chitosan, including low-, medium-, and high-molecular-weight chitosan and chitosan derivatives. Chitosan derivatives are generally produced by chemical modification of the amino or hydroxyl groups of chitosan for the optimization of the physicochemical properties. We found that chitosan and its derivatives had minimal cytotoxicity against KB cells, with 50% lethal doses (LD<sub>50</sub>
s) of ≥750 μg/ml in RPMI 1640 at pH 7.5 or 6.5. These data support previous reports of chitosan’s low cytotoxicity against CCRF-CEM (human lymphoblastic leukemia) and L132 (human embryonic lung) cells that had similar LD<sub>50</sub>
s (<xref rid="B42" ref-type="bibr">42</xref>
, <xref rid="B43" ref-type="bibr">43</xref>
).</p>
<p>We determined that a lower pH of 6.5 enhanced the antileishmanial activities of chitosan and its derivatives against <named-content content-type="genus-species">L. major</named-content>
and <named-content content-type="genus-species">L. mexicana</named-content>
promastigotes and amastigotes by 7 to 20 times compared to the results at pH 7.5. This higher activity of chitosan at the lower pH of 6.5 could be due to its greater ionization (protonation of the amino groups; the pK<sub>a</sub>
of chitosan is ≈6.3). The greater positive charge could increase the antimicrobial activity of chitosan due to interaction with the negatively charged microbial membrane (in accordance with the first postulated mechanism of antimicrobial activity described in the introduction) (<xref rid="B18" ref-type="bibr">18</xref>
, <xref rid="B19" ref-type="bibr">19</xref>
). A higher chitosan activity at lower pH (pH ≈5) has previously been reported against <named-content content-type="genus-species">Escherichia coli</named-content>
and <named-content content-type="genus-species">Salmonella enterica</named-content>
serovar Typhimurium (<xref rid="B44" ref-type="bibr">44</xref>
, <xref rid="B45" ref-type="bibr">45</xref>
).</p>
<p>Our study is the first to show the pH dependence of the antileishmanial activity of chitosan and its derivatives and could explain why reports of the antileishmanial activity of chitosan in the literature have shown such variability, with EC<sub>50</sub>
s ranging from 70 to 240 μg/ml against <named-content content-type="genus-species">L. infantum</named-content>
, <named-content content-type="genus-species">L. amazonensis</named-content>
, and <named-content content-type="genus-species">L. chagasi</named-content>
promastigotes and amastigotes (<xref rid="B29" ref-type="bibr">29</xref>
<xref ref-type="bibr" rid="B30">–</xref>
<xref rid="B34" ref-type="bibr">34</xref>
). For example, in one study, the EC<sub>50</sub>
of chitosan against <named-content content-type="genus-species">L. infantum</named-content>
amastigotes (in PEMs) in RPMI 1640 medium was 100.81 μg/ml, but the pH at which the experiment was conducted was not mentioned (<xref rid="B29" ref-type="bibr">29</xref>
). The influence of pH was also seen when the antileishmanial activities of chitosan (of the different molecular weights) and chitosan derivatives were compared. While the different chitosans and derivatives showed minor differences in their antileishmanial activities at pH 7.5, the derivatives were 3 to 5 times less active than the HMW, MMW, LMW, and fungal chitosan at the lower pH of 6.5. This reduced activity could be due to the smaller number of amino groups on the chitosan derivatives (see <xref ref-type="fig" rid="F8">Fig. 8</xref>
). These derivatives are more soluble at a higher pH and have activities similar to those of chitosan, but at a lower pH, the higher protonation of the chitosan improves the antileishmanial activity significantly (<xref rid="B46" ref-type="bibr">46</xref>
, <xref rid="B47" ref-type="bibr">47</xref>
). Carboxymethyl chitosan had no antileishmanial activity—most of the amino groups on this derivative have been replaced by carboxymethyl moieties, making the molecule negatively charged (<xref rid="B48" ref-type="bibr">48</xref>
).</p>
<fig id="F8" orientation="portrait" position="float"><label>FIG 8</label>
<caption><p>The structures of chitosan (<xref rid="B60" ref-type="bibr">60</xref>
) and its derivatives, chitosan HCl, carboxymethyl chitosan (<xref rid="B61" ref-type="bibr">61</xref>
), chitosan oligosaccharide (<xref rid="B60" ref-type="bibr">60</xref>
), PC-chitosan (republished from reference <xref rid="B28" ref-type="bibr">28</xref>
with permission of the publisher), and chitosan oligosaccharide lactate (<xref rid="B59" ref-type="bibr">59</xref>
).</p>
</caption>
<graphic xlink:href="AAC.01772-19-f0008"></graphic>
</fig>
<p>The higher antileishmanial activity of HMW chitosan compared to the activities of MMW and LMW chitosan mirrors its greater antibacterial activity in another study against <named-content content-type="genus-species">Escherichia coli</named-content>
, <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content>
, and <named-content content-type="genus-species">Staphylococcus aureus</named-content>
(<xref rid="B49" ref-type="bibr">49</xref>
). HMW has a long chain and, therefore, more glucosamine units and possesses more amino groups (<xref ref-type="fig" rid="F8">Fig. 8</xref>
), resulting in more protonated groups (-NH<sub>3</sub>
<sup>+</sup>
) than MMW and LMW chitosan have (<xref rid="B49" ref-type="bibr">49</xref>
), which could explain its greater potency.</p>
<p>We also showed that the antileishmanial activity of chitosan is significantly greater against <named-content content-type="genus-species">L. major</named-content>
-infected PEMs or BMMs than against <named-content content-type="genus-species">L. major</named-content>
-infected differentiated THP-1 cells, in the order PEMs > BMMs > THP-1 cells, underlining the need to take the host cell into consideration when conducting similar experiments (<xref rid="B50" ref-type="bibr">50</xref>
).</p>
<p>In order to understand the potential antiamastigote mechanism(s) of chitosan, we investigated whether the activity of HMW chitosan against the intracellular amastigotes was via direct uptake into the host cell and localization in the parasitophorous vacuole or indirectly via the activation of M1 macrophages, given that the cellular immune responses in cutaneous leishmaniasis play a critical role in self-cure (<xref rid="B51" ref-type="bibr">51</xref>
, <xref rid="B52" ref-type="bibr">52</xref>
).</p>
<p>The activation of M1 macrophages by the Th1 lymphocyte subpopulation, which produces different cytokines, primarily IFN-γ and TNF-α, is crucial for the killing of intracellular <italic>Leishmania</italic>
via the triggering of an oxidative burst, and therefore, the host cells increase the production of ROS and NO, which are responsible for killing of the parasite (<xref rid="B38" ref-type="bibr">38</xref>
, <xref rid="B39" ref-type="bibr">39</xref>
). We found that HMW chitosan stimulated TNF-α production by macrophages, and this would be expected to be an indicator of an M1 macrophage that would have greater leishmanicidal activity. Our results show that chitosan stimulated BMM ROS production with a peak after 4 h and led to significant increases in the TNF-α and NO production after 24 h in a bell-shaped response. Similar findings have been reported showing that HMW chitosan had an <italic>in vitro</italic>
stimulatory effect on NO production in PEMs (from male rats) (<xref rid="B25" ref-type="bibr">25</xref>
) and LMW chitosan stimulated RAW264.7 macrophage TNF-α production (<xref rid="B24" ref-type="bibr">24</xref>
). Another study demonstrated that LMW chitosan induced ROS production in an epithelial, human breast cancer cell line (<xref rid="B53" ref-type="bibr">53</xref>
). The bell-shaped responses are consistent with a study that showed that chitosan stimulated NO and TNF-α production in peritoneal macrophages in a dose-dependent manner and that their levels tended to decrease at higher concentrations of chitosan (320 μg/ml) (<xref rid="B54" ref-type="bibr">54</xref>
). This type of response has also been reported previously for tucaresol for both its immunomodulatory activity and activity against experimental <named-content content-type="genus-species">Leishmania donovani</named-content>
infections, albeit at lower doses (<xref rid="B55" ref-type="bibr">55</xref>
). Despite the observed chitosan-induced ROS and NO production, there was no evidence that this contributed to the antileishmanial activity in our study: the inhibitors that we used to suppress their production had no effect on the ability of chitosan to kill intracellular <italic>Leishmania</italic>
amastigotes (<xref ref-type="fig" rid="F3">Fig. 3</xref>
and <xref ref-type="fig" rid="F5">5</xref>
). This led us to investigate the cellular uptake of HMW chitosan and its relationship to the antileishmanial activity.</p>
<p>The uptake of the large charged molecule of HMW chitosan has not been systematically studied before, and there is no clear evidence of its penetration of cell membranes or of its uptake mechanism. Macrophages are known to take up extracellular materials and plasma by endocytosis. Endocytosis mainly occurs via two different cellular uptake mechanisms, pinocytosis and phagocytosis, where pinocytosis is fluid-phase endocytosis and phagocytosis is the process of engulfing large particles (<xref rid="B56" ref-type="bibr">56</xref>
). Inhibition of pinocytosis (clathrin-mediated endocytosis [CME]) significantly reduced the antileishmanial activity of HMW chitosan. Therefore, in our study, pinocytosis (CME) was considered to be the main mechanism for the uptake of HMW chitosan by BMMs, indicating a direct antileishmanial effect of this molecule against amastigotes. Other studies have previously reported pinocytosis as the pathway for the uptake of chitosan of different molecular weights by HEK293 epithelial cells (<xref rid="B57" ref-type="bibr">57</xref>
). The results of fluorescence imaging in our study showed that in BMMs, HMW chitosan is taken up into the parasitophorous vacuole (PV) where the <italic>Leishmania</italic>
parasites reside, with the labeled chitosan being internalized within 4 h and increasing up to 24 h later. This is consistent with another study where rhodamine isothiocyanate-chitosan (molecular weight of 113 kDa was labelled with RITC) was found to be delivered directly to the U937 macrophage lysosome after 24 h (<xref rid="B58" ref-type="bibr">58</xref>
). The accumulation of chitosan in the PV might be due to chitosan’s relatively high pK<sub>a</sub>
of 6.3 making it more soluble and protonated in the acidic contents of the vacuole. This is consistent with the results of a study using bafilomycin to inhibit acidification and prevent chitosan accumulation within macrophages (<xref rid="B58" ref-type="bibr">58</xref>
).</p>
<p>In summary, our studies indicate that chitosan and its water-soluble derivatives showed antileishmanial activity against both <named-content content-type="genus-species">L. major</named-content>
and <named-content content-type="genus-species">L. mexicana</named-content>
promastigotes and amastigotes in a pH-dependent manner. At pH 6.5, HMW chitosan is more active than MMW and LMW chitosan and chitosan derivatives, in particular those where the amino groups are replaced. In addition, HMW chitosan activated M1 macrophages, stimulating them to produce NO and ROS. However, the antileishmanial activity of chitosan was not due to such immune activation, as an NO inhibitor and an ROS scavenger failed to reduce the antileishmanial activity. Instead, the antileishmanial activity was related to direct uptake of chitosan into the parasitophorous vacuole by pinocytosis (CME). HMW chitosan demonstrated effective <italic>in vitro</italic>
antileishmanial activity with minimal cytotoxicity, and future work will focus on <italic>in vivo</italic>
studies, formulations, and routes of administration.</p>
</sec>
<sec sec-type="materials|methods" id="s4"><title>MATERIALS AND METHODS</title>
<sec id="s4.1"><title>Drugs and chemicals.</title>
<p>Stocks of amphotericin B deoxycholate (5.2 mM [aqueous]) (Fungizone; Gibco, UK) were prepared, aliquoted, and kept at −20°C until use. Chitosan with three different molecular weights and its derivatives were used and are summarized in <xref rid="T4" ref-type="table">Table 4</xref>
and <xref ref-type="fig" rid="F8">Fig. 8</xref>
(<xref rid="B28" ref-type="bibr">28</xref>
, <xref rid="B59" ref-type="bibr">59</xref>
, <xref rid="B60" ref-type="bibr">60</xref>
, <xref rid="B61" ref-type="bibr">61</xref>
). Solutions of chitosan and derivatives were prepared by dissolving 1 g in 100 ml of 1% (vol/vol) acetic acid solution at room temperature with continuous stirring for 24 h until a clear solution was obtained. The pH of the solution was adjusted to approximately 6 by adding sodium hydroxide 2N (NaOH; Sigma, UK) solution and monitoring with a pH meter (Orion model 420A). The chitosan solutions were autoclaved (121°C for 15 min). Phosphorylcholine (PC)-substituted chitosan generated through reductive amination of PC-glyceraldehyde with primary amines of deacetylated chitosan (57 kDa) was kindly provided by F. Winnik (Montreal University, Canada). The percentages of substitution were controlled and determined by nuclear magnetic resonance (NMR) spectroscopy (<xref rid="B28" ref-type="bibr">28</xref>
). Chitosan’s pK<sub>a</sub>
is approximately 6.3, and therefore, the approximate degrees of ionization of chitosan are 61% and 6% at pH 6.5 and 7.5, respectively.</p>
<table-wrap id="T4" orientation="portrait" position="float"><label>TABLE 4</label>
<caption><p>Details of chitosan and its derivatives used in the study</p>
</caption>
<alternatives><table frame="hsides" rules="groups"><colgroup span="1"><col span="1"></col>
<col span="1"></col>
<col span="1"></col>
</colgroup>
<thead><tr><th rowspan="1" colspan="1">Compound (source)</th>
<th rowspan="1" colspan="1">Chitosan mol wt (kDa unless otherwise specified); further description</th>
<th rowspan="1" colspan="1">Supplier</th>
</tr>
</thead>
<tbody><tr><td rowspan="1" colspan="1">HMW (crustacean shells)</td>
<td rowspan="1" colspan="1">310–375</td>
<td rowspan="1" colspan="1">Sigma, UK</td>
</tr>
<tr><td rowspan="1" colspan="1">MMW (crustacean shells)</td>
<td rowspan="1" colspan="1">190–310</td>
<td rowspan="1" colspan="1">Sigma, UK</td>
</tr>
<tr><td rowspan="1" colspan="1">LMW (crustacean shells)</td>
<td rowspan="1" colspan="1">50–190</td>
<td rowspan="1" colspan="1">Sigma, UK</td>
</tr>
<tr><td rowspan="1" colspan="1">Fungal chitosan (white mushroom)</td>
<td rowspan="1" colspan="1">110–150</td>
<td rowspan="1" colspan="1">S. Somavarapu</td>
</tr>
<tr><td rowspan="1" colspan="1">Chitosan oligosaccharide</td>
<td rowspan="1" colspan="1">≤5 Da</td>
<td rowspan="1" colspan="1">S. Somavarapu</td>
</tr>
<tr><td rowspan="1" colspan="1">Chitosan oligosaccharide-lactate</td>
<td rowspan="1" colspan="1">Avg Mn 5; oligosaccharide, 60%</td>
<td rowspan="1" colspan="1">S. Somavarapu</td>
</tr>
<tr><td rowspan="1" colspan="1">Chitosan-HCl</td>
<td rowspan="1" colspan="1">47–65</td>
<td rowspan="1" colspan="1">S. Somavarapu</td>
</tr>
<tr><td rowspan="1" colspan="1">Carboxymethyl chitosan</td>
<td rowspan="1" colspan="1">543.519 Da; level of substitution, 95%</td>
<td rowspan="1" colspan="1">S. Somavarapu</td>
</tr>
<tr><td rowspan="1" colspan="1">PC1-chitosan<xref ref-type="table-fn" rid="T4F1"><sup><italic>a</italic>
</sup>
</xref>
</td>
<td rowspan="1" colspan="1">33; PC mol%, 30</td>
<td rowspan="1" colspan="1">F. Winnik</td>
</tr>
<tr><td rowspan="1" colspan="1">PC2-chitosan<xref ref-type="table-fn" rid="T4F1"><sup><italic>a</italic>
</sup>
</xref>
</td>
<td rowspan="1" colspan="1">108; PC mol%, 20</td>
<td rowspan="1" colspan="1">F. Winnik</td>
</tr>
<tr><td rowspan="1" colspan="1">PC3-chitosan<xref ref-type="table-fn" rid="T4F1"><sup><italic>a</italic>
</sup>
</xref>
</td>
<td rowspan="1" colspan="1">109; PC mol%, 30</td>
<td rowspan="1" colspan="1">F. Winnik</td>
</tr>
</tbody>
</table>
<graphic xlink:href="AAC.01772-19-t0004"></graphic>
</alternatives>
<table-wrap-foot><fn fn-type="other" id="T4F1"><label>a</label>
<p>Phosphorylcholine-substituted chitosan.</p>
</fn>
</table-wrap-foot>
</table-wrap>
</sec>
<sec id="s4.2"><title>Ethics statement.</title>
<p>All animal work was carried out under a UK Home Office project license according to the Animal (Scientific Procedures) Act 1986 and the new European Directive 2010/63/EU. The project license (70/8427) has been reviewed by the LSHTM Animal Welfare and Ethical Review Board prior to submission and consequent approval by the UK Home Office.</p>
</sec>
<sec id="s4.3"><title>Cell lines.</title>
<p>Peritoneal mouse macrophages (PEMs) were obtained from 8- to 12-week-old female CD-1 mice (Charles River Ltd., UK). Two milliliters of a 2% (wt/vol) starch solution in phosphate-buffered saline (PBS; Sigma, UK) was injected intraperitoneally. After 24 h, the animal was sacrificed and the PEMs were harvested by peritoneal lavage with cold RPMI 1640 medium (Sigma, UK) containing 200 units penicillin and 0.2 mg streptomycin per milliliter (PenStrep; Sigma, UK). Subsequently, PEMs were centrifuged at 450 × <italic>g</italic>
at 4°C for 15 min, and then the pellet was resuspended in RPMI 1640 with 10% (vol/vol) heat-inactivated fetal calf serum (HIFCS; Gibco, UK).</p>
<p>Bone marrow-derived macrophages (BMMs) were obtained from femurs of 8- to 12-week-old female BALB/c mice (Charles River Ltd.). Briefly, the bone marrow cells were carefully flushed from the bone with Dulbecco’s modified Eagle’s medium (DMEM; Thermofisher, UK) with 10% (vol/vol) HIFCS, 100 U/ml penicillin, and 100 mg/ml streptomycin (Sigma, UK). Cells were pelleted by centrifugation (450 × <italic>g</italic>
for 10 min) and resuspended in 10 ml DMEM with 10% (vol/vol) HIFCS and 50 ng/ml human macrophage colony-stimulating factor (HM-CSF; Thermofisher, UK). After plating out in T175 flasks (Greiner Bio-One, Stonehouse, UK), BMMs were kept at 37°C, 5% CO<sub>2</sub>
for 7 to 10 days, after which they were harvested, counted, and used.</p>
<p>THP-1 cells are a human leukemic monocyte-like-derived cell line. THP-1 cells were cultured in RPMI 1640 medium supplemented with <sc>l</sc>
-glutamine and 10% HIFCS. THP-1 cells were incubated in RPMI 1640 plus 10% (vol/vol) HIFCS and 20 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma, UK) at 37°C and 5% CO<sub>2</sub>
for 72 h to induce maturation transformation of these monocytes into adherent macrophages (<xref rid="B50" ref-type="bibr">50</xref>
).</p>
<p>Human squamous carcinoma (KB) cells are adherent cells derived from epidermal carcinoma in the mouth. KB cells were cultured in RPMI 1640 medium with 10% HIFCS.</p>
<p>The numbers of cells and macrophages were estimated by counting with a Neubauer hemocytometer by light microscopy (×400 total magnification).</p>
</sec>
<sec id="s4.4"><title>Parasites.</title>
<p>Four <italic>Leishmania</italic>
strains were used, two of which (<named-content content-type="genus-species">L. major</named-content>
[MHOM/SU/73/5ASKH] and <named-content content-type="genus-species">L. mexicana</named-content>
[MNYC/BZ/62/M379], kindly donated by G. Getti [University of Greenwich, UK]) were GFP labeled for the fluorescence microscopy study. They were cultured in Schneider’s insect medium (Sigma, UK) with 23% (vol/vol) HIFCS, 1× penicillin-streptomycin-glutamine (Gibco-Invitrogen) and supplemented with 700 μg/ml G418 (an aminoglycoside antibiotic; Sigma, UK). <named-content content-type="genus-species">L. major</named-content>
(MHOM/SA/85/JISH118) and <named-content content-type="genus-species">L. mexicana</named-content>
(MNYC/BZ/62/M379) were used for other experiments without the G418. Promastigotes were incubated at 26°C, and the maximum passage number used was 7.</p>
</sec>
<sec id="s4.5"><title><italic>In vitro</italic>
cytotoxicity assays.</title>
<p>Resuspended KB cells (4 × 10<sup>4</sup>
/100 μl) were allowed to adhere to the bottom of a 96-well plate overnight and then exposed to specific concentrations of the tested compounds for 72 h at 37°C and 5% CO<sub>2</sub>
in an incubator. Podophyllotoxin (Sigma, UK) was included as a positive control at a starting concentration of 0.05 μM. Cytotoxicity was evaluated by a cell viability assay using resazurin sodium salt solution (Sigma, UK), which was prepared according to the manufacturer’s instructions. Twenty microliters of the resazurin solution was added to each well of the plates, and fluorescence (cell viability) (<xref rid="B62" ref-type="bibr">62</xref>
) was measured over a period of 1 to 24 h using a SpectraMax M3 plate reader (excitation/emission at 530/580 nm and 550-nm cutoff). Results were expressed as follows: percentage of inhibition = (100 – <italic>x</italic>
)% viability (mean ± standard deviation [SD] σ). Cytotoxicity was evaluated in RPMI 1640 at two pHs (at the normal pH of RPMI, pH 7.5, and at a lower pH of 6.5). The pH of RPMI 1640 was reduced from 7.5 to 6.5 by adding 0.05 M acidic buffer 2-<italic>N</italic>
-morpholino ethanesulfonic acid (MES; Sigma, UK). RPMI 1640 plus MES (0.05 M) at pH 6.5 did not show any cytotoxicity to KB cells.</p>
</sec>
<sec id="s4.6"><title><italic>In vitro</italic>
72-h activity of chitosan and its derivatives against extracellular <named-content content-type="genus-species">L. major</named-content>
<italic>and</italic>
<named-content content-type="genus-species">L. mexicana</named-content>
promastigotes.</title>
<p>Promastigotes in RPMI 1640 medium were tested while in the exponential growth phase. The promastigotes were diluted to a density of 5 × 10<sup>6</sup>
promastigotes/ml and then exposed to different concentrations of HMW, MMW, and LMW chitosan, chitosan derivatives, and amphotericin B (positive control) in sterile 96-well flat-bottom culture plates for 72 h at 26°C. The activity of the compounds against promastigotes was evaluated using the resazurin sodium salt solution (Sigma, UK) as described above. pH plays a critical role in the solubility and protonation of chitosan, so the activity against promastigotes was evaluated at two different pHs (pH 7.5 and a lower pH of 6.5 obtained by adding MES). The results were expressed as follows: percentage of inhibition = 100% − <italic>x</italic>
% viability (mean ± SD).</p>
</sec>
<sec id="s4.7"><title><italic>In vitro</italic>
72-h activity of chitosan and its derivatives against intracellular amastigotes of <named-content content-type="genus-species">L. major</named-content>
and <named-content content-type="genus-species">L. mexicana</named-content>
.</title>
<p>One hundred microliters of PEM culture at 4 × 10<sup>5</sup>
cells/ml was dispensed into each well of a 16-well LabTek tissue culture slide (Thermo Fisher, UK) at pH 7.5 or pH 6.5 and incubated for 24 h at 37°C in 5% CO<sub>2</sub>
. After 24 h, the wells were washed with fresh culture medium to remove nonadherent cells. Stationary-phase, low-passage-number <italic>Leishmania</italic>
promastigotes were then added to PEMs at a ratio of 5:1. This infection ratio was previously found to give sufficiently high and reproducible infection levels. Slides were incubated for another 24 h at 34°C to mimic dermal temperatures in 5% CO<sub>2</sub>
. Any free, extracellular parasites were removed by washing the wells with cold culture medium. One slide was fixed with 100% methanol for 2 min and stained with 10% Giemsa stain for 5 min. The number of PEMs infected with <italic>Leishmania</italic>
amastigotes per 100 macrophages was counted under a microscope. All the experiments were conducted at macrophage infection levels of above 80% prior to the addition of chitosan. Chitosan, its derivatives, and amphotericin B solutions at a range of concentrations (in quadruplicate) were added to the wells (100 μl), and the slides were incubated for 72 h at 34°C in 5% CO<sub>2</sub>
. After 72 h, the slides were fixed with 100% methanol for 2 min and stained with 10% Giemsa stain for 5 min. The slides were examined, and the percentages of macrophages infected were determined. The antileishmanial activities of compounds were expressed as the percentages of reduction in infected macrophages compared to the numbers of infected macrophages in untreated control wells (<xref rid="B63" ref-type="bibr">63</xref>
). RPMI 1640 plus MES (0.05 M) at pH 6.5 had no activity against <italic>Leishmania</italic>
amastigotes.</p>
</sec>
<sec id="s4.8"><title>Influence of the origin of the host cell on the <italic>in vitro</italic>
activity of HMW chitosan against <named-content content-type="genus-species">L. major</named-content>
amastigotes.</title>
<p>A further two host cell types, THP-1 and BMMs, were infected with <named-content content-type="genus-species">Leishmania major</named-content>
and the activity of HMW chitosan was assessed. THP-1 cells (cultured in RPMI 1640 plus 10% HIFCS) and BMMs (cultured in DMEM plus 10% HIFCS) were used to assess the host cell dependence of the antileishmanial activity of HMW chitosan (<xref rid="B50" ref-type="bibr">50</xref>
). The experiment was conducted as described above for the assay of chitosan activity against intracellular amastigotes of <named-content content-type="genus-species">L. major</named-content>
and <named-content content-type="genus-species">L. Mexicana</named-content>
at pH 6.5.</p>
</sec>
<sec id="s4.9"><title>The role of HMW chitosan in BMM activation.</title>
<p>We chose BMMs to evaluate the activation effects of HMW chitosan and to study the cell uptake of chitosan as this macrophage population is more homogenous than those of PEMs and THP-1 cells (<xref rid="B64" ref-type="bibr">64</xref>
); both PEMs and BMMs have been reported to have similar acidic pHs of ≈5.5 in parasitophorous vacuoles of <named-content content-type="genus-species">L. amazonensis</named-content>
-infected cells (<xref rid="B65" ref-type="bibr">65</xref>
<xref ref-type="bibr" rid="B66">–</xref>
<xref rid="B67" ref-type="bibr">67</xref>
). One hundred microliters of BMMs (4 × 10<sup>5</sup>
/ml) in DMEM at pH 6.5 was dispensed into each well of 96-well plates (standard clear plates for nitric oxide assay and black-wall, clear-bottom plates for ROS and TNF-α assay) and incubated for 24 h at 37°C in 5% CO<sub>2</sub>
. Plates were washed with DMEM to remove nonadherent macrophages. <named-content content-type="genus-species">L. major</named-content>
at a 1:5 ratio (5 parasites per host cell) was then added to the wells, and the plates were incubated for 24 h at 34°C in 5% CO<sub>2</sub>
to allow infection of the adherent macrophages. After 24 h of incubation with macrophages, the infection rate was more than 80%. The effects of HMW chitosan on BMM activation at pH 6.5 were determined by quantifying the release of TNF-α, ROS, and NO as described below.</p>
<p><bold>(i) Measurement of TNF-α.</bold>
HMW chitosan at concentrations of 1.64, 4.9,14.8, 44.4, 133.3, and 400 μg/ml was added to infected and uninfected macrophages (see “Uptake of chitosan by macrophages” above), and the plates were incubated for 4 or 24 h at 34°C in 5% CO<sub>2</sub>
. Lipopolysaccharides from <named-content content-type="genus-species">Escherichia coli</named-content>
O26:B6 (LPS, 100 ng/ml; Sigma, UK) were used as a positive control and inducer. TNF-α release by the BMMs was measured using a mouse TNF-α enzyme-linked immunosorbent assay (ELISA) kit (product number ab208348; abcam, UK) according to the manufacturer’s instructions, using a SpectraMax M3 microplate reader (wavelength 450 nm).</p>
<p><bold>(ii) Measurement of ROS.</bold>
ROS was measured using a 2′,7′-dichlorofluorescein diacetate (DCFDA) cellular reactive oxygen species detection kit (abcam, UK). Uninfected and infected macrophages were treated with 25 μM DCFDA in PBS for 45 min at 37°C and then washed once in the buffer. The cells were cultured at 34°C in 5% CO<sub>2</sub>
for 0.5, 1, 2, 4, 8, and 24 h, with 1.64, 4.9,14.8, 44.4, 133.3, and 400 μg/ml of HMW chitosan or in the presence of H<sub>2</sub>
O<sub>2</sub>
(25 mM) (Thermofisher, UK) as a positive control, in DMEM plus 10% HIFCS (pH 6.5) in quadruplicate wells. In some experiments, cells were pretreated with a selective inhibitor of ROS, <italic>N</italic>
-acetyl-<sc>l</sc>
-cysteine (NAC) (5 mM; Sigma, UK) for 2 h before the addition of the inducer or chitosan. At 0.5, 1, 2, 4, 8, and 24 h, the plates were read, using a SpectraMax M3 microplate reader (excitation/emission at 485/535 nm).</p>
<p><bold>(iii) Measurement of NO.</bold>
NO was measured using Griess reagent (Thermofisher, UK). HMW chitosan at concentrations of 1.64, 4.9,14.8, 44.4, 133.3, and 400 μg/ml was added to infected and uninfected macrophages, and the plates were incubated at 34°C in 5% CO<sub>2</sub>
for 4 and 24 h. LPS (100 ng/ml) was used as a positive control. In some experiments, cells were pretreated with the selective inhibitor of nitric oxide N<sup>G</sup>
-methyl-<sc>l</sc>
-arginine acetate salt (L-NMMA) (0.4 mM; Sigma, UK) for 2 h before the addition of LPS. NO was quantified according to the kit protocol. Briefly, 150 μl of the cell culture supernatants (particulates were removed by centrifugation) was mixed gently with 150 μl of the Griess reagent in a 96-well plate and the mixture was incubated for 30 min at room temperature. The absorbance was measured using a SpectraMax M3 plate reader (wavelength 548 nm). Sodium nitrite (Sigma, UK) at different concentrations was used to create a standard curve (<xref rid="B68" ref-type="bibr">68</xref>
).</p>
</sec>
<sec id="s4.10"><title>Uptake of chitosan by macrophages.</title>
<p>The uptake of HMW chitosan was evaluated using two methods. The first method used two endocytosis inhibitors: cytochalasin D (1 μg/ml; Sigma, UK), which is a phagocytosis inhibitor, and dynasore (30 μg/ml; Sigma, UK), which inhibits pinocytosis (clathrin-mediated endocytosis [CME]) by blocking the GTPase activity of dynamin (<xref rid="B69" ref-type="bibr">69</xref>
<xref ref-type="bibr" rid="B70">–</xref>
<xref rid="B71" ref-type="bibr">71</xref>
). The second method used dynasore and rhodamine-labeled chitosan (200 kDa; Creative PEGWorks, USA) to track cellular uptake of chitosan over time by fluorescence microscopy.</p>
<p><bold>(i) Activity of chitosan after inhibition of the endocytic pathway of BMMs.</bold>
One hundred microliters of BMM culture (4 × 10<sup>5</sup>
/ml) in DMEM at pH 6.5 or pH 7.5 was dispensed into each well of 16-well LabTek culture slides and infected with stationary-phase <named-content content-type="genus-species">L. major</named-content>
promastigotes. Some of the infected BMMs were pretreated with dynasore (30 μg/ml) or cytochalasin D (1 μg/ml) for 2 h. Subsequently, HMW chitosan was added to each well at concentrations of 1.64, 4.9,14.8, 44.4, 133.3, and 400 μg/ml and macrophages were incubated for 4 or 24 h at 34°C in 5% CO<sub>2</sub>
. After each time point, the slides were examined as described above for the assay of chitosan activity against intracellular amastigotes of <named-content content-type="genus-species">L. major</named-content>
and <named-content content-type="genus-species">L. mexicana</named-content>
. The inhibition activity of the uptake (phagocytosis or pinocytosis) of the two inhibitors was evaluated on a fluorescence plate reader using fluorescent latex beads and pHrodo red dextran (<xref rid="B72" ref-type="bibr">72</xref>
). We showed that cytochalasin caused 94 and 84% inhibition of phagocytosis of fluorescent latex beads (Sigma-Aldrich, UK) after 4 h and 24 h, respectively, and dynasore caused 95 and 90% inhibition of pinocytosis of pHrodo red dextran (<italic>M</italic>
<sub>w</sub>
= 10,000; Thermo Fisher, UK) after 4 h and 24 h, respectively (Table S6 in the supplemental material).</p>
<p><bold>(ii) Microscopic imaging of the cellular uptake of rhodamine-labeled chitosan.</bold>
The qualitative characterization of chitosan uptake of cells was carried out by wide-field microscopy (Nikon Ti-E inverted microscope). Briefly, after deriving BMMs, 500 μl of the BMMs (in DMEM plus 10% HIFCS at pH 6.5, 4 × 10 <sup>4</sup>
macrophages per ml) was seeded in each well of a 4-well LabTek tissue culture slide (Thermo Fisher, UK) and incubated for 24 h at 37°C in 5% CO<sub>2</sub>
. Subsequently, 5 μg/ml of the nuclear dye Hoechst 33342 (excitation/emission at 350/461 nm; Thermofisher, UK) was added and the slides were incubated for 30 min at 37°C in 5% CO<sub>2</sub>
. The macrophages were washed with PBS, and then <named-content content-type="genus-species">L. major</named-content>
-GFP or <named-content content-type="genus-species">L. mexicana</named-content>
-GFP parasites were added at a ratio of 10:1 and further incubated for 24 h at 34°C in 5% CO<sub>2</sub>
(we used a 10:1 ratio, not 5:1 as described above, as in this experiment, different strains of <named-content content-type="genus-species">L. major</named-content>
-GFP and <named-content content-type="genus-species">L. mexicana</named-content>
-GFP were used and the ratio 10:1 was sufficient to obtain a high infection rate). Macrophages were then washed with PBS and 500 μl of LysoTracker deep red (50 nM, excitation/emission at 647/668 nm; Thermo Fisher, UK) was added to each well. The labeled, infected macrophages were then exposed to 30 μg/ml rhodamine-labeled chitosan (200 kDa; Creative PEGWorks, USA) in 500 μl of fresh DMEM plus 10% HIFCS, pH 6.5, and incubated for 4 and 24 h at 37°C with live imaging at each time point. In some experiments, infected BMMs were preincubated with dynasore 30 μg/ml for 2 h before adding rhodamine-labeled chitosan. All the images were collected using a Nikon Ti-E inverted microscope equipped with a 63× objective using Nikon Elements software. Three images for each experiment were then analyzed using ImageJ software. The degree of correlation between pixels in the red and green channels was assessed by using the Colocalization Colormap plugin in the ImageJ software. This plugin enables quantitative visualization of colocalization by calculating the normalized mean deviation product (nMDP) in a color nMDP scale (from −1 to 1): negative indexes (cold colors) refer to no colocalization, while indexes of more than 0 (hot colors) display colocalization and higher numbers refer to greater colocalization (<xref rid="B73" ref-type="bibr">73</xref>
, <xref rid="B74" ref-type="bibr">74</xref>
).</p>
</sec>
<sec id="s4.11"><title>Statistical analysis.</title>
<p>Dose-response curves and EC<sub>50</sub>
s were calculated using GraphPad Prism version 7.02 software, and the corresponding sigmoidal dose-response curves were established by using a nonlinear fit with variable slope models. Results are given as mean values ± SD. EC<sub>50</sub>
s were compared by using extra-sum-of-squares F tests. The <italic>t</italic>
test was used to compare differences between means of two or more groups, and <italic>P</italic>
values of 0.05 were considered statistically significant.</p>
</sec>
</sec>
<sec sec-type="supplementary-material"><title>Supplementary Material</title>
<supplementary-material id="PMC_1" content-type="local-data"><caption><title>Supplemental file 1</title>
</caption>
<media mimetype="application" mime-subtype="pdf" xlink:href="AAC.01772-19-s0001.pdf"></media>
</supplementary-material>
</sec>
</body>
<back><fn-group><fn fn-type="supplementary-material"><p>Supplemental material is available online only.</p>
</fn>
</fn-group>
<ack><title>ACKNOWLEDGMENTS</title>
<p>Alaa Riezk’s doctoral project received funding from the London School of Hygiene and Tropical Medicine (LSHTM) and the Council for At-Risk Academics (CARA, UK).</p>
<p>We are grateful to S. Somavarapu (UCL School of Pharmacy) and K. Van Bocxlaer (University of York) for supply of chemicals and helpful discussions. The authors acknowledge the facilities and the scientific and technical assistance of the LSHTM Wolfson Cell Biology Facility, with specific thanks to E. McCarthy.</p>
</ack>
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