Antigen Presenting Cells Targeting and Stimulation Potential of

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Antigen presenting cells targeting and stimulation potential of lipoteichoic acid functionalized lipo-polymerosome: A chemoimmunotherapeutic approach against intracellular infectious disease Pramod Kumar Gupta, Anil K. Jaiswal, Shalini Asthana, Anuradha Dube, and Prabhat Ranjan Mishra Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm5015156 • Publication Date (Web): 11 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Antigen presenting cells targeting and stimulation potential of lipoteichoic acid functionalized lipo-polymerosome: A chemo-immunotherapeutic approach against intracellular infectious disease

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Pramod K. Guptaa, Anil K. Jaiswalb, Shalini Asthanaa, Anuradha Dubeb and Prabhat R. Mishraa*

5 6

a

7 8

b

9 10

Pharmaceutics Division, Council of Scientific and Industrial Research-Central Drug Research Institute, B 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow, India 226031 Parasitology Division, Council of Scientific and Industrial Research-Central Drug Research Institute, B 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow, India 226031 Email: [email protected], [email protected], [email protected], [email protected], [email protected]

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* Corresponding Author

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Prabhat R. Mishraa Principal Scientist Pharmaceutics Division, Preclinical South PCS 002/011, CSIR-Central Drug Research Institute B 10/1, Sector-10, Jankipuram Extension, Sitapur Road, Lucknow-226031, India Phone :91-522-2771940; 2771942 Fax: 91-522-2771941

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Abstract

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Antigen presenting cells (APC) are well-recognized therapeutic targets for intracellular

35

infectious diseases including visceral leishmaniasis. These targets have raised concerns regarding

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their potential for drug delivery due to over expression of a variety of receptors for pathogen

37

associated molecular pathways after infection. Since, Lipoteichoic acid (LTA), a surface

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glycolipid of gram-positive bacteria responsible for recognition of bacteria by APC receptors

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which also regulate their activation for pro-inflammatory cytokine secretion, provides additive

40

and significant protection against parasite. Here, we report the nanoarchitechture of APC focused

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LTA functionalized Amphotericin B encapsulated lipo-polymerosome (LTA-AmB-L-Psome)

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delivery system mediated by self assembly of synthesized glycol chitosan-stearic acid copolymer

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(GC-SA) and cholesterol lipid, which can activate and target the chemotherapeutic agents to

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Leishmania parasite resident APC. Greater J774A and RAW264.7 macrophage internalization of

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FITC tagged LTA-AmB-L-Psome compared to core AmB-L-Psome was observed by

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FACSCalibur cytometer assessment. This was further confirmed by higher accumulation in

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macrophage rich liver, lung and spleen during biodistribution study. The LTA-AmB-L-Psome

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overcame encapsulated drug toxicity and significantly increased parasite growth inhibition

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beyond commercial AmB treatment in both in vitro (macrophage-amastigote system; IC50,

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0.082±0.009 µg/mL) and in vivo (Leishmania donovani-infected hamsters; 89.25±6.44% parasite

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inhibition) models. Moreover, LTA-AmB-L-Psome stimulated the production of protective

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cytokines like interferon-γ (IFN-γ), interleukin-12 (IL-12), tumor necrosis factor-α (TNF-α) and,

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inducible nitric oxide synthase and nitric oxide with down-regulation of disease susceptible

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cytokines like transforming growth factor-β (TGF-β), IL-10 and IL-4. These data demonstrate

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the potential use of LTA functionalized lipo-polymerosome as a biocompatible lucrative 2


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nanotherapeutic platform for overcoming toxicity and improving drug efficacy along with

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induction of robust APC immune responses for effective therapeutics of intracellular diseases.

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Keywords: Leishmania, amastigotes, cytokines, lipo-polymerosome, biodistribution, toxicity

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1. Introduction

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The professional antigen presenting cells (APC), i.e. monocytes/macrophages, B-cells and

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dendritic cells are major port of survival of parasites into the body and plays an important role in

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disease progression 1. The intracellular parasite utilizes immune evasion mechanisms by down-

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regulation of parasite antigen presentation via the MHC class II antigen pathway and prevents

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activation of oxidative bursts and other microbicidal mechanisms

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eruption of many diseases including tuberculosis, leishmaniasis, cryptococcosis and filariasis.

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Despite numerous advances over the past decades, effective intra-APC disease chemotherapy

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remains challenging. The generalized toxicity of many chemotherapeutic drugs makes it difficult

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to achieve therapeutic concentrations without systemic side effects. Consequently, architecture of

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such drug delivery system is essential that preferentially recognize and bind to specific over

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expressed receptors on infected APC surface, with the hope that the delivery system will be

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internalized and release its contents specifically in to the cell 4. Such drug vehicles could

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potentially open the door to new therapeutic paradigms for practicing medicine that promises to

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eradicate a range of parasitic diseases. Additionally, the toxicity related issues of drug can be

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reduced by targeted delivery to intracellular regions of APC.

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Taking together the immuno-stimulatory tendency of bacteria and its forced engulfment by APC,

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bacterial antigen can be utilized as a component of drug delivery system that will have artificial

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bacteria like identity. Importantly, such delivery system suites as a platform for biomarker

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specific ligand display and affinity selection, raising the possibility that a single vehicle can be

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used both for identification of cell-targeting and immunostimulatory ligand for specific delivery

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of drug.

2, 3

. These events lead to

4


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APC expresses a variety of receptors for pathogen associated molecular pathways (PAMPs) such

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as lipopolysaccharides, glycoproteins and a range of polyanionic molecules, which get over

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expressed after infection. These enable the recognition and phagocytosis of large repertoire of

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pathogenic parasites and subsequent generation of inflammation to eradicate the infection.

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Since, Lipoteichoic acid (LTA) is a common immunostimulatory polyanionic cell-wall

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component of Staphylococci and many gram-positive bacteria, composed of poly(glycerol

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phosphate) attached to the glycolipid anchor β-D -GlcpII-(1→6)-β-D-GlcpI-(1→3)-diacylglycerol

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5, 6

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create a negatively charged network which extends from the membrane to the surface of the

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bacterium and is recognized by involvement of scavenger receptor (SR) particularly type 1 7,

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together with Toll-like receptors (TLRs) especially TLR2 and TLR6, and its co-receptors (CD14,

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CD36), of APC which results in induction of cytokines and co-stimulatory molecules for antigen

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presentation that can modulate T cell polarization. TLRs specific ligands have ability to enhance

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drug efficacy through multiple mechanisms: TLRs stimulation of APC results in increase in

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surface peptide/MHC complexes, co-stimulatory molecules, and cytokine secretion, the three

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signals required for T cell activation and proliferation 8. Additionally, LTA bound to APC can

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interact with circulating antibodies and activate the complement cascade to induce passive

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immune kill phenomenon 9. The rationale for developing this prototype formulation i.e.

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Lipoteichoic acid modified lipopolymerosome (LTA-AmB-L-Psome) is to target the carrier

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system to Antigen Presenting Cells (APCs) which are actually reservoir of Leishmania parasites

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and finally to impart immunostimulatory potential (being component of bacterial cell wall) to

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carrier system so as to evoke Th1 mediated killing of Leishmania parasites. This dual strategy is

. Because of their intrachain phosphodiester groups, LTA collectively with teichoic acids

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likely to improve pharmacokinetics and reduce toxicity to a greater extent by preventing AmB

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exposure to other organs.

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Previously, we have reported Lipo-polymerosome (L-Psome) by spontaneous self-assembly of

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synthesized glycol chitosan-stearic acid copolymer as stable Amphotericin B (AmB) delivery

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system able to reduce toxicity issues 10. In the present study, we have demonstrated better AmB

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chemotherapy for leishmaniasis through immuno-modulation using LTA as specific ligand

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anchored on lipo-polymerosome (LTA-AmB-L-Psome). Furthermore, the developed formulation

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has potential for its application in the condition of VL and intra-APC diseases.

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2. Material and methods

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2.1. Materials

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Glycol

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(dimethylamino)propyl]carbodiimide hydrochloride (EDC), Dimethyl acetamide (DMAc)

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sodium deoxycholate (SDC), stearic acid (SA), cholesterol, fluorescein isothiocynate (FITC),

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Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640

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medium, streptomycin, fetal bovine serum (FBS) and LTA from Bacillus subtilis were purchased

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from Sigma-Aldrich (St. Louis, MO). Water used in all the experiments was obtained by Milli-Q

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Plus 185 water purification system. Methanol and acetonitrile of HPLC grade were provided by

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SD Fine Chem Ltd (Mumbai, India). AmB was kindly provided as a gift sample from Emcure

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pharmaceutical Ltd. (Pune, India). All other reagents were of analytical grade.

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Parasite and cell line culture

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The WHO reference strain of L. donovani (MHOM/IN/80/Dd8) was used for both in vitro and in

146

vivo experiments. Leishmania parasites and the macrophage cell line J774A, RAW264.7 were

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maintained in RPMI-1640 medium (Sigma, USA) supplemented with 10% heat inactivated fetal

chitosan

(GC,

Mw

90

KD,

82.7%

degree

of

deacetylation),

[1-ethyl-3

6


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bovine serum (HIFBS), 100 U/ml penicillin and 100 µg/ml streptomycin at 37 oC in humidified

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atmosphere of 5% (v/v) CO2/air mixture.

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Animals and ethics statements

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For in vivo antileishmanial study, Syrian golden male hamsters (Mesocricetus auratus, 45-50 g),

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for pharmacokinetic and bio-distribution study, male Wistar rats (180-200 g) animal models

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were used while biochemical analysis and tissue histopathology performed in male Swiss mice

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(18-20 g) with prior approval of the Animal Ethics Committee of CSIR-Central Drug Research

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Institute and according to regulations of the Council for the Purpose of Control and Supervision

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of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Govt. of

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India. The Institutional Animal Ethics Committee (IAEC) approval no. is CDRI/2012/38.

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2.2. Preparation of lipoteichoic acid modified lipo-polymerosome (LTA-AmB-L-Psome)

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The core self assembled L-Psome formulation was prepared through a nano-precipitation method

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followed by sonication as illustrated in our previous work

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synthesized glycol chitosan-stearate copolymer through carbodiimide coupling reaction between

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glycol chitosan and stearic acid in molar ratio of 4:1.

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Thereafter, synthesized copolymer (4 mg), AmB (1 mg in 100 µL of DMAc), and cholesterol (1

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mg) was taken in 250 µL of ethanol and were mixed gently. This solution added drop wise in 1

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mL of distilled water under constant stirring for 5 min in 5 mL vial. Obtained dispersion was

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sonicated using probe sonicator (misonix, Germany) at 20% amplitude for 120 sec (15 sec pulse

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on and 5 sec pulse off), followed by dialysis against 1000 mL water for 2 h for the purpose to

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remove un-entrapped AmB and solvents.

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In order to prepare LTA-AmB-L-Psome, the exterior surface of the L-Psome was modified using

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LTA ligand by adsorption method. For this purpose, 5 mg L-Psome was incubated with 1 mL

10

. For this purpose, firstly, we

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LTA solution (at different concentrations from 0.02 to 0.4% w/v) in 10 mM HEPES buffer (pH

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7.4) at 37 oC for 24 h to allow LTA to become electrostatically anchor onto L-Psome surface.

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The excess LTA was removed by washing followed by centrifugation of the resultant

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suspension, and the whole process was repeated thrice to ensure complete removal. The LTA

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amount was optimized by analyzing zeta potential change in LTA-L-Psome dispersion at 25 oC

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as shown in Fig. 1(a).

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2.3. Physicochemical Characterization of L-Psome

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The mean particle size, size distribution and zeta potential of the L-Psome were determined with

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a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) at 25 oC after dilution in

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distilled water.

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The surface morphology of optimized LTA-L-Psome was ascertained using high resolution

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transmission electron microscopy (HR-TEM, Tecnai™ G2 F20, Eindhoven, The Netharlands) 10.

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To quantify LTA anchoring to L-Psome, BCA method for direct estimation of protein was

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performed. In this method, initially LTA decorated L-Psome eluted from sephadax G100 silica

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Gel column then measured by NANODROP 2000 spectrophotometer (Thermo Scientific, USA)

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11-14

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% Conjugation efficiency = Conjugated wt. of LTA × 100 / Total wt. of L-Psome

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The solvent injection method was utilized to load the AmB within LTA-L-Psome, for this AmB

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solution in DMAc was added in LTA-L-Psome with constant stirring. The AmB amount

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encapsulated in LTA-L-Psome was quantified as the amount of un-entrapped drug recovered in

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the supernatant by reverse phase HPLC

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subsequent washings of the formulation. The percentage of drug loading, encapsulation

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efficiency (EE) was calculated using following formulae:

. The coupling efficiency was expressed as the percentage of LTA bound to L-Psome surface

15

after ultracentrifugation at 40,000×g for 30 min and

8


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Loading efficiency = Wt. of (Total – free) AmB in L-Psome × 100/ Wt. of L-Psome

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Encapsulation efficiency = Wt. of (Total – free) AmB in L-Psome × 100/ Wt. of Total AmB

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The release of AmB from formulations was assessed using a dialysis membrane under sink

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conditions. Herein, 2 mg AmB equivalent formulations (LTA-AmB-L-Psome AmB-L-Psome,

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Fungizone, and Ambisome) was kept in dialysis bag, sealed and placed in 200 ml of release

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medium with 2% w/v SDC, under continuous shaking at 100 rpm at 37 °C. A definite volume of

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the release medium was withdrawn at regular time intervals (30 min and 1, 2, 3, 4, 6, 8, 12, 24,

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48, 72 h) and replaced with equal volume of fresh medium. Amount of the AmB in the collected

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samples was analyzed using described HPLC method.

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To understand stability of LTA-AmB-L-Psome, stability study was carried out at different time

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points (1, 7, 15, 30, 90 and 180 days) in PBS at 2-8 0C, 37 0C and room temperature along with

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other formulations i.e. AmB-L-Psome, Ambisome and Fungizone. At each time point, an aliquot

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of each formulation was withdrawn for analysis to observe any change in physicochemical

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parameters such as size and EE of the formulation 10.

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Furthermore, rigid characteristic of LTA-AmB-L-Psome was evaluated through HRTEM study.

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We have observed change in surface morphology of vesicles after storage period of 6 months

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with 10 % w/v BSA and 10 % v/v Wistar rat plasma at 2-8 0C. In continuation to this, rigidity of

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LTA-AmB-L-Psome was also evaluated by analyzing size and PDI variation, when incubated

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with 10 % w/v BSA and 10 % v/v rat plasma at RT under gentle stirring at a concentration of 1

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mg AmB/mL. At predetermined intervals up to 60 min sample was taken out and measurements

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were performed in triplicate.

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2.4. Tagging of AmB with FITC 9


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For in vitro uptake studies, AmB was tagged with FITC as previously reported method

.

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Briefly, AmB (10 mg) and FITC (5 mg) were dissolved in DMAc (2 mL) followed by addition of

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200 µL triethylamine as base catalyst in 5 mL round bottom flask, followed by 2 h stirring at

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room temperature and thereafter 10 mL ethyl acetate was added to precipitate the compound.

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Colloidal precipitate was separated by centrifugation (18,000×g for 10 min) and dried over

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desiccant under vacuum. In order to verify tagging of FITC with AmB (FAmB) TLC analysis

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was carried out using a mobile phase composed of ethyl acetate: methanol (2:3).

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2.5. In vitro uptake by macrophages

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FAmB was encapsulated in the LTA-L-Psome using same procedure as described earlier, in

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short, 1 mg of tagged FAmB was dissolved in 100 µL of DMAc and loaded drop wise in blank

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LTA-L-Psome. Infected J774A and RAW264.7 macrophage cells in 2×105 cells/well were

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placed separately on to 12-well cluster dish and cultivated in 800 µL DMEM supplemented with

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10% FBS and 1% antibiotic and antimycotic. After 24 h the culture medium was replaced with

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fresh culture medium. Cells were incubated for 6 h at 37 oC and 5% CO2 with FITC tagged

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formulations (LTA-FAmB-L-Psome and FAmB-L-Psome) at 10 µg/mL concentration. After

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incubation, cells were transferred in vials and relative fluorescence was measured by

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FACSCalibur (Becton Dickinson, Oxford, UK) at λEX (495 nm) and λEM (525 nm). FACSCalibur

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mediated macrophage uptake study was also carried out for non-infected as well as LTA

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saturated infected J774A and RAW264.7 macrophage cells.

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Macrophage uptake was also assessed by confocal laser scanning microscopy (CLSM). J774A

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cells seeded at a density of 1×105 cells/well on poly-L-lysine-coated glass cover slips (in six-well

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plates), were left for 12 h and then incubated for 4 h with LTA-FAmB-L-Psome and FAmB-L-

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Psome at room temperature in the dark followed by repeated washing and fixed in 10% formalin 10


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in PBS and observed by CLSM (Carl Zeiss LSM5100, Germany) equipped with ×40 objective

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lens. λEX (495 nm) and λEM (525 nm) of FITC were used to analyze tagged AmB.

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2.6. Pharmacokinetic and Bio-distribution study

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Wistar rats (180-200 g) were allocated into the following four treatment groups: IV bolus

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administration of 5 mg/kg AmB-L-Psome (n=6), 5 mg/kg LTA-AmB-L-Psome (n=6), 1 mg/kg

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Fungizone (n=6) and 5 mg/kg AmBisome (n=6). The doses selected for IV administration of

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commercial AmB formulations in present study are in the range for which there is no clinical

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nephrotoxicity for each formulation, based on published reports

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was sampled at 10 min pre-dose and 0.5, 1, 2, 3, 5, 8, 12, 24, 48 and 72 h post-dose of IV bolus

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administrations of AmB formulations, followed by separation of plasma by centrifugation at

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2,500×g for 10 min at 15 oC). The animals were sacrificed at 0.5, 1, 2, 3, 5, 8, 12, 24, 48 and 72

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h post administration of AmB formulations, and liver, spleen, right lung, right kidney and heart

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were harvested for drug analysis. Plasma and tissue samples were stored at -80 oC until drug

252

analysis.

16

. Systemic blood (0.25 mL)

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Samples were analyzed using Shimadzu HPLC system equipped with 10 ATVP binary

254

gradient pumps (Shimadzu, Japan), a Rheodyne model 7125 injector (CA, US) with a 20 µL loop

255

and SPD-M10 AVP UV detector (Shimadzu, Japan). AmB in plasma and tissue samples was

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analyzed following published validated isocratic HPLC method with slight modifications,15

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using column [LichroCART® 250-4, Lichrospher® 100, RP-18e, 5 µm, 250×4 mm (Merck

258

KgaA, 64271 Darmstadt, Germany)] and mobile phase [acetonitrile/10mM KH2PO4 buffer, pH 4

259

(60:40, v/v)] at a flow rate of 1 mL/min. Tissue samples were homogenized in acetonitrile (1 g

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tissue per 2 mL acetonitrile) using IKA T25 digital ULTRA-TURRAX for 3-4 min over an ice

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bath. The filtered aliquots of 20 µL of processed plasma and tissues were used for quantitative 11


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analysis. Calibration curves of AmB were linear in the range of 0-10 µg/mL for plasma and 0-10

263

µg/g for tissue samples. The AmB quantification limit for plasma was 20 ng/mL and for tissue

264

samples was 20 ng/g.

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2.7. In vitro anti-amastigote activity

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The activity of AmB-formulations against intracellular amastigotes was evaluated as per protocol

267

described earlier

268

infected with green fluorescent protein (GFP) expressed promastigotes at multiplicity of 10

269

parasites per macrophage. After 12 h incubation 24-well plates were washed thrice with PBS (pH

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7.4) to remove non-phagocytosed promastigotes and re-supplemented with RPMI-1640 complete

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medium, followed by incubation with LTA-AmB-L-Psome, AmB-L-Psome, Ambisome and

272

Fungizone at different drug concentrations, and with the same amount of blank formulations in

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triplicate. After 48 h incubation, cells were removed, washed in PBS and quantitated by

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FACSCalibur cytometer equipped with a 20 mW argon laser at 488 nm excitation and 515 nm

275

emission followed by data analysis using Kaluza analysis software (Beckman Coulter). The

276

parasite growth inhibition was determined by relative fluorescence levels of drug-treated

277

parasites with that of untreated control parasites and the inhibitory concentrations (IC50 and IC90)

278

were calculated using GraphPad Prism6.

279

2.8. In vivo assay in L. donovani infected hamsters

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The in vivo AmB-formulation efficacy was studied against L. donovani amastigotes in a golden

281

hamster model

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intraperitoneally dosed 1 mg/kg of LTA-AmB-L-Psome, AmB-L-Psome, Fungizone and

283

Ambisome each for 5 consecutive days. Two week post treatment, treated animals were

284

sacrificed and results were compared with the infected untreated control group. Detection of

10

17

. Briefly, J774A macrophages (1×105 cells/well) in 24-well plates were

. After 30 days of established infection, hamsters (n = 5 in each group) were

12


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parasitic load in spleen was carried out by blinded microscopic enumeration with Giemsa-stained

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spleen impression smear. Parasite burdens were calculated by counting the number of

287

amastigotes per 100 macrophage nuclei. The percentage of inhibition (PI) was calculated using

288

the formula:

289

PI= (PP-PT/PP) ×100

290

Where PP and PT are number of amastigotes per 100 macrophage nuclei before and after

291

treatment, respectively.

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2.9. Nitric oxide (NO) determination

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NO content in the splenocytes culture supernatants of treated hamsters was monitored by Griess

294

assay method

295

sulfanilamide 1N HCl and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in H2O] were

296

mixed at 1:1 ratio and incubated for 15 min at room temperature, and the absorbance was

297

determined at 540 nm by microplate reader (DTX 800 multimode detector, Beckman Coulter).

298

Nitrite concentration was calculated with a sodium nitrite standard curve generated for each

299

experiment.

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2.10. Evaluation of Th1/Th2 cytokines and chemokine

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Splenic cell smear of treated and untreated control groups were analyzed for various cytokines

302

(TNF-α, IL-12, IFN-γ, IL-4, IL-10 and TGF-β) and inducible nitric oxide synthase (iNOS) using

303

quantitative real time-PCR (qRT-PCR) technique. Splenocytes mRNA of different hamster

304

groups was isolated using Tri reagent (Invitrogen, USA) followed by cDNA synthesis using first

305

strand cDNA synthesis kit (Fermentas, USA) as per manufacturer’s protocol. qRT-PCR was

306

conducted with initial denaturation at 95 °C for 2 min followed by 40 cycles, each consisting of

307

denaturation at 95 °C for 30s, annealing at 55 °C for 40 s, and extension at 72 °C for 40 s per

18

. Briefly, the culture supernatant and the mixture of Greiss reagent [1%

13


Page 15 of 43

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Biomacromolecules

308

cycle using the iQ5 multicolor real-time PCR system (Bio-Rad, USA). Comparator samples were

309

the cDNAs from infected hamsters. The PCR signals quantification was performed by comparing

310

the cycle threshold (CT) value of the gene of interest with that of reference gene hypoxanthine

311

phosphoribosyltransferase (HPRT). Results were expressed as fold change (FC) of mRNA

312

relative to those in un-stimulated cells.

313

2.11. Toxicity assay

314

The hemolysis and J774A cell cytotoxicity assay was performed in 5 to 20 µg/ml AmB

315

concentrations of LTA-AmB-L-Psome, AmB-L-Psome and their equivalent blank formulations

316

using methods reported previously 15. Subacute toxicity assay was performed in five mice groups

317

(n=3), received LTA-AmB-L-Psome (5mg/kg), AmB-L-Psome (5mg/kg), Ambisome (5mg/kg),

318

Fungizone (1mg/kg) and saline (control group) intravenously in a constant volume of 200 µL

319

daily for 15 days. Twelve hours post treatment, animals were euthanized, and blood was

320

collected by cardiac puncture, centrifuged (2000 × g) and subsequently analyzed for biochemical

321

parameters. Transaminase activities, urea and creatinine were determined using an automatic

322

Hitachi 912 apparatus (Roche Diagnostics Corporation, Indianapolis, IN, USA). Kidney, liver,

323

spleen, lung, heart and colon tissues were collected for histopathological studies as reported in

324

our previous publication 10.

325

2.12. Statistical analysis

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All results are presented as mean±standard deviation (S.D.) of three independent measurements.

327

Data were analyzed by one-way analysis of variance (ANOVA) and p value of

Antigen Presenting Cells Targeting and Stimulation Potential of

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