Hyaluronic Acid-Based Biocompatible Supramolecular Assembly for Sustained Release of Antiretroviral Drug

Journal of Pharmaceutical Sciences xxx (2016) 1e10

Contents lists available at ScienceDirect

Journal of Pharmaceutical Sciences journal homepage: www.jpharmsci.org

Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Hyaluronic Acid-Based Biocompatible Supramolecular Assembly for Sustained Release of Antiretroviral Drug n Puska s 2, Lajos Szente 2, James E.K. Hildreth 1, 3 Byeongwoon Song 1, *, Istva 1

Department of Molecular and Cellular Biology, University of California, Davis, California 95616 CycloLab, Budapest H-1097, Hungary 3 Department of Internal Medicine, Meharry Medical College, Nashville, Tennessee 37208 2

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 December 2015 Revised 14 January 2016 Accepted 27 January 2016

Human immunodeficiency virus (HIV) infection and its associated diseases continue to increase despite the progress in our understanding of HIV biology and the availability of a number of antiretroviral drugs. Adherence is a significant factor in the success of HIV therapy and current HIV treatment regimens require a combination of antiviral drugs to be taken at least daily for the remainder of a patient’s life. A drug delivery system that allows sustained drug delivery could reduce the medical burden and costs associated with medication nonadherence. Here, we describe a novel supramolecular assembly or matrix that contains an anionic polymer hyaluronic acid, cationic polymer poly-L-lysine, and anionic oligosaccharide sulfobutylether-beta-cyclodextrin. HIV reverse transcriptase inhibitors Zidovudine and Lamivudine were successfully encapsulated into the polymer assembly in a noncovalent manner. The physicochemical properties and antiviral activity of the polymer assemblies were studied. The results of this study suggest that the supramolecular assemblies loaded with HIV drugs exert potent antiviral activity and allow sustained drug release. A novel drug delivery formulation such as the one described here could facilitate our efforts to reduce the morbidity and mortality associated with HIV infections and could be utilized in the design of therapeutic approaches for other diseases. © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Keywords: biodegradable polymers controlled release cyclodextrins HIV/AIDS macromolecular drug delivery polymeric drug delivery systems

Introduction The global AIDS pandemic continues to expand despite significant advances in the understanding of human immunodeficiency virus (HIV-1) pathogenesis and the development of highly effective antiviral drugs. Currently, more than 36 million people are infected with HIV-1 worldwide and approximately 2 million more people are infected each year. Effective treatment of HIV-infected individuals requires strict adherence to a multicomponent regimen of antiretroviral agents that must be taken at least daily for the remainder of a patient’s life. Success of the HIV treatment regimen

Abbreviations: API, active pharmaceutical ingredient; CD, cyclodextrin; ELISA, enzyme-linked immunosorbent assay; HA, hyaluronic acid; HIV, human immunodeficiency virus; PBMC, peripheral blood mononuclear cell; PBS, phosphate buffered saline; PL, poly-l-lysine; RLU, relative light unit; SBECD, sulfobutyletherbeta-cyclodextrin. This article contains supplementary material available from the authors by request or via the Internet at http://dx.doi.org/10.1016/j.xphs.2016.01.023. * Correspondence to: Byeongwoon Song (Telephone: þ1-530-752-3628; Fax: þ1530-752-3085). E-mail address: [email protected] (B. Song).

is heavily dependent on patient adherence. This is also true for prevention strategies that utilize antiviral chemotherapy. Nonadherence can lead to emergence of drug resistance and loss of therapeutic effectiveness. Effective prevention requires that the inhibitor be present at the right time, place, duration, and concentration to stop HIV transmission and acquisition. A drug delivery system with the capability of sustained or controlled drug release will significantly reduce the medical burden and costs associated with medication nonadherence. In recent years, the application of supramolecular assembly and nanoparticle technology for optimizing the pharmacologic and therapeutic profiles of existing drugs has gained momentum, thus contributing to enhanced stability, prolonged circulation time, specific delivery to the target tissue, and controlled release of the active pharmaceutical ingredients (APIs).1 Supramolecular assemblies are produced from polymeric precursors, and these structures can be formulated to encapsulate a wide variety of pharmaceuticals.2 The use of natural or biocompatible molecules such as hyaluronic acid (HA) in the design of nanoparticles or polymeric assemblies is increasing because these molecules are biodegradable and are associated with limited, if any, adverse immune responses.

http://dx.doi.org/10.1016/j.xphs.2016.01.023 0022-3549/© 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

2

B. Song et al. / Journal of Pharmaceutical Sciences xxx (2016) 1e10

HA, an anionic polymer of disaccharides, composed of D-glucuronic acid and D-N-acetylglucosamine, is one of the major components of the extracellular matrix and plays a critical role in cell proliferation and migration.3 Proteoglycan aggregates in the extracellular matrix of connective tissues are composed of a long chain of HA as a core surrounded by proteoglycans coupled noncovalently by linker proteins.4 HA and the proteoglycans are both highly negatively charged and provide electrostatic interactions with the basic residues in the linker protein. A recent study suggested that HA-based hydrogels can be successfully exploited as a three-dimensional (3D) artificial extracellular matrix for various tissue engineering applications.5 Polylysines (PLs) are generally regarded as safe by the United States Food and Drug Administration (FDA) and are widely utilized as carriers in a number of applications such as foods and pharmaceuticals. Warren et al.6 have recently demonstrated that PL can be utilized in the design of biodegradable nanogel carriers for the delivery of the nucleoside reverse transcriptase inhibitors. Cyclodextrins (CDs) form inclusion complexes with a wide range of compounds and have been used as carriers in pharmaceutical applications to increase aqueous solubility, dissolution rate, and chemical stability of drugs, as well as to enhance drug permeability through biological membranes.7 CD polymers have also been used in forming complex multicomponent drug delivery systems such as nanoparticles and micelles, and currently two drug delivery systems based on CD polymers have entered clinical phase II trials in cancer treatment.8 In this regard, it is important to note that exposure of HIV to betaCD has been shown to inactivate the virus by depletion of cholesterol.9-11 Based on the analogy of the controlled release of histamine and heparin from proteoglycan aggregates in living tissues during inflammation and allergic reactions,12 Szente et al.13 have developed a supramolecular assembly that includes the anionic polymer HA as a backbone and CD complexed with a cationic surfactant as a linker. This biodegradable polymer assembly was shown to be capable of entrapping both hydrophilic and lipophilic substances and exhibited sustained drug release in vitro.13 In the studies performed herein, we report an HA-based supramolecular assembly as a sustained release system for antiretroviral drugs. To this end, we constructed a supramolecular HA/PL/ CD assembly loaded with an HIV reverse transcriptase inhibitor (Zidovudine or Lamivudine). The physicochemical properties and antiviral activity of the supramolecular assemblies were examined. Our data suggest that the drug-loaded polymer assemblies exhibit potent antiviral activity and allow sustained drug release. Materials and Methods Materials HIV reverse transcriptase inhibitors Zidovudine and Lamivudine were purchased from Sigma-Aldrich (St. Louis, MO). HA was obtained from Pentapharm (Basel, Switzerland; Lot No. H12990001/ 295-03). Sulfobutylether-beta-cyclodextrin-Na salt, USP pharmaceutical grade (Dexolve7), is a product of CycloLab (Budapest, Hungary). Poly-L-lysine was obtained from JNC Corporation (Tokyo, Japan; Lot No. 21450203). Luviquat-Mono-CP was purchased from Fluka/Sigma-Aldrich (Lot No. 4168893/1). A variety of media that included Dulbecco’s modified Eagle’s medium, RPMI 1640 medium, fetal bovine serum, and penicillinstreptomycin were purchased from ThermoFisher Scientific (Waltham, MA). Phytohemagglutinin-M (PHA-M) and interleukin-2 (IL2) were obtained from Sigma-Aldrich. The CellTiter 96® AQueous One Solution Cell Proliferation Assay Kit and Luciferase Assay System were purchased from Promega (Madison, WI).

Cells and Viruses TZM-bl cells are HeLa-derived HIV-1 reporter cells that express the HIV-1 receptor CD4, the co-receptors CXCR4/CCR5, and the HIV-1 long terminal repeat (LTR)-driven firefly luciferase.14 JLTRG cells are T-lymphocyte-derived HIV-1 reporter cells that express the HIV-1 receptor CD4, the co-receptors CXCR4/CCR5, and the HIV1 LTR-driven enhanced green fluorescent protein (EGFP).15 The TZM-bl and JLTRG cell lines were obtained from the NIH AIDS Reagent Program (USA). The T-lymphocyte-derived Jurkat cell line was obtained from the American Type Culture Collection. The TZMbl cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum and 100 U/mL of penicillin-streptomycin at 37 C in a 5% CO2 humidified incubator. The JLTRG and Jurkat cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 100 U/mL of penicillin-streptomycin at 37 C in a 5% CO2 humidified incubator. Human peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque centrifugation from the whole blood obtained from the UCLA CFAR Virology Core Laboratory and cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 100 U/mL of penicillinstreptomycin as described.16 The HIV-1IIIB (CXCR4-tropic) and HIV-1BaL (CCR5-tropic) stocks were purified from the Jurkat-IIIB and PM1-BaL cell lines that have been chronically infected with HIV-1IIIB and HIV-1BaL, respectively. The culture supernatant containing virus was filtered using a 0.45mm filter and the cleared supernatant was subjected to centrifugation for 2 h at 100,000  g. The virus pellets were resuspended in a small volume of phosphate buffered saline (PBS) to achieve a 100-fold concentration. The purified virus stock was aliquoted in small volumes and stored at 80 C. The concentration of the virus stock was determined by assaying the capsid protein p24 by enzyme-linked immunosorbent assay (ELISA) and for infectivity using the TZM-bl cell line. Preparation of Supramolecular Assemblies For Zidovudine/HA/PL/SBECD (sulfobutylether-beta-cyclodextrin) assembly, 8.3 g of SBECD was dissolved in 10 mL of deionized water. To this solution, 0.09 g of poly-L-lysine was added and the resulting solution was stirred at room temperature together with 1.02 g of Zidovudine. The system became slightly opalescent suspension during stirring. Finally, 0.2 g of HA was added and the reaction mixture was stirred for 4 h at room temperature resulting in a slightly opalescent gel. The gel was chilled to 70 C and water was removed by freeze-drying. This procedure resulted in a yield of 9.3 g of white amorphous powder with a Zidovudine content of 10.9% by weight. The same procedure was used for the Lamivudine/ HA/PL/SBECD assembly. In other assemblies, the cationic surfactant hexadecyl(2-hydroxyethyl)dimethylammonium dihydrogen phosphate (also known as Luviquat-Mono-CP) was used instead of poly-L-lysine under the same reaction conditions. Size-Exclusion High-Performance Liquid Chromatography High-performance liquid chromatography (HPLC) measurements were carried out using a Hewlett-Packard 1050 System equipped with ERC-7515B refractive index detector and HewlettPackard 1050 variable wavelength (VW) detector. TSK-GEL G3000SW, silica gelebased column (TosoHaas) (300  7.5 mm), and TSK-GEL SW (75  7.5 mm) guard column were applied. The column temperature was set to 30 C. As an eluent, a water-tomethanol (65:35) mixture containing 1% NaCl was used with a flow rate of 1.0 mL/min. The refractive index detector temperature was

B. Song et al. / Journal of Pharmaceutical Sciences xxx (2016) 1e10

set to 40 C (Fast mode). A VW detector wavelength of 204 nm for HA was applied throughout the studies. Capillary Electrophoresis The capillary electrophoresis (CE) analyses were performed using a Hewlett Packard 3DCE system with Chemstation 6.03 software. The running buffer consisted of 50 mM disodium hydrogen phosphate, 40 mM sodium dodecyl sulfate, and 10 mM disodium tetraborate adjusted to pH 9 with 1 N NaOH solution. A fused silica capillary was used: 75 mm internal diameter, 375 mm outside diameter, 50 cm effective length, and 58.5 cm total length. The electrophoretic parameters used include a voltage of 15 kV and a capillary temperature of 30 C. The electrokinetic injection was conducted at 30 kV in 15 s. Raman Microanalysis Raman mapping spectra were collected using a Horiba JobinYvon LabRAM system coupled with an external 532-nm frequency-doubled ND-YAG laser source and an Olympus BX-40 optical microscope. An objective of 10 magnification was used for optical imaging and spectral acquisition. All samples were investigated in powder form, without any special sample preparation method. The spectra were obtained in the spectral range of 280-1750 cm1 with approximately 3 cm1 resolution. Raman maps were collected with 10 objective (laser spot size: ~3 mm) and 40 mm step size. The measured area varied from 0.8 mm  0.8 mm to 1 mm  1 mm. The reference Raman spectra of the pure antiviral agents were collected with a 100 objective using sufficient acquisition times to achieve adequate signal-to-noise ratio. X-Ray Diffraction The X-ray powder diffraction investigations were performed using a standard normal Cu K-alpha radiation. The reflection peaks were registered in the 2-theta angle range of 5 -40 at room temperature. Differential Scanning Calorimetry The heat flow curves were registered in an Argon atmosphere on a DuPont Thermal Analyzer recording heat flow curves in the temperature range of 300-500 Kelvin. In Vitro Drug Release An in vitro drug release study was conducted using the “dispersed amount technique” as described previously.17 Solid supramolecular polymer-drug formulation was weighted into a temperature-controlled (25 C or 37 C) glass vessel containing either deionized water or 0.9% sodium chloride as dissolution media. Although the system was continuously stirred at 60 rotations/min for 2 days, samples were withdrawn at 0, 1, 4, 8, 12, 24, and 48 h. The samples were filtered across a 0.45-mm pore size filter and the cleared samples were assayed for released drug by HPLC/ UV system, based on previous calibration with free drugs (Zidovudine and Lamivudine) and expressed in percentage of the total input amount of antiviral agents. Cell Viability Test The CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit (Promega) was used to measure the cytotoxicity of drugs or polymer assemblies. This assay system contains a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-

3

(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and the assay is based on the conversion of the MTS tetrazolium compound into a colored formazan product by dehydrogenase enzymes in metabolically active cells,18 thus providing a convenient colorimetric method for determining the number of viable cells. PBS (pH 7.4) was used initially for dissolving the drug-assembly and cell culture medium was used for further diluting the drug-assembly. Cells were seeded in 96-well plates at a density of 104 cells per well. One day after seeding, cells were treated with a series of dilutions of antiretroviral drugs (Zidovudine or Lamivudine) or drug-loaded polymer assemblies. Cells treated with PBS and cells treated with an empty assembly without drug were included as controls. After 48-h incubation at 37 C, 20 mL of the reagent of the CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit was added to the cells. After 30-min incubation at 37 C, absorbance was measured at 490 nm with a Fluostar Omega microplate reader (BMG Labtech, Offenburg, Germany). Infectivity Assays The TZM-bl cells were seeded in 96-well plates at a density of 104 cells per well. One day after initial seeding, cells were treated with a series of dilutions of antiviral drugs (Zidovudine or Lamivudine) or drug-loaded polymer assemblies. Cells treated with PBS and cells treated with an empty assembly without drug were included as controls. At 4 h after drug/assembly treatment, cells were inoculated with HIV-1 (12.5 ng p24 per well). At 48 h after infection, cell lysates were prepared and subjected to the luciferase assay using the Luciferase Assay System (Promega). The relative light units from the luciferase assay reaction were measured using a Fluostar Omega microplate reader (BMG Labtech). The JLTRG cells were seeded in 24-well plates at 105 cells per well in complete culture medium containing free Zidovudine [Z(f)] or Zidovudine-loaded polymer assembly [Z(a)] at a concentration of 10 mg/mL of Zidovudine. Cells were incubated at 37 C for 4 h and then inoculated with HIV-1 (300 ng p24 per well). PBS-treated cells and mock-infected cells were included as controls. Fresh medium was added to the infected culture at days 1 and 4 after infection. GFP expression was analyzed using an EVOS Cell Imaging System (ThermoFisher Scientific) at day 3 and day 6 after infection. Cells were collected at 6 days after infection and fixed with 2% paraformaldehyde, and GFP-positive cells were quantified using an Attune Flow Cytometer (ThermoFisher Scientific). Virus Release Assay PBMCs were stimulated for 2 days with PHA (1 mg/mL). After washing with RPMI 1640 medium, cells were cultured in the complete RPMI 1640 medium supplemented with IL-2 (100 U/mL) along with the antiviral drug (Zidovudine or Lamivudine) or the drug-loaded assembly at a concentration of 10 mg/mL of drug and incubated for 2 h at 37 C. Cells were then incubated with virus (125 ng of p24 per 1.2 mL) for 4 h at 37 C, washed twice with RPMI 1640 medium, resuspended in RPMI 1640 medium supplemented with IL-2 (100 U/mL), and cultured for up to 7 days at 37 C. Supernatants were collected and the virus released from infected cells was measured by quantifying the viral capsid protein p24 using a standard ELISA. The effect of the drug/assembly on virus release was also examined in the Jurkat cells following the same procedure. Statistical Analysis Experiments were performed in triplicate and repeated at least 3 times. Data are presented as means ± SD.

Zidovudine and Lamivudine are FDA-approved nucleoside HIV reverse transcriptase inhibitors that are available in generic form. The structural and physicochemical properties of these antiretroviral compounds have been well characterized, and both compounds have been widely used in a number of preclinical and clinical studies. The supramolecular assemblies loaded with Zidovudine or Lamivudine were formed by self-assembly upon mixing HA with PL and SBECD in water in an appropriate ratio following the procedure described previously.13 The empty assemblies without the API were also generated as controls. The anionic polymer HA was used as a backbone, and the cationic polymer PL and anionic oligosaccharide SBECD were included as potential cross-linkers of the assembly and/or carriers for the API (Zidovudine or Lamivudine). The polymeric assemblies generated in this study are summarized in Table 1. Size exclusion HPLC, UV-VIS spectrophotometry, and CE were used to determine the composition of the supramolecular assemblies; for example, Zidovudine constitutes 11.21% of assembly 4024 and Lamivudine constitutes 29.65% of assembly 4027.

40

Supramolecular Assembly Preparation

Z/HA/PL/SBECD

30

a

20

Results

Absorbance (mAU)

B. Song et al. / Journal of Pharmaceutical Sciences xxx (2016) 1e10

Assembly Components

Composition (%, weight)

4049 4050 4024

93.9/2.2/3.8 94.0/2.3/3.6 11.21/85.58/2.24/ 0.97 11.16/85.24/2.23/ 1.37 29.65/66.46/3.07/ 0.82

4025 4027

SBECD/hyaluronic acid/poly-L-lysine SBECD/hyaluronic acid/Luviquat-Mono-CP Zidovudine/SBECD/hyaluronic acid/Poly-Llysine Zidovudine/SBECD/hyaluronic acid/LuviquatMono-CP Lamivudine/SBECD/hyaluronic acid/poly-Llysine

12

14

16

18

20

Time (min)

c

b

Intensity (cps)

Physicochemical Properties of the Supramolecular Assemblies

Table 1 Polymer Assemblies Generated in This Study

L/HA/PL/SBECD

10

d

250

L/HA/PL/SBECD Z/HA/PL/SBECD

200 150 100 50 10

e Heat Flow (W

g-1)

We first confirmed the integrity of the CD SBECD used in constructing the supramolecular assembly by CE analysis. The fingerprint capillary electropherograms of the starting SBECD (Fig. 1a, middle), SBECD from the Zidovudine-loaded polymer matrix (Fig. 1a, top), and SBECD from the Lamivudine-loaded polymer matrix (Fig. 1a, bottom) demonstrate that SBECD remained intact in the polymer matrix. These results together suggest that the polymer assembly formulation did not cause a change in the physicochemical nature of SBECD. To define the solid state morphologic properties of the supramolecular matrices with the API, Raman microanalyses were performed. Raman mapping has been shown to be suitable to detect traces of pure crystalline API below the detection limit of X-ray powder diffraction.19 The Raman microscopic map of the Zidovudine drug in the polymer matrix without CD showed yellow spots that indicate an inhomogeneity (Fig. 1b). On the other hand, the Raman map of the Zidovudine drug in the polymer matrix with SBECD was homogeneously red (Fig. 1c), suggesting that the antiviral agent is well distributed in the polymer matrix due to the presence of molecular inclusion by SBECD. The detailed mechanism by which SBECD facilitates the homogeneous entrapment of the Zidovudine drug within the polymer matrix is not fully understood and is currently under investigation. We next tested the crystallinity/amorphousness of the supramolecular assembly by X-ray diffraction and differential scanning

SBECD

10

4

0.8

20

2Theta (O)

30

40

Z/HA/PL/SBECD L/HA/PL/SBECD

0.6 0.4 0.2 0.0 300

350

400

450

500

Temperature (OK) Figure 1. Physicochemical properties of the supramolecular assemblies. (a) Capillary electropherograms of the starting SBECD (middle), SBECD within the Zidovudineloaded HA/PL/SBECD assembly (top), and SBECD within the Lamivudine-loaded HA/ PL/SBECD assembly (bottom). Raman microscopy maps of the Zidovudine drug in the polymer assembly without cyclodextrin (b) and the Zidovudine drug in the polymer assembly with SBECD (c) are shown. (d) X-ray diffraction profiles of the HA/PL/SBECD assembly loaded with Zidovudine (red) or Lamivudine (blue). (e) Differential scanning calorimetry profiles of the HA/PL/SBECD assembly loaded with Zidovudine (blue) or Lamivudine (red).

calorimetry. Solid state characterization by X-ray powder diffraction indicates that the polymer assemblies loaded with Zidovudine (Fig. 1d, red) and Lamivudine (Fig. 1d, blue) are amorphous solids, with no signs of any crystallinity. As shown in the DSC scans, the polymer assemblies loaded with Zidovudine (Fig. 1e, blue) and Lamivudine (Fig. 1e, red) are amorphous, and no endothermic heat flow curves appeared during heating in relation to the melting

B. Song et al. / Journal of Pharmaceutical Sciences xxx (2016) 1e10

(4027) using the TZM-bl cell assay (Fig. 2c). An unloaded polymer assembly (4049) was included as a control. In this HIV-1 reporter system, infection of TZM-bl cells with HIV-1 and subsequent expression of the HIV-1 Tat protein results in LTR-driven luciferase expression. The TZM-bl cells were pretreated with serial dilutions of free drug, drug-loaded polymer assembly, or unloaded assembly for 4 h and then inoculated with CXCR4-tropic HIV-1IIIB. At 48 h after infection, cell lysates were prepared from infected cells and subjected to the luciferase assay. The results shown in Figure 2c demonstrate a dose-dependent antiviral activity of Z, L, Z-loaded polymer matrix (4024) and L-loaded polymer matrix (4027), whereas the empty polymer assembly (4049) did not show any detectable antiviral activity. These results suggest that the antiviral drugs formulated into the polymer assembly (4024 and 4027) were effective in viral inhibition. Zidovudine showed relatively more potent antiviral activity compared with Lamivudine and the Zidovudine-loaded assembly showed more potent antiviral activity compared with the Lamivudine-loaded assembly. Because CD4þ T lymphocytes are the major HIV-1 target cells in vivo, we next determined the antiviral activity of the supramolecular assemblies using the T-cell-derived JLTRG cells (Figs. 2d-2g). In this HIV-1 reporter system, infection of JLTRG cells with HIV-1 and subsequent expression of the HIV-1 Tat protein results in LTR-driven EGFP expression, which can be monitored by fluorescence microscopy or flow cytometry. In this experiment, the drug composition within the polymer matrix (Table 1) was taken into account and the equivalent drug concentration (10 mg/mL) was used for the free Zidovudine [Z(f)] and for the Zidovudine drug incorporated into the polymer assembly [Z(a)]. The JLTRG cells were

process of crystalline API in an Argon atmosphere between 300 and 500 K. Overall, these results suggest that the supramolecular assemblies generated in this study are amorphous and lack crystallinity. The Drug-Loaded Supramolecular Assemblies Exhibit Potent Antiviral Activity The HeLa-derived TZM-bl cells14 and the T-cell-derived JLTRG cells15 are HIV-1 reporter cell lines that are widely used for diagnostic assays and for testing vaccine and drug candidates. These reporter cell lines express the HIV-1 receptor CD4, the co-receptors CXCR4/CCR5, and an integrated HIV-1 LTR promoter-driven reporter gene (firefly luciferase in TZM-bl cells and EGFP in JLTRG cells). The potential cytotoxicity of free Zidovudine (Z) or Lamivudine (L), Zidovudine-loaded supramolecular assembly (4024), and Lamivudine-loaded supramolecular assembly (4027) was evaluated in the TZM-bl cells and JLTRG cells using the CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit (Promega). An unloaded supramolecular assembly (4049) was included as a control. The TZM-bl or JLTRG cells were incubated with free drugs, drug-loaded polymer matrices, or unloaded polymer matrix for 48 h at concentrations ranging from 0.5 to 100 mg/mL, and cells were then subjected to the cell viability assay. In the concentration range used in this experiment, free drugs, drug-loaded polymer matrices, or unloaded matrix did not have any adverse effects on the viability of the TZM-bl cells (Fig. 2a) or the JLTRG cells (Fig. 2b). We next tested the antiviral activity of the free drugs (Z or L), Z-loaded polymer assembly (4024), or L-loaded polymer assembly

a

c 0

1.4

0.5

1.2

1

1

2

0.8

5

0.6

10

0.4

20

2500000

HIV-1 Infecon (RLU)

Cell Viability (OD 490 nm)

1.6

2000000 Z 1500000

L

1000000

4049 4024

500000

0

100

Cell Viability (OD 490 nm)

b

4024

L

4027

0.8

0

0.7

0.5

0.6

1

0.5

2

0.4

5

0.3

10

0.8

4

20

100

e

d

4049

0.032 0.16

Drug Concentraon (ug/ml)

0 Z

4027

0

50

0.2

5

Z(a)

Z(f)

f

g

20

0.2

50

0.1

100 0 Z

4024

L

4027

4049

PBS

mock

Figure 2. The drug-loaded supramolecular assemblies exhibit potent antiviral activity. Cell viability was determined in TZM-bl cells (a) and JLTRG cells (b) using the CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit (Promega): the measurements of absorbance at 490 nm at 30 min after incubation with the cell proliferation assay reagent are shown. Antiviral activity was measured in TZM-bl cells (c) and JLTRG cells (d-g). HIV-1IIIB infection in TZM-bl cells was measured at 48 h post-infection and shown as relative light units from the luciferase assay. HIV-1IIIB infection in JLTRG cells was analyzed at 5 days post-infection by fluorescence microcopy. The concentration (mg/mL) of free Zidovudine (Z), free Lamivudine (L), or polymer assembly with embedded Zidovudine (4024) or Lamivudine (4027) is indicated (a-c). An empty polymer assembly without the drug (4049) was included as a control. The Zidovudine and Lamivudine contents in the polymer assembly 4024 and 4027 were approximately 11% and 30% by weight, respectively. For antiviral activity in JLTRG cells (d and e), 10 mg/mL of Zidovudine was applied as a free drug [Z(f)] or as a polymer assembly [Z(a)]. PBS treatment (f) and mock infection (g) were included as controls. Data are shown as means ± SD of 3 experiments.

6

B. Song et al. / Journal of Pharmaceutical Sciences xxx (2016) 1e10

assembly (Figs. 3a and 3b). The antiviral potency of Zidovudine or Lamivudine formulated into the polymer matrices was almost identical to that of free Zidovudine or Lamivudine, respectively. To determine whether the HIV drug-loaded supramolecular assembly can exert antiviral activity against CCR5-tropic virus, experiments were conducted in the TZM-bl system using CCR5-tropic HIV-1BaL (Figs. 3c and 3d). Infection with CCR5-tropic HIV-1BaL was inhibited by both the Zidovudine-loaded polymer assembly (Fig. 3c) and the Lamivudine-loaded polymer assembly (Fig. 3d). The antiviral potency of the drug-loaded assembly was similar to that of the free drug. Cell viability was not affected by treatment with the free drugs or the drug-loaded polymer matrices (Fig. S1). Overall, these results indicate that the drug-loaded supramolecular assemblies exhibit potent antiviral activity against both CXCR4-tropic (HIV-1IIIB) and CCR5-tropic (HIV-1BaL) viruses. We confirmed the antiviral activity of the drug-loaded polymer assembly using the JLTRG system. JLTRG cells were pretreated with the free Zidovudine drug [Z(f)] or the Zidovudine-loaded polymer assembly [Z(a)] for 4 h and then inoculated with HIV-1IIIB. PBStreated cells and mock-infected cells were included as controls. As shown in the experimental scheme (Fig. 4h), fresh medium was added to the infected cultures at days 1 and 4 after infection. Cells were then analyzed by fluorescence microscopy at day 3 and day 6 after infection and by flow cytometry at day 6 after infection. Although HIV-1 infection was efficient in JLTRG cells treated with PBS at 3 days post-infection (Fig. 4c), treatment of cells with free Zidovudine (Fig. 4a) or the Zidovudine-loaded polymer assembly (Fig. 4b) significantly reduced the number of GFP-positive cells at 3 days post-infection. There was an overall increase in the number of GFP-positive cells at 6 days post-infection (Figs. 4d-4f) compared with that observed at 3 days post-infection but the pattern of viral inhibition was similar to that observed at 3 days post-infection. To

pretreated with free Zidovudine [Z(f)] or Zidovudine-loaded polymer assembly [Z(a)] for 4 h and then inoculated with HIV-1IIIB. PBS-treated cells and mock-infected cells were included as controls. At 5 days after infection, cells were analyzed by fluorescence microscopy. Although HIV-1 infection was efficient in JLTRG cells treated with PBS (Fig. 2f), treatment of cells with free Zidovudine (Fig. 2d) or the Zidovudine-loaded polymer assembly (Fig. 2e) significantly reduced the number of GFP-positive cells suggesting viral inhibition by the free drug and the drug-loaded polymer assembly. As expected, GFP-positive cells were not detected in mock-infected JLTRG cells (Fig. 2g). The Drug-Loaded Supramolecular Assemblies Inhibit HIV-1 Infection as Efficiently as the Free Drugs To directly compare the antiviral activity of the drug-loaded polymer matrices with that of the free drugs, we utilized the TZM-bl system, which provides a robust and sensitive detection of HIV-1 infection. Taking into account the compositions of polymer assemblies (Table 1), equivalent concentrations of Zidovudine or Lamivudine were used for the free drug and the drug-loaded assembly. The TZM-bl cells were treated for 4 h with free drugs or the drug-loaded polymer matrices at drug concentrations ranging from 0.2 to 10 mg/mL, and cells were then inoculated with HIV-1IIIB. At 48 h after infection, cell lysates were prepared from infected cells and analyzed by the luciferase assay. The antiviral activity of Zidovudine from the polymer matrix was almost identical to that of free Zidovudine (Fig. 3a) and the antiviral activity of Lamivudine from the polymer matrix was similar to that of free Lamivudine (Fig. 3b). Consistent with the results shown in Figure 2c, Zidovudine showed relatively more potent viral inhibition than Lamivudine, both as a free drug and as a polymer

b

1400000 1200000

Z(f) Z(a)

1000000 800000 600000 400000 200000

HIV-1 Infecon (RLU)

HIV-1 Infecon (RLU)

a

0.2

0.5

1

2

5

L(f) L(a)

1000000 800000 600000 400000 200000

Drug Concentraon (ug/ml)

d

2500000

Z(f) Z(a)

2000000

0

10

1500000 1000000 500000 0

HIV-1 Infecon (RLU)

0

HIV-1 Infecon (RLU)

1200000

0

0

c

1400000

0.2

0.5

1

2

5

10

Drug Concentraon (ug/ml) 2500000

L(f) L(a)

2000000 1500000 1000000 500000 0

0

0.2

0.5

1

2

5

Drug Concentraon (ug/ml)

10

0

0.2

0.5

1

2

5

10

Drug Concentraon (ug/ml)

Figure 3. The drug-loaded supramolecular assemblies inhibit HIV-1 infection as efficiently as the free drugs. The TZM-bl cells were treated with a series of dilutions of Zidovudine (a and c) or Lamivudine (b and d) as a free drug or the drug-loaded assembly and then subjected to infections with CXCR4-tropic HIV-1IIIB (a and b) or CCR5-tropic HIV-1BaL (c and d). At 48 h post-infection, infected cells were subjected to the luciferase assay and the relative light units are shown as a measurement of HIV-1 infection. Z(f), free Zidovudine; Z(a), Zidovudine-loaded HA/PL/SBECD assembly 4024; L(f), free Lamivudine; L(a), Lamivudine-loaded HA/PL/SBECD assembly 4027. Data are shown as means ± SD of 3 experiments.

B. Song et al. / Journal of Pharmaceutical Sciences xxx (2016) 1e10

a

b

Z(f)/d3 d

c

PBS/d3

Z(a)/d3 e

g

35

% GFP-posive Cells

Z(f)/d6

30

7

f

PBS/d6

Z(a)/d6 h

Fresh Medium

Drug Fresh + HIV Medium

25 20

0

15

1

2

3

4

5

6

10 Florescence Microscopy

5 0 Z(f) Z(a) PBS mock

Florescence Microscopy Flow Cytometry

Figure 4. Antiviral activity of the drug-loaded supramolecular assembly in the JLTRG cells. The JLTRG cells were treated with the free Zidovudine drug or the Zidovudine-loaded HA/ PL/SBECD assembly at a concentration of 10 mg/mL of Zidovudine and then inoculated with HIV-1IIIB. GFP-positive cells were analyzed by fluorescence microscopy (a-f) or by flow cytometry (g). Images taken at 3 days post-infection (a-c) and 6 days post-infection (d-f) are shown. Flow cytometry results were obtained at 6 days post-infection and the percentage of GFP-positive cells is shown (g). A schematic diagram of the experiments is shown (h). Z(f), free Zidovudine; Z(a), Zidovudine-loaded HA/PL/SBECD assembly 4024; mock, mock infection control. Data are shown as means ± SD of 3 experiments.

quantify HIV-1-infected JLTRG cells, cells were fixed at 6 days postinfection and subjected to flow cytometry (Fig. 4g). Although PBStreated cells showed ~33% GFP-positive cells, cells treated with either the free Zidovudine drug or the Zidovudine-loaded polymer assembly showed only 6%-7% GFP-positive cells. These results indicate that the Zidovudine drug that formulated into the polymer

assembly was as effective in viral inhibition as the free Zidovudine drug. The viral inhibition capacity of the supramolecular assemblies was also tested in human PBMCs and the T-lymphocyte-derived Jurkat cells. The results of these experiments indicate that both the free drugs (Zidovudine and Lamivudine) and the drug-loaded

8

B. Song et al. / Journal of Pharmaceutical Sciences xxx (2016) 1e10

assembly and the Lamivudine-loaded assembly, respectively. The antiviral potency of the drug-loaded assembly was similar to that of the free drug at each of the drug treatment periods ranging from 10 to 180 min. (Figs. 5a and 5b). A similar pattern of viral inhibition was observed when the TZM-bl cells were pretreated with the drugs/assemblies at a concentration of 10 mg/mL of drug (Fig. S4). The in vitro release of the API from the supramolecular assembly was performed in deionized water or in physiological buffered saline (0.9% NaCl solution) at either 25 C or at 37 C with stirring at 60 rpm (Figs. 5c and 5d). Over a period of 48 h at 25 C, 60% (in water) to approximately 80% (in 0.9% NaCl) of the Lamivudine was released from the polymer matrix. Lamivudine release was somewhat faster in 0.9% NaCl solution (Fig. 5c, red) compared with the drug release in deionized water (Fig. 5c, blue). The NaClinduced acceleration of drug release from the supramolecular assembly suggests that the formation of the drug-loaded supramolecular assembly may be dependent on electrostatic interactions. Although drug release at 37 C (Fig. 5d) was slightly faster than that at 25 C, the pattern of drug release at 37 C was similar to that at 25 C; the release of drug was more efficient in 0.9% NaCl (Fig. 5d, red) compared with that in water (Fig. 5d, blue). Free Lamivudine alone showed a quick dissolution under the identical conditions with a nearly complete release into the medium within 2 min (data not shown). These results suggest that this supramolecular assembly could potentially be utilized in the design of a sustained release drug delivery system. The supramolecular assembly was also generated using the cationic surfactant hexadecyl(2-hydroxyethyl)dimethylammonium

polymer assemblies efficiently suppressed HIV-1 replication in PBMCs and the Jurkat cells (Fig. S2). The Drug-Loaded Supramolecular Assemblies Exhibit Immediate Viral Inhibition and Maintain Sustained Drug Release To determine whether the Zidovudine- or Lamivudine-loaded polymer matrices can exert antiviral activity at earlier time during HIV-1 infection, the drug/assembly treated cells were analyzed at 24 h post-infection instead of 48 h post-infection. The pattern of antiviral activity obtained at 24 h post-infection (Fig. S3) was similar to that obtained at 48 h post-infection (Fig. 3). We next determined whether a short exposure of the HIV-1 target cells to the drug-loaded polymer assembly can confer efficient viral inhibition. The TZM-bl cells were pretreated with free drugs or the drug-loaded polymer assemblies at a concentration of 1 mg/mL of Zidovudine (Fig. 5a) or Lamivudine (Fig. 5b) for 10, 20, 40, 60, 120, or 180 min. Cells were then washed to remove the drugs/assemblies and cells were infected with HIV-1IIIB for 48 h. Exposure of the TZM-bl cells to the Zidovudine-loaded polymer assembly for 10 min conferred approximately 60% viral inhibition compared with the TZM-bl cells treated with PBS as a control (Fig. 5a), whereas a 10-min exposure of the TZM-bl cells to the Lamivudineloaded polymer assembly conferred almost 40% viral inhibition (Fig. 5b). An exposure time-dependent increase in viral inhibition was observed for both the Zidovudine- and Lamivudine-loaded polymer assemblies, thus resulting in approximately 90% and 85% viral inhibition after a 180-min exposure to the Zidovudine-loaded

a

b 2500000

2000000 1500000

Z(f)

1000000

Z(a) PBS

500000

HIV-1 Infecon (RLU)

HIV-1 Infecon (RLU)

2500000

2000000 1500000

L(f)

1000000

L(a)

0

0 10

20

40

60

120

10

180

20

40

60

120

180

1 ug/ml Lamivudine (min)

1 ug/ml Zidovudine (min)

c

d 100 90 80 70 60 50 40 30 20 10 0

in water

Released Lamivudine (%)

Released Lamivudine (%)

PBS

500000

in 0.9% NaCl

0

1

4

8

Time (hours) at

12

25oC

24

48

100 90 80 70 60 50 40 30 20 10 0

in water in 0.9% NaCl

0

1

4

8

Time (hours) at

12

24

48

37oC

Figure 5. The drug-loaded supramolecular assemblies exhibit immediate viral inhibition and maintain sustained drug release (a and b). The TZM-bl cells were pretreated with free drugs or drug-loaded assemblies at a concentration of 1 mg/mL of Zidovudine (a) or Lamivudine (b) for the indicated time ranging from 10 to 180 min. After each treatment, drug/ assembly-containing medium was removed and fresh culture medium containing HIV-1IIIB was added to the cells. At 48 h post-infection, infected cells were subjected to the luciferase assay and the relative light units are shown as a measurement of HIV-1 infection. Z(f), free Zidovudine; Z(a), Zidovudine-loaded HA/PL/SBECD assembly 4024; L(f), free Lamivudine; L(a), Lamivudine-loaded HA/PL/SBECD assembly 4027. Data are shown as means ± SD of 3 experiments (c and d). In vitro release of Lamivudine from the Lamivudineloaded HA/PL/SBECD assembly 4027 was conducted in deionized water (blue) or in 0.9% NaCl solution (red) at either 25 C (c) or 37 C (d) with stirring at 60 rpm. Data are representative of 2 independent experiments.

B. Song et al. / Journal of Pharmaceutical Sciences xxx (2016) 1e10

dihydrogen phosphate (also known as Luviquat-Mono-CP) instead of poly-L-lysine. The compositions of the Luviquat-Mono-CPcontaining assembly were similar to that of the poly-L-lysine-containing assembly (Table 1). The antiviral potency of the Zidovudineloaded Luviquat-Mono-CP assembly (4025) was similar to that of the Zidovudine-loaded poly-L-lysine assembly (4024), whereas the unloaded polymer assembly containing Luviquat-Mono-CP (4050) or poly-L-lysine (4049) did not show any detectable antiviral activity (Fig. S5). These results together suggest that the cationic molecule (poly-L-lysine or Luviquat-Mono-CP) plays a critical role in supramolecular assembly formation, presumably by interacting with the anionic components (HA and SBECD) within the polymer matrix. The hypothetical structure of the supramolecular assembly is depicted in Figure 6. The detailed status of the API (Zidovudine or Lamivudine) within the polymer matrix is not fully understood and is currently under investigation. It is assumed that the API molecules are bound both by inclusion in the CD cavities and by electrostatic attractions between anionic sulfobutylether substituents located on the CD cavity entrance. Discussion The major goal of this study was to design a novel supramolecular assembly that can be utilized as a sustained release antiretroviral drug delivery system. The development of safe, effective, well-tolerated, and acceptable sustained release formulations will improve adherence by simplifying dosing requirements while maintaining consistent and effective drug levels in plasma and HIV target tissues such as central nervous system, cerebrospinal fluid, semen, lymph nodes, reproductive tracts, gastrointestinal tract, and gut-associated lymphoid tissue. Our findings suggest that the anionic polymer HA, cationic polymer PL, and anionic oligosaccharide SBECD form a novel amorphous matrix that efficiently encapsulates the HIV-1 reverse transcriptase inhibitors Zidovudine and Lamivudine. The identical profiles of capillary electropherograms for the starting SBECD and the SBECD within the polymer assembly indicate that SBECD remained intact and was not chemically modified during assembly formation. SBECD seemed to facilitate homogeneous entrapment of Zidovudine within the polymer matrix as demonstrated by Raman microscopy analysis. The amorphous nature of the supramolecular assembly was supported by X-ray diffraction and differential scanning calorimetry. The drug-loaded polymer assemblies showed potent antiviral activity, as measured using two different HIV-1

Figure 6. Hypothetical structure of electrostatically cross-linked HA (blue) with PL (red) and SBECD (gray).

9

infection models. The antiviral potency of the drug formulated into the polymer assembly was equivalent to that of a free drug. It is interesting to find that the drug-loaded assembly showed an immediate and potent viral inhibition while it maintained a longlasting release of the embedded drug. We assume that drug molecules (Zidovudine and Lamivudine) are bound to the anionic CD SBECD via a combination of electrostatic and inclusion phenomena. The anionic SBECD encapsulates basic Zidovudine and Lamivudine by “outer sphere” interaction (electrostatic attraction) and by inclusion complexation. As a component of the supramolecular assembly, SBECD is a multicomponent isomeric mixture and we cannot prove this structural assumption by spectroscopic analysis. However, the Raman mapping data indicated the nearly homogeneous (molecularly dispersed) distribution of drug actives throughout the supramolecular polymer matrix supporting this structural assumption. We assume that the molecular host-guest type complex formation between drugs and SBECD may result in the stabilization or protection of drugs. The drug release from the supramolecular matrix will be affected by several factors. First, the presence of cations and anions in the dissolution media may act as “ion exchanger” causing a physical disassembly of the structure-holding scaffold PLhyaluronan “salt” and thus initiating the drug release. Second, after physical disassembly of the polymer matrix, dilution by water or buffers will result in the dissociation of the non-covalent CD-drug complexes and therefore dilution is the driving force for drug release from CD complex. The antiviral potency observed in the JLTRG system was less than that observed in the TZM-bl system. A number of factors may have contributed to the differences in antiviral potency of the drugs/assemblies in the two HIV infection models. First, the levels of HIV-induced gene expression (luciferase activity) were measured in the TZM-bl system, whereas the numbers of infected cells (GFP-positive cells) were determined in the JLTRG system. Second, the infected TZM-bl cells were cultured for 48 h, whereas the infected JLTRG cells were cultured for 6 days before assessing the drug/assembly-mediated viral inhibition; it is likely that the 6day culture may allow the progeny viruses released in the culture medium to infect other surrounding noninfected cells, while the drugs/assemblies may continue to lose antiviral activity through degradation or inactivation during the long culture period. Overall, the results of this study suggest that the supramolecular assembly composed of HA/PL/SBECD has the desired features of a sustained drug delivery system with long-lasting drug release along with the maintenance of potent and immediate viral inhibition. Considering the slow or sustained release of antiviral drug from the polymer assembly, it was surprising to find that the efficiency of viral inhibition by the drug-loaded polymer assembly was equivalent to that by a free drug. The mechanisms by which the HA/PL/SBECD-based supramolecular assembly can achieve sustained drug release and effective viral inhibition are currently under investigation. An empty assembly without the antiviral drug did not show any significant antiviral activity suggesting that HA, PL, or SBECD used in the polymer assembly is not directly contributing to viral inhibition at the concentrations used in this study. There are a number of potential mechanisms for the effective antiviral activity mediated by the drug-loaded HA/PL/SBECD assembly. It is possible that HA, PL, or SBECD, individually or in combination, may facilitate the attachment of the API-loaded assembly to the cell surface, enhance the penetration of the API through the cell membrane, and/or increase the stability of the API in the culture medium or physiological fluids. HA is a naturally occurring linear polysaccharide in the body and should not elicit any adverse immune responses. PL and SBECD are generally regarded as safe by the FDA and are widely utilized as

10

B. Song et al. / Journal of Pharmaceutical Sciences xxx (2016) 1e10

carriers. The HA/PL/SBECD polymer assembly generated in this study may provide a useful scaffold for incorporating multiple functionalities or moieties that could further aid in specific targeting and/or overcoming biological barriers in vivo. We believe that this novel polymer assembly could serve as the platform for producing drug formulations that reduce the morbidity and mortality associated with HIV infection. Furthermore, such formulations could be utilized in designing improved therapeutic approaches for combating other diseases such as cancer and inflammatory diseases. Conclusion A drug delivery system that allows sustained drug delivery could reduce the medical burden and costs associated with medication nonadherence. Here we describe a novel supramolecular assembly composed of biodegradable polymers (HA/PL/ SBECD). The supramolecular assembly was shown to successfully encapsulate HIV drugs (Zidovudine and Lamivudine). The drugloaded polymer assembly exhibited potent antiviral activity and maintained sustained drug release. This supramolecular assembly could be used in improved therapeutic approaches for HIV/AIDS and other diseases. Acknowledgments This research was supported by the UC Davis Startup Fund (OP 07427). References 1. Prabhu RH, Patravale VB, Joshi MD. Polymeric nanoparticles for targeted treatment in oncology: current insights. Int J Nanomedicine. 2015;10: 1001-1018. 2. Kreuter J. Nanoparticlesda historical perspective. Int J Pharm. 2007;331:1-10. 3. Fraser JR, Laurent TC, Laurent UB. Hyaluronan: its nature, distribution, functions and turnover. J Intern Med. 1997;242:27-33.

4. Neame PJ, Christner JE, Baker JR. Cartilage proteoglycan aggregates. The link protein and proteoglycan amino-terminal globular domains have similar structures. J Biol Chem. 1987;262:17768-17778. 5. Park KM, Yang JA, Jung H, et al. In situ supramolecular assembly and modular modification of hyaluronic acid hydrogels for 3D cellular engineering. ACS Nano. 2012;6:2960-2968. 6. Warren G, Makarov E, Lu Y, et al. Amphiphilic cationic nanogels as braintargeted carriers for activated nucleoside reverse transcriptase inhibitors. J Neuroimmune Pharmacol. 2015;10:88-101. 7. Vecsernyes M, Fenyvesi F, Bacskay I, Deli MA, Szente L, Fenyvesi E. Cyclodextrins, blood-brain barrier, and treatment of neurological diseases. Arch Med Res. 2014;45:711-729. 8. Sheridan C. Proof of concept for next-generation nanoparticle drugs in humans. Nat Biotechnol. 2012;30:471-473. 9. Liao Z, Graham DR, Hildreth JE. Lipid rafts and HIV pathogenesis: virionassociated cholesterol is required for fusion and infection of susceptible cells. AIDS Res Hum Retroviruses. 2003;19:675-687. 10. Campbell SM, Crowe SM, Mak J. Virion-associated cholesterol is critical for the maintenance of HIV-1 structure and infectivity. AIDS. 2002;16:2253-2261. 11. Graham DR, Chertova E, Hilburn JM, Arthur LO, Hildreth JE. Cholesterol depletion of human immunodeficiency virus type 1 and simian immunodeficiency virus with beta-cyclodextrin inactivates and permeabilizes the virions: evidence for virion-associated lipid rafts. J Virol. 2003;77:8237-8248. 12. Rabenstein DL. Heparin and heparan sulfate: structure and function. Nat Prod Rep. 2002;19:312-331. 13. Szente L, Puskas I, Csabai K, Fenyvesi E. Supramolecular proteoglycan aggregate mimics: cyclodextrin-assisted biodegradable polymer assemblies for electrostatic-driven drug delivery. Chem Asian J. 2014;9:1365-1372. 14. Wei X, Decker JM, Liu H, et al. Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob Agents Chemother. 2002;46:1896-1905. 15. Kutsch O, Levy DN, Bates PJ, et al. Bis-anthracycline antibiotics inhibit human immunodeficiency virus type 1 transcription. Antimicrob Agents Chemother. 2004;48:1652-1663. 16. Song B, Cayabyab M, Phan N, et al. Neutralization sensitivity of a simian-human immunodeficiency virus (SHIV-HXBc2P 3.2N) isolated from an infected rhesus macaque with neurological disease. Virology. 2004;322:168-181. 17. Mura P, Faucci MT, Manderioli A, Bramanti G. Influence of the preparation method on the physicochemical properties of binary systems of econazole with cyclodextrins. Int J Pharm. 1999;193:85-95. 18. Berridge MV, Tan AS. Characterization of the cellular reduction of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Arch Biochem Biophys. 1993;303: 474-482. 19. Vajna B, Farkas I, Farkas A, et al. Characterization of drug-cyclodextrin formulations using Raman mapping and multivariate curve resolution. J Pharm Biomed Anal. 2011;56:38-44.