Targeted delivery of doxorubicin to A549 lung cancer cells by CXCR4 antagonist conjugated PLGA nanoparticles

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Contents lists available at ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb

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Research paper

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Targeted delivery of doxorubicin to A549 lung cancer cells by CXCR4 antagonist conjugated PLGA nanoparticles

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Department of Pharmaceutical Technology, Faculty of Pharmacy, Srinakharinwirot University, Nakhonnayok, Thailand Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok, Thailand c Thailand Institute of Nuclear Technology, Nakhonnayok, Thailand d Faculty of Pharmacy, Thammasat University, Pathum Thani, Thailand b

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a r t i c l e

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Chuda Chittasupho a,⇑, Kriengsak Lirdprapamongkol b, Prartana Kewsuwan c, Narong Sarisuta d

i n f o

Article history: Received 30 March 2014 Accepted in revised form 25 June 2014 Available online xxxx

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Keywords: CXCR4 Targeted drug delivery Doxorubicin Lung cancer CXCR4 antagonist

a b s t r a c t Doxorubicin is used to treat a variety of cancers, but dose limiting toxicity or intrinsic and acquired resistance limits its application in many types of cancer. CXCR4 is a chemokine receptor which implicates in metastasis of cancers including lung cancer. LFC131, a peptide inhibitor of CXCR4-ligand binding, is a linear type of low molecular weight CXCR4 antagonist. In this study, we investigated the possibility of using LFC131 conjugated nanoparticles for targeted delivering doxorubicin to CXCR4 expressing lung cancer cells. The LFC131 peptide was conjugated to sodium carboxylmethyl cellulose coated poly (DL-lactic-co-glycolic acid) (PLGA) nanoparticles. Binding and cellular uptake of doxorubicin-loaded LFC131 conjugated nanoparticles (LFC131-DOX NP) in adenocarcinomic human alveolar basal epithelial cells called A549 cells were higher and faster than that of untargeted nanoparticles. The specificity of CXCR4-mediated internalization of LFC131-DOX NPs was confirmed by using free LFC131 peptide or anti-CXCR4 monoclonal antibody. Cell studies suggested that sustained release of doxorubicin afforded by PLGA nanoparticles may enable LFC131-DOX NP as a targeted and controlled release drug delivery system. Ó 2014 Published by Elsevier B.V.

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1. Introduction

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CXCR4, a seven transmembrane G protein coupled receptor (GPCR), is a member of chemokine receptor family. Binding of CXCR4 to its ligand, CXCL12/SDF1, induces chemotaxis and transmigration of leukocytes, and this interaction also plays an important role in metastasis of various cancers [1–10]. CXCR4 expression was found in several types of cancer including lung cancer [11], breast cancer [12], prostate cancer [13], myeloma [14], leukemia

Abbreviations: PLGA, poly(DL-lactic-co-glycolic acid); GPCR, G protein coupled receptor; Mw, molecular weight; EDC, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride; Sulfo-NHS, N-hydroxysulfosuccinimide; MES, 2-(N-morpholino)ethanesulfonic acid sodium salt; BSA, bovine serum albumin; SCMC, sodium carboxymethyl cellulose; DMEM, Dulbecco’s modified eagle media; FBS, fetal bovine serum; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); NP, nanoparticles; DOX, doxorubicin; HPLC, high performance liquid chromatography; DMSO, dimethyl sulfoxide; PBS, phosphate buffer saline; PE, phycoerythrin; DAPI, 40 -6-diamidino-2-phenylindole; mAb, monoclonal antibody; NSCLC, non-small cell lung carcinoma. ⇑ Corresponding author. Department of Pharmaceutical Technology, Faculty of Pharmacy, Srinakharinwirot University, Ongkarak, Nakhonnayok 26120, Thailand. Tel.: +66 3739 5094x21608; fax: +66 3739 5096. E-mail address: [email protected] (C. Chittasupho).

[15], melanoma [16], and pancreatic cancer [17]. CXCR4 is essential for metastasis of cancer cells to CXCL12 expressing organs such as bone. CXCL12 secreted by non-malignant stromal cells induces migration of CXCR4 overexpressing cancer cells and enhances metastasis rate. Targeting CXCR4 with CXCR4 antagonists may block cancer cell migration and prevent metastasis [18,19]. CXCR4 is a critical target for diseases involving CXCR4–CXCL12 axis. The CXCR4 antagonists such as T140, AMD3100 and FC131 can block migration and metastasis of cancer cells [20–23]. The cyclic pentapeptides, FC131, based on the sequence D-TyrArg-Arg-2-Nal-Gly, have shown CXCR4 antagonistic activity [19]. It was reported that LFC131, a linear type of FC131 has a structure containing ligand pharmacophore and showed an antagonistic effect with efficient binding affinity for CXCR4 [19]. In addition to its efficient binding affinity, LFC131 provides functional group to conjugate with carboxylic acid of stabilizing polymer coated on polymeric nanoparticles. Enhanced efficacy of LFC131 therapeutic may be probable by using nanoparticles for multivalent peptide/receptor interactions and sustained, localized release of anti-cancer drugs. Ligand conjugated nanoparticles are designed to provide dual biological activity by encapsulating anti-cancer drug. High affinity

http://dx.doi.org/10.1016/j.ejpb.2014.06.020 0939-6411/Ó 2014 Published by Elsevier B.V.

Please cite this article in press as: C. Chittasupho et al., Targeted delivery of doxorubicin to A549 lung cancer cells by CXCR4 antagonist conjugated PLGA nanoparticles, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.06.020

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binding and selectivity are important properties of targeted therapeutics [24]. Tools for targeting specific cellular populations have been widely explored by using various targeting ligands including antibodies, peptides, and small molecules [25–28]. This strategy can result in an increased therapeutic efficacy and reduced side effects and toxicity [29]. The ideal targeted therapeutics should have low affinity for the targeted receptor expressed in normal cells, but high affinity to receptors on diseased or activated cells. The goal of this study is to develop a drug delivery system for specific targeting CXCR4 expressing cells. Here, we demonstrate an approach to improve binding affinity of doxorubicin-loaded nanoparticles to CXCR4 expressing cells, by using polymeric nanoparticles harboring multiple copies of CXCR4 antagonist. Binding of the multivalent CXCR4 antagonist-conjugated nanoparticles to CXCR4 expressing cells was determined by blocking of targeted nanoparticles using free peptide ligand and CXCR4 antibody. Investigation of the drug release profile and cytotoxic effect of CXCR4targeted nanoparticles in comparison with free drug and untargeted nanoparticles were performed to investigate the possibility of using this targeted NP.

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2. Materials

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LFC131 peptide (Tyr-Arg-Arg-Nal-Gly, Mw = 747.82) was synthesized and characterized by Ward Medic Ltd., Part. Poly(DL-lactic-co-glycolic acid) (50:50) with terminal carboxyl group (PLGA, inherent viscosity 0.22 dL/g) was purchased from Lakeshore Biomaterials (Birmingham, AL, USA). Sodium carboxymethyl cellulose (viscosity = 400–1000 mPa s, 2% in water and degree of substitution = 0.60–0.95) and bovine serum albumin (BSA) were purchased from Sigma–Aldrich (St. Louis, MO, USA). 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (sulfo-NHS) and 2-b mercaptoethanol were purchased from Thermo Fisher Scientific Inc. (Rockford, IL, USA). MES, sodium salt was purchased from EMD Chemicals, Inc. (San Diego, CA, USA) Doxorubicin hydrochloride injection solution was manufactured by Pfizer. A549 cells were purchased from Japanese Collection of Research Bioresources (JRCB) Cell Bank (Tokyo, Japan). Dulbecco’s Modified Eagle Media (DMEM), fetal bovine serum (FBS), penicillin–streptomycin (10,000 U/ml of penicillin and 10,000 lg/ml of streptomycin), and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were obtained from Life Technologies (California, USA). PE anti-mouse IgG, LEAF™ Purified anti-human CD184 (CXCR4), and PE anti-human CD184 (CXCR4) were purchase from BioLegend (California, USA).

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3. Methods

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3.1. Preparation of PLGA nanoparticles

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PLGA nanoparticles (NPs) were prepared by a solvent displacement method at 25 °C [30]. PLGA (30 mg) dissolved in acetone (2 ml) was injected into 15 ml of magnetically stirred (530 rpm) 0.01% sodium carboxymethyl cellulose solution (SCMC), using an infusion pump at a constant flow rate of 10 ml/h. The resulting nanoparticle suspension was continuously stirred to allow evaporation of acetone. Nanoparticles were collected by centrifugation (15,308g, 10 min) at 4 °C and characterized.

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3.2. Preparation of doxorubicin-loaded PLGA nanoparticles (DOX NPs)

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Doxorubicin hydrochloride (4 mg) was converted into free base form by adding 0.01 N NaOH solution (500 ll) [31]. The drug in NaOH solution was then added directly into PLGA solution

(30 mg of PLGA in 2 ml of acetone). The mixture of PLGA and the drug (2.5 ml) was slowly injected into 0.01% sodium carboxymethyl cellulose (15 ml), under magnetic stirring (530 rpm). The dispersion was then centrifuged at 15,308g for 10 min, to remove the unloaded doxorubicin. The resulting DOX NPs were resuspended and characterized.

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3.3. Conjugation of LFC131 peptide to DOX NPs

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The method of LFC131 peptide and SCMC coated DOX NPs conjugation was adapted from Cheng et al. [32]. SCMC coated DOX NPs were buffered using 2-(N-morpholino) ethanesulfonic acid (MES; pH 6.5), and incubated with 100 mM 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and 50 mM Nhydroxysulfosuccinimide (sulfo-NHS) for 15 min at 25 °C. After the carboxylic acids of SCMC on NPs were activated, the NPs were centrifuged at 15,308g to remove the excess EDC/sulfo-NHS. The NPs were redispersed in deionized water and buffered with PBS, pH 7.4. The LFC131 (200 lg) was then added into the medium. The activated carboxyl terminus of SCMC on the surface of DOX NPs was allowed to react with the amino terminus of LFC131 peptide for 12 h at room temperature. Conjugated nanoparticles were recovered by centrifugation (15,308g, 10 min) and washed with purified water. The amount of free LFC131 was quantified by gradient reversed phase HPLC (Perkins Elmer). The LFC131DOX NPs were centrifuged at 15,308g at 4 °C for 10 min. The amount of free LFC131 in the supernatant was measured by a reversed-phase HPLC. All separations were conducted using AerisÒ protein and peptide C18 column. Gradient elution was carried out at constant flow of 1 ml/min, from 17% A to 26% A (corresponding to 83% B to 74% B). Mobile phase compositions were 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B).

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3.4. Characterization of nanoparticles and DOX NPs

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3.4.1. Particle size, size distribution and zeta potential The size, size distribution and zeta potential of nanoparticles were determined by using dynamic light scattering technique (Malvern zetasizer). The PLGA NPs, DOX NPs and LFC131-DOX NPs were diluted in deionized water, pH 7.1 before measurement. The analysis was performed at a scattering angle of 90° and at a temperature of 25 °C.

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3.4.2. Encapsulation efficiency and drug loading The amount of encapsulated doxorubicin in DOX NPs was determined by spectrophotometric method. Freshly prepared DOX NPs (1 ml) were centrifuged at 15,308g for 10 min, to separate DOX NPs and the supernatant. DOX NPs were dissolved in 1 ml of DMSO, the amount of encapsulated doxorubicin was determined by measuring absorbance at 482 nm, using a spectrophotometer (UV-1601 UV–Visible spectrometry Shimadzu, Japan). The drug-entrapment efficiency was presented by the following equation:

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Amount of drug in NPs EE ð%Þ ¼  100 Amount of drug added Drug loading efficiency was determined by measuring the absorbance at 482 nm. The entrapped doxorubicin content in the nanoparticles was calculated from the mass of the incorporated drug using the following equation:

Amount of drug in nanoparticles Drug loading ¼  100 Amount of drug loaded nanoparticles

Please cite this article in press as: C. Chittasupho et al., Targeted delivery of doxorubicin to A549 lung cancer cells by CXCR4 antagonist conjugated PLGA nanoparticles, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.06.020

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3.5. In vitro release of doxorubicin from LFC131-DOX NPs and DOX NPs

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The DOX NPs and LFC131-DOX NPs were centrifuged and redispersed in PBS. Nanoparticle samples (0.2 mg/ml) were incubated in 1 ml phosphate buffer saline (PBS), pH 7.4, in capped vial at 37 °C. At predetermined time interval, samples were centrifuged (15,308g, 10 min), and supernatant was collected, the amount of released doxorubicin in the supernatant was determined by measuring absorbance at 482 nm.

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Amount of released DOX Cumulative DOX released ð%Þ ¼ Amount of total DOX in NP  100

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3.5.1. Cell culture A549 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin–streptomycin, and maintained in a humidified incubator containing an atmosphere of 5% CO2, at 37 °C. 3.5.2. Expression of CXCR4 on A549 cells A549 cells were suspended in PBS containing 1% BSA (1% BSA/ PBS), and incubated for 20 min at room temperature, for non-specific blocking. Cells were then incubated with phycoerythrin (PE) conjugated anti-human CD184 (CXCR4) antibody (10 lg/ml) or PE conjugated anti-human IgG Fc antibody (10 lg/ml) for 1 h at 4 °C. The cells were then washed three times with ice-cold 1%BSA/PBS, and fixed in 4% paraformaldehyde. CXCR4 expression on cell surface was determined using FACSCanto flow cytometer (Becton Dickinson). Ten thousand events were acquired and data were analyzed by FACSDiva software (Becton Dickinson). 3.5.3. Quantification of binding and uptake of nanoparticles to A549 cells A549 cells were seeded in 96-well plate at a cell concentration of 80,000 cells/ml. The cells were grown to confluency in culture medium for 48 h. The culture medium was removed and washed one time with serum-free DMEM. Nanoparticles suspended in serum free DMEM (2 mg/ml, 100 ll) were applied to the cells, and incubated at 37 °C for 5, 15, 30, 60 and 120 min. After the specified time of incubating nanoparticles with A549 cells, the medium containing nanoparticles was removed from each well, and the cells were rinsed three times with cold PBS, and completely dissolved in DMSO. The absorbance of doxorubicin at 482 nm indicating nanoparticles associated with the cells was measured by using a fluorescence plate reader (Beckman). Concentration dependent cell uptake study was conducted by incubating A549 cells with various concentrations of LFC131DOX NP (0.02–10 mg/ml) in serum-free DMEM for 30 min, at 37 °C, 5% CO2. The cells were then washed with cold PBS three times, and completely dissolved in DMSO. The uptake of nanoparticles was measured using a fluorescence plate reader (Beckman). The uptake efficiency was calculated from the following equation [33].

% Uptake efficiency ¼ 245

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Amount of DOX taken up by cells Amount of DOX in NPs added into the cells  100

3.5.4. Fluorescence microscopy study of binding and uptake of nanoparticles A549 cells were seeded in cell culture chamber slide and incubated overnight to allow cell attachment. The cells were then incubated with a suspension of DOX NPs or LFC131-DOX NPs, in serumfree DMEM for 5, 15, 30 and 60 min at 37 °C, 5% CO2. The cells were

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washed three times with PBS to remove unbound nanoparticles, and fixed in 4% paraformaldehyde. To visualize nuclei, cells were incubated with 2 lg/ml DAPI (40 -6-diamidino-2-phenylindole) for 10 min at room temperature, washed three times with PBS, and examined under fluorescence microscope. Fluorescence micrographs were acquired using the DAPI filter set of Nikon Eclipse TS100-F and NIS-Elements, version 4.0 software.

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3.5.5. Blocking of nanoparticles binding to A549 cells A549 cells were incubated with free LFC131 (0, 0.68, 1.36 and 2.72 mg/ml) in serum-free DMEM for 30 min at 37 °C, 5% CO2. The peptide was removed, then the cells were incubated with nanoparticles (2 mg/ml) for 30 min at 37 °C 5% CO2. Cells were washed three times with PBS, and completely dissolved in DMSO. LFC131-DOX NP and DOX NP content bound to the cell surface, or internalized into the cells, were determined by microplate reader (Beckman Coulter DTX 880 Multimode Detector). Various concentrations of anti-CXCR4 monoclonal antibody (anti-CXCR4 mAb) in 1%BSA in PBS (6.725–25 lg/ml) were incubated with A549 cells for 30 min at 37 °C 5% CO2. Anti-CXCR4 mAbs were removed, and the cells were incubated with LFC131DOX NPs, or DOX NPs (2 mg/ml) suspended in serum-free DMEM for further 30 min at 37 °C 5% CO2. After incubation, cells were washed three times with PBS, and completely dissolved in DMSO. Binding of nanoparticles to anti-CXCR4 mAbs treated cells was determined by fluorescence spectrometer (LS55 Fluorescence spectrometer Perkin Elmer).

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3.5.6. Lysosomal trafficking of LFC-131 conjugated nanoparticles A549 cells were seeded 24 h prior to treatment in 8-well cell culture chamber slide. The medium was removed, and cells were washed once with PBS. LFC131-DOX NP or NP was added into the wells and incubated for 1, 2 and 3 h. Cells were washed three times with PBS, and further incubated with Lysotracker Green DND-26 (100 lM) for 30 min at 37 °C. Cells were fixed in 4% paraformaldehyde for 10 min, and observed under an inverted fluorescence microscope (Nikon) equipped with an imaging software (NIS).

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3.6. In vitro cell viability study

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A549 cells were grown in 96-well plate until 80% confluent. DOX NPs or LFC131 DOX NPs were added to the wells at defined concentrations (0.02–10 mg/ml NPs), and incubated for 6 h at 37 °C, 5% CO2. Nanoparticles were removed, and cells were further incubated with culture medium for 18 h. For a control experiment, cells were incubated with doxorubicin for 24 h. The cells were washed three times with PBS, and incubated in culture medium containing MTT (0.5 mg/ml) for 2 h at 37 °C. After incubation, the media were removed, and then 100 ll of DMSO was added to solubilize the formazan crystals formed. The absorbance was measured at 550 nm, and at 482 nm for reference. Percentage of cell viability was calculated as the ratio of mean absorbance of triplicate readings with respect to mean absorbance of control wells.

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3.7. Statistical analysis

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Statistical evaluation of data was performed using an analysis of variance (one-way ANOVA). Newman–Keuls was used as a post hoc test to assess the significance of differences. To compare the significance of the difference between the means of two groups, a t-test was performed; in all cases, a value of p < 0.05 was accepted as significant.

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Please cite this article in press as: C. Chittasupho et al., Targeted delivery of doxorubicin to A549 lung cancer cells by CXCR4 antagonist conjugated PLGA nanoparticles, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.06.020

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4. Results

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DOX-NP Linear FC131-DOX-NP

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4.1. Characterization of PLGA NPs and DOX NPs

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In the preparation of PLGA NPs, sodium carboxymethyl cellulose was used as a surfactant to stabilize nanoparticles in an aqueous suspension, and to increase carboxyl groups on the nanoparticle surface. The characteristics of PLGA NPs, DOX NPs and LFC131-DOX NPs are summarized in Table 1. The encapsulation efficiency and drug loading of DOX NPs and LFC131-DOX NPs are shown in Table 2. The mean size of nanoparticles encapsulating DOX was increased. However, the encapsulation efficiency was decreased after conjugation with LFC131, which may be attributed to the loss of unentrapped doxorubicin available on surface of LFC131-DOX NPs. The surface charge of LFC131-DOX NPs significantly decreased from 46.7 to 37.4 mV, which was a result of surface modification with LFC131 peptide consuming the carboxyl groups on nanoparticle surface.

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4.2. Conjugation of LFC131 peptide to PLGA nanoparticles

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The LFC131 peptides were covalently attached to the carboxylic acid side groups of sodium carboxymethyl cellulose on the nanoparticle surface, using carbodiimide chemistry. The conjugation efficiency was determined by quantifying the unconjugated peptides remaining in the medium after reaction. The percent of LFC131 conjugated to DOX NPs was about 75.6 ± 2.0%. The conjugation reaction was also performed in the absence of EDC to observe any possible adsorption (electrostatic or hydrophobic interaction) of LFC131 peptide to the nanoparticles. The result showed that the amount of peptide conjugated with NP analyzed by RP-HPLC was 22.4 ± 3.6% when peptide was incubated with nanoparticles without activation of COOH.

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4.3. In vitro release of doxorubicin from LFC131-DOX NP and DOX NP

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The release profile of DOX from LFC131-DOX NPs and DOX NPs in PBS, pH 7.4 at 37 °C, was shown in Fig. 1. For untargeted DOX NPs, the initial burst release was observed. The result showed that the amount of drug release increased rapidly, followed by a constant and much slower rate. The first phase is a characteristic of DOX diffusion through the surface of the PLGA nanoparticle, where the second phase corresponds to the diffusion of DOX from the inner polymer matrix. For LFC131-DOX NP, the controlled release of DOX from NP was shown. An initial burst release during the first 2 h represents only 18% of the total DOX encapsulated, where 64% of DOX was released from DOX NP. A period of controlled drug release from LFC131-DOX NP occurred, reaching a value of 50%

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Table 1 Nanoparticle properties at specified formulation points.

PLGA NP DOX NP LFC131-DOX NP *

Effective diameter (nm) [Mean ± SD]

Polydispersity [Mean ± SD]

Zeta potential value (mV) [Mean ± SD]

127 ± 2 396 ± 7 301 ± 10

0.12 ± 0.03 0.24 ± 0.02 0.16 ± 0.03

28.1 ± 2.4 46.7 ± 1.4 37.4 ± 1.4

Values are representative of three experiments.

Table 2 Encapsulation efficiency and drug loading.

DOX NP LFC131-DOX NP *

Encapsulation efficiency (%)

Drug loading (%)

74.1 ± 5.4 41.9 ± 2.1

7.9 ± 0.2 4.5 ± 0.1

Values are representative of three experiments.

% Cumulative release

100 80 60 40 20 0 0

10 20 30 40 50 60 70 80 90 100

Incubation time (hr)



Fig. 1. In vitro release of doxorubicin from DOX NPs ( ) and LFC131-DOX NPs (j) in PBS, pH 7.4 at 37 °C.

after 48 h. The result suggested that modification of DOX NP surface by conjugating with LFC131 peptide significantly reduced the initial release rate, and offered controlled release behavior of this system.

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4.4. CXCR4 expression on A549 cell surface

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The cell surface expression of CXCR4 on A549 cells was evaluated by flow cytometry. Fluorescent intensity of cells stained with PE conjugated anti-CXCR4 was significantly higher than the IgG control (Fig. 2), indicating the presence of CXCR4 protein on surface of A549 cells.

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4.5. Binding of DOX NPs and LFC131-DOX NPs to A549 cells

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Fluorescence spectroscopy was employed to study the binding and uptake of LFC131-DOX NPs to A549 cells compared with DOX NPs. Within the first hour the binding extent of LFC131DOX NPs to A549 cells was found to be significantly higher than DOX NP without targeting peptide (Fig. 3). The fluorescent intensity was increased concomitantly with incubation time, suggesting an increasing amount of nanoparticles bound or taken up by cells along with the incubation time (Fig. 3). The difference in fluorescent intensity of cells incubated with targeted NP at 120 min was not observed. These results may indicate saturation of CXCR4 binding sites on A549 cells. The binding or uptake of LFC131-DOX NPs was faster than that of DOX NPs during the first hour, before reaching saturation at 2 h.

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4.6. Binding and uptake of LFC131-DOX NPs is concentration dependent

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In order to confirm that LFC131-DOX NPs were present in sufficient quantity for maximal cellular uptake, A549 cells were incubated with various nanoparticle concentrations. Binding and uptake of LFC131-DOX NPs increased correspondingly with an increase in nanoparticle concentrations, suggesting a concentration dependent binding of nanoparticles to the cells (Fig. 4A). However, the binding of nanoparticles did not reach the saturation level within the tested concentration range. This may be due to the diffusion of doxorubicin released from nanoparticles that yielded an unsaturated increase in fluorescent intensity. Efficiency of nanoparticle binding and uptake was calculated from the amount of added LFC131-DOX NPs and the amount of LFC131-DOX NPs that were bound and taken up into the cells within 30 min [33]. The calculated binding and uptake efficiency of LFC131-DOX NPs at low added concentrations was greater than

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Please cite this article in press as: C. Chittasupho et al., Targeted delivery of doxorubicin to A549 lung cancer cells by CXCR4 antagonist conjugated PLGA nanoparticles, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.06.020

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LFC131-DOX NP concentration (mg/ml)

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PE

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XC R 4

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PE

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Fig. 2. Expression of cell surface CXCR4 on A549 cells. Each data point is represented as mean ± SD (n = 3); (*p < 0.001 PE-anti CXCR4 vs untreated and PE-IgG).

Mean Fluorescent Intensity

1.0×10 08

DOX NP LFC131-DOX NP

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that of high added concentrations, (Fig. 4B). At higher concentrations of LFC131-DOX NP, cells probably reached saturation uptake efficiency [33]. 4.7. Fluorescence microscopy study of binding and uptake of nanoparticles

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4.8. In vitro targeting of CXCR4 by LFC131-DOX NPs

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Both unlabelled-free LFC131 peptide and anti-CXCR4 mAb were used to demonstrate that LFC131-DOX NPs binding and uptake in A549 cells occurred through a specificity of CXCR4-ligand interac-

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Fig. 4. The dependence of LFC131-DOX NP concentration on the binding to A549 cells.

Drug delivery and release behaviors of LFC131-DOX NP and DOX NP were elucidated by incubating the nanoparticles with A549 cells. At various time points (5–60 min), unbound nanoparticles were washed out, and cells were imaged using fluorescence microscope. As shown in Fig. 5, the red fluorescent intensity of the cells increased with incubation time, and the greatest intensity was observed at the latest time point. These results suggested that doxorubicin was released from the nanoparticles inside the cells, in a time-dependent manner, mainly distributed in the cytoplasm and localized within the nuclei. The fluorescent intensity of cells incubated with LFC131-DOX NPs was clearly higher than that of DOX NPs (Fig. 5), suggesting that conjugation of LFC131 peptide could improve binding and uptake of DOX-loaded nanoparticles to A549 cells.

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Fig. 3. Binding and cellular uptake of DOX NPs and LFC131-DOX NPs in A549 cells. Each data point is represented as mean ± SD (n = 3); (*p < 0.01, **p < 0.001 DOX NP vs LFC131-DOX NP, at each time point).

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% Binding efficiency

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Mean Fluorescent Intensity

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tion. Fig. 6A shows that the free LFC131 peptides could block binding of LFC131-DOX NP to the cells in a dose dependent manner, while the binding of DOX NP was not affected. These results confirmed that LFC131-DOX NPs bind to the same site with the LFC131 peptide. Anti-CXCR4 mAb was used to block CXCR4-ligand binding. Fig. 6B shows that anti-CXCR4 mAb, at a concentration of 25 lg/ ml, could block the binding and uptake of LFC131-DOX NPs to the cells, while the antibody did not affect the binding of DOX NPs. Collectively, the results indicated that LFC131-DOX NPs interact with A549 cells by binding to CXCR4.

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4.9. Lysosomal trafficking of LFC131-DOX NPs

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Lysotracker Green DND-26, a marker for late endosomes and lysosomes, was used to investigate whether the LFC131-DOX NPs localized in late endo-lysosomal compartments. After A549 cells were incubated with LFC131-DOX NPs for 1–3 h, colocalization of doxorubicin (red) and the Lysotracker Green DND-26 (green) was observed, and the extent of colocalization was peaked at 2 h after incubation (Fig. 7), indicating the internalization of LFC131-DOX NPs was occurred via endocytosis, and trafficked to lysosomes. The decreased extent of colocalization at 3 h after incubation, suggested that doxorubicin has escaped from the endo-lysosomal compartment [34].

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4.10. In vitro cell viability study

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LFC131-DOX NPs and DOX NPs were incubated with A549 cells for 6 h to allow a sufficient binding and uptake of the nanoparticles, and then removed out. The cells were further incubated in medium for 18 h before the measurement of cell viability by MTT assay. For positive control, the cells were incubated with free doxorubicin for 24 h. The % cell viability was plotted against log

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Fig. 5. Binding and uptake of (A) DOX NPs and (B) LFC131-DOX NPs to A549 cells and colocalization of released doxorubicin (red) and nuclei (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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concentrations of free DOX, LFC131-DOX NP and DOX-NP. LFC131DOX NP and DOX NP showed cytotoxicity to A549 cells, a reduction in cell viability was observed in a dose dependent manner (Fig. 8A). DOX-NP and LFC131-DOX-NP caused a significant reduction (60% of control) in cell viability at the highest concentration tested (2.5 mg/ml), which is equivalent to 246.5 and 140 lg/ml of DOX entrapped in DOX-NP and LFC131-DOX NP, respectively. Free DOX reduced cell viability to 39% of control at concentration of 500 lg/ml. In addition, cell sensitivity to doxorubicin was greatly increased when the cells were treated with LFC131-DOX NPs and DOX NPs compared with free DOX at concentrations of 30–120 lg/ml doxorubicin entrapped in NPs (Fig. 8B).

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The interaction of CXCR4 and CXCL12 enhances tumor growth, metastasis, and chemotherapeutic resistance of non-small cell lung cancer (NSCLC). Several CXCR4 antagonists have been developed and showed high affinity and antagonistic effect to NSCLC [35–38]. Here, we developed a novel CXCR4-targeted drug delivery system by conjugating CXCR4 antagonist on the surface of PLGA nanoparticles encapsulating doxorubicin, and characterized its binding and uptake, the targeting specificity, and in vitro antitumor activity.

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In this study, the pentapeptide CXCR4 mimetics with antagonistic activity, based on the sequence D-Tyr-Arg-Arg-2-Nal-Gly, was used as a targeting ligand. The peptide ligand was successfully conjugated on PLGA nanoparticle surface by using carbodiimide reaction. Sodium carboxymethyl cellulose (SCMC) was used as a stabilizer of nanoparticles and providing the carboxylic acid for amide linkage. It was hypothesized that the adsorption mechanism of SCMC to PLGA nanoparticle surface would be explained by the hydrogen bond and van der Waal’s force [39]. The high surface energy of PLGA nanoparticles may cause the formation of multilayer SCMC on nanoparticle surface [39]. A certain quantity of SCMC adsorbed or anchored at the nanoparticle surface was not justified in this study. Further investigations could be comparing the zeta potentials of a series of particles prepared with different amounts of SCMC with those of a series of particles prepared with the same amount of methyl cellulose. Encapsulation efficiency of doxorubicin in PLGA nanoparticles was about 74%. The high encapsulation efficiency is due to an increase in non-ionized form of amino group when the pH is above 7.2 [40]. The increase in size of DOX NPs compared with PLGA NPs without drug loaded could be due to an increase in viscosity of acetone/water mixture used to dissolve polymer and the drug before NP formation. In addition, the increase in size of nanoparticles after doxorubicin encapsulation suggested that doxorubicin was incorporated in PLGA nanoparticle matrix. However, after conjugation, the encapsulation efficiency was reduced to 42%, probably due to the release of an ionized form of the drug adsorbed on nanoparticle surface, because during the conjugation reaction, system was buffered to pH 6.5 [40]. The results suggested that % encapsulation efficiency of doxorubicin in LFC131-DOX NPs was attributed to drug incorporated into the matrix of PLGA nanoparticles, with the less extent of drug adsorption. The decrease in hydrodynamic diameter of LFC131-DOX NPs may be attributed to the reduced drug adsorption on nanoparticle surface and the change the brush conformation of SCMC around the particle due to chemical modifications of nanoparticle surface and interactions with the peptide. The reduced drug adsorption on nanoparticle surface was confirmed by the decrease in hydrodynamic diameter of LFC131DOX NPs. The negative charge of peptide-conjugated NPs was reduced compared with unconjugated NPs, which is probably due to neutralization of negative charge on the nanoparticle surface by positive charge of the peptide. The drug release study was conducted to observe the controlled release behavior of the targeted NPs. The DOX NP release profile shows an intermediate and fast release in the first 10 h, after that the drug release rate was much slower and became constant after about 80% of the drug load was released. The burst release of doxorubicin from DOX NP could be a result of the release of the adsorbed drug on nanoparticle surface and the diffusion of the drug through pre-existing pores and channels in the nanoparticles during NP formation process. DOX as a small molecular weight molecules are soluble in aqueous system and can diffuse easily through the porous structure of PLGA nanoparticle upon the hydration of the matrix. The solubility of DOX at pH 7.4 and their partition coefficients can affect the driving force for the release and leads to initial rapid release. The LFC131-DOX NP curve shows a biphasic drug release kinetic. An initial burst release was observed and followed by the second phase which release rate continuously diminished with the time which could be due to the increasing diffusional distance that delayed the drug diffusion from the core of the NP. LFC13-DOX NPs provide a controlled drug release profile suggesting the retaining of the drug during administration and release at the tumor site. The cell studies were carried out to investigate the drug targeting ability of LFC131-DOX NPs. Surface expression of CXCR4 on A549 cells was confirmed in this study, consistent with the

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Fig. 7. Cellular uptake of LFC131-DOX NPs was observed by colocalization of LFC131-DOX NPs and lysosomes using fluorescence microscope. Selected colocalizations of nanoparticles and endo-lysosomes are indicated by the white arrows. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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previous report that A549 is a CXCR4 expressing cell line [2,3,35,41,42]. The surface expression of CXCR4 indicated that A549 cells can be used as a model for characterization of CXCR4targeted nanoparticles. The binding of LFC131-DOX NPs to A549 cells was significantly greater than DOX NP, in a period of 60 min incubation. A further incubation time did not induce a large change in nanoparticle binding and uptake, suggesting that the CXCR4 might be saturated with LFC131-DOX NPs. According to the drug release profile of DOX NP, about 46% of DOX was released after incubated with cells for 5 min. These results suggested that the measured fluorescent intensity of doxorubicin bound or taken up by cells might be a result of passively diffused free doxorubicin. The binding and uptake efficiency of LFC131-DOX NPs in A549 cells is highly efficient until reaching saturation uptake kinetic. The saturation curve is commonly observed for receptor-mediated endocytosis. The time dependent uptake of nanoparticles was confirmed by fluorescence microscopy. Doxorubicin released from LFC131-DOX NPs and DOX NPs was increasingly incorporated into the nuclei of A549 cells, as evidenced by the colocalization of red fluorescence of doxorubicin and blue fluorescence of DAPI-stained nuclei. The fluorescence images demonstrated the significantly

greater binding extent of LFC131-DOX NP compared with the untargeted DOX NPs. These observations indicate that CXCR4-targeting of LFC131-DOX NP is attributed to having more rapidly and more NPs associated with cell surface, which may increase the chance for internalization. The colocalization of doxorubicin with the nuclei suggested that drug was released from nanoparticles and delivered to the site of action. Our results of using unlabelled free LFC131 peptides and antiCXCR4 mAb clearly confirmed that binding and uptake of LFC131-DOX NPs in A549 cells occurred via a CXCR4-ligand specific interaction. The results suggested that CXCR4 antagonist, when attached to nanoparticle surface, showed ability to target CXCR4 expressing cells. Binding of LFC131-DOX NPs to CXCR4 expressing cells may increase a chance to deliver the cytotoxic drug to the intracellular part. The lysosomal trafficking study demonstrated that LFC131-DOX NPs were uptaken by endocytosis and localized in endo-lysosomal compartment of A549 cells within 1–3 h incubation, after that the drug escaped from the lysosomes. This information is important for future development of CXCR4targeting nanoparticles, because enzymatic and chemical degradation in lysosome can affect the delivered drug.

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(MRG5580124), and Srinakharinwirot University. We also acknowledge the Research Center for Drug Discovery and Development, Faculty of Pharmacy, Srinakharinwirot University for providing facilities and equipments used in cell studies.

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The decrease in cell viability after exposure to free doxorubicin, LFC131-DOX NPs, and DOX NPs was observed in a dose dependent manner. The cytotoxicity of doxorubicin-loaded nanoparticles may be caused by either intracellularly released doxorubicin from uptaken nanoparticles, or by the nanoparticle itself. PLGA nanoparticles are known to be non-toxic to A549 cells, thus the observed cytotoxicity due to PLGA was ruled out. The nuclear localization of doxorubicin in the cells treated with LFC131-DOX NPs (Fig. 5) indicated that the use of CXCR4 as target of drug delivery did not alter the localization and site of action of doxorubicin. Thus the observed cytotoxicity of LFC131-DOX NPs was caused by nuclear localization of doxorubicin similar to the cells treated with free doxorubicin.

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In this study, multiple copies of CXCR4 antagonist were designed to express on nanoparticle surface, to increase the efficacy of targeted nanoparticles. Binding and uptake of targeted doxorubicin-loaded nanoparticles in CXCR4 expressing cancer cells was improved by CXCR4-ligand specific interaction. Additionally, the LFC131-conjugated DOX NPs also showed a controlled drug release profile. CXCR4 antagonist peptide was successfully used to target CXCR4 expressing cells with the retained cytotoxicity effect. The LFC131-DOX NPs may lower non-specific cytotoxicity to normal cells compared to the untargeted DOX NP.

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The authors acknowledge the supports from the Office of the Higher Education Commission, the Thailand Research Fund

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