Biodegradable polymeric nanoparticles for oral ...

1 downloads 0 Views 1MB Size Report
Feb 28, 2015 - a Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India b Product Development Cell, National Institute of ...
Colloids and Surfaces B: Biointerfaces 128 (2015) 448–456

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

Biodegradable polymeric nanoparticles for oral delivery of epirubicin: In vitro, ex vivo, and in vivo investigations Mohammad Tariq a , Md. Aftab Alam b , Anu T. Singh c , Zeenat Iqbal a , Amulya K. Panda b , Sushama Talegaonkar a,∗ a

Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India Product Development Cell, National Institute of Immunology, New Delhi, India c Dabur Research Foundation, 22, Site IV, Sahibabad, Ghaziabad, Uttar Pradesh, India b

a r t i c l e

i n f o

Article history: Received 28 September 2014 Received in revised form 15 January 2015 Accepted 22 February 2015 Available online 28 February 2015 Keywords: Epirubicin PLGA NPs Cellular uptake Cellular transport Intestinal transport Oral bioavailability

a b s t r a c t Epirubicin (EPI) is an anthracycline antineoplastic agent, commercially available for intravenous administration only and its oral ingestion continues to remain a challenge. Present investigation is aimed at the development of poly-lactic-co-glycolic acid (PLGA) nanoparticles (NPs) for oral bioavailability enhancement of epirubicin. Developed formulation revealed particle size, 235.3 ± 15.12 nm, zeta potential, −27.5 ± 0.7 mV and drug content (39.12 ± 2.13 ␮g/mg), with spherical shape and smooth surface. Cytotoxicity studies conducted on human breast adenocarcinoma cell lines (MCF-7) confirmed the superiority of epirubicin loaded poly-lactic-co-glycolic acid nanoparticles (EPI-NPs) over free epirubicin solution (EPI-S). Further, flow cytometric analysis demonstrated improved drug uptake through EPI-NPs and elucidated the dominance of caveolae mediated endocytosis for nanoparticles uptake. Transport study accomplished on human colon adenocarcinoma cell line (Caco-2) showed 2.76 fold improvement in permeability for EPI-NPs as compared to EPI-S (p < 0.001) whereas a 4.49 fold higher transport was observed on rat ileum; a 1.8 fold higher (p < 0.01) in comparison to Caco-2 cell lines which confirms the significant role of Peyer’s patches in absorption enhancement. Furthermore, in vivo pharmacokinetic studies also revealed 3.9 fold improvement in oral bioavailability of EPI through EPI-NPs. Henceforth, EPINPs is a promising approach to replace pre-existing intravenous therapy thus providing “patient care at home” © 2015 Published by Elsevier B.V.

1. Introduction Drug molecules from various sources including natural, semisynthetic, synthetic and biotechnology have great curative and prophylactic potential; however, many of these still present a major delivery challenge when administered orally. Most of them either possess poor water solubility or show site specific permeability across the intestine. In addition, they are either substrates for the biological transporters including P-glycoprotein (P-gp) or metabolizing enzyme, cytochrome P450 (CYP450) or for both resulting into a considerable loss owing to their expulsion or first pass metabolism. Additionally, unfavorable physicochemical properties

∗ Corresponding author at: Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi 110062, India. Tel.: +91 98 18453518; fax: +91 11 26059663. E-mail addresses: [email protected], [email protected] (S. Talegaonkar). http://dx.doi.org/10.1016/j.colsurfb.2015.02.043 0927-7765/© 2015 Published by Elsevier B.V.

of drugs along with adverse environment of the gastrointestinal tract obviate their successful oral delivery [1–3]. Most of the anticancer molecules are not untouched by these limitations and therefore are only administered via intravenous route. In spite of the technical hitches, oral administration has always remained a preferred route due to a number of attributes like it being noninvasive, painless, flexibility in designing of dosage form and dosing frequency, avoiding need for hospitalization, nursing care, economical and overall offers a better patient compliance [4,5]. In recent years, the utilization of nanoparticles (NPs) for oral delivery of anticancer drugs has gained considerable attention. Encapsulation of drug molecules within NPs protects them from efflux transporters whereas the small size of particles facilitates its entry across the biological membrane [6]. Nano-systems have revealed a potential to improve drug stability, extend therapeutic effect, reduce degradation, metabolism as well as cellular efflux [7–9]. Amongst the available nano delivery systems, polymeric NPs offer extra advantages over other colloidal carriers with respect to

M. Tariq et al. / Colloids and Surfaces B: Biointerfaces 128 (2015) 448–456

their greater stability, protection of encapsulated drugs and feasibility of modulation of physicochemical characteristics including surface properties, release behavior and biological behavior viz. a viz. targeting, bioadhesion and improved cellular uptake [10–12]. The most accepted and widely exploited class is the biodegradable NPs. Poly-lactic-co-glycolic acid (PLGA) is a food and drug administration (FDA) approved biocompatible and biodegradable polymer and has been widely exploited for encapsulating anticancer drugs [9,13–15]. In addition, drug release rates, size and loading can be easily manipulated to provide further control over drug delivery [16]. Like other nano-systems, PLGA NPs are also taken up by enterocytes and specialized M cells overlaying Peyer’s patches in the small intestine [1,17,18] may results into bypassing of CYP 450 mediated metabolism, P-gp mediated efflux and hepatic first pass metabolism of drugs. Fatma et al., developed PLGA NPs for oral delivery of etoposide, a P-gp substrate drug. They reported a significant reduction in IC50 values against MCF-7 cell line and significant improvement in oral bioavailability in comparison to conventional etoposide formulation [19]. Similar observations were reported by Naseem et al., for etoposide SMEDDS formulation [20]. Moreover, a better pharmacokinetic profile was reported for gemcitabine loaded oral NPs over plain oral solution [1]. Furthermore, Jain et al., demonstrated augmented efficacy of doxorubicin through oral NPs and layersomes in comparison to free solution (administered via oral and intravenous route) against chemically induced breast cancer [21,22]. In addition, Bhardwaj et al., observed a more reduction in tumor burden (induced chemically in Sprague Dawley rat) through oral paclitaxel PLGA NPs when compared with paclitaxel solution in Cremophor EL given via oral and i.v. route [23]. Encouraging outcomes of other studies suggested that nanoparticulate system could be an alternative for the oral delivery of pharmaceutically challenging drug molecules [3,9,24–26]. Epirubicin (EPI) is an analog of doxorubicin and a safer alternative with comparable clinical activities as well as lower side effects at equivalent dose [27]. It is a newer anthracycline antineoplastic molecule which acts by intercalating into DNA strands and suppresses DNA and RNA synthesis. It also prompts DNA breakage by topoisomerase II, which results in cell death. EPI, like doxorubicin has a wide range of clinical applications in diverse types of malignancies including breast cancer, non-Hodgkin’s lymphomas, ovarian cancer, soft-tissue sarcomas, pancreatic cancer, gastric cancer, small-cell lung cancer, and acute leukemia. It is commercially available in an injection form (Ellence, Pfizer) and intravenous administration of the drug leads to its sudden rise and rapid fall in blood, often to sub-therapeutic level hence prompting frequent dosing which is often associated with cumulative adverse effects. Clinical postulation is suggestive of its extended exposure at modest concentration in comparison to its pulsed supply at higher concentration. Researchers are actively investigating nano-systems to improve the delivery of the epirubicin hydrochloride. Epirubicin loaded pullulan acetate NPs exhibited significantly lower IC50 against KB cell line and better safety profile in comparison to epirubicin solution [28]. Li et al., developed epirubicin loaded self-assembled cholesterol-conjugated carboxymethyl curdlan nanoparticles as a novel carrier and reported a higher in vitro cytotoxicity, improved mean residence time (MRT), better safety profile and higher antitumor efficacy in kunning mice [29]. However, oral delivery of EPI, which elicits poor bioavailability, is highly challenging as it is a substrate for both P-gp and CYP450 which are abundantly distributed in the intestine and liver [30–32]. In this perspective, present investigation was aimed at the development of EPI-NPs that could enhance the absorption via endocytic uptake across enterocytes as well as M cells of

449

intestinal epithelium thus evading the first pass metabolism and P-gp mediated efflux. 2. Materials and methods Epirubicin hydrochloride was gifted by Fresenius Kabi Pvt. Ltd, Gurgaon, Haryana, India. PLGA 50:50 (Resomer 503H) was provided ex gratia from Evonik Degussa, India, Pvt. Ltd, Mumbai, India. Polyvinyl alcohol (PVA, MW-25,000), sodium azide, flippin, chlorpromazine (CPZ), Trypsin-EDTA, penicillin streptomycin Amphotericin B mixture (PSA), and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma–Aldrich, Inc. (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM) and Fetal Calf Serum (FCS) were procured from Invitrogen (Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO, HPLC grade) Dichloromethane (DCM, AR grade), acetonitrile (ACN, HPLC grade), methanol (HPLC grade), o-phosphoric acid (HPLC Grade), sodium dihydrogen phosphate dihydrate, potassium dihydrogen phosphate and sodium hydroxide were purchased from SD fine chemical, Mumbai, India. Water was obtained from Milli-Q water purification system (Millipore, MA, USA). 2.1. Preparation of nanoparticles EPI-NPs were prepared by previously reported method with some modification [33]. Briefly, 10 mg of drug was dissolved in 400 ␮L of 0.5% w/v PVA solution (adjusted to pH 3) and 100 mg of PLGA was dissolved in 2 mL DCM. Drug solution was emulsified in polymeric solution under sonication over an ice bath for 60 s (25 W, 40% duty cycles, Sonopuls, Bandelin, Germany). Formed primary emulsion (w/o) was added drop by drop into 8 mL of external aqueous phase (2% w/v PVA) with sonication at 25% amplitude over an ice bath for 2 min, resultant dispersion was subjected to solvent evaporation under mild magnetic stirring (400 rpm) at room temperature. After solvent evaporation, obtained nano-suspension was subjected to centrifugation at 18,000 rpm for 20 min followed by washing of the pellet and subsequently lyophilization for 24 h at −50 ◦ C and 0.015 mbar pressure to achieve a free flowing and easily dispersible lyophilized NPs (Lab Conco., LPYH, Lock 6, USA freeze dryer). 2.2. Particle size and zeta potential The particle size, size distribution (polydispersity index, PDI) and zeta potential analyses of EPI-NPs were performed by using the dynamic light scattering method (DLS) with a computerized inspection system (Malvern Zetasizer, Nano-ZS, Malvern, UK) and analyzed by ‘DTS nano’ software. For analysis, 1 mg of NPs were dispersed in 2 mL of Milli Q water and subjected to size and zeta potential measurement. 2.3. Drug content or drug loading Drug loading of EPI-NPs was determined as reported by Kalaria et al. [9]. Two mg of lyophilized EPI-NPs were dissolved in dimethyl sulfoxide (HPLC, grade) followed by filtration through 0.2 ␮m syringe filter and analyzed by HPLC using Water separation module, e2695 HPLC system with Empower software coupled to UV detector (In house developed method). The separation was achieved at 30 ◦ C with flow rate of 1 mL/min using C18 column, 4.6 × 250 mm, 5 ␮m analytical column (Purosphere, Merck) and detection was made at 233.5 nm. Mobile phase consisted of aqueous (60% v/v) and organic phase (40% v/v). Aqueous phase comprised of 0.1% v/v o-phosphoric acid aqueous solution and organic phase comprised

450

M. Tariq et al. / Colloids and Surfaces B: Biointerfaces 128 (2015) 448–456

of 0.1% o-phosphoric acid solution in ACN and methanol (80:20 v/v) mixture. Drug loading was calculated by using following formula; % Drug loading =

Total amount of the drug entrapped × 100 Total amount of product

2.4. Surface morphology Size and surface morphological analyses of EPI-NPs were performed by transmittance electron microscope (TEM) and scanning electron microscope (SEM). For TEM analysis, particles suspended in Milli Q water were adsorbed on copper grid, 300 meshes followed by samples treatment with salt of a heavy metal viz. 1% uranyl acetate for negative staining, followed by drying. Dried samples were imaged in transmission electron microscope (TOPCON 002B, Tokyo, Japan), analysis was done at an accelerating voltage of 200 kV. For SEM analysis, freeze dried EPI-NPs were mounted on aluminum stubs using double-sided carbon adhesive tape. A circular cover slip was gently placed over the stub to enable even distribution of the sample. They were viewed with an EVO LS 10 (Carl Zeiss, Brighton, Germany) scanning electron microscope operating at an accelerating voltage of 1.0 kV and at high vacuum. Data analysis was done using Smart SEM software program. 2.5. In vitro release study Release study of EPI was performed using dialysis bag method. Two mg of EPI and EPI-NPs equivalent to 2 mg EPI were dispersed in 5 mL of dissolution media and then poured into dialysis bag (MW cut off of 8–10 kDa, Spectra/Por® Spectrum Laboratories, Inc. Rancho Dominguez, CA, USA) and subsequently immersed in 50 mL of dissolution media. In vitro release study was carried out in incubator shaker (SI6R, Shel Lab, Sheldon Mfg. Inc. Ave, Cornelius, OR, USA) under mild stirring (100 rpm) at 37 ± 0.5 ◦ C to maintain the temperature and stirring speed. The dissolution studies were carried out in simulated gastric fluid (pH 1.2) for first 2 h and simulated intestinal fluid (pH 6.5) thereafter for further 48 h. Samples were removed at scheduled time interval (0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, 24 and 48 h) and analyzed by HPLC (In house developed method). 2.6. In vitro cell lines studies 2.6.1. Human breast adenocarcinoma cell line (MCF-7) culture MCF-7 cell line (American Type Culture Collection, Manassas, VA, USA) was exploited to assess the cytotoxic potential of the formulation. Cells were cultured in a DMEM media supplemented with 10% fetal calf serum and 1% antibiotic solution (104 UI/mL of penicillin, and 10 mg/mL streptomycin and 25 ␮g/mL amphotericin B) at 37 ◦ C in 5% CO2 atmosphere. Cells were harvested at 80% confluence by Trypsin-EDTA. 2.6.2. Human colon adenocarcinoma cell line (Caco-2) culture Caco-2 cell line (American Type Culture Collection, Manassas, VA, USA) was grown on 75 cm2 flask in Dulbecco’s modified Eagle medium (DMEM) supplemented with 12% (v/v) fetal bovine serum, 1% (v/v) non-essential amino acids, 1% (v/v) sodium pyruvate, and 1% antibiotic solution (104 UI/mL of penicillin, 10 mg/mL of streptomycin, 25 ␮g/mL of amphotericin B) at 37 ◦ C in a humidified incubator 5% CO2 /95% air atmosphere. Cells were harvested at 80% confluence with Trypsin–EDTA. 2.6.3. In vitro cell viability assay Comparative cell cytotoxicity potential of EPI-S and EPI-NPs was done against MCF-7 cell line by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) colorimetric assay. Harvested

cells were seeded on 96-well flat-bottomed plates (Falcon Plate, Corning Costar, USA) at a density of 5000 cells/well and incubated for 24 h to allow cells adherence. After 24 h growth media was removed from each well and 200 ␮L of media containing different concentration of EPI-S and EPI-NPs were added. After 48 h of incubation, MTT solution (5 mg/mL in PBS diluted to 10 times with media) was added to each well (100 ␮L) then again subjected to 4 h of incubation followed by removal of media. Finally, 100 ␮L of DMSO was added into each well to dissolve formazan crystal followed by measurement of absorbance of each well at 570 nm using a microplate reader (LMR-340 M, Labexim International, Austria). Software Graph Pad/Prism5 was used to process data and to calculate the IC50 values of EPI-S and EPI-NPs [34]. 2.6.4. Cellular uptake Cell uptake studies were conducted at two different concentrations; 2.5 ␮M being lower and 10 ␮M being higher. After 21 days of differentiation, cells were incubated separately with serum free DMEM media (control), EPI-S and EPI-NPs suspension for 4 h at 37 ◦ C. After completion of incubation time, cells were washed, trypsinized for 2 min and neutralized by addition of cold culture medium. Further, cells were centrifuged followed by re-suspension in PBS. Treated samples were transferred to fluorescence activated cell sorter (FACS) tubes (VWR) and analyzed by FACS using BD FACSCantoTM and BD FACSDivaTM software, version 6.1.2 (BD Biosciences, San Jose, CA, USA). This experiment was performed in triplicate for all conditions and a total of 10,000 cells per sample were analyzed. Data was expressed as mean ± SD and compared by applying two tailed paired test using software GraphPad Instat 3 (USA), p < 0.05 was considered as the level of significance. 2.6.5. Mechanism of cellular uptake of EPI-NPs Cellular uptake study of EPI-NPs was performed in the presence of different endocytic inhibitors in order to elucidate its uptake mechanism [35]. Thus, Caco-2 cells were grown separately in four different groups and each group was subjected to prior treatment with specific agent followed by incubation with EPINPs suspension. First group was treated as control i.e. no prior treatment with any agent and their uptake was assumed 100% [3,4]. Second group was pre-treated with 25 ␮M sodium azide for 1 h at 37 ◦ C to inhibit the active transcellular transport [36], third group was subjected to 1 h pre-treatment with 1 ␮g/mL flippin to block caveolae-mediated endocytosis [35,37], similarly fourth group was subjected to pre-treatment with 10 ␮g/mL chlorpromazine to inhibit clathrin mediated endocytosis [38] followed by incubation with EPI-NPs suspension (equivalent to 10 ␮M EPI) for 4 h to carry out cellular uptake study, rest of the procedure was followed as described earlier (Section 2.6.4). Data was expressed as mean ± S.D. and compared by applying Dunnett test (compare control versus all) using software GraphPad Instat 3 (USA), p < 0.05 was considered as the level of significance. 2.6.6. Cellular transport study Cellular transport study was performed according to previous report with slight modification [39]. In brief, harvested cells were seeded on polycarbonate membrane filters (Transwell® cell culture chambers 0.4 ␮m pore size, 1.12 cm2 growth area, Corning Costar, Cambridge, MA, USA) at a density of 105 cells/insert. Initially media was changed every two days for first two weeks and every day thereafter (0.5 mL per insert and 1.5 mL per well). After 3 weeks, cell monolayers were used for transport study. Cell monolayers were rinsed twice with PBS followed by assessment of transepithelial electrical resistance (TEER) by using a Millicell® -ER system (Millipore Corporation, Bedford, MA, USA). Only cell monolayers with TEER values over 300  cm2 were included in the study. Transport

M. Tariq et al. / Colloids and Surfaces B: Biointerfaces 128 (2015) 448–456

of EPI-S and EPI-NPs was studied in the apical to basal direction on Caco-2 cells. To initiate the experiment, 0.5 mL of test solutions (diluted with serum free DMEM) were added to apical side whereas 1.5 mL of serum free DMEM was added to basolateral side. 0.2 mL sample was withdrawn at each time point (15, 30, 45, 60, 75, 90, 120, 150, 180 min) from basolateral side. Collected samples were analyzed by HPLC. Apparent permeability coefficient (Papp) was calculated according to the following equation and expressed in cm/s [40]. Papp =

1 dQ × AC0 dt

where dQ/dt is the rate of drug appearance on the basolateral side, C0 is the initial concentration at the apical side and A is the surface area of monolayer (cm2 ). Data was expressed as mean ± S.D. and compared by applying two tailed paired test using software GraphPad Instat 3 (USA), p < 0.05 was considered as the level of significance. 2.7. Intestinal permeation by gut sac method Male wistar rats, weighing 200 ± 20 g, were obtained from Central Animal House, Jamia Hamdard under the protocol reviewed and approved by the Institutional Animal Ethics Committee of Jamia Hamdard (Approval No. 947, 2013). The animals were kept under standard laboratory conditions (temperature 25 ± 2 ◦ C, relative humidity 55 ± 5%) and were housed in polypropylene cages with free access to standard laboratory diet. Permeation study was carried out as reported with slight modifications [41]. Animals were kept without food for 12 h but water was allowed ad libitum before experimentation. Animals were sacrificed by cervical dislocation under excessive ether anesthesia. Ileum was taken out, washed with saline and one end of segment was ligated with thread while other end was mounted on a port of an in house developed assembly to conduct the study. Sac of each segment was either filled with EPI-S solution (1 mL, 100 ␮g/mL) or EPI-NPs suspension (equivalent to 100 ␮g of EPI in 1 mL) as per the study protocol then incubated into pre-warmed (37 ± 0.5 ◦ C) and pre-oxygenated Tyrode’s buffer (10 mL). Sample (0.5 mL) was collected at different time intervals 15, 30, 45, 60, 75, 90 min and replenished with same volume of fresh Tyrode’s buffer solution. All the samples were analyzed by HPLC. Papp was calculated according to the following equation and expressed in cm/s [40]. Papp =

dQ 1 , × AC0 dt

where dQ/dt is the rate of drug appearance on the basolateral side, C0 is the initial concentration over the apical side and A is the surface area of intestinal tissue (cm2 ). Data was expressed as mean ± S.D. and compared by applying two tailed paired test using software GraphPad Instat 3 (USA) p < 0.05 was considered as the level of significance. 2.8. Pharmacokinetic (PK) study The protocol for PK studies was reviewed and approved by the Institutional Animal Ethics Committee of Jamia Hamdard (Protocol No. 947, 2013). Male albino wistar rats, weight 200 ± 20 g were kept under standard laboratory conditions (temperature 25 ± 2 ◦ C and relative humidity 55 ± 5%) with free access to standard laboratory diet (Lipton feed, Mumbai, India) and water ad libitum. Rats were divided into two groups (n = 6) and each group was treated with EPI at the dose of 10 mg/kg orally. First group was treated with EPI-S while second group received EPI-NPs suspension. Blood sample (0.2 mL) was withdrawn from retro orbital choroid plexus at 0.5, 1, 2, 3, 4, 6, 8, 12, 24, and 48 h under mild

451

anesthesia and collected into EDTA coated blood sampling tubes. Plasma was separated by centrifugation at 5000 rpm for 10 min and stored at −20 ◦ C till further analysis. Drug analysis was carried out using HPLC with slight modification [42]. Data was expressed as mean ± S.D. and compared by applying two tailed paired test using software GraphPad Instat 3 (USA), p < 0.05 was considered as the level of significance. 3. Results and discussion 3.1. Nanoparticles preparation and in vitro characterization EPI is a water soluble anticancer agent hence, double emulsion solvent evaporation method was used for the preparation of EPINPs. Moreover, EPI being basic in nature is more soluble in acidic medium as compared to distilled water. Hence, acidified internal aqueous phase (pH 3) was chosen to improve encapsulation. Generally, hydrophilic drugs show poor entrapment and a tendency to leach out from internal aqueous phase into external aqueous phase due to its lower affinity for lipophilic polymer and organic phase. Therefore, 0.5% w/v PVA was incorporated along with the pH adjustment to enhance the viscosity of internal aqueous phase which can reduce the leaching of drug into external aqueous phase. Size and surface charge of NPs are important factors which decide their fate i.e. cellular uptake and interaction with cell membranes. Generally, particles within a size range of 50–500 nm are considered appropriate for cellular internalization via endocytosis [43–45]. Particle size and size distribution of developed EPI-NPs were measured by Malvern zetasizer at intensity weighted scale. Average hydrodynamic particle size of developed NPs was found to be 235.3 ± 15.12 nm and exhibited narrow polydispersity index, 0.116 ± 0.01 (Fig. 1a). TEM and SEM analyses (Fig. 1b and c) also revealed the size of particles in the range of 150–250 nm which is in agreement with the results of dynamic light scattering. Furthermore, micrographs elucidated smooth surfaced and spherical shaped NPs. Zeta potential of EPI-NPs was found to be −27.5 ± 0.7 (Fig. 1d) that can be attributed to the free carboxylic group of polymer. Drug content of the EPI-NPs was found to be 39.12 ± 2.13 ␮g/mg (3.9% drug loading) which is considered reasonable for hydrophilic drugs [9]. 3.2. In vitro release study In vitro release profiles of EPI demonstrated a cumulative percentage release of 96.23 ± 2.6% from EPI-S in first hour and 80.94 ± 6.39% from EPI-NPs in 48 h (Fig. 2). EPI-NPs showed biphasic release profile initial burst release followed by sustained release. Initially, 21.03 ± 1.66% of total drug was released in first 2 h and remaining thereafter. Initial burst release can be attributed to its high solubility in dissolution media and drug available at/near the surface of the NPs which diffused out quickly while sustained effect might be due to the presence of drug molecules at the center and or in a densely embedded polymer matrix. In addition, hydrophobic nature of the polymer probably restricted the penetration of dissolution medium and the molecules present at the core of NPs have to follow a longer diffusion path in comparison to molecules available at the surface. Sustained release behavior of the formulation favors the therapeutic effect for extended period of time. Further, different in vitro release kinetic models including zero order, first order, Higuchi model, Korsmeyer–Peppas were applied to analyze the release pattern of EPI from EPI-NPs which suggested the Higuchi as a best fit model to explain the release profile (r2 , 0.966). The observations are evidently indicative of the fact that release of drugs is due to matrix diffusion with simultaneous erosion of the controlled system [14]. The release kinetic was further

452

M. Tariq et al. / Colloids and Surfaces B: Biointerfaces 128 (2015) 448–456

Fig. 1. Particles size distribution, zeta potential and morphological analysis of EPI-NPs; (a) intensity weighted size distribution curve; (b) TEM micrograph; (c) SEM micrograph; (d) zeta potential curve.

confirmed by Korsmeyer–Peppas semi empirical model by determining the value of exponent ‘n’ at the linear portion of curve. The value of n was found to be 0.46 which suggests that the developed EPI-NPs followed Fickian diffusion release pattern [46].

retaining their activity for prolonged period of time in the cellular milieu, especially in the nuclear region. On the contrary free drug solution absorbs poorly and washes out quickly owing to its hydrophilic nature [21,28,34].

3.3. In vitro cell viability assay

3.4. Cellular uptake

In vitro anticancer activity of EPI-S and EPI-NPs was assessed in terms of the number of cells survived after the cytotoxic treatment for 48 h (Fig. 3). The IC50 values of EPI-S and EPI-NPs were found to be 1278 ± 70 nM and 824 ± 73 nM, respectively. The difference in IC50 values of EPI-S and EPI-NPs were found to be highly significant (p < 0.001). The observation can be attributed to intracellular trafficking behavior of NPs owing to their small size and sustained drug release from NPs in the cellular vicinity which helps in

Cellular uptake of the NPs is one of the key aspects that decide drug absorption in in vivo. Hence, it was of particular interest to examine whether the developed system would enhance EPI cellular uptake or not. Comparative cell uptake study of EPI-S and EPI-NPs was performed by using FACS at two different concentrations (lower, 2.5 ␮M and higher, 10 ␮M) in Caco-2 cells and the results are depicted in Fig. 4. EPI, being hydrophilic in nature and

Fig. 2. In vitro release profile of EPI-S and EPI-NPs performed by using dialysis bag method, revealing sustained release pattern of EPI-NPs (mean ± SD, n = 3).

Fig. 3. Cell cytotoxicity potential of EPI-S and EPI-NPs against MCF-7 cell lines measured by MTT assay after 48 h incubation. Values were expressed as mean ± SD (n = 3). EPI-NPs showed significantly high cytotoxicity (p < 0.001).

M. Tariq et al. / Colloids and Surfaces B: Biointerfaces 128 (2015) 448–456

453

Fig. 4. Comparative cellular uptake study conducted on Caco-2 cell line model at two different concentration of EPI-S and EPI-NPs, and the data is presented through FACS histogram; (a) control (untreated cell); (b) 2.5 ␮M EPI-S; (c) 2.5 ␮M EPI-NPs; (d) 10 ␮M EPI-S; (e) 10 ␮M EPI-NPs, significantly higher uptake of EPI-NPs (p < 0.001).

a substrate for P-gp exhibited poor cellular uptake. However, FACS uptake studies of EPI-NPs revealed higher fluorescence intensity inside the cells at both the chosen concentrations. Lower concentration of 2.5 ␮M in EPI-S exhibited a fluorescent intensity of 10.63 ± 0.6% while an enhanced value of 26.36 ± 0.5% was obtained in case of EPI-NPs (p < 0.001). On the other hand, at a higher concentration (10 ␮M), EPI-S gave a value of 50.1 ± 2.36%, and a significantly higher value (78.8 ± 1.69%) was obtained by using EPI-NPs (p < 0.001). The enhancement in fluorescence at both concentrations revealed the superiority of developed EPI-NPs [21,34]. However, it is worth noting that at a lower concentration of EPI, enhancement was 2.48 fold high while it was found to be 1.57 fold high at a higher concentration. It can be attributed to the fact that EPI itself is a P-gp substrate which binds with P-gp transporter reversibly thus at a lower concentration relatively more number of free P-gp transporter would be available to efflux out free EPI molecules hence more expulsion of EPI-S while at high EPI concentration more number of P-gp transporter would be occupied by EPI thus less number of free P-gp efflux transporter hence relatively less efflux of EPI-S therefore uptake difference between EPI-S and EPI-NPs is higher at low concentration of EPI than at higher concentration of EPI (p < 0.001).

to 30.88 ± 3.750% (p < 0.01) in the presence of sodium azide which is an active transport inhibitors confirming that uptake of particles is an energy dependent process and our results are corroborating with previous reports [3,36]. Further, cellular uptake was reduced to 76.54 ± 7.8% (p < 0.01) and 59.95 ± 7.06% (p < 0.01) in the presence of chlorpromazine (clathrin mediated endocytosis inhibitor) and flippin (caveolae mediated endocytosis inhibitor), respectively. It indicates that both the mechanisms were involved in uptake of particles however results are showing the dominance of caveolae mediated endocytosis in the cellular uptake of the EPI-NPs. It can

3.5. Mechanism of cellular uptake In order to explore the pathways of cellular uptake of NPs, study was carried out in the presence of specific agents. These agents interfere with a particular phase of endocytosis and block a specific pathway. As shown in Fig. 5, uptake of EPI-NPs was reduced

Fig. 5. Effect of endocytic inhibitors on intracellular uptake of EPI-NPs conducted against Caco-2 cell line after 4 h of incubation (mean ± SD, n = 3), the value of control group was considered as 100%, significant reduction in uptake of EPI-NPs was observed in the presence of inhibitors (p < 0.01).

454

M. Tariq et al. / Colloids and Surfaces B: Biointerfaces 128 (2015) 448–456

be explained in the light of size dependent particles uptake. The average particles size of formulation is 235.3 ± 15.12 nm comprising of both large (≥200 nm) as well as small particles (≤200 nm). Rejman et al., reported that the particles ≤200 nm are preferentially taken up by the clathrin mediated endocytosis while the larger particles by the caveolae mediated endocytosis [47]. Dominancy of caveolae mediated endocytosis can be attributed to presence of higher percentage of larger particles (≥200 nm). Moreover, it would be advantageous as caveolae mediated endocytosis is able to skip lysosomal degradation pathway which can help in further enhancement of bioavailability of drugs [36,48]. 3.6. Cellular transport across Caco-2 cells monolayer Permeability enhancement of drugs across the intestine is one of approaches which are applied to improve oral bioavailability. Various research groups have exploited NPs to improve the permeability, thus oral bioavailability of drugs from diverse class including cancer chemotherapeutics [1,24,48]. In the present study also, similar approach has been utilized in order to improve oral bioavailability of EPI. Hence, to predict EPI absorption across the human intestine, permeation potential of EPI-S and EPI-NPs was evaluated (apical to basolateral) on Caco-2 cell lines monolayer model. Permeation potential was evaluated in terms of Papp. Drug transportation profile of EPI through EPI-S and EPI-NPs across the Caco-2 cell lines was exhibited in Fig. 6a. Higher permeation of EPI through EPI-NPs was observed at each time point and Papp (1.754 ± 0.133 × 10−6 cm/s) was found to be significantly high (p < 0.001) as compared to EPI-S (0.636 ± 0.068 × 10−6 cm/s). Significant improvement in permeability of EPI through EPI-NPs (∼2.76 fold higher) can be attributed to; hydrophobic nature of PLGA polymer would have facilitated the interaction of particles with lipophilic biological membrane thus cellular uptake of particles via endocytosis, and encapsulation of drug provided it protection from P-gp transporter. The plausible reasons for poor permeation of EPI through EPI-S are; its high aqueous solubility and expulsion through P-gp transporters. Results are in corroboration with previous reports [1,12,48]. 3.7. Intestinal permeation study by gut sac method Numerous scientific reports have established a correlation between permeation across the Caco-2 cells and fraction absorbed in human subjects with reference to free drug solution. However, it may not be completely true for NPs as it is a well known fact that Peyer’s patches present in ileum also do impart a significant role in NPs uptake. Hence, to simulate the actual conditions more stringently in situ intestinal permeation studies across rat intestine was also accomplished. In situ intestinal permeation studies can be done in two ways; everted and non-everted gut sac method. Ruan et al. reported the applicability of non-everted rat

Fig. 7. Plasma drug concentrations versus time profiles of EPI in male wistar rats after single oral dose of EPI-S and EPI-NPs (10 mg/kg). Significantly high AUC was achieved with EPI-NPs (p < 0.001, mean ± SD, n = 6).

intestinal sacs method and revealed a good relationship between the permeability of model drugs and their corresponding human absorption data for 11 marketed compounds [41]. In the present study, non-everted intestinal sac method was adopted for the assessment of permeability potential of nanoparticles. Transportation profile of EPI through EPI-S and EPI-NPs across the rat ileum was shown in Fig. 6b. Similar to Caco-2 cell line model, higher permeation of EPI through EPI-NPs was observed at each time point and Papp for EPI-NPs was found to be 2.78 × 10−6 cm/s, significantly high (p < 0.0001, ∼4.49 fold) as compared to EPI-S (0.619 × 10−6 cm/s). Results are in agreement with previous report [4,12]. Further, Papp of EPI through EPI-NPs measured by gut sac method was found to be significantly high (p < 0.001) as compared to measured on Caco-2 cell lines monolayer model, ∼1.8 fold high. It can be attributed to uptake of particles through M cells of Peyer’s patches present in rat ileum. Thus, confirmed the role of Peyer’s patches in bioavailability enhancement. 3.8. Pharmacokinetic studies To further strengthen in vitro and in situ findings, pharmacokinetic parameters were determined in male wistar rats. Pharmacokinetic analysis was performed by non-compartmental (model independent) method. Comparative plasma drug concentration versus time profile of EPI-S and EPI-NPs after single dose oral administration has been depicted in Fig. 7. The values of Cmax and Tmax were identified directly by plasma drug concentration versus time plot. Area under the plasma concentration-time curve (AUC) was calculated using the trapezoidal method (Table 1). Cmax and Tmax of EPI-S were found to be 56.3 ± 5.94 ng/mL and 1.16 ± 0.4 h, respectively while significantly high Cmax , 132.52 ± 12.66 ng/mL (p < 0.001) and Tmax , 3.833 ± 0.4 h (p < 0.01) were attained with EPI-NPs, respectively. As a consequent of higher

Fig. 6. Transepithelial flux of EPI at 37 ◦ C from EPI-S and EPI-NPs across; (a) Caco-2 cells monolayer, apical to basolateral direction (mean ± SD, n = 3), significantly high flux of EPI through EPI-NPs, p < 0.001; (b) across ileum, apical to basolateral direction (mean ± SD, n = 3), significantly high flux of EPI through EPI-NPs, p < 0.001.

M. Tariq et al. / Colloids and Surfaces B: Biointerfaces 128 (2015) 448–456

455

Table 1 Pharmacokinetic parameters of EPI after single oral dose of EPI-S and EPI-NPs (mean ± SD n = 6). Parameters

Cmax (ng/mL)

EPI-S EPI-NPs

56.3 ± 5.94 132.52 ± 12.66*** ***

Tmax (h)

AUC0−t (ng h/mL)

AUC0−∞ (ng h/mL)

Keli (h−1 )

Rel bio

1.16 ± 0.4 3.83 ± 0.4**

512 ± 56.32 1895.28 ± 259.27***

551.32 ± 64.06 2148.6 ± 299.5***

0.064 0.046

3.9 fold

***

**

p < 0.01. *** p < 0.001. Rel bio, relative bioavailability.

Cmax and Tmax and lower elimination rate constant (Table 1) higher AUC0−t 1895.28 ± 259.27 ng h/mL was achieved with EPI-NPs as compared to EPI-S, 512.16 ± 56.32 ng h/mL (p < 0.001). The high Cmax obtained with EPI-NPs can be attributed to the efficient absorption by virtue of their small size and surface properties (hydrophobic surfaces). Both these properties might have facilitated their interaction with enterocytes as well as Peyer’s patches subsequently their uptake thus allowing their entry into the systemic circulation [22,24]. Prolonged Tmax revealed the sustained in vivo release of the drug which is in agreement with the in vitro release profile. Further, it can be understood in term of particles taken up by enterocytes and M cells which releases the drug at lymphatic site and then reaches into the systemic circulation [1,22] Significantly high AUC0–∞ , p < 0.001 (Table 1) observed with EPI-NPs which resulted in ∼3.9 fold increment in oral bioavailability as compared to EPI-S. Higher absorption achieved with NPs might be attributed to the fact that, encapsulation provides protection from hostile environment of GIT, better permeation across the intestine, direct uptake through enterocytes and M cells may result into bypassing of first pass metabolism and P-gp transporters which collectively lead to improved oral bioavailability [1,9,49]. A number of studies have reported a rapid clearance of free epirubicin solution, (marketed injection) thus necessitate high dose and frequent dosing which is undesirable and leads to adverse effects [29,50]. However, developed EPI-NPs exhibited therapeutic plasma drug concentration for extended period of time. Our findings are corroborating with previous reports as they also demonstrated the ability of oral nanoformulation to maintain the plasma drug concentration for extended duration. Paclitaxel loaded nanoparticles administered orally exhibited AUC comparable to Taxol® given by i.v. route, while Yuan et al., reported higher AUC for doxorubicin incorporated PEG-SLN in comparison to doxorubicin solution injected intravenously [3,51]. Thus, EPI-NPs can be a potential candidate to improve oral bioavailability of epirubicin, to achieve therapeutic effect, obviate the need of infusion equipment and hospitalization. However, extensive safety and efficacy studies are required to bring it in clinically viable form.

Acknowledgments Authors are indebted to Fresenius Kabi and Evonik Degussa, Pvt. Ltd, India for providing gift samples of Epirubicin hydrochloride and PLGA 50:50 (Resomer 503 H) respectively. We appreciate the contribution of Jamia Millia Islamia, and sophisticated analytical instrumentation facility, AIIMS, New Delhi India to carry out SEM and TEM analysis. Authors are also thankful to Jamia Hamdard for providing all the facilities to carry out research work. This work is financially supported by Department of Biotechnology, Govt. of India.

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]

[16] [17] [18] [19] [20] [21]

4. Conclusion Conclusively, EPI-NPs were successfully developed and the final formulation demonstrated superior anti-proliferative activity against MCF-7 cell lines. Transport studies conducted on Caco-2 cell monolayer and rat ileum showed a significant improvement in permeation of EPI through EPI-NPs. Elaboration of uptake study in the presence of specific inhibitors revealed the dominance of caveolae mediated endocytosis. Cell uptake, cellular and intestinal transport studies established a significant role of receptor mediated endocytosis and particles uptake by Payer’s patches which revealed that the developed system has immense potential to bypass P-gp mediated efflux as well as CYP450 mediated metabolisms thereby resulting in a marked improvement of 3.9 fold in oral bioavailability when tested in wistar rats. Hence, it is envisaged that PLGA NPs would be a prospective platform for effective oral delivery of epirubicin.

[22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35]

G. Joshi, A. Kumar, K. Sawant, Eur. J. Pharm. Sci. 60 (2014) 80. W. Xu, P. Ling, T. Zhang, J. Drug Deliv. 2013 (2013) 340315. H. Yuan, C.Y. Chen, G.H. Chai, Y.Z. Du, F.Q. Hu, Mol. Pharmacol. 10 (2013) 1865. H.J. Yao, R.J. Ju, X.X. Wang, Y. Zhang, R.J. Li, Y. Yu, L. Zhang, W.L. Lu, Biomaterials 32 (2011) 3285. L.M. Negi, M. Tariq, S. Talegaonkar, Colloids Surf. B: Biointerfaces 111C (2013) 346. W. Sun, S. Mao, Y. Wang, V.B. Junyaprasert, T. Zhang, L. Na, J. Wang, Int. J. Pharm. 386 (2010) 275. B. Sarmento, A. Ribeiro, F. Veiga, D. Ferreira, R. Neufeld, Biomacromolecules 8 (2007) 3054. X. Li, Y. Xu, G. Chen, P. Wei, Q. Ping, Drug Dev. Ind. Pharm. 34 (2008) 107. D.R. Kalaria, G. Sharma, V. Beniwal, M.N. Ravi Kumar, Pharm. Res. 26 (2009) 492. S.A. Galindo-Rodriguez, E. Allemann, H. Fessi, E. Doelker, Crit. Rev. Ther. Drug Carr. Syst. 22 (2005) 419. R.A. Jain, Biomaterials 21 (2000) 2475. C. He, L. Yin, C. Tang, C. Yin, Biomaterials 33 (2012) 8569. C.E. Astete, C.M. Sabliov, J. Biomater. Sci. Polym. Ed. 17 (2006) 247–289. T. Khuroo, D. Verma, S. Talegaonkar, S. Padhi, A.K. Panda, Z. Iqbal, Int. J. Pharm. 473 (2014) 384. K.T. Householder, D.M. DiPerna, E.P. Chung, G.M. Wohlleb, H.D. Dhruv, M.E. Berens, R.W. Sirianni, Int. J. Pharm (2015), http://dx.doi.org/10.1016/ j.ijpharm.2015.01.002. H. Okada, H. Toguchi, Crit. Rev. Ther. Drug Carr. Syst. 12 (1995) 1. A.T. Florence, Pharm. Res. 14 (1997) 259. N. Hussain, V. Jaitley, A.T. Florence, Adv. Drug Deliv. Rev. 50 (2001) 107. S. Fatma, S. Talegaonkar, Z. Iqbal, A.K. Panda, L.M. Negi, D.G. Goswami, M. Tariq, Drug Deliv. (2014) 1. N. Akhtar, S. Talegaonkar, R.K. Khar, M. Jaggi, J. Biomed. Nanotechnol. 9 (2013) 1216. A.K. Jain, N.K. Swarnakar, M. Das, C. Godugu, R.P. Singh, P.R. Rao, S. Jain, Mol. Pharmacol. 8 (2011) 1140. S. Jain, S.R. Patil, N.K. Swarnakar, A.K. Agrawal, Mol. Pharmacol. 9 (2012) 2626. V. Bhardwaj, D.D. Ankola, S.C. Gupta, M. Schneider, C.M. Lehr, M.N. Kumar, Pharm. Res. 26 (2009) 2495. A.K. Jain, N.K. Swarnakar, C. Godugu, R.P. Singh, S. Jain, Biomaterials 32 (2011) 503. V. Zabaleta, G. Ponchel, H. Salman, M. Agueros, C. Vauthier, J.M. Irache, Eur. J. Pharm. Biopharm. 81 (2012) 514. Z. Sezgin-Bayindir, A. Onay-Besikci, N. Vural, N. Yuksel, J. Microencapsul. 30 (2013) 796. D. Ormrod, K. Holm, K. Goa, C. Spencer, Drugs Aging 15 (1999) 389. H.Z. Zhang, F.P. Gao, L.R. Liu, X.M. Li, Z.M. Zhou, X.D. Yang, Q.Q. Zhang, Colloids Surf. B: Biointerfaces 71 (2009) 19. L. Li, F.P. Gao, H.B. Tang, Y.G. Bai, R.F. Li, X.M. Li, L.R. Liu, Y.S. Wang, Q.Q. Zhang, Nanotechnology 21 (2010) 265601. W.T. Bellamy, Annu. Rev. Pharmacol. Toxicol. 36 (1996) 161. L.J. Goldstein, H. Galski, A. Fojo, M. Willingham, S.L. Lai, A. Gazdar, R. Pirker, A. Green, W. Crist, G.M. Brodeur, et al., J. Natl. Cancer Inst. 81 (1989) 116. D.R. Nelson, Biochim. Biophys. Acta 1814 (2011) 14. U. Bilati, E. Allemann, E. Doelker, Eur. J. Pharm. Sci. 24 (2005) 67. F. Gao, L. Li, H. Zhang, W. Yang, H. Chen, J. Zhou, Z. Zhou, Y. Wang, Y. Cai, X. Li, L. Liu, Q. Zhang, Int. J. Pharm. 392 (2010) 254. E. Roger, F. Lagarce, E. Garcion, J.P. Benoit, J. Control Release 140 (2009) 174.

456

M. Tariq et al. / Colloids and Surfaces B: Biointerfaces 128 (2015) 448–456

[36] P. Zhang, G. Ling, J. Sun, T. Zhang, Y. Yuan, Y. Sun, Z. Wang, Z. He, Biomaterials 32 (2011) 5524. [37] P.A. Orlandi, P.H. Fishman, J. Cell Biol. 141 (1998) 905. [38] L.H. Wang, K.G. Rothberg, R.G. Anderson, J. Cell Biol. 123 (1993) 1107. [39] Y.L. Lo, J. Control Release 90 (2003) 37. [40] P. Artursson, J. Karlsson, Biochem. Biophys. Res. Commun. 175 (1991) 880. [41] L.P. Ruan, S. Chen, B.Y. Yu, D.N. Zhu, G.A. Cordell, S.X. Qiu, Eur. J. Med. Chem. 41 (2006) 605. [42] D. Shin, S. Park, O.-S. Kwon, C.-W. Park, K. Han, Y. Chung, J. Pharm. Invest. 43 (2013) 243–249. [43] Y. Liu, T.M. Reineke, J. Am. Chem. Soc. 127 (2005) 3004. [44] L. Kou, J. Sun, Y. Zhai, Z. He, Asian J. Pharm. Sci. 8 (2013) 1.

[45] C.B. Woitiski, R.A. Carvalho, A.J. Ribeiro, R.J. Neufeld, F. Veiga, Strategies toward the improved oral delivery of insulin nanoparticles via gastrointestinal uptake and translocation, BioDrugs 22 (2008) 223–237. [46] N.A. Peppas, Pharm. Acta Helv. 60 (1985) 110. [47] J. Rejman, V. Oberle, I.S. Zuhorn, D. Hoekstra, Biochem. J. 377 (2004) 159. [48] L. Wang, L. Li, Y. Sun, J. Ding, J. Li, X. Duan, Y. Li, V.B. Junyaprasert, S. Mao, Int. J. Pharm. 471 (2014) 391. [49] W. Ke, Y. Zhao, R. Huang, C. Jiang, Y. Pei, J. Pharm. Sci. 97 (2008) 2208. [50] H.B. Tang, L. Li, H. Chen, Z.M. Zhou, H.L. Chen, X.M. Li, L.R. Liu, Y.S. Wang, Q.Q. Zhang, Drug Deliv. 17 (2010) 552. [51] M. Agueros, V. Zabaleta, S. Espuelas, M.A. Campanero, J.M. Irache, J. Control Release 145 (2010) 2.