Targeted Delivery of Doxorubicin-Loaded Poly (ε-caprolactone)-b-Poly (N-vinylpyrrolidone) Micelles Enhances Antitumor Effect in Lymphoma

Background The present study was motivated by the need to design a safe nano-carrier for the delivery of doxorubicin which could be tolerant to normal cells. PCL63-b-PNVP90 was loaded with doxorubicin (6 mg/ml), and with 49.8% drug loading efficiency; it offers a unique platform providing selective immune responses against lymphoma. Methods In this study, we have used micelles of amphiphilic PCL63-b-PNVP90 block copolymer as nano-carrier for controlled release of doxorubicin (DOX). DOX is physically entrapped and stabilized in the hydrophobic cores of the micelles and biological roles of these micelles were evaluated in lymphoma. Results DOX loaded PCL63-b-PNVP90 block copolymer micelles (DOX-PCL63-b-PNVP90) shows enhanced growth inhibition and cytotoxicity against human (K-562, JE6.1 and Raji) and mice lymphoma cells (Dalton's lymphoma, DL). DOX-PCL63-b-PNVP90 demonstrates higher levels of tumoricidal effect against DOX-resistant tumor cells compared to free DOX. DOX-PCL63-b-PNVP90 demonstrated effective drug loading and a pH-responsive drug release character besides exhibiting sustained drug release performance in in-vitro and intracellular drug release experiments. Conclusion Unlike free DOX, DOX-PCL63-b-PNVP90 does not show cytotoxicity against normal cells. DOX-PCL63-b-PNVP90 prolonged the survival of tumor (DL) bearing mice by enhancing the apoptosis of the tumor cells in targeted organs like liver and spleen.


Introduction
Adriamycin or Doxorubicin (DOX) hydrochloride, an anthracycline antibiotics is considered as the most effective chemotherapeutics used for the treatment of cancers including acute lymphoblastic and myelogenous leukemia, sarcomas, pediatric solid tumors, non-Hodgkin's and Hodgkin's lymphoma, neuroblastoma, and carcinomas of breast, ovaries and thyroid. The cytotoxic effects of DOX include DNA double helix intercalation, inhibition of topoisomerase II, production of reactive oxygen species (ROS), mitochondrial dysfunction, induction of p53, and activation of caspases [1,2]. However, its short biological life span, nonspecific distribution, development of drug resistance and severe cardiac toxicity including development of cardiomyopathy, have restricted its success [3]. Several polymer based delivery systems like polymeric micelles [4], synthetic polymer conjugates [5], and antibody targeted carriers [6] have been designed to reduce or alter toxicity in organs like heart, and enhances its potential to the site of drug actions like tumors. Yet the therapeutic efficacy of these formulations has not been demonstrated although modest increase has been reported in certain cases. The versatility of Poly-(e-caprolactone) (PCL) as a model polymer for pharmaceutical formulations along with functionalization features have been demonstrated over the past decade justifies its immense usefulness [7]. PCL modifications could provide better flexibility including modifications in drug release pattern, micellar drug delivery, tissue compatibility and circumvention of multi drug resistance [8]. Amphiphilic block copolymers have both hydrophobic and hydrophilic segments and undergoes self-assembly, which give rise to its typical aqueous solution and dispersion properties.
Amphiphilic block copolymers containing hydrophilic poly (Nvinylpyrrolidone) (PNVP) segment have several biologically important criteria's including high water solubility, low toxicity, biocompatibility, complexation capability, cryo-protectivity, lypoprotectivity and anti biofouling properties. Very few reports of the synthesis and characterization of amphiphilic block copolymers containing a biocompatible hydrophilic poly (N-vinylpyrrolidone) (PNVP) block and a biodegradable and biocompatible hydrophobic poly (e-caprolactone) (PCL) block, prepared via conventional radical polymerization of N-vinylpyrrolidone (NVP) are available in the literature [9][10][11]. Recently, Jeon et al. have reported the synthesis and characterization of well-defined amphiphilic PNVPb-PCL block copolymers prepared through the combination of cobalt-mediated controlled radical polymerization of NVP and controlled ROP of CL [12]. We have recently reported the synthesis of well-defined amphiphilic block copolymers of CL and NVP by combining the controlled ROP of CL and the controlled metal-free xanthate-mediated RAFT polymerization of NVP. Selfassembly behavior of the obtained amphiphilic block copolymers was studied in details using 1 H NMR, TEM, fluorescence spectroscopy, and light scattering [13].
Herein, we report the synthesis of a PCL-based amphiphilic polymeric nano delivery system DOX-PCL 63 -b-PNVP 90 , which is highly efficient in delivering DOX to tumor targets and also showed enhanced performance in DOX resistant forms as well. DOX-PCL 63 -b-PNVP 90 is significantly less toxic compared to free DOX against various cell subsets including lymphocytes, which are specifically susceptible to doxorubicin mediated death. DOX-PCL 63 -b-PNVP 90 prolonged the survival of tumor bearing mice compared to free DOX and restricts the metastasis of lymphoma to other organs. Besides that, DOX-PCL 63 -b-PNVP 90 prevents the accumulation of doxorubicin in targeted organs like heart compared to free DOX, suggesting its unique suitability for therapeutic purposes against lymphoma. Louis, USA, 99%) was dried over calcium hydride (CaH 2 ) for 48 h at room temperature and then distilled under reduced pressure before use. N-Vinylpyrrolidone (Aldrich, St Louis, USA, 99%) was dried over anhydrous magnesium sulfate and distilled under reduced pressure. 2, 2 / -Azobis (isobutyronitrile) (AIBN) (Spectrochem, Mumbai, India, 98%) was recrystallized from methanol. Tetrahydrofuran (THF) (Loba Chemie, Mumbai, India) was dried and fractionally distilled from sodium and benzophenone. Ethanol (Saraya Distilliary, India) was stirred over CaO overnight and distilled over fresh CaO. Potassium O-ethyl xantate was prepared according to our previous work [14]. PCL 63 -b-PNVP 90 was synthesized according to our recently published method [13]. Doxorubicin (Adriamycin) was purchased from Selleckchem (S1208) South Loop West, Suite 225, Houston, TX 77054, USA. RPMI 1640, penicillin and streptomycin were purchased from GIBCO, Invitrogen, Carlsbad, CA and fetal bovine serum from Hyclone, Logan, UT. Hoechst 33342 was purchased from Himedia, India. Annexin V Apoptosis Detection Kit (sc-4252 AK) was purchased from Santa Cruz Biotechnology, Dallas, TX, U.S.A. Other general & fine chemicals unless otherwise stated were purchased from SIGMA-ALDRICH, St. Louis, MO, USA.

Cell Lines and Cell Culture
Dalton lymphoma (DL) was maintained in the peritoneum of AKR (H2k) mice by periodic transfer of tumor cells via intraperitoneal injection [15,16]. Human erytholeukemic cell line K-562, T cell leukemia line JE6.1, and Burkitt lymphoma cell line Raji were kind gift of Dr. Santu Bandyopadhyay, IICB, Kolkata. The cell lines were originally obtained from American Type Culture Collection (ATCC), Manahass, USA. The cells were maintained in RPMI 1640 (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen, Carlsbad, CA), henceforth, called as complete medium. The cell lines used in the study were free from mycoplasma. To generate DOX-resistant (DOX-R) cell lines like K-562/DOX-R, JE6.1/ DOX-R, Raji/DOX-R, and DL/DOX-R, parental K-562, JE6.1, Raji, and DL cells were cultured in complete medium, with increasing concentrations of DOX up to a final concentration of 1 mM at 5% CO 2 , 37uC for one month. Resistant Cells were subsequently cloned by limiting dilution and grown in complete medium. The established cell lines were maintained in complete medium supplemented with 200 nM DOX to maintain the resistant phenotype. All DOX resistance cells were cultured in the DOX free medium for 14 days prior to experiments.

Preparation of DOX-PCL 63 -b-PNVP 90
DOX-loaded polymeric micelles were prepared by dialysis method. In the dialysis method, the copolymer PCL 63 -b-PNVP 90 (20 mg) was dissolved in DMF (2 ml), and a corresponding DOX?HCl (6 mg) with TEA (3 mol eq. to DOX?HCl) were added into the polymer solution. The mixture was stirred at room temperature for 24 h. Then, the final mixture was dialyzed against distilled water using a dialysis membrane [molecular weight cut off (MWCO) = 3500 g mol 21 ] for 8 h. During the first 3 h, the water was exchanged three times (every hour) and then twice during the following 5 h. The dialyzed solution was finally concentrated to 2 mL followed by lyophilisation to yield the solid micelle sample. Morphology and size of the micelles were determined by Transmission Electron Microscopy (TEM) (JOEL, JEM, 2100) operated at an acceleration voltage of 120 kV. The TEM samples were prepared by putting a drop of aqueous block copolymer solution (1 mg/mL) on the carbon coated copper grid followed by the removal of extra solution with a filter paper. The Dynamic Light Scattering Instrument (Malvern Zetasizer Ver. 7.01 Serial Number: MAL1077742) was performed to study the hydrodynamic size (Rh) of the free micelles and DOX-PCL 63 -b-PNVP 90 micelles using 0.5 mg/ml solution at 90 o angle.

Determination of Drug-loading Content (DLC) and Drugloading Efficiency (DLE) for DOX
The DLC was considered as the weight percentage of DOX in the micelle. It was quantified by determining the absorbance at 451 nm using a UV-Vis spectrophotometer (Shimadzu UV-1700). 1 mg of lyophilized sample was dissolved in 2 mL DMF for the UV-Vis measurement. To generate a calibration curve for the DLC calculation of DOX-loaded micelle, DOX solutions of various concentrations were prepared, and the absorbance at 485 nm was measured. DLC and DLE are calculated using the following two formula respectively:Drugloadingcontent(DLC)wt%W eight of drug load Weight of polymer |100, Drugloadingefficiency(DLE)%W Typical Drug Release from DOX-PCL 63 -b-PNVP 90 Micelles 5 mg of lyophilized DOX-loaded polymeric micelle dissolved in 1 ml phosphate buffer saline (PBS) of 6.4/7.4 pH was taken in a dialysis bag with a MWCO of 3500 g mol 21 , which was placed into 20 ml PBS solution. At different intervals, 3.0 ml was removed from the outer aqueous solution and replaced by fresh release medium (PBS solution). The released drug was quantified spectrophotometrically at 483 nm. The test was performed at 37uC.

In-vitro Tumor Cell Growth Inhibition Assay
Growth inhibitory potential of free DOX, PCL 63 -b-PNVP 90 micelle and DOX-PCL 63 -b-PNVP 90 micelle against parental and DOX-resistant DL, K-562, JE6.1, and Raji cells were studied by MTT assay. In a 96-well tissue culture plate 5610 3 cells/well were added and exposed to free DOX, PCL 63 -b-PNVP 90 micelle or, DOX-PCL 63 -b-PNVP 90 micelle solution with serial concentrations of (0.0001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5 mM). Plates were incubated at 37uC, 5% CO 2 , for 48 h. The cell proliferation was measured by CellTiter 96 Non-Radioactive Cell Proliferation Assay (MTT) kit from Promega, USA according to the manufacturer's protocol. The plates were incubated for 4 h with the MTT reagent and absorbance was measured at 570 nm using Synergy HT Multi-Mode Micro plate Reader BioTek, USA. The data presented as the percentage of inhibition of tumor cells and was calculated from the following formula: %GrowthInhibition1 1 À Experimental OD 570 Target OD 490 |100 Where Experimental OD value is the reading of tumor cells exposed to various concentrations of DOX and Target OD value is the corresponding value of tumor cell only cultured in the absence of drug.

DOX-PCL 63 -b-PNVP 90 Cytotoxicity Assay
The lytic activity of DOX-loaded PCL 63 -b-PNVP 90 was measured by 18 h non-radioactive cytotoxicity assay using the CytoTox 96 Non-Radioactive Cytotoxicity assay kit from Promega, USA, which quantitatively measures lactate dehydrogenase (LDH), a stable cytosolic enzyme released upon cell lysis. Target cells (5610 3 ) were added to 96-well tissue culture plate and exposed to serial concentrations (0.0001, 0.0002, 0.0005, 0.001, 0.005, 0.01, 0.05 mM) free DOX, PCL 63 -b-PNVP 90 micelle or, DOX-PCL 63 -b-PNVP 90 micelle solution and incubated for 18 h at 37uC, 5% CO 2 . After incubation, the released LDH in culture supernatants was measured with a 30-minute coupled enzymatic assay, which results in the conversion of a tetrazolium salt (INT) into a red formazan product. The amount of color formed is proportional to the number of lysed cells. Visible wavelength absorbance data at 490 nm were collected using a standard 96-well plate reader. Percent-specific lysis was determined using the following formula: %CytotoxicityẼ xperimental LDH Release (OD 490 ) Maximum LDH Release (OD 490 ) |100:.

Clonogenic Survival Assay
The clonogenic survival assay was performed according to the method described by Nicolaas A P Franken et al. [17] with some modifications. Cells (10 mL from a stock (1610 4 cells/ml) were treated with 0.1 to 5.0 mM of the DOX-PCL 63 -b-PNVP 90 or, free DOX for 24 h. Cells treated with free micelles were used as control. The cells were washed, diluted in RPMI containing 10% (v/v) fetal calf serum, the supplements listed above, and 0.3% noble agar (Difco, Detroit, MI) and plated in 6-well plate in triplicate on soft agar. Once set, the dishes were overlaid with 1.0 ml complete medium and incubated at 37uC. After 10 days, the total number of colonies/plate was counted. Plating efficiency (PE) and surviving fraction (SF) were calculated as mean 6 SD of triplicate by Effect of DOX-PCL 63 -b-PNVP 90 Micelles on Viability of Human Lymphocytes, Monocytes and Dendritic Cell Effect of free DOX or, DOX-PCL 63 -b-PNVP 90 micelles on the proliferation of human lymphocytes, monocytes or dendritic cells (DC) was evaluated by XTT assay. Lymphocytes were collected from peripheral blood by differential centrifugation in Ficoll-Hypaque. Monocytes were isolated by adhering total PBMC by glass adherence. Dendritic cells were generated by incubating monocytes with recombinant GM-CSF and IL-4 for 7 days as described elsewhere. Lymphocytes, monocytes or DC were plated (5610 3 cells/well) in a 96-well plate and exposed to serial concentrations of (0.0001, 0.005, 0.01, 0.05, 0.1, 0.

Fluorescence Microscopy for Doxorubicin Uptake
Parental or doxorubicin resistant K-562 and DL cells were plated in a 24 well plates at concentration of 5610 3 cells per well. Cells were washed twice with PBS and incubated with free doxorubicin or DOX-PCL 63 -b-PNVP 90 micelles with equivalent concentration of doxorubicin (5.0 mM) in complete medium for 4 h at 37uC. Cells were washed twice with ice-cold PBS and fixed with freshly prepared 2% paraformaldehyde for 15 minutes at room temperature. The cells were counterstained with Hoechst stain (for nucleus staining) and mounted on glass microscope slides with a drop of mounting media to reduce fluorescence photo bleaching. The intracellular DOX localization was visualized under a fluorescence microscope Eclipse 80i (Nikon, Japan) (Plan Fluor, 40X, NA 0.75 objective) and images were acquired by DS-Fi 1c CCD camera using imaging software NIS-Elements F 3.2 (Nikon, Japan) at 22uC. Image analysis, merging was performed by using Image-Pro Plus AMS analysis software (Media Cybernetics, Inc. 401 N. Washington Street, Suite 350 Rockville, MD 20850 USA).

Flow Cytometry Analysis for Doxorubicin Uptake
Parental or DOX-resistant (DOX/R) K-562, JE6.1, Raji, and DL cells were plated in 24-well plates at a concentration of 5 610 3 cells per well. Cells were washed twice in PBS, and incubated with free DOX or, DOX-PCL 63 -b-PNVP 90 micelles with equivalent doxorubicin at a concentration of 5.0 mM, in complete RPMI 1640 medium for 4 h at 37uC. Following incubation, the cells were washed twice with PBS and then re-suspended in 500 ml PBS. Fluorescence histograms were then recorded with a BD FACS Calibur flow cytometer (Beckton Dickinson, U.S.A.) in FL3 channel. We analyzed 20,000 events to generate each histogram.

Intracellular DOX Release and Efflux Study
For the time course study, K-562, JE6.1, Raji, and DL cells were incubated with 5.0 mM DOX-PCL 63 -b-PNVP 90 micelles for 12 h. To examine the efflux process, the culture medium containing 5.0 mM DOX-PCL 63 -b-PNVP 90 micelles was replaced by a DOX free medium after 12 h incubation. Cells were harvested at 2, 4, 8, 16, and 24 h later, respectively. The fluorescence of DOX-PCL 63 -b-PNVP 90 micelles in cells was measured using fluorescence plate reader. The determination process was described as above. The conjugate concentration was given by the standard curve.

Murine Lymphoma Model
AKR/J mice were maintained and bred under pathogen-free condition of the central animal house facility of the department. Use of mice was approved by the Institutional Animal Ethics Committee, Banaras Hindu University. All animal experiments were performed according to the National Regulatory Guidelines issued by Committee for the Purpose of Supervision of Experiments on Animals (CPSEA), Ministry of Environment and Forest, Government of India. The animals were euthanized by cervical dislocation to reduce the suffering as minimum as possible and was performed according to AVMA Guidelines on Euthanasia (AVMA 2007). Tumors (DL) in mice were maintained by transplanting fresh tumor cells in PBS (3610 4 cells/mouse) intraperitoneally. All tumor measurements were performed in a blinded fashion. Mice (n = 15/group) were transplanted with tumor and after 96 h (Day 0) were treated with the DOX-PCL 63b-PNVP 90 micelles or, free doxorubicin in PBS and were administered intraperitoneally (3 mg kg -1 body weight). Mice treated with free micelles were used as control. Altogether 9 doses were given in two phases which includes 4 doses (from day 0 to day 4) given every day and remaining 5 booster doses were given from day 10 to day 20 at an interval of 48 h, respectively. The formulation was prepared and validated such that 100 ml of DOX-PCL 63 -b-PNVP 90 micelles contained 3 mg kg 21 body weight of DOX. PBS (100 ml) was used as vehicle control for drug treatment. The tumor volumes (abdominal circumference) and body weights were monitored on a daily basis. The animals (n = 3) were sacrificed when the average abdominal circumference of the control (PBS only) exceeded 15.5 cm. Before sacrifice, tumor cells were collected in the form of ascitic fluid from peritoneum and blood by retro orbital bleeding. Blood films were prepared, fixed (methanol) and stained with Leishman stain to study the effects of DOX on leukocytes. Organs (spleen, liver, heart, lung, and kidney) were dissected out & weighed. Organs were cut into three parts. One portion was preserved in 10% formalin for further analysis and the other two parts were weighed again for DOX distribution and flow cytometry studies. Mice (n = 12/group) were under observation for 50 days when final data collection was made for Kaplan Mayer survival analysis.

Doxorubicin Distribution in Various Organs after Therapy
The tissue blocks (1 mg

Tumor sample analysis
Formalin-fixed, paraffin-embedded primary tissues from liver, lung and spleen were obtained during therapeutic procedures from control, DL tumor bearing mice and the tumor bearing mice undergone indicated therapy. Hematoxylin and eosin-stained sections were examined for regions that contained tumor cells and stroma indicating the extent of metastasis. All studies were conducted using protocols approved by the Institutional Review Board of the University.

Apoptosis Study in Organs after Therapy
Evaluation of apoptotic cell death in tumor, spleen, liver and lung cells by free doxorubicin or, by DOX-PCL 63 -b-PNVP 90 micelles after therapy was assessed by binding FITC-conjugated Annexin V. In a single cell suspension of various organs, the percentages of FITC-conjugated Annexin V-positive cells were analyzed by BD FACS Calibur flow cytometer as described earlier (Beckton Dickinson, U.S.A.). For microscopic study, liver and

Statistical Analysis
Flow cytometry data were analyzed using Flow-Jo software (version 10.0.5; Treestar, Ashland, OR, USA). The mean 6 SD were calculated for each experimental group (n = 3-5). Differences between groups were analyzed by unpaired Student's t-test and one-or two-way ANOVA analysis of variance depending on the requirement. One or two-way ANOVA followed by Holm-Sidak post-hoc multiple comparison tests was used to conduct pair wise comparisons using PRISM statistical analysis software (Graph Pad Software, Inc., San Diego, CA, USA). Significant differences among groups were calculated at P,0.05 or less (*P,0.05, **P,0.01, ***P,0.001, ****P,0.0001 in control versus experimental group). Statistical significance of differences in survival of the mice in different groups was determined by the log-rank test using Graph Pad PRISM software.

Results and Discussion
The present study was motivated by the need to design a safe nano-carrier for the delivery of doxorubicin which could be tolerant to normal cells. PCL 63 -b-PNVP 90 [13] was loaded with doxorubicin (6 mg/ml), and with 49.8% drug loading efficiency; it offers a unique platform providing selective immune responses against lymphoma. The delivery system proved to be a novel strategy, preventing metastasis in a highly aggressive lymphoma besides maintaining a minimum cardiotoxicity.

Preparation and Characterization of Blank and DOX-PCL 63 -b-PNVP 90 Miceller Nanoparticle
Well-defined amphiphilic block copolymer PCL 63 -b-PNVP 90 containing hydrophobic PCL block made of 63 CL unit and  Figure 1A) [13]. Drug-free PCL 63 -b-PNVP 90 micellar nanoparticle and DOX-PCL 63 -b-PNVP 90 micellar nanoparticle were prepared by dialysis method as shown in Figure 1B and 1C. DOX is physically entrapped and stabilized in  Figure  S1 D, the observed DOX release (%) of drug loaded micelles at pH 7.4 and 6.4 are ,19% and ,37%, respectively. The drug release rate increases gradually up to initial 12 h and then becomes leveled off which may due to the strong interaction between drug and PCL hydrophobic block as well as due to the low solubility of the drug in the used PBS media. The observed rapid release of DOX loaded micelles at low pH is due to the presence of NH 2 functional group in DOX. So, the observed accelerated drug release at low pH solution can be considered as an advantage for the antitumor drug delivery system.

Growth Inhibition by DOX-PCL 63 -b-PNVP 90 in Lymphoma Cells
We performed 48 h growth inhibition study by the DOX-PCL 63 -b-PNVP 90 against parental and DOX-resistant human (K-562, JE6.1, Raji) and mice lymphoma cells (DL) (Figure 2). Compared to free doxorubicin (DOX), DOX-PCL 63 -b-PNVP 90 shows significantly higher growth inhibition in a concentration dependent manner against the cells tested. K-562 (6.6262.67 vs. 14.3961.23, p = 0.0227, n = 6), JE6.1 (11.2264.08 vs. 24.7762.69, p = 0.007, n = 6), and DL (9.5264.27 vs. 33.3662.32, p,0.0001, n = 6) responded better with significantly higher growth inhibition compared to free DOX (Figure 2 A, C, E, and G) above the doxorubicin concentration 0.01 mM. Raji appears to be little less sensitive although effect of DOX-PCL 63 -b-PNVP 90 is higher in Raji compared to free DOX (20.6460.625 vs. 31.2560.194, p = 0.022, n = 6) at DOX concentration of 0.5 mM ( Figure 2E). Carrier it-self has no effect on growth inhibition in any of the cell line tested. DOX-resistant variants of the above cell lines   (Figure 2 B, D, F, and H). Free DOX unable to control the tumor cell growth at a concentration of 10 26 M, a concentration in which DOX-PCL 63 -b-PNVP 90 shows remarkably high growth inhibition of all the resistant variants tested. Biodegradable micelles based on amphiphilic block copolymers such as poly (e-caprolactone) (PCL) have emerged as one of the most promising nano system for controlled and site-specific delivery of potent lipophilic anticancer drugs like doxorubicin due to their proven biocompatibility and FDA approval for medical uses [18][19][20][21]. Besides reducing drug induced side effects, these micelles enhance water solubility, bioavailability and increase drug accumulation in tumors via enhanced permeability and retention (EPR) effect [22]. Stimuli (pH, temperature etc.) sensitive biodegradable polymers have recently been developed with faster intracellular drug release feature in tumor cells and ability to reverse multidrug resistance (MDR) in cancer cells [23,24]. Our data shows that DOX-PCL 63b-PNVP 90 works better in lower pH compared to higher pH suggesting its better suitability in acidic environment inside the tumor cells (Figure S1 D). This was supported by the susceptibility of human and murine lymphoma cells against DOX-PCL 63 -b-PNVP 90 with respect to growth inhibition ( Figure 2).

Cellular Cytotoxicity by DOX-PCL 63 -b-PNVP 90 in Lymphoma Cells
The growth inhibition data suggested that DOX-PCL 63 -b-PNVP 90 has significantly higher anti-tumor potential with respect to the reduction in the growth of lymphoma cells from human and mice. We wanted to know whether the growth inhibition is preceded by direct cytotoxicity of the tumor cells. Our results suggest that DOX-PCL 63 -b-PNVP 90 is highly cytotoxic compared to free DOX at each molar concentrations of DOX tested (mean  increased toxicity and need for specific drug release rates during disease evolution [25][26][27]. Our formulation of DOX-PCL 63 -b-PNVP 90 shows significant growth inhibition and cytotoxicity against resistant variants of human and mice lymphoma cells, suggesting broad spectrum usefulness of the compound (Figures 2  and 3).

Cellular Uptake Studies
We generated DOX resistant K-562, JE6.1, Raji and DL by continuous culture of the tumor cells in medium containing IC 50 doses of DOX and selected to test the efficiency of DOX-loaded PCL 63 -b-PNVP 90 in overcoming the DOX resistance. DOX resistance was documented by the huge increase in the IC 50 doses of each cell lines and their tolerance to the doses of DOX, usually causes death of the non-resistant variants (Figures 2 and 3). We next demonstrated whether DOX-PCL 63 -b-PNVP 90 can increase the drug accumulation and retention in normal and resistant variants of the cell lines tested. Free DOX or DOX-PCL 63 -b-PNVP 90 in culture medium was incubated with either resistant or parental variants of K-562, JE6.1, Raji or, DL at concentration of 2.0 mM of DOX for different time intervals from 0 to 30 h. The medium was discarded by washing in PBS and intracellular DOX accumulation was determined by analyses of DOX concentration in the cell lysates, which were normalized to total cellular protein content of the cells. It should be mentioned that the fluorescence intensity of DOX loaded PCL 63 -b-PNVP 90 was similar to that of pure DOX at the same pH and molar concentration. Figure 4A Figure 4 E-H, the DOX incorporation was significantly higher in all the cell lines tested confirming our quantitative determination of DOX incorporation. With DOX-resistant variants, we observed similar results as described by uptake studies (data not shown). We extended our observation of DOX-uptake at microscopic level for visualizing the presence of significantly higher DOX concentration intracellularly in K-562 and DL. As observed in Figure 4I and 4J, the intracellular level of DOX is significantly higher in cells treated with DOX-PCL 63 -b-PNVP 90 compared to free DOX suggesting high drug delivery efficiency of the compound.

Internalization and Intracellular Drug Release Behavior of DOX-PCL 63 -b-PNVP 90
We next demonstrated that DOX could be released from DOX-PCL 63 -b-PNVP 90 in response to the intracellular acidic microenvironment during or after its accumulation in tumor cells. Endocytosis is known as one of the important entry mechanisms for various extracellular materials, particularly nanoparticles, which is energy dependent and can be hindered when incubation is performed at low temperatures (e.g., 4uC instead of 37uC) [28]. We found that free DOX demonstrate significantly higher release behavior compared to DOX-PCL 63 -b-PNVP 90 ( Figure S2). We also studied the DOX release pattern in normal cells like lymphocytes and purified dendritic cells (DC). Our results suggest that intracellular DOX concentration rises to its maximum levels (14.2161.382 mM) between 5-10 h and drop very sharply to a level as low as 3.560.320 mM ( Figure S3). Surprisingly lympho-  cytes retain free DOX compared to DOX-PCL 63 -b-PNVP 90 for longer period of time. DC on the other hand does not retain either free DOX or DOX-PCL 63 -b-PNVP 90 for long. DOX-PCL 63 -b-PNVP 90 is significantly less toxic and exhibits nearly complete tolerance by the normal lymphocytes, dendritic cells and monocytes with very low intracellular retention ( Figure 5). Free doxorubicin on the other hand shows extreme toxicity against lymphocytes and moderate toxicity against monocytes and dendritic cells ( Figure 5). This observation is immensely significant with respect to immune response associated with the drug initiated tumoricidal effect where intact innate and adaptive immune repertoire is must for achieving the desired goals.

Tolerance of Normal Cells to DOX-PCL 63 -b-PNVP 90
Unlike tumor cells, white blood cells from normal human PBMC showed tolerance to DOX-PCL 63 -b-PNVP 90 ( Figure S4 A-C). Viability of cellular fractions like lymphocytes, dendritic cells (DC) and monocytes remain unaltered in the presence of DOX-PCL 63 -b-PNVP 90 . In contrast, free DOX found to be highly cytotoxic against lymphocytes (% viability of lymphocytes 60.8764.44 vs. 93.2662.538, p,0.0001 at concentration 1 mM) and also reduces viability of DC and monocytes as well at comparable molar concentrations of DOX tested ( Figure 5 A-C). We also checked the fate of WBC and RBC upon treatment with free DOX or DOX-PCL 63 -b-PNVP 90 . Our result suggests that in DL tumor bearing mice, WBC number increases significantly (17.2064.12610 3 /ml to 57.6066.13610 3 /ml, control vs. DL) while RBC number decreases (9.560.590610 6 /ml in control to 6.8060.68610 6 /ml in DL). In case of free DOX treatment, WBCs virtually vanishes (5.660.378610 3 /ml) with dramatic reduction in RBC as well. In contrast, with DOX-PCL 63 -b-PNVP 90 treatment, WBC number decreases slightly (8.5061.06610 3 /ml) while RBC numbers remains similar to normal mice (

Induction of Tumor Cell Apoptosis by DOX-PCL 63 -b-PNVP 90
Broad spectrum growth inhibition by DOX-PCL 63 -b-PNVP 90 raises the question whether it also causes apoptosis of tumor cells and if so whether it induces cell death in DOX-resistant tumor cells, which become refractory to any concentrations of DOX. Apoptosis was determined by monitoring changes in cell size and externalization of phosphatidylserine by flow cytometry after exposure to FITC-labeled Annexin V according to the manufacturer's instructions. The cells were harvested, stained with FITClabeled Annexin V and analyzed by flow cytometry using the Cell Quest software program. A minimum of 10,000 cells was analyzed in each case with triplicate determinations. Our results shows that tumor cells behave differently with respect to annexin positivity when treated with DOX-PCL 63 -b-PNVP 90 , exhibiting significantly higher levels of apoptosis compared to DOX alone ( Figure 6A). Annexin positivity in K-562 cells treated with DOX-PCL 63 -b-PNVP 90 is higher (6.4% increase) compared to DOX alone.
Annexin positivity in DL cells increases from 12% in free DOX treatment to 68%, when DL cells were treated with DOX-PCL 63b-PNVP 90 . Similar results were observed in all the other cell lines tested ( Figure 6A). DOX-resistant cells are tolerant to DOX however; remain susceptible to DOX-PCL 63 -b-PNVP 90 mediated apoptosis although percent Annexin positivity is significantly lower compared to the parental variants ( Figure 6B). Percent Annexin positivity in K-562/DOX-R is 19% in cells treated with DOX-PCL 63 -b-PNVP 90 compared to 8% treated with DOX alone. Huge difference was also observed in Annexin V-DOX double positive cells (7% in DOX treatment compared to 24% in DOX-PCL 63 -b-PNVP 90 treatment) ( Figure 6B). Similar results were obtained with other resistant cell lines including DL/DOX-R ( Figure 6B).
We also qualitatively assessed the apoptosis in parental and DOX resistant K-562 and DL to document the events of apoptosis. There is an abundance of Annexin V positive cells in parental K-562 treated with DOX-PCL 63 -b-PNVP 90 which is significantly higher compared to free DOX treatment. Resistant variants of the K-562 exhibits tolerance to free DOX but become susceptible to DOX-PCL 63 -b-PNVP 90 ( Figure S4). Similar results were obtained in DL cells under identical treatment ( Figure S4).

Clonogenic Survival Assay
In addition to the rapid Annexin V assay, a clonogenic survival assay was also employed to assure that we were detecting a true commitment to die and not just early, potentially reversible, changes in the cells treated with either DOX or DOX-PCL 63 -b-PNVP 90 . Our results suggest that with DOX-PCL 63 -b-PNVP 90 treatment, the number of colonies of DL cells become negligible compared to free DOX ( Figure 7A-D). Analysis of survival fraction suggest that with DOX-PCL 63 -b-PNVP 90 treatment, there is a sharp decline compared to free DOX at a concentration of 1-2 mM of DOX ( Figure 7E).

In Vivo Antitumor Activity
Therapeutic efficacy of DOX-PCL 63 -b-PNVP 90 was validated in DL tumor model. Randomly sorted mice (female, 4-6 weeks old) were transplanted with tumor intraperitoneally and were sorted in to 4 groups each with 12 mice and treated with 4 daily injections of (i) phosphate buffer saline (PBS) (ii) free doxorubicin (3 mg/kg of body weight), (iii) vehicle control or (iv) DOX-PCL 63 -b-PNVP 90 (equivalent to 3 mg/kg of doxorubicin dose) after 96 hours post tumor challenge (marked as day 0). A second set of 5 injections were given to the animals from day 10 to day 18 with a gap of 48 h ( Figure 8A). The mice injected with PBS or, vehicle control formed large intraperitoneal ascitis by day 12 post tumor transplant, which continued with increasing size as the day progresses. Mice treated with free DOX also develops tumor and forms similar ascitis at day 20. In contrast, mice treated with DOX-PCL 63 -b-PNVP 90 develops tumor at a significantly slower rate and develops tumor at day 30. All the mice in PBS and vehicle control groups died between day 22 and 24 from tumor progression. Mice in free doxorubicin group did not survive beyond the day 32. In contrast, 4 of 12 mice in the DOX-PCL 63 -b-PNVP 90 group were alive at day 50 when the experiment was doses, 4 doses were given every day (from day 0 to Day 4) and remaining 5 dose were given from day 10 at an interval of 48 h and continued up to day 18. The animals in study responded to the therapy with DOX-PCL 63 -b-PNVP 90 (B). Kaplan-Mayer survival analysis of tumor bearing mice was performed following therapy and was analyzed for the percent survival up to day 50 post tumor transplant by log-rank test using Graph Pad PRISM software (C). Abdominal circumference (D) and body weight (E) are depicted demonstrating the effects of the treatment on the tumor growth of the animals. Representative images of spleen, liver, heart, lung, and kidney from each treatment group (F) and the corresponding weight of excised organ following drug treatment compared to untreated control are shown (G) n = 3. doi:10.1371/journal.pone.0094309.g008 Efficacy of DOX-PCL63-b-PNVP90 Micelle System in Treating Lymphoma  Figure 8D & E). We also determined the weight of important vascularised organs in order to assess the effect of treatment on metastasis. Our results suggests that in liver, where DL tends to get metastasize shows remarkable therapeutic efficacy by reducing the organ weight, ostensibly by therapy with DOX-PCL 63 -b-PNVP 90 (p,0.0001 between DOX and DOX-PCL 63 -b-PNVP 90 ) ( Figure 8F & 8G). Validation of therapeutic efficacy of DOX-PCL 63 -b-PNVP 90 system in DL tumor model resulted in dramatic growth inhibition and prevention of metastasis, indicating the increase in therapeutic index of the formulation. High solubility of DOX-PCL 63 -b-PNVP 90 and effect of tumor derived carboxylesterases could be responsible for release of doxorubicin and reduces its systemic toxicity [29]. Histopathological analysis of liver, lung and spleen clearly indicates the efficacy of the formulation in restricting the metastasis of the organs ( Figure 9A). In liver, infiltrated metastatic lymphoid cell (red circle) are clearly visible in DL mice which are significantly reduced in mice treated with DOX-PCL 63 -b-PNVP 90 . Similarly, in lung normal architecture is lost due to tumor metastasis which is accompanied by the recruitment of neutrophils (red arrow) and evidence of haemorrhage and necrosis (yellow arrow). Upon treatment with DOX-PCL 63 -b-PNVP 90 , mice recovered compared to untreated group (blue arrows). In normal spleen, the capsule is intact (green arrow) throughout the periphery which is disintegrated in tumor bearing mice and not restored in mice treated with DOX only. In DL mice, sub-capsular sinus is infiltrated with malignant cell (black arrow) which is also visible in DOX treated group with capsule invasion by malignant cells. DOX-PCL 63 -b-PNVP 90 treated mice restored the capsular architecture to a large extent ( Figure 9A). Counting of metastatic foci and metastatic field in liver and lung sections clearly suggest significant reduction in metastasis following therapy with DOX-PCL 63 -b-PNVP 90 (p,0.017 and 0.0052 between DOX and DOX-PCL 63 -b-PNVP 90 treatment in liver and lung respectively) ( Figure 9B & C).
Enhanced uptake of DOX-PCL 63 -b-PNVP 90 by Organs in Tumor Bearing Mice Fluorescence spectroscopic analysis of doxorubicin level in heart showed very high accumulation in mice treated with free DOX compared to DOX-PCL 63 -b-PNVP 90 despite no difference in organ weight. Unlike others [30], splenic accumulation of doxorubicin was not significant (0.79060.012 vs. 0.84460.029, p = 0.4667, n = 3) and no difference was observed in weight (data not shown) of the spleen between free DOX and DOX-PCL 63 -b-PNVP 90 treated groups ( Figure 10). This could be due to low concentration of doxorubicin used in our study. Additionally, kidney tissue showed significantly higher accumulation (1.03760.638 vs. 2.72960.011, p,0.0001, n = 3) of DOX-PCL 63 -b-PNVP 90, suggesting easy clearance of the formulation following metabolism. Liver and tumor cells however, showed very high accumulation. Therapy with DOX-PCL 63 -b-PNVP 90 causes change in the accumulation of DOX in the targeted organs. The accumulation of DOX-PCL 63 -b-PNVP 90 was enhanced in liver, kidney (p,0.0001) and lung (p = 0.0012) while in heart, DOX concentration is higher (p,0.0001) compared to DOX-PCL 63 -b-PNVP 90 ( Figure 10A). FACS analysis data supported the observation with major shift in accumulation of DOX in DOX-PCL 63 -b-PNVP 90 treated mice which was diametrically different in case of heart ( Figure 10G). These data suggest that DOX-PCL 63b-PNVP 90 is nontoxic and perhaps causes very less damage to the heart tissue. We also analyzed the apoptosis in single cell suspension derived from spleen, liver and lung tissues following staining with Annexin V of the total cells. Microscopic analysis of total liver cells shows greater Annexin positivity derived from mice treated with DOX-PCL 63 -b-PNVP 90 compared to free DOX (Figure S5 A). FACS analysis of cells derived from liver, spleen and lung showed enhanced apoptosis of cells in the target organs by DOX-PCL 63 -b-PNVP 90 compared to DOX treatment alone (Figure S5 B).
Apoptosis observed in liver and spleen cells presented in Figure  S5C are the mainly in tumor cells present in liver and spleen. No observable apoptosis was observed in control group. In case of DOX loaded micelle group, much more apoptosis is observed in tumor bearing mice. This is further supported by Figure S6, where we observe that free DOX causes cell death in normal mice spleen, liver and lung, significantly higher compared to DOX loaded polymer (normal mice were treated with free DOX or DOX loaded polymer). This indicates the general toxicity of free DOX while DOX-PCL 63 -b-PNVP 90 remain significantly less toxic.

Conclusion
In this study, we have demonstrated the potential application of doxorubicin-loaded poly (e-caprolactone)-b-poly-(N-vinylpyrroli-done) micellar nano-carrier in cancer chemotherapy. Several components of this platform make it an attractive approach for facilitating future therapy in humans. Hydrophilic exterior and hydrophobic interior of our formulation offers entrapment of higher concentrations of doxorubicin to form micelles and get easy access through the cells leading to high efficiency of cellular uptake by endocytosis and subsequent acid responsive release in tumor cells. Assay with normal cells indicates reduced toxicity to WBCs and vital organs like heart while maintaining higher therapeutic efficacy, compared to commercial doxorubicin hydrochloride. DOX-PCL 63 -b-PNVP 90 , compared to free DOX, demonstrated antitumor potential against doxorubicin-resistant lymphoma cells indicating its possible application in drug resistant scenarios. Finally, DOX-PCL 63 -b-PNVP 90 significantly inhibits in-vivo tumor growth in mice model of lymphoma through induction of apoptosis and prevention of metastasis. Designing of DOX-PCL 63b-PNVP 90 opens a new way for treating cancer with higher efficacy and decreased side effects.