Curcumin as a Novel Nanocarrier System for Doxorubicin Delivery to

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Biological and Medical Applications of Materials and Interfaces

Curcumin as a Novel Nanocarrier System for Doxorubicin Delivery to MDR Cancer Cells: In Vitro and In Vivo Evaluation N. Sanoj Rejinold, Jisang Yoo, Sangyong Jon, and Yeu-Chun Kim ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10426 • Publication Date (Web): 01 Aug 2018 Downloaded from http://pubs.acs.org on August 2, 2018

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ACS Applied Materials & Interfaces

Curcumin as a Novel Nanocarrier System for Doxorubicin Delivery to MDR Cancer Cells: In Vitro and In Vivo Evaluation N. Sanoj Rejinold1, Jisang Yoo1, Sangyong Jon2, and Yeu-Chun Kim1* 1

Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST); Daejeon 305-701, Republic of Korea;

2

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea

ABSTRACT Curcumin (CRC) has been widely used as therapeutic agent for various drug delivery applications. In this work, we focused on the applicability of CRC as a nano drug delivery agent for doxorubicin hydrochloride (DOX) (commercially known as Adriamycin®) coated with PEG as an effective therapeutic strategy against multi drug resistant cancer cells. The developed PEG coated CRC/DOX nanoparticles (NPs) (PEG-CRC/DOX NPs) were well localized within the resistant cancer cells inducing apoptosis confirmed by flow-cytometry and DNA fragmentation assays. The PEG-CRC/DOX NPs suppressed the major efflux proteins in DOX resistant cancer cells. The in vivo bio-distribution studies on HCT-8/DOX resistant tumor xenograft showed improved bioavailability of the PEG-CRC/DOX NPs, and thereby suppressed tumor growth significantly compared to the other samples. This study clearly shows that curcumin nanoparticles could deliver DOX efficiently into the MDR cancer cells to have potential therapeutic benefits. Keywords: MDR, p-glycoprotein, Curcumin, Doxorubicin, In vivo tumor suppression

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1. INTRODUCTION Curcumin (CRC), a naturally occurring phytochemical1 has been widely used as a therapeutic agent for various field such as drug delivery,2 wound healing,3 anti-oxidant4 and antiinflammatory research5. However, there has not been much work on the applicability of CRC as a nano delivery carrier to treat the multi drug resistant (MDR) cancer cells except few previous works related to the synthesis, characterization and microbial activities of CRC nanospheres6 and CRC nanoparticles.7 Thus, exploring their drug delivery capability is essential to use them for various applications, where the nanocarrier itself might be therapeutically beneficial. To exemplify, treating MDR cancer cell is challenging, as most of the drug carriers cannot modulate the MDR proteins unlike the CRC does. Elevated expression of p-gp (p-glycoprotein, aka MDR-1) in MDR cancer cells is a major hindrance to achieve maximum therapeutic efficacy.8-11 Even though there are several approaches for treating the solid tumors with MDR, most of them cause severe side effects leading to a low survival rate in the patients.12 In addition, patients have to undergo many diagnosis procedures to follow the progression of the tumor growth, and the treatment regimen can be varied accordingly. Recent developments in cancer nanotechnology have shown that therapy as well as diagnosis with nanotechnology could be much more effective than that of traditional treatments.13-15 Although there are various cancer Nano techniques available, one of the approaches is to use drug nanocrystals16, in which the drug itself can be made into tiny nanoparticles (NPs) without any encapsulating agent achieving a 100% drug composition. 17 However, it is critical to find the proper molecule that can be used as a drug nanocrystal, which has the ability to suppress the p-gp in MDR cancers. On the other hand, CRC is known for its multi targeting capabilities against cancer cells and low toxicity in normal healthy cells.18-20 CRC has also been identified as a p-gp modulator to suppress MDR in cancer.21 However, the major problem associated with the CRC is its poor water solubility and low bioavailability in vivo.22-26 Because CRC has a selective toxicity against cancer cells, it would be great to investigate it as a nano-carrier for anti-cancer therapy. Doxorubicin (DOX) commercially known as Adriamycin™ is well known as an anti-cancer drug against many cancers including breast, prostate, cervical, lung, etc. 27-30 However, its cardiotoxicity after intravenous injection is still a major problem limiting its applicability.

31-32

These toxicity issues associated with

DOX could be avoided by protecting it using CRC NPs. Because CRC is non-toxic and


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selective against cancer cells, it would be an ideal agent to act as a potential MDR nanocarrier for encapsulating DOX within it. PEGylation has been known to help in changing p-gp conformation so that the MDR can be abolished largely.33-36 In addition, PEGylation may enhance the in vivo circulation of the NPs, an added advantage.37 Previously, Zhang et al., (2016) encapsulated CRC in the core of the PEG-DOX micelles as a prodrug approach. There has not been any work on CRC as a nano drug delivery agent to the best of our knowledge. Zhang et al., (2016) made CRC encapsulated PEG modified DOX micelles in which they used PEG conjugated DOX as a nano micelles for CRC delivery, just opposite of our research strategy in this article.38 Our system particularly made for treating MDR cancer cells with low DOX dosage. Moussawi RN et al. (2016) reported CRC conjugated ZnO grain like nanostructures for chemical decontamination, which is again not a similar approach to what we have proposed in our study.39 However, the synthesis of CRC NPs have been previously reported and showed they have excellent cytocompatibility to normal healthy cells.40 Even though, there have been many reports on the co-delivery of CRC and DOX using polymeric NPs41, this is the first report for using CRC as a nanocarrier for drug delivery applications. Thus, the major aim of this work was to investigate CRC as a nanocarrier for loading DOX after coating with PEG for use in cancer cells that highly expressing p-gp in vitro and in vivo. The major research questions we focused on were as follows: 1) How can CRC NPs act as an efficient nanocarrier for DOX for p-gp expressing cancer cells? 2) What are the in vitro and in vivo anticancer effects on p-gp expressing cancer cells? Overall, our work mainly deals with the synthesis, characterization and in vitro assessment of PEG coated and uncoated DOX loaded curcumin nanoparticles in detail. Specific emphasis has been given to the in vivo anti-cancer effects. 2. EXPERIMENTAL SECTION 2.1 Materials. CRC was purchased from Sigma Aldrich (India). Doxorubicin hydrochloride was gifted. The VybrantTM Multidrug Resistance Assay Kit (V-13180) was purchased from USA, Enzo Life Sciences. The eFluxx-ID® Gold multi drug resistance assay kit was purchased from Enzo life sciences, South Korea. Poly ethylene glycol (2000) was purchased from Sigma Aldrich, USA.



2.2 Synthesis of CRC/DOX NPs. To prepare the CRC/DOX NPs, different CRC concentrations were used: 1, 2, and 3 mg/mL. The DOX concentration was fixed at 1 mg/mL. Briefly, 1 mg/mL of ethanolic CRC was mixed with 50 mL water and stirred at 600 rpm for about 10 min. To this solution, 1 mg/mL DOX solution was added gently with very mild stirring at 200 rpm for 10 min. The slight turbidity was indicative of the formation of the CRC/DOX NPs. The whole solution was further stirred at a slightly higher rpm of 600 rpm. The solution of CRC/ DOX NPs was further centrifuged at 13,000 rpm for about 45 min. to separate the ethanolic solution. The residue was then mixed with 10 mL of water to obtain the final DOX/CRC NPs. 2.3 Synthesis of PEGylated CRC/DOX NPs. PEG (molecular weight 2000) was used for this. Briefly, 1mg/mL of (~ 100 µL) PEG-2000 added during the formation of the CR/DOX NPs. To prepare the PEGylated CRC/DOX NPs, different CRC concentrations were used: 1, 2, and 3 mg/mL. The DOX concentration was fixed at 1 mg/mL. Briefly, the ethanolic CRC was initially mixed with 50 mL water (containing 1 mg/mL DOX) and stirred at 600 rpm for about 10 min. The slight turbidity was indicative of the formation of the CRC/DOX NPs. The whole solution was further stirred at a slightly higher rpm of 600 rpm. Once the color changed more turbid reddish/yellow, PEG-2000 solution was added and immediately centrifuged at 13,000 rpm for about 45 min. to separate the ethanolic solution. The residual pellet was then mixed with 10 mL water to obtain the final DOX CRC NPs. 2.4 Synthesis of IR-780 Dye Loaded NPs. For the synthesis, 0.8 mg/mL of IR-780 dye (mixed in 100% ethanol) were used to encapsulate it inside the CRC and PEGylated CRC NPs. To prepare the IR-780 loaded CRC and PEGylated CRC NPs, the ethanolic CRC solution 2 mg/mL was initially added to 50 mL of D.I water and stirred at 600 rpm for about 10 min. Before the turbidity appeared, the IR-780 dye was added until it completely mixed with the CRC NPs. Then, 1 mg/mL of PEG was added to the final solution to separately make the PEG-CRC NPs containing the IR-780 dye. The whole solution was then stirred at a higher rpm of 600 rpm. The solution of NPs was centrifuged at 13,000 rpm for about 45 min. to separate the ethanolic solution. The residue was then mixed with 10 mL of water to obtain the final NPs. 2.5 Cell Culture. NIH-3T3 (mouse embryonic fibroblast cells), HeLa (human cervical cancer cells), NCI-H460 (Human lung carcinoma cells), and HFL1 (Human normal lung cells) were gifted by the department of biological sciences; KAIST. HCT-8 (DOX resistant human colon


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cancer cells) were gifted by POSTECH University, South Korea. They were maintained in DMEM media supplemented with 10% fetal bovine serum (FBS) and incubated at 5% CO2. After reaching confluency, the cells were detached from the flask with trypsin-EDTA. The cell suspension was centrifuged at 3000 rpm for 3 min. and then re-suspended in the growth medium for further studies. 2.6 Evaluation of Cellular Localization Using Confocal Laser Scanning Electron Microscopy (CLSM). For cellular localization studies, 10 µM samples treated with cells with a seeding density of 2 ×104 cells. 48 h later, the harvested cells washed with PBS twice to remove the non-up taken samples. Because the PEGylated CRC/DOX has intense green and red colors, we stained the cells with DAPI (which is blue in color) following the manufacturer’s instructions. Because CLSM gives a better understanding of internalization of NPs, it was used to confirm the cellular uptake of our samples. 2×104 cells seeded on the acid etched cover slips kept in 24 well plates and incubated for 24 h for the cells to attach well. The media removed after a 48 h incubation, and the wells carefully washed with PBS buffer. Then, the particle at a 10-µM concentration added along with the media in triplicate to the wells and incubated for a period of 24 h. After the incubation time, DAPI was added and the cover slips processed for CLSM. The processing involved the washing of the cover slips with PBS and fixing the cells in 3.7% Para Formaldehyde (PFA) followed by a final PBS wash. The cover slips were then air-dried, then mounted onto glass slides with the DPX (Sigma Aldrich) mountant. The slides were then viewed under the CLSM to study the cellular internalization of the samples. 2.7 Evaluation of Cellular Localization by Flow-cytometry. The cellular localization further confirmed by flow-cytometry because DOX has red fluorescence. Briefly, NCI-H460 and HCT-8 MDR+ve cells were seeded on 24 well plates at a density of 2 x104 cells/well. After a 24 h incubation, the cells harvested by trypsin and centrifuged at 5000 rpm for 5 min. The cells then mixed gently with PBS for analysis using flowcytometry. 2.8 Cellular Uptake Mechanism. HCT-8 MDR+ve tumor cells were seeded into 24-well plates at a density of 2.0 × 104cells/well. Once the cells became confluent, they were treated for 1 h with various endocytosis inhibitors such as 30 µM chlorpromazine (CPZ, a clathrin mediated endocytosis blocker), 0.4 M hypertonic sucrose (HS, a lysosomal uptake blocker), 10 mM methyl-β-cyclodextrin (MβCD, a caveolae mediated endocytosis blocker)42, 500mM Sodium azide (SA, ATPase inhibitor), 2.5 M ammonium chloride (NH, lysosome uptake



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inhibitor) and 20 µM 5-(N-ethyl-N-isopropyl)-amiloride (EIPA, macropinocytosis inhibitor) individually. Then, both CRC/DOX NPs and PEG-CRC/DOX NPs (2.5 µM) were added to the wells and incubated for 3 h. To assess the temperature effect on the cellular entry, the cells were incubated with samples at 4 °C for 3 h. After incubation, the cells washed with PBS three times and incubated with 400 µL of lysis buffer for 30 min. The fluorescence of each lysate was measured by spectrofluorophotometer with excitation and emission wavelengths of 490 and 510 nm, respectively.43 2.9 In Vitro Drug Release. In vitro drug release was done at two different pHs 7.4 and 5.5, respectively, to see whether the DOX could be released in a pH dependent manner. To this end, the CRC/DOX NPs and PEG-CRC/DOX NPs mixed with PBS and kept in PBS at pH 7.4 and 5.5. Then, 500 µL aliquots transferred into 1.5 mL eppendorf tubes and kept in a shaking incubator at 37°C. At pre-determined time intervals of 1, 3, 6, 9, 24 and 48 h, the withdrawn samples then centrifuged at 14,000 rpm for 15 min to separate the released DOX and then measured by UV visible spectroscopy. Following equations were used to determine the encapsulation efficiency (EE) and loading efficiency (LE), the. Weight of Drug entrapped in the NPs EE % =

X100 Weight of Initial Drug used for NPs

Weight of Drug entrapped in the NPs LE % =

X100 Weight of NPs

2.10 MTT assay, Live/Dead and Flow Cytometry Assay to Confirm DOX Resistance. The MTT [3-(4,5-Dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium] assay was done in two different cell lines, NCI-H460(MDR-ve) and HCT-8 (MDR+ve), with various concentrations at 2.5, 5 and 10 µM. 96 well plates seeded with 104 cells/well, and once they became confluent, the DOX samples added and incubated for 48h. The MTT solution was added as previously described, 44 and the absorbance was measured by UV-Visible spectroscopy at 570 nm. A live/dead assay used with NCI-H460 and HCT-8 cells with a concentration of 5µM samples following the manufacturer’s instructions. Briefly, the DOX samples were treated with 104 cells/ coverslip in 12 well plates and incubated for 48 h. The cells were then washed gently with PBS two times, and the coverslips were detached with forceps. The EtBr and Calcein


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solutions were added to each coverslip and processed under a microscope to visualize the green/red fluorescence. Finally, flow cytometry assisted cell uptake assay was conducted to visualize the DOX fluorescence in both the NCI-H460 and HCT-8 cells with various concentrations of DOX from 2.5 to 10 µM. To this end, 104 cells were seeded onto 24 well plates, and the samples were added once the cells became 80% confluent. Trypsin was used to detach the cells which were then centrifuged at 5000 rpm for 5 min. The harvested cells were mixed with PBS (400µL) and analyzed for flow cytometry based cellular localization. DOX fluorescence (ex/em 480/590 nm) and Curcumin (ex/em 420/470nm) may have overlap with the dyes used in MTT (570nm) and Live/Dead assay Kit (Calcein ex/em 495/515 nm, EtBr em 600nm). In order to avoid this issue, we did background fluorescence reduction by simply deducting the extra fluorescence coming only from the samples alone (without respective dyes). For this, samples checked without addition of reagent and compared the values with the reagent treated samples. The extra values deducted from without reagent treated samples. Since the background fluorescence were withdrawn by this way, remaining absorption is expected to be from the actual effects of samples.

2.11 Therapeutic Efficacy Evaluation In Vitro. (a) MTT assay: The cells were seeded in 96 well plates as mentioned above. After reaching 80% confluency, the cells were washed with PBS, and the samples (2.5, 5 and 10 µM) were added, and the particles were incubated for about 48 h. The sample treated and untreated cells were harvested and analyzed for the anticancer efficacy. Thus, 5 mg of MTT (Sigma) was dissolved in 1 mL of PBS and filter sterilized. Then, 10 µL of the MTT solution were further diluted to 100 µL with 90 µL of serum–free phenol red free medium. The cells were incubated with 100 µL of the above solution for 4 h to form formazan crystals by mitochondrial dehydrogenases. Then, 100 µL of the solubilization buffer (10% Triton X-100, 0.1N HCl and Isopropyl alcohol) were added to each well and incubated at room temperature for 1 h to dissolve the formazan crystals. The optical density of the solution was measured at a wavelength of 570 nm using a Thermoscientific multi-scan go, Finland with a reported protocol. Triplicate samples were analyzed for each experiment. (b) Apoptosis assay by flowcytometry: One of the important earliest apoptotic features is the



translocation of Phosphatidylserine (PS) from the inner to outer layer of the plasma membrane. Thus, PS translocation can act as an apoptotic marker for many cancer cells. The PS exposure in NCI-H460 and HCT-8 cells was detected with the Annexin V-FITC/PI Vybrant apoptosis assay kit (Molecular probes, Eugene, OR). The cells were seeded in a 12 well plate at a density of 2x104/ well. After reaching 90% confluency, the cells were treated with different concentrations of the samples including the control DOX, CRC, CRC/ DOX NPs and PEGylated CRC/DOX NPs. After treatment with the samples for about 48 h at 37°C, the cells were harvested by trypsinization and washed with PBS for 5 min. followed by centrifugation at 500 g at 4°C. The supernatant was discarded, and the pellet suspended in ice-cold 1X Annexin binding buffer. Then, 2 µL of Annexin V-FITC solution and 0.5 µL of PI (100µg/mL) were added to 100 µL of the cell suspension and mixed gently. The samples were then incubated at room temperature for 15 min. in the dark. After the incubation, 400 µL of ice-cold 1X binding buffer were added, mixed gently, and analyzed by flow cytometry (FACS Aria II (Beckton and Dickinson, San Jose, CA)). Cells in media alone without any NPs (negative control) and cells treated with control NPs were also analyzed in the same manner. Samples were analyzed in triplicates for each experiment. (c) Anti-proliferation and anti-migration assays. For the anti-proliferation studies, a crystal staining protocol was used. Briefly, 2x104 cells/well were seeded on 12 well plates. Once the cells became 80% confluent, the samples including CRC, DOX, CRC/DOX NPs and PEGCRC/DOX NPs (for which the DOX concentration was fixed at 2.5 µM) were added and incubated for 24 h. The cells were washed with PBS, and fresh media was added and incubated for 72 h. The samples were then stained with crystal violet (0.01%v/v), incubated for 15 min. followed by washing 2 times with PBS and quantified by UV Visible spectroscopy. The proliferation was analyzed as follows: % cell proliferation = (λ Crystal Violet of Treated cells/ λ Crystal Violet of Control cells) x100 , where λ Crystal Violet = absorbance of the Crystal Violet.

For the anti-migration assay, the cells were seeded same as above. Once the cells became 80% confluent, uniform scratches were made using 0.1 mL pipette tip. The samples were then added and incubated for 24 and 48 h. The wound closure was measured manually after finding the wound diameter with an optical microscope. The wound closure was measured


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with the following formula:

Wound closure % = (W d of treated sample on day t/ Wd of untreated sample on day 0) x100 Wd = Wound diameter d)

ROS

levels:

ROS

changes

in

both

cells

were

determined

with

2′,

7-

dichlorofluoresceindiacetate (DCFDA) assay. NCI-H460 and HCT cells were seeded onto 96well plates at a density of 1.0 × 104 cells/well, respectively. After an incubation of 24 h, OptiMEM (100 µl) containing different samples were added to each well and incubated for 3 h. Then, DCFDA solution in DMSO was added to each well at a final concentration of 10 µM and incubated for 30 min. Each well was washed with PBS three times, and the green fluorescence intensity was measured with a microplate reader at 490 nm.45 e) DNA fragmentation assay: Quick Apoptotic DNA Ladder Detection Kit (Bio vision, Catalog #: K120) was used for the analysis. After reaching 90% confluency, the cells treated with different concentrations of the samples including the control DOX, CRC, CRC/ DOX NPs and PEGylated CRC/DOX NPs. After treatment with the samples for about 24 h at 37°C, the cells (including the untreated cells) were harvested by trypsinization and washed with PBS for 5 min. The cells were then lyzed with 35 µL TE Lysis Buffer, with a gentle pipetting. After 5 µL Enzyme A Solution added, pellets were mixed by gentle vortex and incubated at 37°C for 10 min. 5µL Enzyme B Solution added into each sample and incubated at 50°C for 30 min. Then 5 µL Ammonium Acetate Solution was added to each sample and mixed well. After adding 50µL isopropanol, mixed well, and kept at –20°C for 10 min, samples Centrifuged for 10 min to precipitate DNA. The supernatant was removed, washed the DNA pellet with 0.5 ml 70% ethanol, removed trace ethanol, and air dried for 10 min at room temperature. DNA pellets were dissolved in 30 µL DNA Suspension Buffer + 5 µL loading star (Dyne Bio, South Korea, Part # A750) (replacement of ethidium bromide) (100 bp DNA used as Marker). The samples were carefully loaded (15-30 µL of the sample onto a 1.2% agarose gel. The gel ran at 5 V/cm for 1-2 hours or until the yellow dye (included in the suspension buffer) run to the edge of the gel. The stained DNA were visualized by transillumination with UV light and photographed. 2.12 Anti-MDR Assays. a) p-gp suppression analysis by VybrantTM Multidrug Resistance Assay (V-13180): MDR cells expressing high levels of p-gp rapidly eliminate nonfluorescent



Calcein AM from the plasma membrane reducing the intake of fluorescent Calcein in the cytosol. Thus, the degree of inhibition of the p-gp activity can be quantitated by measuring the increase in intracellular Calcein fluorescence. To this end, the cells were seeded on 12 well plates at a seeding density of 5x105 cells/well. Verapamil (MDR inhibitor), control Calcein, CRC, DOX, CRC-DOX NPs, and PEG-CRC/DOX NPs were added and analyzed following the manufacturer’s instructions. Calcein fluorescence was measured by Flowcytometry. (b) Suppression of Efflux Proteins Determined by the eFFlux-ID gold Multi Drug Resistance Assay This assay was done according the manufacturer’s instructions to analyze the suppression of two major efflux proteins, MDR-1 and MRP, in DOX resistant HCT-8 cells. Different samples including CRC, DOX, CRC-DOX NPs, and PEG-CRC/DOX NPs that included the inhibitors along with untreated samples were added to 5x105 cells/well. The cells were then analyzed using flow cytometry. In order to avoid the fluorescence overlap between DOX (ex/em 480/590 and eFFlux-ID gold dye (ex/em 530/570nm), we have deducted the background fluorescence from DOX from the samples. 2.13 Stability and Blood Compatibility Studies. Serum stability studies of the different samples were done at two different temperatures (4 and 37°C). Briefly, the samples were treated with 10% serum containing PBS and kept in a shaking incubator at 37°C and inside a refrigerator at 4°C for about 30 days. The size and PDI values were measured at day 1, 3, 7, 14, 21 and 30 to check for differences. Similarly, the blood compatibility tests of various samples were also conducted. CRC, DOX, CRC/DOX NPs and PEG-CRC/DOX NPs(5µM) were diluted with 0.9% saline with a total volume of 200 µL. These solutions were then added onto half of the surface of horse blood agar plates and incubated at 37 °C for 24 h. The clear zone for each NP was checked to observe blood cytotoxicity. 2.14 Tumor Models. Balb/c nude mice were used to develop both tumors model, the NCIH460 and MDR +ve HCT-8 tumor models. The bio-distribution study was done according to the institutional guidelines of the Animal Care Committee of the Korea Advanced Institute of Science and Technology (KAIST). Female BALB/c nude mice (5-week-old) were injected with 5×106 cells/200 µL of NCI-H460 cells subcutaneously into the right hind flank. For the


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HCT-8 tumor model, 5 × 106 cells/100 µL were mixed with 100 µL Matrigel (BD Biosciences), and then, these were subcutaneously injected into the right hind flank. 2.15 In Vivo Bio Distribution and Ex Vivo Imaging. Next, 0.5 mg/kgIR-780 loaded CRC NPs and PEG-CRC NPs were diluted in 0.9% saline and injected into the tail vein. At predetermined time points of 1,3, 6, 24, 48 and 72 h, the mice were anesthetized with isoflurane, and NIR imaging was done with the Xenogen in vivo imaging System (Germany). The mice were sacrificed to collect the tumors and other organs at the end of the experiment. Both in vivo and ex vivo fluorescence levels were measured and quantitated by the NEOimage software.

2.16 In Vivo Therapeutic Efficacy. The in vivo therapeutic efficacy analysis was conducted at KAIST with the approval of the Animal Care Committee of Korea Advanced Institute of Science and Technology (KAIST). The therapeutic efficacy experiment was done with MDR+ve HCT-8 tumor-bearing mice. The mice were randomly divided into five groups including a control group (untreated). Each group was intravenously injected with 200 µL of CRC, DOX, CRC/DOX NPs, and PEG-CRC/DOX NPs (0.5 mg/kg) once a week for 3 weeks. The body weights of all the groups were measured, and the tumor volumes were calculated as follows: V = WL2/2 (W = the longest diameter; L = the shortest diameter).46 The tumor tissue was removed from the mice to weigh them once the therapeutic efficacy experiment was finished. In addition, mouse organs (liver, kidneys, heart, spleen, and lungs) with tumors were collected and stained with hematoxylin and eosin (H&E) to test for toxicity with an optical microscope. TUNEL assay was carried out to find the apoptotic areas in the tumor tissues. 2.17 Tumor Vs Organ detection of DOX using HPLC. 200 µL of DOX, CRC/DOX NPs, and PEG-CRC/DOX NPs (0.5 mg/kg) were injected in HCT-8/DOX bearing tumors via intravenous mode (one time injection). 3 days later, the animals were sacrificed for the detection of HPLC using a standard protocol. 2.18 Analytical determinations: DLS and Zeta potential analysis was done sing Zetasizer, Malvern (UK). TEM analysis was done at Department of Measurement & Analysis, National Nanofab Center , Daejeon South Korea. For this, 200 kV FE-TEM (200 kV accelerating voltage and about 120 µA beam current). Normally, at low magnifications like the image we showed, there is very little beam damage by electrons in materials. For making TEM images,



the nano emulsions were mixed with water (10 µL in 10 mL water) and dropped on the 200 copper mesh, and dried well under room temperature prior to the TEM analysis. FT-IR was done at KARA (KAIST Analysis center for research advancement), KAIST, South Korea. The optical phographs were taken using LEICA DMIL LED microscope and fluorescent images were taken using LEICA DM 2500 Microscope. HPLC was done using YL9100 HPLC and fluorescence reading were done using Spectra Max Gemini XPS (USA). 2.19 Statistical Analysis. Statistical significance of differences in values was calculated by a two-tailed Student's t-test. For multiple comparisons, an ANOVA analysis was performed. P value < 0.05 was considered statistically significant. MTT readings were read using plate reader (make) and HPLC analysis was done using (make).

3. RESULTS AND DISCUSSION 3.1 Synthesis and characterization of CRC/DOX NPs and PEG-CRC/DOX NPs. The stable DOX-CRC NPs were prepared by the solvent evaporation technique. The possible interaction of DOX with CRC could be through two important mechanisms: 1) inter molecular hydrogen bonding, for which the hydroxyl groups of the CRC could interact with the amine groups of the DOX and 2) the strong electrostatic interaction between CRC and DOX because the overall charge of the CRC is negative for which the DOX 47-52 could exert a cationic surface charge causing the two molecules to be bound strongly. Figure S1a-b shows the stable CRC/DOX NPs and the PEGylated form in water. In the case of the PEGylated CRC/DOX NPs, it is easy for the PEG to have a strong hydrogen bonding interaction with the CRC/DOX NPs because there could be exposed hydroxyl functionalities, which is enough to hold the PEG on the CRC/DOX making the PEGylated CRC/DOX NPs. The FT-IR studies clearly show that there were changes in the major stretching frequencies of the CRC, DOX and PEG molecules (Figure S2a-d). The relatively narrow peak at 3420 cm-1 indicates –OH stretching, which was broadened after loading DOX into the CRC NPs, and the further coating by PEG made the –OH stretching wider to 3520 cm-1 shown in Figure S2. The morphology of the CRC/DOX NPs (Figure 1a) and PEG-CRC/DOX NPs (Figure 1b) were visualized by TEM shows a spherical topography, whereas the PEG-


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CRC/DOX NPs showed defined and smooth outer surface indicating may be a smooth coating PEG molecules. The particle size of the synthesized CRC/DOX NPs as well as the PEGylated CRC/DOX NPs was analyzed with a DLS analyzer confirming that the particles are at the optimum size ranges

Curcumin as a Novel Nanocarrier System for Doxorubicin Delivery to

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