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pH-responsive prodrug nanoparticles based on xylan-curcumin conjugate for the efficient delivery of curcumin in cancer therapy
Carbohydrate Polymers 188 (2018) 252–259
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
pH-responsive prodrug nanoparticles based on xylan-curcumin conjugate for the efficient delivery of curcumin in cancer therapy Sauraja, S. Uday Kumarb, Vinay Kumarb, Ruchir Priyadarshia, P. Gopinathb, Yuvraj Singh Negia, a b
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Department of Polymer and Process Engineering, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand-247667, India Nanobiotechnology Laboratory, Centre for Nanotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand-247667, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Xylan Curcumin Prodrug NPs Drug-release Hemocompatibility Cell cytotoxicity
In the present study, novel pH-responsive prodrug nanoparticles based on xylan-curcumin (xyl-cur) conjugate were developed to enhance the therapeutic efficacy of curcumin in cancer therapy. The synthesis of xyl-cur conjugate (prodrug) was confirmed by FT-IR, 1H NMR, UV–vis and fluorescence spectroscopy. The xyl-cur prodrug was subsequently self-assembled in to nanoparticles (xyl-cur prodrug NPs) in an aqueous medium with the average particle size 253 nm and the zeta potential of −18.76 mV. The xyl-cur prodrug NPs were highly pHsensitive in nature and most of the drug was released at lower pH. The interaction of the xyl-cur prodrug NPs with blood components was tested by hemolysis study. The cytotoxic activity of the xyl-cur prodrug NPs against human colon cancer cells (HT-29, HCT-15) demonstrated that the prodrug NPs exhibits greater cytotoxic effect than curcumin. Therefore, these results reveal that xyl-cur prodrug NPs could be a promising candidate for improving the intracellular delivery of curcumin in cancer therapy.
1. Introduction Cancer is one of the most formidable and serious health problems in the world (Song et al., 2016; Wang, Yingsa, & Tian, 2017). According to statistics in the United States, colon cancer is the third most leading causes of deaths in both male and female (Yang et al., 2015). Among the various approaches for cancer treatment, chemotherapy is an indispensable choice for most of the cancers because of its high efficiency. The U.S. Food and Drug Administration (FDA), has approved several chemotherapeutic agents for cancer treatment, but due to their serious side effects and high cost, many plant-based therapeutic agents have also been used as alternatives (Plyduang, Lomlim, Yuenyongsawad, & Wiwattanapatapee, 2014). In recent years, curcumin obtained from the renowned spice turmeric (Curcuma longa) has gained immense interest in the field of pharmaceutical and biotechnology because of its potent antioxidant, anti-inflammatory and anti-cancer properties (Almeida et al., 2017; Anitha et al., 2011). In addition to its anti-cancer activity it also acts as a chemo-sensitizer for reversing multidrug resistance in cancer chemotherapy (Li et al., 2016). Despite of these pharmacological activities, curcumin is still far away from the clinic uses due to its poor bioavailability which is associated with its low absorption, rapid metabolism, low aqueous solubility and chemical instability (Tang et al., 2010).
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To redress these problems, curcumin was delivered through several approaches including, liposomes, micelles, lipid based nanoparticles, polymeric nanoparticles and polymer-drug conjugate, where it was primarily loaded via physical encapsulation or chemical conjugation (Kim et al., 2011). Among these diverse approaches, polymer-drug conjugate (prodrug) is a very popular and widely acceptable approach to improve the aqueous solubility, controlled release and therapeutic efficacy of drug, especially when combined with nanotechnology (Dey & Sreenivasan, 2014). An amphiphilic polymer–drug conjugate is a versatile approach to combine the advantages of both prodrug and nanocarrier. The amphiphilic polymer–drug conjugate not only serves as a prodrug but also forms self-assemble nanoparticles in an aqueous medium (Liu et al., 2017; Wang et al., 2017). Over the last few years, polysaccharides have gained much interest in the field of drug delivery due to their desirable physiochemical and biological properties (Pereira et al., 2013). Compared to synthetic polymers, polysaccharides contains a variety of functional groups that can be used for drug conjugation; in addition they undergo enzymatic or hydrolytic degradation in the biological environment into non-toxic degradation byproducts (Ahmed & Aljaeid, 2016; Basu, Kunduru, Abtew, & Domb, 2015). Xylan is the second most abundant biopolymer of plant origin after cellulose (Peralta, Venkatachalam, Stone, & Pattathil, 2017) and recently used in variety of biomedical applications
Corresponding author. E-mail addresses: [email protected], [email protected] (Y.S. Negi).
https://doi.org/10.1016/j.carbpol.2018.02.006 Received 16 November 2017; Received in revised form 12 January 2018; Accepted 2 February 2018 Available online 07 February 2018 0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
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2.4. Characterization of xyl-cur conjugate (prodrug)
due to its biocompatibility, biodegradability and non-toxic nature (Fonseca Silva, Habibi, Colodette, & Lucia, 2011; Petzold-Welcke, Schwikal, Daus, & Heinze, 2014). In the present study, taking the advantages of nanotechnology as well as prodrug strategy, novel pH responsive prodrug nanoparticles based on xylan-curcumin conjugate were developed for the efficient delivery of curcumin. Towards this aim, first, curcumin was conjugated with hydrophilic xylan via a pH sensitive succinate linker to produce an amphiphilic xyl-cur conjugate (prodrug). Subsequently, the self assembled nanoparticles were prepared by dialysis method and the particle size distribution, morphology, physiological stability, and in-vitro drug release behaviors of xyl-cur prodrug NPs in buffers with different pH values were investigated. Thereafter, the blood compatibility of the xyl-cur prodrug NPs was investigated by hemolytic study. Finally, the cytotoxicity of the xyl-cur prodrug NPs was evaluated against the human colon cancer cells lines HT-29 and HCT-115. All the studies were carried out to evaluate the potential of the xyl-cur prodrug NPs as a promising drug delivery system in cancer therapy.
The FT-IR spectra were obtained with a Perkin Elmer FT-IR C91158 spectrophotometer using KBr disk method in the range 4000–400 cm−1. The 1H NMR spectra of the samples were recorded by Bruker–500 MHz spectrometer in (DMSO-d6) as solvent, with 16 scans. The UV–vis spectra of the samples were measured by the UV–vis spectrophotometer (Schimadu, Japan), after dissolving in DMSO. The absorption intensities were studied at 427 nm, over the range 200–700 nm. Similarly, fluorescence emission spectra of the samples were recorded using Fluorescence spectrophotometer (Hitachi F-4600, Japan) at the excitation wavelength of 427 nm, from the 450–700 nm with excitation and emission slit width of 5 nm. 2.5. Preparation of xyl-cur conjugate (prodrug) nanoparticles (xyl-cur prodrug NPs) For the preparation of xyl-cur prodrug NPs, xyl-cur conjugate (prodrug) solution in DMSO (1 mg/ml) was transferred to a dialysis bag (MWCO 12 kDa). The solution was dialyzed against distilled water for 24 h, (exchange every 4 h to remove the solvent and unbounded drug). After that, the dialyzed solution was passing through (0.45 μm) filter to avoid the large particles, and then it was lyophilized and stored in a dry place for further uses (Cao et al., 2015; Li et al., 2016). The yield of xylcur prodrug NPs obtained was observed to be 87%.
2. Experimental 2.1. Materials Curcumin (≥99%), succinic anhydride (≥99%), pyridine (≥99.5%), N-hydroxysuccinimide (NHS ≥ 97%), and N, N’-dicyclohexylcarbodiimide (DCC ≥ 99%), and 4-Dimethylaminopyridine (DMAP ≥99%), were purchased from Himedia (Mumbai, India). Dialysis membrane (MWCO 12 kDa) was obtained from Sigma-Aldrich (Bangalore, India). The Human colon carcinoma cells (HT-29, HCT-15) were received from National centre for cell science, (Pune, India) and the [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] (MTT ≥ 97.5%), was received from Sigma-Aldrich (Bangalore, India). All other chemicals and solvents were used without purification.
2.6. Particle size and morphological observation The particle size and zeta potential of the xyl-cur prodrug NPs were determined by dynamic light scattering (DLS) (Brookhaven instruments) at a fixed scattering angle of 90°. In detail, 10 mg of lyophilized xyl-cur prodrug NPs sample was dissolved in 10 ml deionized water, followed by ultrasonic treatment, to get the uniform dispersion and the measurements were performed at 25 °C in triplet with a time span of 120 s. The morphology of the xyl-cur prodrug NPs was further analyzed by Field emission scanning electron microscope (FE-SEM) (Mira 3 Tescan), at an acceleration voltage from 10 to 20 kV. Likewise, a drop of the lyophilized xyl-cur prodrug NPs solution in distilled water (1 mg/ml) was placed on a glass slides, air-dried and sputter coated prior to image acquisition.
2.2. Extraction of xylan from corn cob Xylan used in this study, was extracted from corn cob as described previously studies (Fundador, Enomoto-Rogers, Takemura, & Iwata, 2012; Sauraj, Kumar, Gopinath, & Negi, 2017). Briefly, xylan was extracted using 10% NaOH for 24 h at room temperature with a solid to liquor ratio of 1:20 (g/ml) from the holocellulose, which was obtained by the delignification of corn-cob with acidified sodium chlorite solutions at 70 °C for 2 h.
2.7. Determination of drug content For the determination of drug content, a known amount of lyophilized xyl-cur prodrug NPs was dissolved in DMSO and then incubated at 37 °C for 24 h. The amount of curcumin present in xyl-cur prodrug NPs was quantified from a calibration curve of curcumin ranged (5–25 μg/ ml, r2 = 0.996, Limit of Detection = 1.727 μg/ml), established at the same conditions, using UV–vis spectroscopy at 427 nm (Dey, Ambattu, Hari, Rekha, & Sreenivasan, 2015).
2.3. Synthesis of xyl-cur conjugate (prodrug) 2.3.1. Synthesis of curcumin-monosuccinate (cur-monosuccinate) Cur-monosuccinate was synthesized according to previous study (Jain et al., 2014). Curcumin (0.368 g, 1 mmol) was dissolved in 15 ml benzene and then refluxed with succinic anhydride (0.1 g, 1 mmol) in presence of pyridine (1 ml). After refluxing at 80 °C for 24 h, the solvent was removed under reduce pressure and the resulting product was purified by column chromatography using hexane-ethyl acetate as elute to get the final product cur-monosuccinate (Yield = 0.27 g, 52.4%).
2.8. Physiological stability analysis The physiological stability of free curcumin and xyl-cur prodrug NPs, at different physiological pH (5.0 and 7.4), was studied using UV–vis spectroscopy by measuring the changes in the absorbance at 427 nm. Both the free curcumin and xyl-cur prodrug NPs samples were dispersed in PBS buffers of pH 5.0 and 7.4 and incubated at 37 °C. At specified time interval, aliquots were withdrawn and the absorbances were measured at 427 nm (Dey & Sreenivasan, 2014).
2.3.2. Synthesis of xyl-cur conjugate (prodrug) For the synthesis of xyl-cur conjugate, xylan (0.132 g, 1 mmol) and cur-monosuccinate (0.864 g, 2 mmol) were dissolved in 20 ml DMSO, followed by the addition of DCC (0.412 g, 2 mmol) and DMAP (0.116 g, 1 mmol). The reaction mixture was stirred at room temperature for 24 h in dark. After that, the reaction mixture was filtered and then poured into 50 ml ethanol/ethyl ether (1:1 v/v), to precipitate the resulting product. The reaction scheme for the synthesis of xyl-cur conjugate is shown in Fig. 1.
2.9. Drug release study The release of curcumin from the xyl-cur prodrug NPs was performed at different physiological pH (5.0 and 7.4) using dialysis method. Briefly, 10 mg lyophilized powder of xyl-cur prodrug NPs was 253
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Fig. 1. Reaction scheme for the synthesis of cur-monosuccinate and xyl-cur conjugate (prodrug).
dispersed in (10 ml) PBS buffers of pH 5.0 and 7.4 and then transferred to a dialysis bag, which is further kept in a glass vessel containing 90 ml respective media. The release media was incubated at 37 °C with moderate stirring. At particular time intervals, 2 ml of sample was taken out for measurements and the amount of curcumin released was analyzed by UV–vis spectroscopy at 427 nm (Sauraj et al., 2017). Subsequently, 2 ml fresh media was added to incubation bath to maintain the sink condition.
Cellviability (%) =
2.12. Acridine orange-ethidium bromide (AO-EB) staining The AO-EB staining was performed to analyze the apoptotic morphological changes in the nuclei of cells. Briefly, the cells (HT-29, HCT15) were treated with IC50 concentrations of curcumin, cur-monosuccinate and xyl-cur prodrug NPs for 24 h and 48 h, respectively. Thereafter, the growth medium was replaced with PBS containing 10 g/ ml of AO-EB mixture and the cells were incubated for 10–15 min at 37 °C. The cells further rinsed with PBS to remove the excess dyes and visualised immediately by EVOS cell imaging system after staining (Nirmala, Akila, Nadar, Narendhirakannan, & Chatterjee, 2016).
2.10. Blood compatibility The blood compatibility of xyl-cur prodrug Nps was evaluated by hemolysis study. The human blood samples were collect from the blood bank, IITR hospital, Roorkee. For hemolysis study, red blood cells (RBCs) were separated by centrifugation at 1500 rpm for 5 min and then washed with three times with PBS. After that, 100 μl RBCs was added to 10 ml phosphate buffer saline to prepare the stock solution. Then 100 μl RBCs stock solution was incubated with xyl-cur prodrug NPs solutions (0.5–2 mg/ml) at 37 °C for 20 min. After incubation, RBCs were centrifuged at 1500 rpm for 5 min and the supernatants were analyzed by UV–vis spectroscopy at 541 nm (Kumar, Lale, Mahajan, Choudhary, & Koul, 2015). A phosphate buffer saline solution was used as a negative (0% lysis) and distilled water used as a positive controls (100% lysis). The hemolysis percentage (HP) was estimated with the following equation:-
HP(%) =
A sample − Anegative Apositive − Anegative
absorbance of the sample X 100 absorbance of control
2.13. Cell cycle analysis To monitor the effect of curcumin and xyl-cur prodrug NPs on cell proliferation, cell cycle analysis was performed by staining the cells with propidium iodide (PI) and subsequent analysis by flow cytometry. The (1 × 105) HT-29 cells were seeded in 96 plates and then treated with curcumin, cur-monosuccinate and xyl-cur prodrug NPs for 10 h. After treatment, cells were briefly rinsed with PBS and subsequently trypsinized and centrifuged to obtain the cell pellet. The resultant cell pellet was resuspended in 70% ethanol fixative and kept in ice for 10 min. The cells were then treated with 50 (μg/ml) PI, (50 μg/ml) RNase A and (0.05%) TrotonX-100 and incubated in dark for 30 min at 37 °C. The cells were then analyzed by flow cytometer (Amnis Flow sight) for 10,000 events and processed further by IDEAS software to represent the results as histograms (Alam, Panda, & Chauhan, 2012).
X 100
Where, Asample, Anegative and Apositive denote the absorbance of sample, negative and positive controls, respectively. All experiments were carried out in triplicate. 2.11. Cytotoxicity study
2.14. Statistical analysis The cytotoxicity of the curcumin, cur-monosuccinate and xyl-cur prodrug NPs towards HT-29 and HCT-15 cells was evaluated by MTT assay. The cells were seeded in 96-well plates (5 × 103cells/well) and then treated with various concentrations of free curcumin, cur-monosuccinate and xyl-cur prodrug NPs (with an equivalent concentration of curcumin) for 48 h. Thereafter, the growth medium was removed from each well and 10 μl of MTT solution was added to each well and incubated at 37 °C for 3–4 h, the resultant dark-blueformazan crystals were dissolved in DMSO and the absorbance was detected at 574 nm using Biorad plate reader. The experiments were conducted in triplicate and the percent cell viability was calculated using the following equation (Chen et al., 2017)
All experimental data were performed in triplicate, and data were expressed as mean ± standard deviation, where applicable. Data were statistically processed using one way analysis of variance (ANOVA) to assess significant differences among various groups followed by student t-test using GraphPad Prism 6.0, and Origin Pro 8.0 software. 3. Results and discussion 3.1. Synthesis and characterization of xyl-cur conjugate (Prodrug) For the synthesis of xyl-cur conjugate (prodrug), initially cur254
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The synthesis of xyl-cur conjugate (prodrug) was further verified by H NMR spectroscopy. The Fig. 3, depicts the 1H NMR spectrum of the xyl-cur conjugate, the characteristic peaks of xylan between 5.2–3.0 ppm were attributed to the protons of (1–4)-β-D-xylopyranosyl units) (Hansen & Plackett, 2011) and the peaks between 6.1 −7.4 ppm were attributed to the protons of curcumin moiety (Waghela, Sharma, Dhumale, Pandey, & Pathak, 2015). In addition, the peak at 2.4 ppm was attributed to the eCH2 protons of the succinyl group present in the cur-monosuccinate. Appearance of this characteristic peak at 2.4 ppm in the xyl-cur conjugate spectrum, demonstrated the successful synthesis of xyl-cur conjugate (prodrug). The conjugation of curcumin to xylan was further ascertained by UV–vis and fluorescence spectroscopy. The absorption spectra of xylcur conjugate (prodrug) show a broad absorption band at 427 nm similar to the absorption spectrum of curcumin (Fig. 4a), which indicate the presence of curcumin in xyl-cur conjugate (prodrug) (Sarika, James, Kumar, Raj, & Kumary, 2015). The fluorescence emission spectra of xylcur conjugate (Fig. 4b), exhibits a blue shift at 522 nm, compare to curcumin at 550 nm, which attributed to the conjugation between xylan and curcumin (Sarika, James, Nishna, Anil Kumar, & Raj, 2015) 1
Fig. 2. FT-IR spectra of (a) cur-monosuccinate (b) xylan (c) xyl-cur conjugate (prodrug).
3.2. Preparation and characterization of xyl-cur prodrug NPs
monosuccinate was synthesized to introduce the carboxylic acid (eCOOH) functionality, and then conjugated with xylan via carbodiimide chemistry as illustrated in Fig. 1. The FT-IR spectra of curmonosuccinate (Fig. 2a), exhibited an absorption band at 3510 cm−1 attributed to the stretching vibrations of phenolic (eOH) group and the peak at 1732 cm−1 was assigned to the C]O stretching frequencies of succinate group. The peaks at 1605, 1512, and 1272 cm−1 were attributed to the stretching frequencies of C]O (enol) and CeO groups. In the spectrum of xylan (Fig. 2b), band at 3420 cm−1 is attributed to the stretching of −OH group and the sharp band at 1632 cm−1 due to absorbed water, while the peaks between 1465- 1043 cm−1 arise from the stretching and bending vibration of CeO, CeC and CeOH groups. The peak at 890 cm−1 is attributed to the β-glucosidic linkage between the xylose units (Ren, Sun, Liu, Lin, & He, 2007). In addition to the predominant peaks of xylan and cur-monosuccinate, the FT-IR spectrum of xyl-cur conjugate (prodrug) (Fig. 2c), exhibits a new peak at 1735 cm−1 corresponding to the frequency of C]O group which assign the formation of ester bond between xylan and cur-monosuccinate.
The amphiphilic polymers or polymer-drug conjugates could form self-assembled nanoparticles in aqueous medium; it has been already reported by various researchers. Similarly, amphiphilic xyl-cur conjugate (prodrug) formed self-assembled nanoparticles in aqueous medium with the hydrophilic shell of xylan and a hydrophobic core of curcumin. The hydrodynamic size of the xyl-cur prodrug NPs was determined by dynamic light scattering (DLS). The size distribution of the xyl-cur prodrug NPs is shown in Fig. 4c. It can be observed that the prodrug NPs were about 205 nm in size with 108 nm and 343 nm as the low end and high end cutoff, respectively. Furthermore, the morphology of the xyl-cur prodrug NPs was investigated by scanning electron microscopy (SEM). The SEM images of the xyl-cur prodrug NPs (Fig. 4d) show that prodrug NPs were roughly spherical and uniform in size with the small diameter of 160 nm. The variation in size observed between DLS and SEM techniques is due the difference in the procedure condition. The zeta potential of xyl-cur prodrug NPs is also measured by DLS after dispersing the conjugate in aqueous medium and it was observed to
Fig. 3. 1H NMR spectra of xyl-cur conjugate (prodrug).
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Fig. 4. (a) UV–vis and (b) fluorosence spectra of curcumin and xyl-cur conjugate (prodrug) (c) particle size distribution and (d) SEM morphology of xyl-cur conjugate (prodrug) NPs.
show a negative zeta potential of −18.76 mV. The negatively charged surface might be due to the unreacted hydroxyl (eOH) groups of xylan that resides on the outer surface of the nanoparticles after self assembly in an aqueous solution. Thus, the presence of this negative charge on the surface of prodrug NPs may be partly responsible for their stability in aqueous medium due to the interparticle interactions (Liu et al., 2017). The xyl-cur prodrug NPs contained, (24 wt%) drug content as determined by UV-spectroscopy.
condition (pH 7.4) as well as acidic condition (pH 5.0). The release profiles of curcumin (Fig. 5b), shows that about 60–65% of drug was released within 48 h in pH (5.0) while only 40–45% drug was released in pH (7.4). These results indicate that the release rate of curcumin was higher in acidic pH (5.0) than that in physiological pH (7.4). The higher releases rate of curcumin in acidic pH is attributed to the easy breakage of ester linkage. The release study suggested that ester linkage between the xylan and curcumin is more sensitive towards the acidic pH (5.0) compare to basic pH (7.4), which is suitable for the delivery of curcumin to acidic environment of tumor cells (Dey & Sreenivasan, 2015).
3.3. Physiological stability analysis The physiological stability of nanoparticles is an important concern in drug delivery. In order to excess the physiological stability of curcumin and xyl-cur prodrug NPs in PBS (pH 7.4), the change in their absorbance at 427 nm was monitored by UV–vis spectroscopy. As shown in Fig. 5a, xyl-cur prodrug NPs were found to be stable under physiological condition, while the curcumin degrade completely within 6 h. These results indicated that, the stability of curcumin was significantly improved after conjugated with xylan, which could protect curcumin from degradation in alkaline environments (Dey & Sreenivasan, 2014).
3.5. Blood compatibility Hemo-compatibility is an important criterion for safety and biocompatibility of drug-loaded nanoparticles. Therefore, hemolysis study was carried out to evaluate the responses that arise between drugloaded nanoparticle and blood component. Drug-loaded nanoparticles interact with RBCs and damage them. Hemoglobin released from the damage RBCs exhibits a strong absorption peak at 541 nm (Lale, Kumar, Prasad, Bharti, & Koul, 2015). For drug-loaded nanoparticles that do not interact with RBCs, this peak would be negligible. As shown in Fig. 5c, the xyl-cur prodrug NPs exhibited less than 5% hemolytic activity to RBCs even at the highest xyl-cur prodrug NPs concentration of 2 mg/ml, which demonstrate the excellent blood compatibility and suitability for intravenous administration of xyl-cur prodrug NPs.
3.4. Drug release study In order to evaluate the pH responsive behavior of the xyl-cur prodrug NPs, in-vitro drug release study was performed in physiological 256
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Fig. 5. (a) Stability studies of curcumin and xyl-cur prodrug NPs (b) Release profiles of curcumin from the xyl-cur prodrug NPs under different conditions (c) Hemocompatibilty of xyl-cur prodrug NPs.
Fig. 6. Cytotoxicity activity of curcumin, cur-monosuccinate and xyl-cur prodrug NPs againts (a) HCT-15 cells, and (b) HT-29 cells treated with (5–90 μg/ml) curcumin, (5–90 μg/ml) curmonosuccinate, (5–90 μg/ml) xyl-cur prodrug NPs for 48 h.
that xyl-cur prodrug NPs possessed over 2.8 fold lower IC50 value than free curcumin for HT-29 cell line, and over 1.28-fold lower IC50 value than free curcumin for HCT-15 cell line, indicating the significantly enhanced cytotoxicity of xyl-cur prodrug NPs (Table S1, Supplementary data). This might be due to the sustained release of drug molecule from the xyl-cur prodrug NPs, which is in agreement with the in-vitro drug release behavior observations (Fig. 5b). In order to further ascertain the cell viability results, AO/EB staining was carried out to assess the cytotoxicity of drug and drug-polymer conjugates on a qualitative basis. Combination of two dyes AO and EB were used for the study. AO is a cell permeant dye which non-
3.6. Cytotoxicity study The in-vitro cytotoxicity of the free drugs (curcumin and curmonosuccinate) and the xyl-cur prodrug NPs was investigated in HT-29 and HCT-15 colon cancer cells lines by MTT assay. The cells (HT-29 and HCT-15) were treated with different concentrations of xyl-cur prodrug NPs as well as the free drugs for 24 h and 48 h. The cell viability results (Fig. 6a and b) showed that the xyl-cur prodrug NPs and the free drugs exhibited the cytotoxicity in a dosedependent and time-dependent manner and the significant differences in cytotoxicity were observed after 48 h treatment. These results show 257
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Fig. 7. Fluorescence images of live (green) and dead (red) cells (a) HCT-15 cells, and (b) HT-29 cells after treated with IC50 concentration of curcumin, cur-monosuccinate and xyl-cur prodrug NPs for 24 and 48 h. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 8. Cell cyclic analysis of HT-29 cells (a) control (b) curcumin and (c) xyl-cur prodrug NPs after treated with curcumin and xyl-cur prodrug NPs.
3.7. Cell cycle analysis
specifically labels the cell nucleus by intercalating with DNA and also stains cytoplasmic components to certain extent. On the other hand, although EB also intercalates with double stranded DNA, it can do so only in case of cells with compromised membranes (apoptotic cells). As shown in Fig. 7, the controlled cells (labeled with AO alone) appeared to have uniform green nuclei, whereas early apoptotic cells appeared to possess yellow or yellowish green spotted nucleus. In contrast to this, late apoptotic cells with compromised membrane appeared orange in color as their nucleus was stained by both AO and EB. The staining images (Fig. 7a and b) of treated HT-29 and HCT-15 cells clearly indicate that with increase in treatment time span there is consequent increase in apoptotic cells (orange to red cells). Thus, in summary AO-EB staining results corroborated well with the cell viability results and demonstrated the improved therapeutic efficacy of xylcur prodrug NPs as compared to free curcumin and cur-monosuccinate as such.
In order to monitor the effect of xyl-cur prodrug NPs and the free drug (curcumin) on cell proliferation, cell cycle analysis was carried out. It was observed that upon treating cells with xyl-cur prodrug NPs, the cells were predominantly in apoptotic phase (45.1%) as compared to those treated by curcumin (25.9%) and untreated cells (6.14%) as shown in Fig. 8. Also, the G0/G1 cell population also declined to 28.9% and 41.6% upon xyl-cur prodrug NPs and free curcumin treatment, respectively, as compared to 73.7% for untreated cells. This ascertains that although the cells have completed DNA synthesis (S phase) but they could not proceed through subsequent events of karyokinesis due to xyl-cur prodrug NPs mediated destabilization of cell microtubules. The data represents that xyl-cur prodrug NPs efficiently inhibited the growth of the cells and allowed the cells for programmed death.
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4. Conclusion
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In summary, we have successfully developed a pH-sensitive prodrug NPs by directly conjugating the curcumin to the xylan backbone via an acid labile succinate linkage. The prodrug NPs showed a significantly faster drug release at a mildly acidic pH of 5.0, usually present in tumor microenvironments and lysosomes than a physiological pH of 7.4. It is confirmed that the xyl-cur prodrug NPs could efficiently deliver drug to the nucleus of the tumor cells and led to much more cytotoxic effects to HT-29 and HCT-15 human colon cancer cell lines than the parent curcumin. The current study also demonstrated the synergistic advantages of nanocarriers as well as polymer-drug conjugates. This design could be extended to self assembled NP delivery systems for a broad range of anti-cancer drugs by applying the specific linkages between polymer and drug. This novel approach arises the interest of many researchers regarding the design of a new multifunctional drug delivery system. Acknowledgements The authors express their gratitude to Ministry of Human Resource Development (MHRD), New Delhi for financial support to conduct the study. Sincere thanks to Department of Biotechnology, IIT Roorkee for the various biological facilities provided. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.carbpol.2018.02.006. References Ahmed, T. A., & Aljaeid, B. M. (2016). Preparation, characterization, and potential application of chitosan, chitosan derivatives, and chitosan metal nanoparticles in pharmaceutical drug delivery. Drug Design, Development and Therapy, 10, 483–507. http://dx.doi.org/10.2147/DDDT.S99651. Alam, S., Panda, J. J., & Chauhan, V. S. (2012). Novel dipeptide nanoparticles for effective curcumin delivery. International Journal of Nanomedicine, 7, 4207–4222. http://dx.doi.org/10.2147/IJN.S33015. Almeida, E. A. M. S., Bellettini, I. C., Garcia, F. P., Farinácio, M. T., Nakamura, C. V., Rubira, A. F., ... Muniz, E. C. (2017). Curcumin-loaded dual pH- and thermo-responsive magnetic microcarriers based on pectin maleate for drug delivery. Carbohydrate Polymers, 171, 259–266. http://dx.doi.org/10.1016/j.carbpol.2017.05. 034. Anitha, A., Maya, S., Deepa, N., Chennazhi, K. P., Nair, S. V., Tamura, H., & Jayakumar, R. (2011). Efficient water soluble O-carboxymethyl chitosan nanocarrier for the delivery of curcumin to cancer cells. Carbohydrate Polymers, 83(2), 452–461. http://dx.doi. org/10.1016/j.carbpol.2010.08.008. Basu, A., Kunduru, K. R., Abtew, E., & Domb, A. J. (2015). Polysaccharide-based conjugates for biomedical applications. Bioconjugate Chemistry, 26(8), 1396–1412. http://dx.doi.org/10.1021/acs.bioconjchem.5b00242. Cao, Y., Gao, M., Chen, C., Fan, A., Zhang, J., Kong, D., & Zhao, Y. (2015). Triggeredrelease polymeric conjugate micelles for on-demand intracellular drug delivery. Nanotechnology, 26(11), 115101. http://dx.doi.org/10.1088/0957-4484/26/11/ 115101. Chen, Y., Peng, F., Song, X., Wu, J., Yao, W., & Gao, X. (2017). Conjugation of paclitaxel to C-6 hexanediamine-modified hyaluronic acid for targeted drug delivery to enhance antitumor efficacy. Carbohydrate Polymers, 181, 150–158. http://dx.doi.org/10. 1016/j.carbpol.2017.09.017. Dey, S., & Sreenivasan, K. (2014). Conjugation of curcumin onto alginate enhances aqueous solubility and stability of curcumin. Carbohydrate Polymers, 99, 499–507. http://dx.doi.org/10.1016/j.carbpol.2013.08.067. Dey, S., & Sreenivasan, K. (2015). Conjugating curcumin to water soluble polymer stabilized gold nanoparticles via pH responsive succinate linker. Journal of Materials Chemistry B, 3, 824–833. http://dx.doi.org/10.1039/c4tb01731e. Dey, S., Ambattu, L. A., Hari, P. R., Rekha, M. R., & Sreenivasan, K. (2015). Glutathionebearing fluorescent polymer-curcumin conjugate enables simultaneous drug delivery and label-free cellular imaging. Polymer, Brodersen, 25–33. http://dx.doi.org/10. 1016/j.polymer.2015.08.020. Fonseca Silva, T. C., Habibi, Y., Colodette, J. L., & Lucia, L. A. (2011). The influence of the chemical and structural features of xylan on the physical properties of its derived hydrogels. Soft Matter, 7(3), 1090. http://dx.doi.org/10.1039/c0sm00868k.
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pH-responsive prodrug nanoparticles based on xylan-curcumin conjugate for the efficient delivery of curcumin in cancer therapy Sauraja, S. Uday Kumarb, Vinay Kumarb, Ruchir Priyadarshia, P. Gopinathb, Yuvraj Singh Negia, a b
T ⁎
Department of Polymer and Process Engineering, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand-247667, India Nanobiotechnology Laboratory, Centre for Nanotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand-247667, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Xylan Curcumin Prodrug NPs Drug-release Hemocompatibility Cell cytotoxicity
In the present study, novel pH-responsive prodrug nanoparticles based on xylan-curcumin (xyl-cur) conjugate were developed to enhance the therapeutic efficacy of curcumin in cancer therapy. The synthesis of xyl-cur conjugate (prodrug) was confirmed by FT-IR, 1H NMR, UV–vis and fluorescence spectroscopy. The xyl-cur prodrug was subsequently self-assembled in to nanoparticles (xyl-cur prodrug NPs) in an aqueous medium with the average particle size 253 nm and the zeta potential of −18.76 mV. The xyl-cur prodrug NPs were highly pHsensitive in nature and most of the drug was released at lower pH. The interaction of the xyl-cur prodrug NPs with blood components was tested by hemolysis study. The cytotoxic activity of the xyl-cur prodrug NPs against human colon cancer cells (HT-29, HCT-15) demonstrated that the prodrug NPs exhibits greater cytotoxic effect than curcumin. Therefore, these results reveal that xyl-cur prodrug NPs could be a promising candidate for improving the intracellular delivery of curcumin in cancer therapy.
1. Introduction Cancer is one of the most formidable and serious health problems in the world (Song et al., 2016; Wang, Yingsa, & Tian, 2017). According to statistics in the United States, colon cancer is the third most leading causes of deaths in both male and female (Yang et al., 2015). Among the various approaches for cancer treatment, chemotherapy is an indispensable choice for most of the cancers because of its high efficiency. The U.S. Food and Drug Administration (FDA), has approved several chemotherapeutic agents for cancer treatment, but due to their serious side effects and high cost, many plant-based therapeutic agents have also been used as alternatives (Plyduang, Lomlim, Yuenyongsawad, & Wiwattanapatapee, 2014). In recent years, curcumin obtained from the renowned spice turmeric (Curcuma longa) has gained immense interest in the field of pharmaceutical and biotechnology because of its potent antioxidant, anti-inflammatory and anti-cancer properties (Almeida et al., 2017; Anitha et al., 2011). In addition to its anti-cancer activity it also acts as a chemo-sensitizer for reversing multidrug resistance in cancer chemotherapy (Li et al., 2016). Despite of these pharmacological activities, curcumin is still far away from the clinic uses due to its poor bioavailability which is associated with its low absorption, rapid metabolism, low aqueous solubility and chemical instability (Tang et al., 2010).
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To redress these problems, curcumin was delivered through several approaches including, liposomes, micelles, lipid based nanoparticles, polymeric nanoparticles and polymer-drug conjugate, where it was primarily loaded via physical encapsulation or chemical conjugation (Kim et al., 2011). Among these diverse approaches, polymer-drug conjugate (prodrug) is a very popular and widely acceptable approach to improve the aqueous solubility, controlled release and therapeutic efficacy of drug, especially when combined with nanotechnology (Dey & Sreenivasan, 2014). An amphiphilic polymer–drug conjugate is a versatile approach to combine the advantages of both prodrug and nanocarrier. The amphiphilic polymer–drug conjugate not only serves as a prodrug but also forms self-assemble nanoparticles in an aqueous medium (Liu et al., 2017; Wang et al., 2017). Over the last few years, polysaccharides have gained much interest in the field of drug delivery due to their desirable physiochemical and biological properties (Pereira et al., 2013). Compared to synthetic polymers, polysaccharides contains a variety of functional groups that can be used for drug conjugation; in addition they undergo enzymatic or hydrolytic degradation in the biological environment into non-toxic degradation byproducts (Ahmed & Aljaeid, 2016; Basu, Kunduru, Abtew, & Domb, 2015). Xylan is the second most abundant biopolymer of plant origin after cellulose (Peralta, Venkatachalam, Stone, & Pattathil, 2017) and recently used in variety of biomedical applications
Corresponding author. E-mail addresses: [email protected], [email protected] (Y.S. Negi).
https://doi.org/10.1016/j.carbpol.2018.02.006 Received 16 November 2017; Received in revised form 12 January 2018; Accepted 2 February 2018 Available online 07 February 2018 0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
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2.4. Characterization of xyl-cur conjugate (prodrug)
due to its biocompatibility, biodegradability and non-toxic nature (Fonseca Silva, Habibi, Colodette, & Lucia, 2011; Petzold-Welcke, Schwikal, Daus, & Heinze, 2014). In the present study, taking the advantages of nanotechnology as well as prodrug strategy, novel pH responsive prodrug nanoparticles based on xylan-curcumin conjugate were developed for the efficient delivery of curcumin. Towards this aim, first, curcumin was conjugated with hydrophilic xylan via a pH sensitive succinate linker to produce an amphiphilic xyl-cur conjugate (prodrug). Subsequently, the self assembled nanoparticles were prepared by dialysis method and the particle size distribution, morphology, physiological stability, and in-vitro drug release behaviors of xyl-cur prodrug NPs in buffers with different pH values were investigated. Thereafter, the blood compatibility of the xyl-cur prodrug NPs was investigated by hemolytic study. Finally, the cytotoxicity of the xyl-cur prodrug NPs was evaluated against the human colon cancer cells lines HT-29 and HCT-115. All the studies were carried out to evaluate the potential of the xyl-cur prodrug NPs as a promising drug delivery system in cancer therapy.
The FT-IR spectra were obtained with a Perkin Elmer FT-IR C91158 spectrophotometer using KBr disk method in the range 4000–400 cm−1. The 1H NMR spectra of the samples were recorded by Bruker–500 MHz spectrometer in (DMSO-d6) as solvent, with 16 scans. The UV–vis spectra of the samples were measured by the UV–vis spectrophotometer (Schimadu, Japan), after dissolving in DMSO. The absorption intensities were studied at 427 nm, over the range 200–700 nm. Similarly, fluorescence emission spectra of the samples were recorded using Fluorescence spectrophotometer (Hitachi F-4600, Japan) at the excitation wavelength of 427 nm, from the 450–700 nm with excitation and emission slit width of 5 nm. 2.5. Preparation of xyl-cur conjugate (prodrug) nanoparticles (xyl-cur prodrug NPs) For the preparation of xyl-cur prodrug NPs, xyl-cur conjugate (prodrug) solution in DMSO (1 mg/ml) was transferred to a dialysis bag (MWCO 12 kDa). The solution was dialyzed against distilled water for 24 h, (exchange every 4 h to remove the solvent and unbounded drug). After that, the dialyzed solution was passing through (0.45 μm) filter to avoid the large particles, and then it was lyophilized and stored in a dry place for further uses (Cao et al., 2015; Li et al., 2016). The yield of xylcur prodrug NPs obtained was observed to be 87%.
2. Experimental 2.1. Materials Curcumin (≥99%), succinic anhydride (≥99%), pyridine (≥99.5%), N-hydroxysuccinimide (NHS ≥ 97%), and N, N’-dicyclohexylcarbodiimide (DCC ≥ 99%), and 4-Dimethylaminopyridine (DMAP ≥99%), were purchased from Himedia (Mumbai, India). Dialysis membrane (MWCO 12 kDa) was obtained from Sigma-Aldrich (Bangalore, India). The Human colon carcinoma cells (HT-29, HCT-15) were received from National centre for cell science, (Pune, India) and the [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] (MTT ≥ 97.5%), was received from Sigma-Aldrich (Bangalore, India). All other chemicals and solvents were used without purification.
2.6. Particle size and morphological observation The particle size and zeta potential of the xyl-cur prodrug NPs were determined by dynamic light scattering (DLS) (Brookhaven instruments) at a fixed scattering angle of 90°. In detail, 10 mg of lyophilized xyl-cur prodrug NPs sample was dissolved in 10 ml deionized water, followed by ultrasonic treatment, to get the uniform dispersion and the measurements were performed at 25 °C in triplet with a time span of 120 s. The morphology of the xyl-cur prodrug NPs was further analyzed by Field emission scanning electron microscope (FE-SEM) (Mira 3 Tescan), at an acceleration voltage from 10 to 20 kV. Likewise, a drop of the lyophilized xyl-cur prodrug NPs solution in distilled water (1 mg/ml) was placed on a glass slides, air-dried and sputter coated prior to image acquisition.
2.2. Extraction of xylan from corn cob Xylan used in this study, was extracted from corn cob as described previously studies (Fundador, Enomoto-Rogers, Takemura, & Iwata, 2012; Sauraj, Kumar, Gopinath, & Negi, 2017). Briefly, xylan was extracted using 10% NaOH for 24 h at room temperature with a solid to liquor ratio of 1:20 (g/ml) from the holocellulose, which was obtained by the delignification of corn-cob with acidified sodium chlorite solutions at 70 °C for 2 h.
2.7. Determination of drug content For the determination of drug content, a known amount of lyophilized xyl-cur prodrug NPs was dissolved in DMSO and then incubated at 37 °C for 24 h. The amount of curcumin present in xyl-cur prodrug NPs was quantified from a calibration curve of curcumin ranged (5–25 μg/ ml, r2 = 0.996, Limit of Detection = 1.727 μg/ml), established at the same conditions, using UV–vis spectroscopy at 427 nm (Dey, Ambattu, Hari, Rekha, & Sreenivasan, 2015).
2.3. Synthesis of xyl-cur conjugate (prodrug) 2.3.1. Synthesis of curcumin-monosuccinate (cur-monosuccinate) Cur-monosuccinate was synthesized according to previous study (Jain et al., 2014). Curcumin (0.368 g, 1 mmol) was dissolved in 15 ml benzene and then refluxed with succinic anhydride (0.1 g, 1 mmol) in presence of pyridine (1 ml). After refluxing at 80 °C for 24 h, the solvent was removed under reduce pressure and the resulting product was purified by column chromatography using hexane-ethyl acetate as elute to get the final product cur-monosuccinate (Yield = 0.27 g, 52.4%).
2.8. Physiological stability analysis The physiological stability of free curcumin and xyl-cur prodrug NPs, at different physiological pH (5.0 and 7.4), was studied using UV–vis spectroscopy by measuring the changes in the absorbance at 427 nm. Both the free curcumin and xyl-cur prodrug NPs samples were dispersed in PBS buffers of pH 5.0 and 7.4 and incubated at 37 °C. At specified time interval, aliquots were withdrawn and the absorbances were measured at 427 nm (Dey & Sreenivasan, 2014).
2.3.2. Synthesis of xyl-cur conjugate (prodrug) For the synthesis of xyl-cur conjugate, xylan (0.132 g, 1 mmol) and cur-monosuccinate (0.864 g, 2 mmol) were dissolved in 20 ml DMSO, followed by the addition of DCC (0.412 g, 2 mmol) and DMAP (0.116 g, 1 mmol). The reaction mixture was stirred at room temperature for 24 h in dark. After that, the reaction mixture was filtered and then poured into 50 ml ethanol/ethyl ether (1:1 v/v), to precipitate the resulting product. The reaction scheme for the synthesis of xyl-cur conjugate is shown in Fig. 1.
2.9. Drug release study The release of curcumin from the xyl-cur prodrug NPs was performed at different physiological pH (5.0 and 7.4) using dialysis method. Briefly, 10 mg lyophilized powder of xyl-cur prodrug NPs was 253
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Fig. 1. Reaction scheme for the synthesis of cur-monosuccinate and xyl-cur conjugate (prodrug).
dispersed in (10 ml) PBS buffers of pH 5.0 and 7.4 and then transferred to a dialysis bag, which is further kept in a glass vessel containing 90 ml respective media. The release media was incubated at 37 °C with moderate stirring. At particular time intervals, 2 ml of sample was taken out for measurements and the amount of curcumin released was analyzed by UV–vis spectroscopy at 427 nm (Sauraj et al., 2017). Subsequently, 2 ml fresh media was added to incubation bath to maintain the sink condition.
Cellviability (%) =
2.12. Acridine orange-ethidium bromide (AO-EB) staining The AO-EB staining was performed to analyze the apoptotic morphological changes in the nuclei of cells. Briefly, the cells (HT-29, HCT15) were treated with IC50 concentrations of curcumin, cur-monosuccinate and xyl-cur prodrug NPs for 24 h and 48 h, respectively. Thereafter, the growth medium was replaced with PBS containing 10 g/ ml of AO-EB mixture and the cells were incubated for 10–15 min at 37 °C. The cells further rinsed with PBS to remove the excess dyes and visualised immediately by EVOS cell imaging system after staining (Nirmala, Akila, Nadar, Narendhirakannan, & Chatterjee, 2016).
2.10. Blood compatibility The blood compatibility of xyl-cur prodrug Nps was evaluated by hemolysis study. The human blood samples were collect from the blood bank, IITR hospital, Roorkee. For hemolysis study, red blood cells (RBCs) were separated by centrifugation at 1500 rpm for 5 min and then washed with three times with PBS. After that, 100 μl RBCs was added to 10 ml phosphate buffer saline to prepare the stock solution. Then 100 μl RBCs stock solution was incubated with xyl-cur prodrug NPs solutions (0.5–2 mg/ml) at 37 °C for 20 min. After incubation, RBCs were centrifuged at 1500 rpm for 5 min and the supernatants were analyzed by UV–vis spectroscopy at 541 nm (Kumar, Lale, Mahajan, Choudhary, & Koul, 2015). A phosphate buffer saline solution was used as a negative (0% lysis) and distilled water used as a positive controls (100% lysis). The hemolysis percentage (HP) was estimated with the following equation:-
HP(%) =
A sample − Anegative Apositive − Anegative
absorbance of the sample X 100 absorbance of control
2.13. Cell cycle analysis To monitor the effect of curcumin and xyl-cur prodrug NPs on cell proliferation, cell cycle analysis was performed by staining the cells with propidium iodide (PI) and subsequent analysis by flow cytometry. The (1 × 105) HT-29 cells were seeded in 96 plates and then treated with curcumin, cur-monosuccinate and xyl-cur prodrug NPs for 10 h. After treatment, cells were briefly rinsed with PBS and subsequently trypsinized and centrifuged to obtain the cell pellet. The resultant cell pellet was resuspended in 70% ethanol fixative and kept in ice for 10 min. The cells were then treated with 50 (μg/ml) PI, (50 μg/ml) RNase A and (0.05%) TrotonX-100 and incubated in dark for 30 min at 37 °C. The cells were then analyzed by flow cytometer (Amnis Flow sight) for 10,000 events and processed further by IDEAS software to represent the results as histograms (Alam, Panda, & Chauhan, 2012).
X 100
Where, Asample, Anegative and Apositive denote the absorbance of sample, negative and positive controls, respectively. All experiments were carried out in triplicate. 2.11. Cytotoxicity study
2.14. Statistical analysis The cytotoxicity of the curcumin, cur-monosuccinate and xyl-cur prodrug NPs towards HT-29 and HCT-15 cells was evaluated by MTT assay. The cells were seeded in 96-well plates (5 × 103cells/well) and then treated with various concentrations of free curcumin, cur-monosuccinate and xyl-cur prodrug NPs (with an equivalent concentration of curcumin) for 48 h. Thereafter, the growth medium was removed from each well and 10 μl of MTT solution was added to each well and incubated at 37 °C for 3–4 h, the resultant dark-blueformazan crystals were dissolved in DMSO and the absorbance was detected at 574 nm using Biorad plate reader. The experiments were conducted in triplicate and the percent cell viability was calculated using the following equation (Chen et al., 2017)
All experimental data were performed in triplicate, and data were expressed as mean ± standard deviation, where applicable. Data were statistically processed using one way analysis of variance (ANOVA) to assess significant differences among various groups followed by student t-test using GraphPad Prism 6.0, and Origin Pro 8.0 software. 3. Results and discussion 3.1. Synthesis and characterization of xyl-cur conjugate (Prodrug) For the synthesis of xyl-cur conjugate (prodrug), initially cur254
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The synthesis of xyl-cur conjugate (prodrug) was further verified by H NMR spectroscopy. The Fig. 3, depicts the 1H NMR spectrum of the xyl-cur conjugate, the characteristic peaks of xylan between 5.2–3.0 ppm were attributed to the protons of (1–4)-β-D-xylopyranosyl units) (Hansen & Plackett, 2011) and the peaks between 6.1 −7.4 ppm were attributed to the protons of curcumin moiety (Waghela, Sharma, Dhumale, Pandey, & Pathak, 2015). In addition, the peak at 2.4 ppm was attributed to the eCH2 protons of the succinyl group present in the cur-monosuccinate. Appearance of this characteristic peak at 2.4 ppm in the xyl-cur conjugate spectrum, demonstrated the successful synthesis of xyl-cur conjugate (prodrug). The conjugation of curcumin to xylan was further ascertained by UV–vis and fluorescence spectroscopy. The absorption spectra of xylcur conjugate (prodrug) show a broad absorption band at 427 nm similar to the absorption spectrum of curcumin (Fig. 4a), which indicate the presence of curcumin in xyl-cur conjugate (prodrug) (Sarika, James, Kumar, Raj, & Kumary, 2015). The fluorescence emission spectra of xylcur conjugate (Fig. 4b), exhibits a blue shift at 522 nm, compare to curcumin at 550 nm, which attributed to the conjugation between xylan and curcumin (Sarika, James, Nishna, Anil Kumar, & Raj, 2015) 1
Fig. 2. FT-IR spectra of (a) cur-monosuccinate (b) xylan (c) xyl-cur conjugate (prodrug).
3.2. Preparation and characterization of xyl-cur prodrug NPs
monosuccinate was synthesized to introduce the carboxylic acid (eCOOH) functionality, and then conjugated with xylan via carbodiimide chemistry as illustrated in Fig. 1. The FT-IR spectra of curmonosuccinate (Fig. 2a), exhibited an absorption band at 3510 cm−1 attributed to the stretching vibrations of phenolic (eOH) group and the peak at 1732 cm−1 was assigned to the C]O stretching frequencies of succinate group. The peaks at 1605, 1512, and 1272 cm−1 were attributed to the stretching frequencies of C]O (enol) and CeO groups. In the spectrum of xylan (Fig. 2b), band at 3420 cm−1 is attributed to the stretching of −OH group and the sharp band at 1632 cm−1 due to absorbed water, while the peaks between 1465- 1043 cm−1 arise from the stretching and bending vibration of CeO, CeC and CeOH groups. The peak at 890 cm−1 is attributed to the β-glucosidic linkage between the xylose units (Ren, Sun, Liu, Lin, & He, 2007). In addition to the predominant peaks of xylan and cur-monosuccinate, the FT-IR spectrum of xyl-cur conjugate (prodrug) (Fig. 2c), exhibits a new peak at 1735 cm−1 corresponding to the frequency of C]O group which assign the formation of ester bond between xylan and cur-monosuccinate.
The amphiphilic polymers or polymer-drug conjugates could form self-assembled nanoparticles in aqueous medium; it has been already reported by various researchers. Similarly, amphiphilic xyl-cur conjugate (prodrug) formed self-assembled nanoparticles in aqueous medium with the hydrophilic shell of xylan and a hydrophobic core of curcumin. The hydrodynamic size of the xyl-cur prodrug NPs was determined by dynamic light scattering (DLS). The size distribution of the xyl-cur prodrug NPs is shown in Fig. 4c. It can be observed that the prodrug NPs were about 205 nm in size with 108 nm and 343 nm as the low end and high end cutoff, respectively. Furthermore, the morphology of the xyl-cur prodrug NPs was investigated by scanning electron microscopy (SEM). The SEM images of the xyl-cur prodrug NPs (Fig. 4d) show that prodrug NPs were roughly spherical and uniform in size with the small diameter of 160 nm. The variation in size observed between DLS and SEM techniques is due the difference in the procedure condition. The zeta potential of xyl-cur prodrug NPs is also measured by DLS after dispersing the conjugate in aqueous medium and it was observed to
Fig. 3. 1H NMR spectra of xyl-cur conjugate (prodrug).
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Fig. 4. (a) UV–vis and (b) fluorosence spectra of curcumin and xyl-cur conjugate (prodrug) (c) particle size distribution and (d) SEM morphology of xyl-cur conjugate (prodrug) NPs.
show a negative zeta potential of −18.76 mV. The negatively charged surface might be due to the unreacted hydroxyl (eOH) groups of xylan that resides on the outer surface of the nanoparticles after self assembly in an aqueous solution. Thus, the presence of this negative charge on the surface of prodrug NPs may be partly responsible for their stability in aqueous medium due to the interparticle interactions (Liu et al., 2017). The xyl-cur prodrug NPs contained, (24 wt%) drug content as determined by UV-spectroscopy.
condition (pH 7.4) as well as acidic condition (pH 5.0). The release profiles of curcumin (Fig. 5b), shows that about 60–65% of drug was released within 48 h in pH (5.0) while only 40–45% drug was released in pH (7.4). These results indicate that the release rate of curcumin was higher in acidic pH (5.0) than that in physiological pH (7.4). The higher releases rate of curcumin in acidic pH is attributed to the easy breakage of ester linkage. The release study suggested that ester linkage between the xylan and curcumin is more sensitive towards the acidic pH (5.0) compare to basic pH (7.4), which is suitable for the delivery of curcumin to acidic environment of tumor cells (Dey & Sreenivasan, 2015).
3.3. Physiological stability analysis The physiological stability of nanoparticles is an important concern in drug delivery. In order to excess the physiological stability of curcumin and xyl-cur prodrug NPs in PBS (pH 7.4), the change in their absorbance at 427 nm was monitored by UV–vis spectroscopy. As shown in Fig. 5a, xyl-cur prodrug NPs were found to be stable under physiological condition, while the curcumin degrade completely within 6 h. These results indicated that, the stability of curcumin was significantly improved after conjugated with xylan, which could protect curcumin from degradation in alkaline environments (Dey & Sreenivasan, 2014).
3.5. Blood compatibility Hemo-compatibility is an important criterion for safety and biocompatibility of drug-loaded nanoparticles. Therefore, hemolysis study was carried out to evaluate the responses that arise between drugloaded nanoparticle and blood component. Drug-loaded nanoparticles interact with RBCs and damage them. Hemoglobin released from the damage RBCs exhibits a strong absorption peak at 541 nm (Lale, Kumar, Prasad, Bharti, & Koul, 2015). For drug-loaded nanoparticles that do not interact with RBCs, this peak would be negligible. As shown in Fig. 5c, the xyl-cur prodrug NPs exhibited less than 5% hemolytic activity to RBCs even at the highest xyl-cur prodrug NPs concentration of 2 mg/ml, which demonstrate the excellent blood compatibility and suitability for intravenous administration of xyl-cur prodrug NPs.
3.4. Drug release study In order to evaluate the pH responsive behavior of the xyl-cur prodrug NPs, in-vitro drug release study was performed in physiological 256
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Fig. 5. (a) Stability studies of curcumin and xyl-cur prodrug NPs (b) Release profiles of curcumin from the xyl-cur prodrug NPs under different conditions (c) Hemocompatibilty of xyl-cur prodrug NPs.
Fig. 6. Cytotoxicity activity of curcumin, cur-monosuccinate and xyl-cur prodrug NPs againts (a) HCT-15 cells, and (b) HT-29 cells treated with (5–90 μg/ml) curcumin, (5–90 μg/ml) curmonosuccinate, (5–90 μg/ml) xyl-cur prodrug NPs for 48 h.
that xyl-cur prodrug NPs possessed over 2.8 fold lower IC50 value than free curcumin for HT-29 cell line, and over 1.28-fold lower IC50 value than free curcumin for HCT-15 cell line, indicating the significantly enhanced cytotoxicity of xyl-cur prodrug NPs (Table S1, Supplementary data). This might be due to the sustained release of drug molecule from the xyl-cur prodrug NPs, which is in agreement with the in-vitro drug release behavior observations (Fig. 5b). In order to further ascertain the cell viability results, AO/EB staining was carried out to assess the cytotoxicity of drug and drug-polymer conjugates on a qualitative basis. Combination of two dyes AO and EB were used for the study. AO is a cell permeant dye which non-
3.6. Cytotoxicity study The in-vitro cytotoxicity of the free drugs (curcumin and curmonosuccinate) and the xyl-cur prodrug NPs was investigated in HT-29 and HCT-15 colon cancer cells lines by MTT assay. The cells (HT-29 and HCT-15) were treated with different concentrations of xyl-cur prodrug NPs as well as the free drugs for 24 h and 48 h. The cell viability results (Fig. 6a and b) showed that the xyl-cur prodrug NPs and the free drugs exhibited the cytotoxicity in a dosedependent and time-dependent manner and the significant differences in cytotoxicity were observed after 48 h treatment. These results show 257
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Fig. 7. Fluorescence images of live (green) and dead (red) cells (a) HCT-15 cells, and (b) HT-29 cells after treated with IC50 concentration of curcumin, cur-monosuccinate and xyl-cur prodrug NPs for 24 and 48 h. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 8. Cell cyclic analysis of HT-29 cells (a) control (b) curcumin and (c) xyl-cur prodrug NPs after treated with curcumin and xyl-cur prodrug NPs.
3.7. Cell cycle analysis
specifically labels the cell nucleus by intercalating with DNA and also stains cytoplasmic components to certain extent. On the other hand, although EB also intercalates with double stranded DNA, it can do so only in case of cells with compromised membranes (apoptotic cells). As shown in Fig. 7, the controlled cells (labeled with AO alone) appeared to have uniform green nuclei, whereas early apoptotic cells appeared to possess yellow or yellowish green spotted nucleus. In contrast to this, late apoptotic cells with compromised membrane appeared orange in color as their nucleus was stained by both AO and EB. The staining images (Fig. 7a and b) of treated HT-29 and HCT-15 cells clearly indicate that with increase in treatment time span there is consequent increase in apoptotic cells (orange to red cells). Thus, in summary AO-EB staining results corroborated well with the cell viability results and demonstrated the improved therapeutic efficacy of xylcur prodrug NPs as compared to free curcumin and cur-monosuccinate as such.
In order to monitor the effect of xyl-cur prodrug NPs and the free drug (curcumin) on cell proliferation, cell cycle analysis was carried out. It was observed that upon treating cells with xyl-cur prodrug NPs, the cells were predominantly in apoptotic phase (45.1%) as compared to those treated by curcumin (25.9%) and untreated cells (6.14%) as shown in Fig. 8. Also, the G0/G1 cell population also declined to 28.9% and 41.6% upon xyl-cur prodrug NPs and free curcumin treatment, respectively, as compared to 73.7% for untreated cells. This ascertains that although the cells have completed DNA synthesis (S phase) but they could not proceed through subsequent events of karyokinesis due to xyl-cur prodrug NPs mediated destabilization of cell microtubules. The data represents that xyl-cur prodrug NPs efficiently inhibited the growth of the cells and allowed the cells for programmed death.
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4. Conclusion
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In summary, we have successfully developed a pH-sensitive prodrug NPs by directly conjugating the curcumin to the xylan backbone via an acid labile succinate linkage. The prodrug NPs showed a significantly faster drug release at a mildly acidic pH of 5.0, usually present in tumor microenvironments and lysosomes than a physiological pH of 7.4. It is confirmed that the xyl-cur prodrug NPs could efficiently deliver drug to the nucleus of the tumor cells and led to much more cytotoxic effects to HT-29 and HCT-15 human colon cancer cell lines than the parent curcumin. The current study also demonstrated the synergistic advantages of nanocarriers as well as polymer-drug conjugates. This design could be extended to self assembled NP delivery systems for a broad range of anti-cancer drugs by applying the specific linkages between polymer and drug. This novel approach arises the interest of many researchers regarding the design of a new multifunctional drug delivery system. Acknowledgements The authors express their gratitude to Ministry of Human Resource Development (MHRD), New Delhi for financial support to conduct the study. Sincere thanks to Department of Biotechnology, IIT Roorkee for the various biological facilities provided. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.carbpol.2018.02.006. References Ahmed, T. A., & Aljaeid, B. M. (2016). Preparation, characterization, and potential application of chitosan, chitosan derivatives, and chitosan metal nanoparticles in pharmaceutical drug delivery. Drug Design, Development and Therapy, 10, 483–507. http://dx.doi.org/10.2147/DDDT.S99651. Alam, S., Panda, J. J., & Chauhan, V. S. (2012). Novel dipeptide nanoparticles for effective curcumin delivery. International Journal of Nanomedicine, 7, 4207–4222. http://dx.doi.org/10.2147/IJN.S33015. Almeida, E. A. M. S., Bellettini, I. C., Garcia, F. P., Farinácio, M. T., Nakamura, C. V., Rubira, A. F., ... Muniz, E. C. (2017). Curcumin-loaded dual pH- and thermo-responsive magnetic microcarriers based on pectin maleate for drug delivery. Carbohydrate Polymers, 171, 259–266. http://dx.doi.org/10.1016/j.carbpol.2017.05. 034. Anitha, A., Maya, S., Deepa, N., Chennazhi, K. P., Nair, S. V., Tamura, H., & Jayakumar, R. (2011). Efficient water soluble O-carboxymethyl chitosan nanocarrier for the delivery of curcumin to cancer cells. Carbohydrate Polymers, 83(2), 452–461. http://dx.doi. org/10.1016/j.carbpol.2010.08.008. Basu, A., Kunduru, K. R., Abtew, E., & Domb, A. J. (2015). Polysaccharide-based conjugates for biomedical applications. Bioconjugate Chemistry, 26(8), 1396–1412. http://dx.doi.org/10.1021/acs.bioconjchem.5b00242. Cao, Y., Gao, M., Chen, C., Fan, A., Zhang, J., Kong, D., & Zhao, Y. (2015). Triggeredrelease polymeric conjugate micelles for on-demand intracellular drug delivery. Nanotechnology, 26(11), 115101. http://dx.doi.org/10.1088/0957-4484/26/11/ 115101. Chen, Y., Peng, F., Song, X., Wu, J., Yao, W., & Gao, X. (2017). Conjugation of paclitaxel to C-6 hexanediamine-modified hyaluronic acid for targeted drug delivery to enhance antitumor efficacy. Carbohydrate Polymers, 181, 150–158. http://dx.doi.org/10. 1016/j.carbpol.2017.09.017. Dey, S., & Sreenivasan, K. (2014). Conjugation of curcumin onto alginate enhances aqueous solubility and stability of curcumin. Carbohydrate Polymers, 99, 499–507. http://dx.doi.org/10.1016/j.carbpol.2013.08.067. Dey, S., & Sreenivasan, K. (2015). Conjugating curcumin to water soluble polymer stabilized gold nanoparticles via pH responsive succinate linker. Journal of Materials Chemistry B, 3, 824–833. http://dx.doi.org/10.1039/c4tb01731e. Dey, S., Ambattu, L. A., Hari, P. R., Rekha, M. R., & Sreenivasan, K. (2015). Glutathionebearing fluorescent polymer-curcumin conjugate enables simultaneous drug delivery and label-free cellular imaging. Polymer, Brodersen, 25–33. http://dx.doi.org/10. 1016/j.polymer.2015.08.020. Fonseca Silva, T. C., Habibi, Y., Colodette, J. L., & Lucia, L. A. (2011). The influence of the chemical and structural features of xylan on the physical properties of its derived hydrogels. Soft Matter, 7(3), 1090. http://dx.doi.org/10.1039/c0sm00868k.
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