Received Date: July 13, 2017; Accepted Date: August 22, 2017; Published Date: August 28, 2017
Citation: Ahmadi S, Ghasemi-Kasman M, Tabari MG, Kazemi S, Ghasemi S, et al. (2017) Induction of Apoptosis in HeLa Cancer Cells by Ultrasonic-mediated Synthesis of Curcumin-loaded Chitosan-alginate-STPP Nanoparticles. J Nanomed Nanotechnol 8: 455. doi: 10.4172/2157-7439.1000455
Copyright: © 2017 Ahmadi S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Visit for more related articles at Journal of Nanomedicine & Nanotechnology
Natural herbal compounds have been widely introduced as the alternative therapeutic approaches in cancer therapy. Despite potent anti-cancer activity of curcumin, its clinical application has been limited because of low water solubility and resulting poor bioavailability. In this study, we designed a novel ultrasonic assisted method for synthesis of curcumin-loaded chitosan-alginate-sodium tripolyphosphate (CS-ALG-STPP) nanoparticles. Furthermore, the antitumor effects of curcumin-loaded nanoparticles have been evaluated in vitro. Filed emission scanning electron microscopy (FE-SEM) and atomic force microscopy (AFM) were used to characterize the nanoparticles properties. The anti-tumor activity of curcumin-loaded nanoparticles was assessed using MTT and real time PCR. FE-SEM and AFM data revealed the spherical morphology and the average size (<50 nm) of nanoparticles. In vitro cytotoxicity assay suggested that curcumin-loaded CS-ALG-STPP nanoparticles display significant anti-tumor activity compared to free curcumin. Gene expression level analysis showed that nanoparticles significantly increase apoptotic gene expression. Collectively, our results suggest that, curcumin-loaded nanoparticles significantly suppressed proliferation and promoted the induction of apoptosis in cancer cells which might be regarded as an effective alternative strategy for cancer therapy
Cancer; Curcumin; Biodegradable nanoparticles; Antitumor activity; Apoptosis induction
Curcumin is a yellow polyphenol derived from the turmeric rhizome (curcuma longa). A growing number of studies have indicated that curcumin has several beneficial properties such as anti-tumor, anti-oxidant, anti-amyloid, anti-inflammatory, anti-microbial and wound healing effects. Previous studies suggested that curcumin alone or in combination with other anti-cancer drugs possess potent anti-tumor activity on different tumor cell lines including hepatic, prostatic, ovarian, breast, pancreatic and gastric carcinomas [1-14]. Furthermore, it has been reported that curcumin has significant effects on carcinogenesis signaling pathways through angiogenesis inhibition, cell death activation and cell cycle arrest induction. Despite the promising anti-tumor activity of curcumin, its clinical application has been hampered due to rapid systemic elimination and low aqueous solubility that limits the bioavailability of this bioactive compound [13-18]. To overcome these limitations, different approaches have been made to improve curcumin solubility including encapsulation of curcumin in liposome, dendrimers, polymeric nanoparticles, nanogel, cyclodextrin and biodegradable microsphere. Because of hydrophobic nature of curcumin, its encapsulation by natural biocompatible and biodegradable polymers has been introduced as promising strategy for cancer therapy. Alginate and chitosan are two naturally biopolymers that have numerous pharmaceutical and biomedical applications. Alginate (ALG) is a water soluble linear polysaccharide extracted from brown sea weed and alginate nanoparticles can be obtained by adding sodium tripolyphosphate (STPP) and polycationic solution which then result in formation of polyelectrolyte complex. Because of low immunogenicity and nontoxic properties of chitosan (CS), it has been selected as an alternative cationic polymer. There is also an evidence indicating that curcumin can be encapsulated with alginate, chitosan and pluronic composite which could be successfully internalized into the cancer cells. Regarding the necessity of finding an alternative drug delivery system for cancer therapy, the present study was designed to load curcumin with CS, ALG) and STPP nanoparticles by sonication. Afterwards, the effects of curcumin-loaded nanoparticles on proliferation and apoptosis were investigated in cancer cell line.
Curcumin powder was purchased from Merck company, Germany. Chitosan (CS), alginic acid sodium salt (ALG) were obtained from Sigma-Aldrich (St.Louis, Mo). Sodium tripolyphosphate (STPP) was prepared from Daejung, Korea. Culture medium materials and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from ATOCEL (Austria) and Alfa Aesar (USA) respectively.
Preparation of curcumin-loaded CS-ALG-STPP nanoparticles
Curcumin-loaded nanoparticles have been prepared using ultrasonication of CS, ALG and STPP solution. Briefly, 10 mg of chitosan was dissolved in 1% acetic acid to reach a final concentration of 1 mg/mL by magnetic stirring and its pH was adjusted to 4.9. Chitosan solution was stirred by ultrasonic irradiation (Bandelin, Germany) for 1 h. Curcumin powder was then dissolved in a small quantity of ethanol and 1 ml of its solution (1 mg/ml in ethanol) was added in chitosan solution with stirring. Five milligram of sodium alginate were dissolved in distilled water and pH of the suspension was adjusted to 4.6 by HCl 0.5 M. Sodium alginate was added drop wise to this solution and followed by drop wise addition of STPP (0.13% w/v in ultrapure deionized water) with a syringe needle under high-speed stirring for 120 min. The resulting mixture was collected and washed with 10% ethanol and water by centrifugation at 14,000 rpm for 30 min at 4°C.
The surface morphology and size distribution of curcumin-loaded ALG-CS-STPP nanoparticles were characterized by field emission scanning electron microscopy (FE-SEM, Mira 3 XMU, TESCAN, Czech), atomic force microscopy (AFM, Easyscan 2 Flex) and Fouriertransform infrared spectroscopy (FT-IR). The FTIR of curcumin, ALG-CS-TPP and loaded nanoparticles were obtained using FT-IR spectrophotometer (Vector 22 FT-IR, Bruker, Germany).
To determine the amount of curcumin entrapment, highperformance liquid chromatography analysis (HPLC, Knauer, Smartline, Germany) was performed. As we mentioned previously, at final step of nanoparticle preparation, curcumin-CS-ALG-STPP solution was centrifuged and the decanted solution was collected. After washing the mixture with ethanol, supernatant was collected and the amount of remaining curcumin was evaluated. In addition, 1 mg of air-dried nanoparticle was dissolved in 1 mL water and area under the peaks was measured using ezchrome software.
Adsorption isotherms study
Isotherm experiments were performed according to a previous report [19-29]. In brief, different concentrations of curcumin (0.1, 0.15, 0.2, 0.25, 0.3 mg) were mixed with CS-ALG-STPP and the resulting mixture was continuously shaken for 24 h at 25°C. The adsorption capacity of CS-ALG-STPP for curcumin was calculated by the following equation:
C0 is considered as initial concentration of curcumin (mg/ml) and Ce is curcumin concentration in equilibrium state. V and m are total volume of the mixture and total mass of CS-ALG-STPP nanoparticles respectively. qe and Ce values (as dependent variables) were obtained through the experiments. In order to determine the curcumin adsorption onto CS-ALG-STPP nanoparticles and obtaining the maximum uptake (qmax), data were fit to several isotherm equations including Langmuir, Freundich, Temkin, Elovich and Langmuir- Freundlich.
Cell culture assays
To evaluate the cellular uptake of curcumin, human cervical epithelioid carcinoma cell line (HeLa) was purchased from Pasture Institute, Tehran, Iran. Cells were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin at 37°C in CO2 incubator. Cells were treated with 50 μg/ml free curcumin or curcumin-loaded nanoparticles. After 24 h, treated cells were washed with phosphate buffer saline (PBS) and then examined under an inverted fluorescence microscope (Nikon, USA). In order to assess the cytotoxic effects, HeLa cells with density of 7×103 were seeded in 96 well plates and incubated with different concentrations of free and encapsulated curcumin for 48 h. Then, MTT (5 mg/ml) was added to each well and cells were incubated for 4 h at 37°C. For dissolving the blue formazan precipitate, dimethylsulfoxide was added and absorbance was determined at 570 nm by a microplate reader (Biotek, USA). The percentage of cell viability was obtained by the following equation:
All cell culture assessments were performed in triplicate.
Like MTT assay, cells from the HeLa cell line were cultured and incubated with blank nanoparticles, curcumin or curcumin-loaded nanoparticles for 48 h. Then the cells were washed with PBS and fixed with cold 4% PFA for 20 min. After washing with PBS, for enhancing the permeability, 0.2% Triton X-100 was added for 20 min and at final stage, nuclear staining was performed using DAPI (1 μg/ml) (4’, 6-diamidino-2-phenylindole; Santa Cruz, CA) for 25 min and cells were observed under a fluorescence microscope (Nikon, USA).
Quantitative real- time polymerase chain reaction (qRTPCR)
HeLa cells were incubated with blank nanoparticles, curcumin and curcumin-loaded CS-ALG-STPP nanoparticles (50 μg/ml) for 48 h. After that, cells were washed with PBS and collected. RNA was extracted using total RNA extraction kit (Yektatajhiz, Tehran, IRAN) according to the manufacturer’s protocol. cDNA synthesis was performed by (Yektatajhiz, Tehran, IRAN). A total 20 μl of reaction mixture contained 0.4 μl forward primer, 0.4 μl reverse primer, 2 μl cDNA, 10 μl SYBR Green PCR master mix (Yektatajhiz, Tehran, IRAN), 0.4 μl ROX (Yektatajhiz, Tehran, IRAN) and 6.8 μl RNase-free water. GAPDH was used as internal control. Gene expression level was analyzed by Ct method. Table 1 shows the primer sequences and their annealing temperature.
|Langmuir isotherm||qm (mg g-1)||32.467|
|KF(mg1-(1/n) L1/n g-1)||38.448|
|Temkin isotherm||BT(J mol-1)||5.024|
|Elovich isotherm||qm(mg g-1)||0.144|
Table 1: Isotherm constants for adsorption of curcumin onto CS-ALG-TPP nanoparticles.
MTT assay data were analyzed using unpaired student t-test. Analysis of RT-PCR results was performed by one-way ANOVA, followed by Tukey’s post-hoc test. The results are expressed as mean ± SD and p values ≤ 0.05 were considered statistically significant.
Curcumin-loaded CS-ALG-STPP nanoparticles properties
FE-SEM results showed that curcumin-loaded CS-ALG-STPP nanoparticles were spherical, with a mean size ~50 nm which are agglomerated together (Figure 1A). Figure 1B and 1C displays AFM image and its three-dimensional view. Consistent with FE-SEM data, quantification of AFM results also indicated that particles have uniform size distribution and the average size of curcumin-loaded nanoparticles was 50 nm (Figure 1D).
Figure 2 shows the FT-IR spectra of curcumin, CS-ALG and curcumin-loaded CS-ALG-STPP. For curcumin, the peaks at 3478 cm-1 corresponded to amine groups. Furthermore, the peaks at 1512, 1279 and 1029 cm-1 are due to C-O, enol C-O, and C-O-C peaks, respectively. In the spectrum of CS-ALG-TPP nanoparticles, the broad band at 3432 cm–1 corresponds to hydroxyl and amine groups. The peaks near 1628 cm–1 and 1413 cm–1 were attributed to the symmetric and asymmetric stretching vibrations of COO– groups, respectively. Moreover, the bands around 1026 cm–1 belongs to the amid bands. The peak at 1087 cm-1 which is due to the presence of -CH -OH in cyclic alcohol and C-O stretch. After the addition of curcumin to CS-ALGSTPP nanoparticles, the 1626, 1413, and 1087 cm-1 peaks shifted to 1631, 1414 and 1033 cm-1, respectively.
In order to assess the encapsulation efficiency, HPLC analysis was performed on decanted solution, solution after washing with ethanol and curcumin-loaded CS-ALG-STPP nanoparticles mixture in water. By calculating the area under the peaks, the entrapment efficiency for nanoparticles preparation was about 70% (Figure 3).
The adsorption affinity of curcumin and CS-ALG-STPP nanoparticles
Isotherm process was carried out to assess the curcumin loading capacity onto CS-ALG-STPP nanoparticles. Figure 4 depicts the adsorption isotherms for curcumin and CS-ALG-STPP nanoparticles. Experimental results were fit to Langmuir, Freundich, Temkin, Elovich and Langmuir-Freundlich isotherm equations. The adsorption isotherms data for curcumin on CS-ALG-STPP nanoparticles and the equilibrium parameters are presents in Table 1. Regression analysis showed that among the mentioned isotherms, Langmuir-Freundlich isotherm model exhibits high linearity (R2=992).
Figure 4: In vitro assays.A) Bright filed and fluorescence images of free curcumin and curcumin-loaded nanoparticles (50 μg/ml) uptake by HeLa cells. Scale bar: 50 μm B) MTT results showed that curcumin-loaded nanoparticles have suppressed the proliferation of HeLa cells in dose dependent manner compared to free curcumin. *p<0.05 and **p<0.01.
The maximum curcumin uptake (qm) was obtained 243.902 mg of curcumin adsorbed per gram of CS-ALG-STPP nanoparticles. According to this model, intermolecular interactions have important role in adsorption of curcumin onto CS-ALG-STPP nanoparticles.
In vitro assays
Based on the intrinsic fluorescence activity of curcumin, nanoparticle uptake was evaluated in HeLa cells, 48 h after curcuminloaded CS-ALG-STPP nanoparticles incubation. Florescence microscopic images indicated that control cells and those incubated with CS-ALG-STPP as carrier did not show any fluorescence activity. In contrast to control and carrier experimental groups, HeLa cells treated with free curcumin or curcumin nanoparticles (50 μg/ml) robustly exhibited green fluorescence (Figure 5A). In the next step, anti-cancer effect of curcumin nanoparticles was assessed using MTT assay. HeLa cell line was incubated by different doses of free curcumin or curcumin-loaded CS-ALG-STPP nanoparticles (6.25, 12.5, 25, 50 and 100 μg/ml) for 48 h. In contrast to free curcumin, cell viability assay results showed that curcumin-loaded nanoparticles, especially its high dose, has significant inhibitory effect on cell proliferation and this effect is dose dependent (Figure 5B).
The effect of curcumin-loaded nanoparticles on apoptosis -related genes expression
To assess the effect of curcumin-loaded CS-ALG-STPP nanoparticles on apoptosis, HeLa cells were incubated with blank nanoparticles, free curcumin or curcumin-loaded CS-ALG-STPP nanoparticles (50 μg/ml) for 48 h and then nuclear staining was carried out using DAPI and nucleolus changes were observed under fluorescent microscope. Our data showed that untreated and blank nanoparticle receiving cells were stained equably blue fluorescence. In cells treated with curcumin-loaded CS-ALG-STPP nanoparticles, DNA fragmentation, nucleolus pyknosis and bright fluorescence emission, as apoptotic features, were observed. In contrast to curcumin-loaded nanoparticles, curcumin treatment could not induce remarkable changes on nuclear morphology (Figure 6A).
Figure 6: Effect of curcumin-loaded nanoparticles on cell apoptosis.A) Nuclear staining using DAPI showed that nucleolus morphological changes have been occurred in cells under treatment of curcumin-loaded CS-ALG-STPP nanoparticles. Scale bar: 50 μm. B) qRT-PCR data indicated that curcuminloaded CS-ALG-STPP significantly increased the apoptotic gene expression (Bax) and decreased the anti-apoptotic gene expression (Bcl2) compare to carrier and free curcumin experimental groups. *p ≤ 0.05 and ***p ≤ 0.001 nanocurcumin compared to carrier group; ##p ≤ 0.01 and ### p ≤ 0.001 curcumin nanoparticles compared to free curcumin.
In order to determine the effect of curcumin-loaded CS-ALG-STPP nanoparticles on apoptotic gene expression, RT-PCR was performed. RT-PCR data demonstrated that curcumin-loaded CS-ALG-STPP significantly increases the level of Bax gene expression as apoptotic gene marker compared to carrier and free curcumin. Nanoparticles remarkably decreased the Bcl2 gene expression as anti-apoptotic gene marker. In addition, the Bcl2 gene expression level was also significantly decreased in cells incubated with carrier compared to free curcumin group (Figure 6B).
Several evidence has suggested that curcumin exerts potent anti-cancer activity on different cancerous cells . However, because of poor aqueous solubility and low bioavailability, its clinical application has been limited. The development of novel formulation for encapsulation of curcumin attracts strong interest in nanomedicine filed. An ideal nanoparticle should be stable in long term and for circulation in smallest capillary, their size should be lower than 100 nm. Additionally, all the materials for nanoparticle synthesis should be biocompatible. Another important factor in nanoparticle manufacturing is the ability of particles for passing through blood brain barrier .
As aforementioned, nanoparticles size is an important parameter in successful internalization of particles to cancer cells several studies indicated that particle size in the range of 10-100 nm is always ideal for cancer therapy. In our study, FE-SEM and AFM data showed the curcumin-loaded CS-ALG-STPP nanoparticle size is less than 50 nm. A previous study . showed that curcumin-loaded nanoparticles could be synthesized by applying sodium alginate as the cross linker into chitosan solution and the size of prepared particles was 100 nm. Another study showed that the size of curcumin-loaded CS nanoparticles was more than 200 nm . In addition, Niraimathi et al.  used CS and TPP for curcumin loading and particle size was 160 nm. In contrast to the mentioned studies, we could prepare nanoparticles with smaller size. Probably, applying different concentration of CS, ALG, STPP and subsequent ultra-sonication of final solution can result in nanoparticle synthesis with desirable size in cancer therapeutic approach. The encapsulation efficiency for curcumin-loaded CS-ALGSTPP nanoparticles was obtained about 70%. It is also showed the encapsulation efficiency of curcumin-loaded CS-TPP nanoparticles was 75%. However, Das et.al showed that the entrapment efficiency of curcumin-loaded CS-ALG nanoparticles are less than 20%. However, in this study we have achieved higher curcumin loading efficiency which may result from the use of STPP as cross linker [32-38].
Beside the nanoparticle size, the binding affinity between curcumin and CS-ALG-STPP is regarded as a critical factor in nanoparticle manufacturing. Studying the adsorption process using different isotherms model showed that curcumin preferentially adsorbs to CSALG- STPP nanoparticles by intermolecular interaction (Langmuir- Freundlich model). Previous studies have shown that curcumin can be adhered to amine-functionalized mesoporous silica (AAS-MS) nanoparticles using intermolecular interaction of curcumin as well as its interaction with nanoparticles. Due to the hydrophobic structure of curcumin and AAS-MS nanoparticles, hydrophobic attractive forces are regarded as important reason for intermolecular interactions. In next step, the anti-cancer activity of curcumin-loaded CS-ALG-STPP was investigated. MTT assay and qRT-PCR data suggested that in contrast to free curcumin, curcumin-loaded nanoparticles effectively inhibit HeLa cells proliferation and induce apoptosis respectively. Previous reports have suggested that curcumin inhibits cancer cells proliferation and induces cell death, but in our study, free curcumin could not exert significant anti-tumor activity compared to curcuminloaded nanoparticles. This observation may result from very fine dispersion ability of curcumin-loaded CS-ALG-STPP nanoparticles in aqueous solution and the possibility that this property might have been preserved for several months. Unlike to our study, have indicated that curcumin-loaded CS-ALG nanoparticles exhibits very poor solubility in aqueous solution and they have used pluronic F127 composite for enhancing the solubility of nanoparticles. The well-dispersion property and also the small size of nanoparticles may lead to better anti-cancer activity of curcumin-loaded CS-ALG-STPP nanoparticles compared to free curcumin. Similar to the group received curcumin-loaded CSALG- STPP nanoparticles; we have observed that the level of Bcl2 gene expression (as the anti-apoptotic factor) has reduced in cells received blank nanoparticles compared to free curcumin. Previous studies have also demonstrated that chitosan possess anti-cancer activity against several cancer cell lines including oral cancer. It has been shown that the cytotoxic effect of chitosan is mediated through induction of apoptosis in oral cancer cells. Based on these evidences, the significant reduction in Bcl2 gene expression may result from the anti-cancer property of chitosan. However, we did not find any significant difference on Bax gene expression between groups treated by free curcumin and blank nanoparticles. In conclusion, curcumin-loaded nanoparticles were successfully prepared using ultrasonication of biodegradable polymers including chitosan and alginate. Curcumin-loaded CS-ALG-STPP nanoparticles were homogenously dispersed in aqueous solution and ideal size for cancer therapy was obtained. These nanoparticles could be internalized into HeLa cancer cells and incubation of HeLa cells with curcumin-loaded CS-ALG-STPP nanoparticles leads to suppression of proliferation. Additionally, the expression of apoptotic gene was increased in cells treated by curcumin-loaded nanoparticles. These findings may provide a novel therapeutic approach in cancer therapy.
The authors declare no conflict of interest related to this study.
This work was supported by a grant (no: 9503023) from student research committee, Babol University of Medical Sciences, Babol, Mazandaran, Iran.