Artificial Cells, Nanomedicine, and Biotechnology, 2015; Early Online: 1–8 Copyright © 2015 Informa Healthcare USA, Inc. ISSN: 2169-1401 print / 2169-141X online DOI: 10.3109/21691401.2014.982803
ORIGINAL ARTICLE
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PNIPAAm-MAA nanoparticles as delivery vehicles for curcumin against MCF-7 breast cancer cells Vahideh Zeighamian1,2, Masoud Darabi1,3, Abolfazl Akbarzadeh4,5, Mohammad Rahmati-Yamchi1,3, Nosratollah Zarghami1,3, Fariba Badrzadeh1, Roya Salehi4, Fatemeh Sadat Tabatabaei Mirakabad1 & Mortaza Taheri-Anganeh1 1Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz,
Iran, 2Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran, 3Department of Clinical Biochemistry and Laboratory Sciences, Tabriz University of Medical Sciences, Tabriz, Iran, 4Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran, and 5Department of Medical Nanotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran plant Curcuma longa (Basnet and Skalko-Basnet 2011). Curcumin has been used as a therapeutic agent in traditional Indian and Chinese medicine (Mukerjee and Vishwanatha 2009). It exhibits a variety of pharmacological activities that include antimyloid, antioxidant (Ranjbari et al. 2014), and anti-cancer properties, both in vitro and in vivo (Zhou et al. 2011), and can also induce different factors involved in apoptosis in many cancers such as those of the breast and lung (Ranjbari et al. 2014, Zhou et al. 2011). Furthermore, curcumin prevents several cellular pathways, such as pathways that are involved in tumor proliferation and carcinogenesis (Rezaei-Sadabady et al. 2013). Some of the studies have shown that curcumin has inhibitory effects against breast cancer through several signaling pathways (Fallahzadeh et al. 2010). Also, curcumin has not shown cytotoxic effects on normal cells, including mammary epithelial cells, hepatocytes, and fibroblasts, at the dosages required for treatment (Chun et al. 2012). Unfortunately, despite its properties as an effective and safe compound for chemoprevention and cancer therapy (Bisht et al. 2007), curcumin’s therapeutic application is currently limited due to its rapid degradation, poor aqueous solubility (Liu et al. 2013), low bioavailability following oral administration (Ucisik et al. 2013), and poor absorption characteristics (Abas et al. 2006). Different methods have been explored to enhance the water solubility and bioavailability of curcumin, including its conjugates with liposomes, cyclodextrins (CDs), nanoformulations and hydrogels (Dhule et al. 2012), as well as incorporation into phospholipid vesicles (Anand et al. 2010). Recent findings demonstrated that curcumin encapsulated within nanocarriers such as polymer nanoparticles, self-assemblies, and nanoassemblies, can increase cytotoxicity and apoptosis in cancer cells (Yallapu et al. 2012).
Abstract Breast cancer is the most frequently occurring cancer among women throughout the world. Natural compounds such as curcumin hold promise to treat a variety of cancers including breast cancer. However, curcumin’s therapeutic application is limited, due to its rapid degradation and poor aqueous solubility. On the other hand, previous studies have stated that drug delivery using nanoparticles might improve the therapeutic response to anticancer drugs. Poly(N-isopropylacrylamideco-methacrylic acid) (PNIPAAm–MAA) is one of the hydrogel copolymers utilized in the drug delivery system for cancer therapy. The aim of this study was to examine the cytotoxic potential of curcumin encapsulated within the NIPAAm-MAA nanoparticle, on the MCF-7 breast cancer cell line. In this work, polymeric nanoparticles were synthesized through the free radical mechanism, and curcumin was encapsulated into NIPAAm-MAA nanoparticles. Then, the cytotoxic effect of curcumin-loaded NIPAAm-MAA on the MCF-7 breast cancer cell line was measured by MTT assays. The evaluation of the results showed that curcumin-loaded NIPAAm-MAA has more cytotoxic effect on the MCF-7 cell line and efficiently inhibited the growth of the breast cancer cell population, compared with free curcumin. In conclusion, this study indicates that curcuminloaded NIPAAm-MAA suppresses the growth of the MCF-7 cell line. Overall, it is concluded that encapsulating curcumin into the NIPAAm-MAA copolymer could open up new avenues for breast cancer treatment. Keywords: breast cancer, curcumin, drug delivery, PNIPAAm-MAA
Introduction Curcumin is a yellow-colored polyphenol component of turmeric, and is extracted from the rhizome of the
Correspondence: Dr Abolfazl Akbarzadeh, Dr Masoud Darabi, Department of Medical Biotechnology and Medical Nanotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran. Tel/Fax: 984133355789. E-mail: [email protected] (Received 16 October 2014; revised 23 October 2014; accepted 25 October 2014)
1
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2 V. Zeighamian et al. Stimuli-responsive or “smart polymers”, a class of polymer nanoparticles, change their properties in response to environmental stimuli such as temperature, pH, ionic strength, and chemical agents, and have, therefore, considerable efficiency for targeted delivery of cancer therapeutics (Shroff and Vidyasagar 2013). In recent years, hydrogels have been applied in various fields because of their stimulus-responsive behavior (Tian et al. 2008). Poly(Nisopropylacrylamide) (PNIPAAm) is one of the most commonly studied temperature-sensitive polymers (Duan et al. 2012), which shows a low critical solution temperature (LCST) at around 32°C (Jafari et al. 2011). Owing to the formation of self-assembled nanoparticles, consisting of two compartments – an outer hydrophilic corona, and a hydrophobic core, PNIPAAm-based polymers have been considered as one of the promising drug carriers (Mashinchian et al. 2011). As a result, hydrophobic drugs can be loaded inside the inner core and kept safely from leakage from the outer hydrophilic shell (Shroff and Vidyasagar 2013). In the copolymer poly (N-isopropyl acrylamide-co-methacrylic acid) (PNIPAAm–MAA), methacrylic acid (MAA) group possess additional ionized groups, which introduce a pHsensitive phase-transition behavior (Tian et al. 2008). In the present work, we aimed to investigate the anticancer property of nanoformulated curcumin. Hence, we describe the preparation of NIPAAm-MAA nanoparticles for curcumin delivery to cancer cells. In this investigation, the inhibitory effect of curcumin-loaded NIPAAm-MAA nanoparticles on the growth of MCF-7 breast cancer cell lines was analyzed, and its efficacy was compared with that of free curcumin.
Materials and methods Cell culture and cell line The MCF-7 (human breast adenocarcinoma) cell line was obtained from Iran Pasteur Institute. The cells were grown in a RPMI1640 medium (Gibco, Invitrogen, UK) supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen, UK), NaHCO3 (2 mg/ml), 0.08 mg/ml penicillin G, and 0.05 mg/ ml streptomycin (Merck Co, Germany), at 37°C in humidified air containing 5% CO2 and 95% air.
Preparation of chemicals N-isopropylacrylamide (NIPAAm), Methacrylic acid (MAA), Benzoyl proxide (B. P. O), 1,4-dioxan, N, N′-methylene bis acrylamide (MBAm) were purchased from Merck. MTT ((3, 4, 5-dimethylthiazol-2-yl)-2–5-diphenyltetrazolium bromide) for cytotoxicity assay, dimethyl sulfoxide (DMSO) and curcumin (1, 7-bis-(4-hydroxyl-3-methoxyphenyl)-1, 6-hepatadiene-3, 5-Dione), were obtained from Sigma-Aldrich. Trypsin-EDTCA was obtained from Gibco, Invitrogen (UK).
Methods Preparation of the NIPAAm-MAA co-polymer PNIPAAm–MAA particles were synthesized through the free radical mechanism. NIPAAm and MAA were used as the water soluble monomers, and MBAm served as the crosslinking agent. Monomers of NIPAAm and MAA are mixed
together in a molar ratio of 88:12. For preparation of the particles, 8.5 g NIPAAm, 0.3 g MAA and 0.03 g of MBAm were mixed in 20 ml of 1,4- dioxin. The mixture was stirred, and nitrogen gas was passed for 60 min, to remove the dissolved oxygen. After that, the mixture was heated to 70°C in a nitrogen atmosphere. B.P.O (0.17 g) was then added to the solution, to begin the polymerization. The reaction was fulfilled at 70°C for 5 h under N2 gas. In the next step, the copolymer particles were precipitated in ice-cold n-hexane. (Eatemadi et al. 2014a, KarnooshYamchi et al. 2014, Nejati-Koshki et al. 2014, Abbasi et al. 2014b,Ebrahimi et al. 2014, Abbasi et al. 2014c, Ghalhar et al. 2014,Sadat Tabatabaei Mirakabad et al. 2014a, Aval et al. 2014, Zohre et al. 2014, Valizadeh et al. 2014)
Loading of curcumin The process of curcumin loading was carried out using a postpolymerization method. Typically, 100 mg of the freeze-dried NIPAAm-MAA polymeric nanoparticles were dispersed in 10 ml of distilled water and stirred to reform the micelles. 150 ml of curcumin solution in chloroform (CHCl3; 10 mg/ml) was slowly mixed with the polymeric solution and slowly stirred for 15–20 min on low heat, to simultaneously evaporate chloroform and load the curcumin. After that, the curcumin-loaded nanoparticles were lyophilized to dry powder for further use. (Ebrahimnezhad et al. 2013, Pourhassan-Moghaddam et al. 2013, Ahmadi et al. 2014, Davaran et al. 2013, Ghasemali et al. 2013, Sadat Tabatabaei Mirakabad et al. 2014b).
Encapsulation efficiency (EE %) The amount of curcumin entrapped within the NIPAAmMAA nanoparticles was estimated as follows: curcuminloaded nanoparticles were separated from the unentrapped free drug using a NANOSEP (100 kD cut off ) membrane filter, and the amount of unentrapped free curcumin present in the filtrate was quantified by a UV spectrometer (lmax 420 nm) (Shimadzu, Tokyo, Japan). This value was compared with the total amount of curcumin used for loading, to measure the curcumin encapsulation efficiency of the nanoparticles. The percentage of encapsulation (EE %) in the nanoparticles was calculated according to the following equation (Davaran et al. 2014, Kouhi et al. 2014, Abbasi et al. 2014a,Pourhassan-Moghaddam et al. 2014, Eatemadi et al. 2014b,Hosseininasab et al. 2014, Davoudi et al. 2014, Anganeh et al. 2014, Alimirzalu et al. 2014): EE%
([Drug]
tot
[Drug ]free
[Drug ]tot
) 100
Physicochemical characterization Fourier transform infrared The base structure of the nanoparticles and the curcuminloaded NIPAAm-MAA was characterized by fourier transform infrared (FT-IR) spectroscopy (Perkin Elmer series FTIR) using the KBr pellet method.
1H Nuclear Magnetic Resonance study The 1H- Nuclear Magnetic Resonance (NMR) spectrum in real-time (Brucker DRX 300, 400 MHz) was used to analyze
Curcumin against MCF-7 breast cancer cells 3 the chemical structure of NIPAAm- T u f d T. Tetramethylsilane (TMS) was used as an internal reference.
SEM In order to study the morphology and size of the formulated nanoparticles, and the structure of the core shell, scanning electron microscopy (SEM) was performed using the KYKY model EM3200.
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Study of in vitro drug release kinetics To evaluate the kinetics of the in vitro release of the drug from the nanoparticles, experiments were carried out at two temperatures (37°C and 40°C), and at two different pH values (7.4 and 5.8). Briefly, 3 mg of lyophilized curcuminencapsulated nanoparticles were dispersed in 30 ml of phosphate-buffered saline (PBS; pH 7.4) and acetate buffer (pH 5.8). The samples were taken into a shaker incubator maintained at 37°C and 40°C. At designated time points, a 3 mL sample was eliminated from each buffer solution and the volume removed was then replenished with 3 ml of fresh PBS and acetate buffer. The amount of curcumin released into the medium was analyzed using ultraviolet spectrofluorometry, and a standard curve of curcumin concentration was drawn.
In vitro cytotoxicity (MTT assay) After the cells were cultured, the effect of free curcumin and NIPAAm-MAA-encapsulated curcumin on cell growth was examined using 24, 48 and 72-h MTT assays. For this assay, MCF-7 cells were seeded in 96-well culture plates (1 cells/well), and then incubated for 24 h at 37°C in a 5% CO2. Next, the cells were treated with different concentrations (5–70 mM) of free curcumin and curcumin-loaded NIPAAm-MAA for 24, 48 and 72 h, with three replications at every concentration. In order to perform the cytotoxicity assay, stock solutions of curcumin were initially prepared by dissolving the compound in ethanol. Also, cells in the control group were incubated with 200 ml of culture medium containing FBS, without any drugs. The cells were incubated for 24 h; the medium was then exchanged with 200 ml of fresh medium. Following incubation, MTT was dissolved in PBS at a concentration of 2 mg/ml, 50 ml of MTT solution was added, and the cells were incubated for another 4 h. After discarding the content from the wells of the culture plate, 200 ml of DMSO and 25 mL of Sorensen’s glycine buffer were added to wells, and the plates were then shaken. Finally, the absorbance of the formazan product was measured at 570 nm by an ELISA plate reader, with a reference wavelength of 630 nm. ODtot The cell viability is given by: Viability % 100 ODcontrol
Statistical analysis Statistical analysis was performed for each assay by the t-test, using SPSS 16 software and the t-test to measure statistical differences among groups. Statistical significance was considered at the p-values levels of less than 0.05 for all tests.
Results Characterization of the NIPAAm-MAA co-polymer FT-IR spectroscopy can identify the adsorption peaks of the amide and carboxylic acid groups that belong to PNIPAAm-MAA (Figure 1). Formation of the NIPAAmMAA copolymer was indicated by the disappearance of the strong peaks of the vinyl double bonds in the range of 800–1000 cm 1. According to the results of the FT-IR spectrum of the copolymer, the bending frequency of the amide N–H appears at 1520 cm 1, and the absorbance of the amide carbonyl group in NIPAAm emerges at 1660 cm 1. The peak at 1720 cm 1 can be attributed to the carbonyl group of COOH in MAA. The most major bands of MAA are at: 3350 cm 1 [n (OH)], 2950 cm 1 [n (CH3) as, n (CH2) as], and 1080 cm 1 [n (O–C), alcohol), 1040 cm 1 [n (O–C) ester], and 720 [bending (O C–O)]. The FTIR spectra of PNIPAAm-MAA-curcumin indicates the following characteristic signals at: (a) 1520 cm 1, which shows the presence of the C–C group, (b) 1759 cm 1, which is a characteristic peak for C O (enolic), (c) 1250 cm 1, which indicates the C–O stretching, and (d) 3547 cm 1, which displays the presence of the OH group present in the molecule, corresponding to the presence of curcumin encapsulated within the NIPAAm-MAA nanoparticles. Figure 2 shows the 1H NMR spectrum of NIPAAmMAA nanoparticles. The signals pertaining to NIPAAm are found in (ppm) 1.60 (CH2–CH), (ppm) 1.11 (CH3)2CH), (ppm) 3.97(N–CH–CH3)2), (ppm) 2.22 (CH–C O). The signals attributed to methacrylic acid appear at (ppm) 0.9 –C–CH3). A suitable size of nanoparticles is one of the significant properties of hydrogels. The size and morphology of the nanoparticles and the curcumin loaded on poly NIPAAm-MAA are shown in Figure 3. According to the results of the SEM pictures, the size of the NIPAAm-MAA copolymer nanoparticles ranges from 20 nm to 60 nm.
Release kinetics (drug release) and drug-loading efficiency In our studies, the effect of environmental temperatures and pH on the behavior of drug release from the nanoparticles was studied for 220h at 37°C and 40°C, and at various pH levels (7.4 and 5.8). In the conditions mentioned, as depicted in the graph shown in Figure 4, an initial burst release of curcumin was observed within the first two days. The initial burst release from nanoparticles is much faster at higher temperatures and lower pH value. Also, the percentage of release of curcumin at 40°C and a pH of 5.8 is significantly higher than that at 37°C and a pH of 7.4. These results reveal that the curcumin encapsulated in nanoparticles could be released at the tumor cells, because the pH value of the tumor cells is lower than the pH value of the normal tissue; moreover, poly NIPAAm-MAA display reverse thermal and pH-sensitivity properties. In the current work, the drug loading efficiency of the nanoparticles was found to be 89.6%, based on the calculations described previously.
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4 V. Zeighamian et al.
Figure 1. FT-IR diagram of the Structure of (A) NIPAAm-MAA copolymer (B) curcumin-loaded NIPAA-MAA.
Cell viability assay The cytotoxic effects of free curcumin and curcumin-encapsulated nanoparticles were evaluated using the MTT assay at different times. The results of the cytotoxicity assay on the MCF-7 cell line are shown in Figure 5. An analysis of the MTT assay results showed timedependent and dose-dependent decrease in the viability of MCF-7 cells for both free curcumin and curcumin-loaded NIPAAm-MAA nanoparticles. The IC50 values for pure curcumin and curcumin encapsulated in NIPAAm-MAA, on the MCF-7 cell line, are presented in Table I. A comparison of the IC50 values indicated that curcumin-loaded NIPAAm-MAA has more inhibitory effects on cell proliferation than free curcumin.
Discussion Today, the various anticancer drugs available have limited therapeutic potential because of their high toxicity, high cost, and inefficiency in cancer therapy (Shishodia et al. 2007). Therefore, the development of novel agents for the prevention and treatment of breast cancer is highly regarded (Noori and Hassan 2012). Curcumin, as a non-toxic natural product, has various inhibitory effects on the many pathways involved in carcinogenesis and tumor formation (Wilken et al. 2011). In the present study, by using the MCF-7 breast cancer cell line, we sought to investigate whether curcumin-loaded NIPAAm-MAA has inhibitory effects on cell growth. We also
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Curcumin against MCF-7 breast cancer cells 5
Figure 2. 1H NMR spectrum of the NIPAAm-MAA co-polymer.
aimed to examine whether curcumin-loaded NIPAAm-MAA shows more cytotoxicity against cultured cells than free curcumin. Curcumin regulates several targets, and has been applied to increase the sensitization of targeted therapy and to help chemotherapy in treating breast cancer (Jiang et al. 2013). The chemo-preventive properties of curcumin have been studied in multiple preclinical animal models, including breast cancer (Chun et al. 2012). However, the characteristics of low solubility, bioavailability, and stability, as well as rapid degradation when passing through the GI tract, have limited the therapeutic development of curcumin (Bharali et al. 2011). To overcome these major obstacles, various studies have been performed to enhance the clinical efficacy of curcumin through chemical modification or through delivery systems based on nanotechnology (Cridge et al. 2013). Multiple nanopreparations of curcumin, including nanoparticles, solid lipid nanoparticles, micelles, liposomes, nanofibers, and curcumin conjugates, have been demon-
strated to greatly improve curcumin’s therapeutic properties against cancer (Liu et al. 2013). Nano-curcumin, compared to free curcumin, is fully soluble in aqueous media (Bisht et al. 2011). In recent years, nanoparticles have been considered because of their performance in the different fields of biology and medicine (Alimohammadi and Joo 2014). The application of polymeric nanoparticles for drug delivery is being increasingly studied as an approach to overcome various problems associated with the delivery of free drugs (Mukerjee and Vishwanatha 2009). Studies by Bisht et al. indicated that cross-linked and random copolymers of N-isopropylacrylamide (NIPAAm), with N-vinyl-2-pyrrolidone (VP) and poly (ethylene glycol) monoacrylate (PEG-A) nanoparticles can effectively incorporate curcumin (Bharali et al. 2011). Further studies showed that these polymeric nanoparticles provide higher drug release under conditions of acidic pH, as compared to the physiologic pH, indicating their ability to effectively deliver the drug inside the cells (Bansal et al. 2011).
Figure 3. Scanning electron microscopy: (A) NIPAAm-MAA co-polymer (B) Curcumin-loaded NIPAAm-MAA.
6 V. Zeighamian et al. Table I. IC50 values of curcumin and curcumin-loaded NIPAAm-MAA at different times of incubation.
100 90
IC50 values (mM) Mean SD
% release
80 70
Incubation
60
24 h 48 h 72 h
50 40
22.2 1.06 17.1 1.32 7.3 1.15
pH= 7.4, 40°C
20
pH= 5.8, 37°C
10
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32.1 1.11 21.34 1.43 13.7 1.29
Curcumin-loaded NIPAAm-MAA
pH= 7.4, 37°C
30
0
Free Curcumin
pH= 5.8, 40°C 0
50
100
150
200
250
300
time (hours)
Figure 4. Curcumin release from the nanoparticles at different pH levels and temperatures.
In this investigation, we prepared polymeric nanoparticles comprised of NIPAAm and MMA as a thermosensitive polymer, by using the free radical mechanism. The thermosensitive gel NIPAAm is one of the most commonly examined smart systems. On the other hand, the use of methacrylic acid, along with NIPAAm, changes the LCST, in addition to making the hydrogels responsive to both temperature and pH (Roy and Gupta 2003). These nanoparticles are capable of liquidating a wide range of poorly water-soluble drugs (Bisht et al. 2011). Curcumin-loaded NIPAAm-MAA nanoparticles were characterized for their encapsulation efficiency, particle size, surface morphology, and base structure. Our studies showed that NIPAAm-MAA nanoparticles exhibited a drug entrapment efficiency of about 89.6%. According to our work, to determine
the inhibitory effects of free curcumin and curcumin-loaded NIPAAm-MAA on MCF-7, a model for human breast cancer cells, we performed 24, 48 and 72-h MTT assays. The results of this study show that free curcumin and curcumin-loaded NIPAAm-MAA have an anti-proliferative effect on the MCF-7 cell line in a dose-dependent and time-dependent manner. This cytotoxic effect of curcumin on cell growth is consistent with prior reports demonstrating that curcumin affects cell growth in breast cancer cell lines (Kumaravel et al. 2012). However, based on the results of the MTT assay, our study indicates that the curcumin-loaded NIPAAm-MAA has stronger inhibitory effect on MCF-7 cells in comparison with free curcumin. Our results demonstrate that loading curcumin in NIPAAm-MAA nanoparticles improves inhibitory effects of curcumin. In addition, in vitro release studies show that NIPAAm-MAA nanoparticles can efficiently release the drug under conditions of acidic pH and high temperature.
Conclusion In summary, we investigated the anticancer effect of free curcumin and curcumin-loaded NIPAAm-MAA and showed
Figure 5. (A) 24-h MTT assay results. The cytotoxic effects of different concentrations of free curcumin and curcumin-loaded NIPAAm-MAA on the MCF-7 cell line. (B) 48-h MTT assay results. (C) 72-h MTT assay results. (D) The cytotoxic effects of curcumin-loaded NIPAAm-MAA nanoparticles on the MCF-7 cell line at different times. Analysis of the MTT assay results showed a time-dependent decrease in the viability of MCF-7 for curcumin-loaded NIPAAm-MAA nanoparticles.
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Curcumin against MCF-7 breast cancer cells 7 that curcumin encapsulated within nanoparticles has a greater effect on growth in the MCF-7 breast cancer cell line. Cell viability assays demonstrated the significant impact of the curcumin and curcumin-loaded nanoparticles on breast cancer cells. Furthermore, it was found that curcumin-loaded NIPAAm-MAA has lower IC50 values than free curcumin. Overall, it is concluded that NIPAAm-MAA nanoparticles are capable of delivering curcumin over a long-term, thus making them suitable therapeutic agents for cancer therapy. The results of this research suggest that curcumin-loaded NIPAAm-MAA can be used in treating breast cancer. Although our data indicate the inhibitory effect of curcuminloaded NIPAAm-MAA on the proliferation of breast cancer cells, more studies are required to elucidate the underlying mechanisms.
Authors’ contributions AA conceived of the study and participated in its design and coordination. VZ, MD, NZ, and MRY participated in the sequence alignment and drafted the manuscript. All authors read and approved the final manuscript.
Acknowledgements This study is based on an MSc thesis and was supported by the Faculty of Advanced Medical Sciences of Tabriz University of Medical Sciences, project number 92/2-4/2. We hereby thank the Student Research Committee at Tabriz University of Medical Sciences, Tabriz, Iran.
Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.
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ORIGINAL ARTICLE
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PNIPAAm-MAA nanoparticles as delivery vehicles for curcumin against MCF-7 breast cancer cells Vahideh Zeighamian1,2, Masoud Darabi1,3, Abolfazl Akbarzadeh4,5, Mohammad Rahmati-Yamchi1,3, Nosratollah Zarghami1,3, Fariba Badrzadeh1, Roya Salehi4, Fatemeh Sadat Tabatabaei Mirakabad1 & Mortaza Taheri-Anganeh1 1Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz,
Iran, 2Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran, 3Department of Clinical Biochemistry and Laboratory Sciences, Tabriz University of Medical Sciences, Tabriz, Iran, 4Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran, and 5Department of Medical Nanotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran plant Curcuma longa (Basnet and Skalko-Basnet 2011). Curcumin has been used as a therapeutic agent in traditional Indian and Chinese medicine (Mukerjee and Vishwanatha 2009). It exhibits a variety of pharmacological activities that include antimyloid, antioxidant (Ranjbari et al. 2014), and anti-cancer properties, both in vitro and in vivo (Zhou et al. 2011), and can also induce different factors involved in apoptosis in many cancers such as those of the breast and lung (Ranjbari et al. 2014, Zhou et al. 2011). Furthermore, curcumin prevents several cellular pathways, such as pathways that are involved in tumor proliferation and carcinogenesis (Rezaei-Sadabady et al. 2013). Some of the studies have shown that curcumin has inhibitory effects against breast cancer through several signaling pathways (Fallahzadeh et al. 2010). Also, curcumin has not shown cytotoxic effects on normal cells, including mammary epithelial cells, hepatocytes, and fibroblasts, at the dosages required for treatment (Chun et al. 2012). Unfortunately, despite its properties as an effective and safe compound for chemoprevention and cancer therapy (Bisht et al. 2007), curcumin’s therapeutic application is currently limited due to its rapid degradation, poor aqueous solubility (Liu et al. 2013), low bioavailability following oral administration (Ucisik et al. 2013), and poor absorption characteristics (Abas et al. 2006). Different methods have been explored to enhance the water solubility and bioavailability of curcumin, including its conjugates with liposomes, cyclodextrins (CDs), nanoformulations and hydrogels (Dhule et al. 2012), as well as incorporation into phospholipid vesicles (Anand et al. 2010). Recent findings demonstrated that curcumin encapsulated within nanocarriers such as polymer nanoparticles, self-assemblies, and nanoassemblies, can increase cytotoxicity and apoptosis in cancer cells (Yallapu et al. 2012).
Abstract Breast cancer is the most frequently occurring cancer among women throughout the world. Natural compounds such as curcumin hold promise to treat a variety of cancers including breast cancer. However, curcumin’s therapeutic application is limited, due to its rapid degradation and poor aqueous solubility. On the other hand, previous studies have stated that drug delivery using nanoparticles might improve the therapeutic response to anticancer drugs. Poly(N-isopropylacrylamideco-methacrylic acid) (PNIPAAm–MAA) is one of the hydrogel copolymers utilized in the drug delivery system for cancer therapy. The aim of this study was to examine the cytotoxic potential of curcumin encapsulated within the NIPAAm-MAA nanoparticle, on the MCF-7 breast cancer cell line. In this work, polymeric nanoparticles were synthesized through the free radical mechanism, and curcumin was encapsulated into NIPAAm-MAA nanoparticles. Then, the cytotoxic effect of curcumin-loaded NIPAAm-MAA on the MCF-7 breast cancer cell line was measured by MTT assays. The evaluation of the results showed that curcumin-loaded NIPAAm-MAA has more cytotoxic effect on the MCF-7 cell line and efficiently inhibited the growth of the breast cancer cell population, compared with free curcumin. In conclusion, this study indicates that curcuminloaded NIPAAm-MAA suppresses the growth of the MCF-7 cell line. Overall, it is concluded that encapsulating curcumin into the NIPAAm-MAA copolymer could open up new avenues for breast cancer treatment. Keywords: breast cancer, curcumin, drug delivery, PNIPAAm-MAA
Introduction Curcumin is a yellow-colored polyphenol component of turmeric, and is extracted from the rhizome of the
Correspondence: Dr Abolfazl Akbarzadeh, Dr Masoud Darabi, Department of Medical Biotechnology and Medical Nanotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran. Tel/Fax: 984133355789. E-mail: [email protected] (Received 16 October 2014; revised 23 October 2014; accepted 25 October 2014)
1
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2 V. Zeighamian et al. Stimuli-responsive or “smart polymers”, a class of polymer nanoparticles, change their properties in response to environmental stimuli such as temperature, pH, ionic strength, and chemical agents, and have, therefore, considerable efficiency for targeted delivery of cancer therapeutics (Shroff and Vidyasagar 2013). In recent years, hydrogels have been applied in various fields because of their stimulus-responsive behavior (Tian et al. 2008). Poly(Nisopropylacrylamide) (PNIPAAm) is one of the most commonly studied temperature-sensitive polymers (Duan et al. 2012), which shows a low critical solution temperature (LCST) at around 32°C (Jafari et al. 2011). Owing to the formation of self-assembled nanoparticles, consisting of two compartments – an outer hydrophilic corona, and a hydrophobic core, PNIPAAm-based polymers have been considered as one of the promising drug carriers (Mashinchian et al. 2011). As a result, hydrophobic drugs can be loaded inside the inner core and kept safely from leakage from the outer hydrophilic shell (Shroff and Vidyasagar 2013). In the copolymer poly (N-isopropyl acrylamide-co-methacrylic acid) (PNIPAAm–MAA), methacrylic acid (MAA) group possess additional ionized groups, which introduce a pHsensitive phase-transition behavior (Tian et al. 2008). In the present work, we aimed to investigate the anticancer property of nanoformulated curcumin. Hence, we describe the preparation of NIPAAm-MAA nanoparticles for curcumin delivery to cancer cells. In this investigation, the inhibitory effect of curcumin-loaded NIPAAm-MAA nanoparticles on the growth of MCF-7 breast cancer cell lines was analyzed, and its efficacy was compared with that of free curcumin.
Materials and methods Cell culture and cell line The MCF-7 (human breast adenocarcinoma) cell line was obtained from Iran Pasteur Institute. The cells were grown in a RPMI1640 medium (Gibco, Invitrogen, UK) supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen, UK), NaHCO3 (2 mg/ml), 0.08 mg/ml penicillin G, and 0.05 mg/ ml streptomycin (Merck Co, Germany), at 37°C in humidified air containing 5% CO2 and 95% air.
Preparation of chemicals N-isopropylacrylamide (NIPAAm), Methacrylic acid (MAA), Benzoyl proxide (B. P. O), 1,4-dioxan, N, N′-methylene bis acrylamide (MBAm) were purchased from Merck. MTT ((3, 4, 5-dimethylthiazol-2-yl)-2–5-diphenyltetrazolium bromide) for cytotoxicity assay, dimethyl sulfoxide (DMSO) and curcumin (1, 7-bis-(4-hydroxyl-3-methoxyphenyl)-1, 6-hepatadiene-3, 5-Dione), were obtained from Sigma-Aldrich. Trypsin-EDTCA was obtained from Gibco, Invitrogen (UK).
Methods Preparation of the NIPAAm-MAA co-polymer PNIPAAm–MAA particles were synthesized through the free radical mechanism. NIPAAm and MAA were used as the water soluble monomers, and MBAm served as the crosslinking agent. Monomers of NIPAAm and MAA are mixed
together in a molar ratio of 88:12. For preparation of the particles, 8.5 g NIPAAm, 0.3 g MAA and 0.03 g of MBAm were mixed in 20 ml of 1,4- dioxin. The mixture was stirred, and nitrogen gas was passed for 60 min, to remove the dissolved oxygen. After that, the mixture was heated to 70°C in a nitrogen atmosphere. B.P.O (0.17 g) was then added to the solution, to begin the polymerization. The reaction was fulfilled at 70°C for 5 h under N2 gas. In the next step, the copolymer particles were precipitated in ice-cold n-hexane. (Eatemadi et al. 2014a, KarnooshYamchi et al. 2014, Nejati-Koshki et al. 2014, Abbasi et al. 2014b,Ebrahimi et al. 2014, Abbasi et al. 2014c, Ghalhar et al. 2014,Sadat Tabatabaei Mirakabad et al. 2014a, Aval et al. 2014, Zohre et al. 2014, Valizadeh et al. 2014)
Loading of curcumin The process of curcumin loading was carried out using a postpolymerization method. Typically, 100 mg of the freeze-dried NIPAAm-MAA polymeric nanoparticles were dispersed in 10 ml of distilled water and stirred to reform the micelles. 150 ml of curcumin solution in chloroform (CHCl3; 10 mg/ml) was slowly mixed with the polymeric solution and slowly stirred for 15–20 min on low heat, to simultaneously evaporate chloroform and load the curcumin. After that, the curcumin-loaded nanoparticles were lyophilized to dry powder for further use. (Ebrahimnezhad et al. 2013, Pourhassan-Moghaddam et al. 2013, Ahmadi et al. 2014, Davaran et al. 2013, Ghasemali et al. 2013, Sadat Tabatabaei Mirakabad et al. 2014b).
Encapsulation efficiency (EE %) The amount of curcumin entrapped within the NIPAAmMAA nanoparticles was estimated as follows: curcuminloaded nanoparticles were separated from the unentrapped free drug using a NANOSEP (100 kD cut off ) membrane filter, and the amount of unentrapped free curcumin present in the filtrate was quantified by a UV spectrometer (lmax 420 nm) (Shimadzu, Tokyo, Japan). This value was compared with the total amount of curcumin used for loading, to measure the curcumin encapsulation efficiency of the nanoparticles. The percentage of encapsulation (EE %) in the nanoparticles was calculated according to the following equation (Davaran et al. 2014, Kouhi et al. 2014, Abbasi et al. 2014a,Pourhassan-Moghaddam et al. 2014, Eatemadi et al. 2014b,Hosseininasab et al. 2014, Davoudi et al. 2014, Anganeh et al. 2014, Alimirzalu et al. 2014): EE%
([Drug]
tot
[Drug ]free
[Drug ]tot
) 100
Physicochemical characterization Fourier transform infrared The base structure of the nanoparticles and the curcuminloaded NIPAAm-MAA was characterized by fourier transform infrared (FT-IR) spectroscopy (Perkin Elmer series FTIR) using the KBr pellet method.
1H Nuclear Magnetic Resonance study The 1H- Nuclear Magnetic Resonance (NMR) spectrum in real-time (Brucker DRX 300, 400 MHz) was used to analyze
Curcumin against MCF-7 breast cancer cells 3 the chemical structure of NIPAAm- T u f d T. Tetramethylsilane (TMS) was used as an internal reference.
SEM In order to study the morphology and size of the formulated nanoparticles, and the structure of the core shell, scanning electron microscopy (SEM) was performed using the KYKY model EM3200.
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Study of in vitro drug release kinetics To evaluate the kinetics of the in vitro release of the drug from the nanoparticles, experiments were carried out at two temperatures (37°C and 40°C), and at two different pH values (7.4 and 5.8). Briefly, 3 mg of lyophilized curcuminencapsulated nanoparticles were dispersed in 30 ml of phosphate-buffered saline (PBS; pH 7.4) and acetate buffer (pH 5.8). The samples were taken into a shaker incubator maintained at 37°C and 40°C. At designated time points, a 3 mL sample was eliminated from each buffer solution and the volume removed was then replenished with 3 ml of fresh PBS and acetate buffer. The amount of curcumin released into the medium was analyzed using ultraviolet spectrofluorometry, and a standard curve of curcumin concentration was drawn.
In vitro cytotoxicity (MTT assay) After the cells were cultured, the effect of free curcumin and NIPAAm-MAA-encapsulated curcumin on cell growth was examined using 24, 48 and 72-h MTT assays. For this assay, MCF-7 cells were seeded in 96-well culture plates (1 cells/well), and then incubated for 24 h at 37°C in a 5% CO2. Next, the cells were treated with different concentrations (5–70 mM) of free curcumin and curcumin-loaded NIPAAm-MAA for 24, 48 and 72 h, with three replications at every concentration. In order to perform the cytotoxicity assay, stock solutions of curcumin were initially prepared by dissolving the compound in ethanol. Also, cells in the control group were incubated with 200 ml of culture medium containing FBS, without any drugs. The cells were incubated for 24 h; the medium was then exchanged with 200 ml of fresh medium. Following incubation, MTT was dissolved in PBS at a concentration of 2 mg/ml, 50 ml of MTT solution was added, and the cells were incubated for another 4 h. After discarding the content from the wells of the culture plate, 200 ml of DMSO and 25 mL of Sorensen’s glycine buffer were added to wells, and the plates were then shaken. Finally, the absorbance of the formazan product was measured at 570 nm by an ELISA plate reader, with a reference wavelength of 630 nm. ODtot The cell viability is given by: Viability % 100 ODcontrol
Statistical analysis Statistical analysis was performed for each assay by the t-test, using SPSS 16 software and the t-test to measure statistical differences among groups. Statistical significance was considered at the p-values levels of less than 0.05 for all tests.
Results Characterization of the NIPAAm-MAA co-polymer FT-IR spectroscopy can identify the adsorption peaks of the amide and carboxylic acid groups that belong to PNIPAAm-MAA (Figure 1). Formation of the NIPAAmMAA copolymer was indicated by the disappearance of the strong peaks of the vinyl double bonds in the range of 800–1000 cm 1. According to the results of the FT-IR spectrum of the copolymer, the bending frequency of the amide N–H appears at 1520 cm 1, and the absorbance of the amide carbonyl group in NIPAAm emerges at 1660 cm 1. The peak at 1720 cm 1 can be attributed to the carbonyl group of COOH in MAA. The most major bands of MAA are at: 3350 cm 1 [n (OH)], 2950 cm 1 [n (CH3) as, n (CH2) as], and 1080 cm 1 [n (O–C), alcohol), 1040 cm 1 [n (O–C) ester], and 720 [bending (O C–O)]. The FTIR spectra of PNIPAAm-MAA-curcumin indicates the following characteristic signals at: (a) 1520 cm 1, which shows the presence of the C–C group, (b) 1759 cm 1, which is a characteristic peak for C O (enolic), (c) 1250 cm 1, which indicates the C–O stretching, and (d) 3547 cm 1, which displays the presence of the OH group present in the molecule, corresponding to the presence of curcumin encapsulated within the NIPAAm-MAA nanoparticles. Figure 2 shows the 1H NMR spectrum of NIPAAmMAA nanoparticles. The signals pertaining to NIPAAm are found in (ppm) 1.60 (CH2–CH), (ppm) 1.11 (CH3)2CH), (ppm) 3.97(N–CH–CH3)2), (ppm) 2.22 (CH–C O). The signals attributed to methacrylic acid appear at (ppm) 0.9 –C–CH3). A suitable size of nanoparticles is one of the significant properties of hydrogels. The size and morphology of the nanoparticles and the curcumin loaded on poly NIPAAm-MAA are shown in Figure 3. According to the results of the SEM pictures, the size of the NIPAAm-MAA copolymer nanoparticles ranges from 20 nm to 60 nm.
Release kinetics (drug release) and drug-loading efficiency In our studies, the effect of environmental temperatures and pH on the behavior of drug release from the nanoparticles was studied for 220h at 37°C and 40°C, and at various pH levels (7.4 and 5.8). In the conditions mentioned, as depicted in the graph shown in Figure 4, an initial burst release of curcumin was observed within the first two days. The initial burst release from nanoparticles is much faster at higher temperatures and lower pH value. Also, the percentage of release of curcumin at 40°C and a pH of 5.8 is significantly higher than that at 37°C and a pH of 7.4. These results reveal that the curcumin encapsulated in nanoparticles could be released at the tumor cells, because the pH value of the tumor cells is lower than the pH value of the normal tissue; moreover, poly NIPAAm-MAA display reverse thermal and pH-sensitivity properties. In the current work, the drug loading efficiency of the nanoparticles was found to be 89.6%, based on the calculations described previously.
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4 V. Zeighamian et al.
Figure 1. FT-IR diagram of the Structure of (A) NIPAAm-MAA copolymer (B) curcumin-loaded NIPAA-MAA.
Cell viability assay The cytotoxic effects of free curcumin and curcumin-encapsulated nanoparticles were evaluated using the MTT assay at different times. The results of the cytotoxicity assay on the MCF-7 cell line are shown in Figure 5. An analysis of the MTT assay results showed timedependent and dose-dependent decrease in the viability of MCF-7 cells for both free curcumin and curcumin-loaded NIPAAm-MAA nanoparticles. The IC50 values for pure curcumin and curcumin encapsulated in NIPAAm-MAA, on the MCF-7 cell line, are presented in Table I. A comparison of the IC50 values indicated that curcumin-loaded NIPAAm-MAA has more inhibitory effects on cell proliferation than free curcumin.
Discussion Today, the various anticancer drugs available have limited therapeutic potential because of their high toxicity, high cost, and inefficiency in cancer therapy (Shishodia et al. 2007). Therefore, the development of novel agents for the prevention and treatment of breast cancer is highly regarded (Noori and Hassan 2012). Curcumin, as a non-toxic natural product, has various inhibitory effects on the many pathways involved in carcinogenesis and tumor formation (Wilken et al. 2011). In the present study, by using the MCF-7 breast cancer cell line, we sought to investigate whether curcumin-loaded NIPAAm-MAA has inhibitory effects on cell growth. We also
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Curcumin against MCF-7 breast cancer cells 5
Figure 2. 1H NMR spectrum of the NIPAAm-MAA co-polymer.
aimed to examine whether curcumin-loaded NIPAAm-MAA shows more cytotoxicity against cultured cells than free curcumin. Curcumin regulates several targets, and has been applied to increase the sensitization of targeted therapy and to help chemotherapy in treating breast cancer (Jiang et al. 2013). The chemo-preventive properties of curcumin have been studied in multiple preclinical animal models, including breast cancer (Chun et al. 2012). However, the characteristics of low solubility, bioavailability, and stability, as well as rapid degradation when passing through the GI tract, have limited the therapeutic development of curcumin (Bharali et al. 2011). To overcome these major obstacles, various studies have been performed to enhance the clinical efficacy of curcumin through chemical modification or through delivery systems based on nanotechnology (Cridge et al. 2013). Multiple nanopreparations of curcumin, including nanoparticles, solid lipid nanoparticles, micelles, liposomes, nanofibers, and curcumin conjugates, have been demon-
strated to greatly improve curcumin’s therapeutic properties against cancer (Liu et al. 2013). Nano-curcumin, compared to free curcumin, is fully soluble in aqueous media (Bisht et al. 2011). In recent years, nanoparticles have been considered because of their performance in the different fields of biology and medicine (Alimohammadi and Joo 2014). The application of polymeric nanoparticles for drug delivery is being increasingly studied as an approach to overcome various problems associated with the delivery of free drugs (Mukerjee and Vishwanatha 2009). Studies by Bisht et al. indicated that cross-linked and random copolymers of N-isopropylacrylamide (NIPAAm), with N-vinyl-2-pyrrolidone (VP) and poly (ethylene glycol) monoacrylate (PEG-A) nanoparticles can effectively incorporate curcumin (Bharali et al. 2011). Further studies showed that these polymeric nanoparticles provide higher drug release under conditions of acidic pH, as compared to the physiologic pH, indicating their ability to effectively deliver the drug inside the cells (Bansal et al. 2011).
Figure 3. Scanning electron microscopy: (A) NIPAAm-MAA co-polymer (B) Curcumin-loaded NIPAAm-MAA.
6 V. Zeighamian et al. Table I. IC50 values of curcumin and curcumin-loaded NIPAAm-MAA at different times of incubation.
100 90
IC50 values (mM) Mean SD
% release
80 70
Incubation
60
24 h 48 h 72 h
50 40
22.2 1.06 17.1 1.32 7.3 1.15
pH= 7.4, 40°C
20
pH= 5.8, 37°C
10
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32.1 1.11 21.34 1.43 13.7 1.29
Curcumin-loaded NIPAAm-MAA
pH= 7.4, 37°C
30
0
Free Curcumin
pH= 5.8, 40°C 0
50
100
150
200
250
300
time (hours)
Figure 4. Curcumin release from the nanoparticles at different pH levels and temperatures.
In this investigation, we prepared polymeric nanoparticles comprised of NIPAAm and MMA as a thermosensitive polymer, by using the free radical mechanism. The thermosensitive gel NIPAAm is one of the most commonly examined smart systems. On the other hand, the use of methacrylic acid, along with NIPAAm, changes the LCST, in addition to making the hydrogels responsive to both temperature and pH (Roy and Gupta 2003). These nanoparticles are capable of liquidating a wide range of poorly water-soluble drugs (Bisht et al. 2011). Curcumin-loaded NIPAAm-MAA nanoparticles were characterized for their encapsulation efficiency, particle size, surface morphology, and base structure. Our studies showed that NIPAAm-MAA nanoparticles exhibited a drug entrapment efficiency of about 89.6%. According to our work, to determine
the inhibitory effects of free curcumin and curcumin-loaded NIPAAm-MAA on MCF-7, a model for human breast cancer cells, we performed 24, 48 and 72-h MTT assays. The results of this study show that free curcumin and curcumin-loaded NIPAAm-MAA have an anti-proliferative effect on the MCF-7 cell line in a dose-dependent and time-dependent manner. This cytotoxic effect of curcumin on cell growth is consistent with prior reports demonstrating that curcumin affects cell growth in breast cancer cell lines (Kumaravel et al. 2012). However, based on the results of the MTT assay, our study indicates that the curcumin-loaded NIPAAm-MAA has stronger inhibitory effect on MCF-7 cells in comparison with free curcumin. Our results demonstrate that loading curcumin in NIPAAm-MAA nanoparticles improves inhibitory effects of curcumin. In addition, in vitro release studies show that NIPAAm-MAA nanoparticles can efficiently release the drug under conditions of acidic pH and high temperature.
Conclusion In summary, we investigated the anticancer effect of free curcumin and curcumin-loaded NIPAAm-MAA and showed
Figure 5. (A) 24-h MTT assay results. The cytotoxic effects of different concentrations of free curcumin and curcumin-loaded NIPAAm-MAA on the MCF-7 cell line. (B) 48-h MTT assay results. (C) 72-h MTT assay results. (D) The cytotoxic effects of curcumin-loaded NIPAAm-MAA nanoparticles on the MCF-7 cell line at different times. Analysis of the MTT assay results showed a time-dependent decrease in the viability of MCF-7 for curcumin-loaded NIPAAm-MAA nanoparticles.
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Curcumin against MCF-7 breast cancer cells 7 that curcumin encapsulated within nanoparticles has a greater effect on growth in the MCF-7 breast cancer cell line. Cell viability assays demonstrated the significant impact of the curcumin and curcumin-loaded nanoparticles on breast cancer cells. Furthermore, it was found that curcumin-loaded NIPAAm-MAA has lower IC50 values than free curcumin. Overall, it is concluded that NIPAAm-MAA nanoparticles are capable of delivering curcumin over a long-term, thus making them suitable therapeutic agents for cancer therapy. The results of this research suggest that curcumin-loaded NIPAAm-MAA can be used in treating breast cancer. Although our data indicate the inhibitory effect of curcuminloaded NIPAAm-MAA on the proliferation of breast cancer cells, more studies are required to elucidate the underlying mechanisms.
Authors’ contributions AA conceived of the study and participated in its design and coordination. VZ, MD, NZ, and MRY participated in the sequence alignment and drafted the manuscript. All authors read and approved the final manuscript.
Acknowledgements This study is based on an MSc thesis and was supported by the Faculty of Advanced Medical Sciences of Tabriz University of Medical Sciences, project number 92/2-4/2. We hereby thank the Student Research Committee at Tabriz University of Medical Sciences, Tabriz, Iran.
Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.
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