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Preparation, characterization and drug delivery study of a novel nanobiopolymeric multidrug delivery system
Materials Science and Engineering C 73 (2017) 516–524
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Preparation, characterization and drug delivery study of a novel nanobiopolymeric multidrug delivery system Abbas Dadkhah Tehrani ⁎, Masoumeh Parsamanesh Department of Chemistry, Faculty of Science, Lorestan University, Khoramabad, Iran
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Article history: Received 19 October 2016 Received in revised form 8 December 2016 Accepted 20 December 2016 Available online 24 December 2016 Keywords: Biopolymer Anticancer Nanocarrier Drug delivery
a b s t r a c t New nanocarrier for codelivery of curcumin and doxorubicin as the anticancer drugs was synthesized using biocompatible and biodegradable materials. Firstly, an inclusion complex of amylose (Am) and curcumin (CUR) was formed through entrapment of curcumin into the amylose helices. Then the surface of amylose-curcumin (Am-CUR) complex was modified by polycaprolactone (PCL) via esterification reaction between hydroxyl functional groups of amylose and carbonyl groups of PCL. Finally, poly citric acid (PCA) reacted with terminal hydroxyl groups of PCL by esterification reaction. Then, doxorubicin (DOX) reacted with the surface carboxylic acid functional groups of Am-CUR-PCL-PCA through noncovalent interactions to form Am-CUR-PCL-PCA-DOX as a multidrug delivery system. These new synthesized nanomaterials were characterized by spectroscopic measurement methods such as IR spectroscopy, UV–vis spectroscopy, NMR spectroscopy, and scanning electron microscopy. FE-SEM analyses and DLS measurements showed that the hydrodynamic dimensions of Am-CurPCL-PCA were about 50 nm. Due to the presence of ester bonds, the synthesized nanomaterials are pH sensitive. Furthermore, the resulting copolymer was completely water soluble because of the hydrophilic nature of poly citric acid part of copolymer and therefore successfully can be utilized in biomedical applications. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Nowadays, drug delivery systems are playing a significant role in the fields of medical and pharmaceutical sciences. A sustained and controlled drug release is critically important for the effective treatment of many sicknesses, ranging from arthritis to cancer which is one of the major causes of mortality in the world [1,2]. Traditional chemotherapy is an essential approach for cancer therapy. However, its therapeutic efficacy is usually limited by a number of major challenges. For example, the high toxicity of denuded drugs or the drug leakage from traditional drug delivery systems before reaching the tumor site poses a serious challenge to effective treatment. Another challenge is the delivery of water-insoluble drugs. Lots of the hydrophobic drugs showing high potency remain in the pipeline since they suffer from the lack of means for administration in the body. Furthermore, cancer chemotherapeutic agents can unselectively enter into both normal tissues and tumor tissues, which result in distasteful side effects and even death of the patients. The occurrence of multidrug resistance (MDR) phenotypes is one of the other important reasons which lead to chemotherapeutic failure. Therefore, noticeable attention has particularly been focused on anticancer drug delivery systems capable of overcoming the current problems associated with the conventional chemotherapy. Especially, ⁎ Corresponding author. E-mail address: [email protected] (A. Dadkhah Tehrani).
http://dx.doi.org/10.1016/j.msec.2016.12.103 0928-4931/© 2016 Elsevier B.V. All rights reserved.
advanced nanotechnology and nanoscience have been extensively used in the fabrication of controllable drug delivery systems and providing an innovative and promising procedure for circumventing MDR by encapsulating or conjugating chemotherapeutic drugs to nanocarriers [2–4]. A number of drug delivery carriers designed to address these problems including micelles of amphiphilic block copolymers, liposomes, hydrogels, nanoemulsions, nanosuspensions, polymeric nanoparticles, etc. [5–7]. The incorporation of chemotherapeutic agents into polymeric nanoparticles have attracted significant attention in the study of drug delivery systems and has many advantages, consisting of low toxicity, enhanced bioavailability, increased circulation times, high stability in biological systems and break down to biocompatible materials in destination organelles etc. [4,6,8–11]. Selection of polymer is an important step to design a nanocarrier for the successful drug delivery purpose. For this purpose, natural and environmentally friendly polymers involving starch, chitosan, sodium alginate, guar gum, carboxy methyl cellulose and their derivatives were frequently used as raw materials because of their excellent properties [12–14]. Starch, as a promising and renewable natural polymer, has gained increasing attention as a functional material for drug delivery system because of its advantages, such as improving drug solubility and stability, decreasing drug toxicity and side effects, and excellent biocompatibility and storage stability. Moreover, the carriers made of this
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biopolymer, can prolong the residence time by increasing the permeation of loaded drugs [15–19]. However, one of the major practical limitations related to utilization of starch in the drug delivery systems is its hydrophilicity. Also, the quick enzymatic degradation of native starch in biological systems leads to quick release of the drugs from such unmodified starch-based systems. Therefore, chemical modification of starch with hydrophobic units can improve stability of starch as well as degradation of its nanocarrier in vivo [20,21]. Chemical modification of starch biopolymers with various reagents such as isocyanates, anhydrides, and fatty acids and also via graft polymerization of caprolactone, polyethylene oxide and etc. has been reported. PCL is extensively used for controlled drug delivery because of its biocompatibility and biodegradability nature. As reported, PCL hydrophobic nature does not allow the facile release of hydrophobic drugs (from prepared nanomaterials) and its long-term degradation slows down tissue replacement [21–23]. The incorporation of CA to starch-PCL polymers could improve their compatibility and produce polymeric systems with better properties. Furthermore, CA provides worth pendant functionality which can participate in the ester bond formation, enhancing hemocompatibility, balancing the hydrophilicity of the polymeric system, providing hydrogen bonding and additional binding sites for bioconjugation to endow additional functionality [24,25]. Zohreh and coworkers, prepared modified starch polysaccharide by PEG and hydrazine and then coated this modified starch onto the Fe3O4 magnetic nanoparticles. Afterward, they conjugated DOX molecules to this magnetic carrier by hydrazone bond which this linkage is pH-sensitive and can be ruptured in acidic medium and release a large amount of DOX. They showed that the synthesized nanocarrier has good potential for targeted cancer therapy and MRI imaging [26]. In another research, Xiao and co-workers prepared acetylated starch nanocrystals (ASN) using acetic anhydride and loaded DOX by this system. They illustrated
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that DOX-loaded ASN has higher antitumor activity against HeLa human cervical carcinoma cells than free DOX and DOX-loaded in starch nanocrystals [27]. Hamidian and Tavakoli in another work prepared a hydrogel nanocomposite by graft copolymerization of poly (ethylene phthalate) onto starch (starch-g-polyester) and Fe3O4 nanoparticles. They load H3PW12O40 (HPA) by this system because starch-g-poly(ethylene phthalate) have applications in drug delivery [28]. However, single agent therapy has seen limited success in cancer therapy because of the toxicity at high drug dosage, the heterogeneity of cancer cells and the drug resistance. Therefore, combination anti-cancer therapy has been used instead of single-agent therapy because of the synergistic effects to the targeted cancer diseases and prevent drug resistance. A synergistic combination can promote the response rate with lower toxicity compared with single drugs and leads to the higher therapeutic effect [29,30]. For example, Curcumin as a poorly water-soluble nontoxic anticancer drug is now being co-administered with different functional therapeutic agents such as cisplatin, doxorubicin, and paclitaxel in nanomaterials to overcome MDR in the treatment modalities of many multi-drug resistant cancers. A recent study has shown that doxorubicin & curcumin encapsulated in poly-(D,L-lactide-co-glycolide) (PLGA) nanoparticles can efficiently inhibit the development of multidrug resistance (MDR) in leukemia cancer cells. Coadministration of curcumin and paclitaxel can increase cancer cell cytotoxicity by prevention of NF-κB activity and down-regulation of P-gp expression. Published studies also have shown that curcumin is able to increase DOX effectiveness in the cancer cells by increasing cytotoxicity in several cancer cell lines [3,29,31]. Barui and coworkers prepared a tumor vasculature targeting liposomal formulation of a pegylated RGDK-lipopeptide and co-encapsulation of potent anti-cancer drugs such as doxorubicin, and curcumin by this system. They revealed that simultaneous delivery of these potent anti-cancer drugs to tumor vasculature, shows synergistic therapeutic benefit in anti-angiogenic cancer therapy [31]. In another
Scheme 1. Synthesize of Am-CUR-PCL-PCA-DOX nanomaterial.
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research, Li and coworkers modified the surface of starch by stearic acid and glycidyl trimethyl ammonium chloride and loaded apogossypolone (ApoG2) and doxorubicin by this system. They showed that this dualdrug delivery system can evidently reduce the toxicity of DOX and ApoG2 and significantly target cancer cells, therefore it has great potential for the combination and targeting treatment of tumor [16]. The aim of this research was to design and synthesize a new biopolymeric nanocarrier containing two anticancer drugs, curcumin and doxorubicin. To this end, firstly inclusion complex of curcumin with amylose biopolymer was prepared as the core of the system. Then the surface of Am-CUR was modified with polycaprolactone (PCL) and polycitric acid (PCA) respectively to obtain a water-soluble, biocompatible and biodegradable nanomaterial which is suitable for using in the biologic environment. Finally, doxorubicin loaded by the prepared nanomaterial via noncovalent interactions. These newly synthesized nanomaterials were characterized by spectroscopic measurement methods such as IR spectroscopy, UV–vis spectroscopy, NMR spectroscopy, and scanning electron microscopy.
X-ray microanalysis) system with a sufficient sensitivity for detection of corresponding elements atomic numbers. Dried samples used for SEM experiment were coated with a thin layer of gold by sputtering for 15 s.
2.2.7. X-ray diffraction (XRD) The patterns of X-ray diffraction of the synthesized nanomaterials were obtained by a Halland Philips Xpert X-ray powder diffraction (XRD) diffractometer. (Cuk, radiation, λ = 0.154056 nm) at a scanning speed of 2°/min from 10° to 100 (2θ).
2. Materials and methods 2.1. Materials Starch, ε-caprolactone (CP), citric acid (CA), curcumin (CUR), doxorubicin (DOX), dimethylsulfoxide (DMSO), n-hexane, ethanol, tetrahydrofuran (THF) and acetone were purchased from Sigma-Aldrich. A dialysis bag was provided from Spectrum Company with 3.5 kDa MWCO. The other chemicals used in this study were of analytical grade. Deionized water was used in all experiments. 2.2. Instruments 2.2.1. NMR spectroscopy NMR spectra of synthesized nanomaterials were recorded on a Bruker 300 MHz for a proton isotope. The sample was dissolved in DMSO-d6 with the solution concentration of 15% (w/v). The spectrums were recorded at room temperature with a delay time of 10 s, an acquisition time of 2 s and a pulse angle of 30°. 2.2.2. FT-IR The FT-IR analysis was carried out using an FT-IR Bruker-Tensor 320 spectrometer. All of the products were mixed with analytical grade KBr at a weight ratio of 5/200 mg. 2.2.3. UV–vis spectroscopy Absorption spectra of samples in solution were recorded by a Shimadzu UV–visible 1650 PC spectrophotometer with a cell of 1.0 cm path length. 2.2.4. Fluorescence microscopy Emission spectra of products in solution were recorded by a Varian Cary Eclipse fluorescence spectrophotometer using a cell of 1.0 cm path length. 2.2.5. Dynamic light scattering (DLS) and zeta potential analyses The particle size distribution and zeta potential test of synthesized nanomaterials were determined by dynamic light scattering (DLS) by a 90 Plus particle size analyzer equipped with diode laser operating at 658.0 nm. The sample of synthesized nanomaterial was diluted with distilled water to adjust the solid content to 0.05 wt.% and directly placed in the cell. All measurements were carried out at 25 °C. 2.2.6. Field emission scanning electron microscopy (FE-SEM) Morphology and structure of the copolymer investigated using a LEO 440i scanning electron microscope under vacuum at an operating voltage of 10 kV. The instrument equipped with an EDX (energy-dispersive
Fig. 1. FT-IR spectra of (a) Am, (b) CUR, (c) Am-CUR, (d) Am-CUR-PCL, (e) Am-CUR-PCLPCA, (f) DOX and (g) Am-CUR-PCL-PCA-DOX.
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2.2.8. Determination of in vitro drug entrapment efficiency The drug entrapment efficiency was calculated during the preparation of Am-CUR and Am-CUR-PCL-PCA-DOX nanomaterial as follows: Drug entrapment efficiency ¼ ðWeight of drug in nanomaterialÞ=Weight of initial drug 100%
2.3. Methods 2.3.1. Preparation of amylose-curcumin helices (Am-CUR) Firstly, amylose extracted from normal potato starch according to the reported procedure in the literature [32]. Then dried Amylose (0.5 g) and curcumin (0.025 g) were dissolved in 10 ml of DMSO at 90 °C. The obtained orange clear mixture stirred for 24 h at 90 °C. The solvent of reaction evaporated and the product washed with ethanol and water repeatedly. Pure amylose-curcumin complex (Am-CUR) obtained as a dark orange solid. 2.3.2. Preparation of amylose-curcumin-polycaprolactone complex (AmCUR-PCL) 0.1 g of Am-CUR was placed in a polymerization ampoule equipped with a magnetic stirrer and vacuum. Then 1 ml of ε-caprolactone was gradually added to the ampule containing Am-CUR under vacuum and the temperature was raised from 50 to 120 °C during the addition of ε-caprolactone monomers. The mixture was stirred at 120 °C for 4 h. After this time the obtained mixtures was cooled to room temperature, dissolved in DMSO, filtrate and then precipitate in n-hexane. The purified product was obtained after drying as an orange-brown compound. 2.3.3. Preparation of amylose-curcumin-polycaprolactone-polycitric acid complex (Am-CUR-PCL-PCA) Am-CUR-PCL (0.39 g) was dissolved in a mixture of DMSO/THF in a round bottom flask. Then 1.5 g of citric acid was added to this solution gradually and the mixture sonicated for 45 min. Blue silica gel was added to the above solution in an oil bath and mixture was stirred at 70 °C for 48 h. Then cooled to room temperature, filtrated and precipitated in n-hexane. The product obtained after drying as a brown viscose compound. 2.3.4. Preparation of amylose-curcumin-polycaprolactone-polycitric-doxorubicin complex (Am-CUR-PCL-PCA-DOX) Am-CUR-PCL-PCA (0.1 g) was dissolved in 5 ml of phosphate buffered saline (PBS), pH = 7.4. Then doxorubicin hydrochloride (2 ml containing 0.004 g of doxorubicin) was added to this solution. The mixture sonicated for 1 h and stirred at room temperature for 48 h in darkness.
Fig. 2. UV–vis spectra of A: (a) Am, (b) Am-CUR, (c) Am-CUR-PCL, (d) Am-CUR-PCL-PCA and (e) Am-CUR-PCL-PCA-DOX. B: (a) CUR and (b) DOX.
Fig. 3. Florescence spectra of (a) Am-CUR and (b) Am-CUR-PCL-PCA-DOX.
After this time the mixture of reaction centrifuged and the obtained solution filtrate, dried and washed with ethanol. Then For further purification, the product dialyzed against the mixture of distilled water and ethanol to remove unreacted doxorubicin. The final product obtained as a red-orange viscose compound. 2.3.5. In vitro drug release In vitro drug release experiment at different phosphate buffered saline (PBS) solution (pH 7.4 and 5.4) was used to evaluate the release behavior of CUR and DOX from Am-CUR-PCL-PCA-DOX nanomaterial. For this purpose, Am-CUR-PCL-PCA-DOX (0.05 g in 3 ml of PBS) was added to the dialysis bag and then immersed in 40 ml PBS with constant stirring of 100 rpm with the temperature kept at 37 °C. Then at predetermined time intervals, the sample (4 ml) was withdrawn and replaced by the same volume of fresh PBS. The concentration of released CUR and DOX was determined by UV–vis spectrophotometer (428 nm
Fig. 4. Photographs of solutions of (a) Am, (b) Am-CUR, (c) Am-CUR-PCL, (d) Am-CURPCL-PCA and (e) Am-CUR-PCL-PCA-DOX under sunlight.
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for CUR and 483 nm for DOX). The percentage of released DOX and CUR anticancer drugs were calculated according to the following equation: Drug release% ¼ Weight of drug released at time“t” =Total weight of drug loaded in the nanomaterial 100%
3. Result and discussion New hybrid nanomaterial consisting of starch biopolymer and safety materials including caprolactone and citric acid were pre-
pared. Two anticancer drugs, curcumin, and doxorubicin, were loaded by this system. Synthesized nanomaterials were characterized by usual spectroscopy and microscopy methods. For synthesize of these nanomaterials, amylose extracted from starch and forms an inclusion complex with curcumin by V helical conformation (V-type, type I). The surface of amylose modified by caprolactone and citric acid by esterification reactions and finally doxorubicin loaded by synthesized nanomaterials via noncovalent interactions (Scheme 1). New synthesized fluorescence nanomaterials have beneficial properties including biocompatibility and biodegradability and therefore can be used in the different field such as drug delivery and cancer therapy (Scheme 1).
Fig. 5. (a) FE-SEM image of Am-CUR-PCL-PCA-DOX, (b) EDX spectra of Am-CUR-PCL-PCA, (c) EDX spectra of Am-CUR-PCL-PCA-DOX, (d) elemental maps of Am-CUR-PCL-PCA and (e) elemental maps of Am-CUR-PCL-PCA-DOX.
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3.1. Characterization 3.1.1. FT-IR analysis Fourier transform infrared (FT-IR) spectroscopy was used to characterize all of the synthesized products. In all of the spectrums the % of transmittance is plotted as a function of wavenumber (cm−1). The FTIR spectra of all of the products are shown in Fig. 1. Fig. 1a shows the FT-IR spectrum of amylose biopolymer. In this spectrum, the peaks at 3450 cm− 1, 2926 cm−1 and 1000–1300 cm− 1 are attributed to the OH, C\\H and C\\O stretching groups respectively (Fig. 1a). Fig. 1b shows IR spectrum of curcumin anticancer drug. The peaks at 3510 cm−1, 3020 cm−1, 2940 cm−1, 1740 cm−1 and 1627 cm−1 are attributed to the OH, aromatic C\\H, aliphatic C\\H, and C _O groups respectively. In the IR spectrum of Am-CUR, the peaks at 3416 cm−1, 2928 cm− 1 and 1000–1300 cm− 1 are attributed to the O\\H, C\\H and C\\O stretching groups respectively (Fig. 1c). IR spectrum of AmCUR-PCL is shown in Fig. 1d. In this spectrum, absorption bands at 3354 cm−1 and 2935 cm−1 are attributed to the OH and C\\H vibrations respectively. The peak at 2856 cm−1 is attributed to the symmetric C\\H vibrations of caprolactone molecules. Furthermore, a new sharp peak appeared at 1730 cm−1 is corresponding to the C _O vibrations which confirms that caprolactone reacts with the hydroxyl groups of amylose by the esterification reaction. Absorption bands at 3000– 3320 cm− 1, 2937 cm−1 and 1728 cm− 1 in the IR spectrum of AmCUR-PCL-PCA (Fig. 1e) are corresponding to the OH, C\\H and C _O vibrations respectively. Due to the presence of citric acid and formation of more hydrogen bonds, OH peak in this spectrum is wider than the band related to OH peak in Am-CUR-PCL IR spectrum. Doxorubicin could interact with Am-CUR-PCL-PCA by noncovalent interactions such as hydrogen bonding and interaction with the aliphatic chain of caprolactone. Absorption bands at 3421 cm− 1, 2935 cm− 1 and 1724 cm−1 in the IR spectrum of DOX (Fig. 1f) are corresponding to the OH, C\\H and C _O vibrations respectively and absorption bands at 875 cm−1 and 800 cm−1 are attributed to the N\\H wagging. IR spectrum of Am-CUR-PCL-PCA-DOX is shown in Fig. 1g. In this spectrum, absorption bands at 3419 cm−1, 2926 cm−1 and 1724 cm−1 are attributed to the OH, C\\H and C _O vibrations respectively. Presence of DOX in Am-CUR-PCL-PCA-DOX result in decreasing in hydrogen bonding between OH functional groups of Am-CUR-PCL-PCA and therefore result in decreasing in the width of OH peak. 3.1.2. UV–vis spectroscopy and fluorescence microscopy analyses UV–vis experiments were used to evaluate the preparation of these new nanomaterials. Fig. 2A and B are shown the UV–vis spectra of Am, Am-CUR, Am-CUR-PCL, Am-CUR-PCL-PCA, Am-CUR-PCL-PCA-DOX, CUR and DOX. Am shows an absorption peak at 270 nm which corresponds to the n-σ* transition (Fig. 2A.a). Curcumin spectrum has two characteristic peaks at 250 nm and 427 nm which correspond to π-π* and n-π* transitions (Fig. 2B.a). Maximum absorptions appeared in Am-CUR spectrum at around 283 nm and 349 nm are attributed to the n-σ* and π-π* transitions. The new characteristic peak which appears at 349 nm is attributed to the host-guest complex formation between Am and Cur molecules (Fig. 2A.b). The absorption spectrum of AmCUR-PCL displays absorbance bands centered at 260 nm, 318 nm and a shoulder at around 283 nm. These evidences confirm the presence of caprolactone molecules at the surface of Am-CUR complex (Fig. 2A.c). When CA reacts with hydroxyl groups of caprolactone molecules which are located at the surface of Am-CUR-PCL for preparation of Am-CUR-PCL-PCA, only a broad weak peak appeared at 289 nm and other peaks disappeared due to hydrogen bond formation (Fig. 2A.d). Due to the presence of dihydroxyanthraquinone chromophore, the absorption spectrum of DOX, displays characteristic peaks at around 274 and 469 nm (Fig. 2B.b). This anticancer drug could react with the functional groups of Am-CUR-PCL-PCA by noncovalent interactions. As can be seen from the Fig. 2A.e, the absorption spectrum of Am-CUR-PCLPCA-DOX, shows absorbance bands centered at 291 nm and 500 nm.
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The new characteristic peak which is appeared at 500 nm is attributed to the presence of DOX in the synthesized nanomaterials. As mentioned above, we loaded two anticancer drugs by noncovalent interactions in the synthesized nanomaterials. Fluorescence spectroscopy was used for further confirmation of the presence of these anticancer drugs in the system in excitation wavelengths of 427 nm and 469 nm correspond to the CUR and DOX respectively. The room temperature fluorescence spectrum of Am-CUR-PCL-PCA and Am-CUR-PCL-PCA-DOX are shown in Fig. 3. As can be seen from the figure, Am-CUR-PCL-PCA has a maximum emission peak at 511 nm which confirms the presence of curcumin in this product. Am-CUR-PCL-PCADOX has two emission peaks at 511 and 576 nm which are correspond to the CUR and DOX respectively and showed that the prepared nanomaterials successfully loaded two anticancer drugs. Fig. 4 shows the photograph of the solutions of Am, Am-CUR, AmCUR-PCL in DMSO and Am-CUR-PCL-PCA and Am-CUR-PCL-PCA-DOX in water under sunlight. The figure shows that amylose can form a stable inclusion complex with CUR successfully which remains in the system upon the polymerization reactions in the next steps. Am, Am-CUR and Am-CUR-PCL have low solubility in water, but the introduction of water-soluble citric acid molecules into the surface of Am-CUR-PCL by esterification reaction, would alter their surface properties and therefore a water soluble product would be obtained. As can be seen from Fig. 4d and e, Am-CUR-PCL-PCA and Am-CUR-PCL-PCA-DOX have high water solubility, therefore can be used successfully for biomedical applications such as drug delivery and anticancer therapy. 3.1.3. Field emission-scanning electron microscopy (FE-SEM) analysis FE-SEM observations were used for evaluation of the surface morphology of synthesized nanomaterials. FE-SEM image of Am-CUR-PCLPCA-DOX nanoparticles is shown in Fig. 5a. These uniform nanoparticles have a spherical morphology with a mean diameter around 50 nm. Well information about the elements existing in the sample and also the mass concentration of the elements can be determined by Energy dispersive X-ray spectroscopy (EDX). The EDX spectrum and loading content and wt.% of elements of Am-CUR-PCL-PCA and Am-CUR-PCLPCA-DOX also are shown in Fig. 5b and c. Corresponding element mapping images also are shown in Fig. 5d and e. As can be seen from these figures, the Am-CUR-PCL-PCA contains two kinds of elements (C, O) while Am-CUR-PCL-PCA-DOX contains three kinds of elements (C, O, N) indicating the presence of DOX at the surface of Am-CUR-PCL-PCA.
Fig. 6. DLS diagram of (a) Am-CUR-PCL-PCA and (b) Am-CUR-PCL-PCA-DOX.
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Surface charge (mV)
Am-CUR-PCL-PCA Am-CUR-PCL-PCA-DOX
−9.30 −6.20
3.1.4. Dynamic light scattering (DLS) and zeta-potential measurements DLS measurements were used to determine the hydrodynamic dimensions of the Am-CUR-PCL-PCA and Am-CUR-PCL-PCA-DOX. This technique was also used to investigate the ability of aggregate formation of these nanoparticles in solvents. The synthesized nanomaterials are able to form molecular self-assemblies in aqueous solutions, therefore, several particle sizes appeared in their DLS diagrams, can be assigned to their molecular self-assemblies. According to these experiments, due to the self-assembly formation, sizes of Am-CUR-PCL-PCA and Am-CUR-PCL-PCA-DOX were 162 and 106 nm, respectively (Fig. 6). As
can be seen from the figure, DLS experiments show a decrease in the size of Am-CUR-PCL-PCA upon loading of DOX onto their surface since before loading of DOX, the Am-CUR-PCL-PCA nanoparticles self-aggregated by noncovalent interactions such as intermolecular hydrogen bond formation between nanoparticles. These interactions could be reduced the particle sizes due to the presence of DOX in Am-CUR-PCLPCA-DOX. In fact, DOX could increase the intramolecular hydrogen bonds which results in formation of smaller and more monodisperse nanoparticles. The surface charge of Am-CUR-PCL-PCA and Am-CUR-PCL-PCA-DOX were detected by Zeta-potential analysis. As can be seen from Table 1, because of the presence of carboxyl functional groups with the negative charge at the surface of Am-CUR-PCL-PCA, the surface charge of this product was −9.30. But the surface charge of Am-CUR-PCL-PCA-DOX was − 6.20. After interaction of DOX with functional groups of AmCUR-PCL-PCA by noncovalent interaction, due to the presence NH+ 3 groups of DOX, the negative surface charge of Am-CUR-PCL-PCA nanoparticles reduced from −9.30 to −6.20.
Fig. 7. 1H NMR spectrum of (a) Am-CUR-PCL and (b) Am-CUR-PCL-PCA.
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13.04° and 19.99°, which correspond to the V helical amylose conformation (type I). The X-ray diffraction data confirm structural changes in amylose due to the inclusion complex formation with CUR through the amylose V-type polymorph.
Fig. 8. X-ray diffraction patterns of (a) Am and (b) Am-CUR.
3.1.5. NMR analysis The structure of Am-CUR-PCL and Am-CUR-PCL-PCA were evaluated by 1H NMR spectroscopy. Fig. 7a shows representative 1H NMR spectrum of Am-CUR-PCL. The signals which appeared at 3.4–5.6 ppm are attributed to the glycosidic rings of amylose. The signals at 1.2– 3.5 ppm are related to the PCL chains and confirm the presence of PCL chains in the product. Fig. 7b shows the 1H NMR spectrum of AmCUR-PCL-PCA. In this spectrum signals at 3.4–5.5 ppm are related to the glycosidic rings of amylose. The signals for the PCL chains appeared at 1.2–2.5 ppm and 3.9 ppm. The signals of methylene groups of the PCA blocks appeared at 2.6–2.8 ppm as the AB system. All of these data are in agreement with the above-mentioned FT-IR results.
3.1.6. X-ray diffraction The effect of inclusion complex formation on the crystal morphology of amylose biopolymer was investigated by X-ray diffraction. XRD pattern of Am showed obvious characteristic peaks at 2θ = 15.12°, 16.90°, 17.90 and 23.91° (Fig. 8a). XRD pattern of Am-CUR (Fig. 8b) showed high crystallinity with characteristic peaks at 2θ = 7.51°,
3.1.7. In-vitro drug release study The release of CUR and DOX anticancer drugs from synthesized nanomaterials were investigated by regular dialysis against PBS at 37 °C for a period of 72 h. The selected pHs were 5.4 and 7.4 which are the pHs of tumor cells and normal cells respectively. Fig. 9a shows the release of DOX at these two pHs. As can be seen from the figure, after 24 h about 78.163% of DOX was released at pH = 5.4 and 46.39% at pH = 7.4. After 72 h, approximately 81.05% of the drug was released at pH = 5.4 and 55.05% of it was released at pH = 7.4. The release of the encapsulated CUR from the drug-loaded nanomaterials was also investigated at pHs 5.4 and 7.4. Fig. 9b shows the release profile of CUR at these two pHs. As can be seen from the figure, after 24 h about 66.9% of CUR was released at pH = 5.4 and 48.5% at pH = 7.4. After 72 h, about 74.77% of the drug was released at pH = 5.4 and 53.78% of it was released at pH = 7.4. The drug release data confirm that the rate of release is dependence on variation in pH value. The rate of drug release increased by changing the pH from 7.4 to 5.4 for both drugs and is more favorable at acidic pH in both cases since tumor microenvironment is usually acidic. The higher rate of the drug's release from the nanomaterials at acidic pH is due to their lower stability in acidic conditions. Increased hydrophilicity, due to the protonation of polymer branches at acidic pH and also hydrolysis of the ester bonds of PCL and PCA bounded polymers, trigger dissociation of covalence bounds and faster release of the encapsulated drugs [33]. The release data also illustrated that DOX which is absorbed in the surface of synthesized nanomaterial would be released earlier and faster than CUR which is entrapped in the core of the system.
4. Conclusion In this research, amylose biopolymer was extracted from starch and formed inclusion complex with Cur. Then the surface of this complex modified with PCL and PCA respectively. Finally, DOX reacted with surface functional groups of Am-Cur-PCL-PCA by noncovalent interactions. The evidences of the occurrence of chemical modifications of the resulting nanoparticles were checked by FT-IR spectroscopy, UV–vis spectroscopy, scanning electron microscopy, DLS measurements etc. FE-SEM analyses and DLS measurements showed that the hydrodynamic dimensions of Am-Cur-PCL-PCA were about 50 nm. These new nanomaterials has potential application in different field such as drug delivery and cancer therapy.
Fig. 9. In vitro release profiles of DOX (a) and CUR (b) from Am-CUR-PCL-PCA-DOX nanomaterial at 37 °C.
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Preparation, characterization and drug delivery study of a novel nanobiopolymeric multidrug delivery system Abbas Dadkhah Tehrani ⁎, Masoumeh Parsamanesh Department of Chemistry, Faculty of Science, Lorestan University, Khoramabad, Iran
a r t i c l e
i n f o
Article history: Received 19 October 2016 Received in revised form 8 December 2016 Accepted 20 December 2016 Available online 24 December 2016 Keywords: Biopolymer Anticancer Nanocarrier Drug delivery
a b s t r a c t New nanocarrier for codelivery of curcumin and doxorubicin as the anticancer drugs was synthesized using biocompatible and biodegradable materials. Firstly, an inclusion complex of amylose (Am) and curcumin (CUR) was formed through entrapment of curcumin into the amylose helices. Then the surface of amylose-curcumin (Am-CUR) complex was modified by polycaprolactone (PCL) via esterification reaction between hydroxyl functional groups of amylose and carbonyl groups of PCL. Finally, poly citric acid (PCA) reacted with terminal hydroxyl groups of PCL by esterification reaction. Then, doxorubicin (DOX) reacted with the surface carboxylic acid functional groups of Am-CUR-PCL-PCA through noncovalent interactions to form Am-CUR-PCL-PCA-DOX as a multidrug delivery system. These new synthesized nanomaterials were characterized by spectroscopic measurement methods such as IR spectroscopy, UV–vis spectroscopy, NMR spectroscopy, and scanning electron microscopy. FE-SEM analyses and DLS measurements showed that the hydrodynamic dimensions of Am-CurPCL-PCA were about 50 nm. Due to the presence of ester bonds, the synthesized nanomaterials are pH sensitive. Furthermore, the resulting copolymer was completely water soluble because of the hydrophilic nature of poly citric acid part of copolymer and therefore successfully can be utilized in biomedical applications. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Nowadays, drug delivery systems are playing a significant role in the fields of medical and pharmaceutical sciences. A sustained and controlled drug release is critically important for the effective treatment of many sicknesses, ranging from arthritis to cancer which is one of the major causes of mortality in the world [1,2]. Traditional chemotherapy is an essential approach for cancer therapy. However, its therapeutic efficacy is usually limited by a number of major challenges. For example, the high toxicity of denuded drugs or the drug leakage from traditional drug delivery systems before reaching the tumor site poses a serious challenge to effective treatment. Another challenge is the delivery of water-insoluble drugs. Lots of the hydrophobic drugs showing high potency remain in the pipeline since they suffer from the lack of means for administration in the body. Furthermore, cancer chemotherapeutic agents can unselectively enter into both normal tissues and tumor tissues, which result in distasteful side effects and even death of the patients. The occurrence of multidrug resistance (MDR) phenotypes is one of the other important reasons which lead to chemotherapeutic failure. Therefore, noticeable attention has particularly been focused on anticancer drug delivery systems capable of overcoming the current problems associated with the conventional chemotherapy. Especially, ⁎ Corresponding author. E-mail address: [email protected] (A. Dadkhah Tehrani).
http://dx.doi.org/10.1016/j.msec.2016.12.103 0928-4931/© 2016 Elsevier B.V. All rights reserved.
advanced nanotechnology and nanoscience have been extensively used in the fabrication of controllable drug delivery systems and providing an innovative and promising procedure for circumventing MDR by encapsulating or conjugating chemotherapeutic drugs to nanocarriers [2–4]. A number of drug delivery carriers designed to address these problems including micelles of amphiphilic block copolymers, liposomes, hydrogels, nanoemulsions, nanosuspensions, polymeric nanoparticles, etc. [5–7]. The incorporation of chemotherapeutic agents into polymeric nanoparticles have attracted significant attention in the study of drug delivery systems and has many advantages, consisting of low toxicity, enhanced bioavailability, increased circulation times, high stability in biological systems and break down to biocompatible materials in destination organelles etc. [4,6,8–11]. Selection of polymer is an important step to design a nanocarrier for the successful drug delivery purpose. For this purpose, natural and environmentally friendly polymers involving starch, chitosan, sodium alginate, guar gum, carboxy methyl cellulose and their derivatives were frequently used as raw materials because of their excellent properties [12–14]. Starch, as a promising and renewable natural polymer, has gained increasing attention as a functional material for drug delivery system because of its advantages, such as improving drug solubility and stability, decreasing drug toxicity and side effects, and excellent biocompatibility and storage stability. Moreover, the carriers made of this
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biopolymer, can prolong the residence time by increasing the permeation of loaded drugs [15–19]. However, one of the major practical limitations related to utilization of starch in the drug delivery systems is its hydrophilicity. Also, the quick enzymatic degradation of native starch in biological systems leads to quick release of the drugs from such unmodified starch-based systems. Therefore, chemical modification of starch with hydrophobic units can improve stability of starch as well as degradation of its nanocarrier in vivo [20,21]. Chemical modification of starch biopolymers with various reagents such as isocyanates, anhydrides, and fatty acids and also via graft polymerization of caprolactone, polyethylene oxide and etc. has been reported. PCL is extensively used for controlled drug delivery because of its biocompatibility and biodegradability nature. As reported, PCL hydrophobic nature does not allow the facile release of hydrophobic drugs (from prepared nanomaterials) and its long-term degradation slows down tissue replacement [21–23]. The incorporation of CA to starch-PCL polymers could improve their compatibility and produce polymeric systems with better properties. Furthermore, CA provides worth pendant functionality which can participate in the ester bond formation, enhancing hemocompatibility, balancing the hydrophilicity of the polymeric system, providing hydrogen bonding and additional binding sites for bioconjugation to endow additional functionality [24,25]. Zohreh and coworkers, prepared modified starch polysaccharide by PEG and hydrazine and then coated this modified starch onto the Fe3O4 magnetic nanoparticles. Afterward, they conjugated DOX molecules to this magnetic carrier by hydrazone bond which this linkage is pH-sensitive and can be ruptured in acidic medium and release a large amount of DOX. They showed that the synthesized nanocarrier has good potential for targeted cancer therapy and MRI imaging [26]. In another research, Xiao and co-workers prepared acetylated starch nanocrystals (ASN) using acetic anhydride and loaded DOX by this system. They illustrated
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that DOX-loaded ASN has higher antitumor activity against HeLa human cervical carcinoma cells than free DOX and DOX-loaded in starch nanocrystals [27]. Hamidian and Tavakoli in another work prepared a hydrogel nanocomposite by graft copolymerization of poly (ethylene phthalate) onto starch (starch-g-polyester) and Fe3O4 nanoparticles. They load H3PW12O40 (HPA) by this system because starch-g-poly(ethylene phthalate) have applications in drug delivery [28]. However, single agent therapy has seen limited success in cancer therapy because of the toxicity at high drug dosage, the heterogeneity of cancer cells and the drug resistance. Therefore, combination anti-cancer therapy has been used instead of single-agent therapy because of the synergistic effects to the targeted cancer diseases and prevent drug resistance. A synergistic combination can promote the response rate with lower toxicity compared with single drugs and leads to the higher therapeutic effect [29,30]. For example, Curcumin as a poorly water-soluble nontoxic anticancer drug is now being co-administered with different functional therapeutic agents such as cisplatin, doxorubicin, and paclitaxel in nanomaterials to overcome MDR in the treatment modalities of many multi-drug resistant cancers. A recent study has shown that doxorubicin & curcumin encapsulated in poly-(D,L-lactide-co-glycolide) (PLGA) nanoparticles can efficiently inhibit the development of multidrug resistance (MDR) in leukemia cancer cells. Coadministration of curcumin and paclitaxel can increase cancer cell cytotoxicity by prevention of NF-κB activity and down-regulation of P-gp expression. Published studies also have shown that curcumin is able to increase DOX effectiveness in the cancer cells by increasing cytotoxicity in several cancer cell lines [3,29,31]. Barui and coworkers prepared a tumor vasculature targeting liposomal formulation of a pegylated RGDK-lipopeptide and co-encapsulation of potent anti-cancer drugs such as doxorubicin, and curcumin by this system. They revealed that simultaneous delivery of these potent anti-cancer drugs to tumor vasculature, shows synergistic therapeutic benefit in anti-angiogenic cancer therapy [31]. In another
Scheme 1. Synthesize of Am-CUR-PCL-PCA-DOX nanomaterial.
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research, Li and coworkers modified the surface of starch by stearic acid and glycidyl trimethyl ammonium chloride and loaded apogossypolone (ApoG2) and doxorubicin by this system. They showed that this dualdrug delivery system can evidently reduce the toxicity of DOX and ApoG2 and significantly target cancer cells, therefore it has great potential for the combination and targeting treatment of tumor [16]. The aim of this research was to design and synthesize a new biopolymeric nanocarrier containing two anticancer drugs, curcumin and doxorubicin. To this end, firstly inclusion complex of curcumin with amylose biopolymer was prepared as the core of the system. Then the surface of Am-CUR was modified with polycaprolactone (PCL) and polycitric acid (PCA) respectively to obtain a water-soluble, biocompatible and biodegradable nanomaterial which is suitable for using in the biologic environment. Finally, doxorubicin loaded by the prepared nanomaterial via noncovalent interactions. These newly synthesized nanomaterials were characterized by spectroscopic measurement methods such as IR spectroscopy, UV–vis spectroscopy, NMR spectroscopy, and scanning electron microscopy.
X-ray microanalysis) system with a sufficient sensitivity for detection of corresponding elements atomic numbers. Dried samples used for SEM experiment were coated with a thin layer of gold by sputtering for 15 s.
2.2.7. X-ray diffraction (XRD) The patterns of X-ray diffraction of the synthesized nanomaterials were obtained by a Halland Philips Xpert X-ray powder diffraction (XRD) diffractometer. (Cuk, radiation, λ = 0.154056 nm) at a scanning speed of 2°/min from 10° to 100 (2θ).
2. Materials and methods 2.1. Materials Starch, ε-caprolactone (CP), citric acid (CA), curcumin (CUR), doxorubicin (DOX), dimethylsulfoxide (DMSO), n-hexane, ethanol, tetrahydrofuran (THF) and acetone were purchased from Sigma-Aldrich. A dialysis bag was provided from Spectrum Company with 3.5 kDa MWCO. The other chemicals used in this study were of analytical grade. Deionized water was used in all experiments. 2.2. Instruments 2.2.1. NMR spectroscopy NMR spectra of synthesized nanomaterials were recorded on a Bruker 300 MHz for a proton isotope. The sample was dissolved in DMSO-d6 with the solution concentration of 15% (w/v). The spectrums were recorded at room temperature with a delay time of 10 s, an acquisition time of 2 s and a pulse angle of 30°. 2.2.2. FT-IR The FT-IR analysis was carried out using an FT-IR Bruker-Tensor 320 spectrometer. All of the products were mixed with analytical grade KBr at a weight ratio of 5/200 mg. 2.2.3. UV–vis spectroscopy Absorption spectra of samples in solution were recorded by a Shimadzu UV–visible 1650 PC spectrophotometer with a cell of 1.0 cm path length. 2.2.4. Fluorescence microscopy Emission spectra of products in solution were recorded by a Varian Cary Eclipse fluorescence spectrophotometer using a cell of 1.0 cm path length. 2.2.5. Dynamic light scattering (DLS) and zeta potential analyses The particle size distribution and zeta potential test of synthesized nanomaterials were determined by dynamic light scattering (DLS) by a 90 Plus particle size analyzer equipped with diode laser operating at 658.0 nm. The sample of synthesized nanomaterial was diluted with distilled water to adjust the solid content to 0.05 wt.% and directly placed in the cell. All measurements were carried out at 25 °C. 2.2.6. Field emission scanning electron microscopy (FE-SEM) Morphology and structure of the copolymer investigated using a LEO 440i scanning electron microscope under vacuum at an operating voltage of 10 kV. The instrument equipped with an EDX (energy-dispersive
Fig. 1. FT-IR spectra of (a) Am, (b) CUR, (c) Am-CUR, (d) Am-CUR-PCL, (e) Am-CUR-PCLPCA, (f) DOX and (g) Am-CUR-PCL-PCA-DOX.
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2.2.8. Determination of in vitro drug entrapment efficiency The drug entrapment efficiency was calculated during the preparation of Am-CUR and Am-CUR-PCL-PCA-DOX nanomaterial as follows: Drug entrapment efficiency ¼ ðWeight of drug in nanomaterialÞ=Weight of initial drug 100%
2.3. Methods 2.3.1. Preparation of amylose-curcumin helices (Am-CUR) Firstly, amylose extracted from normal potato starch according to the reported procedure in the literature [32]. Then dried Amylose (0.5 g) and curcumin (0.025 g) were dissolved in 10 ml of DMSO at 90 °C. The obtained orange clear mixture stirred for 24 h at 90 °C. The solvent of reaction evaporated and the product washed with ethanol and water repeatedly. Pure amylose-curcumin complex (Am-CUR) obtained as a dark orange solid. 2.3.2. Preparation of amylose-curcumin-polycaprolactone complex (AmCUR-PCL) 0.1 g of Am-CUR was placed in a polymerization ampoule equipped with a magnetic stirrer and vacuum. Then 1 ml of ε-caprolactone was gradually added to the ampule containing Am-CUR under vacuum and the temperature was raised from 50 to 120 °C during the addition of ε-caprolactone monomers. The mixture was stirred at 120 °C for 4 h. After this time the obtained mixtures was cooled to room temperature, dissolved in DMSO, filtrate and then precipitate in n-hexane. The purified product was obtained after drying as an orange-brown compound. 2.3.3. Preparation of amylose-curcumin-polycaprolactone-polycitric acid complex (Am-CUR-PCL-PCA) Am-CUR-PCL (0.39 g) was dissolved in a mixture of DMSO/THF in a round bottom flask. Then 1.5 g of citric acid was added to this solution gradually and the mixture sonicated for 45 min. Blue silica gel was added to the above solution in an oil bath and mixture was stirred at 70 °C for 48 h. Then cooled to room temperature, filtrated and precipitated in n-hexane. The product obtained after drying as a brown viscose compound. 2.3.4. Preparation of amylose-curcumin-polycaprolactone-polycitric-doxorubicin complex (Am-CUR-PCL-PCA-DOX) Am-CUR-PCL-PCA (0.1 g) was dissolved in 5 ml of phosphate buffered saline (PBS), pH = 7.4. Then doxorubicin hydrochloride (2 ml containing 0.004 g of doxorubicin) was added to this solution. The mixture sonicated for 1 h and stirred at room temperature for 48 h in darkness.
Fig. 2. UV–vis spectra of A: (a) Am, (b) Am-CUR, (c) Am-CUR-PCL, (d) Am-CUR-PCL-PCA and (e) Am-CUR-PCL-PCA-DOX. B: (a) CUR and (b) DOX.
Fig. 3. Florescence spectra of (a) Am-CUR and (b) Am-CUR-PCL-PCA-DOX.
After this time the mixture of reaction centrifuged and the obtained solution filtrate, dried and washed with ethanol. Then For further purification, the product dialyzed against the mixture of distilled water and ethanol to remove unreacted doxorubicin. The final product obtained as a red-orange viscose compound. 2.3.5. In vitro drug release In vitro drug release experiment at different phosphate buffered saline (PBS) solution (pH 7.4 and 5.4) was used to evaluate the release behavior of CUR and DOX from Am-CUR-PCL-PCA-DOX nanomaterial. For this purpose, Am-CUR-PCL-PCA-DOX (0.05 g in 3 ml of PBS) was added to the dialysis bag and then immersed in 40 ml PBS with constant stirring of 100 rpm with the temperature kept at 37 °C. Then at predetermined time intervals, the sample (4 ml) was withdrawn and replaced by the same volume of fresh PBS. The concentration of released CUR and DOX was determined by UV–vis spectrophotometer (428 nm
Fig. 4. Photographs of solutions of (a) Am, (b) Am-CUR, (c) Am-CUR-PCL, (d) Am-CURPCL-PCA and (e) Am-CUR-PCL-PCA-DOX under sunlight.
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for CUR and 483 nm for DOX). The percentage of released DOX and CUR anticancer drugs were calculated according to the following equation: Drug release% ¼ Weight of drug released at time“t” =Total weight of drug loaded in the nanomaterial 100%
3. Result and discussion New hybrid nanomaterial consisting of starch biopolymer and safety materials including caprolactone and citric acid were pre-
pared. Two anticancer drugs, curcumin, and doxorubicin, were loaded by this system. Synthesized nanomaterials were characterized by usual spectroscopy and microscopy methods. For synthesize of these nanomaterials, amylose extracted from starch and forms an inclusion complex with curcumin by V helical conformation (V-type, type I). The surface of amylose modified by caprolactone and citric acid by esterification reactions and finally doxorubicin loaded by synthesized nanomaterials via noncovalent interactions (Scheme 1). New synthesized fluorescence nanomaterials have beneficial properties including biocompatibility and biodegradability and therefore can be used in the different field such as drug delivery and cancer therapy (Scheme 1).
Fig. 5. (a) FE-SEM image of Am-CUR-PCL-PCA-DOX, (b) EDX spectra of Am-CUR-PCL-PCA, (c) EDX spectra of Am-CUR-PCL-PCA-DOX, (d) elemental maps of Am-CUR-PCL-PCA and (e) elemental maps of Am-CUR-PCL-PCA-DOX.
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3.1. Characterization 3.1.1. FT-IR analysis Fourier transform infrared (FT-IR) spectroscopy was used to characterize all of the synthesized products. In all of the spectrums the % of transmittance is plotted as a function of wavenumber (cm−1). The FTIR spectra of all of the products are shown in Fig. 1. Fig. 1a shows the FT-IR spectrum of amylose biopolymer. In this spectrum, the peaks at 3450 cm− 1, 2926 cm−1 and 1000–1300 cm− 1 are attributed to the OH, C\\H and C\\O stretching groups respectively (Fig. 1a). Fig. 1b shows IR spectrum of curcumin anticancer drug. The peaks at 3510 cm−1, 3020 cm−1, 2940 cm−1, 1740 cm−1 and 1627 cm−1 are attributed to the OH, aromatic C\\H, aliphatic C\\H, and C _O groups respectively. In the IR spectrum of Am-CUR, the peaks at 3416 cm−1, 2928 cm− 1 and 1000–1300 cm− 1 are attributed to the O\\H, C\\H and C\\O stretching groups respectively (Fig. 1c). IR spectrum of AmCUR-PCL is shown in Fig. 1d. In this spectrum, absorption bands at 3354 cm−1 and 2935 cm−1 are attributed to the OH and C\\H vibrations respectively. The peak at 2856 cm−1 is attributed to the symmetric C\\H vibrations of caprolactone molecules. Furthermore, a new sharp peak appeared at 1730 cm−1 is corresponding to the C _O vibrations which confirms that caprolactone reacts with the hydroxyl groups of amylose by the esterification reaction. Absorption bands at 3000– 3320 cm− 1, 2937 cm−1 and 1728 cm− 1 in the IR spectrum of AmCUR-PCL-PCA (Fig. 1e) are corresponding to the OH, C\\H and C _O vibrations respectively. Due to the presence of citric acid and formation of more hydrogen bonds, OH peak in this spectrum is wider than the band related to OH peak in Am-CUR-PCL IR spectrum. Doxorubicin could interact with Am-CUR-PCL-PCA by noncovalent interactions such as hydrogen bonding and interaction with the aliphatic chain of caprolactone. Absorption bands at 3421 cm− 1, 2935 cm− 1 and 1724 cm−1 in the IR spectrum of DOX (Fig. 1f) are corresponding to the OH, C\\H and C _O vibrations respectively and absorption bands at 875 cm−1 and 800 cm−1 are attributed to the N\\H wagging. IR spectrum of Am-CUR-PCL-PCA-DOX is shown in Fig. 1g. In this spectrum, absorption bands at 3419 cm−1, 2926 cm−1 and 1724 cm−1 are attributed to the OH, C\\H and C _O vibrations respectively. Presence of DOX in Am-CUR-PCL-PCA-DOX result in decreasing in hydrogen bonding between OH functional groups of Am-CUR-PCL-PCA and therefore result in decreasing in the width of OH peak. 3.1.2. UV–vis spectroscopy and fluorescence microscopy analyses UV–vis experiments were used to evaluate the preparation of these new nanomaterials. Fig. 2A and B are shown the UV–vis spectra of Am, Am-CUR, Am-CUR-PCL, Am-CUR-PCL-PCA, Am-CUR-PCL-PCA-DOX, CUR and DOX. Am shows an absorption peak at 270 nm which corresponds to the n-σ* transition (Fig. 2A.a). Curcumin spectrum has two characteristic peaks at 250 nm and 427 nm which correspond to π-π* and n-π* transitions (Fig. 2B.a). Maximum absorptions appeared in Am-CUR spectrum at around 283 nm and 349 nm are attributed to the n-σ* and π-π* transitions. The new characteristic peak which appears at 349 nm is attributed to the host-guest complex formation between Am and Cur molecules (Fig. 2A.b). The absorption spectrum of AmCUR-PCL displays absorbance bands centered at 260 nm, 318 nm and a shoulder at around 283 nm. These evidences confirm the presence of caprolactone molecules at the surface of Am-CUR complex (Fig. 2A.c). When CA reacts with hydroxyl groups of caprolactone molecules which are located at the surface of Am-CUR-PCL for preparation of Am-CUR-PCL-PCA, only a broad weak peak appeared at 289 nm and other peaks disappeared due to hydrogen bond formation (Fig. 2A.d). Due to the presence of dihydroxyanthraquinone chromophore, the absorption spectrum of DOX, displays characteristic peaks at around 274 and 469 nm (Fig. 2B.b). This anticancer drug could react with the functional groups of Am-CUR-PCL-PCA by noncovalent interactions. As can be seen from the Fig. 2A.e, the absorption spectrum of Am-CUR-PCLPCA-DOX, shows absorbance bands centered at 291 nm and 500 nm.
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The new characteristic peak which is appeared at 500 nm is attributed to the presence of DOX in the synthesized nanomaterials. As mentioned above, we loaded two anticancer drugs by noncovalent interactions in the synthesized nanomaterials. Fluorescence spectroscopy was used for further confirmation of the presence of these anticancer drugs in the system in excitation wavelengths of 427 nm and 469 nm correspond to the CUR and DOX respectively. The room temperature fluorescence spectrum of Am-CUR-PCL-PCA and Am-CUR-PCL-PCA-DOX are shown in Fig. 3. As can be seen from the figure, Am-CUR-PCL-PCA has a maximum emission peak at 511 nm which confirms the presence of curcumin in this product. Am-CUR-PCL-PCADOX has two emission peaks at 511 and 576 nm which are correspond to the CUR and DOX respectively and showed that the prepared nanomaterials successfully loaded two anticancer drugs. Fig. 4 shows the photograph of the solutions of Am, Am-CUR, AmCUR-PCL in DMSO and Am-CUR-PCL-PCA and Am-CUR-PCL-PCA-DOX in water under sunlight. The figure shows that amylose can form a stable inclusion complex with CUR successfully which remains in the system upon the polymerization reactions in the next steps. Am, Am-CUR and Am-CUR-PCL have low solubility in water, but the introduction of water-soluble citric acid molecules into the surface of Am-CUR-PCL by esterification reaction, would alter their surface properties and therefore a water soluble product would be obtained. As can be seen from Fig. 4d and e, Am-CUR-PCL-PCA and Am-CUR-PCL-PCA-DOX have high water solubility, therefore can be used successfully for biomedical applications such as drug delivery and anticancer therapy. 3.1.3. Field emission-scanning electron microscopy (FE-SEM) analysis FE-SEM observations were used for evaluation of the surface morphology of synthesized nanomaterials. FE-SEM image of Am-CUR-PCLPCA-DOX nanoparticles is shown in Fig. 5a. These uniform nanoparticles have a spherical morphology with a mean diameter around 50 nm. Well information about the elements existing in the sample and also the mass concentration of the elements can be determined by Energy dispersive X-ray spectroscopy (EDX). The EDX spectrum and loading content and wt.% of elements of Am-CUR-PCL-PCA and Am-CUR-PCLPCA-DOX also are shown in Fig. 5b and c. Corresponding element mapping images also are shown in Fig. 5d and e. As can be seen from these figures, the Am-CUR-PCL-PCA contains two kinds of elements (C, O) while Am-CUR-PCL-PCA-DOX contains three kinds of elements (C, O, N) indicating the presence of DOX at the surface of Am-CUR-PCL-PCA.
Fig. 6. DLS diagram of (a) Am-CUR-PCL-PCA and (b) Am-CUR-PCL-PCA-DOX.
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A. Dadkhah Tehrani, M. Parsamanesh / Materials Science and Engineering C 73 (2017) 516–524 Table 1 The measured Zeta-potential of Am-CUR-PCL-PCA and Am-CUR-PCL-PCA-DOX. Sample name
Surface charge (mV)
Am-CUR-PCL-PCA Am-CUR-PCL-PCA-DOX
−9.30 −6.20
3.1.4. Dynamic light scattering (DLS) and zeta-potential measurements DLS measurements were used to determine the hydrodynamic dimensions of the Am-CUR-PCL-PCA and Am-CUR-PCL-PCA-DOX. This technique was also used to investigate the ability of aggregate formation of these nanoparticles in solvents. The synthesized nanomaterials are able to form molecular self-assemblies in aqueous solutions, therefore, several particle sizes appeared in their DLS diagrams, can be assigned to their molecular self-assemblies. According to these experiments, due to the self-assembly formation, sizes of Am-CUR-PCL-PCA and Am-CUR-PCL-PCA-DOX were 162 and 106 nm, respectively (Fig. 6). As
can be seen from the figure, DLS experiments show a decrease in the size of Am-CUR-PCL-PCA upon loading of DOX onto their surface since before loading of DOX, the Am-CUR-PCL-PCA nanoparticles self-aggregated by noncovalent interactions such as intermolecular hydrogen bond formation between nanoparticles. These interactions could be reduced the particle sizes due to the presence of DOX in Am-CUR-PCLPCA-DOX. In fact, DOX could increase the intramolecular hydrogen bonds which results in formation of smaller and more monodisperse nanoparticles. The surface charge of Am-CUR-PCL-PCA and Am-CUR-PCL-PCA-DOX were detected by Zeta-potential analysis. As can be seen from Table 1, because of the presence of carboxyl functional groups with the negative charge at the surface of Am-CUR-PCL-PCA, the surface charge of this product was −9.30. But the surface charge of Am-CUR-PCL-PCA-DOX was − 6.20. After interaction of DOX with functional groups of AmCUR-PCL-PCA by noncovalent interaction, due to the presence NH+ 3 groups of DOX, the negative surface charge of Am-CUR-PCL-PCA nanoparticles reduced from −9.30 to −6.20.
Fig. 7. 1H NMR spectrum of (a) Am-CUR-PCL and (b) Am-CUR-PCL-PCA.
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13.04° and 19.99°, which correspond to the V helical amylose conformation (type I). The X-ray diffraction data confirm structural changes in amylose due to the inclusion complex formation with CUR through the amylose V-type polymorph.
Fig. 8. X-ray diffraction patterns of (a) Am and (b) Am-CUR.
3.1.5. NMR analysis The structure of Am-CUR-PCL and Am-CUR-PCL-PCA were evaluated by 1H NMR spectroscopy. Fig. 7a shows representative 1H NMR spectrum of Am-CUR-PCL. The signals which appeared at 3.4–5.6 ppm are attributed to the glycosidic rings of amylose. The signals at 1.2– 3.5 ppm are related to the PCL chains and confirm the presence of PCL chains in the product. Fig. 7b shows the 1H NMR spectrum of AmCUR-PCL-PCA. In this spectrum signals at 3.4–5.5 ppm are related to the glycosidic rings of amylose. The signals for the PCL chains appeared at 1.2–2.5 ppm and 3.9 ppm. The signals of methylene groups of the PCA blocks appeared at 2.6–2.8 ppm as the AB system. All of these data are in agreement with the above-mentioned FT-IR results.
3.1.6. X-ray diffraction The effect of inclusion complex formation on the crystal morphology of amylose biopolymer was investigated by X-ray diffraction. XRD pattern of Am showed obvious characteristic peaks at 2θ = 15.12°, 16.90°, 17.90 and 23.91° (Fig. 8a). XRD pattern of Am-CUR (Fig. 8b) showed high crystallinity with characteristic peaks at 2θ = 7.51°,
3.1.7. In-vitro drug release study The release of CUR and DOX anticancer drugs from synthesized nanomaterials were investigated by regular dialysis against PBS at 37 °C for a period of 72 h. The selected pHs were 5.4 and 7.4 which are the pHs of tumor cells and normal cells respectively. Fig. 9a shows the release of DOX at these two pHs. As can be seen from the figure, after 24 h about 78.163% of DOX was released at pH = 5.4 and 46.39% at pH = 7.4. After 72 h, approximately 81.05% of the drug was released at pH = 5.4 and 55.05% of it was released at pH = 7.4. The release of the encapsulated CUR from the drug-loaded nanomaterials was also investigated at pHs 5.4 and 7.4. Fig. 9b shows the release profile of CUR at these two pHs. As can be seen from the figure, after 24 h about 66.9% of CUR was released at pH = 5.4 and 48.5% at pH = 7.4. After 72 h, about 74.77% of the drug was released at pH = 5.4 and 53.78% of it was released at pH = 7.4. The drug release data confirm that the rate of release is dependence on variation in pH value. The rate of drug release increased by changing the pH from 7.4 to 5.4 for both drugs and is more favorable at acidic pH in both cases since tumor microenvironment is usually acidic. The higher rate of the drug's release from the nanomaterials at acidic pH is due to their lower stability in acidic conditions. Increased hydrophilicity, due to the protonation of polymer branches at acidic pH and also hydrolysis of the ester bonds of PCL and PCA bounded polymers, trigger dissociation of covalence bounds and faster release of the encapsulated drugs [33]. The release data also illustrated that DOX which is absorbed in the surface of synthesized nanomaterial would be released earlier and faster than CUR which is entrapped in the core of the system.
4. Conclusion In this research, amylose biopolymer was extracted from starch and formed inclusion complex with Cur. Then the surface of this complex modified with PCL and PCA respectively. Finally, DOX reacted with surface functional groups of Am-Cur-PCL-PCA by noncovalent interactions. The evidences of the occurrence of chemical modifications of the resulting nanoparticles were checked by FT-IR spectroscopy, UV–vis spectroscopy, scanning electron microscopy, DLS measurements etc. FE-SEM analyses and DLS measurements showed that the hydrodynamic dimensions of Am-Cur-PCL-PCA were about 50 nm. These new nanomaterials has potential application in different field such as drug delivery and cancer therapy.
Fig. 9. In vitro release profiles of DOX (a) and CUR (b) from Am-CUR-PCL-PCA-DOX nanomaterial at 37 °C.
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