Curcumin-Loaded Biodegradable Electrospun Fibers

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Curcumin-Loaded Biodegradable Electrospun Fibers: Preparation, Characterization, and Differences in Fiber Morphology a

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Priscilla P. Luz , Marcio L. A. e Silva & Juan P. Hinestroza

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Department of Fiber Science and Apparel Design , Cornell University , Ithaca , New York , USA b

Núcleo de Pesquisas em Ciências Exatas e Tecnológicas, Universidade de Franca, Franca , São Paulo , Brasil Accepted author version posted online: 20 Sep 2013.Published online: 25 Oct 2013.

To cite this article: Priscilla P. Luz , Marcio L. A. e Silva & Juan P. Hinestroza (2013) CurcuminLoaded Biodegradable Electrospun Fibers: Preparation, Characterization, and Differences in Fiber Morphology, International Journal of Polymer Analysis and Characterization, 18:7, 534-544, DOI: 10.1080/1023666X.2013.816207 To link to this article: http://dx.doi.org/10.1080/1023666X.2013.816207

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International Journal of Polymer Anal. Charact., 18: 534–544, 2013 Copyright # Taylor & Francis Group, LLC ISSN: 1023-666X print/1563-5341 online DOI: 10.1080/1023666X.2013.816207

Curcumin-Loaded Biodegradable Electrospun Fibers: Preparation, Characterization, and Differences in Fiber Morphology Priscilla P. Luz,1 Marcio L. A. e Silva,2 and Juan P. Hinestroza1 Downloaded by [Priscilla P. Luz] at 10:04 09 November 2013

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Department of Fiber Science and Apparel Design, Cornell University, Ithaca, New York, USA 2 Nućleo de Pesquisas em Cieˆncias Exatas e Tecnolo´gicas, Universidade de Franca, Franca, Saõ Paulo, Brasil This article relates curcumin incorporation into poly(latic acid) and poly(lactic-co-glycolic acid) fibers by electrospinning. The fibers were characterized by field emission scanning electron microscopy and Fourier transform-infrared spectroscopy, performed in attenuated total reflection, and the actual curcumin content was evaluated by electronic absorption spectroscopy. The poly(latic acid)=curcumin fibers were malleable, had rough surface, and an average diameter of 3.6 mm. On the other hand, poly(lactic-co-glycolic acid)=curcumin fibers were rigid and porous and had an average diameter of 123.6 nm. The bigger diameter of the poly(latic acid) fibers was responsible for a higher percentage of curcumin=milligram of fiber.

INTRODUCTION Fibers and nanofibers have been produced from a wide range of polymers, which make possible their application in many different areas such as filtration, affinity membranes and recovery of metal ions, tissue engineering scaffolds, wound healing, drug delivery systems (DDS), catalyst and enzyme carriers, sensors and energy storage.[1–3] Several techniques can be used for fiber production, but electrospinning, which was first developed in 1930s, is currently the most promising and applied technique to produce continuous fibers from polymer solutions or melts of both natural and synthetic polymers on a large scale. In addition, electrospinning is a versatile and relatively simple and fast process in which a charged fluid jet from a polymer solution or melt undergoes uniaxial stretching in the presence of an applied electric field and is deposited on a collector as fibers with adjustable diameters, from nanometers to few microns, just by controlling the spinning process parameters and physical properties of the solution.[4–6] The fibers obtained by electrospinning offer lots of advantages such as high surface area-to-volume ratios, Submitted 30 May 2013; accepted 8 June 2013. The authors thank FAPESP for the postdoctoral fellowship. This work made use of the LEO 1550 FE SEM facility of the Cornell Center for Materials Research (CCMR) with support from the National Science Foundation Materials Research Science and Engineering Centers (MRSEC) program (DMR 1120296). Correspondence: Priscilla P. Luz, Department of Fiber Science and Apparel Design, Cornell University, Ithaca, New York 14853, USA. E-mail: [email protected]


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porosity with excellent pore interconnection, malleability, and the possibility to fabricate structures with high strength or a wide variety of sizes and shapes.[1,7] Beyond their importance and widespread applications in tissue engineering, electrospun fibers have also been used as DDS and have gained increasing interest in the pharmaceutical area.[8] DDS should be able to lead a biologically active molecule at the desired rate and for the desired duration to the desired target, so as to maintain the drug level in the body at optimum therapeutic concentrations with minimum fluctuation, improving the therapeutic efficacy and safety of drugs.[9,10] Several types of DDS have been proposed along the years, from the most heavily applied, liposomes, to micelles, polymeric micro=nanoparticles, and magnetic nanoparticles.[10,11] The main advantage of electrospun fibers as drug delivery carriers are that they offer site-specific delivery of drugs to the body and exhibit a greater surface area that could allow molecules to diffuse out of the matrix readily due to the highly porous structure.[12] Electrospun polymeric fibers have been used as a carrier for various bioactive molecules. For example, Meng et al.[13,14] have incorporated Febunfen (a nonsteroidal anti-inflammatory drug) into PLGA=chitosan and PLGA=gelatin fibers. Chen et al.[10] developed a controlled release system for titanocene dichloride (a kind of inorganic antitumor agent) based on electrospun fiber and evaluated its vitro antitumor abilities. The possibility offered by the electrospinning method to incorporate dyes, bioactive molecules, and nanoparticles into fiber that can be made from a wide range of polymers, including biocompatible and=or biodegradable ones, has shown electrospun fiber as a promising DDS for anti-inflammatory, chemotherapeutic, antibacterial, and hormone drugs.[7,10,13–15] Biodegradable polymers are those that do not have to be removed after implantation, since they degrade in vitro and in vivo into nontoxic substances that are normal metabolites of the body or into products that can be completely eliminated from the body with or without further metabolic transformations.[9,16] Gelatin (a hydrolyzed form of collagen), chitosan (a deacetylated form of chitin), and other polysaccharides are common naturally biodegradable polymers used for DDS. Commonly applied synthetic polymers are polycaprolactone (PCL), polyethylene glycol (PEG), and the aliphatic polyesters such as poly lactic acid (PLA), poly(glycolic acid) (PGA), and their copolymer PLGA.[9,16] DDS have been widely applied for cancer treatment, for example, but few studies are available in the literature relating the employment of DDS for antiprotozoal drugs. In addition, the available works involve the use of DDS to treat leishmaniasis, malaria, and trypanosomiasis diseases, but even fewer address schistosomiasis.[17] Curcumin or [1,7-bis(4-hydroxy-3methoxyphenyl)-1,6-heptadiene-3,5-dione] (Figure 1) is a natural compound obtained from the rhizomes of Curcuma longa (C. longa). The biological activities of curcumin have been explored, and researchers have found that curcumin exhibits anti-inflammatory, antioxidant, antiviral, anti-infectious, and antitumoral properties.[18–22] Curcumin also possesses in vitro antiprotozoal activity, notably against leishmaniasis, giardiasis, and trypanosomiasis.[23,24] In addition, in vitro and in vivo effects of the oil extract of C. longa and curcumin against S. mansoni have been shown.[25–27]

FIGURE 1 Representation of the curcumin chemical structure obtained by ChemDraw Ultra, version 10.0.

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A small number of works have reported the incorporation of curcumin into biodegradable fibers by electrospinning. For example, Merrell et al.[28] incorporated curcumin into poly (e-caprolactone) nanofibers and explored its antioxidant and anti-inflammatory properties. Suwantong et al.[29] produced curcumin-loaded cellulose acetate fiber and also evaluated its antioxidant activity. Finally, Chen et al.[30] prepared a PLA=curcumin composite membrane by electrospinning and evaluated its anticoagulant property in vitro in order to develop drug-eluting stents to treat in-stent restenosis after stent implantation. The goal of this article is to incorporate curcumin into PLA and PLGA fibers, characterize the fibers, and evaluate differences in fiber morphology due to differences in the solution and electrospinning conditions. MATERIALS AND METHODS Chemicals PLA (Mw ¼ 300,000 g mol1) (Figure 2(a)) was purchased from Polyscience Inc. PLGA (Mw ¼ 50,000–75,000 g mol1) (Figure 2(b)), with a lactic acid=glycolic acid ratio of 85:15, curcumin, acetonitrile, and ethanol were purchased from Sigma Chemical Co. (St. Louis, Mo.). N, N-dimetylformanide (DMF) and methylene chloride (MC) were obtained from Mallinckrodt (Phillipsburg, N.J.). Preparation of PLA=Curcumin and PLGA=Curcumin Solutions PLA (4.2 wt.%) was added to an MC=DMF solvent mixture (1.86 v=v) and stirred with a magnetic stirrer for 4 h at room temperature until a homogeneous solution was obtained. Then, curcumin (2.0 mg mL1) was added to the PLA solution. For the PLGA=curcumin solution, PLGA (26.8 wt.%) was dissolved in acetonitrile at room temperature and under magnetic stirring. After that, the curcumin (8.3 mg mL1) was added to the PLGA solution. Electrospinning The solutions were placed in plastic syringes (6.0 mL) and injected through stainless-steel needles (0.7 and 0.5 mm inner diameter for PLA and PLGA solutions, respectively), which were connected to a high voltage power supply. Voltages of 20 and 13 kV for PLA and PLGA

FIGURE 2 Representation of (a) PLA and (b) PLGA chemical structures obtained by ChemDraw Ultra, version 10.0.


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solutions, respectively, were applied to the electrospinning process with a distance of 15 cm between the needle tips and the collector. A piece of aluminum foil was used as collector, and the feed rate of the electrospinning was kept at 1.2 and 0.9 mL h1 for PLA and PLGA solutions, respectively, by an infusion pump. In the course of electrospinning, the solvents evaporated and only fibers were attached on the aluminum foil.


Characterization The morphology of the electrospun PLA=curcumin and PLGA=curcumin fibers was observed by field emission-scanning electron microscopy (FE-SEM) (Gemini LEO 1550). All samples were coated with a thin layer of carbon in order to reduce charging and produce a conductive surface. The average diameter was determined from FE-SEM images by using Image J software. Fourier transform-infrared (FT-IR) spectroscopy was carried out in attenuated total reflection (ATR) mode using an infrared spectrophotometer (Nicolet Magna IR 560). The spectra were obtained in the range 690–4000 cm1, with a resolution of 2.0 cm1 and 130 scans. Actual Curcumin Content The total amount of curcumin-loaded electrospun PLA and PLGA fibers was determined in triplicate using specimens of approximately 1.0 mg. In the case of PLA=curcumin fibers, each sample was washed four times with ethanol in order to extract curcumin from the fibers. For PLGA=curcumin, the samples were also washed four times with ethanol. However, a visible amount of curcumin remained in the fibers, since a lighter yellowish color was observed. After drying, acetonitrile was added to the samples, leading to the complete dissolution of the PLGA fibers. Then, ethanol was added in order to precipitate PLGA, the resulting suspension was centrifuged, and curcumin was removed with the supernatant. For both fibers, the extracted curcumin was analyzed by absorption spectroscopy by using an ultraviolet-visible spectrophotometer (PerkinElmer Lambda 35). However, the curcumin extracts were concentrated and dilution steps were carried out before analyzing them by absorption spectroscopy. The amount of curcumin was calculated based on the absorbance values and its standard curve (absorbance at 427 nm versus molar concentration) in ethanol. This curve was constructed by the standard addition method using standard solutions of commercial curcumin in ethanol (concentrations 0.6 105 to 1.5 105 mol L1) and their respective absorbance values at 427 nm. RESULTS AND DISCUSSION Characterization The electrospinning of the PLA=curcumin and PLGA=curcumin solutions resulted in the formation of yellow fibers. Images of the yellowish fibers collected over the aluminum foil were acquired using a digital camera (Sony=Cyber-shot W370) and are shown in Figure 3. The yellowish color observed in Figure 3 is characteristic of curcumin, and it was well distributed through the fibers, since there was no visible aggregation of curcumin on the surface.

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FIGURE 3 Photographs of the yellowish fibers produced by electrospinning: (a) PLA=curcumin fibers and (b) PLGA=curcumin fibers (color figure available online).

The surface of PLGA=curcumin fibers collected on the aluminum foil was smoother than the surface of PLA=curcumin fibers and this better recovering was due to the smaller diameter of the PLGA fibers, which was confirmed by FE-SEM analysis. A small sample of each fiber was collected and its morphology (shape and size) was characterized by FE-SEM. The FE-SEM micrographs for PLA=curcumin and PLGA=curcumin fibers are shown in Figure 4(a)–(c) and 4(d)–(f), respectively. The FE-SEM technique revealed the formation of fibers for both polymers, and from the micrographs it is possible to see significant differences between the PLA=curcumin and PLGA=curcumin fibers. The PLA=curcumin fibers were malleable and exhibit a rough surface and an average diameter of 3.6 1.0 mm. On the other hand, the PLGA=curcumin fibers were porous and rigid, and have an average diameter of 123.6 26.8 nm. The PLGA=curcumin fibers were more than 29 times smaller than PLA=curcumin fibers. In addition, the average diameter obtained for curcumin-loaded PLGA electrospun fibers was also smaller than the values available in the literature for curcumin-loaded biodegradable polymeric fibers. For example, Merrell et al.[28] incorporated curcumin into PLC fibers with a broad diameter distribution (200–800 nm), while Suwantong et al.[29] collected curcumin-loaded CA fibers in the range between 314–60 and 340–98 nm, and Chen et al.[30] obtained nanofibers of PLA with average diameters from 756 to 971 nm. As is known, many parameters, including the electric field, solution properties, surface tension, resistivity, charge carried by the jet, and relaxation time can affect the electrospinning process.[31] In this study, the huge difference between the obtained PLA and PLGA fibers can be explained based on the solution properties, and more specifically on the solvent properties, such as dielectric constant and boiling point. The electrospinning process has three stages: (a) jet initiation and its extension along a straight line, (b) the growth of a bending instability and the further elongation of the jet, which allows the jet to become very long and thin, and (c) solidification of the jet into fibers.[31] The dielectric constant and boiling point affect the charge carrying and the elongation of the jet, respectively, and, as a consequence, they affect the fiber size. For PLA fibers, the used solvents were MC (major solvent: 65%) and DMF (35%). In the case of PLGA fibers, pure acetonitrile was used as solvent. The dielectric constants for MC, DMF, and acetonitrile are 8.51, 37.65, and 36.69, respectively.[32,33] As it is possible to see, MC has the lowest dielectric constant, and it is 4.3 times lower than the value for acetonitrile. This means the higher diameter of the PLA fibers was caused by the low dielectric constant of MC, which led to low electric flux density. The

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FIGURE 4 Morphology of the yellow fibers evaluated by FE-SEM: (a)–(c) PLA=curcumin fibers and (d)–(f) PLGA=curcumin fibers (color figure available online).

boiling points of the solvents were also an important contribution to the fiber diameter size. The boiling point values are 40 , 152 , and 82 C for MC, DMF, and acetonitrile, respectively. Even when using a solvent mixture during the electrospinning process for PLA fiber production, the MC boiling point had an important effect, since it was used in higher percentages, and its low boiling point contributed to the bigger PLA fiber size. The volatile solvents affect the fiber elongation stage, since the solvent evaporates before sufficient elongation. The result of this fast evaporation is the formation of larger fibers.[31] The PLA fibers present different sizes (Figure 4(a)) and the same is true of PLGA fibers (Figure 4(d)). It has been recognized that during the traveling of a solution jet from the needle


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onto the metal collector, the primary jet may or may not be split into multiple jets, resulting in different fiber diameters. Comparing the PLA=curcumin fibers produced in this work to the PLA=curcumin fibers obtained by Chen et al.[30] their fibers had a bigger diameter size and were more flexible. The solution properties and the electrospinning conditions (except the needle inner diameter) used in both works are different. All these differences in the process parameters led to fibers with different morphologies. The malleability of the PLA=curcumin fibers obtained in our work might be due to the employment of 35% DMF, which is a nonvolatile solvent. However, the fibers obtained by Chen et al.[30] are rigid, since they used a very volatile solvent mixture.[30] Our PLGA= curcumin fibers, obtained using acetonitrile (more volatile than DMF), were also rigid. The yellow color of the produced fibers is characteristic and a great indicator of curcumin in the fibers. However, the fibers were also characterized by ATR-FT-IR (Figure 5) in order

FIGURE 5 Characterization of the obtained fibers, (a) PLA=curcumin and (b) PLGA=curcumin, by FT-IR using the ATR mode. The PLA and PLGA pure fibers and commercial curcumin were used as standards. For both polymers the inset on the right corresponds to an amplification of a specific wavenumber region (1650–1400 cm1) of their FT-IR spectra. The dashed squares in the graphs and in their insets highlight the presence of a characteristic band of curcumin in the fibers and its absence in the polymer spectra. All graphics were obtained by Origin 8.0 software (color figure available online).



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to not only confirm the curcumin presence in the fibers but also to evaluate the existence or not of chemical interactions between the curcumin and the polymers. PLA and PLGA fibers without curcumin were prepared following the same procedure in order to be used as standard. Commercial curcumin was also used as standard for FT-IR interpretation. According to Figure 5, the PLA=curcumin and PLGA fiber spectra exhibited characteristic bands of PLA, PLGA, and curcumin. Both PLA and PLGA characteristic bands were C ¼ O stretching of aliphatic esther group at 1755 cm1 and C-O stretching at 1268 and 1180 cm1.[34] In addition, the highlighted absorption band at 1505 cm1 corresponded to C ¼ C stretching, which is a characteristic band of curcumin. This absorption band was present in the PLA=curcumin and PLGA=curcumin spectra.[35] As we can see, the features for PLA, PLGA, and curcumin remained the same after the electrospinning process, leading us to conclude the curcumin was only physically attached to the fibers.

Actual Curcumin Content The actual curcumin content in the PLA and PLGA fibers was determined by absorption spectroscopy. The absorbance values, obtained from the absorption spectra, and the curcumin standard curve were used to calculate the percentage of curcumin available in the fibers. The absorption spectra of curcumin extracted from the PLA and PLGA fibers are presented in Figure 6(a) and 6(b), respectively. The standard ethanol solutions of curcumin are shown in Figure 6(c), and their absorbance value at 427 nm was used to plot the standard curve shown in Figure 6(d). As mentioned above the experiment to determine curcumin content was realized in triplicate. As a consequence, three spectra are presented for each fiber (Figure 6(a) and 6(b)). According to the data, the percentage of curcumin per milligram of fiber was 4.0 0.2% for PLA=curcumin fibers and 1.8 0.1% for PLGA=curcumin fibers. As it is possible to see, the percentage of curcumin incorporated in PLA fibers was more than two times higher than the percentage in PLGA fibers and the reason for this difference might be based on the different average diameters. The PLA=curcumin fibers were considerable bigger than the PLGA=curcumin fibers and due the higher diameter the PLA fibers were able to incorporate higher mass of curcumin. Due to the application of a high electrical potential to the PLA=curcumin and PLGA=curcumin solutions during electrospinning, the chemical integrity of curcumin can be questionable. Would curcumin be intact after such a treatment? The first indicative that curcumin was not affected by the electrical potential was the presence of a curcumin band in the ATR-FT-IR spectra (Figure 5(a) and 5(b)). In addition, the determination of the curcumin content in the PLA and PLGA fibers also enabled investigation of the curcumin integrity. Comparing the spectra of curcumin extracted from the fibers (Figure 6(a) and (b)) to the spectra of standard solutions of commercial curcumin (Figure 6(c)), it is possible to see that curcumin was not degraded nor even suffered structural changes due the applied electrical field required for electrospinning, since the same absorption spectrum was observed. Suwantong et al.[29] also verified the curcumin integrity after electrospinning. In their work the curcumin was incorporated in cellulose acetate fibers mats, and they applied proton nuclear magnetic resonance to analyze the curcumin integrity. They also found the curcumin was not affected by the electrical field.


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FIGURE 6 Electronic absorption spectra of curcumin extracted from (a) PLA fibers and (b) PLGA fibers. The three curves for each fiber indicate the triplicate experiments. (c) Absorption measurements of the standard ethanol solutions of commercial curcumin by electronic absorption spectroscopy. (d) Curcumin standard curve in ethanol acquired using the curcumin absorbance values at 427 nm. All graphics were obtained by Origin 8.0 software (color figure available online).

CONCLUSIONS Curcumin was successfully incorporated into PLA and PLGA fibers by electrospinning. The PLA=curcumin fibers were very different from the PLGA=curcumin fibers. The morphological characteristics were specially affected by the solvent properties, such as dielectric constant and boiling point. In both fibers, the curcumin did not suffer any damage during the electrospinning process as confirmed by the spectroscopic properties of curcumin, and the higher diameter size of the PLA=curcumin fibers was responsible for the higher incorporation of curcumin than with PLGA=curcumin fibers. REFERENCES 1. Leunga, V., and F. Ko. 2011. Biomedical applications of nanofibers. Polym. Adv. Technol. 22: 350–365. 2. Jian, F., N. H. Tao, L. Tong, and W. X. Gai. 2008. Applications of electrospun nanofibers. Chin. Sci. Bull. 53: 2265–2286.



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