hydroxyapatite hydrogels: Drug uptake and drug delivery systems

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ScienceDirect Materials Today: Proceedings 5 (2018) 15990–15997

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NN17

Radiation synthesis and characterization of chitosan/hyraluronic acid/hydroxyapatite hydrogels: drug uptake and drug delivery systems Betül Taşdelena*,Sevil Erdoğanb, Bahadır Bekarc a

Çorlu Engineering Faculty, Biomedical Engineering Department, Namık Kemal University, No:13 Tekirdağ 59860, Turkey b

Laborant and Veterinary Programme, Keşan Vocational School, Trakya University, 22800 Keşan, Edirne/TURKEY c

Electricity Programme, Keşan Vocational School, Trakya University, 22800 Keşan, Edirne/TURKEY

Abstract In this work, a new chitosan (CS)/hyaluronic acid (HA)/hydroxyapatite (HAP) hydrogels were synthesized by using gamma rays irradiation technique for oral delivery of drugs. The hydrogelss were characterized using fourier transform infrared spectroscopy (FTIR) and the physicochemical properties of shrimp chitosan was determined with both FTIR and scanning electron microscopy (SEM). The use of the hydrogel samples as a drug delivery system was investigated by an anticancer drug. 5-Fluorouracil (5-FU) was used as a model anticancer drug to investigate the drug uptake and release kinetics of hydrogels. The properties of the hydrogels were evaluated in terms of swelling, drug uptake and release behaviours. The addition of hyaluronic acid and hydroxyapatite in the gel structure improved drug uptake and release capability of the new hydrogels. © 2018 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of 14th International Conference on Nanosciences & Nanotechnologies (NN17). Keywords: Hydrogels; chitosan; hyaluronic acid; hydroxiapatite; drug delivery.

* Corresponding author. Tel.: +0-282-250-2348; fax: +0-282-250-2348. E-mail address: [email protected] 2214-7853 © 2018 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of 14th International Conference on Nanosciences & Nanotechnologies (NN17).

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1. Introduction Chemotherapy and surgical operations are the main treatment methods used for fighting against cancer which is the most serious health problem at the present time. However, it is reported in a study by Wang et al. [1] that some chemotherapeutic drugs have several disadvantages such as toxicity, non-selective distribution and unexpected adverse effects on normal tissues and these disadvantages limited the clinical use of them. As an effective drug delivery system, nanoparticles (NPs) can help to overcome these problems due to their novel properties. There are many studies reporting the use of nanoparticles in biomedical applications including cancer treatment [2,3]. Recent papers revealed that nanoparticles enhance the accumulation of drugs in tumor tissue and control the drug release characteristics [1]. Generally, hydrogels were obtained by cross-linking process of polymers which might be done with physical reactions or with chemical reactions such as free-radical polymerization, high energy irradiation, enzymatic reaction and chemical reaction of complementary groups [4]. Hydrogel production by crosslinking the polymers within an aqueous solution using gamma radiation polymerization technic is highly preferred due to its unique properties. Because no residual monomer that may be toxic is left in the structure, the use of this technic is rather important in terms of biomedical applications. Moreover, radiation processing has two unique advantages such as simultaneous synthesis and sterilization of hydrogel.The use of gamma radiation process to produce cross-linked hydrogels was also reported in another study by Jagur-Grodzinzki [5]. Chitosan is deacetylated form of chitin which is one of the most common biomaterials in nature. Due to its biocompatible, biodegradable and non-toxic properties, chitosan is also widely used in biomedical applications, especially as a carrier of therapeutic agents for drug delivery [6]. Chitosan exhibits a cationic character because of its primary amine groups. This cationic character provides chitosan various properties which makes it one of the best choice for drug release and uptake [7]. Ahmadi et al. [8] reported that cross-linking improves the properties of chitosan such as stability and durability and the chitosan derivatives preperad with different strategies can be a good carrier for pharmaceuticals. In this study, chitosan from shrimp was participated in chitosan/hyaluronic acid/hydroxyapatite hydrogels. Chitosan is commercially obtain from macro crustaceans such as crab and shrimp. The chitosan used in this study, was produced from small shrimp species which are sold as a fish feed in the market. Hyaluronic acid (HA) is a non-sulphated glycosaminoglycan which constitute one of the chief components of the extracellular matrix. In addition to that, it also plays a significant role in cell proliferation and migration and tissue vascularization [9]. Hyaluronic acid has drawn much attention recently as a drug delivery devices. Some studies remarked that hyaluronic acid is one of a number of molecules might be used in gel preparations for drug delivery [10]. Partner molecules include N-isopropylacrylamide, polyacrylic acid, alginic acid and cellulose. Giji [11] suggested that different biophysical properties and pharmacokinetics can be obtained by combining these materials in various forms. Hydroxyapatite is an inorganic compound used in tissue engineering due to its unique properties, such as bioactivity, biocompatibility and osteoconductivity. Commercially available hydroxyapatite is chemically and structurally similar to the mineral phase of human bone. Moreover, it increases the mechanical properties of several polymeric materials. Nanoparticles of hydroxyapatite (HAP) have low crystalline particles exhibiting highly active surface [12]. Because of these properties, nano-HAP has various uses in the medical field as a genetic carrier [13], as a biological medicine for hepatic tumor and in controlled drug delivery [14]. HAP nanoparticles have much higher anti-tumor effect in comparison to the macromolecules showing minimal side effect and it can provide adjuvant effect with chemotheraphy drugs and become less toxic when used together [15]. 5-FU is known as an effective supportive chemotherapy used to treat brain tumor and various cancer types like colorectal, stomach, breast, liver, pancreatic and lung cancers [16]. This study aimed 1) to characterize the shrimp chitosan and chitosan/hyaluronic acid/hydroxyapatite hydrogels synthesized by using gamma rays irradiation technique with fourier transform infrared spectroscopy (FT-IR) and scanning electron microscope (SEM) and also 2) to test the drug uptake and release capacities of this hydrogels as a drug delivery system with 5-Fluorouracil (5-FU).

2. Experimental 2.1. Material Hydroxiapatite, hyaluronic acid and 5-FU (99%) were obtained from Sigma Aldrich Chemical Company. All the reagents mentioned above were used as received.

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Small shrimp species, sold as a fish feed were bought from the market and their shells used for chitosan production. Before used, the shrimps were cleaned and their shells were separated. Then, shrimp shells were dried at 50 °C in an owen for 24 hours.

2.2. Apparatus The concentration of 5-FU was measured by Shimadzu UV-Visible spectrophotometer (Shimadzu UV-2401). The pH was measured using a pH meter (WTW pH 315i). The chemical and surface characteristics of the chitosan and hydrogels produced was characterized using scanning electron microscopy (SEM) (Zeiss EVO® LS 10) and fourier transform infrared spectroscopy (FTIR) (Perkin Elmer ATR and Bruker VERTEX 70 ATR) methods. 2.3. Chitin extraction and chitosan production Dried shrimp shells (17,5 g) was crashed and prepared for chitin isolation. Chitin was extracted in three steps including demineralization, deproteinization and decolorization. In demineralization step, shrimp shells were refluxed with 2M, 250 mL of HCl at 75 °C by stirring for 4 h, at 1000 rpm to remove the minerals from the structure. Then, it was rinsed with distilled water until reaching the pH 7 and filtered. After that, proteins in the structure were removed by refluxing the filtrate with 2M 150 mL NaOH at 65 °C for 18 hours. Later, this alkaline mixture was filtered by rinsing with pure water until a pH of 7 was reached. At the end of this step, the filtrate was subjected to decolorization. For this, filtrate was kept in a mixture consisting of 10 mL kloroform, 20 mL methanol and 40 mL distilled water at room temperature for 20 minutes. After washing and filtering again, isolated chitin sample was put in an owen for drying. As a result of these process, a total of 4,279 g of dry weight chitin was obtained. Chitosan was produced from this dry chitin sample by deacetylation. Deacetylation step was achieved by treating the dry chitin sample with 70% NaOH at 150 °C for 2,5 hours. Then, alkaline mixture was filtered through the filter paper and rinsed with pure water until the NaOH residuals was completely removed from the sample. After reaching a neutral pH, the obtained chitosan sample was taken and dried in the owen at 50°C for 24 hour. 2.4 Preparation of chitosan hydrogels Chitosan hydrogel was synthesized by varied concentration of glutaraldehyde as a cross-linker. Briefly, chitosan (2 g) was dissolved in 1% acetic acid (100 mL) and stirred overnight. Glutaraldehyde was diluted with 1% acetic acid solution to prepare hydrogel of with glutaraldehyde concentration (0.4 wt. %). hyaluronic acid was added to the8 mL chitosan solutions (2 wt.%) in different weight ratios from10 wt.% to 50 wt.%. After

2.4. Synthesis of HA/CS/HAP hydrogel Hydrogel synthesis started by adding HA in different weight ratios (1-3% wt/v) aqueous solution on to the 5 mL of CS (1% w/v) sample solved in 1% acetic acid solution. Later, a certain amount of glutaraldehyde (0.4 wt. %) was added to HA/CS mixture. After that, definite amounts of HAP powder (10 mg (1), 20 mg (2) and 30 mg (3)) were poured into vessels containing 2 mL of HA/CS solution and next, the mixture including all these solutions was stirred at room temperature using a magnetic stirrer. The prepared solutions were put in glass tubes (5 mm inner diameter) and stoppered. Irradiations of all solutions were performed with a Nordion-Canada model JS 9600 model gamma irradiator in GammaPak Ind &Trade Inc under air at 25°C. A total of 25 kGy dose was absorbed (at a dose rate of 3 kGy/h). The crude hydrogels were kept in water used as the extraction solvent at 25 °C. When polymerization completed, cross-linked copolymers were taken out from tubes and the produced hydrogels were divided into pieces of 1 cm. Each gel pieces was put in water for approximately one week period and the water was replaced every other day until remaining no extractable polymer. This extraction process helps to remove residual monomers and uncross-linked polymers from the gel. Extracted gels were stored in vacuum oven (30C) until reaching to a constant dry weight before calculating the gel fraction. The gel fraction was determined with this given formula from the results measured gravimetrically [17]. Gelation % = Wg/Wo x 100% Wg, represents the weight of sample after extraction.Wo represents the weight of sample before extraction.

(1)

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2.5. Swelling Measurements Previously dried hydrogels which have the length of 1 cm and diameter of 5 mm were submerged in vials of 100 mL containing distilled water or aqueous solutions of 5-FU (130 ppm). The vials were put in a temperature-controlled waterbath (250.1C), and the gels were kept in pure water until reaching the equilibrium degree of swelling (for a week). The following equations were used in calculating the mass swelling and equilibrium mass swelling percentages of hydrogels. Mass swelling (%) = [(mt-mo)/ m0] x 100……………………………………………………….......(2) Equilibrium mass swelling (Seq%) = [(m-mo)/mo] x 100…………………………………………(3) m0: the mass of the dry gel, mt : the mass of swollen gel at time t and m: the mass of swollen gel at equilibrium.

2.6. Drug Loading and Release Tests First, 5-FU which is used to test drug loading and release behaviors of hydrogels was prepared in phosphate buffer at pH 7.4 at room temperature. Then, the dry gels were equilibrated in 130 ppm (mg/L) of 5-FU for a week. After a week of incubation, the polymer fragments were taken out of the solution and rinsed with buffer. Later, they were transferred in a vessel containing 10 mL of phosphate buffer (pH 7.4 and 37°C) and placed on a shaker with a constant shaking rate. 3 mL aliquots were taken from the medium from time to time to check the 5-FU release and they were returned to the same vessel to save the constant volume. The amount of 5-FU released from copolymeric hydrogels was determined with a UV spectrophotometer (Shimadzu UV-2401 model) at 266 nm. Percentage release of the drug from hydrogels was determined using the formula given below: % Release = Wt/Wtotal x 100…………………………………………………………………….(4) Wt represents the weight of released drug in water at any time, Wtotal represents the initial total weight of the drug taken by the gel system.

3. Results and Discussion 3.1 FTIR and SEM analysis of hyrogels FT-IR spectrum of pure chitosan hydrogel is shown in Fig. 1a. The peak at 3288 cm-1 is a broad band and corresponding to hydroxyl groups (OH) and hydrogen bond formation that occurred within the chitosan. The characteristic C=N imine peaks can be seen at around 1643.9 cm-1 for the glutaraldehyde cross-linked chitosan hydrogel. This band proves that carbonyl (C=O) group of glutaraldehyde and the amine (-NH2) group of the chitosan are reacted. In Fig. 1b, the sp3 hybridized C-H stretching vibration band from the main peaks in the polymer backbone was recorded in two peaks at 2918.57 and 2872.86 cm-1. The broad band appears at 3273 and 3266 cm-1 are hydroxyl groups (O-H) and hydrogen bond formation that occurred within the uncross-linked hyaluronic acid and chitosan-co-hyaluronic acid hydrogel, respectively. The absence of peak in the 1721 cm-1 region indicated the absence of unreacted carbonyl group. The peak observed at 603 cm-1 represents the vibration of hydroxyl ions in HAP. While, the characteristics bands showing phosphate bending vibration in HAP was located at 1026 and 564 cm-1, the bands representing the phosphate stretching and vibration was observed at 1060.7 and 897.63 cm-1. The absence of the characteristics absorption peak located at 1170 cm-1 indicated that HA/CS gel complex occurred owing to the ionic interaction between the negatively charged HA and positively charged CS amino group. All the characteristic peaks appeared in the IR spectra of HAP indicated that any chemical reaction did not occurred between HAP and the HA/CS. Surface characteristics of shrimp chitosan was determined with SEM analysis (Fig. 2a). Analysis results indicated that shrimp chitosan has firm and smooth surface morphology. Surface morphology of chitosan can vary according to the animal species. Yen et al. [19] reported a surface morphology consisting of nanofibers from crab shells. In another study, porous and fibrous chitin and chitosan surfaces was recorded for insects [20]. Variety in surface morphology expands the use of chitin and chitosan in different areas. In Fig 2b, SEM clearly showed the inorganic hydroxyapatite materials are dispersed in polymer matrices.

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Fig. 1a. FT-IR spectra of shrimp chitosan hydrogel.

Fig. 1b FT-IR spectra of chitosan-hyaluronic acid-hydroxyapatite hydrogel.

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(a) (b) Fig. 2. SEM images of shrimp chitosan (a) and chitosan-hyaluronic acid-hydroxyapatite hydrogel.

3.2 Gelation percent (%) In this study, the hydrogels were synthesized by gamma-ray irradiation. Exposure of the HA/CS/HAP mixture to gamma rays led to the formation of free radicals in aqueous solution. As a result of random collision between free radicals and monomers, the cross-linked HA/CS/HAP hydrogels occurred. Table 1 shows the effects of the gel fraction on the amount of HAP in the prepared hydrogel. Results was revealed that an increase in HAP content leads to an increase in gel fraction. This stems from the ability of HAP to form H bonding by the –OH groups providing a cross-linked structure. Table 1. The effect of HAP content on the gelation percent (%)

Gel name

Gelation (%)

HA/CS/HAP-1 HA/CS/HAP-2 HA/CS/HAP-3

84.5 90.8 94.2

*added amounts of HAP to 2 mL HA/CS solution are 10 mg (1), 20 mg (2) and 30 mg (3) respectively.

3.3. Swelling The synthesized hydrogels have high water retention capacities due to the structures of the chitosan and hyaluronic acid copolymers. In the structure of chitosan and hyaluronic acid, there are amine and hydroxyl groups, which have high ability to make hydrogen bonds. As a result, the water holding ability of these materials is high. Cross-linked chitosan hydrogel with 0.4% glutaraldehyde swells when immersed into the aqueous solution, reaches a maximum swelling of about 537% within 24 hours and reaches equilibrium. The amount of hyaluronic acid added to the chitosan hydrogels has caused a visible increase in swelling rate. Swelling of hydrogels containing 10 wt% of hyaluronic acid was found to be 860%. The reason is that by adding hyaluronic acid the pore size is enlarged, the porous structure gains flexibility and the hyaluronic acid has a high water holding capacity. The addition of HAP content increased the gelation %. Equilibrium swelling of HA/CS/HAP hydrogels were decreased to 557% because the inorganic HAP platelets acted as multifunctional crosslinkers with HA/CS chains linked on them. The higher HAP contents caused to the formation of more densely cross-linked networks, and this was resulted in lower swelling ratios of hydrogels.

3.3 Drug loading and drug release For the investigation of drug uptake behavior of HA/CS/HAP hydrogels, we measured the amount of adsorbate per unit mass of adsorbent (qe) and thus determined the drug uptake capacities of hydrogels. qe values (mg/g) were calculated according to the formula given below [21].

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=

−

∗

/ …………….(5)

In this equation, Ci represents the initial concentration of solution of adsorbate, C represents the equilibrium concentration of solution of adsorbate, Vt represents the volume of solution treated, and M represents the mass of dry adsorbent. Adsorption capacities of 5-FU were found to be increasing from 3.2 to 12.74 mg/g with the addition of hyaluronic acid in the gel structure. Table 2 shows variation of 5-FU uptake with HA content in the gel composite. In the structure of chitosan and hyaluronic acid, there are amine and hydroxyl groups, which have high ability to make hydrogen bonds. As a result, the water holding and drug uptake abilities of the prepared hydrogels is high. Table 2.Variation of 5-FU uptake with HA content in the gel structure.

Gel name

5-FU uptake (mg/g dry gel)

Pure Chitosan HA/CS/HAP-1 HA/CS/HAP-2 HA/CS/HAP-3

3.20 8.52 10.88 12.74

*added amounts of HA (wt %) to 5 mL CS solution are %1 (1), 2% (2) and 3 % (3), respectively. Added amount of HAP to 2 mL HA/CS solution are 30 mg.

Fig. 3 shows the release percent of 5-FU in HA/CS/HAP hydrogels in water (37ºC). As can be observed from Fig. 3, 5-FU release of all gels rapidly increased at the start and later gradually reached the equilibrium value within 24 h. The release 5-FU amount of hydrogels increased depending on the increase in the amount of HAP in gel structure. The reason for this is the increase in cross-linking density due to multi-functional effect of HAP in the gel structure and subsequently, the low free volume in the hydrogel.

5-FU release (mg/g dry gel)

8 HA/CS/HAP-1

7

HA/CS/HAP-2

6

HA/CS/HAP-3

5 4 3 2 1 0 0

500

1000

1500

time (min) Fig. 3 Release profiles of 5-FU from HA/CS/HAP hydrogels prepared with the dose of 48 kGy in phosphate buffer solution (pH 7.4 and 37ºC).

4. Conclusion A new chitosan (CS)/hyaluronic acid (HA)/hydroxyapatite (HAP) hydrogel was successfully synthesized by using gamma rays irradiation technique for oral delivery of drugs. FTIR analysis confirmed that HA/CS/HAP hydrogel was successfully prepared. The amount of hyaluronic acid added to the chitosan hydrogels has caused a visible increase in

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swelling rate (up to 860%). The gelation % increased depending on the addition of HAP content. Besides, the addition of hyaluronic acid and hydroxyapatite in the gel structure improved drug uptake and release capability of the new hydrogels. Adsorption capacities of 5-FU were found to be increasing from 3.2 to 12.74 mg/g with the addition of HA in the gel structure. The release 5-FU amount of hydrogels increased depending on the increase in the amount of HAP in gel structure. The results indicated that the HA/CS/HAP hydrogel may be an appropriate alternative for drug release processes in human body.

Acknowledgements The authors acknowledge Namık Kemal University Scientific Research Project (NKUBAP.00.17.AR.14.14) for funding. References [1] T. Wang, J. Hou, C. Su, L. Zhao, Y. Shi, Journal of Nanobiotechnology 15 (2017) 1-12. [2] D.Peer, J.M. Karp, S. Hong, O.C. Farokhzad, R. Margalit, R. Langer, Nature Nanotechnology 2 (2007) 751-760. [3] J.O. Kim, A.V. Kabanov, T.K. Bronich, Journal of Controlled Release 138 (2009) 197-204. [4] W.E. Hennink, C.F. Nostrum, Advanced Drug Delivery Reviews 54 (2002) 13-36. [5] J. Jagur-Grodzinski, Polymers for Advanced Technologies 21 (2010) 27-47. [6] Y. Yang, S. Wang, Y. Wang, X. Wang, Q. Wang, M. Chen, Biotechnology Advances 32(2014) 1301-1316. [7] A. Bernkop-Schnürch, S. Dünnhaupt, European Journal of Pharmaceutics and Biopharmaceutics 81 (2012) 463-469. [8] F. Ahmadi, Z. Oveisi,S.M. Samani, Z. Amoozgar, Research in Pharmaceutical Sciences 10(2015): 1-16. [9] Y. Parajó, I. d’Angelo, A. Welle, M. Garcia-Fuentes, M.J.Alonso, Drug Delivery 17(2010): 596-604. [10] G. Sadhasivam, A. Muthuvel, A. Pachaiyappan, B. Thangavel, International Journal of Biological Macromolecules 54 (2013) 84-89. [11] S. Fiji, Journal of Bioequivalence & Bioavailability 5 (2013) 209-214. [12] E.I. Abdel-Gawad, S.A. Awad, Nature and Science 8 (2010) 234-244. [13] I.W. Bauer, S.P. Li, Y.C. Han, L.Yuan, M.Z. Yin, 19(2008): 1091-1095. [14] J. Hu, Z.S. Liu, S.L. Tang, Y. He, World Journal of Gastroenterology 13(2007) 2798-2802. [15] M.F.A. Taleb, A. Al Kahtani, S.K. Mohamed, Polym Bull 72 (2015) 725-742. [16] A. Babul Reddy, B. Manjula, T. Jayaramudu, E.R. Sadiku, P. Anand Babu, S. Periyar Selvam, Nano-Micro Letters 8 (2016): 260-269. [17] M.T. Razzak, D. Darwis, Radiation Physics and Chemistry 62 (2001) 107-113. [18] S. Chatterjee, M. Adhya, A.K. Guha, B.P. Chatterjee, Process Biochemistry 40 (2005) 395-400. [19] M.T. Yen, J.H. Yang, J.L. Mau, Carbohydrate Polymers 75 (2009) 15–21. [20] S. Erdogan, M. Kaya, International Journal of Biological Macromolecules 89 (2016) 118–126. [21] M. Sen, C. Uzun,, O. Güven, International Journal of Pharmaceutics 203 (2000) 149-157.