Synthesis of Chitosan Based Drug Delivery System and Evaluation Its Performance In Vitro

Abstract

The aim of the present study is to synthesize, characterize chitosan microspheres that can be used as bone-filling material and drug carrier system, and to evaluate its performance in vitro. For this purpose; chitosan-based microspheres were synthesized by the emulsion cross-linking method and factors such as stirring rate and cross-linker that may affect the size, shape, and drug loading efficiency of microspheres were examined. Antibiotic ciprofloxacin that is used in the treatment of bacterial infection was preferred for drug studies. Furthermore, simulated body fluid (SBF) was prepared to determine the bioactivity of the microspheres, and hydroxyapatite (HA) precipitation on microspheres was followed for 3 weeks in SBF. Microspheres were collected from SBF weekly and characterized using light microscopy, Scanning Electron Microscopy (SEM), and Fourier Transform Infrared Spectroscopy (FTIR). According to the light microscope and SEM analysis, the sphere dimensions decreased as the stirring rate increased and decreased from 102,27 ± 34,58 µm to 16,38 ± 3,26 µm. Low amount of cross-linking agent ( glutaraldehyde (GA)), (NH2: CHO, 10: 1 mol: mol) caused distortion in the shape of microspheres, while the high amount of GA (NH2: CHO, 1:10 mol: mol) caused smooth microspheres, a reduction in the size and drug loading efficiency, and a slowdown in the drug release rate. According to the SEM images of the microspheres incubated with SBF, HA structures began to precipitate on the surfaces after the first week, and these structures became more pronounced in the third week. PO4-3 groups attributed to HA precipitation in FTIR spectrum were more obvious at week 3. In conclusion; antibiotic ciprofloxacin loaded chitosan microspheres were synthesized successfully by the emulsion crosslinking method, and the effect of stirring rate, crosslinking agent GA amounts on microsphere size, shape, and release profiles were demonstrated. Enhanced HA precipitation was shown on chitosan microspheres in bioactivity tests. Hence, chitosan microspheres may be used as bone-filling material that can release biomolecules to damaged sites locally, or as coating to modify the surfaces of implant materials.

Keywords

• Chitosan
• Drug Release
• Bioactivity

Kitosan Esaslı İlaç Taşıyıcı Sistem Üretimi ve In vitro Performansının Belirlenmesi

Öz

Sunulan çalışmanın amacı kemik dolgu malzemesi ve ilaç taşıma sistemi olarak kullanılabilecek kitosan mikro kürelerin üretimi, karakterizasyonu ve performansının in vitro olarak belirlenmesidir. Bu amaçla; kitosan esaslı mikro küreler emülsiyon çarpraz bağlama yöntemi ile üretilmiş olup, mikro kürelerin boyut, şekil ve ilaç yükleme verimliliklerine etki eden karıştırıcı hızı, çapraz bağlama ajanı gibi faktörler incelenmiştir. İlaç çalışmalarında kullanılmak üzere bakteriyel enfeksiyonların tedavisinde kullanılan antibiyotik ciprofloxacin tercih edilmiştir. Ayrıca, hazırlanan mikro kürelerin biyoaktivitelerini test etmek için yapay vücut sıvısı (SBF) hazırlanmış ve 3 hafta boyunca mikro küreler SBF içerisinde bekletilerek hidroksiapatit çökelmesi (HA) takip edilmiştir. SBF içerisine konulan mikro küreler haftalık olarak toplanmış, Taramalı Elektron Mikroskopu (SEM) ve Fourier Dönüşümlü Kızılötesi Spektroskopisi (FTIR) ile analiz edilmiştir. Işık Mikroskopu ve SEM analizlerine göre küre boyutları karıştırma hızı arttıkça azalmış ve 102,27 ± 34,58 µm den 16,38 ± 3,26 µm ye kadar düşmüştür. Düşük miktarda kullanılan çapraz bağlayacı ajan glutaraldehit (GA) (NH2:CHO, 10:1 mol:mol) ise küre şekillerinde bozukluğa neden olurken, yüksek miktarda GA kullanımı (NH2:CHO, 1:10 mol:mol) kürelerin şeklinde iyileşmeye, küre boyutunda ve ilaç yükleme verimliliğininde azalmaya, ilaç salım hızında ise yavaşlamaya neden olmuştur. SBF ile inkübe edilen mikro kürelerin SEM görüntülerine göre HA yapıları 1. haftadan sonra yüzeylerde birikmeye başlamış, bu yapılar 3. haftada yüzeylerde daha belirgin hale gelmiştir. FTIR analizleri de HA çökelmesini işaret eden PO4-3 gruplarının 3. haftada daha belirgin olduğunu göstermiştir. Bu sonuçlara göre antibiyotik ciprofloxacin yüklü kitosan mikro küreler emülsiyon çapraz bağlama yöntemi ile başarılı bir şekilde üretilmiş olup, karıştırma hızı ve çapraz bağlayıcı ajanı GA miktarlarının mikro küre boyut, şekil ve salım profillerine etkisi ortaya konmuştur. Biyoktivite testlerinde kitosan mikro kürelerin, osteoentegrasyonu arttırma potansiyelinin olduğu gösterilmiştir. Dolayısıyla kitosan mikro küreler, kemik doku hasarlarında lokal olarak biyomoleküllerin salımını yapabilen dolgu malzemesi olarak veya implant malzemelerin yüzeylerinin modifikasyonunda kullanılabilir.

Anahtar Kelimeler

• Kitosan
• İlaç Salımı
• Biyoaktivite

References

1. M.J. Rathbone, R. Gurny, Controlled Release Veterinary Drug Delivery- Biological and Pharmaceutical Considerations, 2013. doi:10.1017/CBO9781107415324.004.
2. B.K. Nanjwade, J. Singh, K. Ahmad, F. V. Manvi, Bio-pharmaceuticals: Emerging proniosomes drug delivery, in: Biodegrad. Polym. Process. Degrad. Appl., 2011.
3. A. Seyfoddin, R. Al-Kassas, Biodegradable polymers for controlled delivery of bioactive macromolecules, in: Biodegrad. Polym. Process. Degrad. Appl., 2011.
4. B.G. Zanetti-Ramos, Biopolymers Employed in Drug Delivery, in: Biopolym. Biomed. Environ. Appl., 2011. doi:10.1002/9781118164792.
5. S. Şenel, S.J. McClure, Potential applications of chitosan in veterinary medicine, Adv. Drug Deliv. Rev. (2004). doi:10.1016/j.addr.2004.02.007.
6. A. Di Martino, M. Sittinger, M. V. Risbud, Chitosan: A versatile biopolymer for orthopaedic tissue-engineering, Biomaterials. (2005). doi:10.1016/j.biomaterials.2005.03.016.
7. E. Khor, L.Y. Lim, Implantable applications of chitin and chitosan, Biomaterials. (2003). doi:10.1016/S0142-9612(03)00026-7.
8. M. Dash, F. Chiellini, R.M. Ottenbrite, E. Chiellini, Chitosan - A versatile semi-synthetic polymer in biomedical applications, Prog. Polym. Sci. (2011). doi:10.1016/j.progpolymsci.2011.02.001.

9. K.B. Dias, D. Pereira Da Silva, L.A. Ferreira, R.R. Fidelis, J. Da Luz Costa, A.L. Lopes Da Silva, G.N. Scheidt, Chitin and chitosan: Characteristics, uses and production current perspectives, J. Biotec. Biodivers. (2013).
10. M. Thanou, J.C. Verhoef, H.E. Junginger, Oral drug absorption enhancement by chitosan and its derivatives, Adv. Drug Deliv. Rev. (2001). doi:10.1016/S0169-409X(01)00231-9.
11. I. Wedmore, J.G. McManus, A.E. Pusateri, J.B. Holcomb, A special report on the chitosan-based hemostatic dressing: Experience in current combat operations, J. Trauma - Inj. Infect. Crit. Care. (2006). doi:10.1097/01.ta.0000199392.91772.44.
12. L. Illum, Chitosan and its use as a pharmaceutical excipient, Pharm. Res. (1998). doi:10.1023/A:1011929016601.
13. V.R. Sinha, A.K. Singla, S. Wadhawan, R. Kaushik, R. Kumria, K. Bansal, S. Dhawan, Chitosan microspheres as a potential carrier for drugs, Int. J. Pharm. (2004). doi:10.1016/j.ijpharm.2003.12.026.
14. S.A. Agnihotri, N.N. Mallikarjuna, T.M. Aminabhavi, Recent advances on chitosan-based micro- and nanoparticles in drug delivery, J. Control. Release. (2004). doi:10.1016/j.jconrel.2004.08.010.
15. J. Akbuǧa, G. Durmaz, Preparation and evaluation of cross-linked chitosan microspheres containing furosemide, Int. J. Pharm. (1994). doi:10.1016/0378-5173(94)90344-1.
16. C. Mattu, A. Silvestri, T.R. Wang, M. Boffito, E. Ranzato, C. Cassino, G. Ciofani, G. Ciardelli, Surface-functionalized polyurethane nanoparticles for targeted cancer therapy, Polym. Int. (2016). doi:10.1002/pi.5094.
17. D. Bhattacharya, B. Behera, S.K. Sahu, R. Ananthakrishnan, T.K. Maiti, P. Pramanik, Design of dual stimuli responsive polymer modified magnetic nanoparticles for targeted anti-cancer drug delivery and enhanced MR imaging, New J. Chem. (2016). doi:10.1039/c5nj02504d.
18. D. Hreczuk-Hirst, L. German, R. Duncan, Dextrins as carriers for drug targeting: Reproducible succinoylation as a means to introduce pendant groups, J. Bioact. Compat. Polym. (2001). doi:10.1106/QBKY-E3VM-19K4-3GA5.
19. D. Depan, P.K.C. Venkata Surya, B. Girase, R.D.K. Misra, Organic/inorganic hybrid network structure nanocomposite scaffolds based on grafted chitosan for tissue engineering, Acta Biomater. (2011). doi:10.1016/j.actbio.2011.01.029.
20. Z. Zhe, S. Zhang, S.S. Venkatraman, S. Lei, Growth of hydroxyapatite coating on polymer microspheres, Nanosci. Nanotechnol. Lett. (2011). doi:10.1166/nnl.2011.1204.
21. K.J.L. Burg, S. Porter, J.F. Kellam, Biomaterial developments for bone tissue engineering, Biomaterials. (2000). doi:10.1016/S0142-9612(00)00102-2.
22. B. Leukers, H. Gülkan, S.H. Irsen, S. Milz, C. Tille, M. Schieker, H. Seitz, Hydroxyapatite scaffolds for bone tissue engineering made by 3D printing, in: J. Mater. Sci. Mater. Med., 2005. doi:10.1007/s10856-005-4716-5.
23. S. Teixeira, M.A. Rodriguez, P. Pena, A.H. De Aza, S. De Aza, M.P. Ferraz, F.J. Monteiro, Physical characterization of hydroxyapatite porous scaffolds for tissue engineering, Mater. Sci. Eng. C. (2009). doi:10.1016/j.msec.2008.09.052.
24. S. Onder, A.C. Calikoglu-Koyuncu, K. Kazmanli, M. Urgen, G. Torun Kose, F.N. Kok, Behavior of mammalian cells on magnesium substituted bare and hydroxyapatite deposited (Ti,Mg)N coatings, N. Biotechnol. 32 (2015) 747–755. doi:10.1016/j.nbt.2014.11.006.
25. T. Araştirmalar, A. Pasđnlđ, R. Sami AKSOY, A. Kelimeler, Y. Beden Sıvısı, Yapay Kemik Uygulamaları İçin Hidroksiapatit, Electron. J. Biotechnol. BiyoTeknoloji Elektron. Derg. Çalıgülü U. Electron. J. Biotechnol. (2010).
26. W.K. Lee, S.M. Lee, H.M. Kim, Effect of surface morphology of calcium phosphate on osteoblast-like HOS cell responses, J. Ind. Eng. Chem. (2009). doi:10.1016/j.jiec.2009.09.044.
27. Y. Liu, T. Jiang, Y. Zhou, Z. Zhang, Z. Wang, H. Tong, X. Shen, Y. Wang, Evaluation of the attachment, proliferation, and differentiation of osteoblast on a calcium carbonate coating on titanium surface, Mater. Sci. Eng. C. (2011). doi:10.1016/j.msec.2011.03.003.
28. U. Filipkowska, T. Józwiak, Application of chemically-cross-linked chitosan for the removal of Reactive Black 5 and Reactive Yellow 84 dyes from aqueous solutions, J. Polym. Eng. (2013). doi:10.1515/polyeng-2013-0166.
29. S. Onder, F.N. Kok, K. Kazmanli, M. Urgen, Magnesium substituted hydroxyapatite formation on (Ti,Mg)N coatings produced by cathodic arc PVD technique, Mater. Sci. Eng. C. 33 (2013) 4337–4342. doi:10.1016/j.msec.2013.06.027.
30. A. Ali, S. Ahmed, A review on chitosan and its nanocomposites in drug delivery, Int. J. Biol. Macromol. 109 (2018) 273–286. doi:10.1016/j.ijbiomac.2017.12.078.
31. B. Li, Y. Wang, D. Jia, Y. Zhou, Gradient structural bone-like apatite induced by chitosan hydrogel via ion assembly, J. Biomater. Sci. Polym. Ed. 22 (2011) 505–517. doi:10.1163/092050610X487800.
32. M. Li, Y. Wang, Q. Liu, Q. Li, Y. Cheng, Y. Zheng, T. Xi, S. Wei, In situ synthesis and biocompatibility of nano hydroxyapatite on pristine and chitosan functionalized graphene oxide, J. Mater. Chem. B. 1 (2013) 475–484. doi:10.1039/c2tb00053a.