Fenolik bağlayıcı kullanılarak sentezlenen hidroksiapatit-jelatin kompozit malzemesinin karakterizasyonu ve ilaç salım profilinin incelenmesi
Research on characterization and drug release performance of hydroxyapatite-gelatin composite materials syntheted by using phenolic linker
Yazar: IRMAK BAŞER GÜMÜŞ
Danışman: PROF. DR. GÜLHAYAT SAYGILI
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Mühendisliği Ana Bilim Dalı / Kimya Mühendisliği Bilim Dalı
Derece: Yüksek Lisans, 2018
Konu: Kimya Mühendisliği = Chemical Engineering

Özet:
Hidroksiapatit Ca10(PO4)6(OH)2 formülü ile doğal kemiğin mineral fazıyla biyolojik olarak benzerliklere sahiptir. Jelatin, kollajen, kitosan, nişasta gibi biyobozunur polimerlerin bileşimine katılarak biyomalzemlerin sentezlenmesinde kullanılmaktadır. Jelatin, amino asit gibi fonksiyonel bir grup tarafından doğal biyopolimerdir. Biyolojik olarak bozunabilir, biyolojik olarak uyumlu, suda çözünür ve ucuzdur. Jelatin kollajenin termal, fiziksel veya kimyasal denatürasyonu ile elde edilebilir. Jelatin, hidrofilisitesine bağlı olarak HAp ile büyük uyuma sahiptir ve sulu çözelti içinde iyi homojenize edilebilir. Bu nedenle, HAp ve jelatin biyokompozitin biyoaktif ve biyouyumlu olarak gelişmiş biyomalzeme vermesi beklenmektedir. Bu çalışmada, HAp'in bir matriks olarak jelatin kullanılarak sentezi, biyopolimer konsantrasyon değişiminin nihai yapı üzerindeki etkisi ve hazırlanan kompozitlerin karakterizasyonu incelenmiştir.
Kimyasal bağlayıcıların biyopolimerleri çapraz bağlamak için kullanılması yüksek sitotoksisitesi sebebiyle biyouyumluluğunu engellemektedir. Alternatif çapraz bağlayıcı olarak bitki ve mikroorganizmalardan elde edilen biyobozunur fenolik asit olan tannik asit kullanılmaktadır. Ayrıca, tannik asit diğer kimyasal türevlere kıyasla daha ucuzdur. Fenol asitleri tarafından esterlenen bir merkezi karbonhidrat çekirdeği içeren yüksek moleküler ağırlıklı, polifenolik bir bileşiktir. Tannik asit yüksek bir antioksidan kapasitesine sahiptir ve diğer biyolojik makromoleküllerle de etkileşime girebilmektedir. Birçok çapraz bağlama ajanına göre üstün mekanik özellikler ve su direnci sağlayan, biyopolimerler için oldukça etkili bir çapraz bağlama maddesidir.
Bu çalışmada, SBF (Simulated Body Fluid) ortamında hidroksiapatit-jelatin (HAp-GEL) kompozit malzemesinin üretimi yapılıp püskürtmeli kurutucu yardımıyla 5-Fluorourasil  (5-FU)  ilacının yüklemesi yapılmıştır. Yaş çöktürme yöntemi ile sentezlenen HAp-GEL  kompozitlerinde ağırlıkça (w%w) üç farklı oranda 1:1, 1:2 ve 1:4 jelatin kullanılarak jelatin miktarının ilaç yükleme verimi ve salım profili üzerine etkisi incelenmiştir. Çapraz bağlama ajanı olarak fenolik bağlayıcı olan tannik asit (TA) kullanılmıştır. Kompozitlerdeki TA miktarının ilaç yükleme verimi ve ilaç salım profili üzerine etkisini incelemek amacı ile iki farklı TA oranı %2 ve %5'lik çözeltiler ile HAp-GEL kompozit malzemeleri elde edilmiştir. İlaç salım profillerini incelenmek için Sıfır Derece, Birinci Derece, Higuchi ve Korsmeyer-Peppas olmak üzere dört farklı kinetik model kullanılmıştır.
Elde edilen kompozitlerin yapısal özellikleri enstrümantal analiz cihazları kullanılarak irdelenmiştir. Fourier transform infrared spektroskopisi (FTIR), X- Işını difraktometresi (XRD), Isıl gravimetrik analiz (TGA) ve Taramalı elektron mikroskopu (SEM) cihazlarından alınan sonuçlar ile karakterizasyonu incelenmiştir. Bu çalışma, ilaç salım sistemlerinde kullanılması amacıyla HAp-GEL kompozit malzemesinin biyouygun çapraz bağlayıcı ile hazırlanması gibi güncel bir araştırmaya odaklanmıştır. Yüksek verimde ilaç yükleme yapabilmek için püskürtmeli kurutucu tercih edilmiştir.

Abstract:
Hydroxyapatite has a biological similarity to the mineral phase of the natural bone with the formula Ca10(PO4)6(OH)2. The good adsorption properties of hydroxyapatite provided scaffold for the production of drug delivery systems (DDS) and controlled drug delivery systems. This is due to both the powder and the porous structure of HAp. The porous HAp structures loaded with suitable drug form a system that gives the drug the sensitive area. This feature is used in the development of new DDSs in the treatment and prophylaxis of bone disorders, particularly osteoporosis, bone tumors, and bone metastases. In addition, studies have been conducted on systems that provide important antibiotics directly to hard tissues. In addition to drug delivery systems, attempts are made to use HAp as a carrier for hormones, anti-inflammatory agents and vitamins.
Gelatin is used to synthesize biomaterials by incorporating into the composition of biodegradable polymers such as collagen, chitosan, starch. Gelatin is a natural biopolymer by a functional group such as amino acid. It is biodegradable, biocompatible, water-soluble and inexpensive. Gelatin can be obtained by thermal, physical or chemical denaturation of collagen. Crosslinking of gelatin increases gelatin stability (high gelation and melting temperatures) compared to non-crosslinked particles. Gelatin-based materials prepared without crosslinking are known to be unstable and tend to age. Due to its hydrophilicity, gelatin has a great interest in HAp and can be homogenized well in aqueous solution. Therefore, it is expected that gelatin and HAp will yield bioactive and biocompatible biomaterials with biocomposite. In this study, the synthesis of HAp as a matrix with using gelatin, the effect of biopolymer concentration change on the final structure and the characterization of the prepared composites have been investigated.
Many studies carried out on the preparation methods, physical and chemical properties of HAp-GEL composites, as well as in vitro studies. These HAp-GEL composites prepared by conventional methods are generally in the form of irregular granules, pastes, sponges, and microspheres. Among these different forms, microspheres provided the ideal working environment for many applications at controlled rates over extended periods of time due to their ability to encapsulate more drug molecules and sustained drug release characteristics. HAp-GEL composite microspheres are also used as bone regenerative fillers or as an injectable bone substitute. Therefore, the potential for microspheres to combine their roles in drug delivery and bone repair reduces the time to recovery and regeneration of bone tissue.

Polyphenols make the formation of natural cross-links in biological tissues and make them more resistant to degradation. It has been used in various synthetic and natural crosslinkers both in vivo and in vitro to strengthen collagen fibers in biological tissues such as dentin, tendon, pericardium strips and collagen grafts. In addition, polyphenols contain many phenolic groups capable of forming strong hydrogen bonds. Polyphenols, gelatin-dextran conjugate are obtained as a result of the formation of hydrogen bonds with gelatin.
Glutaraldehyde, a chemical binder, is widely used to cross-link biopolymers in medical applications. However, its cytotoxicity prevents it is biocompatibility. Furthermore, it is difficult to use glutaraldehyde during crosslinking due to its strong odor and low vapor pressure. Various chemicals have been studied as alternative crosslinking agents. These include tannic acid, ferulic, genipine and citric. Tannic acid (TA) is completely biodegradable from plant and microorganisms. It is also cheaper than other chemical derivatives. It is a high molecular weight, a polyphenolic compound containing a central carbohydrate nucleus esterified by phenol acids. Tannic acid has a high antioxidant capacity and can interact with other biological macromolecules. It is a highly effective crosslinking agent for biopolymers which provides superior mechanical properties and water resistance compared to many agents.
Fluorouracil (5-FU) is one of the major anti-metabolites used in various cancers such as stomach, colon, lung, ovary and breast cancer. Given the structural similarity to natural pyrimidines, 5-FU interferes with the nucleic acid synthesis, inhibits DNA synthesis and eventually halts cell growth. The targeted drug dosing of 5-FU is highly dependent on the cancer being treated. In-vitro studies showed cytotoxic effects of 5-FU at a high dose of 50 µg/mL. Because of 5-FU's narrow therapeutic index, its lifecycle in blood and body tissues is limited to a few minutes. As with most chemotherapy drugs, the side effects of 5-FU are also within the boundaries of the region where it is administered. In intravenous administration, 5-FU produces severe toxic effects from a gastrointestinal, hematological, neural, cardiac and dermatological origin. Therefore, site-specific administration of 5-FU provides an effective and safe treatment to reduce systemic side effects.
There are studies in the literature examining the release profile of drug-loaded HAp-GEL composite materials. In this study, hydroxyapatite-gelatin (HAp-GEL) composite material has been produced with a phenolic linker TA in SBF (Simulated Body Fluid) solution, and 5-FU drug was loaded with a spray dryer. In the HAp-GEL composites synthesized by wet chemical method, the effect of the amount of gelatin on drug loading efficiency and release profile by using in three different ratios (w% w) 1:1, 1:2 and 1:4 gelatin was investigated. The tannic acid, the phenolic linker, has been used as the crosslinking agent. Two different TA solutions 2% and 5%  have been used in terms of HAp-GEL composite materials have been obtained to investigate the effect of TA in the composites on drug loading efficiency and drug release profile. Lastly, 5-Fluorouracil (5-FU) has been used as a drug. The drug release profiles have been researched according to four different kinetic models; Zero Degrees, First Degrees, Higuchi and Korsmeyer-Peppas.

The structural properties of the composites have been analyzed using instrumental analysis devices. Fourier transform infrared spectroscopy (FTIR), X-ray diffractometer (XRD), thermal gravimetric analysis (TGA) and scanning electron microscopy (SEM) with devices the results of the characterization have been analyzed. This study focused on a recent study of the preparation of HAp-GEL composite material with a biocompatible crosslinker for use in drug delivery systems. The spray dryer has been preferred for high-efficient drug loading.