Polikaprolakton-polietilen glikol-kitosan bazlı mikrokürecik üretimi ve kontrollü ilaç salınımında kullanılması için ön çalışma
Production of polycaprolactone-polyethylene glycol-chitosan based microspheres and the pre-study of utilization at controlled drug release
Yazar: ELİF NUR HAYTA
Danışman: PROF. DR. FATOŞ YÜKSEL GÜVENİLİR
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2014
Konu: Biyoteknoloji = Biotechnology ; Kimya Mühendisliği = Chemical Engineering

Özet:
Biyopolimerler, üstün biyouyumlulukları ve biyobozunur özellikleri ile birçok alanda kullanılmaktadır. Kolay şekillenebilir olmaları ve uygun mekanik ve fiziksel özellikleri biyopolimerleri ilaç taşıma sistemleri için uygun aday haline getirmektedir. Bu çalışmada kullanılan Polikaprolakton (PKL), Polietilen glikol (PEG) ve kitosan ilaç taşıma sistemlerinde sıklıkla kullanılan polimerler arasındadır. 
İlaç taşıma sistemi, bir terapötik maddenin vücuda alımını sağlayan ve ilacın vücutta salınım hızı, zamanı ve yerini kontrol ederek verimliliğini ve güvenliğini arttıran araç yada formülasyon olarak tanımlanır. İlaç taşıma sistemlerinin konvansiyonel ilaç alım yöntemlerine tercih edilmesinin sebebi daha düşük dozların tedavi için yeterli olması, böylece ilaç yan etkilerinin azaltılması, hassas ilaç etken maddelerin enkapsüle edilerek bozunmaya karşı korunması ve ilaç plazma konsantrasyonunun istenen süre boyunca kararlı halde kalmasıdır. Bu özellikleri bir tek polimerin sağlaması bazı durumlarda mümkün değildir. Bu nedenle birden fazla polimerin karıştırılması gerekir. 
Bu çalışmanın amacı, ilaç taşıma sistemlerinde kullanılabilecek özellikte PKL-PEG-Kitosan bazlı mikrokürecik üreterek ilaç yükleme ve ilaç salınım özelliklerinin belirlenmesidir. 
PEG yüksek biyouyumluluğu ve suda çözünürlüğü ile birçok ilaç taşıma sisteminde kullanılan bir polimerdir. PKL ise poröz yapısıyla çok miktarda ilaç yükleyebilir. Oldukça hidrofobik bir polimer olan PKL, suda çözünmediği için vücutta yavaş bozunur ve ilacın uzun süre mikroküreciklerde hapsolmasını sağlar. Kitosan aktif yüzeyi ile ilaç etken maddelerinin tutuklanmasını arttırmak amacıyla karışıma eklenmiştir.
İlk aşamada, PKL-PEG-Kitosan bazlı mikrokürecik elde etmek için polimer karışımı püskürtmeli kurutucu ile kurutulmuştur. Giriş sıcaklığı ve besleme debisinin üretilen mikroküreciklerin morfolojisi üzerine etkisi incelenmiştir. Bunun için farklı giriş sıcaklığı ve besleme debilerinde üretilen partiküller SEM görüntüleri ve partikül boyut analizi sonuçları ışığında değerlendirilmiştir.  Üretilen mikroküreciklerin kimyasal yapısı FTIR analizi ile belirlenmiştir. En iyi mikrokürecik şekli kurutmanın 120 °C giriş sıcaklığı ve 3 ml/dk besleme debisi ile yapıldığı deneylerde elde edilmiştir.
Çalışmanın ikinci aşaması ilaç yüklü partiküllerin üretilmesidir. Bunun için iki farklı yöntem denenmiştir. Doğrudan ilaç yükleme yönteminde farklı konsantrasyonlarda askorbik asit çözeltileri ile polimer karışımları karıştırılarak kurutucuya beslenmiş ve böylece ilaç yüklü partiküller elde edilmiştir. Dolaylı yüklemede ise üretilmiş boş partiküller askorbik asit çözeltisine eklenerek ilaç yüklemesi sağlanmıştır. Her iki yöntemde de yüklenen ilaç miktarları UV analiz ile belirlenmiştir. En yüksek ilaç yükleme miktarı, dolaylı yükleme yöntemi ile 25 °C sıcaklık, 200 rpm karışma hızı ve partikül konsantrasyonunun 0,5 mg/ml olduğu koşullarda elde edilmiştir. 
Çalışmanın son aşamasında ise yüklü partiküllerin kontrollü ilaç salınımı özellikleri belirlenmiştir. Kütlece  %15' lik askorbik asit çözeltisiyle püskürtmeli kurutma yöntemiyle elde edilen yüklü partiküllerin üç farklı pH ortamında da iki saat sonunda ilaç salınımını tamamladıkları görülmüştür. En yüksek ilaç salınım oranı %93 ile pH 2,8 ortamında elde edilmiştir. Üç ortamda da 2 saatten sonra ilaç miktarı azalmaya başlamıştır. 
Bu çalışmayla elde edilen verilere dayanarak PKL-PEG-kitosan bazlı küreciklerin ilaç taşıma sistemlerinde kullanılmak için uygun olduğu söylenebilir. Ancak, sistemin iyileştirilmesi için çalışmalara devam edilmelidir.

Abstract:
Biopolymers have been  used at various areas due to their excellent biocompatibility and biodegradable properties. Enabling to be formed easily and having convenient mechanical and physical properties have made biopolymers appropriate candidates for drug delivery systems. Polycaprolactone (PCL), polyethylene glycol (PEG) and chitosan which are employed in this study are among the polymers frequently used at drug delivery systems.
Drug delivery system is described as a device or a formulation which provides the administration of a therapeutic agent into body and enhancing its efficiency and reliance by controlling the rate, time and place of release of drug in the body. At drug delivery systems, even low doses of drug are enough for the cure hence the side effects can be minimized, it is possible to protect sensitive active substances from degradation by encapsulation   and to keep the plasma drug concentration at a steady level for a required time period. These are some of the reasons of preferring drug delivery systems to conventional drug administration methods. For some cases, it is impossible to provide these properties with only a polymer. Therefore, more than one polymers should be blended.
The aim of this study is, producing PCL-PEG-chitosan based microspheres for drug delivery systems and determining drug loading and drug release properties. 
PEG is a frequently employed polymer for drug delivery systems due to its biocompatibility and hydrophilicity. It is used to improve the solubility of poorly water soluble drugs. 
PCL can load plenty of drug with its porous structure. As a very hydrophobic polymer, PCL slowly degrades in body and ensures encapsulation of drugs in microspheres for a long time. 
Chitosan is deacylated form of chitin which is the second most abundant natural polymer. It has been added to the blend to enhance the encapsulation of activated substances due to its chemically active surface. Its antitumor, antimicrobial and immune- enhancing properties are among its advantages for employment as drug delivery devices.
At first step, PCL-PEG-chitosan microspheres have been prepared. For this purpose, three polymers have been blended at ratio of 1:1:1 and the ratio has been fixed throughout the study. Then, prepared polymer blend has been spray dried to produce PCL-PEG-chitosan based microspheres. The effects of inlet temperature and flow rate on the morphology of microspheres have been investigated. For this purpose,  the inlet temperatures have been choosen as 120, 135 and 150 °C and feed flow rates have been 3, 6 and 9 ml/min according to capacity of the pump.
The particles produced at different inlet temperatures and flow rates have been morphologically evaluated by SEM images and particle size analysis results. The most appropriate particles which have the best microsphere shape and the littlest particle size, have been gained at experiments of inlet temperature of 120 °C and flow rate of 3 ml/min.
Chemical structure of microspheres has been determined by FTIR analysis. According to FTIR analysis result all of three polymers have been present in composition of the microspheres.  
The second step of the study is producing drug loaded particles. In this case, two different methods have been used. In the method of direct drug loading, the mixture of ascorbic acid solutions at different concentrations (wt 5, 10, 15%) and polymer blend has fed into spray dryer and in this way drug loaded particles have been produced. 
In the method of indirect drug loading, empty microspheres have been added to ascorbic acid solutions and loaded. Effects of loading time, particle concentration, particle size, agitation rate, temperature and concentration of ascorbic acid solution on drug loading have been investigated.
Dried empty particles have been loaded with ascorbic acid for four hours to determine the loading time. After 2 hours, any considerable change at loaded amount has not been observed. Hence, loading time has been settled on 2 hours.
The particle concentrations of 0.5, 1, 1.5 and 2 mg/ ml solution have been tested to specify the most convenient condition for drug loading. The most loading amoun has been gained when the particle concentration was 0,5 mg/ml solution.
After the experiments which the effect of the ascorbic acid concentration was investigated, it has been clearly seen that loading amount of drug has increased with the  increase of concentration of the solution. But according to water solubility of ascorbic acid, 15% (wt)  is the maximum concentration level that has been tested.
Agitation rates of 150, 200 and 250 rpm have been tested. For the particles which have been loaded with 15%  ascorbic acid solution, the most effective loading has been observed when agitation was 200 rpm.
Since ascorbic acid is very sensitive to heat, medium temperature higher than 25 °C has been resulted in drug degradation. Hence, it is decided to run drug loading trials at 25 °C medium temperature. 
In both methods, loaded quantity of ascorbic acid has been determined by UV analysis.  The highest drug loading quantity have been reached by the indirect drug loading method  when the particles obtained via spray drying at 120 °C inlet temperature and 3 ml/min feed flow rate  have been loaded  with the concentartion of  0.5 mg/ml  in 15% ascorbic acid  solution at 25 °C and with  200 rpm agitation rate. 
Drug loading and loading efficiency values for direct drug loading method are very low in comparasion with indirect method. It has been thought that due to heat, light and air sensibility of ascorbic acid, during the spray drying process most of the drug might be degradated. 
At the third and final step, drug release properties of loaded particles have been determined.  Drug loaded particles with the concentration of 0.5 mg/ml have been shaked at 50 rpm and 25 °C in pH 2.8, pH 7.4 and pH 9.6  buffer solutions for 8 hours.
The loaded particles which are produced by spray drying  with 15% (wt) ascorbic acid solution have seemed  to complete drug release at the end of two hours in three different pH medium. The highest cumulative drug release ratio has been  93% obtained at pH 2,8 medium.
With respect to drug release kinetic parameters, it might be said that drug release from PCL-PEG-chitosan microspheres has been as Fick diffusion. 
In three mediums, drug content has started to decrease after two hours. I might be because of drug degradation due to light and air. Also,it has been observed that  degradation was more than the others in alkali medium.
As conclusion, according to data obtained from this study, it can be said that PCL-PEG-chitosan based microspheres are convenient for employment at drug delivery systems. However, studies must be continued to improve the system. 
For furrher studies, cross linker usage might enhance drug loading and loading efficiency. Also, drug loading and release properties could be arranged by changing polymer ratio.
Drug loading capacity of these microspeheres should be proved by loading with other drugs. So that, employement of PCL-PEG-chitosan microspheres as drug delivery devices might be supported more strongly.