Bitki atıklarından hazırlanan ekstraktın kitosan nanopartikülleriyle enkapsülasyonu ve in vitro incelenmesi
Encapsulation of extract prepared from plant wastes with chitosan nanoparticles and in vitro investigation
Yazar: HATİCE SERİN
Danışman: PROF. DR. GÜLNUR ARABACI
Sakarya Üniversitesi / Fen Bilimleri Enstitüsü / Biyomedikal Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2023
Konu: Biyomühendislik = Bioengineering ; Mühendislik Bilimleri = Engineering Sciences

Özet:
Ceviz (Juglans regia L.) ağacı Juglandaceae familyasına ait bir ağaç türüdür. Cevizin meyvesi kadar diğer yapısal bileşenleri olan kabuk, yaprak, zar kısımları da içerdiği komplex bileşenler sayesinde sağlık için önemli bir yere sahiptir. Cevizin iç kısmında bulunan endokarp zarı, içeriğindeki zengin bileşenleri yapılan çalışmalarla ortaya koyulmasına rağmen yeterince faydalanılmayan atık olarak kalmaktadır. Doğal bileşik olarak ceviz zarı ekstresinin, antioksidan ve antiinflamatuar özelliklere sahip, fenolik asitler ve flavonoidler gibi yüksek konsantrasyonlarda biyoaktif bileşikler içerdiği bulunmuştur. Yapılan çalışmalarla bu biyoaktif bileşiklerin, nöroinflamasyonu, oksidatif stresi ve beta-amiloid agregasyonunu azaltmak gibi nörodejeneratif hastalıklarda potansiyel terapötik etkilere sahip olduğu gösterilmiştir.
Nanoteknoloji son zamanların en gözde çalışma alanlarından birini oluşturmaktadır. Kullanım alanı gittikçe genişleyen bu teknoloji sağlık sektöründe, ilaç taşıyıcı sistemlerin geliştirilmesinde ve daha birçok teşhis-tedavi amaçlı yapılan çalışmalarda araştırılmaktadır. Kan-beyin bariyeri (KBB), beyin dokusunu kan kaynaklı toksinler, patojenler ve diğer zararlı maddelerden koruyan bir bariyer sistemidir. Nörodejeneratif hastalıkların tedavisi için ilaçların beyne ulaştırılması, KBB'nin işlevi nedeniyle zordur. Geleneksel ilaçlar, KBB'yi geçemezler veya sınırlı bir geçirgenlik gösterirler. Bu nedenle bilim insanları, KBB'nin geçilmesi için farklı teknikler geliştirmeye çalışmaktadır İlaç taşıyıcı sistemleri uygun taşıyıcı yapı malzemeleri kullanılarak etken maddelerin hedefe taşınmasında umut vadeden yaklaşımlar sunmaktadır. Kitosan, karides ve diğer kabuklu deniz hayvanlarının kabuklarının hidrolizi sonrasında elde edilir. Kitosan, insanlar için biyouyumlu, biyobozunur ve biyoaktif bir polimerdir. Kitosan, yüksek mekanik dayanıklılık ve esnekliğe sahiptir. Ayrıca düşük toksisiteye sahiptir ve biyolojik olarak parçalanabilir, bu nedenle çevre dostudur. Çeşitli endüstriyel uygulamalarda kullanılır. Biyomedikal uygulamalarda kullanılan kitosan, dokularla uyumlu ve biyouyumlu olması nedeniyle yaygın olarak kullanılır.
Bu tez çalışması kapsamında, boyutları 150-300 nm arasında değişen, ceviz endokarp zarından elde edilen özütün enkapsüle edildiği kitosan nanopartikülleri (JP/CSNP) üretilmiş ve nanopartiküllerin karakterizasyon çalışmaları yapılmıştır. İyonik jelasyon tekniği ile üretilen nanoparçacıklara farklı konsantrasyonlarda (seri1: 10 mg/ml, seri2: 5 mg/ml, seri3: 2,5 mg/ml) ekstrakt yüklemesi yapılmıştır. Zeta potansiyel ölçümleri sonucunda +17 mV ve + 44 mV arasında değişen değerler bulunmuştur. Nanopartikül boyutları zetasizer ile ölçülmüş ve boş olan nanopartiküllere (CSNP) ait ortalama partikül boyutu 281 nm olarak bulunmuştur. Bu değerler seri1 nanopartiküller için 197 nm, seri2 nanopartiküller için 194 nm ve seri3 nanopartiküller için 237 nm olarak ölçülmüştür. En yüksek enkapsülasyon oranı % 89 değerle seri2 nanopartiküllerde hesaplanmıştır. Yapılan ilaç salım çalışmaları ile 10. saatin sonunda nanopartiküllere yüklenen ekstraktın % 33-% 12 oranlarında ortama salındığı görülmüştür. Antioksidan aktivitenin nanopartiküllere yüklenen ekstrakt konsantrasyonu ile doğrusal bir artış gösterdiği gözlemlenmiştir. Ayrıca FTIR ve SEM görüntülemeleri ile nanopartiküllerin daha detaylı incelenmeleri sağlanmıştır. Yapılan çalışma ile nörodejeneratif rahatsızlıklarda kullanılmak üzere doğal ilaç potansiyelinde ceviz endokarp özütü enkapsüle edilmiş kitosan nanopartikülleri sentezlenmiştir. Çalışmalar sonucunda üretilen nanopartiküllerin nörodejeneratif rahatsızlıklara karşı umut vadettiği görülmektedir.

Abstract:
Alzheimer's disease is a chronic and progressive neurodegenerative disease that usually occurs in individuals aged 65 and over. The disease is characterized by symptoms such as memory loss, reduced mental abilities, and personality changes due to disruptions in nerve cells in the brain. The exact cause of Alzheimer's disease is unknown, but research shows that genetic and environmental factors play a role in the development of the disease. Currently, there is no complete cure for Alzheimer's disease, but medications and therapies are available for the management of symptoms. This disease affects approximately 50 million people worldwide, and this number is expected to triple by 2050. Treatment strategies for Alzheimer's disease aim to slow or stop the damage to nerve cells caused by the disease.
Walnut (Juglans regia L.) tree is a tree species belonging to the Juglandaceae family. The shell, leaf and membrane parts of the walnut, which are other structural components as well as the fruit, have an important place for health thanks to the complex components they contain. The endocarp membrane in the inner part of the walnut remains as an underutilized waste, although its rich components have been revealed by studies. As a natural compound, walnut shell extract has been found to contain high concentrations of bioactive compounds such as phenolic acids and flavonoids with antioxidant and anti-inflammatory properties. Studies have shown that these bioactive compounds have potential therapeutic effects in neurodegenerative diseases such as reducing neuroinflammation, oxidative stress and beta-amyloid aggregation. Walnut fruit contains high levels of omega-3 fatty acids, which have a strong anti-inflammatory effect. The omega-3 fatty acids contained in the inner membrane of the walnut help protect and improve brain functions. Antioxidants, on the other hand, are important in protecting brain cells against oxidative damage and maintaining brain homeostasis. 
Nanotechnology is one of the most popular fields of study in recent times. This technology, which is increasingly used, is being researched in the health sector, in the development of drug delivery systems and in many other diagnostic-therapeutic studies. These systems provide many advantages such as increasing the bioavailability of drugs, reducing their side effects, enabling drugs to reach the target tissue selectively, increasing the stability of drugs and allowing drugs to stay in the body longer. 
The blood-brain barrier (BBB) is a barrier system that protects brain tissue from blood-borne toxins, pathogens and other harmful substances. Delivery of drugs to the brain for the treatment of neurodegenerative diseases is difficult due to the function of the KBB. Conventional drugs cannot cross the KBB or show limited permeability. For this reason, scientists are trying to develop different techniques for the passage of KBB. Drug delivery systems offer promising approaches in the delivery of active substances to the target by using appropriate carrier building materials. 
Chitosan is obtained after hydrolysis of the shells of shrimp and other shellfish. Chitosan is a biocompatible, biodegradable and bioactive polymer for humans. Chitosan has high mechanical strength and flexibility. It also has low toxicity and is biodegradable, so it is environmentally friendly. It is used in various industrial applications. Chitosan, which is used in biomedical applications, is widely used because it is compatible and biocompatible with tissues. In addition, as a result of studies, it is thought that chitosan can promote the growth and regeneration of nerve cells. This effect makes the use of chitosan in the treatment of neurological injuries and diseases even more attractive.
The effects of walnut endocarp membrane on brain health have been revealed in various studies. Our main goal is to facilitate the transport of walnut material to the target for neurodegenerative disorders in the brain, to increase its utilization by increasing its effects, and to evaluate its potential as a natural medicine by creating drug release profiles for this purpose. JP/CSNPs and empty CSNPs were synthesized by ionic gelation technique by encapsulating the walnut endocarp membrane extract into chitosan nanoparticles. The zeta potential, size analysis, encapsulation rates, drug release profiles, SEM and FTIR analyses and antioxidant determination and characterization processes of the produced CSNP and JP/CSNP nanoparticles were performed. Within the scope of the master thesis study, characterization studies of nanoparticles were carried out by producing chitosan nanoparticles (JP/CSNP) and empty nanoparticles (CSNP) in which the extract obtained from walnut endocarp membrane, varying in size between 150-300 nm, is encapsulated. The nanoparticles produced by the ionic gelation technique were loaded with extract at different concentrations (seri1: 10 mg/ml, seri2: 5 mg/ml, seri3: 2,5 mg/ml). The size and potential measurements of the nanoparticles were carried out with the Zetasizer device. As a result of zeta potential measurements, values varying between +17 mV and + 44 mV were found. As a result of nanoparticle size measurements, the average particle size of the empty nanoparticles (CSNP) was found to be 281 nm. These values were measured as 197 nm for seri1 nanoparticles, 194 nm for seri2 nanoparticles and 237 nm for seri3 nanoparticles. The highest encapsulation rate was calculated in seri2 nanoparticles with a value of 89%. With the drug release studies, it was observed that at the end of the 10th hour, the extract loaded on the nanoparticles was released to the environment at a rate of 33%-12%. While the highest emission value was observed in seri1 nanoparticles, the lowest emission value was observed in seri3 nanoparticles. It is understood that there is a linear relationship between the amount of encapsulated extract and the time-dependent release profile. It was observed that the antioxidant activity increased linearly with the extract concentration loaded on the nanoparticles. In addition, nanoparticles were examined in more detail with FTIR and SEM imaging. In the spectra analyzed by FTIR analysis, it is understood that there is no adverse interaction between the active groups of the walnut skin extract and the functional groups of chitosan and that the extract is encapsulated into nanoparticles. When the CSNP and seri2 JP/CSNP results displayed by SEM are evaluated, it is thought that the particle sizes are higher than the zeta size measurement due to the adhesion of the particle structures and coalescence over time.
As a result of the studies, chitosan nanoparticles encapsulated in walnut endocarp extract have been synthesized with the potential of natural medicine for use in neurodegenerative disorders. It is seen that the nanoparticles produced are promising against neurodegenerative disorders.