M.Sc. THESIS JUNE 2024 BIOMEDICAL APPLICATION OF AN ENZYMATICALLY SYNTHESIZED BIOPOLYESTER ŞENOL BEYAZ Department of Chemical Engineering Chemical Engineering Programme ISTANBUL TECHNICAL UNIVERSITY  GRADUATE SCHOOL M.Sc. THESIS JUNE 2024 BIOMEDICAL APPLICATION OF AN ENZYMATICALLY SYNTHESIZED BIOPOLYESTER Thesis Advisor: Prof. Dr. F. Yüksel GÜVENİLİR Şenol BEYAZ (506211027) Department of Chemical Engineering Chemical Engineering Programme ISTANBUL TECHNICAL UNIVERSITY  GRADUATE SCHOOL YÜKSEK LİSANS TEZİ HAZİRAN 2024 ENZİMATİK OLARAK SENTEZLENMİŞ BİR BİYOPOLİESTERİN BİYOMEDİKAL UYGULAMASI Tez Danışmanı: Prof. Dr. F. Yüksel GÜVENİLİR Şenol BEYAZ (506211027) Kimya Mühendisliği Anabilim Dalı Kimya Mühendisliği Programı İSTANBUL TEKNİK ÜNİVERSİTESİ  LİSANSÜSTÜ EĞİTİM ENSTİTÜSÜ v Thesis Advisor : Prof. Dr. F. Yüksel GÜVENİLİR .............................. Istanbul Technical University Jury Members : Prof. Dr. Didem SALOĞLU DERTLİ .............................. Istanbul Technical University , ............................. Istanbul Technical University Prof. Dr. ORHAN .............................. Istanbul University Şenol BEYAZ, a M.Sc. student of ITU Graduate School student ID 506211027, successfully defended the thesis/dissertation entitled “BIOMEDICAL APPLICATION OF AN ENZYMATICALLY SYNTHESIZED BIOPOLYESTER”, which he prepared after fulfilling the requirements specified in the associated legislations, before the jury whose signatures are below. Date of Submission : 20 May 2024 Date of Defense : 26 June 2024 Asst. Prof. Dr. Yasemin KAPTAN .............................. Beykent University , ............................. Istanbul Technical University vi vii To my beloved wife and precious family, viii ix FOREWORD I would like to express my gratitude and huge respect to my esteemed thesis advisor, Prof. Dr. Fatoş Yüksel GÜVENİLİR, who gave me both academic and life discipline with her mentorship, supported me in every times and kept my motivation and interest alive. I would like to thank and appreciate Dr. Cansu ÜLKER TURAN, who helped me in every way possible with the problems I encountered, both laboratory experiments and methods of the thesis and during my studies. Her valuable comments and evaluations played a major role in determining the roadmap of this study. Also, I would like to express my gratitude and appreciation to Dr. Yasemin KAPTAN for giving me every information that she already knew and for always helping my experiments with pleasure. I will always be indebted to my dear wife İrem GÜVEN BEYAZ, who were always with me in the struggle with the problems I faced during my study and every moments in my life. I am deeply thankful to my dear friend Mete DERVİŞCEMALOĞLU for his kind support. Finally, I would like to thank my entire family, who encouraged me to continue my graduate education and supported me throughout my graduate studies. This study was financially supported by the Istanbul Technical University Research Projects Coordination Department. Grant number : MYL-2022-44072. May 2024 Şenol BEYAZ (Chemical Engineer) x xi TABLE OF CONTENTS Page FOREWORD ............................................................................................................. ix TABLE OF CONTENTS .......................................................................................... xi ABBREVIATIONS ................................................................................................. xiii SYMBOLS ................................................................................................................ xv LIST OF TABLES ................................................................................................. xvii LIST OF FIGURES ................................................................................................ xix SUMMARY ............................................................................................................. xxi ÖZET ....................................................................................................................... xxv 1. INTRODUCTION .................................................................................................. 1 2. LITERATURE REVIEW ...................................................................................... 5 2.1 Biopolymers ....................................................................................................... 5 2.2 Lipase Enzyme ................................................................................................... 7 2.3 Enzyme Immobilization ..................................................................................... 8 2.3.1 Support materials for enzyme immobilization .......................................... 10 2.4 Biopolymer Production .................................................................................... 13 2.4.1 Poly (ω-Pentadecalactone -co-Valerolactone) .......................................... 16 2.4.1.1 ω-Pentadecalactone ............................................................................ 17 2.4.1.2 δ-Valerolactone .................................................................................. 17 2.5 Applications of Biopolymers ........................................................................... 18 2.6 Drug Delivery Systems .................................................................................... 19 2.7 Microspheres .................................................................................................... 22 2.7.1 Emulsion/solvent evaporation method ...................................................... 23 2.8 Oleuropein ........................................................................................................ 25 2.9 Transchalcone ................................................................................................... 26 3. MATERIALS AND METHODS ........................................................................ 29 3.1 Materials ........................................................................................................... 29 3.2 Methods ............................................................................................................ 30 3.2.1 Preparation of support material for the enzyme immobilization .............. 30 3.2.2 Immobilization of CALB .......................................................................... 32 3.2.3 Enzymatic synthesis of poly(ω-pentadecalactone-co-δ-valerolactone) .... 32 3.2.4 Preparation of microspheres ...................................................................... 33 3.2.5 In vitro TC release from loaded microspheres .......................................... 34 3.2.6 Kinetic model fitting ................................................................................. 34 3.2.7 Antibacterial activity test of microspheres ................................................ 35 3.2.8 In vitro cytotoxicity (WST) test of microspheres ..................................... 37 3.3 Characterization Techniques ............................................................................ 38 3.3.1 Gel permeation chromatography (GPC) ................................................... 38 3.3.2 Scanning electron microscope (SEM) ....................................................... 38 3.3.3 Fourier transform infrared spectroscopy (FT-IR) ..................................... 39 3.3.4 Hydrogen nuclear magnetic resonance spectroscopy (1H-NMR) ............. 39 3.3.5 Differential scanning calorimetry (DSC) .................................................. 40 xii 3.3.6 Thermal gravimetric analysis (TGA) ........................................................ 40 3.3.7 X-Ray diffraction analysis (XRD) ............................................................ 40 3.3.8 Ultraviolet (UV) spectrophotometer ......................................................... 40 3.3.9 Dynamic light scattering analysis (DLS) .................................................. 41 4. RESULTS AND DISCUSSION........................................................................... 43 4.1 Synthesis of Poly(ω-pentadecalactone-co-δ-valerolactone) with Immobilized Enzyme ............................................................................................................. 43 4.2 Fabrication of Microspheres ............................................................................. 43 4.3 Characterization of Microspheres ..................................................................... 45 4.3.1 SEM results of microspheres..................................................................... 45 4.3.2 FT-IR results of microspheres ................................................................... 47 4.3.3 DSC results of microspheres ..................................................................... 48 4.3.4 TGA results of microspheres..................................................................... 50 4.3.5 XRD results of microspheres .................................................................... 52 4.4 Antibacterial Activity of Microspheres ............................................................ 53 4.5 In Vitro Cytotoxicity (WST) Test of Microspheres ......................................... 55 4.6 pH Dependent Drug Release from Microspheres ............................................. 56 4.7 Kinetic Modelling and Release Mechanism ..................................................... 59 5. CONCLUSSION AND RECOMMENDATIONS ............................................. 61 REFERENCES ......................................................................................................... 65 APPENDICES .......................................................................................................... 77 CURRICULUM VITAE .......................................................................................... 81 xiii ABBREVIATIONS 1H-NMR : Proton Nuclear Magnetic Resonance 3-APTES : (3-Aminopropyltriethoxysilane 3-APTMS : (3-Aminopropyl)trimethoxysilane 3-GPTMS : (3-Glycidyloxypropyl)trimethoxysilane CALB : Candida antarctica Lipase B DLS : Dynamic Light Scattering DNA : Deoxyribonucleic acid DSC : :Differential Scanning Calorimetry DTG : Derivative of weight loss FT-IR : Fourier Transform Infrared Spectroscopy GPC : Gel Permeation Chromatography Olu : Oleuropein PBS : Phosphate Buffered Saline PCL : Poly(ε-caprolactone) PGA : Polyglycolic acid PHB : Polybutylene terephthalate PHBV : Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) PLA : Poly(lactic acid) PLGA : Poly(lactic-co-glycolic acid) PPL : Porcine pancreatic lipase PVA : Polyvinyl alcohol RHA : Rice Husk Ash RNA : Ribonucleic acid ROCOP : Ring Openning Copolymerization ROP : Ring Openning Polymerization ROS : Reactive Oxygen Species SEM : Scanning Electron Microscope TC : Trans-chalcone TGA : Thermo Gravimetric Analysis XRD : X-Ray diffraction analysis xiv δ-VL : δ-Valerolactone ε-CL : ε-Caprolactone ω-PDL : ω-Pentadecalactone xv SYMBOLS ∆Hm : Melting Enthalpy I1.26 : Integral of Peak at 1.26 ppm in 1H-NMR Spectrum I1.64 : Integral of Peak at 1.64 ppm in 1H-NMR Spectrum I3.65 : Integral of Peak at 3.65 ppm in 1H-NMR Spectrum I4.06 : Integral of Peak at 4.06 ppm in 1H-NMR Spectrum I4.16 : Integral of Peak at 4.16 ppm in 1H-NMR Spectrum KH : Higuchi Dissolution Constant KKP : Korsmeyer-Peppas Release Rate Constant m : Number of δ-Valerolactone Units Mn : Number Average Molecular Weight n : Number of ω-Pentadecalactone Units n : Release Exponent OD : Optical density R2 : Coefficient of determination Q0 : Initial Concentration of Drug Qt : Cumulative % Drug Release at Any Time Qt / Q∞ : Fraction of Drug Released at Any Time t : Time Tg : Glass Transition Temperature Tm : Melting Temperature v/v : Volume to volume ratio W0 : Initial Weight Wt : Dry Weight at Any Time xvi xvii LIST OF TABLES Page Classification of supports used for enzyme immobilization . ................. 11 Table 4.1 : Microsphere formulations, encapsulation efficiencies, mean particle diameters and zeta potentials................................................................... 44 Table 4.2 : Sample codes, Olu and TC ratios in order to PDL-VL copolymers. ...... 46 Table 4.3 : Tm and enthalpy values of PDL-VL, PDL-VL/Olu and TC-loaded PDL- VL/Olu microspheres. ............................................................................. 49 xviii xix LIST OF FIGURES Page Classification of biobased polymers . ...................................................... 6 Free lipase open and closed form . .......................................................... 8 Immobilization methods of enzymes ...................................................... 9 Applications of rice husk for lipase enzyme . ....................................... 12 Immobilization of lipase on APTES/GA-activated silica surface . ....... 13 Synthesis of polyester materials via (a) ROP of cyclic esters; (b) ROCOP of epoxides and cyclic anhydrides . .......................................... 15 Lipase-catalyzed ring-opening polymerization . ................................... 15 Mechanism of lipase-catalyzed ring-opening polymerization of lactones . .................................................................................................. 15 Molecular structure of ω-Pentadecalactone . ......................................... 17 Molecular structure of δ-Valerolactone . ............................................. 18 Applications of biopolymers . ............................................................. 19 Concentration as a function of time after administration . .................. 20 A diagram of the formulation of polymeric carriers for biomedical delivery systems . .................................................................................... 21 Overview of polymeric-based drug delivery systems . ....................... 22 Release mechanisms of biodegradable polymeric drug delivery systems . .................................................................................................. 22 Microspheres . ..................................................................................... 23 Schematic diagram of O/W emulsion solvent evaporation method . .. 24 Formation of microcapsules by single O/W emulsion/solvent evaporation method . ............................................................................... 25 Microspheres created using the Double Emulsion Technique . .......... 25 Molecular structure of oleuropein . ..................................................... 26 Molecular structure of trans-chalcone . ............................................... 27 Figure 3.1 : From left to right: dried rice husk, rice husk ash after combustion. ...... 31 Figure 3.2 : Shaking water bath used in experiments. .............................................. 31 Figure 3.3 : Filtered immobilized enzyme after the immobilization via physical adsorption. ............................................................................................... 32 Figure 3.4 : Apparatus used for polymerization. ...................................................... 33 Figure 3.5 : Incubator that was used for antibacterial activity test. .......................... 35 Figure 3.6 : Left to right: McFarland Densitometer and 0.5 MF adjusted bacteria cultures from each type of bacteria. ........................................................ 36 Figure 3.7 : The scheme followed in the dilution step. ............................................. 36 Figure 3.8 : Microplate Reader and 96-Well Plate. .................................................. 37 Figure 4.1 : SEM images of : PDL-VL, PDL-VL/Olu and TC-loaded PDL-VL/Olu microspheres (Magnification 1000x). ..................................................... 46 Figure 4.2 : FT-IR spectra of (1) Olu, (2) PDL-VL, (3) PDL-VL/Olu microspheres, (4) TC-loaded PDL-VL/Olu microsphes, (5) TC. ................................... 48 xx Figure 4.3 : Melting temperatures of samples: DSC second heating curves, PDL,VL, PDL-VL/Olu, TC-loaded PDL-VL/Olu. ................................. 49 Figure 4.4 : TGA and derivative of weight losses results; PDL-VL (black), PDL- VL/Olu(red), TC-loaded PDL-VL/Olu (blue). ........................................ 51 Figure 4.5 : XRD measurement of : TC-loaded PDL-VL/Olu (blue), PDL-VL/Olu (red), PDL-VL (black). ............................................................................ 52 Figure 4.6 : Antibacterial activity(%) of PDL-VL, PDL-VL/Olu and TC-loaded PDL-VL/Olu microspheres in 105 CFU S. aureus, E.coli cultures. ........ 53 Figure 4.7 : Percentage cellular viability of MCF-7 breast cancer cells treated with TC loaded and Olu added microspheres at 72 hour periods. ................... 55 Figure 4.8 : Cumulative TC release (%) in two different pH media (pH 5.6 and pH 7.4) with different amount of TC-loaded PDL-VL/Olu microspheres. ... 57 Figure 4.9 : Kinetic modelling and release mechanisms calculations in two different pH media (pH 5.6 and pH 7.4) with different amount of TC-loaded PDL- VL/Olu microspheres. ............................................................................. 60 Figure A.1 : Calibration curve of TC in Methanol. ................................................... 78 Figure A.2 : Calibration curve of TC in PBS (pH 7.4). ............................................ 78 Figure A.3 : Calibration curve of TC in acetate buffer solution (pH 5.6). ................ 79 xxi BIOMEDICAL APPLICATION OF AN ENZYMATICALLY SYNTHESIZED BIOPOLYESTER SUMMARY Polymers have played an integral role in advancing drug delivery technology by providing controlled release of therapeutic agents at fixed doses over long periods of time, cyclic dosing, and adjustable release of both hydrophilic and hydrophobic drugs. Modern advances in drug delivery are now based on the rational design of polymers designed to exert different biological functions. Enzyme-based biopolymer syntheses are needed in order to reduce the toxic accumulations caused by these drug systems in the human body, to minimize their side effects, and their effects on the environment. Unlike synthetic polymers, biopolymers produced naturally using enzymes; They are suitable for medical applications due to their biocompatibility, biodegradability, non- toxicity and ability to adsorb bioactive molecules. The use of these biopolymers in drug delivery systems is possible by turning them into materials such as cast films, microspheres, nanoparticles and nanofibers. Various proteins, drugs and proteins can be easily loaded into microspheres. Therefore, in this study, microspheres consisting of biopolymers loaded with antibacterial agents and drugs will be produced. In this study, polypentadelactone-co-valerolactone copolymer synthesized by the immobilized enzyme. In this study, Candida antarctica B lipase was immobilized to rice husk ash, on which surface modifications were applied using the immobilization methods used in previous studies, primarily to be used in enzymatic polymerization reactions. ω- pentadecalactone-co-δ-valerolactone copolymer was produced by ring-opening polymerization using immobilized enzyme from ω-pentadecalactone and δ- valerolactone at different reaction times and temperatures, using monomer ratios of 75-25%. During support preparation and immobilization, RHA was produced by burning rice husks at 600-650°C for 6 hours. The surface of RHA was then modified using a silanization chemical called 3-aminopropyl triethoxysilane (3-APTES), and functional amine (-NH2) groups were added to the surface. Lipase immobilization was achieved by physical adsorption. Previous scientific investigations attempted to optimize novel immobilized lipases using various 3-APTES concentrations and enzyme loading ratios. These research studies are used as references. After copolymers is produced, they will be formed into microspheres and added with oleuropein as an antibacterial agent and loaded with trans-Chalcone as a drug for use in biomedical fields. A drug delivery system with strong mechanical properties, biocompatible, biodegradable, harmless to the environment and living things will be developed by incorporating the copolymer into the polymer-containing microspheres of the drug and antibacterial agent. ω-Pentadecalactone, or pentadecanolide, is a cyclic ester having a 15-carbon backbone. ω-pentadecalactone may have antibacterial and antioxidant properties. Its potential pharmacological qualities make it an attractive candidate for the development xxii of pharmaceutical formulations and nutraceuticals. δ-valerolactone, a lactone, is employed as a chemical intermediate in several processes, such as polyester manufacturing. Polyvalerolactone is a semi-crystalline aliphatic polyester that is hydrophobic. PVL is a well-known biopolymer that has several applications in medication formulation and delivery systems. PVL-based polymers have been employed as antifungal carriers as well as a hydrophobic block in amphiphilic block copolymers for the in vivo administration of chemotherapeutic medications such as daunorubicin (DNR), doxorubicin (DOX), and others. Microspheres are spherically shaped particles that can vary in size from one to a thousand meters. Microspheres are biodegradable, free-flowing particles made up of proteins or synthetic polymers. They are capable of encapsulating small molecules, proteins, peptides, and nucleic acids. They have various advantages over traditional dosage forms, including increased solubility of poorly soluble pharmaceuticals, protection against enzymatic and photolytic degradation, reduced dosing frequency, greater bioavailability, controlled release profile, dose reduction, and drug toxicities. Oleuropein appears to be an effective antibacterial agent. Oleuropein, the major phenolic component of the olive tree, is a chemical found in the fruit in the early stages of ripening, and its level diminishes as the fruit ripens as it is digested. Recent research indicates that oleuropein possesses anticancer, antiviral, antioxidant, and anti- inflammatory properties. Oleuropein will be employed as an addition in this investigation since it is considered to improve antibacterial activity and cell proliferation. Chalcones are open-chain chemicals found naturally in plants. The chemical structure is composed of two aromatic rings separated by a three-carbon α,β- unsaturated carbonyl system.. Trans-chalcone (TC) has grown in popularity in recent years for its biological properties due to its abundance in nature, simplicity of synthesis, and simple structure. TC has been demonstrated to have anticancer effects against a variety of types. TC is also anti-inflammatory, working by reducing the oxidative stress caused by a variety of inflammatory diseases. Many additional compounds are metabolically activated by TC. It has been demonstrated that these substances have estrogenic action. Due to the estrogenic action of xenobiotic chemicals, animals may experience a variety of negative health impacts, including obesity, accelerated female puberty, a decrease in sperm count, altered sexual behavior and reproductive organs, and an increased risk of certain cancers. Controlling the amount of TC treatment and preventing the buildup of TC molecules in the body are therefore crucial. The aim of this study is to develop a new drug delivery system by loading drug and adding antibacterial agent into the bio-based polymeric structure. Microspheres will be obtained by synthesizing a biocompatible, non-toxic and high molecular weight copolymer by using naturally immobilized enzyme to be compatible with the environment and human body. Controlled drug delivery will be carried out by loading a drug and adding an antibacterial agent to this product. Thus, the side effects of the drug will be reduced and its therapeutic properties will be increased. The lack of research in the literature on the use of oleuropein and transchalcone with microspheres for medical reasons adds to the scientific value of the study. The study's uniqueness stems from the lack of literature on the poly(ω-pentadecalactone-co-δ-valerolactone) copolymer produced by enzymatic polymerization. The copolymer synthesized using a biocatalyst will be loaded with oleuropein and trans-chalcone while microspheres are produced and it will be used as medicine. With this mixture, cell biological compatibility will be ensured and the drug will be ensured to reach the desired area at xxiii the desired time. As a result, a new drug delivery system will be created by using natural and synthetic polymers, drugs and antibacterial agents. In the second stage, a ω-pentadecalactone-co-δ-valerolactone copolymer was produced enzymatically using the monomer ratios from earlier research as a reference. The highest molecular weighted sample (Mn = 23722 g/mol) was obtained at 80°C and 24 hour reaction duration with 75% ω-pentadecalactone feed weight ratio and selected for microsphere formation. Therefore, in this work, ω-pentadecalactone-co-δ- valerolactone is synthesized utilizing these values. In the third stage of the study, oleuropein added and transchalcone loaded PDL-VL microspheres was tried to be produced via O/W emulsion method. In order to determine the highest encapsulation efficiency and drug release behavior, combinations of 10, 20 and 40 percent TC, as well as 42.5, 75 and 100 Olu, in proportion to the copolymer mass were examined. It was determined that microspheres produced at 100% Olu:PDL-VL ratio and 20% TC:PDL-VL ratio had the highest Encapsulation Efficiency (%) which it was 81.7 ± 0.5 (%). After microspheres are made, several characterization analysis were applied such as SEM, DSC, TGA, FTIR and XRD in order to understand thermal, mechanical and morphological properties of microspheres. DSC analysis was applied to observe the thermochemical changes of the copolymer and microspheres samples. Melting temperatures and enthalpy values of microspheres were examined according to the previous scientific studies. The fact that no melting peak was observed in both oleuropein and transchalcone samples indicates that PDL-VL/Olu and TC-loaded PDL-VL/Olu microspheres are properly dispersed into the structure as stated in the literature. TGA analyzes were applied in order to analyze the thermal degradation behavior of microspheres and compare with PDL-VL. FT-IR was used as a characterization method to observe the chemical groups indicating the presence of Olu, TC and microspheres. All the characteristic peaks were examined and explained. It was concluded that Olu and TC were encapsulated in the microspheres. In addition to all other analyses, the influence of TC loading on crystallinity and crystalline structures of microspheres was examined using XRD analysis. The Xc values were determined, and distinctive crystalline peaks were investigated. The results were similar with those obtained from the DSC. It can be seen from the SEM images that spherical geometry was found in all microsphere formulations. Antibacterial acitivty tests were also examined and it led to the conclusion that PDL-VL/Olu and TC-loaded PDL-VL/Olu microspheres have antibacterial properties. As a results of cytotoxicity anaylsis, it leads to a reduction in the viability of human breast cancer cell lines (MCF- 7), and therefore it is effective and promising for human breast cancer therapy. In this study, pH dependent drug release experiments were performed with two pH values which was 5.6 and 7.4 in order to see drug release behaviour of microspheres produced with different environments. The microsphere formulations improved the total cumulative release of TC, which reached 91.18 % in pH 5.6 media and 85.89 % in pH 7.4 media. The behavior of microspheres' release was based on pH; the more acidic the release medium, the greater the release. In all cases, TC release was carried out for up to 964 hours. Lastly, the release kinetics of the design points were investigated. When the release rate constants were assessed, it was discovered that the release suited the Korsmeyer-Peppas kinetic model, which had the highest correlation coefficient. After all characterization analysis and drug release behaviour were obtained, it can be concluded that the the results of this study point to a potential use for microspheres in the long-term therapy of disease. And, undoubtedly, much more study will be required to assess cytotoxicity, cell survival, and in vivo pharmacokinetics. xxiv xxv ENZİMATİK OLARAK SENTEZLENMİŞ BİR BİYOPOLİESTERİN BİYOMEDİKAL UYGULAMASI ÖZET Polimerler, terapötik ajanların uzun süreler boyunca sabit dozlarda kontrollü salınımını, döngüsel dozlamayı ve hem hidrofilik hem de hidrofobik ilaçların ayarlanabilir salınımını sağlayarak ilaç dağıtım teknolojisinin geliştirilmesinde bütünleyici bir rol oynamıştır. İlaç dağıtımındaki modern gelişmeler artık farklı biyolojik işlevler gerçekleştirmek üzere tasarlanmış polimerlerin rasyonel tasarımına dayanmaktadır. Bu ilaç sistemlerinin insan vücudunda oluşturduğu toksik birikimlerin azaltılması, yan etkilerinin ve çevreye etkilerinin en aza indirilmesi için enzim bazlı biyopolimer sentezlerine ihtiyaç duyulmaktadır. Sentetik polimerlerden farklı olarak enzimler kullanılarak doğal olarak üretilen biyopolimerler; Biyouyumlulukları, biyolojik olarak parçalanabilirlikleri, toksik olmamaları ve biyoaktif molekülleri adsorbe edebilmeleri nedeniyle tıbbi uygulamalara uygundurlar. Bu biyopolimerlerin ilaç taşıyıcı sistemlerde kullanımı cast film, mikroküre, nanopartikül ve nanolif gibi malzemelere dönüştürülmesiyle mümkündür. Çeşitli proteinler, ilaçlar ve proteinler mikrokürelere kolaylıkla yüklenebilmektedir. Bu nedenle bu çalışmada antibakteriyel madde ve ilaçlarla yüklü biyopolimerlerden oluşan mikroküreler üretilecektir. Bu çalışmada immobilize enzim ile polipentadelakton-ko-valerolakton kopolimeri sentezlendi. Bu çalışmada, öncelikle enzimatik polimerizasyon reaksiyonlarında kullanılmak üzere, önceki çalışmalarda kullanılan immobilizasyon yöntemleri kullanılarak yüzey modifikasyonları uygulanan pirinç kabuğu külüne Candida antarctica B lipazı immobilize edilmiştir. ω-pentadekalakton-ko-δ-valerolakton kopolimeri, %75-25 monomer oranları kullanılarak, farklı reaksiyon süreleri ve sıcaklıklarda, ω- pentadekalakton ve δ-valerolaktondan immobilize edilmiş enzim kullanılarak halka açılması polimerizasyonuyla üretilmiştir. Desteğin hazırlanması ve immobilizasyonu sırasında, pirinç kabuklarının 600-650°C'de 6 saat yakılmasıyla RHA üretilmiştir. Daha sonra RHA'nın yüzeyi, 3-aminopropil trietoksisilan (3-APTES) adı verilen bir silanizasyon kimyasalı kullanılarak değiştirildi ve yüzeye fonksiyonel amin (-NH2) grupları eklenmiştir. Lipaz immobilizasyonu fiziksel adsorpsiyonla sağlandı. Önceki bilimsel araştırmalar, çeşitli 3-APTES konsantrasyonlarını ve enzim yükleme oranlarını kullanarak yeni immobilize lipazları optimize etmeye çalışmıştır. Bu araştırma çalışmaları referans olarak kullanılmaktadır. Kopolimerler üretildikten sonra mikroküreler haline getirilecek ve biyomedikal alanlarda kullanılmak üzere üzerine antibakteriyel madde olarak oleuropein eklenecek ve bu mikroküreler ilaç olarak trans- kalkon ile yüklenecektir. İlacın polimer içeren mikroküreleri ve antibakteriyel ajanın içine kopolimer katılarak güçlü mekanik özelliklere sahip, biyouyumlu, biyolojik olarak parçalanabilen, çevreye ve canlılara zararsız bir ilaç dağıtım sistemi geliştirilecektir. xxvi ω-Pentadekalakton veya pentadekanolid, 15 karbonlu bir omurgaya sahip siklik bir esterdir. ω-pentadekalakton antibakteriyel ve antioksidan özelliklere sahip olabilir. Potansiyel farmakolojik nitelikleri, onu farmasötik formülasyonların ve nutrasötiklerin geliştirilmesi için çekici bir aday haline getirmektedir. Bir lakton olan δ-valerolakton, polyester üretimi gibi çeşitli işlemlerde kimyasal bir ara madde olarak kullanılır. Polivalerolakton, hidrofobik olan yarı kristalli alifatik bir polyesterdir. δ-valerolakton monomerinin polimerleştirilmesiyle üretilir. PVL, ilaç formülasyonu ve dağıtım sistemlerinde çeşitli uygulamalara sahip, iyi bilinen bir biyopolimerdir. PVL bazlı polimerler, daunorubisin (DNR), doksorubisin (DOX) ve diğerleri gibi kemoterapötik ilaçların in vivo uygulanması için amfifilik blok kopolimerlerde hidrofobik bir blokun yanı sıra antifungal taşıyıcılar olarak da kullanılmıştır. Mikroküreler, boyutları bir metreden bin metreye kadar değişebilen küresel şekilli parçacıklardır. Mikroküreler, proteinlerden veya sentetik polimerlerden oluşan biyolojik olarak parçalanabilen, serbest akışlı parçacıklardır. Küçük molekülleri, proteinleri, peptitleri ve nükleik asitleri kapsülleme yeteneğine sahiptirler. Zayıf çözünen farmasötiklerin artan çözünürlüğü, enzimatik ve fotolitik bozunmaya karşı koruma, azaltılmış dozlama sıklığı, daha yüksek biyoyararlanım, kontrollü salım profili, doz azaltımı ve ilaç toksisiteleri dahil olmak üzere geleneksel dozaj formlarına göre çeşitli avantajlara sahiptirler. Oleuropein etkili bir antibakteriyel madde gibi görünmektedir. Zeytin ağacının ana fenolik bileşeni olan oleuropein, olgunlaşmanın erken aşamalarında meyvede bulunan bir kimyasaldır ve meyve olgunlaştıkça sindirildikçe seviyesi azalır. Zeytin meyvelerinin kendine özgü acı tadından sorumludur. Son araştırmalar, oleuropeinin antikanser, antiviral, antioksidan ve antiinflamatuar özelliklere sahip olduğunu göstermektedir. Oleuropein, antibakteriyel aktiviteyi ve hücre proliferasyonunu iyileştirdiği düşünüldüğü için bu araştırmada ilave olarak kullanılacaktır. Kalkonlar bitkilerde doğal olarak bulunan açık zincirli kimyasallardır. Kimyasal yapı, üç karbonlu a,β-doymamış karbonil sistemi ile ayrılan iki aromatik halkadan oluşur. Trans-kalkon (TC), doğada bol miktarda bulunması, sentezinin basitliği ve basit yapısı nedeniyle biyolojik özellikleri nedeniyle son yıllarda popülerlik kazanmıştır. TC'nin çeşitli türlere karşı antikanser etkileri olduğu gösterilmiştir. Trans-kalkonun anti- leishmanial aktivitesi geniş çapta incelenmiştir. TC ayrıca antiinflamatuardır ve çeşitli inflamatuar hastalıkların neden olduğu oksidatif stresi azaltarak çalışır. Birçok ilave bileşik TC tarafından metabolik olarak aktive edilir. Bu maddelerin östrojenik etkiye sahip olduğu kanıtlanmıştır. Ksenobiyotik kimyasalların östrojenik etkisi nedeniyle hayvanlar, obezite, kadınlarda ergenliğin hızlanması, sperm sayısında azalma, cinsel davranışta ve üreme organlarında değişiklik ve bazı kanser risklerinde artış gibi çeşitli olumsuz sağlık etkileriyle karşılaşabilir. TC tedavisi miktarının kontrol edilmesi ve vücutta TC moleküllerinin birikmesinin önlenmesi bu nedenle çok önemlidir. Bu çalışmanın amacı biyo bazlı polimerik yapıya ilaç ve antibakteriyel ajan eklenerek yeni bir ilaç taşıyıcı sistem geliştirmektir. Çevreye ve insan vücuduna uyumlu olacak şekilde doğal olarak immobilize edilmiş enzim kullanılarak biyouyumlu, toksik olmayan ve yüksek molekül ağırlıklı kopolimer sentezlenerek mikroküreler elde edilecektir. Bu ürüne ilaç ve antibakteriyel madde eklenerek kontrollü ilaç dağıtımı gerçekleştirilecek. Böylece ilacın yan etkileri azalacak ve tedavi edici özelliği artacaktır. Oleuropein ve transkalkonun mikrokürelerle tıbbi amaçlarla kullanımına ilişkin literatürde araştırma bulunmaması, çalışmanın bilimsel değerini artırmaktadır. Taşıma sistemi verimliliğini artırmak için doğal ve biyo bazlı sentetik polimerler xxvii birleştirildi. Çalışmanın benzersizliği, enzimatik polimerizasyonla üretilen poli(ω- pentadekalakton-ko-δ-valerolakton) kopolimeriyle ilgili literatür eksikliğinden kaynaklanmaktadır. Biyokatalizör kullanılarak sentezlenecek kopolimer, oleuropein ve trans-kalkon ile yüklenerek mikroküreler üretilecek ve ilaç olarak kullanılacaktır. Bu oluşan karışım ile hücre biyolojik uyumu sağlanacak ve ilacın istenilen zamanda istenilen bölgeye ulaşması sağlanacaktır. Sonuç olarak doğal ve sentetik polimerler, ilaçlar ve antibakteriyel ajanlar kullanılarak yeni bir ilaç dağıtım sistemi oluşturulacaktır. Çalışmanın ikinci aşamasında, daha önceki araştırmalardan elde edilen monomer oranları referans olarak kullanılarak enzimatik olarak bir ω-pentadekalakton-ko-δ- valerolakton kopolimeri üretilmiştir. En yüksek moleküler ağırlıklı numune (Mn = 23722 g/mol) 80°C'de ve 24 saatlik reaksiyon süresinde %75 ω-pentadekalakton besleme ağırlık oranıyla elde edilmiştir ve mikroküre oluşumu için seçilmiştir. Dolayısıyla bu çalışmada bu değerler kullanılarak ω-pentadekalakton-ko-δ- valerolakton sentezlenmiştir. Daha sonra numunenin mikroküre üretiminde kullanılacağı belirlenmiştir. Çalışmanın üçüncü aşamasında oleuropein/transkalkon yüklü PDL-VL mikroküreleri O/W emülsiyon yöntemiyle üretilmilmiştir. En yüksek enkapsülasyon verimliliğini ve ilaç salım davranışını belirlemek amacıyla kopolimer kütlesine orantılı olarak yüzde 10, 20 ve 40 TC'nin yanı sıra 42,5, 75 ve 100 Olu'luk kombinasyonlar incelenmiştir. %100 Olu:PDL-VL oranında ve %20 TC:PDL-VL oranında üretilen mikrokürelerin en yüksek enkapsülasyon verimine (%) sahip olduğu ve 81,7 ± 0,5% olduğu belirlenmiştir. Mikroküreler sentezlendikten sonra PDL-VL, PDL-VL/Olu ve TC yüklü PDL-VL/Olu mikrokürelerinin, termal, mekanik ve morfolojik özelliklerini anlamak amacıyla SEM, DSC, TGA, FTIR ve XRD gibi çeşitli karakterizasyon analizleri uygulanmıştır. Kopolimer ve mikroküre örneklerinin termokimyasal değişimlerini gözlemlemek için DSC analizi uygulanmıştır. PDL-VL, PDL-VL/Olu ve TC yüklü PDL-VL/Olu mikrokürelerinin erime sıcaklıkları ve entalpi değerleri daha önce yapılan bilimsel çalışmalara göre incelenmiştir. Hem oleuropein hem de transkalkon numunelerinde herhangi bir erime zirvesinin gözlenmemesi, PDL- VL/Olu ve TC yüklü PDL-VL/Olu mikrokürelerinin literatürde belirtildiği gibi yapıya düzgün bir şekilde dağıldığını göstermektedir. TC yüklü PDL-VL/Olu ve PDL- VL/Olu mikrokürelerinin termal bozunma davranışını analiz etmek ve PDL-VL ile karşılaştırmak için TGA analizleri uygulanmıştır. FT-IR analizleri, Olu, TC, PDL-VL, PDL-VL/Olu ve TC yüklü PDL-VL/Olu mikrokürelerinin varlığını gösteren kimyasal grupları gözlemlemek için bir karakterizasyon yöntemi olarak kullanılmıştır. Tüm karakteristik zirveler incelenmiştir ve açıklanmıştır. Olu ve TC'nin mikroküreler içerisinde enkapsüle olduğu sonucuna varılmıştır. Diğer tüm analizlere ek olarak, TC yüklemesinin mikrokürelerin kristalliği ve kristal yapıları üzerindeki etkisi XRD analizi kullanılarak incelenmiştir. Xc değerleri belirlenmiştir ve ayırt edici kristal tepe noktaları araştırılmıştır. Sonuçlar DSC'den elde edilenlerle benzer olarak belirlenmiştir. SEM görüntülerinden tüm mikroküre formülasyonlarında küresel geometrinin bulunduğu görülmektedir. Antibakteriyel aktivite testleri de incelenmiştir ve TC yüklü PDL-VL/Olu ve PDL-VL/Olu mikrokürelerinin antibakteriyel özelliklere sahip olduğu sonucuna varılmıştır. Sitotoksisite analizi sonucunda, elde edilen maddenin insan meme kanseri hücre hatlarının (MCF-7) canlılığında azalmaya yol açmakta olduğu görülmüştür ve bu nedenle insan meme kanseri tedavisinde etkili ve umut verici olduğu söylenebilir. Bu çalışmada, farklı ortamlarda üretilen mikrokürelerin ilaç salım davranışlarını görmek amacıyla 5,6 ve 7,4 olmak üzere iki pH değerinde pH'a bağlı ilaç salım xxviii deneyleri yapılmıştır. Mikroküre formülasyonları, pH 5.6 ortamda %91.18'e ve pH 7.4 ortamda %85.89'a ulaşan toplam TC kümülatif salınımını iyileştirmiştir. Mikroküreciklerin salınma davranışı pH'a dayanmaktadır; Salım ortamı ne kadar asidik olursa, salınım da o kadar fazla olur sonucuna varılmıştır. Tüm durumlarda TC salımı 964 saate kadar gerçekleştirilmiştir. Son olarak tasarım noktalarının salınım kinetiği araştırılmıştır. Salınım hızı sabitleri değerlendirildiğinde salınımın en yüksek korelasyon katsayısına sahip olan Korsmeyer-Peppas kinetik modeline uygun olduğu keşfedilmiştir. Tüm karakterizasyon analizleri ve ilaç salınım davranışı elde edildikten sonra, bu çalışmanın sonuçlarının, mikrokürelerin hastalıkların uzun vadeli tedavisinde potansiyel bir kullanıma işaret ettiği sonucuna varılabilir. Ve hiç şüphesiz sitotoksisiteyi, hücre sağkalımını ve in vivo farmakokinetiği değerlendirmek için çok daha fazla çalışmaya ihtiyaç duyulacaktır. 1 1. INTRODUCTION As the environment becomes more polluted, there is a growing interest in developing biodegradable polymers. Aliphatic polyesters are particularly interesting due to their versatile characteristics and diverse synthesis techniques. There are two methods for producing biodegradable polyesters. The first method involves polycondensation of a hydroxyl acid or diol with a diacid, which often results in low molecular weight polymers (<30 kDa). To achieve greater molecular weight polymers, ring-opening polymerization (ROP) is typically used as a second technique. Lactone monomers used in this reaction include ɛ-caprolactone (ɛ-CL), ω-pentadecalactone (ω-PDL), and lactide. Polylactones and PLAs have biocompatibility, mechanical resilience, and biodegradability, making them suitable for biomedical applications such vascular implants, suture materials, and tissue engineering scaffolds. Biodegradable polymers are being studied for their potential to carry and release drugs in the body. Biodegradability allows for regulated drug release over time. Extensive research on enzymatic ROP (eROP) has emerged in response to the increased need for ecologically benign and heavy metal-free goods. Enzymes are not only non-toxic, but also capable of catalyzing specific reactions. This eliminates the need to protect or deprotect functional groups, allowing for selective synthesis of polymers from chiral monomers [1]. Lipases (glycerol ester hydrolase, E.C. 3.1.1.3) catalyze esterification, hydrolysis, and transesterification, producing a variety of useful chemicals under moderate circumstances. These enzymes have unique interfacial kinetics, resulting in strong catalytic activity at the water-oil interface that decreases dramatically in bulk water or oil phases. Uppenberg, Hansen, Patkar, and Jones (1994) initially identified the three- dimensional structure and amino acid sequence of Candida antarctica lipase B. CALB has excellent catalytic activity for both water-soluble and insoluble compounds. CALB's regioselectivity and chiral selectivity enable it to catalyze reactions in the organic phase while maintaining excellent stability [2]. 2 Novel drug delivery technologies have several advantages over standard multi-dose treatment. According to recent trends, micro particulate drug delivery systems are particularly well suited to controlled release and delayed-release oral formulations with low dose dumping, blending flexibility to achieve different release patterns, and reproducible and short gastric residence time [3]. Drugs delivery techniques have advanced, especially those that allow the medicine to work in the intended effect area for a longer period of time under regulated conditions. These cutting-edge drug delivery methods can deliver drugs to a particular place, alter the rate at which they are supplied, and/or extend the therapeutic impact [4]. One of the most advanced techniques for maintaining and regulating pharmacological activity in a particular place is the use of microspheres as drug carriers. To offer temporary embolization, microspheres composed of biodegradable components are used. Theoretically, after serving their therapeutic purpose, they ought should be eliminated from the body without affecting the functionality of other organs [5]. The European Medicines Agency (EMA) has linked the phenolic acid oleeuropein, which is found in large quantities in the leaves and fruits of the Olea europaea plant, to a number of health benefits for people [6]. Because of its well-established anti- inflammatory and antioxidant characteristics, oleeuropein may also have antiviral, antimicrobial, and neuroprotective qualities [7]. When loaded onto microspheres, it can be utilized as an antibacterial agent and in the treatment of diseases [8]. Trans-chalcone (TC), also known as 1,3-diphenyl-2-propen-1-one, is a flavonoid found in plants and a primary precursor to other flavonoids. Depending on the illness model, TC has anti-inflammatory and antioxidant properties. [9] They exhibit many biological activities, including antibacterial, antihelmintic, anticancer, antifungal, antidiabetic and amoebic properties. Previous research indicates that chalcones have potent antileishmanial and antimalarial properties These medications disrupt the parasite's mitochondrial structure and function. Recent research indicates that chalcones reduce parasite respiration and mitochondrial dehydrogenase activity [10]. In this study, it was aimed to produce biocompatible drug loaded and antibacterial agent added microspheres. Oleuropein will be used as an antibacterial agent and trans- chalcone will be used as an drug. Initially, the surface of the support material was silanized with the agent 3-APTES, as reported in earlier investigations. Following that, CALB was immobilized on surface-modified RHA using a physical adsorption 3 method, as previously described [11]. In the second stage of the study, ω- pentadecalactone-co-δ-valerolactone copolymer was effectively synthesized from ω- pentadecalactone and δ-valerolactone with CALB immobilized to RHA, using the techniques and parameters in earlier studies. [11]. In the third stage of the study, oleuropein/transchalcone loaded PDL-VL microspheres was tried to be produced via O/W emulsion method. Microspheres with the highest encapsulation efficiency were identified and used to study drug release behavior. After microspheres are made, several characterization analysis were applied such as scanning electron microscope (SEM), differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), fourier transform infrared spectroscopy (FTIR) and X-Ray diffraction analysis (XRD) in order to understand thermal, mechanical and morphological properties of PDL-VL, PDL-VL/Olu and TC-loaded PDL-VL/Olu microspheres. Antibacterial tests of PDL- VL, PDL-VL/Olu and TC-loaded PDL-VL/Olu microspheres were performed on gram positive Staphylococcus aureus, and gram negative Escherichia coli bacteria. Antibacterial activities (%) for all the samples were determined. The human breast cancer cell (MCF-7) and microspheres loaded with oleuropein at 1:1 (wt%) with copolymer concentrations and TC at 1:5 (wt%) with copolymer concentrations were used for in vitro cytotoxicity tests (WST), where the highest cytotoxicity was observed at these concentrations. In addition, a drug release study was conducted for TC-loaded PDL-VL/Olu microspheres and The drug release profiles were fitted to several kinetic models to better understand the process of drug release. After all characterization analysis and drug release behaviour were obtained, it can be concluded that the the results of this study point to a potential use for microspheres in the long-term therapy of disease. 4 5 2. LITERATURE REVIEW 2.1 Biopolymers Biopolymers are biologically produced polymers as opposed to synthetic ones. Because of their non-toxicity, biocompatibility, biodegradability, and capacity to adsorb bioactive compounds, they are well-suited for use in medical applications. The need for biomaterials and, consequently, biopolymers has grown steadily as a result of the environmental and usage issues associated with the use of synthetic polymers. New approaches have also been developed to produce biomaterials that are even more efficient. Because of this tremendous interest, biopolymers have become increasingly popular as it has become apparent that they have a wide range of uses in the medical industry, including tissue engineering and drug delivery systems [12]. Furthermore, increasing evidence shows that enzyme-mediated catalytic bioprocesses exhibit numerous advantages over traditional synthetic approaches. Additionally, biocatalysts are environmentally friendly as they require minimal use of chemicals and do not produce any toxic byproducts during the reaction. Because, unlike traditional chemical synthesis and physical modification, enzymatic polymerization is an emerging biologically based alternative approach for producing polymeric products [13]. Biopolymers, which are essential to modern sustainable material science, have their origins in prehistoric societies that used natural materials such as plant fibers, animal skins, and resins for a variety of uses. Since the development of contemporary biotechnology and the increased awareness of the need for a sustainable environment, attention has turned to biopolymers made from biomass sources that are renewable, such as microbes, plants, and animals as it can be seen from Figure 2.1. These biopolymers are a varied range of materials with special qualities and uses, generally divided into polysaccharides, proteins, nucleic acids, and biopolyesters [14]. Long chains of sugar molecules called polysaccharides are widely found in nature and play a crucial role in the functioning of living things. One of the prominent polysaccharides is cellulose, which is known to contribute to the strength and stiffness 6 of plant cell walls by serving as their structural basis. Another well-known member of this group is starch, which is used extensively as a staple food by both humans and animals. It is the main energy storage form in plants. Chitin provides these creatures with structural support and defense. It is mostly present in the cell walls of fungi and the exoskeletons of arthropods. Alginate is mainly produced from seaweed and has a variety of uses in the food and pharmaceutical industries. It can be used as a thickening and gelling agent in many kinds of formulations. These polysaccharides, distinguished by their diverse structures and functionalities, play integral roles across a spectrum of applications spanning biomedicine, food science, agriculture, and materials engineering [15]. Proteins, such as collagen, gelatin, and silk fibroin, are made up of amino acids and are useful in medical applications like tissue engineering and drug delivery systems due to their biocompatibility [16]. Nucleic acids, such as DNA and RNA, serve as the blueprint for life and are being researched for utilization in biotechnology and drug delivery. Furthermore, polyhydroxyalkanoates (PHA) and biopolyesters such as poly(lactic aci d) (PLA) provide fascinating alternatives to traditional plastics, providing biodegrada bility and renewability while maintaining mechanical qualities suited for a variety of applications [17]. Classification of biobased polymers [18]. 7 Polyesters, a type of biopolymer, have major applications due to their versatility and eco-friendliness. Poly(lactic acid) (PLA) is a widespread example, generated from renewable resources such as cornstarch or sugarcane [19]. PLA has qualities similar to typical petroleum-based polymers, but it is also biodegradable and compostable under the right conditions, making it ideal for packaging, biomedical applications, and 3D printing [19]. Moreover, Polyhydroxyalkanoates (PHA), a further significant class of biodegradable polyesters, are produced through microbial fermentation of renewable carbon sources such as sugars or lipids [20]. PHA polymers have mechanical characteristics similar to regular plastics, but they can be broken down by microorganisms in a variety of environments, making them interesting candidates for biomedical applications [20]. 2.2 Lipase Enzyme Lipases (EC 3.1.1.3) are triacylglycerol acylhydrolases that break down carboxylic ester linkages and belong to the hydrolase family. These enzymes are classified as serine hydrolases and don't require a cofactor [20]. Lipases primarily hydrolyze triglycerides into fatty acids and glycerol, but can also catalyze esterifications and interesterifications in non-aqueous environments. Lipases have a unique mode of activation at the interface. Lipases typically have an α-helical oligopeptide structure that covers the active site (lid or flap) and prevents substrates from reaching it [21]. When there is no hydrophobic interface, the enzyme's active site remains concealed from the reaction media. This enzyme shape is known as the "closed conformation". In contrast, when a hydrophobic interface occurs, the enzyme undergoes a conformational shift to expose its catalytic triad to the hydrophobic phase. This enzyme conformation shift occurs in the "open conformation". Thus, the activation mechanism of lipase is referred to as "interfacial activation" [21]. These forms are given in Figure 2.2. Lipases catalyze the hydrolysis of ester bonds at the interface of an insoluble substrate and an aqueous phase, where the enzymes remain liquefied under normal circumstances. Pseudomonas aeruginosa, Candida Antarctica B, and Burkholderia glumae had a lid but did not exhibit interfacial activation. Lipases perform several conversion reactions, including esterification, transesterification, interesterification, acidolysis, alcoholysis, and aminolysis [20]. 8 Free lipase open and closed form [22]. Lipase possesses significant selectivity (regio-, enantio-, stereo-, and chemo- selectivity), can catalyze at the interface of aqueous and organic phases, and interacts with a wide range of substrates. Lipase's unique behavior allows for the development of lipase-catalyzed polymerization processes. Lipase-catalyzed ROP of lactones, lactides, and cyclic carbonates can yield aliphatic polyesters or polycarbonates [23]. Lipases are commonly utilized in industry due to their ability to catalyze a wide range of reactions. These unusual catalysts may catalyze reactions across immiscible organic and aqueous phases and maintain catalytic activity in organic solvents. Immobilization can improve the activity, selectivity, and operational stability of enzymes in both aqueous and non-aqueous solvents, allowing for more efficient utilization [11]. Candida antarctica lipase B (CALB) is an efficient and selective lipase capable of catalyzing esterification and transesterification processes [11]. It is immobilized on macroporous acrylic resin and marketed as Novozyme 435®. In this study, free CALB was immobilized on a new support to produce an alternative to Novozyme 435® [23]. 2.3 Enzyme Immobilization Lipases' commercial usage has been limited because to their expensive cost. However, immobilization methods using solid supports may overcome this. Immobilization improves product separation and allows for more flexible enzyme/substrate interactions in different reactor setups. Furthermore, immobilizing enzymes on solid substrates can enhance stability and selectivity [21]. There are several techniques for immobilizing enzymes. As it is shown in Figure 2.3, the most frequent methods are adsorption, trapping, and cross-linking to a support. Adsorption is the process of physically adsorbing an enzyme onto a support material, such as a polymer matrix or 9 inorganic support. Enzyme entrapment occurs when it becomes trapped within a material's lattice structure or polymer ns. While this reduces enzyme leaching and enhances stability, it can also restrict substrate delivery to the active site. Enzymes can be immobilized by cross-linking them to an insoluble support or covalently attaching them to a functionalized support [11]. Immobilization of enzymes on supports (carriers) is a process used to improve the activity, selectivity, and stability of biocatalysts. Furthermore, the usage of solid supports can substantially aid in the extraction of an enzyme from the reaction solution [24]. The activity of immobilized lipase is substantially determined by the enzyme molecules, the immobilization process, the reaction media, and the kind of support. It has been shown that the intensity of enzyme-support, enzyme-substrate (in the reaction media), and support- substrate interactions influences the stability and specificity of biocatalysts. It has also been demonstrated that the reaction media can influence the enzyme-substrate molecular recognition mechanism. Interestingly, a good material for enzyme immobilization can trap substrates by hydrophobic or electrostatic coupling, so contributing to a favorable partition effect of the substrate in the reaction media [24]. Immobilization methods of enzymes [25]. To achieve physical adsorption, immerse the support in an enzyme solution and incubate for several hours. Enzymes are absorbed into the matrix via weak non- specific forces such hydrogen bonding, Van der Waals forces, and hydrophobic interactions. However, mild nonspecific forces can cause reversible enzyme leakage from the matrix by altering the parameters that affect contact strength, such as pH, 10 ionic strength, temperature, or solvent polarity [25]. Covalent binding is a common approach for immobilizing enzymes. It involves creating stable complexes between enzyme functional groups and a support matrix. Enzyme functional groups suitable for covalent coupling include amino, carboxylic, phenolic, sulfhydryl, thiol, imidazole, indole, and hydroxyl groups. Enzyme binding to solid supports involves two stages: activating the surface using linker molecules like glutaraldehyde or carbodiimide, and then covalently attaching the enzyme to the activated support [25]. Cross-linking immobilization involves covalent connection between enzymes without the need of carriers. Cross-linking immobilization strengthens the linkage between enzymes, resulting in improved stability [26]. Entrapment is an irreversible immobilizing process. This approach prevents enzyme leakage by regulating the pore size of the polymeric network, allowing for unrestricted diffusion of reaction contents (substrates or products). This technique prevents denaturation by not allowing enzymes to react with polymers. The entrapment process has several benefits, including increased enzyme loading capacity, cheaper manufacturing costs, improved mechanical stability, and less mass transfer. The encapsulating material may be modified to create an appropriate microenvironment with desired pH, polarity, or amphilicity [26]. Encapsulation is similar to entrapment in that enzymes are enclosed in a polymer matrix. However, the polymer support matrix comprises "pockets" or "pores" to immobilize enzymes. Encapsulated enzymes enhance enzymatic activity by altering hydrophobic interactions, increasing reaction surface area, and boosting intermediate concentration. They are more stable in a range of conditions. They are frequently used in fields including biocatalysis, biosensing, enzyme therapy, biomedicine, and bioremediation [27]. Lipases are often immobilized on supports through adsorption via hydrophobic interactions because it is inexpensive and increases enzyme activity. For example, the most widely used commercial enzyme, Novozyme 435®, is produced by physical adsorption of CALB on porous acrylic resin [23]. 2.3.1 Support materials for enzyme immobilization The immobilized enzyme system's performance relies heavily on the matrix's properties. Ideal support features include compression resistance, hydrophilicity, enzyme inertness, ease of derivatization, biocompatibility, microbiological resistance, 11 and cost-effectiveness [28]. Furthermore, it should have the potential to enhance enzyme activity and specificity [23]. As it can be seen from Table 2.1, supports can be classified as inorganic or organic based on their chemical structure. Organic supports can be separated into natural and synthetic polymers [28]. Classification of supports used for enzyme immobilization [23]. Chemical Composition Example Organic Natural polymers: Polysaccharides (cellulose, dextrans, agar, agarose, chitin, chitosan, alginate), proteins (collagen, albumin) Synthetic polymers: Polystyrene, polyacrylate, polymethacrylates, polyacrylamide, polyamides, vinyl, and allyl-polymers Polysaccharides (cellulose, dextrans, agar, agarose, chitin, chitosan, alginate), proteins (collagen, albumin) Inorganic Zeolites, ceramics, celite, silica, bentonite, clay, alumina, glass, activated carbon Among the support materials which is used for enzyme immobilization, silica is the most extensively utilized support material for lipase immobilization due to its large specific surface area, biocompatibility, and inexpensive cost. Furthermore, the surface could be simply adjusted [23]. As it is written before, the most widely used immobilized lipase is Novozyme 435®, a commercially available immobilized version of CALB. This catalyst is efficient and adaptable, using a porous acrylic resin as the enzyme carrier [29]. Rice beneficiation produces rice husk as a byproduct, accounting for around 23% of the original weight. Rice husk's high silicon concentration makes it a valuable source for producing elementary silicon and other silicon compounds, including silica, silicon carbide, and silicon nitride [30]. Rice husk is a composite substance composed from lignin, cellulose, hemicellulose, and silicon dioxide [31]. Rice husks include 80% organic and 20% inorganic materials. RH's primary elemental components are C (37.05 wt.%), H (8.80 wt.%), N (11.06%), Si (9.01 wt.%), and O (35.03%). RH 12 contains 24.3% hemicellulose, 34.4% cellulose, 19.2% lignin, 18.85% ash, and 3.25 percent other substances [32]. Rice husk is a desirable material for industrial applications due to its high silica concentration (87-97 wt.% SiO2), high porosity, lightweight nature, and large exterior surface area [32]. Figure 2.4 shows the applications of rice husk for lipase enzyme. Rice husk ash is typically formed during the calcination process of rice husks. Rice husk ash is a cost-effective way to immobilize enzymes. It is more efficient, renewable, and includes 91.43% SiO2 [31]. As a result of molecule adsorption, the silica compound in the amorphous state has a tiny particle size and a large surface area. One kind of amorphous silica is silica gel, which may be manufactured via sol-gel. Silica gel is commonly employed as a precursor because of its excellent thermal stability, inability to expand, inertness, and transparency. Silica sol-gel matrices can be utilized to encapsulate enzymes without affecting their structure. Enzymes are stabilized by silica matrix encapsulation, which forms a scaffolding around them to prevent aggregation, dissociation, and destruction [31]. Applications of rice husk for lipase enzyme [33]. As it is shown in Figure 2.5, Silica consists of siloxane (Si-O-Si) and silanol (Si-OH) groups. Silanol groups are scattered on the surface, whereas siloxane groups are found within. Chemical changes are made to the silanol groups on the surface. Organosilane agents modify surfaces by silanization [23]. Among the efficient silanization coupling agents are 3-aminopropyl triethoxysilane (APTES) and 3-glycidyloxypropyl trimethoxysilane (GPTMS); their effective performance is ascribed to the distinct 13 qualities supplied by their bifunctional features [34]. APTES is the most widely used organosilane for functionalizing oxide surfaces. Oxide surfaces include hydroxyl groups (-OH) with high surface energy, allowing for fast interaction and covalent bonding with silane molecules [35]. Density functional theory (DFT) analysis indicates that the protonated amines (-NH3+) in APTES interact strongly with Si surface silanols (-55.2 kcal/mol). The amine head of APTES binds strongly to the silica surface, making its silanols available for covalent condensation with neighboring SiNPs or self-condensation with unreacted APTES molecules. This promotes particle agglomeration at high APTES loading [34]. After activation of silica particles with organosilane agents, enzyme can be covalently immobilized on these particles [23]. Immobilization of lipase on APTES/GA-activated silica surface [36]. 2.4 Biopolymer Production The biopolymer production process is divided into four categories based on the intended outcome and the material utilized. These techniques include chemical synthesis, bacterial synthesis, biopolymer blends, and gathering from renewable resources [37]. Ring-opening polymerization (ROP) of cyclic esters has resulted in biodegradable polyesters, providing a sustainable alternative to standard polyester materials [38]. The ring-opening polymerization (ROP) technique is used to produce large-scale biopolymers such as PLA, PHA polyesters, and cyclic polyesters via chemical processes. This polymerization is selected because it allows exact control of polymer molar mass. A catalyst, such as a metal ion, is required to initiate the polymerization reaction. Ring-opening polymerization often employs metal catalysts such as tin (II) octoate [Sn(Oct)2] or tin(II) butoxide, as well as lithium, magnesium, 14 zinc, and zirconium [37]. Polymers like polycaprolactone and polylactide have been used in many applications. The limited commercial availability of cyclic esters limits the microstructure and characteristics of the resultant polymers. In recent years, ring- opening copolymerization (ROCOP) of epoxides and cyclic anhydrides has arisen as an alternative to ROP of cyclic esters to generate a wider range of polyester materials with different microstructures, characteristics, and uses [38]. Due to the scarcity of group 1A metals such as lithuim and the development of hazardous organometallic intermediates as a result of the reaction, the application of the produced compounds in medical domains has been limited, and several catalysis approaches have been attempted. Using bioresorbable salts, devoid of harmful metallic residues, for the ROP of cyclic lactones is an alternative method for producing aliphatic polyester. These catalysts contain cations such as Na+, K+, Mg+2, Ca+2, Zn+2, and Fe+2. These metals are referred to as "friendly metals". These cations have been examined using a variety of counter ions, including chloride, ionide, oxide, hydroxide, carbonate, acetate, lactate, tartrate, and citrate [23]. Although some chemical catalysts efficiently polymerize lactones and can be employed in food applications, they are unsuitable for biomedical applications. Therefore, it is vital to enhance enzymatic polymerization [23]. Since it does not produce harmful organometallic intermediates and lowers the reaction conditions to a more ideal level, enzymatic ring-opening polymerization has been an effective polymerization technique [23]. Enzymatic ROP (eROP) has been the subject of much research due to the increased interest in ecologically friendly processes and products free of heavy metals. In addition to their non-toxic nature, enzymes are also capable of catalyzing stereoselective and regioselective reactions. Thus, polymers from chiral monomers may be produced selectively and functional groups do not need to be protected or deprotected. Figure 2.6 shows the synthesis of polyester materials via ROP and it can be seen from the Figure 2.7 that which type of materials are used for lipase-catalyzed ROP. While many of the heavy metal catalysts used in ROP cannot be recycled, a variety of enzyme immobilization techniques allow for the recycling of enzymes, particularly lipases, which are the primary biocatalysts utilized in eROP as it be seen from Figure 2.8 [39]. 15 Synthesis of polyester materials via (a) ROP of cyclic esters; (b) ROCOP of epoxides and cyclic anhydrides [38]. Lipase-catalyzed ring-opening polymerization [40]. Mechanism of lipase-catalyzed ring-opening polymerization of lactones [40]. Lipase has been used to accelerate the polymerization of lactides in organic solvents like toluene. In enzymatic polymerization studies, it is preferable to use enzymes like 16 Candida antarctica lipase B (CALB) either in free form or immobilized on acrylic resin (Novozyme 435®). It has been noted that at temperatures where the polymerization reaction occurs, lipase becomes more durable and active when immobilized on organic or inorganic surfaces. Because of these benefits, the immobilization procedure of the enzyme—which also directly boosts efficiency—has become a widely utilized scientific approach for producing biopolymers at a lower cost [37]. 2.4.1 Poly (ω-Pentadecalactone -co-Valerolactone) Aliphatic polyesters are a class of biocompatible and biodegradable polymers with applications in medicine, pharmacology, and the environment. Hence, they have drawn a lot of research interest. [Chang]. According to the research studies, ω- pentadecalactone (ω-PDL), ε-decalactone, δ-valerolactone (δ-VL), and ε-caprolactone (ε-CL) are the most famous and studied ones in order to their properties [41]. The polymerization reactions of macrolactones are primarily driven by entropy due to their low ring strain and bigger size. Traditional chemical techniques struggle to polymerize macrolactones, leaving only a few efficient catalysts [42]. Typically, macrolactones are converted to aliphatic polyesters using organometallic catalysts like stannous octoate and zinc lactate [41]. As a result of using metallic catalysts in the polymerization of these products, some unwanted toxic products would be produced [43]. Using enzymes to catalyze ring-opening polymerization instead of organometallic catalysts eliminates the possibility of hazardous metal contaminants in the final product, making it suitable for biomedical applications [41]. Lipases are commonly utilized in industry due to their ability to catalyze a wide range of reactions. These unusual catalysts may catalyze reactions across immiscible organic and aqueous phases and maintain catalytic activity in organic solvents. Immobilization can improve the activity, selectivity, and operational stability of enzymes in both aqueous and non- aqueous solvents, allowing for more efficient utilization [11]. Candida antarctica lipase B (CALB) has strong catalytic activity for both water-soluble and insoluble compounds. CALB's regioselectivity and chiral selectivity make it an efficient catalyst for organic reactions with high stability. It can be seen from the studies that CALB immobilization on a carrier improved its stability, increased the number of uses, and made separation from the reaction system easier, all of which contributed to a decrease in processing costs [44]. 17 2.4.1.1 ω-Pentadecalactone ω-Pentadecalactone is a fragrance component used in decorative cosmetics, fine scents, shampoos, toilet soaps, face cream, fragrance cream, various toiletries, and non-cosmetic items including home cleansers and detergents [45]. ω- Pentadecalactone, also known as pentadecanolide, is a cyclic ester with a 15-carbon backbone. ω-pentadecalactone may have biological actions including antibacterial and antioxidant effects. Molecular structure of ω-Pentadecalactone is given in Figure 2.9. Its potential pharmacological properties make it a suitable option for the development of pharmaceutical formulations and nutraceutical products. In polymer chemistry, ω- pentadecalactone serves as a monomer for the synthesis of biodegradable polyesters, particularly poly(ω-pentadecalactone) (PPDL). Poly(ω-pentadecalactone) (PPDL) is a semicrystalline polymer derived from pentadecanolide or ω-pentadecalactone (Poly(ω-pentadecalactone), a commercially available macrolactone commonly utilized in the fragrance industry [42]. PPDL, an important poly(ω-hydroxy fatty acid), is a crystalline polymer with 14 methylene groups and in-chain ester group linkage in each repeating unit. It has a high melting point of around 100 °C. Furthermore, PPDL has mechanical qualities similar to linear high-density polyethylene (HDPE). PPDL research is gaining popularity due to its potential for industrial-scale manufacturing, including biodegradability, thermal stability, and mechanical characteristics [46]. Before recent studies with lipase-catalyzed polymerizations, only low molecular weight poly(PDL) was accessible, leading to limited interest in its characteristics and uses. Recently, high molecular weight poly(PDL) was made available using lipase catalysis, prompting a review of its characteristics [47]. Molecular structure of ω-Pentadecalactone [45]. 2.4.1.2 δ-Valerolactone A lactone called δ-valerolactone is used as a chemical intermediary in several processes, including the manufacture of polyesters. When polymerized, this monomer—which is derived from sugars—has characteristics similar to rubber [48, 49]. δ-valerolactone is a renewable monomer that is highly soluble in water. 18 Polyvalerolactone is a semi-crystalline aliphatic polyester that is hydrophobic. It is created by polymerizing δ-valerolactone monomer that the molecular structure is given in Figure 2.10. PVL is a well-established biopolymer with several uses in drug formulation and delivery systems. PVL-based polymers have been used as carriers for antifungal medicines and as a hydrophobic block in amphiphilic block copolymers for in vivo delivery of chemotherapy drugs like daunorubicin (DNR), doxorubicin (DOX), and others [50]. PVL's features include hydrophobicity, crystal structure, low melting point, and minimal cytotoxicity. PVL is also compatible with other polymers. New research and experimental results are required to achieve the best outcomes from the synthesis of these polymers. For this objective, fresh and novel copolymerization syntheses are being tried [51]. Molecular structure of δ-Valerolactone [52]. 2.5 Applications of Biopolymers Recent research has shown that biopolymers have potential applications as materials for medical device manufacture. These biomaterials are best identified by their molecular weight, lubricity, material chemistry, water absorption degradation, form and structure, solubility, hydrophilicity/hydrophobicity, erosion process, and surface energy. Biopolymers are used in a variety of industries, including pharmaceutical encapsulation, food packaging, agriculture, cosmetics, water treatment, biosensors, and data storage (see Figure 2.11). Polysaccharide-based materials are used in several sectors, including films, membranes, fibers, hydrogels, food packaging, sponges, and air gels [53]. Biopolymers are a diverse class of materials obtained from natural sources that provide a wide range of biomedical applications due to their inherent biocompatibility and biodegradability. These polymers have received a lot of interest in drug delivery systems, with chitosan, alginate, gelatin, and hyaluronic acid standing out as promising options for encapsulating and delivering pharmaceuticals precisely and efficiently [54]. Furthermore, biopolymers play an important part in tissue engineering, where scaffolds made of collagen, fibrin, gelatin, and alginate produce a 19 biomimetic milieu that promotes cell proliferation and tissue regeneration [55]. Such scaffolds are useful for the repair and regeneration of a variety of tissues, including bone, cartilage, skin, and heart tissue, opening up new possibilities for regenerative medicine [56]. Furthermore, biopolymer-based wound dressings, which incorporate ingredients like chitosan, alginate, collagen, and hyaluronic acid, assist in wound healing by providing moisture retention, antibacterial activity, and encouraging hemostasis. These dressings treat various wound types while promoting tissue healing and regeneration. [57]. Biopolymers can also be used as absorbable sutures and staples in surgical procedures that need precise tissue approximation. Materials like polyglycolic acid (PGA), and polylactic acid (PLA), and their copolymers provide mechanical strength for wound closure while gradually being reabsorbed by the body, eliminating the need for suture removal operations [58]. Furthermore, biopolymers show promise in orthopedic implants, dental materials, and a wide range of other biomedical applications, demonstrating their versatility and potential to advance medical technologies and treatments [59]. Applications of biopolymers [53]. 2.6 Drug Delivery Systems Drug delivery systems (DDS) are used to make and store drug molecules into tablets or liquids for administration [60]. Traditional dosage forms have a high dose and limited availability, inconsistency, first-pass effect, variation of plasma drug levels, and rapid release of medicinal compounds. Drug delivery systems will tackle the issues 20 through improved performance, protection, patient compliance, and product shelf life [61]. They speed up the delivery of drugs to the precise intended spot in the body, enhancing therapeutic efficacy while decreasing off-target accumulation [60]. Smart drug delivery systems (SDDS) direct the release of active molecules to the target location of action in response to biological or physical stimulation. These systems aim to regulate release kinetics and minimize negative effects. Systems built of stimuli- responsive polymers can alter characteristics based on environmental variables. Minor environmental changes can significantly impact polymer properties, including physical state, shape, solubility, solvent interactions, and hydrophilic/lipophilic balance. Polymers that respond to light, temperature, pH, or redox can be used to create these systems [62]. Drugs can be administered by numerous routes, including oral, buccal, sublingual, nasal, ocular, transdermal, subcutaneous, anal, transvaginal, and intravenous [60]. Figure 2.12 shows the concentration as a function of time after administration for the controlled release. Targeted drug delivery transports therapeutic agents to specified regions without affecting other parts of the body. This method targets specific parts of the body for drug delivery. This improves therapy efficacy while reducing negative effects [63]. Advancements in drug delivery technology impact bioavailability, absorption, and pharmacokinetics. Drug targeting involves four principles: loading the drug, avoiding degradation by bodily fluids, reaching the target region, and releasing the drug at a preset time [63]. Concentration as a function of time after administration [64]. Biodegradable and bioerodible polymers play a significant role in drug delivery [66]. Over the past 30 years, biodegradable polymeric drug delivery systems (DDS) based on aliphatic polyesters, polylactic acid (PLA), polyglycolic acid (PGA), and poly(D,L- lactide-co-glycolide) (PLGA) microspheres have been extensively studied as a 21 formulation approach to protect encapsulated drugs from degradation, enhance bioavailability, and sustain drug release [67]. These classes offer several benefits and are generally well-tolerated in living systems without causing harm. They are non- toxic and physiologically inert. Controlled drug delivery may be achieved using both natural and synthetic polymers, however, synthetic polymers are preferred. Controlled polymerization allows for extremely repeatable structure-function relationships. Some polymers are biodegradable, reducing the risk of accumulating in the body. However, this only applies if the breakdown by-products are non-toxic, do not trigger an immune response, and are below the renal threshold [65]. The most often utilized biodegradable synthetic polymers in drug delivery systems (DDS) are polyesters which include polyglycolide (PGA), polylactide (PLA), and copolymers with specified architecture and chemical composition (lactide to glycolide ratio). The rate of drug release might vary depending on the lactide-to-glycolide ratio. Lactide-rich polymers are very hydrophobic, resulting in delayed degradation and reduced water absorption [64]. There are three main categories of polymeric drug delivery systems; colloidal carriers (micro, nanoparticles, micelles, micro/nano gels), implantable networks or hydrogels, and polymer-drug conjugates as it can be seen from Figure 2.13. Depending on the administration model and desired release method, multiple methods of distribution can be chosen [64]. A diagram of the formulation of polymeric carriers for biomedical delivery systems [65]. 22 Drug release from these systems follows three broad pathways, as it can be seen in Figure 2.14. The therapeutic agent is evenly distributed in all polymer matrices, but drug release is regulated by diffusion, polymer surface erosion, or a combination of both [68]. Achieving continuous release (zero-order release) is challenging due to the decreased concentration and chemical activity of the drug. As the drug is released from the polymer matrix (i) and (iii) in Figure 2.15, its chemical activity or concentration decreases with time, resulting in first-order release rather than zero-order release. Currently, most monolithic biodegradable devices adopt either mechanism (i) or (iii), as indicated in Figure 2.15 [68]. Overview of polymeric-based drug delivery systems [64]. Release mechanisms of biodegradable polymeric drug delivery systems [68]. 2.7 Microspheres Microspheres are spherically shaped particles that range in size from one to a thousand meters and their structures can be seen from Figure 2.16. The term microsphere refers to particles that might be solid or hollow [69]. Microspheres are biodegradable, free- flowing particles composed of proteins or synthetic polymers. A polymer matrix 23 includes distributed drug molecules, and controlled drug release can be achieved by diffusing through the matrix or by degrading the matrix itself [70]. They can encapsulate tiny compounds, proteins, peptides, and nucleic acids. They outperform nanoparticles in terms of translational efficiency and clinical success rate. They provide several advantages over conventional dosage forms, including enhanced solubility of poorly soluble drugs, protection of drugs from enzymatic and photolytic degradation, decreased dosing frequency, improved bioavailability, controlled release profile, dose reduction, and drug toxicities [71]. In terms of efficacy, microspheres have also offer several advantages. The spherical form and tiny particle size facilitate drug administration in the body. Another advantage of small particle sizes is their increased surface area, which works especially well to improve the efficiency of drugs that are not easily soluble in the body. To generate microspheres, various processes can be utilized, including single and double emulsion solvent evaporation, sedimentation polymerization, spray-drying, electrospraying, and phase separation technologies [70]. Microspheres [72]. Microspheres are able to be targeted to a specific area using active or passive targeting approaches. Passive targeting is based on the microspheres' size and general surface features, such as hydrophobicity, surface charge, and non-specific adhesion, which lead them to a given organ. Active targeting, on the other hand, is most commonly connected with ligand-receptor recognition, which is known as secondary active targeting. The increasing number of studies in recent years demonstrating the potential use of microspheres as drug delivery carriers for targeted delivery has captured the interest of researchers throughout the world [71]. 2.7.1 Emulsion/solvent evaporation method Microspheres can be produced by evaporating an organic solvent from dispersed oil droplets containing polymers and drugs [73]. Extensive research has been conducted 24 on the solvent evaporation approach for producing microspheres of biocompatible polymers and copolymers [74]. Microencapsulation using solvent evaporation is commonly utilized in the pharmaceutical industry to create controlled-release formulations. Microencapsulation can be achieved by several solvent evaporation techniques. The hydrophilicity or hydrophobicity of the active molecules determines the most suitable approach for drug encapsulation [75]. The easiest approach for producing drug-loaded microspheres is emulsion/solvent evaporation. The emulsion could either be single or double, depending on the solubility of the polymer and the drug being used. The single O/W (oil-water) emulsion/evaporation process consists of two phases: organic (internal or dispersed) and aqueous (external or continuous) as indicated in Figure 2.17. The organic phase is comprised of polymer and drugs dissolved in an organic solvent, which is then emulsified with water. The aqueous phase is an aqueous solution with high hydrophilic-lipophilic balance (HLB) surfactants; typically, an aqueous polyvinyl alcohol (PVA) solution is employed as the continuous phase [76]. It is waited in the mixing position until the organic solvent evaporates [73]. Schematic diagram of O/W emulsion solvent evaporation method [76]. Figure 2.18 shows the formation of microcapsules by single O/W emulsion/solvent evaporation method. However, when the drug that needs to be encapsulated is insoluble in the organic phase, multiple emulsions are required. The double emulsion technique for microspheres creates several emulsions (w/o/w) and is ideal for water- soluble pharmaceuticals, peptides, proteins, and vaccines. This approach works with both natural and synthetic polymers. The aqueous protein solution is distributed in a 25 lipophilic organic continuous phase. This protein solution may include active components. The continuous phase contains a polymer solution that encapsulates protein from the scattered aqueous phase. The initial emulsion is sonicated before being added to an aqueous solution of polyvinyl alcohol (PVA). This leads to the creation of a double emulsion as it is given in Figure 2.19. The emulsion undergoes solvent removal by either evaporation or extraction [72]. Formation of microcapsules by single O/W emulsion/solvent evaporation method [77]. Microspheres created using the Double Emulsion Technique [72]. Emulsion solvent evaporation has many advantages over other preparation processes, including spray drying, sonication, and homogenization. It requires just basic conditions like ambient temperature and steady stirring. This allows for a stable emulsion while maintaining pharmacological activity [78]. 2.8 Oleuropein Oleuropein, the main component of Olea europea, known as the olive tree, belongs to a very special group of coumarin-like compounds called secoiridoids, which are abundant in oleaceae, gentianales, cornales, and many other plants. Figure 2.20 26 indicates the molecular structer of oleuropein. Oleuropein, the heterosidic ester of elenolic acid and hydroxytyrosol, has many beneficial effects on human health. According to research, it has been observed that oleuropein has various pharmacological properties such as antioxidant, anti-inflammatory, antiatherogenic, anti-cancer, antimicrobial, and antiviral, and for these reasons, it is commercially available as a food supplement in Mediterranean countries. Additionally, research has demonstrated that oleuropein has anti-ischemic, hypolipidemic, and cardioprotective properties against acute adriamycin cardiotoxicity. A study also revealed that oleuropein aids in the prevention of Alzheimer's disease. The phenol components of olive oil, particularly oleuropein, which functions as a free radical scavenger at the skin level, were demonstrated in another study to have a direct antioxidant impact on the skin [79, 80]. Molecular structure of oleuropein [81]. 2.9 Transchalcone Chalcones are open-chained compounds found naturally in plants. As shown in Figure 2.21, their chemical structure consists of two aromatic rings separated by a three- carbon, -unsaturated carbonyl system [82]. Such structure is the primary distinction between chalcones and other flavonoids [83]. By adding functional groups to the aromatic rings, chalcones' chemical structure can be varied. Chalcone molecules act as pigments in plants due to their characteristic, vivid yellow color. Due to their natural abundance, simplicity in manufacture, and biological activity, chalcones have recently drawn interest in terms of biology. It is known that a number of chalcones and chalcone derivatives show anticancer efficacy against a variety of forms of cancer, including breast, colon, ovarian, bladder, and urinary tract cancers, as well as antifungal, anti- tuberculosis, anti-AIDS, and antidiabetic activities. Chalcones can be chemically 27 synthesized by Claisen-Schmidt condensation reaction. The reactants are acetophenone and benzaldehyde, and the reaction is catalyzed by a base, generally NaOH or KOH [84]. Molecular structure of trans-chalcone [84]. 28 29 3. MATERIALS AND METHODS 3.1 Materials Rice husks were kindly supplied by a local rice production facility (Edirne, Turkey) and treated to obtain rice husk ash (RHA). (3-APTES, C9H23NO3Si, 98 %, Sigma- Aldrich), was used in the experiments for surface modification of RHA. During the preparatory preparation stage, rice husks were rinsed with distilled water to eliminate extraneous contaminants. Dried rice husks were burnt for 6 hours at 600-650 °C to produce RHA, which were then employed as a support material for enzyme immobilization. Reactions were catalyzed by immobilized Candida antarctica Lipase B (CALB) enzyme (E.C. 3.1.1.3, recombinant, expressed in Aspergillus niger, Sigma). CALB in free form purchased from Sigma Aldrich was immobilized onto RHA. Surface modification of RHA was achieved by 3-aminopropyl triethoxysilane (3- APTES) (C9H23NO3Si) (Merck) treatment. Acetone (C3H6O, 100 %, Merck) was used as medium in surface modification of RHA. Sodium dihyrogen phosphate monohydrate (NaH2PO4. H2O) (Carlo Erba) and disodium hydrogen phosphate heptahydrate (Na2HPO4. 7H2O) (Merck) were used for preparation of 0.015 M, pH = 7 phospate buffer solution. δ-valerolactone (δ-VL) (C5H8O2, Sigma-Aldrich) and ω- pentadecalactone (ω-PDL) (C15H28O2, Sigma-Aldrich) was used as monomer in polymerization reactions. Toluene (C7H8, 99.8 %, Merck) was used as organic solvent in polymerization reactions. Chloroform (CHCl3, 99 %, Merck) was used to terminate polymerization reactions and as solvent for PPDL-PVL copolymers during microsphere formation. For the termination of polymerization reactions and precipitation of obtained polymers methanol (99% CH3OH) (Emboy) were also used, was respectively purchased from Merck. Chloroform (99%, CHCl3) (Merck) was used to solubilize polymer samples for 1H NMR analysis. Transchalcone (TC, C15H12O, 97%, Sigma-Aldrich), model drug, was purchased from Sigma-Aldrich. During preparation of TC/Oleuropein-loaded microspheres, dichloromethane (CH2Cl2, 99 %, Carlo Erba) was used as solvent of TC, in the internal phase. Poly(vinyl alcohol) (C2H4O)n, Sigma-Aldrich) was used as an organic solvent during the preparation of 30 TC/Oleuropein loaded microspheres. Oleuropein, antibacterial agent, was purchased from natural supplier. Methanol (CH3OH, 99.8 %, Merck) was used in determination of loading efficiency of the microspheres. Potassium di-hydrogen phosphate (KH2PO4, 100 %, Carlo Erba) and di-potassium hydrogen phosphate (K2HPO4, 100 %, Merck), sodium chloride (NaCl, 99.6 %, Carlo Erba), potassium chloride (KCl, 100 %, Merck), disodium phosphate dihydrate (Na2HPO4.2H2O, 100 %, J. T. Baker), sodium dihydrogen phosphate (NaH2PO4, 100 %, Merck), acetic acid (CH3COOH, 99.8 %, Merck), sodium acetate (CH3COONa, 99 %, GPR) and hydrochloric acid (%37 puriss.) (HCl, Riedel-De Haën) were used to prepare buffer solutions for enzyme immobilization and drug release studies. For the preparation of proton nuclear magnetic resonance (1H-NMR) samples, deuterated chloroform (CDCl3, Merck, 99.8%) was used. In antibacterial test for preparation of liquid and solid media for cultured bacteria were Agar-Agar and Nutrient Broth (Merck) were used. Staphylococcus aureus (ATCC 6538) and Escherichia coli (ATCC 8739) bacteria were kindly provided by Istanbul Technical University Food Engineering Department. In WST test, MCF-7 human breast cancer cell was used. WST-1 solution was purchased from Sigma-Aldrich, whereas Eagle’s minimum essential medium (EMEM), fetal bovine serum (FBS), trypsin-EDTA, and Trypan blue were obtained from Gibco BRL and utilized in cell viability assays. All chemicals and reagents were of analytical grade. 3.2 Methods 3.2.1 Preparation of support material for the enzyme immobilization First, rice husks were rinsed with distilled water to eliminate any contaminants. After the rinsing, they dried in an oven at 30 °C overnight. After that, the dried rice husks were burned in oven at between 600-650 °C for 6 hours. The oven's temperature was continually checked and increased by 10°C per minute until the burning temperature was attained. After the burning process was finished, the rice husk ashes (RHA) were transferred to a desiccator to prevent moisture after cooling [11]. Figure 3.1 shows a picture of the processed rice husks. 31 Figure 3.1 : From left to right: dried rice husk, rice husk ash after combustion. Before the immobilization process, the surface of the support material was silanized using the agent 3-APTES as it is described in previous studies [11]. 250 mg RHA was combined with 15% (v/v) 3-APTES in 5 mL acetone. The mixture was incubated in a shaking water bath (Julabo SW22) at 50°C and 160 rpm for 2 hours. Figure 3.2 shows the shaking water bath utilized in the studies. Figure 3.2 : Shaking water bath used in experiments. After the reaction, surface-modified (activated) RHA was filtered and rinsed with distilled water using a vacuum pump (Sartorius stedim 16612) to remove any unreacted chemical substances. Then, dried in oven at 60°C for 2 hours. 0.015 M, pH 7 phosphate buffer was prepared by mixing 29.25 mL 1M NaH2PO4.H2O and 45.75 mL 1M NaH2PO4.7H2O with distilled water and diluting to 1 L with distilled water. The pH of the buffer was adjusted using a pH meter (Inolab TWT). 32 3.2.2 Immobilization of CALB The prepared supports were used for lipase immobilization. CALB was immobilized on surface-modified RHA via a physical adsorption technique, as previously described [11]. CALB and RHA were mixed in 25 mL of 0.015 M pH 7 phosphate buffer with a 2µL/mg enzyme loading ratio for 5 hours at room temperature. At the conclusion of the interval, immobilized lipase was filtered and dried in an oven at 300 °C for 12 hours. Figure 3.3 shows a picture of the filtered enzyme. Figure 3.3 : Filtered immobilized enzyme after the immobilization via physical adsorption. 3.2.3 Enzymatic synthesis of poly(ω-pentadecalactone-co-δ-valerolactone) Immobilized CALB was utilized as a biocatalyst for the copolymerization of pentadecalactone and valeractone. The copolymerization technique was the same as the previous work in which poly(ω-pentadecalactone-co-ε-caprolatone) copolymers were produced [41]. Reactions were carried out in 1 g toluene in a nitrogen environment. The weight ratio of toluene to total monomers was 1:2, with an enzyme concentration of 20%. The reaction mixture was agitated at 120 rpm at 80°C. Copolymers with monomer ratios (75%-25% in weight ratios) were manufactured in 24 hours. In the previous study [37], different monomer ratios and polymerization times were tried and it was decided that these were the most ideal ratios. Therefore, this study proceeded directly through these rate and time parameters. At the end of the process, additional chloroform was used to stop the polymerization. The enzyme was then isolated by filtering, and the chloroform was evaporated in an oven set to 500°C. The copolymer was collected by precipitation in cold methanol. The precipitated 33 copolymer samples were filtered and dried in an oven at 350°C. Figure 3.4 shows pictures of the enzymatic polymerization equipment and glass reactor. Figure 3.4 : Apparatus used for polymerization. 3.2.4 Preparation of microspheres Microspheres were prepared by conventional O/W emulsi