Determination of the tumoricidal effect by in-vitro methods for the acquiring of food-sourced bioactive protein and the formation of active pharmaceutical substance

Abstract

Encapsulation; It is a relatively new technology used for the preservation, stabilization and slow release of food ingredients. However, encapsulation is used in a wide scope such as medicine and cosmetics other than foods. There are several studies in the mentioned areas. However, the number of studies on the encapsulation of protein and peptide-derived APIs with significant therapeutic effects is not high. The BAMLET complex is one of the active medicinal compounds that was discovered in the early 2000s. BAMLET (Bovine Alpha-lactalbumin Made LEthal to Tumors) is a tumoricidal molecular complex of partially unfolded bovine α-LA and multiple oleic acid molecules. Although this class of molecules was shown to be effective in many cancer types, their effectivity was not promising compared to synthetic drug products. In addition to the effect of in-vitro conditions such as production and storage conditions for clinical use of this type of drugs; In vivo conditions (e.g. enzymatic hydrolysis) have negative consequences on the therapeutic effect. It is possible to eliminate these stability problems by encapsulation. Nano-carrier systems have been developed to overcome many of the disadvantages encountered in current cancer treatments, especially the systemic distribution of antitumor agents. These include polymeric nanoparticles; It attracts attention with its small dimensions, ability to release tumor-targeted drugs, easy production, high stability, and biodegradability. Under the lights of these explanations, research was planned to modify the protein purified from skimmed milk and combined with oleic acid by enzymatic methods, to observe the change in tumoricidal properties and to increase its stability. The objectives of this Ph.D. thesis was (i) purification and characterization of α-LA from skimmed milk; (ii) enzyme modification of α-LA and characterization of structural changes; (iii) complexing α-LA and hydrolysed α-LA with oleic acid and examining the tumoricidal activities of the resulting complexes; (iv) encapsulation of the complexes to enhance their (bio)pharmaceutical properties and also in-vitro comparison of their tumoricidal properties. Two different experimental studies (Chapters 3-4) were conducted to achieve the declared purposes. Initially, α-LA protein from skimmed milk was purified, characterized, complexed with oleic acid, and its tumoricidal and functional properties evaluated from a wide-angle perspective (Chapter 3). The protein was then hydrolysed with the help of enzymes and the properties of the new complex were compared with the BAMLET complex (Chapter 3). Based on obtained data from the previous chapter, the characterization and structural modification of the complexes were investigated. In addition, all possible methods were tried for the complexes obtained, but the encapsulation of the complexes was made by choosing the double emulsion (WOW) method. The efficacy of (bio)pharmaceuticals obtained after encapsulation was again examined from a tumoricidal point of view thereafter the structural alterations of protein were discussed (Chapter 4). In Chapter 1, the scope and the main objectives of this present Ph.D. thesis are defined. Right after in Chapter 2, a comprehensive review of the enzymatic modification and functional properties of the α-LA protein is presented. Initially, the compositions of BAMLET-related proteins studied were reported. Following that, the changing methods of protein purification, oleic acid composition, how to form a new complex, and how to increase its functional and tumoricidal properties were evaluated. As the last but is the focal point of this review was examining of all the functional and tumoricidal properties of the BAMLET protein that have been described and studied to date, and methods that could potentially be used to enhance them. In Chapter 3, the α-LA was purified from the skimmed bovine milk, and BAMLET was produced with oleic acid. On the other hand, the purified α-LA was hydrolysed and combined with oleic acid, and the functional properties between the two groups were compared. To produce BAMLET protein, α-LA was obtained from bovine milk. In aggrement with the literature, 0.8 µl g of α-LA was purified from 1 liter of milk. The combination of the protein with oleic acid was carried out by temperature application method. α-LA and BAMLET complexes were identified by Fast Protein Liquid Chromatography (FPLC). For α-LA, a single peak appeared in the chromatogram and a result was obtained at a wavelength of 280 nm as defined in the literature. It was determined that the purification procedure was fulfilled, with α-LA giving a single peak in FPLC and confirming the 14.2 kDa weight of this peak, according to the calibration curve generated with the reference molecular weights. The molecular weight of the BAMLET complex was determined by FPLC and its characterization during the incubation period was determined. α-LA, hydrolysed α-LA, BAMLET and HBAMLET were investigated by Fourier Transform Infrared Spectroscopy (FTIR). In this way, disulfide bonds and amide groups were defined. For HBAMLET, a single distinct peak was detected, while none of the amide groups were observed. A peak corresponding to the esterified group of HBAMLET was determined at 1747.33 cm ˉ ˡ. Characteristic band was observed for BAMLET at 1642 cmˉ ˡ in a characteristic amide I band attributed to the C=O vibration (C=O stretching) of the acetylated units. Additionally, Thermal properties of α-LA were determined by using differential scanning calorimetry (DSC) for PLGA, hydrolysed α-LA, BAMLET and HBAMLET and their nanoparticles. In this way, denaturation peak temperature and denaturation enthalpy of α-LA, BAMLET, hydrolysed α-LA and HBAMLET complexes were determined. As a final point, The cytotoxic character and the anti-cancer activity of BAMLET and of its hydrolysed form were comparatively analyzed in-vitro focusing on breast cancer (MCF7) and prostate cancer cell (DU145) lines. The results showed that the most effective dose of the untreated form of BAMLET decreased the viability of MCF7 and DU145 by 89.17% at 10 µg/mL and 48.10% at 2.14 µg/mL after 24 hours, respectively. After the hydrolyses, the most effective doses were altered, but the anti-cancer effect was improved to 21.96% for MCF7 (6.38 µg/mL) and 32.17% for DU145 (6.38 µg/mL) under the same condition. Interestingly, the BAMLET demonstrated cytotoxic effect on fibroblasts above the concentration of 2.1 µg/mL, but this detrimental effect was vanished after the enzyme treatment of BAMLET. The cell viability was supported by 2.7-fold at 6.38 µg/mL HBAMLET. As conclusion, BAMLET produced from the hydrolysed form of the α-LA was found to be more effective against the cancer cells than its non-hydrolysed form. The HBAMLET was found to be a promising natural anti-cancer product without any toxic effect on fibroblasts. The most substantial differences were observed in the tumoricidal activity properties of BAMLET. It was reported that the anticancer activity increased significantly after enzymatic hydrolysis. Based on the results of this study, the BAMLET and HBAMLET complexes were used as material for the following studies (Chapler 4, 5). Following enzymatic modification of the HBAMLET complex and observation of promising in-vitro changes, both tumoricidal complexes were encapsulated in Chapler 4. For this purpose, the complexes were encapsulated by double emulsion method using poly (lactic-co-glycolic) acid (PLGA) polymer as a carrier. Choosing a polymer as a carrier agent is preferred because it offers many advantages such as high stability, biodegradability, small size, tumor-targeted drug release, and easy production. In this process, the suitability of many polymer materials was tested and the conformity of the PLGA polymer to the configuration allowed to form polymeric nanoparticles (PNPs). Then, the cytotoxic character and the anti-cancer activity of BAMLET and HBAMLET complexes was again examined in-vitro on MCF/ and DU145 cancer cells. The most efficient concentration of HBAMLET was found to be 6.38 µg/mL, at which it decreased the viability of MCF7 and DU145 by 64.63% and 47.7%, respectively. However, BAMLET was less effective and decreased the viability of MCF7 and DU145 at 10 µg/mL and 2.14 µg/mL by 9.6% and 39.5%, respectively. Unfortunately, the BAMLET demonstrated cytotoxic effect on fibroblasts at the concentration higher than 2.14 µg/mL, however HBAMLET was not cytotoxic at the used concentrations, which showed that HBAMLET could be a noticeable natural anti-cancer product. For prolong controlled release of BAMLET products, their double emulsions were prepared.The seven-days release profile of the HBAMLET-PLGA nanoparticle was investigated in-vitro. The PDI value was 0.096, the particle size was 199.3.8 nm and the ζ-potential was determined as -14 mV. When the same criteria are evaluated for the BAMLET complex; while the PDI value was 0.098, the particle size was 22.8 nm and the ζ-potential was determined as -9.46 mV. The highest release rate for BAMLET was observed 65.76% at day 6th, while this rate was observed 73.54% at day 5th for HBAMLET. In addition, the encapsulation efficiency of BAMLET and HBAMLET (72.75% and 84.44%, respectively) was determined. It was concluded that the release of HBAMLET can be controlled in double emulsions and can be released in-vitro. The HBAMLET-loaded double emulsions can be used as an active drug agent of a drug therapy with tumoricidal effect. Finally, Chapter 5 based on the data obtained from the previous parts a comprehensive discussion and results about the deconstruction and complexation of the α-LA protein with oleic acid using the proposed novel therapeutic approach and the potential of this modified form to improve its tumoricidal properties are presented. The stability of the complexes has been increased by forming BAMLET and HBAMLET loaded polimeric nanoparticles with therapeutic effect. The double emulsion method was chosen because of the advantages that nano-carrier systems bring to the systemic distribution of antitumor agents. The efficacy of the study has been proven, and promising recommendations for future research are also provided.