Enhancement of Cancericidal Activity of Bamlet Complexes by Enzymatic Treatment

Enhancement of Cancericidal Activity of Bamlet Complexes by Enzymatic Treatment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhancement of Cancericidal Activity of Bamlet Complexes by Enzymatic Treatment REYHAN KOYUNCU, Gokhan Duruksu, Beraat Ozcelik, Yusufhan Yazir This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2082257/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract BAMLET ( B ovine α -Lactalbumin M ade LE thal to T umors) is a tumoricidal molecular complex of partially unfolded bovine α-lactalbumin 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 this study, the α-lactalbumin was purified from the skimmed cow milk, and BAMLET was produced with oleic acid. 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 89.2% at 10 µg/mL and 48.0% 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 21.9% for MCF7 (6.38 µg/mL) and 32.2% for DU145(6.38 µg/mL) under the same condition. Interestingly, the untreated 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 hydrolised BAMLET. As conclusion, BAMLET produced from the hydrolysed form of the α-lactalbumin was found to be more effective against the cancer cells than its non-hydrolysed form. The hydrolysed BAMLET was found to be a promising natural anti-cancer product without any toxic effect on fibroblasts. Bovine α-lactalbumin BAMLET Breast cancer Prostate cancer Enzymatic hydrolysis Protein purification Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Complexes formed with oleic acid (OA) and α-Lactalbumin (α-LA) have attracted attention due to their therapeutic potential. Many studies have been carried out for years about this complex, which was discovered serendipitously by the Svanborg group (Håkansson et al., 1995 ; Svensson et al., 2003 ; Svenssonet al., 2000 ). α-LA is an acidic (pI around 4.5), (Permyakov & Berliner, 2000 ), globular protein composed of 123 amino acid residues found in milk. The molecular weight of this acidic calcium-binding protein in its native conformation is 14.2 kDa (Svensson et al., 2003 ). The three-dimensional structure of α-LA consists of two domains: the α-domain (residues 1–34 and 86–123), consisting of four α-helices and two short 3₁₀-helices; and the smaller β-domain (residues 35–85), (Casbarra, et al., 2004 ; Spolaore, et al., 2010 ). The native conformation is stabilized by four disulfide bonds (amino acids 6-120, 61–77, 73–91 and 28–111), and the two domains are linked by the calcium-binding loop (Pettersson et al., 2006 ). Either by heating, by the interaction with high concentrations of denaturants or by incubating at low pH, the native α-LA is converted to the partially unfolded protein form, so called the intermediate state or the molten globule state (Casbarra et al., 2004 ; Lassalle et al., 2003 ; Mok et al., 2005 ; Spolaore et al., 2010 ). After the addition of oleic acid to apo-state of α-LA, the formed complex can induce cell death in cancer cells (Svensson et al., 2003 ). α-LA contains conserved amino acid side-chains that can make salt bridges with the carboxylate group of oleic acid. Due to multiple sites with high or low binding affinities in α-LA, the binding of oleic acid to these sites becomes highly complicated (Petitpas et al., 2001 ). Moreover, oligomerization reaction rates are depended on α-LA concentration (Baumann et al., 2012 ). For the production of this protein-fatty acid complex, human milk was used as a first source of α-LA, and the product was named as HAMLET ( H uman A lpha-lactalbumin M ade LE thal to T umor cells), (Svensson et al., 2000 ). While this complex induces an apoptotic mechanism in tumour cells, the healthy cells are resistant to this effect (Delgado et al., Håkansson et al., 1995 ; 2015; Jung et al., 2016 ; Svensson et al., 2003 ; Svensson et al., 2000 ;). Its anti-tumoural activity was shown on more than 40 different lymphoma and cancer cell lines (Hoque et al., 2015 ). It has been found that α-LA from different sources (human, bovine, goat, swine, sheep, equine and camelid) can form OA-complexes that can exhibit biological properties similar to HAMLET (Fontana et al., 2013 ; Pettersson et al., 2006 ; Spolaore et al., 2010 ). In other words, it has been proven that HAMLET-like complexes can also form with other α-LA varieties (Fang et al., 2014 ). For this purpose, bovine milk (BAMLET: B ovine A lpha lactalbumin M ade LE thal to T umor cells), which is more accessible source of milk, was used within the scope of the project. In order to understand the tumoricidal activity of HAMLET, its molecular structure has been studied by researchers (Ho et al., 2012 ; Pettersson et al., 2006 ). Although the sequence variations in α-LA from animal sources exist, other α-LA variants can also bind oleic acid to form complex, as like in human α-LA. BAMLET, on the other hand, can bind high rates of oleic acid residues (5–8 oleic acids per protein molecule) than other protein-lipid complexes. It, therefore, has higher lipid-to-protein ratio than the HAMLET complex, and it might show different properties (Ho et al., 2012 ). In this study, bovine α-LA was fragmented into short peptides in various length with the help of hydrolysing enzymes, and hydrolysed BAMLET was produced subsequently. For this purpose, pepsin and trypsin peptidases were used. The activity of the formed complex was proven on DU145 and MCF7 cancer cell lines. 2. Materials And Methods 2.1 Purification of Bovine α-LA α-LA was purified from skim bovine milk from commercial sources according to the method of Pettersson et al., ( 2006 ) with some modifications. Briefly, casein was removed by acid treatment adjusting the milk to pH 4.0 with HCI, and centrifuging at 4000g for 20 min at 4°C. α-LA was separated in fractional precipitation at the 55% ammonium sulfate saturation, and the precipitate was collected by centrifugation at 8000g for 20 min at 4°C. After dissolving, sample was desalted by dialysis in 0.1 M Tris-HCl buffer at pH 7.5 at 4°C, and ultrafiltrated using Amicon-Ultra 15 Centrifugal Filter Units (Merck-Millipore, Tullagreen, Cork, Ireland) with a molecular weight cut-off of 10 kDa. α-LA was purified by FPLC using size-exclusion (SEC) column (HiPrep Sephacryl S-200 HR) on ÄKTA Prime Plus FPLC (GE Healthcare) by eluting in 10 mM Tris-HCl buffer, pH 7.5 with 10 mM EDTA. Samples were concentrated by the ultrafiltration as described above. The purity was confirmed by SDS-PAGE and the molecular weight was estimated by using the gel filtration chromatogram (HiPrep Sephacryl S-200 HR) with low molecular weight markers (Gel Filtration Cal. Kit Low Molecular Weight, GE Healthcare). The total protein content was measured by SMART bicinchoninic acid assay (BCA) Protein Assay Kit (iNtRON Biotechnology, Sungnam, Korea). After mixing with loading buffer solution (Tris-HCl 0.5 M pH 6.8, glycerol, sodium dodecyl sulfate (SDS), β-mercapto ethanol and Bromophenol Blue), samples (5 µg) were denatured in boiling water for 3 min. The denatured protein was loaded and separated by 12% SDS-polyacrylamide gel (TGX FastCast acrylamide gels; BioRad, Hercules, CA, USA) and run at 120 V for 50 min. Bands were visualized in DNR Bio-Imagining Systems (MF-ChemiBIS 3.2, Jerusalem, Israel). 2.2. Enzymatic Hydrolysis of α-LA For the hydrolysis of α-LA, the method described by Hernández-ledesma & Dávalos ( 2005 ) was used. α-LA (1 mg/mL) was first hydrolysed with trypsin (EC 3.4.21.4.; Type I; 10 900 U/mg protein; Sigma; St. Louis, MO, U.S.A.) in 0.1 M Tris-HCl buffer (pH 7.4) for 24 h at 37°C. Later, the pH was adjusted to pH 2.5 by HCl (1M), and pepsin (EC 3.4.23.1.; 1:60 000, 3400 U/mg protein; Sigma; St. Louis, MO, U.S.A.) was added at the ratio of enzyme-to-substrate by 1:20 (w/w). After the incubation for 2h at 37°C, enzymatic activity was stopped by heating at 95°C for 15 min. 2.3. Preparation of α-LA – oleic acid complexes α-LA at 210 µM in phosphate-buffered saline (PBS) was mixed with oleic acid (1:2, v/v). The mixture was heated to 55°C, and incubated for 15 min at this temperature. Later, the mixture was allowed for 10 min to cool to room temperature. The sample was centrifuged (12.000g, 25°C, 15 min.). Excess oleic acids were removed (Kamijima et al., 2008 ). 2.4. Morphology study For the ultrastructural analysis, scanning electron microscopy (SEM) was used. The samples were sprinkled onto a two-sided adhesive tape and then coated with a thin layer of gold. Morphologic features of samples observed by a scanning electron microscope (Quanta FEG 250) in high vacuum with 5 kV accelerating voltage. 2.5. FTIR study Functional groups in α-LA, hydrolysed α-LA, BAMLET and hydrolysed BAMLET examined by Fourier transform infrared spectroscopy (FTIR) using the attenuated total reflection technique. After freeze-drying the samples were analyzed in the FTIR GX (Perkin-Elmer, Shelton, CT, USA) instrument. All spectra ranged from 200ˉ ˡ cm to 4000ˉ ˡ cm. The spectra were obtained by averaging 16 scans at 4 cmˉ ˡ resolution. 2.6. DSC study Thermal behavior of α-LA hydrolysed α-LA, BAMLET and hydrolysed BAMLET were investigated by a differential scanning calorimeter (DSC-028 Mettler Toledo Inc., Columbus OH). The denaturation temperature range (ΔTd), peak denaturation temperature (Td) and the enthalpy of denaturation (ΔHd) of lyophilised samples were determined. About 5 mg of each sample was weighed in an aluminum pan and hermetically sealed. An empty pan was used as a reference. Samples were scanned at the temperature range of 40–300°C with the rate of 5°C/min. 2.7. Anticancer activity of BAMLET and BAMLET hydrolysates 2.7.1. Cell lines and Cell Culture The analyses of the cancericidal effect of BAMLETs were performed using two cancer cell lines, MCF7 (human breast adenocarcinoma cell line) and DU145 (androgen-independent human prostate cancer cell line). To determine the cytotoxic effect of BAMLETs on normal somatic cells, fibroblast cells derived from foreskin tissue were used (Acemi et al., 2017 ). All cell lines were cultured in the complete RPMI 1640 medium [RPMI 1640 basal medium (Gibco, Paisley, UK) supplemented with 2.0 mM L-glutamine, 0.5 mM sodium pyruvate, 10% fetal bovine serum (Gibco) and 1% Penicillin-Streptomycin solution (Gibco). Cells were cultured under standard culture conditions in a humidified environment of 5% CO 2 at 37°C. Before the cell seeding into the culture plates, cells were detached by 0.25% trypsin-EDTA (Gibco). 2.7.2. Determination of cell viability by WST-1 To determine the effect of BAMLETs on fibroblast, MCF7 and DU145, cells were incubated with different concentrations of BAMLET and hydrolysed-BAMLET in RPMI 1640 complete medium for 24 h. The medium was replaced with RPMI 1640 basal medium supplemented with 10% WST-1 (Roche, Mannheim, Germany), and further incubated for 30 min at 37 °C. The absorbance at 450 nm was measured. The changes in cell numbers were expressed in percentage with respect to control group (medium without BAMLET). 2.8. Lactate dehydrogenase (LDH) activity assay Cytotoxicity was determined with Cytotoxicity Detection Kit LDH (Roche) according to the manufacturer’s instructions (Duruksu & Aciksari, 2018 ). MCF7 and DU145, cells were incubated with different concentrations of BAMLET and hydrolysed-BAMLET in RPMI 1640 complete medium for 24 h. After performing the assay, the absorbance of the collected supernatant was measured at 490 nm and a reference wavelength of 620nm with controls including medium alone. The cell culture without BAMLET or hydrolised BAMLET was used as control. 2.9. Statistical analysis Data were measured in triplicate. All statistical analyses were performed with the use of SPSS 10.0 (SPSS Inc, Chicago, IL, USA). The significance of results was analyzed by means of paired t test. In all analyzed cases, the results were regarded as statistically significant at p < 0.05. 3. Results And Discussions 3.1. Identification of purified α-LA and hydrolysed α-LA α-LA was purified by FPLC method. A protein peak of 14.2 kDa was obtained according to the calibration curve of standard proteins with known molecular mass (Fig. 1 ). In agreement with the results by Svensson et al., ( 2003 ), α-LA was observed as an intense, single band around 14 kDa as shown on SDS-PAGE (Fig. 2 ). Hydrolysis of α-LA with pepsin and trypsin generated peptide fragments with small molecular weight (Table 1 ). Hydrolysed α-LA on the SDS-PAGE showed a faint band around 15 kDa, indicating that the majority of α-LA is hydrolysed by pepsin and trypsin and minor amount of unhydrolysed α-LA still existed as a peptide band of 15 kDa. The digestion of α-LA protein into smaller fragments by the hydrolysing enzymes provided to separate into functional peptides, which was predicted putatively on Table 1 . Many functional groups on α-LA was expected to become available to interact with other molecules by this process (Gómez-Mascaraque et al., 2016 ; Jia et al., 2020 ; Lacroix & Li-Chan, 2014 , Url-1). Table 1 Putative digestion products of α-LA by pepsin or trypsin (Lacroix & Li-Chan, 2014 ; Jia et al., 2020 ; Gómez-Mascaraque, Miralles, Recio & López-Rubio, 2016 , Url-1) Fragment Sequence Pepsin / Trypsin 1–5 EQLTK Trypsin 1–10 EQLTKCEVFR Trypsin 10–17 RELKDLKG Pepsin 12–16 LKDLK Trypsin 16–25 KGYGGVSLPE Pepsin 32–36 HTSGY Pepsin 37–43 DTQAIVQ Pepsin 37–44 DTQAIVQN Pepsin 41–53 IVQNNDSTEYGLF Pepsin 45–50 NDSTEY Trypsin 51–58 GLFQINNK Trypsin 60–79 WCKDDQNPHSSNICNISCDK Pepsin 63–68 DDQNPH Trypsin 90–95 CVKKIL Pepsin 95–104 ILDKVGINY Pepsin 94–98 KILDK Trypsin 94–108 KILDKVGINYWLAHK Trypsin 95–108 ILDKVGINYWLAHK Trypsin 99–104 VGINYW Trypsin 104–117 WLAHKALCSEKLDQ Pepsin 105–111 WLAHKAL Pepsin 105–115 LAHKALCSEKL Pepsin 110–117 LCSEKLDQ Pepsin 115–123 LDQWLCEKL Trypsin 115–122 LDQWLCEK Trypsin 3.2. Characterization of BAMLET and Hydrolysed BAMLET complexes 3.2.1. Molecular weight characterisation In the current study, the heating method was preferred to generate BAMLET because of the high efficient production of BAMLET. The generation of BAMLET by an anion exchange column, which was loaded with oleic acid, has been described previously, but the method by heating a mixture of α-LA and oleic acid generated with higher efficiency (Håkansson et al., 1995 ; Svensson et al., 2003 ; Svensson et al., 2000 ). By heating the α-LA protein, partial denaturation was induced, and the complex formation took place efficiently (Kamijima et al., 2008 ). After the hydrolysis of α-LA, the same procedure for BAMLET was applied to the smaller fragments. SDS-PAGE was insufficient to separate these products, but the single band of BAMLET produced from the α-LA could be detected on the gel (Fig. 2 ). Peptides in hydrolysed BAMLET are mostly smaller than 10 kDa. While hydrolysis-derived peptides formed small aggregates with the effect of low pH (~ 2.5), the cross-links they formed between them consist of disulfide bonds. In addition to disulfide bonds, other intermolecular cross-links were also formed between protein aggregates formed at higher pH values (above 4.5), respectively (Gulzar et al., 2011 ). Observation of a faint band around 15 kDa indicates that new and strong covalent bonds were not formed when hydrolysed BAMLET was formed from hydrolysed α-LA (Gulzar et al., 2011 ), but newly formed disulfide bonds between peptides (Maux et al., 2013 ). During the production of BAMLET, in which the mixture of molecules were heat treated for 15 min., two main forms of BAMLETs were mainly generated with molecular weights of about 25.5 kDa and 51 kDa, which was predicted as the monomeric and dimeric forms of complexes, respectively (Fig. 3 ). The intense bands on SDS-PAGE and the data of SEC analysis of BAMLET indicate that BAMLETs interconnected by disulfide bonds and by intermolecular cross-linkages to form larger insoluble and soluble aggregates. The molecular mass of the monomeric BAMLET was estimated to be approximately 25.5 kDa (Fig. 3 ). In a complementary approach, it confirmed that the SDS-PAGE showed another peak around 51 kDa, a dimeric form of BAMLET. Remarkably, another peak at 14.2 kDa, which refers to α-LA, could be observed to remain as residue in the medium left without interacting with oleic acid (Fig. 3 ). This α-LA residue of BAMLET reaction was left to interact with oleic acid overnight at 55°C. It was observed that BAMLET complexes were formed at various molecular weights (Fig. 4 ). During the synthesis process, high temperature and high pH led some modifications to form high or low binding affinity sites on α-LA. These changes further supported the production of new oligomers after the process. Therefore, oleic acid:α-LA complexes could be formed at different ratios. A continuous complex formation took place between α-LA and excess oleic acid, which cannot be eliminated. The various form of BAMLET complexes formed after processing could be explained by either the formation of peptide aggregates that cannot be dissociated by the SEC eluent or newly formed disulphide bonds between peptides and proteins interconnected by intermolecular cross-links (Maux et al., 2013 ). 3.2.2. Morphological characterisation The SEM micrographs of BAMLET and hydrolysed BAMLET showed that the BAMLET and BAMLET-like complexes have a globular conformation due to the globular structure of α-LA (Fig. 5 ), as stated before (Delgado et al., 2015 ; Mossberg et al., 2010 ). Similarly, the peptide fragments of hydrolysed BAMLET appeared as spherical structures in the SEM image. In general, smaller microparticles were obtained when pepsin and trypsin enzymes were used as hydrolysing substance (mean diameter: 0.192 µm). Particle sizes and polydispersity index (PDI) are extremely important for drug formulations. It has been reported that particles with a diameter of 50–500 nm are known to be suitable for accumulate in tumor cells (Delgado et al., 2015 ). The relatively low polydispersity index (PDI) value of hydrolysed BAMLET indicated that it has a more regular particle size distribution than BAMLET. 3.2.3. FTIR characteristics The FTIR spectrum of α-LA (Fig. 6 a) showed a broad band at around 3000 ~ 3700 cm ˉ ˡ, indicating enhanced hydrogen bonding compared to that of BAMLET seen in Fig. 6 a. This suggests that hydrogen bonding is also involved in the interaction between α-LA and oleic acid. The lipid interaction effect formed by the binding of oleic acid to α-LA is clearly visible in the stretching vibration in the range of 2800 ~ 3000 cm ˉ ˡ (yellow circle). Characteristic bands were observed for both molecules (α-LA and BAMLET) at 1642 cmˉ ˡ (yellow circle) in a characteristic amide I band attributed to the C = O vibration (C = O stretching) of the acetylated units. The decrease in 1408 cm ̄ ˡ is due to the vibration of -OH and -CH (Chang et al., 2016 ). However, although there are many higher or lower wave numbers between the 1000 ~ 1500 cm ˉ ˡ range in both groups, due to the overlap of the amide I region and most spectral bands, these different fluctuations can be attributed to oleic acid binding to α-LA and to the application of heating treatment while BAMLET was formed. The broad band seen in the FTIR spectrum of BAMLET and hydrolysed α-LA in the range 3000 ~ 37000 cm ˉ ˡ was not seen for hydrolysed BAMLET (Fig. 6 b). However, stronger stretch vibration and a wider band are observed in the range of 2800 ~ 3000 cm ˉ ˡ (yellow circle). This situation draws attention to the lipid interaction density between hydrolysed α-LA and oleic acid, which creates hydrolysed BAMLET. The FTIR spectrum of hydrolysed α-LA showed absorption bands at 1615 cm ˉ ˡ (amide I, C ═ O and C ― N stretching), 1410 cm ˉ ˡ and 1320 cm ˉ ˡ (amide II, N ― H deformation and C ― N stretching), (Huang et al., 2012 ). For Hydrolysed BAMLET, while none of the amide groups were observed, a single distinct peak was detected. The peak at 1747.33 cmˉ ˡ corresponds to the esterified group of hydrolysed BAMLET (yellow circle), (Raei et al., 2017 ). BAMLET, on the other hand, has only amide I bonds in this domain (yellow circle, Fig. 6 c). 3.2.4. Thermal characteristics (DSC) The DSC thermograms of BAMLET, hydrolysed BAMLET, α-LA and hydrolysed α-LA were shown in Fig. 7 A and Fig. 7 B respectively. The thermograms of both α-LA and hydrolysed α-LA exhibited a weak melting behavior at around 160°C, which associated with the melting of the network structures (Huang et al., 2012 ). The melting behavior in the network structures continued for the enzyme-treated α-LA, which is corresponding to the thermal denaturations of less stable peptide fragments have been observed at temperatures lower than 200°C (Table 2 ). However, α-LA exhibited a sharp peak. A higher denaturation temperature and denaturation enthalpy of α-LA shows a network structure with improved thermal stability compared to hydrolysed α-LA. It has been announced that, complex proteins are more stable against thermal denaturation (Capitani et al., 2007 ; Chang et al., 2016 ; Huang et al., 2012 ; Timilsena et al., 2015 ). Similar to α-LA and hydrolysed α-LA, both thermograms of BAMLET and hydrolysed BAMLET begin with melting of the network structures; hydrolysed BAMLET exhibited a very different diagram. Hydrolysed BAMLET composed of very small peptides showed only slight crystallinity, attributed to monomerization causing weaker network structure. BAMLET, on the other hand, showed an endothermic peak at higher temperature (Td = 231.44°C). This denaturation temperature could be ascribed to a tendency to dimerisation. The same situation was observed for α-LA at Td = 236.8°C. Table 2 Denaturation temperature range (ΔTd), peak denaturation temperature (Td) and denaturation enthalpy (ΔHd) of CPI and α-LA, hydrolysed α-LA, BAMLET and hydrolysed BAMLET. Materials ΔTd (°C) Td (°C) ΔHd (j/g) α-LA First peak 203.67-219.12 199.33 + 270.7 Secound peak 244.25-235.12 236.8 + 5.16 Hydrolysed α-LA First peak 190.96-183.63 185.22 + 38.96 Secound peak 201.87-190.96 192.15 + 59.28 Third peak 231.78-201.87 204.94 + 101.2 BAMLET First peak 129.68–114.60 116.73 + 50.54 Secound peak 153.65-129.68 142.91 + 32.69 Third peak 183.56-153.65 231.44 + 122.5 Hydrolysed BAMLET First peak 190.8–82.7 143.7 -100.9 3.3. Toxicity of BAMLET products on fibroblast cells LDH activity of BAMLET and hydrolysed BAMLET was estimated for both MCF7 and DU145 cell lines after incubation for 24 h (Fig. 8 ). The toxicity of BAMLET was rather limited for MCF7 cells. At the highest BAMLET concentration (10 µg/µl), less than 6.6% ±1.3 of LDH activity level was observed compared to the control (without BAMLET). On the other hand, the highest toxicity level with 25.6% ±2.2 toxicity was achieved in DU145 cell lines at the BAMLET concentration of 2.14 µg/µl. α-LA has a toxicity effect with 0.83% ±2.2 and 8.8% ±0.9 for MCF7 and DU145, respectively. Compared to α-LA, the BAMLET showed improved toxic effect, but the toxicity level for BAMLET could not be detected higher than 8% and 28% for MCF7 and DU145 over the BAMLET concentration ranging from 1.07 to 10.00 µg/µl. The hydrolysed BAMLET possessed significantly improved toxic effect for both cancer cells. The toxicity levels of hydrolysed BAMLET for MCF7 and DU145 were estimated as 49.2% and 55.2% for the concentration at 6.38 µg/µl of hydrolysed BAMLET compared to the control. The effectivity was improved by almost 6.2-fold and 2-fold against MCF7 and DU145, respectively. In the previous studies with BAMLET, it was shown that two types of cytotoxicity effect of BAMLET could be observed depending on its concentration. Cell apoptosis could be observed at low concentrations, but at high concentrations cell lysis could be detected (Brinkmann et al., 2011 ). In this study, higher BAMLET concentrations were required to observe the cytotoxic effect after 1 hour of incubation. 3.4. Anti-cancer effect of BAMLET and hydrolysed BAMLET products The viability analyses of MCF7 and DU145 cells treated with BAMLET / hydrolysed BAMLET complexes was performed by WST-1. We found that the synthesized complexes showed varying toxicities (Fig. 9 ). Although α-LA demonstrates relatively better cytotoxic effect for the prostate cancer cells (DU145), it also caused to decrease the cell viability of breast cancer cells, MCF7 (Fig. 9 B and Fig. 9 A). After the treatment with BAMLET at the concentration of 2.14 µg/µl for 24 hrs, the viability of DU145 cells were decreased more than 50%. (Fig. 9 B). However, the same BAMLET concentration did not have the same cell viability decreasing effect on MCF7 (Fig. 9 A). The MCF7 viability was slightly decreased to 89.16% at the concentration of 10 µg/µl (Fig. 9 A). After the treatment with the hydrolysing enzymes, tumoricidal effect of BAMLET was significantly improved. It was observed that the formed peptide fragments retained their ability to form a complex with oleic acid possessing a tumoricidal activity, similar to BAMLET. Hydrolysed BAMLET was observed to have improved cytotoxicity at the dose of 6.38 µg/µl for both cancer cells (Fig. 9 C and Fig. 9 D). The effect of BAMLET and hydrolysed BAMLET on somatic cells (fibroblasts) at the specified dosage ranges is quite important for its safely usage. BAMLET supported the cell viability of fibroblast cells until the concentration of 2.14 µg/µl, but the viability suddenly dropped at the concentrations higher than 4.25 µg/µl of BAMLET (Fig. 9 E). Interestingly, the hydrolysed BAMLET did not showed any detrimental effect on the fibroblast cell viability (Fig. 9 F). On the contrary, it supported the cell proliferation. The cell population was enhanced by 2.71-fold at the concentration of 6.38 µg/µl, at which the prostate and breast cancer showed %32.2 and %22 cell viability. BAMLET had a tumoricidal effect of 2.89% (Fig. 9 A) on MCF7, while on DU145 the level of this effect was 51.9% (Fig. 9 B). On the other hand, the hydrolysed BAMLET had a tumoricidal effect of 78.04% and 67.83% on MF7 and DU145 cells, respectively (Fig. 9 C and Fig. 9 D). 4. Conclusion In the current study, the BAMLET was produced from the hydrolysed α-LA fragments, so-called “hydrolysed BAMLET”, with improved cytotoxicity against MCF7 and DU145 cells. Moreover, this hydrolysed BAMLET supported the cell population of somatic cells while decreasing the number of cancer cells. From this aspect, the hydrolysed BAMLET might be an ideal anti-cancer agent to be used in the cancer treatment. The hydrolysis process released the functional groups of α-LA, which were more effective in cellular toxicity against MCF7 and DU145 cells. It is still unclear which released functional peptide fragments of α-LA possessed the toxic effect or which fragments involved in the complex formations with oleic acid. Long-term incubation of α-LA and oleic acid led the formation of complexes with various dimerization rates. The dimerization trend in BAMLET is consistent with the DSC thermograms. Although hydrolysed BAMLET yields a less stable thermogram, an intense lipid interaction between α-LA and oleic acid according to the FTIR spectrum. Moreover, the collection of functional groups in a single peak at 1615 cm ˉ ˡ indicates the presence of structurally esterified groups. BAMLET and hydrolysed BAMLET complexes were identified as significant growth inhibitors on the MCF7 and DU145 cell lines. However, hydrolysed BAMLET is more effective than BAMLET from the point of toxicity. Hydrolysed BAMLET, at the concentration of 6.38 µg/µl where it has a strongest tumoricidal effect on MCF7 and DU145 cancer cells, showed a supportive effect on the viability of fibroblast cells. In the future, BAMLET and hydrolysed BAMLET could be considered for using in drug formulations with the appropriate method. Abbreviations α-LA, alpha lactalbumin; BAMLET, B ovine α -Lactalbumin M ade LE thal to T umors; BCA, bicinchoninic acid; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; FTIR, Fourier transform infrared spectroscopy; DSC, Differential scanning calorimetry; SEM, scanning electron microscopy; LDH, Lactate Dehydrogenase; WST, Water-soluble tetrazolium; PDI, polydispersity index . Declarations Acknowledgments We would like to thank to Evren DEMİRCAN from the Department of Food Engineering at Istanbul Technical University for his technical assistance in DSC and FTIR analyses. The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Declaration of competing interest No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. Credit authorship contribution statement Reyhan Koyuncu : Methodology, Formal analysis, Resources, Investigation, Writing-original draft, Revision, Conceptualization, Project administration. Gokhan Duruksu : Conceptualization, Validation, Reviewing & editing, Supplementary data, Data curation. Beraat Ozcelik : Review & editing, Supervision. Yusufhan Yazır : Investigation, Supervision. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Acemi A, Duruksu G, & Özen F (2017). Cytostatic effects of methanolic extracts of amsonia orientalis decne. On MCF-7 and DU145 cancer cell lines. Notulae Botanicae Horti Agrobotanici Cluj-Napoca , 45 (1), 36–42. https://doi.org/10.15835/nbha45110576 Baumann A, Gjerde A U, Ying M, Svanborg C, Holmsen H, Glomm W R, … Halskau E (2012). HAMLET forms annular oligomers when deposited with phospholipid monolayers. Journal of Molecular Biology , 418 (1–2), 90–102. https://doi.org/10.1016/j.jmb.2012.02.006 Brinkmann C R, Heegaard C W, Petersen T E, Jensenius J C, & Thiel S (2011). The toxicity of bovine α-lactalbumin made lethal to tumor cells is highly dependent on oleic acid and induces killing in cancer cell lines and noncancer-derived primary cells. FEBS Journal , 278 (11), 1955–1967. https://doi.org/10.1111/j.1742-4658.2011.08112.x Casbarra A, Birolo L, Infusini G, Dal Piaz F, Svensson M, Pucci P, Svanborg C, & Marino G (2004). Conformational analysis of HAMLET, the folding variant of human α-lactalbumin associated with apoptosis. 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Microencapsulation of a whey protein hydrolysate within micro-hydrogels: Impact on gastrointestinal stability and potential for functional yoghurt development. Journal of Functional Foods , 26 , 290–300. https://doi.org/10.1016/j.jff.2016.08.006 Håkansson A, Zhivotovsky B, Orrenius S, Sabharwal H, & Svanborg C (1995). Apoptosis induced by a human milk protein. Proceedings of the National Academy of Sciences of the United States of America , 92 (17), 8064–8068. https://doi.org/10.1073/pnas.92.17.8064 Hernández-ledesma B, & Dávalos A (2005). Preparation of Antioxidant Enzymatic Hydrolysates from α-Lactalbumin and β- Lactoglobulin . Identification of Active Peptides by HPLC-MS/MS, (November 2015). https://doi.org/10.1021/jf048626m Ho CS J, Rydstrom A, Manimekalai M S S, Svanborg C, & Grüber G (2012). Low Resolution Solution Structure of HAMLET and the Importance of Its Alpha-Domains in Tumoricidal Activity. PLoS ONE , 7 (12). https://doi.org/10.1371/journal.pone.0053051 Hoque M, Nanduri R, Gupta J, Mahajan S, Gupta P, & Saleemuddin M (2015). Oleic acid complex of bovine α-lactalbumin induces eryptosis in human and other erythrocytes by a Ca2 +-independent mechanism. Biochimica et Biophysica Acta - General Subjects , 1850 (9), 1729–1739. https://doi.org/10.1016/j.bbagen.2015.04.009 Huang G Q, Sun Y T, Xiao J X, & Yang J (2012). Complex coacervation of soybean protein isolate and chitosan. Food Chemistry , 135 (2), 534–539. https://doi.org/10.1016/j.foodchem.2012.04.140 Jia C-li, Hussain N, Joy Ujiroghene O, Pang X-yang, Zhang S-wen, Lu J, Liu L, & Lv J-ping (2020). Generation and characterization of dipeptidyl peptidase-IV inhibitory peptides from trypsin-hydrolyzed α-lactalbumin-rich whey proteins. Food Chemistry , 318 (April 2019), 126333. https://doi.org/10.1016/j.foodchem.2020.126333 Jung S, Lee S, Lee H, Yoon J, & Lee E K (2016). Oleic acid-embedded nanoliposome as a selective tumoricidal agent. Colloids and Surfaces B: Biointerfaces , 146 , 585–589. https://doi.org/10.1016/j.colsurfb.2016.06.058 Kamijima T, Ohmura A, Sato T, Akimoto K, Itabashi M, Mizuguchi M, Kamiya M, Kikukawa T, Aizawa T, Takahashi M, Kawano K, &Demura M (2008). Heat-treatment method for producing fatty acid-bound alpha-lactalbumin that induces tumor cell death. Biochemical and Biophysical Research Communications , 376 (1), 211–214. https://doi.org/10.1016/j.bbrc.2008.08.127 Lassalle M W, Li H, Yamada H, Akasaka K, & Redfield C (2003). Pressure-induced unfolding of the molten globule of all-Ala α-lactalbumin. Protein Science , 12 (1), 66–72. https://doi.org/10.1110/ps.0221303 Lacroix I M E, & Li-Chan E C Y (2014). Isolation and characterization of peptides with dipeptidyl peptidase-IV inhibitory activity from pepsin-treated bovine whey proteins. Peptides , 54 , 39–48. https://doi.org/10.1016/j.peptides.2014.01.002 Maux S Le, Brodkorb A, Croguennec T, Hennessy A A, Bouhallab S, & Giblin L (2013). β-Lactoglobulin-linoleate complexes : In vitro digestion and the role of protein in fatty acid uptake. Journal of Dairy Science , 96 (7), 4258–4268. https://doi.org/10.3168/jds.2013-6682 Mok K H, Nagashima T, Day I J, Hore P J, & Dobson C M (2005). Multiple subsets of side-chain packing in partially folded states of α-lactalbumins. Proceedings of the National Academy of Sciences of the United States of America , 102 (25), 8899–8904. https://doi.org/10.1073/pnas.0500661102 Mossberg A, Mok K H, & Morozova-roche L A (2010). Structure and function of human a -lactalbumin made lethal to tumor cells ( HAMLET ) -type complexes, 277 , 4614–4625. https://doi.org/10.1111/j.1742-4658.2010.07890.x Permyakov E A, & Berliner L J (2000). alpha-Lactalbumin: structure and function. FEBS Letters , 473 (3), 269–274. https://doi.org/10.1016/S0014-5793(00)01546-5 Petitpas I, Grüne T, Bhattacharya A A, & Curry S (2001). Crystal structures of human serum albumin complexed with monounsaturated and polyunsaturated fatty acids. Journal of Molecular Biology , 314 (5), 955–960. https://doi.org/10.1006/jmbi.2000.5208 Pettersson J, Mossberg A, & Svanborg C (2006). a -Lactalbumin species variation, HAMLET formation, and tumor cell death, 345 , 260–270. https://doi.org/10.1016/j.bbrc.2006.04.081 Raei M, Shahidi F, Farhoodi M, Jafari S M, & Rafe A (2017). Application of whey protein-pectin nano-complex carriers for loading of lactoferrin. International Journal of Biological Macromolecules , 105 , 281–291. https://doi.org/10.1016/j.ijbiomac. 2017.07.037 Spolaore B, Pinato O, Canton M, Zambonin M, Polverino De Laureto P, & Fontana A (2010). Α-Lactalbumin Forms With Oleic Acid a High Molecular Weight Complex Displaying Cytotoxic Activity. Biochemistry , 49 (39), 8658–8667. https://doi.org/10.1021/bi1012832 Svensson M, Håkansson A, Mossberg A K, Linse S, & Svanborg C (2000). Conversion of α-lactalbumin to a protein inducing apoptosis. Proceedings of the National Academy of Sciences of the United States of America , 97 (8), 4221–4226. https://doi.org/10.1073/pnas.97.8.4221 Svensson M, Fast J, Mossberg A, Duringer C, Gustafsson L, Hallgren Brooks C L, Berliner L, Linse S, & Svanborg C (2003). α-Lactalbumin unfolding is not sufficient to cause apoptosis , but is required for the conversion to HAMLET ( h uman α -lactalbumin m ade le thal to t umor cells ), 2794–2804. https://doi.org/10.1110/ps.0231003.HAMLET Timilsena Y P, Wang B, Adhikari R, & Adhikari B (2015). Preparation and characterization of chia seed protein isolate-chia seed gum complex coacervates. Food Hydrocolloids , 52 , 554–563. https://doi.org/10.1016/j.foodhyd.2015.07.033 Url-1, https://web.expasy.org/peptide_cutter/ Supplementary Files GraphicalAbstract.tif Highlights.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2082257","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":145459648,"identity":"0594567b-02ac-405f-a88f-14b2e7b78c40","order_by":0,"name":"REYHAN KOYUNCU","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYBACCRiDnxlMMZOgRbKZGaqFjVgtBgeI1SLZ3mMmXVBzON/4OP8xCYYK68QG+d4HeLVI85wxk55x7LDltsPMbBIMZ9ITG9jYDfBqkZPIMZPmbThsYAbSwth2GKiFgMvgWoybQVr+EaFFGqbFgBmkpYEILZI9x4qteY6lG0gcZja2SDiWbtzGloZfi8Tx5o23eWqsDfj7Dz688aHGWraf+Rh+LQwMHEjhk8BAOFqAgP0BYTWjYBSMglEwsgEAir819kWpXfgAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-4383-2483","institution":"Istanbul Teknik Universitesi - Ayazaga Kampusu: Istanbul Teknik Universitesi","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"REYHAN","middleName":"","lastName":"KOYUNCU","suffix":""},{"id":145459649,"identity":"93192e0d-1c2c-4d07-babb-d037823eccad","order_by":1,"name":"Gokhan Duruksu","email":"","orcid":"","institution":"Kocaeli University: Kocaeli Universitesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gokhan","middleName":"","lastName":"Duruksu","suffix":""},{"id":145459650,"identity":"978cf6a3-d486-471a-b725-c5643e19ffe5","order_by":2,"name":"Beraat Ozcelik","email":"","orcid":"","institution":"Istanbul Teknik Üniversitesi: Istanbul Teknik Universitesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Beraat","middleName":"","lastName":"Ozcelik","suffix":""},{"id":145459651,"identity":"586a9a9a-d7dc-4e30-a036-71a357dd471e","order_by":3,"name":"Yusufhan Yazir","email":"","orcid":"","institution":"Kocaeli University: Kocaeli Universitesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yusufhan","middleName":"","lastName":"Yazir","suffix":""}],"badges":[],"createdAt":"2022-09-19 20:12:55","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2082257/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2082257/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":28190822,"identity":"44cca45a-c14e-499d-802a-a51784c8bba6","added_by":"auto","created_at":"2022-10-24 17:37:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":37164,"visible":true,"origin":"","legend":"\u003cp\u003eImage of the α-LA protein obtained in the FPLC.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-2082257/v1/ca8d60e02742da21791965e1.png"},{"id":28191370,"identity":"d47d8177-7090-493c-adcc-8b7f4b70bcdd","added_by":"auto","created_at":"2022-10-24 17:47:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":65969,"visible":true,"origin":"","legend":"\u003cp\u003eSDS-PAGE of α-LA, BAMLET, hydrolysed α-LA and hydrolysed BAMLET.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-2082257/v1/76f50865e3733c5833e39d78.png"},{"id":28190994,"identity":"a236e7e9-ddda-44e4-bed1-3b81be23d8c9","added_by":"auto","created_at":"2022-10-24 17:42:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":43798,"visible":true,"origin":"","legend":"\u003cp\u003eThe FPLC image obtained after BAMLET synthesis.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-2082257/v1/e1fa27f7d14063bc8d622add.png"},{"id":28190823,"identity":"bce7c1e5-7859-41cf-bc7c-0d1435543393","added_by":"auto","created_at":"2022-10-24 17:37:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":43332,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent varieties of BAMLET protein obtained by incubation for a long time(after the incubation for 4 °C, 24 hrs.)\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-2082257/v1/d6e6c18dadba5ab5f1ffc4a0.png"},{"id":28190830,"identity":"ddec9c23-128d-47e4-83f7-4c054e47dd6b","added_by":"auto","created_at":"2022-10-24 17:37:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":308949,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of BAMLET (a) and hydrolysed BAMLET (b) particles, together with their size distributions. Scale bars correspond to 5 µm for BAMLET and 10 µm for hydrolysed BAMLET\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-2082257/v1/b70ad09784d005306852a9ed.png"},{"id":28190828,"identity":"8713eeb0-75b1-4757-a61f-536f98df1c5a","added_by":"auto","created_at":"2022-10-24 17:37:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":151068,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of α-LA and BAMLET (A), hydrolysed α-LA and hydrolysed BAMLET (B) and BAMLET and hydrolysed BAMLET (C).\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-2082257/v1/9375199cae5ace8d91ddb103.png"},{"id":28190995,"identity":"8d7d679f-6005-44c8-a04d-9cca4695681c","added_by":"auto","created_at":"2022-10-24 17:42:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":63819,"visible":true,"origin":"","legend":"\u003cp\u003eDSC patterns of BAMLET and hydrolysed BAMLET(A) and DSC patterns of α-LA and hydrolysed α-LA (B).\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-2082257/v1/cc09be74aef6f51adeec18ba.png"},{"id":28190826,"identity":"2bc1c5b7-1b5d-4f6d-88ee-b8972d52aef4","added_by":"auto","created_at":"2022-10-24 17:37:59","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":24052,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity of BAMLET and hydrolysed BAMLET on MCF7 and DU145 by LDH Activit Assay\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-2082257/v1/8ff7104d3e0ab02a6088de8b.png"},{"id":28190832,"identity":"5a2b03df-8551-4b89-a446-c2ee2ca14391","added_by":"auto","created_at":"2022-10-24 17:37:59","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":41881,"visible":true,"origin":"","legend":"\u003cp\u003eMCF7, DU145 and Fibroblast cells viability after 24 h of incubation with BAMLET and Hydrolysed BAMLET. Effect of BAMLET on breast cancer cell (MCF7) viability (A). Effect of BAMLET on prostate cancer cell (DU145) viability (B). (C) Effect of hydrolysed BAMLET on MCF7 viability (C). Effect of hydrolysed BAMLET on DU145 viability (D). Effect of BAMLET on fibroplast cell viability (E). Effect of hydrolysed BAMLET on fibroblast cell viability (F).\u003c/p\u003e","description":"","filename":"Fig.9.png","url":"https://assets-eu.researchsquare.com/files/rs-2082257/v1/101a6e25990adf6c91185b48.png"},{"id":29037071,"identity":"d83ed097-8b96-4f22-946a-b08e12d00b5d","added_by":"auto","created_at":"2022-11-14 16:11:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1180473,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2082257/v1/b900315a-851d-4199-934e-f7d98a662642.pdf"},{"id":28190993,"identity":"bb9ca06a-e76e-4ea6-84e5-d96276ec5431","added_by":"auto","created_at":"2022-10-24 17:42:59","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":384104,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-2082257/v1/96320a4c2e2aa556b8fecf8b.tif"},{"id":28190991,"identity":"c53650b1-cff4-4e09-a38b-d58ce1b27629","added_by":"auto","created_at":"2022-10-24 17:42:59","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14186,"visible":true,"origin":"","legend":"","description":"","filename":"Highlights.docx","url":"https://assets-eu.researchsquare.com/files/rs-2082257/v1/c2515f6fb6c986cc66641edc.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eEnhancement of Cancericidal Activity of Bamlet Complexes by Enzymatic Treatment\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eComplexes formed with oleic acid (OA) and α-Lactalbumin (α-LA) have attracted attention due to their therapeutic potential. Many studies have been carried out for years about this complex, which was discovered serendipitously by the Svanborg group (H\u0026aring;kansson et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Svensson et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Svenssonet al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). α-LA is an acidic (pI around 4.5), (Permyakov \u0026amp; Berliner, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), globular protein composed of 123 amino acid residues found in milk. The molecular weight of this acidic calcium-binding protein in its native conformation is 14.2 kDa (Svensson et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The three-dimensional structure of α-LA consists of two domains: the α-domain (residues 1\u0026ndash;34 and 86\u0026ndash;123), consisting of four α-helices and two short 3₁₀-helices; and the smaller β-domain (residues 35\u0026ndash;85), (Casbarra, et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Spolaore, et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The native conformation is stabilized by four disulfide bonds (amino acids 6-120, 61\u0026ndash;77, 73\u0026ndash;91 and 28\u0026ndash;111), and the two domains are linked by the calcium-binding loop (Pettersson et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Either by heating, by the interaction with high concentrations of denaturants or by incubating at low pH, the native α-LA is converted to the partially unfolded protein form, so called the intermediate state or the molten globule state (Casbarra et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Lassalle et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Mok et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Spolaore et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). After the addition of oleic acid to apo-state of α-LA, the formed complex can induce cell death in cancer cells (Svensson et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). α-LA contains conserved amino acid side-chains that can make salt bridges with the carboxylate group of oleic acid. Due to multiple sites with high or low binding affinities in α-LA, the binding of oleic acid to these sites becomes highly complicated (Petitpas et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Moreover, oligomerization reaction rates are depended on α-LA concentration (Baumann et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor the production of this protein-fatty acid complex, human milk was used as a first source of α-LA, and the product was named as HAMLET (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003euman \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eA\u003c/span\u003elpha-lactalbumin \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eM\u003c/span\u003eade \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eLE\u003c/span\u003ethal to \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eT\u003c/span\u003eumor cells), (Svensson et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). While this complex induces an apoptotic mechanism in tumour cells, the healthy cells are resistant to this effect (Delgado et al., H\u0026aring;kansson et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; 2015; Jung et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Svensson et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Svensson et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2000\u003c/span\u003e;). Its anti-tumoural activity was shown on more than 40 different lymphoma and cancer cell lines (Hoque et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). It has been found that α-LA from different sources (human, bovine, goat, swine, sheep, equine and camelid) can form OA-complexes that can exhibit biological properties similar to HAMLET (Fontana et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Pettersson et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Spolaore et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In other words, it has been proven that HAMLET-like complexes can also form with other α-LA varieties (Fang et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). For this purpose, bovine milk (BAMLET: \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eB\u003c/span\u003eovine \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eA\u003c/span\u003elpha lactalbumin \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eM\u003c/span\u003eade \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eLE\u003c/span\u003ethal to \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eT\u003c/span\u003eumor cells), which is more accessible source of milk, was used within the scope of the project.\u003c/p\u003e \u003cp\u003eIn order to understand the tumoricidal activity of HAMLET, its molecular structure has been studied by researchers (Ho et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Pettersson et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Although the sequence variations in α-LA from animal sources exist, other α-LA variants can also bind oleic acid to form complex, as like in human α-LA. BAMLET, on the other hand, can bind high rates of oleic acid residues (5\u0026ndash;8 oleic acids per protein molecule) than other protein-lipid complexes. It, therefore, has higher lipid-to-protein ratio than the HAMLET complex, and it might show different properties (Ho et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In this study, bovine α-LA was fragmented into short peptides in various length with the help of hydrolysing enzymes, and hydrolysed BAMLET was produced subsequently. For this purpose, pepsin and trypsin peptidases were used. The activity of the formed complex was proven on DU145 and MCF7 cancer cell lines.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Purification of Bovine α-LA\u003c/h2\u003e \u003cp\u003eα-LA was purified from skim bovine milk from commercial sources according to the method of Pettersson et al., (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) with some modifications. Briefly, casein was removed by acid treatment adjusting the milk to pH 4.0 with HCI, and centrifuging at 4000g for 20 min at 4\u0026deg;C. α-LA was separated in fractional precipitation at the 55% ammonium sulfate saturation, and the precipitate was collected by centrifugation at 8000g for 20 min at 4\u0026deg;C. After dissolving, sample was desalted by dialysis in 0.1 M Tris-HCl buffer at pH 7.5 at 4\u0026deg;C, and ultrafiltrated using Amicon-Ultra 15 Centrifugal Filter Units (Merck-Millipore, Tullagreen, Cork, Ireland) with a molecular weight cut-off of 10 kDa. α-LA was purified by FPLC using size-exclusion (SEC) column (HiPrep Sephacryl S-200 HR) on \u0026Auml;KTA Prime Plus FPLC (GE Healthcare) by eluting in 10 mM Tris-HCl buffer, pH 7.5 with 10 mM EDTA. Samples were concentrated by the ultrafiltration as described above. The purity was confirmed by SDS-PAGE and the molecular weight was estimated by using the gel filtration chromatogram (HiPrep Sephacryl S-200 HR) with low molecular weight markers (Gel Filtration Cal. Kit Low Molecular Weight, GE Healthcare). The total protein content was measured by SMART bicinchoninic acid assay (BCA) Protein Assay Kit (iNtRON Biotechnology, Sungnam, Korea). After mixing with loading buffer solution (Tris-HCl 0.5 M pH 6.8, glycerol, sodium dodecyl sulfate (SDS), β-mercapto ethanol and Bromophenol Blue), samples (5 \u0026micro;g) were denatured in boiling water for 3 min. The denatured protein was loaded and separated by 12% SDS-polyacrylamide gel (TGX FastCast acrylamide gels; BioRad, Hercules, CA, USA) and run at 120 V for 50 min. Bands were visualized in DNR Bio-Imagining Systems (MF-ChemiBIS 3.2, Jerusalem, Israel).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Enzymatic Hydrolysis of α-LA\u003c/h2\u003e \u003cp\u003eFor the hydrolysis of α-LA, the method described by Hern\u0026aacute;ndez-ledesma \u0026amp; D\u0026aacute;valos (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) was used. α-LA (1 mg/mL) was first hydrolysed with trypsin (EC 3.4.21.4.; Type I; 10 900 U/mg protein; Sigma; St. Louis, MO, U.S.A.) in 0.1 M Tris-HCl buffer (pH 7.4) for 24 h at 37\u0026deg;C. Later, the pH was adjusted to pH 2.5 by HCl (1M), and pepsin (EC 3.4.23.1.; 1:60 000, 3400 U/mg protein; Sigma; St. Louis, MO, U.S.A.) was added at the ratio of enzyme-to-substrate by 1:20 (w/w). After the incubation for 2h at 37\u0026deg;C, enzymatic activity was stopped by heating at 95\u0026deg;C for 15 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Preparation of α-LA \u0026ndash; oleic acid complexes\u003c/h2\u003e \u003cp\u003eα-LA at 210 \u0026micro;M in phosphate-buffered saline (PBS) was mixed with oleic acid (1:2, v/v). The mixture was heated to 55\u0026deg;C, and incubated for 15 min at this temperature. Later, the mixture was allowed for 10 min to cool to room temperature. The sample was centrifuged (12.000g, 25\u0026deg;C, 15 min.). Excess oleic acids were removed (Kamijima et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Morphology study\u003c/h2\u003e \u003cp\u003eFor the ultrastructural analysis, scanning electron microscopy (SEM) was used. The samples were sprinkled onto a two-sided adhesive tape and then coated with a thin layer of gold. Morphologic features of samples observed by a scanning electron microscope (Quanta FEG 250) in high vacuum with 5 kV accelerating voltage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. FTIR study\u003c/h2\u003e \u003cp\u003eFunctional groups in α-LA, hydrolysed α-LA, BAMLET and hydrolysed BAMLET examined by Fourier transform infrared spectroscopy (FTIR) using the attenuated total reflection technique. After freeze-drying the samples were analyzed in the FTIR GX (Perkin-Elmer, Shelton, CT, USA) instrument. All spectra ranged from 200ˉ ˡ cm to 4000ˉ ˡ cm. The spectra were obtained by averaging 16 scans at 4 cmˉ ˡ resolution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. DSC study\u003c/h2\u003e \u003cp\u003eThermal behavior of α-LA hydrolysed α-LA, BAMLET and hydrolysed BAMLET were investigated by a differential scanning calorimeter (DSC-028 Mettler Toledo Inc., Columbus OH). The denaturation temperature range (ΔTd), peak denaturation temperature (Td) and the enthalpy of denaturation (ΔHd) of lyophilised samples were determined. About 5 mg of each sample was weighed in an aluminum pan and hermetically sealed. An empty pan was used as a reference. Samples were scanned at the temperature range of 40\u0026ndash;300\u0026deg;C with the rate of 5\u0026deg;C/min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Anticancer activity of BAMLET and BAMLET hydrolysates\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1. Cell lines and Cell Culture\u003c/h2\u003e \u003cp\u003eThe analyses of the cancericidal effect of BAMLETs were performed using two cancer cell lines, MCF7 (human breast adenocarcinoma cell line) and DU145 (androgen-independent human prostate cancer cell line). To determine the cytotoxic effect of BAMLETs on normal somatic cells, fibroblast cells derived from foreskin tissue were used (Acemi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). All cell lines were cultured in the complete RPMI 1640 medium [RPMI 1640 basal medium (Gibco, Paisley, UK) supplemented with 2.0 mM L-glutamine, 0.5 mM sodium pyruvate, 10% fetal bovine serum (Gibco) and 1% Penicillin-Streptomycin solution (Gibco). Cells were cultured under standard culture conditions in a humidified environment of 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. Before the cell seeding into the culture plates, cells were detached by 0.25% trypsin-EDTA (Gibco).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2. Determination of cell viability by WST-1\u003c/h2\u003e \u003cp\u003eTo determine the effect of BAMLETs on fibroblast, MCF7 and DU145, cells were incubated with different concentrations of BAMLET and hydrolysed-BAMLET in RPMI 1640 complete medium for 24 h. The medium was replaced with RPMI 1640 basal medium supplemented with 10% WST-1 (Roche, Mannheim, Germany), and further incubated for 30 min at 37 \u0026deg;C. The absorbance at 450 nm was measured. The changes in cell numbers were expressed in percentage with respect to control group (medium without BAMLET).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Lactate dehydrogenase (LDH) activity assay\u003c/h2\u003e \u003cp\u003eCytotoxicity was determined with Cytotoxicity Detection Kit LDH (Roche) according to the manufacturer\u0026rsquo;s instructions (Duruksu \u0026amp; Aciksari, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). MCF7 and DU145, cells were incubated with different concentrations of BAMLET and hydrolysed-BAMLET in RPMI 1640 complete medium for 24 h. After performing the assay, the absorbance of the collected supernatant was measured at 490 nm and a reference wavelength of 620nm with controls including medium alone. The cell culture without BAMLET or hydrolised BAMLET was used as control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical analysis\u003c/h2\u003e \u003cp\u003eData were measured in triplicate. All statistical analyses were performed with the use of SPSS 10.0 (SPSS Inc, Chicago, IL, USA). The significance of results was analyzed by means of paired t test. In all analyzed cases, the results were regarded as statistically significant at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results And Discussions","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Identification of purified α-LA and hydrolysed α-LA\u003c/h2\u003e \u003cp\u003eα-LA was purified by FPLC method. A protein peak of 14.2 kDa was obtained according to the calibration curve of standard proteins with known molecular mass (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In agreement with the results by Svensson et al., (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), α-LA was observed as an intense, single band around 14 kDa as shown on SDS-PAGE (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Hydrolysis of α-LA with pepsin and trypsin generated peptide fragments with small molecular weight (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Hydrolysed α-LA on the SDS-PAGE showed a faint band around 15 kDa, indicating that the majority of α-LA is hydrolysed by pepsin and trypsin and minor amount of unhydrolysed α-LA still existed as a peptide band of 15 kDa. The digestion of α-LA protein into smaller fragments by the hydrolysing enzymes provided to separate into functional peptides, which was predicted putatively on Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Many functional groups on α-LA was expected to become available to interact with other molecules by this process (G\u0026oacute;mez-Mascaraque et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Jia et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Lacroix \u0026amp; Li-Chan, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Url-1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePutative digestion products of α-LA by pepsin or trypsin (Lacroix \u0026amp; Li-Chan, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Jia et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; G\u0026oacute;mez-Mascaraque, Miralles, Recio \u0026amp; L\u0026oacute;pez-Rubio, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Url-1)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFragment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePepsin / Trypsin\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u0026ndash;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEQLTK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrypsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u0026ndash;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEQLTKCEVFR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrypsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u0026ndash;17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRELKDLKG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePepsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u0026ndash;16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLKDLK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrypsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u0026ndash;25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKGYGGVSLPE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePepsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e32\u0026ndash;36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHTSGY\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePepsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e37\u0026ndash;43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDTQAIVQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePepsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e37\u0026ndash;44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDTQAIVQN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePepsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e41\u0026ndash;53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIVQNNDSTEYGLF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePepsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e45\u0026ndash;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNDSTEY\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrypsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e51\u0026ndash;58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGLFQINNK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrypsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u0026ndash;79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWCKDDQNPHSSNICNISCDK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePepsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e63\u0026ndash;68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDDQNPH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrypsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e90\u0026ndash;95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCVKKIL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePepsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e95\u0026ndash;104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eILDKVGINY\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePepsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e94\u0026ndash;98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKILDK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrypsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e94\u0026ndash;108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKILDKVGINYWLAHK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrypsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e95\u0026ndash;108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eILDKVGINYWLAHK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrypsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e99\u0026ndash;104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVGINYW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrypsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e104\u0026ndash;117\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWLAHKALCSEKLDQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePepsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e105\u0026ndash;111\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWLAHKAL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePepsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e105\u0026ndash;115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLAHKALCSEKL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePepsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e110\u0026ndash;117\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLCSEKLDQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePepsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e115\u0026ndash;123\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLDQWLCEKL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrypsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e115\u0026ndash;122\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLDQWLCEK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTrypsin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Characterization of BAMLET and Hydrolysed BAMLET complexes\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1. Molecular weight characterisation\u003c/h2\u003e \u003cp\u003eIn the current study, the heating method was preferred to generate BAMLET because of the high efficient production of BAMLET. The generation of BAMLET by an anion exchange column, which was loaded with oleic acid, has been described previously, but the method by heating a mixture of α-LA and oleic acid generated with higher efficiency (H\u0026aring;kansson et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Svensson et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Svensson et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). By heating the α-LA protein, partial denaturation was induced, and the complex formation took place efficiently (Kamijima et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAfter the hydrolysis of α-LA, the same procedure for BAMLET was applied to the smaller fragments. SDS-PAGE was insufficient to separate these products, but the single band of BAMLET produced from the α-LA could be detected on the gel (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Peptides in hydrolysed BAMLET are mostly smaller than 10 kDa. While hydrolysis-derived peptides formed small aggregates with the effect of low pH (~\u0026thinsp;2.5), the cross-links they formed between them consist of disulfide bonds. In addition to disulfide bonds, other intermolecular cross-links were also formed between protein aggregates formed at higher pH values (above 4.5), respectively (Gulzar et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Observation of a faint band around 15 kDa indicates that new and strong covalent bonds were not formed when hydrolysed BAMLET was formed from hydrolysed α-LA (Gulzar et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), but newly formed disulfide bonds between peptides (Maux et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). During the production of BAMLET, in which the mixture of molecules were heat treated for 15 min., two main forms of BAMLETs were mainly generated with molecular weights of about 25.5 kDa and 51 kDa, which was predicted as the monomeric and dimeric forms of complexes, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The intense bands on SDS-PAGE and the data of SEC analysis of BAMLET indicate that BAMLETs interconnected by disulfide bonds and by intermolecular cross-linkages to form larger insoluble and soluble aggregates. The molecular mass of the monomeric BAMLET was estimated to be approximately 25.5 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In a complementary approach, it confirmed that the SDS-PAGE showed another peak around 51 kDa, a dimeric form of BAMLET. Remarkably, another peak at 14.2 kDa, which refers to α-LA, could be observed to remain as residue in the medium left without interacting with oleic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This α-LA residue of BAMLET reaction was left to interact with oleic acid overnight at 55\u0026deg;C. It was observed that BAMLET complexes were formed at various molecular weights (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). During the synthesis process, high temperature and high pH led some modifications to form high or low binding affinity sites on α-LA. These changes further supported the production of new oligomers after the process. Therefore, oleic acid:α-LA complexes could be formed at different ratios. A continuous complex formation took place between α-LA and excess oleic acid, which cannot be eliminated. The various form of BAMLET complexes formed after processing could be explained by either the formation of peptide aggregates that cannot be dissociated by the SEC eluent or newly formed disulphide bonds between peptides and proteins interconnected by intermolecular cross-links (Maux et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2. Morphological characterisation\u003c/h2\u003e \u003cp\u003eThe SEM micrographs of BAMLET and hydrolysed BAMLET showed that the BAMLET and BAMLET-like complexes have a globular conformation due to the globular structure of α-LA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), as stated before (Delgado et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Mossberg et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Similarly, the peptide fragments of hydrolysed BAMLET appeared as spherical structures in the SEM image. In general, smaller microparticles were obtained when pepsin and trypsin enzymes were used as hydrolysing substance (mean diameter: 0.192 \u0026micro;m). Particle sizes and polydispersity index (PDI) are extremely important for drug formulations. It has been reported that particles with a diameter of 50\u0026ndash;500 nm are known to be suitable for accumulate in tumor cells (Delgado et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The relatively low polydispersity index (PDI) value of hydrolysed BAMLET indicated that it has a more regular particle size distribution than BAMLET.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3. FTIR characteristics\u003c/h2\u003e \u003cp\u003eThe FTIR spectrum of α-LA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) showed a broad band at around 3000\u0026thinsp;~\u0026thinsp;3700 cm ˉ ˡ, indicating enhanced hydrogen bonding compared to that of BAMLET seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea. This suggests that hydrogen bonding is also involved in the interaction between α-LA and oleic acid. The lipid interaction effect formed by the binding of oleic acid to α-LA is clearly visible in the stretching vibration in the range of 2800\u0026thinsp;~\u0026thinsp;3000 cm ˉ ˡ (yellow circle). Characteristic bands were observed for both molecules (α-LA and BAMLET) at 1642 cmˉ ˡ (yellow circle) in a characteristic amide I band attributed to the C\u0026thinsp;=\u0026thinsp;O vibration (C\u0026thinsp;=\u0026thinsp;O stretching) of the acetylated units. The decrease in 1408 cm ̄ ˡ is due to the vibration of -OH and -CH (Chang et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, although there are many higher or lower wave numbers between the 1000\u0026thinsp;~\u0026thinsp;1500 cm ˉ ˡ range in both groups, due to the overlap of the amide I region and most spectral bands, these different fluctuations can be attributed to oleic acid binding to α-LA and to the application of heating treatment while BAMLET was formed. The broad band seen in the FTIR spectrum of BAMLET and hydrolysed α-LA in the range 3000\u0026thinsp;~\u0026thinsp;37000 cm ˉ ˡ was not seen for hydrolysed BAMLET (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). However, stronger stretch vibration and a wider band are observed in the range of 2800\u0026thinsp;~\u0026thinsp;3000 cm ˉ ˡ (yellow circle). This situation draws attention to the lipid interaction density between hydrolysed α-LA and oleic acid, which creates hydrolysed BAMLET. The FTIR spectrum of hydrolysed α-LA showed absorption bands at 1615 cm ˉ ˡ (amide I, C ═ O and C ― N stretching), 1410 cm ˉ ˡ and 1320 cm ˉ ˡ (amide II, N ― H deformation and C ― N stretching), (Huang et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). For Hydrolysed BAMLET, while none of the amide groups were observed, a single distinct peak was detected. The peak at 1747.33 cmˉ ˡ corresponds to the esterified group of hydrolysed BAMLET (yellow circle), (Raei et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). BAMLET, on the other hand, has only amide I bonds in this domain (yellow circle, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.2.4. Thermal characteristics (DSC)\u003c/h2\u003e \u003cp\u003eThe DSC thermograms of BAMLET, hydrolysed BAMLET, α-LA and hydrolysed α-LA were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB respectively. The thermograms of both α-LA and hydrolysed α-LA exhibited a weak melting behavior at around 160\u0026deg;C, which associated with the melting of the network structures (Huang et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The melting behavior in the network structures continued for the enzyme-treated α-LA, which is corresponding to the thermal denaturations of less stable peptide fragments have been observed at temperatures lower than 200\u0026deg;C (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, α-LA exhibited a sharp peak. A higher denaturation temperature and denaturation enthalpy of α-LA shows a network structure with improved thermal stability compared to hydrolysed α-LA. It has been announced that, complex proteins are more stable against thermal denaturation (Capitani et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Chang et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Huang et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Timilsena et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Similar to α-LA and hydrolysed α-LA, both thermograms of BAMLET and hydrolysed BAMLET begin with melting of the network structures; hydrolysed BAMLET exhibited a very different diagram. Hydrolysed BAMLET composed of very small peptides showed only slight crystallinity, attributed to monomerization causing weaker network structure. BAMLET, on the other hand, showed an endothermic peak at higher temperature (Td\u0026thinsp;=\u0026thinsp;231.44\u0026deg;C). This denaturation temperature could be ascribed to a tendency to dimerisation. The same situation was observed for α-LA at Td\u0026thinsp;=\u0026thinsp;236.8\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDenaturation temperature range (ΔTd), peak denaturation temperature (Td) and denaturation enthalpy (ΔHd) of CPI and α-LA, hydrolysed α-LA, BAMLET and hydrolysed BAMLET.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaterials\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eΔTd (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTd (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eΔHd (j/g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eα-LA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFirst peak\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e203.67-219.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e199.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;270.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSecound peak\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e244.25-235.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e236.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;5.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eHydrolysed α-LA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFirst peak\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e190.96-183.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e185.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;38.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSecound peak\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e201.87-190.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e192.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;59.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThird peak\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e231.78-201.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e204.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;101.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eBAMLET\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFirst peak\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e129.68\u0026ndash;114.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e116.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;50.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSecound peak\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e153.65-129.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e142.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;32.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThird peak\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e183.56-153.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e231.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;122.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHydrolysed BAMLET\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFirst peak\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e190.8\u0026ndash;82.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e143.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-100.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Toxicity of BAMLET products on fibroblast cells\u003c/h2\u003e \u003cp\u003eLDH activity of BAMLET and hydrolysed BAMLET was estimated for both MCF7 and DU145 cell lines after incubation for 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The toxicity of BAMLET was rather limited for MCF7 cells. At the highest BAMLET concentration (10 \u0026micro;g/\u0026micro;l), less than 6.6% \u0026plusmn;1.3 of LDH activity level was observed compared to the control (without BAMLET). On the other hand, the highest toxicity level with 25.6% \u0026plusmn;2.2 toxicity was achieved in DU145 cell lines at the BAMLET concentration of 2.14 \u0026micro;g/\u0026micro;l. α-LA has a toxicity effect with 0.83% \u0026plusmn;2.2 and 8.8% \u0026plusmn;0.9 for MCF7 and DU145, respectively. Compared to α-LA, the BAMLET showed improved toxic effect, but the toxicity level for BAMLET could not be detected higher than 8% and 28% for MCF7 and DU145 over the BAMLET concentration ranging from 1.07 to 10.00 \u0026micro;g/\u0026micro;l.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe hydrolysed BAMLET possessed significantly improved toxic effect for both cancer cells. The toxicity levels of hydrolysed BAMLET for MCF7 and DU145 were estimated as 49.2% and 55.2% for the concentration at 6.38 \u0026micro;g/\u0026micro;l of hydrolysed BAMLET compared to the control. The effectivity was improved by almost 6.2-fold and 2-fold against MCF7 and DU145, respectively.\u003c/p\u003e \u003cp\u003eIn the previous studies with BAMLET, it was shown that two types of cytotoxicity effect of BAMLET could be observed depending on its concentration. Cell apoptosis could be observed at low concentrations, but at high concentrations cell lysis could be detected (Brinkmann et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In this study, higher BAMLET concentrations were required to observe the cytotoxic effect after 1 hour of incubation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Anti-cancer effect of BAMLET and hydrolysed BAMLET products\u003c/h2\u003e \u003cp\u003eThe viability analyses of MCF7 and DU145 cells treated with BAMLET / hydrolysed BAMLET complexes was performed by WST-1. We found that the synthesized complexes showed varying toxicities (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Although α-LA demonstrates relatively better cytotoxic effect for the prostate cancer cells (DU145), it also caused to decrease the cell viability of breast cancer cells, MCF7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). After the treatment with BAMLET at the concentration of 2.14 \u0026micro;g/\u0026micro;l for 24 hrs, the viability of DU145 cells were decreased more than 50%. (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). However, the same BAMLET concentration did not have the same cell viability decreasing effect on MCF7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). The MCF7 viability was slightly decreased to 89.16% at the concentration of 10 \u0026micro;g/\u0026micro;l (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter the treatment with the hydrolysing enzymes, tumoricidal effect of BAMLET was significantly improved. It was observed that the formed peptide fragments retained their ability to form a complex with oleic acid possessing a tumoricidal activity, similar to BAMLET. Hydrolysed BAMLET was observed to have improved cytotoxicity at the dose of 6.38 \u0026micro;g/\u0026micro;l for both cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC and Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD). The effect of BAMLET and hydrolysed BAMLET on somatic cells (fibroblasts) at the specified dosage ranges is quite important for its safely usage. BAMLET supported the cell viability of fibroblast cells until the concentration of 2.14 \u0026micro;g/\u0026micro;l, but the viability suddenly dropped at the concentrations higher than 4.25 \u0026micro;g/\u0026micro;l of BAMLET (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE). Interestingly, the hydrolysed BAMLET did not showed any detrimental effect on the fibroblast cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF). On the contrary, it supported the cell proliferation. The cell population was enhanced by 2.71-fold at the concentration of 6.38 \u0026micro;g/\u0026micro;l, at which the prostate and breast cancer showed %32.2 and %22 cell viability. BAMLET had a tumoricidal effect of 2.89% (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA) on MCF7, while on DU145 the level of this effect was 51.9% (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). On the other hand, the hydrolysed BAMLET had a tumoricidal effect of 78.04% and 67.83% on MF7 and DU145 cells, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC and Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn the current study, the BAMLET was produced from the hydrolysed α-LA fragments, so-called \u0026ldquo;hydrolysed BAMLET\u0026rdquo;, with improved cytotoxicity against MCF7 and DU145 cells. Moreover, this hydrolysed BAMLET supported the cell population of somatic cells while decreasing the number of cancer cells. From this aspect, the hydrolysed BAMLET might be an ideal anti-cancer agent to be used in the cancer treatment. The hydrolysis process released the functional groups of α-LA, which were more effective in cellular toxicity against MCF7 and DU145 cells. It is still unclear which released functional peptide fragments of α-LA possessed the toxic effect or which fragments involved in the complex formations with oleic acid. Long-term incubation of α-LA and oleic acid led the formation of complexes with various dimerization rates. The dimerization trend in BAMLET is consistent with the DSC thermograms. Although hydrolysed BAMLET yields a less stable thermogram, an intense lipid interaction between α-LA and oleic acid according to the FTIR spectrum. Moreover, the collection of functional groups in a single peak at 1615 cm ˉ ˡ indicates the presence of structurally esterified groups.\u003c/p\u003e \u003cp\u003eBAMLET and hydrolysed BAMLET complexes were identified as significant growth inhibitors on the MCF7 and DU145 cell lines. However, hydrolysed BAMLET is more effective than BAMLET from the point of toxicity. Hydrolysed BAMLET, at the concentration of 6.38 \u0026micro;g/\u0026micro;l where it has a strongest tumoricidal effect on MCF7 and DU145 cancer cells, showed a supportive effect on the viability of fibroblast cells. In the future, BAMLET and hydrolysed BAMLET could be considered for using in drug formulations with the appropriate method.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u0026alpha;-LA, alpha lactalbumin; BAMLET, \u003cu\u003eB\u003c/u\u003eovine \u003cu\u003e\u0026alpha;\u003c/u\u003e-Lactalbumin \u003cu\u003eM\u003c/u\u003eade \u003cu\u003eLE\u003c/u\u003ethal to \u003cu\u003eT\u003c/u\u003eumors;\u0026nbsp;BCA, bicinchoninic acid; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate;\u0026nbsp;FTIR, Fourier transform infrared spectroscopy; DSC, Differential scanning calorimetry; SEM, scanning electron microscopy;\u0026nbsp;LDH,\u0026nbsp;Lactate Dehydrogenase; WST, Water-soluble tetrazolium; PDI,\u0026nbsp;polydispersity index\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank to Evren DEMİRCAN from the Department of Food Engineering at Istanbul Technical University for his technical assistance in DSC and FTIR analyses.\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCredit authorship contribution statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReyhan Koyuncu\u003c/strong\u003e: Methodology, Formal analysis, Resources, Investigation, Writing-original draft, Revision, Conceptualization, Project administration. \u003cstrong\u003eGokhan Duruksu\u003c/strong\u003e: Conceptualization, Validation, Reviewing \u0026amp; editing, Supplementary data, Data curation. \u003cstrong\u003eBeraat Ozcelik\u003c/strong\u003e: Review \u0026amp; editing, Supervision. \u003cstrong\u003eYusufhan Yazır\u003c/strong\u003e: Investigation, Supervision.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAcemi A, Duruksu G, \u0026amp; \u0026Ouml;zen F (2017). 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Preparation and characterization of chia seed protein isolate-chia seed gum complex coacervates. \u003cem\u003eFood Hydrocolloids\u003c/em\u003e, \u003cem\u003e52\u003c/em\u003e, 554\u0026ndash;563. https://doi.org/10.1016/j.foodhyd.2015.07.033\u003c/li\u003e\n\u003cli\u003eUrl-1, https://web.expasy.org/peptide_cutter/\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bovine α-lactalbumin, BAMLET, Breast cancer, Prostate cancer, Enzymatic hydrolysis, Protein purification ","lastPublishedDoi":"10.21203/rs.3.rs-2082257/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2082257/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBAMLET (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eB\u003c/span\u003eovine \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eα\u003c/span\u003e-Lactalbumin \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eM\u003c/span\u003eade \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eLE\u003c/span\u003ethal to \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eT\u003c/span\u003eumors) is a tumoricidal molecular complex of partially unfolded bovine α-lactalbumin 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 this study, the α-lactalbumin was purified from the skimmed cow milk, and BAMLET was produced with oleic acid. 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 89.2% at 10 \u0026micro;g/mL and 48.0% at 2.14 \u0026micro;g/mL after 24 hours, respectively. After the hydrolyses, the most effective doses were altered, but the anti-cancer effect was improved to 21.9% for MCF7 (6.38 \u0026micro;g/mL) and 32.2% for DU145(6.38 \u0026micro;g/mL) under the same condition. Interestingly, the untreated BAMLET demonstrated cytotoxic effect on fibroblasts above the concentration of 2.1 \u0026micro;g/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 \u0026micro;g/mL hydrolised BAMLET. As conclusion, BAMLET produced from the hydrolysed form of the α-lactalbumin was found to be more effective against the cancer cells than its non-hydrolysed form. The hydrolysed BAMLET was found to be a promising natural anti-cancer product without any toxic effect on fibroblasts.\u003c/p\u003e","manuscriptTitle":"Enhancement of Cancericidal Activity of Bamlet Complexes by Enzymatic Treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-10-24 17:37:57","doi":"10.21203/rs.3.rs-2082257/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"82e971a5-3f82-4714-9705-987b1f0fc214","owner":[],"postedDate":"October 24th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-11-14T16:11:45+00:00","versionOfRecord":[],"versionCreatedAt":"2022-10-24 17:37:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2082257","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2082257","identity":"rs-2082257","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}