M. T. G. MOYO et al.: HEMOCOMPATIBILITY STUDIES OF LAYER-BY-LAYER POLYELECTROLYTE COMPLEXES ... 525–536 HEMOCOMPATIBILITY STUDIES OF LAYER-BY-LAYER POLYELECTROLYTE COMPLEXES FOR BIO-BASED POLYMERS [TUDIJ HEMOKOMPATIBILNOSTI VE^PLASTNIH POLIELEKTROLITNIH KOMPLEKSOV ZA BIOPOLIMERE Mthabisi Talent George Moyo 1,2 , Terin Adali 2* ,O ðuz Han Edebal 3 , Ece Bayir 5,6 , Aylin ªendemir 4 1 Department of Biomedical Engineering, Faculty of Engineering, Near East University, P.O. Box: 99138, TRNC, Mersin 10 Turkey 2 Department of Medical Biochemistry, Faculty of Medicine, Girne American University, P.O. Box: 99320 Karaman Girne, Turkish Republic of Northern Cyprus (TRNC), Mersin 10 Turkey. 3 Clinical Biochemistry Laboratory, Near East University Hospital, P.O. Box: 99138, Turkish Republic of Northern Cyprus (TRNC), Mersin 10 Turkey 4 Department of Bioengineering, Faculty of Engineering, Ege University, Bornova, 35100 Izmir, Turkey Prejem rokopisa – received: 2023-07-02; sprejem za objavo – accepted for publication: 2023-08-16 doi:10.17222/mit.2023.922 Thrombogenesis is an important issue that causes failure in blood-contacting biomedical devices. This study focuses on hemocompatibility studies of novel, blood-contacting, polyelectrolyte complexes (PECs) for biomedical application designs. PEC films were fabricated from bio-based polymers of silk fibroin (SF), chitosan (CH), and sodium alginate (AL) through the solvent-casting method as well as the layer-by-layer (LbL) technique. Characterization was carried out by Fourier-transform in- frared spectroscopy (FTIR), scanning electron microscopy (SEM), atomic force microscopy (AFM), and differential scanning calorimetry (DSC). FTIR spectra displayed all the layers’ characteristic peaks of SF, CH, and AL. AFM images indicated that the addition of AL as an outer layer increased the surface roughness. DSC analysis suggested that the best thermal stability was observed with the CH outer layer of PECs. SEM micrograph analysis indicated that the morphologies of the PECs were affected by the inclusion of the clopidogrel bisulfate (CLB). Hemocompatibility properties were investigated by a complete blood count (CBC), prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (APTT), platelet ad- hesion, erythrocyte morphology analysis, in vitro cholesterol, and albumin level tests. These hemocompatibility analyses dem- onstrated that the PEC surfaces provide favourable principles to design and develop non-thrombogenic PECs for blood-contact- ing biomedical applications. Keywords: polyelectrolyte complex, hemocompatibility, thrombogenesis Trombogeneza je pomemben pojav, ki povzro~a po{kodbe na biomehanskih napravah v stiku s ~love{ko krvjo. V {tudiji, ki je opisana v tem ~lanku so se avtorji osredoto~ili na raziskavo novih polielektrolitnih kompleksov v stiku s krvjo (PECs, angl.: blood-contacting polyelectrolyte complexes) uporabnih za biomedicinske aplikacije. PEC filme so izdelali iz biopolimerov na osnovi svilenih vlaken (SF, angl.: silk fibroin), hitozana (CH) in natrijevega alginata (AL) s pomo~jo tehnike litja raztopine plast za plastjo (LbL; angl.: Layer-by-Layer). Karakterizacijo so izvedli s Fourierjevo transformacijsko spektroskopijo (FTIR), vrsti~no elektronsko mikroskopijo (SEM), mikroskopijo na atomsko silo (AFM) in vrsti~no diferencialno kalorimetrijo (DSC). FTIR spekter je prikazal karakteristi~ne spektre vseh plasti SF, CH in AL. AFM slike so pokazale, da dodatek AL pove~a povr{insko hrapavost zunanje plasti. DSC analiza nakazuje, da ima najbolj{o termi~no stabilnost zunanja plast PEC na osnovi CH. Medtem ko je SEM mikrografska analiza pokazala, da so na morfologije polielektrolitnih kompleksov vplivali vklju~ki klopidogrel bisulfata (CLB). Hemokompatibilnostne lastnosti so dolo~ili s kompletnim {tevilom krvi (CBC, angl.: complete blood count), protrombinskim ~asom (PT), internacionalnim normaliziranim razmerjem (INR), aktiviranim parcialnim ~asom tromboplastina (APTT), adhezijo plo{~ic, analizo morfologije eritrocitov, in vitro holesterola in testom albuminskega nivoja. Te analize so pokazale, da povr{ine PEC zagotavljajo osnovne ziroma prednostne principe dizajna ter razvoja netrombogenskih polielektrolitnih kompleksov za biomedicinske aplikacije v stiku s krvjo. Klju~ne besede: polielektrolitni kompleks, hemokompatibilnost, trombogeneza 1 INTRODUCTION Hemocompatibility is a challenging problem in blood-contacting biomaterial applications. 1 These non-physiological materials can trigger adverse hemato- logical responses and reactions such as thrombogenicity, embolisms, and blood-related, medical device failure etc. 2 Polyelectrolyte complexes (PECs) have been widely used as films in implants and other therapeutic blood- contacting applications. 3 PECs are a promising approach to the urgent need for designing blood-contacting polymeric applications that have controlled thrombo- genic effects. 4 Thrombogenesis can be activated on any polymeric surface, so elucidating the correlation between material properties and their effect on thrombogenic mechanisms is vital for surface chemistry and biomaterial research. The pursuit of improving hemocompatibility on PEC materials is essential. However, it remains a challenge. 5 Current approaches for developing hemocompatible PEC surfaces are being investigated and they aim to im- prove anti-thrombogenesis and control protein adsorp- tion. 6 A study employed the LbL casting technique to Materiali in tehnologije / Materials and technology 57 (2023) 5, 525–536 525 UDK 591.111.2:543.456 ISSN 1580-2949 Original scientific article/Izvirni znanstveni ~lanek MTAEC9, 57(5)525(2023) *Corresponding author's e-mail: terin.adali@neu.edu.tr (Terin Adali) fabricate Chitosan-poly ( -glutamic acid) biomaterials for the reduction of protein adsorption and enhance the biocompatibility of the biomaterial. 7 Another study used the same LbL solvent-casting method to fabricate SA/CH films for use as transdermal antihistamine drug-delivery patches. 8 Following the success of this trend, a LbL spraying technique to fabricate biomedical coatings from Poly(L-lysine)–hyaluronan was found to be successful in fabricating hemocompatible bio- materials. 9 After multiple trials, the LbL solvent-casting method was used to generate hydrogel films for wound-healing applications using CH/Heparin sodium. 10 These complexes are formed when electrostatic inter- actions between polyanionic and polycationic biopoly- mers bond each layer compactly. 11 LbL assembly is a distinctive method that employs either dipping, spinning, solvent-casting, electromagnetic, or spraying techniques for the formation of custom PEC films with adjustable properties. 12 The advancement of blood-contacting mate- rials design through the LbL technique is a potential ap- proach to overcoming thrombogenicity and therefore im- proving its favorable effect on hemocompatibility. 13 Hemocompatibility and material-surface morphology have a strong and direct correlation based on the type of polymer used. 14 LbL casting is used to modify the surface chemistry of biomaterials by alternatively depositing and adsorbing polyanionic and polycationic biopolymers onto a sub- strate. 15 Oppositely charged materials used as co-poly- mer materials have low protein fouling, which has the potential to improve the biocompatibility. 16 Blood biocompatibility was more achievable in natu- ral-based polymers such as silk fibroin (SF), sodium alginate (AL), and chitosan (CH). 17–21 These bio-based polymer candidates have the potential for remarkable hemocompatibility when cast using the LbL technique. Tailoring these PECs by using the anti-thrombogenic drug may prove to be just as noteworthy. To the best of our knowledge, hemocompatibility studies on tri-layer PEC films prepared by bio-based polymers SF, CH, and AL for use as blood-contacting materials have not been investigated. The novelty of their potential non-thrombo- genic and nonhemolytic properties is the point of our in- terest. CLB is an anti-thrombogenic agent that decreases the risk of strokes and heart attacks in patients with clotting diseases or patients prone to (or that suffered recent) strokes or heart attacks. 22 Its metabolites inhibit platelet aggregation by binding to the receptors of adenosine diphosphate (ADP), a platelet agonist that causes platelet shape change and aggregation. 23,24 Chemically it is methyl (+) -(S)- − (2-chlorophenyl)-6,7-dihydrothieno [3,2-c] pyridine-5(4H)-acetate sulfate as seen in CLB has the potential of being used widely in the surface modifi- cation of biomaterial design to improve their anti-throm- bogenic properties. However, based on a literature sur- vey, limited studies have been carried out on it. Shitole and colleagues aimed to improve the hemo- compatibility of nanofibrous scaffolds, polyurethane/ polyethylene glycol scaffolds by incorporating CLB as an anti-thrombogenic treatment. The results of the hemocompatibility assessment indicated that CLB- treated scaffolds demonstrated excellent hemocom- patibility, exhibiting lower hemolysis, increased albumin, and plasma protein adsorption, while a reduction in fibrinogen adsorption was noted. Further, the platelet ad- hesion was highly suppressed and a significant increase in the coagulation period was observed for CLB incorpo- rated scaffolds. 25 Da Câmara et al. prepared LBL PEC and improved their anti-thrombogenicity by treating them with chondroitin sulfate and glycosaminoglycans heparin. Al- though this study was effective in proving hemocom- patibility, other blood factors like erythrocyte morphol- ogy and CBC were not carried out. 26 Xi et al. fabricated heparin-lysozyme polyelectrolyte multilayers to immobilize on the poly (propylene carbon- ate) surface by LbL assembly with the aim of hemo- compatibility improvement. Combined with the impres- sive results of platelet adhesion, erythrocyte adhesion, and hemolysis, it was concluded that poly(propylene car- bonate)-heparin-lysozyme could be a candidate scaffold material for blood-vessel-tissue engineering due to its high hemocompatibility. 27 Liu et al. developed a paclitaxel/chitosan LbL nano- fiber coating, fabricated with phosphorylcholine groups to improve its hemocompatibility. Their results showed that LbL coatings containing phosphorylcholine groups reduced the platelet adhesion by 6 % and reduced bovine albumin and fibrinogen adsorption by 30–40 %. These results demonstrate that the preparation of the anti- thrombogenic-modified coatings is a simple strategy to improve the hemocompatibility of the polymer complex coatings, has good potential for application in blood-con- tacting materials and devices. 28 Manabe and Nara developed LbL elastic multilayer film complexes and deposited them on the hydrophilic surfaces of blood-contacting medical devices to improve their hemocompatibility by suppressing platelet and pro- tein adhesion. The results demonstrate the possibility of coating biomedical multilayer films onto the surface of medical devices. 29 The objective of this research is to investigate and compare the hemocompatibility properties of PECs as- sembled using the LbL solvent-casting technique as po- tential materials to be used as non-thrombogenic, blood-contacting PECs. The characterization of PECs, and the morphological, physicochemical, and biological properties were analyzed. M. T. G. MOYO et al.: HEMOCOMPATIBILITY STUDIES OF LAYER-BY-LAYER POLYELECTROLYTE COMPLEXES ... 526 Materiali in tehnologije / Materials and technology 57 (2023) 5, 525–536 2 EXPERIMENTAL PART 2.1 Materials Bombyx Mori Silk cocoons were acquired from Lapta, a town in North Cyprus. Medium-molecu- lar-weight CH with an 85 % deacetylation degree and 200 mPa viscosity in 0.1-M acetic acid at 20 °C, (Fluka) as stated by the producer, was used as supplied. Acetic acid (CH3COOH, Aldrich, Germany), calcium chloride (CaCl 2 , Aldrich, Germany), sodium carbonate (Na 2 CO 3 , Aldrich, Germany), methylene bisacrylamide (C 7 N 10 N 2 O 2 , Aldrich, Germany), and ethanol (C 2 H 5 OH, Aldrich, Germany) were used without further purifica- tion. Dialysis membrane (cut off M. W. 12,400) with full average 4 diameters 16 mm and flat width 25 mm capac- ity of ~60 mL/ft was purchased from Aldrich AL (from brown algae Macrocystis pyrifera, medium viscosity) was purchased from Aldrich, Germany. Human venous blood was obtained from the Near East University Hos- pital. Clopidogrel Bisulfate (C 16 H 18 C l NO 6 S 2 , Aldrich, Germany). All other reagents were of analytical grade and purchased from Aldrich. 2.2 Silk Fibroin Purification The degumming process was carried out by adding 2g of SF cocoons in a 0.1-M sodium carbonate solution at 75 °C on a magnetic hot-plate stirrer at a speed of 1.5 min –1 for 6 h. The degummed silk was washed and rinsed thoroughly with deionized water twice after a 3-hour interval. The degummed silk fibers were dried overnight at room temperature. 2.3 Dissolution and Dialysis of Silk Fibroin On a continuously stirring magnetic hot plate, the degummed SF fibers were dissolved in a strong electro- lyte solution of nC 2 H 5 OH:nH 2 O:nCaCl 2 (2: 8: 1) molar ratio at 75 °C. After3do fdialysis in deionized water, the aqueous SF solution was obtained, followed by filtra- tion. 2.4 Dissolution of Chitosan The aqueous CH solution was prepared ata2% (w/v) concentration and dissolved in 0.1-M acetic acid solution aided by a magnetic stirrer. 2.5 Dissolution of Sodium Alginate An aqueous AL solution was prepared with a concen- tration of 2 % (w/v) by adding AL to deionized water and spun until total dissolution. 2.6 Drug Loading CLB was dissolved 51mg/L in deionized water. For every 1 mL of aqueous biopolymer used in the PEC film layering, 3 mg of the dissolved CLB was infused. 2.7 Assembly of LbL PEC Films The solvent-casting method was used to prepare eigh- teen different compositions of PEC films using SF, CH, and AL in glass Petri dishes (5 cm diameter). These op- positely charged biopolymers were cast LbL to assemble a net-charged polyelectrolyte complex. The desired ultrathin LbL films were obtained by alternating the lay- ering of the oppositely charged polymers. Next, 2.5 mL of aqueous biopolymer was drawn into a sterile syringe and discharged onto a sterile glass petri dish then spread evenly across the surface by rotating in dipped circular motions. Each layer was left to dry for 24 h at room tem- perature. Extraction of the PECs was followed by wash- ing in methylene bisacrylamide. 2.8 Swelling Kinetics All the PECs were sampled 2 cm × 2 cm and were in- vestigated by using acetic buffer solution (ABS, pH 4.2), which mimics the pH of healthy wound exudate and phosphate buffer solution (PBS, pH 7.4) that emulates the pH of human venous, arterial and heart capillary blood. The swelling ratios calculated by equation 1: Swelling Ratio (%) = weight weight weight s dry dry − ⎛ ⎝ ⎜ ⎜ ⎞ ⎠ ⎟ ⎟ × 100 % (1) 2.9 Differential Scanning Calorimetry (DSC) Thermoanalysis was performed by utilizing TA In- struments Q20 DSC system to additionally affirm drug-polymer interaction and the mechanical state of PEC. The heating rate was set to 10 °C/min at 25–450 °C, nitrogen flow 50.0 mL/min, 0.2–1 mg samples on an aluminum Hermetic pan. 2.10 Scanning Electron Microscopy (SEM) The microstructural characterization was made with a Thermo Scientific Apreo S using the secondary-electron in lens method, which is used for high-magnification zoom. The morphologies of the dried samples were ana- lyzed with SEM after being sputter-coated with 7 nm of Au/Pd. 2.11 Atomic Force Microscopy (AFM) AFM utilizing the BRUKER dimension edge instru- ment using the tapping mode was used to examine the surface geology and topography. 2.12 Fourier-Transform Infrared Spectroscopy (FTIR) Analysis was carried out using a PerkinElmer FTIR spectrometer with an attenuated total reflection (ATR) function to take the measurements within the range 600–4000 cm –1 . M. T. G. MOYO et al.: HEMOCOMPATIBILITY STUDIES OF LAYER-BY-LAYER POLYELECTROLYTE COMPLEXES ... Materiali in tehnologije / Materials and technology 57 (2023) 5, 525–536 527 2.13 In-vitro Hemocompatibility Assay At the Near East University hospital, fresh human whole blood samples were obtained from a healthy male donor by venipuncture using the vacuum blood-collec- tion system into 2 mL anticoagulated collecting tubes. Two different evacuated tubes were used, one contained 3.6 mg K 2 EDTA as an anti-coagulant, and the other con- tained 3.2 % sodium citrate. In vitro anticoagulation tests, total cholesterol and albumin analysis, in vitro platelet adhesion, and erythrocyte morphology analysis were effective tests to determine the hemocompatibility of PECs according to the ISO 10993. For the mentioned hemocompatibility analyses, the anticoagulated tubes were centrifuged at 1500 min –1 , 15 min for an investiga- tion of each blood-compatibility assay. A CBC analysis was obtained by using uncentrifuged blood and all the anticoagulated blood was used within1ho fcollection. For the analysis of the platelet adhesion and erythrocyte morphology, PEC-A, PEC-C, PEC-D, PEC-E, PEC-1, PEC-3, PEC-4, PEC-5 were used as test samples, while PECs PEC-1, PEC-5, PEC-A, and PEC-E were used as test samples to evaluate the in-vitro anticoagulation tests, total cholesterol, and albumin analysis and CBC. 2.13.1 In-vitro Anticoagulation Analysis For the evaluation of baseline coagulation and mate- rial-blood contacted parameters utilizing the STA Com- pact and fresh venous blood, the blood samples were anticoagulated in sample collection tubes containing 3.2 % sodium citrate at blood to an anticoagulant ratio of 9:1 (v/v). A plasma sample was used to measure the acti- vated partial thrombin time (APTT) and the prothrombin time (PT) of the material contacting blood as blood-clot- ting tests using cephascreen and stago STA-Neoplastine CI Plus commercial reagents. Platelet-rich plasma (PRP) was subsequently transferred to sterile test tubes contain- ing the biomaterials under study and incubated at 37 °C for 60 min. The international normalized ratio (INR) was investigated using equation 2; the international sensitiv- ity index (ISI) as a thromboplastin referencing model to liken all thromboplastins performed. INR depends on the proportion of the patient’s prothrombin time and the typ- ical mean prothrombin time. INR = patient PT mean normal PT ISI ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ (2) where ISI is the international sensitivity index of the thromboplastin tested 2.13.2 Total Cholesterol & Albumin Platelet-rich plasma was placed into a separate Eppendorf tube containing the test biomaterial then incu- bated at 37 °C for 15 min and further shaken on a KS 260 basic flat orbital shaker at 300 min –1 for 20 min. Af- ter shaking the biomaterials were recovered from the se- rum and the suspension-free serum samples were tested for total cholesterol and albumin using a biochemistry autoanalyzer (Abbott Architect, Abbott Laboratories, Di- agnostic Division, Abbott Park, IL). 2.13.3 Complete Blood Count (CBC) For 30 min, specimens submerged in whole blood were shaken at 300 min –1 at room temperature. Complete blood count (erythrocytes, leukocytes, and platelets) of all PEC-contacting and non-PEC-contacting blood sam- ples was determined using a hematology analyzer (Cell Dyn Ruby hematology analyzer Abbott Laboratories, Di- agnostic Division, Abbott Park, IL). 2.13.4 In-vitro Platelet Adhesion PEC samples were incubated in fresh human PRP for 15 min within static conditions at 37 °C and further shaken on a KS 260 basic flat orbital shaker at 300 min –1 for 20 min at room temperature. After shaking, the bio- materials were recovered from the serum and the suspen- sion-free serum samples were repeatedly washed in ul- tra-pure water, after which the direct-drying technique was used with May Grünwald and Giemsa dye. The peripheric screening technique was employed to deter- mine the degree of PEC surface-platelet adherence. Light microscopy was utilized for the investigation of the platelet microparticle formation, amount, adhesion, and morphology. All samples were investigated at 400× mag- nification (450 μ). 2.13.5 Erythrocyte Morphology Analysis Erythrocyte membrane integrity analysis was carried out to investigate any abnormalities of their shape after RBC contact with the PECs. Biomaterial contacting RBC test tubes were shaken at 300 min –1 for 20 min then incubated at 37 °C for 20 min. Peripheral blood smears were stained using the standard Wright-Giemsa Stain Method where the specimen was fixated onto the slides with alcohol. The smears were examined at 400 × mag- nification (450 μ) in 10 different areas. 3 RESULTS 3.1 Derived LBL PEC Film Compositions Compositions of the assembled PEC films are de- tailed in Table 1. Table 1: Compositions of derived PEC films Sample Composition Sample Composition Drug loaded Drug-free PEC-A CH/SF/CH + clb PEC-1 CH/ SF/CH PEC-B SF/CH + clb PEC-2 SF/CH PEC-C SF/CH/SF +clb PEC-3 SF/CH/SF PEC-D AL/CH/SF + clb PEC-4 AL//CH/SF PEC-E AL/SF/AL+ clb PEC-5 AL/SF/AL PEC-F SF/AL + clb PEC-6 SF/AL PEC-G SF + clb PEC-7 SF PEC-H CH + clb PEC-8 CH PEC-I AL + clb PEC-9 AL M. T. G. MOYO et al.: HEMOCOMPATIBILITY STUDIES OF LAYER-BY-LAYER POLYELECTROLYTE COMPLEXES ... 528 Materiali in tehnologije / Materials and technology 57 (2023) 5, 525–536 3.2 Swelling Kinetics Table 2 compares the swelling ratios of the films based on two solvents with different polarities, where all the swelling behaviour is plotted for an average of three trials. Table 2: Optimal swelling ratio (SR%) of PECs in ABS and PBS ABS: pH 4.2 PBS: pH 7.4 Sample Time (min) S.R% Time S.R% PEC-A 35 190.78 15 197.33 PEC-B 10 89.62 10 181.95 PEC-C 10 210.37 20 213.9 PEC-D 10 455.78 35 564.87 PEC-E 5 602.3 55 1253.65 PEC-F 10 109.52 80 188.66 PEC-G 15 52.66 10 131.21 PEC-H 10 51.49 55 108 PEC-I 20 60.53 35 156.66 PEC-1 55 118.26 55 131.21 PEC-2 35 69.05 35 106.66 PEC-3 10 148.53 10 151.44 PEC-4 20 197.33 20 198.66 PEC-5 10 265.7 10 257.33 PEC-6 10 72.85 10 108 PEC-7 10 55.34 10 76.33 PEC-8 25 52.36 25 74.66 PEC-9 15 68.45 15 84.97 3.3 Differential Scanning Calorimetry (DSC) Figure 1 shows analytical differences of the DSC thermograms of all PECs. 3.4 Atomic force microscopy (AFM) Topologies of 8 PEC films were used in the AFM analysis. Figure 2 displays the differences in the topo- graphic features of samples PEC-1, PEC-3, PEC-4, PEC-5, PEC-A, PEC-C, PEC-D, PEC-E. In this study, PEC-3 recorded minimum roughness as Ra = 9.1 nm and maximum roughness in PEC-E as Ra = 113 nm. 3.5 Scanning Electron Microscopy (SEM) The SEM micrographs of all the PECs presented in Figure 3 show their differences in topographic features. M. T. G. MOYO et al.: HEMOCOMPATIBILITY STUDIES OF LAYER-BY-LAYER POLYELECTROLYTE COMPLEXES ... Materiali in tehnologije / Materials and technology 57 (2023) 5, 525–536 529 Figure 2: 3D AFM thermograms of PEC-1, PEC-3, PEC-4, PEC-5, PEC-5, PEC-A, PEC-C, PEC-D, PEC-E Figure 1: DSC analysis of (A) drug-free films and (B) drug-loaded films Drug-free PEC without AL appears smooth, while the CLB addition changed the morphology according to PEC A, PEC-B and PEC-E. 3.6 Fourier-Transform Infrared Spectroscopy (FTIR) The FTIR spectra are shown in Figure 4a for drug-loaded and Figure 4b for drug-free biomacromole- cules. 3.7 In-vitro Anticoagulation, Cholesterol and Albumin Table 3 displays the results of the PT, APTT, and INR values for the control (blood without sample) and test specimen after incubation. Additionally, it displays the results of the blood cholesterol and albumin analyses of PEC-1, PEC-5, PEC-A, and PEC-E. Table 3a shows that PT, PTT, and INR values for the blood in contact with PECs are shorter than the control, indicating that PECs might activate the coagulation process. Table 3b M. T. G. MOYO et al.: HEMOCOMPATIBILITY STUDIES OF LAYER-BY-LAYER POLYELECTROLYTE COMPLEXES ... 530 Materiali in tehnologije / Materials and technology 57 (2023) 5, 525–536 Figure 4: a) FTIR results of drug-free films PEC 1, b) drug-loaded films PECA–I Figure 3: SEM images of drug-free and drug-loaded PECs at 10 μm shows that cholesterol and albumin efflux saturation in all PECs were within range. Table 3: a)Anticoagulation analysis of PEC-1, PEC-5, PEC-A, and PEC-E, b) Blood cholesterol and albumin analysis of PEC-1, PEC-5, PEC-A, and PEC-E a) Sample INR PT (%) PT (Sec) APTT (sec) Control 1.34 65 17.6 31.8 PEC-1 1.09 88 14.4 32.7 PEC-5 1.17 79 15.4 86.1 PEC-A 1.11 85 14.7 57.7 PEC-E 1.07 90 14.2 101.4 b) Sample Cholesterol range (mg/dL) Cholesterol (mg/dL) Albumin range (g/dL) Albumin (g/dL) Control 200 189 3.4–5.4 5.1 PEC-1 200 182 3.4–5.4 4.8 PEC-5 200 176 3.4–5.4 4.5 PEC-A 200 172 3.4–5.4 4.1 PEC-E 200 185 3.4–5.4 4.9 PT: Prothrombin Time, INR: International Normalized Ratio, APTT: Activated Partial Thromboplastin Time 3.8 Complete Blood Count (CBC) Table 4: Complete blood-count analysis of PEC-1, PEC-5, PEC-A, and PEC-E Samples Parameters Range Con- trol PEC-1PEC-5 PEC- A PEC-E WBC (10e3/uL) 3.70–10.1 4.15 4.39 4.30 4.02 4.20 NEU (%) 1.63–6.96 2.25 2.33 2.17 2.13 2.31 LYM (%) 1.09–2.99 1.41 1.54 1.55 1.52 1.47 MONO (%) 0.240–0.790 .385 .414 .365 .290 .395 EOS (%) 0.030–0.440 .042 .048 .055 .062 .065 BASO (%) 0.00–0.080 .061 .058 .055 .021 .063 RBC (10e6/uL) 4.06–4.69 5.92 5.87 5.99 6.21 6.00 HGB (g/dL) 11.0–16.0 17.1 17.2 17.8 18.3 18.0 HCT (%) 37.7–53.7 51.5 51.2 52.2 54.1 52.0 MCV (fL) 81.1–96.0 86.9 87.2 87.0 87.1 86.6 MCH (pg) 27.0–31.2 28.9 29.3 29.7 29.4 30.0 MCHC (g/dL) 31.8–35.4 33.3 33.6 34.1 33.8 34.6 RDW (%) 11.5–14.5 12.0 11.9 11.9 12 11.9 PLT (10e3/uL) 155–366 342 335 337 335 319 MPV (fL) 6.90–10.6 6.41 6.63 6.77 6.78 6.63 The hematology analyzer uses a spectrophotometric method for hemoglobin measurement. The CBC results are shown in Table 4. 3.9 In-vitro Platelet Adhesion Analyses with Peripheric Smear Test The light micrograph demonstrated that no thrombi adhere to the surface of the biomaterials, as shown in M. T. G. MOYO et al.: HEMOCOMPATIBILITY STUDIES OF LAYER-BY-LAYER POLYELECTROLYTE COMPLEXES ... Materiali in tehnologije / Materials and technology 57 (2023) 5, 525–536 531 Figure 6: Wright-Giemsa Stains of PEC-1, PEC-3, PEC-4, PEC-5, PEC-A, PEC-C, PEC-D, PEC-E at 450 × magnification for erythro- cyte morphology analysis Figure 5: Peripheral blood smears for platelet-adhesion analysis of PEC-1, PEC-3, PEC-4, PEC-5, PEC-A, PEC-C, PEC-D, PEC-E at 450 μm Figure 5. The results indicated that the drug-loaded and drug-free PECs could be considered ideal candidates for biomedical applications. No signs of platelet aggrega- tion; however, the biomaterials were very densely stained. 3.10 Erythrocyte Morphology Analysis Direct hemolysis testing was performed as shown in Figure 6. No morphological changes in erythrocytes were noted except in PEC-E, which caused a slight dete- rioration in the erythrocyte morphology. The erythro- cytes physically contacting the surface of biomaterials PEC-A, PEC-1, PEC-3, PEC-4, and PEC-5 exhibited completely normal biconcave morphology. 4 DISCUSSION We would like to acknowledge that there is missing data in our study. During the data-collection process, we encountered technical and financial difficulties with the measurement equipment, which resulted in incomplete data for some variables. Despite our efforts to address these issues promptly, we were unable to recover the missing data. However, it is important to note that the variables consistent with all the experimental analysis are PEC-1, PEC-5, PEC-A and PEC-E. Overall, the re- sults support the robustness of our main conclusions. Eighteen PEC samples with the intended use as blood-contacting biomaterials were fabricated using the LbL casting technique. Drug-loaded and drug-free PECs were evaluated in various characterization and hemocompatibility assays to investigate the effect of electrostatic interaction and surface modification using CLB as an anti-thrombogenic agent. The water-absorption capacity of a biomaterial is an important factor when it is applied as a transdermal patch for wound healing or wound dressing. A biomaterial with excellent water-absorbing properties ab- sorbs exudates and keeps the wound dry, thereby con- serving a proper moist environment that stimulates opti- mal wound healing. 30 In this experiment, PECs absorbed the solvent solution until the maximum swelling was achieved; thereupon the degree of swelling dropped, af- ter which they shrank and degraded. Out of the three biopolymers, the literature suggested that AL has the highest swelling capacity attributed to its randomly dis- tributed hydrophilic units on its polymeric chain. 31 AL has a higher swelling capacity than SF because SF has hydrophobic chains in its backbone. The heavy (H) chain with hydrophobic domains attributes its lower swelling ratio, but remains higher than CH. 32 Literature has shown that of the three biopolymers, CH is the least hydrophilic due to its net positive charge assimilating hydrophobic chains. 33 The highest swelling ratio of 1253.65 % was seen in PEC-E at pH 7.2. This high swelling ratio is at- tributed to the net negative charge of the hydrophilic biopolymer’s SF and AL. AL as an outer layer of the tri-layer PEC has more hydrophilic chains than SF, thus retaining a greater amount of solvent in its polymer chains. The swelling ratio decreased gradually with the number of hydrophilic groups present in the complex. The lowest swelling ratio of 51.49 % was in PEC-H in pH 4.2, which is comprised of CH only. This result is parallel to a study by Liwe et al., where PEC films con- taining AL exhibited excellent swelling characteristics, a lot more CH. 34 The amount of absorbed solvent is closely correlated with the number of hydroxyl groups present in the PEC film structure. Hydroxyl groups are responsible for the formation of hydrogen bonds with water molecules. Generally, the solvent-uptake capacity increases by add- ing a hydrophilic component. In this study, a highly hy- drophilic component, i.e., AL, was added to increase the swelling capacity of the PEC films. The improvement in the PEC film swelling ratio with the increase in the AL content is mainly attributed to the ionic character of AL. Characterization was carried out to investigate the vital- ity of the functional groups. The DSC results suggest that progressively accessible protonated amine groups in CH might bring about carboxylic groups in SF. PEC-B indicated the weakest thermal stability, registered by the least difference in exothermic peaks 130.15 °C and 279.41°C, respectively, among all the peaks. Bond disruption, chain scission, or drug degradation of macromolecules may be a cause of the decrease in thermal stability. 35 An indication of oxi- dative degradation and hydrogen bonding disruption due to a loss of free-amine groups. Strong molecular interac- tions and chemical bonds might be present between SF and CH. There were other nucleation sites such as car- bonyl (–C–O) and amine (–N–H–) groups on SF mole- cules besides the carboxyl (–COOH) and hydroxyl (–OH) groups previously reported that were further in- vestigated using AFM. Topologies of 8 PEC films were used in the AFM analysis. The addition of AL to a complex increases its roughness. The characteristic roughness after the addi- tion of CLB is in PEC-C with a maximum roughness of Ra = 53.6 nm. Thermograms of PEC-A and PEC-E show that the addition of CLB does not increase the surface roughness, which is a favourable trait in this because studies have shown that elevated surface roughness trig- gers blood protein and platelet aggregation. Another study reported that ionic cross-linking utilizing sulfuric acid altered the heterogenicity of CH as an outer layer. 36 Differences in the topologies were additionally investi- gated using SEM. Scanning electron microscopy micro- graphs showed that the increasing number of layers is proportional to PEC thickness, they also showed that the addition of CLB changed the morphology of the biomaterials. In the FTIR spectrum of pure CH and CH-containing PECs, three specific absorption peaks at about 1034 cm –1 , 1602 cm –1 , and 3319 appeared, respectively, corre- M. T. G. MOYO et al.: HEMOCOMPATIBILITY STUDIES OF LAYER-BY-LAYER POLYELECTROLYTE COMPLEXES ... 532 Materiali in tehnologije / Materials and technology 57 (2023) 5, 525–536 sponding to the O-C, C=C stretch, and the sharp C-H stretch. AL displayed similar absorption peaks. Also, three characteristic absorption peaks at 1545 cm –1 , 1642 cm –1 , and 3299 cm –1 were observed in the FTIR spectrum of SF- and SF-containing PECs, which were attributed to double C-C stretching, and sharp, C-H stretching, respectively. A study by Mohammed and Mo- hammed discovered that the FTIR spectrum of pure CLB showed a strong absorbance band due to C=O stretching vibrations at 1752 cm –1 and due to O–H stretching of the hydrogen sulfate moiety around 3012 cm –1 . The band due to aromatic C– H stretching vibrations represented at 3121 cm –1 . The FTIR spectrum included also a broad absorbance band at 2505 cm –1 , which can be attributed to the stretch- ing vibrations of the bonded N–H resulting from salt for- mation between the quaternary nitrogen of CLB and –OH of hydrogen sulfate. The FTIR spectra in Figure 4a can be attributed to the results of pure CLB spectra due to drug loading of the anti-thrombogenic drug. 37 The compatibility of the various PEC films with hu- man blood was evaluated by investigating their ability to alter the coagulation times. In this study, for the first time, to the best of our knowledge, we analyzed the ef- fect of electrostatically assembling PEC films prepared by LbL solvent casting for determining the plasma coag- ulation. According to previous experiments investigating similar parameters, the reference range of PT, APTT and INR are for PT is 11.0–12.5 s; 65–95 % activity (al- though the normal range depends on reagents used for PT), APTT is 30–40 s and INR is 0.8–1.1. 38–43 A study conducted by Qing et al. indicated that the positive charges of CH inhibited activation of the contact system and retarded thrombin generation and blood co- agulation on the fabricated CH films. 44 Another study conducted by Zhang et al. contradicted these results by reporting that blood coagulation is dependent on the mo- lecular weight and deacetylation degree of CH. The hemostatic effects of CH investigated showed that when the molecular weights were high (105–106) and approxi- mate, the coagulation effect of CH improved with a de- crease of the deacetylation degree and achieved a promi- nent level in a moderate degree of deacetylation (68.36 %). With the same degree of deacetylation, the higher the molecular weight of CH, the better the procoagulant effect. 45 The results obtained in these stud- ies led us to select PEC-1 and PEC-A that are electro- statically charged with CH as its outer layers to test. An additional study by Lihong et al. indicated that introduc- ing sulfate groups could not increase PT, it had little ef- fect on coagulation factors in the extrinsic pathway. 46 For the sake of comparison, we investigated this fact using PEC-5 and PEC-E as samples with AL as an outer layer. Our present findings improve understanding of the events leading to blood coagulation on films with CH and AL as outer layers, which will be useful for the fu- ture development of novel CH and AL-based hemostatic materials. Table 4 displays the results of the PT, APTT, and INR values for the control (blood without sample) and test specimen after incubation. The PT, PTT, and INR values for the blood in contact with PECs are shorter than the control, indicating that PECs might acti- vate the coagulation process. An assessment of the change in cholesterol and albu- min levels after 60 minutes of contact with samples PEC-1, PEC-A, PEC-5, and PEC-E was investigated to the best our knowledge these parameters have not been investigated before on PECs of this assembly. Total cho- lesterol levels less than 200 milligrams per deciliter (mg/dL) are considered desirable for adults. 47–50 While the normal range for albumin is about 3.4 g/dL to 5.4 g/dL. 51–53 A thorough literature survey supports that this analy- sis has not been carried out on PECs of this nature be- fore. The number of erythrocytes, leukocytes, and plate- lets was measured before and after the incubation of blood with the biomaterial using a hematology analyzer, which uses flow cytometry. The hematology analyzer utilizes uses a spectrophotometric method for hemoglo- bin measurement. An increase in the platelet release or elevated platelet aggregation on the biomaterial surface is a sign of mate- rial thrombogenicity. 54 Thrombogenicity refers to the tendency of a material in contact with the blood to pro- duce a thrombus or a clot. 55 A decrease in platelet count over time is indicative of a thrombogenic material and anti-thrombogenic materials reduce the activation of platelets. 56 Material surfaces that remain inert to blood contact are considered non-thrombogenic and are pre- ferred for use in biomedical applications. 57 The platelet count of whole blood-contacting all tested PECs re- mained within the normal range and close to the control value of 342 (10e3/uL). Whole blood-contacting PEC-1 and PEC-A separately both recorded a platelet reading of 335 (10e3/uL). Within the realms of this study, these re- sults suggest that CH as an outer layer has no significant impact on the level of platelets, nor does the addition of CLB. Of all the tested PECs, whole blood contacting PEC-5 and PEC-E recorded the highest and lowest platelet readings of 337 (10e3/uL) and 319 (10e3/uL) re- spectively. These findings may indicate that the net nega- tively charged SF and AL tri-layer PECs with AL as an outer layer have significant effects on thrombogenicity. PEC-5 is drug free and has the highest platelet readings, compared to PEC-E with the exact same composition; however, PEC-E is embedded with the antithrombogenic drug, so in this work CLB lowers the levels of platelets in net negatively charged PECs with AL as an outer layer. Furthermore, besides thrombogenicity, blood levels of leukocytes were analyzed to evaluate the leukocyte depletion and/or augmentation responses induced by biomaterial contact. The white blood cell count of the whole blood-contacting all the tested PECs remained M. T. G. MOYO et al.: HEMOCOMPATIBILITY STUDIES OF LAYER-BY-LAYER POLYELECTROLYTE COMPLEXES ... Materiali in tehnologije / Materials and technology 57 (2023) 5, 525–536 533 within normal range and most of the results remained close to the control reading of 4.15 (10e3/uL). Whole blood-contacting PEC-1, PEC-5, PEC-A and PEC-E were 4.39 (10e3/uL), 4.20 (10e3/uL), 4.02 (10e3/uL) and 4.30 (10e3/uL), respectively. PEC-1 and PEC-A have the same biopolymer composition; however, the PEC-A is embedded with the anti-thrombogenic drug and PEC-1 drug-free. Of all the tested PECs, the whole blood-con- tacting PEC-1 and PEC-A recorded the highest and low- est white blood-cell readings, respectively. Although there are some slight differences between the whole-blood specimens incubated with biomaterials, these are not significant. It can be said that neither of these biomaterials has any marked impact on the leuko- cyte levels of the blood. Polycythemia refers to an abnormal increase in the number of red blood cells. Biomaterials that cause polycythemia increase the risk of other health issues, such as blood clots, stroke, heart disease, etc. The RBC count of all the biomaterial contacting whole blood sam- ples exceeded the normal range. This may be attributed to the donor’s high RBC count seeing as the control was 5.92 (10e3/uL) which is above the range. Whole blood- contacting PECs- 1, 5, A, and E were 5.87 (10e3/uL), 5.99 (10e3/uL), 6.21 (10e3/uL) and 6.00 (10e3/uL), re- spectively. These high readings may be due to the inter- actions between blood plasma and biomaterial, leading to water being held by the biomaterial and an increased concentration of RBCs. Understanding the ability of thrombus formation on blood-contacting devices is thought to be crucial because early post-operative platelet adherence might be respon- sible for the formation of thrombi, which has an adverse postoperative outcome. 58–60 Thus, platelet adhesion is re- ported to be an early indicator of the thrombogenicity of blood-contacting implants. 61,62 The exposure of bio- material to the blood can result in an undesired activation of platelets and so lead to thrombotic complications. Thus, the analysis of platelet activation is an important part of the hemocompatibility tests. The contact of blood with foreign surfaces immediately leads to the adsorp- tion of plasma proteins. 63,64 The in vitro peripheric smear test was applied to PRP-immersed biomaterials to analyze their platelet-ad- hesion properties for evaluating the primary hemostasis of the prepared biomaterials. The PEC-A and PEC-E had dense and diffused staining, which suggests that the biomaterials have a lot of open molecules for chemical bonding with other molecules. Taken together, the inter- actions of PEC-E with plasma, they do not induce any significant alteration in the cell-membrane structure and function as well as in cell morphology after both the short- and long-term incubation. Biomaterial interaction with blood may lead to hemolysis, which is accompanied by the release of he- moglobin. 65 Thus, an increased concentration of free he- moglobin in the plasma is a direct indicator of erythro- cytes destruction. The damage of erythrocytes can lead to reduced oxygen transport to tissues and organs in vivo and increased levels of free hemoglobin can induce tox- icity or alter kidney function. 66,67 Thus, hemolysis can be analyzed after direct or indirect blood contact. In direct analysis, blood is incubated with the biomaterial and in- vestigated. 68–70 The Erythrocytes physically contacting the surface of biomaterials PEC-A, PEC-1 and PEC-5 exhibited com- pletely normal biconcave morphology. Biomaterial PEC-E caused abnormal swelling and deformation of erythrocyte membranes furthermore lots of small strayed particles were observed on the slide. 5 CONCLUSIONS These layer-by-layer polyelectrolyte complexes are good candidates to be used as blood-compatible biomaterials for biomedical applications. They show minimal to no interaction with blood cells, or plasma proteins, in terms of affecting their blood levels in vitro. Further in-vitro studies can be carried out with longer in- cubation periods, or on blood, specimens taken from multiple volunteers. 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