X. GU et al.: PREPARATION AND CHARACTERIZATION OF A CipHCl-PLA/PBC/CS COMPOSITE FIBER MEMBRANE ... 341–349 PREPARATION AND CHARACTERIZATION OF A CipHCl-PLA/PBC/CS COMPOSITE FIBER MEMBRANE FOR ANTIBACTERIAL WOUND DRESSING PRIPRAVA IN KARAKTERIZACIJA MEMBRANE IZ KOMPOZITNIH VLAKEN NA OSNOVI CipHCl-PLA/PBC/CS ZA ANTIBAKTERIJSKE PREVEZE PO PO[KODBAH Xiaohua Gu 1,2* , Dasheng Zhang 1 , Siwen Liu 3 , Yan Liu 4 1 College of Innovative Material and Energy, Qiqihar University, Heilongjiang Qiqihar 161006, China 2 College of Innovative Material and Energy, Donghua University, Shanghai 201620, China 3 College of Innovative Material and Energy, Hubei University, Hubei 430062, China 4 School of Energy and Building Environment of Guilin University of Aerospace Technology, Guilin, Guangxi 541004, China Prejem rokopisa – received: 2022-03-08; sprejem za objavo – accepted for publication: 2022-05-09 doi:10.17222/mit.2022.444 In this paper, a polylactic acid (PLA)/polybutylene carbonate (PBC)/chitosan (CS) composite membrane with different mass fractions of ciprofloxacin hydrochloride (CipHCl) drug was prepared through the electrostatic spinning technology. The mor- phology and chemical structure of the resultant CipHCl-PLA/PBC/CS fiber membranes were characterized with scanning elec- tron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). A drug-release test, bacteriostatic test and cytotoxicity test were performed to determine the drug-release properties, bacteriostatic properties and cytotoxicity, respectively. Results show that the CipHCl-PLA/PBC/CS composite fiber membranes have good biocompatibility. When the CipHCl content is 20 w/%, the average fiber diameter is 374 nm. After 192 h, the drug-release rate of the CipHCl-PLA/PBC/CS composite fiber membrane containing 20 w/% of CipHCl reaches 88.2 %. The antibacterial activity of the CipHCl-PLA/PBC/CS composite fiber membrane is much higher than that of the PLA/PBC composite fiber membrane. Besides, the antibacterial activity of the CipHCl-PLA/PBC/CS composite fiber membrane against E. coli is slightly higher than that against S. aureus. Keywords: polylactic acid, polybutylenes carbonate, chitosan, CipHCl, antibacterial activity V ~lanku avtorji opisujejo pripravo kompozitne membrane na osnovi polilakti~ne kisline (PLA), polibutilen karbonata (PBC) in hitozana (CS) z razli~nimi masnimi dele`i zdravila na osnovi kiprofloksiacin-hidroklorida (CipHCl). Njeno pripravo so izvedli s tako imenovano tehnologijo elektrostati~nega nanosa. Morfologijo in kemijsko strukturo iz kompozitnih vlaken izdelane CipHCl-PLA/PBC/CS membrane so okarakterizirali s pomo~jo vrsti~nega elektronskega mikroskopa (SEM), Fourierjeve transformacijske infrarde~e spektroskopije (FTIR), in rentgenske difrakcije (XRD). Izvedli so preizkuse spro{~anja zdravila, bakteriostati~ne teste in teste citotoksi~nosti, da bi dolo~ili lastnosti spro{~anja u~inkovine in bakteriostati~nosti oziroma citotoksi~nosti. Rezultati preiskav in preizkusov so pokazali, da ima kompozitna membrana CipHCl-PLA/PBC/CS dobro biokompatibilnost. Pri vsebnosti CipHCl 20 w/% je povpre~ni premer vlaken 374 nm. Po 192 urah je bila hitrost spro{~anja zdravila kompozitne membrane CipHCl-PLA/PBC/CS pri vsebnosti 20 w/% CipHCl enaka 88,2 %. Antibakterijska aktivnost izdelane vlaknaste membrane CipHCl-PLA/PBC/CS je bila precej vi{ja od kompozitne vlaknaste membrane PLA/PBC. Poleg tega je bila antibakterijska aktivnost izdelane kompozitne vlaknaste membrane CipHCl-PLA/PBC/CS rahlo vi{ja v prisotnosti bakterije E. coli kot pa pri bakteriji S. aureus. Klju~ne besede: polilaktatna kislina, polibutilenski karbonati, hitozan, CipHCl, antibakterijska aktivnost 1 INTRODUCTION Electrospinning is a simple and effective manufactur- ing method for producing fiber structures made from a variety of biopolymers, with diameters ranging from nanometers to micrometers. 1,2 Due to their unique prop- erties such as tunable diameter and pore size, high poros- ity, high surface-to-volume ratio, morphological similar- ity to the extracellular matrix and surface functionality, electrospun fibers have been studied in diverse fields in- cluding drug delivery, filtration, wound dressing, tissue engineering and cell culture. 3–5 In particular, the drug loading technology has attracted more and more atten- tion from drug scientists. For example, Kataria et al. 6 ob- served polyvinyl alcohol (PVA) and sodium alginate electrospun nanofibers loaded with antibiotic cipro- floxacin as transdermal patches in a wound-healing ap- plication. Results showed that the ciprofloxacin-loaded nanofibers reduced the wound healing time compared to the drug-free nanofibers. Chitosan (CS) is a deacetylated derivative of chitin, which can be excavated in large amounts from the extraskeletal waste of crustaceans. The biodegradability, biocompatibility and non-toxic properties of CS make it a safe material for a variety of environmental and bio- medical applications. 7,8 CS has been used in various ap- plications such as hydrogels, membranes, nanofibers, beads, micro/nanoparticles, scaffolds and sponges. Due Materiali in tehnologije / Materials and technology 56 (2022) 4, 341–349 341 UDK 620.168:543.422.3 ISSN 1580-2949 Original scientific article/Izvirni znanstveni ~lanek MTAEC9, 56(4)341(2022) *Corresponding author's e-mail: gxh218@163.com to its biodegradability, predictable degradation rate, anti- bacterial activity, structural integrity, non-toxicity to cells and biocompatibility, CS allows good wound heal- ing, tissue engineering and ophthalmic lens manufactur- ing. 9,10 Because of its low solubility, low stability and low mechanical properties, electrospinning of CS is very difficult to perform. Therefore, it is necessary to mix an- other polymer polylactic acid (PLA) with CS to improve the mechanical properties of CS nanofibers. 11,12 However, polylactic acid exhibits some defects in processability, such as brittleness and poor stiffness, which limit its pro- motion and use as a kind of electrospinning biodegrad- able matrix material. Polybutylene carbonate (PBC) is a new type of biodegradable polyester. PBC has excellent comprehensive properties. It is considered to be one of the most cost-effective and promising materials. 13–15 PBC shows relatively hydrophilic properties and high tensile strength due to its chemical structure. 16 In addition, as PBC is very conducive to cell attachment, its biocom- patability is similar to PLA. 17 Besides, PBC can improve the hard and brittle nature of PLA, and it is well compat- ible with PLA. PBC can be used as a toughener to im- prove the brittleness of PLA. 18 These co-spinning agents are a class of biodegradable polymers with good spinn- ability and good biocompatibility, and are conducive to an efficient application of PLA/PBC/CS composite fiber membranes. Therefore, in this paper a CipHCl-PLA/PBC/CS composite fiber membrane was prepared using the elec- trostatic spinning technology. The properties of the CipHCl-PLA/PBC/CS composite fiber membrane were studied with SEM, XRD, FTIR and a drug-release test. The antibacterial properties of the composite fiber mem- brane against E. coli and S. aureus were legally analyzed with the inhibition zone method, and the antibacterial mechanism was discussed. The composite fiber mem- brane has a high application value in the field of antibac- terial dressings. 2 EXPERIMENTAL PART 2.1 Reagents and apparatus Reagents: polylactic acid (PLA, Mn = 6.08 × 104 Da) and polybutylene carbonate, (PBC, Mn = 7.0 × 105 Da) were purchased from Hisun Biomaterials (Zhejiang Province); chitosan (CS, Mn = 160.9 Da, degree of deacetylation 90 %) was purchased from Xiamen Bio- technology Co., Ltd.; ciprofloxacin hydrochloride (CipHCl) was purchased from Xiamen Yicheng Technol- ogy Co., Ltd.; trifluoroacetic acid (TFA) was purchased from Beijing Coupling Technology Co., Ltd.; the PBS phosphate buffer solution (pH = 7.2–7.4) was purchased from Tianjin Tianli Chemical Reagent Co., Ltd; dimethyl sulfoxide (DMSC, 99.9 %) was purchased from Jinan Yunxiang Chemical Co., Ltd.; peptone, beef extract and agar were purchased from Sinopharm Chemical Reagent Co., Ltd.; the MTT reagent was purchased from Biyun- tian Biotechnology Research Institute of Haimen City. Apparatus: Fourier transform infrared spectrometer (FTIR), Spectrum One model, an American PE com- pany; scanning electron microscope (SEM), S-4300 model, Hitachi, Japan; X-ray diffraction analyzer (XRD), D8 Advance model, Bruker-AXE, Germany; UV-visible spectrophotometer, UV-5100 B model, Shanghai Yuan Analytical Instrument Co., Ltd.; NC ultrasonic cleaner, X. GU et al.: PREPARATION AND CHARACTERIZATION OF A CipHCl-PLA/PBC/CS COMPOSITE FIBER MEMBRANE ... 342 Materiali in tehnologije / Materials and technology 56 (2022) 4, 341–349 Figure 1: Schematic diagram of synthesizing the CipHCl-PLA/PBC/CS composite fiber membrane and its performance KQ5200DE model, Kunshan Ultrasonic Instrument Co., Ltd.; heat-collecting magnetic stirrer, DF-101S model, Jintan Huafeng Instrument Co., Ltd. 2.2 Sample preparation 2.2.1 Preparation of the spinning solutions PLA, PBC and CS were dried in a vacuum drying oven for 24 h at 37 °C. CipHCl (10 w/% and 20 w/%) was sonicated for 0.5 h at room temperature with a small amount of solvent until CipHCl was well dispersed and sealed for future use. According to the previous research of our group, 1 : 2.5 was the best mass ratio of PLA to PBC in the PLA/PBC/CS composite fiber membrane. The dried PLA, PBC and CS were dissolved in TFA to make solutions, and then the dispersed CipHCl solution in different amounts was added to the above polymer dope. Spinning solutions of CipHCl-PLA/PBC/CS were obtained. 2.2.2 Electrospinning The polymer solution was placed in a 10 mL syringe with an inner diameter of 0.41 mm at room temperature. The syringe containing the blend solution was installed into the electrospinning machine. The height of the sy- ringe was adjusted so that the height of the needle and the center position of the aluminum foil receiving plate were on the same horizontal line, and the distance be- tween the needle and the receiving plate was 25 cm. The ejection speed was 0.5 mm/min. The syringe end was connected to the positive pole of the high-voltage DC power supply, and the aluminum foil receiver was con- nected to the negative pole. The positive voltage was 18 kV and the negative voltage was –16 kV. Polymer fi- bers were produced, from the tip of the needle to the grounded current collector, by the action of an external electric field. The samples were labeled as PLA/PBC, PLA/PBC/CS, 10 % CipHCl-PLA/PBC/CS and 20 % CipHCl-PLA/PBC/CS in accordance with the compo- nents. Figure 1 shows the schematic of the formation of the CipHCl-PLA/PBC/CS composite fiber membrane. 2.3 Characterization The chemical structures of the samples were ana- lyzed using FTIR with an ATR accessory in a dry envi- ronment at room temperature. The scanning range was 4000–500 cm –1 and the scanning resolution was less than 0.09 cm –1 . The morphology and microstructures of the samples were observed using SEM with an acceleration voltage of 20 kV. Prior to the analysis, the samples were coated with a thin layer of gold. A D8 Advance type X-ray diffractometer from Bruker-AXE in Germany was used for the X-ray diffraction analysis (XRD) to deter- mine the crystal structure of the electrospun composite fiber membrane. The voltage was 50 kV, the current was 50 mA, the scanning speed was 3 °/min and the scanning range was 10°–60°. 2.4 Performance test of the composite fiber membrane 2.4.1 Drug standard curve equation 0.1 g of CipHCl was finely weighed with an elec- tronic balance and transferred to a 100 mL volume flask. A PBS phosphate buffer solution with a pH value in the range of 7.2–7.4 was added into the flask. The solution was stirred with a glass rod, and then ultrasonic vibration was performed for 30 min. Finally, the solution was fur- ther diluted and configured as (50, 25, 12.50, 6.25, 3.125 and 1.56) μg/mL. The solutions with different concentra- tions were placed in cuvettes. The absorbance tests were carried out with ultraviolet spectrum at a wavelength of 270 nm to measure the absorbance of CipHCl and obtain the standard curve. 2.4.2 Determination of the drug release rate in vitro 5 mg and 10 mg of rectangular drug-loaded fiber- membrane samples were accurately weighed and placed in 100 mL volumetric flasks. To simulate in vitro drug release, the PBS buffer solution was simultaneously added to the flasks and centrifuged with a constant-tem- perature shaker. 5 mL of the solution was taken out after (0.5, 1, 2, 3, 4, 5, 6, 8, 12, 24, 36, 48, 60, 84, 120, 156, and 192) h and put in sample bottles. An ultraviolet spectrophotometer was used to measure the drug concen- trations of the solutions at each time point. The cumula- tive release rate of the drug at different times was calcu- lated as shown in Equation (1). T CVV C M x k xk (%) = + = − ∑kk CipHCL 0 (1) Here, T is the cumulative release rate of the drug (%); x is the number of release media for the replacement; C x is the sample concentration at the x-th time node, in mg/mL; V is the volume of the release medium in mL; V k is the volume sampled at the k-th time node; C k is the concentration of the solution sample taken at the k-th time node, in mg/mL; M CipHCl is the mass of CipHCl in the composite fiber membrane containing CipHCl, in mg. 2.5 Antibacterial performance test According to the GB/T 20944.1-2007 national stan- dard, the in vitro antibacterial activity of two bacterial strains, E. coli and S. aureus, were evaluated with the agar-diffusion method. The composite fiber membrane was cut into a 20 mm diameter membrane and sterilized in a clean bench with ultraviolet light for 30 min prior to using. Luria broth (LB) agar plates containing1×1 0 5 bacterial colony-forming units (CFU/mL) were used for culturing. After 24 h of incubation at 37 °C, the diameter of the zone of inhibition was evaluated, as shown in Equation (2). Y AB A = − × 100 % (2) X. GU et al.: PREPARATION AND CHARACTERIZATION OF A CipHCl-PLA/PBC/CS COMPOSITE FIBER MEMBRANE ... Materiali in tehnologije / Materials and technology 56 (2022) 4, 341–349 343 Here, A is the viable count (cfu/mL) of a blank sam- ple after 24 h of contact with the bacteria; B is the num- ber of viable bacteria (cfu/mL) after 24 h of contact be- tween the sample and the bacteria; Y is the antibacterial rate. 2.6 Cytotoxicity test The extract of a sample was prepared according to the GB/T 16886.12 national standard, that is, the fiber membrane sterilized with irradiation was cut into a 10 mm circular membrane, and dimethylsulfoxide (DMSO) medium was added at a ratio of 6 cm 2 /mL. The extraction stock was obtained by leaching at 37 °C and a 5%CO 2 saturated humidity. Then the extract was added to a 96-well cell culture plate, and L929 cells were inoc- ulated with a cell density of 2.5 × 104 cells/mL. MTT detection was performed after 72 h of incubation in a CO 2 incubator. After a period of time, the status of the cell growth was observed using an optical microscope. The absorbance was measured 4 times at 450 nm with a standard instrument (Bio-Rad, USA).The cell prolifera- tion rate (R) was calculated with the average value of the absorbance, as shown in Equation (3). R OD OD i = × 100 % (3) Here, OD is the absorbance value of the blank control sample, and OD i is the absorbance value of the experi- mental group. Finally, the cytotoxicity levels were deter- mined in accordance with the GB/T 16886.12 cytotox- icity rating standard, as shown in Table 1. Table 1: Cytotoxicity rating criteria R/% 100 75–99 50–74 25–49 1–24 l e v e l01234 evaluation of results qualified qualified to be de- termined disquali- fied disquali- fied 3 RESULTS AND DISCUSSION 3.1 FTIR spectra analysis Figure 2 illustrates the characteristic FTIR spectra of CipHCl, PLA/PBC, PLA/PBC/CS, and CipHCl-PLA/ PBC/CS fiber membranes containing different amounts of CipHCl. It can be seen that the stretching vibration peak of methylene at 2966 cm –1 is a structural character- istic absorption peak attributed to PBC. The stretching vibration peak of C=O at 1732 cm –1 is a structural char- acteristic belonging to the PLA absorption peak. The ad- sorption peak at 1456 cm –1 is ascribed to the saturated C-H bond and the adsorption peak at 1238 cm –1 is as- cribed to the stretching vibration of RCR=O. It can be seen from Figure 2 that the characteristic peaks are gen- erally consistent with the PLA/PBC sample in the PLA/PBC/CS fiber membrane, except that a characteris- tic peak appeares at 1183 cm –1 . This is because -NH 2 in CS bonded with -COOH in PLA to form -NH 3 + . When comparing the FTIR spectra of CipHCl, CipHCl-PLA/ PBC/CS (10 w/%) and CipHCl-PLA/PBC/CS (20 w/%), it can be found that two more absorption peaks are ap- parent at 1626 cm –1 and 1694 cm –1 , ascribed to the struc- tural characteristic absorption peak of NH and C=O in CipHCl, respectively. X. GU et al.: PREPARATION AND CHARACTERIZATION OF A CipHCl-PLA/PBC/CS COMPOSITE FIBER MEMBRANE ... 344 Materiali in tehnologije / Materials and technology 56 (2022) 4, 341–349 Figure 3: SEM micrographs and average diameter distributions of: a) pure PLA/PBC, b) PLA/PBC/CS, c) CipHCl-PLA/PBC/CS with 10 w/% CipHCl, d) CipHCl-PLA/PBC/CS with 20 w/% CipHCl Figure 2: FTIR spectra of pure PLA/PBC sample, PLA/PBC/CS and CipHCl-PLA/PBC/CS composite fiber membranes with different CipHCl drug contents 3.2 SEM image and the fiber diameter distribution analysis Figure 3 shows the SEM images and fiber diameters of PLA/PBC, PLA/PBC/CS, CipHCl-PLA/PBC/CS (10 w/%) and CipHCl-PLA/PBC/CS (20 w/%) composite fiber membranes. It can be seen that the surfaces of all the fibers are relatively smooth without any bead-like structure. The average diameter of the PLA/PBC fiber in Figure 3a is 886 nm, and the average diameter of the PLA/PBC/CS fiber in Figure 3b is 611 nm, indicating that the addition of CS decreased the average diameter of the fiber membrane. When the content of CipHCl in PLA/PBC/CS is 10 w/%, the average fiber diameter is 466 nm as shown in Figure 3. The average diameter is significantly reduced to 374 nm when the CipHCl con- tent is 20 w/%. Besides, the content of drug CipHCl has a significant effect on the fiber diameter. As shown in Figure 3d, the fiber diameter decreases and its distribu- tion becomes more uniform with the increase in the CipHCl content. This is because CipHCl contains more polar groups (hydroxyl groups), which increase the con- ductivity of the polymer dope and lead to a greater de- gree of refinement of the jet stream during the electropinning process. 19 It is more conducive to the loading and release of drug molecules because the gap between the fibers becomes larger. Therefore, it has great potential to be used as a good medical carrier material. 3.3 X-ray diffraction patterns As shown in Figure 4, the XRD patterns of CipHCl, PLA/PBC, PLA/PBC/CS, CipHCl-PLA/PBC/CS (10 w/%) and CipHCl-PLA/PBC/CS (20 w/%) composite fiber membranes explain the crystalline structure and distribution of small drug molecules in the fibrous mem- brane. The PLA/PBC fiber membranes have characteris- tic diffraction peaks at 21.71° and 22.95°, belongiong to PBC and PLA, respectively. Upon the addition of CS, two new 2 peaks appeared at 16.82° and 20.42°, and the characteristic diffraction peaks remain unchanged for PLA/PBC, indicating that the crystal structure did not change and there is no interaction between CS and PLA/PBC. The CipHCl crystal shows the characteristic diffraction peaks at 2 of 11.18°, 19.22°, 23.05°, 24.77° and 26.48°. The characteristic diffraction peaks appear at 11.18° and 26.48° on each drug-loaded fiber membrane. It can be seen that the higher the content of ciprofloxacin hydrochloride, the more pronounced is the peak shape. It can be seen that the addition of CipHCl did not change the crystalline structure of the PLA/PBC/CS fiber mem- brane, indicating that the small drug molecules are uni- formly distributed in the crystalline phase of the poly- mer. 3.4 In vitro drug release analysis Figure 5 shows the UV spectra of CipHCl solutions in the phosphate buffer at different concentrations of (50, 25, 12.50, 6.25, 3.125 and 1.56) μg/mL. Table 2 lists the absorbance of CipHCl at different concentrations. Fig- ure 6 shows the standard curve. The concentration of CipHCl is linearly proportional to the absorbance. Table 2: Absorbance intensity of CipHCl at different concentrations Concentration (μg/mL) Wavelength (nm) Absorbance 1.560 270.87 0.15 3.125 270.76 0.25 6.250 270.58 0.37 12.50 270.02 0.48 25.00 270.09 0.59 50.00 270.02 0.72 The plots from Figure 6 were fitted to the linear Equation (4) based on the regression curve. Y = 0.0563 X + 0.0372 R 2 = 0.9994 (4) X. GU et al.: PREPARATION AND CHARACTERIZATION OF A CipHCl-PLA/PBC/CS COMPOSITE FIBER MEMBRANE ... Materiali in tehnologije / Materials and technology 56 (2022) 4, 341–349 345 Figure 5: UV spectra of CipHCl aqueous solutions with various CipHCl concentrations of (50, 25, 12.50, 6.25, 3.125 and 1.56) μg/mL Figure 4: XRD patterns of CipHCl powder, pure PLA/PBC composite fiber membrane, PLA/PBC/CS composite fiber membrane and CipHCl-PLA/PBC/CS composite fiber membrane with different drug contents Here, Y is the absorbance intensity in the UV spec- trum, and X (μg/mL) is the concentration of CipHCl. As shown in Figure 5, the plots are in good agreement with the fitted curve, indicating a linear relationship between the concentration of CipHCl and the absorbance inten- sity. Thus, the fitted formula was employed as the stan- dard curve for drug release in vitro. Figure 7 shows the release-rate curves for the CipHCl in the CipHCl-PLA/PBC/CS composite fiber membrane with CipHCl contents of 10 w/% and 20 w/%. It can be seen that the release rates of the fiber mem- branes increase with the increase in the drug content. The rate curves in Figure 7 can be divided into three stages. In the initial drug-release stage (from 0–8 h), the drug-release rate was low. The released amount reached 12.5 % and 25.1 % after 8 h for the samples with CipHCl contents of 10 w/% and 20 w/%, respectively. Subse- quently, the released-drug amount raised slowly after 84 h. The released amount reached 66.5 % and 77.2 % for the samples with CipHCl contents of 10 w/% and 20 w/%, respectively, after 84 h. Finally, the release rate turned very slow and the drug-release amount reached the limits of 84.2 % and 88.3 % for the samples with CipHCl contents of 10 w/% and 20 w/%, respectively, af- ter 192 h. According to Table 2, the release rate of the membrane sample with a high CipHCl drug content was higher than that of the sample with a low CipHCl con- tent. In the drug-release system, a high release rate can allow a larger drug amount to reach the target site in a shorter time, achieving the antibacterial effect. So the sample with the 20 w/% CipHCl content is better than the sample with the 10 w/% CipHCl content. 3.5 Antibacterial performance analysis Figures 8a, 8b, 8c and 8d show the pictures of differ- ent composite membranes cultured with E. coli. Figures 8e, 8f, 8g and 8h show the pictures of different mem- branes cultured with S. aureus. The antibacterial width of each sample is shown in Figure 9. As shown in Figures 8a and 8e, there was no bacteriostatic zone around the PLA/PBC fiber membrane, which indicates that the X. GU et al.: PREPARATION AND CHARACTERIZATION OF A CipHCl-PLA/PBC/CS COMPOSITE FIBER MEMBRANE ... 346 Materiali in tehnologije / Materials and technology 56 (2022) 4, 341–349 Figure 6: Standard curve between the UV absorbance intensity and the concentration of CipHCl in phosphate buffer (pH = 7.4) Figure 8: Pictures of different membranes cultured with E. coli and S. aureus Figure 7: In vitro drug release profiles of CipHCl-PLA/PBC/CS com- posite fiber membranes with different CipHCl contents PLA/PBC fiber membrane has almost no antibacterial ef- fect on E. coli and S. aureus. When CS is added (as shown in Figures 8b and 8f), the width of the inhibition zone around the sample increases and the width of the inhibition zone for E. coli and S. aureus is 3.9 mm and 3.7 mm, respectively. Due to its large width of the bacte- ria inhibition zone, it can be concluded that the PLA/ PBC/CS membrane is a material with a good antibacte- rial effect according to the Chinese national standard. As shown in Figures 8c and 8g, the width of the inhi- bition zone for E. coli and S. aureus increases to 8.1 mm and 7.5 mm, respectively, for CipHCl-PLA/PBC/CS with 10 w/% of CipHCl. The width of the inhibition zone for E. coli and S. aureus further increases to 10.5 mm and 10.1 mm, respectively, when the content of CipHCl is 20 w/%. (as shown in Figures 8d and 8h). The CipHCl- PLA/PBC/CS membranes can be defined as a material with an excellent antibacterial effect. The addition of CipHCl significantly improves the antibacterial perfor- mance of the composite fiber membranes. The higher the CipHCl amount, the better is the antibacterial effect. The reason is that the CS macromolecules were adsorbed on the surface of the bacteria to form a polymer film, hin- dering the transport of nutrients into the cells and thus inhibiting the bacteria. The introduction of CipHCl can destroy the morphological structures of the bacteria. This leads to the bacterial death and achieves bacteriostatic ef- fects. 20,21 In addition, it can be concluded that the com- posite fiber membrane has a better antibacterial effect on E. coli than S. aureus. Figure 10 shows the inhibition rates of four compos- ite fiber membranes (PLA/PBC, PLA/PBC/CS, CipHCl- PLA/PBC/CS (10 w/%) and CipHCl-PLA/PBC/CS (20 w/%)) for E. coli and S. aureus detected with the col- ony-recovery method. It can be seen that the antibacterial rates of the four composite fiber membranes for E. coli were (26.8, 78.4, 85.2 and 96.4) %, and the antibacterial rates for S. aureus were (22.9, 76.7, 82.3 and 95.8) %, respectively. These results indicate that the antibacterial activity of the PLA/PBC composite fiber membrane was not obvious and the antibacterial rate was significantly improved after adding CS and CipHCl. The antibacterial effect was better with the increase in the CipHCl drug content. The antibacterial rate of each fiber membrane sample for E. coli was slightly higher than that for S. aureus. The result is consistent with the antibacterial circle experiment. 3.6 Cytotoxicity test In order to study the cellular compatibility of the PLA/PBC/CS and CipHCl-PLA/PBC/CS composite fi- ber membranes, an inverted microscope was used to ob- serve the morphology of the L929 cells on the obtained ultrathin electrospinning film after durations of (1, 3 and 7) d. The results are shown in Figures 11b, 11d and 11f, representing the cell morphologies on the CipHCl- PLA/PBC/CS composite fiber membranes, while Fig- ures 11a, 11c and 11e represent the cell morphologies on the PLA/PBC/CS composite fiber membranes, being the control samples. The cells in the photo are spindly and of a uniform size. They have smooth cell walls, X. GU et al.: PREPARATION AND CHARACTERIZATION OF A CipHCl-PLA/PBC/CS COMPOSITE FIBER MEMBRANE ... Materiali in tehnologije / Materials and technology 56 (2022) 4, 341–349 347 Figure 10: Bacteriostatic rate of PLA/PBC, PLA/PBC/CS and CipHCl-PLA/PBC/CS composite fiber membranes with different drug contents Figure 9: Antibacterial zone width of PLA/PBC, PLA/PBC/CS and CipHCl-PLA/PBC/CS composite fiber membranes with different drug contents Figure 11: Inverted microscopy images of L929 cells grown on PLA/PBC/CS (a, c, e) and CipHCl-PLA/PBC/CS (b, d, f) composite fiber membranes after 1 d (a, b), 3 d (c, d), and 7 d (e, f) growing well. Hence, the composite fiber membrane shows good cellular compatibility and can be used for drug-release applications. As an effective drug carrier, a fiber membrane must be nontoxic and antibacterial. Herein, to explore the cytotoxicity of the prepared fiber membranes, an MTT assay was employed with the L929 cells to detect mem- brane toxicity. The results are shown in Figure 12. The CipHCl-PLA/PBC/CS and PLA/PBC/CS composite membranes showed low cytotoxicity to the L929 cells, and the cellular growth rate was not less than 80 % after (1, 3 and 7) d. According to the GB/T 16886.12, the tox- icity of both tested membranes to cells met the standard of grade 1. The fiber membranes were non-toxic and met the standards of implantable medical materials. There- fore, it can be concluded that the prepared CipHC- PLA/PBC/CS composite fiber membrane is non-toxic. 4 CONCLUSIONS A ciprofloxacin hydrochloride (CipHCl)-polylactic acid (PLA)/polybutylene carbonate (PBC)/chitosan (CS) new component composite fiber membrane was success- fully prepared with the electrostatic spinning technology. The fiber diameter decreased upon the addition of CS and CipHCl. The drug-release test results for the drug-loaded composite fiber membrane showed that the drug-release rate changed. When the content of CipHCl was 20 w/%, the antibacterial widths of E. coli and S. aureus were 10.5 mm and 10.1 mm, and the antibacterial rates were 96.4 % and 95.8 %, respectively. According to the results of the material’s toxicity test and analysis, the prepared CipHCl-PLA/PBC/CS composite fiber mem- brane is non-toxic and meets the standards for implant- able medical materials. It can be concluded that the CipHCl-PLA/PBC/CS composite fiber membrane can be used as a good medical carrier material because of its ex- cellent antibacterial property and excellent drug-release ability. So, it has a great application potential for wound dressings. Acknowledgment The authors gratefully acknowledge the support from the Heilongjiang Provincial Department of Education Project (CLKFKT2021Z3, 145109301), China. 5 REFERENCES 1 Z. Saadi, A. Rasmont, G. Cesar, H. Bewa, L. Benguigui, Fungal deg- radation of poly(l-lactide) in soil and in compost, Journal of Poly- mers and the Environment, 20 (2012), 273–282, doi:10.1007/ s10924-011-0399-9 2 M. M. Cui, L. L. Liu, N. Guo, R. X. Su, F. Ma, Preparation, Cell Compatibility and Degradability of Collagen-Modified Poly(lactic acid), Molecules, 20 (2015) 1, 595–607, doi:10.3390/molecules 20010595 3 J. Fal, K. Bulanda, J. Traciak, J. Sobczak, R. Kuziola, K. Maria, G. Budzik, M. Oleksy, G. ¯yla, Electrical and Optical Properties of Sili- con Oxide Lignin Polylactide (SiO2-L-PLA), Molecules, 25 (2020) 6, 1354, doi:10.3390/molecules25061354 4 Z. Y. Tang, F. L. Fan, Z. Z. Chu, C. L. Fan, Y. Y. Qin, Barrier proper- ties and characterizations of poly(lactic acid)/ZnO nanocomposites, Molecules, 25 (2020) 6, 1310, doi:10.3390/molecules25061310 5 J. Zeng, L. Yang, Q. Liang, Influence of the drug compatibility with polymer solution on the release kinetics of electrospun fiber formula- tion, J. Controlled Release, 105 (2005) 1/2, 43–51, doi:10.1016/ j.jconrel.2005.02.024 6 R. Y. Yang, D. Y. Wang, H. L. Li, Y. He, X. Y. Zheng, M. W. Yuan, M. L. Yuan, Preparation and characterization of bletilla striata polysaccharide/polylactic acid composite, Molecules, 24 (2019) 11, 2104, doi:10.3390/molecules24112104 7 I. Cerkez, A. Sezer, S. K. Bhullar, Fabrication and characterization of electrospun poly(e-caprolactone) fibrous membrane with antibacte- rial functionality, R. Soc. Open Sci., 4 (2017) 2, 160911, doi:10.1098/rsos.160911 8 K. Vidyalakshmi, K. N. Rashmi, T. M. P. K. Siddaramaiah, Studies on formulation and in vitro evaluation of pva/chitosan blend films for drug delivery, J. Macromol. Sci. A, 41 (2004), 1115–1122, doi:10.1081/MA-200026554 9 H. Chi, W. H. Li, C. L. Fan, C. Zhang, L. Li, Y. Y. Qin, M. L. Yuan, Effect of high pressure treatment on poly(lactic acid)/nano-TiO2 composite films, Molecules, 23 (2018) 10, 2621, doi:10.3390/ molecules23102621 10 J. K. Xu, S. Strandman, J. L. X. Zhu, J. Barralet, M. Cerruti, Genipin-crosslinked catechol-chitosan mucoadhesive hydrogels for buccal drug delivery, Biomaterials, 37 (2015), 395, doi:10.1016/ j.biomaterials.2014.10.024 11 P. Taepaiboon, U. Rdthongungsar, P. Supaphol, Drug-loaded electrospun mats of poly(vinyl alcohol) fibres and their release char- acteristics of four model drugs, Nanotechnology, 17 (2006)9 , 2317–2329, doi:10.1088/0957-4484/17/9/041 12 M. S. Austero, A. E. Donius, U. G. K. Wegst, C. L. Schauer, New crosslinkers for electrospun chitosan fibre mats, J. R. Soc. Interface, 9( 2012), 2551–2562, doi:10.1098/rsif.2012.0241 13 E. Fortunati, F. Luzi, D. Puglia, R. Petrucci, J. M. Kenny, L. Torre, Processing of PLA nanocomposites with cellulose nanocrystals ex- tracted from Posidonia oceanica waste: Innovative reuse of coastal plant, Ind. Crops Prod., 67 (2015), 439–447, doi:10.1016/j.indcrop. 2015.01.075 14 A. J. R. Lasprilla, G. A. R. Martinez, B. H. Lunelli, A. L. Jardini, R. Maciel Filho, Poly-lactic acid synthesis for application in biomedical devices – A review, Biotechnol. Adv., 30 (2012) 1, 321–328, doi:10.1016/j.biotechadv.2011.06.019 15 G. Giammona, E. F. Craparo, Biomedical applications of polylactide (PLA) and its copolymers, Molecules, 23 (2018) 4, 980, doi:10.3390/ molecules23040980 X. GU et al.: PREPARATION AND CHARACTERIZATION OF A CipHCl-PLA/PBC/CS COMPOSITE FIBER MEMBRANE ... 348 Materiali in tehnologije / Materials and technology 56 (2022) 4, 341–349 Figure 12: In vitro cytotoxicity of PLA/PBC/CS and CipHCl-PLA/ PBC/CS composite fiber membranes to L929 cells 16 D. Q. Liu, Z. Q. Cheng, et al., Polycaprolactone nanofibres loaded with 20(S)-protopanaxadiol for in vitro and in vivo anti-tumour ac- tivity study, R. Soc. Open Science, 5 (2018), doi:10.1098/rsos. 180137 17 K. Mäenpää, V. Ellä, J. Mauno, M. Kellomäki, R. Suuronen, T. Ylikomi, S. Miettinen, Use of adipose stem cells and polylactide discs for tissue engineering of the temporomandibular joint disc, J. R. Soc., Interface, 7 (2010), 177–188, doi:10.1098/rsif.2009.0117 18 R. Sakai, B. John, M. Okamoto, J. V. Seppälä, J. Vaithilingam, H. Hussein, Fabrication of polylactide-based biodegradable thermoset scaffolds for tissue engineering applications, Macromol. Mater. Eng., 298 (2013), 45–52, doi:10.1002/mame.201100436 19 J. Jeong, M. Ayyoob, J. H. Kim, In situ formation of PLA-grafted alkoxysilanes for toughening a biodegradable PLA stereocomplex thin film, RSC Adv., 9 (2019) 38, 21748–21759, doi:10.1039/ C9RA03299A 20 Y. B. Zhu, Y. Wang, J. Y. Z. Xu, J. H. Chen, L. M. Wang, B. Qi, Enantioselective biosynthesis of l-phenyllactic acid by whole cells of recombinant escherichia coli, Molecules, 22 (2017) 11, 1966, doi:10.3390/molecules22111966 21 W. Shao, S. X. Wang, X. F. Liu, Tetracycline hydrochloride loaded regenerated cellulose composite membranes with controlled release and efficient antibacterial performance, RSC Adv., 6 (2016)4 , 3068–3073, doi:10.1039/C5RA23409C X. GU et al.: PREPARATION AND CHARACTERIZATION OF A CipHCl-PLA/PBC/CS COMPOSITE FIBER MEMBRANE ... Materiali in tehnologije / Materials and technology 56 (2022) 4, 341–349 349