Preparation method and application of alpha-lactalbumin electrospun nanofiber loaded with cefalexin and epicatechin

Abstract

The invention relates to the field of biological medicine, in particular to a preparation method of alpha-lactalbumin electrospun nanofiber loaded with cefalexin and epicatechin and application of the alpha-lactalbumin electrospun nanofiber as wound dressing to killing drug-resistant bacteria. Wound healing interruption caused by multi-drug-resistant bacterial infection is a difficult problem faced by a global medical health system, and due to the existence of drug resistance of antibacterial drugs, the problem may not be solved by a traditional treatment method. In order to solve the problem, the nanofiber prepared from cefalexin (CPL), epigallocatechin (EP) and alpha-lactalbumin (alpha-Lalb) through electrospinning is used as the dressing. Alpha-Lalb can promote migration and proliferation of fibroblasts and synthesis of collagen, scar formation is reduced, and the effect is excellent. CPL and EP have a synergistic inhibition effect on staphylococcus aureus (MRSA) and escherichia coli infection. Mature collagen exists at the wound, the number of inflammatory cytokines is small, vascular expression is increased, and expression of interleukin 6 is low. The dressing can meet individual requirements of infectious wound patients, and a non-invasive and easy-to-operate treatment scheme is provided.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a method for preparing alpha-lactalbumin electrospun nanofibers loaded with cephalexin and epicatechin, and the use of the nanofibers as wound dressings for killing drug-resistant bacteria. Background Art

[0002] Wounds can be caused by accidents, surgery, insect bites, thermal injuries, electrical or chemical burns, congenital defects, diabetic trauma, etc. The epidermal barrier of the skin in the wound is damaged, and the blood or blood clots formed in the early stage of hemostasis provide rich nutrients for the development of invasive bacteria, which will quickly penetrate the wound, proliferate and infect the damaged skin, increase pain, delay the healing process, and may cause extremely serious systemic symptoms. Therefore, unhealed wounds often become more serious or complicated infected wounds, affected by multiple bacteria, and produce a large amount of deadly hydrolyzed protein wound exudate.

[0003] Microbial contamination is the first step in skin infection and can be caused by endogenous or exogenous microbiota. Endogenous microbiota includes Escherichia coli, Staphylococcus aureus, bacilli, and enterococci, among others. However, this type of contamination mainly comes from the surgical process. Exogenous flora mainly includes streptococci and staphylococci, which usually come from the operating room, including surgical instruments, air, and staff. In addition, the number and pathogenicity of microorganisms are also major risk factors. Recently, the number of highly pathogenic microorganisms isolated from postoperative wounds has been increasing, such as multidrug-resistant Escherichia coli (E. coli) and methicillin-resistant Staphylococcus aureus (MRSA), which may be due to the large and unnecessary use of antibiotics.

[0004] Electrospun nanofibers (NFs) have been extensively studied for use as drug delivery, tissue engineering, and skin regeneration, as electrospinning is a facile method to fabricate polymer fibers with unique frameworks. The micro-nano networks can mimic the extracellular matrix and promote cell adhesion and proliferation. α-Lactalbumin (Lalb) is a 14.2 kDa whey protein found in mammalian milk. The main biological function of α-lactalbumin is to control the ability of the mammary gland to synthesize lactose, making it easier to produce milk. In addition, it is a rich source of tryptophan, which is required for the production of the neurotransmitter serotonin. Serotonin can promote the migration and proliferation of keratinocytes and fibroblasts, promote the regeneration of burned skin, and minimize scar formation. α-Lactalbumin can also promote collagen synthesis.

[0005] Epigallocatechin (EP) is a natural compound with potent antibacterial properties that is effective against drug-resistant MRSA and E. coli when used in combination with other antibiotics. Epigallocatechin destroys bacterial cell walls by binding to peptidoglycan, inhibiting the synthesis of E. coli verotoxin and bacterial type II fatty acid synthase, thereby inhibiting drug resistance. Cephalexin (CPL), an antibiotic with the same functional group as penicillin, contains β-lactam and dihydrothiazide, and is a safe antibiotic that can be used to treat a variety of bacterial infections, including skin, respiratory and bone infections. Summary of the invention

[0006] A preparation method and application of α-lactalbumin electrospun nanofibers loaded with cephalexin and epicatechin, wherein the fibers are composed of the following raw materials in parts by weight: 0.5-2 parts of cephalexin, 0.5-2 parts of epicatechin, 1-3 parts of α-lactalbumin, and 4-10 parts of a mixture of ethanol and water (wherein the ethanol content is 50% by mass);

[0007] Preferably, α-lactalbumin, cephalexin and epicatechin, wherein the weight ratio of cephalexin, epicatechin to α-lactalbumin is 1:1:2;

[0008] The method for preparing the fiber comprises the following steps:

[0009] S1: dissolving the raw material components of cephalexin, epicatechin and α-lactalbumin in 50% ethanol and stirring overnight for 12 hours;

[0010] S2: The above solution was loaded into a 10 mL syringe and electrospun at a feed rate of 0.3 mL / h and a high voltage of 18 KV. The syringe was equipped with a 22-gauge flat-head needle, which was connected to the positive electrode of the power supply. The electrospun nanofiber dressing was evenly collected on a collector connected to the negative electrode of the power supply, and the collector was 15 cm away from the needle tip.

[0011] The electrospun nanofibers loaded with cephalexin and epicatechin are used as antibacterial wound dressings to kill bacteria including but not limited to multidrug-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus.

[0012] The beneficial effect of the electrospun nanofibers loaded with cephalexin and epicatechin is that through the electrospinning process, a CPL and EP-loaded α-lactalbumin electrospun nanofiber (CE-Lalb NFs) is conveniently obtained and used as a wound dressing, wherein CPL and EP have obvious synergistic inhibitory effects on MRSA and Escherichia coli, and the α-lactalbumin electrospun nanofibers (Lalb-NFs) can accelerate wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1: Electron microscopic morphology of CE-Lalb NFs of Example 1

[0014] Figure 1 a, b, c, and d are CE-Lalb NFs images of Example 1 taken at different angles under an electron microscope.

[0015] Figure 2 : Antibacterial effect

[0016] in: Figure 2-1 , Figure 2-2 This is a bar graph showing the inhibition rates of CE-Lalb NFs, free EP, free CPL, and free (EP+CPL) against MRSA and E Coli in Example 1. Free means that the drug is not loaded on Lalb-NFs.

[0017] Figure 2-3 It is a bar graph of the total CFU of the control group (blank control group), free (EP+CPL), Lalb-NFs and CE-LalbNFs of Example 1. Free means that the drug is not loaded on Lalb-NFs.

[0018] Figure 2-4 It is a bar graph showing the minimum inhibitory concentrations of EP, EP in combination with EP / CPL, CPL, and CPL in combination with EP / CPL.

[0019] Figure 3 :Wound pathology section

[0020] The control group was a blank control group, the free drug group was a combination of 1 g CPL and 1 g EP not loaded into protein nanofibers, and the CE-Lalb NFs referred to the Example 1 group.

[0021] Figure 4 :Drug-loaded protein fibers promote collagen differentiation

[0022] The control group was a blank control group, CE-Lalb NFs referred to the Example 1 group, and the free drug group was a combination of 1 g CPL and 1 g EP.

[0023] in: Figure 4-1 The electrophoresis diagrams of collagen I, collagen III, and actinin in different groups;

[0024] Figure 4-2 Relative expression of proteins in different groups. DETAILED DESCRIPTION

[0025] CPL: cephalexin, EP: epicatechin, CE-Lalb NFs: α-lactalbumin electrospun nanofibers loaded with cephalexin and epicatechin, Lalb-NFs: α-lactalbumin electrospun nanofibers.

[0026] CD31: platelet-endothelial cell adhesion molecule; IL-6: interleukin-6; VEGF: vascular endothelial growth factor; TGF-β: transforming growth factor-β; α-SMA: α-smooth muscle actin.

[0027] Collagen I: Collagen I, Collagen III: Collagen 3, α-Actinin: Actinin;

[0028] The present invention is further described in detail below with reference to specific examples, but the protection scope of the present invention is not limited thereto:

[0029] Preparation and physicochemical properties of CE-Lalb NFs

[0030] Example 1

[0031] Raw material composition of CE-Lalb NFs:

[0032] Cephalexin 1g, epicatechin 1g, α-lactalbumin 2g, ethanol and water mixture (wherein the ethanol mass content is 50%) 5mL.

[0033] Preparation process:

[0034] 1 g of cephalexin, epicatechin, and 2 g of α-lactalbumin were each dissolved in 5 mL of a mixture of ethanol and water (wherein the ethanol content was 50%) under stirring.

[0035] The above solution was loaded into a 10 mL syringe and electrospun at a feed rate of 0.3 mL / h and a high voltage of 18 KV. The syringe was equipped with a 22-gauge flat-head needle and connected to the positive pole of the power supply. The electrospun nanofibers were uniformly collected on a collector connected to the negative pole of the power supply. The collector was 15 cm away from the needle tip.

[0036] CE-Lalb NFs Fourier transform infrared spectrum: 3400, 1750, 1650, 1520 cm -1 , indicating that it contains the chemical components α-lactalbumin, CPL and EP.

[0037] CE-Lalb NFs surface morphology

[0038] like Figure 1 As shown: the CE-Lalb NFs of Example 1 have a uniform surface, the designed dressing has a good surface morphology, and the fiber diameter is less than 200 nm.

[0039] The water contact angle (WCA) disappeared after 5 seconds, showing excellent wettability, which is conducive to the proliferation and adhesion of cells such as fibroblasts. The release patterns of CPL and EP are both biphasic, and α-lactalbumin can provide sufficient tryptophan to the wound site, thereby allowing the wound to heal rapidly. The excellent wettability of electrospun nanofibers allows the medium to penetrate and improve the release rate.

[0040] Furthermore, the electrospinning uniformity and smoothness of the electrospinning nanofibers, the transparency and viscosity of the electrospinning solution, the solid / liquid weight ratio, the ratio of EP and CPL, the concentration of CPL in the formulation, the uniformity of the electrospinning nanofibers, and whether beads were formed during the electrospinning process were evaluated. The results showed that as the solid / solution ratio increased, the viscosity of the electrospinning solution increased, electrospinning became slightly difficult, and the probability of bead formation increased.

[0041] Synergistic antimicrobial activity

[0042] like Figure 2 As shown in Example 1, the antibacterial test confirmed that cephalexin and epicatechin in CE-Lalb NFs have a synergistic effect. This super strong antibacterial therapy can eliminate pathogens in the wound and make the wound healing process easier and safer.

[0043] α-lactalbumin has no antibacterial properties. In the anti-biological test, the biomass of both pathogens was significantly reduced by electrospun nanofibers. These data all prove that the CE-Lalb NFs of Example 1 have synergistic antibacterial activity against MRSA and Escherichia coli, which is beneficial for the treatment of infected wounds.

[0044] The antibacterial synergy of the combination of free CPL and free EP, and the CE-Lalb NFs of Example 1 against resistant strains of MRSA and E. coli was evaluated using the inhibition zone test, antimicrobial test, CFU measurement and fractional inhibitory concentration index (FICI). The in vitro combined antibacterial effect of the CPL and EP composition was studied by calculating the minimum inhibitory concentrations (MICs) and fractional inhibitory concentration index (FICI) of the CPL and EP composition against E. coli and MRSA. The MICs of the CPL and EP combination were significantly lower than those of CPL and EP alone. The FICI of the CPL and EP combination against MRSA and E. coli were 0.4375 and 0.4782, respectively, both less than 1, indicating that the CPL and EP composition has a synergistic inhibitory effect against these two pathogens.

[0045] FICI = (MIC of EP in EP / CPL combination) / (MIC of EP alone)+(MIC of CPL in EP / CPL combination) / (MIC of CPL alone);

[0046] It is noteworthy that α-lactalbumin does not have any antibacterial properties. At the same time, in the antimicrobial test, the biomass of both pathogens was significantly reduced by electrospun nanofibers. All these data prove that electrospun nanofibers have synergistic antibacterial activity against MRSA and E. coli, which is beneficial for the treatment of infected wounds in vivo.

[0047] Wound healing assay

[0048] like Figure 3 As shown: CE-Lalb NFs of Example 1 accelerate wound healing without leaving scars. Immunohistochemical analysis was performed on the wound tissue collected on the 15th day of the wound healing test to explore the wound healing mechanism. The results showed that the expression of CD31 was high, indicating that the formation of blood vessels was accelerated. The partial expression level of vascular endothelial growth factor (VEGF) showed that the angiogenic ability of α-lactalbumin enhanced angiogenesis. According to statistics, the partial expression level of IL-6 was reduced, indicating that the inflammatory response was alleviated. TGF-β chromosomes, which represent the strength of growth factors, were also highly expressed, promoting the formation of extracellular matrix at the wound. The expression of α-SMA is related to myofibroblasts, which play an important role in wound contraction, but excessive wound contraction can lead to wound formation. Therefore, the expression of α-SMA and α-lactalbumin is quite low, which will lead to reduced or no scar formation after wound healing. These results are consistent with the condition of the wound, indicating that the wound dressing has a super healing ability while reducing scar formation. The drug-loaded fibers based on α-lactalbumin have excellent potential in wound healing, promoting growth factor and collagen synthesis, and reducing scar formation. The overall in vivo therapeutic results showed that CE-Lalb NFs have excellent therapeutic effects on wounds infected with MRSA and E. coli.

[0049] Skin regeneration was observed by hematoxylin-eosin staining. Compared with the control group and the free drug combination (1 g CPL combined with 1 g EP), the CE-Lalb NFs of Example 1 showed good effects on collagen distribution and granulation tissue, wherein the CE-Lalb NFs of Example 1 showed a smaller width of granulation tissue, higher collagen volume and hair follicle fraction.

[0050] like Figure 4As shown, the effects of CE-Lalb NFs of Example 1 and other groups on the ability of collagen synthesis and differentiation. Compared with the free drug group (a combination of 1g EP and 1g CPL) and the blank control, the bands of collagen I and collagen III are denser. Compared with the free drug and the blank control, the relative expression of collagen I and actin is higher in the case of administration of CE-Lalb NFs and Lalb NFs; the ratio of collagen I / collagen III is relatively high; the ratio of collagen I / collagen III in the group administered with CE-Lalb NFs is relatively high, and mature collagen is present. All these data indicate that CE-Lalb NFs of Example 1 has a better healing effect on infected wounds.

[0051] The in vitro degradation of the CE-Lalb NFs of Example 1 was studied by incubating with lysozyme for 48 hours. The degradation percentage was recorded at the designated time points. Compared with the case without lysozyme, the degradation rate of CE-Lalb NFs in the case of lysozyme was higher, which was beneficial for biodegradation, and the results showed that this CE-Lalb NFs had excellent tissue regeneration properties.

[0052] Inflammatory cells were also relatively few in the CE-Lalb NFs treatment group of Example 1. Compared with the control group and the free drug group, the drug-loaded protein fibers showed better healing effects as wound dressings. Figure 4 The natural wound healing ability of α-lactalbumin was confirmed. Masson's trichrome staining further confirmed the collagen content in the healing wound, and a higher collagen distribution was observed, with mature collagen density and regular and layered morphology, indicating a normal wound healing process.

[0053] This CE-Lalb NFs has great benefits for tissue regeneration and healing of infected skin wounds. The promotion of collagen synthesis in wound tissue was confirmed after a few days, which can be verified by Western blotting (WB). This is the reason why CE-Lalb NFs have the ability to quickly heal wounds, which is related to the ability of CE-Lalb NFs to promote collagen. All these data show that the designed CE-Lalb NFs are effective in the healing of infected wounds.

[0054] In CE-Lalb NFs, there is a synergistic effect between the α-lactalbumin electrospun nanofiber carrier, cephalexin and epicatechin. α-lactalbumin has the ability to promote collagen synthesis and tissue adhesion, thereby promoting the proliferation of fibroblasts at the wound site, thereby accelerating the wound healing process.

[0055] Animal wound model test

[0056] The animal wound model was established using balb / c mice, and the experimental results showed that the wound healing rates of CE-Lalb NFs and Lalb NFs in Example 1 were significantly improved. The wound healing rate of Lalb NFs increased at a similar rate, proving that α-lactalbumin can promote fibroblast adhesion and collagen synthesis, thereby accelerating wound healing.

[0057] Cytotoxicity assay

[0058] Standard MTT assay showed that compared with free drugs and dimethyl sulfoxide, CE-Lalb NFs had no toxicity to human embryonic kidney 293 cells and mouse fibroblasts, but significantly improved cell viability.

[0059] Furthermore, CE-Lalb NFs are used as wound dressings, which can meet the needs of infectious wound diseases by providing a non-invasive and easy-to-operate treatment option.

Claims

1. A preparation method and application of α-lactalbumin electrospun nanofibers loaded with cephalexin and epicatechin, wherein the fibers are composed of the following raw materials in parts by weight: 0.5-2 parts of cephalexin, 0.5-2 parts of epicatechin, 1-3 parts of α-lactalbumin, and 4-10 parts of a mixture of ethanol and water (wherein the ethanol content is 50% by mass); The method for preparing the fiber comprises the following steps: S1: dissolving the raw material components of cephalexin, epicatechin and α-lactalbumin in a mixture of ethanol and water (wherein the ethanol content is 50%), and stirring overnight for 12 hours; S2: The above solution was loaded into a 10 mL syringe and electrospun at a feed rate of 0.3 mL / h and a high voltage of 18 KV. The syringe was equipped with a 22-gauge flat-head needle, which was connected to the positive electrode of the power supply. The electrospun nanofiber dressing was evenly collected on a collector connected to the negative electrode of the power supply, and the collector was 15 cm away from the needle tip. The fiber is used as an antibacterial wound dressing to kill bacteria.

2. The electrospun nanofiber loaded with cephalexin and epicatechin prepared according to claim 1, characterized in that: The bacteria targeted include but are not limited to multidrug-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus.

3. The fiber according to claim 1 comprises α-lactalbumin, cephalexin and epicatechin, wherein the weight ratio of cephalexin, epicatechin to α-lactalbumin is 1:1:2.

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