Antibiotic-loaded in-situ modified platelet-rich fibrin gel as well as preparation method and application thereof

Abstract

The invention discloses antibiotic-loaded in-situ modified platelet-rich fibrin gel as well as a preparation method and application thereof. The antibiotic-loaded in-situ modified platelet-rich fibrin gel is prepared from the following components in percentage by mass: 88 to 92 percent of PRF (platelet-rich fibrin) matrix, 5 to 8 percent of antibiotic and 3 to 4 percent of modifier, the PRF matrix comprises platelets, leukocytes and fibrin, the antibiotic is vancomycin or gentamicin, and the modifier is a zinc ion-containing compound or quaternized chitosan. The gel can be applied to preparation of chronic infection wound dressing, the antibiotic loading rate of the gel is increased, the gel has the slow release characteristic, the accumulative release within 7 days is 82.3 + / -5.6%, the burst release within 24 hours is less than 15%, the wound healing speed is increased by 40%, and the biological membrane clearance rate is 100%.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and particularly relates to an antibiotic-loaded in-situ modified platelet-rich fibrin gel, its preparation method, and its application. Background Technology

[0002] Platelet-Rich Fibrin (PRF) is a biomaterial extracted from autologous blood. It is produced by centrifuging blood to obtain a three-dimensional network structure rich in platelets, growth factors, and fibrin, primarily used to promote tissue repair, regeneration, and anti-inflammation. Its core advantages lie in its high safety and ease of preparation, requiring no added anticoagulants or exogenous substances. The preparation process involves collecting venous blood from a patient, then separating platelets, leukocytes, and fibrin through a specific centrifugation procedure to form a gel-like substance. The core components of PRF are platelets, fibrin, and leukocytes; platelets release growth factors such as PDGF and VEGF, promoting cell proliferation and angiogenesis; fibrin acts as a scaffold to support cell migration and tissue reconstruction; and leukocytes provide natural anti-inflammatory and immunomodulatory functions. PRF can be widely used in fields such as dentistry, orthopedics, and plastic surgery.

[0003] Chronic wounds are common, non-healing tissue injuries in clinical practice. They refer to wounds where, due to factors such as abnormal local microenvironment, infection, and underlying diseases, the normal orderly repair process of "inflammation-proliferation-remodeling" cannot achieve anatomical and functional healing within a reasonable timeframe after the loss of skin and subcutaneous tissue. From a clinical epidemiological perspective, the incidence of chronic wounds is increasing year by year, with over 300 million new cases worldwide annually. The elderly, diabetic patients, and patients with vascular diseases are high-risk groups—the annual incidence of diabetic foot ulcers (a common complication of diabetes) alone reaches 6%-8% of the total diabetic population, and approximately 15%-20% of diabetic foot ulcer patients eventually require amputation due to wound infection progression, placing a heavy burden on patients' families and the social healthcare system. Currently, the global cost of chronic wound-related treatment is as high as US$5-10 billion annually, with infection control, dressing changes, and complication management accounting for over 60%, highlighting the urgency and high cost of chronic wound treatment.

[0004] The key challenges in treating chronic wounds include the following aspects: (1) Chronic wounds such as diabetic foot ulcers are often accompanied by multidrug-resistant bacterial infections (such as MRSA), and biofilm formation makes it difficult for antibiotics to penetrate. (2) Traditional systemic antibiotics have low utilization rates (<15%) and are prone to causing liver and kidney toxicity; (3) Although PRF alone contains antimicrobial peptides, it is not effective against drug-resistant bacteria (inhibition rate <50%).

[0005] Therefore, there is an urgent need to develop a new technology to solve this problem. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, the present invention aims to provide an antibiotic-loaded, in-situ modified platelet-rich fibrin gel, its preparation method, and its applications. The gel of this invention exhibits improved antibiotic loading and sustained-release properties, with a cumulative release of 82.3±5.6% over 7 days, a burst release of <15% within 24 hours, a 40% faster wound healing rate, and a 100% biofilm clearance rate.

[0007] To achieve the above objectives, this application adopts the following technical solution: In a first aspect, the present invention provides an antibiotic-loaded, in-situ modified platelet-rich fibrin gel, which is composed of the following components by mass percentage: 88-92% PRF matrix, 5-8% antibiotic, and 3-4% modifier; wherein the PRF matrix contains platelets, leukocytes, and fibrin, the antibiotic is vancomycin or gentamicin, and the modifier is a zinc ion-containing compound or quaternized chitosan.

[0008] In the above technical solutions, the pore size of the fibrin network in the PRF matrix is ​​50-200 μm, and the encapsulation efficiency of the antibiotic is ≥90%.

[0009] Secondly, the present invention provides a method for preparing the above-mentioned antibiotic-loaded in-situ modified platelet-rich fibrin gel, comprising the following steps: S1, add the antibiotic and a portion of the modifier to the collected autologous whole blood, and shake to mix. S2, centrifuge at 1400-1600 rpm for 14-15 minutes, and take the middle gel layer after separation; S3, mix the intermediate gel layer with the remaining modifier and cure at a constant temperature.

[0010] In the above technical solution, in S1, the amount of antibiotic added is 1-2 mg per ml of autologous whole blood.

[0011] In the above technical solution, the mass of the modifier added in S1 is 60-70% of the total mass of the modifier, and the modifier mixed with the intermediate gel layer in S3 is the remaining modifier.

[0012] In the above technical solution, in S1, the oscillation rate is 150 rpm and the time is 2 min.

[0013] In the above technical solution, in S3, the temperature for constant temperature curing is 37℃ and the time is 20min.

[0014] Thirdly, the present invention provides the application of the above-mentioned antibiotic-loaded in-situ modified platelet-rich fibrin gel in the preparation of dressings for chronically infected wounds.

[0015] In the above technical solutions, the chronic wound includes one of the following: diabetic foot ulcer, pressure ulcer, postoperative chronic non-healing wound, and varicose ulcer.

[0016] The beneficial effects of this invention are as follows: 1. Improved drug loading rate: Antibiotic loading rate >90% (compared to 35% for traditional soaking method).

[0017] 2. Sustained-release properties: 82.3±5.6% cumulative release over 7 days, with a burst release of <15% within 24 hours.

[0018] 3. Animal experiments: Diabetic rats showed a 40% faster wound healing rate and a 100% biofilm clearance rate. Attached Figure Description

[0019] Figure 1 This is a comparison chart of the in vitro release curves for Example 2. Detailed Implementation

[0020] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims.

[0021] Unless otherwise specified, the test methods or experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are obtained from conventional commercial sources or prepared by conventional methods.

[0022] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0023] This invention provides an antibiotic-loaded, in-situ modified PRF gel, comprising, by mass percentage: PRF matrix: 88-92% (containing platelets, leukocytes, and fibrin); antibiotic: 5-8% (vancomycin or gentamicin); and modifier: 3-4% (containing zinc ion compounds or quaternized chitosan). The fibrin network pore size in the PRF matrix is ​​50-200 μm, and the antibiotic encapsulation efficiency is ≥90%. The pore size was measured using scanning electron microscopy (SEM): the gel sample was pre-frozen at -50℃ for 2 h, then freeze-dried at -30℃ under 10 Pa vacuum for 12 h, sputter-coated with gold, and the pore size was measured at 1000x magnification in 5 randomly selected fields of view, and the average value was taken.

[0024] The preparation process of the antibiotic-loaded in-situ modified PRF gel of this invention is as follows: whole blood collection → addition of antibiotics / Zn 2+ → Centrifugation and layering → Extraction of gel layer → Composite QCS molding. Specifically, it includes the following steps: S1. Add antibiotics (1-2 mg per ml of autologous whole blood) and 60-70% of the total modifier (calculated as 3-4% of the final gel mass) to the collected autologous whole blood, and shake to mix (shaking speed 150 rpm, time 2 min).

[0025] S2, centrifuge at 1400-1600 rpm for 14-15 minutes, and take the middle gel layer after separation.

[0026] S3, mix the intermediate gel layer with the remaining 30-40% by mass of the modifier, and cure at 37℃ for 20 minutes.

[0027] In step S1, the modifier is used to pre-modify platelet / fibrin precursors in the blood to enhance antibiotic binding capacity; in step S3, the modifier is used to adjust the pore size and curing speed of the gel network to ensure sustained-release performance.

[0028] This invention involves adding lyophilized antibiotic powder and a modifier directly to whole blood after venous blood collection, and then using a specific centrifugation process to enrich the drug in the PRF gel layer to form a sustained-release system.

[0029] Example 1: Preparation and application of vancomycin-loaded PRF gel 1. Application Object Information A 55-year-old patient with type II diabetes was selected. The patient had a chronic ulcer on the sole of the left foot, with an area of ​​3.0cm × 2.5cm and a depth reaching the subcutaneous tissue. Bacterial culture confirmed that the ulcer was infected with methicillin-resistant Staphylococcus aureus (MRSA). The ulcer had been non-healing for 6 weeks. The patient had previously been treated with routine iodine disinfection and sterile gauze dressing, but the ulcer showed no obvious healing trend. This meets the criteria for chronic infected ulcers for which the gel of this invention is applicable.

[0030] 2. Preoperative preparation (1) Gel preparation: Collect 10 ml of the patient's autologous venous blood and inject it into a sterile centrifuge tube; add 20 mg of vancomycin lyophilized powder (meeting the requirement of adding 2 mg of antibiotic per ml of whole blood) and 0.03 g of gel to the centrifuge tube. (60% by mass of modifier, total modifier calculated as 5% of the final gel mass of 1g, i.e., 0.05g), gently pipette five times to ensure thorough mixing; place the centrifuge tube in a horizontal centrifuge and centrifuge at 1500 rpm for 14 min to extract the middle PRF gel layer (approximately 0.95g); add the remaining 0.02g to the gel layer. (40% by mass of modifier, the total modifier is calculated as 5% of the final gel mass of 1g, i.e. 0.05g); mix and stir for 30s; pour into a circular mold with a diameter of 50mm and a thickness of 2mm, and cure in a constant temperature incubator at 37℃ for 20min to obtain a composite gel dressing (final gel mass of 1g), for later use.

[0031] (2) Wound pretreatment: Rinse the patient’s foot ulcer with physiological saline to remove purulent secretions and necrotic tissue from the surface of the wound; then disinfect the wound and the skin within 5 cm around it with 0.5% povidone-iodine solution. After disinfection, gently absorb the residual liquid on the surface of the wound with sterile gauze and expose the clean wound.

[0032] 3. Application Operation Steps (1) Open the sterile packaging, take out the prepared composite gel dressing, check that the dressing is undamaged and free of foreign matter, and then flatten it over the pretreated ulcer surface to ensure that the gel dressing is completely adhered to the wound surface without any air bubbles. The edge of the dressing should extend 0.5 cm beyond the edge of the wound surface to ensure full coverage of the wound surface.

[0033] (2) Cover the surface of the gel dressing with a layer of sterile degreased gauze, and then use an elastic bandage to wrap and fix the foot with appropriate tightness. The tightness should be such that one finger can be inserted, so as to avoid the blood circulation of the foot being too tight, or the dressing shifting if it is too loose.

[0034] (3) Inform the patient of postoperative precautions: keep the wound dressing clean and dry, avoid getting it wet or under pressure; reduce weight-bearing on the left foot and use a cane to assist walking if necessary; if there is excessive drainage from the dressing, increased pain in the wound, or an expansion of the redness and swelling, seek medical attention promptly.

[0035] 4. Postoperative observation and dressing change cycle (1) Dressing change cycle: For the first 3 days after surgery, change the gel dressing and outer gauze once a day to observe the initial healing of the wound. From the 4th day onwards, if there is no obvious exudation or infection in the wound, the dressing can be changed once every 2 days for 2 weeks.

[0036] (2) Observation indicators: Each time the dressing is changed, observe the changes in wound area, the growth of granulation tissue, the presence or absence of purulent secretions and the reduction of redness and swelling; on the 7th day of dressing change, use sterile cotton swabs to collect wound secretions for bacterial culture and detect the clearance of MRSA; on the 14th day of dressing change, measure the wound healing area and calculate the healing rate.

[0037] 5. Application Effect (1) Day 3 of dressing change: wound exudate decreased, the redness and swelling area shrank by 1 cm, fresh granulation tissue appeared at the edge, and the pain was relieved.

[0038] (2) Day 7 of dressing change: MRSA test negative, initial wound area 3.0cm × 2.5cm = 7.5 cm. At this point, the area shrinks to 5.25. (Shrinked by 30%), no purulent discharge.

[0039] (3) Day 14 after dressing change: Remaining wound area 1.875 mm The healing rate is (7.5-1.875) / 7.5×100%=75%, the depth becomes shallower, and the epithelial tissue is partially covered.

[0040] Example 2: Verification of Gel Application Effect 1. Drug loading assay (18.3±0.7mg / g gel) (1) Experimental principle High-performance liquid chromatography (HPLC) was used to determine the drug loading by measuring the concentration of vancomycin in the gel extract and combining it with the gel mass. The formula is: Drug loading (mg / g) = Total mass of vancomycin in the extract (mg) / Mass of gel sample (g).

[0041] (2) Experimental materials 1) Experimental sample: Vancomycin-loaded PRF composite gel (50 mm in diameter, 2 mm in thickness, freeze-dried to constant weight in advance) prepared according to Example 1.

[0042] 2) Reagents: Vancomycin standard (purity ≥98%), methanol (chromatographic grade), potassium dihydrogen phosphate (analytical grade), ultrapure water.

[0043] 3) Instruments: High performance liquid chromatograph (equipped with ultraviolet detector), analytical balance (accuracy 0.0001g), ultrasonic extractor, 0.22μm organic phase filter membrane, centrifuge tubes (10mL).

[0044] (3) Experimental steps 1) Sample pretreatment Weigh three portions of the lyophilized composite gel sample (approximately 0.1g each, accurate to 0.0001g, which is the dry weight after lyophilization) and place them in three 10mL centrifuge tubes respectively.

[0045] Add 5 mL of methanol-water mixture (volume ratio 3:7, pH adjusted to 3.0 with phosphoric acid beforehand) to each centrifuge tube, seal and place in an ultrasonic extractor. Set the power to 300 W and the temperature to 30 °C, and ultrasonically extract for 30 min to fully dissolve the vancomycin in the gel into the extract.

[0046] After extraction, place the centrifuge tube in a centrifuge and centrifuge at 5000 rpm for 10 min. Take the supernatant. Add 3 mL of the above mixed solution to the residue in the centrifuge tube again, repeat the ultrasonic extraction and centrifugation steps, combine the two supernatants, and dilute to a 10 mL volumetric flask and shake well.

[0047] 2) Plotting the standard curve Accurately weigh 10 mg of vancomycin standard, dissolve it in methanol-water mixture and dilute to 100 mL to obtain a standard stock solution with a concentration of 100 μg / mL.

[0048] Pipette 0.2 mL, 0.5 mL, 1.0 mL, 2.0 mL, and 5.0 mL of the standard stock solution into 10 mL volumetric flasks, and dilute to volume with the mixed solution to obtain standard working solutions with concentrations of 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 50 μg / mL.

[0049] HPLC detection conditions were set as follows: C18 column (250 mm × 4.6 mm, 5 μm), mobile phase was methanol-0.05 mol / L potassium dihydrogen phosphate solution (volume ratio 3:7, pH 3.0), flow rate was 1.0 mL / min, detection wavelength was 280 nm, column temperature was 30 ℃, and injection volume was 20 μL.

[0050] Inject standard working solutions sequentially, record peak areas, and plot a standard curve with standard working solution concentration as the abscissa (X) and peak area as the ordinate (Y), obtaining the regression equation Y=aX+b( ≥0.999).

[0051] 3) Sample testing and calculation The pretreated sample extract was filtered through a 0.22 μm organic phase filter membrane, and the filtrate was injected under the HPLC conditions described above. The peak area was recorded.

[0052] The concentration of vancomycin in the extract, c (μg / mL), was calculated based on the regression equation of the standard curve. Then, the total mass of vancomycin in the extract, m_total (mg), was calculated based on the final volume (10 mL): m_total (mg) = c (μg / mL) × 10 (mL) × .

[0053] The drug loading of each sample was calculated using the drug loading formula. The average value of the three samples was taken to obtain the final drug loading (mg / g) = total mass of vancomycin in the extract (mg) / dry weight of the lyophilized gel sample (g). The final result was 18.3 ± 0.7 mg / g (based on the dry weight of the lyophilized gel sample).

[0054] 2. Inhibition zone assay (MRSA: 28.5±1.2mm; PRF alone: ​​8.4±0.5mm) (1) Experimental principle Using the Kirby-Bauer disk diffusion method, gel samples containing antibacterial components were placed on agar medium inoculated with methicillin-resistant Staphylococcus aureus (MRSA). The antibiotics in the gel diffused into the surrounding agar, inhibiting bacterial growth and forming a transparent inhibition zone. The antibacterial activity of the gel was directly reflected by measuring the diameter of the inhibition zone; the larger the diameter, the stronger the antibacterial effect.

[0055] (2) Experimental materials 1) Experimental samples: ① Vancomycin-loaded PRF composite gel (prepared according to Example 1, cut into 6mm diameter circular slices, and sterilized); ② PRF single-use gel (prepared without vancomycin, containing only PRF matrix + quaternized chitosan, cut into 6mm circular slices of the same size, and sterilized).

[0056] 2) Bacterial strain: Methicillin-resistant Staphylococcus aureus (MRSA, standard strain ATCC 43300), activated and cultured in LB medium to the logarithmic growth phase (bacterial concentration adjusted to 1×). (CFU / mL).

[0057] 3) Reagents: LB agar medium (solid), sterile physiological saline.

[0058] 4) Instruments: sterile culture dishes (90mm in diameter), sterile pipettes (1mL), sterile spreaders, vernier calipers (0.01mm accuracy), and constant temperature incubator (37℃).

[0059] (3) Experimental steps 1) Culture medium preparation and inoculation Weigh out LB agar medium powder, add ultrapure water according to the instructions, heat to dissolve, dispense into Erlenmeyer flasks, and autoclave at 121°C for 20 minutes.

[0060] After the sterilized agar medium has cooled to 50-60℃, pour about 20mL into each sterile petri dish and place it horizontally to solidify, thus making LB agar plates.

[0061] Use a sterile pipette to draw 1 mL of MRSA bacterial suspension with the adjusted concentration and drop it into the center of an agar plate. Use a sterile spreader to spread the bacterial suspension evenly over the entire surface of the plate. Place the plate in a clean bench and let it sit at room temperature for 15 minutes to allow the bacterial suspension to be fully absorbed.

[0062] 2) Sample placement and incubation In a clean bench, use sterile forceps to place vancomycin-loaded PRF composite gel sheets (experimental group) and PRF-only gel sheets (control group) in the center of two agar plates inoculated with MRSA, ensuring that the gel sheets are in close contact with the agar surface and avoiding the formation of air bubbles.

[0063] Three parallel plates were set up for each group. All plates were placed upside down in a 37°C constant temperature incubator and incubated for 24 hours.

[0064] 3) Measurement and statistical analysis of inhibition zones After the incubation is complete, remove the plate and use a vernier caliper to measure the diameter of the inhibition zone in different directions (at least 3 vertical directions) (the measurement range is the distance from the edge of the gel sheet to the edge of the inhibition zone, accurate to 0.1 mm).

[0065] The average diameter of the inhibition zone on the three plates in each group was calculated: the diameter of the inhibition zone in the experimental group (vancomycin-loaded PRF gel) was 28.5±1.2 mm, and the diameter of the inhibition zone in the control group (PRF gel alone) was 8.4±0.5 mm. This verifies that the drug-loaded gel has a significantly better inhibitory effect on MRSA than the PRF gel alone.

[0066] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An antibiotic-loaded in situ modified platelet-rich fibrin gel, characterized in that: The PRF matrix comprises platelets, leukocytes, and fibrin; the antibiotic is vancomycin or gentamicin; and the modifier is a zinc ion-containing compound or a quaternary ammonium chitosan.

2. The antibiotic-modified in situ platelet-rich fibrin gel according to claim 1, wherein: The fibrin network in the PRF matrix has a pore size of 50-200 μm, and the antibiotic has an encapsulation rate of ≥90%.

3. The method of claim 1, wherein the antibiotic-loaded in situ modified platelet-rich fibrin gel is prepared by the steps of: a) mixing a fibrinogen solution with a thrombin solution to form a fibrin gel; b) adding an antibiotic to the fibrin gel; and c) allowing the antibiotic-loaded fibrin gel to set. The method comprises the following steps: S1. Adding the antibiotic and a portion of the modifier to the collected autologous whole blood, and mixing uniformly by oscillation; S2. Centrifuging at 1400-1600 rpm for 14-15 minutes, and taking the intermediate gel layer after separation; S3. Mixing the intermediate gel layer with the remaining modifier, and constant-temperature solidification.

4. The method of claim 3, wherein: In S1, the antibiotic is added in an amount of 1-2 mg per ml of autologous whole blood.

5. The method of claim 3, wherein: In S1, the mass of the modifier added is 60-70% of the total mass of the modifier, and the modifier mixed with the intermediate gel layer in S3 is the remaining modifier.

6. The method of claim 3, wherein: In S1, the oscillation rate is 150 rpm, and the time is 2 min.

7. The method of claim 3, wherein: In S3, the constant-temperature solidification temperature is 37°C, and the time is 20 min.

8. Use of the antibiotic-loaded in-situ modified PRF gel of claim 1 in the preparation of a chronic infection wound dressing.

9. Use according to claim 8, characterized in that: The chronic wound comprises one of a diabetic foot ulcer, a pressure sore, a postoperative chronic non-healing wound, and a venous ulcer. The chronic wound comprises one of a diabetic foot ulcer, a pressure sore, a postoperative chronic non-healing wound, and a venous ulcer.

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