A CS-HA nanoparticle cross-linked collagen shield and a preparation method thereof

Abstract

The application is suitable for the technical field of collagen materials, and provides a CS-HA nanoparticle crosslinked collagen protein shield and a preparation method thereof.The application proposes that type I collagen protein extracted from bovine Achilles tendon is used as a shield preparation material, and CS-HA nanoparticles are used as a crosslinking agent after being activated by EDCM, so that multiple crosslinking sites can be provided, crosslinking efficiency is increased, and the advantages of no crosslinking agent residue, high biological safety and the like are achieved.In addition, the CS-HA nanoparticles can effectively promote the repair of corneal injury, inhibit bacteria and improve the bioavailability of delivered drugs.On this basis, the added PRAM2 plays pharmacological effects such as anti-inflammatory and antioxidant effects.The application realizes the performance improvement of the traditional collagen protein shield, plays the advantages of the collagen protein shield as a corneal bandage, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of collagen materials, and particularly relates to a CS-HA nanoparticle crosslinked collagen protein shield and a preparation method thereof. BACKGROUND

[0002] Cataract surgery and refractive surgery are the two most common surgeries in ophthalmology, although the safety of the surgery is very high, but after the surgery, there are still not less than 2% of the patients who have irregular wound healing complication patterns. In addition, corneal disease is the second most common cause of blindness worldwide, and if not properly treated, it can lead to serious corneal complications and vision loss. Reducing inflammation and promoting epithelial wound healing are crucial for scar-free corneal recovery without vision complications for postoperative and corneal disease patients.

[0003] Corneal collagen shields are developed as corneal bandage lenses and are currently suitable for postoperative and traumatic and non-traumatic corneal protection. Like standard bandage contact lenses, soft shields promote ocular healing by providing a protective barrier on the surface of the eye, but unlike bandage lenses, corneal collagen shields are absorbable. As the collagen shield gradually degrades, the released collagen helps to lubricate the eye, promote epithelial and stromal healing, neutralize collagenase and reduce corneal inflammation. Collagen shields pre-soaked with drugs can act as a corneal prodrug reservoir, resulting in higher drug concentrations, prolonged residence time and enhanced bioavailability, thereby reducing corneal epithelial toxicity and enhancing patient compliance. Therefore, frequent instillation and hospitalization time of special groups can be reduced. However, the application of collagen shields in ocular drug delivery is limited for various reasons. The main limitations include 1) low transparency, lack of visual clarity, causing discomfort to patients; 2) the use of crosslinking agents poses a safety hazard; 3) collagen shields only promote the improvement of drug bioavailability for a limited time. Therefore, how to overcome the limitations of collagen shields while achieving repair, anti-inflammatory and antibacterial effects will be of great significance to improve their clinical application and meet the growing treatment needs of patients. SUMMARY

[0004] The purpose of the present application is to provide a CS-HA nanoparticle crosslinked collagen protein shield and a preparation method thereof, which aims to solve the problems raised in the above background.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0006] A preparation method of a CS-HA nanoparticle crosslinked collagen protein shield, the preparation method comprising the following steps:

[0007] Step 1, the type I collagen solution extracted from bovine Achilles tendon, crosslinking agent EDCM (1-[3-(dimethylamino) propyl]-3-ethyl carbodiimide methiodide) and the CS-HA nanoparticle solution after ultrasonic treatment are mixed thoroughly and poured into a mold (the CS-HA nanoparticle activated by EDCM serves as a crosslinking agent, can provide multiple crosslinking sites, increase crosslinking efficiency and has the advantages of no crosslinking agent residue and high biological safety), and the sample is solidified at room temperature; the collagen and the CS-HA nanoparticle are mixed at a volume ratio of 2:1; the molar ratio of EDCM to collagen is 1:1;

[0008] Step 2, the sample is placed in a 5 mM EDCM solution for secondary crosslinking, and the crosslinking time is 24 h; after secondary crosslinking, the sample is soaked in PBS and repeatedly washed with sterile PBS to remove reaction residues.

[0009] Further, the preparation steps of the CS-HA nanoparticle are as follows:

[0010] Step a, dissolve CS (chitosan) in acetic acid solution to obtain a CS solution with a concentration of 0.5 mg / mL, adjust the pH to 5.0 by NaOH solution, and filter the solution with a 0.22 μm filter membrane; dissolve HA (hyaluronic acid) in ultrapure water to obtain a HA solution with a concentration of 0.5 mg / mL, and filter the solution with a 0.22 μm filter membrane;

[0011] Step b, place the CS solution on a magnetic stirrer, stir at a speed of 500 r / min at room temperature, the mass percentage concentration ratio of CS to HA is 4:2, slowly add an appropriate amount of HA solution to the CS solution, and continue stirring for 1 h to obtain a stable CS-HA nanoparticle solution;

[0012] Step c, centrifuge the obtained solution to collect the nanoparticles, the centrifugal rate for collecting the nanoparticles is 12000 g, the centrifugal time is 10 min, and the nanoparticles are washed with PBS and then centrifuged again, repeated for 3 times to remove free CS and HA.

[0013] A CS-HA nanoparticle crosslinked corneal collagen shield prepared by the preparation method of the CS-HA nanoparticle crosslinked collagen shield.

[0014] A PRAM2-loaded CS-HA nanoparticle crosslinked corneal collagen shield prepared by the preparation method of the CS-HA nanoparticle crosslinked collagen shield, wherein when the CS-HA nanoparticle is prepared, PRAM2 (atractylodes rhizome polysaccharide) with a concentration of 0.25 mg / mL is added to the CS solution, and the mass molar concentration ratio of CS, HA and PRAM2 is 4:2:1.

[0015] Compared with the prior art, the present application has the beneficial effects that:

[0016] 1、The CS-HA nanoparticles prepared by the present application not only can play the role of drug controlled release, but also can play the effects of bacteriostasis, lubrication and promotion of repair, and the preparation method is simple and easy to implement.

[0017] 2、In the CS-HA nanoparticle cross-linked corneal collagen protein shield loaded with PRAM2 prepared by the present application, PRAM2 is used for eye safety and plays multiple pharmacological effects in corneal repair.

[0018] 3、The CS-HA nanoparticle cross-linked collagen protein shield has good porous air permeability, can significantly improve the wearing comfort of the patient; the CS-HA nanoparticles can release the drug into the eye for a long time, improve the bioavailability of the drug, and improve the drug loading / sustained release capacity; there is no cross-linking agent residue, the in-vivo application is safe, and it is an excellent candidate for corneal injury repair materials. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is the characterization of the nanoparticles; wherein (A) is a transmission electron microscope picture of the CS-HA nanoparticles loaded with PRAM2; (B) is the particle size of the CS-HA nanoparticles and the CS-HA nanoparticles loaded with PRAM2.

[0020] Figure 2 is the preparation of the collagen protein shield; wherein (A) is the light transmittance of the CS-HA nanoparticle cross-linked collagen protein shield; (B) is the physical state before and after the cross-linking of the collagen protein.

[0021] Figure 3 is a scanning electron microscope picture of the CS-HA nanoparticle cross-linked collagen protein shield.

[0022] Figure 4 is an enzyme degradation resistance experiment of the collagen protein shield cross-linked by EDCM and EDCM combined with CS-HA nanoparticles.

[0023] Figure 5 is an in-vitro anti-inflammatory and anti-oxidation experiment of the CS-HA nanoparticle cross-linked collagen protein shield and the PRAM2 / CS-HA nanoparticle cross-linked collagen protein shield; wherein (A) and (B) are respectively the detection of the pro-inflammatory cytokines (TNF-α and IL-6) secreted by RAW 264.7 cells using ELISA; (C)-(F) are respectively the level statistics of ROS, SOD, GSH and MDA.

[0024] Figure 6 is the cell compatibility of the CS-HA nanoparticle cross-linked collagen protein shield.

[0025] Figure 7Drug release of PRAM2 loaded collagen shield crosslinked with EDCM, EDCM combined with CS-HA nanoparticles.

[0026] Figure 8 Photos of collagen shield. DETAILED DESCRIPTION

[0027] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and not used to limit the present application.

[0028] The specific implementation of the present application is described in detail below with reference to specific embodiments.

[0029] Example 1, preparation and characterization of CS-HA nanoparticles and PRAM2 loaded CS-HA nanoparticles;

[0030] Preparation of CS-HA nanoparticles: 100 mg of CS was dissolved in 200 mL of 0.1M acetic acid solution to make the concentration of CS 0.5 mg / mL, and the CS was fully dissolved by overnight stirring. The PH of CS was adjusted to 5.0 with 2M NaOH solution, and the solution was filtered with a 0.22 μm filter membrane to remove impurities. 20 mL of CS solution was taken and placed in a beaker on a magnetic stirrer to stir at a speed of 500 r / min. 10 mL of HA solution was slowly added to the CS solution under constant stirring, and the mass percentage concentration ratio was CS:HA = 4:2. At this time, the solution showed a light blue opalescence, and the nanoparticles were stabilized by continuous stirring for 1 h. After 1 h, the solution was centrifuged at high speed (10 min, 12000 g) at 4°C to collect the nanoparticles, which were washed with PBS and then centrifuged repeatedly for 3 times to remove free CS and HA.

[0031] Preparation of PRAM2 loaded CS-HA nanoparticles: PRAM2 loaded CS-HA nanoparticles were prepared by adding 50 mg of PRAM2 to the CS solution to make the concentration of PRAM2 0.25 mg / mL. 100 mg of HA was dissolved in 200 mL of ultrapure water to make the concentration of HA 0.5 mg / mL, and the solution was filtered with a 0.22 μm filter membrane to remove impurities. 20 mL of CS and PRAM2 mixed solution was taken and placed in a beaker on a magnetic stirrer to stir at a speed of 500 r / min. 10 mL of HA solution was slowly added to the mixed solution under constant stirring, and the mass percentage concentration ratio was CS:HA:PRAM2 = 4:2:1. At this time, the solution showed a light blue opalescence, and the nanoparticles were stabilized by continuous stirring for 1 h. After 1 h, the solution was centrifuged at high speed (10 min, 12000 g) at 4°C to collect the nanoparticles, which were washed with PBS and then centrifuged repeatedly for 3 times to remove free PRAM2.

[0032] The particle size, particle size distribution and zeta potential of the nanoparticles were measured by Malvern particle size analyzer, and the morphology of the nanoparticles was observed by transmission electron microscopy. Figure 1 As shown in Table 1 , the particle size of PRAM2-loaded CS-HA nanoparticles is about 170 nm, and they are evenly distributed and have a regular spherical shape.

[0033] Table 1: Nanoparticle size, size distribution, zeta potential, and drug loading

[0034]

[0035] Example 2: Preparation and characterization of CS-HA nanoparticle cross-linked corneal collagen shield;

[0036] 1) A 1.65% (w / w) solution of type I collagen extracted from bovine Achilles tendon and a sonicated CS-HA nanoparticle solution were mixed in a 2:1 volume ratio. Then, 1% (w / v) of the crosslinker EDCM was added at a molar ratio (EDCM:collagen = 1:1) and poured into a mold. The plate was stored at 4°C to remove air bubbles. The sample was then placed in a 5 mM EDCM solution for a secondary crosslinking period of 24 hours. After secondary crosslinking, the sample was soaked in PBS for 1 hour to remove the mold and repeatedly rinsed with sterile PBS to remove any reaction residue.

[0037] 2) Scanning electron microscopy (SEM);

[0038] The surface characteristics of the collagen shield were examined using SEM. The sample was mounted on an aluminum stub using double-sided tape and then electrically conductively coated with gold.

[0039] The results are as follows Figure 3 As shown, the collagen shield cross-linked by CS-HA nanoparticles can form a good structure with good porosity and good air permeability.

[0040] 3) Transparency measurement;

[0041] The collagen shield was cross-linked with EDCM and EDCM + CS-HA nanoparticles in a cuvette, and the transparency of the collagen shield was measured at 600 nm using a UV-visible spectrophotometer with distilled water as a reference.

[0042] The results are as follows Figure 2 As shown in Table 2, Figure 2 (A) shows that collagen has good light transmittance after being cross-linked with CS-HA nanoparticles, and the numbers can be clearly seen through the collagen shield. The cross-linking efficiency is high. After adding EDCM and CS-HA nanoparticles, collagen can quickly coagulate. Figure 2The results are shown in Table 2, which shows that the bovine tendon extract type I collagen shields crosslinked with EDCM have good light transmittance, and the addition of CS-HA nanoparticles for crosslinking slightly reduces the light transmittance of the shield, but does not have a significant effect. In addition, the addition of PRAM2 to the collagen shield does not affect the light transmittance.

[0043] Table 2: Light transmittance of collagen shields

[0044]

[0045] 4) Swelling ratio and mechanical properties determination;

[0046] Different collagen shields were placed in a simulated tear solution at pH 7.4 in a petri dish, kept at 32°C to simulate physiological conditions. After 12 h, when the equilibrium point was reached, the collagen shields were removed and the excess surface moisture was carefully removed with filter paper. The swollen collagen shields were re-weighed and the swelling ratio was calculated as follows:

[0047]

[0048] where W0is the initial weight of the collagen shield, W 12 is the weight of the collagen shield swollen for 12 h.

[0049] The tensile strength of the collagen shields was evaluated using a universal testing machine. The original shields were cut into 30 x 10 mm strips and fixed vertically between two clamps. They were then pulled at a speed of 2 mm / s and the force required to break the collagen shield was recorded. The tensile strength was then calculated as follows:

[0050] Tensile strength (g / mm 2 ) = collagen shield breaking force (g) / collagen shield cross-sectional area (mm 2 );

[0051] The results are shown in Table 3, which shows that the collagen shields crosslinked with CS-HA have a better crosslinking density, and as the crosslinking density increases, the polymer network becomes more hydrophobic and its water absorption capacity decreases. The increase in crosslinking density is closely related to the mechanical properties, with the breaking force and tensile strength determining the ability to resist tearing and withstand handling during treatment. The addition of CS-HA nanoparticles as a crosslinking agent significantly improves the breaking force and tensile strength of the collagen shield compared to the collagen shield crosslinked with EDCM alone. The addition of PRAM2 does not affect the swelling ratio and mechanical properties of the collagen, indicating that the addition of PRAM2 does not affect the crosslinking effect of the CS-HA nanoparticles.

[0052] Table 3: Swelling ratio and mechanical properties of collagen shields

[0053]

[0054] 6) Antifungal activity in vitro;

[0055] The agar cup diffusion method was used to test the antifungal efficacy. C. albicans was cultured on glucose agar. At the time of solidification, 1 cm holes were made and the three prepared collagen shields were added. After three days of incubation of the plates at 28°C, the concentric inhibition zones around the holes were evaluated in millimeters.

[0056] The results, shown in Table 4, show that the addition of CS-HA nanoparticles and PRAM2-loaded CS-HA nanoparticles crosslinked collagen shields have better antibacterial properties, indicating that CS-HA nanoparticles and PRAM2 endow the collagen shields with antibacterial capacity, significantly improving the antibacterial effect of the collagen shields.

[0057] Table 4: Antifungal activity of collagen shields

[0058]

[0059] 7) Anti-inflammatory and antioxidant effects in vitro;

[0060] Collagen shields were incubated with RAW264.7 macrophages to evaluate the anti-inflammatory and antioxidant properties of each component. Macrophages were stimulated with lipopolysaccharide at a concentration of 10 pg / mL for 12 h and RAW264.7 macrophages were exposed to collagen shields for 8 h. Finally, the concentration of inflammatory cytokines (TNF-a, IL-6, IL-1 b and IL-10) was quantified by ELISA. The levels of superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione (GSH) and total ROS of the cells were evaluated by commercial kits and flow cytometry analysis.

[0061] The results, shown in Table 5, indicate that collagen shields are able to inhibit the secretion of TNF-a and IL-6 in LPS-induced RAW264.7 inflammatory cells. It also inhibits the secretion of ROS, increases the levels of antioxidant enzymes SOD and GSH and reduces the levels of MDA. The addition of PRAM2 significantly improves the anti-inflammatory and antioxidant effects of the collagen shields, which will contribute to the corneal repair action of the collagen shields. Figure 5

[0062] 7) Enzymatic degradation test of collagen shields;

[0063] Collagen shields were exposed to collagenase type I (from Clostridium histolyticum) and the residual mass percentage was measured over time. The solution was changed every 8 h to maintain sufficient collagenase activity. After blotting the surface of the shield with filter paper, the samples were weighed at different time intervals. The residual mass percentage of the samples was calculated from the ratio of the initial sample weight to the weight at each time point.

[0064] The results, shown in Table 6, show that the collagen shields are degraded over time, with a residual mass percentage of 50% after 24 h.​Figure 4 As shown, the collagen shield cross-linked by CS-HA nanoparticles had a better cross-linking effect, better stability and slower degradation rate than the shield cross-linked with EDCM alone, indicating its increased resistance.

[0065] 8) In vitro cytocompatibility of collagen shield;

[0066] The cytotoxicity of collagen shields was evaluated using the MTT assay on human corneal epithelial cells (HCEC). Cells were exposed to different amounts of shields and cultured for an additional 24 hours, followed by incubation with MTT for 4 hours. The absorbance was measured at 570 nm, and cell viability was calculated using the following formula:

[0067]

[0068] The results are as follows Figure 6 As shown, the collagen shield cross-linked by EDCM and CS-HA nanoparticles has high application safety. The use of EDCM and CS-HA nanoparticles as cross-linking agents can effectively avoid the toxicity problem of traditional chemical cross-linking agent residues.

[0069] 9) In vitro release experiment of PRAM2-loaded collagen shield;

[0070] The cross-linked shielding layer was cut into equal pieces, weighed, and placed in a centrifuge tube containing 2 mL of STF. The samples were placed in a shaker maintained at 32°C and continuously shaken at 300 rpm. At set time intervals, 500 μL of liquid was removed from each tube and replaced with fresh STF. The released drug content was determined using the sulfuric acid-phenol method.

[0071] The results are as follows Figure 7 As shown, compared to the burst release of the drug (PRAM2) directly loaded into the collagen shield, the release rate of the drug loaded into CS-HA is slower. This may be mainly due to the dual sustained-release structure formed by the nanoparticles and collagen, as well as the increased cross-linking density. Overall, the collagen shield with CS-HA nanoparticles as a cross-linker has enhanced the sustained-release performance of the gel, which is beneficial for clinical application.

[0072] The above are only preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the concept of the present invention. These should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A method for preparing a CS-HA nanoparticle crosslinked corneal collagen shield, characterized by, The method comprises the following steps: Step 1, mix the type I collagen solution extracted from bovine Achilles tendon, EDCM and the CS-HA nanoparticle solution after ultrasonic treatment, and pour into a mold, and solidify the sample at room temperature; The CS-HA nanoparticles are activated by EDCM and used as a crosslinking agent; the preparation steps of the CS-HA nanoparticles are as follows: Step a, dissolve chitosan CS in an acetic acid solution, adjust the pH to 5.0 by a NaOH solution, and filter the solution by a 0.22 μm filter membrane; dissolve hyaluronic acid HA in ultrapure water, and filter the solution by a 0.22 μm filter membrane; Step b, place the CS solution on a magnetic stirrer, slowly add an appropriate amount of HA solution to the CS solution drop by drop under continuous stirring, and continuously stir for 1 h to obtain a stable CS-HA nanoparticle solution; Step c, centrifuge the obtained solution to collect the nanoparticles, and wash with PBS for 3 times; Step 2, place the sample in an EDCM solution for secondary crosslinking, and then immerse the sample in PBS, and repeatedly rinse with sterile PBS to remove reaction residues; When the CS-HA nanoparticles are prepared, 0.25 mg / mL of atractylodes rhizome polysaccharide PRAM2 is added to the CS solution.

2. The process for the preparation of CS-HA nanoparticle crosslinked corneal collagen shield according to claim 1, characterized in that, In step 1, the collagen and the CS-HA nanoparticles are mixed at a volume ratio of 2:1; and the molar ratio of EDCM to collagen is 1:

1.

3. The method of claim 1, wherein the CS-HA nanoparticle-crosslinked corneal collagen shield is prepared by the steps of: In step 2, the concentration of the EDCM solution for secondary crosslinking is 5 mM, and the crosslinking time is 24 h.

4. The process for the preparation of CS-HA nanoparticle crosslinked corneal collagen shield according to claim 1, characterized in that, In step a, the concentrations of the CS solution and the HA solution are both 0.5 mg / mL.

5. The method of claim 1, wherein the CS-HA nanoparticle crosslinked corneal collagen shield is prepared by the steps of: In step b, the magnetic stirring rate is 500 r / min, and the mass percentage concentration ratio of CS to HA is 4:

2.

6. The process for the preparation of CS-HA nanoparticle crosslinked corneal collagen shield according to claim 1, wherein, In step c, the centrifugal rate for collecting the nanoparticles is 12000 g, the centrifugal time is 10 min, and the free CS and HA are removed by centrifugation after washing with PBS for 3 times.

7. A CS-HA nanoparticle crosslinked corneal collagen shield loaded with PRAM2 prepared by the method of claim 1-6. When the CS-HA nanoparticles are prepared, the mass molar concentration ratio of CS, HA and PRAM2 is 4:2:

1. In step 1, the type I collagen solution extracted from bovine Achilles tendon, EDCM and the CS-HA nanoparticle solution after ultrasonic treatment are mixed and poured into a mold, and the sample is solidified at room temperature; The CS-HA nanoparticles are activated by EDCM and used as a crosslinking agent; the preparation steps of the CS-HA nanoparticles are as follows: Step a, dissolve chitosan CS in an acetic acid solution, adjust the pH to 5.0 by a NaOH solution, and filter the solution by a 0.22 μm filter membrane; dissolve hyaluronic acid HA in ultrapure water, and filter the solution by a 0.22 μm filter membrane; Step b, place the CS solution on a magnetic stirrer, slowly add an appropriate amount of HA solution to the CS solution drop by drop under continuous stirring, and continuously stir for 1 h to obtain a stable CS-HA nanoparticle solution; Step c, centrifuge the obtained solution to collect the nanoparticles, and wash with PBS for 3 times; Step 2, place the sample in an EDCM solution for secondary crosslinking, and then immerse the sample in PBS, and repeatedly rinse with sterile PBS to remove reaction residues; When the CS-HA nanoparticles are prepared, 0.25 mg / mL of atractylodes rhizome polysaccharide PRAM2 is added to the CS solution. In step 1, the collagen and the CS-HA nanoparticles are mixed at a volume ratio of 2:1; and the molar ratio of EDCM to collagen is 1:

1. In step 2, the concentration of the EDCM solution for secondary crosslinking is 5 mM, and the crosslinking time is 24 h. In step a, the concentrations of the CS solution and the HA solution are both 0.5 mg / mL. In step b, the magnetic stirring rate is 500 r / min, and the mass percentage concentration ratio of CS to HA is 4:

2. In step c, the centrifugal rate for collecting the nanoparticles is 12000 g, the centrifugal time is 10 min, and the free CS and HA are removed by centrifugation after washing with PBS for 3 times. When the CS-HA nanoparticles are prepared, the mass molar concentration ratio of CS, HA and PRAM2 is 4:2:1.

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