Extraction process of jellyfish extract and preparation process of drug-loaded collagen microneedle preparation

Abstract

The invention belongs to the technical field of extraction and utilization of collagen, and particularly relates to an extraction process of a jellyfish extract and a preparation process of a drug-loaded collagen microneedle preparation. The extraction process of the jellyfish extract comprises the following steps: cutting jellyfish into strips, removing salt, removing impure protein, removing fat, removing pigment, homogenizing, continuing enzymolysis, salting out, dialyzing and drying to obtain the enzyme-soluble collagen. The preparation process of the drug-loaded collagen microneedle preparation comprises the following steps: dissolving soluble collagen in water, preparing precipitate after centrifugation into a jellyfish collagen gel solution, mixing the jellyfish collagen gel solution with a drug solution, and adding the mixture into a microneedle mold to obtain the drug-loaded collagen microneedle. The extraction process of the jellyfish extract, provided by the invention, is low in extraction cost, simple to operate and high in extraction rate, and the extracted enzyme-soluble collagen is high in purity; the extracted enzyme-soluble collagen is used for preparing a drug-loaded collagen microneedle preparation, and has the advantages of good biocompatibility and strong drug-loading capability.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction and utilization of collagen, and specifically relates to an extraction process of a jellyfish extract and a preparation process of a drug-loaded collagen microneedle preparation. Background Art

[0002] Jellyfish collagen is a bioactive ingredient extracted from marine jellyfish, with good hydrophilicity and biocompatibility. It can effectively lock in moisture, promote skin moisturizing, whitening, enhance elasticity, and fight aging. In addition, jellyfish collagen maintains a good triple helix structure during the extraction process, which makes its absorption and action in the skin more stable.

[0003] Microneedle technology stimulates the skin's self-repair mechanism by creating tiny puncture channels on the skin, thereby promoting the production of collagen and elastic fibers. This technology is often used in the field of beauty to improve skin texture, reduce wrinkles and enhance skin elasticity. Combining jellyfish collagen with microneedle technology can deliver jellyfish collagen directly to the dermis of the skin, thereby enhancing its effectiveness. This method not only provides deep nourishment, but also promotes the regeneration of collagen through mechanical stimulation, making the skin firmer and smoother. It has the characteristics of high efficiency, high safety, strong durability, good biocompatibility, abundant resources and extraction convenience, antioxidant and anti-aging. However, the current microneedle technology still has the disadvantages of difficult operation, long recovery period, difficult care, and high cycle frequency.

[0004] At present, the traditional collagen extraction methods include acid extraction, alkaline extraction, enzymatic extraction, neutral salt extraction, and hot water extraction, but each has its limitations. Although the acid extraction can extract a complete triple helix structure, the extraction rate is low; alkaline extraction can also retain a relatively complete structure, but it is currently mainly used in fish meat extraction, but the collagen extraction rate obtained is still low; enzymatic extraction is specific, and most of the extraction is carried out by combining with acid, and it has resistance to proteases other than collagenase; although the neutral salt extraction and hot water extraction are relatively simple and convenient to operate, the extraction rate still needs to be improved.

[0005] CN119235742A discloses an extraction process of jellyfish extract, which is prepared through the steps of pre-treatment cleaning, desalting, acid-base treatment, homogenization, enzymolysis, centrifugation, etc., and ultrafiltration concentration. The supernatant obtained by the pre-treatment is separated and the concentrated liquid is retained; the concentrated liquid is freeze-dried to obtain a jellyfish extract solid product rich in collagen and mucin. The use of ultrafiltration concentration technology in the protein purification process will cause thermal denaturation loss of the protein, and since macromolecules such as proteins are easily deposited on the surface of the filter membrane, the membrane pores are gradually blocked, and the membrane needs to be frequently cleaned or replaced, which increases the maintenance cost.

[0006] CN119183373A discloses a collagen microneedle preparation, which comprises the steps of preparing a microneedle raw material solution having a collagen content of 5% by mass or less in the aqueous solution by modulating a raw material containing collagen and one or more pH adjusters at a temperature below 37°C, adjusting the pH to 3-9, and filling the microneedle raw material solution into a casting mold and drying the mold. When other solvents are added, the collagen solution that has not been centrifuged will show stratification, that is, the protein and other components are unevenly distributed in the solution, thereby further increasing the manufacturing cost of the microneedles, reducing the drug loading capacity, and causing poor biocompatibility. Summary of the invention

[0007] The technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art and provide an extraction process for jellyfish extract, which has low extraction cost, simple operation, high extraction rate, and high purity of the extracted enzyme-soluble collagen; the extracted enzyme-soluble collagen is used to prepare a drug-loaded collagen microneedle preparation, which has good biocompatibility and strong drug loading capacity.

[0008] The extraction process of the jellyfish extract of the present invention comprises the following steps: 1) Take the pickled jellyfish, clean it and separate the cap tissue; 2) Soak the jellyfish cap tissue in pure water, changing the liquid several times, with the liquid volume ≥ 40 times the mass of the jellyfish; 3) Soak in NaOH solution for 4 to 6 days, changing the solution several times during the period, with the liquid volume ≥ 40 times the mass of the jellyfish. After the soaking, wash with pure water; 4) Place H 2 O 2 Soak in solution for 6-24 hours, with the amount of liquid ≥40 times the mass of the jellyfish. After soaking, rinse with pure water; 5) Soak in isopropanol for 6-24 hours, with the amount of liquid ≥40 times the mass of the jellyfish. After soaking, rinse with pure water; 6) Weigh and homogenize, add 3.5~7.0wt% pepsin, solid-liquid ratio 1:8~1:15, react for 6~24h; 7) Filter with 300~500 mesh filter; 8) Take the supernatant, centrifuge, collect the supernatant and place it in a 3000~5000Da dialysis bag. 2 HPO 4 The solution was dialyzed for 1 to 6 days, with multiple changes of liquid, and the liquid volume was ≥ 40 times the mass of the jellyfish; 9) Take the gel in the dialysis bag, centrifuge it, take the precipitate and redissolve it in 2.5~5mol / L acetic acid, add NaCl to a concentration of 3.5~6.0mol / L, and salt out for 2~5 days; 10) Centrifuge again, take the precipitate, redissolve it in 4.5-7.5 mol / L acetic acid, place it in a 3000-5000Da dialysis bag, dialyze for 1-3 days, change the liquid several times, and the liquid volume should be ≥40 times the mass of the jellyfish; 11) Dialyze in deionized water for 2 to 7 days, change the liquid several times, the liquid volume should be ≥ 40 times the mass of jellyfish, freeze-dry at -20℃~-80℃ to obtain enzyme-soluble collagen.

[0009] The concentration of the sodium hydroxide solution in step 3) is 1.0 mol / L to 4.5 mol / L.

[0010] Step 4) H 2 O 2 The mass concentration of the solution is 4.5%~9%.

[0011] Step 8) The centrifugal speed is 4000-8000 rpm, the centrifugal time is 60-90 min, and the Na 2 HPO 4 The solution concentration is 2.4~7.2mol / L.

[0012] The centrifugal speed in step 9) is 4000-8000 rpm, and the centrifugal time is 30-90 min.

[0013] The speed of the second centrifugation in step 10) is 3000-8000 rpm, and the centrifugation time is 60-90 min.

[0014] The preparation process of the drug-loaded collagen microneedle preparation specifically comprises the following steps: 1) Dissolve 80-500 mg of extracted enzyme-soluble collagen in 10-50 mL of ultrapure water, vortex and centrifuge horizontally; 2) After the centrifugation of step 1), ultrapure water was added to the precipitate to prepare a 100 μg / mL to 500 μg / mL jellyfish collagen solution; 3) Prepare 1 mL of jellyfish collagen solution and 500 μL of 100 μg / mL~500 μg / mL drug solution, mix them, add them into the microneedle mold, vacuum filter, dry and shape them to obtain drug-loaded collagen microneedles.

[0015] The centrifugal speed in step 1) is 3000-7000 rpm, and the centrifugal time is 10-30 min.

[0016] Step 3) includes the drugs MD13, IB-367, tranexamic acid, vitamin E or vitamin C.

[0017] The vacuum filtration time of step 3) is 10 to 30 minutes.

[0018] Specifically, the extraction process of the jellyfish extract comprises the following steps: 1) Take the yellow pickled jellyfish, clean it and separate the cap tissue; 2) Trim the jellyfish umbrella tissue into strips of approximately 2 cm in length, width and height by 1 cm and 0.5 cm, and soak them in pure water for 3 days to remove salt. During this period, change the liquid several times, every 8 hours, and the liquid volume should be ≥40 times the mass of the jellyfish; 3) Soak in 1.0~4.5mol / L NaOH solution for 4~6 days to remove impurities and proteins. Change the solution several times every 8 hours. The liquid volume should be ≥40 times the weight of the jellyfish. After soaking, wash with pure water 3 times. 4) Place at 4.5~9wt%H 2 O 2 Soak in solution for 6-24 hours, with the amount of liquid ≥40 times the mass of the jellyfish. After soaking, rinse with pure water three times; 5) Soak in isopropanol for 6-24 hours to remove fat. The amount of liquid should be ≥40 times the mass of the jellyfish. After soaking, rinse with pure water 3 times. 6) After draining the water, weigh and homogenize, add 3.5-7.0wt% pepsin, the solid-liquid ratio is 1:8-1:15, and react for 6-24h; 7) Filter with 300~500 mesh filter; 8) Take the supernatant, centrifuge for 60-90 min at 4000-8000 rpm, collect the supernatant and place it in a 3000-5000 Da dialysis bag with 2.4-7.2 mol / L Na 2 HPO 4 The solution was dialyzed for 1 to 6 days, with multiple changes of solution every 8 hours, and the liquid volume was ≥ 40 times the mass of the jellyfish; 9) Take the gel in the dialysis bag and centrifuge it at 4000-8000 rpm for 30-90 min. Take the precipitate and redissolve it in 2.5-5 mol / L acetic acid. Add NaCl to a concentration of 3.5-6.0 mol / L and salt out for 2-5 days. 10) Centrifuge again at a speed of 3000-8000rpm for 60-90min, take the precipitate, redissolve it in 4.5-7.5mol / L acetic acid, place it in a 3000-5000Da dialysis bag, and dialyze for 1-3 days, changing the liquid several times, every 12h, with the liquid volume ≥40 times the mass of the jellyfish; 11) Dialyze in deionized water for 2 to 7 days, change the liquid several times, every 12 hours, the liquid volume should be ≥ 40 times the mass of jellyfish, freeze-dry at -20℃~-80℃ to obtain white enzyme-soluble collagen (PSC).

[0019] Specifically, the preparation process of the drug-loaded collagen microneedle preparation comprises the following steps: 1) Dissolve 80-500 mg of the enzyme-soluble collagen prepared in the above steps in 10-50 mL of ultrapure water, vortex to fully dissolve it, and centrifuge it at 3000-7000 rpm in a horizontal centrifuge for 10-30 min; remove the bubbles in the collagen solution.

[0020] 2) After the centrifugation of step 1), ultrapure water is added to the precipitate to prepare a jellyfish collagen solution with a mass concentration of 100 μg / mL to 500 μg / mL; 3) Take 1 mL of the jellyfish collagen solution of step 2) and 500 μL of 100 μg / mL~500 μg / mL of drugs IB-367, MD13, drug tranexamic acid, vitamin E or vitamin C, mix them, and drop them into the PDMS mold. Vacuum filter for 10~30 minutes, remove the bubbles and repeat the process until the bubbles in the mold are completely removed; then place it in a 37°C oven to dry and concentrate, replenish the solution every 5-6 hours until the temperature is lowered after molding and continuous solidification is achieved, take the prepared microneedles, cut off the needle-free area on the edge, and note that the steps of taking the molded microneedles should be slow to prevent damage to the overall structure of the needle tip, and obtain drug-loaded collagen microneedles.

[0021] The present invention combines jellyfish collagen with microneedle technology to deliver jellyfish collagen directly to the dermis of the skin, thereby improving its effect. This method can not only provide deep nourishment, but also promote the regeneration of collagen through mechanical stimulation, making the skin tighter and smoother. In addition, collagen microneedles can also carry other drugs for the treatment of other diseases.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) The extraction process of the jellyfish extract of the present invention has low cost, simple operation, high extraction rate, and high protein purity of the extracted collagen.

[0023] (2) The preparation process of the drug-loaded collagen microneedle preparation of the present invention is to prepare the drug-loaded collagen microneedle preparation by compatibility of the enzyme-soluble collagen extracted by the present invention with the corresponding drug, which has a high drug loading rate and good compatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the SDS-PAGE gel electrophoresis diagram of enzyme-soluble collagen of different concentrations prepared in Example 1.

[0025] Figure 2 This is a comparison chart of ultraviolet spectrum scanning of the enzyme-soluble collagen prepared in Example 1 and fish collagen.

[0026] Figure 3 This is the SEM image of the enzyme-soluble collagen prepared in Example 1.

[0027] Figure 4These are SEM images of the drug-loaded collagen microneedle preparation prepared in Example 5 at different magnifications.

[0028] Figure 5 This is a graph showing the results of a hemolysis experiment on the drug-loaded collagen microneedle preparation prepared in Example 5.

[0029] Figure 6 This is a graph showing the results of a cytotoxicity experiment on the drug-loaded collagen microneedle preparation prepared in Example 6. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific embodiments.

[0031] The pickled jellyfish used in the following examples and comparative examples are commercially available from Qingdao fishermen, and the collagen content is 90%-95%. The entire process of each embodiment of the present invention is kept in a 4°C environment and continuously stirred with a magnetic stirrer. The soaking described in the present invention is complete immersion.

[0032] The PDMS mold used in the present invention is a polydimethylsiloxane (PDMS) microneedle mold with a needle length of 350-750 μm, an array of 15×15 needles, and a size of 13.5×13.5 mm.

[0033] The sources of the chemicals used below are as follows: NaOH (AR): Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.; Acetic acid: Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.; Pepsin: Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.; Isopropyl alcohol: Shanghai Aladdin Biochemical Technology Co., Ltd.; H 2 O 2 Solution: Chongqing Chuandong Chemical Group Co., Ltd.; Na 2 HPO 4 :Shanghai Aladdin Biochemical Technology Co., Ltd.; Drug MD13: MedChemExpress (MCE) biological reagent company; Tranexamic acid: Shanghai MacLean Biochemical Technology Co., Ltd.; Vitamin E: Shanghai MacLean Biochemical Technology Co., Ltd.; Vitamin C: Shanghai MacLean Biochemical Technology Co., Ltd.; Fish collagen: Shanghai Aladdin Biochemical Technology Co., Ltd.; IB-367 antimicrobial peptide: synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0034] Example 1 The extraction process of the jellyfish extract comprises the following steps: 1) Take the yellow pickled jellyfish, clean it and separate the cap tissue; 2) Trim the jellyfish umbrella tissue into strips of approximately 2 cm in length, width and height by 1 cm and 0.5 cm, and soak them in pure water for 3 days to remove salt. During this period, change the liquid several times, every 8 hours, and the liquid volume should be ≥40 times the mass of the jellyfish; 3) Soak in 2.0 mol / L NaOH solution for 5 days to remove impurities and proteins. Change the solution several times every 8 hours. The liquid volume should be ≥ 40 times the weight of the jellyfish. After soaking, wash with pure water 3 times. 4) Place in 6wt%H 2 O 2 Soak in the solution for 8 hours, with the liquid volume ≥40 times the mass of the jellyfish. After soaking, rinse with pure water 3 times; 5) Soak in isopropanol for 12 hours to remove fat. The liquid volume should be ≥ 40 times the mass of the jellyfish. After soaking, rinse with pure water 3 times; 6) After draining the water, weigh and homogenize, add 5.0wt% pepsin, the solid-liquid ratio is 1:10, and react for 12h; 7) Filter with 400 mesh filter; 8) Take the supernatant, centrifuge for 70 minutes at 6000 rpm, collect the supernatant and place it in a 4000 Da dialysis bag with 5.0 mol / L Na 2 HPO 4 The solution was dialyzed for 3 days, with multiple changes of solution every 8 hours, and the liquid volume was ≥40 times the mass of the jellyfish; 9) Take the gel in the dialysis bag and centrifuge it at 6000 rpm for 60 min. Take the precipitate and redissolve it in 3.5 mol / L acetic acid. Add NaCl to a concentration of 5.0 mol / L and salt it out for 3 days. 10) Centrifuge again at 5000 rpm for 70 min, take the precipitate, redissolve it in 5.5 mol / L acetic acid, place it in a 4000 Da dialysis bag, and dialyze for 2 days, changing the liquid several times, every 12 hours, with the liquid volume ≥ 40 times the mass of the jellyfish; 11) Dialyze in deionized water for 3 days, change the liquid several times, every 12 hours, the liquid volume should be ≥ 40 times the mass of jellyfish, freeze-dry at -20℃ to obtain white enzyme-soluble collagen (PSC).

[0035] The white enzyme-soluble collagen prepared above was added with pure water to prepare samples with concentrations of 4 mg / mL, 2 mg / mL, and 1 mg / L, respectively. The SDS-PAGE gel electrophoresis diagrams of the samples are shown in FIG. Figure 1 As shown; Figure 1M is a protein marker; 1, 2, and 3 are enzyme-soluble collagens with concentrations of 4 mg / mL, 2 mg / mL, and 1 mg / mL, respectively. Figure 1 It can be seen that the enzyme-soluble collagen of jellyfish extracted by the present invention has high protein purity.

[0036] The white enzyme-soluble collagen prepared above was compared with the commercially available fish collagen by UV spectroscopy, and the comparison chart is as follows: Figure 2 As shown in the figure, A is the ultraviolet spectrum scanning diagram of the enzyme-soluble collagen prepared above, and B is the ultraviolet spectrum scanning diagram of fish collagen. Figure 2 It can be seen that the enzyme-soluble collagen prepared by the present invention is the same as fish collagen.

[0037] The SEM image of the white enzyme-soluble collagen prepared above is as follows: Figure 3 As shown by Figure 3 It can be seen that the uneven and larger pore structure inside the extracted jellyfish enzyme-soluble collagen under the electron microscope is conducive to the attachment of drugs and can support the transport of large molecule drugs.

[0038] Example 2 The extraction process of the jellyfish extract comprises the following steps: 1) Take the yellow pickled jellyfish, clean it and separate the cap tissue; 2) Trim the jellyfish umbrella tissue into strips of approximately 2 cm in length, width and height by 1 cm and 0.5 cm, and soak them in pure water for 3 days to remove salt. During this period, change the liquid several times, every 8 hours, and the liquid volume should be ≥40 times the mass of the jellyfish; 3) Soak in 1.0 mol / L NaOH solution for 6 days to remove impurities and proteins. Change the solution several times every 8 hours. The liquid volume should be ≥ 40 times the weight of the jellyfish. After soaking, wash with pure water 3 times. 4) Place at 4.5wt%H 2 O 2 Soak in solution for 24 hours, with the amount of liquid ≥ 40 times the mass of the jellyfish. After soaking, rinse with pure water three times; 5) Soak in isopropanol for 6 hours to remove fat. The liquid volume should be ≥ 40 times the mass of the jellyfish. After soaking, rinse with pure water 3 times; 6) After draining the water, weigh and homogenize, add 3.5wt% pepsin, the solid-liquid ratio is 1:8, and react for 24h; 7) Filter with 300 mesh filter; 8) Take the supernatant, centrifuge for 60 min at 4000 rpm, collect the supernatant and place it in a 3000 Da dialysis bag with 2.4 mol / L Na 2 HPO 4The solution was dialyzed for 6 days, with multiple changes of solution every 8 hours, and the liquid volume was ≥40 times the mass of the jellyfish; 9) Take the gel in the dialysis bag and centrifuge it at 4000 rpm for 90 min. Take the precipitate and redissolve it in 2.5 mol / L acetic acid. Add NaCl to a concentration of 3.5 mol / L and salt out for 5 days. 10) Centrifuge again at 3000 rpm for 90 min, take the precipitate, redissolve it in 4.5 mol / L acetic acid, place it in a 3000 Da dialysis bag, and dialyze for 1 day, changing the liquid several times, every 12 hours, with the liquid volume ≥ 40 times the mass of the jellyfish; 11) Dialyze in deionized water for 2 days, change the liquid several times, every 12 hours, the liquid volume should be ≥ 40 times the mass of jellyfish, freeze-dry at -80℃ to obtain white enzyme-soluble collagen (PSC).

[0039] Example 3 The extraction process of the jellyfish extract comprises the following steps: 1) Take the yellow pickled jellyfish, clean it and separate the cap tissue; 2) Trim the jellyfish umbrella tissue into strips of approximately 2 cm in length, width and height by 1 cm and 0.5 cm, and soak them in pure water for 3 days to remove salt. During this period, change the liquid several times, every 8 hours, and the liquid volume should be ≥40 times the mass of the jellyfish; 3) Soak in 4.5 mol / L NaOH solution for 4 days to remove impurities and proteins. Change the solution several times every 8 hours. The liquid volume should be ≥ 40 times the weight of the jellyfish. After soaking, wash with pure water 3 times. 4) Place in 9wt%H 2 O 2 Soak in the solution for 6 hours, with the liquid volume ≥40 times the mass of the jellyfish. After soaking, rinse with pure water 3 times; 5) Soak in isopropanol for 24 hours to remove fat. The liquid volume should be ≥ 40 times the mass of the jellyfish. After soaking, rinse with pure water 3 times; 6) After draining the water, weigh and homogenize, add 7.0wt% pepsin, the solid-liquid ratio is 1:15, and react for 6h; 7) Filter with 500 mesh filter; 8) Take the supernatant, centrifuge for 90 minutes at 8000 rpm, collect the supernatant and place it in a 5000 Da dialysis bag with 7.2 mol / L Na 2 HPO 4 The solution was dialyzed for 1 day, with multiple changes of solution every 8 hours, and the liquid volume was ≥40 times the mass of the jellyfish; 9) Take the gel in the dialysis bag and centrifuge it at 8000 rpm for 30 min. Take the precipitate and redissolve it in 5 mol / L acetic acid. Add NaCl to a concentration of 6.0 mol / L and salt it out for 2 days. 10) Centrifuge again at 8000 rpm for 60 min, take the precipitate, redissolve it in 7.5 mol / L acetic acid, place it in a 5000 Da dialysis bag, and dialyze for 3 days, changing the liquid several times, every 12 hours, with the liquid volume ≥40 times the mass of the jellyfish; 11) Dialyze in deionized water for 7 days, change the liquid several times, every 12 hours, the liquid volume should be ≥ 40 times the mass of jellyfish, freeze-dry at -30℃ to obtain white enzyme-soluble collagen (PSC).

[0040] Example 4 The extraction process of the jellyfish extract comprises the following steps: 1) Take the yellow pickled jellyfish, clean it and separate the cap tissue; 2) Trim the jellyfish umbrella tissue into strips of approximately 2 cm in length, width and height by 1 cm and 0.5 cm, and soak them in pure water for 3 days to remove salt. During this period, change the liquid several times, every 8 hours, and the liquid volume should be ≥40 times the mass of the jellyfish; 3) Soak in 2.5 mol / L NaOH solution for 5 days to remove impurities and proteins. Change the solution several times every 8 hours. The liquid volume should be ≥ 40 times the weight of the jellyfish. After soaking, wash with pure water 3 times. 4) Place in 6wt%H 2 O 2 Soak in the solution for 8 hours, with the liquid volume ≥40 times the mass of the jellyfish. After soaking, rinse with pure water 3 times; 5) Soak in isopropanol for 10 hours to remove fat. The liquid volume should be ≥ 40 times the mass of the jellyfish. After soaking, rinse with pure water 3 times. 6) After draining the water, weigh and homogenize, add 5.0wt% pepsin, the solid-liquid ratio is 1:12, and react for 10h; 7) Filter with 300 mesh filter; 8) Take the supernatant, centrifuge for 80 min at 5000 rpm, collect the supernatant and place it in a 3500 Da dialysis bag with 4.0 mol / L Na 2 HPO 4 The solution was dialyzed for 3 days, with multiple changes of solution every 8 hours, and the liquid volume was ≥40 times the mass of the jellyfish; 9) Take the gel in the dialysis bag and centrifuge it at 5000 rpm for 40 min. Take the precipitate and redissolve it in 4.0 mol / L acetic acid. Add NaCl to a concentration of 5.0 mol / L and salt out for 4 days. 10) Centrifuge again at 5000 rpm for 70 min, take the precipitate, redissolve it in 6.0 mol / L acetic acid, place it in a 3500 Da dialysis bag, and dialyze for 2 days, changing the liquid several times, every 12 hours, with the liquid volume ≥40 times the mass of the jellyfish; 11) Dialyze in deionized water for 3 days, change the liquid several times, every 12 hours, the liquid volume should be ≥ 40 times the mass of jellyfish, freeze-dry at -20℃ to obtain white enzyme-soluble collagen (PSC).

[0041] Example 5 The preparation process of the drug-loaded collagen microneedle preparation specifically comprises the following steps: 1) Dissolve 200 mg of the enzyme-soluble collagen prepared in Example 1 in 30 mL of ultrapure water, vortex to fully dissolve it, and centrifuge at 5000 rpm for 20 min to remove bubbles in the collagen solution.

[0042] 2) After the centrifugation of step 1), ultrapure water was added to the precipitate to prepare a jellyfish collagen solution with a mass concentration of 300 μg / mL.

[0043] 3) Take 1 mL of the jellyfish collagen solution from step 2) and 500 μL of 300 μg / mL drug MD13, mix them, and drop them into the PDMS mold. Vacuum filter for 20 minutes, remove the bubbles and repeat the process until the bubbles in the mold are completely removed; then place it in a 37°C oven to dry and concentrate, replenish the solution every 5 hours until the temperature is lowered after molding and it continues to solidify, take the prepared microneedles, cut off the needle-free area on the edge, and note that the step of taking the molded microneedles should be slow to prevent damage to the overall structure of the needle tip, and obtain the collagen microneedle preparation loaded with MD13.

[0044] The SEM image of the MD13-loaded collagen microneedle preparation prepared above is as follows: Figure 4 As shown, Figure 4 The left image is magnified 110 times, and the right image is magnified 150 times. Figure 4 It can be seen that the tip of the collagen microneedle presented in the SEM image of the jellyfish collagen microneedle under the electron microscope is sharp, without obvious fractures, which is more conducive to piercing the surface of the skin for drug treatment.

[0045] The hemolysis experimental data of the collagen microneedle preparation loaded with MD13 prepared above is shown in the figure. Figure 5 As shown by Figure 5 It can be seen that the hemolysis rate of the collagen microneedle preparation immersion solution is less than 5%, indicating that the product has very little destructive effect on red blood cells. When the product is used for related experiments or treatments, the degree of red blood cell destruction is low, thereby reducing the adverse reactions or side effects caused.

[0046] The hemolysis experimental data of the collagen microneedle preparation loaded with MD13 prepared above is as follows: the drug-loaded collagen microneedle preparation prepared in Example 5 was added to a complete culture medium to dissolve into a 200 μg / mL sample solution, and then diluted into samples of different concentrations of 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL, and the hemolysis rates of the samples of different concentrations were measured, with the sample concentration as the horizontal axis and the measured hemolysis rates as the vertical axis, as shown in FIG. Figure 5 shown.

[0047] Hemolysis rate refers to the degree of rupture or dissolution of red blood cells, usually expressed as a percentage. It shows the hemolytic activity of different drugs on red blood cells. The higher the hemolysis rate, the more serious the rupture of red blood cells.

[0048] Depend on Figure 5 It can be seen that the hemolysis rates of the drug-loaded collagen microneedle preparations of different concentrations prepared by the present invention are all less than 5%, indicating that the drug-loaded collagen microneedle preparation has very little destructive effect on red blood cells under experimental conditions. When the preparation is used for relevant experiments or treatments, the degree of red blood cell destruction is low, thereby reducing the adverse reactions or side effects caused.

[0049] The steps for detecting the hemolysis rate are as follows: (1) Preparation of red blood cell suspension: First, collect fresh blood from the orbital cavity of male C57bl / 6 mice into an anticoagulant tube. Then, centrifuge the blood to remove the supernatant and retain the blood cell pellet.

[0050] (2) Diluting the red blood cell suspension: dilute the above blood cell sediment with PBS to a concentration of 5wt% to obtain a diluted red blood cell suspension.

[0051] (3) The drug-loaded microneedle solutions prepared above with concentrations of 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL were respectively taken as sample groups, and physiological saline was used as a negative control group, and distilled water was used as a positive control group, and mixed with equal volumes of the diluted red blood cell suspension to obtain a mixture; (4) Incubation: Incubate the mixture at 37°C for 30 min to observe the occurrence of hemolysis.

[0052] (5) Centrifugation and observation: After the incubation, the mixture was centrifuged to separate the supernatant and the precipitate.

[0053] (6) Determination of hemolysis rate: Take 100 μL of each sample into a 96-well plate, use a spectrophotometer to measure the absorbance of the supernatant at a wavelength of 540 nm, and calculate the hemolysis rate. The hemolysis rate can be calculated using the formula: Hemolysis rate = [(absorbance of sample group − absorbance of negative control group) / (absorbance of positive control group − absorbance of negative control group)] × 100% hemolysis rate; The absorbance of the negative control group is the absorbance of the supernatant of the red blood cell suspension treated with physiological saline, the absorbance of the drug-loaded microneedle sample group is the absorbance of the supernatant of step (6), and the absorbance of the positive control is the absorbance of the supernatant of the red blood cell suspension treated with distilled water.

[0054] Example 6 The preparation process of the drug-loaded collagen microneedle preparation specifically comprises the following steps: 1) 80 mg of the enzyme-soluble collagen prepared in Example 2 was dissolved in 10 mL of ultrapure water, vortexed to fully dissolve it, and centrifuged at 3000 rpm for 30 min in a horizontal centrifuge to remove bubbles in the collagen solution.

[0055] 2) After the centrifugation of step 1), ultrapure water was added to the precipitate to prepare a jellyfish collagen solution with a mass concentration of 100 μg / mL; 3) Take 1 mL of the jellyfish collagen solution in step 2) and 500 μL of 100 μg / mL IB-367, mix them, and drop them into the PDMS mold. Vacuum filter for 10 minutes, remove the bubbles and repeat the process until the bubbles in the mold are completely removed; then place it in a 37°C oven to dry and concentrate, replenish the solution every 5 hours until the temperature is lowered after molding and it continues to solidify. Take the prepared microneedles and cut off the needle-free area on the edge. Note that the step of taking the molded microneedles should be slow to prevent damage to the overall structure of the needle tip, and obtain the collagen microneedle preparation loaded with IB-367.

[0056] Example 7 The preparation process of the drug-loaded collagen microneedle preparation specifically comprises the following steps: 1) Dissolve 150 mg of the enzyme-soluble collagen prepared in Example 3 above in 50 mL of ultrapure water, vortex to fully dissolve it, and centrifuge at 7000 rpm for 10 min in a horizontal centrifuge to remove bubbles in the collagen solution.

[0057] 2) After the centrifugation of step 1), ultrapure water was added to the precipitate to prepare a jellyfish collagen solution with a mass concentration of 500 μg / mL; 3) Take 1 mL of the jellyfish collagen solution in step 2) and 500 μL of 500 μg / mL tranexamic acid, mix them, and drop them into the PDMS mold. Vacuum filter for 30 minutes, remove the bubbles and repeat the process until the bubbles in the mold are completely removed; then place it in a 37°C oven to dry and concentrate, replenish the solution every 6 hours until the temperature is lowered after molding and it continues to solidify, take the prepared microneedles, cut off the needle-free area on the edge, and pay attention to taking the molded microneedles slowly to prevent damage to the overall structure of the needle tip, and obtain the collagen microneedle preparation loaded with tranexamic acid.

[0058] Example 8 The preparation process of the drug-loaded collagen microneedle preparation specifically comprises the following steps: 1) Dissolve 100 mg of the enzyme-soluble collagen prepared in Example 4 above in 20 mL of ultrapure water, vortex to fully dissolve it, and centrifuge at 4000 rpm for 15 min in a horizontal centrifuge to remove bubbles in the collagen solution.

[0059] 2) After the centrifugation of step 1), ultrapure water was added to the precipitate to prepare a jellyfish collagen solution with a mass concentration of 200 μg / mL; 3) Take 1 mL of the jellyfish collagen solution in step 2) and 500 μL of 200 μg / mL vitamin C, mix them, and drop them into the PDMS mold. Vacuum filter for 15 minutes, remove the bubbles and repeat the process until the bubbles in the mold are completely removed; then place it in a 37°C oven to dry and concentrate, replenish the solution every 6 hours until the temperature is lowered after molding and it continues to solidify. Take the prepared microneedles and cut off the needle-free area on the edge. Note that the step of taking the molded microneedles should be slow to prevent damage to the overall structure of the needle tip, and obtain the collagen microneedle preparation loaded with vitamin C.

[0060] Comparative Example 1 A process for extracting jellyfish extract comprises the following steps: 1) Take the yellow pickled jellyfish, clean it and separate the cap tissue; 2) Trim the jellyfish umbrella tissue into strips of approximately 2 cm in length, width and height by 1 cm and 0.5 cm, and soak them in pure water for 3 days to remove salt. During this period, change the liquid several times, every 8 hours, and the liquid volume should be ≥40 times the mass of the jellyfish; 3) Soak in 2.0 mol / L NaOH solution for 5 days to remove impurities and proteins. Change the solution several times every 8 hours. The liquid volume should be ≥ 40 times the weight of the jellyfish. After soaking, wash with pure water 3 times. 4) Place in 6wt%H 2 O 2 Soak in the solution for 8 hours, with the liquid volume ≥40 times the mass of the jellyfish. After soaking, rinse with pure water 3 times; 5) After draining the water, weigh and homogenize, add 5.0wt% pepsin, the solid-liquid ratio is 1:10, and react for 12h; 6) Filter through 400 mesh filter; 7) Take the supernatant, centrifuge for 70 minutes at 6000 rpm, collect the supernatant and place it in a 4000 Da dialysis bag with 5.0 mol / L Na 2 HPO 4 The solution was dialyzed for 3 days, with multiple changes of solution every 8 hours, and the liquid volume was ≥40 times the mass of the jellyfish; 8) Centrifuge again at 5000 rpm for 70 min, take the precipitate, redissolve it in 5.5 mol / L acetic acid, place it in a 4000 Da dialysis bag, and dialyze for 2 days, changing the liquid several times, every 12 hours, with the liquid volume ≥ 40 times the mass of the jellyfish; 9) Dialyze in deionized water for 3 days, change the liquid several times, 12 hours / time, the liquid volume should be ≥40 times the mass of jellyfish, freeze-dry at -20℃ to obtain white enzyme-soluble collagen (PSC).

[0061] Comparative Example 2 A process for extracting jellyfish extract comprises the following steps: 1) Take the yellow pickled jellyfish, clean it and separate the cap tissue; 2) Trim the jellyfish umbrella tissue into strips of approximately 2 cm in length, width and height by 1 cm and 0.5 cm, and soak them in pure water for 3 days to remove salt. During this period, change the liquid several times, every 8 hours, and the liquid volume should be ≥40 times the mass of the jellyfish; 3) Soak in 2.0 mol / L NaOH solution for 5 days to remove impurities and proteins. Change the solution several times every 8 hours. The liquid volume should be ≥ 40 times the weight of the jellyfish. After soaking, wash with pure water 3 times. 4) After draining the water, weigh and homogenize, add 5.0wt% pepsin, the solid-liquid ratio is 1:10, and react for 12h; 5) Filter through 400 mesh filter; 6) Take the supernatant, centrifuge for 70 minutes at 6000 rpm, collect the supernatant and place it in a 4000 Da dialysis bag with 5.0 mol / L Na 2 HPO 4 The solution was dialyzed for 3 days, with multiple changes of solution every 8 hours, and the liquid volume was ≥40 times the mass of the jellyfish; 7) Take the gel in the dialysis bag and centrifuge it at 6000 rpm for 60 min. Take the precipitate and redissolve it in 3.5 mol / L acetic acid. Add NaCl to a concentration of 5.0 mol / L and salt out for 3 days. 8) Centrifuge again at 5000 rpm for 70 min, take the precipitate, redissolve it in 3.5 mol / L acetic acid, place it in a 4000 Da dialysis bag, and dialyze for 2 days, changing the liquid several times, every 12 hours, with the liquid volume ≥40 times the mass of the jellyfish; 9) Dialyze in deionized water for 3 days, change the liquid several times, 12 hours / time, the liquid volume should be ≥40 times the mass of jellyfish, freeze-dry at -20℃ to obtain white enzyme-soluble collagen (PSC).

[0062] Comparative Example 3 A preparation process of a non-drug-loaded fish collagen microneedle preparation specifically comprises the following steps: 1) Dissolve 100 mg of fish collagen peptide powder in 50 mL of ultrapure water, vortex to fully dissolve it, and centrifuge at 3500 rpm for 40 minutes to remove bubbles in the collagen solution.

[0063] 2) After the centrifugation of step 1), ultrapure water was added to the precipitate to prepare a fish collagen solution with a mass concentration of 400 μg / mL.

[0064] 3) Take 1 mL of the fish collagen solution from step 2) and drop it into the PDMS mold. Vacuum filter for 20 minutes, remove the bubbles and repeat the process until the bubbles in the mold are completely removed; then place it in a 32°C oven to dry and concentrate, replenish the solution once every 6 hours until it is formed and then the temperature is lowered and continuously solidified. Take the prepared microneedles and cut off the needle-free area on the edge. Note that the step of taking the formed microneedles should be slow to prevent damage to the overall structure of the needle tip, and obtain the fish collagen microneedle preparation.

[0065] (I) The collagen extraction rates of Examples 1 to 4 and Comparative Examples 1 and 2 were measured and calculated. The results are shown in Table 1. The extraction rate calculation formula is: (weight of freeze-dried jellyfish collagen / weight of initial jellyfish) × 100%.

[0066] Table 1 Test results

[0067] As can be seen from Table 1, the extraction rate calculated based on dry matter in the present invention is relatively high, while in Comparative Example 1, compared with Example 1, the lack of isopropanol treatment results in fat inside the jellyfish, so that the jellyfish tissue cannot be completely dissolved in the enzymatic hydrolyzate of Comparative Example 1, and the jellyfish extract is not subjected to acetic acid redissolution and salting-out treatment in the subsequent conditions, lacking the key step of jellyfish collagen purification, so the collagen extraction rate is greatly reduced. Compared with Example 2, Comparative Example 2 lacks H 2 O 2The degreasing and depigmenting treatment with isopropyl alcohol makes the resulting collagen not pure enough and contains too many impurities inside the jellyfish, which affects the subsequent important enzymatic hydrolysis process, resulting in no collagen precipitation in the subsequent salting out, greatly reducing the extraction rate.

[0068] (ii) The collagen microneedle preparations prepared in Examples 5-8 and Comparative Example 3 were dissolved in water to prepare collagen solutions with a concentration of 200 μg / mL, and the hemolysis rates thereof were tested respectively. The test results are shown in Table 2.

[0069] Table 2 Test results

[0070] It can be seen from Table 2 that the hemolysis rates of the microneedle preparations prepared in Examples 5-8 of the present invention are all ≤5%, which are all at a safe level. However, in Comparative Example 3, fish collagen is used, and the impurity content in the fish collagen is slightly higher than that in jellyfish collagen. Therefore, the microneedles prepared with 200 μg / mL fish collagen solution have a hemolysis rate higher than 5% of the jellyfish collagen microneedles with the same mass concentration, which exceeds the safety level.

[0071] (III) Cytotoxicity assay (CCK-8 method) In this experiment, the IB-367-loaded collagen microneedle preparation prepared in Example 6 was used to prepare a Co@IBM-367 MN sample group with a concentration of 100µg / mL, and the same steps as in Example 6 were used but without adding IBM-367, i.e., a blank group of the collagen microneedle preparation without drug loading, which was the Co-MN blank group. The 100µg / mL Co@IBM-367 MN was diluted into samples with different concentration gradients, and the concentrations after dilution were 12.5µg / mL, 25µg / mL, and 50µg / mL, respectively.

[0072] The following experiments were conducted using the above Co@IBM-367 MN sample group and Co-MN blank group: RAW264.7 and HUVEC cells in the logarithmic growth phase were digested and adjusted to approximately 5×10 6 100 µL / well was inoculated into a 96-well plate, and an equal volume of PBS was added to the edge wells. The plate was placed in a 37°C, 5% CO 2The cells were cultured in an incubator. After the cells adhered to the wall, the culture supernatant was discarded, and the DMEM complete medium infusion of the Co-MN blank group, the DMEM complete medium infusion of the Co@IBM-367 MN sample group, and samples of different concentration gradients were added to a 96-well plate. Five replicate wells were set up for each group and incubated. After 24h and 48h, CCK-8 solution was added to the DMEM complete medium at 10µL / well, and the cells were incubated in the incubator for 4h. The absorbance of RAW and HUVEC cells at 24h and 48h was measured at 450nm using a multifunctional microplate reader to calculate the cell survival rate: cell survival rate = [(absorbance of the sample group – absorbance of the blank group) / (absorbance of the solvent control – absorbance of the blank group)] × 100%. The solvent control absorbance refers to the absorbance inside the well without drug treatment, that is, only the culture medium and cells, and the blank group absorbance refers to the absorbance inside the well without cells and only the culture medium.

[0073] Finally, a graph was drawn with the cell survival rate as the vertical axis and the degree of each experimental group as the horizontal axis. Figure 6 , The left side shows the toxicity experiment on RAW264.7 cells, and the right side shows the experiment on HUVEC cells.

[0074] Depend on Figure 6 It can be seen that the 24h survival rate of cells treated with pure collagen microneedles (Co-MN group) is 96%~120%, and the 48h survival rate is 130%~150%; in the Co@IBM-367MN sample group, the survival rates of both cells decrease with increasing concentration. Therefore, the present invention has low toxicity to other cells and good biocompatibility.

Claims

1. A process for extracting jellyfish extract, characterized in that: The following steps are involved: 1) Take the pickled jellyfish, clean it and separate the cap tissue; 2) Soak the jellyfish cap tissue in pure water, changing the liquid several times, with the liquid volume ≥ 40 times the mass of the jellyfish; 3) Soak in NaOH solution for 4 to 6 days, changing the solution several times during the period, with the liquid volume ≥ 40 times the mass of the jellyfish. After the soaking, wash with pure water; 4) Soak in H2O2 solution for 6-24 hours, with the liquid volume ≥40 times the mass of the jellyfish. After soaking, rinse with pure water; 5) Soak in isopropanol for 6-24 hours, with the amount of liquid ≥40 times the mass of the jellyfish. After soaking, rinse with pure water; 6) Weigh and homogenize, add 3.5~7.0wt% pepsin, solid-liquid ratio 1:8~1:15, react for 6~24h; 7) Filter with 300~500 mesh filter; 8) Take the supernatant, centrifuge, collect the supernatant and place it in a 3000~5000Da dialysis bag, dialyze with Na2HPO4 solution for 1~6 days, change the solution several times, and the liquid volume should be ≥40 times the mass of the jellyfish; 9) Take the gel in the dialysis bag, centrifuge it, take the precipitate and redissolve it in 2.5~5mol / L acetic acid, add NaCl to a concentration of 3.5~6.0mol / L, and salt out for 2~5 days; 10) Centrifuge again, take the precipitate, redissolve it in 4.5-7.5 mol / L acetic acid, place it in a 3000-5000Da dialysis bag, and dialyze for 1-3 days, changing the liquid several times, with the liquid volume ≥40 times the mass of the jellyfish; 11) Dialyze in deionized water for 2 to 7 days, change the liquid several times, the liquid volume should be ≥ 40 times the mass of jellyfish, freeze-dry at -20℃~-80℃ to obtain enzyme-soluble collagen.

2. The extraction process of jellyfish extract according to claim 1, characterized in that: The concentration of the sodium hydroxide solution in step 3) is 1.0 mol / L to 4.5 mol / L.

3. The extraction process of the jellyfish extract according to claim 1, characterized in that: The mass concentration of the H2O2 solution in step 4) is 4.5%~9%.

4. The extraction process of the jellyfish extract according to claim 1, characterized in that: In step 8), the centrifugal speed is 4000-8000 rpm, the centrifugal time is 60-90 min, and the concentration of the Na2HPO4 solution is 2.4-7.2 mol / L.

5. The extraction process of the jellyfish extract according to claim 1, characterized in that: The centrifugal speed in step 9) is 4000-8000 rpm, and the centrifugal time is 30-90 min.

6. The extraction process of jellyfish extract according to claim 1, characterized in that: The speed of the second centrifugation in step 10) is 3000-8000 rpm, and the centrifugation time is 60-90 min.

7. A preparation process of a drug-loaded collagen microneedle preparation, characterized in that: The enzyme-soluble collagen used in the preparation process is extracted by the extraction process of the jellyfish extract according to any one of claims 1 to 6, and the specific preparation process comprises the following steps: 1) Dissolve 80-500 mg of extracted enzyme-soluble collagen in 10-50 mL of ultrapure water, vortex and centrifuge horizontally; 2) After the centrifugation of step 1), ultrapure water was added to the precipitate to prepare a 100 μg / mL to 500 μg / mL jellyfish collagen solution; 3) Prepare 1 mL of jellyfish collagen solution and 500 μL of 100 μg / mL~500 μg / mL drug solution, mix them, add them into the microneedle mold, vacuum filter, dry and shape them to obtain drug-loaded collagen microneedles.

8. The preparation process of the drug-loaded collagen microneedle preparation according to claim 7, characterized in that: The centrifugal speed in step 1) is 3000-7000 rpm, and the centrifugal time is 10-30 min.

9. The preparation process of the drug-loaded collagen microneedle preparation according to claim 7, characterized in that: Step 3) The drugs IB-367, MD13, tranexamic acid, vitamin E or vitamin C.

10. The preparation process of the drug-loaded collagen microneedle preparation according to claim 7, characterized in that: The vacuum filtration time of step 3) is 10 to 30 minutes.

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