A nanoparticle microneedle for assisted reproduction and a preparation method thereof

Abstract

The present invention provides a nanoparticle microneedle for assisted reproduction and a preparation method thereof, belonging to the field of reproductive medical technology. The preparation method of the nanoparticle microneedle of the present invention comprises the following steps: (1) mixing a drug solution for assisted reproduction and a silk fibroin solution, freezing, and obtaining nanoparticles; (2) pouring the nanoparticle solution into a membrane for centrifugation, and after forming, continuing to add a water-soluble polymer solution to form a matrix layer to obtain the nanoparticle microneedle. The microneedle tip of the present invention can be quickly implanted into the skin to ensure that the drug enters the human body. At the same time, it can prevent the drug from being degraded by enzymes in the skin, maintain the stability of the drug, and increase the relative bioavailability of the drug to 180%, which can reduce the dosage and improve the tolerance of patients receiving ART.

Description

Technical Field

[0001] The present invention relates to the field of reproductive medical technology, and in particular to a nanoparticle microneedle for assisted reproduction and a preparation method thereof. Background Art

[0002] Infertility is caused by disorders of the male or female reproductive system. It is estimated that 48 million couples and 186 million people suffer from infertility worldwide. To address the problem of infertility, various forms of assisted reproductive technology (ART) have been clinically used since 1978. Through ART interventions, such as in vitro fertilization, more than 8 million children have been born worldwide. In the ART regimen, gonadotropin-releasing hormone (GnRH) analogs (including antagonists and agonists) are used to control luteinizing hormone secretion to improve pregnancy rates.

[0003] At present, gonadotropin-releasing hormone analogs are mostly administered by subcutaneous injection. For example, triptorelin is a GnRH agonist with high biological efficacy and has been widely used in ART. At present, the commercially available triptorelin is usually a long-acting injection suspension, but the harmful effects of long-acting sustained-release preparations on early pregnancy embryos must be treated with caution. Therefore, in the current clinical program for ART treatment, triptorelin (0.1 mg) must be administered by subcutaneous injection every day, and women who are preparing for pregnancy need to go to medical institutions for injection every day, which seriously affects women's physical and mental health. At the same time, after subcutaneous injection, the polypeptide will be degraded by enzymes in the skin, resulting in low bioavailability. Therefore, the drug dose at the time of administration exceeds the effective drug dose, which puts women preparing for pregnancy at potential risks.

[0004] Microneedle (MN) delivery is an excellent alternative to subcutaneous injections because it is painless and can be self-administered. MNs (diameter = 50-2000 μm) allow painless penetration of the stratum corneum, facilitating transdermal delivery of biomolecules. In particular, various types of drug-loaded polymer MNs have been extensively studied due to their sufficient drug loading capacity and biodegradability. Nevertheless, drug-loaded MNs are not sufficient to address the problem of enzymatic degradation of peptides in the skin.

[0005] Nanoparticles (NPs) have the function of protecting drugs from enzyme attacks. Polymer microneedles encapsulating nanoparticles (NPs-MNs) have been widely used in the treatment of diabetes, cancer, and skin diseases. In the process of developing NP-MNs, NPs are usually first prepared using one polymer, then freeze-dried and dispersed in another polymer (usually water-soluble) to prepare MNs. However, high temperature and interactions between different polymers are not conducive to drug stability. Therefore, it is urgent to provide a nanoparticle microneedle for assisted reproduction that can effectively solve the problems of poor drug stability and low drug bioavailability.

[0006] Silk fibroin is a natural polymer with biocompatibility and biodegradability. It has abundant production and unique material properties, making it a candidate material for biomedical applications. Silk fibroin is an amphiphilic protein composed of a hydrophobic heavy chain (~391kDa), a hydrophilic light chain (~25kDa) and the glycoprotein P25. The amino acid sequences (primary structure) in the heavy and light chains can form different secondary structures through hydrogen bonds, van der Waals forces or hydrophobic interactions. When the α-helix and random helix forms in the formed secondary structure are dominant, silk fibroin can self-assemble in water and avoid the use of organic solvents. When the highly repeated hexapeptide GAGAGS motif in the hydrophobic chain forms a β-folded structure under induction conditions, silk fibroin has mechanical strength and sustained release. Therefore, silk fibroin is the preferred natural polymer for the preparation of nanoparticles and microneedles. Summary of the invention

[0007] The purpose of the present invention is to provide a nanoparticle microneedle for assisted reproduction and a preparation method thereof. The nanoparticles of the present invention are based on silk fibroin and are used to deliver drugs for assisted reproduction. At the same time, silk fibroin is used as the polymer of the needle tip part, avoiding the use of other polymers. In the present invention, a water-soluble polymer is used to prepare the base part of the microneedle. When the microneedle is inserted into the skin, the water-soluble polymer contacts the tissue fluid and dissolves rapidly. The microneedle tip can be implanted subcutaneously to ensure that the drug enters the human body. At the same time, the drug can be prevented from being degraded by enzymes in the skin, maintaining the stability of the drug, and increasing the relative bioavailability of the drug to 180%, which can reduce the dosage and improve the tolerance of patients receiving ART.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for preparing nanoparticle microneedles for assisted reproduction, comprising the following steps:

[0010] (1) mixing a drug solution for assisted reproduction and a silk fibroin solution, and freezing the mixture to obtain nanoparticles;

[0011] (2) Pour the nanoparticle solution into a membrane and centrifuge it. After forming, continue to add a water-soluble polymer solution to form a matrix layer to obtain the nanoparticle microneedle.

[0012] Preferably, the molecular weight of the silk fibroin is 10-100 kDa, the concentration of the silk fibroin solution is 6-12% (w / v); the volume ratio of the drug solution to the silk fibroin solution is (0.5-2):(0.5-2); the mass ratio of the drug to the silk fibroin is (2-5):60.

[0013] Preferably, the mixing method in step (1) is: injecting the drug solution into the silk fibroin solution in a stirring state at a uniform speed, and continuing to stir and mix; the stirring speed is 200-400 rpm, the stirring temperature is 20-25°C; the total stirring time is 20-40 min; the freezing temperature is -30--10°C, and the time is 1-3 h.

[0014] Preferably, the concentration of silk fibroin in the nanoparticle solution is 6-12% (w / v); the volume of the nanoparticle solution poured into the mold accounts for 2 / 3-5 / 6 of the volume of the needle tip part of the mold.

[0015] Preferably, the centrifugal speed in step (2) is 2000-3000 rpm, the time is 20-40 min, and the temperature is 8-12°C; the temperature during the molding is 23-27°C, and the time required for the molding is 18-26 h.

[0016] Preferably, the water-soluble polymer includes one or more of polyacrylic acid, polyvinyl pyrrolidone and polyvinyl alcohol; the concentration of the water-soluble polymer solution is 30-50wt%, and the molecular weight is ≤250kDa.

[0017] Preferably, the matrix layer is formed by adding a water-soluble polymer, centrifuging at 2500-3200 rpm for 20-40 min at 8-12° C., and drying to form the matrix layer.

[0018] Preferably, the drug is a gonadotropin-releasing hormone analog, including a gonadotropin-releasing hormone receptor agonist and a gonadotropin-releasing hormone receptor inhibitor.

[0019] Preferably, the gonadotropin-releasing hormone receptor agonist includes one of triptorelin, leuprolide, goserelin, buserelin and histrelin; and the gonadotropin-releasing hormone receptor inhibitor includes one of cetrorelix, ganirelix, degarelix and abarelix.

[0020] The present invention also provides a nanoparticle microneedle prepared by the above preparation method.

[0021] The present invention provides a nanoparticle microneedle for assisted reproduction and a preparation method thereof. The technical principle of the present invention is as follows Figure 1As shown. The present invention uses silk fibroin for the first time as both a polymer material for preparing nanoparticles and a supporting material for microneedle tips, avoiding the use of other polymers; a solvent casting method is used to prepare microneedle tips loaded with nanoparticles, and then a soluble polymer is used to prepare the backing of the microneedle, so that after the microneedle is inserted into the skin, the polymer contacts the tissue fluid and dissolves rapidly, allowing the needle tip to be implanted subcutaneously to release the drug. The nanoparticles based on silk fibroin of the present invention are used to deliver drugs for assisted reproduction, which can prevent the drug from being degraded by enzymes in the skin, maintain the stability of the drug, increase the relative bioavailability of the drug to 180%, reduce the dosage, and improve the tolerance of patients receiving ART. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a technical schematic diagram of the nanoparticle microneedle drug delivery of the present invention.

[0023] Figure 2 This is a transmission electron microscope image of the nanoparticles prepared in Example 1.

[0024] Figure 3 This is a stereomicroscope image of the nanoparticle microneedles prepared in Example 1.

[0025] Figure 4 Schematic diagram of the mechanical properties test of the nanoparticle microneedle of Example 1.

[0026] Figure 5 This is a graph showing the mechanical properties test results of a single nanoparticle microneedle of Example 1.

[0027] Figure 6 This is a diagram of the pores formed after the nanoparticle microneedle of Example 1 is inserted into the skin.

[0028] Figure 7 The results are the test results of the effects of the triptorelin solution and the nanoparticle solution prepared in Example 1 on drug stability; wherein A is the triptorelin solution, and B is the nanoparticle solution prepared in Example 1.

[0029] Figure 8 These are the in vitro skin permeability test results of the drug-containing silk fibroin film and the nanoparticle microneedles prepared in Example 1; wherein A is the drug-containing silk fibroin film, and B is the nanoparticle microneedles prepared in Example 1.

[0030] Fig. 9 Schematic diagram of the process of microneedle drug delivery of nanoparticles prepared in Example 1; wherein, I is an image of the skin of rats during microneedle drug delivery, II is an image of the skin after peeling off the polyacrylic acid matrix of the microneedles, and III is an image of the skin 1 day after drug delivery.

[0031] Fig.10 These are the test results of the effects of subcutaneous injection and microneedle administration of the nanoparticles prepared in Example 1 on the pharmacokinetics of triptorelin.

[0032] Fig.11 The changes in LH in rat plasma before and after administration of the nanoparticle microneedles prepared in Example 1.

[0033] Fig.12 The changes of E2 in rat plasma before and after administration of the nanoparticle microneedles prepared in Example 1. DETAILED DESCRIPTION

[0034] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] Example 1

[0036] Taking the gonadotropin-releasing hormone receptor agonist triptorelin as an example, the present invention provides a nanoparticle microneedle for assisted reproduction, and the specific preparation process is as follows:

[0037] Take 100kDa lyophilized silk protein powder and dissolve it in water to prepare a 6% (w / v) silk protein solution. According to the mass ratio of triptorelin to silk protein of 1:30, prepare a triptorelin solution with the same volume as the silk protein. At 25°C, inject the triptorelin solution into the silk protein solution at a uniform speed with a syringe, and stir continuously at 300rpm for 30min to mix the two solutions evenly. After freezing the mixed solution at -20°C for 2h, thaw it at 4°C, and then centrifuge it at 25°C at 10000rpm for 30min, and take the precipitate to obtain nanoparticles.

[0038] Using the concentration of silk fibroin as a standard, the nanoparticles are redispersed in water so that the concentration of silk fibroin in the solution is 6% (w / v) to obtain a nanoparticle solution. The nanoparticle solution is poured into the microneedle mold, and the volume of the solution poured into the mold accounts for 2 / 3 of the total volume of the mold. After centrifugation at 2800 rpm for 30 minutes at 10°C, the mold is placed at 25°C for 20 hours to dry and form the nanoparticles, and a 35wt% polyacrylic acid solution (molecular weight = 250kDa) is added, and centrifuged at 2800 rpm for 30 minutes at 10°C to allow the solution to completely enter the mold and expel bubbles, finally forming a mold as shown below. Figure 1 The needle-shaped structure is shown in . Then, it is placed in a dryer at 25°C for 72 hours and demolded to obtain nanoparticle microneedles.

[0039] Example 2

[0040] Taking triptorelin as an example, the present invention provides a nanoparticle microneedle for assisted reproduction, and the specific preparation process is as follows:

[0041] Take 10kDa lyophilized silk fibroin powder and dissolve it in water to prepare a 12% (w / v) silk fibroin solution. According to the mass ratio of triptorelin to silk fibroin of 1:12, prepare a triptorelin solution with the same volume as the silk fibroin. At 25°C, inject the triptorelin solution into the silk fibroin solution at a uniform speed with a syringe, and stir continuously at 400rpm for 30min to mix the two solutions evenly. The mixed solution was frozen at -20°C for 2h and then thawed, and centrifuged at 10000rpm for 30min at 25°C to obtain nanoparticles.

[0042] Using the concentration of silk fibroin as a standard, the nanoparticles are redispersed in water so that the concentration of silk fibroin in the solution is 12% (w / v) to obtain a nanoparticle solution. The nanoparticle solution is poured into the microneedle mold, and the volume of the solution poured into the mold accounts for 2 / 3 of the total volume of the mold. After centrifugation at 2800rpm for 30 minutes at 10°C, the mold is placed at a temperature of 27°C for 26 hours to dry and form the nanoparticles, and a polyvinyl alcohol solution with a concentration of 50wt% (molecular weight = 200kDa) is added, and centrifuged at 2800rpm for 30 minutes at 10°C to allow the solution to completely enter the mold and discharge bubbles. After that, it is placed in a dryer at 25°C for 72 hours, and demolded to obtain nanoparticle microneedles.

[0043] Example 3

[0044] Taking triptorelin as an example, the present invention provides a nanoparticle microneedle for assisted reproduction, and the specific preparation process is as follows:

[0045] Take 50kDa lyophilized silk protein powder and dissolve it in water to prepare a silk protein solution with a concentration of 8% (w / v). According to the mass ratio of triptorelin to silk protein of 1:20, prepare a triptorelin solution with the same volume as the silk protein. At 23°C, inject the triptorelin solution into the silk protein solution at a uniform speed with a syringe, and stir continuously at 400rpm for 20min to mix the two solutions evenly. The mixed solution was frozen at -20°C for 2h and then thawed, and centrifuged at 10000rpm for 30min at 25°C to obtain nanoparticles.

[0046] Using the concentration of silk fibroin as a standard, the nanoparticles are redispersed in water so that the concentration of silk fibroin in the solution is 8% (w / v) to obtain a nanoparticle solution. The nanoparticle solution is poured into the microneedle mold, and the volume of the solution poured into the mold accounts for 5 / 6 of the total volume of the mold. After centrifugation at 2800 rpm for 30 minutes at 10°C, the mold is placed at a temperature of 27°C for 18 hours to dry and form the nanoparticles, and a polyvinyl pyrrolidone solution (molecular weight = 250 kDa) with a concentration of 50wt% is added, and centrifuged at 2800 rpm for 30 minutes at 10°C to allow the solution to completely enter the mold and discharge bubbles. After that, it is placed in a dryer at 25°C for 72 hours, and demolded to obtain nanoparticle microneedles.

[0047] Comparative Example 1

[0048] Take 100kDa lyophilized silk fibroin powder and dissolve it in water to prepare a 6% (w / v) silk fibroin solution. According to the mass ratio of triptorelin to silk fibroin of 1:12, prepare a triptorelin solution with the same volume as the silk fibroin. At 25°C, inject the triptorelin solution into the silk fibroin solution at a uniform speed with a syringe, and stir continuously at 300rpm for 30min to mix the two solutions evenly. The mixed solution was frozen at -20°C for 2h and then thawed, and centrifuged at 10000rpm for 30min at 25°C to obtain nanoparticles.

[0049] Using the concentration of silk fibroin as a standard, the nanoparticles are redispersed in water so that the concentration of silk fibroin in the solution is 6% (w / v) to obtain a nanoparticle solution. The nanoparticle solution is poured into the microneedle mold, and the volume of the solution poured into the mold accounts for 2 / 3 of the total volume of the mold. After centrifugation at 2800rpm for 30min at 10°C, the mold is placed at 25°C for 20h to dry and form the nanoparticles, and a polyacrylic acid solution (molecular weight = 250kDa) with a concentration of 35wt% is added. Centrifugation is carried out at 2800rpm for 30min at 10°C to allow the solution to completely enter the mold and discharge bubbles. After that, it is placed in a dryer at 25°C for 72h and demolded to obtain nanoparticle microneedles.

[0050] Comparative Example 2

[0051] Take 100kDa lyophilized silk fibroin powder and dissolve it in water to prepare a 6% (w / v) silk fibroin solution. According to the mass ratio of triptorelin to silk fibroin of 1:15, prepare a triptorelin solution with the same volume as the silk fibroin. At 25°C, inject the triptorelin solution into the silk fibroin solution at a uniform speed with a syringe, and stir continuously at 300rpm for 30min to mix the two solutions evenly. The mixed solution was frozen at -20°C for 2h and then thawed, and centrifuged at 10000rpm for 30min at 25°C to obtain nanoparticles.

[0052] Using the concentration of silk fibroin as a standard, the nanoparticles are redispersed in water so that the concentration of silk fibroin in the solution is 6% (w / v) to obtain a nanoparticle solution. The nanoparticle solution is poured into the microneedle mold, and the volume of the solution poured into the mold accounts for 2 / 3 of the total volume of the mold. After centrifugation at 2800rpm for 30min at 10°C, the mold is placed at 25°C for 20h to dry and form the nanoparticles, and a polyacrylic acid solution (molecular weight = 250kDa) with a concentration of 40wt% is added, and centrifuged at 2800rpm for 30min at 10°C to allow the solution to completely enter the mold and discharge bubbles. After that, it is placed in a dryer at 25°C for 72h, and demolded to obtain nanoparticle microneedles.

[0053] Test Example 1

[0054] 1. The properties of the nanoparticles prepared in each embodiment and comparative example were tested. The results are shown in Table 1.

[0055] Table 1 Properties of the nanoparticles in each embodiment and comparative example

[0056]

[0057] As can be seen from Table 1, when the molecular weight of silk fibroin is 10 kDa to 100 kDa and the concentration is 6 to 12% (w / v), and the mass ratio of triptorelin to silk fibroin is (2 to 5):60, stable nanoparticles can be prepared.

[0058] 2. Detect the morphological characteristics of the nanoparticles and nanoparticle microneedles of Example 1. The specific process is as follows:

[0059] The morphology of the nanoparticles of Example 1 was detected using a transmission electron microscope. The detection results are as follows: Figure 2 As shown. Figure 2 It can be seen that the nanoparticles are spherical in shape, uniform in size and well dispersed.

[0060] The morphology of the nanoparticle microneedles in Example 1 was detected using a stereo microscope. The detection results are as follows: Figure 3 As shown. Figure 3It can be seen that the nanoparticle microneedle is conical in shape, complete in morphology, with a sharp tip, a height of 600 μm, and a base diameter of 300 μm.

[0061] 3. Test the mechanical properties of the nanoparticle microneedles of Example 1. The specific process is as follows:

[0062] Place the single nanoparticle microneedle prepared in Example 1 with the tip facing upward on the test table of the texture analyzer (such as Figure 4 The P / 5 probe was used to detect the breaking force of the microneedle tip. The probe was moved vertically toward the microneedle at a speed of 0.1 mm / s. The distance between the sensor and the tip was initially 5 mm, the trigger force was 5 g, and the sensor was moved 0.4 mm from the microneedle tip to the patch backing to test the breaking force of the microneedle. The results are shown in Figure 5 As shown. Figure 5 It can be seen that the breaking force of the nanoparticle microneedle can reach over 110mN / needle, exceeding 100mN / needle, which can meet the strength requirements for insertion into human skin.

[0063] After the microneedle is inserted into the ex vivo pig skin and stained with methylene blue, a uniform and complete microchannel can be produced (e.g. Figure 6 shown).

[0064] 4. Detect the effect of the nanoparticles of Example 1 on drug stability. The specific process is as follows:

[0065] Rat skin tissue fluid was taken and divided into two groups, A and B, and equal volumes of triptorelin solution and nanoparticle solution prepared in Example 1 were added respectively. The two groups were incubated at 37°C for 0 min, 5 min, 30 min, 1 h, 2 h, 4 h, and 6 h. After taking out part of the tissue fluid at each time point, aprotinin and methanol were immediately added to the tissue fluid to terminate the reaction, the drug concentration at each time point was measured, and the percentage of remaining drug was calculated. The test results are shown in Figure 2. Figure 7 shown.

[0066] from Figure 7 It can be seen that the triptorelin solution was rapidly enzymatically hydrolyzed in the skin tissue fluid, degrading about 26% in 5 minutes, and the drug in the tissue fluid was completely degraded by 6 hours. However, the nanoparticles were degraded slowly in the tissue fluid, especially only 12% in 1 hour, and the amount of drug in the tissue fluid was still about 26% by 6 hours, indicating that the nanoparticles can effectively protect the drug from degradation by enzymes in the skin tissue fluid, which is conducive to maintaining the stability of the drug.

[0067] 5. Detect the in vitro skin permeability of the nanoparticle microneedles of Example 1. The specific process is as follows:

[0068] The triptorelin nanoparticle solution was filled into a mold of the same size as the microneedle but without pinholes, and after drying and molding, it was demoulded to make a drug-containing silk fibroin film as a control. Using a vertical Franz diffusion cell, pig skin loaded with a drug-containing silk fibroin film (Group A) and pig skin with nanoparticle microneedles of Example 1 (Group B) were placed on a diffuser, the temperature was set to 32°C, the speed of the rotor in the receiving chamber was 600rpm, and 200μL of liquid was drawn from the sampling port at 0.5, 1, 2, 4, 8, 24, 48, and 72h, respectively, and the same volume of receiving solution was supplemented at the same time. The drug concentration at each time point was measured, and the cumulative release amount was calculated. The test results are shown in Figure 8 shown.

[0069] from Figure 8 It can be seen that the silk fibroin film containing drugs (Group A) can only penetrate 1% in 72 hours, because the triptorelin with a large molecular weight and water solubility can hardly penetrate the skin by passive diffusion. After being prepared into microneedles (Group B), it can overcome the barrier of the stratum corneum of the skin, and the microneedles can penetrate about 65% in 24 hours.

[0070] 6. Detect the effect of nanoparticle microneedle administration in Example 1 on the pharmacokinetics and bioavailability of triptorelin. The specific process is as follows:

[0071] Sprague–Dawley rats (weight 200±10 g) were randomly divided into two groups, 8 in each group. After shaving their backs, one group was subcutaneously injected with triptorelin at a dose of 9 μg / kg, and the other group was administered with the nanoparticle microneedle prepared in Example 1 at a dose of 9 μg / kg. The process of microneedle administration is as follows Fig. 9 As shown, I is the skin image of rats during microneedle administration, II is the skin image after removing the polyacrylic acid matrix of the microneedles, and III is the skin image 1 day after administration. Fig. 9 It can be seen that when microneedles are used to deliver drugs, only skin tissue fluid seeps out, and when the microneedle tips are implanted in the body, the microneedles have almost no irritating effect on the skin of rats.

[0072] Blood was collected before administration and at 0.5, 1, 2, 4, 8, 24, and 48 hours after administration, and immediately centrifuged at 3500 rpm for 5 minutes in a 4°C centrifuge. The supernatant was collected to determine the concentration of triptorelin in plasma and calculate the pharmacokinetic parameters and relative bioavailability. The test results are shown in Table 2, and Fig.10 shown.

[0073] Table 2 Pharmacokinetic parameters after microneedle administration and subcutaneous injection in rats (n=8)

[0074] parameter Microneedle drug delivery Subcutaneous injection <![CDATA[C max (ng / mL)]]> 5.51±1.12 10.54±0.60 <![CDATA[T max (h)]]> 0.50±0.00 0.50±0.00 <![CDATA[T 1 / 2 (h)]]> 8.39±0.22 2.11±0.05 <![CDATA[AUC 0-t (mg / mL)]]> 16.01±2.19 8.65±0.41

[0075] from Fig.10It can be seen that the drug in the microneedle reaches a peak at 0.5h, then slowly decreases, and can be released continuously for 48h. The drug in the subcutaneous injection group reaches a peak, then decreases rapidly, and is undetectable at 24h. According to calculations, the half-life of the nanoparticle microneedle is prolonged, the AUC is increased, and the relative bioavailability can reach more than 180%, which effectively improves the relative bioavailability of triptorelin and lays the foundation for reducing the frequency and dosage of medication.

[0076] 7. Verify the "ignition effect" and "down-regulation" effects of the nanoparticle microneedles of Example 1. The specific process is as follows:

[0077] Rats with similar estrogen levels (weight 200±10g) were randomly divided into two groups, 8 in each group. After shaving their backs, one group was injected subcutaneously at 9μg / kg, and the other group was administered with microneedles at 9μg / kg. Blood was collected before and 0.5, 1, 2, 4, 8, 24, and 48 hours after administration, and immediately centrifuged at 3500rpm for 5 minutes in a 4℃ centrifuge. The supernatant was taken and the levels of LH and E2 in plasma were measured. The test results are as follows: Fig.11 and Fig.12 shown.

[0078] from Fig.11 It can be seen that the concentration of LH in the nanoparticle microneedle group and the subcutaneous injection group first increased, then continued to decrease, and was lower than the normal level at 2 hours. The microneedle group could continue to suppress the level of LH to 48 hours, while the LH in the subcutaneous injection group began to increase at 48 hours.

[0079] from Fig.12 It can be seen that the E2 concentration of rats in the subcutaneous injection group reached a peak at 2 hours and decreased to below the normal level at 4 hours; while the E2 concentration of the microneedle group reached a peak at 4 hours and decreased to below the normal level at 8 hours, which should be related to the sustained release effect of the microneedles.

[0080] In summary, the nanoparticle microneedles of Example 1 can induce an "ignition effect" and a "down-regulation" effect in female mice, and the down-regulation effect can be maintained for 48 hours, which is better than the subcutaneous injection group.

[0081] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing nanoparticle microneedles for assisted reproduction, characterized in that: The steps include: (1) mixing a drug solution for assisted reproduction and a silk fibroin solution, and freezing the mixture to obtain nanoparticles; (2) pouring the nanoparticle solution into a membrane and centrifuging it. After forming, a water-soluble polymer solution is added to form a matrix layer to obtain the nanoparticle microneedles; When the molecular weight of the silk fibroin is 10-50 KDa, the mass ratio of the drug to the silk fibroin is 1:12-20; when the molecular weight of the silk fibroin is 100 KDa, the mass ratio of the drug to the silk fibroin is 1:30; The mixing method of step (1) is: injecting the drug solution into the silk fibroin solution in a stirring state at a uniform speed, and continuing to stir and mix; the stirring speed is 200-400 rpm, the stirring temperature is 20-25° C.; the total stirring time is 20-40 min; the freezing temperature is -30--10° C., and the time is 1-3 h; The concentration of the silk fibroin solution is 6-12% w / v; The drug is triptorelin.

2. The preparation method according to claim 1, characterized in that: The volume ratio of the drug solution to the silk fibroin solution is 0.5-2:0.5-2.

3. The preparation method according to claim 2, characterized in that: In the nanoparticle solution, the concentration of silk fibroin is 6-12% w / v; the volume of the nanoparticle solution poured into the mold accounts for 2 / 3-5 / 6 of the volume of the needle tip part of the mold.

4. The preparation method according to claim 3, characterized in that: The centrifugal speed in step (2) is 2000-3000 rpm, the time is 20-40 min, and the temperature is 8-12° C.; the temperature during the molding is 23-27° C., and the time required for the molding is 18-26 h.

5. The preparation method according to claim 4, characterized in that: The water-soluble polymer includes one or more of polyacrylic acid, polyvinyl pyrrolidone and polyvinyl alcohol; the concentration of the water-soluble polymer solution is 30-50wt%, and the molecular weight is ≤250kDa.

6. The preparation method according to claim 5, characterized in that: The formation process of the matrix layer is as follows: after adding the water-soluble polymer, centrifuging at 2500-3200 rpm for 20-40 minutes at 8-12° C., and forming the matrix layer after drying.

7. A nanoparticle microneedle prepared by the preparation method according to any one of claims 1 to 6.

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