Reversible long-acting hydrogel capable of being cured in situ in human body cavity and application of reversible long-acting hydrogel

Abstract

The invention discloses reversible long-acting hydrogel capable of being cured in situ in a human body cavity, the reversible long-acting hydrogel comprises hydrogel and a reversible reagent, after the hydrogel swells, long-acting blocking is achieved, after the reversible reagent is added, the hydrogel is degraded, and reversible recovery is achieved. According to the invention, 10wt%-60wt% of a polyethylene glycol derivative acid solution and 1wt%-10wt% of a polylysine alkaline solution are subjected to a cross-linking effect, and a formed cross-linked network can prevent cells from passing but cannot prevent body fluid and salt ions from passing, so that the long-acting contraception agent can be applied to seminiferous ducts and fallopian tubes to realize a long-acting contraception effect. The hydrogel has a certain degradation period and can meet the requirements of different contraception periods, when a patient has the fertility requirement in advance, a reversible reagent can be injected in situ to degrade the gel to achieve cavity restoration, and the hydrogel is fast in degradation, safe to use and good in biocompatibility.

Description

Technical Field

[0001] The invention relates to A61L and the field of medical devices, and in particular to a reversible long-acting hydrogel capable of in-situ curing in a human body cavity and an application thereof. Background Art

[0002] Male contraceptive technology is mainly achieved through condoms and vasectomy. The former will reduce the pleasure of sexual life, and the latter has irreversible disadvantages. Existing long-term contraceptive measures include tubal or vas deferens embolization surgery, which causes necrosis and fibrosis of the inner wall of the fallopian tube or vas deferens, forming scars to achieve permanent contraception, but the blockage is irreversible. If there is a need for reproduction again, surgery is required to re-establish the connection of the fallopian tube or vas deferens. In addition, chitosan or carboxymethyl cellulose can be injected to achieve physical isolation and spermicidal effects, but it can only achieve short-term contraception, not long-term contraception.

[0003] Chinese invention patent CN201510044582.8 discloses a contraceptive and microbicidal dual-function vaginal composite thermosensitive hydrogel preparation and its preparation method. The drug in the gel exists in the body in the form of microspheres, which can prolong the retention time of the drug in the treatment site. It has the functions of contraception and HIV pre-exposure prevention, is non-irritating to the vaginal mucosa, and has a good spermicidal effect, but cannot achieve a long-term contraceptive effect. Chinese invention patent CN201711291469.5 discloses a thermo-hydrogel sustained-release veterinary drug injection for animal contraception and its preparation method. The injection preparation is composed of an amphiphilic block copolymer, a contraceptive active ingredient and a solvent. The sustained-release period of a single injection can range from several weeks to several months, but there is a problem of minimally invasiveness and poor safety for human application. Summary of the invention

[0004] In order to develop a reversible long-acting contraceptive method, the first aspect of the present invention provides a reversible long-acting hydrogel that can be solidified in situ in a human body cavity, wherein the reversible long-acting hydrogel comprises a hydrogel and a reversible agent, and the mass ratio of the hydrogel to the reversible agent is 1:(4-6). After the hydrogel swells, long-term blockage is achieved, and after the reversible agent is added, the hydrogel is degraded to achieve reversible recanalization.

[0005] As a preferred embodiment, the mass ratio of the hydrogel to the reversible reagent is 1:5.

[0006] As a preferred embodiment, the raw materials for preparing the hydrogel include polyethylene glycol derivative solution and poly-lysine solution, and the volume ratio of the polyethylene glycol derivative solution to the poly-lysine solution is 1:(0.5-2).

[0007] As a preferred embodiment, the volume ratio of the polyethylene glycol derivative solution to the poly-lysine solution is 1:(0.5-1).

[0008] As a preferred embodiment, the volume ratio of the polyethylene glycol derivative solution to the poly-lysine solution is 1:1.

[0009] As a preferred embodiment, the mass concentration of the polyethylene glycol derivative solution is 10%-60%, and the pH of the polyethylene glycol derivative solution is 4-6.

[0010] As a preferred embodiment, the mass concentration of the polyethylene glycol derivative solution is 20%-40%, and the pH of the polyethylene glycol derivative solution is 4-6.

[0011] As a preferred embodiment, the mass concentration of the polyethylene glycol derivative solution is 40%, and the pH of the polyethylene glycol derivative solution is 5.

[0012] As a preferred embodiment, the mass concentration of the poly-lysine solution is 1%-10%, and the pH of the poly-lysine solution is 8-10.

[0013] As a preferred embodiment, the mass concentration of the poly-lysine solution is 1%-6%, and the pH of the poly-lysine solution is 8-9.

[0014] As a preferred embodiment, the mass concentration of the poly-lysine solution is 2%-5%, and the pH of the poly-lysine solution is 8.7-8.84.

[0015] As a preferred embodiment, the reversible reagent is a lysine solution with a mass concentration of 5-50%, and the pH of the reversible reagent is 4-6.

[0016] As a preferred embodiment, the reversible reagent is a lysine solution with a mass concentration of 20%, and the pH of the reversible reagent is 4.3.

[0017] As a preferred embodiment, the polyethylene glycol derivative in the polyethylene glycol derivative solution is an aldehyde-terminated multi-arm polyethylene glycol derivative, the number of arms of the multi-arm polyethylene glycol derivative is 4-8, and the molecular weight of the polyethylene glycol derivative is 5000-400000 Da.

[0018] As a preferred embodiment, the number of arms of the multi-arm polyethylene glycol derivative is 6 to 8. The molecular weight of the polyethylene glycol derivative is 10000 to 15000 Da.

[0019] As a preferred embodiment, the derivative group in the polyethylene glycol derivative is selected from at least one of N-hydroxysuccinimide ester, alkane aldehyde, and aromatic aldehyde.

[0020] As a preferred embodiment, the polylysine in the polylysine solution is at least one selected from trilysine, tetralysine, pentalysine, and epsilon-polylysine.

[0021] As a preferred embodiment, the weight average molecular weight of the poly-lysine is 2000-4000; preferably, the weight average molecular weight of the poly-lysine is 3000.

[0022] As a preferred embodiment, the molar ratio of the aldehyde groups in the aldehyde-terminated multi-arm polyethylene glycol derivative to the amino groups in the polylysine is (0.2-2):1.

[0023] As a preferred embodiment, the molar ratio of the aldehyde groups in the aldehyde-terminated multi-arm polyethylene glycol derivative to the amino groups in the polylysine is (0.8-2):1.

[0024] As a preferred embodiment, the molar ratio of the aldehyde groups in the aldehyde-terminated multi-arm polyethylene glycol derivative to the amino groups in the polylysine is 1.5:1.

[0025] The present invention adopts 10wt%-60wt% polyethylene glycol derivative acidic solution and 1wt%-10wt% polylysine alkaline solution to crosslink, and the formed crosslinked network can block the passage of cells, but will not block the passage of body fluids and salt ions, and can be applied to the vas deferens and fallopian tubes to achieve long-term contraceptive effect. When there is a need for fertility, the third component can be added to quickly degrade, and the contraceptive effect is safe and painless, and easy to use. The possible reason is guessed that the crosslinked network pore size generated by the preferred polyethylene glycol derivative acidic solution and polylysine alkaline solution is appropriate, and the pore size of the crosslinked network is smaller than the diameter of sperm protein 3-5μm, but the pore size of the crosslinked network will not block the passage of body fluids and salt ions, and can meet normal physiological needs.

[0026] The inventors further discovered that the polyethylene glycol derivative is an aldehyde-terminated polyethylene glycol derivative, and the molar ratio of the aldehyde group in the polyethylene glycol derivative to the amino group of polylysine is (0.8-1.5):1. The formed hydrogel can achieve rivets at the injection site through positive swelling after being injected into the body cavity, and cells are not likely to penetrate. When the preferred molar ratio is exceeded, the hydrogel is in negative swelling. After being injected into the body cavity, it is easy to shift, and protein cells diffuse through the gaps. When used for blocking, contraception is likely to fail.

[0027] The second aspect of the present invention provides an application of a reversible long-acting hydrogel that can be solidified in situ in a human body cavity, which is applied to long-acting contraceptive products.

[0028] As a preferred embodiment, the application method of the reversible long-acting hydrogel is: use a double mixer to inject equal volumes of polyethylene glycol derivative solution and polylysine solution into the cavity, or pre-mix equal volumes of polyethylene glycol derivative solution and polylysine solution and inject them into the cavity before they solidify, and then use it.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The reversible long-acting hydrogel of the present invention can be solidified in situ in the human body cavity. A 10wt%-60wt% polyethylene glycol derivative acidic solution and a 1wt%-10wt% polylysine alkaline solution are cross-linked to form a cross-linked network that can block the passage of cells but does not block the passage of body fluids and salt ions. The hydrogel can be applied to the vas deferens and fallopian tubes to achieve a long-term contraceptive effect.

[0031] (2) The reversible long-acting hydrogel of the present invention can be solidified in situ in the human body cavity. The molar ratio of the aldehyde group in the polyethylene glycol derivative to the amino group of polylysine is (0.8-1.5):1. After the hydrogel is injected into the body cavity, it can achieve riveting at the injection site through positive swelling and is not easy for cells to penetrate.

[0032] (3) The reversible long-acting hydrogel of the present invention can be solidified in situ in the human body cavity. The acidic solution of 10wt%-60wt% polyethylene glycol derivative and the alkaline solution of 1wt%-10wt% polylysine can solidify in situ to form a hydrogel and continue to swell to achieve stable blockage of the cavity.

[0033] (4) The reversible long-acting hydrogel of the present invention can be solidified in situ in the human body cavity and has a certain degradation cycle, which can meet the needs of different contraceptive cycles.

[0034] (5) The present invention is a reversible long-acting hydrogel that can be solidified in situ in the human body cavity. When the patient changes his plan and has the need to have children in advance, a reversible agent can be injected in situ to degrade the gel to restore the cavity. The hydrogel degrades quickly, is safe to use, and has good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the 24h swelling ratio of 8-arm polyethylene glycol derivative solution to poly-lysine solution at different molar ratios;

[0036] Figure 2 is the 24h swelling ratio of the 6-arm polyethylene glycol derivative solution and the poly-lysine solution at different molar ratios;

[0037] Figure 3 The reversible long-acting hydrogel prepared in Example 12 was stained with methylene blue and then injected into a 1 mm inner diameter hose (left picture), and a blank hose (right picture).

[0038] Figure 4 The degradation curves of the reversible long-acting hydrogels prepared in Examples 4, 7, and 8 at 60°C.

[0039] Figure 5 This is a picture of the results under a microscope after the reversible long-acting hydrogel prepared in Example 12 was added to a culture medium containing serum and cells and incubated in an air oscillator at 37° C. for 48 hours;

[0040] Figure 6 This is a photo of the newly prepared reversible long-lasting hydrogel in Example 12 (left picture) and the photo after rapid degradation for 1 hour (right picture). DETAILED DESCRIPTION

[0041] Example

[0042] A reversible long-acting hydrogel that can be cured in situ in a human body cavity. The reversible long-acting hydrogel comprises a hydrogel and a reversible reagent. The raw materials for preparing the hydrogel comprise a polyethylene glycol derivative solution and a polylysine solution. The volume ratio of the polyethylene glycol derivative solution to the polylysine solution is 1:1.

[0043] The polyethylene glycol derivative solution is a polyethylene glycol derivative solution prepared with a 0.02M phosphate buffer solution with a pH of 3.6; the pH of the polyethylene glycol derivative solution is 4-6. The polyethylene glycol derivative is purchased from Jiankai.

[0044] The poly-lysine solution is a poly-lysine solution prepared with water, and the poly-lysine is purchased from Aladdin, item number P303210.

[0045] The specific raw materials for preparation are shown in Table 1:

[0046] Table 1

[0047]

[0048]

[0049] Performance Testing

[0050] 1. Curing time: Pre-warm the polyethylene glycol derivative solution and poly-lysine solution in a vial at 37°C in a water bath, then mix the polyethylene glycol derivative solution and poly-lysine solution in equal volumes, start timing, and after shaking for 2 seconds, use the tilt method to observe the time when the solution loses fluidity, which is the curing time.

[0051] 2. Hydrogel pH: Take the solidified gel, add purified water 10 times the mass of the gel, let it stand at room temperature for 24 hours, and then test the pH of the extract to obtain the gel pH.

[0052] 3. Accelerated degradation experiment: The prepared hydrogel was immersed in PBS buffer solution with a volume 10 times the mass of the gel and placed in a 60°C oven. The gel state was observed and the extract was replaced with new extract every 7 days, and the time for the hydrogel to be completely degraded was recorded.

[0053] The test results are shown in Table 2.

[0054] Table 2

[0055]

[0056]

[0057] The 24h swelling rates of 8-arm polyethylene glycol derivative solution and poly-lysine solution at different molar ratios are shown in Figure 2. Figure 1 ; The 24h swelling rate of 6-arm polyethylene glycol derivative solution and poly-lysine solution at different molar ratios is shown in Figure 2 The degradation curves of the reversible long-acting hydrogels prepared in Examples 4, 7, and 8 at 60°C are shown in Figure 4 In the figure: A. Example 4; B. Example 7; C. Example 8.

[0058] From the data, we can see that for the hydrogel formed by 8-arm PEG, when the molar ratio of aldehyde group: amino group is less than 0.8, the hydrogel is in negative swelling. If injected into the body cavity, it is easy to shift and the substance diffuses through the gap. When used as a blockage of the vas deferens, contraceptive failure is likely to occur. When the molar ratio of aldehyde group: amino group is greater than 0.8, the swelling rate is positive. If injected into the body cavity, the rivet at the injection site can be achieved through positive swelling, and it is not easy for cells to pass through. At the same aldehyde group: amino group molar ratio, the greater the solid content of the overall hydrogel, the greater its swelling rate.

[0059] For the hydrogel formed by 6-arm PEG, its swelling law is consistent with that of 8-arm PEG. As the molar ratio of aldehyde group:amino group increases, the swelling rate will increase, but within the studied ratio range, it is negative.

[0060] 4. Rapid degradation experiment: Prepare a reversible reagent: a lysine aqueous solution with a mass concentration of 20%, and the pH of the reversible reagent is 4.3.

[0061] The reversible long-acting hydrogel prepared in Example 12 was added with 5 times the mass of the reversible reagent and placed in a 37° C. water bath shaker. The gel was completely degraded within 3 hours.

[0062] Example 12: Newly prepared reversible long-lasting hydrogel (left), photo after rapid degradation for 1 hour (right), the gel completely turns into liquid after 3 hours. Figure 6 .

[0063] 5. Hydrogel cytotoxicity test: The polyethylene glycol derivative solution and polylysine solution of Example 12 were mixed in equal volumes in a 15 mL sterile centrifuge tube, and then a serum- and penicillin-streptomycin-containing culture medium 10 times the mass of the gel was added, and the mixture was shaken in an air shaker at 37°C for 72 hours. The cytotoxicity of the gel was detected by the MTT method according to the requirements of GB / T16886.5. The cell survival rate of Example 12 was 99.68%, which proved that the vas deferens embolization hydrogel had good biocompatibility.

[0064] 6. Characterization of hydrogel mesh size: The polyethylene glycol derivative solution of Examples 11 and 12 and the polylysine solution were mixed in equal volumes in a 15 mL sterile centrifuge tube, and then a medium containing serum and cells 10 times the mass of the gel was added and incubated in an air oscillator at 37°C for 48 h. After the gel was stained with methylene blue, no cells were observed to enter the gel under a microscope (see Figure 5 ), which can prove the barrier effect of vas deferens embolization hydrogel on cells.

[0065] The reversible long-acting hydrogel prepared in Example 11 was dyed with methylene blue and then injected into a 1 mm inner diameter hose (left picture). The blank hose (right picture) was Figure 3 .

[0066] The sources of serum, penicillin-streptomycin, culture medium, and cells are shown in Table 3.

[0067] Table 3

[0068]

Claims

1. A reversible long-lasting hydrogel that can be cured in situ in a human body cavity, characterized in that: The reversible long-acting hydrogel comprises a hydrogel and a reversible agent, and the mass ratio of the hydrogel to the reversible agent is 1:(4-6).

2. The reversible long-acting hydrogel that can be cured in situ in a human body cavity according to claim 1, characterized in that: The raw materials for preparing the hydrogel include polyethylene glycol derivative solution and poly-lysine solution, and the volume ratio of the polyethylene glycol derivative solution to the poly-lysine solution is 1:(0.5-2).

3. The reversible long-acting hydrogel that can be cured in situ in a human body cavity according to claim 2, characterized in that: The mass concentration of the polyethylene glycol derivative solution is 10%-60%, and the pH of the polyethylene glycol derivative solution is 4-6.

4. The reversible long-acting hydrogel that can be cured in situ in a human body cavity according to claim 2, characterized in that: The mass concentration of the poly-lysine solution is 1%-10%, and the pH of the poly-lysine solution is 8-10.

5. The reversible long-acting hydrogel that can be cured in situ in a human body cavity according to claim 2, characterized in that: The reversible reagent is a lysine solution with a mass concentration of 5-50%, and the pH of the reversible reagent is 4-6.

6. The reversible long-acting hydrogel capable of in-situ curing in a human body cavity according to claim 2, characterized in that: The polyethylene glycol derivative in the polyethylene glycol derivative solution is an aldehyde-terminated multi-arm polyethylene glycol derivative, the number of arms of the multi-arm polyethylene glycol derivative is 4-8, and the molecular weight of the polyethylene glycol derivative is 5000-400000Da.

7. The reversible long-acting hydrogel capable of in-situ curing in a human body cavity according to claim 6, characterized in that: The derivative group in the polyethylene glycol derivative is selected from at least one of N-hydroxysuccinimide ester, alkane aldehyde and aromatic aldehyde.

8. The reversible long-acting hydrogel capable of in-situ curing in a human body cavity according to claim 6, characterized in that: The polylysine in the polylysine solution is selected from at least one of trilysine, tetralysine, pentalysine and epsilon-polylysine.

9. The reversible long-acting hydrogel capable of in-situ curing in a human body cavity according to claim 8, characterized in that: The molar ratio of the aldehyde group in the aldehyde-terminated multi-arm polyethylene glycol derivative to the amino group in the polylysine is (0.2-2):

1.

10. An application of the reversible long-acting hydrogel capable of in-situ curing in a human cavity according to any one of claims 1 to 9, characterized in that: Used in long-acting contraceptive products.

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