Lactobacillus-loaded injectable temperature-sensitive hydrogel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedical materials, and discloses injectable temperature-sensitive hydrogel loaded with lactobacillus as well as a preparation method and application of the injectable temperature-sensitive hydrogel. The preparation method comprises the following steps: carrying out surface modification on bacterial cellulose to obtain bacterial cellulose containing carbon-carbon double bonds; grafting the bacterial cellulose containing the carbon-carbon double bond with a poly (N-isopropylacrylamide) high-molecular side chain to obtain a temperature-sensitive high-molecular brush; the temperature-sensitive polymer brush is dispersed in deionized water, and injectable temperature-sensitive hydrogel is obtained; and uniformly mixing the injectable temperature-sensitive hydrogel with a phosphate buffer salt solution containing the lactobacillus to obtain the injectable temperature-sensitive hydrogel loaded with the lactobacillus. The injectable hydrogel loaded with the lactobacillus can promote conversion between fluidity and in-situ stability through a thermal phase change behavior to realize efficient combination of easy injection and long retention, and can adjust a tissue microenvironment by utilizing the loaded lactobacillus, so that rapid repair of endometrial injury is facilitated, and the injectable hydrogel loaded with the lactobacillus has a wide application prospect.

Description

Technical Field

[0001] The present application relates to the technical field of biomedical materials, and in particular to an injectable thermosensitive hydrogel loaded with lactobacilli, and a preparation method and application thereof. Background Art

[0002] The endometrium is the site of embryo implantation and is usually divided into a functional layer and a basal layer. The functional layer plays a key role in successful pregnancy. However, endometrial damage caused by uterine inflammatory diseases or intrauterine operations (such as abortion, intrauterine contraceptive device placement, and hysteroscopic surgery) usually leads to poor regeneration of the functional layer, which may eventually lead to embryo implantation failure, resulting in uterine infertility. This reason accounts for about 15-20% of infertile women. Therefore, functional layer regeneration is the top priority for endometrial damage repair.

[0003] Intrauterine injection of sodium hyaluronate hydrogel to form a physical barrier is a common method for treating endometrial damage. However, due to the relatively low fluidity of the cross-linked polymer network, sodium hyaluronate hydrogel not only has a high injection pressure and difficulty in injection, but also tends to form an uneven distribution in the uterine cavity, making it difficult to cover the most severely damaged parts (deep recesses in the endometrium). At the same time, the in situ stability of the cross-linked polymer network is also easily weakened by the humid environment in the uterine cavity, resulting in a shorter retention time (usually less than 7 days), which is difficult to match the time span required for endometrial repair (the longest duration of the proliferation phase in a normal menstrual cycle is 21 days). In general, high-fluidity hydrogels usually require a relatively flexible structure, but this structure will have a negative impact on its in situ stability. At present, commercial sodium hyaluronate hydrogels still have difficulty in achieving a precise combination of high fluidity and high in situ stability, which is not conducive to providing an effective barrier function to meet clinical needs; even worse, sodium hyaluronate hydrogels lack sufficient bioactive ingredients to promote the regeneration of the functional layer, which easily leads to poor repair of endometrial damage.

[0004] Lactobacillus has been widely used as a functional component of hydrogels to promote tissue repair due to its excellent tissue microenvironment regulation effect, and has broad application prospects in vagina, skin and intestines. Since the application of lactobacillus has high requirements for its survival rate and biological activity, the ideal hydrogel carrier should accurately combine excellent biocompatibility, excellent fluidity and in situ stability to achieve uniform distribution and long-term retention of lactobacillus in the uterine cavity; in addition, the ideal hydrogel carrier should have a loose porous network to provide loading space and material exchange channels for lactobacillus.

[0005] Based on this, in the field of endometrial damage repair, developing a single hydrogel that integrates high fluidity, long retention, and microenvironmental controllability remains a huge challenge. Summary of the invention

[0006] The present application provides an injectable thermosensitive hydrogel loaded with lactobacillus, and a preparation method and application thereof, aiming to solve the technical problems of the imbalance between fluidity and in situ stability of existing injectable hydrogels, and the difficulty in promoting efficient repair of endometrial damage.

[0007] In order to achieve the above objectives, the present application adopts the following technical solutions.

[0008] In a first aspect of the present application, a method for preparing an injectable thermosensitive hydrogel loaded with lactobacilli is provided, comprising:

[0009] S1, surface modification of bacterial cellulose to obtain bacterial cellulose containing carbon-carbon double bonds;

[0010] S2, grafting the bacterial cellulose containing carbon-carbon double bonds with a poly(N-isopropylacrylamide) polymer side chain to obtain a temperature-sensitive polymer brush;

[0011] S3, dispersing the thermosensitive polymer brush in deionized water to obtain an injectable thermosensitive hydrogel;

[0012] S4, mixing the injectable thermosensitive hydrogel and the phosphate buffered saline solution containing lactobacilli evenly to obtain the injectable thermosensitive hydrogel loaded with lactobacilli.

[0013] Preferably, the surface modification in step S1 specifically includes:

[0014] The bacterial cellulose dispersion is added into an aqueous solution of a silane coupling agent containing a carbon-carbon double bond, and reacted at room temperature to obtain bacterial cellulose containing a carbon-carbon double bond.

[0015] The solid content of the bacterial cellulose dispersion is 0.6-1.0 wt %; the concentration of the aqueous solution of the silane coupling agent containing carbon-carbon double bonds is 1.0-2.0 mg mL -1 .

[0016] The corresponding relationship between the amount of the bacterial cellulose dispersion and the volume of the aqueous solution of the silane coupling agent containing a carbon-carbon double bond is 0.30-0.40 g mL -1 .

[0017] Further preferably, the silane coupling agent containing a carbon-carbon double bond includes at least one of vinyl triethoxysiloxane, vinyl trimethoxysiloxane, γ-methacryloxypropyl trimethoxysilane or vinyl-tri(2-methoxyethoxy)silane.

[0018] Preferably, in step S2, the step of grafting poly(N-isopropylacrylamide) polymer side chains comprises: dispersing bacterial cellulose containing carbon-carbon double bonds in a solvent, then adding N-isopropylacrylamide monomer to disperse evenly, and then adding an initiator to react at 60-70° C. under an inert atmosphere.

[0019] Further preferably, the mass ratio of the N-isopropylacrylamide monomer in step S2 to the bacterial cellulose dispersion in step S1 is (4.0-16.0):12.5.

[0020] Further preferably, the solvent is at least one of tetrahydrofuran, N,N-dimethylformamide, 1,4-dioxane or deionized water;

[0021] The initiator is at least one of azobisisobutyronitrile, azobisisobutyramidine hydrochloride or potassium persulfate;

[0022] The concentration of the N-isopropylacrylamide monomer is 0.10-0.40 g mL -1 , the concentration of initiator is 0.75~3.00mg mL -1 .

[0023] Preferably, in step S3, the corresponding relationship between the amount of the thermosensitive polymer brush and the volume of deionized water is 0.01-0.03 g mL -1 .

[0024] Preferably, in step S4, the lactobacillus is in a phosphate buffered saline solution with a concentration of 10 6 ~10 7 CFU mL -1 ;

[0025] The volume ratio of the phosphate buffered saline solution containing lactobacillus to the injectable thermosensitive hydrogel is (0.05-0.2):1.

[0026] The second aspect of the present application provides an injectable thermosensitive hydrogel loaded with lactobacillus prepared by the above preparation method.

[0027] The third aspect of the present application provides the use of the above-mentioned injectable thermosensitive hydrogel loaded with lactobacillus in a drug for treating endometrial damage.

[0028] Compared with the prior art, the beneficial effects of this application are:

[0029] The present application prepares a thermosensitive polymer brush by grafting surface-modified bacterial cellulose with a poly(N-isopropylacrylamide) polymer side chain, which is then dispersed in deionized water to obtain an injectable thermosensitive hydrogel, which is mixed with a phosphate buffered saline solution containing lactobacillus to obtain an injectable thermosensitive hydrogel loaded with lactobacillus. The high fluidity, long retention and microenvironmental controllability are integrated into a single hydrogel, which can not only promote the conversion of fluidity and in situ stability through thermal phase change behavior, achieving an efficient combination of easy injection and long retention, but also utilize the lactobacillus loaded thereon to regulate the tissue microenvironment, which is beneficial to the rapid repair of endometrial damage, and has broad application prospects.

[0030] The injectable thermosensitive hydrogel loaded with lactobacillus prepared in the present application, firstly, based on the good thermosensitivity of the poly(N-isopropylacrylamide) side chain, the hydrogel prepared in the present application can exhibit excellent fluidity and low injection pressure at 25°C (room temperature), which is conducive to achieving uniform distribution in the uterine cavity, and at 37°C (body temperature), a solid three-dimensional nanonetwork can be constructed through thermal phase change behavior, which is conducive to the long-term retention of the hydrogel in the moist environment of the uterine cavity to provide a stable physical barrier effect; secondly, the three-dimensional nanonetwork has interconnected pores, which provides loading space and material exchange channels for lactobacillus, which is conducive to maintaining the biological activity of lactobacillus; thirdly, lactobacillus can reduce inflammation and fibrosis, and regulate macrophage polarization, thereby effectively regulating the microenvironment and promoting the rapid repair of endometrial damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present application, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0032] Figure 1 Fourier transform infrared spectra of the bacterial cellulose sample containing carbon-carbon double bonds and the temperature-sensitive polymer brush sample provided in Example 1;

[0033] Figure 2 Scanning electron micrographs of the surface morphology of (a) bacterial cellulose sample, (b) bacterial cellulose sample containing carbon-carbon double bonds, (c) thermosensitive polymer brush sample, and (d) injectable thermosensitive hydrogel sample loaded with lactobacillus provided in Example 1;

[0034] Figure 3 This is a graph showing the transmittance test results of the bacterial cellulose sample, the injectable thermosensitive hydrogel sample and the injectable thermosensitive hydrogel sample loaded with lactobacillus provided in Example 1 at 808 nm at different temperatures;

[0035] Figure 4 The injectable thermosensitive hydrogel sample provided in Example 1, the injectable thermosensitive hydrogel sample loaded with lactobacillus, and the injection resistance diagram of commercial sodium hyaluronate hydrogel at different injection rates;

[0036] Figure 5 The figure is a test result of the retention rate of different types of hydrogels in the rat uterus on different days;

[0037] Figure 6 The figure shows the test results of the effects of different types of hydrogels on endometrial thickness, glandular number and fibrosis ratio in a rat uterine injury model. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0039] In the following description of this embodiment, the terms "include", "comprising", "having" and "containing" are all open terms, meaning including but not limited to.

[0040] In the following description of this embodiment, the term "and / or" is used to describe the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, B exists alone, and A and B exist at the same time. A and B can be singular or plural. The character " / " generally indicates that the associated objects before and after are in an "or" relationship.

[0041] In the following description of this embodiment, the term "at least one" refers to one or more, and "plurality" refers to two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b or c", or "at least one of a, b and c", can all represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0042] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0043] Those skilled in the art should understand that in the following description of the embodiments of the present application, the order of serial numbers does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0044] Those skilled in the art will appreciate that the numerical ranges in the embodiments of the present application are to be construed as specifically disclosing each intermediate value between the upper and lower limits of the scope. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present application. The upper and lower limits of these smaller ranges may be independently included or excluded in the scope.

[0045] Unless otherwise specified, the technical / scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this application belongs. Although this application only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of this application. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0046] The present application prepares a thermosensitive polymer brush by grafting surface-modified bacterial cellulose with a poly(N-isopropylacrylamide) polymer side chain, and then disperses the brush in deionized water to obtain an injectable thermosensitive hydrogel, which is mixed with a phosphate buffered saline solution containing lactobacilli to obtain an injectable thermosensitive hydrogel loaded with lactobacilli, integrating high fluidity, long retention and microenvironmental regulation into a single hydrogel. Specifically, the preparation method of the injectable thermosensitive hydrogel loaded with lactobacilli of the present application includes:

[0047] S1, surface modification of bacterial cellulose to obtain bacterial cellulose containing carbon-carbon double bonds;

[0048] In the present application, the surface modification specifically includes:

[0049] The bacterial cellulose dispersion with a solid content of 0.6-1.0 wt% is added into an aqueous solution of a silane coupling agent containing a carbon-carbon double bond, and reacted at room temperature to obtain bacterial cellulose containing a carbon-carbon double bond.

[0050] In the present application, the silane coupling agent containing a carbon-carbon double bond includes at least one of vinyl triethoxysiloxane, vinyl trimethoxysiloxane, γ-methacryloxypropyl trimethoxysilane or vinyl-tri(2-methoxyethoxy)silane. In the present application, the silane coupling agent containing a carbon-carbon double bond is dissolved in water, wherein the concentration of the silane coupling agent containing a carbon-carbon double bond is 1.0 to 2.0 mg mL -1 .

[0051] In the present application, the corresponding relationship between the amount of the bacterial cellulose dispersion and the volume of the aqueous solution of the silane coupling agent containing carbon-carbon double bonds is 0.30-0.40 g mL -1 Preferably, the amount of bacterial cellulose dispersion is 12.50 g, and the amount of the aqueous solution of the silane coupling agent containing a carbon-carbon double bond is 0.04 L.

[0052] S2, grafting the bacterial cellulose containing carbon-carbon double bonds with a poly(N-isopropylacrylamide) polymer side chain to obtain a temperature-sensitive polymer brush;

[0053] In the present application, the step of grafting poly(N-isopropylacrylamide) polymer side chains comprises: dispersing bacterial cellulose containing carbon-carbon double bonds in a solvent, then adding N-isopropylacrylamide monomer to disperse evenly, then adding an initiator, and reacting at 60-70° C. under an inert atmosphere. The inert atmosphere is a nitrogen atmosphere.

[0054] The solvent is at least one of tetrahydrofuran, N,N-dimethylformamide, 1,4-dioxane or deionized water; and the initiator is at least one of azobisisobutyronitrile, azobisisobutyramidine hydrochloride or potassium persulfate.

[0055] In the present application, the preferred mass ratio of N-isopropylacrylamide monomer to the bacterial cellulose dispersion in step S1 is (4.0-16.0):12.5.

[0056] In the present application, the concentration of the N-isopropylacrylamide monomer is preferably 0.10 to 0.40 g mL -1 , the concentration of initiator is 0.75~3.00mg mL -1 .

[0057] S3, dispersing the thermosensitive polymer brush in deionized water to obtain an injectable thermosensitive hydrogel;

[0058] In the present application, the preferred relationship between the amount of the thermosensitive polymer brush and the volume of deionized water is 0.01 to 0.03 g mL -1 , preferably 0.02 g mL -1 .

[0059] S4, mixing the injectable thermosensitive hydrogel and the phosphate buffered saline solution containing lactobacilli evenly to obtain the injectable thermosensitive hydrogel loaded with lactobacilli.

[0060] In the present application, it is preferred that the lactobacillus is in a phosphate buffered saline solution with a concentration of 10 6 ~10 7 CFU mL -1 ; The preferred volume ratio of the phosphate buffered saline solution containing lactobacilli to the injectable thermosensitive hydrogel is (0.05-0.2):1.

[0061] The injectable thermosensitive hydrogel loaded with lactobacillus prepared in the present application has high fluidity, long retention and microenvironmental regulation. It can not only promote the conversion of fluidity and in-situ stability through thermal phase change behavior, realize the efficient combination of easy injection and long retention, but also use the lactobacillus loaded therein to regulate the tissue microenvironment, which is conducive to the rapid repair of endometrial damage.

[0062] Specifically, based on the good temperature sensitivity of the poly(N-isopropylacrylamide) side chain, the hydrogel prepared in the present application can exhibit excellent fluidity and low injection pressure at 25°C (room temperature), which helps to achieve uniform distribution in the uterine cavity, and at 37°C (body temperature), a solid three-dimensional nanonetwork can be constructed through thermal phase change behavior, which is conducive to the long-term retention of the hydrogel in the moist environment of the uterine cavity to provide a stable physical barrier effect. In addition, the three-dimensional nanonetwork of the hydrogel of the present application has interconnected pores, which provides load space and material exchange channels for lactobacilli, which is conducive to maintaining the biological activity of lactobacilli; lactobacilli can reduce inflammation and fibrosis, and regulate macrophage polarization, thereby effectively regulating the microenvironment and promoting endometrial repair.

[0063] The injectable thermosensitive hydrogel loaded with lactobacillus of the present application can be used to prepare a drug for treating endometrial damage, or used as a drug for treating endometrial damage to treat endometrial damage.

[0064] The present application is further described below through specific examples.

[0065] Example 1

[0066] This embodiment provides a method for preparing an injectable thermosensitive hydrogel loaded with lactobacilli, comprising the following steps:

[0067] S1: 12.50 g bacterial cellulose dispersion (solid content 0.8 wt%) and 0.04 L aqueous solution containing vinyl triethoxysiloxane (concentration 1.25 mg mL -1) were mixed evenly, and the reaction was continued under stirring at room temperature for 24 hours. The obtained product was centrifuged, and then washed with deionized water and tetrahydrofuran three times each to obtain bacterial cellulose containing carbon-carbon double bonds.

[0068] S2: Disperse the bacterial cellulose containing carbon-carbon double bonds obtained in S1, N-isopropylacrylamide monomer, and azobisisobutyronitrile in 0.04 L of tetrahydrofuran to prepare a solution, wherein the concentration of N-isopropylacrylamide monomer is 0.20 g mL -1 , the concentration of azobisisobutyronitrile is 1.50 mg mL -1 The solution was stirred and ultrasonically dispersed for 30 minutes each, and then nitrogen was introduced for pre-protection for 30 minutes, and then stirred and reacted at 65°C for 24 hours. The obtained product was centrifuged and washed with deionized water for 5 times to obtain a thermosensitive polymer brush.

[0069] S3: Disperse the thermosensitive polymer brush into deionized water to obtain an injectable thermosensitive hydrogel, wherein the ratio of the thermosensitive polymer brush to deionized water is 0.02 g mL -1 ;

[0070] S4: Add phosphate buffered saline solution containing lactobacilli (the concentration of lactobacilli is 10 7 CFU mL -1 ) was mixed with an injectable thermosensitive hydrogel in a volume ratio of 0.1:1.0 to obtain an injectable thermosensitive hydrogel loaded with lactobacillus.

[0071] Embodiment 2-7

[0072] Compared with Example 1, the silane coupling agent and reaction time in S1, the solvent, initiator type, N-isopropylacrylamide monomer concentration, initiator concentration, polymerization reaction time, polymerization reaction temperature and other parameters in Example 2-7 were adjusted. The specific conditions are shown in Table 1.

[0073] Table 1 Related parameters for preparing injectable thermosensitive hydrogel loaded with lactobacillus in Examples 2-7

[0074]

[0075]

[0076] The structure of the injectable thermosensitive hydrogel loaded with lactobacilli prepared in Example 1 was characterized as follows:

[0077] (1) Fourier transform infrared spectrum

[0078] The bacterial cellulose containing carbon-carbon double bonds and the thermosensitive polymer brush prepared in Example 1 were characterized by infrared spectroscopy. The results are as follows: Figure 1 shown.

[0079] according to Figure 1 It can be seen that compared with bacterial cellulose containing carbon-carbon double bonds, the temperature-sensitive polymer brush has a higher -1 Two characteristic peaks were shown, originating from N–H and C=O bonds, respectively, confirming the existence of poly(N-isopropylacrylamide) polymer side chains.

[0080] (2) Elemental analysis

[0081] The bacterial cellulose containing carbon-carbon double bonds and the temperature-sensitive polymer brush prepared in Example 1 were characterized by elemental analysis. The results are shown in Table 2.

[0082] Table 2 Elemental analysis of bacterial cellulose containing carbon-carbon double bonds and thermosensitive polymer brushes in Example 1

[0083]

[0084] According to the results in Table 2, the side chain content (X) of the poly(N-isopropylacrylamide) polymer of the thermosensitive polymer brush was calculated. The specific calculation formula is as follows:

[0085]

[0086] Among them, A, B and C represent the N element content of the thermosensitive polymer brush, the relative atomic mass of N and the relative molecular mass of the structural unit in the poly(N-isopropylacrylamide) polymer side chain, respectively; from the data in Table 2, it can be obtained that the poly(N-isopropylacrylamide) polymer side chain content is 60wt%.

[0087] (3) Scanning electron microscope photos

[0088] Scanning electron microscope photographs were taken of the bacterial cellulose, the bacterial cellulose containing carbon-carbon double bonds, the thermosensitive polymer brush, and the injectable thermosensitive hydrogel loaded with lactobacilli in the preparation method of Example 1 to observe the microstructure. The results are as follows: Figure 2 shown. Figure 2 In the figure, the four scanning electron micrographs are scanning electron micrographs of the surface morphology of (a) bacterial cellulose sample, (b) bacterial cellulose sample containing carbon-carbon double bonds, (c) thermosensitive polymer brush sample and (d) injectable thermosensitive hydrogel sample loaded with lactobacillus.

[0089] according to Figure 2 (a)-(c) show that after modification with silane coupling agent and graft polymerization reaction, the obtained thermosensitive polymer brush still retains a good one-dimensional structure. Figure 2(d) shows that after the thermosensitive polymer brush aqueous dispersion is blended with lactobacillus, the resulting injectable thermosensitive hydrogel loaded with lactobacillus has a three-dimensional nanonetwork structure formed by overlapping polymer brushes, and the lactobacillus is evenly distributed in the interconnected pores, achieving effective loading of lactobacillus.

[0090] (4) UV-visible spectrum

[0091] The transmittance of the bacterial cellulose, the injectable thermosensitive hydrogel and the injectable thermosensitive hydrogel loaded with lactobacillus in the preparation method of Example 1 at 808 nm at different temperatures was tested by UV-visible spectrophotometer. The test results are shown in FIG. Figure 3 shown.

[0092] according to Figure 3 It can be seen that compared with bacterial cellulose, the permeability of injectable thermosensitive hydrogel and injectable thermosensitive hydrogel loaded with lactobacillus is significantly reduced in the range of 32-34°C, indicating that the side chain of poly(N-isopropylacrylamide) polymer undergoes thermal phase change behavior, which helps to form a rigid three-dimensional nanonetwork, thereby achieving a precise combination of fluidity and in situ stability.

[0093] The performance test of the injectable thermosensitive hydrogel loaded with lactobacilli prepared in Example 1 was carried out, specifically including:

[0094] (I) Injection resistance test

[0095] The injection resistance of the injectable thermosensitive hydrogel and the injectable thermosensitive hydrogel loaded with lactobacillus in the preparation method of test example 1, as well as the commercial sodium hyaluronate hydrogel at different injection rates, is shown in the injection resistance diagram Figure 4 Data are presented as means plus or minus standard deviations (n=3).

[0096] according to Figure 4 It can be seen that at room temperature, when the injection rate is 0.5 mL min -1 When the injection speed was increased to 2 mL min, the injection pressure of the injectable thermosensitive hydrogel and the injectable thermosensitive hydrogel loaded with lactobacillus (5.17 and 5.07 kPa, respectively) was significantly lower than that of the sodium hyaluronate hydrogel (30.30 kPa). -1 When the injection pressure of the injectable thermosensitive hydrogel and the injectable thermosensitive hydrogel loaded with lactobacillus increased slightly to 7.93 and 7.90 kPa, respectively, while the injection pressure of the sodium hyaluronate hydrogel increased greatly to 70.70 kPa. The above results show that benefiting from the flexible structure of the poly(N-isopropylacrylamide) polymer side chain at room temperature, the injectable thermosensitive hydrogel loaded with lactobacillus has excellent fluidity, which helps to reduce the difficulty of injection and promote its uniform distribution in the uterine cavity.

[0097] (II) Retention rate of hydrogel in rat uterus at different days

[0098] After the injectable thermosensitive hydrogel loaded with lactobacillus prepared in Example 1 and the commercial sodium hyaluronate hydrogel were injected into the rat uterus, the retention rate of the hydrogel samples was evaluated by quantitative analysis of the fluorescence intensity calculated by small animal in vivo imaging software at 1, 3, 5, 7, 14 and 21 days; the data were presented in the form of mean plus or minus standard deviation (n=3), and the fluorescent dye was CY5.5-active ester. The test results are shown in Figure 5 shown.

[0099] according to Figure 5 It can be seen that the retention rate of the injectable thermosensitive hydrogel loaded with lactobacillus on the 7th day (40%) was significantly higher than that of the sodium hyaluronate hydrogel (10%). Even on the 21st day, the injectable thermosensitive hydrogel loaded with lactobacillus was still retained in the rat uterus, while the sodium hyaluronate hydrogel group had almost no obvious residue, indicating that the side chain of the poly(N-isopropylacrylamide) polymer can promote the formation of a strong three-dimensional nanonetwork through thermal phase transition behavior at 37°C, thereby improving the in-situ stability of the hydrogel to achieve long-term retention.

[0100] (III) Effects of hydrogel on endometrial thickness, gland number and fibrosis ratio in rat uterine injury model

[0101] The injectable thermosensitive hydrogel prepared in Example 1, the injectable thermosensitive hydrogel loaded with lactobacillus, and the commercial sodium hyaluronate hydrogel were injected into the damaged rat uterus to observe their effects on endometrial repair; wherein, the blank group was a normal rat uterus, and the control group was a rat uterus injury without subsequent treatment. The intervention time for all groups was 7 days. The test results are as follows: Figure 6 shown.

[0102] Figure 6 In the figure, (a) and (b) are HE staining and Masson staining images of rat endometrial injury after intervention in different groups, respectively. The upper and lower scale bars are 300 μm and 30 μm, respectively; (c) is the quantitative analysis of endometrial thickness, which was statistically analyzed by one-way analysis of variance and Dunnett's multiple comparisons, and the data are presented in the form of mean plus minus standard deviation (n=9); (d) is the quantitative analysis of gland number, which was statistically analyzed by one-way analysis of variance and Dunnett's multiple comparisons, and the data are presented in the form of mean plus minus standard deviation (n=9); (e) is the quantitative analysis of collagen relative area coverage, which was statistically analyzed by one-way analysis of variance and Dunnett's multiple comparisons, and the data are presented in the form of mean plus minus standard deviation (n=9).

[0103] according to Figure 6 (a) It can be seen that the functional layer cell arrangement of the injectable thermosensitive hydrogel group loaded with lactobacillus is similar to that of the blank group, while the sodium hyaluronate group and the injectable thermosensitive hydrogel group have irregular functional layer tissue cell arrangement similar to that of the control group. Figure 6 (c) and (d) show that the endometrial thickness and the number of regenerated endometrial glands in the injectable thermosensitive hydrogel group loaded with lactobacillus were almost the same as those in the blank group, showing an efficient repair effect. Figure 6 Masson staining in (b) shows that the collagen structure of the injectable thermosensitive hydrogel group loaded with lactobacilli is also similar to that of the blank group. Figure 6 As shown in (e), compared with the sodium hyaluronate group and the injectable thermosensitive hydrogel group, the fibrosis area of ​​the injectable thermosensitive hydrogel group loaded with lactobacillus was significantly reduced. The above results show that the injectable thermosensitive hydrogel group loaded with lactobacillus has excellent biological activity, exhibits excellent tissue microenvironment regulation, and is conducive to the efficient repair of endometrial damage.

[0104] Although the present application has been described in detail in general terms and in specific embodiments in this specification, it is obvious to those skilled in the art that some modifications or improvements may be made to the present application. Therefore, these modifications or improvements made without departing from the spirit of the present application are within the scope of protection claimed in the present application.

Claims

1. A method for preparing an injectable thermosensitive hydrogel loaded with lactobacilli, characterized in that: include: S1, surface modification of bacterial cellulose to obtain bacterial cellulose containing carbon-carbon double bonds; S2, grafting the bacterial cellulose containing carbon-carbon double bonds with a poly(N-isopropylacrylamide) polymer side chain to obtain a temperature-sensitive polymer brush; S3, dispersing the thermosensitive polymer brush in deionized water to obtain an injectable thermosensitive hydrogel; S4, mixing the injectable thermosensitive hydrogel and the phosphate buffered saline solution containing lactobacilli evenly to obtain the injectable thermosensitive hydrogel loaded with lactobacilli.

2. The preparation method according to claim 1, characterized in that: The surface modification in step S1 specifically includes: The bacterial cellulose dispersion is added into an aqueous solution of a silane coupling agent containing a carbon-carbon double bond, and reacted at room temperature to obtain bacterial cellulose containing a carbon-carbon double bond; The solid content of the bacterial cellulose dispersion is 0.6-1.0 wt %; the concentration of the aqueous solution of the silane coupling agent containing carbon-carbon double bonds is 1.0-2.0 mg mL -1 ; The corresponding relationship between the amount of the bacterial cellulose dispersion and the volume of the aqueous solution of the silane coupling agent containing a carbon-carbon double bond is 0.30-0.40 g mL -1 .

3. The preparation method according to claim 2, characterized in that: The silane coupling agent containing a carbon-carbon double bond includes at least one of vinyl triethoxysiloxane, vinyl trimethoxysiloxane, γ-methacryloxypropyl trimethoxysilane or vinyl-tri(2-methoxyethoxy)silane.

4. The preparation method according to claim 1, characterized in that: In step S2, the step of grafting poly(N-isopropylacrylamide) polymer side chains includes: dispersing bacterial cellulose containing carbon-carbon double bonds in a solvent, then adding N-isopropylacrylamide monomer to disperse evenly, and then adding an initiator to react at 60-70° C. under an inert atmosphere.

5. The preparation method according to claim 4, characterized in that: The mass ratio of the N-isopropylacrylamide monomer in step S2 to the bacterial cellulose dispersion in step S1 is (4.0-16.0):12.

5.

6. The preparation method according to claim 4, characterized in that: The solvent is at least one of tetrahydrofuran, N,N-dimethylformamide, 1,4-dioxane or deionized water; The initiator is at least one of azobisisobutyronitrile, azobisisobutyramidine hydrochloride or potassium persulfate; The concentration of the N-isopropylacrylamide monomer is 0.10-0.40 g mL -1 , the concentration of initiator is 0.75~3.00mg mL -1 .

7. The preparation method according to claim 1, characterized in that: In step S3, the corresponding relationship between the amount of the thermosensitive polymer brush and the volume of deionized water is 0.01-0.03 g mL -1 .

8. The preparation method according to claim 1, characterized in that: In step S4, the concentration of the lactobacillus in the phosphate buffered saline solution is 10 6 ~10 7 CFU mL -1 ; The volume ratio of the phosphate buffered saline solution containing lactobacillus to the injectable thermosensitive hydrogel is (0.05-0.2):

1.

9. The injectable thermosensitive hydrogel loaded with lactobacilli prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the injectable thermosensitive hydrogel loaded with lactobacilli according to claim 9 in a drug for treating endometrial damage.

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