Novel oral delivery microcarrier with intestinal adhesion function and preparation method thereof

Abstract

The invention discloses a novel oral delivery microcarrier with an intestinal adhesion function and a preparation method thereof, and the preparation method specifically comprises the following steps: preparing methacrylic anhydride gelatin, o-nitrobenzaldehyde group gelatin, a photoinitiator, a color indicator and a drug to be loaded into a mixed solution, and preparing microspheres by using a microfluidic instrument, and irradiating with an ultraviolet light source until curing to obtain the novel oral delivery microcarrier with the intestinal adhesion function. According to the invention, the limitation of the existing traditional single material is overcome, the compounding ratio of GelMA to GelNB is optimized, and the stability and biological activity of the microspheres are ensured; drug molecules are successfully and uniformly entrapped in the microspheres through an advanced microsphere preparation microfluidic technology; the targeting delivery efficiency of the medicine is improved, the healing and regeneration of tissues are promoted, and a new strategy and a new tool are provided for disease treatment and tissue engineering application.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a novel oral delivery microcarrier with intestinal adhesion function and a preparation method thereof. Background Art

[0002] At present, sustained-release microspheres have shown great potential in the field of biomedicine. However, most sustained-release microspheres do not have intestinal adhesion function and have a short residence time in the body. Even if some have a certain adhesion ability, the adhesion effect on wet surfaces is poor. Therefore, improving the adhesion ability of sustained-release microspheres on wet surfaces in vivo is the key to achieving efficient treatment.

[0003] Ortho-nitrosobenzaldehyde-modified gelatin (GelNB), by introducing ortho-nitrosobenzaldehyde groups, not only enhances the chemical stability and biocompatibility of gelatin, but also opens up new avenues for its application in drug delivery systems. With its improved physicochemical properties, GelNB has become a preferred material for constructing tissue engineering scaffolds and drug carriers. However, GelNB alone still faces limitations in achieving efficient drug controlled release and promoting good adhesion to tissue interfaces.

[0004] Methacrylate anhydride gelatin (GelMA) hydrogel has good biocompatibility, controllable degradation, and is easy to functionalize. Therefore, the present invention focuses on designing and synthesizing a hybrid microsphere system of GelMA and GelNB with tissue adhesion properties, which is optimized for drug-loaded sustained-release applications to meet complex therapeutic needs, such as enhancing intestinal tissue adhesion, promoting tissue integration, and achieving long-term controllable drug release. Summary of the invention

[0005] The purpose of the present invention is to provide a novel oral delivery microcarrier with intestinal adhesion function and a preparation method thereof in view of the deficiencies of the prior art.

[0006] To achieve the above object, the present invention adopts the following technical scheme: a method for preparing a novel oral delivery microcarrier with intestinal adhesion function, comprising the following steps:

[0007] S1, prepare a mixed solution of methacrylic anhydride gelatin, o-nitrosobenzaldehyde grouped gelatin, photoinitiator, color indicator, water and the drug to be loaded, so that the final concentrations of methacrylic anhydride gelatin, o-nitrosobenzaldehyde grouped gelatin, photoinitiator and color indicator are 40 mg / mL, 10 mg / mL, 5 mg / mL and 1 mg / mL respectively;

[0008] S2. The mixed solution is prepared into microspheres, and irradiated with an ultraviolet light source until solidified, thereby obtaining a novel oral delivery microcarrier with intestinal adhesion function.

[0009] Preferably, in the step (S1), methacrylic anhydride-modified gelatin, o-nitrosobenzaldehyde-modified gelatin, a photoinitiator, a color indicator, water and the drug to be loaded are placed in a water bath at 37° C. to prepare a mixed solution; the drug to be loaded is preferably an aqueous solution.

[0010] Preferably, in step (S1), light is avoided throughout the process.

[0011] Furthermore, in the step (S2), the mixed solution is prepared into microspheres by placing the mixed solution in a light-proof container as the water phase; taking 20 mg / mL of droplet-generated oil as the oil phase; setting the water phase and oil phase flow rates to 1 mL / h and 4 mL / h, respectively, and loading them on a microfluidic instrument connected to a 100 μm cross droplet chip to prepare microspheres.

[0012] Furthermore, in step (S2), the intensity of the ultraviolet light source is 28.8 mW / cm 2 , wavelength is 340-380nm.

[0013] Preferably, the wavelength of the ultraviolet light source is 365 nm.

[0014] Furthermore, in the step (S2), after the ultraviolet light source is irradiated until solidified, the following steps are also included:

[0015] Aspirate the droplet-generating oil;

[0016] Add drop-breaker to submerge the top layer of microspheres, centrifuge, aspirate the supernatant, and repeat once;

[0017] Add water twice as much as the demulsifier, pipette and beat, centrifuge, aspirate the supernatant, and repeat adding water twice as much as the demulsifier and pipetting.

[0018] Furthermore, the centrifugal speed is 600 RPM.

[0019] The present invention also provides a novel oral delivery microcarrier with intestinal adhesion function prepared by the above preparation method.

[0020] The beneficial effects of the present invention are as follows:

[0021] (1) The present invention overcomes the limitations of existing traditional single materials, optimizes the composite ratio of GelMA and GelNB, and ensures the stability and biological activity of the microspheres.

[0022] (2) The present invention successfully and uniformly encapsulates drug molecules in microspheres through advanced microsphere preparation microfluidic technology.

[0023] (3) The present invention not only improves the targeted delivery efficiency of drugs, but also promotes tissue healing and regeneration, providing new strategies and tools for disease treatment and tissue engineering applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0025] Figure 1 : Photorheological plot of a novel oral delivery microcarrier with intestinal adhesion function;

[0026] Figure 2 :Light microscopic image of a novel oral delivery microcarrier with intestinal adhesion function;

[0027] Figure 3 :Electron microscopic image of a novel oral delivery microcarrier with intestinal adhesion function;

[0028] Figure 4 :Adhesion performance diagram of a novel oral delivery microcarrier with intestinal adhesion function;

[0029] Figure 5 :Drug sustained release diagram of a novel oral delivery microcarrier with intestinal adhesion function (taking dexamethasone as an example);

[0030] Figure 6 :Biocompatibility of a novel oral delivery microcarrier with intestinal adhesion function (in vitro);

[0031] Figure 7 : Biocompatibility of a novel oral delivery microcarrier with intestinal adhesion function (in vivo);

[0032] Figure 8 :Application of a novel oral delivery microcarrier with intestinal adhesion function in a radiation enteritis model (in vitro);

[0033] Fig. 9 : Application of a novel oral delivery microcarrier with intestinal adhesion function in a radiation enteritis model (in vivo); wherein, (A) is a schematic diagram of representative small intestine lengths of mice in each group, and (B) is a statistical diagram of small intestine lengths of mice in each group. DETAILED DESCRIPTION

[0034] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0035] The present invention is further described with reference to specific examples. The following examples are only for explaining the present invention and are not intended to limit the contents thereof.

[0036] The invention prepares a novel oral delivery microcarrier with intestinal adhesion function.

[0037] Using commercial methacrylic anhydride gelatin and o-nitrosobenzaldehyde grouped gelatin mentioned in patent CN114931629A, a novel oral delivery microcarrier with intestinal adhesion function is prepared by microfluidic instrumentation. The specific method is as follows:

[0038] S1. The methacrylic anhydride gelatin and the o-nitrosobenzaldehyde grouped gelatin are mixed, and a photoinitiator and a drug to be loaded are added, and heated in a water bath to form a mixed solution;

[0039] S2. The mixed solution is prepared into microspheres using a microfluidic device and irradiated with an ultraviolet light source to solidify them into a new type of oral delivery microcarrier with intestinal adhesion function.

[0040] Preferably, the S1 step is specifically implemented as follows: methacrylic anhydride gelatin (GelMA) and o-nitrosobenzaldehyde grouped gelatin (GelNB) (preferably at a final concentration of 40 mg / mL and 10 mg / mL) are added to purified water, and then a photoinitiator phenyl-2,4,6-trimethylbenzoylphosphinate lithium (LAP) (preferably at a final concentration of 5 mg / mL) is added, and a color indicator brilliant blue (preferably at a final concentration of 1 mg / mL) is added, and the required loaded drug (preferably a drug aqueous solution) is added as needed, and the mixed system is heated in a water bath (preferably at 37° C.) to form a mixed solution system. Preferably, light is avoided throughout the process as much as possible.

[0041] Preferably, the S2 step is specifically implemented as follows: the obtained mixed solution system is placed in a light-proof container and loaded into a microfluidic instrument (preferably transferred to a syringe with a diameter of 4.7 mm and a capacity of 1 mL) as the aqueous phase. In addition, a droplet-generated oil (preferably at a concentration of 20 mg / mL, preferably transferred to a syringe with a diameter of 4.7 mm and a capacity of 1 mL) is selected as the oil phase. The preferred water-to-oil flow ratio is 1 mL / h: 4 mL / h. Use a 100 μm cross droplet chip to connect the already set microfluidic instrument, turn on the microfluidic instrument to prepare microspheres, and collect the prepared, temporarily uncured microspheres into an ep tube, and place it at an intensity of 28.8 mW / cm 2Under a UV light source, solidify it for 1 minute to obtain a novel oral delivery microcarrier with intestinal adhesion function. The wavelength of the near-ultraviolet light source is 340-380nm, and it is further preferred that the wavelength of the near-ultraviolet light source is 365nm. The droplet-generated oil in the ep tube is sucked out as much as possible; add drop-breaker demulsifier to immerse the top layer of microspheres, and centrifuge at a speed of 600RPM, suck out the supernatant, and repeat this step; add pure water and blow it gently, transfer it to a new clean ep tube, centrifuge it again, suck out the supernatant again, add pure water and blow it gently.

[0042] Example 1: Preparation of a novel oral delivery microcarrier with intestinal adhesion function

[0043] (Take the preparation of 1mL solution as an example) Methacrylic anhydride gelatin (0.04g), o-nitrosobenzaldehyde grouped gelatin (0.01g), 50mg / mL phenyl-2,4,6-trimethylbenzoylphosphinate lithium (LAP) (0.1ml), 10mg / mL brilliant blue (0.1ml) and 1.5mg / mL dexamethasone solution (0.75ml). Dissolve the mixture in a 37°C water bath to form a mixed solution system. Protect from light throughout the process. Among them, o-nitrosobenzaldehyde grouped gelatin is prepared according to the method mentioned in CN114931629A.

[0044] Transfer the mixed solution to a syringe with a diameter of 4.7 mm and a capacity of 1 mL as the water phase, and wrap the syringe with tin foil to protect it from light. Select another droplet-generating oil with a concentration of 20 mg / mL, and transfer it to a syringe with a diameter of 4.7 mm and a capacity of 1 mL as the oil phase. Adjust the water-oil flow ratio of the microfluidic instrument to 1 mL / h:4 mL / h, use a 100 μm cross droplet chip to connect the already set microfluidic instrument, turn on the microfluidic instrument to prepare microspheres, and collect the prepared, yet unsolidified microspheres into a 1.5 mL ep tube and place it at an intensity of 28.8 mW / cm 2 The near-ultraviolet light source is placed under a UV light source and cured for 1 minute to obtain a novel oral delivery microcarrier with intestinal adhesion function. The wavelength of the near-ultraviolet light source is 340-380 nm; more preferably, the wavelength of the near-ultraviolet light source is 365 nm.

[0045] The microdroplet-generated oil in the ep tube was sucked out as much as possible, and an appropriate amount of drop-breaker demulsifier was added (immersing the top layer of the microspheres), and the mixture was centrifuged at 600RPM, and the supernatant was sucked out, and the above steps were repeated. Then, pure water (twice the volume of the demulsifier) ​​was added and gently blown, and the mixture was transferred to a new clean ep tube, centrifuged again, and the supernatant was sucked out again, and pure water (twice the volume of the demulsifier) ​​was added and gently blown. A novel oral delivery microcarrier with intestinal adhesion function dispersed in pure water was obtained. It was stored at 4°C for further use.

[0046] Example 2: Photorheological properties of novel oral delivery microcarriers with intestinal adhesion function

[0047] Using an MCR302 rheometer, at 25°C (room temperature), the light intensity was set to 28.8 mW / cm 2 The material was measured using a UV light source. The results are shown in Figure 1 .

[0048] Example 3: Light microscopic morphology of a novel oral delivery microcarrier with intestinal adhesion function

[0049] The microspheres obtained in Example 1 were placed in a 10 cm culture dish and some pure water was added to make them more dispersed. The culture dish was placed under an optical microscope (Nikon, Japan) and the microspheres were photographed. It can be seen that the diameter of the microspheres is about 150 μm. The results are as follows Figure 2 shown.

[0050] Example 4: Electron microscopic morphology of a novel oral delivery microcarrier with intestinal adhesion function

[0051] The microspheres obtained in Example 1 were dehydrated with ethanol solutions of gradient concentrations (including six concentrations of 30%, 50%, 70%, 80%, 90% and 95%, expressed as volume fractions), and each concentration was treated for 15 minutes, and then treated once with 100% ethanol for 20 minutes. Finally, new 100% ethanol was used. The samples were placed in 100% ethanol to end the pretreatment for the day. Critical point drying: Drying was performed in a Hitachi HCP-2 critical point dryer. Coating and observation. The treated samples were observed in a Hitachi SU-8010 scanning electron microscope. The results are shown in the figure. Figure 3 shown.

[0052] Example 5: Determination of the adhesion properties of a novel oral delivery microcarrier with intestinal adhesion function

[0053] Methacrylic anhydride gelatin solution (final concentration 50 mg / mL) and methacrylic anhydride gelatin / o-nitrosobenzaldehyde grouped gelatin mixed solution (final concentrations 40 mg / mL and 10 mg / mL, respectively) were smeared on an amino glass slide and heated at an intensity of 28.8 mW / cm 2 The material was cured under ultraviolet light for 1 minute. The material has been connected to the isothiocyanate group of fluorescein isothiocyanate (FITC) to achieve fluorescent labeling. The maximum absorption wavelength of FITC is 490-495nm, and the maximum emission wavelength is 520-530nm. Therefore, it is excited by blue light (488) and photographed with a green light (518) filter.

[0054] Then they were placed in PBS, simulated gastric fluid, and simulated intestinal fluid, and photographed using a fluorescence microscope at the same time. The light intensity represented the remaining amount of material remaining on the amino-treated glass slide, which indirectly characterized the adhesion ability of the material. Figure 4 .

[0055] Example 6: Determination of sustained release performance of a novel oral delivery microcarrier with intestinal adhesion function (taking dexamethasone as an example)

[0056] The standard curve of dexamethasone concentration was drawn using an ELISA instrument. The maximum wavelength of the dexamethasone absorption peak was 240 nm. The standard curve was y = 15.18x + 0.0905, R 2 =0.9999.

[0057] Weigh a certain mass of microspheres, place them in an ep tube, add 200 μl of PBS / gastric juice (SGF) / intestinal juice (SIF), place the ep tube in a metal bath at 37°C, and at regular intervals, draw 100 μl (as needed) of the solution with a pipette, measure with an enzyme reader, and refill the ep tube to 200 μl.

[0058] The data measured by the microplate reader were converted into drug concentration using the standard curve, and the sustained-release curve was drawn. Figure 5 .

[0059] This model experiment shows that the microcarrier can safely pass through the gastric juice (SGF) environment and enter the intestinal (SIF) environment for release, thus providing more possibilities for the treatment of intestinal diseases.

[0060] Example 7: Biocompatibility of a novel oral delivery microcarrier with intestinal adhesion function (in vitro)

[0061] NCM460 is a colon mucosal cell line purchased from Shanghai Cell Bank and cultured in DMEM containing 10% fetal bovine serum and 100 mg / mL penicillin-streptomycin at 37°C in a 5% CO2 incubator.

[0062] The microspheres in Example 1 were added to DMEM and filtered to obtain a microsphere extract (DMEM). The obtained extract was used to culture NCM460 in a 96-well plate at a seeding density of 3000 cells / well (named mDex group). At the same time, pure DMEM (named Blank group), DMEM containing dexamethasone (concentration of 64 μM, named Dex group), and DMEM extract of microspheres without drug loading (named mH2O group) were used for cell culture.

[0063] Cell viability was measured using the CCK-8 assay. CCK-8 reagent (Yeasen) was diluted to 1:10 (v / v) in DMEM (Gibco), and 100 μL of CCK-8 dilution was added to each well, incubated at 37°C for 1 h, and measured at 450 nm using a microplate reader (Thermo MK3, USA).

[0064] like Figure 6 The CCK-8 assay results showed that there was no significant difference in the cell proliferation ability of the four groups of cells on the third day (P>0.05), indicating that the microspheres have good biocompatibility in vitro.

[0065] Example 8: Biocompatibility of a novel oral delivery microcarrier with intestinal adhesion function (in vivo)

[0066] Eight-week-old male C57 BL / 6 (C57) mice were used for the experiments. All experiments were performed in strict accordance with the institutional guidelines of the Animal Care and Use Committee of the Run Run Shaw Hospital, Zhejiang University School of Medicine, and in compliance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (ethical code SRRSH202107106).

[0067] Mice were divided into 4 groups, the same as in Example 7, with 6 mice in each group. All mice were adaptively fed for one week. Blank group, Dex group, mH2O group and mDex group were gavaged every day, and the gavage contents were purified water, dexamethasone aqueous solution (64 μM), microspheres without drug loading, and microspheres loaded with dexamethasone. The gavage volume was 200 μL. The gavage was continued for 7 days, and the mice were killed on the 8th day.

[0068] The heart, liver, spleen, lung, kidney and brain of each group of mice were removed and subjected to H&E staining.

[0069] The obtained H&E sections were scanned using an imaging device to observe the tissue damage of mice in each group. Figure 7 As shown, there was no significant difference among the four groups, and the new oral delivery microcarrier had good biocompatibility.

[0070] Example 9: Application of a novel oral delivery microcarrier with intestinal adhesion function in a radiation enteritis model (in vitro)

[0071] The NCM460 cell line and the purified extract were the same as those in Example 7.

[0072] NCM460 was cultured in 96-well plates with a seeding density of 3000 cells / well. The cells were divided into 5 groups, namely Blank group, NC group, Dex group, mH2O group and mDex group. Among them, the cells in the NC group, Dex group, mH2O group and mDex group received a one-time 6Gy dose of radiation from the irradiator. The cells in the Blank group, NC group, Dex group, mH2O group and mDex group were given pure DMEM, pure DMEM, DMEM containing dexamethasone (concentration of 64μM, named Dex group), DMEM pure extract of microspheres without drug loading (named mH2O group), and DMEM pure extract of microspheres loaded with dexamethasone (named mDex group) for cell culture.

[0073] Cell viability was measured using the CCK-8 assay. CCK-8 reagent (Yeasen) was diluted to 1:10 (v / v) in DMEM (Gibco), and 100 μL of CCK-8 dilution was added to each well, incubated at 37°C for 1 h, and measured at 450 nm using a microplate reader (Thermo MK3, USA).

[0074] like Figure 8 The CCK-8 assay results showed that among the four groups of cells receiving radiation, the mDex group had better cell proliferation ability on the third day (P<0.05), indicating that the new oral delivery microcarrier can achieve relief of the in vitro model of radiation enteritis.

[0075] Example 10: Application of a novel oral delivery microcarrier with intestinal adhesion function in a radiation enteritis model (in vivo)

[0076] The mice were the same as in Example 8.

[0077] Mice were divided into 5 groups, namely Blank group, NC group, Dex group, mH2O group and mDex group, with 6 mice in each group. All mice were adaptively raised for one week. Except for the Blank group, the other 4 groups of mice received a one-time 6Gy dose of radiation using an irradiator, and the irradiation range was the abdomen of the mice. Thereafter, the Blank group, NC group, Dex group, mH2O group and mDex group were gavaged every day, and the gavage contents were purified water, purified water, dexamethasone aqueous solution (64μM), microspheres without drug loading, and microspheres loaded with dexamethasone. The gavage volume was 200μL. Gavage was performed continuously for 7 days, and the mice were killed on the 8th day.

[0078] The small intestines of mice in each group were taken out and the length of the small intestines was compared. Fig. 9 , indicating that this novel oral delivery microcarrier can achieve relief in the in vivo model of radiation enteritis.

[0079] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for preparing a novel oral delivery microcarrier with intestinal adhesion function, characterized in that: The following steps are involved: S1, prepare a mixed solution of methacrylic anhydride gelatin, o-nitrosobenzaldehyde grouped gelatin, photoinitiator, color indicator, water and the drug to be loaded, so that the final concentrations of methacrylic anhydride gelatin, o-nitrosobenzaldehyde grouped gelatin, photoinitiator and color indicator are 40 mg / mL, 10 mg / mL, 5 mg / mL and 1 mg / mL respectively; S2. The mixed solution is prepared into microspheres, and irradiated with an ultraviolet light source until solidified, thereby obtaining a novel oral delivery microcarrier with intestinal adhesion function.

2. The preparation method according to claim 1, characterized in that: In the step (S1), methacrylic anhydride-treated gelatin, o-nitrosobenzaldehyde-treated gelatin, a photoinitiator, a color indicator, water and a drug to be loaded are placed in a water bath at 37° C. to prepare a mixed solution.

3. The preparation method according to claim 1, characterized in that: In the step (S1), light is avoided throughout the process.

4. The preparation method according to claim 1, characterized in that: In the step (S2), the mixed solution is prepared into microspheres by placing the mixed solution in a light-proof container as the water phase; taking 20 mg / mL of droplet-generated oil as the oil phase; setting the flow rates of the water phase and the oil phase to 1 mL / h and 4 mL / h, respectively, and loading the mixture into a microfluidic instrument connected to a 100 μm cross droplet chip to prepare the microspheres.

5. The preparation method according to claim 1, characterized in that: In the step (S2), the intensity of the ultraviolet light source is 28.8 mW / cm 2 , wavelength is 340-380nm.

6. The preparation method according to claim 4, characterized in that: In the step (S2), after the ultraviolet light source is irradiated until solidified, the following steps are also included: Aspirate the droplet-generating oil; Add drop-breaker to submerge the top layer of microspheres, centrifuge, aspirate the supernatant, and repeat once; Add twice the amount of water as the demulsifier, pipette and centrifuge, remove the supernatant, and repeat the process of adding water and pipetting.

7. The preparation method according to claim 6, characterized in that: The centrifugal speed was 600 RPM.

8. A novel oral delivery microcarrier with intestinal adhesion function prepared by the preparation method according to any one of claims 1 to 7.

Images