Dual release microspheres, methods of making and using the same

Abstract

The application discloses a double-release microsphere, which comprises: a PLGA microsphere loaded with a drug and an alginate microcapsule, wherein the alginate microcapsule is an alginate microcapsule loaded with stem cell exosomes or / and stem cell-derived vesicles. The application further discloses a preparation method of the double-release microsphere, which comprises: loading stem cell exosomes or / and stem cell-derived vesicles into and preparing the alginate microcapsule by a W / O emulsification method, wherein strontium ions are used as a crosslinking agent in the preparation process; loading the drug and the alginate microcapsule into PLGA and preparing the double-release microsphere by an S / O / W emulsification method. The application further discloses application of the double-release microsphere in preparation of a medicine for treating spinal cord injury. The preparation method is simple and easy to control. When the double-release microsphere is applied in preparation of the medicine for treating spinal cord injury, the treatment substances wrapped in the inner and outer layers of the double-release microsphere are released into tissues at different times, play a treatment role, and finally promote repair and regeneration of the damaged tissues in a synergistic manner.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bioengineering, in particular to a double-release microsphere and a preparation method and application thereof. BACKGROUND

[0002] Traumatic spinal cord injury (SCI) refers to the temporary or permanent functional damage of spinal cord tissue caused by external force. Although the incidence of the disease is increasing, there is still a lack of effective treatment in clinical practice. One of the reasons is that the pathological process of SCI is quite complex, and the pathological microenvironment is extremely unfavorable for cell survival. In a short time after injury, the spinal cord becomes a highly inflammatory state, and the continuous release of inflammatory cytokines leads to the amplification of the inflammatory cascade and begins to induce a large number of active oxygen, causing the injury to spread from the original area to the surrounding normal spinal cord tissue. In addition, insufficient angiogenesis and the formation of inhibitory extracellular matrix further hinder the connection of the spinal cord circuit.

[0003] For diseases with complex pathological processes, a single treatment measure cannot meet the full regeneration of SCI. It is very important to adopt a multi-faceted treatment method. For example, the current single treatment measure, the Chinese patent with publication number CN111437268A discloses a preparation method of MicroRNA micro-nano spheres and its application in spinal cord injury repair. A poly (lactic-co-glycolic acid) micro-nano sphere solution is prepared by using a multiple emulsion method; a polyetherimide aqueous solution is added to the poly (lactic-co-glycolic acid) micro-nano sphere solution, and the mixture is fully oscillated and mixed to form a polyetherimide-modified poly (lactic-co-glycolic acid) micro-nano sphere composite solution; different MicroRNAs are loaded onto the microspheres to form a MicroRNAs microsphere composite.

[0004] Because the main disease factors and treatment needs of different SCI pathological processes are different, timely and flexible treatment measures are needed. The severe inflammatory response formed immediately after injury can lead to the occurrence of secondary injury, resulting in a large number of nerve cell deaths. In addition, the lack of angiogenesis during the tissue regeneration stage also hinders the repair of the tissue. Therefore, there is an urgent need for a composite microsphere that can simultaneously load multiple therapeutic drugs. SUMMARY

[0005] The purpose of the present application is to provide a double-release microsphere and a preparation method and application thereof. The preparation method provided by the present application is simple and easy to control. When used in the preparation of drugs for treating spinal cord injury, the therapeutic substances wrapped in the inner and outer layers of the double-release microsphere are released into the tissue at different times, exerting a therapeutic effect, and ultimately synergistically promoting the repair and regeneration of damaged tissue.

[0006] The present application provides the following technical solutions:

[0007] A dual-release microsphere comprises: a PLGA microsphere loaded with a drug and an alginate microcapsule, wherein the alginate microcapsule is an alginate microcapsule loaded with stem cell exosomes and / or stem cell-derived vesicles.

[0008] Based on different loading modes of therapeutic substances in microspheres, the dual-release microspheres provided by the present invention sequentially release drugs encapsulated in PLGA and stem cell exosomes or stem cell extruded vesicles with angiogenesis-promoting effects encapsulated in alginate microcapsules.

[0009] The molecular weight of the PLGA is between 5kDa and 30kDa. The physicochemical properties of PLGA significantly influence the release behavior of the drug contained therein. When the molecular weight of PLGA is too small, it is prone to glass transition at room temperature, the microspheres are prone to deformation and denaturation, and are difficult to store. This also results in excessively rapid drug release, making it impossible to meet the needs of sustained inflammation regulation in the early stages of SCI. When the molecular weight is too large, the degradation rate of PLGA in the microspheres slows, resulting in the inability to release the outer layer drug and the vesicles in the alginate in a timely manner. Therefore, the present invention limits the molecular weight of PLGA to between 5kDa and 30kDa.

[0010] The mass ratio of the PLGA, drug, and alginate microcapsules is 10-8:5-2:2-1. By adjusting the mass ratio of the three, the outer drug layer and the angiogenic vesicles in the microcapsules are released in the early and late stages of spinal cord injury, respectively. Preferably, the ratio of PLGA, drug, and alginate microcapsules is 10:2:1.

[0011] The drug is selected from steroid drugs, nonsteroidal anti-inflammatory drugs, antibiotics, proteins or polypeptide drugs.

[0012] The present invention also provides a method for preparing dual-release microspheres, which comprises the following steps:

[0013] (1) loading the stem cell exosomes and / or stem cell-derived vesicles into an alginate solution containing stem cell exosomes and / or stem cell-derived vesicles by a W / O emulsification method to prepare alginate microcapsules, wherein strontium ions are used as a cross-linking agent during the preparation process;

[0014] (2) Drugs and alginate microcapsules were loaded into PLGA by the S / O / W emulsification method to prepare dual-release microspheres.

[0015] In step (1), the preparation methods of the stem cell exosomes and stem cell-derived vesicles are respectively:

[0016] The stem cell culture supernatant was collected and subjected to refrigerated centrifugation to obtain stem cell exosomes;

[0017] The stem cells were squeezed and filtered through filter membranes of different sizes and the stem cell-derived vesicles were collected.

[0018] The preparation method further comprises: low-oxygen pretreatment of the stem cells before preparation; the low-oxygen pretreatment is under the conditions of 1-5% oxygen volume percentage, 2-8% carbon dioxide volume percentage, and 30-37 DEG C temperature; and the stem cells are selected from embryonic stem cells, mesenchymal stem cells or induced pluripotent stem cells. The present application promotes the angiogenic promotion of the exosomes or extruded vesicles of the stem cells by culturing the stem cells under low-oxygen conditions. When the low-oxygen conditions are used, the oxygen concentration needs to be maintained, so the present application limits the oxygen volume percentage to 1-5%.

[0019] Preferably, the freezing centrifugation is under the conditions of a centrifugal force not less than 100000g, a centrifugation time of 3-4h, and a temperature of 0-4 DEG C.

[0020] In step (1), the alginate solution concentration is 1-5% when the alginate microcapsules are prepared.

[0021] In step (1), the stem cell exosomes or stem cell extruded vesicles are loaded and encapsulated in the alginate microcapsules by W / O emulsification; a crosslinking agent is added to crosslink the alginate during the emulsification; preferably, the crosslinking agent is strontium chloride, because strontium ions have the effects of promoting angiogenesis and regulating inflammation; the alginate solution concentration has an influence on the release of the contents, and preferably, the alginate solution concentration is 1% when the alginate microcapsules are prepared.

[0022] In step (2), the PLGA, the drug and the alginate microcapsules are dissolved and then prepared by S / O / W emulsification, and the PLGA concentration after dissolution is 20-200mg / mL.

[0023] In step (2), the steroid drugs, non-steroidal anti-inflammatory drugs, antibiotics, proteins or polypeptides and other drugs are encapsulated in the PLGA microspheres together with the alginate microcapsules by S / O / W emulsification. In addition, the PLGA concentration has a great influence on the release of the contents of the microspheres when the double-release microspheres are prepared, and preferably, the PLGA concentration is 100mg / mL. Preferably, the ratio of PLA to PGA in the PLGA is 50:50.

[0024] The present application also provides a use of the above double-release microspheres in the preparation of a drug for treating spinal cord injury.

[0025] In the double-release microspheres provided by the present application, the drug is encapsulated in the PLGA microspheres, can play an inflammation regulation effect in the acute stage of SCI; then, the stem cell exosomes or stem cell extruded vesicles encapsulated in the alginate microcapsules start to release and participate in angiogenesis in the later stage; through the synergistic effect of the two treatment approaches, the microenvironment of the spinal cord injury can be effectively improved, and the regeneration of the spinal cord nerves can be promoted.

[0026] Compared with the prior art, the main advantages of the present application include:

[0027] 1) The stem cell hypoxic exosome or hypoxic extrusion vesicle prepared by the present application has stronger effect of promoting angiogenesis;

[0028] 2) The double-release property of the double-release microspheres provided by the present application can release corresponding therapeutic substances in time at different disease stages, participate in the improvement of specific case environment and promote tissue regeneration;

[0029] 3) The preparation method provided by the present application is simple and easy to control;

[0030] 4) The double-release microspheres prepared by the present application have significant effect of promoting spinal cord injury regeneration, which proves that they have good application prospect and research value. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The morphology of the stem cell hypoxic exosome and the stem cell hypoxic extrusion vesicle;

[0032] Figure 2 The promotion effect of the stem cell hypoxic exosome and the stem cell hypoxic extrusion vesicle on the tube formation ability and migration ability of human umbilical cord vein endothelial cells;

[0033] Figure 3 The morphology and element distribution results of the alginate microcapsule loaded with the stem cell hypoxic exosome and the stem cell hypoxic extrusion vesicle and using strontium as a crosslinking agent;

[0034] Figure 4 The morphology of the double-release PLGA microspheres loaded with the alginate microcapsule and the methylprednisolone drug;

[0035] Figure 5 The release results of the methylprednisolone and the hypoxic extrusion vesicle in the double-release PLGA microspheres;

[0036] Figure 6 The BBB score of the motor function of the spinal cord injury rats in the double-release microsphere system;

[0037] Figure 7 The influence of the double-release microsphere system on the distribution of NF and GFAP in the spinal cord nerve tissue of the spinal cord injury rats. DETAILED DESCRIPTION

[0038] The present application will be further described below in conjunction with the drawings and specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The operation methods not specified in the following examples are usually performed according to the conventional conditions or the conditions recommended by the manufacturers.

[0039] 1. Preparation of stem cell hypoxic exosomes and stem cell hypoxic extruded vesicles:

[0040] Mesenchymal stem cells (MSCs) were cultured in BC-T4 medium containing 5% Helios UltraGRO-Advanced in an environment of 1% oxygen.

[0041] Hypoxic exosomes (hypo-Exo) were collected from the cell culture medium by ultracentrifugation (100,000 × g, 70 min, 4°C).

[0042] MSCs were passed through filters with pore sizes of 5, 1, and 0.4 μm, and then prepared into hypoxic extruded vesicles (hypo-NVs) using a liposome extruder. The suspension after extrusion and filtration was ultracentrifuged again (100,000 x g, 70 min, 4°C), and the precipitate was collected to obtain hypo-NVs.

[0043] like Figure 1 As shown in the figure, both hypo-Exo and hypo-NV are vesicles with a particle size of about 200 nm and a typical cup-shaped structure.

[0044] 2. Effects of stem cell hypoxia exosomes and stem cell hypoxia extruded vesicles on HUVECs tube formation and migration ability:

[0045] The effect of Matrigel on the tube-forming ability of HUVECs was investigated: first, the 48-well cell culture plate and Matrigel were pre-cooled at 4°C overnight, and the subsequent bonding operation was performed on ice; 100 μL of Matrigel was added to each well of the culture plate, and 300 μL of cell culture medium containing hypo-Exo or hypo-NV (30 μg / mL) was added to each well, and 3×10 4 A small amount of HUVECs suspension was inoculated into each well according to the cell number; after appropriate shaking, the cells were incubated at 37°C for 4 h, and the tube formation was observed.

[0046] Detect the migration ability of HUVECs using Transwell plate: resuspend HUVECs to 1×10 5 cells / mL, and 100 μL was added to the upper chamber of the Transwell; at the same time, hypo-Exo or hypo-NV at a concentration of 30 μg / mL was administered to the upper chamber; another 500 μL of cell culture medium was added to the lower chamber; 6 hours later, the Transwell membrane was fixed with 4% paraformaldehyde for 10 minutes, the cells on the upper membrane were gently scraped off, and the cells on the lower membrane were stained with crystal violet for 10 minutes.

[0047] like Figure 2 As shown in the results, hypo-Exo and hypo-NV have similar promoting effects on the tube-forming ability and migration ability of HUVECs.

[0048] 3. Preparation of alginate microcapsules:

[0049] The surfactant Span 80 was dispersed in isooctane at a concentration of 5%, and then mixed with 20 mL of 1% alginate solution containing hypo-NV; the mixture was emulsified at 12,000 rpm, 3 mL of 30% Tween 80 was added, 10 mL of 2% strontium chloride was added dropwise; then 40 mL of isopropanol was added to harden the microcapsules and separate the microcapsules from the organic phase; after washing with isopropanol and water, the microcapsules were collected by centrifugation at 2000 rpm for 5 min; the collected alginate microcapsules were dispersed with 1% chitosan solution, shaken for 10 min, washed with water 3 times and freeze-dried.

[0050] As shown in Figure 3 , the alginate microcapsule particle size is about 5 μm, and the interior has a distribution of strontium element.

[0051] 4. Preparation of double-release microspheres:

[0052] S / O / W emulsification method was used to prepare the sustained-release microspheres: PLGA was dissolved in dichloromethane to make its concentration 100 mg / mL; methylprednisolone MP and alginate microcapsule NV-Alg were dispersed in the DCM solution, PLGA: MP: NV-Alg = 10:2:1; under stirring, the dispersion was added dropwise to 1% polyvinyl alcohol aqueous solution, and then the mixture was transferred to a vacuum drying oven to remove DCM; washed with water 3 times, freeze-dried.

[0053] The blank microspheres without MP and hypo-NV are denoted as Alg@PLGA. The microspheres containing only MP are denoted as Alg@MP-PLGA. The microspheres carrying only hypo-NV are denoted as NV-Alg@PLGA. When the microspheres are loaded with both MP and hypo-NV, they are denoted as NV-Alg@MP-PLGA.

[0054] As shown in Figure 4 , the double-release microspheres loaded with MP and hypo-NV. The particle size is about 40 μm.

[0055] As shown in Figure 5 , the release results of MP and hypo-NV in the double-release microspheres of MP and hypo-NV. A large amount of MP is released into the medium within 48 hours, while hypo-NV is released from 7 days, and the different release behaviors of the two are in line with the treatment means required by the disease progression of SCI.

[0056] 5. Animal experiments:

[0057] (1) Establishment of spinal cord injury (SCI) model:

[0058] Take the weight of about 220 g of female SD rats, after complete anesthesia, shave the back hair. The back skin is disinfected with iodophor, and the muscle on both sides of the ridge is separated after the fascia is exposed. Cut off the T9, T10 dorsal lamina of the spine with forceps, expose the spinal cord, and completely cut off the spinal cord; according to the experimental group design, the corresponding material is transplanted to the spinal cord incision, and the paraspinal muscle and skin are sutured in turn.

[0059] (2) On the 7th, 14th, 21st, and 28th days after modeling, the rats were subjected to Basso Beattie Bresnahan (BBB) test and scoring, respectively, to estimate the recovery of the rats.

[0060] As shown in Figure 6 , in all experimental groups, the double-release microspheres NV-Alg@MP-PLGA promoted the rapid recovery of motor function in the early stage of spinal cord injury rats, and achieved the best effect of motor function recovery.

[0061] As shown in Figure 7 , through immunostaining of neuronal marker NF, in all experimental groups, the double-release microspheres NV-Alg@MP-PLGA had the most obvious effect on neuron regeneration.

[0062] In addition, it should be understood that, after reading the above description of the present application, those skilled in the art can make various modifications or modifications to the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

Claims

1. A dual release microsphere characterized in that, The double-release microspheres comprise: PLGA microspheres loaded with a drug and alginate microcapsules, wherein the alginate microcapsules are alginate microcapsules loaded with stem cell exosomes or / and stem cell-derived vesicles; The molecular weight of the PLGA is between 5k Da and 30k Da; The mass ratio of the PLGA, the drug and the alginate microcapsules is 10-8:5-2:2-1; The drug is selected from a steroidal drug, a non-steroidal anti-inflammatory drug or an antibiotic; The preparation methods of the stem cell exosomes and the stem cell-derived vesicles are respectively: The culture medium supernatant of stem cells is collected, frozen and centrifuged to obtain stem cell exosomes; The stem cells are extruded and filtered through different size filter membranes to collect stem cell-derived vesicles.

2. A method of preparing the dual release microspheres of claim 1, characterized in that, The preparation method comprises the following steps: (1) alginate solution containing stem cell exosomes or / and stem cell-derived vesicles is prepared by W / O emulsification method to load the stem cell exosomes or / and stem cell-derived vesicles into alginate microcapsules, and strontium ions are used as cross-linking agents during the preparation process; (2) the drug and the alginate microcapsules are loaded into PLGA by S / O / W emulsification method to prepare double-release microspheres.

3. The method of claim 2, wherein the dual release microspheres are prepared by, The stem cells are subjected to hypoxic pretreatment before preparation; the hypoxic pretreatment conditions are: oxygen volume percentage of 1% to 5%, carbon dioxide volume percentage of 2% to 8%; and the stem cells are selected from embryonic stem cells, mesenchymal stem cells or induced pluripotent stem cells.

4. The method of claim 2, wherein the dual release microspheres are prepared by the process of: In step (1), the alginate solution concentration is 1% to 5% when preparing the alginate microcapsules.

5. The method of claim 2, wherein the dual release microspheres are prepared by the steps of: In step (2), the PLGA, the drug and the alginate microcapsules are dispersed and then prepared by S / O / W emulsification method, and the PLGA concentration after dissolution is 20 to 200 mg / mL.

6. Use of the double-release microspheres of claim 1 in the preparation of a drug for treating spinal cord injury.

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