Dual-release microsphere as well as preparation method and application thereof

Abstract

The invention discloses a dual-release microsphere which comprises a PLGA (poly (lactic-co-glycolic acid)) microsphere loaded with a medicine and an alginic acid microcapsule, and the alginic acid microcapsule is an alginic acid microcapsule loaded with stem cell exosome or / and stem cell derived vesicles. The invention also discloses a preparation method of the dual-release microspheres, which comprises the following steps: loading stem cell exosomes or / and stem cell derived vesicles through a W / O emulsification method to prepare alginic acid microcapsules, and using strontium ions as a cross-linking agent in the preparation process; and loading the drug and the alginic acid micro-capsule into PLGA through an S / O / W emulsification method to prepare the dual-release microspheres. The invention also discloses an application of the dual-release microsphere in preparation of drugs for treating spinal cord injury. The preparation method provided by the invention is simple and easy to control; when the double-release microsphere is applied to preparation of drugs for treating spinal cord injury, the treatment substances wrapped in the inner layer and the outer layer in the double-release microsphere are released into tissues at different time to play a treatment role, and finally the repair and regeneration of the damaged tissues are promoted through the synergistic effect.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a dual-release microsphere and a preparation method and application thereof. Background Art

[0002] Traumatic spinal cord injury (SCI) refers to temporary or permanent functional damage to spinal cord tissue caused by external force. Despite the increasing incidence of the disease, there is still a lack of effective treatment options in clinical practice. One of the reasons is that the pathological process of SCI is quite complex, and its pathological microenvironment is extremely unfavorable for cell survival. Within a short period of time after the injury, the spinal cord will become highly inflammatory. The continuous release of inflammatory cytokines will lead to the amplification of the inflammatory cascade and begin to induce the production of a large amount of reactive oxygen species, 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 spinal cord circuits.

[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 method for preparing MicroRNA micro-nanospheres and their application in spinal cord injury repair. The polylactic acid-glycolic acid copolymer micro-nanosphere solution is prepared by double emulsion method; the polyetherimide aqueous solution is added to the polylactic acid-glycolic acid copolymer micro-nanosphere solution and fully shaken and mixed to form a polyetherimide-modified polylactic acid-glycolic acid copolymer micro-nanosphere complex solution; different MicroRNAs are loaded onto the microspheres to form a MicroRNAs microsphere complex.

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

[0005] The purpose of the present invention is to provide a dual-release microsphere and a preparation method and application thereof; the preparation method provided by the present invention 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 dual-release microspheres are released into the tissue at different times to exert a therapeutic effect, and ultimately synergistically promote the repair and regeneration of damaged tissues.

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

[0007] A dual-release microsphere comprises: a PLGA microsphere loaded with drugs and alginate microcapsules, wherein the alginate microcapsules are alginate microcapsules 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 5k Da and 30k Da. The physical and chemical properties of PLGA have a great influence on the release behavior of the drug in it. When the molecular weight of PLGA is too small, it is easy to undergo glass transition at room temperature, the microspheres are easy to deform and denature, and are difficult to preserve, and the drug is released too quickly, which cannot meet the continuous inflammation regulation in the early stage of SCI; when the molecular weight is too large, the degradation rate of PLGA in the microspheres slows down, resulting in the inability to release the outer layer of drugs and the vesicles in the alginate in time; therefore, the present invention limits the molecular weight of PLGA to between 5k Da and 30k Da.

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

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

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

[0013] (1) loading 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 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 culture supernatant of stem cells was collected and subjected to refrigerated centrifugation to obtain stem cell exosomes;

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

[0018] The preparation method also includes: pre-treating the stem cells with hypoxia before preparation; the conditions of the hypoxia pre-treatment are: 1% to 5% by volume of oxygen, 2% to 8% by volume of carbon dioxide, and a temperature of 30 to 37°C; the stem cells are selected from embryonic stem cells, mesenchymal stem cells, or induced pluripotent stem cells. The present invention cultured stem cells under hypoxic conditions to promote the role of their exosomes or extruded vesicles in promoting angiogenesis. When treating under hypoxic conditions, it is necessary to maintain a suitable oxygen concentration, so the present invention limits the oxygen volume percentage to 1% to 5%.

[0019] Preferably, the conditions of the refrigerated centrifugation are: centrifugal force not less than 100,000 g, centrifugal time 3 to 4 h, and temperature 0 to 4°C.

[0020] In step (1), the concentration of alginate solution is 1% to 5% when preparing alginate microcapsules.

[0021] In step (1), stem cell exosomes or stem cell extruded vesicles are loaded and encapsulated in alginate microcapsules by W / O emulsification method; during the emulsification process, a cross-linking agent is added to cross-link the alginate; preferably, strontium chloride is used as the cross-linking agent, because strontium ions have the effects of promoting angiogenesis and regulating inflammation; the concentration of the alginate solution will affect the release of the contents, and preferably, when preparing the alginate microcapsules, the concentration of the alginate solution is 1%.

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

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

[0024] The present invention also provides an application of the dual-release microspheres in preparing a medicine for treating spinal cord injury.

[0025] In the dual-release microspheres provided by the present invention, the drug is encapsulated in the PLGA microspheres, which can exert an inflammatory regulatory effect in the acute phase of SCI; subsequently, the stem cell exosomes or stem cell extruded vesicles encapsulated in the alginate microcapsules begin to release and participate in angiogenesis in the later stage; through the synergistic effect of the two treatment pathways, the spinal cord injury microenvironment can be effectively improved and the regeneration of spinal nerves can be promoted.

[0026] Compared with the prior art, the present invention has the following main advantages:

[0027] 1) The stem cell hypoxic exosomes or hypoxic extruded vesicles prepared by the present invention have a stronger effect of promoting angiogenesis;

[0028] 2) The dual-release properties of the dual-release microspheres provided by the present invention can release corresponding therapeutic substances in a timely manner at different stages of the disease, participate in the improvement of the specific case environment and promote tissue regeneration;

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

[0030] 4) The dual-release microspheres prepared by the present invention have a significant effect on promoting the regeneration of spinal cord injury, proving that they have good application prospects and research value. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The morphological characteristics of stem cell hypoxic exosomes and stem cell hypoxic extruded vesicles;

[0032] Figure 2 The promoting effects of stem cell hypoxic exosomes and stem cell hypoxic extruded vesicles on the tube-forming ability and migration ability of human umbilical vein endothelial cells;

[0033] Figure 3 The morphology and element distribution results of alginate microcapsules loaded with stem cell hypoxic exosomes and stem cell hypoxic extruded vesicles with strontium as a cross-linking agent;

[0034] Figure 4 The morphological characteristics of dual-release PLGA microspheres loaded with alginate microcapsules and methylprednisolone drugs;

[0035] Figure 5 The release results of methylprednisolone in dual-release PLGA microspheres and hypoxic extrusion vesicles;

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

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

[0038] The present invention will be further described below in conjunction with the accompanying drawings and specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. The operating methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[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 filter membranes with pore sizes of 5 μm, 1 μm and 0.4 μm, and hypoxic extruded vesicles (hypo-NV) were prepared using a liposome extruder. The suspension after extrusion and filtration was ultracentrifuged again (100,000×g, 70 min, 4° C.), and the precipitate was collected to obtain hypo-NV.

[0043] like Figure 1 As shown, 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 hypoxic exosomes and stem cell hypoxic extruded vesicles on HUVECs tube formation and migration ability:

[0045] Matrigel was used to examine the effect of HUVECs on tube formation: first, the 48-well cell culture plate and Matrigel were precooled 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 with the cell number of 1:1; 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 Transwell; at the same time, hypo-Exo or hypo-NV at a concentration of 30 μg / mL was given to the upper chamber; another 500 μL of cell culture medium was added to the lower chamber; after 6 hours, 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, 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 a 1% alginate solution containing hypo-NV; the mixture was emulsified at 12,000 rpm, 3 mL of 30% Tween 80 was added, and 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 a 1% chitosan solution, shaken for 10 min, washed with water 3 times and then freeze-dried.

[0050] like Figure 3 As shown, the particle size of alginate microcapsules is about 5 μm, and strontium is distributed inside.

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

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

[0053] Blank microspheres without MP and hypo-NV were denoted as Alg@PLGA. Microspheres containing only MP were denoted as Alg@MP-PLGA. Microspheres carrying only hypo-NV were denoted as NV-Alg@PLGA. When microspheres were loaded with both MP and hypo-NV, they were denoted as NV-Alg@MP-PLGA.

[0054] like Figure 4 The figure shows dual-release microspheres loaded with MP and hypo-NV, with a particle size of about 40 μm.

[0055] like Figure 5 The figure shows the release results of MP and hypo-NV in the dual-release microspheres of MP and hypo-NV. A large amount of MP was released into the medium within 48 hours, while hypo-NV began to be released from 7 days. The different release behaviors of the two correspond to the treatment methods required for the progression of SCI.

[0056] 5. Animal experiments:

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

[0058] Female SD rats weighing about 220 g were taken and their back hair was shaved after complete anesthesia. The back skin was disinfected with iodine and incised with a scalpel; after exposing the fascia, the muscles on both sides of the crest were separated. The dorsal lamina of the T9 and T10 spines were cut with forceps to expose the spinal cord and completely cut off the spinal cord; according to the experimental group design, the corresponding materials were transplanted into the spinal cord incision, and the paravertebral muscles and skin were sutured in sequence.

[0059] (2) The rats were subjected to Basso Beattie Bresnahan (BBB) ​​test and scoring on days 7, 14, 21, and 28 after modeling to estimate the neurological recovery of the rats.

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

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

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

Claims

1. A dual-release microsphere, characterized in that: The dual-release microspheres include: PLGA microspheres loaded with drugs and alginate microcapsules, wherein the alginate microcapsules are alginate microcapsules loaded with stem cell exosomes and / or stem cell-derived vesicles.

2. The dual-release microspheres according to claim 1, characterized in that: The molecular weight of the PLGA is between 5k Da and 30k Da.

3. The dual-release microspheres according to claim 1, characterized in that: The mass ratio of the PLGA, the drug and the alginate microcapsule is 10-8:5-2:2-1.

4. The dual-release microspheres according to claim 1, characterized in that: The drug is selected from steroidal drugs, non-steroidal anti-inflammatory drugs, antibiotics, proteins or polypeptide drugs.

5. A method for preparing the dual-release microspheres according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: (1) loading 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; (2) The drug and alginate microcapsules were loaded into PLGA by S / O / W emulsification method to prepare dual-release microspheres.

6. The method for preparing dual-release microspheres according to claim 5, characterized in that: In step (1), the preparation methods of the stem cell exosomes and stem cell-derived vesicles are respectively: The culture supernatant of stem cells was collected and subjected to refrigerated centrifugation to obtain stem cell exosomes; The stem cells were squeezed and filtered through filters of different sizes and the stem cell-derived vesicles were collected.

7. The method for preparing dual-release microspheres according to claim 6, characterized in that: The stem cells are subjected to hypoxia pretreatment before preparation; the conditions of the hypoxia pretreatment are: 1% to 5% by volume of oxygen, 2% to 8% by volume of carbon dioxide; the stem cells are selected from embryonic stem cells, mesenchymal stem cells or induced pluripotent stem cells.

8. The method for preparing dual-release microspheres according to claim 5, characterized in that: In step (1), the concentration of the alginate solution is 1% to 5% when preparing alginate microcapsules.

9. The method for preparing dual-release microspheres according to claim 5, characterized in that: In step (2), PLGA, drug and alginate microcapsules are dispersed and prepared by S / O / W emulsification method, and the concentration of PLGA after dissolution is 20-200 mg / mL.

10. Use of the dual-release microspheres according to any one of claims 1 to 4 in preparing a drug for treating spinal cord injury.

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