Drug-loaded microgels for regulating behavior of neural stem cells and preparation method thereof
The drug-loaded microgels prepared using microfluidic technology, combined with ISP peptides, solved the problem of restricted migration of neural stem cells in bulk hydrogels, achieving the maintenance of stemness and differentiation of neural stem cells toward neurons, and improving cell culture results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHAOXING RES INST OF ZHEJIANG UNIV
- Filing Date
- 2023-11-28
- Publication Date
- 2026-07-07
Smart Images
Figure CN117695444B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a drug-loaded microgel that can regulate the behavior of neural stem cells and its preparation method, belonging to the field of medical materials. Background Technology
[0002] Following a stroke, the inflammatory response stimulates astrocyte proliferation, leading to the production of glial scars to prevent damage to surrounding normal cells and tissues from the severe inflammatory response. However, these glial scars can also hinder subsequent nerve regeneration. Currently, the main approach to promoting nerve regeneration is through biomaterials loaded with neural stem cells.
[0003] Neural stem cells have limited sources; laboratory researchers typically extract them from pregnant mice, and in vitro passage is limited, with the first three passages generally considered to be stem-like neural stem cells. Under in vitro culture conditions, the maintenance of stemness and cell differentiation of neural stem cells are difficult to regulate, especially their differentiation behavior. Neural stem cells typically differentiate into glial cells when cultured in differentiation media, making it difficult to differentiate into neurons. Therefore, researchers have developed various biocompatible materials for the in vitro culture of neural stem cells and for regulating their cell behavior.
[0004] Hydrogel materials, due to their extracellular matrix-like properties, are commonly used as carriers for in vitro cell culture and in vivo cell delivery. However, because of the dense cross-linked network of bulk hydrogels, neural stem cells on the outer layer of the hydrogel generally have difficulty migrating deep into the bulk hydrogel. When neural stem cells are dispersed in bulk hydrogels, cell migration is restricted, intercellular interactions are weakened, and it is difficult to regulate their cell behavior. Compared to bulk hydrogels, microgels have a larger specific surface area, which can improve the rate of nutrient transport within microgels. Moreover, microgels have a high porosity, providing cells with adhesion sites and space.
[0005] Therefore, this invention prepares a drug-loaded microgel that can regulate the cell behavior of neural stem cells. After lyophilization, the microgel containing grafted cell adhesion peptides adsorbs and loads intracellular Sigma peptides (ISP). ISP peptides, derived from the conserved wedge region of the PTPσ receptor (protein tyrosine phosphatase σ), can effectively bind to the PTPσ receptor on cells and further regulate the stemness and differentiation behavior of neural stem cells within the microgel. Summary of the Invention
[0006] This invention provides a drug-loaded microgel capable of regulating the behavior of neural stem cells and its preparation method. A microfluidic technique is used to prepare a microgel grafted with cell adhesion peptides. After lyophilization, the microgel is loaded with ISP peptides for co-culturing of neural stem cells, thereby regulating their stemness and differentiation behavior.
[0007] The technical solution adopted in this invention is as follows:
[0008] A drug-loaded microgel capable of regulating neural stem cell behavior is disclosed, wherein the drug-loaded microgel is a microgel system loaded with an ISP peptide. The drug-loaded microgel can regulate the stemness and differentiation of neural stem cells. The amino acid sequence of the ISP peptide is RKKRRQRRRCDMAEHMERLKANDSLKLSQEYESI.
[0009] Its preparation method and cell culture process may include the following steps:
[0010] 1) Prepare a microgel prepolymer solution, wherein the concentration of the microgel polymer material with double bonds is 5-20 wt%, the concentration of the cell adhesion peptide with thiol groups is 1-5 mg / mL, and the concentration of the photoinitiator is 0.5-1 wt%.
[0011] 2) Microgels were prepared by initiating crosslinking of the prepolymer solution using microchannels and ultraviolet light irradiation;
[0012] 3) Wash the microgel sequentially with n-hexane, anhydrous ethanol, and water;
[0013] 4) Immerse the lyophilized microgel in an ISP peptide solution to adsorb the ISP peptide-loaded microgel. Centrifuge and discard the supernatant to obtain the ISP peptide-loaded microgel. The subsequent cell culture steps are as follows:
[0014] 5) Seed neural stem cells on drug-loaded microgels and cultured them in differentiation medium at 37°C and 5% CO2 for 3-5 days.
[0015] The microgel polymer material mentioned in step 1) is at least one of the following: grafted double bond fibroin, grafted double bond gelatin, grafted double bond collagen, grafted double bond keratin, and reactive oxygen species responsive terminal double bond hyperbranched polymer (HBPAK).
[0016] The cell adhesion polypeptide mentioned in step 1) is at least one of RGD, IKVAV, YIGSR, and REDV. The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphite.
[0017] Step 2) The ultraviolet light power is 50-400 mW / cm². 2 The initiation time is 0.5-2 minutes.
[0018] Furthermore, in step 2), microgels with a diameter of 50-300 μm are prepared using microchannels. The aqueous phase flow rate is 50-100 μL / h, and the oil phase flow rate is 8-14 mL / h.
[0019] Furthermore, in step 2), the microchannel aqueous phase is water, the oil phase is liquid paraffin, the surfactant in the oil phase is Span 80, and the concentration of the surfactant in the oil phase is 5-20 v.
[0020] The incubation conditions for the ISP peptide described in step 4) are as follows: add an excess of ISP peptide to the lyophilized microgel and incubate at 37°C and 100 rpm for 1-3 hours; the concentration of ISP peptide is 2-20 μM when used for cell culture, and 0.5-5 mM when used for SEM electron microscopy observation and drug release testing.
[0021] Furthermore, in step 4), the centrifugation speed is 5000 rpm and the time is 10 min.
[0022] In step 5), the concentration of microgel is 1-20 mg / mL.
[0023] Furthermore, in step 5), the seeding density of neural stem cells is 3-20 weeks / well. The neural stem cell differentiation culture medium consists of 96 v% DMEM / F12, 20 ng / mL EGF, 20 ng / mL FGF, 1 x B-27TM-supplement, 1 x N2-supplement, and 1% fetal bovine serum.
[0024] The beneficial effects of this invention are as follows:
[0025] This invention prepares microgels grafted with cell adhesion peptides via microfluidics, and then uses a simple freeze-drying adsorption method to prepare microgels loaded with ISP peptides. Applying these drug-loaded microgels to neural stem cell culture helps maintain the stemness of neural stem cells and promotes their differentiation into neurons. This invention is simple and easy to implement, and holds promise for applications in the in vitro culture of neural stem cells and the design of organoid materials. Attached Figure Description
[0026] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided:
[0027] Figure 1 Microscopic images of microgels prepared for microchannels;
[0028] Figure 2 Particle size distribution of microgels prepared for microchannels;
[0029] Figure 3The amount of cell adhesion peptides grafted onto the microgel prepared for microfluidic channels;
[0030] Figure 4 Electron micrographs of the lyophilized microgels and drug-loaded microgels;
[0031] Figure 5 The drug release curve of the drug-loaded microgel;
[0032] Figure 6 Laser confocal microscopy image of extracted neural stem cells.
[0033] Figure 7 The effect of microgels on the stemness of neural stem cells;
[0034] Figure 8 The effect of microgels on neural stem cell differentiation. Detailed Implementation
[0035] The technical solutions of the present invention are further illustrated below with reference to embodiments, but these embodiments are not intended to limit the present invention.
[0036] Example 1
[0037] A 5% aqueous solution of gelatin (GelMA with a double bond grafting rate of approximately 95%) grafted with double bonds was prepared. 2 mg / mL of IKVAV peptide with thiol groups (amino acid sequence: CGGIKVAV) was added. Lithium phenyl-2,4,6-trimethylbenzoyl phosphite was used as the photoinitiator at a concentration of 0.5 wt%. The three solutions were mixed thoroughly and ultrasonically removed to obtain the aqueous phase solution for the microfluidic channel. A liquid paraffin solution containing 5% Span 80 was prepared, mixed thoroughly, and ultrasonically removed to obtain the oil phase solution for the microfluidic channel.
[0038] GelMA microgels (abbreviated as G) were prepared using microfluidics, with an aqueous phase flow rate of 60 μL / h and an oil phase flow rate of 12 mL / h. The microfluidic channels were then subjected to UV crosslinking via a UV chamber with a UV light power of 360 mW / cm². 2 The curing time was 1 min. The microgels were collected and washed three times each with hexane, anhydrous ethanol, and water to obtain GelMA microgels. Microscopic observation and imaging were performed, and the particle size distribution was analyzed. The particle size was found to be approximately 177.7 ± 16.9 μm. Figure 1 and Figure 2 As shown.
[0039] The GelMA microgels were freeze-dried and placed in an excess of 12.5 μM ISP peptide solution. They were incubated at 37 °C and 100 rpm for 1 h, and then centrifuged at 5000 rpm for 10 min to obtain ISP-loaded GelMA microgels (abbreviated as G / ISP), which were used for cell culture.
[0040] Under the same conditions, lyophilized GelMA microgels were placed in an excess of 1.39 mM ISP peptide solution to prepare ISP-loaded GelMA microgels (abbreviated as G / ISP), which were used for cell SEM electron microscopy observation and drug release testing.
[0041] Example 2
[0042] A 5% aqueous solution of double-bonded gelatin (GelMA, with a double-bond grafting rate of approximately 95%) was prepared. 5% of a reactive oxygen species-responsive terminal double-bonded hyperbranched polymer (HBPAK) was added, along with 4 mg / mL of a thiol-containing IKVAV peptide (amino acid sequence: CGGIKVAV). Lithium phenyl-2,4,6-trimethylbenzoyl phosphite was used as the photoinitiator at a concentration of 0.5 wt%. These four solutions were mixed thoroughly and ultrasonically removed to remove air bubbles, forming the aqueous phase solution for the microfluidic channel. A liquid paraffin solution containing 5% Span 80 was prepared, mixed thoroughly, and ultrasonically removed to remove air bubbles, forming the oil phase solution for the microfluidic channel.
[0043] HBPAK & GelMA microgels (H&G) were prepared using microfluidics with an aqueous phase flow rate of 60 μL / h and an oil phase flow rate of 10 mL / h. The microfluidic channels were then subjected to UV crosslinking via a UV chamber with a UV light power of 360 mW / cm². 2 The curing time was 1 min. The microgels were collected and washed three times each with n-hexane, anhydrous ethanol, and water to obtain HBPAK & GelMA microgels. Microscopic observation and imaging were performed, and the particle size distribution was analyzed. The particle size was found to be approximately 146.1 ± 20.0 μm. Figure 1 and Figure 2 As shown.
[0044] HBPAK & GelMA microgels were freeze-dried and placed in an excess of 12.5 μM ISP peptide solution. They were incubated at 37 °C and 100 rpm for 1 h, and then centrifuged at 5000 rpm for 10 min to obtain ISP-loaded HBPAK & GelMA microgels (abbreviated as H&G / ISP), which were used for cell culture.
[0045] Under the same conditions, lyophilized HBPAK & GelMA microgels were placed in an excess of 1.39 mM ISP peptide solution to prepare ISP-loaded HBPAK & GelMA microgels (abbreviated as H&G / ISP), which were used for cell SEM electron microscopy observation and drug release testing.
[0046] Example 3
[0047] To determine the IKVAV peptide concentration in the microgel, CGGIKVAV was first labeled with FITC. Specifically, 0.1 mg / mL FITC was reacted with 1 mg / mL CGGIKVAV (PBS solution) at 37°C and 200 rpm for 12 h. The reaction product was dialyzed in water and then lyophilized. The peptide concentration was determined using a BCA kit, and a standard curve of peptide fluorescence intensity versus concentration was plotted. Microgels were prepared using the FITC-CGGIKVAV peptide under the conditions described above, and the IKVAV concentration on the microgel was quantified by measuring the fluorescence intensity of the microgel. Figure 3 a is the standard curve of fluorescence intensity-concentration of FITC-CGGIKVAV peptide. The calculated grafting concentrations of the peptide on G and H&G microgels were 12.72 ± 0.19 mg / g and 18.95 ± 0.21 mg / g, respectively. Figure 3 b) The peptide concentration is close to that reported in the literature, which is beneficial for cell adhesion.
[0048] Example 4
[0049] Four types of microgels were lyophilized, and their surface morphology was observed using scanning electron microscopy (SEM). Figure 4 As shown, both G and H&G microgels were found to be distributed as individual microgels. The particle size of the lyophilized microgels was smaller than that in aqueous solution, and they exhibited a spindle shape. The G microgel had more porous structures and smaller pore sizes (approximately 20 μm) than the H&G microgel. After drug loading, both microgels exhibited an aggregated state during lyophilization, with virtually no individual microgel distribution, and a relatively reduced number of porous structures on the microgels.
[0050] Example 5
[0051] To better determine the drug release profile of the ISP peptide in the microgel, the ISP peptide concentration was increased to 1.39 mM to prepare ISP peptide-loaded microgels. The calculated ISP peptide loading concentrations on the G and H&G microgels were 114.5 ± 6.9 mg / g and 125.9 ± 17.1 mg / g, respectively. Then, 10 mg of the ISP peptide-loaded microgel was placed in 2 mL of PBS solution, and the amount of released peptide was determined using a BCA kit. Figure 5 As shown, the ISP peptide was gradually released from the microgel into PBS, and the release was almost complete in 4.5 h, exhibiting a slow release effect.
[0052] Example 6
[0053] Neural stem cells were extracted according to the literature. Before cell extraction, PBS and neural stem cell basal culture medium (Table 1) were prepared and pre-cooled on ice. The instruments used were sterilized in advance and stored in a laminar flow hood for later use. The extraction process was completed in the laminar flow hood.
[0054] First, pregnant mice (C57BL / 6 mice) at 16 days of gestation were euthanized by cervical dislocation and sterilized by immersion in 75% ethanol. After removing the pregnant mice, the abdominal skin was cut open, the uterus was dissected, and the fetus was removed and placed in a clean culture dish. The fetus was decapitated and quickly transferred to a culture dish containing pre-chilled PBS. Under a stereomicroscope, the whole brain tissue was extracted using microforceps, and the surface vascular membrane was dissected. The separated brain tissue was immediately placed in pre-chilled neural stem cell basal culture medium and kept on ice. The collected brain tissue was minced with scissors and filtered through a 70 μm cell filter. The cell filtrate was collected and placed in neural stem cell basal culture medium for suspension culture at 37°C and 5% CO2. After 2-3 days of culture, the neural stem cells proliferated and aggregated into cell spheroids, which were then digested and passaged using Accutase. Before use, the neural stem cells were validated for stem cell stemness using a Nestin protein antibody. Figure 6 As shown, the cultured cell spheroids are of similar diameter and evenly distributed in the field of view. After staining, the blue cell nuclei (DAPI) and red Nestin cells are visible. + The cells completely overlapped, indicating that the extracted nerve cells were neural stem cells, which can be used in subsequent neural stem cell experiments.
[0055] Table 1. Culture media used for neural stem cell culture
[0056]
[0057] Example 7
[0058] First, the stemness of neural stem cells in different microgels was evaluated. 0.4 mg of microgel was added to a 96-well plate and sterilized by UV irradiation for 30 min. Neural stem cells were then cultured at 10 × 10⁻⁶ wells. 4 Cells were seeded at a density of 1 cell per well into microgel-containing plates and cultured for 4 days at 37°C and 5% CO2.
[0059] After culture, the culture medium was discarded, and cells were fixed overnight at 4°C with 4% paraformaldehyde. Cell membranes were permeabilized with 0.1% Triton X-100 at 4°C for 10 min, followed by incubation with 3% BSA at 37°C for 90 min. To assess the retention of stemness of neural stem cells in the microgel, cells were stained. Neural stem cells were labeled with mouse Nestin antibody (1:2000) and incubated overnight at 4°C. Afterward, staining was performed using CY3-labeled goat anti-mouse IgG secondary antibody at room temperature for 1 h. The nuclei were then stained with 4',6-diamidinyl-2-phenylindole (DAPI) at room temperature for 10 min. Finally, laser confocal scanning microscopy was used to image the cells and microgel, and Z-stack scanning was performed.
[0060] The results are as follows Figure 7 As shown, cells spread well on the TCPS wells, but none of the cells exhibited red Nestin fluorescence, indicating that the cells no longer possess the stemness of neural stem cells. Compared to TCPS, cells on the G microgel showed a filamentous adhesion distribution, with significant Nestin staining at the areas where cells were relatively clustered. After the addition of ISP peptide, the cells largely lacked filamentous structures and were distributed as neurospheres with an average diameter of approximately 90 μm. Each neurosphere exhibited Nestin staining. + The morphology is similar to that of neurospheres in suspension culture. Therefore, the addition of ISP peptides to G microgels is beneficial for maintaining the stemness of neural stem cells. Compared to G microgels, which are prone to cell adhesion, although IKVAV peptides are grafted onto H&G microgels, the presence of hydrophilic PEG chains in HBPAK results in a neurosphere distribution within the cells. Similarly, each neurosphere is Nestin. + The diameter of the cells was approximately 30 μm, indicating that the cells in the H&G microgel possessed stem cell characteristics. Statistical analysis of Z-stack scanning revealed that neurospheres were relatively evenly distributed in the H&G microgel, while on the G and G / ISP microgels, cells / cell spheres were only distributed near the TCPS substrate. The introduction of the ISP peptide increased the distribution height of neurospheres on the H&G microgel, and statistical results showed that the ISP peptide increased the diameter of neurospheres on the H&G microgel to approximately 45 μm. However, the number of neurospheres per field of view decreased, indicating that the ISP peptide enhanced the stem cell characteristics of the cells in the H&G microgel. Overall, compared to the TCPS well plate, the H&G microgel effectively maintained the stem cell characteristics of neural stem cells, and the addition of the ISP peptide further enhanced the maintenance of stem cell characteristics in both G and H&G microgels.
[0061] Example 8
[0062] Furthermore, the differentiation behavior of neural stem cells in different microgels was evaluated. 0.4 mg of microgel was added to a 96-well plate and sterilized by UV irradiation for 30 min. Neural stem cells were then cultured at 10 × 10⁻⁶ wells. 4 Cells were seeded at a density of 1 cell per well into microgel-containing plates and cultured for 4 days at 37°C and 5% CO2.
[0063] After culture, the culture medium was discarded, and cells were fixed overnight at 4°C with 4% paraformaldehyde. Cell membranes were permeabilized with 0.1% Triton X-100 at 4°C for 10 min, followed by incubation with 3% BSA at 37°C for 90 min. Differentiated neurons were labeled with mouse β-tublin antibody (1:400), and differentiated glial cells were labeled with rabbit GFAP antibody (1:1000), and incubated overnight at 4°C. Subsequently, mouse β-tublin antibody was conjugated with CY3-labeled goat anti-mouse IgG secondary antibody, and rabbit GFAP antibody was conjugated with FITC-labeled goat anti-rabbit IgG secondary antibody, and incubated at room temperature for 1 h for staining. Cell nuclei were then stained with 4',6-diamidindo-2-phenylindole (DAPI) at room temperature for 10 min. Finally, cells and microgels were imaged using a laser confocal scanning microscope.
[0064] The results are as follows Figure 8 As shown, neurons are marked in red (β-tubulin). + Astrocytes are marked as green (GFAP). + As can be seen, cells adhering and spreading on both TCPS and G microgels exhibit green fluorescence, while on the G microgel, only the central areas where cells are relatively aggregated show weak β-tubulin. + Staining indicated that neural stem cells differentiated into astrocytes in both groups. Neurospheres on the G / ISP microgel also contained β-tubulin. + and GFAP + The staining indicates that cells in the neurospheres may differentiate into neurons or astrocytes, suggesting that the ISP peptide increases the likelihood of neural stem cells differentiating into neural stem cells on G microgels. Neurospheres on both H&G and H&G / ISP microgels stained red with β-tubulin. + The absence of green GFAP fluorescence indicates that the neurospheres are differentiating towards neurons. Overall, compared to TCPS (Potentially Transformed Plates) and G microgels, H&G microgels are more conducive to the differentiation of neural stem cells towards neurons, and the addition of ISP peptides can simultaneously promote the differentiation of neural stem cells towards neurons in both G and H&G microgels.
Claims
1. A drug-loaded microgel capable of regulating the behavior of neural stem cells, characterized in that, The drug-loaded microgel is a microgel system loaded with ISP peptides; the drug-loaded microgel can regulate the stemness and differentiation of neural stem cells; the amino acid sequence of the ISP peptide is RKKRRQRRRCDMAEHMERLKANDSLKLSQEYESI. The drug-loaded microgel is obtained by first preparing a microgel and then placing it in an ISP peptide solution. The microgel polymer material used in the preparation of the microgel is at least one of the following: grafted double-bond fibroin, grafted double-bond gelatin, grafted double-bond collagen, grafted double-bond keratin, and reactive oxygen species-responsive terminal double-bond hyperbranched polymer HBPAK. A thiol-containing cell adhesion peptide is added simultaneously during the preparation of the microgel. The cell adhesion peptide is at least one of RGD, IKVAV, YIGSR, and REDV. A photoinitiator is added during the preparation of the microgel. The microgel is prepared using a microfluidic channel. The lyophilized microgel is placed in an excess of ISP peptide solution to obtain a microgel loaded with ISP peptides.
2. The drug-loaded microgel capable of regulating neural stem cell behavior according to claim 1, characterized in that, When preparing microgels, the concentration of microgel polymer material is 5-20 wt%, and the concentration of cell adhesion peptides is 1-5 mg / mL.
3. The drug-loaded microgel capable of regulating neural stem cell behavior according to claim 1, characterized in that, The photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphite is added during the preparation of the microgels, the concentration of the photoinitiator is 0.5-1 wt%, the UV light power is 50-400 mW / cm 2 , and the initiation time is 0.5-2 min.
4. The drug-loaded microgel capable of regulating neural stem cell behavior according to claim 1, characterized in that, Microgels with diameters of 50-300 μm were prepared using microfluidics; the aqueous phase flow rate was 50-100 μL / h, and the oil phase flow rate was 8-14 mL / h.
5. The drug-loaded microgel capable of regulating neural stem cell behavior according to claim 4, characterized in that, When preparing microgels, the aqueous phase of the microchannel is water, the oil phase is liquid paraffin, the surfactant in the oil phase is Span 80, and the concentration of the surfactant in the oil phase is 5-20 v.
6. The drug-loaded microgel capable of regulating neural stem cell behavior according to claim 4, characterized in that, After the microgel preparation was completed, the microgel was washed sequentially with hexane, anhydrous ethanol, and water.
7. The drug-loaded microgel capable of regulating neural stem cell behavior according to claim 1, characterized in that, The lyophilized microgels were placed in an excess of ISP peptide solution and incubated at 37°C and 100 rpm for 1-3 hours. After centrifugation, the supernatant was discarded. The concentration of ISP peptides was 2-20 μM for cell culture and 0.5-5 mM for SEM electron microscopy observation and drug release testing.
8. The drug-loaded microgel capable of regulating neural stem cell behavior according to claim 1, characterized in that, When applied, the extracted neural stem cells are seeded in 96-well plates containing drug-loaded microgels at a concentration of 1-20 mg / mL, with a cell density of 3-20 w / well, and cultured in differentiation medium for 3-5 days under conditions of 37°C 5% CO2, wherein the differentiation medium composition is 96v% DMEM / F12, 20 ng / mL EGF, 20 ng / mL FGF, 1 x B-27 TM -supplement, 1 x N2-supplement, 1% fetal bovine serum.