Freezing cell drug delivery system for promoting bone repair and preparation method thereof

Abstract

The invention is applicable to the technical field of biological medicines, and provides a frozen cell drug delivery system for promoting bone repair and a preparation method thereof, and the preparation method comprises the following steps: preparing cells into frozen cells by adopting a liquid nitrogen freezing method; incubating the frozen cells with a DHA solution, and performing ultrasonic treatment at a fixed time point to obtain a mixed solution; and centrifuging the mixed solution, and collecting the precipitate to obtain the frozen cell drug delivery system. The frozen macrophages are used for enhancing the intercellular effect, the DHA is integrated into the frozen cell membrane to activate a PPAR gamma channel to maintain the metabolic balance of the macrophages, so that the continuous intercellular effect is supported, the prepared FrozCell-DHA can reduce inflammation and promote bone repair at the same time, and a new thought is provided for preparation of the osteoporosis treatment medicine.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a frozen cell drug-carrying system for promoting bone repair and a preparation method thereof. Background Art

[0002] Osteoporosis is closely related to chronic inflammation, especially in the process of age-related bone loss, inflammation is considered to be an important driving factor. This phenomenon is called "inflammatory aging", which affects the function of macrophages in bone tissue, especially their ability to clear apoptotic cells, i.e. efferocytosis. The weakening of efferocytosis ability not only leads to the persistence of inflammation, but also aggravates the development of osteoporosis. Therefore, enhancing the sustained efferocytosis function of macrophages has become a new strategy for the treatment of osteoporosis.

[0003] Among the technologies related to enhancing efferocytosis, apoptotic cell injection therapy is a widely studied method. By injecting apoptotic cells into the body, macrophages can be induced to efferocytose, thereby clearing the apoptotic cell residues in the inflammatory microenvironment and promoting the resolution of inflammation. However, traditional apoptotic cell injection therapy has shortcomings in targeting and durability, which limits its clinical application. In recent years, researchers have tried to use frozen cells as a substitute for apoptotic cells to enhance the efferocytosis of macrophages. Frozen cells are usually prepared by liquid nitrogen freezing, which can retain the integrity of the cell membrane to a certain extent and show the characteristics of late apoptosis. However, there are still some problems with the existing frozen cell preparation technology. First, the degradation and metabolites of frozen cells in the body may trigger an inflammatory response and affect the treatment effect; in addition, frozen cells themselves cannot effectively alleviate the metabolic stress generated by macrophages during continuous efferocytosis, resulting in a decrease in efferocytosis efficiency.

[0004] Docosahexaenoic acid (DHA) is an important ω-3 long-chain polyunsaturated fatty acid with significant anti-inflammatory and antioxidant effects. It can reduce the endoplasmic reticulum stress of macrophages by activating the PPARγ pathway, thereby improving the sustainability and efficiency of efferocytosis. Based on this, by integrating DHA into frozen macrophages (FrozCell), it is expected to solve the key problems in traditional efferocytosis enhancement therapy and provide a new solution for the treatment of osteoporosis. Summary of the invention

[0005] The purpose of the embodiments of the present invention is to provide a method for preparing a frozen cell drug-carrying system for promoting bone repair, aiming to solve the problems raised in the above-mentioned background technology.

[0006] The embodiment of the present invention is implemented as follows: a method for preparing a frozen cell drug-carrying system for promoting bone repair comprises the following steps:

[0007] S1: Prepare cells into frozen cells (FrozCell) by liquid nitrogen freezing method;

[0008] S2: incubating the frozen cells obtained in step S1 with a docosahexaenoic acid (DHA) solution, and performing ultrasonic treatment at a fixed time point to allow DHA to be integrated into the cell membrane to obtain a mixed solution;

[0009] S3: Centrifuge the mixed solution obtained in step S2 and collect the precipitate to obtain a frozen cell drug delivery system (FrozCell-DHA).

[0010] Preferably, in step S1, the cells are Raw 264.7 cells.

[0011] Preferably, in step S1, the liquid nitrogen freezing time is 12-24 hours.

[0012] Preferably, in step S2, the concentration of the docosahexaenoic acid solution is 100 μM.

[0013] Preferably, in step S2, the incubation temperature is 37° C. and the time is 1.8-2.2 h.

[0014] Preferably, in step S2, the fixed time points for ultrasonic treatment are when incubating for 30 min, 60 min, 90 min and 120 min, and the ultrasonic time is 25-35 s.

[0015] Preferably, in step S3, the centrifugation parameters are 800-1200 g, 3-8 min.

[0016] Another object of an embodiment of the present invention is to provide a frozen cell drug-carrying system for promoting bone repair, which is prepared by the above-mentioned preparation method.

[0017] Another object of the embodiment of the present invention is to provide a frozen cell drug-carrying system for promoting bone repair and its application in the preparation of drugs for treating osteoporosis.

[0018] The present invention provides a method for preparing a frozen cell drug delivery system for promoting bone repair. By combining frozen macrophages (FrozCell) with docosahexaenoic acid (DHA), DHA can be integrated into the frozen cell membrane, significantly reducing endoplasmic reticulum stress. In addition, DHA helps maintain the metabolic balance of macrophages by activating the PPARγ pathway, thereby supporting sustained efferocytosis. Compared with traditional apoptotic cell therapy, the FrozCell-DHA prepared by the preparation method provided in the present invention provides a more stable and efficient strategy, which can promote bone repair while reducing inflammation, and provides a new idea for the preparation of osteoporosis treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A graph showing the results of cell viability assays of living cells and FrozCell provided in an embodiment of the present invention;

[0020] Figure 2 A flow cytometry result diagram of live cells and FrozCell cell death status provided in an embodiment of the present invention;

[0021] Figure 3 Transmission electron microscope images of living cells, FrozCell and FrozCell-DHA provided in the embodiments of the present invention;

[0022] Figure 4 Fluorescence microscope images of the cell efferocytosis-promoting effect of FrozCell and FrozCell-DHA provided in the embodiments of the present invention;

[0023] Figure 5 A flow cytometry test result diagram of the cell burial promoting effect of FrozCell and FrozCell-DHA provided in an embodiment of the present invention;

[0024] Figure 6 A graph showing the anti-inflammatory effect test results of FrozCell and FrozCell-DHA provided in an embodiment of the present invention;

[0025] Figure 7 A graph showing the results of DHA reducing the expression of endoplasmic reticulum stress-related genes provided by an embodiment of the present invention;

[0026] Figure 8 A result diagram of the recovery of cell efferocytosis after using a PPARγ activator provided in an embodiment of the present invention;

[0027] Fig. 9 Fluorescence microscope images of the effects of DHA, FrozCell and FrozCell-DHA provided in the embodiments of the present invention on the mitotic ability of BMDM in osteoporotic mice;

[0028] Fig.10 Micro-CT results of DHA, FrozCell and FrozCell-DHA in treating osteoporosis provided by the embodiments of the present invention;

[0029] Fig.11 A flow chart for preparing a frozen cell drug-carrying system for promoting bone repair provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0031] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0032] Example 1: A frozen cell (FrozCell), the preparation method of which is as follows Fig.11 As shown, the specific steps include:

[0033] Step 1: Raw 264.7 cells (from American Type Culture Collection ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C and 5% CO. 2 A moist environment;

[0034] Step 2: Raw 264.7 cells were suspended in serum-free cell cryopreservation solution and immediately transferred to liquid nitrogen for 12 h;

[0035] Step 3: Wash the frozen cells with phosphate buffered saline (PBS) and centrifuge to prepare FrozCell.

[0036] Calcein / PI cell viability assay was performed according to the manufacturer's instructions (Beyotime) and the results were as follows Figure 1 As shown, according to Figure 1 It can be seen that after freezing in liquid nitrogen for 12 h, live-dead staining showed that FrozCell was completely dead;

[0037] The cell death status of FrozCell cells was evaluated using the AnnexinV-FITC / PI apoptosis detection kit (Yeasen) and analyzed by flow cytometry. The results were as follows Figure 2 As shown, according to Figure 2 It can be seen that 90.0% of FrozCells are in the late apoptotic state.

[0038] Example 2: A frozen cell drug delivery system (FrozCell-DHA) for promoting bone repair, the preparation method of which is as follows Fig.11 As shown, the specific steps include:

[0039] Step 1: Incubate the FrozCell prepared in Example 1 with a DHA solution (100 μM) at 37° C. for 2 h, and perform ultrasonic treatment for 30 s at 30 min, 60 min, 90 min, and 120 min after incubation, respectively, to allow DHA to be integrated into the cell membrane, thereby obtaining a mixed solution;

[0040] Step 2: Centrifuge the mixed solution at 1000 g for 5 min and collect the precipitate to obtain FrozCell-DHA.

[0041] Performance Analysis:

[0042] The results of observing the living cells, the FrozCell prepared in Example 1, and the FrozCell-DHA prepared in Example 2 using a transmission electron microscope are as follows: Figure 3 As shown, according to Figure 3 It can be seen that there is a membrane-wrapped nuclear structure in FrozCell, the cytoplasmic content is negligible, the surface of FrozCell-DHA is obviously smoother and more complete, and there are significantly fewer small vesicles in the surrounding environment.

[0043] The in vitro biological performance test of FrozCell-DHA prepared in Example 2 was performed:

[0044] Cell efferocytosis assay: Raw 264.7 cells were used as model cells, and 2×10 5 The cells / well were seeded in a 12-well plate at a density of 100 cells / well. After being treated with LPS (1 μg / mL) for 24 h, FrozCell and FrozCell-DHA were labeled with CM-DILRed fluorescent probe (Yeason) and added to Raw 264.7 cells at a ratio of 5:1 for 12 h. The cells were washed three times with PBS and collected for flow cytometry analysis. Raw 264.7 cells were labeled with CellTracker Green CMFDA (Yeason). After being treated with LPS (1 μg / mL) for 24 h, CM-DIL-labeled FrozCell and FrozCell-DHA were added to Raw 264.7 cells at a ratio of 5:1 for 12 h. The cells were washed three times with PBS to identify the cells that were not engulfed. Fluorescence microscopy was used to image the cells and the fluorescence intensity was quantitatively analyzed using ImageJ software. The results are shown in the figure. Figure 4 , Figure 5 As shown, according to Figure 4 It can be seen that the green fluorescently labeled Raw 264.7 cells took up the red fluorescently labeled FrozCell and FrozCell-DHA; Figure 5It can be seen that in the single-stage efferocytosis, the efferocytosis index (EI, percentage of fluorescent cells) of FrozCell is 13.15%, and the EI of FrozCell-DHA is 21.28%. In comparison, the two-stage efferocytosis significantly increased the EI of FrozCell to 64.33%, and the EI of FrozCell-DHA increased to 76.05%, indicating that the efferocytosis ability of macrophages is significantly enhanced after the initial uptake, there is a significant difference between the single-stage and two-stage efferocytosis, and FrozCell-DHA almost doubles the EI in the single-stage efferocytosis.

[0045] Anti-inflammatory effect detection: Raw 264.7 was used as a model cell, induced with 1 μg / mL LPS for 24 hours, and DHA, FrozCell and FrozCell-DHA were added respectively. Raw 264.7 cells without LPS treatment were used as negative control group; after 24 hours of action, qRT-PCR was used to detect the relative mRNA expression levels of inflammation-related genes IL-1β, TNF-α, IL-6, Arg-1 and IL-10. The results are as follows Figure 6 As shown, according to Figure 6 It can be seen that the expression of pro-inflammatory genes (IL-1β, TNF-α, IL-6) in both the FrozCell group and the FrozCell-DHA group was significantly inhibited, while the expression of anti-inflammatory genes (Arg-1 and IL-10) was significantly upregulated.

[0046] DHA alleviates endoplasmic reticulum stress through the PPARγ pathway: Raw 264.7 was used as a model cell and siRNA transfection was performed using Transfect-Mate (GenePharma) to establish Raw 264.7 Pr- / Pr- Cells were analyzed by qRT-PCR to detect wild-type Raw264.7 cells and Raw 264.7 Pr- / Pr- The mRNA levels of ER stress-related genes, including CHOP, Xbp1s, and Grp78, were measured after cells phagocytosed FrozCell and FrozCell-DHA. Figure 7 , Figure 8 As shown, according to Figure 7 It can be seen that FrozCell-mediated efferocytosis upregulated the expression of ER stress-related genes (CHOP, Grp78, Xbp1s), while FrozCell-DHA treatment reduced the expression of ER stress-related genes, and the ER stress level increased significantly after inhibiting PPARγ; Figure 8 It can be seen that after treatment with the PPARγ activator rosiglitazone, Raw 264.7 Pr- / Pr- The reduced efferocytosis in the cells was restored.

[0047] The FrozCell-DHA prepared in Example 2 was used to treat an osteoporosis mouse model to study its therapeutic effect in treating osteoporosis. The modeling process was as follows:

[0048] Eight-week-old female C57BL / 6J mice were selected and an osteoporosis model was established by ovariectomy. The OVX group underwent bilateral ovarian removal, while the sham group retained both ovaries and only removed the adipose tissue around the ovarian tissue that was equivalent to the weight of the ovary. The OVX group was further randomly divided into PBS, DHA, FrozCell, and FrozCell-DHA treatment groups (n=5 in each group) and received tail vein injections once a week.

[0049] Bone marrow-derived macrophages (BMDM) were isolated from mice in the Sham, OVX, DHA, FrozCell, and FrozCell-DHA groups, and BMDM were seeded in 24-well plates at a density of 5×10 5 To evaluate the sustained efferocytosis, apoptotic cells labeled with CellTracker Green CMFDA were added to BMDM at a ratio of 5:1 for 45 min and then washed with PBS. The results were shown in Fig. 9 As shown, according to Fig. 9 It can be seen that compared with the Sham group, the BMDM burial ability in the OVX group was significantly reduced, while FrozCell significantly increased the BMDM burial ability, and the addition of DHA further improved the BMDM burial ability;

[0050] After 8 weeks of treatment, the mice were anesthetized and euthanized, and femoral tissue samples were collected. The samples were immediately immersed in 4% paraformaldehyde for 48 h. The femurs of the experimental mice were scanned by Micro-CT using a standardized cylindrical sample container. The following parameters were set: 70 kV, 200 μA, 300 ms integration time, 1 mm aluminum filter, and 30 μm spatial resolution. Representative images of bone tissue were generated using Dataviewer software. Bone mineral density (BMD), bone volume fraction (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were quantitatively analyzed using CTAn software. The results are shown in Figure 2. Fig.10 As shown, according to Fig.10 It can be seen that the trabecular structure of the femur in the OVX group was severely damaged. Both FrozCell and FrozCell-DHA treatments could improve bone loss. The FrozCell-DHA group showed excellent bone repair and regeneration capabilities. Quantitative analysis of trabecular parameters confirmed that FrozCell and FrozCell-DHA significantly promoted bone formation and FrozCell-DHA could restore better quality trabeculae.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a frozen cell drug-carrying system for promoting bone repair, characterized in that: The following steps are involved: S1: Prepare cells into frozen cells by liquid nitrogen freezing method; S2: incubating the frozen cells obtained in step S1 with the docosahexaenoic acid solution, and performing ultrasonic treatment at a fixed time point to allow the docosahexaenoic acid to be integrated into the cell membrane to obtain a mixed solution; S3: Centrifuge the mixed solution obtained in step S2 and collect the precipitate to obtain a frozen cell drug delivery system.

2. The method for preparing the frozen cell drug-carrying system for promoting bone repair according to claim 1, characterized in that: In step S1, the cells used are Raw 264.7 cells.

3. The method for preparing the frozen cell drug-carrying system for promoting bone repair according to claim 1, characterized in that: In step S1, the liquid nitrogen freezing time is 12-24 hours.

4. The method for preparing the frozen cell drug-carrying system for promoting bone repair according to claim 1, characterized in that: In step S2, the concentration of the docosahexaenoic acid solution is 100 μM.

5. The method for preparing the frozen cell drug-carrying system for promoting bone repair according to claim 1, characterized in that: In step S2, the incubation temperature is 37° C. and the incubation time is 1.8-2.2 h.

6. The method for preparing the frozen cell drug-carrying system for promoting bone repair according to claim 1, characterized in that: In step S2, the fixed time points for the ultrasonic treatment are 30 min, 60 min, 90 min and 120 min of incubation, and the ultrasonic time is 25-35 s.

7. The method for preparing the frozen cell drug-carrying system for promoting bone repair according to claim 1, characterized in that: In step S3, the centrifugation parameters are 800-1200 g, 3-8 min.

8. A frozen cell drug-carrying system for promoting bone repair, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the frozen cell drug-carrying system for promoting bone repair as claimed in claim 8 in the preparation of drugs for treating osteoporosis.

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