Construction and application of drug-loaded frozen dental pulp stem cells for promoting cell burial effect

Abstract

The invention is applicable to the technical field of nano materials and medicines, and provides construction and application of drug-loaded frozen dental pulp stem cells for promoting a cell burial effect. Through a liquid nitrogen repeated quick-freezing technology, dead cells which have complete cell morphology and reserve cell membrane functional protein are successfully prepared, and the dead cells have good biocompatibility due to loss of a proliferation function, and can play good cell burial promotion and anti-inflammatory functions at the same time. When being used as a drug carrier, especially when shikonin and other Chinese herbal medicines and derived carbon dots are delivered, the drug stability can be kept, and a better regulation effect can be achieved after the drug is swallowed by macrophages, so that the functions of the macrophages with dysfunctional intercellular effect in a long-term inflammatory environment are improved, the inflammatory microenvironment is regulated, and the anti-inflammatory effect of the drug is improved. A new direction is provided for developing a treatment strategy aiming at chronic inflammatory diseases and senescence diseases.

Description

Technical Field

[0001] The present invention belongs to the technical fields of nanomaterials and medicine, and particularly relates to the construction and application of drug-loaded cryopreserved dental pulp stem cells that promote efferocytosis. Background Art

[0002] Efferocytosis refers to the process by which phagocytes clear apoptotic cells. Hundreds of millions of cells in the human body undergo apoptosis every day, and these apoptotic cells must be rapidly cleared to avoid secondary necrosis and the release of cytotoxic substances, which can lead to inflammatory damage. Among the phagocytes involved in efferocytosis, macrophages have the most significant effect. After efferocytosis, macrophages transform from a pro-inflammatory type to an anti-inflammatory type and release anti-inflammatory factors. Impairment of the efferocytosis process can lead to the accumulation of apoptotic cells, thereby triggering chronic inflammation and other clinical diseases, such as inflammatory and aging-related diseases like rheumatoid arthritis and osteoporosis. Therefore, treatment strategies that exert anti-inflammatory and anti-aging effects by enhancing the efferocytosis process are expected to become effective interventions for extending healthy lifespan and provide new ideas and methods for the treatment of chronic inflammation and aging-related diseases.

[0003] Traditional Chinese medicine is a precious wealth of our country, and many active ingredients of Chinese herbal medicines have multiple effects such as anti-inflammatory, antioxidant, and immunomodulatory effects. However, the clinical application of Chinese herbal medicines is limited by low solubility, poor permeability, poor gastrointestinal absorption, and low bioavailability. Therefore, improving the bioavailability and targeting of Chinese herbal medicines is the focus of current research and development of new drug delivery carriers.

[0004] As a new type of drug delivery carrier, stem cells have the ability to promote wound healing and tissue repair, regulate immunity and inflammation, and have targeting properties. Stem cells can migrate to specific areas of acute injury through "homing" delivery, enabling cells to migrate along chemical gradients. However, after living stem cells enter the body, there are heterogeneity and immunogenicity, which may trigger immune rejection reactions, and there are also adverse risks such as inducing tumors and thrombus formation. For this reason, the present invention proposes the construction and application of drug-loaded cryopreserved dental pulp stem cells that promote efferocytosis. Summary of the Invention

[0005] The purpose of the present invention is to provide the construction and application of drug-loaded cryopreserved dental pulp stem cells that promote efferocytosis, aiming to solve the problems raised in the above background art.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A construction method of drug-loaded cryopreserved dental pulp stem cells that promote efferocytosis, comprising the following steps:

[0008] Step 1: Extract primary human dental pulp stem cells, culture the dental pulp stem cells using standard cell culture techniques, collect the 3rd to 8th generation dental pulp stem cells with good growth status, resuspend them through centrifugation and cell cryopreservation solution, adjust the cell density, after repeated freezing with liquid nitrogen, wash three times with phosphate buffer solution, and centrifuge to obtain cryopreserved dental pulp stem cells;

[0009] Step 2: Mix the cryopreserved dental pulp stem cells obtained in Step 1 with the drug solution, and place them on a constant temperature shaker at 37°C and shake and incubate for 2 h;

[0010] Step 3: Centrifuge the solution after incubation in Step 2, wash three times with phosphate buffer solution to obtain drug-loaded cryopreserved dental pulp stem cells.

[0011] Further, in Step 1, the cell density is 5×10 6 ~1×10 7 / mL.

[0012] Further, in Step 1, the method of repeated freezing with liquid nitrogen is to freeze in liquid nitrogen for 24 h, then place it at 4°C to thaw for 30 min, and then place it in liquid nitrogen again to freeze for 24 h.

[0013] Further, the preparation method of the drug solution is: weigh 500 μg of the drug, dissolve it in 1 mL of PBS to prepare the drug solution.

[0014] Further, the ratio of the drug to the cryopreserved dental pulp stem cells is 500 μg / 1×10 7 .

[0015] Further, the drug is one of quinone Chinese herbal medicines, polyphenol Chinese herbal medicines or carbon dots derived from the above Chinese herbal medicines. Quinone Chinese herbal medicines include but are not limited to juglone, tanshinone, emodin, alizarin, aloe-emodin, lawsone, hypericin, etc.; Polyphenol Chinese herbal medicines include but are not limited to quercetin, kaempferol, tea polyphenols, resveratrol, curcumin, theaflavin, proanthocyanidins, naringenin, silybin, apigenin, gallic acid, caffeic acid, ferulic acid, syringaresinol, coniferyl alcohol, resveratrol, polydatin, etc.

[0016] Further, in Step 1 and Step 3, the centrifugation parameters are both centrifugation at 1500 rpm for 5 min.

[0017] A drug-loaded cryopreserved dental pulp stem cell for promoting efferocytosis obtained according to the above construction method.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] Through the repeated cryopreservation technology with liquid nitrogen, the present invention successfully prepared dead cells (LNT-DPSC) with intact cell morphology and retaining cell membrane functional proteins. These dead cells have good biocompatibility due to the loss of proliferation function, and at the same time can exert good functions in promoting efferocytosis and anti-inflammation, which provides new tools and models for in vitro studies on the mechanisms of various inflammation-related diseases. When LNT-DPSC is used as a drug carrier, especially when delivering traditional Chinese herbs and their derived carbon dots, it can maintain the stability of the drug and play a better regulatory role after being phagocytosed by macrophages, thereby improving the function of macrophages with dysregulated efferocytosis in the long-term inflammatory environment and regulating the inflammatory microenvironment, providing a new direction for the development of treatment strategies for chronic inflammatory diseases and senile diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a flow chart for constructing drug-loaded cryopreserved dental pulp stem cells of the present invention.

[0021] Figure 2 It is a live / dead cell staining result diagram of dental pulp stem cells cultured normally and cryopreserved dental pulp stem cells.

[0022] Figure 3 It is a representative image of drug-loaded cryopreserved dental pulp stem cells.

[0023] Figure 4 It is a scanning electron microscope image of dental pulp stem cells cultured normally and cryopreserved dental pulp stem cells.

[0024] Figure 5 It is an apoptosis detection result diagram of dental pulp stem cells cultured normally and cryopreserved dental pulp stem cells.

[0025] Figure 6 It is a fluorescence probe staining result diagram of Raw264.7 cells phagocytosing cryopreserved dental pulp stem cells.

[0026] Figure 7 It is a flow cytometry detection result diagram of cryopreserved dental pulp stem cells promoting efferocytosis of Raw264.7 cells.

[0027] Figure 8 It is an expression result diagram of efferocytosis-related genes TIM4, MERTK, AXL, TYRO3 and MFGE8 of cryopreserved dental pulp stem cells promoting Raw264.7 cells.

[0028] Figure 9 It is a regulatory effect diagram of cryopreserved dental pulp stem cells on the expression of inflammation-related genes of M1 type Raw264.7 cells. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] For a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the following provides a detailed description of the technical solution of the present invention, but it should not be construed as a limitation on the scope of implementation of the present invention.

[0030] The following describes the specific implementation of the present invention in detail in conjunction with specific embodiments.

[0031] An embodiment of the present invention provides a method for constructing a drug-loaded cryopreserved dental pulp stem cell (LNT-DPSC) that promotes efferocytosis, and its flowchart is as Figure 1 shown, including the following steps:

[0032] Step 1: Prepare LNT-DPSC by liquid nitrogen freezing;

[0033] Select healthy permanent teeth (without caries, periodontal disease, and periapical disease) extracted from adults aged 18-22 years due to impacted or orthodontic extraction, and place them in α-MEM medium containing 2% penicillin-streptomycin for later use; under sterile conditions, take out the dental pulp, remove about 3 mm of dental pulp tissue at the root apex, and repeatedly rinse the dental pulp tissue with phosphate buffered saline (PBS) containing 2% penicillin-streptomycin. After cutting it into pieces, add α-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, place the tissue mass in a T25 culture flask, and culture it at 37 °C and 5% CO 2 under saturated humidity conditions; after 7 days, observe the adhesion of the dental pulp tissue mass and the outgrowth of cells under a microscope. When the cells reach 80-90% confluence, passage them, and then continue to culture them with α-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, which is the 1st generation of hDPSCs (human dental pulp stem cells). Collect the 3rd to 8th generations of hDPSCs, resuspend them with cell cryopreservation solution after centrifuging at 1500 rpm for 5 min, adjust the cell density to 5×10 6 ~1×10 7 / mL, then immediately immerse the cell suspension in liquid nitrogen and freeze it for 24 h, then place it at 4 °C and thaw it for 30 min, put it back into liquid nitrogen for freezing for 24 h, take it out, wash it three times with PBS, and centrifuge it at 1500 rpm for 5 min to obtain LNT-DPSC.

[0034] Step 2: Prepare the drug-loaded cryopreserved dental pulp stem cells;

[0035] Weigh 500 μg of shikonin and curcumin-derived carbon dots respectively, dissolve them in 1 mL of PBS to prepare a shikonin solution and a curcumin-derived carbon dot solution, and use the 1×10 7LNT-DPSCs were resuspended in shikonin solution and curcumin-derived carbon dots solution respectively, placed on a constant temperature shaker at 37 °C and incubated with shaking for 2 h, centrifuged at 1500 rpm for 5 min, and washed three times with PBS to obtain shikonin-loaded dental pulp stem cells (LNT-DPSC / SH) and curcumin-derived carbon dots-loaded dental pulp stem cells (LNT-DPSC / Cur-CDs).

[0036] Representative images of shikonin-loaded dental pulp stem cells (LNT-DPSC / SH) and curcumin-derived carbon dots-loaded dental pulp stem cells (LNT-DPSC / Cur-CDs) are shown as Figure 3 follows.

[0037] In the embodiment of the present invention, as a novel drug delivery carrier, LNT-DPSCs can load shikonin and curcumin-derived carbon dots into them through co-incubation. On the one hand, it improves the bioavailability and targeting of shikonin and curcumin-derived carbon dots, which helps to better exert the therapeutic effect of Chinese herbal medicines. On the other hand, through targeting macrophage efferocytosis, it promotes the transformation of macrophages from a pro-inflammatory phenotype to an anti-inflammatory phenotype. It not only provides a new tool and model for in vitro study of macrophage efferocytosis and inflammation regulation, but also provides an experimental basis for exploring the treatment mechanism of inflammatory diseases.

[0038] Based on the excellent drug-loading ability of dental pulp stem cells, shikonin and curcumin-derived carbon dots can be replaced by quinone Chinese herbal medicines, polyphenol Chinese herbal medicines or one of the above Chinese herbal medicine-derived carbon dots. Quinone Chinese herbal medicines include but are not limited to juglone, tanshinone, emodin, alizarin, aloe-emodin, lawsone, hypericin, etc.; Polyphenol Chinese herbal medicines include but are not limited to quercetin, kaempferol, tea polyphenols, resveratrol, curcumin, theaflavin, proanthocyanidins, naringenin, silybin, apigenin, gallic acid, caffeic acid, ferulic acid, syringaresinol, coniferyl alcohol, resveratrol, polydatin, etc. It can be used for in vitro study of the mechanisms of efferocytosis disorder-related diseases (such as cardiovascular system diseases, respiratory system diseases, digestive system diseases, immune system diseases, etc.).

[0039] Experimental methods and results:

[0040] 1. Cell staining and fluorescence microscopy observation:

[0041] Normal cultured hDPSCs (LIVE-DPSC) and dental pulp stem cells (LNT-DPSC) were stained using the Beyotime Calcein-AM / PI staining kit, and observed under a fluorescence microscope after staining. As Figure 2 shown, red fluorescence represents dead cells, and green fluorescence represents live cells. It can be seen that almost all dental pulp stem cells died after repeated quick-freezing in liquid nitrogen.

[0042] 2. Scanning electron microscopy observation:

[0043] After the preparation of LNT-DPSC, the LNT-DPSC obtained in step 1 and the normally cultured hDPSCs were collected separately, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded; after washing once with PBS, it was fixed with 4% paraformaldehyde at room temperature for 30 min; washed twice with PBS, 10 min each time, and then successively immersed in 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% ethanol solutions for gradient dehydration treatment, 3 min each time; after dehydration, it was dropped on a silicon wafer and placed in a critical point dryer for drying; after drying, the sample was fixed with conductive glue and placed on the sample stage of an ion sputtering instrument for gold spraying for 30 s, and then observed using a scanning electron microscope. The scanning electron micrographs of LIVE-DPSC and LNT-DPSC are as Figure 4 shown. Compared with LIVE-DPSC, LNT-DPSC has a complete overall structure and a spherical structure similar to that of living cells, but the cell surface of LNT-DPSC is slightly rough.

[0044] 3. Detection of cell apoptosis:

[0045] The LIVE-DPSC and LNT-DPSC were detected for apoptosis using the Annexin V / PI apoptosis kit from Beyotime. The results are as Figure 5 shown. Most of the LNT-DPSC showed AnnexinⅤ+PI+(UR region), followed by AnnexinⅤ+PI-(LR region), that is, late apoptotic cells / necrotic cells and early apoptotic cells respectively. Phosphatidylserine (PS) is one of the important phospholipid components of the cytoplasmic membrane. Along with the occurrence of apoptosis, PS flips from the inner side of the cell membrane to the outer side of the cell membrane, presenting a specific "eat me" signal. There are various receptors on the surface of macrophages that can recognize PS, and the receptors can specifically bind to PS on the surface of apoptotic cells, thus initiating the phagocytosis process of macrophages. Therefore, it is expected to use LNT-DPSC to achieve precise drug delivery to macrophages and at the same time enhance the efferocytosis of macrophages.

[0046] 4. Macrophage phagocytosis experiment:

[0047] Raw264.7 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and cultured under the conditions of 37 °C, 5% CO 2 saturated humidity. After induction with 1 μg / mL lipopolysaccharide (LPS) for 6 h, Raw264.7 cells were labeled with CellTracker TM GreenCMFDA (green), and LNT-DPSC was labeled with CellTracker CM-Dil (red).

[0048] Co-culture the CellTracker CM-Dil-labeled LNT-DPSC with the CellTracker TM Green CMFDA-labeled Raw264.7 cells. As shown in the +LNT-DPSC group, a small amount of LNT-DPSC was phagocytosed by Raw264.7 cells after 12 h. Co-culture the unlabeled LNT-DPSC with the CellTracker Figure 6 Green CMFDA-labeled Raw264.7 for 12 h, then remove the LNT-DPSC and at intervals of 6 h, and then co-culture again with the CellTracker CM-Dil-labeled LNT-DPSC for 12 h. The results are as TM shown in the ++LNT-DPSC group. The phagocytosis of Raw264.7 cells stimulated by LNT-DPSC for the first time was further enhanced, and more LNT-DPSC could be phagocytosed, indicating that multiple stimulations of LNT-DPSC can promote the continuous efferocytosis ability of macrophages. Figure 6

[0049] 5. Detection of macrophage efferocytosis index:

[0050] Figure 7 For the results of the detection of macrophage efferocytosis index, LNT-DPSC was labeled with CellTracker CM-Dil, and the efferocytosis index of Raw264.7 cells was the number of positive cells / total number of Raw264.7 cells. When LNT-DPSC was added for the first time (+LNT-DPSC), the efferocytosis index was 17.75%. When LNT-DPSC was added for the second time (++LNT-DPSC), the efferocytosis index further increased to 24.61%, indicating that LNT-DPSC can promote the continuous efferocytosis of macrophages and further enhance phagocytosis.

[0051] 6. Gene expression analysis:

[0052] Add 1 μg / mL LPS to Raw264.7 cells to induce for 6 h, then add LNT-DPSC. After 24 h or 48 h, remove the culture medium, wash three times with PBS, add fresh culture medium, and collect the cells after 6 h for RT-qPCR detection. The results are as Figure 8 shown. After adding LNT-DPSC, the expressions of efferocytosis-related genes TIM4, MERTK, AXL, TYRO3, and MFGE8 were significantly up-regulated. As Figure 9 ​As shown, compared with the LPS group, the expressions of pro-inflammatory related genes TNF-α, IL-1β and IL-6 in the +LNT-DPSC group were significantly down-regulated, and the expression of the anti-inflammatory related gene IL-10 was up-regulated. It indicates that LNT-DPSC can not only promote macrophage efferocytosis, but also has excellent anti-inflammatory effects.

[0053] The above are only the preferred embodiments of the present invention. It should be noted that for those skilled in the art, without departing from the concept of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicability of the patent.

Claims

1. A method for constructing drug-loaded frozen dental pulp stem cells that promotes the cell burial effect, characterized in that: The following steps are involved: Step 1: Extract primary human dental pulp stem cells, culture dental pulp stem cells using standard cell culture techniques, collect dental pulp stem cells from the 3rd to 8th generations with good growth status, adjust the cell density by centrifugation and resuspending in cell freezing solution, freeze repeatedly with liquid nitrogen, wash three times with phosphate buffer, and centrifuge to obtain frozen dental pulp stem cells; Step 2: Mix the frozen dental pulp stem cells obtained in step 1 with the drug solution and incubate them on a 37°C constant temperature shaker for 2 hours; Step 3: Centrifuge the solution after incubation in step 2, wash three times with phosphate buffer, and obtain drug-loaded frozen dental pulp stem cells.

2. The construction method according to claim 1, characterized in that: In step 1, the cell density was 5×10 6 ~1×10 7 / mL.

3. The construction method according to claim 1, characterized in that: In step 1, the liquid nitrogen repeated freezing method is to freeze in liquid nitrogen for 24 hours, then thaw at 4° C. for 30 minutes, and then freeze in liquid nitrogen again for 24 hours.

4. The construction method according to claim 1, characterized in that: The preparation method of the drug solution is as follows: 500 μg of drug is weighed and dissolved in 1 mL of PBS to prepare the drug solution.

5. The construction method according to claim 4, characterized in that: The ratio of the drug to frozen dental pulp stem cells is 500 μg / 1×10 7 .

6. The construction method according to claim 5, characterized in that: The drug is a quinone Chinese herbal medicine, a polyphenol Chinese herbal medicine or one of the above Chinese herbal medicine-derived carbon dots. The quinone Chinese herbal medicine includes but is not limited to juglone, danshenquinone, rhamnolide, madder, aloe-rhamnolide, anacardin, and hypericin; the polyphenol Chinese herbal medicine includes but is not limited to quercetin, kaempferol, tea polyphenols, resveratrol, curcumin, theaflavins, proanthocyanidins, naringenin, silybin, apigenin, gallic acid, caffeic acid, ferulic acid, eugenol, coniferol, resveratrol, and polydatin.

7. The construction method according to claim 1, characterized in that: In step 1 and step 3, the centrifugation parameters are both 1500 rpm for 5 min.

8. A drug-loaded frozen dental pulp stem cell that promotes cell burial effect obtained according to the construction method of any one of claims 1 to 7.

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