Freezing treatment cell as well as preparation method and application thereof

Abstract

The invention is applicable to the technical field of biological medicines, and provides a frozen cell as well as a preparation method and application thereof. According to the invention, frozen human umbilical cord mesenchymal stem cells (CK-MSCs) are adopted as a carrier of mitoxantrone (MTX), so that accurate delivery and pH-sensitive controlled release of drugs at tumor sites are realized, the treatment effect is remarkably improved, and the side effects of traditional chemotherapy are greatly reduced. The freezing treatment enables the human umbilical cord mesenchymal stem cells to lose the multiplication capacity, so that the tumor formation risk is eliminated, and the treatment safety is ensured. By adjusting the immune response in the tumor microenvironment, the CK-MTX not only directly inhibits the tumor, but also enhances the anti-tumor immune defense of the body. The freezing treatment method disclosed by the invention can be applied to other types of cells, and the frozen cells can be compatible with other medicines, so that revolutionary breakthrough is brought to tumor treatment, and a new way is opened up for treatment of other diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a cryo-treated cell and a preparation method and application thereof. Background Art

[0002] In the field of medicine, precision medicine and the optimization of drug delivery systems have always been hot topics and difficulties in research. Although traditional chemotherapy and radiotherapy have achieved remarkable results in the treatment of various malignant tumors, these methods have encountered significant limitations and challenges in practical applications. Chemotherapy is administered systemically so that the drug reaches the tumor site through the blood circulation. However, since the drug will inevitably be distributed in normal tissues during systemic circulation, it will cause a series of serious side effects, such as bone marrow suppression, nausea, vomiting, hair loss, etc. These side effects not only affect the patient's quality of life, but also limit the dose of the drug, thereby reducing the therapeutic effect. Although radiotherapy can effectively kill tumor cells, its accuracy is limited and it often causes damage to the surrounding normal tissues, leading to radiation dermatitis, bone marrow suppression and other serious side effects. In addition, the targeting of radiotherapy is not ideal, and it is difficult to avoid the impact on non-tumor tissues.

[0003] Prostate cancer, as one of the most common malignant tumors in men, also faces the above challenges in its treatment. In this context, existing drug delivery systems face the problems of insufficient precise targeting and limited controlled release ability in tumor treatment. Traditional drug delivery methods often find it difficult to achieve efficient accumulation of drugs at the tumor site, resulting in excessive distribution of drugs in normal tissues, thereby increasing the probability of toxic side effects. In recent years, cell-based drug delivery systems have attracted widespread attention due to their unique biocompatibility and potential targeting capabilities. Although some studies have begun to explore the use of stem cells as drug carriers to improve the efficiency of targeted drug delivery by utilizing their natural tumor tropism, active stem cells as drug carriers still face significant challenges in their application, such as tumorigenic risks, ethical issues, and limited drug loading capacity. These problems limit the clinical application of active stem cells and make it difficult to meet the needs of efficient anti-cancer treatment. For this reason, a cryo-treated cell, a preparation method, and an application thereof are proposed. Summary of the invention

[0004] The purpose of the present invention is to provide a cryo-treated cell and a preparation method and application thereof, aiming to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for preparing cryotreated cells, wherein the cryotreated cells are used as a drug delivery system, the drug delivery system includes CK-MTX, and the preparation method of CK-MTX is:

[0007] Preparation of hUC-MSCs: First, the hUC-MSCs were digested with trypsin to separate and collect the cells. The collected cells were transferred to a 15 ml centrifuge tube and centrifuged at 500 g for 5 minutes, and the supernatant was discarded;

[0008] Preparation of CK-MSCs: hUC-MSCs were resuspended in PBS, and then the cell suspension was transferred to a 2 ml cell cryotube, centrifuged again at 500 g for 5 minutes, the supernatant was discarded, and the cryotube was frozen at -80 °C for 24 hours;

[0009] Preparation of CK-MTX: The frozen CK-MSCs were mixed with 500 μg / ml MTX solution and incubated at 4°C for 24 hours. After the incubation, the cells were collected and centrifuged, the supernatant was discarded, and the cells were resuspended in PBS to obtain the final drug delivery system.

[0010] The CK-MSCs were stored at -80°C for a short term of up to one year and stored in liquid nitrogen for a long term.

[0011] A frozen cell prepared according to the method for preparing frozen cells described above.

[0012] A use of the above-described cryotreated cells in the preparation of medicines.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention adopts cryopreserved human umbilical cord mesenchymal stem cells (CK-MSCs) as a carrier of the chemotherapy drug mitoxantrone (MTX), achieving accurate delivery of the drug at the tumor site and pH-sensitive controlled release, thereby significantly improving the therapeutic effect and greatly reducing the side effects of traditional chemotherapy. Cryopreservation causes umbilical cord mesenchymal stem cells to lose their ability to proliferate, eliminates the risk of tumor formation, and ensures the safety of treatment. By regulating the immune response in the tumor microenvironment, CK-MTX not only directly inhibits tumors, but also enhances the body's anti-tumor immune defense. The good drug loading capacity and release stability of CK-MSCs further improve the effectiveness and stability of drug delivery. The cryopreservation method of the present invention is not limited to human umbilical cord mesenchymal stem cells, but can also be applied to other types of cells; in terms of drug loading, cryopreserved cells are not limited to loading MTX, but can also be compatible with other drugs other than MTX. Therefore, the present invention not only brings a revolutionary breakthrough in tumor treatment, but also opens up new ways for the treatment of other diseases, showing significant clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1Characterization of the morphological and biological characteristics of CK-MSCs; a is the characterization of surface markers of hUC-MSCs using flow cytometry, b is the quantitative analysis of flow cytometry data, c is the growth curve of hUC-MSCs measured by CCK-8 method, d is the cell cycle distribution analysis of hUC-MSCs using flow cytometry, e is the quantitative analysis of cell cycle data, and f is the observation of the multipotential differentiation potential of hUC-MSCs using a microscope, showing the chondrogenic, adipogenic and osteogenic differentiation abilities from left to right.

[0016] Figure 2 The effects of freezing treatment strategy on hUC-MSCs and their potential as targeted drug carriers; a is the optical microscope and confocal microscope bright field imaging (Bright field) images of hUC-MSCs and CK-MSCs, b is the confocal microscope image of Calcein-AM / PI double staining, c is the trypan blue staining of hUC-MSCs and CK-MSCs under optical microscope, d is the flow cytometry image of hUC-MSCs and CK-MSCs, e is Figure 2 d is the quantitative result, f is the effect of freezing treatment strategy on the proliferation ability of hUC-MSCs determined by CCK8 experiment, g is the scanning electron microscope (SEM) image of hUC-MSCs and CK-MSCs, and h is the transmission electron microscope (TEM) image of hUC-MSCs and CK-MSCs.

[0017] Figure 3 The drug loading capacity and release characteristics of CK-MSCs loaded with MTX; wherein, a is the confocal microscope image of CK-MSCs loaded with MTX, b is the high performance liquid chromatography (HPLC) graph, c is the standard curve, d is the change of the drug loading amount of CK-MSCs in different concentrations of MTX solution with the increase of MTX concentration, e is the change of the drug loading rate of CK-MSCs with the increase of MTX concentration, f is the Zeta potential measurement of CK-MSCs and CK-MTX, g is the HPLC analysis of MTX release of CK-MTX under in vitro conditions, h is the standard curve of the release experiment, and i is the drug release curve of CK-MTX under different pH conditions.

[0018] Figure 4 The protein expression changes of hUC-MSCs and CK-MSCs; a is Coomassie Brilliant Blue staining of CK-MSCs and hUC-MSCs, b is Western blot analysis of CXCR4 and β-actin in CK-MSCs and hUC-MSCs, c is Figure 4b is the quantitative analysis, d is the Western blot analysis of CCR2 and β-tubulin in CK-MSCs and hUC-MSCs, and e is Figure 4 d is the quantitative analysis, f is the volcano plot of differentially expressed proteins, g is the heat map of differentially expressed proteins, h is the Gene Ontology (GO) analysis, i is the pathways involved in the downregulated proteins, j is the key protein expression ratio analysis, k is the KEGG pathway analysis, l is the pathways involved in the downregulated KEGG pathways, and m is the relative quantitative analysis of key proteins.

[0019] Figure 5 is the in vitro inhibitory effect of CK-MTX on tumor cells; a is high-content imaging, b is scanning electron microscopy (SEM) image, c is flow cytometry analysis, d is Figure 5 The quantitative analysis of c, e is the scratch test, and f is Figure 5 e is the quantitative analysis, g is the invasion assay, and h is Figure 5 Quantitative analysis in g, i is a 24h CCK-8 cell viability experiment, j is a 48h CCK-8 cell viability experiment, and k is a 72h CCK-8 cell viability experiment.

[0020] Figure 6 is the antitumor efficacy of CK-MTX in the tumor-bearing mouse model; wherein, a is H&E staining analysis, b is the fluorescence imaging result, c is the quantitative result of the distribution of CK-Cy5.5 in tumor-bearing mice, d is the change of fluorescence intensity of each organ over time (I-tumor, II-kidney, III-liver, IV-heart, V-lung, VI-spleen), e is the fluorescence intensity time curve of each organ (I-tumor, II-kidney, III-liver, IV-heart, V-lung, VI-spleen), f is the outline of the animal experiment, g is mouse weight monitoring, h is survival analysis, i is a photo of mouse tumor tissue, and j is the volume change curve.

[0021] Figure 7 is the effect of CK-MTX on immune-related markers in the tumor microenvironment; wherein, a is the expression of multiple immune markers in tumor tissues of different treatment groups, b is the quantitative result of CD3-positive T cells, c is the quantitative result of CD4-positive T cells, d is the quantitative result of CD8-positive T cells, e is the CD8 / CD4 signal intensity ratio, f is the quantitative analysis of HMGB1, g is the quantitative result of Granzyme B, h is the TUNEL staining result, i is the CRT staining analysis, j is the quantitative analysis of FOXP3, k is the quantitative result of GR-1-positive myeloid-derived suppressor cells, l is the expression analysis of PD-L1, m is the quantitative expression result of SDF-1, and n is the Ki67 staining analysis.

[0022] Figure 8For the systemic toxicity and safety evaluation of CK-MTX; a is the H&E staining images of the main organs (heart, liver, spleen, lung and kidney) of NC, CK-MSCs, MTX and CK-MTX groups, b is the quantitative analysis of heart tissue, c is the quantitative result of liver tissue, d is the number of germinal centers in spleen tissue, e is the quantitative analysis of alveolar area, and f is the glomerular diameter analysis of kidney tissue.

[0023] Fig. 9 This is an in vivo animal efficacy evaluation of CK-MTX; wherein, a is the H&E staining image of tumor tissue under different treatment groups (NC, CK-MSCs, MTX, CK-MTX), b is the quantitative ratio of pathological nuclear division figures in tumor tissue, c is the TNF-α concentration analysis, d is the IFN-γ concentration analysis, e is the IL-1β concentration analysis, f is the IL-6 concentration analysis, and g is the IL-8 concentration analysis. DETAILED DESCRIPTION

[0024] 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 used to limit the present invention.

[0025] Human umbilical cord mesenchymal stem cells (hUC-MSCs) not only inherit the general characteristics of mesenchymal stem cells (MSCs), but also show superior targeting and biosafety due to their unique source. Therefore, one embodiment of the present invention provides an innovative drug delivery system using frozen human umbilical cord mesenchymal stem cells (CK-MSCs) as a drug carrier for the preparation of drugs for treating prostate cancer. By loading mitoxantrone (MTX) into CK-MSCs, the present invention can achieve precise targeted delivery of MTX, providing a new idea for the treatment of prostate cancer.

[0026] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0027] Example 1, preparation and characterization of CK-MSCs;

[0028] 1) Preparation of hUC-MSCs;

[0029] Cell digestion and collection: After trypsin digestion, hUC-MSCs were collected into a 15 ml centrifuge tube and centrifuged at 500 g (relative centrifugal force, RCF) for 5 minutes, and the supernatant was discarded.

[0030] 2) Preparation of CK-MSCs;

[0031] Cell resuspension and freezing: After resuspending hUC-MSCs with PBS, transfer the cell suspension to a 2 ml cell cryopreservation tube. Centrifuge again at 500g for 5 minutes, discard the supernatant, and store the cryopreservation tube at -80℃ for 24 hours.

[0032] Storage of CK-MSCs: short-term storage (within 1 year) at -80°C, long-term storage in liquid nitrogen.

[0033] 3) Preparation of CK-MTX;

[0034] Treatment and incubation: CK-MSCs were mixed with 500 μg / ml MTX solution and incubated at 4°C for 24 hours. After the incubation, the cells were collected and centrifuged, the supernatant was discarded, and the cells were resuspended in PBS to obtain CK-MTX cells for subsequent use.

[0035] Figure 1 The morphological and biological characteristics of CK-MSCs were characterized. Flow cytometry was used to characterize the surface markers of hUC-MSCs, and the markers analyzed included CD73, CD90, CD105 (positive markers) and CD34, CD45, HLA-DR (negative markers). Figure 1 As can be seen in a, hUC-MSCs highly express CD73, CD90 and CD105, and lowly express CD34, CD45, HLA-DR, CD11b and CD19. This shows that the expression of surface markers of hUC-MSCs is consistent with the characteristics of mesenchymal stem cells. Further quantitative analysis of flow cytometry data ( Figure 1 The results showed that hUC-MSCs showed consistent surface marker expression in multiple experimental samples, verifying the stability and uniformity of the cell population. The growth curve of hUC-MSCs was determined by CCK-8 method ( Figure 1 c), observe the proliferation ability of cells. It can be seen from the figure that hUC-MSCs present a typical logarithmic growth phase, indicating that these cells have good proliferation ability. Flow cytometry was used to analyze the cell cycle distribution of hUC-MSCs ( Figure 1 The results showed that most cells were in the G0 / G1 phase, indicating that hUC-MSCs were in an active proliferation state, while some cells were in a quiescent phase or preparing to divide. Quantitative analysis of cell cycle data ( Figure 1 e), further confirming that most hUC-MSCs remain in the G0 / G1 phase, which is consistent with good proliferation ability. The multipotent differentiation potential of hUC-MSCs was observed using a microscope ( Figure 1The results showed that hUC-MSCs successfully differentiated into osteocytes, adipocytes and chondrocytes under appropriate culture conditions. It can be seen that hUC-MSCs not only have typical surface markers, but also retain multipotent differentiation potential, laying the foundation for subsequent research.

[0036] Example 2: Effects of cryotherapy strategies on hUC-MSCs and their potential as targeted drug carriers;

[0037] Figure 2 The effects of cryotherapy on hUC-MSCs were demonstrated and their potential as targeted drug delivery vehicles was evaluated. Bright field images of optical microscopy and confocal microscopy showed the morphological changes of hUC-MSCs before and after cryotherapy. Figure 2 (a). The optical microscope image in the upper part shows that the untreated hUC-MSCs have intact morphology, while the CK-MSCs after freezing treatment are slightly enlarged, but the overall structure is still preserved. The bright field imaging in the lower part further shows that the cell surface of CK-MSCs is slightly rougher than that of hUC-MSCs, but the cell outline is clearly visible. This shows that although the freezing treatment strategy leads to a decrease in cell activity, CK-MSCs still retain a relatively complete cell structure. The confocal microscope image of Calcein-AM / PI double staining shows the activity state of the cells ( Figure 2 b). In the figure, the green Calcein-AM dye marks live cells, while the red PI dye marks dead cells. The results showed that all cells in the CK-MSCs group were stained red by PI, indicating that the cells had lost their activity. In contrast, most cells in the hUC-MSCs group were green, showing higher activity. This indicates that the freezing treatment strategy leads to the loss of activity of CK-MSCs. Trypan blue staining under an optical microscope also shows the activity status of the cells ( Figure 2 c). The results showed that the cells in the hUC-MSCs group were not stained blue, while the cells in the CK-MSCs group were stained blue. This shows that the freezing treatment strategy leads to the loss of activity of CK-MSCs. Flow cytometry images show the quantitative analysis of cell apoptosis status ( Figure 2 CK-MSCs group showed a higher proportion of apoptotic cells, further verifying that the freezing treatment strategy significantly increased the apoptosis rate of cells. The quantitative results ( Figure 2 Figure e) shows that the proportion of late apoptotic cells in the CK-MSCs group was much higher than that in the hUC-MSCs group, and the difference was statistically significant. This result supports the loss of cell activity and increased apoptosis caused by the freezing treatment strategy. The CCK8 experiment was used to determine the effect of the freezing treatment strategy on the proliferation ability of hUC-MSCs. Figure 2Figure f shows that the proliferation ability of CK-MSCs is significantly lower than that of hUC-MSCs, and there is almost no proliferation activity within a few days after freezing. This result indicates that CK-MSCs lose their proliferation ability after freezing. Scanning electron microscopy (SEM) images further observed the microstructure of hUC-MSCs and CK-MSCs ( Figure 2 The results showed that the surface structure of CK-MSCs still maintained the overall cell structure compared with hUC-MSCs, which provided a basis for its use as a drug carrier. Transmission electron microscopy (TEM) images showed the internal structure of hUC-MSCs and CK-MSCs ( Figure 2 (h) As can be seen from the figure, the main organelles of the internal structure of CK-MSCs are still clearly visible, indicating the structural stability of the cells after freezing treatment.

[0038] Example 3: Drug loading capacity and release characteristics of CK-MSCs loaded with MTX;

[0039] Figure 3 The process of CK-MSCs loading with MTX and its drug loading and release characteristics are demonstrated. Confocal microscopy images show that CK-MSCs are successfully loaded with MTX ( Figure 3 (a). Dio green fluorescence labels the cell membrane of CK-MSCs, while MTX is labeled by its own red fluorescence. In the Merge diagram, it can be seen that the red MTX is mainly concentrated in the cytoplasmic region of the cell, which is in sharp contrast with the green Dio dye, indicating that MTX successfully enters and resides in CK-MSCs. This shows that CK-MSCs have the potential to be used as drug carriers. The high-performance liquid chromatography (HPLC) diagram shows the main peak of MTX in the CK-MSCs cell lysate ( Figure 3 b), proving the presence of MTX in cells. The standard curve shows the linear relationship between different concentrations of MTX in HPLC detection ( Figure 3 c), to further quantify the drug loading in the cells. Figure 3 Figure d shows that the drug loading of CK-MSCs in different concentrations of MTX solution increases with the increase of MTX concentration, reaching the highest value at a concentration of 500 μg / mL. This indicates that CK-MSCs can efficiently absorb and maintain a higher concentration of MTX. Figure 3 Figure e shows that the drug loading rate gradually decreases with the increase of MTX concentration, which means that at higher concentrations, the absorption of MTX by CK-MSCs reaches saturation, resulting in a decrease in relative drug loading efficiency. Zeta potential measurement shows ( Figure 3In (f), the potential of CK-MTX was significantly lower than that of CK-MSCs, which indicates that the CK-MTX particles loaded with MTX are more stable and suitable for transmission in the systemic circulation in vivo. Further HPLC analysis showed the release of MTX from CK-MTX under in vitro conditions ( Figure 3 The peak in the figure indicates that MTX is gradually released from CK-MTX after a period of time. The standard curve of the release experiment once again confirmed the linear relationship between CK-MTX and MTX release under different conditions ( Figure 3 h), which provides a basis for calculating the cumulative release. Figure 3 Figure i shows the drug release curve of CK-MTX under different pH conditions. The results show that in an acidic environment (pH 5.5), the release rate of MTX is significantly faster than that in a neutral environment (pH 7.4), and the release amount is significantly increased. This property indicates that CK-MTX can achieve rapid drug release in the acidic microenvironment of the tumor, enhance the chemotherapy effect, and reduce drug release in normal tissues, thereby reducing side effects.

[0040] Summary: This example demonstrates the stability, drug loading and release characteristics of CK-MSCs under different pH conditions after loading with MTX. The results show that the targeted drug release characteristics of CK-MTX in the tumor microenvironment provide strong support for its use as a chemotherapeutic drug carrier.

[0041] Example 4: Proteomic analysis of hUC-MSCs and CK-MSCs;

[0042] Figure 4 The expression of key proteins in hUC-MSCs and CK-MSCs is shown. Coomassie Brilliant Blue staining shows significant differences in protein expression between CK-MSCs and hUC-MSCs ( Figure 4 In (a), it can be observed that there are obvious changes in band intensity and position between the two cell types, indicating that the freezing treatment strategy leads to extensive changes in the protein expression profile of CK-MSCs. Western blot analysis shows ( Figure 4 b), the expression of chemokine receptor CXCR4 was significantly upregulated in CK-MSCs after freezing treatment, and this result was quantitatively analyzed ( Figure 4 c) was further verified, and the upregulation of CXCR4 suggested that the cryotherapy strategy may enhance the migration and immunomodulatory ability of CK-MSCs. Similarly, the expression of CCR2 in CK-MSCs was also significantly upregulated ( Figure 4 d), its quantitative analysis ( Figure 4This observation is further supported by the fact that the upregulation of CCR2 may indicate that the cryotherapy strategy enhances the responsiveness of CK-MSCs to inflammatory signals, which contributes to their potential application as drug delivery vehicles. The volcano plot of differentially expressed proteins revealed significant expression changes of a large number of proteins between CK-MSCs and hUC-MSCs ( Figure 4 f), the volcano plot shows the distribution of significantly up-regulated and down-regulated proteins, further demonstrating the effect of freezing treatment strategy on protein expression. The heat map shows the clustering of these differentially expressed proteins ( Figure 4 Figure g) shows that the protein expression profiles of CK-MSCs involved in multiple biological processes such as metabolism, gene expression regulation, cytoskeleton, immune response, and autophagy have undergone systematic changes after freezing treatment, emphasizing the significant changes in the expression levels of key proteins before and after freezing treatment. Gene Ontology (GO) analysis revealed ( Figure 4 h), the upregulated proteins in CK-MSCs were mainly involved in biological processes such as oxidative phosphorylation, cellular respiration, nucleic acid metabolism, and protein localization. These upregulated biological processes may indicate that CK-MSCs maintain basic cellular functions by enhancing metabolic activity. The downregulated proteins were mainly involved in pathways such as glycolysis and metabolite transport ( Figure 4 i), suggesting that CK-MSCs may adapt to environmental changes after freezing through metabolic reprogramming, which also reflects the remodeling of their energy metabolism pathways. Analysis of key protein expression ratios showed ( Figure 4 In the study, important biological processes such as oxidative phosphorylation, fatty acid metabolism and glycosylation were significantly activated in CK-MSCs. This analysis suggests that CK-MSCs may adapt to the new cell living environment by activating these key metabolic pathways after freezing. KEGG pathway analysis further revealed that ( Figure 4 In the KEGG table, the upregulated pathways in CK-MSCs mainly include energy metabolism-related pathways such as oxidative phosphorylation, thermal thermogenesis, and fatty acid degradation. These upregulated pathways indicate that CK-MSCs are more dependent on efficient energy generation pathways to maintain their metabolic needs after freezing. The downregulated KEGG pathways involve MAPK signaling pathways and glycolysis / gluconeogenesis ( Figure 4 l), indicating that the freezing treatment strategy may induce cells to enter a metabolic energy-saving mode by inhibiting these pathways. The relative quantitative analysis of key proteins further supports the changes in the above pathways ( Figure 4 m), especially in pathways such as thermal thermogenesis, glycosaminoglycan biosynthesis, fatty acid degradation, and O-glycosylation. Multiple key proteins were significantly upregulated in CK-MSCs, indicating that these pathways play an important role in the metabolic adaptation of cells after cryotreatment.

[0043] Summary: Proteomic analysis showed that the freezing treatment strategy had a significant impact on the metabolic pathways and protein expression of CK-MSCs. In particular, the activation of key biological processes such as oxidative phosphorylation, fatty acid metabolism and glycosylation emphasized the metabolic reprogramming of CK-MSCs after freezing treatment, which provides a new biological basis for its potential as a drug delivery vehicle.

[0044] Example 5, in vitro anti-tumor effect of CK-MTX;

[0045] Figure 5 Demonstrated the inhibitory effect of CK-MTX on tumor cells in vitro. High-content imaging ( Figure 5 (a) shows the results of CK-MTX targeting tumor cells RM-1 at different time points. The cells showed obvious MTX staining, indicating that CK-MTX effectively delivered the drug to the target cells. Scanning electron microscopy (SEM) images further show ( Figure 5 In (b), the close contact between CK-MTX and tumor cells is more significant than that in other treatment groups, indicating that CK-MTX has stronger targeting. This close cell-to-cell contact may be the key to its enhanced drug delivery efficiency. Flow cytometry analysis ( Figure 5 c) and its quantitative results ( Figure 5 (d) shows that the uptake of MTX by RM-1 cells in the CK-MTX group was significantly higher than that in other groups, and the positive rate of MTX reached 87.4%, which further verified the efficient drug delivery ability of CK-MTX. Figure 5 e), CK-MTX significantly inhibited the migration ability of tumor cells. The CK-MTX-treated group had almost no migration within 24 hours, and this result was confirmed by quantitative analysis ( Figure 5 f) further confirmed that the migration rate of the CK-MTX group was significantly lower than that of the other groups. Invasion experiments showed that ( Figure 5 g), CK-MTX significantly inhibited the invasion ability of tumor cells. The quantitative results ( Figure 5 Middle h) showed that the number of cell invasion in the CK-MTX treatment group was significantly reduced, further proving its strong anti-invasion ability. CCK-8 cell viability experiment showed ( Figure 5 In i, j, and k, i-24h, j-48h, and k-72h), CK-MTX significantly inhibited the growth of tumor cells RM-1 better than other treatment groups. With the increase of concentration, the cell viability of the CK-MTX group decreased significantly, showing its strong tumor inhibitory effect.

[0046] Example 6, in vivo anti-tumor effect of CK-MTX;

[0047] Figure 6The results showed that CK-MSCs had no obvious toxic damage to the main organs (heart, liver, spleen, lung, and kidney) of tumor-bearing mice. H&E staining analysis showed the histological structure of the main organs (heart, liver, spleen, lung, and kidney) of mice in different treatment groups. Figure 6 The results showed that compared with the control group (NC), no obvious pathological damage was observed in the main organs of the CK-MSCs-treated group, indicating that CK-MSCs have less toxicity to normal tissues and have good biosafety. Fluorescence imaging results show the temporal dynamic distribution of CK-Cy5.5 in tumor-bearing mice ( Figure 6 (b), the accumulation of CK-Cy5.5 in the tumor site reached a peak at 4 hours and decreased significantly after 24 hours, indicating that CK-MTX has good tumor targeting and rapid clearance properties. The quantitative results of the distribution of CK-Cy5.5 in tumor-bearing mice showed ( Figure 6 In (c), the fluorescence intensity of CK-Cy5.5 at the tumor site was significantly higher than that of the Cy5.5 group, confirming its excellent tumor targeting ability. The changes in the fluorescence intensity of each organ (tumor, kidney, liver, heart, lung, spleen) over time showed that ( Figure 6 In (d), the fluorescence intensity of CK-Cy5.5 is higher in the tumor and lower in other organs, indicating that it is enriched in the tumor. The fluorescence intensity time curve of each organ shows ( Figure 6 In the figure (e), the fluorescence intensity of CK-Cy5.5 in the tumor reached the highest level 4 hours after injection, and was significantly stronger than Cy5.5 and decreased more slowly, indicating that it remained in the tumor for a longer time. The animal experiment outline showed that the drug was administered through the tail vein on the 8th, 11th, and 14th days, and then observed for two weeks until the 28th day. The weight monitoring of mice showed ( Figure 6 g), the weight of mice in the CK-MTX group changed little during the whole experiment, and no significant weight loss was observed, which further indicated that the systemic toxicity of CK-MTX was low. Survival analysis showed that ( Figure 6 h), CK-MTX significantly prolonged the survival of tumor-bearing mice, and the survival rate was significantly higher than that of other treatment groups. This shows that CK-MTX has a significant effect in delaying tumor progression and prolonging the survival of mice. Photo of mouse tumor tissue ( Figure 6 In the figure, each column represents a biological replicate, that is, each of the four groups has 5 tumor tissues displayed) and volume change curves ( Figure 6 Middle j) shows that the tumor volume of mice in the CK-MTX treatment group was significantly reduced, and the tumor inhibition effect was significantly better than that in other treatment groups.

[0048] Example 7, immunomodulatory effect of CK-MTX;

[0049] Figure 7The effects of CK-MTX on immune-related markers in the tumor microenvironment were demonstrated. The expression of multiple immune markers (CD3, CD4, CD8, HMGB1, Granzyme B, Tunel, CRT, FOXP3, GR-1, PD-L1, SDF-1, Ki67) in tumor tissues of different treatment groups showed that ( Figure 7 In a), the infiltration of CD3, CD4 and CD8 T cells in the CK-MTX-treated group increased significantly, indicating that CK-MTX promoted the infiltration of immune cells in the tumor microenvironment. In addition, the expression of ICD-related indicators such as CRT, HMGB1 and Granzyme B was also significantly upregulated, suggesting that CK-MTX induced immune cell activation and tumor cell killing. At the same time, the expression of tumor invasion indicator SDF-1 and growth indicator Ki67 in the CK-MTX-treated group was significantly downregulated, suggesting that CK-MTX weakened tumor invasiveness. Immunosuppressive indicators such as FOXP3, PD-L1 and GR-1 were also downregulated, suggesting that CK-MTX relieved immunosuppression. The quantitative results of CD3-positive T cells showed ( Figure 7 In b), the CD3 signal in the CK-MTX treatment group was significantly higher than that in other groups, further confirming the promoting effect of CK-MTX on T cell infiltration. The quantitative results of CD4 positive T cells showed ( Figure 7 In (c), the CD4 signal increased significantly in the CK-MTX group, indicating its potential in activating helper T cells. The quantitative results of CD8 positive T cells showed ( Figure 7 In the CK-MTX group, the CD8 signal increased significantly, indicating that it has a significant activation effect on cytotoxic T cells. The increased CD8 / CD4 signal intensity ratio in the CK-MTX group shows an active immune environment ( Figure 7 e). Quantitative analysis of HMGB1 further confirmed the significant increase in HMGB1 expression in the CK-MTX treatment group ( Figure 7 f), indicating that it induced the immunogenic death of tumor cells. The quantitative results of Granzyme B showed ( Figure 7 In g), the expression of Granzyme B in the CK-MTX group was significantly upregulated, supporting its role in enhancing the killing activity of immune cells. Tunel staining results showed that ( Figure 7 In the CK-MTX treatment group, apoptosis of tumor cells increased significantly, confirming its potent anti-tumor activity. CRT staining analysis showed ( Figure 7 In the CK-MTX group, the expression of CRT was significantly increased, which further supports its role in enhancing anti-tumor immune response by inducing immunogenic cell death. Quantitative analysis of FOXP3 showed that ( Figure 7In the middle j), the proportion of regulatory T cells (Tregs) in the CK-MTX treatment group was significantly reduced, indicating that it may enhance the anti-tumor immune response by inhibiting immunosuppressive cells. The quantitative results of GR-1 positive myeloid-derived suppressor cells showed that ( Figure 7 In the middle k), CK-MTX treatment significantly reduced GR-1 positive cells in the tumor, further supporting its immunomodulatory effect. PD-L1 expression analysis showed ( Figure 7 In the CK-MTX group, the expression of PD-L1 was significantly decreased, indicating that it may enhance the immune response by reducing the expression of immunosuppressive molecules. The quantitative expression results of SDF-1 showed that ( Figure 7 The expression of SDF-1 was decreased in the CK-MTX treatment group, which may help to reduce the immunosuppression in the tumor microenvironment. Ki67 staining analysis showed that ( Figure 7 In n), CK-MTX treatment significantly reduced the proliferation capacity of tumor cells, further supporting its antitumor efficacy.

[0050] Example 8, safety assessment of CK-MTX;

[0051] Figure 8 The results of HE staining of CK-MTX on the main organs of mice (heart, liver, spleen, lung, and kidney) were shown to evaluate its systemic toxicity. The H&E staining images showed the histomorphological changes of the main organs (heart, liver, spleen, lung, and kidney) in the NC, CK-MSCs, MTX, and CK-MTX groups ( Figure 8 (a) The results showed that the heart, liver, spleen, lungs, and kidneys of the NC, CK-MSCs, and CK-MTX groups had normal tissue structures, with no significant signs of tissue damage. However, the liver, spleen, and lungs of the MTX group showed slight structural abnormalities, while the CK-MTX group showed fewer pathological changes in each organ. Quantitative analysis of heart tissue showed ( Figure 8 In b), the ratio of the total area of ​​the gaps between myocardial cells in the CK-MTX group was significantly lower than that in the MTX group, indicating that CK-MTX caused less damage to the heart tissue. The quantitative results of liver tissue showed that ( Figure 8 In the middle (c), the liver sinusoid area of ​​the CK-MTX group was relatively normal, indicating that its protective effect on liver tissue was better than that of the MTX group. The number of germinal centers in spleen tissue showed ( Figure 8 In (d), the number of germinal centers in the CK-MTX group was significantly higher than that in the other groups, suggesting that it has a stronger effect on activating the immune system. Quantitative analysis of alveolar area showed that ( Figure 8In the middle (e), the alveolar area of ​​the CK-MTX group was normal, further supporting its protective effect on lung tissue. The glomerular diameter analysis of kidney tissue showed ( Figure 8 Middle (f), the glomerular diameter of the CK-MTX group was relatively normal, indicating that the damage to the kidney was relatively mild.

[0052] Summary: CK-MTX treatment effectively inhibited tumor growth while causing significantly less damage to normal tissues than the group using MTX alone. H&E staining of major organs and its quantitative analysis further supported CK-MTX as a safer and more effective tumor treatment strategy. These results indicate that CK-MTX can target tumors while maximally protecting normal tissues, demonstrating its potential in clinical applications.

[0053] Example 9, in vivo efficacy evaluation of CK-MTX in animals;

[0054] Fig. 9 The results of HE staining of mouse tumor tissue by CK-MTX were shown to evaluate its therapeutic effectiveness. The H&E staining images showed the pathological changes of tumor tissues under different treatment groups (NC, CK-MSCs, MTX, CK-MTX) ( Fig. 9 (a). The tumor cells in the NC group and the CK-MSCs group were loosely arranged, with larger nuclei and increased pathological mitotic figures. In contrast, the pathological mitotic figures in the tumor tissues of the MTX group and the CK-MTX group were significantly reduced, especially in the CK-MTX group, indicating its significant effect in inhibiting tumor cell proliferation. Quantitative analysis of the proportion of pathological mitotic figures in tumor tissues ( Fig. 9 In (b), the proportion of pathological mitotic figures in the CK-MTX group was significantly lower than that in the other groups, indicating that CK-MTX has a stronger activity in inhibiting tumor cell proliferation. Analysis of TNF-α concentration showed ( Fig. 9 In the middle (c), the TNF-α level in the serum of mice in the CK-MTX group was significantly increased, indicating its role in inducing tumor cell apoptosis and inflammatory response. Analysis of IFN-γ concentration showed that ( Fig. 9 In (d), the IFN-γ level in the serum of mice in the CK-MTX group was significantly higher than that in the other groups, further supporting its role in enhancing anti-tumor immune response. Analysis of IL-1β concentration showed ( Fig. 9 In the middle (e), the IL-1β levels in the MTX group and the CK-MTX group were significantly increased, suggesting that these two groups have a certain role in inducing inflammatory responses in the tumor microenvironment. Analysis of IL-6 concentrations showed that ( Fig. 9 f), the IL-6 level in the serum of mice in the CK-MTX group was significantly higher than that in the other groups, suggesting that it may further enhance anti-tumor activity by promoting inflammatory response. Analysis of IL-8 concentration showed that ( Fig. 9(g), the IL-8 level in the serum of mice in the CK-MTX group was significantly increased, which may be related to its role in attracting and activating immune cells to enhance anti-tumor response.

[0055] Conclusion: CK-MTX not only directly inhibits tumor growth by inhibiting tumor cell proliferation (such as decreased pathological nuclear division figures), but also further enhances its anti-tumor effect by enhancing inflammatory response and immune activation in the tumor microenvironment (such as increased TNF-α, IFN-γ, IL-6 and IL-8). These results further support the potential of CK-MTX as a potent and multi-mechanistic anti-tumor treatment.

[0056] Conclusion: The present invention uses cryotreated human umbilical cord mesenchymal stem cells (CK-MSCs) as an innovative drug delivery system for drug carriers. Through carefully designed CK-MSCs preparation and characterization, and in-depth exploration of the effects of cryotreatment strategies on hUC-MSCs, we have successfully constructed this drug carrier with targeting potential. Further studies have verified the efficiency and accuracy of CK-MSCs as a drug delivery system from multiple dimensions such as drug loading capacity, in vitro and in vivo anti-tumor effects, immunomodulatory functions, and safety assessments. By loading MTX into CK-MSCs, the present invention can achieve precise targeted delivery of MTX, providing a new solution for the treatment of prostate cancer. Therefore, the present invention can achieve precise targeted delivery of drugs, and achieve controlled release in the tumor microenvironment through a pH-sensitive release mechanism, thereby significantly improving the therapeutic effect and reducing side effects.

[0057] The cryotherapy method of the present invention is not limited to human umbilical cord mesenchymal stem cells, but can also be applied to other types of cells; in terms of drug loading, the cryotherapy cells are not limited to loading MTX, but can also be compatible with other drugs besides MTX. Therefore, the present invention not only brings a revolutionary breakthrough in tumor treatment, but also opens up a new way for the treatment of other diseases.

[0058] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A method for preparing frozen cells, characterized in that: The frozen cells are used as a drug delivery system, the drug delivery system includes CK-MTX, and the preparation method of CK-MTX is: Preparation of hUC-MSCs: First, the hUC-MSCs were digested with trypsin to separate and collect the cells. The collected cells were transferred to a 15 ml centrifuge tube and centrifuged at 500 g for 5 minutes, and the supernatant was discarded; Preparation of CK-MSCs: hUC-MSCs were resuspended in PBS, and then the cell suspension was transferred to a 2 ml cell cryotube, centrifuged again at 500 g for 5 minutes, the supernatant was discarded, and the cryotube was frozen at -80 °C for 24 hours; Preparation of CK-MTX: The frozen CK-MSCs were mixed with 500 μg / ml MTX solution and incubated at 4°C for 24 hours. After the incubation, the cells were collected and centrifuged, the supernatant was discarded, and the cells were resuspended in PBS to obtain the final drug delivery system. The CK-MSCs were stored at -80°C for a short term of up to one year and stored in liquid nitrogen for a long term.

2. A frozen cell prepared according to the method for preparing frozen cells according to claim 1.

3. Use of the frozen cells according to claim 2 in the preparation of medicines.

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