A malignant brain glioma live cell delivery system, and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a malignant brain glioma living cell delivery system and a preparation method and application thereof, which comprises a delivery system, the delivery system is a drug-loaded system formed by macrophages loading albumin-bound paclitaxel, and the albumin-bound paclitaxel is phagocytosed by macrophages and endocytosed into cells. The application aims to provide a living cell delivery system based on MΦ, which can utilize the natural targeting ability of MΦ to accurately deliver nab-PTX to target tissues, increase the accumulation of drugs in tumors and enhance the anti-tumor effect, thereby improving the treatment effect and reducing side effects. The core of the method of the application is to utilize the natural endocytosis ability and tumor targeting ability of MΦ, and the advantage of crossing physiological barriers, carry nab-PTX, and play a therapeutic role through the enrichment and drug release of nab-PTX in the tumor microenvironment. In summary, the application can be applied in the field of breaking through the blood-brain barrier and enhancing the anti-tumor effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a live cell delivery system for malignant gliomas, its preparation method, and its application. Background Technology

[0002] Traditional drug delivery systems struggle to achieve precise targeted delivery to lesions within the complex physiological environment of the body, often resulting in uneven drug distribution and significant toxic side effects. Macrophages, as innate immune cells, possess chemotactic migration, phagocytosis, and excellent tissue penetration capabilities, enabling them to migrate specifically to areas of inflammation and tumor lesions. Developing live-cell drug delivery systems based on these biological characteristics of macrophages can effectively improve drug targeting, reduce toxic side effects, and enhance therapeutic efficacy, showing broad application prospects in the field of cancer treatment.

[0003] Albumin-bound paclitaxel is a well-established nano-antitumor drug with good antitumor activity against various tumors. It can also induce macrophage polarization towards the M1 type, thereby achieving immunomodulatory effects. However, when used alone to treat central nervous system tumors, albumin-bound paclitaxel suffers from limitations due to the unique physiological structure of the brain, resulting in low drug distribution and insufficient tissue penetration. Therefore, developing a drug delivery system that can efficiently deliver albumin-bound paclitaxel to tumor tissue has significant clinical value and application prospects. Summary of the Invention

[0004] The purpose of this invention is to provide a live-cell delivery system for malignant gliomas, its preparation method, and its application. This aims to address the shortcomings of albumin-bound paclitaxel, a well-established nano-antitumor drug with good antitumor activity against various tumors. Furthermore, this drug can induce macrophage polarization towards the M1 type, thereby achieving immunomodulatory effects. However, when albumin-bound paclitaxel is used alone to treat central nervous system tumors, it suffers from low drug distribution and insufficient tissue penetration due to the unique physiological structure of the brain.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a live cell delivery system for malignant glioma and its preparation method and application, comprising a delivery system, wherein the delivery system is a drug loading system formed by loading albumin-bound paclitaxel onto macrophages, wherein the albumin-bound paclitaxel is phagocytosed and internalized into the cells by macrophages.

[0006] As a malignant glioma live cell delivery system and its preparation method and application according to the present invention, preferably, the albumin-bound paclitaxel can induce macrophages to polarize to the M1 type.

[0007] The macrophages retain their original chemotactic migration ability, can cross the blood-brain barrier and specifically accumulate in the tumor microenvironment;

[0008] After the macrophages endocytosed albumin-bound paclitaxel, their cell morphology and organelle structures remained intact, and their cell state was stable.

[0009] The delivery system can gradually release intracellular albumin-bound paclitaxel over time, and the drug release process is time-dependent. The released albumin-bound paclitaxel can enter tumor cells.

[0010] A method for preparing a live cell delivery system for malignant glioma includes the following steps: co-incubating macrophages with albumin-bound paclitaxel, achieving drug endocytosis through the phagocytic action of macrophages, and then removing the free albumin-bound paclitaxel to obtain the live cell delivery system.

[0011] The concentration of albumin-bound paclitaxel used for incubation was 5 μg / mL to 30 μg / mL, with the concentration of albumin-bound paclitaxel used for incubation being 30 μg / mL. The co-incubation time of the macrophages and albumin-bound paclitaxel was 6 hours, at which time the macrophages reached saturation with albumin-bound paclitaxel. Free albumin-bound paclitaxel was then removed by washing with PBS.

[0012] The application of a live-cell delivery system for malignant gliomas in the preparation of drugs for treating malignant gliomas, wherein the malignant glioma is glioblastoma. The live-cell delivery system is used in combination with radiotherapy, chemotherapy, and immunotherapy to prepare drugs for treating malignant gliomas.

[0013] Compared with the prior art, the beneficial effects of the present invention are:

[0014] The present invention aims to provide a live cell delivery system based on MΦ, which can utilize the natural targeting ability of MΦ to accurately deliver nab-PTX to the target tissue, increase the accumulation of the drug in the tumor and enhance its anti-tumor effect, thereby improving the therapeutic effect and reducing side effects.

[0015] The core of this invention lies in utilizing the natural endocytic and tumor-targeting capabilities of MΦ, as well as its advantage in crossing physiological barriers, to carry nab-PTX and exert a therapeutic effect through its accumulation and drug release in the tumor microenvironment.

[0016] In summary, this invention enables applications in the fields of crossing the blood-brain barrier and enhancing anti-tumor efficacy. Attached Figure Description

[0017] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0018] Figure 1 This is a schematic diagram of the fluorescence image of nab-PTX / MΦ provided in the embodiments of this application, wherein: red fluorescence comes from the cell membrane, green fluorescence comes from FITC-nab-PTX (fluorescently labeled with isothiocyanate), and blue fluorescence comes from the cell nucleus. The scale bar is 20 μm.

[0019] Figure 2 The activity of MΦ after treatment with nab-PTX is provided in the embodiments of this application; wherein: (A) is the activity of MΦ after 24h of treatment; (B) is a schematic diagram of the activity of MΦ after 48h of treatment.

[0020] Figure 3 The nab-PTX / MΦ migration test provided for the embodiments of this application;

[0021] Where: (A) Schematic diagram of the Transwell model; (B) and (C) are Transwell models of blank MΦ and nab-PTX / MΦ, respectively.

[0022] Migration assay; scale bar 100 μm; (D) Schematic diagram of quantitative analysis of the number of migrating cells.

[0023] Figure 4 Fluorescence images of nab-PTX distribution in MΦ cells at different time points provided in the embodiments of this application;

[0024] Wherein: green fluorescence comes from FITC-nab-PTX, blue fluorescence comes from the cell nucleus, and the scale bar is a schematic diagram at 40μm.

[0025] Figure 5 The embodiments of this application provide an analysis of the endocytosis of nab-PTX at different time points (MΦ).

[0026] Among them: (A) ImageJ software analysis of MΦ endocytosis of nab-PTX; (B) UPLC-MS quantitative analysis of intracellular nab-PTX.

[0027] Concentration diagram.

[0028] Figure 6 The transmission electron microscope image of MΦ provided in the embodiments of this application;

[0029] Wherein: (A) and (B) are blank MΦ, nab-PTX / MΦ, respectively, and the scale bar is 5μm.

[0030] Figure 7 This is a schematic diagram showing the distribution of FITC-nab-PTX within MΦ at different time points provided in the embodiments of this application, with a scale bar of 40 μm.

[0031] Figure 8Analysis of nab-PTX release at different times MΦ provided in the embodiments of this application;

[0032] Among them: (A) ImageJ software was used to analyze the release of nab-PTX in MΦ; (B) Ultra-high performance liquid chromatography combined with mass spectrometry was used.

[0033] A schematic diagram of quantitative analysis of the concentration of released nab-PTX using an up-concentration liquid chromatography-mass spectrometry (UPLC-MS).

[0034] Figure 9 Images of nab-PTX / MΦ co-cultured with tumor cells for 24 hours, provided in this application embodiment;

[0035] Wherein: blue fluorescence comes from the cell nucleus, green fluorescence comes from FITC-nab-PTX, and the scale bar is a schematic diagram at 50 μm.

[0036] Figure 10 The polarization state of MΦ under different processing conditions provided in the embodiments of this application;

[0037] Among them: (A) Flow cytometry detection of CD86 expression levels in MΦ under different treatment conditions; (B) Different treatment conditions,

[0038] Immunofluorescence image of CD86 expressed by MΦ, green fluorescence from CD86 antibody, blue fluorescence from cell nucleus, scale bar is 40μm; (C) Immunofluorescence image analyzed by ImageJ software; (D) Schematic diagram of the expression levels of IL-1β, IL-6 and TNF-α mRNA detected by real-time PCR under different treatment conditions. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Please see Figure 1-10 The present invention provides the following technical solution: a live cell delivery system for malignant glioma and its preparation method and application, comprising a delivery system, wherein the delivery system is a drug loading system formed by macrophages loading albumin-bound paclitaxel, wherein the albumin-bound paclitaxel is phagocytosed and internalized into the cells by macrophages;

[0041] The albumin-bound paclitaxel can induce macrophages to polarize to the M1 type;

[0042] The macrophages retain their original chemotactic migration ability, can cross the blood-brain barrier and specifically accumulate in the tumor microenvironment;

[0043] After the macrophages endocytosed albumin-bound paclitaxel, their cell morphology and organelle structures remained intact, and their cell state was stable.

[0044] The delivery system can gradually release intracellular albumin-bound paclitaxel over time, and the drug release process is time-dependent. The released albumin-bound paclitaxel can enter tumor cells.

[0045] A method for preparing a live cell delivery system for malignant glioma includes the following steps: co-incubating macrophages with albumin-bound paclitaxel, achieving drug endocytosis through the phagocytic action of macrophages, and then removing the free albumin-bound paclitaxel to obtain the live cell delivery system.

[0046] The concentration of albumin-bound paclitaxel used for incubation was 5 μg / mL to 30 μg / mL, with the concentration of albumin-bound paclitaxel used for incubation being 30 μg / mL. The co-incubation time of the macrophages and albumin-bound paclitaxel was 6 hours, at which time the macrophages reached saturation with albumin-bound paclitaxel. Free albumin-bound paclitaxel was then removed by washing with PBS.

[0047] The application of a live-cell delivery system for malignant gliomas in the preparation of drugs for treating malignant gliomas, wherein the malignant glioma is glioblastoma. The live-cell delivery system is used in combination with radiotherapy, chemotherapy, and immunotherapy to prepare drugs for treating malignant gliomas.

[0048] Example 1

[0049] Preparation and application of albumin-bound paclitaxel based on macrophage release:

[0050] 1. Preparation and evaluation of nab-PTX / MΦ:

[0051] like Figure 1 As shown, the cell membrane of macrophages (MΦ) was stained with red fluorescence using DiI. Using FITC-labeled nab-PTX, a clear green fluorescent signal emitted by FITC-nab-PTX was observed, indicating that nab-PTX was uniformly dispersed in the cytoplasm of MΦ cells. This demonstrates that nab-PTX is effectively taken up and loaded by MΦ cells.

[0052] To determine the optimal concentration of nab-PTX, the cell viability of macrophages (MΦ) was first assessed under different concentrations of nab-PTX. Cytotoxicity to MΦ cells was evaluated after treatment with different concentrations of nab-PTX for 24 and 48 hours.

[0053] like Figure 2 As shown, at lower concentrations (5-30 μg / mL), nab-PTX had no significant effect on the viability of macrophages (MΦ); however, at higher concentrations (40-100 μg / mL), the viability of MΦ cells was significantly reduced. Therefore, a concentration of 30 μg / mL of nab-PTX was selected for co-incubation with MΦ cells.

[0054] To evaluate the migration and chemotaxis capabilities of nab-PTX / MΦ, experiments were conducted using a Transwell model. Among these experiments, GBM cells...

[0055] Cells were placed in the lower chamber, while untreated blank MΦ and nab-PTX / MΦ were seeded in the upper chamber, respectively. The migration and chemotaxis abilities of nab-PTX / MΦ were evaluated by observing whether they could pass through the pores and migrate to the lower chamber.

[0056] like Figure 3 As shown, there was no significant difference in the migration and chemotaxis abilities of nab-PTX / MΦ compared to the blank MΦ. This indicates that nab-PTX at the selected concentration does not affect the migration and chemotaxis abilities of MΦ.

[0057] 2. MΦ's internalization of nab-PTX:

[0058] First, MΦ and FITC-nab-PTX were co-cultured in a cell culture incubator for different times (0, 1, 2, 4, 6, 8, 12 h). After culture, the cells were washed twice with PBS to remove free nab-PTX. Subsequently, the cells were imaged under a laser scanning confocal microscopy (LSCM) and the images were analyzed using ImageJ software.

[0059] like Figure 4 As shown, after MΦ and nab-PTX were co-incubated for different periods of time, the intensity of the green fluorescence expressed by the endocytosed FITC-nab-PTX gradually increased with the extension of time.

[0060] like Figure 5 As shown in Figure A, endocytosis of nab-PTX by macrophages (MΦ) occurred within 1 hour of co-incubation. At 4 hours, over 50% of MΦ cells contained varying levels of nab-PTX; this proportion increased to 95% at 6 hours, and remained thereafter until 12 hours. These results indicate a time-dependent endocytosis of nab-PTX by MΦ. The fluorescence intensity at 6 hours showed no statistically significant difference compared to 8 and 12 hours, suggesting that endocytosis of nab-PTX by MΦ almost reached saturation at 6 hours. Therefore, a nab-PTX / MΦ mixture was prepared by co-incubating MΦ with a concentration of 30 μg / mL for 6 hours for subsequent experiments.

[0061] To further quantify the intracellular Nab-PTX content in MΦ cells, the Nab-PTX / MΦ ratio was determined after lysis and incubation for different times (0, 2, 4, 6 h). The lysates were collected and centrifuged at 8000 rpm for 5 min. The supernatant was collected, dissolved in methanol, mixed, and centrifuged again at 8000 rpm for 10 min. Finally, the supernatant was extracted, and the Nab-PTX content was determined using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS).

[0062] like Figure 5 As shown in Figure B, quantitative analysis further confirmed that the content of nab-PTX in MΦ cells gradually increased over time, with the amount of endocytosed nab-PTX being approximately 18 μg / 10⁶ cells.

[0063] like Figure 6 Transmission electron microscopy images show that, compared with blank MΦ, the cell morphology of nab-PTX / MΦ remained unchanged, and organelles (such as mitochondria, endoplasmic reticulum, cell membrane, and nucleus) were not damaged. This indicates that the endocytosis of nab-PTX has no significant effect on the cell structure of MΦ, and MΦ can stably endocytose nab-PTX.

[0064] 3. MΦ releases nab-PTX:

[0065] The prepared nab-PTX / MΦ was cultured for different times (12, 24, 48, 72 h), with the medium changed every other day. The images were then imaged under LSCM and analyzed using ImageJ software.

[0066] like Figure 7 As shown, after endocytosis, MΦ can release nab-PTX; with the extension of incubation time, the green fluorescence signal in the nab-PTX / MΦ cell gradually weakens.

[0067] like Figure 8 As shown in Figure A, the release of nab-PTX exhibits a time-dependent effect, with the intracellular fluorescence signal showing a rapid decline over time. After 24 hours, approximately 40% of nab-PTX is released extracellularly by MΦ. After 48 hours, this proportion increases to 80%. However, after 72 hours, the release amount does not increase significantly, indicating that the release of nab-PTX by MΦ is close to its limit after 48 hours.

[0068] To quantitatively analyze the release of nab-PTX, nab-PTX / MΦ was incubated in a cell culture incubator for different times (12, 24, 48 h). After incubation, the supernatant from the culture dish was collected and centrifuged at 8000 rpm for 5 min. The supernatant was collected, dissolved in methanol, and mixed well. Subsequently, it was centrifuged at 8000 rpm for 10 min. Finally, the supernatant was collected, and the release of nab-PTX was determined by ultra-high performance liquid chromatography-tandem mass spectrometry.

[0069] Figure 8 As shown in B, after 48 hours, MΦ can release approximately 6 μg / mL of nab-PTX.

[0070] The FITC-nab-PTX / MΦ and tumor cells were co-cultured and analyzed. After co-culturing the prepared FITC-nab-PTX / MΦ and tumor cells for 24 h, the images were obtained under LSCM.

[0071] like Figure 9 As shown, a significant green fluorescence signal was observed in both MΦ and GBM cells, indicating that nab-PTX was successfully released from MΦ and entered GBM cells. This result demonstrates that nab-PTX / MΦ can effectively release nab-PTX and deliver it into tumor cells, providing a basis for further research on its therapeutic effects.

[0072] 4. Evaluate the polarization state of nab-PTX / MΦ:

[0073] LPS can induce MΦ to polarize towards the M1 type. Using MΦ with added LPS as a positive control group, the polarization status of the three groups of MΦ (blank MΦ, nab-PTX / MΦ, LPS / MΦ) was assessed and compared.

[0074] 1) Flow cytometry detection of the M1 biomarker CD86:

[0075] Three cell lines—blank MΦ, nab-PTX / MΦ, and LPS / MΦ—were fixed with flow cytometry fixative. After washing twice with PBS, the PE-labeled flow cytometry antibody CD86 was added, and staining was performed at room temperature in the dark for 30 min. Finally, the cells were washed twice with PBS, resuspended, and analyzed by flow cytometry to assess CD86 expression in the three MΦ groups. Figure 10 As shown in Figure A, the percentage of CD86-positive cells was 8.8% in the blank MΦ group, while it was 70% and 85.7% in the nab-PTX / MΦ group and the LPS / MΦ group, respectively.

[0076] 2) Immunofluorescence staining analysis of the M1 biomarker CD86:

[0077] Three groups of MΦ cells (blank MΦ, nab-PTX / MΦ, LPS / MΦ) cultured under different conditions were prepared. Cells were fixed with 4% PFA for 15 min, washed twice with PBS, and blocked with 5% BSA for 1 h. Subsequently, after washing twice with PBS, CD86 primary antibody was added, and the cells were stained overnight at 4°C in the dark. The next day, cells were washed three times with TBST, and Alexa Flour 488-labeled secondary antibody was added, followed by staining at room temperature in the dark for 1 h. Cells were washed three times again with TBST, and the nuclei were stained with DAPI. Images were then imaged under LSCM and analyzed using ImageJ software.

[0078] like Figure 10 As shown in B and 10C, the expression level of CD86 in the nab-PTX / MΦ group and the LPS / MΦ group was significantly higher than that in the blank MΦ group.

[0079] 3) Detection of M1-related gene expression by quantitative real-time PCR:

[0080] RNA was extracted from three groups of MΦ (blank MΦ, nab-PTX / MΦ, and LPS / MΦ) to ensure the quality and purity of the extracted RNA. The RNA was reverse transcribed into cDNA according to standard procedures. Then, quantitative real-time PCR analysis was performed. The expression levels of relevant genes were quantitatively analyzed using the ΔΔCT method.

[0081] like Figure 10 As shown in Figure D, higher levels of IL-1β, IL-6, and TNF-α mRNA expression were detected in the nab-PTX / MΦ and LPS / MΦ groups; and the expression levels of these specific mRNAs in the nab-PTX / MΦ group were higher than those in the blank MΦ group, but lower than those in the LPS / MΦ group.

[0082] The results of the above three methods confirm that nab-PTX can induce MΦ to polarize towards the M1 type, making nab-PTX / MΦ effector cells with M1 type characteristics, indicating the potential role of nab-PTX / MΦ in anti-tumor immune regulation.

[0083] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A live cell delivery system for malignant gliomas, comprising a delivery system, characterized in that, The delivery system is a drug delivery system formed by loading albumin-bound paclitaxel onto macrophages, wherein the albumin-bound paclitaxel is phagocytosed and internalized into the cells by the macrophages.

2. The live cell delivery system for malignant glioma according to claim 1, characterized in that: The albumin-bound paclitaxel can induce macrophages to polarize to the M1 type.

3. The live cell delivery system for malignant glioma according to claim 1, characterized in that: The macrophages retain their original chemotactic migration ability, enabling them to cross the blood-brain barrier and specifically accumulate in the tumor microenvironment.

4. The live cell delivery system for malignant glioma according to claim 1, characterized in that: After the macrophages endocytosed albumin-bound paclitaxel, their cell morphology and organelle structures remained intact, and their cell state was stable. The delivery system can gradually release intracellular albumin-bound paclitaxel over time, and the drug release process is time-dependent. The released albumin-bound paclitaxel can enter tumor cells.

5. A method for preparing a live cell delivery system for malignant gliomas, as described in any one of claims 1 to 4, characterized in that, Includes the following steps: Macrophages were co-incubated with albumin-bound paclitaxel, and the drug was internalized by phagocytosis of macrophages. The free albumin-bound paclitaxel was then removed to obtain the live cell delivery system.

6. The method for preparing a live cell delivery system for malignant glioma according to claim 5, characterized in that: The concentration of albumin-bound paclitaxel used in the incubation is 5 μg / mL to 30 μg / mL, and the concentration of albumin-bound paclitaxel used in the incubation is 30 μg / mL.

7. The method for preparing a live cell delivery system for malignant glioma according to claim 6, characterized in that: The macrophages were incubated with albumin-bound paclitaxel for 6 hours, at which time the macrophages reached saturation in endocytosis of albumin-bound paclitaxel.

8. The method for preparing a live cell delivery system for malignant glioma according to claim 5, characterized in that: Free albumin-bound paclitaxel was removed by PBS rinsing.

9. The use of a live cell delivery system for malignant glioma in the preparation of drugs for treating malignant glioma, as described in any one of claims 1 to 4, characterized in that: The malignant glioma is glioblastoma.

10. The application of the live cell delivery system for malignant glioma according to claim 9 in the preparation of drugs for treating malignant glioma, characterized in that: The live cell delivery system is used in combination with radiotherapy, chemotherapy, and immunotherapy to prepare drugs for the treatment of malignant gliomas.

Images