A method for improving the solid tumor enrichment ability of red blood cell carriers

Abstract

The present invention discloses a method for improving the solid tumor enrichment ability of red blood cell carriers, specifically: a small molecule compound RRx-001 is loaded into the red blood cell carrier, RRx-001 causes the PS molecules on the surface of the red blood cells to be everted, which can mediate the targeted adhesion of red blood cells to tumor vascular endothelial cells expressing PSR protein, thereby achieving active targeting of tumors. In addition, the present invention transforms red blood cells by RRx-001, thereby enhancing the ability of red blood cells to produce NO, dilates tumor blood vessels by NO, and increases blood flow in tumor blood vessels. The present invention transforms red blood cells by RRx-001 so that they have the performance of both tumor targeting and generating NO to dilate tumor blood vessels, thereby achieving effective enrichment of chemotherapy drugs at the tumor site, and is applicable to all solid tumors, so the present invention is of great significance for drug delivery to tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of targeted drug delivery, and specifically relates to a method for transforming red blood cells by using the small molecule compound RRx-001, thereby improving the solid tumor targeting ability of red blood cell carriers and thereby increasing their enrichment effect at the tumor site. Background Art

[0002] Malignant tumors have long posed a serious threat to human health. Currently, chemotherapy is the most common treatment for malignant tumors. However, the inherent resistance and poor targeting of chemotherapy drugs often lead to significant toxic side effects. Therefore, the development of drug delivery systems with excellent targeting and biocompatibility is crucial.

[0003] Barriers to drug delivery to solid tumors are one of the important reasons why solid tumors are difficult to cure. A major reason for the drug delivery to solid tumors is the microenvironment of solid tumors, among which the abnormal vascular system is an important component of the tumor microenvironment. The vascular system of tumors is usually tortuous and dysfunctional, which brings great difficulties to drug delivery to solid tumors.

[0004] Cell carriers, primarily red blood cells (erythrocytes), white blood cells (leukocytes), and stem cells, are widely used due to their excellent lung tumor targeting, low immunogenicity, and biocompatibility. Erythrocytes (erythrocytes) have become a research hotspot due to their simple structure, widespread availability, and excellent biocompatibility. However, as drug carriers, erythrocytes also have certain drawbacks. For example, they lack tumor targeting capabilities and can generally only be used to target lung tumors through their natural lung homing. Summary of the Invention

[0005] In response to the problem that red blood cell carriers have no tumor targeting ability, the present invention aims to develop a strategy to improve the tumor targeting ability of red blood cells, achieve effective enrichment of chemotherapy drugs at the tumor site, and thus reduce the toxic side effects of chemotherapy.

[0006] The technical solutions of the present invention are as follows:

[0007] In a first aspect, the present invention provides the use of RRx-001 to enhance the ability of erythrocyte carriers to accumulate solid tumors. By loading RRx-001 into erythrocyte carriers, erythrocytes loaded with both the drug and RRx-001 accumulate in large quantities at the site of the solid tumor, achieving precise delivery of chemotherapy drugs such as doxorubicin. In one embodiment of the present invention, the solid tumor is a breast tumor. Erythrocytes loaded with both the chemotherapy drug and RRx-001 accumulate in large quantities at the tumor site in a time-dependent manner, increasing over time. In contrast, erythrocytes loaded only with the chemotherapy drug accumulate only in small quantities at the tumor site.

[0008] The inventors conducted in-depth research on how RRx-001 improves the targeting ability of red blood cell carriers and revealed its mechanism. Specifically, after entering red blood cells, RRx-001 will specifically bind to hemoglobin, subsequently causing a series of biochemical reactions between hemoglobin and red blood cells, stimulating the externalization of phosphatidylserine (PS) on the red blood cell membrane to the cell membrane surface (under normal circumstances, the PS on the red blood cell membrane is located in the inner leaflet). The hypoxic and inflammatory microenvironment of the tumor will upregulate the expression of PS receptors (PSR) on vascular endothelial cells. Therefore, through the specific targeting effect of PS on the red blood cell surface and PSR of tumor vascular endothelial cells, the red blood cell carriers can adhere to the tumor site, thereby improving their enrichment ability at the tumor site.

[0009] Furthermore, loading RRx-001 into erythrocyte carriers not only imbues the erythrocytes with the targeting group PS, enhancing their tumor-targeting capabilities, but also generates NO, which dilates tumor blood vessels. This dilation of tumor vessels increases blood flow. The synergistic effect of tumor targeting and NO-generated vasodilation further enhances the enrichment of the modified erythrocyte carriers at the tumor site. The mechanism is as follows: when RRx-001-loaded erythrocytes reach the hypoxic tumor site, oxyhemoglobin releases oxygen to become deoxyhemoglobin, which then acquires nitrite reductase activity, mediating NO production. RRx-001 enhances the nitrite reductase activity of deoxyhemoglobin, further increasing NO production through its facilitation.

[0010] The second aspect of the present invention provides a method for improving the solid tumor enrichment ability of red blood cell carriers, specifically: loading RRx-001 into red blood cell carriers, and loading the model chemotherapy drug DOX for relevant verification.

[0011] Preferably, the above method includes the following steps:

[0012] S1, RRx-001 was loaded into red blood cells using hypotonic dialysis;

[0013] S2. Store the red blood cells loaded with RRx-001 in a preservation solution.

[0014] More preferably, in the above method, step S1 includes:

[0015] S11. Mix the red blood cells with the drug and RRx-001, place them in a dialysis bag, and dialyze them against a hypotonic buffer solution containing glucose (19.68-20.18 mM), Na2HPO4·12H2O (9.77-10.05 mM), NaH2PO4·2H2O (9.61-10.26 mM), MgCl2·6H2O (3.94-4.18 mM), ATP·Na2 (2.17-2.37 mM), and glutathione (2.93-3.25 mM).

[0016] S12. Transfer the dialysis bag to a hypertonic buffer containing 10% PIGPA-NaCl, ATP·Na2 (2.17-2.37 mM), and glutathione (2.93-3.25 mM). PIGPA-NaCl contains sodium pyruvate (90.88-109.05 mM), inosine (96.94-100.67 mM), glucose (95.88-100.92 mM), NaH2PO4·2H2O (32.05-38.46 mM), adenine (4.44-5.18 mM), and NaCl (12-13% w / v). Dilute PIGPA-NaCl tenfold as the stock solution to obtain 10% PIGPA-NaCl as the working solution.

[0017] S13, centrifugation and washing to obtain red blood cells loaded with RRx-001 and drugs.

[0018] More preferably, in the above method, the preservation solution includes NaCl (8.0-8.5% w / v), inosine (9.69-10.07 mM), adenine (0.44-0.52 mM), sodium pyruvate (10.00-10.91 mM), ATP·Na2 (2.17-2.37 mM), glutathione (2.93-3.25 mM), glucose (29.77-20.28 mM), and NaH2PO4·2H2O (3.20-3.85 mM). The homemade preservation solution of the present invention can effectively delay morphological changes and death of red blood cells during storage.

[0019] The beneficial technical effects of the present invention are:

[0020] 1. The present invention uses the small molecule compound RRx-001 to transform the red blood cell carrier, effectively solving the problem of red blood cells' lack of tumor targeting and providing a new tool for solid tumor drug delivery.

[0021] 2. The erythrocyte vector obtained by the modification strategy of the present invention achieves efficient tumor targeting through the specificity between PS on the surface of erythrocytes and PSR upregulated by tumor vascular endothelial cells, so it has broad-spectrum tumor targeting.

[0022] 3. The red blood cell carrier obtained by the modification strategy of the present invention not only has tumor targeting performance, but also has the performance of promoting the generation of NO to dilate tumor blood vessels. Through the synergistic effect of tumor targeting and the generation of NO to dilate tumor blood vessels, while giving the red blood cell carrier tumor targeting, it also improves the unfavorable factors for drug delivery and targeting in the tumor microenvironment, dilates tumor blood vessels, increases blood flow in tumor blood vessels, further increases the enrichment effect of drugs at the tumor site, and realizes the precise delivery of tumor chemotherapy drugs.

[0023] 4. The present invention provides a preservation solution suitable for preserving red blood cells loaded with RRx-001 and chemotherapy drugs, which can effectively delay the morphological changes and death of red blood cells during the preservation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Optical microscopy images of erythrocytes loaded with doxorubicin and RRx-001 by hypotonic dialysis;

[0025] Figure 2 Scanning electron micrographs of erythrocytes loaded with doxorubicin and RRx-001 by hypotonic dialysis;

[0026] Figure 3 The morphological changes of red blood cells loaded with doxorubicin and RRx-001 after being stored in the preservation solution for different time periods;

[0027] Figure 4 This figure shows the experimental results of the accumulation of red blood cells loaded with doxorubicin and RRx-001 at the 4T1 tumor site in tumor-bearing mice over time;

[0028] Figure 5 The figure shows the experimental results of PS protein expression on the surface of red blood cells loaded with doxorubicin and RRx-001;

[0029] Figure 6 The graph shows the results of the adhesion experiment of red blood cells loaded with doxorubicin and RRx-001 to simulated tumor vascular endothelial cells;

[0030] Figure 7 The graph shows the experimental results of NO in red blood cells loaded with doxorubicin and RRx-001 under different conditions;

[0031] In the figure, RBC (Red Blood Cell): red blood cells, RD (RBC @ DOX): red blood cells loaded with DOX, RR (RBC @ RRx-001): red blood cells loaded with RRx-001, RDR (RBC @ DOX&RRx-001): red blood cells loaded with DOX and RRx-001 at the same time. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be described clearly and completely below in conjunction with the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. The term "comprise" and any variations thereof in the description and claims of the present invention are intended to cover non-exclusive inclusions.

[0034] If no specific techniques or conditions are specified in the following examples, the procedures were carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product instructions; if no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0035] Example 1

[0036] Taking the chemotherapy drug doxorubicin (DOX) as an example, this example uses hypotonic dialysis to prepare red blood cells loaded with doxorubicin or RRx-001 simultaneously or individually. The specific steps are as follows:

[0037] (1) Collection and processing of red blood cells.

[0038] Fresh whole blood from BALB / c mice was collected by eyeball sampling into anticoagulant blood collection tubes. The blood was centrifuged at 400g for 10 minutes at 4°C. The upper plasma layer and the middle buffy coat were discarded. The upper, pale yellow liquid is plasma, the middle, opaque white layer is the buffy coat (containing white blood cells and platelets), and the lower, dark red, opaque layer is red blood cells. The red blood cells were suspended in 10 volumes of pre-chilled 1× PBS (pH 7.4) and centrifuged at 400g for 10 minutes at 4°C. The supernatant was discarded and the red blood cells were washed three times. The red blood cells were resuspended in 1× PBS (pH 7.4) to obtain a 70% hematocrit red blood cell suspension and stored at 4°C.

[0039] (2) The chemotherapy drugs doxorubicin and / or RRx-001 are loaded into red blood cells using a low-osmotic dialysis drug loading method.

[0040] After mixing the red blood cell suspension with DOX and RRx-001, place the mixture in a dialysis bag (MWCO = 3500). Dialyze the mixture against hypotonic buffer at 4°C for 30 min. Transfer the dialysis bag containing DOX, RRx-001, and red blood cells to hypertonic buffer and dialyze for 30 min at 37°C. Centrifuge at 400 g for 10 min at 4°C and wash three times with PBS to remove unencapsulated DOX and RRx-001. The DOX- and RRx-001-loaded red blood cells were then stored at 4°C. The hypotonic buffer used in this example consisted of 20 mM glucose, 10 mM Na₂HPO₄·12H₂O, 10 mM NaH₂PO₄·2H₂O, 4 mM MgCl₂·6H₂O, 2 mM ATP·Na₂, and 3 mM glutathione. The hypertonic buffer used in this example consisted of 10% PIGPA-NaCl (100mM sodium pyruvate, 100mM inosine, 100mM glucose, 35mM NaH2PO4·2H2O, 5mM adenine, and 12-13% w / v NaCl), 2mM ATP·Na2, and 3mM glutathione. The drug-loaded groups were prepared using the same method as described above, with only the desired drug added and the other drug replaced with a corresponding volume of saline. DOX-loaded and RRx-001-loaded erythrocytes were prepared. The DOX loading in each erythrocyte prepared in this example was 283.5±29.5μg per 3×10 9 The loading amount of RRx-001 was 8.7±2.1 μg per 3×10 9 Red blood cells.

[0041] The morphology of red blood cells and red blood cells loaded with DOX and RRx-00 were characterized by optical microscopy and scanning electron microscopy. Figure 1 and Figure 2 The results show that the extracted red blood cells in this example are in good condition, showing a biconcave disc shape. Moreover, most of the red blood cells loaded with the chemotherapy drugs doxorubicin and RRx-001 using the low-osmotic dialysis drug loading method still retain the biconcave shape.

[0042] In steps (1) and (2), the preservation solution used for the storage of erythrocytes, DOX-loaded erythrocytes, and RRx-001-loaded erythrocytes was 8.0% w / v NaCl, 10 mM inosine, 0.5 mM adenine, 10 mM sodium pyruvate, 2.2 mM ATP·Na2, 3 mM glutathione, 25 mM glucose, and 3.5 mM NaH2PO4·2H2O. The cell morphology at different storage times was analyzed using an optical microscope. The results are shown in Figure 2. Figure 3As shown, after one day of storage, the morphology of red blood cells began to gradually change, but some still maintained a well-defined biconcave, round shape. On the second day of storage, only a very small number of red blood cells died, and the black background fragments represent these dead red blood cell fragments. The vast majority of red blood cells remained alive, and some still maintained a well-defined biconcave, round shape. Compared to storage in PBS, storage in a homemade red blood cell preservation solution effectively slowed the deformation and death of red blood cells loaded with the chemotherapy drug doxorubicin, allowing more red blood cells to survive longer and maintain their biconcave, round shape.

[0043] Example 2

[0044] Using DOX-only loaded erythrocytes as a control, this example tested the ability of erythrocytes loaded with DOX and RRx-001 to target solid tumors through in vivo experiments, including the following steps:

[0045] (1) BALB / c mice were subcutaneously inoculated with 1×10 6 4T1 cells were used to construct a 4T1 mouse tumor model;

[0046] (2) After the drug-loaded erythrocyte carriers were prepared, erythrocytes loaded with DOX only (RD-DiR) and erythrocytes loaded with both DOX and RRx-001 (RDR-DiR) were labeled with DiR, and RD-DiR or RDR-DiR was injected into the tail vein of mice bearing subcutaneous tumors of mouse breast cancer cells 4T1. At different time points, the distribution of erythrocytes in the mice was observed using a small animal imaging device, and the aggregation of erythrocytes at the tumor site was analyzed.

[0047] The results are as follows Figure 4 As shown, RBCs loaded with both DOX and RRx-001 accumulated in large numbers at the tumor site in a time-dependent manner, increasing over time. In contrast, RBCs without RRx-001 (i.e., loaded only with DOX) accumulated only a small amount at the tumor site. This suggests that loading RRx-001 enables RBCs to target tumors, successfully delivering the chemotherapy drug doxorubicin precisely to the tumor site.

[0048] Example 3

[0049] This case investigated the mechanism by which RRx-001 modifies red blood cells and improves their tumor-targeting ability, specifically including the following two aspects:

[0050] (1) Characterization of PS protein on the surface of erythrocytes loaded with DOX and RRx-001.

[0051] The detection principle is as follows: Annexin V is a member of the intracellular protein annexin family and can selectively bind to phosphatidylserine in a calcium ion-dependent manner. PS externalization can be detected by flow cytometry using Annexin V labeled with FITC (Annexin V-FITC).

[0052] The detection method is as follows: the red blood cells and erythrocytes loaded with DOX and RRx-001 are diluted to 2×10 7 After that, the cells were incubated with Annexin V-FITC at room temperature in the dark for 15 min, washed twice with PBS, and the FITC fluorescence intensity of different groups was detected by flow cytometer. The maximum absorption wavelength of FITC was 490 nm and the excitation wavelength was 525 nm.

[0053] The results are as follows Figure 5 As shown: Compared with red blood cells, the amount of PS on the surface of red blood cells loaded with DOX and RRx-001 increased significantly, indicating that the loading of RRx-001 significantly increased the expression of PS on the surface of RDR.

[0054] (2) Characterization of the adhesion ability of DOX- and RRx-001-loaded red blood cells to tumor vascular endothelial cells.

[0055] Normal mouse vascular endothelial cells were activated using conditioned medium from mouse breast cancer 4T1 cells to simulate tumor endothelial cells. The medium was removed, and red blood cells loaded with DOX and RRx-001 were added to the activated endothelial cells. After incubation for a specified period, the cells were washed three times with PBS. The adhesion of the red blood cells loaded with DOX and RRx-001 to the activated mouse vascular endothelial cells was observed under bright field laser confocal microscopy.

[0056] The results are as follows Figure 6 As shown in the results, DOX- and RRx-001-loaded erythrocytes adhered extensively to activated endothelial cells, while erythrocytes showed little adhesion to activated endothelial cells. Furthermore, DOX- and RRx-001-loaded erythrocytes showed only a small amount of adhesion to unactivated endothelial cells. These results indicate that DOX- and RRx-001-loaded erythrocytes have the ability to target tumor endothelial cells.

[0057] Example 4

[0058] In this study, the NO production capacity of erythrocytes loaded with DOX and RRx-001 was tested under different conditions.

[0059] In this example, DAF-FM DA was used for detection. The detection principle is as follows: DAF-FM DA can pass through the cell membrane. After entering the cell, it can be catalyzed by intracellular esterase to form DAF-FM, which cannot pass through the cell membrane. DAF-FM itself has only very weak fluorescence, but after binding with NO, it can produce strong fluorescence with an excitation wavelength of 495 nm and an emission wavelength of 515 nm.

[0060] The assay involved treating RBCs loaded with DOX and RRx-001, RRx-001, and RBCs with different conditions. These conditions involved creating a hypoxic environment using anaerobic bags and sealed boxes for 0.5 hours, with the degree of hypoxia indicated by a hypoxia indicator. Furthermore, the effect of adding nitrite was further investigated by manipulating the substrate. NO production was determined under normoxic conditions with or without substrate, and under hypoxic conditions with or without substrate. NO production was then measured using a NO detection kit. Specifically, RBCs were resuspended in DAF-FM DA and incubated at 37°C for 20 minutes. The cells were then washed three times with PBS to remove any unincorporated DAM-FM DA, and analyzed by flow cytometry.

[0061] Test results are shown in Figure 7 :like Figure 7 As shown in a, the NO production under normoxic and hypoxic conditions was compared: compared with the NO produced under normoxic conditions, the NO of erythrocytes loaded with DOX and RRx-001 increased significantly after 0.5 h of hypoxia, which was also the case in erythrocytes and erythrocytes loaded with RRx-001, indicating that hypoxia can increase the NO production of erythrocytes; Figure 7 As shown in Figure b, the NO production of different drug-loaded erythrocyte materials under the same conditions was compared: compared with erythrocytes, the NO production of erythrocytes loaded with DOX and RRx-001 was significantly increased, which was also the case with erythrocytes loaded with RRx-001, indicating that the loading of RRx-001 can enhance the ability of RBC to produce NO; Figure 7 As shown in Figure c, under the same conditions, whether adding substrates further promotes NO production: after adding nitrite, the NO production of erythrocytes, erythrocytes loaded with RRx-001, and erythrocytes loaded with DOX and RRx-001 increased significantly, indicating that NO production can be further increased by adding substrates.

[0062] In summary, the present invention significantly improves the ability of red blood cells to target solid tumors through RRx-001, explains and verifies its mechanism of action, and lays a theoretical and experimental foundation for the application of red blood cell carriers in targeting tumors; at the same time, RRx-001 can also improve the ability of red blood cells to produce NO, and further increase the production of NO under conditions of hypoxia and the addition of the substrate nitrite, which has a positive effect on dilating tumor blood vessels; the present invention increases the enrichment effect of red blood cell carriers at the tumor site through the synergistic effect of tumor targeting and the generation of NO to dilate tumor blood vessels, thereby achieving precise delivery of chemotherapy drugs.

[0063] It should be noted that the above embodiments are only part of the embodiments of the present invention rather than all the embodiments, and are only used to illustrate the technical solutions of the present invention rather than to limit them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. Use of red blood cells loaded with RRx-001 and doxorubicin in the preparation of a drug for treating solid tumors, characterized in that: The RRx-001 improves the enrichment ability of red blood cells in solid tumors, and the solid tumors are breast tumors. The method for preparing red blood cells loaded with RRx-001 and doxorubicin comprises the following steps: S1, RRx-001 and doxorubicin were loaded into erythrocytes using hypotonic dialysis; S2. The red blood cells loaded with RRx-001 and doxorubicin are preserved in a preservation solution; the preservation solution includes 8.0-8.5% w / v NaCl, 9.69-10.07 mM inosine, 0.44-0.52 mM adenine, 10.00-10.91 mM sodium pyruvate, 2.17-2.37 mM ATP·Na2, 2.93-3.25 mM glutathione, 29.77-20.28 mM Glucose and 3.20-3.85 mM NaH2PO4·2H2O.

2. The use according to claim 1, characterized in that Step S1 includes: S11, mixing red blood cells with doxorubicin and RRx-001, placing in a dialysis bag and dialyzing in a hypotonic buffer; S12, transfer the dialysis bag to a hypertonic buffer solution for dialysis; S13. Centrifuge and wash to obtain red blood cells loaded with RRx-001 and doxorubicin.

3. The use according to claim 2, characterized in that The hypotonic buffer solution includes 19.68-20.18 mM Glucose, 9.77-10.05 mM Na2HPO4·12H2O, 9.61-10.26 mM NaH2PO4·2H2O, 3.94-4.18 mM MgCl2·6H2O, 2.17-2.37 mM ATP·Na2 and 2.93-3.25 mM glutathione.

4. The use according to claim 2, characterized in that The hypertonic buffer comprises 10% PIGPA-NaCl, 2.17-2.37 mM ATP·Na2 and 2.93-3.25 mM glutathione, wherein PIGPA-NaCl comprises 90.88-109.05 mM sodium pyruvate, 96.94-100.67 mM inosine, 95.88-100.92 mM Glucose, 32.05-38.46 mM NaH2PO4·2H2O, 4.44-5.18 mM adenine and 12-13% w / v NaCl.

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