A deformed red blood cell preparation targeting liver and its preparation method and application

Abstract

The application discloses a deformable red blood cell preparation targeting liver and a preparation method and application thereof. The deformable red blood cell preparation comprises red blood cells and a deformable agent combined on the red blood cell membrane. The deformable agent comprises a CpG oligonucleotide. The deformable red blood cell preparation of the application highly targets liver by using the natural immune homing property of deformable red blood cells, and can reduce the toxic side effects of drugs and increase the safety of drug administration. Meanwhile, the deformable red blood cell preparation of the application has a simple preparation process, and raw materials are economical and easy to obtain, and the preparation process does not need to depend on complex equipment and is easy to industrialize.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine and materials, in particular to a deformed red blood cell preparation targeting liver and a preparation method and application thereof. BACKGROUND

[0002] In the treatment of tumors, many drugs and treatment methods can have side effects on the liver, which is a key medical challenge. The liver is the main organ for drug metabolism and excretion, and is therefore susceptible to chemotherapy, radiotherapy and other targeted therapies. These treatments can cause abnormal liver function, hepatitis, liver fibrosis or other more serious complications. In particular, in combination therapy using multiple drugs, the risk of liver toxicity can increase. In addition, certain tumor treatments can interact with the patient's existing liver disease or other complications, increasing the risk of liver damage.

[0003] In the study of drug delivery, targeting rate is a key indicator, and low targeting rate limits the effective accumulation of drugs at the lesion site. Currently, drug delivery is mainly focused on the field of nanotechnology, and a study in 2016 showed that only about 0.7% of the dose of nanoparticles can be delivered to solid tumors. At present, by controlling the size, modifying the protein, etc., the targeting efficiency of some nanomaterials has been improved, but most of the nanomedicine systems are still limited by biodistribution, and off-target effects of drugs can also cause high liver toxicity.

[0004] Based on the defects of nanodelivery, some researchers have begun to focus on using autologous cells to transport therapeutic substances. Cell materials are generally micron-sized and do not penetrate cells to cause side effects, and their natural biological origin ensures high compatibility in the body, reducing immune reactions and toxicity. These two characteristics make red blood cells an ideal drug delivery carrier, which can improve the bioavailability of drugs and reduce potential side effects.

[0005] Therefore, it is urgent to obtain a stable red blood cell preparation that can target the liver. SUMMARY

[0006] The primary purpose of the present application is to overcome the shortcomings and deficiencies of the prior art and provide a deformed red blood cell preparation and a preparation method thereof.

[0007] Another purpose of the present application is to provide the application of the deformed red blood cell preparation described above.

[0008] The purpose of the present application is achieved by the following technical solutions:

[0009] A deformed red blood cell preparation comprises red blood cells and a deforming agent bound to the red blood cell membrane.

[0010] Further, the deformant comprises CpG oligonucleotide. Red blood cell membrane expresses Toll-like receptor 9 (TLR9); TLR9 can recognize specific unmethylated CpG motifs universally existing in microbial genome, and the CpG oligonucleotide can be combined with red blood cell by specific recognition to make the red blood cell deform.

[0011] Further, the CpG oligonucleotide comprises oligodeoxynucleotide containing unmethylated CpG motifs.

[0012] The preparation method of the deformant red blood cell preparation comprises the following steps:

[0013] Incubating the red blood cell with the deformant to obtain the deformant red blood cell preparation.

[0014] Further, the ratio of the red blood cell to the deformant is 10 5 ~ 10 8 cells: 10 pmol ~ 1 μmol; further 10 6 ~ 10 8 : 10 pmol ~ 40 nmol.

[0015] Further, the deformant comprises CpG oligonucleotide.

[0016] Further, the incubation condition is 37℃ shaker incubation for 2 ~ 4 hours.

[0017] The application of the deformant red blood cell preparation in preparing a platinum-containing drug clearing agent.

[0018] Further, the platinum-containing drug comprises at least one of cisplatin, carboplatin and oxaliplatin.

[0019] The application of the deformant red blood cell preparation in preparing a pharmaceutical composition for treating tumor; the pharmaceutical composition comprises the deformant red blood cell preparation and the platinum-containing drug.

[0020] Further, the tumor is liver tumor.

[0021] Further, the platinum-containing drug comprises at least one of cisplatin, carboplatin and oxaliplatin.

[0022] The present application has the following advantages and effects relative to the prior art:

[0023] Compared with existing technologies, the liver-targeting deformable erythrocyte formulation of the present invention carries a deformable agent. On the one hand, the deformable agent binds to erythrocytes through receptors and does not enter tissue cells, reducing side effects. On the other hand, it can also clear small molecule chemotherapy drugs that bind to DNA, reducing drug toxicity. In addition, the deformable erythrocyte formulation can accumulate in the liver, achieving targeted therapy to the liver. Furthermore, the preparation process of the liver-targeting deformable erythrocyte formulation of the present invention is simple, the raw materials are economical and readily available, it does not rely on complex equipment, and it is easy to industrialize. Attached Figure Description

[0024] Figure 1 This is a schematic diagram illustrating the principle of a modified red blood cell preparation.

[0025] Figure 2 This is a scanning electron microscope image of the deformed erythrocyte preparation of Example 1 of the present invention;

[0026] Figure 3 This is a graph showing the detection results of CD47 and TLR9 in red blood cells treated with CpG oligonucleotides;

[0027] Figure 4 This is a fluorescent staining image of band 3 protein in erythrocytes treated with CpG oligonucleotides.

[0028] Figure 5 This is a calcein-AM fluorescence staining image of erythrocytes treated with CpG oligonucleotides;

[0029] Figure 6 This is a flow cytometry plot of CD47 in erythrocytes treated with CpG oligonucleotides;

[0030] Figure 7 This is a gel electrophoresis image of the detection of cisplatin and CpG oligonucleotide binding in Example 2;

[0031] Figure 8 This is a graph showing the results of the L-02 cytotoxicity experiment in Example 2, which tested the effect of the deformed erythrocyte preparation on reducing cisplatin toxicity in vitro.

[0032] Figure 9 This is a graph showing the results of detecting intracellular Pt levels using inductively coupled plasma mass spectrometry in Example 2.

[0033] Figure 10 This is an ex vivo fluorescence imaging of normal organs and tumors after intravenous injection of Cy5.5-grafted deformed erythrocyte preparation in Example 3.

[0034] Figure 11 This is a quantitative result graph of the intravenous injection of a nano-gold modified deformed red blood cell preparation in Example 3.

[0035] Figure 12These are tumor photographs and relative tumor weight results of mice treated with the deformed red blood cell preparation in Example 3.

[0036] Figure 13 This is a graph showing the ALT and AST levels in the serum of mice after treatment with the deformed erythrocyte preparation in Example 3. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0038] Example 1: Preparation of Deformed Red Blood Cell Preparation

[0039] The denatured erythrocyte preparation in this embodiment involves binding CpG oligonucleotides to the erythrocyte membrane, as shown in the schematic diagram. Figure 1 As shown. The CpG oligonucleotides used in this embodiment are ODN type A or B, and undergo full-chain sulfurization treatment.

[0040] (1) Centrifuge 3000g of blood from healthy adults for 10 minutes. Remove the plasma and brownish-yellow coating, and wash three times with 1×PBS buffer (pH 7.3) to obtain red blood cells.

[0041] (2) Separate red blood cells (RBCs) and CpG oligonucleotides (CpG oligonucleotide to red blood cell ratio: 10-100 pmol / 10). 6 The cells were incubated at 37°C for 2–4 hours while being gently shaken to obtain a red blood cell-CpG oligonucleotide-conjugated modified red blood cell preparation.

[0042] The scanning electron microscope image of the drug-loaded erythrocytes prepared in Example 1 is shown below. Figure 2 As shown, they are meteor hammer shaped, with uniform red blood cell size and a particle size of about 2 to 4 μm.

[0043] After we incubated red blood cells with CpG oligonucleotides, as follows Figure 3 As shown, CpG oligonucleotides can bind to erythrocytes, leading to changes in erythrocyte structure and the redistribution of CD47 and TLR9. TLR9-positive and CpG-negative cells exhibited uniform distribution of TLR9 and CD47 on the cell surface, while TLR9-positive and CpG-positive cells showed membrane alterations and aggregation of TLR9 and CD47. These findings suggest that CD47 undergoes conformational changes upon CpG binding to erythrocytes.

[0044] The expression of cell membrane receptors in the deformed erythrocytes of Example 1 was detected, such as... Figure 4 As shown in the figure (scale bar, 2 μm), the confocal image reveals the expression of band 3 protein after CpG treatment. After calcein-AM staining (results as shown in the figure), the expression was further enhanced. Figure 5As shown in the figure, this indicates that binding to CpG oligonucleotides preserves the integrity and activity of the RBC membrane; the results of flow cytometry detection of CD47 antibody on deformed erythrocytes are as follows. Figure 6 As shown, the proportion of CD47 negative cells increased after the addition of CpG oligonucleotides, indicating that the binding of CpG oligonucleotides to red blood cells leads to the loss of CD47 detection, which is beneficial for clearance by the immune system.

[0045] Example 2: In vitro biological evaluation

[0046] (1) Binding ability of cisplatin (CDDP) and CpG oligonucleotides

[0047] The CpG oligonucleotides used in Example 1 were ODN type A or B, and underwent full-chain sulfurization treatment. The CpG oligonucleotides used in Example 1 were prepared at a loading concentration of 1.3 μM, mixed according to CDDP:CpG oligonucleotide molar ratios of 1:1, 2:1, 5:1, and 10:1, and then subjected to agarose gel electrophoresis. Figure 7 As shown, the concentration of CpG oligonucleotides decreases with increasing CDDP concentration, indicating that CpG oligonucleotides can effectively bind to CDDP.

[0048] (2) Neutralization capacity of deformed erythrocyte preparations against CDDP in in vitro cell experiments

[0049] The neutralizing capacity of different quantities of deformed erythrocyte preparations against CDDP in vitro was determined using CCK-8 assay. The specific experimental procedure was as follows: Human normal hepatocytes (L-02) (Zhejiang Meisen Cell Technology Co., Ltd.) were seeded in 96-well plates at a concentration of 5000 cells / well in RPMI-1640 medium (CellorLab CB004). After 12 hours, the medium was removed, and different concentrations of cisplatin and deformed erythrocyte preparation solutions were added (concentration design as follows). Figure 8 (As shown). Cells were incubated in a humidified incubator at 37°C and 5% CO2. Afterwards, the culture medium was removed, and the cells were washed three times with PBS. Finally, CCK-8 reagent was added, and the cells were incubated at 37°C for 2 hours. Cell viability was determined by measuring absorbance at 450 nm using a microplate reader.

[0050] The results are as follows Figure 8 As shown, the presence of the erythropoietin preparation prevented platinum-based drugs from killing normal cells, and cell viability was positively correlated with the concentration of the erythropoietin preparation. Pretreatment with the erythropoietin preparation significantly improved cell viability. The mechanism by which cell damage is reduced may be that the erythropoietin preparation captures CDDP and reduces intracellular Pt levels.

[0051] We further validated this mechanism by detecting intracellular Pt levels 24 hours after cisplatin addition. Intracellular Pt levels were detected using inductively coupled plasma mass spectrometry. The specific steps were as follows: L-02 cells were cultured at 5 × 10⁶ cells per well. 5 The cells were seeded at a density of [missing information - likely a specific density] into 6-well plates containing RPMI-1640 medium. After cell adhesion, the cells were treated with a deformed erythrocyte preparation (10 [missing information - likely a specific concentration]). 5 Cell pretreatment: After co-incubating the cells with the deformed erythrocyte preparation for 2 hours, the culture medium was either replaced or retained, and then CDDP (2.5 μg / mL) was added. -1 After 24 hours, cells were collected, and all cells from each well were digested into a suspension. Then, concentrated nitric acid was added to digest the cells. Finally, the intracellular Pt concentration was detected by ICP-MS.

[0052] The results are as follows Figure 9 As shown, in the experiment of culture medium preservation, the deformed erythrocyte preparation reduced the intracellular Pt level of L-02 cells compared with the group without the deformed erythrocyte preparation; when the culture medium was refreshed, no significant difference was observed between the groups with and without the deformed erythrocyte preparation, indicating that the CpG oligonucleotide binding on erythrocytes is stable and did not detach and be bound or endocytosed by L-02 cells.

[0053] Example 3: In vivo biological evaluation

[0054] (1) In vivo imaging to verify the liver-targeting ability of deformed erythrocyte preparations

[0055] 4T1 cells (Shanghai Bogu Biotechnology Co., Ltd.) were seeded in the groin area of ​​Balb / c mice (Vitol Dermatology). The mice were randomly divided into five groups (n=3): 1-hour, 2-hour, 4-hour, 12-hour, and 24-hour groups, for imaging studies. 100-200 μL of autologous blood was collected from the mice by centrifugation and washing to collect red blood cells. RBCs (10T) were then analyzed. 8 Cells were mixed with 40 nmol CpG oligonucleotide-Cy5.5 in 1 mL of physiological saline and then incubated at 37 °C on a shaker for 2 hours. After incubation and subsequent washing and centrifugation, 100 μL of autologous RBCs treated with CpG oligonucleotides were injected via tail vein.

[0056] The animals were then anesthetized, and the distribution and aggregation of fluorescence in the mice were observed using an in vivo imaging instrument at 1, 2, 4, 12, and 24 hours after injection. Immediately after fluorescence imaging of each group of mice, the blood was cleared by cardiac perfusion with PBS. Various organs, including tumors, were collected from the mice for ex vivo organ fluorescence imaging.

[0057] The results are as follows Figure 10As shown, in mouse in vivo imaging, fluorescence is concentrated in the liver region of the upper abdominal cavity, while in ex vivo organ imaging at specific time points, deformed erythrocyte preparations show high targeting of the liver.

[0058] To quantify the targeting rate of deformed erythrocyte formulations, we used polyethylene glycol-modified AuNP with SH-PEG-NHS molecules. In short, PEG molecules were added to the AuNP solution at a molar ratio of 5000:1. After reacting at 37°C for 1-2 hours, centrifugation was performed to precipitate the polyethylene glycol-modified AuNP. After washing the AuNP with PBS, it was co-incubated with RBC-CpG for 1-4 hours to modify the deformed erythrocyte formulation. Quantification was then performed using gold nanoparticles to modify the deformed erythrocyte material, and the quantitative results are shown below. Figure 11 As shown, 87.36% of the material was targeted to the liver in major organs.

[0059] (2) Validation of toxicity reduction efficacy in animals

[0060] 4T1 cells were orally implanted into the mammary glands of Balb / c mice. The mice were randomly divided into five groups (n=5): a saline group, a CDDP group, a group receiving CDDP after one hour of pre-enrichment with STS (sodium thiosulfate), a group receiving CDDP after one hour of pre-enrichment with deformed erythrocytes, a group receiving CDDP after four hours of pre-enrichment with deformed erythrocytes, and a group receiving CDDP after eight hours of pre-enrichment with deformed erythrocytes. The toxicity-attenuating effect was studied. The CDDP dose was 5.0 mg / (kg·BW).

[0061] First, autologous blood was collected from mice, centrifuged, washed, and red blood cells were collected. The RBCs (10T) were then processed according to the prescribed ratio. 8 Cells were incubated with 40 nmol CpG oligonucleotides at 37°C for 2 hours with gentle agitation. After incubation and subsequent washing, the cells were ready for use. The pre-enriched cells were administered via tail vein injection of STS or deformed erythrocyte preparation (100 μL) according to grouping, while the saline and CDDP groups received an equal volume of saline; this constituted one treatment. Treatment was performed every 3 days (treatment refers to the injection of CDDP after each injection of deformed erythrocyte enrichment preparation or STS), with a treatment cycle of 12 days. Figure 12 As shown, the mouse's weight was recorded during treatment, and the tumor was collected at the end of treatment.

[0062] To verify the hepatoprotective effect of the deformed erythrocyte preparation, biochemical tests were performed on alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice. Figure 13 As shown, the expression values ​​of CDDP in the CDDP group were 2.1 and 1.6 times higher than those in the saline group, respectively, indicating chronic hepatocellular injury; the expression values ​​of CDDP in the CDDP group injected 8 hours after pre-enrichment of deformed red blood cells were 1.6 and 1.4 times lower than those in the CDDP group, respectively, indicating a toxicity reduction effect.

[0063] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A deformed erythrocyte preparation, characterized in that: Including red blood cells and deformants bound to the red blood cell membrane; The deforming agent is a CpG oligonucleotide; The CpG oligonucleotides are oligodeoxynucleotides containing unmethylated CpG motifs.

2. The method for preparing the deformed erythrocyte preparation according to claim 1, characterized in that... Includes the following steps: Red blood cells were co-incubated with a deforming agent to obtain a deformed red blood cell preparation.

3. The preparation method according to claim 2, characterized in that: The ratio of red blood cells to deforming agent is 10. 5 ~10 8 Cells: 10 pmol to 1 μmol.

4. The preparation method according to claim 3, characterized in that: The co-incubation conditions are as follows: incubation at 37°C on a shaker for 2–4 hours.

5. The use of the deformed erythrocyte preparation of claim 1 in the preparation of platinum-containing drug scavengers.

6. The use of the deformed erythrocyte preparation of claim 1 in the preparation of a pharmaceutical composition for treating tumors, characterized in that: The pharmaceutical composition comprises the deformed erythrocyte preparation of claim 1 and a platinum-containing drug.

7. The application according to claim 6, characterized in that: The tumor in question is a tumor in the liver.

8. The application according to claim 6, characterized in that: The platinum-containing drugs mentioned include at least one of cisplatin, carboplatin, and oxaliplatin.

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