Tissue engineered red blood cell as well as preparation method and application thereof

Abstract

The invention provides a tissue-engineered red blood cell as well as a preparation method and application thereof. The tissue-engineered red blood cell is prepared by the following method: taking red blood cells, culturing the red blood cells in a culture medium containing recombinant erythropoietin, then adding TAT peptide lactoferrin nanoparticles, and incubating to obtain the tissue-engineered red blood cell. The preparation method comprises the following steps: firstly, adding TAT peptide in the construction process of LF-IONPs to prepare TAT peptide lactoferrin nanoparticles, and then incubating the TAT peptide lactoferrin nanoparticles with erythrocytes in a culture system containing recombinant erythropoietin to obtain the tissue-engineered erythrocytes. Therefore, when the TAT peptide lactoferrin nanoparticles enter red blood cells through phagocytosis, the activity, high survival rate and normal function of the red blood cells are ensured, so that the prepared tissue-engineered red blood cells have brittleness, form, in-vivo tracing and half-life period similar to those of normal red blood cells, thereby having the basis of in-vivo medication.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically anti-tumor drugs, and particularly relates to a tissue-engineered red blood cell, its preparation method, and its application. Background Technology

[0002] Lactoferrin (LF) is a natural iron-binding glycoprotein belonging to the transferrin family. With a molecular weight of 80 kDa, it is primarily expressed and secreted by mammary epithelial cells and is widely found in the milk, saliva, and tears of mammals. Lactoferrin not only participates in iron transport but also possesses powerful biological functions such as broad-spectrum antibacterial, antioxidant, anticancer, and immune system modulation, and is considered a novel antibacterial and anticancer drug. However, currently, in vivo administration of lactoferrin to alleviate disease is only used in newborns.

[0003] Lactoferrin nanoparticles (LF-IONPs) have demonstrated unique technological advantages and potential applications in cancer therapy. These advantages are reflected in several aspects, including structural composition, physicochemical properties, and preparation and characterization methods. Based on these characteristics, LF-IONPs have broad application prospects in cancer treatment and drug delivery. However, in vivo administration of LF-IONPs carries certain risks. The complex in vivo microenvironment leads to potential off-target effects. Furthermore, the in vivo dosage is relatively insufficient. Given the characteristic of drug release in acidic environments, LF-IONPs require release within the strongly acidic tumor microenvironment, making the release conditions relatively demanding and limiting their application value.

[0004] Immune checkpoint inhibitors have become a major treatment strategy for certain tumors (such as liver cancer), but their efficacy is relatively low. Dual immune checkpoint inhibitors have improved efficacy, but the efficacy is still less than 50%, and they bring significant side effects. The core issue lies in the strong immunosuppressive microenvironment within liver cancer tissue, in which myeloid suppressor cells (MDSCs) and erythroid progenitor cells (EPCs) are important inducers of immune tolerance. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide tissue-engineered red blood cells, their preparation method and application, wherein the tissue-engineered red blood cells can release lactoferrin in tumor tissue with high targeting and high efficiency, effectively reverse the immune tolerance of tumor tissue, and improve the anti-tumor efficacy.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] The first aspect of this invention provides TAT ​​peptide lactoferrin nanoparticles, which are prepared by the following method: taking an iron oxide nanoparticle solution, sonicating it, then adding a lactoferrin solution and a TAT peptide solution, and incubating it to obtain the TAT peptide lactoferrin nanoparticles.

[0008] In some embodiments, the mass ratio of the iron oxide nanoparticles, lactoferrin, and TAT peptide is (0.3–0.6):(0.2–0.4):(0.2–1.2).

[0009] In some embodiments, the frequency of the ultrasonic treatment is 20 kHz to 200 kHz, preferably 40 kHz to 200 kHz.

[0010] In some embodiments, the power of the ultrasonic treatment is 100W to 200W.

[0011] In some embodiments, the ultrasonic treatment time is 10 min to 20 min.

[0012] In some embodiments, the incubation time is 10h to 15h.

[0013] The second aspect of this invention provides the application of the TAT peptide lactoferrin nanoparticles described above in the preparation of tissue-engineered erythrocytes.

[0014] The third aspect of this invention provides a method for preparing tissue-engineered red blood cells, comprising the following steps: taking red blood cells, culturing them in a culture medium containing recombinant erythropoietin, then adding TAT peptide lactoferrin nanoparticles as described above, incubating, and obtaining the tissue-engineered red blood cells.

[0015] In some embodiments, the concentration of the recombinant erythropoietin in the culture medium is 5 IU / ml to 15 IU / ml; preferably 8 IU / ml to 12 IU / ml.

[0016] In some embodiments, the culture medium is StemSpan SFE II medium.

[0017] In some embodiments, the density of the red blood cells is 0.8 × 10⁻⁶. 7 ~1.5×10 7 The preferred value is 1×10 7 ~1.2×10 7 .

[0018] In some embodiments, the concentration of the TAT peptide lactoferrin nanoparticles in the erythrocyte culture system is 10 μg / ml to 160 μg / ml; preferably 20 μg / ml to 80 μg / ml; more preferably 40 μg / ml to 80 μg / ml.

[0019] In some embodiments, the incubation time is 2 hours to 5 hours.

[0020] In some embodiments, the red blood cells are human red blood cells or mouse red blood cells.

[0021] The fourth aspect of this invention provides tissue-engineered red blood cells prepared by the preparation method described above.

[0022] The fifth aspect of this invention provides the application of tissue-engineered red blood cells as described above in the preparation of antitumor drugs.

[0023] The sixth aspect of this invention provides an antitumor drug, the active ingredient of which comprises tissue-engineered red blood cells as described above.

[0024] In some embodiments, the active ingredient further includes an immune checkpoint inhibitor; preferably, the immune checkpoint inhibitor is a PD-1 antibody and / or a PD-L1 antibody.

[0025] This invention provides a method for preparing tissue-engineered red blood cells through research and optimization. First, TAT peptide is added during the construction of LF-IONPs to prepare TAT peptide lactoferrin nanoparticles. Then, these TAT peptide lactoferrin nanoparticles are incubated with red blood cells in a culture system containing recombinant erythropoietin to obtain the tissue-engineered red blood cells. This method ensures that the TAT peptide lactoferrin nanoparticles can enter red blood cells through phagocytosis while maintaining the activity, high survival rate, and normal function of the red blood cells. The resulting tissue-engineered red blood cells possess similar fragility, morphology, in vivo traceability, and half-life to normal red blood cells, as well as good biocompatibility, thus providing a basis for in vivo drug administration.

[0026] The tissue-engineered red blood cells of this invention can circulate to tumor tissues like normal red blood cells after entering the body. As the tissue-engineered red blood cells undergo in situ hemolysis in the tumor, they can release a large amount of lactoferrin in the tumor tissue. The high concentration of lactoferrin effectively reverses the function of MDSC and EPC in the tumor microenvironment, improves the immune tolerance of the tumor, and thus effectively exerts an anti-tumor effect. It can also improve the efficacy of immune checkpoint inhibitors against tumors (especially liver cancer). Attached Figure Description

[0027] Figure 1 Safety experiments of TAT-LF-IONPs tissue-engineered mouse (A) and human (B) erythrocytes.

[0028] Figure 2 The retention time of TAT-LF-IONPs tissue-engineered mouse erythrocytes in healthy and tumor-bearing mice.

[0029] Figure 3 Surgical procedures for a mouse orthotopic liver cancer model.

[0030] Figure 4 The results of detecting the inhibition of T cell proliferation and function by PMN-MDSC and M-MDSC in the spleen (A) and tumor (B) of TAT-LF-IONPs mice after erythrocyte intervention.

[0031] Figure 5 Results of detection of T cell proliferation and function inhibition by PMN-MDSC and M-MDSC in peripheral blood (A) and tumors (B) after TAT-LF-IONPs human erythrocyte intervention.

[0032] Figure 6 Results of PMN-MDSC and M-MDSC ratio detection in tumor tissue, peripheral blood and spleen of mice after in vivo transfusion of TAT-LF-IONPs tissue-engineered red blood cells into hepatocellular carcinoma mice.

[0033] Figure 7 Results of T cell function inhibition by PMN-MDSC and M-MDSC in tumor tissue (A) and spleen (B) of hepatocellular carcinoma mice after in vivo transfusion of TAT-LF-IONPs tissue-engineered erythrocytes.

[0034] Figure 8 Survival time of mice after in vivo transfusion of TAT-LF-IONPs tissue-engineered red blood cells into liver cancer mice.

[0035] Figure 9 The effect of in vivo infusion of TAT-LF-IONPs tissue-engineered red blood cells into hepatocellular carcinoma mice in combination with PD-1 antibody.

[0036] Figure 10 The results of lactoferrin release from TAT-LF-IONPs tissue-engineered mouse (A) and human (B) erythrocytes under acidic conditions were obtained using the ultrasound-activated IONPs protocol.

[0037] Figure 11 To adjust the concentration of TAT peptide lactoferrin nanoparticles, the results of lactoferrin release from TAT-LF-IONPs tissue-engineered mouse (A) and human (B) erythrocytes under acidic conditions were obtained.

[0038] Figure 12 The release rate of lactoferrin from TAT-LF-IONPs-RBCs prepared with different mass ratios of iron oxide nanoparticles, lactoferrin, and TAT peptides.

[0039] Figure 13 The release amount of lactoferrin from TAT-LF-IONPs-RBCs prepared under different incubation and culture systems. Detailed Implementation

[0040] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0041] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0042] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0043] The term "and / or" as used in this invention describes the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can represent three situations: A exists alone, A and B exist simultaneously, and B exists alone.

[0044] Some embodiments of the present invention relate to a TAT peptide lactoferrin nanoparticle, characterized in that it is prepared by the following method: taking an iron oxide nanoparticle solution, sonicating it, then adding a lactoferrin solution and a TAT peptide solution, incubating it, and obtaining the TAT peptide lactoferrin nanoparticle.

[0045] Through research, this invention incorporates TAT ​​peptides during the construction of LF-IONPs to prepare TAT peptide lactoferrin nanoparticles, which can be safely taken up by erythrocytes via TAT peptides.

[0046] In some embodiments, the iron oxide nanoparticles can be prepared using conventional techniques in the art.

[0047] In some embodiments, the iron oxide nanoparticles are prepared by the following method: FeSO4 solution and FeCl3 solution are mixed in a suitable ratio, then PEG400 is added and stirred; then NaOH solution is slowly added dropwise to the mixed solution while stirring, and a black precipitate appears, thus obtaining the iron oxide nanoparticles.

[0048] In some embodiments, the following steps are also included: after the reaction is complete, centrifugation, washing, and drying are performed to obtain iron oxide nanoparticles.

[0049] This invention also discovered that the ultrasonic activation frequency of iron oxide nanoparticles and the mass ratio of iron oxide nanoparticles, lactoferrin, and TAT peptides significantly affect the lactoferrin release from the prepared tissue-engineered erythrocytes during the preparation of TAT peptide lactoferrin nanoparticles. Optimization of suitable ultrasonic frequency and raw material mass ratio resulted in improved lactoferrin release from the prepared tissue-engineered erythrocytes.

[0050] Some embodiments of the present invention relate to a method for preparing tissue-engineered red blood cells, comprising the following steps: taking red blood cells, culturing them in a culture medium containing recombinant erythropoietin, then adding TAT peptide lactoferrin nanoparticles as described above, incubating, and obtaining the tissue-engineered red blood cells.

[0051] Tissue-engineered red blood cells (RBCs) technology involves modifying red blood cells using gene editing, synthetic biology, and nanotechnology to give them specific functions. For example, drugs, biomolecules, or gene carriers can be encapsulated within or attached to the surface of red blood cells to achieve targeted drug delivery. Some technologies utilize stem cell sources such as induced pluripotent stem cells (iPSCs) or hematopoietic stem cells (HSCs) to culture and differentiate into red blood cells in vitro. However, tissue-engineered red blood cells constructed from in vitro induced stem cells still face the following challenges: (1) low production efficiency and high cost, limiting their large-scale production and clinical application; (2) functional validation: the functionality of engineered red blood cells, such as drug release efficiency and cell activity, requires further in-depth research and validation to ensure their safety and efficacy; (3) immune response: although red blood cells have good biocompatibility, they may still trigger immune responses in certain situations, requiring further research into immune exemption mechanisms. Therefore, to achieve their clinical application, challenges such as production efficiency, functional validation, and immune responses still need to be overcome. However, constructing tissue-engineered red blood cells using nanotechnology requires addressing issues such as penetration, drug targeting, and red blood cell biocompatibility before these cells can be clinically applied.

[0052] Furthermore, this invention has discovered that the incubation system of TAT peptide lactoferrin nanoparticles and erythrocytes is crucial for ensuring the activity and normal function of erythrocytes. After extensive research and screening, it was found that incubation in StemSpan SFE II medium containing a suitable concentration (5 IU / ml–15 IU / ml) of recombinant erythropoietin can simultaneously allow the TAT peptide lactoferrin nanoparticles to enter erythrocytes via phagocytosis while maintaining the activity and normal function of the erythrocytes. This results in tissue-engineered erythrocytes with similar fragility, morphology, in vivo traceability, and half-life to normal erythrocytes, as well as good biocompatibility, thus providing a basis for in vivo drug administration.

[0053] The tissue-engineered red blood cells of this invention can circulate to tumor tissues like normal red blood cells after entering the body. As the tissue-engineered red blood cells undergo in situ hemolysis in the tumor, they can release a large amount of lactoferrin in the tumor tissue. The high concentration of lactoferrin effectively reverses the function of MDSC and EPC in the tumor microenvironment, improves the immune tolerance of the tumor, and thus effectively exerts an anti-tumor effect. It can also improve the efficacy of immune checkpoint inhibitors against tumors (especially liver cancer).

[0054] The following description is based on specific embodiments.

[0055] Example 1

[0056] This embodiment provides a method for preparing tissue-engineered red blood cells, the method comprising the following steps:

[0057] 1. Synthesis of iron oxide nanoparticles

[0058] 1.1 Solution Preparation: Dissolve 2.78g of FeSO4·7H2O in ddH2O (deionized water), stir until completely dissolved, and then bring the volume to 50ml to prepare a 0.2M FeSO4 solution. Dissolve 10.82g of FeCl3·6H2O in ddH2O, stir until completely dissolved, and then bring the volume to 100ml to prepare a 0.4M FeCl3 solution. Prepare 100ml of 3M NaOH solution for later use.

[0059] [Preparation of 100ml 3M NaOH Solution] Weighing NaOH solid: Accurately weigh 12.0g of NaOH solid using a balance. Since NaOH solid is corrosive and hygroscopic, it should be weighed in a small beaker. Dissolving NaOH solid: Add an appropriate amount of distilled water to the beaker, then add the weighed NaOH solid and stir with a glass rod until fully dissolved. A large amount of heat will be released during dissolution; therefore, it needs to be cooled to room temperature. Transferring the solution: Transfer the cooled NaOH solution to a 100ml volumetric flask using a glass rod. Washing the beaker and glass rod: Wash the beaker and glass rod 2-3 times with a small amount of distilled water, transferring the washings to the volumetric flask as well to ensure all solute is transferred. Making up to volume: Add distilled water to the volumetric flask until the liquid level is 1-2cm below the graduation mark. Then, use a dropper to add distilled water dropwise until the meniscus of the solution is tangent to the graduation mark. Shake well: Seal the volumetric flask, invert it repeatedly, and shake to mix the solution thoroughly. Bottle and label: Transfer the prepared NaOH solution to a reagent bottle, affix a label indicating the solution name, concentration, preparation date, and other information. Note: Strictly follow the operating procedures during preparation to ensure the accuracy and safety of the solution. Also, because NaOH solution is highly corrosive, handle it with care.

[0060] [Preparation of 100ml 1M Sodium Citrate Solution] Weighing: Accurately weigh 29.41g of sodium citrate using an electronic balance and place it in a clean beaker. Dissolving: Add an appropriate amount of deionized water to the beaker and stir with a glass rod until the sodium citrate is completely dissolved. Transferring: Transfer the dissolved sodium citrate solution to a 100ml volumetric flask using a glass rod. Washing: Wash the beaker and glass rod 2-3 times with a small amount of deionized water, transferring all the washings to the volumetric flask. Making up to volume: Add deionized water to the volumetric flask until the liquid level is 1-2cm below the graduation mark. Then, use a dropper to add deionized water dropwise until the meniscus of the solution is tangent to the graduation mark. Shaking: Stopper the volumetric flask and repeatedly invert it to mix the solution.

[0061] 1.2. Reaction Procedure: Mix 40 ml of FeSO4 solution and 80 ml of FeCl3 solution at a volume ratio of 1:2, add 8 ml of 5% (w / v) PEG400, and place the mixture in a three-necked flask. Stir at room temperature. Slowly add NaOH solution dropwise to the mixture while stirring, carefully observing the changes in the solution. As NaOH solution is added, a black precipitate will gradually appear in the solution, forming preliminary iron oxide nanoparticles. During the dropwise addition, continuously monitor the pH value of the solution to ensure it remains stable between 9 and 13. If citrate is used, add the citrate solution before or simultaneously with the addition of NaOH solution, and continue stirring for a period of time to allow the reaction to proceed fully.

[0062] 1.3 Post-processing: After the reaction, the reaction solution was transferred to a centrifuge tube and centrifuged at 8000-10000 rpm for 5-10 minutes (10000 rpm for 5 minutes in this example) to precipitate the nanoparticles. The supernatant was discarded, and the precipitate was washed repeatedly with deionized water and anhydrous ethanol to remove residual reagents and impurities. After washing, the precipitate was centrifuged again, and the washing and centrifugation steps were repeated 3-5 times (3 times in this example). The washed nanoparticles were dried in an oven at 60-80℃ for 12-24 hours (20 hours at 60℃ in this example) to obtain dried iron oxide nanoparticle powder.

[0063] 2. The binding (conjugation) of iron oxide nanoparticles with TAT peptides and lactoferrin.

[0064] 2.1 Solution Preparation: Accurately weigh 25 mg EDC and 25 mg NHS into a clean beaker. Add an appropriate amount of deionized water to the beaker and stir with a glass rod until EDC and NHS are completely dissolved. Pipette 70 μl of 1M NaOH solution into the beaker. Transfer the solution from the beaker to a 10 ml volumetric flask. Wash the beaker and glass rod 2-3 times with a small amount of deionized water, transferring all the washings into the volumetric flask. Add deionized water to the volumetric flask until the liquid level is about 1-2 cm below the 5.07 ml mark. Then, use a dropper to add deionized water dropwise until the concave meniscus of the solution is tangent to the 5.07 ml mark. Stopper the volumetric flask and invert it repeatedly to mix the solution. EDC and NHS are unstable in aqueous solution and easily hydrolyze, so it is best to prepare the solution immediately before use. Also, NaOH solution is corrosive; handle with care to avoid contact with skin and eyes.

[0065] 2.2 Ultrasonic activation: Take 507 μl of the above solution, add 0.5 mg of synthesized iron oxide nanoparticles to it, and sonicate at 20 kHz and 100 W for 15 minutes to activate the carboxyl groups on the surface of the nanoparticles.

[0066] 2.3 Protein conjugation: For the above solution, add 125 μl of 3 mg / ml lactoferrin and 125 μl of 3 mg / ml TAT peptide solution, and incubate overnight at room temperature. Purify the solution by centrifugation at 12,000 g for 1 hour, and redisperse it three times in phosphate-buffered saline (PBS) to obtain purified nanoparticles (TAT peptide lactoferrin nanoparticles, TAT-LF-IONPs).

[0067] 3. Construction of tissue-engineered red blood cells

[0068] 3.1 Blood Collection from Mouse Heart: Anesthesia and Fixation: Deep anesthesia is typically achieved using isoflurane inhalation. The mouse is then fixed in a supine position on the operating table, ensuring full exposure of the thoracic cavity. Puncture Site Location: The puncture site is located in the 4th-5th intercostal space on the left side of the sternum, corresponding to the left ventricle of the heart. The puncture location is confirmed by palpating the point of strongest heartbeat. Puncture Procedure: A 22-25G sterile needle (approximately 0.4-0.5 mm in diameter) connected to an EDTA blood collection tube is inserted into the thoracic cavity at a 30-45° angle. Blood will automatically flow into the syringe when the needle tip enters the heart chamber; slow aspiration is necessary to prevent cardiac collapse. Blood Volume Control: Approximately 200 μl of blood is collected per 25g mouse, with an interval of at least 2 weeks to allow for animal recovery. Terminal Treatment: Euthanasia is performed via cervical dislocation. Mouse peripheral blood erythrocytes were collected: whole blood was centrifuged (1500×g, 10 minutes), and the plasma and leukocyte layer were discarded. The erythrocytes were washed three times with sterile physiological saline and diluted with 1 ml of StemSpanSFE II medium containing 10 IU / ml mouse recombinant erythropoietin (abcam, catalog number ab276979). The cells were counted using a cell counting chamber.

[0069] 3.2 Construction of TAT-LF-IONPs tissue-engineered erythrocytes (TAT-LF-IONPs-RBCs, abbreviated as tissue-engineered erythrocytes)

[0070] With 1×10 per hole 7 Cells were seeded at a density of 100 μl in 48-well plates and cultured in StemSpan SFE II medium containing 10 IU / ml recombinant mouse erythropoietin (purchased from Abcam, catalog number ab276979). The cells were cultured in a cell culture incubator at 37°C and 5% CO2. Red blood cells were incubated with low-frequency (100 rpm) shaking using 10 μg / ml solutions of FeCl3, FeSO4, LF, IONPs, and TAT-LF-IONPs (stock solution concentration 10 mg / ml). After 3 hours of culture, red blood cells were adsorbed onto TAT-LF-IONPs nanoparticles using a medium-intensity (0.3-0.5T) magnetic rack for 15-30 minutes. The supernatant red blood cells were then incubated a second time. After centrifuging the TAT-LF-IONPs-RBCs adsorbed by the magnetic rack (1500×g, 10 min), the supernatant was discarded. The red blood cells were washed three times with sterile physiological saline, diluted with 100 μl of sterile physiological saline, and counted using a cell counting chamber.

[0071] Preparation of tissue-engineered human erythrocytes: Tissue-engineered human erythrocytes were prepared according to the same method as in 3.2 above for constructing tissue-engineered mouse erythrocytes. The human erythrocytes were derived from peripheral blood of healthy individuals. Recombinant human erythropoietin (purchased from Kirin Brewery Company Limited, Takasaki Pharmaceutical, catalog number S20010076) was added to StemSpan SFE II medium.

[0072] The following indicators of the tissue-engineered mouse erythrocytes and tissue-engineered human erythrocytes obtained were tested.

[0073] 1. Red blood cell agglutination test

[0074] TAT-LF-IONPs tissue-engineered erythrocytes obtained under the above conditions were diluted with PBS to a concentration of 3 × 10⁻⁶. 9 / ml, with ordinary red blood cells from the same source as control cells, were added to a U-bottom 96-well plate at 100μl per well. The ordinary red blood cell group was added to the donkey serum group as a positive control, and the ordinary red blood cell group was added to the PBS group as a negative control.

[0075] 2. Red blood cell fragility test

[0076] Hypotonic solutions were prepared using sterile physiological saline and sterile water for injection, with sodium chloride concentrations of 0.85, 0.80, 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, and 0.20. Sterile water for injection served as a positive control, and physiological saline as a negative control. 10 TAT-LF-IONP tissue-engineered erythrocytes were obtained using the above conditions. 7 One sample of normal red blood cells was used as a control. The cells were resuspended in 1 ml of the above liquid, allowed to stand at room temperature for 30 minutes, centrifuged at 3000g for 3 minutes, and 200 μl of the supernatant was collected. The absorbance (Abs) at 414 nm was measured using an ELISA reader. The hemolysis rate was calculated as: Abs sample / Abs positive control x 100%.

[0077] 3. In vitro shear stress test

[0078] 10 TAT-LF-IONPs tissue-engineered erythrocytes obtained under the above conditions 7 One control group of red blood cells was prepared. The cells were resuspended in 1 ml of fetal bovine serum and placed in a 48-well plate. The cells were incubated at 37°C with shaking at 12 rpm (simulating normal blood vessel shear stress conditions in vivo) for 0.5 h. The morphology of the red blood cells was photographed, and phenomena such as red blood cell deformation, aggregation, and rupture were observed.

[0079] like Figure 1As shown, A represents the results of tissue-engineered mouse erythrocytes, and B represents the results of tissue-engineered human erythrocytes. Figure 1 It can be seen that the tissue-engineered mouse erythrocytes and tissue-engineered human erythrocytes prepared by this invention showed no obvious erythrocyte aggregation, and their cell fragility was basically the same as that of ordinary erythrocytes. The results of the in vitro shear stress experiment were also similar to those of ordinary erythrocytes. The results indicate that the tissue-engineered erythrocytes of this invention have good safety.

[0080] 4. In vivo tracer experiment

[0081] 10 tissue-engineered red blood cells were taken 10 CFSE-stained red blood cells were used as control cells and injected via tail vein into both hepatocellular carcinoma mice and normal mice. The proportion of CFSE-positive red blood cells in peripheral blood, spleen, and tumors was measured by flow cytometry at 1, 7, 14, and 21 days post-injection.

[0082] Flow cytometry ( Figure 2 The results showed no significant difference between the two groups, with a half-life of about 3 weeks, indicating that the survival time of the tissue-engineered red blood cells of the present invention TAT-LF-IONPs is consistent with that of transfused red blood cells and does not affect the lifespan of red blood cells.

[0083] Example 2

[0084] Myeloid suppressor cells (MDSCs) are important inducers of immune tolerance. This example examines the inhibitory effect of tissue-engineered erythrocytes prepared in Example 1 on the immunosuppressive function of MDSCs in vitro.

[0085] I. Experimental Methods

[0086] 1. Construction of a mouse orthotopic hepatocellular carcinoma model

[0087] 1.1 Preparation and transport of Hepa1-6 cell matrix suspension: After resuscitation, Hepa1-6 cells were routinely cultured and passaged to passages 3-5. Hepa1-6 cells in the logarithmic growth phase were digested, centrifuged, and prepared into a 20 μl system containing 1 × 10⁻⁶ cells. 5 A mixture of 10 μL cells, 10 μL PBS, and 10 μL of matrix gel. All instruments that come into contact with the matrix gel should be pre-cooled at 4°C. The prepared Hepa1-6 cell matrix gel suspension should be transported on ice.

[0088] 1.2 Procedure for in situ tumorigenesis of hepatocellular carcinoma: 10 to 15 minutes before surgery, mice were anesthetized by intraperitoneal injection of 160 μL / 25 g 1% phenobarbital. The animals' response to anesthesia was observed—mice should have slow, shallow breathing and show no response to squeezing of their hind legs. The abdominal skin of the mice was shaved with a razor, and fine hairs adhering to the skin were wiped away with gauze moistened with 70% ethanol to prevent hair from entering the peritoneum. The animals were fixed in a supine position on the lid of a 100 mm culture dish. Placing cotton swabs on the back of the mice could better expose the liver. Disinfect the skin with povidone-iodine. Make a 1 cm midline abdominal incision below the xiphoid process using sterile surgical scissors. Make another 1 cm incision below the xiphoid process along the linea alba. Gently pull the middle lobe of the liver onto the peritoneum using a saline-moistened cotton swab, placing it on the peritoneal surface. Place a saline-moistened cotton swab on the dorsal side of the middle lobe of the mouse liver. Using a 0.5 ml insulin injection needle, aspirate 20 μL of Hepa1-6 cells mixed with matrix gel and slowly inject the cell suspension into the subcapsule of the left lobe of the liver, near the lower edge of the liver. After injection, withdraw the needle along the direction of insertion and apply pressure with a cotton swab for 1 minute to stop bleeding. Before suturing, observe the injection site until there is no bleeding or leakage, then remove the cotton swab, reposition the liver, and intermittently suture the peritoneum and skin incision with 5-0 sutures. Wipe away blood from the skin with sterile gauze. Place the animal in a lateral recumbent position on a warm blanket until the anesthesia wears off (30 to 60 minutes). Once the mice have recovered and are able to turn over on their own, place them in a sterilized new cage.

[0089] Figure 3 Surgical procedures for a mouse orthotopic liver cancer model.

[0090] 2. Extraction of mouse spleen mononuclear cells

[0091] 2.1 Sacrifice of mice: Mice were euthanized by cervical dislocation after anesthesia and then immersed in 75% alcohol for 5 minutes;

[0092] 2.2 Spleen removal: The mouse was placed in a right lateral decubitus position. The skin and subcutaneous muscle were cut open about 1 cm along the left costal arch with scissors. The dark red strip-shaped mouse spleen was lifted with forceps. The connective tissue of the spleen mesentery was cut with scissors and the spleen was removed.

[0093] 2.3 Grinding: Place the removed spleen in a 100mm culture dish containing 5ml of pre-cooled PBS, and crush the spleen on crushed ice using the end of a 1ml syringe plunger;

[0094] 2.4 Filtration: Place a 70-micron filter on a 50 ml centrifuge tube, use a Pasteur tube to aspirate the cell suspension from the petri dish, and filter the suspension through the 70-micron filter. Add PBS to the filtered cell suspension to a final volume of 10 ml, and centrifuge at 4°C, 500 g, for 5 min.

[0095] 2.5 Red blood cell lysis: After centrifugation, discard the supernatant, add 1 ml of red blood cell lysis buffer, gently pipette to mix, lyse at room temperature for 1 min, and then add 9 ml of PBS to stop the red blood cell lysis.

[0096] 2.6 Centrifugation: Centrifugation conditions were set to 4℃, 500g, 5min. The supernatant was discarded after centrifugation.

[0097] 2.7 Wash once: Add 5 ml of pre-cooled PBS to the precipitate to resuspend the precipitate, and set the centrifugation conditions to 4℃, 500g, 5min.

[0098] 2.8 After centrifugation, add 10 ml of PBS, mix well by pipetting to obtain a suspension of spleen mononuclear cells, and calculate the total number of spleen mononuclear cells after cell density counting.

[0099] 3. Extraction of mononuclear cells from mouse liver cancer tissue

[0100] 3.1 Sacrifice the mice: After anesthetizing the mice, euthanize them by dislocation of the cervical spine and then soak them in 75% alcohol for 5 minutes.

[0101] 3.2 Liver Removal: The mouse was placed in a supine position. The skin and subcutaneous muscle were cut open about 2 cm along the linea alba with scissors. The dark red, lobed liver of the mouse was lifted with forceps. The connective tissue of the mesentery at the upper and lower ends of the liver was cut with scissors and the liver was removed. The liver was then divided into the liver tumor and the adjacent liver along the edge of the liver cancer.

[0102] 3.3 Mesh Digestion: Place the removed liver tumor in a 100mm culture dish containing 5ml of pre-chilled DMEM medium. Using scissors on crushed ice, mince the tissue to 1mm³. Collect the tissue suspension into a 50ml centrifuge tube. Set the centrifuge to 4℃, 500g, 5min. Discard the supernatant after centrifugation. Add 10ml of DMEM medium containing 0.05% IV collagenase and incubate at 37℃ in a shaker for 30 minutes in the dark.

[0103] 3.4 Filtration: Place a 70-micron filter on a 50 ml centrifuge tube, use a Pasteur tube to aspirate the digested cell suspension, and filter the suspension through the 70-micron filter. Add PBS to the filtered cell suspension to a final volume of 40 ml, and centrifuge at 4°C, 500 g, for 5 min.

[0104] 3.5 Red blood cell lysis: After centrifugation, discard the supernatant, add 3 times the volume of precipitate erythrocyte lysis buffer, gently pipette to mix, lyse at room temperature for 1 min, then add PBS to stop the lysis, centrifuge at 4℃, 500g for 5 min, and discard the supernatant after centrifugation.

[0105] 3.6 Preparation of Percoll single-cell separation solutions of different concentrations: 100% Percoll working solution is prepared by mixing sterile PBS at a concentration of 10 times with Percoll stock solution at a ratio of 1:9; 70% Percoll is prepared by mixing 100% Percoll working solution and sterile isotonic PBS at a ratio of 7:3; 30% Percoll is prepared by mixing 100% Percoll working solution and PBS at a ratio of 3:7.

[0106] 3.7 Density gradient centrifugation: Add 3 ml of 70% Percoll to the bottom of a 15 ml centrifuge tube, resuspend the lysed red blood cell suspension in 30% Percoll, and slowly add 7 ml of the suspension to the top of the 70% Percoll using a sterile pipette. Centrifuge at 18°C, 600 g for 20 minutes.

[0107] 3.8 Aspirate mononuclear cells: After centrifugation, use a 1ml pipette to remove the cell cluster at the top of the centrifuge tube, then replace the pipette tip with a new one to aspirate the mononuclear cells in the white membrane layer in the center of the centrifuge tube and place them into a new 15ml centrifuge tube.

[0108] 3.9 Centrifugation: Add 10 ml of PBS to mononuclear cells in the white membrane layer, and set the centrifugation conditions to 4℃, 500g, 5 min. After centrifugation, discard the supernatant.

[0109] 3.10 Wash once: Add 5 ml of pre-cooled PBS to the precipitate to resuspend the precipitate, and set the centrifugation conditions to 4℃, 500g, 5min.

[0110] 3.11 After centrifugation, add 10 ml of PBS, mix well by pipetting to obtain a liver mononuclear cell suspension, count the cell density and calculate the total number of liver cancer tissue mononuclear cells in the suspension.

[0111] 4. Extraction of human peripheral blood mononuclear cells (PBMCs)

[0112] 4.1 Take 2-5 ml of fresh peripheral venous blood (within 2 hours) from liver cancer patients collected in EDTA anticoagulant tubes, centrifuge at 18°C ​​and 2000 rpm for 8 minutes, and then centrifuge again at 9°C and 9°C.

[0113] 4.2 After centrifugation, aspirate the upper plasma layer, place it in an EP tube, label it and record the time, and freeze it at -80℃ for later use;

[0114] 4.3 Take blood cells and add them to a 15ml centrifuge tube. Then, rinse the anticoagulant tube with PBS and aspirate the blood cells and PBS into the 15ml centrifuge tube (the ratio of blood cells to PBS is approximately 1:2). Mix well.

[0115] 4.4 Take another 15ml centrifuge tube, add lymphocyte separation medium (the ratio of lymphocyte separation medium to blood cell diluent is approximately 1:2), hold the centrifuge tube at a 45° angle, and slowly add the mixture along the wall using a pipette, keeping the blood and separation medium clearly separated, and avoiding excessive force that may cause turbidity; (this step is crucial)

[0116] 4.5 Centrifugation: 18℃, 2000r / min, 20min, increment 5, decrement 0;

[0117] 4.6 After 30 minutes, gently pick up the cell and you will see that it is divided into four layers, from the bottom layer to the top: red blood cells, lymphocyte separation solution, mononuclear cell white membrane layer, plasma and PBS. Use a pipette to rotate and aspirate the middle white membrane layer (make sure to aspirate it completely) into a 15ml centrifuge tube, add PBS to 14ml and mix well.

[0118] 4.7 Centrifugation: 18℃, 1600rpm, 8min, increment 9, decrement 9;

[0119] 4.8 Discard the supernatant, keep the precipitate, add 4 ml of PBS or 1640 culture medium, and resuspend the PBMC with a pipette;

[0120] 4.9 Centrifugation: 18℃, 1600rpm, 8min, increment 9, decrement 9;

[0121] 4.10 The precipitate obtained is peripheral blood mononuclear cells. The cells are resuspended in 1 ml PBS for counting.

[0122] 4.11 Flow Cycling MDSC (Human CD11b) + HLA-DR - / low CD33 + ) and T(CD3 + The cells were used for subsequent experiments.

[0123] 5. Extraction of mononuclear cells from human liver cancer tissue

[0124] 5.1 Specimen collection: After verifying the patient's information, the specimen was collected in the operating room after the liver cancer resection and placed in 1640 complete culture medium.

[0125] 5.2 Mincing and Digestion: Place the removed liver cancer tissue in a 100mm culture dish containing 5ml of pre-chilled DMEM medium. Using scissors on crushed ice, mince the tissue to a size of 1mm³. Collect the tissue suspension into a 50ml centrifuge tube. Set the centrifuge to 4℃, 500g, 5min. Discard the supernatant after centrifugation. Add 10ml of DMEM medium containing 0.05% IV collagenase and incubate at 37℃ in a shaker for 30 minutes in the dark.

[0126] 5.3 Filtration: Place a 70-micron filter on a 50 ml centrifuge tube, use a Pasteur tube to aspirate the digested cell suspension, and filter the suspension through the 70-micron filter. Add PBS to the filtered cell suspension to a final volume of 40 ml, and centrifuge at 4°C, 500 g, for 5 min.

[0127] 5.4 Red blood cell lysis: After centrifugation, discard the supernatant, add 3 times the volume of precipitate erythrocyte lysis buffer, gently pipette to mix, lyse at room temperature for 1 min, then add PBS to stop red blood cell lysis, centrifuge at 4℃, 500g for 5 min, and discard the supernatant after centrifugation.

[0128] 5.5 Preparation of Percoll single-cell separation solutions of different concentrations: 100% Percoll working solution is prepared by mixing sterile PBS at a concentration of 10 times with Percoll stock solution at a ratio of 1:9; 70% Percoll is prepared by mixing 100% Percoll working solution and sterile isotonic PBS at a ratio of 7:3; 30% Percoll is prepared by mixing 100% Percoll working solution and PBS at a ratio of 3:7.

[0129] 5.6 Density gradient centrifugation: Add 3 ml of 70% Percoll to the bottom of a 15 ml centrifuge tube, resuspend the lysed red blood cell suspension in 30% Percoll, and slowly add 7 ml of the suspension to the top of the 70% Percoll using a sterile pipette. Centrifuge at 18°C, 600 g for 20 minutes.

[0130] 5.7 Aspirating Mononuclear Cells: After centrifugation, use a 1ml pipette to remove the cell clump at the top of the centrifuge tube, then replace the pipette tip with a new one to aspirate the mononuclear cells in the white membrane layer from the center of the centrifuge tube and transfer them to a new 15ml centrifuge tube.

[0131] 5.8 Centrifugation: Add 10 ml of PBS to mononuclear cells in the white membrane layer, and set the centrifugation conditions to 4℃, 500g, 5 min. After centrifugation, discard the supernatant.

[0132] 5.9 Wash once: Add 5 ml of pre-cooled PBS to the precipitate to resuspend the precipitate, and centrifuge at 4°C, 500 g, for 5 min.

[0133] 5.10 After centrifugation, add 10ml of PBS, mix well by pipetting to obtain a liver mononuclear cell suspension, count the cell density and calculate the total number of liver cancer tissue mononuclear cells in the suspension.

[0134] 6. TAT-LF-IONPs tissue-engineered erythrocyte intervention MDSC

[0135] 6.1 Intervention under mouse peripheral blood environmental conditions: MDSCs from the spleen of hepatocellular carcinoma mice were sorted by flow cytometry. Tissue-engineered red blood cells and control red blood cells incubated in each group of Example 1 were resuspended in RPMI 1640 complete medium (containing 20% ​​fetal bovine serum) and co-cultured directly at a ratio of 1:10. After 60 minutes, the cells were centrifuged at 1500g for 10 minutes, and the supernatant was collected to obtain the granulocyte layer. The cells were centrifuged at 3500g for 5 minutes, and the supernatant was discarded to obtain the MDSC precipitate. The MDSCs after intervention were obtained using RPMI 1640 complete medium (containing 20% ​​fetal bovine serum) and counted by cell counting chamber.

[0136] 6.2 Intervention under acidic tumor microenvironment conditions in mice: RPMI 1640 medium was adjusted to a pH of 6.4-6.5 using MES buffer. MDSCs from mouse hepatocellular carcinoma tumors were sorted by flow cytometry. Both tissue-engineered red blood cells and control red blood cells were resuspended in RPMI 1640 complete medium (containing 20% ​​fetal bovine serum) and co-cultured at a 1:10 ratio. Centrifugation was performed at 1500g for 10 minutes at 10, 20, and 40 minutes, and the supernatant was collected to obtain the granulocyte layer. The granulocyte layer was then centrifuged at 3500g for 5 minutes, the supernatant was discarded, and the MDSC precipitate was obtained. The MDSCs after intervention were obtained using RPMI 1640 complete medium (containing 20% ​​fetal bovine serum) and counted using a cell counting chamber.

[0137] 6.3 Intervention under human peripheral blood environmental conditions: Peripheral blood MDSCs from hepatocellular carcinoma patients were sorted by flow cytometry. The above-mentioned tissue-engineered red blood cells and control red blood cells were resuspended in RPMI 1640 complete medium (containing 20% ​​fetal bovine serum) and co-cultured directly at a ratio of 1:10. After 60 minutes, the cells were centrifuged at 1500g for 10 minutes, and the supernatant was collected to obtain the granulocyte layer. The cells were centrifuged at 3500g for 5 minutes, and the supernatant was discarded to obtain the MDSC precipitate. The MDSCs after intervention were obtained using RPMI 1640 complete medium (containing 20% ​​fetal bovine serum) and counted by cell counting chamber.

[0138] 6.4 Intervention under acidic tumor microenvironment conditions: RPMI 1640 medium was adjusted to a pH of 6.4-6.5 using MES buffer. MDSCs from liver cancer patients were sorted by flow cytometry. Both tissue-engineered red blood cells and control red blood cells were resuspended in RPMI 1640 complete medium (containing 20% ​​fetal bovine serum) and co-cultured at a 1:10 ratio. Centrifugation was performed at 1500g for 10 minutes at 10, 20, and 40 minutes, and the supernatant was collected to obtain the granulocyte layer. The granulocyte layer was then centrifuged at 3500g for 5 minutes, the supernatant was discarded, and the MDSC precipitate was obtained. The MDSCs after intervention were obtained using RPMI 1640 complete medium (containing 20% ​​fetal bovine serum) and counted using a cell counting chamber.

[0139] 7. Immunosuppressive function of MDSCs treated with TAT-LF-IONPs tissue-engineered erythrocytes

[0140] 7.1 T cell proliferation inhibition experiment

[0141] (1) CFSE staining of T cells

[0142] 1) Preparation and dispensing of CFSE solution: Dissolve 500 μg of CFSE lyophilized powder in 180 μl of anhydrous DMSO and then dispense into containers and freeze at -30℃;

[0143] 2) Prepare a single-cell suspension (500 μl, total 1.75 x 10⁻⁶ cells). 7 (cells);

[0144] 3) Adjust cell concentration: Add 1.5 ml of 1xPBS to 500 μl of single-cell suspension to make 2 ml of 8x10⁻¹ PBS. 6 / ml of single-cell suspension;

[0145] 4) Prepare CFSE working solution: Dilute the CFSE stock solution with 1 x PBS at room temperature to a 2x concentration (50 μM) working solution, for example: add 1 ml PBS to 2 μl of stock solution;

[0146] 5) CFSE staining: Add an equal volume of CFSE working solution (2ml) to the cell suspension (2ml), mix well, and incubate at room temperature (37℃) in the dark for 15min, mixing once every 7-8min.

[0147] 6) Prepare 5 times the volume (20 ml) of 4℃ complete culture medium and place it on ice for later use; after incubation, add 5 times the volume of 4℃ complete culture medium and incubate on ice in the dark for 5 min to stop staining [the action should be quick];

[0148] 7) Centrifuge at room temperature (300g, 5min), and wash cells: wash cells twice with 2ml of complete culture medium;

[0149] 8) Prepare samples for flow cytometry to detect CFSE staining rate: Pipette some cell fluid into a flow cytometry tube and add an appropriate amount of 1xPBS, then flow cytometry.

[0150] 9) Adjust the cell concentration to the required concentration for co-culture (1.75 x 10⁻⁶). 6 / ml): 1.75x10 7 1.75 x 10 6 / ml = 10ml, that is, dilute the original cell suspension to 10ml and set aside for use.

[0151] (2) Co-culture of MDSCs and T cells

[0152] 1) Co-culture system 200 μl;

[0153] 2) Cell concentration: 1.75 x 10⁻⁶ 6 / ml;

[0154] 3) The volume ratio of the two cell types is 1:1 (MDSC:T);

[0155] 4) Set up 3 duplicate holes;

[0156] 5) In the co-culture system, add appropriate neutralizing antibodies and isotype controls according to specific experimental needs;

[0157] 6) Culture for 3 days and then detect T cell proliferation by flow cytometry.

[0158] ① Transfer cells from each well of the 96-well plate to a flow cytometry tube, add 1 ml of 1xPBS, and centrifuge (1500 rpm for 5 min, 5°C up and 5°C down);

[0159] ② Discard the Upper Purification;

[0160] ③ Prepare antibody solutions (CD4-FITC, CD8-APC) in the dark, with 0.25 μl of antibody required for each sample;

[0161] ④ Add 50 μl of antibody solution to each flow cytometry tube, protect from light, and stain for 20 min;

[0162] ⑤ Wash antibody: Add 1xPBS to each flow cytometry tube and centrifuge (1500 rpm, 5 min, up 5, down 5);

[0163] ⑥ Discard the supernatant, add a certain volume of 1xPBS solution (200μl) to each tube, and run the flow cytometer.

[0164] 7.2 Levels of IFN-γ in culture supernatant

[0165] The levels of IFN-γ in the culture supernatant of each group were detected using ELISA kits (human, ThermoFisher, catalog number: KHC4021; mouse, Invitrogen, catalog number: 88-7314-88).

[0166] II. Experimental Results

[0167] Figure 4 Spleen of TAT-LF-IONPs mice after erythrocyte intervention ( Figure 4 A) and tumors ( Figure 4 B) The ability of PMN-MDSCs and M-MDSCs to inhibit T cell proliferation and function. Figure 4 It is known that, among the tissue-engineered erythrocytes incubated with FeCl3, FeSO4, LF, IONPs, and TAT-LF-IONPs in Example 1, only the tissue-engineered erythrocytes constructed with TAT-LF-IONPs were able to inhibit the immunosuppressive function of tumor tissue MDSCs, and T cell proliferation and function could be restored. Figure 4 TAT-LF-IONPs-mediated human erythrocytes have a certain blocking effect on the immunosuppressive function of tumor immune microenvironment MDSCs, but have no significant effect on the immunosuppressive function of peripheral blood MDSCs, suggesting that TAT-LF-IONPs-engineered erythrocytes mainly function in the tumor microenvironment.

[0168] Figure 5 Peripheral blood for TAT-LF-IONPs human erythrocyte intervention ( Figure 5 A) and tumors ( Figure 5 B) The ability of PMN-MDSC and M-MDSC to inhibit T cell proliferation and function.

[0169] The results showed that the ability of tumor MDSCs treated with tissue-engineered red blood cells in this invention to inhibit T cell proliferation and function was reduced, indicating that tissue-engineered red blood cells can improve the inhibitory effect of MDSCs on T cells in the tumor immune microenvironment.

[0170] Example 3

[0171] This embodiment studies the effect of in vivo infusion of TAT-LF-IONPs tissue-engineered erythrocytes prepared in Example 1 on inhibiting MDSC.

[0172] I. Experimental Methods

[0173] 1. TAT-LF-IONPs tissue-engineered erythrocytes injected via tail vein into mice with liver cancer (10...) 10 One sample of liver cancer tissue mononuclear cells and spleen cells were extracted, with ordinary red blood cells as a control, following the same method as in Example 2.

[0174] 2. Flow cytometry detection of MDSCs: Targeting hepatocellular carcinoma (HCC) tissue mononuclear cells and spleen cells, flow cytometry is used to detect MDSCs, PMN-MDSCCD11b. + Ly6G high Ly6C + and M-MDSC CD11b + Ly6G int Ly6C + .

[0175] 3. MDSC function test: The ability of MDSC in the tumor and spleen of the above-mentioned liver cancer mouse to inhibit T cell proliferation was tested according to the same method as in Example 2. The results showed that the tumor of TAT-LF-IONPs tissue-engineered erythrocyte liver cancer mouse had a decreased ability to inhibit T cell immunosuppression, and the ability of spleen MDSC to inhibit T cell proliferation showed a decreasing trend.

[0176] II. Experimental Results

[0177] 1. In vivo infusion of TAT-LF-IONPs tissue-engineered erythrocytes into hepatocellular carcinoma mice reduced tumor MDSCs.

[0178] like Figure 6 As shown, after in vivo transfusion of TAT-LF-IONPs tissue-engineered erythrocytes into hepatocellular carcinoma mice, the proportions of PMN-MDSCs and M-MDSCs in tumor tissue, peripheral blood, and spleen all decreased, with the decrease in tumor tissue showing a statistically significant difference. This indicates that TAT-LF-IONPs tissue-engineered erythrocytes can inhibit the proportion of MDSCs in tumor tissue.

[0179] 2. The immunosuppressive ability of MDSCs to inhibit T cell function decreased after in vivo transfusion of TAT-LF-IONPs tissue-engineered erythrocytes into hepatocellular carcinoma mice.

[0180] Figure 7 After in vivo infusion of TAT-LF-IONPs tissue-engineered red blood cells into liver cancer mice, tumor tissue was obtained. Figure 7 A) and spleen ( Figure 7 B) Results of detection of T cell function inhibition by heavy PMN-MDSC and M-MDSC. It can be seen that after in vivo transfusion of TAT-LF-IONP tissue-engineered red blood cells into hepatocellular carcinoma mice, the ability of tumor MDSC to inhibit T cell proliferation and function was significantly reduced, and the ability of spleen MDSC to inhibit T cell proliferation and function showed a decreasing trend.

[0181] Example 4

[0182] This embodiment studies the therapeutic effect of TAT-LF-IONPs tissue-engineered erythrocytes prepared in Example 1 on liver cancer.

[0183] A mouse orthotopic hepatocellular carcinoma model was constructed using the same method as described in Example 2. After the orthotopic hepatocellular carcinoma model was established, 10 TAT-LF-IONP tissue-engineered red blood cells were injected weekly via the tail vein. 8 10 units (diluted in 200 μl PBS), twice in total, were injected via tail vein into the vein using ordinary red blood cells. 8 One mouse (200 μl PBS dilution) and 100 μg LF-IONPs (200 μl PBS dilution) were used as controls. Body weight, time to reach the benevolent endpoint, and liver tumor burden were recorded in each group of mice with liver cancer.

[0184] The results showed that the TAT-LF-IONPs tissue-engineered erythrocytes of this invention slowed the weight loss, prolonged the survival time, and reduced the liver tumor burden in the hepatocellular carcinoma mice treated with this invention. Figure 8 The results demonstrate that the TAT-LF-IONPs tissue-engineered red blood cells of this invention have a good therapeutic effect on liver cancer.

[0185] Example 5

[0186] This embodiment studies the enhancing effect of TAT-LF-IONPs tissue-engineered erythrocytes prepared in Example 1 on the efficacy of PD-1 antibody.

[0187] A mouse orthotopic liver cancer model was constructed using the same method as described in Example 2. TAT-LF-IONP tissue-engineered erythrocytes were injected via the tail vein into the liver cancer mice. 8 Count 100 mice were used, with ordinary red blood cells serving as a control. Both groups were simultaneously administered PD-1 antibody via intraperitoneal injection at a dose of 200 μg once. Body weight, time to reach the benevolent endpoint, and liver tumor burden were recorded for each group of mice with liver cancer.

[0188] The results showed that, compared with the combination of ordinary red blood cells and PD-1 antibody, the combination of TAT-LF-IONPs tissue-engineered red blood cells and PD-1 antibody had a better therapeutic effect on liver cancer, and the survival time of mice was further prolonged. Figure 9 The results showed that the TAT-LF-IONPs tissue-engineered erythrocytes of this invention enhanced the therapeutic effect of PD-1 antibody.

[0189] Example 6

[0190] Study on LF release from tissue-engineered erythrocytes using TAT-LF-IONPs.

[0191] We investigated the factors influencing the release of lactoferrin from tissue-engineered erythrocytes using TAT-LF-IONPs. We found that the ultrasonic activation frequency of the iron oxide nanoparticles and the concentration of the TAT-LF-IONP nanoparticles in the erythrocyte incubation system significantly affected the lactoferrin release from the prepared tissue-engineered erythrocytes. We optimized the appropriate ultrasonic frequency and the incubation concentration of the TAT-LF-IONP nanoparticles. The relevant findings are as follows.

[0192] 1. Ultrasonic activation optimization

[0193] Except for the ultrasound treatment conditions for IONPs, the other operations were the same as in Example 1. Different ultrasound activation protocols for IONPs were set: ultrasound frequencies were set to 20Hz, 40Hz, 100Hz, and 200Hz; power was set to 100W and 200W; and intervention times were 10 minutes, 15 minutes, and 20 minutes. The release of lactoferrin from TAT-LF-IONPs tissue-engineered erythrocytes prepared under various protocols was detected.

[0194] The detection method was as follows: TAT-LF-IONPs tissue-engineered erythrocytes were cultured in RPMI 1640 medium for 20 minutes under pH 6.4-6.5 conditions, and the release of lactoferrin from TAT-LF-IONPs tissue-engineered erythrocytes was detected using a human lactoferrin transfer protein (LTF / LF) ELISA kit (Sangon Biotech, D711315).

[0195] The results are as follows Figure 10 As shown, the ultrasonic frequency has a significant impact on the release of lactoferrin from TAT-LF-IONPs tissue-engineered erythrocytes. The preferred ultrasonic frequency is 40Hz to 200Hz, which results in higher lactoferrin release from the constructed TAT-LF-IONPs tissue-engineered erythrocytes. The ultrasonic power has no significant effect on the release of lactoferrin from TAT-LF-IONPs tissue-engineered erythrocytes. Ultrasonic treatment for 10 to 20 minutes is acceptable, with 15 minutes yielding the best results.

[0196] 2. Optimization of TAT-LF-IONPs concentration.

[0197] Except for the concentration of TAT-LF-IONPs in the erythrocyte incubation system, the other procedures were the same as in Example 1. Erythrocytes were treated with TAT-LF-IONPs at concentrations of 10 μg / ml, 20 μg / ml, 40 μg / ml, 80 μg / ml, and 160 μg / ml, respectively. The lactoferrin release from TAT-LF-IONPs-engineered erythrocytes prepared using each method was measured using the same method as above.

[0198] The results showed that lactoferrin release was positively correlated with the concentration of TAT-LF-IONPs intervention, but the release did not continue to increase after reaching 80 μg / ml. Figure 11 Therefore, the preferred TAT-LF-IONPs intervention concentration is 20 μg / ml to 80 μg / ml; more preferably, it is 40 μg / ml to 80 μg / ml.

[0199] Example 7

[0200] Research on the preparation method of TAT-LF-IONPs tissue-engineered erythrocytes.

[0201] We investigated the factors affecting the success rate and performance of TAT-LF-IONPs tissue-engineered erythrocyte preparation.

[0202] 1. Mass ratio of iron oxide nanoparticles, lactoferrin, and TAT peptide

[0203] Except for the amount of TAT peptide used, the other operations were the same as in Example 1. For the IONPs solution obtained in 2.2, 125 μl of lactoferrin at 3 mg / ml and 125 μl of TAT peptide solutions at 1 mg / ml, 3 mg / ml, and 9 mg / ml (mass ratios of iron oxide nanoparticles, lactoferrin, and TAT peptide were 0.5:0.375:0.125, 0.5:0.375:0.375, and 0.5:0.375:1.125) were added respectively to construct TAT-LF-IONPs, and then human TAT-LF-IONPs-RBCs were prepared. The amount of lactoferrin released from the TAT-LF-IONPs-RBCs prepared under various raw material ratio schemes was detected.

[0204] The detection method was as follows: TAT-LF-IONPs-RBCs were cultured in RPMI 1640 medium for 20 minutes at a pH of 6.4-6.5, and then the release of lactoferrin from TAT-LF-IONP tissue-engineered erythrocytes was detected using a human lactoferrin transfer protein (LTF / LF) ELISA kit (Sangon Biotech, D711315).

[0205] The results are as follows Figure 12 As shown, the mass ratio of iron oxide nanoparticles, lactoferrin, and TAT peptide has a significant impact on the release of lactoferrin from the subsequently prepared TAT-LF-IONPs-RBCs. The preferred mass ratio of iron oxide nanoparticles, lactoferrin, and TAT peptide is (0.3–0.6):(0.2–0.4):(0.2–1.2). Within this mass ratio range, the TAT-LF-IONPs-RBCs prepared from TAT-LF-IONPs exhibit a higher release of lactoferrin.

[0206] 2. Red blood cell incubation medium

[0207] Except for the different red blood cell culture medium, the other operations were the same as in Example 1. This example screened several culture media, and the following two media are used as examples for comparison. Human red blood cells were cultured using Hyclone (GE) RPMI-1640 medium (catalog number SH30809.01) or StemSpan SFE II medium (catalog number 09655), respectively. TAT-LF-IONPs were added, and the cells were incubated with low-frequency shaking to construct TAT-LF-IONPs-RBCs. The survival rate of TAT-LF-IONPs-RBCs prepared in different culture media was detected. The results are as follows: Figure 13 As shown, the survival rate of TAT-LF-IONPs-RBCs obtained by incubation in StemSpan SFE II medium was higher than that in 1640 medium.

[0208] However, in clinical applications, higher survival rates are desired. Therefore, this invention further investigated the TAT-LF-IONPs and erythrocyte incubation culture system. Through extensive screening and optimization, it was ultimately found that using StemSpan SFE II medium containing 10 IU / ml recombinant human erythropoietin as the incubation medium, the survival rate of TAT-LF-IONPs-RBCs can reach approximately 98%. Figure 13 ).

[0209] The above results indicate that the mass ratio of iron oxide nanoparticles, lactoferrin, and TAT peptides in the preparation of TAT-LF-IONPs, as well as the selection of culture conditions for the incubation system of TAT-LF-IONPs and erythrocytes, significantly affect the survival rate and lactoferrin release of TAT-LF-IONPs tissue-engineered erythrocytes.

[0210] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0211] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A TAT peptide lactoferrin nanoparticle, characterized in that, The TAT peptide lactoferrin nanoparticles were prepared by the following method: an iron oxide nanoparticle solution was taken, sonicated, and then lactoferrin solution and TAT peptide solution were added and incubated to obtain the TAT peptide lactoferrin nanoparticles.

2. The TAT peptide lactoferrin nanoparticles as described in claim 1, characterized in that, The mass ratio of the iron oxide nanoparticles, lactoferrin, and TAT peptide is (0.3–0.6):(0.2–0.4):(0.2–1.2); and / or, The frequency of the ultrasonic treatment is 20kHz to 200kHz.

3. The application of the TAT peptide lactoferrin nanoparticles as described in any one of claims 1 to 2 in the preparation of tissue-engineered erythrocytes.

4. A method for preparing tissue-engineered red blood cells, characterized in that, The procedure includes the following steps: taking red blood cells, culturing them in a culture medium containing recombinant erythropoietin, then adding TAT peptide lactoferrin nanoparticles as described in any one of claims 1 to 2, incubating, and obtaining the tissue-engineered red blood cells.

5. The preparation method according to claim 4, characterized in that, The concentration of the recombinant erythropoietin in the culture medium is 5 IU / ml to 15 IU / ml; and / or, The culture medium is StemSpan SFE II medium.

6. The preparation method according to claim 5, characterized in that, The density of the red blood cells is 0.8 × 10⁻⁶. 7 ~1.5×10 7 ; and / or, The concentration of the TAT peptide lactoferrin nanoparticles in the erythrocyte culture system is 10 μg / ml to 160 μg / ml; preferably 20 μg / ml to 80 μg / ml; more preferably 40 μg / ml to 80 μg / ml.

7. Tissue-engineered red blood cells prepared by the preparation method according to any one of claims 4 to 6.

8. The use of tissue-engineered red blood cells as described in claim 7 in the preparation of antitumor drugs.

9. An antitumor drug, characterized in that, The active ingredient of the drug comprises tissue-engineered red blood cells as described in claim 7.

10. The antitumor drug as described in claim 9, characterized in that, The active ingredient also includes an immune checkpoint inhibitor; preferably, the immune checkpoint inhibitor is a PD-1 antibody and / or a PD-L1 antibody.

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