Mediterranean anemia treatment method based on stem cell technology and application

Abstract

The invention discloses a thalassemia treatment method based on a stem cell technology and application. The thalassemia treatment method comprises the following steps: collecting a peripheral blood sample of a patient, and carrying out stem cell isolated culture; constructing a nucleic acid aptamer by combining a vascular cell adhesion molecule-1 and a stromal cell-derived factor-1 in a bone marrow microenvironment; connecting a nucleic acid aptamer, a cell adhesion promoting peptide and a stem cell growth factor, and constructing a homing guiding reagent; preparing nano liposome and nano polymer particles; the nucleic acid aptamer is connected to the surfaces of the nano-liposome and the nano-polymer particles, and is grafted with a temperature-responsive material to obtain the nano-carrier with a responsive release function; stem cell growth factors and platelet-derived growth factors are mixed with gelatin microspheres to form a slow release system; the cultured stem cells are mixed with a homing guide reagent, a nano-carrier and a slow release system, and are transplanted into the body of a patient through intramedullary injection. The stem cell homing efficiency can be improved, and the treatment effect on thalassemia is improved.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to thalassemia treatment methods and applications based on stem cell technology. Background Technology

[0002] Thalassemia, a common hereditary blood disorder, seriously threatens human health. This disease is mainly caused by defects in globin genes, leading to impaired globin chain synthesis and chronic hemolytic anemia. Currently, the main clinical treatments for thalassemia include blood transfusion and iron chelation therapy. Blood transfusion can temporarily relieve the patient's anemia symptoms, but long-term transfusion can easily lead to complications such as iron overload, damaging the function of vital organs such as the heart and liver, and greatly affecting the patient's quality of life and lifespan. Although iron chelation therapy can reduce the harm of iron overload to a certain extent, it cannot fundamentally solve the root cause of thalassemia.

[0003] Currently, traditional treatments for thalassemia have certain shortcomings. Traditional methods use single-factor sustained-release technology, and the nanocarriers have limited functions, resulting in low stem cell homing efficiency and an inability to create a suitable survival environment for stem cells, thus limiting the success rate of stem cell transplantation. Summary of the Invention

[0004] This application provides a treatment method and application for thalassemia based on stem cell technology to solve at least one of the above-mentioned technical problems.

[0005] In a first aspect, this application provides a treatment method for thalassemia based on stem cell technology, comprising:

[0006] Peripheral blood samples were collected from the patient for stem cell isolation and culture.

[0007] Nucleic acid aptamers were constructed by combining vascular cell adhesion molecule-1 (VCAM-1) and stromal cell-derived factor-1 (SDF-1) in the bone marrow microenvironment.

[0008] A homing guidance reagent was constructed by connecting a nucleic acid aptamer, a cell adhesion-promoting peptide (RGD peptide), and a stem cell growth factor (SCF).

[0009] Preparation of nanoliposomes and nanopolymer particles;

[0010] Nucleic acid aptamers were attached to the surface of nanoliposomes and nanopolymer particles and grafted with temperature-responsive materials to obtain nanocarriers with responsive release functions.

[0011] Stem cell growth factor (SCF) and platelet-derived growth factor (PDGF) are mixed with gelatin microspheres to form a sustained-release system;

[0012] The cultured stem cells are mixed with homing guidance reagents, nanocarriers and sustained-release systems, and then transplanted into the patient via intramedullary injection.

[0013] This application overcomes the limitations of traditional single-target nucleic acid aptamer screening by simultaneously targeting vascular cell adhesion molecule-1 (VCAM-1) and stromal cell-derived factor-1 (SDF-1) in the bone marrow microenvironment. By constructing a composite nanocarrier with both targeting and intelligent release functions, it overcomes the single-function limitations of traditional nanocarriers. Using a dual-factor combination of SCF and PDGF loaded on gelatin microspheres, key regulatory molecules are screened, and a sustained-release system is constructed, overcoming the limitations of traditional single-factor sustained-release technologies and creating a suitable survival environment for stem cells.

[0014] Secondly, this application provides a homing guide reagent, comprising:

[0015] Nucleic acid aptamers are constructed based on vascular cell adhesion molecule-1 (VCAM-1) and stromal cell-derived factor-1 (SDF-1) in the bone marrow microenvironment.

[0016] Cell adhesion-promoting peptide (RGD peptide), RGD peptide linked to nucleic acid aptamers;

[0017] Stem cell growth factor (SCF) is a protein that links a nucleic acid aptamer to an RGD peptide.

[0018] Thirdly, this application provides a nanocarrier, comprising:

[0019] Nanoliposomes;

[0020] Nanopolymer particles;

[0021] Nucleic acid aptamers connect the surfaces of nanoliposomes and nanopolymer particles;

[0022] Temperature-responsive materials, and the surface of temperature-responsive materials grafted onto nanoliposomes and nanopolymer particles.

[0023] Fourthly, this application provides a sustained-release system, comprising:

[0024] Stem cell growth factor (SCF);

[0025] Platelet-derived growth factor (PDGF);

[0026] Gelatin microspheres, gelatin microspheres mixed with stem cell growth factor (SCF) and PDGF.

[0027] Fifthly, this application provides a stem cell therapy composition comprising:

[0028] The second aspect is the homing guidance reagent;

[0029] The third aspect of nanocarriers;

[0030] The fourth aspect is the sustained-release system;

[0031] Isolated and cultured stem cells. Attached Figure Description

[0032] Figure 1 A schematic flowchart illustrating a stem cell-based thalassemia treatment method provided in an embodiment of this application;

[0033] Figure 2 This is a schematic diagram of a process for constructing nucleic acid aptamers provided in an embodiment of this application;

[0034] Figure 3 A schematic diagram of a process for constructing a homing guide reagent provided in an embodiment of this application;

[0035] Figure 4 This is a schematic diagram of a process for obtaining nanocarriers provided in an embodiment of this application;

[0036] Figure 5 A schematic diagram of a process for forming a sustained-release system provided in an embodiment of this application;

[0037] Figure 6 This is a schematic diagram of a process for stem cell homing guidance and targeted delivery provided in an embodiment of this application. Detailed Implementation

[0038] The terms “comprising” and “having”, and any variations thereof, in the specification, claims, and accompanying drawings of this application are intended to cover non-exclusive inclusion, such that a process, method, composition, or application that includes a series of steps or modules is not necessarily limited to those steps, modules, or components that are explicitly listed, but may include other steps or modules that are not explicitly listed or that are inherent to such processes, methods, compositions, or applications.

[0039] It should also be noted that in some alternative embodiments, the functions / actions indicated may not appear in the order shown in the figures. For example, depending on the functions / actions involved, the two figures shown consecutively may actually occur simultaneously or may sometimes be performed in reverse order.

[0040] In this application, the specific term "exemplary" means "used as an example, embodiment, or illustration." Any embodiment illustrated "exemplarily" is not necessarily to be construed as superior to or better than other embodiments.

[0041] Traditional methods have limitations in guiding stem cell homing, failing to accurately identify key molecules in the bone marrow microenvironment, resulting in low homing efficiency and difficulty in effectively restoring hematopoietic function, thus leading to poor treatment outcomes for thalassemia. Secondly, conventional carrier materials cannot simultaneously achieve good biocompatibility, precise targeting, and intelligent responsiveness, easily damaging stem cells during transport and hindering precise engraftment, affecting treatment efficacy and potentially causing side effects. Furthermore, traditional methods lack sufficient regulation of the stem cell microenvironment, lacking a mechanism for the continuous and stable release of regulatory molecules, failing to create a suitable survival environment for stem cells, thus limiting the success rate of stem cell transplantation.

[0042] To address at least one of the aforementioned problems, embodiments of this application provide a treatment method for thalassemia based on stem cell technology. Please refer to... Figure 1 , Figure 1 A schematic flowchart of a stem cell-based thalassemia treatment method provided in this application embodiment includes the following steps:

[0043] S101. Collect peripheral blood samples from patients for stem cell isolation and culture.

[0044] Patients with thalassemia undergo physical examinations, including complete blood counts and genetic testing to determine the type and severity of thalassemia. Liver and kidney function tests, cardiopulmonary function tests, and other examinations assess the patient's overall health. Peripheral blood samples are collected for subsequent stem cell isolation, with strict aseptic technique followed during the collection process. The peripheral blood samples are centrifuged to obtain a mononuclear cell layer rich in hematopoietic stem cells, which is then seeded into culture flasks for culture.

[0045] A complete blood count (CBC) can quickly assess the degree of anemia and abnormalities in blood cells. Combined with genetic testing, it can identify the mutation type (e.g., α or β thalassemia) and severity (e.g., β0 / β0 severe anemia), providing a molecular biological basis for developing personalized stem cell therapy plans. Liver, kidney, and cardiopulmonary function tests can screen for patients eligible for stem cell transplantation, reducing treatment risks. Strict aseptic techniques during peripheral blood collection prevent sample contamination.

[0046] In one implementation, the peripheral blood sample collected from the patient is 5mL-10mL, and EDTA-K2 anticoagulant is used after collection to prevent blood clotting.

[0047] In one implementation, a peripheral blood sample is added to a centrifuge tube containing lymphocyte separation medium and centrifuged. After centrifugation, a mononuclear cell layer rich in hematopoietic stem cells is collected, and the separated stem cells are seeded into a culture flask containing IMDM medium for culture.

[0048] In one implementation, the volume ratio of peripheral blood sample to lymphocyte separation liquid is 1:1-1.5.

[0049] In one implementation, peripheral blood samples are added to centrifuge tubes containing lymphocyte separation medium and centrifuged at 18℃-20℃ and 2000r / min-2500r / min for 20-30 min. Then, 15%-17% fetal bovine serum and 15ng / mL-20ng / mL stem cell growth factor (SCF) are added to IMDM medium. The tubes are then incubated at 37℃ with 5%-8% CO2, with the medium changed every 2-3 days for 5-7 days. Standardized parameters (temperature, centrifugation force, and medium formulation) ensure reproducibility and meet the aseptic requirements for clinical-grade cell preparation. Furthermore, optimized gradient centrifugation and precise control of nutrient factors significantly improve cell purity and viability.

[0050] S102. Construct nucleic acid aptamers by combining vascular cell adhesion molecule-1 (VCAM-1) and stromal cell-derived factor-1 (SDF-1) in the bone marrow microenvironment.

[0051] In one implementation, please refer to Figure 2 , Figure 2 A schematic diagram of a process for constructing nucleic acid aptamers provided in this application embodiment includes the following steps:

[0052] S201. Construct a single-stranded DNA random library.

[0053] S202. Incubate a single-stranded DNA random library with vascular cell adhesion molecule-1 (VCAM-1) and stromal cell-derived factor-1 (SDF-1) in the bone marrow microenvironment to screen for nucleic acid sequences that specifically bind to the target molecules.

[0054] By simultaneously targeting vascular cell adhesion molecule-1 (VCAM-1) and stromal cell-derived factor-1 (SDF-1) in the bone marrow microenvironment, the limitations of traditional single-target nucleic acid aptamer screening can be overcome, thereby significantly improving the specific recognition ability of hematopoietic stem cell homing microenvironment.

[0055] S203. Cyclic screening, elution and amplification of nucleic acid sequences to obtain nucleic acid aptamers with specific recognition functions for vascular cell adhesion molecule-1 (VCAM-1) and stromal cell-derived factor-1 (SDF-1).

[0056] In one implementation, the single-stranded DNA random library includes 10^14-10^15 different sequences, and the length of the single-stranded DNA random library is 30-80 nucleotides.

[0057] By limiting the size and length of the single-stranded DNA random library, screening efficiency can be significantly improved.

[0058] In one implementation, the incubation temperature is 37°C and the incubation time is 1-2 hours.

[0059] An incubation temperature of 37°C can simulate the in vivo environment and accelerate the specific binding of antigen and antibody. An incubation time of 1-2 hours can balance reaction efficiency and molecular stability. The combination of these two factors can reduce the risk of non-specific binding caused by prolonged incubation at low temperatures.

[0060] In one implementation, nucleic acid sequences are screened, eluted, and amplified in 8-15 cycles to obtain nucleic acid aptamers that specifically recognize VCAM-1 and SDF-1.

[0061] For example, the specific process of S102 includes:

[0062] After 8-15 rounds of screening, elution, and amplification, the first screening involved incubating a constructed random library of single-stranded DNA containing 10^14 different sequences and 50 nucleotides in length with an excess of vascular cell adhesion molecule-1 and stromal cell-derived factor-1 from the bone marrow hematopoietic microenvironment at 37°C for 1.5 h. Elution was then performed using a low-rigidity buffer (containing 50 mM NaCl, 10 mM Tris-HCl pH 7.5, and 1 mM MgCl2) to remove unbound nucleic acids. The bound nucleic acids were then amplified by PCR to obtain the first-round screening products. Starting from the second round of screening, the NaCl concentration in the elution buffer was decreased by 10 mM each time, while the incubation temperature was increased by 0.5°C. The second-round elution buffer consisted of 40 mM NaCl, 10 mM Tris-HCl pH 7.5, and 1 mM MgCl2, with an incubation temperature of 37.5°C. This gradual increase in screening rigor was repeated for the subsequent seven screening, elution, and amplification cycles, ultimately yielding nucleic acid aptamers capable of specifically recognizing key molecules.

[0063] It has the following advantages:

[0064] 1. The temperature was gradually increased from 37℃ to 41℃ to simulate fluctuations in the physiological environment, and aptamers that could still bind stably under complex in vivo conditions were screened to ensure the reliability of practical applications.

[0065] 2. By progressively increasing the screening stringency (reducing the NaCl concentration in the buffer by 10 mM and increasing the incubation temperature by 0.5 °C in each round), the specificity and affinity of nucleic acid aptamers can be significantly improved. Through 8-15 rounds of progressive screening, low-affinity sequences are gradually eliminated, ultimately yielding highly efficient aptamers that recognize only key targets (such as biomarkers of the bone marrow hematopoietic microenvironment), with dissociation constants (KD) reaching the nM level.

[0066] 3. By progressively increasing the screening stringency (reducing the NaCl concentration in the buffer by 10 mM and increasing the incubation temperature by 0.5 °C in each round), the specificity and affinity of nucleic acid aptamers were significantly improved.

[0067] S103. Connect nucleic acid aptamers, cell adhesion-promoting peptides (RGD peptides), and stem cell growth factors (SCF) to construct a homing guidance reagent.

[0068] In one implementation, please refer to Figure 3 , Figure 3 A schematic flowchart for constructing a homing guide reagent provided in this application embodiment includes the following steps:

[0069] S301. Purification of nucleic acid aptamers based on high performance liquid chromatography (HPLC).

[0070] The selected nucleic acid aptamers were purified using high performance liquid chromatography (HPLC) to remove impurities and unbound nucleic acids.

[0071] For example, the specific process of S301 is as follows:

[0072] The crude nucleic acid aptamers obtained from screening were dissolved in an aqueous solution containing 0.1% trifluoroacetic acid (TFA) to achieve a concentration of 1 mg / mL-5 mg / mL. The solution was then filtered through a 0.22 μm filter to remove insoluble impurities. A C18 reversed-phase column was used, with mobile phase A being an aqueous solution containing 0.1% TFA and mobile phase B being an acetonitrile solution containing 0.1% TFA. A linear gradient elution program was set, with the initial mobile phases being 95% A and 5% B, gradually changing to 50% A and 50% B over 30 min at a flow rate of 1 mL / min. The detection wavelength was set to 260 nm. The nucleic acid aptamer solutions corresponding to the elution peaks were collected and subjected to vacuum freeze-drying to remove organic solvents and water, yielding nucleic acid aptamers with a purity of over 95%.

[0073] Filtration using a 0.22 μm filter membrane effectively retains particles larger than 220 nm, while a solution containing 0.1% TFA acts as an ion-pairing reagent, neutralizing the negative charge of the nucleic acid aptamer's phosphate backbone and enhancing its retention in a C18 reversed-phase column. Combined with gradient elution (5-50% acetonitrile), the target aptamer can be precisely separated from sequence defective or incompletely folded secondary structure products, resulting in a final purity exceeding 95%.

[0074] S302. Based on the chemical coupling method, the cell adhesion-promoting peptide (RGD peptide) is reacted with the purified nucleic acid aptamer at room temperature.

[0075] The cell adhesion-promoting peptide (Arginine-Glycine-Aspartic Acid peptide, RGD peptide) was linked to a nucleic acid aptamer via chemical coupling and the reaction was carried out at room temperature.

[0076] For example, the specific process of linking RGD peptides and nucleic acid aptamers via chemical coupling is as follows:

[0077] The purified nucleic acid aptamers were dissolved in 100 mM (millimoles per liter) of 2-morpholinoethanesulfonic acid. In an acid (MMES) buffer (pH 6.0), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added at a molar ratio of 4:1 (EDC:NHS) and 1:10 (Nucleic acid aptamer:EDC). The mixture was activated at room temperature for 30 min to attach active carboxyl groups to the surface of the aptamer. The RGD peptide was dissolved in the same MES buffer and added to the activated aptamer solution at a molar ratio of 1:4 (Nucleic acid aptamer:RGD peptide). The mixture was stirred slowly at room temperature for 2.5 h. After the reaction was complete, unreacted RGD peptide and other small molecule impurities were removed by gel filtration chromatography to obtain the conjugated product of RGD peptide and aptamer.

[0078] It has the following advantages:

[0079] 1. MES buffer (pH 6.0) provides the optimal reaction environment for the EDC / NHS system (EDC has the highest activity at pH 4.5-7.5), and significantly improves the carboxyl activation efficiency (activation rate greater than 90%) by forming a stable NHS ester intermediate (with a half-life extended to several hours).

[0080] 2. A molar ratio of EDC to NHS of 4:1 balances activation and side reactions (such as EDC hydrolysis), while a molar ratio of nucleic acid aptamer to EDC of 1:10 ensures sufficient activation of the carboxyl site (avoiding low coupling rate due to insufficient activation). An excess of RGD peptide (aptamer:RGD = 1:4) drives the reaction in the forward direction, increasing the coupling rate to over 80%.

[0081] S303. Connects stem cell growth factor (SCF) to the reacted nucleic acid aptamer to form a homing guide reagent.

[0082] After the reaction was completed, SCF was ligated to nucleic acid aptamers to obtain homing guidance reagents.

[0083] Compared to traditional methods that rely solely on single-factor guidance, the covalent coupling of dual-target nucleic acid aptamers (VCAM-1 / SDF-1) with RGD peptides and SCF allows for a synergistic effect between the nucleic acid aptamers and peptides / factors, significantly improving homing efficiency.

[0084] In one implementation, the molar ratio of nucleic acid aptamer to RGD peptide is 1:3-5, and the reaction is carried out at room temperature for 2.5-3.5 hours.

[0085] In one implementation, after the reaction is complete, SCF and nucleic acid aptamers are linked at a molar ratio of 1:1.5-2.0, and the reaction time is 3.5-4.5 h.

[0086] Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology precisely screens nucleic acid aptamers from a random single-stranded DNA library that can recognize key molecules in the bone marrow hematopoietic microenvironment. By linking RGD peptides to nucleic acid aptamers, the adhesion between stem cells and cells in the bone marrow microenvironment is enhanced, making it easier for stem cells to colonize at the target site. The addition of SCF also provides nutritional support and differentiation signals for stem cells during homing, promoting their differentiation into normal hematopoietic cells. This precisely guides stem cell homing and promotes their differentiation, improving the distribution and function of stem cells in the patient's body, helping to restore normal hematopoietic function, and significantly enhancing the efficacy of stem cell therapy for thalassemia.

[0087] For example, the specific process for connecting SCF to the reacted nucleic acid aptamer is as follows:

[0088] The obtained RGD peptide-nucleic acid aptamer conjugate was dissolved in phosphate-buffered saline (PBS) at pH 7.4. An appropriate amount of maleimide modification reagent was added to modify some amino groups on the nucleic acid aptamer with maleimide groups. After reacting for 1 h, unreacted modification reagent was removed by dialysis. SCF was dissolved in PBS containing 10 mM dithiothreitol (DTT) and incubated at room temperature for 30 min to reduce the disulfide bonds in the SCF molecule, exposing thiol groups. The treated SCF solution was added to the modified nucleic acid aptamer solution at a molar ratio of SCF to nucleic acid aptamer of 1:1.5, and reacted at room temperature for 3.5 h. The SCF and nucleic acid aptamer were linked through the specific reaction between the maleimide groups and thiol groups. After the reaction, unreacted SCF was removed by ultrafiltration centrifugation to obtain the SCF-nucleic acid aptamer conjugate product.

[0089] It has the following advantages:

[0090] 1. Maleimide groups and the thiol groups (-SH) exposed by reduced SCF form stable thioether bonds (CS) through Michael addition reaction. The reaction is highly specific (efficiency greater than 90% at pH 7.0-7.5), avoiding non-specific cross-linking.

[0091] 2. The 1:1.5 molar ratio of SCF to nucleic acid aptamer ensures full exposure of the coupling site. Ultrafiltration centrifugation can accurately retain high molecular weight complexes (aptamer-RGD-SCF > 30 kDa) and remove unreacted SCF (molecular weight approximately 18 kDa), resulting in a product purity > 95%.

[0092] S104. Preparation of nanoliposomes and nanopolymer particles.

[0093] Nanoliposomes and nanopolymer particles have good biocompatibility and modifiability, and can effectively encapsulate stem cells, protect their activity during transport, and reduce damage to stem cells from the external environment.

[0094] In one implementation, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol are dissolved in chloroform, and the chloroform is removed by evaporation in a rotary evaporator to form a uniform lipid film. A buffer solution containing stem cells is added, followed by sonication to form nanoliposomes.

[0095] In one implementation, polylactic acid-glycolic acid copolymer is used as a raw material. The polylactic acid-glycolic acid copolymer is dissolved in dichloromethane, and then an aqueous solution containing stem cells is added. Nanopolymer particles are prepared by emulsification-solvent evaporation method to obtain nanopolymer particles.

[0096] In one implementation, the molar ratio of DSPC to cholesterol is 3.5-4.0:1. The chloroform is removed by evaporation in a rotary evaporator at 40℃-50℃, followed by ultrasonic treatment for 10-15 minutes at an ultrasonic power of 200W-300W.

[0097] The high phase transition temperature (Tm≈55℃) of DSPC synergistically works with the rigid cyclic structure of cholesterol to form a dense bilayer during evaporation at 40-50℃, reducing liposome membrane fluidity and minimizing drug leakage (encapsulation efficiency increased by 15-20%). A 200-300W ultrasonic power combined with a 10-15 minute processing time effectively breaks down liposome aggregates to uniform nanoparticle sizes (typically <200nm) while avoiding excessive ultrasonication that could lead to lipid oxidation or structural damage.

[0098] In one implementation, a solution with a concentration of 100 mg / mL to 150 mg / mL is prepared by dissolving polylactic acid-glycolic acid copolymer in dichloromethane. An aqueous solution containing stem cells is then added and stirred to emulsify the solution. The stirring speed is controlled at 1000 r / min to 1500 r / min, and the emulsification time is 1 h to 2 h.

[0099] In one implementation, the surface of the prepared nanoliposomes and nanopolymer particles is modified with an immunomodulator such as interleukin-10 (IL-10).

[0100] The modified nanocarrier can regulate the local immune microenvironment, reduce the incidence of immune rejection, promote stem cell colonization and differentiation, and improve treatment efficacy during the delivery of stem cells to the bone marrow microenvironment.

[0101] S105. Nucleic acid aptamers are attached to the surface of nanoliposomes and nanopolymer particles, and grafted with temperature-responsive materials to obtain nanocarriers with responsive release functions.

[0102] In one implementation, please refer to Figure 4 , Figure 4 A schematic diagram of a process for obtaining nanocarriers provided in this application embodiment includes the following steps:

[0103] S401. Nucleic acid aptamers are covalently linked to the surfaces of nanoliposomes and nanopolymer particles, and the reaction is allowed to proceed overnight to obtain nanocarriers with targeted molecular modification.

[0104] By combining nucleic acid aptamers with temperature-responsive materials onto the surface of nanocarriers, a composite carrier with both targeting and intelligent release functions is formed, breaking through the single-function limitation of traditional nanocarriers.

[0105] S402. Grafting temperature-responsive materials onto nanocarriers with targeted molecular modification yields nanocarriers possessing both targeted molecular modification and temperature-responsive release functions.

[0106] In one implementation, the temperature-responsive material is poly(N-isopropylacrylamide) (PNIPAM).

[0107] By using PNIPAM as the temperature-sensitive material and restricting its binding to the support via a free radical grafting reaction, the uncontrollability of conventional physical adsorption is overcome. Furthermore, grafting can form stable covalent bonds, which, compared to electrostatic adsorption or hydrophobic interactions in traditional methods, significantly improves the accuracy of temperature-responsive release.

[0108] In one implementation, the mass ratio of nanoliposomes and nanopolymer particles to nucleic acid aptamers is 15-17:1, and the reaction is carried out overnight at 4℃-6℃.

[0109] In one implementation, the grafting reaction temperature is 55-58℃, and the grafting reaction time is 4-6 hours.

[0110] By modifying the surface of nanocarriers with targeting molecules such as nucleic acid aptamers, and through targeted modification and responsive functionalization, these nanocarriers specifically recognize and target the bone marrow microenvironment, precisely delivering and engrafting stem cells, thus achieving precise targeted transport. By introducing the temperature-responsive material PNIPAM, the nanocarriers can intelligently control the release of stem cells based on local temperature changes after reaching the bone marrow microenvironment. This improves the targeted engraftment efficiency and delivery accuracy of stem cells in the bone marrow, reduces cell loss in non-target tissues, ultimately enhancing therapeutic efficacy and reducing potential side effects.

[0111] S106. Stem cell growth factor (SCF) and platelet-derived growth factor (PDGF) are mixed with gelatin microspheres to form a sustained-release system.

[0112] In one implementation, please refer to Figure 5 , Figure 5 A schematic diagram of a process for forming a sustained-release system provided in this application embodiment includes the following steps:

[0113] S501. Stem cell growth factor (SCF) and platelet-derived growth factor (PDGF) are prepared into stem cell growth factor solution and platelet-derived growth factor solution, respectively.

[0114] In one implementation, the concentration of the stem cell growth factor solution is 15 ng / mL to 17 ng / mL, and the concentration of the platelet-derived growth factor solution is 7.5 ng / mL to 9.0 ng / mL.

[0115] S502. Add the stem cell growth factor solution and platelet-derived growth factor solution to the buffer solution respectively to obtain the regulated buffer solution.

[0116] SCF and platelet-derived growth factor (PDGF) were weighed and prepared into a solution, which was then added to a buffer solution to obtain a regulated buffer solution. By combining SCF and PDGF at specific concentrations (15-17 ng / mL SCF, 7.5-9.0 ng / mL PDGF), the limitations of single-factor sustained release were overcome, achieving a synergistic effect of two factors.

[0117] S503. The regulated buffer solution is mixed with gelatin microspheres, and the gelatin microspheres are cured through a cross-linking reaction to form a sustained-release system.

[0118] The regulated buffer solution was mixed with biodegradable gelatin microspheres, and the microspheres were solidified through a cross-linking reaction to form a sustained-release system. Its release performance was tested in a simulated physiological environment at 37°C to ensure the slow release of the regulatory molecules over 8 days. This sustained-release system, through gradient release of SCF and PDGF, can continuously promote stem cell proliferation and differentiation, as well as vascular microenvironment reconstruction.

[0119] By loading gelatin microspheres with a dual-factor combination of SCF and PDGF, key regulatory molecules were screened and a sustained-release system was constructed, overcoming the limitations of traditional single-factor sustained-release technology and creating a suitable environment for stem cell survival.

[0120] By considering the bone marrow hematopoietic microenvironment, we screened for SCF and PDGF, which play a key regulatory role in stem cell homing and colonization, aiming to regulate stem cell growth, differentiation, and migration. Using biodegradable gelatin microspheres as carriers, we encapsulated the regulatory molecules, which were then solidified through a cross-linking reaction to form a sustained-release system. During stem cell transplantation, this system was injected into the patient along with the stem cells, slowly releasing the regulatory molecules over a prolonged period. This provides continuous and stable microenvironmental support for stem cell homing and colonization, ensuring a suitable survival environment for stem cells in vivo, promoting better stem cell function, and contributing to a higher success rate of stem cell transplantation.

[0121] In one implementation, the volume ratio of the regulated buffer solution to the gelatin microspheres is 1:2.5-3.3.

[0122] For example, the specific process of S503 is as follows:

[0123] Gelatin was dissolved in distilled water to prepare a 5% (w / v) solution, which was heated to 50°C to ensure complete dissolution. This solution was then slowly added dropwise to a mixture containing liquid paraffin and Span-80 (as an emulsifier). Emulsification was carried out at 1000 rpm for 30 min to form a water-in-oil (w / o) emulsion. An appropriate amount of glutaraldehyde (a crosslinking agent) was added to the emulsion at a molar ratio of glutaraldehyde to gelatin of 1:100. The mixture was stirred at 37°C for 2 h to allow the gelatin microspheres to crosslink and solidify. The crosslinked gelatin microspheres were collected by centrifugation and washed three times with anhydrous ethanol to remove residual oil phase and emulsifier. The gelatin microspheres were then dispersed in the conditioning solution prepared in step seven (containing 15 ng / mL SCF and 7.5 ng / mL...). In a PBS solution of PDGF, the volume ratio of the regulatory solution to gelatin microspheres was 1:2.5. The mixture was stirred and adsorbed at room temperature for 1 hour to load the regulatory molecules onto the gelatin microspheres, forming a sustained-release system. Finally, its release performance was tested in a simulated physiological environment at 37°C to ensure that the regulatory molecules could be slowly released within 8 days.

[0124] It has the following advantages:

[0125] 1. The volume ratio of the solution (SCF+PDGF) to the microspheres was adjusted to 1:2.5, combined with the porous structure of gelatin (specific surface area > 50m²). 2 / g), with a loading efficiency of over 80%, and maintains molecular activity through electrostatic interaction.

[0126] 2. Under simulated physiological conditions (37℃, pH 7.4), gelatin microspheres release regulatory molecules through a swelling-diffusion mechanism, with a cumulative release of approximately 70-85% over 8 days, meeting the continuous signaling requirements for tissue repair.

[0127] S107. The cultured stem cells are mixed with homing guidance reagents, nanocarriers and sustained-release systems, and transplanted into the patient via intraosseous injection.

[0128] S107 can be understood as a specific implementation method for stem cell homing guidance and targeted delivery. For one implementation method, please refer to... Figure 6 , Figure 6 A schematic diagram of a stem cell homing guidance and targeted delivery process provided in this application embodiment includes the following steps:

[0129] S601. Incubate the cultured stem cells with a homing guidance reagent.

[0130] The cultured and expanded stem cells were mixed with homing guidance reagent and incubated to allow the homing guidance reagent to fully act on the stem cells.

[0131] S602. Mix the incubated stem cells, nanocarriers, and sustained-release system to form a mixed product.

[0132] In one implementation, the cultured and expanded stem cells are mixed with homing guidance reagent at a volume ratio of 1:7.5-8.5, and the incubation time is 45 min-55 min.

[0133] In one implementation, the mixed volume ratio of the incubated stem cells, the nanocarrier with responsive release function, and the sustained-release system is 3-4:1:1.

[0134] S603. The mixed product is transplanted into the patient via intraosseous injection.

[0135] The mixed products are injected into the patient's body via intraosseous injection. The injection volume is determined based on the patient's weight and condition, generally 5-10 mL, to complete the stem cell transplant. After transplantation, weekly blood tests are performed, and bone marrow aspiration is conducted every 2-4 weeks.

[0136] For example, the stem cell-based thalassemia treatment method provided in this application includes:

[0137] Patients with thalassemia undergo a thorough physical examination, including complete blood count and genetic testing to determine the type and severity of thalassemia, as well as liver and kidney function, cardiopulmonary function, and other examinations to assess their overall health, all while strictly adhering to aseptic procedures.

[0138] 5 mL of peripheral blood samples were collected from patients using EDTA-K2 anticoagulant for subsequent stem cell isolation. The collected samples were added to centrifuge tubes containing lymphocyte separation medium at a sample-to-separation medium volume ratio of 1:1. The samples were centrifuged at 18°C ​​and 2000 rpm for 20 min to collect a mononuclear cell layer rich in hematopoietic stem cells. The cells were then seeded into culture flasks containing IMDM medium supplemented with 15% fetal bovine serum and 15 ng / mL LSCCF. The flasks were incubated at 37°C and 5% CO2, with the medium changed every 2 days for 5 days.

[0139] A randomized single-stranded DNA library containing 10^14 different sequences, each 30 nucleotides in length, was constructed and incubated with vascular cell adhesion molecule-1 and stromal cell-derived factor-1 from the bone marrow hematopoietic microenvironment at 37°C for 1 hour. After 8 rounds of screening, elution, and amplification, specific aptamers were obtained.

[0140] The nucleic acid aptamer was purified by high performance liquid chromatography. The RGD peptide was chemically coupled at room temperature for 2.5 h at a molar ratio of 1:3 to the nucleic acid aptamer. Then, the SCF was coupled to the nucleic acid aptamer at a molar ratio of 1:1.5 for 3.5 h to obtain the homing guide reagent.

[0141] DSPC and cholesterol were weighed at a molar ratio of 3.5:1 and dissolved in chloroform. The chloroform was then removed by evaporation at 40°C using a rotary evaporator to form a uniform lipid film. Next, a buffer solution containing stem cells was added, and the mixture was sonicated at 200W for 10 min to form nanoliposomes. Then, polylactic acid-glycolic acid copolymer was dissolved in dichloromethane to prepare a solution with a concentration of 100 mg / mL. Finally, an aqueous solution containing expanded stem cells (after stem cell isolation and culture) was added, and the mixture was emulsified at 1000 rpm using an emulsification-solvent evaporation method for 1 h to prepare nanopolymer particles.

[0142] The screened nucleic acid aptamers were reacted with nanoliposomes and nanopolymer particles at a mass ratio of 15:1 overnight at 6°C to attach them to the surface of the nanoliposomes and nanopolymer particles. Subsequently, the temperature-responsive material poly-N-isopropylacrylamide was grafted onto the nanocarrier with the targeted molecule modification at 55°C for 4 hours to obtain a nanocarrier with responsive release function.

[0143] SCF and PDGF were weighed, and SCF was prepared into a solution with a concentration of 15 ng / mL and PDGF was prepared into a solution with a concentration of 7.5 ng / mL. The solution was added to a buffer solution and mixed with gelatin microspheres at a volume ratio of 1:2.5. The gelatin microspheres were cured through a cross-linking reaction to form a sustained-release system. The system was tested at 37°C to simulate a physiological environment to ensure that the regulatory molecules could be slowly released within 8 days.

[0144] The cultured and expanded stem cells were mixed with homing guidance reagent at a volume ratio of 1:7.5 and incubated for 45 minutes. Then, the incubated stem cells were mixed with a responsive release nanocarrier and a sustained-release system at a volume ratio of 3:1:1. Stem cell transplantation was performed by intraosseous injection of 5-10 mL into the patient. Post-transplantation, routine blood tests were conducted weekly, and bone marrow aspiration was performed every two weeks. Liver and kidney function, as well as immune function indicators, were monitored. Patients were given prophylactic antibiotics and antiviral drugs for three weeks, and nutritional support was provided based on their condition to ensure adequate intake of protein, vitamins, and other nutrients.

[0145] In the homing guidance reagent preparation stage, SELEX technology is used to screen nucleic acid aptamers and connect them to cell adhesion-promoting peptides and stem cell growth factors, precisely guiding stem cells to hom and promoting their differentiation. During the preparation of targeted carrier materials, nanoliposomes and nanopolymer particles are targeted and functionally responsively endowed, precisely delivering and engrafting stem cells. In the preparation of the microenvironment regulation solution, key regulatory molecules are screened and a sustained-release system is constructed to create a suitable environment for stem cell survival. By screening and modifying specific nucleic acid aptamers, preparing targeted carrier materials, and developing a microenvironment regulation solution, the efficacy of stem cell therapy for thalassemia is effectively improved, achieving a good therapeutic effect for thalassemia.

[0146] In one implementation, after S107, the thalassemia treatment method based on stem cell technology provided in this application embodiment further includes:

[0147] After transplantation, a complete blood count is performed weekly to observe the recovery of blood cell count and morphology, assess the colonization and differentiation of stem cells, and monitor liver and kidney function and immune function indicators. During this period, patients are given prophylactic antibiotics and antiviral drugs, and nutritional support is provided according to their physical condition to ensure that they consume sufficient protein, vitamins and other nutrients.

[0148] This application also provides a homing guide reagent, comprising:

[0149] Nucleic acid aptamers are constructed based on vascular cell adhesion molecule-1 (VCAM-1) and stromal cell-derived factor-1 (SDF-1) in the bone marrow microenvironment.

[0150] Cell adhesion-promoting peptide (RGD peptide), RGD peptide linked to nucleic acid aptamers.

[0151] Stem cell growth factor (SCF) is a protein that links a nucleic acid aptamer to an RGD peptide.

[0152] The combined action of RGD peptide and SCF can significantly enhance the directional migration ability of hematopoietic stem cells and the homing efficiency of the bone marrow microenvironment.

[0153] This application also provides a nanocarrier, comprising:

[0154] Nanoliposomes.

[0155] Nanopolymer particles.

[0156] Nucleic acid aptamers connect the surfaces of nanoliposomes and nanopolymer particles.

[0157] Temperature-responsive materials, and the surface of temperature-responsive materials grafted onto nanoliposomes and nanopolymer particles.

[0158] Nanocarriers significantly improve the precision and safety of treatment through the synergistic effect of multiple functions.

[0159] This application also provides a sustained-release system, including:

[0160] Stem cell growth factor (SCF).

[0161] Platelet-derived growth factor (PDGF).

[0162] Gelatin microspheres, gelatin microspheres mixed with stem cell growth factor (SCF) and PDGF.

[0163] By loading gelatin microspheres with a dual-factor combination of SCF and PDGF, key regulatory molecules were screened and a sustained-release system was constructed, overcoming the limitations of traditional single-factor sustained-release technology and creating a suitable environment for stem cell survival.

[0164] This application also provides a stem cell therapy composition, comprising:

[0165] Homing guide reagent.

[0166] Nanocarriers.

[0167] Sustained-release system.

[0168] Isolated and cultured stem cells.

[0169] By integrating homing agents, nanocarriers, and sustained-release systems, this method addresses the pain points of low homing rates and insufficient microenvironment support in traditional stem cell transplantation. The stem cell therapy composition, administered via intramedullary injection, directly targets the lesion, achieving targeted stem cell homing, microenvironment remodeling, and long-term functional maintenance, while reducing systemic side effects.

[0170] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them.

Claims

1. A treatment method for thalassemia based on stem cell technology, characterized in that, include: Peripheral blood samples were collected from the patient for stem cell isolation and culture. Nucleic acid aptamers were constructed by combining vascular cell adhesion molecule-1 (VCAM-1) and stromal cell-derived factor-1 (SDF-1) in the bone marrow microenvironment. By connecting the nucleic acid aptamer, the cell adhesion-promoting peptide (RGD peptide), and the stem cell growth factor (SCF), a homing guidance reagent is constructed. Preparation of nanoliposomes and nanopolymer particles; The nucleic acid aptamer is attached to the surface of the nanoliposomes and the nanopolymer particles, and then grafted with a temperature-responsive material to obtain a nanocarrier with responsive release function. The stem cell growth factor (SCF) and platelet-derived growth factor (PDGF) are mixed with gelatin microspheres to form a sustained-release system; The cultured stem cells are mixed with the homing guidance reagent, the nanocarrier, and the sustained-release system, and then transplanted into the patient via intramedullary injection.

2. The method according to claim 1, characterized in that, The nucleic acid aptamer was constructed by combining the vascular cell adhesion molecule-1 (VCAM-1) and the stromal cell-derived factor-1 (SDF-1) in the bone marrow microenvironment, comprising: Constructing a single-stranded DNA random library; The single-stranded DNA random library was incubated with the vascular cell adhesion molecule-1 (VCAM-1) and the stromal cell-derived factor-1 (SDF-1) in the bone marrow microenvironment to screen for nucleic acid sequences that specifically bind to the target molecules; The nucleic acid sequence is cyclically screened, eluted, and amplified to obtain the nucleic acid aptamer that specifically recognizes the vascular cell adhesion molecule-1 (VCAM-1) and the stromal cell-derived factor-1 (SDF-1).

3. The method according to claim 2, characterized in that, The single-stranded DNA random library includes 10^14-10^15 different sequences, and the length of the single-stranded DNA random library is 30-80 nucleotides.

4. The method according to claim 2, characterized in that, The incubation temperature is 37℃, and the incubation time is 1-2 hours.

5. The method according to claim 1, characterized in that, The homing guidance reagent is constructed by connecting the nucleic acid aptamer, the cell adhesion-promoting peptide (RGD peptide), and the stem cell growth factor (SCF), comprising: The nucleic acid aptamers were purified using high-performance liquid chromatography (HPLC). The cell adhesion-promoting peptide (RGD peptide) was reacted with the purified nucleic acid aptamer at room temperature using a chemical coupling method. The stem cell growth factor (SCF) is connected to the nucleic acid aptamer after the reaction to form the homing guide reagent.

6. The method according to claim 5, characterized in that, The molar ratio of the nucleic acid aptamer to the cell adhesion-promoting peptide (RGD peptide) is 1:3-5, and the reaction time between the nucleic acid aptamer and the cell adhesion-promoting peptide (RGD peptide) is 2.5-3.5 hours.

7. The method according to claim 5, characterized in that, The molar ratio of the stem cell growth factor (SCF) to the nucleic acid aptamer is 1:1.5-2.0, and the reaction time between the stem cell growth factor (SCF) and the nucleic acid aptamer is 3.5-4.5 h.

8. The method according to claim 1, characterized in that, The nucleic acid aptamer is attached to the surface of the nanoliposomes and the nanopolymer particles, and then grafted with the temperature-responsive material to obtain the nanocarrier with responsive release function, comprising: The nucleic acid aptamer was covalently linked to the surface of the nanoliposomes and the nanopolymer particles, and the reaction was carried out overnight to obtain the nanocarrier with targeted molecule modification. By grafting a temperature-responsive material onto the nanocarrier that has undergone targeted molecular modification, a nanocarrier with both targeted molecular modification and temperature-responsive release functions is obtained.

9. The method according to claim 8, characterized in that, The mass ratio of the nanoliposomes and nanopolymer particles to the nucleic acid aptamer is 15-17:1, and the reaction temperature of the nanoliposomes and nanopolymer particles with the nucleic acid aptamer is 4-6℃.

10. The method according to claim 8, characterized in that, The grafting reaction temperature is 55-58℃, and the grafting reaction time is 4-6 hours.

11. The method according to claim 1, characterized in that, The stem cell growth factor (SCF) and the platelet-derived growth factor (PDGF) are mixed with the gelatin microspheres to form the sustained-release system, comprising: The stem cell growth factor (SCF) and the platelet-derived growth factor (PDGF) were respectively formulated into stem cell growth factor solution and platelet-derived growth factor solution; The stem cell growth factor solution and the platelet-derived growth factor solution were respectively added to a buffer solution to obtain the regulated buffer solution; The regulated buffer solution is mixed with the gelatin microspheres, and the gelatin microspheres are cured through a cross-linking reaction to form the sustained-release system.

12. The method according to claim 11, characterized in that, The concentration of the stem cell growth factor solution is 15 ng / mL-17 ng / mL, and the concentration of the platelet-derived growth factor solution is 7.5 ng / mL-9.0 ng / mL.

13. The method according to claim 11, characterized in that, The volume ratio of the adjusted buffer solution to the gelatin microspheres is 1:2.5-3.

3.

14. The method according to claim 1, characterized in that, The cultured stem cells are mixed with the homing guidance reagent, the nanocarrier, and the sustained-release system, and then transplanted into the patient via intraosseous injection, including: The cultured stem cells were mixed with the homing guidance reagent and incubated. The incubated stem cells, the nanocarrier, and the sustained-release system are mixed to form a mixed product; The mixture was transplanted into the patient via intraosseous injection.

15. The method according to claim 14, characterized in that, The volume ratio of the cultured stem cells to the homing guide reagent is 1:7.5-8.5, and the incubation time is 45-55 minutes.

16. The method according to claim 14, characterized in that, The volume ratio of the incubated stem cells, the nanocarrier, and the sustained-release system is 3-4:1:

1.

17. A homing guidance reagent, characterized in that, include: Nucleic acid aptamers, which are constructed based on the vascular cell adhesion molecule-1 (VCAM-1) and the stromal cell-derived factor-1 (SDF-1) in the bone marrow microenvironment; Cell adhesion-promoting peptide (RGD peptide), wherein the RGD peptide is linked to the nucleic acid aptamer; Stem cell growth factor (SCF), wherein the SCF links the nucleic acid aptamer and the RGD peptide.

18. A nanocarrier, characterized in that, include: Nanoliposomes; Nanopolymer particles; Nucleic acid aptamers, wherein the nucleic acid aptamers connect the surfaces of the nanoliposomes and the nanopolymer particles; A temperature-responsive material, wherein the temperature-responsive material is grafted onto the surface of the nanoliposomes and the nanopolymer particles.

19. A sustained-release system, characterized in that, include: Stem cell growth factor (SCF); Platelet-derived growth factor (PDGF); Gelatin microspheres, wherein the gelatin microspheres are mixed with the stem cell growth factor (SCF) and the PDGF.

20. A stem cell therapy composition, characterized in that, include: The homing guidance reagent as described in claim 17; The nanocarrier as described in claim 18; The sustained-release system as described in claim 19; Isolated and cultured stem cells.

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