Preparation process of pH-responsive biomimetic nanodrug

Abstract

The application relates to the fields of biological medicine and nanomaterial technology, and discloses a preparation process of a pH response type biomimetic nanodrug, which comprises synthesizing pH response nanoparticles with uniform particle size in a microfluidic chip, and co-printing the nanoparticles with biological ink into a personalized support or patch through an integrated platform. The process realizes in-situ mixing of the nanoparticles, spatial gradient drug loading and precise construction of a biomimetic structure, and through the microfluidic-3D biological printing integrated platform, a pH response type biomimetic nanodrug with the following characteristics is prepared: highly uniform particle size, precisely controllable drug release, personalized structure adaptation, excellent biocompatibility and enhanced immune escape ability. The obtained product can release drugs in a controllable manner in an acidic microenvironment, and has the functions of tissue repair and local treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and nanomaterials technology, specifically a pH-responsive biomimetic nanomedicine preparation process. Background Technology

[0002] pH-responsive nanomedicine delivery systems have become a research hotspot in the biomedical field in recent years due to their ability to achieve targeted drug release in the slightly acidic environment of lesions such as tumors. Among the existing technologies, various pH-responsive systems based on polymers, mesoporous materials, or inorganic nanoparticles have been developed, but there is still room for improvement in terms of the controllability of the preparation process, batch repeatability, and biomimetic functional integration.

[0003] Patent CN114672031B discloses a pH-responsive polymer nanomedicine and its preparation method. It uses a modified lignin-grafted polymer as a carrier, and by improving the solubility of lignin in organic solvents and the solubility of the grafted polymer in water, an amphiphilic structure is constructed to achieve nano-self-assembly. While this method exhibits good biocompatibility and pH responsiveness, its preparation relies on multiple chemical modification and solvent replacement processes, resulting in a lengthy process. Furthermore, it lacks biomimetic interface design, making it difficult to effectively simulate the immune escape and long-circulation characteristics of natural cell membranes, thus limiting its in vivo delivery efficiency and targeting precision.

[0004] Patent CN113181137B discloses a pH-responsive nanoparticle-mediated RNA nanomedicine and its preparation method. The nanoparticles are obtained by mixing a circRNA overexpression vector with cross-linked PAMAM and PEG-b-PEICA followed by dialysis and ultrafiltration. This technology achieves targeted delivery to tumor cells and inhibits tumor growth, exhibiting good biocompatibility. However, the preparation process mainly relies on traditional separation methods such as dialysis and ultrafiltration. The particle size distribution is greatly affected by operating conditions, making batch-to-batch consistency control difficult. Furthermore, the system lacks an integrated biomimetic membrane structure and therefore lacks effective ability to circumvent complex physiological barriers (such as the mononuclear phagocytic system), limiting its potential for long-term circulation and deep tumor penetration in vivo.

[0005] While the aforementioned existing technologies have made some progress in pH-responsive drug release, further optimization is needed in terms of standardization of preparation processes, control of particle size uniformity, and integration of biomimetic functions. This invention proposes a pH-responsive biomimetic nanomedicine preparation process, aiming to achieve continuous and highly uniform formulation production through a combination of microfluidics and 3D printing technologies. Furthermore, by incorporating a cell membrane biomimetic encapsulation strategy, it endows the nanomedicine with excellent immune escape capabilities and targeted enrichment properties, making it particularly suitable for personalized medical scenarios such as local treatment of complex wounds or post-tumor surgery. Summary of the Invention

[0006] This invention provides a pH-responsive biomimetic nanomedicine preparation process. By constructing a microfluidic-3D bioprinting integrated platform, pH-responsive nanoparticles continuously synthesized in a microfluidic chip are directly mixed with bio-ink, and personalized scaffolds or patches are co-printed based on a three-dimensional model of a patient's lesion, realizing the integrated manufacturing of local drug delivery and tissue repair functions.

[0007] This invention provides a pH-responsive biomimetic nanomedicine preparation process, comprising the following steps: S10: In a microfluidic chip, an organic phase solution containing a pH-responsive polymer and an aqueous phase solution containing a drug active ingredient are injected into two inlet channels of the chip, respectively, to form a stable liquid-liquid interface in the intersection region of a T-shaped or flow focusing structure. By adjusting the flow rate ratio of the two phases, the nucleation and growth process of nanoparticles is controlled, and a pH-responsive nanoparticle suspension with a particle size distribution coefficient of less than 0.15 is obtained. S20: The pH-responsive nanoparticle suspension is directly introduced into the nanoparticle storage hopper of the 3D bioprinter through a sterile connection tube at the outlet end of the microfluidic chip. The storage hopper has a built-in magnetic stirring device to maintain the uniform dispersion of the nanoparticles. S30: The bio-ink raw material and the cross-linking precursor solution are loaded separately into the independent printhead of the 3D bioprinter. The bio-ink contains natural polymer materials, extracellular matrix components and optional live cell suspension. The cross-linking precursor solution is one of calcium ions, photoinitiators or enzyme cross-linking agents. S40: Reconstruct a three-dimensional digital model of the patient's lesion area based on medical imaging data, set the geometric configuration, porosity, layer thickness and nanoparticle loading density parameters of the stent or patch, and generate G-code control instructions; S50: Start the 3D bioprinter and simultaneously extrude the pH-responsive nanoparticle suspension in the nanoparticle storage tank and the bio-ink in the bio-ink printhead. During the deposition process, the nanoparticles and bio-ink are mixed in situ through a coaxial nozzle or a parallel multi-nozzle structure. After each printing layer is deposited, cross-linking conditions are applied immediately to solidify the bio-ink and ultimately construct a personalized scaffold or patch with spatial gradient drug distribution and biomimetic microstructure.

[0008] According to the present invention, the microfluidic chip is made of polydimethylsiloxane or thermoplastic polymer material, with an internal channel width of 50-300 μm and a depth of 30-200 μm. The confluence region is designed as a flow focusing structure. The flow rate of the organic phase is controlled at 10-100 μL / min, the flow rate of the aqueous phase is controlled at 50-500 μL / min, and the flow rate ratio of the two phases is maintained between 1:2 and 1:10, so as to regulate the nanoparticle size in the range of 80-250 nm.

[0009] In step S10, the pH-responsive polymer is selected from at least one of poly(β-amino ester), poly(L-histidine), poly(acrylic acid) grafted chitosan, or poly(N-isopropylacrylamide-co-methacrylic acid), and its concentration in the organic phase is 1-10 mg / mL.

[0010] In step S10, the organic solvent is at least one of ethanol, acetone, acetonitrile or tetrahydrofuran, and the aqueous phase is deionized water, phosphate buffer or aqueous solution containing surfactant, wherein the surfactant is poloxamer 188, Tween 80 or lecithin, and the concentration is 0.01-0.5wt%.

[0011] In step S10, the active pharmaceutical ingredient is doxorubicin, paclitaxel, gemcitabine, siRNA, miRNA, or a protein-based therapeutic agent, and its initial concentration in the aqueous phase is 0.1-5 mg / mL.

[0012] In step S20, the outlet end of the microfluidic chip is connected to the nanoparticle storage hopper of the 3D bioprinter through a polytetrafluoroethylene (PTFE) tube. The inner diameter of the tube is 0.2-1.0 mm, the length is less than 50 cm, the temperature of the storage hopper is maintained at 4-10℃, and the magnetic stirring speed is 200-800 rpm.

[0013] In step S30, the natural polymer material in the bio-ink is sodium alginate, gelatin, hyaluronic acid, collagen, fibroin, or decellularized matrix hydrogel, with a concentration of 2-15 wt%; the extracellular matrix component is laminin, fibronectin, or chondroitin sulfate, with an addition amount of 0.1-2 mg / mL; and the live cell suspension is mesenchymal stem cells, fibroblasts, or keratinocytes, with a cell density of 1×10⁻⁶ cells / mL. 6 -5×10 7 per mL.

[0014] In step S30, the crosslinking precursor solution is a calcium chloride solution with a concentration of 50-200 mmol / L; or an Irgacure2959 photoinitiator solution with a concentration of 0.05-0.5 wt%; or a transglutaminase solution with a concentration of 1-10 U / mL.

[0015] In step S40, the medical imaging data comes from CT, MRI or optical coherence tomography, the three-dimensional reconstruction software uses Mimics, 3DSlicer or Simpleware, the scaffold porosity is set to 60%-90%, the layer thickness is 100-500μm, and the nanoparticle loading density is controlled at 0.5%-10% by volume.

[0016] In step S50, the 3D bioprinter uses a pneumatic drive or screw extrusion system with a printing pressure of 10-100 kPa and a printing speed of 1-10 mm / s; the inner diameter of the coaxial nozzle is 100-400 μm and the outer diameter is 200-600 μm; the spacing between the parallel multi-nozzle is 0.5-2 mm.

[0017] In step S50, the crosslinking conditions are as follows: if calcium ion crosslinking is used, an atomizing nozzle is set below the printing platform to spray a 50-200 mmol / L calcium chloride solution in real time; if photocrosslinking is used, a 365 nm LED light source is integrated next to the print head, with a light intensity of 5-20 mW / cm². 2 Exposure time is 1-10 seconds per layer; if enzyme crosslinking is used, the crosslinking precursor solution and bio-ink are premixed in a Y-type mixer and printed immediately.

[0018] This invention provides a pH-responsive biomimetic nanomedicine composite scaffold or patch, which is prepared by the process described in any embodiment of the first aspect. It comprises a three-dimensional porous bio-ink matrix and pH-responsive nanoparticles uniformly dispersed in the matrix. The nanoparticles remain stable at pH 7.4 and swell or dissociate at pH 5.0-6.5 to release the encapsulated drug.

[0019] The pH-responsive nanoparticles are further coated with a cell membrane biomimetic coating. The cell membrane is derived from red blood cells, cancer cells, or macrophages, extracted by differential centrifugation and hypotonic lysis, and then extruded or sonicated to form vesicles. The vesicles are then incubated with the nanoparticles at 4°C for 12-24 hours to complete the coating.

[0020] The encapsulation process of the cell membrane biomimetic coating is completed in the membrane fusion module downstream of the microfluidic chip. This module contains a serpentine mixing channel with a length of 10-50 cm, a width of 100-300 μm, and a flow rate of 20-200 μL / min, ensuring full contact between the membrane vesicles and nanoparticles.

[0021] The 3D bioprinter is equipped with an online monitoring system, including a high-speed camera and a laser particle size analyzer, to monitor the dispersion state of the nanoparticle suspension and the printing linewidth in real time, and to provide feedback to adjust the printing parameters.

[0022] A buffer storage unit with a capacity of 0.5-5 mL is set between the microfluidic chip and the 3D bioprinter to balance flow fluctuations and ensure the stability of continuous feeding.

[0023] The bio-ink is degassed before printing, with a vacuum of -80 to -100 kPa and a processing time of 5 to 15 minutes, to eliminate the impact of air bubbles on printing accuracy.

[0024] After printing, the personalized scaffolds or patches are post-treated in a 37°C, 5% CO2 incubator for 1-24 hours to promote cell adhesion and matrix remodeling.

[0025] The pH-responsive polymer is a poly(β-amino ester), which is synthesized by Michael addition reaction of 1,4-butanediol diacrylate and 4-aminobutanol. The weight-average molecular weight is 5,000-20,000 Daltons, and the content of tertiary amine groups is 0.8-2.5 mmol per gram of polymer.

[0026] The microfluidic chip is manufactured using soft photolithography. The master mold is formed by spin-coating SU-8 photoresist onto a silicon wafer and exposing it to ultraviolet light. Polydimethylsiloxane prepolymer and curing agent are mixed at a mass ratio of 10:1 and then poured. The mixture is cured at 60-80℃ for 2-4 hours. After peeling, holes are drilled and plasma-bonded to a glass substrate.

[0027] The motion control system of the 3D bioprinter uses a stepper motor or a servo motor, with a positioning accuracy of ±5μm and a Z-axis stacking error of less than 10μm.

[0028] The nanoparticle storage silo is equipped with a liquid level sensor and a temperature controller. When the liquid level is lower than the set threshold, the microfluidic chip replenishment program is automatically triggered, and the temperature fluctuation is controlled within ±0.5℃.

[0029] The mixing ratio of the bio-ink to the nanoparticle suspension is precisely controlled by a flow meter, with a mixing ratio of 1:0.01 to 1:0.5 by volume, ensuring that the drug loading is within the therapeutic window.

[0030] The outer surface of the support or patch may be further coated with a pH-responsive film, which is formed by the self-assembly of chitosan and sodium alginate layer by layer, with 3-10 layers and a thickness of 20-100 nm for each layer.

[0031] The entire process is carried out in an ISO 5 clean environment, and all liquid delivery pipelines use disposable sterile consumables to avoid cross-contamination.

[0032] The microfluidic chip integrates an online filtration unit at its outlet with a pore size of 0.22 μm to remove aggregated particles and ensure the monodispersity of nanoparticles entering the 3D printing system.

[0033] The surface of the 3D printing platform is coated with poly-L-lysine or fibronectin to enhance the adhesion of the initial printing layer and prevent structural collapse.

[0034] The personalized scaffold or patch is suitable for post-breast cancer surgery wounds, diabetic foot ulcers, or burn wounds. Its shape perfectly matches the lesion contour, and the drug release kinetics and tissue regeneration rate are synergistically regulated.

[0035] Fluorescent markers such as DiR or FITC are introduced during the preparation of the pH-responsive nanoparticles to facilitate subsequent in vivo tracking and distribution evaluation.

[0036] The survival rate of live cells in the bio-ink after printing is higher than 85%, as verified by Calcein-AM / PI double staining.

[0037] The entire integrated platform is centrally controlled and managed by a central controller. Microfluidic flow rate, printing path, crosslinking timing, and environmental parameters are all controlled by LabVIEW or Python scripts, achieving fully automated operation.

[0038] Compared with the prior art, the beneficial effects of the present invention are: The pH-responsive biomimetic nanomedicine composite scaffold or patch of the present invention is prepared by the process described in any embodiment of the first aspect, and therefore possesses the technical characteristics of highly uniform particle size, precise and controllable drug release, personalized structural adaptation, excellent biocompatibility, and enhanced immune escape ability. Detailed Implementation

[0039] This invention provides a pH-responsive biomimetic nanomedicine preparation process. The microfluidic-3D bioprinting integrated platform on which the preparation process is based mainly includes a microfluidic chip, sterile connecting tubing, buffer storage unit, nanoparticle storage tank, 3D bioprinter body, multi-nozzle printhead assembly, crosslinking execution module, online monitoring system, central controller, and printing platform.

[0040] The microfluidic chip is equipped with an organic phase inlet, an aqueous phase inlet, and an outlet. The outlet is connected to the buffer storage unit and the nanoparticle storage bin in sequence through a sterile connecting pipe. The 3D bioprinter body is equipped with an independently controlled multi-nozzle printhead assembly, including a first printhead for extruding bio-ink, a second printhead for extruding cross-linking precursor solution, and a third printhead for delivering nanoparticle suspension; The cross-linking execution module is configured as an atomizing nozzle, an LED light source, or a Y-type mixer, depending on the cross-linking type. The online monitoring system consists of a high-speed camera and a laser particle size analyzer, which collects fluid state and structural parameters in real time during the printing process; The central controller is connected to the flow control pump of the microfluidic chip, the motion system of the 3D bioprinter body, the cross-linking execution module and the online monitoring system via data cables to achieve fully automated coordination of the entire process.

[0041] The technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples, so as to ensure that those skilled in the art can fully understand and implement the present invention.

[0042] Example 1: The pH-responsive polymer was polyβ-amino ester (5 mg / mL); the microfluidic organic phase flow rate was 50 μL / min, and the aqueous phase flow rate was 250 μL / min; the scaffold porosity was 75%, the layer thickness was 300 μm; the nanoparticle loading density was 5%; and calcium ion crosslinking was used (100 mmol / L). Preparation process: Microfluidic synthesis of pH-responsive nanoparticles → sterile delivery to 3D printer → loading of bio-ink and cross-linking agent → 3D model reconstruction → simultaneous printing and mixing → cross-linking and curing → finished product.

[0043] Example 2: The pH-responsive polymer was poly-L-histidine (5 mg / mL), and the rest of the formulation and process were the same as in Example 1; Preparation process: Same as in Example 1 (polymer substitution).

[0044] Example 3: Organic phase flow rate 10 μL / min, aqueous phase flow rate 20 μL / min (flow rate ratio 1:2), the rest of the formulation and process are the same as in Example 1; Preparation process: Same as in Example 1 (flow rate parameter adjusted).

[0045] Example 4: Organic phase flow rate 100 μL / min, aqueous phase flow rate 1000 μL / min (flow rate ratio 1:10), other formulations and processes are the same as in Example 1; Preparation process: Same as in Example 1 (flow rate parameter adjusted).

[0046] Example 5: Nanoparticle loading density 0.5%, other formulations and processes are the same as in Example 1; Preparation process: Same as in Example 1 (load density adjusted).

[0047] Example 6: Nanoparticle loading density 10%, other formulations and processes are the same as in Example 1; Preparation process: Same as in Example 1 (load density adjusted).

[0048] Example 7: Crosslinking method is photocrosslinking (365 nm LED, 10 mW / cm²). 2 The remaining formulas and processes are the same as in Example 1; Preparation process: Same as in Example 1 (with crosslinking method adjusted).

[0049] Example 8: The surface of the nanoparticles is coated with red blood cell membranes, and the rest of the formulation and process are the same as in Example 1; Preparation process: Same as in Example 1 (with the addition of a cell membrane coating step).

[0050] Comparative Example 1: Nanoparticles were prepared using the conventional dialysis method; the scaffold was manually cast; the rest of the formulation was the same as in Example 1; Preparation process: Synthesize nanoparticles by dialysis → mix with bio-ink → manually cast → cross-link and cure → finished product.

[0051] Comparative Example 2: The polymer was ordinary polyethylene glycol (without pH response); the rest of the process was the same as in Example 1; Preparation process: Same as in Example 1 (the polymer is replaced with a non-responsive polymer).

[0052] Test method: Nanoparticle and drug testing: dynamic light scattering method for particle size and distribution coefficient; high performance liquid chromatography for drug encapsulation and release rate; evaluation of pH response specificity.

[0053] Scaffold performance testing: compressive modulus was measured using a mechanical testing machine; pore structure uniformity was observed using scanning electron microscopy; cell viability was measured using the MTT assay.

[0054] Bionic performance testing: Detecting the immune escape ability after cell membrane coating; verifying the in vivo targeted enrichment effect.

[0055] The test data comparisons are shown in Table 1 and Table 2.

[0056] Table 1. Comparison of nanoparticle size, distribution coefficient, drug encapsulation efficiency, and 24-hour release rate at pH 7.4. Test Project Nanoparticle size (nm) Distribution coefficient Drug encapsulation rate (%) pH 7.4 24h release rate (%) Example 1 160 0.12 85 12 Example 2 150 0.11 83 10 Example 3 220 0.14 81 13 Example 4 90 0.1 87 11 Example 5 160 0.12 84 10 Example 6 170 0.13 82 14 Example 7 155 0.11 86 12 Example 8 180 0.13 83 9 Comparative Example 1 280 0.25 65 25 Comparative Example 2 150 0.12 80 45 Table 2 Comparison of release rate, cell viability, and compressive modulus at pH 6.0 for 24 hours Test Project Release rate (%) at pH 6.0 over 24 hours Cell viability (%) Compression modulus (kPa) Example 1 65 88 25 Example 2 68 87 23 Example 3 62 86 27 Example 4 70 89 22 Example 5 60 90 24 Example 6 66 85 26 Example 7 67 88 28 Example 8 63 91 25 Comparative Example 1 50 75 15 Comparative Example 2 52 86 24 Examples 1-8 showed a nanoparticle distribution coefficient ≤0.14 and significant differences in pH-responsive release, far superior to the comparative examples. Comparative example 1 showed uneven particle size and low encapsulation efficiency due to traditional processes, while comparative example 2 showed no pH-responsive characteristics. This demonstrates that microfluidics + 3D printing + pH-responsive polymers are key to efficient and controllable preparation.

[0057] Polymers with different pH responses are all compatible (Examples 1-2); the microfluidic flow rate ratio is increased (Examples 3→1→4), the nanoparticle size is reduced and the distribution is more uniform; the release controllability can be guaranteed in the range of 0.5%-10% loading density; cell membrane coating (Example 8) improves biocompatibility and immune escape ability.

[0058] The embodiments achieve uniform nanoparticle size and high drug encapsulation efficiency; precise pH response, stable under physiological conditions, and efficient drug release in acidic lesion environments; 3D printing to construct personalized biomimetic structures that fit the lesion contour; excellent biocompatibility with cell survival rate ≥85%; and good batch stability of the integrated process, making it suitable for industrialization.

[0059] Compared to the traditional dialysis method (Comparative Example 1), the encapsulation efficiency of the example was improved by 31%, and the pH response release difference was improved by 26%; compared to the non-pH-responsive system (Comparative Example 2), the pH-selective release difference of the example was improved by 27%, solving the industry problem of uneven particle size and uncontrolled release of traditional nanomedicines.

[0060] In summary, the process described in this invention, through integrated preparation and pH-responsive design, can achieve efficient and controllable preparation of biomimetic nanomedicines with different parameter combinations, making it suitable for personalized medical scenarios such as local tumor treatment and wound repair.

[0061] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pH-responsive biomimetic nanomedicine preparation process, characterized in that, Includes the following steps: S10: In a microfluidic chip, an organic phase solution containing a pH-responsive polymer and an aqueous phase solution containing a drug active ingredient are injected into two inlet channels of the chip, respectively, to form a stable liquid-liquid interface in the intersection region of a T-shaped or flow focusing structure. By adjusting the flow rate ratio of the two phases, the nucleation and growth process of nanoparticles is controlled, and a pH-responsive nanoparticle suspension with a particle size distribution coefficient of less than 0.15 is obtained. S20: The pH-responsive nanoparticle suspension is directly introduced into the nanoparticle storage hopper of the 3D bioprinter through a sterile connection tube at the outlet end of the microfluidic chip. The storage hopper has a built-in magnetic stirring device to maintain the uniform dispersion of the nanoparticles. S30: The bio-ink raw material and the cross-linking precursor solution are loaded separately into the individual printheads of the 3D bioprinter; S40: Reconstruct a three-dimensional digital model of the patient's lesion area based on medical imaging data, set the geometric configuration, porosity, layer thickness and nanoparticle loading density parameters of the stent or patch, and generate G-code control instructions; S50: Start the 3D bioprinter and simultaneously extrude the pH-responsive nanoparticle suspension in the nanoparticle storage tank and the bio-ink in the bio-ink printhead. During the deposition process, the nanoparticles and bio-ink are mixed in situ through a coaxial nozzle or a parallel multi-nozzle structure. After each printing layer is deposited, cross-linking conditions are applied immediately to solidify the bio-ink and ultimately construct a personalized scaffold or patch with spatial gradient drug distribution and biomimetic microstructure.

2. The pH-responsive biomimetic nanomedicine preparation process according to claim 1, characterized in that, The bio-ink contains natural polymer materials, extracellular matrix components, and optional live cell suspensions.

3. The pH-responsive biomimetic nanomedicine preparation process according to claim 1, characterized in that, The crosslinking precursor solution is one of calcium ions, photoinitiators, or enzyme-based crosslinking agents.

4. The pH-responsive biomimetic nanomedicine preparation process according to claim 1, characterized in that, In step S10, the microfluidic chip is made of polydimethylsiloxane or thermoplastic polymer material, with an internal channel width of 50-300 μm and a depth of 30-200 μm. The confluence region is a flow focusing structure, the organic phase flow rate is 10-100 μL / min, the aqueous phase flow rate is 50-500 μL / min, and the two-phase flow rate ratio is 1:2 to 1:

10.

5. The pH-responsive biomimetic nanomedicine preparation process according to claim 1, characterized in that, In step S10, the pH-responsive polymer is selected from at least one of poly(β-amino ester), poly(L-histidine), poly(acrylic acid) grafted chitosan, or poly(N-isopropylacrylamide-co-methacrylic acid), and its concentration in the organic phase is 1-10 mg / mL.

6. The pH-responsive biomimetic nanomedicine preparation process according to claim 1, characterized in that, In step S30, the natural polymer material in the bio-ink is sodium alginate, gelatin, hyaluronic acid, collagen, fibroin, or decellularized matrix hydrogel, with a concentration of 2-15 wt%; the extracellular matrix component is laminin, fibronectin, or chondroitin sulfate, with an addition amount of 0.1-2 mg / mL; and the live cell suspension is mesenchymal stem cells, fibroblasts, or keratinocytes, with a cell density of 1×10⁻⁶ cells / mL. 6 -5×10 7 per mL.

7. The pH-responsive biomimetic nanomedicine preparation process according to claim 1, characterized in that, In step S40, the porosity of the scaffold or patch is 60%-90%, the layer thickness is 100-500μm, and the nanoparticle loading density is 0.5%-10% by volume.

8. The pH-responsive biomimetic nanomedicine preparation process according to claim 1, characterized in that, In step S50, the crosslinking conditions are as follows: if calcium ions are used for crosslinking, an atomizing nozzle is set below the printing platform to spray 50-200 mmol / L calcium chloride solution in real time. If photocrosslinking is used, a 365nm LED light source is integrated next to the printhead, with a light intensity of 5-20mW / cm². 2 Exposure time is 1-10 seconds per layer; if enzyme crosslinking is used, the crosslinking precursor solution and bio-ink are premixed in a Y-type mixer and printed immediately.

9. The pH-responsive biomimetic nanomedicine preparation process according to claim 1, characterized in that, The pH-responsive nanoparticles are coated with a cell membrane biomimetic coating. The cell membrane is derived from red blood cells, cancer cells, or macrophages. The coating process is completed in a membrane fusion module downstream of the microfluidic chip. This module contains a serpentine mixing channel with a length of 10-50 cm, a width of 100-300 μm, and a flow rate of 20-200 μL / min.

10. A pH-responsive biomimetic nanomedicine composite scaffold or patch, characterized in that, The preparation of a pH-responsive biomimetic nanomedicine by the process described in any one of claims 1-9 comprises a three-dimensional porous bio-ink matrix and pH-responsive nanoparticles uniformly dispersed in the matrix, wherein the nanoparticles remain stable at pH 7.4 and swell or dissociate at pH 5.0-6.5.