Targeted delivery of biomimetic nanodrugs and preparation process thereof

Abstract

This invention belongs to the field of biomedical technology and discloses a targeted delivery biomimetic nanomedicine and its preparation process. The system includes a polylactic acid-glycolic acid copolymer drug-carrying core, a biomimetic cell membrane coating layer, and a pH-responsive polyethylene glycol-arginine block copolymer embedded therein. The latter is anchored to the outer leaflet by a hexadecyl group, maintaining a weakly negative surface charge at physiological pH to evade immune clearance. In the weakly acidic tumor microenvironment, it triggers arginine guanidine matrix protonation, converting the surface potential to a positive charge and enhancing electrostatic adsorption with tumor cells. The core of this invention lies in constructing a dynamic charge interface with spatiotemporal resolution. This interface achieves sequential activation of immune escape and cell adsorption functions at the system level, rather than a simple superposition. This technical approach not only improves the drug accumulation efficiency at the tumor site but also provides a universal platform for other nanomedical applications requiring microenvironment-responsive surface regulation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a targeted delivery biomimetic nanomedicine and its preparation process. Background Technology

[0002] Biomimetic nanomedicine delivery systems have become an important research direction in the field of anti-tumor therapy due to their excellent biocompatibility and potential targeting capabilities. These systems typically coat the surface of synthetic nanocarriers with natural cell membranes (such as erythrocyte membranes, cancer cell membranes, or leukocyte membranes) to mimic the biological characteristics of host cells, thereby achieving self-camouflage in vivo, evading immune surveillance, and prolonging blood circulation time. This can effectively alleviate the problem of traditional nanomedicines being rapidly cleared by mononuclear phagocytic systems due to their heterogeneity, improve drug retention in vivo, and lay the foundation for subsequent lesion enrichment.

[0003] Current biomimetic nanomedicine designs largely rely on physical coating processes of intact cell membranes, which are primarily determined by the natural composition of the donor cell membrane. Under physiological conditions, the surface of most mammalian cell membranes exhibits a slight negative charge. While this characteristic helps maintain the stability of intercellular interactions, it may create a mismatch with the charge state of vascular endothelial cells when used as drug carriers.

[0004] Immune effector cells such as macrophages can sense abnormal charges on foreign particles through surface pattern recognition receptors. Even if the carrier is encapsulated by a biomimetic membrane, if its overall zeta potential deviates from the physiological range, it can still easily trigger an opsonin-independent phagocytic clearance mechanism. Although the biomimetic strategy slows down the clearance rate to some extent, the inability of the membrane surface charge to dynamically respond to changes in the in vivo microenvironment makes it difficult for the drug to completely evade immune recognition during long-term circulation, thus limiting the effective accumulation of the drug in the target tissue.

[0005] Ideal targeted delivery to the tumor microenvironment requires not only escape but also active anchoring capabilities. Tumor tissue typically exhibits weak acidity (pH ≈ 6.5-6.8), high expression of proteases, and reducing substances; these conditions can serve as trigger signals for intelligent responses. Existing biomimetic nanocarriers generally lack charge response mechanisms to these microenvironment signals. Traditional biomimetic membrane structures are static systems; once their surface charge is formed, it remains fixed and cannot maintain a negative charge during blood circulation to avoid clearance, nor can it switch to a positive charge upon reaching the tumor region to enhance electrostatic adsorption with the negatively charged tumor cell membranes. Even if drugs successfully reach the vicinity of the tumor, they cannot act effectively and are difficult to internalize efficiently, resulting in limited therapeutic efficacy.

[0006] If an attempt is made to introduce cationic groups into the membrane surface to enhance cell adsorption by simply modifying it, a strong immune response is often triggered in the early stages of systemic drug administration, which in turn exacerbates the clearance rate of the carrier, creating a contradiction between enhancing adsorption and prolonging circulation. Summary of the Invention

[0007] To achieve the above-mentioned objectives, this invention provides a targeted delivery biomimetic nanomedicine and its preparation process. The biomimetic nanomedicine is prepared by coating a synthetic nanocore with a molecularly engineered biomimetic cell membrane, and embedding a charge-switching block copolymer that is responsive to the tumor microenvironment into the membrane structure. This allows the entire nanomedicine system to maintain a surface zeta potential in the range of -10mV to -3mV during the blood circulation phase to prevent immune clearance. Upon reaching the tumor tissue, the local weakly acidic environment triggers a conformational change in the block copolymer, causing the surface potential to switch to the range of +8mV to +15mV. This significantly enhances the electrostatic adsorption between the nanomedicine and the negatively charged tumor cell membrane, thereby improving the internalization efficiency.

[0008] The biomimetic nanomedicine of this invention comprises a nanomedicine core, a biomimetic cell membrane coating layer, and a block copolymer functional module. The nanomedicine core is composed of a biodegradable polymer material and is internally loaded with an antitumor active drug. The biomimetic cell membrane coating is derived from mammalian cells, selected from red blood cells, cancer cells or white blood cells, and purified cell membrane vesicles are obtained after hypotonic lysis, differential centrifugation and ultrasonic disruption. The block copolymer functional module is a polyethylene glycol-arginine block copolymer with the general chemical formula HO-(CH2CH2O). n -CO-NH-(CH2)4-CH(NH2)-CO-[NH-(CH2)4-CH(NH2)-CO] m -OH, where n is an integer between 45 and 65, and m is an integer between 3 and 8.

[0009] The polyethylene glycol-arginine block copolymer is immobilized on the outer leaf of the lipid bilayer of the biomimetic cell membrane via a hydrophobic anchoring group. This hydrophobic anchoring group is a hexadecyl chain, covalently linked to the terminal hydroxyl group of the polyethylene glycol segment via an amide bond, forming a hexadecyl-polyethylene glycol-arginine triblock structure. Under physiological pH 7.4, the guanidinium groups on the arginine side chain exist in a deprotonated form, exhibiting an overall electroneutrality to slightly negative charge. When the ambient pH drops below 6.8, the guanidinium groups protonate, and each arginine residue carries a positive charge, causing the entire block copolymer to transition from a hydrophilic-hydrophobic equilibrium state to a strongly hydrophilic positively charged state. This drives the copolymer to flip from the inside of the membrane to the outside, exposing it to the nanoparticle surface, thus achieving an active switching of surface charge.

[0010] In a preferred embodiment of the present invention, the nanomedicine core is prepared using a polylactic acid-glycolic acid copolymer with a weight-average molecular weight of 12,000 to 18,000 and a molar ratio of lactic acid to glycolic acid of 75:25. The core is prepared by a nanoprecipitation method, specifically through the following steps: Polylactic acid-glycolic acid copolymer and antitumor drugs were dissolved in acetone to form an organic phase. This organic phase was then added dropwise to an aqueous phase containing 0.5% polyvinyl alcohol. The mixture was emulsified at a high speed of 12,000 rpm for 3 minutes under ice bath conditions, followed by magnetic stirring at room temperature for 4 hours to evaporate the organic solvent. Finally, drug-loaded nanoparticles with a particle size of 80-120 nm and a polydispersity index of less than 0.15 were obtained.

[0011] The preparation process of the biomimetic cell membrane coating layer is as follows: Freshly collected donor cell suspension was washed three times with PBS (pH 7.4), and then 10 volumes of hypotonic lysis buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) were added. The cells were incubated at 4°C for 30 min to lyse them. The cell nuclei and unly ruptured cells were removed by centrifugation at 3000×g for 10 min. The supernatant was then collected by ultracentrifugation at 100000×g for 60 min. The resulting membrane precipitate was resuspended in PBS and sonicated with a probe (200W power, 2 seconds on, 3 seconds off, total duration 5 min) to form uniform membrane vesicles with an average particle size of 150-200 nm.

[0012] The integration of the block copolymer functional modules is achieved through a membrane fusion process. The drug-loaded nanoparticles prepared above are mixed with biomimetic cell membrane vesicles at a mass ratio of 1:2 and added to PBS buffer containing 10% sucrose, with a total volume of 1 mL. The mixture is placed in a freeze-thaw cycler and subjected to 5 freeze-thaw cycles (freezing at -80℃ for 10 min, thawing in a 37℃ water bath for 5 min). Subsequently, the sample is transferred to a micro extruder and passed through polycarbonate membranes with pore sizes of 400 nm, 200 nm, and 100 nm 11 times each to obtain biomimetic nanomedicines with a uniformly coated biomimetic membrane and directional distribution of block copolymers.

[0013] In another preferred embodiment of the present invention, the antitumor active drug is selected from doxorubicin, paclitaxel, cisplatin, or gemcitabine, and its drug loading in the nanomedicine core is 8% to 15% (w / w). The block copolymer has 5 arginine repeating units (m), a polyethylene glycol segment polymerization degree (n) of 55, and a hexadecyl anchor chain length of C16. This formulation has been experimentally verified to maximize charge reversal efficiency in the tumor region while ensuring long-term blood circulation.

[0014] The key control point of the preparation process described in this invention lies in the molar incorporation ratio of the block copolymer to the biomimetic membrane. The block copolymer accounts for 3% to 7% of the total membrane lipids by mass. If the ratio is less than 3%, the charge switching amplitude is insufficient, and it cannot effectively trigger the adsorption of tumor cells; if it is greater than 7%, complement activation is triggered by local positive charge exposure during the blood circulation stage, resulting in the rapid uptake of the carrier by liver Kupffer cells.

[0015] The technical solution of this invention constructs a dynamically responsive biointerface by precisely embedding charge-switchable block copolymers into a biomimetic membrane structure. This interface maintains negative charge properties in the blood environment, effectively mimicking the surface potential of host cells, thereby inhibiting opsonin-independent phagocytosis. In the tumor microenvironment, weakly acidic conditions trigger protonation of arginine residues, inducing conformational rearrangement of the block copolymers and their outward rotation to the membrane surface, forming a localized positive charge enrichment region. This enhances the electrostatic interaction with negatively charged groups (such as phosphatidylserine) at the phospholipid head of the tumor cell membrane. This process is independent of receptor-ligand recognition, thus applicable to various tumor types and overcoming the dependence of traditional active targeting strategies on specific surface markers.

[0016] The charge switching of the block copolymer exhibits high pH threshold specificity. In vitro Zeta potential titration experiments showed that when the ambient pH decreased from 7.4 to 6.8, the surface potential underwent a steep transition with a slope of -18 mV / pH unit, indicating its keen responsiveness to the weakly acidic tumor microenvironment, while remaining unresponsive to normal tissue (pH 7.2-7.4), ensuring the spatial specificity of charge switching. Furthermore, this switching process is irreversible; once positive charge exposure is completed in the tumor region, even if the carrier returns to normal tissue via the bloodstream, it will not revert to a negatively charged state, thus avoiding the risk of immune recognition caused by repeated switching.

[0017] The preparation process of this invention further includes a purification step to remove unbound free block copolymers and membrane fragments. The purification is performed using size exclusion chromatography with Sephadex G-100 packing material, a mobile phase of PBS buffer containing 5% glucose (pH 7.4), and a flow rate of 0.5 mL / min. The elution peaks at 12 to 15 min are collected, corresponding to intact biomimetic nanomedicines with particle sizes of 130-170 nm. High-performance liquid chromatography (HPLC) analysis shows that the free drug content in the final product is less than 0.5%, and the residual block copolymer content is less than 1%, meeting preclinical formulation standards.

[0018] Compared with the prior art, the beneficial effects of the present invention are: This invention resolves the performance paradox of existing biomimetic nanomedicines in balancing prolonged circulation and enhanced targeting through molecular design and process integration. Its core lies in constructing a dynamic charge interface with spatiotemporal resolution, which achieves sequential activation of immune escape and cell adsorption functions at the system level, rather than a simple superposition. This technical approach not only improves the drug accumulation efficiency at tumor sites but also provides a universal platform for other nanomedical applications requiring microenvironment-responsive surface regulation. Detailed Implementation

[0019] This invention provides a targeted delivery biomimetic nanomedicine and its preparation process. The core of this invention lies in constructing a dynamic biomimetic interface with microenvironment-responsive charge switching capabilities. This allows the nanomedicine to maintain an immune-inert negatively charged surface during blood circulation to evade immune clearance, and at the tumor lesion site, the surface potential is reversed to a positive state through local weak acidity, thereby enhancing the electrostatic adsorption between the nanomedicine and the tumor cell membrane and improving cell internalization efficiency. This technical solution uses molecular engineering to precisely embed a pH-responsive block copolymer into a biomimetic cell membrane structure, achieving spatiotemporal controllable conversion of surface charge. This fundamentally solves the performance paradox of existing biomimetic nanomedicines in prolonging circulation and enhancing targeting. The following detailed description of the technical solution of this invention, along with specific embodiments and comparative examples, will ensure that those skilled in the art can fully understand and implement this invention.

[0020] Example 1: The nanomedicine core is a polylactic acid-glycolic acid copolymer (weight average molecular weight 15000, lactic acid:glycolic acid = 75:25), loaded with doxorubicin (drug loading 12%), with a core particle size of 100 nm; the biomimetic cell membrane is derived from erythrocytes, with membrane vesicles having a particle size of 180 nm; the block copolymer is hexadecyl-polyethylene glycol-arginine (n=55, m=5), accounting for 5% of the membrane lipid mass; the overall particle size is 150 nm; Preparation process: Drug-loaded cores are prepared by nanoprecipitation; cell membrane vesicles are obtained by hypotonic lysis; drug-loaded cores and membrane vesicles are mixed at a 1:2 ratio, subjected to 5 freeze-thaw cycles and extruded through a three-pore membrane; purified by Sephadex G-100 chromatography.

[0021] Example 2: Block copolymer n=45, other parameters are the same as in Example 1; Preparation process: Same as in Example 1.

[0022] Example 3: Block copolymer n=65, other parameters are the same as in Example 1; Preparation process: Same as in Example 1.

[0023] Example 4: Block copolymer m=3, other parameters are the same as in Example 1; Preparation process: Same as in Example 1.

[0024] Example 5: Block copolymer m=8, other parameters are the same as in Example 1; Preparation process: Same as in Example 1.

[0025] Example 6: The biomimetic cell membrane is derived from tumor cells, and the other parameters are the same as in Example 1; Preparation process: Same as in Example 1 (cell membrane extraction process is consistent).

[0026] Example 7: Paclitaxel loaded with drug (10% loading), other parameters are the same as in Example 1; Preparation process: Same as in Example 1 (the organic solvent is adapted to the solubility of paclitaxel).

[0027] Example 8: The block copolymer accounted for 3% of the membrane lipid mass, and the other parameters were the same as in Example 1; Preparation process: Same as in Example 1.

[0028] Comparative Example 1: The nanomedicine core is the same as in Example 1; the biomimetic cell membrane is derived from red blood cells (without block copolymer embedding); the overall particle size is 150 nm; Preparation process: The drug-loaded core is fused with the membrane vesicle, without the addition of block copolymers, and the rest is the same as in Example 1.

[0029] Comparative Example 2: Only polylactic acid-glycolic acid copolymer drug-loaded core (same as Example 1), without biomimetic membrane and block copolymer; particle size 100 nm; Preparation process: The nanoprecipitation method is used to prepare the sample, followed by direct purification, without the membrane fusion step.

[0030] Test method: Physicochemical performance testing: Particle size and polydispersity index were tested using a dynamic light scattering instrument; potential values ​​at different pH levels were measured using a Zeta potentiometer; and drug loading and free drug residues were detected by high performance liquid chromatography.

[0031] Biological performance testing: Tumor-bearing mice were injected via tail vein, and plasma drug concentration-time curves were measured (half-life was calculated); the drug accumulation rate in tumor tissue was detected 24 hours after administration; and the internalization efficiency of tumor cells was analyzed by flow cytometry.

[0032] Safety testing: Detection of liver and kidney function indicators in mice; observation of the accumulation of nanomedicines in normal tissues; assessment of serum stability (24-hour particle size change rate).

[0033] Test data comparison table 1: Test Project pH 7.4 Zeta potential (mV) pH 6.5 Zeta potential (mV) Plasma half-life (hours) Example 1 -7 11 12.3 Example 2 -6 9 10.5 Example 3 -8 10 13.8 Example 4 -7 8 11.8 Example 5 -5 15 9.2 Example 6 -6 12 10.8 Example 7 -7 11 11.5 Example 8 -8 9 12.6 Comparative Example 1 -8 -8 6.8 Comparative Example 2 -15 -12 2.1 Test data comparison table 2: Test Project Tumor accumulation rate (%) 24-hour serum particle size change rate (%) Tumor cell internalization rate (%) Example 1 8.7 6 65 Example 2 7.2 7 58 Example 3 7.8 5 60 Example 4 6.5 6 55 Example 5 9.5 8 72 Example 6 9 7 68 Example 7 8.2 6 62 Example 8 6.8 5 56 Comparative Example 1 4.9 9 32 Comparative Example 2 2.3 15 25 Examples 1-8 achieved pH-responsive charge reversal through block copolymers, with a plasma half-life ≥9.2 hours and a tumor accumulation rate ≥6.5%; Comparative Example 1 had no charge switching and an accumulation rate of only 4.9%, while Comparative Example 2 had no biomimetic membrane and a half-life of only 2.1 hours, demonstrating that charge switching and biomimetic membranes synergistically ensure long circulation and targeting.

[0034] As the block copolymer n increases (Examples 2→1→3), the plasma half-life is prolonged but the charge reversal amplitude decreases slightly; as m increases (Examples 4→1→5), the charge reversal effect is enhanced (+8mV→+11mV→+15mV), the tumor accumulation rate is increased but the half-life is shortened; the proportion of block copolymer needs to be controlled at 3%-7%, too low (Example 8) will result in insufficient targeting, and too high will easily induce immune clearance.

[0035] Example 6: The biomimetic membrane was replaced with tumor cells, and the accumulation rate reached 9.0% due to enhanced homologous targeting. Example 7: The drug loading was replaced with paclitaxel, and the performance was stable, confirming the compatibility of the process with different hydrophobic drugs.

[0036] Examples 1-8 show excellent serum stability (particle size change rate ≤8%) and no significant liver and kidney function damage; the charge switching is pH specific (reversal only at pH ≤ 6.8), avoiding misactivation of normal tissues, thus balancing safety and targeting.

[0037] Compared to traditional nanoparticles (Comparative Example 2), the tumor accumulation rate of Examples 1-8 was increased by 3-4 times and the half-life was extended by 5-6 times, which solved the inherent contradiction between long circulation and targeting.

[0038] The biomimetic nanomedicine described in this invention achieves a balance between long circulation and efficient targeting through the synergistic design of pH-responsive charge switching and biomimetic membrane camouflage. Different parameter combinations can meet the requirements of biomedical applications and are suitable for the precision treatment of various tumor types.

[0039] 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 targeted delivery biomimetic nanomedicine, characterized in that, include: The core of the nanomedicine is composed of biodegradable polymer materials and is loaded with anti-tumor active drugs. A biomimetic cell membrane coating layer is applied to the outer surface of the nanomedicine core. A block copolymer functional module is embedded in the outer leaflet of the lipid bilayer of the biomimetic cell membrane coating.

2. The targeted delivery of biomimetic nanomedicine according to claim 1, characterized in that, The biomimetic cell membrane coating is derived from mammalian cells, selected from red blood cells, cancer cells, or white blood cells.

3. The targeted delivery of biomimetic nanomedicine according to claim 1, characterized in that, The block copolymer functional module is a hexadecyl-polyethylene glycol-arginine triblock copolymer, whose general chemical formula is C1. 16 H 33 -CO-NH-(CH2CH2O) n -CO-NH-(CH2)4-CH(NH2)-CO-[NH-(CH2)4-CH(NH2)-CO] m -OH, where n is an integer between 45 and 65, and m is an integer between 3 and 8.

4. The targeted delivery of biomimetic nanomedicine according to claim 1, characterized in that, The nanomedicine core is prepared using polylactic acid-glycolic acid copolymer, with a molar ratio of lactic acid to glycolic acid of 75:

25.

5. The targeted delivery biomimetic nanomedicine according to claim 1, characterized in that, The antitumor active drug is selected from one of doxorubicin, paclitaxel, cisplatin or gemcitabine, and its loading in the nanomedicine core is 8% to 15% (w / w).

6. The targeted delivery of biomimetic nanomedicine according to claim 3, characterized in that, The block copolymer functional module is inserted into the lipid bilayer of the biomimetic cell membrane coating through a hexadecyl hydrophobic anchoring chain, and maintains the guanidine deprotonated state of the arginine side chain at physiological pH 7.4, while it undergoes protonation at pH ≤ 6.8, driving its conformational flip and exposing it to the particle surface.

7. The targeted delivery biomimetic nanomedicine according to claim 1, characterized in that, The biomimetic cell membrane coating was obtained through hypotonic lysis, differential centrifugation, and ultrasonic disruption.

8. The targeted delivery of biomimetic nanomedicine according to claim 5, characterized in that, The hypotonic lysis buffer is a mixed solution of 10 mM Tris-HCl and 1 mM EDTA with a pH of 8.

0.

9. The targeted delivery of biomimetic nanomedicine according to claim 1, characterized in that, In the block copolymer functional module, the number of repeating arginine units m is 5, and the degree of polymerization of polyethylene glycol segments n is 55.

10. A preparation process for targeted delivery of biomimetic nanomedicines as described in any one of claims 1 to 9, characterized in that, Includes the following steps: Drug-loaded nanoparticles and biomimetic cell membrane vesicles were mixed at a mass ratio of 1:2 and added to PBS buffer containing 10% sucrose. Five freeze-thaw cycles were performed, and then the mixture was passed through polycarbonate membranes with pore sizes of 400 nm, 200 nm and 100 nm 11 times each to complete membrane fusion and directional distribution of block copolymers.