Homologous membrane nanotargeting anti-sepsis acute kidney injury drug delivery system

Abstract

This invention relates to the fields of biomedical engineering and nanomedicine delivery technology, specifically disclosing a homologous membrane nano-targeted drug delivery system for treating acute kidney injury with sepsis. The system includes: preparation of solid silicon spheres; mesoporization and etching; drug loading; preparation of homologous cell membranes; encapsulation and fusion; characterization and quality control. This invention utilizes KTP-modified RBCM vesicles to provide homologous surface proteins / receptors, achieving highly selective recognition and endocytosis of renal tubular cells, significantly improving drug accumulation and bioavailability in diseased kidney tissue, thereby enhancing therapeutic efficacy and reducing systemic exposure. The hollow, mesoporous structure provides high drug loading capacity and controllable release sites. The outer homologous membrane inhibits early leakage under physiological conditions and promotes drug release in inflammatory, acidic microenvironments, or intracellular environments, combining the advantages of sustained release and targeted activation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering and nanomedicine delivery technology, specifically relating to a homologous membrane nano-targeted antiseptic acute kidney injury drug delivery system. Background Technology

[0002] Septic acute kidney injury (SKI) is a severe and critical illness caused by a systemic inflammatory response syndrome triggered by a serious infection. It has a rapid onset and progression, with patients experiencing a sharp decline in kidney function within a short period. This is accompanied by serious complications such as the accumulation of metabolic products and acid-base imbalance, resulting in a persistently high mortality rate. Furthermore, it is highly likely to progress to chronic kidney disease or even end-stage renal disease, placing a heavy burden on the healthcare system and patients' families. Current clinical treatment primarily relies on supportive interventions such as fluid resuscitation and renal replacement therapy. There is a lack of specific drugs that can precisely target the site of kidney injury, highlighting the urgent need to develop efficient drug delivery systems to fill this clinical gap.

[0003] The development of nanocarrier technology has provided a new direction for targeted therapy of septic acute kidney injury. Among them, mesoporous silica nanoparticles have become an important research direction for drug delivery carriers due to their large specific surface area, controllable mesoporous structure and good biocompatibility. However, existing nanomedicine delivery systems still face several key technical bottlenecks when applied to the treatment of septic acute kidney injury: First, insufficient targeting specificity. Traditional nanocarriers often rely on passive targeting or simple surface modification to achieve kidney enrichment, making it difficult to accurately identify damaged kidney tissue and target cells. This results in a large distribution of drugs in non-target organs, reducing treatment efficiency and easily causing off-target toxicity. Second, poor biocompatibility and in vivo circulation stability. After entering the bloodstream, nanocarriers are easily recognized and cleared by the immune system, resulting in short circulation times and difficulty in effectively reaching the site of kidney injury. Furthermore, some carrier materials may trigger inflammatory reactions, exacerbating kidney tissue damage. Third, insufficient controlled drug release capability. The pathological microenvironment of septic acute kidney injury is characterized by acidity and high protease levels. Existing carriers cannot achieve precise and controllable drug release in this specific microenvironment, leading to premature leakage or insufficient release, and failing to exert a sustained therapeutic effect. Fourth, limited drug loading and delivery efficiency. The cavity structure and mesoporous channel design of traditional carriers are unreasonable, resulting in low drug loading. Moreover, problems such as drug aggregation and reduced activity easily occur during loading, further restricting the therapeutic effect.

[0004] To improve targeting and biocompatibility, some studies have attempted to modify nanocarriers using cell membrane coating technology. However, existing technologies still have significant drawbacks: Firstly, the selection of cell membrane sources lacks specificity, and carriers coated with non-homologous cell membranes are prone to immune rejection, making it difficult to achieve precise targeting through the specific recognition mechanisms of homologous cells. Secondly, cell membrane coating processes are immature, often resulting in incomplete coating and poor membrane stability, leading to membrane detachment during in vivo circulation and loss of targeting ability and biocompatibility. Furthermore, in the cell membrane preparation process, existing methods struggle to obtain uniformly sized and stably active cell membrane vesicles, resulting in low fusion efficiency with nanocarriers and further impacting the overall performance of the drug delivery system. Moreover, existing preparation techniques lack precise control over drug loading conditions, leading to large fluctuations in encapsulation efficiency and drug loading, and lack controlled-release design tailored to the pathological microenvironment of septic acute kidney injury, failing to guarantee effective drug concentration at the target site.

[0005] In summary, existing drug delivery systems for treating septic acute kidney injury suffer from numerous shortcomings, including poor targeting specificity, insufficient biocompatibility, weak cyclic stability, inadequate controlled drug release capabilities, and immature manufacturing processes, which severely restrict their clinical translation and application. Therefore, developing a drug delivery system with homologous targeting capabilities, good biocompatibility, precise controlled release characteristics, and a stable manufacturing process is of great significance for improving the therapeutic efficacy of septic acute kidney injury.

[0006] In response to this, this application proposes a homologous membrane nano-targeted antiseptic acute kidney injury drug delivery system to address the aforementioned problems. Summary of the Invention

[0007] The purpose of this invention is to provide a homologous membrane nano-targeted drug delivery system for treating septic acute kidney injury, in order to solve the problems mentioned in the background art.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A method for preparing a drug for targeting septic acute kidney injury using homologous cell membranes, comprising:

[0010] Step S1: Prepare solid silica spheres, the step of which includes introducing tetraethyl orthosilicate (TEOS) into a mixed solution of alcohol and water containing an alkaline catalyst and hydrolyzing and condensing it under stirring conditions to obtain the solid silica spheres;

[0011] Step S2: Using the solid silica spheres as templates, cationic surfactants and organic amines are used as auxiliaries to introduce mesoporous structures on the surface of the solid silica spheres under heating and stirring conditions, and hollow mesoporous silica nanoparticles (MSNPs) are obtained by alkaline etching or dissolution treatment.

[0012] Step S3: Contact the hollow mesoporous silica nanoparticles with the drug solution (Res) to be delivered and allow the drug to diffuse into the cavity and mesoporous channels of the MSNPs under light-shielded oscillation / ultrasound-assisted means to obtain drug-loaded nanocarriers (MSNPs@Res).

[0013] Step S4: Preparation of cell membrane derivatives from erythrocytes, namely erythrocyte membrane (RBCM): including cell culture, collection, repeated freeze-thaw lysis, differential centrifugation to enrich cell membrane components, quantitative confirmation by protein, and extrusion of RBCM-derived vesicles of uniform size through graded polycarbonate membranes (e.g., 0.45, 0.2, 0.1 μm), and modification of erythrocyte membrane vesicles with KTP to obtain KRBCM-derived vesicles;

[0014] Step S5: The drug-loaded nanocarrier and the KRBCM-derived vesicles are coated in a cholesterol-containing mixture through an extrusion / membrane fusion operation to obtain targeted nanodelivery particles (MSNPs@Res-KRBCM) coated with a homologous cell membrane.

[0015] Step S6: Perform aseptic treatment, characterization and quality assessment on the targeted nanoparticles coated with homologous cell membranes to obtain the final drug delivery formulation.

[0016] Preferably, in step S1, the alkaline catalyst is ammonia water, and tetraethyl orthosilicate is added in steps at room temperature and stirred for 6 to 20 hours to obtain solid silica spheres with uniform particle size.

[0017] Preferably, in step S2, the surfactant used to construct the mesoporous and cavity structures is hexadecyltrimethylammonium bromide (CTAB), the auxiliary agent is triethanolamine, the etchant is a saturated sodium carbonate solution, and the template surfactant is removed by acid or ethanol elution to obtain template-free hollow mesoporous silica nanoparticles.

[0018] Preferably, in step S3, the loading of drug Res is achieved by shaking at room temperature in the dark for 12–36 h. After loading is completed, the supernatant is separated by centrifugation and the content of unloaded drug in the supernatant is determined by ultraviolet spectrophotometry or high performance liquid chromatography to calculate the encapsulation efficiency and drug loading.

[0019] Preferably, the specific procedure for preparing RBCM vesicles in step S4 includes: collecting cells when the cell confluence is about 70% to 90%, washing with PBS, adding lysis buffer and incubating on ice, performing three freeze-thaw lysis cycles in liquid nitrogen at room temperature, first removing cell debris and nuclear components by low-speed differential centrifugation, then enriching membrane components by high-speed differential centrifugation (e.g., 4°C, 12000 rpm, about 20 to 30 min), and quantitatively confirming the amount of membrane protein using the BCA method, finally obtaining uniformly sized RBCM vesicles by extrusion from 0.45 to 0.2 to 0.1 μm, and modifying erythrocyte membrane vesicles with KTP to obtain KRBCM-derived vesicles;

[0020] The KTP modification includes the following steps:

[0021] KTP pretreatment: Select KTP with a purity ≥95% (sequence KCSAVPLC, molecular weight ~1.2kDa), dissolve it in PBS buffer (pH 7.4) to prepare a 1mg / mL solution, add N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (KTP:NHS:EDC molar ratio 1:1.2:1.5), and activate at room temperature for 30min;

[0022] Coupling reaction: The activated KTP solution was mixed with the RBCM vesicle suspension (membrane protein concentration 1 mg / mL) prepared in S4 at a volume ratio of 1:10, and incubated at 4°C in the dark with shaking for 2 h, during which the rotation speed was maintained at 200 rpm.

[0023] Purification: After the reaction, centrifuge at 4℃ and 12000rpm for 30min, discard the supernatant, wash the precipitate twice with PBS to remove unbound free KTP, and obtain KTP-modified RBCM vesicles (KRBCM).

[0024] Validation: The concentration of membrane proteins was quantified by BCA method, and the content of unbound KTP was determined by high performance liquid chromatography (HPLC). The modification efficiency was calculated (modification efficiency = (additional KTP amount - free KTP amount) / additional KTP amount × 100%, target modification efficiency ≥ 85%).

[0025] Preferably, the coating conditions in step S5 are as follows: 0.2 mg of equivalent membrane protein and 1 mg of MSNPs@Res are mixed at a mass ratio, and 0.5% to 10% (w / w, relative to the membrane protein or the total system) of cholesterol is added to enhance membrane stability. After filtration through 0.22 μm, the mixture is subjected to at least 1 to 3 stepwise extrusions to achieve complete coating and obtain targeted nanodelivery particles MSNPs@Res-KRBCM coated with homologous cell membranes.

[0026] Preferably, the characterization in step S6 includes encapsulation efficiency, drug loading, particle size distribution, surface potential, in vitro release curve, and homologous targeting verification.

[0027] The characterization also includes: dynamic light scattering (DLS) to determine particle size and PDI, zeta potential measurement, transmission electron microscopy (TEM) to observe the coating structure, in vitro pH-sensitive or enzymatic release experiments to demonstrate controlled release characteristics in sepsis, inflammation-associated microenvironments (e.g., acidic pH or protease-containing environments), and isotopic controls using erythrocytes to verify that cellular uptake is significantly higher than that of heterologous cells.

[0028] Another aspect of this application is to provide a homologous membrane nano-targeted drug delivery system for treating septic acute kidney injury, comprising:

[0029] A solid silica sphere preparation module is used to introduce a silicon source into an alkaline alcohol and water system and obtain solid silica spheres through hydrolysis and condensation.

[0030] The mesoporization and etching module is used to introduce a mesoporous template onto the surface of the solid silica sphere, form a mesoporous shell through sol-gel growth, and remove the template through alkaline etching, dissolution, and acid and alcohol elution to obtain hollow mesoporous silica nanoparticles (MSNPs).

[0031] The drug loading module is used to contact the hollow mesoporous silica nanoparticles with a drug solution and form a drug-loaded nanocarrier by means of diffusion promotion through oscillation and ultrasound, and to separate the supernatant and measure the unloaded drug to calculate the encapsulation rate.

[0032] The homologous cell membrane preparation module is used for cell culture, collection, lysis, differential centrifugation to enrich membrane components, protein quantification, and preparation of RBCM vesicles by extrusion through porous membranes. KRBCM-derived vesicles are obtained by modifying erythrocyte membrane vesicles with KTP.

[0033] The coating and fusion module is used to perform membrane fusion and extrusion coating of the drug-loaded nanocarrier and KRBCM vesicles in a cholesterol-containing mixture to obtain MSNPs@Res-KRBCM;

[0034] The characterization and quality control module is used to detect the particle size, zeta potential, encapsulation efficiency, in vitro release, biocompatibility, and homologous targeting of the MSNPs@Res-KRBCM, and is used for aseptic processing and formulation preservation to obtain the final formulation.

[0035] Compared with the prior art, the beneficial effects of the present invention are:

[0036] (1) The present invention uses the cell membrane of red blood cells to encapsulate the vesicles, which can enhance biocompatibility and reduce immune clearance, and achieve immune escape and long circulation. The KTP-modified RBCM vesicles can provide homologous surface proteins / receptors to achieve highly selective recognition and endocytosis of renal tubular cells, significantly improving the accumulation and bioavailability of drugs in diseased kidney tissue, thereby enhancing the therapeutic effect and reducing systemic exposure. The hollow, mesoporous structure provides high drug loading and controllable release sites. The homologous membrane coating inhibits early leakage under physiological conditions and promotes drug release in inflammatory / acidic microenvironment or intracellular environment, taking into account the advantages of both sustained release and targeted activation.

[0037] (2) By optimizing the loading and coating process, the present invention improves the drug encapsulation rate and drug loading, while preserving the natural biological function of membrane proteins, so that the formulation exhibits good compatibility, immune concealment and better therapeutic indicators (such as a decrease in inflammatory factors and improvement in renal function) in vitro and in vivo, thereby enhancing its clinical translation potential. Attached Figure Description

[0038] Figure 1 This is a block diagram of the homologous membrane nano-targeted antiseptic acute kidney injury drug delivery system of the present invention. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1:

[0041] Please see Figure 1 As shown, the homologous membrane nano-targeted drug delivery system for treating septic acute kidney injury includes:

[0042] 1.1 Preparation of solid silica spheres:

[0043] First, 6 mL of ammonia solution was added to a mixture of 150 mL of anhydrous ethanol and 3 mL of water to create a weakly alkaline environment. Then, 4.5 mL of tetraethyl orthosilicate was rapidly added to the mixture, and the reaction was carried out at 400 rpm at room temperature for 7 hours. Next, 3 mL of tetraethyl orthosilicate was added, and the reaction was continued for another 15 hours to synthesize solid silica spheres. The reaction solution was centrifuged at 10,000 rpm for 15 minutes, washed twice with ethanol and ultrapure water, and the collected solid was identified as solid silica spheres (SiO2). The obtained SiO2 was resuspended in 40 mL of ultrapure water for later use.

[0044] 1.2 Preparation of mesoporous silica nanoparticles (MSNPs):

[0045] 1.5 g of hexadecyltrimethyl bromide, 53.4 μL of triethanolamine, and 64.5 mL of water were dissolved completely by vigorous stirring at 80 °C. Then, 10 mL of the above SiO2 suspension was added, and the mixture was stirred continuously for 20 min. Next, 450 μL of tetraethyl orthosilicate was added, and the mixture was incubated for 1 h. After cooling to 50 °C, 9 mL of saturated sodium carbonate solution was added, and the silica sphere template was etched for 2 h. Hollow silica spheres were obtained by centrifugation at 15000 rpm for 15 min and washed twice with ethanol and ultrapure water, respectively. The obtained hollow silica spheres were dispersed in 100 mL of hydrochloric acid (1 M) / ethanol solution (v / v = 1:10) and stirred at 60 °C for 2 h to remove hexadecyltrimethyl bromide. Hollow mesoporous silica nanoparticles were obtained by centrifugation again at 15000 rpm for 15 min. The above process was repeated twice to ensure complete removal of hexadecyltrimethyl bromide.

[0046] 1.3 Preparation of KTP-modified erythrocyte membrane (RBCM) (KRBCM):

[0047] 1.3.1 Cell Culture: Red blood cell lines were purchased from the Shanghai Experimental Cell Center, Chinese Academy of Sciences (derived from the ATCC cell bank in the United States). They were cultured in DMEM medium (Gibco, USA) containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator.

[0048] 1.3.2 After discarding the supernatant, RBCs with 80% cell confluence and good growth status were rinsed twice with 2 mL of PBS.

[0049] 1.3.3 Add 600 μL of cell lysis buffer to the above RBCs, incubate on ice for 2 min, scrape off the cells with a cell scraper and collect them into a 1.5 mL EP tube;

[0050] 1.3.4 The collected RBCs were subjected to three freeze-thaw cycles at liquid ammonia and room temperature, and then centrifuged at 4°C and 1000 rpm for 10 min. The supernatant was collected.

[0051] 1.3.5 After centrifuging the supernatant at 4℃ and 12000rpm for 30min, discard the supernatant. The resulting precipitate is RBCM.

[0052] 1.3.6 Resuspend RBCM in an appropriate amount of PBS to obtain an RBCM suspension, and quantify its protein content using the BCA method;

[0053] 1.3.7 RBCM was sequentially extruded through 0.45, 0.2, and 0.1 μm polycarbonate porous membranes to obtain RBCM-derived vesicles with uniform size;

[0054] 1.3.8 KTP modification of RBCM vesicles to obtain KRBCM vesicles.

[0055] 1.4 Preparation of homologous cell membrane-targeting nano-formulations:

[0056] To load Res into MSNPs, 40 mg of MSNPs were added to 10 mL of Res / ethanol solution (4 mg / mL) and dispersed by sonication. The solution was then incubated at 220 rpm for 24 h at room temperature in the dark to allow Res to diffuse sufficiently into the cavities and mesoporous channels of the MSNPs. After centrifugation, the supernatant and MSNPs@Res precipitate were collected separately. The supernatant was diluted 100-fold, and the unloaded drug content in the supernatant was determined by UV spectrophotometry to calculate the drug encapsulation efficiency and drug loading.

[0057] Encapsulation efficiency = (WT - WF) / WT × 100% (T: dosage, F: drug content in supernatant);

[0058] Drug loading = (WT - WF) / Ws × 100% (S: total amount of carrier + drug);

[0059] Subsequently, 0.2 mg KRBCM and 1 mg MSNPs@Res were mixed and extruded using a sterile syringe and a 0.22 μm filter membrane to obtain MSNPs@Res-KRBCM.

[0060] In addition, cholesterol is added to the outer membrane to enhance the biocompatibility and stability of the cell membrane.

[0061] Example 2:

[0062] A method for preparing a drug for targeting septic acute kidney injury using homologous cell membranes includes the following steps:

[0063] S1 (solid silica spheres): Stöber-type synthesis, TEOS is added stepwise to obtain solid SiO2 spheres.

[0064] S2 (Forming hollow mesoporous MSNPs): Using CTAB / triethanolamine as a template, the shell is formed by stirring at 80°C, followed by alkaline etching and acid / ethanol elution to obtain template-free hollow mesoporous MSNPs.

[0065] S3 (Drug Loading → MSNPs@Res): 40 mg MSNPs were mixed with 10 mL Res / ethanol (4.0 mg / mL). -1 Shake for 24 hours, then centrifuge to collect MSNPs@Res.

[0066] S4 (RBCM preparation): Membrane vesicles were prepared from erythrocytes, followed by freeze-thaw lysis, differential centrifugation, BCA quantification, and three-stage extrusion (0.45 / 0.2 / 0.1 μm).

[0067] S5 (Coated → MSNPs@Res-KRBCM): 0.2 mg of membrane protein was mixed with 1.0 mg of MSNPs@Res, 2% (w / w) cholesterol was added, filtered through a 0.22 μm filter, and extruded twice to obtain coated particles.

[0068] S6 (Characteristics and Quality): Characterization and QC of DLS, zeta potential, TEM, HPLC, in vitro release, cellular uptake, cytotoxicity, ELISA (inflammatory factors), etc.

[0069] S1 – Synthesis of solid SiO2 spheres:

[0070] Reagents and quantities: 150 mL anhydrous ethanol, 3 mL deionized water, 6 mL ammonia, and TEOS were added in two portions: 4.5 mL initially, and stirred at 400 rpm for 7 h at room temperature; then 3.0 mL TEOS was added, and stirring was continued for 15 h.

[0071] Post-processing: Centrifuge at 10,000 rpm for 15 min, wash twice with ethanol / ultrapure water, and finally resuspend in 40 mL of ultrapure water to obtain solid silica spheres (record the mass of the solid for the next step of use).

[0072] S2 – Hollow mesoporous MSNPs:

[0073] Reagents and quantities: CTAB 1.5g, triethanolamine 53.4μL, deionized water 64.5mL, dissolved by vigorous stirring at 80℃. Add 10mL A (resuspended solution from the previous step), stir for 20min, then add 450μL TEOS, incubate for 1h, cool to 50℃, add 9mL saturated Na2CO3 solution for etching for 2h.

[0074] Centrifuge at 15000 rpm for 15 min, and wash twice with ethanol / ultrapure water. Then place the product in 100 mL (1M HCl / ethanol v / v=1:10) at 60 °C and stir for 2 h to remove the template. Repeat twice to ensure removal of CTAB. Centrifuge and wash to obtain MSNPs.

[0075] Product dry weight: Weigh approximately 40.0 mg of the dried product (for the next loading step).

[0076] S3 – Drug Loading (MSNPs@Res):

[0077] Loading conditions: Add 40.0 mg MSNPs to 10.0 mL Res / ethanol (4.0 mg / mL) -1Therefore, the total dosage WT = 10.0 mL × 4.0 mg·mL -1 =40.0mg), dispersed by sonication for 5 minutes, and shaken at room temperature in the dark for 24 hours (220rpm).

[0078] Centrifugation: 12000 rpm × 10 min, collect the supernatant and preserve it to determine the unloaded drug content (WF), wash the precipitate once and dry it or prepare MSNPs@Res directly.

[0079] Determination of supernatant: HPLC.

[0080] HPLC conditions: C18 reversed-phase column (150 × 4.6 mm, 5 μm), mobile phase acetonitrile:water (0.1% formic acid) = 60:40, flow rate 1.0 mL·min -1 The detection wavelength was 306 nm (typical detection wavelength for resveratrol), and the injection volume was 20 μL.

[0081] Test results (Example 2): The residual drug WF in the supernatant was measured to be 5.00 mg (that is, the concentration measured in the supernatant is equivalent to a total of 5.00 mg, which is obtained after dilution and conversion).

[0082] Encapsulation efficiency and drug loading calculation:

[0083] Wherein: drug dosage WT=40.00mg; unloaded supernatant WF=5.00mg; total amount of carrier + drug Ws=40.00mg (original mass of carrier) + (WT−WF) (actual loaded drug amount).

[0084] Calculation steps:

[0085] Calculate the amount of drug already loaded = WT − WF = 40.00 − 5.00 = 35.00 mg.

[0086] Encapsulation efficiency = (WT − WF) / WT × 100% = 35.00 / 40.00 × 100% = 0.875 × 100% = 87.50%.

[0087] Total mass Ws = 40.00 + 35.00 = 75.00 mg.

[0088] Drug loading = (WT−WF) / Ws×100% = 35.00 / 75.00×100% = 46.67%.

[0089] S4 – Preparation of KRBCM (Red Blood Cell Source):

[0090] Cell culture: Red blood cells were cultured in DMEM + 10% FBS at 37°C and 5% CO2 until approximately 80% confluenced for collection.

[0091] Extraction procedure (parameters and steps): Discard the supernatant, wash twice with PBS (2 mL), add 600 μL of cell lysis buffer (incubate on ice for 2 min), scrape off the cells with a cell scraper and transfer them into a 1.5 mL EP tube; freeze and thaw three times alternately with liquid nitrogen and room temperature; centrifuge at 4 °C and 1000 rpm for 10 min and collect the supernatant; then centrifuge at 4 °C and 12000 rpm for 30 min, discard the supernatant, and retain the precipitate as the membrane component (RBCM).

[0092] Protein quantification: Membrane protein concentration was determined by the BCA method and adjusted to 1.0 mg / mL. -1 Preservation; Vesicles were prepared by sequential extrusion of polycarbonate membrane (0.45→0.2→0.1μm), and the final vesicles were designated as RBCM vesicles;

[0093] KTP-modified RBCM vesicles: Take 1 mL of RBCM vesicle suspension (membrane protein concentration 1 mg / mL) and add 0.1 mL of activated KTP solution (1 mg / mL) (KTP:membrane protein mass ratio 1:10, sequence KCSAVPLC). Incubate at 4℃ and 200 rpm for 2 h with shaking. Centrifuge at 12000 rpm and 4℃ for 30 min, wash twice with PBS, and collect the precipitate as KRBCM vesicles. HPLC determination shows that the free KTP content is 0.01 mg, and the modification efficiency is calculated as (0.1 mg - 0.01 mg) / 0.1 mg × 100% = 90%. Flow cytometry detection shows that the fluorescence positivity rate of FITC-KTP-labeled KRBCM vesicles is 82%.

[0094] S5 Coating: Mix 0.2 mg KRBCM membrane protein with 1 mg MSNPs@Res, add 2% cholesterol, filter at 0.22 μm, and extrude twice in stages to obtain MSNPs@Res-KRBCM.

[0095] Yield: From initial 10 7 Each cell can obtain approximately 2.0 mg of total membrane proteins, of which 0.2 mg is used for coating.

[0096] S5, Encapsulation (MSNPs@Res-KRBCM):

[0097] Preparation and conditions: Mix 1.0 mg MSNPs@Res (the above-mentioned loaded product) with 0.2 mg equivalent membrane protein at a mass ratio of 1:0.2, add 2% cholesterol (w / w, relative to membrane protein), push through a 0.22 μm filter membrane with a sterile syringe, and then squeeze twice through a 0.45 / 0.2 / 0.1 μm polycarbonate membrane with a 200 μL syringe to complete membrane coating / fusion.

[0098] Coating verification: TEM was used to observe the coating layer (outer membrane thin layer), and SDS-PAGE and silver staining or WB were used to confirm the retention of membrane protein characteristic bands; BCA was used to determine the relative retention rate of outer membrane proteins after coating.

[0099] S6 – Characterization, In Vitro Release, and Cellular Experiments:

[0100] Particle size (DLS):

[0101] MSNPs (template-free, hollow mesoporous): average particle size 70 nm, PDI 0.12.

[0102] MSNPs@Res: Average particle size 80 nm, PDI 0.15.

[0103] MSNPs@Res-KRBCM (coated): average particle size 95nm, PDI 0.18.

[0104] ζ potential: MSNPs−25.0mV; MSNPs@Res−18.0mV; MSNPs@Res-KRBCM−8.0mV (membrane protein coverage makes the surface potential close to neutral).

[0105] TEM: Hollow mesoporous structure and uniform membrane coating (membrane thickness approximately 8–12 nm) were observed.

[0106] In vitro release (dialysis, 37°C, pH 7.4 and pH 5.5) – cumulative release (24h):

[0107] At pH 7.4: MSNPs@Res cumulative release was 34.0%; MSNPs@Res-KRBCM cumulative release was 28.0%.

[0108] At pH 5.5 (simulating an acidic environment of inflammation / lysosomes): MSNPs@Res 62.0%; MSNPs@Res-KRBCM 69.0%.

[0109] Method description: Transfer to dialysis bag (MWCO 3.5kDa), place in 100mL release medium (PBS, pH adjusted), rotate at 37℃ (100rpm), take samples at regular intervals and quantify by HPLC, and replenish with an equal volume of fresh medium when taking samples.

[0110] Cellular uptake (flow cytometry or fluorescence microscopy): FITC-labeled nanoparticles (or Res chromophores) were used in erythrocytes. After incubation for 4 hours, the percentage of positive cells was counted by flow cytometry: MSNPs@Res uptake rate was 42%; MSNPs@Res-KRBCM uptake rate was 68% (homogeneous membranes significantly enhanced targeted uptake).

[0111] Cytotoxicity (CCK-8, 24h, treatment dose corresponding to MSNPs 50 μg / mL) -1 The cell viability was >92% for both MSNPs@Res and MSNPs@Res-KRBCM, indicating good biocompatibility.

[0112] Inflammatory factor inhibition (LPS-stimulated erythrocyte model, LPS 1 μg·mL) -1 After 24 hours of incubation, TNF-α was measured by ELISA.

[0113] Positive control (LPS only): TNF-α = 1000 pg / mL -1 (Setting a baseline).

[0114] Free Res treatment (40 μg·mL) -1 (Equivalent): TNF-α = 650 pg·mL -1 (Reduced by 35.0%).

[0115] MSNPs@Res: TNF-α = 500 pg·mL -1 (Reduced by 50.0%).

[0116] MSNPs@Res-KRBCM:TNF-α=360pg·mL -1 (Reduced by 64.0%).

[0117] Assay method: ELISA according to the kit instructions, triple replicates, average value and standard deviation are given; the values ​​here are representative averages.

[0118] As can be seen from the above, using red blood cell membrane coating can enhance biocompatibility and reduce immune clearance, enabling immune escape and long circulation. Using KTP to modify RBCM vesicles can provide homologous surface proteins / receptors, enabling highly selective recognition and endocytosis of renal tubular cells, significantly improving drug accumulation and bioavailability in diseased kidney tissue, thereby enhancing therapeutic effects and reducing systemic exposure.

[0119] Example 3:

[0120] To improve drug diffusion efficiency, ultrasound-intermittent oscillation (3 min ultrasound + 4 h oscillation × 3 times) was used on the drug loading (S3); the dosing conditions were the same (10 mL × 4 mg·mL). -1 ).

[0121] In the coating (S5), cholesterol was increased to 4% (w / w), and the number of squeezes was increased to 3 to improve coating integrity.

[0122] For membrane preservation and processing, 4℃ PBS buffering with the addition of 0.02% NaN3 (only for short-term preservation; long-term cryopreservation is required) was used to reduce protein degradation and improve the preservation of the membrane characteristic protein library.

[0123] Specific parameters and measurements (Example 3):

[0124] The dosage WT = 40.00 mg (same as above); the supernatant WF was measured to be 3.30 mg (less residue after optimization).

[0125] Calculation bit by bit (encapsulation efficiency and drug loading):

[0126] The amount of drug loaded = WT − WF = 40.00 − 3.30 = 36.70 mg.

[0127] Encapsulation rate = 36.70 / 40.00 × 100% = 0.9175 × 100% = 91.75% (approximately 91.8%).

[0128] Total mass Ws = 40.00 + 36.70 = 76.70 mg.

[0129] Drug loading = 36.70 / 76.70×100% = 0.4786×100% = 47.86% (approximately 47.9%).

[0130] DLS / ζ / TEM:

[0131] MSNPs: average particle size 65 nm, PDI 0.11.

[0132] MSNPs@ResC: 75nm, PDI 0.14.

[0133] MSNPs@Res-KRBCM: 100nm, PDI 0.16.

[0134] ζ potential: −26mV→−17mV→−7mV (the trend is the same as in Example 2).

[0135] Protein retention (SDS-PAGE quantification): approximately 86% (Example 3, superior to 78% in Example 2).

[0136] In vitro release (24-hour cumulative):

[0137] pH7.4: MSNPs@Res32.0%; MSNPs@Res-KRBCM26.0%.

[0138] pH5.5: MSNPs@Res64.0%; MSNPs@Res-KRBCM71.0%.

[0139] Cellular uptake: MSNPs@Res uptake rate 45%; MSNPs@Res-KRBCM uptake rate 74%.

[0140] Inhibition of inflammatory factors (same as LPS model, ELISA TNF-α):

[0141] MSNPs@Res: TNF-α = 480 pg·mL -1 (Reduced by 52.0%)

[0142] MSNPs@Res-KRBCM:TNF-α=340pg·mL -1 (Reduced by 66.0%).

[0143] Cytotoxicity: CCK-8 cell viability was >90%.

[0144] Comparison inside small animals

[0145] Model: A mouse model of septic AKI was established by intraperitoneal injection of LPS or cecal ligation and puncture (CLP). BUN / SCr was measured 24 h later. There were 6 mice in each experimental group.

[0146] Representative results (24h):

[0147] Model control (untreated): BUN = 80 mg·dL -1 SCr = 2.00 mg·dL -1 .

[0148] MSNPs@Res treatment: BUN = 60 mg·dL -1 SCr = 1.40 mg·dL -1 .

[0149] MSNPs@Res-KRBCM (Optimization in Example 2): BUN = 48 mg·dL -1 SCr = 1.10 mg·dL -1 .

[0150] These figures indicate that targeted delivery after encapsulation can further improve the recovery of renal function markers in vivo.

[0151] Encapsulation efficiency / drug loading: The unloaded drug content (WF) in the supernatant is quantified by HPLC according to the formula.

[0152] Three parallel samples (n=3) were taken in the experiment, and the average and standard deviation were given.

[0153] DLS / ζ potential: The sample was filtered through 0.22 μm and measured at 25 °C. The size report shows the average particle size (Z-average) and PDI, and the average was taken from three replicates.

[0154] TEM: Negative staining or freeze-cross section observation, using 80–120kV voltage to take pictures, and measuring film thickness and particle size (statistics >100 particles).

[0155] In vitro release: Dialysis method, the sample is placed in a 3.5kDa MWCO dialysis bag, 37℃, 100rpm, and samples are taken at regular intervals (e.g., 0.5h, 1h, 2h, 4h, 8h, 24h). The cumulative release amount of the sample is quantified by HPLC (the same volume is replenished each time).

[0156] Cellular uptake: Nanoparticles were labeled with fluorescent labels or dyes, and the percentage of positive cells and median fluorescence intensity (MFI) were read by flow cytometry. The results were statistically analyzed with n=3.

[0157] ELISA / Cytotoxicity: Perform according to the kit instructions (standard curve), repeat with triple replicates and provide mean and standard deviation.

[0158] Animal indicators: Blood samples were collected to measure BUN / SCr (using commercial clinical test reagents or biochemical analyzers), with at least n=6 in each group. Statistical analysis was performed using t-tests or ANOVA (p<0.05 was considered significant).

[0159] The comprehensive data are shown in Table 1 below (Examples 2, 3 and Comparative Examples: MSNPs@Res without coating).

[0160] Table 1 lists only the main indicators for the final comparison (24h data / key quality characteristics), and the values ​​are the representative averages of the above experiments (n≥3).

[0161] Table 1

[0162] Indicators / Groups Comparative example: MSNPs@Res (without membrane) Example 2: MSNPs@Res-KRBCM (Conventional) Example 3: MSNPs@Res-KRBCM (Optimized) Dosage (WT, mg) 40.00 40.00 40.00 Unloaded WF (mg) in the supernatant 8.00 5.00 3.30 Packaging ratio (%) 80.00% 87.50% 91.75% Drug loading (%) 28.57% 46.67% 47.86% Average particle size (after loading) (nm) 80 95 100 PDI 0.15 0.18 0.16 Zeta potential (mV) −18.0 −8.0 −7.0 Membrane protein retention rate (%) — 78% 86% Red blood cell uptake rate (%) 42 68 74 In vitro drug release (pH 7.4, 24h) (%) 34.0 28.0 26.0 In vitro drug release (pH 5.5, 24h) (%) 62.0 69.0 71.0 <![CDATA[LPS-TNF-α(pg·mL -1 )]]> 500 360 340 <![CDATA[Mouse BUN (mg·dL -1 , 24 h)]]> 60 48 48 (Example 3 is slightly better) <![CDATA[Mouse SCr (mg·dL -1 , 24 h)]]> 1.40 1.10 1.10

[0163] As shown in the table above, selective homologous targeting: KRBCM coating significantly increased the uptake of nanoparticles by erythrocytes (Example 2: 42%→68%; Example 3: 45%→74%), indicating that the surface proteins / receptors provided by the homologous cell membrane can enhance the recognition and endocytosis of renal tubular cells.

[0164] Improving the in vivo and in vitro therapeutic window and reducing early leakage: Membrane coating reduces drug release at physiological pH (7.4) but increases release in acidic environments (pH 5.5), which is beneficial for achieving stronger drug release under inflammatory or intracellular lysosomal conditions, demonstrating the advantages of controlled release.

[0165] Both drug loading and encapsulation efficiency are robustly guaranteed: By optimizing the loading and coating process (Example 3), the encapsulation efficiency is increased from 87.5% to 91.75%, and the drug loading is increased from 46.67% to 47.86% (a reasonable range), enhancing drug loading efficiency while maintaining particle stability.

[0166] Improved biocompatibility and efficacy indicators: Cell survival rate was higher than 90% after the carrier was coated with a membrane, and BUN / SCr was significantly improved in the in vivo mouse model (compared to the uncoated group), demonstrating the balance between safety and functionality of the formulation.

[0167] As can be seen from the above, hollow and mesoporous structures provide high drug loading capacity and controllable release sites. The outer homologous membrane not only inhibits early leakage under physiological conditions, but also promotes drug release in inflammatory / acidic microenvironments or intracellular environments, thus taking into account the advantages of both sustained release and targeted activation.

[0168] By optimizing the loading and coating processes, the drug encapsulation rate and drug loading capacity can be improved while preserving the natural biological functions of membrane proteins. This allows the formulation to exhibit good compatibility, immune concealment, and better therapeutic indicators (such as a decrease in inflammatory factors and improvement in renal function) in vitro and in vivo, thereby enhancing its clinical translation potential.

[0169] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a drug for targeting septic acute kidney injury using homologous cell membranes, characterized in that, Includes the following steps: Step S1: Prepare solid silica spheres, the step of which includes introducing tetraethyl orthosilicate into a mixed solution of alcohol and water containing an alkaline catalyst and hydrolyzing and condensing it under stirring conditions to obtain the solid silica spheres; Step S2: Using the solid silica spheres as templates, cationic surfactants and organic amines are used as auxiliaries to introduce mesoporous structures on the surface of the solid silica spheres under heating and stirring conditions, and hollow mesoporous silica nanoparticles are obtained by alkaline etching or dissolution treatment. Step S3: The hollow mesoporous silica nanoparticles are brought into contact with the drug solution to be delivered, and the drug diffuses into the cavity and mesoporous channels of the MSNPs under light-protected vibration and ultrasonic assistance to obtain a drug-loaded nanocarrier. Step S4: Preparation of cell membrane derivatives from red blood cells: including cell culture, collection, repeated freeze-thaw lysis, differential centrifugation to enrich cell membrane components, quantitative confirmation by protein and obtaining uniformly sized RBCM-derived vesicles by stepwise polycarbonate membrane extrusion, and modification of red blood cell membrane vesicles by KTP to obtain modified KRBCM-derived vesicles; Step S5: The drug-loaded nanocarrier and the modified KRBCM-derived vesicles are coated in a cholesterol-containing mixture through extrusion and membrane fusion to obtain targeted nanodelivery particles coated with homologous cell membranes. Step S6: Perform aseptic treatment, characterization and quality assessment on the targeted nanoparticles coated with homologous cell membranes to obtain the final drug delivery formulation.

2. The method for preparing a drug for targeting septic acute kidney injury using homologous cell membranes according to claim 1, characterized in that, In step S1, the alkaline catalyst is ammonia water, and tetraethyl orthosilicate is added in steps at room temperature and stirred for 6 to 20 hours to obtain solid silica spheres with uniform particle size.

3. The method for preparing a drug for targeting septic acute kidney injury using homologous cell membranes according to claim 1, characterized in that, In step S2, the surfactant used to construct the mesoporous and cavity structures is hexadecyltrimethylammonium bromide, the auxiliary agent is triethanolamine, the etchant is saturated sodium carbonate solution, and the template surfactant is removed by acid or ethanol elution to obtain template-free hollow mesoporous silica nanoparticles.

4. The method for preparing a drug for targeting septic acute kidney injury using homologous cell membranes according to claim 1, characterized in that, In step S3, the loading of drug Res is achieved by shaking at room temperature in the dark for 12–36 h. After loading is completed, the supernatant is separated by centrifugation and the content of unloaded drug in the supernatant is determined by ultraviolet spectrophotometry or high performance liquid chromatography to calculate the encapsulation efficiency and drug loading.

5. The method for preparing a drug for targeting septic acute kidney injury using homologous cell membranes according to claim 1, characterized in that, The specific procedure for preparing RBCM vesicles in step S4 includes: collecting cells when the cell confluence is about 70% to 90%, washing with PBS, adding lysis buffer and incubating on ice, performing three freeze-thaw lysis cycles in liquid nitrogen at room temperature, first removing cell debris and nuclear components by low-speed differential centrifugation, then enriching membrane components by high-speed differential centrifugation, and quantitatively confirming the amount of membrane protein using the BCA method, and finally obtaining uniformly sized RBCM vesicles by extrusion at 0.45, 0.2, and 0.1 μm in sequence, and then modifying erythrocyte membrane vesicles with KTP to obtain modified KRBCM-derived vesicles; The KTP modification includes the following steps: KTP pretreatment: Select KTP with a purity ≥95% and sequence KCSAVPLC, dissolve it in PBS buffer to prepare a 1 mg / mL solution, add N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and activate at room temperature for 30 min; Coupling reaction: The activated KTP solution and RBCM vesicle suspension were mixed at a volume ratio of 1:10 and incubated at 4°C in the dark with shaking for 2 hours, during which the rotation speed was maintained at 200 rpm. Purification: After the reaction, centrifuge at 4℃ and 12000rpm for 30min, discard the supernatant, wash the precipitate twice with PBS to remove unbound free KTP, and obtain KTP-modified KRBCM vesicles; Validation: The concentration of membrane proteins was quantified using the BCA method, and the content of unbound KTP was determined by high performance liquid chromatography. The modification efficiency was calculated as follows: modification efficiency = (amount of added KTP - amount of free KTP) / amount of added KTP × 100%.

6. The method for preparing a drug for targeting septic acute kidney injury using homologous cell membranes according to claim 1, characterized in that, The coating conditions in step S5 are as follows: 0.2 mg of equivalent membrane protein and 1 mg of MSNPs@Res are mixed at a mass ratio, 0.5% to 10% cholesterol is added to enhance membrane stability, and after filtration through 0.22 μm, the mixture is subjected to at least 1 to 3 stepwise extrusions to achieve complete coating and obtain targeted nanodelivery particles MSNPs@Res-KRBCM coated with homologous cell membranes.

7. The method for preparing a drug for targeting septic acute kidney injury using homologous cell membranes according to claim 1, characterized in that, The characterization in step S6 includes encapsulation efficiency, drug loading, particle size distribution, surface potential, in vitro release curve, and homologous targeting verification; The characterization also includes: dynamic light scattering to determine particle size and PDI, zeta potential measurement, transmission electron microscopy to observe the coating structure, in vitro pH-sensitive or enzymatic release experiments to demonstrate controlled release characteristics in sepsis- and inflammation-associated microenvironments, and isotopic controls using erythrocytes to verify that cellular uptake is significantly higher than that of heterologous cells.

8. A homologous membrane nano-targeted drug delivery system for treating septic acute kidney injury, characterized in that, include: A solid silica sphere preparation module is used to introduce a silicon source into an alkaline alcohol and water system and obtain solid silica spheres through hydrolysis and condensation. The mesoporization and etching module is used to introduce a mesoporous template onto the surface of the solid silica sphere, form a mesoporous shell through sol-gel growth, and remove the template through alkaline etching, dissolution, and acid and alcohol elution to obtain hollow mesoporous silica nanoparticles. The drug loading module is used to contact the hollow mesoporous silica nanoparticles with a drug solution and form a drug-loaded nanocarrier by means of diffusion promotion through oscillation and ultrasound, and to separate the supernatant and measure the unloaded drug to calculate the encapsulation rate. The homologous cell membrane preparation module is used for cell culture, collection, lysis, differential centrifugation to enrich membrane components, protein quantification, and preparation of RBCM vesicles by extrusion through porous membranes. KRBCM-derived vesicles are obtained by modifying erythrocyte membrane vesicles with KTP. The coating and fusion module is used to perform membrane fusion and extrusion coating of the drug-loaded nanocarrier and the modified KRBCM-derived vesicles in a cholesterol-containing mixture to obtain MSNPs@Res-KRBCM. The characterization and quality control module is used to detect the particle size, zeta potential, encapsulation efficiency, in vitro release, biocompatibility, and homologous targeting of the MSNPs@Res-KRBCM, and is used for aseptic processing and formulation preservation to obtain the final formulation.

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