Photo-activated myocardial targeting nano capsule for heart failure

Abstract

The invention discloses a photo-activated myocardial targeting nano capsule for heart failure, and belongs to the technical field of biological medicines and nano materials. The nanometer capsule-like body comprises an inner core which is a nanometer capsule-like body unit, is obtained by nanocrystallization of a plant capsule-like body film and retains photoreaction activity; the shell is a bionic coating layer derived from a myocardial cell membrane. The invention also provides a preparation method of the nano capsule-like body and application of the nano capsule-like body in preparation of medicines for treating pressure load related cardiomyopathy (such as heart failure, cardiac hypertrophy and fibrosis). According to the nanometer cystoid, myocardial targeted enrichment is achieved through myocardial cell membrane coating, ATP is controllably generated under specific red light irradiation, myocardial cell energy metabolism defects are repaired, the mitochondrial function is improved, and therefore myocardial hypertrophy and fibrosis are effectively inhibited on cell and animal models, and the heart function is improved. The invention provides a novel energy metabolism repairing strategy which is triggered on demand and controllable in dosage for heart failure treatment.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and nanomaterials technology, specifically relating to a photoactivated myocardial-targeting nanothylakoid for heart failure, its preparation method, and its application in the preparation of products or drugs for treating stress-related cardiomyopathy. Background Technology

[0002] Heart failure (HF) is often caused by sustained pressure loads (such as hypertension and valvular stenosis), leading to myocardial hypertrophy, fibrosis, and ventricular remodeling. Extensive research and clinical evidence suggest that myocardial energy deficiency and mitochondrial dysfunction are core pathological factors in the development of HF, manifesting as insufficient ATP production, abnormal mitochondrial membrane potential, increased oxidative stress, and decreased energy substrate utilization efficiency. Current drug treatments primarily achieve benefits through neurohumoral regulation, hemodynamic improvement, and indirect metabolic influences, but a strategy that can achieve exogenous, controllable, and on-demand energy metabolism repair locally in the myocardium is generally lacking.

[0003] In recent years, various patented technology routes related to plant thylakoids / nanothylakoids have emerged, including but not limited to: 1) Oral anti-aging / organ failure direction: For example, a class of "oral thylakoid products for combating organ failure and aging" has been disclosed, emphasizing that oral absorption promotes NAD+ related enzymes associated with thylakoids. + De novo synthesis and other systemic mechanisms are used for anti-aging and organ failure (CN121313755A). This type of technology mainly focuses on oral systemic metabolic regulation, but it does not solve the problems of targeted residence and photodosimetrically controllable triggering of local myocardial energy defects in stress-induced heart failure.

[0004] 2) Cross-species cell membrane delivery of cellular components: Existing patents are based on cell membrane-based cross-species cellular component delivery biomaterials as the core framework, targeting a very wide range of cells, and explicitly including thylakoid vesicles derived from chloroplasts as one of the deliverable cellular components. This type of approach focuses on broad-spectrum coverage of delivery methods, but it is not built around a pressure-overload heart failure treatment system (local drug delivery + red light dosimetry + cardiac function endpoints).

[0005] 3) Applications in ROS production / oxygen supply / antibacterial or superficial tissues: Using nano-thylakoids in photodynamic / antibacterial hydrogels, dental bleaching, etc. (CN121129743A, CN120788971A, CN118048292A). This approach emphasizes ROS production / oxygen production / antibacterial or bleaching effects, which are not equivalent to the reduction of oxidative stress and improvement of mitochondrial efficiency and energy metabolism required in the pathology of chronic remodeling in heart failure.

[0006] In summary, there is currently a lack of therapeutic systems that can achieve local enrichment and retention in the myocardium and enable on / off energy output through exogenous red light dosimetry. Existing thylakoid-related technologies are mostly limited to oral systemic delivery, broad-spectrum delivery, or ROS production applications, and lack a systematic and repeatable dosing and activation parameter system for pressure-related heart failure (such as the TAC model). Summary of the Invention

[0007] This invention primarily addresses the myocardial energy deficiency and mitochondrial dysfunction present in the above-mentioned stress-related heart failure / myocardial hypertrophy / myocardial fibrosis processes, providing a therapeutic solution that can locally enrich the myocardium and achieve on-demand, dose-controllable energy metabolism repair through exogenous red light.

[0008] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One objective of this invention is to provide a photoactivated myocardial-targeting nanovesicle for heart failure, which is a core-shell structured biomimetic membrane vesicle comprising: The core is a nanothylakoid unit, which is obtained by nano-sizing of plant-derived thylakoid membranes and retains photoreactivity; The outer shell is a biomimetic coating layer derived from cardiomyocyte membranes, which covers the surface of the core. The mass ratio of the biomimetic coating layer derived from the cardiomyocyte membrane to the nanothylakoid unit is 0.1:1 to 10:1; the hydrated particle size of the photoactivated cardiomyocyte-targeting nanothylakoid is 30-200 nm, and the zeta potential is -30 mV to +10 mV.

[0009] Preferably, the mass ratio of the biomimetic coating layer derived from the cardiomyocyte membrane to the nanothylakoid unit is 0.5:1 to 2:1.

[0010] Preferably, the hydrated particle size of the photoactivated myocardial-targeting nanothylakoids is 60-120 nm.

[0011] Preferably, the zeta potential of the photoactivated myocardial-targeting nanothylakoid is -25 mV to -10 mV.

[0012] The second objective of this invention is to provide a method for preparing the aforementioned photoactivated myocardial-targeting nanothylakoids, comprising the following steps: S1, Preparation of nanothylakoid units: The plant-derived thylakoid membrane suspension was subjected to ultrasonic disruption and extrusion homogenization, then purified by ultracentrifugation, and the purified precipitate was resuspended in the first buffer system to obtain the working solution of nanothylakoid units. S2, Preparation of a biomimetic coating layer derived from cardiomyocyte membrane: Isolate cell membrane components from cardiomyocytes and resuspend them in a second buffer system to obtain a cardiomyocyte membrane suspension; S3, the myocardial cell membrane suspension from step S2 is mixed and incubated with the working solution of the nano-thylakoid unit from step S1, and then the myocardial cell membrane is coated onto the surface of the nano-thylakoid unit by extrusion to form a biomimetic membrane vesicle with a core-shell structure.

[0013] Preferably, in step S1, the ultrasonic crushing power is 50-500 W and the time is 0.5-10 min; the extrusion homogenization involves passing the material through a polycarbonate membrane with a pore size of 50-200 nm, and the number of extrusions is 3-21; the centrifugal force of the ultracentrifugation is 50,000-150,000 × g and the time is 30-120 min.

[0014] Preferably, in step S1, the first buffer system contains HEPES, MgCl2 and ascorbate, and has a pH of 7.2-7.8.

[0015] Preferably, in step S2, the concentration of the myocardial cell membrane suspension is 0.5-5 mg / mL.

[0016] Preferably, in step S3, the volume ratio of the myocardial cell membrane suspension to the working solution of the nano-thylakoid unit is 0.5:1 to 2:1; the incubation temperature is 4-25℃, and the incubation time is 10-60 min; the extrusion method involves sequentially extruding the mixed and incubated materials through a polycarbonate membrane with decreasing pore size, wherein the decreasing pore size order is 400 nm, 200 nm, and 100 nm, and the number of extrusions per stage is 3-15 times.

[0017] A third objective of this invention is to provide the application of the aforementioned photoactivated myocardial-targeting nanothylakoids or the aforementioned preparation method in the preparation of products for treating stress-related cardiomyopathy, wherein the stress-related cardiomyopathy includes heart failure, myocardial hypertrophy, myocardial fibrosis, or ventricular remodeling; and the products include drugs.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1) Red light-controlled triggering of on-demand energy repair enables dosage control and on / off treatment. This invention uses nano-thylakoid units (NTUs) that retain the function of photoreactive membranes as the core and establishes a red light activation parameter system to directly link the material's energy output with the light dose, thereby overcoming the shortcomings of existing metabolic interventions that are "difficult to adjust in intensity and difficult to switch on and off". Experimental results show that intracellular ATP levels in the MM-NTU(+) group significantly increased under red light stimulation, while the enhancement in the dark state was limited, proving that it has the functional characteristics of exogenous controllable triggering.

[0019] 2) Cardiac cell membrane coating significantly enhances myocardial targeted uptake and local effective concentration, reducing the risk of off-target distribution. Biomimetic coating of cardiomyocyte membranes, with the outer layer retaining homologous membrane proteins and lipid structures, enhances membrane fusion and endocytosis with cardiomyocytes, improving local enrichment and delivery efficiency in the myocardium. Flow cytometry results showed that the uptake rate of MM-NTU in cardiomyocytes was approximately 18.9%, significantly higher than other membrane-derived coating systems. In vivo imaging showed that the material was mainly localized in the cardiac region and had good retention, suggesting reduced systemic off-target exposure.

[0020] 3) Establish quantifiable stability time windows and release criteria to improve formulation reproducibility and commercialization feasibility. This invention uses ATP output, D1 / D2 stability, and changes in Zeta potential over time as quality control criteria, clearly defining the functional window: energy output capacity begins to decline after approximately 16 hours of continuous illumination, decreases after approximately 7 days of dark storage, and remains relatively stable for the first 72 hours in the dark. These results provide an operable dosimetric basis for dosing frequency and illumination protocols, and are beneficial for batch-to-batch release and industrial-scale quality control.

[0021] 4) Evidence of mitochondrial protection and anti-remodeling efficacy was generated in stress-related pathologies, with clear organ-level endpoints. In the Ang II injury model, MM-NTU(+) restored mitochondrial membrane potential, improved mitochondrial ultrastructure, and downregulated hypertrophy / fibrosis markers such as β-MyHC, Col1A1, Tgfb1, and Myh7. In the TAC stress overload model, it further improved cardiac function indicators such as EF% and FS% and reduced fibrosis and hypertrophy. This demonstrates that the present invention not only increases ATP but also effectively inhibits adverse remodeling in the progression of stress overload-related heart failure, possessing clear preclinical translational value. Attached Figure Description

[0022] Figure 1 The figure shows the characterization results of the MM-NTU prepared in Example 1 of this invention.

[0023] Figure 2 This is a graph showing the functional output and stability evaluation results of the MM-NTU under red light activation in Embodiment 2 of the present invention.

[0024] Figure 3 This is a diagram showing the effect of MM-NTU in the Ang II-induced H9C2 cell damage model in Example 3 of the present invention.

[0025] Figure 4 This is a diagram showing the therapeutic effect of the TAC rat model in Example 3 of the present invention.

[0026] Figure 5This is a diagram showing the myocardial histological staining results of the TAC rat model in Example 3 of the present invention. Detailed Implementation

[0027] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0028] This invention addresses the myocardial energy deficiency and mitochondrial dysfunction present in stress-related heart failure, myocardial hypertrophy, and myocardial fibrosis. It provides a treatment solution for energy metabolism repair that can be locally enriched in the myocardium and triggered on demand with controllable dosage via exogenous red light. The closed-loop treatment system is constructed primarily through the following technical means: 1) Plant-derived thylakoid membranes were prepared as nanothylakoid units (NTUs) to retain their photoreaction-related membrane functions; 2) MM-NTU was constructed using biomimetic coating of cardiomyocyte membrane (MM) to enhance cardiomyocyte uptake and improve intracellular fate; 3) Set up a red light activation parameter system (wavelength / power density / irradiation duration / frequency) to achieve exogenous controllable triggering, thereby enhancing ATP production, stabilizing mitochondrial function, and inhibiting myocardial hypertrophy and fibrosis.

[0029] The following provides the range of structural and process parameters that can be repeatedly implemented, as well as the preferred solutions.

[0030] 1. Product Technical Solution: Myocardial-targeted, red light-activated biomimetic nanothylakoid system (MM-NTU) The MM-NTU described in this invention is a core-shell type biomimetic membrane vesicle structure, which comprises the following components: (A) Nanothylakoid unit (NTU): Membrane nanovesicles / nanomembranes obtained by separating and nano-sizing the thylakoid membrane of plant chloroplasts, serving as the core, containing a photoreactive membrane protein complex and a lipid bilayer; (B) Cardiac cell membrane coating (MM): derived from the cell membrane components of cardiomyocytes (containing membrane protein and lipid bilayer), used to coat (A) to form the outer biomimetic membrane; (C) Buffer system / stabilizing system: used to maintain thylakoid membrane activity and colloidal stability, including but not limited to HEPES, MgCl2, ascorbate, etc.; (D) Cryoprotectants: including but not limited to DMSO, trehalose, etc., for cryopreservation and thawing.

[0031] 1.1 Component ratio (applicable scope) The mass ratio of MM to NTU (based on membrane protein mass) is 0.1:1 to 10:1, preferably 0.5:1 to 2:1; The volume ratio of MM to NTU is 0.5:1 to 2:1, preferably 1:1; The concentration of MM suspension is 0.5-5 mg / mL, preferably 2 mg / mL; NTU concentration can be expressed as NTA count (particles / mL) or as membrane protein concentration (mg / mL). For ease of industrial-scale quality control, NTA count is preferred as the release indicator.

[0032] 1.2 Product Structure and Physicochemical Properties Morphological characterization: The complete vesicle structure is visible under transmission electron microscopy (TEM), and the outer edge boundary is clearer after encapsulation; the outer layer retains the characteristic proteins of cardiomyocyte membrane, thereby achieving selective uptake / membrane fusion of cardiomyocytes.

[0033] Hydrated particle size (Dynamic Light Scattering DLS / Nanoparticle Tracking Analysis NTA): 30-200 nm, preferably 60-120 nm; Zeta potential: -30 mV to +10 mV, preferably -25 mV to -10 mV, depending on the stability of the system dispersion; Dispersion stability: Short-term colloidal stability at room temperature or 4°C ≥24-72 h (depending on buffer system and concentration); Activity retention: In protective systems containing ascorbate, the phototriggered function can be maintained within a certain time window (which can be verified by the expression level of D1 / D2 proteins or the amount of ATP output).

[0034] 1.3 Raw material sources and pretreatment Plant materials: tender spinach leaves are preferred; alternatively, lettuce, cabbage, Arabidopsis thaliana, green algae, or wheat seedlings, which are rich in chloroplasts and thylakoids, can also be used.

[0035] Cell membrane raw materials: Cardiomyocyte sources include, but are not limited to, H9C2 cells, primary cardiomyocytes, iPSC-derived cardiomyocytes, or cell membranes derived from myocardial tissue.

[0036] 2. NTU preparation steps (implementable process) (1) Thylakoid membrane separation: Thylakoid membrane suspension was obtained under low temperature and light-protected conditions, following the chloroplast separation and thylakoid membrane extraction process.

[0037] (2) Nanoparticles (ultrasound): The thylakoid membrane suspension is subjected to water bath ultrasound or probe ultrasound for 0.5-10 min, preferably 1-3 min, and optimally 2 min; the ultrasound power is 50-500 W (adjusted according to the equipment), with the aim of not significantly increasing the temperature and not damaging the membrane proteins, preferably 200-400 W.

[0038] (3) Extrusion homogenization: The ultrasonically sonicated suspension is extruded through a polycarbonate membrane with a pore size of 50-200 nm, preferably 100 nm; the number of extrusions is 3-21, preferably 9-15; the operating temperature is 4-25℃, preferably 4-10℃ to protect the membrane activity.

[0039] (4) Ultracentrifugation purification: the centrifugation force is 50,000-150,000×g, preferably 80,000-120,000×g, and optimally 100,000×g; the centrifugation time is 30-120 min, preferably 45-90 min, and optimally 60 min; collect the precipitate and resuspend it.

[0040] (5) Resuspension buffer system: The buffer system contains HEPES-KOH 1-50 mM (preferably 10 mM), MgCl2 (preferably 10 mM), ascorbate 1-50 mM (preferably 10 mM), and pH preferably 7.2-7.8.

[0041] (6) Counting and concentration characterization: NTA was used to determine the particle concentration (particles / mL) as an indicator for subsequent drug addition and batch release.

[0042] (7) Cryopreservation (recommended): Add DMSO or other cryopreservation system, with a final concentration of DMSO of 5-15%, preferably 10%; cryopreservation conditions are -80℃; thaw on ice before use and wash with a buffer system 2-3 times to remove DMSO.

[0043] 3. Preparation steps of cardiomyocyte membrane (MM) (1) Cardiac cells were collected, washed with PBS, and then lysed; (2) Separate membrane components using differential centrifugation or a commercial cell membrane / membrane protein extraction kit; (3) Membrane resuspension: Resuspend in deionized water or isotonic buffer, and adjust the membrane concentration to 0.5-5 mg / mL, with the optimal concentration being 2 mg / mL; (4) Short-term storage: Store at -80℃ for short-term storage or prepare and use immediately (recommended).

[0044] 4. MM-NTU encapsulation assembly steps (1) Mixed incubation: Mix MM suspension and NTU suspension at a volume ratio of 0.5:1-2:1, preferably 1:1; incubation time is 10-60 min, preferably 20-40 min, and most preferably 30 min; incubation temperature is 4-25℃, preferably 4-10℃ to protect membrane activity.

[0045] (2) Multi-stage extrusion coating: a stable coating structure is formed by extruding 400 nm, 200 nm and 100 nm polycarbonate films in sequence, with each stage of extrusion being 3-15 times, preferably 5-11 times.

[0046] (3) Removal of free membrane and large aggregates: Use low speed centrifugation, centrifugation force 5,000-20,000×g, preferably 8,000-12,000×g; centrifugation time 3-10 min, the optimal centrifugation time is 10,000×g for 5 min; take the supernatant / resuspend to obtain MM-NTU working solution.

[0047] (4) Final particle size control: By combining “extrusion aperture + extrusion times + centrifugation conditions”, the particle size is adjusted to the preferred range of 60-120 nm.

[0048] 5. Methods of Use / Treatment Techniques 5.1 Indications / Uses The MM-NTU described in this invention is used for stress-related cardiomyopathy, including but not limited to: heart failure (HF), myocardial hypertrophy, myocardial fibrosis, and ventricular remodeling; it can also be used for metabolic improvement in diseases related to myocardial mitochondrial dysfunction (as a secondary use).

[0049] 5.2 Administration method and dosage (1) In vivo administration method: local myocardial injection is preferred (multiple points in the ventricular wall / subepicardial); coronary perfusion and intravenous injection can be selected as subordinate regimens, which need to be combined with dose and distribution assessment.

[0050] (2) Single injection volume: 20-200 µL, preferably 50-150 µL, optimal 100 µL / injection.

[0051] (3) Frequency of administration: once every 2-14 days, preferably once every 3-7 days, and optimally once every 3 days or once a week (determined according to the model and efficacy window).

[0052] (4) Dosage expression: Measured by the number of NTA particles per dose (particles / dose) or the amount of membrane protein (µg / dose). The number of particles is 10. 8 -10 12 particles / time, preferably 10 9 -10 11particles / session; protein content is 1-500 µg / session, preferably 10-200 µg / session.

[0053] 5.3 Red light activation parameters To achieve controllable external triggering, transthoracic red light irradiation was set: Wavelength: 620-700 nm, preferably 650-670 nm, optimal 660 nm; Power density: 10-200 mW / cm³ 2 Preferred value: 30-80 mW / cm 2 Optimal 50 mW / cm 2 ; Spot diameter: 5-15 mm, preferably 5 mm; Single irradiation duration: 1-30 min, preferably 5-15 min, optimal 10 min; Irradiation frequency: once every 1-7 days, preferably once every 3 days; Key points of operation: Shave hair, apply light coupling gel, cover eyes before irradiation, and use a constant temperature table to control body temperature to avoid heat damage.

[0054] 5.4 In vitro / in vivo efficacy evaluation indicators and experimental methods (1) In vitro energy metabolism and mitochondrial function: ATP content was measured using an ATP assay kit (comparing dark and light conditions before and after time curves); mitochondrial membrane potential was detected using JC-1 staining; ROS level was detected using DCFH-DA probe flow cytometry; cell hypertrophy / fibrosis markers (β-MyHC, etc.) were detected using qPCR / Western blot, or ANP / BNP were detected by ELISA.

[0055] (2) Cell uptake and membrane fusion / intracellular fate: Fluorescent labels (cell membrane DiI / material membrane DiO, etc.) were used to observe uptake, membrane fusion and line scanning by confocal microscopy; Lysosomal tracers such as LysoTracker were used to observe the colocalization of materials and lysosomes and to perform quantitative analysis.

[0056] (3) Animal model efficacy endpoint (pressure load model): a pressure load heart failure model was established by transverse aortic coarctation (TAC); cardiac function parameters such as EF% / FS% / LVID were assessed by echocardiography; myocardial hypertrophy and fibrosis were assessed by histological staining (H&E, Masson, WGA); heart weight ratio (HW / BW, HW / TL) was measured; biocompatibility was assessed by H&E staining of major organs, combined with serum biochemical / inflammatory indicators when necessary.

[0057] 6. Quality Control and Release Standards To ensure batch consistency, it is recommended to set the following quality control indicators: Particle size: 60-120 nm (preferred), optimal 80-100 nm; Morphology: TEM showed that the vesicles were intact and there was no obvious aggregation; Encapsulation validation: Western blot detection of myocardial membrane characteristic proteins / membrane markers; Particle concentration: NTA count (particles / mL) within the set window; Functional approval: The difference in ATP output before and after red light activation reaches the preset threshold.

[0058] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0059] Example 1: Preparation and Property / Coating Verification of MM-NTU 1) Preparation of NTU (1) Take plant tissue rich in chloroplast thylakoids (preferably fresh spinach leaves), remove the petioles, rinse quickly 2–3 times with pre-cooled deionized water at 4°C and blot dry the surface moisture; operate at 4°C and in the dark throughout the process, and pre-cool the centrifuge rotor and each buffer solution for later use. Add the spinach leaves to chloroplast separation buffer at a ratio of 1 g: 5–10 mL (preferably 1:8) for homogenization. The preferred chloroplast separation buffer is: 0.33 M sorbitol, 50 mM HEPES-KOH (pH 7.5–7.8, preferably pH 7.6), 2 mM EDTA, 1 mM MgCl2 and 5 mM ascorbate, and 0.1% (w / v) BSA can be added if necessary. Homogenization is performed at a low speed and for a short time, preferably 5–15 s × 2–3 times (with 10 s intervals to avoid temperature rise). The resulting homogenate is filtered through two layers of gauze supplemented with a 40–100 μm (preferably 60 μm) nylon mesh, and the filtrate is collected as crude chloroplast extract. The crude chloroplast extract is then centrifuged at 1,000 × g for 5 min at 4°C, the precipitate is discarded and the supernatant is retained; then it is centrifuged again at 3,000–6,000 × g for 5–10 min (preferably 4,000 × g for 7 min) to collect the green chloroplast precipitate. To improve purity, the chloroplast precipitate can be gently resuspended in pre-cooled washing buffer and the above 3,000–6,000 × g centrifugation step can be repeated once. The obtained chloroplast precipitate was then lysed using a hypotonic method to release the thylakoid membrane: resuspended in thylakoid lysis buffer and incubated on ice for 2–10 min (preferably 5 min). The thylakoid lysis buffer was preferably 10 mM HEPES-KOH (pH 7.5) and 5 mM MgCl2 (optionally 0–50 mM NaCl was added). After lysis, MgCl2 could be added to achieve a final concentration of 5–10 mM to promote thylakoid membrane structural stability. The lysed suspension was then centrifuged at 6,000–10,000 × g for 10–15 min (preferably 8,000 × g for 10 min) at 4 °C to collect the thylakoid membrane precipitate. The supernatant was discarded, and the thylakoid membrane was gently resuspended in thylakoid resuspension / preservation buffer to obtain a thylakoid membrane suspension. The thylakoid resuspension / preservation buffer was preferably 10 mM HEPES-KOH (pH 7.2–7.8, preferably pH 7.6), 10 mM MgCl2, and 10 mM ascorbate. To reduce the background of soluble proteins and improve batch consistency, the thylakoid membrane suspension can be washed once by centrifugation at 8,000×g for 10 min and then resuspended for later use.

[0060] (2) Nanoparticles of thylakoid membrane suspension: ultrasonic treatment under ice bath conditions (preferably 2 min), followed by homogenization by extrusion through polycarbonate membrane (preferably 100 nm membrane, extrusion 9–15 times, preferably 12 times) to obtain NTU.

[0061] (3) The suspension after extrusion is purified by ultracentrifugation (preferably 100,000×g, 60 min), the supernatant is discarded, and the precipitate is resuspended in a protective buffer system (HEPES+MgCl2+ascorbate, pH 7.2–7.8) to obtain NTU working solution.

[0062] (4) The particle concentration (particles / mL) recorded by NTA is used as the basis for subsequent formulation and dosing conversion.

[0063] 2) Extraction of myocardial cell membrane (MM) (1) Take H9C2 cells (purchased from Procyno Corporation, catalog number CL-0089), wash with PBS and lyse them; separate membrane components by differential centrifugation or membrane extraction kit.

[0064] (2) Resuspend and quantify the membrane components, and adjust the membrane protein concentration to 2 mg / mL (preferred).

[0065] 3) Assembly of MM-NTU (1) Mix MM and NTU at a volume ratio of 1:1 and incubate at 4°C for 30 min.

[0066] (2) The mixture is extruded through 400 / 200 / 100 nm membranes in sequence (5–11 times per stage) to promote membrane fusion and stable coating.

[0067] (3) Centrifuge at 10,000×g for 5 min to remove the free membrane and large aggregates, and take the supernatant to obtain MM-NTU.

[0068] 4) Identification results (corresponding to) Figure 1 ) (1) DLS showed that the original thylakoid membrane particles had a diameter of about 1047 nm; after nano-sizing and myocardial membrane coating, the MM-NTU particles had a diameter of about 87 nm.

[0069] (2) TEM shows that MM-NTU is a well-dispersed and uniform nanovesicle structure.

[0070] (3) Zeta potential shows that the surface electrical properties change after coating; it remains relatively stable for the first 72 hours under dark conditions.

[0071] (4) Western blot showed that MM-NTU simultaneously expressed the thylakoid marker protein AtpB and the myocardial marker protein Na. + / K + -ATPase and Connexin 43 proved successful encapsulation.

[0072] Example 1 Conclusion: This example established a repeatable optimal preparation process and completed key quality control (particle size, morphology, potential, membrane markers), and proved that it can obtain a stable MM-NTU product.

[0073] Example 2: Controllable Energy Output and Functional Time Window under Red Light Activation 1) Experimental grouping and treatment H9C2 cells were cultured under standard conditions (37°C, 5% CO2) and seeded into culture plates to achieve approximately 70%–80% confluence at the time of treatment. Cells were divided into a control group (Ctrl, with an equal volume of carrier buffer), an MM-NTU dark group (MM-NTU, Dark), and an MM-NTU light group (MM-NTU, Light). MM-NTU working solution was added to the treatment groups, with the preferred amount of MM-NTU being 1 × 10⁻⁶ particles. 4 –1×10 6 particles / cells, further preferred at 5×10 4 –5×10 5 The optimal particle / cell ratio is 2 × 10⁻⁶. 5 Particles / cell; or 0.5–50 μg / mL, preferably 2–20 μg / mL, based on membrane protein content. After addition, mix gently and incubate at 37°C for 1–12 h, preferably 4–8 h, optimally 6 h; after incubation, gently wash 1–3 times with PBS to remove untaken particles and replace with fresh culture medium.

[0074] 2) Red light activation parameters The dark-state group was cultured in complete darkness; the red-light group was irradiated with red light after incubation and washing, with the preferred wavelength of the red light being 620–700 nm, more preferably 650–670 nm, and optimally 660 nm; the preferred power density was 10–200 mW / cm². 2 Further optimization of 30–80 mW / cm 2 Optimal 50 mW / cm 2 The preferred duration of a single irradiation session is 1–30 min, further preferably 5–15 min, with 10 min being optimal. After irradiation, energy metabolism indicators such as ATP should be detected immediately or at a set time point according to the experimental design.

[0075] 3) Functional output and stability evaluation (corresponding to) Figure 2 ) (1) Under red light stimulation, the ATP in the MM-NTU(+) group was significantly higher than that in the dark / control group, proving that the energy metabolism enhancement effect can be triggered by exogenous control.

[0076] (2) Under continuous light conditions, the ATP production capacity decreased after 16 hours of continuous irradiation; under dark storage conditions, the ATP production capacity decreased after 7 days.

[0077] (3) During continuous illumination for 0–32 h, D1 / D2 gradually decreased; during dark conditions for 0–7 days, D1 / D2 remained relatively stable, supporting the material’s characteristic of being more stable in dark storage.

[0078] Example 2 Conclusion: This example demonstrates that the MM-NTU prepared by the present invention has red light controllable trigger output, and clearly provides a key time window (continuous illumination and dark storage) that can be used for dosimetric design.

[0079] Example 3: Validation of therapeutic efficacy in pressure overload-related cardiomyopathy To avoid having too many examples, this example uses in vivo TAC as the main evidence chain, and in the same example, in vitro Ang II mitochondria and hypertrophy indicators are added as mechanistic support.

[0080] A. The therapeutic effect of the TAC rat stress overload model (corresponding to) Figure 4 , 5 ) (1) A rat pressure overload model was established using transverse aortic coarctation (TAC). After anesthesia (intraperitoneal injection of sodium pentobarbital), a small incision was made in the upper sternum or the left second intercostal space to expose the aortic arch. A calibration needle (preferably 18–22G) was inserted between the brachiocephalic artery and the left common carotid artery. The aorta and the calibration needle were ligated simultaneously with silk suture (preferably 5-0), and then the calibration needle was removed to form a standardized coarctation. The control group (Con) underwent only thoracotomy and aortic dissection without ligation. Postoperatively, routine analgesia and anti-infection treatment were administered, and echocardiography was performed at predetermined time points to confirm ventricular remodeling and decreased systolic function caused by pressure overload. Animals were randomly divided into a control group (Con), a TAC group, a TAC+MM-NTU(–) group, and a TAC+MM-NTU(+) group. Among them, MM-NTU(–) was without red light activation, and MM-NTU(+) was MM-NTU with red light triggering energy output. Intramyocardial injection is preferred, with multiple injections at the left ventricular free wall (or near the apex). The preferred single injection volume is 20–200 μL, more preferably 50–150 μL, and optimally approximately 100 μL. The preferred dosing frequency is once every 3 days. The single dose of MM-NTU is 1 × 10⁻⁶ particles. 9 –1×10 11 particles / time, further optimized to 5×10 9 –5×10 10Particles / dose; or 10–200 μg / dose, preferably 50–150 μg / dose, based on membrane protein content. The TAC+MM-NTU(+) group underwent transthoracic red light irradiation (preferably 660 nm, 30–80 mW / cm²) according to a pre-defined protocol after each dose. 2 (5–15 min), the TAC+NTU(–) group did not receive red light irradiation or were treated in a dark state; at the end of the treatment cycle, cardiac function parameters such as LVIDd, EF%, and FS% were assessed by echocardiography, and the degree of myocardial hypertrophy and fibrosis was evaluated in combination with the heart weight / tibia length ratio (HW / TL) and histological staining.

[0081] (2) Intramyocardial injection is performed according to the above protocol, preferably once every 3 days.

[0082] (3) Echocardiography was used to assess LVIDd, HW / TL, EF%, FS%, etc.; the results showed that the TAC group had significant systolic function decline and remodeling, the MM-NTU(+) group had significantly improved EF% and FS% and partially restored structural parameters, while the NTU(–) group did not show significant improvement.

[0083] (4) Histological H&E showed myofibrosis disorder and interstitial widening in the TAC group; Masson's method showed significant fibrosis; WGA showed increased cross-sectional area of ​​cardiomyocytes. MM-NTU(+) treatment significantly alleviated the above changes and reduced CVF.

[0084] (5) Heart failure-related indicators ANP / BNP / NT-proBNP and inflammatory factors (IL-6, TNF-α) were significantly elevated in the TAC group and significantly decreased in the MM-NTU(+) group.

[0085] B. Validation of Ang II-induced H9C2 damage and mitochondrial hypertrophy (corresponding) Figure 3 ) (1) Angiotensin II (Ang II)-induced H9C2 cardiomyocyte injury model: H9C2 cells were cultured to approximately 70%–80% confluence and then treated with Ang II working solution for 24–48 h. The final concentration of Ang II was preferably 0.1–5 μM, and more preferably 0.5–2 μM. The experimental groups included: control group (Ctrl, treated with an equal volume of carrier), Ang II model group (AngII), Ang II+MM-NTU(–) group and Ang II+MM-NTU(+) group, where MM-NTU(+) was red light-activated MM-NTU, and MM-NTU(–) was control material without red light activation or lacking light-triggered output. The amount of MM-NTU used was 1×10⁻⁶ particles. 4 –1×10 6particles / cells, further preferred at 5×10 4 –5×10 5 The optimal particle / cell ratio is 2 × 10⁻⁶. 5 Particles / cell; or 0.5–50 μg / mL, preferably 2–20 μg / mL, based on membrane protein content. After Ang II induction, MM-NTU was added to the corresponding treatment group and incubated at 37°C for 1–12 h, preferably 4–8 h, optimally 6 h; subsequently, red light irradiation (preferably 660 nm, 30–80 mW / cm²) was performed consistently using the TAC / in vitro light-triggered protocol. 2 The JC-1 mitochondrial membrane potential, β-MyHC, Col1A1, Tgfb1, Myh7 and other indicators were measured (5–15 min) to evaluate the protective effect.

[0086] (2) JC-1: Ang II leads to a decrease in MMP; MM-NTU(+) significantly restores the red / green ratio.

[0087] (3) β-MyHC and qPCR: Ang II upregulated β-MyHC and Col1A1, Tgfb1, and Myh7; MM-NTU(+) significantly inhibited it.

[0088] (4) Mitochondrial cristae breakage, swelling, and vacuolization were observed in the TEMAng II group; the mitochondrial structure of the MM-NTU(+) group was significantly improved.

[0089] Example 3 Conclusion: This example demonstrates, starting from the functional-structural endpoint of TAC in vivo, that MM-NTU(+) can improve stress-induced cardiac function decline and remodeling, and further confirms its mitochondrial protective and anti-hypertrophy / anti-fibrotic molecular effects using the Ang II in vitro model.

[0090] Note: The specific experimental results and corresponding figures above were obtained from the optimized / optimal parameters.

[0091] In summary, this invention successfully constructed a photoactivated myocardial-targeting nanothylakoid for heart failure. This nanothylakoid achieves targeted enrichment in the myocardium through biomimetic encapsulation of the myocardial cell membrane and controllably outputs energy under red light irradiation, repairing metabolic defects in myocardial cells. Ultimately, its significant efficacy in improving cardiac function and inhibiting ventricular remodeling was demonstrated in a pressure-overload heart failure model. This invention provides a novel strategy with clinical translational potential for the treatment of heart failure.

[0092] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A photoactivated myocardial-targeting nanothylakoid for heart failure, characterized in that, It is a biomimetic membrane vesicle with a core-shell structure, including: The core is a nanothylakoid unit, which is obtained by nano-sizing of plant-derived thylakoid membranes and retains photoreactivity; The outer shell is a biomimetic coating layer derived from cardiomyocyte membranes, which covers the surface of the core. The mass ratio of the biomimetic coating layer derived from the cardiomyocyte membrane to the nanothylakoid unit is 0.1:1 to 10:1; the hydrated particle size of the photoactivated cardiomyocyte-targeting nanothylakoid is 30-200 nm, and the zeta potential is -30 mV to +10 mV.

2. The photoactivated myocardial-targeting nanothylakoid according to claim 1, characterized in that, The mass ratio of the biomimetic coating layer derived from the cardiomyocyte membrane to the nanothylakoid unit is 0.5:1 to 2:

1.

3. The photoactivated myocardial-targeting nanothylakoid according to claim 1, characterized in that, The hydrated particle size of the photoactivated myocardial-targeting nanothylakoids is 60-120 nm.

4. The photoactivated myocardial-targeting nanothylakoid according to claim 1, characterized in that, The zeta potential of the photoactivated myocardial-targeting nanothylakoids is -25 mV to -10 mV.

5. The method for preparing photoactivated myocardial-targeting nanothylakoids according to any one of claims 1-4, characterized in that, Includes the following steps: S1, Preparation of nanothylakoid units: The plant-derived thylakoid membrane suspension was subjected to ultrasonic disruption and extrusion homogenization, then purified by ultracentrifugation, and the purified precipitate was resuspended in the first buffer system to obtain the working solution of nanothylakoid units. S2, Preparation of a biomimetic coating layer derived from cardiomyocyte membrane: Isolate cell membrane components from cardiomyocytes and resuspend them in a second buffer system to obtain a cardiomyocyte membrane suspension; S3, the myocardial cell membrane suspension from step S2 is mixed and incubated with the working solution of the nano-thylakoid unit from step S1, and then the myocardial cell membrane is coated onto the surface of the nano-thylakoid unit by extrusion to form a biomimetic membrane vesicle with a core-shell structure.

6. The preparation method according to claim 5, characterized in that, In step S1, the ultrasonic crushing power is 50-500 W and the time is 0.5-10 min; the extrusion homogenization involves passing the material through a polycarbonate membrane with a pore size of 50-200 nm, and the number of extrusions is 3-21; the centrifugal force of the ultracentrifugation is 50,000-150,000 × g and the time is 30-120 min.

7. The preparation method according to claim 5, characterized in that, In step S1, the first buffer system contains HEPES, MgCl2 and ascorbate, and has a pH of 7.2-7.

8.

8. The preparation method according to claim 5, characterized in that, In step S2, the concentration of the myocardial cell membrane suspension is 0.5-5 mg / mL.

9. The preparation method according to claim 5, characterized in that, In step S3, the volume ratio of the myocardial cell membrane suspension to the working solution of the nano-thylakoid unit is 0.5:1 to 2:1; the incubation temperature is 4-25℃, and the incubation time is 10-60 min; the extrusion method involves sequentially extruding the mixed and incubated materials through a polycarbonate membrane with decreasing pore size, wherein the decreasing pore size order is 400 nm, 200 nm, and 100 nm, and the number of extrusions per stage is 3-15 times.

10. The application of the photoactivated myocardial-targeting nanothylakoids according to any one of claims 1-4 or the preparation method according to any one of claims 5-9 in the preparation of products for treating stress-related cardiomyopathy, characterized in that, The pressure-related cardiomyopathy includes heart failure, myocardial hypertrophy, myocardial fibrosis, or ventricular remodeling; the product includes a drug.

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