Hydrogen sulfide donor delivery system for spinal cord injury blood spinal cord barrier repair as well as preparation method and application of hydrogen sulfide donor delivery system

Abstract

The invention relates to the technical field of biomedicine nanotechnology, and discloses a hydrogen sulfide donor delivery system for spinal cord injury and blood spinal cord barrier repair as well as a preparation method and application of the hydrogen sulfide donor delivery system. The delivery system takes mesoporous polydopamine nanoparticles MPDA as a core carrier, the surface of the MPDA is covalently modified with targeted polypeptide c (RGDyK), a hydrogen sulfide donor SPRC is loaded in mesopores of the MPDA, and SPRC-coated MPDA-RGD composite nanoparticles are formed. The application provides an innovative solution for targeted nano treatment of spinal cord injury, and has a wide clinical transformation prospect.

Description

Technical Field

[0001] This application relates to the field of biomedical nanotechnology, and more specifically, to a hydrogen sulfide donor delivery system for repairing the blood-spinal cord barrier in spinal cord injury, its preparation method, and its application. Background Technology

[0002] Disruption of the blood-spinal barrier (BSCB) following spinal cord injury (SCI) is a key pathological step leading to secondary injury and neurological deficits. Currently, there is a lack of effective treatment strategies in clinical practice that can precisely target the injury site and synergistically address multiple pathological mechanisms. Existing hydrogen sulfide (H2S) donors, such as NaHS, suffer from uncontrollable release and potential cytotoxicity. While the endogenous H2S donor S-propynyl-L-cysteine ​​(SPRC) can upregulate cystathionine-γ-lyase (CSE) expression, thereby promoting endogenous H2S production and is considered safer, it still faces technical bottlenecks such as poor targeting, short in vivo circulation time, and rapid metabolic clearance.

[0003] Therefore, if a delivery system with good targeting, controllable release of hydrogen sulfide, and high biocompatibility can be developed, it will provide new ideas for overcoming the limitations of existing donors in the treatment of spinal cord injury. Summary of the Invention

[0004] The purpose of this invention is to provide a hydrogen sulfide donor delivery system (SPRC@MPDA-RGD) for repairing the blood-spinal barrier in spinal cord injury, its preparation method, and its applications. The core of this system lies in constructing a multifunctional integrated nanoplatform that specifically repairs the spinal cord injury barrier (BSCB) through active targeting, controlled release, and synergistic effects of multiple mechanisms. It particularly addresses the problems of low drug targeting efficiency, the inability of single therapies to cope with complex pathological environments, and the significant side effects of traditional H2S donors in existing technologies.

[0005] To achieve the above-mentioned objectives, this application provides the following technical solution: In a first aspect, this application provides a hydrogen sulfide donor delivery system for repairing the blood-spinal barrier in spinal cord injury. The delivery system uses mesoporous polydopamine nanoparticles (MPDA) as the core carrier. The surface of the MPDA is covalently modified with a targeting peptide c (RGDyK), and the mesopores of the MPDA are loaded with a hydrogen sulfide donor (SPRC) to form SPRC@MPDA-RGD composite nanoparticles.

[0006] Secondly, this application provides a method for preparing the delivery system described in the first aspect, comprising the following steps: Step 1: MPDA carrier synthesis Dopamine hydrochloride and F127 were mixed in a solvent, and a pore expander and a catalyst were added. The emulsion polymerization reaction was carried out at room temperature. After the reaction was completed, the product was collected by centrifugation, washed, and MPDA nanoparticles were obtained. Step 2: Preparation of MPDA-RGD The c(RGDyK) solution was added to the MPDA solution, the reaction was stirred, and the mixture was purified by centrifugation to obtain MPDA-RGD nanoparticles. Step 3: SPRC Drug Loading SPRC was dissolved in a solvent, MPDA-RGD nanoparticles were added, and the mixture was dispersed with ultrasonic assistance. The mixture was stirred in the dark at room temperature, and unloaded SPRC was removed by dialysis or centrifugation. After freeze-drying, the SPRC@MPDA-RGD nanodelivery system was obtained.

[0007] Furthermore, in step one, the solvent is a mixture of deionized water and ethanol; the pore-expanding agent is trimethylbenzene; and the catalyst is ammonia.

[0008] Furthermore, the feed ratio of the dopamine hydrochloride monomer, F127, pore expander and catalyst is 0.15g:0.1g:160μL:400μL.

[0009] Furthermore, in step two, c(RGDyK) and MPDA are respectively added to deionized water to prepare solutions, and then the c(RGDyK) solution is added to the MPDA solution, with the mass ratio of c(RGDyK) to MPDA in the mixed solution being 10:1.

[0010] Furthermore, in step three, the solvent is methanol; the concentration of the SPRC solution is 1-5 mg / mL.

[0011] Furthermore, in step three, the feeding ratio of MPDA-RGD nanoparticles to SPRC solution is 2-10 mg: 10 mL.

[0012] Thirdly, this application provides the use of the delivery system described in the first aspect in the preparation of a medicament for the prevention and / or treatment of blood-spinal cord barrier repair after spinal cord injury.

[0013] This application has the following beneficial effects: 1. Improve drug delivery accuracy by targeting damaged blood-spinal cord barriers with peptide-targeted design; 2. Combining hydrogen sulfide signaling regulation and antioxidant function, it enhances therapeutic efficacy through multi-pathway synergistic action; 3. The nanocarrier is biodegradable, reducing systemic toxic side effects.

[0014] In summary, this invention provides an innovative solution for targeted nanotherapy of spinal cord injury and has broad prospects for clinical translation. Attached Figure Description

[0015] Figure 1 MPDA transmission electron microscopy imaging, scale bar: 100 nm; Figure 2 Hydrodynamic diameter distributions of MPDA, SPRC@MPDA, and SPRC@MPDA-RGD measured by dynamic light scattering (DLS) technology; Figure 3 Hydrodynamic dimensions of SPRC@MPDA-RGD dispersed in PBS for 7 days; Figure 4 Zeta potentials of MPDA, SPRC@MPDA, and SPRC@MPDA-RGD; Figure 5 : Evaluation of CAT-like activity of different nanoparticles by UV-Vis spectroscopy; Figure 6 Evaluation of the antioxidant capacity of different nanoparticles in scavenging hydroxyl radicals (·OH); Figure 7 : For superoxide anion (·O2) - The antioxidant capacity of different nanoparticles was assessed. Figure 8 Reduces Evans Blue exudation and protects the blood-spinal cord barrier; Figure 9 Improved neurological function: Significantly improved hindlimb motor function score (BMS score); Figure 10 It reduces pathological damage and regulates the levels of inflammatory factors (reducing IL-1β, IL-6, and TNF-α, and increasing IL-4 and IL-10). Figure 11 Immunofluorescence of barrier-related proteins in mouse microvascular endothelial cells was significantly restored under the action of drugs after glucose-oxygen deprivation; Figure 12 Increase the hydrogen sulfide content in the spinal cord; Figure 13 It activates the ferrophagy pathway and reduces intracellular iron levels. Detailed Implementation

[0016] The technical solutions and effects of this application will be further described in detail below with reference to the embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely for explaining the invention and are not intended to limit the invention.

[0017] This invention discloses a hydrogen sulfide donor delivery system for repairing the blood-spinal cord barrier in spinal cord injury, its preparation method, and its application.

[0018] The system utilizes mesoporous polydopamine nanoparticles (MPDA) to carry SPRC drugs, and further functionalizes them with c(RGDyK) peptides to ultimately form a hydrogen sulfide delivery system, SPRC@MPDA-RGD. This delivery system achieves specific drug delivery to the spinal cord injury site by actively targeting endothelial cells at the injury site. It also efficiently repairs the damaged blood-spinal cord barrier after injury by activating the endogenous hydrogen sulfide signaling pathway and scavenging reactive oxygen species, thus promoting functional recovery after spinal cord injury. This system primarily addresses the problems of poor drug targeting, low bioavailability, and difficulty in simultaneously addressing multiple pathological mechanisms (such as oxidative stress, ferrophagy, and inflammatory responses) in existing spinal cord injury treatments, offering advantages such as precise delivery, synergistic effects, and high biocompatibility.

[0019] In terms of system composition, SPRC@MPDA-RGD uses mesoporous polydopamine nanoparticles as the core scaffold, with c(RGDyK) targeting peptides covalently modified on their surface, and SPRC loaded internally as a hydrogen sulfide donor. The preparation process includes: dissolving dopamine hydrochloride and F127 copolymers in a deionized water-ethanol mixed solvent, adding pore-expanding agent trimethylbenzene (TMB) and ammonia water as catalysts, and forming MPDA nanoparticles with a mesoporous structure through emulsion polymerization. After centrifugation and washing, the carrier material is obtained. Subsequently, surface functionalization is performed using c(RGDyK) peptides. MPDA nanoparticles are mixed with the c(RGDyK) peptide solution, and the targeting peptide is covalently grafted onto the MPDA surface through a Michael addition reaction to obtain the MPDA-RGD complex, which is then purified by centrifugation for later use. Finally, SPRC is dissolved in methanol and added to MPDA-RGD nanoparticles. Efficient loading of SPRC in high specific surface area mesoporous structures is achieved through ultrasonic dispersion and continuous stirring. The final product is purified by dialysis to obtain the SPRC@MPDA-RGD nanodelivery system.

[0020] This system relies on the active targeting mediated by the c(RGDyK) peptide to specifically recognize α-reactive protein overexpressed in endothelial cells after spinal cord injury. v β3 integrin enables the accumulation of nanoparticles at lesion sites. Once inside cells, the release of SPRC upregulates the expression of endogenous CSE enzymes, promotes the generation of endogenous hydrogen sulfide, and simultaneously, the polydopamine shell effectively scavenge reactive oxygen species, synergistically inhibiting lipid peroxidation and ferroptosis. Mechanistic studies show that SPRC@MPDA-RGD can activate the PI3K / Akt / mTOR signaling pathway, inhibit ferritin phagocytosis mediated by nuclear receptor coactivator 4, and reduce ferritin degradation and free Fe. 2+This release of iron maintains intracellular iron homeostasis and alleviates endothelial cell ferroptosis. In animal models, the system significantly restored the integrity of the blood-spinal cord barrier, reduced local inflammatory responses, and promoted the recovery of neurological function.

[0021] The advantages of this invention are: firstly, by targeting the damaged blood-spinal cord barrier with peptides, the precision of drug delivery is improved; secondly, by combining hydrogen sulfide signaling regulation and antioxidant function, the therapeutic efficacy is enhanced through multi-pathway synergy; and thirdly, the nanocarrier is biodegradable, reducing systemic toxicity. This invention provides an innovative solution for targeted nanotherapy of spinal cord injury and has broad prospects for clinical translation.

[0022] Example 1. Core components and structural design of nanodelivery systems Core carrier – mesoporous polydopamine nanoparticles (MPDA): Size and morphology: Synthesized using an optimized template method, transmission electron microscopy (TEM) revealed monodisperse spherical nanoparticles with a regular mesoporous structure. The hydration kinetic diameter was controlled to approximately 218.5 ± 8.7 nm, a size favorable for long-term in vivo circulation and targeting of damaged BSCBs.

[0023] Material Functions: The polydopamine backbone endows it with excellent biocompatibility and biodegradability. Its abundant phenolic hydroxyl and amino groups on the surface give it powerful enzyme-mimicking activity, effectively scavenging hydrogen peroxide (H₂O₂), hydroxyl radicals (·OH), and superoxide anions (·O₂). 2- It contains multiple ROS such as α, β, and γ, which directly combat oxidative stress at the damaged site.

[0024] Therapeutic load – S-propynyl-L-cysteine ​​(SPRC): Drug loading and encapsulation efficiency: SPRC is loaded into the mesopores of MPDA via diffusion, achieving continuous and mild intracellular endogenous H2S generation.

[0025] Targeting module – c(RGDyK) cyclic peptide: Connection method: c(RGDyK) undergoes a Michael addition reaction with the quinone group on the surface of MPDA through its terminal thiol group, achieving a covalent and stable connection to form the final SPRC@MPDA-RGD.

[0026] Targeting mechanism: c(RGDyK) can specifically and with high affinity recognize and bind to integrin α, which is highly expressed on neovascular endothelial cells after spinal cord injury. v β3. This design enables the nanosystem to actively target diseased BSCBs.

[0027] Characterization parameters: After functionalization, the hydration diameter of the nanoparticles increased slightly (to about 269.3 nm), and the Zeta potential changed from about -27.8 mV at MPDA to about -12.2 mV, indicating that the peptide was successfully modified.

[0028] 2. Preparation process of SPRC@MPDA-RGD Step 1: MPDA carrier synthesis 0.15 g of dopamine hydrochloride (Aladdin Reagent, China) and 0.1 g of F127 (Sigma-Aldrich, USA) were mixed in a 1:1 mixture of 20 mL of deionized water and ethanol and stirred at 350 rpm for 3 hours. Then, 160 μL of pore-expanding agent trimethylbenzene (TMB) (Aladdin Reagent, China) was added, and the mixture was sonicated for 2 minutes. Next, 400 μL of ammonia solution (Guangzhou Chemical Reagent Factory, China) was added. After stirring at 350 rpm for 2 hours, the mixture was centrifuged at 15,000 rpm and washed multiple times with ethanol and water to obtain MPDA nanoparticles.

[0029] Step 2: c(RGDyK) targeted functionalization (preparation of MPDA-RGD) c(RGDyK) (Gier Biotech, China) was modified onto the surface of MPDA via a Michael addition reaction. The specific steps were as follows: c(RGDyK) and MPDA were separately dissolved in deionized water to prepare solutions. Then, the c(RGDyK) solution was added to the MPDA solution, with a mass ratio of c(RGDyK) to MPDA of 10:1. The mixture was stirred at 350 rpm for 24 hours. The product was obtained by centrifugation (15000 rpm, 10 minutes) and washed three times with deionized water.

[0030] Step 3: SPRC drug loading (preparation of SPRC@MPDA-RGD) SPRC (MCE, USA) was dissolved in methanol to prepare a 1-5 mg / mL solution in 10 mL. 2-10 mg of MPDA-RGD was added, and the mixture was sonicated for 1 hour. The mixture was then stirred at 350 rpm for 24 hours, followed by dialyzing with 500 mL of deionized water (molecular weight cutoff 3500 Daltons) for 3 days. The product was collected for later use. Unloaded SPRC was removed by dialysis or centrifugation, and the final SPRC@MPDA-RGD nanodelivery system was obtained after freeze-drying.

[0031] 3. Analysis of Mechanism of Action and Technological Advantages (1) Active targeting and enrichment capabilities SPRC@MPDA-RGD, with its surface c(RGDyK) peptide, specifically recognizes and binds to the injured spinal cord region in the systemic circulation after intravenous injection to express α. vβ3 integrin is efficiently enriched in vascular endothelial cells at lesion sites.

[0032] (2) Dual synergistic treatment mechanism Endogenous H2S Regulation: After the nanoparticles are internalized by cells, SPRC is slowly released from the mesopores, upregulating the expression of cystathionine-γ-lyase (CSE), promoting the body's own production of physiological levels of H2S, and increasing the hydrogen sulfide content in the spinal cord. Figure 12 This avoids the toxic side effects of exogenous donors.

[0033] Highly efficient ROS removal: The MPDA framework acts as a broad-spectrum ROS scavenger, directly neutralizing excess ROS at the damaged site and reducing oxidative damage.

[0034] (3) Core molecular pathways H2S activates the PI3K / Akt / mTOR intracellular signaling pathway and significantly inhibits nuclear receptor coactivator 4 (NCOA4)-mediated ferritin phagia. Inhibition of ferritin phagia prevents the release of large amounts of free Fe. 2+ The release of these substances helps maintain intracellular iron homeostasis. Ultimately, by blocking the chain of "ferritin autophagy → iron overload → lipid peroxidation," the study effectively inhibits ferroptosis in vascular endothelial cells, thus protecting and repairing the integrity of BSCBs—a key mechanism.

[0035] 4. Performance Verification of SPRC@MPDA-RGD In a mouse model of spinal cord contusion, mice were anesthetized with 2% isoflurane, their skin was prepared and disinfected, and a laminectomy was performed at the T10 vertebra to expose the spinal cord. Then, a spinal cord impactor was used to induce spinal cord injury in mice with parameters of speed 3.0 m / s, depth 1 mm, compression time 0.5 s, and impactor diameter 1 mm. Mice in the sham surgery group underwent laminectomy only at the same level and did not induce spinal cord injury.

[0036] After the model was established, mice were divided into six groups: sham-operated group, spinal cord injury group, spinal cord injury + MPDA, spinal cord injury + SPRC, spinal cord injury + SPRC@MPDA, and spinal cord injury + SPRC@MPDA-RGD. Intravenous injection of SPRC@MPDA-RGD significantly restored BSCB function. Reduce Evans Blue exudation and protect the blood-spinal barrier. Figure 8 ); Improved neurological function: Significantly improved hindlimb motor function score (BMS score) Figure 9 ); Reduce pathological damage and regulate inflammatory factor levels (reduce IL-1β, IL-6, and TNF-α, and increase IL-4 and IL-10). Figure 10).

[0037] Meanwhile, these results were also well validated in standardized in vitro cell experiments. When mouse microvascular endothelial cells (bend.3) were subjected to oxidative stress simulation, the immunofluorescence of barrier-related proteins, which had been reduced due to glucose-oxygen deprivation, was restored under drug treatment. Figure 11 ), activates the ferrophagy pathway and reduces intracellular iron levels ( Figure 13 ).

[0038] Therefore, the hydrogen sulfide donor delivery system (SPRC@MPDA-RGD) provides a highly efficient, safe, and promising nanomedicine platform for addressing the clinical challenges of spinal cord injury treatment.

[0039] In summary, the beneficial effects of this invention are: 1. Precise targeting: Through active targeting mediated by c(RGDyK) peptide, the hydrogen sulfide donor delivery system (SPRC@MPDA-RGD) greatly increases the concentration of the drug at the lesion site, improves efficacy and reduces systemic side effects.

[0040] 2. Synergistic Mechanism: The system has both endogenous H2S signal regulation (upregulating CSE through SPRC) and highly efficient broad-spectrum antioxidant function (clearing ROS through MPDA), providing synergistic treatment through multiple pathways.

[0041] 3. Safe and controllable: It adopts an endogenous H2S generation mode, which is more in line with physiological state; the MPDA carrier has high biocompatibility and is degradable.

[0042] 4. Highly innovative: For the first time, the mechanism of "ferroautophagy" is linked to the repair of BSCB in the treatment of SCI with nanomedicine, providing a brand-new strategy and idea for nanotherapy of spinal cord injury.

[0043] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A hydrogen sulfide donor delivery system for repairing the blood-spinal barrier in spinal cord injury, characterized in that, The delivery system uses mesoporous polydopamine nanoparticles (MPDA) as the core carrier. The surface of the MPDA is covalently modified with a targeting peptide c (RGDyK), and the mesopores of the MPDA are loaded with a hydrogen sulfide donor (SPRC), forming SPRC@MPDA-RGD composite nanoparticles.

2. The method for preparing the delivery system according to claim 1, characterized in that, Includes the following steps: Step 1: MPDA carrier synthesis Dopamine hydrochloride and F127 were mixed in a solvent, and a pore expander and a catalyst were added. The emulsion polymerization reaction was carried out at room temperature. After the reaction was completed, the product was collected by centrifugation, washed, and MPDA nanoparticles were obtained. Step 2: Preparation of MPDA-RGD The c(RGDyK) solution was added to the MPDA solution, the reaction was stirred, and the mixture was purified by centrifugation to obtain MPDA-RGD nanoparticles. Step 3: SPRC Drug Loading SPRC was dissolved in a solvent, MPDA-RGD nanoparticles were added, and the mixture was dispersed with ultrasonic assistance. The mixture was stirred in the dark at room temperature, and unloaded SPRC was removed by dialysis or centrifugation. After freeze-drying, the SPRC@MPDA-RGD nanodelivery system was obtained.

3. The method for preparing the delivery system according to claim 2, characterized in that, In step one, the solvent is a mixture of deionized water and ethanol; the pore-expanding agent is trimethylbenzene; and the catalyst is ammonia.

4. The method for preparing the delivery system according to claim 3, characterized in that, The feed ratio of the dopamine hydrochloride monomer, F127, pore expander and catalyst is 0.15g:0.1g:160μL:400μL.

5. The method for preparing the delivery system according to claim 2, characterized in that, In step two, c(RGDyK) and MPDA are added to deionized water to prepare solutions, and then the c(RGDyK) solution is added to the MPDA solution. The mass ratio of c(RGDyK) to MPDA in the mixed solution is 10:

1.

6. The method for preparing the delivery system according to claim 2, characterized in that, In step three, the solvent is methanol; the concentration of the SPRC solution is 1-5 mg / mL.

7. The method for preparing the delivery system according to claim 6, characterized in that, In step three, the feeding ratio of MPDA-RGD nanoparticles to SPRC solution is 2-10 mg: 10 mL.

8. Use of the delivery system of claim 1 in the preparation of a medicament for the prevention and / or treatment of blood-spinal cord barrier repair after spinal cord injury.

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