Polydopamine / drug nanocomposite particles, preparation method and application thereof

Abstract

The application discloses a kind of polydopamine / drug nano composite particles and preparation method and application thereof.The polydopamine / drug nano composite particles in aqueous solution, using the synchronous process of alkaline drug molecule crystallization and dopamine oxidative polymerization, green synthesis nano drug.The polydopamine / drug nano composite particles of the application have: (1) high drug loading efficiency, simple preparation method; (2) have excellent active oxygen scavenging capacity; (3) response active oxygen controlled release drug; (4) the active oxygen of synthesis nano drug retains the activity of drug, such as nanofasudil nano drug can inhibit ROCK signal pathway; (5) good biocompatibility; can significantly reduce the ROS level in diabetic retinopathy model, reduce the expression of inflammatory factors, improve vascular leakage and inhibit neovascularization.The preparation process of the application is simple, the reaction condition is mild, and the biocompatibility is good, which provides a new nano drug delivery strategy for the treatment of diabetic retinopathy.

Description

Technical Field

[0001] This invention relates to a nanomedicine, specifically to a polydopamine / drug nanocomposite particle, its preparation method, and its application in the treatment of diabetic retinopathy, etc., belonging to the field of nanomedicine and ophthalmic disease treatment technology. Background Technology

[0002] Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes, caused by metabolic disorders induced by long-term hyperglycemia. Its pathological features include retinal vascular leakage, ischemia and hypoxia, neuroinflammation, and pathological neovascularization. It is estimated that about one-third of diabetic patients worldwide suffer from DR, with about 10% progressing to proliferative diabetic retinopathy (PDR), a vision-threatening condition and the leading cause of blindness among working-age individuals. Current clinical treatments include laser photocoagulation, anti-vascular endothelial growth factor (VEGF) injections, and vitrectomy. While these methods can slow disease progression, they have limitations such as significant trauma, high recurrence rates, and high costs, and cannot fundamentally reverse the oxidative damage and inflammatory microenvironment of retinal tissue.

[0003] Studies have shown that the pathogenesis of diabetic retinopathy (DR) is closely related to oxidative stress caused by excessive accumulation of reactive oxygen species (ROS). Under hyperglycemic conditions, dysfunction of the mitochondrial electron transport chain, activation of the polyol pathway, and accumulation of advanced glycation end products (AGEs) can all promote ROS bursts, thereby activating inflammatory pathways such as nuclear factor-κB (NF-κB), upregulating the expression of cytokines such as VEGF and interleukin-6 (IL-6), and exacerbating blood-retinal barrier disruption and neurovascular unit damage. Furthermore, ROS can also promote endothelial cell apoptosis and pericyte migration by activating the Rho-associated coiled-coil kinase (ROCK) signaling pathway, further aggravating retinal microvascular dysfunction. Therefore, a dual intervention strategy targeting ROS clearance combined with ROCK pathway inhibition may become a new direction for overcoming current therapeutic bottlenecks.

[0004] Fasudil, as a Rho kinase (ROCK) inhibitor, has been proven to alleviate various vascular-related complications. However, its clinical application is primarily in the form of derivatized salts, which suffer from poor targeting, short half-life, and limited duration of efficacy, thus restricting its effectiveness in treating diabetic retinopathy. The endogenous neurotransmitter dopamine can undergo oxidative polymerization in a weakly alkaline environment, assembling into size-controllable porous nanoparticles (PDAs). These PDAs possess excellent biocompatibility, antioxidant activity, drug loading capacity, and responsive release capabilities, making them ideal materials for nanomedicine. Therefore, developing a PDA-based fasudil nanodelivery system, combining its dual functions of ROS scavenging and ROCK signaling pathway inhibition, holds promise for providing a more efficient and safer treatment strategy for diabetic retinopathy.

[0005] This invention achieves a green synthesis of fasudil nanomedicines by controlling the crystallization and dopamine polymerization of fasudil through solution acidity / alkalinity regulation. This controlled release of fasudil also provides antioxidant effects. Currently, no research reports have combined dopamine oxidation and fasudil crystallization processes to synthesize nanomedicines, nor their application in the treatment of diabetic retinopathy (DR). Furthermore, similar alkaline drugs (dissolved in acidic solutions, crystallizing or precipitating in neutral or alkaline solutions), including doxorubicin hydrochloride, imiquimod, and lidocaine, can also utilize similar synthetic nanomedicines for the treatment of oxidative stress or drug-related diseases. Summary of the Invention

[0006] The purpose of this invention is to provide a polydopamine / drug nanocomposite particle, its preparation method, and its application. The aim is to combine the scavenging of reactive oxygen species (ROS) and inhibition of rockburst (ROK), protecting endothelial and nerve cell function, and inhibiting angiogenesis, thereby improving the pathological damage of diabetic retinopathy.

[0007] To achieve the above objectives, the present invention provides the following technical solution: The preparation method of polydopamine / drug nanocomposite particles includes the following steps: S1: Dissolve dopamine hydrochloride and an alkaline drug in water to obtain a mixed solution of dopamine and the drug; S2: Add an alkaline solution to the above dopamine and drug mixture under stirring conditions to adjust the pH of the solution to alkaline. React at room temperature for 4-48 hours to obtain the reaction product. S3: After centrifuging, washing, and freeze-drying the reaction product, polydopamine / drug nanocomposite particles are obtained.

[0008] The solubility of the alkaline drug is pH-dependent, meaning it can dissolve in acidic aqueous solutions and crystallize or precipitate in neutral or alkaline aqueous solutions. The alkaline drug includes, but is not limited to, fasudil, doxorubicin hydrochloride, imiquimod, or lidocaine.

[0009] In step S2, the pH of the dopamine and drug mixture solution is adjusted to 8.0~11.0.

[0010] The alkaline solution can be Tris-HCl buffer, ammonia, sodium hydroxide solution, or sodium carbonate solution, etc.

[0011] In the dopamine and drug mixture solution, the concentration of dopamine is 0.1~10 mg / mL.

[0012] In the dopamine and drug mixture solution, the concentration of the alkaline drug is 0.1~5 mg / mL, and the mass ratio of the alkaline drug to dopamine is 1:0.2~1:20.

[0013] The present invention also provides polydopamine-based nanomedicines prepared by any of the above preparation methods.

[0014] The nanomedicine has a particle size of 50 to 500 nm.

[0015] This invention also provides the application of the above-mentioned nanomedicines in the preparation of drugs that respond to or release reactive oxygen species or drugs for the treatment of oxidative stress-related diseases.

[0016] The present invention further provides the application of the fasudil nanomedicine in the preparation of therapeutic drugs for diabetic retinopathy, and the application of the fasudil nanomedicine in the preparation of therapeutic drugs for diseases related to the ROCK pathway or oxidative stress.

[0017] Performance and applications of polydopamine-based fasudil nanomedicines: 1. The polydopamine-based fasudil nanoparticles prepared in this invention have a diameter of approximately 50-500 nm and are spherical; 2. The successful synthesis of fasudil nanomedicine was verified by Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and elemental mapping analysis. 3. Polydopamine-based fasudil nanomedicines possess the ability to scavenge H2O2 and O2. •- It has the ability to neutralize reactive free radicals such as DPPH, protecting endothelial cells from oxidative stress damage; 4. Cell co-incubation experiments showed that polydopamine-based fasudil nanomedicines can be taken up by cells via the classical endocytic pathway and release the drug in response to reactive oxygen species degradation; 5. In vitro functional experiments showed that fasudil nanomedicine based on polydopamine can stabilize tight junctions of endothelial cells and inhibit cell migration and tube formation; 6. Western blot experiments confirmed that it exerts anti-angiogenic and angiogenic protective effects by inhibiting signaling molecules such as ROCK1 / pMLC and LIMK1 / Cofilin; 7. In a streptozotocin (STZ)-induced mouse model of diabetes, polydopamine-based fasudil nanomedicine can reduce retinal ROS levels, decrease the expression of inflammatory factors (TNF-α, IL-6, IL-1β), reduce vascular leakage, inhibit angiogenesis, and improve retinal function through electroretinography (ERG). Beneficial effects

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1) This invention uses a one-pot auto-oxidative polymerization method to synthesize polydopamine-based nanomedicines. The reaction is carried out in an aqueous solution, without the need for exogenous crosslinking agents, organic solvents, or high temperature and pressure. The process is simple, highly controllable, and suitable for industrial scale-up preparation. 2) The nanomedicine synthesized by the drug crystallization / dopamine polymerization method used in this invention is applicable to other basic drug molecules or their salt forms, such as doxorubicin hydrochloride, imiquimod, lidocaine, etc. The synthesis process is simple and has a wide range of applications. 3) The prepared polydopamine-based fasudil nanomedicine has excellent sustained-release properties and reactive oxygen species responsiveness, enabling precise drug release; 4) Fasudil nanomedicine based on polydopamine has multiple functions such as anti-oxidation, anti-inflammation and anti-angiogenesis, which can effectively improve the multifactorial pathological damage associated with diabetic retinopathy. 5) The nanosystem has good biocompatibility, and animal experiments have shown that it has no significant toxicity to retinal tissue and is highly safe; 6) It provides a promising new strategy for nanomedicine delivery in the clinical treatment of diabetic retinopathy. Attached Figure Description

[0019] Figure 1 Scanning electron microscope image of polydopamine-based fasudil nanomedicine (NanoFA); Figure 2 This is a particle size distribution diagram of NanoFA; Figure 3 A diagram illustrating the ability of NanoFA to scavenge reactive oxygen species; Figure 4 Fluorescence micrographs of NanoFA scavenging intracellular reactive oxygen species Figure 5 Transmission electron microscopy image of the endocytosis process of NanoFA nanoparticles; Figure 6 Figure for NanoFA protecting endothelial cell tight junctions; Figure 7 A graph comparing ERG data between the NanoFA treatment group and the control group; Figure 8 For the determination of NanoFA's toxicity to vascular endothelial cells; Figure 9 Biocompatibility assay of NanoFA in the retina; Figure 10 The image shows the fasudil release curve for NanoFA. Detailed Implementation

[0020] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to these embodiments.

[0021] Example 1: Synthesis of polydopamine-based fasudil nanomedicine (NanoFA) 20 mg of dopamine hydrochloride and 5 mg of fasudil were dissolved in 20 mL of deionized water, and then Tris-HCl buffer (1.5 M, pH 8.8) was added. The mixture was then magnetically stirred continuously at 1000 rpm for 24 hours at room temperature. During the reaction, the solution gradually changed from colorless and transparent to a dark brown suspension, indicating the formation of nanoparticles.

[0022] After the reaction, the mixture was centrifuged at 15,000 rpm for 15 minutes, the precipitate was collected, and washed three times each with deionized water and anhydrous ethanol to remove unreacted monomers, free drug, and buffer salts. Finally, the product was redispersed in 5 mL of deionized water, filtered through a microporous membrane for sterilization, and then freeze-dried to obtain NanoFA nanoparticle powder. Scanning electron microscopy revealed that NanoFA exhibited a spherical shape (see attached image). Figure 1 The hydrated particle size was determined using dynamic light scattering (see attached). Figure 2 The results indicate that the obtained NanoFA consists of nanoparticles with a particle size of 50–500 nm, which can be dispersed in aqueous solution. Sulfur elemental analysis revealed that the drug loading of fasudil in the nanomedicine was 28%.

[0023] Example 2: Evaluation of the antioxidant properties of NanoFA H2O2 removal experiment The H2O2 scavenging ability of NanoFA was quantitatively analyzed by detecting the absorbance change at 560 nm of the peroxymolybdate complex formed by the reaction of H2O2 with molybdate using the ammonium molybdate colorimetric method. The results showed that NanoFA exhibited significant H2O2 scavenging activity within the concentration range of 0–1.6 mg / mL, with the scavenging rate increasing in a dose-dependent manner, reaching a maximum scavenging rate of over 85%, indicating that it can effectively alleviate H2O2 accumulation in oxidative stress microenvironments.

[0024] Superoxide anion (O2⁻) scavenging experiment Based on the riboflavin-methionine-NBT photoreduction system, the scavenging effect of NanoFA on O2⁻ was evaluated by measuring the absorbance change of blue formazan generated by the reduction of NBT by O2⁻ at 560 nm. The experiment showed that NanoFA significantly inhibited the NBT photoreduction reaction at concentrations of 0.4–1.6 mg / mL, with an O2⁻ scavenging rate of up to 78%, confirming its effective neutralization of O2⁻ generated by mitochondrial electron leakage or NADPH oxidase activation.

[0025] DPPH free radical scavenging experiment The radical scavenging ability of NanoFA was analyzed by utilizing the attenuation of the characteristic absorption peak of DPPH methanol solution at 517 nm. The results showed that NanoFA could rapidly scavenge DPPH radicals within 20 min, and the scavenging efficiency increased with increasing concentration, exceeding 90% at 1.6 mg / mL, highlighting the strong antioxidant properties of the PDA nanocarrier itself. (See attached image) Figure 3 NanoFA can effectively scavenge various reactive oxygen free radical molecules, exhibiting excellent antioxidant effects.

[0026] Example 3: NanoFA endocytosis experiment bEnd3 cells were co-incubated with NanoFA for 4 hours, then washed with PBS and collected by centrifugation. The distribution of the nanomedicine within the cells was observed using transmission electron microscopy (TEM). (See attached image.) Figure 5 As shown, nanomedicines enter cells via endocytosis and are distributed in lysosomes and cytoplasm.

[0027] Example 4: The role of NanoFA in scavenging intracellular reactive oxygen species The DCFH-DA fluorescent probe method was used to detect intracellular reactive oxygen species (ROS) levels. bEnd.3 cells were divided into five groups: control group (normal culture), H2O2 group (treated with 100 μM H2O2), Fasudil + H2O2 group (100 μg / mL), PDA + H2O2 group, and NanoFA + H2O2 group (100 μg / mL + 100 μM H2O2). After 24 hours of treatment, cells were washed with PBS and then stained with DCFH-DA for 30 minutes. After staining, the green fluorescence of the cells was observed using a fluorescence microscope. Figure 4 As shown, H2O2 treatment significantly enhanced intracellular DCF fluorescence intensity, indicating the successful establishment of an oxidative stress model. NanoFA treatment significantly inhibited H2O2-induced ROS production, reducing fluorescence intensity, and its scavenging effect was superior to the Fasudil-only group.

[0028] Example 5: Evaluation of Animal Models To evaluate the therapeutic effect of NanoFA nanoparticles on diabetic retinopathy, a streptozotocin (STZ)-induced diabetic mouse model was established. After successful modeling, mice were randomly divided into 5 groups (Control group, diabetic control group, free Fasudil treatment group, PDA treatment group, and NanoFA treatment group). The drugs were administered once via intravitreal injection (1 mg / mL), and the observation period was 4 weeks. After retinal injection of NanoFA in diabetic mice, ROS levels, inflammatory factors, vascular leakage, and angiogenesis were measured. Compared with the control group, the NanoFA treatment group showed restoration of tight junctions in endothelial cells and oscillatory potentials in electroretinography, and decreased ROCK protein expression levels (e.g., ...). Figure 6 and Figure 7 ). Example

[0029] Safety evaluation of NanoFA prepared by this invention 1) Cytotoxicity test This experiment used the CCK-8 assay to evaluate the cytotoxic effect of NanoFA nanoparticles on mouse brain microvascular endothelial cells (bEnd.3). Five × 10³ bEnd.3 cells were seeded in each well of a 96-well plate and cultured for 24 hours. NanoFA nanoparticles were diluted with complete culture medium to five concentration gradients: 0, 10, 40, 80, and 160 μg / mL. 200 μL of drug-containing medium was added to each well, and the cells were cultured for 48 hours. After incubation, the supernatant was discarded, and 100 μL of fresh serum-free culture medium was added to each well, followed by 10 μL of CCK-8 reagent. The cells were incubated in the dark for 2 hours. Finally, the absorbance at 450 nm was measured using a microplate reader. Figure 8As shown, even at the highest concentration (160 μg / mL), the survival rate of bEnd.3 cells remained above 75% after 24 and 48 hours of NanoFA treatment, indicating that NanoFA nanoparticles have no significant toxicity to bEnd.3 cells within the experimental concentration range and have good biocompatibility.

[0030] 2) In vivo safety evaluation Retinal tissue was isolated after treatment, frozen sections were prepared, and stained with hematoxylin and eosin (HE). Compared with the blank control group, the retinal structures of all layers (including ganglion cell layer, inner nuclear layer, outer nuclear layer, and photoreceptor cell layer) in the NanoFA treatment group remained intact, with no disordered cell arrangement or destruction of the laminar structure observed (see Appendix). Figure 9 ).

[0031] Example 7: Drug release behavior of NanoFA NanoFA was dispersed in PBS solution or PBS solution containing hydrogen peroxide (100 μM) (pH 7.4) and then incubated in a shaker at 37°C. At different time points, 500 μL of the supernatant was collected and fresh solution was added. The release of fasudil in the supernatant was detected using high-performance liquid chromatography-mass spectrometry. Figure 10 As shown, NanoFA can slowly release fasudil encapsulated within it in PBS solution, exhibiting a good sustained-release effect; when hydrogen peroxide is added to the solution, the release rate of fasudil is significantly accelerated, indicating that nanomedicines can respond to reactive oxygen species degradation and accelerate drug release.

[0032] Example 8: Synthesis of nano-doxacin particles, nano-imiquimod particles, and nano-lidocaine particles Referring to the synthesis method of NanoFA, other basic drug molecules can be used to synthesize nanomedicines, such as nano-doxorubicin, nano-imiquimod, and nano-lidocaine.

[0033] 20 mg of dopamine hydrochloride and 8 mg of doxorubicin hydrochloride were dissolved in 20 mL of deionized water, and Tris-HCl buffer (1 M, pH 8.8) was added to adjust the pH of the solution to 8.5. The solution was then magnetically stirred continuously at 1000 rpm for 24 hours at room temperature. As the reaction proceeded, the solution turned into a dark brown suspension. After centrifugation, washing with water, and lyophilization, nano-doxorubicin particles were obtained.

[0034] 20 mg of dopamine hydrochloride and 5 mg of imiquimod were dissolved in 20 mL of deionized water, and Tris-HCl buffer (1.5 M, pH 8.8) was added to adjust the pH of the solution to 8.5. The solution was then magnetically stirred continuously at 1000 rpm for 24 hours at room temperature. As the reaction proceeded, the solution turned into a dark brown suspension. After centrifugation, washing with water, and lyophilization, nano-imiquimod particles were obtained.

[0035] Dissolve 20 mg of dopamine hydrochloride and 10 mg of lidocaine in 30 mL of deionized water, add ammonia, and adjust the pH of the solution to 8.5. Then, continuously stir magnetically at 1000 rpm for 24 hours at room temperature. As the reaction proceeds, the solution turns into a dark brown suspension. After centrifugation, washing with water, and lyophilization, lidocaine nanoparticles are obtained.

[0036] The basic drug crystallization / dopamine polymerization synthesis method used in this invention is applicable to the preparation of nanomedicines for different types of basic drugs (including their salt derivatives).

[0037] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0038] 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 polydopamine / drug nanocomposite particles, comprising the following steps: S1: Dissolve dopamine hydrochloride and an alkaline drug in water to form a mixed solution of dopamine and the drug; S2: Under stirring conditions, add an alkaline solution to the above dopamine and drug mixture to adjust the pH of the dopamine and drug mixture to alkaline, and react at room temperature for 4-48 hours to obtain the reaction product; S3: Centrifuge, wash, and freeze-dry the reaction product to obtain polydopamine / drug nanocomposite particles.

2. The preparation method according to claim 1, characterized in that: The solubility of the alkaline drug is pH-dependent, meaning it can dissolve in acidic aqueous solutions and crystallize or precipitate in neutral or alkaline aqueous solutions. The alkaline drug includes, but is not limited to, fasudil, doxorubicin hydrochloride, imiquimod, and lidocaine.

3. The preparation method according to claim 2, characterized in that: In step S2, the pH of the dopamine and drug mixture solution is adjusted to 8.0~11.

0.

4. The preparation method according to claim 1, 2, or 3, characterized in that: Alkaline solutions include, but are not limited to, Tris-HCl buffer solution, ammonia, sodium hydroxide solution, and sodium carbonate solution.

5. The preparation method according to claim 2, characterized in that: In the mixed solution of dopamine and drug, the concentration of dopamine is 0.1~10 mg / mL; the concentration of the alkaline drug is 0.1~5 mg / mL, and the mass ratio of drug to dopamine is 1:0.2~1:

20.

6. Polydopamine / drug nanocomposite particles prepared by any one of the preparation methods described in claims 2 to 5.

7. The polydopamine / drug nanocomposite particles according to claim 6, characterized in that: The particle size of the nanocomposite particles is 50 to 500 nm.

8. The use of the polydopamine / drug nanocomposite particles according to claim 6 or 7 in the preparation of a drug for treating diabetic retinopathy, wherein the alkaline drug is fasudil.

9. The use of the polydopamine / drug nanocomposite particles according to claim 6 or 7 in the preparation of therapeutic drugs for ROCK pathway or oxidative stress-related diseases, wherein the alkaline drug is fasudil.

10. The use of the polydopamine / drug nanocomposite particles according to claim 6 or 7 in the preparation of drugs that respond to or release reactive oxygen species or drugs for the treatment of oxidative stress-related diseases.

Images