A carrier-free nanodelivery material based on hydrophobic indole alkaloids
By self-assembling hydrophobic indole alkaloids with hydrophilic nucleic acids to form nanoparticles, the problems of low drug loading and low encapsulation efficiency were solved, achieving efficient delivery of siRNA, inhibiting vascular endothelial growth factor expression, and improving vision in patients with nAMD.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
- Filing Date
- 2026-07-01
- Publication Date
- 2026-08-04
AI Technical Summary
Existing nanodelivery systems suffer from problems such as low drug loading, low encapsulation efficiency, and complex synthesis processes when treating age-related macular degeneration (nAMD).
A carrier-free nanomaterial is formed by self-assembling hydrophobic indole alkaloids and hydrophilic nucleic acid molecules. The nanoparticles are formed by non-covalent interactions such as π-π conjugation, hydrogen bonding, and electrostatic interaction to deliver siRNA to target and degrade vascular endothelial growth factor mRNA.
It achieves high drug loading and high encapsulation efficiency in nano-delivery, and significantly improves vision in nAMD patients by downregulating vascular endothelial growth factor gene expression, inhibiting angiogenesis.
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedicine, specifically to a carrier-free nanodelivery material based on hydrophobic indole alkaloids. Background Technology
[0002] Age-related macular degeneration (AMD) is a serious blinding eye disease caused by degenerative changes in the retinal tissue of the macular region of the eye, resulting in progressive loss of central vision. Clinically, it can be divided into dry AMD and wet AMD. Wet AMD is also known as neovascular age-related macular degeneration (nAMD).
[0003] Current research has identified subretinal choroidal neovascularization (CNV) as the core pathological basis of nAMD. Vascular endothelial growth factor (VEGF) is a key factor inducing angiogenesis and vascular exudation. Blocking the binding of VEGF to its receptor (VEGFR) or downregulating VEGF expression can inhibit CNV, promote the regression of immature neovascularization, and thus improve vision in nAMD patients. Therefore, targeting VEGF and VEGFR is a current research focus in designing treatments for nAMD. Currently, the first-line clinical treatment for nAMD involves intravitreal injection of anti-VEGF antibodies or fusion protein drugs. These drugs bind to VEGF and block related pathways, inhibiting angiogenesis and showing some efficacy in treating nAMD.
[0004] Some researchers have proposed that introducing siRNA (siVEGF) targeting the VEGF gene into cells can bind to and activate the RNA-induced silencing complex (RISC), thereby targeting and degrading mRNA complementary to siVEGF, ultimately preventing mRNA translation, downregulating VEGF expression, and effectively reducing the number of new blood vessels. Although siRNA can provide long-term and effective treatment for nAMD at the gene level, current nanodelivery systems suffer from low drug loading, low encapsulation efficiency, and complex synthesis processes. Summary of the Invention
[0005] To address the aforementioned technical deficiencies, this application proposes a carrier-free nanodelivery material based on hydrophobic indole alkaloids, which solves the problems of low drug loading, low encapsulation efficiency, and complex synthesis process in nanodelivery systems.
[0006] To achieve the above objectives, this application provides a carrier-free nanodelivery material based on hydrophobic indole alkaloids, which is formed by the self-assembly of hydrophobic indole alkaloids and hydrophilic nucleic acid molecules. A carrier-free nanomaterial based on hydrophobic indole alkaloids is prepared by the following steps: S1. Dissolve hydrophobic indole alkaloids in an ethanol / water mixture, adjust the pH value, stir, and obtain a hydrophobic indole alkaloid solution. In the above reaction process, the pH value is adjusted to protonate the hydrophobic indole alkaloids; S2. Add the hydrophobic indole alkaloid solution dropwise to the hydrophilic nucleic acid reagent, sonicate, stir, and dialyze to obtain carrier-free nanodelivery materials based on hydrophobic indole alkaloids.
[0007] In the above reaction process, the protonated hydrophobic indole alkaloids react with negatively charged hydrophilic nucleic acids and undergo electrostatic adsorption. The hydrophobic indole alkaloids and hydrophilic nucleic acids self-assemble through hydrophobic interactions and π-π stacking. The free hydrophobic indole alkaloids and hydrophilic nucleic acids are removed by dialysis.
[0008] Furthermore, the hydrophobic indole alkaloid is evodiamine.
[0009] Furthermore, the hydrophilic nucleic acid is siRNA.
[0010] Furthermore, in the ethanol / water mixed solution, the ethanol content is 30-50 wt%.
[0011] Furthermore, the ratio between the hydrophobic indole alkaloid and the ethanol / water mixed solution is 5-10 mg: 1 mL.
[0012] Furthermore, the mass ratio of the hydrophobic indole alkaloid to the hydrophilic nucleic acid is 2-4:1.
[0013] Furthermore, the ultrasonic processing parameters are: frequency: 20-40kHz, power: 100-160W, and time: 3-5min.
[0014] Furthermore, the dialysis is performed at least three times, with each dialysis session lasting at least 20 minutes, and the molecular weight cutoff is 150-800 kDa.
[0015] Furthermore, the particle size of the carrier-free nanodelivery material based on hydrophobic indole alkaloids is 80-150 nm.
[0016] In summary, this application has the following beneficial effects: This application presents a carrier-free nanodelivery material based on hydrophobic indole alkaloids. This nanodelivery material primarily utilizes the self-assembly of evodiamine and siRNA through non-covalent interactions such as π-π conjugation, hydrogen bonding, hydrophilicity / hydrophobicity, and electrostatic interactions to form nanoparticles, thus creating a carrier-free nanodelivery material. After entering cells, this carrier-free nanodelivery material exhibits two effects: firstly, evodiamine downregulates the expression of hypoxia-inducible factor HIF-1, thereby downregulating the initiation of the vascular endothelial growth factor (VEGF) gene and reducing the expression level of VEGF mRNA; secondly, siRNA binds to and activates an RNA-induced silencing complex, subsequently targeting and degrading VEGF mRNA complementary to siRNA, thereby preventing VEGF mRNA translation. Ultimately, this approach achieves both downregulation of VEGF gene transcription and inhibition of VEGF mRNA translation, fundamentally suppressing VEGF expression. The synergistic inhibition of transcription and translation provides a comprehensive treatment approach. The carrier-free nanodelivery material based on hydrophobic indole alkaloids prepared in this application has a simple synthesis process, high drug loading capacity, high encapsulation efficiency, and good effect on age-related macular degeneration. Detailed Implementation
[0017] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the embodiments of this application. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0018] The raw materials involved in the specific embodiments of this application are: evodiamine selected from Dest Biotechnology, No. 518-17-2; siRNA selected from Shenzhen Xinbosheng Biotechnology Co., Ltd., Catalog No. SI-5000, Specification 1mg / mL.
[0019] Example 1 A carrier-free nanomaterial based on hydrophobic indole alkaloids is prepared by the following steps: S1. Dissolve 80 mg of evodiamine in 10 mL of ethanol / water mixed solution (ethanol content is 40 wt%), adjust the pH to 6 with 0.1 M HCl, and stir (80 rpm, 30 min) to obtain evodiamine solution. S2. Evodiaein solution was slowly added dropwise to siRNA reagent at a dropping rate of 0.5 mL / min. The mass ratio of evodiaein to siRNA was 2:1. The mixture was sonicated at 4℃ (frequency: 25 kHz, power: 100 W, time: 5 min), stirred at room temperature (200 rpm, time: 2 h), and then dialyzed 5 times with a molecular weight cutoff of 150 kDa. Each dialysis session lasted 2 h, thus obtaining carrier-free nanodelivery materials based on hydrophobic indole alkaloids.
[0020] Example 2 A carrier-free nanomaterial based on hydrophobic indole alkaloids is prepared by the following steps: S1. Dissolve 80 mg of evodiamine in 10 mL of ethanol / water mixed solution (ethanol content is 40 wt%), adjust the pH to 6 with 0.1 M HCl, and stir (80 rpm, 30 min) to obtain evodiamine solution. S2. Evodiaein solution was slowly added dropwise to siRNA reagent at a rate of 0.5 mL / min. The mass ratio of evodiaein to siRNA was 3:1. The mixture was sonicated at 4℃ (frequency: 25 kHz, power: 130 W, time: 5 min), stirred at room temperature (200 rpm, time: 2 h), and then dialyzed 5 times with a molecular weight cutoff of 150 kDa. Each dialysis session lasted 2 h, thus obtaining carrier-free nanodelivery materials based on hydrophobic indole alkaloids.
[0021] Example 3 A carrier-free nanomaterial based on hydrophobic indole alkaloids is prepared by the following steps: S1. Dissolve 80 mg of evodiamine in 10 mL of ethanol / water mixed solution (ethanol content is 40 wt%), adjust the pH to 6 with 0.1 M HCl, and stir (80 rpm, 30 min) to obtain evodiamine solution. S2. Evodiaein solution was slowly added dropwise to siRNA reagent at a dropping rate of 0.5 mL / min. The mass ratio of evodiaein to siRNA was 4:1. The mixture was sonicated at 4℃ (frequency: 25 kHz, power: 160 W, time: 5 min), stirred at room temperature (200 rpm, time: 2 h), and then dialyzed 5 times with a molecular weight cutoff of 150 kDa. Each dialysis session lasted 2 h, thus obtaining carrier-free nanodelivery materials based on hydrophobic indole alkaloids.
[0022] Compare with Example 1 A carrier-free nanomaterial based on hydrophobic indole alkaloids is prepared by the following steps: S1. Dissolve 80 mg of evodiamine in 10 mL of ethanol / water mixed solution (ethanol content is 40 wt%), adjust the pH to 6 with 0.1 M HCl, and stir (80 rpm, 30 min) to obtain evodiamine solution. S2. Evodiaein solution was slowly added dropwise to siRNA reagent at a dropping rate of 0.5 mL / min. The mass ratio of evodiaein to siRNA was 5:1. The mixture was sonicated at 4℃ (frequency: 25 kHz, power: 130 W, time: 5 min), stirred at room temperature (200 rpm, time: 2 h), and then dialyzed 5 times with a molecular weight cutoff of 150 kDa. Each dialysis session lasted 2 h to obtain carrier-free nanodelivery material based on hydrophobic indole alkaloids.
[0023] Compare with Example 2 A carrier-free nanomaterial based on hydrophobic indole alkaloids is prepared by the following steps: S1. Dissolve 80 mg of evodiamine in 10 mL of ethanol / water mixed solution (ethanol content is 40 wt%), adjust the pH to 6 with 0.1 M HCl, and stir (80 rpm, 30 min) to obtain evodiamine solution. S2. Evodiaein solution was slowly added dropwise to siRNA reagent at a dropping rate of 0.5 mL / min. The mass ratio of evodiaein to siRNA was 3:1. The mixture was sonicated at 4℃ (frequency: 25 kHz, power: 250 W, time: 5 min), stirred at room temperature (200 rpm, time: 2 h), and then dialyzed 5 times with a molecular weight cutoff of 150 kDa. Each dialysis session lasted 2 h, thus obtaining carrier-free nanodelivery materials based on hydrophobic indole alkaloids.
[0024] Compare with Example 3 A carrier-free nanomaterial based on hydrophobic indole alkaloids is prepared by the following steps: S1. Dissolve 80 mg of evodiamine in 10 mL of ethanol / water mixed solution (ethanol content is 40 wt%) to obtain evodiamine solution; S2. Evodiaein solution was slowly added dropwise to siRNA reagent at a dropping rate of 0.5 mL / min. The mass ratio of evodiaein to siRNA was 2:1. The mixture was sonicated at 4℃ (frequency: 25 kHz, power: 100 W, time: 5 min), stirred at room temperature (200 rpm, time: 2 h), and then dialyzed 5 times with a molecular weight cutoff of 150 kDa. Each dialysis session lasted 2 h to obtain carrier-free nanodelivery materials based on hydrophobic indole alkaloids.
[0025] Performance testing The particle size, drug loading and encapsulation efficiency of the carrier-free nanodelivery materials prepared in Examples 1-3 and Control Examples 1-3 were tested.
[0026] Particle size measurement: The average particle size of the carrier-free nanodelivery materials prepared in Examples 1-3 and Control Examples 1-3 was measured using a laser particle size analyzer; Drug loading measurement: 0.2 mL of each of the carrier-free nanodelivery materials prepared in Examples 1-3 and Control Examples 1-3 were diluted, and the absorbance at 260 nm was measured using a UV spectrophotometer. The siRNA content and drug loading rate were calculated. Drug loading rate = siRNA content in carrier-free nanomaterials / total amount of carrier-free nanomaterials; Encapsulation efficiency measurement: 0.2 mL of each of the carrier-free nanodelivery materials prepared in Examples 1-3 and Control Examples 1-3 were diluted, and the absorbance at 260 nm was measured using a UV spectrophotometer. The siRNA content and encapsulation efficiency were calculated. Encapsulation efficiency = siRNA content in carrier-free nanodelivery material / amount of siRNA used. The test results are shown in Table 1. Table 1 ; As shown in Table 1, the carrier-free nanodelivery materials prepared in the embodiments of this application have high drug loading and high encapsulation efficiency, especially the carrier-free nanodelivery material prepared in Example 2, which has the highest drug loading and encapsulation efficiency. Compared with Example 2, the mass ratio of evodiamine to siRNA in Control Example 1 was adjusted to 5:1, which greatly increased the content of evodiamine. The test results showed that compared with Example 2, its particle size was larger, and the drug loading and encapsulation efficiency were relatively low. The test results were not as good as those of Example 2, indicating that the mass ratio of evodiamine to siRNA in this application is the optimal range. Compared with Example 2, the ultrasonic power of Control Example 2 increased by 250W. The test results showed that the particle size of Control Example 2 was larger, and the drug loading and encapsulation efficiency were relatively low. The test results were not as good as those of Example 2, indicating that the preparation conditions of this application are the optimal preparation conditions. Compared with Example 1, Control Example 3 did not have a pH adjustment step. The test results showed that the drug loading and encapsulation efficiency were lower than those of Example 1. The test results were not as good as those of Example 1, indicating that the preparation conditions of this application are the optimal preparation conditions.
[0027] In summary, the carrier-free nanodelivery materials prepared in the embodiments of this application have excellent drug loading and encapsulation efficiency, especially Example 2, which is the best embodiment of this application.
[0028] The above description is merely an example and illustration of the concept of this application. Those skilled in the art can make various modifications or additions to the specific embodiments described or use similar methods to replace them, as long as they do not deviate from the inventive concept or exceed the scope defined in the claims, they should all fall within the protection scope of this application.
Claims
1. A carrier-free nanomaterial based on hydrophobic indole alkaloids, characterized in that, This carrier-free nanodelivery material is formed by the self-assembly of hydrophobic indole alkaloids and hydrophilic nucleic acid molecules; A carrier-free nanomaterial based on hydrophobic indole alkaloids is prepared by the following steps: S1. Dissolve hydrophobic indole alkaloids in an ethanol / water mixture, adjust the pH value, stir, and obtain a hydrophobic indole alkaloid solution. S2. Add the hydrophobic indole alkaloid solution dropwise to the hydrophilic nucleic acid reagent, sonicate, stir, and dialyze to obtain carrier-free nanodelivery materials based on hydrophobic indole alkaloids.
2. The carrier-free nanodelivery material based on hydrophobic indole alkaloids according to claim 1, characterized in that, The hydrophobic indole alkaloid is evodiamine.
3. The carrier-free nanodelivery material based on hydrophobic indole alkaloids according to claim 1, characterized in that, The hydrophilic nucleic acid is siRNA.
4. The carrier-free nanodelivery material based on hydrophobic indole alkaloids according to claim 1, characterized in that, The ethanol / water mixture contains 30-50 wt% ethanol.
5. The carrier-free nanodelivery material based on hydrophobic indole alkaloids according to claim 1, characterized in that, The ratio between the hydrophobic indole alkaloid and the ethanol / water mixture is 5-10 mg: 1 mL.
6. The carrier-free nanodelivery material based on hydrophobic indole alkaloids according to claim 1, characterized in that, The mass ratio of the hydrophobic indole alkaloid to the hydrophilic nucleic acid is 2-4:
1.
7. The carrier-free nanodelivery material based on hydrophobic indole alkaloids according to claim 1, characterized in that, The ultrasonic processing parameters are: frequency: 20-40kHz, power: 100-160W, and time: 3-5min.
8. The carrier-free nanodelivery material based on hydrophobic indole alkaloids according to claim 1, characterized in that, The dialysis procedure shall be performed at least three times, with each dialysis session lasting at least 20 minutes, and the molecular weight cutoff shall be 150-800 kDa.
9. The carrier-free nanodelivery material based on hydrophobic indole alkaloids according to claim 1, characterized in that, The particle size of the carrier-free nanodelivery material is 80-150 nm.