A self-assembled nanodrug of cynarin-quercetin, a preparation method and application thereof

Abstract

The application discloses a kind of chrysanthemum constituent-quercetin self-assembled nano-drugs, preparation method and application;The chrysanthemum constituent-quercetin self-assembled nano-drugs of the application is realized by hydrogen bond and π-π conjugation effect supersmolecular self-assembly by chrysanthemum constituent and quercetin, on the basis of improving the water solubility of chrysanthemum constituent, formed the functional synergistic nanostructure, its antiviral activity is significantly improved;Its raw material chrysanthemum constituent, quercetin is derived from natural medicinal plant extract, without chemical auxiliary agent, economic and easy to get, the side effect of nano-drug prepared is low, and drug safety is high, and it has very application prospect.

Description

Technical Field

[0001] This invention relates to a self-assembled nanomedicine, particularly to an araliain-quercetin self-assembled nanomedicine, and also to a method for preparing the above-mentioned self-assembled nanomedicine and its application in the preparation of anti-respiratory syncytial virus drugs. Background Technology

[0002] Respiratory syncytial virus (RSV) belongs to the genus Orthopneumovirus in the family Pneumoviridae. The virus particle has a diameter of 100-1000 nm, a filamentous envelope, and contains a single-stranded negative-sense RNA genome (approximately 15.2 kb long), encoding 11 viral proteins. It is transmitted through aerosols, droplets, and close contact, with an incubation period of 3-8 days. After infection, RSV replicates in the nasopharynx and upper respiratory tract epithelium, and can even spread to the small bronchioles or alveoli of the lower respiratory tract. It is a highly contagious virus posing a significant risk to public health, particularly to infants, young children, the elderly, and patients with chronic diseases. In high-risk groups, RSV can cause severe clinical symptoms such as pneumonia, acute respiratory distress syndrome, and acute respiratory failure, and is a leading cause of hospitalization and even death. Its high variability and resulting immune escape pose significant challenges to the development of vaccines and antiviral drugs.

[0003] Currently, safe and effective antiviral drugs are still lacking in clinical practice. Existing RSV prevention products include live attenuated vaccines, protein / subunit vaccines, mRNA vaccines, and monoclonal antibodies. Arexvy, Abrysvo, mRESVIA, Synagis, Beyfortus, and Clesrovimab have been approved for marketing, but all have certain shortcomings: Arexvy carries an increased risk of GBS after vaccination and requires a two-year booster shot; Abrysvo also has GBS warnings and preterm birth risks, and maternal antibodies provide less than 30% protection for infants; mRESVIA showed delayed VAERD signaling in infant studies; Synagis has a half-life of only 20 days, requiring monthly injections and incurring high costs; Beyfortus has shown insufficient efficacy in some infants and has reported GBS; Clesrovimab is still in the observational stage. Overall, existing products still have significant limitations in terms of safety, duration of protection, accessibility, and cost. Therefore, developing new, safe, and effective antiviral drugs remains a critical need. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a cynarin-quercetin self-assembled nanomedicine with strong stability, good drug-like properties, and significant anti-respiratory syncytial virus activity, and to provide a method for preparing the above-mentioned self-assembled nanomedicine and its application in the preparation of anti-respiratory syncytial virus drugs.

[0005] Technical Solution: The cynarin-quercetin self-assembled nanomedicine of the present invention is formed by the π-π stacking of the aromatic rings of quercetin and cynarin to form a hydrophobic core, and the hydroxyl groups between molecules are tightly bound by hydrogen bonds. This multi-layered non-covalent interaction collectively constructs the stable structure of the nanocomposition.

[0006] The cynarin is a polyphenolic acid cynarin, with the following structure: .

[0007] The structure of the self-assembled nanomedicine is as follows: .

[0008] The preparation method of the above-mentioned cynarin-quercetin self-assembled nanomedicine includes the following steps: cynarin and quercetin are dissolved in an organic solvent to prepare a stock solution of 10 mmol / mL. 100 μL of the stock solution is added to 10 mL of ultrapure water and ultrasonically treated to obtain the cynarin-quercetin self-assembled nanomedicine, which achieves supramolecular self-assembly through hydrogen bonding and π-π conjugation.

[0009] The molar ratio of cynarin to quercetin is 5:1 to 1:5, preferably 1:1.

[0010] The organic solvent is dimethyl sulfoxide.

[0011] The aforementioned cynarin-quercetin self-assembled nanomedicine can be used in the preparation of drugs against respiratory syncytial virus.

[0012] The virus in question is RSV-A2 strain.

[0013] Invention Principle: Traditional drugs struggle to address viral infectious diseases and their complex complications due to their limited target range, high toxicity, and poor water solubility of natural products. This invention leverages the inherent advantages of the polyphenolic structures of cynarin and quercetin, natural products. Through hydrogen bonding and π-π conjugation, intermolecular interactions occur between the two, forming a novel supramolecular nanostructure distinct from simple mixtures. This structural synergy overcomes the limitations of existing technologies in terms of single-effect drug use and physicochemical defects, resulting in a significant improvement in efficacy.

[0014] Specifically, self-assembled nanomedicines, through the introduction of quercetin and its stable binding via π-π conjugation, achieve a bimolecular, multi-target synergistic effect. This synergistic structure significantly enhances the overall therapeutic effect, overcoming the limitations of single drugs in immunomodulation and overall treatment of viral infections. Simultaneously, building upon improved water solubility of cynarin, self-assembled nanomedicines utilize the high specific surface area and suitable nano-delivery size resulting from particle sizeization to significantly improve its bioavailability, overcoming the inherent challenges of poor water solubility and low absorption rate of cynarin. Through direct antiviral action and efficient intracellular delivery, self-assembled nanomedicines enable the drug to reach viral replication targets at higher effective concentrations, thus significantly enhancing its antiviral efficacy and potentially effectively reducing viral pulmonary inflammatory infiltration and lung damage, demonstrating enormous development potential.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The cynarin-quercetin self-assembled nanomedicine of the present invention has a very significant anti-respiratory syncytial virus (A2 strain) activity (EC). 50 =8.42μM, SI=33.49); (2) The raw material, cynarin, is derived from natural medicinal plant extracts, which are economical and readily available. The self-assembled nanomedicine prepared has low side effects and high drug safety (CC). 50 =286.6μM); (3) The cynarin-quercetin self-assembled nanomedicine of the present invention achieves supramolecular self-assembly of cynarin and quercetin through hydrogen bonding and π-π conjugation. On the basis of improving the water solubility of cynarin, a functionally synergistic nanostructure is formed, and its antiviral activity is significantly improved; (4) The self-assembled nanomedicine of the present invention has the advantage of good stability. The Zeta potential (-43.87 mV) shows that it provides strong electrostatic repulsion, which can effectively resist the aggregation of particles in solution and physiological conditions, greatly ensuring the integrity of the drug in vivo and the persistence of in vivo efficacy, and has good drug-like properties. Attached Figure Description

[0016] Figure 1 The half-maximal toxicity concentration (CC) of cynarin 50 ); Figure 2 The antiviral efficacy of cynarin and self-assembled nanomedicine; where a is the half-maximal effective concentration (EC50) of cynarin. 50 b represents the half-maximal effective concentration (MCI) of the positive control drug ribavirin, and c represents the inhibitory effect of cyperipherin and self-assembled nanomedicine on the mRNA level of RSV N protein. Figure 3 The material properties of self-assembled nanomedicines are shown in Figure 1; where a represents appearance and Tyndall effect, b represents particle size distribution and Zeta potential, c represents fluorescence spectrum, and d represents Fourier transform infrared spectrum. Figure 4This is a schematic diagram illustrating the interaction between cynarin and quercetin. Detailed Implementation

[0017] The present invention will now be described in detail with reference to specific embodiments.

[0018] Example 1 The preparation method of the cynarin-quercetin nano-self-assembled drug of the present invention is as follows: Citronella (Cyn) and quercetin (Que) were dissolved in dimethyl sulfoxide (DMSO) at a molar ratio of 1:1 to obtain a stock solution of 10 mmol / mL. 100 μL of the stock solution was added to 10 mL of ultrapure water and ultrasonically treated to obtain citronella-quercetin nano-self-assembled drug CQN.

[0019] The appearance and Tyndall effect were observed, and the particle size, PDI and Zeta potential of the self-assembled drug were detected using a Malvern particle size analyzer Zetasizer Pro.

[0020] Fluorescence and Fourier transform infrared spectra of cynarin, quercetin, a 1:1 molar ratio mixture of cynarin and quercetin (Cyn+Que), and cynarin-quercetin nano-self-assembled drugs were detected using an Edinburgh FLS1000 (UK) and a Thermo Fisher Scientific Nicolet iS20 (USA). Figure 3 The figure shows that c compares the fluorescence intensity of Cyn, Que, Cyn+Que, and CQN. As shown in the figure, the fluorescence intensity of CQN is much higher than that of Cyn+Que, indicating that the molecules have transformed from a free state in solution into a tightly aggregated nanostructure, which is very likely due to intermolecular interactions. Figure 3 The infrared spectra of Cyn, Que, Cyn+Que, and CQN were compared. The results showed that the stretching vibration region of the hydroxyl (-OH) group (3500-3000 cm⁻¹) was... -1 In the CQN, the shape and position of the -OH peak changed relative to Cyn and Que, strongly suggesting the formation of hydrogen bonds between cynarin and quercetin molecules. The C=C or C=O vibrational region (1700-1500 cm⁻¹) -1 In ), CQN is at 1600 cm -1 The positions and intensities of nearby characteristic peaks showed significant shifts in peak shape, intensity, or wavenumber, confirming the occurrence of π-π conjugation and intermolecular hydrogen bonding, which in turn drove the formation of supramolecular structures.

[0021] like Figure 4 As shown, cynarin and quercetin are bound by hydrogen bonds and π-π conjugation, interacting with each other and forming a self-assembled nanomedicine.

[0022] In summary, the cynarin-quercetin compound successfully achieved supramolecular self-assembly through non-covalent interactions such as hydrogen bonding and π-π conjugation, forming a novel nano-aggregate structure with altered electronic structure and optical properties.

[0023] Example 2 Cell proliferation assay (CCK-8 assay) to determine drug cytotoxicity (CC50). 1. Cell Culture Human alveolar basal epithelial cells (A549) for lung cancer were seeded into 96-well plates containing 10% FBS 1640 medium, with 7000 cells per well and cultured at 37°C in a 5% CO2 incubator for 24 hours.

[0024] 2. Prepare cynarin dilution solution Artichokein was dissolved in DMSO to prepare a 10 mM stock solution. Artichokein was serially diluted with 1640 medium containing 10% FBS and added to 96-well plates at 100 μL / well. The plates were then incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0025] 3. Cell viability detection Discard the solution in each well, add 200 μL of 1640 medium containing 5% CCK-8 solution and 10% FBS to each well, and incubate in the dark at 37°C in a 5% CO2 incubator for 3 hours. Measure the absorbance at 450 nm using a microplate reader and calculate the percentage of viable cells in each treatment group relative to the control group.

[0026] like Figure 1 As shown, the half-maximal toxicity concentration (IC50) of cynarin is 286.6 μM, indicating that this compound, as a natural product, has low toxicity and high drug safety.

[0027] Example 3 Quantitative detection of the inhibitory effect of drugs on respiratory syncytial virus (RSV) using real-time quantitative polymerase chain reaction (RT-qPCR) (EC50) 1. Cell Culture Human alveolar basal epithelial cells (A549) for lung cancer were seeded into 24-well plates containing 10% FBS 1640 medium, with 1 x 10 cells per well. 5 Cells per 500 μL of culture medium were incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0028] 2. Prepare virus-containing cynarin / ribavirin / self-assembly diluent The target drug was serially diluted in 1640 medium containing 10% FBS and respiratory syncytial virus (RSV), and A549 cells were infected with the drug at an MOI of 0.5. The original medium in each well was discarded, and the diluted solution was added to 24-well plates at 500 μL / well. The plates were then incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0029] 3. Total RNA extraction and RT-qPCR quantification Total RNA was isolated using a FreeZol kit. RNA integrity was verified using a NANODROP ONE analyzer. Complementary DNA (cDNA) was synthesized using a HisyGo RT Red SuperMix for qPCR (+gDNA Wiper). Quantitative real-time PCR was performed using a 2*Universal SYBR Green real-time PCR detection system. Data analysis was performed using QuantStudio real-time PCR software.

[0030] .

[0031] like Figure 2 As shown in a and b, the half-maximal effective concentration (WMC) of cynarin was 8.42 μM, lower than that of the positive control drug ribavirin (WMC) of 10.38 μM, indicating that cynarin has higher activity against RSV than ribavirin. Furthermore, the selectivity index (SI = CC) of cynarin... 50 / EC 50 The value was 33.49, far exceeding the generally accepted critical value of 10, demonstrating that cynarin has a high safety window and is safe and effective against RSV. Figure c shows the significant inhibitory effect of cynarin and its nanocomposite on RSV viral replication. Compared with the untreated virus model group (Mock), cynarin inhibited the mRNA expression level of viral N protein by approximately 60%, exhibiting good antiviral activity. The inhibition efficiency of the nanocomposite formed by supramolecular self-assembly was higher than 85%, and its activity was about 50% higher than that of cynarin. The significant improvement in efficacy mainly stems from the unique advantages brought by the nanocarrier: First, nano-sizing fundamentally solves the problem of poor water solubility of cynarin, improving the bioavailability of the drug; second, the size of the cynarin nanocomposite (254.7 nm) endows it with efficient intracellular delivery capability, enabling the drug to be more effectively taken up by target cells, thereby achieving a higher effective concentration in the intracellular environment of viral replication; finally, the introduction of quercetin and supramolecular synergistic effect in the composition structure may further enhance the inhibition of viral replication, ultimately achieving a significant anti-RSV effect that surpasses that of free drugs.

[0032] Therefore, the cynarin-quercetin self-assembled nanomedicine of the present invention achieves supramolecular self-assembly of cynarin and quercetin through hydrogen bonding and π-π conjugation. On the basis of improving the water solubility of cynarin, a functionally synergistic nanostructure is formed, and its antiviral activity is significantly enhanced. Its raw materials, cynarin and quercetin, are derived from natural medicinal plant extracts, without chemical additives, are economical and readily available, and the resulting nanomedicine has low side effects, high drug safety, and great application prospects.

Claims

1. A self-assembled nanomedicine of cynarin and quercetin, characterized in that, The self-assembled nanomedicine is prepared by dissolving cynarin and quercetin in dimethyl sulfoxide at a molar ratio of 1:1 to form a stock solution. The stock solution is then added to ultrapure water and subjected to ultrasonic treatment to obtain the cynarin-quercetin self-assembled nanomedicine. The structure of the self-assembled nanomedicine is as follows: .

2. The cynarin-quercetin self-assembled nanomedicine according to claim 1, characterized in that, The self-assembled nanomedicine was prepared by adding 100 μL of the stock solution to 10 mL of ultrapure water and sonicating it to obtain the araliain-quercetin self-assembled nanomedicine.

3. The use of the cynarin-quercetin self-assembled nanomedicine of claim 1 in the preparation of a drug for treating respiratory syncytial virus.

4. The application according to claim 3, characterized in that, The virus in question is RSV-A2 strain.

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