Ultrasonic-assisted self-assembled resveratrol nanocrystal as well as preparation method and application thereof

Abstract

The invention discloses an ultrasonic-assisted self-assembled resveratrol nanocrystal as well as a preparation method and application thereof, and belongs to the technical field of anti-pneumonia drugs. According to the resveratrol nanocrystal prepared by the method, the particle size is 181.5 + / -2.27 nm, the PDI is 0.08 + / -0.07, the potential is-12.6 + / -0.85 mV, and the resveratrol nanocrystal has reliable stability under the condition that a stabilizer is not used and can be maintained for at least one week; compared with free resveratrol, the crystal form of the resveratrol nanocrystal is changed, and the problems of resveratrol solubility (saturation solubility is increased by 35.1 times compared with free resveratrol), in-vitro uptake (in-vitro uptake is increased by 3.5 times compared with free resveratrol) and lung retention can be solved. Meanwhile, the resveratrol nanocrystal shows anti-inflammatory, antioxidant and antiviral activities superior to those of free resveratrol, and comprehensively shows that the resveratrol nanocrystal has safety and effectiveness in the field of aerosol inhalation treatment of RSV pneumonia.

Description

Technical Field

[0001] The invention relates to the technical field of anti-pneumonia drugs, and in particular to an ultrasound-assisted self-assembled resveratrol nanocrystal and a preparation method and application thereof. Background Art

[0002] Viral pneumonia is an inflammatory disease that affects the lower respiratory tract and is characterized by viral infection and lung inflammation. It is mainly caused by respiratory viruses such as influenza virus, coronavirus, and respiratory syncytial virus. Respiratory syncytial virus (RSV) is a filamentous, enveloped, negative-sense, single-stranded RNA virus. It is a common pathogen that often causes pneumonia in infants and young children. Pneumonia caused by severe RSV infection is the leading cause of death in children under 2 years of age. Unfortunately, there is currently a lack of safe small molecule drugs and effective vaccines for infants and young children infected with RSV.

[0003] Resveratrol (3,4',5-trihydroxystilbene) is a natural compound synthesized by plants under external influences. It has anti-inflammatory, antioxidant, anti-aging and neuroprotective effects. It is usually found in grapes, peanut shells and mulberries, and is currently widely used as a dietary supplement. Recent studies have shown that resveratrol has broad-spectrum antiviral activity, including against DNA and RNA viruses. Various in vitro and in vivo studies have confirmed that resveratrol has antiviral activity against RSV infection. It can inhibit RSV-induced TRIF and TBK12 protein expression, reduce IL-6, IFN-γ levels and RSV titers in the lungs. In addition, resveratrol can bind to heparin sulfate proteoglycans (HSPGs) of host cells to fight RSV infection. Resveratrol can target multiple sites, and its safety makes it an ideal candidate for the treatment of infant RSV pneumonia. However, its poor water solubility (30μg / mL) and low bioavailability (<1%) hinder its application.

[0004] In recent years, the research on nanocrystal drugs has gradually become a hot topic in nanoformulation research. Nanocrystal drugs are pure drug particles with a particle size of 1 to 1000 nm, which do not require carrier materials. Drug nanocrystals can enhance adhesion to biofilms with their huge specific surface area, while increasing the saturated solubility and dissolution of the drug and improving bioavailability. Nanocrystal technology can be used as an intermediate preparation technology and is widely used in the preparation of preparations for various routes of administration (oral, intravenous, pulmonary, transdermal and ocular administration, etc.).

[0005] Compared with traditional treatment methods, inhalation drug delivery has attracted more and more attention in recent years. It is a local drug delivery method for lung diseases, which can directly and quickly increase the drug concentration in the treatment area and reduce the systemic concentration. However, pulmonary inhalation drug delivery also faces problems such as rapid drug clearance and frequent drug delivery. There are three main barriers to pulmonary inhalation drug delivery: mucociliary clearance, phagocytosis of pulmonary macrophages, and the action of pulmonary surfactant. Compared with other nanoformulation technologies, nanocrystal technology has unique advantages in pulmonary drug delivery, such as high drug loading, good safety, reduced macrophage clearance, and enhanced mucus penetration. Summary of the invention

[0006] The purpose of the present invention is to provide an ultrasound-assisted self-assembled resveratrol nanocrystal and its preparation method and application, so as to solve the problems of poor water solubility and low bioavailability of resveratrol, and to provide a new resveratrol nanocrystal that is safe, stable, water-soluble and has good therapeutic effect.

[0007] To achieve the above object, the present invention provides a method for preparing ultrasound-assisted self-assembled resveratrol nanocrystals, the steps of which are as follows:

[0008] S1, dissolving resveratrol in an organic solvent to obtain a resveratrol organic phase;

[0009] S2. Rapidly inject the resveratrol organic phase obtained in step S1 into deionized water under ultrasonic conditions, and continue ultrasonication for 5 minutes.

[0010] Preferably, the organic solvent in step S1 is dimethyl sulfoxide.

[0011] Preferably, the concentration of resveratrol in the organic solvent in step S1 is 20 mM.

[0012] Preferably, the concentration of the ultrasound-assisted self-assembled resveratrol nanocrystals prepared in step S2 is 400 μM.

[0013] Preferably, the ultrasound intensity in step S2 is 100 Hz.

[0014] Preferably, in step S2, the volume ratio of the organic phase to deionized water is 1:25-100.

[0015] Preferably, in step S2, the volume ratio of the organic phase to deionized water is 1:50.

[0016] Resveratrol nanocrystals prepared by the method for preparing ultrasound-assisted self-assembled resveratrol nanocrystals as described above.

[0017] A use of the resveratrol nanocrystals as described above in antiviral and anti-inflammatory activities, wherein the virus is respiratory syncytial virus, and inflammation is caused by respiratory syncytial virus.

[0018] A use of the resveratrol nanocrystals as described above in preparing a drug for treating pneumonia, wherein the pneumonia is caused by respiratory syncytial virus.

[0019] Therefore, the ultrasound-assisted self-assembled resveratrol nanocrystals and their preparation method and application provided by the present invention have the following specific technical effects:

[0020] (1) The ultrasound-assisted self-assembled resveratrol nanocrystals prepared by the present invention are regular spherical in shape, 181.5±2.27 nm, with a zeta potential of -12.6±0.85 mV and a PDI of 0.08±0.07. They have reliable stability without the use of a stabilizer and can be maintained for at least one week;

[0021] (2) The ultrasound-assisted self-assembled resveratrol nanocrystals prepared by the present invention have significantly improved water solubility of 1793.8±36.8μg / mL compared with untreated resveratrol (water solubility of 47.9±1.97μg / mL); and have good solubility in a slightly acidic inflammatory environment;

[0022] (3) The ultrasound-assisted self-assembled resveratrol nanocrystals prepared by the present invention have a stronger affinity for the protein receptor on RSV. Compared with resveratrol, they show a significantly improved inhibitory effect on RSV both in vitro and in vivo, and the inhibitory effect on RSV is dose-dependent; they also have good in vitro anti-inflammatory and antioxidant activities;

[0023] (4) The ultrasound-assisted self-assembled resveratrol nanocrystals prepared by the present invention are more conducive to cell uptake. The cell uptake at 37°C is four times that at 4°C, which can improve the retention in the lungs. Atomized inhalation is safe and effective in treating RSV pneumonia.

[0024] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0026] Figure 1is the investigation result of the resveratrol nanocrystals obtained by different preparation methods in Example 1 of the present invention, wherein Aa is the TEM image of the resveratrol nanocrystals prepared by the existing anti-solvent precipitation method, Ab is the TEM image of the resveratrol nanocrystals prepared by the stirring-assisted anti-solvent precipitation method, Ac is the TEM image of the resveratrol nanocrystals prepared by the high-speed shear-assisted anti-solvent precipitation method, and Ad is the TEM image of the resveratrol nanocrystals prepared by the ultrasonic-assisted anti-solvent precipitation method; Ba is the particle size distribution of the resveratrol nanocrystals prepared by the anti-solvent precipitation method, Bb is the particle size distribution of the resveratrol nanocrystals prepared by the stirring-assisted anti-solvent precipitation method, Bc is the particle size distribution of the resveratrol nanocrystals prepared by the high-speed shear-assisted anti-solvent precipitation method, and Bd is the particle size distribution of the resveratrol nanocrystals prepared by the ultrasonic-assisted anti-solvent precipitation method;

[0027] Figure 2 The results of the particle size, PDI (A) and potential (B) of resveratrol nanocrystals prepared by different organic solvents in Example 1 of the present invention are shown;

[0028] Figure 3 The results of investigating the particle size, PDI (A) and potential (B) of resveratrol nanocrystals prepared at different ratios of organic phase to aqueous phase in Example 1 of the present invention are shown;

[0029] Figure 4 The results of the particle size, PDI (A) and potential (B) of resveratrol nanocrystals prepared at different resveratrol concentrations in Example 1 of the present invention are shown;

[0030] Figure 5 TEM image (A), particle size distribution diagram (B), potential change diagram (C), laser irradiation photo (D), DSC test result (E) and XRD test result (F) of the resveratrol nanocrystals prepared in Example 3 of the present invention;

[0031] Figure 6 The results of the investigation of the physicochemical properties of the resveratrol nanocrystals prepared in Example 3 of the present invention, wherein A is the change in particle size of the resveratrol nanocrystals at different temperatures and time; B is the change in zeta potential of the resveratrol nanocrystals at different temperatures and time; C is the saturated solubility of the resveratrol nanocrystals in different solvents; D is the physical formula and method used by scientists in the past 15 years to improve the solubility of resveratrol; E is the in vitro cumulative release of resveratrol; the data are expressed as mean ± SEM, representing the results of three independent experiments, *P<0.05, **P<0.01, ***P<0.001;

[0032] Figure 7The results of the in vitro antiviral effect investigation of resveratrol nanocrystals prepared in Example 3 of the present invention, wherein A is a typical RVS infected host cell image; B is a fluorescence image and; C and D are fluorescence quantification of RSV protein expression during RSV infection using immunofluorescence detection; EC50 values ​​of resveratrol (E) and resveratrol nanocrystals (F); G is a schematic diagram of RSV entering the host cell; H is a schematic diagram of the host cell cycle during RSV infection, including attachment, invasion, replication and release stages; I is the EC50 values ​​of resveratrol and resveratrol nanocrystals in different RSV infection cycles. Effect of nanocrystals on RSV proliferation; J is the relative expression of RSV protein mRNA in A549 cells cultured with 25 μM resveratrol or resveratrol nanocrystals for 24 hours determined by RT-qPCR; K is the molecular docking of resveratrol and M protein receptor; L is the heat map of docking binding energy; data are expressed as mean ± SEM and represent the results of three independent experiments. Compared with the control group, ##P<0.01, ##P<0.001; compared with the model group or the indicated groups, *P<0.05, **P<0.01, ***P<0.001;

[0033] Figure 8 The results of the antiviral stage investigation of the resveratrol nanocrystals prepared in Example 3 of the present invention, wherein A is the antiviral result of the adsorption stage; B is the antiviral result of the entry stage; C is the antiviral result of the replication stage; D is the antiviral result of the release stage; the data are expressed as mean ± SEM, representing the results of three independent experiments, compared with the indicated groups, *P<0.05, **P<0.01, ***P<0.001;

[0034] Fig. 9 It is the molecular docking simulation result of the oligomers (dimer, trimer and tetramer) of resveratrol and the protein receptor in Example 3 of the present invention;

[0035] Fig.10: These are the results of the in vitro anti-inflammatory and antioxidant activities of the resveratrol nanocrystals prepared in Example 3 of the present invention, wherein A is a schematic diagram of LPS-induced RAW264.7 cells; B is the level of NO after LPS-induced RAW264.7 cells were treated with resveratrol or resveratrol nanocrystals; CF is the flow cytometry analysis of the expression of CD86 and CD206 in LPS-induced RAW264.7 cells treated with resveratrol or resveratrol nanocrystals; GJ is the expression of IL-6, IL-1B and TNF-α in the supernatant of LPS-induced RAW264.7 cells; KN is qRT-PCR analysis of the expression of IL-6, IL-1B and TNF-α mRNA in RAW264.7 cells induced by resveratrol or resveratrol nanocrystals; Fluorescence images of RAW264.7 cells infected with resveratrol or resveratrol nanocrystals (O) and quantitative changes in ROS (P); Data are expressed as mean ± SEM and represent the results of three independent experiments; Compared with the control group, by t test, ##P<0.01, ##P<0.001; Compared with the model group or the indicated groups, *P<0.05, **P<0.01, ***P<0.001;

[0036] Fig.11 The results of the absorption of resveratrol and resveratrol nanocrystals by A549 cells in Example 3 of the present invention, wherein A is the result of fluorescence microscopy observation; B is the average fluorescence intensity of resveratrol and resveratrol nanocrystals entering cells; the cellular uptake of resveratrol (C) and resveratrol nanocrystals (D) at different temperatures, times (E) and different endocytosis inhibitors (F); the data are expressed as mean ± SEM and represent the results of three independent experiments; compared with the indicated groups, *P<0.05, **P<0.01, ***P<0.001;

[0037] Fig.12 The results of the investigation of lung retention in mice after aerosol inhalation of resveratrol nanocrystals in Example 3 of the present invention, wherein A is the in vivo imaging photos and average fluorescence intensity of different organs of Cy7.5-labeled resveratrol nanocrystals after 1 hour, 3 hours, and 6 hours (B); C is the H&E staining section of the tissue; compared with the indicated groups, **P<0.01, ***P<0.001;

[0038] Fig.13The in vivo treatment results of viral pneumonia in RSV-infected mice in Example 3 of the present invention, wherein A is a virus-infected mouse pneumonia model and a schematic diagram of drug administration; B is the appearance of fresh lung tissue; C is the change in mouse weight; D is the RT-qPCR determination of the viral load in the lungs of different groups of mice; E is the RT-qPCR determination of the changes in pro-inflammatory factors in different groups of mice; F is a micro-CT image of the lung microenvironment of different groups of mice before and after virus infection; G is a lung function test of different groups of mice; H is a representative H&E staining image of the lungs of different groups of mice (scale bar, 200 μm); blue arrows indicate bronchiectasis, yellow arrows indicate lung consolidation, and black arrows indicate inflammatory infiltration; N=8, **P<0.01, ***P<0.001, and t-test was used for comparison with the groups shown;

[0039] Fig.14 The micro-CT images of the lung microenvironment of different groups of mice before and after virus infection in Example 3 of the present invention are shown, wherein A is a graph showing changes in expiratory time; B is a graph showing changes in inspiratory time; C is a graph showing changes in inspiratory peak flow; D is a graph showing changes in expiratory peak flow; E is a graph showing changes in tidal volume; F is a graph showing changes in expiratory volume; G is a graph showing changes in relaxation time; and H is a graph showing changes in 50% expiratory flow. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0041] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0042] The resveratrol used in the examples was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. The protein primers for F, G, M, NS1, NS2, IL-6, IL-1β, and TNF-α were purchased from Shanghai Sangon Biotech Co., Ltd. CY7.5 NHS was purchased from Lumiprobe Corporation (USA). Trizo, Hi-Scrper Mix for qPCR and gDNAEraser (R223), and SYBR Green PCR MasterMix (Q331-02) were purchased from Vazyme Co., Ltd. (Nanjing, China). Dulbecco's Modified Eagle Medium (DMEM, C11995500BT / 8123412) and fetal bovine serum (FBS) were purchased from Gibco Invitrogen. Phosphate Buffered Saline (PBS) was provided by Servicebio. Lipopolysaccharide (LPS), DAPI, NO detection kit, reactive oxygen species detection kit, and Radio-Immunoprecipitation Assay (RIPA) were purchased from Beyotime Biotechnology Co., Ltd.

[0043] The human respiratory syncytial virus A2 strain was purchased from the Institute of Virology, Wuhan University (A2 strain, Wuhan, China). Human laryngeal epithelial cells (HEp-2), human lung cancer cells (A549), and mouse macrophages (RAW 264.7) were purchased from ATCC (Manassas, VA, USA). In all cell experiments, resveratrol was formulated in DMEM containing 0.01% DMSO.

[0044] BALB / c mice (female, 6 weeks old, weighing 16 - 18 g) were purchased from Shanghai Bikai Biotechnology Co., Ltd. (Shanghai, China, SCXKK). (Shanghai, China; SCXK (Hu) 2018 - 0006). All animal experiments were approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine (202312A054).

[0045] XRD analysis was performed using a D8 QUEST X-ray diffractometer (Bruker Corporation, Germany) to generate CuKa radiation at 40 KV and 30 mV. The scanning range of the diffraction angle (2θ) was from 5° to 60°, and the scanning speed was 5° / min. When performing DSC analysis, 6 mg of the powder was placed in an aluminum pan, sealed, and protected with nitrogen. The temperature was raised from 25°C to 600°C at a rate of 10°C per minute and a flow rate of 30 mL per minute. The DSC instrument (DZ-DSC300C, Nanjing, China) was used to evaluate the thermal stability.

[0046] Other instruments, equipment, reagents, and materials not specified were also obtained through commercial channels, and the test methods not detailed were conventional methods in this field.

[0047] Example 1

[0048] Studies have shown that the greater the absolute value of the zeta potential, the smaller the polydispersity index (PDI) (PDI < 0.3), the more stable the nanocrystals, and the better the dispersibility. Therefore, the present invention uses particle size, PDI and zeta potential as standards and adopts a single factor method to obtain the optimal preparation process of resveratrol nanocrystals. The specific steps are as follows:

[0049] (1) Investigate the influence of different external forces

[0050] ① No external force:

[0051] 25 mg of resveratrol was dissolved in 10 mL of dimethyl sulfoxide (DMSO) to obtain a resveratrol organic phase with a concentration of 2.5 mg / mL. The resveratrol organic phase was directly and rapidly injected into deionized water to obtain resveratrol nanocrystals. The hydrodynamic size, PDI and potential of the obtained resveratrol nanocrystals were measured at 25°C using a Malvern Zetasizer ZS-90 dynamic light scattering (DLS) instrument. The results are shown in Figure 1 Aa and Figure 1 As shown in Ba.

[0052] ②Stirring assistance:

[0053] 25 mg of resveratrol was dissolved in 10 mL of dimethyl sulfoxide (DMSO) to obtain a resveratrol organic phase with a concentration of 2.5 mg / mL. The obtained resveratrol organic phase was quickly injected into deionized water under stirring (600 rpm) and then kept for 5 minutes to obtain resveratrol nanocrystals. The hydrodynamic size, PDI and potential of the obtained resveratrol nanocrystals were measured at 25°C using a Malvern Zetasizer ZS-90 dynamic light scattering (DLS) instrument. The results are as follows: Figure 1 Ab and Figure 1 Bb is shown.

[0054] ③ High-speed shearing assistance:

[0055] 25 mg of resveratrol was dissolved in 10 mL of dimethyl sulfoxide (DMSO) to obtain a resveratrol organic phase with a concentration of 2.5 mg / mL. The obtained resveratrol organic phase was quickly injected into deionized water under high-speed shear (9000 rpm) conditions and then kept for 5 minutes to obtain resveratrol nanocrystals. The hydrodynamic size, PDI and potential of the obtained resveratrol nanocrystals were measured at 25°C using a Malvern Zetasizer ZS-90 dynamic light scattering (DLS) instrument. The results are as follows: Figure 1 Ac and Figure 1 As shown in Bc.

[0056] ④Ultrasound-assisted:

[0057] 25 mg of resveratrol was dissolved in 10 mL of dimethyl sulfoxide (DMSO) to obtain a resveratrol organic phase with a concentration of 2.5 mg / mL. Under ultrasonic (100 Hz) conditions, the obtained resveratrol organic phase was quickly injected into deionized water and then kept for 5 minutes to obtain resveratrol nanocrystals (resveratrol nanocrystals). The hydrodynamic size, PDI and potential of the obtained resveratrol nanocrystals were measured at 25°C using a Malvern Zetasizer ZS-90 dynamic light scattering (DLS) instrument. The results are as follows: Figure 1 Ad and Figure 1 Bd.

[0058] The statistical results are shown in Table 1.

[0059] Table 1 Results of investigation of different external forces (n=3)

[0060] external force Particle size (nm) PDI Potential (mv) No external force 751±38.0 0.186±0.101 -8.77±0.92 Stirring 468.2±26.9 0.435±0.039 -12.2±0.79 High speed shear 425.2±70.9 0.391±0.063 -11.9±0.49 Ultrasound 178.1±5.68 0.137±0.037 -13.4±1.52

[0061] From Table 1, Figure 1 As can be seen from A and B, compared with the resveratrol nanocrystals prepared by stirring-assisted antisolvent precipitation and high-speed shear-assisted antisolvent precipitation, the resveratrol nanocrystals prepared under ultrasonic conditions are regular spherical ( Figure 1 Ad, Bd), the particle size is 178.1±5.68nm, the PDI is 0.137±0.037, and the Zeta potential is -13.4±1.52mV.

[0062] This indicates that the high energy of ultrasound overcomes the dynamic and stable interactions between monomers, and the directional flow of sound waves overcomes the disordered arrangement of macromolecules, which can induce the monomers to arrange in order and form a dynamically stable nanostructure. That is, ultrasound-assisted antisolvent precipitation is more conducive to the orderly interaction between molecules. Therefore, ultrasound was used in the following steps.

[0063] (2) Investigation of the most suitable organic phase type

[0064] 0.23 mg of resveratrol was dissolved in 100 μL of methanol, ethanol, DMSO and tetrahydrofuran to obtain resveratrol organic phases with a concentration of 20 mM. The obtained different resveratrol organic phases were quickly injected into 5 mL of deionized water, and then continuously ultrasonicated for 5 minutes to obtain resveratrol nanocrystals (resveratrol nanocrystals).

[0065] The particle size, PDI and potential of the obtained resveratrol nanocrystals were measured using a DLS instrument at 25°C. The results are shown in Table 2. Figure 2 shown.

[0066] Table 2 Results of organic phase investigation (n=3)

[0067] Organic phase type Particle size (nm) PDI Potential (mv) Methanol 2686.3±267 1 -7.3±0.26 Ethanol 309.5±38.3 0.402±0.079 -7.11±0.01 DMSO 178.1±5.68 0.137±0.037 -13.4±1.52 Tetrahydrofuran 308.9±3.55 0.354±0.132 -7.3±0.47

[0068] Table 2 and Figure 2 The results show that when the organic solvent is dimethyl sulfoxide, the particle size and PDI of the prepared resveratrol nanocrystals are the smallest, and the absolute value of the potential is the largest, indicating that the resveratrol nanocrystals under this condition are the most stable.

[0069] (3) Investigate the optimal ratio of organic phase to aqueous phase

[0070] 1.2, 0.9, 0.45, 0.23, 0.12 mg of resveratrol were dissolved in 500, 200, 100, 50, 25 μL of DMSO to obtain a series of resveratrol organic phases with a concentration of 10 mM. Different volumes of resveratrol organic phases were quickly injected into 5 mL of deionized water, and the ratio of organic phase to aqueous phase was 1:10, 1:25, 1:50, 1:100, 1:200, respectively, and then ultrasonicated continuously for 5 minutes to obtain resveratrol nanocrystals.

[0071] The particle size, PDI and potential of the obtained resveratrol nanocrystals were measured using a DLS instrument at 25°C. The results are shown in Table 3 and Figure 3 As shown, when the ratio of organic phase to aqueous phase is 1:50, the particle size and PDI of the prepared resveratrol nanocrystals are the smallest, and the absolute value of the potential is the largest, indicating that the resveratrol nanocrystals under this condition are the most stable.

[0072] Table 3 Investigation results of the ratio of organic phase to aqueous phase (n=3)

[0073] Proportion Particle size (nm) PDI Potential (mv) 1:10 1784±460 1 -7.3±0.26 1:25 300.1±6.90 0.402±0.079 -7.11±0.01 1:50 178.1±5.68 0.137±0.037 -13.4±1.52 1:100 301.4±10.6 0.101±0.028 -11.4±0.95 1:200 512.5±10.6 0.354±0.132 -7.3±0.47

[0074] (4) Final resveratrol concentration investigation

[0075] 1.8, 0.9, 0.45, 0.23, 0.12 mg of resveratrol were dissolved in 100 μL of DMSO to obtain resveratrol organic phases with concentrations of 80, 40, 20, 10, and 5 mM, respectively. The obtained resveratrol organic phases with different concentrations were quickly injected into 5 mL of deionized water, and then ultrasonicated for 5 minutes continuously, and finally resveratrol nanoparticles with concentrations of 1600, 800, 400, 200, and 100 μM were obtained, that is, resveratrol nanocrystals.

[0076] The particle size, PDI and potential of the obtained resveratrol nanocrystals were measured using a DLS instrument at 25°C. Figure 4 As shown in Table 4, when the final drug concentration is 400 μM, the particle size and PDI of the prepared resveratrol nanocrystals are the smallest, and the absolute value of the potential is the largest, indicating that the resveratrol nanocrystals under this condition are the most stable.

[0077] Table 4 Investigation results of final drug concentration (n=3)

[0078] concentration Particle size (nm) PDI Potential (mv) 1600 443.1±46.4 0.434±0.010 -4.62±0.33 800 450.6±15.9 0.435±0.045 -5.96±0.60 400 181.5±2.27 0.084±0.075 -12.6±0.85 200 178.1±5.68 0.137±0.037 -13.4±1.52 100 178.2±1.52 0.118±0.016 -12.99±0.45

[0079] Therefore, according to the above investigation results, the best preparation method of resveratrol nanocrystals is determined as follows: using DMSO as the organic phase, dissolving resveratrol in the organic phase (20mM), and rapidly injecting the organic phase into deionized water (aqueous phase) under ultrasound (100Hz), with the volume ratio of the organic phase to the aqueous phase being 1:50, and continuing ultrasound for 5 minutes after the injection. Finally, resveratrol nanocrystals with a concentration of 400μM were obtained.

[0080] Embodiment 2

[0081] Resveratrol nanocrystals are prepared by the method of the prior art, and the specific steps are as follows:

[0082] 2.5 mg of resveratrol was dissolved in 1 mL of DMSO to obtain a resveratrol organic phase with a concentration of 10 mM. 100 μL of the obtained organic phase was quickly injected into 5 mL of deionized water to obtain resveratrol nanoparticles.

[0083] TEM image of the prepared resveratrol nanoparticles Figure 1 The particle size distribution diagram is shown in Aa. Figure 1 B. From the TEM image, it can be seen that the resveratrol nanoparticles are in an aggregated state and have poor dispersion. From DLS, it can be seen that the particle size of the resveratrol nanoparticles is about 751±38.1nm, which indicates that the resveratrol nanoparticles prepared by the anti-solvent precipitation method are easy to aggregate and have poor dispersion.

[0084] Embodiment 3

[0085] The optimal process obtained in Example 1 was used to prepare resveratrol nanocrystals, and the specific steps were as follows:

[0086] 0.45 mg of resveratrol was dissolved in 100 μL of DMSO to obtain a resveratrol organic phase with a concentration of 20 mM. Under ultrasonic conditions (100 Hz), the obtained resveratrol organic phase was quickly injected into 5 mL of deionized water and then maintained for 5 minutes to obtain resveratrol nanocrystals.

[0087] (1) The obtained resveratrol nanocrystals were characterized, and the results were as follows:

[0088] The morphology of resveratrol nanocrystals was investigated by TEM. Figure 5 As shown in A, resveratrol nanocrystals are regular spheres with a particle size of about 150 nm.

[0089] The particle size, PDI and potential changes of resveratrol nanocrystals were investigated by DLS. Figure 5 As shown in Figure B, the particle size of resveratrol nanocrystals is 181.5±2.27nm, the zeta potential is -12.6±0.85mV, and the PDI is 0.08±0.07. The research report pointed out that when PDI<0.3, the greater the absolute value of the zeta potential, the more uniform the particle size distribution.

[0090] Under laser irradiation, resveratrol nanocrystals showed the Tyndall effect, confirming that resveratrol molecules formed resveratrol nanocrystals ( Figure 5 D).

[0091] The results of DSC detection are as follows Figure 5 As shown in E, the main endothermic peak of resveratrol was observed to be 273.52°C and the initial melting temperature was 267.13°C, while the main endothermic peak of resveratrol nanocrystals was 267.42°C and the initial melting temperature was 258.49°C. Compared with resveratrol, the melting point of resveratrol nanocrystals decreased slightly, indicating that the lattice energy was reduced and the lattice changed after the formation of resveratrol nanocrystals.

[0092] The crystallographic changes of resveratrol nanocrystals were further studied by XRD analysis. The XRD spectrum of resveratrol showed sharp diffraction peaks, indicating that it was in a good crystalline state ( Figure 5 F). In contrast, resveratrol nanocrystals maintained diffraction peaks at 6.65°, 13.23°, 16.54°, 19.20°, and 28.31°, indicating that they still exist in the form of crystals. However, the new characteristic diffraction peaks that appeared in resveratrol nanocrystals, especially at 27.41°, 31.80°, 45.65°, and 56.54°, indicate that the crystal structure of the prepared resveratrol nanocrystals has changed. This indicates that the stacking form between small molecules in resveratrol nanocrystals has changed, which may be affected by the dissolution effect and ultrasound.

[0093] (2) Physical and chemical properties of resveratrol nanocrystals

[0094] The particle size and potential were measured by DLS. Figure 6 As shown in A and B, the particle size and potential of resveratrol nanocrystals remained stable over seven days. This indicates that the synthesized resveratrol nanocrystals have reliable stability without the use of stabilizers and can be maintained for at least one week.

[0095] The saturation solubility method was used to compare the saturation solubility of resveratrol and resveratrol nanocrystals in different solvents. The specific method is as follows: an excess of resveratrol / resveratrol nanocrystal powder was dissolved in various solvents (water, ethanol, DMSO and 1% SDS) to a final volume of 4 mL. The mixture was then incubated in a thermostatic oscillator at 37±2°C for 48 hours to evaluate its solubility. After the incubation, the precipitate was centrifuged and the resveratrol concentration in the supernatant was quantified using fluorescence spectrophotometry.

[0096] The results are as follows Figure 6 As shown in Figure C, compared with resveratrol (water solubility of 47.9±1.97μg / mL), the water solubility of resveratrol nanocrystals (1793.8±36.8μg / mL) increased by 35.1 times (P<0.001). In addition, the solubility in methanol increased by 4.5 times, the solubility in DMSO increased by 2.1 times, and the solubility in 1% SDS increased by 1.8 times. This shows that the prepared resveratrol nanocrystals can significantly improve the water solubility of resveratrol. Compared with other studies, the resveratrol nanocrystals prepared by this method have obvious advantages (the results of the literature survey are shown in Figure 6 D).

[0097] Studies have shown that poorly soluble substances are often difficult to dissolve in biological fluids due to solubility and particle size limitations, which greatly reduces their efficacy. Therefore, PBS buffer solution containing 1% SDS was used as the dissolution medium for in vitro evaluation of the dissolution of drugs and nanocrystals. Figure 6 As shown in E, after 48 hours at a pH of 7.4, the cumulative dissolution rate of resveratrol was only 17.8%, while the dissolution rate of resveratrol nanocrystals was 41.6%. This shows that the small particle size, high solubility and excellent water dispersibility of resveratrol nanocrystals contribute to the slow and continuous dissolution of active molecules. In addition, under slightly acidic inflammatory conditions (pH 5.5), resveratrol nanocrystals showed rapid drug dissolution within 0-4 hours, with a cumulative dissolution rate of 58.1%, which then gradually increased to 78.9% within 48 hours. Compared with the dissolution rates of 28.1% (4 hours) and 41.2% (48 hours) in an environment with a pH of 7.4 when there is no inflammation, this dissolution curve is significantly higher, indicating that resveratrol nanocrystals have a strong ability to effectively dissolve drugs in a slightly acidic inflammatory environment.

[0098] (3) In vitro viral inhibition effect of resveratrol nanoparticles

[0099] The cell morphology was observed by inverted microscope. Figure 7As shown in A, cells infected with RSV showed obvious cytopathic effects (CPE) and syncytia. Immunofluorescence analysis of RSV infection (MOI = 0.1) showed that the fluorescence intensity of the resveratrol and resveratrol nanocrystal treatment groups decreased, indicating that RSV proliferation was inhibited ( Figure 7 In particular, compared with the free resveratrol administration group, the inhibitory effect of resveratrol nanocrystals on RSV proliferation was more significant (P<0.001) ( Figure 7 C), which may be due to different levels of cellular uptake of resveratrol ( Figure 7 D). Figure 7 The qRT-PCR experiments in E and F confirmed the consistent results, revealing that the inhibitory effects of resveratrol and resveratrol nanocrystals on RSV were dose-dependent. 50 ) is 31.8μM, and resveratrol nanocrystals is 21.3μM. It is worth noting that when the concentration is 100μM (lower than the saturated solubility of resveratrol nanocrystals), the inhibitory effect is close to 90%, while the same concentration of free resveratrol (dissolved in 0.5% DMSO) has an inhibitory effect on RSV of only 60%. In addition, the selectivity index (SI) calculated based on the 50% cytotoxic concentration (CC50) value found that the antiviral effect of resveratrol nanocrystals (SI=25.8) was significantly improved compared with free resveratrol (SI=16).

[0100] like Figure 7 As shown in G, the viral life cycle involves multiple key stages. Initially, RSV attaches to receptors on the apical surface of ciliated epithelial cells through G protein. Subsequently, the fusion protein (F) promotes the fusion of the viral envelope with the host membrane, resulting in the release of the nucleocapsid into the cytoplasm of the host cell. Subsequently, the mRNA is translated and replicated within the host cell. Finally, the matrix protein (M) and non-domain proteins (NS1 and NS2) interact with the complex, allowing RSV to be released from the cell by budding. In order to elucidate the potential antiviral mechanism of resveratrol nanocrystals, the effects of resveratrol and resveratrol nanocrystals on the four stages of the viral life cycle (attachment, invasion, replication and release) were investigated, and the results are shown in Figure 2. Figure 7 H and Figure 8 As shown). At all stages of RSV entry into host cells, resveratrol and resveratrol nanocrystals showed concentration-dependent inhibitory effects on RSV increment. During the virus invasion and replication stages, there was a significant difference in the inhibitory ability of resveratrol and resveratrol nanocrystals on RSV mRNA expression (P<0.001) ( Figure 7 I).

[0101] Similarly, the same results were observed when the expression of RSV-related proteins was inhibited. G protein and F protein are extracellular RSV proteins, and M protein, NS1 protein and NS2 protein are intracellular RSV proteins. Figure 7 The results in J showed that compared with free resveratrol, resveratrol nanocrystals can significantly inhibit the replication of RSV proteins (M, NS1 and NS2) in cells, thereby affecting the increase of RSV genome. Molecular docking simulations reached a consistent conclusion.

[0102] As we all know, the oligomers produced by the oxidation of resveratrol are collectively called vinblastine, which is an antitoxin produced by plants in response to environmental stress. In order to further simulate resveratrol nanocrystals, molecular docking simulations were performed using resveratrol oligomers (dimers, trimers and tetramers) and protein receptors. Figure 7 K and Fig. 9 As shown, the binding energy of resveratrol to M, NS1 and NS2 protein receptors (-6.6, -7.1, -6.9 kcal / mol, respectively) was significantly increased.

[0103] The study found that resveratrol interacted with the M receptors ALA53, LYS50, ILE49, VAL5, ASP46, MET1, and PHE0 via hydrogen bonds, Pi alkyl, and Pi-Pi amino acid residues. Importantly, the binding energy of the protein-receptor interaction decreased with increasing numbers of resveratrol molecules, suggesting an increased potency ( Figure 7 These results further support the view that resveratrol nanocrystals formed by self-assembly of multiple small molecules have a stronger affinity for the protein receptor on RSV, thereby inhibiting the replication of RSV.

[0104] (4) Investigate the anti-inflammatory and antioxidant activities of resveratrol nanocrystals in vitro

[0105] The inflammatory model was established by stimulating Raw 264.7 cells with LPS ( Fig.10 The effects of different doses (high, 50 μM; medium, 25 μM; low, 12.5 μM) of resveratrol and resveratrol nanocrystals on cellular NO levels were evaluated ( Fig.10 B). The results showed that resveratrol nanocrystals significantly reduced cellular NO levels in a dose-dependent manner compared to resveratrol alone. In addition, resveratrol nanocrystals significantly reduced intracellular NO concentrations compared to free resveratrol.

[0106] To further explore the anti-inflammatory properties of resveratrol nanocrystals, flow cytometry was used to detect the polarization of macrophages. Fig.10As shown in CF, LPS stimulation led to a significant increase in CD86 expression compared with normal Raw 264.7 cells and promoted the polarization of macrophages from M0 to M1. Compared with the LPS group, each treatment group inhibited the expression of CD86 to varying degrees, among which the inhibition rate of the resveratrol nanocrystal group was half that of the resveratrol group. In addition, compared with the resveratrol group, the resveratrol nanocrystal group not only promoted the polarization of macrophages from M1 to M2, but also increased the expression of CD206 from 0.35% to 4.59%. These findings suggest that resveratrol nanocrystals can enhance the immunomodulatory effects of drugs, leading to anti-inflammatory responses.

[0107] ELISA and RT-qPCR were used to detect the expression of pro-inflammatory cytokines in cells. Fig.10 As shown in GJ, after LPS stimulation, the levels of IL-6, IL-1β, and TNF-α cytokines were significantly increased, indicating that the cell model is effective. Compared with resveratrol, resveratrol nanocrystals significantly reduced the expression of these cytokines. In addition, RT-qPCR results showed that the expression of IL-6 and TNF-α mRNA was significantly reduced after resveratrol nanocrystal treatment ( Fig.10 of KN).

[0108] RSV was used to induce ROS production in Raw 264.7 cells to evaluate the pharmacological differences between resveratrol and resveratrol nanocrystals in alleviating oxidative stress. Fig.10 As shown in O and P, RSV stimulation of A549 cells resulted in excessive ROS production, while the amount of ROS production was reduced when the same concentration (25 μM) of resveratrol or resveratrol nanocrystals was applied. These findings suggest that nanoscale resveratrol nanocrystals not only have antiviral effects, but also can inhibit RSV-induced ROS.

[0109] (5) Cellular absorption of resveratrol nanocrystals

[0110] Fluorescence microscopy was used to observe the effects of resveratrol and resveratrol nanocrystals on cell uptake. Fig.11 As shown in A and B, compared with free drug, resveratrol nanocrystals are more conducive to cellular uptake (green fluorescence). Since cellular uptake is affected by many factors, different incubation times, concentrations, temperatures and inhibitors were tested. The results showed that in the resveratrol group and the resveratrol nanocrystal group, the concentration-dependent ( Fig.11 C and D) and time dependence ( Fig.11 E) showed an increase in cellular uptake. Specifically, the intracellular content of resveratrol gradually increased over time, showing a time-dependent manner, and after 6 hours, there was no significant change in the intracellular content.

[0111] Since low temperature may inhibit the activity of cellular transporters, the effect of temperature on cellular absorption was investigated by decreasing the temperature from 37°C to 4°C. Fig.11 As shown in C, for the resveratrol group, no significant difference was observed between 4°C and 37°C, indicating that absorption mainly depends on passive diffusion. In contrast, for the resveratrol nanocrystal group ( Fig.11 D), the uptake at 37°C was four times that at 4°C. The obvious increase from low to high temperature suggests that the uptake of resveratrol nanocrystals may involve transporters and may be related to energy-dependent processes.

[0112] The uptake mechanism of resveratrol nanocrystals was investigated by adding different inhibitors ( Fig.11 F). Cellular internalization occurs through multiple pathways, and the typical effects of inhibitors on the uptake of resveratrol nanocrystals were investigated, including the clathrin inhibitor chlorpromazine (10 μg / mL), the caveolin inhibitors nystatin and methyl-β-cyclodextrin (30 μg / mL), the macrocytosis inhibitor amiloride (30 μg / mL), and the mitosis inhibitor colchicine (40 μg / mL). Compared with the control group without the addition of inhibitors, the presence of chlorpromazine resulted in the most significant decrease in uptake (50%). This indicates that the cellular uptake pathway of resveratrol nanocrystals is a lectin-mediated endocytosis process.

[0113] (6) Biodistribution and biocompatibility of aerosol inhaled resveratrol nanocrystals

[0114] Aerosol inhalation is a method of direct pulmonary drug delivery. Due to its high compliance, it is particularly suitable for the treatment of respiratory infections, especially in children. Cy7.5-labeled resveratrol and resveratrol nanocrystals were delivered into the lungs of mice using a micro-atomizer, and their biodistribution was investigated using fluorescence imaging technology. Fig.12 The results described in A and B of the Figures show that the deposition levels in the lungs varied at 1, 3, and 6 hours after administration compared to the free Cy7.5 dye group. Notably, there was no significant difference in fluorescence intensity between the resveratrol group and the resveratrol nanocrystal group at 1 hour. Over time, the fluorescence intensity of the resveratrol group decreased, while the resveratrol nanocrystal group maintained a consistent intensity, indicating that resveratrol nanocrystals improved lung retention and have the potential to enhance the treatment of RSV pneumonia. In addition, histological analysis ( Fig.12 C) showed no obvious abnormalities or lesions in the lungs or other tissues, indicating that the aerosolized inhaled nano-sized resveratrol nanocrystals were well tolerated. The results support the safety and efficacy of aerosolized inhaled resveratrol nanocrystals in the treatment of RSV pneumonia.

[0115] (7) Pharmacodynamic evaluation of resveratrol nanocrystals in vivo

[0116] Resveratrol nanocrystals have significant therapeutic effects on viral inhibition and anti-inflammatory responses in vitro. The in vivo antiviral effects of resveratrol and resveratrol nanocrystals on a mouse model of pneumonia infected with RSV were then evaluated. Fig.13 As shown in A, mice were infected with RSV and administered via the pulmonary route using a micronebulizer once a day. After three consecutive days of treatment, the mice were euthanized to assess viral load and inflammation levels. Fig.13 Analysis of lung tissue images in B showed that RSV-infected mice exhibited pulmonary capillary congestion and edema (indicated by the red arrows) with a dark red appearance compared to the control group that was not infected with RSV. The lung tissue in the ribavirin-treated group had a basically normal appearance, with milder pulmonary capillary congestion and edema, indicating less lung tissue damage. In contrast, the free resveratrol group exhibited mild pulmonary capillary congestion and edema, while the combined resveratrol group showed a normal lung tissue appearance with a light pink color and no obvious pulmonary capillary congestion, edema, or hemorrhage. Fig.13 As shown in Figure C, each treatment group showed different abilities to inhibit weight loss compared to the control group without RSV infection. Interestingly, the resveratrol group was significantly more effective than the resveratrol nanocrystal group in this regard. Fig.13 D and E further showed that compared with resveratrol alone, resveratrol nanocrystals can more effectively inhibit the viral load in lung lavage fluid (50%, P<0.001) and reduce the expression of proinflammatory factors IL-6 and TNF-α. It is worth noting that although the dosage of the resveratrol nanocrystal group was only 1 mg / kg, which is one-fifth of the dosage of ribavirin (5 mg / kg), they showed considerable efficacy in both antiviral and anti-inflammatory aspects. Ribavirin is a broad-spectrum antiviral drug that is not specific to RSV, so it cannot effectively reduce the viral load of RSV when taken at a dose of 1 mg / kg in vivo. The significant reduction in viral titers and inflammation levels confirms the outstanding advantages of resveratrol nanocrystals in the treatment of viral pneumonia.

[0117] Each mouse was scanned twice by micro-CT before and after RSV infection. The model group showed typical symptoms of viral pneumonia, such as severe bronchiectasis, inflammatory infiltration, and exudation. In addition, compared with the control group, no obvious diffuse white consolidation (yellow arrows) or bronchiectasis (blue arrows) were observed in the lungs after taking resveratrol nanocrystals ( Fig.13 F). Fig.13 G and Fig.14 As shown in the results, lung function is a clear indicator of lung damage in RSV-infected pneumonia mice. Compared with the free drug group, resveratrol nanocrystals significantly reduced bronchoconstriction, expiratory pause, maximum inspiratory volume, and maximum expiratory volume in mice (P<0.001). Histopathological analysis of lung tissue ( Fig.10H) also obtained similar results, showing that the lung lesions in the resveratrol nanocrystal treatment group were alleviated, with only mild alveolar collapse and inflammatory infiltration (black arrows), while severe lung damage was observed in the model group. In conclusion, the use of resveratrol nanocrystals in RSV-induced viral pneumonia is not only effective in antiviral and anti-inflammatory effects, but also protects lung function and provides good prevention.

[0118] Therefore, the resveratrol nanocrystals prepared by the present invention are regular spherical, 181.5±2.27nm, with a zeta potential of -12.6±0.85mV and a PDI of 0.08±0.07. They have reliable stability without the use of a stabilizer and can be maintained for at least one week. Compared with untreated resveratrol (water solubility of 47.9±1.97μg / mL), they have significantly improved water solubility of 1793.8±36.8μg / mL; they have good solubility in a slightly acidic inflammatory environment; compared with resveratrol, they show significantly improved inhibitory effects on RSV both in vitro and in vivo, and the inhibitory effects on RSV are dose-dependent; they also have good in vitro anti-inflammatory and antioxidant activities; they are more conducive to cell uptake, and the cell uptake at 37°C is four times that at 4°C, which can improve the retention in the lungs, and atomization inhalation is safe and effective in treating RSV pneumonia.

[0119] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing ultrasound-assisted self-assembled resveratrol nanocrystals, characterized in that: Here are the steps: S1, dissolving resveratrol in an organic solvent to obtain a resveratrol organic phase; S2. Rapidly inject the resveratrol organic phase obtained in step S1 into deionized water under ultrasonic conditions, and continue ultrasonication for 5 minutes.

2. The method for preparing ultrasound-assisted self-assembled resveratrol nanocrystals according to claim 1, characterized in that: The organic solvent in step S1 is dimethyl sulfoxide.

3. The method for preparing ultrasound-assisted self-assembled resveratrol nanocrystals according to claim 1, characterized in that: In step S1, the concentration of resveratrol in the organic solvent is 20 mM.

4. The method for preparing ultrasound-assisted self-assembled resveratrol nanocrystals according to claim 1, characterized in that: The ultrasonic intensity in step S2 is 100 Hz.

5. The method for preparing ultrasound-assisted self-assembled resveratrol nanocrystals according to claim 1, characterized in that: In step S2, the volume ratio of the organic phase to deionized water is 1:25-100.

6. The method for preparing ultrasound-assisted self-assembled resveratrol nanocrystals according to claim 1, characterized in that: The volume ratio of the organic phase to deionized water in step S2 is 1:

50.

7. The method for preparing ultrasound-assisted self-assembled resveratrol nanocrystals according to claim 1, characterized in that: The concentration of the ultrasound-assisted self-assembled resveratrol nanocrystals prepared in step S2 is 400 μM.

8. Resveratrol nanocrystals prepared by the method for preparing ultrasound-assisted self-assembled resveratrol nanocrystals according to any one of claims 1 to 7.

9. A use of resveratrol nanocrystals as claimed in claim 8 in antiviral and anti-inflammatory activities, characterized in that: The virus is respiratory syncytial virus, and the inflammation is caused by respiratory syncytial virus.

10. Use of the resveratrol nanocrystals as claimed in claim 8 in preparing a drug for treating pneumonia, characterized in that: The pneumonia was caused by respiratory syncytial virus.

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