Self-assembled nanoparticles, methods of making and using the same
By preparing self-assembled nanoparticles of isozygoflavin and isochlorogenic acid C, the problems of strong drug resistance and poor drug efficacy in the treatment of MRSA infection were solved, and synergistic inhibition of MRSA and wound healing effects were achieved.
Patent Information
- Application Number
- CN202610161607.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-04
- Publication Date
- 2026-05-29
AI Technical Summary
In the treatment of MRSA infection, strong drug resistance and poor drug efficacy, as well as poor water solubility and low bioavailability, limit its clinical application.
Nanoparticles are formed by the self-assembly of isochlorogenic acid C and isochlorogenic acid C. By utilizing the synergistic effect of the two, nanoparticles are prepared to synergistically inhibit MRSA, improve antibacterial effect and reduce the risk of drug resistance.
It significantly enhanced the inhibitory effect on MRSA, promoted wound healing of methicillin-resistant bacteria infections, and reduced the risk of drug resistance.
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Figure CN122097267A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to a self-assembled nanoparticle, its preparation method, and its application. Background Technology
[0002] Methicillin-resistant Staphylococcus aureus (MRSA), a typical "superbug," has spread widely worldwide since its discovery in 1961, becoming one of the major challenges in clinical infection control. This strain carries the SCCmec cassette chromosome, which encodes the penicillin-binding protein PBP2a, reducing its binding affinity to β-lactam antibiotics. This leads to resistance to most penicillin antibiotics, with a resistance rate exceeding 90%. Furthermore, resistance is enhanced through multiple mechanisms, including the production of β-lactamases and activation of efflux pump systems, severely limiting clinical treatment options.
[0003] MRSA infection can cause skin and soft tissue infections, and in severe cases, can progress to fatal complications such as sepsis, pneumonia, and endocarditis. Clinical data shows that the average hospital stay for patients with MRSA infection is three times that of ordinary patients, and the 30-day mortality rate can reach 34%. Globally, MRSA accounts for over 30% of hospital-acquired infections, placing a heavy burden on healthcare systems. Currently, glycopeptide antibiotics such as vancomycin and daptomycin are commonly used to treat MRSA infections, but MRSA strains with reduced vancomycin sensitivity have emerged, making the development of novel antimicrobial drugs urgently needed.
[0004] Natural active ingredients, due to their unique antibacterial mechanisms and low likelihood of inducing drug resistance, have become an important direction for the development of anti-MRSA drugs. Isorhynchonine, a flavonoid isolated from the traditional Chinese medicine *Artemisia*, has been proven to have significant antibacterial activity against *Staphylococcus aureus*, providing a potential candidate ingredient for anti-MRSA treatment. Isochlorogenic acid C, an important isomer of chlorogenic acid, also possesses excellent antibacterial activity. Studies have shown that it can resensitize drug-resistant *Escherichia coli* to multiple antibiotics and produce synergistic antibacterial effects with antibiotics, demonstrating promising application prospects in combating drug-resistant bacterial infections. However, isorhynchonine suffers from inherent drawbacks such as poor water solubility, low bioavailability, and rapid in vivo metabolism. When used alone, it is difficult to achieve effective antibacterial concentrations, and excessive dosage can easily cause toxic side effects, limiting its clinical translation. To address the delivery challenges of natural active ingredients, nanodelivery systems are widely used. Among them, carrier-free self-assembled nanoparticles, due to their lack of the need for additional carriers, high biosafety, and high drug loading rate, can effectively improve drug solubility and stability, and enhance antibacterial effects, making them a hot research topic in antibacterial nanomedicine development. Summary of the Invention
[0005] The purpose of this invention is to address the problems of strong drug resistance and poor efficacy in the treatment of MRSA infection in existing technologies, and to provide a self-assembled nanoparticle, its preparation method, and its application. This self-assembled nanoparticle achieves a synergistic effect between isochlorogenic acid C and iso-zellin, enhancing the antibacterial effect and promoting wound healing in methicillin-resistant bacteria infections.
[0006] To achieve the above objectives, the first aspect of the present invention provides a self-assembled nanoparticle, which is formed by the self-assembly of isochlorogenic acid C and isochlorogenic acid C through non-covalent bonding to form a supramolecular structure, and the particle size of the self-assembled nanoparticle is 459~825nm and the Zeta potential is -14.8mV~-35.2mV.
[0007] Preferably, the self-assembled nanoparticles have a particle size of 459~550nm and a Zeta potential of -25mV~-35.2mV.
[0008] Preferably, in the self-assembled nanoparticles, the molar ratio of isochlorogenic acid C to isochlorogenic acid C is 0.1~10:1, more preferably 0.5~2:1.
[0009] A second aspect of the present invention provides a method for preparing the self-assembled nanoparticles described above, the method comprising: dissolving isochlorogenic acid and isochlorogenic acid C in an organic solvent to obtain an organic phase, adding water to the organic phase, stirring to obtain a pre-assembled mixture, and then allowing the pre-assembled mixture to stand.
[0010] Preferably, the organic solvent is selected from at least one of ethanol, methanol, and dimethyl sulfoxide.
[0011] Preferably, the molar ratio of the organic solvent, the isochlorogenic acid C, and the isochlorogenic acid C is (600~700):(0.1~10):1.
[0012] Preferably, the volume ratio of the water to the organic solvent is 1 to 10:1.
[0013] Preferably, the stirring conditions include: a stirring speed of 300~700 r / min and a stirring time of 30~60 min.
[0014] Preferably, the conditions for settling include: a temperature of 1~10℃ and a time of 20~48h.
[0015] A third aspect of the present invention provides the application of the aforementioned self-assembled nanoparticles in the preparation of antibacterial drugs.
[0016] Preferably, the antibacterial drug kills methicillin-resistant bacteria.
[0017] This invention utilizes a self-assembly technique to prepare nanoparticles from isochlorogenic acid C and isochlorogenic acid C, aiming to achieve synergistic inhibition of MRSA, enhance antibacterial efficacy, and reduce the risk of drug resistance. Based on this, an isochlorogenic acid C self-assembled nanoparticle is developed, leveraging the synergistic antibacterial effects of both and the advantages of a nanodelivery system to address issues such as strong drug resistance and poor drug efficacy in the treatment of MRSA infections.
[0018] This invention, based on the self-assembly characteristics of small active molecules in traditional Chinese medicine, discovers and confirms through various techniques that EUP and IAC can undergo supramolecular self-assembly to form stable nanomedicines (EUP-IACs). These nanomedicines significantly enhance the inhibitory effect on MRSA USA 300 through the synergistic effect of EUP and IAC. This invention provides new ideas and strategies for optimizing the carrier-free self-assembled nanomaterial preparation of traditional Chinese medicine and expanding its application in the antibacterial field. Attached Figure Description
[0019] Figure 1 Tyndall effect diagram of EUP-IACs A1 prepared in Example 1.
[0020] Figure 2 The particle size distribution diagram is shown for EUP-IACs A1 prepared in Example 1.
[0021] Figure 3 The zeta potential diagram of EUP-IACs A1 prepared in Example 1.
[0022] Figure 4 FT of EUP-IACs A1 and monomers prepared in Example 1 IR spectrum.
[0023] Figure 5 The XRD patterns of EUP-IACs A1 and monomers prepared in Example 1 are shown.
[0024] Figure 6 The image shows the H-NMR spectrum of a single EUP.
[0025] Figure 7 The image shows the H-NMR spectrum of the monomeric IAC.
[0026] Figure 8 The H-NMR spectrum of EUP-IACs A1 prepared in Example 1.
[0027] Figure 9 Heatmaps showing the growth inhibition of MRSA USA 300 by different formulations.
[0028] Figure 10 The minimum inhibitory concentrations (MICs) of different formulations against MRSA USA 300 are given.
[0029] Figure 11 Image of an animal wound showing the skin healing process in mice.
[0030] Figure 12 This is a schematic diagram showing the results of skin healing in mice.
[0031] Figure 13 This is a statistical chart showing the results of skin healing in mice.
[0032] Figure 14 This is a statistical graph showing the bacterial load in mouse skin tissue.
[0033] Figure 15 This is a plate plot showing the bacterial load in mouse skin tissue. Detailed Implementation
[0034] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.
[0035] The endpoints and any values of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of the various ranges, the endpoint values of the various ranges and individual point values, and individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.
[0036] The self-assembled nanoparticles of the present invention are formed by the self-assembly of isozygoflavin and isochlorogenic acid C through non-covalent bonding to form supramolecular structures, and the particle size of the self-assembled nanoparticles is 459~825nm, and the zeta potential is -14.8mV~-35.2mV.
[0037] In a preferred embodiment, the self-assembled nanoparticles have a particle size of 459~550nm and a Zeta potential of -25mV~-35.2mV.
[0038] In this invention, the Fourier transform infrared spectrum of the self-assembled nanoparticles ( Figure 4 It can be seen that at 3414cm -1 Near the same location, the relative intensity of the OH absorption peak of the self-assembled nanoparticles significantly decreased and shifted noticeably. This redshift of the OH stretching vibration peaks is closely related to the formation of hydrogen bonds, indicating that isochlorogenic acid C and isochlorogenic acid C form a stable hydrogen-bonded complex structure through oxygen-hydrogen bonding (OH···O=C). Furthermore, the sample showed positive results in the 3100–3000 cm⁻¹ range. -1 Band, 839cm -1The absorption peaks of nearby CH showed varying degrees of shift and intensity changes, indicating that in addition to hydrogen bonding interactions, isoenzein and isochlorogenic acid C may also exhibit π–π stacking interactions between aromatic rings. In summary, isoenzein and isochlorogenic acid C form a stable composite structure through hydrogen bonding and π–π stacking interactions.
[0039] In this invention, the XRD pattern of the self-assembled nanoparticles ( Figure 5 As can be seen, the peak of EUP-ICAs A1 in the 10°~30° range simultaneously covers the characteristic diffraction range of EUP and ICA, indicating that the complex retains some crystal structure features of EUP and ICA, possibly due to intermolecular interactions that altered the arrangement of the original crystal form.
[0040] In this invention, the nuclear magnetic resonance hydrogen spectrum of the self-assembled nanoparticles shows that ( Figures 6-8 The EUP has active hydrogen peaks (10.220, 12.051 ppm), hydroxyl (-OH) hydrogen, and aromatic hydrogen peaks (6.6-7.6 ppm), indicating that the EUP contains a multi-substituted aromatic ring. The alkyl hydrogen peak (1.008 ppm) is characteristic of saturated hydrocarbon groups. The active hydrogens (hydroxyl hydrogen, 10.22, 12.05 ppm) of the EUP disappear in the complex, indicating that these active hydrogens may have formed intermolecular hydrogen bonds with electronegative groups (such as carbonyl C=O) in the ICA molecule.
[0041] In this invention, the molar ratio of isoenzyme to isochlorogenic acid C in the self-assembled nanoparticles can be 0.1~10:1, preferably 0.5~2:1. In this invention, when the molar ratio of isoenzyme to isochlorogenic acid C is within the above range (especially the preferred range), the self-assembled nanoparticles exhibit superior antibacterial properties.
[0042] The method for preparing self-assembled nanoparticles according to the present invention includes: dissolving isochlorogenic acid C and isochlorogenic acid C in an organic solvent to obtain an organic phase, then adding water to the organic phase, stirring to obtain a pre-assembled mixture, and then allowing the pre-assembled mixture to stand.
[0043] In this invention, the organic solvent can be at least one of ethanol, methanol and dimethyl sulfoxide, preferably ethanol.
[0044] In the method described in this invention, the molar ratio of the organic solvent, isochlorogenic acid, and isochlorogenic acid C can be (600~700):(0.1~10):1, preferably (620~690):(0.2~8):1, and more preferably (650~680):(0.5~2):1. In the method described in this invention, when the molar ratio of the organic solvent, isochlorogenic acid, and isochlorogenic acid C is within the above range, the isochlorogenic acid and isochlorogenic acid C are more uniformly dispersed in the organic solvent, resulting in more uniform self-assembled nanoparticles.
[0045] In the method described in this invention, the volume ratio of water to organic solvent can be 1~10:1, preferably 2~8:1, and more preferably 3~5:1. In the method described in this invention, when the volume ratio of water to organic solvent is within the above range, the formed self-assembled nanoparticles are more uniform and possess superior antibacterial properties.
[0046] In the method described in this invention, the stirring conditions may include: a stirring speed of 300-700 r / min and a stirring time of 30-60 min. In the method described in this invention, when the stirring conditions are within the above range, the formed self-assembled nanoparticles are more uniform and possess superior antibacterial properties.
[0047] In the method described in this invention, the settling conditions may include a temperature of 1~10℃ and a time of 20~48h. When the settling conditions are within the above range, the formed self-assembled nanoparticles are more uniform and exhibit superior antibacterial properties.
[0048] The self-assembled nanoparticles described in this invention can be used in the preparation of antibacterial drugs. Preferably, the antibacterial drug kills methicillin-resistant bacteria.
[0049] This invention utilizes a self-assembly technique to prepare nanoparticles from isochlorogenic acid C and isochlorogenic acid C, aiming to achieve synergistic inhibition of MRSA, enhance antibacterial efficacy, and reduce the risk of drug resistance. Based on this, an isochlorogenic acid C self-assembled nanoparticle is developed, leveraging the synergistic antibacterial effects of both and the advantages of a nanodelivery system to address issues such as strong drug resistance and poor drug efficacy in the treatment of MRSA infections.
[0050] This invention, based on the self-assembly characteristics of small active molecules in traditional Chinese medicine, discovers and confirms through various techniques that EUP and IAC can undergo supramolecular self-assembly to form stable nanomedicines (EUP-IACs). These nanomedicines significantly enhance the inhibitory effect on MRSA USA 300 through the synergistic effect of EUP and IAC. This invention provides new ideas and strategies for optimizing the carrier-free self-assembled nanomaterial preparation of traditional Chinese medicine and expanding its application in the antibacterial field.
[0051] The following examples further illustrate the self-assembled nanoparticles, their preparation method, and applications according to the present invention. These examples are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures; however, the scope of protection of the present invention is not limited to the following examples.
[0052] Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods in the art.
[0053] Unless otherwise specified, all experimental materials used in the following examples are commercially available.
[0054] In the following examples and comparative examples, the experimental instruments were: ultrasonic cleaner (Kunshan Jielimei Ultrasonic Instrument Co., Ltd., model: KS-5200E), electric heating drying oven (Shanghai Senxin Experimental Instrument Co., Ltd., model: DGG-9203A); electronic balance (Shanghai Hengping Balance Scientific Instrument Co., Ltd., model: AF224); multifunctional microplate reader (Gene, USA, model: Bio-TekSynergy2); infrared spectrometer (Thermo Fisher Scientific Nicolet iS20, USA), nuclear magnetic resonance spectrometer (Bruker Avance Ⅲ 400MHz, Germany), and X-ray diffractometer (Rigaku SmartLabSE, Japan).
[0055] Experimental reagents: isochlorogenic acid C and anhydrous ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd., MRSA USA 300 (ATCC BAA-1717) and SPF-grade male ICR mice.
[0056] Example 1 Accurately weigh 3.3 mg EUP and 5.2 mg IAC (molar ratio 1:1), add 400 μL of anhydrous ethanol, sonicate to dissolve, add 1.6 mL of water, stir at 500 r / min for 30 min, and let stand at 4℃ for 24 h to obtain EUP-IACs solution, denoted as A1.
[0057] Example 2 Accurately weigh 0.3 mg EUP and 5.2 mg IAC (molar ratio 0.1:1), add 400 μL of anhydrous ethanol, sonicate to dissolve, add 1.6 mL of water, stir at 500 r / min for 30 min, and let stand at 4℃ for 24 h to obtain EUP-IACs solution, denoted as A2.
[0058] Example 3 Accurately weigh 33 mg EUP and 5.2 mg IAC (molar ratio 10:1), add 400 μL of anhydrous ethanol, sonicate to dissolve, add 1.6 mL of water, stir at 500 r / min for 30 min, and let stand at 4℃ for 24 h to obtain EUP-IACs solution, denoted as A3.
[0059] Example 4 Accurately weigh 1.5 mg EUP and 5.2 mg IAC (molar ratio 0.5:1), add 400 μL of anhydrous ethanol, sonicate to dissolve, add 1.6 mL of water, stir at 500 r / min for 30 min, and let stand at 4℃ for 24 h to obtain EUP-IACs solution, denoted as A4.
[0060] Example 5 Accurately weigh 6.6 mg EUP and 5.2 mg IAC (molar ratio 2:1), add 400 μL of anhydrous ethanol, sonicate to dissolve, add 1.6 mL of water, stir at 500 r / min for 30 min, and let stand at 4℃ for 24 h to obtain EUP-IACs solution, denoted as A5.
[0061] Example 6 Accurately weigh 3.3 mg EUP and 5.2 mg IAC (molar ratio 1:1), add 350 μL of anhydrous ethanol, sonicate to dissolve, add 0.4 mL of water, stir at 300 r / min for 60 min, and let stand at 1℃ for 48 h to obtain EUP-IACs solution, denoted as A6.
[0062] Example 7 Accurately weigh 3.3 mg EUP and 5.2 mg IAC (molar ratio 1:1), add 408 μL of anhydrous ethanol, sonicate to dissolve, add 4 mL of water, stir at 700 r / min for 30 min, and let stand at 10℃ for 20 h to obtain EUP-IACs solution, denoted as A7.
[0063] Example 8 Accurately weigh 0.1 mg EUP and 5.2 mg IAC (molar ratio 0.03:1), add 400 μL of anhydrous ethanol, sonicate to dissolve, add 1.6 mL of water, stir at 500 r / min for 30 min, and let stand at 4℃ for 24 h to obtain EUP-IACs solution, denoted as A8.
[0064] Example 9 Accurately weigh 66 mg EUP and 5.2 mg IAC (molar ratio 20:1), add 400 μL of anhydrous ethanol, sonicate to dissolve, add 1.6 mL of water, stir at 500 r / min for 30 min, and let stand at 4℃ for 24 h to obtain EUP-IACs solution, denoted as A9.
[0065] Example 10 This embodiment is implemented according to the method described in Embodiment 1, except that the amount of water used is 0.1 mL, resulting in an EUP-IACs solution, denoted as A10.
[0066] Example 11 This embodiment is implemented according to the method described in Embodiment 1, except that the amount of water used is 8 mL, resulting in an EUP-IACs solution, denoted as A11.
[0067] Example 12 This embodiment is implemented according to the method described in Embodiment 1, except that the stirring speed is 200 r / min and the time is 90 min, resulting in an EUP-IACs solution, denoted as A12.
[0068] Example 13 This embodiment is implemented according to the method described in Embodiment 1, except that the stirring speed is 900 r / min and the time is 10 min, resulting in an EUP-IACs solution, denoted as A13.
[0069] Example 14 This embodiment is implemented according to the method described in Embodiment 1, except that the standing conditions include: temperature of 30°C and time of 10 hours, to obtain EUP-IACs solution, denoted as A14.
[0070] Example 15 This embodiment is implemented according to the method described in Embodiment 1, except that the standing conditions include: temperature of 30°C and time of 50h, to obtain EUP-IACs solution, denoted as A15.
[0071] Comparative Example 1 This comparative example was carried out according to the method described in Example 1, except that water was not added, resulting in an EUP-IACs solution, denoted as D-A1.
[0072] Comparative Example 2 This comparative example was carried out according to the method described in Example 1, except that no stirring was performed, and an EUP-IACs solution was obtained, denoted as D-A2.
[0073] Comparative Example 3 This comparative example was carried out according to the method described in Example 1, except that no standing was performed, and an EUP-IACs solution was obtained, denoted as D-A3.
[0074] Test Example 1 In this test example, the particle size distribution, polydispersity index (PDI), and zeta potential of EUP-IACs A1~A15 and D-A1~D-A3 were determined using a Malvern laser particle size analyzer. Each sample was measured three times. Results are as follows: Figures 1-3 As shown in Table 1.
[0075] Figure 1 To investigate the Tyndall effect of the EUP-IACs self-assembled nanoparticle solution, the obtained traditional Chinese medicine EUP-IACs self-assembled nanoparticle solution was placed in the sample cell of a dynamic light scattering instrument to test its particle size, PDI, and zeta potential. The results are as follows: Figure 2-3 As shown, the particle size is 506.3 nm, the PDI is 0.374, the zeta potential is -28.4 mV, and the nanoparticles are uniform in size.
[0076] Table 1
[0077] As shown in Table 1, the nanoparticles prepared under the preferred conditions have a more uniform and stable particle size than those in other groups.
[0078] Test Example 2 Weigh appropriate amounts of freeze-dried EUP, IAC, and EUP-IACs A1 powders, mix them separately with dry KBr fine powder, grind and mix evenly, and then compress them into uniform thin tablets using a tablet press. Fourier transform infrared spectroscopy was used to analyze the samples at 400–4000 cm⁻¹. -1 Its infrared absorption spectrum was measured within the wavenumber range. The results are as follows: Figure 4 As shown.
[0079] To preliminarily explore the binding sites of self-assembled nanoparticles, FT was performed. IR spectroscopy analysis. The lyophilized nanoparticle powder was analyzed using FT. The Fourier transform infrared spectra of monomers, nanoparticles, and physical mixtures were measured using an IR spectrometer, with wavenumbers ranging from 4000 to 400 cm⁻¹. -1 ,like Figure 4 As shown. At 3414cm -1 The relative intensity of the nearby OH absorption peaks decreased significantly and shifted noticeably. This red shift of the OH stretching vibration peaks is closely related to the formation of hydrogen bonds, indicating that isochlorogenic acid C and isochlorogenic acid C form a stable hydrogen-bonded complex structure through oxygen-hydrogen bonding (OH···O=C). Furthermore, the sample showed absorption peaks in the 3100–3000 cm⁻¹ range. -1Band, 839cm -1 The absorption peaks of nearby CH showed varying degrees of shift and intensity changes, indicating that in addition to hydrogen bonding interactions, isoenzein and isochlorogenic acid C may also exhibit π–π stacking interactions between aromatic rings. In summary, isoenzein and isochlorogenic acid C form a stable composite structure through hydrogen bonding and π–π stacking interactions.
[0080] Test Example 3 X-ray diffraction was used to analyze the crystal structure and phase composition of freeze-dried powders of EUP, IAC, and EUP-IACs A1. The incident light source was Cu target Kα rays (λ = 1.5406 Å). The diffraction angle range was set to 2θ = 10-90°, the step size was 0.02°, and the scanning speed was 1° / min. X-ray diffraction patterns were acquired. The results are as follows: Figure 5 As shown.
[0081] To explore the crystal types and structures of nanoparticles, lyophilized nanoparticle powder was used, and XRD patterns of monomers, nanoparticles, and physically mixed nanoparticles were determined using an X-ray diffractometer. The scan rate was set to 0.5° / min, and the range was 10–90°. Figure 5 As shown, EUP exhibits sharp and high-intensity diffraction peaks in the 10°–30° range, indicating that EUP itself is a well-crystallized substance. The diffraction peaks essentially disappear after 30°, reflecting the absence of characteristic diffraction at the high 2θ angle. ICA's peaks are less sharp and wider, indicating relatively lower crystallinity. The peaks of EUP-ICAs A1 in the 10°–30° range simultaneously cover the characteristic diffraction ranges of both EUP and ICA, suggesting that the complex retains some crystal structure characteristics of both EUP and ICA. This may be due to the presence of amorphous components in the complex or enhanced scattering caused by intermolecular interactions. EUP shows "multiple sharp single peaks," ICA shows "broad raised peaks," while EUP-ICAs shows "high-intensity, wide-wave peaks," indicating that the complex retains some structural characteristics of both, possibly due to intermolecular interactions altering the original crystal arrangement.
[0082] Test Example 4 Precisely weighed EUP-IACs A1 and precisely weighed sodium p-hydroxybenzoate (internal standard) were uniformly mixed in a deuterated reagent to obtain a sample solution. The sample solution was then scanned using a nuclear magnetic resonance (NMR) spectrometer to obtain a hydrogen NMR spectrum. The results are as follows: Figures 6-8 As shown.
[0083] Depend on Figures 6-8It can be seen that EUP has active hydrogen peaks (10.220, 12.051 ppm), hydroxyl (-OH) hydrogen, and aromatic hydrogen peaks (6.6~7.6 ppm), indicating that EUP contains multi-substituted aromatic rings. The alkyl hydrogen peak (1.008 ppm) is a characteristic of saturated hydrocarbon groups. The active hydrogens (hydroxyl hydrogen, 10.22, 12.05 ppm) of EUP disappear in the complex, indicating that these active hydrogens may have formed intermolecular hydrogen bonds with electronegative groups (such as carbonyl C=O) in the ICA molecule.
[0084] Test Example 5 Antibacterial activity assay of nanoparticles and monomers: The inhibition rate of MRSA USA300 in vitro was determined by microbroth dilution. Bacteria were cultured overnight in nutrient broth at 37°C with a shaking speed of 150 rpm. 100 μL of sample (monomer and nanoparticle A1) was injected into a 96-well plate using the broth dilution method, followed by an equal volume of 100 μL of bacterial suspension (2 × 10⁻⁶). 6 CFU was added to 96-well plates to a final volume of 200 μL. The three diluted sample concentration gradients were 8, 2, 1, 0.5, 0.25, and 0.125 μmol / L. Gentamicin sulfate was used as a positive control, and nutrient broth was used as a solvent control. The 96-well plates were incubated at 37°C for 18–24 h. The minimum drug concentration at which no bacterial growth was observed visually was determined as the MIC, and the combination index (FIC) was calculated. Results are as follows: Figure 9 , Figure 10 As shown in Table 2.
[0085] like Figure 9 and Figure 10 As shown, the antibacterial effect of EUP-IACs A1 was determined by the micro-broth dilution method. The synergistic effect of EUP-IACs A1 was judged by calculating the combination index (FIC). When the combination index was less than 0.5, it indicated that the two had a synergistic antibacterial effect. When the FIC was greater than 2, it indicated that the two had an antagonistic effect. The FIC of EUP-IACs A1 was 0.375, indicating that the two had a synergistic antibacterial effect.
[0086] Table 2
[0087] As shown in Table 2, A1 has the best synergistic antibacterial effect.
[0088] Test Example 6 The effect of nanoparticles and monomers on promoting the healing of skin excision wounds in mice infected with methicillin-resistant bacteria: 1. Establishment and drug administration of a mouse model of skin excision wound infected with methicillin-resistant bacteria. Male ICR mice weighing 18-22 g were selected and fed at 22-25 °C daily. After one week of observation with no deaths or abnormal behavior, the mice were randomly divided into 6 groups of 6 mice each. After hair removal from the back, the mice were injected intraperitoneally with ursodeoxycholic acid (15%, 5 mL / kg). A circular hole was made in the back of each mouse using a punch, and a full-thickness skin sample with a diameter of 10.0 mm was excised. 50 μL of ursodeoxycholic acid (OD200) was applied to the wound. 600 =0.1) Staphylococcus aureus bacterial suspension, infected for 24 h, with PBS as blank control, EUP group (4 μM 100 μL EUP solution / mouse / day), IAC group (4 μM 100 μL IAC solution / mouse / day), and EUP-IACs A1 group (4 μM 100 μL EUP-IACs A1 / mouse / day), administered continuously for 2 weeks. The wound healing of mice was photographed and the mice were weighed daily.
[0089] 2. Sampling and pharmacodynamic evaluation After administration, the mice were euthanized, and the skin tissue at the wound site was removed using sterile scissors. 50 mg of mouse skin tissue was homogenized with 1 mL of PBS. 50 μL of the diluted solution was evenly spread onto a Staphylococcus aureus solid selection dish and incubated at 37°C for 18 h. The final viable colony count was calculated based on the dilution.
[0090] 3. Wound healing effect: like Figures 11-13 As shown, compared with the control group and the model group, the wound area of the EUP, IAC, and EUP-IACs A1 groups was reduced. The wound area of the EUP-IACs A1 group was smaller than that of the single-drug EUP and IAC groups, indicating that the EUP-IACs A1 nanoparticles have the effect of promoting wound healing, and the effect is more significant compared with single drugs.
[0091] 4. Tissue bacterial load: like Figures 14-15 As shown, compared with the control, the bacterial load in the skin tissue of the EUP, IAC, and EUP-IACs A1 groups was significantly reduced. The bacterial load in the skin tissue of the EUP-IACs A1 group was also lower than that of the single-drug EUP and IAC groups, indicating that the EUP-IACs A1 nanoparticles can inhibit bacterial growth at the wound site, which may promote wound healing. Moreover, the effect is more significant compared with single-drug therapy.
[0092] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.
Claims
1. A self-assembled nanoparticle, characterized in that, The self-assembled nanoparticles are formed by the self-assembly of isozygoflavin and isochlorogenic acid C through non-covalent bonding to form supramolecular structures. The particle size of the self-assembled nanoparticles is 459~825nm, and the zeta potential is -14.8mV~-35.2mV.
2. The self-assembled nanoparticles according to claim 1, characterized in that, The self-assembled nanoparticles have a particle size of 459~550nm and a zeta potential of -25mV~-35.2mV.
3. The self-assembled nanoparticles according to claim 1 or 2, characterized in that, In the self-assembled nanoparticles, the molar ratio of isochlorogenic acid C to isochlorogenic acid C is 0.1~10:1, preferably 0.5~2:
1.
4. A method for preparing self-assembled nanoparticles according to any one of claims 1-3, characterized in that, The method includes: dissolving isochlorogenic acid C and isochlorogenic acid C in an organic solvent to obtain an organic phase, adding water to the organic phase, stirring to obtain a pre-assembled mixture, and then allowing the pre-assembled mixture to stand.
5. The method according to claim 4, characterized in that, The organic solvent is selected from at least one of ethanol, methanol and dimethyl sulfoxide.
6. The method according to claim 4 or 5, characterized in that, The molar ratio of the organic solvent, the isochlorogenic acid C, and the isochlorogenic acid C is (600~700):(0.1~10):
1.
7. The method according to claim 4, characterized in that, The volume ratio of water to organic solvent is 1 to 10:
1.
8. The method according to claim 4, characterized in that, The stirring conditions include: a stirring speed of 300~700 r / min and a stirring time of 30~60 min.
9. The method according to claim 4, characterized in that, The conditions for settling include: a temperature of 1~10℃ and a time of 20~48h.
10. The use of the self-assembled nanoparticles according to any one of claims 1-3 in the preparation of antibacterial drugs; Preferably, the antibacterial drug kills methicillin-resistant bacteria.