Lemon peel-derived lipid nanoparticles and application thereof in anti-tumor and anti-inflammatory drug delivery

Abstract

The invention relates to the technical field of nano-medicines, and particularly discloses lemon peel-derived lipid nanoparticles and application thereof in anti-tumor and anti-inflammatory drug delivery. According to the invention, lemon peel is used as a raw material, a natural lipid component is obtained by adopting an ultrasonic-assisted method, and the lemon peel-derived lipid nanoparticles (LP LNPs) with uniform particle size and high encapsulation efficiency are successfully prepared by adopting a solvent injection method. The method is simple and convenient in process and low in cost, raw materials are easy to obtain, and the prepared LP LNPs have good colloidal stability and biocompatibility. A subsequent characterization experiment proves that the LP LNPs has higher cell uptake efficiency and good biological safety. And then adriamycin or naringenin is wrapped in LP LNPs, so that the stability and bioavailability of the medicine can be improved, and finally, stronger anti-tumor and anti-inflammatory effects are realized. Therefore, the polymer has an important potential application value and a wide development prospect in the field of drug delivery.

Description

TECHNICAL FIELD

[0001] The application relates to the field of nanomedicine, and particularly discloses a lemon peel-derived lipid nanoparticle and application thereof in anti-tumor and anti-inflammatory drug delivery. BACKGROUND

[0002] In recent decades, nanomedicine, as an interdisciplinary field combining nanoscience, nanotechnology and life science, has become one of the most productive research directions in the world. A large number of studies have confirmed that nanomedicine has shown significant advantages in improving the bioavailability and safety of free drugs, and can achieve precise drug delivery, thereby greatly improving the therapeutic effect. Therefore, it lays an important foundation for developing advanced drug delivery systems (DDS) with high specificity, high efficiency and individualization, and has key significance for promoting the treatment process of various diseases.

[0003] In the drug delivery system, the selection of a suitable drug carrier is a key factor determining the application effect. At present, a variety of types of carriers have been reported, such as micelles, liposomes and the like. The composition materials of these carriers are widely sourced from natural or artificially synthesized organic and inorganic materials. However, although the existing drug delivery systems have achieved certain results, they still face many challenges to be solved in practical application, including: (1) difficulty in effectively crossing the biological barrier and achieving specific targeted drug delivery; (2) difficulty in large-scale production, which is difficult to meet the actual clinical application needs; (3) problems such as unstable pharmacological properties and further verification of biological safety, which seriously restrict the popularization and application of the drug delivery system.

[0004] In recent years, lipid nanoparticles have been widely concerned and developed due to their excellent performance. This kind of spherical vesicle is composed of a single or multiple phospholipid bilayers, and the typical components are cationic lipids, auxiliary lipids, cholesterol and pegylated lipids. It has good dispersibility, can enhance the bioavailability of poorly soluble drugs, improve the stability of unstable drugs, can also carry hydrophilic polypeptides, siRNA and other nucleic acid drugs, and has strong transmembrane ability, excellent physical stability, high drug protection effect, constant blood drug concentration, controllable drug release and positioning targeting, which is a very potential drug delivery carrier.

[0005] The preparation materials of the existing lipid nanoparticles are mostly dependent on artificial synthesis or non-natural sources, which not only increases the production cost, but also may cause biological safety risks for some synthetic materials, further limiting their application in the clinical field. Lemons are widely planted fruits, and the peel thereof is rich in lipid components. At present, the development and utilization of lemon peel-derived lipids are mostly concentrated in the fields of food and daily chemicals, and there are few reports on the preparation of lipid nanoparticles from lemon peel-derived lipids and the application thereof in anti-tumor and anti-inflammatory drug delivery. SUMMARY

[0006] The application adopts an ultrasonic-assisted method to successfully extract lemon peel lipids, and adopts a solvent injection method to prepare lemon peel source lipid nanoparticles (LP LNPs) with uniform particle size and high encapsulation efficiency. The method extracts lipids and prepares LP LNPs with good biocompatibility at low cost. Subsequent characterization experiments prove that the LP LNPs have higher cell uptake efficiency and good biological safety. Subsequently, doxorubicin (Doxorubicin) or naringin (Naringin) is wrapped in the LP LNPs, which can improve the drug stability and bioavailability, and ultimately achieve stronger anti-tumor and anti-inflammatory effects. Therefore, it has important potential application value and broad development prospects in the field of drug delivery.

[0007] The preparation method of the lemon peel source lipid nanoparticles is as follows: (1) Fresh lemons are purchased from a fruit store and their peels are peeled off, frozen at -80℃ for 24h, and the lemon peel tissue is pulverized into powder. Then, anhydrous ethanol is added to the powder to fully immerse the powder, and ice bath ultrasonic is performed for 0.5h. The obtained lemon peel crude extract is centrifuged, and the supernatant after centrifugation is removed and concentrated by a rotary evaporator under reduced pressure to obtain a crude product.

[0008] The mass-volume ratio of the frozen and pulverized lemon peel to anhydrous ethanol is 1:2.5; the centrifugation conditions are 4℃, 4000rpm, and 10min.

[0009] (2) Dichloromethane is added to dissolve the lipid crude product, and after nitrogen blowing to remove dichloromethane, vacuum drying treatment is performed to obtain lemon peel lipids, which are stored at -80℃ for standby.

[0010] The mass-volume ratio of the lemon peel lipid crude product to dichloromethane is 1:3, and the vacuum drying treatment time is 12h.

[0011] The composition of the obtained lemon peel lipids includes fatty acids (48.1%), phosphatidylcholine (13.7%), triglycerides (8.8%), sphingosine (2.9%), diglycerides (2.7%), phosphatidylethanolamine (2.2%), ceramide (2.1%), phosphatidylserine (2.1%), (O-acyl)-omega-hydroxy fatty acid (1.9%), lysophosphatidylmethanol (1.5%), and other lipid components (14%).

[0012] (3) The lemon peel lipids obtained in step (1) are dissolved with anhydrous ethanol, and the lipid ethanol solution is injected into deionized water under stirring of a magnetic stirrer to obtain lemon peel source lipid nanoparticles (LP LNPs).

[0013] The concentration of the lipid ethanol solution is 10 mg / mL, the volume ratio of the lipid ethanol solution to deionized water is 1:3-1:11, the stirring speed is 1500 rpm, and the stirring time is 5 min.

[0014] Preferably, the volume ratio of the lipid ethanol solution to deionized water is 1:9.

[0015] The lemon peel-derived lipid nanoparticles prepared by the above method are used for delivering an anti-tumor or anti-inflammatory drug, and the anti-tumor or anti-inflammatory drug is doxorubicin or naringin.

[0016] The specific method for delivering an anti-tumor or anti-inflammatory drug is that the obtained lemon peel lipid is dissolved in anhydrous ethanol; another doxorubicin (DOX) is dissolved in DMSO and uniformly mixed with the lipid ethanol solution, and the mixed solution is injected into deionized water under stirring of a magnetic stirrer to obtain lemon peel-derived lipid nanoparticles loaded with doxorubicin (DOX-LP LNPs).

[0017] The concentration of the lipid ethanol solution is 20 mg / mL, the concentration of the doxorubicin DMSO solution is 1-20 mg / mL, the added amount is 100 μL, the volume ratio of the mixed solution to deionized water is 1:9, the stirring speed is 1500 rpm, and the stirring time is 5 min.

[0018] Preferably, the concentration of the doxorubicin DMSO solution is 10 mg / mL.

[0019] Or the obtained lemon peel lipid and naringin are jointly added into anhydrous ethanol, and the lipid-naringin ethanol solution is injected into deionized water under stirring of a magnetic stirrer to obtain lemon peel-derived lipid nanoparticles loaded with naringin (NG-LP LNPs).

[0020] In the lipid-naringin ethanol solution, the concentration of the lipid is 15 mg / mL, the concentration of the naringin is 0.1-2 mg / mL, the volume ratio of the ethanol solution to deionized water is 1:9, the stirring speed is 1500 rpm, and the stirring time is 5 min.

[0021] Preferably, the concentration of the naringin is 1 mg / mL.

[0022] The present application has the following beneficial effects: (1) The extraction process of the present application is simple, low in cost, and the raw material is easy to obtain, and the prepared LP LNPs have good colloidal stability and biocompatibility.

[0023] (2) The subsequent characterization experiments prove that, compared with other citrus-derived lipid nanoparticles, the LP LNPs have higher cell uptake efficiency and good biological safety.

[0024] (3) Doxorubicin (Doxorubicin) and naringin (Naringin) are wrapped in LP LNPs, which can improve the stability and bioavailability of drugs, and ultimately achieve stronger anti-tumor and anti-inflammatory effects. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 . Pie chart of the omics analysis of the lemon peel lipid extracted in Example 1.

[0026] Figure 2 . Tyndall effect diagram of the LP LNP solution prepared in Example 2.

[0027] Figure 3 . Particle size distribution diagram of the LP LNPs prepared in Example 2.

[0028] Figure 4 . Zeta potential diagram of the LP LNPs prepared in Example 2.

[0029] Figure 5 . TEM image of the LP LNPs prepared in Example 2.

[0030] Figure 6 . Particle size distribution diagram of the DOX-LP LNPs prepared in Example 4.

[0031] Figure 7 . Zeta potential diagram of the DOX-LP LNPs prepared in Example 4.

[0032] Figure 8 . Ultraviolet absorption spectrum diagram of the DOX-LP LNPs prepared in Example 4.

[0033] Figure 9 . Stability of the DOX-LP LNPs prepared in Example 4 in H2O.

[0034] Figure 10 . Stability of the DOX-LP LNPs prepared in Example 4 in PBS.

[0035] Figure 11 . Stability of the DOX-LP LNPs prepared in Example 4 in FBS.

[0036] Figure 12 . Particle size distribution diagram of the NG-LP LNPs prepared in Example 6.

[0037] Figure 13 . Zeta potential diagram of the NG-LP LNPs prepared in Example 6.

[0038] Figure 14. UV absorption spectra of NG-LP LNPs prepared in Example 6.

[0039] Figure 15 . Stability of NG-LP LNPs prepared in Example 6 in H2O.

[0040] Figure 16 . Stability of NG-LP LNPs prepared in Example 6 in PBS.

[0041] Figure 17 . Stability of NG-LP LNPs prepared in Example 6 in FBS.

[0042] Figure 18 . In vitro cytotoxicity results plot of different concentrations of LP LNPs prepared in Example 2.

[0043] Figure 19 . Hemolysis experiment results plot of different concentrations of LP LNPs prepared in Example 2.

[0044] Figure 20 . Confocal microscope fluorescence imaging of CR LNPs in 4T1 cells.

[0045] Figure 21 . Confocal microscope fluorescence imaging of CR LNPs in MCF-7 cells.

[0046] Figure 22 . Fluorescence intensity quantitative analysis of CR LNPs in 4T1 cells.

[0047] Figure 23 . Fluorescence intensity quantitative analysis of CR LNPs in MCF-7 cells.

[0048] Figure 24 . Fluorescence images of live / dead staining of 4T1 cells treated with DOX-LP LNPs prepared in Example 4.

[0049] Figure 25 . Fluorescence images of live / dead staining of MCF-7 cells treated with DOX-LP LNPs prepared in Example 4.

[0050] Figure 26 . Flow cytometry analysis of PI staining of 4T1 cells after treatment with DOX-LP LNPs prepared in Example 4.

[0051] Figure 27 . Flow cytometry analysis of PI staining of MCF-7 cells after treatment with DOX-LP LNPs prepared in Example 4.

[0052] Figure 28The relative cell viability of 4T1 cells treated with DOX-LP LNPs prepared in Example 4.

[0053] Figure 29 The relative cell viability of MCF-7 cells treated with DOX-LP LNPs prepared in Example 4.

[0054] Figure 30 Example 6: NG-LP LNPs prepared with ABTS +UV absorption spectrum after free radical incubation.

[0055] Figure 31 ABTS of NG-LP LNPs prepared in Example 6 +Quantitative analysis of clearance capacity.

[0056] Figure 32 Example 6 prepared NG-LP LNPs and DPPH UV absorption spectrum after free radical incubation.

[0057] Figure 33 DPPH of NG-LP LNPs prepared in Example 6 Quantitative analysis of clearance capacity.

[0058] Figure 34 UV absorption spectrum of NG-LP LNPs prepared in Example 6 after incubation with hydroxyl radicals.

[0059] Figure 35 Quantitative analysis of the hydroxyl radical scavenging ability of the NG-LP LNPs prepared in Example 6.

[0060] Figure 36 Confocal fluorescence imaging of DCFH-DA in Raw264.7 cells after treatment with NG-LP LNPs prepared in Example 6. Detailed Implementation

[0061] The present invention will be further described in detail below with reference to specific embodiments, but this should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the invention are within the scope of the present invention. Experimental methods and reagents not specified in the embodiments are performed according to conventional conditions in the art.

[0062] Example 1: Extraction of lipids from lemon peel

[0063] (1) Purchase fresh lemons from a fruit store and peel off their peels. Freeze them at -80℃ for 24 hours. Crush the lemon peel tissue into powder. Add anhydrous ethanol to the lemon peel powder at a mass-volume ratio of 1:2.5 (g / mL). Stir until the powder is completely soaked. Then, sonicate under ice bath conditions for 0.5 hours.

[0064] (2) The crude lemon peel extract obtained in step (1) was centrifuged. The centrifugation conditions were set as follows: temperature 4℃, speed 4000 rpm, centrifugation time 10 min. After centrifugation, the supernatant was removed and concentrated under reduced pressure using a rotary evaporator to obtain crude lemon peel lipid product.

[0065] (3) Take the crude lemon peel lipid obtained in step (2) and add it to dichloromethane at a mass-volume ratio of 1:3 (g / mL) to dissolve it. After complete dissolution, remove the dichloromethane by nitrogen purging. Then, vacuum dry the residue for 12 h to obtain lemon peel lipid. Store it at -80℃ for later use.

[0066] To investigate the lipid composition of lemon peel, a systematic analysis was performed using lipidomics mass spectrometry. (See attached image.) Figure 1 The pie chart shows the omics analysis of lipids in lemon peel. The results indicate that the top 10 lipid components account for approximately 86% of the total lipid content. The main lipid categories and their relative contents are as follows: fatty acids (FA, 48.1%), phosphatidylcholine (PC, 13.7%), triglycerides (TG, 8.8%), sphingosine (So, 2.9%), diglycerides (DG, 2.7%), phosphatidylethanolamine (PE, 2.2%), ceramides (Cer, 2.1%), phosphatidylserine (Ps, 2.1%), (O-acyl)-ω-hydroxy fatty acids (OAHFA, 1.9%), lysophosphatidylethanolamine (LPMe, 1.5%), and other lipid components (Others, 14%).

[0067] Example 2: Preparation of lemon peel-derived lipid nanoparticles (LP LNPs): The lemon peel lipids obtained in Example 1 were dissolved in anhydrous ethanol to prepare a stock solution with a concentration of 10 mg / mL. Using a magnetic stirrer at 1500 rpm, the lipid ethanol solution was rapidly injected into deionized water at a volume ratio of 1:9, and the mixture was stirred continuously for 5 min to obtain lemon peel-derived lipid nanoparticles (LP LNPs).

[0068] As attached Figure 2 As shown, under the preparation conditions of a concentration of 1 mg / mL, the obtained LP LNPs exhibited a clear Tyndall effect, indicating that they possess highly dispersed colloidal properties. (See attached image)Figure 3 The dynamic light scattering (DLS) results of the LPLNPs show that the particle size of the LPLNPs is 61.59 nm; the polydispersity index (PDI) is 0.185, which indicates that the LPLNPs have the optimal dispersion performance in the aqueous solution. The Figure 4 The zeta potential measurement results of the LPLNPs show that the potential value of the LPLNPs is -34.3 mV. The negative potential characteristic indicates that the LPLNPs are negatively charged on the surface, and the absolute value of the potential is high, which indicates that the LPLNPs have good dispersion stability in the solution system and can effectively inhibit the aggregation of particles. The Figure 5 The representative TEM micrograph of the LPLNPs shows that the micro-morphology of the LPLNPs is close to a regular sphere, and the particle size distribution range is basically consistent with the results obtained by the DLS test.

[0069] Example 3

[0070] To explore the effect of the volume ratio of the lipid ethanol solution to deionized water on the properties of the lipid nanoparticles (LPLNPs), the following method was used to perform the experiment: the lipid extracted in Example 1 was dissolved in anhydrous ethanol to prepare a lipid ethanol solution; 1 mL of the lipid ethanol solution was rapidly injected into deionized water of different volumes, and the mixing system was stirred at a speed of 1500 rpm for 5 min, and finally different concentrations of lipid nanoparticles (LPLNPs) were prepared. The particle size and dispersion coefficient (PDI) of each group of lipid nanoparticles were detected, and the results of the effect of the volume ratio of the lipid ethanol solution to deionized water on the particle size of the lipid nanoparticles are shown in Table 1.

[0071] Table 1 Effect of the volume ratio of the lipid ethanol solution to deionized water on the particle size of the lipid nanoparticles (LPLNPs)

[0072] As shown in Table 1, within the experimentally defined volume ratio range, the particle size of LP LNPs exhibited a continuous and significant decreasing trend as the volume ratio of lipid ethanol solution to deionized water gradually decreased. Increasing the aqueous phase ratio allowed the injected lipid ethanol solution to be sufficiently diluted by the aqueous phase. During the formation of nanoparticles in the aqueous phase, the nucleation sites of lipid molecules were more dispersed, effectively preventing particle aggregation and ultimately leading to a significant reduction in the particle size of the formed lipid nanoparticles. Furthermore, the dispersion index (PDI) showed a significant decreasing trend; however, when the volume ratio continued to decrease to 1:11, the PDI slightly rebounded to 0.186. This indicates that increasing the aqueous phase ratio can improve the dispersion uniformity of nanoparticles, but excessive dilution may result in some lipid molecules failing to assemble sufficiently, leading to a slight widening of the particle size distribution. Therefore, the LP LNPs prepared at a volume ratio of 1:9 exhibited the best overall performance and met the performance requirements for subsequent applications of lipid nanoparticles.

[0073] Example 4: Preparation of doxorubicin-loaded lemon peel-derived lipid nanoparticles (DOX-LP LNPs): 20 mg of the lemon peel lipid obtained in Example 1 was dissolved in 1 mL of anhydrous ethanol, followed by the addition of 1 mg of doxorubicin (DOX) dissolved in 100 μL of DMSO. The mixture was rapidly injected into deionized water at a volume ratio of 1:9, and stirred continuously at 1500 rpm for 5 minutes to ensure uniform dispersion, ultimately yielding doxorubicin-loaded lemon peel-derived lipid nanoparticles (DOX-LP LNPs). Characterization experiments were performed after dilution with deionized water to a lipid concentration of 1 mg / mL, as shown in the attached figure. Figure 6 As shown, the hydrated particle size of DOX-LP LNPs is 74.63 nm, and the PDI is 0.164, which is slightly larger than that of unloaded LP LNPs. (See attached image) Figure 7 The Zeta potential measurements showed that the potential of DOX-LP LNPs was -19.3 mV, and there was no significant difference in the Zeta potential of LP LNPs before and after DOX loading, indicating that the drug loading process did not significantly affect the surface charge properties of LP LNPs. (See attached image) Figure 8 The UV-Vis spectrophotometric analysis results showed that DOX-LP LNPs exhibited a characteristic UV absorption peak at 489 nm, which perfectly matched the characteristic absorption peak of free DOX, further confirming that DOX had been successfully loaded into LP LNPs. To evaluate the colloidal stability of DOX-LP LNPs, this study monitored the changes in hydrated particle size and PDI during incubation in three different media (H2O, PBS, and FBS) for 5 days. (See attached image)Figures 9-11 As shown, no obvious particle size increase or PDI rise was observed for DOX-LP LNPs in the three media throughout the incubation period, indicating that DOX-LP LNPs exhibited excellent colloidal stability in aqueous media and physiologically relevant media.

[0074] Example 5

[0075] To investigate the effect of the loading concentration of doxorubicin on the properties of the lipid nanoparticles (DOX-LP LNPs), the following method was used for the experiment: the lipids extracted in Example 1 were dissolved in anhydrous ethanol to form a 2 mg / mL lipid ethanol solution; then different weights of DOX dissolved in 100 μL of DMSO were added to the solution. The mixed solution was quickly injected into deionized water at a volume ratio of 1:9, and the system was continuously stirred at a speed of 1500 rpm for 5 min to ensure uniform dispersion, finally obtaining citrus peel-derived lipid nanoparticles (DOX-LP LNPs) with different DOX loading concentrations. The particle size and polydispersity index (PDI) of each group of lipid nanoparticles were detected, and the results of the effect of the DMSO solution concentration of DOX on the particle size of the nanoparticles are shown in Table 2.

[0076] Table 2 Effect of DMSO solution concentration of DOX on particle size of lipid nanoparticles (DOX-LP LNPs)

[0077] As can be seen from the data in Table 2, within the DOX concentration range set in the experiment, as the DMSO solution concentration of DOX increased, the particle size of DOX-LP LNPs showed a continuous increasing trend. Since DOX molecules are embedded in the structure of the lipid nanoparticles, as the DOX concentration increases, the number of drug molecules loaded per nanoparticle increases, resulting in an expansion of the core volume of the nanoparticles, and the particle size increases accordingly. When the DOX concentration increased from 1 mg / mL to 10 mg / mL, the PDI decreased from 0.237 to 0.173, indicating that the uniformity of dispersion improved; when the concentration further increased to 20 mg / mL, the PDI significantly rose to 0.314, the uniformity decreased, and the solution became turbid after standing. This is because high-concentration DOX destroys the ordered assembly structure of the lipid molecules, causing nanoparticle aggregation and leading to a decrease in system stability. Therefore, when the DMSO solution concentration of DOX is 10 mg / mL, the DOX-LP LNPs prepared have the best overall performance, which can meet the core performance requirements of drug delivery systems for drug-loaded nanoparticles.

[0078] Example 6: Preparation of naringenin-loaded citrus peel-derived lipid nanoparticles (NG-LP LNPs) Take the lemon peel lipid 15 mg obtained in the preceding Example 1 and naringin 1 mg, and add them together in 1 mL of anhydrous ethanol, and stir to completely dissolve the two, to obtain a lipid-naringin ethanol solution; using a magnetic stirrer, inject the ethanol solution into deionized water at a volume ratio of 1:9 at a speed of 1500 rpm, and continue stirring for 5 min until the system is uniformly dispersed, to prepare naringin-loaded lemon peel-derived lipid nanoparticles (NG-LP LNPs). Since naringin has strong hydrophobicity, as shown in FIG. 1, the hydrated particle size of the NG-LP LNPs is about 58.31 nm (PDI = 0.202), which is slightly lower than that of the LP LNPs. The zeta potential measurement result shown in FIG. 2 is -38.3 mV, which is almost the same as that of the LP LNPs. As shown in the UV-visible spectrum in FIG. 3, the NG-LP LNP has an overlapping absorption signal in the characteristic absorption region of naringin, which indicates that naringin has been successfully loaded into the lipid nanoparticles. In addition, the stability evaluation result shown in FIG. 4 shows that in the three different media (H2O, PBS and FBS), the hydrated particle size and PDI of the NG-LP LNPs remain basically stable within 5 days, without significant changes, indicating that it has good colloidal stability. Figure 12 Figure 13 Figure 14 Figures 15-17

[0079] Example 7

[0080] To explore the effect of the loading concentration of naringin on the properties of the lipid nanoparticles (NG-LP LNPs), the following method was used for the experiment: the lipid extracted in Example 1 was dissolved with different weights of naringin in anhydrous ethanol to prepare a lipid-naringin mixed ethanol solution. The lipid concentration in the solution was 15 mg / mL, and the naringin concentration was 0.1-2 mg / mL. The mixed solution was quickly injected into deionized water at a volume ratio of 1:9, and stirred at a speed of 1500 rpm for 5 min to ensure uniform dispersion of the system, to finally prepare NG-LP LNPs with different loading concentrations of naringin. The particle size, dispersion coefficient (PDI) and system stability of each group of NG-LP LNPs were detected, and the effect of the naringin concentration on the particle size is shown in Table 3.

[0081] Table 3 Effect of naringin concentration on the particle size of the lipid nanoparticles (NG-LP LNPs)

[0082] ​​​​As shown in Table 3, within the experimentally defined naringenin concentration range, the particle size of NG-LPLNPs exhibited a continuous and significant increasing trend with increasing naringenin concentration. This is because naringenin molecules embed into the hydrophobic core of lipid nanoparticles or adsorb onto the particle surface. With increasing naringenin concentration, the number of drug molecules loaded per unit nanoparticle increases, leading to an expansion of the nanoparticle core volume or a thickening of the surface modification layer, ultimately resulting in a continuous increase in particle size. Furthermore, the dispersion index (PDI) results indicate that at a concentration of 1 mg / mL, the assembly coordination between naringenin and lipid molecules is optimal; however, at 2 mg / mL, excess naringenin exceeds the effective loading capacity of the lipids, resulting in a significantly wider particle size distribution. Excess unloaded free naringenin molecules are prone to sedimentation or precipitation in the solution, leading to decreased system stability. Therefore, when the naringenin concentration is 1 mg / mL, the prepared NG-LPLNPs exhibit moderate particle size, optimal dispersion uniformity, and good system stability, meeting the core performance requirements for subsequent applications of drug-loaded nanoparticles.

[0083] Example 8: Biosafety of LP-LNPs prepared in Example 2: 3T3 cells were planted at 1×10⁻⁶ cells per well. The culture medium was seeded at a density of [insert density here] into 96-well plates and incubated at 37°C for 12 hours. Then, the medium was replaced with serum-free fresh medium containing different concentrations of LP LNPs, and incubated for another 24 hours. Afterward, 10 μL of CCK-8 solution was added to each well, and after incubation for 1 hour, the absorbance was measured at 450 nm using a microplate reader. (See attached image.) Figure 18 As shown, there was no significant difference in cell viability compared to the blank control group; even under high concentration treatment of 100 μg / mL, the cell viability remained above 80%, which confirms that LP LNPs have good in vitro biocompatibility.

[0084] The hemolysis assay was used to evaluate the biocompatibility of LP LNPs. Blood samples were centrifuged at 1000 rpm for 10 minutes, and the supernatant was carefully discarded. The precipitated red blood cells were washed with PBS and resuspended in PBS. The red blood cells were incubated with different concentrations of LP LNPs at 37°C for 1 hour. Red blood cells incubated with deionized water served as a positive control, and those incubated with PBS served as a negative control. The mixture was then centrifuged again at 1000 rpm for 10 minutes. The mixture was photographed, and the absorbance of the supernatant was measured at 540 nm using a microplate reader to calculate the hemolysis rate. (See attached figure.) Figure 19As shown, the hemolytic effect of LP LNPs on red blood cells was negligible, with a hemolysis rate of only 1.3% at the maximum test concentration (350 pg / mL), indicating excellent in vitro blood safety of LP LNPs.

[0085] Example 9: Effective cellular uptake of LP-LNPs prepared in Example 2: Three citrus-derived lipid nanoparticles (orange peel-derived lipid nanoparticles and grapefruit peel-derived lipid nanoparticles were prepared in the same way as in Example 1 and Example 2, except that the lemon peel was replaced by orange peel or grapefruit peel) (CR LNPs) were labeled with coumarin 6 (C6) as a fluorescent dye. C6-labeled CR LNPs (C6-CR LNPs, 50 pg / mL) were co-incubated with different cell lines (4T1 cells and MCF-7 cells) for 4 hours. After incubation, the cells were washed with PBS for 3 times and fixed with 4% paraformaldehyde. The nuclei were stained with DAPI (5 pg / mL), and then the cellular uptake of C6-CR LNPs was observed by confocal laser scanning microscopy, in which the green fluorescent signal corresponds to CR LNPs, and the blue fluorescent signal (DAPI staining) is used to mark the cell nucleus, and the mean fluorescence intensity (Mean) is quantitatively analyzed using ImageJ software. As shown in FIG. 6A and FIG. 6B, C6-labeled LP LNPs were effectively internalized by both 4T1 and MCF-7 cells, and the strongest green fluorescent signal was detected in the above-mentioned cells. Figures 20-21 Further quantitative analysis results shown in FIG. 6C and FIG. 6D show that LP LNPs exhibit significantly higher cellular uptake efficiency, while the cellular uptake efficiency of orange peel-derived lipid nanoparticles (OP LNPs) and grapefruit peel-derived lipid nanoparticles (GP LNPs) is relatively low. Figures 22-23

[0086] Example 10: In vitro anti-tumor effect of DOX-LP LNPs prepared in Example 4:

[0087] The killing effect of DOX-LP LNPs on tumor cells was evaluated by live / dead cell double staining (Calcein-AM / PI). After blank LP LNPs, free doxorubicin (DOX) and DOX-LP LNPs were co-incubated with 4T1 cells and MCF-7 cells for 12 h, the cell staining was observed by laser confocal microscopy: in which the red fluorescence of PI (propidium iodide) represents dead cells, and the green fluorescence of Calcein-AM represents live cells. As shown in FIG. 7A and FIG. 7B, DOX-LP LNPs showed the strongest killing effect on 4T1 cells and MCF-7 cells, and the number of dead cells was significantly higher than that of blank LP LNPs and free DOX. Figures 24-25 ​The results showed that the red fluorescence signal intensity was significantly strongest in the DOX-LP LNPs-treated group, while the green fluorescence signal intensity was significantly weakest. This staining result indicates that the tumor cell mortality rate was highest in the DOX-LP LNPs-treated group. To further quantify cell viability, flow cytometry was used to detect the cell survival status in each group. (See attached image) Figures 26-27 As shown, under the same drug concentration conditions, DOX-LPLNPs exhibited more significant cytotoxic effects on both 4T1 cells and MCF-7 cells than the free DOX group, indicating that drug delivery via LP LNPs can enhance the killing effect on tumor cells.

[0088] Cell death rate was quantitatively determined using the CCK-8 assay to further verify the in vitro antitumor therapeutic effect of DOX-LP LNPs. 4T1 cells and MCF-7 cells were cultured at 1×10⁻⁶ cells per well. Cells were seeded at a density of [insert density here] into 96-well plates and cultured at 37°C for 12 hours. The medium was then replaced with fresh serum-free medium containing different concentrations of LP LNPs, DOX, and DOX-LP LNPs. After incubation for 24 hours, 10 μL of CCK-8 solution was added to each well, and incubation continued for 1 hour. The absorbance of each well was measured at 450 nm using a microplate reader. (See attached image.) Figures 28-29 As shown, with increasing drug concentration, the survival rate of tumor cells in both the free DOX treatment group and the DOX-LP LNPs treatment group gradually decreased, exhibiting a clear concentration-dependent characteristic. The half-maximal inhibitory concentration (IC50) was calculated. 50 It can be seen that DOX-LP LNPs have an IC50 effect on 4T1 cells and MCF-7 cells. 50 The concentrations were 0.93 μg / mL and 0.30 μg / mL, respectively; and at the same drug concentration, the cell viability of the DOX-LPLNPs treatment group was significantly lower than that of the free DOX treatment group. These results indicate that LP LNPs, as a DOX delivery carrier, can effectively enhance the killing efficiency of DOX against tumor cells and significantly strengthen the in vitro antitumor activity of DOX.

[0089] Example 11: In vitro antioxidant and ROS scavenging capabilities of NG-LP LNPs prepared in Example 6

[0090] Given that naringenin has been proven to possess definite antioxidant activity, this study further evaluated the antioxidant capacity of NG-LP LNPs through in vitro experiments. Currently, probes such as ABTS, DPPH, and TMB are widely used to detect the reactive oxygen species (ROS) scavenging ability of nanomaterials. The principle of the ABTS assay is as follows: ABTS generates a blue-green cationic free radical (ABTS) under the oxidation of potassium persulfate (K2S2O8). This study used this method to evaluate the antioxidant effect of LP LNPs. (See attached image.) Figures 30-31 Experimental results showed that as the concentration of NG-LP LNPs increased, the color of the reaction solution gradually faded from blue-green to colorless; correspondingly, the intensity of the characteristic absorption peak of ABTS at 734 nm wavelength decreased significantly. When the concentration of NG-LP LNPs was as low as 40 μg / mL, more than 90% of the ABTS in the system... + Free radicals are neutralized, and the above results indicate that LP LNPs possess strong antioxidant capabilities. To further verify the antioxidant performance of NG-LP LNPs, this study evaluated their free radical scavenging activity in the same solvent system (e.g., anhydrous ethanol) using the DPPH assay. (See attached...) Figures 32-33 As shown, DPPH radicals exhibit a distinct absorption peak at 517 nm. However, after interaction with NG-LP LNPs, the absorbance of this peak significantly decreases, and the degree of absorbance reduction is positively correlated with the concentration of NG-LP LNPs, indicating that the scavenging effect of NG-LP LNPs on DPPH radicals is concentration-dependent. Furthermore, this study used the TMB assay to evaluate the effect of NG-LP LNPs on hydroxyl radicals (…). Scavenging activity of OH). (See attached image) Figures 34-35 The results showed that with increasing NG-LP LNP concentration, its effect on... The ability to scavenge OH radicals shows a gradually increasing trend; it is noteworthy that even under the condition that the concentration of NG-LP LNPs is only 20 μg / mL, the nanomaterial can still scavenge more than 60% of the hydroxyl radicals in the system.

[0091] Reactive oxygen species (ROS) was detected by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The specific steps are as follows: after RAW264.7 cells were cultured overnight, they were pretreated with 1000 μM hydrogen peroxide (H2O2) for 2 hours to induce oxidative stress. After the end of pretreatment, they were incubated with LP LNPs, naringin and NG-LP LNPs for 4 hours. Subsequently, the cells were incubated with 10 μM DCFH-DA at 37 °C in the dark for 30 minutes. After the cells were washed with phosphate buffer solution (PBS), the intracellular ROS level was evaluated by confocal laser scanning microscopy and flow cytometry analysis. Figure 36 The confocal microscope imaging results of the present application show that the green fluorescence signal intensity of the cell group treated with NG-LP LNPs is significantly weaker than that of other control groups, indicating that NG-LP LNPs can effectively scavenge intracellular ROS, thereby reducing the oxidative stress damage of cells.

Claims

1. A method of preparing a citrus peel-derived lipid nanoparticle, characterized by, The preparation method of the lemon peel-derived lipid nanoparticles is as follows: (1) freeze the lemon peel at -80℃ for 24 h, then crush it into powder; add anhydrous ethanol to the frozen and crushed lemon peel, and perform ice-bath ultrasonication; centrifuge the obtained crude extract of lemon peel, and remove the supernatant by rotary evaporation under reduced pressure to obtain a crude lipid product; (2) dissolve the crude lipid product in dichloromethane, remove the dichloromethane by nitrogen blowing, and then vacuum dry to obtain lemon peel lipid, which is stored at -80℃ for standby; (3) dissolve the lemon peel lipid obtained in step (2) in anhydrous ethanol, and inject the lipid ethanol solution into deionized water under magnetic stirring to obtain lemon peel-derived lipid nanoparticles LP LNPs.

2. The method for preparing lemon peel-derived lipid nanoparticles as described in claim 1, characterized in that, In step (1), the mass-volume ratio of the frozen and crushed lemon peel to anhydrous ethanol is 1:2.5, and the ice-bath ultrasonication time is 0.5 h.

3. The method for preparing lemon peel-derived lipid nanoparticles as described in claim 1, characterized in that, In step (1), the centrifugation conditions of the crude extract of lemon peel are 4℃, 4000 rpm, and 10 min.

4. The method for preparing lemon peel-derived lipid nanoparticles as described in claim 1, characterized in that, In step (2), the mass-volume ratio of the crude lipid product of lemon peel to dichloromethane is 1:3, and the vacuum drying time is 12 h.

5. The method for preparing lemon peel-derived lipid nanoparticles as described in claim 1, characterized in that, In step (2), the obtained lemon peel lipid has a composition by mass percentage of: fatty acids 48.1%, phosphatidylcholine 13.7%, triglyceride 8.8%, sphingosine 2.9%, diglyceride 2.7%, phosphatidylethanolamine 2.2%, ceramide 2.1%, phosphatidylserine 2.1%, (O-acyl)-ω-hydroxy fatty acid 1.9%, lysophosphatidylmethanol 1.5%, and other lipid components 14%.

6. The method for preparing lemon peel-derived lipid nanoparticles as described in claim 1, characterized in that, In step (3), the concentration of the lipid ethanol solution is 10 mg / mL, the volume ratio of the lipid ethanol solution to deionized water is 1:3-1:11, the stirring speed is 1500 rpm, and the stirring time is 5 min.

7. Lemon peel-derived lipid nanoparticles prepared by the method of any one of claims 1-7.

8. Use of a lipid nanoparticle of lemon peel origin prepared according to the method of any one of claims 1 to 7, characterized in that, The lemon peel-derived lipid nanoparticles are used for delivering an anti-tumor or anti-inflammatory drug.

9. The use of lemon peel-derived lipid nanoparticles according to claim 9, characterized in that, The anti-tumor or anti-inflammatory drug is doxorubicin or naringenin.

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