Pogostemon cablin alcohol lipid cubic liquid crystal nanoparticles as well as preparation method and application thereof
By designing core-shell structures of patchouli alcohol lipid cubic liquid crystal nanoparticles, the problems of intestinal inflammation and damage caused by chemotherapy drugs were solved, achieving targeted delivery and sustained release of drugs, and significantly improving the treatment effect of chemotherapy-induced intestinal injury.
Patent Information
- Application Number
- CN202411634692.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-15
- Publication Date
- 2026-05-15
AI Technical Summary
Chemotherapy drugs such as irinotecan can cause intestinal inflammation and diarrhea, especially the delivery of the drug to the site of inflammation faces challenges such as poor absorption, low bioavailability, and the influence of biological environmental factors.
Patchouli lipid cubic liquid crystal nanoparticles are designed with a core-shell structure, consisting of a shell composed of monooleate glycerol, polyethylene glycol 400, poloxamer 407, and D(+)-glucose, and loaded with patchouli alcohol. The particle size is 220nm~250nm and the zeta potential is -18mV~-19mV. They are used to target chemotherapy-induced intestinal injury sites.
It significantly reduced the rate of weight loss in a chemotherapy-induced intestinal injury model mouse, inhibited small intestinal shortening, and reduced the expression of inflammatory cytokines and myosin light chain kinase, demonstrating good safety and sustained-release effect.
Smart Images
Figure CN122031394A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of biomedical technology, specifically to a patchouli alcohol lipid cubic liquid crystal nanoparticle, its preparation method, and its application. Background Technology
[0002] Chemotherapy is an important treatment for cancer, especially for patients with advanced cancer. It exerts cytotoxic effects on cancer cells through multiple mechanisms, including direct killing and immunostimulation. However, the side effects of chemotherapy, such as intestinal damage and severe diarrhea, limit its widespread clinical application. Irinotecan (CPT-11) is a first-line drug for the treatment of colorectal cancer, but approximately 40% of patients experience severe diarrhea during treatment, which not only threatens the patient's life but may also affect the efficacy of chemotherapy.
[0003] Patchouli alcohol, as a component of traditional Chinese medicine, possesses various medicinal values, including anti-inflammatory effects. It can effectively inhibit inflammatory factors and shows potential application prospects in reducing intestinal inflammation and alleviating intestinal damage. However, the delivery of the drug to the site of inflammation faces challenges such as poor absorption, low bioavailability, and the influence of in vivo environmental factors.
[0004] Lipid-cubic liquid crystal nanoparticles, as an emerging nanosystem, offer a potential solution to these problems. Composed of bio-derived amphiphilic molecules, they possess biocompatibility and biodegradability. This nanodelivery system can enhance drug solubility, prolong drug circulation time in vivo, and slow drug release rates. Furthermore, the small size of the nanocarriers allows for accumulation on inflamed and damaged tissues through enhanced permeability and retention, thereby enabling more effective targeting of diseased tissues. Summary of the Invention
[0005] Based on this, the researchers of this application selected patchouli alcohol, an effective component of traditional Chinese medicine, as the active ingredient and developed a patchouli alcohol lipid cubic liquid crystal nanoparticle for treating intestinal inflammation and damage caused by chemotherapy drugs. It has good stability and better efficacy than patchouli alcohol alone, while also showing good safety.
[0006] The technical solution of this application includes the following:
[0007] A patchouli alcohol lipid cubic liquid crystal nanoparticle, the components of which include glyceryl monooleate, polyethylene glycol 400, poloxamer 407, D(+)-glucose and patchouli alcohol;
[0008] The patchouli alcohol lipid cubic liquid crystal nanoparticles have a core-shell structure, wherein the shell is a framework structure composed of the glyceryl monooleate, polyethylene glycol 400, poloxamer 407, and D(+)-glucose.
[0009] The core is the patchouli alcohol.
[0010] In one embodiment, the weight ratio of the glyceryl monooleate, the polyethylene glycol 400, and the poloxamer 407 is (8-12):(4-6):1; and / or
[0011] The patchouli alcohol constitutes 2% to 3% by weight in the lipid cubic liquid crystal nanoparticles; and / or
[0012] The weight ratio of poloxamer 407 to D(+)-glucose is 1:(10~15).
[0013] In one embodiment, the lipid cubic liquid crystal nanoparticles have at least one of the following characteristics:
[0014] (1) Particle size is 220nm~250nm;
[0015] (2) The polydispersity index is 0.1~0.3; and
[0016] (3) The Zeta potential is -18mV to -19mV.
[0017] A method for preparing patchouli alcohol lipid cubic liquid crystal nanoparticles as described above includes the following steps:
[0018] The patchouli alcohol was dissolved in polyethylene glycol 400 to prepare a first solution;
[0019] The monooleic glycerol ester is heated to melt it, and the melted monooleic glycerol ester is kept at 40℃~50℃ while the first solution is added and mixed to obtain the first mixture.
[0020] The poloxamer 407 was dissolved in a D(+)-glucose solution to prepare a second solution;
[0021] The first mixture is added to the second solution at 750 rpm to 1000 rpm to prepare the second mixture.
[0022] The second mixture was homogenized to obtain the patchouli alcohol lipid cubic liquid crystal nanoparticles.
[0023] In one embodiment, the weight ratio of the glyceryl monooleate, the polyethylene glycol 400, and the poloxamer 407 is (8~12):(4~6):1; and / or;
[0024] The patchouli alcohol constitutes 2% to 3% by weight in the lipid cubic liquid crystal nanoparticles; and / or
[0025] The weight ratio of poloxamer 407 to D(+)-glucose is 1:(10~15).
[0026] In one embodiment, the preparation method includes at least one of the following features:
[0027] (1) The weight ratio of patchouli alcohol to polyethylene glycol 400 is 1:(8~12);
[0028] (2) The weight ratio of the monooleic glyceride to the first solution is (1.8~2.2):1;
[0029] (3) The weight-to-volume ratio of the first mixture to the second solution is (0.55~0.65) g: 10 mL; and
[0030] (4) The weight-to-volume ratio of the poloxamer 407 and the D(+)-glucose solution is (35~45) mg: 10 mL;
[0031] (5) The mass concentration of the D(+)-glucose solution is 4%~6%.
[0032] In one embodiment, the homogenization pressure is 400 bar to 600 bar, and the number of homogenization cycles is 5 to 15.
[0033] The application of the lipid cubic liquid crystal nanoparticles described above in the preparation of a drug delivery system for chemotherapeutic intestinal injury.
[0034] The application of the lipid cubic liquid crystal nanoparticles described above in the preparation of a drug for treating chemotherapy-induced intestinal injury.
[0035] Furthermore, the chemotherapy-induced bowel injury includes irinotecan-induced chemotherapy-induced bowel injury.
[0036] Details of one or more embodiments of this application are set forth in the following description, and other features, objects, and advantages of this application will become apparent from the specification and its claims. Attached Figure Description
[0037] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0038] Figure 1 This is a particle size distribution diagram of patchouli alcohol lipid cubic liquid crystal nanoparticles in Example 2 of this application.
[0039] Figure 2 This is a transmission electron microscope image of patchouli alcohol lipid cubic liquid crystal nanoparticles in Example 2 of this application.
[0040] Figure 3 This is a polarized light microscope image of patchouli alcohol lipid cubic liquid crystal nanoparticles in Example 2 of this application.
[0041] Figure 4 The Fourier transform infrared spectroscopy test results are shown in Embodiment 2 of this application. Figure 4 Image A is the Fourier transform infrared spectrum of patchouli alcohol. Figure 4 Image B is the Fourier transform infrared spectrum of patchouli alcohol lipid cubic liquid crystal nanoparticles. Figure 4 C in the image represents the Fourier transform infrared spectrum of glyceryl monooleate.
[0042] Figure 5 This is a graph showing the in vitro stability evaluation results of patchouli alcohol lipid cubic liquid crystal nanoparticles in Example 2 of this application. Figure 5 In the figure, A represents the statistical results of in vitro particle size stability experiments. Figure 5 B represents the statistical results of the in vitro release experiment.
[0043] Figure 6 This is a graph showing the statistical results of weight changes in mice in Example 3 of this application, including the control group, model group, patchouli alcohol group (PA), and patchouli alcohol lipid cubic liquid crystal nanoparticle group (PA-LCN).
[0044] Figure 7 This is a statistical result diagram of the small intestine length of mice in the normal group (control), model group (model), patchouli alcohol group (PA), and patchouli alcohol lipid cubic liquid crystal nanoparticle group (PA-LCN) in Example 3 of this application.
[0045] Figure 8 This is a graph showing the statistical results of dsDNA content in peritoneal lavage fluid of mice in Example 4 of this application, including the normal group (control), model group (model), patchouli alcohol group (PA), and patchouli alcohol lipid cubic liquid crystal nanoparticle group (PA-LCN).
[0046] Figure 9 This is a graph showing the statistical results of inflammatory cytokines (IL-1β) and myosin light chain kinase (MLCK) in the ileum tissue of mice in Example 5 of this application, including the control group, model group, patchouli alcohol group (PA), and patchouli alcohol lipid cubic liquid crystal nanoparticle group (PA-LCN). Figure 9 Figure A shows the statistical results of inflammatory cytokines (IL-1β). Figure 9Figure B shows the statistical results of myosin light chain kinase (MLCK).
[0047] Figure 10 This is an immunohistochemical image showing the effects of patchouli alcohol group (control), model group (model), patchouli alcohol group (PA), and patchouli alcohol lipid cubic liquid crystal nanoparticle group (PA-LCN) on the expression of apoptosis-related proteins in the ileum tissue of mice in Example 6 of this application. Figure 10 The first row shows the immunohistochemical images of the anti-apoptotic protein BCL in each group. Figure 10 The second row in the middle shows the immunohistochemical images of the pro-apoptotic protein BAX in each group.
[0048] Figure 11 The results show the in vivo safety evaluation of mice in the normal group (control), model group (model), patchouli alcohol group (PA), and patchouli alcohol lipid cubic liquid crystal nanoparticle group (PA-LCN) in Example 7 of this application. Detailed Implementation
[0049] The present application is further described below with reference to embodiments and examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Furthermore, it should be understood that after reading the teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the protection scope of the appended claims.
[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
[0051] the term:
[0052] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:
[0053] As used herein, the terms “and / or,” “or / and,” and “and / or” encompass any one of two or more of the related listed items, as well as any and all combinations of the related listed items, including any two related listed items, any more related listed items, or a combination of all the related listed items.
[0054] In this application, terms such as "further," "preferred," and "optional" are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of this application.
[0055] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.
[0056] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, optional numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.
[0057] In this application, weight can be a well-known unit of mass in the chemical industry, such as μg, mg, g, or kg.
[0058] In this application, terms related to particle characterization include "particle size", "D50", "polydispersity index", "Zeta potential", and "particle size distribution", which have the following meanings:
[0059] Particle size: refers to the maximum length or diameter of a particle or nanoparticle, usually measured in nanometers (nm) or micrometers (μm).
[0060] D50 (Median Particle Size): In a particle size distribution, particles smaller than this size account for half of the total number of particles, and particles larger than this size also account for half of the particle size values.
[0061] Polydispersity Index (PDI): A parameter that represents the width or uniformity of particle size distribution. A PDI close to 0 indicates monodispersity (all particles are the same size), while a higher PDI value indicates a wider particle size distribution, meaning the particles are less uniform in size.
[0062] Zeta potential is a parameter characterizing the surface charge state of particles in a colloid, usually expressed in millivolts (mV). A high absolute value of zeta potential (e.g., exceeding 20 mV) generally indicates strong repulsive forces between particles, which helps to improve the stability of the colloid.
[0063] Particle Size Distribution (PSD): Statistical data describing the range of drug particle sizes; it can be a continuous or discrete distribution.
[0064] In this application, "homogenization" is a process of distributing different components in a mixture or multiphase system more evenly, "homogenization pressure" refers to the pressure applied during the homogenization process to reduce the particle size, usually measured in bar, and "homogenization times" refers to the number of times the mixture is passed through a homogenizer, affecting the particle size and distribution in the final mixture.
[0065] Patchouli alcohol is a natural compound extracted from the traditional Chinese medicine patchouli. In traditional Chinese medicine, patchouli is used to treat symptoms such as colds, abdominal distension, and vomiting. Modern pharmacological studies have confirmed that patchouli alcohol has good effects in reducing intestinal inflammation and alleviating intestinal damage, and has extremely broad application prospects in reducing the side effects of chemotherapy drugs for gastrointestinal cancer.
[0066] For inflammatory bowel diseases, drugs need to reach the site of inflammation to be more effective. However, drugs are affected by various barriers and environmental factors in the body. For example, when administered orally, drugs need to overcome the intestinal mucosal barrier, pH changes from the stomach (pH 1.3–1.8) to the colon (pH 6–8), and the influence of various enzymes, digested food, and microorganisms. Drug delivery to the site of inflammation faces challenges such as poor stability, poor absorption, and low bioavailability.
[0067] One aspect of this application provides patchouli alcohol lipid cubic liquid crystal nanoparticles, the components of which include glyceryl monooleate, polyethylene glycol 400, poloxamer 407, D(+)-glucose, and patchouli alcohol. The patchouli alcohol lipid cubic liquid crystal nanoparticles have a core-shell structure, wherein the shell is a framework structure jointly composed of the glyceryl monooleate, polyethylene glycol 400, poloxamer 407, and D(+)-glucose, and the core is the patchouli alcohol. These patchouli alcohol lipid cubic liquid crystal nanoparticles exhibit good stability and a sustained-release effect compared to free patchouli alcohol. Furthermore, these lipid cubic liquid crystal nanoparticles demonstrate excellent efficacy against chemotherapy-induced intestinal injury, significantly reducing the expression of anti-apoptotic proteins in ileal tissue, while reducing the expression of pro-apoptotic proteins shows good anti-inflammatory effects and good safety.
[0068] In one embodiment, patchouli alcohol accounts for 2% to 3% by weight in the patchouli alcohol lipid cubic liquid crystal nanoparticles.
[0069] In one embodiment, the weight ratio of glyceryl monooleate, polyethylene glycol 400 and poloxamer 407 is (8~12):(4~6):1.
[0070] In one embodiment, the weight ratio of glyceryl monooleate, polyethylene glycol 400, and poloxamer 407 is 10:5:1.
[0071] In one embodiment, the weight ratio of poloxamer 407 to D(+)-glucose is 1:(10~15).
[0072] In one embodiment, the weight ratio of poloxamer 407 to D(+)-glucose is 1:12.5.
[0073] In one embodiment, the patchouli alcohol lipid cubic liquid crystal nanoparticles have at least one of the following characteristics:
[0074] (1) Particle size is 220 nm to 250 nm;
[0075] (2) The polydispersity index is 0.1~0.3; and
[0076] (3) The Zeta potential is -18mV to -19mV.
[0077] In this application, particle size refers to the average particle size unless otherwise specified.
[0078] Preferably, the particle size of the patchouli alcohol lipid cubic liquid crystal nanoparticles is 220nm~250nm, more preferably 225nm~240nm, and examples of particle sizes include 220nm, 230nm, 240nm, and 250nm.
[0079] Preferably, the polydispersity index of the patchouli alcohol lipid cubic liquid crystal nanoparticles is 0.1 to 0.3, more preferably 0.1 to 0.2, and examples of polydispersity indices such as 0.1, 0.2, and 0.3 are given.
[0080] Preferably, the Zeta potential is -18mV to -19mV, for example -18mV, -18.73mV, -19mV, etc.
[0081] In another aspect, this application provides a method for preparing the patchouli alcohol lipid cubic liquid crystal nanoparticles described above, comprising the following steps:
[0082] The first solution was prepared by dissolving patchouli alcohol in polyethylene glycol 400.
[0083] The monooleate glycerol is heated to melt it, and the melted monooleate glycerol is kept at 40℃~50℃ while the first solution is added and mixed to obtain the first mixture.
[0084] A second solution was prepared by dissolving poloxamer 407 in D(+)-glucose solution.
[0085] At 750 rpm to 1000 rpm, the first mixture is added to the second solution and mixed to obtain the second mixture;
[0086] The second mixture was homogenized to obtain patchouli alcohol lipid cubic liquid crystal nanoparticles.
[0087] In one embodiment, the weight ratio of glyceryl monooleate, polyethylene glycol 400 and poloxamer 407 is (8~12):(4~6):1.
[0088] In one embodiment, patchouli alcohol accounts for 2% to 3% by weight in the patchouli alcohol lipid cubic liquid crystal nanoparticles.
[0089] In one embodiment, the weight ratio of poloxamer 407 to D(+)-glucose is 1:(10~15).
[0090] Optionally, the weight ratio of patchouli alcohol to polyethylene glycol 400 is 1:(8~12).
[0091] Optionally, the weight ratio of glyceryl monooleate to the first solution is (1.8~2.2):1.
[0092] Optionally, the weight-to-volume ratio of the first mixture and the second solution is (0.55~0.65) g: 10 mL.
[0093] Optionally, the weight-to-volume ratio of poloxamer 407 to D(+)-glucose solution is (35~45) mg: 10 mL.
[0094] Optionally, the mass concentration of the D(+)-glucose solution is 4% to 6%.
[0095] The molten glyceryl monooleate is kept at 40℃~50℃ to maintain its molten state. Optionally, the holding temperature is 40℃~50℃, preferably 45℃.
[0096] Preferably, the homogenization pressure is 400 bar to 800 bar, and the number of homogenization cycles is 5 to 15.
[0097] In one embodiment, the homogenization pressure can be 400 bar to 800 bar, more preferably 500 bar, and the number of homogenization cycles can be 5 to 15, more preferably 10.
[0098] In another aspect, this application provides the use of the patchouli alcohol lipid cubic liquid crystal nanoparticles described above in the preparation of a drug delivery system for chemotherapy-induced intestinal injury.
[0099] In another aspect, this application provides the use of the patchouli alcohol lipid cubic liquid crystal nanoparticles described above in the preparation of a medicament for treating chemotherapy-induced intestinal injury.
[0100] Furthermore, the above text refers to chemotherapy-induced bowel injury, including but not limited to irinotecan-induced chemotherapy-induced bowel injury.
[0101] The patchouli alcohol lipid cubic liquid crystal nanoparticles of this application have good in vitro stability and exhibit sustained-release effects compared to free drugs.
[0102] The patchouli alcohol lipid cubic liquid crystal nanoparticles of this application have a therapeutic effect on a mouse model of chemotherapy-induced intestinal injury, significantly reducing the rate of weight loss, effectively inhibiting small intestinal shortening, reducing intestinal permeability, and decreasing the expression of inflammatory cytokines and myosin light chain kinase in ileal tissue.
[0103] Furthermore, the patchouli alcohol lipid cubic liquid crystal nanoparticles of this application can significantly reduce the content of dsDNA in the peritoneal lavage fluid of a mouse model of chemotherapy-induced intestinal injury.
[0104] Furthermore, the patchouli alcohol lipid cubic liquid crystal nanoparticles of this application increase the expression of anti-apoptotic proteins in mouse ileum tissue and reduce the expression of pro-apoptotic proteins, showing good anti-inflammatory effects and good safety.
[0105] The following are some specific examples.
[0106] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines given in this application document first, or refer to experimental manuals or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer.
[0107] The raw materials and reagents involved in the following specific embodiments can be obtained commercially or prepared by those skilled in the art using known methods.
[0108] Example 1
[0109] This embodiment is an example of the preparation of patchouli alcohol lipid cubic liquid crystal nanoparticles, including the following steps:
[0110] Accurately weigh 60 mg of patchouli alcohol and dissolve it in 0.6 g of polyethylene glycol 400. Slowly add the 0.6 g of polyethylene glycol 400 containing 60 mg of patchouli alcohol to 1.2 g of melted monooleate glycerol, while maintaining the temperature in a water bath at 45°C. Add the 1.2 g of monooleate glycerol containing 0.6 g of polyethylene glycol 400 and 60 mg of patchouli alcohol, melted at 45°C, to 30 mL of a 5% glucose solution containing 0.12 g of poloxamer 407, and stir at high speed for 5 min. Homogenize the solution 10 times using a high-pressure homogenizer at a homogenization pressure of 500 bar to obtain patchouli alcohol lipid cubic liquid crystal nanoparticles.
[0111] Example 2
[0112] This embodiment is a characterization of the patchouli alcohol lipid cubic liquid crystal nanoparticles of Example 1, including:
[0113] (1) Determination of morphology, particle size, polydispersity index and zeta potential of patchouli alcohol lipid cubic liquid crystal nanoparticles.
[0114] Patchouli lipid cubic liquid crystal nanoparticles were prepared according to the method in Example 1, and the particle size, polydispersity index and zeta potential were determined using a laser particle size analyzer (Zetasizer Nano ZS90, Malvern, UK).
[0115] As shown in Table 1, the particle size of the patchouli alcohol lipid cubic liquid crystal nanoparticles is 233.30±2.72 nm, the polydispersity index is 0.161±0.037, and the zeta potential is -18.73±0.25 mV.
[0116] like Figure 1 As shown, the patchouli alcohol lipid cubic liquid crystal nanoparticles have a relatively uniform particle size distribution, with a D50 of around 255 nm.
[0117] Table 1
[0118]
[0119] Patchouli alcohol lipid cubic liquid crystal nanoparticles were prepared according to the method in Example 1 and observed using transmission electron microscopy (TEM). The TEM image is shown below. Figure 2 As shown in the figure, the patchouli alcohol lipid cubic liquid crystal nanoparticles exhibit good dispersibility.
[0120] (2) Polarized light image of patchouli alcohol lipid cubic liquid crystal nanoparticles
[0121] Patchouli alcohol lipid cubic liquid crystal nanoparticles were prepared according to the method in Example 1 and observed under an optical microscope equipped with a polarizing lens. Figure 3 Polarized light microscopy image of patchouli alcohol lipid cubic liquid crystal nanoparticles. Figure 3 The scale bar for A in the middle is 100 μm. Figure 3 The scale bar for B in the middle is 10 μm. According to... Figure 3 The results of polarized light microscopy show that patchouli alcohol lipid cubic liquid crystal nanoparticles have crystalline properties.
[0122] (3) Fourier transform infrared spectrum of patchouli alcohol lipid cubic liquid crystal nanoparticles
[0123] Patchouli lipid cubic liquid crystal nanoparticles were prepared according to the method in Example 1. Fourier transform infrared spectroscopy was used to detect the presence of patchouli alcohol, glyceryl monooleate, and patchouli lipid cubic liquid crystal nanoparticles.
[0124] Figure 4 The results are from Fourier transform infrared spectroscopy tests. Figure 4 Image A is the Fourier transform infrared spectrum of patchouli alcohol. Figure 4 Image B is the Fourier transform infrared spectrum of patchouli alcohol lipid cubic liquid crystal nanoparticles. Figure 4 C represents the Fourier transform infrared spectrum of monooleoglycerate. (Example:) Figure 4 As shown, patchouli alcohol was successfully encapsulated in patchouli alcohol lipid cubic liquid crystal nanoparticles.
[0125] (4) Evaluation of in vitro stability and release
[0126] 1) Stability evaluation
[0127] Patchouli alcohol lipid cubic liquid crystal nanoparticles were prepared according to the method in Example 1 and stored at room temperature. The particle size of the nanoparticles was measured for 7 consecutive days to observe whether there was a significant change in particle size.
[0128] 2) Release evaluation
[0129] Patchouli alcohol lipid cubic liquid crystal nanoparticles were prepared according to the method in Example 1. 1 mL of the solution was placed into a dialysis bag with a molecular weight cutoff of 14 K. Artificial gastric fluid and artificial small intestinal fluid (both purchased from Shanghai Yuanye Biotechnology Co., Ltd.) were used as dissolution media. The dialysis bag was placed in a large amount of dissolution media and incubated on a shaker at 37°C and 100 rpm. At each set time point, 1 mL of release medium was collected, and 1 mL of fresh release medium was added. The drug concentration at each time point was determined by GC-MS (Agilent Technologies, USA), and the in vitro release curve of the drug was finally calculated.
[0130] 3) Evaluation Results
[0131] Figure 5 The results are the in vitro stability evaluation results of patchouli alcohol lipid cubic liquid crystal nanoparticles. Figure 5 Statistical results of in vitro stability experiments and A-type stability test. Figure 5 B represents the statistical results of the in vitro release experiment. For example... Figure 5 As shown in Figure A, patchouli alcohol lipid cubic liquid crystal nanoparticles exhibit good stability. Figure 5 As shown in Figure B, the nanoparticles are released more slowly than the free drug in artificial gastric fluid within 2 hours, but more quickly than the free drug in artificial small intestinal fluid after 2 hours.
[0132] Example 3
[0133] This embodiment is an in vivo pharmacodynamic evaluation of the patchouli alcohol lipid cubic liquid crystal nanoparticles of Example 1, including the following steps:
[0134] SPF-grade C57 / BL6 mice were randomly divided into four groups: control group, model group, patchouli alcohol group (PA), and patchouli alcohol lipid cubic liquid crystal nanoparticle group (PA-LCN). Except for the control group, the other groups were intraperitoneally injected with irinotecan hydrochloride 200 mg / kg on days 1, 3, and 5 to induce chemotherapy-induced intestinal injury. Throughout the induction period, daily changes in mouse body weight were recorded, and fecal characteristics and diarrhea were observed.
[0135] When the animal experiment cycle reached its end, the mice were euthanized, their entire intestines were dissected and separated, rinsed with phosphate buffer solution, filtered dry with filter paper, and the length of the small intestine was measured and photographed.
[0136] Figure 6 This is a statistical result of the changes in body weight of mice in the control group, model group, patchouli alcohol group (PA), and patchouli alcohol lipid cubic liquid crystal nanoparticle group (PA-LCN). Figure 7 This figure shows the statistical results of small intestinal length in mice from the control group, model group, patchouli alcohol group (PA), and patchouli alcohol lipid cubic liquid crystal nanoparticle group (PA-LCN). Figures 6-7 As shown, patchouli alcohol lipid cubic liquid crystal nanoparticles had the best therapeutic effect on chemotherapy-induced intestinal injury model mice, significantly reducing the rate of weight loss and effectively inhibiting small intestinal shortening.
[0137] Example 4
[0138] This embodiment analyzes the dsDNA content in the peritoneal lavage fluid of mice after administration of patchouli alcohol lipid cubic liquid crystal nanoparticles, including:
[0139] Mice were induced and drugged according to the method described in Example 3. At the end of the animal experiment, the mice were euthanized, and their peritoneal cavities were flushed with phosphate buffered solution to obtain peritoneal lavage fluid. The supernatant was collected by centrifugation and repeated twice to obtain cell-depleted peritoneal lavage fluid. The dsDNA content in the mouse peritoneal lavage fluid was detected using a dsDNA content detection kit (purchased from Shanghai Yisheng Biotechnology Co., Ltd.).
[0140] Figure 8 The graph shows the statistical results of dsDNA content in peritoneal lavage fluid from mice in the control group, model group, patchouli alcohol group (PA), and patchouli alcohol lipid cubic liquid crystal nanoparticle group (PA-LCN). Figure 8 As shown, patchouli alcohol lipid cubic liquid crystal nanoparticles have the most significant effect in reducing the dsDNA content in mouse peritoneal lavage fluid.
[0141] Example 5
[0142] This embodiment describes the q-PCR detection of ileal tissue after administration of patchouli alcohol lipid cubic liquid crystal nanoparticles, including:
[0143] Mice were induced and drugged according to the method described in Example 3. At the end of the animal experiment period, the mice were euthanized, and their entire intestines were dissected. 20 mg of ileum tissue was weighed, and changes in inflammatory cytokines and myosin light chain kinase (MLCK) were detected using real-time quantitative PCR (q-PCR).
[0144] Figure 9 This is a graph showing the statistical results of inflammatory cytokines (IL-1β) and myosin light chain kinase (MLCK) in the ileum tissue of mice in the control group, model group, patchouli alcohol group (PA), and patchouli alcohol lipid cubic liquid crystal nanoparticle group (PA-LCN). Figure 9 Figure A shows the statistical results of inflammatory cytokines (IL-1β). Figure 9 Figure B shows the statistical results of myosin light chain kinase (MLCK).
[0145] like Figure 9 As shown, patchouli alcohol lipid cubic liquid crystal nanoparticles showed the best effect in downregulating the mRNA levels of inflammatory cytokine (IL-1β) and myosin light chain kinase (MLCK).
[0146] Example 6
[0147] This embodiment is an immunohistochemical study of the ileum after administration of patchouli alcohol lipid cubic liquid crystal nanoparticles, including:
[0148] Mice were induced and drugged according to the method in Example 3. At the end of the animal experiment cycle, the mice were euthanized, the ileum was dissected and separated, fixed with 4% paraformaldehyde, embedded in paraffin to prepare slides, dewaxed, stained with apoptosis protein-related antibodies, and photographed using a tissue imaging system (TCS-SP8, Leica Germany) for observation and evaluation.
[0149] Figure 10 Immunohistochemical images showing the effects of patchouli alcohol group (PA), patchouli alcohol lipid cubic liquid crystal nanoparticle group (PA-LCN) on the expression of apoptosis-related proteins in the ileum tissue of mice. Figure 10 Image A shows the immunohistochemical images of the anti-apoptotic protein BCL in each group. Figure 10 In Figure B, the immunohistochemical images of the pro-apoptotic protein BAX in each group are shown.
[0150] like Figure 10As shown, in the untreated chemotherapy-induced intestinal injury model group (irinotecan group), compared with the normal group mice (phosphate buffered solution group), the expression of the anti-apoptotic protein BCL in the ileum tissue was significantly decreased, while the expression of the pro-apoptotic protein BAX was significantly increased. However, in the ileum tissue of chemotherapy-induced intestinal injury model mice treated with free patchouli alcohol and patchouli alcohol lipocubic liquid crystal nanoparticles, the expression of the anti-apoptotic protein BCL was significantly increased, especially in the lipocubic liquid crystal nanoparticle treatment group.
[0151] Example 7
[0152] This embodiment evaluates the in vivo safety of patchouli alcohol lipid cubic liquid crystal nanoparticles, including:
[0153] Mice were induced and drugged according to the method described in Example 3. At the end of the animal experiment period, the mice were euthanized, and their hearts, livers, spleens, lungs, and kidneys were removed, weighed, and their organ coefficients calculated. The hearts, livers, spleens, lungs, and kidneys were fixed in 4% paraformaldehyde solution, embedded in paraffin, and slides were prepared. After dewaxing, the slides were stained with hematoxylin and eosin and photographed using a tissue imaging system (TCS-SP8, Leica Germany) for observation and evaluation.
[0154] Figure 11 This presents the in vivo safety evaluation results for mice in the control group, model group, patchouli alcohol group (PA), and patchouli alcohol lipid cubic liquid crystal nanoparticle group (PA-LCN). Figure 11 As shown, no obvious lesions were found in the pathological sections of the major organs in each drug administration group, demonstrating that the free drug and patchouli alcohol lipid cubic liquid crystal nanoparticles have good biosafety.
[0155] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0156] The embodiments described above merely illustrate several implementation methods of this application and should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Furthermore, it should be understood that after reading the above teachings, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the protection scope of the appended claims. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.
Claims
1. A patchouli alcohol lipid cubic liquid crystal nanoparticle, characterized in that, Its ingredients include glyceryl monooleate, polyethylene glycol 400, poloxamer 407, D(+)-glucose and patchouli alcohol; The patchouli alcohol lipid cubic liquid crystal nanoparticles have a core-shell structure, wherein the shell is a framework structure composed of the monooleic glycerol ester, the polyethylene glycol 400, the poloxamer 407 and the D(+)-glucose, and the core is the patchouli alcohol.
2. The patchouli alcohol lipid cubic liquid crystal nanoparticles according to claim 1, characterized in that, The weight ratio of the glyceryl monooleate, the polyethylene glycol 400, and the poloxamer 407 is (8~12):(4~6):1; and / or The patchouli alcohol constitutes 2% to 3% by weight in the lipid cubic liquid crystal nanoparticles; and / or The weight ratio of poloxamer 407 to D(+)-glucose is 1:(10~15).
3. The patchouli alcohol lipid cubic liquid crystal nanoparticles as described in claim 1 or 2, characterized in that, It has at least one of the following characteristics: (1) Particle size is 220nm~250nm; (2) The polydispersity index is 0.1~0.3; and (3) The Zeta potential is -18mV to -19mV.
4. The method for preparing patchouli alcohol lipid cubic liquid crystal nanoparticles according to any one of claims 1 to 3, characterized in that, Includes the following steps: The patchouli alcohol was dissolved in polyethylene glycol 400 to prepare a first solution; The monooleic glycerol ester is heated to melt it, and the melted monooleic glycerol ester is kept at 40℃~50℃ while the first solution is added and mixed to obtain the first mixture. The poloxamer 407 was dissolved in a D(+)-glucose solution to prepare a second solution; The first mixture is added to the second solution at 750 rpm to 1000 rpm to prepare the second mixture. The second mixture was homogenized to obtain the patchouli alcohol lipid cubic liquid crystal nanoparticles.
5. The preparation method according to claim 4, characterized in that, The weight ratio of the glyceryl monooleate, the polyethylene glycol 400, and the poloxamer 407 is (8~12):(4~6):1; and / or The patchouli alcohol constitutes 2% to 3% by weight in the lipid cubic liquid crystal nanoparticles; and / or The weight ratio of poloxamer 407 to D(+)-glucose is 1:(10~15).
6. The preparation method according to claim 4 or 5, characterized in that, Includes at least one of the following features: (1) The weight ratio of patchouli alcohol to polyethylene glycol 400 is 1:(8~12); (2) The weight ratio of the monooleic glyceride to the first solution is (1.8~2.2):1; (3) The weight-to-volume ratio of the first mixture to the second solution is (0.55~0.65) g: 10 mL; (4) The weight-to-volume ratio of the poloxamer 407 and the D(+)-glucose solution is (35~45) mg: 10 mL; as well as (5) The mass concentration of the D(+)-glucose solution is 4%~6%.
7. The preparation method according to claim 4 or 5, characterized in that, The homogenization pressure is 400 bar to 600 bar, and the number of homogenization cycles is 5 to 15.
8. A drug delivery system for chemotherapy-induced intestinal injury, characterized in that, Including the patchouli alcohol lipid cubic liquid crystal nanoparticles as described in any one of claims 1 to 3.
9. The use of the patchouli alcohol lipid cubic liquid crystal nanoparticles according to any one of claims 1 to 3 or the drug delivery system for chemotherapy-induced intestinal injury according to claim 8 in the preparation of a medicament for treating chemotherapy-induced intestinal injury.
10. The application according to claim 9, characterized in that, The chemotherapy-induced bowel injury includes irinotecan-induced chemotherapy-induced bowel injury.