Emodin nano preparation based on copper ions as well as preparation method and application of emodin nano preparation

Abstract

On the first aspect, the emodin nano preparation based on the copper ions is provided, emodin is used as a copper ion carrier, and emodin and the copper ions synergistically play a role in inhibiting tumor cell activity; on the second aspect, the invention provides a preparation method of the anti-tumor nano preparation, the particle size of the preparation is controlled to be 100-200nm, the distribution and residence time of the active ingredients in tumor tissues are improved, and the preparation has tumor targeting property. And on the third aspect, the ROS content of the emodin in tumor cells is increased by utilizing the photodynamic effect, and the activity of the tumor cells is inhibited.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to a copper ion-based rhubarb nano preparation and a preparation method and application thereof. Background Art

[0002] Copper is a common metal element in organisms. It is closely related to the occurrence, development, metastasis and tumor immune microenvironment of tumors. In the past two years, scholars have proposed that intracellular copper overload can induce copper death in tumor cells. The main mechanism of "copper death" is that excessive intracellular copper directly binds to the acylated components of the tricarboxylic acid cycle (TCA), leading to the aggregation of acylated proteins and the loss of iron-sulfur cluster proteins, which in turn triggers protein toxic stress and ultimately causes cell death. Copper death can also induce immunogenic death (ICD), releasing damage-associated molecular patterns (DAMPs), such as calreticulin (CRT), high-mobility group box 1 (HMGB1) and adenosine triphosphate (ATP). Damage-associated molecules promote immune cells to secrete immune factors, activate T lymphocytes to kill tumor cells, inhibit tumor growth, and enhance anti-tumor efficacy.

[0003] Based on the anti-tumor treatment strategy of copper ion concentration, the drugs disulfiram and ilisimol have been carried out in phase II clinical trials, which are used for glioblastoma and melanoma respectively. Due to the strong toxicity and side effects of copper carriers, the treatment effect of clinical studies is not good.

[0004] Aloe-emodin (AE) is a natural small molecule compound and one of the active ingredients of aloe vera. It has multiple biological activities such as antibacterial, antiviral, and antitumor. It is widely used in the fields of medicine and cosmetics and has good safety. Studies have found that it has antitumor activity against nervous system tumors, lung squamous cell carcinoma, liver cancer cells, colon cancer cells, melanoma, etc., and can be used as an antitumor therapeutic molecule. However, AE has poor solubility in water, which is not conducive to the delivery and absorption of drugs.

[0005] Therefore, there is an urgent need to develop a new copper ion-based targeted anti-tumor preparation to improve the specificity of copper ion therapy while reducing side effects. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a copper ion-based rhubarb nano preparation and a preparation method and application thereof.

[0007] In a first aspect of the present invention, a copper ion-based emodin nanoformulation (NPs) is provided. The nanoformulation comprises copper ions and emodin, and emodin is a copper ion carrier.

[0008] Rhein can coordinate with multivalent metal ions, such as iron, magnesium, and copper, and enhance its antioxidant activity through coordination. After coordination, Rhein acts as a copper ion carrier, delivering copper ions to tumor cells in a targeted manner; after coordination, copper ions increase the solubility of Rhein in water, further promoting the delivery carrier function of Rhein.

[0009] As a preferred embodiment, the emodin is aloe-emodin.

[0010] As a preferred embodiment, the emodin is derived from a traditional Chinese medicine extract.

[0011] As a preferred embodiment, the Chinese medicine extract is aloe extract.

[0012] As a preferred embodiment, the molar ratio of aloe-emodin to copper ions is 1:1-1:10.

[0013] As a preferred embodiment, the molar ratio of aloe-emodin to copper ions is 1:2.

[0014] As a preferred embodiment, the copper ions are derived from copper dichloride.

[0015] As a preferred embodiment, the nanoformulation further comprises 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and the molar ratio of rhamnosin to 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000 is 1:0.8.

[0016] As a preferred embodiment, the nanoformulation further comprises 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000-folic acid (DSPE-PEG2000-FA), and the molar ratio of rhamnosin to 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000-folic acid is 1:0.2.

[0017] As a preferred embodiment, the particle size of the nanoparticles is 100nm~200nm.

[0018] The carboxyl groups of DSPE-PEG2000 and DSPE-PEG2000-FA form amide bonds with the amino groups, so that folic acid is combined with the rhein-copper complex as a ligand, and the rhein-copper complex forms a nanosphere form, thereby improving the stability of the preparation solution.

[0019] The second aspect of the present invention provides a method for preparing a copper ion-based rhubarb nanoformulation, comprising the following steps: Step S1: dissolving emodin (AE), copper dichloride (CuCl2), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000-folic acid (DSPE-PEG2000-FA) in a dimethyl sulfoxide (DMSO) solution to obtain solution 1; Step S2: dispersing solution 1 in water to obtain nano-mixed solution 2; Step S3: centrifuging the nano-mixed solution 2 to obtain nano-particle precipitate 3; Step S4: dispersing the nanoparticle precipitate 3 in water to prepare a rhein nano preparation.

[0020] As a preferred embodiment, the dispersion method in step S2 is ultrasound, the ultrasound power is 100w, and the ultrasound time is 5min.

[0021] As a preferred embodiment, the centrifugal speed in step S3 is 16000 rpm and the centrifugal time is 20 min.

[0022] The EPR effect is the high permeability and retention effect of solid tumors. Due to the abundant blood vessels, wide gaps between blood vessel walls, poor structural integrity, and lack of lymphatic return in solid tumor tissues, molecules or particles of a certain size tend to accumulate in tumor tissues. The present invention utilizes the particle size characteristics of nanoparticles to improve the distribution and retention time of nanoparticles in tumor tissues, and has tumor targeting.

[0023] The third aspect of the present invention provides the application of the copper ion-based emodin nanoformulation in anti-tumor.

[0024] As a preferred solution, the application step is: after the nano preparation enters the tumor cells, a laser with a wavelength of 450nm is used to irradiate the tumor tissue to promote the release of copper ions from emodin.

[0025] As a preferred embodiment, the laser irradiation method is to connect the tumor tissue via an optical fiber instrument to achieve irradiation.

[0026] As a preferred embodiment, the tumor tissue is lung tumor tissue.

[0027] AE is photosensitivity, and its maximum absorption wavelength is 450nm. Under 450nm wavelength light irradiation, emodin increases the content of reactive oxygen species (ROS) in tumor cells through photodynamic effect, induces endoplasmic reticulum stress, thereby triggering ICD, enhancing anti-tumor immune response, and inhibiting anti-tumor activity. In addition, under 450nm wavelength light irradiation, emodin is promoted to release copper ions, which promote copper death and ICD in tumor cells, inhibiting anti-tumor activity.

[0028] Through the above technical solutions, the present invention produces the following technical effects: (1) Using rhamnosine as a carrier, copper ions are targeted and transported to tumor tissues through coordination, thereby inhibiting the activity of tumor cells.

[0029] (2) Copper ions coordinate with rheum officinale, increasing its solubility in water, improving its absorbability, and improving its ability to transport copper ions as a carrier.

[0030] (3) The present invention utilizes the particle size characteristics of the nanoformulation to improve the distribution and retention time of the nanoformulation in tumor tissues, thereby achieving tumor targeting.

[0031] (4) Through the photodynamic effect, the ROS content of emodin in tumor cells is increased, inhibiting the anti-tumor cell activity; through the photodynamic effect, emodin is promoted to release copper ions, inducing copper death in tumor cells and inhibiting tumor cell activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the morphology of the nanoformulation of rheum officinale characterized by transmission electron microscopy. Figure 1 The arrows in the middle indicate nanoparticles.

[0033] Figure 2 This is the DLS particle size distribution diagram of the nanoformulation.

[0034] Figure 3 These are the results of X-ray electron spectroscopy determination of copper in nanoformulations.

[0035] Figure 4 These are the results of X-ray electron spectroscopy measurements of carbon, oxygen and copper in nanoformulations.

[0036] Figure 5 The standard curve for encapsulation efficiency determination by microplate reader.

[0037] Figure 6 The results of the photodynamic experiment of nanoformulations. AE represents solution 1; NPs represents solution 2; CU represents solution 3; SOSG represents the control solution.

[0038] Figure 7 These are the results of the mouse lung cancer cell (LLC) activity inhibition test.

[0039] Figure 8 The results of measuring the ROS content in mouse lung cancer cells (LLC), where DAPI is the fluorescence of cell nucleus staining; ROS is the green fluorescence of reactive oxygen species; Merge is the combination of the two fluorescences.

[0040] Fig. 9Figure 5 is the fluorescence intensity of nanoformulation uptake by mouse lung cancer cells (LLC) at different time points.

[0041] Fig.10 This is a graph showing the change trend of fluorescence intensity over time in mouse lung cancer cells (LLC).

[0042] Fig.11 This is a graph showing the aggregation trend of nanoformulations at tumor sites in mice. DETAILED DESCRIPTION

[0043] In order to make the above-mentioned objects, features and advantages of the present invention more clearly understood, embodiments are described below.

[0044] Example 1: Preparation and characterization of nanoparticles (NPs) 1. Sample preparation AE was dissolved in DMSO to prepare a 100 mmol / L AE solution.

[0045] Dissolve CuCl2 in DMSO to prepare a 100 mmol / L CuCl2 solution.

[0046] DSPE-PEG2000 was dissolved in DMSO to prepare a 100 mmol / L DSPE-PEG2000 solution.

[0047] DSPE-PEG2000-FA was dissolved in DMSO to prepare a 100 mmol / L DSPE-PEG2000-FA solution.

[0048] AE solution, CuCl2 solution, DSPE-PEG2000 solution, and DSPE-PEG2000-FA solution were mixed in a volume ratio of 1:2:0.8:0.2 to prepare solution 1. Solution 1 was dispersed in water under ultrasound, with a volume ratio of solution 1 to water of 0.4:1, an ultrasound power of 100w, and an ultrasound time of 5 min to obtain a nanomixed solution; the nanomixed solution was centrifuged to obtain nanoparticle precipitation, with a centrifugal speed of 16000rpm and a centrifugal time of 20 min, and an appropriate amount of water was added for dispersion to obtain a nanopreparation.

[0049] The nanoparticles were centrifuged to obtain nanoparticles precipitate, which were vacuum dried for 24 hours to obtain solid powder.

[0050] 2. Characterization methods The morphology of the nanoformulation was characterized by transmission electron microscopy. The NPs solution sample was dropped onto a molybdenum mesh and dried under a baking lamp for 10 minutes. The dried NPs-loaded molybdenum mesh was cleaned on the surface with an ear bulb and placed in a sample rod for photography.

[0051] The DLS particle size of the nanoformulation was measured by Nano-ZS90 nanoparticle size-potential analyzer. NPs of appropriate concentration were dispersed in 1 mL of ultrapure water at room temperature. The dispersed NPs solution was then placed in a clean sample cell to measure the hydrated particle size and distribution index (PDI) of the NPs.

[0052] The copper ion loading in the solid powder was determined by X-ray electron spectroscopy. The NPs solution was centrifuged (12000 rpm) to obtain the precipitate, which was vacuum dried for 24 hours to obtain the solid powder. The solid powder was placed on aluminum foil and pressed into a sample with a thickness of 1 mm. The sample was fixed on a sample stand to determine the copper ion content in the sample.

[0053] The AE loading in the NPs solution was determined by an ELISA instrument, and a standard curve for AE was established: 54 mg of AE was accurately weighed and dissolved in anhydrous ethanol in a small beaker, and then transferred to a 5 mL volumetric flask to make a fixed volume to prepare an AE solution with a concentration of 1.08 mg / mL. A certain volume of the mother solution was diluted to different concentrations (0.55, 0.27, 0.135, 0.0675, 0.03375, 0.016875, 0 mg / mL), and the absorbance value at a wavelength of about 430 nm was measured by an ELISA instrument. The AE concentration was used as the horizontal axis and the absorbance value (OD) was used as the vertical axis to draw a standard AE standard curve. The NPs solutions prepared under the same conditions from three different batches and diluted by different times (sample 1 was diluted 4 times, sample 2 was diluted 2 times, and sample 3 was diluted 0 times) were measured by an ELISA instrument at the corresponding wavelength. The AE concentration was determined by the standard curve.

[0054] 3. Test results and analysis Transmission electron microscopy characterization results are shown in Figure 1 As shown, the NPs are spherical.

[0055] The DLS particle size determination results are as follows Figure 2 As shown, the average hydrated particle size value of NPs is 143.9 nm; the particle distribution index (PDI) is 0.159, indicating that the particle size distribution is relatively concentrated and the uniformity of the nanoparticles is relatively high.

[0056] X-ray electron spectroscopy determination Figure 3 , Figure 4 As shown, the determination of Cu in rheum officinale nanoparticles 2+ The atomic number of Cu 2+ Cu, O, and C atoms 2+ The ratio was 19.1%, indicating that the NPs were successfully loaded with copper.

[0057] The standard curve of the ELISA instrument is as follows Figure 5As shown, the measured absorbance of NPs was substituted into the standard curve, and the encapsulation efficiency of NPs was obtained to be approximately 37.28%.

[0058] Table 1 NPs encapsulation efficiency test results

[0059] Example 2: Photodynamic experiment of nanoformulation 1. Sample preparation Solution 1 preparation: Disperse emodin in 1 mL of aqueous solution to make the emodin content reach 10 μmol / L, and add 5 μL of 5 mmol / L singlet oxygen fluorescence probe (SOSG) solution with methanol as solvent. Solution 1 is represented by AE.

[0060] Solution 2 preparation: The nanoformulation of Example 1 was dispersed in 1 mL of aqueous solution, the emodin content reached 10 μmol / L, the copper ion concentration reached 20 μmol / L, and 5 μL of 5 mmol / L singlet oxygen fluorescence probe (SOSG) solution with methanol as solvent was added. Solution 2 was represented by NPs.

[0061] Solution 3 preparation: Disperse copper ions in 1 mL of aqueous solution to make the emodin content reach 20 μmol / L, and add 5 μL of 5 mmol / L singlet oxygen fluorescence probe (SOSG) solution with methanol as solvent. Solution 3 is represented by CU.

[0062] Preparation of control solution: Add 5 μL of 5 mmol / L singlet oxygen fluorescence probe (SOSG) solution with methanol as solvent to 1 mL of water. The control solution is represented by SOSG.

[0063] 2. Test methods Solution 1 was irradiated with a 450 nm laser for 0, 30, 60, 120, and 180 seconds, respectively. The fluorescence intensity of the solution was detected by a fluorescence spectrophotometer at an excitation / emission wavelength of 504 / 525 nm.

[0064] Solution 2 was irradiated with a 450 nm laser for 0, 30, 60, 120, and 180 seconds, respectively. The fluorescence intensity of the solution was detected by a fluorescence spectrophotometer at an excitation / emission wavelength of 504 / 525 nm.

[0065] Solution 3 was irradiated with a 450 nm laser for 0, 30, 60, 120, and 180 seconds, respectively. The fluorescence intensity of the solution was detected by a fluorescence spectrophotometer at an excitation / emission wavelength of 504 / 525 nm.

[0066] The control solution was irradiated with a 450 nm laser for 0, 30, 60, 120, and 180 seconds, respectively. The fluorescence intensity of the solution was detected by a fluorescence spectrophotometer at an excitation / emission wavelength of 504 / 525 nm.

[0067] 3. Test results and analysis like Figure 6 As shown, under 450 nm laser irradiation, solution 2 released the most singlet oxygen, indicating that 450 nm light irradiation promoted the release of copper ions from rhamnosus-emodin and enhanced the photodynamic properties of the nanoformulation.

[0068] Example 3: Experiment on the inhibition of the activity of mouse lung cancer cells by rheumatoid arthritis nanoparticle preparation 1. Test methods Mouse lung cancer cells (LLC cells) in the logarithmic phase were digested and counted, and 5×10 3 Cells / well were inoculated in a 96-well plate and cultured for 24 h. Rheum officinale and the nanoformulation prepared in Example 1 were added according to the grouping method in Table 1 to make the concentration of Rheum officinale reach the concentration in Table 1. The culture was continued for 7 h. The illumination group was illuminated with 450 nm at 0.1 W / cm 2 The cells were irradiated with high intensity laser for 3 min. After the irradiation, the cells were placed in a cell culture incubator for 17 h. Diluted 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) solution was added to each well of all groups and incubated in the incubator for 4 h. The absorbance of each well at 562 nm was measured by an ELISA instrument. The emodin group was represented by AE, the nanoformulation group was represented by NPs, the emodin + irradiation group was represented by AE + L, and the emodin nanoformulation + irradiation group was represented by NPs + L.

[0069] 2. Test results and analysis The results are as follows Figure 7 As shown in the figure, when the AE concentration is the same, NPs+L has a stronger killing effect on tumor cells than NPs; when irradiated with 450 nm laser, NPs+L releases rhamnosin and copper ions, which synergistically inhibit the activity of tumor cells, so NPs+L has a stronger killing effect on tumor cells.

[0070] When the concentration of AE was 10 μmol / L, NPs had a stronger killing effect on tumor cells than AE. Since the emodin in NPs coordinated copper ions, the combination of the two could inhibit the activity of tumor cells.

[0071] Table 2 Mouse lung cancer cell activity inhibition test

[0072] Case study 4: Effect of rhein nanoparticle preparation on ROS content in mouse lung cancer cells 1. Test methods LLC cells were cultured at 2×10 5 Cells / well were seeded into 12-well plates and cultured overnight. Complete culture medium containing certain concentrations of CuCl2 (10 μmol / L), AE (AE content: 4.354 μmol / L), and AE@Cu NPs (IC50 of AE@Cu NPs, equivalent to AE content: 4.354 μmol / L) was added and laser (450 nm, 0.1 W / cm 2 ) Irradiate for 3 minutes, continue to incubate in the incubator for 17 hours before collecting the sample. Wash with PBS 3 times, add 200ul 4% paraformaldehyde to each well for light-proof fixation for 15 minutes, wash with PBS twice, add DCFHDA probe (10 μM) for staining for 30 minutes, wash with PBS twice, take out the slide, turn it upside down on a slide with DAPI dye, and take fluorescence images under a confocal microscope the next day.

[0073] 2. Test results and analysis The results are as follows Figure 8 As shown in the figure, when the concentration of AE is the same, NPs+L causes tumor cells to produce more ROS than NPs; and under laser irradiation, the ROS content in tumor cells of NPs+L is higher than that of AE+L, indicating that AE and Cu 2+ After coordination, its photodynamic effect is enhanced, so NPs+L has a stronger photodynamic effect.

[0074] Example 5: Experimental study on the uptake of nanoformulations by mouse lung cancer cells (LLC cells) 1. Test methods Cy5.5 (a near-infrared fluorescent cyanine dye, Ex=673 nm, Em=707 nm, which can be used to label proteins, antibodies, peptides, nucleic acid molecules, nanoparticles, etc.) was co-encapsulated with NPs to assemble new nanoparticles (Cy5.5@NPs). LLC cells in the logarithmic growth phase were taken and cultured at 1×10 5 Each well was inoculated in a laser confocal culture dish and incubated overnight. Then, the culture medium was discarded and the cells were incubated with a medium containing Cy5.5@NPs for 0h, 1h, 4h, and 7h, and the samples were collected. The nanoparticle uptake in each group of cells was detected by flow cytometry.

[0075] 2. Test results and analysis like Fig. 9 , Fig.10As shown in the figure, the fluorescence intensity in LLC cells increases with the increase of treatment time, and the fluorescence intensity in cells after 7 h of treatment is nearly 40 times that after 0 h of treatment. This shows that the nanoparticles can be enriched in tumor cells and play a role in inhibiting tumor activity.

[0076] Example 6: Biodistribution and metabolism of nanoformulations 1. Test methods A subcutaneous tumor model was established in healthy male C57BL / 6 mice aged 6 weeks. Three mice with uniform tumor growth were selected and injected with NPs (NPs@Cy7.5) labeled with Cy7.5 (a near-infrared (NIR) fluorescent anthocyanin dye with an emission peak (Emmax) greater than 800nm ​​and strong tissue penetration, and its marker is often used for small animal in vivo imaging) through the tail vein. The mice were monitored by fluorescence imaging using the small animal in vivo imaging system (IVIS) at 1 h, 4 h, 7 h, 10 h, 24 h, and 36 h after injection. The mice were killed 36 h later, and the main organs (heart, liver, spleen, lung, kidney) and tumor tissues were separated for in vitro fluorescence imaging.

[0077] 2. Test results and analysis like Fig.11 As shown in Figure 2, 1 h after injection of NPs@Cy7.5, the total fluorescence value of the mouse tumor site was 1.076×10 9 p / s / cm 2 / sr. As time went on, the fluorescence intensity of the tumor site continued to increase, and the total fluorescence value at 24 h was 1.219×10 9 p / s / cm 2 / sr, the total fluorescence value at 36 h was 1.615×10 9 p / s / cm 2 The above research results fully demonstrate that NPs can target tumor sites, quickly accumulate and retain in tumor sites, and exert targeted tumor inhibition effects.

[0078] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A copper ion-based rhubarb nanoparticle preparation, characterized in that: The nano preparation comprises copper ions and rhein, and rhein is a copper ion carrier.

2. The nanoformulation according to claim 1, characterized in that The emodin is derived from a traditional Chinese medicine extract, and the traditional Chinese medicine extract is an aloe vera extract.

3. The nanoformulation according to claim 2, characterized in that The molar ratio of aloe-emodin to copper ions is 1:1-1:

10.

4. The nanoformulation according to claim 3, characterized in that The molar ratio of aloe-emodin to copper ions is 1:

2.

5. The nanoformulation according to claim 4, characterized in that The nanoformulation also includes 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000 and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000-folic acid, and the molar ratio of rhamnosin:1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000:1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000-folic acid is 1:0.8:0.

2.

6. The nanoformulation according to claim 5, characterized in that The particle size of the nanoparticles is 100nm~200nm.

7. A method for preparing a copper ion-based rhubarb nanoformulation, characterized in that: The following steps are involved: Step S1: dissolving aloe-emodin, copper dichloride, 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000, and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000-folic acid in a dimethyl sulfoxide solution to obtain a solution 1; Step S2: dispersing solution 1 in water to obtain nano-mixed solution 2; Step S3: centrifuging the nano-mixed solution 2 to obtain nano-particle precipitate 3; Step S4: dispersing the nanoparticle precipitate 3 in water to prepare a rhein nano preparation.

8. The preparation method according to claim 7, characterized in that: The dispersion method in step S2 is ultrasound, the ultrasound power is 100w, and the ultrasound time is 5 minutes; the centrifugal speed in step S3 is 16000 rpm, and the centrifugal time is 20 minutes.

9. The application of copper ion-based emodin nanoformulation in anti-tumor, characterized in that: The application steps are: after the nano preparation enters the tumor cells, a laser with a wavelength of 450nm is used to irradiate the tumor tissue.

10. The use according to claim 9, characterized in that: The laser irradiation is performed close to the tumor tissue through a fiber optic instrument.

Images