A nano drug delivery system and its preparation method and application

Abstract

The present invention discloses a nano drug delivery system and a preparation method and application thereof, wherein the nano drug delivery system comprises chlorin e6 and theophylline derivative d17. The nano drug delivery system prepared by the scheme of the present invention synthesizes two drugs, chlorin e6 and theophylline derivative d17, with different action mechanisms and encapsulates them in one nanoparticle, adopts a dual-path combination of chemical drug therapy and photodynamic therapy to treat tumors, can release two drugs in the tumor microenvironment and under specific near-infrared light, can also adjust the action target, control the time and can concentrate at the tumor cells, effectively reduce the toxic side effects of photosensitizers, improve solubility, and reduce adverse reactions of the body, providing a new scheme and theoretical basis for the development of clinical anti-tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the field of nano-biomaterials, and in particular relates to a nano-drug delivery system and a preparation method and application thereof. Background Art

[0002] Tumors are new organisms formed when a cell in a local tissue loses normal regulation of its growth at the genetic level under the influence of various carcinogenic factors, leading to abnormal clonal proliferation. Cancer is one of the main causes of death for people around the world and a major obstacle to improving life expectancy.

[0003] The traditional way to treat tumors is chemotherapy, which is to take anti-tumor drugs orally or intravenously, and transmit the drugs to the lesion site through the systemic circulation to kill tumor cells. However, most chemotherapy drugs have no selective specificity for tumor tissues, and can also cause damage to normal tissues, causing great pain to patients. Secondly, due to various biological barriers in the human body, only a small amount of chemotherapy drugs can reach the tumor site, resulting in low bioavailability and poor treatment effect. In addition, tumor resistance caused by changes in the tumor microenvironment, abnormal expression of resistance-related proteins, and regulation of resistance-related genes seriously restricts the effectiveness of chemotherapy. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a nano drug delivery system.

[0005] The present invention also provides a method for preparing the nano drug delivery system.

[0006] The present invention also proposes the application of the nano drug delivery system.

[0007] According to one aspect of the present invention, a nano drug delivery system is provided, wherein the nano drug delivery system comprises chlorin e6 and theophylline derivative d17; the structure of theophylline derivative d17 is shown in formula (I):

[0008]

[0009]

[0010] In some embodiments of the present invention, the particle size of the nano drug delivery system is 80-100 nm.

[0011] In some embodiments of the present invention, the mass addition ratio of the dihydrochlorin e6 and theophylline derivative d17 is (1-2): (1-4).

[0012] According to a second aspect of the present invention, a method for preparing the above-mentioned nano drug delivery system is proposed, the method comprising the following steps: ultrasonically treating dihydrochlorin e6 and theophylline derivative d17, and then magnetically stirring them to obtain the obtained system.

[0013] In some embodiments of the present invention, the concentration of the dihydrochlorin e6 is 8-12 mg / ml.

[0014] In some embodiments of the present invention, DMSO is selected as the solvent for dissolving chlorin e6.

[0015] In some embodiments of the present invention, the concentration of theophylline derivative d17 is 8-12 mg / ml.

[0016] In some embodiments of the present invention, DMSO is selected as the solvent for dissolving theophylline derivative d17.

[0017] In some embodiments of the present invention, the ultrasonic time is 40-80 min, and the ultrasonic temperature is 15-25°C.

[0018] In some embodiments of the present invention, the frequency of the ultrasound is 40KHz and the power is 300W.

[0019] In some embodiments of the present invention, the method further comprises the step of filtering the product obtained by magnetic stirring.

[0020] In some embodiments of the present invention, the filtration is performed by ultrafiltration.

[0021] In some embodiments of the present invention, the particle size of the ultrafiltration membrane used in the ultrafiltration is 80-120KD.

[0022] In some embodiments of the present invention, the ultrafiltration centrifugation speed is 3000-5000 rpm and the time is 6-15 min.

[0023] In the third aspect of the present invention, an application of the nano drug delivery system is proposed, wherein the application is application in the preparation of anti-tumor drugs.

[0024] In some embodiments of the present invention, the tumor is lung cancer.

[0025] In some embodiments of the present invention, the method of using the anti-tumor nano drug delivery system is as follows: the nano drug delivery system is introduced into the body, and when the enrichment of the nano drug delivery system at the tumor site reaches a maximum, the tumor site is irradiated with laser.

[0026] In some embodiments of the present invention, the wavelength of the laser is 630-680 nm, and the irradiation time is 0.5-30 min.

[0027] In some embodiments of the present invention, the laser light is infrared light.

[0028] In some embodiments of the present invention, the introduction method is tail vein injection.

[0029] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the nano drug delivery system prepared by the scheme of the present invention synthesizes and encapsulates two drugs, dihydrochlorin e6 and theophylline derivative d17, with different mechanisms of action in a nanoparticle, and uses chemotherapy and photodynamic therapy to treat tumors in a dual-pathway combination. The two drugs can be released in the tumor microenvironment and in a specific near-infrared light irradiation, and the target of action can be adjusted, the time can be controlled, and the drug can be concentrated at the tumor cells, effectively reducing the toxic and side effects of the photosensitizer, improving solubility, and reducing adverse reactions of the body, providing new ideas and theoretical basis for the development of clinical anti-tumor drugs. At the same time, the near-infrared light used is harmless to the body, and the release of the photosensitizer Ce6 can be controlled by adjusting the action site and action time of the near-infrared light from the outside world, generating ROS to kill tumor cells or tissues. The targeting of the photosensitizer Ce6 is increased, the solubility and bioavailability of the chemotherapy drug d17 are improved, and the toxic and side effects of the chemotherapy drug d17 on normal cells or tissues are reduced. The nano drug delivery system prepared by the scheme of the present invention is prepared by self-assembly of dihydrochlorin e6 and d17, has a simple preparation method, low cost and significant effect, and provides a new scheme for preparing tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0031] Figure 1 This is a graph showing the particle size distribution results of the self-assembled nano drug delivery system with different concentrations in the test examples of the present invention;

[0032] Figure 2 is the ultraviolet spectrum of the nano drug delivery system in the test example of the present invention;

[0033] Figure 3 This is a graph showing the killing effect test results of non-small cell lung cancer cells H460 in the test example of the present invention;

[0034] Figure 4 This is a graph showing the killing effect test results of non-small cell lung cancer cell A549 in the test example of the present invention;

[0035] Figure 5 The graphs are the results of the effect of the nano drug delivery system on tumors in the test examples of the present invention, wherein A is a graph showing the weight change of mice, B is a graph showing the volume change of mouse tumors, C is a graph showing the mass change of tumors, and D is a graph showing the mass change of tumors after quantification; E is a graph showing the results of H&E staining;

[0036] Figure 6 These are the H&E staining results of the main organs (heart, liver, spleen, lung, and kidney) of different groups of tumor-bearing nude mice after treatment. DETAILED DESCRIPTION

[0037] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0038] Example 1 A nano drug delivery system

[0039] This example prepares a nano drug delivery system, and the specific preparation process is as follows:

[0040] (1) Solution preparation: Chlorin e6 (Ce6) was dissolved in DMSO to prepare a stock solution with a concentration of 10 mg / ml; compound d17 was dissolved in DMSO to prepare a stock solution with a concentration of 10 mg / ml.

[0041] (2) Synthesis of nanomedicine: The self-assembled nanomedicine was synthesized by ultrasonic precipitation method. The prepared dihydrochlorin e6 and d17 were added into a 5 mL glass bottle in the ratio of d17 to dihydrochlorin e6 (2:1) (i.e., the added amount was 100 μL:50 μL). The mixture was placed in a 1.5 mL EP tube and ultrasonicated at 20°C for 60 min.

[0042] (3) Transfer the sonicated solution to a 2 mL glass volumetric flask, add ultrapure water to make the volume 1 mL, and stir with a magnetic stirrer for 30 min.

[0043] (4) After stirring, the mixture was transferred to a 100KD ultrafiltration tube and centrifuged at 4200 rpm for 15 min to obtain d17@Ce6 self-assembled nanoparticles.

[0044] Example 2

[0045] This example prepares a nano drug delivery system, which is different from Example 1 only in that the ratio of d17 to dihydrochlorin e6 is 1:2 (ie, the added amount is 50 μL:100 μL).

[0046] Example 3

[0047] This example prepares a nano drug delivery system, which is different from Example 1 only in that the ratio of d17 to dihydrochlorin e6 is 1:1 (ie, the added amount is 50 μL:50 μL).

[0048] Example 4

[0049] This example prepares a nano delivery system, which is different from Example 1 only in that the ratio of d17 to dihydrochlorin e6 is 3:1 (ie, the added amount is 150 μL:50 μL).

[0050] Example 5

[0051] This example prepares a nano drug delivery system, which is different from Example 1 only in that the ratio of d17 to dihydrochlorin e6 is 4:1 (ie, the added amount is 200 μL:50 μL).

[0052] Test example

[0053] This test example tests the performance of the nano drug delivery system prepared in Examples 1-5.

[0054] 1. Physical and chemical determination

[0055] (1) Particle size detection

[0056] The morphology of the nano drug delivery system prepared in Examples 1-5 was detected using a transmission electron microscope, and the specific steps were as follows:

[0057] 1) 100 μL of the prepared nano drug delivery system solution prepared in Example 1-5 was aspirated and dropped onto a copper grid for transmission electron microscopy.

[0058] 2) Place the copper mesh after the sample is dropped in a drying oven to dry.

[0059] 3) After the nano drug delivery system solution on the copper mesh is completely dried, it is observed and photographed using a transmission electron microscope.

[0060] (2) Spectral detection

[0061] The ultraviolet-visible light spectrum of the nano drug delivery system prepared in Example 1 was detected by ultraviolet-visible spectrophotometry (UV-Vis).

[0062] 1) Pipette 100 μL of the prepared d17-ce6 NPs solution into 1 mL of deionized water, sodium chloride solution, DMSO and 0.2% SDS solution respectively.

[0063] 2) Transfer the test solution prepared in step (1) to the test dish of the UV spectrophotometer.

[0064] 3) Set the spectrophotometer scanning wavelength range to 200-900nm, adjust to zero, and click Start.

[0065] (3) Particle size detection

[0066] The particle size range of the nano drug delivery system prepared in Example 1 was characterized using a state light scattering instrument.

[0067] Test results such as Figure 1-2 As shown in the figure, it can be seen that only when the ratio of d17 to dihydrochlorin e6 is 2:1, can it self-assemble in aqueous solution to form core-shell nanoparticles. The nanoparticles are spherical, regular in shape, and evenly distributed. The average particle size of the nanoparticles characterized by a dynamic light scattering instrument is 89.33±0.58nm. The drug loading rate of compound d17 is 72.97%, and the encapsulation rate is 71.41%; the drug loading rate of Ce6 is 27.03%, and the encapsulation rate is 52.89%.

[0068] 2. Determination of anti-tumor effect

[0069] (1) H460 (purchased from ATCC) and A549 non-small cell lung cancer cells (purchased from ATCC) were cultured in 1640 complete medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin) at a stable 37°C and 5% CO 2 Concentration cell culture incubator.

[0070] (2) After the cells are in good condition after 2-3 generations of culture and the growth density is 80%-90%, H460 and A549 cells are taken out and placed in a cell ultra-clean operation table that has been disinfected by ultraviolet irradiation for 30 minutes. The culture medium supernatant is aspirated, the cells are washed twice with PBS, and an appropriate amount of trypsin is added and placed in a cell culture incubator for digestion for 2-3 minutes. When the cells become a quicksand, two times the volume of trypsin is added to terminate the digestion with complete culture medium. The cell suspension is then transferred to a 15 mL centrifuge tube and centrifuged at 1000 rpm in a flat-angle centrifuge at room temperature for 3 minutes. The supernatant is discarded, the cell pellet is resuspended with complete culture medium, and the cell pellet is plated in a 96-well plate at a density of 2000-4000 viable cells per 100 μL of complete culture medium.

[0071] (3) Theophylline derivative d17 solutions with a concentration gradient of 0, 0.0625, 0.625, 1.25, 2.5, 5, 10 and 20 μM were prepared using 1640 complete medium; Ce6 solutions with a concentration gradient of 0, 0.03125, 0.0625, 0.625, 1.25, 2.5, 5 and 10 μM were prepared using 1640 complete medium; d17-Ce6 NPs solutions containing equal amounts of d17 and Ce6 in the above concentration gradients were prepared using 1640 complete medium. H460 and A549 cells were cultured overnight in a 96-well plate until the cells were completely attached and the cell density was appropriate. The supernatant was discarded and 100 μL of theophylline derivatives d17, Ce6 and d17-Ce6 NPs solutions of different concentrations were added for co-incubation (respectively, d17 group, Ce6 group, Ce6 + light group, d17@Ce6 nanoparticle group and d17@Ce6 nanoparticle group (nanodrug delivery system prepared in Example 1) + light group), and 4 replicate wells were set for each group and cultured in a cell culture incubator.

[0072] (4) After H460 and A549 cells were co-cultured with different concentrations of theophylline derivatives d17, Ce6 and d17-Ce6 NPs for 72 h (the illumination group was treated with 650 nm near-infrared light for 30 s), complete culture medium containing 10% CCK-8 solution was prepared in advance, the cell supernatant of the 96-well plate was aspirated, and 100 μL of complete culture medium containing 10% CCK-8 solution was added to each well. Another group of 10% CCK-8 complete culture medium without cells and theophylline derivatives was added as a blank control (Ab). After incubation for 0.5 h, the absorbance of each well at 450 nm was detected using a multifunctional microplate reader to calculate the cell activity.

[0073] The results are as follows Figure 3-4 As shown in the figure, it can be seen that the survival rate of tumor cells gradually decreases with the increase of the treatment concentration of d17-Ce6 NPs solution. At the same time, the d17-Ce6 NPs solution + light irradiation group shows better tumor cell killing effect, indicating that nanomedicines can achieve better tumor cell killing effect under light.

[0074] 3. Animal experiments

[0075] (1) Enrichment of Nano-drug Delivery Systems

[0076] (1) Subcutaneous tumor formation: Human lung cancer A549 cells in the logarithmic growth phase were digested with 0.25% trypsin, collected by centrifugation, resuspended in PBS, and plated at 2×10 6 The density of cells / mouse was inoculated subcutaneously in nude mice;

[0077] (2) Random grouping: When the subcutaneous tumor grows to about 80 mm 3At d17, the cells were randomly divided into 4 groups: control group, d17 group, d17-Ce6NPs+light-irradiation group, d17 and Ce6 combined administration+light-irradiation group;

[0078] (3) Preparation of drugs: Dissolve compound d17 with DMSO and then dilute it with sterile water to a final concentration of 666 μg / mL; Dissolve photosensitizer Ce6 with DMSO and then dilute it with sterile water to a final concentration of 333 μg / mL; Dilute d17-Ce6 NPs with sterile water to a concentration of 666 μg / mL and 333 μg / mL for d17 and Ce6, respectively; Prepare a mixed solution of compound d17 and Ce6 with sterile water, wherein the concentrations of compound d17 and Ce6 are 666 μg / mL and 333 μg / mL, respectively;

[0079] (4) Tail vein injection and illumination: The drug prepared in step (3) was administered to each nude mouse at 200 μL according to the group. The control group was given 200 μL PBS solution. Four hours after administration, the subcutaneous tumor sites of all nude mice in the d17-Ce6 nanoparticles + illumination group and the d17 / Ce6 combined administration (d17 and Ce6 concentrations were 666 μg / mL and 333 μg / mL, respectively) + illumination group were illuminated with a strong flashlight for 20 min.

[0080] (5) Tumor measurement and body weight: Starting from the first day of tail vein injection, the mice were weighed every two days using an electronic scale and the length and width of the subcutaneous tumor were measured using a vernier caliper to calculate the tumor volume. The formula is: Tumor volume (mm 3 ) = length × width × width / 2;

[0081] (6) Dissection: When the diameter of the subcutaneous tumor of the nude mouse exceeds 15 mm, the nude mouse is killed by cervical dislocation, the tumor is dissected, weighed and photographed, and the heart, liver, spleen, lung, kidney and tumor tissue sections of the nude mouse are taken for H&E staining. The specific staining steps are as follows:

[0082] (1) Fixation: The tumor tissues and major organs (heart, liver, spleen, lung, and kidney) of each group were immersed in 10-fold volume of 4% paraformaldehyde and fixed for 48-72 h;

[0083] (2) Dehydration: After removal, place the tissue in an embedding box marked with a pencil and perform programmed dehydration in a fully enclosed biological tissue dehydrator;

[0084] (3) Embedding: Embed the dehydrated tissue with 70°C liquid paraffin;

[0085] (4) Sectioning: The embedded wax block is sectioned with a paraffin tissue slicer (the slice thickness is 3-5 μm), and then the slices are spread, mounted, and baked;

[0086] (5) Dewaxing: Place the sections in xylene for dewaxing, then switch to fresh xylene and repeat twice, leaving them in the solution for 10 min each time.

[0087] (6) Hydration: Soak the dewaxed sections in 100%, 95%, and 75% anhydrous ethanol for 5 min, and finally in distilled water;

[0088] (7) Staining: First stain with hematoxylin solution for 2 min, wash off the excess stain with distilled water, then stain with eosin solution for 1 min, and rinse slowly with water for 2 s;

[0089] (8) Sealing: Add a small amount of xylene to make it transparent, seal the coverslip with neutral gum, and observe and photograph under an inverted microscope.

[0090] The experimental results are as follows Figure 5-6 As shown, from Figure 5 As can be seen from A, there was no significant change in the body weight of nude mice in the compound d17 group; although the body weight of mice in the d17-Ce6 NPs + light group showed a continuous downward trend on the 2nd, 4th and 6th days after treatment, it gradually returned to the pre-treatment level from the 10th to the 14th day; Figure 5 As can be seen from Figure B, the growth rate of the tumor volume of mice in the nano drug delivery system prepared in Example 1 + light irradiation group was significantly lower than that in the other groups; Figure 5 It can be seen from Figure C that the tumors of mice in the nano drug delivery system prepared in Example 1 + light irradiation group were significantly smaller than those in other groups; Figure 5 It can be seen from D that the growth rate of the tumor volume of mice in the nano drug delivery system prepared in Example 1 + light irradiation group is significantly lower than that in other groups; Figure 5 As can be seen from Figure E, the tumor cells in the d17-Ce6 NPs+light irradiation group were damaged, with irregular morphology and shrunken nuclei, while the tumor cells in the other groups showed no obvious damage and no other inflammatory and immune responses occurred.

[0091] from Figure 6 As can be seen from A in the figure, no obvious cell damage and morphological changes were observed among the groups; Figure 6 It can be seen from the BE in that there was no significant difference in AST, ALT, CRE, and BUN levels in the d17-Ce6 NPs+light group compared with the control group (P>0.05). This indicates that d17-Ce6 NPs have no obvious liver and kidney toxicity. The results of the in vivo safety experiment show that theophylline derivative d17-Ce6 NPs is relatively safe and does not produce any immune response or induce inflammatory response.

[0092] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A nano drug delivery system, characterized in that: The nano drug delivery system comprises dihydrochlorin e6 and theophylline derivative d17; the structure of theophylline derivative d17 is shown in formula (I): ； （I） The mass addition ratio of the chlorin e6 and the theophylline derivative d17 is (1-2): (1-4); The preparation method of the nano drug delivery system comprises the following steps: subjecting dihydrochlorin e6 and theophylline derivative d17 to ultrasonic treatment and then subjecting them to magnetic stirring to obtain the drug.

2. The nano drug delivery system according to claim 1, characterized in that The particle size of the nano drug delivery system is 80-100 nm.

3. A method for preparing a nano drug delivery system according to any one of claims 1 to 2, characterized in that: The method comprises the following steps: subjecting the dihydrochlorin e6 and theophylline derivative d17 to ultrasonic treatment and then subjecting them to magnetic stirring to obtain the product.

4. The method according to claim 3, characterized in that The ultrasonic time is 40-80 minutes, and the temperature is 15-25°C.

5. The method according to claim 3, characterized in that: The method further comprises the step of filtering the product obtained by magnetic stirring.

6. The method according to claim 5, characterized in that The filtration is carried out by ultrafiltration, and the particle size of the ultrafiltration membrane used in the ultrafiltration is 80-120KD.

7. The method according to claim 5, characterized in that The filtration method adopts ultrafiltration centrifugation, the rotation speed of the centrifugation is 3000-5000rpm, and the time is 6-15min.

8. Use of the nano drug delivery system according to any one of claims 1 to 2 in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that: The tumor includes lung cancer.

10. The use according to claim 8, characterized in that: The method for using the nano drug delivery system is as follows: the nano drug delivery system is introduced into the body, and when the enrichment amount of the nano drug delivery system at the tumor site reaches the maximum, the tumor site is irradiated with laser.

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