Y-type polyethylene glycol conjugate nanoparticle as well as synthesis method and application thereof

Abstract

The invention discloses a Y-type polyethylene glycol conjugate nanoparticle as well as a synthesis method and application thereof. The synthesis method of the Y-type polyethylene glycol conjugate nanoparticle comprises the following steps: combining Y-type polyethylene glycol with adriamycin to synthesize a Y-type PEG-DOX conjugate; and dissolving the Y-type PEG-DOX conjugate in a PBS (Phosphate Buffer Solution), and carrying out self-assembly under ultrasonic oscillation to form the Y-type polyethylene glycol conjugate nanoparticles. The application mainly refers to application of the Y-type polyethylene glycol conjugate nanoparticle in preparation of drugs for treating cancers, especially application of the Y-type polyethylene glycol conjugate nanoparticle in preparation of drugs for treating pancreatic cancer. The nanoparticles synthesized by the method can improve the water solubility of DOX, improve the in-vivo and in-vitro stability of drugs, improve the enrichment of DOX at tumor sites and reduce the toxicity of the whole body, thereby improving the treatment effect on solid tumors.

Description

Technical Field

[0001] The present application belongs to the field of medical technology, and in particular relates to a Y-shaped polyethylene glycol conjugate nanoparticle and a synthesis method and application thereof. Background Art

[0002] Pancreatic cancer is a highly malignant tumor with a low survival rate and difficult treatment. Since pancreatic cancer is often discovered in the late stage, treatment options are limited, and the effects of conventional treatments such as surgery, radiotherapy, and chemotherapy are usually unsatisfactory. Therefore, the development of new treatment strategies is urgently needed. Doxorubicin (DOX), as a highly effective chemotherapy drug, is widely used due to its strong anti-tumor activity. However, the systemic toxicity of DOX, especially its cardiotoxicity, severely limits its clinical use. In addition, DOX has poor water solubility and is widely distributed in the body, requiring a cosolvent to improve its solubility, but a cosolvent may induce other adverse reactions.

[0003] This application aims to solve the application limitations of DOX in tumor treatment by developing a Y-shaped polyethylene glycol conjugate nanoparticle to improve the water solubility of DOX, improve the in vivo and in vitro stability of the drug, increase the enrichment of DOX at the tumor site and reduce systemic toxicity, thereby improving the therapeutic effect on solid tumors. Summary of the invention

[0004] Based on the first aspect of the present application, a Y-shaped polyethylene glycol conjugate nanoparticle is provided, which has the following structural formula:

[0005]

[0006] Based on the second aspect of the present application, a method for synthesizing the above-mentioned Y-shaped polyethylene glycol conjugate nanoparticles is provided, comprising:

[0007] Y-shaped polyethylene glycol (Y-shaped PEG-ALD) was combined with doxorubicin to synthesize Y-shaped PEG-DOX conjugate;

[0008] The Y-type PEG-DOX conjugate is dissolved in a PBS solution, and the Y-type polyethylene glycol conjugate nanoparticles (Y-type PEG-DOX NPs) are formed by self-assembly through ultrasonic oscillation.

[0009] In at least one embodiment, the method of combining Y-shaped polyethylene glycol with doxorubicin comprises:

[0010] Doxorubicin was dissolved in dimethyl sulfoxide (DMSO), and sodium cyanoborohydride was added to obtain solution 1;

[0011] Dissolve Y-type polyethylene glycol in dimethyl sulfoxide to obtain solution 2;

[0012] The solution 2 is added to the solution 1, stirred in the dark at 38-42° C. for 12-36 h, dialyzed and dried to obtain the Y-type PEG-DOX conjugate.

[0013] Preferably, the temperature of the light-proof stirring is 40° C., and the stirring time is 24 hours. The dialysis is performed using a dialysis bag with a molecular weight of 1000, and the dialysis time is 2 days. The drying is rotary evaporation drying.

[0014] In at least one embodiment, the conditions of the ultrasonic oscillation are: power of 20 to 30 kW, cyclic ultrasonic operation, each ultrasonic time is 10 to 40 seconds, each interval is 1 to 10 seconds, and the total ultrasonic treatment time is 2 to 20 minutes.

[0015] Preferably, the ultrasonic oscillation conditions are: power 25 kW, ultrasonic operation is performed cyclically, each ultrasonic time is 30 s, each interval is 5 s, and the total ultrasonic treatment time is 5 min.

[0016] In at least one embodiment, the pH of the PBS solution is 7.4.

[0017] Based on the third aspect of the present application, there is provided the use of the above-mentioned Y-shaped polyethylene glycol conjugate nanoparticles in the preparation of drugs for treating cancer.

[0018] Specifically, the cancer is a solid tumor, such as pancreatic cancer. The drug at least comprises the Y-shaped polyethylene glycol conjugate nanoparticles, and may further comprise pharmaceutically acceptable excipients.

[0019] Compared with the prior art, this application can achieve the following beneficial effects:

[0020] 1. The polyethylene glycol-doxorubicin conjugate synthesized by chemical coupling uses Y-shaped polyethylene glycol to couple DOX and is prepared into nanoparticles by self-assembly technology, which has good water solubility and stability.

[0021] 2. The application of nanoparticles in anti-cancer drugs, especially anti-pancreatic cancer drugs, can increase the drug enrichment in tumor tissues by enhancing the permeability retention effect (EPR effect), and can effectively release doxorubicin in pancreatic cancer cells, showing excellent anti-tumor activity, significantly slowing down the growth of tumor volume, and reducing toxicity to normal tissues and systemic toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Chemical structure formula of Y-type polyethylene glycol conjugate nanoparticles.

[0023] Figure 2 : NMR spectrum of PEG-DOX conjugate.

[0024] Figure 3 : DLS detection results of particle size.

[0025] Figure 4 : Fluorescence microscopy observation results.

[0026] Figure 5 :The content of DOX uptake by cells in different time periods was detected by ELISA (excitation light).

[0027] Figure 6 : Cell apoptosis rate.

[0028] Figure 7 : Distribution of DOX and PEG-DOX in tumors and important organs of tumor-bearing mice.

[0029] Figure 8 : Tumor inhibitory effect.

[0030] Fig. 9 : Changes of tumor volume and mouse body weight. DETAILED DESCRIPTION

[0031] The present application will be described in detail below with reference to the exemplary embodiments or experimental examples in the accompanying drawings. However, it should be understood that the present application can be implemented in a variety of different forms and should not be construed as being limited to the embodiments described herein. These embodiments are provided here to make the disclosure of the present application more complete and to fully convey the concept of the present application to those skilled in the art.

[0032] Example 1. Synthesis of Y-type PEG-DOX conjugates.

[0033] In order to synthesize the Y-type PEG-DOX conjugate, 50 mg of DOX was first dissolved in an appropriate amount of DMSO, and 20 mg of sodium cyanoborohydride (NaBH3CN) was added; the reducing agent can provide milder reducing conditions, thereby promoting the stable coupling between DOX and Y-type PEG-ALD; then 100 mg of Y-type PEG-ALD (mw = 2000) was weighed and dissolved in DMSO, and the Y-type PEG-ALD solution was slowly added to the DOX solution. Stir at 40°C in the dark for 24 hours to ensure that the reaction is fully carried out. After the reaction is completed, stop stirring and dialyze the reaction system using a dialysis bag with a molecular weight of 1000 for 2 days. After the dialysis is completed, the final product, Y-type PEG-DOX conjugate (82 mg), is obtained by rotary evaporation and drying, and its chemical structure is as shown Figure 1 The structure of the product is shown in 1 H-NMR was used to verify the results. Figure 2 As shown, through 1The structure of the synthesized PEG-DOX conjugate was effectively verified by detailed analysis of the H-NMR spectrum. First, the aromatic ring and olefin protons of the DOX molecule showed characteristic peaks in the chemical shift range of 7-8ppm, which was consistent with the spectrum of the standard DOX. Figure 1 The results showed that DOX maintained its core structural characteristics in the conjugate. At the same time, the proton characteristic peak of the -CH2-CH2- repeating unit of the PEG segment was also clearly presented near 3.5ppm, indicating that the PEG structure was successfully introduced into the conjugate. After 2 days, the dialysate was detected by an ELISA instrument (Ex / Em=480nm / 520nm) and no fluorescence absorption was found, indicating that there was no free DOX in the product. In summary, 1 The H-NMR spectrum results verified the coupling structure of PEG and DOX.

[0034] Example 2: Self-assembly of Y-shaped PEG-DOX NPs

[0035] The Y-type PEG-DOX conjugate synthesized in Example 1 was dissolved in an appropriate amount of PBS (pH 7.4) solution, and gradually self-assembled to form Y-type PEG-DOX NPs by ultrasonic oscillation. The conditions of ultrasonic oscillation were: power 25 kW, cyclic ultrasonic operation, each ultrasonic time was 30 s, each interval was 5 s, and the total ultrasonic treatment time was 5 min. The particle size of the nanoparticles was characterized by dynamic light scattering (DLS), which was monitored once a week. The particle size results are shown in Figure 3 As shown. The results of three different treatment times showed that the self-assembly process of Y-type PEG-DOX NPs was fast and stable, and the particle size of the obtained nanoparticles was maintained at about 10nm, with a relatively uniform distribution. More importantly, the particle size characterization results for three consecutive weeks were consistent, all maintained at about 10nm, indicating that the nanoparticle system has good size stability. This stable and small-sized nanoparticle is extremely advantageous in drug delivery systems, and can utilize the enhanced permeation and retention effect (EPR effect) to accumulate more efficiently in tumor tissues, thereby further improving the therapeutic effect.

[0036] Example 3: Drug release and cellular uptake

[0037] DOX and the PEG-DOX NPs prepared in Example 2 were used to perform drug release experiments and cell uptake experiments respectively according to conventional methods. In the drug release experiment, the Y-shaped PEG-DOX NPs showed a longer DOX release behavior.

[0038] However, in the cell uptake experiment, Y-shaped PEG-DOX NPs were able to enter pancreatic cancer cells more efficiently and effectively. Figure 4 The fluorescence microscopy results Figure 5 The results of DOX uptake by cells at different time periods were detected by an ELISA instrument. Figure 5 The results show that the uptake rate of PEG-DOX NPs is greater than that of DOX, whether it is 2 hours or 4 hours. Figure 4 It was further shown that after 2 hours of incubation, the Y-type PEG-DOX NPs were mainly distributed in the cell nucleus, while DOX was distributed in the cytoplasm. As the incubation time increased (4 hours), DOX began to enter the cell nucleus, and the Y-type PEG-DOX NPs basically completely entered the cell nucleus, showing strong cellular uptake performance.

[0039] Example 4: Cell proliferation inhibition experiment

[0040] DOX and PEG-DOX NPs prepared in Example 2 were taken and the cytotoxicity of PEG-DOX NPs was evaluated using the conventional CCK-8 method (cell counting kit-8). The results are as follows: Figure 6 As shown. The results showed that both PEG-DOX NPs and DOX exhibited concentration-dependent inhibitory effects on pancreatic cancer cell proliferation. Although the initial cell uptake rate of PEG-DOX NPs was slower than that of DOX, as time went on, the concentration of DOX accumulated in pancreatic cancer cells by PEG-DOX NPs was higher, thereby enhancing the inhibitory effect on cell proliferation.

[0041] Example 5: In vivo tumor enrichment effect

[0042] In order to evaluate the enrichment effect of PEG-DOX NPs in living tumors, an animal experiment was conducted using a pancreatic cancer tumor-bearing mouse model. The tumor-bearing mice were divided into two groups, the DOX group and the PEG-DOX NPs group. Each group of mice was injected with drugs through the tail vein, and the important tissues and tumors of the animals were removed 2 hours later for in vivo imaging to observe the distribution of DOX. The results are shown in Figure 2. Figure 7 The results showed that the enrichment degree of PEG-DOX NPs group in tumor tissue was significantly higher than that of DOX group.

[0043] Example 6: In vivo tumor inhibition effect

[0044] In order to evaluate the anti-tumor effect of PEG-DOX NPs in vivo, an animal experiment was conducted using a pancreatic cancer tumor-bearing mouse model. The tumor-bearing mice were divided into three groups: saline group, DOX group, and PEG-DOX NPs group. Each group of mice was injected with drugs through the tail vein, and the tumor volume and body weight changes were measured regularly. The results are shown in Figure 2. Figure 8 and Fig. 9 shown.

[0045] The results showed that the tumor volume of mice in the PEG-DOX NPs group increased significantly slower than that in the saline group and the DOX group, indicating that PEG-DOX NPs had a more excellent effect in inhibiting tumor growth in vivo. In addition, the weight loss of mice in the PEG-DOX NPs group was significantly less than that in the DOX group, indicating that PEG-DOX NPs reduced the systemic toxicity of DOX and had better safety.

[0046] The above experimental results show that the Y-shaped polyethylene glycol conjugate nanoparticles of the present application exhibit excellent drug release and anti-tumor effects both in vivo and in vitro, have good targeting and controllability, and are expected to become an efficient and low-toxic tumor treatment method. They have the potential for actual clinical application and are expected to provide new treatment options for pancreatic cancer patients.

Claims

1. A Y-shaped polyethylene glycol conjugate nanoparticle, characterized in that: It has the following chemical structure:

2. The method for synthesizing Y-shaped polyethylene glycol conjugate nanoparticles according to claim 1, characterized in that: include: Y-shaped polyethylene glycol was combined with doxorubicin to synthesize Y-shaped PEG-DOX conjugate; The Y-shaped PEG-DOX conjugate is dissolved in a PBS solution, and the Y-shaped polyethylene glycol conjugate nanoparticles are formed by self-assembly through ultrasonic oscillation.

3. The synthesis method according to claim 2, characterized in that Methods for conjugating Y-shaped polyethylene glycol to doxorubicin include: Doxorubicin was dissolved in dimethyl sulfoxide, and sodium cyanoborohydride was added to obtain solution 1; Dissolve Y-type polyethylene glycol in dimethyl sulfoxide to obtain solution 2; The solution 2 is added to the solution 1, stirred in the dark at 38-42° C. for 12-36 h, dialyzed and dried to obtain the Y-type PEG-DOX conjugate.

4. The synthesis method according to claim 2, characterized in that The conditions of the ultrasonic oscillation are: The power is 20-30 kW, and the ultrasonic operation is performed cyclically. The ultrasonic time for each time is 10-40 seconds, and the interval between each time is 1-10 seconds. The total ultrasonic treatment time is 2-20 minutes.

5. The synthesis method according to claim 3, characterized in that: The dialysis was performed using a dialysis bag with a molecular weight of 1000, and the dialysis time was 2 days.

6. The synthesis method according to claim 3, characterized in that: The drying is rotary evaporation drying.

7. The synthesis method according to claim 1, characterized in that: The pH of the PBS solution is 7.

4.

8. Use of the Y-shaped polyethylene glycol conjugate nanoparticles according to claim 1 or the Y-shaped polyethylene glycol conjugate nanoparticles prepared by the synthesis method according to any one of claims 2 to 7 in the preparation of drugs for treating cancer.

9. The use according to claim 8, characterized in that The cancer is pancreatic cancer.

10. The use according to claim 8, characterized in that The medicine comprises the Y-shaped polyethylene glycol conjugate nanoparticles and pharmaceutically acceptable excipients.

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