CN116999575A - Preparation of mitoxantrone nano-drug and application of mitoxantrone nano-drug in pancreatic cancer targeted therapy - Google Patents

Preparation of mitoxantrone nano-drug and application of mitoxantrone nano-drug in pancreatic cancer targeted therapy Download PDF

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CN116999575A
CN116999575A CN202310944361.0A CN202310944361A CN116999575A CN 116999575 A CN116999575 A CN 116999575A CN 202310944361 A CN202310944361 A CN 202310944361A CN 116999575 A CN116999575 A CN 116999575A
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mitoxantrone
drug
nano
hyaluronic acid
mito
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施裕新
刘峰君
陈天佑
徐智阳
施楠楠
刘晓玉
唐一丹
周粟
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SHANGHAI PUBLIC HEALTH CLINICAL CENTER
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Abstract

The invention relates to preparation of mitoxantrone nano-drugs and application of mitoxantrone nano-drugs in pancreatic cancer targeted therapy, and belongs to the technical field of biological medicines. Adding mitoxantrone hydrochloride solution into sodium hyaluronate solution, dialyzing, and freeze-drying to obtain mitoxantrone-hyaluronic acid nano-drug powder; mitoxantrone and hyaluronic acid are combined to form a mitoxantrone-hyaluronic acid nano-targeting drug. The invention provides an application of mitoxantrone-hyaluronic acid nano-drug in preparing pancreatic cancer therapeutic drug; the mitoxantrone-hyaluronic acid nano-drug has obvious inhibition effect on pancreatic tumors; has the CD44 receptor targeting function, can effectively reduce the toxicity of the medicine and simultaneously enhance the treatment effect on pancreatic cancer tumors.

Description

Preparation of mitoxantrone nano-drug and application of mitoxantrone nano-drug in pancreatic cancer targeted therapy
Technical Field
The invention relates to preparation of mitoxantrone nano-drugs and application of mitoxantrone nano-drugs in pancreatic cancer targeted therapy, and belongs to the technical field of biological medicines.
Background
Pancreatic cancer is one of the most lethal malignant tumors in the digestive system, has high invasiveness and metastasis, and the number of deaths per year exceeds 20 ten thousand. Due to the lack of typical clinical manifestations of early pancreatic cancer, diagnosis is difficult, 80% of patients are already advanced at the time of diagnosis, losing the best opportunity for surgery. Therefore, chemotherapy plays a very important role, but has poor therapeutic effects and serious side effects, and clinical application is greatly limited. In order to improve the current situation, researchers have developed various polymer nanoparticles as drug carriers, the diameters of which are generally about 10-200nm, and utilize various advantages of the polymer nanoparticles, including targeting, stimulus response release and the like, so as to effectively improve the therapeutic effect of tumors. How to improve the survival rate of pancreatic cancer patients is always a difficult problem for domestic and external scholars. Due to unsmooth drainage of pancreatic cancer lymph fluid, nano-drugs with proper size can enter the tumor through small blood vessels and relatively unsmooth cell gaps by utilizing high-permeation long retention and targeting effects, stay in the tumor for a period of days, and are passively and actively enriched to a relatively high concentration. The research of polymer nanoparticles for treating pancreatic cancer is not common at present and is still in the research stage. Hyaluronic Acid (HA) is an acidic mucopolysaccharide, is a main component of extracellular matrix, HAs a structure of a repeating unit formed by D-glucuronic acid and N-acetylglucosamine, HAs good water solubility and biocompatibility, and can be biodegraded. More importantly, the hyaluronic acid also has the targeting property of the CD44 receptor, can be accumulated and accumulated in various malignant tumor tissues over-expressed by the CD44 receptor, and can increase the local drug content, so that the hyaluronic acid is widely studied and applied to the field of nano drug delivery. Mitoxantrone hydrochloride (mitotone HCl, MITO), which inhibits nucleic acid synthesis by binding to DNA molecules, resulting in cell death, is a cell cycle nonspecific drug. On the basis of the prior research work, the invention constructs the MITO@HA nano medicine which is synthesized by taking hyaluronic acid as a targeting medicine carrier and wrapping mitoxantrone and is used for pancreatic cancer anti-tumor treatment (as shown in figure 1).
Disclosure of Invention
The invention aims to solve the technical problems of how to prepare mitoxantrone nano-drugs and how to apply the nano-drugs in pancreatic cancer targeted therapy.
In order to achieve the aim of the invention, the invention provides a preparation method of mitoxantrone nano-drug, which comprises the steps of adding mitoxantrone hydrochloride solution into sodium hyaluronate solution, dialyzing and freeze-drying to obtain mitoxantrone-hyaluronic acid nano-drug powder.
The invention provides a mitoxantrone-hyaluronic acid nano-drug, which comprises mitoxantrone and hyaluronic acid, wherein the mitoxantrone and the hyaluronic acid are combined to form the mitoxantrone-hyaluronic acid nano-targeting drug.
The invention provides an application of mitoxantrone-hyaluronic acid nano-drug in preparing pancreatic cancer therapeutic drug.
Preferably, the dosage form of the medicine comprises tablets, powder, granules, capsules, oral liquid, injection or sustained release agent.
The present invention provides a therapeutic system for treating pancreatic cancer, comprising: a drug delivery system; the drug administration system contains mitoxantrone-hyaluronic acid nano-drugs.
Compared with the prior art, the invention has the following beneficial effects:
in the invention, HA is selected to synthesize hyaluronic acid/mitoxantrone/MITO@HA nano-drug, compared with the traditional chemotherapy drug, the polymer nano-particle loaded antitumor drug HAs the advantages that: (1) targeted therapy; (2) enhance accumulation in tumors. MITO@HA nano-drug can enhance the anti-tumor curative effect and reduce the toxic and side effects by actively targeting CD44 receptor and EPR effect (high permeability and retention effect enhanced permeability and retention effect of solid tumor) on the surface of pancreatic cancer tumor.
Compared with the MITO group, MITO@HA inhibits the G0/G1 phase retardation, increased apoptosis, decreased cell cloning, cell migration and invasion of pancreatic cancer cell cycle. The animal experiment results show that: the MITO@HA nano-drug has obvious inhibition effect on pancreatic tumors. The biological safety of the MITO@HA nano particles is further verified through blood biochemical index detection, so that the damage of a chemotherapeutic drug to animals can be effectively reduced, and the anti-tumor effect of the chemotherapeutic drug can be enhanced. The MITO@HA nano particles with the CD44 receptor targeting effect can effectively reduce toxicity of a drug system, enhance the treatment effect on pancreatic cancer tumors and provide possibility for clinical targeting treatment of pancreatic cancer.
Drawings
FIG. 1 is a schematic diagram of the in vitro and in vivo anti-tumor effect study of MITO@HA.
Abbreviations in the figures: MITO: mitoxantrone; HA: hyaluronic acid.
FIG. 2 is a graph showing experimental results related to the expression of CD44 in pancreatic cancer cells.
FIG. 3 is a graph showing the results of verification experiments on completion of MITO@HA nano-drug preparation.
Fig. 4 is a graph of experimental results related to in vitro study of the MITO@HA nano-drug.
Fig. 5 is a graph of experimental results related to in vivo study of the mito@ha nano-drug.
FIG. 6 is a graph showing experimental results related to in vivo toxicity evaluation of MITO@HA nano-drugs.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments accompanied with the accompanying drawings are described in detail as follows:
the invention provides a preparation method of mitoxantrone nano-drug, which comprises the steps of adding mitoxantrone hydrochloride solution into sodium hyaluronate solution, dialyzing, and freeze-drying to obtain mitoxantrone-hyaluronic acid nano-drug powder.
The invention provides a mitoxantrone-hyaluronic acid nano-drug, which comprises mitoxantrone and hyaluronic acid, wherein the mitoxantrone and the hyaluronic acid are combined to form the mitoxantrone-hyaluronic acid nano-targeting drug.
The invention provides an application of mitoxantrone-hyaluronic acid nano-drug in preparing pancreatic cancer therapeutic drug.
The dosage forms of the medicine comprise tablets, powder, granules, capsules, oral liquid, injection or sustained release agent.
The present invention provides a therapeutic system for treating pancreatic cancer, comprising: a drug delivery system; the drug administration system contains mitoxantrone-hyaluronic acid nano-drugs.
Examples
1. Preparation of MITO@HA nano-drug:
300mg of sodium hyaluronate was weighed out by a balance, dissolved in 50ml of deionized water, and stirred for 10min to allow for adequate dissolution. Then 30mg mitoxantrone hydrochloride is weighed and dissolved in 5ml deionized water, added dropwise to the sodium hyaluronate solution (3-4 drops/min) until dissolution is complete, and stirred overnight. The extra free drug was removed by dialysis (MWCO 3500 Da) for 24 hours, and finally the solution was lyophilized to give a mito@ha powder. All the processes need to be protected from light. Particle size, drug Loading (DLC) and Drug Loading (DLE) of mito@ha were determined by uv spectroscopy and inductively coupled plasma atomic emission spectrometry. DLC and DLE are calculated by the following formula:
DLC (wt.%) = (mass of nanoparticle-loaded drug)/(total drug amount-nanoparticle mass) ×100%
DLE (% by weight) = (mass of nanoparticle loaded drug)/(mass of total drug dosed) ×100%
2. Expression of CD44 in pancreatic cancer cells:
determining the expression level of the PANC-1 pancreatic cancer cells CD44 by a flow cytometer; the results showed (as in panel a of fig. 2) that 99.9% of pancreatic cancer cells significantly overexpressed CD44. Pancreatic cancer cells and tissue immunohistochemistry all showed high expression of CD44. (see fig. 2B and C).
3. Related experiments for MITO@HA nanomedicine application:
3.1 verification experiment of completion of MITO@HA nano-drug preparation:
first, different concentrations (10. Mu.g/ml, 15. Mu.g/ml, 25. Mu.g/ml, 35. Mu.g/ml, 50. Mu.g/ml, 75. Mu.g/ml) were prepared with mitoxantrone hydrochloride standard aqueous solutions, and the ultraviolet absorption spectrum was measured by an ultraviolet spectrophotometer with 610nm as the maximum absorption peak, and the linear relationship between the different concentrations of the drug and the absorbance was determined as a standard curve (see FIG. 3, panels A and B). By measuring the absorbance of MITO@HA at 610nm, the encapsulation rate and drug loading of MITO@HA were calculated to be 45.82 + -1.72% and 4.15+ -0.82%, respectively (as shown in FIG. 3, panel C). Indicating that hyaluronic acid and mitoxantrone successfully self-assemble by electrostatic interactions. Provides basis for the subsequent active targeting treatment of pancreatic cancer. Then, morphological characteristics and particle size distribution of the nano-ions were analyzed. The average hydration particle size of MITO@HA measured by a dynamic light scattering nano laser particle size MITO@HA instrument is 51.4+/-2.3 nm, and the particle size in a transmission electron microscope image is consistent with the particle size measured by particle size analysis. Meanwhile, the high-voltage electrostatic coating has higher electrostatic stability on the surface of MITO@HA zeta potential (-26.1+/-3.2 mV) (shown as a D chart in figure 3).
3.2 screening MITO@HA effective concentration:
CCK-8 method pancreatic cancer cells were treated in vitro with MITO and MITO@HA at different concentrations of 1 μg/mL, 2.5 μg/mL, 5 μg/mL, 20 μg/mL, 50 μg/mL, respectively, and the MITO@HA group was found to inhibit proliferation of cells more significantly than the MITO group (see E-panel in FIG. 3). The concentration of μg/mL was converted to μmol/L and seven different concentrations of mito@ha were evaluated for treatment of pancreatic cancer cells at 0, 1, 2 and 3 days by efficacy and toxicity, and the results showed: the most suitable concentration and time for MITO@HA is 0.5. Mu. Mol/L and 2 days (as shown in FIG. 3, panel F).
3.3 in vitro study-related experiments:
pancreatic cancer cell experiments showed that: the most suitable therapeutic concentration and time for MITO@HA is 0.5. Mu. Mol/L and 2 days; compared with the MITO group, MITO@HA has cell cycle G0/G1 phase retardation, increased apoptosis, reduced cell clonality, cell migration and invasion inhibition on pancreatic cancer.
3.3.1 cell cycle experiments:
cell cycle experimental results (as shown in figure 4, panel a) showed that the average percentages of the control, MITO and mito@ha groups were 24.91±1.42%, 39.37±1.27% and 42.67±0.944%, respectively, indicating that the nanoparticles affected the circulating activity of PANC-1 pancreatic cancer cells, resulting in a majority of cells at the G0/G1 phase block (P < 0.0001).
3.3.2 apoptosis experiments:
apoptosis experimental results are shown (as shown in panel B of fig. 4); the average percentages of total apoptosis rate (early apoptosis rate Q4 and late apoptosis rate Q2) for the Control, MITO and mito@ha groups were 3.01±0.12%, 5.30±0.49% and 7.28±0.59%, respectively (P < 0.0001). MITO@HA has the greatest effect on apoptosis of PANC-1 pancreatic cancer cells.
3.3.3 migration and invasion experiments of cells:
results of cell migration and invasion (shown in figure 4, panel C), the average migration and invasion rates of PANC-1 cells in the control group were 280.75±18.51%, consistent with the rapid metastasis characteristics of pancreatic cancer. Mobility of the mito@ha group and the MITO group were significantly lower than that of the control group (P < 0.0001), respectively. The difference between the MITO group and the mito@ha group (p= 0.3539) was not statistically significant. Also, the cell invasion rates of the mito@ha group and the MTO group were significantly reduced compared to the control group. The above results indicate that MITO has a significant inhibitory effect on the tumor growth process. In addition, hyaluronic acid HA can target and recognize CD44 receptor in pancreatic cancer, and HA-coated MITO can also tightly adhere to the CD44 receptor, and target and inhibit migration and invasion of tumor cells.
3.3.4 cell cloning experiments
Cell cloning results are shown (as shown in panel D of fig. 4); the average percentages of cell clones for each of the control, MITO and MITO@HA groups were 100.00.+ -. 3.56%, 47.02.+ -. 9.53% and 45.02.+ -. 7.38%, respectively. The cloning number of PANC-1 pancreatic cancer cells in the MITO@HA group is minimal and is obviously lower than that of the control group and the MITO group. Thus, we believe that hyaluronic acid HA targets the recognition of CD44 receptor in pancreatic cancer and plays the most important role in the killing of pancreatic cancer cells. In addition, the constructed MITO@HA nano-drug target is concentrated in the plasma of pancreatic cancer cells to release MITO, so that the G0/G1 cell cycle of pancreatic cancer is obviously blocked, the apoptosis of tumor cells is promoted, the cell cloning is obviously reduced, and the cell migration and invasion are inhibited.
3.4. Related experiments for in vivo studies:
animal experiment results show that the MITO@HA nano-drug has remarkable inhibition effect on pancreatic tumors. The tumor growth curve of the pancreatic cancer nude mice (shown as a graph A in fig. 5) shows that the tumor volume of the nude mice in the control group grows fastest, and the tumor growth speed of the MITO@HA group is slower, which indicates that the tumor inhibition effect is remarkable. The tumor mass of each group of nude mice tumors (as shown in panel B of FIG. 5) showed that the average tumor mass of the control group was (2313.22 + -94.61) g and the tumor mass of the MITO@HA group was (1480.64 + -242.95) g; (p=0.0052).
To further verify the anti-tumor effect of targeting tumors, we performed fixation, sectioning, HE staining of tumor tissue (as shown in figure 5, panel C). The results showed that compared to the control, the MITO@HA group showed significantly large area of liquidized necrosis, whereas the control did not show significant tumor necrosis. The results show that the MITO@HA group has obvious inhibition effect on tumors and good tumor treatment effect. The reason for this is that MITO@HA allows more drug to reach the tumor tissue by EPR action (high permeability and retention effect enhanced permeability and retention effect of solid tumors), while HA fuses with the cell membrane, engulfs lipids into the cells and increases uptake by tumor cells. In vivo anti-tumor experiments further prove that the surface modified HA can further promote the accumulation and uptake of MITO@HA at tumor sites through passive and active targeting, so that the effect of inhibiting tumors is better exerted.
3.5. In vivo toxicity assessment experiment
During the treatment, the biochemical index results of each group of nude mice are shown (as shown in figure 6), and important parameters such as leucocytes, erythrocytes, PLT, ALT, AST, TBIL, ALB, CK, CK, CK-MB, LDH, LDB1, urs, creatinine and UA; the MITO@HA groups all showed normal, indicating that the MITO@HA groups had good security. The biological safety of the MITO@HA nano particles is further verified through the detection of blood biochemical indexes, so that the damage of the chemotherapeutic drugs to animals can be effectively reduced, and meanwhile, the anti-tumor effect of the chemotherapeutic drugs can be enhanced through the MITO@HA. In conclusion, the MITO@HA nano particles with the CD44 receptor targeting effect can effectively reduce the toxicity of a drug system, improve the treatment effect of pancreatic cancer tumors and provide possibility for clinical targeting treatment of pancreatic cancer.
As shown in fig. 1, the research diagram of the anti-tumor effect of MITO@HA is shown;
as shown in fig. 2, a graph of experimental results related to the expression of CD44 in pancreatic cancer cells is shown. Wherein, a diagram: flow cytometry showed that 99% of pancreatic cancer cells expressed CD44. Panel B and C show high CD44 expression (brown) in pancreatic cancer cells and tissues.
As shown in FIG. 3, a graph of the results of verification experiments on completion of MITO@HA nano-drug preparation is shown. Wherein, the A graph-D graph is the characterization of MITO and MITO@HA. Panel E shows that MITO and MITO@HA alter cell viability at different concentrations. Panel F shows that the difference between the survival rate and the inhibition rate of pancreatic cancer cells at different concentrations and different treatment times of MITO@HA is statistically significant (statistical P value).
As shown in fig. 4, the experimental result diagram related to the in vitro study of the MITO@HA nano-drug is shown; wherein panels A-D are comparisons of cell cycle changes. Under the microscope, PANC-1 apoptosis of the control group, the MTIO@HA group and the MITO group is increased, cell migration and invasion are reduced, and cell cloning is reduced. The difference in cell function of each group was statistically significant (P-value was statistically significant).
As shown in fig. 5, the experimental result diagram related to the in vivo study of the MITO@HA nano-drug is shown; panel A shows that the MITO@HA nanomedicine group slowed down the tumor growth curve of pancreatic cancer animals compared to the control group. Panel B shows that the MITO@HA nano-drug group has a lighter tumor weight after 26 days of tumor growth. HE staining in panel C shows that mito@ha causes massive necrosis of the tumor. The differences between the groups were statistically significant (P-value).
As shown in FIG. 6, the experimental results of the in vivo toxicity evaluation of MITO@HA nano-drug are shown.
Compared with the control group, the MITO@HA group blood is normal, the liver and kidney function index is normal, and the myocardial zymogram is normal.
While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims (5)

1. A preparation method of mitoxantrone nano-drug is characterized in that mitoxantrone hydrochloride solution is added into sodium hyaluronate solution, and mitoxantrone-hyaluronic acid nano-drug powder is obtained through dialysis and freeze-drying.
2. The mitoxantrone-hyaluronic acid nano-drug is characterized by comprising mitoxantrone and hyaluronic acid, wherein the mitoxantrone and the hyaluronic acid are combined to form the mitoxantrone-hyaluronic acid nano-targeting drug.
3. An application of mitoxantrone-hyaluronic acid nano-drug in preparing pancreatic cancer therapeutic drug.
4. The use according to claim 3, wherein the pharmaceutical dosage form comprises a tablet, powder, granule, capsule, oral liquid, injection or sustained release formulation.
5. A therapeutic system for treating pancreatic cancer, comprising: a drug delivery system; the drug administration system contains mitoxantrone-hyaluronic acid nano-drugs.
CN202310944361.0A 2023-07-28 2023-07-28 Preparation of mitoxantrone nano-drug and application of mitoxantrone nano-drug in pancreatic cancer targeted therapy Pending CN116999575A (en)

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