CN111763315B - Polyester compound, nano medicine taking polyester compound as carrier and application of nano medicine - Google Patents
Polyester compound, nano medicine taking polyester compound as carrier and application of nano medicine Download PDFInfo
- Publication number
- CN111763315B CN111763315B CN202010474210.XA CN202010474210A CN111763315B CN 111763315 B CN111763315 B CN 111763315B CN 202010474210 A CN202010474210 A CN 202010474210A CN 111763315 B CN111763315 B CN 111763315B
- Authority
- CN
- China
- Prior art keywords
- drug
- nano
- compound
- osteosarcoma
- polyester
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
- C08G69/44—Polyester-amides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
- A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
- A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/14—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof
- C07C227/18—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof by reactions involving amino or carboxyl groups, e.g. hydrolysis of esters or amides, by formation of halides, salts or esters
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C229/34—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton containing six-membered aromatic rings
- C07C229/36—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton containing six-membered aromatic rings with at least one amino group and one carboxyl group bound to the same carbon atom of the carbon skeleton
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Polymers & Plastics (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a polyester compound, a nano medicine taking the polyester compound as a carrier, and application of the medicine. The nanomaterial 8P4 used in the invention is used for encapsulating the compound Apatinib to prepare the nano-drug (8P 4-Apa), so that the nano-drug can target the tumor part of osteosarcoma, has better drug delivery capacity on osteosarcoma stem cells with poor traditional drug effects, remarkably improves the therapeutic effect of the compound Apatinib to induce apoptosis of osteosarcoma stem cells, has no obvious cytotoxicity under the application concentration, and can reduce the toxic and side effects of Apatinib on normal cells. The nano-drug 8P4-Apa has the advantages of targeting osteosarcoma, small side effect and the like, and has good application prospect and wide development space in the field of osteosarcoma clinical treatment.
Description
Technical Field
The invention belongs to the technical field of biological medicine. More particularly relates to a preparation method of a nano-drug and application thereof in treating osteosarcoma.
Background
Osteosarcoma is a highly malignant bone tumor with a high incidence in teenagers or children under 20 years of age, and current clinical treatments include surgical excision and post-operative combination chemotherapy with various cytotoxic drugs such as doxorubicin, cisplatin, ifosfamide and methotrexate. About 70% of patients can be cured, however, clinical treatment is limited for patients with advanced stages of metastasis and recurrent osteosarcoma.
Angiogenesis is critical for tumor growth, invasion and metastasis, and osteosarcoma tissue promotes angiogenesis by generating and releasing various pro-angiogenic factors, of which vascular endothelial growth factors are a very important class. Currently, the scientific community believes that VEGFR-2 is one of the most predominant receptors in the process of angiogenesis mediated by VEGF-A. The cell signaling pathway mediated by VEGFR-2 has been the subject of choice in anti-angiogenic therapies. Some VEGFR-2 Tyrosine Kinase Inhibitors (TKIs) have been approved by the us FDA for the treatment of clinical osteosarcomas, such as sorafenib He Yiwei limus. Apatinib (Apatinib, apa) is also a class of VEGFR-2 TKIs, with half-inhibitory concentration IC for VEGFR-2 50 Can reach 1nM, which greatly prolongs the overall survival and progression-free survival of patients with refractory advanced gastric cancer. Recently, two reports have shown that Apatinib has demonstrated superior inhibition of progression of inoperable advanced osteosarcoma than chemotherapy in a clinical phase two trial study.
In order to ensure that the tumor site can reach enough drug concentration, the improvement of targeting efficiency in TKI treatment becomes a primary goal. Maintenance of the dosage of Apatinib is critical for the efficacy of patients with advanced osteosarcoma, however, unfortunately, the toxic side effects of Apatinib in clinical applications often lead to down-regulation of the dosage and even drug withdrawal, which contradiction makes suppression of osteosarcoma by Apatinib difficult to achieve. Furthermore, there is growing evidence that insufficient exposure doses of tyrosine kinase inhibitor targets also readily lead to the development of drug resistance, and that the mechanism of drug resistance development is independent of acquired somatic mutation of TKI target genes. Osteosarcoma Stem Cells (OSCs) are a subset of tumor cells with self-renewal capacity and can helpTumor heterogeneity is maintained and new tumors are produced. OSCs can form tumor balls in serum-free medium and can form tumors in mice. Traditional methods of treatment may result in non-OSC S Death (e.g., apoptosis) of tumor cells, but has limited effect on OSCs. Under treatment-induced stress, the viability of OSCs is associated with rapid activation of pro-survival mechanisms. Eradicating OSC S The treatment effect of TKI is greatly improved, and even osteosarcoma can be cured.
Meanwhile, the nanotechnology has rapidly entered the field of tumor treatment, and active or passive targeted treatment by taking a nanomaterial as a carrier, even gene therapy, not only can reduce toxic and side effects caused by nonspecific distribution of chemotherapeutic drugs, but also can target tumor stem cells, resist multidrug resistance and improve the anticancer efficacy of the drugs. Among them, stimulus-responsive polymer systems (including temperature, light, redox, pH response) have been widely used in biomedical fields for the preparation of nanocarriers, wherein pH-responsive controlled release systems, which have specific lactone and peptide bond groups in their polymer molecular structures, can be widely used in the delivery of anticancer drugs and in tumor therapy in response to slightly acidic microenvironments in tumors. The polyester polymer is an important biomedical nanomaterial, has the advantages of good biocompatibility, excellent biodegradability, no toxicity, no harm, low cost, wide application prospect and wide development space in the field of drug delivery, and the like. The direct polycondensation method is one of the main methods for synthesizing polyester polymers in the prior art, but the direct polycondensation method still has the defects of difficult control of reaction temperature, pressure, time, vacuum degree and other conditions. Therefore, the research on the polyester material with simple synthetic method, good controllability of synthetic conditions and certain excellent properties of synthetic products is one of important conditions for promoting the application of the polymer to tumor treatment.
The combination of Apatinib and the nano-drug delivery system can effectively inhibit osteosarcoma stem cells, thereby overcoming the drug resistance of osteosarcoma. Drug delivery systems based on nanoparticle design have been widely demonstrated to significantly improve the bioavailability and tumor targeting efficiency of Apatinib. The polymer nano particles with the polyester amide skeleton are designed, and have the characteristics of excellent biocompatibility, improved tumor accumulation efficiency, high drug loading capacity, capability of realizing controlled release in tumor microenvironment and the like. In order to achieve the aim of accurate targeted drug delivery to osteosarcoma stem cells, nano drug particles are optimized, so that the therapeutic effect of Apatinib in osteosarcoma can be greatly improved. We have initially studied the therapeutic effects of Apatinib on osteosarcoma stem cells and provided a possible mechanistic explanation for their failed therapeutic strategies. Furthermore, it was found that the Apatinib nano-drug can inhibit the self-renewal capacity of osteosarcoma stem cells and tumors formed by osteosarcoma stem cells in vivo and in vitro. In addition, the 8P4-Apa nano-drug has obvious advantages in toxicity tolerance compared with Apatinib free drug. Therefore, the development of the nano-drug with good biocompatibility and targeted tumor cells based on Apatinib research has important significance in the treatment of malignant osteosarcoma.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of poor targeting, large side effect and incapacity of inhibiting osteosarcoma stem cells of the existing Apatinib, provides a nano medicament, realizes medicament 8P4-Apa encapsulation by a nano precipitation method, prepares a nano medicament-carrying system with pH responsiveness, and has application prospect as 8P4-Apa nano medicament. The 8P4-Apa nano-drug can realize high-efficiency load of Apatinib, has strong sensitivity to slightly acidic tumor environment, can effectively deliver the drug into osteosarcoma cells and osteosarcoma stem cells, can rapidly release and induce osteosarcoma stem cells to apoptosis in the tumor cells, and opens up a new way for effective treatment of osteosarcoma.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a polyester compound having the structural formula:
wherein n=2 to 50.
The present invention provides a process for preparing the polyester compound, comprising the steps of:
s1, preparing a polymer I, di-p-nitrophenyl sebacate through condensation reaction;
s2, preparing a polymer II through solid-liquid reaction, namely diphenyl alanine butyl diester di-p-toluenesulfonate;
s3, obtaining a Phe-PEA polymer, namely 8-Phe-4, through solution polycondensation, wherein the polymers I and II are prepared by the following steps of 1:1 in anhydrous dimethyl sulfoxide (DMSO), stirring and mixing uniformly, and keeping the temperature at 120 ℃, wherein a certain amount of triethylamine is added dropwise to obtain a uniform yellow solution;
and S4, maintaining the yellow solution at 80 ℃ overnight in a static state, adding pre-cooled ethyl acetate to precipitate a final product, eluting with methanol, and drying under vacuum to obtain the polyester 8P4 compound.
The invention also provides a nano-drug taking the polyester compound as a carrier, wherein the nano-drug comprises the polyester 8P4 compound and the drug Apatinib.
The invention also provides a method for preparing the nano-drug, which comprises the following steps:
s5, respectively dissolving Apatinib, a polyester 8P4 compound and a stabilizer in an organic solvent which is mutually soluble with water, and uniformly mixing the three according to a certain proportion to form an oil phase;
s6, slowly and uniformly dripping the oil phase into the water phase under intense stirring to obtain the nano-drug with uniform and stable particle size.
Preferably, the stabilizer is distearoyl phosphatidylethanolamine-polyethylene glycol 2000.
Preferably, the particle size of the obtained stable nano-drug is 80-500nm.
Preferably, the particle size of the nano-drug is 110nm.
The invention also provides an application of the nano-drug in treating osteosarcoma.
The invention has the beneficial effects that the nano-particles with controllable particle size are prepared by coating the anti-angiogenesis drugs with the polyester polymer material with the phenylalanine phenylethylamine with the corresponding pH as a framework, and the nano-particles can be accumulated in tumor tissues through high permeability and high retention (EPRefect) of the tumor tissues, thereby realizing the passive targeting effect. Then the acid microenvironment of tumor tissues is utilized to accelerate the rupture of ester bonds and peptide bonds in the nano particles and the release of the drug, thereby playing a role in targeted treatment of cancers and having extremely high clinical application value.
Drawings
FIG. 1A shows a Transmission Electron Microscope (TEM) photograph of an NP nano-drug entrapped with a drug 8P4-Apa prepared according to the present invention; FIG. 1B shows a Dynamic Light Scattering (DLS) diagram of the present invention for preparing blank nanoparticles and 8P4-Apa nanomedicines; FIG. 1C shows the release profile of the 8P4-Apa nano-drug prepared by the present invention under different pH conditions.
FIG. 2A is a diagram showing the uptake of nano-drugs by osteosarcoma cells in an embodiment of the present invention; FIG. 2B shows the uptake of nano-drugs by osteosarcoma stem cells cultured in vitro in osteosarcoma according to an embodiment of the present invention; fig. 2C shows the concentration of nanomedicine at tumor sites in vivo: diR dye and nanomaterial 8P4 are coated on the tumor-bearing naked tail vein injection and then are changed at intervals.
FIG. 3A shows IC50 values for the nanopharmaceutical 8P4-Apa and the free drug Apatinib versus 143B and SJSA1 cells of the invention; FIG. 3B shows tumor spheres formed by in vitro osteosarcoma stem cells of the nano-drug 8P4-Apa and the free drug Apatinib inhibited 143B and SJSA1 cells; fig. 3C shows that the nano-drug 8P4-Apa and the free drug Apatinib promote apoptosis of tumor cells formed by osteosarcoma stem cells in vitro in 143B and SJSA1 cells.
FIG. 4A shows nanomedicine inhibiting in situ animal model in vivo growth of 143B cell osteosarcoma stem cell formation; FIG. 4B is a tumor size comparison; FIG. 4C shows the Ki67 immunohistochemical staining results for tumor tissue.
Detailed Description
The present invention will be further described with reference to the accompanying drawings, wherein the following examples are provided on the premise of the present technical solution, and detailed embodiments and specific operation procedures are given, but the scope of the present invention is not limited to the examples.
The invention relates to a polyester compound, which has the following structural formula:
wherein n=2 to 50.
The present invention provides a process for preparing the polyester compound, comprising the steps of:
s1, preparing a polymer I, di-p-nitrophenyl sebacate through condensation reaction;
s2, preparing a polymer II, namely diphenylalanine butyl diester di-p-toluenesulfonate through a solid-liquid reaction;
s3 is subjected to solution polycondensation reaction to obtain a Phe-PEA polymer, namely 8-Phe-4, wherein polymers I and II are prepared by the following steps of 1:1 in anhydrous dimethyl sulfoxide (DMSO), stirring and mixing uniformly, and keeping the temperature at 120 ℃, wherein a certain amount of triethylamine is added dropwise to obtain a uniform yellow solution;
and S4, maintaining the yellow solution at 80 ℃ overnight under a static state, adding pre-cooled ethyl acetate to precipitate a final product, eluting with methanol, and drying under vacuum to obtain the polyester 8P4 compound.
The invention also provides a nano-drug taking the polyester compound as a carrier, wherein the nano-drug comprises the polyester 8P4 compound and the drug Apatinib.
The invention also provides a method for preparing the nano-drug, which comprises the following steps:
s5, respectively dissolving Apatinib, a polyester 8P4 compound and a stabilizer in an organic solvent which is mutually soluble with water, and uniformly mixing the three according to a certain proportion to form an oil phase;
s6, slowly and uniformly dripping the oil phase into the water phase under intense stirring to obtain the nano-drug with uniform and stable particle size.
Preferably, the stabilizer is distearoyl phosphatidylethanolamine-polyethylene glycol 2000.
Preferably, the particle size of the obtained stable nano-drug is 80-500nm.
Preferably, the particle size of the nano-drug is 110nm.
The invention also provides an application of the nano-drug in treating osteosarcoma.
Example 1 preparation of a pH responsive drug-loaded 8P4-Apa nanosystem
(1) When preparing the drug-loaded 8P4 nano-particles, 8P4 is dissolved in DMSO to form oil phase 1 with a concentration of 40mg/mL. Apatinib drug was dissolved in DMSO in oil phase 2 at a drug concentration of 10mg/mL. Stabilizer DSPE-PEG2000 was dissolved in DMSO at a concentration of 20mg/mL to form oil phase 3.
(2) The appropriate volume of 1,2,3 oil phases were taken and thoroughly mixed to give DSPE-PEG2000 of about 40% by weight of the total mass of 8P4, apatinib, and added dropwise to deionized water stirred at 2000 r/m. The volume ratio of the oil phase to the water phase was 1:6. The free organic solution DMSO was removed by centrifugation three times using an ultrafiltration tube with a molecular weight cut-off of 100,000Da and finally resuspended in PBS. DLS detection shows that the particle size of the drug-loaded nanoparticle is 100-150nm; TEM images can observe that nanoparticles are in a uniformly dispersed spherical structure.
(3) Transferring the prepared 8P4-Apa nano particles into a dialysis bag, boiling the dialysis bag in pure water for 2-3 minutes in advance, sealing the dialysis bag, immersing the dialysis bag in phosphate buffer solution with pH of 5.0 and pH of 7.4, oscillating at 100rpm/min of a constant-temperature shaking table with the temperature of 37 ℃, taking liquid at 0.25,0.5,1,2,4,6,8, 10, 12, 24, 48, 72, 96, 120 and 144 hours respectively, supplementing buffer solution with the same volume and corresponding pH, detecting the release content of Apatinib by using high performance liquid chromatography, calculating the cumulative release percentage of the medicine and drawing a cumulative release graph. As shown in fig. 1.
Example 2 drug delivery Capacity of Polymer 8P4
Coumarin 6 (C) using fluorescent substance 6 2.0 ug/mL) was loaded into 8P4 nanoparticles to obtain 8P4-C 6 And (3) nanoparticles.
(1) 143B (ATCC accession No. HCC-1143, human, epithelial-like adherence) in logarithmic phase of growthGrowth) and SJSA1 cells (ATCC accession number CRL-1469, humanized, epithelial-like adherent growth), digested with pancreatin, blown off into single cell suspensions, counted, inoculated into 6 well plates, 1.5×10 per well 5 Individual cells. Culturing in incubator for 24 hr, and adding pure C after cell adhesion 6 Solution and 8P4-C 6 After incubation of the solutions at 37℃for 1 hour, respectively, nuclei were stained with DAPI after fixation with 4% paraformaldehyde. Fluorescence intensity was recorded using a fluorescence microscope and flow cytometer.
(2) 143B and SJSA1 cells in the logarithmic growth phase were digested with pancreatin, blown off into single cell suspensions, counted, inoculated into a low-adhesion 96-well culture plate, and 10ng/mL of basic fibroblast growth factor (bFGF), 10ng/mL of human epidermal growth factor (hEGF), and 1% B27 serum-free additive were added using DMEM/F12 medium to prepare a stem cell pellet medium. After 7 days in a 37℃incubator, compact and morphologically regular tumor spheres (stem cells) were obtained for the experiment. Respectively adding C 6 Free pharmaceutical solution and 8P4-C 6 The solution was brought to a final concentration of 200ng/ml. After 1 hour of incubation at 37℃respectively, fixation with 4% paraformaldehyde was carried out for 15 minutes. Fluorescence intensity was recorded using a fluorescence microscope and flow cytometer.
(3) Obtaining tumor balls (stem cells) by the method in the step 2, centrifugally collecting at a low speed of 500rpm/min, digesting for about 10min by using 5% pancreatic protein digestive enzyme while gently blowing, and passing through a 200-mesh metal sterile filter screen to obtain stem cell suspension, and adjusting the cell concentration to 10 7 /mL, ready for in situ oncology.
The experiment adopts 4-week-old BALB/c male nude mice, which are purchased from Nanjing university model animal institute, and have weight of 18-20 g, and are fed in separate cages under the condition of no Special Pathogen (SPF) for 12 hours, alternately illuminated and eaten freely. The nude mice are randomly divided into two groups, 3 nude mice are respectively numbered, and the in-situ injection of the front end of the tibia of the right hind limb of the nude mice is carried out for 10 7 50. Mu.L of cell suspension/mL. The tumor size is monitored periodically until the tumor grows to 500mm 3 100mL of physiological saline, 100mL of physiological saline in which 10ug of near infrared Dye (DiR) was dissolved, and an equal amount of NPDiR were intravenously injected through the tail of the mice, respectively. Respectively 1 hour and 4 hours after injectionMice were anesthetized with a small animal live imaging system and fluorescent signals were captured at 8 hours, 12 hours and 24 hours. After observation, mice were sacrificed and the heart, lung, liver, spleen, kidney and tumor tissues of the main organs were taken for in vitro imaging.
Experimental results:
as shown in FIG. 2, both the tumor ball and the animal model which are derived from the osteosarcoma cells show that the fluorescence intensity of the cells after the cells are subjected to the nano-encapsulation effect is obviously enhanced, which indicates that the 8P4 nano-carrier has better targeting osteosarcoma capability.
Example 38 effect of P4-Apa nanomaterials on proliferation of osteosarcoma cells was compared.
The influence of different concentrations of 8P4-Apa nanoparticles and Apatinib free drugs on the proliferation of osteosarcoma cells and the formation of tumor balls by osteosarcoma stem cells is detected through a cell viability test.
(1) 143B and SJSA1 cells in an exponentially growing phase were digested with pancreatin, blown off into single cell suspensions, counted, and plated into 96-well plates. After culturing for 24h in an incubator, after the cells are attached, 8P4-Apa nano particles and Apatinib free drugs with different concentrations are added, and after culturing for 48h in the incubator, the cell viability is evaluated according to the CellTiter-Glo luminous cell viability assay kit instructions.
(2) 143B and SJSA1 cells in a logarithmic growth phase are inoculated in a low-adhesion 96-well plate, after being cultured for 5 days in a 37 ℃ incubator, single, compact and regular tumor balls are selected for experiments, and the independent 8P4-Apa nano-drugs with different concentrations and equal concentrations are respectively added into DMSO. After 48 hours of action, the number of tumor balls was determined by microscopic counting.
Experimental results:
as shown in FIG. 3, the inhibition effect of the 8P4-Apa nano-drug on the proliferation of the osteosarcoma cell line and the osteosarcoma stem cell-derived tumor globus is obviously superior to that of the Apatinib free drug. The method for calculating the drug action relationship comprises the following steps: survival = (experimental group value-blank group value)/(control group value-blank group value)
Example 48 effect of P4-Apa nanomedicines on osteosarcoma apoptosis was compared.
143B and SJSA1 cells in an exponentially growing phase were digested with pancreatin, blown off into single cell suspensions, counted, and plated into 96-well plates. Culturing in an incubator for 24 hours, adding 8P4-Apa nano particles and Apatinib free medicines with different concentrations after cells are attached, and culturing in the incubator for 24 hours. Cells were collected and stained with FITC-labeled Annexin V and Propidium Iodide (PI) for 15 min, respectively, and flow cytometry was used to determine apoptosis.
Experimental results:
as shown in FIG. 3, the 8P4-Apa nano-drug has significantly better effect on promoting apoptosis of osteosarcoma cell lines than Apatinib free drug.
Example 58 inhibition of osteosarcoma Stem cells by P4-Apa nanomaterials in animal models
Tumor ball culturing bone sarcoma cell 143B to obtain bone sarcoma stem cell, culturing for 5 days, and making into 1×10 with PBS 7 Single cell suspensions were injected in situ into the anterior end of the right hind limb tibia of female nude mice, 50 μl each. The condition of the mice was observed daily, and the long diameter (a) and the short diameter (b) of the tumors were measured with a vernier caliper according to the formula V (mm) 3 ) Tumor volume was calculated by = (a×b2)/2. When the tumor volume reaches 80-100 mm 3 At this time, animals were randomly divided into 3 groups of 8 animals each. NPs of a nanomaterial blank control group respectively; an equal concentration of Apatinib free drug; the concentration of 8P4-Apa was equal, and the administration was continued 3 times every other day. 7 days after the end of the administration, the mice were sacrificed by cervical dislocation, the tumors were stripped, weighed, compared to the tumor growth size, and the expression of Ki67 was examined by immunohistochemistry.
Experimental results:
as shown in FIG. 4, the results show that the 8P4-Apa nano-drug has significantly better effect of inhibiting the growth of osteosarcoma stem cell-derived tumors than other experimental groups, and the 8P4-Apa nano-drug has the effect of inhibiting the self-renewal capacity of osteosarcoma stem cells in vivo.
Finally, it is noted that in some embodiments of the invention, the osteosarcoma cells are 143B cells and SJSA1 cells, and the osteosarcoma stem cells are osteosarcoma stem cell nodules formed by 143B cells and SJSA1 cells under dry culture conditions.
Various modifications and variations of the present invention will be apparent to those skilled in the art in light of the foregoing teachings and are intended to be included within the scope of the following claims.
Claims (5)
1. A method for preparing nano-medicine with polyester compound as carrier is characterized in that,
s1, respectively dissolving Apatinib, a polyester 8P4 compound and a stabilizer in an organic solvent which is mutually soluble with water, and uniformly mixing the three according to a certain proportion to form an oil phase;
s2, slowly and uniformly dripping the oil phase into the water phase under intense stirring to obtain the nano-drug with uniform and stable particle size;
the structural formula of the polyester 8P4 compound is as follows:
wherein n=2 to 50;
the preparation method of the polyester 8P4 compound comprises the following steps:
s1, preparing a polymer I, di-p-nitrophenyl sebacate through condensation reaction;
s2, preparing a polymer II through solid-liquid reaction, namely diphenyl alanine butyl diester di-p-toluenesulfonate;
s3, obtaining a Phe-PEA polymer, namely 8-Phe-4, through solution polycondensation, wherein the polymers I and II are prepared by the following steps of 1:1 in anhydrous dimethyl sulfoxide (DMSO), stirring and mixing uniformly, and keeping the temperature at 120 ℃, wherein a certain amount of triethylamine is added dropwise to obtain a uniform yellow solution;
and S4, maintaining the yellow solution at 80 ℃ overnight in a static state, adding pre-cooled ethyl acetate to precipitate a final product, eluting with methanol, and drying under vacuum to obtain the polyester 8P4 compound.
2. The method for preparing a nano-drug using a polyester compound as a carrier according to claim 1, wherein the stabilizer is distearoyl phosphatidylethanolamine-polyethylene glycol 2000.
3. The method for preparing a nano-drug using a polyester compound as a carrier according to claim 1, wherein the obtained stable nano-drug has a particle size ranging from 80 to 500nm.
4. The method for preparing a nano-drug using a polyester compound as a carrier according to claim 1, wherein the particle size of the nano-drug is 110nm.
5. Use of the nano-drug prepared by the preparation method of any one of claims 1 to 4 for preparing a drug for treating osteosarcoma.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010474210.XA CN111763315B (en) | 2020-05-29 | 2020-05-29 | Polyester compound, nano medicine taking polyester compound as carrier and application of nano medicine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010474210.XA CN111763315B (en) | 2020-05-29 | 2020-05-29 | Polyester compound, nano medicine taking polyester compound as carrier and application of nano medicine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111763315A CN111763315A (en) | 2020-10-13 |
| CN111763315B true CN111763315B (en) | 2023-07-25 |
Family
ID=72719925
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010474210.XA Active CN111763315B (en) | 2020-05-29 | 2020-05-29 | Polyester compound, nano medicine taking polyester compound as carrier and application of nano medicine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111763315B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114272212A (en) * | 2021-12-04 | 2022-04-05 | 中山大学孙逸仙纪念医院 | Rac1 inhibitor nano particle targeting prostate cancer, preparation method and application |
| CN114306618B (en) * | 2022-01-14 | 2024-03-29 | 中山大学 | Application of polyester amide compound in preparation of medicine for preventing and/or treating blood system diseases |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110804178A (en) * | 2019-10-17 | 2020-02-18 | 中山大学 | Nano drug-loaded system with glutathione responsiveness and preparation method and application thereof |
| CN110922587A (en) * | 2019-12-05 | 2020-03-27 | 中山大学 | Preparation method of nano-drug and application of nano-drug in treatment of osteosarcoma |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CZ302830B6 (en) * | 2009-12-15 | 2011-11-30 | Ústav makromolekulární chemie AV CR, v.v.i. | High-molecular polymeric carriers of medicaments derived from dendrimers and conjugates thereof with medicaments for treating especially solid tumors |
-
2020
- 2020-05-29 CN CN202010474210.XA patent/CN111763315B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110804178A (en) * | 2019-10-17 | 2020-02-18 | 中山大学 | Nano drug-loaded system with glutathione responsiveness and preparation method and application thereof |
| CN110922587A (en) * | 2019-12-05 | 2020-03-27 | 中山大学 | Preparation method of nano-drug and application of nano-drug in treatment of osteosarcoma |
Non-Patent Citations (2)
| Title |
|---|
| Chen, X ; Zhao, LL ; Kang, Y.Significant Suppression of Non-small-cell Lung Cancer by Hydrophobic Poly(ester amide) Nanoparticles with High Docetaxel Loading.《Frontiers in Pharmacology》.2018,第9卷第1-11页. * |
| Targeting Super-Enhancers via Nanoparticle-Facilitated BRD4 and CDK7 Inhibitors Synergistically Suppresses Pancreatic Ductal Adenocarcinoma;Huang, CS ; You; XR ; Dai, CL;《Advanced science》;20200216;第7卷(第7期);第1-13页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111763315A (en) | 2020-10-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Saneja et al. | Development and evaluation of long-circulating nanoparticles loaded with betulinic acid for improved anti-tumor efficacy | |
| Fu et al. | Aspartic acid-based modified PLGA–PEG nanoparticles for bone targeting: In vitro and in vivo evaluation | |
| Yang et al. | Dual-targeting hybrid nanoparticles for the delivery of SN38 to Her2 and CD44 overexpressed human gastric cancer | |
| CN107669632B (en) | Drug carrier, micelle, drug preparation, preparation method and application thereof | |
| Zhang et al. | Improving anti-tumor activity of curcumin by polymeric micelles in thermosensitive hydrogel system in colorectal peritoneal carcinomatosis model | |
| Wu et al. | Novel simvastatin-loaded nanoparticles based on cholic acid-core star-shaped PLGA for breast cancer treatment | |
| Li et al. | Reduction breakable cholesteryl pullulan nanoparticles for targeted hepatocellular carcinoma chemotherapy | |
| WO2006041613A2 (en) | Nanoparticles for targeting hepatoma cells | |
| Tao et al. | Paclitaxel-loaded tocopheryl succinate-conjugated chitosan oligosaccharide nanoparticles for synergistic chemotherapy | |
| CN111763315B (en) | Polyester compound, nano medicine taking polyester compound as carrier and application of nano medicine | |
| WO2021057007A1 (en) | Rapamycin nanoscale sustained-release agent and preparation method thereof | |
| CN108175860A (en) | A kind of poly curcumin thio-2 acid copolymer prodrug nano-micelle of esterase response type and its preparation method and application | |
| Hou et al. | Dual-responsive polyphosphazene as a common platform for highly efficient drug self-delivery | |
| Lu et al. | Micellar nanoparticles inhibit the postoperative inflammation, recurrence and pulmonary metastasis of 4T1 breast cancer by blocking NF-κB pathway and promoting MDSCs depletion | |
| Fan et al. | Dual oligopeptides modification mediates arsenic trioxide containing nanoparticles to eliminate primitive chronic myeloid leukemia cells inside bone marrow niches | |
| CN101708337B (en) | Preparation method of human serum albumin nano granules coated with oxaliplatin | |
| CN108339124B (en) | Preparation method and application of two-stage brain-targeted polymer micelle drug delivery system | |
| Liu et al. | Thermosensitive selenium hydrogel boosts antitumor immune response for hepatocellular carcinoma chemoradiotherapy | |
| Zu et al. | Docetaxel-loaded bovine serum albumin nanoparticles conjugated docosahexaenoic acid for inhibiting lung cancer metastasis to bone | |
| CN109432051B (en) | Targeting nanoparticle with anti-ovarian cancer activity and preparation and application thereof | |
| Yadav et al. | Chondroitin sulphate decorated nanoparticulate carriers of 5-fluorouracil: development and in vitro characterization | |
| CN107266384A (en) | N carboxyl inner-acid anhydride monomers and polyaminoacid based on 2 aminohexadecanoic acids and preparation method thereof | |
| CN109953974B (en) | Preparation method of enzyme-reduction dual-responsiveness hyaluronic acid-polypropylene sulfide copolymer nanocapsule | |
| CN110511387B (en) | Hyaluronic acid-g-polytyrosine-lipoic acid copolymer, polypeptide nanoparticles, and preparation methods and applications thereof | |
| CN115089561B (en) | Prodrug nanoparticle coated by cell membrane, preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |