CN110742874B - Polymer-coated cisplatin nanoparticle and preparation method and application thereof - Google Patents

Polymer-coated cisplatin nanoparticle and preparation method and application thereof Download PDF

Info

Publication number
CN110742874B
CN110742874B CN201910923324.5A CN201910923324A CN110742874B CN 110742874 B CN110742874 B CN 110742874B CN 201910923324 A CN201910923324 A CN 201910923324A CN 110742874 B CN110742874 B CN 110742874B
Authority
CN
China
Prior art keywords
cisplatin
polymer
solution
preparation
polyethyleneimine
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
Application number
CN201910923324.5A
Other languages
Chinese (zh)
Other versions
CN110742874A (en
Inventor
张宇
尹湉
何海冰
苟靖欣
唐星
李孝文
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenyang Pharmaceutical University
Original Assignee
Shenyang Pharmaceutical University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Shenyang Pharmaceutical University filed Critical Shenyang Pharmaceutical University
Priority to CN201910923324.5A priority Critical patent/CN110742874B/en
Publication of CN110742874A publication Critical patent/CN110742874A/en
Application granted granted Critical
Publication of CN110742874B publication Critical patent/CN110742874B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Nanotechnology (AREA)
  • Biomedical Technology (AREA)
  • Inorganic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention belongs to the technical field of medicine preparation, and particularly relates to a polymer-coated cisplatin nanoparticle and a preparation method and application thereof. The method comprises the steps of mixing a cisplatin solution and a polyethyleneimine solution, and performing crosslinking to obtain a cisplatin nanoparticle core solution; and mixing the cisplatin nanoparticle core solution with a polymer solution, and compounding to obtain the polymer-coated cisplatin nanoparticle. The polymer-coated cisplatin nanoparticle prepared by the invention has high encapsulation efficiency (more than 95%), has high drug-loading rate up to 16.43% and can realize slow release. The polymer-coated cisplatin nanoparticle prepared by the invention can overcome the problems of high plasma protein binding rate and high renal toxicity of the existing cisplatin commercial preparation, and has important pharmaceutical significance. Animal experiments prove that the preparation can realize good drug effect, and the tumor inhibition rate reaches 80.95%.

Description

Polymer-coated cisplatin nanoparticle and preparation method and application thereof
Technical Field
The invention relates to the technical field of medicine preparation, in particular to a polymer-coated cisplatin nanoparticle and a preparation method and application thereof.
Background
According to the global cancer statistics report of 2018, lung cancer is the most common cancer type, accounting for 11.6% of the total number, and is one of the main causes of cancer death. There are over 1400 million new cancer cases per year, and this figure is expected to increase to over 2100 million by 2030. Chemotherapy is currently one of the clinically effective treatments. The chemotherapeutic drug can kill cancer cells and inhibit the growth and reproduction of the cancer cells, but the chemotherapeutic drug usually has extremely high cytotoxicity and lacks selectivity, and has the function of killing normal cells while killing tumor cells.
Cisplatin (cissplatin) is a broad-spectrum antitumor drug widely applied and can form intrachain crosslinking with DNA to induce apoptosis. Since its FDA approval was obtained in 1978, it has been used to treat a variety of tumors, including non-small cell lung cancer (NSCLC), Small Cell Lung Cancer (SCLC), ovarian cancer, testicular cancer, neuroblastoma, and the like. However, the binding rate of cisplatin and plasma protein can reach 65% -98%, and the medicament combined with the plasma protein has no curative effect. Currently, commercially available cisplatin injection and cisplatin preparation for injection are rapidly distributed in plasma after administration, and bind to plasma protein to reduce the drug effect. Meanwhile, the combined medicine can be distributed and accumulated in the kidney and the intestinal tract, and adverse reactions such as renal toxicity and gastrointestinal toxicity are caused.
Disclosure of Invention
The invention aims to provide a polymer-coated cisplatin nanoparticle, and a preparation method and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of polymer-coated cisplatin nanoparticles, which comprises the following steps:
mixing the cisplatin solution with the polyethyleneimine solution, and crosslinking to obtain a cisplatin nanoparticle core solution;
and mixing the cisplatin nanoparticle core solution with a polymer solution, and compounding to obtain the polymer-coated cisplatin nanoparticle.
Preferably, the number average molecular weight of polyethyleneimine in the polyethyleneimine solution is 800-100000 g/mol.
Preferably, the mass concentration of the cisplatin solution is 0.5-2 mg/mL, and the mass concentration of the polyethyleneimine solution is 0.5-10 mg/mL.
Preferably, the mass ratio of the cisplatin in the cisplatin solution to the polyethyleneimine in the polyethyleneimine solution is 0.2-0.6: 1.
Preferably, the crosslinking is carried out under the condition of stirring, the stirring speed is 200-800 r/min, the time is 1-4 h, and the temperature is 60-80 ℃.
Preferably, the polymer in the polymer solution comprises polyglutamic acid grafted polyethylene glycol, polyaspartic acid grafted polyethylene glycol, polyglutamic acid block polyethylene glycol or polyaspartic acid block polyethylene glycol, and the number average molecular weight of the polymer in the polymer solution is 10000-100000 g/mol; the concentration of the polymer solution is 1-20 mg/mL.
Preferably, the mass ratio of the polyethyleneimine in the polyethyleneimine solution to the polymer in the polymer solution is 1: 0.5-10.
Preferably, the compounding is carried out under the condition of stirring, the stirring speed is 200-800 r/min, the time is 2-6 h, and the temperature is 20-30 ℃.
The invention provides the polymer-coated cisplatin nanoparticle prepared by the preparation method in the technical scheme.
The invention provides application of the polymer-coated cisplatin nanoparticles in the technical scheme in antitumor drugs.
The invention provides a preparation method of polymer-coated cisplatin nanoparticles, which comprises the steps of mixing a cisplatin solution with a polyethyleneimine solution, and carrying out crosslinking to obtain a cisplatin nanoparticle core solution; and mixing the cisplatin nanoparticle core solution with a polymer solution, and compounding to obtain the polymer-coated cisplatin nanoparticle. According to the invention, through the coordination bond between polyethyleneimine and cisplatin and the cross-linking effect of a hydrogen bond, the cisplatin nanoparticle core with positive charges is obtained, and then the cisplatin nanoparticle core is adsorbed on the surface of the cisplatin nanoparticle core through the electrostatic action of a polymer, so that the polymer-coated cisplatin nanoparticle is obtained.
The polymer-coated cisplatin nanoparticles prepared by the invention can avoid the combination of free drugs and plasma proteins and slow down the distribution of combined drugs in blood as a intravenous injection preparation, so that the polymer-coated cisplatin nanoparticles can realize long circulation in blood, and the blood concentration is 25.59 times of that of an equal-dose cisplatin solution, namely the preparation obviously improves the bioavailability of the drugs, can overcome the problems of high plasma protein combination rate and high renal toxicity of the existing cisplatin commercial preparations, and has important pharmaceutical significance. Animal experiments prove that the preparation can realize good drug effect, and the tumor inhibition rate reaches 80.95%.
The polymer-coated cisplatin nanoparticle prepared by the invention has high encapsulation efficiency (more than 95 percent) and high drug-loading rate (up to 16.43 percent).
The invention adopts biodegradable high molecular material-polyethyleneimine cross-linked drug to form nanoparticle inner core (cisplatin nanoparticle core), then adopts polymer as coating layer to avoid phagocytosis of macrophage, and the obtained polymer coated cisplatin nanoparticle delays drug release through gradual hydrolysis and falling off of the coating layer after administration, thereby achieving the purpose of slow release.
Drawings
FIG. 1 is a schematic diagram of the preparation process of polymer-coated cisplatin nanoparticles of the present invention;
FIG. 2 is a graph of particle size and potential for the cisplatin nanoparticle core and the polymer-coated cisplatin nanoparticle prepared in example 1;
FIG. 3 shows the in vitro drug release of cisplatin solution, cisplatin nanoparticle core, and polymer-coated cisplatin nanoparticle;
fig. 4 is a result of uptake of cisplatin nanoparticle cores and polymer-coated cisplatin nanoparticles by a549 cells;
FIG. 5 is a graph showing the effect of nanoparticles (cisplatin solution, cisplatin nanoparticle core, and polymer-coated cisplatin nanoparticles) on cytotoxicity of A549 cells and MCF-7 cells; wherein A is cytotoxicity of the nanoparticles to A549 cell culture for 48 h; b is the cytotoxicity of the nanoparticles to MCF-7 cell culture for 48 h; c is the cytotoxicity of the nanoparticles to A549 cells and MCF-7 cells for 24h, 48h and 72 h;
FIG. 6 shows the therapeutic effect of nanoparticles (cisplatin solution, Ca/PEI nanoparticles, cisplatin nanoparticle core and polymer-coated cisplatin nanoparticles) in mice with tumor-bearing C57BL/6 in axilla; wherein A is the change of the relative tumor volume of the mouse; b is the weight change of the mice; c is the comparison of the physiological index urea nitrogen of the mice; d is comparison of mouse physiological index creatinine; e is mouse H & E staining results.
Detailed Description
The invention provides a preparation method of polymer-coated cisplatin nanoparticles, which comprises the following steps:
mixing the cisplatin solution with the polyethyleneimine solution, and crosslinking to obtain a cisplatin nanoparticle core solution;
and mixing the cisplatin nanoparticle core solution with a polymer solution, and compounding to obtain the polymer-coated cisplatin nanoparticle.
In the present invention, unless otherwise specified, all the starting materials required for the preparation are commercially available products well known to those skilled in the art.
The cisplatin solution and the polyethyleneimine solution are mixed and crosslinked to obtain the cisplatin nanoparticle core solution. In the present invention, the number average molecular weight of polyethyleneimine in the polyethyleneimine solution is preferably 800 to 100000g/mol, and most preferably 20000 to 30000 g/mol. In the invention, the cisplatin solution is preferably prepared by dissolving cisplatin in distilled water, and the mass concentration of the cisplatin solution is preferably 0.5-2 mg/mL, and more preferably 0.8-1.5 mg/mL; the polyethyleneimine solution is preferably prepared by dissolving Polyethyleneimine (PEI) in distilled water, and the mass concentration of the polyethyleneimine solution is preferably 0.5-10 mg/mL, more preferably 2-8 mg/mL, and most preferably 5-6 mg/mL. In the invention, the mass ratio of cisplatin in the cisplatin solution to polyethyleneimine in the polyethyleneimine solution is preferably 0.2-0.6: 1, preferably 0.4-0.6: 1, and most preferably 0.6: 1. The invention can ensure that the cisplatin nanoparticle core has high encapsulation efficiency and higher drug-loading rate by further controlling the mass ratio of the cisplatin to the polyethyleneimine, thereby facilitating the subsequent coating process. The mixing process is not particularly limited, and the cisplatin solution and the polyethyleneimine solution can be uniformly mixed by adopting a method well known by the technical personnel in the field.
In the invention, the crosslinking is preferably carried out under the condition of stirring, the stirring speed is preferably 200-800 r/min, more preferably 300-600 r/min, the time is preferably 1-4 h, more preferably 2-3 h, and the temperature is preferably 60-80 ℃, more preferably 65-75 ℃. The crosslinking is preferably carried out under the condition of keeping out of the sun, so that the influence of light on the cisplatin (the cisplatin has poor light stability) is avoided. In the cross-linking process, cisplatin and polyethyleneimine form a coordinate bond and a hydrogen bond, and a cisplatin nanoparticle core is formed through cross-linking, wherein the cisplatin nanoparticle core is marked as NP-I (Pt/PEI).
After the cisplatin nanoparticle solution is obtained, the cisplatin nanoparticle core solution and the polymer solution are mixed and compounded to obtain the polymer-coated cisplatin nanoparticle, which is marked as NP-II. In the present invention, the polymer solution is preferably obtained by dissolving a polymer in distilled water; the polymer in the polymer solution preferably comprises polyglutamic acid grafted polyethylene glycol, polyaspartic acid grafted polyethylene glycol, polyglutamic acid block polyethylene glycol or polyaspartic acid block polyethylene glycol, and more preferably polyglutamic acid grafted polyethylene glycol; the number average molecular weight of the polymer is preferably 10000-100000 g/mol, and more preferably 40000-60000 g/mol; the number average molecular weight of the polyethylene glycol in the polymer is preferably 1000-20000 g/mol, and more preferably 3000-6000 g/mol.
In the invention, the concentration of the polymer solution is preferably 1-20 mg/mL, and more preferably 5-15 mg/mL. In the invention, the mass ratio of the polyethyleneimine in the polyethyleneimine solution to the polymer in the polymer solution is preferably 1: 0.5-10, more preferably 1: 2-8, and most preferably 1: 2-4. In the invention, taking a polymer as polyglutamic acid grafted polyethylene glycol as an example, when the mass ratio of the polymer to polyethyleneimine is less than 0.5:1, a glutamic acid monomer is tightly attached to the surface of NP-I, negative charges are far less than positive charges, the whole NP-II is still positively charged, but the polymer cannot cover all the surfaces of NP-I, and a plurality of NP-I can be wrapped in the same coating layer, so that the NP-I is aggregated, and the particle size is remarkably increased; when the mass ratio of the polymer to the polyethyleneimine is 2-4: 1, the increase of the polymer can reduce the amount of positive charges, the overall potential of NP-II begins to reverse to negative, the potential of NP-II is close to 0mv, the electrostatic action between the coating layer and NP-I is strongest, and the particle size is smallest; when the mass ratio of the polymer to the polyethyleneimine is increased to be more than 4:1, a part of the glutamic acid monomer is attached to the surface of the NP-I, and the other part of the glutamic acid monomer extends outwards, so that the NP-II nanoparticles are negatively charged, and the particle size of the NP-II is slightly increased. The invention can ensure that the polymer is successfully coated on the cis-platinum nanoparticle core and control the potential and the particle size of the polymer-coated cis-platinum nanoparticle by further controlling the mass ratio of the polymer to the polyethyleneimine.
In the invention, the mixing process is preferably to drop the polymer solution into the cisplatin nanoparticle core solution under a stirring state, and the dropping rate is preferably 0.5-1.5 d/s, and more preferably 0.8-1.2 d/s.
In the invention, the compounding is preferably carried out under the condition of stirring, the stirring speed is preferably 200-800 r/min, more preferably 400-600 r/min, the time is preferably 2-6 h, more preferably 4-5 h, and the temperature is preferably 20-30 ℃, more preferably 25-26 ℃. In the invention, the complexing is carried out under the condition of keeping out of the sun, so that the influence of light on the cisplatin (the light stability of the cisplatin is poor) is avoided. In the compounding process, the polymer is adsorbed on the surface of the cisplatin nanoparticle core through electrostatic action, so that the stability of the cisplatin nanoparticle is improved.
After the compounding is completed, the obtained system is preferably subjected to centrifugal separation (separation of uncoated polymer) to obtain the polymer-coated cisplatin nanoparticle, which is marked as NP-II. The centrifugal separation is preferably carried out by ultrafiltration, and the conditions of ultrafiltration are not particularly limited in the present invention, and the uncoated polymer can be separated by a process well known to those skilled in the art.
FIG. 1 is a schematic diagram of the preparation process of polymer-coated cisplatin nanoparticles of the present invention; as can be seen from the figure, the cisplatin nanoparticle core NP-I with positive charges is obtained through the cross-linking effect of the coordination bond and the hydrogen bond formed between the polyethyleneimine and the cisplatin, and then is adsorbed on the surface of the cisplatin nanoparticle core through the electrostatic effect of the polymer, so that the polymer-coated cisplatin nanoparticle NP-II is obtained.
The invention provides the polymer-coated cisplatin nanoparticle prepared by the preparation method in the technical scheme. The invention utilizes cisplatin and polyethyleneimine to prepare cisplatin nanoparticle cores, and then adds polymers as coating materials to improve the stability of the cisplatin nanoparticle cores, so that the obtained polymer-coated cisplatin nanoparticles have high encapsulation efficiency (more than 95 percent) and high drug loading rate (up to 16.43 percent).
The invention provides application of the polymer-coated cisplatin nanoparticles in the technical scheme in antitumor drugs. The polymer-coated cisplatin nanoparticle preparation is preferably used for anti-tumor treatment as an intravenous injection preparation, and after intravenous injection, the polymer-coated cisplatin nanoparticle preparation is delivered to a tumor part in a targeted manner, and can generate a remarkable inhibition effect on lung cancer through Enhanced Permeability and Retention (EPR) accumulation.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Respectively dissolving cisplatin and polyethyleneimine in distilled water to obtain cisplatin solution (2mg/mL) and polyethyleneimine solution (5 mg/mL); the mass ratio of the cisplatin to the polyethyleneimine is 3: 5; mixing the cisplatin solution and the polyethyleneimine solution at 65 ℃ in a dark place, and crosslinking for 2 hours under the stirring condition of 500r/min to obtain a cisplatin nanoparticle core (marked as NP-I) solution; wherein, the encapsulation rate of the cisplatin nanoparticle core is 96.70 percent;
dissolving polyglutamic acid grafted polyethylene glycol with distilled water, dropwise adding the obtained polyglutamic acid grafted polyethylene glycol solution (5mg/mL) into the cisplatin nanoparticle core solution at the speed of 1d/s, compounding for 4 hours at the temperature of 25 ℃ and at the speed of 500r/min in the dark, and carrying out ultrafiltration on the obtained system to obtain the polymer-coated cisplatin nanoparticle (marked as NP-II) with the drug-loading rate of 16.43%.
Wherein the number average molecular weight of the polyglutamic acid grafted polyethylene glycol is 35000g/mol, and the number average molecular weight of the polyethylene glycol is 5000 g/mol; the mass ratio of the polyethyleneimine to the polyglutamic acid grafted polyethylene glycol is 1: 2.
Performance testing
1) Particle size and potential measurements were performed on the cisplatin nanoparticle core (NP-I) and the polymer-coated cisplatin nanoparticle (NP-II) prepared in example 1, and the specific results are shown in FIG. 2. As can be seen, the particle size of NP-I was 45.75nm and that of NP-II was 41.79nm (the particle size was slightly reduced due to the compression action of the polymer coating); the potential of NP-I is 25.90mv, the potential of NP-II is reduced to 11.10mv, which fully proves the success of the polymer coating layer on the cisplatin nanoparticle core.
2) The in vitro release of cisplatin solution, NP-I and NP-II was examined by the dialysis bag method, and the results are shown in FIG. 3.
As can be seen, under the condition of pH 5.0, the cisplatin solution can be completely released within 2 h; NP-I is lack of a coating layer, so the release speed is high and can reach 53.27 percent; because of the coverage of the polyglutamic acid grafted polyethylene glycol coating layer, the release speed of NP-II is obviously slower than that of NP-I, which fully proves that the polymer polyglutamic acid grafted polyethylene glycol coating layer can obviously delay the release of the drug.
3) Uptake condition of human lung adenocarcinoma cell A549 cell to NP-I and NP-II nanoparticles
After the nanoparticles are given to the cells, the obtained cells are resuspended in PBS with the pH value of 7.2-7.4, and the number of cells which are fluorescently labeled in a certain number of cells is determined by using a flow cytometry, namely the number of cells which take up the nanoparticles. The uptake of the fluorescent labeled nanoparticles by the a549 cells was measured by a flow cytometry, and the quantitative results of the uptake are shown in fig. 4.
As can be seen from the figure, after NP-I and NP-II are administered to A549 cells for 2 hours, the intracellular uptake of NP-I is obviously higher than that of NP-II, because the NP-I with positive charge is easier to adsorb with the cell membrane with negative charge through electrostatic interaction to promote the endocytosis of the cells, and the NP-II with reduced positive charge and the cells have reduced electrostatic interaction, so the endocytosis of the cells is reduced. The NP-II is respectively incubated for 1h, 2h and 4h under the PBS condition of pH 6.5 and pH 7.4, then added into A549 cells, and the culture is continued for 2 h. When the intracellular uptake is measured, the intracellular uptake is increased compared with that of the non-incubated NP-II, because the NP-II has the defects that the coating layer is reduced under the acidic condition, the potential is increased, the adsorption effect with a negatively charged cell membrane is enhanced, and the cellular uptake is increased. The uptake increased with longer incubation time, also indicating a gradual detachment of the NP-II coating.
4) Cytotoxicity of NP-I and NP-II nanoparticles on human lung adenocarcinoma cell A549 cell and human breast cancer cell MCF-7 cell
The principle of the 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide (MTT) method for determining cytotoxicity is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-purple crystalline formazan, and the formazan is deposited in the cells, but dead cells do not have the function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and absorbance of formazan is measured at 490nm wavelength by enzyme-labeling instrument, which can indirectly reflect the number of living cells, and the amount of MTT crystal formed is directly proportional to the number of cells within a certain range. The cytotoxicity of NP-I, NP-II on A549 cells and MCF-7 cells in vitro was determined by the MTT method, and the results are shown in FIG. 5.
As can be seen, NP-I showed stronger cytotoxicity than NP-II after 48h of culture, because the release of the cisplatin nanoparticle nucleus drug was delayed by the NP-II coating layer, which is also shown by the in vitro release and can be proved by the effect of culture time on cell survival rate. With increasing culture time, cell viability decreased and NP-II decreased more significantly. At 24h, the IC of NP-I is lower because NP-I lacks a coating layer and is more easily taken up by cells and releases the drug rapidly in the cells, NP-II takes up slowly and releases the drug slowly in the cells50The values were lower than NP-II. At 48h, NP-I and NP-II are all taken up by cells, the factors influencing the cell survival rate are only the release rate of the medicament in the cells, the release rate of NP-II is lower than that of NP-I, and therefore the IC of NP-II50IC with values still lower than NP-I50Value, but the difference between the two was less than 24 h. When the culture time reaches 72h, all the medicines of the ingested nanoparticles are almost released, so that the IC of the two medicines is50There was no significant difference in the values. The incubation time of the nanoparticles is in negative correlation with the survival rate of cells, and the NP-II nanoparticles are proved to have a certain slow release effect.
5) Pharmacokinetic analysis for investigating NP-II nano particle in animal body
SD rats (approximately 200g in body weight) were injected into the tail vein at a dose of 3.5mg/kg (in cisplatin). The rats were bled from the posterior ocular venous plexus at predetermined time points and plasma concentrations of cisplatin were determined by inductively coupled plasma mass spectrometry. The results of the pharmacokinetic experiments are shown in table 1.
Table 1 pharmacokinetic parameter results
Figure BDA0002218265790000081
Wherein, AUC(0-72h): curve area at time of drug from 0 to 72 h; t1/2β: the half-life of the drug; cmax: peak concentration; CL, clearance; vzApparent volume of distribution.
Cisplatin distributes rapidly in various tissues and organs after intravenous injection, resulting in very low plasma concentrations and AUC. However, the binding rate of cisplatin and plasma protein is high, and the cisplatin needs longer time to be distributed in blood, so that the elimination half-life is obviously prolonged. After the coating layer of polyglutamic acid grafted polyethylene glycol is added, the adsorption of protein to NP-II can be avoided, the circulation time of the nanoparticles in blood is prolonged, the blood concentration and AUC are improved, the blood concentration is 25.59 times that of a cisplatin solution set, and the AUC is 5.99 times that of the cisplatin solution set, and meanwhile, due to the slow release effect, only a small amount of medicine is released into the blood to be combined with plasma protein, so that the half-life period of NP-II is shortest. NP-I can reduce the rapid distribution of the drug in the plasma, so the blood concentration and AUC are higher than those of the cisplatin solution, but the NP-I is released faster than NP-II, the drug combined with plasma protein is increased, and the half life is slightly higher than that of NP-II.
In addition, the clearance rate and apparent volume distribution value of NP-II are respectively reduced by 0.29 times and 0.053 times compared with the cisplatin solution. Therefore, NP-II can obviously improve blood concentration, reduce plasma clearance rate and apparent distribution volume, and really realize 'long circulation' of the preparation.
6) The anti-tumor effect of the polyglutamic acid grafted polyethylene glycol coated cisplatin nanoparticle (NP-II) on axillary tumor-bearing C57BL/6 mice is examined
In order to evaluate the antitumor effect of the nanoparticles in vivo, cisplatin solution and NP-II nanoparticles of the same drug dose were injected via tail vein, and the results are shown in fig. 6. A in FIG. 6 reflects the change of the relative tumor volume of the mice, and the graph can show that the relative tumor volume of the control group (control) and the calcium nanoparticle group (Ca/PEI) is increased sharply, which proves that the Ca/PEI nanoparticles have little inhibition effect on the tumor growth. NP-I had a high tumor suppression rate, but all mice in this group died on day 8; compared with the cisplatin solution group, the NP-II has obviously improved tumor inhibition rate. In FIG. 6, B reflects changes in body weight of mice, with a decrease in cisplatin group body weight associated with gastrointestinal toxicity caused by cisplatin, and NP-I body weight continued to decrease and died all at day 8, with no apparent change in other groups. The nephrotoxicity of the preparation is evaluated by biochemical indexes of serum urea nitrogen and creatinine (such as C, D in figure 6), the indexes of the cisplatin solution group are obviously increased compared with the control group, and the cisplatin solution group has certain nephrotoxicity, while the NP-II group has no obvious difference from the control group, so that the NP-II is considered not to cause obvious nephrotoxicity.
After the in vivo efficacy study was completed, tissues including tumors were H & E stained (E in fig. 6). Tumor sections of mice given NP-II showed significant tumor necrosis with no significant damage to the liver, kidney, spleen. Therefore, the NP-II has good safety and effectiveness in the process of treating the tumor.
Example 2
Respectively dissolving cisplatin and polyethyleneimine in distilled water to obtain cisplatin solution (2mg/mL) and polyethyleneimine solution (5 mg/mL); the mass ratio of the cisplatin to the polyethyleneimine is 3: 5; mixing the cisplatin solution and the polyethyleneimine solution at 65 ℃ in a dark place, and crosslinking for 3 hours under the stirring condition of 600r/min to obtain a cisplatin nanoparticle core solution;
dissolving polyglutamic acid grafted polyethylene glycol with distilled water, dripping the obtained polyglutamic acid grafted polyethylene glycol solution (5mg/mL) into the cisplatin nanoparticle core solution at the speed of 1d/s, compounding for 3h at the temperature of 25 ℃ and the speed of 600r/min in the dark, and performing ultrafiltration on the obtained system to obtain the polymer-coated cisplatin nanoparticle.
Wherein the number average molecular weight of the polyglutamic acid grafted polyethylene glycol is 35000g/mol, and the number average molecular weight of the polyethylene glycol is 5000 g/mol; the mass ratio of the polyethyleneimine to the polyglutamic acid grafted polyethylene glycol is 1: 4.
Example 3
Respectively dissolving cisplatin and polyethyleneimine in distilled water to obtain cisplatin solution (2mg/mL) and polyethyleneimine solution (5 mg/mL); the mass ratio of the cisplatin to the polyethyleneimine is 3: 5; mixing the cisplatin solution and the polyethyleneimine solution at 65 ℃ in a dark place, and crosslinking for 3 hours under the stirring condition of 400r/min to obtain a cisplatin nanoparticle core solution;
dissolving polyglutamic acid grafted polyethylene glycol with distilled water, dripping the obtained polyglutamic acid grafted polyethylene glycol solution (5mg/mL) into the cisplatin nanoparticle core solution at the speed of 1d/s, compounding for 3h at the temperature of 25 ℃ and at the speed of 400r/min in the dark, and performing ultrafiltration on the obtained system to obtain the polymer-coated cisplatin nanoparticle.
Wherein the number average molecular weight of the polyglutamic acid grafted polyethylene glycol is 35000g/mol, and the number average molecular weight of the polyethylene glycol is 5000 g/mol; the mass ratio of the polyethyleneimine to the polyglutamic acid grafted polyethylene glycol is 1: 6.
The embodiment of the invention provides a polymer-coated cisplatin nanoparticle, and a preparation method and application thereof. The polymer-coated cisplatin nanoparticle prepared by the invention can avoid the combination of free drugs and plasma proteins as a intravenous injection preparation, slows down the distribution of the combined drugs in blood, enables the polymer-coated cisplatin nanoparticle to realize long circulation in the blood, has blood concentration 25.59 times of that of an equal-dose cisplatin solution, can overcome the problems of high plasma protein combination rate and high renal toxicity of the existing cisplatin commercial preparation, and has important pharmaceutical significance. Animal experiments prove that the preparation can realize good drug effect, and the tumor inhibition rate reaches 80.95%. The polymer-coated cisplatin nanoparticle prepared by the invention has high encapsulation efficiency (more than 95 percent) and high drug-loading rate (up to 16.43 percent). After the polymer-coated cisplatin nanoparticle prepared by the invention is administrated, the release of the medicament is delayed through gradual hydrolysis and falling of a coating layer, so that the aim of slow release is fulfilled.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. A preparation method of polymer-coated cisplatin nanoparticles is characterized by comprising the following steps:
mixing the cisplatin solution with the polyethyleneimine solution, and crosslinking to obtain a cisplatin nanoparticle core solution;
and mixing the cisplatin nanoparticle core solution with a polymer solution, and compounding to obtain the polymer-coated cisplatin nanoparticle.
2. The method according to claim 1, wherein the polyethyleneimine in the polyethyleneimine solution has a number-average molecular weight of 800 to 100000 g/mol.
3. The preparation method according to claim 1, wherein the cisplatin solution has a mass concentration of 0.5 to 2mg/mL, and the polyethyleneimine solution has a mass concentration of 0.5 to 10 mg/mL.
4. A production method according to any one of claims 1 to 3, wherein the mass ratio of cisplatin in the cisplatin solution to polyethyleneimine in the polyethyleneimine solution is 0.2 to 0.6: 1.
5. The preparation method according to claim 1, wherein the crosslinking is carried out under stirring conditions, wherein the stirring speed is 200-800 r/min, the time is 1-4 h, and the temperature is 60-80 ℃.
6. The preparation method according to claim 1, wherein the polymer in the polymer solution comprises polyglutamic acid grafted polyethylene glycol, polyaspartic acid grafted polyethylene glycol, polyglutamic acid block polyethylene glycol or polyaspartic acid block polyethylene glycol, and the number average molecular weight of the polymer in the polymer solution is 10000-100000 g/mol; the concentration of the polymer solution is 1-20 mg/mL.
7. The preparation method according to claim 1 or 6, wherein the mass ratio of the polyethyleneimine in the polyethyleneimine solution to the polymer in the polymer solution is 1: 0.5-10.
8. The preparation method according to claim 1, wherein the compounding is carried out under stirring conditions, wherein the stirring speed is 200-800 r/min, the time is 2-6 h, and the temperature is 20-30 ℃.
9. The polymer-coated cisplatin nanoparticle prepared by the preparation method of any one of claims 1-8.
10. The polymer-coated cisplatin nanoparticle of claim 9, in its application in preparing antineoplastic agent.
CN201910923324.5A 2019-09-27 2019-09-27 Polymer-coated cisplatin nanoparticle and preparation method and application thereof Active CN110742874B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910923324.5A CN110742874B (en) 2019-09-27 2019-09-27 Polymer-coated cisplatin nanoparticle and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910923324.5A CN110742874B (en) 2019-09-27 2019-09-27 Polymer-coated cisplatin nanoparticle and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN110742874A CN110742874A (en) 2020-02-04
CN110742874B true CN110742874B (en) 2021-12-24

Family

ID=69277259

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910923324.5A Active CN110742874B (en) 2019-09-27 2019-09-27 Polymer-coated cisplatin nanoparticle and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN110742874B (en)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102813937A (en) * 2012-06-12 2012-12-12 天津大学 Hydrophobic drug containing polyelectrolyte complex, its preparation method and application thereof
CN104812369B (en) * 2012-10-26 2017-11-24 心悦生医股份有限公司 Core-shell particle and its manufacture method and the composition containing core-shell particle
CN107028873A (en) * 2017-03-31 2017-08-11 福州大学 A kind of hydrogel medicine-carried system for loading cis-platinum and preparation method thereof

Also Published As

Publication number Publication date
CN110742874A (en) 2020-02-04

Similar Documents

Publication Publication Date Title
Song et al. Methoxypoly (ethylene glycol)‐block‐poly (L‐glutamic acid)‐loaded cisplatin and a combination with iRGD for the treatment of non‐small‐cell lung cancers
Yang et al. Dual-targeting hybrid nanoparticles for the delivery of SN38 to Her2 and CD44 overexpressed human gastric cancer
CN103920153A (en) Chitosan-modified pH responsive medicine-loading controlled release material and preparation method thereof
CN108144067B (en) Tetravalent platinum compound-bicyclic double-bond amphiphilic polymer prodrug, nano micelle, preparation method and application thereof
Song et al. Erythrocyte-biomimetic nanosystems to improve antitumor effects of paclitaxel on epithelial cancers
JP4991563B2 (en) Dosage form in which hydrophobic anticancer agent is encapsulated inside bile acid-chitosan complex and method for producing the same
CN108178803B (en) Preparation of drug-loaded cinnamaldehyde-glucan polymer self-assembled nanoparticles and anti-tumor application thereof
CN103357022A (en) Bifunctional polyethylene glycol and adriamycin conjugate and preparation method thereof
Zhu et al. Reduction-responsive core-crosslinked hyaluronic acid-b-poly (trimethylene carbonate-co-dithiolane trimethylene carbonate) micelles: synthesis and CD44-mediated potent delivery of docetaxel to triple negative breast tumor in vivo
WO2022052413A1 (en) Drug-loaded polymer vesicle having asymmetric membrane structure, preparation method therefor, and application thereof in preparation of drugs for treating acute myeloid leukemia
CN102600082B (en) Preparation method of tumor targeting nano-preparation
CN113018251A (en) Dual-drug controlled release system with pH and glutathione dual responses and preparation method thereof
TWI374742B (en)
Cheng et al. Cisplatin-cross-linked and oxygen-resupply hyaluronic acid-based nanocarriers for chemo-photodynamic therapy
CN101708337A (en) Preparation method of human serum albumin nano granules coated with oxaliplatin
CN109464676A (en) A kind of preparation method and product of the photosensitive targeted nano granule of chitosan oligosaccharide
CN102793671A (en) Human recombinant epidermal growth factor (hrEGF)-modified cisplatin-loaded polymeric nanoparticles and preparation method and application thereof
CN110742874B (en) Polymer-coated cisplatin nanoparticle and preparation method and application thereof
Zhu et al. Combined immunochemotherapy achieving targeted co-delivery of chlorogenic acid and doxorubicin by sialic acid-modified liposomes enhances anti-cancer efficacy
WO2020053445A1 (en) Drug delivery sytem for platinum-based drugs
CN115844822A (en) Oral drug-loaded micelle composition and preparation method thereof
CN105037739A (en) Reduced sensitive type polymer with effect of arginine membrane penetration as well as preparation method and application of reduced sensitive type polymer
CN109276720B (en) Metal-organic complex nano material and preparation method and application thereof
CN109666087B (en) Cyclodextrin derivative and preparation method and application thereof
CN112274495A (en) H2O2Preparation method and application of self-supply type calcium peroxide loaded curcumin nanoparticles

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