CN110974973A - Cationic poly-prodrug polymer, preparation method and application thereof - Google Patents
Cationic poly-prodrug polymer, preparation method and application thereof Download PDFInfo
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- CN110974973A CN110974973A CN201911363620.0A CN201911363620A CN110974973A CN 110974973 A CN110974973 A CN 110974973A CN 201911363620 A CN201911363620 A CN 201911363620A CN 110974973 A CN110974973 A CN 110974973A
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- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
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Abstract
A cationic poly-prodrug polymer, a preparation method and application thereof. The preparation method of the cationic poly-prodrug polymer PCB-L-X comprises the following steps: and bonding polycarboxyl betaine methacrylate (PCB) and the micromolecular drug X through a single bond or a connecting molecule containing a bifunctional group to obtain the PCB-L-X loaded with the micromolecular drug. The PCB-L-X has positive charges, can load gene therapy medicines to form a combined delivery carrier, realizes the common loading of the gene therapy medicines and small molecule medicines, and is used for preparing cooperative treatment medicines for related diseases caused by gene abnormal expression. The use of cationic polymers and hydrophobic polymers is effectively reduced by the PCB-L-X, the drug loading rate of the combined delivery carrier can be effectively improved, the metabolic burden of a patient is reduced, and the safety is high.
Description
Technical Field
The invention relates to the technical field of drug-loaded polymer molecules, in particular to a cationic predrug polymer molecule, and a preparation method and application thereof.
Background
In the treatment of genetic diseases, malignant tumors, neurodegenerative diseases, cardiovascular diseases or infectious diseases and the like, the curative effect of single drug treatment is not ideal. Therefore, the combination of drugs is mostly used in clinic, such as by combining gene therapy drugs and small molecule drugs aiming at different targets to exert synergistic effect to treat diseases. However, the application of the two drugs is severely limited by their own shortcomings, wherein the small molecule drugs have short blood half-life and poor tissue selectivity; gene therapy drugs are generally poor in stability, short in blood half-life, tissue selective and poorly accessible. The combined delivery carrier simultaneously entraps two drugs, can overcome the defects of the two drugs, and delivers the two drugs into the same cells according to a certain proportion, thereby improving the synergistic drug effect of the two drugs. Therefore, the development of safe and efficient gene therapy drug and small molecule drug combination delivery vehicles is the key to synergistic therapy.
At present, a triblock polymer is commonly used for carrying out combined delivery of gene therapy medicines and small molecule medicines, wherein in the triblock polymer, a hydrophobic block loads hydrophobic small molecule medicines, a cationic block adsorbs the gene therapy medicines, and a hydrophilic block is used for maintaining the stability of a system. However, most of the materials in the triblock polymer are inert materials, so that the carrier drug loading rate is low, the metabolic burden of a patient is increased, and a great obstacle is brought to practical application.
Therefore, a new material needs to be developed to improve the drug loading rate of the combined delivery carrier of the gene therapy drug and the small molecule drug and realize safe and efficient treatment of diseases.
Disclosure of Invention
In view of the above, the present invention is directed to a cationic poly-prodrug polymer, a preparation method and applications thereof, which are intended to at least partially solve at least one of the above-mentioned technical problems.
In order to achieve the purpose, the technical scheme of the invention is as follows:
as an aspect of the present invention, there is provided a cationic poly prodrug polymer, which is PCB-L-X, having a structure represented by the following formula (1);
wherein, X is a micromolecular drug containing hydroxyl, amino or carboxyl;
when X is a small molecule drug containing hydroxyl or amino, L is a single bond;
when X is a small molecule drug containing carboxyl, L is a connecting molecule containing a bifunctional group;
wherein the polymerization degrees m and n are respectively any integer between 1 and 100.
As another aspect of the present invention, there is also provided a method of preparing the cationic poly prodrug polymer as described above:
when X of PCB-L-X is a small molecule drug containing hydroxyl or amino and L is a single bond, the preparation method comprises the following steps:
adding PCB into a first organic solvent, adding a catalyst and X, and reacting to obtain a mixed solution containing polymer PCB-L-X;
performing first dialysis on the mixed solution containing the polymer PCB-L-X to obtain a PCB-L-X polymer;
when X of PCB-L-X is a small molecule drug containing carboxyl and L is a connecting molecule containing a bifunctional group, the preparation method comprises the following steps:
adding X into a first organic solvent, adding a catalyst and PCB-L, and reacting to obtain a mixed solution containing polymer PCB-L-X;
and carrying out first dialysis on the mixed solution containing the polymer PCB-L-X to obtain the polymer PCB-L-X.
As a further aspect of the invention, there is also provided the use of a cationic predrug polymer as described above in the preparation of a combination delivery vehicle.
As a further aspect of the present invention, there is also provided a method of preparing a combination delivery vehicle comprising the steps of:
self-assembling the cationic poly-prodrug polymer to obtain the PCB-L-X of the nano-particles;
compounding the PCB-L-X of the nano-particles with a gene therapy drug to obtain a combined delivery carrier;
preferably, the ratio of PCB-L-X of the nano particles to N/P of the gene therapy medicine is (0.01-100) to 1;
preferably, the compounding time is 0.01 to 12 hours;
preferably, the compounding temperature is 4 ℃ to 60 ℃.
In yet another aspect of the present invention, there is provided a combination delivery vehicle prepared by the method of making a combination delivery vehicle as described above.
As a further aspect of the present invention, there is also provided the use of a combination delivery vector as described above for the preparation of a medicament for the treatment of a disease associated with aberrant gene expression;
preferably, the related diseases comprise one or more of malignant tumor and neurodegenerative disease;
preferably, the malignant tumor is one of breast cancer, ovarian cancer, liver cancer, lung cancer, colon cancer, esophageal cancer, stomach cancer, colorectal cancer, nasopharyngeal carcinoma, brain tumor, cervical cancer, leukemia and bone cancer;
preferably, the neurodegenerative disease is one of alzheimer's disease, parkinson's disease, huntington's disease, amyotrophic lateral sclerosis, brain injury, and cerebral ischemia.
Based on the technical scheme, the invention has one or part of the following beneficial effects:
(1) compared with the prior art that a triblock polymer is used as a delivery carrier, the invention adopts PCB-L-X as the delivery carrier, and the PCB and the micromolecule drug X are bonded through a single bond or a connecting molecule containing a bifunctional group to obtain the PCB-L-X loaded with the micromolecule drug, and the PCB-L-X has positive charges and can load gene therapy drugs to form a combined delivery carrier, thereby realizing the common loading of the gene therapy drugs and the micromolecule drugs;
(2) the invention effectively reduces the use of carrier materials of cationic polymer and hydrophobic polymer, can effectively improve the drug loading rate of the combined delivery carrier, reduces the metabolic burden of patients and has high safety;
(3) the combined delivery carrier has good biocompatibility, simple components, convenient operation, low cost and convenient later popularization and application.
Drawings
FIG. 1 is a graph comparing drug loading results for the combination delivery vehicles prepared according to example 2 of the present invention and comparative example 1;
FIG. 2 is a graph showing the drug release results of the combination delivery vehicle prepared in example 2 of the present invention;
FIG. 3 is a graph comparing cytotoxicity results for different N/P ratios of combination delivery vehicles prepared in examples 2, 4-7 and comparative examples 1-5 of the present invention;
FIG. 4 is a graph comparing the results of the cellular level of therapeutic effect of the combination delivery vehicles prepared in example 2 of the present invention and comparative example 1.
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.
As an aspect of the present invention, there is provided a cationic poly prodrug polymer, which is PCB-L-X, having a structure represented by the following formula (1);
wherein, X is a micromolecular drug containing hydroxyl, amino or carboxyl;
when X is a small molecule drug containing hydroxyl or amino, L is a single bond;
when X is a small molecule drug containing carboxyl, L is a connecting molecule containing a bifunctional group;
wherein the polymerization degrees m and n are respectively any integer between 1 and 100;
preferably, the polymerization degrees m and n are respectively any integer between 5 and 50;
preferably, the polymerization degrees m and n are each an arbitrary integer of 10 to 30.
In the embodiment of the invention, L is one of ethylenediamine, ethylene glycol and propylenediamine;
x is one of adriamycin, camptothecin, retinoic acid, paclitaxel, doxorubicin, simvastatin, calicheamicin, topotecan, cytarabine, curcumin, epigallocatechin gallate, cyclophosphamide and cyclosporine A.
As another aspect of the present invention, there is also provided a method for preparing the cationic poly-prodrug polymer as described above,
when X of PCB-L-X is a small molecule drug (X-NH) containing hydroxyl or amino2And X-OH), when L is a single bond, the preparation method comprises the following steps:
adding PCB into a first organic solvent, adding a catalyst and X, and stirring for reaction to obtain a mixed solution containing polymer PCB-L-X;
performing first dialysis on the mixed solution containing the polymer PCB-L-X, and freeze-drying to obtain a PCB-L-X polymer;
when X of PCB-L-X is small molecule drug (X-COOH) containing carboxyl and L is connecting molecule containing bifunctional group, the preparation method comprises the following steps:
adding X into a first organic solvent, adding a catalyst and PCB-L, and stirring for reaction to obtain a mixed solution containing polymer PCB-L-X;
and (3) carrying out first dialysis on the mixed solution containing the polymer PCB-L-X, and freeze-drying to obtain the PCB-L-X polymer.
In the embodiment of the invention, the PCB-L is prepared by the following steps:
adding the PCB into a first organic solvent, adding a catalyst and L, and reacting to obtain a PCB-L mixed solution;
dialyzing the mixed solution of the PCB-L, and freeze-drying to obtain the PCB-L;
wherein the adding molar ratio of the PCB to the L is 1 to (1-200);
the adding molar ratio of the PCB-L to the X is 1: 1-200;
when X is a hydroxyl-or amino-containing micromolecule drug, the adding molar ratio of PCB to X is 1: 1-200.
In an embodiment of the invention, the catalyst comprises one of 4-Dimethylaminopyridine (DMAP)/1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), thionyl chloride, Dicyclohexylcarbodiimide (DCC)/4-Dimethylaminopyridine (DMAP), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS);
wherein the adding molar ratio of the catalyst to the PCB is 1: 1-500;
wherein the first organic solvent is dimethyl sulfoxide;
wherein the reaction temperature is 10-60 ℃; preferably, the reaction temperature is 20-50 ℃; preferably, the reaction temperature is 25 ℃ to 37 ℃;
wherein the reaction time is 4-48 hours; preferably, the reaction time is 12 to 36 hours;
wherein the step of performing first dialysis on the mixed solution containing the polymer PCB-L-X comprises the steps of adding the mixed solution containing the polymer PCB-L-X into a dialysis bag, and performing dialysis for 12 to 48 hours by taking a dialysis organic solvent and/or water as an external phase;
wherein the cut-off molecular weight of the first dialysis is 1000 Da-14000 Da;
wherein, the dialysis organic solvent used in the first dialysis is dimethyl sulfoxide, ethanol, methanol or N, N-dimethylformamide.
As a further aspect of the invention, there is also provided the use of a cationic predrug polymer as described above in the preparation of a combination delivery vehicle.
As a further aspect of the present invention, there is also provided a method of preparing a combination delivery vehicle comprising the steps of:
self-assembling the cationic poly-prodrug polymer to obtain the PCB-L-X of the nano-particles;
compounding the PCB-L-X of the nano-particles with a gene therapy drug to obtain a combined delivery carrier;
preferably, the ratio of PCB-L-X of the nano particles to N/P of the gene therapy medicine is (0.01-100) to 1; preferably, the N/P ratio of the PCB-L-X of the nano particles to the gene therapy medicine is (0.2-30) to 1; preferably, the N/P ratio of the PCB-L-X of the nano particles to the gene therapy medicine is (0.5-20) to 1;
preferably, the compounding time is 0.01 to 12 hours; preferably, the compounding time is 0.1 to 6 hours; preferably, the compounding time is 0.5 to 3 hours;
preferably, the compounding temperature is 4-60 ℃; preferably, the compounding temperature is 20-50 ℃; preferably, the compounding temperature is 25 ℃ to 37 ℃.
In an embodiment of the present invention, when X is hydrophilic, the self-assembly step specifically includes: dissolving PCB-L-X in the assembled water phase, and stirring to obtain PCB-L-X of the nano-particles;
when X is hydrophobic, the self-assembly step specifically comprises: dissolving PCB-L-X in a second organic solvent, dropwise adding an assembly water phase into the second organic solvent, stirring, and performing second dialysis to remove the second organic solvent to obtain PCB-L-X of the nanoparticles;
wherein the second organic solvent comprises one of ethanol, methanol, propanol, dimethyl sulfoxide and dimethylformamide;
wherein the mass-volume ratio of the PCB-L-X to the second organic solvent or the assembled water phase is 1/(0.01-100) mg/mL; preferably, the mass-volume ratio of the PCB-L-X to the second organic solvent or the assembled water phase is 1/(0.1-50) mg/mL; preferably, the mass-volume ratio of the PCB-L-X to the second organic solvent or the assembled water phase is 1/(0.5-5) mg/mL;
wherein the self-assembling aqueous phase comprises one of water, phosphate buffer (pH 7.4), and physiological saline;
wherein the self-assembly time is 0.01-6 hours; preferably, the self-assembly time is 0.1 to 6 hours; preferably, the self-assembly time is 0.5 to 3 hours;
wherein the second dialysis step specifically comprises: taking the second dialysis water phase as an external phase, and carrying out the dialysis for 0.01 to 24 hours; preferably, the second dialysis is performed for 0.1 to 6 hours; preferably, the second dialysis is performed for 0.5 to 3 hours.
Wherein the second dialysis water phase comprises one of water, phosphate buffer (pH 7.4) and physiological saline; preferably, the second dialysis aqueous phase is physiological saline.
In yet another aspect of the present invention, there is provided a combination delivery vehicle prepared by the method of making a combination delivery vehicle as described above.
As a further aspect of the present invention, there is also provided the use of a combination delivery vector as described above for the preparation of a medicament for the treatment of a disease associated with aberrant gene expression;
preferably, the related diseases comprise one or more of malignant tumor and neurodegenerative disease;
preferably, the malignant tumor is one of breast cancer, ovarian cancer, liver cancer, lung cancer, colon cancer, esophageal cancer, gastric cancer, carcinoma of large intestine, nasopharyngeal carcinoma, brain tumor, cervical cancer, leukemia, and bone cancer;
preferably, the neurodegenerative disease is one of alzheimer's disease, parkinson's disease, huntington's disease, amyotrophic lateral sclerosis, brain injury, and cerebral ischemia.
The cationic polyprodrug polymer, the preparation method and the application thereof are further illustrated by the following specific examples and the attached drawings.
Example 1: preparation of combined delivery carrier of gene therapeutic drug and small molecule drug camptothecin
6mg of cyanoisopropyl dithiobenzoate, 311.1mg of carboxybetaine methacrylate (CB) and 1.5mg of azobisisobutyronitrile were dissolved in methanol, and oxygen was removed from the reaction flask and the mixture, and the mixture was stirred at 60 ℃ under nitrogen for 24 hours. Dialyzing with water as external phase for 24 hr, and freeze drying to obtain polycarboxyl betaine methacrylate (PCB) with theoretical degree of polymerization of 5050)。
40mg of polycarboxybetaine methacrylate (PCB) with a degree of polymerization of 5050) 255.8mg of 4-dimethylaminopyridine and 481.8mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimides hydrochloride are dissolved in dimethyl sulphoxide. 303.8mg of Camptothecin (CPT) is dissolved in dimethyl sulfoxide, and then the mixture is dropwise added into the solution to be stirred and reacted for 48 hours at the temperature of 25 ℃. Dialyzing the product with dimethyl sulfoxide and water as external water phase for 24h, and freeze drying for 48h to obtain final PCB25-CPT25And (3) obtaining the product.
At 25 ℃, the cationic poly-prodrug polymer molecule PCB25-CPT25Dissolved in dimethyl sulfoxide to a concentration of 1 mg/mL. Under stirring, 400. mu.L of the organic phase was added dropwise to 2mL of physiological saline, and stirring was continued for 30 min. Dialyzing with physiological saline as external water phase for 6h to obtain camptothecin-loaded carrier. The camptothecin-loaded carrier and the gene therapy drug Plk1 siRNA are mixed according to the N/P of 5/1, and the mixture is kept still for 30min, thus obtaining the combined delivery carrier of the gene therapy drug and the small molecule drug camptothecin in the embodiment 1.
Example 2: preparation of combined delivery carrier of gene therapy medicine and small molecule medicine simvastatin
Polycarboxybetaine methacrylate (PCB) with a theoretical degree of polymerization of 50 was prepared using the method for preparing PCB described in example 150)。
400mg of polycarboxybetaine methacrylate (PCB) having a degree of polymerization of 5050) 255.8mg of 4-dimethylaminopyridine and 518.5mg of dicyclohexylcarbodiimide were dissolved in dimethyl sulfoxide. 730.2mg Simvastatin (SIM)30) Dissolving in dimethyl sulfoxide, dripping the mixture dropwise, and reacting at 25 deg.C for 48 hr under stirring. The product is respectively taken dimethyl sulfoxide and water as the externalDialyzing the water phase for 24h, and freeze-drying for 48h to obtain the final PCB20-SIM30And (3) obtaining the product.
At 25 ℃, the cationic poly-prodrug polymer molecule PCB20Simvastatin (SIM)30) Dissolved in dimethyl sulfoxide to a concentration of 1 mg/mL. Under stirring, 600. mu.L of the organic phase was added dropwise to 2mL of physiological saline, and stirring was continued for 30 min. And dialyzing for 6 hours by taking normal saline as an external water phase to obtain the simvastatin-loaded carrier. And mixing the simvastatin-loaded vector with the gene therapy drug let-7b antisense oligonucleotide according to the N/P of 5/1, and standing for 30min to obtain the combined delivery vector of the gene therapy drug and the small molecule drug simvastatin in the embodiment 2.
Example 3: preparation of combined delivery carrier of gene therapy medicine and small molecular medicine tretinoin
Polycarboxybetaine methacrylate (PCB) with a theoretical degree of polymerization of 100 was prepared using the method for preparing PCB described in example 1100)。
200mg of polycarboxybetaine methacrylate (PCB) with a degree of polymerization of 100100) 319.7mg of 4-dimethylaminopyridine and 602.2mg of 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride are dissolved in dimethyl sulphoxide. 52.4mg of Ethylenediamine (EA) was dissolved in dimethyl sulfoxide, and the mixture was added dropwise thereto, followed by stirring at 25 ℃ for 48 hours. Dialyzing the product with dimethyl sulfoxide and water as external water phase for 24h, and freeze drying for 48h to obtain final PCB50-EA。
262.1mg of tretinoin (Retinoic acid, RA), 319.7mg of 4-dimethylaminopyridine and 602.2mg of 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride are dissolved in dimethyl sulfoxide. PCB of 226mg50After EA is dissolved in dimethyl sulfoxide, the mixed solution is dropwise added, and the reaction is carried out for 24 hours under the condition of 37 ℃. Dialyzing the product with dimethyl sulfone and water as external water phase for 48h, and freeze drying for 48h to obtain final PCB50-EA-RA。
Cationic poly-prodrug polymer PCB (printed Circuit Board) at 37 DEG C50EA-RA was dissolved in dimethyl sulfoxide at a concentration of 2 mg/mL. Under the condition of stirring200. mu.L of the organic phase was added dropwise to 2mL of physiological saline, and the mixture was stirred for 30 min. And dialyzing for 8h by using normal saline as an external water phase to obtain the tretinoin-loaded carrier. The tretinoin-loaded vector and the gene therapy drug Sox9siRNA were mixed according to the N/P of 10/1, and left to stand for 30min to obtain the combined delivery vector of the gene therapy drug and the small molecule drug tretinoin of example 3.
Examples 4 to 7: preparation of combined delivery carrier of gene therapeutic drugs and small molecule drug simvastatin with different N/P
The combined delivery vehicle of the gene therapy drug and the small molecule drug simvastatin of examples 4-7 was prepared by the method described in example 2.
The difference is that the ratio N/P of the simvastatin-loaded vector and the gene therapy drug in examples 4-7 is 1: 1, 3: 1, 8: 1 and 10: 1, respectively.
Comparative example 1: physical embedding carrier
Polycarboxybetaine methacrylate (PCB) with a theoretical degree of polymerization of 50 was prepared using the method for preparing PCB described in example 150)。
400mg of polycarboxybetaine methacrylate (PCB) having a degree of polymerization of 5050) 255.8mg of 4-dimethylaminopyridine and 481.8mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimides hydrochloride are dissolved in dimethyl sulphoxide. 178.2mg of 1-Hexanol (1-Hexanol) was dissolved in dimethyl sulfoxide, and the mixture was added dropwise thereto, followed by stirring at 25 ℃ for 48 hours. Dialyzing the product with dimethyl sulfoxide and water as external water phase for 24h, and freeze drying for 48h to obtain final PCB25-H25And (3) obtaining the product.
At 25 deg.C, mixing PCB25-H25Dissolved in dimethyl sulfoxide to a concentration of 1 mg/mL. Simvastatin was dissolved in dimethyl sulfoxide at a concentration of 1 mg/mL. Taking 400 mu L of PCB under stirring25-H25And 80 mu L of simvastatin organic phase are mixed evenly, 2mL of physiological saline is dripped into the mixture, and the mixture is stirred for 30 min. And dialyzing for 6 hours by taking normal saline as an external water phase to obtain the simvastatin-loaded carrier. According to the N/P of 5/1, the simvastatin-loaded vector and the gene therapy drug letAnd 7b, mixing the antisense oligonucleotides, and standing for 30min to obtain the physical entrapment combined delivery vector of the gene therapy medicament and the small molecular medicament.
Comparative examples 2 to 5: preparation of physical entrapment combined delivery carrier for gene therapy medicines and small molecular medicine simvastatin with different N/P
The physical entrapment combined delivery carrier of the gene therapy medicine and the small molecule medicine simvastatin in the comparative examples 2 to 5 is prepared by adopting the method of the comparative example 1.
The difference is that the ratio N/P of the simvastatin-loaded vector and the gene therapy drug in comparative examples 2-5 is 1: 1, 3: 1, 8: 1 and 10: 1, respectively.
And (3) performance testing:
test 1: carrier rate of small molecule drug
An ultraviolet-visible spectrophotometer is respectively adopted to measure the carrier rate of the simvastatin physically entrapped in the comparative example 1 and the carrier rate of the simvastatin in the combined delivery carrier of the gene therapy medicament and the small molecule medicament in the example 2, as shown in figure 1, compared with the carrier of the simvastatin physically entrapped, the combined delivery carrier of the gene therapy medicament and the small molecule medicament prepared in the example 2 of the invention effectively improves the medicament loading rate of the medicament.
And (3) testing 2: drug delivery in combination with delivery vehicle
The simvastatin release effect of the combination delivery vehicle prepared in example 2 was measured using High Performance Liquid Chromatography (HPLC). 3mL of the carrier solution was put into a dialysis bag having a molecular weight of 3500, and the bag was put into a 100mL flask, and 80mL of a phosphate buffer and 10U of a phosphate buffer containing esterase were added to the flask, respectively. The vial was placed in a constant temperature shaker at 37 ℃ with the rotation speed adjusted to 100rpm, and 2mL of samples were taken from the vial at preset times of 0h, 10min, 40min, 1h, 4h, 17h, 22h, and 30 h. After the sample is taken, 2mL of the corresponding buffer is added. The simvastatin concentration was calculated from the standard curve and the release profile was plotted. As a result, as shown in FIG. 2, the combination delivery vehicles of the present invention release substantially no drug in the absence of esterase, which is added to facilitate controlled drug release. The release results show that the combined delivery carrier prepared in the example 2 has stable physiological conditions and esterase responsiveness controlled release capacity, improves the concentration of the drug in cells, and lays a foundation for the maximum exertion of the curative effect of the drug.
And (3) testing: cytotoxicity of combination delivery vehicles
Good biocompatibility is the premise of the application of the gene transfection reagent, and the experiment adopts a neural stem cell model to investigate the cytotoxicity of the combined delivery vector under different N/P conditions.
The neural stem cells are divided into 1 × 104The density of (2) was inoculated in a 96-well plate and cultured for 24 hours. The combined delivery vehicles of examples 2 and 4-7 and comparative examples 1-5, which had different N/P ratios, were added to the culture medium at a gene therapy drug concentration of 1. mu.g/mL, and cultured for 24 hours, respectively. mu.L of 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide was added to each well and incubated at 37 ℃ for 4 h. The medium was removed, 100. mu.L of dimethyl sulfoxide was added, and the mixture was incubated at 37 ℃ for 5min to dissolve MTT crystals. And (3) measuring the absorbance value by adopting an enzyme-linked immunosorbent assay detector under the condition that the wavelength is 490nm, and calculating the cell survival rate by taking the untreated cells as reference. The results are shown in FIG. 3, where the combined delivery vector of the present invention survived more than 80% of cells under different N/P conditions, indicating that the gene transfection reagent has good biocompatibility.
And (4) testing: cellular level efficacy results of combination delivery vehicle
Simvastatin and let-7b antisense oligonucleotide can synergistically regulate exogenous neural stem cells, promote the exogenous neural stem cells to secrete brain-derived growth factors, and can treat neurodegenerative diseases Alzheimer's disease.
The neural stem cells are divided into 1 × 105The cells were inoculated in 6-well plates and cultured for 24 hours. The combined delivery vehicle containing 4. mu.g of the gene therapeutic agent prepared in example 2 and having an N/P ratio of 5/1 was added to the medium and cultured at 37 ℃ for 7 days. Collecting the culture medium, and determining the level of the brain-derived neurotrophic factor in the culture medium by adopting an ELISA kit. As shown in FIG. 4, the combined delivery vehicle of the present invention can effectively stimulate the neural stem cells to secrete the brain-derived growth factor, compared with the simvastatin-physically entrapped vehicle of comparative example 1And (4) adding the active ingredients.
In addition, the embodiment and the corresponding test of the invention show that the simvastatin and the let-7b antisense oligonucleotide loaded on the combined delivery carrier have application prospect in the medicine for treating the neurodegenerative disease Alzheimer's disease through the synergistic effect. It is understood that if the known small molecule drugs and gene therapy drugs having synergistic effect on other diseases such as malignant tumor, other neurodegenerative diseases and the like are selected, the same combined delivery effect can be achieved through the combined delivery vector of the invention, so that the combined delivery vector can be applied to the treatment drugs of the related diseases caused by abnormal expression of genes such as malignant tumor, other neurodegenerative diseases and the like.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A cationic polyprodrug polymer, wherein the cationic polyprodrug polymer is PCB-L-X and has a structure represented by the following formula (1);
wherein, X is a micromolecular drug containing hydroxyl, amino or carboxyl;
when X is a small molecule drug containing hydroxyl or amino, L is a single bond;
when X is a small molecule drug containing carboxyl, L is a connecting molecule containing a bifunctional group;
wherein the polymerization degrees m and n are respectively any integer between 1 and 100.
2. The cationic polyprodrug polymer of claim 1,
l is one of ethylenediamine, ethylene glycol and propylenediamine;
x is one of adriamycin, camptothecin, retinoic acid, paclitaxel, doxorubicin, simvastatin, calicheamicin, topotecan, cytarabine, curcumin, epigallocatechin gallate, cyclophosphamide and cyclosporine A.
3. A process for preparing the cationic polyprodrug polymer of claim 1 or 2, wherein:
when X of PCB-L-X is a small molecule drug containing hydroxyl or amino and L is a single bond, the preparation method comprises the following steps:
adding PCB into a first organic solvent, adding a catalyst and X, and reacting to obtain a mixed solution containing polymer PCB-L-X;
performing first dialysis on the mixed solution containing the polymer PCB-L-X to obtain a PCB-L-X polymer;
when X of PCB-L-X is a small molecule drug containing carboxyl and L is a connecting molecule containing a bifunctional group, the preparation method comprises the following steps:
adding X into a first organic solvent, adding a catalyst and PCB-L, and reacting to obtain a mixed solution containing polymer PCB-L-X;
and carrying out first dialysis on the mixed solution containing the polymer PCB-L-X to obtain the polymer PCB-L-X.
4. The method of claim 3, wherein said PCB-L is prepared by the steps of:
adding the PCB into a first organic solvent, adding a catalyst and L, and reacting to obtain a PCB-L mixed solution;
dialyzing the mixed solution of the PCB-L to obtain the PCB-L;
wherein the adding molar ratio of the PCB to the L is 1 to (1-200);
the adding molar ratio of the PCB-L to the X is 1: 1-200;
when X is a hydroxyl-or amino-containing micromolecule drug, the adding molar ratio of PCB to X is 1: 1-200.
5. The method of claim 3 or 4, wherein said catalyst comprises one of 4-dimethylaminopyridine/1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, thionyl chloride, dicyclohexylcarbodiimide/4-dimethylaminopyridine, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide;
wherein the adding molar ratio of the catalyst to the PCB is 1: 1-500;
wherein the first organic solvent is dimethyl sulfoxide;
wherein the reaction temperature is 10-60 ℃, and the reaction time is 4-48 hours;
wherein the step of performing the first dialysis on the mixed solution containing the polymer PCB-L-X comprises the steps of adding the mixed solution containing the polymer PCB-L-X into a dialysis bag, and performing dialysis for 12 to 48 hours by taking a dialysis organic solvent and/or water as an external phase;
wherein the cut-off molecular weight of the first dialysis is 1000 Da-14000 Da;
wherein, the dialysis organic solvent used in the first dialysis is dimethyl sulfoxide, ethanol, methanol or N, N-dimethylformamide.
6. Use of a cationic polyprodrug polymer according to claim 1 or 2 for the preparation of a combination delivery vehicle.
7. A method of making a combination delivery vehicle comprising the steps of:
self-assembling the cationic polyprodrug polymer of claim 1 or 2 to give PCB-L-X of nanoparticles;
compounding the PCB-L-X of the nano-particles with a gene therapy drug to obtain a combined delivery carrier;
preferably, the ratio of PCB-L-X of the nano particles to N/P of the gene therapy medicine is (0.01-100) to 1;
preferably, the compounding time is 0.01 to 12 hours;
preferably, the compounding temperature is 4 ℃ to 60 ℃.
8. The method of preparing the combination delivery vehicle of claim 7, wherein when X is hydrophilic, the self-assembling step comprises: dissolving PCB-L-X in the assembled water phase, and stirring to obtain PCB-L-X of the nano-particles;
when X is hydrophobic, the self-assembly step specifically comprises: dissolving PCB-L-X in a second organic solvent, dropwise adding an assembly water phase into the second organic solvent, stirring, and performing second dialysis to remove the second organic solvent to obtain PCB-L-X of the nanoparticles;
wherein the second organic solvent comprises one of ethanol, methanol, propanol, dimethyl sulfoxide and dimethylformamide;
wherein the mass-volume ratio of the PCB-L-X to the second organic solvent or the assembled water phase is 1/(0.01-100) mg/mL;
wherein the self-assembling aqueous phase comprises one of water, phosphate buffer, and physiological saline;
wherein the self-assembly time is 0.01-6 hours;
wherein the second dialysis step specifically comprises: taking the second dialysis water phase as an external phase, and carrying out the dialysis for 0.01 to 24 hours;
wherein the second dialysis water phase comprises one of water, phosphate buffer solution and physiological saline.
9. A combination delivery vehicle prepared by the method of claim 7 or 8.
10. Use of a combination delivery vector according to claim 9 for the manufacture of a medicament for the treatment of a disease associated with aberrant gene expression;
preferably, the related diseases comprise one or more of malignant tumor and neurodegenerative disease;
preferably, the malignant tumor is one of breast cancer, ovarian cancer, liver cancer, lung cancer, colon cancer, esophageal cancer, stomach cancer, colorectal cancer, nasopharyngeal carcinoma, brain tumor, cervical cancer, leukemia and bone cancer;
preferably, the neurodegenerative disease is one of alzheimer's disease, parkinson's disease, huntington's disease, amyotrophic lateral sclerosis, brain injury, and cerebral ischemia.
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| CN106267229A (en) * | 2016-08-12 | 2017-01-04 | 南开大学 | A kind of Liver targeting carries structure of platinum nano-prodrug and preparation method thereof |
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| CN106267229A (en) * | 2016-08-12 | 2017-01-04 | 南开大学 | A kind of Liver targeting carries structure of platinum nano-prodrug and preparation method thereof |
| CN107970453A (en) * | 2017-12-05 | 2018-05-01 | 北京林业大学 | A kind of double targeted delivery methods of the pectin nano-particle of modified with folic acid |
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