CN109608646B - Nano-carrier for targeted therapy of myocardial injury and application - Google Patents
Nano-carrier for targeted therapy of myocardial injury and application Download PDFInfo
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Abstract
The invention discloses a nano-carrier for targeted treatment of myocardial injury and application thereof, belonging to the technical field of biomedicine. The nano-composite of the invention carries out targeted repair on heart injury after myocardial infarction: not only can reduce the side effect of tanshinone IIA on other histiocytes, but also can ensure that tanshinone IIA is properly released.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a nano-carrier for targeted therapy of myocardial injury and application thereof.
Background
Myocardial infarction, which is the leading cause of mortality and morbidity worldwide, can result in the loss of up to 10 million myocardial cells. Due to the very limited ability of the myocardium to regenerate after injury, the lost cells are replaced by fibrotic scar tissue, resulting in heart failure. Although surgical treatment reduces mortality associated with myocardial infarction, cardiac function is impaired because infarcted cardiomyocytes cannot be regenerated. Despite the extensive efforts made to prevent, limit or treat heart failure, to date, there is no treatment that can successfully restore the function of the injured heart.
It has been reported that small drug molecules have the potential to be used in myocardial generation. Over the past few years, they have been found to be particularly effective in relieving symptoms of myocardial infarction, angina pectoris, arrhythmia, hypertension and other cardiovascular diseases, as tanshinone IIA, which contains a estrogen ring (phytoestrogen), extracted from salvia miltiorrhiza, is of great interest, and is widely used by chinese medical practitioners for anti-oxidation, anti-inflammation and anti-hyperplasia.
In acute myocardial ischemia, the ischemic myocardium produces atrial peptides to reduce stress on the left ventricular wall, thereby inducing repair and healing of myocardial infarction. Atrial peptides can be expressed by both cardiomyocytes and non-myocytes, such as cardiac fibroblasts, by binding to specific receptors.
The application of micro-nano carrier materials and nano technology in the treatment of cardiovascular diseases is a relatively new field, and the micro-nano carrier materials and the nano technology are a promising treatment and diagnosis method. These carriers can be used as delivery carriers for short-lived cytokines, growth factors or water-soluble molecular drugs, and due to the material properties and tunable properties, the main advantages of the delivery carrier system are: the payload protects, prolongs and enhances the drug treatment effect, reduces the drug management frequency, thereby improving the compliance of patients. The nano micelle can provide stable shell protection for the treatment medicament without being degraded, has good biocompatibility and targeting property, and is often used as a medicament carrier.
Traditional drug therapy and surgical therapy have seriously reduced the mortality rate of myocardial infarction, but the infarcted cardiomyocytes cannot regenerate, thereby impairing the function of the heart. In addition, stem cells transplanted in ischemic heart tissue are affected by apoptosis and necrosis due to oxidative and inflammatory microenvironments of the infarct zone, severely limiting the therapeutic efficiency of stem cell transplantation. The traditional nano material has no specificity when carrying drugs, which causes a certain toxic and side effect on normal cells.
Therefore, a nano carrier which can load tanshinone IIA and treat myocardial damage in a targeted manner is needed to be prepared, so that the toxic and side effects of tanshinone IIA are relieved, and the release rate of tanshinone IIA is controlled.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a nano-carrier for targeted treatment of myocardial damage and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that: a preparation method of a nano carrier loaded with tanshinone IIA sequentially comprises the following steps:
s1) dissolving the carboxylated polylactic acid-glycolic acid into dichloromethane, adding N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and activating to generate polylactic acid-glycolic acid-N-hydroxysuccinimide; after the activation reaction, sequentially carrying out glacial ethyl ether precipitation, dialysis (dialysis for removing redundant N-hydroxysuccinimide) and vacuum drying; the polylactic acid-glycolic acid-N-hydroxysuccinimide is abbreviated as PLG-NHS, and the PLG-NHS is a polymer with the structure shown in the formula (I);
wherein n is more than or equal to 2;
s2) dissolving PLG-NHS into chloroform, adding amino polyethylene glycol carboxyl and N, N-diisopropylethylamine, and activating to generate polylactic acid-glycolic acid-polyethylene glycol; after the activation reaction, the reaction mixture was subjected to ice-methanol precipitation and ice-methanol washing (to remove excess NH) in this order2-PEG-COOH) and vacuum drying; polylactic acid-glycolic acid-polyethylene glycol is abbreviated as PLGA-PEG, PLGA-PEG is a polymer with a structure shown in formula (II), amino polyethylene glycol carboxyl is abbreviated as NH2-PEG-COOH；
Wherein n is more than or equal to 2, and m is more than or equal to 1;
s3) dissolving atrial peptides into a phosphate buffer solution to prepare an atrial peptide solution; adding the atrial natriuretic peptide solution into PLGA-PEG solution (deionized water as solvent) with adjusted pH value, and reacting at room temperature to generate polylactic acid-glycolic acid-polyethylene glycol-atrial natriuretic peptide; after the reaction is finished, transferring the mixture into an ultrafiltration centrifugal tube for ultrafiltration (removing unreacted polymers and polypeptides), and then freeze-drying the mixture; polylactic acid-glycolic acid-polyethylene glycol-atrial natriuretic peptide is abbreviated as PLGA-PEG-ANP, PLGA-PEG-ANP is a polymer with the structure shown in the formula (III), and in the PLGA-PEG-ANP, amino at the tail end of atrial natriuretic peptide and hydroxyl in PLGA-PEG are subjected to dehydration condensation reaction;
wherein n is more than or equal to 2, m is more than or equal to 1, and ANP represents atrial natriuretic peptide.
As an improvement of the technical proposal, in the step S1, the mass ratio of the polylactic acid-glycolic acid, the N-hydroxysuccinimide and the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is (0.1-1) to (1-10) 1, the ratio of the total volume of the dichloromethane, the polylactic acid-glycolic acid, the N-hydroxysuccinimide and the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the volume of the dichloromethane is 5-20: 1, the activation reaction time is 4-12 h, the ratio of the total volume of the dichloromethane, the polylactic acid-glycolic acid, the N-hydroxysuccinimide and the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the volume of the glacial ethyl ether is 1 (10-20), the dialysis is carried out by adopting a dialysis bag with the molecular weight cutoff of 500-3500, the vacuum drying temperature is 40-60 ℃, and the vacuum drying time is 12-24 hours.
Preferably, the mass ratio of polylactic acid-glycolic acid, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 0.25:2:1, the ratio of the total volume of dichloromethane, polylactic acid-glycolic acid, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the volume of dichloromethane is 10:1, the time of the activation reaction is 8h, a dialysis bag with a molecular weight cut-off of 1000 is used for dialysis, and the temperature of vacuum drying is 40 ℃.
As an improvement of the technical scheme, in the step S2, the mass ratio of PLGA-NHS to N, N-diisopropylethylamine is 1 (1-10), the activation reaction time is 4-12 h, the ratio of the total volume of PLGA-NHS, N-diisopropylethylamine, trichloromethane and amino polyethylene glycol carboxyl to the volume of trichloromethane is (5-20) to 1, the ratio of the total volume of PLGA-NHS, N-diisopropylethylamine, trichloromethane and amino polyethylene glycol carboxyl to the volume of glacial methanol is 1 (10-20), the vacuum drying temperature is 40-60 ℃, and the vacuum drying time is 12-24 h.
Preferably, in step S2, the mass ratio of PLGA-NHS to N, N-diisopropylethylamine is 1:1, the activation reaction time is 6h, the ratio of the total volume of PLGA-NHS, N-diisopropylethylamine, chloroform and aminopolyethylene glycol carboxyl to chloroform volume is 10:1, and the temperature of vacuum drying is 40 ℃.
As an improvement of the technical scheme, in step S3, the mass ratio of the atrial natriuretic peptide to the PLGA-PEG is 1 (1-10), the pH value of the PLGA-PEG solution is 6-7, the pH value of the phosphate buffer solution is less than 7.2, the reaction time at normal temperature is 2-6 h, the rotational speed of ultrafiltration is 12000rpm, the ultrafiltration time is 30min, and the freeze drying time is 24-48 h.
Preferably, in step S3, the ratio of the amount of atrial natriuretic peptide to PLGA-PEG is 1:5, and the ultrafiltration centrifugal tube is MWCO 10000.
In addition, the invention also provides a nano-carrier for targeted therapy of myocardial injury, wherein the nano-carrier is polylactic acid-glycolic acid-polyethylene glycol-atrial natriuretic peptide or medicinal acid addition salt thereof prepared by the preparation method, the nano-carrier is a nano micelle, and the diameter of the nano-carrier is 20-80 nm.
In addition, the invention also provides a preparation method of the nano compound loaded with tanshinone IIA, which comprises the following steps: the tanshinone IIA and the nano-carrier are used as raw materials, a thin film hydration method is adopted to prepare the nano-composite loaded with the tanshinone IIA, and the diameter of the nano-composite is 20-80 nm.
As an improvement of the technical scheme, the nano-carrier and the tanshinone IIA are dissolved in acetone, and are mixed and then are subjected to rotary evaporation until a film is formed; after the film is dried, phosphate buffer solution redissolving, water bath heating and dialysis (removing unencapsulated tanshinone IIA by dialysis) are carried out in sequence to obtain a nano compound loaded with tanshinone IIA, wherein the abbreviation of the nano compound is PLGA-PEG-ANP/TAN;
the mass ratio of the nano carrier to the tanshinone IIA is (1-10): 1, the feed-liquid ratio of the tanshinone IIA to the acetone is 8 (1-10) mg/ml, the rotary evaporation temperature is 40-60 ℃, the water bath heating temperature is 40-80 ℃, the water bath heating time is 4-12 hours, a dialysis bag with the molecular weight cutoff of 500-3500 is adopted for dialysis, and the freeze drying time is 24-48 hours; preferably, the mass ratio of the nano-carrier to the tanshinone IIA is 5:1, the feed-liquid ratio of the tanshinone IIA to the acetone is 8:5mg/ml, the rotary evaporation temperature is 40-60 ℃, and dialysis is carried out by using a dialysis bag with the molecular weight cut-off of 3500.
In addition, the invention also provides a nano composite prepared by the preparation method of the nano composite.
In addition, the invention also provides application of the nano-composite in preparing a medicament for targeted treatment of myocardial ischemia injury.
In addition, the invention also provides a medicine containing the nano-composite, which also contains pharmaceutically acceptable auxiliary materials.
The invention has the beneficial effects that: the invention provides a nano-carrier for targeted therapy of myocardial injury and application, the nano-carrier takes polyethylene glycol and polylactic acid-glycollic acid as raw materials, PLGA-PEG is generated through reaction, atrial natriuretic peptide ANP with a myocardial targeted function is grafted, tanshinone IIA (TAN) is loaded on the nano-carrier to form a nano-composite with the diameter of 20-80 nm, and the nano-composite carries out targeted repair on heart injury after myocardial infarction: not only can reduce the side effect of tanshinone IIA on other histiocytes, but also can ensure that tanshinone IIA is properly released; the nanometer carrier loaded with tanshinone IIA medicine can be injected into human body intravenously to repair and regenerate damaged myocardial tissue.
Drawings
FIG. 1 shows PLGA-PEG prepared according to an embodiment of the present invention1H.NMR chart;
FIG. 2 is a diagram of critical micelles of PLGA-PEG-ANP prepared according to an embodiment of the present invention;
FIG. 3 is a transmission electron microscope image of the nano-carrier and the nano-composite prepared by the embodiment of the invention, wherein 3A is the transmission electron microscope image of the nano-carrier, and 3B is the transmission electron microscope image of the nano-composite;
FIG. 4 is a graph showing tanshinone IIA release;
fig. 5 shows the effect of nanocarriers and nanocomplexes on cell viability.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the following detailed description and accompanying drawings.
Example 1
The embodiment provides a preparation method of a nano carrier loaded with tanshinone IIA, which sequentially comprises the following steps:
s1) dissolving the carboxylated polylactic acid-glycolic acid into dichloromethane, adding N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and activating to generate polylactic acid-glycolic acid-N-hydroxysuccinimide; after the activation reaction, sequentially carrying out glacial ethyl ether precipitation, dialysis and vacuum drying; the polylactic acid-glycolic acid-N-hydroxysuccinimide is abbreviated as PLG-NHS, and the PLG-NHS is a polymer with the structure shown in the formula (I);
wherein n is more than or equal to 2;
s2) dissolving PLG-NHS into chloroform, adding amino polyethylene glycol carboxyl and N, N-diisopropylethylamine, and activating to generate polylactic acid-glycolic acid-polyethylene glycol; after the activation reaction, carrying out ice-methanol precipitation, ice-methanol cleaning and vacuum drying in sequence; polylactic acid-glycolic acid-polyethylene glycol is abbreviated as PLGA-PEG, PLGA-PEG is a polymer with a structure shown in formula (II), amino polyethylene glycol carboxyl is abbreviated as NH2-PEG-COOH；
Wherein n is more than or equal to 2, and m is more than or equal to 1;
s3) dissolving atrial peptides into a phosphate buffer solution to prepare an atrial peptide solution; adding the atrial natriuretic peptide solution into the PLGA-PEG solution with the adjusted pH value, and reacting at normal temperature to generate polylactic acid-glycolic acid-polyethylene glycol-atrial natriuretic peptide; after the reaction is finished, transferring the mixture into an ultrafiltration centrifugal tube for ultrafiltration, and then freeze-drying the mixture; polylactic acid-glycolic acid-polyethylene glycol-atrial natriuretic peptide is abbreviated as PLGA-PEG-ANP, PLGA-PEG-ANP is a polymer with the structure shown in the formula (III), and in the PLGA-PEG-ANP, amino at the tail end of atrial natriuretic peptide and hydroxyl in PLGA-PEG are subjected to dehydration condensation reaction;
wherein n is more than or equal to 2, m is more than or equal to 1, and ANP represents atrial natriuretic peptide;
in step S1, the mass ratio of polylactic acid-glycolic acid, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride was 0.25:2:1, the ratio of the total volume of dichloromethane, polylactic acid-glycolic acid, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the volume of dichloromethane was 10:1, the time of activation reaction was 8 hours, the ratio of the total volume of dichloromethane, polylactic acid-glycolic acid, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the volume of iced ether was 1:15, dialysis was performed using a dialysis bag having a molecular weight cut-off of 1000, the temperature of vacuum drying was 40 ℃, the vacuum drying time is 24 h;
in step S2, the mass ratio of PLGA-NHS to N, N-diisopropylethylamine is 1:1, the activation reaction time is 6h, the ratio of the total volume of PLGA-NHS, N-diisopropylethylamine, trichloromethane and amino polyethylene glycol carboxyl to the volume of trichloromethane is 10:1, the ratio of PLGA-NHS, N-diisopropylethylamine, trichloromethane and amino polyethylene glycol carboxyl to the volume of glacial methanol is 1:15, the vacuum drying temperature is 40 ℃, and the vacuum drying time is 24 h;
in step S3, the mass ratio of atrial natriuretic peptide to PLGA-PEG is 1:5, the pH value of PLGA-PEG solution is 6-7, the pH value of phosphate buffer solution is less than 7.2, the time of normal temperature reaction is 2h, the ultrafiltration time is 30min at the rotation speed of 12000rpm, the model of ultrafiltration centrifugal tube is MWCO10000, and the freeze drying time is 24 h.
In addition, this embodiment also provides a preparation method of the nano-composite loaded with tanshinone IIA, which includes the following steps: dissolving the nano-carrier and tanshinone IIA in acetone, mixing, and performing rotary evaporation until a film is formed; after the film is dried, phosphate buffer solution redissolving, water bath heating and dialysis are sequentially carried out, so that the nano compound loaded with the tanshinone IIA is obtained, wherein the abbreviation of the nano compound is PLGA-PEG-ANP/TAN;
the mass ratio of the nano-carrier to the tanshinone IIA is 5:1, the feed liquid ratio of the tanshinone IIA to the acetone is 8:5mg/ml, the rotary evaporation temperature is 40 ℃, the water bath heating time is 12 hours, a dialysis bag with the molecular weight cutoff of 3500 is adopted for dialysis, and the freeze drying time is 36 hours.
Example 2
The embodiment provides a preparation method of a nano carrier loaded with tanshinone IIA, which is different from embodiment 1 in that:
1) in step S1, the mass ratio of polylactic acid-glycolic acid, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride was 0.1:10:1, the ratio of the total volume of dichloromethane, polylactic acid-glycolic acid, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the volume of dichloromethane was 5:1, the time of activation reaction was 4 hours, the ratio of the total volume of dichloromethane, polylactic acid-glycolic acid, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the volume of iced ether was 1:10, dialysis was performed using a dialysis bag having a molecular weight cut-off of 500, the temperature of vacuum drying was 50 ℃, the vacuum drying time is 19 h;
2) in step S2, the mass ratio of PLGA-NHS to N, N-diisopropylethylamine is 1:5, the activation reaction time is 4h, the ratio of the total volume of PLGA-NHS, N-diisopropylethylamine, trichloromethane and amino polyethylene glycol carboxyl to the volume of trichloromethane is 5:1, the ratio of the total volume of PLGA-NHS, N-diisopropylethylamine, trichloromethane and amino polyethylene glycol carboxyl to the volume of glacial methanol is 1:10, the vacuum drying temperature is 50 ℃, and the vacuum drying time is 17 h;
3) in step S3, the mass ratio of atrial natriuretic peptide to PLGA-PEG is 1:1, the pH value of PLGA-PEG solution is 6-7, the pH value of phosphate buffer solution is less than 7.2, the time of normal temperature reaction is 4h, the ultrafiltration time is 30min at the rotation speed of 12000rpm, the model of ultrafiltration centrifugal tube is MWCO10000, and the freeze drying time is 35 h.
In addition, this embodiment also provides a preparation method of the nano-composite loaded with tanshinone IIA, which is different from embodiment 1 in that: the mass ratio of the nano-carrier to the tanshinone IIA is 1:1, the feed liquid ratio of the tanshinone IIA to the acetone is 8:1mg/ml, the rotary evaporation temperature is 50 ℃, the water bath heating temperature is 60 ℃, the water bath heating time is 8 hours, a dialysis bag with the molecular weight cutoff of 2500 is adopted for dialysis, and the freeze drying time is 24 hours.
Example 3
The embodiment provides a preparation method of a nano carrier loaded with tanshinone IIA, which is different from embodiment 1 in that:
1) in step S1, the mass ratio of polylactic acid-glycolic acid, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride was 1:1:1, the ratio of the total volume of dichloromethane, polylactic acid-glycolic acid, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the volume of dichloromethane was 20:1, the time for activation reaction was 12 hours, the ratio of the total volume of dichloromethane, polylactic acid-glycolic acid, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the volume of ice was 1:20, dialysis was performed using a dialysis bag having a molecular weight cutoff of 3500, the temperature for vacuum drying was 60 ℃, the vacuum drying time is 12 h;
2) in step S2, the mass ratio of PLGA-NHS to N, N-diisopropylethylamine is 1:10, the activation reaction time is 12h, the ratio of the total volume of PLGA-NHS, N-diisopropylethylamine, trichloromethane and amino polyethylene glycol carboxyl to the volume of trichloromethane is 20:1, the ratio of the total volume of PLGA-NHS, N-diisopropylethylamine, trichloromethane and amino polyethylene glycol carboxyl to the volume of glacial methanol is 1:20, the vacuum drying temperature is 60 ℃, and the vacuum drying time is 12 h;
3) in step S3, the mass ratio of atrial natriuretic peptide to PLGA-PEG is 1:10, the pH value of PLGA-PEG solution is 6-7, the pH value of phosphate buffer solution is less than 7.2, the time of normal temperature reaction is 6h, the ultrafiltration time is 30min at the rotation speed of 12000rpm, the model of ultrafiltration centrifugal tube is MWCO10000, and the freeze drying time is 48 h.
In addition, this embodiment also provides a preparation method of the nano-composite loaded with tanshinone IIA, which is different from embodiment 1 in that: the mass ratio of the nano-carrier to the tanshinone IIA is 10:1, the feed liquid ratio of the tanshinone IIA to the acetone is 8:10mg/ml, the rotary evaporation temperature is 60 ℃, the water bath heating temperature is 80 ℃, the water bath heating time is 4 hours, a dialysis bag with the molecular weight cut-off of 500 is adopted for dialysis, and the freeze drying time is 48 hours.
Example 4
The embodiment provides a preparation method of a nano carrier loaded with tanshinone IIA, which is different from embodiment 1 in that:
1) in step S1, the mass ratio of polylactic acid-glycolic acid, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride was 1:10:1, the ratio of the total volume of dichloromethane, polylactic acid-glycolic acid, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the volume of dichloromethane was 13:1, the time for activation reaction was 8 hours, the ratio of the total volume of dichloromethane, polylactic acid-glycolic acid, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the volume of glacial ethyl ether was 1:16, a dialysis bag having a molecular weight cut-off of 1000 was used, the temperature for vacuum drying was 40 ℃, the vacuum drying time is 24 h;
2) in step S2, the mass ratio of PLGA-NHS to N, N-diisopropylethylamine is 1:6, the activation reaction time is 6h, the ratio of the total volume of PLGA-NHS, N-diisopropylethylamine, trichloromethane and amino polyethylene glycol carboxyl to the volume of trichloromethane is 14:1, the ratio of the total volume of PLGA-NHS, N-diisopropylethylamine, trichloromethane and amino polyethylene glycol carboxyl to the volume of glacial methanol is 1:13, the vacuum drying temperature is 40 ℃, and the vacuum drying time is 24 h;
3) in step S3, the mass ratio of atrial natriuretic peptide to PLGA-PEG is 1:7, the pH value of PLGA-PEG solution is 6-7, the pH value of phosphate buffer solution is less than 7.2, the time of normal temperature reaction is 6h, the ultrafiltration time is 30min at the rotation speed of 12000rpm, the model of ultrafiltration centrifugal tube is MWCO10000, and the freeze drying time is 48 h.
In addition, this embodiment also provides a preparation method of the nano-composite loaded with tanshinone IIA, which is different from embodiment 1 in that: the mass ratio of the nano-carrier to the tanshinone IIA is 8:1, the feed liquid ratio of the tanshinone IIA to the acetone is 8:7mg/ml, the rotary evaporation temperature is 60 ℃, the water bath heating temperature is 80 ℃, the water bath heating time is 4 hours, a dialysis bag with the molecular weight cutoff of 3500 is adopted for dialysis, and the freeze drying time is 48 hours.
Example 5
The embodiment provides a preparation method of a nano carrier loaded with tanshinone IIA, which is different from embodiment 1 in that:
in step S1, the mass ratio of polylactic acid-glycolic acid, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 0.1:1: 1;
in addition, this embodiment also provides a preparation method of the nano-composite loaded with tanshinone IIA, which is different from embodiment 1 in that: the mass ratio of the nano-carrier to the tanshinone IIA is 9: 1.
Characterization of nanocarriers and nanocomposites
1) Polylactic acid-glycolic acid-polyethylene glycol (PLGA-PEG) prepared in the embodiment of the invention is dissolved in deuterated chloroform for nuclear magnetic hydrogen spectrum characterization. As shown in FIG. 1, the characteristic peak at 5.25ppm was attributed to (-O-CH- (CH)3) -) methine hydrogen; the characteristic peak at 4.85ppm was attributed to (-O-CH)2-methylene hydrogen in O-; the characteristic peak at 3.7ppm was attributed to (-CH)2-CH2-methylene hydrogen in O-; characteristic at 1.6ppmThe peak was attributed to the methyl hydrogen of (-O-CH- (CH3) -). As can be seen, the present examples successfully synthesized PLGA-PEG.
2) Pyrene was prepared with acetone to a concentration of 6X 10-7Preparing the polylactic acid-glycolic acid-polyethylene glycol-atrial natriuretic peptide (PLGA-PEG-ANP) prepared by the embodiment of the invention into continuous solutions with different concentrations by using deionized water as a solvent; taking 20 EP tubes, adding an equal amount of pyrene solution into each EP tube, and naturally volatilizing acetone overnight in a ventilation kitchen; sequentially adding 1mL of PLGA-PEG-ANP solution with different concentrations into each EP, carrying out ultrasonic treatment for 30min, placing in a constant-temperature water bath at 60 ℃ for 30min, carrying out constant temperature overnight after the temperature is reduced to 40 ℃, and then respectively measuring the fluorescence spectrograms of the PLGA-PEG-ANP solution with different concentrations by using a fluorescence spectrophotometer; the excitation wavelength of fluorescence scanning is set to be 337nm, the scanning range is 360-450 nm, the scanning speed is set to be 1200nm/min, the excitation slit is set to be 2.5nm, and the emission slit is set to be 10 nm.
As shown in FIG. 2, the CMC value of PLGA-PEG-ANP obtained from the intersection point was 0.0836mg/mL, and it can be seen that PLGA-PEG-ANP can form micelles in an aqueous solution, and the lowest concentration of micelle formation was 0.0836 mg/mL.
3) The polylactic acid-glycolic acid-polyethylene glycol-atrial natriuretic peptide/tanshinone IIA (PLGA-PEG-ANP/TAN, nano compound) prepared in the embodiment of the invention and polylactic acid-glycolic acid-polyethylene glycol-atrial natriuretic peptide (PLGA-PEG-ANP, nano carrier) before drug loading are subjected to transmission electron microscope characterization.
As shown in FIG. 3, both PLGA-PEG-ANP and PLGA-PEG-ANP/TAN micelles exhibited a spherical or elliptical geometry with a diameter of 20-80 nm. In addition, the micelle particle size of the nanocomposite is significantly increased; it can be seen that PLGA-PEG-ANP can form spherical micelles and can successfully entrap tanshinone IIA as a drug.
4) Accurately weighing 10mg of the nano-composite prepared in the embodiment of the invention, placing the nano-composite in a dialysis bag with an interception molecule 3500, fastening two ends of the dialysis bag by a rope, placing the dialysis bag in 10mL of PBS release liquid containing different concentrations, and oscillating in a shaking table at room temperature (25-30 ℃) for 24 hours, wherein the slow release liquid is Tween-80 containing 0.1%. Timing, respectively taking 5mL of release solution at preset time points, namely 0.5, 1, 2, 4, 8, 12 and 24h, in an EP (taking 5mL aims at better releasing the drug at the later time), then adding an equal amount of PBS solution containing Tween-80 in an equal volume to perform a subsequent release experiment (samples which can be accumulated for several time periods are measured together) under the same conditions, detecting the concentration of tanshinone IIA in the extracted supernatant by using UV-VIS, and calculating the cumulative release percentage of tanshinone IIA; each sample was done in triplicate and the results are expressed as mean and standard deviation.
As shown in fig. 4, the release rate of the drug, Shentong IIA, is fast, up to 85% in the first 12 hours, and after 12 hours, the release rate of Shentong IIA begins to slow and become gentle with time. Therefore, the PLGA-PEG-ANP nano-carrier has good slow release effect.
5) The cell activity detection is carried out on the nano-carrier PLGA-PEG-ANP and the nano-compound PLGA-PEG-ANP/TAN prepared by the embodiment of the invention by a CCK-8 method. The cells used in this experiment were fibroblasts (3T3 cells), and the culture medium used for culturing the cells was DMEM containing 10% fetal bovine serum and 1% diabody (mixture of penicillin and streptomycin), and the culture conditions were 37 ℃ and CO2An incubator with a concentration of 5%; in the process of culture, the culture solution is changed for the cells every two days, and the purpose of changing the culture solution for the cells is to add new nutrient substances for the cells, remove non-adherent cells and metabolites of the cells; adding PLGA-PEG-ANP and PLGA-PEG-ANP/TAN culture medium solutions with different concentrations into a 96-well plate, then placing the 96-well plate into an incubator, adding a CCK-8 reagent after culturing for 1 day, adding the CCK-8 reagent according to the proportion of 1:10, namely adding 10 mu L of CCK-8 reagent into 100 mu L of culture solution, and continuing culturing for 2-4 hours; the absorbance of each well was read using a microplate reader at a wavelength of 450 nm.
As shown in FIG. 5, each experimental group always showed concentration dependence on 3T3 cells, and when the concentration is lower than 0.5mg/mL, PLGA-PEG-ANP showed very little cytotoxicity, and the cell survival rate was all over 80%; when the concentration is more than 0.5mg/mL, the cell survival rate of the PLGA-PEG-ANP and PLGA-PEG-ANP/TAN nano-micelle is reduced, and the PLGA-PEG-ANP/TAN nano-micelle is reduced more. PLGA-PEG-ANP showed very little cytotoxicity at concentrations below 0.5mg/mL, whereas the usual intravenous concentrations were all below 0.5 mg/mL; therefore, the nano-carrier PLGA-PEG-ANP has better biocompatibility.
Finally, it should be noted that the above embodiments are intended to illustrate the technical solutions of the present invention and not to limit the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A preparation method of a nano-carrier for targeted therapy of myocardial injury is characterized by sequentially comprising the following steps:
s1) dissolving the carboxylated polylactic acid-glycolic acid into dichloromethane, adding N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and activating to generate polylactic acid-glycolic acid-N-hydroxysuccinimide; after the activation reaction, sequentially carrying out glacial ethyl ether precipitation, dialysis and vacuum drying; the polylactic acid-glycolic acid-N-hydroxysuccinimide is abbreviated as PLG-NHS, and the PLG-NHS is a polymer with the structure shown in the formula (I);
wherein n is more than or equal to 2;
s2) dissolving PLG-NHS into chloroform, adding amino polyethylene glycol carboxyl and N, N-diisopropylethylamine, and activating to generate polylactic acid-glycolic acid-polyethylene glycol; after the activation reaction, carrying out ice-methanol precipitation, ice-methanol cleaning and vacuum drying in sequence; polylactic acid-glycolic acid-polyethylene glycol is abbreviated as PLGA-PEG, PLGA-PEG is a polymer with a structure shown in formula (II), amino polyethylene glycol carboxyl is abbreviated as NH2-PEG-COOH；
Wherein n is more than or equal to 2, and m is more than or equal to 1;
s3) dissolving atrial peptides into a phosphate buffer solution to prepare an atrial peptide solution; adding the atrial natriuretic peptide solution into the PLGA-PEG solution with the adjusted pH value, and reacting at normal temperature to generate polylactic acid-glycolic acid-polyethylene glycol-atrial natriuretic peptide; after the reaction is finished, transferring the mixture into an ultrafiltration centrifugal tube for ultrafiltration, and then freeze-drying the mixture; polylactic acid-glycolic acid-polyethylene glycol-atrial natriuretic peptide is abbreviated as PLGA-PEG-ANP, PLGA-PEG-ANP is a polymer with the structure shown in the formula (III), and in the PLGA-PEG-ANP, amino at the tail end of atrial natriuretic peptide and hydroxyl in PLGA-PEG are subjected to dehydration condensation reaction;
wherein n is more than or equal to 2, m is more than or equal to 1, and ANP represents atrial natriuretic peptide.
2. The method for producing the nanocarrier according to claim 1, wherein in step S1, the ratio of the amounts of the polylactic acid-glycolic acid, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is (0.1-1): 1-10): 1, the ratio of the total volume of the dichloromethane, polylactic acid-glycolic acid, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the volume of dichloromethane is 5-20: 1, the time for the activation reaction is 4-12 hours, the ratio of the total volume of the dichloromethane, polylactic acid-glycolic acid, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the volume of iceberg ether is 1: (10-20), the dialysis is carried out by adopting a dialysis bag with the molecular weight cutoff of 500-3500, the vacuum drying temperature is 40-60 ℃, and the vacuum drying time is 12-24 hours.
3. The method for preparing the nanocarrier of claim 1, wherein in step S2, the mass ratio of PLGA-NHS to N, N-diisopropylethylamine is 1 (1-10), the activation reaction time is 4-12 h, the ratio of the total volume of PLGA-NHS, N-diisopropylethylamine, chloroform and aminopolyethylene glycol carboxyl to the volume of chloroform is (5-20): 1, the ratio of the total volume of PLGA-NHS, N-diisopropylethylamine, chloroform and aminopolyethylene glycol carboxyl to the volume of glacial methanol is 1 (10-20), the vacuum drying temperature is 40-60 ℃, and the vacuum drying time is 12-24 h.
4. The method for preparing the nanocarrier of claim 1, wherein in step S3, the mass ratio of the atrial natriuretic peptide to the PLGA-PEG is 1 (1-10), the pH value of the PLGA-PEG solution is 6-7, the pH value of the phosphate buffer is <7.2, the reaction time at room temperature is 2-6 h, the rotational speed of the ultrafiltration is 12000rpm, the ultrafiltration time is 30min, and the freeze-drying time is 24-48 h.
5. The nano-carrier for targeted therapy of myocardial injury is polylactic acid-glycolic acid-polyethylene glycol-atrial natriuretic peptide or a medicinal acid addition salt thereof prepared by the preparation method of any one of claims 1 to 4, and is a nano micelle, and the diameter of the nano carrier is 20 to 80 nm.
6. A preparation method of a nano compound loaded with tanshinone IIA is characterized by comprising the following steps: the tanshinone IIA and the nano-carrier of claim 5 are used as raw materials, a thin film hydration method is adopted to prepare the nano-composite loaded with the tanshinone IIA, and the diameter of the nano-composite is 20-80 nm.
7. The method of claim 6, wherein the nanocarrier and tanshinone IIA are dissolved in acetone, mixed and rotary evaporated until a thin film is formed; after the film is dried, phosphate buffer solution redissolving, water bath heating and dialysis are sequentially carried out, so that the nano compound loaded with the tanshinone IIA is obtained, wherein the abbreviation of the nano compound is PLGA-PEG-ANP/TAN;
the mass ratio of the nano-carrier to the tanshinone IIA is (1-10): 1, the feed-liquid ratio of the tanshinone IIA to the acetone is 8 (1-10) mg/ml, the rotary evaporation temperature is 40-60 ℃, the water bath heating temperature is 40-80 ℃, the water bath heating time is 4-12 h, and a dialysis bag with the molecular weight cutoff of 500-3500 is adopted for dialysis.
8. A nanocomposite produced by the production method according to claim 7.
9. Use of the nanocomposite of claim 8 in the preparation of a medicament for the targeted treatment of myocardial ischemic injury.
10. A medicament comprising the nanocomposite of claim 8, further comprising a pharmaceutically acceptable excipient.
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| WO2008141326A1 (en) * | 2007-05-14 | 2008-11-20 | Tyco Healthcare Group Lp | Biodegradable peptide releasing polymers |
| CN103110567A (en) * | 2013-03-11 | 2013-05-22 | 上海中医药大学附属普陀医院 | Preparation method of nanometer drug delivery system carrying tanshinone IIA and application thereof |
| CN103142489A (en) * | 2012-11-28 | 2013-06-12 | 深圳市健元医药科技有限公司 | Human alpha-atrial natriuretic peptide sustained release microsphere preparation and preparation method thereof |
| CN107789632A (en) * | 2017-09-06 | 2018-03-13 | 哈尔滨理工大学 | A kind of active Brain targeting nanoscale medicine delivery system of T7 peptides modification and preparation method thereof |
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| WO2008141326A1 (en) * | 2007-05-14 | 2008-11-20 | Tyco Healthcare Group Lp | Biodegradable peptide releasing polymers |
| CN103142489A (en) * | 2012-11-28 | 2013-06-12 | 深圳市健元医药科技有限公司 | Human alpha-atrial natriuretic peptide sustained release microsphere preparation and preparation method thereof |
| CN103110567A (en) * | 2013-03-11 | 2013-05-22 | 上海中医药大学附属普陀医院 | Preparation method of nanometer drug delivery system carrying tanshinone IIA and application thereof |
| CN107789632A (en) * | 2017-09-06 | 2018-03-13 | 哈尔滨理工大学 | A kind of active Brain targeting nanoscale medicine delivery system of T7 peptides modification and preparation method thereof |
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