CN111228486B - Carbonylation polycaprolactone-based photodynamic therapy prodrug and preparation method and application thereof - Google Patents

Carbonylation polycaprolactone-based photodynamic therapy prodrug and preparation method and application thereof Download PDF

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CN111228486B
CN111228486B CN202010025619.3A CN202010025619A CN111228486B CN 111228486 B CN111228486 B CN 111228486B CN 202010025619 A CN202010025619 A CN 202010025619A CN 111228486 B CN111228486 B CN 111228486B
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polycaprolactone
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郎美东
王申春
王秀丽
申抒展
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Abstract

The invention discloses a carbonylation polycaprolactone-based photodynamic therapy prodrug, which has the following structure:
Figure DDA0002362333340000011
wherein x is 10 to 40 and y is 10 to 40. The carbonylation polycaprolactone-based photodynamic therapy prodrug is an amphiphilic polymer and can be prepared into micelles, and cell experiments prove that compared with pure 5-aminolevulinic acid, the prodrug micelles have higher conversion amount into protoporphyrin and better killing property on tumor cells.

Description

Carbonylation polycaprolactone-based photodynamic therapy prodrug and preparation method and application thereof
Technical Field
The invention belongs to a carbonylation polycaprolactone-based photodynamic therapy prodrug, and particularly relates to a carbonylation polycaprolactone-based photodynamic therapy prodrug, a preparation method and application thereof.
Background
Photodynamic therapy (PDT) is a new technique for diagnosing and treating diseases by using Photodynamic effect. 5-Aminolevulinic acid (ALA) is a second generation porphyrin photosensitizer developed in recent years. ALA is absorbed in cells and converted into protoporphyrin IX (PpIX) with a strong photosensitivity effect, and PpIX generates a photodynamic reaction after being activated by illumination with specific wavelength to generate singlet oxygen with a cell killing effect, so that tumor cells are killed. ALA has been widely used clinically as the first photosensitizer prodrug approved by the U.S. Food and Drug Administration (FDA) for topical PDT. However, ALA is a hydrophilic zwitterion, poorly soluble in lipids, unstable at physiological pH, and not conducive to its further conversion to PpIX, which greatly limits its use in photodynamic therapy.
Poly (epsilon-caprolactone) (PCL) is a biodegradable biomedical polymer material with good biocompatibility, passes FDA certification and is an important tissue engineering material and a drug controlled release carrier. The functional group of the epsilon-caprolactone is functionalized, and the reactive active functional group is introduced, so that the polyester can be further chemically and biologically modified, and the biomedical field of the polyester material can be widened.
In conclusion, the carbonylation polycaprolactone-based photodynamic therapy prodrug prepared by combining PCL and ALA is beneficial to improving the stability of ALA, improving the capability of converting ALA into PpIX and improving the effect of photodynamic therapy.
Disclosure of Invention
In order to overcome the defects of poor lipid solubility, instability and the like of ALA, the invention aims to provide a carbonylation polycaprolactone-based photodynamic therapy prodrug, which can improve the amount of ALA converted into PpIX, can improve the killing property on tumor cells and further improve the effect of photodynamic therapy.
The second purpose of the invention is to provide a preparation method of the carbonylation polycaprolactone-based photodynamic therapy prodrug.
The third purpose of the invention is to provide the application of the carbonylation polycaprolactone-based photodynamic therapy prodrug in preparing anticancer drugs.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a carbonylation polycaprolactone-based photodynamic therapy prodrug, which has the following structure:
Figure BDA0002362333320000021
x＝10～40,y＝10～40。
the second aspect of the present invention provides a preparation method of the carbonylation polycaprolactone-based photodynamic therapy prodrug, comprising the following steps:
adding a polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD) and hydroxyethylhydrazine into a mixed solution of methanol and chloroform at a molar ratio of 1 (2-5), reacting at room temperature, dialyzing the obtained reaction solution, and obtaining a polymer of the polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD) after being grafted with hydroxyethylhydrazine;
under the protection of argon, dissolving a compound CALAA in dry DMF, adding pentafluorophenol and Dicyclohexylcarbodiimide (DCC), reacting for 2h at room temperature, adding a polymer obtained by inoculating polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD) into hydroxyethylhydrazine, wherein the molar ratio of the polymer obtained by inoculating polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD) into hydroxyethylhydrazine to the compound CALAA, pentafluorophenol and DCC is 1 (1.01-1.5) to 1.01 (1.01-1.5), reacting at room temperature, concentrating and settling reaction liquid to obtain a polymer mPEG-b-P (CL-co-CALAOPD);
under the protection of argon, dissolving a polymer mPEG-b-P (CL-co-CALAPAOPD) by using dry dichloromethane, adding palladium carbon, and introducing hydrogen, wherein the mass ratio of the palladium carbon to the polymer mPEG-b-P (CL-co-CALAPAOPD) is 1: (5-10), reacting at room temperature to obtain the carbonylation polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD).
In the mixed solution of methanol and chloroform, the volume ratio of chloroform to methanol is 1: (2-3).
The preparation method of the polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD) comprises the following steps:
under the protection of argon, polyethylene glycol monomethyl ether 2000(mPEG2000), 4-carbonyl-epsilon-caprolactone (OPD) and epsilon-caprolactone (epsilon-CL) in a molar ratio of 1 (10-40) to 10-40 are mixed, the mixture is vacuumized, dried toluene is added as a solvent, a catalyst stannous isooctanoate is added, the molar ratio of stannous isooctanoate to the sum of monomer 4-carbonyl-epsilon-caprolactone (OPD) and epsilon-caprolactone (epsilon-CL) is 1 (100-200), and the mixture is continuously reacted for 1-48 hours at the temperature of 80-100 ℃ to obtain the polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD).
In the polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD), a caprolactone unit (epsilon-CL) accounts for 40-80% and a 4-carbonyl-epsilon-caprolactone (OPD) unit (OPD) accounts for 20-60% in terms of mole number.
The preparation method of the compound CALALA comprises the following steps:
dissolving 5-aminolevulinic acid hydrochloride into a saturated sodium bicarbonate aqueous solution, dropwise adding benzyl chloroformate under the condition of ice-water bath, wherein the molar ratio of the 5-aminolevulinic acid hydrochloride to the benzyl chloroformate is 1 (1.1-1.5), reacting overnight at normal temperature, concentrating the reaction solution, filtering, washing with diethyl ether, and drying to obtain the compound CALAA.
The third aspect of the invention provides an application of the carbonylation polycaprolactone-based photodynamic therapy prodrug in preparing an anticancer drug.
The cancer is skin cancer (especially skin squamous carcinoma).
The carbonylation polycaprolactone-based photodynamic therapy prodrug is an amphiphilic polymer and can be prepared into a carbonylation polycaprolactone-based photodynamic therapy prodrug micelle; the carbonylation polycaprolactone-based photodynamic therapy prodrug micelle takes a polyethylene glycol monomethyl ether (mPEG2000) segment as a hydrophilic shell, and takes a chain segment randomly copolymerized by 4-carbonyl-epsilon-caprolactone (OPD) and epsilon-caprolactone (epsilon-CL) as a hydrophobic inner core; compared with the pure photosensitizer precursor 5-aminolevulinic acid (ALA) with the same concentration, the amount of the carbonylation polycaprolactone-based photodynamic therapy prodrug micelle converted into protoporphyrin is increased; compared with the pure photosensitizer precursor 5-aminolevulinic acid (ALA) with the same concentration, the carbonylation polycaprolactone-based photodynamic therapy prodrug micelle has higher killing property on cancer cells.
Due to the adoption of the technical scheme, the invention has the following advantages and beneficial effects:
the carbonylation polycaprolactone-based photodynamic therapy prodrug is an amphiphilic polymer and can be prepared into micelles, and cell experiments prove that compared with pure 5-aminolevulinic acid (ALA), the prodrug micelles are higher in conversion amount into protoporphyrin and better in killing property on tumor cells.
Drawings
FIG. 1 is a drawing of the compound CALALA1H NMR spectrum.
FIG. 2 shows the preparation of polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD)1H NMR spectrum.
FIG. 3 shows the polymer of polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD) after being grafted with hydroxyethylhydrazine1H NMR spectrum.
FIG. 4 is a schematic representation of the carbonylation of polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD)1H NMR spectrum.
FIG. 5 is a DLS map of micelles of the carbonylated polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD).
FIG. 6 is a TEM image of a micelle of carbonylated polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD).
FIG. 7 is a graphical representation of the results of an intracellular protoporphyrin PpIX assay of a carbonylation polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD) micelle.
FIG. 8 is a graph showing the results of an intracellular singlet oxygen ROS assay of a carbonylation polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD) micelle.
FIG. 9 is a graph showing the results of the cytotoxicity test of the carbonylated polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD) micelle.
FIG. 10 is a graph showing the results of a kill test of the micelle of the carbonylated polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD).
FIG. 11 is a schematic representation of tumor growth in mice.
FIG. 12 is a graph showing the body curves of a mouse tumor.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Example 1
Preparation of CAALA:
Figure BDA0002362333320000041
adding 1.68g of 5-aminolevulinic acid hydrochloride, 0.84g of sodium bicarbonate and 20mL of deionized water into a 50mL two-neck flask in sequence, after the raw materials are completely dissolved, dropwise adding 1.7g of benzyl chloroformate under the condition of ice-water bath, reacting at normal temperature overnight, concentrating and filtering to remove unreacted benzyl chloroformate and solvent, washing the obtained white solid with methanol, and drying to constant weight to obtain the compound CALAL. FIG. 1 is a drawing of the compound CALALA1H NMR spectrum.
Example 2
Synthesis of polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD)
Figure BDA0002362333320000042
x＝20，y＝30。
Vacuumizing a 25mL reaction eggplant bottle for three times, adding 1g of polyethylene glycol monomethyl ether 2000(mPEG2000), 1.28g of 4-carbonyl-epsilon-caprolactone (OPD) and 1.71g of epsilon-caprolactone (epsilon-CL) under the protection of argon, vacuumizing for 2h, adding 10mL of dry toluene and a catalyst stannous isooctanoate Sn (Oct)20.101g, reacted at 90 ℃ for 24 h. And (3) settling a reaction product in the glacial ethyl ether, performing suction filtration on a sediment by using a sand core funnel, and performing vacuum drying to constant weight to obtain the polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD). FIG. 2 shows the preparation of polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD)1H NMR spectrum.
The preparation method of the carbonylated polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD) comprises the following steps:
Figure BDA0002362333320000051
x＝20，y＝30。
and (3) inoculating hydroxyethylhydrazine: 2g of polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD) and 0.32g of hydroxyethylhydrazine are weighed and added into a mixed solution of 20ml of methanol and chloroform (volume ratio is 9:4) to react for 24 hours at room temperature. Dialyzing the obtained reaction solution in methanol for 12h by using a dialysis bag with the molecular weight cutoff of 3500, changing methanol every 4h, finally settling and recovering by using ether, and drying to obtain a white solid. FIG. 3 shows the polymer of polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD) after being grafted with hydroxyethylhydrazine1H NMR spectrum.
Preparation of carbonylated polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD):
under the protection of argon, 0.837g CAALA is dissolved in 10mL of dry DMF, then 0.581g pentafluorophenol and 0.651g dicyclohexylcarbodiimide DCC are added, after the reaction is carried out for 2h at room temperature, 1g polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD) is added to the polymer after being added with the hydroxyethylhydrazine, the reaction is carried out for 12h at room temperature, the reaction solution is concentrated and settled in a large amount of ice anhydrous ether, and the polymer mPEG-b-P (CL-co-CAALAOPD) is obtained.
The deprotection method comprises the following steps: adding 0.5g of polymer mPEG-b-P (CL-co-CALAPAOPD) into a polymerization bottle filled with argon, dissolving the polymer mPEG-b-P (CL-co-CALAPAOPD) with 2mL of dry dichloromethane, adding 0.1g of palladium carbon, introducing hydrogen, reacting at room temperature for 4h, filtering to remove the palladium carbon, settling in ice anhydrous ether, and drying to obtain the carbonylation polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD). FIG. 4 is a schematic representation of the carbonylation of polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD)1H NMR spectrum.
Example 3
Synthesis of polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD)
Figure BDA0002362333320000061
x＝30，y＝20。
Vacuumizing a 25mL reaction eggplant bottle for three times, adding 1g of polyethylene glycol monomethyl ether 2000(mPEG2000), 1.92g of 4-carbonyl-epsilon-caprolactone (OPD) and 1.14g of epsilon-caprolactone (epsilon-CL) under the protection of argon, vacuumizing for 2h, adding 10mL of dry toluene and a catalyst stannous isooctanoate Sn (Oct)20.101g, reacted at 90 ℃ for 24 h. And (3) settling a reaction product in the glacial ethyl ether, performing suction filtration on a sediment by using a sand core funnel, and performing vacuum drying to constant weight to obtain the polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD).
The preparation method of the carbonylated polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD) comprises the following steps:
Figure BDA0002362333320000071
x＝30，y＝20。
and (3) inoculating hydroxyethylhydrazine: 1g of polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD) and 0.438g of hydroxyethylhydrazine are weighed and added into a mixed solution of 20ml of methanol and chloroform (volume ratio is 9:4) to react for 24 hours at room temperature. Dialyzing the obtained reaction solution in methanol for 12h by using a dialysis bag with the molecular weight cutoff of 3500, changing methanol every 4h, finally settling and recovering by using ether, and drying to obtain a white solid.
Preparation of carbonylated polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD):
under the protection of argon, 1.147g of CAALA is dissolved in 10mL of dry DMF, then 0.796g of pentafluorophenol and 0.891g of dicyclohexylcarbodiimide DCC are added, after the reaction is carried out for 2h at room temperature, 1g of polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD) is added to the polymer after being inoculated with hydroxyethylhydrazine, the reaction is carried out for 12h at room temperature, the reaction solution is concentrated and settled in a large amount of ice anhydrous ether, and the polymer mPEG-b-P (CL-co-CAALAOPD) is obtained.
The deprotection method comprises the following steps: adding 0.5g of polymer mPEG-b-P (CL-co-CALAPAOPD) into a polymerization bottle filled with argon, dissolving the polymer mPEG-b-P (CL-co-CALAPAOPD) with 2mL of dry dichloromethane, adding 0.1g of palladium carbon, introducing hydrogen, reacting at room temperature for 4h, filtering to remove the palladium carbon, settling in ice anhydrous ether, and drying to obtain the carbonylation polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD).
Example 4
Synthesis of polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD)
Figure BDA0002362333320000081
x＝10，y＝40。
Vacuum-pumping 25mL reaction eggplant bottle for three times, adding 1g of polyethylene glycol monomethyl ether 2000(mPEG2000), 0.64g of 4-carbonyl-epsilon-caprolactone (OPD) and 2.28g of epsilon-caprolactone (epsilon-CL) under the protection of argon, vacuum-pumping for 2h, adding 10mL of dry toluene and catalyst stannous isooctanoate Sn (Oct)20.101g, reacted at 90 ℃ for 24 h. And (3) settling a reaction product in the glacial ethyl ether, performing suction filtration on a sediment by using a sand core funnel, and performing vacuum drying to constant weight to obtain the polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD).
The preparation method of the carbonylated polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD) comprises the following steps:
Figure BDA0002362333320000082
x＝10，y＝40。
and (3) inoculating hydroxyethylhydrazine: 1g of polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD) and 0.177g of hydroxyethylhydrazine are weighed and added into a mixed solution of 20ml of methanol and chloroform (volume ratio is 9:4) to react for 24 hours at room temperature. Dialyzing the obtained reaction solution in methanol for 12h by using a dialysis bag with the molecular weight cutoff of 3500, changing methanol every 4h, finally settling and recovering by using ether, and drying to obtain a white solid.
Preparation of carbonylated polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD):
under the protection of argon, 0.462g of CAALA is dissolved in 10mL of dry DMF, then 0.321g of pentafluorophenol and 0.359g of dicyclohexylcarbodiimide DCC are added, after the reaction is carried out for 2h at room temperature, 1g of polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD) is added to the polymer after being added with hydroxyethylhydrazine, the reaction is carried out for 12h at room temperature, the reaction solution is concentrated and settled in a large amount of ice anhydrous ether, and the polymer mPEG-b-P (CL-co-CAALAOPD) is obtained.
The deprotection method comprises the following steps: adding 0.5g of polymer mPEG-b-P (CL-co-CALAPAOPD) into a polymerization bottle filled with argon, dissolving the polymer mPEG-b-P (CL-co-CALAPAOPD) with 2mL of dry dichloromethane, adding 0.1g of palladium-carbon, introducing hydrogen, reacting at room temperature for 4h, filtering to remove the palladium-carbon, settling in ice anhydrous ether, and drying to obtain the carbonylation polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-CALAOPD).
Example 5
Synthesis of polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD)
Figure BDA0002362333320000091
x＝25，y＝25。
Vacuum-pumping 25mL reaction eggplant bottle for three times, adding 1g of polyethylene glycol monomethyl ether 2000(mPEG2000), 1.6g of 4-carbonyl-epsilon-caprolactone (OPD) and 1.425g of epsilon-caprolactone (epsilon-CL) under the protection of argon, vacuum-pumping for 2h, adding 10mL of dry toluene and catalyst stannous isooctanoate Sn (Oct)20.101g, reacted at 90 ℃ for 24 h. And (3) settling a reaction product in the glacial ethyl ether, performing suction filtration on a sediment by using a sand core funnel, and performing vacuum drying to constant weight to obtain the polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD).
The preparation method of the carbonylated polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD) comprises the following steps:
Figure BDA0002362333320000101
x＝25，y＝25。
and (3) inoculating hydroxyethylhydrazine: 1g of polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD) and 0.382g of hydroxyethylhydrazine are weighed and added into a mixed solution of 20ml of methanol and chloroform (volume ratio is 9:4) to react for 24 hours at room temperature. Dialyzing the obtained reaction solution in methanol for 12h by using a dialysis bag with the molecular weight cutoff of 3500, changing the methanol every 4h, finally settling and recovering by using ether, and drying to obtain a white solid.
Preparation of carbonylated polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD):
under the protection of argon, 0.776g of CAALA is dissolved in 10mL of dry DMF, then 0.693g of pentafluorophenol and 0.776g of dicyclohexylcarbodiimide DCC are added, reaction is carried out at room temperature for 2h, 1g of polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD) is added to the polymer after being inoculated with hydroxyethylhydrazine, reaction is carried out at room temperature for 12h, the reaction solution is concentrated, and the polymer mPEG-b-P (CL-co-CAALAOPD) is obtained by sedimentation in a large amount of ice anhydrous ether.
The deprotection method comprises the following steps: adding 0.5g of polymer mPEG-b-P (CL-co-CALAPAOPD) into a polymerization bottle filled with argon, dissolving the polymer mPEG-b-P (CL-co-CALAPAOPD) with 2mL of dry dichloromethane, adding 0.1g of palladium carbon, introducing hydrogen, reacting at room temperature for 4h, filtering to remove the palladium carbon, settling in ice anhydrous ether, and drying to obtain the carbonylation polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD).
Example 6
Preparation of carbonylation polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD) micelle:
completely dissolving 200mg of the carbonylated polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD) in 2.5mL of DMF solution, dropwise adding the solution into 10mL of double distilled water, stirring for 2h, transferring the solution into a dialysis bag (with molecular weight cut-off (MWCO) ═ 3500) for dialysis for 48h, changing water every 12h to remove the organic solvent, and finally fixing the volume to 20mL (with micelle concentration of 10 mg/mL). FIG. 5 is a DLS diagram of the carbonylation polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD) micelle, and it can be seen that the mPEG-b-P (CL-co-ALAOPD) micelle has a particle size of about 160nm and is uniform in size; FIG. 6 is an SEM image of micelles of the carbonylated polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD), which is seen to be rod-shaped.
Example 7
PPIX fluorescence kinetics study of carbonylation polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD) micelle
Cells in logarithmic growth phase were seeded into 6-well plates, and when approximately 90% of the cells were confluent, serum-containing medium was aspirated and washed twice with PBS. Adding 3mL of serum-free medium without medicine into the blank control group; the pure ALA group is prepared by adding 3mL serum-free culture medium containing 5mM ALA; the carbonylation polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD) micelle group is prepared by adding mPEG-b-P (CL-co-ALAOPD) micelles, diluting with a serum-free culture medium to 3mL of a mixed solution containing ALA with the final concentration of 5mM, and wrapping 3 groups with tinfoil paper and culturing in the dark for 24 h. The relative amount of intracellular PpIX was determined by flow cytometry. The results are shown in FIG. 7, and FIG. 7 is a graph showing the results of intracellular protoporphyrin PpIX test of the carbonylated polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD) micelle, wherein the amount of intracellular PpIX of the micelle of the pure ALA and the prodrug is increased compared with that of the normal cells, and the prodrug mPEG-b-P (CL-co-ALAOPD) micelle group is higher than that of the ALA group, namely the prodrug mPEG-b-P (CL-co-ALAOPD) micelle can generate more PpIX.
Example 8
Intracellular ROS assay
Cells in logarithmic growth phase were inoculated into 6-well culture plates, cultured until about 90% of the cells were fused, the serum-containing culture medium was aspirated, and washed twice with PBS. The blank control group is added with 3mL of serum-free medium without medicine; the pure ALA group is prepared by adding 3mL serum-free culture medium containing 5mM ALA; the mPEG-b-P (CL-co-ALAOPD) micelle group is prepared by adding 3mL of mixed culture medium prepared by diluting mPEG-b-P (CL-co-ALAOPD) micelle with serum-free culture medium to ALA final concentration of 5mM, wrapping 3 groups with tinfoil paper, culturing in dark for 24h, and irradiating with 635nm red light for 5 min. After irradiation, each group was cultured for 4 hours. According to 1:1000, the DCFH-DA reagent was diluted in serum-free medium to a final concentration of 10 umol/L. Three groups of cell culture fluid were removed and 3mL of diluted DCFH-DA was added. Incubate at 37 ℃ for 20min in a cell culture box. Cells were washed three times with serum-free medium to remove DCFH-DA well without entering the cells. The cells were collected and examined by flow cytometry. As shown in FIG. 8, FIG. 8 is a graph showing the results of ROS test for intracellular singlet oxygen of the prodrug mPEG-b-P (CL-co-ALAOPD) micelle, and it can be seen that the intracellular ROS amount is increased in the case of adding pure ALA and mPEG-b-P (CL-co-ALAOPD) micelle compared with normal cells, and the prodrug mPEG-b-P (CL-co-ALAOPD) micelle is slightly higher than that of ALA micelle, i.e. the prodrug mPEG-b-P (CL-co-ALAOPD) micelle can generate more ROS.
Example 9
Dark toxicity and killing test of cells
Cells in logarithmic growth phase were seeded into 96-well culture plates, and when approximately 90% of the cells were fused, serum-containing medium was aspirated and washed twice with PBS.
In the case of cell dark toxicity, cells in the logarithmic growth phase were inoculated into 96-well culture plates, and when approximately 90% of the cells were fused, serum-containing culture medium was aspirated and washed twice with PBS. Adding non-medicated serum-free culture medium 100 μ L into the blank control group, and culturing in dark place; adding 100 μ L of 0.1mM ALA, 1mM ALA and 10mM ALA in 0.1mM ALA light-shielding group, 1mM ALA light-shielding group and 10mM ALA light-shielding group respectively; 0.1mM mPEG-b-P (CL-co-ALAOPD) micelle light-resistant group, 1mM mPEG-b-P (CL-co-ALAOPD) micelle light-resistant group, 10mM mPEG-b-P (CL-co-ALAOPD) micelle light-resistant group: each of these was added 100. mu.L of a mixed solution prepared by diluting mPEG-b-P (CL-co-ALAOPD) micelles with serum-free medium to a final ALA concentration of 0.1mM, 1mM, or 10 mM. After each group was wrapped with tinfoil paper and cultured for 48h in the dark, the cell survival rate was measured in CCK 8. The results of the cell dark toxicity test are shown in FIG. 9, and it can be seen from the figure that under the condition of keeping out of the light, the mPEG-b-P (CL-co-ALAOPD) micelle has no cytotoxicity, is a safe biological material, and has the advantage of small dark toxicity as a photosensitizer prodrug.
Cells in logarithmic growth phase were seeded into 96-well culture plates, and when approximately 90% of the cells were fused, serum-containing medium was aspirated and washed twice with PBS. Adding 100 μ L of serum-free medium containing ALA0.1mM, 1mM, 2mM, 5mM, 10mM into ALA component; the mPEG-b-P (CL-co-ALAOPD) micelle group was added to 100. mu.L each of a mixed solution prepared by diluting mPEG-b-P (CL-co-ALAOPD) micelles with serum-free medium to a final ALA concentration of 0.1mM, 1mM, 2mM, 5mM and 10mM in the solution. And packaging each group with tinfoil paper, culturing for 24h in the dark, irradiating with 635nm red light for 5min, and culturing for 24h, and detecting the cell survival rate with CCK 8. The cell killing test results are shown in FIG. 10, and it can be seen that mPEG-b-P (CL-co-ALAOPD) micelles have cell killing property, and the cell killing effect of the mPEG-b-P (CL-co-ALAOPD) micelles is better than that of pure ALA under the same MLA concentration.
Example 10
Animal experiments
Establishing a mouse tumor model:
the establishment method of the squamous carcinoma tumor model comprises the following steps: purchasing 20 female nude mice, about 6w, adaptively feeding for 1 week, and injecting 0.2mL of 2 x 10-containing solution into each nude mouse6Physiological saline of individual squamous carcinoma cells. When the tumor volume is increased to 15mm3An antitumor experiment was performed.
In vivo antitumor study:
mice were randomized into 3 groups:
the group A is a group of pictures without any treatment;
group B Free ALA group: locally injecting 200 μ L of 10mM sterilized ALA solution;
group C prodrug mPEG-b-P (CL-co-ALAOPD) micelle group A200. mu.L of mPEG-b-P (CL-co-ALAOPD) micelles containing ALA in the same amount as that of the pure drug group were locally injected.
Adding medicine, keeping away from light for 4h, anesthetizing the mouse, irradiating the tumor part with 635nm red light to ensure that the light spot covers the whole tumor and the normal tissue with the power density of 75mW/cm and the power density of 3-5mm around the tumor2The irradiation time was 5min each. Mouse body weight and subcutaneous tumor size, including length and width, were recorded every 2 days, and tumor volume was calculated according to the following formula:
V(mm3)＝1/2×length(mm)×width(mm)×width(mm)
and simultaneously photographing and recording the growth condition of the tumor and the health condition of the nude mouse, and drawing a survival curve.
The growth conditions of the mice and the tumors are shown in fig. 11, wherein A is a control group, B is an ALA group, and C is a carbonylation polycaprolactone-based photodynamic therapy prodrug mPEG-B-P (CL-co-ALAOPD) micelle group, in the experiment, the back tumors of the mice in the group A are increased on the sixth day and the 12 th day, and the back tumors of the mice in the group B, C are obviously reduced; FIG. 12 is a graph showing the tumor body curves of mice, and the antitumor effect of the micelle of the carbonylated polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD) is better than that of pure ALA under the same ALA concentration.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (9)

1. A carbonylation polycaprolactone-based photodynamic therapy prodrug is characterized in that the structure is as follows:
Figure FDA0003481511880000011
x＝10～40，y＝10～40。
2. a method of preparing the carbonylated polycaprolactone-based photodynamic therapy prodrug as claimed in claim 1, comprising the steps of:
adding a polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD) and hydroxyethylhydrazine into a mixed solution of methanol and chloroform at a molar ratio of 1 (2-5), reacting at room temperature, dialyzing the obtained reaction solution, and obtaining a polymer of the polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD) after being grafted with hydroxyethylhydrazine;
under the protection of argon, dissolving a compound CALAA in dry DMF, adding pentafluorophenol and dicyclohexylcarbodiimide, reacting for 2 hours at room temperature, adding a polymer obtained by inoculating polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD) into hydroxyethylhydrazine, wherein the molar ratio of the polymer obtained by inoculating polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD) into hydroxyethylhydrazine to the compound CALAA, pentafluorophenol and DCC is 1 (1.01-1.5) to 1.01-1.5, reacting at room temperature, concentrating and settling a reaction solution to obtain a polymer mPEG-b-P (CL-co-ALAOPD);
under the protection of argon, dissolving a polymer mPEG-b-P (CL-co-CALAPAOPD) by using dry dichloromethane, adding palladium carbon, and introducing hydrogen, wherein the mass ratio of the palladium carbon to the polymer mPEG-b-P (CL-co-CALAPAOPD) is 1: (5-10), reacting at room temperature to obtain a carbonylation polycaprolactone-based photodynamic therapy prodrug mPEG-b-P (CL-co-ALAOPD);
wherein the CAALA has the following structure:
Figure FDA0003481511880000021
3. the method according to claim 2, wherein the volume ratio of chloroform to methanol in the mixed solution of methanol and chloroform is 1: (2-3).
4. The preparation method according to claim 2, wherein the preparation method of the polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD) comprises the following steps:
under the protection of argon, polyethylene glycol monomethyl ether 2000, 4-carbonyl-epsilon-caprolactone and epsilon-caprolactone in a molar ratio of 1 (10-40) to 10-40 are mixed, vacuum pumping is carried out, dry toluene is added as a solvent, a catalyst stannous isooctanoate is added, the molar ratio of stannous isooctanoate to the sum of monomer 4-carbonyl-epsilon-caprolactone and epsilon-caprolactone is 1 (100-200), and the reaction is continuously carried out for 1-48 h at the temperature of 80-100 ℃ to obtain the polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD).
5. The preparation method of claim 2, wherein the polycaprolactone-based amphiphilic copolymer mPEG-b-P (CL-co-OPD) comprises 40 to 80 mole percent of caprolactone units and 20 to 60 mole percent of 4-carbonyl-epsilon-caprolactone units.
6. The process of claim 2, wherein the CABALA compound is prepared by a process comprising the steps of:
dissolving 5-aminolevulinic acid hydrochloride into a saturated sodium bicarbonate aqueous solution, dropwise adding benzyl chloroformate under the condition of ice-water bath, wherein the molar ratio of the 5-aminolevulinic acid hydrochloride to the benzyl chloroformate is 1 (1.1-1.5), reacting overnight at normal temperature, concentrating the reaction solution, filtering, washing with diethyl ether, and drying to obtain the compound CALAA.
7. Use of the carbonylated polycaprolactone-based photodynamic therapy prodrug as claimed in claim 1 for the preparation of an anticancer drug.
8. Use according to claim 7, wherein said cancer is skin cancer.
9. Use according to claim 8, wherein said skin cancer is squamous cell skin cancer.
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