CN112294759B - Docetaxel polymer nano injection and preparation method thereof - Google Patents
Docetaxel polymer nano injection and preparation method thereof Download PDFInfo
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Abstract
The invention provides a docetaxel polymer nano injection, which comprises the following components: modified docetaxel, a photosensitizer and a pH sensitive block; the modified docetaxel is coated and modified by a shell cross-linked polymer; the modified docetaxel and the photosensitizer are connected through an acid-sensitive bond; the pH sensitive block is coated outside the photosensitizer. The invention also provides a preparation method of the docetaxel polymer nano injection. The docetaxel has high water solubility, high stability, difficult sudden release, systemic toxic and side effects and slow release, and the treatment effect on tumor cells can be effectively improved by combining chemotherapy and phototherapy.
Description
Technical Field
The invention relates to the technical field of medicines, and particularly relates to a docetaxel polymer nano injection and a preparation method thereof.
Background
Taxanes have important roles in the treatment of various solid tumors; docetaxel is a second-generation semi-synthetic taxane derivative, has about twice the efficacy of paclitaxel in microtubule depolymerization, can inhibit cell division, and is mainly used for treating tumor diseases such as advanced breast cancer, non-small cell lung cancer and the like.
However, docetaxel has poor water solubility and is almost insoluble in water, and Tween80 is adopted for solubilization and ethanol for assisting dissolution in the prior art to prepare a pharmaceutical preparation; due to the existence of Tween80, the preparation can cause serious adverse reactions such as anaphylactic reaction, neurotoxicity and the like in the clinical use process; after docetaxel enters a body, the docetaxel is distributed and non-selectively reaches tissues and organs of the whole body, so that toxic and side effects of the whole body are brought, toxicity of a nervous system and an immune system is easily caused, especially toxicity of the immune system such as neutropenia is easily caused, physical and mental health of a patient is greatly injured, and clinical application of the docetaxel is limited; in addition, docetaxel has limited stability and must be used within 4 hours after clinical formulation, limiting the administration of docetaxel.
At present, the problem of poor water solubility of docetaxel is solved by coating a water-soluble polymer micelle on the surface of docetaxel in the prior art, but the coating stability is still limited, the docetaxel can be released suddenly during placement to cause low stability and inconvenience in administration, and the docetaxel is easy to release suddenly after entering a body to bring toxic and side effects to the whole body; in addition, it is released too quickly after entering the body, and has poor therapeutic effect on tumor cells.
Disclosure of Invention
The first purpose of the invention is to provide a docetaxel polymer nano injection, which has higher stability while enabling docetaxel to have higher water solubility, is not easy to release suddenly to bring systemic toxic and side effects, can be released slowly, and can effectively improve the treatment effect on tumor cells by chemotherapy and phototherapy combined treatment.
The second object of the present invention is to provide a method for preparing a docetaxel polymer nano injection, which is used for preparing the docetaxel polymer nano injection.
The embodiment of the invention is realized by the following technical scheme:
a docetaxel polymer nano injection comprises the following components: modified docetaxel, a photosensitizer and a pH sensitive block; the modified docetaxel is coated and modified by a shell cross-linked polymer; the modified docetaxel and the photosensitizer are connected through an acid-sensitive bond; the pH sensitive block is coated outside the photosensitizer.
Further, the photosensitizer is chlorin.
Further, the PH sensitive block is PCL-acrylamide-PEG.
Further, the shell cross-linked polymer is PPhe-PLys-PEG, wherein PLys is cross-linked by disulfide bonds.
Further, the acid sensitive bond is an orthoester bond.
Further, the molecular weight of the docetaxel polymer is 41000-48000.
The modified docetaxel is prepared by loading docetaxel drug into a shell cross-linked polymer to form the modified docetaxel, so that the water solubility of the docetaxel is obviously enhanced; the stability of the medicament is improved, and the slow release of the docetaxel is realized;
one end of the PCL-acylamide-PEG is provided with a hydrophilic chain segment, and the other end of the PCL-acylamide-PEG is provided with a block of a hydrophobic chain segment; the block takes a hydrophobic chain segment as a core to carry out entrapment of a photosensitizer, and takes a hydrophilic chain segment as a shell to have obvious solubilization effect on the medicine; and PEG is used as a shell, so that the recognition and phagocytosis of phagocytes can be effectively avoided, the circulation time in vivo is prolonged, and the targeting effect is enhanced;
however, the endocytosis of the cells to the nano-drug can be reduced by surface PEGylation, so that the PCL and the PEG are connected by adopting an amido bond to form a PH sensitive block PCL-acylamide-PEG, and the PH sensitive block is hydrolyzed to separate the PEG under the environment of PH 6-7 outside the tumor cells, so that the endocytosis of the tumor cells to the rest of the drug is improved;
after the rest of the medicine is taken into the tumor cells by the tumor cells, under the environment of PH 4-5 in the tumor cells, the acid-sensitive bond orthoester bond is hydrolyzed to separate the photosensitizer, and at the moment, the tumor parts are irradiated by visible light to initiate the kinetic effect of the photosensitizer, so that the tumor cells are killed to a certain extent in advance;
the subsequent modified docetaxel is slowly released in the tumor cells, so that the action time of the docetaxel on the tumor cells is prolonged, and the treatment effect on the tumor cells is effectively improved.
The invention adopts docetaxel and photosensitizer to carry together, so that the docetaxel and the photosensitizer are used for combined treatment, namely chemotherapy and phototherapy are combined for treatment, and the inhibition effect on tumor cells can be effectively enhanced.
The molecular weight of the docetaxel polymer is 41000-48000, passive targeting of EPR effect can be realized, tumor cells can be targeted for treatment, and systemic toxic and side effects are avoided.
According to the invention, a layer of shell cross-linked polymer is coated on the surface of docetaxel, and a layer of PH sensitive block is further coated on the outer surface of a drug, so that double-layer coating is formed, the coating property of the drug is enhanced, and the drug burst release can be avoided.
A preparation method of docetaxel polymer nano injection comprises the following steps:
s1: preparing a PPhe-PLys-PEG shell cross-linked polymer in advance;
s2: dissolving docetaxel and a PPhe-PLys-PEG shell cross-linked polymer in a methanol solution according to a mass ratio of 1: 2-3, heating to 20-40 ℃, reacting for 10-12 h, and evaporating the solvent to obtain modified docetaxel;
s3: dissolving modified docetaxel and a photosensitizer in THF (tetrahydrofuran) under the condition of argon, dropwise adding 10-20 drops of toluenesulfonic acid, stirring and reacting for 2-4 hours at 35-45 ℃, adding water according to the mass ratio of 1: 1-2 for mixing, dialyzing, and removing an organic solvent to obtain a substance A, wherein the substance A is the photosensitizer-loaded modified docetaxel with acid sensitivity;
s4: and dissolving the prepared substance A and the PH sensitive block in methanol, reacting for 10-12 h at room temperature, centrifuging at 6000-7000 rpm for 5min, precipitating, taking supernatant, adding water for injection, stirring and mixing, and filtering through a microporous filter membrane to obtain the docetaxel polymer nano injection.
Further, the mass ratio of the modified docetaxel to the photosensitizer added in the step S3 is 1: 1-2.
Further, the mass ratio of the substance A to the PH sensitive block in the step S4 is 1: 1-3.
Further, the average particle size of the docetaxel polymer nano injection is 20-80 nm.
Further, the preparation method of the PPhe-PLys-PEG shell cross-linked polymer in the step S1 includes: will CH3O-PEG-NH2Dissolving in DMF, stirring, adding Lys (Z) -NCA under the protection of nitrogen, reacting at 50 ℃ for 48h, adding Phe-NCA, reacting at 50 ℃ for 48h, adding water according to the proportion of 1:2, mixing, dialyzing, removing the organic solvent, and freeze-drying to obtain the PPhe-PLys-PEG shell cross-linked polymer.
Further, the preparation method of the PCL-acylamide-PEG comprises the following steps: adding polyethylene glycol and polycaprolactone diol into tetrahydrofuran, and stirring for complete dissolution; adding hydrazine hydrate, stirring uniformly, adding a sodium cyanide catalyst, and stirring at room temperature for reacting for 2-4 h; then adding a boron tribromide catalyst, and stirring and reacting for 5-6 h at room temperature; putting the product into a dialysis bag with the molecular weight cutoff of 3500, and dialyzing for 48 h; and (4) removing residual tetrahydrofuran by rotary evaporation, and drying in vacuum until the weight is constant to obtain the PCL-acylamide-PEG.
The technical scheme of the embodiment of the invention at least has the following advantages and beneficial effects:
according to the invention, the modified docetaxel surface loaded with a photosensitizer is coated with a PH sensitive block to obtain the docetaxel polymer nano injection, so that the water solubility is enhanced, and adverse reactions caused by solubilization of Tween80 and ethanol are avoided; meanwhile, the stability is improved, and the toxic and side effects of the whole body caused by burst release are avoided; and the slow-release drug can be slowly released, and the chemotherapy and phototherapy are combined for treatment, so that the treatment effect on tumor cells can be effectively improved.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The docetaxel polymer nano injection and the preparation method thereof provided by the embodiments of the present invention are specifically described below.
Example 1
The embodiment provides a docetaxel polymer nano injection, which comprises the following components: modified docetaxel, chlorin and PCL-acylamide-PEG; the modified docetaxel is coated and modified by a shell cross-linked polymer; the modified docetaxel and the chlorin are connected through an ortho ester bond; the PCL-acylamide-PEG is coated outside the chlorin.
The embodiment also provides a preparation method of the docetaxel polymer nano injection, which comprises the following steps:
s1: preparing a PPhe-PLys-PEG shell cross-linked polymer in advance;
s2: dissolving docetaxel and PPhe-PLys-PEG shell cross-linked polymer in a methanol solution according to the mass ratio of 1:2.5, heating to 30 ℃, reacting for 11h, and evaporating the solvent to obtain modified docetaxel;
s3: dissolving modified docetaxel and chlorin in THF (tetrahydrofuran) according to the mass ratio of 1:1, dropwise adding 10 drops of toluenesulfonic acid, stirring and reacting at 35 ℃ for 4 hours, adding water according to the mass ratio of 1:2, mixing, dialyzing, and removing an organic solvent to obtain a substance A, wherein the substance A is modified docetaxel loaded with a photosensitizer and having acid sensitivity;
s4: dissolving the prepared substance A and PCL-acylamide-PEG in methanol according to the mass ratio of 1:2, reacting for 12h at room temperature, centrifuging at 6600rpm for 5min, precipitating, taking supernatant, adding water for injection, stirring and mixing, and filtering through a microporous filter membrane to obtain the docetaxel polymer nano injection.
Example 2
The embodiment provides a docetaxel polymer nano injection, which comprises the following components: modified docetaxel, chlorin and PCL-acylamide-PEG; the modified docetaxel is coated and modified by a shell cross-linked polymer; the modified docetaxel and the chlorin are connected through an ortho ester bond; the PCL-acylamide-PEG is coated outside the chlorin.
The embodiment also provides a preparation method of the docetaxel polymer nano injection, which comprises the following steps:
s1: preparing a PPhe-PLys-PEG shell cross-linked polymer in advance;
s2: dissolving docetaxel and PPhe-PLys-PEG shell cross-linked polymer in a methanol solution according to the mass ratio of 1:3, heating to 35 ℃, reacting for 10 hours, and evaporating the solvent to obtain modified docetaxel;
s3: dissolving modified docetaxel and chlorin in THF (tetrahydrofuran) according to the mass ratio of 1:2, dropwise adding 20 drops of toluenesulfonic acid, stirring and reacting at 45 ℃ for 3 hours, adding water according to the mass ratio of 1:2, mixing, dialyzing, and removing an organic solvent to obtain a substance A, wherein the substance A is modified docetaxel loaded with a photosensitizer and having acid sensitivity;
s4: dissolving the prepared substance A and PCL-acylamide-PEG in methanol according to the mass ratio of 1:1, reacting for 10h at room temperature, centrifuging at 6200rpm for 5min, precipitating, taking supernatant, adding water for injection, stirring and mixing, and filtering through a microporous filter membrane to obtain the docetaxel polymer nano injection.
Example 3
The embodiment provides a docetaxel polymer nano injection, which comprises the following components: modified docetaxel, chlorin and PCL-acylamide-PEG; the modified docetaxel is coated and modified by a shell cross-linked polymer; the modified docetaxel and the chlorin are connected through an ortho ester bond; the PCL-acylamide-PEG is coated outside the chlorin.
The embodiment also provides a preparation method of the docetaxel polymer nano injection, which comprises the following steps:
s1: preparing a PPhe-PLys-PEG shell cross-linked polymer in advance;
s2: dissolving docetaxel and PPhe-PLys-PEG shell cross-linked polymer in a methanol solution according to a mass ratio of 1:2, heating to 25 ℃, reacting for 12 hours, and evaporating the solvent to obtain modified docetaxel;
s3: dissolving modified docetaxel and chlorin in THF (tetrahydrofuran) according to a mass ratio of 1:1.5, dropwise adding 15 drops of toluenesulfonic acid, stirring and reacting at 40 ℃ for 3 hours, adding water according to a mass ratio of 1:1.5, mixing, dialyzing, and removing an organic solvent to obtain a substance A, wherein the substance A is modified docetaxel loaded with a photosensitizer and having acid sensitivity;
s4: dissolving the prepared substance A and PCL-acylamide-PEG in methanol according to the mass ratio of 1:3, reacting for 12h at room temperature, centrifuging at 6800rpm for 5min, precipitating, taking supernatant, adding water for injection, stirring and mixing, and filtering through a microporous filter membrane to obtain the docetaxel polymer nano injection.
Example 4
The embodiment provides a docetaxel polymer nano injection, which comprises the following components: modified docetaxel, chlorin and PCL-acylamide-PEG; the modified docetaxel is coated and modified by a shell cross-linked polymer; the modified docetaxel and the chlorin are connected through an ortho ester bond; the PCL-acylamide-PEG is coated outside the chlorin.
The embodiment also provides a preparation method of the docetaxel polymer nano injection, which comprises the following steps:
s1: preparing a PPhe-PLys-PEG shell cross-linked polymer in advance;
s2: dissolving docetaxel and PPhe-PLys-PEG shell cross-linked polymer in a methanol solution according to a mass ratio of 1:2, heating to 30 ℃, reacting for 11 hours, and evaporating the solvent to obtain modified docetaxel;
s3: dissolving modified docetaxel and chlorin in THF (tetrahydrofuran) according to a mass ratio of 1:1.8, dropwise adding 16 drops of toluenesulfonic acid, stirring and reacting at 42 ℃ for 4 hours, adding water according to a mass ratio of 1:2, mixing, dialyzing, and removing an organic solvent to obtain a substance A, wherein the substance A is modified docetaxel loaded with a photosensitizer and having acid sensitivity;
s4: dissolving the prepared substance A and PCL-acylamide-PEG in methanol according to the mass ratio of 1:2, reacting for 11h at room temperature, centrifuging at 6500rpm for 5min, precipitating, taking supernatant, adding water for injection, stirring and mixing, and filtering through a microporous filter membrane to obtain the docetaxel polymer nano injection.
Example 5
The embodiment provides a docetaxel polymer nano injection, which comprises the following components: modified docetaxel, chlorin and PCL-acylamide-PEG; the modified docetaxel is coated and modified by a shell cross-linked polymer; the modified docetaxel and the chlorin are connected through an ortho ester bond; the PCL-acylamide-PEG is coated outside the chlorin.
The embodiment also provides a preparation method of the docetaxel polymer nano injection, which comprises the following steps:
s1: preparing a PPhe-PLys-PEG shell cross-linked polymer in advance;
s2: dissolving docetaxel and PPhe-PLys-PEG shell cross-linked polymer in a methanol solution according to a mass ratio of 1:3, heating to 40 ℃, reacting for 10 hours, and evaporating the solvent to obtain modified docetaxel;
s3: dissolving modified docetaxel and chlorin in THF (tetrahydrofuran) according to a mass ratio of 1:1.6, dropwise adding 12 drops of toluenesulfonic acid, stirring and reacting at 45 ℃ for 2 hours, adding water according to a mass ratio of 1:1 for mixing, dialyzing, and removing an organic solvent to obtain a substance A, wherein the substance A is modified docetaxel loaded with a photosensitizer and having acid sensitivity;
s4: dissolving the prepared substance A and PCL-acylamide-PEG in methanol according to the mass ratio of 2:3, reacting for 12h at room temperature, centrifuging at 7000rpm for 5min, precipitating, taking supernatant, adding water for injection, stirring and mixing, and filtering through a microporous filter membrane to obtain the docetaxel polymer nano injection.
Example 6
The embodiment provides a docetaxel polymer nano injection, which comprises the following components: modified docetaxel, chlorin and PCL-acylamide-PEG; the modified docetaxel is coated and modified by a shell cross-linked polymer; the modified docetaxel and the chlorin are connected through an ortho ester bond; the PCL-acylamide-PEG is coated outside the chlorin.
The embodiment also provides a preparation method of the docetaxel polymer nano injection, which comprises the following steps:
s1: preparing a PPhe-PLys-PEG shell cross-linked polymer in advance;
s2: dissolving docetaxel and PPhe-PLys-PEG shell cross-linked polymer in a methanol solution according to a mass ratio of 1:3, heating to 20 ℃, reacting for 12 hours, and evaporating the solvent to obtain modified docetaxel;
s3: dissolving modified docetaxel and chlorin in THF (tetrahydrofuran) according to a mass ratio of 1:1.4, dropwise adding 10 drops of toluenesulfonic acid, stirring and reacting at 40 ℃ for 3 hours, adding water according to a mass ratio of 1:2, mixing, dialyzing, and removing an organic solvent to obtain a substance A, wherein the substance A is modified docetaxel loaded with a photosensitizer and having acid sensitivity;
s4: dissolving the prepared substance A and PCL-acylamide-PEG in a mass ratio of 1:1 in methanol, reacting at room temperature for 10 hours, centrifuging at 6000rpm for 5 minutes, precipitating, taking supernate, adding water for injection, stirring and mixing, and filtering through a microporous filter membrane to obtain the docetaxel polymer nano injection.
Comparative example 1
The comparative example provides a docetaxel polymer injection comprising the following components: modified docetaxel and chlorin; the modified docetaxel is coated and modified by a PPhe-PLys-PEG shell cross-linked polymer; the modified docetaxel and the chlorin are connected through an orthoester bond.
The comparative example also provides a preparation method of the docetaxel polymer injection, which comprises the following steps:
s1: preparing a PPhe-PLys-PEG shell cross-linked polymer in advance;
s2: dissolving docetaxel and PPhe-PLys-PEG shell cross-linked polymer in a methanol solution according to the mass ratio of 1:2.5, heating to 30 ℃, reacting for 11h, and evaporating the solvent to obtain modified docetaxel;
s3: dissolving modified docetaxel and chlorin in THF (tetrahydrofuran) under the condition of argon, dropwise adding 10 drops of toluenesulfonic acid, stirring and reacting for 4 hours at 35 ℃, adding water according to the mass ratio of 1:2, mixing, dialyzing, removing the organic solvent, adding absolute ethyl alcohol, stirring and mixing, and filtering through a microporous membrane to obtain the docetaxel polymer nano injection.
Comparative example 2
The comparative example provides a docetaxel polymer injection, which comprises the following components: modified docetaxel; the modified docetaxel is coated and modified by PPhe-PLys-PEG shell cross-linked polymer.
The comparative example also provides a preparation method of the docetaxel polymer injection, which comprises the following steps:
s1: preparing a PPhe-PLys-PEG shell cross-linked polymer in advance;
s2: dissolving docetaxel and PPhe-PLys-PEG shell cross-linked polymer in a methanol solution according to the mass ratio of 1:2.5, heating to 30 ℃, reacting for 11h, and evaporating the solvent to obtain modified docetaxel; adding water for injection, stirring and mixing, and filtering by a microporous filter membrane to obtain the docetaxel polymer nano injection.
Comparative example 3
The comparative example provides a docetaxel polymer injection, which comprises the following components: docetaxel and PCL-acylamide-PEG; the docetaxel is encapsulated in PCL-acylamide-PEG.
The comparative example also provides a preparation method of the docetaxel polymer injection, which comprises the following steps:
dissolving docetaxel and PCL-acylamide-PEG in a methanol solution according to a mass ratio of 1:3, heating to 40 ℃, reacting for 10h, evaporating the solvent, adding water for injection, stirring and mixing, and filtering through a microporous filter membrane to obtain the docetaxel polymer nano injection.
Comparative example 4
The comparative example provides a docetaxel polymer injection comprising the following components: docetaxel, chlorin, and PCL-acylamide-PEG; the docetaxel and the chlorin are encapsulated in PCL-acylamide-PEG.
The comparative example also provides a preparation method of the docetaxel polymer injection, which comprises the following steps:
dissolving docetaxel, chlorin and PCL-acylamide-PEG in a methanol solution according to a mass ratio of 1:1:3, heating to 35 ℃ for reaction for 11h, evaporating the solvent, adding water for injection, stirring and mixing, and filtering through a microporous filter membrane to obtain the docetaxel polymer nano injection.
Experimental example 1
Solubility test:
standing the docetaxel polymer nano injection prepared in the examples 1-6 and the docetaxel polymer injection prepared in the comparative examples 2 and 4 at room temperature for 0-48 h to observe the condition of the injection; the results are shown in Table 1.
TABLE 1 docetaxel polymer nano-injection
As can be seen from Table 1, the docetaxel polymer nano injection prepared in the embodiments 1-6 of the invention has high stability, and no precipitate is separated out after standing for 48 hours; the docetaxel polymer injection prepared in the comparative examples 2 and 4 has low stability, and precipitates begin to appear after standing for 8 hours; the docetaxel polymer injection prepared in the comparative example 2 is only subjected to primary docetaxel modification, and does not use PCL-acylamide-PEG for secondary coating; the docetaxel polymer injection prepared in the comparative example 4 only coats PCL-acylamide-PEG, and docetaxel is not modified; the prepared docetaxel polymer nano injection coats the PCL-acylamide-PEG on the surface of the modified docetaxel loaded with the photosensitizer, so that the modified docetaxel loaded with the photosensitizer and coated with the PCL-acylamide-PEG forms double-layer coating, the coating property of the medicine is enhanced, and the sudden release of the medicine is avoided, so that the medicine has higher stability after redissolution and is convenient to apply.
Experimental example 2
In vitro cytotoxicity experiments:
1. tumor cell culture
Breast tumor cell (MCF-7): culturing in RPMI 1640 medium containing 10% fetal bovine serum and 1% streptomycin mixture at 37 deg.C and 5% CO2And carrying out subculture once every 2-3 days.
2. In vitro cytotoxicity assay
Digesting the mammary tumor cell suspension in logarithmic growth phase, inoculating 100 μ l of the suspension into each well of a 96-well plate, and adjusting the density of the mammary tumor cells to 5 × 103Wells/well, marginal wells filled with sterile PBS; placing at 37 ℃ and 5% CO2The culture box is incubated for 24 hours until cell monolayers are paved on the bottom of a hole (a 96-hole flat-bottom plate), 10 mul of docetaxel polymer nano injection prepared in examples 1-6 and comparative examples 1-4 with concentration gradients of 20 mug/ml, 50 mug/ml, 80 mug/ml and 100 mug/ml is respectively added, and each concentration gradient of each sample of the examples is provided with 6 compound holes; setting the concentration gradient to 20. mu.g/ml, 50. mu.g/ml, 80. mu.g/ml, 100. mu.g/mlTaking 10 mul of docetaxel injection as a control group, and arranging 6 compound wells for each concentration gradient of a control group sample; setting 6 cell suspension holes without docetaxel polymer nanometer injection as medicine-free sets; set up 6 wells with medium only as blank; the cell plate after adding the drug is placed at 37 ℃ and 5% CO2The culture was terminated after 48h incubation in the incubator, 10. mu.l of CCK-8 reagent was added to each well, and the mixture was incubated at 37 ℃ with 5% CO2Incubating for 1-4 h in the incubator; the OD of each well was measured at a wavelength of 450nm, and the average OD of each 6 wells was calculated, and the results are shown in Table 2.
The cell survival rate is (the OD value of the drug-added group-the blank group OD value)/(the OD value of the drug-not-added group-the blank group OD value) × 100%
TABLE 2 mean survival rates of breast tumor cells
As can be seen from table 2, the survival rates of breast tumor cells of the docetaxel polymer nano-injections prepared in examples 1 to 6 are significantly lower than those of the docetaxel polymer injections prepared in examples 1 to 4 and the commercially available docetaxel injection, that is, the inhibition rates of the docetaxel polymer nano-injections prepared in examples 1 to 6 on breast tumor cells are higher than those of the docetaxel polymer injections prepared in examples 1 to 4 and the commercially available docetaxel injection on breast tumor cells; the docetaxel polymer injection prepared in the comparative example 1 is not coated with PCL-acylamide-PEG on the surface, the docetaxel polymer injection prepared in the comparative example 2 is not coated with photosensitizer medicine or PCL-acylamide-PEG on the surface, the docetaxel polymer injection prepared in the comparative example 3 is not coated with photosensitizer medicine, and the docetaxel polymer injection prepared in the comparative example 4 is not modified; according to the docetaxel polymer nano injection prepared by the invention, docetaxel is modified by coating a PPhe-PLys-PEG shell cross-linked polymer on the surface, and is loaded with a photosensitizer at the same time, and then coated with PCL-acylamide-PEG on the surface; therefore, the docetaxel and the photosensitizer generate a combined treatment effect, and meanwhile, the docetaxel and the photosensitizer have double coating, the stability is improved, the docetaxel is not easy to burst, and the PEG is used as a shell, so that the recognition and phagocytosis of phagocytic cells can be effectively avoided, the in vivo circulation time is prolonged, the targeting effect is enhanced, the modified docetaxel can be slowly released, and the anti-tumor performance of the medicine is comprehensively improved.
Experimental example 3
In vivo antitumor activity assay:
1. establishment of mouse mammary gland tumor animal model
(1) Experiment mouse: 66 nude mice have no specific pathogen level, are half male and half female, and are 4-8 weeks old.
(2) Preparing a breast tumor cell suspension: digesting the mammary tumor cell suspension in logarithmic growth phase, washing with serum-free RPMI 1640 culture solution, centrifuging, counting, and preparing into 1 × 10 serum-free RPMI 1640 culture solution or sterile physiological saline7A suspension of breast tumor cells in ml.
(3) Inoculating breast tumor cells: sterilizing the skin of a nude mouse in a sterile environment, and injecting 0.2ml of breast tumor cell suspension into the skin of the underarm part of the right front limb of the nude mouse by using a syringe; the inoculated nude mice were bred in a sterile environment.
(4) Establishing a mouse mammary tumor animal model: 3 mutually perpendicular diameters of each nude mouse tumor body are measured at regular time every day, and the average diameter of the tumor body is calculated, and the tumor body is used as a mouse mammary tumor animal model when the tumor body grows to have an average diameter of 4 mm.
2. In vivo antitumor Activity test
Preparing docetaxel polymer nano injection and commercially available docetaxel injection with the concentration of 10mg/ml prepared in examples 1-6 and comparative examples 1-4, injecting the docetaxel nano injection and the commercially available docetaxel injection into experimental mice set as mouse mammary tumor animal models in a tail vein manner, injecting 6 experimental mice into each group, and administering once a day for 7 days continuously; and a group of tail vein injection physiological saline is arranged in an experimental mouse set as a mouse mammary tumor animal model to be used as a blank group; the test mice were sacrificed the next day after the administration was stopped, tumor masses were removed, the tumor masses were weighed after removing other tissues, and the average tumor inhibition rate of each dose group of each test group was calculated, and the results are shown in table 3.
Tumor inhibition rate (tumor weight of blank group-tumor weight of administered group)/tumor weight of blank group × 100%
TABLE 3 mean tumor inhibition
As can be seen from Table 3, the docetaxel polymer nano-injections prepared in examples 1 to 6 have significantly higher antitumor activity than docetaxel polymer injections and commercially available docetaxel injections prepared in comparative examples 1 to 4; the docetaxel polymer injection prepared in the comparative example 1 is not coated with PCL-acylamide-PEG on the surface, the docetaxel polymer injection prepared in the comparative example 2 is not coated with photosensitizer medicine or PCL-acylamide-PEG on the surface, the docetaxel polymer injection prepared in the comparative example 3 is not coated with photosensitizer medicine, and the docetaxel polymer injection prepared in the comparative example 4 is not modified; according to the docetaxel polymer nano injection prepared by the invention, docetaxel is modified by coating a PPhe-PLys-PEG shell cross-linked polymer on the surface, and is loaded with a photosensitizer at the same time, and then coated with PCL-acylamide-PEG on the surface; therefore, the docetaxel and the photosensitizer generate a combined treatment effect, and meanwhile, the docetaxel and the photosensitizer have double coating, the stability is improved, the docetaxel is not easy to burst, and the PEG is used as a shell, so that the recognition and phagocytosis of phagocytic cells can be effectively avoided, the in vivo circulation time is prolonged, the targeting effect is enhanced, the modified docetaxel can be slowly released, and the anti-tumor performance of the medicine is comprehensively improved.
Experimental example 4
Respectively dissolving the docetaxel polymer nano injection prepared in the example 1-6 and the docetaxel polymer injection prepared in the comparative example 4 in an aqueous solution with the pH of 7 for 2 hours, adjusting the pH of the aqueous solution to 5, heating the aqueous solution to 37 ℃, and recording the cumulative release rate of the drug for 0-72 hours; the results are shown in Table 4.
TABLE 4 cumulative drug release rate
As can be seen from table 3, the docetaxel polymer nano-injections prepared in examples 1 to 6 were slowly released, while the docetaxel polymer injection prepared in comparative example 4 was rapidly released; docetaxel of docetaxel polymer injection prepared in comparative example 4 was not modified; according to the docetaxel polymer nano injection prepared by the invention, docetaxel is modified by coating a PPhe-PLys-PEG shell cross-linked polymer on the surface, so that the stability is improved, the docetaxel is not easy to release suddenly, and the slow release of the docetaxel is realized, so that the in-vivo action time is prolonged, and the anti-tumor performance of the medicine is improved.
In conclusion, the docetaxel has better water solubility through modification, and can be directly dissolved by water for injection to prepare injection, so that toxic and side effects of the whole body caused by the fact that the injection is prepared by solubilization of Tween80 and ethanol-assisted dissolution are avoided; the docetaxel polymer nano injection has high stability, does not precipitate after standing for 48 hours, and does not bring systemic toxic and side effects due to burst release; the docetaxel polymer nano injection realizes the slow release of docetaxel by modifying docetaxel, and carries a photosensitizer for combined treatment, thereby having better anti-tumor performance.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A docetaxel polymer nano injection comprises the following components: modifying docetaxel, chlorin and PCL-acylamide-PEG blocks; the modified docetaxel is coated and modified by a shell cross-linked polymer; the modified docetaxel and the chlorin are connected through an ortho ester bond; the PCL-acylamide-PEG block is coated outside the chlorin; the shell cross-linked polymer is PPhe-PLys-PEG, wherein the PLys is cross-linked by disulfide bonds.
2. The docetaxel polymer nano-injection as claimed in claim 1, wherein the docetaxel polymer has a molecular weight of 41000-48000.
3. The preparation method of the docetaxel polymer nano-injection as claimed in any of claims 1 to 2, which comprises the following steps:
s1: preparing a PPhe-PLys-PEG shell cross-linked polymer in advance;
s2: dissolving docetaxel and a PPhe-PLys-PEG shell cross-linked polymer in a methanol solution according to a mass ratio of 1: 2-3, heating to 20-40 ℃, reacting for 10-12 h, and evaporating the solvent to obtain modified docetaxel;
s3: dissolving modified docetaxel and chlorin in THF (tetrahydrofuran) under the condition of argon, dropwise adding 10-20 drops of toluenesulfonic acid, stirring and reacting for 2-4 hours at 35-45 ℃, then adding water according to the mass ratio of 1: 1-2, mixing, dialyzing, and removing an organic solvent to obtain a substance A, wherein the substance A is modified docetaxel loaded with chlorin and having acid sensitivity;
s4: dissolving the prepared substance A and the PCL-acylamide-PEG block in methanol, reacting for 10-12 h at room temperature, centrifuging at 6000-7000 rpm for 5min, precipitating, taking supernate, adding water for injection, stirring and mixing, and filtering through a microporous filter membrane to obtain the docetaxel polymer nano injection.
4. The method for preparing the docetaxel polymer nano injection as claimed in claim 3, wherein the modified docetaxel and the chlorin are added in the step S3 in a mass ratio of 1: 1-2.
5. The method for preparing docetaxel polymer nano-injection according to claim 3, wherein the mass ratio of the substance A to the PCL-acylamide-PEG block in the step S4 is 1: 1-3.
6. The method for preparing the docetaxel polymer nano injection as claimed in claim 3, wherein the docetaxel polymer nano injection has an average particle size of 20 to 80 nm.
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