CN114177136B - Amphiphilic block copolymer nano drug-loaded micelle for injection - Google Patents
Amphiphilic block copolymer nano drug-loaded micelle for injection Download PDFInfo
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- CN114177136B CN114177136B CN202010957145.6A CN202010957145A CN114177136B CN 114177136 B CN114177136 B CN 114177136B CN 202010957145 A CN202010957145 A CN 202010957145A CN 114177136 B CN114177136 B CN 114177136B
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- drug
- block copolymer
- amphiphilic block
- micelle
- fullerene
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Abstract
The invention relates to the field of drug carriers, and relates to a micelle material with pH response characteristics, which is prepared by chemically embedding hydrophobic fullerene, hydrophilic group polylactic acid and polyacrylic acid. The material has simple preparation process, strong micelle hydrophilicity and good drug carrying capacity on insoluble hydrophobic drugs, and is a good carrier for preparing injection dosage forms. The drug-loaded micelle prepared by the material has good pH response under the action of focal cell microenvironment and lysosomes, and can release drugs more easily; in addition, animal experiments show that the vorinostat micelle prepared by the material has the pharmacodynamics effect of obviously reducing peripheral bladder cancer cells compared with the traditional emulsion on bladder cancer model mice.
Description
Technical Field
The invention belongs to the field of preparation of drug micelle preparations, and particularly relates to a new amphiphilic block copolymer drug-loaded micelle and a preparation method thereof.
Background
The segmented copolymer micelle is a novel drug nano-carrier which is rapidly developed in recent years. In aqueous solution, the hydrophobic blocks in the amphiphilic block copolymer form the inner core, the hydrophilic blocks form the outer shell, and the block copolymer micelle with a spherical inner core-outer shell structure is formed. The block copolymer micelle has the following advantages as a drug carrier: (1) The Critical Micelle Concentration (CMC) is low, the thermodynamic stability and the kinetic stability are high, the CMC is not easy to break even under CMC, and the dilution resistance is high; (2) Self-assembling in water solution to form micelle, the inner core can be loaded with hydrophobic medicine, and the hydrophilic surface is not easy to be identified and acquired by reticuloendothelial system (RES), so that the circulation time of the medicine in blood is prolonged; (3) The EPR effect can penetrate through the capillary wall of the tumor part to enter the tumor tissue, and accumulate at the tumor part, so that the targeted drug release is achieved, and the toxic and side effects of the drug are reduced.
With the development of polymer material science and nanotechnology, more and more segmented copolymer micelles are used as drug carriers, and some of the drug carriers have entered clinical trial stages and even are approved for clinical use. In order to improve the treatment possibility to the greatest extent and reduce the toxic and side effects of the medicine, the construction of micelle structures of various stimulus-responsive block copolymers, such as temperature-responsive, pH-responsive, light-responsive, magnetic field-responsive, ultrasonic-responsive, chemical and bioactive molecule-responsive and the like, is realized through flexible design of the polymer structure. However, the common stimulus-responsive micelles such as pH, light, magnetic field and ultrasound have the problems of low drug utilization rate, limited drug release behavior control and the like, and the application of the chemical and biological active molecule-responsive micelles is limited, so that the targeted controlled release effect is poor.
Fullerene (also known as C) 60 ～ 85 Fullerenes as photosensitizers are used in photodynamic therapy (photodynamic therapy, PDT) of tumors. For example, application number "CN201711066996.6" discloses fullerene and polyaza ligand transition metal complexes for magnetic resonance imaging and photodynamic therapy, with high relaxation rates, efficient magnetic resonance effects and high accuracyThe magnetic resonance imaging diagnosis and photodynamic therapy function of the composition can be used for preparing magnetic resonance imaging contrast agents, antioxidants and photodynamic therapeutic agents. The application number of CN201310138295.4 is that magnetic water-soluble polyethylene glycol fullerene (C60-Fe) 3 O 4 PEG 2000) is mixed with an anti-tumor drug as a carrier for the drug using ultrasound to meet the requirements of photodynamic therapy. Most of the similar patents use the photosensitivity of fullerenes in combination with paramagnetic metal ions to provide therapeutic sensitivity, and to increase nuclear magnetic resonance and imaging effects, and do not primarily use fullerenes as drug delivery vehicles for poorly soluble drugs.
In addition, since fullerenes are hydrophobic compounds and are difficult to dissolve in water, hydrophilic excipients are often used to disperse them. For example, patent 201410006420.0 "a preparation method and application of self-agglomerating drug-loaded sustained-release microsphere of implantable fullerene polylactic acid" uses synthesized fullerene polylactic acid as drug-loaded sustained-release microsphere for photodynamic therapy. However, the objective of this patent is not to use the drug-loaded microsphere as a directly injectable preparation, and the side chain polylactic acid has a large molecular weight, a high degree of polymerization, and a molecular weight up to 1,0000-1,8000, so that the high molecular weight is difficult to transmit and release drugs through cell membranes or blood brain barriers, and large agglomeration easily occurs in an aqueous phase, and the produced microsphere is difficult to stably and uniformly disperse in the aqueous phase, and is difficult to be clinically used as an injectable preparation. According to the patent, the microspheres are required to be prepared as a solid, rather than as a liquid formulation, after lyophilization.
Disclosure of Invention
The photosensitivity of the fullerene can be used as a targeting drug carrier for photodynamic therapy, or the fullerene can be easily gathered around inflammatory lesions to remove free radicals, and can be used as a targeting drug carrier for anti-inflammatory drugs, and meanwhile, the problem that the drugs pass through cell membranes and blood brain barriers is solved, so that the drug dosage form for injection is prepared. The size of the polymer micelle is smaller than that of the microsphere and the emulsion, and the small particles can avoid the recognition and phagocytosis of macrophages in the human body after entering the human body, so that the polymer micelle has excellent tissue permeability and long in-vivo residence time, can enable the medicine to effectively reach a target point, has the effect of controlling the release of the medicine, and is more suitable for preparing injectable medicine preparations.
In order to solve the problems, the invention utilizes the hydrophobicity of fullerene to increase the affinity with hydrophobic drugs in the hydrophobic part of a drug-carrying carrier, introduces meta-acid hydrophilic group acrylic acid as a pH response segment in the design, introduces polylactic acid with certain molecular weight as a hydrophilic segment, and simultaneously controls the molecular weight of a polylactic acid side chain and the molecular weight of a polyacrylic acid side chain not to be too large, thereby facilitating the formation of micelles with smaller particle size, and finally forming a new micelle carrier, namely the fullerene and a hydrophilic block to prepare the amphiphilic pH-sensitive stimulus-responsive micelle.
The drug-loaded micelle has the following characteristics:
1. the hydrophobic part of the drug-loaded micelle is a fullerene part, can be affinitized with hydrophobic indissolvable drug molecules, and utilizes the photosensitivity and free radical affinity of the fullerene, so that the targeting of the micelle to a focus is improved; the polyacrylic acid and polylactic acid become hydrophilic parts, so that the water solubility of the micelle is increased.
2. The drug-loaded micelle adopts low molecular weight polyacrylic acid and polylactic acid side chains, and the molecular weight of the copolymer is controlled to be less than 10000, so that the drug-loaded micelle can be cleared by kidneys and can be discharged out of the body through normal body metabolic pathways, and potential toxicity caused by accumulation of amphiphilic block copolymers in the body is avoided.
2. The acrylic acid part of the drug-loaded micelle has pH response characteristic, and after the carrier is easy to form micelle with weak acid and weak alkaline hydrophobic drugs, the drug can be released in the pH environment around the focus in vivo, and the amphiphilic block copolymer can be discharged out of the body through a normal metabolic pathway after drug release, so that potential toxicity caused by accumulation in the body is avoided, the drug utilization rate can be improved, and the synthesis method is simplified.
The amphiphilic block copolymer drug-loaded micelle disclosed by the invention consists of an amphiphilic block copolymer and a hydrophobic drug, wherein the hydrophobic drug is embedded in a hydrophobic inner core formed by the amphiphilic block copolymer, and the amphiphilic block copolymer has the following structural formula:
wherein the number of carbon atoms of the fullerene may be 60, 70; lactic acid molecule n=1 to 30; acrylic acid molecule m=1 to 20; the average molecular weight of the copolymer is 960-4000.
The content of the hydrophobic drug in the drug-loaded micelle is 3-10wt%.
The particle size of the drug-loaded micelle of the amphiphilic block copolymer is 70-300 nm.
The polyacrylic acid-fullerene-polylactic acid copolymer prepared by the method can be dissolved in organic solvents such as methanol, ethanol, pyridine, DMSO, acetone and the like, and can be dissolved in hot water and alkaline water. The self-agglomeration drug-carrying slow-release microsphere can be effectively used for micelle carriers of weak acid and weak base type insoluble drugs, the preparation method is simple, the preparation conditions are easy to meet, the characteristics of fullerene can not be damaged, and innovation on micelle carrier materials is realized.
The steps of copolymer synthesis and micelle preparation are as follows:
(1) Low molecular weight polyacrylic acid is prepared. Adding silver sulfate and deionized water, starting stirring, heating to 70 ℃ in a water bath, slowly dropwise adding acrylic acid and hydrogen peroxide for 4 hours, reacting, and simultaneously controlling the pH=5-6 of the system by dropwise adding sodium hydroxide solution. The precipitate was dried in vacuo at 30 ℃ to give a pale yellow solid, low molecular weight polyacrylic acid PAA.
(2) Preparing the bromine-substituted low molecular weight polyacrylic acid. And (3) taking the polyacrylic acid in the step (1), sequentially adding ethyl 2-bromoisobutyrate (EBiB), cuBr, pentamethyldiethylenetriamine (PMEDTA) and acetone, and carrying out freeze-thawing and degassing. The ampoule bottle is sealed by nitrogen, reacted for 5 hours at 60 ℃, cooled by cold water, and then put into liquid nitrogen to terminate the reaction. After the polymer is diluted by tetrahydrofuran, impurities such as copper ions and the like are removed by neutral alumina column chromatography, tetrahydrofuran solution is concentrated, the polymer is precipitated twice in methanol/water (V/V=1/1) to obtain a white viscous product, and the white viscous product is placed at 40 ℃ for vacuum drying for 4 hours to obtain the bromine-substituted low molecular weight polyacrylic acid PAA-Br.
(3) Preparing fullerene terminated low molecular weight polyacrylic acid. Adding the PAA-Br in the step (2) into a flask, adding sarcosine, fullerene and an organic solvent, repeatedly vacuumizing, introducing nitrogen for three times to deoxidize, stirring at 30-60 ℃ until the end-capped polyacrylic acid is completely dissolved, and heating to 80-100 ℃ to react for 24-36h; after cooling to room temperature, 50-75ml of methanol is used for precipitation, centrifugal separation is carried out at 3000-8000rpm, and the Fullerene terminated polyacrylic acid (Fullerene-PAA) is obtained after drying under reduced pressure at room temperature.
(4) Preparing the hydroxy fullerene polyacrylic acid. Adding the Fullerene terminated polyacrylic acid obtained in the step (3) into a flask, adding p-hydroxybenzaldehyde and myoammoniumalcohol, heating to 80-120 ℃ with an organic solvent under stirring, magnetically stirring for 24 hours, rapidly cooling to room temperature, precipitating the reaction liquid methanol, centrifuging the precipitate at 3000-8000rpm, and drying under reduced pressure at room temperature to obtain the Fullerene polyacrylic acid (Fullerene-OH-PAA) with hydroxyl groups.
(5) And (3) taking the Fullerene-OH-PAA, the D, L-lactide and the stannous octoate in the step (4), adding a certain amount of toluene into a flask, slowly heating to 60-100 ℃ under magnetic stirring, reacting for 36-72h, and pouring the reaction liquid into methanol for precipitation. And (3) centrifugally separating the precipitate at 3000-8000rpm, and drying the precipitate at room temperature under reduced pressure for 8-10h to obtain the polyacrylic acid-Fullerene-polylactic acid amphiphilic block copolymer PAA-Fullerene-PLA.
(6) Dissolving the medicine and the copolymer obtained in the step (5) in a cosolvent, evaporating the cosolvent to form a film, adding deionized water or pH buffer solution under stirring or shaking, high-speed shearing or homogenizing, dispersing the copolymer to obtain an amphiphilic block copolymer medicine-carrying micelle, filtering and dialyzing the mixed solution to remove residual impurities and bacteria to obtain an amphiphilic block copolymer medicine-carrying micelle solution, and filtering and freeze-drying to obtain the amphiphilic block copolymer medicine-carrying micelle.
According to the preparation method of the amphiphilic block copolymer drug-loaded micelle, the cosolvent can be used for well dissolving the fullerene hydrophobic block, the hydrophilic block and the insoluble hydrophobic drug of the amphiphilic block copolymer, the specific effect is different according to the different hydrophobic drugs, and the cosolvent can be one or more of methanol, ethanol, tetrahydrofuran, acetone, pyridine, 1N, N-dimethylformamide or triethylamine and 4-dioxane.
In the preparation method of the amphiphilic block copolymer drug-loaded micelle, in the step (4), deionized water or distilled water is dropwise added into the solution at the dropwise adding rate of 10-20 s/drop, a filter membrane is adopted when the mixed solution is filtered, and the pore diameter of the filter membrane is 0.20-0.45 mu m.
In the preparation method of the amphiphilic block copolymer drug-loaded micelle, in the step (6), the addition amount of the hydrophobic drug is related to the drug-loaded amount of the drug-loaded micelle, the addition amount of the hydrophobic drug is determined according to the requirement of the drug-loaded amount in practical application, and the addition amount of the hydrophobic drug is 1-60% of the mass of the amphiphilic block copolymer.
According to the preparation method of the amphiphilic block copolymer PAA-Fullerene-PLA drug-loaded micelle, the hydrophobic drug is determined according to actual application requirements, and can be weak acid and weak alkaline compounds such as vorinostat, doxorubicin and retinoic acid, or one or more insoluble natural compounds such as engelhardin, houttuynin, angelica lactone and pulsatilla saponin A.
Further preferably, the molar ratio of the monomer lactide to the dihydroxyl initiator in the step (5) is 27-78:1, and the monomer lactide is one or more of L-lactide or D-lactide.
The amphiphilic block copolymer is a drug-loaded micelle with carrier targeting intracellular drug release, and is characterized by comprising the amphiphilic block copolymer and a hydrophobic drug, wherein the hydrophobic drug is wrapped in a hydrophobic core formed by a hydrophobic fullerene block of the amphiphilic block copolymer.
Further preferably, the particle size of the drug-loaded micelle is 40-200nm.
The drug release mechanism of the amphiphilic block copolymer drug-loaded micelle is as follows:
when PMMA-Fullerene-PLA micelle enters a human body, the pH value of plasma is 7.1-7.5, the pH value is larger than pKa, and the electron rearrangement of Fullerene structural fragments of the copolymer becomes deprotonation, and the micelle is dissociated, so that the entrapped medicine is released. In vitro drug release experiments showed faster release of vorinostat by drug-loaded micelles at ph=5.5 compared to ph=7.4 and 6.8. These results demonstrate that such pH-responsive polymers are very promising as carriers for hydrophobic drugs.
Compared with the prior art, the invention has the following outstanding beneficial technical effects:
(1) The preparation method is simple, the preparation conditions are easy to meet, the physical and chemical stability is good, the raw material sources are rich, the cost is low, and the characteristics of the fullerene are not damaged;
(2) The invention enriches the types of drug-loaded micelles, and the fullerene-containing microspheres have photosensitivity after self-agglomeration and can be used for photodynamic therapy;
(3) After micelle dissociation, the fullerene hydrophobic block as a hydrophobic core is converted into a hydrophilic block, and the whole amphiphilic block copolymer is converted into a hydrophilic polymer. The amphiphilic block copolymer is converted into a hydrophilic polymer after drug release, can be dissolved in a human body, has a relative molecular mass of less than 10000, can be cleared by kidneys and can be discharged out of the human body through a normal body metabolic pathway, so that potential toxicity caused by accumulation of the amphiphilic block copolymer in the human body is avoided.
(4) The polyacrylic acid-Fullerene-polylactic acid PAA-Fullerene-PLA self-agglomeration drug-loaded sustained-release microsphere prepared by the invention has uniform particle size and high drug loading capacity up to 55%, has wide applicable drug range, can uniformly disperse one or more of a plurality of insoluble drugs such as vorinostat, doxorubicin, taxol, retinoic acid or insoluble natural compounds such as engelhardin, houttuynin, angelica lactone, pulsatilla saponin A and the like in aqueous solution, obviously improves the concentration in the solution, is easier to prepare into injectable preparations, and is innovation on drug carriers.
Drawings
FIG. 1 is a schematic diagram of the structure of PAA-Fullerene-PLA
FIG. 2 is a PAA-C prepared in example 1 60 -PLA nuclear magnetic profile hydrogen spectrum.
FIG. 3 is a PAA-C prepared in example 1 60 PLA time-of-flight mass spectrometry.
FIG. 4 is a distribution diagram of the particle size of the blank micelle prepared in example 2.
Fig. 5 is a distribution diagram of the particle size of vorinostat micelles prepared in example 2.
Fig. 6 is a drug release profile of the drug-loaded micelle of example 2.
FIG. 7 is an image of the live bladder of each group of mice after drug administration.
FIG. 8 is a comparison of peripheral blood G-MDSCs level test data from mice in each group.
Detailed Description
The amphiphilic block copolymer drug-loaded micelle and the preparation method thereof are further described below by way of examples.
Example 1
In this example, an amphiphilic block copolymer PAA-C was prepared 60 PLA, as in fig. 1, of the formula:
(1) Adding 10mg of silver sulfate and deionized water into a reaction kettle provided with a reflux condenser, a stirrer and a thermometer, starting stirring, heating to 70 ℃ by heating in a water bath, slowly dripping 100g of acrylic acid and 20g of hydrogen peroxide for 4 hours, reacting while dripping 10mol/L of sodium hydroxide solution, controlling the pH value of a system to be 5-6, preserving heat for 4 hours after finishing the reaction to obtain light yellow viscous liquid, washing precipitate with deionized water after filtering, adding 200ml of water to be heated to 70-80 ℃ for dissolving, adding absolute ethyl alcohol for precipitation, cooling for refining, and freeze-drying for 24 hours to obtain low molecular weight polyacrylic acid with the average molecular weight of 700-1200 and m=10-15. The yield is 80-90%.
(2) A100 ml ampoule was charged with a magnetic stirrer, followed by 0.29ml of ethyl 2-bromoisobutyrate (EBiB) (2 mmol), 0.22g of CuBr (1.5 mmol), 0.32ml of Pentamethyldiethylenetriamine (PMEDTA) (1.5 mmol), 11.5g (about 13 mmol) of the low molecular weight polyacrylic acid obtained in (1) and 20ml of acetone in this order, and the mixture was degassed three times by freeze-thawing. The ampoule bottle is sealed by nitrogen, reacted for 5 hours at 60 ℃, cooled by cold water, and then put into liquid nitrogen to terminate the reaction. After diluting the polymer with tetrahydrofuran, removing impurities such as copper ions through neutral alumina column chromatography, concentrating tetrahydrofuran solution, precipitating the polymer twice in methanol/water (V/v=1/1) to obtain white viscous product, and vacuum drying at 40 ℃ for 4h. Thus obtaining the bromine end capped low molecular weight polyacrylic acid (PAA-Br). The yield is 65-75%.
(3) Taking 1.5g of bromine-terminated polyacrylic acid in the step (2) to a 200ml two-neck flask, adding 50-100mg of sarcosine and 144mg of fullerene and 100ml of toluene, repeatedly vacuumizing and introducing nitrogen for three times to remove oxygen, stirring at 30-60 ℃ until the terminated polyacrylic acid is completely dissolved, and heating to 80-100 ℃ to react for 24-36h; cooling to room temperature, precipitating with 50-75ml methanol, centrifuging at 3000-8000rpm, and drying at room temperature under reduced pressure to obtain fullerene terminated polyacrylic acid. The yield is 75-85%. The average molecular weight is 1500-2000 as determined by a QX-08 type gas phase permeameter (VPO). m=10-15. The yield is 65-75%.
(4) Taking 300mg of the fullerene terminated polyacrylic acid of the above (3) to a two-neck flask with a condenser, adding 50mg of p-hydroxybenzaldehyde, 50mg of myoamino alcohol and 50ml of toluene, heating to 80-120 ℃ under stirring, magnetically stirring for 24 hours, rapidly cooling to room temperature, precipitating the reaction solution with 100ml of methanol, centrifuging the precipitate at 3000-8000rpm, and drying at room temperature under reduced pressure to obtain the fullerene (C) 60 -OH-PAA). The yield is 75-80%.
(5) 100mg (C) 60 -OH-PAA), 200mgD, L-lactide and 5-8mg stannous octoate are put into a flask, 15-20ml toluene is added, the temperature is slowly increased to 60-100 ℃ under magnetic stirring, and after 36-72 hours of reaction, the reaction solution is poured into methanol to be cooled and precipitated. And (3) centrifugally separating the precipitate at 3000-8000rpm, dissolving the polymer in hot water, cooling and precipitating with methanol twice for refining, and drying at 40 ℃ under reduced pressure for 8-10h to obtain the polyacrylic acid-fullerene-polylactic acid amphiphilic block copolymer. The average molecular weight is 3000-3800, m=10-15, n=20-25 as determined by QX-08 type gas phase permeameter (VPO). The yield is 75-80%.
PAA-C prepared in this example 60 Nuclear magnetic spectrum hydrogen spectrum of PLA is shown in fig. 2, and mass spectrum is shown in fig. 3. Combined with the VPO measurement result, the present practice is explainedExample successful preparation of amphiphilic Block copolymer PAA-C 60 -PLA。
Example 2: preparation of blank micelle and drug-loaded micelle Using the amphiphilic Block copolymer prepared in example 1
(1) Preparation of blank micelles
The amphiphilic block copolymer PAA-C prepared in example 1 60 PLA is precisely weighed to 50mg and dissolved in 10ml of methanol, the solution is oscillated to be completely dissolved, the methanol solution is added into a 50ml rotary steam bottle, the methanol is evaporated to be dry to form a film, then 5ml of pure water is added, and the solution is fully oscillated and hydrated by ultrasonic waves, and a blank micelle solution with the thickness of 0.45 mu m can be obtained. The particle size distribution of the blank micelle was shown in FIG. 4, and the average particle size was 199.9nm, and the distribution coefficient (PDI) was 0.280.
(2) Determination of critical micelle concentration CMC for blank micelle
Critical micelle concentration determination blank micelles without drug were prepared. A5X 104mol/L pyrene solution was prepared with acetone, and 20. Mu.l was added to an ampoule and air-dried for 12 hours. Then adding blank micelles with different concentrations into air-dried ampoule bottles, wherein the concentrations are 6×10 respectively -1 、3×10 -1 、6×10 -2 、3×10 -2 、6×10 -3 、3×10 -3 、6×10 -4 、3×10 -4 、6×10 -5 、3×10 -5 、6×10 -6 And 3X 10 -6 mg/mL, and mixed for 24h. Fluorescence intensities at 333nm excitation wavelength of 373nm and 384nm were detected by I 373 /I 384 The ratio calculates the micelle critical concentration (CMC). I 373 /I 384 The ratio of (a) reflects the change in the form of micelle present in the aqueous solution (single molecule state and micelle state). The critical micelle concentration is 67.42 mug/mL, and the micelle can keep good stability when being applied in vitro and in vivo.
(3) Micelle drug loading and encapsulation efficiency experiment
The amphiphilic block copolymer PAA-C prepared in example 1 60 Accurately weighing 50mg of PLA, accurately weighing 100mg of excessive vorinostat, adding into 20ml of methanol, shaking for 2 hr, filtering with 0.45 μm microporous membrane, evaporating to dryness in 50ml rotary evaporator to form film, and adding 5ml of pureAnd (3) sufficiently ultrasonically oscillating and hydrating water, passing through a microporous filter membrane with the thickness of 0.45 mu m to obtain micelle solution with the maximum drug loading capacity, and finally quantifying into a 5ml volumetric flask. 500. Mu.L of the micelle was taken and destroyed by adding an equal volume of methanol precisely for 1 hour. And then 200 mu L of each liquid before and after the destruction is added into a sample injection bottle, the content of vorinostat in the micelle liquid and the liquid after the destruction by methanol is detected by high performance liquid phase respectively, and the Encapsulation Efficiency (EE) and the maximum drug loading rate (DL) are calculated. The calculation formula is as follows:
EE% = (concentration of vorinostat in the micelle liquid before disruption/concentration of vorinostat in the liquid after disruption 2 x) 100%
DL% = (concentration of vorinostat in post-disruption liquid 2-concentration of vorinostat in pre-disruption micelle liquid) 5/50 x 100%
Calculated EE% = 95.4%, DL% = 55%, the micelle has good encapsulation efficiency of 95.4% for vorinostat and the maximum drug loading is 55%, namely, the micelle concentration of 10mg/ml can lead the concentration of vorinostat in the aqueous solution to be as high as 5.5mg/ml, has good drug loading, and improves the hydrophobic state of vorinostat which is almost insoluble in water.
(4) Preparation of drug-loaded micelles
The amphiphilic block copolymer PAA-C prepared in example 1 60 PLA precisely weighing 50mg, precisely weighing 10mg of vorinostat, putting into 20ml of methanol, sufficiently oscillating for 2 hours, filtering the methanol into a 50ml rotary steaming bottle by using a 0.45 mu m microporous filter membrane, evaporating the methanol to form a film, then adding 5ml of pure water, sufficiently oscillating and hydrating the film by ultrasound, filtering the film by using the 0.45 mu m microporous filter membrane, and quantifying the film into a 5ml volumetric bottle to obtain the micelle solution with the concentration of vorinostat of 2 mg/ml. The particle size distribution of the drug-loaded micelle is shown in FIG. 5, the average particle size is 176.3nm, and the distribution coefficient (PDI) is 0.254.
Example 3
Micelle drug release assay: taking 0.5ml of the drug-loaded micelle prepared in the example 2 (4), adding the drug-loaded micelle into a 2kDa dialysis bag, placing the dialysis bag into a 50ml centrifuge tube, adding 5ml of dialysis medium, and carrying out 10 times of internal and external volume difference to prepare a sink condition, and oscillating at 100rpm and 37 ℃. The dialysis media was divided into 3 groups: phosphate Buffer (PBS) +10% foetal calf serum at pH 7.4; PBS at pH 6.8; pH5.5 PBS. Each medium contained 1mol/L sodium salicylate. Wherein, pH7.4 Phosphate Buffer (PBS) +10% fetal bovine serum simulates blood environment, pH6.8 PBS simulates tumor extracellular slightly acidic environment, and pH5.5 PBS group simulates lysosomal condition.
The dialysis medium was collected at 0.5h, 1h, 2h, 4h, 6h, 9h, 12h, 24h, 36h, 48h, respectively, and replaced. 200 μl of the collected dialysis medium was added to a sample bottle, and the vorinostat content was detected by HPLC, and a release curve was drawn.
As in fig. 6.
The results show that the micelle is released quickly within 8 hours, and after 8 hours, the micelle tends to be stable, and under the condition of pH5.5, the micelle is quickly decomposed, so that the drug release is quick. And the release speed is faster at pH6.4 than pH7.4, which shows that the micelle can have better controlled release effect on vorinostat in tumor microenvironment. The pH change affects the drug release of the drug-loaded micelle, and the prepared drug-loaded micelle shows good pH-sensitive response characteristics in an in-vivo environment and can realize the controlled release of the entrapped drug.
Example 4
Preliminary stability investigation: to examine the stability of formulations of different concentration formulations to meet the intravenous injection grade requirements, micelles of formulations 1, 2, and 3 were prepared according to the method of example 2 (4).
1. Preserving at 4 DEG C
From the above data, it was demonstrated that the stability of micelles was good under low temperature conditions.
2. Preserving at 30 ℃):
the data show that the amphiphilic block copolymer vorinostat micelle can keep better stability in the condition of 4-30 ℃ within 6 months, but the micelle particle size change is larger in the condition of 30 ℃ in the period of 6 months, which indicates that the micelle is more stable at the low temperature of 4 ℃.
Example 5
Comparison of the anti-tumor effect of the amphiphilic Block copolymer vorinostat micelle and vorinostat lecithin emulsion injection in model mice
Preparing vorinostat emulsion: 20mg of vorinostat is dissolved in 10ml of soybean oil, 20mg of egg yolk lecithin is added, and the mixture is mixed by a shearing machine, and a small amount of phosphate buffer solution with pH of 7.4 is added to prepare colostrum, and the colostrum is homogenized at a speed of 3000-10000 rpm for 10 minutes to 30 minutes and repeated for 3-5 times. Finally homogenizing in a high-pressure homogenizer for 1-2 times under 800-1000kg pressure to collect emulsion, and diluting the emulsion to 100ml with pH7.4 phosphate buffer solution to obtain 2mg/ml vorinostat emulsion; in addition, 2mg/ml vorinostat micelles were formulated as in formulation 3 described in example 3.
30 healthy C3H/He female mice which are 8 weeks old and have no specific pathogen (specific pathogen free, SPF) are selected, MBT-2 mouse bladder cancer cells are subcutaneously injected to construct a C3H/He mouse bladder cancer model, 10 mice are grouped into groups, and A group is set as a blank control group. Group B was set as vorinostat emulsion group (2 mg/ml) and group C was set as vorinostat micelle group (2 mg/ml).
Establishing an animal model: (1) female C3H/He female mice were selected for 30, 8 week old, 17-20g weight of staining method, and given regular diet during feeding for 12H of circadian rhythm. (2) Isoflurane is inhaled into the anesthetized mice, and the oxygen concentration is adjusted to enable the mice to be in a shallow anesthetizing state, and an electric blanket is arranged on the mice in the anesthetizing process to keep the body temperature of the mice. (3) After anesthesia, the mice were supine, the limbs were fixed, the indwelling needles were fixed by wiping with a paraffin cotton ball, inserting the mice into the urinary bladder through the urethra after lubrication, and the depth was about 5 mm. (4) Sucking urine in the bladder out, and flushing the urine with PBS for 3 times; 100 μl of 0.1mol/L HCl solution was injected, stagnant for 15-20s, rapidly neutralized with NaOH solution of the same concentration and volume to fully neutralize the hydrochloric acid and finally aspirated, and rinsed 3 times with PBS solution again. (5) After evacuating the bladder, 100. Mu.l of a 0.1mol/ml poly L-lysine solution (molecular weight 70000-150000) was poured, left for 20min, and then gently withdrawn. (6) Lightly perfusing the pretreated MBT-2 cell suspension (2X 106/ml) into the bladder, and standing for 2h; after 2 hours, the remaining needle hose is pulled out, and oxygen is introduced to wake up the mice.
After MBT-2 was successfully transplanted into mice, the corresponding fluorescence signal was observed by the small animal fluorescence imaging device after about 10min by intraperitoneal injection of an aqueous solution of the luciferase substrate D-potassium luciferin salt, and the intensity of the fluorescence signal and the number of cancer cells were positively correlated. The mice were observed daily for diet and mental status after planting. The living mouse imaging results were observed at 5 th, 10 th and 14 th days when the plantation diary was 0 d. FIG. 7 is an image of the live bladder after each group of 14 days of drug administration.
Administration: the vorinostat emulsion and micelle preparation doses were 50mg/kg intravenously every 48h for group B and group C mice, respectively, and the group a was a blank control group, which was sacrificed at 14 d.
Identification and isolation of G-MDSCs: after C3H/He mice were sacrificed at 14d, they were dissected and spleened after 75% ethanol sterilization. After washing 3 times with sterile physiological saline, the spleen was placed in a mortar and sufficiently ground. Filtering with 200 mesh sieve, and cleaning with sterile physiological saline for 3 times. The CD11b and Ly6G mouse fluorescent antibody (working concentration is 1:50) is adopted to mark G-MDSCs in spleen cells, the spleen cells are incubated for 30min at room temperature in a dark place, then the spleen cells are washed 3 times by Phosphate Buffer (PBS), and the spleen cells are placed in a flow cytometer for analysis, identification, separation and purification.
Results: flow cytometry results show that the in-situ bladder cancer animal model of mice transplanted with MBT-2 in group A has an oncological rate of 80%; the G-MDSCs levels in the circulatory system were significantly reduced in group C mice compared to group B (p=0.000). Conclusion that in the study of the bladder perfusion treatment of the mouse model, the tumor inhibition effect of the micelle group is higher than that of the emulsion group and the control group (P < 0.05), which shows that the pH response of the micelle and the targeting of the micelle are superior to those of the vorinostat emulsion group. FIG. 7 is an image of the live bladder after each group of 14 days of drug administration; FIG. 8 is a comparison of peripheral blood G-MDSCs level test data from mice in each group.
Example 6
The method for preparing an excessive amount of drug micelle of example 2 (3) was changed to doxorubicin, taxol, retinoic acid, engelhardin, houttuynin, angelicalactone, pulsatilla saponin a and the like in the amounts shown in the following table, and the solvent in which the drug was dissolved in the step was replaced according to the following table. And finally, detecting the content of each drug in the liquid after the destruction of the micelle liquid and the methanol by using a high-efficiency liquid phase, and calculating the Encapsulation Efficiency (EE) and the maximum Drug Loading (DL), wherein the results are shown in the following table.
Experimental results show that the copolymer has different degrees of affinity for medicines with different structural types, has higher affinity for compounds with naphthenes and long fatty chains, such as taxol, retinoic acid and the like, can be widely applied to various insoluble medicines, has high medicine carrying capacity, can obviously improve the medicine concentration in solution compared with a pure aqueous solution, is easier to prepare into injectable preparations, and is a good medicine carrier.
While the basic principles, principal features and advantages of the present invention have been described in the foregoing examples, it will be appreciated by those skilled in the art that the present invention is not limited by the foregoing examples, but is merely illustrative of the principles of the invention, and various changes and modifications can be made without departing from the scope of the invention, which is defined by the appended claims.
Claims (14)
1. The amphiphilic block copolymer is characterized by comprising two hydrophilic segments and a hydrophobic segment, wherein the hydrophobic segment is a fullerene segment, the hydrophilic segment is polylactic acid and polyacrylic acid, the average molecular weight of the amphiphilic block copolymer is 800-10000, and the structure formula is as follows:
wherein n=2 to 50; m=2 to 50.
2. The process for preparing an amphiphilic block copolymer according to claim 1, wherein hydroxylated fullerene-containing polyacrylic acid, D, L-lactide and stannous octoate are placed in a flask, a certain amount of toluene is added, the temperature is slowly raised to 60-100 ℃ under magnetic stirring, reaction is carried out for 36-72h, reaction liquid is poured into methanol for precipitation, the precipitate is centrifugally separated at 3000-8000rpm, and the precipitate is dried at room temperature under reduced pressure for 8-10h, so that the polyacrylic acid-fullerene-polylactic acid amphiphilic block copolymer is obtained.
3. The process for preparing an amphiphilic block copolymer according to claim 2, wherein the preparation method of the hydroxylated fullerene-containing polyacrylic acid is as follows: adding p-hydroxybenzaldehyde, myoammoniol and a certain amount of toluene into a two-neck flask, heating to 80-120 ℃ under stirring, magnetically stirring for 24-72 h, precipitating with a certain amount of methanol, centrifuging, separating, and drying under reduced pressure to obtain the hydroxylated fullerene polyacrylic acid.
4. The process for preparing an amphiphilic block copolymer according to claim 3, wherein the process for preparing fullerene terminated polyacrylic acid comprises the following steps: the bromine-capped low molecular weight polyacrylic acid is chemically combined with a fullerene to form a fullerene-capped polyacrylic acid.
5. The process for preparing the amphiphilic block copolymer according to claim 4, wherein the preparation process comprises the steps of taking a small amount of silver sulfate and deionized water, stirring and making water bath, slowly dropwise adding acrylic acid and hydrogen peroxide, adding sodium hydroxide solution, controlling pH of a system to be 5-6, drying precipitate to obtain low molecular weight polyacrylic acid, sequentially adding 2-bromoisobutyric acid ethyl ester, copper bromide, pentamethyldiethylenetriamine, acetone and nitrogen for sealing, reacting, adding liquid nitrogen for terminating reaction, removing impurities, refining to obtain a white viscous product, and vacuum drying to obtain the bromine-substituted low molecular weight polyacrylic acid.
6. The drug-loaded micelle formed by the amphiphilic block copolymer according to claim 1, which consists of the amphiphilic block copolymer and a hydrophobic drug.
7. The drug-loaded micelle formed by the amphiphilic block copolymer according to claim 6, wherein the content of the hydrophobic drug in the drug-loaded micelle is 3-10wt%.
8. The drug-loaded micelle formed by the amphiphilic block copolymer according to claim 6, wherein the particle size of the drug-loaded micelle is 70-300 nm.
9. The drug-loaded micelle formed by the amphiphilic block copolymer according to claim 6, wherein the hydrophobic drug is vorinostat, doxorubicin, retinoic acid, engelhardin, houttuynin, angelica lactone, pulsatilla saponin a.
10. The method for preparing the drug-loaded micelle according to any one of claims 6 to 9, wherein the hydrophobic drug and the amphiphilic block copolymer are dissolved in a cosolvent, the cosolvent is evaporated to form a film, deionized water or a pH buffer solution is added for dispersion, filtration and dialysis are carried out, and the amphiphilic block copolymer drug-loaded micelle is obtained after filtration and freeze drying, wherein the cosolvent is one or any mixture of methanol, ethanol, tetrahydrofuran, acetone, pyridine, N, N-dimethylformamide, triethylamine and 1, 4-dioxane.
11. The method for preparing drug-loaded micelles of claim 10, wherein the dispersion is by stirring, shaking, high-speed shearing, or homogenizing.
12. The method for preparing drug-loaded micelles of claim 10, wherein the pore size of the filter membrane used for filtering is 0.20-0.45 μm.
13. Use of a drug-loaded micelle formed by the amphiphilic block copolymer according to any one of claims 6-9 in the preparation of a micelle formulation for injection.
14. Use of the micelle preparation for injection according to claim 13 for preparing anti-tumor, anti-inflammatory and anti-infective drugs.
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