CN114177136A - Injectable amphiphilic block copolymer nano drug-loaded micelle - Google Patents
Injectable amphiphilic block copolymer nano drug-loaded micelle Download PDFInfo
- Publication number
- CN114177136A CN114177136A CN202010957145.6A CN202010957145A CN114177136A CN 114177136 A CN114177136 A CN 114177136A CN 202010957145 A CN202010957145 A CN 202010957145A CN 114177136 A CN114177136 A CN 114177136A
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- Prior art keywords
- drug
- fullerene
- micelle
- block copolymer
- amphiphilic block
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- 229940079593 drug Drugs 0.000 title claims abstract description 114
- 239000000693 micelle Substances 0.000 title claims abstract description 108
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Abstract
The invention discloses an injectable amphiphilic block copolymer nano drug-loaded micelle, which relates to the field of drug carriers. The material has simple preparation process, strong micelle hydrophilicity and good drug loading capacity on insoluble hydrophobic drugs, and is a good carrier for preparing injection preparations. The drug-loaded micelle prepared by the material has better pH response under the actions of a focus cell microenvironment and lysosomes, and can release drugs more easily; in addition, animal experiments show that the vorinostat micelle prepared by the material has a pharmacodynamic effect of remarkably reducing peripheral bladder cancer cells on a bladder cancer model mouse compared with the traditional emulsion.
Description
Technical Field
The invention belongs to the field of preparation of medicinal micelle preparations, and particularly relates to a novel amphiphilic block copolymer medicine-carrying micelle and a preparation method thereof.
Background
The block copolymer micelle is a novel drug nano-carrier which is rapidly developed in recent years. In an aqueous solution, the hydrophobic block in the amphiphilic block copolymer forms a core, and the hydrophilic block forms a shell, so that a block copolymer micelle with a spherical core-shell structure is formed. The block copolymer micelle as a drug carrier has the following advantages: (1) the Critical Micelle Concentration (CMC) is low, the thermodynamic and kinetic stability is high, the CMC is not easy to damage even below the CMC, and the dilution resistance is high; (2) the micelle is formed by self-assembly in an aqueous solution, the inner core can be loaded with hydrophobic drugs, and the hydrophilic surface ensures that the drugs are not easily identified and obtained by a reticuloendothelial system (RES), so that the circulation time of the drugs in blood is prolonged; (3) the size is small, the distribution is narrow, the particle size is generally between 10 nm and 200nm, and the particle size can be prevented from being cleared by the kidney; (4) can penetrate through the capillary wall of a tumor part to enter a tumor tissue through an EPR effect and accumulate at the tumor part, thereby achieving targeted drug release and reducing the toxic and side effects of the drug.
With the development of polymer material science and nanotechnology, more and more block copolymer micelles are used as drug carriers, and some drug carriers have entered clinical trial stage or even been approved for clinical use. In order to improve the treatment possibility and reduce the toxic and side effect of the medicine to the maximum extent, the construction of a plurality of different stimulus response type block copolymer micelle structures is realized through the flexible design of a polymer structure, such as a temperature response type, a pH response type, a light response type, a magnetic field response type, an ultrasonic response type, a chemical and biological active molecule response type and the like. However, the common stimuli-responsive micelles such as pH, light, magnetic field, ultrasound and the like have the problems of low drug utilization rate, limited control of drug release behavior and the like, and the application of the chemically and biologically active molecule-responsive micelles is limited, and the targeted controlled release effect is not good.
Fullerene (Fullerene), also known as C60-85, is used as a photosensitizer for photodynamic therapy (PDT) treatment of tumors. For example, application No. CN201711066996.6 discloses a fullerene and polyaza ligand transition metal complex for magnetic resonance imaging and photodynamic therapy, which has high relaxivity, high-efficiency magnetic resonance effect, and high-accuracy magnetic resonance imaging diagnosis and photodynamic therapy effects, and can be used for preparing magnetic resonance imaging contrast agents, antioxidants and photodynamic therapy agents. The application number "CN 201310138295.4" is that the paramagnetic performance of fullerene and ferroferric oxide is utilized to prepare magnetic water-soluble polyethylene glycol fullerene (C60-Fe3O4-PEG2000), and the magnetic water-soluble polyethylene glycol fullerene is mixed with an anti-tumor drug by using an ultrasonic method to be used as a carrier of the drug, so as to meet the requirements of photodynamic therapy. Most of the similar patents utilize the photosensitivity of fullerene, and combine with paramagnetic metal ions to achieve the sensitivity of phototherapy and increase the nuclear magnetic resonance and imaging effects, and the purpose of the fullerene is not to mainly use fullerene as a drug release carrier of a poorly soluble drug.
In addition, since fullerene is a hydrophobic compound and is hardly soluble in water, it is often dispersed with a hydrophilic excipient. For example, in patent 201410006420.0, "a method for preparing an implantable fullerene polylactic acid self-agglomerated drug-carrying sustained release microsphere and its application", a synthetic fullerene polylactic acid is used as a drug-carrying sustained release microsphere for photodynamic therapy. However, the drug-loaded microsphere is not used as a preparation which can be directly injected, and the polylactic acid with the side chain has large molecular weight and high polymerization degree, and the molecular weight reaches 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 is easy to occur in a water phase, and the generated microsphere is difficult to stably and uniformly disperse in the water phase and is difficult to be used clinically as an injection preparation. The microspheres described therein need to be freeze-dried to prepare a solid, rather than a liquid, formulation.
Disclosure of Invention
How to effectively utilize the photosensitivity of the fullerene can be used as a photodynamic therapy targeting drug carrier, or the fullerene is easy to gather around an inflammation focus to remove free radicals and can be used as a targeting drug carrier of an anti-inflammatory drug, and meanwhile, the problem that the drug passes through cell membranes and blood brain barriers is solved, and a 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 a human body after entering the human body, so that the polymer micelle has excellent tissue permeability and long in-vivo retention time, can enable the medicine to effectively reach a target spot, has the effect of controlling the release of the medicine, and is more suitable for preparing an injectable medicinal preparation.
In order to solve the problems, the invention utilizes the hydrophobicity of fullerene as a hydrophobic part of a drug-carrying carrier to increase the affinity with hydrophobic drugs, introduces a meta-acid hydrophilic group acrylic acid as a pH response segment in the design, then introduces polylactic acid with a 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, so that micelles with smaller particle sizes are conveniently formed, and finally a new micelle carrier is formed, namely the fullerene and a hydrophilic block are prepared into an amphiphilic pH sensitive stimulus response type micelle.
The drug-loaded micelle has the following characteristics:
1. the hydrophobic part of the drug-loaded micelle is a fullerene part, can be compatible with hydrophobic and insoluble drug molecules, and utilizes the photosensitivity and the free radical affinity of fullerene, thereby improving the targeting property of the micelle to a focus; and the polyacrylic acid and polylactic acid parts 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, controls the molecular weight of the copolymer to be less than 10000, so that the drug-loaded micelle can be cleared by the kidney and can be discharged out of the body through a normal organism metabolic pathway, thereby avoiding potential toxicity caused by accumulation of the amphiphilic block copolymer in the body.
2. The acrylic acid part of the drug-loaded micelle has pH response characteristics, after the carrier is easy to form a colloid with weak acid, weak base and hydrophobic drugs, the drug can be released in the pH environment around the focus in vivo, and after the drug is released, the amphiphilic block copolymer can be discharged out of the body through a normal metabolic pathway, so that potential toxicity caused by accumulation in the body is avoided, the utilization rate of the drug can be improved, and the synthesis method is simplified.
The amphiphilic block copolymer drug-loaded micelle disclosed by the invention is composed of an amphiphilic block copolymer and a hydrophobic drug, wherein the hydrophobic drug is embedded in a hydrophobic core formed by the amphiphilic block copolymer, and the structural formula of the amphiphilic block copolymer is as follows:
wherein, the number of carbon atoms of the fullerene can be 60, 70; a lactic acid molecule n is 1-30; acrylic acid molecules m are 1-20; the average molecular weight of the copolymer is 960-4000.
According to the amphiphilic block copolymer drug-loaded micelle, the content of hydrophobic drugs in the drug-loaded micelle is 3-10 wt%.
The amphiphilic block copolymer drug-loaded micelle has the particle size of 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-aggregation drug-loaded sustained-release microspheres 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 the innovation on the micelle carrier material is realized.
The steps of copolymer synthesis and micelle preparation are as follows:
(1) preparing low molecular weight polyacrylic acid. Adding silver sulfate and deionized water, starting stirring, heating the water bath to 70 ℃, starting to slowly dropwise add acrylic acid and hydrogen peroxide for 4 hours, reacting while dropwise adding a sodium hydroxide solution, and controlling the pH value of the system to be 5-6. The precipitate was dried in vacuo at 30 ℃ to give a pale yellow solid, low molecular weight polyacrylic acid, PAA.
(2) Preparing bromine-substituted low molecular weight polyacrylic acid. And (2) taking the polyacrylic acid obtained in the step (1), sequentially adding ethyl 2-bromoisobutyrate (EBiB), CuBr, pentamethyl diethylenetriamine (PMEDTA) and acetone, and freeze-melting and degassing. Sealing ampoule with nitrogen, reacting at 60 deg.C for 5 hr, cooling with cold water, and adding into liquid nitrogen to stop reaction. After the polymer is diluted by tetrahydrofuran, impurities such as copper ions and the like are removed through neutral alumina column chromatography, then 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 bromine-substituted low molecular weight polyacrylic acid PAA-Br.
(3) Preparing the fullerene terminated low molecular weight polyacrylic acid. Putting the PAA-Br obtained in the step (2) into a flask, adding sarcosine, fullerene and an organic solvent, repeatedly vacuumizing and introducing nitrogen for three times to remove oxygen, stirring at the temperature of 30-60 ℃ until the end-capped polyacrylic acid is completely dissolved, and heating to 80-100 ℃ to react for 24-36 hours; cooling to room temperature, precipitating with 50-75ml methanol, centrifuging at 3000-8000rpm, and drying under reduced pressure at room temperature to obtain Fullerene-terminated polyacrylic acid (Fullerene-PAA).
(4) Preparing hydroxyfullerene polyacrylic acid. And (3) putting the Fullerene-terminated polyacrylic acid obtained in the step (3) into a flask, adding p-hydroxybenzaldehyde and sarcosinol, heating to 80-120 ℃ under stirring and keeping the temperature, magnetically stirring for 24 hours, then rapidly cooling to room temperature, precipitating reaction liquid by methanol, centrifugally separating the precipitate at 8000rpm of 3000, and drying under reduced pressure at room temperature to obtain the Fullerene polyacrylic acid with hydroxyl (Fullerene-OH-PAA).
(5) And (3) putting the Fullerene-OH-PAA, D, L-lactide and stannous octoate in the step (4) into a flask, adding a certain amount of toluene, slowly heating to 60-100 ℃ under magnetic stirring, reacting for 36-72h, and pouring the reaction liquid into methanol for precipitation. And (3) centrifuging and separating the precipitate at 8000rpm of 3000-.
(6) Dissolving the drug and the copolymer obtained in the step (5) in a cosolvent, evaporating the cosolvent to form a film, stirring or oscillating, adding deionized water or pH buffer solution under high-speed shearing or mixing by a homogenizer, dispersing the copolymer to obtain the amphiphilic block copolymer drug-loaded micelle, filtering the mixed solution after dialysis to remove residual impurities and bacteria to obtain an amphiphilic block copolymer drug-loaded micelle solution, and filtering, freezing and drying to obtain the amphiphilic block copolymer drug-loaded micelle.
According to the preparation method of the amphiphilic block copolymer drug-loaded micelle, the cosolvent only needs to be capable of well dissolving the fullerene hydrophobic block, the hydrophilic block and the insoluble hydrophobic drug of the amphiphilic block copolymer, and specifically can be different due to 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 dripped into the solution at a dripping rate of 10-20 s/droplet, and a filter membrane is adopted when the mixed solution is filtered, wherein the aperture 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 adding amount of the hydrophobic drug is related to the drug-loaded amount of the drug-loaded micelle, the adding amount of the hydrophobic drug is determined according to the requirement of the drug-loaded amount in practical application, and the adding 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 the actual application requirements, and can be weak-acid weak-alkaline compounds such as vorinostat, adriamycin, doxorubicin and retinoic acid, and can also be one or more of insoluble natural compounds such as engelhardoside, houttuynin, angelica lactone and pulsatilla saponin A.
Further preferably, the feeding molar ratio of the monomer lactide to the dihydroxy 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 a carrier targeting intracellular drug release, and is characterized in that the drug-loaded micelle consists of 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 medicine-carrying micelle is 40-200 nm.
The drug release mechanism of the amphiphilic block copolymer drug-loaded micelle is as follows:
when the PMMA-Fullerene-PLA micelle enters a human body, the pH value of blood plasma is 7.1-7.5, the pH value is greater than pKa, the electron rearrangement of the Fullerene structural segment of the copolymer is changed into deprotonation, and the micelle is dissociated, so that the entrapped drug is released. In vitro drug release experiments showed that drug loaded micelles released vorinostat faster at pH5.5 compared to pH7.4 and 6.8. These results indicate that such pH-responsive polymers are very potential 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 can not be damaged;
(2) the invention enriches the types of the drug-loaded micelle, contains fullerene which has photosensitivity after self-agglomeration into microspheres, and can be used for photodynamic therapy;
(3) after the micelle is dissociated, the fullerene hydrophobic block serving as the 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 hydrophilic polymer after releasing medicine, can be dissolved in vivo, has small enough relative molecular mass (<10000), can be cleared by kidney, and can be discharged out of body through normal organism metabolic pathway, thereby avoiding potential toxicity caused by accumulation of the amphiphilic block copolymer in vivo.
(4) The polyacrylic acid-Fullerene-polylactic acid PAA-Fullerene-PLA self-aggregation drug-loaded sustained-release microspheres prepared by the invention have uniform particle size and wide application range, the drug-loaded sustained-release microspheres can be used for uniformly dispersing a plurality of insoluble drugs such as vorinostat, adriamycin, doxorubicin, paclitaxel and retinoic acid or one or more insoluble natural compounds such as engelhardoside, houttuynin, angelica lactone and pulsatilla saponin A in an aqueous solution, the concentration of the solution is obviously improved, the microspheres are easier to prepare into injectable preparations, and the microspheres are innovation on drug carriers.
Drawings
FIG. 1 is a schematic structural diagram of PAA-Fullerene-PLA
FIG. 2 is a nuclear magnetic spectrum hydrogen spectrum of PAA-C60-PLA prepared in example 1.
FIG. 3 is a PAA-C60-PLA time-of-flight mass spectrum prepared in example 1.
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 micelles of example 2.
FIG. 7 is an image of live bladders after drug administration in each group of mice.
FIG. 8 is a graph showing data comparison between the peripheral blood G-MDSCs level measurements in mice of each group.
Detailed Description
The amphiphilic block copolymer drug-loaded micelle and the preparation method thereof of the present invention are further described below by examples.
Example 1
In this example, an amphiphilic block copolymer PAA-C60-PLA was prepared, as shown in FIG. 1, and has the following structural formula:
(1) adding 10mg of silver sulfate and deionized water into a reaction kettle provided with a reflux condenser tube, a stirrer and a thermometer, starting stirring, heating a water bath to 70 ℃, starting slowly dropwise adding 100g of acrylic acid and 20g of hydrogen peroxide for 4 hours, dropwise adding 10mol/L of sodium hydroxide solution during the reaction, controlling the pH value of the system to be 5-6, preserving the temperature for 4 hours after the reaction is ended to obtain light yellow viscous liquid, washing the precipitate with deionized water after filtering, then adding 200ml of water, heating to 70-80 ℃, dissolving, adding absolute ethyl alcohol for precipitation, cooling and refining, and freeze-drying for 24 hours to obtain low molecular weight polyacrylic acid, wherein the average molecular weight is 700-. The yield is 80-90%.
(2) A100 ml ampoule was charged with a magnetic stirrer, followed by sequentially adding 0.29ml of 2-bromoethyl isobutyrate (EBiB) (2mmol), 0.22g of CuBr (1.5mmol), and 0.32ml of Pentamethyldiethylenetriamine (PMEDTA) (1.5mmol), and freeze-thawing 11.5g (about 13mmol) of the low-molecular weight polyacrylic acid obtained in (1) and 20ml of acetone three times. Sealing ampoule with nitrogen, reacting at 60 deg.C for 5 hr, cooling with cold water, and adding into liquid nitrogen to stop reaction. The polymer was diluted with tetrahydrofuran, purified by neutral alumina column chromatography to remove impurities such as copper ions, and then the tetrahydrofuran solution was concentrated, and the polymer was precipitated twice in methanol/water (V/V-1/1) to give a white viscous product, which was dried under vacuum at 40 ℃ for 4 hours. Thus obtaining the bromine-terminated low-molecular-weight polyacrylic acid (PAA-Br). The yield is 65-75%.
(3) Taking 1.5g of bromine-terminated polyacrylic acid (2) to a 200ml two-neck flask, adding 50-100mg of sarcosine, 144mg of fullerene and 100ml of toluene, repeatedly vacuumizing, 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 24-36 h; cooling to room temperature, precipitating with 50-75ml methanol, centrifuging at 3000-8000rpm, and drying under reduced pressure at room temperature to obtain fullerene terminated polyacrylic acid. The yield is 75-85%. The average molecular weight of the sample was determined to be 1500-. And m is 10-15. The yield is 65-75%.
(4) And (3) putting 300mg of fullerene-terminated polyacrylic acid into a two-neck flask with a condensing tube, adding 50mg of p-hydroxybenzaldehyde, 50mg of sarcosine and 50ml of toluene, stirring, keeping the temperature, heating to 80-120 ℃, magnetically stirring for 24h, then rapidly cooling to room temperature, precipitating the reaction solution by using 100ml of methanol, centrifugally separating the precipitate by using 3000-8000rpm, and drying at room temperature under reduced pressure to obtain the fullerene (C60-OH-PAA) with hydroxyl. The yield is 75-80%.
(5) 100mg (C60-OH-PAA), 200mgD, L-lactide and 5-8mg stannous octoate are taken to be put into a flask, 15-20ml of toluene is added, the temperature is slowly raised to 60-100 ℃ under magnetic stirring, reaction is carried out for 36-72h, and then reaction liquid is poured into methanol for cooling and precipitation. Centrifuging at 3000-. The average molecular weight of the product is 3000-3800, M is 10-15, and n is 20-25, measured by QX-08 gas phase permeameter (VPO). The yield is 75-80%.
The nuclear magnetic spectrum hydrogen spectrum and the mass spectrum of the PAA-C60-PLA prepared in the example are shown in FIG. 2 and FIG. 3 respectively. The results of VPO measurement are combined to show that the amphiphilic block copolymer PAA-C60-PLA is successfully prepared in the example.
Example 2: preparation of blank micelle and drug-loaded micelle from amphiphilic Block copolymer prepared in example 1
(1) Preparation of blank micelles
50mg of amphiphilic block copolymer PAA-C60-PLA prepared in example 1 is precisely weighed, dissolved in 10ml of methanol, oscillated to be completely dissolved, the methanol solution is added into a 50ml rotary steaming bottle, the methanol is evaporated to form a film, then 5ml of pure water is added, and the mixture is fully ultrasonically oscillated and hydrated to 0.45 mu m to obtain a blank micelle solution. The particle size distribution of the blank micelle is shown in FIG. 4, and the average particle size is 199.9nm, and the distribution coefficient (PDI) is 0.280.
(2) CMC (critical micelle concentration) of blank micelle is determined
Critical micelle concentration determination a blank micelle was prepared without drug. 5X 104mol/L pyrene solution is prepared by acetone, 20 mu L of the solution is added into an ampoule bottle for air drying for 12 h. Then blank micelles with different concentrations are added into the air-dried ampoule bottles, the concentrations are respectively 6X 10-1, 3X 10-1, 6X 10-2, 3X 10-2, 6X 10-3, 3X 10-3, 6X 10-4, 3X 10-4, 6X 10-5, 3X 10-5, 6X 10-6 and 3X 10-6mg/mL, and the mixture is mixed for 24 h. Fluorescence intensities at 373nm and 384nm at an excitation wavelength of 333nm were measured, and micelle critical concentration (CMC) was calculated from the I373/I384 ratio. The change in the ratio of I373/I384 reflects the change in the form of micelles present in aqueous solution (monomolecular state and micellar 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 experiments
50mg of amphiphilic block copolymer PAA-C60-PLA prepared in example 1 is precisely weighed, 100mg of excessive drug vorinostat is precisely weighed, the drug vorinostat is put into 20ml of methanol and fully oscillated for 2 hours, the drug vorinostat is filtered by a 0.45 mu m microporous membrane to a 50ml rotary evaporation bottle to be evaporated to dryness to form a film, then 5ml of pure water is added, the mixture is fully oscillated and hydrated by ultrasound, the micelle solution with the maximum drug loading can be obtained by the 0.45 mu m microporous membrane, and finally the solution is quantified to a 5ml volumetric flask. 500. mu.L of the micelle was taken and disrupted by adding methanol of the same volume precisely for 1 hour. And then adding 200 mu L of liquid before and after the destruction into a sample injection bottle, respectively detecting the content of vorinostat in the micelle liquid and the liquid after the destruction of methanol by high performance liquid chromatography, and calculating the Entrapment Efficiency (EE) and the maximum Drug Loading (DL). The calculation formula is as follows:
EE% (vorinostat concentration in micellar solution before disruption/vorinostat concentration in solution after disruption) × 100%
DL% (vorinostat concentration in post-disruption liquid 2-vorinostat concentration in pre-disruption micellar liquid) 5/50%
The calculated EE percent is 95.4 percent and DL percent is 55 percent, the micelle has a good entrapment rate of 95.4 percent on the vorinostat, and the maximum drug loading is 55 percent, namely, the micelle concentration of 10mg/ml can ensure that the concentration of the vorinostat in an aqueous solution is as high as 5.5mg/ml, the good drug loading is realized, and the hydrophobic state of the vorinostat which is almost insoluble in water is improved.
(4) Preparation of drug-loaded micelles
50mg of amphiphilic block copolymer PAA-C60-PLA prepared in example 1 is precisely weighed, 10mg of vorinostat is precisely weighed, the weighed product is placed into 20ml of methanol, the mixture is fully oscillated for 2 hours, the obtained product is filtered by a 0.45 mu m microporous membrane to be dried in a 50ml rotary evaporation bottle to form a film, 5ml of pure water is added, the obtained product is fully ultrasonically oscillated and hydrated, the obtained product is filtered by the 0.45 mu m microporous membrane, and the obtained product is quantified to a 5ml volumetric flask, so that a micelle solution with the vorinostat concentration of 2mg/ml can be obtained. 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
Micellar 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, putting the dialysis bag into a 50ml centrifuge tube, adding 5ml of dialysis medium, making a leak groove condition by 10 times of difference between the internal volume and the external volume, and oscillating at 37 ℃ at 100 rpm. The dialysis media were divided into 3 groups: phosphate Buffered Saline (PBS) pH7.4 + 10% fetal bovine serum; pH6.8PBS; PBS (pH5.5PBS). Each group of media contained 1mol/L sodium salicylate. Wherein, pH7.4 Phosphate Buffer Solution (PBS) + 10% fetal bovine serum simulates blood environment, pH6.8PBS simulates micro-acid environment outside tumor cells, and pH5.5PBS simulates endoenzyme condition.
Collecting the dialysis medium at 0.5h, 1h, 2h, 4h, 6h, 9h, 12h, 24h, 36h and 48h respectively, and replacing the dialysis medium. 200 μ l of the collected dialysis medium was added to a sample bottle, and the release curve was plotted by HPLC for vorinostat content.
As shown in fig. 6.
The result shows that the micelle releases quickly in 8h and becomes stable after 8h, and under the condition of pH5.5, the micelle is decomposed quickly and the drug is released quickly. And the release speed is higher at pH6.4 than at pH7.4, which shows that the micelle can have better controlled release effect on the vorinostat in a tumor microenvironment. The change of the pH value influences the drug release of the drug-loaded micelle, the prepared drug-loaded micelle shows good pH sensitive response characteristic in the in vivo environment, and the control release of the entrapped drug can be realized.
Example 4
And (3) carrying out primary stability investigation: in order to examine the stability of formulations with different concentrations to meet the intravenous injection level requirements, micelles of formulations 1, 2 and 3 were prepared according to the method of example 2 (4).
1. Storing at 4 deg.C
From the above data, it is demonstrated that the stability of micelles is good under low temperature conditions.
2. And (3) storage at 30 ℃:
the data show that the amphiphilic block copolymer vorinostat micelle can keep good stability at 4-30 ℃ within 6 months, but the micelle particle size change is large at 30 ℃ within 6 months, which shows that the micelle is more stable at 4 ℃ at low temperature.
Example 5
Comparison of anti-tumor effects of amphiphilic block copolymer vorinostat micelle and vorinostat lecithin emulsion injection in model mice
Preparing vorinostat emulsion: dissolving vorinostat 20mg in soybean oil 10ml, adding egg yolk lecithin 20mg, mixing with shearing machine, adding small amount of phosphate buffer solution with pH7.4 to prepare primary emulsion, homogenizing at 3000-10000 rpm for 10-30 min, and repeating for 3-5 times. Finally homogenizing for 1-2 times in a high-pressure homogenizer under the pressure of 800-1000kg, collecting the emulsion, and diluting the emulsion to 100ml by using a phosphate buffer solution with pH7.4 to obtain the vorinostat emulsion of 2 mg/ml; in addition, vorinostat micelles of 2mg/ml were prepared according to formulation 3 described in example 3.
30 healthy C3H/He female mice of 8-week-old free from Specific Pathogen (SPF) grade were selected, and after a C3H/He mouse bladder cancer model was constructed by subcutaneously injecting MBT-2 mouse bladder cancer cells, they were divided into groups of 10 mice each, and group A was set as a blank control group. The group B was vorinostat emulsion group (2mg/ml) and the group C was vorinostat micelle group (2 mg/ml).
Establishing an animal model: 30 female C3H/He female mice are selected, the mice are 8-week-old and are marked by a staining method with the weight of 17-20g, and the mice are given regular diet and circadian rhythm of 12h in the breeding process. ② isoflurane is inhaled to anaesthetize the mouse, oxygen concentration is adjusted to make the mouse in a shallow anaesthetization state, and an electric blanket is padded during anaesthetization to keep the body temperature of the mouse. And thirdly, after anesthesia, the mouse lies on the back, the four limbs are fixed, an indwelling hose of a BD venous indwelling needle is used, the mouse is wiped and lubricated by a paraffin cotton ball and then is inserted into the bladder through the urethra with the depth of about 5mm, and the indwelling needle is fixed. Fourthly, urine in the bladder is sucked out and washed for 3 times by PBS; 100. mu.l of 0.1mol/L HCl solution are poured in, the solution is left standing for 15-20s, the hydrochloric acid is quickly and fully neutralized by NaOH solution with the same concentration and volume, finally the solution is sucked out, and the solution is washed 3 times by PBS solution again. Fifthly, after the bladder is evacuated, 100 mu L of 0.1mol/ml poly L-lysine solution (molecular weight 70000-. Sixthly, gently infusing the pretreated MBT-2 cell suspension (2X106/ml) into the bladder, and standing for 2 hours; after 2h, the indwelling needle hose is pulled out, and oxygen is introduced to revive the mouse.
After MBT-2 is successfully transplanted to a mouse, a luciferase substrate D-luciferin potassium salt aqueous solution is injected into the abdominal cavity, and a corresponding fluorescent signal can be observed by a small animal fluorescence imaging device after about 10min, wherein the intensity of the fluorescent signal is in positive correlation with the number of cancer cells. Mice were observed daily for diet and mental status after planting. The results of in vivo imaging of mice were observed at 5 th, 10 th and 14 th days, respectively, with the date of planting being 0 d. FIG. 7 is an image of live bladder after 14 days of administration of each group.
Administration: mice in group B and group C were injected intravenously with vorinostat emulsion and micelle formulation at a dose of 50mg/kg every 48h, and mice in group A were sacrificed at 14d after a blank control group was injected with physiological saline.
Identification and isolation of G-MDSCs: after killing the C3H/He mice at 14d, their spleens were dissected and harvested after 75% ethanol sterilization. After washing 3 times with sterile physiological saline, the spleen was put in a mortar and ground thoroughly. After filtering through a 200-mesh screen, the mixture was washed with sterile physiological saline for 3 times. G-MDSCs in spleen cells are marked by CD11b and Ly6G mouse fluorescent antibodies (working concentration is 1:50), incubated for 30min at room temperature in the dark, washed 3 times by Phosphate Buffer Solution (PBS), and placed in a flow cytometer for analysis, identification, separation and purification.
As a result: flow cytometry results show that the tumor formation rate of the mouse in situ bladder cancer animal model transplanted with the MBT-2 in the group A is 80 percent; the circulating levels of G-MDSCs were significantly reduced in mice in group C compared to group B (P ═ 0.000). The conclusion is that in the mouse model bladder perfusion treatment research, the tumor inhibition effect of the micelle group is higher than that of the emulsion group and the control group (P is less than 0.05), which indicates that the pH response type of the micelle and the targeting property of the micelle are better than those of the vorinostat emulsion group. FIG. 7 is an image of a live bladder after 14 days of administration of each group; FIG. 8 is a graph showing data comparison between the peripheral blood G-MDSCs level measurements in mice of each group.
Example 6
In the method for preparing the micelle of the excessive drug according to example 2(3), the drugs in the step are replaced by adriamycin, doxorubicin, paclitaxel, retinoic acid, engelhardoside, houttuynin, angelica lactone, pulsatilla saponin a and the like according to the amount of the following table, and the solvents for dissolving the drugs in the step are replaced according to the following table. And finally, detecting the content of each drug in the liquid after the micelle liquid and the methanol are damaged by using a high performance liquid, and calculating the Entrapment Efficiency (EE) and the maximum Drug Loading (DL), wherein the result is shown in the following table.
The experimental result shows that the copolymer has affinity with different structural types of drugs in different degrees, and has higher affinity with compounds with naphthene and long aliphatic chains, such as paclitaxel and retinoic acid, so that the copolymer can be widely applied to various insoluble drugs, has high drug loading, can obviously improve the drug concentration in a solution compared with a simple aqueous solution, is easier to prepare into an injectable preparation, and is a good drug carrier.
The foregoing embodiments illustrate the principles, principal features and advantages of the invention, and it will be understood by those skilled in the art that the invention is not limited to the foregoing embodiments, which are merely illustrative of the principles of the invention, and that various changes and modifications may be made therein without departing from the scope of the principles of the invention.
Claims (9)
1. The amphiphilic block copolymer drug-loaded micelle disclosed by the invention is composed of an amphiphilic block copolymer and a hydrophobic drug, wherein the hydrophobic drug is embedded in a hydrophobic core of a fullerene fragment formed by the amphiphilic block copolymer, and the hydrophilic part is composed of polylactic acid and an acrylic acid group. The structural formula of the amphiphilic block copolymer is as follows:
2. a process for the preparation of amphiphilic block copolymers according to claim 1, characterized in that bromine terminated low molecular weight polyacrylic acid is chemically combined with Fullerene to form Fullerene terminated polyacrylic acid (Fullerene-PAA).
3. The preparation process of the amphiphilic block copolymer as claimed in claim 1, wherein the Fullerene-terminated polyacrylic acid as claimed in claim 1 is prepared by adding p-hydroxybenzaldehyde, sarcosine 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, and drying under reduced pressure to obtain the hydroxylated Fullerene polyacrylic acid (Fullerene-OH-PAA).
4. The preparation process of the amphiphilic block copolymer as claimed in claim 1, wherein the hydroxylated Fullerene polyacrylic acid (Fullerene-OH-PAA) as claimed in claim 3, 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, and after reaction for 36-72h, the reaction solution is poured into methanol for precipitation. Centrifuging at 8000rpm under 3000-;
more preferably, the feeding molar ratio of the monomer lactide to the dihydroxy initiator is 27-78: 1, and the monomer lactide is one or more of L-lactide or D-lactide.
5. The bromine-terminated low molecular weight polyacrylic acid of claim 2 prepared by taking a small amount of silver sulfate and deionized water, stirring and bathing, slowly adding acrylic acid and hydrogen peroxide dropwise, adding sodium hydroxide solution, and controlling the system pH to 5-6. Drying the precipitate to obtain the low molecular weight polyacrylic acid. Then, ethyl 2-bromoisobutyrate (EBiB), copper bromide CuBr, Pentamethyldiethylenetriamine (PMEDTA), and acetone were added in this order, sealed with nitrogen, reacted, and poured into liquid nitrogen to terminate the reaction. Removing impurities, refining to obtain a white viscous product, and vacuum drying to obtain bromine substituted low molecular weight polyacrylic acid PAA-Br.
6. Amphiphilic block copolymer PAA-Fullerene-PLA as claimed in claims 1-5, characterized in that the number of carbon atoms of the Fullerene may be 60, 70; the number of lactic acid molecules n is 2-50; acrylic acid molecules m is 2-50; the average molecular weight of the copolymer is 800-10000;
more preferably, the fullerene has 60 carbon atoms, and the lactic acid molecule n is 2 to 30; the acrylic acid molecule m is 2-30, and the average molecular weight is 800-5000;
the preparation method of the amphiphilic block copolymer drug-loaded micelle is characterized in that the cosolvent can be one or more of tetrahydrofuran, acetone, pyridine, 1N, N-dimethylformamide or triethylamine and 4-dioxane.
7. The preparation method of the drug-loaded micelle of the amphiphilic block copolymer as claimed in claims 1 to 6, characterized in that after the hydrophobic drug is mixed with the copolymer, the copolymer is dissolved by the cosolvent as claimed in claim 6, after the solvent is evaporated to dryness, a proper amount of water or buffer solution is added, and the micelle is prepared by methods such as ultrasound, homogenizer mixing, high-speed shearing machine mixing, etc., the micelle solution is filtered by a filter membrane, and the aperture of the filter membrane is 0.2 to 0.45 μm.
8. The dosage of the amphiphilic block copolymer of claims 1-6 can be determined according to the actual application requirements of hydrophobic drugs, and can be drug carriers of weak acid and weak base compounds such as vorinostat, adriamycin, daunorubicin, retinoic acid and the like, and can also be drug carriers of insoluble natural compounds such as engelhardtia roxburghi glycoside, houttuynin, angelica lactone, astragaloside A and the like.
9. The drug-loaded micelle of claims 7-8, which is prepared into injectable micelle preparation for the treatment fields of tumor resistance, inflammation resistance, infection resistance and the like.
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