CN111407740A - Albumin nanoparticles capable of activating and releasing drugs by ultrasound, and preparation method and application thereof - Google Patents
Albumin nanoparticles capable of activating and releasing drugs by ultrasound, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an albumin nanoparticle capable of activating and releasing a drug by ultrasound, a preparation method and application thereof, wherein the albumin nanoparticle capable of activating and releasing the drug by ultrasound uses micromolecules containing azo groups as a chemical cross-linking agent, uses albumin as a nanoparticle framework to encapsulate an anti-cancer drug, and is prepared by a desolvation method; the particle diameter of the nano-particles is 50-300nm under normal physiological conditions, and the distribution is uniform and stable. Under the action of ultrasonic waves acceptable to organisms, azo bonds of the cross-linking agent can be broken, so that the albumin nano particle structure is damaged, the drug coated in the albumin nano particle structure is quickly released, the release amount of the drug can be regulated and controlled through the time and the strength of the ultrasonic waves, and the maximum release amount can reach 70%. By means of the strong penetrability of the ultrasound, the albumin nano-particles can realize the rapid and controllable release of the medicine at the tumor tissue.
Description
Technical Field
The invention belongs to the field of new biomedical materials, and particularly relates to albumin drug nanoparticles capable of activating drug release by ultrasound, a preparation method thereof, and application thereof in releasing drugs through ultrasound.
Background
Albumin is a serum protein with the most abundant content in blood, and is often selected as a drug carrier due to its characteristics of high water solubility, good stability, high biocompatibility and the like, and a large number of amino acid disabilities exist in the primary structure of albumin, so that binding sites are provided for some small molecules (ligands), and the albumin can effectively load different kinds of drugs. Human Serum Albumin (HSA) and Bovine Serum Albumin (BSA) have been the most studied carriers for drug delivery. HAS is a soluble single-chain globular protein with a molecular weight of 67kDa and consists of 585 amino acids, and HAS many drug binding sites, and can be bound with salicylic acid, warfarin, diazepam, penicillin and penicillin. BSA is a globular heart-shaped protein, has the molecular weight of 66kDa, and has the advantages of no toxicity, low antigenicity, biodegradability and the like. The BSA peptide chain consists of 583 amino acid residues, provides rich drug-loading sites, also contains 35 cysteine residues, one sulfydryl and the balance of disulfide bonds, and plays an important role in maintaining the spatial structure of proteins.
Chemotherapy is one of the indispensable important means for treating common tumors, but the low targeting property and the large toxic and side effects in the treatment process greatly prevent the further clinical application of the chemotherapy. The nano-drug delivery system can effectively reduce the toxic and side effects of the drug, improve the solubility of the hydrophobic drug, improve the pharmacokinetics of the drug, enhance the passive targeting capability of the solid tumor through the EPR effect (high permeability and retention effect), and improve the enrichment of the drug at the tumor.
Azo group-containing small molecules are generally used as radical thermal initiators for polymerization reactions, and are widely used in the industries of chemical industry, construction, medicine, food, agriculture and the like. The commonly used are oil-soluble Azobisisobutyronitrile (AIBN), Azobisisoheptonitrile (ABVN), and the like, and water-soluble azobisisobutyramidine hydrochloride (AIBA), 4' -azobis (4-cyanovaleric acid) (ACVA), and the like. The azo initiator generally has a symmetrical structure, is decomposed by an azo bond when being heated to generate corresponding carbon free radicals and nitrogen, almost all the reactions are first-order reactions, only one free radical is formed, and the azo initiator is not easy to react with a solvent and impurities and is relatively stable at normal temperature.
Most of the preparation methods of albumin nano particles have mild and simple conditions, the traditional methods comprise an emulsion method, a solvent removal method and a heating gel method, and the methods developed in recent years comprise a nano spray drying method, a self-assembly method, a nab-technology and the like. In order to stabilize the nanoparticles when redispersed, the nanoparticles are usually crosslinked by aldehyde-containing crosslinking agents such as glutaraldehyde to obtain stable nanoparticles, and because the aldehyde-containing crosslinking agents may generate toxic and side effects in vivo, it has also been reported that glutathione or cysteine is used to form albumin nanoparticles by the action of sulfydryl and disulfide bonds. The albumin drug nanoparticles are stable under normal physiological conditions, the drug release rate is less than 20%, the albumin drug nanoparticles have the drug slow release effect under acidic or reducing conditions, but the rapid and controllable release of the drug cannot be completed under physiological environments.
In a word, the prior art has the problem that the albumin nanoparticles can not controllably and rapidly release the drugs under physiological conditions, can only slowly release the drugs under the endogenous stimulation of acidity or reducibility and the like, and meanwhile, the defects that the traditional light-controlled release has poor tissue penetrability, causes low drug release amount and the like need to be solved urgently.
Disclosure of Invention
The invention aims to provide albumin nano particles capable of activating drug release by ultrasound, a preparation method and application thereof; the nano particles have the characteristics of uniform and stable particle size distribution, can be damaged under the action of ultrasound to quickly release the entrapped drug, and the drug release amount can be controlled according to the time and the intensity of the ultrasound, so that the aim of quickly and controllably releasing the drug at the tumor in vivo is fulfilled.
In order to achieve the purpose, the albumin nano particles which are loaded with the anticancer drugs and can activate the drug release by ultrasound are prepared by a desolvation method, micromolecules with azo groups are used as a chemical cross-linking agent, and functional groups such as hydroxyl, amino, carboxyl and the like on two sides of the azo groups are cross-linked with amino acid residues of albumin to form stable nano particles, the azo groups can be broken under the action of ultrasound with strong penetrability, so that the structure of the nano particles is damaged, the release amount of the drugs can be further controlled according to the time and the intensity of ultrasound, and the purpose of rapidly and controllably releasing the drugs at the tumor positions in vivo is achieved. Specifically, the following technical scheme is adopted:
the invention provides an albumin nanoparticle capable of activating drug release by ultrasound, which comprises albumin molecules, an anticancer drug and a micromolecular chemical cross-linking agent containing azo groups;
preferably, the albumin molecule is any one or a mixture of more than two of human serum albumin, recombinant human serum albumin, bovine serum albumin, ovalbumin or animal serum albumin;
preferably, the anticancer drug is one or a mixture of more than two of doxorubicin hydrochloride, paclitaxel, docetaxel, 10-hydroxycamptothecin, curcumin, cisplatin, 5-fluorouracil, methotrexate and mitoxantrone hydrochloride.
Preferably, the azo group-containing small-molecule chemical crosslinking agent has the following structural general formula:
wherein:
R1and R2Independently of one another from-CH3、-COOH、-NO2and-CN, wherein n is an integer from 0 to 5;
R3is that
One of (1);
R4a substituent selected from: -H, -OH, -COOH, -NH2、-NHX、-OCH3、-NH-(CH2)m-COOH，-CO-(CH2)m–NH2、
Wherein X is- (CH)2)mOH、-CH((CH2)mOH)2or-C ((CH)2)mOH)3And m is an integer of 1 to 3.
The azo group-containing small-molecule chemical crosslinking agent is more preferably 4,4' -azobis (4-cyanovaleric acid), azobisisobutyramidine hydrochloride, dimethyl azobisisobutyrate, azobisisopropylimidazoline hydrochloride, azobisisobutyrocarboxamide, azodihydroxyethyl imidazoline propane hydrochloride, azobis (1-imino-1-pyrrolidinyl-2-ethylpropyl) hydrochloride, azobis [ 2-methyl-N- (1, 1-2-hydroxymethyl-2-hydroxyethyl) propionamide ], azobis [ 2-methyl-N- (2-hydroxyethyl) propionamide, and azobis (N-2-carboxyethyl-2-methylpropionamidine) hydrate.
A second aspect of the present invention provides a method for preparing nanoparticles described in the first aspect, comprising the following steps:
(1) mixing cross-linking agent molecules with an activating reagent in a solvent, and reacting at 5-50 ℃ and preferably 20-40 ℃ for 0.5-6h and preferably 1-3h to obtain a cross-linking agent active solution;
(2) preparing an albumin molecule aqueous solution, dropwise adding an anticancer drug solution according to a certain proportion, stirring for 1-60min, adding an alkaline substance to adjust the pH of the solution to 7-10.5, preferably 8-10, and then adding an organic solvent according to a certain proportion at a constant speed under stirring to perform a desolvation process to obtain a drug-loaded albumin nanoparticle suspension;
(3) dropwise adding the solution obtained in the step (1) into the aqueous solution obtained in the step (2), carrying out light-shielding reaction for more than 6 hours at the temperature of 5-50 ℃, preferably 20-40 ℃, carrying out crosslinking solidification, and carrying out dialysis dehydration treatment to obtain stable albumin nanoparticles.
Preferably, in the step (2), the concentration of albumin molecules in the aqueous solution is 1-200mg/m L, preferably 10-100mg/m L, the concentration of the anticancer drug is 1-50mg/m L, preferably 1-20mg/m L, and the added alkaline substance is any one or a mixture of more than two of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate and sodium bicarbonate.
Preferably, the organic solvent added in the step (2) is one or a mixture of more than two of methanol, ethanol, acetone, 1.4-dioxane, dimethyl sulfoxide, n-propanol, isobutanol, polyethylene glycol and tetrahydrofuran, the rate of adding the organic solvent is 0.1-5m L/min, the more preferred rate is 1-4m L/min, the volume ratio of the volume of the added organic solvent to the volume of the albumin aqueous solution is (0.1-10): 1, and the more preferred volume ratio is (1-3): 1.
Preferably, in the step (1), the activating reagent is one or a mixture of more than two of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 1, 3-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride;
preferably, in the step (1), the solvent is one or a mixture of more than two of methanol, ethanol, acetone, 1, 4-dioxane, dimethyl sulfoxide, n-propanol, isobutanol, polyethylene glycol, tetrahydrofuran and dimethylformamide, the concentration of the azo group-containing small-molecule chemical cross-linking agent in the solvent is 1-100mg/m L, preferably 10-50mg/m L, and the concentration of the activating reagent in the solvent is 1-200mg/m L, preferably 10-100mg/m L.
Preferably, the chemical crosslinker molecules added in step (3) in step (1) have a final concentration in aqueous solution of 1-50mg/m L, preferably 1-10mg/m L.
Preferably, the dialysis temperature in step (3) is 0-40 ℃, preferably 4-30 ℃; a cut-off for dialysis of 500Da to 100kDa, preferably 3000Da to 12 kDa; the dehydration treatment is high speed centrifugation, freeze drying, spray drying, vacuum drying or reduced pressure distillation.
A fourth aspect of the present invention provides the use of a nanoparticle according to the first aspect described above for the release of a drug by the action of ultrasound.
Preferably, the ultrasound used to release the albumin nanoparticle drug by ultrasound is within an ultrasonic power and ultrasonic frequency range acceptable for living organisms.
Preferably, the ultrasonic power is 0-3W/cm2More preferably 1.5 to 2.5W/cm2(ii) a Preferably, the ultrasonic frequency is 0.5 to 10MHz, more preferably 0.5 to 2 MHz.
The invention has the beneficial effects that:
1. according to the albumin nano particles capable of activating drug release by ultrasound, the novel chemical cross-linking agent with azo groups can break azo bonds under the action of ultrasound to generate free radicals, so that the nano particles are damaged to release the entrapped drug, the release amount of the drug is related to the time and the intensity of ultrasound, and the purpose of controllable drug release is achieved;
2. the ultrasound is used as an activation mode harmless to human bodies, compared with the traditional light-operated release, the tissue penetrability is stronger, the released medicine can be regulated and controlled through the time and power of the ultrasound, the purpose of controllable release is achieved, and the control is facilitated;
3. the nano particles provided by the invention are prepared by a desolvation-chemical crosslinking method, so that the toxicity of the medicine to normal tissues is avoided, the distribution of the nano particle size is uniform and controllable within the range of 50-300nm, the dispersibility is good, and the large-scale preparation is facilitated;
4. under the condition of ultrasound, the maximum release amount of the drug can reach 70%, and under the condition of no ultrasound, the nano particles are kept relatively stable, so that the method has a better application prospect.
5. The nano-particles provided by the invention have excellent killing effect on cancer cells after being activated by ultrasonic.
Drawings
FIG. 1 is a transmission electron microscope image of ultrasonically activatable pharmaceutical albumin BSA-ACVA nanoparticles.
FIG. 2 is a hydrated particle size distribution of BSA-ACVA-DOX nanoparticles.
FIG. 3 is an ultraviolet-visible spectrum (UV-Vis) of BSA-ACVA-DOX nanoparticles.
FIG. 4 is the curve of the drug release amount of BSA-ACVA-DOX and BSA-GA-DOX nanoparticles with the change of ultrasonic time (1MHz, 2W/cm)2)。
FIG. 5 is the curve (1MHz, 3min) of the release of BSA-ACVA-DOX and BSA-GA-DOX nanoparticles with the variation of ultrasonic power.
FIG. 6 is an "off-on" controlled drug release profile (1MHz, 2W/cm) of BSA-ACVA-DOX nanoparticles2)。
FIG. 7 shows the dark toxicity of BSA-ACVA-DOX nanoparticles and the ultrasound toxicity (1MHz, 2W/cm) at different times2)。
FIG. 8 shows the dark toxicity of BSA-GA-DOX nanoparticles and the ultrasound toxicity at different times (1MHz, 2W/cm)2)。
Detailed Description
The present invention will be described in detail below with reference to examples in order to make objects, technical solutions, and advantages of the present invention more apparent, but the present invention is not limited to the following examples.
An albumin drug nanoparticle capable of activating drug release by ultrasound is characterized in that albumin molecules are used as a nanoparticle framework, an anticancer drug is coated by a desolvation-chemical crosslinking method, small molecules with azo groups are used as a chemical crosslinking agent, the small molecules can be broken under the action of ultrasound to generate free radicals, the albumin drug nanoparticle is prepared, the albumin drug nanoparticle can activate and release the drug controllably under the action of ultrasound, and the release amount of the drug is related to the time and the intensity of ultrasound.
Example 1
Preparation of chemically cross-linking agent molecular active ester
Weighing 50mg of 4,4 '-azobis (4-cyanovaleric acid) in a 25m L flask, adding anhydrous dimethyl sulfoxide 2m L, magnetically stirring until the solution is completely dissolved, sequentially adding 50mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 80mg of N-hydroxysuccinimide under the stirring condition until the solution is completely dissolved, and reacting at 37 ℃ for two hours under the dark condition to obtain a colorless and clear 4,4' -azobis (4-cyanovaleric acid) active ester solution.
Example 2
Preparation of ultrasonic activatable albumin nano particle
Preparing nano particles (BSA-ACVA) based on bovine serum albumin by a desolvation-chemical crosslinking method, weighing 90mg of bovine serum albumin powder, dissolving the bovine serum albumin powder in 3m L of ultrapure water, dropwise adding 0.1 mol/L of sodium hydroxide aqueous solution, adjusting the pH value of the solution to 9.0, continuously stirring for one hour, dropwise adding 6m L of absolute ethyl alcohol at a constant speed of 4m L/min, after the desolvation process is finished, slowly adding a crosslinking agent solution of 80 mu L for crosslinking and curing, continuously reacting overnight at 37 ℃ in a dark condition to obtain a stable albumin nano particle solution, carrying out rotary evaporation on the obtained albumin nano suspension at 37 ℃ to remove the ethyl alcohol, carrying out centrifugal separation at 12000rpm, washing and precipitating with deionized water, removing the uncrosslinked albumin and redundant crosslinking agent by the deionized water, repeating for three times, finally dispersing with a certain amount of the deionized water, and carrying out freeze drying to obtain the bovine serum albumin nano particles.
The transmission electron microscope picture of the obtained ultrasound-activatable drug albumin BSA-ACVA nano particle is shown in figure 1, and the nano particle has a special spherical structure, the particle size is about 200nm, and the dispersibility is good.
Example 3
Preparation of ultrasonic activatable medicinal albumin nano particle
Preparing drug-loaded bovine serum albumin nanoparticles (BSA-ACVA-DOX) by a desolvation-chemical crosslinking method, weighing 90mg of bovine serum albumin powder, dissolving the bovine serum albumin powder in 3m L ultrapure water, adding 6.0mg of aspergillus hydrochloride powder, stirring by magnetic force until the bovine serum albumin powder is completely dissolved, dropwise adding 0.1 mol/L of sodium hydroxide aqueous solution, adjusting the pH value of the solution to 9.0, continuously stirring for one hour, dropwise adding 6m L of absolute ethyl alcohol at a constant speed of 4m L/min, after the desolvation process is completed, slowly adding 80 mu L of a crosslinking agent solution for crosslinking and curing, continuously reacting overnight at 37 ℃ in a dark condition to obtain a stable albumin nanoparticle solution, carrying out rotary evaporation on the obtained albumin nano suspension at 37 ℃ to remove the ethyl alcohol, carrying out centrifugal separation at 12000rpm, washing and precipitating by deionized water, removing the uncrosslinked albumin and redundant crosslinking agent by the deionized water, repeating the steps for three times, finally dispersing by a certain amount of deionized water, and carrying out freeze drying to obtain.
The hydrated particle diameter (Rh), polydispersity index (PDI) and Zeta potential of the nanoparticles were determined by dynamic light scattering analysis, and the nanoparticle solution was diluted to 0.1mg/m L at the time of measurement.
The hydrated particle size distribution of the obtained BSA-ACVA-DOX nanoparticles is shown in FIG. 2, which shows that the particle size of the albumin drug nanoparticles is about 170nm after DOX coating.
The ultraviolet-visible light spectrum (UV-Vis) of the obtained BSA-ACVA-DOX nanoparticles is shown in FIG. 3, and a characteristic absorption peak of DOX exists at 479nm, which indicates that DOX is successfully coated in the albumin nanoparticles.
Example 4
Can activate the drug release of the drug albumin nano-particles under different ultrasonic time
Dispersing a certain amount of lyophilized BSA-ACVA-DOX nanoparticles in ultrapure water to prepare 0.2mg/m L solution, placing the solution in a 1m L sealed centrifugal tube, placing the whole centrifugal tube under an ultrasonic probe, filling the gap between the probe and the centrifugal tube with a special ultrasonic coupling agent, and filling the gap with 1MHz, 2W/cm2Selecting different times (0,0.5,1.0,3.0,5.0 and 10.0min) for carrying out ultrasound under the ultrasound condition, cleaning redundant ultrasound coupling agent outside the centrifuge tube after the ultrasound is finished, centrifuging for 15min under the condition of 12000rpm, taking supernatant liquid for diluting, measuring the absorbance at 480nm, calculating the concentration of DOX in a release medium according to a standard curve of the DOX, and calculating the drug release degree under different ultrasound times to draw a drug release curve, wherein the figure is shown in figure 4.
The change curve (1MHz, 2W/cm) of the drug release amount of the obtained ultrasound-activatable drug albumin BSA-ACVA-DOX and glutaraldehyde cross-linked BSA-GA-DOX nano particles with the change of the ultrasound time2) As shown in FIG. 4, with the increase of the ultrasonic time, the BSA-ACVA-DOX coated DOX is gradually and rapidly released, the maximum release amount can reach 70%, which indicates that the nano particles are successfully destroyed under the ultrasonic condition, and under the condition of no ultrasonic condition, the nano particles are relatively stable, and the release amount is less than5％。
Example 5
Can activate the drug release of the drug albumin nano-particles under different ultrasonic powers
Dispersing a certain amount of lyophilized BSA-ACVA-DOX nanoparticles in ultrapure water to prepare a 0.2mg/m L solution, placing the solution in a 1m L closed centrifugal tube, placing the whole centrifugal tube under an ultrasonic probe, filling a gap between the probe and the centrifugal tube with a special ultrasonic coupling agent, and selecting different ultrasonic powers (0,0.5,1.0,1.5,2.0 and 2.5W/cm) by taking 1MHz and 3min as ultrasonic time2) And (3) carrying out ultrasound, after the ultrasound is finished, cleaning the redundant ultrasound coupling agent outside the centrifuge tube, centrifuging for 15min at 12000rpm, taking supernatant liquor, diluting, measuring the absorbance at 480nm, and calculating the concentration of DOX in a release medium according to a standard curve of DOX so as to calculate the drug release degree under different ultrasound powers and draw a drug release curve, which is shown in figure 5.
The curve (1MHz, 3min) of the drug release amount of the obtained ultrasound-activatable drug albumin BSA-ACVA-DOX and glutaraldehyde-crosslinked BSA-GA-DOX nanoparticles along with the change of an ultrasound power table is shown in FIG. 5, the release amount of DOX is gradually increased along with the gradual increase of the ultrasound power, which indicates that the damage degree of the BSA-ACVA-DOX is continuously increased, so the drug release amount of the nanoparticles can be regulated and controlled through the time and the power of ultrasound.
Example 6
Controllable drug release of activatable pharmaceutical albumin nanoparticles
Dispersing a certain amount of freeze-dried BSA-ACVA-DOX nano particles in ultrapure water to prepare a 0.2mg/m L solution, placing the solution in a 1m L closed centrifugal tube, placing the whole centrifugal tube under an ultrasonic probe, and filling a gap between the probe and the centrifugal tube with a special ultrasonic coupling agent to measure the off-on controllable drug release capacity of the nano particles by using 1MHz and 2W/cm2Under the ultrasonic condition, the ultrasonic treatment is stopped for 5 hours after the ultrasonic treatment is carried out for 1min, the ultrasonic treatment is stopped for 5 hours after the ultrasonic treatment is carried out for 2min, and the drug release degrees before and after the ultrasonic treatment at each stage are respectively calculated to draw a drug release curve, which is shown in fig. 6.
The obtained ultrasound activatable pharmaceutical albuminOff-on controlled drug release profile (1MHz, 2W/cm) of BSA-ACVA-DOX nanoparticles2) As shown in fig. 6, it is demonstrated that the nanoparticles release the drug rapidly only under the ultrasonic condition and remain relatively stable without ultrasonic, so that the start and stop of the drug release can be effectively controlled by the ultrasonic shutdown.
Example 7
MTT method for detecting ultrasonic toxicity of ultrasonic activatable drug albumin nano particle
Collecting MCF-7 cells growing in logarithmic phase, pouring out culture solution, digesting with 0.25% trypsin for 1-2 min, stopping digestion with serum-containing culture solution 2m L, centrifuging at 1000rpm for 5min, pouring out supernatant, adding fresh serum-containing culture solution 2m L, making into single cell suspension, adjusting cell density to 5 x 103Pieces/m L, gently and uniformly blown, inoculated into a 96-well plate, each well has a size of 100 mu L, placed at 37 ℃ and 5% CO2After culturing for 24 hours in the incubator, respectively adding culture media containing BSA-ACVA-DOX nano particles with different concentrations, 100 mu L per well, and enabling the drug concentrations to be 0, 60, 100, 140 and 200 mu g/m L, wherein the cell suspension is added, the drug-containing culture solution is added, the ultrasonic treatment is not carried out to be a control group, the cell suspension is added, the drug is not added, the ultrasonic treatment is carried out to be a blank group, the culture is continued for 1 hour, the ultrasonic treatment is carried out on the experimental group for different times, each group comprises 4 multiple wells, the experimental group is continuously placed at 37 ℃ and 5% CO, and the temperature of each group is controlled to be 5% CO2The cells are cultured in an incubator for 24 hours, 5 mg/L MTT solution is added, the incubator is continuously cultured for 4 hours, after the MTT solution is poured out, DMSO with the concentration of 100m L is added into each hole, blue-purple formazan is fully dissolved and uniformly mixed, an enzyme-labeling instrument is used for detecting OD values of each hole at 570nm and 630nm, and the survival rate of each group of cells is calculated, which is shown in figure 7.
The ultrasound toxicity of the obtained ultrasound-activatable drug albumin BSA-ACVA-DOX nanoparticles is shown in FIG. 7 (1MHz, 2W/cm)2) The result shows that the BSA-ACVA-DOX nanoparticles which are not subjected to ultrasonic treatment have lower cytotoxicity, and the survival rate of cells is remarkably reduced after 1min and 3min of ultrasonic treatment, so that the evidence that the BSA-ACVA-DOX nanoparticles in the cells are damaged by the ultrasonic treatment to cause DOX to be released so as to kill cancer cells is provided.
Comparative example 1
Preparation of glutaraldehyde cross-linked drug albumin nano-particle
Preparing drug-loaded bovine serum albumin nanoparticles (BSA-GA-DOX) by a desolvation-chemical crosslinking method, weighing 90mg of bovine serum albumin powder, dissolving the bovine serum albumin powder in 3m L ultrapure water, adding 6.0mg of doxorubicin hydrochloride, completely dissolving the drug-loaded bovine serum albumin powder under magnetic stirring, dropwise adding 0.1 mol/L of sodium hydroxide aqueous solution, adjusting the pH value of the solution to 9.0, continuously stirring for one hour, dropwise adding 6m L of absolute ethyl alcohol at a constant speed of 4m L/min, after the desolvation process is completed, slowly adding 8% of glutaraldehyde solution 100 mu L for crosslinking and curing, continuously reacting at 37 ℃ in a dark condition for overnight to obtain stable albumin nanoparticles, rotationally evaporating the obtained albumin nano suspension at 37 ℃ to remove the ethyl alcohol, centrifugally separating the albumin suspension at 12000rpm, washing and precipitating the albumin suspension with deionized water, removing the uncrosslinked albumin and redundant crosslinking agent with the deionized water, repeating the steps for three times, finally dispersing the albumin nanoparticles with a certain amount of deionized water, and freeze-drying to obtain the glutaraldehyde crosslinked drug albumin.
Comparative example 2
Drug release of glutaraldehyde cross-linked drug albumin nanoparticles at different ultrasound times
Dispersing a certain amount of lyophilized BSA-GA-DOX nanoparticles in ultrapure water to obtain 0.2mg/m L solution, placing in a 1m L sealed centrifugal tube, placing the whole centrifugal tube under an ultrasonic probe, filling the gap between the probe and the centrifugal tube with a special ultrasonic coupling agent, and filling with 1MHz, 2W/cm2Selecting different times (0,0.5,1.0,3.0,5.0 and 10.0min) for carrying out ultrasound under the ultrasound condition, cleaning redundant ultrasound coupling agent outside the centrifuge tube after the ultrasound is finished, centrifuging for 15min under the condition of 12000rpm, taking supernatant liquid for diluting, measuring the absorbance at 480nm, calculating the concentration of DOX in a release medium according to a standard curve of the DOX, and calculating the drug release degree under different ultrasound times to draw a drug release curve.
Comparative example 3
Drug release of glutaraldehyde cross-linked drug albumin nanoparticles under different ultrasonic powers
Taking a certain amount of lyophilized BSA-GA-DOX nanoDispersing the particles in ultrapure water to obtain 0.2mg/m L solution, placing in 1m L sealed centrifugal tube, placing the whole centrifugal tube under ultrasonic probe, filling the gap between the probe and the centrifugal tube with special ultrasonic coupling agent, and selecting different ultrasonic powers (0,0.5,1.0,1.5,2.0, 2.5W/cm) with 1MHz and 3min as ultrasonic time2) And (3) carrying out ultrasound, after the ultrasound is finished, cleaning the redundant ultrasound coupling agent outside the centrifuge tube, centrifuging for 15min at 12000rpm, taking supernatant liquor, diluting, measuring the absorbance at 480nm, and calculating the DOX concentration in a release medium according to a standard curve of the DOX so as to calculate the drug release degree under different ultrasound powers and draw a drug release curve.
Comparative example 4
MTT method for detecting ultrasonic toxicity of glutaraldehyde cross-linked drug albumin nanoparticles
Collecting MCF-7 cells growing in logarithmic phase, pouring out culture solution, digesting with 0.25% trypsin for 1-2 min, stopping digestion with serum-containing culture solution 2m L, centrifuging at 1000rpm for 5min, pouring out supernatant, adding fresh serum-containing culture solution 2m L, making into single cell suspension, adjusting cell density to 5 x 103Pieces/m L, gently and uniformly blown, inoculated into a 96-well plate, each well has a size of 100 mu L, placed at 37 ℃ and 5% CO2After culturing for 24 hours in the incubator, respectively adding culture media containing BSA-GA-DOX nano particles with different concentrations, 100 mu L per well, and enabling the drug concentrations to be 0, 60, 100, 140 and 200 mu g/m L, wherein the cell suspension is added, the drug-containing culture solution is added, the ultrasound treatment is not carried out to be a control group, the cell suspension is added, the drug is not added, the ultrasound treatment is carried out to be a blank group, the culture is continued for 1 hour, the ultrasound treatment is carried out on the experimental group for different times, 4 multiple wells of each group are continuously placed at 37 ℃ and 5% CO, and the temperature of each group is controlled to be 5% CO2After the MTT solution is poured out, 100m L of DMSO is added into each hole, after the blue-violet formazan is fully dissolved and uniformly mixed, an enzyme-labeling instrument is used for detecting the OD value of each hole at 570nm and 630nm, the survival rate of each group of cells is calculated, and the result is shown in figure 8.
The ultrasonic toxicity of the obtained ultrasonic activatable drug albumin BSA-GA-DOX nano particle is shown in figure 8 (1MHz, 2W/cm)2) Without sayingThe BSA-GA-DOX nanoparticles subjected to ultrasonic treatment have low cytotoxicity, and the survival rate of cells is not remarkably reduced after 1min and 3min of ultrasonic action, so that the BSA-GA-DOX nanoparticles in the cells are relatively stable under the ultrasonic action, and the DOX is not rapidly released, so that the cell killing property is low.
In summary, it can be seen from comparison of comparative examples 1 to 4 and examples 1 to 7 of the present application that the ultrasound-activatable drug albumin BSA-ACVA-DOX nanoparticles remain relatively stable under normal conditions compared to glutaraldehyde-crosslinked BSA-GA-DOX nanoparticles, and can be effectively destroyed under ultrasound with high tissue penetration, thereby rapidly releasing the coated anticancer drug, and further, the released drug can be controlled by adjusting the time and power of ultrasound, so as to achieve the purpose of controlled release. In a cell killing experiment on human breast cancer cells MCF-7, the ultrasound-activatable drug albumin BSA-ACVA-DOX nano particles have lower cytotoxicity. Compared with BSA-GA-DOX nanoparticles crosslinked by glutaraldehyde, the BSA-ACVA-DOX nanoparticles are destroyed under the action of ultrasound to release the coated anticancer drug, and have higher cancer cell killing property.
The applicant states that the product and the detailed preparation method of the present invention are illustrated by the above examples, but the present invention is not limited to the above product and the detailed preparation method, i.e. the present invention is not meant to be implemented by relying on the above product and the detailed preparation method. Persons skilled in the art should understand that the persons and improvements of the present invention, the equivalent substitution of each raw material and the addition of auxiliary components, the selection of specific modes, etc. of the product of the present invention all fall within the protection scope and the disclosure scope of the present invention.
Claims (10)
1. An albumin drug nanoparticle capable of activating drug release by ultrasound is characterized by comprising albumin molecules, anticancer drugs and chemical cross-linking molecules; the albumin molecule is used as a nano particle framework, and the micromolecule containing azo groups is used as a chemical cross-linking agent to coat the anti-cancer drug.
2. The drug delivery albumin nanoparticle activated by ultrasound according to claim 1, wherein the albumin molecule is any one or a mixture of two or more of human serum albumin, recombinant human serum albumin, bovine serum albumin, ovalbumin, or animal serum albumin; preferably, the anticancer drug is one or a mixture of more than two of doxorubicin hydrochloride, paclitaxel, docetaxel, 10-hydroxycamptothecin, curcumin, cisplatin, 5-fluorouracil, methotrexate and mitoxantrone hydrochloride.
3. The ultrasonically activatable drug-releasable albumin drug nanoparticle of claim 1, wherein the azo group-containing small molecule chemical crosslinker has the following general structural formula:
wherein:
R1and R2Independently of one another from-CH3、-COOH、-NO2and-CN, wherein n is an integer from 0 to 5;
R3is selected from
One of (1);
R4one selected from the following substituents: -H, -OH, -COOH, -NH2、-NHX、-OCH3、-NH-(CH2)m-COOH，-CO-(CH2)m–NH2、
Wherein X is selected from- (CH)2)mOH、-CH((CH2)mOH)2and-C ((CH)2)mOH)3M is an integer of 1 to 3;
the azo group-containing small-molecule chemical crosslinking agent is more preferably 4,4' -azobis (4-cyanovaleric acid), azobisisobutyramidine hydrochloride, dimethyl azobisisobutyrate, azobisisopropylimidazoline hydrochloride, azobisisobutyrocarboxamide, azodihydroxyethyl imidazoline propane hydrochloride, azobis (1-imino-1-pyrrolidinyl-2-ethylpropyl) hydrochloride, azobis [ 2-methyl-N- (1, 1-2-hydroxymethyl-2-hydroxyethyl) propionamide ], azobis [ 2-methyl-N- (2-hydroxyethyl) propionamide, and azobis (N-2-carboxyethyl-2-methylpropionamidine) hydrate.
4. The method for preparing the ultrasonically activatable drug-releasing albumin drug nanoparticles as claimed in claim 1, comprising the steps of:
(1) mixing a micromolecular chemical crosslinking agent containing azo groups with an activating reagent in a solvent, and reacting at 5-50 ℃ and preferably 20-40 ℃ for 0.5-6h and preferably 1-3h to obtain a crosslinking agent active solution;
(2) preparing an albumin molecule aqueous solution, dropwise adding an anticancer drug solution according to a certain proportion, stirring for 1-60min, adding an alkaline substance to adjust the pH of the solution to 7-10.5, preferably 8-10, and then adding an organic solvent according to a certain proportion at a constant speed under stirring to perform a desolvation process to obtain a drug-loaded albumin nanoparticle suspension;
(3) dropwise adding the solution obtained in the step (1) into the aqueous solution obtained in the step (2), carrying out light-shielding reaction for more than 6 hours at the temperature of 5-50 ℃, preferably 20-40 ℃, carrying out crosslinking solidification, and carrying out dialysis dehydration treatment to obtain stable albumin nanoparticles.
5. The preparation method of the drug nanoparticles of albumin with ultrasonic-activatable drug release according to claim 4, wherein in step (2), the concentration of albumin molecules in the aqueous solution is 1-200mg/m L, preferably 10-100mg/m L, the concentration of the anticancer drug is preferably 1-50mg/m L, more preferably 1-20mg/m L, the added alkaline substance is preferably one or a mixture of two or more of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate and sodium bicarbonate, the added organic solvent in step (2) is preferably one or a mixture of two or more of methanol, ethanol, acetone, 1.4-dioxane, dimethyl sulfoxide, n-propanol, isobutanol, polyethylene glycol and tetrahydrofuran, the added organic solvent is preferably 0.1-5m L/min, the added organic solvent is preferably 1-4m L/min, the ratio of the volume of the added organic solvent to the volume of the aqueous solution is preferably 0.1-10, and the volume of the aqueous solution is preferably 1-10.
6. The preparation method of the drug nanoparticles capable of ultrasonically activating the drug releasing albumin according to claim 4, wherein in the step (1), the activating reagent is one or a mixture of more than two of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 1, 3-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, preferably, in the step (1), the solvent is one or a mixture of more than two of methanol, ethanol, acetone, 1, 4-dioxane, dimethyl sulfoxide, N-propanol, isobutanol, polyethylene glycol, tetrahydrofuran and dimethylformamide, preferably, the azo group-containing small molecule chemical cross-linking agent has a concentration of 1-100mg/m L, preferably 10-50mg/m L, and the activating reagent has a concentration of 1-200mg/m L, preferably 10-100mg/m L.
7. The method for preparing the drug nanoparticles capable of ultrasonically activating the drug releasing albumin according to claim 4, wherein in the step (3), the final concentration of the chemical cross-linking agent molecules in the aqueous solution in the step (1) is 1-50mg/m L, preferably 1-10mg/m L, the dialysis treatment temperature is 0-40 ℃, preferably 4-30 ℃, the dialysis molecular weight cut-off is preferably 500Da-100kDa, preferably 3000Da-12kDa, and the dehydration treatment method is preferably selected from high-speed centrifugation, freeze drying, spray drying, vacuum drying or reduced pressure distillation.
8. Use of albumin drug nanoparticles according to claim 1 for drug release by ultrasound.
9. Use according to claim 8, wherein the ultrasound means is in a range of acceptable ultrasound power and ultrasound frequency for the living organism.
10. Use according to claim 8 or 9, wherein the ultrasonic power is 0-3W/cm2More preferably 1.5 to 2.5W/cm2(ii) a The ultrasonic frequency is 0.5-10MHz, and more preferably 0.5-2 MHz.
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| CN113616617A (en) * | 2021-07-09 | 2021-11-09 | 大连理工大学 | Modified albumin nano particle capable of releasing NO gas by ultrasonic, preparation method and application thereof |
| CN113651959A (en) * | 2021-07-14 | 2021-11-16 | 中山大学 | Nano drug loading system based on amino acid-hydroxy acid copolymer and preparation method and application thereof |
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| CN113616617A (en) * | 2021-07-09 | 2021-11-09 | 大连理工大学 | Modified albumin nano particle capable of releasing NO gas by ultrasonic, preparation method and application thereof |
| CN113651959A (en) * | 2021-07-14 | 2021-11-16 | 中山大学 | Nano drug loading system based on amino acid-hydroxy acid copolymer and preparation method and application thereof |
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