CN113368237B - Nano composite material with photo-thermal response controllable drug release and preparation method thereof - Google Patents
Nano composite material with photo-thermal response controllable drug release and preparation method thereof Download PDFInfo
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Abstract
The invention provides a nano composite material with photothermal response controllable drug release and a preparation method thereof, and solves the problems that the existing composite material with photothermal response controllable drug release uses gold nanorods as a photothermal conversion material, and has the defects of low light stability, large toxicity and high cost. The nano composite material comprises silver nanorods, a mesoporous silica layer and a temperature-sensitive layer from inside to outside; the silver nanorods are used as a photothermal response core, and the length-diameter ratio of the silver nanorods is (3); the mesoporous silicon dioxide layer is used as an intermediate drug-loaded layer and grows on the surface of the silver nanorod in situ; the temperature-sensitive layer is used as a mesoporous switch and wraps the surface of mesoporous silica, and the low critical solution temperature of the temperature-sensitive layer is 39-45 ℃.
Description
Technical Field
The invention belongs to the technical field of nano composite materials, and particularly relates to a nano composite material with photo-thermal response and controllable drug release and a preparation method thereof.
Background
The mesoporous silica nano-particle is a novel inorganic polymer, and has huge specific surface area and pore volume, adjustable pore diameter and large drug loading capacity. The protein can enter cells in an endocytosis mode, is prevented from being captured by lysosomes, and has good biocompatibility; the surface of the material is modified by functional groups, so that the functions of active targeting, controllable medicine and sustained release can be realized. Thus, mesoporous silica nanoparticles have gradually become a carrier for drug delivery.
The photo-thermal stimulation response drug control drug delivery system generates photo-thermal conversion under the illumination of specific wavelength, and the environmental temperature is increased, so that the drug is released. The light source includes ultraviolet light, visible light, and near infrared light. The light source irradiation can cause the change of the structural hierarchy of the nano composite material, the switching change is generated, the medicine can be released, some medicine carrying systems cause the temperature change due to the illumination, the form change of the temperature sensitive material is caused, the switching function is realized, and the medicine release can also be controlled. Two main factors of photothermal response are a laser light source and a photothermal conversion material with higher photothermal conversion efficiency; although there are many methods of controlling drug release using ultraviolet rays, the most promising is near infrared light because of weak ultraviolet light penetration ability due to strong dispersion of soft tissue and cell damage caused by ultraviolet rays, which limits the application thereof. There are four main categories of near-infrared photothermal conversion materials: semiconductor nano-materials, organic polymer materials, carbon-based nano-materials and noble metal nano-materials. At present, the most studied and widely applied are precious metal nano materials, the plasma resonance effect on the surface of the precious metal nano material can absorb the energy of near infrared light and convert the energy into heat energy, and the precious metal nano material is a good photo-thermal conversion material and widely applied to the photo-thermal treatment of tumors; the noble metal material mainly comprises gold, palladium, platinum and the like; document 1, "xiahuiting, design, synthesis and biological application of a drug controlled release system based on gold nanorods, a master thesis of Nanjing stamp and Electricity university, 2018," reports that the gold nanorods are used as a photo-thermal core, a silica thin layer is used as a shell, and an anticancer drug adriamycin and a bubble generation precursor ammonium bicarbonate are loaded to realize photo-thermal response controlled release under near infrared. Document 2, "Liudan pellet, preparation of gold nanorod composite material and study on photo-thermal and drug-loading performance thereof, master thesis of Henan university, 2020" reports that gold nanorods are used as cores, silicon dioxide is used as drug-loading parts, and manganese dioxide nanosheets are used as pH and glutathione response parts, so that the photo-thermal response controllable drug-release material is prepared.
The above publications all use gold nanorods as a photothermal conversion material, but the gold nanorods have low light stability, the thermal conversion efficiency is reduced after long-time laser irradiation, and gold ions with high toxicity are released after the shell material is degraded, and the gold nanoparticles have high cost and limited development and application.
Disclosure of Invention
The invention aims to solve the defects of low light stability, high toxicity and high cost of the existing composite material with controlled drug release through photo-thermal response, which uses gold nanorods as a photo-thermal conversion material, and provides the nanocomposite material with controlled drug release through photo-thermal response and the preparation method thereof.
In order to achieve the purpose, the technical solution provided by the invention is as follows:
a nano composite material with photo-thermal response controllable drug release is characterized in that: the nano-silver/mesoporous silica composite material comprises silver nanorods, a mesoporous silica layer and a temperature-sensitive layer from inside to outside;
the silver nanorods are used as a photothermal response core, and the length-diameter ratio of the silver nanorods is (3);
the mesoporous silicon dioxide layer is used as an intermediate drug-loaded layer and grows on the surface of the silver nanorod in situ;
the temperature-sensitive layer is used as a mesoporous switch and wraps the surface of mesoporous silica, and the low critical solution temperature of the temperature-sensitive layer is 39-45 ℃.
Further, in order to improve the drug loading capacity, the specific surface area of the mesoporous silica layer is 900-1200 m2·g-1The pore volume is 3-5 cm3·g-1The aperture is 2-5 nm.
Furthermore, the nano composite material can perform photo-thermal conversion on near infrared light with the wavelength of 780-850 nm.
The invention also provides application of the nano composite material with photo-thermal response controllable drug release in preparation of a drug-loaded composition for treating diseases.
The drug-loaded composition is characterized by comprising the nano composite material with photothermal response controllable drug release and a loaded drug.
Further, the drug-carried drug is an antibacterial drug, an anti-inflammatory drug, an anti-tumor drug or a bone growth promoting drug.
The preparation method of the medicine carrying composition is characterized by comprising the following steps:
1) Preparation of AgNR
Preparing by adopting a polyol method to obtain AgNR;
2) Preparation of AgNR @ MSN
Growing mesoporous silica on the AgNR surface obtained in the step 1) in situ by adopting a sol-gel method to obtain AgNR @ MSN;
3) Loaded with drugs
3.1 Dispersing AgNR @ MSN obtained in the step 2) in an organic solvent containing the medicine, performing ultrasonic treatment to uniformly mix the AgNR @ MSN, and placing the AgNR @ MSN in a constant-temperature shaking table for oscillation to fully carry the medicine; the organic solvent can be ethanol;
3.2 AgNR @ MSN after drug loading is centrifugally separated and washed to remove the drug attached to the surface;
4) Wrapping temperature sensitive material
4.1 ) dispersing AgNR @ MSN subjected to drug loading in the step 3) in distilled water, and uniformly dispersing to prepare a solution E;
4.2 Slowly dropping the solution E into the temperature-sensitive material suspension, continuously stirring at room temperature, after fully coating, centrifugally separating, and collecting precipitates to obtain the drug-carrying composition.
Further, the temperature-sensitive material used in the step 4) is NIPAAm-co-NMA, and the preparation method comprises the following steps:
s1, dissolving poly-N-isopropyl acrylamide and hydroxymethyl acrylamide in distilled water, and violently stirring to uniformly mix reaction raw materials;
s2, dropwise adding an ammonium persulfate solution into the mixed solution of the S1 under ice bath, after completely mixing, dropwise adding a sodium metabisulfite solution, polymerizing for 2-3 h at 0-5 ℃, and then continuously reacting for 15-19 h at room temperature;
s3, after the reaction is finished, transferring the reaction liquid to a dialysis bag, dialyzing with distilled water, and removing unreacted raw material monomers; and (4) carrying out vacuum freeze-drying on the dialyzed product, and collecting a sample to obtain NIPAAm-co-NMA.
Further, the step 1) specifically comprises:
1.1 AgNO) to3Dissolving in glycol to obtain a standby solution A; dissolving PVP and KBr in ethylene glycol to obtain reaction liquid B; wherein, in the standby solution A, agNO3The concentration of (A) is 12-15 mg/ml, such as 120mg AgNO3Dissolving the mixture in 8-10 mL of glycol to obtain a standby reaction solution A; in the standby solution B, the concentration of PVP is 12-15 mg/ml, the mass ratio of PVP to KBr is 30-120: 1, for example: dissolving 120mg of PVP and 1-4 mg of KBr in 8-10 mL of glycol to obtain a standby reaction solution B;
1.2 ) dripping the standby solution A and the standby solution B into glycol with the temperature of 140-160 ℃ at the injection speed of 0.5-1.5 mL/min, stirring and reacting for 1-3 h at the temperature, standing and cooling to room temperature, centrifuging and washing to obtain AgNR, and storing in dark place for standby;
further, the step 2) is specifically as follows:
2.1 Dispersing the silver nanorods prepared in the step 1) in chloroform to prepare a suspension solution; dropwise adding the suspension solution into aqueous solution of Cetyl Trimethyl Ammonium Bromide (CTAB), and violently stirring to obtain homogeneous oil-in-water microemulsion; heating the obtained microemulsion in a water bath at 65-70 ℃ to remove trichloromethane to obtain a standby solution C;
sequentially adding cetyl trimethyl ammonium bromide and NaOH aqueous solution into distilled water, and violently stirring at room temperature to obtain a standby solution D;
2.2 Dropwise adding the standby solution C obtained in the step 2.1) into the standby solution D, heating to 70-85 ℃, adding tetraethyl orthosilicate (TEOS) for reaction under vigorous stirring, and centrifuging, washing and heating to remove hexadecyl trimethyl ammonium bromide after the reaction is finished to obtain AgNR @ MSN.
The invention has the advantages that:
1. the silver nanorod is prepared by a polyol method, mesoporous silica is coated on the outer layer of the silver nanorod by a sol-gel method, then a drug is loaded in the mesopores, and finally a layer of temperature sensitive material is coated for hole sealing to serve as a temperature response switch, so that controllable drug release under photo-thermal response is realized. The invention takes the silver nano-rod as the photothermal conversion material to convert the light energy into the heat energy, and when the temperature of the system reaches the lowest conversion temperature of the composite material, the outer temperature sensitive copolymer shrinks, so that the medicine can be quickly released.
2. The surface of the silver nanorod (the length-diameter ratio is within a range of 5-3; the silver nanorods are used as the photothermal conversion material, so that the cost and the toxicity are reduced, the photostability is improved, and the silver ions are slowly released to achieve a sterilization effect.
3. The method for preparing the drug-loaded composition has simple process and convenient condition control; and the drug loading experiment shows that the nano composite material with photo-thermal response controllable drug release has large drug loading amount and obvious controlled release effect.
4. The temperature-sensitive material selected by the invention can still keep the shape and the sensitive temperature response characteristic in the aqueous solution; after the temperature-sensitive material is wrapped on the MSN outer layer, compared with a single temperature-sensitive material, the low critical solution temperature of the temperature-sensitive material is still unchanged.
Drawings
FIG. 1 is a scanning electron microscope image of silver nanorods manufactured in example 1 of the present invention.
FIG. 2 is a transmission electron microscope image of the mesoporous silica composite material prepared in example 1 of the present invention.
FIG. 3 is a temperature rise curve of the nanocomposite material of the same concentration under different power near infrared irradiation in example 4 of the present invention.
FIG. 4 is a graph showing the cumulative release of the drug (tetracycline) at different temperatures in example 5 of the present invention.
FIG. 5 is a graph of the cumulative release of the drugs under the excitation of NIR light from the silver nanorod composite material in example 6 of the invention.
FIG. 6 is a graph showing the temperature dependence of the nanocomposite and the temperature sensitive material according to example 7 of the present invention on light having a wavelength of 500 nm;
fig. 7 shows the results of the bacteriostatic test of example 8, wherein a is the test result of the bacteriostatic circle of escherichia coli (e.coli) and b is the test result of the bacteriostatic circle of staphylococcus aureus (s.aureus);
FIG. 8 is a graph showing the sterilization curves of the 9AgNR @ MSN @ NIPAm-co-NMA material for Staphylococcus aureus and Escherichia coli according to the example of the present invention.
Detailed Description
The invention is described in further detail below with reference to the following figures and specific examples:
example 1:
the method comprises the following steps: and (4) preparing the silver nanorods.
120mg of AgNO is taken3Dissolving in 8mL of ethylene glycol to obtain a standby reaction solution A, and dissolving 120mg of PVP and 2.0mg of KBr in 8mL of ethylene glycol to obtain a standby reaction solution B. And dropwise adding the reaction liquid A and the reaction liquid B into 10mL of ethylene glycol with the temperature of 140 ℃ at the injection speed of 0.5mL/min by using a double-hole syringe, and continuously stirring and reacting for 1h at the temperature. Standing and cooling to room temperature, 10000 r/min, centrifuging for 10min, washing with distilled water for 3 times, re-dispersing the sample into 10mL of pure water, and keeping the sample away from light for later use, wherein a scanning electron microscope picture is shown in figure 1, the product is a silver nanorod, and the length-diameter ratio is 1-5.
Step two: preparing the mesoporous silicon dioxide with the silver nano-rod as the core.
And (4) dispersing the silver nanorods (AgNR) prepared in the step one in chloroform to prepare a suspension solution of 5 mg/mL. 3mL of the suspension was added dropwise to 10mL of an aqueous solution containing 0.2g of cetyltrimethylammonium bromide (CTAB), and vigorously stirred to obtain a homogeneous oil-in-water microemulsion. Heating the obtained microemulsion in water bath at 65-70 deg.C for 30min, removing chloroform to obtain AgNR @ CTAB solution, which is called as standby solution C.
0.05g of CTAB and 0.7mL of a 2mol/L NaOH aqueous solution were sequentially added to 85mL of distilled water, and vigorously stirred at room temperature to obtain a solution D. Slowly dripping 10mL of the standby solution C into the standby solution D, heating the reaction mixture to 70 ℃, adding 1.35mL of tetraethyl orthosilicate (TEOS for short) into the reaction solution under vigorous stirring, reacting for 3h, after the reaction is finished, centrifugally collecting the product, washing for 3 times by using distilled water, heating in a muffle furnace to remove a template agent CTAB, collecting the product, and obtaining AgNR @ MSN, wherein a transmission electron microscope picture is shown in figure 2.
Step three: and (4) loading the medicine.
Dispersing 20mg AgNR @ MSN into 10mL ethanol containing 2mg/mL medicine or a corresponding solvent, performing ultrasonic treatment to uniformly mix the solution, and placing the solution in a constant-temperature shaking table to oscillate for 24 hours to fully carry the medicine on the nano composite material. And (3) centrifugally separating the nanoparticles after carrying the medicine by using a high-speed centrifuge, collecting the nanoparticles after carrying the medicine, washing the nanoparticles for 2 times by using ethanol or a corresponding solvent, and removing the medicine attached to the surface of the material.
Step four: and (3) preparing and wrapping the temperature-sensitive material.
Respectively dissolving 0.08mol of poly-N-isopropylacrylamide (NIPAAm) and 0.01mol of hydroxymethyl acrylamide (NMA) in 72mL of distilled water, and violently stirring to uniformly mix the reaction raw materials. Under ice bath, 4mL of additive with the concentration of 6X 10 is slowly dropped4mol/L ammonium persulfate solution, after being completely mixed, 4mL of ammonium persulfate solution with the concentration of 6X 10-4mol/L sodium metabisulfite solution. First, polymerization was carried out at 0 to 5 ℃ for 2 hours, and then, the reaction was continued at room temperature for 15 hours. After the reaction, the mixture was transferred to a dialysis bag and dialyzed with distilled water to remove the unreacted raw material monomer. The dialyzed product was lyophilized under vacuum and the sample collected and labeled NIPAAm-co-NMA.
10mg of nano AgNR @ MSN is dispersed in 10mL of distilled water to prepare 1mg/mL solution, and the solution is uniformly dispersed under magnetic stirring. Then, 2mg/mL NIPAAm-co-NMA suspension was slowly added dropwise to the above system, and stirring was continued at room temperature for 24h. After the reaction is finished, centrifugal separation is carried out by a high-speed centrifuge, and precipitate is collected and marked as AgNR @ MSN @ NIPAAm-co-NMA, namely the nano composite material loaded with the drug.
Example 2:
the method comprises the following steps: and (4) preparing the silver nanorods.
120mg of AgNO is taken3Dissolving in 9mL of ethylene glycol to obtain a reaction solution A, and dissolving 120mg of PVP and 2.8mg of KBr in 9mL of ethylene glycol to obtain a reaction solution B. And dropwise adding the reaction liquid A and the reaction liquid B into 10mL of ethylene glycol with the temperature of 150 ℃ at the injection speed of 1mL/min by using a double-hole syringe, and continuously stirring and reacting for 2 hours at the temperature. Standing and cooling to room temperature, 10000 r/min, centrifuging for 10min, washing with distilled water for 3 times, re-dispersing the sample into 10mL of pure water, and keeping away from light for later use.
Step two: preparing the mesoporous silicon dioxide with the silver nano-rod as the core.
And (4) dispersing the silver nanorods (AgNR) prepared in the step one in chloroform to prepare a suspension solution of 5 mg/mL. 3mL of the suspension was added dropwise to 10mL of an aqueous solution containing 0.25g of cetyltrimethylammonium bromide (CTAB), and vigorously stirred to obtain a homogeneous oil-in-water microemulsion. Heating the obtained microemulsion in water bath at 65-70 deg.C for 30min, removing chloroform to obtain AgNR @ CTAB solution, which is called as standby solution C. 0.05g of CTAB and 0.7mL of a 2mol/L NaOH aqueous solution were sequentially added to 85mL of distilled water, and vigorously stirred at room temperature to obtain a solution D. Slowly and dropwise adding 10mL of the standby solution C into the standby solution D, heating the reaction mixture to 80 ℃, adding 1.35mL of tetraethyl orthosilicate (TEOS for short) into the reaction solution under vigorous stirring, reacting for 3 hours, after the reaction is finished, centrifugally collecting a product, washing the product for 3 times by distilled water, heating a muffle furnace to remove a template agent CTAB, and collecting the product, wherein the product is marked as AgNR @ MSN.
Step three: and (4) loading the medicine.
Dispersing 20mg AgNR @ MSN into 10mL ethanol containing 2mg/mL medicine or corresponding solvent, performing ultrasonic treatment to uniformly mix the mixture, and placing the mixture in a constant-temperature shaking table to vibrate for 24 hours to fully carry the medicine on the nano composite material. And (3) centrifugally separating the nanoparticles after carrying the medicine by using a high-speed centrifuge, collecting the nanoparticles after carrying the medicine, washing the nanoparticles for 2 times by using ethanol or a corresponding solvent, and removing the medicine attached to the surface of the material.
Step four: and (3) preparing and wrapping the temperature-sensitive material.
0.08mol of poly-N-isopropylacrylamide (short for NIPAAm) and 0.02mol of hydroxymethyl acrylamide (short for NMA) are respectively dissolved in 72mL of distilled water and stirred vigorously to mix the reaction raw materials uniformly. Under ice bath, 4mL of additive with the concentration of 6X 10 is slowly dropped4mol/L ammonium persulfate solution, after being completely mixed, 4mL of ammonium persulfate solution with the concentration of 6X 10-4mol/L sodium metabisulfite solution. First, polymerization was carried out at 0 to 5 ℃ for 2.5 hours, and then, the reaction was continued at room temperature for 17 hours. After the reaction, the mixture was transferred to a dialysis bag and dialyzed with distilled water to remove the unreacted raw material monomer. The dialyzed product was lyophilized under vacuum and the sample collected and labeled NIPAAm-co-NMA.
10mg of nano AgNR @ MSN is dispersed in 10mL of distilled water to prepare 1mg/mL solution, and the solution is uniformly dispersed under magnetic stirring. Then, 2mg/mL NIPAAm-co-NMA suspension is slowly added dropwise to the system, and stirring is continued for 24h at room temperature. After the reaction is finished, centrifugal separation is carried out by a high-speed centrifuge, and precipitate is collected and marked as AgNR @ MSN @ NIPAAm-co-NMA, namely the nano composite material loaded with the drug.
Example 3:
the method comprises the following steps: and (4) preparing the silver nanorods.
120mg of AgNO is taken3Dissolving in 10mL of ethylene glycol to obtain a standby reaction solution A, and dissolving 120mg of PVP and 3.0mg of KBr in 10mL of ethylene glycol to obtain a standby reaction solution B. The reaction solution A and the reaction solution B were added dropwise to 10mL of 160 ℃ ethylene glycol at an injection rate of 1.5mL/min with a double-channel syringe, and the reaction was continued for 3 hours with stirring at that temperature. Standing and cooling to room temperature, 10000 r/min, centrifuging for 10min, washing with distilled water for 3 times, re-dispersing the sample into 10mL of pure water, and keeping away from light for later use.
Step two: preparing the mesoporous silicon dioxide with the silver nano-rod as the core.
And (4) dispersing the silver nanorods (AgNR) prepared in the step one in chloroform to prepare a suspension solution of 5 mg/mL. 3mL of the suspension was added dropwise to 10mL of an aqueous solution containing 0.3g of cetyltrimethylammonium bromide (CTAB), and vigorously stirred to obtain a homogeneous oil-in-water microemulsion. Heating the obtained microemulsion in water bath at 65-70 deg.C for 30min, removing chloroform to obtain AgNR @ CTAB solution, which is called as standby solution C. 0.05g CTAB and 0.7mL of 2mol/L NaOH aqueous solution were added to 85mL of distilled water in this order, and stirred vigorously at room temperature to obtain a solution D. Slowly dripping 10mL of the standby solution C into the standby solution D, heating the reaction mixture to 85 ℃, adding 1.35mL of tetraethyl orthosilicate (TEOS for short) into the reaction solution under vigorous stirring, reacting for 3h, after the reaction is finished, centrifugally collecting a product, washing for 3 times by using distilled water, heating in a muffle furnace to remove a template agent CTAB, and collecting the product, wherein the product is marked as AgNR @ MSN.
Step three: and (4) loading the medicine.
Dispersing 20mg AgNR @ MSN into 10mL ethanol containing 2mg/mL medicine or a corresponding solvent, performing ultrasonic treatment to uniformly mix the solution, and placing the solution in a constant-temperature shaking table to oscillate for 24 hours to fully carry the medicine on the nano composite material. And (3) carrying out centrifugal separation on the nanoparticles after carrying the drugs by using a high-speed centrifuge, collecting the nanoparticles after carrying the drugs, washing the nanoparticles for 2 times by using ethanol or a corresponding solvent, and removing the drugs attached to the surface of the material.
Step four: and (3) preparing and wrapping the temperature-sensitive material.
0.08mol of poly-N-isopropylacrylamide (short for NIPAAm) and 0.03mol of hydroxymethyl acrylamide (short for NMA) are respectively dissolved in 72mL of distilled water and stirred vigorously to mix the reaction raw materials uniformly. Slowly dropping 4mL of additive with the concentration of 6X 10 under ice bath4mol/L ammonium persulfate solution, after being completely mixed, 4mL of ammonium persulfate solution with the concentration of 6 multiplied by 10 is slowly dripped4mol/L sodium metabisulfite solution. First, the polymerization was carried out at 0 to 5 ℃ for 3 hours, and then, the reaction was continued at room temperature for 19 hours. After the reaction, the reaction mixture was transferred to a dialysis bag and dialyzed against distilled water to remove the unreacted raw material monomers. The dialyzed product was lyophilized under vacuum and the sample collected and labeled NIPAAm-co-NMA.
10mg of nano AgNR @ MSN is dispersed in 10mL of distilled water to prepare 1mg/mL solution, and the solution is uniformly dispersed under magnetic stirring. Then, 2mg/mL NIPAAm-co-NMA suspension was slowly added dropwise to the above system, and stirring was continued at room temperature for 24h. After the reaction is finished, centrifugal separation is carried out by a high-speed centrifuge, and precipitate is collected and marked as AgNR @ MSN @ NIPAAm-co-NMA, namely the nano composite material loaded with the drug.
Example 4:
the procedure of examples 1 to 3 was followed to omit the drug loading step, prepare a nanocomposite, and test the photothermal response properties thereof:
2mL of water-based suspension with the concentration of the nano composite material being 0.5mg/mL is prepared, near-infrared laser with the wavelength of 808nm is selected, and the intensities are respectively set as 1, 1.5 and 2W/cm-2Pure water was used as a blank control in the experiment. Under the irradiation of near-infrared laser with wavelength of 808nm, continuous irradiation time is 10min per minute, the temperature of suspension is measured by a thermocouple thermodetector, the temperature of the suspension is accurately read and recorded, and a relation graph between irradiation time and temperature is obtained, as shown in fig. 3. The temperature of the suspension of the nanocomposite rapidly increases in a very short time, eventually reaching a plateau. The rate of temperature rise increases with increasing laser power, for example: at 2W/cm-2The laser radiation can lead the solution to be lifted to 62.1 ℃ within 10 min; therefore, the material has good photo-thermal conversion capability.
Example 5:
temperature response release experiment after nanocomposite drug loading:
10mg of the photothermal response nanocomposite material loaded with the drug tetracycline (taking loaded tetracycline as an example) are respectively filled into a dialysis bag with the length of 5cm, the dialysis bag is placed into a centrifuge tube with the proper size filled with 10mL of buffer solution, and the centrifuge tube is placed into a constant temperature shaking table for in vitro drug release. The experiment was set up with two control groups at 37 ℃ and 42 ℃ and 3 replicates per group were run. At different time points, 0.5mL of each dialysate was removed while an equal volume of PBS solution was replenished. The uv absorbance of the dialysate was measured at different time points and the cumulative drug release rate was calculated to obtain the cumulative drug release curve, as shown in fig. 4. As can be seen from FIG. 4, the drug release behavior is significantly influenced by the temperature, at 37 ℃, the drug release is slow due to the wrapping of the outer temperature-sensitive copolymer, and the cumulative release rate of tetracycline within 14 days is only 14.5%. When the temperature was raised from 37 ℃ to 42 ℃, the cumulative drug release increased significantly to 79.3%. The results indicate that the composite material can achieve controlled release of the drug in response to temperature.
Example 6:
drug release experiments under near infrared light irradiation:
weighing 10mg of tetracycline-loaded nanocomposite, putting the nanocomposite into a 5 cm-long dialysis bag (model MW 8-14K), clamping both ends with a clamp, placing the dialysis bag in a centrifuge tube containing 10mL buffer solution, and performing centrifugation at wavelength of 808nm NIR (1.5W-cm-2) Laser irradiation is carried out at intervals of 2 hours, irradiation is carried out for a certain time, 0.5mL of dialysate is taken at the end of each 2 hours, 0.5mL of PBS solution is supplemented at the same time, the drug release condition is measured, and the release rate is calculated. As can be seen from FIG. 5, tetracycline has significant drug release behavior after each irradiation with near-infrared laser. After three times of irradiation with near-infrared light, it was found that its cumulative release rate gradually increased to 34.7%. The light energy is converted into heat energy due to the surface plasma resonance effect of the silver nanorods, the system temperature reaches the lowest transition temperature of the composite material, and the outer layer temperature sensitive copolymer shrinks, so that the medicine is rapidly released. The result proves that the composite material has the drug release property of near infrared light-stimulated drug release. According to the mode in the embodiment, the drug release experiment can be carried out under the near infrared light with the wavelength of 780-850 nm, so as to achieve the purpose of drug release.
Example 7:
the procedure of examples 1 to 3 was followed to omit the drug loading step, prepare nanocomposite and temperature sensitive material, and perform experiments on their low critical solution temperature:
preparing 2mL of water-based suspension with the concentration of 2% (mass fraction) of the nano composite material, measuring the low critical dissolution temperature of the water-based suspension by using a variable temperature ultraviolet spectrophotometer, firstly putting a test sample into a sample seat connected with a temperature control circulating groove, and measuring the transmittance of the sample to light with the wavelength of 500nm along with the curve of the temperature. The lower critical solution temperature is defined as the temperature at which the original transmittance of the solution decreases by 10%.
Similarly, 2mL of water-based suspension with the concentration of 2% (mass fraction) of temperature-sensitive material (NIPAAm-co-NMA) is prepared, and the transmittance of the sample to light with the wavelength of 500nm is measured, along with the curve of the temperature. The result is shown in fig. 6, which shows that the trend of the temperature-changing ultraviolet curve of the nanocomposite is consistent with that of the temperature-sensitive material, and shows that the temperature-sensitive material still maintains the original low critical solution temperature after being wrapped on the outer layer, and the low critical solution temperature of the nanocomposite and the temperature-sensitive material is 40.8 ℃, namely when the temperature is lower than 40.8 ℃, the temperature-sensitive material is in a swelling form, hydrophilic property, transparent form and high light transmittance, and when the temperature is higher than 40.8 ℃, the temperature-sensitive material is in a shrinking form, hydrophobic property, opaque and high light transmittance. The temperature-sensitive material can block the silicon dioxide mesoporous air hole in a swelling state, and the air hole is opened in a contraction state, so that the temperature-sensitive material is used as a temperature-controllable sealing agent.
By adjusting the proportion of poly-N-isopropyl acrylamide and hydroxymethyl acrylamide, temperature-sensitive materials with different low critical solution temperatures in the range of 39-45 ℃ can be prepared.
Example 8:
the step of example 1 was followed to carry out bacteriostatic experiments on the nanocomposite loaded with tetracycline drug. The concentration of the adopted material particles is 170 mu g/mL, and the density of the experimental bacteria is 5 multiplied by 105pieces/mL, with a wet filter paper sheet as a blank control. Samples of the experimental group and the blank control group are respectively taken and lightly stuck on a bacterial contamination plate. And (4) covering the cover of the watch glass, inverting the watch glass to culture in a constant-temperature bacterial incubator at 42 ℃, and taking out the watch glass after 17-20 hours to observe results. As shown in fig. 7, a is a bacteriostatic circle experimental result of escherichia coli (e.coli), wherein a clear escherichia coli bacteriostatic circle can be observed in the area a1 by using the wetted filter paper as a blank control and the areas a2 and a3 as an intergroup control, and the size of the bacteriostatic circle is determined to be 3.2 ± 0.3mm by a vernier caliper; b, the graph is the experimental result of the inhibition zone of staphylococcus aureus (S.aureus), the area b1 also takes wet filter paper as a blank control, the areas b2 and b3 are group controls, the obvious inhibition zone of staphylococcus aureus can be observed, and the size of the inhibition zone is determined to be 4.6 +/-0.4 mm by a vernier caliper; comparing the two figures, we can find that the silver nanorod composite material AgNR @ MSN @ NIPAm-co-NMA is applied to the large intestine rodThe bacteria and staphylococcus aureus have obvious growth inhibition effect, and the growth inhibition effect on staphylococcus aureus is more obvious.
Example 9:
sterilization curve experiment of the composite material:
the concentration of the adopted composite material particles is 170 mug/mL, and the density of the control bacteria is 5 multiplied by 10 during the experiment5One per mL. Respectively co-culturing the composite material with escherichia coli or staphylococcus aureus, taking a small amount of bacterial liquid to dilute 100 times by using a culture medium at time points of 1, 2, 4, 8 hours and the like, then dripping 0.5mL of bacterial-containing culture liquid on the surface of an agar culture medium, coating by using a coating rod to ensure that the bacterial liquid is as uniform as possible, placing a solid flat plate in a constant-temperature shaking table in an inverted manner, setting the temperature to be 42 ℃, incubating for 24 hours at a rotating speed of 180 revolutions per minute, counting bacterial colonies, repeatedly setting three agar plates for each material concentration, and calculating by adopting the following formula:
number of colonies (CFU/mL) = dilution factor number of colonies/inoculum size
FIG. 8 is the sterilization curve of AgNR @ MSN @ NIPAm-co-NMA material for golden yellow grape ball and Escherichia coli. As can be seen from the figure, the AgNR @ MSN @ NIPAm-co-NMA material has obvious inhibition effect on the growth of both Escherichia coli and Staphylococcus aureus within 8h, and shows stronger antibacterial performance. The composite material has more obvious growth inhibition on staphylococcus aureus, and the number of colonies observed at experimental nodes is obviously less than that of escherichia coli colonies.
While the invention has been described with reference to specific embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.
Claims (4)
1. A drug-loaded composition characterized by: the drug-loaded composition comprises a nano composite material with photo-thermal response controllable drug release and a loaded drug;
the photothermal response controlled-release nanocomposite comprises silver nanorods, a mesoporous silica layer and a temperature-sensitive layer from inside to outside;
the silver nanorod is used as a photothermal response inner core, and the length-diameter ratio of the silver nanorod is 5-3;
the mesoporous silicon dioxide layer is used as an intermediate drug-loaded layer and grows on the surface of the silver nanorod in situ;
the temperature-sensitive layer is used as a mesoporous switch and wraps the surface of mesoporous silica, and the low critical solution temperature is 39-45 ℃;
a preparation method of a medicine-carrying composition comprises the following steps:
the drug-loaded composition comprises a nano composite material with photo-thermal response controllable drug release and a loaded drug;
1) Preparing AgNR by adopting a polyol method to obtain AgNR;
1.1 AgNO) to3Dissolving in glycol to obtain a standby solution A; dissolving PVP and KBr in ethylene glycol to obtain reaction liquid B;
1.2 The standby solution A and the standby solution B are simultaneously dripped into glycol with the temperature of 140-160 ℃ at the injection speed of 0.5-1.5 mL/min, and after the mixture is stirred and reacts for 1-3 h at the temperature, the mixture is kept stand and cooled to the room temperature, and is centrifuged and washed to obtain AgNR;
2) Preparing AgNR @ MSN, and growing mesoporous silica on the AgNR surface obtained in the step 1) in situ by adopting a sol-gel method to obtain the AgNR @ MSN;
2.1 Dispersing the silver nanorods prepared in the step 1) in chloroform to prepare a suspension solution; dropwise adding the suspension solution into aqueous solution of hexadecyl trimethyl ammonium bromide, and violently stirring to obtain homogeneous oil-in-water microemulsion; heating the obtained microemulsion in a water bath at 65-70 ℃ to remove trichloromethane to obtain a standby solution C;
sequentially adding cetyl trimethyl ammonium bromide and NaOH aqueous solution into distilled water, and violently stirring at room temperature to obtain a standby solution D;
2.2 Dropwise adding the standby solution C obtained in the step 2.1) into the standby solution D, heating to 70-85 ℃, adding tetraethyl orthosilicate for reaction under vigorous stirring, and after the reaction is finished, centrifuging, washing and heating to remove hexadecyl trimethyl ammonium bromide to obtain AgNR @ MSN;
3) Loaded with drugs
3.1 Dispersing the AgNR @ MSN obtained in the step 2) in an organic solvent containing the medicine, performing ultrasonic treatment to uniformly mix the AgNR @ MSN, and placing the mixture in a constant-temperature shaking table for oscillation to fully carry the medicine;
3.2 AgNR @ MSN after drug loading is centrifugally separated and washed to remove the drug attached to the surface;
4) Wrapping temperature sensitive material
4.1 The temperature-sensitive material is NIPAAm-co-NMA, and the preparation method comprises the following steps:
s1, dissolving poly N-isopropyl acrylamide and hydroxymethyl acrylamide in distilled water, and violently stirring to uniformly mix reaction raw materials;
s2, under ice bath, dropwise adding an ammonium persulfate solution into the mixed solution of the S1, after completely mixing, dropwise adding a sodium metabisulfite solution, polymerizing for 2-3 h at the temperature of 0-5 ℃, and then continuously reacting for 15-19 h at room temperature;
s3, after the reaction is finished, transferring the reaction liquid to a dialysis bag, dialyzing with distilled water, and removing unreacted raw material monomers; freeze-drying the dialyzed product in vacuum, and collecting a sample to obtain NIPAAm-co-NMA;
4.2 AgNR @ MSN after the medicine loading in the step 3) is dispersed in distilled water uniformly to prepare a solution E;
4.3 Slowly dropping the solution E into the temperature-sensitive material suspension, continuously stirring at room temperature, after fully coating, centrifugally separating, and collecting precipitates to obtain the drug-carrying composition.
2. The drug-loaded composition of claim 1, wherein: the specific surface area of the mesoporous silicon dioxide layer is 900-1200 m2·g-1The pore volume is 3-5 cm3·g-1The aperture is 2-5 nm.
3. The drug-loaded composition of claim 1, wherein:
the nano composite material can perform photo-thermal conversion on near infrared light with the wavelength of 780-850 nm.
4. The drug-loaded composition of claim 1, wherein:
the drug to be carried is an antibacterial drug, an anti-inflammatory drug, an anti-tumor drug or a bone growth promoting drug.
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| CN102319435A (en) * | 2011-06-20 | 2012-01-18 | 复旦大学 | Mesoporous silica compound particles containing gold nanorods, preparation method and application thereof |
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