CN114452400A - Composite material nanosphere and preparation method and application thereof - Google Patents
Composite material nanosphere and preparation method and application thereof Download PDFInfo
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- CN114452400A CN114452400A CN202210112380.2A CN202210112380A CN114452400A CN 114452400 A CN114452400 A CN 114452400A CN 202210112380 A CN202210112380 A CN 202210112380A CN 114452400 A CN114452400 A CN 114452400A
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6925—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a microcapsule, nanocapsule, microbubble or nanobubble
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Abstract
The application discloses a composite material nanosphere and a preparation method and application thereof. The composite material nanosphere comprises core particles, a metal nanoshell layer, a mercapto acid modification layer and a polyethyleneimine compound modification layer; the core particles are film-forming substances and coated with fluorocarbon compounds; the metal nano shell layer is coated on the surface of the core particle; the mercapto acid modification layer is connected to the metal shell layer through a mercapto group; the polyethylene imine compound layer is connected to the mercaptan acid modification layer through an amido bond. The composite material nanosphere has multiple functions of photothermal therapy, macrophage gene transfection enhancement, ultrasonic imaging enhancement and the like.
Description
Technical Field
The application relates to a composite material nanosphere and a preparation method and application thereof, belonging to the technical field of biomedicine.
Background
In recent years, immunotherapy has significantly improved the prognosis of cancer patients, especially in late-stage patients. Inhibition of tumor-associated macrophage (TAMs) polarization is one of the important pathways for tumor immunotherapy. TAMs are natural immune effector cell populations that infiltrate tumor tissue, accounting for approximately 5-72% of the cells of the entire tumor tissue. TAMs can be induced to different phenotypes by different factors, one of which is M1 type TAMs with anti-tumor effects, and the other is M2 type TAMs that promote tumor proliferation and metastasis. M2 type TAMs play an important role in tumor immunity, and can regulate inflammatory responses, adaptive immunity, clear debris, promote angiogenesis, repair tissue remodeling, stimulate angiogenesis, cause abnormal angiogenesis, inhibit recruitment of anti-tumor T cells, and induce proliferation and metastasis of tumor cells. Therefore, inhibiting the polarization of M2 type TAMs provides a new strategy for tumor therapy.
Small interfering RNA (siRNA) has wide application prospect in the treatment of various diseases, particularly in the treatment of tumors, because siRNA can inhibit target genes of any type of cancer cells. In addition, siRNA synthesis is relatively easy, production cost is low, development period is short, treatment range is wide, and the siRNA has a wide prospect in cancer treatment. However, various biological barriers inside and outside the cell limit their widespread clinical use. In order to successfully introduce siRNA into cells and obtain sufficient therapeutic index, researchers have designed various carriers, such as lipids, lipoidal materials (lipid-like materials), polymers, polypeptides, exosomes, inorganic nanoparticles, and the like. However, transfection of siRNA into human immune cells is still very difficult. Macrophages act as immune cells and are difficult to transfect with siRNA into macrophages even with mature transfection agents because they recognize foreign nucleic acids, destroy them by enzymatic degradation and generation of reactive oxygen species. Since the composition, size, shape and surface functionalization of nanoparticles can be flexibly and precisely tailored, they have great advantages in siRNA delivery. Nevertheless, the transfection efficiency of various nano biomaterials on human macrophages is basically between 60% and 80%, which is improved compared with that of commercial transfection reagents (50-60%), but is still not satisfactory. Therefore, the preparation of a nano material capable of further improving the transfection efficiency of macrophage gene, biosafety and low toxicity is still an important subject to be researched.
In addition to immunotherapy, gene therapy, photodynamic therapy, and photothermal therapy (PTT), emerging therapies in cancer treatment also exhibit encouraging therapeutic effects. Photothermal therapy utilizes the photothermal effect of a photothermal transfer agent to convert light energy into heat, thereby increasing ambient temperature to trigger cancer cell death. Among the many different treatments, photothermal therapy has many advantages in that it can precisely control the temperature of the target region by adjusting the intensity of the light given, the time, the area of the light given, and the concentration of the drug, thereby avoiding damage to normal tissues around the tumor. In addition, photothermal therapy is an efficient, noninvasive and safe treatment method, and is suitable for various types of cancers. Among numerous photothermal therapy nanomaterials, the unique optical properties and biosafety of gold nanoparticles are one of the main application materials in photothermal therapy. The near infrared absorption wavelength of the gold nanoparticles can be controlled by changing the shape and size of the gold nanoparticles, and the gold nanoparticles have various functional modifications, so that a preparation basis is provided for realizing the multifunctionality of the material. Although the gold nanoparticles have good photothermal effect, the photothermal conversion efficiency reported so far is between 20 and 40 percent, and if the photothermal conversion efficiency can be further improved, the photothermal treatment effect is expected to be further enhanced.
Currently, single treatment modalities are still limited in efficacy in cancer, and combination treatments of multiple treatment modalities are better. The combination of photothermal therapy and gene therapy is receiving increasing attention. Meanwhile, if the visualization tracking of the nanoparticles in the treatment can be realized, the real-time monitoring of the photothermal treatment range becomes possible, unnecessary damage to surrounding normal tissues in the treatment is reduced, and the application of the multifunctional nano platform in the cancer treatment is further promoted.
Disclosure of Invention
According to one aspect of the present application, a composite nanosphere is provided, which has various functions of photothermal therapy, enhanced macrophage gene transfection, ultrasound-enhanced imaging, and the like.
A composite material nanosphere comprises core particles, a metal nanoshell layer, a mercapto acid modification layer and a polyethyleneimine compound modification layer;
the core particles are film-forming substances and coated with fluorocarbon compounds;
the film forming material is at least one of polylactic acid and polylactic acid derivative copolymer;
the metal in the metal nanoshell layer is selected from at least one of inorganic noble metals with photo-thermal properties;
the mercapto acid in the mercapto acid modification layer is selected from at least one of linear mercapto acid compounds;
the polyethyleneimine compound in the polyethyleneimine compound modification layer is selected from at least one of branched polyethyleneimine compounds;
the metal nano shell layer is coated on the surface of the core particle;
the mercapto acid modification layer is connected to the metal shell layer through a mercapto group;
the polyethylene imine compound layer is connected to the mercaptan acid modification layer through an amido bond.
Optionally, the fluorocarbon compound has 5 to 6 carbon atoms.
Optionally, the fluorocarbon compound is selected from at least one of perfluoro-n-pentane and perfluorohexane.
Optionally, the polylactic acid is selected from a levolactic acid;
the polylactic acid derivative copolymer is at least one selected from polylactic acid-glycolic acid, polyethylene glycol monomethyl ether-polylactic acid copolymer and polyethylene glycol-polylactic acid copolymer.
Optionally, the metal in the metal nanoshell layer is selected from at least one of gold, silver, platinum, and palladium.
Optionally, the mercapto acid compound is selected from at least one of 6-mercaptohexanoic acid and 11-mercaptoundecanoic acid.
The mercapto acid modification layer can increase the binding capacity of the post-polyethyleneimine compound on one hand, and has the effect of antiserum on enhancing the stability of the gene carried by the nanoparticles on the other hand.
The polyethyleneimine compound modification layer not only has the function of carrying siRNA, but also can realize the escape of intracellular lysosomes of nanoparticles through the proton osmosis effect to play a role in gene therapy.
Optionally, the polyethyleneimine-based compound is at least one selected from branched polyethyleneimine, branched oligoethyleneimine, branched polyvinylamine, and branched polyallylamine.
Optionally, the metal nanoshell layer is a layered structure formed by metal nanoparticles adsorbed on the surface of the inner core particle through electrostatic interaction.
Optionally, the particle size of the metal nanoparticles is 20-50 nm.
Optionally, the composite nanospheres have a particle size of 80-600 nm.
Perfluoro-n-pentane is a low-boiling liquid fluorocarbon compound, can be gasified at the temperature of more than 30 ℃, enhances the ultrasonic enhanced imaging capability on the one hand, and can enhance lysosome escape and improve gene silencing efficiency by realizing phase change in a tumor region on the other hand.
The gold nanoparticles form a nano shell layer on the surface of the nanosphere, so that the nano particle has the efficiency of photo-thermal conversion, and the nano particle has the efficacy of photo-thermal treatment and tumor killing.
According to another aspect of the present application, there is provided a method for preparing the composite nanosphere, the method comprising the steps of:
(S1) encapsulating the fluorocarbon compound in the film-forming substance to obtain inner core particles;
(S2) coating a metal nanoshell layer on the surface of the core particle to obtain a metal nanoshell layer coated core particle;
(S3) sequentially modifying a mercapto acid modification layer and a polyethyleneimine compound modification layer on the surface of the metal nano shell layer to obtain the composite material nanosphere.
Optionally, the (S1) includes: and (3) carrying out ultrasonic treatment on the solution I containing the film forming substance and the fluorocarbon compound, adding the surfactant solution II into the solution I, carrying out ultrasonic treatment on the solution II, and stirring the solution I to obtain the core particles.
Optionally, the (S2) includes: mixing the core particles with polyallylamine hydrochloride solution III, stirring II to obtain core particles with positive charges, mixing the core particles with the positive charges with metal nano seed liquid, adding metal salt solution IV and reducing agent solution V, and stirring III to obtain the core particles coated by the metal nano shell.
Optionally, the (S3) includes: and stirring IV a reaction extraction liquid containing mercapto acid, an activating agent, a catalyst and a polyethyleneimine compound solution VI with a solution containing the inner core particles coated by the metal nano shell to obtain the composite material nano sphere.
Alternatively, (S1), the solvent of the solution I is at least one selected from dichloromethane, chloroform and ethyl acetate;
in the solution I, the proportion of the film-forming substance, the fluorocarbon compound and the solvent is 60-100 mg: 100-300. mu.L: 2-6 mL;
optionally, in the solution II, the surfactant is selected from at least one of polyvinyl alcohol and polyacrylic acid;
optionally, in the solution II, the concentration of the surfactant is 1-3 wt%;
optionally, the ratio of the film forming substance to the solution II is 60-100 mg: 20-30 ml;
optionally, the ultrasonic I power is 100-;
the power of the ultrasonic II is 300-500W, and the time is 2-3 minutes;
stirring I time is 4-5 hours.
Alternatively, (S2), the polyallylamine hydrochloride concentration in the solution III is 0.5 to 2.0 weight percent;
optionally, the ratio of the film forming substance to the solution III is 60-100 mg: 20-30 ml;
optionally, the stirring II time is 20-40 minutes;
optionally, the metal salt in the metal salt solution IV is selected from at least one of tetrachloroauric acid dihydrate, tetrachloroauric acid trihydrate, and tetrachloroauric acid tetrahydrate;
optionally, the metal salt concentration in the metal salt solution IV is 0.5-2.0 wt%;
optionally, the reducing agent in the reducing agent solution V is selected from hydroxylamine hydrochloride;
optionally, the concentration of the reducing agent in the reducing agent solution V is 1-2 wt%;
optionally, the ratio of film-forming material, metal salt solution IV and reducing agent solution V is 60-100 mg: 2-3 mL: 400-600 μ L;
optionally, the stirring time of the stirring III is 20-40 min;
optionally, the metal nano-seed solution is obtained by the following steps:
diluting a metal salt solution VII, adding a stabilizer solution VIII, stirring V, adding a reducing agent solution VIIII, and stirring VI;
optionally, the metal salt in the metal salt solution VII is selected from at least one of tetrachloroauric acid dihydrate, tetrachloroauric acid trihydrate and tetrachloroauric acid tetrahydrate, and the concentration is 0.5-2.0 wt%;
the stabilizer in the stabilizer solution VIII is selected from sodium citrate, and the concentration is 0.5-2.0 wt%;
the reducing agent in the reducing agent solution VIIII is selected from sodium borohydride and has the concentration of 10-100 mM;
optionally, the ratio of the film-forming substance, the metal salt solution VII, the stabilizer solution VIII and the reducing agent solution VIIII is 60-100 mg: 800-: 500-1000. mu.L: 250-400 μ L.
Optionally, (S3), the solvent in the solution VI is at least one selected from dichloromethane, chloroform and ethyl acetate
Alternatively, in said solution VI, the ratio of mercaptoacid, activator, catalyst, polyethyleneimine compound and solvent is 50-70 mg: 50-60 mg: 30-40 mg: 400-600 mg: 2-4 ml;
optionally, the activator is selected from EDC;
the catalyst is selected from NHS;
optionally, the ratio of the film-forming substance to the reaction extract is 60-100 mg: 300-;
optionally, the stirring IV is for a period of 10 to 12 hours.
According to another aspect of the present application, there is provided a pharmaceutical composition comprising an siRNA solution and a carrier solution;
the volume ratio of the carrier solution to the siRNA solution is 1: 5-1: 15
siRNA is loaded on a carrier;
the carrier is selected from at least one of the composite material nanospheres and the composite material nanospheres prepared according to the preparation method.
Preferably, the concentration of the carrier solution is 0.5-2 mg/mL;
the concentration of the siRNA solution is 50-200 mug/mL.
According to another aspect of the present application, there is provided a method of preparing the above composition, the method comprising the steps of:
and mixing the carrier solution and the siRNA solution to obtain the composition.
According to another aspect of the present application, there is provided the composite nanosphere, the composite nanosphere obtained according to the above preparation method, the pharmaceutical composition, and the use of the pharmaceutical composition obtained according to the above preparation method in preparation of at least one of photothermal therapy drugs, gene therapy drugs, and ultrasound imaging agents.
In order to overcome the defects in the prior art, the application provides an ultrasonic visual multifunctional nano-carrier with photothermal therapy and macrophage gene transfection enhancement, a preparation method thereof and application in biomedicine.
A preparation method and application of an ultrasonic visual multifunctional nano-carrier with photothermal therapy and macrophage gene transfection enhancement.
The nano-carrier prepared by the invention has the multiple functions of integrating photothermal therapy, gene therapy and ultrasonic enhanced imaging. For cells which are difficult to transfect, such as macrophages, the nano-carrier prepared by the invention can realize about 90% of gene transfection efficiency, has the function of antiserum and is superior to the existing commercial transfection reagent. Meanwhile, the prepared nanoparticles have higher photo-thermal conversion efficiency than common gold nanorods, can realize the effect of enhancing ultrasonic imaging while generating photo-thermal effect under the stimulation of 808nm laser, and realize the visual tracking of a nano carrier.
The gold nanoshell layer coated by the nano-carrier has a photothermal effect which is superior to that of a conventional gold nanorod, the photothermal conversion efficiency can reach 59%, and the photothermal treatment of tumor cells is realized;
the nano carrier carries PFP nano liquid drop with the power of 0.5W/cm2The 808nm laser can realize liquid-gas phase change after being irradiated for 5 minutes, and plays a role in ultrasonic enhanced imaging;
the nano-carrier can obviously improve the gene transfection efficiency of macrophages, realizes the gene transfection efficiency of about 90 percent of THP-1 cells and has the function of antiserum;
the nano-carrier has better biocompatibility and low toxicity, and can achieve the treatment effect at lower concentration.
The ultrasonic visual multifunctional nano-carrier with photothermal therapy and macrophage gene transfection enhancement has the beneficial effects of preparation and application in biomedicine, and comprises the following components:
the preparation process of the nano carrier PGMP is simple, convenient to operate, low in required equipment requirement, strong in controllability, low in energy consumption, easy to produce, easy to obtain used raw materials, economical and practical; the biological compatibility and the biological safety are better; can be used for gene transfection of macrophage, photothermal therapy of tumor cells and photothermal triggered ultrasonic enhanced imaging; the compound has good antiserum effect, high macrophage gene transfection rate, strong photothermal treatment effect and good ultrasonic enhanced imaging effect. The beneficial effects that this application can produce include:
the composite material nanosphere is simple in preparation process, convenient to operate, low in required equipment requirement, strong in controllability, low in energy consumption, easy to produce, easy to obtain used raw materials, economical and practical; the biological compatibility and the biological safety are better; can be used for gene transfection of macrophage, photothermal therapy of tumor cells and photothermal triggered ultrasonic enhanced imaging; the compound has good antiserum effect, high macrophage gene transfection rate, strong photothermal treatment effect and good ultrasonic enhanced imaging effect.
Drawings
FIG. 1(a) is a transmission electron microscope of the PGMP nanocarrier synthesized in example 2, and FIG. 1(b) is a scanning electron microscope of the PGMP nanocarrier synthesized in example 2;
FIG. 2(a) is a particle size distribution diagram of the PGMP nanocarrier synthesized in example 2 and FIG. 2(b) is a zeta potential diagram of the PGMP nanocarrier synthesized in example 2;
FIG. 3 is a graph of UV-Vis spectra of PGMP nanocarriers synthesized in example 2 at different concentrations;
FIG. 4 is a graph of photothermal temperature curves of PGMP nanocarriers synthesized in example 2 under different power 808nm laser irradiation;
FIG. 5 is a graph showing the photothermal conversion efficiency of the PGMP nanocarrier synthesized in example 2 under laser irradiation;
FIG. 6 is an image of the PGMP nanocarrier synthesized in example 2 under laser irradiation with ultrasound enhanced imaging;
FIG. 7 shows the cell activity of macrophages treated with different concentrations of PGMP nanoparticles synthesized in example 2 for 24h according to the CCK8 method;
FIG. 8 is a flow cytometry method for detecting the gene transfection efficiency of the PGMP nano-carrier and Lipo6000 synthesized in example 2 on macrophages, wherein the transfection efficiency of the blank group in FIG. 8 is 0; the transfection efficiency of the naked siRNA group was 17.7%; the transfection efficiency of the Lipo-siRNA group is 50.8%; the transfection efficiency of the PGMP-siRNA group was 89.1%; the transfection efficiency of the PGMP-siRNA + 10% FBS group was 87.6%.
FIG. 9 shows the CCK8 method for detecting the cell activity of A549 cells after being treated with PGMP nanocarriers (with and without laser irradiation) synthesized in example 2 for 24 h.
Detailed Description
The present invention is described in detail below by way of examples, which are provided for further illustration only and are not to be construed as limiting the scope of the present invention. The reagents and test equipment used are commercially available unless otherwise indicated.
As one embodiment, the application provides an ultrasonic visual multifunctional nano-carrier with photothermal therapy and macrophage gene transfection enhancement, which is shown by PGMP-siRNA; the former P is the abbreviation of poly (lactic-co-glycolic acid) (PLGA) encapsulated perfluoro-n-pentane (PFP) nanoparticles, namely PFP @ PLGA; g is the abbreviation of a gold shell layer; m is the abbreviation of 11 mercaptoundecanoic acid; the latter P is shorthand for branched polyethylenimine; the siRNA is target siRNA required to be carried in gene therapy.
The preparation method of the ultrasonic visual multifunctional nano-carrier with photothermal therapy and macrophage gene transfection enhancement comprises the following steps:
1) preparing polylactic acid-glycollic acid entrapped perfluoro-n-pentane nanoparticles:
proportionally adding 60-100mg of polylactic acid-glycolic acid white powder into 2-6mL of dichloromethane, and ultrasonically dispersing in an ultrasonic cleaner with power of 30-50Hz until the polylactic acid-glycolic acid white powder is fully dissolved. Adding 100-; dropwise adding the mixed solution into 20-30ml of pre-precooled 2 wt% polyvinyl alcohol aqueous solution, carrying out ultrasonic crushing again for 2-3 minutes by using an ultrasonic crusher with power of 300-13000W under ice bath, carrying out magnetic stirring for 4-5 hours at room temperature, centrifuging the mixed solution by using a low-temperature high-speed centrifuge (10000-13000rpm, 15-20min and 4 ℃), washing, and dispersing the precipitate in 5ml of pure water to obtain the poly (lactic-glycolic acid) -encapsulated perfluoro-n-pentane nanoparticle aqueous solution.
2) Preparing perfluorinated n-pentane nanoparticles coated by a gold nanoshell layer;
and (2) adding 20-30mL of pre-precooled 1 wt% polyallylamine hydrochloride solution into the prepared perfluorinated n-pentane-loaded polylactic acid-glycolic acid nanoparticles, magnetically stirring at room temperature for 20-40 minutes, centrifuging and washing by using a low-temperature high-speed centrifuge, dispersing the precipitate in 5mL of pure water to obtain the perfluorinated n-pentane-loaded polylactic acid-glycolic acid nanoparticles with positive potential, and storing in a refrigerator at 4 ℃ for later use. Adding 800-. Adding the gold nanoparticle solution into the prepared polylactic acid-glycollic acid entrapped perfluoro-n-pentane nanoparticle solution with the positive potential, magnetically stirring for 20-40 minutes, adding 2-3mL of 1 wt% tetrachloroauric acid aqueous solution again, stirring for 10-30 minutes, adding 400-plus-600 mu L of 1 wt% hydroxylamine hydrochloride aqueous solution, continuously magnetically stirring for 20-40 minutes, and carrying out low-temperature high-speed centrifugation (10000-plus-13000 rpm, 15-20min, 4 ℃) and washing to obtain a precipitate, namely the perfluoro-n-pentane nanoparticle coated with the gold nanosheet layer;
3) preparing PGMP nano particles;
weighing 11-mercaptoundecanoic acid powder 50-70mg, adding into chloroform 2-4ml for full dissolution, adding EDC 50-60mg and NHS solid powder 30-40mg under ice bath, magnetically stirring for 15min, adding branched polyethyleneimine solution 400-600mg into the solution, and magnetically stirring for 24 hr; adding the above solution into 50-100ml of triple distilled water, stirring for 1 hour, collecting the extractive solution, and storing at 4 deg.C for use. Dissolving the nanoparticles coated by the gold nanoshell layer obtained in the step 2) in 50mL of aqueous solution, adding 300-fold 600 mu L of the extract, magnetically stirring for 10-12 hours, centrifuging at a high speed at a low temperature (7000-fold 10000rpm, 15-20min and 4 ℃) and washing to obtain the sulfydryl polyethyleneimine modified gold nanoshell coated perfluoro-n-pentane nanoparticles abbreviated as PGMP.
4) Preparing PGMP-siRNA nanoparticles:
preparing the PGMP nanoparticles prepared in the step 3) into a concentration of 1mg/mL by using enzyme-free water, preparing the siRNA into a concentration of 100 mu g/mL by using enzyme-free water, and mixing the PGMP solution: siRNA solution ═ 1: 5-1: 15 (volume ratio) to obtain the PGMP-siRNA nanoparticle solution.
In a specific embodiment of the present application, the pre-cooling is pre-cooling at 4 ℃; the room temperature was 25 ℃.
Polylactic acid-glycolic acid was purchased from Shanghai Yunye Biotech limited and has a molecular weight of 7000-;
polyvinyl alcohol was purchased from Sigma-Aldrich and has a molecular weight of 30000-70000;
polyallylamine hydrochloride was purchased from Mecanne and had a molecular weight of 15000;
branched polyethylenimine is available from Biotech, Inc., of Keli research, Beijing, and has a molecular weight of 1800.
The siRNA is purchased from Gima organisms, the code is A06001, and the siRNA with a fluorescent group CY5 is the siRNA with the fluorescent group CY5 added at the 5' end of a sense strand of the siRNA.
Lipo6000 was purchased from bi yun tian technologies, inc.
Example 1
1) Preparing polylactic acid-glycollic acid entrapped perfluoro-n-pentane nanoparticles:
60mg of polylactic acid-glycolic acid white powder was added to 2mL of dichloromethane in proportion and ultrasonically dispersed in an ultrasonic cleaner with a power of 30Hz until sufficiently dissolved. Adding 100 mu L of perfluoro-n-pentane into the solution, and carrying out ultrasonic crushing for 2 minutes by adopting an ultrasonic crusher with the power of 100W under ice bath; dropwise adding the mixed solution into 20ml of pre-cooled 2 wt% polyvinyl alcohol aqueous solution, carrying out ultrasonic crushing again for 2 minutes by using an ultrasonic crusher with the power of 300W under ice bath, carrying out magnetic stirring for 4 hours at room temperature, centrifuging the mixed solution by using a low-temperature high-speed centrifuge (10000rpm, 15min, 4 ℃), washing, and dispersing the precipitate in 5ml of pure water to obtain the poly (lactic-co-glycolic acid) -coated perfluoro-n-pentane nanoparticle aqueous solution.
2) Preparing perfluorinated n-pentane nanoparticles coated by a gold nanoshell layer;
and (2) adding 20mL of pre-precooled 1 wt% polyallylamine hydrochloride solution into the prepared polylactic acid-glycolic acid nanoparticle loaded with perfluoro-n-pentane, magnetically stirring for 20 minutes at room temperature, centrifuging and washing by using a low-temperature high-speed centrifuge, and dispersing the precipitate in 5mL of pure water to obtain the polylactic acid-glycolic acid encapsulated perfluoro-n-pentane nanoparticle with positive potential, and storing in a refrigerator at 4 ℃ for later use. Adding 800 mu L of 1 wt% tetrachloroauric acid aqueous solution into 100mL of pure water, magnetically stirring at room temperature for 1 minute, adding 500 mu L of 1 wt% sodium citrate aqueous solution, magnetically stirring for 1 minute, adding 250 mu L of 100mM sodium borohydride aqueous solution, and magnetically stirring at room temperature overnight to obtain the gold nano-seed solution. Adding the gold nanoparticle solution into the prepared polylactic acid-glycolic acid entrapped perfluoro-n-pentane nanoparticle solution with the positive potential, magnetically stirring for 20 minutes, adding 2mL of 1 wt% aqueous solution of tetrachloroauric acid again, stirring for 10 minutes, adding 400 mu L of 1 wt% aqueous solution of hydroxylamine hydrochloride, continuously magnetically stirring for 20 minutes, centrifuging at a high speed at a low temperature (10000rpm, 15min, 4 ℃), and washing to obtain a precipitate, namely the perfluoro-n-pentane nanoparticle coated with the gold nanosheet layer; 3) preparing PGMP nano particles;
weighing 11-mercaptoundecanoic acid powder 50mg, adding into chloroform 2ml, dissolving, adding EDC 50mg and NHS 30mg solid powder under ice bath, magnetically stirring for 15min, adding branched polyethyleneimine 400mg into the solution, and magnetically stirring for 24 hr; the above solution was added to 50ml of triple distilled water and stirred for 1 hour, and the extract solution was collected and stored at 4 ℃ for further use. Dissolving the nanoparticles coated with the gold nanoshell layer obtained in the step 2) in 50mL of aqueous solution, adding 300 microliter of the extract, magnetically stirring for 10 hours, centrifuging at a low temperature and a high speed (7000rpm, 15min and 4 ℃) and washing to obtain the sulfydryl polyethyleneimine modified gold nanoshell coated perfluoro-n-pentane nanoparticles abbreviated as PGMP.
4) Preparing PGMP-siRNA nanoparticles:
preparing the PGMP nanoparticles prepared in the step 3) into a concentration of 1mg/mL by using enzyme-free water, preparing the siRNA into a concentration of 100 mu g/mL by using enzyme-free water, and mixing the PGMP: siRNA ═ 1: 5, and dissolving in proportion to obtain the PGMP-siRNA nanoparticle solution.
Example 2
1) Preparing polylactic acid-glycollic acid entrapped perfluoro-n-pentane nanoparticles:
in proportion, 100mg of polylactic acid-glycolic acid white powder was added to 4mL of methylene chloride and dispersed by ultrasound in an ultrasonic cleaner with a power of 50Hz until sufficiently dissolved. Adding 200 mu L of perfluoro-n-pentane into the solution, and carrying out ultrasonic crushing for 3 minutes by adopting an ultrasonic crusher with the power of 160W under ice bath; dropwise adding the mixed solution into 25ml of pre-precooled 2 wt% polyvinyl alcohol aqueous solution, carrying out ultrasonic crushing again for 2 minutes by using an ultrasonic crusher with the power of 400W under ice bath, carrying out magnetic stirring for 4 hours at room temperature, centrifuging the mixed solution by using a low-temperature high-speed centrifuge (13000rpm, 20min and 4 ℃), washing, and dispersing the precipitate in 5ml of pure water to obtain the poly (lactic-co-glycolic acid) encapsulated perfluoro-n-pentane nanoparticle aqueous solution.
2) Preparing perfluorinated n-pentane nanoparticles coated by a gold nanoshell layer;
adding 25mL of pre-precooled 1 wt% polyallylamine hydrochloride solution into the prepared polylactic acid-glycolic acid nanoparticle loaded with perfluoro-n-pentane, magnetically stirring for 20 minutes at room temperature, centrifuging and washing by using a low-temperature high-speed centrifuge, and dispersing the precipitate in 5mL of pure water to obtain the polylactic acid-glycolic acid encapsulated perfluoro-n-pentane nanoparticle with positive potential, and storing in a refrigerator at 4 ℃ for later use. Adding 1000 mu L of 1 wt% tetrachloroauric acid aqueous solution into 100mL of pure water, magnetically stirring at room temperature for 1 minute, adding 1000 mu L of 1 wt% sodium citrate aqueous solution, magnetically stirring for 1 minute, adding 300 mu L of 100mM sodium borohydride aqueous solution, and magnetically stirring at room temperature overnight to obtain the gold nano-seed solution. Adding the gold nanoparticle solution into the prepared polylactic acid-glycolic acid entrapped perfluoro-n-pentane nanoparticle solution with the positive potential, magnetically stirring for 30 minutes, adding 3mL of 1 wt% tetrachloroauric acid aqueous solution again, stirring for 20 minutes, adding 500 mu L of 1 wt% hydroxylamine hydrochloride aqueous solution, continuing to magnetically stir for 30 minutes, centrifuging at a high speed at a low temperature (10000rpm, 20min, 4 ℃), and washing to obtain a precipitate, namely the perfluoro-n-pentane nanoparticle coated with the gold nanosheet layer; 3) preparing PGMP nano particles;
weighing 11-mercaptoundecanoic acid powder 60mg, adding into chloroform 3ml, dissolving, adding solid powder 55mg EDC and 35mg NHS under ice bath, magnetically stirring for 15min, adding branched polyethyleneimine solution 500mg into the solution, and magnetically stirring for 24 hr; the above solution was added to 100ml of triple distilled water and stirred for 1 hour, and the extract solution was collected and stored at 4 ℃ for further use. Dissolving the nanoparticles coated with the gold nanoshell layer obtained in the step 2) in 50mL of aqueous solution, adding 500 microliter of the extract, magnetically stirring for 10 hours, centrifuging at a low temperature and a high speed (7000rpm, 20min and 4 ℃) and washing to obtain the sulfydryl polyethyleneimine modified gold nanoshell coated perfluoro-n-pentane nanoparticles abbreviated as PGMP.
4) Preparing PGMP-siRNA nanoparticles:
preparing the PGMP nanoparticles prepared in the step 3) into a concentration of 1mg/mL by using enzyme-free water, preparing the siRNA into a concentration of 100 mu g/mL by using enzyme-free water, and mixing the PGMP: siRNA ═ 1: 10, namely obtaining the PGMP-siRNA nanoparticle solution.
Example 3
1) Preparing polylactic acid-glycollic acid entrapped perfluoro-n-pentane nanoparticles:
80mg of polylactic acid-glycolic acid white powder was added to 6mL of dichloromethane in proportion and ultrasonically dispersed in an ultrasonic cleaner with a power of 50Hz until sufficiently dissolved. Adding 300 mu L of perfluoro-n-pentane into the solution, and carrying out ultrasonic crushing for 3 minutes by using an ultrasonic crusher with the power of 200W under ice bath; dropwise adding the mixed solution into 30ml of pre-cooled 2 wt% polyvinyl alcohol aqueous solution, carrying out ultrasonic crushing for 3 minutes again by using an ultrasonic crusher with the power of 500W under ice bath, carrying out magnetic stirring for 5 hours at room temperature, centrifuging the mixed solution by using a low-temperature high-speed centrifuge (13000rpm, 20min, 4 ℃), washing, and dispersing precipitates in 5ml of pure water to obtain the poly (lactic-co-glycolic acid) -entrapped perfluoro-n-pentane nanoparticle aqueous solution.
2) Preparing perfluorinated n-pentane nanoparticles coated by a gold nanoshell layer;
and adding 30mL of pre-precooled 1 wt% polyallylamine hydrochloride solution into the prepared polylactic acid-glycolic acid nanoparticle loaded with perfluoro-n-pentane, magnetically stirring for 40 minutes at room temperature, centrifuging and washing by using a low-temperature high-speed centrifuge, and dispersing the precipitate in 5mL of pure water to obtain the polylactic acid-glycolic acid-loaded perfluoro-n-pentane nanoparticle with positive potential, and storing in a refrigerator at 4 ℃ for later use. Adding 800 mu L of 1 wt% tetrachloroauric acid aqueous solution into 120mL of pure water, magnetically stirring at room temperature for 1 minute, adding 1000 mu L of 1 wt% sodium citrate aqueous solution, magnetically stirring for 1 minute, adding 400 mu L of 100mM sodium borohydride aqueous solution, and magnetically stirring at room temperature overnight to obtain the gold nano-seed solution. Adding the gold nanoparticle solution into the prepared polylactic acid-glycolic acid entrapped perfluoro-n-pentane nanoparticle solution with the positive potential, magnetically stirring for 40 minutes, adding 3mL of 1 wt% tetrachloroauric acid aqueous solution again, stirring for 30 minutes, adding 600 mu L of 1 wt% hydroxylamine hydrochloride aqueous solution, continuing to magnetically stir for 40 minutes, centrifuging at a low temperature and a high speed (13000rpm, 20min, 4 ℃), washing, and obtaining a precipitate, namely the perfluoro-n-pentane nanoparticle coated with the gold nanosheet layer; 3) preparing PGMP nano particles;
weighing 11-mercaptoundecanoic acid powder 70mg, adding into chloroform 4ml, dissolving, adding solid powder 60mg EDC and 40mg NHS under ice bath, magnetically stirring for 15min, adding branched polyethyleneimine solution 600mg into the solution, and magnetically stirring for 24 hr; the above solution was added to 100ml of triple distilled water and stirred for 1 hour, and the extract solution was collected and stored at 4 ℃ for further use. Dissolving the nanoparticles coated by the gold nanoshell layer obtained in the step 2) in 50mL of aqueous solution, adding 600 microliter of the extract, magnetically stirring for 12 hours, centrifuging at a low temperature and a high speed (10000rpm, 20min and 4 ℃) and washing to obtain the sulfydryl polyethyleneimine modified gold nanoshell coated perfluoro-n-pentane nanoparticles abbreviated as PGMP.
4) Preparing PGMP-siRNA nanoparticles:
preparing the PGMP nanoparticles prepared in the step 3) into a concentration of 1mg/mL by using enzyme-free water, preparing the siRNA into a concentration of 100 mu g/mL by using enzyme-free water, and mixing the PGMP: siRNA ═ 1: 15, and obtaining the PGMP-siRNA nanoparticle solution.
Example 4
The PGMP described in example 4 was prepared as in example 2;
the PGMP-siRNA was prepared by replacing the siRNA in example 2 with siRNA carrying a fluorophore CY5 in the same manner as in example 2;
the Lipo-siRNA was prepared in the same manner as in example 2, except that the siRNA in example 2 was replaced with siRNA with a fluorophore CY5, and PGMP was replaced with Lipo 6000.
The transmission electron microscope and the scanning electron microscope of the PGMP nanocarrier prepared in example 2 are shown in fig. 1(a) and fig. 1(b), respectively, which show that the PGMP nanoparticles are spherical and uniform in size, the gold nanoparticles are coated on the surface of the nanospheres to form a gold nanoshell layer, and the diameter of the gold nanoparticles is within the range of 20-50 nm. The PGMP nano-carrier has a particle size range of 80-600nm as measured by Malvern laser particle sizer, as shown in FIG. 2(a), a zeta potential range of 30-45mV, and a potential of-10 to-20 mV after siRNA attachment, as shown in FIG. 2 (b). Absorption spectra of the PGMP nano-carriers with different concentrations are shown in FIG. 3 measured by an ultraviolet-visible spectrophotometer, and the PGMP nano-carriers have stronger ultraviolet absorption intensity within the range of 600-900 nm. Detecting the photo-thermal temperature rise curve of the PGMP nano carrier: taking 1.0mL of PGMP water solution with the concentration of 100 mu g/mL in a quartz cuvette, and adjusting the laser power density to be 0.25W/cm respectively2、0.5W/cm2、0.75W/cm2、1W/cm2Measuring the temperature change curve of the nano solution under 808nm laser irradiation for 0-15min, and recording the temperature by using an infrared temperature recorder, wherein the maximum temperature can reach 56 ℃ as shown in figure 4. The photothermal conversion efficiency of the PGMP nano carrier is about 59% (see figure 5) and is obtained by calculation of a photothermal conversion efficiency formula, and the photothermal conversion efficiency is higher than that of the common gold nanorod reported at present. The PGMP nano-carrier is expected to be used for photo-thermal treatment of cancer.
Ultrasonic enhanced imaging detection of PGMP nanocarriers in vitro: latex tubes with an inner diameter of 4mm were embedded in 1.5% agarose gel, PGMP nanocarrier solutions of different concentrations (3mg/ml,1mg/ml,0.5mg/ml,0.25mg/ml) were applied with and without laser (808nm, 0.5W/cm)25min), quickly injecting into a latex tube simulating a blood vessel, and controlling a constant current cable to circulate in the tube to perform ultrafiltration by a pumpAnd performing acoustic imaging, wherein the experimental control group is set to be a PBS solution. An ultrasonic imaging system is adopted for processing, and under a conventional gray scale mode, the frequency of the transducer is set to be 10.0MHz, and the frequency of the linear array probe is 8-11 MHz. The results (see fig. 6) show that the nano-carrier prepared by the invention has the effect of enhancing ultrasonic imaging under the condition of no laser irradiation, and the enhancement effect is further enhanced after the laser irradiation.
The toxicity of the PGMP nano-carrier to the human-derived macrophage THP-1 under different concentrations is determined: the in vitro toxicity of the PGMP nano-carrier on the macrophage is detected by a CCK8 method for characterization. Taking THP-1 cells with good logarithmic growth phase state at 2X 105Inoculating the culture medium into a 96-well plate, inducing by adopting PMA (phorbol ester) with the concentration of 200nM for 24h, adding culture mediums containing PGMP nano-carriers with different concentrations into the 96-well plate inoculated with macrophages, setting 5 repeated wells for each concentration, culturing in an incubator at 37 ℃ for 24h, adding 10 mu L of CCK8 solution into each well, continuously culturing in the incubator at 37 ℃ for 30min-2h until the color of the culture medium is changed into orange yellow, setting the wavelength to be 450nM by adopting an enzyme linked immunosorbent assay detector, shaking for 30s before detection, and measuring the absorbance value of each well. The results of CCK8 show that PGMP nanocarriers have no significant effect on macrophage activity at a concentration of 120. mu.g/mL, as shown in FIG. 7. In addition, siRNA with the fluorescent group CY5 was used for gene transfection experiment flow cytometry detection of macrophages: taking THP-1 cells with good logarithmic growth state at 2X 106Inoculating the wells into 6-well plate, inducing with PMA with concentration of 200nM for 24h, adding the materials of each component into the pore plate of the macrophage according to different groups (blank group, naked siRNA group (120nM), Lipo-siRNA group (120nM), PGMP-siRNA (120nM) + 10% FBS group), culturing at 37 ℃ for 6h, pancreatin digestion, PBS washing, using 500 u L PBS solution to suspend in the flow cell tube for machine detection, the results show, PGMP-siRNA group, PGMP-siRNA + 10% FBS group transfection rate basically consistent, all in about 90%, far higher than Lipo-siRNA group and naked siRNA group (see figure 8, blank group transfection rate of 0; naked siRNA group transfection rate of 17.7%, Lipo-siRNA group transfection rate of 50.8%; PGMP-siRNA group transfection rate of 89.1%; PGMP-siRNA + 10% FBS group transfection rate of 87.6%). Illustrating the preparation of the inventionThe PGMP nano-carrier has better biological safety, can realize the antiserum effect and improve the gene transfection efficiency of macrophages.
Determining the toxicity of the PGMP nano-carrier to the non-small cell lung cancer A549 cells under different concentrations: the in vitro toxicity and photothermal therapy of PGMP and PGMP + PTT on A549 cells were characterized by the CCK8 method. A549 cells with good logarithmic growth phase state are taken at the ratio of 1 × 105After the cells are inoculated in a 96-well plate for 24 hours, adding culture media containing PGMP nano-carriers with different concentrations into the 96-well plate inoculated with A549 cells, setting 4 repeated wells for each concentration, continuously incubating the cells in an incubator at 37 ℃ for 24 hours with or without laser irradiation, adding 10 mu L of CCK8 solution into each well, continuously culturing the cells in the incubator at 37 ℃ for 30min to 2 hours until the color of the culture media is changed into orange yellow, setting the wavelength to be 450nm by adopting an enzyme linked immunosorbent assay (ELISA) detector, shaking the cells for 30s before detection, and measuring the absorbance value of each well. Experiments prove that (see figure 9) PGMP nano-carriers have no obvious influence on the activity of A549 cells within the concentration of 120 mu g/mL and are applied to 808nm laser (the power is 0.75W/cm)2And 15min), the proliferation of the A549 cells can be directly influenced along with the increase of the concentration of the nano-carrier disclosed by the invention after irradiation, and the cell proliferation inhibition rate of an illumination group is increased by about 70% compared with that of a non-illumination group, which indicates that the PGMP nano-carrier can be effectively used for photo-thermal treatment of tumors. The results show that the ultrasonic visual multifunctional nano-carrier with photothermal therapy and macrophage gene transfection enhancement, which is prepared by the invention, can realize the implementation of a synergistic treatment mode combining ultrasonic enhanced imaging and tumor photothermal therapy with immunocyte gene therapy, is a great innovation of cancer treatment medicines, and has excellent use value.
Although the present application has been described with reference to a few embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.
Claims (10)
1. The composite material nanosphere is characterized by comprising core particles, a metal nanoshell layer, a mercapto acid modification layer and a polyethyleneimine compound modification layer;
the core particles are film-forming substances and coated with fluorocarbon compounds;
the film forming material is at least one of polylactic acid and polylactic acid derivative copolymer;
the metal in the metal nanoshell layer is selected from at least one of inorganic noble metals with photo-thermal properties;
the mercapto acid in the mercapto acid modification layer is selected from at least one of linear mercapto acid compounds;
the polyethyleneimine compound in the polyethyleneimine compound modification layer is selected from at least one of branched polyethyleneimine compounds;
the metal nano shell layer is coated on the surface of the core particle;
the mercapto acid modification layer is connected to the metal shell layer through a mercapto group;
the polyethylene imine compound layer is connected to the mercaptan acid modification layer through an amido bond.
2. The composite nanoball of claim 1, wherein the number of carbon atoms of the fluorocarbon-based compound is 5-6;
preferably, the fluorocarbon compound is selected from at least one of perfluoro-n-pentane and perfluorohexane;
preferably, the polylactic acid is selected from the group consisting of l-polylactic acid;
the polylactic acid derivative copolymer is at least one selected from polylactic acid-glycolic acid, polyethylene glycol monomethyl ether-polylactic acid copolymer and polyethylene glycol-polylactic acid copolymer;
preferably, the metal in the metal nanoshell layer is selected from at least one of gold, silver, platinum and palladium;
preferably, the mercapto acid compound is selected from at least one of 6-mercaptohexanoic acid and 11-mercaptoundecanoic acid;
preferably, the polyethyleneimine-based compound is at least one selected from branched polyethyleneimine, branched oligoethyleneimine, branched polyvinylamine, and branched polyallylamine;
preferably, the metal nano shell layer is a layered structure formed by metal nanoparticles adsorbed on the surface of the inner core particle through electrostatic action;
preferably, the particle size of the metal nanoparticles is 20-50 nm;
preferably, the particle size of the composite material nanosphere is 80-600 nm.
3. The method for preparing the composite nanosphere according to any of claims 1-2, wherein the method comprises the following steps:
(S1) encapsulating the fluorocarbon compound in the film-forming substance to obtain inner core particles;
(S2) coating a metal nanoshell layer on the surface of the core particle to obtain a metal nanoshell layer coated core particle;
(S3) sequentially modifying a mercapto acid modification layer and a polyethyleneimine compound modification layer on the surface of the metal nano shell layer to obtain the composite material nanosphere.
4. The method according to claim 3, wherein the (S1) includes: performing ultrasonic treatment on a solution I containing a film forming substance and a fluorocarbon compound, adding a surfactant solution II, performing ultrasonic treatment on the solution II, and stirring the solution I to obtain core particles;
preferably, the (S2) includes: mixing and stirring II core particles and polyallylamine hydrochloride solution III to obtain core particles with positive charges, mixing the core particles with the positive charges with metal nano seed liquid, adding metal salt solution IV and reducing agent solution V, and stirring III to obtain the core particles coated by the metal nano shell;
preferably, the (S3) includes: and stirring IV a reaction extraction liquid containing mercapto acid, an activating agent, a catalyst and a polyethyleneimine compound solution VI with a solution containing the inner core particles coated by the metal nano shell to obtain the composite material nano sphere.
5. The method according to claim 5, wherein (S1), the solvent of solution I is at least one selected from dichloromethane, chloroform and ethyl acetate;
in the solution I, the proportion of the film forming substance, the fluorocarbon compound and the solvent is 60-100 mg: 100-300. mu.L: 2-6 mL;
preferably, in the solution II, the surfactant is selected from at least one of polyvinyl alcohol and polyacrylic acid;
preferably, in the solution II, the concentration of the surfactant is 1-3 wt%;
preferably, the ratio of the film-forming substance to the solution II is 60-100 mg: 20-30 ml;
preferably, the power of the ultrasonic I is 100-;
the power of the ultrasonic II is 300-500W, and the time is 2-3 minutes;
stirring I time is 4-5 hours.
6. The method of claim 4, wherein (S2), in said solution III, the concentration of said polyallylamine hydrochloride is 0.5-2.0 wt%;
preferably, the ratio of the film-forming substance to the solution III is 60-100 mg: 20-30 ml;
preferably, the stirring II time is 20-40 minutes;
preferably, the metal salt in the metal salt solution IV is selected from at least one of tetrachloroauric acid dihydrate, tetrachloroauric acid trihydrate, and tetrachloroauric acid tetrahydrate;
preferably, the metal salt concentration in the metal salt solution IV is 0.5-2.0 wt%;
preferably, the reducing agent in the reducing agent solution V is selected from hydroxylamine hydrochloride;
preferably, the concentration of the reducing agent in the reducing agent solution V is 1-2 wt%;
preferably, the ratio of film-forming substance, metal salt solution IV and reducing agent solution V is 60-100 mg: 2-3 mL: 400-;
preferably, the stirring time of the stirring III is 20-40 min;
preferably, the metal nano seed solution is obtained by the following steps:
diluting a metal salt solution VII, adding a stabilizer solution VIII, stirring V, adding a reducing agent solution VIIII, and stirring VI;
preferably, the metal salt in the metal salt solution VII is at least one selected from tetrachloroauric acid dihydrate, tetrachloroauric acid trihydrate and tetrachloroauric acid tetrahydrate, and the concentration is 0.5-2.0 wt%;
the stabilizer in the stabilizer solution VIII is selected from sodium citrate, and the concentration is 0.5-2.0 wt%;
the reducing agent in the reducing agent solution VIIII is selected from sodium borohydride and has the concentration of 10-100 mM;
optionally, the ratio of the film-forming substance, the metal salt solution VII, the stabilizer solution VIII and the reducing agent solution VIIII is 60-100 mg: 800-: 500-1000. mu.L: 250-400 μ L.
7. The method according to claim 5, wherein (S3), the solvent in the solution VI is at least one selected from the group consisting of dichloromethane, chloroform and ethyl acetate
Preferably, in the solution VI, the ratio of mercaptoacid, activator, catalyst, polyethyleneimine compound and solvent is 50-70 mg: 50-60 mg: 30-40 mg: 400-600 mg: 2-4 ml;
preferably, the activator is selected from EDC;
the catalyst is selected from NHS;
preferably, the ratio of the film-forming substance to the reaction extract is 60-100 mg: 300-;
preferably, the stirring IV is carried out for a period of 10 to 12 hours.
8. A pharmaceutical composition comprising a siRNA solution and a carrier solution;
the volume ratio of the carrier solution to the siRNA solution is 1: 5-1: 15
siRNA is loaded on a carrier;
the carrier is at least one selected from the composite nanospheres according to any one of claims 1 to 2 and the composite nanospheres prepared by the preparation method according to any one of claims 3 to 7.
9. A process for preparing a pharmaceutical composition according to claim 8, comprising the steps of:
and mixing the carrier solution and the siRNA solution to obtain the composition.
10. The composite nanosphere according to any one of claims 1 to 2, the composite nanosphere obtained by the preparation method according to any one of claims 3 to 7, the pharmaceutical composition according to claim 8, and the pharmaceutical composition obtained by the preparation method according to claim 9, wherein the pharmaceutical composition is used for preparing at least one of photothermal therapy drugs, gene therapy drugs and ultrasonic imaging agents.
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