CN110859822A - Synthesis method of nano preparation - Google Patents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- 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
- A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5123—Organic compounds, e.g. fats, sugars
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5169—Proteins, e.g. albumin, gelatin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55588—Adjuvants of undefined constitution
Abstract
The invention relates to a method for preparing a nano preparation. Comprising 1) CuCl2·2H2Preparing an O aqueous solution; 2) GdCl3·6H2Preparing O aqueous solution, 3) synthesizing Gd (CuS @ Flu @ TPE) nano-particles by a one-pot method, 4) synthesizing Gd (CuS @ Flu @ TPE @ α cd47@ RGD nano-particles, RGD targeting molecules and PLGA-Hyd-PEG carriers to help efficiently target tumor lesions by a two-step double emulsion method, and influenzaThe virus activates the whole immune system by utilizing strong immune activation function to cause immune response; the CD47 antibody shields immune cell inhibitory molecules on the surface of the tumor to prevent the tumor from immune escape; the visual guidance of the AIE probe and the Gd and CuS realizes the light-operated release of tumor antigens, further activates the killing efficiency of immune cells, induces the organism to generate the immunological memory function, and achieves the aim of efficiently and thoroughly treating tumors.
Description
Technical Field
The invention relates to Gd CuS @ Flu @ TPE @ α cd47@ RGD nano diagnosis and treatment agent successfully prepared by a protein-mediated biomimetic simulation strategy.
Background
Currently, tumors are one of the most threatening diseases to humans in the world. Immunotherapy achieves the effect of treating cancer by activating the human immune system and killing cancer cells and tumor tissues by means of autoimmune function. Therefore, immunotherapy is one of the most effective methods for treating tumors.
Influenza virus as foreign body can stimulate the whole body immune system to produce cytokine storm (such as interferon, tumor necrosis factor and various interleukins, etc.), and make dendritic cell (DC cell, presenting tumor antigen) and macrophage (macrophage) having important function in the course of anti-tumor therapy
Cells, phagocytic tumor cells) and natural killer cells (NK cells, killer tumor cells), etc., are activated in large quantities, and subsequently the proliferation efficiency of T cells is greatly improved. The composite nanometer preparation based on the influenza virus can activate the immune function of the organism systemically, cause all-round killing and elimination of the tumor, and compared with the traditional tumor treatment method, the strategy ensures that the tumor treatment is more efficient.
In order to ensure that the medicine can accurately reach the tumor part, ensure the medicine concentration of the tumor part and avoid adverse reaction caused by overlarge medicine concentration, the tumor targeting carrier can be used for achieving the purpose of accurate treatment. At present, the carrier systems studied at home and abroad mainly comprise: (1) a macromolecular carrier system (2), a magnetic drug preparation (3), a multi-targeting preparation (4) and a microparticle carrier system. Wherein the PLGA-Hyd-PEG carrier can efficiently target tumor focus.
RGD targeting molecules are short peptides containing arginine-glycine-aspartic acid, and can mediate the targeted therapy of tumors. The antitumor drug and the delivery system thereof can increase the tumor active targeting property of the drug by the modification of RGD targeting molecules, thereby achieving more effective, accurate and safe treatment.
In the diseased state, tumor cells can escape immune surveillance of the body, leading to tumor development and metastasis. The protein CD47 is taken as a friend or foe identification signal molecule and is closely related to the escape of tumor cells from immune phagocytosis. The protein CD47 antibody is used for shielding immune cell inhibitory molecules on the surface of the tumor to prevent the tumor immune escape, and the protein CD47 antibody is a very effective new way for tumor immunotherapy.
The photothermal therapy (PTT) is a novel tumor therapy method, under the irradiation of external near infrared light, a photothermal agent at a tumor part absorbs the near infrared light and converts the near infrared light into heat, so that the temperature of the tumor part is rapidly increased to more than 48 ℃, and cancer cells can be killed within a few minutes. The photothermal therapy process has less side effect, low systemic toxicity, no damage to normal tissue and great clinical application potential. If the photothermal therapy and the tumor immunotherapy are combined, the tumor therapy effect is greatly enhanced, and the tumor therapy is more thorough.
The prepared medicine has the following advantages: 1) RGD targeting molecules and PLGA-Hyd-PEG carriers can help efficiently target tumor lesions; 2) influenza viruses can activate the systemic immune system using a strong immune activation function, resulting in an immune response; 3) the CD47 antibody shields immune cell inhibitory molecules on the surface of the tumor, so that the tumor immune escape can be effectively prevented; 4) the visual guidance of the AIE probe and the Gd and CuS realizes the light-operated release of tumor antigens, further activates the killing efficiency of immune cells, induces the organism to generate the immunological memory function, and achieves the aim of efficiently and thoroughly treating tumors.
Disclosure of Invention
The invention relates to a method for successfully synthesizing Gd CuS @ Flu @ TPE @ α cd47@ RGD tumor combined treatment nano-preparation through strategy bionic simulation.
The technical scheme of the invention is a method for synthesizing a nano preparation, which comprises the following steps:
1) cupric chloride dihydrate (CuCl)2·2H2O) powder is added into ultrapure water and then dissolved by ultrasonic to obtain CuCl with the concentration of 0.005-0.02M2·2H2An aqueous solution of O;
2) gadolinium chloride hexahydrate powder (GdCl)3·6H2O) adding 1mL of ultrapure water, and ultrasonically dissolving to obtain GdCl with the concentration of 0.025-0.1M3·6H2An aqueous solution of O;
3) the method for synthesizing Gd CuS @ Flu @ TPE nano-particles by using the one-pot method comprises the following steps:
(1) placing 2.5-10ml single-mouth bottle with concentration of 0.005-0.02mg/ml flu @ TPE solution on a water bath magnetic stirrer at 37 ℃, and then dropwise adding the prepared CuCl by using a disposable dropper under the stirring condition2·2H2O and GdCl3·6H2Continuously stirring the solution O after the dropwise addition is finished;
(2) preparing a 1M sodium hydroxide (NaOH) solution, adding 0.25-1mL of the NaOH solution into the reaction system, and adjusting the final pH value of the solution to 8-10, wherein the solution becomes clear and transparent dark blue;
(3) sodium sulfide nonahydrate (Na) with the concentration of 242.16mg/mL is prepared2S·9H2O) solution, and 0.4mL of the solution is added into the reaction system, and the solution turns brown;
(4) stirring and reacting for 4h in a water bath environment at 37 ℃, taking out the reaction liquid, pouring the reaction liquid into a dialysis bag with the molecular weight cutoff of 8000-;
(5) putting the dialyzed solution into a refrigerator at minus 80 ℃ for overnight, and transferring the solution into a freeze dryer for freeze drying to obtain light green flocculent Gd (CuS @ Flu @ TPE) nano particles;
(6) the Gd is CuS @ Flu @ TPE @ α cd47@ RGD nano-particles are synthesized by a two-step double emulsion method.
The Gd is synthesized by a two-step double emulsion method, namely CuS @ Flu @ TPE @ α cd47@ RGD nano-particles, and the method specifically comprises the following steps:
a) PLGA-Hyd-PEG-RGD is dissolved in dichloromethane to be used as an oil phase (O) so as to contain Gd; the CuS @ Flu @ TPE multi-modal virus nanoparticle and CD47 antibody complex solution was used as an internal aqueous phase (W1);
b) adding W1 into O, and performing ultrasonic treatment for 1min at 60W to form water-in-oil (W1/O) emulsion;
c) adding W1/O into 1.5% PVA water solution, and performing ultrasonic treatment for 7min at 100W to obtain water-in-oil-in-water (W1/O/W2) type double emulsion.
The invention has the advantages that: 1) RGD targeting molecules and PLGA-Hyd-PEG carriers can help efficiently target tumor lesions; 2) influenza viruses can activate the systemic immune system using a strong immune activation function, resulting in an immune response; 3) the CD47 antibody shields immune cell inhibitory molecules on the surface of the tumor, so that the tumor immune escape can be effectively prevented; 4) the visual guidance of the AIE probe and the Gd and CuS realizes the light-operated release of tumor antigens, further activates the killing efficiency of immune cells, induces the organism to generate the immunological memory function, and achieves the aim of efficiently and thoroughly treating tumors.
Detailed Description
The present invention is further illustrated by the following examples.
Example 1:
the method for synthesizing the Gd, CuS @ Flu @ TPE @ α cd47@ RGD tumor combined treatment nano preparation comprises the following specific steps:
(1) 4.25mg of copper chloride dihydrate (CuCl) was accurately weighed2·2H2O) powder, 5mL of ultrapure water was added and dissolved by ultrasonic to obtain 0.005M CuCl2·2H2And (4) O aqueous solution.
(2) 9.3mg of gadolinium chloride hexahydrate powder (GdCl) was weighed out accurately3·6H2O), adding 1mL of ultrapure water, and ultrasonically dissolving to obtain GdCl with the concentration of 0.025M3·6H2And (4) O aqueous solution.
(3) The method for synthesizing Gd CuS @ Flu @ TPE nano-particles by using the one-pot method comprises the following steps:
1) placing 2.5ml single-mouth bottle with concentration of 0.005mg/ml flu @ TPE solution on a water bath magnetic stirrer at 37 ℃, and then dropwise adding the prepared CuCl by using a disposable dropper under the stirring condition2·2H2O and GdCl3·6H2Continuously stirring the solution O for three minutes after the dropwise addition;
2) preparing a 1M sodium hydroxide (NaOH) solution, adding 0.25mL of the NaOH solution into the reaction system, and adjusting the final pH value of the solution to 8, wherein the solution becomes clear, transparent and dark blue;
3) prepared concentration of 242.16mg/mL sodium sulfide nonahydrate (Na)2S·9H2O) solution, and 0.4mL of the solution is added into the reaction system, and the solution turns brown;
4) stirring and reacting for 2h in a water bath environment at 37 ℃, taking out the reaction liquid, pouring the reaction liquid into a dialysis bag with the molecular weight cutoff of 8000-;
5) and (3) putting the dialyzed solution into a refrigerator at-80 ℃ for overnight, and transferring the solution into a freeze dryer for freeze drying to obtain light green flocculent Gd (CuS @ Flu @ TPE) nano particles.
(4) The method for synthesizing Gd CuS @ Flu @ TPE @ α cd47@ RGD nanoparticles by using the two-step double emulsion method comprises the following steps:
1) PLGA-Hyd-PEG-RGD is dissolved in dichloromethane to be used as an oil phase (O) so as to contain Gd; the solution of the CuS @ Flu @ TPE multi-modal virus nanoparticle and CD47 antibody complex served as the internal aqueous phase (W1).
2) W1 was added to O and sonicated for 1min at 60W to form a water-in-oil (W1/O) emulsion.
3) Adding W1/O into 1.5% PVA water solution, and performing ultrasonic treatment for 7min at 100W to obtain water-in-oil-in-water (W1/O/W2) type double emulsion.
Example 2:
the method for synthesizing the Gd, CuS @ Flu @ TPE @ α cd47@ RGD tumor combined treatment nano preparation comprises the following specific steps:
(1) 9.5mg of cupric chloride dihydrate (CuCl) was accurately weighed2·2H2O) powder, 5mL of ultrapure water was added and dissolved by ultrasonic to obtain 0.01M CuCl2·2H2And (4) O aqueous solution.
(2) 18.6mg of gadolinium chloride hexahydrate powder (GdCl) was weighed out accurately3·6H2O), 1mL of ultrapure water was added thereto and the mixture was ultrasonically dissolved to obtain GdCl having a concentration of 0.05M3·6H2And (4) O aqueous solution.
(3) The method for synthesizing Gd CuS @ Flu @ TPE nano-particles by using the one-pot method comprises the following steps:
1) placing 5ml of single-mouth bottle with the concentration of 0.01mg/m flu @ TPE solution on a water bath magnetic stirrer at 37 ℃, and then dropwise adding the prepared CuCl by using a disposable dropper under the stirring condition2·2H2O and GdCl3·6H2Continuously stirring the solution O for three minutes after the dropwise addition;
2) preparing a 1M sodium hydroxide (NaOH) solution, adding 0.5mL of the NaOH solution into the reaction system, and adjusting the final pH value of the solution to 9, wherein the solution becomes clear, transparent and dark blue;
3) sodium sulfide nonahydrate (Na) with the concentration of 242.16mg/mL is prepared2S·9H2O) solution, and 0.4mL of the solution is added into the reaction system, and the solution turns brown;
4) stirring and reacting for 4h in a water bath environment at 37 ℃, taking out the reaction liquid, pouring the reaction liquid into a dialysis bag with the molecular weight cutoff of 8000-;
5) and (3) putting the dialyzed solution into a refrigerator at-80 ℃ for overnight, and transferring the solution into a freeze dryer for freeze drying to obtain light green flocculent Gd (CuS @ Flu @ TPE) nano particles.
(4) The method for synthesizing Gd CuS @ Flu @ TPE @ α cd47@ RGD nanoparticles by using the two-step double emulsion method comprises the following steps:
1) PLGA-Hyd-PEG-RGD is dissolved in dichloromethane to be used as an oil phase (O) so as to contain Gd; the solution of the CuS @ Flu @ TPE multi-modal virus nanoparticle and CD47 antibody complex served as the internal aqueous phase (W1).
2) W1 was added to O and sonicated for 1min at 60W to form a water-in-oil (W1/O) emulsion.
3) Adding W1/O into 1.5% PVA water solution, and performing ultrasonic treatment for 7min at 100W to obtain water-in-oil-in-water (W1/O/W2) type double emulsion.
Example 3:
the method for synthesizing the Gd, CuS @ Flu @ TPE @ α cd47@ RGD tumor combined treatment nano preparation comprises the following specific steps:
(1) 17mg of copper chloride dihydrate (CuCl) was accurately weighed2·2H2O) powder, 5mL of ultrapure water was added and dissolved by ultrasonic to obtain CuCl with a concentration of 0.02M2·2H2And (4) O aqueous solution.
(2) 37.2mg of gadolinium chloride hexahydrate powder (GdCl) was accurately weighed3·6H2O), 1mL of ultrapure water was added thereto and the mixture was ultrasonically dissolved to obtain GdCl having a concentration of 0.1M3·6H2And (4) O aqueous solution.
(3) The method for synthesizing Gd CuS @ Flu @ TPE nano-particles by using the one-pot method comprises the following steps:
1) placing 10ml of single-mouth bottle with the concentration of 0.02mg/ml flu @ TPE solution on a water bath magnetic stirrer at 37 ℃, and then dropwise adding the prepared CuCl by using a disposable dropper under the stirring condition2·2H2O and GdCl3·6H2Continuously stirring the solution O for three minutes after the dropwise addition;
2) preparing a 1M sodium hydroxide (NaOH) solution, adding 1mL of the NaOH solution into the reaction system, and adjusting the final pH value of the solution to 10, wherein the solution becomes clear and transparent dark blue;
3) sodium sulfide nonahydrate (Na) with the concentration of 484.32mg/mL is prepared2S·9H2O) solution, and 0.4mL of the solution is added into the reaction system, and the solution turns brown;
4) stirring and reacting for 8h in a water bath environment at 37 ℃, taking out the reaction liquid, pouring the reaction liquid into a dialysis bag with the molecular weight cutoff of 8000-;
5) and (3) putting the dialyzed solution into a refrigerator at-80 ℃ for overnight, and transferring the solution into a freeze dryer for freeze drying to obtain light green flocculent Gd (CuS @ Flu @ TPE) nano particles.
(4) The method for synthesizing Gd CuS @ Flu @ TPE @ α cd47@ RGD nanoparticles by using the two-step double emulsion method comprises the following steps:
1) PLGA-Hyd-PEG-RGD is dissolved in dichloromethane to be used as an oil phase (O) so as to contain Gd; the solution of the CuS @ Flu @ TPE multi-modal virus nanoparticle and CD47 antibody complex served as the internal aqueous phase (W1).
2) W1 was added to O and sonicated for 1min at 60W to form a water-in-oil (W1/O) emulsion.
3) Adding W1/O into 1.5% PVA water solution, and performing ultrasonic treatment for 7min at 100W to obtain water-in-oil-in-water (W1/O/W2) type double emulsion.
Claims (2)
1. A method for synthesizing a nano preparation is characterized by comprising the following steps:
1) cupric chloride dihydrate (CuCl)2·2H2O) powder is added into ultrapure water and then dissolved by ultrasonic to obtain CuCl with the concentration of 0.005-0.02M2·2H2An aqueous solution of O;
2) gadolinium chloride hexahydrate powder (GdCl)3·6H2O) adding 1mL of ultrapure water, and ultrasonically dissolving to obtain GdCl with the concentration of 0.025-0.1M3·6H2An aqueous solution of O;
3) the method for synthesizing Gd CuS @ Flu @ TPE nano-particles by using the one-pot method comprises the following steps:
(1) placing 2.5-10ml single-mouth bottle with concentration of 0.005-0.02mg/ml flu @ TPE solution on a water bath magnetic stirrer at 37 ℃, and then dropwise adding the prepared CuCl by using a disposable dropper under the stirring condition2·2H2O and GdCl3·6H2Continuously stirring the solution O after the dropwise addition is finished;
(2) preparing a 1M sodium hydroxide (NaOH) solution, adding 0.25-1mL of the NaOH solution into the reaction system, and adjusting the final pH value of the solution to 8-10, wherein the solution becomes clear and transparent dark blue;
(3) sodium sulfide nonahydrate (Na) with the concentration of 242.16mg/mL is prepared2S·9H2O) solution, and 0.4mL of the solution is added into the reaction system, and the solution turns brown;
(4) stirring and reacting for 4h in a water bath environment at 37 ℃, taking out the reaction liquid, pouring the reaction liquid into a dialysis bag with the molecular weight cutoff of 8000-;
(5) putting the dialyzed solution into a refrigerator at minus 80 ℃ for overnight, and transferring the solution into a freeze dryer for freeze drying to obtain light green flocculent Gd (CuS @ Flu @ TPE) nano particles;
(6) the Gd is CuS @ Flu @ TPE @ α cd47@ RGD nano-particles are synthesized by a two-step double emulsion method.
2. The method for synthesizing the nano-preparation according to claim 1, wherein the Gd is CuS @ Flu @ TPE @ α cd47@ RGD nano-particles synthesized by a two-step double emulsion method, and the method comprises the following steps:
a) PLGA-Hyd-PEG-RGD is dissolved in dichloromethane to be used as an oil phase (O) so as to contain Gd; the CuS @ Flu @ TPE multi-modal virus nanoparticle and CD47 antibody complex solution was used as an internal aqueous phase (W1);
b) adding W1 into O, and performing ultrasonic treatment for 1min at 60W to form water-in-oil (W1/O) emulsion;
c) adding W1/O into 1.5% PVA water solution, and performing ultrasonic treatment for 7min at 100W to obtain water-in-oil-in-water (W1/O/W2) type double emulsion.
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