Preparation method of photo-thermal response type gold nanorod-bone cement composite material
By preparing photothermally responsive gold nanorod-bone cement composite materials, the problems of uncontrollable heat and non-degradable materials in bone tumor treatment were solved, achieving precise local temperature control, immunogenic cell death and implantation stability, and promoting bone repair and immune response.
Patent Information
- Application Number
- CN202511612050.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-06
- Publication Date
- 2026-01-02
AI Technical Summary
Existing bone repair materials have problems such as uncontrollable local heat, poor targeting, inability of thermotherapy equipment to accurately control temperature, and non-degradability of materials when treating bone tumors, leading to tissue damage and prosthesis loosening.
A photothermal responsive gold nanorod-bone cement composite material was prepared. Through the preparation, surface modification, and embedding of gold nanorods into polymethyl methacrylate bone cement, precise local temperature control and immunogenic cell death were achieved, combined with optimization of the bone microenvironment.
It achieves precise control of local temperature, avoids tissue damage, effectively induces immunogenic cell death, activates anti-tumor immune response, and provides sufficient mechanical support and implantation stability.
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Figure CN121243472A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bone cement preparation technology, and particularly to a method for preparing a photothermal responsive gold nanorod-bone cement composite material. Background Technology
[0002] Currently, widely used bone repair materials in clinical practice, such as polymethyl methacrylate (PMMA) bone cement and calcium phosphate (CPC) bone cement, are mainly used for fracture filling and support. However, these materials have limitations in treating bone tumors or inducing immunotherapy. Furthermore, bone cement generates a large amount of heat during curing (local polymerization temperatures >80°C), which is transient and uncontrollable, potentially causing damage to surrounding tissues. Additionally, these materials are generally non-degradable, which may lead to prosthesis loosening.
[0003] Photothermal responsive materials, especially gold nanorods (GNRs), are near-infrared photothermal converters that can absorb light energy at specific wavelengths and convert it into heat. However, current systemic administration of gold nanorods suffers from poor targeting, hepatotoxicity, and poor local retention in bone metastases. Conventional hyperthermia devices (radiofrequency / microwave) also cannot precisely control temperature, have difficulty penetrating bone, and exhibit uneven temperature distribution, making it impossible to achieve precise sub-high temperature (42–45°C) control at bone metastases and thus hindering their effective application in the treatment of bone metastases.
[0004] In recent years, a technology combining implantable bone cement with nano-thermosensitive materials has gradually emerged, but the following technical problems still exist: Focusing on temperature control or hyperthermia, there is a lack of designs that clearly induce immunogenic cell death (ICD). Most of them are spherical particles, and there are still limitations in temperature control and thermal response speed; The mechanical properties of the thermotherapy material and bone cement are not well integrated, and the thermal simulation and tissue safety after implantation are not fully considered. Summary of the Invention
[0005] To overcome the deficiencies of the prior art, this invention provides a method for preparing a photothermal responsive gold nanorod-bone cement composite material that combines mechanical stability and photothermal response characteristics, effectively solves the problem of precise temperature control in local sub-high temperatures of bone metastases, efficiently induces immunogenic cell death (ICD), overcomes the inhibition of immunogenic cell death by the bone microenvironment, and activates a systemic anti-tumor immune response.
[0006] This invention is achieved through the following technical solution: A method for preparing a photothermally responsive gold nanorod-bone cement composite material, characterized by comprising the following steps: 1) Preparation of gold nanorods: (1) Synthesis of gold seed solution Take 5.00 ± 0.05 mL of cetyltrimethylammonium bromide solution with a concentration of 0.18-0.22 M in a water bath at 25 ± 0.5 °C, inject 4.5-5.5 mL of chloroauric acid solution with a concentration of 0.45-0.55 mM; use a pipette to quickly inject 0.55-0.65 mL of ice-precooled sodium borohydride solution with a concentration of 9.5-10.5 mM within 2 seconds, immediately start the vortex oscillator at a speed of 2400-2600 rpm for vigorous mixing for 115-125 seconds, then form a uniform brown-yellow colloid, and then stand for 170-190 minutes at 25 ± 1 °C in the dark to allow the excess reducing agent to fully decompose; (2) Growth solution construction Add 1.5-2.0 mL of silver nitrate solution with a concentration of 3.8-4.2 mM to 45-55 mL of cetyltrimethylammonium bromide solution with a concentration of 0.18-0.22 M, and stand for 12-18 minutes at 25 ± 1 °C in the dark; add 45-55 mL of chloroauric acid diluent with a concentration of 0.8-1.2 mM, then inject 0.68-0.72 mL of ascorbic acid solution with a concentration of 78.5-79 mM, and gently shake until the solution is transparent white; use electrodes calibrated with pH 1.68 and 4.01 standard buffers, respectively, to add 0.09-0.11 M hydrochloric acid dropwise to accurately adjust the pH to 2.70 ± 0.03, which is the growth solution; (3) Nanorod growth control Inject 11.8-12.2 μL of gold seed solution into the above growth solution, and use a polytetrafluoroethylene stirring rod to gently stir at a speed of 55-65 rpm for 25-35 seconds; stand for 170-190 minutes in a dark environment at 25 ± 1 °C to obtain a gray-blue colloid solution with a surface plasmon resonance characteristic peak at 808 ± 3 nm; (4) Gradient centrifugation purification First centrifugation: centrifuge at 7500-8500 rpm for 12-18 minutes at 25 ± 1 °C to remove spherical by-products; Second centrifugation: centrifuge the supernatant obtained from the first centrifugation at 11500-12500 rpm for 18-22 minutes at 4 ± 0.5 °C, collect the obtained precipitate and resuspend it with cetyltrimethylammonium bromide solution with a concentration of 0.09-0.11 mM; then treat with 38-42 W water bath ultrasonic for 28-32 seconds to make the particles fully and uniformly dispersed, and obtain a single-dispersed gold nanorod dispersion.
[0007] (5) Surface ligand exchange (thiol-PEG) First, perform surfactant replacement pretreatment: Take 0.9-1.1 mL of the above gold nanorod dispersion, add 9-11 times the volume of phosphate buffer solution to remove free PEG based on the volume of the filtered sample liquid;
[0008] Secondly, the thiol-PEG is covalently grafted According to the ratio of 4.5-5.5 μg of mercapto polyethylene glycol carboxyl per 1 OD 808 of gold nanorods, slowly add 9.5-10.5 mg / mL deoxyphosphate buffer solution to the above gold nanorod dispersion; then oscillate at 180-220 rpm under nitrogen protection, 25±1℃, light protection for 11.5-12.5 hours to complete the ligand exchange; Third, sterile purification Use a tangential flow filtration system to replace the buffer, then: First stage: use 9-11 times the volume of phosphate buffer to wash and remove free PEG based on the volume of the filtered sample liquid; Second stage: use 4.5-5.5 times the volume of water for injection to wash; Finally, sterilely dispense through a 0.21-0.23 μm PVDF filter membrane to obtain the final product of polyethylene glycol modified gold nanorods; 2) Disperse the above polyethylene glycol modified gold nanorods in the liquid monomer of polymethyl methacrylate to form a mixture, wherein the weight percentage of polyethylene glycol modified gold nanorods in the above mixture is 0.9-1.1%; use an ultrasonic homogenization device to treat at room temperature for 25-35 minutes to ensure uniform dispersion of the nanorods in the liquid phase; introduce the mixed above mixture into a predetermined amount of polymethyl methacrylate powder, wherein the weight ratio of polymethyl methacrylate powder to liquid monomer of polymethyl methacrylate is 1.8-2.2:1; quickly and thoroughly mix with a sterile stirring rod until the mixture is uniform in state and moderate in viscosity; 3) Solidification and shaping: after injecting the above mixture into a predetermined position or mold, keep it stable at physiological temperature, and it will solidify and shape within 3-5 minutes.
[0009] The preferred scheme comprises the following steps: 1) Preparation of gold nanorods: (1) Gold seed solution synthesis Take 5.00 mL concentration of 0.2 M cetyltrimethylammonium bromide solution in a water bath at 25 ℃, inject 5.0 mL concentration of 0.5 mM chloroauric acid solution; using a syringe in 2 seconds fast injection of 0.60 mL of ice pre-cooled 10 mM sodium borohydride solution, immediately start vortex mixer at 2500 rpm speed mixing 120 seconds, form a uniform brown yellow colloid, then at 25 ℃, avoid light conditions for 180 minutes, make the excess reductant fully decomposed; (2) Growth solution construction In 50 mL concentration of 0.2 M cetyltrimethylammonium bromide solution, add 1.80 mL concentration of 4 mM silver nitrate solution, 25 ℃ conditions avoid light for 15 minutes; add 50 mL concentration of 1 mM chloroauric acid diluent, then inject 0.70 mL concentration of 78.8 mM ascorbic acid solution, gentle shaking until the solution is transparent white; using the electrode calibrated by pH 1.68 and 4.01 standard buffer, add 0.1 M hydrochloric acid drop by drop, adjust the pH to 2.70 accurately, which is the growth solution; (3) Nanorod growth control Into the above growth solution, inject 12.0 μL gold seed solution, use a polytetrafluoroethylene stirring rod to gently stir at 60 rpm for 30 seconds; in a 25 ℃ light-free environment, stand for 180 minutes to obtain a gray-blue colloidal solution with surface plasmon resonance characteristic peak at 808±3 nm; (4) Gradient centrifugation purification First centrifugation: centrifuge at 8000 rpm for 15 minutes at 25 ℃ to remove spherical by-products; Second centrifugation: centrifuge the supernatant obtained by the first centrifugation at 12000 rpm for 20 minutes at 4 ℃, collect the obtained precipitate and resuspend it with 0.1 mM cetyltrimethylammonium bromide solution; then treat it with 40 W water bath ultrasonic for 30 seconds to make the particles fully and uniformly dispersed, thus obtaining a monodispersed gold nanorod dispersion.
[0010] (5) Surface ligand exchange (thiol-PEG) First, surfactant replacement pretreatment: Take 1.0 mL of the above gold nanorod dispersion, add 1:1 volume ratio of pre-cooled acetone at -20 ℃, vortex mix immediately and then centrifuge at 14000 rpm for 15 minutes at 4 ℃ to precipitate the gold nanorods and remove free cetyltrimethylammonium bromide; after discarding the supernatant, resuspend the precipitate with 1.0 mL of 0.5 mM cetyltrimethylammonium bromide solution to maintain the dispersion stability of the gold nanorods; repeat the above acetone precipitation and resuspension process twice.
[0011] Secondly, the mercapto-PEG is covalently grafted According to the proportion of 5.0 μg mercapto polyethylene glycol carboxyl per 1 OD 808 The deoxy phosphate buffer solution of 10 mg / mL is slowly added to the above gold nanorod dispersion liquid in the proportion of 5.0 μg mercapto polyethylene glycol carboxyl per 1 OD; then, under the conditions of nitrogen protection, 25 DEG C, and light protection, the liquid is oscillated at a speed of 200 revolutions per minute for 12 hours, so as to complete the ligand exchange; Thirdly, sterile purification The tangential flow filtration system is used for buffer replacement, and then: In the first stage, the free PEG is removed by flushing with 10 times the volume of phosphate buffer based on the volume of the filtered sample liquid; In the second stage, 5 times the volume of water for injection is flushed; Finally, the product polyethylene glycol modified gold nanorod is obtained by sterile packaging through a 0.22 μm PVDF filter membrane; 2) The above polyethylene glycol modified gold nanorod is dispersed in the liquid monomer of polymethyl methacrylate to form a mixture, wherein the weight percentage of the polyethylene glycol modified gold nanorod in the above mixture is 1.0%; an ultrasonic homogenization device is used to treat at room temperature for 30 minutes to ensure uniform dispersion of the nanorod in the liquid phase; the mixed above mixture is introduced into a predetermined amount of polymethyl methacrylate powder, and the weight ratio of the polymethyl methacrylate powder to the liquid monomer of polymethyl methacrylate is 2:1; a sterile stirring rod is used for rapid and thorough mixing until the mixture is uniform in state and moderate in viscosity; 3) Solidification forming: after the above mixture is injected into a predetermined position or mold, it is kept stable at a physiological temperature, and solidification forming can be completed within 3-5 minutes.
[0012] The chloroauric acid diluent and the ascorbic acid solution are prepared on demand.
[0013] When the water bath ultrasonic treatment is performed, the probe is 5 millimeters away from the bottom of the tube.
[0014] The beneficial effects of the present application are: The present application uniformly embeds gold nanorods (GNRs) into a polymethyl methacrylate (PMMA) cement matrix, solving the problems of easy migration of free nanorods and poor bone targeting.
[0015] The present application can precisely control the local temperature and avoid damage to the surrounding tissues.
[0016] The present application can effectively induce immunogenic cell death (ICD) and activate the anti-tumor immune response.
[0017] The present application can provide sufficient mechanical support and ensure the stability after implantation.
[0018] The application can optimize the bone microenvironment, promote bone repair and immune response. BRIEF DESCRIPTION OF DRAWINGS
[0019] The application will be further described below with reference to the drawings: Figure 1 is a nanorod size electron microscope image; Figure 2 is an ultraviolet-visible spectrum (UV-Vis) image; Figure 3 is a bone cement compressive strength graph of different load nanorod concentrations; Figure 4 is a heating curve graph of different concentrations and with or without nanorods; Figure 5 is an ICD factor HMGB1 graph obtained by qRT-pcr experiment; Figure 6 is an ICD factor CRT graph obtained by qRT-pcr experiment; Figure 7 is a protein band graph of HMGB1 and CRT obtained by wb experiment; Figure 8 is a CRT protein band quantification graph obtained by wb experiment; Figure 9 is a HMGB1 protein band quantification graph obtained by wb experiment; Figure 10 is a survival rate graph of different concentrations tested by CCK-8; Figure 11 is an inhibition rate graph of different concentrations tested by CCK-8. DETAILED DESCRIPTION
[0020] In order to more clearly understand the above-mentioned purposes, features and advantages of the present application, the present application will be further described in detail below through specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.
[0021] In the following description, many specific details are set forth in order to provide a thorough understanding of the present application, however, the present application can also be implemented in other ways different from those described herein, therefore, the protection scope of the present application is not limited by the specific embodiments disclosed below.
[0022] The preparation method of the photothermal response type gold nanorod-cement composite material, Example 1 comprises the following steps: 1) Preparation of gold nanorods: (1) Gold seed solution synthesis Take 4.95 mL of 0.22 M concentration of cetyltrimethylammonium bromide solution (CTAB) in a 24.5 °C water bath, inject 5.5 mL of 0.45 mM concentration of chloroauric acid solution (diluted from chloroauric acid stock solution (HAuCl4·3H2O) (10 mM)); use a syringe to quickly inject 0.65 mL of ice-precooled 9.5 mM concentration of sodium borohydride (NaBH4) solution in 2 seconds, immediately start the vortex oscillator at 2600 rpm to mix vigorously for 115 seconds, form a uniform brown-yellow colloid, then stand at 26 °C in the dark for 170 minutes to allow the excess reducing agent to decompose completely; (2) Growth solution construction In 55 mL of 0.18 M concentration of cetyltrimethylammonium bromide solution, add 2.0 mL of 3.8 mM concentration of silver nitrate solution, stand at 26 °C in the dark for 12 minutes; add 55 mL of 0.8 mM concentration of chloroauric acid diluent (prepared from 10 mM stock solution), then inject 0.72 mL of 78.5 mM concentration of ascorbic acid solution (freshly prepared), gently shake until the solution is transparent white; using electrodes calibrated with pH 1.68 and 4.01 standard buffers, add 0.11 M hydrochloric acid dropwise to accurately adjust the pH to 2.67, which is the growth solution; (3) Nanorod growth control Inject 11.8 μL of gold seed solution into the above growth solution, use a polytetrafluoroethylene stirring rod to gently stir at 65 rpm for 25 seconds; stand in a 26 °C dark environment for 170 minutes to obtain a gray-blue colloid solution with a surface plasmon resonance (LSPR) characteristic peak at 808±3 nm; (4) Gradient centrifugation purification First centrifugation: centrifuge at 7500 rpm for 18 minutes at 26 °C to remove spherical by-products; Second centrifugation: centrifuge the supernatant obtained from the first centrifugation at 12500 rpm for 18 minutes at 3.5 °C, collect the obtained precipitate, and resuspend it with 0.11 mM concentration of cetyltrimethylammonium bromide solution to maintain the stability of the nanorod dispersion; then treat with 38 W water bath ultrasonic for 32 seconds (ice bath condition, probe distance from tube bottom 5 mm), make the particles fully and uniformly dispersed, the obtained dispersion liquid is detected by ultraviolet-visible spectrophotometer, the optical density (OD808) at 808 nm is 10.0±0.2, indicating that a monodispersed gold nanorod dispersion liquid is obtained; (5) Surface ligand exchange (thiol-PEG) First, surfactant replacement pretreatment Take 0.9 mL of the above gold nanorod dispersion, add 1:1 volume ratio of pre-cooled acetone at -18°C, vortex mix immediately and centrifuge at 14500 rpm for 12 minutes at 3.5°C to precipitate the gold nanorods and remove free cetyltrimethylammonium bromide (CTAB); after discarding the supernatant, resuspend the precipitate with 1.1 mL of 0.45 mM cetyltrimethylammonium bromide (CTAB) solution to maintain short-term dispersion stability of the nanorods; then repeat the above "acetone precipitation-CTAB resuspension" step twice to further reduce CTAB residues and provide conditions for subsequent PEG (polyethylene glycol) covalent grafting; Secondly, covalent grafting of thiol-PEG Add 9.5 mg / mL deoxyphosphate buffer solution (pH 7.5) to the above gold nanorod dispersion at a ratio of 5.5 μg of thiol polyethylene glycol carboxyl (HS-PEG-COOH) per 1 OD 808 Unit, then oscillate at 180 rpm for 12.5 hours under nitrogen protection, 26°C, and light-avoiding conditions to complete ligand exchange; Third, sterile purification Use a tangential flow filtration system (100 kDa molecular weight cut-off) for buffer replacement, then: First stage: use 9 volumes of phosphate buffer (PBS) (pH 7.3) to flush and remove free PEG based on the filtered sample liquid volume; Second stage: use 5.5 volumes of water for injection to flush; Finally, sterilely dispense through a 0.21 m PVDF filter membrane to obtain the final product, PEG-modified gold nanorods; 2) Disperse the above PEG-modified gold nanorods in the liquid monomer of polymethyl methacrylate to form a mixture, with the PEG-modified gold nanorods accounting for 1.1% of the weight percentage of the above mixture; use an ultrasonic homogenization device (300 W power) to treat at room temperature for 25 minutes to ensure uniform dispersion of the nanorods in the liquid phase; introduce the mixed above mixture into a predetermined amount of polymethyl methacrylate powder, with a weight ratio of polymethyl methacrylate powder to liquid monomer of polymethyl methacrylate of 2.2:1; quickly and thoroughly mix with a sterile stirring rod until the mixture is uniform in state and moderate in viscosity; 3) Solidification and shaping: after injecting the above mixture into a predetermined position or mold, keep it stable at physiological temperature (36.5-37.5°C) to allow free radical polymerization to occur, and solidify and shape within 3 minutes to obtain a composite solid with a dense structure and stable mechanical properties.
[0023] Example Two 1) Preparation of gold nanorods: (1) Gold seed solution synthesis Take 5.05 mL of 0.18 M concentration of cetyltrimethylammonium bromide solution (CTAB) in a 25.5 °C water bath, inject 4.5 mL of 0.55 mM concentration of chloroauric acid solution (HAuCl4·3H2O) (diluted from chloroauric acid stock solution (10 mM)), use a pipette to quickly inject 0.55 mL of ice-precooled 10.5 mM concentration of sodium borohydride (NaBH4) solution within 2 seconds, immediately start the vortex oscillator at 2400 rpm to mix vigorously for 125 seconds, form a uniform brown-yellow colloid, then stand at 24 °C in the dark for 190 minutes to allow the excess reducing agent to decompose completely; (2) Growth solution construction Add 1.5 mL of 4.2 mM concentration of silver nitrate solution to 45 mL of 0.22 M concentration of cetyltrimethylammonium bromide solution, stand at 24 °C in the dark for 18 minutes; add 45 mL of 1.2 mM concentration of chloroauric acid diluent (freshly prepared from 10 mM stock solution), then inject 0.68 mL of 79 mM concentration of ascorbic acid solution (freshly prepared), gently shake until the solution turns transparent white; using electrodes calibrated with pH 1.68 and 4.01 standard buffers, add 0.09 M hydrochloric acid dropwise to accurately adjust the pH to 2.73, which is the growth solution; (3) Nanorod growth control Inject 12.2 μL of gold seed solution into the above growth solution, use a polytetrafluoroethylene stirring rod to gently stir at 55 rpm for 35 seconds; stand in a 24 °C dark environment for 190 minutes to obtain a gray-blue colloidal solution with a surface plasmon resonance (LSPR) characteristic peak at 808 ± 3 nm; (4) Gradient centrifugation purification First centrifugation: centrifuge at 8500 rpm for 12 minutes at 24 °C to remove spherical by-products; Second centrifugation: centrifuge the supernatant obtained from the first centrifugation at 11500 rpm for 22 minutes at 4.5 °C, collect the obtained precipitate, and resuspend it with 0.09 mM concentration of cetyltrimethylammonium bromide solution to maintain the stability of the nanorod dispersion; then treat with a 42 W water bath ultrasonic for 28 seconds (ice bath condition, probe distance from tube bottom 5 mm), to make the particles fully and uniformly dispersed, the obtained dispersion was detected by ultraviolet-visible spectrophotometer, the optical density (OD808) at 808 nm was 10.0 ± 0.2, indicating that a monodispersed gold nanorod dispersion was obtained; (5) Surface ligand exchange (thiol-PEG) First, surfactant replacement pretreatment Take 1.1 mL of the above gold nanorod dispersion, add 1:1 volume ratio of pre-cooled acetone at -19°C, vortex mix immediately and then centrifuge at 13500 rpm for 18 minutes at 4.5°C to precipitate the gold nanorods and remove free cetyltrimethylammonium bromide (CTAB); after discarding the supernatant, resuspend the precipitate with 0.9 mL of 0.55 mM cetyltrimethylammonium bromide (CTAB) solution to maintain short-term dispersion stability of the nanorods; then repeat the above "acetone precipitation-CTAB resuspension" step twice to further reduce CTAB residues and provide conditions for subsequent PEG covalent grafting; Second, covalent grafting of thiol-PEG Add 10.5 mg / mL deoxyphosphate buffer solution (pH 7.3) to the above gold nanorod dispersion at a ratio of 4.5 μg of thiol polyethylene glycol carboxyl (HS-PEG-COOH) per 1 OD 808 Then, under nitrogen protection, oscillate at 220 rpm for 11.5 hours at 24°C in the dark to complete ligand exchange; Third, sterile purification Use a tangential flow filtration system (100 kDa molecular weight cut-off) for buffer replacement, and then: First stage: flush with 11 volumes of phosphate buffer (PBS) (pH 7.5) based on the liquid volume of the filtered sample to remove free PEG; Second stage: flush with 4.5 volumes of water for injection; Finally, sterilely dispense through a 0.23 μm PVDF filter membrane to obtain the final product, PEG-modified gold nanorods; 2) Disperse the above PEG-modified gold nanorods in the liquid monomer of polymethyl methacrylate to form a mixture, with the PEG-modified gold nanorods accounting for 0.9% of the weight percentage of the above mixture; use an ultrasonic homogenization device (300W power) to treat at room temperature for 35 minutes to ensure uniform dispersion of the nanorods in the liquid phase; introduce the mixed above mixture into a predetermined amount of polymethyl methacrylate powder, with a weight ratio of polymethyl methacrylate powder to liquid monomer of polymethyl methacrylate of 1.8:1; mix quickly and thoroughly with a sterile stirring rod until the mixture is uniform in state and moderate in viscosity; 3) Solidification and shaping: after injecting the above mixture into a predetermined position or mold, keep it stable at physiological temperature (36.5-37.5°C) to allow free radical polymerization to occur, and solidify and shape within 5 minutes to obtain a composite solid with a dense structure and stable mechanical properties.
[0024] Example Three 1) Preparation of gold nanorods: (1) Gold seed solution synthesis Take 5.00 mL of 0.2 M cetyltrimethylammonium bromide solution (CTAB) in a 25 °C water bath, inject 5.0 mL of 0.5 mM chloroauric acid solution (diluted from 10 mM HAuCl4·3H2O stock solution); use a syringe to quickly inject 0.60 mL of 10 mM sodium borohydride (NaBH4) solution pre-cooled with ice within 2 seconds, immediately start the vortex oscillator at 2500 rpm to mix vigorously for 120 seconds, form a uniform brown-yellow colloid, then stand at 25 °C in the dark for 180 minutes to allow the excess reducing agent to decompose completely; (2) Growth solution construction Add 1.80 mL of 4 mM silver nitrate solution to 50 mL of 0.2 M cetyltrimethylammonium bromide solution, stand at 25 °C in the dark for 15 minutes; add 50 mL of 1.0 mM chloroauric acid diluent (freshly prepared from 10 mM stock solution), then inject 0.70 mL of 78.5-79 mM ascorbic acid solution (freshly prepared), gently shake until the solution turns transparent white; use electrodes calibrated with pH 1.68 and 4.01 standard buffers to add 0.1 M hydrochloric acid dropwise to adjust the pH to 2.70 precisely, which is the growth solution; (3) Nanorod growth control Inject 12.0 μL of gold seed solution into the above growth solution, use a polytetrafluoroethylene stirring rod to gently stir at 60 rpm for 30 seconds; stand in a 25 °C dark environment for 180 minutes to obtain a gray-blue colloidal solution with a surface plasmon resonance (LSPR) characteristic peak at 808±3 nm; (4) Gradient centrifugation purification First centrifugation: centrifuge at 8000 rpm for 15 minutes at 25 °C to remove spherical by-products; Second centrifugation: centrifuge the supernatant obtained from the first centrifugation at 12000 rpm for 20 minutes at 4 °C, collect the precipitate, and resuspend it with 0.1 mM cetyltrimethylammonium bromide solution to maintain the stability of the nanorod dispersion; then treat with 40W water bath ultrasonic for 30 seconds (ice bath condition, probe distance from tube bottom 5 mm), make the particles fully and uniformly dispersed, the obtained dispersion is detected by ultraviolet-visible spectrophotometer, the optical density (OD808) at 808 nm is 10.0±0.2, indicating that the monodisperse gold nanorod dispersion is obtained; (5) Surface ligand exchange (thiol-PEG) First, surfactant replacement pretreatment Take 1.0 mL of the above gold nanorod dispersion and add acetone pre-cooled at -20℃ at a volume ratio of 1:1. After vortex mixing, immediately centrifuge at 14000 rpm for 15 minutes at 4℃ to precipitate the gold nanorods and remove free hexadecyltrimethylammonium bromide (CTAB). After discarding the supernatant, resuspend the precipitate in 1.0 mL of 0.5 mM hexadecyltrimethylammonium bromide (CTAB) solution to maintain the short-term dispersion stability of the nanorods. Then repeat the above "acetone precipitation-CTAB resuspension" step twice to further reduce CTAB residue and provide conditions for subsequent PEG covalent grafting. Secondly, thiol-PEG covalent grafting per 1 OD 808 Add 10 mg / mL of deoxyphosphate buffer (pH 7.4) to the above gold nanorod dispersion at a ratio of 5 μg of mercaptopolyethylene glycol carboxyl group (HS-PEG-COOH); then shake at 200 rpm for 12 hours under nitrogen protection, 25°C, and in the dark to complete ligand exchange. Third, aseptic purification Buffer replacement was performed using a tangential flow filtration system (100 kDa molecular weight cutoff), followed by: First stage: Based on the volume of the filtered sample liquid, rinse with 10 times the volume of phosphate-buffered saline (PBS) (pH 7.4) to remove free PEG; Second stage: Rinse with 5 times the volume of water for injection; Finally, the product was aseptically dispensed through a 0.22 μm PVDF filter membrane to obtain polyethylene glycol modified gold nanorods. 2) The above-mentioned polyethylene glycol modified gold nanorods were dispersed in the liquid phase monomer of polymethyl methacrylate to form a mixture, wherein the weight percentage of polyethylene glycol modified gold nanorods in the mixture was 1%; the mixture was treated with an ultrasonic homogenizer (300W power) at room temperature for 30 minutes to ensure that the nanorods were uniformly dispersed in the liquid phase; the mixed mixture was introduced into a predetermined amount of polymethyl methacrylate powder, wherein the weight ratio of polymethyl methacrylate powder to the liquid phase monomer of polymethyl methacrylate was 2:1; the mixture was quickly and thoroughly mixed with a sterile stirring rod until the mixture was homogeneous and had a suitable viscosity; 3) Curing and molding: After the above mixture is injected into the predetermined part or mold, it is kept in a stable position under physiological temperature (36.5-37.5℃) to undergo free radical polymerization reaction and be cured and molded within 4 minutes to obtain a composite solid with dense structure and stable mechanical properties.
[0025] verify: I. For exampleFigure 1 As shown, the size of the nanorods was observed by electron microscopy, which confirmed that the size of the nanorods was the best aspect ratio.
[0026] II. As shown in Figure 2 As shown, the modified nanorods and unmodified nanorods, it can be confirmed that the best absorbance of the nanorods is between 795-810 nm, which proves that the use of 808 nm near-infrared laser can make the nanorods warm up to the best.
[0027] III. As shown in Figure 3 As shown, the mechanical test of bone cement, the test standard is ISO 5833, through the standard mechanical test of bone cement, it is proved that the 1.0% nanorod mixed into the bone cement has no or little change on the compression resistance of the bone cement.
[0028] IV. As shown in Figure 4 As shown, the thermal performance comparison of different concentrations and with or without nanorods, the nanorods made at different concentrations and with or without nanorods were tested for warming up, and the warming efficiency and stability were ensured after five tests.
[0029] V. Immunogenic cell death (ICD), experimental indicators: calreticulin (CRT), high mobility group protein B1 (HMGB1): As shown in Figure 5 and Figure 6 The cell model selected is the mouse breast cancer cell line (4T1); The light condition is 808 nm laser (1.0 W / cm²), irradiation for 10 min (maintained at about 42°C); it is proved that the material after warming up can indeed cause ICD in 4T1 cells at different temperatures, and then RNA is extracted for polymerase chain reaction (PCR).
[0030] As shown in Figures 7 to 9 , Model establishment: the mouse breast cancer cell line (4T1) was cultured to the logarithmic growth phase, then 6-8 week old BALB / c mice were selected, the mice were anesthetized and fixed, and a 30 gauge needle was used to punch through the middle of the patellar ligament into the femur, and the cell number was 10ul 1x10 6 After the injection of the mouse breast cancer cell line (4T1) was completed, the artificial light cycle was observed, and the mouse state was observed closely, and the mice grew to 50mm 3 Start intervention; Material implantation: drill hole injection of GNRs-cement composite (volume 10 μL); Photothermal therapy: observe the state of the mice after implantation, and irradiate within 1-2 days, 808 nm laser irradiation (1.0 W / cm², 10 min / time, once a day); Results: After 12 days of treatment, the mice were sacrificed by carbon dioxide asphyxiation, then the femur tumor was taken out, the protein was extracted, then electrophoresis, membrane transfer, blocking, incubation of antibody, and then development were carried out to observe the target protein expression of each sample to verify the optimal condition 42℃, 10min experiment.
[0031] Six, biocompatibility and test material toxicity: cell viability detection kit (CCK-8): As Figures 10 to 11 shown, in order to ensure the biological safety of the material, the cell viability detection kit (CCK-8) experiment was carried out, the mouse osteoblast cell line (MC3T3-E1) cells were selected for culture to the logarithmic growth phase, then after treatment, the cells were counted by using a cell counting plate, 7ml of non-centrifuged cell liquid was taken, centrifuged, the supernatant was discarded, the cells were obtained, resuspended with serum-containing culture medium, then 96-well plates were plated, 100ul of cell-containing culture medium was added to each well, after 24h of culture, the number of cells was observed under a microscope, the culture medium was removed, and serum-free culture medium was added, because the bone cement is a medical grade and has been verified to have good biocompatibility, only the concentration of nanorods was changed, the culture medium was aspirated, then the concentration was 0.15, 0.08, 0.04, 0.02, 0.01, 0.005, and 100ul of serum-free culture medium was added, the test time was 24h, and Figure 6 , the results showed that when the concentration was 0.15, cell inhibition began to occur, and the survival rate did not decrease too much, therefore, it was concluded that the safest concentration was between 0.08 and 0.1.
[0032] In the present application, the terms "first", "second", "third" are only for the purpose of description, and cannot be understood as indicating or implying relative importance; the term "a plurality of" refers to two or more, unless otherwise explicitly limited. The terms "mounting", "connecting", "connecting", "fixing" and the like should be understood in a broad sense, for example, "connecting" can be fixed connection, or detachable connection, or integrally connected; "connected" can be directly connected, or indirectly connected through an intermediate medium. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.
[0033] In the description of the present application, it should be understood that the terms "up", "down", "left", "right", "front", "back" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the purpose of facilitating the description of the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the devices or units referred to must have a particular direction, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application.
[0034] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "a specific embodiment", and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in one or more embodiments or examples.
[0035] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.
[0036] In addition to the technical features described in the specification, the remaining technical features are known to those skilled in the art.
Claims
1. A method for preparing a photothermally responsive gold nanorod-bone cement composite material, characterized in that, Includes the following steps: 1) Preparation of gold nanorods: (1) Synthesis of gold seed solution Take 5.00±0.05 mL of 0.18-0.22 M hexadecyltrimethylammonium bromide solution and keep it at a constant temperature of 25±0.5℃ in a water bath. Inject 4.5-5.5 mL of 0.45-0.55 mM chloroauric acid solution. Using a pipette, quickly inject 0.55-0.65 mL of 9.5-10.5 mM sodium borohydride solution pre-cooled with ice within 2 seconds. Immediately start a vortex mixer and mix vigorously at a speed of 2400-2600 rpm for 115-125 seconds to form a homogeneous brownish-yellow colloid. Then, let it stand at 25±1℃ in the dark for 170-190 minutes to allow the excess reducing agent to decompose completely. (2) Construction of growth solution Add 1.5-2.0 mL of 3.8-4.2 mM silver nitrate solution to 45-55 mL of 0.18-0.22 M hexadecyltrimethylammonium bromide solution, and let stand in the dark at 25±1℃ for 12-18 minutes. Add 45-55 mL of 0.8-1.2 mM chloroauric acid diluent, followed by 0.68-0.72 mL of 78.5-79 mM ascorbic acid solution, and gently shake until the solution is transparent white. Using an electrode calibrated with standard buffer solutions at pH 1.68 and 4.01, add 0.09-0.11 M hydrochloric acid dropwise to precisely adjust the pH to 2.70±0.03, which is the growth solution. (3) Nanorod growth control Inject 11.8-12.2 μL of gold seed solution into the above growth solution, and gently stir with a polytetrafluoroethylene stir bar at a speed of 55-65 rpm for 25-35 seconds; let it stand in a light-protected environment at 25±1℃ for 170-190 minutes to obtain a gray-blue colloidal solution with a surface plasmon resonance characteristic peak located at 808±3 nm. (4) Gradient centrifugation purification First centrifugation: Centrifuge at 7500-8500 rpm for 12-18 minutes at 25±1℃ to remove spherical byproducts; Secondary centrifugation: The supernatant obtained from the first centrifugation is centrifuged at 11500-12500 rpm for 18-22 minutes at 4±0.5℃. The precipitate is collected and resuspended in a 0.09-0.11 mM cetyltrimethylammonium bromide solution. Then, it is ultrasonically treated in a 38-42 W water bath for 28-32 seconds to ensure that the particles are fully and uniformly dispersed, thus obtaining a monodisperse gold nanorod dispersion.
2. (5) Surface ligand exchange (thiol-PEG) First, a surfactant displacement pretreatment is performed: Take 0.9–1.1 mL of the above gold nanorod dispersion and add acetone pre-cooled at -18 to -20 °C at a volume ratio of 1:
1. Vortex mix and immediately centrifuge at 13,500–14,500 rpm for 12–18 minutes at 4 ± 0.5 °C to precipitate the gold nanorods and remove free hexadecyltrimethylammonium bromide. After discarding the supernatant, resuspend the precipitate in 0.9–1.1 mL of a 0.45–0.55 mM hexadecyltrimethylammonium bromide solution to maintain the dispersion stability of the gold nanorods. Repeat the above acetone precipitation and resuspension process twice.
3. Secondly, thiol-PEG covalent grafting per 1 OD 808 Add 9.5-10.5 mg / mL of deoxyphosphate buffer solution to the above gold nanorod dispersion at a ratio of 4.5-5.5 μg of mercaptopolyethylene glycol carboxyl group; then shake at 180-220 rpm for 11.5-12.5 hours under nitrogen protection, 25±1℃, and in the dark to complete ligand exchange. Third, aseptic purification Buffer replacement was performed using a tangential flow filtration system, and then... First stage: Based on the volume of the filtered sample liquid, rinse with 9-11 times the volume of phosphate buffer to remove free PEG; Second stage: Rinse with 4.5-5.5 times the volume of water for injection; Finally, the product was aseptically dispensed through a 0.21-0.23 μm PVDF filter membrane to obtain polyethylene glycol modified gold nanorods. 2) Disperse the above-mentioned polyethylene glycol-modified gold nanorods in the liquid phase monomer of polymethyl methacrylate to form a mixture, wherein the weight percentage of polyethylene glycol-modified gold nanorods in the mixture is 0.9-1.1%; treat with an ultrasonic homogenizer at room temperature for 25-35 minutes to ensure that the nanorods are uniformly dispersed in the liquid phase; introduce the above-mentioned mixture into a predetermined amount of polymethyl methacrylate powder, wherein the weight ratio of polymethyl methacrylate powder to the liquid phase monomer of polymethyl methacrylate is 1.8-2.2:1; mix quickly and thoroughly with a sterile stirring rod until the mixture is homogeneous and has a suitable viscosity; 3) Curing and shaping: After injecting the above mixture into the predetermined part or mold, keep the position stable under physiological temperature conditions and cure and shape within 3-5 minutes.
4. The preparation method of the photothermal responsive gold nanorod-bone cement composite material according to claim 1, characterized in that, Includes the following steps: 1) Preparation of gold nanorods: (1) Synthesis of gold seed solution Take 5.00 mL of 0.2 M hexadecyltrimethylammonium bromide solution and keep it at a constant temperature of 25 °C in a water bath. Add 5.0 mL of 0.5 mM chloroauric acid solution. Use a pipette to quickly add 0.60 mL of 10 mM sodium borohydride solution that has been pre-cooled with ice within 2 seconds. Immediately start a vortex mixer and mix vigorously at a speed of 2500 rpm for 120 seconds to form a homogeneous brownish-yellow colloid. Then let it stand at 25 °C in the dark for 180 minutes to allow the excess reducing agent to decompose completely. (2) Construction of growth solution Add 1.80 mL of 4 mM silver nitrate solution to 50 mL of 0.2 M hexadecyltrimethylammonium bromide solution, and let stand at 25 °C in the dark for 15 minutes; add 50 mL of 1 mM chloroauric acid diluent, followed by 0.70 mL of 78.8 mM ascorbic acid solution, and gently shake until the solution is transparent white; using an electrode calibrated with standard buffer solutions at pH 1.68 and 4.01, add 0.1 M hydrochloric acid dropwise to precisely adjust the pH to 2.70, which is the growth solution; (3) Nanorod growth control 12.0 μL of gold seed solution was injected into the above growth solution, and the mixture was gently stirred at 60 rpm for 30 seconds using a polytetrafluoroethylene stir bar. The mixture was then allowed to stand in a light-protected environment at 25°C for 180 minutes to obtain a gray-blue colloidal solution with a surface plasmon resonance characteristic peak located at 808±3 nm. (4) Gradient centrifugation purification First centrifugation: Centrifuge at 8000 rpm for 15 minutes at 25°C to remove spherical byproducts; Secondary centrifugation: The supernatant obtained from the first centrifugation was centrifuged at 12,000 rpm for 20 minutes at 4°C. The precipitate was collected and resuspended in a 0.1 mM cetyltrimethylammonium bromide solution. Subsequently, the mixture was ultrasonically treated in a 40 W water bath for 30 seconds to ensure that the particles were fully and uniformly dispersed, thus obtaining a monodisperse gold nanorod dispersion.
5. (5) Surface ligand exchange (thiol-PEG) First, a surfactant displacement pretreatment is performed: Take 1.0 mL of the above gold nanorod dispersion and add acetone pre-cooled at -20℃ at a volume ratio of 1:
1. After vortex mixing, immediately centrifuge at 14000 rpm for 15 minutes at 4℃ to precipitate the gold nanorods and remove free hexadecyltrimethylammonium bromide. After discarding the supernatant, resuspend the precipitate in 1.0 mL of 0.5 mM hexadecyltrimethylammonium bromide solution to maintain the dispersion stability of the gold nanorods. Repeat the above acetone precipitation and resuspension process twice.
6. Secondly, thiol-PEG covalent grafting per 1 OD 808 Add 10 mg / mL of deoxyphosphate buffer solution to the above gold nanorod dispersion at a ratio of 5.0 μg / mL mercaptopolyethylene glycol carboxyl group; then shake at 200 rpm for 12 hours under nitrogen protection, 25°C, and in the dark to complete ligand exchange. Third, aseptic purification Buffer replacement was performed using a tangential flow filtration system, and then... First stage: Based on the volume of the filtered sample liquid, rinse with 10 times the volume of phosphate buffer to remove free PEG; Second stage: Rinse with twice the volume of water for injection; Finally, the product was aseptically dispensed through a 0.22 μm PVDF filter membrane to obtain polyethylene glycol modified gold nanorods. 2) The above-mentioned polyethylene glycol modified gold nanorods are dispersed in the liquid phase monomer of polymethyl methacrylate to form a mixture, wherein the weight percentage of polyethylene glycol modified gold nanorods in the mixture is 1.0%; the mixture is treated with an ultrasonic homogenizer at room temperature for 30 minutes to ensure that the nanorods are uniformly dispersed in the liquid phase; the mixed mixture is introduced into a predetermined amount of polymethyl methacrylate powder, wherein the weight ratio of polymethyl methacrylate powder to the liquid phase monomer of polymethyl methacrylate is 2:1; the mixture is quickly and thoroughly mixed with a sterile stirring rod until the mixture is homogeneous and has a suitable viscosity; 3) Curing and shaping: After injecting the above mixture into the predetermined part or mold, keep the position stable under physiological temperature conditions and cure and shape within 3-5 minutes.
7. The method for preparing the photothermal responsive gold nanorod-bone cement composite material according to claim 1 or 2, characterized in that, Both the chloroauric acid diluent and the ascorbic acid solution were prepared fresh for each use.
8. The method for preparing the photothermal responsive gold nanorod-bone cement composite material according to claim 1 or 2, characterized in that, When performing water bath ultrasonic treatment, the probe should be 5 mm away from the bottom of the tube.