CN111097042A - Azide group modified Janus nano particle and preparation method and application thereof - Google Patents
Azide group modified Janus nano particle and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses azide group modified Janus nanoparticles and a preparation method and application thereof. The invention takes azide group modified silicon dioxide nano particles with different particle diameters as a main body, uses a Pickering emulsion method to prepare nano particles/paraffin composite spheres, and then uses azide group modified Janus nano particles. The Janus nano particle can be used for preparing a biologically-functionalized Janus nano platform, biologically-functionalized molecules such as functional proteins, antibodies or small molecular compounds are modified on the surface of the Janus nano particle modified by the azide groups, the biologically-functionalized Janus nano platform capable of being selectively and specifically combined with specific areas of different types of cells is constructed, and the biologically-functionalized Janus nano particle has higher biologically-functionalized modification efficiency. The prepared Janus nano platform can effectively avoid the mutual interference among different functional molecules and improve the targeting affinity; can be applied to biomedical research.
Description
Technical Field
The invention belongs to the technical field of biological materials, and particularly relates to azide group modified Janus nanoparticles and a preparation method and application thereof.
Background
The tumor immunotherapy is to enhance the immunity against tumor by mobilizing the immune system of the body, thereby inhibiting and killing tumor cells. Compared with the traditional therapies such as chemotherapy, radiotherapy and the like, the tumor immunotherapy has the advantages of small toxic and side effect, strong broad spectrum, low tumor recurrence rate and the like. However, in patients with malignant tumors, tumor cells can inhibit the activity of immune cells in various ways, escape immune surveillance of the body, and enable immune responses to be in a tolerant state. Therefore, how to activate the host's anti-tumor immune system, block immune evasion mechanism, inhibit and activate key sites on the pathway becomes the current research hotspot. At present, various immune checkpoint inhibitors are approved by FDA and used for clinical treatment, but the application of the inhibitors is limited due to the low positive response rate of the clinical treatment, the enhancement of side effects caused by the combination of various immune checkpoints and the like. At present, researches show that the immune check point is combined with the nano material, so that the multivalent antibody delivery can be realized, the administration dosage and the systemic toxicity can be reduced, and the activity of related immune cells can be restored to realize efficient immune system response. However, the simultaneous and uniform modification of multiple targeting molecules on the nanomaterial can cause mutual interference between molecular signals, so that the affinity between the targeting molecules and cells is reduced, and the specific targeting ability is weakened. Therefore, there is an urgent need to develop a bio-functionalized anisotropic nanomaterial to improve the above problems.
Janus particles are anisotropic materials with different physical or chemical properties on both sides. Its asymmetry makes it possess special properties not possessed by homogeneous particles, and can be used as an ideal model for providing multifunctional modification and avoiding intermolecular mutual interference. At present, methods for preparing Janus particles mainly comprise a microfluid method, selective surface modification, a limited region phase separation method and the like, but due to the problems of high preparation cost, low yield, large prepared particle size and the like, related performance research and further application research of Janus particles are limited. Therefore, it is very urgent to develop a simple and effective method for making Janus particles have specific biological functions to prepare a large amount of biologically functionalized Janus particles, and to study the characteristics of the particles themselves and to develop their applications in the research of tumor immunotherapy.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a preparation method of azide group modified Janus nanoparticles. The method takes azide group modified silicon dioxide nano-particles with different particle sizes as a main body, uses a Pickering emulsion method to prepare nano-particles/paraffin composite spheres, and prepares the azide group modified Janus nano-particles by a series of click chemistry reactions or silane coupling agent modification and other methods.
The invention also aims to provide azide group modified Janus nanoparticles prepared by the preparation method.
The invention further aims to provide application of the azide group modified Janus nanoparticles. The azide group modified Janus nano particle can be used for preparing a biologically functionalized Janus nano platform, and is characterized in that the surface of the azide group modified Janus nano particle is modified with biologically functionalized molecules such as functional proteins, antibodies or small molecular compounds, and the like, so that the biologically functionalized Janus nano platform capable of being selectively and specifically combined with specific regions of different types of cells is constructed.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a preparation method of azide group modified Janus nanoparticles comprises the following steps:
(1) silicon dioxide (SiO) modified by azide group2-N3) Preparing the nano-particle/paraffin composite ball: SiO by using Pickering emulsion method2-N3Dispersing the nanoparticles in water, adding paraffin wax, didodecyldimethylammonium bromide (DDAB), and homogenizing to obtain SiO2-N3The nano particles are distributed in a single layer at a paraffin/water interface, and SiO is obtained by cooling and filtering2-N3Nanoparticle/paraffin composite spheres;
(2) preparation of azide group modified Janus nanoparticles: for the SiO obtained in the step (1)2-N3And chemically modifying the exposed surface of the nano particle/paraffin wax composite ball, and washing to remove the paraffin wax to obtain the azide group modified Janus nano particle.
SiO as described in step (1)2-N3The size of the nanoparticles is preferably 150-2000 nm.
The SiO2-N3The nanoparticles are preferably prepared by the following steps:
(A) preparing silicon dioxide nano-particles with different particle sizes;
(B) preparing silicon dioxide nano particles modified by chloropropyl silane coupling agent;
(C) preparing silicon dioxide nano particles modified by azide groups.
The SiO2-N3When the particle size of the nano-particles is 150-450 nm, the preparation method of the silicon dioxide nano-particles in the step (A) is preferably as follows: mixing and stirring ethanol, ultrapure water and ammonia water, adding tetraethoxysilane for reaction, centrifuging, removing the upper reaction liquid, cleaning and drying to obtain the silicon dioxide nano-particles.
The ammonia water and the tetraethoxysilane are preferably mixed according to a volume ratio of 53-95: 47-53, and more preferably 56-90: 50.
The ethanol is used as a reaction medium, and the dosage of the ethanol is preferably as follows: the volume ratio of the ethyl orthosilicate is 800-860: 47-53; more preferably according to it: and (4) calculating the proportion of the ethyl orthosilicate in the volume ratio of 830: 50.
The water is used as a reaction medium, and the amount of the water is preferably as follows: the volume ratio of the ethyl orthosilicate is 14-20: 47-53; more preferably according to it: the volume ratio of the ethyl orthosilicate is 17: 50.
The stirring time is preferably 2 hours.
The reaction conditions are preferably in an oil bath at 40 ℃ for overnight reaction.
The centrifugation condition is preferably 5000-10000 rpm for 8-12 min; more preferably, the centrifugation is carried out at 6000 to 9000rpm for 10 min.
The cleaning is preferably repeated by using ultrapure water and ethanol.
The drying is preferably vacuum drying.
The SiO2-N3When the particle size of the nanoparticle is 2000nm, the method for preparing the silica nanoparticle described in the step (a) is preferably:
① mixing ethanol, ultrapure water, potassium chloride, and ammonia water, and stirring;
② adding mixed solution of ethanol and ethyl orthosilicate, reacting, centrifuging, discarding the upper layer reaction solution, cleaning, and drying to obtain silicon dioxide nanoparticles.
The ethanol used in steps ① and ② is preferably used as a reaction medium, the ethanol used in step ① is preferably used in a ratio of 50 to 70mL of ethyl orthosilicate to 6.04g, more preferably in a ratio of 60mL of ethyl orthosilicate to 6.04g, and the ethanol used in step ② is preferably used in a ratio of 30 to 40mL of ethyl orthosilicate to 6.04g, more preferably in a ratio of 35mL of ethyl orthosilicate to 6.04 g.
The water used in step ① is preferably used as a reaction medium in a ratio of 6-8 mL/6.04 g of ethyl orthosilicate, and more preferably in a ratio of 7 mL/6.04 g of ethyl orthosilicate.
The amount of the potassium chloride used in step ① is preferably calculated according to a mass ratio of ethyl orthosilicate to ethyl orthosilicate of 0.1-0.3: 6.04, and more preferably according to a mass ratio of ethyl orthosilicate to 0.2: 6.04.
The amount of the ammonia water used in step ① is preferably calculated according to a ratio of 5-7 mL to 6.04g of tetraethoxysilane, and more preferably according to a ratio of 6mL to 6.04g of tetraethoxysilane.
The stirring conditions described in step ① are preferably stirring in a 40 ℃ oil bath for 1 hour.
The adding speed of the mixed solution in the step ② is preferably 5-6 mL/h, and more preferably 5.5 mL/h.
The centrifugation conditions are preferably 3000rpm for 10 minutes.
The cleaning is preferably repeated by using ultrapure water and ethanol.
The drying is preferably vacuum drying.
Step (B) is preferably as follows: and (B) dispersing the silicon dioxide nano-particles obtained in the step (A) in anhydrous toluene, then adding a chloropropyl silane coupling agent, reacting, and removing the unreacted silane coupling agent to obtain the chloropropyl silane coupling agent modified silicon dioxide nano-particles.
The anhydrous toluene is used as a reaction medium, and the volume consumption (mL) of the anhydrous toluene is preferably 40-60 times of the mass (g) of the silicon dioxide nanoparticles; more preferably 50 times.
The chloropropyl silane coupling agent in the step (B) is preferably 3-chloropropyl trimethoxy silane.
The volume consumption (mL) of the chloropropyl silane coupling agent is preferably 1.3-1.6 times of the mass (g) of the silicon dioxide nanoparticles; more preferably 1.375 to 1.5 times.
The dispersion is preferably placed in an ultrasonic cleaning machine for 30 minutes of ultrasonic dispersion.
The reaction conditions are preferably stirring reaction at 120 ℃ for 12 hours.
The unreacted silane coupling agent is removed by centrifugation with ethanol.
The centrifugation conditions are preferably 9000rpm for 10 minutes.
SiO in step (C)2-N3Preparing nano particles: dispersing the chloropropyl silane coupling agent modified silicon dioxide nano particles obtained in the step (B) in N, N-dimethylformamide, then adding sodium azide, reacting, centrifuging, discarding supernatant, and removing unreacted sodium azide to obtain SiO2-N3And (3) nanoparticles.
The volume consumption (mL) of the N, N-dimethylformamide is preferably 10-20 times of the mass (g) of the silica nanoparticles modified by the chloropropyl silane coupling agent; more preferably 15 times.
The mass of the sodium azide is preferably 0.3-0.5 times of that of the silicon dioxide nano particles modified by the chloropropyl silane coupling agent; more preferably 0.4 times.
The dispersion is preferably placed in an ultrasonic cleaning machine for 30 minutes of ultrasonic dispersion.
The reaction conditions are preferably stirring reaction at 80 ℃ for 24 hours.
The centrifugation condition is preferably 2000-10000 rpm for 8-12 minutes; more preferably 3000 to 9000rpm for 10 minutes.
The above-mentioned method for removing unreacted sodium azide is preferably repeated washing with ultrapure water.
The paraffin wax in the step (1) is preferably preheated and melted at 80 ℃.
SiO as described in step (1)2-N3The ratio of the nano particles to the paraffin is preferably 1: 5-1: 8 by mass.
The final volume of the water in the step (1) is preferably 2.0-2.5 mL; more preferably 2.0 to 2.4 mL.
The final concentration of DDAB in the step (1) is preferably 10-60 mg/L; more preferably 10 to 35 mg/L.
The homogenizing condition in the step (1) is preferably 8000-10000 rpm for 1-2 min; more preferably at 10000rpm for 2 min.
The cooling in step (1) is preferably slow cooling at room temperature.
The room temperature is preferably 10-30 ℃; more preferably 24 to 26 ℃.
The filtration in step (1) is preferably slow filter paper filtration.
The chemical modification described in step (2) is preferably a surface modification of the silica particles by a "click chemistry" reaction or a silane coupling agent modification method.
The solvent for washing and removing paraffin in the step (2) is preferably n-hexane or dichloromethane; more preferably n-hexane.
The azide group modified Janus nano particle is prepared by the method.
The azide group-modified Janus nanoparticles are preferably spherical with uniform size.
The particle size of the azide group modified Janus nano particle is preferably 150-2000 nm.
The azide group modified Janus nano particle is applied to preparation of a biologically functionalized Janus nano platform.
A biologically-functionalized Janus nano-platform is obtained by performing biological functional modification on the azide group-modified Janus nano-particles.
The azide group modified Janus nanoparticle is preferably subjected to biological functional modification, and the method comprises the following steps:
(I) activation of the molecule modified by biofunctionalization modification: uniformly mixing dimethyl sulfoxide solution containing a substance A with molecules modified by biological functionalization modification or PBS buffer solution containing the molecules modified by biological functionalization modification, incubating, ultrafiltering and centrifuging, and removing unreacted substance A to obtain activated molecules modified by biological functionalization modification;
(II) preparation of biologically functionalized Janus nano-platform: and (3) mixing the activated molecule modified by biological functionalization modification obtained in the step (I) with the Janus nano particles modified by the azide group, stirring, and centrifuging to obtain the biological functionalized Janus nano platform.
The molecule modified by the biological functionalization modification is preferably at least one of functional protein, antibody, small molecule compound and other biological functionalization molecules which can specifically target specific regions on the surfaces of different types of cells; more preferably at least one of horse ferritin, rhodamine, bovine serum albumin and Anti-SIRPa antibody.
The substance A in the step (I) is preferably at least one of DBCO-PEG4-Ma1 and DBCO-PEG4-NHS Ester.
The molar weight ratio of the substance A to the molecule modified by the biological functional modification in the step (I) is preferably 1: 50.
The incubation condition in the step (I) is preferably incubation for 12 hours at 4-30 ℃.
The centrifugation conditions described in step (I) are preferably 6000rpm for 20 minutes.
The stirring condition in the step (II) is preferably 4-30 ℃ for 1 hour.
The centrifugation conditions described in step (II) are preferably 8000rpm for 10 minutes.
The quantity of the molecules modified by the biological functional modification can be controlled by controlling the quality of the molecules modified by the biological functional modification.
The number of molecules modified by the biological functional modification increases with the mass of the molecules modified by the biological functional modification.
The molecular number modified by the biological functional modification is preferably SiO2-N3The number of grafts of the modified molecule by surface biofunctionalization.
The specific choice of the molecule modified by the biofunctionalization modification can be determined according to the specific biofunctionalization function to be realized.
The application of the biologically-functionalized Janus nano platform in biomedical research.
The biomedicine is preferably tumor immunotherapy.
Compared with the prior art, the invention has the following advantages and effects:
1. due to the anisotropic structure of the Janus particles, compared with homogeneous particles, the Janus nanoparticles prepared by the invention can effectively avoid the mutual interference among different functional molecules and improve the targeting affinity when multifunctional biological modification is carried out.
2. The Janus nano particles prepared by the invention take silicon dioxide nano particles modified by azide groups as subjects, carry out biological functional modification through efficient click chemistry reaction, and show more excellent biological functional modification efficiency.
3. The Janus nano-platform prepared by the invention has good biocompatibility, can be selectively and specifically combined with specific areas of tumor cells and immune cells, improves the interaction between the tumor cells and the immune cells, mobilizes the immune reaction of an organism, and expands the application of Janus particles in the aspect of biomedical research.
4. The invention can realize the controllable production of particle structure, size and biological function by controlling the feeding speed, the reactant concentration and the species of biological functionalized molecules.
Drawings
FIG. 1 is an infrared spectrum and a particle size distribution diagram of different nanoparticles; wherein (a) is 150nm SiO2And 150nmSiO2-N3An infrared spectrum of (1); (b) is SiO with different sizes2-N3Particle size distribution diagram of (c).
FIG. 2 shows SiO films of different sizes2-N3Transmission electron microscopy images of; wherein (a) is 150nm SiO2-N3Transmission electron microscopy images of; (b) is 450nm SiO2-N3Transmission electron microscopy images of; (c) is 2000nm SiO2-N3Transmission electron micrograph (D).
FIG. 3 is a scanning electron micrograph of modified silica particle/paraffin composite spheres based on azide groups of different sizes: wherein (a) is a scanning electron microscope image of the 150nm azide group modified silica particle/paraffin composite sphere when the scale is 10 mu m; (b) is a scanning electron microscope image of the 150nm azide group modified silicon dioxide particle/paraffin composite sphere when the scale is 1 mu m; (c) is a scanning electron microscope image of the silica particle/paraffin wax composite sphere modified by 450nm azide groups when the ruler is 10 mu m; (d) is a scanning electron microscope image of the silica particle/paraffin wax composite sphere modified by 450nm azide groups when the scale is 2 mu m; (e) is a scanning electron microscope image of the silica particle/paraffin wax composite sphere modified by 450nm azide groups when the ruler is 10 mu m; (f) is a scanning electron microscope image of the 450nm azide group modified silica particle/paraffin composite sphere when the scale is 2 μm.
FIG. 4 is SiO2-N3And different PEGs2000PEG-SiO prepared by adding Saline2-N3Thermogravimetric analysis of (a).
FIG. 5 is SiO2-N3-ferritin and PEG-SiO2-N3-high resolution transmission electron microscopy images and corresponding elemental analysis images of ferritin biofunctionalized Janus nano-platforms; wherein (a) is PEG-SiO2-N3-high resolution transmission electron microscopy images (left) and corresponding elemental analysis images (right) of ferritin biofunctionalized Janus nano-platforms; (b) is SiO2-N3High resolution transmission electron microscopy images (left) and corresponding elemental analysis images (right) of ferritin.
FIG. 6 is a graph of absorbance at 412nm and corresponding fit for various concentrations of cysteine standards; wherein (a) is an absorbance detection graph at 412nm for cysteine standards of different concentrations; (b) fitting curves for cysteine standards at different concentrations and their absorbances.
FIG. 7 shows FITC-SiO2-N3And FITC-SiO2-N3-Rhodamine bio-functionalized Janus nano-platform fluorescence microscopy images; wherein (a) is FITC-SiO2-N3Fluorescence microscopy images of (a); (b) is FITC-SiO2-N3Rhodamine bio-functionalized Janus nano-platform fluorescence microscopy images.
Fig. 8 is a BCA standard graph.
FIG. 9 is SiO of FITC labeled Transferrin (Tf-FITC), FITC labeled homogeneously modified Transferrin and BSA2-N3Particles (Mixed NPs), FITC-labeled Transferrin-SiO2-N3BSA biofunctional Janus nano-platforms (Janus NPs) vs B16F10 tumor cell dissociation constant maps; wherein (a) is a Tf-FITC vs. B16F10 tumor cell dissociation constant map; (b) dissociation constant maps of Mixed NPs and Janus NPs on B16F10 tumor cells.
FIG. 10 shows FITC-labeled Transferrin-SiO2-N3-BSA biofunctionalizationJanus nano platform (Janus NPs), FITC marked SiO uniformly modified Transferrin and BSA2-N3Particles (Mixed NPs), FITC-labeled SiO2-N3Mean fluorescence intensity statistics were tested for the ability of nanoparticles (Free NPs), PBS to target B16F10 tumor cells.
FIG. 11 is a graph showing the results of measurements of the phagocytic capacity of tumor cells induced by macrophages with different particles; wherein A is a blank control group, and B is SiO for uniformly modifying Transferrin and Anti-SIRPa2-N3Nanoparticles (Mixed Transferrin-SiO)2-N3SIRPa NPs) group, C is Transferrin-SiO2-N3-SIRPa bio-functionalized Janus nano platform (Janus transferrin-SiO)2-N3-SIRPa NPs).
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1: 150nm azide group-based modified Silica (SiO)2-N3) Preparation of PEG-SiO2-N3-ferritin biofunctionalized Janus nano platform
1、150nm SiO2-N3The preparation of (1):
(1) 83mL of absolute ethyl alcohol, 1.7mL of ultrapure water and 5.6mL of ammonia water (28%, the same applies hereinafter) are mixed and stirred for 2 hours, 5mL of ethyl orthosilicate (liquid, analytically pure, the same applies hereinafter) is added to react in an oil bath kettle at 40 ℃ overnight, the reaction liquid at the upper layer is removed after centrifugation is carried out at 9000rpm for 10 minutes, and then the ultrapure water and the ethanol are used for repeatedly cleaning and vacuum drying to obtain the silicon dioxide nano particles.
Obtained SiO2The infrared spectrum of the nanoparticles is shown in fig. 1 (a): at 942cm-1The bending vibration peak of Si-OH appears at 1100cm-1The stretching vibration peak of Si-O-Si appears, which indicates that the SiO is successfully prepared2And (3) nanoparticles.
(2) And (2) dispersing 0.2g of the silica nanoparticles obtained in the step (1) in 10mL of anhydrous toluene, placing the mixture in an ultrasonic cleaning machine for ultrasonic dispersion for 30 minutes, adding 275 mu L of 3-chloropropyltrimethoxysilane, stirring at 120 ℃ for reaction for 12 hours, and after the reaction is finished, centrifuging with ethanol (9000rpm for 10 minutes) to wash away unreacted silane coupling agent to obtain the 3-chloropropyltrimethoxysilane modified silica nanoparticles.
(3) And (3) dispersing 0.1g of the silica particles of the 3-chloropropyltrimethoxysilane obtained in the step (2) in 1.5mLN, N-dimethylformamide, placing in an ultrasonic cleaning machine for ultrasonic dispersion for 30 minutes, then adding 0.04g of sodium azide, stirring at 80 ℃ for reaction for 24 hours, after the reaction is finished, centrifuging at 9000rpm for 10 minutes, then discarding the supernatant, and repeatedly cleaning with ultrapure water to remove the unreacted sodium azide to obtain the azide group modified silica nanoparticles.
Prepared SiO2-N3The transmission electron micrograph and the particle size distribution of the particles are shown in fig. 2(a) and fig. 1(b), respectively: prepared SiO2-N3The average particle diameter of the particles is 150nm, and the particles are uniform spheres.
SiO2-N3The infrared spectrum results are shown in FIG. 1 (a): at 2100cm-1The characteristic peak of azide group appears, which indicates that SiO is successfully prepared2-N3And (3) granules.
SiO2-N3The results of elemental analysis are shown in Table 1, and the grafting density of azide groups on the surface of the silica particles was 3.1 azide groups/nm2。
TABLE 1 SiO2-N3Results of elemental analysis
2. Preparation of 150nm azide group modified silica particles/paraffin wax composite spheres:
15mg of SiO having an average particle diameter of 150nm2-N3Dispersing the granules in 2.16mL of water, adding 120mg of paraffin, preheating at 80 deg.C to melt the paraffin, adding 0.24mL of 100mg/L didodecyldimethylammonium bromide (DDAB), homogenizing at 10000rpm for two minutes to make SiO2-N3The nanometer particles are distributed in a single layer at a paraffin/water interface, slowly cooled at room temperature, and then filtered by slow filter paperTo obtain SiO2-N3Nanoparticle/paraffin composite spheres.
The scanning electron microscope result of the 150nm azide group modified silica particle/paraffin composite sphere is shown in fig. 3(a) and (b): SiO 22-N3The nano particles are partially embedded in the paraffin and are uniformly and monodispersely distributed on the surface of the paraffin sphere.
3. Silane coupling agent modified polyethylene glycol (PEG)2000-preparation of salene):
taking 2g of mPEG2000Vacuum drying-OH (methoxy-polyethylene glycol-hydroxy, molecular weight 2000) at 90 deg.C for 3 hr, adding 4mL anhydrous pyridine, stirring well, adding 320 μ L isopropyltriethoxysilane isocyanate, and stirring at 70 deg.C under nitrogen for 24 hr. After the reaction is finished, the PEG is prepared by ether precipitation2000-Saline。
4、PEG-SiO2-N3-preparation of ferritin biofunctionalized Janus nano-platforms:
(1) 0.1g of SiO2-N3Dispersing the nano-particle/paraffin wax composite ball in 9mL of absolute ethyl alcohol, and respectively adding 1mL, 4mL and 8mL of PEG-containing composite ball2000Ethanol solution of Saline (1mg/mL), mixed at room temperature and shaken for 1 hour, filtered through slow filter paper, and washed with dichloromethane to remove paraffin, to obtain Janus particles (PEG-SiO) PEGylated with one hemisphere having azide group and with one hemisphere having azide group2-N3)。
Testing of different PEGs by thermogravimetric analysis2000PEG-SiO prepared by adding Saline2-N3The results of the test are shown in FIG. 4, with different PEGs2000PEG-SiO prepared by adding Saline2-N3The grafting density of surface PEG is shown in Table 2, and the subsequent reactions all use the grafting density of 1.32PEG group/nm2PEG-SiO of2-N3。
TABLE 2 different PEGs2000PEG-SiO prepared by adding Saline2-N3Grafting Density of surface PEG
(2) 6 μ L of the suspension containing DBCO-PEG4-dimethyl sulfoxide solution of NHSEster (available from click chemistry tools) (20mg/mL) and 2mL phosphate buffer containing Ferritin (Ferritin) (0.1M, pH 7.2) (Ferritin and DBCO-PEG)4Mass ratio of-NHSEster is 1:50), incubating for 12 hours at 10-30 ℃, and removing unreacted DBCO-PEG by ultrafiltration and centrifugation (6000rpm, 30 minutes)4-NHS to DBCO-PEG4-Ferritin。
(3) Mixing the products prepared in the step (1) and the step (2), stirring for 1 hour at 10-30 ℃, centrifuging for 10 minutes at 8000rpm, reserving the supernatant (supernatant 1), and precipitating the lower layer to obtain the prepared PEG-SiO2-N3-ferritin biofunctionalized Janus nano-platforms.
PEG-SiO2-N3The high resolution transmission electron microscopy and corresponding elemental analysis results of ferritin bio-functionalized Janus nano-platforms are shown in fig. 5 (a): SiO 22-N3The spherical surface of the side of the ball is covered by a ferritin sphere.
5. Azide group modified silica particles (SiO) for modifying equine ferritin2-N3-ferritin) preparation:
mixing 10mg of SiO2-N3The particles were dispersed in 1mL of water, and 100. mu.L of DBCO-PEG was added4Stirring at room temperature for 4 hours under conditions of-Ferritin (1mg/mL) to obtain SiO2-N3-ferritin。
SiO2-N3High resolution transmission electron microscopy and corresponding elemental analysis of ferritin is shown in figure 5 (b): SiO 22-N3Is covered by a ferritin sphere.
Example 2: based on 450nm SiO2-N3Preparation of PEG-SiO2-N3-ferritin biofunctionalized Janus nano platform
1、450nm SiO2-N3The preparation of (1):
(1) mixing 83mL of ethanol, 1.7mL of ultrapure water and 9mL of ammonia water, stirring for 2 hours, adding 5mL of ethyl orthosilicate, reacting in an oil bath kettle at 40 ℃ overnight, centrifuging at 6000rpm for 10 minutes, removing upper-layer reaction liquid, repeatedly cleaning with the ultrapure water and the ethanol, and performing vacuum drying to obtain silicon dioxide nanoparticles;
(2) the preparation of 3-chloropropyltrimethoxysilane modified silica nanoparticles is as in example 1.
(3) The procedure for preparation of azide group-modified silica nanoparticles was the same as in example 1.
Prepared SiO2-N3The transmission electron microscope images and the particle size distributions of the nanoparticles are shown in fig. 2(b) and fig. 1(b), respectively: SiO 22-N3The average particle diameter of the nano particles is 450nm, and the nano particles are uniform spheres.
2. Preparation of 450nm azide group modified silica particles/paraffin wax composite spheres:
mixing 36mg of SiO2-N3Dispersing the granules in 2mL of water, adding 214mg of paraffin wax, preheating at 80 deg.C to melt the paraffin wax, adding 0.4mL of 200mg/L DDAB, homogenizing at 10000rpm for two minutes to make SiO2-N3The nanometer particles are distributed in a single layer at a paraffin/water interface, slowly cooled at room temperature, and filtered by slow filter paper to obtain SiO2-N3Nanoparticle/paraffin composite spheres.
Scanning electron microscope results of 450nm azide group modified silica particles/paraffin composite spheres are shown in fig. 3(c) and (d): SiO 22-N3The particles are partially embedded in the paraffin and are uniformly and monodispersely distributed on the surface of the paraffin sphere.
3. Silane coupling agent modified polyethylene glycol (PEG)2000Salene) was prepared in the same manner as in example 1.
4、PEG-SiO2-N3The procedure for the preparation of the ferritin biofunctionalized Janus nano-platform was as in example 1.
Example 3: based on 2000nm SiO2-N3Preparation of FITC-SiO2-N3Rhodamine biofunctionalized Janus nano platform
1、2000nm SiO2-N3The preparation of (1):
(1) mixing 60mL of ethanol, 7mL of ultrapure water, 0.2g of potassium chloride and 6mL of ammonia water, stirring for 1 hour at 40 ℃ in an oil bath kettle, adding a mixed solution of 35mL of ethanol and 6.04g of tetraethoxysilane into the reaction solution at the speed of 5.5mL/h, stirring for 6 hours at 10-30 ℃, centrifuging for 10 minutes at 3000rpm after the reaction is finished, removing the upper-layer reaction solution, repeatedly cleaning with the ultrapure water and the ethanol, and drying in vacuum to obtain the silicon dioxide nanoparticles.
(2) And (2) dispersing 0.2g of the silicon dioxide nano-particles obtained in the step (1) in 10mL of anhydrous toluene, placing the silicon dioxide nano-particles in an ultrasonic cleaner for ultrasonic dispersion for 30 minutes, then adding 300 mu L of 3-chloropropyltrimethoxysilane, stirring at 120 ℃ for reaction for 12 hours, and after the reaction is finished, centrifuging the mixture by using ethanol (centrifuging the mixture at 3000rpm for 10 minutes) to wash away unreacted silane coupling agent to prepare the 3-chloropropyltrimethoxysilane modified silicon dioxide nano-particles.
(3) And (3) dispersing 0.1g of the 3-chloropropyltrimethoxysilane modified particles obtained in the step (2) in 1.5mL of N, N-dimethylformamide, placing the N, N-dimethylformamide in an ultrasonic cleaning machine for ultrasonic dispersion for 30 minutes, then adding 0.04g of sodium azide, stirring and reacting for 24 hours at 80 ℃, centrifuging at 3000rpm for 10 minutes after the reaction is finished, and then washing the unreacted sodium azide with water to obtain 2000nm azide group modified silicon dioxide particles.
Prepared SiO2-N3The transmission electron microscope images and the particle size distributions of the nanoparticles are shown in fig. 2(c) and fig. 1 (b): SiO 22-N3The average particle size of the nanoparticles was 2000nm, and the nanoparticles were uniform spheres.
2. Preparation of 2000nm azide group modified silica particles/paraffin wax composite spheres:
36mg of 2000nm SiO2-N3Dispersing the granules in 1.6mL of water, adding 180mg of paraffin, preheating at 80 ℃ to melt the paraffin, adding 0.4mL of 200mg/L DDAB, and homogenizing at 10000rpm for two minutes to make SiO2-N3The nanometer particles are distributed in a single layer at a paraffin/water interface, slowly cooled at room temperature, and filtered by slow filter paper to obtain SiO2-N3Nano granule/paraffin wax composite ball
The scanning electron microscope results of the 2000nm nano azide group modified silica particle/paraffin composite sphere are shown in fig. 3(e) and (f): SiO 22-N3The particles are partially embedded in the paraffin and are single on the surface of the paraffin ballThe dispersion is evenly distributed.
3. Preparation of sulfhydrylated fluorescein isothiocyanate labeled bovine serum albumin and sulfhydrylated rhodamine labeled bovine serum albumin:
(1) bovine Serum Albumin (BSA), 2mg of which was dissolved in 5mL of carbonate buffer (0.1M, pH 9.0), and Fluorescein Isothiocyanate (FITC), 1mg of which was dissolved in the buffer, were stirred at room temperature for 12 hours in the absence of light, and after the reaction was completed, unreacted FITC was removed by ultrafiltration and centrifugation (6000rpm, 20 minutes), whereby fluorescein isothiocyanate-labeled bovine serum albumin (BSA-FITC) was obtained.
(2) 2mg of Rhodamine (Rhodamine), 1.4mg of carbodiimide (EDC) and 1.05mg of N-hydroxysuccinimide (NHS) were dissolved in 5mL of PBS (pH 7.2 at 0.01M), and after stirring at room temperature for 2 hours, 6mg of BSA was added to the solution, and after the reaction was completed, the solution was stirred at room temperature in the dark for 24 hours, and after the completion of the reaction, unreacted Rhodamine was removed by ultrafiltration centrifugation (6000rpm, 20 minutes), whereby Rhodamine-labeled bovine serum albumin (BSA-Rhodamine) was obtained.
(3) After the completion of the reaction, unreacted 2-iminothiolane hydrochloride was removed by ultrafiltration (6000rpm, 20 minutes) to prepare thiolated BSA-FITC, 100 μ L of a PBS solution (0.01M pH 7.2) (0.01M chemically indicates 0.01 mol/L (abbreviated as M)) containing 2-iminothiolane hydrochloride (1mg/mL) was added to 900 μ L of PBS (0.01M pH 7.2), and the resulting solution was incubated at room temperature for 1 hour.
(4) After dissolving 2mg of BSA-Rhodamine in 900 μ L of PBS (0.01M pH 7.2), 100 μ L of a PBS solution of 2-iminothiolane hydrochloride (1mg/mL) (0.01M pH 7.2) was added and incubated at room temperature for 1 hour, and after completion of the reaction, unreacted 2-iminothiolane hydrochloride was removed by ultrafiltration centrifugation (6000rpm, 20 minutes), thereby obtaining a thiolated BSA-Rhodamine.
(5) The degree of thiolation of BSA-FITC and BSA-Rhodamine was detected using Ellman reagent:
① cysteine standards were prepared at different concentrations and tested for UV absorbance at 412nm to obtain a standard curve after fitting, the results are shown in FIG. 6.
② testing the UV absorbance at 412nm of thiolated BSA-FITC and thiolated BSA-Rhodamine resulted in 16 and 10 thiolated lysine residues for BSA-FITC and BSA-Rhodamine, respectively.
4、FITC-SiO2-N3Preparation of Rhodamine biofunctionalized Janus nano-platform:
(1) 0.1g of SiO2-N3Dispersing the nano-particle/paraffin wax composite ball in 2mL of water, adding 100 mu L (10mg/mL) of DBCO-PEG4-Ma1 (available from click chemistry tools) in dimethylsulfoxide, mixed and shaken at room temperature for 1 hour, filtered through slow filter paper, and washed with n-hexane to remove paraffin, yielding Janus particles with maleimide on one hemisphere and azide groups on one hemisphere.
(2) Mixing the product obtained in the step (1) with thiolated BSA-FITC and stirring overnight to obtain Janus particles (FITC-SiO) with one hemispherical bonded fluorescein isothiocyanate2-N3)。
Prepared FITC-SiO2-N3The fluorescence microscope results of (a) are shown in FIG. 7 (a): FITC-SiO2-N3One hemisphere has green fluorescence.
(3) 2mgDBCO-PEG4-Ma1 in 100 μ L dimethylsulfoxide to give solution 1; 2mg BSA-Rhodamine was dissolved in 2mL phosphate buffer (0.1M, pH 9.0) to give solution 2; mixing the solution 1 and the solution 2, incubating for 12 hours at 10-30 ℃, and performing ultrafiltration centrifugation (6000rpm, 20 minutes) to remove unreacted DBCO-PEG4Ma1 to obtain DBCO-PEG4-Rhodamine。
(4) Mixing the product obtained in the step (2) and the product obtained in the step (3) at 10-30 ℃, and stirring for 1 hour to obtain FITC-SiO2-N3Rhodamine biofunctionalized Janus nano-platform.
FITC-SiO2-N3Fluorescence microscopy results for Rhodamine biofunctionalized Janus nano-platforms are shown in fig. 7 (b): one hemisphere with green fluorescence and one hemisphere with red fluorescence.
Example 4: based on 150nm SiO2-N3Preparation of Transferrin-SiO2-N3-BSA biofunctionalized Janus nano-platform
1、150nm SiO2-N3The procedure of preparation was the same as in example 1.
2. The preparation process of the 150nm nano azide group modified silica particle/paraffin composite sphere is the same as that of example 1.
3、Transferrin-SiO2-N3-preparation of BSA biofunctional Janus nano-platforms:
(1) 0.1g of SiO having an average particle diameter of 150nm2-N3The particles/paraffin wax composite spheres are dispersed in 2mL of water, and 100 mu L of DBCO-PEG with the concentration of 10mg/mL is added4-Ma1 in dimethylsulfoxide, mixed at room temperature and shaken for 1 hour, filtered through slow filter paper, and washed with n-hexane to remove paraffin, yielding Janus particles with maleamide on one hemisphere and azide groups on one hemisphere.
(2) 2mg of Transferrin (Transferrin) was dissolved in 900. mu.L of PBS (pH 7.2 at 0.01M), 100. mu.L of a PBS solution of 2-iminothiolane hydrochloride was added, the mixture was incubated at room temperature for 1 hour, and after completion of the reaction, unreacted 2-iminothiolane hydrochloride was removed by ultrafiltration and centrifugation (6000rpm, 20 minutes), whereby thiolated Transferrin was obtained.
(3) Mixing the Janus particles obtained in the step (1) with a series of sulfhydrylation Transferrin with different mass obtained in the step (2), stirring overnight, centrifuging and cleaning to obtain Janus particles (Transferrin-SiO) with Transferrin on one hemisphere2-N3)。
(4) 6 μ L of the suspension containing DBCO-PEG4Dissolving a dimethylsulfoxide solution (20mg/mL) of Ma1 and 1mg of thiolated Bovine Serum Albumin (BSA) in 1mL of PBS (0.01M pH 7.2), incubating at 10-30 ℃ for 12 hours, and removing unreacted DBCO-PEG by ultrafiltration centrifugation (6000rpm, 20 minutes)4Ma1 to obtain DBCO-PEG4-BSA。
(5) Transferring the Transferrin-SiO obtained in the step (3)2-N3A series of DBCO-PEG with different qualities obtained in the step (4)4Mixing with BSA, stirring at 10-30 ℃ for 1 hour, centrifuging at 8000rpm for 10 minutes, and cleaning to obtain Transferrin-SiO2-N3-BSA biofunctionalized Janus nano-platforms.
(6) Testing of step (3) and step (5) Using the BCA kitThe protein content of the supernatant was centrifuged, and the BCA standard curve was measured as shown in fig. 8, where y is 0.0013x +0.0117, and SiO was calculated2-N3The grafting density and the grafting proportion of the surface Transferrin and BSA, and the test results are shown in table 3.
TABLE 3 SiO2-N3Grafting density and grafting proportion of surface transferrin and BSA
Example 5: based on 150nm SiO2-N3Preparation of Transferrin-SiO2-N3-SIRPa bio-functionalized Janus nano-platform
1、Transferrin-SiO2-N3The procedure of preparation was the same as in example 4.
2、Transferrin-SiO2-N3Preparation of SIRPa biofunctionalized Janus nano-platform:
(1) 100 μ L of the mixture containing DBCO-PEG4The dimethylsulfoxide solution of-NHS (10mg/mL) was mixed homogeneously with 10. mu.g of Anti-SIRPa antibody, incubated at 4 ℃ for 12 hours, and subjected to ultrafiltration centrifugation (6000rpm, 20 minutes) to remove unreacted DBCO-PEG4-NHS to DBCO-PEG4-SIRPa。
(2) Adding DBCO-PEG obtained in the step (1)4Addition of 1mg of transferrin-SiO to SIRPa2-N3Mixing uniformly, stirring for 12 hours at 4 ℃, centrifuging for 10 minutes at 8000rpm, and cleaning to obtain the Transferrin-SiO2-N3-SIRPa bio-functionalized Janus nano platform (Janus Transferrin-SiO)2-N3-SIRPa NPs)。
Comparative example 1 preparation of FITC-labeled transferrin (Tf-FITC):
after 5mg of transferrin was dissolved in 900. mu.L of PBS (0.1M, pH 7.2), 100. mu.L of a dimethylsulfoxide solution containing FITC (5 mg/mL) was added thereto, and the mixture was stirred at room temperature for 12 hours in the dark, and subjected to ultrafiltration centrifugation (6000rpm for 20 minutes) to remove unreacted FITC, Tf-FITC was obtained.
Comparative example 2 FITC-labeled SiO2-N3Nanoparticles (Free)NPs) preparation:
(1) taking 50mg of SiO with the average grain diameter of 150nm2-N3Dissolving in 2.5mL of N, N-dimethylformamide, placing in an ultrasonic cleaning machine for ultrasonic dispersion for 30 minutes, adding 0.1 mu L of propargylamine, mixing, and sequentially dropwise adding 5 mu L of 1.245210-2mol/L copper sulfate solution and 5 mu L2.49x10-2mixing and stirring the sodium ascorbate solution in mol/L for 8 hours, and after the reaction is finished, centrifuging and cleaning the mixture by using 0.05% (w/v) EDTA and water (8000rpm,10 minutes) to prepare propargylamine-labeled SiO2-N3And (3) nanoparticles.
(2) Marking propargylamine obtained in the step (1) with SiO2-N3Dissolving the nanoparticles in 1mL of pH 9 carbonate buffer (0.1M, pH 9), ultrasonically dispersing for 10min in an ultrasonic cleaner, adding 100. mu.L of FITC solution with concentration of 0.07mg/mL, stirring at room temperature for 6 hours, and after the reaction is finished, centrifugally cleaning to remove unreacted FITC to obtain FITC-labeled SiO2-N3And (3) nanoparticles.
Comparative example 3 FITC-labeled SiO homogeneously modified Transferrin and BSA2-N3Preparation of particles (Mixed NPs):
(1)DBCO-PEG4the procedure for the preparation of BSA was the same as in example 4.
(2) 6 μ L of the suspension containing DBCO-PEG4-Ma1 in dimethylsulfoxide (20mg/mL) was mixed with 100mg of thiolated transferrin (100 mg/mL) in 1mL PBS (0.01M, pH ═ 7.2) and incubated at room temperature for 12 hours, and the unreacted DBCO-PEG was removed by ultrafiltration centrifugation (6000rpm, 20 minutes)4Ma1 to obtain DBCO-PEG4-Transferrin。
(3) 10mg of FITC-labeled SiO prepared in comparative example 2 were taken2-N3Uniformly dispersing the DBCO-PEG obtained in the step (1) in water4BSA with DBCO-PEG obtained in step (2)4After all of Transferrin was added, the mixture was stirred at room temperature for 1 hour, and after completion of the reaction, the mixture was centrifuged (8000rpm,10 minutes) to discard the supernatant, thereby obtaining Mixed NPs.
Comparative example 4 FITC-labeled Transferrin-SiO2-N3Preparation of BSA biofunctional Janus nano-platforms (Janus nps):
(1) FITC-labeled SiO2-N3The procedure of (1) was the same as in comparative example 2.
(2) The preparation of FITC-labeled Janus NPs was performed as described in example 4.
Comparative example 5 SiO uniformly modified with Transferrin and Anti-SIRPa2-N3Granules (Mixed Transferrin-SiO)2-N3-preparation of SIRPa NPs):
(1)DBCO-PEG4the procedure for the preparation of Transferrin is the same as in comparative example 3.
(2)DBCO-PEG4The preparation process of SIRPa is the same as that of example 5.
(3) Taking 10mg of SiO2-N3Uniformly dispersing in water, adding the products of the step (1) and the step (2), stirring for 12 hours at 4 ℃, centrifuging (8000rpm,10 minutes) after the reaction is finished, and discarding the supernatant to obtain the MixedTransfern-SiO2-N3-SIRPa NPs。
Comparative example 6 amino-modified silica nanoparticles (SiO)2-NH2) PEG-SiO as main body2-N3-ferritin biofunctionalized Janus nano-platform preparation:
1、SiO2-NH2the preparation of (1):
(1)SiO2the procedure for preparing nanoparticles was the same as in example 1.
(2) And (2) dispersing 0.2g of the silicon dioxide nano particles obtained in the step (1) in 10mL of anhydrous toluene, placing the silicon dioxide nano particles in an ultrasonic cleaner for ultrasonic dispersion for 30 minutes, then adding 125 mu L of 3-aminopropyltriethoxysilane, stirring and reacting for 24 hours at 80 ℃, and after the reaction is finished, centrifugally (8000rpm for 10 minutes) cleaning to remove unreacted silane coupling agent to obtain the silicon dioxide nano particles with the modified surface amino groups.
2、PEG-SiO2-NH2-preparation of ferritin biofunctionalized Janus nano-platforms:
(1) the preparation process of the surface amino modified silica nanoparticle/paraffin composite sphere was the same as in example 2.
(2) Dispersing 0.1g of the surface amino modified silica nanoparticle/paraffin composite spheres of step (1) in 9mL of absolute ethanol, and adding 8mL of the aqueous solution containing the surface amino modified silica nanoparticle/paraffin composite spheresPEG2000Ethanol solution of Saline (1mg/mL), mixed at room temperature and shaken for 1 hour, filtered through slow filter paper, and washed with dichloromethane to remove paraffin, to obtain Janus particles (PEG-SiO) PEGylated in one hemisphere with amino group in one hemisphere2-NH2)。
(3) The PEG-SiO of the step (2)2-NH2Stirring with ferritin at room temperature for 24 hr, centrifuging at 8000rpm for 10min, collecting supernatant (supernatant 2), and precipitating as PEG-SiO with surface amino modified silica nanoparticles as main body2-NH2-ferritin biofunctionalized Janus nano-platforms.
Effect example 1: FITC-labeled Transferrin (Tf-FITC), FITC-labeled uniformly modified Transferrin, and SiO of BSA2-N3Nanoparticles (Mixed NPs), FITC-labeled Transferrin-SiO2-N3BSA biofunctionalized Janus Nano-platform (Janus NPs) dissociation constant test for mouse melanoma cells (B16F10 tumor cells) (purchased from American type culture Collection ATCC)
Inoculate 5X10 per well in 24-well plates4Individual cells/500. mu.L DMEM medium (containing 10% primary bovine serum) in CO2Incubator (37 ℃, CO)2Concentration of 5%), DMEM medium was aspirated after 12 hours of culture, Tf-FITC (obtained in comparative example 1), Mixed NPs (obtained in comparative example 3), Janus NPs (obtained in comparative example 4) were added at different concentrations, respectively (specific concentrations of each group are shown in abscissa of fig. 9), after 1 hour, the average fluorescence intensity was measured using a flow cytometer, and dissociation constants were determined using Scatchard analysis fitting, according to the following formula:
wherein B is the bound receptor concentration, F is the free ligand concentration, KDAs dissociation constant, BmaxMaximum saturation concentration of ligand binding site, KDThe value is equal to the absolute value of the inverse slope of the fitted line.
The results of Tf-FITC, Mixed NPs, Janus NPs on B16F10 tumor cell dissociation constant test are shown in FIG. 9: dissociation constants were 249nM, 0.66nM, and 0.15nM, respectively, indicating that the Janus NPs have stronger targeting affinity than the Mixed NPs.
Effect example 2: FITC-labeled SiO homogeneously modified Transferrin and BSA2-N3Nanoparticles (MixedNTPs), FITC-labeled SiO2-N3Nanoparticles (Free NPs), FITC-labeled Transferrin-SiO2-N3BSA biofunctionalized Janus Nano platform (Janus NPs) test for B16F10 tumor cell targeting ability
Firstly, 5X10 of each pore is planted on a 24-pore plate4Individual cells/500. mu.L DMEM (containing 10% primary bovine serum) in CO2Incubator (37 ℃, CO)2Concentration of 5%), DMEM medium was aspirated after 12 hours of culture, FreeNPs (obtained in comparative example 2), Mixed NPs (obtained in comparative example 3), Janus NPs (obtained in comparative example 4), and PBS were added at the same concentrations, respectively, and after 1 hour, the average fluorescence intensity was measured using a flow cytometer.
The test results are shown in fig. 10: janus NPs can specifically target the surface of B16F10 tumor cells, and have better targeting affinity compared with homogeneous particles (Mixed NPs).
Effect example 3: Transferrin-SiO2-N3-SIRPa bio-functionalized Janus nano platform (Janus transferrin-SiO)2-N3-SIRPa NPs), SiO uniformly modifying Transferrin and Anti-SIRPa2-N3Nanoparticles (Mixed Transferrin-SiO)2-N3SIRPa NPs) induce phagocytosis of tumor cells by macrophages
(1) Macrophage extraction and staining:
c57BL/6 mice (purchased from Splakeda laboratory animals Co., Ltd., Hunan province) were bled from the eyeballs, the carotid arteries were cut off to remove the leg bones after the bleeding was completed, bone marrow was blown out from the leg bones into DMEM medium with a 2.5mL syringe, cells were collected by centrifugation at 450 Xg for 5min, then 1mL of erythrocyte lysate was added to resuspend the cells, the cells were left at 4 ℃ for 10min, lysis was stopped with 10-12mL of 1 XPBS, cells were collected by centrifugation at 450 Xg for 5min, and finally DMEM medium containing M-CSF cytokine (10ng/mL) was added to culture for 7 days to obtain mature BMDMs. Mature macrophages were stained with eFluor670 dye (ex eBioscience, cat # 65-0840) to obtain eFluor670 dye-labeled macrophages (eFluor670-BMDM) (the specific staining procedure was performed according to the product instructions).
(2) B16F10 tumor cell staining:
the B16F10 tumor cells were stained using a CFDA SE cell proliferation and tracer detection kit (purchased from beyond, cat # C0051) to obtain CFSE dye-labeled B16F10 tumor cells (CFSE-B16F10), and the specific staining procedure was performed according to the product instructions.
(3) Phagocytic potency assay for tumor cells by macrophages
5X10 per well on a 24-well plate4An amount of eFluor670-BMDM added to 0.5mL of DMEM medium containing 10ng/mLIL-4 and 10ng/mLIL-13 in CO2Incubator (37 ℃, CO)2Concentration of 5%), after 24 hours of culture, DMEM medium was aspirated and inoculated at 2X 10 per well5CFSE-B16F10 with the addition of 0.5mL of 0.2nM Janus Transferrin-SiO2-N3SIRPa NPs (obtained in example 5), Mixed Transferrin-SiO2-N3SIRPa NPs (obtained in comparative example 5), using a blank without any addition of particles as control, in CO2Incubator (37 ℃, CO)2Concentration of 5%), after 4 hours of culture, the percentage of tumor cells phagocytosed by macrophages was tested using a flow cytometer.
The test results are shown in fig. 11: janus Transferrin-SiO2-N3-SIRPa NPs compared to MixedTransferrin-SiO2-N3SIRPa NPs, have better ability to induce macrophages to phagocytose tumor cells.
Effect example 4: biological functionalization efficiency comparison experiment of Janus nano platform taking azide group modified silicon dioxide nano particles as main body
The BCA kit was used to test the protein content of supernatant 1 (obtained in example 1) and supernatant 2 (obtained in comparative example 5), and the BCA standard curve was determined as shown in fig. 8, where y is 0.0013x +0.0117, and PEG-SiO could be calculated2-NH2-ferritin and PEG-SiO2-N3-ferritin surface ironTotal mass of protein bonding and bonding efficiency.
The test results are shown in table 4: PEG-SiO prepared by taking silicon dioxide nano-particles modified by azide groups as main bodies2-N3-ferritin biofunctionalized Janus nano-platforms, due to the use of efficient "click chemistry" reactions for biofunctionalized modification compared to SiO2-NH2PEG-SiO prepared as main body2-N3The ferriritin biological functionalized Janus nano platform has higher protein bonding efficiency and shows more excellent biological functionalization performance.
TABLE 4 PEG-SiO2-NH2-ferritin and PEG-SiO2-N3Comparison of ferritin grafting efficiency on ferricin surface
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A preparation method of azide group modified Janus nanoparticles is characterized by comprising the following steps:
(1)SiO2-N3preparing the nano-particle/paraffin composite ball: SiO by using Pickering emulsion method2-N3Dispersing the nanoparticles in water, adding paraffin and DDAB, homogenizing to obtain SiO2-N3The nano particles are distributed in a single layer at a paraffin/water interface, and SiO is obtained by cooling and filtering2-N3Nanoparticle/paraffin composite spheres;
(2) preparation of azide group modified Janus nanoparticles: for the SiO obtained in the step (1)2-N3Chemically modifying the exposed surface of the nano-particle/paraffin wax composite ball, washing to remove the paraffin wax to obtain the azide group modifiedJanus nanoparticles.
2. The method for preparing azide-group-modified Janus nanoparticles according to claim 1, wherein the method comprises the following steps:
SiO as described in step (1)2-N3The size of the nano particles is 150-2000 nm;
the SiO2-N3The nanoparticles are prepared by the following steps:
(A) preparing silicon dioxide nano-particles with different particle sizes;
(B) preparing silicon dioxide nano particles modified by chloropropyl silane coupling agent;
(C) preparing silicon dioxide nano particles modified by azide groups;
the chemical modification in the step (2) is to modify the surface of the silica particles by a click chemistry reaction or a silane coupling agent modification method.
3. The method for preparing azide-group-modified Janus nanoparticles according to claim 1, wherein the method comprises the following steps:
preheating and melting the paraffin wax in the step (1) at 80 ℃;
SiO as described in step (1)2-N3The nano particles and the paraffin are mixed according to the mass ratio of 1: 5-1: 8;
the final volume of the water in the step (1) is 2.0-2.5 mL; further 2.0-2.4 mL;
the final concentration of DDAB in the step (1) is 10-60 mg/L; further 10-35 mg/L;
homogenizing at 8000-10000 rpm for 1-2 min in the step (1); homogenizing at 10000rpm for 2 min;
the cooling in the step (1) is slow cooling at room temperature;
the room temperature is 10-30 ℃; further 24-26 ℃;
the filtration in the step (1) is slow filter paper filtration;
the solvent for washing and removing paraffin in the step (2) is n-hexane or dichloromethane; further, n-hexane was used.
4. An azide group modified Janus nanoparticle obtained by the preparation method of any one of claims 1-3.
5. The azide-modified Janus nanoparticle according to claim 4, wherein: the azide group modified Janus nano particles are spherical and uniform in size, and the particle size is 150-2000 nm.
6. Use of the azide-modified Janus nanoparticles of claim 4 or 5 in the preparation of a biologically functionalized Janus nano-platform.
7. A biologically-functionalized Janus nano-platform, comprising: the azide-modified Janus nanoparticle is obtained by performing biological functional modification on the azide-modified Janus nanoparticle in claim 4 or 5.
8. The biofunctionalized Janus nano-platform according to claim 7, wherein:
the molecule modified by the biological functional modification is at least one of functional protein, antibody, small molecule compound and other biological functional molecules which can specifically target specific areas on the surfaces of different types of cells.
9. Use of the biofunctionalized Janus nano-platform of claim 7 or 8 in biomedical research.
10. Use according to claim 9, characterized in that: the biomedicine is tumor immunotherapy.
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