WO2022188013A1 - 化学发光介导的纳米颗粒、自组装聚集体及其制备方法和应用 - Google Patents

化学发光介导的纳米颗粒、自组装聚集体及其制备方法和应用 Download PDF

Info

Publication number
WO2022188013A1
WO2022188013A1 PCT/CN2021/079615 CN2021079615W WO2022188013A1 WO 2022188013 A1 WO2022188013 A1 WO 2022188013A1 CN 2021079615 W CN2021079615 W CN 2021079615W WO 2022188013 A1 WO2022188013 A1 WO 2022188013A1
Authority
WO
WIPO (PCT)
Prior art keywords
nanoparticles
cross
chemiluminescence
nanoparticle
linking agent
Prior art date
Application number
PCT/CN2021/079615
Other languages
English (en)
French (fr)
Inventor
史海斌
毛秋莲
Original Assignee
苏州大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 苏州大学 filed Critical 苏州大学
Priority to PCT/CN2021/079615 priority Critical patent/WO2022188013A1/zh
Publication of WO2022188013A1 publication Critical patent/WO2022188013A1/zh

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials

Definitions

  • the invention belongs to the technical field of nanoparticle self-assembly, and in particular relates to a method for preparing chemiluminescence-mediated nanoparticle self-assembly aggregates, the aggregates prepared by the method, and the preparation of the aggregates based on photothermal therapy (photothermal therapy). therapy, PTT) in the application of antitumor drugs.
  • photothermal therapy photothermal therapy
  • Nanoparticles with different sizes, structures, and morphologies exhibit different surface effects and quantum effects, and then exhibit different optical, electrical, magnetic, mechanical, and chemical properties. It has important research and application value in the fields of clinical diagnostic materials and so on. Therefore, the controllable self-assembly of nanoparticles has always been a research hotspot in the field of nanotechnology.
  • the traditional self-assembly of nanoparticles is mainly through the spontaneous organization or aggregation of nanoparticles into a thermodynamically stable, uniformly distributed structure with special properties.
  • the process mainly relies on two factors to form a self-assembled system: one is to form a stable nano-self-assembled system through non-covalent bonds (especially hydrogen bonds); the other is to achieve the lowest energy level of the nano-system Spontaneous self-assembly.
  • Commonly used nanoparticle self-assembly methods mainly include solvent evaporation method, magnetoelectric optical induction method, interface method and template method.
  • the nanoself-assembled systems obtained by these traditional methods often exhibit poor reproducibility and stability due to relying on non-covalent bonds or inter-charge interactions, which greatly restricts the application of materials in biological systems.
  • the penetration depth of external irradiation is limited, which cannot induce cross-linking of photosensitizers well for deep tumors.
  • it is also subject to the conditions of external irradiation to a certain extent, so that the overall aggregation efficiency is not very high. .
  • the present invention develops a simple, fast, green, stable and controllable nanoparticle self-assembly method.
  • This method is not only suitable for the self-assembly between the same nanoparticles, but also for the hybrid assembly between different nanoparticles, which provides a new strategy and means for the preparation of multifunctional nanocomposites.
  • a chemiluminescence-mediated nanoparticle comprising a nanoparticle with a cross-linking agent, luminol and a nano-particle with a cross-linking agent.
  • a chemiluminescence-mediated nanoparticle system includes nanoparticles with a cross-linking agent and luminol, nanoparticles with a cross-linking agent, an oxidant, iron ions, and water.
  • the weight ratio of the nanoparticles with the cross-linking agent to the nanoparticles with the cross-linking agent and luminol is 1:(0.8-1.2), preferably 1:1.
  • a chemiluminescence-mediated nanoparticle self-assembly aggregate is obtained by cross-linking nanoparticles with a cross-linking agent, luminol, and nanoparticles with a cross-linking agent.
  • chemiluminescence-mediated nanoparticles are composed of nanoparticles with a cross-linking agent, luminol, and nanoparticles with a cross-linking agent, and the cross-linking and self-assembly between nanoparticles does not require external light.
  • hydrogen peroxide iron ions
  • the present invention uses nanoparticles with cross-linking agents and luminol, nanoparticles with cross-linking agents, oxidants, iron ions, water
  • the composition of the chemiluminescence-mediated nanoparticle system enables the preparation of nanoparticle self-assembled aggregates without exogenous light (excitation).
  • the invention discloses the application of the above-mentioned chemiluminescence-mediated nanoparticles or the chemiluminescence-mediated nanoparticle system in the preparation of nanoparticle self-assembled aggregates without exogenous illumination.
  • the invention uses chemiluminescence to trigger the crosslinking of the photosensitizer without external excitation, which is particularly novel, is a simple, fast and controllable new method for self-assembly of nanoparticles, and has great research and application value.
  • the crosslinking agent is different.
  • the crosslinking agent in the nanoparticle with crosslinking agent and luminol is crosslinking agent.
  • the cross-linking agent A, the cross-linking agent in the nanoparticles with the cross-linking agent is the cross-linking agent B; the cross-linking agent A is preferably methacrylic acid, and the cross-linking agent B is preferably tetrathiazole.
  • the chemical structural formula of the crosslinking agent is as follows: .
  • the preparation method of the above-mentioned chemiluminescence-mediated nanoparticle self-assembled aggregate includes the following steps.
  • a cross-linking agent is modified on the surface of the nanoparticles to obtain nanoparticles with a cross-linking agent.
  • step (3) Cross-linking the nanoparticle with a crosslinking agent in step (1) and the nanoparticle with a crosslinking agent and luminol in step (2) to obtain a chemiluminescence-mediated nanoparticle self-assembly aggregate.
  • the nanoparticles are one or more of metal nanoparticles, inorganic non-metallic nanoparticles, organic nanoparticles, and inorganic-organic hybrid nanoparticles;
  • the oxidant is hydrogen peroxide.
  • amino polyethylene glycol is modified on the surface of nanoparticles, and then reacted with a cross-linking agent to obtain nanoparticles with cross-linking agent; amino polyethylene glycol is modified on the surface of nanoparticles, and then reacted with cross-linking agent, lu Minol reaction to obtain nanoparticles with cross-linking agent, luminol.
  • the preparation method of the above-mentioned chemiluminescence-mediated nanoparticle self-assembled aggregate is as follows.
  • the alcohol thiol and the amino polyethylene glycol thiol are stirred at room temperature for 12-48 hours, centrifuged by ultrafiltration, and resuspended in water to obtain a mother solution of PEG-modified amino-functionalized nanoparticles.
  • Luminol 1: 2 ⁇ 15
  • Nanoparticles with cross-linking agent methacrylic acid are added to the luminol solution, stirred at room temperature for 2 to 36 hours, and subjected to ultrafiltration and centrifugation to obtain nanoparticles with cross-linking agent and luminol.
  • the nanoparticles with cross-linking agent prepared in step 2) and the nanoparticles with cross-linking agent and luminol prepared in step 4) were respectively resuspended in water, and then the two solutions (nanoparticles Equal weight ratio) was added into the H 2 O solution containing H 2 O 2 and Fe 3+ and reacted for 15-90 min to obtain nanoparticle self-assembled aggregates.
  • the nanoparticles in step (1) are selected from any one of metal nanoparticles, inorganic non-metallic nanoparticles, organic nanoparticles, and inorganic-organic hybrid nanoparticles or any ratio thereof more preferably, the metal nanoparticles are gold nanoparticles, the inorganic non-metallic nanoparticles are iron tetroxide nanoparticles, and the organic nanoparticles are polydopamine nanoparticles.
  • the methoxy polyethylene glycol thiol in step (1) is polyethylene glycol whose two ends are modified by methoxy groups and sulfhydryl groups respectively, which is selected from M-PEG 2000 Any one of -SH, M-PEG 5000 -SH, M-PEG 10000 -SH, M-PEG 20000 -SH or a mixture thereof in any ratio; more preferably, the methoxy polyethylene glycol thiol is M-PEG 5000 -SH.
  • the amino polyethylene glycol thiol in step (1) is polyethylene glycol whose two ends are modified by amino groups and sulfhydryl groups respectively, which is selected from NH 2 -PEG 2000 -SH, Any one of NH 2 -PEG 5000 -SH, NH 2 -PEG 10000 -SH, NH 2 -PEG 20000 -SH or a mixture thereof in any proportion; more preferably, the amino polyethylene glycol thiol is NH 2 -PEG 5000 -SH.
  • the invention discloses the application of the above-mentioned chemiluminescence-mediated nanoparticles or a chemiluminescence-mediated nanoparticle system in the preparation of nanoparticle self-assembled aggregates without exogenous illumination; the above-mentioned chemiluminescence-mediated nanoparticles or chemiluminescence-mediated nanoparticle systems
  • Nanomedicine is a medicine for treating tumors, preferably a photothermal therapy medicine.
  • the present invention has the following advantages compared with the prior art.
  • the photosensitive cross-linking agents tetrathiazole and methacrylic acid with small volume are used for the first time, and the precipitation of nanoparticles due to the excessively large volume of the cross-linking agent is successfully avoided.
  • the self-assembly degree of the nanoparticles can be effectively regulated, thereby realizing the controllable self-assembly of the nanomaterials.
  • luminescent luminol was used to induce crosslinking of UV-sensitive cross-linking agents in vivo or in vitro, so that gold nanoparticles can be effectively aggregated, thereby further enhancing the sensitivity of tumor photoacoustic imaging and tumor photothermal therapy.
  • the self-assembly method of the present invention has the characteristics of simplicity, speed, stability, controllability, and environmental protection, which greatly saves the preparation time, and is a universal new method for the rapid preparation of multifunctional nanomaterials. application space.
  • FIG. 1 is a schematic diagram of the modification process of nanoparticles 1 with a cross-linking agent in Example 3.
  • FIG. 1 is a schematic diagram of the modification process of nanoparticles 1 with a cross-linking agent in Example 3.
  • FIG. 2 is a schematic diagram of the modification process of gold nanoparticles 2 modified with crosslinking agent methacrylic acid in Example 4.
  • FIG. 2 is a schematic diagram of the modification process of gold nanoparticles 2 modified with crosslinking agent methacrylic acid in Example 4.
  • FIG. 3 is a schematic diagram of the modification process of gold nanoparticles 3 carrying a chemiluminescence source luminol and a photosensitive cross-linking agent in Example 5.
  • FIG. 3 is a schematic diagram of the modification process of gold nanoparticles 3 carrying a chemiluminescence source luminol and a photosensitive cross-linking agent in Example 5.
  • Figure 4 is a TEM image of gold nanoparticles with different modifications.
  • FIG. 5 is a material characterization diagram of chemiluminescence imaging of gold nanoparticles 3 carrying a chemiluminescence source luminol and a photosensitive crosslinking agent in Example 6.
  • FIG. 5 is a material characterization diagram of chemiluminescence imaging of gold nanoparticles 3 carrying a chemiluminescence source luminol and a photosensitive crosslinking agent in Example 6.
  • FIG. 6 is a schematic diagram of the chemiluminescence-mediated nanoparticle self-assembly process in Example 7.
  • FIG. 6 is a schematic diagram of the chemiluminescence-mediated nanoparticle self-assembly process in Example 7.
  • Example 7 shows the TEM images of the gold nanoparticles before and after the self-assembly induced by ultraviolet light irradiation, and the changes of ultraviolet absorption and particle size before and after cross-linking in Example 8.
  • FIG. 8 shows the photothermal properties of the gold nanoparticles in Example 9 before and after the cross-linking induced by a chemiluminescence source.
  • FIG. 9 is a study on the toxicity to cells and photothermal treatment effect of gold nanoparticles before and after self-assembly induced by a chemiluminescence source in Example 10.
  • FIG. 9 is a study on the toxicity to cells and photothermal treatment effect of gold nanoparticles before and after self-assembly induced by a chemiluminescence source in Example 10.
  • Figure 10 shows the reaction cross-linking (uncross-linked) of the nanoparticles 1 (tAu) with the cross-linking agent in Example 3 of the control group and the gold nanoparticles 2 (mAu) modified with the cross-linking agent methacrylic acid in Example 4.
  • Figure 11 shows that gold nanoparticles 1 (tAu) alone or gold nanoparticles 2 (mAu) alone do not aggregate under luminol chemiluminescence conditions.
  • Figure 12 shows direct irradiation of the solution (unshielded) and indirect irradiation (shielded) through the wall of a 1.5 mL centrifuge tube (tAu+mAu).
  • the nanoparticles with cross-linking agent prepared by the present invention and the prepared nanoparticles with cross-linking agent/luminol constitute chemiluminescence-mediated nanoparticles, which can self-assemble to obtain aggregates without exogenous illumination .
  • the present invention provides a method for the preparation of chemiluminescence-mediated nanoparticle self-assembled aggregates, in which self-luminescence is applied to the cross-linking of photosensitizers in this way without external excitation, with easy It is an excellent method in the self-assembly technology of nanoparticles due to its advantages of modification and controllability.
  • Example 1 Synthesis and characterization of cross-linking agent tetrathiazole.
  • Example 2 Preparation of gold nanoparticles and PEG modification of their surfaces: Add 1wt% chloroauric acid solution (0.6 mL) to ultrapure water (100 mL), heat to boiling and add 1wt% sodium citrate solution (3 mL), after the system turns wine red, continue to boil for 30 min to obtain a gold nanoparticle stock solution.
  • Example 4 PEG end modification on the surface of gold nanoparticles
  • Example 5 Carrying chemiluminescence luminol: As shown in Figure 3, the gold nanoparticles 2 (mAu) mother solution obtained in Example 4 was mixed with an aqueous solution of luminol (1 mg of gold nanoparticles corresponds to 10 mg of luminol) After mixing, shaking for 20 h, and ultrafiltration centrifugation (5000 rpm ⁇ 10 min) for 3 times, gold nanoparticles 3 carrying chemiluminescence source luminol and photosensitive cross-linking agent were obtained, which were mAu/Lu, and the mother liquor was prepared. , 4mg/mL.
  • Figure 4 is a TEM image of gold nanoparticles with different modifications.
  • a is the electron microscope image of PEG-modified amino-functional gold nanoparticles
  • b is the electron microscope image of the gold nanoparticles 1 (tAu) with crosslinking agent obtained in Example 3
  • c is the electron microscope image obtained in Example 4
  • d is the electron microscope of gold nanoparticles 3 (mAu/Lu) carrying chemiluminescence source luminol and photosensitive cross-linking agent obtained in Example 5 It can be seen from the figure that the size distribution of these four gold nanoparticles is relatively uniform (about 20 nm). Too large to cause nanoparticle precipitation and aggregation.
  • Example 6 The luminescence intensity and signal of gold nanoparticles 3 carrying chemiluminescence source luminol and photosensitive crosslinking agent: Gold nanoparticles 3 were resuspended in ultrapure water, and at the same time, a 1 mL aqueous solution containing 200 mM H 2 O 2 and 100 mM Fe 3+ (potassium ferricyanide) was prepared, and the chemiluminescence source luminol and the photosensitive cross-linking agent were added to it. The gold nanoparticles 3 were left standing for 0-90 min without external excitation light source.
  • luminol can emit light with a wavelength of about 450 nm in the environment of H 2 O 2 and Fe 3+ in vitro; as shown in Figures 5b and 5c, with the prolongation of standing time, the luminescence intensity of gold nanoparticles 3 gradually weakened.
  • Example 7 Self-assembly of gold nanoparticles mediated by chemiluminescence source: As shown in Figure 6, equal amounts of gold nanoparticles 1 ( tAu ) prepared in Example 3 and gold nanoparticles prepared in Example 5 were taken. Particle 3 ( mAu/Lu ), wherein the weight of gold nanoparticles 1 and gold nanoparticles 3 is the same, and both are 50 ⁇ g. Added to the aqueous solution containing 200 mM H 2 O 2 and 100 mM Fe 3+ , the total volume was 1 mL, and the cross-linking reaction time was 45 min to obtain UV-mediated nanoparticle self-assembled aggregates.
  • Example 8 Changes in particle size distribution and UV absorption before and after self-assembly of gold nanoparticles: take equal amounts of gold nanoparticles 1 ( tAu ) prepared in Example 3 and gold nanoparticles 3 prepared in Example 5 ( mAu/Lu ), in which the weight of gold nanoparticles 1 and gold nanoparticles 3 is the same, and both are 50 ⁇ g. Added to the aqueous solution containing 200 mM H 2 O 2 and 100 mM Fe 3+ , the total volume was 1 mL, and the cross-linking reaction time was 45 min to obtain UV-mediated nanoparticle self-assembled aggregates.
  • the TEM, particle size distribution (DLS) and UV absorption were tested, and the sampling time was 0 min, 15 min, and 30 min.
  • the hydrated particle size of gold nanoparticles (gold nanoparticle reaction mother solution) before luminol luminescence is about 50 nm
  • its electron microscope (TEM) is a circular particle with a uniform size of about 20 nm.
  • TEM electron microscope
  • the gold nanoparticles begin to aggregate gradually, and the maximum hydrated particle size can be about 280 nm.
  • the electron microscope aggregates and aggregates into very large particles (Fig. 7c).
  • Figure 7e shows the change of UV absorption of gold nanoparticles after light-mediated aggregation.
  • the maximum UV absorption of gold nanoparticles before luminol luminescence is around 523 nm, and the maximum UV absorption of gold nanoparticles after luminol luminescence produces chemiluminescence at 523 nm. After that, lifting occurred, and after 30 min, except for an absorption peak around 530 nm, the maximum UV absorption shifted to the right between 700 nm and 800 nm.
  • Example 9 Study on photothermal properties of gold nanoparticles after photo-mediated self-assembly: take 4 mL of aqueous solution containing 200 mM H 2 O 2 , 100 mM Fe 3+ , 200 ⁇ g/mL tAu and 200 ⁇ g/mL mAu/Lu 4 tubes, each tube of 1 mL, were allowed to stand for 0 min, 15 min, 30 min, and 45 min for cross-linking reaction, respectively, to obtain UV-mediated nanoparticle self-assembled aggregates, and their photothermal properties were measured. As shown in Figure 8a, the abscissa is the irradiation time.
  • the self-assembled aggregates of gold nanoparticles were irradiated by near-infrared laser (808 nm, 1 W/cm 2 ).
  • the abscissa is the static crosslinking reaction time.
  • the photothermal heating effect is the most obvious at 45 min, and the temperature can be increased by 28.6 °C.
  • the crosslinking reaction time is 60min, the photothermal heating effect is similar to that of 45min.
  • the total volume was 1 mL, and the total concentration of gold nanoparticles was 0, 50, 100, 200, 400 and 600 ⁇ g/mL, respectively, after 45 min of reaction , after being irradiated by 808 nm near-infrared laser (10 min), as shown in Figure 8c, the abscissa is the irradiation time, and the temperature increases with the increase of the concentration of gold nanoparticles. As shown in Figure 8d, the concentration of 600 ⁇ g/mL is the most obvious heating effect, and the temperature rises by about 40 °C. All indicated that gold nanoparticles had better photothermal effect after light-mediated aggregation.
  • Example 2 The materials of Examples 2, 3, 4, and 5 (400 ⁇ g/mL) were tested in the same way. After 600s of irradiation, the temperature rises were all lower than 10°C. (mixing in equal amounts) the temperature rises to 9.45201°C.
  • the mother solution of gold nanoparticles 2 (mAu) obtained in Example 4 was mixed with an aqueous solution of luminol (1 mg of gold nanoparticles corresponds to 12 mg of luminol), and after shaking for 20 h, it was centrifuged by ultrafiltration (5000 rpm ⁇ 10 min). After 3 times, the mother solution of gold nanoparticles 3-1 ( mAu/Lu-1 ) carrying chemiluminescence source luminol and photosensitive cross-linking agent was obtained.
  • Example 10 Cytotoxicity and photothermal treatment effect of gold nanoparticles after chemiluminescent light-mediated self-assembly: Cytotoxicity test method: Mouse breast cancer cells (4T1) were cultured in 96-well plates (density 8000 cells/well) After 24 hours of incubation, 0, 25, 50, 100, 200 ⁇ g/mL of the materials of Examples 3, 4, and 5 were added to each well and incubated for 24 hours to measure MTT. As can be seen from Figure 9a, amino-functionalized gold nanoparticles modified with PEG at different concentrations had very low toxicity to mouse breast cancer cells (4T1) at 24 h.
  • Intracellular photothermal therapy experimental method mouse breast cancer cells (4T1) were cultured in a 96-well plate (density 8000 cells/well), and after 24 hours of incubation, they were divided into six groups (PBS group, PBS+NIR group, tAu+ mAu group, tAu+mAu+NIR group, tAu+mAu/Lu group, tAu+mAu/Lu+NIR group), PBS means only cells without any material treatment; tAu means adding gold nanoparticles prepared in Example 3 1 ; mAu refers to adding gold nanoparticles 2 prepared in Example 4, mAu/Lu refers to adding gold nanoparticles 3 prepared in Example 5; NIR refers to near-infrared light irradiation at 808 nm (1 W/cm 2 , 10 min); MTT was measured after 24 h of cell growth; all without exogenous UV light, in the group containing gold nanoparticles, the total amount of
  • the cell viability in the tAu+mAu/Lu+NIR group was significantly lower than that in the other groups, indicating that the method of the present invention can realize the self-assembly of gold nanoparticles in situ in cells, and has better photothermal properties
  • the therapeutic effect can effectively kill tumor cells.
  • Intracellular photothermal therapy experimental method live&dead: Mouse breast cancer cells (4T1) were cultured in 6-well plates and divided into 6 groups (PBS group, PBS+NIR group, tAu+mAu group, tAu+mAu+NIR group , tAu+mAu/Lu group, tAu+mAu/Lu+NIR group), PBS means only cells without any material treatment; tAu means adding gold nanoparticles 1 prepared in Example 3; mAu means adding in Example 4 Prepared gold nanoparticles 2 , mAu/Lu refers to adding gold nanoparticles 3 prepared in Example 5, in the group containing gold nanoparticles, the total amount of gold nanoparticles is 50 ⁇ g/mL/well; NIR refers to 808 Near-infrared light irradiation at nm (1 W/cm 2 , 10 min); NIR refers to near-infrared light irradiation at 808 n
  • the self-assembly of gold nanoparticles can be induced by ultraviolet light irradiation in cells. After near-infrared light irradiation, it has a good photothermal treatment effect and can effectively kill tumor cells.
  • the gold nanoparticles did not aggregate, indicating that the simple reaction environment would not cause the gold nanoparticles 1 (tAu) prepared in Example 3 and 2 (tAu) and the gold nanoparticles 2 ( mAu) prepared in Example 4. ) gather.
  • the aggregation was observed by particle size (placement time was 30 min), as shown in Figure 11.
  • a is the direct irradiation of the solution (unshielded)
  • b is the indirect illumination (shielded) through the wall of a 1.5mL centrifuge tube.
  • the unshielded 405 nm laser can produce gold nanoparticles 1 (tAu) with crosslinking agent in Example 3 and gold nanoparticles 2 (mAu) modified with crosslinking agent methacrylic acid in Example 4.
  • Cross-linking as shown in Figure a, large aggregates can be formed in 15 minutes, and basically all agglomerate in 30 minutes; and the cross-linking efficiency of Figure b after shading is significantly reduced, not as good as the chemiluminescence-mediated gold nanoparticle in Example 8.
  • the particle aggregation effect is good, the hydrated particle size at 15 minutes is 95nm, and the hydrated particle size at 30 minutes is 200nm.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Plasma & Fusion (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Electrochemistry (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Medicinal Preparation (AREA)

Abstract

一种化学发光介导的纳米颗粒、自组装聚集体及其制备方法和应用。制备方法包括以下步骤：1)在纳米颗粒表面上修饰PEG；2)在PEG末端氨基上修饰光敏感交联剂；3)在修饰好光敏感交联剂表面吸附鲁米诺分子成为化学发光源和4)化学发光介导的纳米颗粒自组装。该方法首次使用了非外照发光源诱导光敏感的交联剂交联，从而使得金纳米颗粒聚集。获得的化学发光介导的纳米颗粒自组装聚集体具有较低的毒性和较好的光热治疗效果，适合于开发成一种基于光热疗法的抗肿瘤药物，具有重要的科研及经济价值。

Description

[根据细则37.2由ISA制定的发明名称]　化学发光介导的纳米颗粒、自组装聚集体及其制备方法和应用 技术领域
本发明属于纳米颗粒自组装技术领域，具体涉及化学发光介导的纳米颗粒自组装聚集体的制备方法，通过该方法制备的聚集体，以及该聚集体在制备基于光热疗法（photothermal therapy，PTT）的抗肿瘤药物中的应用。
 背景技术
众所周知，纳米颗粒的性质与其尺寸、结构、形态有着密切的关系。具有不同尺寸、结构、形态的纳米颗粒会呈现出不同的表面效应和量子效应，进而表现出不同的光学、电学、磁学、力学和化学性质，这一特性在信息存储器、生物传感器、纳米器件、临床诊断材料等领域中具有重要的研究和应用价值。因此，纳米颗粒的可控自组装一直以来都是纳米技术领域中的研究热点。
传统的纳米颗粒（尤其是金属纳米颗粒）自组装主要是通过纳米颗粒自发地组织或聚集成一种热力学稳定、分布均一、性能特殊的结构的过程。该过程主要依靠两个因素来形成自组装体系：其一是通过非共价键（特别是氢键）作用来形成稳定的纳米自组装体系；其二是纳米体系为了达到最低能级所产生的自发自组装。常用的纳米颗粒自组装方法主要包括溶剂挥发法、磁电光诱导法、界面法及模板法等。然而，由于依赖非共价键作用或电荷间作用，通过这些传统方法所获得的纳米自组装体系往往呈现出较差的重复性和稳定性，大大制约了材料在生物体系中的应用。
为了克服传统方法的缺点，小分子交联剂的使用应运而生，现有技术利用外部刺激，例如酸性细胞外pH，癌症相关的酶和光诱导来组装小的纳米颗粒，以便将表面等离子体共振转移到近红外区域进行光热疗法；尽管在过去的十年中，基于光响应分子（例如生色团，螺吡喃，偶氮苯）的异构化或二聚化，已经进行了大量关于光触发自组装的体外研究。但其在体内的应用很少被研究，这可能是由于光响应分子合成复杂和紫外线的组织穿透力有限。与此同时，在体内，外部照射穿透深度有限，对于深部的肿瘤并不能很好的诱导光敏剂发生交联，同时还一定程度上受到外部照射的条件要求，使得整体聚集效率并不是很高。
 技术问题
为了克服上述现有技术中存在的问题，本发明开发出一种简单、快速、绿色、稳定、可控的纳米颗粒自组装方法。该方法不仅适用于同种纳米颗粒之间的自组装，而且适用于不同纳米颗粒之间的杂化组装，为多功能纳米复合材料的制备提供了新的策略与手段。
 技术解决方案
本发明采用如下技术方案：一种化学发光介导的纳米颗粒，包括带有交联剂、鲁米诺的纳米颗粒与带有交联剂的纳米颗粒。
一种化学发光介导的纳米颗粒体系，包括带有交联剂和鲁米诺的纳米颗粒、带有交联剂的纳米颗粒、氧化剂、铁离子、水。优选的，带有交联剂的纳米颗粒与带有交联剂、鲁米诺的纳米颗粒的重量比为1∶（0.8～1.2），优选为1∶1。
一种化学发光介导的纳米颗粒自组装聚集体，由带有交联剂、鲁米诺的纳米颗粒与带有交联剂的纳米颗粒交联得到。
本发明以带有交联剂、鲁米诺的纳米颗粒与带有交联剂的纳米颗粒组成化学发光介导的纳米颗粒，纳米颗粒间的交联自组装无需外源光照，在氧化剂（比如过氧化氢）、铁离子存在下进行，与肿瘤细胞内常规环境近似；本发明以带有交联剂和鲁米诺的纳米颗粒、带有交联剂的纳米颗粒、氧化剂、铁离子、水组成化学发光介导的纳米颗粒体系，可以在无外源光照（激发）下制备纳米颗粒自组装聚集体。本发明公开了上述化学发光介导的纳米颗粒或者化学发光介导的纳米颗粒体系在无外源光照下制备纳米颗粒自组装聚集体中的应用。
本发明在没有外部激发的情况下，利用化学发光触发光敏剂的交联，显得尤为新颖，是一种简单、快速、可控的纳米颗粒自组装新方法，具有重大的研究及应用价值。
本发明带有交联剂、鲁米诺的纳米颗粒与带有交联剂的纳米颗粒中，交联剂不同，比如带有交联剂、鲁米诺的纳米颗粒中的交联剂为交联剂A，带有交联剂的纳米颗粒中的交联剂为交联剂B；交联剂A优选为甲基丙烯酸，交联剂B优选为四噻唑。
本发明中，交联剂的化学结构式如下：
Figure 450634dest_path_image001
。
鲁米诺的化学结构式如下：
Figure 385092dest_path_image002
。
上述化学发光介导的纳米颗粒自组装聚集体的制备方法，包括以下步骤。
（1）在纳米颗粒表面修饰交联剂，得到带有交联剂的纳米颗粒。
（2）在纳米颗粒表面修饰交联剂与鲁米诺，得到带有交联剂、鲁米诺的纳米颗粒。
（3）将步骤（1）带有交联剂的纳米颗粒、步骤（2）带有交联剂、鲁米诺的纳米颗粒交联，得到化学发光介导的纳米颗粒自组装聚集体。
本发明中，纳米颗粒为金属纳米颗粒、无机非金属纳米颗粒、有机纳米颗粒、无机-有机杂化纳米颗粒中的一种或几种；氧化剂为过氧化氢。
本发明中，在纳米颗粒表面修饰氨基聚乙二醇，再与交联剂反应，得到带有交联剂的纳米颗粒；在纳米颗粒表面修饰氨基聚乙二醇，再与交联剂、鲁米诺反应，得到带有交联剂、鲁米诺的纳米颗粒。
具体的，上述化学发光介导的纳米颗粒自组装聚集体的制备方法，为以下步骤。醇；优选的：按照纳米颗粒∶甲氧基聚乙二醇硫醇∶氨基聚乙二醇硫醇=1~2∶20∶20的质量比，向纳米颗粒原液中加入甲氧基聚乙二醇硫醇和氨基聚乙二醇硫醇，于室温搅拌12～48小时，经超滤离心、加水重悬，得到经PEG修饰的氨基功能化纳米颗粒的母液。
2）在PEG末端氨基上修饰交联剂四噻唑；优选的：按照交联剂四噻唑∶碳二胺∶N-羟基琥珀酰亚胺=1∶2~15∶2~15的质量比加入DMSO 中，再加入步骤1）获得的经PEG修饰的氨基功能化纳米颗粒的母液，于室温搅拌2~5小时，经超滤离心，得到带有交联剂的纳米颗粒；交联剂四噻唑如下：
Figure 581719dest_path_image003
。
3）在PEG末端氨基上修饰交联剂甲基丙烯酸；优选的：按照交联剂甲基丙烯酸∶碳二胺∶N-羟基琥珀酰亚胺=1∶2~15∶2~15的质量比加入乙醇中，再加入步骤1）获得的经PEG修饰的氨基功能化纳米颗粒的母液，于室温搅拌2~5小时，经超滤离心，得到带有交联剂甲基丙烯酸的纳米颗粒；交联剂甲基丙烯酸如下：
Figure 601627dest_path_image004
。
4）在带有交联剂甲基丙烯酸的纳米颗粒表面搭载鲁米诺；优选的：按照带有交联剂甲基丙烯酸的纳米颗粒∶鲁米诺= 1∶2~15的质量比，将带有交联剂甲基丙烯酸的纳米颗粒加入鲁米诺溶液中，于室温搅拌2~36小时，经超滤离心，得到带有交联剂、鲁米诺的纳米颗粒。
5）化学发光介导的纳米颗粒自组装得到化学发光介导的纳米颗粒自组装聚集体；优选的：将步骤2）制备的带有交联剂的纳米颗粒、步骤4）制备的带有交联剂、鲁米诺的纳米颗粒在过氧化氢、铁离子存在下，交联自组装得到化学发光介导的纳米颗粒自组装聚集体；带有交联剂的纳米颗粒与带有交联剂、鲁米诺的纳米颗粒的重量比为1∶（0.8～1.2），优选为1∶1。
作为一个实施例，将步骤2）制备的带有交联剂的纳米颗粒、步骤4）制备的带有交联剂、鲁米诺的纳米颗粒分别加水重悬，然后将两种溶液（纳米颗粒等重量比）加入含有H 2O 2、Fe 3+的H 2O溶液中，反应15~90 min，得到纳米颗粒自组装聚集体。
现有技术中的纳米颗粒大都适用于本发明的制备方法，能够最终实现化学发光介导的纳米颗粒自组装。优选的，在上述制备方法中，步骤（1）中所述纳米颗粒选自金属纳米颗粒、无机非金属纳米颗粒、有机纳米颗粒、无机-有机杂化纳米颗粒中的任意一种或其任意比例的混合物，更优选的，所述金属纳米颗粒为金纳米颗粒，所述无机非金属纳米颗粒为四氧化三铁纳米颗粒，所述有机纳米颗粒为聚多巴胺纳米颗粒。
优选的，在上述制备方法中，步骤（1）中所述甲氧基聚乙二醇硫醇为两个末端分别由甲氧基和巯基修饰的聚乙二醇，其选自M-PEG 2000-SH、M-PEG 5000-SH、M-PEG 10000-SH、M-PEG 20000-SH中的任意一种或其任意比例的混合物；更优选的，所述甲氧基聚乙二醇硫醇为M-PEG 5000-SH。
优选的，在上述制备方法中，步骤（1）中所述氨基聚乙二醇硫醇为两个末端分别由氨基和巯基修饰的聚乙二醇，其选自NH 2-PEG 2000-SH、NH 2-PEG 5000-SH、NH 2-PEG 10000-SH、NH 2-PEG 20000-SH中的任意一种或其任意比例的混合物；更优选的，所述氨基聚乙二醇硫醇为NH 2-PEG 5000-SH。
本发明公开了上述化学发光介导的纳米颗粒或者化学发光介导的纳米颗粒体系在无外源光照下制备纳米颗粒自组装聚集体中的应用；上述化学发光介导的纳米颗粒或者化学发光介导的纳米颗粒体系或者化学发光介导的纳米颗粒自组装聚集体在制备纳米药物中的应用。纳米药物为治疗肿瘤的药物，优选为光热治疗药物。
 有益效果
由于上述技术方案的运用，本发明与现有技术相比具有如下优点。
（1）本发明中首次使用了体积较小的光敏感交联剂四噻唑和甲基丙烯酸，成功避免了由于交联剂体积过大而导致的纳米颗粒沉淀。
（2）通过设计交联剂的用量与反应时间，可有效调控纳米颗粒的自组装程度，从而实现纳米材料的可控自组装。
（3）利用化学发光源照射实现相同或不同种类纳米颗粒的自组装，大大扩展了自组装技术的适用范围。
（4）首次使用发光的鲁米诺来诱导紫外敏感性交联剂在体内或体外交联，从而使得金纳米颗粒可以有效的聚集，从而进一步增强肿瘤光声成像和肿瘤光热治疗的敏感性。
（5）本发明的自组装方法具有简便、快捷、稳定、可控和绿色环保等特点，极大地节约了制备时间，是一种快速制备多功能纳米材料的普适性新方法，具有广阔的应用空间。
 附图说明
图1为实施例3中带有交联剂的纳米颗粒 1修饰过程的示意图。
图2为实施例4中交联剂甲基丙烯酸修饰的金纳米颗粒 2修饰过程的示意图。
图3为实施例5中搭载化学发光源鲁米诺与光敏感交联剂的金纳米颗粒3修饰过程的示意图。
图4为不同修饰的金纳米粒TEM图。
图5为实施例6中搭载化学发光源鲁米诺与光敏感交联剂的金纳米颗粒 3的化学发光成像的材料表征图。
图6为实施例7中化学发光介导的纳米颗粒自组装过程的示意图。
图7为实施例8中金纳米颗粒经紫外光照射诱导自组装前后TEM图像及交联前后的紫外吸收和粒径变化情况。
图8为实施例9中金纳米颗粒经化学发光源诱导交联前后的光热性能研究。
图9为实施例10中金纳米颗粒经化学发光源诱导自组装前后对细胞的毒性和光热治疗效果研究。
图10为对照组实施例3中带有交联剂的纳米颗粒1(tAu)和实施例4中交联剂甲基丙烯酸修饰的金纳米颗粒2(mAu)反应交联（未交联）。
图11为单独的金纳米颗粒1（tAu）或者单独的金纳米颗粒2（mAu）在鲁米诺化学发光条件下不会发生聚集。
图12为直接照射溶液（未经遮挡）和透过1.5mL离心管壁间接照射（遮挡）（tAu+mAu）。
 本发明的实施方式
本发明制备的带有交联剂的纳米颗粒、制备的带有交联剂/鲁米诺的纳米颗粒组成化学发光介导的纳米颗粒，在无需外源光照条件下，可以自组装得到聚集体。具体而言，本发明提供了一种化学发光介导的纳米颗粒自组装聚集体的制备方法，在没有外部激发的情况下将自发光在这种方式下应用于光敏剂的交联，具有易于修饰、可控等优点，为纳米颗粒自组装技术中的一种优异方法。
下文将结合附图和具体实施例来进一步阐述本发明。应当理解的是，这些实施例仅用于解释和说明本发明中的技术方案，而并非旨在限制本发明的范围。此外，除非另有说明，下列实施例中所使用的材料、试剂、仪器等均可通过商业手段获得；除非另有说明，实施例涉及的具体方法都为本领域常规方法。
HAuCl 4 ·4H 2O      阿拉丁上海试剂有限公司。
柠檬酸三钠·3H2O  国药集团化学试剂有限公司。
巯基-聚乙二醇-甲氧基(SH-PEG 5000-M) 北京键凯科技有限公司。
巯基-聚乙二醇-氨基(SH-PEG 5000-NH 2) 北京键凯科技有限公司。
浓盐酸           国药集团化学试剂有限公司。
4-甲酰基苯甲酸    百灵威科技有限公司。
苯磺酰肼         百灵威科技有限公司。
苯胺            百灵威科技有限公司。
吡啶              国药集团化学试剂有限公司。
亚硝酸钠          国药集团化学试剂有限公司。
二甲基亚砜        国药集团化学试剂有限公司。
乙醇             上海凌峰化学试剂有限公司。
乙酸乙酯         上海凌峰化学试剂有限公司。
石油醚           上海凌峰化学试剂有限公司。
N-羟基琥珀酰亚胺（NHS）  阿拉丁上海试剂有限公司。
1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)   阿拉丁上海试剂有限公司。
30%H 2O 2           国药集团化学试剂有限公司。
铁氰化钾          国药集团化学试剂有限公司。
鲁米诺            阿拉丁上海试剂有限公司。
ME204 电子分析天平       梅特勒-拖利多上海有限公司。
KQ-100KD 超声清洗仪     昆山市超声仪器有限公司。
Direct Q5 超纯水系统      美国 Merk Millipore 公司。
Agilent 1260 高效液相色谱仪           美国安捷伦公司。
Alphal-4LSCplus RC6 冷冻干燥机        美国阿尔法公司。
Tecnai G2 spirit BioTwin 透射电子显微镜        美国 FEI 公司。
Lambda 750 UV-vis-NIR 紫外光谱仪     美国 PerkinElmer 公司。
Nano ZS90 DLS 纳米粒度仪             英国马尔文有限公司。
WFH-204B 手提式紫外分析仪        杭州奇威仪器有限公司。
Hei-VAP Presision ML/G3 旋转蒸发仪       德国 Heidolph 公司。
D-405nm-HS-1W-15031117 激光器       长春新产业光电技术有限公司。
MDL-N-808 nm-10W 激光器            长春新产业光电技术有限公司。
Ax5 Series 热成像仪                   美国FLIR公司。
实施例1：交联剂四噻唑的合成与表征。
Figure 541901dest_path_image005
。
（1）向盛有4-甲酰基苯甲酸（1.50 g，10 mmol）的圆底烧瓶中加入65 mL的乙醇溶液，随后加入苯磺酰肼于室温搅拌30 min。反应结束后，用100 mL 超纯水沉淀产物，随后抽滤干燥，可得腙加和产物2.7 g，收率2.7 g（88.8%）。称取1.2 g腙加和产物溶于1.5 mL吡啶溶液中，制成溶液A。
（2）向苯胺（0.46 mL，5 mmol）中滴加NaNO 2 (0.17 g，5 mmol)溶液（15 mL），制成溶液B。
（3）将溶液B加入溶液A中，于0℃搅拌6 h，待反应结束后，低压旋蒸蒸发掉溶剂，加入100 mL水沉淀产物，再抽滤、干燥，得到4-（2-苯基-2H-四唑-5-基）苯甲酸的粗产物，经硅胶柱层析（乙酸乙酯：石油醚=2：1）提纯后，得到0.524 g目标产物交联剂四噻唑，产率为70%。 1H-NMR (400 MHz, DMSO)： δ 8.34-8.28 (m, 2H), 8.22-8.14(m, 4H), 7.72(t, J=7.5Hz, 2H), 7.65(t, J=7.3Hz,1H)。
实施例2：金纳米颗粒的制备及其表面的PEG修饰：向超纯水（100 mL）中加入1wt%的氯金酸溶液（0.6 mL），加热至沸腾后加入1wt%的柠檬酸钠溶液（3 mL），待体系变成酒红色后，继续煮沸30 min，得到金纳米颗粒原液。
待冷却后，向其中依次加入M-PEG 5000-SH（20 mg）和NH 2-PEG 5000-SH（20 mg），于室温搅拌24 h。经超滤离心（5000 rpm × 10 min）3次，用超纯水重悬，得到PEG修饰的氨基功能化金纳米颗粒母液3mL，浓度为3.8mg/mL。
实施例3：金纳米颗粒表面PEG末端修饰交联剂四噻唑：将交联剂四噻唑∶碳二胺∶N-羟基琥珀酰亚胺=1∶10∶10的质量比加入1.4mL 二甲基亚砜中，室温活化20分钟，得到活化好的交联剂溶液。
如图1所示，取1mL实施例2中制得的经PEG修饰的氨基功能化金纳米颗粒母液，向其中加入上述活化好的交联剂溶液（含10.5 mg 的交联剂），于室温搅拌反应3h。经超滤离心（5000 rpm × 10 min）3次后，得到带有交联剂的金纳米颗粒1，为tAu，制得母液，为4mg/mL。
实施例4：金纳米颗粒表面PEG末端修饰交联剂甲基丙烯酸：将交联剂甲基丙烯酸∶碳二胺∶N-羟基琥珀酰亚胺=1∶10∶10的质量比加入1.4mL 乙醇中，室温活化20分钟，得到活化好的交联剂溶液。
如图2所示，取1mL实施例2中制得的经PEG修饰的氨基功能化金纳米颗粒母液，向其中加入上述活化好的交联剂溶液（含2 mL甲基丙烯酸），室温搅拌反应3 h。经超滤离心（5000 rpm × 10 min）3次后，得到经交联剂甲基丙烯酸修饰的金纳米颗粒2，为mAu, 制得母液，4mg/mL。
实施例5：搭载化学发光鲁米诺：如图3所示，将实施例4中得到的金纳米颗粒2(mAu) 母液与鲁米诺水溶液（1 mg 金纳米颗粒对应10 mg 鲁米诺）混合，振荡20 h后，经超滤离心（5000 rpm × 10 min）3次后，得到搭载化学发光源鲁米诺与光敏感交联剂的金纳米颗粒 3,为mAu/Lu，制得母液，4mg/mL。
图4为不同修饰的金纳米粒TEM图。其中，a为PEG修饰的氨基功能化金纳米颗粒的电镜图，b为实施例3中得到的带有交联剂的金纳米颗粒1（tAu）的电镜图，c为实施例4中得到的带有交联剂的金纳米颗粒2（mAu）的电镜图，d为实施例5中得到的搭载化学发光源鲁米诺与光敏感交联剂的金纳米颗粒3（mAu/Lu）的电镜图，可以看出这四种金纳米颗粒的尺寸分布较为均匀（约为20 nm），本发明的颗粒在溶液中的分散性较好，不会聚集，也不会因为表面修饰交联剂体积过大而导致纳米颗粒沉淀聚集。
实施例6：搭载化学发光源鲁米诺与光敏感交联剂的金纳米颗粒 3 的发光强度及信号：将实施例5中制得的搭载化学发光源鲁米诺与光敏感交联剂的金纳米颗粒 3用超纯水重悬，同时准备含有200mM H 2O 2、100mM Fe 3+（铁氰化钾）的1mL水溶液，向其中加入搭载化学发光源鲁米诺与光敏感交联剂的金纳米颗粒 3，静置0~90 min，期间没有外界激发光源。测试经交联剂甲基丙烯酸修饰的金纳米颗粒2、搭载化学发光源鲁米诺与光敏感交联剂的金纳米颗粒 3 、鲁米诺的发光强度；如图5a所示，为屏蔽激发光源后接收到的荧光强度值，搭载化学发光源鲁米诺与光敏感交联剂的金纳米颗粒（mAu/Lu）与单纯鲁米诺（Lu）荧光信号重合。说明体外H 2O 2和Fe 3+的环境中可以使鲁米诺发出波长在450 nm左右的光；如图5b、5c所示，随着静置时间的延长，金纳米颗粒 3的发光强度逐渐减弱。
实施例 7：化学发光源介导的金纳米颗粒自组装：如图6所示，取等量的实施例3中制得的金纳米颗粒 1 （tAu ）和实施例5中制得的金纳米颗粒 3 （mAu/Lu ），其中金纳米颗粒 1 、金纳米颗粒 3重量一致，均为50μg。加入含200 mM H 2O 2和100 mM Fe 3+ 的水溶液中，总体积为1mL，交联反应时间45 min，得到紫外光介导的纳米颗粒自组装聚集体。
 实施例 8：金纳米颗粒自组装前后的粒径分布和紫外吸收的变化：取等量的实施例3中制得的金纳米颗粒 1 （tAu ）和实施例5中制得的金纳米颗粒 3 （mAu/Lu ），其中金纳米颗粒 1 、金纳米颗粒 3重量一致，均为50μg。加入含200 mM H 2O 2和100 mM Fe 3+ 的水溶液中，总体积为1mL,交联反应时间45 min，得到紫外光介导的纳米颗粒自组装聚集体。测试其TEM、粒径分布（DLS）和紫外吸收情况，取样时间为0 min、15 min、30 min。如图7a~7c所示，鲁米诺发光前的金纳米颗粒（金纳米颗粒反应母液）水合粒径约为50 nm，其电镜（TEM）为分散的，大小均一的20nm左右的圆形颗粒（图7a），发光产生化学反应后15 min的金纳米颗粒开始逐渐聚集，水合粒径最大可以为280 nm左右，其电镜如图7b，呈逐渐聚集状态，而30 min后的水合粒径最大约为400 nm左右，其电镜成团聚集，可以聚集成很大的颗粒（图7c）。此外，纳米颗粒的聚集程度随反应时间的延长而增强（见图7d）。图7e是金纳米颗粒光介导聚集后的紫外吸收变化，鲁米诺发光前的金纳米颗粒紫外吸收最大在523 nm左右，鲁米诺发光产生化学发光后15 min的最大紫外吸收在523 nm之后发生了抬起，而30 min后除了在530 nm左右有一个吸收峰以外，最大紫外吸收右移到了700 nm-800 nm之间。
实施例9：金纳米颗粒光介导自组装后光热特性研究：取4mL含200 mM H 2O 2、100 mM Fe 3+、200 μg/mL tAu 和 200 μg/mL mAu/Lu 水溶液，分4管，每管1mL，分别静置交联反应0 min、15 min、30 min、45 min，得到紫外光介导的纳米颗粒自组装聚集体，测其光热特性。如图8a所示，横坐标为照射时间，金纳米颗粒自组装聚集体经近红外激光照射（808 nm，1 W/cm 2），照射时间越长，温度升高越大。如图8b所示，横坐标为静置交联反应时间，随着交联反应时间的延长，在45 min时，光热升温效果最明显，温度可升高28.6℃。交联反应时间在60min时，光热升温效果与45min差不多。
取等量实施例3中制得的金纳米颗粒 1 （tAu ）和实施例5中制得的金纳米颗粒 3 （mAu/Lu ），其中金纳米颗粒 1 、金纳米颗粒 3重量一致。加入含200 mM H 2O 2和100 mM Fe 3+ 的水溶液中，总体积为1mL,其中金纳米颗粒总浓度分别为0，50，100，200，400和600 μg/mL，在反应45min后，经808 nm近红外激光照射（10 min）后，如图8c所示，横坐标为照射时间，随着金纳米颗粒浓度的增大，温度升高越大。如图8d所示，浓度为600 μg/mL是升温效果最为明显，温度升高约40℃。均表明金纳米颗粒光介导聚集后具有较好的光热效应。
同样的方法测试了实施例2、3、4、5的材料（400 μg/mL），照射600s后，升温全部低于10℃，具体的，tAu升温8.58929℃、mAu升温9.21509℃、tAu+mAu（等量混合）升温9.45201℃。
将实施例4中得到的金纳米颗粒2(mAu) 母液与鲁米诺水溶液（1 mg 金纳米颗粒对应12mg 鲁米诺）混合，振荡20 h后，经超滤离心（5000 rpm × 10 min）3次后，得到搭载化学发光源鲁米诺与光敏感交联剂的金纳米颗粒 3-1 （mAu/Lu-1 ）母液。取1mL含200 mM H 2O 2、100 mM Fe 3+、200 μg/mL tAu 和 200 μg/mL mAu/Lu-1 水溶液置入一个反应管，静置交联反应45 min，经近红外激光照射（808 nm，1 W/cm 2）600s后，温度可升高24.7℃。
取1mL含200 mM H 2O 2 、100 mM Fe 3+、180 μg/mL tAu 和 220 μg/mL mAu/Lu水溶液置入一个反应管，静置交联反应45 min，经近红外激光照射（808 nm，1 W/cm 2）600s后，温度可升高22.1℃。
取1mL含200 mM H 2O 2 、100 mM Fe 3+、250 μg/mL tAu 和 150 μg/mL mAu/Lu水溶液置入一个反应管，静置交联反应45 min，经近红外激光照射（808 nm，1 W/cm 2）600s后，温度可升高20.3℃。
实施例10：金纳米颗粒化学发光光介导自组装后的细胞毒性及光热治疗效果：细胞毒性实验方法：小鼠乳腺癌细胞（4T1）在96孔板中（密度8000个/孔）培养，孵育24 h后，分别向各个孔中加入0，25，50，100，200 μg/mL的实施例3、4、5的材料孵育24h后，测MTT。从图9a中可以看出，不同浓度经PEG修饰的氨基功能化金纳米颗粒对小鼠乳腺癌细胞（4T1）在24 h具有很低的毒性。
细胞内光热治疗实验方法：小鼠乳腺癌细胞（4T1）在96孔板中（密度8000个/孔）培养，孵育24 h后，分为六组（PBS组，PBS+NIR组，tAu+mAu组，tAu+mAu+NIR组，tAu+mAu/Lu组，tAu+mAu/Lu+NIR组），PBS指只有细胞不加任何材料处理；tAu指加入实施例3中制得的金纳米颗粒 1；mAu指加入实施例4中制得的金纳米颗粒 2，mAu/Lu指加入实施例5中制得的金纳米颗粒 3；NIR指808 nm的近红外光照射（1 W/cm 2，10 min）；细胞生长24 h后测MTT；全部没有外源紫外光照，含有金纳米粒子的组别中，金纳米粒子总量为50 μg/mL/孔。如图9b所示，tAu+mAu/Lu+NIR组细胞存活率较其他组别明显下降，说明本发明的方法能够在细胞内原位实现金纳米颗粒的自组装，并具有较好的光热治疗效果，能够有效杀死肿瘤细胞。
细胞内光热治疗实验方法（live&dead）：小鼠乳腺癌细胞（4T1）在6孔板中培养，分为6组（PBS组，PBS+NIR组，tAu+mAu组，tAu+mAu+NIR组，tAu+mAu/Lu组，tAu+mAu/Lu+NIR组），PBS指只有细胞不加任何材料处理；tAu指加入实施例3中制得的金纳米颗粒 1；mAu指加入实施例4中制得的金纳米颗粒 2，mAu/Lu指加入实施例5中制得的金纳米颗粒 3，含有金纳米粒子的组别中，金纳米粒子总量为50 μg/mL/孔；NIR指808 nm的近红外光照射（1 W/cm 2，10 min）；NIR指808 nm的近红外光照射（1 W/cm 2，10 min）；细胞生长24 h后用live&dead染色试剂盒染色30 min，荧光显微镜下10倍拍照；全部没有外源紫外光照。如图9c所示，金纳米颗粒在细胞内经紫外光照射会诱导自组装，经近红外光照射后，具有较好的光热治疗效果，能够有效杀死肿瘤细胞。
取等量的实施例3中制得的金纳米颗粒 1 （tAu ）和实施例4中制得的金纳米颗粒 2 （mAu ），其中金纳米颗粒 1 、金纳米颗粒 2重量一致，均为50μg。加入含200 mM H 2O 2和100 mM Fe 3+ 的水溶液中，总体积为1mL，放置时间30 min，测试其透射电镜TEM，取样时间为0 min、15 min和30 min。如图10，金纳米颗粒并未发生聚集，说明单纯的反应环境并不会使实施例3中2制得的金纳米颗粒 1 （tAu ）和实施例4中制得的金纳米颗粒2（mAu）聚集。用粒径观察其聚集情况（放置时间30 min），如图11。
分别取50μg的实施例3中制得的金纳米颗粒1（tAu）和50μg实施例4中制得的金纳米颗粒2（mAu），分别加入含200 mM H 2O 2和100 mM Fe 3+ 的水溶液中，各自再加入化学发光剂鲁米诺30μg，总体积为1mL,放置30 min，用粒径观察其聚集情况，如图11，都小于100nm，可以发现单独的金纳米颗粒1（tAu）或者单独的金纳米颗粒2（mAu）在鲁米诺化学发光条件下不会发生聚集，以PEG修饰的氨基功能化金纳米颗粒（Au）作对照。将50μg实施例5中制得的金纳米颗粒3（mAu/Lu）加入含200 mM H 2O 2和100 mM Fe 3+ 的水溶液中，总体积为1mL,放置30 min，用粒径观察也无聚集情况。
取等量的实施例3中制得的金纳米颗粒 1 （tAu ）和实施例4中制得的金纳米颗粒 2 （mAu ），其中金纳米颗粒 1 、金纳米颗粒 3重量一致，均为50μg，加入水溶液中，总体积为1mL；配置两份一样的体系，一份用405nm激光器0.5w/cm 2照射不同时间，另一份用405nm激光器0.5w/cm 2透过1.5mL离心管照射不同时间。如图12，其中a图为直接照射溶液（未经遮挡），b为透过1.5mL离心管壁间接照射（遮挡）。可以发现，未经遮挡的405nm激光器可使实施例3中带有交联剂的金纳米颗粒1（tAu）和实施例4中经交联剂甲基丙烯酸修饰的金纳米颗粒2（mAu）发生交联，如图a所示，15min即可形成较大的聚集体，30min时基本全部团聚；而遮挡后的b图的交联效率明显降低，不如实施例8中化学发光介导的金纳米颗粒聚集效果好，15分钟时的水合粒径95nm，30分钟时的水合粒径在200nm。

Claims (10)

  1. 一种化学发光介导的纳米颗粒，其特征在于，包括带有交联剂、鲁米诺的纳米颗粒与带有交联剂的纳米颗粒。
  2. 根据权利要求1所述化学发光介导的纳米颗粒，其特征在于，在纳米颗粒表面修饰氨基聚乙二醇，再与交联剂反应，得到带有交联剂的纳米颗粒；在纳米颗粒表面修饰氨基聚乙二醇，再与交联剂、鲁米诺反应，得到带有交联剂、鲁米诺的纳米颗粒。
  3. 一种化学发光介导的纳米颗粒体系，其特征在于，包括带有交联剂和鲁米诺的纳米颗粒、带有交联剂的纳米颗粒、氧化剂、铁离子、水。
  4. 根据权利要求3所述化学发光介导的纳米颗粒体系，其特征在于，所述氧化剂为过氧化氢。
  5. 一种化学发光介导的纳米颗粒自组装聚集体，其特征在于，所述化学发光介导的纳米颗粒自组装聚集体由带有交联剂、鲁米诺的纳米颗粒与带有交联剂的纳米颗粒交联得到。
  6. 根据权利要求5所述化学发光介导的纳米颗粒自组装聚集体，其特征在于，交联剂的化学结构式如下：
    Figure 892161dest_path_image001
    。
  7. 根据权利要求5所述化学发光介导的纳米颗粒自组装聚集体，其特征在于，纳米颗粒为金属纳米颗粒、无机非金属纳米颗粒、有机纳米颗粒、无机-有机杂化纳米颗粒中的一种或几种。
  8. 权利要求5所述化学发光介导的纳米颗粒自组装聚集体的制备方法，其特征在于，包括以下步骤：
    （1）在纳米颗粒表面修饰交联剂，得到带有交联剂的纳米颗粒；
    （2）在纳米颗粒表面修饰交联剂与鲁米诺，得到带有交联剂、鲁米诺的纳米颗粒；
    （3）将步骤（1）带有交联剂的纳米颗粒、步骤（2）带有交联剂、鲁米诺的纳米颗粒交联，得到化学发光介导的纳米颗粒自组装聚集体。
  9. 根据权利要求8所述化学发光介导的纳米颗粒自组装聚集体的制备方法，其特征在于，所述交联在氧化剂、铁离子存在下进行；所述交联无需外源光照。
  10. 权利要求1所述化学发光介导的纳米颗粒或者权利要求3所述化学发光介导的纳米颗粒体系在无外源光照下制备纳米颗粒自组装聚集体中的应用；权利要求1所述化学发光介导的纳米颗粒或者权利要求3所述化学发光介导的纳米颗粒体系或者权利要求5所述化学发光介导的纳米颗粒自组装聚集体在制备纳米药物中的应用。
PCT/CN2021/079615 2021-03-08 2021-03-08 化学发光介导的纳米颗粒、自组装聚集体及其制备方法和应用 WO2022188013A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/079615 WO2022188013A1 (zh) 2021-03-08 2021-03-08 化学发光介导的纳米颗粒、自组装聚集体及其制备方法和应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/079615 WO2022188013A1 (zh) 2021-03-08 2021-03-08 化学发光介导的纳米颗粒、自组装聚集体及其制备方法和应用

Publications (1)

Publication Number Publication Date
WO2022188013A1 true WO2022188013A1 (zh) 2022-09-15

Family

ID=83227322

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/079615 WO2022188013A1 (zh) 2021-03-08 2021-03-08 化学发光介导的纳米颗粒、自组装聚集体及其制备方法和应用

Country Status (1)

Country Link
WO (1) WO2022188013A1 (zh)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102520195A (zh) * 2011-12-30 2012-06-27 天津市协和医药科技集团有限公司 一种嗜铬粒蛋白a化学发光免疫分析试剂盒及其制备方法
CN105424921A (zh) * 2015-11-06 2016-03-23 上海师范大学 功能化碳纳米管-铂-鲁米诺纳米复合材料及制备与应用
CN106066324A (zh) * 2016-05-30 2016-11-02 济南大学 一种电致化学发光生物传感器标记物的制备方法及应用
EP3338799A1 (en) * 2008-11-25 2018-06-27 AlderBio Holdings LLC Antibodies to il-6 and use thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3338799A1 (en) * 2008-11-25 2018-06-27 AlderBio Holdings LLC Antibodies to il-6 and use thereof
CN102520195A (zh) * 2011-12-30 2012-06-27 天津市协和医药科技集团有限公司 一种嗜铬粒蛋白a化学发光免疫分析试剂盒及其制备方法
CN105424921A (zh) * 2015-11-06 2016-03-23 上海师范大学 功能化碳纳米管-铂-鲁米诺纳米复合材料及制备与应用
CN106066324A (zh) * 2016-05-30 2016-11-02 济南大学 一种电致化学发光生物传感器标记物的制备方法及应用

Similar Documents

Publication Publication Date Title
Gao et al. A COF-based nanoplatform for highly efficient cancer diagnosis, photodynamic therapy and prognosis
Xu et al. Integration of IR‐808 sensitized upconversion nanostructure and MoS2 nanosheet for 808 nm NIR light triggered phototherapy and bioimaging
Chen et al. NIR-II light activated photodynamic therapy with protein-capped gold nanoclusters
Shan et al. Upconverting organic dye doped core-shell nano-composites for dual-modality NIR imaging and photo-thermal therapy
Ren et al. Light-activated oxygen self-supplied starving therapy in near-infrared (NIR) window and adjuvant hyperthermia-induced tumor ablation with an augmented sensitivity
US6514481B1 (en) Magnetic nanoparticles for selective therapy
Lin et al. A dual-responsive nanocapsule via disulfide-induced self-assembly for therapeutic agent delivery
Su et al. BODIPY@ carbon dot nanocomposites for enhanced photodynamic activity
Liu et al. Fluorescence-enhanced covalent organic framework nanosystem for tumor imaging and photothermal therapy
Xu et al. Tuning the properties of atomically precise gold nanoclusters for biolabeling and drug delivery
Ding et al. A NO/ROS/RNS cascaded-releasing nano-platform for gas/PDT/PTT/immunotherapy of tumors
Benyettou et al. Microwave assisted nanoparticle surface functionalization
RU2546663C2 (ru) Комплексное соединение металл-сален и способ его получения
CN111978556B (zh) 一种硒化zif-67的制备方法及应用
Cai et al. Polypyrrole-coated UCNPs@ mSiO 2@ ZnO nanocomposite for combined photodynamic and photothermal therapy
CN110194822B (zh) 一种基于单臂TPE分子的温敏型双荧光Pdots的制备及应用
Wang et al. Polyetherimide functionalized carbon dots with enhanced red emission in aqueous solution for bioimaging
Zou et al. A glutathione responsive pyrrolopyrrolidone nanotheranostic agent for turn-on fluorescence imaging guided photothermal/photodynamic cancer therapy
Xia et al. Near-infrared organic fluorescent nanoparticles for long-term monitoring and photodynamic therapy of cancer
CN110893237A (zh) 铜钯合金纳米颗粒和自噬抑制剂在制备基于光热效应杀伤肿瘤的药物或试剂盒中的应用
Dang et al. Galactose conjugated boron dipyrromethene and hydrogen bonding promoted J-aggregates for efficiently targeted NIR-II fluorescence assistant photothermal therapy
CN110960697A (zh) 一种两性离子修饰树状大分子包裹硫化铜纳米颗粒/pDNA复合物的制备方法
WO2022257855A1 (zh) 一种可实现靶向光热和化学协同治疗的h-BN/MoS2纳米探针及其制备方法和应用
Zhao et al. Magnetic mesoporous silica nanoparticles mediated redox and pH dual-responsive target drug delivery for combined magnetothermal therapy and chemotherapy
Kang et al. Flexible human serum albumin nanocapsules to enhance drug delivery and cellular uptake for photodynamic/chemo cancer therapy

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21929495

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21929495

Country of ref document: EP

Kind code of ref document: A1