CN112823809A - Chlorin e 6-nanogold compound for tumor photothermal synergistic photodynamic therapy and preparation method thereof - Google Patents

Chlorin e 6-nanogold compound for tumor photothermal synergistic photodynamic therapy and preparation method thereof Download PDF

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CN112823809A
CN112823809A CN201911139470.5A CN201911139470A CN112823809A CN 112823809 A CN112823809 A CN 112823809A CN 201911139470 A CN201911139470 A CN 201911139470A CN 112823809 A CN112823809 A CN 112823809A
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马宁宁
马超
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Jiangsu Normal University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/338—Polymers modified by chemical after-treatment with inorganic and organic compounds

Abstract

A chlorin e 6-nanogold compound for tumor photothermal synergistic photodynamic therapy and a preparation method thereof specifically comprise the following steps: preparing hollow spike-shaped nano gold through a displacement reaction of a chloroauric acid compound and silver nanoparticles; synthesizing thioctic acid end polyethylene glycol cross-linked dihydroporphin e6 functional molecules; the prepared nanogold is used as a carrier, and lipoic acid polyethylene glycol methoxyl and LA-PEG-Ce6 in a certain proportion are coupled on the surface of the nanogold at the same time, so that a chlorin e 6-nanogold compound with double functions of near infrared absorption and photosensitization is constructed. The compound has the advantages of low cost, simple preparation method, strong stability, good biocompatibility and the like. Tumor cell experiments further prove that the GNS-Ce6 compound has a remarkable effect of improving the uptake efficiency of chlorin e6 and nanogold; the mediated photothermal and photodynamic therapy can effectively reduce the survival rate of cancer cells.

Description

Chlorin e 6-nanogold compound for tumor photothermal synergistic photodynamic therapy and preparation method thereof
Technical Field
The invention relates to the fields of chemical synthesis and biomedicine, in particular to a chlorin e 6-nanogold compound for tumor photothermal synergistic photodynamic therapy and a preparation method thereof.
Background
Cancer is one of the major diseases that endanger human health, and has received more and more attention from scientific research and medical workers. The traditional treatment means of malignant tumor mainly comprises chemotherapy, radiotherapy, operation treatment and the like. At present, more interdisciplinary disciplines are concerned with the development and utilization of new strategies for diagnosis and treatment, combining the advantages and disadvantages of various treatment methods.
Photodynamic therapy (PDT), as a potential novel therapeutic approach, is mostly used for the treatment of cancer and other malignant diseases, and mainly consists of three elements: a light source, tissue oxygen, and a photosensitizer. The photosensitizer has no toxicity to cells under the condition of no light source irradiation, and molecular oxygen can be converted into singlet oxygen under the irradiation of a light source with a specific excitation wavelength: (1O2) Thereby achieving the purpose of killing cancer cells. Compared to traditional chemotherapy and radiotherapy, photodynamic therapy with localized illumination has significantly selective and fewer side effects. However, photodynamic therapy also has a number of drawbacks: (1) the distribution selectivity of the photosensitizer in biological tissues is low, and the photosensitizer cannot be efficiently enriched in tumors; (2) as the local molecular oxygen in the tissue is consumed, the tumor is in a hypoxic environment to terminate the production of singlet oxygen. The two points greatly limit the potential application of photodynamic therapy in clinic. Therefore, there is an urgent need to combine other therapeutic approaches in photodynamic therapy to develop a novel synergistic therapeutic strategy.
In order to overcome the defects of photodynamic therapy, a plurality of researchers skillfully combine the advantages of the nano materials and introduce the nano materials into the field of nano medicine. Particularly, gold nanomaterials are one of the hot spots in nano-medical research due to the advantages of good biocompatibility, easy surface modification, unique optical physicochemical properties and the like. The gold nano structure, especially the gold nano rod, gold nano star, gold nano cage and other nano gold with near infrared absorption performance can absorb and convert near infrared light to generate high heat to ablate tumor, so that the gold nano structure becomes a good photo-thermal absorption reagent, and opens up a new direction for the application of the gold nano structure in the photo-thermal treatment field. The gold nano material can be enriched in tumor lesion tissues in a multi-selective manner due to the Enhanced Permeation and Retention (EPR) effect, and is often used as a passive targeting transport carrier for genes, proteins, medicaments and the like. In addition, the gold nano material modified by water-soluble macromolecules such as polyethylene glycol can realize long-acting circulation in blood and is not easy to aggregate.
Synergistic treatment strategies combining nanomaterials, photothermal effects and photodynamic therapy have been developed. Researchers design that photosensitizers are loaded on the surface of a nano structure, and after the photosensitizers act with cancer cells or tumor tissues, combined treatment is carried out under the irradiation of exciting light with single wavelength or two different wavelengths, so that a stronger tumor killing effect is achieved. The design scheme solves the problem of low tumor enrichment rate of the photosensitizer, and can achieve the purpose of synergy by means of the unique optical property of the nano structure. At present, gold nanostructures connected with photosensitizer loads are mostly concentrated on nanorods, and other structures are still rarely reported. More gold nanostructures with excellent near-infrared absorption effect are developed, and the possibility of designing photo-thermal and photodynamic treatment strategies is provided as much as possible.
Disclosure of Invention
The invention relates to a nano compound with near infrared absorption and photosensitization functions, which is designed by taking hollow spike-shaped nano gold as a carrier and loading a certain proportion of chlorin e6 photosensitizer, so as to solve the scientific problem that the uptake rate of the photosensitizer and the nano gold in tumor cells or tissues is low.
Specifically, the invention provides a preparation method of a chlorin e 6-nanogold compound for tumor photothermal synergistic photodynamic therapy, which comprises the following steps:
s1: preparing hollow spike-shaped nanogold through a displacement reaction of a chloroauric acid compound and silver nanospheres;
s2: synthesizing thioctic acid end polyethylene glycol crosslinked chlorin e6 functional molecule, namely LA-PEG-Ce 6;
s3: and simultaneously coupling a certain proportion of lipoic acid terminated polyethylene glycol methoxy group LA-PEG-OMe and LA-PEG-Ce6 to the surface of the nanogold prepared in the step S1 to obtain a chlorin e 6-nanogold compound.
Further, the step S1 specifically includes:
s11: adding a newly prepared silver nitrate solution into deionized water, stirring and boiling, quickly adding a sodium citrate solution, keeping boiling for a period of time, and cooling to room temperature to obtain silver nanoparticles;
s12: centrifuging and washing the silver nanoparticles, then suspending and dispersing the silver nanoparticles in ultrapure water, slowly dropwise adding the silver nanoparticles into a chloroauric acid solution, shaking and uniformly mixing the solution, gradually changing the color of the solution from bright yellow to light gray, then adding an ascorbic acid solution, rapidly and uniformly mixing the solution, and standing the mixture to obtain the hollow spike-shaped nanogold stock solution.
Further, the step S2 specifically includes:
s21: completely dissolving Ce6 in dimethylformamide, then adding 1-ethyl-3- (3-trimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, uniformly mixing, and stirring and activating for 1h at room temperature in a dark place to obtain a mixed solution;
s22: mixing LA-PEG2K-NH2Adding the mixed solution, stirring overnight, dialyzing with water, centrifuging, and collecting supernatant to obtain LA-PEG2K-stock solution of Ce 6.
Further, the reaction molar ratio of Ce6, EDC and NHS is 1: 10: 20.
further, the step S3 specifically includes: firstly, dissolving LA-PEG-OMe in ultrapure water to prepare a storage solution for later use; taking the nanogold stock solution, sequentially adding LA-PEG-OMe stock solution and LA-PEG-Ce6 stock solution, and reacting at room temperature in the dark overnight; after the reaction solution is centrifuged for 2 times, unreacted substances are removed, and the reaction solution is suspended in ultrapure water for later use.
The invention also provides a chlorin e 6-nanogold compound prepared by the preparation method.
Compared with the prior art, the invention has the beneficial effects that:
the hollow spike-shaped nano gold carrier adopted by the invention has the following advantages: the surface is rough, the specific surface area is large, and the loading capacity of the photosensitizer is large; the nano-silver dye has a strong absorption peak in a near-infrared band and strong light stability; the preparation cost is low, the steps are simple, the operation is easy, and the like;
the lipoic acid terminated polyethylene glycol is covalently combined with the nanogold through disulfide bonds, the reaction condition is mild, and the stability is stronger; meanwhile, a certain amount of LA-PEG-OMe and LA-PEG-Ce6 are coupled to the surface of the nanogold, so that the interference of a space structure between Ce6 molecules is avoided, the compound is endowed with better dispersibility in an aqueous solution or a culture medium, and the nonspecific adsorption effect in vivo circulation is reduced;
ce6 is loaded on the surface of the nano-gold, can maximally improve the uptake efficiency of cancer cells, and is long-acting gathered on tumor focuses by utilizing the permeation and retention effects of the nano-gold and is not easy to be discharged out of the body;
the unique photo-thermal conversion effect of the nano-gold can effectively enhance the photodynamic curative effect, promote the generation of singlet oxygen and achieve the aim of reducing the survival rate of cancer cells;
the nanogold-photosensitizer compound prepared by the invention can be used as an exploration research platform, and a more effective synergetic photodynamic therapy strategy can be developed by exploring nano material carriers with different functions.
Drawings
FIG. 1 is a diagram of the UV-VIS absorption spectrum of hollow spike-shaped nanogold;
FIG. 2 shows the laser (2W/cm) of nano-gold at 808nm2) A time-dependent temperature profile under irradiation;
FIG. 3 UV-VISIBLE ABSORPTION SPECTRUM (a) and Absorbance (at 404 nm) of Ce6 dispersed in DMF as a function of Ce6 concentration (b);
FIG. 4 is fluorescence imaging of KB oral cancer cells after GNS-Ce6, Ce6, GNS treatment;
FIG. 5 shows fluorescence intensity of a singlet oxygen fluorescent probe (SOSG) detecting GNS-Ce6 after PDT and PTT irradiation;
FIG. 6 is a graph of Ce6, GNS and GNS-Ce6 treated and treated at photodynamic light (PDT, 650nm, 0.02W/cm)23min) and photothermal (PTT, 808nm, 2.4W/cm)25min) survival of KB cells after irradiation.
The specific implementation mode is as follows:
the technical method in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Examples
Example 1
Preparing and characterizing the hollow spike-shaped nanogold: prepared by the replacement reaction of chloroauric acid and silver nanoparticles. The method comprises the following specific steps: (1) preparing silver nanoparticles with the particle size of about 30nm by a sodium citrate reduction method: taking newly prepared 180 mu L of 1% AgNO3Adding the solution into 25mL of deionized water, stirring and boiling, quickly adding 5mL of 1% sodium citrate solution, keeping boiling for 30min, and cooling to room temperature; (2) after 5mL of silver nanoparticles were centrifuged, washed, resuspended in 4mL of water and HAuCl was slowly added dropwise4Solution (140. mu.L 1% HAuCl)4Diluting the solution in 1mL of ultrapure water), shaking and uniformly mixing, and gradually changing the color of the solution from bright yellow to light gray. Then adding 1mL of 10mM ascorbic acid solution, quickly mixing uniformly, and standing to obtain the hollow spike-shaped nano gold stock solution with the average particle size of 50 nm. (3) And (3) characterization of nanogold: the ultraviolet-visible absorption spectrum of the hollow spike-shaped nano gold is measured and is shown in figure 1, and the nano gold has a strong near infrared absorption peak at 808nm and shows a strong light heat absorption effect. As shown in FIG. 2, 50. mu.g/mL of the nanogold dispersion was subjected to a laser (2W/cm) at 808nm2) The temperature of the nano gold can rise by about 50 ℃ within 5min after irradiation for different time, which proves the good near infrared absorption effect of the nano gold.
Lipoic acid end polyethylene glycol crosslinked chlorin e6 (LA-PEG)2K-synthesis of Ce 6): utilizing carboxyl group in Ce6 and LA-PEG2K-NH2EDC-NHS reaction of middle amino. Specifically, 15mg of Ce6 was completely dissolved in 4mL of Dimethylformamide (DMF), and then 39mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and 57.8mg of N-hydroxysuccinimide (NHS) were added thereto and mixed well, that is, the reaction molar ratio of Ce6, EDC and NHS was 1: 10: 20, stirring and activating for 1h at room temperature in the dark. Adding 5mg of LA-PEG2K-NH2Adding the mixed solution, stirring overnight, dialyzing with water, centrifuging, and collecting supernatant to obtain LA-PEG2K-stock solution of Ce 6. In addition, 1mg/mL of DMF stock solution of Ce6 was prepared, different amounts of the stock solution were diluted in 3mL of DMF, the UV-VIS absorption spectrum of Ce6 was measured, the absorbance value of Ce6 at 404nm at different concentrations was determined, and the Ce6 concentration-absorbance value was plottedThe calibration curve, see FIGS. 3(a) and (b); simultaneously, taking a proper amount of LA-PEG2KDissolving the-Ce 6 storage solution in 3mL of DMF, and quantifying the content of Ce6 according to the measured ultraviolet-visible absorption spectrum to obtain LA-PEG2KThe final concentration of Ce6 was 0.16 ug/uL.
Construction of chlorin e 6-Nanogold Complex (GNS-Ce 6): lipoic acid polyethylene glycol methoxyl (LA-PEG-OMe) and LA-PEG-Ce6 are coupled on the GNS surface at the same time, and the content of Ce6 on the composite surface is quantified. The method comprises the following specific steps: firstly, LA-PEG-OMe is dissolved in ultrapure water to prepare a 1mg/mL storage solution for later use. And taking 2mL of the nanogold stock solution, sequentially adding 36uL of LA-PEG-OMe stock solution and 900uL of LA-PEG-Ce6 stock solution, and reacting at room temperature in the dark overnight. After the reaction solution is centrifuged for 2 times, unreacted substances are removed, and the reaction solution is suspended in ultrapure water for later use. The UV absorption spectrum of the GNS-Ce6 composite is consistent before and after the composite is dispersed in PBS containing 10% fetal calf serum for 24h, which shows that the composite has good dispersibility. In addition, the GNS-Ce6 composite was successively subjected to PTT (808nm, 2.4W/cm)210min) and PDT (650nm, 0.02W/cm)2And 5min) before and after laser irradiation, the ultraviolet absorption spectra are basically consistent, and the good optical stability is seen.
Example 2
To compare the advantages of the designed nanocomposites, nanogold (abbreviated as GNS) modified with LA-PEG-OMe alone was selected as a negative control. The preparation method is basically the same as that of the embodiment 1, and specifically, 2mL of newly prepared nanogold stock solution is added with 180uL LA-PEG-OMe storage solution, shaking reaction is carried out at room temperature overnight, supernatant is removed by centrifugation, and the nanogold stock solution is obtained by resuspending in ultrapure water.
Example 3
GNS-Ce6 tumor cell uptake fluorescence imaging assay: KB oral cancer cells are inoculated on a confocal special culture plate, incubated in an incubator at 37 ℃ for 24h, then the original culture medium is sucked off, washed twice with PBS, DMEM cell culture medium containing GNS (50 mu g/mL), GNS-Ce6(50 mu g/mL) and equivalent Ce6(0.6 mu g/mL) is added, incubated for 24h, the original culture medium is discarded, and washed twice with PBS. Adding a proper amount of Hoechst 33342 cell nucleus staining solution, fully covering adherent cells, incubating in an incubator at 37 ℃ for 20min, discarding the staining solution, washing with PBS for 2-3 times, and performing confocal fluorescence imaging and bright field imaging detection. The imaging results are shown in fig. 4, in which the nucleus fluoresces blue and Ce6 fluoresces red. Fluorescence imaging shows that the GNS-Ce6 group cells exhibited stronger red fluorescence signals than the Ce6 treated group, which indicates that Ce6 loading on GNS surface caused their endocytosis rate to be significantly increased. Bright field imaging also indicated that the number of black particles in cells of the GNS-Ce6 group was significantly increased compared to the GNS treated group, indicating that the functionalization of Ce6 also greatly facilitated the uptake of GNS by cells, and that the construction of this complex had a dual effect.
Example 4
To further illustrate the role and mechanism of GNS-Ce 6-mediated hyperthermic effect in tumor photodynamic therapy, the singlet oxygen production and cancer cell survival in GNS-Ce6 solution were examined. The method comprises the following specific steps:
detecting the content of singlet oxygen of the GNS-Ce6 solution by using the SOSG singlet oxygen probe after different light treatments: 4 groups of 50 mu g/mL GNS-Ce6 solutions were taken, and an appropriate amount of SOSG singlet oxygen probe working solution (working concentration 10 mu M) was added to each group. Then, the following treatments are respectively carried out on each group: (1) GNS-Ce 6; (2) GNS-Ce6 laser at 808nm (PTT, 2.4W/cm)2) Irradiating for 5 min; (3) GNS-Ce6 laser at 650nm (PDT, 0.02W/cm)2) Irradiating for 6 min; (4) GNS-Ce6 was first irradiated with 808nm laser for 5min, followed by 650nm laser for 6 min. Subsequently, the fluorescence spectra of each set were measured using a fluorescence spectrophotometer, and the relative fluorescence intensity was recorded (fig. 5). As shown in FIG. 5, compared with the GNS-Ce6+ PDT treated group, the singlet oxygen produced by the GNS-Ce6+ PTT + PDT group is significantly increased, indicating that the high thermal effect induced by GNS-Ce6 can increase the solution temperature, and greatly enhance the singlet oxygen content produced by single photodynamic irradiation.
And (3) detecting the survival rate of the cancer cells: filling the edge of a 96-well plate with sterile PBS, dispersing a proper amount of KB oral cancer cells in 100 mu L DMEM medium, inoculating the cells in the 96-well plate, and placing the cells in 5% CO2Incubating overnight in an incubator at 37 ℃ until the cell confluence is about 70%, setting 13 control groups, and respectively carrying out the following treatments: (1) PBS; (2) ce 6; (3) GNS-Ce 6; (4) PDT; (5) ce6+ PDT; (6) GNS + PDT; (7) GNS-Ce6+ PDT; (8) PTT; (9) GNS + PTT; (10) ce6+ PTT;(11) GNS-Ce6+ PTT; (12) GNS-Ce6+ PTT + PDT; (13) GNS + Ce6+ PTT + PDT, three replicate wells per group, and zero-adjusted wells for medium. Wherein the concentration of Ce6 in each group is 0.6 mu g/mL, and the Ce6 in each group is incubated with cells for 24 hours; GNS-Ce6 was 50. mu.g/mL in concentration and incubated with cells for 24 h; PDT (650nm, 0.02W/cm)2，3min)，PTT(808nm，2.4W/cm25 min). Subsequently, the original medium in the 96-well plate was discarded, PBS was washed 2 times, 100. mu.L of the new medium was replaced, 10. mu.L of MTT stock solution (5mg/mL) was added to each well, the culture was incubated at 37 ℃ for 4 hours in an incubator, the medium containing MTT was discarded, 150. mu.L of dimethyl sulfoxide (DMSO) was added thereto, the mixture was placed in a microplate reader and shaken at a medium speed for 10 minutes to dissolve the crystals sufficiently, the light absorption at OD 570nm of each well was measured, and the results were recorded as shown in FIG. 6. It can be seen that compared with the Ce6+ PDT group, the cell survival rate of the GNS-Ce6+ PDT group is reduced from 90% to 69%, and the Ce6 loaded on the GNS surface increases the endocytosis rate of the photosensitizer Ce6, so that the photodynamic therapy effect is enhanced; similarly, the survival rate of the cells of the GNS-Ce6+ PTT group is reduced from 86% to 54% compared with the GNS + PTT group, namely the cellular uptake rate of the Ce6 functionalized GNS is obviously enhanced, so that the photothermal treatment effect is remarkable; obviously, the introduction of PTT treatment in the GNS-Ce6+ PDT group reduced cell survival from 69% to 18%, i.e. the most effective killing of tumor cells was achieved in combination with photothermal treatment in GNS-Ce 6-mediated photodynamic therapy.

Claims (6)

1. A preparation method of a chlorin e 6-nanogold compound for tumor photothermal synergistic photodynamic therapy is characterized by comprising the following steps:
s1: preparing hollow spike-shaped nano gold through a displacement reaction of a chloroauric acid compound and silver nanoparticles;
s2: synthesizing thioctic acid end polyethylene glycol crosslinked chlorin e6 functional molecule, namely LA-PEG-Ce 6;
s3: and simultaneously coupling a certain proportion of lipoic acid terminated polyethylene glycol methoxy group LA-PEG-OMe and LA-PEG-Ce6 to the surface of the nanogold prepared in the step S1 to obtain a chlorin e 6-nanogold compound.
2. The method according to claim 1, wherein the step S1 specifically includes:
s11: adding a newly prepared silver nitrate solution into deionized water, stirring and boiling, quickly adding a sodium citrate solution, keeping boiling for a period of time, and cooling to room temperature to obtain silver nanoparticles;
s12: centrifuging and washing the silver nanoparticles, then suspending and dispersing the silver nanoparticles in ultrapure water, slowly dropwise adding the silver nanoparticles into a chloroauric acid solution, shaking and uniformly mixing the solution, gradually changing the color of the solution from bright yellow to light gray, then adding an ascorbic acid solution, rapidly and uniformly mixing the solution, and standing the mixture to obtain the hollow spike-shaped nanogold stock solution.
3. The method according to claim 1, wherein the step S2 specifically includes:
s21: completely dissolving Ce6 in dimethylformamide, then adding 1-ethyl-3- (3-trimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, uniformly mixing, and stirring and activating for 1h at room temperature in a dark place to obtain a mixed solution;
s22: mixing LA-PEG2K-NH2Adding the mixed solution, stirring overnight, dialyzing with water, centrifuging, and collecting supernatant to obtain LA-PEG2K-storage of Ce6, quantitative by uv-vis absorption spectrum.
4. The preparation method according to claim 3, wherein the reaction molar ratio of Ce6, EDC and NHS is 1: 10: 20.
5. the method according to claim 1, wherein the step S3 specifically includes: firstly, dissolving LA-PEG-OMe in ultrapure water to prepare a storage solution for later use; taking the nanogold stock solution, sequentially adding LA-PEG-OMe stock solution and LA-PEG-Ce6 stock solution, and reacting at room temperature in the dark overnight; after the reaction solution is centrifuged for 2 times, unreacted substances are removed, and the reaction solution is suspended in ultrapure water for later use.
6. A chlorin e 6-nanogold complex prepared by the preparation method of any one of the preceding claims.
CN201911139470.5A 2019-11-20 2019-11-20 Chlorin e 6-nanogold compound for tumor photothermal synergistic photodynamic therapy and preparation method thereof Pending CN112823809A (en)

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李晨露等: "金纳米星负载二氢卟吩e6对肺癌A549 细胞的光动力效应", 《中国肿瘤生物治疗杂志》 *
马宁宁: "金纳米材料在肿瘤治疗中的应用及其对细胞自噬的调控", 《万方学位论文》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114452406A (en) * 2022-03-16 2022-05-10 安徽工程大学 Antibacterial material and preparation method and application thereof
CN114452406B (en) * 2022-03-16 2023-05-23 安徽工程大学 Antibacterial material and preparation method and application thereof
CN115252775A (en) * 2022-05-11 2022-11-01 华南师范大学 Tumor nano-drug based on polyethyleneimine and Prussian blue as well as preparation method and application thereof
CN115252775B (en) * 2022-05-11 2024-02-09 华南师范大学 Tumor nano-drug based on polyethyleneimine and Prussian blue, and preparation method and application thereof

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