CN113143884B - Lignin nanoparticles for photothermal-immunotherapy and preparation method thereof - Google Patents
Lignin nanoparticles for photothermal-immunotherapy and preparation method thereof Download PDFInfo
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- CN113143884B CN113143884B CN202110378211.9A CN202110378211A CN113143884B CN 113143884 B CN113143884 B CN 113143884B CN 202110378211 A CN202110378211 A CN 202110378211A CN 113143884 B CN113143884 B CN 113143884B
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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A—HUMAN NECESSITIES
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- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55505—Inorganic adjuvants
Abstract
The invention provides a lignin nanoparticle for photothermal-immunotherapy and a preparation method thereof, wherein the preparation method comprises the following steps: (1) Uniformly dispersing lignosulfonate and indocyanine green in pure water to obtain a mixed solution; (2) Under the condition of stirring at normal temperature, dropwise adding an aluminum chloride solution into the mixed solution, and then carrying out a light-resistant reaction; (3) And after the reaction is finished, carrying out solid-liquid separation to obtain the LS-Al-ICG nanoparticles. The LS-Al-ICG nanoparticles provided by the invention have uniform particle size and good dispersibility and stability; has good photothermal conversion effect in vitro. LS-Al-ICG nanoparticle solution with appropriate concentration has good biological safety, more than 70% of tumor cells can be killed by combining with 808nm laser irradiation, and LS-Al-ICG nanoparticles have excellent anti-tumor effect in vitro and can stimulate immune response. The lignin nanoparticles prepared by the invention can be used for preparing antitumor drugs in photothermal-immunotherapy.
Description
Technical Field
The invention belongs to the field of nanoparticle preparation, and particularly relates to a lignin nanoparticle for photothermal-immunotherapy and a preparation method thereof.
Background
Combination photothermal therapy and immunotherapy show great potential in the treatment of cancer. Among them, immune adjuvants induce innate or adaptive immune responses by activating Antigen Presenting Cells (APCs), and have become promising tools for cancer immunotherapy. Commonly used immunological adjuvants include alum (aluminum salt), lipopolysaccharide (LPS), oligonucleotide (CpG), layered Double Hydroxide (LDH), and polyinosinic acid (poly I: C). The aluminum salt used as the immunologic adjuvant has the history of more than 80 years, and has the advantages of low cost, convenient use and no toxicity. The combination of photothermal therapy and immunoadjuvant-based nano-drug delivery systems play a synergistic role in causing immunogenic cell death. Thus, combination therapy of nanoparticle immunoadjuvants and photosensitizers is considered a promising approach to activate the immune system.
Lignin (Lignin) is a natural aromatic polymer derived from wood, and is a polymer compound having a three-dimensional structure formed by connecting phenylpropane structural units through an ether bond and a carbon-carbon bond. It is estimated that only about 5-10% of lignin can be fully used as value added products, such as concrete fillers, dispersants and additives. The lignin has the characteristics of environmental friendliness, renewability, biodegradability, low toxicity and the like, a more complex and clearly defined structure can be easily constructed based on a natural structure of the lignin, and the obtained lignin polymer can be assembled into various forms with unique properties, such as elastomers, hydrogels, aerogels, nanoparticles and the like. The Lignosulfonate (LS) is a lignin derivative, and researches show that the lignosulfonate can be used as a carrier, a surfactant, a reducing agent, a stabilizing agent and the like, has the characteristics of sustainability, low price, biodegradability, nontoxicity, specific combination, complexation and the like compared with other protein carriers and inorganic carriers, and has wide application prospects in the aspect of nanoparticle preparation.
Therefore, a novel LS-Al-ICG nanoparticle is provided for photothermal-immunotherapy, and the nanoparticle forms a final self-assembly nanoparticle by virtue of complexation of sulfonic acid groups of indocyanine green (ICG) and Lignosulfonate (LS) and inorganic metal aluminum ions and action of a surfactant and a reducing agent of lignosulfonate, so that the purpose of treating tumors to the greatest extent by combining photothermal therapy and immunotherapy is achieved.
Disclosure of Invention
Based on the previous research, the invention provides lignin nanoparticles for photothermal-immunotherapy and a preparation method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for preparing lignin nanoparticles for photothermal-immunotherapy, comprising the steps of:
(1) Uniformly dispersing Lignosulfonate (LS) and indocyanine green (ICG) in pure water to obtain a mixed solution;
(2) Aluminum chloride (AlCl) is stirred at normal temperature 3 ) Dropwise adding the solution into the mixed solution, and then reacting in a dark place;
(3) And after the reaction is finished, carrying out solid-liquid separation to obtain the LS-Al-ICG nanoparticles.
Further, in the step (1), the mass ratio of the indocyanine green to the lignosulfonate is 1:20 to 40, preferably 1:20 to 30.
In the step (2), the aluminum chloride solution is an aqueous solution having an aluminum chloride content of 0.2 to 0.5% (preferably 0.2 to 0.3%). The mass ratio of the aluminum chloride to the lignosulfonate in the aluminum chloride solution is 1:1.
in the step (1), the mass fraction of the lignosulfonate in the prepared mixed solution is 0.2-0.5% (preferably 0.2-0.3%).
In the step (2), the reaction time is 8-12 hours.
In the step (3), the solid-liquid separation can be performed by centrifugation or filtration.
In the invention, the lignosulfonate can be calcium lignosulfonate, sodium lignosulfonate and the like, and preferably is calcium lignosulfonate.
The product prepared by the method can be characterized by a Transmission Electron Microscope (TEM), dynamic Light Scattering (DLS), ultraviolet visible absorption spectrum (UV-Vis) and photo-thermal conversion temperature rise test.
The invention also provides the lignin nanoparticles prepared by the method.
The lignin nanoparticles provided by the invention can be used for photothermal-immunotherapy and preparation of antitumor drugs.
Based on the characteristics of LS, the photosensitizer ICG and the aluminum salt serving as the immunologic adjuvant are combined, LS-Al-ICG nanoparticles with uniform particle size are prepared in a one-step method, and are passively targeted to tumor tissues after intravenous injection, so that the purpose of photo-thermal and immunologic synergistic tumor treatment is achieved.
The LS-Al-ICG nano-particles prepared by the invention have uniform particle size and good dispersity and stability; has good photothermal conversion effect in vitro. The LS-Al-ICG nanoparticle solution with a proper concentration has good biological safety, and can kill more than 70% of tumor cells by combining with 808nm laser irradiation; LS-Al-ICG nano-particles can cause the immunogenic death of tumor cells, and the secretion of immune related proteins CRT and HMGB1 is increased, which shows that the LS-Al-ICG nano-particles have excellent anti-tumor effect in vitro and can stimulate the immune response. Therefore, the invention is predicted to have good anti-tumor effect in vivo, and can be used for preparing anti-tumor drugs for photothermal-immune combination treatment of tumors.
Drawings
FIG. 1 is a transmission electron microscope image of LS-Al-ICG nanoparticles of an embodiment of the present invention.
FIG. 2 shows particle size, potential and PDI of LS-Al-ICG nanoparticles of an embodiment of the present invention.
FIG. 3 is the ultraviolet-visible absorption spectrum (UV-Vis) of LS-Al-ICG nanoparticles of an example of the present invention.
FIG. 4 is a photo-thermal image of LS-Al-ICG nanoparticles of an embodiment of the present invention.
FIG. 5 is a graph showing the photothermal temperature increase of LS-Al-ICG nanoparticles of an embodiment of the present invention.
FIG. 6 is a schematic diagram of the synthesis of nanoparticles of LS-Al-ICG.
FIG. 7 in vitro cytotoxicity profiles of LS-Al-ICG nanoparticles.
FIG. 8 laser confocal image of live/dead cell staining of LS-Al-ICG nanoparticles.
FIG. 9 is a graph of in vitro immunoprotein detection of LS-Al-ICG nanoparticles.
Detailed Description
The technical solution of the present invention is further described below with specific examples, but the scope of the present invention is not limited thereto.
Example 1
1. Material
Calcium lignosulfonate (more than or equal to 96.0%, china, shanghai Mecang Biotech limited Co.); anhydrous aluminum chloride (greater than or equal to 99.0%, china, shanghai alading biochem-technological corporation); indocyanine green (not less than 75.0%, china, shanghai alatin biochemistry science and technology limited).
2. Method of producing a composite material
1. Preparation of
Uniformly dispersing 40mg of calcium Lignosulphonate (LS) and 1.5mg of indocyanine green (ICG) in 15mL of pure water to obtain a mixed solution; 40mg of aluminum chloride (AlCl) was added by a syringe pump under stirring at room temperature 3 ) Adding a solution prepared by 15mL of pure water dropwise into the mixed solution, and then reacting for 12 hours in a dark place; and centrifuging for 2-3 times after the reaction is finished, and adding pure water for dispersion to obtain the LS-Al-ICG nanoparticles.
2. Characterization of
2.1 topography Observation
Diluting the nanoparticles, performing ultrasonic dispersion, dropping 50 μ L of liquid onto a copper mesh overnight, naturally drying, and observing the morphology by a Transmission Electron Microscope (TEM) as shown in FIG. 1.
2.2 particle size potential detection
The nanoparticles were diluted and ultrasonically dispersed, and the particle size, potential and PDI thereof were detected by Dynamic Light Scattering (DLS), with the results shown in fig. 2.
2.3 ultraviolet visible absorption Spectrum
The absorption of the nanoparticles at 300-1000nm was measured by uv-vis absorption spectroscopy and the results are shown in fig. 3.
2.4 photothermal conversion ability
The nanoparticles were diluted to different concentrations, irradiated by a 808nm laser for 5 minutes, the thermal imaging camera took thermal imaging pictures at 30 second intervals, and photo-thermal temperature rise curves were drawn as shown in fig. 4 and 5, respectively.
3. In vitro cytological evaluation
3.1 cytotoxicity
The CCK-8 method is adopted to investigate the killing effect of LS-Al-ICG nanoparticles with different concentrations on 4T1 cells, and the biological safety and the in vitro anti-tumor effect of the nanoparticles are investigated, and the result is shown in figure 7.
3.2 live/dead cell staining
The in vitro antitumor effect of the nanoparticles was examined by using live/dead cell staining kit in combination with laser confocal microscopy (CLSM), and the concentration of the nanoparticles was quantified by ICG (16 μ g/mL), with the results shown in fig. 8.
3.3 DAMP immune protein assay
Calreticulin (CRT) and high mobility group protein B1 (HMGB 1) antibodies are adopted to incubate the 4T1 cells treated by the LS-Al-ICG nanoparticles, the expression of the two proteins is examined by combining a laser confocal microscope, the concentration of the nanoparticles is quantified by ICG (16 mu g/mL), and the result is shown in figure 9.
The results obtained show that: the LS-Al-ICG nano-particles prepared by the invention have uniform particle size and good dispersity and stability; has good photothermal conversion effect in vitro. The results of in vitro cytotoxicity and live/dead cell staining evaluation show that the LS-Al-ICG nanoparticle solution with proper concentration has good biological safety, and more than 70% of tumor cells can be killed by combining 808nm laser irradiation; meanwhile, the detection result of the DAMP immune protein shows that the DAMP immune protein can cause the immunogenic death of tumor cells, and the secretion of immune related proteins CRT and HMGB1 is increased, which shows that the LS-Al-ICG nano-particle has excellent anti-tumor effect in vitro and can stimulate the immune response. Therefore, the present invention is predicted to have a good antitumor effect in vivo as well, and can be used for photothermal-immune combination therapy of tumors.
Claims (5)
1. A preparation method of lignin nanoparticles for photothermal-immunotherapy is characterized by comprising the following steps:
(1) Uniformly dispersing lignosulfonate and indocyanine green in pure water to obtain a mixed solution; the mass ratio of the indocyanine green to the lignosulfonate is 1:20 to 30 percent; the mass fraction of the lignosulfonate in the prepared mixed solution is 0.2-0.5%;
(2) Under the condition of stirring at normal temperature, dropwise adding an aluminum chloride solution into the mixed solution, and then carrying out a light-resistant reaction; the aluminum chloride solution is an aqueous solution with the aluminum chloride content of 0.2-0.5%; the mass ratio of the mass of the aluminum chloride in the aluminum chloride solution to the mass of the lignosulfonate is 1:1;
(3) After the reaction is finished, performing solid-liquid separation to obtain LS-Al-ICG nanoparticles.
2. The method according to claim 1, wherein in the step (2), the reaction is carried out for 8 to 12 hours while avoiding light.
3. The method of claim 1, wherein the lignosulfonate is calcium lignosulfonate or sodium lignosulfonate.
4. Lignin nanoparticles prepared according to the method of any one of claims 1 to 3.
5. The application of the lignin nanoparticles as claimed in claim 4 in preparing antitumor drugs in photothermal-immunotherapy.
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