CN111330006B - Chlorin nano photosensitizer and preparation method and application thereof - Google Patents
Chlorin nano photosensitizer and preparation method and application thereof Download PDFInfo
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- CN111330006B CN111330006B CN202010149420.1A CN202010149420A CN111330006B CN 111330006 B CN111330006 B CN 111330006B CN 202010149420 A CN202010149420 A CN 202010149420A CN 111330006 B CN111330006 B CN 111330006B
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- 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
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- 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
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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Abstract
The invention discloses a preparation method of a chlorin nano photosensitizer, which comprises the following steps: mixing a chlorin photosensitizer and tris (hydroxymethyl) aminomethane in an organic solvent, and under the electrostatic interaction, carrying out self-assembly on the chlorin photosensitizer and the tris (hydroxymethyl) aminomethane to form the chlorin nano photosensitizer; the chlorin photosensitizer comprises a chlorin e6, a chlorin e6 copper compound, a chlorin e6 zinc compound and a chlorin e6 manganese compound. The invention also discloses the chlorin nano photosensitizer prepared by the method and application thereof in photodynamic therapy. The chlorin nano photosensitizer is amphiphilic, can be rapidly self-assembled in a water system and biological body fluid to form nanoparticles with uniform and stable size, is favorable for high-selectivity tumor enrichment, and improves the phototherapeutic effect.
Description
Technical Field
The invention relates to the technical field of photosensitizers, in particular to a chlorin nano photosensitizer and a preparation method and application thereof.
Background
Photodynamic therapy (PDT), as an emerging tumor therapy modality, is clinically used for treating many diseases, including tumors, particularly superficial tumors (esophageal tumors, bladder tumors, melanoma, and the like), and has the advantages of high selectivity, small side effects, strong selectivity, and the like. Photosensitizers play an important role in photodynamic therapy, in which photosensitizers are injected into tumor patients and accumulate in tumor tissues. Then near-infrared laser is adopted for irradiation, the excited photosensitive drug reacts with oxygen in the tissue to generate singlet oxygen and other active oxygen substances, so that apoptosis or necrosis of cells is caused, and the target of tumor targeted therapy is finally realized.
In the past 50 years, the first generation of photosensitizers represented by hematoporphyrin (HpD) have been developed for clinical use, such as photoporphyrin (Photofrin), Photofrin (Photogem), and oncophotofrin (Photofrin), among others. However, these HpD products generally have the disadvantages of complex composition, poor tissue selectivity, weak absorption in the red region, shallow treatment depth, large dose or light dose required for treatment, and significant skin phototoxicity after treatment. In view of this, in recent years, the second-generation photosensitizers represented by chlorins photosensitizers have been attracting attention. The dihydroporphin photosensitizer has a single and definite structure, the absorption capacity in a red light region or a near infrared region is obviously enhanced, and the accumulation of the dihydroporphin photosensitizer in tumor tissues can be further improved by modifying the dihydroporphin photosensitizer (such as hydrophilicity, hydrophobicity, charge regulation and the like). Among them, Verteporfin (Verteporfin), Temoporfin (Temoporfin), and talaporfin (Talaphorfin) have been successfully used in clinical practice. Although the second generation photosensitizers compensate to some extent for the deficiencies of the first generation photosensitizers, it is still difficult to achieve the desired effect of high selective accumulation of tumors. Although photosensitizer molecules are continuously developed, the photosensitizer molecules have certain defects, for example, most of the photosensitizer molecules are difficult to dissolve in water, aggregation is formed in water due to the hydrophobic interaction among the molecules, direct intravenous injection cannot be performed, the targeting property of a tumor part is not high, phototoxicity is easily generated when the photosensitizer molecules accumulate on skin, and the like. In recent years, studies have found that a photosensitizer nano-delivery system can overcome the drawbacks of the above-mentioned photosensitizers, and also enhance the accumulation of the photosensitizers at tumor sites by a high osmotic long retention (EPR) effect. Therefore, the development of high-performance nano-photosensitizers is an important direction in the field of light therapy at present.
CN103169968A discloses an albumin-based hydrophobic chlorin photosensitizer nano-drug preparation, which is composed of albumin and a hydrophobic chlorin photosensitizer only, does not contain other cross-linking agents or auxiliary materials, and can avoid adverse clinical reactions caused by the use of the cross-linking agents; the used hydrophobic chlorin photosensitizer has simple structure, does not contain functional groups such as hydroxyl, alkoxy, amino and the like for improving the water solubility of the chlorin photosensitizer, is easy to synthesize, and can effectively absorb and utilize therapeutic light sources from a superficial layer to a deeper tissue part. CN102397545A discloses a nano photosensitizer drug delivery system for photodynamic therapy, which is composed of chitosan, chlorin e6 and single-walled carbon nanotubes, wherein the single-walled carbon nanotubes are loaded with chlorin e6 by non-covalent bond bonding, and the outer layer of the obtained chlorin e 6-single-walled carbon nanotube composite is wrapped with chitosan. Currently, photosensitizer nano-delivery systems can overcome the drawbacks of the above photosensitizers, but also enhance the accumulation of photosensitizers at tumor sites through the EPR effect. But also has the defects of complex preparation process, poor controllability and the like, and has the problems of less selectable photosensitizer and the like.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of a chlorin nano photosensitizer, the preparation method is very simple and convenient, and the prepared nano photosensitizer has amphipathy, can be rapidly self-assembled in a water system and biological body fluid to form nano particles with uniform and stable size, is beneficial to high-selectivity tumor enrichment, and improves the phototherapeutic effect.
As can be seen from the chemical structural formulas of the chlorin photosensitizer and tris (hydroxymethyl) aminomethane, the carboxyl group of the chlorin photosensitizer and the amino group of the tris (hydroxymethyl) aminomethane can be bonded by electrostatic interaction. Based on the above, the invention provides a preparation method of a chlorin nano photosensitizer, which comprises the following steps:
mixing a chlorin photosensitizer and tris (hydroxymethyl) aminomethane in an organic solvent, and under the electrostatic interaction, carrying out self-assembly on the chlorin photosensitizer and the tris (hydroxymethyl) aminomethane to form a chlorin nano photosensitizer (Trisporfin); the chlorin photosensitizer includes, but is not limited to, compounds containing carboxyl groups, such as chlorin e6, chlorin e6 copper compounds, chlorin e6 zinc compounds, chlorin e6 manganese compounds, and the like.
Further, the organic solvent includes, but is not limited to, anhydrous methanol, tetrahydrofuran, dichloromethane, N-dimethylformamide.
Further, the chlorin photosensitizer is preferably chlorin e6(Ce 6). Due to the three carboxyl groups of the chlorin e6 molecule, the photosensitizer chlorin e6 and tris (hydroxymethyl) aminomethane are theoretically in a molar ratio of 1: 3. However, since tris (hydroxymethyl) aminomethane is inexpensive and easy to remove, tris (hydroxymethyl) aminomethane can be used in excess in order to sufficiently combine chlorin e6 and tris (hydroxymethyl) aminomethane.
Further, the preparation method specifically comprises the following steps:
s1, weighing a chlorin photosensitizer and tris (hydroxymethyl) aminomethane according to a ratio, dissolving the chlorin photosensitizer and the tris (hydroxymethyl) aminomethane in an organic solvent, mixing, and performing ultrasonic treatment to fully combine the chlorin photosensitizer and the tris (hydroxymethyl) aminomethane in the solvent through electrostatic interaction;
s2, removing the solvent by spin drying, adding deionized water, continuing to perform ultrasonic treatment, then removing redundant tris (hydroxymethyl) aminomethane by dialysis, and performing freeze drying to obtain the chlorin nano photosensitizer.
Further, the time of the sonication is 5 minutes to 1 hour.
Further, the organic solvent is removed by an evaporation device, such as a rotary evaporator, at a certain temperature and rotation speed.
Further, dialyzing for more than 24 hours by adopting a dialysis bag with the molecular weight of 100-3000.
The invention also provides a chlorin nano photosensitizer prepared by the method, wherein the structural formula of the chlorin nano photosensitizer is as follows:
wherein m and n are positive integers.
The invention also provides a nano micelle solution of the chlorin nano photosensitizer, which is prepared by the following steps:
dissolving a chlorin nano photosensitizer in a solvent, carrying out spin drying, adding deionized water under the ultrasonic condition, continuing to carry out ultrasonic treatment for a period of time, and removing the free chlorin photosensitizer through a water system filter membrane (the pore diameter is 0.22 micrometer or 0.45 micrometer) to obtain a nano micelle solution of the chlorin nano photosensitizer.
The invention also provides application of the chlorin nano photosensitizer in photodynamic therapy.
The invention has the beneficial effects that:
the invention utilizes electrostatic interaction to lead the carboxyl-containing chlorin photosensitizer and tris (hydroxymethyl) aminomethane to generate self-assembly in an organic solvent, thereby leading the hydrophilic tris (hydroxymethyl) aminomethane segment into the chlorin photosensitizer molecule and stabilizing the hydrophobic chlorin photosensitizer. Compared with the prior art, the preparation method greatly simplifies the preparation process of the photosensitizer, and the obtained nanometer photosensitizer Trisporfin has uniform and stable size in a water system and biological fluid, is beneficial to high-selectivity tumor enrichment, and improves the effect of light therapy.
Drawings
FIG. 1 is a drawing of the nano photosensitizer Trisporfin1H NMR spectrum;
FIG. 2 is a diagram of the state of chlorin e6 (left) and the nano photosensitizer Trisporfin (right) in water;
FIG. 3 is a graph showing the distribution of the nanoparticle size of Trisporfin as a photosensitizer measured by a dynamic light scattering instrument;
FIG. 4 is a particle size distribution diagram of the nano photosensitizer Trisporfin measured by a transmission electron microscope;
FIG. 5 is a UV-visible absorption spectrum of chlorin e6 and the nanophotosensitizer Trisporfin;
FIG. 6 is a fluorescence emission spectrum of chlorin e6 and the nanophotosensitizer Trisporfin;
FIG. 7 shows the particle size variation of the photosensitizer Trisporfin measured by a dynamic light scattering instrument;
FIG. 8 shows the quenching of DPBF by the photosensitizer Trisporfin under light;
FIG. 9 is the dark cytotoxicity of chlorin e6 and the nanophotosensitizer Trisporfin;
FIG. 10 is the cytotoxicity of chlorin e6 and the nanophotosensitizer Trisporfin.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used therein are commercially available without otherwise specified.
Example 1
Tris (hydroxymethyl) aminomethane (5 mg) and chlorin e6(5 mg) were weighed into a round-bottomed flask, and 5 ml of anhydrous methanol was added thereto, followed by sonication for 30 minutes to sufficiently bind them by electrostatic interaction. And (3) removing the organic solvent by using a rotary evaporator (at 40 ℃ at 30 rpm), adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 minutes, and dialyzing for 24 hours by using a dialysis bag with the molecular weight of 100-500 to remove the redundant tris (hydroxymethyl) aminomethane. The compound Trisporfin was obtained after lyophilization.
Weighing the compound Trisporfin (5 mg), adding 5 ml of anhydrous methanol, carrying out rotary drying by a rotary evaporator (30 r/min, 40 ℃), adding ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 min, and removing free chlorin e6 by using a water system filter membrane (0.22 micron) to obtain a clear Trisporfin nano micelle solution.
Of the nano-photosensitizer Trisporfin1The H NMR spectrum is shown in FIG. 1.
Example 2
Tris (hydroxymethyl) aminomethane (10 mg) and chlorin e6(5 mg) were weighed into a round-bottomed flask, and 5 ml of anhydrous tetrahydrofuran was added thereto, followed by sonication for 60 minutes to sufficiently bind them by electrostatic interaction. And (3) removing the organic solvent by using a rotary evaporator (at the temperature of 50 ℃ at the speed of 30 revolutions per minute), adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 minutes, and dialyzing for 24 hours by using a dialysis bag with the molecular weight of 100-500 to remove the redundant tris (hydroxymethyl) aminomethane. The compound Trisporfin is obtained after freeze-drying by a freeze-dryer.
Weighing the compound Trisporfin (5 mg), adding the Trisporfin into 5 ml of anhydrous methanol, carrying out rotary drying by a rotary evaporator (30 r/min, 40 ℃), adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 min, removing free chlorin e6 by using a water system filter membrane (the pore diameter is 0.45 micron), and finally obtaining a clear Trisporfin nano micelle solution.
Example 3
Tris (hydroxymethyl) aminomethane (20 mg) and chlorin e6(5 mg) were weighed into a round-bottomed flask, and 5 ml of methylene chloride was added thereto and sonicated for 30 minutes to sufficiently bind the two by electrostatic interaction. The organic solvent was removed by rotary evaporation (100 rpm, 35 ℃ C.), 5 ml of deionized water was added under sonication for 60 minutes and dialyzed against a dialysis bag of molecular weight 1500 for 12 hours to remove excess tris (hydroxymethyl) aminomethane. The compound Trisporfin is obtained after freeze-drying by a freeze-dryer.
Weighing the compound Trisporfin (10 mg), adding 10 ml of anhydrous methanol, spin-drying by a rotary evaporator (30 r/min, 50 ℃), adding 10 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 min, removing free chlorin e6 by a water-system filter head (the pore diameter is 0.22 micron), and finally obtaining a clear Trisporfin nano micelle solution.
Example 4
Tris (hydroxymethyl) aminomethane (15 mg) and chlorin e6(15 mg) were weighed into a round-bottomed flask, and 15 ml of tetrahydrofuran was added thereto and sonicated for 60 minutes to allow them to be bonded sufficiently by electrostatic interaction. And (3) removing the organic solvent by using a rotary evaporator (at the temperature of 50 ℃ at 100 rpm), adding 15 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 45 minutes, and dialyzing for 12 hours by using a dialysis bag with the molecular weight of 100-500 to remove the redundant tris (hydroxymethyl) aminomethane. The compound Trisporfin was obtained after lyophilization.
Weighing the compound Trisporfin (15 mg), adding 10 ml of anhydrous methanol, carrying out rotary drying by a rotary evaporator (60 r/min, 50 ℃), adding 10 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 min, removing free chlorin e6 by using a water-system filter head (the pore diameter is 0.45 micron), and finally obtaining a clear Trisporfin nano micelle solution.
Example 5
Tris (hydroxymethyl) aminomethane (5 mg) and chlorin e6(5 mg) were weighed into a round-bottomed flask, and 5 ml of N, N-dimethylformamide was added thereto and sonicated for 30 minutes to sufficiently bind the two by electrostatic interaction. And (3) removing the organic solvent by using a rotary evaporator (at 40 ℃ at 30 rpm), adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 minutes, and dialyzing for 24 hours by using a dialysis bag with the molecular weight of 100-500 to remove the redundant tris (hydroxymethyl) aminomethane. The compound Trisporfin is obtained after freeze-drying by a freeze-dryer.
Weighing the compound Trisporfin (5 mg), adding 5 ml of anhydrous methanol, carrying out rotary drying by a rotary evaporator (30 r/min, 60 ℃), adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 min, removing free chlorin e6 by a water system filter head (the pore diameter is 0.22 micron), and finally obtaining a clear Trisporfin nano micelle solution.
Example 6
Tris (hydroxymethyl) aminomethane (5 mg) and chlorin e6(5 mg) were weighed into a round-bottomed flask, and 5 ml of anhydrous methanol was added thereto, followed by sonication for 30 minutes to sufficiently bind them by electrostatic interaction. And (3) removing the organic solvent by using a rotary evaporator (at 30 r/min and 20 ℃ C.) in a rotary manner, adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 min, and dialyzing for 24 h by using a dialysis bag with the molecular weight of 100-500 to remove the redundant tris (hydroxymethyl) aminomethane. The compound Trisporfin is obtained after freeze-drying by a freeze-dryer.
Weighing the compound Trisporfin (5 mg), adding the Trisporfin into 5 ml of anhydrous methanol, carrying out rotary drying by a rotary evaporator (30 r/min, 60 ℃), adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 min, removing free chlorin e6 by using a water system filter membrane (the pore diameter is 0.22 micron), and finally obtaining a clear Trisporfin nano micelle solution.
Example 7
Weighing tris (hydroxymethyl) aminomethane (5 mg) and dissolving in tetrahydrofuran (5 ml) in round-bottomed flask 1, dissolving chlorin e6(5 mg) in anhydrous methanol (5 ml) in round-bottomed flask 2, slowly dropping 2 into 1 under ultrasonic condition, and continuing ultrasonic for 30 min to make the two combined by electrostatic interaction. And (3) removing the organic solvent by using a rotary evaporator (at 30 r/min and 20 ℃ C.) in a rotary manner, adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 min, and dialyzing for 24 h by using a dialysis bag with the molecular weight of 100-500 to remove the redundant tris (hydroxymethyl) aminomethane. The compound Trisporfin is obtained after freeze-drying by a freeze-dryer.
Weighing the compound Trisporfin (5 mg), adding the Trisporfin into 5 ml of anhydrous methanol, carrying out rotary drying by a rotary evaporator (30 r/min, 60 ℃), adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 min, removing free chlorin e6 by using a water system filter membrane (the pore diameter is 0.22 micron), and finally obtaining a clear Trisporfin nano micelle solution.
Example 8
Weighing tris (hydroxymethyl) aminomethane (5 mg) and dissolving in 5 ml of anhydrous methanol in round-bottomed flask 1, dissolving chlorin e6(5 mg) in 5 ml of anhydrous methanol in round-bottomed flask 2, slowly dropping 2 into 1 under ultrasonic condition, and continuing ultrasonic for 30 minutes to make the two combined by electrostatic interaction. And (3) removing the organic solvent by using a rotary evaporator (at 30 r/min and 20 ℃ C.) in a rotary manner, adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 min, and dialyzing for 24 h by using a dialysis bag with the molecular weight of 100-500 to remove the redundant tris (hydroxymethyl) aminomethane. The compound Trisporfin is obtained after freeze-drying by a freeze-dryer.
Weighing the compound Trisporfin (5 mg), adding the Trisporfin into 5 ml of anhydrous methanol, carrying out rotary drying by a rotary evaporator (30 r/min, 60 ℃), adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 min, removing free chlorin e6 by using a water system filter membrane (the pore diameter is 0.22 micron), and finally obtaining a clear Trisporfin nano micelle solution.
Example 9
Weighing tris (hydroxymethyl) aminomethane (5 mg) and dissolving in 5 ml dichloromethane in round bottom flask 1, dissolving chlorin e6(5 mg) in 5 ml anhydrous methanol in round bottom flask 2, slowly dropping 2 into 1 under ultrasonic condition, and continuing ultrasonic for 30 min to make the two combined by electrostatic effect. And (3) removing the organic solvent by using a rotary evaporator (at 30 r/min and 20 ℃ C.) in a rotary manner, adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 min, and dialyzing for 24 h by using a dialysis bag with the molecular weight of 100-500 to remove the redundant tris (hydroxymethyl) aminomethane. The compound Trisporfin is obtained after freeze-drying by a freeze-dryer.
Weighing the compound Trisporfin (5 mg), adding the Trisporfin into 5 ml of absolute ethyl alcohol, carrying out rotary drying by a rotary evaporator (30 r/min, 60 ℃), adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 min, removing free chlorin e6 by using a water system filter membrane (the pore diameter is 0.45 micron), and finally obtaining a clear Trisporfin nano micelle solution.
Example 10
Weighing tris (hydroxymethyl) aminomethane (5 mg) and dissolving in 5 ml dichloromethane in round bottom flask 1, dissolving chlorin e6(5 mg) in 5 ml anhydrous methanol in round bottom flask 2, slowly dropping 2 into 1 under ultrasonic condition, and continuing ultrasonic for 30 min to make the two combined by electrostatic effect. And (3) removing the organic solvent by using a rotary evaporator (at 30 r/min and 20 ℃ C.) in a rotary manner, adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 min, and dialyzing for 24 h by using a dialysis bag with the molecular weight of 100-500 to remove the redundant tris (hydroxymethyl) aminomethane. The compound Trisporfin is obtained after freeze-drying by a freeze-dryer.
Weighing the compound Trisporfin (5 mg), adding the Trisporfin into 5 ml of anhydrous methanol, carrying out rotary drying by a rotary evaporator (30 r/min, 60 ℃), adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 min, removing free chlorin e6 by using a water system filter membrane (the pore diameter is 0.22 micron), and finally obtaining a clear Trisporfin nano micelle solution.
Example 11
Weighing tris (hydroxymethyl) aminomethane (5 mg) and dissolving in 5 ml dichloromethane in round bottom flask 1, dissolving chlorin e6(5 mg) in 5 ml anhydrous methanol in round bottom flask 2, slowly dropping 2 into 1 under ultrasonic condition, and continuing ultrasonic for 10 min to make the two combined by electrostatic interaction. And (3) removing the organic solvent by using a rotary evaporator (at 30 r/min and 20 ℃ C.) in a rotary manner, adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 10 minutes, and dialyzing for 24 hours by using a dialysis bag with the molecular weight of 100-500 to remove the redundant tris (hydroxymethyl) aminomethane. The compound Trisporfin is obtained after freeze-drying by a freeze-dryer.
Weighing the compound Trisporfin (5 mg), adding the Trisporfin into 5 ml of anhydrous methanol, carrying out rotary drying by a rotary evaporator (30 r/min, 60 ℃), adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 10 min, removing free chlorin e6 by using a water system filter membrane (the pore diameter is 0.22 micron), and finally obtaining a clear Trisporfin nano micelle solution.
Example 12
Weighing tris (hydroxymethyl) aminomethane (5 mg) and dissolving in 5 ml dichloromethane in round bottom flask 1, dissolving chlorin e6(5 mg) in 5 ml anhydrous methanol in round bottom flask 2, slowly dropping 2 into 1 under ultrasonic condition, and continuing ultrasonic for 20 min to make the two combined by electrostatic interaction. The organic solvent was removed by rotary evaporation (30 rpm, 20 degrees celsius) and 5 ml of deionized water was added under sonication for 20 minutes and dialyzed against a dialysis bag of molecular weight 1500 for 12 hours to remove excess tris (hydroxymethyl) aminomethane. The compound Trisporfin is obtained after freeze-drying by a freeze-dryer.
Weighing the compound Trisporfin (5 mg), adding the Trisporfin into 5 ml of anhydrous methanol, carrying out rotary drying by a rotary evaporator (30 r/min, 60 ℃), adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 20 min, removing free dihydroporphin 6 through a water-based filter head membrane (the pore diameter is 0.45 micron), and finally obtaining a clear Trisporfin nano micelle solution.
Example 13
Tris (hydroxymethyl) aminomethane (20 mg) and chlorin e6(5 mg) were weighed into a round bottom flask, 25 ml of dichloromethane was added, and the mixture was sonicated for 30 minutes to allow the two to bind well by electrostatic interaction. And (3) removing the organic solvent by using a rotary evaporator (at the temperature of 35 ℃ at 100 rpm), adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 minutes, and dialyzing for 24 hours by using a dialysis bag with the molecular weight of 100-500 to remove the redundant tris (hydroxymethyl) aminomethane. The compound Trisporfin was obtained after lyophilization.
Weighing the compound Trisporfin (5 mg), adding the Trisporfin into 5 ml of anhydrous methanol, carrying out rotary drying by a rotary evaporator (30 r/min, 50 ℃), adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 min, removing free chlorin e6 by using a water system filter membrane (the pore diameter is 0.22 micron), and finally obtaining a clear Trisporfin nano micelle solution.
Example 14
Tris (hydroxymethyl) aminomethane (20 mg) and chlorin e6(10 mg) were weighed into a round-bottomed flask, and 5 ml of N, N-dimethylformamide was added thereto and sonicated for 30 minutes to sufficiently bind the two by electrostatic interaction. And (3) removing the organic solvent by using a rotary evaporator (at 40 ℃ at 30 rpm), adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 minutes, and dialyzing for 24 hours by using a dialysis bag with the molecular weight of 100-500 to remove the redundant tris (hydroxymethyl) aminomethane. The compound Trisporfin is obtained after freeze-drying by a freeze-dryer.
Weighing the compound Trisporfin (5 mg), adding the Trisporfin into 5 ml of N, N-dimethylformamide, carrying out rotary drying by a rotary evaporator (30 r/min, 40 ℃), adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 30 min, removing free dihydroporphin 6 by a water-system filter membrane (the pore diameter is 0.22 micron), and finally obtaining a clear Trisporfin nano micelle solution.
Example 15
Tris (hydroxymethyl) aminomethane (20 mg) and chlorin e6(5 mg) were weighed into a round-bottomed flask, and 5 ml of N, N-dimethylformamide was added thereto, followed by sonication for 5 minutes to sufficiently bind them by electrostatic interaction. The organic solvent was removed by rotary evaporation (30 rpm, 60 degrees celsius) and 5 ml of deionized water was added under sonication for 5 minutes and dialyzed against a dialysis bag of molecular weight 1500 for 36 hours to remove excess tris (hydroxymethyl) aminomethane. The compound Trisporfin is obtained after freeze-drying by a freeze-dryer.
Weighing the compound Trisporfin (5 mg), adding the Trisporfin into 5 ml of N, N-dimethylformamide, carrying out rotary drying by a rotary evaporator (30 r/min, 70 ℃), adding 5 ml of deionized water under the ultrasonic condition, continuing the ultrasonic treatment for 5 min, removing free chlorin e6 by using a water system filter membrane (the pore diameter is 0.22 micron), and finally obtaining a clear Trisporfin nano micelle solution.
Example 16
Tris (hydroxymethyl) aminomethane (20 mg) and chlorin e6(5 mg) were weighed into a round-bottomed flask, and 5 ml of N, N-dimethylformamide was added thereto, followed by sonication for 5 minutes to sufficiently bind them by electrostatic interaction. And (3) removing the organic solvent by using a rotary evaporator (60 revolutions per minute, 60 ℃, and the like) in a rotary manner, adding 5 ml of deionized water under the ultrasonic condition, continuing ultrasonic treatment for 5 minutes, and dialyzing for 24 hours by using a dialysis bag with the molecular weight of 100-500 to remove the redundant tris (hydroxymethyl) aminomethane. The compound Trisporfin was obtained after lyophilization.
Weighing the compound Trisporfin (5 mg), adding the Trisporfin into 5 ml of N, N-dimethylformamide, carrying out rotary drying by a rotary evaporator (60 r/min, 60 ℃ C.), adding 5 ml of deionized water under the ultrasonic condition, continuing to carry out ultrasonic treatment for 5 min, removing free chlorin e6 by using a water system filter membrane (the pore diameter is 0.22 micron), and finally obtaining a clear Trisporfin nano micelle solution.
FIG. 2 shows the state of chlorin e6 and the nano photosensitizer Trisporfin in water. As can be seen from the figure, the photosensitizer Trisporfin obtained by electrostatic interaction has significantly improved water solubility compared to chlorin e 6.
The nanometer photosensitizer Trisporfin is prepared by a thin film dispersion method, and is characterized by utilizing a dynamic light scattering instrument (DLS) and a transmission electron microscope. As can be seen from fig. 3 and 4, the nano photosensitizer Trisporfin has a uniform nano size.
The target product was further characterized by its spectral properties, see FIGS. 5-7, with the maximum absorption wavelength of Trisporfin, a nano-photosensitizer, at 664 nm. The fluorescence quantum yield of the nano photosensitizer Trisporfin is 0.35 by taking zinc phthalocyanine (ZnPc) as a reference.
In order to further investigate the singlet oxygen generation efficiency of Trisporfin under the illumination condition, 1, 3-diphenyl isobenzofuran (DPBF) was quantitatively tested using a singlet oxygen probe, and zinc phthalocyanine (ZnPc) was used as a reference, and the obtained results are shown in fig. 8. DPBF can react with singlet oxygen efficiently, thereby causing the 415nm absorption peak to be reduced obviously. The 415nm absorption peak of the Trisporfin solution was found to quench significantly over time, calculated as a 0.38 singlet oxygen quantum yield for Trisporfin.
Previous related experiments have tested the ability of the nanophotosensitizer Trisporfin to produce singlet oxygen. In order to further explore the lethality of the nano photosensitizer to the tumor cells, the toxicity of the nano photosensitizer Trisporfin to 4T1 tumor cells under non-illumination and illumination conditions was studied by using the MTT method, and the obtained results are shown in FIGS. 9-10. From the figure, it can be seen that the nano photosensitizer Trisporfin has better cell killing capability.
The test results show that the nano photosensitizer Trisporfin prepared by the invention has uniform and stable size in a water system and biological fluid and has better cell killing capability.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (6)
1. A nano-micelle solution of a chlorin nano photosensitizer is characterized in that the nano-micelle solution is prepared by the following steps:
dissolving a chlorin nano photosensitizer in an organic solvent, after spin-drying, adding deionized water under the ultrasonic condition, continuing to perform ultrasonic treatment, and passing through a water system filter membrane to obtain a nano micelle solution of the chlorin nano photosensitizer;
the preparation method of the chlorin nano photosensitizer comprises the following steps of mixing the chlorin photosensitizer and tris (hydroxymethyl) aminomethane in an organic solvent, and carrying out self-assembly on the chlorin photosensitizer and the tris (hydroxymethyl) aminomethane under the electrostatic interaction to form the chlorin nano photosensitizer; the chlorin photosensitizer comprises chlorin e6, chlorin e6 copper compound, chlorin e6 zinc compound and chlorin e6 manganese compound;
the preparation method of the chlorin nano photosensitizer comprises the following specific steps:
s1, weighing a chlorin photosensitizer and tris (hydroxymethyl) aminomethane according to a ratio, dissolving the chlorin photosensitizer and the tris (hydroxymethyl) aminomethane in an organic solvent, mixing, and performing ultrasonic treatment to fully combine the chlorin photosensitizer and the tris (hydroxymethyl) aminomethane in the solvent through electrostatic interaction;
s2, removing the solvent by spin drying, adding deionized water, continuing to perform ultrasonic treatment, then removing redundant tris (hydroxymethyl) aminomethane by dialysis, and performing freeze-drying to obtain the chlorin nano photosensitizer;
the organic solvent comprises anhydrous methanol or N, N-dimethylformamide.
2. The nanomicelle solution of chlorin nanophotosensitizers according to claim 1, wherein the chlorin nanophotosensitizer is chlorin e6, and the molar ratio of tris (hydroxymethyl) aminomethane to chlorin e6 is not less than 3.
3. The nanomicelle solution of chlorin nanophotosensitizers of claim 1, wherein the organic solvent is removed using an evaporation apparatus.
4. The nanomicelle solution of chlorin nanophotosensitizers according to claim 1, wherein the dialysis is carried out for 24 hours or more using a dialysis bag having a molecular weight of 100 to 3000.
5. The nanomicelle solution of chlorin nanophotosensitizers according to claim 1, wherein the chlorin nanophotosensitizer has a structural formula of:
6. use of a nanomicelle solution of a chlorin nanophotosensitizer according to any one of claims 1-5 for the preparation of a photodynamic therapy medicament.
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