CN110538324B

CN110538324B - Photothermal compound based on iridoid amido color reaction and preparation method and application thereof

Info

Publication number: CN110538324B
Application number: CN201810531484.0A
Authority: CN
Inventors: 闫学海; 刘亚美; 邢蕊蕊; 邹千里; 赵鲁阳
Original assignee: Institute of Process Engineering of CAS
Current assignee: Institute of Process Engineering of CAS
Priority date: 2018-05-29
Filing date: 2018-05-29
Publication date: 2021-04-30
Anticipated expiration: 2038-05-29
Also published as: CN110538324A

Abstract

The invention relates to a photo-thermal compound based on iridoid molecular amino chromogenic reaction, a preparation method and application thereof. The components of the photothermal complex comprise iridoid molecules and molecules containing primary amine functional groups; the preparation method of the related photo-thermal compound comprises the steps of carrying out nucleophilic attack color development reaction on a hemiacetal structure of an iridoid molecule and primary amine functional groups, and carrying out self-assembly on generated color products into particles through hydrophobic effect or forming hydrogel and other forms through hydrogen bond effect; the photothermal composite has a remarkable photothermal conversion effect; the photo-thermal compound has the advantages of uniform size, good stability, injectability and the like, and the preparation method is simple, mature and strong in repeatability, and other photo-thermal conversion materials are not required to be added; the photo-thermal compound has good biocompatibility and biodegradability, and has wide application prospect in the biomedical fields of inflammation diminishing and antibiosis, tissue repair, tumor treatment and the like.

Description

Photothermal compound based on iridoid amido color reaction and preparation method and application thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to a photo-thermal compound and a preparation method and application thereof. In particular to a photothermal compound based on iridoid amido color reaction, a preparation method and application thereof in the biomedical fields of inflammation diminishing and antibiosis, tissue repair, tumor treatment and the like.

Background

The photothermal complex has wide application in the field of biomedicine, including anti-inflammatory and antibacterial, tissue repair, anti-tumor treatment and the like. Photothermal therapy is characterized by high spatio-temporal selectivity and low systemic damage. The photothermal complex plays an important role in photothermal therapy, after the photothermal complex absorbs light with specific wavelength, the light energy is converted into heat energy, and the generated high temperature has a killing effect on tumor cells. Although photothermal complexes are continuously developed, certain problems still exist in the photothermal complexes, such as poor targeting at tumor sites, poor in vivo circulation stability and the like.

As tumor tissue angiogenesis is exuberant, forming leaky vasculature, nanoparticles can extravasate from this vascular region, passively enriching into tumor tissue through enhanced permeability and retention Effects (EPR). Therefore, the nano photothermal complex has important application in the fields of tumor diagnosis, imaging and treatment; the injectable hydrogel can be used for minimally invasive treatment, has controllable degradation performance and treatment characteristics of one-time injection and multiple treatments, and is widely applied to biomedicine, and meanwhile, the hydrogel is widely applied to the field of preparation of anti-inflammatory and antibacterial agents and tissue repair preparations.

The photothermal material can be roughly divided into an inorganic photothermal material and an organic photothermal material, the inorganic photothermal material mainly comprises gold materials (gold nano-dots, gold nano-rods, gold nano-cages and the like), metal compounds and two-dimensional materials (graphene oxide, black phosphorus and the like), the preparation methods of the inorganic photothermal materials are mature, the stability is good, and the development of the inorganic photothermal materials in photothermal therapy is limited by the potential biotoxicity of the inorganic photothermal materials; the organic photothermal material mainly comprises polymers (polypyridine, polydopamine and the like) and organic dye molecules (photosensitizer, cyanine and the like), and the polymer photothermal agent has the defects of complex preparation method and complex biodegradation process; organic dye molecules (photosensitizers, cyanines, etc.) are good in biocompatibility and degradable, but cannot form stable nanoparticles in aqueous solution by themselves, and thus require a nano-drug delivery system for loading. CN105816877A discloses a nanoparticle with a photothermal effect, which is prepared by taking a molecule with primary amino group as a framework material and genipin as a cross-linking agent, preparing nanoparticles by using an inverse microemulsion method, then performing surface modification by using PEG, and carrying a photothermal conversion material indocyanine green. The patent takes nanoparticles prepared by crosslinking genipin with primary amino molecules as carriers of photo-thermal medicine indocyanine green, the effect of the nanoparticles is only the effect of loading and delivering photo-thermal agent indocyanine green, and the photo-thermal effect of the nanoparticles is realized by the photo-thermal agent indocyanine green. Although the nano delivery particle improves and enhances the stability and tumor accumulation capacity of the photothermal conversion material indocyanine green, the problems of drug leakage in-vivo circulation and the like are inevitable.

Disclosure of Invention

Surprisingly, the inventor of the present invention found that iridoid molecules and molecules containing primary amine functional groups undergo nucleophilic attack color development reaction to form a composite, and the composite has photothermal conversion performance without carrying other photothermal conversion materials. The present invention has been completed based on the unexpected finding that iridoid molecules themselves do not have significant photothermal conversion properties.

The invention aims to provide a photothermal complex based on iridoid molecular amino chromogenic reaction, a preparation method thereof and application of the photothermal complex in preparation of anti-inflammatory antibacterial, tissue repair and anti-tumor photothermal treatment preparations. The photothermal composite of the invention has the advantages of injectability, good stability, simple and mature preparation process, strong repeatability and no need of adding other light absorption materials. When the photothermal complex is used for preparing tumor imaging and photothermal treatment preparations, the photothermal complex has the advantages of good biocompatibility, high tumor enrichment rate and high photothermal conversion efficiency, can be doped with chemotherapeutic drugs, anti-inflammatory and antibacterial drugs and tissue repair promoting factors, and is used for preparing biological medicinal preparations for anti-inflammatory and antibacterial drugs, tissue repair, tumor treatment and the like.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect: the invention provides an iridoid molecule amino chromogenic photothermal complex, which is characterized in that the photothermal complex is formed by the reaction of iridoid molecules and molecules containing primary amine functional groups;

preferably, the iridoid molecule is one or a mixture of more than two of genipin, geniposide acid, aucubin (aglycone) and paederoside (aglycone);

preferably, the primary amine functional group-containing molecule comprises a molecule such as a polypeptide, a protein, a polysaccharide, a primary amine modified polymer, or the like, for biocompatibility reasons; more preferably, the molecule containing primary amine functional group is one or a mixture of any two or more of polylysine, collagen, albumin, protamine, chitosan, dopamine, primary amine modified polylactic acid, primary amine modified polyethylene glycol, primary amine modified polycaprolactone and the like;

the iridoid molecules are not limited to the molecules listed in the invention, and other iridoids capable of undergoing an amino color reaction with molecules containing primary amine functional groups can be used;

the primary amine molecules described herein are not limited to the molecules listed herein, and other molecules containing primary amine functionality are possible.

The photothermal complex of the invention is formed by the nucleophilic attack color development reaction of the hemiacetal structure of the iridoid molecule and a primary amino functional group, and the generated color product can be self-assembled into particles through hydrophobic effect or form hydrogel through hydrogen bond effect, and the like.

In a second aspect: the present invention provides a method for producing the photothermal composite of the first aspect, comprising the steps of:

(1) pre-dissolving molecules containing primary amine functional groups and iridoid molecules, adding the pre-dissolved molecules and the iridoid molecules into an aqueous solution, and uniformly mixing;

(2) adjusting the pH value of the mixed solution obtained in the step (1) to 3-10, preferably 3.5-7.5;

(3) reacting the mixed solution obtained in the step (2) at 5-80 ℃, preferably 20-70 ℃ for 3-168 hours, preferably 12-72 hours to obtain a photothermal complex;

in step (1) of the present invention, in the preparation of the hydrophobic iridoid molecule and the molecule containing a primary amine functional group, the solvent is preferably: one or a mixture of more than two of phosphate buffer solution, ethanol and hexafluoroisopropanol solution; if hydrophilic iridoid molecules and primary amine-containing molecules are prepared, the hydrophilic iridoid molecules and the primary amine-containing molecules are directly added into water for dissolving and mixing.

The final prepared photothermal complex aqueous solution in step (1) of the present invention has a primary amine functional group-containing molecule concentration of 0.1-200mg/mL, preferably 0.5-20mg/mL, and more preferably 1-10mg/mL by volume mass of the aqueous solution. The volume mass concentration of the iridoid molecules in the aqueous solution is 0.01-50mg/mL, preferably 0.1-20mg/mL, and more preferably 1-10 mg/mL.

In a third aspect: the particle size of the photo-thermal compound can be regulated, the particle size is regulated by changing the initial concentration, the proportion and the reaction temperature of iridoid molecules and primary amine-containing molecules in the preparation process, and the particle size distribution of the prepared photo-thermal compound can be changed between 20nm and 1 mu m, preferably between 40 and 200 nm. The photothermal hydrogel provided by the invention has injectable properties.

In a fourth aspect, as a preferred embodiment of the present invention, the method for preparing the photothermal composite comprises the steps of:

(1) preparing aqueous solutions of iridoid molecules and molecules containing primary amine functional groups respectively; wherein, when the iridoid molecules and the molecules containing primary amine are hydrophobic molecules, the hydrophobic molecules are pre-dissolved in ethanol and hexafluoroisopropanol solution when preparing the aqueous solution of the iridoid molecules and the molecules containing primary amine; when the molecules are hydrophilic molecules, the hydrophilic iridoid molecules and the molecules containing primary amine are added into water or phosphate buffer salt solution for dissolving without adding other organic solvents when preparing the aqueous solution of the molecules;

wherein the volume mass concentration of the iridoid molecules in the aqueous solution is 1-10mg/mL, and the volume mass concentration of the primary amine-containing molecules in the aqueous solution is 1-10 mg/mL.

(2) And (2) adjusting the pH value of the mixed solution obtained in the step (1) to 3.5-7.5.

(3) And (3) reacting the mixed solution obtained in the step (2) at the temperature of between 20 and 70 ℃ for 12 to 72 hours to obtain the photothermal composite.

In a fifth aspect, the present invention provides the photothermal composite of the second aspect, wherein the photothermal effect of the photothermal composite is derived from an amino color development product of an iridoid molecule and a primary amine molecule, rather than by adding other photothermal conversion materials.

In a sixth aspect, the invention also provides the use of a photothermal complex as described in the first aspect for the preparation of a formulation for tumor imaging and photothermal therapy.

In a seventh aspect, the present invention provides a doped photothermal composite, which is characterized by comprising the photothermal composite of the first aspect to the fourth aspect and a dopable component.

In an eighth aspect, the doped optical composite according to the seventh aspect, wherein the dopable component is any one or a mixture of two or more of chemotherapeutic drugs, anti-inflammatory and antibacterial drugs, and tissue repair-promoting factors.

Preferably, the anti-inflammatory and antibacterial drug is any one or a mixture of more than two of silver ions, silver particles, streptomycin, gentamicin, cephalothin, vancomycin and analogues thereof;

preferably, the chemotherapeutic drug is any one or a mixture of more than two of adriamycin, paclitaxel, docetaxel, curcumin and analogues thereof;

preferably, the factor for promoting tissue repair is any one or a mixture of more than two of vascular endothelial growth factor, platelet-derived growth factor, epidermal growth factor, transforming growth factor beta, basic fibroblast growth factor and insulin-like growth factor;

the method for producing a photothermal composite according to a ninth aspect, a seventh aspect, or an eighth aspect, comprises the steps of:

(1) pre-dissolving primary amine-containing molecules and iridoid molecules, adding the mixture into an aqueous solution, and uniformly mixing, wherein the volume mass concentration of the primary amine-containing molecules in the aqueous solution is 0.1-200mg/mL, preferably 0.5-20mg/mL, and more preferably 1-10 mg/mL. The volume mass concentration of the iridoid molecules in the aqueous solution is 0.01-50mg/mL, preferably 0.1-20mg/mL, and more preferably 1-10 mg/mL.

(2) Adding a dopable component into the aqueous solution prepared in the step (1), wherein the volume mass concentration of the dopable component in the solution is 0.01-100mg/mL, preferably 0.1-20 mg/mL;

(3) adjusting the pH value of the solution in the step (2) to 3-10, preferably 3.5-7.5;

(4) and (4) reacting the solution obtained in the step (3) at 5-80 ℃, preferably 20-70 ℃ for 3-168 hours, preferably 12-72 hours to finally obtain the photothermal composite doped with the active ingredient.

In a tenth aspect, the present invention also provides the use of the doped photothermal composite of the seventh or eighth aspect in the preparation of an anti-inflammatory, antibacterial, tissue repair formulation.

Compared with the prior art, the invention has the advantages that:

(1) the preparation method of the photo-thermal compound has universality and is suitable for amino color development reaction of a series of iridoid and primary amine;

(2) the photo-thermal compound obtained by the invention has uniform appearance, adjustable size and adjustable absorption wavelength.

(3) The preparation method of the photo-thermal compound is simple, mature and strong in repeatability, and the photo-thermal compound is prepared by only mixing and reacting iridoid molecular solution and primary amine-containing molecular solution, and other materials with light absorption properties are not required to be added in the preparation process.

(4) The photothermal complex of the invention shows good blood circulation stability in vivo experiments; the photothermal nano-composite has an EPR effect, and the enrichment of the photothermal nano-composite in tumor parts in vivo experiments is improved; after the photo-thermal nano-composite is enriched at a tumor part, the position of the tumor can be displayed through imaging, the optimal treatment time can be judged, and photo-thermal treatment can be further carried out.

(5) The photo-thermal compound has wide application prospect in the biomedical fields of anti-inflammation and antibiosis, tissue repair, anti-tumor treatment and the like.

Drawings

FIG. 1 shows a photograph of the photothermal composite obtained in example 1 on the left and a photograph of the photothermal hydrogel obtained in example 2 on the right;

FIG. 2 is a graph showing a distribution of particle sizes of the photothermal composite obtained in example 1;

FIG. 3 is a transmission electron microscope photograph of the photothermal composite obtained in example 3;

FIG. 4 is a scanning electron micrograph of the photothermal composite obtained in example 4;

FIG. 5 is a graph showing the temperature rise curves of the photothermal composite obtained in example 5 and blank water under irradiation of 660nm laser (1 w);

fig. 6 is a graph showing the ultraviolet absorption of the photothermal composite obtained in example 6;

FIG. 7 is a TEM image of the doxorubicin-doped photothermal hydrogel obtained in example 8;

FIG. 8 is a photograph of dark toxicity of Hela cells of the photothermal complex obtained in example 9;

fig. 9 is a graph showing a temperature rise curve of the photothermal composite obtained in example 10 in living animals.

Detailed Description

The invention is described in detail below with reference to the figures and the embodiments. The following examples are only illustrative of the present invention, and the scope of the present invention shall include the full contents of the claims, not limited to the examples.

Example 1

(1) Pre-dissolving phenylalanine dipeptide into hexafluoroisopropanol solution to prepare solution with volume mass concentration of 100 mg/mL; preparing a genipin aqueous solution with the volume mass concentration of 0.5 mg/mL.

(2) Adding 10 mu L of hexafluoroisopropanol solution of the phenylalanine dipeptide into 1mL of genipin aqueous solution, uniformly mixing by ultrasonic waves, and reacting at 70 ℃ for 24 hours to obtain the photo-thermal particles.

The physical diagram of the photothermal particles obtained in this example is shown in fig. 1a, and the particle size distribution diagram is shown in fig. 2; the phenylalanine dipeptide and genipin are adopted for crosslinking and self-assembling to form the photo-thermal particles, and the photo-thermal particles are dark blue and have good dispersibility.

Example 2

(1) 5mg of collagen was dissolved in 2mL of 0.2M acetic acid solution, and vortexed for 6h until the collagen was completely dissolved. Preparing a genipin aqueous solution with the volume mass concentration of 1 mg/mL.

(2) 500uL of the genipin aqueous solution was added to 500uL of the collagen solution, and the mixture was shaken to mix the two solutions uniformly. The photothermal hydrogel was formed after standing at 37 ℃ for 24 hours.

The physical diagram of the photothermal hydrogel obtained in this example is shown in FIG. 1 b.

Example 3

(1) Pre-dissolving histidine-phenylalanine dipeptide into hexafluoroisopropanol solution to prepare solution with volume mass concentration of 100 mg/mL; preparing a genipin aqueous solution with the volume mass concentration of 0.5 mg/mL.

(2) Adding 10 mu L of hexafluoroisopropanol solution of the histidine-tryptophan dipeptide into 1mL of genipin aqueous solution, uniformly mixing by ultrasonic waves, and reacting at 70 ℃ for 24 hours to obtain the photo-thermal particles.

The transmission electron microscope image of the photothermal particles obtained in this example is shown in FIG. 3. The photo-thermal nano-drug formed by crosslinking and self-assembling histidine-phenylalanine dipeptide and genipin has good dispersity and the average grain diameter is 200 nm.

Example 4

(1) Preparing an albumin aqueous solution with the volume mass concentration of 50 mg/mL; preparing a genipin ethanol solution with the volume mass concentration of 50 mg/mL.

(2) 200. mu.L of the above ethanol solution of genipin was added to 100. mu.L of an aqueous albumin solution to precipitate albumin particles, followed by reaction at 70 ℃ for 24 hours to obtain photothermal particles.

(3) The photothermal pellet solution was centrifuged and washed, and then 1mL of ultrapure water was added for resuspension.

The scanning electron micrograph of the photothermal particles obtained in this example is shown in fig. 4.

Example 5

(1) Preparing a hexafluoroisopropanol solution of histidine-tryptophan dipeptide with the volume mass concentration of 100 mg/mL; preparing a genipin aqueous solution with the volume mass concentration of 0.5 mg/mL.

(2) And adding 10 mu L of the dipeptide solution into 1mL of the genipin aqueous solution, uniformly mixing by ultrasonic waves, and reacting at 70 ℃ for 24 hours to obtain the photo-thermal particles.

The temperature rise profile of the photothermal composite obtained in this example and blank water under 660nm laser (1w) irradiation is shown in FIG. 5.

Example 6

(1) Pre-dissolving phenylalanine dipeptide into hexafluoroisopropanol solution to prepare solution with volume mass concentration of 100 mg/mL; preparing the aucubin water solution with the volume mass concentration of 0.5 mg/mL.

(2) Adding 10 μ l of hexafluoroisopropanol solution of phenylalanine dipeptide into 1mL of aucubin aqueous solution, mixing uniformly by ultrasonic wave, and reacting at 70 deg.C for 24 hr to obtain photothermal complex.

The ultraviolet absorption pattern of the photothermal particles obtained in this example is shown in fig. 6.

Example 7

(1) Pre-dissolving phenylalanine dipeptide into hexafluoroisopropanol solution, wherein the volume mass concentration of the phenylalanine dipeptide is 100 mg/mL; preparing a genipin aqueous solution with the volume mass concentration of 0.5 mg/mL; and preparing streptomycin water solution with volume mass concentration of 50 mg/mL.

(2) And adding 10 mu l of the phenylalanine dipeptide into 1mL of genipin aqueous solution, uniformly mixing by ultrasonic waves, adding 10 mu l of streptomycin aqueous solution into the mixed solution, and reacting for 24 hours at 70 ℃ to obtain the streptomycin-doped photo-thermal compound.

Example 8

(1) 5mg of collagen was dissolved in 2mL of 0.2M acetic acid solution, and vortexed and shaken for 6 hours until the collagen was completely dissolved. Preparing a mixed aqueous solution of genipin and adriamycin with the volume mass concentration of 1 mg/mL.

(2) 500uL of the mixed aqueous solution of genipin and adriamycin was added to 500uL of the collagen solution, and the mixture was shaken to mix the two solutions uniformly. Standing at 37 deg.C for 24 hr to form photothermal hydrogel doped with chemotherapeutic adriamycin.

FIG. 7 shows a TEM image of the photothermal hydrogel obtained in this example.

Example 9

In vitro cytotoxicity experiments were performed using the phenylalanine dipeptide genipin photothermal particles obtained in example 1 as an example. Hela cells with the density of 10000 are inoculated into a 96-well plate and cultured overnight at 37 ℃, samples with a series of concentrations are added to be incubated with the cells for 48 hours, and the killing degree of the cells is measured by using an MTT reagent at different administration concentrations, as shown in figure 8, photothermal particles have no obvious toxicity to the cells, which indicates that the material has high biocompatibility.

Example 10

And (4) carrying out temperature rise imaging on the living animal body. Selecting female BALB/c-nu nude mice of 4-6 weeks, and taking 100 μ L of the nude mice with the density of 5 × 10⁶Hela cells were injected into the hind leg of nude mice when tumors grew to about 100mm³The experiment was started. 200 μ L of the histidine-tryptophan dipeptide-genipin photothermal complex obtained in example 5 was injected intravenously, and compared with a blank nude mouse without drug injection, 24h post-exposure imaging was performed at elevated temperature.

As shown in fig. 9, the photothermal particle-injected group showed an increased ability to increase the temperature at the tumor site compared to the drug-non-injected blank group.

The applicant states that the product and the detailed preparation method of the present invention are illustrated by the above examples, but the present invention is not limited to the above product and the detailed preparation method, i.e. the present invention is not meant to be implemented by relying on the above product and the detailed preparation method. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims

1. The application of the photothermal complex prepared based on iridoid molecule amino chromogenic reaction in preparing preparations for tumor imaging and tumor treatment through photothermal conversion is characterized in that the photothermal complex is a complex formed by the chromogenic reaction of iridoid molecules and molecules containing primary amine functional groups,

the iridoid molecules are one or a mixture of more than two of genipin, geniposide, aucubin or aglycone, and paederoside or aglycone,

the molecule containing the primary amine functional group is polypeptide, protein and polysaccharide.

2. The use according to claim 1, wherein the molecule containing primary amine functional group is one or a mixture of any two or more of polylysine, collagen, albumin, protamine, chitosan, and dopamine.

3. The use according to claim 1 or 2, the photothermal composite being prepared by a preparation method comprising the steps of:

(1) pre-dissolving molecules containing primary amine functional groups and iridoid molecules, adding the pre-dissolved molecules into an aqueous solution, and uniformly mixing;

(2) adjusting the pH value of the mixed solution obtained in the step (1) to 3-10;

(3) and (3) reacting the mixed solution obtained in the step (2) at the temperature of 5-80 ℃ for 3-168 hours to obtain the photo-thermal compound.

4. The use according to claim 3, wherein the solvent used in the pre-dissolving in step (1) is one or a mixture of any two or more of water, phosphate buffered saline, ethanol, and hexafluoroisopropanol solution.

5. The use according to claim 3, wherein the mixed solution in step (2) has a volume/mass concentration of the primary amine functional group-containing molecule of 0.1-200mg/mL and a volume/mass concentration of the iridoid molecule of 0.01-50 mg/mL.

6. The use according to claim 1, wherein the photothermal composite further comprises a dopant component, and the dopant component is any one or a mixture of two or more of an anti-inflammatory and antibacterial drug, a chemotherapeutic drug, and a tissue repair-promoting factor.

7. The use of claim 6, wherein the chemotherapeutic agent is any one or a mixture of more than two of adriamycin, paclitaxel, docetaxel and curcumin.

8. Use according to claim 1, characterized in that it comprises a step of heating by optical excitation.