阅读说明：本技术 一种Ce6衍生物、其纳米制剂及其制备方法和应用 (Ce6 derivative, nano preparation thereof, and preparation method and application thereof ) 是由 栾玉霞 秦晓晗 于 2021-01-08 设计创作，主要内容包括：本发明提供一种Ce6衍生物、其纳米制剂及其制备方法和应用。所述Ce6衍生物,其命名为TCe6,结构式如下式所示,本发明以TCe6与CHC共组装构建TCe6/CHC NPs,解决了PDT中氧气不足和效率低下的问题,实现了高效的光动力治疗。(The invention provides a Ce6 derivative, a nano preparation thereof, a preparation method and application thereof. The Ce6 derivative is named as TCe6, the structural formula is shown as the following formula, and TCe6 and CHC are jointly assembled to construct TCe6/CHC NPs, so that the problem of oxygen deficiency in PDT is solvedThe problem of low efficiency and the realization of high-efficiency photodynamic therapy.)

1. A Ce6 derivative is named as TCe6, and the structural formula is as follows:

2. a method for producing the Ce6 derivative of claim 1, comprising: ce6 was subjected to esterification reaction with TPGS.

3. The method of manufacturing according to claim 2, comprising: NHS and DCC are dripped into Ce6 solution, the solution is stirred under the inert protective atmosphere to obtain Ce6-COOH solution, DMAP is introduced into the Ce6-COOH solution, and the reaction is carried out under the inert protective atmosphere.

4. Use of the Ce6 derivative of claim 1 in the preparation of a pharmaceutical carrier.

5. A drug delivery system which is prepared from the Ce6 derivative of claim 1 or comprises the Ce6 derivative of claim 1.

6. A nano-formulation comprising the Ce6 derivative of claim 1 and at least one drug.

7. The nano-formulation according to claim 6, wherein the drug is a drug having an endogenous oxygen conserving property.

8. TCe6/CHC NPs comprising the Ce6 derivative of claim 1 and CHC.

9. The process of claim 8 for the preparation of TCe6/CHC NPs comprising co-assembling TCe6 and CHC in solution.

10. Use of the Ce6 derivative of claim 1 or the nanoformulation of claims 6 to 7 or the TCe6/CHC NPs of claim 8 in the preparation of an anti-neoplastic drug or in the preparation of a photodynamic therapy drug.

Technical Field

The invention relates to the technical field of medicines, and particularly relates to a Ce6 derivative, a nano preparation thereof, and a preparation method and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Photodynamic therapy (PDT) uses photosensitizers and oxygen to generate singlet oxygen to kill tumors, and spatial and temporal control treatment is achieved by laser. PDT as a minimally invasive therapy can selectively act on tumor sites and greatly reduce side effects compared to traditional therapies such as surgery, chemotherapy and radiation. However, the hypoxic microenvironment of tumors and poor water solubility of traditional photosensitizers are two major problems that compromise the advantages of PDT anti-tumor therapy.

Chlorin e6 is a photosensitizer widely used for tumor growth in photodynamic therapy (PDT), but its hydrophobic characteristics and hypoxia in the tumor microenvironment greatly impair its therapeutic efficacy, and thus, there is an urgent need to solve this problem to achieve more excellent therapeutic efficacy. Much current research has overcome the limitations of hypoxia by increasing the oxygen supply in tumors, primarily through the use of external oxygen supply or in situ generation strategies. However, oxygen regulation for PDT is not ideal due to premature leakage of oxygen and insufficient oxygen production efficiency.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a Ce6 derivative TCe6, which improves the solubility of Ce6 and enables the Ce6 derivative to have excellent amphiphilic performance. The invention further provides Ce6/CHC NPs, which are prepared by co-assembling Ce6 derivatives TCe6 and alpha-cyano-4-hydroxycinnamate (CHC), and the Ce6/CHC NPs can be prepared into a nano preparation, so that the combination of improving the solubility of a photosensitizer and improving the tumor hypoxia microenvironment is realized, the problems of insufficient oxygen and low efficiency in PDT can be effectively solved, and the efficient photodynamic therapy is realized.

Specifically, the technical scheme of the invention is as follows:

in a first aspect of the present invention, the present invention provides a Ce6 derivative, named TCe6, having the following structural formula:

the Ce6 derivative TCe6 can improve the solubility of Ce6, increase the generation of ROS and enhance the curative effect of photodynamic therapy.

In a second aspect of the present invention, the present invention provides a process for the preparation of the Ce6 derivative TCe6, which comprises: performing esterification reaction on chlorin E6(Ce6) and vitamin E succinate polyethylene glycol (TPGS).

Vitamin E succinate polyethylene glycol (D-alpha-tocopherol succinate, abbreviated as TPGS, CAS #: 9002-96-4, formula (C)₂H₄O)_nC₃₃H₅₄O₅Is prepared by esterifying natural Vitamin E Succinate (VES) and polyethylene glycol (PEG), and the molecular weight of the natural Vitamin E Succinate (VES) depends on the polymerization degree n of the PEG. In an embodiment of the invention, the TPGS is D- α -tocopheryl polyethylene glycol 1000 succinate, i.e. the carboxyl group of VES is esterified with PEG1000, abbreviated as TPGS1000, which has a relative molecular weight of about 1513.

In some embodiments of the invention, the method comprises: dropwise adding N-hydroxysuccinimide (NHS) and Dicyclohexylcarbodiimide (DCC) into the Ce6 solution, stirring under an inert protective atmosphere to obtain a carboxyl activated Ce6 solution (Ce 6-COOH solution for short), introducing TPGS and 4-Dimethylaminopyridine (DMAP) into the Ce6-COOH solution, and reacting under the inert protective atmosphere.

In an embodiment of the present invention, the preparation method involves the following reaction scheme:

in some embodiments of the present invention, the method activates the carboxyl group of Ce6 at a stirring temperature of-10 to 10 ℃, preferably 0 ℃, for a stirring time of 18 to 30 hours, preferably 24 hours.

In some embodiments of the invention, the temperature of the reaction of TPGS with Ce6 is room temperature (10-30 ℃) and the reaction time is 42-54h, preferably 48 h.

In some embodiments of the invention, the solvent of the NHS solution, DCC solution, DMAP solution, Ce6 solution, TPGS solution is anhydrous N, N-Dimethylformamide (DMF).

In some embodiments of the invention, the molar ratio of Ce6 and TPGS is 1-3:1-2, preferably 2: 1.

the amount of NHS, DCC, DMAP used will have an effect on the extent of the reaction in some embodiments of the invention, and the reaction is more complete and the yield is relatively better when the molar ratio of Ce6, TPGS, NHS, DCC, DMAP is 2:1:4:4:2 in some embodiments of the invention.

In order to obtain more pure TCe6, in one or more embodiments of this embodiment, purification and refinement processes can be performed, such as removing solvent from the reacted material to obtain a crude product, and purifying the crude product by dialysis, preferably by DMF dialysis and water dialysis, respectively. After completion of dialysis, the dialyzed solution was freeze-dried to obtain TCe6 powder.

In a third aspect of the invention, the invention provides the use of the Ce6 derivative TCe6 in the preparation of a pharmaceutical carrier.

In a fourth aspect of the invention, the invention provides a pharmaceutical carrier or drug delivery system, which is prepared from TCe6, a Ce6 derivative, or comprises TCe6, a Ce6 derivative.

In a fifth aspect of the invention, the invention provides a nano-formulation comprising the Ce6 derivative TCe6 and at least one drug.

In the embodiment of the invention, in the nano-preparation, the Ce6 derivative TCe6 can be used as a drug carrier, and the carried drug is a drug with oxygen saving performance (a drug for reducing the consumption of endogenous oxygen by cells). During metabolism, the produced lactic acid enters the tumor cells from the tumor microenvironment via monocarboxylate transporter 1(MCT1), and is rapidly oxidized by consuming oxygen to produce adenosine triphosphate. Therefore, the MCT1 receptor inhibitor can inhibit the metabolism of lactic acid and save endogenous oxygen. Thus, in some embodiments of the invention, the drug is an MCT1 receptor inhibitor, such as ethyl (E) -2-cyano-3- (4-hydroxyphenyl) acrylate and/or α -cyano-4-hydroxycinnamate (CHC), and the like.

In some embodiments of the invention, the nanoformulation is an intravenous injection.

In a sixth aspect of the invention, the invention provides TCe6/CHC NPs comprising the Ce6 derivatives TCe6 and CHC, which are obtained by co-assembly of TCe6 and CHC in aqueous solution. The co-assembly, i.e., TCe6 and CHC, spontaneously forms ordered structures or nanoaggregates (or also referred to as nanoassemblies).

In some embodiments of the invention, TCe6/CHC NPs are prepared by a process comprising: and mixing and stirring the TCe6 solution and the CHC solution, adding the mixed solution into water, and stirring to spontaneously form nano-aggregates.

In some embodiments of the invention, TCe6/CHC NPs can be obtained after spontaneous formation of nanoaggregates, by membrane filtration of the resulting solution dialyzed against water in the dark by means of dialysis bags (molecular weight cut-off: 1000 Da).

In some embodiments of the invention, the mass ratio of TCe6 to CHC is 1: 1-10, especially when the mass ratio of TCe6 to CHC is 1:3, the effect is better.

In the embodiment of the invention, TCe6/CHC NPs have uniform morphology, good particle size and less than 200nm, and can be prepared into intravenous preparations.

In a seventh aspect of the present invention, the present invention provides an application of the Ce6 derivative TCe6 of the first aspect, or the nano-formulation of the fifth aspect, or the TCe6/CHC NPs of the sixth aspect, in the preparation of an anti-tumor drug or in the preparation of a photodynamic therapy drug.

In some embodiments of the present invention, the anti-tumor drug is a drug for treating colon cancer

In some embodiments of the invention, TCe6/CHC NPs have excellent photodynamic properties that can enhance the effect of phototherapy, and in embodiments of the invention, TCe6/CHC NPs can generate ROS not only through Ce 6-mediated photodynamic ability, but also through TPGS.

The TCe6/CHC NPs of the invention can be accumulated at the tumor site through EPR effect, and have strong tumor inhibiting effect. In some embodiments of the invention, the TCe6/CHC NPs formulation showed strong cytotoxicity to cancer cells in cellular experiments, and the formulation was biocompatible and low in dark toxicity; in animal experiments, TCe6/CHC NPs show excellent tumor inhibition effect, and the tumor inhibition rate is as high as 95%.

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

the novel Ce6 derivative (TCe6) is synthesized for the first time, and the novel Ce6 derivative and CHC are assembled together for the first time to form the nano-particle TCe6/CHC NPs, so that the defects of strong hydrophobicity of Ce6 and difficult drug delivery in vivo are overcome, the generation of ROS can be increased, endogenous oxygen can be enhanced and saved, the tumor hypoxia microenvironment is improved, and the phototherapy curative effect is increased. TCe6/CHC NPs can be accumulated in tumor sites through EPR effect, have good ROS generation capacity, show stronger cytotoxicity under the action of light, have good biocompatibility and low dark toxicity, have excellent tumor inhibition effect, and are particularly suitable for photodynamic therapy.

In addition, the TCe6/CHC NPs have good stability, uniform shape, good particle size of less than 200nm, can be accumulated in tumor sites through EPR effect, are favorable for intravenous injection, are easy to transport and store, and provide favorable conditions for industrial storage.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application. Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a nuclear magnetic spectrum of TCe6 in example 2 of the present invention;

FIG. 2 is a UV spectrum of TCe6/CHC NPs of example 3 of this invention;

FIG. 3 is a graph showing the results of in vitro cytotoxicity experiments of TCe6/CHC NPs of example 4 of the present invention, wherein the left graph shows the cytotoxicity results under the action of light, and the right graph shows the cytotoxicity results in the absence of light (dark);

FIG. 4 is a graph of the results of the in vitro ROS assay for TCe6/CHC NPs of example 5 of the present invention;

FIG. 5 is a graph of the results of in vivo antitumor experiments with TCe6/CHC NPs of example 6 of the present invention; the left graph is a curve chart of tumor volume change, and the right graph is a graph of tumor weight and tumor inhibition rate.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or starting materials used in the present invention can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present invention can be used in a conventional manner in the art or in accordance with the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1Molecular synthesis of TCe6

An amount of Ce6 was precisely weighed on an analytical balance, dissolved in anhydrous N, N-Dimethylformamide (DMF) and placed in a 50mL round bottom flask. A certain amount of NHS and DCC are weighed, dissolved in anhydrous DMF, added into the stirring Ce6 solution and reacted for 24 hours under the protection of nitrogen and at the temperature of 0 ℃. Removing impurities from the activated Ce6-COOH film, placing the activated Ce6-COOH film in a 50mL round-bottom flask, weighing a certain amount of DMAP and TPGS, dissolving the DMAP and TPGS in anhydrous DMF, adding the DMAP and TPGS into the stirring Ce6-COOH, and reacting for 48h under the conditions of nitrogen protection and normal temperature. Wherein the molar ratio of Ce6, TPGS, NHS, DCC and DMAP is 2:1:4:4: 2. After the reaction, the crude product was purified by DMF dialysis and water dialysis (cut-off: 1500Da) respectively, and the final TCe6 solution was freeze-dried to give TCe6 in the form of a dark green solid, TCe6 pure product in 51.92% yield.

Example 2Hydrogen nuclear magnetic resonance (1H-NMR) spectroscopy identified the chemical structure of TCe 6.

Weighing TCe6 about 5mg, dissolving deuterated dimethyl sulfoxide (DMSO-d6) and placing in a nuclear magnetic tube, measuring the nuclear magnetic resonance hydrogen spectrum by a 400MHz nuclear magnetic resonance hydrogen spectrometer, and recording the chemical shift value (ppm) of the compound. The results are shown in fig. 1, and the nuclear magnetic results can confirm that the newly synthesized molecules have peaks of raw material molecules Ce6 and TPGS, and confirm the successful synthesis of TCe 6; similarly, the UV results in the left panel of FIG. 2 aided in confirming the successful synthesis of TCe 6.

Example 3Preparation of TCe6/CHC NPs.

Precisely weighing about 5mg of TCe6, and dissolving in 50 mu LDMF; precisely weighing 15mg of CHC, dissolving in 150 mu LDMF, fully and uniformly mixing the TCe6 solution and the CHC solution, stirring for 15min, dropwise adding the obtained mixed solution into 4mL of stirred water, spontaneously forming nano aggregates, removing the organic solvent from the obtained preparation by a dialysis method (cut-off molecular weight of a dialysis bag: 1000Da), and filtering through a 0.8nm filter membrane to remove impurities to obtain the final preparation. The UV results are shown in the right panel of FIG. 2, and show the simultaneous appearance of characteristic peaks for TCe6 and CHC in the prepared TCe6/CHC NPs, confirming the successful preparation of the formulation.

The TCe6/CHC NPs prepared were used in examples 4-6 described below.

Example 4TCe6/CHC NPs in vitro cytotoxicity assay

1. Culture of cells

The colon cancer line CT26 cells were selected as the study subject. CT26 cells were cultured in RPMI 1640 medium containing 10% Fetal Bovine Serum (FBS), 1% streptomycin/penicillin at 37 deg.C in a humidified environment of 5% CO 2. And (5) when the cells grow to high density, carrying out passage, proportionally transferring the cells into a culture bottle, continuously culturing the cells and counting the cells. In the latter assay, CT26 cells were incubated with glucose-free RPMI 1640 (containing 10X 10 cells)^-3M lactic acid) was incubated at 37 ℃, and the medium was called lactic acid 1640 medium.

2. Experiment of cytotoxicity

CT26 cells were collected in logarithmic growth phase and diluted to 3X 10 with lactic acid 1640 medium⁴one/mL. The preparations of the target compounds Ce6, CHC, TCe6, TCe6/CHC NPs to be tested were each diluted with the medium to (the concentration of Ce6 is 1.0,2.0,4.0,6.0,8.0, 10.0. mu.g/mL; the concentration of CHC is 0.7, 1.4, 2.8, 4.2, 5.6, 7.0. mu.g/mL). Cells were cultured at 6X 10³After adding the concentration of each well (200 mu L) into a 96-well plate, adhering the cells to the wall overnight, discarding the culture medium, adding 200 mu L of target compound solution with different concentrations, setting 3 multiple wells for each concentration, setting the wells without drugs as a negative control group, setting the wells without cells as a blank control group, and incubating all the groups at 37 ℃ for 6 h. Then the drug-containing medium was discarded and replaced with fresh lactic acid 1640 medium, and the Ce6 group, the TCe6 group and the TCe6/CHC NPs group were irradiated with 660nm laser (all power was 100 mW/cm)²The irradiation time was 2 min). To examine the dark toxicity of Ce6, TCe6 and TCe6/CHC NPs themselves, dark toxic wells were simultaneously established, and all treatments were identical except without laser irradiation. And continuing to incubate for 20h, adding 10 mu L of MTT solution (5mg/mL) into each well, incubating for 4h, then discarding liquid in each well, adding 100 mu L of DMSO into each well for dissolution, measuring the absorbance at 490nm by using a microplate reader, and calculating the inhibition rate according to the following formula:

wherein A is_{Negative of}Denotes the absorbance of the wells of cells not treated with the drug, A_{Sample (I)}Indicates the absorbance of the wells of the cells treated with each sample, A_{Blank space}Represents the absorbance of wells that were not seeded with cells and were not drug treated.

The experimental results of CT26 cytostatic rate of different samples at different concentrations are shown in figure 3, under the irradiation of light, TCe6/CHC NPs show excellent cytotoxicity, and under the condition of no light, the dark toxicity is low, and the biocompatibility is good.

Example 5TCe6/CHC NPs ROS production assay in vitro

1. Culture of cells

The colon cancer line CT26 cells were selected as the study subject. CT26 cells were cultured in RPMI 1640 medium containing 10% Fetal Bovine Serum (FBS), 1% streptomycin/penicillin at 37 ℃ in a humidified environment of 5% CO 2. And (5) when the cells grow to high density, carrying out passage, proportionally transferring the cells into a culture bottle, continuously culturing the cells and counting the cells. In the latter assay, CT26 cells were incubated with glucose-free RPMI 1640 (containingIs 10 x 10^-3M lactic acid) was incubated at 37 ℃, and the medium was called lactic acid 1640 medium.

2. In vitro ROS assay

ROS probe 2', 7' -dichlorofluorescein diacetate (DCFH-DA) was used to assess Ce 6-mediated ROS production in PDT. The fluorescent DCF oxidized by ROS can be measured by flow cytometry. CT26 cells were plated at 2X 10 per well⁵The cells were seeded at a density in 6-well plates and after 24 hours of incubation, the cells were cultured in fresh medium containing equal amounts of Ce6 (3. mu.g/mL), TPGS (10.9. mu.g/mL), TCe6/CHC NPs. After 6 hours of incubation, cells were washed, incubated with 20. mu.g/mL DCFH-DA in RPMI glucose-free 1640 medium (FBS-free) for 20 minutes, followed by a 660nm laser at 100mW/cm²Is irradiated for 2 minutes. And meanwhile, a dark group of hatches are set, and all treatments are consistent except that laser irradiation is not carried out.

As can be seen from fig. 4, the excellent photodynamic properties of TCe6/CHC NPs, which can generate ROS not only through Ce 6-mediated photodynamic ability, but also have the ability to generate additional ROS through TPGS, were confirmed by combining TPGS with Ce 6.

Example 6TCe6/CHC NPs in vivo antitumor assay

Balb/c mice are subjected to axillary tumor bearing (80 ten thousand 4T1 cells each), the tumors grow to about 80mm3, the mice are randomly divided into 4 groups, 5 mice in each group are respectively injected with physiological saline (NS), Ce6, TCe6 and TCe6/CHC NPs intravenously, and are irradiated with 660nm excitation light (100 mW/cm) 6h after injection²) For 10 min. Changes in tumor volume were recorded during treatment. After 6 treatments, mice were sacrificed, tumors were removed, weighed and tumor inhibition rate was calculated.

Wherein, w_NSIs the mean tumor weight, w, of mice in the saline group_{Sample (I)}Is the average tumor weight of the other groups of mice.

As can be seen in figure 5, TPGS amplifies ROS generated by Ce6, the tumor volume growth of mice in the TCe6 group is slower than that of mice in the Ce6 group, CHC is used as a photosensitizer to save oxygen, and the EPR effect of a nano preparation is utilized to enable more drugs to be accumulated at the tumor part, so that the tumor inhibition effect of the TCe6/CHC NPs preparation is obviously better than that of the TCe6 group, and the tumor inhibition rate is as high as 95%. The preparation is proved to greatly improve the anti-tumor effect.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.