阅读说明：本技术 一种萜类小分子组装的氧化还原响应光敏药物的制备方法 (Preparation method of terpene micromolecule assembled redox response photosensitive drug ) 是由 杨鑫 程建军 王舒 李欣瑜 于 2020-06-03 设计创作，主要内容包括：一种萜类小分子组装的氧化还原响应光敏药物的制备方法,属于生物医药材料领域,具体方案如下：一种萜类小分子组装的氧化还原响应光敏药物的制备方法,包括以下步骤：步骤一：以天然萜类小分子和二硫代羧酸作为反应物,通过酯缩合反应脱水生成氧化还原功能基二硫修饰的天然萜类小分子；步骤二：将氧化还原功能基二硫修饰的天然萜类小分子和Ce6采用超声共沉淀法制备得到萜类小分子组装的氧化还原响应光敏药物。本发明采用简单的共组装方式,制备了新型天然萜类小分子介导的无载体纳米复合光敏药物,不仅消除了纳米载体的潜在安全性和抗癌惰性问题,同时降低药物因突释所导致的潜在毒副作用,更重要的是有望实现安全的协同光化联合抗肿瘤治疗。(A preparation method of a redox response photosensitive drug assembled by terpene micromolecules belongs to the field of biological medicine materials, and the specific scheme is as follows: a preparation method of a terpene small molecule assembled redox response photosensitive drug comprises the following steps: the method comprises the following steps: taking natural terpene micromolecules and dithiocarboxylic acid as reactants, and dehydrating the reactants through ester condensation reaction to generate natural terpene micromolecules modified by oxidation-reduction functional group disulfide; step two: the natural terpene micromolecules modified by the oxidation-reduction functional group disulfide and Ce6 are prepared into the oxidation-reduction response photosensitive drug assembled by the terpene micromolecules by an ultrasonic coprecipitation method. The invention adopts a simple co-assembly mode to prepare the novel natural terpene micromolecule mediated carrier-free nano composite photosensitive drug, thereby not only eliminating the potential safety and anticancer inertia problems of a nano carrier, but also reducing the potential toxic and side effects of the drug caused by burst release, and more importantly, being expected to realize safe synergistic photochemical combined antitumor treatment.)

1. A preparation method of a terpene small molecule assembled redox response photosensitive drug is characterized by comprising the following steps:

the method comprises the following steps: taking natural terpene micromolecules and dithiocarboxylic acid as reactants, and dehydrating the reactants through ester condensation reaction to generate natural terpene micromolecules modified by oxidation-reduction functional group disulfide;

step two: the natural terpene micromolecules modified by the oxidation-reduction functional group disulfide and Ce6 are prepared into the oxidation-reduction response photosensitive drug assembled by the terpene micromolecules by an ultrasonic coprecipitation method.

2. The method for preparing a photosensitive drug with a terpene small molecule assembly and a redox response according to claim 1, wherein the photosensitive drug comprises: in the first step, the molecular structural formula of the dithiocarboxylic acid is shown as

Wherein n is more than or equal to 2.

3. The method for preparing a terpene small molecule-assembled redox-responsive photosensitive drug as claimed in claim 1 or 2, wherein: in step one, the dithiocarboxylic acid comprises one or more of 2-2 ' dithiodiacetic acid, 4-4 ' dithiodibutyric acid and 6-6 ' dithiodihexanoic acid.

4. The method for preparing a photosensitive drug with a terpene small molecule assembly and a redox response according to claim 1, wherein the photosensitive drug comprises: in step one, Dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DMAP) are added to the reactants as catalysts.

5. The method for preparing a photosensitive drug with a terpene small molecule assembly and a redox response according to claim 1, wherein the photosensitive drug comprises: in the first step, the molar ratio of the natural terpene micromolecules to the dithiocarboxylic acid is 1: 1-5.

6. The method for preparing a photosensitive drug with a terpene small molecule assembly and a redox response as claimed in claim 4, wherein the photosensitive drug comprises: in the first step, the molar ratio of the natural terpene micromolecules to the DCC and the DMAP is 1: 1.2-2.0: 1.2-3.0.

7. The method for preparing a photosensitive drug with a terpene small molecule assembly and a redox response according to claim 1, wherein the photosensitive drug comprises: in the first step, the time of the ester condensation reaction is 15-48 h.

8. The method for preparing a photosensitive drug with a terpene small molecule assembly and a redox response according to claim 1, wherein the photosensitive drug comprises: in the first step, dichloromethane is added into reactants to dissolve natural terpene micromolecules, anhydrous N, N-dimethylformamide DMF is added to dissolve dithiocarboxylic acid, the ratio of the natural terpene micromolecules to the dichloromethane is 0.1-50mg:1ml, and the ratio of the dithiocarboxylic acid to the DMF is 0.1-75mg:1 ml.

9. The method for preparing a photosensitive drug with a terpene small molecule assembly and a redox response according to claim 1, wherein the photosensitive drug comprises: in the first step, after the ester condensation reaction is finished, extracting the reaction solution, then performing rotary evaporation concentration and vacuum freeze drying, and separating and purifying by forward silica gel column chromatography to obtain the natural terpene micromolecules modified by the oxidation-reduction functional group disulfide.

10. The method for preparing a photosensitive drug with a terpene small molecule assembly and a redox response according to claim 1, wherein the photosensitive drug comprises: the second step comprises the following steps:

step 1, dissolving a natural terpene micromolecule modified by a redox functional group disulfide in dimethyl sulfoxide (DMSO) to obtain a sample solution I, dissolving Ce6 in DMSO to obtain a sample solution II, wherein the molar concentration of the sample solution I is 10-50 mmol/L, the molar concentration of the sample solution II is 10-50 mmol/L, and uniformly mixing the sample solution I and the sample solution II in a volume ratio of 0.5-8: 1 to obtain a sample solution III;

and 2, adding a NaOH solution into distilled water to obtain a mixed solution I, adding a sample liquid III into the mixed solution I under the action of ultrasonic waves to obtain a mixed solution II, continuously performing ultrasonic waves on the mixed solution II for 0.1-25 min, centrifuging, removing a supernatant, and washing with water to remove an organic reagent to obtain the terpene micromolecule assembled redox response photosensitive drug, wherein the molar ratio of NaOH to Ce6 in the mixed solution II is 2-4: 1, the volume ratio of DMSO to distilled water is 0.2-1: 10.

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to a preparation method of a redox response photosensitive drug assembled by terpene small molecules.

Background

The natural terpene micromolecules have huge prospect in the aspect of biomedicine anticancer due to the unique physiological activities such as anticancer activity, anti-immune activity and antibacterial activity, as well as special biocompatibility, biodegradability and biological safety, and also have huge advantages in the combined treatment of tumors by using the natural terpene micromolecules with anticancer activity. Although terpene small molecules with self-assembly function are applied to synergistic photochemical anticancer as a nano construction system to replace great curative effect, the traditional preparation method (such as an emulsion solvent volatilization method) is complicated, and the prepared synergistic antitumor photosensitive drug lacks a stimulus response release function, so that the synergistic antitumor photosensitive drug cannot effectively release active drug molecules to exert curative effect under physiological conditions and lacks a certain release control capability, and on the other hand, a compound drug system lacking stimulus response may have drug burst release behavior to cause potential toxic effect.

Disclosure of Invention

The invention aims to provide a preparation method of a terpene micromolecule assembled redox response photosensitive drug, which can realize a tumor microenvironment redox response release function and is safe and efficient to resist cancer by combined chemotherapy-photodynamic therapy.

The purpose of the invention is realized by the following technical scheme:

a preparation method of a terpene small molecule assembled redox response photosensitive drug comprises the following steps:

the method comprises the following steps: taking natural terpene micromolecules and dithiocarboxylic acid as reactants, and dehydrating the reactants through ester condensation reaction to generate natural terpene micromolecules modified by oxidation-reduction functional group disulfide;

step two: the natural terpene micromolecules modified by the oxidation-reduction functional group disulfide and Ce6 are prepared into the oxidation-reduction response photosensitive drug assembled by the terpene micromolecules by an ultrasonic coprecipitation method.

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

1) the novel natural terpene micromolecule mediated carrier-free nano composite photosensitive drug is prepared by adopting a simple co-assembly mode, and the problems of potential safety and anticancer inertia of a nano carrier are solved;

2) the introduction of natural terpenoid micromolecules ensures that the nano composite photosensitive medicine has excellent biocompatibility and biological safety;

3) the anticancer activity of the natural terpene micromolecules further realizes the possibility of safe synergistic chemotherapy-photodynamic therapy combined antitumor therapy; the active terpene micromolecules are combined with photodynamic therapy to construct the synergistic anti-tumor nano composite photosensitive drug, so that the defect of low bioavailability of pure photosensitizer and terpene micromolecules can be overcome, and the tumor targeting of the drug is improved based on permeability and retention Effect (EPR).

4) In consideration of the characteristics of high-concentration Glutathione (GSH) in a tumor microenvironment, the disulfide bond with oxidation-reduction stimulation response is grafted on the natural terpene micromolecules, so that the controlled release of the medicament can be realized, the toxic and side effects caused by the burst release of the medicament are reduced, the curative effect of the medicament is improved, and the bioavailability is improved.

Drawings

FIG. 1 is a reaction equation of steroid natural terpene micromolecules and 3, 3' -dithiodipropionic acid, wherein R is a linear or branched alkyl group with more than or equal to 5 carbon atoms, including saturated alkyl and unsaturated alkyl;

FIG. 2 is a molecular structural formula of ergosterol (ergosterol);

FIG. 3 is a molecular structural formula of beta-sitosterol (beta-sitosterol);

FIG. 4 shows the molecular structural formula of Stigmasterol;

FIG. 5 is a reaction equation of pentacyclic triterpenoid natural terpene micromolecules and 3, 3' -dithiodipropionic acid, wherein R is a straight chain or branched chain alkyl group with more than or equal to 0 carbon atom;

FIG. 6 shows a structural formula of a pentacyclic triterpene natural terpene micromolecule;

FIG. 7 shows a structural formula II of a pentacyclic triterpene natural terpene micromolecules;

FIG. 8 is a molecular structural formula of Betulonic acid (Betulonic acid);

FIG. 9 shows the molecular structural formula of Glycyrrhetinic acid (Glycyrrhetinic acid);

FIG. 10 shows the molecular structure of Ursolic Acid (UA);

FIG. 11 shows a molecular structural formula of Betulinic acid (Betulinic acid);

FIG. 12 is a diagram of the redox functional group disulfide modified Stigmasterol Stigmasterol-S-S-COOH¹H-NMR chart;

FIG. 13 shows the preparation of redox functional group disulfide-modified Ursolic acid Ursolic acid-S-S-COOH¹H-NMR chart;

FIG. 14 is a Scanning Electron Microscope (SEM) picture of UAD-Ce6 NPs;

FIG. 15 shows UV-VISIBLE ABSORPTION SPECTRUM of UAD, Ce6, and UAD-Ce6 NPs;

fig. 16 is an in vitro release profile of UAD-Ce6NPs at different concentrations of GSH and pH, with free Ce6 used as a control and error bars representing standard deviation (n-3);

FIG. 17 is a graph of 4T1 cell viability at different concentrations of UAD-Ce6NPs under light versus no light (dark);

FIG. 18 shows the change of tumor volume ratio of tumor-bearing female Balb-c mice treated with UAD-Ce6NPs and Ce6 in light and no light, respectively;

FIG. 19 is a graph showing the change of body weight of tumor-bearing mice in the UAD-Ce6NPs drug treatment period of 14 days.

Detailed Description

The technical solutions of the present invention are further described below with reference to the drawings and the specific embodiments, but not limited thereto, and modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the scope of the technical solutions of the present invention.