CN113041361B - Diagnosis and treatment integrated material responding to HDAC and CTSL and preparation method and application thereof - Google Patents

Diagnosis and treatment integrated material responding to HDAC and CTSL and preparation method and application thereof Download PDF

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CN113041361B
CN113041361B CN202110202874.5A CN202110202874A CN113041361B CN 113041361 B CN113041361 B CN 113041361B CN 202110202874 A CN202110202874 A CN 202110202874A CN 113041361 B CN113041361 B CN 113041361B
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ctsl
hdac
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袁友永
马迪
宗庆瑜
俞方舟
肖炫
姚旺
杨蕊梦
江新青
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Guangzhou First Peoples Hospital
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Abstract

The invention belongs to the technical field of biomedicine and fluorescence detection, and particularly relates to a diagnosis and treatment integrated material responding to HDAC and CTSL as well as a preparation method and application thereof. The material contains an antitumor drug Cy-NH 2 And has a large number of bonds with specific response of HDAC and CTSL, and generates enzyme response in tumor cells with high expression of HDAC and CTSL to break, thereby the antitumor drug Cy-NH is obtained 2 The medicament can be quickly released and placed in tumor cells, and has the advantage of targeted medicament delivery; furthermore, Cy-NH 2 The compound has the advantages of capability of imaging tumor cells under the excitation of specific wavelengths and huge clinical application potential.

Description

Diagnosis and treatment integrated material responding to HDAC and CTSL and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological medicine and fluorescence detection, particularly belongs to the technical field of anti-tumor drugs and tumor detection, and more particularly relates to a diagnosis and treatment integrated material with HDAC and CTSL response, and a preparation method and application thereof.
Background
Currently, the focus of cancer treatment programs is to improve the pharmacokinetics of candidate drugs, and over the past decades, tumor-targeted drug delivery strategies have been extensively studied in chemotherapy to minimize adverse effects that interfere with normal cellular function, thereby enhancing the therapeutic efficacy of cancer. The traditional approach is to use targeting ligands to selectively act on the overexpressed receptors or transporters on the surface of cancer cells, and further to use the tumor microenvironment of the case, such as acidic pH, Reactive Oxygen Species (ROS), and overexpressed enzymes in or around the cancer cells, to release anticancer drugs with restored cytotoxicity.
Histone Deacetylases (HDACs), epigenetic enzymes, are of widespread interest in oncology due to their important role in various biological processes. HDACs regulate chromatin structure and function by deacetylation of lysine residues on amino groups.
Upregulation of cysteine cathepsin l (ctsl) is considered to be a hallmark of cancer multistage progression and metastasis. Thus, increased activity and localization of CTSL is of prognostic and diagnostic value clinically.
Therefore, it is of great significance for anticancer drug development and cancer treatment by selectively killing human cancer cell lines having high HDAC and CTSL activities.
Disclosure of Invention
The invention aims to provide a material with HDAC and CTSL responses, a preparation method and application thereof, the material achieves the aim of treating cancer by generating double-enzyme response to HDAC and CTSL in tumor cells to release a medicament in a targeted manner, and has the advantages of targeting and high efficiency.
Based on the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides an HDAC and CTSL responsive diagnosis and treatment integrated material, the molecular structure of which is
Figure BDA0002948830900000021
Wherein n is 45.
The diagnosis and treatment integrated material with HDAC and CTSL response contains an antitumor drug Cy-NH 2 And has a large number of HDAC, CTSL-specific responsesThe corresponding amide bond has the characteristic of generating enzyme response and breaking in tumor cells highly expressing HDAC and CTSL, thereby converting Cy-NH 2 Quickly released into tumor cells to increase the free Cy-NH in the tumor cells 2 At a concentration of Cy-NH 2 Can generate a large amount of active oxygen in tumor cells to kill the tumor cells, and has the advantage of targeting; furthermore, Cy-NH 2 The compound has the advantages of capability of imaging tumor cells under the excitation of specific wavelengths and huge clinical application potential.
In a second aspect, the present invention provides a method for preparing the HDAC/CTSL responsive diagnosis and treatment integrated material, comprising the following steps:
(1) fluorescent molecule Cy-NH 2 Synthesizing;
(2) synthesis of Boc-Lys (Ac) -Cy:
the Cy-NH prepared in the step (1) 2 Performing amide reaction with Boc-Lys (Ac) -OH for 15-30 h at room temperature according to the molar ratio (1.5-2.5) of 1, and extracting, separating and purifying to obtain Boc-Lys (Ac) -Cy;
(3) synthesis of Lys (Ac) -Cy:
removing Boc from Boc-Lys (Ac) -Cy prepared in the step (2) in trifluoroacetic acid to prepare Lys (Ac) -Cy;
(4)PEG 2000 -synthesis of lys (ac) -Cy:
PEG after carboxyl activation 2000 mixing-COOH and Lys (Ac) -Cy according to the molar ratio of 1 (1-2), stirring and reacting for 3-5 h at room temperature in a dark place to obtain PEG 2000 -Lys (Ac) -Cy, which is a diagnosis and treatment integrated material responding to HDAC and CTSL.
Cy-NH 2 As a common fluorescent molecule, the toxicity of the fluorescent molecule is rarely studied, and the research team of the inventor firstly discovers the fluorescent molecule Cy-NH 2 Has extremely strong killing effect on cells, but is caused by free Cy-NH 2 Has no selectivity to tumor cells, therefore, the invention uses dipeptide, mPEG to Cy-NH 2 Modifying the modified Cy-NH 2 Has high selectivity to tumor cells, and endows the tumor cells with enzyme response capability and blood circulation prolonging capability.
Further, the fluorescent molecule Cy-NH in the step (1) 2 The synthesis process of (A) is as follows:
①.Cy-NO 2 the synthesis of (2):
dissolving IR780, m-nitrophenol and DIPEA (dimethyl Diphenyl Ether) in DMF according to the molar ratio of 1 (1-1.5) to (0.1-0.2), reacting for 10-20 h at room temperature, extracting by an organic solvent, concentrating, separating out and drying to obtain Cy-NO 2 ；
②.Cy-NH 2 The synthesis of (2):
reacting Cy-NO 2 With excess SnCl 2 ·2H 2 Dissolving O and concentrated hydrochloric acid in methanol, reacting at 60-80 deg.C for 10-14 h, extracting with organic solvent, concentrating, separating out, and drying to obtain Cy-NH 2 。
Further, IR780 in step (r) is commercially available or prepared by the following method:
synthesis of 1-ethyl-2, 3, 3-trimethyl-3H-indole
Mixing 2,3, 3-trimethyl-3H-indole and iodoethane according to a molar ratio of 1 (1-2) in an organic solvent, reacting for 20-28H at 70-80 ℃, concentrating, washing and drying a reaction solution to obtain 1-ethyl-2, 3, 3-trimethyl-3H-indole;
II.2 Synthesis of 2-chloro-3-hydroxymethylene-1-cyclohexene-1-carbaldehyde
Adding POCl 3 And (3) mixing with cyclohexanone according to the volume ratio of (2-4): 1, mixing the raw materials in an organic solvent, reacting at the temperature of-4-1 ℃ for 20-30 min, reacting at the temperature of 40-50 ℃ for 4-6 h, cooling, carrying out solid-liquid separation, collecting precipitate, and drying to obtain 2-chloro-3-hydroxymethylene-1-cyclohexene-1-formaldehyde;
synthesis of IR780
2-chloro-3-hydroxymethylene-1-cyclohexene-1-formaldehyde, 1-ethyl-2, 3, 3-trimethyl-3H-indole and sodium acetate trihydrate are mixed according to a molar ratio of 1: (1.5-2.5): (1.5-2.5), reacting for 1-3 h at 120-140 ℃ in an inert atmosphere to obtain a crude product, and washing, concentrating and drying the crude product to obtain the IR 780.
Further, the specific process of removing Boc-Lys (Ac) -Cy from trifluoroacetic acid in the step (3) to prepare Lys (Ac) -Cy comprises the following steps:
and dissolving Boc-Lys (Ac) -Cy in DCM, mixing with trifluoroacetic acid for reaction until the Boc-Lys (Ac) -Cy completely reacts, neutralizing with acid and alkali, concentrating an organic phase, and drying to obtain Lys (Ac) -Cy.
In a third aspect, the invention provides a double-enzyme response drug-loaded nanoparticle, wherein the main active ingredient of the double-enzyme response drug-loaded nanoparticle is the diagnosis and treatment integrated material with HDAC and CTSL response.
Preferably, the particle size of the double-enzyme response drug-loaded nanoparticle is 90-120 nm, and due to the EPR effect of a tumor part, particles of about 100nm are easy to gather and permeate at the tumor part, so that a large amount of Cy-NH is released at the tumor part 2 And the anti-tumor effect is improved.
In a fourth aspect, the invention provides a preparation method of the double-enzyme response drug-loaded nanoparticle, which comprises the following steps:
the diagnosis and treatment integrated material with HDAC and CTSL responses is dispersed in dimethyl sulfoxide and water in sequence, and then dialyzed by a 3500D specification dialysis bag and filtered by a 0.22 mu m filter to prepare the double-enzyme response drug-loaded nanoparticles.
PEG in diagnosis and treatment integrated material structure with HDAC and CTSL response 2000 Being a hydrophilic part, Cy-NH 2 The material is a hydrophobic part and can be self-assembled in a water phase to form nanoparticles, dimethyl sulfoxide is removed by using a 3500D-standard dialysis bag, large particles are removed by using a 0.22-micron filter, and the particle size of the prepared double-enzyme response drug-loaded nanoparticles is 90-120 nm.
In a fifth aspect, the invention provides an application of the double-enzyme response drug-loaded nanoparticle in preparing anticancer drugs.
Further, the double-enzyme response drug-loaded nanoparticle enables the nanoparticle to disintegrate through HDAC and CTSL double-enzyme response in tumor cells, and Cy-NH is released into the tumor cells 2 Promoting the generation of active oxygen in tumor cells, inhibiting the growth of the tumor cells and achieving the aim of treating the cancer.
In conclusion, the fluorescent molecule Cy-NH is found for the first time in the invention 2 Killing tumor cells, and modifying to obtain the product with anti-tumor effectThe HDAC and CTSL responsive diagnosis and treatment integrated material of (1); in addition, the diagnosis and treatment integrated material with HDAC and CTSL response prepared by the invention is self-assembled to form nanoparticles, so that drug-loaded nanoparticles can be conveniently gathered and permeated at a tumor part, and the drug release is specifically responded to the high-expression sites of HDAC and CTSL of tumor tissues, so that a large amount of Cy-NH is released at the tumor part 2 The composition has a killing effect on tumor tissues and improves the anti-tumor effect; in addition, the material also has an imaging effect, can generate fluorescence under a specific excitation wavelength, and is convenient for real-time monitoring of tumor tissues in the treatment process; in addition, the material also has good biocompatibility and degradability, and has a huge clinical application prospect.
Drawings
FIG. 1 shows PEG 2000 -a schematic scheme of the synthesis of Lys (Ac) -Cy;
FIG. 2 shows Cy-NH 2 A schematic diagram of a synthetic route;
FIG. 3 is a diagram of Boc-Lys (Ac) -Cy 1 H NMR chart;
FIG. 4 is of Lys (Ac) -Cy 1 H NMR chart;
FIG. 5 shows PEG 2000 -a GPC profile of lys (ac) -Cy;
FIG. 6 is a particle size plot of PLC NPs;
FIG. 7 shows PEG 2000 -fluorescence masking profile of lys (ac) -Cy;
FIG. 8 is a graph showing the recovery of fluorescence of PLC NPs under different enzymatic reactions;
FIG. 9 is a graph of fluorescence intensity of PLC NPs as a function of enzyme time;
FIG. 10 shows the detection of free Cy-NH by MTT 2 Cytotoxicity profiles of PLC NPs on different cell lines;
FIG. 11 shows free Cy-NH 2 IC of PLC NPs on different cell lines 50 A histogram of values;
FIG. 12 is a diagram of confocal detection of PLC NPs intracellular fluorescence recovery;
FIG. 13 is a graph showing recovery of intracellular fluorescence in the presence of confocal detection of PLC NPs plus inhibitor;
FIG. 14 is a confocal measurement of PLC NPs intracellular ROS levels;
FIG. 15 shows intracellular ROS levels following confocal detection of PLC NPs plus inhibitor;
FIG. 16 is a diagram of confocal measurements of membrane potential of different cell lines;
FIG. 17 is a graph of the change in membrane potential levels in different cell lines due to confocal detection of PLC NPs;
figure 18 is a graph of the therapeutic efficacy of dual enzyme response drug loaded nanoparticles in vivo;
FIG. 19 is a graph showing the change in body weight of mice in each experimental group in an in vivo treatment experiment.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. The raw materials used in the following examples are all commercially available general-purpose products unless otherwise specified.
Example 1PEG 2000 -Lys (Ac) -Cy and preparation and characterization thereof
The embodiment provides diagnosis and treatment integrated material PEG responding to HDAC and CTSL 2000 -Lys(Ac)-Cy，PEG 2000 The molecular structure of-Lys (Ac) -Cy is:
Figure BDA0002948830900000051
wherein n is 45.
The diagnosis and treatment integrated material PEG with HDAC and CTSL response 2000 the-Lys (Ac) -Cy is prepared by the following method, the synthetic route is shown in figure 1, and the specific preparation process is as follows:
mono, Cy-NH 2 Synthesis of (2)
Cy-NH 2 The synthesis process mainly comprises two processes, wherein the first process is as follows: synthesis of Cy-NO from IR780, m-nitrophenol, DIPEA (N, N-diisopropylethylamine) 2 The process of (2); the second process is as follows: from Cy-NO 2 With SnCl 2 Synthesis of Cy-NH 2 The process of (1).
In the first process, the IR780 and m-nitrophenol can be prepared by using commercial products or by themselves, and in this example, both the IR780 and m-nitrophenol are prepared by themselves.
Cy-NH 2 Synthetic route of (1)As shown in fig. 2, the specific synthesis process is as follows:
1. synthesis of 1-ethyl-2, 3, 3-trimethyl-3H-indole
A set of reflux device is set up, a condenser pipe is connected with circulating cooling water, 5mL (0.32mmol) of 2,3, 3-trimethyl-3H-indole, 3.7mL (0.47mmol) of ethyl iodide and 50mL of acetonitrile are added into a 250mL round bottom flask under the protection of nitrogen, the round bottom flask is placed into an oil bath pot, the temperature of the oil bath pot is set to be 75 ℃, and the mixture is heated and refluxed for 24 hours under the stirring; after the reaction is finished, evaporating the solvent under reduced pressure, carrying out ultrasonic washing on the crude product by using normal hexane and diethyl ether respectively until the obtained solid becomes red powder, and carrying out vacuum drying to obtain the 1-ethyl-2, 3, 3-trimethyl-3H-indole.
2. Synthesis of 2-chloro-3-hydroxymethylene-1-cyclohexene-1-formaldehyde
40mL of DCM (dichloromethane) and 40mL of DMF (N, N-dimethylformamide) are sequentially added into a 250mL round-bottom flask, and the mixture is stirred in ice bath; 36mL of POCl 3 Placing the mixture in a constant-pressure dropping funnel, slowly dropping the mixture into the system, adding 10.6mL of cyclohexanone into the system, carrying out ice-bath reaction for 30min, and carrying out reflux reaction for 5h at 45 ℃; after the reaction is finished, slowly dripping the reaction solution on 400g of ice, and standing overnight after finishing dripping; and (4) carrying out suction filtration, collecting yellow solid, and carrying out vacuum drying to obtain the 2-chloro-3-hydroxymethylene-1-cyclohexene-1-formaldehyde.
3. Synthesis of IR780
2g (11.58mmol) of 2-chloro-3-hydroxymethylene-1-cyclohexene-1-formaldehyde, 7.3g (23.2mmol) of 1-ethyl-2, 3, 3-trimethyl-3H-indole and 2.4g (23.2mmol) of sodium acetate trihydrate are sequentially added into a 250mL round-bottom flask and reacted for 2 hours at the temperature of 130 ℃ under the protection of nitrogen by refluxing; after the reaction is finished, evaporating the solvent under reduced pressure to obtain a crude product, dissolving the crude product in 100mL of anhydrous DCM, carrying out liquid separation washing three times by using saturated KI, adding anhydrous magnesium sulfate, and carrying out suction filtration; and (3) concentrating DCM containing the crude product to 10mL, slowly dropwise adding the concentrated solution into 200mL of anhydrous ether while stirring in an ice bath for precipitation, continuously stirring for 30min after dropwise adding is finished, fully separating out the product, performing suction filtration, and performing vacuum drying to obtain IR 780.
4. Synthesis of m-nitrophenol
In a 250mL round-bottom flask, 3.75g (22.5mmol) of m-nitrobenzoic acid and 3.9g (22.5mmol) of m-chloroperoxybenzoic acid are dissolved in 120mL of ethanol aqueous solution, the volume ratio of ethanol to water in the ethanol aqueous solution is 2:1, and the reaction is carried out for 6h at room temperature; after the reaction is finished, concentrating to remove most of ethanol, and extracting with ethyl acetate; evaporating the spin-drying solvent under reduced pressure, mixing the silica gel with a sample, passing through a column, and eluting according to the volume ratio of n-hexane to ethyl acetate of 6:1, 5:1 and 4: 1; and (3) collecting the part eluted first, concentrating and drying to obtain the m-nitrophenol because the polarity of the target product is low.
5、Cy-NO 2 Synthesis of (2)
In a 500mL round-bottom flask, 7.7g (12.01mmol) of IR780, 1.9g (13.65mmol) of m-nitrophenol, 2.5mL (1.25mmol) of DIPEA were dissolved in 200mL of DMF and reacted at room temperature for 16 h; after completion of the reaction, 100mL of DCM was added to the reaction mixture, and 200mL of H was added 2 O, extracting and separating by using a separating funnel, collecting an organic phase, washing the organic phase twice by using water, drying by using anhydrous magnesium sulfate, and collecting the organic phase; concentrating the collected organic phase, dripping into ethyl glacial ether to separate out precipitate, and performing suction filtration and vacuum drying to obtain Cy-NO 2 。
6、Cy-NH 2 Synthesis of (2)
In a 500mL round-bottom flask, 3g of Cy-NO was taken 2 、18.3g SnCl 2 ·2H 2 Dissolving O and 19.3mL of concentrated hydrochloric acid in 200mL of methanol, and stirring the mixed solution at 70 ℃ for reaction overnight; after the reaction is finished, taking reaction liquid, spin-drying methanol, sequentially adding water and dichloromethane for extracting and separating liquid, and filtering out redundant SnCl 2 Drying the organic phase with anhydrous magnesium sulfate, spin-drying, dripping the concentrated organic phase into a large amount of diethyl ether to precipitate, vacuum-filtering, and vacuum-drying to obtain Cy-NH 2 。
Synthesis of di, Boc-Lys (Ac) -Cy
The reaction was carried out in a 10mL round-bottomed flask, 209.14mg (1equiv) Boc-Lys (Ac) — OH, 487.24mg (1.5equiv) HATU, 207.87. mu.L (1.5equiv) DIPEA were dissolved in 9.67mL anhydrous DMF, and the reaction was stirred for 30min under ice bath conditions; subsequently, 200mg (2equiv) of Cy-NH was added 2 Dissolved in 6.66mL of anhydrous DMF and added dropwise to the reaction solutionThe system is reacted at 37 ℃ overnight; after the reaction is finished, extracting the reaction solution by using saturated saline solution and dichloromethane, washing until a water layer is colorless, collecting an organic phase, drying the collected dichloromethane phase by using anhydrous magnesium sulfate to remove water, and removing the solvent by rotary evaporation to obtain a crude product; purifying the crude product by a silica gel column, carrying out gradient elution by using a mixed solution of DCM and methanol according to the volume ratio of DCM to MeOH of 50:1, 40:1 and 30:1 in sequence, collecting blue eluate, concentrating and drying to obtain Boc-Lys (Ac) -Cy.
NMR Hydrogen Spectroscopy on Boc-Lys (Ac) -Cy: ( 1 H NMR) analysis, as shown in FIG. 3, of Boc-Lys (Ac) -Cy 1 H NMR spectrum letters mark the protic hydrogens assigned to Boc-Lys (Ac) -Cy. Wherein, Cy-NH 2 The characteristic peaks of (A) appear at 2.718ppm, 4.288ppm, 6.135ppm, 6.637ppm, 6.923ppm, 7.151ppm, 7.472ppm, 7.814ppm, 8.620ppm and 8.820 ppm; 4.466ppm peak belongs to methine beside amido bond, and the other methylene peaks on amino acid have chemical shifts of 0.907ppm, 1.648ppm, 1.767ppm and 3.033ppm respectively due to different structural environments; whereas 4.244ppm was assigned to the methylene peak on the indole ring and 6.135ppm was assigned to the protic hydrogen of the olefinic bond.
Preparation of Lys (Ac) -Cy
The reaction was carried out in a 10mL round-bottomed flask, 254.8mg of Boc-Lys (Ac) -Cy was taken, dissolved by adding 8mL of DCM, and then TFA (trifluoroacetic acid) was added dropwise slowly, and the reaction was stirred at room temperature for 20min until the Boc-Lys (Ac) -Cy reaction was completed by Thin Layer Chromatography (TLC); and then slowly dropwise adding saturated sodium carbonate into the reaction liquid, separating the liquid until no bubbles are generated, collecting an organic phase, spin-drying the solvent, and performing vacuum drying to obtain Lys (Ac) -Cy.
NMR Hydrogen Spectroscopy of Lys (Ac) -Cy: ( 1 H NMR) analysis, as shown in FIG. 4, of Lys (Ac) -Cy 1 The H NMR spectrum is letter-labeled with the protic hydrogen ascribed to Lys (Ac) -Cy. 1 H NMR was substantially similar to Boc-Lys (Ac) -Cy except for the reduction in methyl numbers of 1.239ppm to 1.471 ppm. Wherein, Cy-NH 2 The characteristic peaks of (A) appear at 2.718ppm, 4.288ppm, 6.135ppm, 6.637ppm, 6.923ppm, 7.151ppm, 7.472ppm, 7.814ppm, 8.620ppm and 8.820 ppm; 4.466ppmThe peak of (A) is attributed to methine beside an amido bond, and the other methylene peaks on the amino acid have chemical shifts of 0.907ppm, 1.648ppm, 1.767ppm and 3.033ppm respectively due to different structural environments.
Four, PEG 2000 Synthesis of-Lys (Ac) -Cy
The reaction was carried out in a 10mL round-bottomed flask, 423.6mg PEG-COOH 2000 Dissolve in 2mL DCM, add 131. mu.L oxalyl chloride to the above system, stir at room temperature under nitrogen for 2h to activate the carboxyl group. Then, pumping oxalyl chloride in the reaction system by an oil pump; PEG-COOH obtained by activating the above carboxyl group 2000 Added dropwise to 144.3mg Lys (Ac) -Cy, 50. mu.L of TEA was added, and the reaction was stirred for 3 hours with exclusion of light. Spin-drying the solvent, and vacuum-drying to obtain PEG 2000 -Lys(Ac)-Cy。
For PEG 2000 Gel chromatography of-Lys (Ac) -Cy showed a GPC chart as shown in FIG. 5, from which it can be seen that PEG 2000 Efflux time of-Lys (Ac) -Cy is less than that of PEG 2000 Shows that Lys (Ac) -Cy was successfully attached to PEG.
Example 2 Dual enzyme responsive drug-loaded nanoparticles and preparation thereof
In this embodiment, the diagnosis and treatment integrated material PEG responded by HDAC and CTSL in embodiment 1 2000 the-Lys (Ac) -Cy is used as a main raw material to prepare the double-enzyme response drug-loaded nanoparticle, the nanoparticle enhances the aggregation and the penetration of the nanoparticle in tumor tissues due to the EPR effect of the tumor part, and the diagnosis and treatment integrated material PEG with the response of HDAC and CTSL is improved 2000 -targeting and therapeutic effect of lys (ac) -Cy to tumor cells.
The specific preparation process of the double-enzyme response drug-loaded nanoparticle is as follows:
adding 10.0mg of PEG 2000 dispersing-Lys (Ac) -Cy in 1.0mL of dimethyl sulfoxide, dropwise adding 10mL of ultrapure water while stirring, continuing stirring for 2h after dropwise adding, transferring the obtained particle solution into a dialysis bag (MWCO 3500), dialyzing in the ultrapure water for 24h to remove the dimethyl sulfoxide, and filtering by using a 0.22 mu m filter to remove large particles to obtain the double-enzyme response drug-loaded nanoparticles which are marked as PLC NPs.
Due to PEG 2000 -Lys (Ac) -Cy molecular structure containsHydrophilic group PEG 2000 And a hydrophobic group Cy-NH 2 So that PEG 2000 -lys (ac) -Cy is capable of self-assembling in the aqueous phase to form nanoparticulate PLC NPs.
The particle size of the dual-enzyme response drug-loaded nanoparticle PLC NPs is detected by using a Dynamic Light Scattering (DLS) instrument, the particle size distribution of the dual-enzyme response drug-loaded nanoparticle PLC NPs is shown in figure 6, and it can be seen that most of the PLC NPs have particle sizes concentrated within 90-120 nm, and the number of the PLC NPs with particle sizes of about 100nm is large.
Example 3 enzyme response mechanism of double-enzyme response drug-loaded nanoparticles and antitumor efficacy thereof
This example will illustrate the enzyme response mechanism of the dual enzyme response drug-loaded nanoparticle PLC NPs of example 2 and its anti-tumor efficacy in three ways.
Enzyme response mechanism of one-enzyme-response drug-loaded nanoparticle PLC NPs and two-enzyme-response drug-loaded nanoparticle PLC NPs
In this example, the fluorescence spectrum change of the drug-loaded nanoparticles responded by PLC NPs through the expected spectrum change is monitored by a fluorescence spectrophotometer, and the drug release mechanism of PLC NPs after the HDAC and CTSL dual-enzyme responses is studied.
(1)PEG 2000 Fluorescence masking of-Lys (Ac) -Cy Bienzyme-responsive amphiphilic polymers PEG monitoring with a fluorescence spectrophotometer 2000 Fluorescence masking of amphiphilic polymers of the-Lys (Ac) -Cy Bizyme response and with free Cy-NH 2 As a control material.
Cy-NH in free form 2 And modified to an amphiphilic polymer PEG 2000 The fluorescence masking of-Lys (Ac) -Cy is shown in FIG. 7, from which it can be seen that Cy-NH 2 Is modified to PEG 2000 after-Lys (Ac) -Cy, fluorescence is masked.
(2) Enzymatic properties of double-enzyme-responsive drug-loaded nanoparticles
The enzyme response of the PLC NPs double-enzyme-responsive drug-loaded nanoparticles was monitored with a fluorescence spectrophotometer. The drug release mechanism effect of the nanoparticle PLC NPs after HDAC and CTSL response was investigated by expected spectral changes, PLC NPs absorption and fluorescence spectral monitoring. The specific test method is as follows:
test group reaction buffer (20mM Tris-HCl-buffer, pH8.0) was prepared and DTT (1mM), glycerol (10%, w/w), NaCl (0.1M), HDAC (0.5mg/mL), Trypsin (1.25mg/mL, 500BAEE units per mL) was added, wherein Trypsin functions similar to CTSL, so the present invention performs CTSL enzymatic hydrolysis assay with Trypsin.
The control buffer contained no or only one enzyme (HDAC-CTSL-, HDAC + CTSL-, HDAC-CTSL +), and the other components or conditions were the same as those of the experimental reaction buffer. Wherein HDAC-CTSL-indicates that neither HDAC nor CTSL is contained in the reaction buffer of the control group; HDAC + CTSL-indicates that the control buffer contained HDAC but not CTSL; HDAC-CTSL + indicates that the control buffer contained CTSL but not HDAC.
PLC NPs mother liquor is diluted to Cy-NH in the buffer solution of the test group and the buffer solution of the three control groups respectively 2 The mixture was incubated at 37 ℃ for 12 hours at a concentration of 0.2 mg/mL. 10. mu.L of the sample solution was diluted 100-fold with DMSO, and the fluorescence spectrum was measured with a fluorescence spectrophotometer. Detection conditions are as follows: the wavelength of the exciting light is 755nm, the width of the exciting slit is 5, and the width of the emission slit is 5 nm.
As shown in FIG. 8, the effect of PLC NPs on different enzymatic hydrolyzations is shown, and it can be seen that only by adding HDAC and CTSL enzymes at the same time, the nanoparticles will release Cy-NH in response 2 The fluorescence was recovered, but the fluorescence was not recovered only with the HDAC or CTSL single enzyme, indicating that PLC NPs specifically respond to both HDAC and CTSL enzymes.
(3) Analysis of influence of enzymolysis time on fluorescence of double-enzyme-response drug-loaded nanoparticles
And (2) preparing a reaction buffer solution containing HDAC and CTSL, adding the nanoparticle PLC NPs into the buffer solution, and monitoring the fluorescence intensity change of the PLC NPs under different enzymolysis time by using a fluorescence spectrophotometer. The specific test method is as follows:
reaction buffer (20mM Tris-HCl-buffer, pH8.0) was prepared and DTT (1mM), glycerol (10%, v/v), NaCL (0.1M), HDAC (0.5mg/mL), Trypsin (1.25mg/mL, 500BAEE units per mL) was added. Dissolving PLC NPs in buffer to Cy-NH 2 The concentration was 0.2mg/mL and the volume of the solution was 1 mL. Reacting for corresponding time (0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 10h and 12h)Then, 10. mu.L of the sample solution was diluted 100-fold with DMSO, and the fluorescence spectrum was measured with a fluorescence spectrophotometer. Detection conditions are as follows: the wavelength of the exciting light is 755nm, the width of the exciting slit is 5, and the width of the emission slit is 5 nm.
The fluorescence intensity of the nanoparticles at different enzymatic hydrolysis times is shown in fig. 9, and it can be seen from the figure that, after adding two enzymes, HDAC and CTSL, the fluorescence recovery increases with the increase of the incubation time, and the fluorescence intensity is strongest at 12h, indicating that the fluorescence recovery of the nanoparticles is time-dependent.
In-vitro cell anti-tumor effect test of double-enzyme response drug-loaded nanoparticle PLC NPs
This section will illustrate the in vitro cell anti-tumor effect of the dual-enzyme response drug-loaded nanoparticle PLC NPs from the four aspects as follows.
(1) Killing effect of double-enzyme response drug-loaded nanoparticle PLC NPs on MEF, MCF-7 and 4T1 cells
MEF, MCF-7 and 4T1 cells as experimental objects, wherein MEF is normal cells, MCF-7 and 4T1 cells are tumor cells, the cells are incubated by culture media containing PLC NPs drugs with different concentrations to form an experimental group, and free Cy-NH is added 2 (free Cy-NH 2 ) The culture medium is used as a control group, the activity of the incubated cells is detected by an MTT method, so that the killing effect of the double-enzyme response drug-loaded nanoparticle PLC NPs on MEF, MCF-7 and 4T1 cells is analyzed, and the specific test process is as follows.
MEF, MCF-7, 4T1 cells and different Cy-NH 2 Mixing double-enzyme response drug-loaded nanoparticles (PLC NPs) with concentration gradients (20 mu g/mL, 10 mu g/mL, 5 mu g/mL, 2.50 mu g/mL, 1.25 mu g/mL, 0.63 mu g/mL, 0.32 mu g/mL, 0.16 mu g/mL, 0.08 mu g/mL, 0.04 mu g/mL), diluting the PLC NPs with serum-free DMEM medium twice to obtain culture medium containing PLC NPs drugs with different concentrations, replacing the original culture medium of a 96-well plate with the culture medium containing PLC NPs drugs, 37 ℃, and 5% CO 2 Under the condition, the medicine is incubated for 24 hours; each group is provided with 6 multiple holes, and the NPs without adding PLC are used as a blank control group; to add free Cy-NH 2 The drug was used as a control group, and after the incubation time was over, the thiazole blue (MTT) reagent was dissolved inPBS to a concentration of 0.5mg/mL, the drug-containing medium in the 96-well plate was replaced with MTT-containing PBS solution at 37 ℃ with 5% CO 2 Incubating for 4h under the condition; absorbing PBS containing MTT reagent, adding 0.15mL DMSO into each hole to dissolve formazan crystals, oscillating for 30min, and detecting the 490nm/570nm absorption value by a micropore detector; and calculating the cell activity of the experimental group according to the ratio of the absorption values of the experimental group and the blank control group.
The test results are shown in FIG. 10, IC 50 As shown in Table 1 and FIG. 11, PLC NPs killed MCF7 and 4T1 tumor cells more than normal MEF cells, while free Cy-NH 2 The killing of the three cell lines has no obvious difference, which indicates that the PLC NPs have high selectivity and IC on tumor cells 50 The results further confirm the above test results.
TABLE 1free Cy-NH 2 IC of PLC NPs on different cell lines 50 Value of value
Figure BDA0002948830900000111
(2) Drug release effect of double-enzyme response drug-loaded nanoparticle PLC NPs in tumor cells
Three cell lines of MEF, MCF-7 and 4T1 are selected for researching drug Cy-NH of double-enzyme response drug-loaded nano-particles 2 The effect of intracellular internalization and release. With PLC NPs ([ Cy-NH ] 2 ]2.0 μ g/mL) were used as experimental materials, and three cell lines of MEF, MCF-7, and 4T1 were set as three experimental groups, respectively. Respectively culturing the three cell lines and the PLC NPs for 0.5h, 2h, 6h and 12h, washing off particles or medicines which are not taken up, observing the fluorescence recovery condition of the nanoparticles by a laser confocal scanning microscope, as shown in figure 12, the fluorescence intensity of the MCF-7 cell line and the fluorescence intensity of the 4T1 cell line are obviously enhanced along with the increase of the incubation time, and the fluorescence intensity in the MEF cell line is not obviously changed, which shows that the double-enzyme response medicine-carrying nanoparticles can effectively convey Cy-NH 2 And can increase Cy-NH in tumor cells 2 And (4) enriching.
Subsequently, the three cell lines are cultured together with the nanoparticle PLC NPs and the inhibitor for 0.5h, 2h, 6h and 12h, wherein the inhibitor is (1) the HDAC inhibitor: HDACi (1 μ M), (2) CTSL inhibitors: CTSLi (0.2 μ M), (3) HDAC inhibitors and CTSL inhibitors: HDACI (1. mu.M) + CTSLi (0.2. mu.M), while no inhibitor was added as a control, the rest of the test methods were the same as above. The experimental results are shown in fig. 13, and it can be clearly seen that, compared to the control group, the fluorescence of the cells is obviously reduced after the inhibitor is added, thus proving the enzyme responsiveness of the nanoparticle PLC NPs of the present invention.
(3) ROS levels of dual-enzyme response drug-loaded nanoparticle PLC NPs in different cell lines
And observing the ROS levels of the double-enzyme response drug-loaded nanoparticles in different cell lines by using a laser confocal scanning microscope. As with the drug release experiment, three cell lines of MEF, MCF-7 and 4T1 are set as three groups of experimental groups. PLC NPs and the three groups of cells were incubated for 4h, and the three groups of cells were stained with ROS probes (DCFH-DA; 2',7' -Dichlorodihydrofluorescein diacetate) and 4', 6-diamidino-2-phenylindole (DAPI) with ROS in green and blue nuclei.
The experimental results are shown in fig. 14, in the three experimental groups, ROS (green) is mainly enriched around cell nucleus (blue), and red fluorescence in MCF-7 and 4T1 groups is obviously stronger than that in MEF group, which proves that the double-enzyme response drug-loaded nanoparticles can effectively transport Cy-NH 2 And increases intracellular generation of ROS.
Subsequently, PLC NPs, the three groups of cells, and inhibitors were incubated for 4h, and the added inhibitors were divided into two groups, the first group to which HDAC inhibitors were added: HDACi (1 μ M); the second group added CTSL and HADC inhibitors: HDACI (1. mu.M) + CTSLi (0.2. mu.M); the control group is not added with the inhibitor, and the other test treatment modes are the same as the above; the experimental results are shown in fig. 15, and it can be seen that, compared to the control group, the fluorescence of the three groups of cells is obviously reduced after the inhibitor is added, and the fluorescence reduction phenomenon is most obvious in 4T1, which proves the enzyme responsiveness of the double-enzyme response drug-loaded nanoparticle.
(4) Influence of double-enzyme response drug-loaded nanoparticle PLC NPs on membrane potentials of different cell lines
Double enzyme response carrier observed by laser confocal scanning microscopeEffect of drug nanoparticles on membrane potential of different cell lines. Setting an adding drug group and a control group, wherein the culture medium of the adding drug group contains PLC NPs ([ Cy-NH ] 2 ]2.0 μ g/mL) and a control group without PLC NPs drug.
And (3) co-culturing three cell lines of MEF, MCF-7 and 4T1 for 4h by using the two drug-added culture media and the control culture medium respectively, washing the tumor cells for three times by using 1 XPBS (phosphate buffer solution) after the uptake is finished, removing unabsorbed double-enzyme response drug-loaded nanoparticles, carrying out probe staining on the cells according to the JC-1 kit instruction, and observing the fluorescence intensity in the cells by using a laser confocal scanning microscope after the staining is finished. As shown in FIG. 16, the fluorescence intensities of the three cell lines in the control group were substantially similar. In the drug-added group, the experimental results are shown in fig. 17, and it can be seen that the fluorescence intensity of two cell lines, i.e., MCF-7 and 4T1, JC-1 is reduced or even disappears, while the fluorescence intensity in MEF is substantially unchanged, which indicates that the membrane potential of tumor cells can be destroyed by the double-enzyme response drug-loaded nanoparticles without destroying the membrane potential of normal cells.
Animal experiment for anti-tumor effect of three-enzyme response drug-loaded nanoparticle PLC NPs
20 BALB/C nude mice, in which 4T1 subcutaneous tumor models were implanted, were randomly divided into 4 groups of 5 mice each. Tail vein injection of 100. mu.L PBS and 100. mu.L free Cy-NH 2 (4.0mg/kg)、100μL PLC NPs([Cy-NH 2 ]2.0mg/kg) and 100 μ L PLC NPs ([ Cy-NH) 2 ]4.0mg/kg), the drug was administered every two days, and the mice were subjected to the 21d treatment experiment. The tumor volume was measured with a caliper every two days throughout the treatment and the weight change of the mice in each experimental group was examined. The formula for tumor volume is as follows: volume (mm) 3 ) 0.5 x length x width 2 . Wherein, PLC NPs ([ Cy-NH ] 2 ]The treatment group was designated PLC NPs (2) ([ Cy-NH) 2.0mg/kg) 2 ]4.0mg/kg) treatment group was designated PLC NPs (4).
The results are shown in FIG. 18, and the tumors grew rapidly in the PBS group and the PLC NPs (2) group. free Cy-NH 2 The group and PLC NPs (4) have certain inhibition effect on tumor growth; Cy-NH in the free state 2 (free Cy-NH 2 ) Can enter tumor part to destroy tumor cell mitochondrion and kill tumor. The PLC NPs (4) has a more obvious inhibition effect on tumor growth, because the double-enzyme response drug-loaded nanoparticles can effectively transport Cy-NH 2 Increasing Cy-NH in tumor cells 2 Concentration, realizing the treatment effect on the tumor. And free Cy-NH 2 In contrast, PLC NPs (4) can generate HDAC and CTSL double enzyme response in tumor cells to cause granule disintegration process, trigger Cy-NH 2 The rapid release can realize the tumor targeting and generate more active oxygen, thereby obtaining better treatment effect. As shown in FIG. 19, the free Cy-NH was removed during the entire treatment period 2 Outside the group, the body weight of each group of mice does not obviously change, and the fact that the double-enzyme response drug-loaded nano material has good biocompatibility is proved.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims (10)

1. An HDAC and CTSL response diagnosis and treatment integrated material is characterized in that the molecular structure of the material is as follows:
Figure FDA0003705713140000011
wherein n is 45.
2. The method for preparing an HDAC/CTSL responsive integrated diagnosis and treatment material according to claim 1, comprising the steps of:
(1) fluorescent molecule Cy-NH 2 Synthesizing;
(2) synthesis of Boc-Lys (Ac) -Cy:
the Cy-NH prepared in the step (1) 2 Performing amide reaction with Boc-Lys (Ac) -OH for 15-30 h at room temperature according to the molar ratio (1.5-2.5) of 1, and extracting, separating and purifying to obtain Boc-Lys (Ac) -Cy;
(3) synthesis of Lys (Ac) -Cy:
removing Boc from the Boc-Lys (Ac) -Cy prepared in the step (2) in trifluoroacetic acid to prepare Lys (Ac) -Cy;
(4)PEG 2000 -synthesis of lys (ac) -Cy:
PEG after carboxyl activation 2000 mixing-COOH and Lys (Ac) -Cy according to the molar ratio of 1 (1-2), stirring and reacting for 3-5 h at room temperature in a dark place to obtain PEG 2000 -Lys (Ac) -Cy, which is a diagnosis and treatment integrated material responding to HDAC and CTSL.
3. The method according to claim 2, wherein the fluorescent molecule of step (1) is Cy-NH 2 The synthesis process of (A) is as follows:
①.Cy-NO 2 the synthesis of (2):
dissolving IR780, m-nitrophenol and DIPEA (dimethyl Diphenyl Ether) in DMF according to the molar ratio of 1 (1-1.5) to (0.1-0.2), reacting for 10-20 h at room temperature, extracting by an organic solvent, concentrating, separating out and drying to obtain Cy-NO 2 ；
②.Cy-NH 2 The synthesis of (2):
reacting Cy-NO 2 With excess SnCl 2 ·2H 2 Dissolving O and concentrated hydrochloric acid in methanol, reacting at 60-80 deg.C for 10-14 h, extracting with organic solvent, concentrating, separating out, and drying to obtain Cy-NH 2 。
4. The method of claim 3, wherein IR780 in step (i) is commercially available or prepared by the following method:
synthesis of 1-ethyl-2, 3, 3-trimethyl-3H-indole
Mixing 2,3, 3-trimethyl-3H-indole and iodoethane according to a molar ratio of 1 (1-2) in an organic solvent, reacting for 20-28H at 70-80 ℃, concentrating, washing and drying a reaction solution to obtain 1-ethyl-2, 3, 3-trimethyl-3H-indole;
II.2 Synthesis of 2-chloro-3-hydroxymethylene-1-cyclohexene-1-carbaldehyde
POCl is added 3 And (3) mixing with cyclohexanone according to the volume ratio of (2-4): 1 in organic solventsMixing, reacting at-4-1 ℃ for 20-30 min, reacting at 40-50 ℃ for 4-6 h, cooling, performing solid-liquid separation, collecting precipitate, and drying to obtain 2-chloro-3-hydroxymethylene-1-cyclohexene-1-formaldehyde;
synthesis of IR780
2-chloro-3-hydroxymethylene-1-cyclohexene-1-formaldehyde, 1-ethyl-2, 3, 3-trimethyl-3H-indole and sodium acetate trihydrate according to a molar ratio of 1: (1.5-2.5): (1.5-2.5), reacting for 1-3 h at 120-140 ℃ in an inert atmosphere to obtain a crude product, and washing, concentrating and drying the crude product to obtain the IR 780.
5. The method of claim 2, wherein the step (3) of separating Boc-Lys (Ac) -Cy from Boc in trifluoroacetic acid to obtain Lys (Ac) -Cy comprises:
and dissolving Boc-Lys (Ac) -Cy in DCM, mixing with trifluoroacetic acid for reaction until the Boc-Lys (Ac) -Cy completely reacts, neutralizing with acid and alkali, concentrating an organic phase, and drying to obtain Lys (Ac) -Cy.
6. The double-enzyme response drug-loaded nanoparticle is characterized in that the main effective component of the double-enzyme response drug-loaded nanoparticle is the diagnosis and treatment integrated material responding to HDAC and CTSL according to claim 1.
7. The double-enzyme-response drug-loaded nanoparticle of claim 6, wherein the particle size of the nanoparticle is 90-120 nm.
8. The preparation method of the double-enzyme response drug-loaded nanoparticle of claim 6 or 7, which is characterized by comprising the following steps:
the diagnosis and treatment integrated material with HDAC and CTSL responses is dispersed in dimethyl sulfoxide and water in sequence, and then dialyzed by a 3500D specification dialysis bag and filtered by a 0.22 mu m filter to prepare the double-enzyme response drug-loaded nanoparticles.
9. Use of the double enzyme-responsive drug-loaded nanoparticle of claim 6 or 7 for the preparation of an anti-cancer drug.
10. The use of claim 9, wherein the bi-enzyme responsive drug-loaded nanoparticle is prepared by generating HDAC and CTSL bi-enzyme response in tumor cells, so that the nanoparticle is disintegrated and Cy-NH is released into the tumor cells 2 The mitochondria of the tumor cells are destroyed, active oxygen is promoted to be generated in the tumor cells, the growth of the tumor cells is inhibited, and the aim of treating the cancer is achieved.
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