CN108524946B

CN108524946B - Ternary compound nano-drug, preparation method thereof and application thereof in preparation of light controlled release nano-delivery system

Info

Publication number: CN108524946B
Application number: CN201810302324.9A
Authority: CN
Inventors: 殷黎晨; 王金慧
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2018-04-04
Filing date: 2018-04-04
Publication date: 2020-09-25
Anticipated expiration: 2038-04-04
Also published as: CN108524946A

Abstract

The invention discloses a ternary complex nano-drug, a preparation method thereof and application thereof in preparing a light controllable release nano-delivery system. The photosensitizer contained in the compound can generate Reactive Oxygen Species (ROS) under the control of exogenous red light, so that lysosome escape mediated by photochemical internalization (PCI) and polymer degradation promoted by light are realized, and the aims of reducing toxic and side effects of materials, improving transfection efficiency and enhancing tumor gene treatment effect are finally realized.

Description

Ternary compound nano-drug, preparation method thereof and application thereof in preparation of light controlled release nano-delivery system

Technical Field

The invention belongs to the fields of high polymer material technology and pharmacy, and relates to a preparation method and application of a light controllable release nano delivery system.

Background

Polyethyleneimine (PEI) is a commercial gene vector, and since its structure contains a large number of positively charged amino groups, it can effectively carry negatively charged nucleic acids, thereby forming a stable complex for further delivery into cells. However, the high-molecular cationic polymer has a large positive charge, the cytotoxicity of the material is not negligible, and the nucleic acid material encapsulated in the high-molecular cationic polymer is difficult to release, so that the transfection effect is limited. The degradable cationic polymer is developed, so that the contradiction can be solved, the nucleic acid entrapment and the intracellular release of the nucleic acid entrapment are realized, and the cytotoxicity of the high molecular material is reduced. The existing stimulation response degradable cationic polymers mostly adopt endogenous stimulation, such as Glutathione (GSH), acidic environment and the like. However, endogenous stimuli are poorly regulated. Therefore, the development of a new delivery system for developing a high molecular cationic polymer gene vector with controllable exogenous stimulation, high biological safety, small toxic and side effects and high transfection efficiency is a very important means for realizing effective gene therapy.

Disclosure of Invention

The invention provides a ternary compound nano-drug, in particular to a photosensitizer contained in the compound which can generate Reactive Oxygen Species (ROS) under the control of exogenous red light, so as to realize lysosome escape mediated by photochemical internalization (PCI) and polymer degradation promoted by light, and finally realize the purposes of reducing toxic and side effects of materials, improving transfection efficiency and enhancing the tumor gene therapy effect.

The invention adopts the following technical scheme:

a ternary complex nano-drug comprises a thioketal bond cross-linked low molecular weight PEI-based polymer, a photosensitizer-connected hyaluronic acid polymer and a nucleic acid drug.

The invention provides a thioketal bond crosslinked low molecular weight PEI-based polymer, which has a structure shown in a formula I,

wherein R is dendritic PEI with the molecular weight of less than 5 kDa; n is more than or equal to 20 and less than or equal to 500, and m is more than or equal to 4 and less than or equal to 24.

The invention also discloses a thioketal crosslinking agent which has the following chemical structural formula:

the invention provides a preparation method of a thioketone bond crosslinked low molecular weight PEI-based polymer, which comprises the following steps:

(1) reacting cysteamine, methoxypropene and acryloyl chloride serving as raw materials to prepare a thioketal cross-linking agent;

(2) the thioketal crosslinking agent and PEI are used as raw materials to react to prepare the thioketal bond crosslinked low molecular weight PEI-based polymer (TK-PEI).

The invention provides a preparation method of a thioketal crosslinking agent, which comprises the following steps:

(1) cysteamine, methoxy propylene and acryloyl chloride are taken as raw materials to react to prepare the thioketal cross-linking agent.

In the technical scheme, in the step (1), cysteamine and trifluoroacetic acid are used as raw materials to prepare a compound 1, the compound 1 and methoxypropene are used as raw materials to prepare a compound 2, the compound 2 is deprotected by sodium hydroxide to prepare a compound 3, and finally the compound 3 and acryloyl chloride are used as raw materials to prepare the thioketal cross-linking agent.

In the technical scheme, in the step (1), the molar ratio of the cysteamine to the trifluoroacetic acid is 1: 1.9-2.1; the molar ratio of the compound 1 to the methoxypropene is 2.5-2.6: 1; the mol ratio of the compound 3 to the acryloyl chloride is 1: 3.

In the technical scheme, in the step (2), the TK-PEI is prepared by taking a thioketal crosslinking agent and PEI as raw materials through Michael addition reaction.

In the technical scheme, the mass ratio of the thioketal crosslinking agent to the PEI is 1: 1.3-1.4.

In the above-mentioned embodiments, the PEI used in the present invention comprises dendritic PEI having a molecular weight of less than 5 kDa, such as PEI (600Da), PEI (1800 Da).

The method for preparing TK-PEI of the invention can be specifically exemplified as follows:

(1) cysteamine (20.0 g, 176.0 mmol, 1 eq) was dissolved in methanol (400 mL) containing triethylamine (35.8 g, 353.9 mmol, 2 eq.) Ethyl trifluoroacetate (26.2 g, 184.8 mmol, 1.05 eq) was added and the reaction solution was stirred at room temperature overnight then acetic acid was added and the pH adjusted to 6 the solution was extracted with ethyl acetate (3 × 100 mL), the organic layers combined, dried over MgSO 2₄And (5) drying. The solution was filtered and rotary evaporated. The residue was further purified by silica gel column chromatography using n-hexane/ethyl acetate (8/1) as eluent to give compound 1, deuterated chloroform as a nuclear magnetic resonance;

(2) compound 1 (14.2 g, 82.0 mmol, 2.5 equivalents) and PTSA (0.2 g, 1.1 mmol, 0.03 equivalents) were dissolved in benzene (250 mL) and stirred at room temperature for 10 min. Molecular sieve (5 a, 100.0 g) was added and the mixture was stirred for a further 10 minutes. 2-Methoxypropylene (2.3 g, 32.6 mmol, 1 eq.) was then added and the mixture was stirred at room temperature for 4 h. The solution was filtered and concentrated by rotary evaporation to give compound 2, deuterated chloroform nuclear-magnetized;

(3) compound 2 (10.0 g, 25.8 mmol) was deprotected in NaOH solution (1M, 150 mL) at room temperature for 5 h. Extracting the solution with dichloromethane (5 × 100 mL), collecting the aqueous phase, and removing the solvent by rotary evaporation to obtain compound 3, wherein deuterated DMSO is used for nuclear magnetic resonance;

(4) compound 3 (0.3 g, 1.6 mmol, 1 equiv.) is dissolved in dichloromethane (30 mL) containing triethylamine (0.9 g, 9.6mmol, 6 equiv.) at 0 deg.C. Acryloyl chloride (0.4 g, 4.8 mmol, 3 equivalents) was then added dropwise to the mixture at 0 ℃. The solution was extracted with ethyl acetate (3 × 100 mL), the aqueous phase collected and purified by silica gel chromatography (n-hexane/ethyl acetate, 8/1) to give compound 4, the thioketal crosslinker, deuterated DMSO to nucleate nuclei;

(5) compound 4 (25.0 mg) and PEI (600Da) (33.0 mg) were dissolved in 1 mL of methanol and stirred at 45 ℃ under a dark nitrogen atmosphere for 48 hours. The mixture was dialyzed against deionized water (deionized water, MWCO =1 kDa) for 2 days and lyophilized to give TK-PEI, deuterated deuterium heavy water to nuclear magnetic resonance, and Gel Permeation Chromatography (GPC) to determine molecular weight.

The specific reaction described above can be represented as follows:

the invention discloses a Hyaluronic Acid Polymer (HAP) connected with a photosensitizer, wherein the main chain is hyaluronic acid, the side chain is connected with the photosensitizer, and the molecular weight of the hyaluronic acid is 7-500 kDa and has a structure shown in a formula II;

wherein R is₂Is a photosensitizer unit; preferably, the photosensitizer is a carboxyl porphyrin derivative, a carboxyl chlorophyll porphyrin derivative or a dihydroporphin derivative, and the structure can be represented as follows:

the invention discloses a preparation method of a hyaluronic acid polymer connected with a photosensitizer, which comprises the following steps of adding acetic anhydride into a mixture of hyaluronic acid and pyridine, and reacting to obtain acetylated hyaluronic acid; mixing photosensitizer such as pheophytin a (Pha), carbodiimide (EDC) and 4-Dimethylaminopyridine (DMAP), stirring, adding acetylated hyaluronic acid, and reacting carboxyl of the photosensitizer with hydroxyl of hyaluronic acid to obtain hyaluronic acid polymer connected with the photosensitizer.

In the technical scheme, the molar ratio of acetic anhydride to hyaluronic acid is 147:1, and the mass ratio of the photosensitizer to the acetylated hyaluronic acid is 20: 1.

The method for preparing HAP of the present invention may be specifically exemplified by:

hyaluronic acid (0.5 g, 86.2. mu. mol) and pyridine (1.5 mL, 18.6 mmol) were dissolved in 20mL formamide, and the mixture was stirred at room temperature for 1 hour. Acetic anhydride (1.2 mL, 12.7 mmol) was added, stirred for further 24 hours, and dialyzed against deionized water (MWCO =1 kDa) for 2 days. Obtaining acetylated HA after freeze drying; pha (1 eq), EDC (1 eq) and DMAP (1.5 eq) were dissolved in anhydrous DMSO and the mixture was stirred for 6 hours. The acetylated HA (25.0 mg) was then added to the mixture and stirred at room temperature for a further 24 hours. HAP was obtained after dialysis with deionized water (MWCO =1 kDa) for 2 days and freeze-drying, and the photosensitizer content in HAP was measured by ultraviolet-visible (UV-Vis) spectroscopy.

The nucleic acid drug of the invention is selected from DNA, RNA, oligonucleotides or polynucleotides.

The invention also discloses a preparation method of the ternary complex nano-drug, which comprises the following steps of mixing and incubating the thioketone bond cross-linked low molecular weight PEI-based polymer solution and the nucleic acid drug solution, then adding the hyaluronic acid polymer solution connected with the photosensitizer, and incubating again to obtain the ternary complex nano-drug. The method specifically comprises the following steps:

dissolving TK-PEI in DEPC water, mixing the TK-PEI with a nucleic acid solution dissolved in advance according to different mass ratios, and incubating in a water bath at 37 ℃ for 30min to obtain a binary compound; adding HAP dissolved in DEPC water according to different mass ratios, and incubating in water bath at 37 deg.C for 60min to obtain the final nanometer medicine.

In the above technical scheme, the nucleic acid drug is selected from DNA, RNA, oligonucleotide or polynucleotide.

In the technical scheme, the DNA is p53 plasmid DNA and can express protein of p 53.

In the ternary complex nano-drug, the mass ratio of TK-PEI to the nucleic acid drug is (0.1-10) to 1, the preferred mass ratio is (0.5-2.5) to 1, and the more preferred mass ratio is 1 to 1.

In the ternary complex nano-drug of the invention, the mass ratio of the HAP to the nucleic acid drug is (0.1-10) to 1, the preferred mass ratio is (0.5-5) to 1, and the more preferred mass ratio is 2.5 to 1.

The particle size of the ternary complex nano-drug is 100-1000 nm, the preferred particle size is 100-300 nm, and the more preferred particle size is 100-150 nm.

The Zeta potential of the ternary complex nano-drug is-20-50 mV, the preferred Zeta potential is 5-30 mV, and the more preferred Zeta potential is 5-10 mV.

The invention also discloses application of the TK-PEI and/or HAP in preparation of a nucleic acid drug carrier.

The invention further discloses application of the TK-PEI, HAP or ternary complex nano-drug in preparation of gene drugs, in particular application in preparation of a light controllable release nano-gene drug delivery system.

The main advantages of the invention are:

(1) the TK-PEI entraps a binary compound formed by nucleic acid drugs and serves as an inner core, and has the following advantages and effects: the nucleic acid drug can be stably entrapped, and the endocytosis of the nucleic acid drug is effectively promoted; secondly, the structure of the polyamino can improve the escape capacity of lysosomes and enhance the nuclear entry amount of nucleic acid drugs through the proton sponge effect; thirdly, the thioketal bond contained can be broken under the external stimulation, the toxic and side effects of the material are reduced, and the release of the accounting drug is promoted; fourthly, the synthetic method is simple and controllable; the raw materials are cheap, so that the cost is saved;

(2) the ternary composite nano-particles obtained by the invention have the following advantages and effects: hyaluronic acid adsorbed on the outer layer can enhance the serum stability of the compound, and improve the specific endocytosis of tumor cells by actively targeting the tumor cells; the photosensitizer can generate ROS under the condition of short-time illumination, so that lysosome escape of nanoparticles is further promoted through a PCI effect, and meanwhile, the degradation of TK-PEI can be controlled, so that the effects of reducing toxic and side effects and enhancing nucleic acid drug release are achieved; the photosensitizer can generate a lethal dose ROS under the long-time illumination condition, and the photodynamic therapy (PDT) of the tumor is realized;

(3) the ternary composite nano-particles disclosed by the invention can play an obvious role in transfecting tumor cells or normal cells in different cell models, and further improve the gene transfection effect through illumination; can realize the synergic tumor gene therapy and photodynamic therapy in vivo.

Drawings

FIG. 1 shows the preparation of example 4¹H NMR spectrum;

FIG. 2 is a schematic representation of example ditk-PEI¹H NMR spectrum;

FIG. 3 is a UV-Vis spectrum of example six;

FIG. 4 is a GPC chart before and after treatment of hydrogen peroxide water in example II and comparative example;

FIG. 5 is a graph showing the particle size of the DNA encapsulated in the second example and the first comparative example;

FIG. 6 is a graph of the potentials after DNA encapsulation in example two and comparative example one;

FIG. 7 is a graph of DNA encapsulation after pretreatment with hydrogen peroxide of different concentrations for example two and comparative example one;

FIG. 8 is a graph of the particle size change in serum over time for ternary and binary complexes;

FIG. 9 is a graph of gene transfection of complexes on B16F10 cells, example two, comparative example one, and PEI 25k after encapsulation of the DNA;

FIG. 10 is a photograph of gene transfection on B16F10, HeLa, COS-7 cells before and after illumination with ternary and binary complexes;

FIG. 11 is a plot of fluorescence of lysosomal escape before and after ternary complex illumination on B16F10 cells;

FIG. 12 is a graph of fluorescence of DNA release before and after illumination with ternary complex on B16F10 cells;

FIG. 13 is a graph of the cytotoxicity of materials treated with hydrogen peroxide solution of example two, comparative example one, PEI 25k and PEI 600 on B16F10 cells;

FIG. 14 is a graph of the tumor inhibition curves of ternary complexes encapsulating model DNA (luc) and therapeutic DNA (p53), respectively, under short and long light conditions in a B16F10 tumor model.

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 under conventional conditions or conditions recommended by the manufacturers. In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Example one

(1) Cysteamine (20.0 g, 176.0 mmol, 1 eq) was dissolved in methanol (400 mL) containing triethylamine (35.8 g, 353.9 mmol, 2 eq.) Ethyl trifluoroacetate (26.2 g, 184.8 mmol, 1.05 eq) was added and the reaction solution was stirred at room temperature overnight then acetic acid was added and the pH adjusted to 6 the solution was extracted with ethyl acetate (3 × 100 mL), the organic layers combined, dried over MgSO 2₄And (5) drying. The solution was filtered and rotary evaporated and the residue was further purified by silica gel column chromatography using n-hexane/ethyl acetate (8/1) as eluent to give compound 1, of the formula:

(2) compound 1 (14.2 g, 82.0 mmol, 2.5 equivalents) and PTSA (0.2 g, 1.1 mmol, 0.03 equivalents) were dissolved in benzene (250 mL) and stirred at room temperature for 10 min. Molecular sieve (5 a, 100.0 g) was added and the mixture was stirred for a further 10 minutes. 2-Methoxypropylene (2.3 g, 32.6 mmol, 1 eq.) was then added and the mixture was stirred at room temperature for 4 h. The solution was filtered and concentrated by rotary evaporation to give compound 2, of the formula:

(3) compound 2 (10.0 g, 25.8 mmol) was deprotected in NaOH solution (1M, 150 mL) at room temperature for 5 h. The solution was extracted with dichloromethane (5X 100 mL), the aqueous phase collected and, after removal of the solvent by rotary evaporation, compound 3 was obtained, of the formula:

(4) compound 3 (0.3 g, 1.6 mmol, 1 equiv.) is dissolved in dichloromethane (30 mL) containing triethylamine (0.9 g, 9.6mmol, 6 equiv.) at 0 deg.C. Acryloyl chloride (0.4 g, 4.8 mmol, 3 equivalents) was then added dropwise to the mixture at 0 ℃. The solution was extracted with ethyl acetate (3 × 100 mL), the aqueous phase collected and purified by silica gel chromatography (n-hexane/ethyl acetate, 8/1) to give compound 4, of the formula:

deuterated DMSO adopts nuclear magnetic resonance, and the figure 1 is a nuclear magnetic diagram of a compound 4.

Example two

Compound 4 (25.0 mg) and PEI (600Da) (33.0 mg) were dissolved in 1 mL of methanol and stirred at 45 ℃ under a dark nitrogen atmosphere for 48 hours. The mixture was dialyzed against deionized water (deionized water, MWCO =1 kDa) for 2 days and lyophilized to give compound 5 (TK-PEI), deuterated deuterium-water-base nuclear magnetic resonance, fig. 2 is the nuclear magnetic resonance image of compound 5, fig. 4 is the GPC image of compound 5, with a molecular weight of 11 kDa.

EXAMPLE III

Compound 4 (25.0 mg) and PEI (1800Da) (33.0 mg) were dissolved in 1 mL of methanol and stirred at 45 ℃ under a dark nitrogen atmosphere for 48 hours. The mixture was dialyzed against deionized water (deionized water, MWCO =1 kDa) for 2 days and lyophilized to give compound 5 (TK-PEI) with a molecular weight of 18 kDa.

Example four

Compound 4 (25.0 mg) and PEI (1800Da) (66.0 mg) were dissolved in 1 mL of methanol and stirred at 45 ℃ under a dark nitrogen atmosphere for 48 hours. The mixture was dialyzed against deionized water (deionized water, MWCO =1 kDa) for 2 days and lyophilized to give compound 5 (TK-PEI) with a molecular weight of 20 kDa.

EXAMPLE five

Compound 4 (25.0 mg) and PEI (600Da) (66.0 mg) were dissolved in 1 mL of methanol and stirred at 45 ℃ under a dark nitrogen atmosphere for 48 hours. The mixture was dialyzed against deionized water (deionized water, MWCO =1 kDa) for 2 days and lyophilized to give compound 5 (TK-PEI) with a molecular weight of 12 kDa.

Comparative example 1

(1) 1, 7-heptanediamine (0.3 g, 1.6 mmol, 1 equiv.) is dissolved in dichloromethane (30 mL) containing triethylamine (0.9 g, 9.6mmol, 6 equiv.) at 0 deg.C. Acryloyl chloride (0.4 g, 4.8 mmol, 3 equivalents) was then added dropwise to the mixture at 0 ℃. The solution was extracted with ethyl acetate (3 × 100 mL), the aqueous phase collected and purified by silica gel chromatography (n-hexane/ethyl acetate, 8/1) to give compound 6, of the formula:

(2) compound 6 (25.0 mg) and PEI (600Da) (33.0 mg) were dissolved in 1 mL of methanol and stirred at 45 ℃ under a dark nitrogen atmosphere for 48 hours. The mixture was dialyzed against deionized water (deionized water, MWCO =1 kDa) for 2 days and lyophilized to give compound 7 (NK-PEI) of the formula:

EXAMPLE six

Hyaluronic acid (0.5 g, 86.2. mu. mol) and pyridine (1.5 mL, 18.6 mmol) were dissolved in 20mL formamide, and the mixture was stirred at room temperature for 1 hour. Acetic anhydride (1.2 mL, 12.7 mmol) was added, stirred for further 24 hours, and dialyzed against deionized water (MWCO =1 kDa) for 2 days. Obtaining acetylated HA after freeze drying; pha (1 eq), EDC (1 eq) and DMAP (1.5 eq) were dissolved in anhydrous DMSO and the mixture was stirred for 6 hours. The acetylated HA (25.0 mg) was then added to the mixture and stirred at room temperature for a further 24 hours. After dialysis with deionized water (MWCO =1 kDa) for 2 days and freeze-drying, the photosensitizer-conjugated hyaluronic acid polymer HAP was obtained, and fig. 3 is a graph of the HAP uv-vis spectrum.

EXAMPLE seven

Hyaluronic acid (0.5 g, 86.2. mu. mol) and pyridine (1.5 mL, 18.6 mmol) were dissolved in 20mL formamide, and the mixture was stirred at room temperature for 1 hour. Acetic anhydride (1.2 mL, 12.7 mmol) was added, stirred for further 24 hours, and dialyzed against deionized water (MWCO =1 kDa) for 2 days. Obtaining acetylated HA after freeze drying; pha (1 eq), EDC (1 eq) and DMAP (1.5 eq) were dissolved in anhydrous DMSO and the mixture was stirred for 6 hours. The acetylated HA (25.0 mg) was then added to the mixture and stirred at room temperature for a further 24 hours. HAP was obtained after dialysis against deionized water (MWCO =1 kDa) for 2 days and freeze-drying.

Example eight

Hyaluronic acid (0.5 g, 86.2. mu. mol) and pyridine (1.5 mL, 18.6 mmol) were dissolved in 20mL formamide, and the mixture was stirred at room temperature for 1 hour. Acetic anhydride (1.2 mL, 12.7 mmol) was added, stirred for further 24 hours, and dialyzed against deionized water (MWCO =1 kDa) for 2 days. Obtaining acetylated HA after freeze drying; chlorin Ce6 (1 eq), EDC (1 eq) and DMAP (1.5 eq) were dissolved in anhydrous DMSO and the mixture was stirred for 6 hours. The acetylated HA (25.0 mg) was then added to the mixture and stirred at room temperature for a further 24 hours. HAP was obtained after dialysis against deionized water (MWCO =1 kDa) for 2 days and freeze-drying.

Example nine

Hyaluronic acid (0.5 g, 86.2. mu. mol) and pyridine (1.5 mL, 18.6 mmol) were dissolved in 20mL formamide, and the mixture was stirred at room temperature for 1 hour. Acetic anhydride (1.2 mL, 12.7 mmol) was added, stirred for further 24 hours, and dialyzed against deionized water (MWCO =1 kDa) for 2 days. Obtaining acetylated HA after freeze drying; carboxyporphyrin (1 equivalent), EDC (1 equivalent) and DMAP (1.5 equivalents) were dissolved in anhydrous DMSO and the mixture was stirred for 6 hours. The acetylated HA (25.0 mg) was then added to the mixture and stirred at room temperature for a further 24 hours. HAP was obtained after dialysis against deionized water (MWCO =1 kDa) for 2 days and freeze-drying.

Example ten

And (3) preparing, characterizing and performing the nano-drug assembled by taking the TK-PEI of the second embodiment and the HAP of the sixth embodiment as carriers.

Aqueous DEPC solutions were prepared in the concentrations of TK-PEI and DNA (plasmid DNA, a plasmid containing luciferase or p53 expression extracted from E.coli) of example II at 1mg/mL and 0.1mg/mL, respectively. The TK-PEI/DNA was mixed in different weight ratios and the mixture was vortexed for 10 seconds and then incubated at 37 ℃ for 30 minutes to form the TK-PEI/DNA complex.

Preparation example an aqueous DEPC solution having a six HAP concentration of 1mg/mL was prepared. The mixture was mixed according to different weight ratios of HAP/binary complex above, vortexed for 10 seconds and then incubated at 37 ℃ for 60 minutes to form a HAP/TK-PEI/DNA ternary complex.

The particle size and potential of the ternary complex were evaluated using Dynamic Light Scattering (DLS) at different weight ratios of the complex mixed.

The EB solution was mixed with DNA in a weight ratio of 10: 1. And incubated at room temperature for 1 hour. The examples were then added to EB/DNA mixtures at different weight ratios TK-PEI/DNA, the mixtures were further incubated at room temperature for 30 minutes, and their fluorescence intensity (λ ex = 510 nm, λ em = 590 nm) was measured quantitatively by a microplate reader for DNA encapsulation efficiency.

B16F10 cells at 2.5 × 10 per well⁴Each was inoculated into a 96-well plate, and then cultured in DMEM medium containing 10% FBS for 24 hours. The medium was then replaced with serum-free DMEM and the binary complex was added at a concentration of 0.3 μ g DNA per well. After 4 hours of incubation at 37 ℃, the medium was removed and replaced with fresh medium followed by a further 20 hours of incubation. Luciferase expression was measured using a luciferase kit, and cellular protein concentration was measured using a BCA kit, and gene transfection efficiency thereof was evaluated.

B16F10 cells at 2.5 × 10 per well⁴Each was inoculated into a 96-well plate, and then cultured in DMEM medium containing 10% FBS for 24 hours. Adding the ternary complex according to the concentration of 0.3 mug DNA/each hole. After 4 hours incubation at 37 ℃, the medium was removed, replaced with fresh medium, and the cells were divided into two groups, one with light (661 nm, 8 min) and one without light, followed by further incubation for 20 hours. Luciferase expression was measured using a luciferase kit, and cellular protein concentration was measured using a BCA kit, and gene transfection efficiency thereof was evaluated.

The cell internalization and distribution of the complex can be further observed by confocal laser scanning microscopy B16F10 cells at 1.5 × 10 per well⁴Each was inoculated into a 24-well plate, and after culturing in DMEM medium containing 10% FBS for 24 hours, the medium was changed to serum-free medium, and the complex was added at a concentration of 1. mu.g YOYO-1-DNA/well. Incubated at 37 ℃ for 4 hours. Washed three times with sodium heparin-containing PBS and stained with Lysotracker-Red (200 nM) for 1 hour; washing with PBS containing heparin sodium for three times, and fixing with 4% paraformaldehyde for 15 min; followed by three washes with PBS containing sodium heparin followed by staining with DAPI (5. mu.g/mL) for 10 min; finally, sealing the piece by using glycerol. The fluorescence intensity was observed by a confocal laser microscope.

B16F10 cells at 2.5 × 10 per well⁴One was inoculated into a 96-well plate and cultured for 24 hours. Then replaced with serum-free medium and different final concentrations of hydrogen peroxide pre-treated polymer were added. After incubation at 37 ℃ for 4 hours, the medium was discarded and replaced with 10% FBS DMEM for 20 hours. Cell viability was determined by the MTT method.

Male C57BL/6 mice were injected subcutaneously on the left side with 1 × 10⁶And B16F10 cells. When the tumor volume reaches 80mm³At the time, mice are randomly divided into 4 groups (n = 6), receiving intratumoral injection of phosphate buffer or various nanoparticles (3 mg DNA/kg, injection volume-50 μ L) on

days

1, 3, 5, 7 and 9. Group 1 received injections of phosphate buffer. Group 3 received injections of TK-PEI/HAP/luc nanoparticles.

Groups

2 and 4 received TK-PEI/HAP/p53 nanoparticles. Animals in group 2 were irradiated 4 hours after injection (20 mW/cm)²661 nm) for 8 minutes, while animals in

groups

3 and 4 were irradiated (20 mW/cm) for 4 hours²661 nm) 8 minutes post injection and 24 hours 30 minutes post injection. Tumor size was monitored every other day.

The TK-PEI of example II is replaced by the NK-PEI of comparative example I, and the binary and ternary complexes formed by assembly are compared, and the results are as follows:

FIG. 4 is a GPC chart before and after treatment with hydrogen peroxide water of example two and comparative example two, and it is found from FIG. 4 that example two and comparative example one of the present invention have similar molecular weights (10 to 12 kDa). Compared with the first comparative example, the retention time of the second example after the hydrogen peroxide treatment is obviously prolonged, which shows that the hydrogen peroxide treatment causes the molecular weight of the second example to be reduced.

FIG. 5 is a graph showing the particle size of the DNA encapsulated in the second example and the first comparative example, and it is found from FIG. 5 that TK-PEI of the present invention as a carrier has a good nano particle size distribution of about 100 to 150 nm.

FIG. 6 is a graph showing the potential after wrapping DNA in example two and comparative example one, and it is found from FIG. 6 that the example two and comparative example one change from negative charge to positive charge of about 10 to 20mV at a mass ratio of 0.5 to nucleic acid drug, and that the example two and comparative example one compound well with nucleic acid drug.

FIG. 7 is a DNA encapsulation graph of ethidium bromide treatment after DNA encapsulation of example two and comparative example one, and it is found from FIG. 7 that the encapsulation efficiency of up to nearly 90% can be achieved when the mass ratio of the DNA drug to the nucleic acid drug is 0.5 for example two and comparative example one, which proves that the nucleic acid drug can be well encapsulated in both example two and comparative example one; by treating the second example and the first comparative example with different concentrations of hydrogen peroxide, it can be found in fig. 7 that the DNA entrapment rate of the second example is obviously reduced, while the DNA entrapment rate of the first comparative example is not changed, which indicates that the second example has hydrogen peroxide-responsive degradation.

FIG. 8 is a graph showing the stability of the binary complex (TK-PEI/DNA) and the ternary complex (HAP/TK-PEI/DNA) in serum, and it can be seen from FIG. 8 that the particle size of the binary complex is significantly increased and the particle size of the ternary complex is kept stable with time. Indicating that the ternary complex has greater stability in serum.

FIG. 9 shows the gene transfection of the complex after encapsulation of DNA by B16F10 cells, example II, comparative example I and 25k PEI, and it can be seen from FIG. 9 that example II possesses similar transfection efficiency when compared to comparative example I at the same mass ratio as the nucleic acid drug.

Fig. 10 shows gene transfection before and after illumination after the ternary complex and the binary complex wrap DNA on B16F10 cells, and it can be found from fig. 10 that when the ternary complex is subjected to short illumination, the transfection effect of the ternary complex is significantly improved compared with that of non-illumination, demonstrating that the ternary complex has good photoactivation, promotes photodynamic membrane rupture, and is beneficial for the complex to escape from endosomes.

FIG. 11 is a fluorescence diagram of lysosome escape before and after illumination of ternary complex on B16F10 cells, and from FIG. 11, it can be found that after illumination, overlap of red and green fluorescence is reduced, which proves that more complexes escape from endosomes, and thus gene transfection is facilitated.

FIG. 12 is a fluorescence diagram of DNA release from complexes before and after illumination of ternary complex on B16F10 cells, and it can be seen from FIG. 12 that after illumination, the overlap of red and green fluorescence decreases, demonstrating that more DNA is released from complexes into cytoplasm, which is beneficial for promoting gene transfection.

Fig. 13 shows the cytotoxicity of the polymer materials on B16F10 cells, example two, comparative example one, 25k PEI and 600 PEI after 24 hours of hydrogen peroxide pretreatment, and from fig. 13, it can be seen that example two and 600 PEI have similar cell viability rates, which can be maintained above 90% when the polymer concentration reaches 0.1mg/mL, while the cell viability rate of comparative example one shows significant cytotoxicity as the polymer concentration increases. The embodiment proves that the responsive degradation of the two pairs of hydrogen peroxide can improve the biocompatibility of the material and reduce the toxic and side effects.

FIG. 14 is a graph of tumor inhibition curves for ternary complex encapsulated mode DNA (luc) and therapeutic DNA (p53) under short and long light conditions on a B16F10 tumor model. It can be seen from figure 14 that the best inhibition of tumor growth can be achieved by combining the photodynamic therapy effect of therapeutic DNA and long light compared to gene therapy alone or photodynamic therapy alone.

The ternary light controllable release nano delivery system containing the photosensitizer and the thioketal sensitive bond, provided by the invention, has the advantages that the designed ternary compound with red light response can be used as a carrier of a nucleic acid drug, has good stability, biodegradability and photoactivatability, and has good application in a nucleic acid drug delivery system.

Claims

1. A ternary complex nano-drug, characterized in that the ternary complex nano-drug comprises thioketone bond cross-linked low molecular weight PEI based polymer, hyaluronic acid polymer connected with photosensitizer, nucleic acid drug;

the thioketal bond crosslinked low molecular weight PEI-based polymer has a structure shown in formula I;

wherein n is more than or equal to 20 and less than or equal to 500, and m is more than or equal to 4 and less than or equal to 24;

the hyaluronic acid polymer connected with the photosensitizer has a structure shown in a formula II;

wherein R is₂Is a photosensitizer unit.

2. The ternary complex nano-drug according to claim 1, wherein the preparation method of the ternary complex nano-drug comprises the steps of mixing and incubating a thioketal bond-crosslinked low molecular weight PEI-based polymer solution with a nucleic acid drug solution, adding a photosensitizer-linked hyaluronic acid polymer solution, and incubating again to obtain the ternary complex nano-drug;

wherein R is₂Is a photosensitizer unit.

3. The ternary complex nanopharmaceutical of claim 2, wherein said nucleic acid drug is selected from DNA, RNA, oligonucleotides or polynucleotides; the mass ratio of the thioketal bond crosslinked low molecular weight PEI-based polymer to the nucleic acid drug is (0.1-10) to 1; the mass ratio of the hyaluronic acid polymer connected with the photosensitizer to the nucleic acid medicine is (0.1-10) to 1.

4. A thioketal bond-crosslinked low molecular weight PEI-based polymer characterized in that said thioketal bond-crosslinked low molecular weight PEI-based polymer has a structure represented by formula I,

wherein n is more than or equal to 20 and less than or equal to 500, and m is more than or equal to 4 and less than or equal to 24.

5. The thioketal-crosslinked low molecular weight PEI-based polymer of claim 4 wherein said method of preparing said thioketal-crosslinked low molecular weight PEI-based polymer comprises the steps of:

(2) the thioketal crosslinking agent and PEI are used as raw materials to react to prepare the thioketal bond crosslinked low molecular weight PEI-based polymer.

6. Use of the thioketal-crosslinked low molecular weight PEI-based polymer of claim 4 for the preparation of a nucleic acid drug carrier; use of the thioketal-crosslinked low molecular weight PEI-based polymer of claim 4 or the ternary complex nano-drug of claim 1 for the preparation of a gene-based drug.