OXIDATION-RESPONSIVE WATER-SOLUBLE CATIONIC PILLARARENE, AND PREPARATION METHOD AND APPLICATION THEREOF

Abstract

Disclosed are an oxidation-responsive water-soluble cationic pillararene, and a preparation method and application thereof, the oxidation-responsive water-soluble cationic pillararene is a cyclic small-molecule nucleic acid vector, and is obtained by quaternization reaction of 4-methylborate and tertiary amine-modified pillararene. According to the invention, a delivery vector with a high positive charge density is obtained through small molecule synthesis, the delivery vector is capable of being tightly complexed with negatively charged nucleic acid substances to form a nano-composite, and after the nano-composite enters cells, vector positive charges fall off to release the nucleic acid substances in an oxidation environment, and nucleic acid is efficiently translated and expressed. The delivery vector has the characteristics of simplicity and convenience in synthesis, high delivery efficiency and good biological safety, and has a good application prospect.

Claims

1. An oxidation-responsive water-soluble cationic pillararene, comprising the following structure: ##STR00006## in the above formula: X is an integer from 1 to 4, R.sub.1 and R.sub.2 are independently the following segments respectively, and R.sub.3 and R.sub.4 are independently H and C1-C6 alkyl or acyl respectively; ##STR00007## R.sub.5 and R.sub.6, and R.sub.7 and R.sub.8 are independently C1-C6 alkyl or aryl respectively; R.sub.9 and R.sub.10 are independently H and C1-C20 alkyl or aryl respectively; and anions are bromide ions or chloride ions.

2. The oxidation-responsive water-soluble cationic pillararene according to claim 1, wherein the pillararene is prepared by reacting a pillararene containing primary amino, secondary amino or tertiary amine with boric acid benzyl or borate benzyl.

3. The oxidation-responsive water-soluble cationic pillararene according to claim 2, wherein the pillararene is a copolymerized pillar[5]arene, and a copolymerization ratio is 1:4, that is, x=1 or 4, and 2:3, that is, x=2 or 3.

4. The oxidation-responsive water-soluble cationic pillararene according to claim 3, wherein the copolymerized pillar[5]arene comprises a pillar[5]arene obtained by copolymerization cyclization reaction according to a reaction monomer molar ratio of 2:3, with an obtained product x=2 or 3; and according to a reaction monomer molar ratio of 1:4, with an obtained product x=1 or 4.

5. The oxidation-responsive water-soluble cationic pillararene according to claim 1, wherein R.sub.5 and R.sub.6, and R.sub.7 and R.sub.5 are methyl or ethyl, R.sub.9 is methyl and ethyl, and R.sub.10 is C6-20 alkyl or aryl.

6. The oxidation-responsive water-soluble cationic pillararene according to claim 3, wherein R.sub.5 and R.sub.6, and R.sub.7 and R.sub.5 are methyl or ethyl, R.sub.9 is methyl and ethyl, and R.sub.10 is C6-20 alkyl or aryl.

7. The oxidation-responsive water-soluble cationic pillararene according to claim 4, wherein R.sub.5 and R.sub.6, and R.sub.7 and R.sub.5 are methyl or ethyl, R.sub.9 is methyl and ethyl, and R.sub.10 is C6-20 alkyl or aryl.

8. A method for preparing an oxidation-responsive water-soluble cationic pillararene according to claim 1, wherein the method is specifically as follows: 1-(2-haloethoxy)-4-methoxybenzene and 4-alkoxymethyl(ethyl)oxybenzene react together under the condition of Lewis acid catalyst to obtain copolymerized pillar[5]arene obtained by copolymerization according to molar ratios of 2:3 and 1:4, then react with dimethylamine and diethylamine to obtain a copolymerized pillar[5]arene substituted with tertiary amine, and then react with boric acid benzyl bromide, borate benzyl bromide, boric acid benzyl chloride or borate benzyl chloride to obtain the pillararene compound.

9. A method for preparing an oxidation-responsive water-soluble cationic pillararene according to claim 2, wherein the method is specifically as follows: 1-(2-haloethoxy)-4-methoxybenzene and paraformaldehyde or trioxane react together under the condition of Lewis acid catalyst to obtain a cyclic pillar[n]arene, wherein n=5 to 15, then react with dimethylamine and diethylamine to obtain a pillar[n]arene substituted with tertiary amine, and then react with boric acid benzyl bromide, borate benzyl bromide, boric acid benzyl chloride or borate benzyl chloride to obtain the pillararene compound.

10. A method for preparing an oxidation-responsive water-soluble cationic pillararene according to claim 2, wherein a structure of the water-soluble pillararene prepared is as follows: ##STR00008##

Description

DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1A shows the oxidation reaction of compound 1; and FIG. 1B is a .sup.1HNMR spectra of a compound 1 in Embodiments 1, 2 and 3 under oxidation conditions;

[0030] FIG. 2 shows particle sizes and Zeta potentials of nano-composites with different N/P formed by complexing a compound 1 in Embodiment 4 of the present invention with a plasmid DNA;

[0031] FIG. 3 is a gel retardation electrophoretogram of the nano-composites with different N/P formed by complexing the compound 1 in Embodiment 4 of the present invention with the plasmid DNA;

[0032] FIG. 4 is a gel retardation electrophoretogram under oxidation conditions of the nano-composite formed by complexing the compound 1 in Embodiment 4 of the present invention with the plasmid DNA;

[0033] FIG. 5A shows particle sizes under oxidation conditions of the nano-composite formed by complexing the compound 1 in Embodiment 4 of the present invention with the plasmid DNA; and FIG. 5B is a diagram of corresponding Zeta potentials change;

[0034] FIG. 6 is a transmission electron micrograph of the nano-composite formed by complexing the compound 1 in Embodiment 4 of the present invention with the plasmid DNA;

[0035] FIG. 7 is a cytotoxicity diagram of a compound 1 in Embodiment 5 of the present invention at different concentrations;

[0036] FIG. 8 is an effect diagram of cell transfection under different N/P conditions of a nano-composite formed by combining a compound 1 in Embodiment 6 of the present invention with the plasmid DNA;

[0037] FIG. 9 is an effect diagram of cell transfection under different N/P conditions of a nano-composite formed by complexing a compound 1 in Embodiment 7 of the present invention with an mRNA; and

[0038] FIG. 10 is an effect diagram of cell transfection under oxidation conditions of a nano-composite formed by complexing a compound 1 in Embodiment 8 of the present invention with the DNA.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0039] The present invention provides some specific embodiments, but the present invention is not limited by these embodiments.

Embodiment 1

Synthesis of Compound 1

[0040] 1-methoxy-4-hexadecylbenzene (1.74 g, 5.00 mmol) and 4-bis(2-bromoethoxy)benzene (6.48 g, 20.0 mmol) were placed in 80 mL of 1,2-dichloroethane, then added with boron trifluoride diethyl ether (3.20 mL, 25 mM), and stirred at room temperature for 2 hours. After reaction, the reaction solution was poured into methanol to separate out a large number of white solids and filtered to obtain a precipitate, and the precipitate was dissolved in dichloromethane and then filtered again to remove insoluble substances. The dichloromethane solution was washed with water twice to obtain an organic phase, and the organic phase was dried with anhydrous sodium sulfate and then spin-dried to obtain a crude product. The crude product was subjected to column chromatography, a mobile phase was petroleum ether/ethyl acetate=50:1 (R.sub.f=0.50), and a product compound 3 was obtained by spin drying, which was a white powdery solid (0.85 g, 10%).

[0041] The compound 3 (1.64 g, 1.00 mmol) and excess diethylamine (7.50 g, 100 mmol) were added into 100 mL of absolute ethanol, and heated and stirred at 80 C. for reflux reaction for 24 hours. After reaction, the solvent was removed by rotary evaporation, and 200 mL of 1 M sodium hydroxide solution was poured into the mixture and stirred for 1 hour. Subsequently, the reaction solution was fully extracted with ethyl acetate, and an organic phase was spin-dried to obtain a dark yellow oily compound 2 (1.55 g, 98%).

[0042] The compound 2 (0.49 g, 0.30 mM) and 4-(bromomethyl)benzeneboronic acid pinacol ester (0.78 g, 2.64 mmol) were dissolved in 25 mL of acetonitrile, and heated and stirred at 75 C. for reflux reaction for 24 hours. After reaction, the mixed solution obtained from the reaction was concentrated to 2.0 mL, added with excessive ether, and filtered to collect a white precipitate separated out, and the precipitate was fully washed with ether, and dried in a vacuum oven to obtain a white precipitate, which was a product compound 1 (0.98 g, 81.6%).

##STR00005##

[0043] Structure detection data of the compound 1 were as follows:

[0044] .sup.1H NMR (400 MHZ, D.sub.2O, 298 K) (ppm): 7.80-7.73 (m, 16H), 7.54-7.45 (m, 16H), 6.97-6.63 (m, 10H), 4.79-4.58 (m, 16H), 4.48-4.38 (m, 16H), 4.30-4.25 (t, 2H), 3.86-3.77 (m, 16H), 3.68 (s, 3H), 3.65-3.50 (s, 10H), 3.50-3.42 (m, 32H), 1.46-1.39 (m, 48H), 1.23 (s, 96H), 0.71-0.41 (m, 31H). .sup.13C NMR (600 MHZ, CD.sub.3OD, 298 K) (ppm): 151.45, 136.54, 135.77, 133.44, 133.01, 131.50, 117.47, 85.54, 75.81, 70.92, 63.47, 58.05, 55.57, 33.05, 30.79, 30.46, 25.27, 25.03, 23.72, 14.47, 8.96. HR-MS: [M8Br].sup.8+ m/z determined as 422.8757, [M7Br].sup.7+ m/z determined as 494.5405. A melting point was 162.4 C. to 162.9 C.

Embodiment 2

H.sub.2O.sub.2 Responsiveness of Compound

[0045] A certain amount of compound 1 was dissolved in D.sub.2O (1 mM), and dropwise added with a small amount of hydrogen peroxide to reach a final concentration of 10 mM. Under oxidation conditions, the compound 1 reacted quickly and produced quinone, and the quinone was converted into p-hydroxybenzyl alcohol in water. In this process of change, corresponding proton peaks a, b and c could be observed in .sup.1H NMR, which proved that the compound 1 made a redox response. Detection results were as shown in FIG. 1B.

Embodiment 4

Preparation and Characterization of Nano-Composite of Compound 1 and Plasmid DNA

[0046] A certain amount of compound 1 was dissolved in an HEPES buffer solution (pH=7.4, 10 mM) at a concentration of 2.0 mg/ml, and a plasmid DNA was also diluted to a concentration of 40 g/ml with the HEPES buffer solution at the same time. After the compound was diluted to a corresponding concentration according to a corresponding N/P molar ratio, the compound was quickly added into the plasmid DNA solution according to a volume ratio of 1:1, and vortexed and vibrated for 30 seconds and then allowed to stand for 30 minutes, so as to obtain a series of nano-composites with different N/P.

[0047] Particle size and potential of nano-composite: a proper amount of the series of nano-composites with different N/P prepared above were placed in a sample pool, particle sizes and Zeta potentials of nano-composite solutions with different N/P were measured by using a dynamic light scattering instrument, and each sample was repeatedly experimented for 3 times to obtain an average value. As shown in FIG. 2, the particle sizes of the nano-composites were about 70 nm to 100 nm, and the zeta potentials were 20 mV to 30 mV.

[0048] Gel retardation experiment of nano-composite: 1.0% agarose gel (containing 2 g/ml gelred) was prepared and placed in a 1TAE buffer solution, and 20 L of the nano-composites with different N/P to be tested was added into a gel pore respectively. 20 L of pure plasmid DNA at the same concentration was used as a control, and a voltage of 120 mV was applied to electrophoresis for 30 minutes. After electrophoresis, the gel was placed in a gel imaging system to shoot, and results were as shown in FIG. 3, which showed that the compound 1 wrapped with the DNA could well retard the migration of DNA, thus effectively protecting the DNA during gene delivery.

[0049] Gel retardation experiment of nano-composite under oxidation conditions: a nano-composite with N/P of 15 was incubated in H.sub.2O.sub.2 solutions with different concentrations at 37 C. for 30 minutes, and then, in the same way, 1.0% agarose gel (containing 2 g/ml gelred) was prepared and placed in a 1TAE buffer solution, and 20 L of the incubated nano-composites was added into a gel pore respectively. 20 L of pure plasmid DNA at the same concentration was used as a control, and a voltage of 120 mV was applied to electrophoresis for 30 minutes. After electrophoresis, the gel was placed in a gel imaging system to shoot, and results were as shown in FIG. 4, which showed that the nano-composites could effectively release the DNA under oxidation conditions.

[0050] Changes of particle size and potential of nano-composite under oxidation conditions: the nano-composite with N/P of 15 was incubated in H.sub.2O.sub.2 solutions with different concentrations at 37 C. for 30 minutes, a proper amount of the incubated nano-composites were placed in a sample pool, particle sizes of nano-composite solutions with different N/P were measured by using a dynamic light scattering instrument, and each sample was repeatedly experimented for 3 times to obtain an average value. The nano-composite with N/P of 15 was incubated in 1.0 mM H.sub.2O.sub.2 at 37 C., a proper amount of sample was taken in different time points and placed in a sample pool, point positions were measured by using a dynamic light scattering instrument, and each sample was repeatedly experimented for 3 times to obtain an average value. Results were as shown in FIG. 5A and FIG. 5B, with the increase of concentration of hydrogen peroxide, the particle size of the nano-composite was gradually increased, and a tight bond was dissociated; and with the extension of time, a surface potential of the nano-composite was changed from positive to negative, and an electrostatic attraction with the negatively charged DNA disappeared.

[0051] Projection electron microscope observation experiment of nano-composite: the nano-composite with N/P of 15 was dropwise added onto a 300-mesh copper mesh, and then negatively stained with phosphotungstic acid. After the liquid was suck-dried with an edge of filter paper, the nano-composite was naturally dried at room temperature, and then the nano-composite on the copper mesh was observed with a transmission electron microscope. As shown in FIG. 6, the compound 1 was combined with the plasmid DNA to form a nearly spherical nano-particle with regular morphology and a particle size of about 80 nm, which was basically consistent with the results of dynamic light scattering detection.

Embodiment 5

[0052] Cytotoxicity experiment of compound 1: the cytotoxicity evaluation of the compound 1 was characterized by a CCK8 kit. Cells were cultured in vectors of compounds 1 at different concentrations, and a traditional polymer gene vector PEI was used as a control. The cells were incubated for 48 hours, and after culture, the culture medium was discarded and a diluted CCK8 reagent was added to continuously incubate the cells for 1 hour to 2 hours. A light absorbance value at a wavelength of 450 nm was measured with a microplate reader, which was compared with that of the control group, and a survival ratio of the cells was calculated. Results were as shown in FIG. 7, with the increase of concentration, the survival rate of the cells under the treatment of PEI was decreased sharply, while the survival rate of the cells under the treatment of the compound 1 was decreased slowly, which was significantly higher than that of the PEI, thus proving that the compound 1 had higher biological safety in the measured concentration range.

Embodiment 6

[0053] Luciferase gene transfection experiment: A549 cells were cultured in a 96-well plate with a cell density of 15000 cells/well and 200 L of culture medium in each well, and then cultured in an incubator with 5% CO.sub.2 and 95% humidity at 37 C. for 24 hours, and then the culture medium was discarded and replaced with a fresh serum-free medium. The compound 1 was complexed with a luciferase gene plasmid to form nano-composites with different N/P, added into the culture medium, and incubated at 37 C. for 4 hours, and then the culture medium wad discarded again and replaced with a fresh culture medium to continue culture for 48 hours. After culture, the culture medium was discarded and added with 20 L of 1 cell lysate, and after lysis for 10 minutes, 5 L of supernatant was taken and added with 20 L of luciferase substrate. A chemiluminescence intensity was measured with a chemiluminescence detector, and a protein concentration was measured with a Bradford protein detection kit. Three repeated wells were measured in parallel for each group of data to obtain an average value, and the chemiluminescence intensity was normalized by the protein concentration to obtain a luminescence intensity per mg of protein (RLU/mg protein). Results were as shown in FIG. 8, and compared with the commonly used non-viral gene vector PEI, the transfection efficiency of the compound 1 was improved by 1 to 2 orders of magnitude, indicating that the compound 1 was a more efficient gene vector.

Embodiment 7

[0054] Green fluorescent protein mRNA transfection experiment: RAW264.7 cells were cultured in a glass-bottom culture dish with a radius of 15 mm, with a cell density of 25000 cells/dish, and added with 1.5 mL of culture medium, and the cells were cultured in an incubator with 5% CO.sub.2 and 95% humidity at 37 C. for 24 hours. Subsequently, the culture medium in the culture dish was replaced with a serum-free medium, and added with a composite of the prepared compound 1 and a green fluorescent protein mRNA to incubate at 37 C. for 4 hours, and then the culture medium was discarded again and replaced with a fresh culture medium to continuously culture for 48 hours. After culture, the expression of the green fluorescent protein was observed with a laser confocal microscope, and at an excitation wavelength of 488 nm and an emission wavelength of 510 nm to 540 nm, all photos were taken under an objective lens of 10 times and a light intensity of shooting for all samples was kept the same. Results were as shown in FIG. 9, and compared with the PEI, the compound 1 loaded with the mRNA could realize higher transfection efficiency.

Embodiment 8

[0055] Cell transfection experiment of nano-composite under oxidation conditions: A549 cells were cultured in a 96-well plate, with a cell density of 15000 cells/well and 200 L of culture medium in each well, and cultured in an incubator with 5% CO.sub.2 and 95% humidity at 37 C. for 24 hour. The culture medium was discarded and replaced with a fresh culture medium, and the culture medium was added with H.sub.2O.sub.2 to prepare high-oxidation environment media at different concentrations (5 M, 10 M, 20 M, 50 M and 100 M) to simulate a high oxidation microenvironment of tumors. A compound 1 and a luciferase gene plasmid were added for combination to form nano-composites with different N/P, and the nano-composites were added into the culture medium to incubate at 37 C. for 4 hours. The culture medium was discarded again and replaced with a fresh culture medium to continuously culture for 48 hours. After culture, the culture medium was discarded and added with 20 L of 1 cell lysate, and after lysis for 10 minutes, 5 L of supernatant was taken and added with 20 L of luciferase substrate. A chemiluminescence intensity was measured with a chemiluminescence detector, and a protein concentration was measured with a Bradford protein detection kit. Three repeated wells were measured in parallel for each group of data to obtain an average value, and the chemiluminescence intensity was normalized by the protein concentration to obtain a luminescence intensity per mg of protein (RLU/mg protein). Results were as shown in FIG. 10, which showed that the expression efficiency of the plasmid DNA loaded by the compound 1 was improved in the oxidation environment.