A gene carrier based on cationic porphyrin covalent organic framework, a preparation method thereof and application thereof

Abstract

The application provides a gene carrier based on a cationic porphyrin covalent organic framework, which is prepared from a porphyrin monomer shown in formula (I) and a cationic monomer shown in formula (II). The application solves the problem that a porphyrin small molecule is prone to aggregation, active oxygen and heat generated are reduced, and the treatment effect is affected by introducing a stable framework structure of a COF. Meanwhile, the COF nanoparticles prepared in the application can carry a gene drug and have a good gene silencing effect. The COF nanoparticles or the COF nanoparticles carrying a gene drug provided in the application can be efficiently accumulated at a tumor site through an EPR effect, and have good photoacoustic imaging, gene therapy and photothermal photodynamic therapy functions, and the gene combined photothermal photodynamic therapy guided by photoacoustic imaging can effectively inhibit the growth of a tumor.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, in particular to a gene carrier based on cationic porphyrin covalent organic framework, a preparation method thereof and application thereof. BACKGROUND

[0002] Gene therapy is to deliver therapeutic genes such as DNA, mRNA, siRNA, etc. into cells to exert its biological effect through gene expression, thereby directly treating diseases at the gene level. Gene therapy has always been highly expected by researchers, and after fully understanding the genetic and molecular basis of diseases, gene therapy is expected to cure almost all diseases. However, the existing gene carriers still have some defects, and better gene carriers still need to be developed (see K. Kostarelos, A. D. Miller. Chemical Society Reviews, 2005, 34: 970-994.). For example, adenovirus vectors have potential mutations, gene recombination and immunogenicity; and most cationic materials can be toxic and can harm the body; and the stability of many existing liposome materials still needs to be improved. Therefore, it is still very necessary to develop efficient, non-toxic and clinically applicable drug delivery technology.

[0003] Compared with general small molecule chemotherapy drugs, phototherapy can overcome cell resistance and precisely treat tumor sites. Porphyrin, as a good photosensitizer and photothermal agent, can simultaneously generate reactive oxygen species and heat under single-wavelength light, avoiding the damage to normal cells caused by the extension of treatment time or the increase of laser power due to the insufficient effect of photothermal or photodynamic alone, so it has always been concerned in the field of phototherapy. However, porphyrin small molecules are prone to aggregation, which reduces the generation of reactive oxygen species and heat, affecting the treatment effect. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a gene carrier based on cationic porphyrin covalent organic framework, a preparation method thereof and application thereof. The gene carrier provided by the present application contains a porphyrin structure and can effectively carry siRNA, and has good photoacoustic imaging, gene therapy and photothermal and photodynamic therapy functions.

[0005] The present application provides a gene carrier based on cationic porphyrin covalent organic framework, which is prepared from a porphyrin monomer represented by formula (I) and a cationic monomer represented by formula (II):

[0006]

[0007] In formula (I), R is selected from H or C1-C6 alkyl; Ar is selected from substituted or unsubstituted phenyl, biphenyl or naphthyl.

[0008]

[0009] In formula (II), 1≤n≤5, X is halogen; R1 and R2 are independently selected from hydrogen or C1-C6 alkyl.

[0010] The present application prepares a covalent organic framework (COF) by reacting a porphyrin monomer shown in formula (I) and a cationic monomer shown in formula (II), solves the problem that porphyrin small molecules are prone to aggregation, resulting in a decrease in active oxygen and heat, and affecting the treatment effect, by introducing the stable framework structure of COF. The COF synthesized from the porphyrin monomer and the cationic monomer has a long-range ordered structure and stable crystalline state, can position and control organic molecules in two or three dimensions, avoids the contact between small molecules due to π-π interaction or hydrophobic interaction, and further improves the photothermal and photodynamic treatment capacity. Moreover, the high crystallinity and long-range ordered structure of COF make its morphology not easily affected when it is compounded with a gene carrier; the chemical stability of COF is good, the modular synthesis method can accurately regulate the structure of COF at the molecular level, and the diversity of monomers is conducive to the construction of multifunctional nanocarriers; the specific surface area of COF is high, and the porous structure is also conducive to the loading of nucleic acids. At the same time, the COF nanoparticles prepared in the present application can load gene drugs and have good gene silencing effect. The COF nanoparticles or the COF nanoparticles loaded with gene drugs provided in the present application can be efficiently accumulated at the tumor site through the enhanced permeability and retention effect (EPR, Enhanced Permeability and Retention Effect), have good photoacoustic imaging, gene therapy and photothermal photodynamic therapy functions, and the photoacoustic imaging guided gene combined photothermal photodynamic therapy can effectively inhibit the growth of tumors.

[0011] The present application also provides a preparation method of the gene carrier.

[0012] The porphyrin monomer shown in formula (I) and the cationic monomer shown in formula (II) are reacted to obtain a cationic porphyrin-based covalent organic framework;

[0013] The cationic porphyrin-based covalent organic framework is subjected to molecular etching to obtain a cationic porphyrin-based covalent organic framework gene carrier.

[0014] In the present application, in the porphyrin monomer shown in formula (I), R is preferably selected from H or methyl; Ar is preferably selected from a phenyl group. Specifically, the porphyrin monomer is a 5,10,15,20-tetra(4-aminophenyl)porphyrin monomer, and its structural formula is as follows:

[0015]

[0016] In the cationic monomer of formula (II), n is 1, 2, 3, 4 or 5, X is preferably chlorine or bromine; R1and R2are preferably selected from hydrogen or methyl.

[0017] Specifically, the cationic monomer is selected from any one of formula (II-1) to formula (II-7):

[0018]

[0019] In some specific embodiments, the cationic monomer of formula (II) is prepared by the following method:

[0020] The compound of formula (III) and the compound of formula (IV) are reacted to obtain the cationic monomer of formula (II):

[0021]

[0022] Specifically, the compound of formula (III) and the compound of formula (IV) are reacted in the presence of a catalyst and an organic medium. In some specific embodiments, the catalyst is a basic catalyst, including but not limited to potassium carbonate, sodium carbonate and the like; the organic medium includes but is not limited to N,N-dimethylformamide and the like. In some specific embodiments, the molar ratio of the compound of formula (III), the compound of formula (IV) and the catalyst is preferably 1:(1-5):(1-3), more preferably 1:3:1.5; the volume-molar ratio of the organic medium and the compound of formula (III) is (2-10) mL:1 mol, more preferably 5 mL:1 mol. The reaction is preferably carried out in a thick-walled reaction bottle, preferably under stirring, the temperature of the reaction is preferably 30-50°C, more preferably 40°C; the time of the reaction is preferably 48-96 h, more preferably 72 h. After the reaction is completed, the obtained reaction mixture is preferably subjected to centrifugal treatment, the speed of the centrifugal treatment is preferably 5000-15000 rpm, more preferably 10000 rpm; the time of the centrifugal treatment is preferably 5-15 min, more preferably 10 min. After the centrifugal treatment, the obtained reaction product is dried to obtain the cationic monomer.

[0023] After the cationic monomer is obtained, it is reacted with a porphyrin monomer. Taking the cationic monomer synthesized by 5,10,15,20-tetra(4-aminophenyl)porphyrin monomer and 3-bromopropyltrimethylammonium bromide and 2,5-dihydroxyterephthaldehyde as an example, the reaction process is as follows:

[0024]

[0025] The cationic monomer and the porphyrin monomer are reacted in the presence of a catalyst and an organic medium. In some specific embodiments, the catalyst is an acid catalyst, including but not limited to organic acids such as acetic acid, preferably acetic acid with a concentration of 1-5 mol / L, more preferably 3 mol / L. The organic medium includes but is not limited to o-dichlorobenzene, n-butanol, etc., preferably a mixture of o-dichlorobenzene and n-butanol, and the volume ratio of o-dichlorobenzene to n-butanol is preferably (1-2):(1-2), more preferably 1:1. In some specific embodiments, the molar ratio of the cationic monomer to the porphyrin monomer is preferably (1-3):1, more preferably 2:1. The volume-molar ratio of the organic medium to the porphyrin monomer is preferably (0.25-2) mL:1 mol, more preferably (0.5-1.5) mL:1 mol; the volume-molar ratio of the catalyst to the porphyrin monomer is preferably (100-300) μL:1 mol, more preferably 100 μL:1 mol. In some specific embodiments, the reaction is preferably carried out in a sealed tube, and the reaction time in the sealed tube is preferably 3-7 days, more preferably 3 days; the reaction temperature in the sealed tube is preferably 100-130°C, more preferably 120°C. After the reaction is completed, the obtained reaction mixture is preferably subjected to centrifugal treatment, and the centrifugal treatment rate is preferably 5000-10000 rpm, more preferably 10000 rpm, and the centrifugal treatment time is preferably 5-15 min, more preferably 10 min.

[0026] After obtaining the cationic porphyrin-based covalent organic framework, molecular etching is performed to obtain nanoparticles, which are cationic porphyrin-based covalent organic framework-based gene carriers.

[0027] In some specific embodiments, the molecular etching of the cationic porphyrin-based covalent organic framework specifically includes:

[0028] The cationic porphyrin-based covalent organic framework is mixed with aniline and subjected to ultrasonic treatment, and the power of the ultrasonic treatment is 100-500 W and the time is 20-60 min.

[0029] Specifically, the cationic porphyrin-based covalent organic framework is first mixed with an organic medium, and after the addition of aniline, ultrasonic treatment is performed, preferably in a cell crusher. In some specific embodiments, the power of the ultrasonic treatment is preferably 100-500 W, more preferably 200-400 W; the time of the ultrasonic treatment is preferably 20-60 min, more preferably 25-50 min, more preferably 30 min. In some specific embodiments, the molar ratio of aniline to the imine bond of COF is preferably (0.015-0.5):1, more preferably 0.02:1.

[0030] After the ultrasonic treatment, the obtained reaction mixture is subjected to centrifugal treatment and washed with ethanol, the rate of the centrifugal treatment is preferably 5000-10000 rpm, more preferably 10000 rpm. The time of the centrifugal treatment is preferably 5-30 min, more preferably 20 min, and the number of centrifugal washing is 3 times.

[0031] In some specific embodiments, the ultrasonic treatment is repeated, and finally subjected to centrifugal treatment, the rate of the centrifugal treatment is preferably 5000-10000 rpm, more preferably 10000 rpm. The time of the centrifugal treatment is preferably 5-30 min, more preferably 20 min. After the ultrasonic dispersion, the gene carrier based on cationic porphyrin covalent organic framework is obtained.

[0032] In some specific embodiments, the particle size of the gene carrier based on cationic porphyrin covalent organic framework is preferably 10-300 nm, more preferably 20-250 nm, and most preferably 50-200 nm.

[0033] The gene carrier provided by the present application contains a porphyrin structure, can generate reactive oxygen species and heat at the same time under single wavelength light irradiation, and can be used for killing tumor cells. Based on this, the present application provides the use of the gene carrier in the preparation of an anti-tumor drug.

[0034] The gene carrier prepared by the present application is suitable for accumulating in the tumor site through the EPR effect, can carry a gene drug, and has good gene silencing effect. Based on this, the present application provides an anti-tumor drug, which comprises the gene carrier and the gene drug loaded on the gene carrier. In some specific embodiments, the gene drug can be small interfering RNA used for gene silencing, including but not limited to siPD-L1. In some specific embodiments, the mass ratio of the gene drug to the gene carrier is preferably 1:10-50, more preferably (20-40):1, and most preferably 40:1.

[0035] The present application prepares a covalent organic framework (COF) by reacting a porphyrin monomer shown in formula (I) and a cationic monomer shown in formula (II), solves the problem that the active oxygen and heat generated by the porphyrin monomer are reduced due to the aggregation of the porphyrin monomer itself, and the treatment effect is affected, by introducing the stable framework structure of the COF. The COF synthesized by the porphyrin monomer and the cationic monomer has a long-range ordered structure and a stable crystalline state, can position and control organic molecules in two dimensions or three dimensions, avoids the contact between small molecules due to the π-π interaction or hydrophobic interaction, and further improves the photothermal and photodynamic treatment capacity. Meanwhile, the COF nanoparticles prepared in the present application can carry a gene drug, and have a good gene silencing effect. The COF nanoparticles or the COF nanoparticles carrying a gene drug provided in the present application can be efficiently accumulated at a tumor site through an EPR effect, have good photoacoustic imaging, gene therapy and photothermal and photodynamic therapy functions, and the photoacoustic imaging guided gene combined photothermal and photodynamic therapy can effectively inhibit the growth of a tumor. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 XRD pattern of the cationic porphyrin-based covalent organic framework gene carrier obtained in Example 1;

[0037] Figure 2 Scanning electron microscope photo of the cationic porphyrin-based covalent organic framework gene carrier obtained in Example 1;

[0038] Figure 3 Photoacoustic signal of the cationic porphyrin-based covalent organic framework gene carrier obtained in Example 1 at different times at a tumor site;

[0039] Figure 4 Tumor weight of a mouse after treatment of the cationic porphyrin-based covalent organic framework gene carrier obtained in Example 1. DETAILED DESCRIPTION

[0040] The technical solutions of the present application will be described clearly and completely in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0041] The reagents in the following examples are all purchased from the market.

[0042] Example 1

[0043] Into a thick-walled reaction bottle, 58.14 mg of 2,5-dihydroxyterephthaldehyde, 72.45 mg of potassium carbonate and 274.6 mg of 3-bromopropyl trimethylammonium bromide were sequentially added, 5 mL of N,N-dimethylformamide was added, and after nitrogen protection, 40°C oil bath stirring was performed for 3 days. After the reaction was completed, centrifugation was performed at a rate of 10000 rpm for 10 min. After drying in a vacuum oven, the cationic monomer was obtained.

[0044] 23.16 mg of the cationic monomer, 14.85 mg of 5,10,15,20-tetrakis(4- aminophenyl)porphyrin, 0.5 mL of o-dichlorobenzene, 0.5 mL of n-butanol, and 0.1 mL of 3M acetic acid were mixed, the tube was sealed, and the reaction was performed at 120°C for 3 days. After the reaction was completed, centrifugation was performed at a rate of 10000 rpm for 10 min, and COF nanoparticles were obtained.

[0045] 10 mg of the COF nanoparticles were mixed with 10 mL of DMF, 220 μL of aniline was added, and ultrasonic treatment was performed using a cell crusher at a power of 300 W for 30 min. After the ultrasonic treatment was completed, centrifugation was performed and ethanol was used for washing, the centrifugation rate was 10000 rpm, the centrifugation time was 20 min, and the centrifugation washing was performed 3 times. This preparation process was repeated several times. Finally, centrifugation was performed at a rate of 10000 rpm for 20 min. After ultrasonic dispersion, a cationic porphyrin-based covalent organic framework gene carrier was prepared.

[0046] The cationic porphyrin-based covalent organic framework gene carrier obtained in Example 1 was subjected to XRD analysis, and the results are shown in Figure 1 . Figure 1 FIG. 1 is an XRD spectrum of the cationic porphyrin-based covalent organic framework gene carrier obtained in Example 1. The XRD spectrum shows a high-intensity sharp absorption peak, indicating the high crystallinity of the COF, and proving the successful synthesis of the organic framework.

[0047] The cationic porphyrin-based covalent organic framework gene carrier prepared in Example 1 was analyzed by scanning electron microscopy, and the scanning electron microscope image is shown in Figure 2 . Figure 2 FIG. 2 is a scanning electron microscope image of the cationic porphyrin-based covalent organic framework gene carrier obtained in Example 1, and the results show that the average size of the cationic porphyrin-based covalent organic framework gene carrier prepared in Example 1 is about 80 nm.

[0048] Example 2

[0049] In the experiment, a CT26 tumor model was selected, and about 20 g of Balb / C nude mice were used, and 1×10 6The cells were inoculated in the armpit of the mouse at a density, and when the tumor volume reached 200mm 3 When the tumor volume reached 200mm Figure 3 , Figure 3 The photoacoustic signals of the cationic porphyrin-based covalent organic framework gene carrier obtained in Example 1 at different times in the tumor site. The photoacoustic imaging results show that the maximum accumulation is formed in the tumor site 12 hours after tail vein injection, which guides the best treatment time. The tumor site can be treated by light irradiation 12 hours after tail vein injection to achieve the best treatment effect.

[0050] Example 3

[0051] The cationic porphyrin-based covalent organic framework gene carrier prepared in Example 1 and siPD-L1 were mixed at a mass ratio of 40:1 at room temperature, and then uniformly blown and beaten with a pipette gun. After standing at room temperature for 15 min, the siPD-L1-loaded cationic porphyrin-based covalent organic framework gene carrier was obtained.

[0052] Example 4

[0053] The CT26 tumor model was selected for the in vivo treatment experiment:

[0054] A Balb / C mouse with a weight of about 20g was used, and the CT26 cells were inoculated in the armpit of the mouse at a density of 1.0×10 6 The cells were inoculated in the armpit of the mouse at a density, and when the tumor volume reached 100mm 3 When the tumor volume reached 100mm

[0055] PBS group: when the tumor volume reached 100mm 3 PBS buffer was injected into the mouse through the tail vein, and the injection dose was 200μL per administration, and the concentration was 1mg / mL. The drug was given every two days, and a total of 3 times.

[0056] Material group (COF NPs group): When the tumor volume grows to 100 mm 3 The cationic porphyrin-based covalent organic framework gene carrier prepared in Example 1 was injected into the mice through the tail vein when the tumor volume grew to 100 mm, the injection dose was 200 μL per administration, the concentration was 1 mg / mL, and the administration was performed every two days for a total of 3 times.

[0057] Photothermal therapy group (COF NPs+Laser+Vc group): When the tumor volume grows to 100 mm 3 The cationic porphyrin-based covalent organic framework gene carrier prepared in Example 1 was injected into the mice through the tail vein when the tumor volume grew to 100 mm, the injection dose was 200 μL per administration, the concentration was 1 mg / mL, and the administration was performed every two days for a total of 3 times. 25 μL of Vc solution (25 μmol / kg) was injected into the tumor before each administration to consume the reactive oxygen species (ROS) generated by the laser irradiation in the tumor, so as to achieve only photothermal therapy. When the tumor accumulation reached the maximum, a 671 nm laser was used for light irradiation treatment, the power was 1.5 W / cm 2 , and the irradiation time was 10 min.

[0058] Photodynamic therapy group (COF NPs+Laser+Cooling group): When the tumor volume grows to 100 mm 3 The cationic porphyrin-based covalent organic framework gene carrier prepared in Example 1 was injected into the mice through the tail vein when the tumor volume grew to 100 mm, the injection dose was 200 μL per administration, the concentration was 1 mg / mL, and the administration was performed every two days for a total of 3 times. When the tumor accumulation reached the maximum, a 671 nm laser was used for light irradiation treatment, the power was 1.5 W / cm 2 , and the irradiation time was 10 min. The treatment was performed in a cycle mode of 30 s irradiation and 30 s cooling to achieve only photodynamic treatment under the premise of constant temperature.

[0059] Photothermal and photodynamic combined therapy group (COF NPs+Laser group): When the tumor volume grows to 100 mm 3 The cationic porphyrin-based covalent organic framework gene carrier prepared in Example 1 was injected into the mice through the tail vein when the tumor volume grew to 100 mm, the injection dose was 200 μL per administration, the concentration was 1 mg / mL, and the administration was performed every two days for a total of 3 times. When the tumor accumulation reached the maximum, a 671 nm laser was used for light irradiation treatment, the power was 1.5 W / cm 2 , and the irradiation time was 10 min.

[0060] Gene therapy group (COF / siPD-L1 NPs group): When the tumor volume grows to 100 mm 3When the siPD-L1 loaded cationic porphyrin-based covalent organic framework gene carrier prepared in Example 3 was injected into the mice through the tail vein, the injection dose was 200 μL per administration, the concentration was 1 mg / mL, and the administration was performed every two days for a total of 3 times.

[0061] The combined photothermal photodynamic gene therapy group (COF / siPD-L1 NPs+Laser group): when the tumor volume reached 100 mm 3 When the siPD-L1 loaded cationic porphyrin-based covalent organic framework gene carrier prepared in Example 3 was injected into the mice through the tail vein, the injection dose was 200 μL per administration, the concentration was 1 mg / mL, and the administration was performed every two days for a total of 3 times; when the tumor accumulation reached the maximum, a 671 nm laser was used for light treatment, the power was 1.5 W / cm 2 , and the irradiation time was 10 min.

[0062] After 14 days, the tumors of the mice in each group were weighed, and the results are shown in Figure 4 , Figure 4 The tumor weight after treatment of the mice with the cationic porphyrin-based covalent organic framework gene carrier obtained in Example 1. The results show that the pure material has almost no inhibitory effect on the tumor. The photothermal group and the photodynamic group have therapeutic effects, and the combination has better therapeutic effect. The pure gene group has a certain therapeutic effect, and the three combinations achieve the best tumor inhibition effect.

[0063] The siPD-L1 loaded cationic porphyrin-based covalent organic framework gene carrier prepared in Example 3 was used to treat 4T1, B16F10 and HeLa and other tumor models, and the treatment effect was good.

[0064] The above description of the disclosed embodiments enables one of ordinary skill in the art to make or use the application. Various modifications to these embodiments will be readily apparent to those of ordinary skill in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Accordingly, the application is not to be limited to these embodiments shown herein but is to be accorded the broadest scope consistent with the principles and novel features disclosed herein.

Claims

1. A gene vector based on a cationic porphyrin covalent organic framework, prepared from a 5,10,15,20-tetra(4-aminophenyl)porphyrin monomer and a cationic monomer of formula (II): Formula (II); In formula (II), 1≤n≤5, X is a halogen; R1 and R2 are independently selected from hydrogen or C1~C6 alkyl groups; Its preparation method includes the following steps: 5, 10, 15, 20-tetra(4-aminophenyl)porphyrin monomers and the cationic monomers shown in formula (II) were reacted to obtain a cationic porphyrin covalent organic framework. The aforementioned cationic porphyrin covalent organic framework was molecularly etched to obtain a gene vector based on the cationic porphyrin covalent organic framework. The molecular etching based on the cationic porphyrin covalent organic framework specifically includes: The mixture of the cationic porphyrin covalent organic framework and aniline was subjected to ultrasonic treatment, with a power of 100-500 W and a duration of 20-60 min.

2. The gene vector according to claim 1, characterized in that, The cationic monomer is selected from any one of formulas (II-1) to (II-7): Formula (II-1); Equation (II-2); Formula (II-3); Equation (II-4); Equation (II-5); Equation (II-6); Equation (II-7).

3. The gene vector according to claim 1, characterized in that, Its particle size is 10~300 nm.

4. A method for preparing the gene vector according to claim 1, comprising the following steps: 5, 10, 15, 20-tetra(4-aminophenyl)porphyrin monomers and the cationic monomers shown in formula (II) were reacted to obtain a cationic porphyrin covalent organic framework. The aforementioned cationic porphyrin covalent organic framework was molecularly etched to obtain a gene vector based on the cationic porphyrin covalent organic framework. The molecular etching based on the cationic porphyrin covalent organic framework specifically includes: The mixture of the cationic porphyrin covalent organic framework and aniline was subjected to ultrasonic treatment, with a power of 100-500 W and a duration of 20-60 min.

5. The preparation method according to claim 4, characterized in that, The cationic monomer represented by formula (II) is prepared by the following method: The compound shown in formula (III) reacts with the compound shown in formula (IV) to give the cationic monomer shown in formula (II): Formula (III); Formula (IV).

6. The preparation method according to claim 4, characterized in that, The molar ratio of 5, 10, 15, 20-tetra(4-aminophenyl)porphyrin monomer to the cationic monomer shown in formula (II) is 1: (1~3).

7. The use of the gene vector according to any one of claims 1 to 3 in the preparation of antitumor drugs.

8. An antitumor drug, characterized in that, Includes the gene vector as described in any one of claims 1 to 3 and the gene drug loaded on the gene vector.

9. The antitumor drug according to claim 8, characterized in that, The mass ratio of the gene drug to the gene vector is 1:(10~50).

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