阅读说明：本技术 一种水溶性阳离子卟啉在制备pdt纳米光敏剂中的应用 (Application of water-soluble cationic porphyrin in preparation of PDT nano photosensitizer ) 是由 朱莉娜 楚俊卿 孔德明 王东霞 于 2020-05-25 设计创作，主要内容包括：本发明公开一种水溶性阳离子卟啉在制备PDT纳米光敏剂中的应用,本发明利用水溶性阳离子卟啉化合物——四碘化5,10,15,20-四[1-甲基-1-哌啶基乙氧基苯基]卟啉(TMPipEOPP)4<Sup>+</Sup>·4I<Sup>-</Sup>(以下简称TMPipEOPP)与一种新型DNA纳米结构相结合,并应用于制备具有生物兼容性好的可识别肿瘤细胞的纳米PDT光敏剂。(The invention discloses an application of water-soluble cationic porphyrin in preparing PDT nano photosensitizer, which utilizes water-soluble cationic porphyrin compound-tetraiodide 5,10,15, 20-tetra [ 1-methyl-1-piperidyl ethoxy phenyl]Porphyrin (TMPipEOPP)4 + ·4I ‑ (TMPipEOPP for short) is combined with a novel DNA nano structure and is applied to preparing nano PDT photosensitizer which has good biocompatibility and can identify tumor cells.)

1. A method for preparing PDT nano photosensitizer by water-soluble cationic porphyrin, which is characterized by comprising the following steps:

(1) four oligonucleotide sequences A1, A2, P1 and P2 at a concentration of 1. mu.M were added to a mixture containing 25mM MgCl₂And 10mM Tris-HCl buffer solution (pH 7.0),forming a mixture A; placing mixture A in gene amplification apparatus (PCR), heating to 95 deg.C for 5min, and incubating on ice for 10 min; namely preparing a DNA nano lantern;

(2) diluting 2. mu. M P3-KRAS in 10mM Tris-HCl buffer (pH 7.0) and 10mM KCl to form a mixture B; placing the mixture B into a gene amplification instrument, heating to 95 ℃ for 5min, and then quickly cooling to 25 ℃ for incubation for 0.5 h; then, adding 2 mu M of TMPipEOPP into the mixture B, and fully and uniformly mixing to obtain a composite photosensitizer TMPipEOPP/P3-KRAS;

(3) mixing the prepared 1 mu M DNA nano lantern with 2 mu M composite photosensitizer TMPipEOPP/P3-KRAS to form a mixture C; placing the mixture C into a gene amplification instrument, and keeping the mixture C at 37 ℃ for incubation for 1 h; after incubation, 2mM Na was added₂EDTA to form a mixture D, and putting the mixture D into a gene amplification instrument to continuously keep the temperature at 37 ℃ for 1 h; then, centrifuging the obtained mixture D for 15min at the temperature of 4 ℃ and the rotating speed of 14000 rpm; washing with ultrapure water for three times to obtain a nano-composite photosensitizer TMPipEOPP/P3-KRAS-DNA nano lantern, DNA-NPs for short, and suspending the DNA-NPs in 10mM Tris-HCl buffer solution (pH 7.0) for later use;

(4) the formation of the above nanocomposite photosensitizer was verified by ultraviolet-visible spectroscopy, Dynamic Light Scattering (DLS), and Zetapotential, transmission electron microscopy.

2. The application of the water-soluble cationic porphyrin compound in preparing PDT nano photosensitizer is characterized in that the structural formula of the water-soluble cationic porphyrin compound is as follows:

the chemical name is as follows: tetraiodo 5,10,15, 20-tetrakis {4- [2- (1-methyl-1-piperidine) ethoxy ] phenyl } porphyrin, abbreviated as TMPipEOPP.

3. An application experiment method of a water-soluble cationic porphyrin compound in the preparation of PDT nano photosensitizer is characterized by comprising the following steps:

(301) carrying out uptake and confocal fluorescence imaging on the DNA-NPs through the cells;

(302) detecting singlet oxygen produced in living cells;

(303) cytotoxicity experiments were performed.

4. An experimental method for the application of water-soluble cationic porphyrin compound in the preparation of PDT nano photosensitizer as claimed in claim 3, wherein the step (301) is as follows: incubating DNA-NPs (wherein the concentration of TMPipEOPP is 0.5. mu.M) with HeLa cells for 1h, 4h, 8h, washing with PBS 3 times to remove unabsorbed drug, and fixing HeLa cells with 4% paraformaldehyde for 15 min; confocal laser imaging was performed using an Olympus IX-81 microscope, with 458nm selected as the diode-pumped laser excitation wavelength.

5. An experimental method for the application of water-soluble cationic porphyrin compound in the preparation of PDT nano photosensitizer as claimed in claim 3, wherein the step (302) is specifically as follows: 2',7' -dichlorofluoroxanthate diacetate (DCFH-DA) as an indicator of singlet oxygen in HeLa cells; DNA-NPs (wherein the concentration of TMPipEOPP is 0.5. mu.M) were incubated with HeLa cells for 4h, washed 3 times with PBS to remove unabsorbed drugs, and then serum-free medium containing 1. mu.L of DCFH-DA was incubated with HeLa cells for 30min and washed 3 times with PBS to remove unabsorbed DCFH-DA; irradiating with laser with wavelength of 690nm for 5 min; finally fixed with 4% paraformaldehyde for CLSM analysis, excitation wavelength 488 nm.

6. An experimental method for the application of water-soluble cationic porphyrin compound in the preparation of PDT nano photosensitizer as claimed in claim 3, wherein the step (303) is implemented by inoculating 5 × 10 per well in 96-well plate³Incubating HeLa cells for 24 h; removing the original culture medium, and replacing with new culture medium containing DNA-NPs (wherein TMPipEOPP concentration is 0.5 μ M); after incubation for 4h in the novel medium, the cells were washed 3 times with PBS, the medium without drug was replaced and incubated at 650nm andirradiating for 3.5min with 690nm laser, and culturing for 24 hr; then, 10. mu.L of MTT (5mg/mL) was added to each well and incubated with HeLa cells for 4 hours, the medium containing MTT was removed, and 100. mu.L of DMSO was added to each well to dissolve formazan crystals and absorbance at 490nm of the dissolved formazan crystals was measured using a plate reader.

Technical Field

The invention belongs to the fields of photodynamic therapy (PDT), chemical analysis and biochemistry, and particularly relates to application of water-soluble cationic porphyrin in preparation of a PDT nano photosensitizer.

Background

Cancer is a general term for a series of related malignant tumors, which can occur almost anywhere in the human body, and may be related to genetic factors and long-term exposure to carcinogens, often manifesting as significant weight loss, pain, weakness, etc. The specific etiology of cancer is not well defined, but to date, there have been many methods of treating cancer. The following are common: surgical treatment, chemotherapy, radiotherapy, photodynamic treatment and photothermal mechanical treatment. Among them, photodynamic therapy (PDT) is a non-toxic drug or dye therapy administered systemically or locally to a patient with a disease, and when a latent period has elapsed, a specific lesion is irradiated with red visible light of near infrared wavelength in the presence of oxygen, resulting in the production of certain toxic substances, which in turn lead to cell death and tissue destruction. From this, three main components for implementing PDT are: suitable Photosensitizers (PSs), light and oxygen, none of which are acceptable. Due to the specificity and selectivity of PDT, its use as a cancer therapy is particularly attractive because one reactive oxygen species (singlet oxygen) can be generated directly on the lesion, leading to cell destruction. And has become the object of extensive research because of its advantages of small wound, low toxicity, repeated action, no interference with traditional treatment, etc. The good water solubility provides guarantee for the application of the compound in the biological field. However, the photosensitizer applied clinically at present has the defects of low cell uptake, strong dark toxicity, poor targeting, short light wave absorption length (low tissue penetration capacity and reduction of potential treatment of deep tumors) and the like. Only if the defects are overcome, the water-soluble cationic porphyrin photosensitizer can be really further widely applied to the treatment of cancers.

DNA is genetic material that encodes, stores, and transfers biological information, and is also one of four biological macromolecules within biological cells. In recent years, with the rapid development of DNA nanotechnology, a variety of DNA nanostructures (including DNA tetrahedrons, DNA octahedrons, DNA nanotubes, DNA origami, etc.) with delicate designs and precise structural characterization emerge in succession, and this DNA nanotechnology has become an important technical means in the field of self-assembly due to the precise controllability of its nanoscale, and has shown important application prospects in the fields of nanomaterial assembly preparation, nanoreactors, biosensing, controlled drug release, etc. Studies have shown that many DNA nanostructures enter cells easily and have high cellular uptake efficiency. The reference can be found in Li j, Fan c.h., peih, et al, adv.mater, 2013,25, 4386-. Based on the DNA nano-structures, the method provides a wide prospect for developing multifunctional intelligent drug delivery nano-carriers.

At present, various porphyrin compounds such as Ce6 and TMPyP4 have been reported to be widely used as photosensitizers because porphyrin compounds have high light absorption capacity and react under the excitation of high energy to release energy to realize the transfer of electrons. References can be found, for example, in Hua l., Nagao k., chem.rev.2016,116, 6184-6261. Due to the fact that the planar sizes of a porphine ring and a G-tetrad of the porphyrin compound are matched with each other and the pi-pi stacking effect exists, the porphyrin can promote the formation of the G-tetrad and can generate a stabilizing effect on the G-tetrad. A water-soluble cationic porphyrin, TMPipEOPP, has been reported to specifically recognize the G-quadruplex in the presence of single-stranded DNA and double-stranded DNA. The references can be found in Zhu l. -n., Zhao s. -j., Wu b., et al, PloS One,2012,7, e 35586. Thus, the method lays an important foundation for the water-soluble cationic porphyrin TMPiPEOPP as a PDT photosensitizer taking G-quadruplex as a target spot.

Disclosure of Invention

The invention aims to overcome the defects of passive targeting, poor biocompatibility and the like existing in the existing photosensitizer, and provides a method for utilizing a water-soluble cationic porphyrin compound, namely 5,10,15, 20-tetra {4- [2- (1-methyl-1-piperidine) ethoxy]Phenyl } porphyrin (TMPiPEOPP for short) is combined with DNA nano lantern (DNA nano structure), and is applied to preparing nano PDT photosensitizer which has good biocompatibility and can identify tumor cells. The invention utilizes G-quadruplex KRAS (with the sequence of AGGGCGGTGTGGGAAGAGGGAAGAGGGGGAGG and the molar extinction coefficient of 341000 L.mol)^-1·cm^-1) The specific combination of the two forms the TMPipEOPP/KRAS composite photosensitizer, and the photosensitizer has certain targeting property. Introducing DNA nano structure into the composite photosensitizer, extending 25 bases (sequence: TTTTTGTTTTTGTTTTTTTTTTTTT) from one section of KRAS sequence to become P3-KRAS, and performing base complementary pairing,the sequence extended from P3-KRAS can be complemented, paired and self-assembled with the sequence extended from the DNA nanostructure to form a novel nano-composite photosensitizer with EPR effect, so that the supramolecular nano-composite photosensitizer can stably exist and be enriched in tumor cells, and the photodynamic effect of the supramolecular nano-composite photosensitizer in the tumor cells is further researched.

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

a method for preparing PDT nano photosensitizer by water-soluble cationic porphyrin comprises the following steps:

(1) four oligonucleotide sequences A1, A2, P1 and P2 at a concentration of 1. mu.M were added to a mixture containing 25mM MgCl₂And 10mM Tris-HCl buffer (pH 7.0) to form a mixture A; placing mixture A in gene amplification apparatus (PCR), heating to 95 deg.C for 5min, and incubating on ice for 10 min; namely preparing a DNA nano lantern;

(2) diluting 2. mu. M P3-KRAS in 10mM Tris-HCl buffer (pH 7.0) and 10mM KCl to form a mixture B; placing the mixture B into a gene amplification instrument, heating to 95 ℃ for 5min, and then quickly cooling to 25 ℃ for incubation for 0.5 h; then, adding 2 mu M of TMPipEOPP into the mixture B, and fully and uniformly mixing to obtain a composite photosensitizer TMPipEOPP/P3-KRAS;

(3) mixing the prepared 1 mu M DNA nano lantern with 2 mu M composite photosensitizer TMPipEOPP/P3-KRAS to form a mixture C; placing the mixture C into a gene amplification instrument, and keeping the mixture C at 37 ℃ for incubation for 1 h; after incubation, 2mM Na was added₂EDTA to form a mixture D, and putting the mixture D into a gene amplification instrument to continuously keep the temperature at 37 ℃ for 1 h; then, centrifuging the obtained mixture D for 15min at the temperature of 4 ℃ and the rotating speed of 14000 rpm; washing with ultrapure water for three times to obtain a nano-composite photosensitizer TMPipEOPP/P3-KRAS-DNA nano lantern, DNA-NPs for short, and suspending the DNA-NPs in 10mM Tris-HCl buffer solution (pH 7.0) for later use.

(4) The formation of the above nanocomposite photosensitizer was verified by ultraviolet-visible spectroscopy, Dynamic Light Scattering (DLS), and Zetapotential, transmission electron microscopy.

Also provides an application of the water-soluble cationic porphyrin compound in preparing PDT nano photosensitizer, wherein the structural formula of the water-soluble cationic porphyrin compound is as follows:

the chemical name is as follows: tetraiodo 5,10,15, 20-tetrakis {4- [2- (1-methyl-1-piperidine) ethoxy ] phenyl } porphyrin, abbreviated as TMPipEOPP.

An application experiment method of a water-soluble cationic porphyrin compound in the preparation of PDT nano photosensitizer comprises the following steps:

(301) carrying out uptake and confocal fluorescence imaging on the DNA-NPs through the cells;

(302) detecting singlet oxygen produced in living cells;

(303) cytotoxicity experiments were performed.

Further, the step (301) is specifically as follows: incubating DNA-NPs (wherein the concentration of TMPipEOPP is 0.5. mu.M) with HeLa cells for 1h, 4h, 8h, washing with PBS 3 times to remove unabsorbed drug, and fixing cells with 4% paraformaldehyde for 15 min; confocal laser imaging was performed using an Olympus IX-81 microscope, with 458nm selected as the diode-pumped laser excitation wavelength.

Further, the step (302) is specifically as follows: 2',7' -dichlorofluoroxanthate diacetate (DCFH-DA) as an indicator of singlet oxygen in living cells; DNA-NPs (wherein the concentration of TMPipEOPP is 0.5. mu.M) were incubated with HeLa cells for 4h, washed 3 times with PBS to remove unabsorbed drugs, and then serum-free medium containing 1. mu.L of DCFH-DA was incubated with HeLa cells for 30min and washed 3 times with PBS to remove unabsorbed DCFH-DA; irradiating with laser with wavelength of 690nm for 5 min; finally fixed with 4% paraformaldehyde for CLSM analysis, excitation wavelength 488 nm.

Further, step (303) was carried out by seeding each well of a 96-well plate with 5 × 10³Incubating HeLa cells for 24 h; removing the original culture medium, and replacing with new culture medium containing DNA-NPs (wherein TMPipEOPP concentration is 0.5 μ M); in a novel culture mediumAfter incubation for 4h, washing with PBS 3 times, changing new culture medium without drug, irradiating with 650nm and 690nm lasers for 3.5min, and further culturing for 24 h; 10 μ LMTT (5mg/mL) was then added to each well for 4h of incubation with cells, the MTT-containing medium was removed, and 100 μ LDMSO was added to each well to solubilize the formazan crystals and its absorbance at 490nm was measured with a plate reader.

Compared with the prior art, the DNA nano lantern prepared by 4 oligonucleotides and the porphyrin/G-quadruplex compound are assembled by base complementary pairing of a specific partial sequence to form the nano composite photosensitizer DNA-NPs, and the technical scheme of the invention has the beneficial effects that:

1. the invention constructs the nano PDT photosensitizer DNA-NPs based on the combination and cooperation of 5 oligonucleotide chains and porphyrin molecules. It has low dark toxicity and high phototoxicity to cells; the raw materials and products for preparing the nano composite photosensitizer are all substances with good water solubility, good biocompatibility, easy metabolic decomposition, low toxic and side effects, simple preparation method, easy synthesis, high synthesis efficiency and lower cost.

2. The DNA-NPs prepared by the method are composed of 5 oligonucleotides and porphyrin, can be prepared in a water solvent, are constructed through electrostatic interaction and supermolecular interaction, and are simple in method and easy to synthesize.

3. The size of the DNA-NPs prepared by the method is about 100nm, the DNA-NPs have obvious EPR (permeation and retention) effect, and the internalization of the nanoparticles in the tumor cells is easily promoted through endocytosis.

4. The absorption of the window of near-infrared light of the DNA-NPs prepared in the present invention is greatly enhanced, and the porphyrin/G-quadruplex complex can be well assembled on the DNA nanostructure, and the wavelength of the excitation light is red-shifted from the transparent window boundary to the inside (about 700nm), thereby showing excellent PDT efficiency.

5. The DNA-NPs prepared in the invention can show good photosensitive activity without in vivo release, namely, the whole nanoparticles can directly play PDT effect in tumor cells. Under light conditions, exhibit a rather high photo-cytotoxicity, which also demonstrates its potential for good photodynamic therapy.

Drawings

FIG. 1 is a UV-VIS spectrum of the DNA nanocapyrase, TMPipEOPP/P3-KRAS and DNA-NPs of the present invention.

FIG. 2 is a graph of Dynamic Light Scattering (DLS) of DNA-NPs of the present invention.

FIGS. 3a, 3b and 3c are confocal fluorescence images of the DNA-NPs taken up by Hela cells for 1h, 4h and 8h, respectively.

FIG. 4 is a confocal fluorescence image of singlet oxygen production by DNA-NPs in Hela cells according to the present invention. Wherein the excitation wavelength is 690 nm.

FIG. 5 is a graph showing the dark cytotoxicity of DNA-NPs in the present invention.

FIG. 6 is a graph showing cytotoxicity of DNA-NPs of the present invention at two excitation wavelengths.

FIG. 7 is a molecular structure diagram of a cationic water-soluble cationic porphyrin TMPipEOPP according to the present invention.

Detailed Description

The invention is described in further detail below with reference to the figures and specific examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The oligonucleotide sequences used in the present invention are shown in Table 1, and are ordered from Biotechnology engineering (Shanghai) Co., Ltd;