CN110642865A

CN110642865A - Application of high-charge cationic porphyrin in preparation of PDT nano photosensitizer

Info

Publication number: CN110642865A
Application number: CN201910864722.4A
Authority: CN
Inventors: 朱莉娜; 楚俊卿; 李永晖
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2019-09-09
Filing date: 2019-09-09
Publication date: 2020-01-03
Anticipated expiration: 2039-09-09
Also published as: CN110642865B

Abstract

The invention discloses an application of high-charge cationic porphyrin in preparing PDT nano photosensitizer, which utilizes eight-charge water-soluble porphyrin compound-octaiodide 5,10,15, 20-tetra {4- [2- [1, 4-dimethyl-4- (2-hydroxyethyl) piperazine-1-yl]Ethoxy radical]Phenyl } porphyrin (OMHEPzEOPP)⁸⁺·8I^‑The (OMHEPzEOPP) is combined with a polypeptide nano-carrier and applied to preparing a nano PDT photosensitizer capable of specifically identifying tumor cells.

Description

Application of high-charge cationic porphyrin in preparation of PDT nano photosensitizer

Technical Field

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

Background

Cancer has been a intractable disease from ancient times to present, and seriously threatens human health and social development. Common cancer treatments are: surgical treatment, chemotherapy, radiotherapy, ultrasound treatment, gene therapy, immunotherapy, magnetic hyperthermia, photothermal therapy, photodynamic therapy, and the like. At present, the traditional methods such as radiotherapy, chemotherapy and the like in the treatment method of cancer have some disadvantages. Not only can cancer tissues and cells be killed in the treatment process, but also normal organs can be disabled so as to cause serious side effects, and the tumor is multidrug-resistant and drug-resistant after long-term administration. However, promising photodynamic therapy does not harm normal tissues because it selectively destroys malignant cells (producing a therapeutic effect only in the illuminated areas). References can be found, for example, in VanS.D., Mashayekhi V., De B.S.H., et al, Oncology Photodynamic Therapy: BasicPrincles, Current Clinical Status and Future Directions [ J ]. cancer, 2017, 9. And has attracted more and more attention because of its advantages of small wound, low toxicity, repeated action, no interference to traditional treatment, etc.

Photodynamic therapy is a highly selective or targeted drug-mechanical combination technology, which is a novel technology generated after infiltration and fusion of clinical medicine, optics and optoelectronics. The clinical research and application of the photodynamic therapy in China have been in the history of nearly thirty years, and a large number of disease species and cases are treated. Its main mechanism of action relies on Photosensitizers (PS), which selectively irradiate the lesion with light of appropriate wavelength, in the presence of oxygen, to generate singlet oxygen through the PS molecule (¹O₂) Or Reactive Oxygen Species (ROS), induce cytotoxicity, leading to cell death and tissue destruction. Various water-soluble cationic porphyrins such as TMPyP4 have been reported to be widely used as photosensitizers, because porphyrin compounds have high light absorption capability and react to release energy under the excitation of high energy to realize electron transfer. The reference can be found in C.Zhou, chem.Commun.2011,47, 2982-. 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 by overcoming the defects, the water-soluble cationic porphyrin photosensitizer can be really and further widely applied to the treatment of cancers and tumors.

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. OMHEPzEOPP has good water solubilityIt is reported to specifically recognize G-quadruplexes in the presence of single-stranded DNA, double-stranded DNA. The literature references may be found in Zhang L.M., cuiY.X., Zhu L.N., Chu J.Q., Kong D.M.C. functional proteins with large side arms as resources light scattering ratio probes for specific identification of nucleic acid G-quatrilexes [ J.M. ]]Nucleic Acids Res, 2019,47:2727-2738, which lays an important foundation for the eight-charge water-soluble cationic porphyrin OMHEPzEOPP as a PDT photosensitizer with a G-quadruplex as a target and is more than four-charge water-soluble cationic porphyrin (TMPipEOPP)⁴⁺·4I^-The combination effect with G-quadruplexes is better. At the same time, by means of a G-quadruplex AS1411 (sequence GGTGGTGGTGGTTGGTGGTGGTGGTGGTGG, molar extinction coefficient of 250800 L.mol)^-1·cm^-1) Can specifically identify nucleolin over-expressed on the surface of tumor cells, so that the compound photosensitizer has certain targeting property. Nanometer material polypeptide is introduced into the photosensitizer, and self-assembly is carried out through coordination and electrostatic non-covalent interaction to form the supermolecule nanometer composite photosensitizer with EPR effect, so that the supermolecule nanometer composite photosensitizer can stably exist and be enriched in tumor cells, and the photodynamic effect of the supermolecule nanometer composite photosensitizer in the tumor cells is further researched.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide application of high-charge cationic porphyrin in preparing PDT nano photosensitizer, wherein the invention utilizes eight-charge water-soluble porphyrin compound, namely

octaiodide

5,10,15, 20-tetra {4- [2- [1, 4-dimethyl-4- (2-hydroxyethyl) piperazin-1-yl]Ethoxy radical]Phenyl } porphyrin (OMHEPzEOPP)⁸⁺·8I^-The (OMHEPzEOPP) is combined with a polypeptide nano-carrier and applied to preparing a nano PDT photosensitizer capable of specifically identifying tumor cells.

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

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

(101) fluorenylmethoxycarbonyl-L-histidine (Fmoc-H) at 1mg/mL and 0.5mM ZnCl₂Self-assembly in aqueous solution to form nano-particlesSpherical structure, designated Fmoc-H/Zn²⁺；

(102) To the Fmoc-H/Zn prepared above²⁺Adding OMHEPzEOPP dissolved in 2M Tris-HCl buffer solution to make the final concentration of OMHEPzEOPP be 1mg/mL to obtain a mixture; mixing the mixture, centrifuging in a centrifuge, removing supernatant, and resuspending with Tris-HCl buffer to obtain resuspension solution as Fmoc-H/Zn²⁺/OMHEPzEOPP；

(103) Dissolving 0.5. mu.M AS1411 in 20mM Tris-HCl buffer, adding Fmoc-H/Zn AS the above-mentioned resuspension²⁺Adding ultrapure water and 20mM KCl solution into the OMHEPzEOPP simultaneously, mixing uniformly, placing the mixture into a centrifugal machine for centrifugation, removing supernatant, and resuspending the mixture by using Tris-HCl buffer solution to finally obtain the supermolecular nano composite photosensitizer which is recorded as Fmoc-H/Zn²⁺/OMHEPzEOPP/AS1411；

(104) The formation of the supramolecular nanocomposite photosensitizer was verified by ultraviolet-visible spectroscopy, Dynamic Light Scattering (DLS) and Zeta potential, transmission electron microscopy.

Further, the rotation speed in the centrifugation in the steps (102) and (103) is 14000r, the centrifugation time is 20min, and the centrifugation is carried out at 4 ℃.

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

the chemical name is as follows: octa-

iodinated

5,10,15, 20-tetrakis {4- [2- [1, 4-dimethyl-4- (2-hydroxyethyl) piperazin-1-yl ] ethoxy ] phenyl } porphyrin.

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

(401) Fmoc-H/Zn of supermolecule nano-composite photosensitizer through cell pair²⁺the/OMHEPzEOPP/AS 1411 carries out the uptake and confocal fluorescence imaging; Fmoc-H/Zn²⁺The OMHEPzEOPP/AS1411 (wherein the OMHEPzEOPP concentration is 0.5 mu M) and HeLa cells are incubated for 1h, 4h, 12h and 24h,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.

(402) Detecting singlet oxygen produced in living cells; 2',7' -dichlorofluoroxanthate diacetate (DCFH-DA) as an indicator of singlet oxygen in living cells; Fmoc-H/Zn²⁺OMHEPzEOPP/AS1411 (wherein OMHEPzEOPP concentration is 0.5. mu.M) was incubated with HeLa cells for 4h, washed 3 times with PBS to remove non-ingested drug, and then washed 3 times with PBS to remove non-ingested DCFH-DA after incubating cells with a serum-free medium containing 1. mu.L DCCFH-DA for 30 min; irradiating with laser with wavelength of 690nm for 5 min; finally fixed with 4% paraformaldehyde for CLSM analysis, excitation wavelength 488 nm.

(403) Cytotoxicity experiments were performed. Inoculate 5X 10 per well in 96-well plates³Incubating HeLa cells for 24 h; removing the original culture medium, and replacing with new culture medium containing Fmoc-H/Zn²⁺OMHEPzEOPP/AS1411 (wherein the OMHEPzEOPP concentration is 0.5. mu.M); incubating in fresh medium for 4 hr, washing with PBS for 3 times, changing fresh medium without drug, irradiating with 650nm and 690nm lasers for 3.5min, and further culturing for 24 hr; then 10. mu.L of MTT (5mg/mL) was added to each well and incubated with the cells for 4h, the medium containing MTT was removed, and 100. mu.L of DMSO was added to each well to dissolve formazan crystals and its absorbance at 490nm was measured with a plate reader.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

1. the invention constructs a supermolecular nano-composite photosensitizer Fmoc-H/Zn through combination and cooperation based on short peptide, porphyrin photosensitizer, coordination between AS1411 and metal zinc ions and electrostatic non-covalent interaction²⁺OMHEPzEOPP/AS 1411. It has low dark toxicity and high phototoxicity to cells; the raw materials and products for preparing the supermolecule nano composite photosensitizer are all substances with good water solubility, good biocompatibility, easy metabolic decomposition, low toxic and side effects, simple preparation method and lower cost.

2. The nano-composite photosensitizer Fmoc-H/Zn obtained by the invention²⁺the/OMHEPzEOPP/AS 1411 has good EPR effect and is easy to penetrate through a tumor cell membrane to enter the interior of a tumor cell. Moreover, the aptamer AS1411 with the surface modified with the tumor marker nucleolin has a remarkable targeting effect on tumor cells and is gathered on the surface of the tumor cells. Under light conditions, exhibit a rather high photo-cytotoxicity, which also demonstrates its potential for good photodynamic therapy.

3. Fmoc-H/Zn prepared in the invention²⁺the/OMHEPzEOPP/AS 1411 is composed of simple amino acid, porphyrin, metal zinc ions and a DNA chain, can be prepared in an aqueous solvent, and is constructed through coordination and supermolecule action, and the method is simple.

4. Fmoc-H/Zn prepared in the invention²⁺the/OMHEPzEOPP/AS 1411 has the size of about 100nm, has a remarkable EPR (retention) effect and is easy to enter the interior of tumor cells.

5. The prepared nano-composite photosensitizer is gradually decomposed into an effective component G-quadruplex/porphyrin under the action of intracellular GSH, and the nano-composite photosensitizer is promoted to permeate from the outside of the membrane to the inside of the membrane so as to reach permeation balance, so that the concentration of the effective component and the existence time of the effective component in the cell are obviously increased.

6. The active ingredient G-quadruplex/porphyrin released from the nano composite photosensitizer has high-efficiency photosensitization activity, including singlet oxygen yield and red shift of exciting light (700 nm), so that the PDT (photodynamic therapy) efficiency is high.

Drawings

FIG. 1 shows a high charge cationic porphyrin (OMHEPzEOPP) of the present invention⁸⁺·8I^-The molecular structure of (1).

FIG. 2 is a molecular structural diagram of fluorenylmethoxycarbonyl-L-histidine (Fmoc-H) in the present invention.

FIG. 3 is Fmoc-H/Zn according to the invention²⁺Dynamic Light Scattering (DLS) plot of/OMHEPzEOPP/AS 1411.

FIGS. 4a to 4d are Fmoc-H/Zn in the present invention, respectively²⁺And the results of confocal fluorescence images of the/OMHEPzEOPP/AS 1411 and HeLa cells after incubation for 1h, 4h, 12h and 24 h.

FIG. 5 shows Fmoc-H/Zn in HeLa cells of the present invention²⁺the/OMHEPzEOPP/AS 1411 produces confocal fluorescence images of singlet oxygen.

FIG. 6 is Fmoc-H/Zn in the present invention²⁺the/OMHEPzEOPP/AS 1411 cell dark toxicity profile.

FIG. 7 is Fmoc-H/Zn in the present invention²⁺Cytotoxicity profile of/OMHEPzEOPP/AS 1411 at 650nm and 690nm excitation.

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.

AS1411[GGTGGTGGTGGTTGTGGTGGTGGTGG]Ordering by Biotechnology (Shanghai) Limited; AS1411 is G-quadruplex DNA, and the molar extinction coefficient is 250800 L.mol^-1·cm^-1。

fluorenylmethoxycarbonyl-L-histidine (Fmoc-H) is an amphiphilic amino acid, ordered by Mecanne Biochemical reagents, Inc., Shanghai, see FIG. 2.

Example 1

Preparation of PDT nano photosensitizer:

(1)Fmoc-H/Zn²⁺the preparation of (1):

dissolving fluorenylmethoxycarbonyl-L-histidine (hereinafter, Fmoc-H) in 50mM HCl to prepare an Fmoc-H solution having a concentration of 10 mg/mL; then, Fmoc-H solution and ZnCl are added₂The solution was added to water to prepare ZnCl at a final concentration of 0.5mM₂And 1mg/mL of an aqueous mixture of Fmoc-H; adjusting the pH value of the mixture to be neutral by adding 1M Tris solution, and self-assembling to form nano spherical particles Fmoc-H/Zn²⁺。

(2)Fmoc-H/Zn²⁺Preparation of/OMHEPzEOPP:

dissolving porphyrin OMHEPzEOPP in a Tris solution with the concentration of 50mg/mL 2M to serve as a stock solution of porphyrin; to the Fmoc-H/Zn prepared above²⁺Adding OMHEPzEOPP dissolved in 2M Tris-HCl buffer solution to make the final concentration of OMHEPzEOPP be 1mg/mL to obtain a mixture;mixing the mixture uniformly, placing the mixture in a centrifuge for centrifugation, removing supernatant, and carrying out heavy suspension by using Tris-HCl buffer solution to obtain heavy suspension, namely the prepared nanosphere particle Fmoc-H/Zn²⁺/OMHEPzEOPP。

Tris is Tris (hydroxymethyl) aminomethane in powder form, soluble in water to make a solution, where the pH is adjusted with HCl, i.e. hydrochloric acid solution, and used as a buffer solution.

(3)Fmoc-H/Zn²⁺Preparation of/OMHEPzEOPP/AS 1411:

dissolving 0.5. mu.M AS1411 in 20mM Tris-HCl buffer, adding Fmoc-H/Zn AS the above-mentioned resuspension²⁺Adding ultrapure water and 20mM KCl solution into the OMHEPzEOPP simultaneously, mixing uniformly, placing the mixture into a centrifugal machine for centrifugation, removing supernatant, and resuspending the mixture by using Tris-HCl buffer solution to finally obtain the PDT nano photosensitizer Fmoc-H/Zn²⁺OMHEPzEOPP/AS 1411. Referring to fig. 3, it was confirmed that the PDT nano photosensitizer has a particle size of about 117 ± 5nm using Dynamic Light Scattering (DLS). Dynamic Light Scattering (DLS) plots were obtained by measuring hydrodynamic size distribution using a Zetasizer Nano ZS (Malvern Instruments, UK).

Example 2

(1) Cellular uptake and confocal microscopy.

(A) And (5) culturing the cells. The incubator is maintained at 37 ℃ and 5% CO₂HeLa cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody.

(B) Adding the nano composite photosensitizer. Fmoc-H/Zn²⁺The cells were incubated with OMHEPzEOPP/AS1411 (in which the OMHEPzEOPP concentration was 0.5. mu.M) and Hela cells for 1h, 4h, 12h, and 24h, and then labeled AS No. 1,2, 3, and 4, respectively, and washed 3 times with PBS to remove the unabsorbed drug, and then fixed with 4% paraformaldehyde for 15 min.

(C) Experimental results were obtained using confocal laser imaging. Confocal laser imaging was performed using an Olympus IX-81 microscope, with 458nm selected as the diode-pumped laser excitation wavelength. Obtaining the nano composite photosensitizer Fmoc-H/Zn²⁺Confocal fluorescence microscopy images of/OMHEPzEOPP/AS 1411 after 1h, 4h, 12h and 24h of co-incubation with HeLa cells. (see FIGS. 4a to 4d)

(2) Singlet oxygen produced in living cells is detected.

(B) Adding a nano-composite photosensitizer and a singlet oxygen indicating probe. Fmoc-H/Zn²⁺OMHEPzEOPP/AS1411 (wherein OMHEPzEOPP concentration is 0.5. mu.M) was incubated with HeLa cells for 4h, washed 3 times with PBS to remove non-ingested drug, and then washed 3 times with PBS to remove non-ingested DCFH-DA after incubating the cells with a serum-free medium containing 1. mu.L of DCFH-DA for 30 min; irradiating with laser with wavelength of 690nm for 5 min; finally, cells were fixed with 4% paraformaldehyde for 15 min.

(C) Experimental results were obtained using confocal laser imaging. Confocal laser imaging was performed using an Olympus IX-81 microscope with an excitation wavelength of 488 nm. Obtaining the nano composite photosensitizer Fmoc-H/Zn²⁺Confocal fluorescence microscopy images of singlet oxygen production in HeLa cells by/OMHEPzEOPP/AS 1411. See FIG. 5, where the excitation wavelength is 690 nm.

(3) And (4) performing cytotoxicity experiments.

(A) And (5) culturing the cells. The incubator is maintained at 37 ℃ and 5% CO₂HeLa cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody. Then, 5X 10 of the seed per well was inoculated in a 96-well plate³HeLa cells were incubated for 24 h.

(B) Cell culture under dark conditions. After 24H incubation, the original medium was removed and replaced with a fresh medium containing Fmoc-H/Zn²⁺OMHEPzEOPP/AS1411 (with OMHEPzEOPP concentrations of 0.05, 0.25, 0.5, 1, 2.5, 5 μ M); after incubation in fresh medium for 4h, the cells were washed 3 times with PBS and incubated for 24 h.

(C) Cell culture under light conditions. After 24H incubation, the original medium was removed and replaced with a fresh medium containing Fmoc-H/Zn²⁺OMHEPzEOPP/AS1411 (wherein the OMHEPzEOPP concentration is 0.5. mu.M); incubating in fresh medium for 4 hr, washing with PBS 3 times, changing fresh medium without drug, irradiating with 650nm and 690nm lasers for 3.5min, and further culturing for 24 hr.

(D) Cytotoxicity is obtained under light and dark conditions by using a microplate reader. The original medium was removed, 10. mu.L of thiazole blue (MTT) (5mg/mL) was added to each well and incubated with the cells for 4h, the medium containing MTT was removed, 100. mu.L of DMSO was added to each well to dissolve formazan crystals, after 15min, absorbance at 490nm was measured using a microplate reader, and the cell survival rate was calculated. Referring to fig. 6 and 7, the concentrations of OMHEPzEOPP in fig. 6 were 0.05, 0.25, 0.5, 1, 2.5, 5 μ M, respectively. In FIG. 7, the concentration of OMHEPzEOPP was 0.5. mu.M.

Example 3

Preparation of a high-charge cationic porphyrin:

the chemical name of the high-charge cationic porphyrin is as follows: octa-iodinated 5,10,15, 20-tetrakis {4- [2- [1, 4-dimethyl-4- (2-hydroxyethyl) piperazin-1-yl ] ethoxy ] phenyl } porphyrin. (abbreviated as OMHEPzEOPP) is shown in figure 1.

(1) Synthesis of 5,10,15, 20-tetrakis [4- (2-bromoethoxy) phenyl ] porphyrin

5,10,15, 20-tetrakis (4-hydroxyphenyl) porphyrin 0.23mM, 36mM K was added to a three-necked flask₂CO₃40mL of N, N-dimethylformamide, and stirring the mixture evenly by magnetic force at normal temperature. Then, under the condition of keeping out of the light, 23mM 1, 2-dibromoethane is diluted by 10mLDMF, and is dropwise added into a three-neck flask through a constant-pressure separating funnel, and the mixture is magnetically stirred uniformly at normal temperature. After the addition was complete, the reaction temperature was adjusted to 60 ℃ and stirred for 8 h. And after the reaction is finished, cooling the temperature to room temperature, carrying out vacuum filtration, and removing potassium carbonate to obtain purple crystals.

(2) Synthesis of 5,10,15, 20-tetrakis {4- [2- [4- (2-hydroxyethyl) piperazin-1-yl ] ethoxy ] phenyl } porphyrin

In a three-neck flask, 20mM N- (2-hydroxyethyl) piperazine, 20mM K₂CO₃40mL of N, N-dimethylformamide, and stirring the mixture evenly by magnetic force at normal temperature. Then under the protection of nitrogen and at normal temperature, 10mL of the solution is dissolved with 0.1mM of 5,10,15, 20-tetra [4- (2-bromoethoxy) phenyl]The solution of porphyrin in N, N-dimethylformamide is added dropwise into a three-neck flask through a constant-pressure separating funnel and stirred uniformly. After the dropwise addition, the reaction temperature was adjusted to 60 ℃ and heated for 72h, and the whole reaction was carried out under nitrogen protection. After the reaction is finished, the temperature is reduced to room temperature, and the pressure is reducedSuction filtration is carried out, and potassium carbonate is removed to obtain purple solid.

(3) Synthesis of 5,10,15, 20-tetrakis {4- [2- [1, 4-dimethyl-4- (2-hydroxyethyl) piperazin-1-yl ] ethoxy ] phenyl } porphyrin [1] octaiodide

Into a three-necked flask was added 0.046mM of 5,10,15, 20-tetrakis {4- [2- [4- (2-hydroxyethyl) piperazin-1-yl group]Ethoxy radical]Phenyl } porphyrin, 35mL CHCl₃And stirring uniformly. Under the condition of keeping out of light, 0.16M CH₃I with 5mL CHCl₃After dispersion, the mixture is added into a three-mouth bottle dropwise through a constant-pressure separating funnel, the dropping speed is controlled to be about 5s one drop, and the mixture is stirred and mixed uniformly by magnetic force at normal temperature. After the dropwise addition, the reaction temperature is adjusted to 40 ℃ for 24 hours, and the reaction process needs to be protected from light and nitrogen. After the reaction is finished, carrying out vacuum filtration to obtain a filter cake, and obtaining a mauve solid.

The present invention is not limited to the above-described embodiments. The foregoing description of the specific embodiments is intended to describe and illustrate the technical solutions of the present invention, and the above specific embodiments are merely illustrative and not restrictive. Those skilled in the art can make many changes and modifications to the invention without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for preparing PDT nano photosensitizer by using high-charge cationic porphyrin is characterized by comprising the following steps:

(101) fluorenylmethoxycarbonyl-L-histidine (Fmoc-H) at 1mg/mL and 0.5mM ZnCl₂Self-assembly in aqueous solution to form a nanospherical structure, denoted Fmoc-H/Zn²⁺；

(103) Dissolve 0.5. mu.M AS1411 in 20mM TrThe above-mentioned heavy suspension Fmoc-H/Zn was added to is-HCl buffer²⁺Adding ultrapure water and 20mM KCl solution into the OMHEPzEOPP simultaneously, mixing uniformly, placing the mixture into a centrifugal machine for centrifugation, removing supernatant, and resuspending the mixture by using Tris-HCl buffer solution to finally obtain the supermolecular nano composite photosensitizer which is recorded as Fmoc-H/Zn²⁺/OMHEPzEOPP/AS1411；

2. A method of preparing PDT nano photosensitizer with high charge cationic porphyrin according to claim 1, wherein the centrifugation in steps (102) and (103) is carried out at 14000r rotation speed and 20min rotation time, and at 4 ℃.

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

the chemical name is as follows: octa-iodinated 5,10,15, 20-tetrakis {4- [2- [1, 4-dimethyl-4- (2-hydroxyethyl) piperazin-1-yl ] ethoxy ] phenyl } porphyrin.

4. An application experiment method of an eight-charge water-soluble porphyrin compound in the preparation of PDT nano photosensitizer is characterized by comprising the following steps:

(401) Fmoc-H/Zn of supermolecule nano-composite photosensitizer through cell pair²⁺the/OMHEPzEOPP/AS 1411 carries out the uptake and confocal fluorescence imaging;

(402) detecting singlet oxygen produced in living cells;

(403) cytotoxicity experiments were performed.

5. An eight-charge water-soluble porphyrin compound as defined in claim 4An application experiment method in preparing PDT nano photosensitizer is characterized in that the step (401) is as follows: Fmoc-H/Zn²⁺After incubating OMHEPzEOPP/AS1411 (wherein OMHEPzEOPP concentration is 0.5 μ M) with HeLa cells for 1h, 4h, 12h, 24h, 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.

6. An experimental method for the application of eight-charge water-soluble porphyrin compound in the preparation of PDT nano-photosensitizer as claimed in claim 4, wherein the step (402) is as follows: 2',7' -dichlorofluoroxanthate diacetate (DCFH-DA) as an indicator of singlet oxygen in living cells; Fmoc-H/Zn²⁺OMHEPzEOPP/AS1411 (wherein OMHEPzEOPP concentration is 0.5. mu.M) was incubated with HeLa cells for 4h, washed 3 times with PBS to remove non-ingested drug, and then washed 3 times with PBS to remove non-ingested DCFH-DA after incubating the cells with a serum-free medium containing 1. mu.L of DCFH-DA for 30 min; irradiating with laser with wavelength of 690nm for 5 min; finally fixed with 4% paraformaldehyde for CLSM analysis, excitation wavelength 488 nm.

7. An experimental method for the application of eight-charge water-soluble porphyrin compound in the preparation of PDT nano photosensitizer as claimed in claim 4, wherein the step (403) is as follows: inoculate 5X 10 per well in 96-well plates³Incubating HeLa cells for 24 h; removing the original culture medium, and replacing with new culture medium containing Fmoc-H/Zn²⁺OMHEPzEOPP/AS1411 (wherein the OMHEPzEOPP concentration is 0.5. mu.M); incubating in fresh medium for 4 hr, washing with PBS for 3 times, changing fresh medium without drug, irradiating with 650nm and 690nm lasers for 3.5min, and further culturing for 24 hr; then 10. mu.L of MTT (5mg/mL) was added to each well and incubated with the cells for 4h, the medium containing MTT was removed, and 100. mu.L of DMSO was added to each well to dissolve formazan crystals and its absorbance at 490nm was measured with a plate reader.