Amphiphilic porphyrin derivative as well as preparation method and application thereof

Abstract

The invention discloses an amphiphilic porphyrin derivative and a preparation method and application thereof.The preparation method comprises the steps that hydrogen bromide and hemin serve as raw materials, a porphyrin intermediate is prepared through deferrization and bromination addition, the porphyrin intermediate and diethylene glycol are subjected to etherification and esterification reaction, and the hydrophilic porphyrin derivative is obtained. And finally, carrying out transesterification on the hydrophilic porphyrin derivative and an alcohol compound to obtain the amphiphilic porphyrin derivative. Through the mode, two diethylene glycol hydrophilic side chains and two n-butyl ester hydrophobic side chains are introduced to a porphyrin ring to prepare the amphiphilic porphyrin derivative, so that the amphiphilic porphyrin derivative can be better taken by cells; the preparation method is simple, safe in synthesis operation and convenient in purification step; the porphyrin derivative is small in particle size, has amphipathy, can quickly enter cells, has high photosensitivity under near-infrared light, and has a more excellent photodynamic therapy effect on tumor cells compared with traditional commercially available porphyrin drugs.

Description

Technical Field

[0001] The invention relates to the technical field of porphyrin derivatives, in particular to an amphiphilic porphyrin derivative, a preparation method and application thereof. Background Art

[0002] Photodynamic therapy (PDT) is an emerging treatment strategy that has been gradually applied to clinical practice since the late 1970s. It has excellent targeting and minimal damage to normal tissues. It has developed rapidly in recent decades and has achieved remarkable results. This therapy has been approved by the US Food and Drug Administration (FDA) as another option for treating superficial cancers. The principle is that when the photosensitizer material is injected into the body, it will not work without specific light irradiation, but will produce a large amount of reactive oxygen under specific light irradiation, thereby destroying biological components such as DNA, RNA and protein, effectively killing tumor cells and bacteria, and thus exerting a therapeutic effect. The photodynamic effect is only produced in the area that needs to be treated. This on-demand excitation feature makes photodynamic therapy have good biosafety. In addition, the excitation light used has a low energy density and is only used to activate the photosensitizer, with almost no damage to normal tissue structures. Therefore, compared with traditional treatments such as radiotherapy and chemotherapy, in the face of some difficult-to-treat cancers and infectious diseases caused by drug-resistant bacteria, photodynamic therapy not only has fewer toxic and side effects, but also has significant therapeutic effects and a shorter cycle. Although photodynamic therapy has achieved certain success in clinical applications, it is still subject to certain limitations in the treatment of cancer. The therapeutic effect of photodynamic therapy is related to factors such as the type of photosensitizer, its concentration in the tumor microenvironment, and the time between administration and light irradiation. Some photosensitizers need to reach a certain high concentration to have a therapeutic effect. In addition, they are not easily degraded by the human body, and are easily accumulated and retained in normal tissues for a long time, resulting in skin photosensitivity for a period of time after treatment (see literature: Lucky, SS; Soo, KC; Zhang, Y., Nanoparticles in Photodynamic Therapy. Chem. Rev. 2015, 115, 1990-2042.).

[0003] Porphyrin and its derivatives, as N-heterocyclic compounds widely present in nature, play a key role in natural biological processes and have proved their practicality in biomedical applications, so they are called "pigments of life". The unique macrocyclic structure of porphyrin enables it to show targeting in cancer cells. As a unique molecule with multiple diagnostic and therapeutic functions, porphyrin derivatives have great advantages in the diagnosis and treatment of cancer (see literature: Dolmans, DEJGJ; Fukumura, D.; Jain, RK, Photodynamic therapy for cancer. Nat. Rev. Cancer 2003, 3, 380-387.). Porphyrin materials have good photophysical and photochemical properties, and the light absorption range extends from the ultraviolet region to the near-infrared region. The typical feature of porphyrin materials is that under light, porphyrin can transfer energy to oxygen in the surrounding environment, activate it, and then produce reactive oxygen and play a therapeutic role. More importantly, studies have shown that porphyrins, as the first generation of photosensitizers, not only show strong efficacy in photodynamic therapy, but also have potential in medical imaging such as magnetic resonance (MR) imaging, photoacoustic (PA) imaging, fluorescence (FL) imaging and positron emission tomography (PET). However, the application of porphyrin materials in vivo still faces many challenges, such as poor water solubility, low bioavailability, difficult metabolism and skin phototoxicity, which will hinder its wider application (see literature: Rajora, MA; Lou, JWH; Zheng, G., Advancing porphyrin's biomedical utility supramolecular chemistry. Chem. Soc. Rev 2017, 46, 6433-6469.). Therefore, the modification and performance improvement of porphyrin materials have become an important research topic in the field of photodynamic therapy. Summary of the invention

[0004] The present invention provides an amphiphilic porphyrin derivative, a preparation method and application thereof, which solve the problems of low efficiency, high injection dosage, difficulty in being degraded by the human body, high cost, complex method and difficult operation in the purification or preparation process of porphyrin and other problems existing in the prior art of commercially available porphyrin photosensitizers.

[0005] In order to solve the above technical problems, a technical solution adopted by the present invention is:

[0006] Provided is an amphiphilic porphyrin derivative, the structural formula of the amphiphilic porphyrin derivative is:

[0007]

[0008] In a preferred embodiment of the present invention, the porphyrin ring of the amphiphilic porphyrin derivative is equipped with two diethylene glycol-based hydrophilic side chains and two n-butyl ester-based hydrophobic side chains, and the structural formula of the amphiphilic porphyrin derivative is:

[0009]

[0010] Provided is a method for preparing an amphiphilic porphyrin derivative, comprising the steps of: using hydrogen bromide and hemin chloride as raw materials, obtaining a porphyrin intermediate through deferrification and bromination addition, then subjecting the intermediate to etherification and esterification with diethylene glycol to obtain a hydrophilic porphyrin derivative, and finally subjecting the hydrophilic porphyrin derivative to an ester exchange reaction with an alcohol compound to obtain an amphiphilic porphyrin derivative.

[0011] In a preferred embodiment of the present invention, the alcohol compound is a combination of one or more of methanol, ethanol, n-propanol, n-butanol, n-pentanol, n-hexanol, n-heptanol, n-octanol, n-nonanol, n-decanol, n-undecanol and n-dodecanol.

[0012] Provided is an application of an amphiphilic porphyrin derivative, including: application of the amphiphilic porphyrin derivative to tumor cells.

[0013] In a preferred embodiment of the present invention, the amphiphilic porphyrin derivative is used for HT-29 colon cancer cells.

[0014] In a preferred embodiment of the present invention, the amphiphilic porphyrin derivative is applied to HT-29 colon cancer cells under light irradiation.

[0015] In a preferred embodiment of the present invention, the amphiphilic porphyrin derivative is applied to HT-29 colon cancer cells at a concentration of 2 μM of the amphiphilic porphyrin derivative and a light parameter of 660 nm and 100 mW / cm 2 , under the condition of illumination for 2 to 3 minutes.

[0016] In a preferred embodiment of the present invention, the application of the amphiphilic porphyrin derivative to monitor drug uptake in tumor cells

[0017] The beneficial effects of the present invention are:

[0018] First, two diethylene glycol-based hydrophilic side chains and two n-butyl ester-based hydrophobic side chains were introduced into the porphyrin ring to prepare an amphiphilic porphyrin derivative, which can be better taken up by cells.

[0019] Second, the preparation method is simple, the synthesis operation is safe, and the purification steps are convenient;

[0020] Third, this porphyrin derivative has a small particle size and is amphiphilic. It can quickly enter cells and has high photosensitivity under near-infrared light. Compared with traditional commercially available porphyrin drugs, it has a better photodynamic therapy effect on tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work, among which:

[0022] Figure 1 This is a synthetic route diagram of the amphiphilic porphyrin derivative prepared in Example 1 of the present invention.

[0023] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the amphiphilic porphyrin derivative prepared in Example 1 of the present invention.

[0024] Figure 3 This is the carbon nuclear magnetic resonance spectrum of the amphiphilic porphyrin derivative prepared in Example 1 of the present invention.

[0025] Figure 4 It is the mass spectrum of the amphiphilic porphyrin derivative prepared in Example 1 of the present invention.

[0026] Figure 5 This is the infrared spectrum of the amphiphilic porphyrin derivative prepared in Example 1 of the present invention.

[0027] Figure 6 It is the ultraviolet absorption spectrum of the amphiphilic porphyrin derivative prepared in Example 1 of the present invention.

[0028] Figure 7 This is a particle size spectrum of the amphiphilic porphyrin derivative prepared in Example 1 of the present invention in aqueous solution.

[0029] Figure 8 It is a bar graph showing the cytotoxicity of the amphiphilic porphyrin derivative prepared in Example 1 of the present invention on HT-29 colon cancer cells as the concentration of the amphiphilic porphyrin derivative changes.

[0030] Fig. 9 It is a bar graph showing the change of the fluorescence emission peak intensity of the amphiphilic porphyrin derivative prepared in Example 1 of the present invention at 625 nm in HT-29 colon cancer cells as a function of the concentration of the amphiphilic porphyrin derivative.

[0031] Fig.10It is a bar graph showing the photodynamic therapy effects of the amphiphilic porphyrin derivative prepared in Example 1 of the present invention and some commercially available porphyrins on HT-29 colon cancer cells.

[0032] Fig.11 This is a flow chart showing the photodynamic therapy effect of the amphiphilic porphyrin derivative prepared in Example 1 of the present invention on HT-29 colon cancer cells. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] According to one embodiment of the present application, an amphiphilic porphyrin derivative is provided, and the structural formula of the amphiphilic porphyrin derivative is:

[0035]

[0036] In the examples of this specification, the porphyrin ring of the porphyrin derivative is equipped with two diethylene glycol hydrophilic side chains and two n-butyl ester hydrophobic side chains, and the structural formula of the porphyrin derivative is:

[0037]

[0038] According to one embodiment of the present application, a method for preparing an amphiphilic porphyrin derivative is provided, which is characterized in that it comprises the steps of: using hydrogen bromide and hemin chloride as raw materials, obtaining a porphyrin intermediate by deferrification and bromination addition, then subjecting the intermediate to etherification and esterification with diethylene glycol to obtain a hydrophilic porphyrin derivative, and finally subjecting the hydrophilic porphyrin derivative to an ester exchange reaction with an alcohol compound to obtain an amphiphilic porphyrin derivative.

[0039] In the embodiments of this specification, the mass ratio of the hemin chloride and the hydrogen bromide is not limited by the present invention, as long as the effect of the present invention can be achieved. Optionally, the mass ratio of the hemin chloride and the hydrogen bromide is 1:2.5-15. More preferably, the hydrogen bromide is a hydrogen bromide acetic acid solution, and the mass ratio of hydrogen bromide and acetic acid in the hydrogen bromide acetic acid solution is 1:2-15. The hydrogen bromide is a hydrogen bromide acetic acid solution with a mass fraction of 33%, and the mass fraction ratio of the hemin chloride and the hydrogen bromide acetic acid solution with a mass fraction of 33% is 1:40-100. The present invention uses hydrogen bromide as a raw material, and other substances cannot be used as a substitute. During the bromination reaction, only hydrogen bromide can be used to undergo a substitution reaction (bromine).

[0040] In the embodiments of this specification, the de-ferrification and bromination addition reactions are carried out under anhydrous and oxygen-free conditions and under the protection of an inert gas. Optionally, the inert gas is nitrogen. Light is avoided during the de-ferrification and bromination addition reactions. The reaction temperature of the de-ferrification and bromination addition reactions is room temperature. The reaction duration of the de-ferrification and bromination addition reactions is 16 to 24 hours. After the de-ferrification and bromination addition reactions are completed, a porphyrin intermediate is obtained, and the obtained porphyrin intermediate is also post-treated. The post-treatment specifically includes: distillation under reduced pressure, dissolving with acetone, transferring to anhydrous ether, and vacuum filtration of the precipitated solid. The mass ratio of hemin to the acetone is 1:2.5 to 15. The mass ratio of hemin to the anhydrous ether is 1:100 to 500. The chemical formula of the porphyrin intermediate is:

[0041] The chemical formula of the hydrophilic porphyrin derivative is:

[0042] In the examples of this specification, the etherification and esterification reactions are carried out under anhydrous and oxygen-free conditions and under the protection of an inert gas. Optionally, the inert gas is nitrogen. The etherification and esterification reactions are first ultrasonically reacted and then reacted at room temperature. The duration of the ultrasonic reaction is 0.5 to 3 hours, and the duration of the room temperature reaction is 10 to 40 hours.

[0043] In the embodiments of this specification, after the etherification and esterification reactions are completed, post-treatment is also included. The post-treatment specifically includes: adding a saturated sodium bicarbonate aqueous solution for neutralization reaction, then adding n-butanol and a saturated sodium chloride aqueous solution for extraction, retaining the organic phase, extracting the aqueous phase with n-butanol, combining the organic phases, washing with water, and drying the organic layer with anhydrous sodium sulfate.

[0044] In the embodiments of this specification, the porphyrin intermediate is further subjected to etherification and esterification reaction with diethylene glycol, and concentrated sulfuric acid is added. The concentrated sulfuric acid is added dropwise. Optionally, the mass fraction of the concentrated sulfuric acid is 70-99%, and the mass ratio of the porphyrin intermediate to the concentrated sulfuric acid is 1:1-5. In addition to using concentrated sulfuric acid during the esterification reaction, inorganic acids such as phosphoric acid and formic acid can also be added as catalysts to accelerate the esterification reaction rate.

[0045] In the embodiments of this specification, the mass ratio of the porphyrin intermediate, the diethylene glycol, and the concentrated sulfuric acid is not limited by the present invention, as long as the effect of the present invention can be achieved. Optionally, the mass ratio of the porphyrin intermediate to the diethylene glycol is 1:30-100.

[0046] In the embodiments of this specification, the present invention does not limit the type of the alcohol compound, as long as the effect of the present invention can be achieved. Optionally, the alcohol compound is a combination of one or more of methanol, ethanol, n-propanol, n-butanol, n-pentanol, n-hexanol, n-heptanol, n-octanol, n-nonanol, n-decanol, n-undecyl alcohol and n-dodecyl alcohol.

[0047] In the present embodiment, an ester exchange reaction was carried out, specifically, ethyl acetate and saturated sodium chloride aqueous solution were added for extraction, liquid separation, the organic phase was retained, the aqueous phase was extracted with ethyl acetate again, the organic phases were combined, washed with deionized water, and the organic layer was dried with anhydrous sodium sulfate. Filtered. The filtrate was concentrated under reduced pressure and then separated and purified by silica gel column chromatography [eluent: methanol: ethyl acetate: n-hexane (9:81:10)].

[0048] The present invention can obtain a low-cost, easy-to-prepare amphiphilic porphyrin derivative. The prepared amphiphilic porphyrin derivative can better penetrate the cell membrane and enter the cell to be taken up by the cell because it has both hydrophilicity and hydrophobicity. At the same time, due to the excellent photophysical properties of porphyrin, it can also be used for fluorescent imaging of tumor cells, and the drug uptake in tumor cells can be monitored in real time to determine whether it has successfully entered the cell.

[0049] The amphiphilic porphyrin derivatives of the present invention have a more excellent photodynamic therapy effect. On the one hand, due to their amphiphilicity and moderate particle size, they can improve the cell uptake rate. On the other hand, their cytotoxicity is not strong in the absence of light and low concentration. After incubation for 24 hours at a concentration below 2 μM, the cell survival rate is still above 95%. Under light, the amphiphilic porphyrin derivatives of the present invention can be used at a concentration of 2 μM, light parameters of 660 nm, 100 mW / cm 2 , within 2 to 3 minutes of illumination, it exerts a far superior effect than other commercially available porphyrins (hematoporphyrin (Hp), protoporphyrin (PpIX), hematoporphyrin monomethyl ether (HMME), chlorin e6 (Ce6) and tetrakis (4-carboxyphenyl) porphyrin (TCPP)). Commercially available porphyrins at a concentration of 2μM, 660nm, 100mW / cm 2 , when irradiated for 2 to 3 minutes, it is almost non-toxic to HT-29 colon cancer cells, but the cytotoxicity of the amphiphilic porphyrin derivative of the present invention reaches nearly 80%. The reduction of irradiation time shortens the treatment time, and the reduction of concentration makes the drug more easily metabolized by the human body, reducing the harm of the drug to the human body.

[0050] The present invention broadens the application of amphiphilic porphyrin derivatives in fluorescence detection. Through the fluorescent color discernible by naked eyes, it can quickly detect whether the drug has entered the cell. It can be distinguished without the use of large instruments, and the operation is convenient and the cost is low.

[0051] The technical solutions provided by various embodiments of the present application are described in detail below in conjunction with the accompanying drawings.

[0052] Embodiment 1:

[0053] Provided is a method for preparing an amphiphilic porphyrin derivative. The preparation route is shown in Figure 1 , including the steps of:

[0054] (1) In a nitrogen atmosphere, 3.0276 g of hemin chloride (5 mmol) and 45 ml of 33% hydrogen bromide acetic acid solution (274.8 mmol) were added to a flask, stirred and reacted for about 20 hours at room temperature in the dark, and then concentrated under reduced pressure to remove excess acetic acid. 15 ml of acetone was added to the flask to dissolve the product, and the dissolved product was transferred to 750 ml of anhydrous ether to obtain a purple-black precipitate, which was filtered. The filtered precipitate was dried in a vacuum environment to obtain a porphyrin intermediate. Finally, 2.9624 g of the porphyrin intermediate was obtained with a yield of 90.92%.

[0055] (2) In a nitrogen atmosphere, the porphyrin intermediate obtained above was mixed with 30 ml of diethylene glycol (316.1 mmol). After it was completely dissolved, 0.35 ml of concentrated sulfuric acid was added, and the mixture was reacted in ultrasound for 45 min. Then, it was stirred for 24 h in the dark. After the stirring was completed, 7 ml of saturated sodium bicarbonate solution and excess sulfuric acid were added to the solution. After neutralization, 250 ml of n-butanol and 250 ml of saturated sodium chloride solution were added for extraction. The organic phase was washed three times with deionized water to remove the remaining diethylene glycol. The organic phase after extraction was dried with anhydrous sodium sulfate, and then filtered under reduced pressure to obtain a crude product, which was then purified by silica gel column chromatography (ethyl acetate: methanol = 4: 1). Finally, a reddish brown oily product hydrophilic porphyrin derivative was obtained, with a total of 0.8072 g and a yield of 20.01%.

[0056] (3) In a nitrogen atmosphere, 0.6035 g (0.634 mmol) of the hydrophilic porphyrin derivative prepared above was mixed with 45 ml of n-butanol, and the mixture was placed in a water bath at 60°C to completely dissolve the hydrophilic porphyrin derivative. Then, 0.6241 g of potassium carbonate (4.51 mmol) was added to the solution, and the mixture was stirred for 4 h under light-proof conditions to allow the reaction to be fully achieved. After the stirring was completed, 10 ml of hydrochloric acid solution (24 mmol) was added to the solution to neutralize the remaining potassium carbonate. The neutralized mixture was mixed with 250 ml of ethyl acetate and 250 ml of sodium chloride solution, and extracted. The obtained organic layer was washed with deionized water and then dried over anhydrous sodium sulfate. The dried product was purified by silica gel column chromatography (ethyl acetate: methanol = 9:1) to obtain a reddish brown oily product amphiphilic porphyrin derivative, a total of 0.1575 g, with a yield of 27.98%, and its molecular formula is: C 50 H 70 N 4 O 10 .

[0057] Amphiphilic porphyrin derivatives: 1 H NMR (400 MHz, CDCl 3 ,ppm): δ10.60–10.50(m,2H,meso-H),10.12,10.11(s,2H,meso-H),6.24–6.16(m,2H,O CH ),4.42(t,J=7.8Hz,4H, CH 2 CH 2 COO), 4.12–4.04 (m, 4H, CH 2 CH 2 CH 2 CH 3 ),3.97–3.88,3.87–3.82,3.81–3.75,3.69–3.67,3.64–3.54(m,16H,CHO CH 2 CH 2 O CH 2 CH 2 OH),3.72,3.70(s,6H, CH 3 ),3.67,3.66(s,6H, CH 3 ),3.30(t,J=7.9,4H,CH 2 CH 2 COO), 2.29(d, J = 6.6 Hz, 6H, CH CH 3 ),1.51–1.45(m,4H,CH 2 CH 2 CH 2 CH 3 ),1.23–1.16(m,4H,CH 2 CH 2CH 2 CH 3 ),0.73(t,J=7.4Hz,6H,CH 2 CH 2 CH 2 CH 3 ),-3.70(s,2H,pyrrole-H). 13 C NMR (100 MHz, CDCl 3 ,ppm)δ173.32,173.30,139.72–136.14(16C,C pyrrole),98.61,98.29,96.97,96.43,73.85,73.79,72.54,72.50,72.46 ,72.42,70.86,68.66,64.58,61.85,61.80,37.17,30.59,29.75,29.36,27 .25,25.43,25.38,25.30,22.73,21.90,19.01,14.17,13.59,11.87,11.79 ,11.73,11.66.FTIR(cm-1):3422,3310,2956,2926,2866,1728,1665,1607 1451,1418,1378,1348,1268,1228,1162,1088,1063,988,944,903,887,833,790,741,734,708,692,678.HRMS(ESI,positive):calcd for C 50 H 70 N 4 O 10 [M+H] + :887.5170.Found:887.5175.UV-vis:λ max ,nm:398,503,535,572,624.ElementalAnal.Calcd for C 50 H 70 N 4 O 10 (%): C, 67.70; H, 7.95; N, 6.32. Found: C, 67.63; H, 7.88; N, 6.41.

[0058] like Figure 2 As shown, Figure 2 : is the hydrogen nuclear magnetic resonance spectrum of the amphiphilic porphyrin derivative provided in this embodiment, and all proton-related peaks have been marked in the figure, wherein the chemical shift at -3.70 ppm is the characteristic peak of NH in the porphyrin ring of the amphiphilic porphyrin derivative. Figure 3 As shown, Figure 3 is the carbon NMR spectrum of the amphiphilic porphyrin derivative provided in this embodiment. Figure 4 As shown, Figure 4 is the mass spectrum of the amphiphilic porphyrin derivative provided in this embodiment. Figure 5 As shown, Figure 5 is the infrared spectrum of the amphiphilic porphyrin derivative provided in this embodiment, 3310cm -1 The position is the characteristic stretching vibration peak of the NH of the porphyrin ring of the amphiphilic porphyrin derivative. Figure 6 As shown, Figure 6 : is the ultraviolet absorption spectrum of the amphiphilic porphyrin derivative provided in this embodiment, with a maximum absorption peak at 400nm and four weak absorption Q bands at 500-650nm, which are 502nm, 536nm, 574nm and 625nm respectively. Figure 7 As shown, Figure 7 It is a particle size spectrum of the amphiphilic porphyrin derivative in aqueous solution. The amphiphilic porphyrin derivative can be dissolved in water and has a small particle size.

[0059] Depend on Figure 2 , 3, 4, 5, 6, 7, it can be seen that the structure of the amphiphilic porphyrin derivative is correct.

[0060] Embodiment 2:

[0061] Cytotoxicity and cellular uptake of amphiphilic porphyrin derivatives on HT-29 colon cancer cells. Experimental methods: Prepare cell culture medium (89% RPMI-1640 medium, 10% serum and 1% double antibody) for use; After multiple passages and medium changes, HT-29 cells showed good cell viability; Prepare dimethyl sulfoxide (DMSO) solution of amphiphilic porphyrin derivatives with a concentration of 10mM for use; Prepare cell culture medium containing amphiphilic porphyrin derivatives by calculation, with concentration gradients of 0, 1, 2, 4, 6, 8, 10, 20 and 40μM, respectively, to ensure that the volume ratio of DMSO solution in the culture medium is always less than 0.4% to prevent DMSO from damaging cells and avoid experimental errors.

[0062] 96-well plate seeding method: Take out the cell culture flask containing tumor cells from the 37°C carbon dioxide cell culture incubator, use an inverted microscope to observe whether the cells are in good condition, and then put it in the biosafety cabinet for use. Use a disposable sterile pipette to aspirate the cell culture fluid in the cell culture flask, and add 2-3mL PBS aqueous solution to wash the cell surface. After removing the PBS aqueous solution, use a disposable sterile pipette to aspirate 1.5-2mL of trypsin to digest the cells, place them in a 37°C carbon dioxide cell culture incubator for 5-7 minutes to digest them completely, place them in 3-4ml culture fluid to neutralize the trypsin, centrifuge, and resuspend. The total number of cells measured by the cell counter is approximately 6×10 6 indivual.

[0063] The mixed cell culture medium was taken with a pipette and distributed into a 96-well plate in an 8×6 array, with 200 μL added to each well. The number of cells per well was calculated to be approximately 10^4. 200 μL of PBS aqueous solution was added to the outermost circle of the well plate. Subsequently, the well plate was incubated in a carbon dioxide cell culture incubator at 37°C for 24 hours. After the incubation was completed, the cell culture medium in the well plate was taken with a pipette and washed with 200 μL of PBS aqueous solution. Then, under light-proof conditions, 200 μL of amphiphilic porphyrin derivative cell culture medium of different concentrations was added to each well, and 8 groups of concentration gradients (0, 1, 2, 4, 6, 8, 10, 20 and 40 μM) were set, and each group of concentration gradients contained 6 wells to conduct 6 parallel experiments.

[0064] Then, the well plate was placed in a carbon dioxide cell culture incubator at 37° C. for 24 hours. After the incubation, the cell status of the blank group (0 μM) was observed using an inverted microscope. After confirming that the cells grew to about 80-90%, the CCK-8 cell viability test was performed.

[0065] CCK-8 cell viability test method: Prepare a 10% CCK-8 cell culture solution for use. Use a pipette to draw the cell culture solution in the well plate, and then wash each well with 200 μL PBS aqueous solution. Then, add 100 μL of CCK-8 cell culture solution to each well under light-proof conditions. Place the well plate in a carbon dioxide cell culture incubator at 37°C and incubate for 20 to 30 minutes, and use an enzyme reader to detect the optical density (OD) value at 450nm.

[0066] In order to test the cell uptake of amphiphilic porphyrin derivatives, after measuring the cell viability, use a pipette to remove the excess CCK-8 cell culture medium in the well plate, and wash each well with 200 μL PBS aqueous solution. Subsequently, 200 μL of cell culture medium was added to each well, and finally a microplate reader was used to detect the characteristic fluorescence emission peak intensity of the amphiphilic porphyrin derivative at 625 nm. After the test is completed, the cells on the well plate are imaged with a fluorescence microscope.

[0067] like Figure 8 As shown, Figure 8 : is a bar graph showing the cytotoxicity of the amphiphilic porphyrin derivatives to HT-29 colon cancer cells in this example as the concentration of the amphiphilic porphyrin derivatives changes. Figure 8 As shown, the amphiphilic porphyrin derivatives have low toxicity to HT-29 cells. When the concentration of the amphiphilic porphyrin derivatives is 2 μM, the cell survival rate can reach about 95%.

[0068] like Fig. 9 As shown, Fig. 9 6 is a bar graph showing the change in the fluorescence emission peak intensity of the amphiphilic porphyrin derivative at 625 nm in HT-29 colon cancer cells as a function of the concentration of the amphiphilic porphyrin derivative. The characteristic fluorescence emission peak of the amphiphilic porphyrin derivative at 625 nm is used to semi-quantitatively compare the uptake of the amphiphilic porphyrin derivative by tumor cells. Fig. 9 As shown, the fluorescence intensity of the amphiphilic porphyrin derivatives in HT-29 colon cancer cells increases with the increase of the concentration of the amphiphilic porphyrin derivatives, indicating that the higher the concentration, the higher the fluorescence intensity of the amphiphilic porphyrin derivatives in the tumor cells. It also shows that the amphiphilic porphyrin derivatives can be taken up by HT-29 colon cancer cells, and the uptake increases with the increase of the concentration of the amphiphilic porphyrin derivatives.

[0069] Embodiment 3:

[0070] The photodynamic cytotoxicity of the amphiphilic porphyrin derivatives and other commercially available porphyrins was determined. Fig.10 As shown, Fig.10 The amphiphilic porphyrin derivative obtained in Example 1 and five other commercially available porphyrins (hematoporphyrin (Hp), protoporphyrin (PpIX), hematoporphyrin monomethylether (HMME), chlorin e6 (Ce6) and tetrakis (4-carboxyphenyl) porphyrin (TCPP)). The drug concentration is 2 μM. The uptake time is 3 hours, and the photodynamic cytotoxicity test of the drug on HT-29 colon cancer cells is carried out. The laser parameters are 660nm, 100mW / cm 2 The illumination time was 2 minutes, and 4 parallel samples were tested in each group. Fig.10 As shown, commercially available porphyrins were detected at a concentration of 2 μM, 660 nm, and 100 mW / cm 2 , when irradiated with light for 2 minutes, it has almost no toxicity to HT-29 colon cancer cells, but the cytotoxicity of the amphiphilic porphyrin derivatives of the present invention reaches nearly 80%.

[0071] like Fig.11 As shown, the photodynamic effect of amphiphilic porphyrin derivatives was analyzed by flow cytometry. The drug concentration was 2 μM and incubated in HT-29 colon cancer cells for 3 h. The illumination group was irradiated with 660 nm, 100 mW / cm 2 The cells were irradiated with light for 10 minutes. Under the condition of no drug and no light, the cell survival rate was 99.88%. Under the condition of simple light or single drug addition, the cell survival rate was 98.70% and 98.33%. Under the combined action of amphiphilic porphyrin derivatives and light, the cell survival rate dropped to 24.07%.

[0072] The present invention uses hemin chloride as a raw material, and obtains a novel amphiphilic porphyrin derivative containing two diethylene glycol hydrophilic side chains and two n-butyl ester hydrophobic side chains through deferrification, bromination addition, etherification and esterification reactions, with a total yield of 27.98%. The raw materials are cheap and easy to obtain during the preparation process, and the synthesis operation is simple, convenient and safe. Tests have found that the amphiphilic porphyrin derivative of the present invention is more easily taken up by cells, has red fluorescence, and has strong targeting. The results of cytotoxicity experiments show that when the concentration of the amphiphilic porphyrin derivative is 2 μM, the survival rate of HT-29 colon cancer cells is more than 80%, proving that the cytotoxicity is very low. Under light conditions, it has a cytotoxicity that is superior to other commercially available porphyrins, and is expected to be used in photodynamic therapy, fluorescence imaging and other fields. In summary, the low-cost amphiphilic porphyrin derivative of the present invention has an excellent photodynamic therapy effect, and low concentrations can achieve rapid targeted treatment of tumors. At the same time, as a fluorescent probe, it is expected to achieve more convenient and efficient detection in practical applications, expanding the application of porphyrin derivatives in photodynamic therapy and bioimaging.

[0073] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. An amphiphilic porphyrin derivative, characterized in that: The structural formula of the amphiphilic porphyrin derivative is:

2. The amphiphilic porphyrin derivative according to claim 1, characterized in that The porphyrin ring of the amphiphilic porphyrin derivative is equipped with two diethylene glycol-based hydrophilic side chains and two n-butyl ester-based hydrophobic side chains. The structural formula of the amphiphilic porphyrin derivative is:

3. A method for preparing an amphiphilic porphyrin derivative, characterized in that: The method comprises the following steps: using hydrogen bromide and hemin chloride as raw materials, preparing a porphyrin intermediate through deferrification and bromination addition, then carrying out etherification and esterification reaction with diethylene glycol to obtain a hydrophilic porphyrin derivative, and finally carrying out an ester exchange reaction between the hydrophilic porphyrin derivative and an alcohol compound to obtain an amphiphilic porphyrin derivative.

4. The preparation method according to claim 3, characterized in that: The alcohol compound is a combination of one or more of methanol, ethanol, n-propanol, n-butanol, n-pentanol, n-hexanol, n-heptanol, n-octanol, n-nonanol, n-decanol, n-undecanol and n-dodecanol.

5. An application of an amphiphilic porphyrin derivative, characterized in that: include: Application of the amphiphilic porphyrin derivative to tumor cells.

6. The use of the amphiphilic porphyrin derivative according to claim 5, characterized in that: The amphiphilic porphyrin derivative is used for HT-29 colon cancer cells.

7. The use of the amphiphilic porphyrin derivative according to claim 6, characterized in that: The amphiphilic porphyrin derivative is used on HT-29 colon cancer cells during light irradiation.

8. The use of the amphiphilic porphyrin derivative according to claim 7, characterized in that: The amphiphilic porphyrin derivative is applied to HT-29 colon cancer cells at a concentration of 2 μM of the amphiphilic porphyrin derivative and a light parameter of 660 nm and 100 mW / cm 2 , under the condition of illumination for 2 to 3 minutes.

9. The use of the amphiphilic porphyrin derivative according to claim 5, characterized in that: The amphiphilic porphyrin derivative is used to monitor drug uptake in tumor cells.

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