An amphiphilic porphyrin derivative, a preparation method and application thereof

Abstract

The application discloses an amphiphilic porphyrin derivative, a preparation method and application thereof, and relates to the technical field of medicine, wherein hydrogen bromide and hematin chloride are used as raw materials, a porphyrin intermediate is prepared through deferrization and bromination addition, then etherification and esterification reactions are carried out on the porphyrin intermediate and diethylene glycol to obtain a hydrophilic porphyrin derivative, finally, the hydrophilic porphyrin derivative is subjected to an ester exchange reaction with an alcohol compound to obtain the amphiphilic porphyrin derivative. Through the above method, two diethylene glycol hydrophilic side chains and two n-butyl ester hydrophobic side chains are introduced on the porphyrin ring, and the amphiphilic porphyrin derivative is prepared, so that the porphyrin derivative can be better taken up by cells; the preparation method is simple, the synthesis operation is safe, and the purification step is convenient; the porphyrin derivative has small particle size and has amphiphilicity, can quickly enter cells, has high photosensitivity under near-infrared light, and has more excellent photodynamic therapy effect on tumor cells compared with traditional commercially available porphyrin drugs.

Description

Technical Field

[0001] This invention relates to the field of porphyrin derivative technology, and in particular to an amphiphilic porphyrin derivative, its preparation method and application. Background Technology

[0002] Photodynamic therapy (PDT), as an emerging treatment strategy, has been gradually applied clinically since the late 1970s. With its superior targeting and minimal damage to normal tissues, it has developed rapidly in recent decades and achieved significant results. This therapy has been approved by the U.S. Food and Drug Administration (FDA) as another option for treating superficial cancers. Its principle is that when a photosensitizer is injected into the body, it will not function without specific light irradiation. However, under specific light irradiation, it generates a large amount of reactive oxygen species, which damage biological components such as DNA, RNA, and proteins, effectively killing tumor cells and bacteria, thereby exerting a therapeutic effect. The photodynamic effect is generated only at the site of treatment, and this on-demand activation characteristic makes PDT have good biocompatibility. Furthermore, the excitation light energy density used is low, only used to activate the photosensitizer, with almost no damage to normal tissue structures. Therefore, compared with traditional radiotherapy and chemotherapy, PDT is not only less toxic and side-effect-free, but also has significant therapeutic effects and a shorter treatment cycle for some difficult-to-treat cancers and infectious diseases caused by drug-resistant bacteria. Although photodynamic therapy has achieved some success in clinical applications, its use in cancer treatment remains limited. 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 require sufficiently high concentrations to be effective. Furthermore, they are not easily degraded by the body and tend to accumulate and remain in normal tissues for extended periods, leading to a period of post-treatment skin photosensitivity (see: Lucky, SS; Soo, KC; Zhang, Y., Nanoparticles in Photodynamic Therapy. Chem. Rev. 2015, 115, 1990-2042.).

[0003] Porphyrins and their derivatives, as widely distributed N-heterocyclic compounds in nature, play a crucial role in natural biological processes and have demonstrated their practicality in biomedical applications, thus earning the title of "pigments of life." The unique macrocyclic structure of porphyrins enables them to exhibit targeting in cancer cells. Porphyrin derivatives, as unique molecules with multiple diagnostic and therapeutic functions, offer significant advantages in cancer diagnosis and treatment (see: Dolmans, DEJGJ; Fukumura, D.; Jain, RK, Photodynamic therapy for cancer. Nat. Rev. Cancer 2003, 3, 380-387.). Porphyrin materials possess excellent photophysical and photochemical properties, with light absorption extending from the ultraviolet region to the near-infrared region. A typical characteristic of porphyrin materials is that under light irradiation, porphyrins can transfer energy to oxygen in the surrounding environment, activating it and generating reactive oxygen species to exert therapeutic effects. More importantly, research has shown that porphyrins, as first-generation photosensitizers, not only exhibit powerful efficacy in photodynamic therapy but also show 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, difficulty in metabolism, and skin phototoxicity, which hinder their wider application (see: 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] This invention provides an amphiphilic porphyrin derivative, its preparation method, and its application, solving the problems of low efficiency, high injection dosage, and difficulty in human degradation of existing commercially available porphyrin photosensitizers, as well as the high cost, complex methods, and difficult operation in the purification or preparation process of porphyrins.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] An amphiphilic porphyrin derivative is provided, wherein the structural formula of the amphiphilic porphyrin derivative is as follows:

[0007]

[0008] In a preferred embodiment of the present invention, the amphiphilic porphyrin derivative has two diethylene glycol hydrophilic side chains and two n-butyl ester hydrophobic side chains assembled on its porphyrin ring, and the structural formula of the amphiphilic porphyrin derivative is as follows:

[0009]

[0010] A method for preparing an amphiphilic porphyrin derivative is provided, comprising the following steps: using hydrogen bromide and heme chloride as raw materials, a porphyrin intermediate is prepared by deferrooxidation and bromination addition, followed by etherification and esterification reactions with diethylene glycol to obtain a hydrophilic porphyrin derivative, and finally, the hydrophilic porphyrin derivative is reacted with an alcohol compound by transesterification to obtain the amphiphilic porphyrin derivative.

[0011] In a preferred embodiment of the present invention, the alcohol compound is 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] An application of an amphiphilic porphyrin derivative is provided, including: the application of the amphiphilic porphyrin derivative to tumor cells.

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

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

[0015] In a preferred embodiment of the present invention, the application of the amphiphilic porphyrin derivative to HT-29 colon cancer cells is carried out at a concentration of 2 μM, with optical parameters of 660 nm and 100 mW / cm². 2 Under conditions of 2-3 minutes of light exposure.

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

[0017] The beneficial effects of this invention are:

[0018] First, two diethylene glycol hydrophilic side chains and two n-butyl ester hydrophobic side chains were introduced onto 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, these porphyrin derivatives have small particle size and are amphiphilic, enabling them to quickly enter cells and exhibit high photosensitivity under near-infrared light. Compared to traditional commercially available porphyrin drugs, they have a superior photodynamic therapy effect on tumor cells. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. 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 effort, wherein:

[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 1H NMR spectrum of the amphiphilic porphyrin derivative prepared in Example 1 of this invention.

[0024] Figure 3 This is the carbon NMR spectrum of the amphiphilic porphyrin derivative prepared in Example 1 of this invention.

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

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

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

[0028] Figure 7 This is the particle size distribution of the amphiphilic porphyrin derivative prepared in Example 1 of this invention in an aqueous solution.

[0029] Figure 8 This is a bar chart showing the cytotoxicity of the amphiphilic porphyrin derivative prepared in Example 1 of this invention in HT-29 colon cancer cells as a function of the concentration of the amphiphilic porphyrin derivative.

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

[0031] Figure 10This is a bar chart showing the photodynamic therapy effects of the amphiphilic porphyrin derivatives prepared in Example 1 of this invention and some commercially available porphyrins on HT-29 colon cancer cells.

[0032] Figure 11 This is a flow cytometry diagram showing the photodynamic therapy effect of the amphiphilic porphyrin derivative prepared in Example 1 of this invention on HT-29 colon cancer cells. Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

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

[0035]

[0036] In the embodiments described in this specification, the porphyrin derivative has two diethylene glycol hydrophilic side chains and two n-butyl ester hydrophobic side chains assembled on its porphyrin ring. The structural formula of the porphyrin derivative is as follows:

[0037]

[0038] According to one embodiment of this application, a method for preparing an amphiphilic porphyrin derivative is provided, characterized by the following steps: using hydrogen bromide and heme chloride as raw materials, a porphyrin intermediate is prepared by deferrooxidation and bromination addition, followed by etherification and esterification reactions with diethylene glycol to obtain a hydrophilic porphyrin derivative, and finally, the hydrophilic porphyrin derivative is reacted with an alcohol compound through an ester exchange reaction to obtain the amphiphilic porphyrin derivative.

[0039] In the embodiments of this specification, the mass ratio of heme chloride to 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 heme chloride to hydrogen bromide is 1:2.5 to 15. More preferably, the hydrogen bromide is a hydrobromic acid solution, and the mass ratio of hydrogen bromide to acetic acid in the hydrobromic acid solution is 1:2 to 15. The hydrogen bromide is a 33% hydrobromic acid solution, and the mass ratio of heme chloride to the 33% hydrobromic acid solution is 1:40 to 100. The present invention uses hydrogen bromide as a raw material, and other substances cannot be used as substitutes. Only hydrogen bromide can be used to undergo the substitution reaction (bromination) during the bromination reaction.

[0040] In the embodiments of this specification, the iron removal 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. The iron removal and bromination addition reactions are conducted in the dark. The reaction temperature for the iron removal and bromination addition reactions is room temperature. The reaction duration for the iron removal and bromination addition reactions is 16–24 hours. After the iron removal and bromination addition reactions are completed, a porphyrin intermediate is obtained, and the process further includes post-treatment of the obtained porphyrin intermediate. The post-treatment specifically includes: vacuum distillation, dissolution in acetone, transfer to anhydrous diethyl ether, and vacuum filtration of the precipitated solid. The mass ratio of heme chloride to acetone is 1:2.5–15. The mass ratio of heme chloride to anhydrous diethyl ether is 1:100–500. The chemical formula of the porphyrin intermediate is:

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

[0042] In the embodiments described in 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 performed first by sonication, followed by reaction at room temperature. The duration of the sonication 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, a post-treatment is also included. The post-treatment specifically includes: adding a saturated sodium bicarbonate aqueous solution to neutralize the reaction, then adding n-butanol and a saturated sodium chloride aqueous solution for extraction, retaining the organic phase, extracting the aqueous phase again 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 etherification and esterification reactions of the porphyrin intermediate with diethylene glycol further include the addition of concentrated sulfuric acid. 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 concentrated sulfuric acid, inorganic acids such as phosphoric acid and formic acid can be added as catalysts to accelerate the esterification reaction rate.

[0045] In the embodiments described in 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 desired effect of the present invention can be achieved. Optionally, the mass ratio of the porphyrin intermediate to the diethylene glycol is 1:30 to 100.

[0046] In the embodiments of this specification, the types of alcohol compounds are not limited by the present invention, as long as they can achieve the effects of the present invention. Optionally, the alcohol compounds are one or more combinations of methanol, ethanol, n-propanol, n-butanol, n-pentanol, n-hexanol, n-heptanol, n-octanol, n-nonanol, n-decanol, n-undecanol, and n-dodecanol.

[0047] In this illustrative example, a transesterification reaction was performed. Specifically, ethyl acetate and a saturated sodium chloride aqueous solution were added for extraction, followed by separation. The organic phase was retained, and the aqueous phase was extracted again with ethyl acetate. The organic phases were combined, washed with deionized water, and the organic layer was dried over anhydrous sodium sulfate. The mixture was then filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography [eluent: methanol: ethyl acetate: n-hexane (9:81:10)].

[0048] This invention provides a low-cost, easy-to-prepare amphiphilic porphyrin derivative. Due to its dual hydrophilic and hydrophobic properties, the derived amphiphilic porphyrin derivative can better permeate cell membranes and enter cells for uptake. Furthermore, the excellent photophysical properties of porphyrins allow for fluorescence imaging of tumor cells, enabling real-time monitoring of drug uptake within tumor cells and determining whether the drug has successfully entered the cell.

[0049] The amphiphilic porphyrin derivative of this invention exhibits superior photodynamic therapy effects. On one hand, its amphiphilicity and moderate particle size enhance cellular uptake. On the other hand, it demonstrates low cytotoxicity even in the absence of light and at low concentrations; after 24 hours of incubation at concentrations below 2 μM, cell viability remains above 95%. Under light conditions, the amphiphilic porphyrin derivative of this invention can be used at a concentration of 2 μM, with light parameters of 660 nm and 100 mW / cm². 2 Within 2-3 minutes of light exposure, it exhibits significantly superior efficacy compared to other commercially available porphyrins (hematoporphyrin (Hp), protoporphyrin (PpIX), hematoporphyrin monomethyl ether (HMME), chlorin e6 (Ce6), and tetracarboxyphenylporphyrin (TCPP)). Commercially available porphyrins show superior efficacy at a concentration of 2 μM, 660 nm, and 100 mW / cm². 2 When exposed to light for 2-3 minutes, the drug exhibits almost no toxicity to HT-29 colon cancer cells. However, the amphiphilic porphyrin derivative of this invention achieves cytotoxicity close to 80%. Reducing the light exposure time shortens the treatment period, while lowering the concentration makes the drug more easily metabolized by the body, thus reducing harm to the human body.

[0050] This invention expands the application of amphiphilic porphyrin derivatives in fluorescence detection. The fluorescence color, discernible to the naked eye, allows for rapid detection of drug entry into cells. It eliminates the need for large instruments, making it convenient and cost-effective.

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

[0052] Example 1:

[0053] A method for preparing an amphiphilic porphyrin derivative is provided; the preparation route is described in [reference needed]. Figure 1 The steps include:

[0054] (1) Under a nitrogen atmosphere, 3.0276 g of heme chloride (5 mmol) and 45 ml of 33% acetic acid hydrobromide solution (274.8 mmol) were added to a flask. The mixture was stirred and reacted at room temperature in the dark for about 20 h, 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 diethyl ether to obtain a purplish-black precipitate, which was filtered. The filtered precipitate was dried under vacuum to obtain a porphyrin intermediate. Finally, 2.9624 g of the porphyrin intermediate was obtained, with a yield of 90.92%.

[0055] (2) Under a nitrogen atmosphere, the porphyrin intermediate obtained above was mixed with 30 ml of diethylene glycol (316.1 mmol). After complete dissolution, 0.35 ml of concentrated sulfuric acid was added, and the mixture was sonicated for 45 min, followed by stirring under light-protected conditions for 24 h. After stirring, 7 ml of saturated sodium bicarbonate solution and excess sulfuric acid were added to the solution for neutralization. Then, 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 extracted organic phase was dried with anhydrous sodium sulfate, and the crude product was obtained by vacuum filtration. The crude product was then purified by silica gel column chromatography (ethyl acetate: methanol = 4:1) to finally obtain a reddish-brown oily hydrophilic porphyrin derivative, totaling 0.8072 g, with a yield of 20.01%.

[0056] (3) Under 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 placed in a 60°C water bath to completely dissolve the hydrophilic porphyrin derivative. Then, 0.6241 g (4.51 mmol) of potassium carbonate was added to the solution, and the mixture was stirred for 4 h under light-protected conditions to allow the reaction to proceed fully. After stirring, 10 ml of hydrochloric acid solution (24 mmol) was added to the solution to neutralize the remaining potassium carbonate. The neutralized mixture was then mixed with 250 ml of ethyl acetate and 250 ml of sodium chloride solution, extracted, and the resulting organic layer was washed with deionized water and dried with 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, an amphiphilic porphyrin derivative, totaling 0.1575 g, with a yield of 27.98%. Its molecular formula is: C 50 H 70 N4O 10 .

[0057] Amphiphilic porphyrin derivatives: 1 H NMR (400MHz, CDCl3, 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 = 7.8 Hz, 4H, CH2 CH2COO), 4.12–4.04 (m, 4H, CH2 CH2CH2CH3),3.97–3.88,3.87–3.82,3.81–3.75,3.69–3.67,3.64–3.54(m,16H,CHO CH2CH2 O CH2CH2 OH), 3.72, 3.70 (s, 6H), CH3 ),3.67,3.66(s,6H, CH3 ),3.30(t,J=7.9,4H,CH2 CH2 COO), 2.29 (d, J = 6.6 Hz, 6H, CH CH3 ), 1.51–1.45 (m, 4H, CH2) CH2 CH2CH3),1.23–1.16(m,4H,CH2CH2 CH2 CH3),0.73(t,J=7.4Hz,6H,CH2CH2CH2 CH3 ), -3.70(s,2H,pyrrole-H). 13C NMR (100MHz, CDCl3, 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 N4O 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 N4O 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 This is the 1H NMR spectrum of the amphiphilic porphyrin derivative provided in this embodiment. All proton-related peaks are marked in the figure. The chemical shift at -3.70 ppm is the characteristic NH peak in the porphyrin ring of the amphiphilic porphyrin derivative. Figure 3 As shown, Figure 3 This is the carbon NMR spectrum of the amphiphilic porphyrin derivative provided in this embodiment. Figure 4 As shown, Figure 4 This is the mass spectrum of the amphiphilic porphyrin derivative provided in this embodiment. Figure 5 As shown, Figure 5 This is the infrared spectrum of the amphiphilic porphyrin derivative provided in this embodiment, 3310 cm⁻¹. -1The peak at this location represents the characteristic NH stretching vibration of the porphyrin ring in amphiphilic porphyrin derivatives. (Example:) Figure 6 As shown, Figure 6 This is the UV absorption spectrum of the amphiphilic porphyrin derivative provided in this embodiment. It shows a maximum absorption peak at 400 nm and four weak absorption Q bands in the 500-650 nm range, at 502 nm, 536 nm, 574 nm, and 625 nm. Figure 7 As shown, Figure 7 This is a particle size distribution of amphiphilic porphyrin derivatives in aqueous solution. These amphiphilic porphyrin derivatives are soluble in water and have relatively small particle sizes.

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

[0060] Example 2:

[0061] Cytotoxicity and uptake of amphiphilic porphyrin derivatives on HT-29 colon cancer cells. Experimental methods: Cell culture medium (89% RPMI-1640 medium, 10% serum, and 1% penicillin-dextrin antibiotics) was prepared for use. After multiple passages and medium changes, HT-29 cells exhibited good cell viability. A 10 mM dimethyl sulfoxide (DMSO) solution of the amphiphilic porphyrin derivative was prepared for use. Cell culture media containing the amphiphilic porphyrin derivative were prepared with concentration gradients of 0, 1, 2, 4, 6, 8, 10, 20, and 40 μM, ensuring that the volume ratio of DMSO solution in the culture medium was always less than 0.4% to prevent DMSO from damaging the cells and to avoid experimental errors.

[0062] 96-well plate seeding method: Remove the cell culture flask containing tumor cells from the 37°C CO2 cell culture incubator. Observe the cell condition using an inverted microscope, then place it in a biosafety cabinet for later use. Use a disposable sterile pipette to aspirate the cell culture medium from the flask and add 2-3 mL of PBS aqueous solution to wash the cell surface. After removing the PBS aqueous solution, use a disposable sterile pipette to aspirate 1.5-2 mL of trypsin to digest the cells. Incubate at 37°C in a CO2 cell culture incubator for 5-7 minutes until complete digestion. Add 3-4 mL of culture medium to neutralize the trypsin, centrifuge, and resuspend. The total cell count, measured by a cell counter, is approximately 6 × 10⁶ cells / mL. 6 indivual.

[0063] The mixed cell culture medium was pipetted into 96-well plates (8×6 array), with 200 μL added to each well, resulting in approximately 10^4 cells per well. 200 μL of PBS was added to the outermost ring of the wells. The plates were then incubated at 37°C in a CO2 cell culture incubator for 24 hours. After incubation, the cell culture medium was aspirated from the wells and washed with 200 μL of PBS. Then, under light-protected conditions, 200 μL of amphiphilic porphyrin derivative cell culture medium at different concentrations was added to each well, creating eight concentration gradients (0, 1, 2, 4, 6, 8, 10, 20, and 40 μM), with each gradient comprising six wells for six replicate experiments.

[0064] Next, the well plate was placed in a CO2 cell culture incubator at 37°C for 24 hours. After incubation, the cell status of the blank group (0 μM) was observed using an inverted microscope. Once the cells were confirmed to have grown to about 80-90%, the CCK-8 cell viability test could be performed.

[0065] CCK-8 cell viability assay method: Prepare a 10% CCK-8 cell culture medium for use. Use a pipette to aspirate the cell culture medium from the wells of the plate, then wash each well with 200 μL of PBS aqueous solution. Next, under light-protected conditions, add 100 μL of CCK-8 cell culture medium to each well. Incubate the plate in a 37°C CO2 cell culture incubator for 20–30 minutes, and measure the optical density (OD) value at 450 nm using a microplate reader.

[0066] To test the uptake of the amphiphilic porphyrin derivative by cells, after measuring cell viability, excess CCK-8 cell culture medium in the well plate was aspirated using a pipette, and each well was washed with 200 μL of PBS aqueous solution. Subsequently, 200 μL of cell culture medium was added to each well, and the intensity of the characteristic fluorescence emission peak of the amphiphilic porphyrin derivative at 625 nm was detected using a microplate reader. After the test, fluorescence imaging of the cells in the well plate was performed using a fluorescence microscope.

[0067] like Figure 8 As shown, Figure 8 This is a bar chart showing the cytotoxicity of the amphiphilic porphyrin derivative against HT-29 colon cancer cells in this embodiment as a function of the concentration of the amphiphilic porphyrin derivative. Figure 8 As shown, the amphiphilic porphyrin derivatives exhibit low toxicity to HT-29 cells, and the cell survival rate can still reach about 95% at a concentration of 2 μM.

[0068] like Figure 9 As shown, Figure 9This is a bar graph showing the change in 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 was used to semi-quantitatively compare the uptake of the amphiphilic porphyrin derivative by tumor cells. Figure 9 As shown, the fluorescence intensity of amphiphilic porphyrin derivatives in HT-29 colon cancer cells increases continuously with increasing concentration, indicating that the higher the concentration, the higher the fluorescence intensity of amphiphilic porphyrin derivatives in tumor cells. This also demonstrates that amphiphilic porphyrin derivatives can be taken up by HT-29 colon cancer cells, and the amount taken up increases with increasing concentration.

[0069] Example 3:

[0070] The photodynamic cytotoxicity of amphiphilic porphyrin derivatives and other commercially available porphyrins was determined. For example... Figure 10 As shown, Figure 10 The drug used was the amphiphilic porphyrin derivative obtained in Example 1, along with five other commercially available porphyrins (hematoporphyrin (Hp), protoporphyrin (PpIX), hematoporphyrin monomethylether (HMME), chlorin e6 (Ce6), and tetracarboxyphenyl porphyrin (TCPP)). All drugs were used at a concentration of 2 μM. The uptake time was 3 hours, and photodynamic cytotoxicity testing was performed on HT-29 colon cancer cells. Laser parameters were 660 nm and 100 mW / cm². 2 The light exposure time was 2 minutes, and each group had 4 parallel samples tested. Figure 10 As shown, commercially available porphyrins at a concentration of 2 μM, 660 nm, and 100 mW / cm²... 2 When exposed to light for 2 minutes, it has almost no toxicity to HT-29 colon cancer cells, but the amphiphilic porphyrin derivative of the present invention has a cytotoxicity of nearly 80%.

[0071] like Figure 11 As shown, the photodynamic effects of the amphiphilic porphyrin derivative were analyzed by flow cytometry. The drug concentration was 2 μM, and it was incubated in HT-29 colon cancer cells for 3 h. The light group used 660 nm and 100 mW / cm². 2 The cells were exposed to light for 10 minutes. Under no-drug and no-light conditions, the cell survival rate was 99.88%. Under light-only or drug-only conditions, the cell survival rates were 98.70% and 98.33%, respectively. Under the combined effects of the amphiphilic porphyrin derivative and light, the cell survival rate decreased to 24.07%.

[0072] This invention uses heme chloride as a raw material and, through deferrohydration, bromination, etherification, and esterification reactions, yields a novel amphiphilic porphyrin derivative containing two diethylene glycol hydrophilic side chains and two n-butyl ester hydrophobic side chains, with a total yield of 27.98%. The raw materials are inexpensive and readily available, and the synthesis process is simple, convenient, and safe. Testing revealed that the amphiphilic porphyrin derivative of this invention is more easily taken up by cells, exhibits red fluorescence, and demonstrates strong targeting. Cytotoxicity experiments showed that at a concentration of 2 μM, HT-29 colon cancer cells maintained a survival rate of over 80%, demonstrating very low cytotoxicity. Under light irradiation, it also exhibits superior cytotoxicity compared to other commercially available porphyrins, showing promise for applications in photodynamic therapy, fluorescence imaging, and other fields. In summary, the low-cost amphiphilic porphyrin derivative of this invention possesses excellent photodynamic therapy effects, achieving rapid targeted treatment of tumors even at low concentrations. At the same time, as a fluorescent probe, it is expected to enable more convenient and efficient detection in practical applications, expanding the application of porphyrin derivatives in photodynamic therapy and bioimaging.

[0073] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. An amphiphilic porphyrin derivative, characterized in that, The amphiphilic porphyrin derivative has two diethylene glycol hydrophilic side chains and two n-butyl ester hydrophobic side chains assembled on its porphyrin ring. The structural formula of the amphiphilic porphyrin derivative is as follows: 。

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