CN118908965A - Synthesis and application of pH activated photosensitizer - Google Patents
Synthesis and application of pH activated photosensitizer Download PDFInfo
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Abstract
The invention belongs to the technical field of biological detection and biological medicine, and particularly relates to synthesis and application of a pH activated photosensitizer. In order to construct an NIR-II activated photosensitizer, the invention takes rhodamine derivatives as raw materials to synthesize the pH activated photosensitizer, and the NIR-II activated photosensitizer does not have fluorescence emission capability and can be activated by illumination after H+ stimulation, so that a large amount of active oxygen and fluorescence emission are generated. Wherein, the generated active oxygen can destroy proteins, DNA and lipid in cells, so that the cells die and the tumor cells are killed; the fluorescence emission can generate stronger fluorescence signals in the NIR-II region, and can carry out NIR-II living imaging on cancerous parts. Therefore, the pH activated photosensitizer can provide a new thought for the design of the activated photosensitizer, and has important reference significance for realizing diagnosis and treatment integration and overcoming the cancer difficulty.
Description
Technical Field
The invention belongs to the technical field of biological detection and biological medicine, and particularly relates to synthesis and application of a pH activated photosensitizer.
Background
Along with the continuous improvement of life quality, health problems are increasingly valued by people. In recent years, cancer has become a focus of medical care. Current methods of treating cancer are mainly radiation therapy, chemotherapy and surgical excision. However, these treatments have more or less drawbacks, which make the treatment result unsatisfactory. Photodynamic therapy (PDT) is a non-invasive diagnosis and treatment method, and has the advantages of small surgical trauma, excellent selectivity, small side effect, capability of cooperating with surgical treatment, capability of eliminating hidden lesions and the like, and is widely applied to the aspect of cancer treatment and is concerned by students.
PDT is a new treatment for diseases based on the interaction of light, photosensitizers and oxygen. Photosensitizers, which are one of the key elements of PDT treatment, are photoactive substances capable of absorbing light energy of a specific wavelength and converting it into chemical energy, thereby initiating a series of chemical reactions. The activated photosensitizer can only generate fluorescent effect and active oxygen under the stimulation of specific biological markers in tumor cells, and can directly kill the tumor cells and reduce toxic and side effects on normal tissues by selectively activating the photosensitizer to control the generation of active oxygen in the tumor cells, so that the tumor cells can be detected more accurately and treated more accurately. Thus, photosensitizers that can be specifically activated by biomarkers in tumors are of great interest.
The wavelength of near infrared light is between visible light and mid infrared light, the wavelength is 700-2500 nm, and the near infrared light is subdivided into different optical windows according to the wavelength. For example, the early near infrared (NIR-I) wavelength is studied in the range of 700-900 nm, and more near infrared (NIR-II) wavelengths are studied in recent years in the range of 900-1700 nm. Compared with the visible light and near infrared first-region imaging, the near infrared second-region imaging has the advantages of small adverse reaction, high imaging speed, low self-biological fluorescence background and the like. Due to the characteristic that the scattering intensity of light decreases exponentially with the increase of wavelength, near infrared two-region imaging has more excellent tissue penetrating capability and spatial resolution compared with visible light and near infrared one-region imaging. Due to the complexity of the tumor microenvironment, the adjuvant effects of single photodynamic therapy treatment on tumor elimination and immunotherapy are often diminished. Therefore, if the advantages of the NIR-II imaging and the activated photosensitizer can be combined to develop the NIR-II activated photosensitizer, the diagnosis and treatment effects of photodynamic therapy are expected to be further enhanced.
In addition, since tumor tissue is more acidic than normal tissue, tumor tissue can serve as a target for pH-activated photosensitizers. In conclusion, if H + can be further utilized as a biomarker in cancer cells to activate the photosensitizer based on the NIR-II activated photosensitizer, the application prospect is wide.
Disclosure of Invention
In order to overcome the defects in the prior art, the rhodamine derivative is used as a raw material to construct the pH activated photosensitizer, and the pH activated photosensitizer is a diagnosis and treatment integrated dosage form, can provide a new strategy for diagnosis and treatment integration of cancer treatment work, and has wide development prospect.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the first aspect of the invention provides a pH activated photosensitizer, which has the following structural formula:
According to a second aspect of the present invention, there is provided a method for preparing the pH-activated photosensitizer according to the first aspect, the method comprising the steps of:
S1, dissolving a compound 2b in acetic acid, adding 2,2' -trithiophene-5-formaldehyde, refluxing at 100-120 ℃ for 8-15 hours, and performing reduced pressure cooling concentration and column chromatography purification after reaction to obtain a compound 2b+3t;
S2, dissolving a compound 2b+3t in dichloromethane, adding NHS and EDC, refluxing and stirring for 4-7 hours at the temperature of 35-50 ℃, adding amantadine and triethylamine, continuously refluxing and stirring for 8-15 hours at the temperature of 35-50 ℃, and concentrating under reduced pressure and purifying by column chromatography after reaction to obtain the pH activated photosensitizer.
The active photosensitizer 1O2 can be generated only in the presence of specific biomarkers, and can reduce damage to normal tissues to the greatest extent. The photosensitizer without heavy metal atoms has the advantages of low toxicity, long triplet state service life, good light stability, low cost and the like. When the fluorescence emission range of the photosensitizer is in the near infrared two region (NIR-II) (900-1700 nm), it can be used for living organism imaging. Compared with visible light and near infrared one-region imaging, the near infrared two-region imaging has the advantages of small adverse reaction, high imaging speed, deep tissue penetration, high image contrast, low self-biological fluorescence background and the like. According to the invention, a thiophene structure is added to the rhodamine derivative through modification of the rhodamine derivative, so that the photosensitizer without heavy metal atoms is synthesized. The synthesized photosensitizer shows good singlet oxygen generating capacity through experimental detection of hairstyles. The photosensitizer is further modified on the basis to obtain the pH activated photosensitizer. By monitoring the change of the fluorescence emission spectrum of the activated photosensitizer, the activated photosensitizer is found to have good fluorescence emission capability in the NIR-II region. The research results provide a new idea for the diagnosis and treatment integration of cancers.
Preferably, in S1, the molar ratio of the compound 2b to 2,2':5', 2' -trithiophene-5-carbaldehyde is between 0.8 and 1.0:2.0-3.0.
Preferably, the column chromatography purification of S1 is performed with dichloromethane: methanol=200: 1-100; v/v (more preferably 11 v/v) is the eluting solvent.
Preferably, in S2, the molar ratio of the compounds 2b+3t, NHS, EDC to amantadine is between 0.06 and 0.09:0.1-0.2:0.06-0.09:0.2-0.3.
Preferably, in S2, the concentration of the compound 2b+3t in dichloromethane is 40-60mg/20mL.
Preferably, in S2, NHS and EDC are added under light-protected conditions.
Preferably, the column chromatography purification of S2 is performed with dichloromethane: ethanol = 100:1-100: v/v (more preferably 5 v/v) is the eluting solvent.
The third aspect of the invention provides an application of the pH activated photosensitizer in the second aspect in preparing a tumor diagnosis and treatment integrated preparation.
The activated photosensitizer synthesized by the method can generate a large amount of active oxygen and fluorescent emission through illumination after H + is stimulated, so that tumor cells are killed, a stronger fluorescent signal can be generated in an NIR-II region, and an NIR-II living body imaging is carried out on a cancerous part, so that the activated photosensitizer is an integrated tumor diagnosis and treatment dosage form with wide application prospect.
Compared with the prior art, the invention has the beneficial effects that:
According to the invention, rhodamine derivatives are used as raw materials, and a pH activated photosensitizer is constructed, and the NIR-II region activated photosensitizer does not have fluorescence emission capability and can be activated only by illumination after H + is stimulated, so that a large amount of active oxygen and fluorescence emission are generated. Wherein, the generated active oxygen can destroy proteins, DNA and lipid in cells, so that the cells die and the tumor cells are killed; the fluorescence emission can generate stronger fluorescence signals in the NIR-II region, and can carry out NIR-II living imaging on cancerous parts. Therefore, the pH activated photosensitizer is a diagnosis and treatment integrated dosage form, can provide a new thought for the design of the activated photosensitizer, and has important reference significance for realizing diagnosis and treatment integration and overcoming the cancer difficulty.
Drawings
FIG. 1 is a 1 H NMR spectrum of 2b+t.
FIG. 2 is a 1 H NMR chart of 2b+2t.
FIG. 3 is a 1 H NMR chart of 2b+3t.
FIG. 4 is a 13 C NMR chart of 2b+t.
FIG. 5 is a 13 C NMR chart of 2b+2t.
FIG. 6 is a 13 C NMR chart of 2b+3t.
Fig. 7 is a HRMS diagram of 2b+t.
Fig. 8 is an HRMS diagram of 2b+2t.
Fig. 9 is an HRMS diagram of 2b+3t.
FIG. 10 is an ultraviolet absorbance spectrum of 10. Mu.M photosensitizer in 10% DMSO/H 2 O;
FIG. 11 is a graph showing fluorescence emission spectra of DCFH in a solution at different illumination times after 2b+t is added; (b) Adding a fluorescence emission spectrum chart of DCFH in the solution after 2b+2t under different illumination time; (c) Adding a fluorescence emission spectrum of DCFH in the solution after 2b+3t under different illumination time; (d) Comparison of fluorescence emission intensity of DCFH at 525nm with time.
FIG. 12 is a graph showing the ultraviolet absorption spectrum of DPBF in solution after 2b+t addition at different illumination times; (b) Ultraviolet absorption spectrum of DPBF in solution after 2b+2t is added under different illumination time; (c) Ultraviolet absorption spectrum of DPBF in solution after 2b+3t is added under different illumination time; (d) Comparison of the absorption intensity of DPBF at 410nm with respect to the time of illumination.
FIG. 13 is a 1 H NMR chart of a pH activated photosensitizer.
FIG. 14 is a 13 C NMR chart of a pH activated photosensitizer.
Fig. 15 is an HRMS diagram of a pH activated photosensitizer.
FIG. 16 shows (a) the change in fluorescence intensity of DCFH in solution before pH activated photosensitizer plus H + at different illumination times; (b) The fluorescence intensity of DCFH in the solution after the pH activated photosensitizer is added with H + changes under different illumination time; (c) A comparison plot of fluorescence emission intensity of DCFH at 525nm in solution before and after addition of H + over time; a. the numbers in panel b are pH values.
FIG. 17 is a fluorescence emission spectrum of pH activated photosensitizers at different H + levels.
Detailed Description
The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.
Example 1: synthesis and characterization of photosensitizers
1. Synthesis of photosensitizers
The synthetic reaction formula of the photosensitizer is shown as follows:
(1) Synthesis of Compound 2b+t:
compound 2b (199.9 mg,0.42 mmol) was added to a 100mL round bottom flask, dissolved in 10mL acetic acid, thiophenal (58 mg,0.52 mmol) was added to the round bottom flask, and the resulting reaction mixture was heated at 110℃for reaction for 12h. After the completion of the reaction, the reaction mixture was concentrated under reduced pressure, and the obtained product was purified by column chromatography (dichloromethane: methanol=400:1-100; 5 v/v) to obtain a purple target product (yield: 156mg, yield: 65.16%). The nuclear magnetism and mass spectrum information of the product is as follows:
1H NMR(600MHz,Chloroform-d)δ7.96(d,J＝7.7Hz,1H),7.63(td,J＝7.5,1.1Hz,1H),7.57-7.52(m,2H),7.37(d,J＝5.1Hz,1H),7.24-7.19(m,2H),7.10(dd,J＝5.1,3.6Hz,1H),6.49(d,J＝8.9Hz,1H),6.44(d,J＝2.5Hz,1H),6.35(dd,J＝8.9,2.6Hz,1H),3.37(q,J＝7.1Hz,4H),2.92(dddd,J＝15.7,6.7,4.7,1.6Hz,1H),2.69(dddd,J＝15.9,9.0,4.9,2.0Hz,1H),2.04(ddd,J＝16.0,6.3,4.7Hz,1H),1.77-1.69(m,2H),1.63(ddd,J＝16.0,8.4,4.7Hz,1H),1.18(t,J＝7.0Hz,6H).13C NMR(151MHz,CDCl3)δ170.22,152.58,152.43,149.54,147.18,140.71,134.58,129.38,129.10,128.71,128.18,127.83,127.33,126.60,125.12,123.66,118.39,108.96,108.25,105.04,97.48,44.57,27.64,23.03,22.11,12.73.ESI-MS(M)+[m/z]470.17844, The calculation result is 470.17844, m=c 29H28NO3S+.
(2) Synthesis of Compound 2b+2t:
Compound 2b (463 mg,0.97 mmol) was added to a 100mL round bottom flask, dissolved in 20mL acetic acid, 2-dithiophene-5-acetaldehyde (376 mg,1.94 mmol) was added to the round bottom flask and the resulting reaction mixture was refluxed overnight at 110 ℃. After the completion of the reaction, the mixture was concentrated under reduced pressure, and the obtained product was purified by column chromatography (dichloromethane: methanol=200:1-100:7v/v) to obtain the objective product (yield: 318mg, yield: 50.27%) in blue. The nuclear magnetism and mass spectrum information of the product is as follows:
1H NMR(600MHz,Chloroform-d)δ8.01(d,J＝7.7Hz,1H),7.65-7.60(m,2H),7.58-7.53(m,1H),7.25-7.21(m,3H),7.20-7.16(m,2H),7.03(dd,J＝5.1,3.6Hz,1H),6.57(d,J＝8.9Hz,1H),6.54(d,J＝2.4Hz,1H),6.43(dd,J＝9.1,2.5Hz,1H),3.41(q,J＝7.1Hz,4H),2.91(d,J＝16.3Hz,1H),2.73(t,J＝12.4Hz,1H),2.15-2.09(m,1H),1.82-1.72(m,3H),1.20(t,J＝7.1Hz,6H).13C NMR(151MHz,CDCl3)δ169.84,139.47,137.38,134.17,131.47,129.49,128.98,128.16,127.44,126.24,125.03,124.40,124.10,124.04,97.28,44.85,27.69,23.50,21.91,12.75.ESI-MS(M)+[m/z]552.16632, The calculation result is 552.16616, m=c 33H30NO3S2 +.
(3) Synthesis of Compound 2b+3t
Compound 2b (457 mg,0.96 mmol) was added to a 100mL round bottom flask, dissolved with 20mL acetic acid, 2':5', 2' -trithiophene-5-carbaldehyde (552 mg,2.11 mmol) was added to the round bottom flask, and the resulting reaction mixture was then refluxed overnight at 110 ℃. After the reaction, the mixture was concentrated under reduced pressure, and the obtained product was purified by column chromatography (dichloromethane: methanol=200:1-100; 11 v/v) to obtain a green target product (yield: 387mg, yield: 54.90%). The nuclear magnetism and mass spectrum information of the product is as follows:
1H NMR(600MHz,Chloroform-d)δ7.99(d,J＝7.7Hz,1H),7.63(t,J＝7.4Hz,1H),7.57-7.52(m,2H),7.24-7.22(m,1H),7.18(td,J＝8.6,4.3Hz,4H),7.12(d,J＝3.8Hz,1H),7.09(d,J＝3.8Hz,1H),7.02(dd,J＝5.1,3.6Hz,1H),6.53(d,J＝8.9Hz,1H),6.49(d,J＝2.5Hz,1H),6.39(dd,J＝8.9,2.6Hz,1H),3.39(q,J＝7.1Hz,4H),2.91(d,J＝16.3Hz,1H),2.75-2.67(m,1H),2.08(dd,J＝14.6,6.5Hz,1H),1.74-1.67(m,3H),1.19(t,J＝7.1Hz,6H).13C NMR(151MHz,CDCl3)δ129.44,128.85,128.09,124.78,124.64,124.51,123.95,44.71,27.75,23.29,22.00,12.75.ESI-MS(M)+[m/z]634.15388, The calculation result is 634.15388, m=c 37H32NO3S3 +.
2. Characterization of photosensitizers
(1) Test method
1) Nuclear magnetic resonance testing: the structure of the compound was measured by dissolving the compound in deuterated chloroform (CDCl 3) at room temperature using an AVANCE III type nuclear magnetic resonance apparatus manufactured by Bruce, germany, setting the frequency to 400/600 MHz.
2) Mass spectrometry test: a Q Exactive HF-X type liquid chromatography mass spectrometer manufactured by the United states Sieimer is used for determining the molecular weight of the target product.
3) Fluorescence emission spectrum test: the FLS980 type steady-state fluorescence spectrometer produced by Edinburgh in England is adopted to measure the fluorescence emission intensity of a target product under different experimental conditions, fluorescent emission spectrograms under different conditions are drawn, and the spectrograms are analyzed to obtain the fluorescence characteristics of each compound. Wherein the sensitivity is that the signal-to-noise ratio of the water Raman peak is more than 12000:1, the transient wavelength range is 300-1700nm.
4) Ultraviolet absorption spectrum test: and (3) measuring the ultraviolet absorption intensity of a target product under different experimental conditions by adopting an ultraviolet-2700 i type ultraviolet-visible spectrophotometer manufactured by Shimadzu corporation, drawing ultraviolet absorption spectrograms under different conditions, and analyzing the spectrograms to obtain the generation condition of singlet oxygen in each compound. Quartz cuvettes (width=1 cm) were used for all spectrophotometric and fluorometric measurements.
(2) Test results
1) 1H NMR、13 C NMR and mass spectrometry characterization: the prepared compounds 2b+t, 2b+2t and 2b+3t were dissolved in deuterated chloroform, respectively, and the structure of 2b+t, 2b+2t and 2b+3t was confirmed by 1 H NMR (fig. 1-3), 13 C NMR (fig. 4-6) and mass spectrometry (fig. 7-9).
2) Spectrometry: by measuring the photophysical properties of the three photosensitizers in solution (FIG. 10), the absorption wavelength of the photosensitizers shifted redly with increasing amount of thiophene in the compound. Wherein, the maximum absorption wavelength of 2b+t is 575nm, the maximum absorption wavelength of 2b+2t is 610nm, and the maximum absorption wavelength of 2b+3t is 632nm.
3) Detection of active oxygen: the compound 2'-7' -Dichlorofluorescein (DCFH) was used as an indicator for detecting active oxygen in solution. When active oxygen is generated in the system, DCFH without fluorescence is oxidized, and emits significant fluorescence at 525 nm. 10 μM 2b+t, 2b+2t, and 2b+3t were dissolved in 5mL DMSO/pbs=1 containing 5 μM DCFH: 9 (v/v). The mixture was then placed in a test tube, irradiated with 3mW cm -2 of LED lamp (400-800 nm), and the change in fluorescence intensity of the sample at 525nm was recorded with a fluorescence spectrometer.
As can be seen from an analysis of fig. 11 (a) (b) (c), as the illumination time increases, the fluorescence emission intensity of the three photosensitizers at 525nm increases continuously, and then gradually becomes stable, which indicates that active oxygen is generated in all of the three photosensitizers. As can be seen from an analysis of fig. 11 (d), the amounts of active oxygen generated by the three photosensitizers with an increase in illumination time are in order from more to less: 2b+2t > 2b+3t > 2b+t.
4) Testing of singlet oxygen: the amount of singlet oxygen produced in the solution was measured using the compound 1, 3-Diphenylisobenzofuran (DPBF) as an indicator. When singlet oxygen is generated in the solution, DPBF is oxidized by the singlet oxygen, resulting in a decrease in absorbance of the solution at 410 nm. 10. Mu.M of 2b+t, 2b+2t and 2b+3t were dissolved in 5mL DMSO/PBS=1:9 (v/v) containing 25. Mu.M DPBF. The mixture was then placed in a test tube, irradiated with 3mW cm -2 of LED light (400-800 nm), and the absorbance change at 410nm of the sample was recorded with an ultraviolet-visible spectrophotometer.
From an analysis of fig. 12 (a) (b) (c), it is known that the ultraviolet absorption intensity of the three photosensitizers at 410nm is continuously reduced as the illumination time is continuously increased, and eventually becomes stable. This phenomenon suggests that all three photosensitizers generate a large amount of singlet oxygen upon illumination. As can be seen from an analysis of fig. 12 (d), the amounts of singlet oxygen generated by these three photosensitizers with increasing illumination time were in order from more to less: 2b+3t > 2b+t > 2b+2t.
5) Theoretical calculation: in order to explore the relationship between the photophysical properties and the structure of the photosensitizers, the HOMO-LUMO orbital energy levels and orbital compositions of the front molecular orbitals were calculated using the density functional theory using the B3LYP method, selecting the 6-31G group, and were shown in tables 1 and 2.
From the data of HOMO and LOMO in tables 1 and 1, it can be seen that the HOMO-LUMO bandgap of 2b+t is 2.447eV, the HOMO-LUMO bandgap of 2b+2t is 2.175eV, and the HOMO-LUMO bandgap of 2b+3t is 1.981eV in the three compounds. The maximum energy gap value of 2b+t and the minimum energy gap value of 2b+3t indicate that the energy required by the electron transition of the molecule of the 2b+t compound is higher, the energy required by the electron transition of the molecule of the 2b+3t compound is lower, the excitation is easier, and the excitation wavelength is longer.
TABLE 1 energy level values of HOMO-LUMO orbitals of three photosensitizers
TABLE 2 HOMO-LUMO orbitals of three photosensitizers
Example 2: synthesis and characterization of pH activated photosensitizers
1. Synthesis of pH activated photosensitizers
The synthetic reaction formula of the pH activated photosensitizer is shown below:
The specific synthesis method comprises the following steps: compound 2b+3t (55 mg,0.075 mmol) was added to a 100mL round bottom flask, after dissolution with 20mL of methylene chloride, NHS (11.85 mg,0.103 mmol) and EDC (14.4 mg,0.075 mmol) were added to the round bottom flask in the absence of light, then stirring at 40℃for 5h under reflux, then amantadine (36 mg,0.238 mmol) and triethylamine were added to the round bottom flask after stirring, stirring at 40℃continued overnight under reflux, after the reaction, the resulting reaction was concentrated under reduced pressure using a rotary evaporator, and the product was purified by column chromatography (methylene chloride: ethanol=100:1-100:5) to give the desired product as a yellow solid, pH-activated photosensitizer (yield 14.7mg, yield 67.88%). The nuclear magnetism and mass spectrum information of the product is as follows:
1H NMR(400MHz,Chloroform-d)δ7.69(dd,J＝6.7,1.6Hz,1H),7.36(d,J＝2.1Hz,1H),7.35-7.25(m,2H),7.15(dd,J＝5.1,1.1Hz,1H),7.10(dd,J＝5.8,3.9Hz,2H),7.06(d,J＝3.9Hz,1H),7.03(q,J＝3.8Hz,2H),6.97-6.95(m,1H),6.93(dd,J＝7.5,1.9Hz,1H),6.44(d,J＝8.8Hz,1H),6.31(d,J＝2.5Hz,1H),6.21(dd,J＝8.9,2.6Hz,1H),3.29(q,J＝7.1Hz,4H),2.86(dt,J＝16.0,5.6Hz,1H),2.70-2.60(m,1H),2.32(d,J＝11.8Hz,3H),2.14(d,J＝11.8Hz,3H),2.05(ddd,J＝16.1,6.5,4.4Hz,1H),1.96-1.89(m,3H),1.67(ddt,J＝18.1,10.0,5.0Hz,3H),1.53(q,J＝13.9,13.1Hz,6H),1.12(t,J＝7.0Hz,6H).13C NMR(101MHz,CDCl3)δ169.67,152.52,151.25,148.72,144.44,140.16,137.74,137.21,136.44,136.32,132.15,131.51,129.76,128.66,128.51,128.02,127.94,124.66,124.57,124.39,124.20,124.10,123.83,123.81,123.74,122.56,122.36,116.84,113.03,108.26,107.89,98.17,67.47,59.35,53.53,44.38,39.30,36.59,30.08,28.24,23.34,22.38,12.75.ESI-MS(M+H)+[m/z]767.27972, The calculation result is 767.27997, m=c 47H46N2O2S3.
2. Characterization of pH-activated photosensitizer:
1) 1H NMR、13 C NMR and mass spectrometry characterization: through modification of 2b+3T structure and purification, new compound pH activated photosensitizer is obtained. The structure of the pH-activated photosensitizer was confirmed by dissolving this photosensitizer in deuterated chloroform, detected by 1 H NMR (fig. 13), 13 CNMR (fig. 14) and mass spectrometry (fig. 15).
2) Reactive oxygen testing of pH activated photosensitizer before and after marker addition: active oxygen was measured at various illumination times (min) before and after the addition of the pH-activated photosensitizer to the markers, as shown in FIG. 16. As can be seen from the figure, the fluorescence of the activated photosensitizer is significantly enhanced after the marker is added, indicating that this activated photosensitizer can be successfully activated, thereby generating a large amount of active oxygen.
3) NIR-II region profile of pH activated photosensitizer: the fluorescence emission spectrum of the activated photosensitizer in the NIR-II region was investigated as shown in FIG. 17. As can be seen from an analysis of FIG. 17, the fluorescence emission intensity in the NIR-II region of the activated photosensitizer increases with increasing H + content. This demonstrates that this activated photosensitizer has good fluorescence imaging potential.
In summary, three photosensitizers are synthesized by modifying rhodamine derivatives and controlling the quantity of the linked thiophenes. According to the heavy atomic effect, the coupling of spin orbitals is enhanced due to the existence of sulfur atoms in the three photosensitizers, so that intersystem crossing is promoted, and more active oxygen can be generated. Experiments show that the photosensitizer 2b+3t with the largest thiophene content generates the largest singlet oxygen, and the work provides a new thought for constructing the photosensitizer without heavy metals.
Meanwhile, the pH activated photosensitizer is constructed by modifying the photosensitizer 2b+3t, the activated photosensitizer maintains the characteristics of rhodamine dye, and a large amount of singlet oxygen can be generated through ring opening after the rhodamine dye is specifically activated by tumor markers, so that the pH activated photosensitizer has the advantages of high stability, easiness in synthesis, high biocompatibility and the like. Experiments show that the activated photosensitizer can generate stronger fluorescence emission in an NIR-II region, can be used for carrying out NIR-II living body imaging on cancerous parts, provides a new strategy for diagnosis and treatment integration of cancer treatment work, and has wide development prospect.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.
Claims (9)
1. A pH-activated photosensitizer, characterized in that the structural formula of the pH-activated photosensitizer is as follows:
2. the method for preparing a pH activated photosensitizer according to claim 1, characterized in that the preparation method comprises the following steps according to the following reaction scheme:
S1, dissolving a compound 2b in acetic acid, adding 2,2' -trithiophene-5-formaldehyde, refluxing at 100-120 ℃ for 8-15 hours, and performing reduced pressure cooling concentration and column chromatography purification after reaction to obtain a compound 2b+3t;
S2, dissolving a compound 2b+3t in dichloromethane, adding NHS and EDC, refluxing and stirring for 4-7 hours at the temperature of 35-50 ℃, adding amantadine and triethylamine, continuously refluxing and stirring for 8-15 hours at the temperature of 35-50 ℃, and concentrating under reduced pressure and purifying by column chromatography after reaction to obtain the pH activated photosensitizer.
3. The method for preparing a pH activated photosensitizer according to claim 2, wherein in S1, the molar ratio of the compound 2b to 2,2':5', 2' -trithiophene-5-carbaldehyde is 0.8-1.0:2.0-3.0.
4. The method for preparing a pH activated photosensitizer as set forth in claim 2, wherein the column chromatography purification of S1 is performed with methylene chloride: methanol=200: 1-100; v/v is the eluting solvent.
5. The method for preparing a pH activated photosensitizer according to claim 2, wherein in S2, the molar ratio of the compound 2b+3t, NHS, EDC to amantadine is 0.06-0.09:0.1-0.2:0.06-0.09:0.2-0.3.
6. The process for preparing a pH-activated photosensitizer according to claim 2, wherein in S2 the concentration of 2b+3t in dichloromethane is 40-60mg/20mL.
7. The method for preparing a pH activated photosensitizer according to claim 2, wherein NHS and EDC are added to S2 in the absence of light.
8. The method for preparing a pH activated photosensitizer as set forth in claim 2, wherein S2 said column chromatography purification is performed with methylene chloride: ethanol = 100:1-100: v/v is the eluting solvent.
9. The application of the pH activated photosensitizer in preparing a tumor diagnosis and treatment integrated preparation as claimed in claim 1.
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