CN115215756B - Organic fluorescent probe with endoplasmic reticulum targeting function, and preparation method and application thereof - Google Patents

Organic fluorescent probe with endoplasmic reticulum targeting function, and preparation method and application thereof Download PDF

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CN115215756B
CN115215756B CN202210698640.9A CN202210698640A CN115215756B CN 115215756 B CN115215756 B CN 115215756B CN 202210698640 A CN202210698640 A CN 202210698640A CN 115215756 B CN115215756 B CN 115215756B
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CN115215756A (en
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唐本忠
张云娇
高蒙
张敏杰
金鑫
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South China University of Technology SCUT
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Abstract

The invention belongs to the technical field of antitumor drugs, and discloses an organic fluorescent probe with endoplasmic reticulum targeting and a preparation method and application thereof. The structural formula of the organic fluorescent probe with endoplasmic reticulum targeting is shown as the formulaI. The invention also discloses a preparation method of the organic fluorescent probe. The fluorescent probe can quickly enter cells, target the endoplasmic reticulum through being inserted into a hydrophobic cavity of endoplasmic reticulum protein, has imaging capability of quickly targeting the endoplasmic reticulum, induces oxidative stress in cancer cells through chemotherapy, causes death of immune prototype cells, improves immunogenicity of tumor cells, and further causes persistent anti-tumor immune response of organisms. The organic fluorescent probe is used for endoplasmic reticulum fluorescent imaging and/or preparing products of tumor cell immune response.

Description

Organic fluorescent probe with endoplasmic reticulum targeting function, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of antitumor drugs, and particularly relates to an organic fluorescent probe with endoplasmic reticulum targeting and a preparation method and application thereof.
Background
In recent years, with the advent of immunotherapy, an anticancer therapy capable of stimulating immunogenic apoptosis of cancer cells and inducing an effective antitumor immune response in tumor tissues has attracted a great deal of attention in the oncology research community, and this death mode is called Immunogenic Cell Death (ICD). Immunogenic cell death is characterized by tumor cells that release damage-associated molecular patterns (e.g., calreticulin, high mobility group 1 protein, etc.) outside the cell upon stimulation by some drug, and allow Dendritic Cells (DCs) to phagocytose themselves and thereby stimulate DC maturation. The DCs then present tumor-associated antigens to T cells in the lymph nodes, thereby initiating adaptive anti-tumor immunity.
ICD inducers currently fall into two main categories: type I, which does not target the endoplasmic reticulum directly, but which is stimulated by indirect induction of endoplasmic reticulum stress effects to produce ICD-related risk signals (e.g., most of the traditional chemotherapeutics: oxaliplatin, anthracyclines, etc.); type II, actively targets the endoplasmic reticulum and focuses on endoplasmic reticulum production of ROS to induce cell death and ICD-related immunogenic expression (e.g., hypericin-based photodynamic therapy, ER-targeted iridium or platinum complexes). Since endoplasmic reticulum stress is more effective at inducing ICD, type II ICD inducers have proven to be more effective. Recently developed photosensitizers, photothermal agents, targeted to the endoplasmic reticulum to induce ICD, but they typically require complex synthesis and continuous illumination. Although chemotherapeutic organic molecules have been the leading drug approved by the FDA (about 75%) for the past few years, small organic molecules have not been reported as type II ICD inducers. Therefore, the development of small organic molecules as type II ICD inducers targeting the endoplasmic reticulum is attractive and has very good clinical application prospect.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention aims to provide an organic fluorescent probe with endoplasmic reticulum targeting capability, and a preparation method and application thereof. The organic fluorescent probe can enter cells rapidly, efficiently induce endoplasmic reticulum stress, effectively induce immunogenic cell death of tumor cells, and further can cause lasting anti-tumor immune response in vivo. The organic fluorescent probe is used for endoplasmic reticulum fluorescent imaging and/or preparing type II ICD inducer targeting endoplasmic reticulum. The organic fluorescent probe is also used for preparing medicines for tumor cell immune response.
The aim of the invention is achieved by the following technical scheme:
an organic fluorescent probe with endoplasmic reticulum targeting, which has a structural formula of formula I:
wherein R is 1 、R 2 、R 3 、R 4 Independently hydrogen, C 1-12 Alkyl, halogen, C 1-12 Alkyloxy, C 1-12 Alkylthio, C 1-12 Alkylamino, halogen-substituted C 1-12 Alkyl is more than or equal to 0 and less than or equal to 8.
The alkyl is a straight or branched chain alkyl.
Preferably, R 1 、R 2 Independently hydrogen, C 1-6 Alkyl, C substituted by halogen 1-6 An alkyl group; r is R 3 、R 4 Independently hydrogen, halogen, C 1-3 Alkyl, C 1-3 Alkyloxy, C 1-3 Alkylthio, C 1-3 Alkylamino, halogen-substituted C 1-3 Alkyl is more than or equal to 1 and less than or equal to 6.
R 1 、R 2 Independently C 1-4 Alkyl, C substituted by halogen 1-4 An alkyl group; r is R 3 、R 4 Independently hydrogen, n is more than or equal to 1 and less than or equal to 4.
Further preferably, the fluorescent probe has the structure of formula II or formula III:
wherein the compound of formula II is named SA-Cbl and the compound of formula III is named SA-DPR.
The preparation method of the organic fluorescent probe with endoplasmic reticulum targeting comprises the following steps: reacting a compound of formula IV with a compound of formula V under the catalysis of an alkaline compound to generate a compound of formula I, namely an organic fluorescent probe with endoplasmic reticulum targeting;
the compound of formula IV has the structure
The compound of formula V has the structure
In the formula IV and the formula V, R 1 、R 2 、R 3 、R 4 As defined above.
Preferably, the alkaline compound is more than one of potassium carbonate or sodium carbonate.
A compound of formula IV, the molar ratio of the compound of formula V to the basic compound being between 0.5 and 2:0.5 to 2:0.5 to 10.
The reaction takes an organic solvent as a reaction medium, wherein the organic solvent is more than one of acetonitrile, ethanol, methanol, DMSO or DMF.
The reaction is carried out under the condition of avoiding light; under a protective atmosphere.
The reaction equation of the preparation method of the organic fluorescent probe with endoplasmic reticulum targeting is as follows:
the invention provides application of the organic fluorescent probe in endoplasmic reticulum fluorescence imaging, and the organic fluorescent probe is used as an endoplasmic reticulum fluorescence imaging agent.
The invention provides application of the organic fluorescent probe in preparing a reagent for triggering oxidative stress of endoplasmic reticulum ROS and inducing immune prototype cell death.
The invention provides application of the organic fluorescent probe in protein recognition. The protein is bovine serum albumin.
The invention provides application of the organic fluorescent probe in preparing a medicine for tumor cell immune response.
The organic fluorescent probe provided by the invention has fluorescent property based on intramolecular charge transfer, has imaging capability of rapidly targeting an endoplasmic reticulum, can rapidly induce intracellular Reactive Oxygen Species (ROS) to rise in cells, induces Unfolded Protein Reaction (UPR), enables tumor cells to effectively release damage related molecular patterns, effectively improves immunogenicity of the tumor cells, and causes persistent anti-tumor immune response of organisms. The invention verifies in vitro and in vivo by B16F10 cells (mouse skin melanoma cells), obtains good anti-tumor immune response effect, and provides an exploration direction for developing an efficient induction immunogenic cell death inducer.
Compared with the prior art, the invention has the following advantages and effects:
1. compared with the traditional endoplasmic reticulum targeting probe, the organic fluorescent probe provided by the invention has the capability of entering cells rapidly by inserting an endoplasmic reticulum protein hydrophobic cavity to target the endoplasmic reticulum, can dye the endoplasmic reticulum within 1 minute, and has good selectivity.
2. The organic fluorescent probe directly induces endoplasmic reticulum oxidative stress through the chemotherapy effect so as to activate cell immune prototype cell death, and has better effect compared with the traditional immune prototype cell death chemotherapy inducer.
Drawings
FIG. 1 is a schematic representation of the targeting of probe SA-Cbl to the endoplasmic reticulum and induction of immunogenic cell death;
FIG. 2 shows the UV-visible absorption spectra and the corresponding fluorescence spectra of (A, B) SA-Cbl, (C, D) SA-A or (E, F) SA-DPR at different pH values; A. c, E is an absorption spectrum, B, D, F is a fluorescence spectrum;
FIG. 3 is a graph showing the ultraviolet-visible light absorption spectra, fluorescence spectra, and corresponding maximum emission wavelength and corresponding fluorescence intensity as a function of water content for (A, B) SA-Cbl, (C, D) SA-A and (E, F) SA-DPR in different ratios of DMSO/water mixtures; A. c, E is the absorption spectrum;
FIG. 4 (A) fluorescence spectra of SA-Cbl blended with different kinds of proteins; (B) Fluorescence spectra of SA-Cbl and BSA at different concentrations or (C) SA-Cbl and BSA heated at different temperatures for 10 minutes; (D) Fluorescence spectra of SA-Cbl blended with endoplasmic reticulum proteins with different concentrations; (E) Fluorescence spectra of SA-DPR and BSA with different concentrations after blending; (F) fluorescence spectra after blending SA-A and BSA or HSA;
FIG. 5 is a confocal laser scanning microscope image of SA-Cbl treated B16F10 cells co-stained with commercial dye (A) DCFH-DA, ER-Tracker Red or (B) Fluo-3 AM; (C) Monitoring protein expression in cells treated by SA-Cbl with different concentrations through Western blot; (D) LDH release profile of cells after 1 hour of treatment with SA-Cbl; (E) B16F10 cells treated with SA-Cbl were co-stained with Phalidin-FITC, and fixed cell staining Phalidin-FITC was set as positive control;
FIG. 6 is a confocal laser scanning microscope image of SA-DPR treated B16F10 cells, which were co-stained with commercial dye DCFH-DA, ER-Tracker Red, taken at different times after SA-DPR addition;
FIG. 7 is a confocal laser scanning microscope image of SA-A treated B16F10 cells co-stained with commercial dye DCFH-DA, ER-Tracker Red;
FIG. 8 is a laser confocal image of SA-Cbl incubated with Annexin V-FITC of the apoptosis kit with PI in B16F10 cells for various times;
FIG. 9 shows the viability of B16F10 cells after (A) SA-Cbl, (B) SA-A, (C) SA-DPR treatment;
FIG. 10 is ICD markers in SA-Cbl or DOX treated B16F10 cells; immunofluorescent staining of B16F10 extracellular (a) CRT or (B) HMGB 1; (C) flow cytometry results of CRT expression in B16F10 cells; concentration of (D) HMGB1 or (E) ATP in the cell culture supernatant;
confocal images of B16F10 cells treated with SA-Cbl at different concentrations (CellTracker Geen staining) incubated with DC (CellTracker Deep Red staining) for 24 hours in FIG. 11 (A); (B) CD40 + ，MHC II + DC or (C) CD80 + ，CD86 + Flow cytometry analysis plots and histograms of DCs;
FIG. 12 (A) is a schematic diagram of the evaluation of in vivo induced immunogenicity of SA-Cbl using a prophylactic tumor vaccination model; (B) tumor growth trend after inoculation of live cancer cells; photographs of tumors (C) excised from mice after 4 days and (D) volume bar charts;
FIG. 13 drainage of (A) CD80 in lymph nodes from C57BL/6 mice in the control, DOX or SA-Cbl group + ，CD86 + DC, isolation of (B) DC, (C) T cells, (D) CD8 from tumors + T cells, (E) CD8 + T EM (F) quantitative analysis of the percentages of regulatory T cells, (G) myeloid-derived suppressor cells and NK cells (H);
FIG. 14 is a nuclear magnetic hydrogen spectrum of SA-Cbl; FIG. 15 is a nuclear magnetic carbon spectrum of SA-Cbl;
FIG. 16 is a nuclear magnetic hydrogen spectrum of SA-DPR; FIG. 17 is a nuclear magnetic carbon spectrum of SA-DPR;
FIG. 18 is a nuclear magnetic resonance hydrogen spectrum of SA-A; FIG. 19 is a nuclear magnetic resonance spectrum of SA-A;
FIG. 20 is a nuclear magnetic hydrogen spectrum of MS-Cbl; FIG. 21 is a nuclear magnetic carbon spectrum of MS-Cbl;
FIG. 22 is a nuclear magnetic hydrogen spectrum of HA-Cbl; FIG. 23 shows the nuclear magnetic carbon spectrum of HA-Cbl.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
Example 1
(1) Preparation of SA-Cbl:
5-bromoacetyl-2-hydroxybenzaldehyde (243 mg,1.0 mmol) and chlorambucil (304 mg,1.0 mmol) were weighed separately, dissolved in 5mL of acetonitrile, potassium carbonate (166 mg,1.2 mmol) was added and stirred at room temperature in the dark for 4h, and the progress of the reaction was monitored by thin plate chromatography. After the substrate 5-bromoacetyl-2-hydroxybenzaldehyde had disappeared, the reaction solution was filtered to remove potassium carbonate, and the filtrate was distilled under reduced pressure to obtain a crude product, followed by ethyl acetate: petroleum ether=1:4 eluent was separated by column to give the product (200 mg, 43% yield). The structural characterization-related data are as follows: 1 H NMR(400MHz,CDCl 3 )δ(ppm):11.48(s,1H),9.97(s,1H),8.22(d,J＝2.0Hz1H),8.11-8.08(d,J＝10.8Hz,1H),7.14-7.08(m,3H),6.75-6.73(d,J＝8.0Hz,2H),5.31(s,2H),3.71-3.64(m,8H),2.67-2.63(t,J 1 ＝15.2Hz,J 2 ＝7.6Hz,2H),2.53-2.49(t,J 1 ＝14.8Hz,J 2 ＝7.6Hz,2H),2.02-1.98(m,2H)； 13 C NMR(125MHz,CDCl 3 )δ(ppm):196.20,189.74,173.00,165.63,144.30,135.96,134.43,130.47,129.75,126.67,120.12,118.48,112.14,65.45,53.58,40.49,33.79,33.09,26.71.HRMS(ESI + ):calcd.for[C 23 H 25 Cl 2 NO 5 +H + ]465.1110,found 465.1192.
(2) Preparation of SA-DPR:
5-bromoacetyl-2-hydroxybenzaldehyde (243 mg,1.0 mmol) and 3- [4- (dimethylamino) phenyl ] propionic acid (193 mg,1.0 mmol) were weighed separately, dissolved in 5mL of acetonitrile, potassium carbonate (166 mg,1.2 mmol) was added and stirred at room temperature in the dark for 4h, and the progress of the reaction was monitored by thin plate chromatography. After the completion of the reaction, the reaction solution was filtered to remove potassium carbonate, and the filtrate was distilled under reduced pressure to obtain a crude product, followed by ethyl acetate: petroleum ether=1:5 eluent was separated by column to give the product (180 mg, 51% yield). The structural characterization-related data are as follows:
1 H NMR(600MHz,CDCl 3 )δ(ppm):11.48(s,1H),9.95(s,1H),8.22(s,1H),8.07-8.09(d,J＝8.4Hz,1H),),7.11-7.07(m,3H),6.71(s,2H),5.29(s,2H),2.93(s,8H),2.79-2.77(t,J 1 ＝15.0Hz,J 2 ＝7.8Hz,2H)； 13 C NMR(125MHz,CDCl 3 )δ(ppm):196.39,190.03,172.73,165.76,149.40,136.15,134.63,129.04,128.38,126.85,120.25,118.59,113.12,65.71,40.94,35.98,30.01.HRMS(ESI + ):calcd.for[C 11 H 10 O 5 +Na + ]245.0420,found 245.0421.
(3) Preparation of SA-A:
5-bromoacetyl-2-hydroxybenzaldehyde (243 mg,1.0 mmol) and acetic acid (60 mg,1.0 mmol) were weighed separately, dissolved in 5mL of acetonitrile, potassium carbonate (166 mg,1.2 mmol) was added and stirred at room temperature for 4 hours in the absence of light, and the progress of the reaction was monitored by thin plate chromatography. After the completion of the reaction, the reaction solution was filtered to remove potassium carbonate, and the filtrate was distilled under reduced pressure to obtain a crude product, followed by ethyl acetate: petroleum ether=1:5 eluent was separated by column to give the product (150 mg, 68% yield). The structural characterization-related data are as follows:
1 H NMR(400MHz,CDCl 3 )δ(ppm):11.49(s,1H),9.98(s,1H),8.23-8.22(d,J＝1.6Hz,1H),8.11-8.09(d,J＝8.8Hz,1H),7.11-7.08(d,J＝8.8Hz,1H),5.31(s,2H),2.24(s,3H)； 13 C NMR(125MHz,CDCl 3 )δ(ppm):δ196.27,189.71,170.46,165.69,136.00,134.47,126.68,120.16,118.53,65.63,20.59.HRMS(ESI + ):calcd.for[C 20 H 21 NO 5 +H + ]356.1492,found 356.1491.
(4) Preparation of MS-Cbl:
methyl 5- (2-bromoacetyl) -2-hydroxybenzoate (273 mg,1.0 mmol) and 3- [4- (dimethylamino) phenyl ] propanoate (193 mg,1.0 mmol) were weighed separately, dissolved in 5mL of acetonitrile, potassium carbonate (166 mg,1.2 mmol) was added and stirred at room temperature for 6h in the absence of light, and the progress of the reaction was monitored by thin plate chromatography. After the completion of the reaction, the reaction solution was filtered to remove potassium carbonate, and the filtrate was distilled under reduced pressure to obtain a crude product, followed by ethyl acetate: petroleum ether=1:4 eluent was separated by column to give the product (450 mg, 91% yield). The structural characterization-related data are as follows:
1 H NMR(400MHz,CDCl 3 )δ(ppm):11.29(s,1H),8.45(s,1H),8.04-8.05(d,J＝8.8Hz,1H),7.06-7.12(m,3H),6.66-6.68(d,J＝8.3Hz,2H),5.29(s,2H),4.00(s,3H),3.61-3.72(m,8H),2.61-2.65(t,J 1 ＝7.5Hz,J 2 ＝15.2Hz,2H),2.50(t,J 1 ＝7.4Hz,J 2 ＝14.8Hz,2H),1.97-2.01(t,J 1 ＝7.2Hz,J 2 ＝14.8Hz,2H). 13 C NMR(125MHz,CDCl 3 )δ(ppm):190.21,173.23,170.02,165.95,144.44,135.11,131.01,130.71,129.95,126.14,118.64,112.43,112.31,77.51,77.19,76.87,65.69,53.76,52.96,40.66,33.99,33.31,26.92.HRMS(ESI + ):calcd.for[C 24 H 27 Cl 2 NO 6 +H + ]496.1288,found 496.1284.
(5) Preparation of HA-Cbl:
2-bromo-1- [ 4-hydroxy-3- (hydroxymethyl) phenyl ] ethan-1-one (273 mg,1.0 mmol) and 3- [4- (dimethylamino) phenyl ] propionic acid (193 mg,1.0 mmol) were weighed separately, dissolved in 5mL of acetonitrile, potassium carbonate (166 mg,1.2 mmol) was added and stirred at room temperature in the dark for 6h, and the progress of the reaction was monitored by thin plate chromatography. After the completion of the reaction, the reaction solution was filtered to remove potassium carbonate, and the filtrate was distilled under reduced pressure to obtain a crude product, followed by ethyl acetate: petroleum ether=1:4 eluent was separated by column to give the product (405 mg, 87% yield). The structural characterization-related data are as follows:
1 H NMR(400MHz,CDCl 3 )δ(ppm):8.28(s,1H),7.73-7.75(d,J＝8.5Hz,1H),7.62(s,1H),7.09-7.11(d,J＝8.6Hz,2H),6.90-6.92(d,J＝8.5Hz,1H),6.63-6.65(d,J＝8.6Hz,2H),5.25(s,2H),4.90(s,2H),3.72-3.60(m,8H),2.60-2.64(t,J 1 ＝7.5Hz,J 2 ＝15.2Hz,2H),2.48-2.51(t,J 1 ＝7.4Hz,J 2 ＝14.8Hz,2H),2.05-1.90(m,2H). 13 C NMR(125MHz,CDCl 3 )δ(ppm):190.82,173.42,161.54,144.27,130.55,129.78,129.69,127.88,126.36,124.86,116.83,112.17,77.34,77.23,77.02,76.70,65.66,64.32,53.60,40.51,33.82,33.19,26.72.HRMS(ESI + ):calcd.for[C 23 H 27 Cl 2 NO 6 +H + ]468.1339,found 468.1335.
wherein SA-A does not target the endoplasmic reticulum, MS-Cbl does not fluoresce with HA-Cbl and is unable to induce cell death in the cytoimmune prototype.
FIG. 1 is a schematic representation of the targeting of probe SA-Cbl to the endoplasmic reticulum and induction of immunogenic cell death.
FIG. 14 is a nuclear magnetic hydrogen spectrum of SA-Cbl; FIG. 15 is a nuclear magnetic carbon spectrum of SA-Cbl; FIG. 16 is a nuclear magnetic hydrogen spectrum of SA-DPR; FIG. 17 is a nuclear magnetic carbon spectrum of SA-DPR; FIG. 18 is a nuclear magnetic resonance hydrogen spectrum of SA-A; FIG. 19 is a nuclear magnetic resonance spectrum of SA-A; FIG. 20 is a nuclear magnetic hydrogen spectrum of MS-Cbl; FIG. 21 is a nuclear magnetic carbon spectrum of MS-Cbl; FIG. 22 is a nuclear magnetic hydrogen spectrum of HA-Cbl; FIG. 23 shows the nuclear magnetic carbon spectrum of HA-Cbl.
Example 2
Fluorescent Property testing of SA-Cbl, sA-A, SA-DPR at different pH
SA-Cbl, sA-A, SA-DPR were added to Britton-Robinson buffer solutions of different pH to give final concentrations of 10. Mu.M, and their absorption spectra and fluorescence spectra were measured. FIG. 2 shows the UV-visible absorption spectra and the corresponding fluorescence spectra of SA-Cbl (A, B), sA-A (C, D), SA-DPR (E, F) at different pH values, with probe concentrations of 10. Mu.M and excitation wavelength of 373nm. The Stokes shift of the three compounds is above 100 nm. The absorption spectrum of three molecules at 310nm and 380nm is obviously increased along with the increase of pH, because the phenolic hydroxyl groups in the molecules are gradually abstracted into oxygen anions along with the increase of pH, and the molecules start charge transfer in the molecules, so that conjugation is increased, absorption is increased, and the fluorescence intensity of the three molecules is also gradually increased along with the increase of pH.
Fluorescence property test of SA-Cbl, sA-A, SA-DPR in DMSO/Water
SA-Cbl, sA-A and SA-DPR were dissolved in DMSO/water solutions of different volume ratios, respectively, to give a final concentration of 10. Mu.M, and the fluorescence emission of the compounds under these solvent systems was examined by means of appropriate excitation light. FIG. 3 shows the ultraviolet-visible light absorption spectra of (A, B) SA-Cbl, (C, D) SA-A and (E, F) SA-DPR in DMSO/water mixtures in different ratios, fluorescence spectra and corresponding curves of maximum emission wavelength (black line) and corresponding fluorescence intensity (red line) as a function of water content, probe concentrations of 10. Mu.M, excitation wavelength of 373nm. With increasing water content, the fluorescence of the compounds all show a tendency to increase before decrease, because the polarity increases after addition of the aqueous solution, inhibiting proximity effects, thus increasing the fluorescence of the molecules, and subsequently fluorescence decreases because the non-radiative transitions are increasingly dominant after further increases in polarity.
Fluorescent Property test of SA-Cbl, sA-A, SA-DPR after binding to different proteins
FIG. 4 (A) SA-Cbl and different kinds of proteins (1 mg mL) -1 ) Fluorescence spectrum after blending; (B) SA-Cbl and BSA at different concentrations or (C) SA-Cbl and BSA heated at different temperatures for 10 minutes (1 mg mL) -1 ) Fluorescence spectrum after blending; excitation wavelength is 373nm, and SA-Cbl concentration is 10 μm; (D) Fluorescence spectra of SA-Cbl and endoplasmic reticulum proteins with different concentrations are mixed, and the concentration of the SA-Cbl is 10 mu M; (E) Fluorescence spectrum after blending SA-DPR and BSA with different concentrations, excitation wavelength is 373nm, and concentration of SA-DPR is 10 mu M; (F) SA-A and BSA or HSA (1 mg mL) -1 ) The fluorescence spectrum after blending is used for obtaining the fluorescent spectrum,the excitation wavelength was 365nm and the concentration of SA-A was 10. Mu.M.
SA-Cbl is mixed with different protein solutions, SA-Cbl has fluorescence enhancement effect with Bovine Serum Albumin (BSA), under the effect of 1mg/mL bovine serum albumin, the fluorescence signal of SA-Cbl is enhanced by about 7 times, the emission wavelength is also blue-shifted from 490nm to 450nm, and no obvious fluorescence enhancement effect is generated after the SA-Cbl is blended with other proteins including human serum albumin. After blending with BSA heated at different temperatures, it was found that the fluorescence gradually decreased as the heating temperature increased. The reason that SA-Cbl can selectively illuminate bovine serum albumin is that SA-Cbl can selectively enter the hydrophobic cavity of BSA, resulting in spatial structure locking, limiting its non-radiative transition, and thus fluorescence enhancement. The heated BSA is gradually denatured with the rise of temperature, and the SA-Cbl can not be carried any more due to the change of the cavity environment, so that the effect of enhancing fluorescence is not generated any more. Similarly, the fluorescence intensity of SA-DPR also increased with increasing BSA concentration, but the effect was not as pronounced as that of SA-Cbl, but no response after interaction of SA-A with different proteins, indicating that the size of the molecule and protein cavity were not matched. In addition, SA-Cbl also has fluorescence enhancement effect under the action of endoplasmic reticulum protein, which proves that SA-Cbl can be inserted into the hydrophobic cavity of endoplasmic reticulum.
Example 3
SA-Cbl is used in cells to image the endoplasmic reticulum and induce endoplasmic reticulum stress.
B16F10 cells or HeLa cells were cultured at 1X 10 5 The concentration of individual cells/dish was seeded on a confocal dish and after 24 hours the medium was replaced with DMEM.
For endoplasmic reticulum co-localization experiments with intracellular ROS detection, after incubation for 30 min with 1. Mu.M ER-Tracker Red and 10. Mu.M DCFH-DA, the cells were washed 3 times with PBS, followed by 50. Mu.M SA-Cbl, and immediately observed under confocal conditions, one photograph was taken every 30 seconds. For SA-Cbl, the excitation wavelength is 405nm, and the collection band is 410-513nm; for ER-Tracker Red, excitation wavelength is 543nm, collection band is 550-733nm, and for DCFH (2 ',7' -dichlorofluorescein diacetate, probe), excitation wavelength is 488nm, collection band is 513-530nm.
For calcium detection, after incubation for 30 min with 5. Mu.M calcium fluorescent probe Fluo-3 AM added to the medium, the cells were washed 3 times with PBS, followed by 50. Mu.M SA-Cbl and immediately observed under confocal conditions, taking a photograph every 30 seconds. For Fluo-3, the excitation wavelength was 488nm and the collection band was 513-600nm.
For co-staining experiments with phalloidin, B16F10 living cells were allowed to act at 50. Mu.M SA-Cbl for 1 hour, and then phalloidin-FITC dye was added thereto, followed by incubation for 30 minutes, and then cell morphology was observed under confocal microscope. The control group was first fixed with paraformaldehyde for 10 minutes, then incubated with phalloidin for 30 minutes, and then observed under confocal conditions. For phalloidin-FITC, the excitation wavelength was 488nm and the collection band was 513-550nm.
For the LDH (lactate dehydrogenase) release assay, B16F10 cells were assayed at 1×10 4 The density of individual cells/wells was seeded in 96-well plates, after 24 hours the medium was changed to 200. Mu.L of DMEM, 50. Mu.M SA-Cbl was added, and 20. Mu.L of LDH releasing reagent provided by the kit was added to the positive control. After one hour of incubation, 500g of the supernatant was centrifuged for 5 minutes, and 120. Mu.L of each was taken out for LDH concentration measurement. According to the operation of the specification, preparing an LDH detection working solution, adding 60 mu L of the detection working solution into each hole, uniformly mixing, carrying out light-shielding reaction for 30 minutes, and measuring the absorbance at 490 nm.
FIG. 5 is a confocal laser scanning microscope image of SA-Cbl treated B16F10 cells co-stained with commercial dye (A) DCFH-DA, ER-Tracker Red or (B) Fluo-3 AM; SA-Cbl concentration of 10. Mu.M, DCFH-DA concentration of 10. Mu.M, ER-Tracker Red concentration of 1. Mu.M; fluo-3 AM was 5. Mu.M; (C) Monitoring protein expression in cells treated by SA-Cbl with different concentrations through Western blot; (D) LDH release profile of cells after 1 hour of treatment with SA-Cbl; (E) B16F10 cells treated with SA-Cbl were co-stained with Phalidin-FITC, fixed cell staining Phalidin-FITC was set as positive control, SA-Cbl was at a concentration of 10. Mu.M, and the scale was 20. Mu.m.
SA-Cbl can enter cells rapidly within 1 minute and selectively target the endoplasmic reticulum, with co-localization coefficients with ER-tracker reaching 0.94 (FIG. 5 (A)). Intracellular ROS levels indicated by DCFH-DA increased significantly, while the emission intensity of ER-Tracker Red decreased significantly after 10 min incubation with SA-Cbl, and furthermore, free calcium ions transferred from the endoplasmic reticulum to the cytoplasm under the action of SA-Cbl were verified by the calcium ion fluorescent probe Fluo-3 (FIG. 5 (B)). These results indicate that SA-Cbl can effectively induce ER stress. The principle of SA-Cbl induced endoplasmic reticulum stress was further studied. A concentration-dependent increase in PERK phosphorylation and elf2α phosphorylation was observed by SA-Cbl treatment (FIG. 5 (C)), indicating that unfolded protein response occurred under endoplasmic reticulum stress. Furthermore, after SA-Cbl treatment, leakage of intracellular Lactate Dehydrogenase (LDH) into the outside of the cell was observed (fig. 5 (D)). In addition, after SA-Cbl was added to the medium, phalidin-FITC was added immediately, and the cells were fixed as a positive control, and Phalidin-FITC was observed to be able to enter the cells and stain F-actin (FIG. 5 (E)). These results indicate that the SA-Cbl effect induces an increase in cell membrane permeability.
Example 4
SA-DPR and SA-A are used intracellular to image the endoplasmic reticulum and induce intracellular ROS rise. For endoplasmic reticulum co-localization experiments with intracellular ROS detection, after adding 1. Mu.M ER-Tracker Red and 10. Mu.M DCFH-DA to B16F10 cell culture medium for 30 min co-incubation, cells were washed 3 times with PBS followed by 50. Mu.M SA-DPR or SA-A and immediately observed under confocal imaging, one photograph was taken every 30 seconds. For SA-DPR or SA-A, the excitation wavelength is 405nm, and the collection band is 410-513nm; for ER-Tracker Red, the excitation wavelength is 543nm, the collection band is 550-733nm, and for DCF, the excitation wavelength is 488nm, the collection band is 513-530nm. The co-localization coefficient of SA-DPR and ER-tracker reached 0.88, whereas SA-A did not target the cytoplasmic network. Under the action of SA-DPR and SA-A, intracellular ROS are obviously increased, and ER-Tracker signals are reduced, which indicates that cell stress is caused (FIG. 6 and FIG. 7).
FIG. 6 is a confocal laser scanning microscope image of SA-DPR treated B16F10 cells, which were co-stained with commercial dye DCFH-DA, ER-Tracker Red, taken at different times after SA-DPR addition; SA-DPR concentration is 10. Mu.M, DCFH-DA concentration is 10. Mu.M, ER-Tracker Red concentration is 1. Mu.M; the scale bar is 20 μm.
FIG. 7 is a confocal laser scanning microscope image of SA-A treated B16F10 cells co-stained with commercial dye DCFH-DA, ER-Tracker Red; the concentration of SA-A was 10. Mu.M; the concentration of DCFH-DA was 10. Mu.M; ER-Tracker Red at a concentration of 1. Mu.M; the scale bar is 20 μm.
Example 5
B16F10 cells were grown at 1X 10 5 The density of individual cells/wells was seeded in copolymer Jiao Min, after 24 hours the medium was changed to DMEM, and after 15 minutes of Annexin V-FITC/PI working solution in the apoptosis kit, 50 μm SA-Cbl was added and the real-time fluorescence signal was observed under confocal microscopy. In the co-staining experiments with SA-Cbl, annexin V-FITC and PI, it was found (FIG. 8) that SA-Cbl was able to stain cell membranes with Annexin V-FITC at 15 min and to cause PI to enter the nucleus at 25 min, indicating that SA-Cbl induced cell death by the apoptotic pathway.
FIG. 8 is a laser confocal image of SA-Cbl incubated with Annexin V-FITC of the apoptosis kit with PI in B16F10 cells for various times; the concentration of SA-Cbl was 10. Mu.M, and the scale was 20. Mu.m.
Example 6
B16F10 cells were grown at 1X 10 4 The density of individual cells/wells was seeded in 96-well plates, after 24 hours the medium was changed to DMEM, and after 50 μm each of the three compounds SA-Cbl, SA-a, SA-DPR of formula I was added, incubation was continued for 24 hours. The medium was then aspirated, 100. Mu.L of CCK-8 working solution was added to each well, and after incubation for 2 hours, the absorbance at 450nm was measured using an ELISA reader. SA-Cbl had a remarkable effect of killing cancer cells after 24 hours, and had an IC50 of about 50. Mu.M (FIG. 9).
FIG. 9 shows the viability of B16F10 cells after (A) SA-Cbl, (B) SA-A and (C) SA-DPR treatment.
Example 7
B16F10 cells were grown at 5X 10 4 Density of individual cells/well on a 14mm slide in a 24 well plate, after 24 hours the medium was changed to DMEM and 50. Mu.M SA-Cbl or 2. Mu.g/mL DOX was added for several times, the cells were fixed with 4% paraformaldehyde for 5 minutes and washed with PBSWashing 3 times, blocking with 5% bovine serum albumin in PBS at 37deg.C for 30 min, then sucking off blocking solution, adding primary antibody (1:500 dilution) and incubating overnight at 4deg.C, sucking off primary antibody dilution the next day, washing 3 times with blocking solution, adding secondary antibody (1:500 dilution) and incubating at 37deg.C for 1 hr, then washing off secondary antibody, washing 3 times with PBS, taking out slide and back-fastening on cover glass, and observing under confocal microscope. For immunofluorescence channels, the excitation wavelength is 633nm and the collection band is 640-740nm.
FIG. 10 is ICD markers in SA-Cbl or DOX treated B16F10 cells; immunofluorescent staining of B16F10 extracellular (A) CRT or (B) HMGB1, SA-Cbl concentration of 50. Mu.M, DOX concentration of 2. Mu.g/mL, and scale bar of 20. Mu.m; (C) flow cytometry results of CRT expression in B16F10 cells; concentration of (D) HMGB1 or (E) ATP in the cell culture supernatant; DOX was found to be 2. Mu.g/mL.
In the placebo group, CRT immunofluorescence was weak because normal cellular calreticulin is mainly inside the cell and antibodies were more difficult to enter the cell. While SA-Cbl and DOX can obviously induce cell CRT to evert after acting on B16F10 cells for 24 hours (figure 10A), red fluorescent signals are all on cell membranes, which indicates that intracellular calreticulin has been transported to the cell membranes by the cells, and plays a role in presenting antigens to DC cells. Similarly, HMGB1 is expressed in the nucleus itself, and its distribution sites are transferred from the nucleus to the cytoplasm and cell membrane after SA-Cbl and DOX action (fig. 10B). SA-Cbl induced the signal of CRT and HMGB1 eversion more pronounced than DOX at 2. Mu.g/mL. A more quantitative assessment of CRT exposure by flow cytometry confirmed a dose-dependent induction of immune prototype cell death by SA-Cbl (fig. 10C). Dose-dependent release of HMGB1 and HSP90 was also confirmed by Western Blot (fig. 10D). About 2.5-fold ATP release was also observed after SA-Cbl treatment, which was much higher than in DOX group (FIG. 10E).
Example 8
SA-Cbl treated B16F10 cells activated dendritic cells.
FIG. 11 copolymerization of SA-Cbl treated B16F10 cells (CellTracker Geen staining) at various concentrations (A) after 24 hours of co-incubation with DC (CellTracker Deep Red staining)Focal image, cellTracker Geen and CellTracker Deep Red concentration of 1 μm, scale 20 μm; (B) CD40 + ，MHC II + DC or (C) CD80 + ，CD86 + Flow cytometry analysis plots and histograms of DCs;
B16F10 cells and DCs were stained with CellTracker Green and CellTracker Deep Red, respectively, and then incubated together to observe interactions between treated cancer cells and DCs. The results indicate that after SA-Cbl treatment, B16F10 cells tended to co-localize with DCs, indicating that DCs were approaching and phagocytosing B16F10 cells. DC maturation markers CD40, CD80, CD86, and major histocompatibility complex class II (MHC II) were then analyzed by flow cytometry for the SA-Cbl group as compared to the control and DOX groups. CD40 in SA-Cbl group + ，MHC II + The DC duty cycle (50.7%) was about 2.5 times higher than the control (20.2%) and about 1.2 times higher than the DOX (41.9%). For CD80 + ，CD86 + DC, SA-Cbl group was 69.3% higher than DOX group (62.7%) and control group (45.5%). These results indicate that SA-Cbl treated dying cancer cells can effectively promote DC maturation.
Example 9
C57BL/6J mice were randomly assigned to 3 groups, designated control, SA-Cbl, and DOX, respectively. Each group contained 6 mice. For the SA-Cbl and DOX groups, B16F10 cells were treated with 50. Mu.M SA-Cbl or 2. Mu.g/mL DOX for 24 hours and used as cancer vaccines. The cancer vaccine was injected subcutaneously in SA-Cbl and DOX mice groups 0 and 8, respectively (containing 3X 10 6 100 μl sterile PBS per cell) elicits an immune response. As a control, an equal volume of sterile PBS was injected into mice in the control group. On day 15, groups 3 mice were vaccinated with 1X 10 in the left armpit 6 Living B16F10 cells. Tumor growth was monitored in 3 groups of mice by measuring tumor volume. Rapid tumor growth was observed in the control group and slow tumor growth in the SA-Cbl group, mice were sacrificed on day 29 and tumors were removed, while a significant difference in final tumor volume was observed in the DOX group, but a better tumor growth inhibition effect was achieved in the SA-Cbl group (FIG. 12). This result demonstrates that the SA-Cbl group cancer vaccine can effectively enhance mininessAbility of mice to resist cancer cell attack.
FIG. 12 (A) is a schematic diagram of the evaluation of in vivo induced immunogenicity of SA-Cbl using a prophylactic tumor vaccination model; (B) tumor growth trend after inoculation of live cancer cells; photographs of tumors (C) excised from mice after 4 days and (D) volume bar charts.
Example 10
Immune cell analysis in mice. Spleen, lymph nodes and tumors were collected from mice sacrificed on day 29 for flow cytometry analysis of immune cells therein. The first key step in achieving anti-tumor immunity is to activate DCs. FIG. 13 shows the drainage of (A) CD80 in lymph nodes from C57BL/6 mice in the control, DOX or SA-Cbl group + ，CD86 + DC, isolation of (B) DC, (C) T cells, (D) CD8 from tumors + T cells, (E) CD8 + T EM (F) quantitative analysis of the percentages of regulatory T cells, (G) myelogenous suppressor cells and NK cells (H).
As shown in FIG. 13, CD80 from the SA-Cbl group of lymph nodes + ，CD86 + The DC proportion (60.0%) of (c) was significantly higher than that of the control group (43.5%). These results indicate that the cancer vaccine of the SA-Cbl group performs well in promoting DC maturation in vivo. We further analyzed immune cells in tumors. The activated DCs in the SA-Cbl group were 5.4 times higher than in the control group, indicating that the SA-Cbl vaccine increased the degree of infiltration of DCs at the tumor site. Total T cells, CD8, extracted from the tumor were then analyzed + T cells and CD3 + ，CD8 + ，CD62L - ，CD44 + Effector memory T cells (T) EM ) Is a concentration of (3). The total T cell concentration of the SA-Cbl group was 1.5 times higher, CD8, compared to the control group + T cells are 2.3 times higher, T EM 1.7 times higher. Expression of regulatory T cells (tregs) and Myeloid Derived Suppressor Cells (MDSCs) that can cause immune down regulation have also been studied. The concentration of these two immunodownregulated cells in the SA-Cbl group was 2.5-fold and 2.2-fold lower, respectively, than in the control group, indicating that the mouse immunosuppressive tumor microenvironment was improved. NK cells also play an important role in adaptive immune responses. The results show that the NK cell expression induced by the cancer vaccine of the SA-Cbl group is higher than that of the control group1.9 times. From the results of these in vivo experiments, SA-Cbl showed excellent effects in inducing anti-tumor immunity and immune memory effect.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (2)

1. Use of an organic fluorescent probe with endoplasmic reticulum targeting in endoplasmic reticulum fluorescent imaging, characterized in that the organic fluorescent probe with endoplasmic reticulum targeting has the structural formula I:
wherein R is 1 、R 2 Independently C 1-12 Alkyl, C substituted by halogen 1-12 Alkyl, R 3 、R 4 Hydrogen is more than or equal to 0 and less than or equal to 8;
the alkyl is a straight or branched chain alkyl.
2. Use of an organic fluorescent probe with endoplasmic reticulum targeting for protein recognition, characterized in that the protein is bovine serum albumin;
the structural formula of the organic fluorescent probe with endoplasmic reticulum targeting is shown as formula I:
wherein R is 1 、R 2 Independently C 1-12 Alkyl, C substituted by halogen 1-12 Alkyl, R 3 、R 4 Hydrogen is more than or equal to 0 and less than or equal to 8;
the alkyl is a straight or branched chain alkyl.
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