Engineered cell and use

Abstract

An engineered cell and a use, belonging to the technical field of fine chemical engineering. A photosensitive compound is loaded into a phagocyte, a bacterium in a host is captured by utilizing the innate immunity of the cell, and thus by means of light treatment, the bacterium is killed. Compared with the limitation that a conventional photosensitizer cannot target a living bacterium, the cell formulation can precisely capture a bacterium by means of a macrophage and precisely "deliver" a photosensitive dye to the surface of the bacterium. Therefore, the light treatment can directly act on the surface of the bacterium without damaging an autologous tissue in a patient and has high sterilization efficiency and good treatment safety.

Description

A type of engineered cells and their applications Technical Field

[0001] The present invention belongs to the field of fine chemicals, and specifically relates to a type of engineered cells and applications. Background Art

[0002] Pathogenic bacteria are a hot topic in modern life science and medical research. For example, methicillin-resistant Staphylococcus aureus (MRSA) resistance is becoming increasingly severe, leading to intracellular survival and recurrent infection, resulting in significant hospital-acquired infections and mortality. The evolution of multidrug-resistant (MDR) bacteria, invasive procedures, and overuse of implantable monitoring technologies are direct contributors to these serious infections. Furthermore, the immunocompromised state of critically ill patients, including those in critically ill ICUs, those with congenital immunodeficiency, and those undergoing chemotherapy, makes it difficult to effectively prevent and eliminate bacterial infections. Currently, antibiotics are the standard clinical treatment. However, for critically ill patients, lesions are often located in vital organs such as the brain and spinal cord, leading to rapid disease progression. Conventional antibiotics are slow to take effect and fail to effectively curb infection progression. Furthermore, in immunocompromised patients, long-term, high-dose antibiotic use can cause severe side effects in patients already in poor health. Therefore, treating bacterial infections in immunocompromised patients remains a significant challenge, and precise, efficient, and universally applicable treatments are urgently needed to combat malignant bacterial infections.

[0003] Photodynamic therapy has a good antibacterial effect in vitro. Its advantage is that the reactive oxygen species (ROS) produced by photosensitizers under light conditions can effectively kill malignant bacteria and other microorganisms. This method has high efficiency and broad spectrum. However, these photosensitizers represented by organic fluorophores cannot target bacteria active in the host body. Currently, it is only possible to apply large doses of photosensitizers at the site of infection and achieve maximum sterilization by increasing the light treatment conditions. This bottleneck limits the practical application of photodynamic therapy, and a large number of "off-target" photosensitizers cause damage to the body itself.

[0004] Summary of the Invention

[0005] The present invention provides an engineered cell and its application.

[0006] The present invention provides an engineered cell, comprising a phagocyte; the phagocyte contains at least one photosensitizing dye having a photodynamic therapy effect.

[0007] An engineered cell, wherein the lysosome of the phagocyte contains at least one photosensitizing dye with photodynamic therapy effect.

[0008] An engineered cell, wherein the photosensitizing dye is a photosensitizing dye with lysosome targeting function.

[0009] An engineered cell, wherein the photosensitizing dye is a fluoroboron pyrrole photosensitizing dye, an indocyanine green photosensitizing dye, a porphyrin derivative or a dihydrochlorin photosensitizing dye with a lysosome targeting function.

[0010] The present invention also provides a class of photosensitizing dyes having photodynamic therapy effects, having structures of Formulas I-IV:

[0011] Among them, R is independent of each other. or R3.

[0012] R1 is H, CH3, n is an integer from 0 to 18, m is an integer from 0 to 18, Y - is an anion selected from BF4 - 、Cl - Br - , I - 、NO 3- 、SO4 2- 、ClO4 - 、CH3COO - 、CH3SO3 - CF3SO3 - .

[0013] R2 is Br or I.

[0014] R is independent of each other or R3.

[0015] R3 and R4 are independent of each other. wherein X is independently O or N.

[0016] The photosensitizing dye provided by the present invention has stable cell lysosome targeting capability.

[0017] The engineered cells are used in the preparation of drugs for treating bacterial infections.

[0018] The engineered cells are used in the preparation of drugs for drug-resistant bacterial infections.

[0019] The photosensitizing dye provided by the present invention has good antibacterial efficiency against intracellular multidrug-resistant bacteria.

[0020] The present invention also provides a preparation comprising the engineered cells.

[0021] The present invention also provides a method for preparing a cell preparation, which is obtained by incubating phagocytes with a solution of a photosensitizing dye having a photodynamic therapy effect and removing the residual photosensitizing dye in the culture solution that has not been taken up by the cells.

[0022] The drug-loaded macrophages can be used in the future in the form of macrophages isolated from healthy human bodies.

[0023] The macrophages are selected from bone marrow-derived macrophages, microglia, liver Kupffer cells, alveolar macrophages, renal mesangial cells, osteoclasts, splenic red pulp macrophages, monocyte-derived macrophages, and macrophages induced and differentiated from adipose stem cells or bone marrow stem cells. After being loaded with a photosensitive drug compound in vitro, they are re-injected intravenously into the patient as a reagent.

[0024] The beneficial effects of the present invention are as follows: This type of photosensitive compound targeting cell lysosomes can accurately target and stably remain in the lysosomes of living cells, and has a good photodynamic effect in the acidic lysosomal environment. The photosensitive compound is loaded into phagocytes, and the innate immune ability of the cells is used to capture bacteria in the host body, and then the bacteria are killed through phototherapy. Compared with the limitation that traditional photosensitizers cannot target living bacteria, the cell preparation proposed in the present invention can use macrophages to accurately capture bacteria and accurately "deliver" the photosensitive dye to the surface of the bacteria. Therefore, light therapy can act directly on the surface of bacteria without damaging the patient's own tissues, with high sterilization efficiency and good treatment safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG1 is a nuclear magnetic resonance hydrogen spectrum characterization diagram of the photosensitizing drug compound F1.

[0026] FIG2 is a high-resolution mass spectrum of the photosensitizing drug compound F1.

[0027] FIG3 is the ultraviolet absorption and fluorescence emission spectra of the photosensitizing drug compound F1.

[0028] FIG4 is a pH response graph of the photosensitizing drug compound F1.

[0029] FIG5 is a graph showing detection of active oxygen solvents by the photosensitizing drug compound F1 under a 740 nm light source.

[0030] FIG6 is a graph showing the light / dark toxicity of the photosensitizing drug compound F1 in macrophages under a 740 nm light source.

[0031] FIG7 is a graph showing detection of active oxygen solvents of photosensitizing drug compound F1 under a 740 nm light source at simulated tissue thickness.

[0032] Figure 8 is a graph showing the phototoxicity evaluation of the photosensitizing drug compound F1 in macrophages at different simulated tissue thicknesses under a 740 nm light source.

[0033] Figure 9 is a graph showing the phototoxicity evaluation of the photosensitizing drug compound F1 in macrophages at different illumination times under a 740 nm light source.

[0034] Figure 10 is a graph showing the phototoxicity evaluation of the photosensitizing drug compound F1 in macrophages at different light doses under a 740 nm light source.

[0035] Figure 11 is an evaluation graph of the retention of photosensitizing drug compound F1 in macrophages under different illumination times under a 740 nm light source.

[0036] FIG12 is a graph showing detection of active oxygen solvents in the drug-loaded macrophage preparation under a 740 nm light source.

[0037] FIG13 is a diagram showing the lysosomal localization of the photosensitive drug compound F1 in the drug-loaded macrophage preparation.

[0038] Figure 14 is a fluorescence imaging image of bacteria captured by drug-loaded macrophage preparations

[0039] Figure 15 shows fluorescence imaging of bacterial inactivation of drug-loaded macrophage preparations under different illumination times at 740 nm.

[0040] FIG16 is a graph showing the evaluation of the intracellular antibacterial efficiency of the drug-loaded macrophage preparation under a 740 nm light source.

[0041] FIG17 is a safety assessment diagram of the drug-loaded macrophage preparation under a 740 nm light source.

[0042] FIG18 is a graph showing the evaluation of the intracellular antibacterial efficiency of the drug-loaded macrophage preparation under a simulated sunlight light source.

[0043] FIG19 is a graph showing the in vivo antibacterial efficiency evaluation of the drug-loaded macrophage preparation at the mouse level under sunlight irradiation.

[0044] FIG20 is a survival curve of the drug-loaded macrophage preparation curing systemic bacterial infection in mice.

[0045] FIG21 is a weight tracing curve showing that the drug-loaded macrophage preparation cured systemic bacterial infection in mice. DETAILED DESCRIPTION

[0046] A lysosome-targeted small molecule photosensitizer is designed and synthesized, and loaded onto the lysosomes of phagocytes in vitro to obtain a cell preparation. The cell preparation is then delivered to patients with severe bacterial infections, using the natural chemotaxis of phagocytes to accurately capture free bacteria in the patient's body, and efficiently kill the captured bacteria through the photodynamic effect. This cell preparation not only replenishes the patient with active immune cells, but also uses the inherent targeting of phagocytes to bacteria to accurately implement photodynamic therapy on the surface of bacteria, which can accurately and quickly cure malignant bacterial infections in critically ill patients. The present invention proposes a class of lysosome-targeted photosensitizers and cell preparations that can be used for precise anti-malignant bacterial treatment, which will provide a non-destructive, efficient and original treatment plan for malignant bacterial infections in critically ill patients in hospitals.

[0047] The following non-limiting examples may enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way. Any person skilled in the art who, within the technical scope disclosed herein, makes equivalent substitutions or modifications based on the technical solutions and inventive concepts of the present invention falls within the scope of protection of the present invention.

[0048] In the following examples, unless otherwise specified, all reagents used can be prepared by conventional methods or purchased from commercial sources.

[0049] The drug-loaded macrophages select photosensitizing drug compounds as the antibacterial core of the photodynamic therapy (PDT) effect. On the one hand, the photosensitizing drug compounds stably target macrophage lysosomes, enabling delivery of the photosensitizing drug compounds to bacteria within a single lysosome at a minimal distance, maximizing the PDT effect. On the other hand, macrophages are naturally capable of capturing bacteria, enabling precise eradication of MDR bacterial infections without damaging healthy tissue.

[0050] The macrophages of the present invention can be isolated from tissues or preferentially differentiated ex vivo from blood monocytes (also referred to herein as "monocyte-derived macrophages"), bone marrow precursor cells (also referred to herein as "bone marrow-derived macrophages"), or from any other possible precursors by using any differentiation method known to those skilled in the art.

[0051] The present invention provides an engineered cell, comprising a phagocyte; the phagocyte contains at least one photosensitizing dye having a photodynamic therapy effect.

[0052] Some specific engineered cells include phagocytes; the lysosomes of the phagocytes contain at least one photosensitizing dye with photodynamic therapy effect.

[0053] Some specific engineered cells include phagocytes; the lysosomes of the phagocytes contain at least one of the compounds of Formula I to Formula IV;

[0054] Among them, R is independent of each other. or R3.

[0055] R1 is H, CH3, n is an integer from 0 to 18, m is an integer from 0 to 18, Y - is an anion selected from BF4 - 、Cl - Br - , I - 、NO 3- 、SO4 2- 、ClO4 - 、CH3COO- 、CH3SO3 - CF3SO3 - ;

[0056] R2 is Br or I.

[0057] R3 and R4 are independent of each other. wherein X is independently O or N.

[0058] Some specific engineered cells include phagocytes; the lysosomes of the phagocytes contain at least one of the compounds of Formula I to Formula IV;

[0059] Wherein, R1 is H, CH3, n is an integer from 0 to 10, m is an integer from 0 to 10, Y - is an anion selected from BF4 - 、Cl - Br 、 I - 、NO 3- 、SO4 2- 、ClO4 - 、CH3COO - 、CH3SO3 - CF3SO3 - ;

[0060] R2 is Br or I;

[0061] R3 and R4 are independent of each other. wherein X is independently O or N.

[0062] Some specific engineered cells include phagocytes; the lysosomes of the phagocytes contain at least one of the compounds of Formula I to Formula IV;

[0063] Wherein, R1 is H, CH3, n is an integer from 0 to 6, and m is an integer from 0 to 6;

[0064] R2 is 1.

[0065] R3 and R4 are independent of each other. wherein X is independently O or N.

[0066] Some specific engineered cells include phagocytes; the lysosomes of the phagocytes contain at least one of the compounds of Formula I to Formula IV;

[0067] Wherein, R1 is H, CH3, n is an integer of 0-5, m is an integer of 0-5; and R2 is 1.

[0068] R3 and R4 are independent of each other. wherein X is independently O or N.

[0069] Some specific engineered cells include phagocytes; the lysosomes of the phagocytes contain at least one of the compounds of Formula I to Formula IV.

[0070] Wherein, R1 is H, CH3, R2 is 1.

[0071] R3 and R4 are independent of each other. wherein X is independently O or N.

[0072] In some specific engineered cells, the phagocytes are macrophages.

[0073] In some specific engineered cells, the macrophages are selected from bone marrow-derived macrophages or monocyte-derived macrophages.

[0074] A preparation comprising the engineered cells.

[0075] A method for preparing a cell preparation comprises incubating phagocytes with a photosensitive dye concentration having a cell survival rate of more than 60%.

[0076] A specific method for preparing a cell preparation comprises incubating phagocytes with a solution of a compound of Formula I to Formula IV at a concentration of 0.1 to 20 μM to remove residual compounds.

[0077] A specific method for preparing a cell preparation comprises incubating phagocytes with a solution of a compound of Formula I to Formula IV at a concentration of 0.1 to 20 μM for 10 minutes to 10 hours to remove residual compounds.

[0078] A specific method for preparing a cell preparation comprises incubating phagocytes with a solution of a compound of Formula I to Formula IV at a concentration of 0.1 to 20 μM for 30 minutes to 5 hours to remove residual compounds.

[0079] A specific method for preparing a cell preparation comprises incubating phagocytes with a solution of a compound of Formula I to Formula IV at a concentration of 0.1 to 20 μM for 1 to 3 hours to remove residual compounds.

[0080] A specific method for preparing a cell preparation comprises incubating phagocytes with a culture medium solution containing a compound of Formula I to Formula IV at a concentration of 0.1 to 10 μM to remove residual compounds.

[0081] A specific method for preparing a cell preparation comprises incubating phagocytes with a culture medium solution of a compound of Formula I to Formula IV at a concentration of 0.1 to 1 μM for 1 to 3 hours, and then washing away the compound remaining in the culture medium with PBS.

[0082] The excitation light source of the photosensitizing dye of the present invention is a light source corresponding to the maximum absorption wavelength of the photosensitizing dye, a light source with a sunlight-like spectrum, or sunlight.

[0083] The excitation light source used in treatment of the engineered cells and cell preparations provided by the present invention is a light source corresponding to the maximum absorption wavelength of the photosensitizing dye contained therein, a light source with a sunlight-like spectrum, or sunlight.

[0084] A specific acquisition method: macrophages are cultured for 24 hours. Then they are incubated with photosensitizing drugs at 37°C for 3 hours under 5% CO2. The cells are washed three times with PBS. The drug-loaded macrophages are obtained by trypsinization. The cells are dispersed in ethanol and placed at room temperature for 20 minutes. The above mixture is centrifuged at 1000 rpm for 5 minutes to collect the supernatant. The concentration of the dye taken up by the drug-loaded macrophages is calculated as follows: the fluorescence intensity of the photosensitizing drug in the supernatant is measured on a fluorescence spectrophotometer. The fluorescence intensity is converted to the corresponding concentration using a standard curve between fluorescence intensity and ethanol concentration of the photosensitizing drug. Subsequently, the average molar amount of the photosensitizing drug in each cell (nsingle cell) is

[0085] c is the concentration of the photosensitizer in ethanol; V is the volume of the photosensitizer in ethanol solution; and N is the number of cells in the test.

[0086] The loading method of the drug-loaded macrophages is as follows: the photosensitive drug compound is dissolved in macrophage culture medium, and the compound is co-incubated with the macrophages at 37° C. in a 5% CO 2 atmosphere for 1-3 hours. PBS is used three times to completely remove the unloaded photosensitive drug compound.

[0087] The drug-loaded macrophages have the potential to innately capture bacteria into lysosomes and deliver photosensitive drug compounds to the bacteria at a minimal distance within a single lysosome, maximizing the PDT effect and limiting potential side effects.

[0088] Preparation of drug-loaded macrophages: Incubate macrophages with a 0.1-1 μM photosensitive drug compound in a culture medium solution at 37°C in a 5% CO2 atmosphere for 1-3 hours. Repeat three times with PBS to completely remove any unloaded photosensitive drug compound.

[0089] The present invention also provides a method for preparing compounds of formula I to formula IV, which are prepared by condensation, substitution or clik reaction using the parent dye fluoroboron pyrrole (BDP) of the compound of formula I, the parent dye indocyanine green (ICG) of the compound of formula II, the parent dye porphyrin derivative (HpD) of the compound of formula III, and the parent dye dihydrochlorin e6 (Ce6) of the compound of formula IV as raw materials.

[0090] BDP, ICG, HpD, and Ce6 were synthesized with reference to related substances reported in existing literature (Coord. Chem. Rev. 399, 213024 (2019); Eur. J. Med. Chem. 46, 2037-2042 (2011); Results Phys. 3, 100092 (2021); J. Photochem. Photobiol. B, Biol. 150, 38-43 (2015); Faraday Discuss. 170, 59 (2014)). The method specifically includes the following steps:

[0091] 1) Under argon, copper sulfate pentahydrate, sodium ascorbate, compound E-1, and compound R-1 were dissolved in 10 mL of a mixture of an organic solvent and water. The mixture was reacted at room temperature for 2 h, and the endpoint was determined by TLC. The mixture was diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation. Compound F was then purified by silica gel column chromatography.

[0092] 2) Compound E-2 and Compound R-2 were dissolved in 10 mL of N,N-dimethylformamide and reacted at 80°C. The end point was determined by TLC. The mixture was diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation. Compound F was then purified by silica gel column chromatography.

[0093] 3) Under argon, compound G (J, L) was dissolved in an organic solvent, and then EDCI, triethylamine, and R were added sequentially; the mixture was stirred at room temperature for at least 3 h; most of the solvent was removed, and the residue was poured into diethyl ether to precipitate a solid, which was filtered and dried to obtain compound I (K, M).

[0094] Wherein, the definitions of R1-R4 are the same as those in the above general structural formula.

[0095] The heating reflux described in step 1 is preferably refluxed for more than 12 hours.

[0096] The organic solvent in step 2 is selected from DMF, ethanol, tetrahydrofuran or a mixture thereof.

[0097] The organic solvent in step 3 is selected from DMF, acetonitrile, tetrahydrofuran or a mixture thereof. The molar ratio of compound E to EDCI is more preferably 1:1.2.

[0098] The organic solvent in step 4 is selected from DMF, ethanol, tetrahydrofuran or a mixture thereof. The reaction time at room temperature is preferably more than 1 hour.

[0099] Example 1

[0100] Synthesis route of photosensitizing drug compound (F1):

[0101] (1) Synthesis of intermediate C1

[0102] Dissolve A1 (500 mg, 1.26 mmol) in 10 ml of dichloromethane (DCM). Add dropwise a DCM solution of B1 (569 mg, 2.53 mmol), stir at room temperature, and monitor by TLC. Wash three times with water, dry over anhydrous sodium sulfate, remove the solvent by distillation under reduced pressure, and purify by silica gel column chromatography to obtain intermediate C1.

[0103] (2) Synthesis of intermediate E1

[0104] Dissolve C1 (500 mg, 0.772 mmol) and D1 (338 mg, 1.55 mmol) in 5 ml of toluene. Add 0.25 ml of piperidine and 0.25 ml of glacial acetic acid dropwise at a time. Stir and reflux to remove the water produced during the reaction. Monitor by TLC. Cool the reaction mixture to room temperature, pour it into 20 ml of DCM, wash three times with water, dry it over anhydrous sodium sulfate, remove the solvent by distillation under reduced pressure, and purify it by silica gel column chromatography. This yields intermediate E1.

[0105] (3) Synthesis of Compound F1

[0106] Under argon, copper sulfate pentahydrate (11.46 mg, 0.048 mmol), sodium ascorbate (9.44 mg, 0.047 mmol), intermediate E1 (100 mg, 0.095 mmol), and compound R1 (53 mg, 0.286 mmol) were dissolved in a mixture of 3 mL of N,N-dimethylformamide and water. The mixture was allowed to react at room temperature for 2 h, and the endpoint was determined by TLC. The mixture was diluted with 10 mL of DCM, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation. Compound F1 was then purified by silica gel column chromatography.

[0107] Example 2

[0108] The synthesis method is as shown in Example 1. replace Synthesis of compound F2.

[0109] Example 3

[0110] Synthesis of compound F3

[0111] Compound E2 (100 mg, 0.1 mmol) and compound R3 (52 mg, 0.25 mmol) were dissolved in 10 mL of N,N-dimethylformamide and reacted at 80°C. The endpoint was determined by TLC. The mixture was diluted with DCM, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Compound F3 was then purified by silica gel column chromatography to obtain the product.

[0112] Example 4

[0113] The synthesis method is as shown in Example 3. replace Synthesis of compound F4.

[0114] Example 5

[0115] The synthesis method is as shown in Example 1. replace Synthesis of compound F5.

[0116] Example 6

[0117] The synthesis method is as shown in Example 1. replace Synthesis of compound F6.

[0118] Example 7

[0119] The synthesis method is as shown in Example 6. replace Synthesis of compound F7.

[0120] Example 8

[0121] The synthesis method is as shown in Example 7. replace Synthesis of compound F8.

[0122] Example 9

[0123] The synthesis method is as shown in Example 8. replace Synthesis of compound F9.

[0124] Example 10

[0125] The synthesis method is as shown in Example 9. replace Synthesis of compound F10.

[0126] Example 11

[0127] Synthesis of compound I1

[0128] Under argon, compound G1 (200 mg, 0.334 mmol) was dissolved in an organic solvent, and then EDCI (160.36 mg, 0.836 mmol), triethylamine (101.58 mg, 1 mmol), and R4 (133.23 mg, 0.836 mmol) were added in sequence; stirred at room temperature for 12 h; most of the solvent was removed, and the residue was poured into diethyl ether to precipitate a solid, which was filtered and dried to obtain compound I1.

[0129] Example 12

[0130] The synthesis method is as shown in Example 11. replace Synthesis of compound I2.

[0131] Example 13

[0132] Synthesis of compound I3

[0133] Under argon, compound G1 (200 mg, 0.334 mmol) was dissolved in an organic solvent, and then triethylamine (101.58 mg, 1 mmol) and R6 (173.25 mg, 0.836 mmol) were added in sequence; stirred at room temperature for 12 h; most of the solvent was removed, and the residue was poured into diethyl ether to precipitate a solid, which was filtered and dried to obtain compound I3.

[0134] Example 14

[0135] The synthesis method is as shown in Example 13. replace Synthesis of compound I4.

[0136] Example 15

[0137] Synthesis of compound K1

[0138] Under argon, compound J1 (200 mg, 0.321 mmol) was dissolved in an organic solvent, and then triethylamine (97.50 mg, 0.964 mmol) and R6 (127.89 mg, 0.803 mmol) were added in sequence; the mixture was stirred at room temperature for 12 h; most of the solvent was removed, and the residue was poured into diethyl ether to precipitate a solid, which was filtered and dried to obtain compound K1.

[0139] Example 16

[0140] The synthesis method is as shown in Example 15. replace Synthesis of compound K2.

[0141] Example 17

[0142] The synthesis method is as shown in Example 16. replace Synthesis of compound K3.

[0143] Example 18

[0144] The synthesis method is as shown in Example 17. replace Synthesis of compound K4.

[0145] Example 19

[0146] The synthesis method is as shown in Example 15. replace Synthesis of compound M1.

[0147] Example 20

[0148] The synthesis method is as shown in Example 19. replace Synthesis of compound M2.

[0149] Example 21

[0150] The synthesis method is as shown in Example 20. replace Synthesis of compound M3.

[0151] Example 22

[0152] The synthesis method is as shown in Example 21. replace Synthesis of compound M4.

[0153] Example 23

[0154] The following compounds were prepared according to the method of Example 1, and their structures were identified by mass spectrometry.

[0155] Example 24

[0156] Method for loading drug-loaded macrophages: Raw264.7 cells in the exponential phase were plated on a 6-well plate at 5×10 4 The cells were cultured for 24 h, and then incubated with 250 nM F1 for 3 h at 37° C. and 5% CO 2 . PBS was used three times to completely remove the unloaded photosensitizing drug compounds.

[0157] Method for calculating the dosage of drug-loaded macrophages: Raw264.7 cells in exponential growth phase were seeded on 6-well plates at a density of 5×10 4 The cells were grown and cultured for 24 hours. They were then incubated with 250nM F1 for 3 hours at 37°C and 5% CO2. The cells were washed three times with PBS. Drug-loaded macrophages were obtained by trypsinization. The cells were dispersed in ethanol and placed at room temperature for 20 minutes. The above mixture was centrifuged at 1000rpm for 5 minutes to collect the supernatant. The concentration of the dye taken up by the cells was calculated as follows: the fluorescence intensity of F1 in the supernatant was measured on a fluorescence spectrophotometer. The standard curve between the fluorescence intensity and concentration in ethanol conforms to the fluorescence intensity-concentration (μM) standard curve formula: FI=25.56+182.55×c. The supernatant fluorescence intensity was converted to the corresponding concentration. Subsequently, the average concentration of F1 in each cell (nsingle cell) was calculated according to the formula:

[0158] Example 25

[0159] The photosensitizing drug compound F1 was dissolved in DMSO to obtain a 5 mM solution. This solution was further diluted with DCM to obtain a 5 μM test solution. The absorption spectrum was measured using an Agilent 8453 UV-visible spectrophotometer, and the fluorescence spectrum was measured using an Agilent 8453 spectrometer. Figure 3 shows the UV absorption and fluorescence emission spectra of the photosensitizing drug compound F1 synthesized in Example 1. As can be seen from the figure, F1 exhibits strong absorption in the near-infrared region of 500 nm to 800 nm, and the extinction coefficient in DCM is 168580 M. -1 cm -1 This absorption feature indicates that deep tissues are efficiently excited in the near-infrared region. At the same time, the F1 also shows that the detectable radiation ranges from 710nm to 900nm, which can be used to track the distribution of stained cells.

[0160] Example 26

[0161] Lysosomal acidic response: Photosensitizer compound F1 was dissolved in DMSO to obtain a 5 mM solution. This solution was further diluted with DCM to a 5 μM test solution. Dilute hydrochloric acid solution or sodium hydroxide solution was added to the test solution to adjust the test pH. Fluorescence spectra were measured using an Agilent 8453 spectrometer. As can be seen in Figure 4, when the pH is greater than 5.0, this near-infrared emission gradually weakens, and when the pH is less than 5.0, this near-infrared emission gradually increases. This result is conducive to the controllable acidity of photosensitizer compound F1 in achieving the PDT effect in lysosomes.

[0162] Example 27

[0163] In vitro ROS detection:

[0164] Spectral test: The photosensitizing drug compound F1 was dissolved in DMSO to obtain a 5 mM solution. The solution was further diluted with DCM to a 5 μM test solution. 1,3-diphenylisobenzofuran (DPBF) was used as the singlet oxygen ( 1 O2) production indicator. The absorption spectrum was measured by an Agilent 8453 UV-visible spectrophotometer. Under a 740nm light source and an ultra-low power intensity of 5mW / cm2, the absorption of DPBF at 418nm decreased significantly within 100s (Figure 5). Compared with the decrease in DPBF absorption in a solution without 0.1% trifluoroacetic acid (TFA), the PDT effect of the photosensitizing drug compound F1 ( 1 The O2 generation efficiency was pH-dependent, and the DPBF absorption decreased significantly only in the solution containing 0.1% trifluoroacetic acid (TFA). This indicates that the PDT effect of the photosensitizing drug compound F1 is fully activated under acidic conditions.

[0165] Cell test: The prepared drug-loaded macrophage preparation was evenly dispersed on the confocal culture dish, and dichlorodihydrofluorescein-acetoacetate (H2DCFDA) was used as an indicator of the generation of reactive oxygen species. The control group was H2DCFDA-stained RAW264.7 cells and drug-loaded macrophages, and then subjected to illumination or non-illumination operation. Prepare the staining solution: Take 0.5μL of 10mM H2DCFDA and add it to 1mL PBS. Staining operation: Wash the cell culture dish with PBS three times and then add the staining solution. Cover with tin foil and culture in the dark for 20 minutes. Wash with PBS three times, add fresh DMEM culture medium for subsequent illumination or light-proof operation. Fluorescence imaging was tested by Olympus Fluoview FV1000. H2DCFDA was excited at 488nm, and the collection band was: 510-560nm. As shown in Figure 12, under a 740nm light source, 30mW / cm 2After 10 minutes of ultra-low power intensity treatment, green fluorescence was shown in the macrophages compared with the control group, proving that the photosensitive drug compound F1 loaded by the macrophages produced reactive oxygen species under light irradiation.

[0166] Example 28

[0167] MTT test of drug-loaded macrophage preparations: Raw264.7 cells were seeded in 96-well plates (3×10 3 Cells were placed on the plate and cultured for 24 hours. Staining operation: the control group was 2 μL DMSO added to 1 mL fresh DMEM medium; the experimental group was 2 μL photosensitizer stock solution added to 5 tubes of 1 mL fresh DMEM medium, with final concentrations of 120 nM, 310 nM, 620 nM, 930 nM, and 1250 nM, respectively. The cells in the well plate were washed 3 times with PBS and incubated with different concentrations of F1 fresh medium for 3 hours. The cells were then washed three times with PBS. Dark group: the well plate was covered with tin foil and cultured for 24 hours; light group: the well plate was illuminated at 740 nm (30 mW / cm 2 ) light for 15 minutes. After irradiation, the cells were incubated for an additional 24 hours. Relative cell viability was measured by MTT assay. As shown in Figure 6, in the dark state, the cell viability was nearly 100%, indicating that F1 did not activate reactive oxygen species in the dark state and was non-toxic to drug-loaded macrophages. Under light conditions, the cell viability decreased depending on the increase in F1 dose. This indicates that F1 activates reactive oxygen species under light and its toxicity to drug-loaded macrophages depends on the drug concentration.

[0168] Example 29

[0169] Reactive oxygen species spectrum test of photosensitizer F1 in simulated tissues of different thicknesses: photosensitizer compound F1 was dissolved in DMSO to obtain a 5 mM solution. The solution was further diluted with DCM to a 5 μM test solution. 1,3-diphenylisobenzofuran (DPBF) was used as the singlet oxygen ( 1 The absorption spectrum was measured using an Agilent 8453 UV-visible spectrophotometer under a 740 nm light source and a 5 mW·cm -2 At ultra-low power intensities, the absorption change of DPBF at 418nm decreases with increasing tissue thickness (Figure 7). This indicates that tissue thickness affects light source transmittance, and thus ROS release. However, F1 still exhibits high ROS generation capacity at a thickness of 1 cm. This demonstrates that the photosensitizing drug compound F1 possesses a highly effective PDT effect and is suitable for in vivo treatment.

[0170] Example 30

[0171] MTT test of drug-loaded macrophage preparations at different tissue thicknesses: Raw264.7 cells were seeded in 96-well plates (3×10 3 Cells) and cultured for 24 hours. Staining operation: 2μL photosensitizer stock solution was added to 6 tubes of 1mL fresh DMEM culture medium, with a final concentration of 250nM. The cells in the well plate were washed 3 times with PBS and incubated with fresh F1 culture medium for 3 hours. Then the cells were washed three times with PBS. The control group was not covered with muscle tissue for light operation; the experimental group: chicken tissue of different thicknesses was covered on the well plate. At 740nm (30mW / cm 2 ) light for 15 minutes. After irradiation, the cells were incubated for another 24 hours. The relative cell viability was measured by MTT assay. As shown in Figure 8, in the dark state, the cell survival rate was nearly 100%, indicating that F1 was not activated by reactive oxygen species in the dark state and was non-toxic to drug-loaded macrophages. Under light conditions, the cell survival rate was dependent on the increase in the thickness of the covered chicken tissue. However, F1 still showed high cytotoxicity at a thickness of 1 cm. This shows that F1 is sufficient to induce an efficient PDT effect even when the light source's ability to penetrate is weakened.

[0172] Example 31

[0173] MTT test of drug-loaded macrophage preparations under different illumination times: Raw264.7 cells were seeded in 96-well plates (3×10 3 Cells were placed on the plate and cultured for 24 hours. Staining procedure: 2 μL of photosensitizer stock solution was added to 6 tubes of 1 mL of fresh DMEM medium, with a final concentration of 250 nM. The cells in the well plate were washed 3 times with PBS and incubated with fresh F1 medium for 3 hours. The cells were then washed three times with PBS. The control group was covered with tin foil to avoid light; the experimental group: 2 ) light for different durations. After irradiation, the cells were incubated for an additional 24 hours. Relative cell viability was measured using the MTT assay. As shown in Figure 9, cell viability decreased with increasing illumination time, indicating that the PDT effect induced by F1 is dependent on illumination time.

[0174] Example 32

[0175] MTT test of drug-loaded macrophage preparations under different light doses: Raw264.7 cells were seeded in 96-well plates (3×10 3cells) and cultured for 24 hours. Staining operation: 2 μL of photosensitizer stock solution was added to 6 tubes of 1 mL of fresh DMEM culture medium, with a final concentration of 250 nM. The cells in the well plate were washed 3 times with PBS and incubated with fresh culture medium containing F1 for 3 hours. The cells were then washed three times with PBS. The control group was wrapped in tin foil to avoid light; the experimental group: irradiated with different light powers under 740 nm light for 15 minutes. After irradiation, the cells were incubated for another 24 hours. The relative cell viability was measured by MTT assay. As shown in Figure 10, with the increase of light dose, the cell survival rate decreased successively. This shows that the PDT effect induced by F1 is dependent on the light dose.

[0176] Example 33

[0177] Test of the retention of photosensitizing drugs in macrophages after light irradiation: The prepared drug-loaded macrophage preparation was evenly dispersed on the confocal culture dish and the light was irradiated at 740nm (30mW / cm 2 ) light for different durations. Fluorescence imaging was performed using an Olympus Fluoview FV1000. 635nm laser excitation was used, and signal acquisition was performed in the 620-675nm range. As shown in Figure 11, with prolonged illumination, the reactive oxygen species produced by F1 damage and kill cells. However, F1 fluorescence remains intracellular, demonstrating that F1 is stably retained in cells and does not leak out even after cell damage.

[0178] Example 34

[0179] Lysosomal targeting of photosensitive drug compound F1 in drug-loaded macrophages: The prepared drug-loaded macrophage preparation (prepared according to Example 24) was evenly dispersed on a confocal culture dish, and DND-26 was used as a lysosomal localization indicator. Prepare the staining solution: Take 1 μL of 0.5 mM DND-26 and add it to 1 mL of PBS. Staining operation: Wash the cell culture dish with PBS three times and then add the staining solution. Cover with tin foil and culture in the dark for 20 minutes. Wash with PBS three times and add fresh DMEM culture medium for subsequent operations. Fluorescence imaging was tested using an Olympus Fluoview FV1000. 488 nm and 635 nm lasers were excited simultaneously, and the signal acquisition range was selected from 510-550 nm and 620-675 nm. As can be seen from Figure 13, the fluorescence distribution of the photosensitive drug compound F1 is highly consistent with that of DND-26, indicating that F1 specifically targets lysosomes.

[0180] Example 35

[0181] Evaluation of the ability of drug-loaded macrophages to capture bacteria: The prepared drug-loaded macrophage preparation (prepared according to Example 24) was evenly dispersed on a confocal culture dish and the cells were plated with about 10 5CFU of MRSA expressing green fluorescent protein were inoculated with drug-loaded macrophages for 30 minutes. Fluorescence imaging was performed using an Olympus Fluoview FV1000. Simultaneous excitation was achieved using 488nm and 635nm lasers, with signal acquisition in the 510-550nm and 620-675nm ranges. As shown in Figure 14, the fluorescence distribution of the photosensitizing drug compound F1 is highly consistent with that of MRSA expressing green fluorescent protein, demonstrating that drug-loaded macrophages were able to successfully capture the bacteria and deliver them to the lysosomes, where the photosensitizing drug F1 resides. This maximizes the photodynamic effect.

[0182] Example 36

[0183] Fluorescence imaging test of intracellular antibacterial ability of drug-loaded macrophages: The prepared drug-loaded macrophage preparation (prepared according to Example 24) was evenly dispersed on a confocal culture dish and the cells were fixed with about 10 5 CFU of MRSA expressing green fluorescent protein were inoculated with drug-loaded macrophages for 30 minutes. The cells were then illuminated at various times under 740nm light. Fluorescence imaging was performed using an Olympus Fluoview FV1000. 488nm and 635nm lasers were used for simultaneous excitation, with signal acquisition in the 510-550nm and 620-675nm ranges. As shown in Figure 15, the fluorescence of the MRSA expressing green fluorescent protein gradually disappears, and the cell outlines gradually disintegrate. This demonstrates that drug-loaded macrophages can successfully capture bacteria and kill them under illumination.

[0184] Example 37

[0185] Intracellular antibacterial test of drug-loaded macrophage preparation: The prepared drug-loaded macrophage preparation (prepared according to Example 24) was evenly dispersed on a 6-well plate. 5 CFU of MRSA infects drug-loaded macrophages for 30 minutes. Irradiate with 740 nm or simulated sunlight for 15 minutes. Incubate with 100 μg / mL gentamicin sulfate (Sigma-Aldrich, G1914) for 1 hour to kill extracellular bacteria. After incubation, rinse the cells with PBS and irradiate with 740 nm (30 mW / cm 2 ) or simulated sunlight (80mW / cm 2 ) for 15 min. 0.5% Triton X-100 (Sigma-Aldrich, X100) was added to dissolve the bacterial solution. 1 , 10 2 , 10 3 , 10 4The cells were diluted by multiple dilutions, and 10 μl of each sample was plated on TSB agar (Sigma-Aldrich, 22092 and BD Biosciences, 214010). Bacteria were counted after incubation at 37°C for 12 hours. The plate culture images and statistical results shown in Figures 16 and 18 show rapid bacterial proliferation in the control group. The experimental group completely eliminated intracellular bacteria. This demonstrates the significance of the strategy for constructing drug-loaded macrophage preparations. The drug-loaded therapy efficiency based on RAW264.7 cells is much higher than that of free photosensitizer molecules.

[0186] Example 38

[0187] Safety assessment of drug-loaded macrophage preparations: The prepared drug-loaded macrophage preparation (prepared according to Example 24) and normal cells COS-7 were uniformly dispersed on the same confocal culture dish at a ratio of 1:1 (v:v). Prepare AM / PI staining reagent: Take 0.5μL of 4mM Calcein-AM and 16mM PI and add them to 1mL PBS. Staining operation: Wash the cell culture dish 3 times with PBS and then add the staining solution. Cover with tin foil and culture in the dark for 20 minutes. Wash 3 times with PBS, add fresh DMEM culture medium and incubate at 30mW / cm2 under 740nm light. 2 Irradiation was performed for 15 minutes. Fluorescence imaging was tested using an Olympus Fluoview FV1000. 488nm and 559nm lasers were used for simultaneous excitation, and the signal acquisition ranges of 510-550nm and 560-625nm were selected. The results in Figure 17 show that the green fluorescence in the cytoplasm of drug-loaded macrophages (round cells) decreased, the red fluorescence in the nucleus increased, and the cells gradually underwent apoptosis, while the strong green fluorescence in the cytoplasm of adjacent COS-7 cells (spindle cells marked by broken lines) showed survival. This indicates that the photodynamic effect of the photosensitizing drug compound F1 is highly efficient and the generated ROS work within the drug-loaded macrophages, and is therefore safe for the host.

[0188] Example 39

[0189] In vivo antibacterial testing of drug-loaded macrophage formulations under sunlight: Female C57BL / 6 mice, 6-7 weeks old, were purchased from the Shanghai Laboratory Animal Center of Dalian Medical University. The animal experiments performed in this work were approved by the Dalian Medical University Animal Care and Use Committee. A mouse immunodeficiency model was established using intraperitoneal administration of cyclophosphamide (CY). Briefly, C57BL / 6 mice were intraperitoneally injected with 100 mg / kg CY for three consecutive days for bacterial infection. Blood routine analysis of leukocytes and lysozymes was performed to assess the development of immunodeficiency and recovery of health in the mice.

[0190] For the construction of systemic bacterial infection model in mice: mice were anesthetized with 10% chloral hydrate intraperitoneally. All mice were hairless. 50 μl of stained MRSA suspension (1×10 8 CFU) were inoculated into the blood system of mice.

[0191] For in vivo antimicrobial data collection: 2 h after MRSA injection, mice were randomly divided into five groups. One group contained 2 × 10 6 Each mouse was intravenously injected with a drug-loaded macrophage preparation. After bacterial injection, the mice were exposed to sunlight (120 mW / cm 2 The mice were irradiated with PBS, blank macrophages, and compound F1 at the same dose for 3 days. The blood of the mice was collected immediately after 3 days. 1 , 10 2 , 10 3 , 10 4 The cells were diluted in multiples, and 10 μl was taken from each group and spread on TSB agar (Sigma-Aldrich, 22092 and BD Biosciences, 214010). Bacteria were counted after incubation at 37°C for 12 hours. From the plate culture pictures and statistical results shown in Figure 19, the control group showed that the bacterial content in the blood remained at a high level. The experimental group completely eliminated the bacteria in the blood. From the mouse survival curve in Figure 20, the mice treated with drug-loaded macrophage preparations and light remained alive and gradually recovered their weight (Figure 21); while the control group mice injected with only PBS, only injected with photosensitizers or without light all died within a short period of time, indicating the high efficiency of the strategy of constructing drug-loaded macrophage preparations for treatment under sunlight. It provides a new and convenient idea for clinical treatment.

[0192] Example 40

[0193] Table 1 Antibacterial efficiency of other photosensitizing drug compounds

[0194] Referring to the methods of Examples 25, 27, 34 and 37, compounds F6, F8, F9, I1, K1 and M1 were also tested for absorption spectra, reactive oxygen species release capacity, lysosomal targeting and antibacterial ability. The specific test results are given in the following table.

Claims

1. An engineered cell, characterized in that: Includes phagocytes; The phagocytes contain at least one photosensitizing dye having a photodynamic therapy effect.

2. An engineered cell according to claim 1, characterized in that: The lysosomes of the phagocytes contain at least one photosensitizing dye having a photodynamic therapy effect.

3. The engineered cell according to claim 2, characterized in that The photosensitizer dye is a photosensitizer dye with lysosome targeting function.

4. The engineered cell according to claim 3, characterized in that The photosensitizing dye is a fluoroboropyrrole photosensitizing dye, an indocyanine green photosensitizing dye, a porphyrin derivative or a dihydrochlorin photosensitizing dye having a lysosome targeting function.

5. An engineered cell according to claim 2, characterized in that: The photosensitizing dye having a photodynamic therapy effect is selected from at least one of the compounds represented by formula I-IV; Among them, R is independent of each other. or R3; R1 is H or CH3; n is an integer from 0 to 18, m is an integer from 0 to 18, Y - is an anion selected from BF4 - , Cl - Br - ,I - 、NO 3- 、SO4 2- 、ClO4 - 、CH3COO - 、CH3SO3 - CF3SO3 - ; R2 is Br or I; R3 and R4 are independent of each other. or Wherein, X is independently O or N.

6. The engineered cell according to claim 5, characterized in that R1 is H, CH3, n is an integer from 0 to 10, m is an integer from 0 to 10, Y - is an anion selected from BF4 - , Cl - Br - ,I - 、NO 3- 、SO4 2- 、ClO4 - 、CH3COO - 、CH3SO3 - CF3SO3 - ; R2 is Br or I; R3 and R4 are independent of each other. or Wherein, X is independently O or N.

7. The engineered cell according to claim 6, characterized in that R1 is H, CH3, n is an integer from 0 to 6, and m is an integer from 0 to 6; R2 is 1; R3 and R4 are independent of each other. or wherein X is independently O or N.

8. The engineered cell according to claim 7, characterized in that R1 is H, CH3, n is an integer from 0 to 5, m is an integer from 0 to 5; R2 is 1; R3 and R4 are independent of each other. Wherein, X is independently O or N.

9. The engineered cell according to claim 8, characterized in that R1 is H, CH3, R2 is 1.

10. The engineered cell according to any one of claims 1 to 9, characterized in that: The phagocytic cells are macrophages.

11. The engineered cell according to claim 10, characterized in that The macrophages are selected from bone marrow-derived macrophages, microglia, liver Kupffer cells, alveolar macrophages, renal mesangial cells, osteoclasts, spleen red pulp macrophages, monocyte-derived macrophages, and macrophages induced and differentiated from adipose stem cells or bone marrow stem cells.

12. The engineered cell according to any one of claims 1 to 9, characterized in that: The excitation light source of the photosensitizing dye is a light source corresponding to the maximum absorption wavelength of the photosensitizing dye, a light source of a sunlight-like spectrum, or sunlight.

13. A preparation, characterized in that The invention comprises the engineered cell according to any one of claims 1 to 9.

14. A method for preparing a cell preparation, characterized in that: The cell preparation is obtained by incubating phagocytes with a photosensitizing dye solution having a concentration of 0.1 to 20 μM and having a photodynamic therapy effect, and removing the residual photosensitizing dye that is not taken up by the cells.

15. The preparation method according to claim 14, characterized in that: The concentration of the photosensitizing dye solution is 0.1 to 10 μM; The incubation time of phagocytes is 0.5-3h.

16. Use of the engineered cell according to any one of claims 1 to 9 in the preparation of a drug for treating bacterial infection.

17. The use according to claim 16, characterized in that The engineered cells are used for preparing drugs against drug-resistant bacteria.

18. Use of the compound of formula I in the preparation of photodynamic therapy materials or drugs: in, R is independent of each other. or R3; R1 is H, CH3, n is an integer from 0 to 18, m is an integer from 0 to 18, Y - is an anion selected from BF4 - , Cl - Br - ,I - 、NO 3- 、SO4 2- 、ClO4 - 、CH3COO - 、CH3SO3 - CF3SO3 - ; R2 is Br or I; R3 and R4 are independent of each other. or Wherein, X is independently O or N.

19. The use according to claim 18, characterized in that R1 is H, CH3, n is an integer from 0 to 10, and m is an integer from 0 to 10; R2 is Br or I; R3 and R4 are independent of each other. or Wherein, X is independently O or N.

20. The use according to claim 19, characterized in that R1 is H, CH3, n is an integer from 0 to 6, and m is an integer from 0 to 6; R2 is 1; R3 and R4 are independent of each other. Wherein, X is independently O or N.

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