Use of pegylated indocyanine dye conjugated paclitaxel prodrug type compounds in anti-tumor cells

Abstract

This invention belongs to the field of pharmaceutical science, specifically relating to the application of a PEGylated indocyanine dye-conjugated paclitaxel prodrug compound in antitumor cells. The application of this PEGylated indocyanine dye-conjugated paclitaxel prodrug compound in antitumor cells is characterized by the following structure: after targeting tumor cells, this compound generates a large amount of reactive oxygen species (ROS) under near-infrared light irradiation, causing mitochondrial damage to tumor cells, thereby exerting cytotoxic effects while simultaneously combining photodynamic therapy to kill tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical science, specifically relating to the application of PEGylated indocyanine dye-coupled paclitaxel prodrug compound in antitumor cells. Background Technology

[0002] Cancer treatment is a global challenge. Following traditional treatments such as surgery, chemotherapy, and radiotherapy, photodynamic therapy (PDT) is the fourth type of cancer treatment. It boasts advantages such as being non-invasive or minimally invasive, highly selective, and capable of synergistic effects with other treatments, making it a new research direction and hot topic in the field of cancer treatment. Its principle is as follows: a photosensitizer selectively concentrates at the target site, enters tumor cells via ingestion, and is activated by light of an appropriate wavelength, generating cytotoxic reactive oxygen species (ROS), thereby selectively killing tumor cells. As one of the three key elements of PDT, the photosensitizer is the most crucial, playing a decisive role in promoting the clinical development of PDT. Summary of the Invention

[0003] The purpose of this invention is to provide an application of a PEGylated indocyanine dye-coupled paclitaxel prodrug compound in antitumor cells.

[0004] To achieve the above objectives, the technical solution of the present invention is as follows:

[0005] Application of a PEGylated indocyanine dye-coupled paclitaxel prodrug compound in antitumor cells, the compound having the following structure:

[0006]

[0007] Application of the compound in combination with near-infrared light irradiation in promoting mitochondrial damage in tumor cells.

[0008] The application of the compound in combination with near-infrared light irradiation to promote mitochondrial damage in tumor cells, thereby killing tumor cells.

[0009] The compound is HP-PTX; the near-infrared light irradiation conditions are 808 nm and 1 W / cm². 2 , irradiate for 5 minutes.

[0010] The tumor cells were mouse breast cancer cells 4T1 and human breast cancer cells MCF-7.

[0011] Advantages of this invention:

[0012] Paclitaxel is a cytotoxic antitumor drug. HP-PTX, as a prodrug of paclitaxel, targets tumor cells via organic anion transporting polypeptides (OATP). Under near-infrared light irradiation, it induces tumor cells to produce reactive oxygen species, leading to mitochondrial damage and thus killing tumor cells. HP-PTX selectively targets tumor cells, reducing damage to normal tissues. It kills tumor cells through both cytotoxic and photodynamic therapies, thus offering the advantage of synergistic efficacy and reduced toxicity. Attached Figure Description

[0013] Figure 1 CLSM imaging of HP-PTX accumulation in MCF-7 cells and subcellular co-localization of Mito-TrackerGreen in MCF-7 cells.

[0014] Figure 2 Flow cytometry was used to assess the uptake of HP-PTX by MCF-7 cells.

[0015] Figure 3 Quantitative analysis of HP-PTX uptake in MCF-7 cells

[0016] Figure 4 Flow cytometry was used to evaluate the uptake mechanism of HP-PTX in cancer cells after pretreatment with BSP (400 μM) for 30 minutes.

[0017] Figure 5 Quantitative analysis of the mechanism of HP-PTX uptake in MCF-7 cells

[0018] Figure 6 Mitochondrial fluorescence colocalization assay was used to analyze the correlation intensity of HP-PTX in MCF-7 cells.

[0019] Figure 7 CLSM imaging of HP-PTX accumulation in 4T1 tumor cells and subcellular co-localization of Mito-TrackerGreen in 4T1 cells.

[0020] Figure 8 Flow cytometry was used to assess the uptake of HP-PTX by 4T1 cells.

[0021] Figure 9 Quantitative analysis of HP-PTX uptake by 4T1 cells

[0022] Figure 10 CLSM imaging of HP-PTX accumulation in LO-2 cancer cells and subcellular co-localization of Mito-TrackerGreen in LO-2 cells.

[0023] Figure 11Flow cytometry was used to assess the uptake of HP-PTX by LO-2 cells.

[0024] Figure 12 Quantitative analysis of HP-PTX uptake in .LO-2 cells

[0025] Figure 13 .ROS Generation Level Detection

[0026] Figure 14 .ROS-induced mitochondrial damage

[0027] Figure 15 .CCK-8 test

[0028] Figure 16 Real-time near-infrared fluorescence images in MCF-7 tumor-bearing mice after intravenous injection of IR808-1 and HP-PTX.

[0029] Figure 17 Quantitative evaluation of the mean fluorescence intensity of IR808-1 and HP-PTX

[0030] Figure 18 Near-infrared fluorescence images of tumors and tissues in MCF-7 tumor-bearing mice after 12 hours of IR808-1 treatment.

[0031] Figure 19 Near-infrared fluorescence images of tumors and tissues in MCF-7 tumor-bearing mice 12 hours after HP-PTX treatment.

[0032] Figure 20 Quantitatively evaluate the mean fluorescence intensity of tumors and tissues 12 h after treatment with IR808-1 and HP-PTX.

[0033] Figure 21 Frozen sections of major organs from MCF-7 tumor-bearing mice after 4 hours of IR808-1 treatment.

[0034] Figure 22 Frozen sections of major organs from MCF-7 tumor-bearing mice 4 hours after HP-PTX treatment.

[0035] Figure 23 .Schematic diagram of the treatment process for MCF-7 tumors

[0036] Figure 24 Ex vivo images of tumors in MCF-7 tumor-bearing mice from different drug administration groups (n=3)

[0037] Figure 25 Tumor volume and body weight change curves for each treatment group

[0038] Figure 26 Tumor inhibition rate in each treatment group

[0039] Figure 27 H&E staining of tumors in mice of each treatment group

[0040] Figure 28 Blood routine tests of mice in each treatment group Detailed Implementation

[0041] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0042] The HP-PTX compounds in the following examples were prepared according to existing techniques, and the cell lines used were all purchased from Sigma-Aldrich.

[0043] Example

[0044] 1. In vitro cellular uptake and its mechanism

[0045] To observe the selective accumulation of HP-PTX in cancer and normal cells, one normal cell line (LO-2) and two cancer cell lines (4T1 and MCF-7) were injected with 1×10⁻⁶ cells per cell line. 4 Cells were seeded at density per well overnight. Different concentrations of HP-PTX (5, 10, 20, 40 μM) were added to each cell line and incubated for 1 h. Then, cells were stained with MitoTracker Greeen for 15 min. Cells were then stained with Hoechst 33342 for 15 min. Cells were then observed under a fluorescence microscope (see [link to relevant documentation]). Figure 1 , 7, 10).

[0046] 4T1, MCF-7, and LO-2 cells were seeded in 6-well plates. After 1 hour, different concentrations of HP-PTX (5, 10, 20, and 40 μM) were added to each cell line and incubated for 1 hour. Cells were then digested with trypsin. Finally, intracellular fluorescence intensity was detected by flow cytometry (see [link to relevant documentation]). Figure 2 , 3, 8, 9, 11, 12).

[0047] In addition, flow cytometry was used to investigate the mechanism of HP-PTX uptake by tumor cells. MCF-7 cells were seeded into 6-well plates and incubated for 24 h, followed by incubation with 400 μM BSP (an OATP competitive inhibitor) for 30 min, and then treated with HP-PTX (20 μM) for 4 h. Cells were digested with trypsin, and the intensity of intracellular fluorescence was detected by flow cytometry (see [link to study]). Figure 4 ,5).

[0048] Organic anion transporting polypeptides (OATP) are transport peptides highly expressed in tumor cells such as liver cancer, breast cancer, and lung cancer. Indocyanine dyes can target tumor cells via OATP and enter the cells via active transport. Due to the high transmembrane negative potential of mitochondria, the positively charged indocyanine dyes can further target and accumulate in the mitochondria of tumor cells. We used three cell types: breast cancer cells 4T1, MCF-7, and human liver cancer cells LO-2 to study the targeted uptake mechanism of HP-PTX by tumor cells. The results showed that after co-incubation with cells, the fluorescence intensity of HP-PTX in MCF-7, 4T1, and LO-2 cells was 31.94 times that of the control group. Figure 3 ), 3.34 times ( Figure 9 ) and 1.89 times ( Figure 12 This indicates that HP-PTX selectively targets tumor cells, with MCF-7 cells exhibiting the strongest uptake capacity due to the high expression of the organic anion transport peptide OATP on their surface. Therefore, the mechanism of HP-PTX uptake in MCF-7 cells was further investigated using the OATP competitive inhibitor sulfobromine protein (BSP). Figure 4 The results showed that after BSP pretreatment, the intracellular fluorescence intensity of HP-PTX was significantly reduced when co-incubated with cells, thus proving that the targeted uptake of HP-PTX by tumor cells is mediated by OATP.

[0049] 2. In vitro ROS generation

[0050] MCF-7 cells were seeded in 48-well plates and incubated for 12 h. PTX (2 μM) or HP-PTX (1 μM, 808 nm, 1 W / cm²) was then added. 2 Incubate for 1 h with either irradiated or unirradiated fluorescein diacetate (DCFH-DA) for 5 min. Then, treat MCF-7 cells with 2'7'-dichlorofluorescein diacetate (DCFH-DA) (2 μL) for 30 min. Finally, observe MCF-7 cells using a fluorescence microscope (CLSM) to assess intracellular ROS levels (see [link to CLSM]). Figure 13 ).

[0051] 3. ROS-induced mitochondrial damage

[0052] MCF-7 cells were co-incubated with HP-PTX (2 μM) in 6-well plates for 1 h, and then treated with 808 nm, 1 W / cm². 2One well was irradiated with laser for 5 min, while the other well was left unirradiated. MCF-7 cells were then digested with trypsin and centrifuged at 4°C. MCF-7 cells were fixed with 2.5% glutaraldehyde. Subsequently, the MCF-7 cells were placed in gelatin and fixed at 60°C for 24 h, then sectioned into 70 nm sections. The sections were then fixed onto a copper grid using a Formvar membrane (300 mesh). Staining was performed with 2% uranyl acetate for 30 min. Then, staining was performed with 3% lead citrate for 30 min. Finally, mitochondrial damage was examined using transmission electron microscopy (see [link to study]). Figure 14 ).

[0053] Depend on Figure 13 and 14 The levels of reactive oxygen species (ROS) in HP-PTX-induced MCF-7 cells were detected using the DCFH-DA reactive oxygen species assay kit. The results showed that after co-incubation with HP-PTX at 808 nm and 1 W / cm², the levels of reactive oxygen species (ROS) in MCF-7 cells were significantly reduced. 2 Under laser irradiation, tumor cells can produce a large amount of ROS; then, the degree of mitochondrial damage in MCF-7 cells was observed using biological transmission electron microscopy. The results showed that the inner mitochondrial cristae and matrix of the unirradiated HP-PTX group were well developed, while the mitochondrial matrix was completely lost after 808nm laser irradiation, indicating that the ROS generated after irradiation caused mitochondrial damage.

[0054] 4. CCK-8 Experiment

[0055] PTX, HP-PTX, and HP-PTX (808nm, 1W / cm²) were evaluated using a CCK-8 assay kit. 2 Cytotoxicity was observed (5 min). MCF-7 cells (5 × 10⁶ cells / well) were seeded in 96-well plates. 3 Cells were collected (cells / well) and incubated overnight in a cell culture incubator containing 5% CO2. 100 μL of culture medium containing different concentrations of PTX or HP-PTX (0.125, 0.25, 0.5, 1, 2 μM) was added and incubated for 24 h. Then, fresh culture medium containing 10% CCK-8 was added and incubated for 30 min. Finally, the absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated using the following formula (see [link to microplate reader]). Figure 15 Cytotoxicity % = (Experimental group OD value - PBS group OD value) / (Control group OD value - PBS group OD value)

[0056] Depend on Figure 15 As can be seen, the CCK-8 method was used to evaluate PTX and HP-PTX (808nm, 1W / cm²). 2Antiproliferative activity of PTX, HP-PTX and HP-PTX (808nm, 1W / cm) against MCF-7 cells (5 min, laser irradiation and no irradiation). 2 IC of group (5 min) 50 The values ​​were 0.508±0.38μM, 0.595±0.93μM and 0.478±0.85μM, respectively. Without laser irradiation, HP-PTX had weaker anti-tumor cell proliferation activity than PTX. After laser irradiation, HP-PTX had increased anti-tumor cell proliferation activity and was superior to PTX. This can be attributed to HP-PTX inducing ROS production after irradiation, which led to mitochondrial damage and affected cell proliferation.

[0057] 5. Near-infrared imaging for tumor targeting

[0058] MCF-7 cell suspension (10) was injected subcutaneously under aseptic conditions. 7 A BALB / c nude mouse model bearing tumors was established using individual cells. When the tumor volume reached approximately 50 mm², the tumor was successfully induced. 3 Mice were randomly divided into two groups (n=3 per group) and intravenously injected with IR808-1 and HP-PTX (1 mg / kg / 100 μL), respectively. In vivo images were captured at different intervals (0, 2, 4, 8, and 12 h) using the IVIS SPECTRUM imaging system and analyzed using Living Image software. After 12 h, mice were sacrificed, and tissues, organs, and tumors were collected from each mouse. The tumor-targeting effect of HP-PTX was evaluated using the IVIS SPECTRUM imaging system (see [link to IVIS SPECTRUM imaging system]). Figures 16-20 ).

[0059] 6. Frozen slicing

[0060] When the tumor volume is approximately 50 mm 3 BALB / c nude mice were randomly divided into an IR808-1 group and an HP-PTX group (n=1 in each group). Indocyanine dye IR808-1 and HP-PTX (1 mg / kg / 100 μL) were administered intravenously. Organs and tissues were collected 4 h later. Tissues were embedded using OTC embedding medium and sectioned (5 μm). Finally, sections were placed on slides and stained with Hoechst 33342. Data were collected and examined using a fluorescence microscope (see [link to relevant documentation]). Figure 21 ,twenty two).

[0061] Depend on Figures 16-22To investigate their in vivo tumor-targeting capabilities, BALB / c nude mice carrying MCF-7 tumors were intravenously injected with indocyanine dye IR808-1 and HP-PTX (1 mg / kg). The biodistribution of IR808-1 and HP-PTX in the mice was examined at different time points. Results showed that IR808-1 did not exhibit significant fluorescence at the tumor site, while HP-PTX showed strong fluorescence, reaching its peak intensity after 4 hours, demonstrating that HP-PTX can selectively target tumor tissue in vivo. Twelve hours after administration, the mice were sacrificed, dissected, and their tissues and organs were collected to assess the levels of IR808-1 and HP-PTX in different organs. Results indicated that HP-PTX remained significantly present at the tumor site 12 hours after administration, while IR808-1 was largely metabolized.

[0062] In the frozen section experiment, two BALB / c nude mice were injected with IR808-1 and HP-PTX (1 mg / kg). Four hours later, they were sacrificed, dissected, and their tissues and organs were collected for frozen section analysis. The results showed that the test compound was mainly metabolized by the liver and excreted by the kidneys. Meanwhile, the fluorescence intensity of the tumor site in the HP-PTX group was still higher than that of IR808-1.

[0063] 7. In vivo antitumor activity experiment

[0064] When the tumor volume reaches approximately 50 mm 3 Mice were randomly divided into four groups: blank control group, PTX group, HP-PTX group without light, and HP-PTX group with light (808nm, 1W / cm²). 2 Each administration was performed for 5 minutes (n=3 per group). The PTX group received PTX (10 mg / kg / 100 μL) intravenously, while the HP-PTX groups without light and under light received HP-PTX (10 mg / kg / 100 μL) intravenously. Administered every other day, and tumor size was measured every two days using calipers (see [link to relevant documentation]). Figures 23-26 Mice were administered the drug for 8 days, observed for 7 days, and then sacrificed 14 days later. Tumor tissue was collected, carefully weighed, and photographed. Tumor volume was calculated (volume = length × width). 2 ×0.5).

[0065] 8. In vivo safety evaluation

[0066] BALB / c nude mice were randomly divided into three groups: a control group, a PTX group, and an HP-PTX group (n=3 in each group). The PTX group received intravenous injections of PTX (10 mg / kg / 100 μL) every two days. The HP-PTX groups (no light exposure and light exposure) received intravenous injections of HP-PTX (10 mg / kg / 100 μL) every two days. The control group received an equal volume of physiological saline. After three injections, blood was collected from the mice's eyes and centrifuged at 4°C and 1200 rpm for 5 min. Complete blood counts were performed using mouse serum (see [link to relevant documentation]). Figure 28 ).

[0067] In addition, tumors were removed from mice in the drug-treated group 14 days later and fixed with 4% paraformaldehyde. Tumor damage was observed by H&E staining.

[0068] Depend on Figures 23-28 As can be seen, BALB / c nude mice were inoculated with MCF-7 cells, and on day 10 after cell inoculation, they were intravenously injected with PBS, PTX, and HP-PTX (808nm, 1W / cm). 2 (5 min, laser irradiation and no irradiation). All mice were administered the drug once every other day for 8 days, followed by 7 days of monitoring. Results showed that the HP-PTX (laser irradiation) group exhibited the best tumor inhibition rate (69.52%) under combined chemotherapy and photodynamic therapy. Simultaneously, blood routine tests were performed on the PTX and HP-PTX groups, indicating that HP-PTX had minimal toxicity in vivo and was relatively safe. H&E staining of tumor tissues from each treatment group showed that HP-PTX significantly killed tumor cells under light irradiation. These results indicate that HP-PTX possesses photodynamic therapy (PDT) activity, and its anti-tumor effect is enhanced under light irradiation through the combined effects of cytotoxicity and PDT.

Claims

1. The use of a PEGylated indocyanine dye conjugated paclitaxel prodrug type compound in anti-tumor cells, characterized in that: The compound has the following structure.

2. Use according to claim 1, characterized in that: Application of the compound in combination with near-infrared light irradiation in promoting mitochondrial damage in tumor cells.

3. Use according to claim 2, characterized in that: The application of the compound in combination with near-infrared light irradiation to promote mitochondrial damage in tumor cells, thereby killing tumor cells.

4. Use according to claim 3, characterized in that: The compound is HP-PTX, the near-infrared light irradiation condition is 808 nm, and the intensity is 1 W / cm 2 Irradiation for 5 min.

5. The application according to any one of claims 1-4, characterized in that: The tumor cells were mouse breast cancer cells 4T1 and human breast cancer cells MCF-7.

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