Photosensitizer and tntp-rapamycin carrier-free self-assembled nano platform, and preparation method and application thereof

Abstract

This invention provides a photosensitizer and a carrier-free self-assembled nanoplatform of TNTP-rapamycin, along with their preparation method and applications. The photosensitizer TNTP is used as a novel photosensitizer to establish the TNTP-rapamycin carrier-free self-assembled nanoplatform. This integrates TNTP and the poorly water-soluble small-molecule anticancer drug rapamycin into the same nanoplatform, achieving synergy between photodynamic therapy (PDT) and chemotherapy, overcoming the limitations of single-treatment. This is a very promising combination therapy strategy. In vitro experiments have demonstrated that it can be well taken up by cells. Compared to a single TNTP self-assembled nanoplatform, TNTP-Ra NPs combined with photodynamic therapy and chemotherapy exhibit enhanced anticancer effects.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a photosensitizer and a carrier-free self-assembled nanoplatform for TNTP-rapamycin, as well as its preparation method and application. Background Technology

[0002] Photodynamic therapy (PDT) has shown promise as an adjunctive treatment in clinical practice due to its advantages such as being non-invasive, highly spatiotemporally selective, and having low toxicity. However, the efficiency of current clinical PDT applications is hampered by several factors, primarily the fluorescence quenching (ACQ) and reduced ROS generation caused by the aggregation of traditional organic phosphoric acid (PS) in biological systems, as well as the limitations imposed by the inherent hypoxia and immunosuppressive properties of the complex tumor microenvironment (TME).

[0003] Photosensitizers with aggregation-induced emission (AIE) effects have attracted widespread attention as a next-generation photosensitive material (PS). These photosensitizers not only endow PS with strong fluorescence properties, making them easier to track, but also enhance their ROS-generating capacity, resulting in a more potent antitumor effect. More importantly, type I photosensitizers are less dependent on oxygen and generate more cytotoxic free radical ROS. Therefore, exploring type I photosensitizers with high-performance ROS generation is essential for enhancing the antitumor efficacy of phototherapy (PDT). Nevertheless, the development of photosensitizers with type I ROS-generating capabilities is still in its early stages and requires further research and significant effort.

[0004] One of the key drivers of hypoxia and immunosuppression in tumor microenvironment (TME) is the intense aerobic glycolysis in cancer cells, known as the "Warburg effect." Metabolic reprogramming is often mediated by oncogenic signals, among which mammalian rapamycin (mTOR) signaling, as a central signaling pathway controlling the relationship between tumor metabolism and immunity, has become a popular target in anti-tumor therapy research in recent years. mTOR activation stimulates glucose uptake and aerobic glycolysis, thereby exacerbating tumor hypoxia and immunosuppression. Rapamycin (Ra) is a secondary metabolite secreted by Streptomyces spp., first discovered by scientists in 1975 from the soil of Easter Island, Chile. The FDA approved the first mTOR inhibitor in 1999 as an immunosuppressive drug to prevent transplant rejection. Rapamycin's specific inhibitory effect on mTOR is considered to have broad clinical significance. However, its poor water solubility, poor targeting ability, and low bioavailability severely affect its therapeutic efficacy.

[0005] In recent years, an increasing number of AIEgens have been developed for type I PDT, but the hydrophobicity of their large conjugated structure remains a major obstacle to practical application. Delivery via amphiphilic carriers or hydrophilic modification of AIEgens inevitably leads to trade-offs, resulting in problems such as short emission wavelengths, weak ROS generation capacity, and purification difficulties. Some photosensitizers with amphiphilic characteristics have also been reported to self-assemble in water to form carrier-free nanoparticles; however, the self-assembly of nanoparticles using only photosensitizers is a relatively singular therapeutic modality with certain limitations. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a photosensitizer.

[0007] Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned photosensitizer.

[0008] Another technical problem to be solved by the present invention is to provide a carrier-free self-assembled nanoplatform of TNTP-rapamycin containing the above-mentioned photosensitizer.

[0009] Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned TNTP-rapamycin carrier-free self-assembled nanoplatform.

[0010] The technical problem to be solved by the present invention is to provide the application of the above-mentioned TNTP-rapamycin carrier-free self-assembly nanoplatform.

[0011] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0012] A photosensitizer, TNTP, has the following chemical structural formula:

[0013]

[0014] Preferably, the photosensitizer described above is a purplish-red solid. 1H NMR (400MHz, DMSO-d6) δ8.76(d,J=6.7Hz,2H),8.13(d,J=6.7Hz,2H),7.95(d,J=15. 8Hz,1H),7.58-7.50(m,2H),7.11-7.02(m,4H),6.94(dd,J=9.2,6.8Hz,5H),6.82-6. 74(m,2H),4.54(t,J=6.8Hz,2H),4.44(dd,J=5.8,2.7Hz,2H),4.38(dd,J=5.6,2.7H z,2H),3.75(s,6H),2.40(t,J=7.1Hz,2H),2.18(p,J=7.0Hz,2H).MALDI-TOF-MS:m / z calcd for C36H34N2O7S2,M 670.795,found[M+H]671.256.

[0015] The above-mentioned photosensitizer is prepared by synthesizing the zwitterionic AIEgen-TNTP with a D-π-A structure through a three-step reaction. The specific steps are as follows:

[0016] (1) Pre-synthesized picolinium;

[0017] (2) Obtaining intermediate aldehyde TNT via Suzuki-Miyaura coupling reaction

[0018] (Thieno[3,4-b]-1,4-dioxin-5-carboxaldehyde,

[0019] 7-[4-[bis(4-methoxyphenyl)amino]phenyl]-2,3-dihydro-(ACI)), yield 82%;

[0020] (3) The intermediate aldehyde further reacted with the pre-synthesized picolinium via a knoevenagel condensation reaction to obtain the target compound TNTP in a moderate yield.

[0021] pass 1 H NMR and MALDI-TOF-MS were used to characterize and verify the target product at each step.

[0022] The application of an intermediate aldehyde TNT in the preparation of photosensitizers, wherein the intermediate aldehyde TNT has the following chemical structural formula:

[0023]

[0024] Preferably, in the application of the above-mentioned intermediate aldehyde TNT, the preparation method of the intermediate aldehyde TNT is as follows:

[0025] (1) 4,4'-dimethoxy-4”-boronic acid triphenylamine, 5-bromo-2-(3,4-vinyldioxythiophene) formaldehyde, chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) and potassium carbonate were dissolved in 1,4-dioxane aqueous solvent and refluxed at 90°C under N2 protection;

[0026] (2) After the reaction is complete, the reaction is cooled to room temperature, water and dichloromethane are added and stirred vigorously. The mixture is then extracted using a separatory funnel, the lower organic phase is collected and dried with anhydrous sodium sulfate.

[0027] (3) The dried crude product was purified by gradient elution using a silica gel column with petroleum ether / ethyl acetate as the eluent. The pure product was obtained at a volume ratio of 10:1 and dried to obtain an orange-yellow solid.

[0028] A carrier-free self-assembled nanoplatform containing the above-mentioned photosensitizer, TNTP-Ra NPs, is prepared by the following method: Rapamycin (Ra) is weighed and dissolved in TNTP (in DMSO) by ultrasonication; then the above solution is added to 9 mL of distilled water and ultrasonication is maintained; the obtained TNTP-Ra NPs solution is filtered through a membrane, placed in a dialysis bag, and dialyzed with deionized water to remove DMSO; finally, the TNTP-Ra NPs solution is obtained by ultrafiltration concentration.

[0029] Preferably, in the above-mentioned carrier-free self-assembled nanoplatform for TNTP-rapamycin, the TNTP-Ra NPs exhibit a characteristic UV absorption peak of rapamycin at 280 nm and a characteristic UV absorption peak of TNTP at 515 nm.

[0030] Preferably, the above-mentioned TNTP-rapamycin carrier-free self-assembled nanoplatform has TNTP-Ra NPs with a uniform spherical structure and an average nanoparticle size of 96.66±11.89 nm.

[0031] The specific steps for preparing the above-mentioned carrier-free self-assembled nanoplatform of TNTP-rapamycin are as follows:

[0032] (1) In DMSO, weigh rapamycin (Ra) and dissolve it in TNTP by sonication;

[0033] (2) Add the solution described in step (1) to distilled water and maintain sonication;

[0034] (3) The obtained TNTP-Ra NPs solution was filtered through a membrane, placed in a dialysis bag, and dialyzed with deionized water to remove DMSO; the final TNTP-Ra NPs solution obtained after ultrafiltration concentration was stored at 4-8℃ for further use.

[0035] Preferably, in the above-mentioned method for preparing the carrier-free self-assembled nanoplatform of TNTP-rapamycin, the membrane filtration is a 220nm membrane filtration, and the molecular weight cutoff of the dialysis bag is 8000-14000.

[0036] The above-mentioned TNTP-rapamycin carrier-free self-assembled nanoplatform is used in the preparation of anticancer drugs or anticancer detection reagents.

[0037] Beneficial effects:

[0038] The aforementioned photosensitizer, as a novel photosensitizer, is used to establish a carrier-free self-assembled nanoplatform of TNTP-rapamycin. This TNTP-rapamycin carrier-free self-assembled nanoplatform innovatively constructs a carrier-free multifunctional self-assembled nanoplatform containing both the photosensitizer TNTP and rapamycin, simultaneously overcoming ACQ, hypoxia, and the immunosuppressive tumor microenvironment. It cleverly utilizes zwitterionic AIEgen-TNTP to self-assemble into spherical nanoparticles (TNTPNPs) in water through a simple preparation method. Furthermore, the water-insoluble small-molecule anticancer drug rapamycin is loaded through hydrophobic interactions to form a multifunctional self-assembled nanoplatform (TNTN-Ra NPs). As a carrier-free self-assembled multifunctional nanoplatform integrating fluorescence imaging, chemotherapy, and photodynamic therapy, it exhibits excellent water stability, biosafety, and tumor cell uptake capacity. The multifunctional self-assembled nanoplatform (TNTP-Ra NPs) showed enhanced antitumor effects in the H460 lung cancer model compared to nanoparticles containing only a single drug.

[0039] In summary, this application integrates TNTP and the poorly water-soluble small molecule anticancer drug rapamycin into the same nanoplatform, realizing the collaboration between PDT and chemotherapy, overcoming the limitations of monotherapy. This is a very promising combination therapy strategy, and it also provides useful reference and inspiration for improving the formulation design of small molecule anticancer drugs with poor water solubility, poor targeting ability, and low bioavailability. Attached Figure Description

[0040] Figure 1 The overall synthetic route of amphiphilic AIEgen-TNTP;

[0041] Figure 2 1 H NMR spectra of Pic;

[0042] Figure 3MALDI-TOF-MS spectrum of Pic;

[0043] Figure 4 1 H NMR spectra of TNT;

[0044] Figure 5 MALDI-TOF-MS spectrum of TNT;

[0045] Figure 6 1 H NMR spectra of TNTP;

[0046] Figure 7 MALDI-TOF-MS spectrum of TNTP;

[0047] Figure 8 The UV-VIS absorption spectrum of TNTP-Ra NPs;

[0048] Figure 9 The morphology, particle size distribution, zeta potential distribution, and stability of TNTP-Ra NPs in aqueous solution and FBS at different time points are described, where (A) particle size distribution; (B) zeta potential distribution; (C) morphology of TNTP-Ra NPs; and (D) stability in aqueous solution and FBS at different time points.

[0049] Figure 10 Generate type determination for ROS of TNTP-Ra NPs;

[0050] Figure 11 For tumor cells to take up TNTP-Ra NPs;

[0051] Figure 12 The in vitro antitumor activity of TNTP-Ra NPs;

[0052] Figure 13 For in vitro ROS generation of TNTP-Ra NPs;

[0053] Figure 14 This study aimed to assess the in vivo antitumor activity of TNTP-Ra NPs. Detailed Implementation

[0054] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0055] The NCI-H460 (human large cell lung cancer) involved in the examples was purchased from Pronosai, China, in May 2023. Contact number: 400-999-2100.

[0056] Example 1

[0057] 1. Experimental instruments and reagents

[0058] Experimental instruments: 400MHz liquid nuclear magnetic resonance spectrometer (Ascend 400, Bruker), matrix-assisted laser dissociation ionization-time-of-flight mass spectrometer (Maldi-TOF, Bruker).

[0059] Experimental reagents: 4-methylpyridine (CAS108-89-4, Shanghai Mairui Biochemical Technology Co., Ltd.), 1,3-propanesulfonyl lactone (CAS1120-71-4, Shanghai Bide Pharmaceutical Technology Co., Ltd.), dichloromethane (CAS75-09-2, Tianjin Jiangtian Chemical Technology Co., Ltd.), deuterated methanol (CAS1455-13-6, Shanghai Aladdin Biochemical Technology Co., Ltd.), 4,4'-dimethoxy-4”-boronic acid triphenylamine (compound 1, CAS201802-29-1, Shanghai Bide Pharmaceutical Technology Co., Ltd.), 5-bromo-2-(3,4-vinyldioxythiophene)carboxaldehyde (compound 2, CAS... 852054-42-3, Shanghai Bid Pharmaceutical Technology Co., Ltd.), Chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (XPhos Pd G2, CAS1310584-14-5, Shanghai Mairui Biochemical Technology Co., Ltd.), Potassium carbonate (CAS 584-08-7, Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.), 1,4-dioxane (CAS 54841-74-6, Tianjin Jiangtian Chemical Technology Co., Ltd.), Anhydrous sodium sulfate (CAS15124-09-1, Tianjin Jiangtian Chemical Technology Co., Ltd.), Petroleum ether (CAS 64742-49-0, Tianjin Jiangtian Chemical Technology Co., Ltd.), Ethyl acetate (CAS141-78-6, Tianjin Jiangtian Chemical Technology Co., Ltd.), Deuterated chloroform (CAS 865-49-6, Shanghai Aladdin Biochemical Technology Co., Ltd.), deuterated dimethyl sulfoxide (CAS2206-27-1, Shanghai Aladdin Biochemical Technology Co., Ltd.), tetrahydropyrrole (CAS123-75-1, Shanghai Mairui Biochemical Technology Co., Ltd.), methanol (CAS 67-56-1, Tianjin Jiangtian Chemical Technology Co., Ltd.), tetrahydrofuran (CAS109-99-9, Tianjin Jiangtian Chemical Technology Co., Ltd.).

[0060] 2. Test methods

[0061] Synthetic compound Picolinium (Pic)

[0062] 4-Methylpyridine (93.13 g / mol, 2.0 g, 21.48 mmol, 2.1 eq.) and 1,3-propanesulfonyl lactone (122.14 g / mol, 1.22 g, 9.99 mmol, 1.0 eq.) were added to dichloromethane and refluxed at 100 °C under N2 protection. After 2 h, the reaction solution was cooled to room temperature. The precipitate was collected by filtration, washed with dichloromethane, and finally dried in an oven to obtain a white solid.

[0063] Synthetic intermediate aldehyde TNT (CAS1384897-26-0)

[0064] Compound 1 (349.18 g / mol, 349.19 mg, 1.00 mmol, 2.0 eq), compound 2 (249.08 g / mol, 124.54 mg, 0.50 mmol, 1.0 eq), XPhos Pd G2 (784.79 g / mol, 39.24 mg, 0.05 mmol, 0.1 eq.), and potassium carbonate (138.21 g / mol, 138.21 mg, 1.00 mmol, 2.0 eq.) were dissolved in a mixed solvent of 1,4-dioxane / water (v / v 5:1) and refluxed at 90 °C under N2 protection. After 2 h, the reaction was cooled to room temperature, and after adding an appropriate amount of water and dichloromethane and stirring vigorously, the mixture was extracted using a separatory funnel. The lower organic phase was collected and dried over anhydrous sodium sulfate. The crude product, after being dried by rotary evaporation, was purified by gradient elution using a silica gel column with petroleum ether / ethyl acetate as the eluent. Finally, the pure product was obtained at a v / v ratio of 10:1 and dried by rotary evaporation to obtain an orange-yellow solid.

[0065] Synthesis of target product TNTP

[0066] Intermediate aldehydes TNT (473.55 g / mol, 194.12 mg, 0.41 mmol, 1.0 eq.), Pic (215.27 g / mol, 133.47 mg, 0.62 mmol, 1.5 eq.), and tetrahydropyrrole (71.12 g / mol, ρ 0.8 g / cm³) were added. 3 110 μL (1.23 mmol, 3.0 eq.) was dissolved in a methanol / tetrahydrofuran (v / v 3:1) mixture and refluxed at 75 °C under N2 protection. After 1 hour, the reaction was cooled to room temperature, and the solvent was evaporated to dryness using a rotary evaporator. The product was purified by gradient elution with dichloromethane / methanol as the eluent, and finally purified at a v / v 10:1 ratio to obtain a pure product, which was then evaporated to dryness to give a purple-red solid.

[0067] 3. Test Results

[0068] According to Figure 1 The synthesis was carried out according to the steps shown, and the compound synthesized in each step was obtained through... 1 The characterization was verified by H NMR and MALDI-TOF-MS.

[0069] The compound Picolinium (Pic) was synthesized in 78% yield. 1 H NMR (400MHz, Methanol-d4) δ8.86–8.80(m,2H),7.93(d,J=6.2Hz,2H),4.76(d,J=14.7Hz,2H),2.84(t,J=6.9Hz,2H),2.68(s,3H),2.42(p,J=7.1Hz,2H). (See Figure 2 )MALDI-TOF-MS:m / z calcd for C9H13NO3S,M215.062,found[M+H]216.064.(See Figure 3 )

[0070] The intermediate aldehyde TNT was synthesized in 82% yield. 1 H NMR(400MHz,Chloroform-d)δ9.78(s,1H),7.52–7.42(m,2H),7.02–6.94(m,4H), 6.83–6.70(m,6H),4.29(dd,J=6.0,2.4Hz,2H),4.27–4.21(m,2H),3.70(s,6H).(See Figure 4 )MALDI-TOF-MS: m / z calcd for C36H34N2O7S2,M473.543, found M 473.255. (See Figure 5 )

[0071] The target product TNTP was synthesized in 56% yield. 1H NMR (400MHz, DMSO-d6) δ8.76(d,J=6.7Hz,2H),8.13(d,J=6.7Hz,2H),7.95(d,J= 15.8Hz,1H),7.58–7.50(m,2H),7.11–7.02(m,4H),6.94(dd,J=9.2,6.8Hz,5H), 6.82–6.74(m,2H),4.54(t,J=6.8Hz,2H),4.44(dd,J=5.8,2.7Hz,2H),4.38(dd, J=5.6, 2.7Hz, 2H), 3.75 (s, 6H), 2.40 (t, J=7.1Hz, 2H), 2.18 (p, J=7.0Hz, 2H). (See Figure 6 )MALDI-TOF-MS: m / z calcd for C36H34N2O7S2,M 670.795, found[M+H]671.256.(See Figure 7 )

[0072] Example 2

[0073] 1. Experimental instruments and reagents

[0074] Experimental instruments: Cary 60 UV-Vis spectrophotometer (CARY 60, Agilent), nanoparticle size analyzer (Zetasizer Nano ZS, Malvern), field emission transmission electron microscope (Tecnai G2F20, Philips GmbH, Netherlands).

[0075] Experimental reagents: Rapamycin (CAS 53123-88-9, Solarbio), DMSO (CAS 847778-77-2, Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0076] 2. Test methods

[0077] A method for preparing a carrier-free self-assembled nanoplatform of TNTP-rapamycin (TNTP-Ra NPs) was described. Rapamycin (Ra, 4.0 mg) was weighed and dissolved in 1 mL of TNTP (2 mg / mL in DMSO) by sonication for approximately 2 minutes. The solution was then added to 9 mL of distilled water and sonicated for another 2 minutes. The resulting TNTP-Ra NPs solution was filtered through a 220 nm membrane, placed in a dialysis bag (molecular weight cutoff = 8000-14000), and dialyzed with deionized water for 24 hours to remove DMSO. The final TNTP-Ra NPs solution, after ultrafiltration concentration, was stored at 4-8 °C for further use. Qualitative and quantitative analyses of the TNTP-Ra NPs were performed using a UV-Vis spectrophotometer. The morphology of the nanoparticles was studied using transmission electron microscopy (TEM). The particle size, zeta potential distribution, and stability at different time points in aqueous solution and FBS were monitored using a Zetasizer Nano ZS nanometer.

[0078] 3. Test Results

[0079] Such as UV-VIS spectral results ( Figure 8 As shown in the figure, compared with TNTP in DMSO, the optical properties of TNTP NPs / TNTP-Ra NPs in the 400-650 nm range did not change significantly. Compared with TNTP NPs, TNTP-Ra NPs showed a characteristic UV absorption peak of rapamycin at 280 nm, indicating the successful preparation of the self-assembled nanocomposite. A standard curve was plotted at the maximum absorption wavelength of 515 nm for quantitative analysis of TNTP. Figure 9 As shown, TEM results revealed that TNTP-Ra NPs possess a uniform spherical structure. Further Zetasizer Nano ZS analysis showed that the average particle size of the nanoparticles was 96.66 nm (PDI 0.123), with a zeta potential of -24.8 mV. The nanoparticles were stable in aqueous solution and FBS, and the increase in particle size in the stock solution after 7 days was negligible.

[0080] Example 3

[0081] 1. Experimental instruments and reagents

[0082] Test instruments:

[0083] Fully automated multifunctional microplate testing platform (FlexStation3, Molecular Devices).

[0084] Test reagents:

[0085] 9,10-Anthracene dimethyl-bis(methylene)dimalonic acid (ABDA, CAS 307554-62-7, Maclean's), hydroxyphenyl fluorescein (HPF, CAS 359010-69-8, Shanghai Maokang Biotechnology Co., Ltd.), dihydrorhodamine 123 (DHR 123, CAS109244-58-8, Tianjin Xiens Biochemical Technology Co., Ltd.), dihydroporphyrin E6 (Ce6, CAS19660-77-6, Maclean's), D-PBS (White Shark).

[0086] 2. Test methods

[0087] Using 9,10-anthratridiyl-bis(methylene)dimalonic acid (ABDA) as an indicator, the following was performed: 1 Measurement of O2 generation. ABDA (10 μM) solution was mixed with TNTP NPs, TNTP-Ra NPs, and Ce6 (2 μM). The solution was subjected to white light, 100 mW cm⁻¹. 2 After irradiation, the absorption spectrum of the indicator was monitored in the range of 350-450 nm. The decrease in absorbance at 380 nm relative to the initial value was recorded to represent the decomposition rate of ABDA.

[0088] The amount of ·OH generated was measured using hydroxyphenyl fluorescein (HPF) as an indicator. HPF (10 μM) was added to TNTP NPs, TNTP-RaNPs, and Ce6 (2 μM). The solutions were subjected to white light, 100 mW cm⁻¹. 2 After irradiation, the fluorescence signal of the indicator was monitored in the range of 500-550 nm, with an excitation wavelength of 490 nm. The increase in fluorescence intensity at 515 nm relative to the initial value was recorded to represent the generation rate of OH·.

[0089] The O2 test was conducted using dihydrorhodamine 123 (DHR 123) as an indicator. - Measurements were performed. DHR 123 (10 μM) was added to TNTP NPs, TNTP-RaNPs, and Ce6 (2 μM). The solutions were subjected to light (white light, 250 mW cm⁻¹). 2 After irradiation, the fluorescence signal of the indicator was monitored in the range of 500-550 nm, with an excitation wavelength of 490 nm. The increase in fluorescence intensity at 525 nm relative to the initial value was recorded as the expression for O2. - The production rate.

[0090] 3. Test Results

[0091] Photosensitizer-triggered phototransfer (PDT) mechanisms primarily involve two types to generate various reactive oxygen species (ROS): Type I mechanisms, which generate free radicals through electron transfer, and Type II mechanisms, which generate singlet oxygen through energy transfer. To characterize the nature of the ROS generated by TNTP-Ra NPs, ABDA, HPF, and DHR123 were used for distribution detection. 1 O2, ·OH and ·O2 - The generation of . For example Figure 10 As shown, under white light (250mW cm⁻¹) 2 Under irradiation, the fluorescence intensity of the blank control remained weak after irradiation. Compared with the Ce6 group, HPF and DHR123 showed stronger fluorescence emission in the presence of TNTPNPs and TNTP-Ra NPs, demonstrating excellent ·OH and ·O2 fluorescence. - Generation capacity. However, under the same conditions, ABDA showed no significant change in uptake in the presence of both TNTP NPs and TNTP-Ra NPs. These results suggest that TNTP NPs and TNTP-Ra NPs primarily generate type I ROS, rather than type II. Typically, type I photosensitizers are less dependent on O2 and generate more cytotoxic free radical ROS.

[0092] Example 4

[0093] 1. Experimental instruments and reagents

[0094] Experimental instrument: Super-resolution confocal / STEDmicroscopy (SP8, Leica).

[0095] Test reagents: Endoplasmic reticulum green fluorescent probe (ER-Tracker Green, Beyotime Biotechnology), NCI-H460 [H460] cell culture medium (CM-0299, Wuhan Pronosei Life Science Technology Co., Ltd.).

[0096] 2. Test methods

[0097] H460 cells were seeded in confocal imaging dishes and cultured for 24 hours per dish to allow for adequate cell adhesion. The culture medium was then replaced with fresh medium containing TNTP-Ra NPs (TNTP, 16 μM). After incubation for 1 / 3 / 6 / 9 / 12 hours, the cells were washed three times with PBS and then stained with the commercial dye ER-Tracker Green (1 μM) at 37°C for 30 minutes. Cell imaging was performed using a CLSM. For TNTP-Ra NPs, the excitation wavelength was 514 nm, and the emission filter was 660–800 nm; for ER-Tracker Green, the excitation wavelength was 488 nm, and the emission filter was 500–570 nm.

[0098] 3. Test Results

[0099] CLSM was used to monitor the uptake and accumulation of TNTP-Ra NPs by tumor cells after different incubation cycles (1 / 3 / 6 / 9 / 12h). Figure 11 As shown, a significant red fluorescence signal was recorded after 1 hour of incubation, revealing the excellent cellular uptake of TNTP-Ra NPs. With increasing incubation time, the maximum fluorescence signal intensity was observed until 6 hours, and strong fluorescence intensity was still observed after 12 hours of incubation, indicating the excellent tumor accumulation ability of TNTP-Ra NPs. This also demonstrates that TNTP-Ra NPs possess strong fluorescence properties, making them easier to track.

[0100] Example 4

[0101] 1. Experimental instruments and reagents

[0102] Experimental instrument: Fully automated multifunctional microplate testing platform (FlexStation3, Molecular Devices).

[0103] Test reagent: CCK-8 reagent (white shark).

[0104] 2. Test methods

[0105] NCI-H460 cells in logarithmic growth phase were seeded in 96-well plates and cultured for 24 hours in a cell culture incubator at 37°C and 5% CO2. Then, TNTP NPs / TNTP-Ra NPs at different concentration gradients (TNTP, 0 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM) were added, resulting in four groups with four replicates per concentration. TNTP NPs (Dark) and TNTP-Ra NPs (Dark) were incubated in the dark for 24 hours. TNTP NPs (Laser) and TNTP-Ra NPs (Laser) groups were irradiated with white light (100 mW cm⁻¹).-2 After 3 minutes, the cells were incubated at 37°C for 18 hours. The original culture medium was then removed, and 100 μl of fresh cell culture medium (containing 10% CCK8) was added to each well (avoiding air bubbles). The culture plate was then placed in an incubator for further incubation. Finally, the absorbance was measured at 450 nm using a microplate reader to calculate cell viability.

[0106] 3. Test Results

[0107] To confirm the anticancer effect of TNTP-Ra NPs, their cytotoxicity was assessed using the CCK8 assay, with the NCI-H460 cell line used as a model. Figure 12 As shown, the TNTP NPs (Dark) group after 24 hours of incubation exhibited considerably low cytotoxicity, indicating good biocompatibility. The TNTP-Ra NPs (Dark) group, due to the chemotherapeutic effect of rapamycin, showed some cytotoxicity compared to TNTP NPs (Dark) at (TNTP, 0-32 μM). Under light irradiation, both TNTP NPs (Laser) and TNTP-RaNPs (Laser) produced strong tumor-killing effects due to the PDT effect. Due to the co-assembly of Ra, TNTP-RaNPs (Laser) showed stronger cytotoxicity than TNTP NPs (Laser) at the same concentration. This indicates that TNTP-Ra NPs can achieve synergistic effects of PDT and chemotherapy in vitro, overcoming the limitations of single-treatment.

[0108] Example 5

[0109] 1. Experimental instruments and reagents

[0110] Experimental instrument: Super-resolution confocal / STEDmicroscopy (SP8, Leica).

[0111] Test reagent: 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, Beyotime Biotechnology).

[0112] 2. Test methods

[0113] NCI-H460 cells were seeded in confocal imaging dishes and cultured for 24 hours to allow for sufficient cell adhesion. They were then incubated with TNTP NPs / TNTP-Ra NPs (16 μM) for 6 hours, washed three times with D-PBS, and stained with DCFH-DA (10 μM) at 37°C for 30 minutes. The light-illuminated group was incubated for 3 minutes followed by 10 minutes of light exposure before being imaged using CLSM. The excitation wavelength was 488 nm. For DCFH-DA, the emission filter was 500-550 nm; for TNTP NPs / TNTP-Ra NPs, the emission filter was 660-800 nm.

[0114] 3. Test Results

[0115] NCI-H460 cells were stained using a cellular ROS detection probe (DCFH-DA). DCFH-DA detects intracellular ROS and then produces green fluorescence. Confocal images showed that bright green fluorescence was detected in the NCI-H460 cell group treated with TNTP NPs and TNTP-Ra NPs under laser irradiation. However, rather weak green fluorescence was observed in the group without laser irradiation. Figure 13 This indicates that TNTP-Ra NPs can produce a good PDT effect in vitro.

[0116] Example 6: Anticancer effect of TNTP-Ra NPs on human large cell lung cancer H460 tumor-bearing mice

[0117] 1. Experimental animals

[0118] SPF-grade male BALB / c nude mice were purchased from Beijing Spaford Biotechnology Co., Ltd.

[0119] 2. Test methods

[0120] H460 cells were subcutaneously injected into the lower right back and axillary region of male BALB / C (nu / nu) nude mice in complete cell culture medium. The H460 tumor-bearing mice were randomly divided into 5 groups (n=4). The tumors were allowed to grow to approximately 100 mm². 3 The following treatments were administered to patients on the left and right sides respectively: (1) control group, (2) TNTP NPs (Dark), (3) TNTP-Ra NPs (Dark), (4) TNTP NPs (Laser), (5) TNTP-Ra NPs (Laser) (TNTP 1mg / mL, 20μL). Six hours after intratumoral injection, the Laser group was treated with white light (300mW cm⁻¹). 2Each mouse tumor was irradiated for 5 minutes continuously. The injection was repeated every 2 days for 10 consecutive days. The length and width of the tumor and the mouse's weight were recorded before each injection during the treatment period. Tumor volume was calculated using the formula: Volume = Length × Width. 2 / 2(mm 3 On day 11, mice were sacrificed, and tumors and major organs (heart, liver, spleen, lungs, and kidneys) from each group of mice were collected and fixed for subsequent analysis.

[0121] 3. Test Results

[0122] In vivo tumor growth inhibition was performed for 10 consecutive days to evaluate the anti-cancer effect. Figure 14 A) and weight change monitoring ( Figure 14 (B) To assess drug safety. Tumor size continued to increase in both the control and TNTP NPs (Dark) groups, and body weight showed no significant changes, indicating that only TNTP NPs exhibited negligible dark toxicity. Conversely, treatment with TNTP-Ra NPs under dark conditions significantly inhibited tumor growth because the released Ra exerted a CHT effect to suppress tumor cell growth. For the TNTP NPs group under laser irradiation, tumor growth was also greatly inhibited due to PDT. The best tumor-suppressing effect was observed with TNTP-Ra NPs under laser irradiation, with almost complete inhibition of tumor growth and minimal impact on body weight changes, indicating good biocompatibility of the nanomedicine.

[0123] The experimental results of the above embodiments demonstrate that the TNTP-Ra NPs in this application, as a multifunctional diagnostic and therapeutic nanoplatform integrating fluorescence imaging, chemotherapy, and photodynamic therapy, overcomes the shortcomings of rapamycin, such as poor water solubility, poor targeting ability, and low bioavailability. Based on carrier-free self-assembly technology, TNTP-Ra NPs exhibit excellent water stability, biosafety, and tumor cell uptake ability. In the H460 lung cancer model, it shows enhanced anti-tumor effects compared to nanoparticles containing only a single drug. In vitro experiments have demonstrated that it can be well taken up by cells. Compared to a single TNTP self-assembled nanoplatform, TNTP-Ra NPs combined with photodynamic therapy (PDT) and chemotherapy (CHT) have enhanced anti-cancer effects.

[0124] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. All technical improvements and modifications made by those skilled in the art based on the technical solution of the present invention are considered to be within the scope of protection of the present invention.

Claims

1. A carrier-free self-assembled nanoparticle containing a photosensitizer TNTP-rapamycin, characterized in that: Named TNTP-Ra NPs, the photosensitizer TNTP has the following chemical structural formula: The TNTP-Ra NPs solution was prepared by the following method: rapamycin was weighed and dissolved in TNTP; then the solution was added to distilled water; the resulting TNTP-Ra NPs solution was filtered through a membrane, placed in a dialysis bag, and dialyzed with deionized water. Finally, after ultrafiltration and concentration, TNTP-rapamycin carrier-free self-assembled nanoparticles were obtained.

2. The TNTP-rapamycin vectorless self-assembling nanoparticle of claim 1, wherein: The TNTP-Ra NPs exhibit a characteristic UV absorption peak of rapamycin at 280 nm and a characteristic UV absorption peak of TNTP at 515 nm.

3. The TNTP-rapamycin vectorless self-assembling nanoparticle of claim 1, wherein: The TNTP-Ra NPs have a uniform spherical structure and the average particle size of the nanoparticles is 96.66±11.89 nm.

4. The method of producing TNTP-rapamycin nanocarriers without a carrier according to any one of claims 1 to 3, characterized in that: The specific steps are as follows: (1) In a DMSO environment, weigh rapamycin and dissolve it in photosensitizer TNTP by sonication; (2) Add the solution described in step (1) to 9 mL of distilled water and maintain sonication; (3) The obtained TNTP-Ra NPs solution was filtered through a membrane, placed in a dialysis bag, and dialyzed with deionized water to remove DMSO; the TNTP-Ra NPs particles obtained after ultrafiltration concentration were stored at 4-8℃.

5. The method for preparing carrier-free self-assembled TNTP-rapamycin nanoparticles according to claim 4, characterized in that: The photosensitizer TNTP is prepared as follows: (1) A pre-synthesized picolinium having the following chemical structural formula: ; (2) The intermediate aldehyde TNT was obtained by the Suzuki–Miyaura coupling reaction, and the intermediate aldehyde TNT has the following chemical structural formula: ; (3) The intermediate aldehyde TNT undergoes a knoevenagel condensation reaction with the pre-synthesized picolinium to obtain the target compound TNTP.

6. The method for preparing carrier-free self-assembled TNTP-rapamycin nanoparticles according to claim 5, characterized in that: The preparation method of the intermediate aldehyde TNT is as follows: (1) 4,4'-dimethoxy-4''-boronic acid triphenylamine, 5-bromo-2-(3,4-vinyldioxythiophene) formaldehyde, chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) and potassium carbonate were dissolved in 1,4-dioxane aqueous solvent and refluxed at 90°C under N2 protection; (2) After the reaction is complete, the reaction is cooled to room temperature, water and dichloromethane are added and stirred vigorously, then extracted with a separatory funnel, the lower organic phase is collected and dried with anhydrous sodium sulfate; (3) The dried crude product was purified by gradient elution using a silica gel column with petroleum ether / ethyl acetate as the eluent. The pure product was obtained at a volume ratio of 10:1 and dried to obtain an orange-yellow solid.

7. The method for preparing carrier-free self-assembled TNTP-rapamycin nanoparticles according to claim 4, characterized in that: The membrane filtration is a 220nm membrane filtration, and the molecular weight cutoff of the dialysis bag is 8000-14000 Da.

8. The use of the TNTP-rapamycin carrier-free self-assembled nanoparticles according to any one of claims 1-3 in the preparation of anticancer drugs or anticancer detection reagents.

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