A DNA damaging prodrug, nanoparticle, preparation method and application

Abstract

The present invention relates to the field of biomedicine technology, and discloses a DNA damage prodrug, nanoparticles, a preparation method and an application. The DNA damage prodrug CSP of the present invention is a photosensitizer pyropheophorbide a (PPa) and a DNA damage agent chlorambucil (Cb) as a simulated drug, bridged by a GSH-responsive disulfide bond. The nano drug of the present invention is prepared by a nanoprecipitation method using the DNA damage prodrug of the present invention and the PARP inhibitor Olaparib. The present invention can realize the GSH-triggered release of chlorambucil and Olaparib in a high GSH concentration tumor microenvironment, thereby enhancing DNA damage and DNA damage repair dysfunction, respectively, and promoting the effect of chemotherapy-photodynamic synergistic therapy for breast cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a DNA damaging prodrug, nanoparticles, a preparation method and application thereof. Background Art

[0002] Currently, breast cancer (BC) is one of the most common malignant tumors. Due to its high morbidity and mortality, it seriously threatens the health and life of women all over the world. Among the many breast cancer treatments that have been studied recently, photodynamic therapy (PDT) has attracted much attention due to its non-invasive, high precision, good controllability and local effectiveness. It has a bright future. Photodynamic therapy is a new method of treating tumors, precancerous lesions, proliferative skin diseases, and vascular diseases using photosensitizer molecules and laser activation; after the photosensitizer molecules are internalized by tumor cells, they will be excited under the irradiation of lasers of a specific wavelength, thereby producing a large amount of singlet oxygen ( 1 O 2 ) and other reactive oxygen species (ROS). The generation of reactive oxygen species can cause rapid oxidative damage to key biomacromolecules such as DNA, proteins, and unsaturated lipids, leading to damage to key organelles such as the nucleus and mitochondria, and ultimately causing tumor cell death.

[0003] However, the therapeutic effect of photodynamic therapy is still unsatisfactory due to the inherent properties of photosensitizers, the complexity of the tumor microenvironment, and the DNA damage repair mechanism present in tumor cells. The main reason for the limited practical application of photodynamic therapy is the insufficient distribution and retention of photosensitizers, because most photosensitizers have low water solubility and poor hydrophobicity. Nanocarriers can not only increase the solubility of photosensitizers, but also provide suitable size and surface properties to prolong blood circulation, thereby promoting the distribution and retention of photosensitizers in tumor sites through the high permeability and retention effect (EPR) of solid tumors. In addition, high levels of glutathione (GSH) in the tumor microenvironment will consume a large amount of reactive oxygen species generated by the excited photosensitizer, thereby reducing the therapeutic efficiency of PDT. Furthermore, tumor cells have a complex set of DNA repair mechanisms, which tumor cells will immediately activate complex cell repair mechanisms to combat DNA damage.

[0004] Among them, Olaparib (Ola) has been approved by the US Food and Drug Administration for the treatment of recurrent ovarian cancer and advanced breast cancer, which can cause death by disrupting the DNA repair pathway. Chlorambucil is a type of nitrogen mustard DNA alkylating agent and one of the earliest DNA damaging agents used in chemotherapy of lymphoma and some solid tumors. The N, N-bis(2-chloroethyl)amine group in chlorambucil can react rapidly with nucleic acids to alkylate genomic DNA, leading to cell apoptosis. Therefore, integrating the functions of pyropheophorbide a (PPa), PARP inhibitor (Ola) and chlorambucil into a multifunctional nanomedicine will provide a new option for significantly improving the efficacy of synergistic chemotherapy-photodynamic therapy by enhancing DNA damage and inhibiting DNA damage repair function. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a DNA damaging prodrug, nanoparticles, preparation method and application, which can achieve GSH-triggered release of chlorambucil and olaparib in a high GSH tumor microenvironment, thereby enhancing DNA damage and inhibiting DNA damage repair function, respectively, and promoting the effect of chemotherapy-photodynamic synergistic therapy for breast cancer.

[0006] The present invention solves the above technical problems by the following technical means:

[0007] In a first aspect, the present invention discloses a DNA damaging prodrug, wherein the structural formula of the prodrug is shown in Formula I:

[0008]

[0009] Furthermore, the preparation method of the prodrug comprises the following steps:

[0010] S1, dissolving chlorambucil (Cb) in anhydrous dichloromethane, and then adding 4-dimethylaminopyridine (DMAP) and N, N-dicyclohexylcarbodiimide (DCC) to obtain a mixture 1;

[0011] S2, adding bis(2-hydroxyethyl) disulfide (SS) in anhydrous dichloromethane (DCM) to mixture 1 to obtain mixture 2;

[0012] S3, stirring the mixture 2 at room temperature, cooling the reaction solution mixture and precipitating, filtering, and concentrating and purifying the filtrate by rotary evaporation to obtain product 1 (Cb-SS);

[0013] S4, dissolving pyropheophorbide a (PPa) in anhydrous dichloromethane (DCM), then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) under stirring, and stirring to react;

[0014] S5. Add product 1 (Cb-SS) to the stirred reaction in step S4, stir the reaction at room temperature, and then concentrate and purify the mixture by rotary evaporation to obtain the prodrug (CSP).

[0015] Furthermore, the molar ratio of chlorambucil, 4-dimethylaminopyridine, N,N-dicyclohexylcarbodiimide and bis(2-hydroxyethyl)disulfide is 1:1.2:1.2:2.

[0016] Furthermore, the molar ratio of the product 1 to pyropheophorbide a is 1.2:1.

[0017] In a second aspect, the present invention also discloses the use of the above-mentioned DNA damaging prodrug in the preparation of a chemotherapy-photodynamic therapy combined reagent drug or an anti-breast cancer drug.

[0018] In a third aspect, the present invention also discloses the use of the above-mentioned DNA damaging prodrug in the preparation of photoacoustic imaging products or the preparation of fluorescence imaging products.

[0019] In a fourth aspect, the present invention further discloses a DNA damaging nanoparticle, wherein the nanoparticle comprises a nanoparticle CSP NP and a nanoparticle CSP / Ola NP, wherein the nanoparticle CSPNP is a prodrug and a DSPE-PEG 2000 The nanoparticle CSP / Ola NP is prepared by self-assembly, and the above-mentioned prodrug is combined with DSPE-PEG 2000 It is prepared by self-assembly of olaparib (Ola).

[0020] In a fifth aspect, the present invention also discloses a method for preparing DNA damaging nanoparticles, wherein the nanoparticles include nanoparticles CSP NP and nanoparticles CSP / Ola NP. The method for preparing the nanoparticles CSP NP comprises the following steps: 2000 Dissolved in dimethyl sulfoxide, respectively, to obtain prodrug solution, DSPE-PEG 2000 Solution; take prodrug solution and DSPE-PEG 2000 The solutions were mixed and dispersed in water and stirred to obtain mixture three; dimethyl sulfoxide (DMSO) was removed by dialysis, and then the solution was filtered to remove free prodrug to obtain nanoparticles CSP NP.

[0021] Furthermore, the preparation method of the nanoparticle CSP / OlaNP comprises the following steps: 2000 Dissolved in dimethyl sulfoxide, respectively, to obtain prodrug solution, olaparib (Ola) solution, DSPE-PEG 2000solution; the above solution was mixed and dispersed in water and stirred to obtain a mixture of four; dimethyl sulfoxide (DMSO) was removed by dialysis, and then the solution was filtered to remove the unencapsulated reagent to obtain nanoparticles CSP / Ola NP.

[0022] Further, in the preparation of the nanoparticle CSP NP, the prodrug and DSPE-PEG 2000 The mass ratio of DSPE-PEG is 1:0%-20%; in the preparation of the nanoparticles CSP / Ola NP, the prodrug solution, DSPE-PEG 2000 The mass ratio of solution to olaparib (Ola) was 1:0.15:0.45.

[0023] Beneficial effects of the present invention:

[0024] 1. The chlorambucil of the present invention is combined with pyropheophorbide a through a disulfide bond to form a smart response prodrug (Cb-SS-PPa, referred to as CSP) with dispersion stability; the prodrug CSP is combined with DSPE-PEG 2000 It can self-assemble into nanoparticles CSPNP, and the nanoparticles CSP NP can be further coated with olaparib to construct nanodrug CSP / Ola NP; this new type of nanoparticle has unique properties and has better efficacy in enhancing TNBC chemotherapy-photodynamic therapy.

[0025] 2. The PEGylation of the present invention can prolong the circulation time of CSP / Ola NP in the blood and increase its accumulation in the tumor area through passive targeting; when CSP / Ola NP is internalized by cancer cells, the disulfide bonds of Cb-SS-PPa can achieve GSH-triggered release of chlorambucil and olaparib in a high-GSH tumor microenvironment, thereby enhancing DNA damage and destroying DNA damage repair function, respectively.

[0026] 3. In the present invention, under the synergistic effect of chlorambucil and olaparib, the oxidative damage induced by pyropheophorbide a during PDT is further amplified, and the purpose of enhancing chemo-photodynamic therapy is ultimately achieved by promoting cell apoptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a diagram of the synthesis and preparation method of the DNA damaging prodrug of the present invention;

[0028] Figure 2 It is the Cb-SS-PPa of the DNA damaging prodrug of the present invention. 1 H-NMR spectrum;

[0029] Figure 3 It is the Cb-SS-PPa of the DNA damaging prodrug of the present invention. 13 C-NMR spectrum;

[0030] Figure 4 is the HRMS graph of Cb-SS-PPa of the DNA damaging prodrug of the present invention;

[0031] Figure 5 is a measurement graph of CSP NP after dynamic light scattering in the present invention;

[0032] Figure 6 The present invention contains different DSPE-PEG 2000 Particle size and PDI diagram of the formulated nanoparticles;

[0033] Figure 7 1 is a graph showing the particle size and potential distribution of the drug-loaded nanoparticles CSP NP and CSP / Ola NP of the present invention;

[0034] Figure 8 is a TEM image of the morphological change of the nanoparticle CSP NP of the present invention in response to GSH;

[0035] Fig. 9 Graphs showing storage stability and serum stability of the drug-loaded nanoparticles CSP NP and CSP / Ola NP of the present invention;

[0036] Fig.10 is the UV-visible spectra of the prodrug CSP and CSP NP of the present invention;

[0037] Fig.11 The present invention produces a singlet in vitro 1 O 2 Ability test result chart;

[0038] Fig.12 3 is a graph showing the results of investigating the cellular uptake ability of PPa, CSP, CSP NP and CSP / Ola NP of the present invention;

[0039] Fig.13 This is a diagram showing the results of investigating the cellular uptake mechanism of the CSP / Ola NP of the present invention;

[0040] Fig.14 is a graph of the ability to produce ROS in cells of the present invention;

[0041] Fig.15 This is a graph showing the results of the detection of GSH content in cells of the present invention;

[0042] Fig.16 is a graph showing the results of an immunofluorescence assay of the present invention;

[0043] Fig.17 is a Western blot of the present invention;

[0044] Fig.18This is a graph showing the detection results of cell survival rate under the condition of no laser irradiation of the present invention;

[0045] Fig.19 is a graph showing the detection result of cell survival rate under the laser irradiation condition of the present invention;

[0046] Fig. 20 It is the quantitative analysis of cell cycle distribution under the laser irradiation conditions of the present invention;

[0047] Fig.21 This is a diagram showing the results of the cell apoptosis assay of the present invention. DETAILED DESCRIPTION

[0048] The present invention will be described in detail below with reference to the accompanying drawings and embodiments:

[0049] Embodiment 1,

[0050] This example is the preparation of DNA damaging prodrug (CSP)

[0051] refer to Figure 1 As shown, the specific preparation method is as follows:

[0052] 1. Synthesis of Cb-SS: Chlorambucil (Cb, 608 mg, 2.0 mmol) was dissolved in anhydrous dichloromethane, and then 4-dimethylaminopyridine (DMAP, 293 mg, 2.4 mmol) and N, N-dicyclohexylcarbodiimide (DCC, 495 mg, 2.4 mmol) were added at 0°C. After 0.5 hours, the mixture was added with bis(2-hydroxyethyl) disulfide (SS, 616 mg, 4.0 mmol) in anhydrous DCM. The resulting mixture was stirred at room temperature overnight. The reaction solution mixture was cooled and precipitated. After filtration, the filtrate was concentrated by rotary evaporation. The crude product was purified by silica gel column chromatography to obtain Cb-SS (yield: 67.5%) 1 H NMR (CDCl 3 ,400MHz): δ(ppm)=7.12-7.01(d,2H,-CH-),6.67-6.56(d,2H,-CH-),4.42-4.30(t,2H,-CH-) 2 -),3.92-3.80(m,2H,-CH 2 -),3.74-3.66(m,4H,-CH 2 -),3.66-3.57(m,4H,-CH 2 -), 2.95-2.83(m,4H,-CH 2 -),2.60-2.52(t,2H,-CH 2 -),2.38-2.30(t,2H,-CH 2-),1.96-1.87(m,2H,-CH 2 -). 13 C-NMR (CDCl 3 ,100MHz): δ (ppm) = 173.5, 144.4, 130.2, 129.7, 112.3, 62.2, 60.3, 53.6, 41.7, 40.6, 37.1, 34.0, 33.5, 26.7. HRMS (ESI): m / z calcd forC 18 H 28 Cl 2 NO 3 S 2 + [M+H] + 440.0882,found440.0878.

[0053] 2. Synthesis of CSP: Dissolve pyropheophorbide a (PPa) (50 mg, 0.09 mmol) in anhydrous dichloromethane, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDCI (100 mg, 0.53 mmol) and DMAP (65 mg, 0.53 mmol) at 0°C with stirring. After stirring for 0.5 hours, add Cb-SS (50 mg, 0.11 mmol). The mixture is stirred at room temperature overnight. After rotary evaporation and concentration, the crude product is purified by silica gel column chromatography to obtain a black solid prodrug CSP (yield: 82%). Figure 2-Figure 4 As shown, 1 H-NMR (CDCl 3 ,400MHz): δ(ppm)=9.50(s,1H,-CH-),9.38(s,1H,-CH-),8.55(s,1H,-CH-),8.05-7.95(m,1H,-CH-),7.12-7.0 0(d,2H,-CH-),6.66-6.58(d,2H,-CH-),6.32-6.25(d,1H,-CH-),6.20-6.13(d,1H,-CH-),5.31-5.21(d,1H,-CH 2 -),5.15-5.05(d,1H,-CH 2 -),4.53-4.44(m,1H,-CH-),4.39-4.33(t,2H,-CH 2 -),4.32-4.25(m,1H,-CH-),3.97-3.83(m,2H,-CH 2 -),3.72-3.65(m,8H,-CH 2 -),3.64-3.56(m,7H,-CH2 -,-CH 3 ),3.41(s,3H,-CH 3 ),3.24(s,3H,-CH 3 ),2.79-2.80(m,4H,-CH 2 -),2.60-2.51(m,3H,-CH 2 -), 2.38-2.27(m,3H,-CH 2 -),2.05-1.98(m,1H,-CH-),1.96-1.85(m,2H,-CH 2 -),1.84-1.77(m,3H,-CH 2 -),1.73-1.63(t,3H,-CH 3 ). 13 C-NMR (CDCl 3 ,100MHz): δ(ppm)=196.3,173.5,173.4,171.4,160.3,155.2,150.8,149.0,145.0, 144.4,141.6,137.9,136.2,136.1,135.9,131.6,130.5,129.7,129.2,122.6,112.3 ,106.0,104.1,97.2,93.0,62.2,60.3,53.6,51.7,51.7,50.0,48.1,41.7,40.5,37 .1,34.0,33.5,31.0,29.9,26.7,23.1,19.5,17.4,12.2,12.1,11.3.HRMS(ESI):m / z calcd for C 51 H 60 Cl 2 N 5 O 5 S 2 + [M+H] + 956.3407, found 956.3419.

[0054] Embodiment 2,

[0055] This example is the preparation of CSPNP-15% in the nanoparticle CSPNP, and the steps include: 2000 Dissolved in dimethyl sulfoxide at a concentration of 2 mg / mL. To obtain NP-15%, CSP (1 mL) and DSPE-PEG 2000(335 μL) was mixed and dispersed in water (10 mL). The mixture was stirred for 2 hours. Then, the mixture was dialyzed for 3 hours to remove dimethyl sulfoxide DMSO. The solution was filtered (particle size 220 nm) to remove free CSP. The obtained NP-15% was stored at 4°C for further use. NP-15% was named CSPNP.

[0056] It should be noted here that the method of this embodiment can be used to prepare NPs with other ratios.

[0057] Embodiment 3,

[0058] This example is a preparation of nanoparticles CSP / Ola NP, the steps include: prodrug CSP, Ola and DSPE-PEG 2000 Dissolve in DMSO at a concentration of 2 mg / mL. CSP (1 mL), olaparib Ola (450 μL) and DSPE-PEG 2000 (150 μL) was mixed and dispersed in water (10 mL), and the mixture was stirred for 2 hours. Then, the mixture was dialyzed for 3 hours to remove dimethyl sulfoxide (DMSO). The solution was filtered (particle size 220 nm) to remove free CSP and Ola to obtain CSP / Ola NPs.

[0059] Embodiment 4,

[0060] This example is a study and verification of the self-assembly behavior of CSP, such as Figure 5 As shown: The particle size of the prepared CSPNP (0%) is about 80 nm as measured by dynamic light scattering (DLS). Transmission electron microscopy (TEM) observations show that the harvested CSPNP (0%) is irregularly spherical in shape, showing good self-assembly properties.

[0061] Embodiment 5,

[0062] This example uses nanoprecipitation to prepare CSP NPs with different formulations, DSPE-PEG 2000 The contents of 0%, 5%, 10%, 15% and 20% were respectively. Then, the particle size of the obtained CSP NPs was characterized by DLS. Figure 6 As shown, after PEGylation, with the DSPE-PEG 2000 With the increase of DSPE-PEG content, the average particle size of CSPNPs increased from 77nm to 107nm. 2000 The CSPNP with a content of 15% has the smallest polymer dispersibility index PDI. Therefore, this application will select this component ratio to prepare Ola-loaded nanoparticles CSP / OlaNP.

[0063] Embodiment 6,

[0064] In this example, the particle size and morphology of the obtained NP-0%~20% were characterized by DLS and TEM. The particle size distribution and zeta potential were measured by dynamic light scattering (DLS) Zetasizernano zsp instrument (Malvern instruments Led).

[0065] like Figure 7 As shown, the results showed that the particle size of CSPNP (15%) was about 105nm. After encapsulation of Ola, the particle size of CSP / Ola NP slightly increased to 110nm. In addition, the zeta potentials of CSP NP and CSP / Ola NP were -24.16mV and -12.10mV, respectively. The negative charge contributed to the stability of the obtained nanoparticles in serum.

[0066] like Figure 8 As shown, after 48 h of treatment with 100 mM GSH, the structure of CSPNPs gradually changed from spherical to irregular flakes, and the destruction of the nanoparticles can be attributed to the response of intracellular glutathione to disulfide bonds.

[0067] like Fig. 9 As shown, it can be seen that the nanomedicine of the present invention has good storage stability, and the particle size remains stable after storage at 4 degrees for 20 days; and the nanomedicine has good serum stability, and the particle size is smaller after incubation in 10% serum, indicating its good serum tolerance.

[0068] from Fig.10 As shown, the ultraviolet absorption of the nanomedicine formed by the present invention is red-shifted. Compared with free CSP, the characteristic peaks of CSPNP and CSP / OlaNP have an obvious shift. However, the peak shift is restored after being dissolved in DMSO, which means that the nanoparticles are disintegrated due to the destruction of the intermolecular interaction.

[0069] Embodiment 7,

[0070] This example is a singlet generated in vitro by the present invention 1 O 2 Specifically, DPBF was used as a ROS indicator to monitor the generation of ROS. First, a CSP / Ola NP (or free PPa, CSP, CSPNP) aqueous solution (3 mL) with a concentration of 1 μM equivalent PPa was prepared. Then, 60 μL of DPBF in DMSO (5 mM) was added. The UV-visible spectrum of the resulting mixture was measured. The mixture was illuminated by a 660 nm laser (20 mW / cm 2 ) After irradiation, the UV-visible spectrum of the mixture was measured every 10 s, and the absorption of DPBF at 417 nm decreased, reflecting the generation rate of ROS.

[0071] like Fig.11 As shown, by using DPBF as singlet oxygen 1 O 2 Indicators to monitor singlet oxygen 1 O 2 The generation capacity of the prodrug CSP is that the rate of decrease of the absorption value of DPBF at 417 nm can reflect the generation rate of reactive oxygen species. 1 O 2 The ability of PPa is stronger than that of PPa, indicating that structural modification of photosensitizers can increase the singlet oxygen 1 O 2 The ability of CSPNP and CSP / OlaNP to generate singlet oxygen 1 O 2 The ability of singlet oxygen is comparable to that of nanoparticles, which is the strongest. 1 O 2 The ability may be due to the increased water solubility of the photosensitizer, which provides a basic guarantee for improving photodynamic therapy.

[0072] Embodiment 8,

[0073] In this example, the fluorescence imaging of nanoparticles can be realized by using photosensitizers. In this example, MDA-MB-231 cells were inoculated into 96-well plates for culture. The cells were treated with free PPa, CSP, CSPNP, and CSP / OlaNP (0.2 μM equivalent PPa) for 4 hours, and then Hoechst 33342 (λ ex =346nm,λ em =460nm) to stain the cell nucleus for 10 minutes. After washing the cells, the intracellular fluorescence was observed using a high-content analysis system. Fig.12 As shown, the red fluorescence of free PPa is less and the fluorescence intensity is low, indicating that its cellular uptake is low; the red fluorescence of the prodrug CSP is more and the fluorescence intensity is stronger than that of PPa, indicating that the chemically modified photosensitizer has stronger cellular uptake ability; the nanoparticles CSPNP and CSP / OlaNP prepared by nanotechnology have stronger red fluorescence, further enhancing the cellular uptake ability.

[0074] Embodiment 9,

[0075] In order to evaluate the endocytic pathway of nanomedicine, common endocytic inhibitors were used. MDA-MB-231 cells were seeded into 6-well plates and cultured. The cells were pretreated with chlorpromazine (31 μM), genistein (275 μM), nocodazole (16 μM) and cytochalasin D (2 μM) for 0.5 h, respectively. CSP / Ola NPs (0.2 μM) were added and incubated with inhibitors or at 4 °C. After 4 h, the cells were washed and collected. The fluorescence intensity was measured by flow cytometer (BD, USA). Untreated cells were used as controls. Fig.13 As shown, the cell uptake was reduced by about 90% at 4 degrees, indicating that it was mainly energy-dependent cell uptake. It was also found that the fluorescence intensity of cell uptake was greatly reduced by cytochalasin D and genistein, so the cell uptake of nanoparticles was mainly through macropinocytosis (54%) and clathrin-mediated (65%) endocytosis.

[0076] Embodiment 10

[0077] This example is about the ability of cells to produce ROS: MDA-MB-231 cells were inoculated into 96-well plates and cultured. After 24 hours of drug treatment (0.8 μM), the cells were stained with DCFH-DA for 30 minutes. Then, the cells were washed and irradiated with a 660 nm laser (18 mW / cm 2 ) for 2 minutes. ex =488nm,λ em =525nm) to observe the fluorescence of DCF in cells. Fig.14 As shown, almost no green fluorescence was observed in cells treated with PBS, Cb, and Ola, indicating that Cb or Ola alone may have no therapeutic effect without a photosensitizer. In addition, due to the poor water solubility of PPa, the green fluorescence found in PPa-treated cells was negligible. In contrast, cells treated with CSPNPs and CSP / Ola NPs showed obvious green fluorescence, indicating that a large amount of ROS was generated upon light irradiation, which was attributed to their nanostructure formation by self-assembly and the subsequent high cellular uptake.

[0078] Embodiment 11,

[0079] This example is to detect the glutathione GSH level in MDA-MB-231 cells to study the potential ability of nanoparticles to regulate cellular GSH levels. The specific method is: MDA-MB-231 cells are planted in a 6 cm culture dish. The cells are treated with different concentrations (1.2 μM). After 24 hours of culture, the cells are collected and tested using a total glutathione detection kit (Biyuntian Biotechnology Co., Ltd.).

[0080] The results showed that Fig.15As shown, CSPNPs and CSP / OlaNPs showed significant GSH inhibition (about 35%) compared with the control group. In contrast, the GSH inhibition rates of the Cb, Ola, and PPa groups were only 5%, 0%, and 15%, respectively. It is particularly noteworthy that the prodrug CSP had a moderate inhibitory effect on GSH. The significant reduction in GSH levels in the CSPNPs and CSP / OlaNPs groups may be due to the stimulatory response of GSH consumption by disulfide bonds after cellular internalization. These results indicate that CSPNPs and CSP / OlaNPs improve drug delivery efficiency and contribute to improved cellular internalization, ROS generation, and GSH consumption.

[0081] Embodiment 12

[0082] This example evaluates the expression of γ-H2AX by immunofluorescence detection. MDA-MB-231 cells were inoculated into 96-well plates and cultured. The cells were treated with different concentrations (0.2 μM). After 24 hours, the culture medium was replaced with fresh complete culture medium. The cells were illuminated with a 660 nm laser (18 mW / cm 2 ) irradiated the cells for 2 minutes and then cultured for 4 hours. Then, the DNA damage of the cells was detected using the γ-H2AX immunofluorescence detection kit.

[0083] The results are as follows Fig.16 As shown, weak green immunofluorescence was observed in the free Cb, Ola, PPa or CSP groups, indicating that these agents had poor ability to damage DNA. Notably, CSP NPs induced significant expression of γ-H2AX, implying that the DNA damaging agent Cb encapsulated in CSPNPs played a role. In contrast, CSP / OlaNPs had the highest expression of γ-H2AX, indicating that CSP / OlaNPs had a synergistic effect in inhibiting DNA damage and its repair, which was due to the inherent functions of the co-loaded Cb and Ola, respectively.

[0084] Embodiment 13

[0085] This example is an experiment to detect the expression of γ-H2AX using Western blotting: MDA-MB-231 cells were seeded and cultured in a 6 cm culture dish. The cells were treated with different doses (0.2 μM). After 24 hours, the culture medium was replaced with fresh complete culture medium. The cells were illuminated with a 660 nm laser (18 mW / cm 2 ) The cells were irradiated for 2 min or in the dark and then incubated for another 4 h. The extracted proteins were used for Western blot analysis

[0086] The results are as follows Fig.17As shown, there was almost no expression of γ-H2AX in the blank control group, free Cb, and Ola groups, while there was a certain expression of γ-H2AX in the PPa and CSP groups, and the CSP group had significantly more. More importantly, the expression of γ-H2AX in CSPNP and CSP / OlaNP was the highest, proving that the prepared nanomedicine significantly enhanced the ability to damage DNA.

[0087] Embodiment 14

[0088] This example uses the MTT method to detect the cytotoxicity of different drugs. Specifically, MDA-MB-231 cells were inoculated into 96-well plates for culture. Cells were treated with gradient culture solutions of different concentrations. After 24 hours, the culture medium was replaced with fresh complete culture medium. Illumination group: 660nm laser (18mW / cm 2 ) were irradiated for 2 min. Then, 20 μL MTT solution (5 mg / mL) was added to each well and incubated for 4 h. Cell viability was assessed by absorbance at 570 nm using a microplate reader (Bio-Tek, Winooski, VT, USA). Fig.18 and Fig.19 As shown in the figure, it was found that under light-proof conditions, at a concentration of 0.4 μM, the cell survival rate was greater than 90%. However, under laser irradiation, the cell survival rate was significantly reduced, especially under 0.1 μM conditions, the cell survival rates of CSPNP and CSP / OlaNP were about 60% and 40%, respectively, which were significantly lower than the free PPa and prodrug CSP groups (greater than 90%), which was due to the high cellular uptake of nanomedicines and high ROS production concentration.

[0089] Embodiment 14

[0090] This example is a cell cycle distribution detection experiment, specifically: MDA-MB-231 cells were seeded and cultured in a 6 cm culture dish. The cells were treated with different doses (0.1 μM). After 24 hours, the culture medium was replaced with fresh complete culture medium. Illumination group: 660 nm laser (18 mW / cm 2 ) irradiated the cells for 90 seconds. After the cells were further cultured for 24 hours, the cell cycle distribution was detected using a cell cycle detection kit. Fig. 20As shown in the figure, from the results of cell cycle distribution, it can be seen that the cells in the blank control group, free Cb, Ola, Pa and CSP prodrug groups are mainly in the G0 / G1 phase (~55%) and S phase (~30%), and only ~15% of the cells are in the G2 / M phase. After CSP / OlaNP treated MDA-MB-231 cells under laser irradiation, the proportion of cells in the G2 / M phase increased significantly to 55.29%, and the proportion of cells in the G0 / G1 phase and S phase decreased significantly to about 30.69% and 14.02%. These results show that CSP / OlaNP can induce mitotic G2 / M phase cell cycle arrest and lead to apoptosis of cancer cells.

[0091] Example 15

[0092] This example is a cell apoptosis detection experiment, specifically: MDA-MB-231 cells were seeded and cultured in a 6 cm culture dish. The cells were treated with different doses (0.2 μM). After 24 hours, the culture medium was replaced with fresh complete culture medium. Illumination group: 660 nm laser (18 mW / cm 2 ) irradiated the cells for 2 minutes. After the cells were further cultured for 24 hours, cell apoptosis was detected using the Annexin V-FITC cell apoptosis detection kit. Fig.21 As shown, from the results of cell apoptosis assay, it can be seen that similar conclusions as those in the above examples were obtained.

[0093] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention. The techniques, shapes, and structural parts not described in detail in the present invention are all known technologies.

Claims

1. A DNA damaging prodrug, It is characterized in that The structural formula of the prodrug is shown in Formula I: Formula Ⅰ.

2. A method for preparing a DNA damaging prodrug, It is characterized in that The following steps are involved: S1, dissolving chlorambucil in anhydrous dichloromethane, and then adding 4-dimethylaminopyridine and N,N-dicyclohexylcarbodiimide to obtain a mixture 1; S2, adding bis(2-hydroxyethyl) disulfide in anhydrous dichloromethane to mixture 1 to obtain mixture 2; S3, stirring the mixture 2 at room temperature, cooling the reaction solution mixture and precipitating, filtering, and concentrating and purifying the filtrate by rotary evaporation to obtain product 1; S4, dissolving pyropheophorbide a in anhydrous dichloromethane, then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine under stirring, and stirring for reaction; S5. Add product 1 to the stirred reaction in step S4, stir and react at room temperature, and then concentrate and purify by rotary evaporation to obtain the prodrug.

3. A method for preparing a DNA damaging prodrug according to claim 2, Features: The molar ratio of chlorambucil, 4-dimethylaminopyridine, N,N-dicyclohexylcarbodiimide and bis(2-hydroxyethyl)disulfide is 1:1.2:1.2:

2.

4. A method for preparing a DNA damaging prodrug according to claim 2, Features: The molar ratio of the product 1 to pyropheophorbide a is 1.2:

1.

5. Use of the DNA damaging prodrug as claimed in claim 1 in the preparation of a chemotherapy-photodynamic therapy combined reagent drug or an anti-breast cancer drug.

6. Use of the DNA damaging prodrug according to claim 1 in preparing a photoacoustic imaging product or a fluorescent imaging product.

7. A DNA damaging nanoparticle, Features: The nanoparticles include nanoparticles CSP NP and nanoparticles CSP / Ola NP, wherein the nanoparticles CSP NP are the prodrug according to claim 1 and DSPE-PEG 2000 The nanoparticle CSP / Ola NP is prepared by self-assembly, and the prodrug according to claim 1 and DSPE-PEG 2000 and olaparib by self-assembly.

8. A method for preparing DNA damaging nanoparticles, Features: The nanoparticles include nanoparticles CSP NP and nanoparticles CSP / Ola NP. The preparation method of the nanoparticles CSP NP includes the following steps: 2000 Dissolved in dimethyl sulfoxide, respectively, to obtain prodrug solution, DSPE-PEG 2000 Solution; take prodrug solution and DSPE-PEG 2000 The solution is mixed and dispersed in water, stirred to obtain a mixture three; dialyzed to remove dimethyl sulfoxide, and then filtered to remove the free prodrug to obtain nanoparticles CSP NP; the preparation method of the nanoparticles CSP / Ola NP comprises the following steps: the prodrug according to claim 1, olaparib, DSPE-PEG 2000 Dissolved in dimethyl sulfoxide respectively to obtain prodrug solution, olaparib solution, DSPE-PEG 2000 Solution; take prodrug solution, olaparib solution, DSPE-PEG 2000 The solutions were mixed and dispersed in water and stirred to obtain a mixture of four; dimethyl sulfoxide was removed by dialysis, and then the solution was filtered to remove the unencapsulated reagent to obtain nanoparticles CSP / Ola NP.

9. The method for preparing DNA damaging nanoparticles according to claim 8, Features: In the preparation of the nanoparticle CSP NP, the prodrug is reacted with DSPE-PEG 2000 The mass ratio of the prodrug, DSPE-PEG is 1:0%-20%; in the preparation of the nanoparticle CSP / OlaNP, the prodrug, DSPE-PEG 2000 The mass ratio of olaparib is 1:0.15:0.45.

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