NADP < + >-encapsulated reduction response type nano material as well as preparation method and application thereof

Abstract

The invention provides an NADP < + >-encapsulated reduction response type nano material as well as a preparation method and application thereof for the first time. The nano material is NADP < + > and a reduction response type polymer containing disulfide bonds. The NADP < + > can inhibit ADP-ribosylation, but the clinical application of the NADP < + > is limited due to poor membrane permeability; the polymer can encapsulate NADP < + > into cells and dissociate in a high-concentration glutathione environment of tumor cells, so that the NADP < + > is released, the concentration of the NADP < + > in the tumor cells is remarkably increased, ADP-ribosylation is prevented, and DNA damage repair is inhibited. And the growth of tumor cells can be synergistically inhibited by the combined action of the compound and a commercially available PARP inhibitor Olaparib. The preparation method provided by the invention is simple and feasible, and provides more thoughts and means for clinical application of NADP +.

Description

Technical Field

[0001] The invention relates to a reduction-responsive nano material encapsulating NADP+ and a preparation method and application thereof, belonging to the technical field of nano biomaterials. Background Art

[0002] Poly (ADP-ribose) polymerase inhibitors (PARPi) have significantly changed the treatment landscape for various cancers by targeting the genetic vulnerabilities of cancer cells. These drugs inhibit the PARP enzyme, preventing DNA damage repair in cancer cells, especially those with BRCA1 / 2 mutations or homologous recombination (HR) repair defects, thereby inducing cell death. This mechanism of action makes PARPi particularly effective against cancers with widespread genetic vulnerabilities, including ovarian and breast cancer. However, the clinical effects of PARPi are not satisfactory in all cases. Even in the presence of BRCA1 / 2 mutations or HR repair defects, many tumors still do not show a significant response to PARPi treatment. This difference in efficacy reflects the key limitations of PARPi application and highlights the urgent need to increase the sensitivity of cancer cells to these inhibitors. Addressing this challenge requires not only in-depth analysis of the molecular mechanisms of PARPi resistance, but also the development of innovative strategies to overcome resistance and thereby expand the range of cancers that can benefit from PARPi treatment.

[0003] NADP+ (nicotinamide adenine dinucleotide phosphate) is a key cofactor of cellular metabolism and plays a vital role in redox reactions necessary for lipid and nucleic acid synthesis. In addition to its metabolic function, recent studies have shown that NADP+ also plays a key role in regulating PARylation, an important process for DNA damage repair. As a natural PARP inhibitor, this function of NADP+ provides new ideas for improving the efficacy of PARP inhibitors in cancer treatment and a potential strategy for enhancing the sensitivity of cancer cells to these drugs. However, the clinical development of NADP+ faces significant challenges, mainly due to its inherent physicochemical properties. Due to the presence of phosphate groups, NADP+ carries a significant negative charge, which makes it difficult to penetrate the cell membrane. This membrane impermeability constitutes a major barrier to its entry into cells, limiting the application of NADP+ to directly regulate PARylation in cells, thereby hindering its therapeutic utility. Overcoming this limitation is crucial to advance the application of NADP+ in cancer treatment. This will not only help to develop its potential as an endogenous PARP inhibitor, but may also further enhance the therapeutic efficacy of PARPi and expand its clinical application range in oncology.

[0004] Nanoparticle drug delivery systems are a revolutionary approach in the field of drug delivery, offering many advantages that enhance the efficacy and specificity of therapeutic agents. These systems exploit the unique properties of nanoparticles, such as their small size, large surface area, and the ability to be functionalized with targeting ligands, to improve the bioavailability, stability, and controlled release of drugs. By encapsulating therapeutic agents in nanoparticles, these drugs can be protected from degradation, their solubility can be improved, and their targeted delivery to specific tissues or cells can be facilitated, thereby minimizing off-target effects and reducing systemic toxicity. In addition, nanoparticles can be designed to respond to specific stimuli in the tumor microenvironment, such as pH, temperature, or redox conditions, so that the encapsulated drug is released precisely at the site of action. This targeted and controlled delivery is particularly advantageous for cancer treatment, where achieving high local concentrations of drugs in tumors while sparing healthy tissues is critical to maximizing therapeutic efficacy and minimizing adverse reactions. Given these advantages, nanoparticle-based delivery systems offer a promising solution to overcome the challenge of NADP+ membrane impermeability. By encapsulating NADP+ in reduction-responsive nanoparticles, its entry into cancer cells can be facilitated, making it a natural PARP inhibitor and enhancing the efficacy of PARP inhibitors. Summary of the invention

[0005] The present invention provides a reduction-responsive nanomaterial encapsulating NADP+, and a preparation method and application thereof, aiming to promote the intracellular delivery of NADP+. By adding disulfide bonds to the nanoparticle structure, these NPs can be selectively released in the reducing environment of tumor cells. This strategic design ensures that after the cells take up the nanoparticles, the concentration of intracellular NADP+ is significantly increased, negatively regulating DNA damage-induced PARylation and disrupting the DNA damage repair process. In addition, NADP+ is co-encapsulated with the chemically synthesized PARP inhibitor Olaparib, and the inhibitory effect on tumor cells is significantly enhanced through synergistic effects. Through this innovative method, the clinical application challenges caused by NADP+ due to its poor membrane permeability are solved, and it has broad clinical application potential.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for preparing a reduction-responsive nanomaterial encapsulating NADP+, comprising the following steps:

[0008] DSB (bis(2-hydroxyethyl) disulfide, 0.20 mmol, 30.8 mg) and CHAT (1,2,4,5-cyclohexanetetracarboxylic dianhydride, 0.21 mmol, 44.8 mg) were suspended in 5 mL of anhydrous DMF (N,N-dimethylformamide), and the mixture was magnetically stirred for 24 h;

[0009] Add mPEG5000-OH (2 mmol, 100 mg) and stir for another 24 hours;

[0010] The reaction mixture was dialyzed against deionized water and then freeze-dried under reduced pressure to obtain 112 mg of white product P1;

[0011] Preparation of nanomaterials of different components: NP-N component [NADP+ (1 mg) and P1 (5 mg)], NP-N&RB component [NADP+ (1 mg), Rhodamine B (0.5 mg), and P1 (5 mg)], and NP-N&O component [NADP+ (1 mg), Olaparib (0.5 mg), and P1 (5 mg)] were dissolved in 1 mL of DMSO, respectively, mixed and stirred for 15 min;

[0012] Deionized water (10 mL) was added dropwise to each mixture and stirring was continued for 24 h;

[0013] The mixture was dialyzed against deionized water (MWCO: 3500Da) to remove any unbound drug for another 24 hours. Finally, the supernatant was collected by centrifugation at 4000 rpm for 5 minutes.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] (1) The clinical application of NADP+ is severely limited by its poor membrane permeability. The present invention introduces an innovative approach to improve the efficacy of PARPi in cancer treatment by using reduction-responsive nanoparticles loaded with NADP+. By increasing the intracellular concentration of NADP+, these nanoparticles effectively inhibit DNA damage-induced PARylation, thereby inhibiting the DNA repair process and sensitizing cancer cells to PARPi.

[0016] (2) The co-encapsulation of NADP+ and the chemically synthesized PARP inhibitor Olaparib in these nanoparticles represents a new strategy for synergistic therapeutic effects.

[0017] (3) This method shows significant clinical potential for addressing the major challenge of PARPi resistance, which is common in many cancers. It is expected to expand the therapeutic scope of PARPi to cover previously difficult-to-treat cancer types, thereby promoting the development of precision cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is the abstract figure of the present invention, which is a schematic diagram of the design of a reduction-responsive nanoparticle encapsulating NADP+ and its cell interaction.

[0020] Figure 2 It is a schematic diagram of the synthesis of reduction-responsive nanoparticles encapsulating NADP+ in the present invention. Figure 2 (A) Synthesis route of the disulfide-containing reduction-responsive polymer (P1). DSB and CHAT were reacted in anhydrous DMF, and then mPEG5000-OH was added. The mixture was dialyzed and freeze-dried to obtain the white product P1. (B) The molecular structure of NADP+ (nicotinamide adenine dinucleotide phosphate) includes a negatively charged phosphate group and a positively charged nicotinamide group. (C) Schematic diagram of the synthesis and reduction-responsive release of nanoparticles (NP-N) that capture NADP+ via hydrophobic interactions and electrostatic complexation mechanisms.

[0021] Figure 3 Schematic diagram of the characterization of reduction-responsive nanoparticles encapsulating NADP+ in the present invention. (A) Typical dynamic light scattering (DLS) results of NP-N. (B) Representative transmission electron microscopy (TEM) images of NP-N. Scale bar, 200 nm. (C) Particle size of NP-N after 7 days of incubation in PBS. (D) NADP+ release curves of NP-N under different conditions.

[0022] Figure 4 The present invention is to optimize the preparation conditions of the reduction-responsive nanoparticles encapsulating NADP+. The size (A), PDI (B), potential (C) and NADP+ loading (D) of the nanoparticles are evaluated for different mass ratios of NADP+ to polymer.

[0023] Figure 5 Schematic diagram of the cellular uptake experiment of reduction-responsive nanoparticles encapsulating NADP+ and rhodamine B in the present invention, wherein the cells were treated with nanoparticles labeled with rhodamine B (NP-N&RB) and analyzed using immunofluorescence staining, flow cytometry and NADP+ level measurement at different time points.

[0024] Figure 6 This is the result of cellular uptake of reduction-responsive nanoparticles encapsulating NADP+ and rhodamine B in the present invention. Figure 6 In the figure, (A) Confocal laser scanning microscopy (CLSM) images of HeLa cells were captured after incubation with NP-N&RB at three different time points: 1, 5, and 10 h. Cell nuclei were stained with DAPI. Scale bar, 10 μm. (B) Flow cytometry was used to quantitatively evaluate the uptake of NP-N&RB by HeLa cells. (C) The relative levels of NADP+ in HeLa cells were measured after incubation with NADP+ or NP-N&RB at three different time points (1, 5, and 10 h).

[0025] Figure 7 In the present invention, nanoparticles loaded with NADP+ have an inhibitory effect on PARylation. Figure 7 Figure 2. (A) NADP+-loaded nanoparticles inhibited DNA damage-induced PARylation. Cells were treated with 5 mM methyl methanesulfonate (MMS). Dot blotting was performed to examine PAR levels at the indicated time points after DNA damage. (B) PARylation was detected by immunofluorescence staining with anti-PAR antibodies. Representative cells at different time points are shown. (C-D) After 30 minutes of MMS treatment, the DNA damage marker γH2AX was detected by immunofluorescence staining (C) and Western blotting (D).

[0026] Figure 8 NADP+-loaded nanoparticles sensitize cancer cells to PARP inhibitors. Figure 8 (A) Schematic diagram of the experimental flow. (B) OVCAR5 was subjected to monoclonal formation assay. Cells were treated with olaparib or NP-N&O in culture medium at 37°C. (C) Real-time cell proliferation assay of OVCAR5 cells treated with the indicated conditions.

[0027] Fig. 9 NADP+-loaded nanoparticles sensitize cancer cells to PARP inhibitors. Fig. 9 Real-time cell proliferation assay of OVCAR3 cells (A) and OVCAR8 cells (B) treated with olaparib or NP-N&O for the indicated times.

[0028] Fig.10 Effects of NADP+ and olaparib-loaded nanoparticles on cancer cells. Fig.10In the figure, (A) Western blot analysis of DNA damage marker γH2AX after 3 days of treatment under the indicated conditions. (BD) Flow cytometric analysis of apoptosis in OVCAR5 cells after 3 days of treatment with the indicated conditions. Cells were stained with Annexin V-FITC and propidium iodide (PI) to distinguish live cells, early apoptotic cells, and late apoptotic / necrotic cells. (E) Western blot analysis of apoptosis marker cleaved caspase 3 after 3 days of treatment under the indicated conditions. (F) Fluorescent staining of apoptosis detected by GreenNuc Caspase-3 Detection Kit in OVCAR5 cells after 3 days of treatment under the indicated conditions. Scale bar, 10 μm. DETAILED DESCRIPTION

[0029] The following specific examples further illustrate the present invention. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The various reagents, reaction conditions, detection methods, etc. used in the following examples are, unless otherwise stated, considered as reagents, reaction conditions and detection methods conventionally used in the art.

[0030] Obviously, the described embodiments are only some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The experimental materials used in the following examples, unless otherwise specified, can be purchased from conventional biochemical reagent companies. The present invention is further described below in conjunction with the examples.

[0032] Example 1

[0033] In order to encapsulate NADP+, the present invention synthesized a reduction-responsive polymer P1 ( Figure 2 A). During nanoprecipitation with NADP+, P1 captures NADP+ through hydrophobic interaction and electrostatic complexation ( Figure 2 B), assembled into spherical nanoparticles (designated as NP-N). Figure 2 As shown in C, NP-N was designed to enter cancer cells via endocytosis and subsequently decompose in response to the high concentration of glutathione (GSH) unique to cancer cells. The synthesized nanoparticles were optimized by evaluating the weight ratio of NADP+ to P1 ( Figure 4 ). At a ratio of 0.2, NP-N showed relatively uniform morphology and particle size. Dynamic light scattering (DLS) ( Figure 3 A) and transmission electron microscopy (TEM) ( Figure 3 B) analysis showed that these spherical NP-N had a uniform diameter of 111 nm. The encapsulation efficiency of NADP+ was 9.78% ( Figure 4D). Subsequently, the stability of NP-N was evaluated by incubation in PBS for 7 days, and particle size measurement showed significant stability ( Figure 3 C). The in vitro release kinetics of NP-N under different conditions were further explored using high performance liquid chromatography (HPLC). Conditions simulating normal tissue (pH 7.4) and acidic tumor microenvironment (pH 5.0) were used, as well as 10 mM GSH solution to simulate the elevated GSH level in tumor cells. Figure 3 As shown in D, in the presence of 10 mM GSH, NADP+ released from NP-N was close to 75% after 48 h. These findings not only highlight the sensitivity of NP-N to GSH, but also emphasize that it can be rapidly decomposed within tumor cells, facilitating the targeted delivery and release of NADP+.

[0034] Example 2

[0035] This example is used to illustrate the results of cell uptake of reduction-responsive nanoparticles encapsulating NADP+ and rhodamine B.

[0036] To study the uptake of nanoparticles by tumor cells, e.g. Figure 5 As shown, HeLa cells were treated with rhodamine B-labeled NPs (NP-N&RB) for 1, 5, and 10 h. Using confocal laser scanning microscopy (CLSM), we observed that the red fluorescence intensity in the cells increased over time within 10 h ( Figure 6 A). This result was further supported by flow cytometry analysis, which showed a significant increase in the red fluorescence signal during the first few hours, indicating rapid internalization of the nanoparticles ( Figure 6 B). NP-N&RB uptake was also quantified by measuring intracellular NADP+ levels, which showed a gradual increase in NADP+ levels over time ( Figure 6 C). Notably, cells exposed to NP-N&RB exhibited significantly higher NADP+ levels compared to cells treated with free NADP+. These results highlight the effectiveness of nanoparticle-based delivery systems in enhancing cellular uptake of NADP+, demonstrating their potential for targeted cancer therapy.

[0037] Example 3

[0038] This example is used to illustrate the effect of NADP+-loaded nanoparticles (NP-N) on DNA damage-induced PARylation.

[0039] HeLa cells were pre-incubated with NP-N for 24 h and then treated with methyl methanesulfonate (MMS) for 30 min to induce DNA damage, and the resulting PARylation levels were examined. Both dot blot and immunofluorescence assays showed that PARylation levels were significantly increased in control cells without nanoparticle pretreatment (0 min), and these PARylations were rapidly degraded within 5 min. In contrast, pretreatment with nanoparticles increased the intracellular level of NADP+ and significantly inhibited PARylation ( Figure 7 AB). Correspondingly, after 30 min of MMS treatment, cells pre-incubated with nanoparticles showed significantly higher levels of the DNA damage marker γH2AX compared to the control group: the fluorescence intensity increased significantly ( Figure 7 C), the number of foci per nucleus was significantly higher than that in the control group. Western blot results showed that the H2AX level remained unchanged, while the γH2AX content increased ( Figure 7 D). This suggests that nanoparticle pretreatment impedes DNA damage repair by inhibiting PARylation. These findings suggest that nanoparticles can effectively inhibit cellular PARylation, thereby hindering the DNA damage repair mechanism.

[0040] Example 4

[0041] This example is used to illustrate the effect of NADP+-loaded nanoparticles on the sensitivity of cancer cells to PARP inhibitors.

[0042] Different ovarian cancer cell lines show significant differences in their sensitivity to PARPi, even in the absence of obvious HR defects. Previous studies have shown that overexpression of the NADK enzyme, which catalyzes the conversion of intracellular NAD+ to NADP+, can increase the sensitivity of cells to PARP inhibitors. Here, to investigate whether exogenous supplementation of NADP+ via nanoparticles can also enhance the sensitivity of cells to PARP inhibitors, we treated OVCAR5 cells with nanoparticles encapsulating NADP+ alone (NP-N) or in combination with the clinically used PARP inhibitor Olaparib (NP-N&O). Subsequently, the proliferation capacity of tumor cells was assessed using real-time cell analysis (RTCA) and colony formation assays ( Figure 8 A). The results showed that the cytotoxic effects of treatment with Olaparib and NP-N alone were limited. However, the combination of Olaparib and NP-N significantly increased the cytotoxic effects on cells. Notably, cells treated with nanoparticles encapsulating NADP+ and Olaparib also showed significant anticancer activity ( Figure 8BC). In addition, we treated OVCAR3 and OVCAR8 cells with NP-N&O and found that encapsulated NADP+ enhanced the cytotoxic effect of olaparib in both cell lines ( Fig. 9 ). These findings suggest that NADP+-loaded nanoparticles can sensitize cancer cells to PARP inhibitors, highlighting their potential for use in cancer therapy.

[0043] Example 5

[0044] This example is used to illustrate the mechanism of action of nanoparticles loaded with NADP+ in killing tumor cells.

[0045] Cells treated with nanoparticles loaded with NADP+ and olaparib (NP-N&O) showed a significant increase in γH2AX levels compared with the control and olaparib-treated groups ( Fig.10 A). This suggests that NP-N&O hinders DNA repair, leading to the accumulation of DNA damage in cells. Further analysis of apoptosis by flow cytometry showed that the proportion of apoptotic cells in the NP-N&O treatment group (44.2%) was significantly higher than that in the control group (6.06%) and the olaparib treatment group (24.1%) ( Fig.10 BD). Accordingly, caspase 3, a key executor of the apoptotic pathway responsible for decomposing cellular components and promoting cell death, was significantly activated in the NP-N&O treated group ( Fig.10 EF). These findings suggest that NP-N&O inhibits PARylation, thereby preventing DNA repair in cells, leading to the accumulation of DNA damage and ultimately cell death. This highlights the potential of NP-N&O as an effective strategy to enhance the efficacy of PARP inhibitors in cancer therapy by exploiting the mechanism by which accumulation of unrepaired DNA damage induces apoptosis.

[0046] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claim involved.

[0047] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for preparing a nanomaterial, characterized in that: The nano material is obtained by reacting NADP+ and a reduction-responsive polymer containing a disulfide bond in a solvent, and the particle size is about 110 nm.

2. The nanomaterial according to claim 1, characterized in that: The nano material has both nano material properties and reduction responsiveness.

3. The method for preparing a nanomaterial according to any one of claims 1 to 2, characterized in that: The method comprises the following steps: (1) To synthesize the reduction-responsive polymer P1, DSB (bis(2-hydroxyethyl) disulfide, 0.20 mmol, 30.8 mg) and CHAT (1,2,4,5-cyclohexanetetracarboxylic dianhydride, 0.21 mmol, 44.8 mg) were suspended in 5 mL of anhydrous DMF (N,N-dimethylformamide), and the mixture was magnetically stirred for 24 h; (2) Add mPEG 5000 -OH (2 mmol, 100 mg), and stirred for another 24 hours; (3) The reaction mixture was dialyzed against deionized water and then freeze-dried under reduced pressure to obtain 112 mg of a white product P1; (4) Nanomaterials with different components were prepared: NP-N component [NADP+ (1 mg) and P1 (5 mg)], NP-N&RB component [NADP+ (1 mg), Rhodamine B (0.5 mg), and P1 (5 mg)], and NP-N&O component [NADP+ (1 mg), Olaparib (0.5 mg), and P1 (5 mg)] were dissolved in 1 mL of DMSO, respectively, mixed, and stirred for 15 min; (5) Deionized water (10 mL) was added dropwise to each mixture and stirring was continued for 24 h; (6) The mixture was dialyzed against deionized water (MWCO: 3500Da) to remove any unbound drug, and this process continued for another 24 hours. Finally, the supernatant was collected by centrifugation at 4000 rpm for 5 minutes.

4. Use of the reduction-responsive nanomaterial encapsulating NADP+ according to claim 1 in the preparation of a drug for treating tumors.

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