Nano preparation for synergistic chemotherapy of natural product as well as preparation method and application of nano preparation

Abstract

The invention discloses a nano preparation for natural product synergistic chemotherapy as well as a preparation method and application of the nano preparation, nanoparticles are formed by self-assembly of an SN38 prodrug sensitive to redox and galangin in a carrier-free manner, and can also be co-assembled nanoparticles modified by polyethylene glycol or entrapped with hydrophobic fluorescent molecules. According to the SN38 prodrug, a fluorene methanol side chain is coupled to an SN38 molecule through a redox-sensitive disulfide bond, so that a prodrug structure with a responsive release characteristic is constructed. The construction of the nano system not only significantly reduces the off-target toxicity of SN38 and improves the tolerance dose of SN38, but also enhances the SN38-mediated chemotherapy effect by the sensibilization effect of galangin, significantly improves the treatment efficiency, and realizes high-efficiency and low-toxicity cancer treatment. The invention provides a novel and synergistic prodrug nano drug delivery platform, and a new strategy is provided for precise treatment of refractory tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of sensitizing drugs for tumor chemotherapy, and specifically relates to a nano preparation of natural products for enhancing chemotherapy, and a preparation method and application thereof. Background Art

[0002] 7-ethyl-10-hydroxycamptothecin (SN38) is a topoisomerase I inhibitor that can induce DNA damage and apoptosis in tumor cells by interfering with processes such as DNA replication, transcription, recombination, and repair. Although SN38 has potent antitumor activity, its poor physicochemical properties, including low water solubility, inefficient oral absorption, and severe systemic toxicity, have hindered its clinical translation. Low oral bioavailability means that it can only be administered intravenously, and poor water solubility also makes this process difficult.

[0003] In recent years, numerous attempts have been made to load SN38 into nanocarriers to overcome its shortcomings. However, the rigid chemical structure of such molecules often results in low drug encapsulation efficiency and premature drug leakage from the nanocarriers, resulting in suboptimal drug delivery efficiency and a high risk of toxicity. These limitations have significantly hindered the clinical translation of SN38-loaded nanoparticles. Therefore, designing a highly efficient and low-toxic drug delivery system for SN38 delivery remains a hot topic of research. Prodrug strategies are also gaining increasing attention as a viable approach to overcome the shortcomings of traditional chemotherapeutic drugs. Well-designed prodrugs can significantly improve the poor physicochemical properties of the parent drug, thereby enhancing its oral absorption and bioavailability. Most importantly, rational prodrug design can minimize adverse reactions associated with the parent drug, such as reducing toxicity to normal tissues, improving therapeutic safety, and enhancing patient tolerability. However, while the prodrug strategy offers great promise, it also presents numerous challenges. An ideal anticancer prodrug should exhibit good chemical stability before reaching its target. More importantly, within the tumor, the prodrug should be selectively and rapidly activated to release the drug.

[0004] However, prodrug activation requires at least a minimum effective concentration of the free drug, which greatly limits its clinical application. Therefore, how to fully unleash the anti-tumor potential of SN38 is a key issue that needs to be addressed in the construction of prodrug-based drug delivery methods. Summary of the Invention

[0005] The purpose of the present invention is to design and synthesize a disulfide-bridged SN38-9-fluorenylmethanol small molecule prodrug, prepare a prodrug self-assembled nano drug delivery system and a prodrug and galangin co-assembled nano drug delivery system, and its application in drug delivery. The stability of the prodrug self-assembled nanoparticles and the galangin and prodrug co-assembled nanoparticles, drug release, and the effect of galangin on the SN38 sensitization effect in cytotoxicity, pharmacokinetics, tissue distribution and pharmacodynamics are explored to provide a new, efficient and safe SN38-9-fluorenylmethanol small molecule prodrug nanoparticles and their preparation method and application for precise cancer chemotherapy for tumor treatment.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] A natural product nanoformulation for enhancing chemotherapy, wherein the nanoformulation is a polyethylene glycol-modified SN38-9-fluorenylmethanol small molecule prodrug and galangin co-assembled nanoparticles, or a hydrophobic fluorescent substance-encapsulated SN38-9-fluorenylmethanol small molecule prodrug and galangin co-assembled nanoparticles; the SN38-9-fluorenylmethanol small molecule prodrug is formed by connecting SN38, camptothecin compounds and their derivatives with 9-fluorenylmethanol through disulfide bonds and other sensitive bonds.

[0008] In the above technical solution, further, the sensitive bond is a pH sensitive bond or a reducing environment sensitive bond, the pH sensitive bond is a hydrazone bond or a carbonate bond, and the reducing environment sensitive bond is a monosulfide bond, a disulfide bond, a disulfide bond or a metalloprotease sensitive bond.

[0009] In the above technical solution, further, the SN38-9-fluorenylmethanol small molecule prodrug is formed by connecting SN38, camptothecin compounds and their derivatives with 9-fluorenylmethanol through a disulfide bond, and its structural formula is:

[0010]

[0011] The present invention also discloses a method for preparing the above-mentioned natural product synergistic chemotherapy nanoformulation, comprising the following steps:

[0012] dissolving SN38-9-fluorenylmethanol small molecule prodrug, galangin and polyethylene glycol modifier, or SN38-9-fluorenylmethanol small molecule prodrug, galangin and polyethylene glycol modifier, and a hydrophobic fluorescent substance in an organic solvent;

[0013] The solution is added dropwise into water, and the SN38-9-fluorenemethanol small molecule prodrug and galangin spontaneously form uniform nanoparticles. The organic solvent in the preparation is then removed by rotary evaporation to obtain a natural product synergistic chemotherapy nanoparticle preparation that does not contain any organic solvent.

[0014] The present invention also discloses a SN38-9-fluorenylmethanol small molecule prodrug self-assembled nanoparticle, wherein the self-assembled nanoparticle is a SN38-9-fluorenylmethanol small molecule prodrug self-assembled nanoparticle modified with a polyethylene glycol modifier, or a SN38-9-fluorenylmethanol small molecule prodrug self-assembled nanoparticle encapsulating a hydrophobic fluorescent substance; the SN38-9-fluorenylmethanol small molecule prodrug is obtained by connecting SN38, SN38, camptothecin compounds and derivatives thereof, and 9-fluorenylmethanol through a disulfide bond.

[0015] The present invention also discloses a method for preparing the SN38-9-fluorenylmethanol small molecule prodrug self-assembled nanoparticles as described above, comprising the following steps:

[0016] Dissolving the SN38-9-fluorenylmethanol small molecule prodrug and the polyethylene glycol modifier, or the SN38-9-fluorenylmethanol small molecule prodrug, the polyethylene glycol modifier, and the fluorescent substance in an organic solvent to obtain a mixed solution;

[0017] The mixed solution is added dropwise to water, and the SN38-9-fluorenemethanol small molecule prodrug spontaneously forms uniform nanoparticles. The organic solvent in the preparation is removed by rotary evaporation to obtain SN38-9-fluorenemethanol small molecule prodrug self-assembled nanoparticles without any organic solvent.

[0018] In the above technical solution, further, the polyethylene glycol modifier includes one or more of PCL-PEG, DSPE-PEG, DSPE-SS-PEG, PLGA-PEG or PE-PEG; the molecular weight of the polyethylene glycol modifier is 1000-5000; the hydrophobic fluorescent substance includes one or more of coumarin-6, rhodamine, DiR, DiI, Cy3, Cy5 and Cy7; the organic solvent includes one or more of tetrahydrofuran, ethanol, methanol and dimethyl sulfoxide.

[0019] In the above technical solution, further, the mass ratio of the SN38-9-fluorenylmethanol small molecule prodrug modified by the polyethylene glycol modifier and galangin in the co-assembled nanoparticles is (0.1-10):1:(0.1-0.3);

[0020] The mass ratio of the SN38-9-fluorenylmethanol small molecule prodrug, the galangin, the polyethylene glycol modifier and the hydrophobic fluorescent substance in the SN38-9-fluorenylmethanol small molecule prodrug and galangin co-assembled nanoparticles encapsulating the hydrophobic fluorescent substance is (0.1-10):1:(0.1-0.3):(0.02-0.06);

[0021] The mass ratio of SN38-9-fluorenylmethanol small molecule prodrug, hydrophobic fluorescent substance and polyethylene glycol modifier in the SN38-9-fluorenylmethanol small molecule prodrug self-assembled nanoparticles encapsulating the hydrophobic fluorescent substance is 1:(0.02~0.06):(0.1~0.3); the mass ratio of SN38-9-fluorenylmethanol small molecule prodrug and polyethylene glycol modifier in the SN38-9-fluorenylmethanol small molecule prodrug self-assembled nanoparticles modified with polyethylene glycol is 1:(0.1~0.3).

[0022] The present invention also discloses a method for synthesizing the SN38-9-fluorenylmethanol small molecule prodrug, comprising the following steps:

[0023] S1, dehydrating dithiodiacetic acid I to obtain an acid anhydride compound II;

[0024] S2. In the presence of a catalyst, the acid anhydride compound II undergoes an esterification reaction with 9-fluorenylmethanol to obtain an intermediate product III;

[0025] S3. Under the action of a catalyst, the intermediate product III undergoes an esterification reaction with SN38 to obtain SN38-9-fluorenylmethanol small molecule prodrug IV; the reaction formula is as follows:

[0026]

[0027] In the above technical solution, further, in S1, the temperature of the dehydration reaction is 20°C to 25°C; the time of the dehydration reaction is 2h to 4h;

[0028] In S2, the molar ratio of the acid anhydride compound II to the 9-fluorenylmethanol is (1-2): (1-2); the catalyst is DMAP; the molar ratio of the acid anhydride compound II to DMAP is 1: (0.1-1); the temperature of the esterification reaction is 20° C. to 25° C., and the time of the esterification reaction is 12 h to 24 h;

[0029] In S3, the molar ratio of the intermediate product III to SN38 is (1-2): (1-2); the catalyst is DMAP, EDCI and HOBT; the molar ratio of the intermediate product III, DMAP, EDCI and HOBT is 1: (0.1-1): (1-2): (1-2); the temperature of the esterification reaction is 20°C to 25°C, and the time of the esterification reaction is 24h to 48h.

[0030] The present invention also discloses a use of the above-mentioned natural product synergistic chemotherapy nanoformulation or the above-mentioned SN38-9-fluorenemethanol small molecule prodrug self-assembled nanoparticles in a drug delivery system.

[0031] The present invention also discloses a use of the above-mentioned natural product synergistic chemotherapy nanoformulation or the above-mentioned SN38-9-fluorenemethanol small molecule prodrug self-assembled nanoparticles in the preparation of anti-tumor drugs.

[0032] The present invention also discloses a use of the above-mentioned natural product synergistic chemotherapy nanoformulation or the above-mentioned SN38-9-fluorenemethanol small molecule prodrug self-assembled nanoparticles in an injection, oral administration or local administration system.

[0033] This invention combines prodrug nanoassembly with chemotherapy sensitization to develop a new chemotherapy regimen to fully release the chemotherapy of SN38, with the following beneficial effects:

[0034] (1) Design and synthesis of a small molecule prodrug of SN38-9-fluorenemethanol containing a disulfide bridge. The disulfide bond has dual redox sensitivity and is rapidly broken under highly reducing conditions in tumor cells, thereby achieving tumor response release of SN38, significantly improving the anti-tumor effect of SN38 while reducing toxic side effects, achieving a "reduced toxicity" effect.

[0035] (2) For the first time, a carrier-free hybrid nanoassembly of galangin and SN38 oxidation-sensitive prodrug was constructed. The SN38 prodrug with a π-conjugated Fmoc structure was easily co-assembled with the chemical sensitizer galangin into stable nanoparticles. The prepared galangin-mediated prodrug co-assembled nanoparticles can be used for precise cancer chemotherapy for tumor treatment, accurately achieving SN38 "synergy", improving the therapeutic efficiency of SN38 prodrug, opening an ultra-low-dose chemotherapy window for SN38, and solving the problem of delayed and insufficient prodrug activation.

[0036] (3) Tumor-specific prodrug design and precise hybrid nanoassembly effectively controlled the off-target toxicity of SN38.

[0037] (4) The one-step nanoprecipitation method is simple, safe, non-toxic and has no side effects. It is easy to industrialize and can achieve efficient encapsulation of SN38. The prepared nanoparticles have uniform particle size and have the characteristics of synchronous drug delivery. After accumulating in large quantities in targeted tumor tissues through the EPR effect, they specifically release chemotherapy drugs under the conditions of reducing the microenvironment at the tumor site, thereby playing a synergistic sensitization role in chemotherapy, and have good application prospects in tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The disulfide bridged SN38-9-fluorene methanol of Example 1 of the present invention 1 HNMR spectrum.

[0039] Figure 2 This is the mass spectrum of the disulfide-bridged SN38-9-fluorenemethanol of Example 1 of the present invention.

[0040] Figure 3 This is a transmission electron microscopy image of the FSSN nanoparticles of Example 2 of the present invention.

[0041] Figure 4 This is a transmission electron microscopy image of the FSSN / GA nanoparticles of Example 3 of the present invention.

[0042] Figure 5 This is a graph showing the particle size-storage time of the PEG-modified small molecule prodrug nanoparticles of Example 6 of the present invention.

[0043] Figure 6 This is a graph showing the in vitro SN38 release test of the FSSN / GA nanoparticles of Example 7 of the present invention.

[0044] Figure 7 This is a graph showing the in vitro galangin release test of the FSSN / GA nanoparticles of Example 7 of the present invention.

[0045] Figure 8 4T1 cytotoxicity graphs of the GA solution, SN38 solution, FSSN / GA solution of Example 8, FSSN nanoparticles of Example 2, and FSSN / GA nanoparticles of Example 3 of the present invention.

[0046] Figure 9 CT26 cytotoxicity graphs of the GA solution, SN38 solution, FSSN / GA solution of Example 8, FSSN nanoparticles of Example 2, and FSSN / GA nanoparticles of Example 3 of the present invention.

[0047] Figure 10 3T3 cytotoxicity graphs of the SN38 solution of Example 8, the FSSN nanoparticles of Example 2, and the FSSN / GA nanoparticles of Example 3 of the present invention.

[0048] Figure 11 These are cell uptake diagrams of the Cy3-containing solution of Example 9 of the present invention, the Cy3-PEG-modified FSSN nanoparticles (FSSN-Cy3 nanoparticles) prepared in Example 4, and the Cy3-PEG-modified FSSN / GA nanoparticles (FSSN / GA-Cy3 nanoparticles) prepared in Example 5.

[0049] Figure 12 The blood drug concentration-time curves of the DiR solution of Example 10 of the present invention, the DiR-modified FSSN nanoparticles (DiR-FSSN nanoparticles) prepared in Example 4, and the DiR-modified FSSN / GA nanoparticles (DiR-FSSN / GA nanoparticles) prepared in Example 5 are shown.

[0050] Figure 13This is a tissue distribution diagram of the DiR solution of Example 11 of the present invention, the DiR-modified FSSN nanoparticles (DiR-FSSN nanoparticles) prepared in Example 4, and the DiR-modified FSSN / GA nanoparticles (DiR-FSSN / GA nanoparticles) prepared in Example 5.

[0051] Figure 14 This is the in vivo antitumor effect of the nanoparticles of Example 12 of the present invention on 4T1 orthotopic tumor-bearing mice.

[0052] Figure 15 This is the in vivo anti-tumor effect of the nanoparticles of Example 12 of the present invention on CT26 in situ tumors. DETAILED DESCRIPTION

[0053] The present invention will be further described below with reference to specific examples, but the present invention is not limited thereto in any way.

[0054] The present invention discloses that the inventors have discovered that inserting a tumor-sensitive chemical linker, such as a disulfide bond, into a conjugate can effectively manage the off-target toxicity of anticancer drugs through tumor-selective prodrug activation. The inventors also discovered that the BCL2 protein family plays a decisive role in the anti-apoptotic process of cells. This protein can inhibit cell death caused by a variety of cytotoxic factors. Overexpression of BCL2 can enhance the resistance of tumor cells to many cytotoxins. The inventors have found that galangin can selectively bind to the BCL2 family anti-apoptotic proteins to inhibit their expression. At the same time, galangin can significantly enhance the anti-tumor effect of SN38.

[0055] Therefore, the present inventors arrived at the following present invention based on these findings.

[0056] The present invention first designs a disulfide-bridged SN38-9-fluorenylmethanol small molecule prodrug. The SN38-9-fluorenylmethanol small molecule prodrug is a compound of SN38, camptothecin compounds and their derivatives, and 9-fluorenylmethanol connected by a disulfide bond. Its structural formula is:

[0057]

[0058] Specifically, the SN38-9-fluorenylmethanol small molecule prodrug designed in the present invention connects SN38 and 9-fluorenylmethanol with a π-electron conjugated structure through a disulfide bond that breaks in response to reduction. It can stably exist in the form of a prodrug in the blood circulation and normal tissues, and quickly break under the abnormally high reducing environment in tumor cells, thereby achieving the tumor-responsive release of SN38, significantly improving the anti-tumor effect of SN38 while reducing toxic side effects, achieving a "toxicity reduction" effect.

[0059] The present invention also discloses a method for synthesizing the SN38-9-fluorenylmethanol small molecule prodrug according to any embodiment of the present invention, comprising the following steps:

[0060] S1. Dehydrating dithiodiacetic acid I to obtain an acid anhydride compound II.

[0061] In a specific embodiment, step S1 specifically includes the following steps: dissolving dithiodiacetic acid in acetic anhydride, and performing a dehydration reaction at 20° C. to 25° C. for 2 h to 4 h to obtain anhydride compound II.

[0062] S2. Under the action of a catalyst, the acid anhydride compound II undergoes an esterification reaction with 9-fluorenylmethanol to obtain an intermediate product III.

[0063] In one embodiment, the molar ratio of the acid anhydride compound II to 9-fluorenylmethanol is (1-2):(1-2).

[0064] In one embodiment, the catalyst is DMAP; the molar ratio of the acid anhydride compound II to DMAP is 1:(0.1-1).

[0065] In a specific embodiment, step S2 specifically includes the following steps: dissolving the acid anhydride compound II in dichloromethane, adding DMAP, carrying out esterification reaction at 20°C to 25°C and stirring for 12h to 24h, and obtaining the intermediate product III by column chromatography separation.

[0066] S3. Under the action of a catalyst, the intermediate product III undergoes an esterification reaction with SN38 to obtain SN38-9-fluorenemethanol small molecule prodrug IV.

[0067] In one embodiment, the molar ratio of intermediate III to SN38 is (1-2): (1-2).

[0068] In a specific embodiment, the catalyst is DMAP, EDCI and HOBt; the molar ratio of the intermediate product III, DMAP, EDCI and HOBt is 1:(0.1-1):(1-2):(1-2).

[0069] In a specific embodiment, step S3 specifically includes the following steps: dissolving the intermediate product III, EDCI, HOBt and DMAP in dichloromethane, ice bathing for 2 hours, and then adding SN38 to carry out esterification reaction at 20°C to 25°C for 24 hours to 48 hours. The resulting product is separated and purified by preparative liquid phase to obtain SN38-9-fluorenemethanol small molecule prodrug IV.

[0070] In a specific embodiment, steps S1 to S3 are all performed under a nitrogen protective atmosphere.

[0071] Specifically, the synthetic reaction formula of SN38-9-fluorenylmethanol small molecule prodrug is as follows:

[0072]

[0073] The present invention also discloses a natural product synergistic chemotherapy nanoformulation as in any embodiment of the present invention, wherein the self-assembled nanoparticles are SN38-9-fluorenemethanol small molecule prodrug self-assembled nanoparticles modified with a polyethylene glycol modifier, or SN38-9-fluorenemethanol small molecule prodrug self-assembled nanoparticles encapsulating a hydrophobic fluorescent substance.

[0074] Furthermore, the preparation method of SN38-9-fluorenylmethanol small molecule prodrug self-assembled nanoparticles comprises the following steps:

[0075] S1. Dissolve the SN38-9-fluorenemethanol small molecule prodrug and polyethylene glycol modifier, or the SN38-9-fluorenemethanol small molecule prodrug, polyethylene glycol modifier and fluorescent substance in an organic solvent to obtain a mixed solution.

[0076] S2. The mixed solution is added dropwise to water, and the SN38-9-fluorenemethanol small molecule prodrug spontaneously forms uniform nanoparticles. The organic solvent in the preparation is removed by rotary evaporation to obtain SN38-9-fluorenemethanol small molecule prodrug self-assembled nanoparticles without any organic solvent.

[0077] Furthermore, the polyethylene glycol modifier includes one or more of PCL-PEG, DSPE-PEG, DSPE-SS-PEG, PLGA-PEG or PE-PEG.

[0078] Furthermore, the molecular weight of the polyethylene glycol modifier is 1000-5000.

[0079] Furthermore, the hydrophobic fluorescent substance includes one or more of coumarin-6, rhodamine, DiR, DiI, Cy5, Cy3 and Cy7.

[0080] Furthermore, the organic solvent includes one or more of tetrahydrofuran, ethanol, methanol and dimethyl sulfoxide. Preferably, the organic solvent is a mixed solvent of tetrahydrofuran and ethanol.

[0081] Furthermore, the mass ratio of SN38-9-fluorenylmethanol small molecule prodrug, hydrophobic fluorescent substance and polyethylene glycol modifier in the SN38-9-fluorenylmethanol small molecule prodrug self-assembled nanoparticles encapsulating the hydrophobic fluorescent substance is 1: (0.02~0.06): (0.1~0.3).

[0082] Furthermore, the mass ratio of SN38-9-fluorenylmethanol small molecule prodrug to polyethylene glycol modifier in the polyethylene glycol modifier-modified SN38-9-fluorenylmethanol small molecule prodrug self-assembled nanoparticles is 1:(0.1~0.3).

[0083] The present invention also discloses a natural product synergistic chemotherapy nanoformulation as in any embodiment of the present invention, wherein the co-assembled nanoparticles are SN38-9-fluorenylmethanol small molecule prodrug modified with polyethylene glycol modifier and galangin co-assembled nanoparticles, or SN38-9-fluorenylmethanol small molecule prodrug encapsulated with hydrophobic fluorescent substance and galangin co-assembled nanoparticles. Specifically, in order to further improve the anti-tumor effect of SN38, galangin is used as a chemosensitizer to construct a carrier-free hybrid nanoassembly with SN38 oxidation-sensitive prodrug for the first time. In this assembly process, the SN38 prodrug with a π-conjugated Fmoc structure is easily co-assembled with the chemosensitizer galangin into stable nanoparticles. The prepared galangin-mediated prodrug co-assembled nanoparticles can be used for precise cancer chemotherapy for tumor treatment, accurately achieving SN38 "synergy", improving the therapeutic efficiency of the SN38 prodrug, opening an ultra-low dose chemotherapy window for SN38, and solving the problems of delayed and insufficient prodrug activation.

[0084] The present invention also discloses a method for preparing a natural product synergistic chemotherapy nanoformulation according to any embodiment of the present invention, comprising the following steps:

[0085] S1. Dissolve SN38-9-fluorenylmethanol small molecule prodrug, galangin and polyethylene glycol modifier, or SN38-9-fluorenylmethanol small molecule prodrug, galangin and polyethylene glycol modifier, and a hydrophobic fluorescent substance in an organic solvent.

[0086] S2. The mixed solution is added dropwise to water, and the SN38-9-fluorenemethanol small molecule prodrug and galangin spontaneously form uniform nanoparticles. The organic solvent in the preparation is then removed by rotary evaporation to obtain a natural product synergistic chemotherapy nanoparticle preparation that does not contain any organic solvent.

[0087] In a specific embodiment, the polyethylene glycol modifier includes one or more of PCL-PEG, DSPE-PEG, DSPE-SS-PEG, PLGA-PEG or PE-PEG.

[0088] In one embodiment, the molecular weight of the polyethylene glycol modifier is 1000-5000.

[0089] In a specific embodiment, the hydrophobic fluorescent substance includes one or more of coumarin-6, rhodamine, DiR, DiI, Cy5, Cy3, and Cy7.

[0090] In a specific embodiment, the organic solvent includes one or more of tetrahydrofuran, ethanol, methanol and dimethyl sulfoxide; the preferred organic solvent is tetrahydrofuran.

[0091] In a specific embodiment, the mass ratio of SN38-9-fluorenylmethanol small molecule prodrug modified with polyethylene glycol modifier and galangin in the co-assembled nanoparticles is (0.1~10):1:(0.1~0.3).

[0092] In a specific embodiment, the mass ratio of SN38-9-fluorenylmethanol small molecule prodrug, galangin, polyethylene glycol modifier and hydrophobic fluorescent substance in the SN38-9-fluorenylmethanol small molecule prodrug and galangin co-assembled nanoparticles encapsulating the hydrophobic fluorescent substance is (0.1~10):1:(0.1~0.3):(0.02~0.06).

[0093] The present invention also discloses a natural product synergistic chemotherapy nanoformulation as in any embodiment of the present invention, or the use of the SN38-9-fluorenemethanol small molecule prodrug self-assembled nanoparticles in any embodiment of the present invention in a drug delivery system.

[0094] The present invention also discloses a natural product synergistic chemotherapy nanoformulation as in any embodiment of the present invention, or the use of the SN38-9-fluorenemethanol small molecule prodrug self-assembled nanoparticles in any embodiment of the present invention in the preparation of anti-tumor drugs.

[0095] The present invention also discloses a natural product synergistic chemotherapy nanoformulation as in any embodiment of the present invention, or the use of the SN38-9-fluorenemethanol small molecule prodrug self-assembled nanoparticles in any embodiment of the present invention in an injection, oral administration or local administration system.

[0096] Specifically, the present invention combines prodrug nanoassembly with chemotherapy sensitization to develop a new chemotherapy regimen to fully release the chemotherapy potential of SN38. The synthesized SN38-9-fluorenylmethanol small molecule prodrug is prepared into prodrug self-assembled nanoparticles and prodrug co-assembled nanoparticles with galangin. The present invention controls the off-target toxicity of SN38 well through tumor-specific prodrug design and precise hybrid nanoassembly; the prepared nanoparticles have a small particle size and good uniformity and stability, and have the characteristics of synchronous drug delivery. After accumulating in large quantities in targeted tumor tissues through the EPR effect, they specifically release chemotherapy drugs under the conditions of reducing the microenvironment at the tumor site, thereby playing a synergistic sensitization chemotherapy role; at the same time, the preparation process is simple, safe, has no toxic side effects, is easy to industrialize, and realizes efficient encapsulation of SN38, which has good application prospects in drug delivery systems, tumor treatment, injection, oral administration or local administration systems.

[0097] The following are specific embodiments

[0098] Example 1 Synthesis of SN38-9-fluorenemethanol small molecule prodrug

[0099] Under nitrogen protection, 2mmol of dithiodiacetic acid was placed in a 100mL eggplant-shaped flask and dissolved with 6mL of acetic anhydride. The mixture was magnetically stirred at 25°C for 2h, and 30mL of toluene was added. The toluene and excess acetic anhydride were removed by distillation under reduced pressure. 2mmol of the obtained product was dissolved in 15mL of dichloromethane, and 2mmol of 9-fluorene methanol and 0.2mmol of DMAP solution were added. The mixture was magnetically stirred at 25°C for 12h. The intermediate product was separated and purified using a dichloromethane-methanol elution system. Finally, 1mmol of the intermediate product, 2mmol of EDCI and 2mmol of HOBt were placed in an ice bath at 0°C for 2h, and then 1mmol of SN38 and 0.2mmol of DMAP were added. The mixture was reacted at 25°C for 48h. The final product was separated and purified by preparative liquid chromatography with a yield of 31.2%.

[0100] Using MS and 1 The product structure was confirmed by H-NMR. Figure 1-Figure 2 shown.

[0101] Example 2 Preparation of PEG-modified SN38-9-fluorenylmethanol small molecule prodrug self-assembled nanoparticles

[0102] Accurately weigh DSPE-PEG 2k 2 mg of SN38-9-fluorenemethanol and 8 mg of the SN38-9-fluorenemethanol small molecule prodrug of Example 1 were dissolved in 500 μL of tetrahydrofuran and diluted with 500 μL of tetrahydrofuran. The mixed solution was slowly added dropwise to 4 mL of deionized water under stirring, spontaneously forming uniform FSSN nanoparticles. The organic solvent was removed by rotary evaporation at 25°C. The particle size and morphology of the prepared self-assembled nanoparticles were measured by transmission electron microscopy. The results are shown in Figure 2. Figure 3 ,Transmission electron microscopy images show that the drug-loaded nanoparticles are uniform spherical with a particle size of about 120nm.

[0103] Example 3 Preparation of PEG-modified SN38-9-fluorenylmethanol small molecule prodrug and galangin co-assembled nanoparticles

[0104] Accurately weigh DSPE-PEG 2k2 mg of SN38-9-fluorenylmethanol small molecule prodrug from Example 1, 2.30 mg of galangin, and 1.70 mg of galangin were dissolved in 200 μL of tetrahydrofuran. The mixed solution was slowly added dropwise to 2 mL of deionized water under stirring, spontaneously forming uniform FSSN / GA nanoparticles. The organic solvent was removed by rotary evaporation at 25°C. The particle size and morphology of the prepared co-assembled nanoparticles were measured by transmission electron microscopy. The results are shown in Figure 2. Figure 4 ,Transmission electron microscopy images show that the drug-loaded nanoparticles are uniform spherical with a particle size of about 120nm.

[0105] Example 4 Preparation of Cy3-PEG Modified SN38-9-Fluorenecarbinol Small Molecule Prodrug Self-Assembled Nanoparticles

[0106] Accurately weigh Cy3-DSPE-PEG 2k 0.5 mg of SN38-9-fluorenylmethanol and 2 mg of the SN38-9-fluorenylmethanol small molecule prodrug prepared in Example 1 were dissolved in 200 μL of tetrahydrofuran. The mixed solution was slowly added dropwise to 2 mL of deionized water with stirring, spontaneously forming uniform Cy3-PEG-modified FSSN nanoparticles (FSSN-Cy3 nanoparticles). The organic solvent was removed by rotary evaporation at 25°C.

[0107] Example 5 Preparation of DiR-modified SN38-9-fluorenylmethanol small molecule prodrug and galangin co-assembled nanoparticles

[0108] Accurately weigh 1 mg of DiR and dissolve it in 100 μL of tetrahydrofuran. Dissolve 2.30 mg of the SN38-9-fluorenylmethanol small molecule prodrug prepared in Example 1 and 1.70 mg of galangin in 160 μL of tetrahydrofuran. Add 40 μL of DiR solution and stir. The resulting mixture is then slowly added dropwise to 2 mL of deionized water. Uniform DiR-modified FSSN / GA nanoparticles (DiR-FSSN / GA nanoparticles) spontaneously form. Remove the organic solvent by rotary evaporation at 25°C.

[0109] Example 6 Colloidal Stability Test of PEG-Modified SN38-9-Fluorenylmethanol Small Molecule Prodrug Nanoparticles

[0110] 1 mL of each of the prodrug nanoparticles prepared in Example 2 and Example 3 was taken out and added to 20 mL of phosphate buffered saline (PBS, pH 7.4) containing 10% fetal bovine serum. The mixture was incubated at 37°C for 12 h, and the particle size changes were measured by dynamic light scattering at predetermined time points (0 h, 2 h, 4 h, 6 h, 8 h, and 12 h). The results are shown in FIG. Figure 5As shown in the figure, the nanoparticle colloid has good stability and the particle size does not change significantly within 12 hours. That is, in the presence of inorganic salts, the constructed SN38 prodrug nanoparticles can exist in a relatively stable form, which is conducive to more drugs being enriched in the tumor site through blood circulation and achieving better therapeutic effects.

[0111] Example 7 In vitro release test of PEG-modified SN38-9-fluorenylmethanol small molecule prodrug and galangin co-assembled nanoparticles

[0112] (1) Release of SN38

[0113] Using PBS buffer at pH 7.4 containing 20% ​​tetrahydrofuran as the release medium, the in vitro release of SN38 in nanoparticles co-assembled with SN38-9-fluorenylmethanol small molecule prodrug and galangin was investigated. 0.82 mL of FSSN / GA nanoparticles (SN38 content of 200 μg / mL) prepared in Example 3 was added to 30 mL of the release medium. Samples were taken at set time points at 37°C, and the concentration of released SN38 was determined by high performance liquid chromatography. A certain concentration of dithiothreitol (DTT, 10 mM) was added to the release medium to investigate the release of the nanoparticles under reducing conditions. The results are shown in FIG. Figure 6 As shown, the disulfide-bridged nanoparticles are reduction-responsive and can quickly release SN38 under the action of DTT.

[0114] (2) Release of galangin

[0115] Using PBS buffer at pH 7.4 containing 20% ​​tetrahydrofuran as the release medium, the in vitro release of galangin from nanoparticles co-assembled with SN38-9-fluorenemethanol small molecule prodrug and galangin was investigated. 125 μL of FSSN / GA nanoparticles prepared in Example 3 (with a galangin content of 200 μg / mL) were added to 30 mL of the release medium. Samples were taken at set time points at 37°C, and the concentration of released galangin was determined by high performance liquid chromatography. A certain concentration of dithiothreitol (DTT, 10 mM) was added to the release medium to investigate the release of the nanoparticles under reducing conditions. The results are shown in FIG. Figure 7 As shown in the figure, the disulfide-bridged nanoparticles are reduction-responsive and can quickly release galangin under the action of DTT.

[0116] Example 8 Cytotoxicity of PEG-modified SN38-9-fluorenylmethanol small molecule prodrug nanoparticles

[0117] The MTT assay was used to investigate the toxicity of PEG-modified SN38 prodrug self-assembled nanoparticles to mouse breast cancer cells (4T1), mouse colon cancer cells (CT26), and mouse fibroblasts (3T3). 4T1 cells and CT26 cells were cultured at 2×103 The cells were seeded in a 96-well culture plate at a density of 100 cells / well. After culturing for 12 hours, the cells were treated with different concentrations of GA solution, SN38 solution, FSSN / GA solution, FSSN nanoparticles prepared in Example 2, and FSSN / GA nanoparticles prepared in Example 3 (same concentration of SN38 and / or GA) for 48 hours. 5 mg / mL of MTT (20 μL / well) was then added and incubated at 37°C for 4 hours. The culture medium was replaced with DMSO (200 μL / well) to dissolve the generated formazan. Finally, the ultraviolet absorbance at 490 nm was measured using a microplate reader. Using 3T3 cells as a cell model, the same method was used to verify the cytotoxicity of SN38 solution, FSSN nanoparticles prepared in Example 2, and FSSN / GA nanoparticles prepared in Example 3 to normal cells.

[0118] The results are as follows Figure 8 and Figure 9 As shown in Figure 2, when combined with non-toxic concentrations of GA, the cytotoxicity of the FSSN / GA nanoparticle-treated group was significantly higher than that of the FSSN nanoparticle-treated group, confirming the sensitizing effect of GA on FSSN nanoparticles. It is worth noting that compared with the FSSN / GA solution, FSSN nanoparticles exhibited stronger in vitro antitumor activity, mainly due to their efficient cellular uptake and rapid intracellular drug release. In addition, as Figure 10 As shown, FSSN NPs and FSSN / GA NPs showed less cytotoxicity towards 3T3 cells, highlighting their good biocompatibility.

[0119] Example 9 Cellular Uptake of PEG-Modified SN38-9-Fluorenylmethanol Small Molecule Prodrug Nanoparticles

[0120] 4T1 cells were cultured at 2 × 10 5 Cells were seeded at a density of 1 μg / well in 12-well plates and incubated for 24 hours. The culture medium was then replaced with fresh medium containing Cy3 solution, Cy3-PEG-modified FSSN nanoparticles (FSSN-Cy3 nanoparticles) prepared in Example 4, and Cy3-PEG-modified FSSN / GA nanoparticles (FSSN / GA-Cy3 nanoparticles) prepared in Example 5 (Cy3 concentration in each medium was 4 μg / mL), and incubation was continued for 0.5 hours or 2 hours, respectively. After washing, fixation, or digestion, the cells were analyzed using a flow cytometer (BD, East Rutherford, NJ, USA).

[0121] like Figure 11As shown, the uptake efficiency of Cy3-PEG-modified FSSN nanoparticles and Cy3-PEG-modified FSSN / GA nanoparticles by 4T1 cells was found to be much higher than that of the solution, and this efficiency was time-dependent. Importantly, there was no significant difference between Cy3-PEG-modified FSSN nanoparticles and Cy3-PEG-modified FSSN / GA nanoparticles, indicating that nanoparticle co-assembly did not hinder cellular uptake of the nanoparticles. The nanoparticles were effectively internalized into cells via concentration-independent endocytosis. Therefore, the highly stable Cy3-PEG-modified FSSN / GA nanoparticles and Cy3-PEG-modified FSSN nanoparticles exhibited significantly higher cellular uptake efficiency than the Cy3 solution, facilitating tumor cell uptake of the prodrug nanoparticles and subsequently resulting in a superior tumor inhibitory effect.

[0122] Example 10 Pharmacokinetic Study of PEG-Modified SN38-9-Fluorenylmethanol Small Molecule Prodrug Nanoparticles

[0123] Sprague-Dawley rats weighing 200-220 g were randomly divided into three groups, each consisting of six rats. The in vivo pharmacokinetic behavior of DiR-modified FSSN nanoparticles (DiR-FSSN nanoparticles) prepared in Example 4 and DiR-PEG-modified FSSN / GA nanoparticles (DiR-FSSN / GA nanoparticles) prepared in Example 5 were investigated. DiR solution, DiR-modified FSSN nanoparticles, and DiR-modified FSSN / GA nanoparticles were injected intravenously via the tail vein at a dose of 1 mg / kg of DiR. Blood samples (0.5 mL) were collected from the ophthalmic vein at 2, 5, 15, 30, 1, 2, 4, 8, and 12 hours after injection. Plasma was obtained by centrifugation (8000 rpm, 3 min), and DiR plasma concentrations were quantified using a microplate reader with excitation at 748 nm and emission at 790 nm.

[0124] The plasma drug concentration-time curves of these compounds are as follows Figure 12 As shown, the DiR solution is rapidly cleared from the body in vivo. In contrast, DiR-modified FSSN nanoparticles and DiR-modified FSSN / GA nanoparticles have a longer circulation time in the blood, resulting in a higher AUC at the same DiR dose compared to the DiR solution. In addition, compared with DiR-modified FSSN nanoparticles, DiR-modified FSSN / GA nanoparticles exhibited better pharmacokinetic behavior, which may be related to their enhanced stability. This shows that DiR-modified FSSN / GA nanoparticles can effectively prolong the circulation time of SN38 in the blood and exhibit unique advantages in in vivo administration, which may be conducive to the accumulation of drugs in tumor tissues mediated by the enhanced permeability and retention (EPR) effect, thereby fully exerting the drug efficacy.

[0125] Example 11 Tissue Distribution Experiment of DiR-Modified SN38-9-Fluorenemethanol Small Molecule Prodrug Nanoparticles

[0126] 4T1 cell suspension was inoculated into BALB / c mice. When the tumor volume reached 400 mm 3 At 1 mg / kg, mice were injected via the tail vein with a DiR solution, DiR-modified FSSN nanoparticles prepared in Example 4 (DiR FSSN nanoparticles), and DiR-modified FSSN / GA nanoparticles prepared in Example 5 (DiR-FSSN / GA nanoparticles). Tumors and major organs (heart, liver, spleen, lung, and kidney) were removed. Fluorescence intensity was then analyzed using an IVIS imaging system.

[0127] like Figure 13 As shown, the fluorescence signals of DiR-modified FSSN nanoparticles and DiR-modified FSSN / GA nanoparticles were much stronger than those of the DiR solution and were primarily distributed in the liver and tumor tissues. Notably, at 12 hours after treatment with an equal dose of DiR, the fluorescence intensity of DiR-PEG-modified FSSN / GA nanoparticles at the tumor site was stronger than that of DiR-modified FSSN nanoparticles. The stronger fluorescence intensity of DiR-modified FSSN / GA nanoparticles may be related to their superior colloidal stability and in vivo circulation advantages. This suggests that prolonged circulation time can promote the accumulation of prodrug nanoassemblies in tumors through the EPR effect.

[0128] Example 12 In vivo antitumor experiment of PEG-modified SN38-9-fluorenylmethanol small molecule prodrug nanoparticles

[0129] The anti-tumor effect of nanoparticles was evaluated using 4T1 tumor-bearing female BALB / c mice as an in vivo model. 100 μL of 4T1 cell suspension (5×10 7 / mL) to establish a tumor-bearing model. When the tumor volume reached 100mm 3 Mice were randomly divided into six groups, each containing five mice: a control group (PBS), GA solution, FSSN / GA solution, SN38 solution, FSSN nanoparticles prepared in Example 2, and FSSN / GA nanoparticles prepared in Example 3. Dosing was performed every other day for a total of five doses, with each dose containing the same concentration of SN38 (4 mg / kg) and / or GA (25 mg / kg). Tumor volume and body weight were monitored daily. Mice were sacrificed after the final treatment, and tumor tissue was obtained.

[0130] like Figure 14As shown, compared with the control group, the FSSN / GA solution and the SN38 solution exhibited comparable, moderate tumor growth delays. The SN38 solution exhibited significant body weight loss at day 12, indicating significant systemic toxicity. This suggests that the prodrug strategy can largely avoid the off-target toxicity of SN38, and that galangin-mediated chemosensitization is expected to enhance the therapeutic efficacy of the SN38 prodrug. Furthermore, compared with FSSN nanoparticles, FSSN / GA nanoparticles exhibited significant tumor growth inhibition. This suggests that the optimal antitumor efficacy of FSSN / GA nanoparticles is attributed to the GA-mediated sensitization and the multiple therapeutic advantages of the prodrug nanosystem, including prolonged blood circulation, efficient cellular uptake, and redox-sensitive drug release. Finally, the therapeutic safety of the nanoparticles was preliminarily investigated. Compared with the GA solution, the FSSN and FSSN / GA nanoparticle groups did not cause significant weight loss after the end of treatment, as assessed by changes in mouse body weight, demonstrating a good safety profile during treatment.

[0131] like Figure 15 As shown, based on the promising therapeutic effects of the dual nanococktail observed in an orthotopic 4T1 tumor model, we expanded our studies to an orthotopic CT26-Luc colorectal tumor model to further evaluate its efficacy. Tumor progression was monitored by bioluminescence imaging, providing real-time assessment of tumor suppression across treatment groups. Mice intravenously injected with FSSN / GA NAs demonstrated the strongest antitumor response, as evidenced by a significant decrease in bioluminescence signal over time. In terms of survival, FSSN / GA NAs demonstrated a significant survival advantage, with a median survival of 41 days in the treated group, significantly longer than in any other group. In contrast, mice treated with FSSN NAs alone had a median survival of only 24 days, indicating that the addition of GA to NAs significantly enhanced the therapeutic effect through GA-mediated chemosensitization and improved tumor accumulation. Notably, the commercially available irinotecan liposomal Onivyde produced only a modest survival benefit compared to the PBS control, with median survival of 15 days versus 8 days, respectively. In conclusion, the prolonged survival in the FSSN / GA NAs treatment group highlights the potential to improve outcomes in an aggressive colorectal tumor model.

[0132] The above results indicate that the galangin-mediated prodrug nanoassembly of the present invention can be used as an efficient and safe combined treatment model, which can effectively alleviate the systemic toxicity of SN38 while enhancing the anti-tumor effect, and can be used for precision cancer chemotherapy for tumor treatment.

[0133] The above-described embodiments merely represent several implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A natural product nanoformulation for enhancing chemotherapy, characterized in that: The nanoformulation is a nanoparticle co-assembled by a polyethylene glycol-modified SN38-9-fluorenylmethanol small molecule prodrug and galangin, or a nanoparticle co-assembled by a hydrophobic fluorescent substance-encapsulated SN38-9-fluorenylmethanol small molecule prodrug and galangin; The SN38-9-fluorenylmethanol small molecule prodrug is obtained by connecting SN38, camptothecin compounds and their derivatives with 9-fluorenylmethanol through disulfide bonds and other sensitive bonds.

2. The natural product synergistic chemotherapy nanoformulation according to claim 1, characterized in that: The sensitive bond is a pH sensitive bond or a reducing environment sensitive bond, the pH sensitive bond is a hydrazone bond or a carbonate bond, and the reducing environment sensitive bond is a monosulfide bond, a disulfide bond, a disulfide bond or a metalloproteinase sensitive bond.

3. A method for preparing the natural product synergistic chemotherapy nanoformulation according to claim 1, characterized in that: The following steps are involved: Dissolving SN38-9-fluorenylmethanol small molecule prodrug, galangin and polyethylene glycol modifier, or SN38-9-fluorenylmethanol small molecule prodrug, galangin and polyethylene glycol modifier, and a hydrophobic fluorescent substance in an organic solvent, and then diluting with ethanol to obtain a mixed solution; The mixed solution is added dropwise to water, and the SN38-9-fluorenylmethanol small molecule prodrug and galangin spontaneously form uniform nanoparticles. The organic solvent in the preparation is then removed by rotary evaporation to obtain SN38-9-fluorenylmethanol small molecule prodrug and galangin co-assembled nanoparticles that do not contain any organic solvent.

4. The preparation method according to claim 3, characterized in that The PEG modifier includes one or more of PCL-PEG, DSPE-PEG, DSPE-SS-PEG, PLGA-PEG or PE-PEG; The hydrophobic fluorescent substance includes one or more of coumarin-6, rhodamine, DiR, DiI, Cy3, Cy5 and Cy7; The organic solvent includes one or more of tetrahydrofuran, ethanol, methanol and dimethyl sulfoxide; The mass ratio of the SN38-9-fluorenylmethanol small molecule prodrug modified by the polyethylene glycol modifier and galangin in the co-assembled nanoparticles is (0.1-10):1:(0.1-0.3); The mass ratio of the SN38-9-fluorenylmethanol small molecule prodrug, the galangin, the polyethylene glycol modifier and the hydrophobic fluorescent substance in the SN38-9-fluorenylmethanol small molecule prodrug and galangin co-assembled nanoparticles encapsulating the hydrophobic fluorescent substance is (0.1-10):1:(0.1-0.3):(0.02-0.06).

5. The preparation method according to claim 3, characterized in that The synthesis method of the SN38-9-fluorenylmethanol small molecule prodrug comprises the following steps: S1, dehydrating dithiodiacetic acid I to obtain an acid anhydride compound II; S2. In the presence of a catalyst, the acid anhydride compound II undergoes an esterification reaction with 9-fluorenylmethanol to obtain an intermediate product III; S3. Under the action of a catalyst, the intermediate product III undergoes an esterification reaction with SN38 to obtain SN38-9-fluorenylmethanol small molecule prodrug IV; the reaction formula is as follows:

6. The preparation method according to claim 5, characterized in that In S1, the temperature of the dehydration reaction is 20°C to 25°C; the time of the dehydration reaction is 2h to 4h; In S2, the molar ratio of the acid anhydride compound II to the 9-fluorenylmethanol is (1-2): (1-2); the catalyst is DMAP; the molar ratio of the acid anhydride compound II to DMAP is 1: (0.1-1); the temperature of the esterification reaction is 20° C. to 25° C., and the time of the esterification reaction is 12 h to 24 h; In S3, the molar ratio of the intermediate product III to SN38 is (1-2): (1-2); the catalyst is DMAP, EDCI and HOBT; the molar ratio of the intermediate product III, DMAP, EDCI and HOBT is 1: (0.1-1): (1-2): (1-2); the temperature of the esterification reaction is 20°C to 25°C, and the time of the esterification reaction is 24h to 48h.

7. A SN38-9-fluorenylmethanol small molecule prodrug self-assembled nanoparticle, characterized in that: The self-assembled nanoparticles are SN38-9-fluorenylmethanol small molecule prodrug self-assembled nanoparticles modified with a polyethylene glycol modifier, or SN38-9-fluorenylmethanol small molecule prodrug self-assembled nanoparticles encapsulating a hydrophobic fluorescent substance; The SN38-9-fluorenylmethanol small molecule prodrug is formed by connecting SN38 and 9-fluorenylmethanol via a disulfide bond, and its structural formula is: Preferably, the method for preparing the SN38-9-fluorenylmethanol small molecule prodrug self-assembled nanoparticles comprises the following steps: dissolving the SN38-9-fluorenylmethanol small molecule prodrug and a polyethylene glycol modifier, or the SN38-9-fluorenylmethanol small molecule prodrug, a polyethylene glycol modifier, and a fluorescent substance in an organic solvent to obtain a mixed solution; The mixed solution is added dropwise to water, and the SN38-9-fluorenylmethanol small molecule prodrug spontaneously forms uniform nanoparticles, and the organic solvent in the preparation is removed by rotary evaporation to obtain SN38-9-fluorenylmethanol small molecule prodrug self-assembled nanoparticles without any organic solvent; Preferably, the polyethylene glycol modifier includes one or more of PCL-PEG, DSPE-PEG, DSPE-SS-PEG, PLGA-PEG or PE-PEG; the molecular weight of the polyethylene glycol modifier is 1000 to 5000; the hydrophobic fluorescent substance includes one or more of coumarin-6, rhodamine, DiR, DiI, Cy3, Cy5 and Cy7; the organic solvent includes one or more of tetrahydrofuran, ethanol, methanol and dimethyl sulfoxide; Preferably, the mass ratio of SN38-9-fluorenylmethanol small molecule prodrug, hydrophobic fluorescent substance and polyethylene glycol modifier in the SN38-9-fluorenylmethanol small molecule prodrug self-assembled nanoparticles encapsulating hydrophobic fluorescent substance is 1:(0.02~0.06):(0.1~0.3); the mass ratio of SN38-9-fluorenylmethanol small molecule prodrug and polyethylene glycol modifier in the SN38-9-fluorenylmethanol small molecule prodrug self-assembled nanoparticles modified with polyethylene glycol is 1:(0.1~0.3).

8. Use of the natural product synergistic chemotherapy nanoformulation according to claim 1 or the SN38-9-fluorenylmethanol small molecule prodrug self-assembled nanoparticles according to claim 7 in a drug delivery system.

9. Use of the natural product synergistic chemotherapy nanoformulation according to claim 1 or the SN38-9-fluorenylmethanol small molecule prodrug self-assembled nanoparticles according to claim 7 in the preparation of anti-tumor drugs.

10. Use of the natural product synergistic chemotherapy nanoformulation according to claim 1, or the SN38-9-fluorenemethanol small molecule prodrug self-assembled nanoparticles according to claim 7 in an injection, oral administration or local administration system.

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