A small molecule prodrug nanoparticle of podophyllotoxin-9-fluorenyl alcohol, its preparation method, and its application.

Abstract

This invention discloses a podophyllotoxin-9-fluorenylmethanol small molecule prodrug nanoparticle, its preparation method, and its applications. The podophyllotoxin-9-fluorenylmethanol small molecule prodrug co-assembled nanoparticles are either polyethylene glycol-modified podophyllotoxin-9-fluorenylmethanol small molecule prodrug co-assembled with gossypol, or podophyllotoxin-9-fluorenylmethanol small molecule prodrug co-assembled with gossypol and loaded with hydrophobic fluorescent substances. The podophyllotoxin-9-fluorenylmethanol small molecule prodrug is prepared by linking podophyllotoxin and its compounds with 9-fluorenylmethanol via disulfide bonds. This invention is the first to construct a carrier-free hybrid nanoassembly of gossypol and PPT oxidation-sensitive prodrug. Gossypol-mediated prodrug co-assembled nanoparticles can be used for precision cancer chemotherapy in tumor treatment, precisely achieving PPT "enhancement," improving the therapeutic efficiency of PPT prodrugs, opening an ultra-low-dose chemotherapy window for PPT, and solving the problems of delayed and insufficient prodrug activation.

Description

Technical Field

[0001] This invention belongs to the field of sensitizing drugs for tumor chemotherapy, specifically relating to a podophyllotoxin-9-fluorenylmethanol small molecule prodrug nanoparticle, its preparation method, and its application. Background Technology

[0002] Podophylotoxin (PPT) is a natural aryl lignin extracted from the roots and rhizomes of plants in the Podophyllum genus. PPT exhibits potent antitumor activity by binding to tubulin and inhibiting the formation of the mitotic spindle during cell division; studies have shown that podophylotoxin has a relatively ideal inhibitory effect on various tumors. However, PPT has poor water solubility and produces severe off-target toxicity, both of which significantly limit its application. For these reasons, it has not yet been used in the clinical treatment of cancer.

[0003] In recent years, to overcome the shortcomings of podophyllotoxin (PPT), medicinal chemists have attempted to synthesize a series of similar compounds for the treatment of clinical cancers such as lung cancer, breast cancer, and leukemia, such as etoposide (VP-16) and teniposide (VM-26). However, their efficacy and severe toxic side effects remain limitations. Therefore, designing an efficient and low-toxicity drug delivery system for podophyllotoxin delivery remains a research hotspot. Prodrug strategies are an effective method to improve the delivery efficiency of anticancer drugs. Structural modification of PPT through prodrug strategies can effectively improve its poor solubility and severe toxic side effects. Meanwhile, nanotechnology, by effectively prolonging the circulation time of drugs in vivo and enhancing antitumor effects, has also greatly improved drug delivery efficiency in the field of drug delivery. Therefore, integrating prodrug strategies and nanotechnology into a single system has become a significant trend in promoting more efficient delivery of anticancer drugs.

[0004] However, prodrug activation requires reaching a minimum effective concentration of free drug, which greatly limits its clinical application. Therefore, fully leveraging the anti-tumor potential of prodrug-based drugs is a crucial issue that urgently needs to be addressed in developing prodrug-based drug delivery methods. Summary of the Invention

[0005] The purpose of this invention is to design and synthesize a small-molecule prodrug of podophyllotoxin-9-fluorenylmethanol containing disulfide bonds, prepare a self-assembled nanomedicine delivery system of the prodrug and a co-assembled nanomedicine delivery system of the prodrug and gossypol, and their application in drug delivery. The stability, drug release, and effects of gossypol on the sensitizing effect of podophyllotoxin in terms of cytotoxicity, pharmacokinetics, tissue distribution, and pharmacodynamics of the self-assembled nanoparticles of the prodrug and the co-assembled nanoparticles of the gossypol and prodrug are investigated. This aims to provide a novel, highly efficient, and safe small-molecule prodrug nanoparticle of podophyllotoxin-9-fluorenylmethanol, its preparation method, and its application in precision chemotherapy for tumor treatment.

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

[0007] A co-assembled nanoparticle of a podophyllotoxin-9-fluorenylethanol small molecule prodrug, wherein the co-assembled nanoparticle is a polyethylene glycol-modified podophyllotoxin-9-fluorenylethanol small molecule prodrug co-assembled with gossypol, or a podophyllotoxin-9-fluorenylethanol small molecule prodrug loaded with a hydrophobic fluorescent substance co-assembled with gossypol; the podophyllotoxin-9-fluorenylethanol small molecule prodrug is formed by linking podophyllotoxin and its compounds with 9-fluorenylethanol via disulfide bonds, and its structural formula is as follows:

[0008]

[0009] This invention also discloses a method for preparing the above-mentioned podophyllotoxin-9-fluorenylethanol small molecule prodrug co-assembled nanoparticles, comprising the following steps:

[0010] Podophyllotoxin-9-fluorenylmethanol small molecule prodrug, gossypol and polyethylene glycol modifier, or podophyllotoxin-9-fluorenylmethanol small molecule prodrug, gossypol and polyethylene glycol modifier, and hydrophobic fluorescent substance are dissolved in an organic solvent and then diluted with ethanol to obtain a mixed solution.

[0011] The mixed solution was added dropwise to water, and podophyllotoxin-9-fluorenylethanol small molecule prodrug and gossypol spontaneously formed uniform nanoparticles. Then, the organic solvent in the formulation was removed by rotary evaporation to obtain podophyllotoxin-9-fluorenylethanol small molecule prodrug co-assembled nanoparticles without any organic solvent.

[0012] The present invention also discloses a self-assembled nanoparticle of podophyllotoxin-9-fluorenylethanol small molecule prodrug, wherein the self-assembled nanoparticle is a self-assembled nanoparticle of podophyllotoxin-9-fluorenylethanol small molecule prodrug modified with a polyethylene glycol modifier, or a self-assembled nanoparticle of podophyllotoxin-9-fluorenylethanol small molecule prodrug loaded with a hydrophobic fluorescent substance; wherein the podophyllotoxin-9-fluorenylethanol small molecule prodrug is formed by linking podophyllotoxin and its compounds with 9-fluorenylethanol through disulfide bonds.

[0013] This invention also discloses a method for preparing podophyllotoxin-9-fluorenylethanol small molecule prodrug self-assembled nanoparticles as described above, comprising the following steps:

[0014] Podophyllotoxin-9-fluorenylmethanol small molecule prodrug is dissolved in an organic solvent with a polyethylene glycol modifier, or podophyllotoxin-9-fluorenylmethanol small molecule prodrug is dissolved in a polyethylene glycol modifier and a fluorescent substance to obtain a mixed solution;

[0015] The mixed solution was added dropwise to water, and the podophyllotoxin-9-fluorenylethanol small molecule prodrug spontaneously formed uniform nanoparticles. The organic solvent in the formulation was removed by rotary evaporation to obtain podophyllotoxin-9-fluorenylethanol small molecule prodrug self-assembled nanoparticles without any organic solvent.

[0016] The polyethylene glycol modifier includes one or more of TPGS, DSPE-PEG, PLGA-PEG, and 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, Cy5, and Cy7; the organic solvent includes one or more of tetrahydrofuran, ethanol, methanol, and dimethyl sulfoxide; the encapsulated hydrophobic... In the self-assembled nanoparticles of podophyllotoxin-9-fluorenylethanol small molecule prodrug, the mass ratio of podophyllotoxin-9-fluorenylethanol small molecule prodrug, hydrophobic fluorescent substance, and polyethylene glycol modifier is 1:(0.02-0.06):(0.1-0.3); in the self-assembled nanoparticles of podophyllotoxin-9-fluorenylethanol small molecule prodrug modified with polyethylene glycol modifier, the mass ratio of podophyllotoxin-9-fluorenylethanol small molecule prodrug to polyethylene glycol modifier is 1:(0.1-0.3).

[0017] The present invention also discloses the application of the above-mentioned podophyllotoxin-9-fluorenylethanol small molecule prodrug co-assembled nanoparticles or the above-mentioned podophyllotoxin-9-fluorenylethanol small molecule prodrug self-assembled nanoparticles in a drug delivery system.

[0018] The present invention also discloses the application of the above-mentioned podophyllotoxin-9-fluorenylethanol small molecule prodrug co-assembled nanoparticles or the above-mentioned podophyllotoxin-9-fluorenylethanol small molecule prodrug self-assembled nanoparticles in the preparation of antitumor drugs.

[0019] The present invention also discloses the application of the above-mentioned podophyllotoxin-9-fluorenylethanol small molecule prodrug co-assembled nanoparticles or the above-mentioned podophyllotoxin-9-fluorenylethanol small molecule prodrug self-assembled nanoparticles in injection, oral or topical drug delivery systems.

[0020] This invention combines prodrug nanoassembly with chemotherapy sensitization to develop a new chemotherapy regimen to fully release the chemotherapy potential of PPT. Implementing the embodiments of this invention will have the following beneficial effects: (1) A small molecule prodrug containing disulfide bond bridging, podophyllotoxin-9-fluorenylmethanol, is designed and synthesized. The disulfide bond has dual redox sensitivity. Under high reduction conditions in tumor cells, the disulfide bond breaks rapidly, thereby realizing the tumor response release of PPT, significantly improving the anti-tumor effect of PPT while reducing toxic side effects, achieving the effect of "reduced toxicity". (2) For the first time, a carrier-free hybrid nanoassembly of gossypol and PPT oxidation-sensitive prodrug is constructed. The PPT prodrug with π-conjugated Fmoc structure is easily co-assembled with the chemical sensitizer gossypol into stable nanoparticles. The prepared gossypol-mediated prodrug co-assembled nanoparticles can be used for precision cancer chemotherapy in tumor treatment, accurately realizing PPT "enhancement", improving the therapeutic efficiency of PPT prodrug, opening an ultra-low dose chemotherapy window for PPT, and solving the problems of delayed and insufficient prodrug activation. (3) Tumor-specific prodrug design and precise hybrid nanoassembly effectively controlled the off-target toxicity of PPT. (4) The one-step nanoprecipitation method is simple, safe, and has no toxic side effects, making it easy to industrialize and achieve efficient PPT encapsulation. The prepared nanoparticles have uniform particle size and synchronous drug delivery characteristics. After accumulating in large quantities in tumor tissue through the EPR effect, they specifically release chemotherapeutic drugs under the reduced microenvironment conditions at the tumor site, thereby playing a synergistic role in sensitizing chemotherapy and showing good application prospects in tumor treatment. Attached Figure Description

[0021] Figure 1 The disulfide-bridged podophyllotoxin-9-fluorenyl alcohol of Example 1 of this invention 1 HNMR spectrum.

[0022] Figure 2 This is the mass spectrum of disulfide-bridged podophyllotoxin-9-fluorenylethanol in Example 1 of the present invention.

[0023] Figure 3 This is a transmission electron microscope image of the PSSF nanoparticles in Example 2 of the present invention.

[0024] Figure 4 This is a transmission electron microscope image of the PSSF / GSP nanoparticles in Example 3 of the present invention.

[0025] Figure 5 This is a particle size-storage time diagram of the PEG-modified small molecule prodrug nanoparticles of Example 6 of the present invention.

[0026] Figure 6 This is a diagram of the in vitro PPT release test of PSSF / GSP nanoparticles in Example 7 of the present invention.

[0027] Figure 7This is a diagram of the in vitro gossypol release test of PSSF / GSP nanoparticles in Example 7 of the present invention.

[0028] Figure 8 The graph shows the 4T1 cytotoxicity of the GSP solution, PPT solution, PSSF / GSP solution of Example 8, PSSF nanoparticles of Example 2, and PSSF / GSP nanoparticles of Example 3 of this invention.

[0029] Figure 9 The graphs show the RM-1 cytotoxicity of the GSP solution, PPT solution, PSSF / GSP solution of Example 8, PSSF nanoparticles of Example 2, and PSSF / GSP nanoparticles of Example 3 of this invention.

[0030] Figure 10 3T3 cytotoxicity diagrams of the PPT solution of Example 8, the PSSF nanoparticles of Example 2, and the PSSF / GSP nanoparticles of Example 3 of this invention.

[0031] Figure 11 This is a cellular uptake diagram of the Cy7-containing solution of Example 9, the Cy7-PEG modified PSSF nanoparticles (PSSF-Cy7 nanoparticles) prepared in Example 4, and the Cy7-PEG modified PSSF / GSP nanoparticles (PSSF / GSP-Cy7 nanoparticles) prepared in Example 5.

[0032] Figure 12 The blood drug concentration-time curves are for the Cy7 solution of Example 10, the Cy7-PEG modified PSSF nanoparticles (PSSF-Cy7 nanoparticles) prepared in Example 4, and the Cy7-PEG modified PSSF / GSP nanoparticles (PSSF / GSP-Cy7 nanoparticles) prepared in Example 5.

[0033] Figure 13 The images show the tissue distribution of the Cy7 solution of Example 11, the Cy7-PEG modified PSSF nanoparticles (PSSF-Cy7 nanoparticles) prepared in Example 4, and the Cy7-PEG modified PSSF / GSP nanoparticles (PSSF / GSP-Cy7 nanoparticles) prepared in Example 5.

[0034] Figure 14 This is a diagram of the in vivo antitumor experiment of nanoparticles in Example 12 of the present invention. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments, but this does not limit the present invention in any way.

[0036] This invention discloses the inventors' discovery that inserting tumor-stimuli-sensitive chemical linkers, such as disulfide bonds, into conjugates 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 induced by various cytotoxic factors, and overexpression of BCL2 can enhance the resistance of tumor cells to many cytotoxins. Furthermore, the inventors' research found that gossypol can selectively bind to BCL2 family anti-apoptotic proteins to inhibit their expression, and gossypol can also significantly enhance the antitumor effect of PPT.

[0037] Therefore, the present invention obtained by the inventors based on these insights is as follows.

[0038] This invention first designs a small-molecule prodrug of podophyllotoxin-9-fluorenylethanol containing disulfide bonds. The podophyllotoxin-9-fluorenylethanol small-molecule prodrug is formed by linking podophyllotoxin and its compounds with 9-fluorenylethanol via disulfide bonds, and its structural formula is as follows:

[0039]

[0040] Specifically, the podophyllotoxin-9-fluorenylmethanol small molecule prodrug designed in this invention connects podophyllotoxin with 9-fluorenylmethanol, which has a π-electron conjugated structure, through a disulfide bond that breaks in a reduction response. It can exist stably in the form of a prodrug in blood circulation and in normal tissues, but breaks rapidly in the abnormally high reducing environment in tumor cells, thereby achieving tumor-responsive release of PPT. This significantly improves the anti-tumor effect of PPT while reducing toxic side effects, achieving a "reduced toxicity" effect.

[0041] This invention also discloses a method for synthesizing a small molecule prodrug of podophyllotoxin-9-fluorenyl alcohol as described in any embodiment of this invention, comprising the following steps:

[0042] S1. 4,4'-Dithiodibutyric acid I undergoes a dehydration reaction to obtain an anhydride compound II.

[0043] In one specific embodiment, step S1 specifically includes the following steps: dissolving 4,4'-dithiodibutyric acid in acetic anhydride and carrying out a dehydration reaction at 20°C to 25°C for 2 to 4 hours to obtain anhydride compound II.

[0044] S2. Under the action of a catalyst, acid anhydride compound II undergoes an esterification reaction with 9-fluorenylmethanol to give intermediate product III.

[0045] In one specific embodiment, the molar ratio of acid anhydride compound II to 9-fluorenylmethanol is (1-2):(1-2).

[0046] In one specific embodiment, the catalyst is DMAP; the molar ratio of acid anhydride compound II to DMAP is 1:(0.1~1).

[0047] In one specific embodiment, step S2 specifically includes the following steps: dissolving acid anhydride compound II in dichloromethane, adding DMAP, and carrying out an esterification reaction at 20°C to 25°C with stirring for 12 to 24 hours, and obtaining intermediate product III by column chromatography.

[0048] S3. Under the action of a catalyst, intermediate product III undergoes an esterification reaction with podophyllotoxin to obtain podophyllotoxin-9-fluorenylmethanol small molecule prodrug IV.

[0049] In one specific embodiment, the molar ratio of intermediate product III to podophyllotoxin is (1-2):(1-2).

[0050] In one specific embodiment, the catalyst is DMAP, EDCI and HOBt; the molar ratio of intermediate III, DMAP, EDCI and HOBt is 1:(0.1~1):(1~2):(1~2).

[0051] In one specific embodiment, step S3 specifically includes the following steps: dissolving intermediate product III, EDCI, HOBt and DMAP in dichloromethane, incubating on ice for 2 hours, then adding podophyllotoxin and performing an esterification reaction at 20°C to 25°C for 24 to 48 hours, and purifying the obtained product by preparative liquid phase separation to obtain podophyllotoxin-9-fluorenylmethanol small molecule prodrug IV.

[0052] In one specific embodiment, steps S1-S3 are all performed under a nitrogen protective atmosphere.

[0053] Specifically, the synthetic reaction formula for the podophyllotoxin-9-fluorenylmethanol small molecule prodrug is as follows:

[0054]

[0055] The present invention also discloses a self-assembled nanoparticle of podophyllotoxin-9-fluorenylethanol small molecule prodrug as described in any embodiment of the present invention. The self-assembled nanoparticle is a self-assembled nanoparticle of podophyllotoxin-9-fluorenylethanol small molecule prodrug modified with a polyethylene glycol modifier, or a self-assembled nanoparticle of podophyllotoxin-9-fluorenylethanol small molecule prodrug loaded with a hydrophobic fluorescent substance.

[0056] Furthermore, the preparation method of podophyllotoxin-9-fluorenylmethanol small molecule prodrug self-assembled nanoparticles includes the following steps:

[0057] S1. Dissolve the podophyllotoxin-9-fluorenylmethanol small molecule prodrug with a polyethylene glycol modifier, or the podophyllotoxin-9-fluorenylmethanol small molecule prodrug with a polyethylene glycol modifier and a fluorescent substance in an organic solvent to obtain a mixed solution.

[0058] S2. The mixed solution is added dropwise to water, and the podophyllotoxin-9-fluorenylethanol small molecule prodrug spontaneously forms uniform nanoparticles. The organic solvent in the formulation is removed by rotary evaporation to obtain podophyllotoxin-9-fluorenylethanol small molecule prodrug self-assembled nanoparticles without any organic solvent.

[0059] Furthermore, the polyethylene glycol modifier includes one or more of TPGS, DSPE-PEG, PLGA-PEG, and PE-PEG.

[0060] Furthermore, the molecular weight of the polyethylene glycol modifier is between 1000 and 5000. Preferably, the molecular weight of the polyvinyl alcohol is 1000, 2000, or 5000. More preferably, the molecular weight of the polyvinyl alcohol is 2000.

[0061] Furthermore, the hydrophobic fluorescent substances include one or more of coumarin-6, rhodamine, DiR, DiI, Cy5, and Cy7.

[0062] Furthermore, the organic solvent includes one or more of tetrahydrofuran, ethanol, methanol, and dimethyl sulfoxide. Preferably, the organic solvent is a mixture of tetrahydrofuran and ethanol.

[0063] Furthermore, in the self-assembled nanoparticles of podophyllotoxin-9-fluorenylmethanol small molecule prodrug loaded with hydrophobic fluorescent material, the mass ratio of podophyllotoxin-9-fluorenylmethanol small molecule prodrug, hydrophobic fluorescent material, and polyethylene glycol modifier is 1:(0.02~0.06):(0.1~0.3).

[0064] Furthermore, the mass ratio of podophyllotoxin-9-fluorenylmethanol small molecule prodrug to polyethylene glycol modifier in the self-assembled nanoparticles of podophyllotoxin-9-fluorenylmethanol small molecule prodrug is 1:(0.1-0.3).

[0065] This invention also discloses a co-assembled nanoparticle of podophyllotoxin-9-fluorenylethanol small molecule prodrug as described in any embodiment of this invention. The co-assembled nanoparticle is a polyethylene glycol-modified podophyllotoxin-9-fluorenylethanol small molecule prodrug co-assembled with gossypol, or a podophyllotoxin-9-fluorenylethanol small molecule prodrug loaded with a hydrophobic fluorescent substance co-assembled with gossypol. Specifically, to further improve the antitumor effect of PPT, this invention uses gossypol as a chemical sensitizer to construct a carrier-free hybrid nanoassembly with a PPT oxidation-sensitive prodrug for the first time. In this assembly process, the PPT prodrug with a π-conjugated Fmoc structure readily co-assembles with the chemical sensitizer gossypol into stable nanoparticles. The prepared gossypol-mediated prodrug co-assembled nanoparticles can be used for precision cancer chemotherapy in tumor treatment, precisely achieving PPT "enhancement," improving the therapeutic efficiency of the PPT prodrug, opening an ultra-low-dose chemotherapy window for PPT, and solving the problems of delayed and insufficient prodrug activation.

[0066] This invention also discloses a method for preparing podophyllotoxin-9-fluorenylmethanol small molecule prodrug co-assembled nanoparticles as described in any embodiment of this invention, comprising the following steps:

[0067] S1. Dissolve the podophyllotoxin-9-fluorenylmethanol small molecule prodrug, gossypol and polyethylene glycol modifier, or the podophyllotoxin-9-fluorenylmethanol small molecule prodrug, gossypol and polyethylene glycol modifier, and hydrophobic fluorescent substance in an organic solvent, and then dilute with ethanol to obtain a mixed solution.

[0068] S2. The mixed solution is added dropwise to water, and the podophyllotoxin-9-fluorenylethanol small molecule prodrug and gossypol spontaneously form uniform nanoparticles. Then, the organic solvent in the formulation is removed by rotary evaporation to obtain podophyllotoxin-9-fluorenylethanol small molecule prodrug co-assembled nanoparticles without any organic solvent.

[0069] In one specific embodiment, the polyethylene glycol modifier includes one or more of TPGS, DSPE-PEG, PLGA-PEG, and PE-PEG.

[0070] In one specific embodiment, the molecular weight of the polyethylene glycol modifier is 1000-5000. Preferably, the molecular weight of the polyvinyl alcohol is 1000, 2000, and 5000. More preferably, the molecular weight of the polyvinyl alcohol is 2000.

[0071] In one specific embodiment, the hydrophobic fluorescent material includes one or more of coumarin-6, rhodamine, DiR, DiI, Cy5, and Cy7.

[0072] In one specific embodiment, the organic solvent includes one or more of tetrahydrofuran, ethanol, methanol, and dimethyl sulfoxide; preferably, the organic solvent is a mixture of tetrahydrofuran and ethanol.

[0073] In one specific embodiment, the mass ratio of podophyllotoxin-9-fluorenylmethanol small molecule prodrug modified with polyethylene glycol modifier to gossypol co-assembled nanoparticles is 1:(0.1-10):(0.1-0.3).

[0074] In one specific embodiment, the mass ratio of podophyllotoxin-9-fluorenylmethanol small molecule prodrug loaded with hydrophobic fluorescent substance, gossypol, polyethylene glycol modifier and hydrophobic fluorescent substance in the co-assembled nanoparticles of podophyllotoxin-9-fluorenylmethanol small molecule prodrug loaded with hydrophobic fluorescent substance is 1:(0.1-10):(0.1-0.3):(0.02-0.06).

[0075] The present invention also discloses the application of podophyllotoxin-9-fluorenylethanol small molecule prodrug co-assembled nanoparticles, as described in any embodiment of the present invention, or podophyllotoxin-9-fluorenylethanol small molecule prodrug self-assembled nanoparticles, in a drug delivery system.

[0076] The present invention also discloses the application of podophyllotoxin-9-fluorenylethanol small molecule prodrug co-assembled nanoparticles, as described in any embodiment of the present invention, or podophyllotoxin-9-fluorenylethanol small molecule prodrug self-assembled nanoparticles, in the preparation of antitumor drugs.

[0077] The present invention also discloses the application of podophyllotoxin-9-fluorenylethanol small molecule prodrug co-assembled nanoparticles, as described in any embodiment of the present invention, or podophyllotoxin-9-fluorenylethanol small molecule prodrug self-assembled nanoparticles, in injection, oral, or local drug delivery systems.

[0078] Specifically, this invention combines prodrug nanoassembly with chemotherapy sensitization to develop a novel chemotherapy regimen, fully releasing the chemotherapeutic potential of podophyllotoxin-9-fluorenylmethanol. The synthesized podophyllotoxin-9-fluorenylmethanol small molecule prodrug is prepared into prodrug self-assembled nanoparticles and co-assembled with gossypol into prodrug nanoparticles. This invention effectively controls the off-target toxicity of PPT through tumor-specific prodrug design and precise hybrid nanoassembly. The prepared nanoparticles have small particle size, good uniformity and stability, and possess simultaneous drug delivery characteristics. After accumulating in large quantities in tumor tissue through the EPR effect, they specifically release chemotherapeutic drugs under the reduced microenvironment conditions at the tumor site, thereby exerting a synergistic sensitizing effect. Simultaneously, the preparation process is simple, safe, and has no toxic side effects, making it easy to industrialize and achieving efficient PPT encapsulation. It has good application prospects in drug delivery systems, tumor treatment, injection, oral administration, or local administration systems.

[0079] The following are specific embodiments.

[0080] Example 1: Synthesis of Podophyllotoxin-9-fluorenyl alcohol small molecule prodrug

[0081] Under nitrogen protection, 2 mmol of 4,4'-dithiodibutyric acid was placed in a 100 mL round-bottom flask and dissolved in 6 mL of acetic anhydride. The mixture was magnetically stirred at 25 °C for 2 h. 30 mmol of toluene was added, and the toluene and excess acetic anhydride were removed by vacuum distillation. 2 mmol of the resulting product was dissolved in 15 mL of dichloromethane, and 2 mmol of 9-fluorenemethanol and 0.2 mmol of DMAP solution were added. The mixture was magnetically stirred at 25 °C for 12 h. The intermediate product was obtained by separation and purification using a dichloromethane-methanol elution system. Finally, 1 mmol of the intermediate product, 2 mmol of EDCI, and 2 mmol of HOBt were incubated at 0 °C on ice for 2 h. Then, 1 mmol of podophyllotoxin and 0.2 mmol of DMAP were added, and the reaction was carried out at 25 °C for 48 h. The final product was purified by preparative liquid chromatography, with a yield of 61.2%.

[0082] Using MS and 1 The structure of the product was confirmed by H-NMR, and the results are as follows: Figures 1-2 As shown.

[0083] Example 2: Preparation of PEG-modified podophyllotoxin-9-fluorenyl alcohol small molecule prodrug self-assembled nanoparticles

[0084] Accurately weigh DSPE-PEG 2k 2 mg of podophyllotoxin-9-fluorenyl alcohol small molecule prodrug from Example 1 and 8 mg of podophyllotoxin-9-fluorenyl alcohol were dissolved in 500 μL of tetrahydrofuran, then diluted with 500 μL of anhydrous ethanol. The mixture was slowly added dropwise to 4 mL of deionized water with stirring, spontaneously forming uniform PSSF nanoparticles. The organic solvent was removed by rotary evaporation at 25 °C. The particle size and morphology of the prepared self-assembled nanoparticles were determined by transmission electron microscopy, and the results are as follows: Figure 3 Transmission electron microscopy images show that the drug-loaded nanoparticles are uniform spherical with a particle size of about 86 nm.

[0085] Example 3: Preparation of PEG-modified podophyllotoxin-9-fluorenyl alcohol small molecule prodrug co-assembled with gossypol nanoparticles

[0086] Accurately weigh DSPE-PEG 2k 2 mg of podophyllotoxin-9-fluorenylmethanol small molecule prodrug from Example 1, 1.92 mg of gossypol, and 6.08 mg of fluorescein were dissolved in 500 μL of tetrahydrofuran, then diluted with 500 μL of anhydrous ethanol. The mixture was then slowly added dropwise to 4 mL of deionized water with stirring, spontaneously forming uniform PSSF / GSP nanoparticles. The organic solvent was removed by rotary evaporation at 25 °C. The particle size and morphology of the prepared co-assembled nanoparticles were determined by transmission electron microscopy, and the results are as follows: Figure 4Transmission electron microscopy images show that the drug-loaded nanoparticles are uniform spherical with a particle size of about 84 nm.

[0087] Example 4: Preparation of Cy7-PEG modified podophyllotoxin-9-fluorenyl alcohol small molecule prodrug self-assembled nanoparticles

[0088] Accurately weigh Cy7-DSPE-PEG 2k 2 mg of podophyllotoxin-9-fluorenylmethanol small molecule prodrug prepared in Example 1 and 8 mg of podophyllotoxin-9-fluorenylmethanol were dissolved in 500 μL of tetrahydrofuran, then diluted with 500 μL of anhydrous ethanol. The mixture was slowly added dropwise to 4 mL of deionized water with stirring, spontaneously forming uniform Cy7-PEG modified PSSF nanoparticles (PSSF-Cy7 nanoparticles). The organic solvent was removed by rotary evaporation at 25 °C.

[0089] Example 5: Preparation of Cy7-PEG modified podophyllotoxin-9-fluorenylmethanol small molecule prodrug co-assembled with gossypol nanoparticles

[0090] Accurately weigh Cy7-DSPE-PEG 2k 2 mg of podophyllotoxin-9-fluorenylmethanol small molecule prodrug prepared in Example 1, 1.92 mg of podophyllotoxin, and 6.08 mg of gossypol were dissolved in 500 μL of tetrahydrofuran, then diluted with 500 μL of anhydrous ethanol. While stirring, the mixture was slowly added dropwise to 4 mL of deionized water, spontaneously forming uniform Cy7-PEG-modified PSSF / GSP nanoparticles (PSSF / GSP-Cy7 nanoparticles). The organic solvent was removed by rotary evaporation at 25 °C.

[0091] Example 6: Colloidal stability test of PEG-modified podophyllotoxin-9-fluorenylmethanol small molecule prodrug nanoparticles

[0092] One mL of each of the prodrug nanoparticles prepared in Examples 2 and 3 was added to 20 mL of phosphate-buffered saline (PBS, pH 7.4) and incubated at 37°C for 24 h. The particle size change was measured by dynamic light scattering at predetermined time points (0 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h). The results are as follows: Figure 5 As shown, the nanoparticles exhibit good colloidal stability, with no significant change in particle size within 24 hours. This indicates that the constructed podophyllotoxin prodrug nanoparticles can exist in a relatively stable form in the presence of inorganic salts, which is beneficial for more drugs to accumulate at the tumor site via blood circulation, thereby achieving better therapeutic effects.

[0093] Example 7: In vitro release assay of PEG-modified podophyllotoxin-9-fluorenylmethanol small molecule prodrug co-assembled with gossypol nanoparticles

[0094] (1) Release of podophyllotoxin

[0095] The in vitro release of podophyllotoxin from podophyllotoxin-9-fluorenylmethanol small molecule prodrug co-assembled with gossypol nanoparticles was investigated using PBS buffer (pH 7.4) containing 30% anhydrous ethanol as the release medium. 0.82 mL of PSSF / GSP nanoparticles (podophyllotoxin content 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 podophyllotoxin was determined by high-performance liquid chromatography (HPLC). Certain concentrations of dithiothreitol (DTT, 0 mM, 1 mM, 5 mM, 10 mM) were added to the release medium to investigate the release of the nanoparticles under reducing conditions. The results are as follows: Figure 6 As shown, the disulfide-bridged nanoparticles exhibit reduction responsiveness and can rapidly release podophyllotoxin under the action of DTT.

[0096] (2) Release of gossypol

[0097] The in vitro release of gossypol from podophyllotoxin-9-fluorenylmethanol small molecule prodrug co-assembled with gossypol was investigated using PBS buffer (pH 7.4) containing 20% ​​tetrahydrofuran as the release medium. 125 μL of PSSF / GSP nanoparticles (gossypol content 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 gossypol was determined by high-performance liquid chromatography (HPLC). Certain concentrations of dithiothreitol (DTT, 0 mM, 1 mM, 5 mM, 10 mM) were added to the release medium to investigate the release of the nanoparticles under reducing conditions. The results are as follows: Figure 7 As shown, the disulfide-bridged nanoparticles exhibit reduction responsiveness and can rapidly release gossypol under the action of DTT.

[0098] Example 8: Cytotoxicity of PEG-modified podophyllotoxin-9-fluorenylmethanol small molecule prodrug nanoparticles

[0099] The MTT assay was used to investigate the cytotoxicity of PEG-modified podophyllotoxin prodrug-assembled nanoparticles against mouse breast cancer cells (4T1), mouse prostate cancer cells (RM-1), and mouse fibroblasts (3T3). 4T1 and RM-1 cells were cultured at 2 × 10⁻⁶ cells / cells. 3Cells were seeded at a density of [number] cells / well in 96-well plates and cultured for 12 h. Then, cells were treated for 48 h with different concentrations of GSP solution, PPT solution, PSSF / GSP solution, PSSF nanoparticles prepared in Example 2, and PSSF / GSP nanoparticles prepared in Example 3 (with the same concentrations of PPT and / or GSP). Then, 5 mg / mL MTT (20 μL / well) was added, and the cells were incubated at 37°C for 4 h. The culture medium was then replaced with DMSO (200 μL / well) to dissolve the generated formazan. Finally, the UV absorbance at 490 nm was measured using a microplate reader. Using 3T3 cells as a cell model, the cytotoxicity of PPT solution, PSSF nanoparticles prepared in Example 2, and PSSF / GSP nanoparticles prepared in Example 3 to normal cells was verified using the same method.

[0100] The results are as follows Figure 8 and Figure 9 As shown, when bound to a non-toxic concentration of GSP, the cytotoxicity of the PSSF / GSP nanoparticle treatment group was significantly higher than that of the PSSF nanoparticle treatment group, thus confirming the sensitizing effect of GSP on PSSF nanoparticles. Notably, compared to the PSSF / GSP solution, PSSF nanoparticles exhibited stronger in vitro antitumor activity, mainly due to their efficient cellular uptake and rapid intracellular drug release. Furthermore, as... Figure 10 As shown, PSSF nanoparticles and PSSF / GSP nanoparticles exhibited low cytotoxicity to 3T3 cells, highlighting their good biocompatibility.

[0101] Example 9: Cellular uptake of PEG-modified podophyllotoxin-9-fluorenyl alcohol small molecule prodrug nanoparticles

[0102] 4T1 cells were fed at a rate of 2 × 10 5 Cells were seeded at a density of [number] cells / well in 12-well plates and incubated for 24 h. The medium was then replaced with fresh medium containing Cy7 solution, Cy7-PEG modified PSSF nanoparticles prepared in Example 4 (PSSF-Cy7 nanoparticles), and Cy7-PEG modified PSSF / GSP nanoparticles prepared in Example 5 (PSSF / GSP-Cy7 nanoparticles) (Cy7 concentration of 250 ng / mL for each medium), and incubated for another 0.5 h or 2 h, respectively. After washing, fixing, or digestion, the cells were analyzed using flow cytometry (BD, East Rutherford, NJ, USA).

[0103] like Figure 11As shown, 4T1 cells exhibited significantly higher uptake efficiency of Cy7-PEG-modified PSSF nanoparticles and Cy7-PEG-modified PSSF / GSP nanoparticles than the solution formulation, and this uptake was time-dependent. Importantly, there was no significant difference between Cy7-PEG-modified PSSF nanoparticles and Cy7-PEG-modified PSSF / GSP nanoparticles, indicating that nanoparticle co-assembly does not hinder cellular uptake. Nanoparticles can be effectively internalized into cells via non-concentration-dependent endocytosis. Therefore, the well-stable Cy7-PEG-modified PSSF / GSP nanoparticles and Cy7-PEG-modified PSSF nanoparticles demonstrated significantly higher cellular uptake efficiency than the Cy7 solution formulation, which is beneficial for tumor cells to uptake prodrug nanoparticles, thereby producing a better tumor-suppressive effect.

[0104] Example 10: Pharmacokinetic Study of PEG-Modified Podophyllotoxin-9-fluorenylmethanol Small Molecule Prodrug Nanoparticles

[0105] SD rats weighing 200g–220g were randomly divided into three groups of six each. The pharmacokinetic behavior of Cy7-PEG modified PSSF nanoparticles (PSSF-Cy7 nanoparticles) prepared in Example 4 and Cy7-PEG modified PSSF / GSP nanoparticles (PSSF / GSP-Cy7 nanoparticles) prepared in Example 5 was studied in vivo. Cy7 solution, Cy7-PEG modified PSSF nanoparticles, and Cy7-PEG modified PSSF / GSP nanoparticles were administered intravenously via tail vein injection to rats at a dose of 2 mg / kg of Cy7. Ocular venous blood samples (0.1 mL) were collected from rats at 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 12 h post-injection. Plasma was obtained by centrifugation (8000 rpm, 3 min), and the plasma concentration of Cy7 was quantified using a microplate reader with excitation at 750 nm and emission at 773 nm.

[0106] The plasma drug concentration-time curves of these compounds are as follows: Figure 12As shown, the Cy7 solution was rapidly cleared from the body within 4 hours. In contrast, Cy7-PEG-modified PSSF nanoparticles and Cy7-PEG-modified PSSF / GSP nanoparticles exhibited longer circulation times in the blood, resulting in higher AUCs at the same Cy7 dose compared to the Cy7 solution. Furthermore, Cy7-PEG-modified PSSF / GSP nanoparticles demonstrated better pharmacokinetic behavior compared to Cy7-PEG-modified PSSF nanoparticles, which may be related to their enhanced stability. This indicates that Cy7-PEG-modified PSSF / GSP nanoparticles can effectively prolong the circulation time of PPT in the blood, exhibiting unique advantages in in vivo administration. This may facilitate drug accumulation in tumor tissues mediated by enhanced permeability and retention (EPR) effects, thereby maximizing drug efficacy.

[0107] Example 11: Tissue distribution experiment of PEG-modified podophyllotoxin-9-fluorenylmethanol small molecule prodrug nanoparticles

[0108] 4T1 cell suspension was inoculated into BALB / c mice. When the tumor volume reached 400 mm², the cells were cultured. 3 Mice were injected via tail vein with Cy7 solution, Cy7-PEG modified PSSF nanoparticles (PSSF-Cy7 nanoparticles) prepared in Example 4, and Cy7-PEG modified PSSF / GSP nanoparticles (PSSF / GSP-Cy7 nanoparticles) prepared in Example 5, all at a dose of 2 mg / kg of Cy7. Mice were sacrificed at 4 h, 12 h, and 12 h after injection, and tumors and major organs (heart, liver, spleen, lung, and kidney) were harvested. Fluorescence intensity was then analyzed using an IVIS imaging system.

[0109] like Figure 13 As shown, the fluorescence signals of Cy7-PEG modified PSSF nanoparticles and Cy7-PEG modified PSSF / GSP nanoparticles were significantly stronger than those of the Cy7 solution, and they were mainly distributed in the liver, kidneys, and tumor tissues. Notably, under the same dose of Cy7, at 12 h, the fluorescence intensity of Cy7-PEG modified PSSF / GSP nanoparticles at the tumor site was stronger than that of Cy7-PEG modified PSSF nanoparticles. The stronger fluorescence intensity of Cy7-PEG modified PSSF / GSP nanoparticles may be related to their better colloidal stability and in vivo circulation advantage. This indicates that prolonged circulation time can promote the aggregation of prodrug nanoassemblies in tumors through the EPR effect.

[0110] Example 12: In vivo antitumor experiment of PEG-modified podophyllotoxin-9-fluorenylmethanol small molecule prodrug nanoparticles

[0111] The antitumor effect of nanoparticles was evaluated using 4T1 tumors in female BALB / c mice as an in vivo model. 100 μL of 4T1 cell suspension (5 × 10⁶ cells / mL) was subcutaneously injected into the right hind limb of BALB / c mice. 7 A tumor-bearing model was established using a tumor volume of 100 mm² / mL. 3 Mice were randomly divided into 6 groups of 5 mice each: control group (PBS), GSP solution, PSSF / GSP solution, PPT solution, PSSF nanoparticles prepared in Example 2, and PSSF / GSP nanoparticles prepared in Example 3. Administered every other day for a total of 5 times, each dose containing the same concentration of PPT (4 mg / kg) and / or GSP (25 mg / kg). Tumor volume and body weight were monitored daily. Mice were sacrificed after the last treatment to obtain tumor tissue.

[0112] like Figure 14 As shown, compared with the control group, both PSSF / GSP solution and PPT solution exhibited comparable, moderate tumor growth delay. The PPT solution showed a 12-day survival rate of only 40%, indicating significant systemic toxicity. This suggests that the prodrug strategy can largely avoid the off-target toxicity of podophyllotoxin, while gossypol-mediated chemosensitization holds promise for improving the therapeutic efficiency of podophyllotoxin prodrugs. Furthermore, compared with PSSF nanoparticles, PSSF / GSP nanoparticles significantly inhibited tumor growth. The optimal antitumor efficacy of PSSF / GSP nanoparticles is attributed to GSP-mediated sensitization and the multiple therapeutic advantages of the prodrug nanosystem, including prolonged blood circulation time, efficient cellular uptake, and redox-sensitive drug release. Finally, the therapeutic safety of the nanoparticles was preliminarily investigated. Compared with the GSP solution, changes in mouse body weight indicated that neither the PSSF nanoparticle nor the PSSF / GSP nanoparticle group caused a significant decrease in mouse body weight after treatment, demonstrating good safety during treatment.

[0113] The above results indicate that the gossypol-mediated prodrug nanoassembly of the present invention can serve as a highly efficient and safe combination therapy modality, effectively alleviating the systemic toxicity of PPT while enhancing anti-tumor effects, and can be used for precision cancer chemotherapy in tumor treatment.

[0114] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing podophyllotoxin-9-fluorenylethanol small molecule prodrug co-assembled nanoparticles, characterized in that, The podophyllotoxin-9-fluorenylmethanol small molecule prodrug is composed of podophyllotoxin and its compounds linked to 9-fluorenylmethanol via a disulfide bond, and its structural formula is as follows: ； The preparation method of the podophyllotoxin-9-fluorenyl alcohol small molecule prodrug co-assembled nanoparticles includes the following steps: Podophyllotoxin-9-fluorenylmethanol small molecule prodrug, gossypol, and polyethylene glycol modifier, or podophyllotoxin-9-fluorenylmethanol small molecule prodrug, gossypol, polyethylene glycol modifier, and hydrophobic fluorescent substance are dissolved in an organic solvent and then diluted with ethanol to obtain a mixed solution; wherein the mass ratio of podophyllotoxin-9-fluorenylmethanol small molecule prodrug, gossypol, and polyethylene glycol modifier is 1:(0.1~10):(0.1~0.3); The mixed solution was added dropwise to water, and podophyllotoxin-9-fluorenylethanol small molecule prodrug and gossypol spontaneously formed uniform nanoparticles. Then, the organic solvent in the formulation was removed by rotary evaporation to obtain podophyllotoxin-9-fluorenylethanol small molecule prodrug co-assembled nanoparticles without any organic solvent. The organic solvent includes one or more of tetrahydrofuran, ethanol, methanol, and dimethyl sulfoxide.

2. The preparation method according to claim 1, characterized in that, When the mixed solution is prepared by dissolving podophyllotoxin-9-fluorenylmethanol small molecule prodrug, gossypol, polyethylene glycol modifier, and hydrophobic fluorescent substance in an organic solvent and then diluting with ethanol, the mass ratio of podophyllotoxin-9-fluorenylmethanol small molecule prodrug, gossypol, polyethylene glycol modifier, and hydrophobic fluorescent substance is 1:(0.1~10):(0.1~0.3):(0.02~0.06).

3. The preparation method according to claim 1 or 2, characterized in that, The polyethylene glycol modifier is DSPE-PEG; The polyethylene glycol modifier has a molecular weight of 2000; The hydrophobic fluorescent substance includes one or more of coumarin-6, rhodamine, DiR, DiI, Cy5, and Cy7.

4. The preparation method according to claim 3, characterized in that, The method for synthesizing the podophyllotoxin-9-fluorenyl alcohol small molecule prodrug includes the following steps: S1. 4,4'-Dithiodibutyric acid I undergoes a dehydration reaction to yield an anhydride compound II; S2. Under the action of a catalyst, the acid anhydride compound II undergoes an esterification reaction with 9-fluorenylmethanol to obtain intermediate product III; S3. Under the action of a catalyst, the intermediate product III undergoes an esterification reaction with podophyllotoxin to obtain podophyllotoxin-9-fluorenylmethanol small molecule prodrug IV; the reaction formula is as follows: 。 5. The preparation method according to claim 4, characterized in that, In S1, the temperature of the dehydration reaction is 20℃~25℃; the time of the dehydration reaction is 2h~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℃~25℃, and the time of the esterification reaction is 12h~24h; In S3, the molar ratio of intermediate product III to podophyllotoxin is (1~2):(1~2); the catalyst is DMAP, EDCI and HOBT; the molar ratio of intermediate product III, DMAP, EDCI and HOBT is 1:(0.1~1):(1~2):(1~2); the temperature of the esterification reaction is 20℃~25℃, and the time of the esterification reaction is 24h~48h.

6. The application of podophyllotoxin-9-fluorenylmethanol small molecule prodrug co-assembled nanoparticles prepared by the preparation method as described in claim 1 in the preparation of drug delivery systems.

7. The application of podophyllotoxin-9-fluorenylmethanol small molecule prodrug co-assembled nanoparticles prepared by the preparation method as described in claim 1 in the preparation of antitumor drugs.

8. The application of podophyllotoxin-9-fluorenylmethanol small molecule prodrug co-assembled nanoparticles prepared by the preparation method as described in claim 1 in the preparation of injection, oral or topical drug delivery systems.

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