Polymer vesicle stabilized drug-iodized oil emulsion, method of making and use thereof

Abstract

The application discloses a polymer vesicle-stabilized drug-iodized oil emulsion and a preparation method and application thereof, to realize safe and efficient transarterial chemoembolization (TACE) treatment of hepatocellular carcinoma in situ. The drug-iodized oil emulsion system has the following advantages: simple preparation, convenient size adjustment, excellent stability, near-zero order and sustained controllable drug release rate, low blood drug concentration after transarterial embolization, and the retention time and retention amount of the drug in the liver are superior to those of free drug-iodized oil emulsion used in clinical treatment, the tumor can be completely eradicated, angiogenesis can be effectively inhibited, and adverse reactions can be reduced. In general, the polymer vesicle-stabilized drug-iodized oil emulsion has the advantages of high stability and sustained controllable drug release, and can greatly improve the embolization chemotherapy effect of hepatocellular carcinoma, and provide a safe and efficient TACE strategy.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine technology, specifically relating to a polymer vesicle-stabilized drug-iodized oil emulsion, its preparation method, and its application. More particularly, it relates to a polymer vesicle-stabilized doxorubicin-iodized oil microemulsion and its preparation method, which can be used in the transarterial chemoembolization treatment of hepatocellular carcinoma. Background Technology

[0002] Hepatocellular carcinoma (HCC) is often diagnosed at an advanced stage, with a mortality-to-morbidity ratio as high as 0.91, seriously threatening human health. Transarterial chemoembolization (TACE) is the standard and first-line treatment for patients with advanced HCC. It blocks arterial blood supply to HCC, inducing tumor necrosis, and allows for local chemotherapy. Lipiodol-based formulations are the most popular TACE technique due to their good deposition in HCC, effective distal tumor vascular embolization, and rapid metabolism by the normal liver. Lipiodol-based TACE typically delivers chemotherapeutic drugs (such as doxorubicin hydrochloride, DOX) and a crude emulsion of iodized oil via the artery, exerting a certain anti-HCC effect and prolonging patient survival. However, it is important to note that the drug-iodized oil emulsion has poor physical stability and rapid phase separation, significantly reducing retention at the tumor site and accelerating drug release into the bloodstream, leading to poor clinical efficacy and severe systemic toxicity. Existing technologies involve dissolving drugs in low-boiling-point solvents to obtain drug solutions, then preparing drug nanoparticles. These nanoparticles are then mixed with iodized oil injection solution and ultrasonically dispersed in a water bath to obtain a highly stable homogeneous chemoembolic agent composed of chemotherapy drug nanoparticles and iodized oil, which can be stored for three weeks. Another existing technology discloses dissolving and thoroughly mixing iodized oil injection solution and drug molecules under high pressure with rapid stirring. After depressurization, the small molecule drug is dispersed in iodized oil to prepare a uniformly mixed drug-iodized oil solvent, which shows no significant sedimentation after two weeks. However, the existing methods for preparing chemotherapy drug nanoparticles-iodized oil involve unsafe operations such as high pressure, and the product stability needs improvement, with sedimentation occurring even within a storage period of less than one month. Therefore, obtaining a highly stable drug-iodized oil emulsion to achieve continuous and controllable drug release is crucial for achieving efficient TACE treatment of HCC. Summary of the Invention

[0003] The purpose of this invention is to disclose a highly stable polymer vesicle-stabilized drug-iodized oil emulsion with continuously controllable drug release, its preparation method, and its application. Specifically, it discloses a disulfide crosslinked polymer vesicle-stabilized doxorubicin-iodized oil emulsion, its preparation method, and its application. Using a simple method, the product obtained has a stability of at least 60 days without sedimentation, which was unexpected.

[0004] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0005] A polymer vesicle-stabilized drug-iodized oil emulsion comprises a polymer vesicle nanomedicine and iodized oil; the polymer vesicle nanomedicine comprises an amphiphilic block polymer and a small molecule drug.

[0006] In this invention, the amphiphilic block polymer is an existing polymer with the following chemical structural formula:

[0007]

[0008] The amphiphilic block polymer is denoted as PEG-P(TMC-DTC), corresponding to the structural units, where x and y represent repeating units. In this amphiphilic block polymer, the molecular weight of PEG is 2000–8000 Da; the molecular weight of P(TMC-DTC) is 2.0–6.0 times that of PEG; and the molecular weight of PDTC is 10%–30% of that of P(TMC-DTC). P(TMC-DTC) has the following structure:

[0009]

[0010] The PDTC chain segment is a y-chain segment unit.

[0011] In this invention, the small molecule drug is doxorubicin hydrochloride, epirubicin hydrochloride, or maytansin, preferably doxorubicin hydrochloride (DOX); the iodized oil is commercially available and is a conventional raw material for drug-iodized oil emulsions. The polymer vesicle doxorubicin nanomedicine of this invention is obtained by self-assembly and cross-linking of an amphiphilic block polymer. Its outer shell is polyethylene glycol (PEG), and the film layer is a reversibly cross-linked hydrophobic polycarbonate, achieving efficient and stable loading of DOX through hydrophilic-hydrophobic interactions.

[0012] The preparation method of the above-mentioned polymer vesicle-stabilized doxorubicin-iodized oil emulsion is as follows: an amphiphilic block polymer and a small molecule drug are assembled to form a polymer vesicle nanomedicine, which is then mixed with iodized oil to form the polymer vesicle-stabilized drug-iodized oil emulsion; specifically, the polymer vesicle nanomedicine is dispersed in a buffer solution to form a polymer vesicle nanomedicine solution, which is then mixed with iodized oil to form the polymer vesicle-stabilized drug-iodized oil emulsion. Preferably, the volume ratio of the polymer vesicle nanomedicine solution to iodized oil is 1:(1-5), more preferably 1:(2-4); the concentration of the polymer vesicle nanomedicine solution is 6-18 mg / mL.

[0013] The polymer vesicle nanodrug solution and iodized oil are mixed through a three-way valve to form a polymer vesicle-stabilized drug-iodized oil emulsion, wherein the droplet size of the emulsion is 10-70 μm and the drug release rate is close to zero order.

[0014] This invention discloses the application of the aforementioned polymer vesicle-stabilized drug-iodized oil emulsion in the preparation of antitumor drugs, preferably anti-hepatocellular carcinoma drugs, specifically transarterial chemoembolization (TACE) anti-hepatocellular carcinoma drugs. Specifically, the application of the aforementioned polymer vesicle-stabilized drug-iodized oil emulsion in the preparation of TACE for hepatocellular carcinoma. When the polymer vesicle-stabilized drug-iodized oil emulsion of this invention is used in TACE for hepatocellular carcinoma, the emulsion can block the blood supply to the hepatocellular carcinoma while simultaneously releasing polymer vesicle nanoparticles and small molecule anticancer drugs locally, exerting its effect through a dual action.

[0015] This invention discloses the application of the above-mentioned polymer vesicle-stabilized drug-iodized oil emulsion in improving the stability of drug-iodized oil emulsion.

[0016] The polymer vesicle-stabilized drug-iodized oil emulsion of the present invention is composed of polymer vesicles, a drug, and iodized oil, wherein the vesicles are obtained by polymer assembly and crosslinking. The preparation method of the polymer vesicle-stabilized drug-iodized oil emulsion of the present invention can be as follows:

[0017] (1) Using PEG-P (TMC-DTC) as raw material, disulfide cross-linked polymer vesicles loaded with small molecule drugs were prepared by pH gradient method, namely polymer vesicle nanomedicines.

[0018] (2) The above polymer vesicle nanomedicine was mixed with iodized oil to prepare a polymer vesicle-stabilized doxorubicin-iodized oil emulsion.

[0019] Specifically: A DMF solution of PEG-P (TMC-DTC) is added to a buffer solution at 40 °C and mixed thoroughly. Na₂HPO₄ solution is then added to adjust the pH to 7–8. A small molecule drug solution (preferably an aqueous solution) is then added, and after overnight incubation, dialyzed to prepare polymer vesicle nanomedicine. A three-way valve is then used to thoroughly mix the polymer vesicle nanomedicine with iodized oil at a volume ratio of 1:3, which easily prepares a polymer vesicle-stabilized drug-iodized oil emulsion.

[0020] The polymer vesicles in this invention are reduction-sensitive, reversibly cross-linked, and biodegradable polymer vesicles; the polymer is PEG-P (TMC-DTC), wherein the hydrophobic blocks of TMC and DTC are randomly arranged. The vesicle membrane is reversibly cross-linked, biodegradable, and highly compatible PTMC, with a dithiopentyl ring structure in the side chain similar to the human body's natural antioxidant lipoic acid, which can spontaneously form reduction-sensitive, reversible cross-links. This not only provides high stability in blood but also enables rapid intracellular decross-linking, quickly releasing drugs into target cells.

[0021] This invention discloses the application of the above-mentioned polymer vesicle-stabilized doxorubicin-iodized oil emulsion in transarterial chemoembolization of hepatocellular carcinoma.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] 1. The polymer vesicle-stabilized doxorubicin-iodized oil emulsion disclosed in this invention has high stability and continuous and controllable drug release, effectively overcoming the defects of existing drug-iodized oil emulsions such as poor physical stability and rapid drug release.

[0024] 2. The polymer vesicle-stabilized doxorubicin-iodized oil emulsion disclosed in this invention has a long retention time in the liver, less systemic drug exposure, and good safety, which is superior to the free doxorubicin-iodized oil emulsion used in clinical practice.

[0025] 3. The polymer vesicle-stabilized doxorubicin-iodized oil emulsion disclosed in this invention has a significant anti-hepatocellular carcinoma effect. In a rat orthotopic hepatocellular carcinoma model, administration of Ps-DOX / L emulsion via artery can completely eradicate the tumor, effectively inhibit angiogenesis, and no adverse reactions occur.

[0026] 4. The polymer vesicle-stabilized doxorubicin-iodized oil emulsion of the present invention has many advantages, including simple preparation, good injectability, high stability, sustained and controllable drug release, long retention time at the tumor site, low systemic drug exposure, significant tumor growth inhibition effect, and good safety. Therefore, this polymer vesicle-stabilized doxorubicin-iodized oil emulsion is expected to provide a novel transarterial chemoembolization regimen for intermediate and advanced hepatocellular carcinoma. Attached Figure Description

[0027] Figure 1 The particle size distribution diagrams are for (A) the nanomedicine Ps-DOX in Example 1 and (B) the nanomedicine ncPs-DOX in Example 2.

[0028] Figure 2 The static stability and microstructure of Ps-DOX / L emulsions prepared at different Ps concentrations in Example 3 are shown in the figure. The scale bar is 50 µm.

[0029] Figure 3 The static stability and centrifugal stability of the Ps-DOX / L and free DOX / L emulsions (A) in Example 3 during storage at 25 ºC for 60 days are shown in Figure 3.

[0030] Figure 4 The images shown are CLSM images of the Ps-DOX / L and free DOX / L emulsions in Example 3, with a scale bar of 50 µm.

[0031] Figure 5 The images show the static stability and microscopic images of the ncPs-DOX / L, Ps+DOX / L and ncPs+DOX / L emulsions in Example 3 at the same Ps and DOX concentrations. The scale bar is 50 µm.

[0032] Figure 6 The relationship between viscosity and shear rate of Ps-DOX / L emulsion and iodized oil in Example 3 is shown.

[0033] Figure 7 The in vitro DOX release of Ps-DOX / L and free DOX / L emulsions in Example 4 is shown (n = 3).

[0034] Figure 8 The in vitro anti-HCC activity of Ps-DOX and Ps-DOX / L in Example 5 (n = 4). (A) Rat N1S1, (B) Mouse H22, (C) Mouse Hepa 1-6, (D) Human HepG2 and (E) Human SMMC-7721 HCC cells after incubation with Ps-DOX or free DOX for 48 h. (F) Survival rate of rat N1S1 cells after incubation with Ps-DOX / L emulsion, blank Ps / L emulsion and Ps for 72 h.

[0035] Figure 9 The values ​​represent the plasma DOX concentrations after arterial embolization of Ps-DOX / L and free DOX / L in Example 7.

[0036] Figure 10 (A) Ex vivo DOX fluorescence imaging after arterial embolization of Ps-DOX / L and free DOX / L in Example 8, and (B) DOX content in major organs at 24 and 72 h after embolization.

[0037] Figure 11 The effect of Ps-DOX / L emulsion on TACE treatment in rats with orthotopic N1S1 HCC in Example 9 (n = 5). The untreated group (n = 4), iodized oil, and free DOX / L emulsion served as control groups (DOX: 500 μg / rat; iodized oil: 0.2 mg / kg). (A) Flowchart of model construction and treatment, (B) MRI images of rats in different treatment groups, (C) changes in tumor volume, and (D) changes in body weight.

[0038] Figure 12 Changes in aspartate aminotransferase (AST) and alanine aminotransferase (ALT).

[0039] Figure 13 Histological and TUNEL analyses of tumor sections from rats (A) in different treatment groups during day 7 in Example 9. Red circles indicate vacuolar iodized oil deposition. Scale bar: 50 μm.

[0040] Figure 14 IHC analysis of tumor sections from rats in different treatment groups on day 7 in Example 9 (A). Scale bar is 50 μm. (B) Quantitative analysis of VEGF and (C) CD31 expression. Detailed Implementation

[0041] As an example, this invention uses doxorubicin, an amphiphilic block polymer, and iodized oil as raw materials. The amphiphilic block polymer is self-assembled and loaded with the drug to form polymeric vesicles of doxorubicin nanomedicine. These vesicles are then thoroughly mixed with iodized oil to prepare a polymeric vesicle-stabilized doxorubicin-iodized oil emulsion. This invention utilizes DOX-loaded disulfide cross-linked biodegradable polymeric vesicles (Ps-DOX) to form a uniform, highly stable water-in-oil (W / O) microemulsion (Ps-DOX / L) with iodized oil, promoting efficient and prolonged DOX retention in the liver, thereby completely eliminating in situ N1S1 HCC in rats.

[0042] The present invention will be further described below with reference to the accompanying drawings and embodiments. All raw materials involved are existing products, and the specific preparation operations and tests are conventional techniques. For example, the preparation operations of drug-loaded vesicles and empty vesicles are existing techniques. In the following embodiments, the amphiphilic block polymer is PEG5k-P (TMC15k-DTC2k); doxorubicin hydrochloride (DOX, 99%) and iodized oil (poppy seed iodine oil injection, Youliying) are both commercially available raw materials.

[0043] Example 1: Preparation of DOX-loaded disulfide crosslinked polymer vesicles (Ps-DOX)

[0044] Ps-DOX was prepared by loading DOX into disulfide-crosslinked polymer vesicles based on PEG-P (TMC-DTC) copolymer using a pH gradient method. In short, 5 mL of PEG-P (TMC-DTC) DMF solution (40 mg / mL) was added to 45 mL of citrate buffer (pH 4.0, 10 mM) at 40 ºC. After stirring at 400 rpm for 2 min, Na2HPO4 solution was added to adjust the pH to 7.6. Then, 5 mL of DOX aqueous solution (10 mg / mL) was added. After incubation in a shaker at 100 rpm and 37 ºC for 12 h, the mixture was dialyzed against phosphate buffer (PB, pH 7.4, 10 mM) (MWCO: 3500 Da) for 6 h to obtain Ps-DOX. The average particle size was measured to be 58 nm by DLS, with a particle size distribution of 0.11. The encapsulation efficiency of DOX was measured to be 78.0% and the drug loading was 15.5 wt.% by UV-Vis spectroscopy. (See attached image.) Figure 1 A is the particle size distribution diagram of Ps-DOX.

[0045] Example 2: Preparation of non-crosslinked polymer vesicles (ncPs-DOX) encapsulated with DOX

[0046] Non-crosslinked polymeric vesicles were prepared based on PEG-PTMC copolymers, and ncPs-DOX were obtained by loading DOX using a pH gradient method. In short, 5 mL of PEG-PTMC in DMF solution (40 mg / mL) was added to 45 mL of citrate buffer (pH 4.0, 10 mM) at 40 ºC. After stirring at 400 rpm for 2 min, Na2HPO4 solution was added to adjust the pH to 7.6, and then 5 mL of DOX aqueous solution (10 mg / mL) was added. The mixture was incubated in a shaker at 100 rpm and 37 ºC for 12 h, followed by dialyzing with phosphate buffer (PB, pH 7.4, 10 mM) (MWCO: 3500 Da) for 6 h to prepare ncPs-DOX. The average particle size was 56 nm and the particle size distribution was 0.15, as measured by DLS. The encapsulation efficiency of DOX was 71.5%, and the drug loading was 14.3 wt.%. Figure 1 B is the particle size distribution diagram of ncPs-DOX.

[0047] Example 3: Preparation and Characterization of Ps-DOX / L and ncPs-DOX / L Emulsions

[0048] Iodized oil was mixed with different DOX formulations at an oil-to-water ratio of 3:1 using a three-way valve to form various emulsions, with iodized oil as the oil phase and PB solutions of different DOX formulations as the aqueous phase. Ps-DOX with Ps concentrations of 6, 12, and 18 mg / mL (corresponding to DOX concentrations of 1.1, 2.2, and 3.3 mg / mL, respectively) was mixed with iodized oil to form Ps-DOX / L emulsions with different Ps and DOX contents. Similarly, ncPs-DOX (Ps: 12 mg / mL) and DOX solution (2.2 mg / mL) were mixed with iodized oil to prepare ncPs-DOX / L and free DOX / L emulsions. A mixture of empty vesicles (Ps or ncPs: 12 mg / mL) and DOX solution (2.2 mg / mL) was mixed with iodized oil to obtain Ps+DOX / L or ncPs+DOX / L emulsions. The morphology and structure of different emulsions were observed using fluorescence microscopy or confocal laser scanning microscopy (CLSM). The physical stability of Ps-DOX / L and free DOX / L emulsions was observed by centrifugation at 25 ºC (12000 rpm, 2 min) or storage for 60 days. All experiments involving DOX were conducted in the dark.

[0049] Appendix Figure 2The static stability and microstructure of Ps-DOX / L emulsions prepared at different Ps concentrations were investigated. As the Ps concentration increased from 6 to 12 and 18 mg / mL, the average droplet size of the Ps-DOX / L microemulsion decreased from 44 ± 23 μm to 33 ± 8 μm and 14 ± 4 μm, respectively, indicating that Ps has good emulsifying properties. In the following examples, Ps-DOX / L microemulsions with a size of 33 ± 8 μm, i.e., prepared with Ps-DOX at a concentration of 12 mg / mL, were used.

[0050] Appendix Figure 3 A indicates that the Ps-DOX / L microemulsion remained stable during 60 days of storage at 25 ºC, without phase separation or precipitation, while the free DOX / L emulsion rapidly separated into two layers within 5 min. Furthermore, the Ps-DOX / L microemulsion maintained a uniform and stable dispersion after centrifugation at 12000 rpm for 2 min (see attached image). Figure 3 B). Existing technologies have disclosed that PLGA nanoparticles coated with DOX dispersed in iodized oil are no longer homogeneous after one day, as seen in the images. Even with complex methods to first prepare DOX nanoparticles and then disperse them in iodized oil, precipitation occurs in less than 30 days. However, the present invention unexpectedly remains stable after 60 days of storage at 25 ºC (actually exceeding 60 days, no phase separation or precipitation was observed as of the date of this application). In particular, it maintains a uniform and stable dispersion even after centrifugation at 12000 rpm for 2 minutes. This unexpected technical effect is beyond people's imagination.

[0051] Subsequently, the structure and morphology of the Ps-DOX / L and free DOX / L emulsions were further observed using CLSM. (See attached image.) Figure 4 As shown, the Ps-DOX / L microemulsion exhibits a typical W / O structure, with spherical droplets uniformly dispersed in iodized oil, and DOX evenly distributed within the droplets. In contrast, the free DOX / L emulsion presents large and irregular droplets.

[0052] Emulsions of ncPs-DOX / L, Ps+DOX / L, and ncPs+DOX / L were prepared and compared under the same Ps and DOX concentrations. (See attached image) Figure 5 The results showed that ncPs-DOX / L, Ps+DOX / L and ncPs+DOX / L emulsions were more stable than free DOX / L, but phase separation occurred within 5 days. This indicates that both disulfide crosslinked polymer vesicles and stable DOX encapsulation are key to the formation of stable DOX-iodized oil microemulsions.

[0053] The viscosities of Ps-DOX / L emulsion and iodized oil at 37 °C were determined using a rotational rheometer, with shear rates ranging from 0.1 to 1000 s⁻¹. -1Rheological results show that when the shear rate increases from 0 to 1000 s⁻¹, -1 At this time, iodized oil exhibits typical Newtonian fluid behavior with little viscosity change. In contrast, Ps-DOX / L emulsion displays typical non-Newtonian shear thinning characteristics, showing a significant viscosity change when the shear rate increases to 400 s⁻¹. -1 At that time, its viscosity decreased to a level comparable to iodized oil, indicating that it has good injectability (see appendix). Figure 6 ).

[0054] Example 4: In vitro DOX release experiment of Ps-DOX / L and free DOX / L emulsions

[0055] To investigate DOX release from Ps-DOX / L and free DOX / L emulsions, 0.1 mL of freshly prepared Ps-DOX / L or free DOX / L emulsion (DOX: 2 mg / mL) was placed in a drug release bag with a molecular weight of 12000 Da and incubated in 5 mL PB at 37 ºC for release. At predetermined time points, 1 mL of the released solution was removed, and 1 mL of fresh release medium was immediately added. The DOX content in the samples was measured using fluorescence spectroscopy, and the results are expressed as the mean ± standard deviation (SD) of three replicate studies. In vitro drug release studies showed that the Ps-DOX / L microemulsion exhibited no burst release, and the DOX release behavior within 15 days was close to zero-order release. This release behavior is beneficial for embolization, maximizing the embolic effect while preventing drug migration into the bloodstream. In contrast, the free DOX / L emulsion showed a faster drug release rate and burst release behavior, releasing approximately 17% of the DOX within 2 hours (within the specified time). Figure 7 ).

[0056] Example 5: In vitro cytotoxicity test of Ps-DOX and Ps-DOX / L emulsion

[0057] Five different HCC cell lines—rat N1S1 cells, human HepG2 and SMMC-7721 cells, and mouse H22 and Hepa 1-6 cells—were used to evaluate the in vitro anti-HCC activity of Ps-DOX and free DOX. N1S1, HepG2, and Hepa 1-6 cells were cultured in DMEM medium, while SMMC-7721 and H22 cells were cultured in RPMI-1640 medium. All media were supplemented with 10% FBS and 1% penicillin / streptomycin (100 IU / mL). 80 μL of cells were seeded in 96-well plates (5 × 10⁻⁶). 3Cells were cultured in wells (number of cells per well) for 24 h, then 20 μL of Ps-DOX or free DOX in PBS was added, and incubated at 37 ºC for 48 h, with the DOX concentration in each well ranging from 0.001 to 20 μg / mL. Subsequently, 10 µL of CCK-8 solution was added to suspension cells (N1S1, H22, Hepa 1-6) and incubated for another 4 h, with absorbance measured at 450 nm using a microplate reader. For adherent cells (HepG2, SMMC-7721), 10 µL of MTT in PBS (5 mg / mL) was added to each well and incubated for 4 h. The culture medium was then carefully removed, and 150 µL of DMSO was added to dissolve the generated formazan crystals. The absorbance at 570 nm was measured for each well. Cell viability was calculated by comparing the absorbance of the experimental groups with that of cells cultured only in PBS.

[0058] The results showed that Ps-DOX had 13.4 times higher anti-HCC activity in rat N1S1 HCC cells than free DOX (see attached image). Figure 8 A). In mouse H22 and Hepa 1-6 HCC cells, as well as human HepG2 and SMMC-7721 HCC cells, the anti-HCC activity of Ps-DOX was 2.3-6.5 times higher than that of free DOX (see appendix). Figure 8 (BE and Table 1) The enhanced anti-HCC activity of Ps-DOX demonstrates that Ps-DOX can effectively release DOX within tumor cells.

[0059]

[0060] The anti-HCC activity of Ps-DOX / L microemulsion was further investigated using rat N1S1 cells, with Ps / L emulsion and blank Ps as controls. Due to the high viscosity of the emulsion, it was difficult to prepare a homogeneous stock solution in a 96-well plate. Therefore, 0.45 mL of cells (2 × 10⁻⁶ cells / well) was used. 4 Cell suspension (number of cells / well) was seeded into 24-well plates and incubated for 24 h in a cell culture incubator. Then, different doses of emulsion or Ps were added to each well, and the volume of each well was brought up to 0.5 mL with fresh culture medium. After incubation at 37 ºC for 72 h, 50 µL of CCK-8 solution was added, and incubation was continued for 4 h. Subsequently, 100 µL of the solution was transferred from each well to a 96-well plate, and the absorbance at 450 nm was measured. The experiment was performed in quadruplicate, and cell viability was calculated using the above method. The results showed that the anti-HCC activity of Ps-DOX / L emulsion was dose-dependent, with an IC50 value of [missing value]. 50 The concentration was 2.29 μg / mL, while neither the Ps / L emulsion nor Ps showed cytotoxicity (see attached image). Figure 8 F).

[0061] Example 6: Construction of an orthotopic N1S1 HCC model in rats

[0062] All animal experiments and procedures were conducted in accordance with the experimental protocols approved by the Experimental Animal Center of Soochow University and the Animal Care and Use Committee of Soochow University. All animal studies used SD rats (350-380 g). To establish an orthotopic N1S1 rat model, 75 μL of N1S1 cell suspension (6 × 10⁻⁶ cells) was injected into the left lateral lobe of the liver of each rat. 6 Tumor growth and size were monitored using magnetic resonance imaging (MRI) with a 3.0-T MRI scanner. Five minutes prior to imaging, each rat was injected with gadotetrate dimeglumine via the tail vein. All rats were lightly anesthetized to obtain stable and accurate images. Tumor volume (V) was calculated based on the maximum diameter (L), minimum diameter (S), and number of slices (N) of the axial imaging as follows:

[0063]

[0064] Example 7: Pharmacokinetic Study of Ps-DOX / L in Orthotopic N1S1 HCC Rats Following Embolization

[0065] The average tumor volume is approximately 400 mm. 3 N1S1 orthotopic HCC rats underwent transcatheter embolization to investigate the pharmacokinetics and biodistribution of Ps-DOX / L and free DOX / L emulsions in vivo. In the pharmacokinetic studies, the DOX dose was 500 μg / rat, and the iodized oil dose was 0.2 mg / kg (n = 3). Blood samples were collected via the orbital cavity at 0.05, 0.08, 0.17, 0.33, 0.67, 1.5, 3, 5, 9, 12, 24, and 48 h post-embolization and rapidly centrifuged (3000 rpm, 15 min). 15 μL of plasma was collected from each sample and 700 μL of DMSO was added. The mixture was incubated in the dark for 24 h to extract DOX. After centrifugation, the supernatant was collected, and the DOX concentration in the plasma was determined by fluorescence microscopy. The area under the curve (AUC) was calculated using the trapezoidal method. 0-48h (Attached) Figure 9 The results showed that after embolization with free DOX / L emulsion, the plasma DOX concentration rapidly reached 2.74 ± 0.23 μg / mL, AUC 0-48h The concentration was 20.70 ± 1.10 μg·h / mL. In contrast, the plasma DOX concentration in rats embolized with Ps-DOX / L emulsion remained low, reaching its highest level at 5 min at 0.38 ± 0.06 μg / mL, with an AUC of 20.70 ± 1.10 μg·h / mL. 0-48hThe concentration was 9.51 ± 0.66 μg·h / mL, which was 7.2 and 2.2 times lower than that of the free DOX / L group, respectively. These results indicate that Ps-DOX / L emulsion can significantly reduce the release of DOX into the bloodstream, reducing systemic drug exposure, which is beneficial for embolization and reducing systemic toxicity.

[0066] Example 8: Biodistribution of Ps-DOX / L emulsion in in situ N1S1 HCC rats

[0067] The retention of Ps-DOX / L or free DOX / L emulsions in the liver of N1S1 HCC rats and their distribution to other organs were observed using ex vivo imaging. When the tumor volume reached approximately 400 mm... 3 Rats (n = 3) were embolized with either Ps-DOX / L or free DOX / L emulsions, with a DOX dose of 200 μg / rat and an iodized oil dose of 0.2 mg / kg. At 24 and 72 h post-embolization, major organs were collected from the sacrificed rats, and DOX fluorescence imaging was performed using a near-infrared fluorescence imaging system (IVIS Lumina II, excitation wavelength 488 nm, emission wavelength 560 nm). To further determine the DOX content in each organ, different organs were homogenized with 1 mL of 1% Triton X-100 (IKA T25), then 4 mL of DMSO was added, incubated at 4 ºC for 48 h, and the supernatant was collected by centrifugation at 8000 rpm for 10 min for fluorescence measurement. In vitro DOX fluorescence images showed that at 24 and 72 h post-embolization, the Ps-DOX / L microemulsion was mostly retained in the liver, especially in the left lobe of the liver inoculated with tumors, exhibiting significant DOX fluorescence (see attached image). Figure 10 A). In rats treated with free DOX / L, although DOX fluorescence in the liver was strong at 24 h post-embolization, it faded rapidly and became undetectable at 72 h. Furthermore, significant DOX fluorescence was observed in the heart and kidneys 24 h after free DOX / L embolization, consistent with the rapid release of the drug into the bloodstream from the free DOX / L emulsion. Quantitative analysis further demonstrated that the Ps-DOX / L microemulsion significantly increased DOX retention in the liver; at 72 h post-embolization, its retention in the liver was 3.6-14.2 times higher than in other organs, and 3.2 times higher than that of the free DOX / L emulsion (see appendix). Figure 10 B).

[0068] Example 9: Therapeutic effect of Ps-DOX / L microemulsion in orthotopic rat N1S1 homologous HCC model

[0069] The anti-HCC efficacy of Ps-DOX / L emulsion in rats bearing orthotopic N1S1 HCC was evaluated using a DOX dose of 500 µg / rat and an iodized oil dose of 0.2 mg / kg. Free DOX / L emulsion, iodized oil, and untreated groups were used as controls. The efficacy was evaluated when the average tumor volume reached 400 mm. 3 Rats bearing tumors were randomly divided into four groups. After anesthesia, different preparations were administered via gastroduodenal artery catheterization, defined as day 0. The PBS group consisted of 5 rats, while the other groups each contained 6 rats. One rat from each group was randomly selected for histological and immunohistochemical (IHC) analysis. MRI scans were performed on rats in each group on days 0, 3, 7, and 14 to measure tumor size (see attached image). Figure 11 A). Rats were weighed every other day and compared with their initial weight on day 0. The results showed that Ps-DOX / L emulsion embolization effectively reduced tumor size, with 40% and 100% of rats showing complete tumor eradication on days 7 and 14, respectively. Free DOX / L emulsion only inhibited tumor growth to a certain extent, and iodized oil showed no significant inhibitory effect on tumor growth. Importantly, no significant toxicity or weight loss was observed in any group (see appendix). Figure 11 BD).

[0070] On days 3, 7, and 14 post-embolization, blood was collected from the orbital sinus of three rats in each group (1 mL from each rat). Three healthy rats were selected as controls. After precipitation overnight at 4ºC, 200 μL of serum was collected by centrifugation. Liver-related biochemical indicators, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT), were detected using a biochemical analyzer. Figure 12 The results showed that in rats embolized with free DOX / L, ALT and AST levels were significantly elevated on day 3, returning to normal levels on day 7, indicating some liver function impairment. In contrast, in the Ps-DOX / L group, ALT and AST levels remained consistently low and within the normal range from day 3 to day 14 after embolization, indicating that the Ps-DOX / L emulsion has high safety. (See attached image) Figure 12 ).

[0071] On day 7, one rat from each group was randomly sacrificed, and its tumors and major organs were harvested for histological and IHC analysis. Tumor and organ samples were immediately fixed in formalin, embedded in paraffin, and cut into 5 μm thick sections, which were then stained with hematoxylin and eosin (H&E). Tumor sections were stained with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and 4,6-diamino-2-phenylindole (DAPI) to distinguish apoptotic cells. To analyze tumor angiogenesis, tumor sections were further labeled with anti-CD31 rabbit pAb, anti-VEGFA rabbit pAb, and HRP-labeled goat anti-rabbit IgG antibody to label CD31 and vascular endothelial growth factor (VEGF). H&E, TUNEL, and IHC images were captured using an inverted fluorescence microscope (Olympus BX41). H&E-stained tumor sections showed significant vacuolar iodized oil deposition in rats embolized with iodized oil, free DOX / L, or Ps-DOX / L emulsions (see attached image). Figure 13 This indicates that different iodized oil formulations can be effectively distributed from the hepatic artery to the tumor site. Ps-DOX / L emulsion induced significant tumor necrosis and marked cell atrophy. TUNEL assays showed that in rats treated with Ps-DOX / L emulsion, tumor cells underwent extensive apoptosis, while tumor cells in the free DOX / L and iodized oil groups showed no significant apoptosis (see attached image). Figure 13 ).

[0072] As attached Figure 14 As shown in A and B, VEGF expression was significantly upregulated in rat tumor sections embolized with iodized oil and free DOX / L, consistent with findings in clinical studies. Conversely, VEGF levels in the Ps-DOX / L group were significantly lower than those in the iodized oil and free DOX / L groups. Furthermore, CD31 expression in tumor sections of the Ps-DOX / L group was also significantly downregulated compared to the iodized oil and free DOX / L groups (see attached image). Figure 14 (A, C). The above results demonstrate that Ps-DOX / L not only induces apoptosis and necrosis in HCC, but also effectively inhibits angiogenesis.

Claims

1. A polymer vesicle-stabilized drug-iodized oil emulsion, characterized in that, The invention comprises polymeric vesicle nanomedicines and iodized oil; the polymeric vesicle nanomedicines include amphiphilic block polymers and small molecule drugs; the amphiphilic block polymer is PEG-P (TMC-DTC); the small molecule drugs include doxorubicin hydrochloride, epirubicin hydrochloride, or maytansin; the amphiphilic block polymer and the small molecule drugs are assembled to form polymeric vesicle nanomedicines, which are then mixed with iodized oil to form a polymeric vesicle-stabilized drug-iodized oil emulsion; in the amphiphilic block polymer, the molecular weight of the PEG segment is 2000–8000 Da; the molecular weight of the hydrophobic segment is 2.0–6.0 times the molecular weight of the PEG segment; and the molecular weight of the PDTC segment is 10%–30% of the total molecular weight of the hydrophobic segment.

2. The method for preparing the polymer vesicle-stabilized drug-iodized oil emulsion according to claim 1, characterized in that, Amphiphilic block polymers and small molecule drugs are assembled to form polymer vesicle nanomedicines, which are then mixed with iodized oil to form a polymer vesicle-stabilized drug-iodized oil emulsion.

3. The method for preparing the polymer vesicle-stabilized drug-iodized oil emulsion according to claim 2, characterized in that, Polymer vesicle nanomedicines are dispersed in a buffer solution and then mixed with iodized oil to form a polymer vesicle-stabilized drug-iodized oil emulsion.

4. The method for preparing the polymer vesicle-stabilized drug-iodized oil emulsion according to claim 2, characterized in that, In the emulsion, the droplet size is 10–100 μm.

5. The use of the polymer vesicle-stabilized drug-iodized oil emulsion of claim 1 in the preparation of antitumor drugs.

6. An antitumor drug, wherein the active ingredient is the polymer vesicle-stabilized drug-iodized oil emulsion as described in claim 1.

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