Preparation method of polymer nanorod with controllable and adjustable length-diameter ratio and application of polymer nanorod in preparation of antitumor drugs

Abstract

The invention provides a preparation method of a polymer nanorod with a controllable and adjustable length-diameter ratio. The invention further provides a preparation method of the anti-tumor nano-drug, the anti-tumor nano-drug is prepared by the preparation method of the polymer nano-rod with the controllable and adjustable length-diameter ratio, and the paclitaxel nano-drug is prepared by the method. The three-step method adopted by the invention is based on a traditional emulsification method, special instruments and equipment are not needed, large-scale production is easy to realize, and the batch yield is relatively high; the preparation conditions are mild, and the drug activity can be effectively protected; the method is low in energy consumption, economical and efficient. According to the method, the length-diameter ratio of the nanorod can be controllably adjusted on the basis of not changing the raw material composition of the nanorod, the consistency of other parameters in the subsequent biological behavior research is favorably kept, the influence of mixed factors is reduced, and the reliability and repeatability of the research can be improved. Besides, when the length-diameter ratio of the nano-carrier is adjusted, only the process of heating fracture in the last step needs to be changed, so that the cost can be greatly saved, customized production is facilitated, or preclinical simple preparation is realized.

Description

A method for preparing polymer nanorods with controllable aspect ratio and their application in the preparation of antitumor drugs. Technical Field

[0001] This invention relates to the field of nanomaterials, and specifically to a method for preparing polymer nanorods with controllable aspect ratio. Background Technology

[0002] Cancer remains one of the leading causes of death worldwide, and chemotherapy is one of the main methods of clinical cancer treatment. However, most anti-tumor drugs have poor water solubility and low bioavailability, resulting in limited therapeutic effects on tumor tissues. Furthermore, chemotherapy can produce severe toxicity and side effects on other normal tissues and cells, which not only impairs the patient's bodily functions but also leads to drug resistance, seriously affecting treatment outcomes.

[0003] In 1978, Marty first used nanoparticles as drug carriers. Nanocarriers refer to micro-delivery systems with sizes ranging from 1 to 1000 nanometers. They can be used to encapsulate, protect, and precisely deliver drugs to specific sites within the body, offering advantages such as controlled release, targeting, high efficiency, low toxicity, and high stability. Among these, using biodegradable polymer materials to prepare nanoparticles as drug carriers allows for the regulation of drug release behavior, improves the biocompatibility of nanocarriers, and ensures safety for in vivo application, making them one of the most widely used carrier materials in clinical practice.

[0004] The design parameters of nanomedicine carriers directly influence their in vivo behavior and ultimate therapeutic effect. Size and shape are primary factors, jointly regulating the carrier's blood circulation time, penetration depth into tumor tissue, and the manner of cellular uptake. Secondly, surface properties are crucial; the carrier's surface charge, hydrophilicity / hydrophobicity, and whether it has been modified with PEGylation or targeting ligands (such as antibodies or peptides) directly affect its stability in the bloodstream, immune escape capabilities, and specific recognition and binding to tumor cells. Finally, drug loading and release characteristics, including drug load, encapsulation efficiency, and drug release kinetics (such as whether it is pH-, enzyme-, or photoresponsive smart release), determine the effective drug dose and therapeutic window ultimately reaching the lesion. Therefore, the design of nanocarriers is a multi-parameter synergistic optimization system engineering project, aiming to balance a series of potentially mutually restrictive delivery links—"long circulation," "deep penetration," "strong endocytosis," and "precise drug release"—to maximize the therapeutic index.

[0005] In recent years, research on the regulation of nanoparticle morphology has received widespread attention. For example, Champion et al. found that macrophages have more difficulty taking up anisotropic rod-shaped non-spherical nanoparticles than spherical nanoparticles. This ability of non-spherical nanoparticles to evade macrophage uptake demonstrates their long-circulation characteristics, revealing their potential application in tumor targeting. However, the rules governing the uptake of nanoparticles of different shapes by tumor cells remain highly controversial. For instance, Xiongfeng Cao et al. synthesized three Pt / Au nanoparticle alloys with different shapes: nanospheres with a size of 85 × 85 nm and an aspect ratio of ≈1.0, nanorods with a size of 116 × 38 nm and an aspect ratio of ≈3.05, and bipyramidal nanoparticles with a size of 99 × 38 nm and an aspect ratio of ≈2.61. The results showed that the bipyramidal nanoparticles with an aspect ratio of approximately 2.61 were more effectively taken up by tumor cells and had stronger tumor sphere penetration (Xiongfeng Cao et al., Biomaterials research, 2025). Libing Fu et al. prepared upconversion nanoparticles with aspect ratios of 1, 2, 3, and 4, and found that neuronal cells showed the highest uptake efficiency for nanoparticles with an aspect ratio of 2. Some studies found that cells uptake both the rate and amount of spherical nanoparticles higher than non-spherical nanoparticles, while other studies showed that non-spherical nanoparticles were taken up by tumor cells more quickly and in greater quantities. Other studies found that gold nanorods with high aspect ratios exhibited higher cytotoxicity than nanorods with low aspect ratios. Since nanorods possess both a long and short axis, their dimensions and aspect ratio can influence cellular uptake, tissue targeting, in vivo delivery, and in vivo therapeutic effects. Therefore, the aspect ratio is also an important factor affecting the structure-activity relationship of nanomedicines.

[0006] Currently, the preparation processes for non-spherical nanoparticles from polymer raw materials include microfluidics, non-wetting template methods, thin film stretching methods, and template assembly methods. Microfluidics relies on precise microscale fluid control, resulting in low throughput and difficulty in large-scale scaling. It also requires precision injection pumps, high-precision temperature controllers, UV light sources, and alignment systems, leading to significant overall equipment investment. Furthermore, most microfluidic methods rely on UV curing, requiring the polymer to possess photosensitive groups (such as acrylates and methacrylates), limiting the application of non-photosensitive materials (such as biodegradable polymers like PLGA and PLLA). Non-wetting template methods utilize low-surface-energy fluorinated polyether (PFPE) elastomers as mold materials, leveraging their non-wetting properties to control particle size, morphology, and composition. However, the cost of silicon master templates and PFPE materials is high. Additionally, this method requires the precursor to have good flowability before curing, limiting the application of high-viscosity polymers such as high-molecular-weight PLGA. Thin film stretching utilizes the viscoelasticity of polymers, deforming them through mechanical stretching. Each film requires individual processing, making true continuous production difficult. Template-based assembly methods offer a unique approach to preparing complex, hierarchical, and anisotropic non-spherical nanoparticles through the synergistic effect of template spatial confinement and interparticle interactions. However, they are highly dependent on the precision and reproducibility of the template, and the preparation of large-area uniform templates is challenging. Most of the aforementioned methods require complex and expensive specialized instruments and are difficult to scale up.

[0007] Poly(lactic-co-glycolic) acid (PLGA) is an important biomedical polymer material and a pharmaceutical excipient approved by the U.S. Food and Drug Administration (FDA), exhibiting good biodegradability and biocompatibility. Furthermore, the conventional emulsification method for preparing PLGA nanoparticles is simple and easily scaled up for industrial production, and has already been applied in manufacturing practice. However, PLGA nanoparticles prepared by traditional emulsification processes typically spontaneously form spherical shapes. Therefore, previous research on optimizing PLGA nanoparticles has primarily focused on improving their particle size, surface charge, and surface grafting modifications; research on the shape control and applications of these nanoparticles remains limited.

[0008] Bhide et al. prepared PLGA nanoparticles using standard nanoprecipitation and combined it with mechanical film stretching to obtain non-spherical nanoparticles with major diameters of 128.1±23.1 nm to 510.7±114.6 nm, minor diameters of 58.1±12.0 nm to 140.1±31.3 nm, and aspect ratios of 1.7±0.3 to 5.1±0.8. They also developed PLGA-based artemisinin methyl ether-loaded nanorods to improve intravenous delivery of antimalarial drugs, where the PLGA molecular weight is 4000–15000. However, this literature only investigated the preparation process of nanorods with different aspect ratios using film stretching, and in the drug release and cytotoxicity studies, only compared the differences between spherical and rod-shaped nanocarriers, without investigating the drug release and cytotoxicity of nanocarriers with different aspect ratios. Furthermore, the data in this literature indicates that drug leakage occurs when spherical particles are stretched to prepare rod-shaped particles, resulting in a decrease in drug loading (which can be reduced by more than half from the original drug loading).

[0009] Kaplan et al. prepared spherical PLGA nanoparticles using a double-emulsion solvent evaporation method (W / O / W). Subsequently, they used a modified film stretching method, heating and mechanically stretching the nanoparticles in PVA / glycerol films to obtain spherical nanoparticles with a major diameter of 163.2 ± 0.7 nm and an aspect ratio of 1.0; rod-shaped nanoparticles with a major diameter of 312.9 ± 5.4 nm and an aspect ratio of 4.0 ± 0.5; and elliptical disk-shaped nanoparticles with a major diameter of 251.4 ± 4.7 nm and an aspect ratio of 7.5 ± 0.5. The PLGA used had a molecular weight of 35 kDa. Using human serum albumin as a model drug, they investigated the encapsulation efficiency and in vitro release behavior of nanoparticles with different shapes. They found that while nanoparticle shape affected drug release rate and cellular uptake efficiency, it did not show a significant difference in tumor-targeted accumulation as described in this study. Therefore, shape design can serve as a tool for regulating the performance of nanocarriers, but it needs to be optimized in conjunction with specific disease models and the characteristics of the nanomaterials.

[0010] Patent application number: 201610232509.8, invention title: A uniform non-spherical polymer particle with its preparation method and uses. This invention discloses a two-step "emulsion-deformation" method. Based on emulsification, disodium hydrogen phosphate and / or sodium dihydrogen phosphate are used as deformation initiators. The method comprehensively considers the effects of the concentration of disodium hydrogen phosphate and / or sodium dihydrogen phosphate, the properties of the polymer itself, the mass concentration of the polymer in the oil phase, and the size of the emulsion droplets. This yields non-spherical polymer particles with uniform particle size, a short diameter of 100 nm to 30 μm, a long diameter of 1 to 60 μm, and an aspect ratio of 2 to 40. However, this method is difficult to prepare nanoparticles with both long and short diameters below 1 μm, and it is difficult to controllably adjust the aspect ratio of the nanorods without changing the raw material composition. This limits further research on the influence of PLGA nanoparticle shape on drug activity, in vivo delivery, efficacy, and safety.

[0011] Currently, there is no method based on emulsification to prepare non-spherical PLGA drug-loaded nanoparticles with controllable aspect ratios of less than 1 μm without altering the raw material composition of the nanorods or relying on complex instruments. Summary of the Invention

[0012] To this end, this invention provides a three-step method of "emulsion preparation-deformation-heat fracture" for preparing polymer nanorods with controllable aspect ratio. PLGA-loaded paclitaxel nanoparticles with different aspect ratios were used for targeted therapy of CT26 colorectal cancer. The effects of nanoparticle aspect ratio on its targeting behavior, cellular uptake, and tumor-suppressing effect were compared, and the optimal aspect ratio was screened. The results demonstrate that the nanoparticle aspect ratio can affect the tumor targeting, cellular uptake, and anti-tumor effect of nanoparticles, and is an important parameter determining the therapeutic efficacy of nanomedicines.

[0013] In a first aspect, the present invention provides a method for preparing polymer nanorods with controllable aspect ratio, comprising the following steps:

[0014] (a) Preparation of the oil phase: The polymer is dissolved in an organic solvent to prepare the oil phase;

[0015] (b) Preparation of emulsion: Dissolve the emulsifier in water to prepare aqueous phase 1; emulsify the oil phase and aqueous phase 1 by ultrasonic emulsification to obtain an oil-in-water emulsion;

[0016] (c) Dissolve the deformation initiator in an emulsifier solution to prepare aqueous phase 2; mix aqueous phase 2 with the prepared oil-in-water emulsion, remove the organic solvent to solidify the droplets, and ultrasonically wash to obtain a nanoparticle suspension;

[0017] (d) The prepared nanoparticle suspension is heated to break it apart, and then washed and dried to obtain polymer nanorods with uniform particle size and different aspect ratios.

[0018] Preferably, the organic solvent in step (a) is dichloromethane or a mixture of dichloromethane and acetone or dichloromethane and methanol or dichloromethane and dimethyl sulfoxide or dichloromethane and acetonitrile, and the polymer is polylactic acid-polyglycolic acid copolymer, or polylactic acid copolymer or polylactic acid-polyglycolic acid-polyethylene glycol copolymer, and the polymer has a mass concentration of 20 mg / mL in the oil phase;

[0019] Preferably, the emulsifier in step (b) is polyvinyl alcohol, the concentration of the emulsifier in aqueous phase 1 is 1.5%-4.0%, the ultrasonic conditions are ice bath, and the ultrasonic power is 30-200W for 5 seconds, followed by a 5-second pause, with a total duration of 2-10 minutes.

[0020] Preferably, the deformation initiator in step (c) is disodium hydrogen phosphate, the emulsifier solution is a polyvinyl alcohol aqueous solution with a mass concentration of 1.5%-5.0%, and the organic solvent is removed by stirring at 500 rpm for 4 h at room temperature to allow the organic solvent to evaporate; the ultrasonic washing conditions are as follows: centrifuge the nanoparticles obtained by solidifying the droplets, collect the nanoparticle precipitate, add pure water for washing, ultrasonically disperse, then centrifuge again, add pure water for ultrasonic dispersion, adjust the concentration, and obtain a nanoparticle suspension with a concentration of 1 mg / mL;

[0021] Preferably, the heating temperature in step (d) is 25℃-65℃, the heating time is 5 min-60 min, and the aqueous phase system is pure water during heating.

[0022] More preferably, the organic solvent in step (a) is a mixed organic solvent of dichloromethane:acetone = 4:1;

[0023] More preferably, in step (b), the concentration of the emulsifier in the aqueous phase 1 is 1.5%, the ultrasonic power is 120W, and the total ultrasonic duration is 2 min.

[0024] More preferably, the emulsifier solution in step (c) is a 3% (w / w) aqueous solution of polyvinyl alcohol.

[0025] In a second aspect, the present invention provides a method for preparing antitumor nanomedicines, which is prepared by the above-described method for preparing polymer nanorods with controllable aspect ratio, the difference being that an antitumor drug is added to the oil phase in step (a).

[0026] The antitumor drugs are hydrophobic and include paclitaxel, docetaxel, demineralized doxorubicin, gemcitabine, etoposide, vincristine, vinorelbine, temozolomide, carmustine, camptothecin, retinoic acid, all-trans retinoic acid, and cabazitaxel.

[0027] In a third aspect, the present invention provides a paclitaxel nanomedicine prepared by the above-described method for preparing antitumor nanomedicines. The paclitaxel nanomedicine has a major diameter of 118-820 nm, a minor diameter of 66-191 nm, an aspect ratio of 1-12, and a drug loading rate of 1.7%-4.3%.

[0028] Preferably, the paclitaxel nanomedicine has a major diameter of 250.03±94.26 nm, a minor diameter of 92.58±24.40 nm, an aspect ratio of 2.75±0.85, and a drug loading rate of 2.5%.

[0029] A fourth aspect of the present invention provides a method for preparing paclitaxel nanomedicine, comprising the following steps:

[0030] This invention provides a method for preparing the aforementioned paclitaxel nanomedicine, comprising the following steps:

[0031] (a) Preparation of oil phase: Polylactic acid-polyglycolic acid copolymer and paclitaxel are dissolved in a mixed organic solvent of dichloromethane:acetone = 4:1 to prepare an oil phase with a polymer concentration of 20 mg / mL and a paclitaxel concentration of 0.8 mg / mL-1.2 mg / mL; preferably, the paclitaxel concentration is 1.0 mg / mL, and the drug loading rate at this concentration is 2.5%.

[0032] (b) Preparation of emulsion: The emulsifier polyvinyl alcohol was dissolved in water to prepare aqueous phase 1. The concentration of the emulsifier in aqueous phase 1 was 1.5%. The oil phase and aqueous phase 1 were emulsified by ultrasonic emulsification. The ultrasonic conditions were ice bath. The ultrasonic power was 120W. The ultrasonic power was 5 seconds per 5 seconds, paused for 5 seconds, and the total duration was 2 minutes to obtain an oil-in-water emulsion.

[0033] (c) Dissolve the deformation initiator disodium hydrogen phosphate in an emulsifier solution, which is a 3% (w / w) polyvinyl alcohol aqueous solution, to prepare aqueous phase 2; mix aqueous phase 2 with the prepared oil-in-water emulsion and stir at 500 rpm for 4 h at room temperature to allow the organic solvent to evaporate; ultrasonic washing conditions are as follows: centrifuge the nanoparticles obtained by solidifying the droplets, collect the nanoparticle precipitate, add pure water for washing, ultrasonically disperse, then centrifuge again, add pure water for ultrasonic dispersion, adjust the concentration, and obtain a nanoparticle suspension with a concentration of 1 mg / mL;

[0034] (d) The prepared nanoparticle suspension was heated to break it apart. The heating temperature was 25℃-65℃ and the heating time was 5 min-60 min. After washing and drying, polymer nanorods with uniform particle size and different aspect ratios were obtained.

[0035] Preferably, the heating time is 5-10 minutes.

[0036] A fifth aspect of the present invention provides the use of paclitaxel nanomedicine in the preparation of medicaments for treating breast cancer, lung cancer, colorectal cancer, or melanoma.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] This invention's three-step method is based on the traditional emulsification method, requiring no special equipment, easily scalable for large-scale production, and achieving high batch yields. The preparation conditions are mild, effectively protecting drug activity; it is energy-efficient, economical, and highly effective. In the third step, heating and fracture to control the aspect ratio of the nanocarrier, our method does not alter the nanocarrier's composition, surface properties, or drug loading rate. Therefore, it helps maintain consistency of other parameters in subsequent biological behavior studies, reducing the influence of confounding factors and improving the reliability and reproducibility of the research. Furthermore, this invention's method only requires changing the final heating and fracture process when adjusting the nanocarrier's aspect ratio, which can significantly save costs and facilitate customized production or simple preclinical preparation. Attached Figure Description

[0039] Figure 1. Flowchart of the process for preparing polymer nanorods with different aspect ratios using the thermal fracture method.

[0040] Figure 2. Effect of different aqueous phase systems on the degree of nanorod fracture upon heating (a: unheated group, b: pure water heating group, c: 1.5% PVA heating group containing 150 mM Na2HPO4·12H2O, d: 1% PBS heating group, e: 150 mM Na2HPO4·12H2O heating group, heating time: 10 min, heating temperature: 60 ℃, scale bar: 1 μm)

[0041] Figure 3. Effect of heating at different temperatures for 5 min on the degree of thermal breakage of nanorods (a: 25℃, b: 43℃, c: 55℃, scale bar: 1 μm).

[0042] Figure 4. Effect of different heating times at the same heating temperature on the degree of thermal fracture of nanorods (scale bar: 1 μm)

[0043] Figure 5. Thermal fracture of nanomedicines with different paclitaxel concentrations after 5 min at different heating temperatures (a: 0.8 mg / mL, 25℃; b: 1.0 mg / mL, 25℃; c: 1.2 mg / mL, 25℃; d: 0.8 mg / mL, 49℃; e: 1.0 mg / mL, 49℃; f: 1.2 mg / mL, 49℃; scale bar: 1 μm)

[0044] Figure 6. Effect of heating time at 45 °C on the degree of thermal fracture of paclitaxel nanomedicine.

[0045] Figure 7. Effect of heating at different temperatures for 10 min on the thermal breakage of paclitaxel nanomedicine (a: 25 °C, b: 38 °C, c: 40 °C, d: 43 °C, e: 46 °C, f: 65 °C; scale bar: 1 μm).

[0046] Figure 8. Scanning electron microscopy images of paclitaxel nanomedicines with different aspect ratios. Scale bar: 1 μm

[0047] Figure 9. Paclitaxel nanomedicine and Taxol ® In vitro cumulative drug release graph (n=3)

[0048] Figure 10. Uptake of paclitaxel nanomedicines with different aspect ratios by RAW264.7 macrophages. a: Laser confocal microscopy; b: Flow cytometry results; **, p < 0.01; ***, p < 0.001

[0049] Figure 11. Laser confocal microscopy images of CT-26 cells uptake of paclitaxel nanomedicines with different aspect ratios.

[0050] Figure 12 Flow cytometry results of CT-26 cells uptake of paclitaxel nanomedicines with different aspect ratios (n=3)

[0051] Figure 13. Toxicity of paclitaxel nanomedicines with different aspect ratios to CT-26 cells (n=6); *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001

[0052] Figure 14. In vivo imaging observation of the distribution and tumor targeting of paclitaxel nanomedicines with different aspect ratios in tumor-bearing mice. a: In vivo imaging at different time points; b: Statistical results of average fluorescence intensity at tumor sites in live animals; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, no significant difference.

[0053] Figure 15. Ex vivo imaging and mean fluorescence intensity of nanoparticles in major organs and tumor sites. a: Ex vivo fluorescence imaging of nanoparticles in major organs; b: Mean fluorescence intensity of nanoparticles in major organs; c: Fluorescence imaging of nanoparticles in tumor sites; d: Mean fluorescence intensity of nanoparticles in tumor sites; n=3; *, p<0.05; ***, p<0.001

[0054] Figure 16. Evaluation of the in vivo tumor-suppressing effect of nanoparticles: a: Experimental flowchart; b: Tumor growth curve of CT-26 tumor-bearing mice; c: Tumor image after dissection; d: Body weight change curve of tumor-bearing mice; e: Ex vivo tumor weight; *, p < 0.05; **, p < 0.01; ***, p < 0.001; n=5

[0055] Figure 17. Safety evaluation of nanoparticles: a: Serum biochemical indicators in mice (ALT: alanine aminotransferase; AST: aspartate aminotransferase; UREA: urea; UA: uric acid); b: Pathological sections and H&E staining images of major organs. Detailed Implementation

[0056] Example 1: A method for preparing polymer nanorods with controllable aspect ratio according to the present invention.

[0057] PVA storage solution preparation (3% PVA): Dissolve 30 g of polyvinyl alcohol (PVA) in 1000 mL of deionized water under heating conditions to prepare a 3% PVA aqueous solution.

[0058] Oil phase preparation: Weigh 10 mg PLGA into a 2 mL centrifuge tube, add 0.5 mL of mixed oil phase (dichloromethane: acetone = 4:1), dissolve it completely, and store it in a 4 °C refrigerator for later use.

[0059] Preparation of aqueous phase 1: Dilute the PVA stock solution with deionized water to a concentration of 1.5% PVA aqueous solution in 2 mL.

[0060] Preparation of aqueous phase 2: Take 5 mL of 3% PVA stock solution and place it in a sample bottle. Stir on a magnetic stirrer and weigh a certain amount of disodium hydrogen phosphate powder. Slowly add the powder and stir until completely dissolved. This is aqueous phase 2 containing the deformation inducer disodium hydrogen phosphate.

[0061] Emulsion preparation: The oil phase was added to the aqueous phase 1, and the mixture was ultrasonically disrupted for 2 min (5 s for sonication, 5 s for rest, total time 2 min) under ice bath conditions using an ultrasonic cell disruptor at 60% power (120 W) to prepare an oil-in-water emulsion.

[0062] Curing: Pour the prepared emulsion into aqueous phase 2 and stir at 500 rpm for more than 4 hours to allow the dichloromethane in the emulsion to completely evaporate and the emulsion droplets to solidify into solid nanoparticles.

[0063] Washing: Collect the solidified nanoparticle suspension in a centrifuge tube, centrifuge (40,000 g, 10 min), remove the supernatant, add deionized water, and use an ultrasonic cleaner to thoroughly disperse the precipitate. Centrifuge again, repeating this washing process 4 times. After the final centrifugation to remove the supernatant, add a certain amount of deionized water and use an ultrasonic cleaner to thoroughly disperse the precipitate. This is the washed nanoparticle suspension. Adjust the concentration to obtain a nanoparticle suspension with a concentration of 1 mg / mL.

[0064] Heating fracture: Take 5 mL of nano suspension and place it in a sample bottle. Under magnetic stirring (500 rpm), heat it in a water bath at a certain temperature. After the temperature of the nano suspension reaches the set temperature, start timing according to the set time. After heating is completed, immediately place it in an ice water bath for rapid cooling.

[0065] Washing: After centrifugation (40,000 g, 10 min) to remove the supernatant, add a small amount of deionized water and use an ultrasonic cleaner to thoroughly disperse the polymer nanorods with different aspect ratios.

[0066] The prepared nanorod suspension was dropped onto the sample stage and allowed to dry naturally at room temperature. Then, it was sputter-coated with gold. The surface morphology of the nanorods was observed using a scanning electron microscope. The images were processed using ImageJ image analysis software to determine the major and minor diameters of the nanorods. The average major diameter, average minor diameter, and aspect ratio (AR, AR = major diameter / minor diameter) of the nanorods were statistically analyzed.

[0067] Figure 1 shows the process flow diagram for preparing polymer nanorods with different aspect ratios using the thermal fracture method.

[0068] Example 2: Process screening test of a method for preparing polymer nanorods with controllable aspect ratio according to the present invention.

[0069] 1. Effect of aqueous system on the thermal fracture of blank polymer nanorods

[0070] As shown in Figure 2, different aqueous phase systems affect the degree of thermal fracture of polymer nanorods. Table 2 shows that without thermal fracture, the aspect ratio of the nanorods is 11.22. Under heating conditions of 10 min and 60 °C, most of the nanorods in the pure water heating group become spherical. The secondary deformation in the 1.5% PVA group containing 150 mM Na2HPO4·12H2O is not significant, with an aspect ratio of 8.38. The 150 mM Na2HPO4·12H2O nanorods become short rods, with an aspect ratio decreasing to 4.44. The aspect ratio of the nanorods in the 1% PBS group decreases significantly to 2.99. Therefore, controlling the composition of the aqueous phase system during thermal fracture can effectively regulate the aspect ratio of the nanorods.

[0071] The results above show that the residual inorganic salt Na2HPO4 and PVA in the system after the nanorods are solidified will inhibit the thermal breakage of the nanorods. Therefore, we determined a three-step process to control the aspect ratio of the nanorods: collect the solidified nanorods by centrifugation, wash them twice with pure water by ultrasonic dispersion, centrifuge again, adjust the concentration of the nanorods to 1 mg / mL by pure water dispersion, and then perform thermal breakage.

[0072] Table 1. Sizes of nanorods after heating in different aqueous phase systems.

[0073]

[0074] 2. Effect of heating temperature on the thermal fracture of blank polymer nanorods

[0075] As shown in Figure 3, the heating temperature affects the degree of fracture of the nanorods. After the nanorods fractured in a water bath at 25℃ and 43℃ for 5 minutes, the shape change was not obvious; when the temperature was further increased to 55℃, the nanorods became spherical or spherical.

[0076] 3. Effects of heating temperature and time on the thermal fracture of blank polymer nanorods

[0077] As shown in Figure 4, both heating temperature and time affect the change in the aspect ratio of polymer nanorods.

[0078] At lower temperatures (41 °C and 43 °C), as the heating time increased from 5 min to 60 min, most nanorods in the field of view broke into shorter rods, but the overall morphology change was small. Even with extended heating time, it was difficult to achieve a significant transformation from nanorods to nanospheres at lower temperatures. At higher temperatures, the nanorods fractured more rapidly. For example, at 45 °C, the nanorods fractured into spheres after 30 min of heating. However, excessively high temperatures cause the nanorods to rapidly transform into spheres in a short time, which is not conducive to obtaining rod-shaped structures with intermediate aspect ratios. For example, heating at 55 °C for 5 min resulted in spherical fracture (Figure 3c). Therefore, the optimal temperature range for preparing blank polymer nanorods with different aspect ratios is 25 °C - 47 °C.

[0079] With a constant heating time, the aspect ratio of the nanorods can be adjusted by changing the heating temperature. For example, as shown in Figure 4, when the heating time is 5 min, the nanorods prepared at a heating temperature of 47 °C are short rods, while those prepared at a heating temperature of 45 °C are long rods. Furthermore, when heated for 30 min or 60 min, even when the temperature is reduced to 45 °C, the nanorods have essentially transformed into spheres.

[0080] Similarly, by adjusting the heating time while keeping the heating temperature constant, the aspect ratio of the nanorods can also be controlled. For example, by keeping the heating temperature constant at 45 °C and extending the heating time from 5 min to 10 min and then to 30 min, the nanorods break from long rods into short rods and then into spheres. Therefore, the effective control of nanorod morphology depends on the synergistic optimization of heating temperature and heating time, with temperature-driven thermodynamic control being more critical.

[0081] Example 3: Preparation method of the antitumor nanomedicine of the present invention

[0082] PVA storage solution preparation (3% PVA): Dissolve 30 g of polyvinyl alcohol (PVA) in 1000 mL of deionized water under heating conditions to prepare a 3% PVA aqueous solution.

[0083] Oil phase preparation: Weigh 10 mg PLGA into a 2 mL centrifuge tube, weigh a certain amount of antitumor drug, add 0.5 mL of mixed oil phase (dichloromethane: acetone = 4:1), dissolve it completely, and store it in a 4 °C refrigerator for later use.

[0084] Preparation of aqueous phase 1: Dilute the PVA stock solution with deionized water to a concentration of 1.5% PVA aqueous solution in 2 mL.

[0085] Preparation of aqueous phase 2: Take 5 mL of 3% PVA stock solution and place it in a sample bottle. Stir on a magnetic stirrer and weigh a certain amount of disodium hydrogen phosphate powder. Slowly add the powder and stir until completely dissolved. This is aqueous phase 2 containing the deformation inducer disodium hydrogen phosphate.

[0086] Emulsion preparation: The oil phase was added to the aqueous phase 1, and the mixture was ultrasonically disrupted for 2 min (5 s for sonication, 5 s for rest, total time 2 min) under ice bath conditions using an ultrasonic cell disruptor at 60% power (120 W) to prepare an oil-in-water emulsion.

[0087] Curing: Pour the prepared emulsion into aqueous phase 2 and stir at 500 rpm for more than 4 hours to allow the dichloromethane in the emulsion to completely evaporate and the emulsion droplets to solidify into solid nanoparticles.

[0088] Washing: Collect the solidified nanoparticle suspension in a centrifuge tube, centrifuge (40,000 g, 10 min), remove the supernatant, add deionized water, and use an ultrasonic cleaner to thoroughly disperse the precipitate. Centrifuge again, repeating this washing process four times. After the final centrifugation to remove the supernatant, add a certain amount of deionized water and use an ultrasonic cleaner to thoroughly disperse the precipitate. This is the washed nanoparticle suspension, which should be stored at 4 °C for later use. Adjust the concentration to obtain a nanoparticle suspension with a concentration of 1 mg / mL.

[0089] Heating fracture: Take 5 mL of nano suspension and place it in a sample bottle. Under magnetic stirring (500 rpm), heat it in a water bath at a certain temperature. After the temperature of the nano suspension reaches the set temperature, start timing according to the set time. After heating is completed, immediately place it in an ice water bath for rapid cooling.

[0090] Washing: After centrifugation (40,000 g, 10 min) to remove the supernatant, add a small amount of deionized water and use an ultrasonic cleaner to thoroughly disperse the antitumor nanomedicines with different aspect ratios.

[0091] The process flow is the same as that for the blank polymer nanorods in Example 1 (Figure 1), except that hydrophobic drugs are added to the oil phase. The hydrophobic drugs include paclitaxel, docetaxel, demineralized doxorubicin, gemcitabine, etoposide, vincristine, vinorelbine, temozolomide, carmustine, camptothecin, retinoic acid, all-trans retinoic acid, and cabazitaxel.

[0092] Example 4: Process screening test of the preparation method of paclitaxel nanomedicine of the present invention.

[0093] Paclitaxel nanomedicine was prepared using the preparation method described in Example 3.

[0094] 1. Heating fracture of nanomedicines prepared with different paclitaxel concentrations

[0095] We prepared drug-loaded nanorods using oil-phase paclitaxel concentrations of 0.8 mg / mL, 1.0 mg / mL, and 1.2 mg / mL. The paclitaxel loading rates of the nanorods were determined by high-performance liquid chromatography (HPLC), and were 1.75±0.18%, 2.45±0.21%, and 4.28±0.23%, respectively. The fracture and deformation of the paclitaxel nanorods after heating at 25 °C and 49 °C for 5 min were also investigated, and the parameters are shown in Table 2.

[0096] Table 2. Aspect ratio and changes of nanomedicines with different paclitaxel concentrations at different heating temperatures.

[0097]

[0098] Note: For the same paclitaxel concentration, the aspect ratio of nanomedicines was compared at different heating temperatures. ***, p < 0.001

[0099] As shown in Figure 5, the drug concentration affects the aspect ratio (AR) of the nanorods. At the same temperature, the change in AR of the nanorods decreases with increasing paclitaxel concentration in the formulation. Table 2 presents a quantitative analysis of the AR of nanomedicines with different paclitaxel concentrations at different heating temperatures. The results show that at a paclitaxel concentration of 0.8 mg / mL, the AR of the 49℃ heating group (2.04 ± 0.59) is significantly lower than that of the 25℃ heating group (12.95 ± 4.58), with an AR change value (ΔAR) of 10.91. This change value is approximately twice that of the 1.0 mg / mL group (ΔAR = 5.36) and the 1.2 mg / mL group (ΔAR = 4.35), indicating that at lower drug concentrations, the nanomedicines are more sensitive to heating temperature and exhibit more significant morphological changes. Therefore, after loading the nanorods with drugs, further optimization of their heating fracture temperature and time is needed.

[0100] 2. Effect of different heating times on the thermal fracture of paclitaxel nanomedicine

[0101] Figure 6 shows representative microscopic images of paclitaxel nanomedicines heated at 45°C for different times. Similar to the case of blank nanorods, changing the heating time can adjust the aspect ratio of the nanorods after thermal breakage. A longer heating time results in a shorter aspect ratio, indicating a greater degree of thermal breakage. However, at the same heating temperature, extending the heating time prolongs the preparation time and increases the risk of drug leakage. On the other hand, too short a heating time leads to insufficient heat transfer, resulting in uneven heating and difficulty in precisely controlling the process flow. To avoid drug leakage, accelerate the preparation process, and ensure heating uniformity, a heating time range of 5-10 min is preferred.

[0102] 3. Effect of different heating temperatures on the thermal fracture of paclitaxel nanomedicine

[0103] Figure 7 shows representative microscopic images of paclitaxel nanomedicines heated at different temperatures (25 °C – 65 °C) for 10 min. Table 3 shows the quantitative analysis of the particle size and aspect ratio of paclitaxel nanomedicines prepared at different heating temperatures. It can be seen that, similar to the case of blank nanorods, by adjusting the heating temperature while keeping the heating time constant, the aspect ratio of the nanorods after heating and breakage can be adjusted.

[0104] Table 3. Particle size and aspect ratio of paclitaxel nanomedicines prepared at different heating temperatures

[0105]

[0106] Example 5: Preparation and characterization of the paclitaxel nanomedicine of the present invention

[0107] 1. Morphological characterization of paclitaxel nanomedicines with different aspect ratios

[0108] First, a series of paclitaxel nanomedicines with different aspect ratios were prepared using the method described in Example 4, and the results are shown in Figure 8. Increasing the heating temperature reduced the long diameter of the nanorods, while the short diameter showed little change, and the aspect ratio decreased, demonstrating that the developed method primarily controls the aspect ratio of the nanorods by inducing nanorod breakage through heating. Scanning electron microscopy images of the nanoparticles were measured using ImageJ software, and the long diameter, short diameter, and their ratio were statistically analyzed, as shown in Table 4. Based on this, we used the aspect ratio of the nanoparticles as a classification standard, defining them as NP-AR10 (AR=10.08±3.11), NP-AR6 (AR=6.06±1.82), NP-AR3 (AR=2.75±0.85), and NP-AR1 (AR=1.14±0.04), respectively. The above four paclitaxel nanomedicines with different aspect ratios were applied to anti-tumor therapy. Colorectal cancer CT26 cells were selected as a model to study the effect of the aspect ratio of nanorods on anti-tumor efficacy, and to elucidate the biological effects of shape and the mechanism of tumor suppression.

[0109] Table 4. ImageJ statistical analysis of particle size parameters of paclitaxel nanomedicines with different aspect ratios (n=100)

[0110]

[0111] 2. Particle size, potential and drug loading of paclitaxel nanomedicines with different aspect ratios

[0112] The hydrated particle size and zeta potential of each group of paclitaxel-loaded nanoparticles were determined using a Malvern laser particle size analyzer. As shown in Table 5, the hydrated diameter of the nanoparticles gradually decreased with decreasing aspect ratio. The average particle size of the quasi-spherical NP-AR1 nanoparticles was 250 nm, slightly larger than the aspect ratios measured by scanning electron microscopy in Table 4. This is because the Malvern particle size analyzer measures the hydrated particle size, which is usually larger than the results of electron microscopy measurements when the nanoparticles are dry. This trend is consistent with the results in the literature. The average particle size of other nanoparticles differed significantly from their aspect ratios. This is because the Malvern particle size analyzer primarily calculates the average particle size based on the Brownian motion of the nanoparticles, assuming they are spherical. Therefore, it cannot obtain the dimensions of anisotropic nanorods in both aspect ratios. As shown in Table 5, the surface charge measurement results showed that the zeta potential of all samples was negative. This is because the PLGA raw material is carboxyl-terminated, and the surface of the prepared nanoparticles is rich in negatively charged carboxyl groups. The negative surface charge is beneficial for the nanoparticles to maintain good dispersibility and stability. Precisely weighed lyophilized nanorod powder was dissolved in a certain volume of acetonitrile solution to demulsify and release the drug paclitaxel. The paclitaxel concentration was detected using high-performance liquid chromatography (HPLC), and the drug loading rate of the nanorods was calculated. As shown in Table 5, the drug loading rates of the four paclitaxel nanoparticles were similar, indicating that there was almost no drug leakage during the process of heating and breaking down high aspect ratio nanorods into low aspect ratio nanorods. Therefore, the heating-breaking method developed in this invention does not change the surface potential, drug loading performance, or other physicochemical parameters of the paclitaxel-loaded nanoparticles during the adjustment of their aspect ratio, which is beneficial for eliminating the influence of changes in other parameters in subsequent structure-activity relationship studies.

[0113] Table 5. Hydration size, Zeta potential, and drug loading rate of paclitaxel nanomedicines with different length-to-diameter ratios (n=3)

[0114]

[0115] The following efficacy tests demonstrate the beneficial effects of this invention.

[0116] Experimental Example 1: In vitro drug experiments of paclitaxel nanomedicines with different aspect ratios of the present invention

[0117] 1. Drug release experiment

[0118] To investigate the effect of nanoparticle aspect ratio on drug release behavior, this invention employs dialysis combined with high-performance liquid chromatography (HPLC) to determine drug concentration. The in vitro drug release kinetics of paclitaxel nanomedicines with different aspect ratios were systematically evaluated under simulated normal body fluid conditions at 37 °C and pH 7.4, using the clinical formulation Taxol as an example. ®As a positive control, as shown in Figure 9, all four paclitaxel nanoparticles with varying aspect ratios were slowly released within 24 hours, with a cumulative release rate of approximately 50% in the first 12 hours. No significant burst release was observed, and the aspect ratio of the nanoparticles had little effect on their release behavior within the first 12 hours. At 48 hours, the paclitaxel nanoparticles were nearly completely released, but at this point, Taxol... ® The cumulative release rate of the four paclitaxel nanomedicines was significantly lower than that of NP-AR3 and NP-AR10, while there was no significant difference in the cumulative release rate of the four paclitaxel nanomedicines with different aspect ratios at 48 h. This may be because the four paclitaxel nanomedicines were prepared using the same carrier material, and the three paclitaxel nanomedicines with lower aspect ratios were all derived from the fragmentation of the paclitaxel nanomedicine with the highest aspect ratio, resulting in similar drug loading rates and similar drug distribution in the carrier. Therefore, the drug release behavior of the four paclitaxel nanomedicines was also similar.

[0119] 2. Uptake of paclitaxel nanomedicines with different aspect ratios by phagocytes

[0120] To investigate the immune escape behavior of nanoparticles with different aspect ratios, this invention further evaluated the internalization efficiency and kinetic changes of paclitaxel nanoparticles with different aspect ratios in RAW264.7 macrophages. Nanoparticles were labeled with the fluorescent dye Cy5 and then co-incubated in macrophage culture plates to allow macrophages to take up the nanoparticles. After a certain incubation time, untaken nanoparticles were washed away. The uptake behavior of nanoparticles was observed using laser confocal scanning microscopy, and the average fluorescence intensity of nanoparticles taken up by cells was quantitatively examined using flow cytometry. As shown in Figure 10, after 4 h of incubation, the red fluorescence intensity of NP-AR1 in RAW264.7 cells was significantly higher than that in other groups. Furthermore, flow cytometry results further indicated that after 4 h and 16 h of incubation, the uptake of NP-AR1 by RAW264.7 cells was significantly higher than that in other groups. NP-AR10 had the lowest average uptake efficiency in macrophages, and the amount of nanoparticles taken up by cells increased with prolonged incubation time.

[0121] These results indicate that the aspect ratio of PLGA nanoparticles significantly affects their phagocytic efficiency by macrophages. Spherical NP-AR1 nanoparticles are more readily internalized by RAW264.7 cells, while nanoparticles with higher aspect ratios are more likely to evade macrophage uptake. Therefore, increasing the aspect ratio of nanoparticles may be beneficial in prolonging their in vivo circulation time.

[0122] 3. Tumor cell uptake of paclitaxel nanomedicines with different aspect ratios

[0123] To investigate the uptake efficiency of CT26 tumor cells for nanoparticles with different aspect ratios, laser confocal microscopy and flow cytometry were used to qualitatively and quantitatively evaluate the internalization behavior of paclitaxel nanoparticles with different aspect ratios in CT26 cells.

[0124] As shown in Figure 11, the laser confocal microscopy results revealed significant differences in fluorescence intensity of paclitaxel nanoparticles with different aspect ratios in CT26 cells. The NP-AR1 group exhibited the strongest red fluorescence, indicating the highest cellular uptake efficiency; NP-AR3 was second strongest, while the fluorescence signals of the NP-AR6 and NP-AR10 groups were significantly weaker. These results indicate a clear aspect ratio-dependent cellular uptake of the nanoparticles, with the uptake efficiency trending from highest to lowest as NP-AR1 > NP-AR3 > NP-AR6 > NP-AR10.

[0125] To further quantify the level of cellular internalization, flow cytometry was used to detect the uptake of paclitaxel nanoparticles by CT26 cells after different incubation times (4, 6, and 16 h) with varying aspect ratios. As shown in Figure 12, the uptake of nanoparticles by cells increased with prolonged incubation time. At the same incubation time, the uptake of nanoparticles by cells was affected by the aspect ratio. After co-incubation with NP-AR1 for 4 h, the percentage of positive cells uptaken by nanoparticles in the NP-AR1 group was 76.8%, significantly higher than that in the NP-AR3 (65.9%), NP-AR6 (47.2%), and NP-AR10 (39.4%) groups. At 16 h, the percentage of positive cells uptaken by nanoparticles was 99.3% in the NP-AR1 group, 98.6% in the AR3 group, 98.4% in the AR6 group, and 97.8% in the AR10 group. The average fluorescence intensity after cellular uptake of nanoparticles also followed this pattern: NP-AR1 > NP-AR3 > NP-AR6 > NP-AR10. This demonstrates that the aspect ratio of PLGA nanorods significantly affects their uptake efficiency by tumor cells. Nanoparticles with smaller aspect ratios (such as NP-AR1 and NP-AR3) exhibit higher tumor cell internalization capabilities.

[0126] 4. Cytotoxicity test

[0127] To evaluate the inhibitory effects of free paclitaxel (PTX) and PLGA nanoparticles with different aspect ratios on tumor cells, this invention used the MTT assay to detect the survival rate of CT26 cells after 48 h of drug treatment and to evaluate the effect of paclitaxel nanoparticles with different aspect ratios on CT26 cell proliferation. The experimental results are shown in Figure 13. Compared with the free drug, the nanoparticles showed a significantly enhanced tumor-inhibiting effect. The nanoparticle NP-AR10, with the highest aspect ratio, exhibited lower tumor-inhibiting effects at all concentrations than nanoparticles with other aspect ratios due to less efficient tumor cell uptake. Specifically, NP-AR1 and NP-AR3 showed significantly better tumor-inhibiting effects than the NP-AR10 group at concentrations of 0.8 μg / mL, 2 μg / mL, and 5 μg / mL.

[0128] In summary, nanoparticles with relatively small aspect ratios (NP-AR1 and NP-AR3) exhibited higher cytotoxicity in CT26 cells. This result is consistent with the results of cellular uptake, indicating that nanoparticles with smaller aspect ratios are more easily taken up by tumor cells and therefore have better tumor-suppressing effects at the in vitro cellular level.

[0129] Experimental Example 2: Evaluation of the in vivo antitumor effect of paclitaxel nanomedicine

[0130] 1. In vivo tissue distribution of paclitaxel nanomedicines with different aspect ratios

[0131] To further investigate the in vivo tissue distribution behavior of paclitaxel nanomedicines with different aspect ratios, this invention constructed a CT26 tumor-bearing mouse model, labeled each group of nanoparticles with DiD fluorescent dye, and dynamically monitored and evaluated the biodistribution and tumor targeting efficiency of the fluorescent nanoparticles in the mice after tail vein injection using an in vivo imaging system.

[0132] As shown in Figure 14, NP-AR3 exhibited a strong fluorescence signal at the tumor site immediately after injection and maintained a high level for the next 12 hours, demonstrating excellent tumor targeting ability. While the fluorescence intensity of NP-AR6 at the tumor site was slightly lower than that of NP-AR3 at 0 h, it remained high throughout the observation period, indicating good tumor accumulation ability. In contrast, NP-AR1 and NP-AR10 showed relatively weak fluorescence signals at the tumor site within 48 h. To further verify the in vivo imaging results, major organs and tumor tissues were harvested 24 h after injection for ex vivo fluorescence imaging analysis. As shown in Figure 15, the liver and spleen showed the strongest fluorescence signals, while non-target organs such as the heart, lungs, and kidneys showed weaker fluorescence signals. The ex vivo tumor tissue imaging results further confirmed that the NP-AR3 group retained a significant fluorescence signal after 24 h, significantly superior to NP-AR1, NP-AR6, and NP-AR10.

[0133] In vivo tumor targeting results showed that NP-AR3 exhibited the highest targeting efficiency, likely due to the combined effects of macrophage and tumor cell uptake. In vitro cell experiments indicated that as the aspect ratio increased, the uptake of nanoparticles by both macrophages and tumor cells decreased. Therefore, NP-AR3, with its moderate aspect ratio, demonstrated better in vivo tumor targeting efficacy due to its ability to evade macrophage uptake and relatively good tumor cell uptake capacity.

[0134] In summary, the aspect ratio of PLGA nanoparticles has a crucial impact on their in vivo distribution behavior and tumor targeting efficiency. NP-AR3, with a moderate aspect ratio, achieves a better balance between blood circulation stability and tumor accumulation capacity, exhibiting more ideal in vivo delivery characteristics.

[0135] 2. Evaluation of the in vivo antitumor efficacy and safety of paclitaxel nanomedicines with different aspect ratios

[0136] To further compare the antitumor effects of PLGA nanoparticles with different aspect ratios in vivo, this invention systematically evaluated the therapeutic efficacy and systemic biosafety of paclitaxel nanomedicines with different aspect ratios in a CT26 tumor-bearing mouse model. Mice in each group were injected via tail vein with nanoformulations of different aspect ratios or clinical paclitaxel injection (Taxol). ® The drug was administered every 3 days for 14 days (as shown in Figure 16a). The changes in tumor length and width over time were monitored using vernier calipers, and the tumor volume was calculated and a tumor growth curve was plotted.

[0137] As shown in Figure 16b, the tumor volume growth curve results indicate that all paclitaxel nanomedicines can inhibit tumor growth to a certain extent, and the inhibitory effect is significantly better than that of free paclitaxel injection (Taxol). ® Among them, the NP-AR3 and NP-AR6 groups showed the most significant tumor-suppressing effects throughout the entire treatment cycle, with tumor volume consistently maintained at a low level, significantly superior to NP-AR1, NP-AR10, and Taxol. ® Groups. Figures 16c and 16e show tumor tissue images and tumor weight measurements at the terminal stage. These results further confirm that the NP-AR3 group had the lowest tumor weight, followed by NP-AR6, both of which were significantly lower than NP-AR10 and Taxol. ® Group.

[0138] The above results highlight the crucial role of geometry in regulating the delivery efficiency and therapeutic efficacy of nanomedicines. NP-AR3 nanoparticles with a moderate aspect ratio can both evade macrophage uptake and clearance to some extent and better promote tumor cell uptake, thus exhibiting better in vivo tumor targeting capabilities and exerting a stronger anti-tumor therapeutic effect.

[0139] During the treatment period, mouse weight changes were monitored. After treatment, mice were anesthetized, and blood samples were collected using the orbital venous plexus sampling method. After serum separation, biochemical indicators such as liver and kidney function were detected using a biochemical analyzer. Major organs, including the liver, spleen, lungs, and kidneys, were dissected, fixed in paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) to analyze histopathological changes and evaluate the biosafety of the nanoparticles. The results, as shown in Figure 16d, showed that the weight of mice in all treatment groups remained stable throughout the experimental period without significant decrease, indicating good tolerability of all formulations. As shown in Figure 17a, liver function (ALT, AST) and kidney function (BUN, CRE) indicators in all groups were within the normal physiological range and showed no significant difference compared to the PBS control group, indicating that the nanoparticle formulation did not cause significant hepatotoxicity or nephrotoxicity. Furthermore, as shown in Figure 17b, no significant histopathological changes were observed in the H&E-stained sections of the major organs, indicating that the constructed nanoplatform has good biocompatibility.

[0140] In summary, PLGA nanoparticles with different aspect ratios all exhibited good antitumor activity without inducing systemic toxicity, and the aspect ratio of the nanoparticles significantly affected their antitumor therapeutic effects. In the CT26 tumor-bearing mouse model, the NP-AR3 nanoparticle with a moderate aspect ratio achieved an optimal balance between evading macrophage uptake and promoting tumor cell uptake, thus exhibiting better in vivo tumor targeting effects and superior antitumor therapeutic capabilities.

[0141] The above results demonstrate the importance of aspect ratio optimization in tumor-targeted nanodelivery systems. The aspect ratio of the nanoparticle carrier is a key indicator that directly determines the anti-tumor therapeutic effect of nanoparticles by influencing immune clearance, tumor cell uptake, and tumor targeting. This invention emphasizes the significant impact of the aspect ratio as a physical parameter on anti-tumor therapy using non-spherical nanocarriers, providing important experimental evidence for constructing anti-tumor nanodelivery systems that combine high efficiency and safety.

Claims

1. A method for preparing polymer nanorods with controllable aspect ratio, characterized in that: It includes the following steps: (a) preparing an oil phase: dissolving the polymer in an organic solvent to prepare an oil phase; (b) preparing an emulsion: dissolving an emulsifier in water to prepare an aqueous phase 1; emulsifying the oil phase and aqueous phase 1 using ultrasonic emulsification to obtain an oil-in-water emulsion; (c) dissolving a deformation initiator in an emulsifier solution to prepare an aqueous phase 2; mixing the aqueous phase 2 with the prepared oil-in-water emulsion, removing the organic solvent to solidify the droplets, and ultrasonically washing to obtain a nanoparticle suspension; (d) heating the prepared nanoparticle suspension to break it down, and then washing and drying it to obtain polymer nanorods with uniform particle size and different aspect ratios.

2. The method for preparing polymer nanorods with controllable aspect ratio according to claim 1, characterized in that: The organic solvent in step (a) is dichloromethane, or a mixture of dichloromethane and acetone, dichloromethane and methanol, dichloromethane and dimethyl sulfoxide, or dichloromethane and acetonitrile; the polymer is polylactic acid-polyglycolic acid copolymer, or polylactic acid copolymer, or polylactic acid-polyglycolic acid-polyethylene glycol copolymer; the polymer concentration in the oil phase is 20 mg / mL. The emulsifier in step (b) is polyvinyl alcohol; the concentration of the emulsifier in aqueous phase 1 is 1.5%-4.0%; the ultrasonic conditions are an ice bath, with ultrasonication every 5 seconds at 30-200W power, followed by a 5-second pause, for a total duration of 2-10 min. The deformation initiator in step (c) is disodium hydrogen phosphate; the emulsifier solution is a polyvinyl alcohol aqueous solution with a mass concentration of 1.5%-5.0%; the organic solvent is removed by stirring at 500 rpm at room temperature for 4 minutes. h causes the organic solvent to evaporate; the ultrasonic washing conditions are as follows: centrifuge the nanoparticles obtained by solidifying the droplets, collect the nanoparticle precipitate, add pure water for washing, ultrasonically disperse, then centrifuge again, add pure water for ultrasonic dispersion, adjust the concentration, and obtain a nanoparticle suspension with a concentration of 1 mg / mL; the heating temperature in step (d) is 25℃-65℃, the heating time is 5 min-60 min, and the aqueous phase system is pure water during heating.

3. The method for preparing polymer nanorods with controllable aspect ratio according to claim 2, characterized in that: The organic solvent in step (a) is a mixed organic solvent of dichloromethane and acetone in a ratio of 4:1; the concentration of the emulsifier in aqueous phase 1 in step (b) is 1.5%, the ultrasonic power is 120W, and the total ultrasonic duration is 2 min; the emulsifier solution in step (c) is a polyvinyl alcohol aqueous solution with a mass concentration of 3%.

4. A method for preparing an antitumor nanomedicine, characterized in that: It is prepared by the method for preparing polymer nanorods with controllable aspect ratio as described in claim 1 or 2, wherein an antitumor drug is added to the oil phase in step (a).

5. The method for preparing antitumor nanomedicine according to claim 4, characterized in that: The antitumor drugs mentioned include hydrophobic drugs.

6. The method for preparing antitumor nanomedicine according to claim 5, characterized in that: The hydrophobic drugs mentioned include paclitaxel, docetaxel, demineralized doxorubicin, gemcitabine, etoposide, vincristine, vinorelbine, temozolomide, carmustine, camptothecin, retinoic acid, all-trans retinoic acid, and cabazitaxel.

7. A paclitaxel nanomedicine, characterized in that: It is prepared by the method of preparing antitumor nanomedicine according to any one of claims 4-6, wherein the paclitaxel nanomedicine has a major diameter of 118-820 nm, a minor diameter of 66-191 nm, an aspect ratio of 1-12, and a drug loading rate of 1.7%-4.3%.

8. The paclitaxel nanomedicine according to claim 7, characterized in that: The paclitaxel nanomedicine has a major diameter of 250.03±94.26 nm, a minor diameter of 92.58±24.40 nm, an aspect ratio of 2.75±0.85, and a drug loading rate of 2.5%.

9. A method for preparing the paclitaxel nanomedicine according to claim 7 or 8, characterized in that: It includes the following steps: (a) Preparing the oil phase: dissolving polylactic acid-polyglycolic acid copolymer and paclitaxel in a mixed organic solvent of dichloromethane:acetone = 4:1 to prepare the oil phase, with a polymer concentration of 20 mg / mL and a paclitaxel concentration of 0.8 mg / mL-1.2 mg / mL; preferably, the paclitaxel concentration is 1.0 mg / mL, and the drug loading rate at this concentration is 2.5%; (b) Preparing the emulsion: dissolving the emulsifier polyvinyl alcohol in water to prepare aqueous phase 1, with an emulsifier concentration of 1.5% in aqueous phase 1; emulsifying the oil phase and aqueous phase 1 by ultrasonic emulsification under ice bath conditions, at 120W power, for 5 seconds of ultrasonication followed by a 5-second pause, for a total duration of 2 minutes, to obtain an oil-in-water emulsion; (c) dissolving the deformation initiator disodium hydrogen phosphate in the emulsifier solution, with the emulsifier solution having a mass concentration of 3%. Aqueous phase 2 is prepared by mixing a polyvinyl alcohol aqueous solution with the prepared oil-in-water emulsion and stirring at 500 rpm for 4 h at room temperature to allow the organic solvent to evaporate. The ultrasonic washing conditions are as follows: centrifuge the nanoparticles obtained by solidifying the droplets, collect the nanoparticle precipitate, add pure water for washing, ultrasonically disperse, then centrifuge again, add pure water for ultrasonic dispersion, adjust the concentration to obtain a nanoparticle suspension with a concentration of 1 mg / mL; (d) heat the obtained nanoparticle suspension to break it up, the heating temperature is 25℃-65℃, the heating time is 5 min-60 min, and then wash and dry to obtain polymer nanorods with uniform particle size and different aspect ratios; preferably, the heating time in (d) is 5 min-10 min.

10. Use of the paclitaxel nanomedicine of claim 7 or 8 in the preparation of medicaments for treating breast cancer, lung cancer, colorectal cancer, cervical cancer, ovarian cancer, liver cancer, head and neck cancer, and melanoma.

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