Hyaluronic acid-modified CPD12C15-loaded PLGA (poly (lactic-co-glycolic acid)) nano preparation as well as preparation method and application thereof

Abstract

The invention relates to a drug nano delivery system, in particular to a hyaluronic acid modified CPD12C15 loaded PLGA (poly (lactic-co-glycolic acid)) nano preparation as well as a preparation method and application thereof. The nano preparation comprises PLGA, CPD12C15 and hyaluronic acid, PLGA forms a nuclear layer structure, CPD12C15 is encapsulated in the nuclear layer structure of PLGA, hyaluronic acid forms a modification layer covering the surface of the nuclear layer structure through electrostatic adsorption, and the structure of CPD12C15 is # imgabs0. When the nano preparation is combined with copper ions for use, the aerobic glycolysis pathway of pancreatic cancer cells can be remarkably inhibited, and the pancreatic cancer cells can be effectively inhibited. Meanwhile, cell dryness and drug resistance are inhibited, and the in-vivo tumor inhibition rate reaches 66.10% and is superior to that of a clinical first-line drug gemcitabine; moreover, adverse reactions such as myelosuppression are not caused, and the safety is remarkably improved. The invention provides an efficient and safe new strategy for pancreatic cancer treatment.

Description

Technical Field

[0001] The present application relates to the field of medicine, and in particular to a hyaluronic acid-modified PLGA nanoformulation loaded with CPD12C15, a preparation method thereof, and an application thereof. Background Art

[0002] Any discussion of the prior art throughout the specification should not be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0003] Pancreatic cancer is a malignant digestive tract tumor with a dismal prognosis, characterized by insidious onset and rapid progression. Current clinical data indicate a five-year survival rate of less than 10%, with nearly 470,000 deaths, ranking among the top ten leading causes of death from malignant tumors worldwide. Due to the relatively hidden location of the pancreas in the human body, early diagnosis is limited. Furthermore, pancreatic cancer has a complex tumor microenvironment, including a dense tumor stroma and distorted tissue architecture, resulting in low blood flow and high intratumoral pressure, limiting drug accumulation at the tumor site.

[0004] Currently, clinical treatments for pancreatic cancer primarily include surgery, chemotherapy, radiotherapy, immunotherapy, and targeted therapy. However, approximately 80% of patients are diagnosed with advanced disease or have distant metastases, making surgical resection impossible. Surgery, while a potential cure, is only suitable for less than 10% of patients. Gemcitabine (GEM) is a first-line chemotherapy drug for pancreatic cancer, primarily targeting tumor cells in the G1 / S phase through its metabolites. While GEM plays an important role in pancreatic cancer treatment, its clinical use faces two major challenges: first, the development of drug resistance during treatment; second, GEM treatment is often associated with adverse reactions such as bone marrow suppression and hepatotoxicity, which limit its clinical effectiveness. Research has shown that tumor cells often exhibit abnormal energy metabolism, known as the "Warburg effect." In normal cells, energy is primarily generated through aerobic oxidation of glucose; however, tumor cells, even under aerobic conditions, primarily generate energy through glycolysis. This altered metabolic pattern not only provides the energy necessary for tumor cell proliferation but also creates a microenvironment conducive to tumor growth.

[0005] Disulfiram (DSF) is an FDA-approved drug for alcohol abstinence. Recent studies have shown that it exhibits anti-tumor potential in combination with copper ions in the treatment of various tumors. However, DSF's poor water solubility, rapid metabolism, and neurotoxicity limit its application in the anti-tumor field.

[0006] Traditional drugs often face challenges during treatment, such as low bioavailability, poor targeting, and significant toxic side effects. With the advancement of drug delivery technologies, various novel delivery systems have been developed to improve drug efficacy and safety. However, for solid tumors such as pancreatic cancer, existing delivery strategies still face numerous challenges due to their dense tumor stroma and complex tumor microenvironment.

[0007] In summary, the development of new pancreatic cancer treatment strategies, especially those that can overcome the limitations of existing treatment methods, is of great significance to improving the prognosis and quality of life of pancreatic cancer patients. Summary of the Invention

[0008] The purpose of the present invention is to provide a novel anti-pancreatic cancer drug delivery system in order to solve some problems existing in existing pancreatic cancer treatment drugs.

[0009] Specifically, the technical problems that the present invention attempts to solve include at least one of the following: the drug resistance problem that may arise from the pancreatic cancer treatment drug gemcitabine (GEM); the poor water solubility and low bioavailability of disulfiram (DSF) drugs in the body; the neurotoxicity safety issues that may be caused by DSF drugs; the targeted delivery efficiency of drugs in pancreatic cancer tissue needs to be improved; and the toxic side effects such as bone marrow suppression that may be caused by pancreatic cancer treatment drugs.

[0010] The hyaluronic acid-modified PLGA nanoformulation (HPC-NP) loaded with CPD12C15 provided by the present invention, or its combined use with copper ions, may achieve at least one of the following technical effects: it helps to improve the in vitro release characteristics of CPD12C15 and can exhibit better release performance under specific pH conditions; hyaluronic acid modification can enhance the targeting ability of the nanoformulation to pancreatic cancer cells expressing CD44 receptors, which helps to improve the accumulation of drugs at the tumor site; HPC-NP combined with copper ions can show a good inhibitory effect on pancreatic cancer cells and can show a lower IC in specific cell lines. 50 value; in animal experiments, the combined use of HPC-NPs and copper ions can show a certain inhibitory effect on pancreatic cancer without causing severe adverse reactions such as bone marrow suppression; HPC-NPs combined with copper ions can have a certain effect on the aerobic glycolysis pathway of pancreatic cancer cells, and can also affect factors related to cell stemness and drug resistance.

[0011] According to the experimental data of the present invention, when the nanoformulation is used in combination with copper ions, the IC values of the tested PANC-1 and HS766T pancreatic cancer cell lines are significantly higher than those of the conventional nanoformulation. 50 values can be as low as approximately 0.13 μM and 0.28 μM, respectively, while the IC values of GEMs tested under the same conditions 50The values were approximately 27.98 μM and 21.36 μM. In the established animal model, the tumor inhibition rate of the HPC-NP combined with copper ions group was approximately 66.10%, while that of the GEM group was approximately 45.95%.

[0012] In summary, the present invention provides a novel hyaluronic acid-modified PLGA nanoformulation loaded with CPD12C15, which may help to enhance the anti-pancreatic cancer efficacy and improve safety, providing a potential new direction for the treatment of pancreatic cancer.

[0013] Specifically, the present invention provides the following technical solutions.

[0014] In a first aspect of the present invention, a hyaluronic acid-modified PLGA nanoformulation loaded with CPD12C15 is provided, wherein the nanoformulation comprises PLGA, CPD12C15, and hyaluronic acid, wherein the PLGA forms a core layer structure, the CPD12C15 is encapsulated within the PLGA core layer structure, and the hyaluronic acid forms a modified layer covering the surface of the core layer structure by electrostatic adsorption, wherein the structure of CPD12C15 is:

[0015]

[0016] This core-shell structure design helps improve the water solubility and stability of CPD12C15 while providing a basis for targeted delivery.

[0017] In some embodiments of the present invention, the mass ratio of CPD12C15 to PLGA is 1:2 to 1:6, preferably 1:2 to 1:4, and more preferably 1:2. The mass ratio of CPD12C15 to PLGA directly affects drug loading efficiency and release characteristics. Experimental data show that a 1:2 ratio can achieve a high drug loading capacity (approximately 17%) while maintaining a good encapsulation efficiency (approximately 70%). This helps maximize drug content while minimizing the amount of carrier material used, thereby improving drug delivery efficiency.

[0018] In some embodiments of the present invention, the hyaluronic acid binds to the nanoparticle surface through electrostatic interactions with positively charged groups on the PLGA surface. This electrostatic adsorption modification method, which eliminates the need for complex chemical coupling reactions, is simple to operate and highly reproducible. It also maintains the structural integrity of the hyaluronic acid molecules, ensuring their specific recognition of the CD44 receptor and enhancing the nanoparticle's targeting of pancreatic cancer cells.

[0019] In some embodiments of the present invention, the molecular weight of the hyaluronic acid is 5,000-250,000 Daltons, preferably 6,000-240,000 Daltons, and more preferably 6,000 Daltons. The molecular weight of hyaluronic acid significantly affects the stability and targeting ability of the nanoparticles. Experimental studies have shown that hyaluronic acid with a lower molecular weight (6,000) can form nanoparticles with a more uniform particle size distribution, avoiding the aggregation and precipitation of nanoparticles caused by higher molecular weight hyaluronic acid, while still maintaining targeted binding ability to the CD44 receptor.

[0020] In some embodiments of the present invention, the nanoformulation has a particle size of 50-200 nm, preferably 100-150 nm, and more preferably 130-150 nm. This particle size range offers multiple advantages: Firstly, it is larger than the renal filtration threshold (approximately 10 nm), preventing rapid elimination; secondly, it is smaller than 200 nm, effectively utilizing the EPR effect of tumor tissue for passive targeting. In particular, a particle size of 130-150 nm is more conducive to maintaining blood circulation stability and tumor tissue accumulation, while also facilitating CD44-mediated cellular endocytosis.

[0021] In some embodiments of the present invention, the nanoformulation has a zeta potential of -5 to -30 mV, preferably -15 to -20 mV, and more preferably -16 to -18.5 mV. An appropriately negative potential imparts excellent dispersion stability to the nanoparticles. Experimental studies have shown that a potential range of -16 to -18.5 mV can effectively prevent nanoparticle aggregation in physiological environments and prolong their blood circulation time. Furthermore, this moderate negative charge helps reduce nonspecific cellular uptake and enhance CD44 targeting specificity.

[0022] In some embodiments of the present invention, the nanoformulation has an encapsulation efficiency of 50%-85%, preferably 60-80%, more preferably 65-75%, and most preferably 69-72%. This high encapsulation efficiency reflects the effectiveness of the preparation process and significantly reduces drug waste. Experiments have shown that an encapsulation efficiency of 69-72% enables efficient loading and controlled release of CPD12C15, demonstrating the excellent encapsulation capability of the emulsified solvent evaporation method for the hydrophobic drug CPD12C15.

[0023] In some embodiments of the present invention, the drug loading of the nanoparticle formulation is 10%-25%, preferably 16-18%, and more preferably 17-18%. A higher drug loading can reduce the amount of carrier material used and reduce potential toxicity. Experimental data show that a drug loading of 17-18% can ensure sufficient drug dosage while maintaining the stability and uniformity of the nanoparticles, providing a reasonable drug delivery system for clinical applications.

[0024] In some embodiments of the present invention, the nanoparticles have a particle size of 120-125 nm, a Zeta potential of -15 to -18 mV, an encapsulation efficiency of 69%-71%, and a drug loading of 16%-18%. This set of parameters represents the characteristics of a relatively optimized nanoparticle formulation. In vitro experiments have shown that nanoparticles with these characteristics exhibit optimal cellular uptake efficiency and anti-tumor activity, with an IC of 1. 50 The value (about 0.13 μM) was significantly lower than that of other preparation conditions; at the same time, the nanoparticles with this set of characteristic parameters showed the best tumor targeting and anti-tumor effect in in vivo experiments, with a tumor inhibition rate of up to 66.10%.

[0025] In a second aspect of the present invention, a method for preparing the hyaluronic acid-modified CPD12C15-loaded PLGA nanoformulation described in the first aspect is provided, comprising:

[0026] CPD12C15 and PLGA were dissolved in an organic solvent to form an organic phase;

[0027] Dissolving the cationic surfactant in the aqueous phase and mixing it with the organic phase to form an emulsion;

[0028] The organic solvent is removed to form PLGA nanoparticles;

[0029] The PLGA nanoparticles are brought into contact with a hyaluronic acid aqueous solution, so that the hyaluronic acid is modified on the surface of the PLGA nanoparticles by electrostatic adsorption, thereby obtaining a hyaluronic acid-modified PLGA nanoformulation loaded with CPD12C15.

[0030] This two-step preparation process can effectively avoid the solubility problem of hyaluronic acid in organic solvents and ensure the uniformity and stability of surface modification.

[0031] In some embodiments of the present invention, the organic solvent is selected from one or a mixture of dichloromethane, chloroform, ethyl acetate or acetone, preferably dichloromethane.

[0032] In some embodiments of the present invention, the cationic surfactant is selected from dodecyltrimethylammonium bromide (DTAB), cetyltrimethylammonium bromide (CTAB) or a combination thereof; preferably dodecyltrimethylammonium bromide (DTAB).

[0033] In some embodiments of the present invention, the mass ratio of CPD12C15 to PLGA is 1:2 to 1:6, preferably 1:2 to 1:4, and more preferably 1:2.

[0034] In some embodiments of the present invention, the volume ratio of the organic phase to the aqueous phase is 1:2 to 1:5, preferably 1:2.

[0035] In some embodiments of the present invention, the concentration of PLGA is 5-25 mg / mL, preferably 10-20 mg / mL, and more preferably 15 mg / mL.

[0036] In some embodiments of the present invention, the concentration of the cationic surfactant is 0.5%-2.5%, preferably 0.5%-1.5%, and more preferably 0.5%.

[0037] In some embodiments of the present invention, the molecular weight of the hyaluronic acid is 5,000-250,000 Daltons, preferably 6,000-240,000 Daltons, and more preferably 6,000 Daltons.

[0038] In some embodiments of the present invention, the concentration of the hyaluronic acid is 0.05-2 mg / mL, preferably 0.1-1.5 mg / mL, and preferably 0.1 mg / mL.

[0039] In some embodiments of the present invention, the organic phase and the aqueous phase are mixed by one or more of ultrasound, high-speed stirring, or high-pressure homogenization.

[0040] In some embodiments of the present invention, the organic solvent is removed by volatilization under stirring at room temperature, distillation under reduced pressure, or dialysis. The volatilization under stirring at room temperature is simple to operate, facilitates the slow formation of nanoparticles, and can obtain nanoparticles with more regular morphology and more uniform distribution.

[0041] In some embodiments of the present invention, the stirring time at room temperature after mixing to form the emulsion is 3-8 hours, preferably 4-6 hours, and more preferably 5 hours. Sufficient stirring time helps the organic solvent to completely volatilize, ensures the stability of the nanoparticle structure, and avoids the toxicity that may be caused by residual organic solvent.

[0042] In some embodiments of the present invention, the stirring time after mixing the PLGA nanoparticles with the hyaluronic acid aqueous solution is 0.5-2 hours, preferably 1 hour. An appropriate stirring time can ensure that the hyaluronic acid is fully adsorbed on the surface of the nanoparticles while avoiding structural damage that may be caused by excessive stirring.

[0043] In some embodiments of the present invention, the prepared nanoformulation is filtered using a microporous membrane having a pore size of 0.22-0.45 μm. The filtration step helps remove possible large particle aggregates and impurities, thereby improving the uniformity and stability of the nanoformulation.

[0044] In some embodiments of the present invention, the organic phase is added dropwise to the aqueous phase under ultrasonic conditions at a power of 100-500 W for 2-5 minutes, preferably 3 minutes. Appropriate ultrasonic conditions facilitate the formation of nanoparticles with uniform particle size distribution, improving the repeatability and controllability of the preparation.

[0045] For example, in one specific embodiment, the method includes:

[0046] a) dissolving CPD12C15 and PLGA in an organic solvent (such as dichloromethane) to form an organic phase;

[0047] b) dissolving dodecyltrimethylammonium bromide (DTAB) in the aqueous phase (e.g., at a DTAB concentration of 0.5%);

[0048] c) adding the organic phase dropwise to the aqueous phase under ultrasonic conditions to form colostrum (e.g., the volume ratio of the oil phase to the aqueous phase is 1:2, and the concentration of PLGA is 15 mg / mL);

[0049] d) stirring at room temperature to volatilize the organic solvent to form PLGA nanoparticles;

[0050] e) adding the above PLGA nanoparticles dropwise into a hyaluronic acid aqueous solution (for example, a hyaluronic acid having a molecular weight of 6,000 and a concentration of 0.1 mg / mL), stirring and electrostatically adsorbing to obtain a hyaluronic acid-modified PLGA nanoformulation loaded with CPD12C15.

[0051] In the third aspect of the present invention, a pharmaceutical composition is provided, which at least comprises the nanoformulation described in the first aspect.

[0052] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable copper salt, which is selected from one or more of copper gluconate, copper chloride, and copper sulfate, preferably copper gluconate.

[0053] In some embodiments of the present invention, the molar ratio of the nanoformulation to copper ions is 0.5:1 to 1.5:1, preferably 1:1.

[0054] In a fourth aspect, the present invention provides a medical kit comprising: a first container containing the nanoformulation according to any one of claims 1 to 4; and a second container containing a pharmaceutically acceptable copper salt solution; the two containers are designed for simultaneous, separate, or sequential administration. Furthermore, the kit includes instructions for directing a dosing regimen.

[0055] In a third aspect, the present invention provides the use of the hyaluronic acid-modified CPD12C15-loaded PLGA nanoformulation described in the first aspect in the preparation of a drug for treating pancreatic cancer. The drug exerts an anti-tumor effect by inhibiting the aerobic glycolysis pathway of pancreatic cancer cells.

[0056] In some embodiments of the present invention, the drug is used to treat gemcitabine-resistant pancreatic cancer. Experiments have shown that the nanoformulation can significantly inhibit the expression of the multidrug resistance protein MRP1, potentially overcoming pancreatic cancer's resistance to conventional chemotherapy drugs.

[0057] In some embodiments of the present invention, the nanoformulation is used in combination with copper ions, wherein the copper ions are divalent copper ions. The interaction between CPD12C15 and divalent copper ions can enhance its activity in inhibiting PDK, thereby synergistically inhibiting the aerobic glycolysis pathway of pancreatic cancer cells.

[0058] In some embodiments of the present invention, the divalent copper ion is copper ion provided by copper chloride, copper sulfate, or copper gluconate, preferably copper ion provided by copper gluconate. Copper gluconate has good biocompatibility, is suitable for oral administration, and can reduce the irritation of copper ions to the gastrointestinal tract.

[0059] In some embodiments of the present invention, the molar ratio of the nanoformulation to copper ions is 0.5:1 to 1.5:1, preferably 1:1. Experimental data show that a molar ratio of 1:1 can achieve a better synergistic anti-tumor effect, ensuring sufficient activity while reducing the potential toxic effects of copper ions.

[0060] In some embodiments of the present invention, the nanoformulation is administered intravenously at a dose of 5-15 mg / kg, preferably 10 mg / kg, calculated as CPD12C15; the copper ion is administered orally in the form of copper gluconate at a dose of 1-5 mg / kg, preferably 2 mg / kg, calculated as copper gluconate. Intravenous administration can improve the bioavailability of the nanoformulation, while oral administration facilitates the absorption and utilization of the copper ion.

[0061] In some embodiments of the present invention, the nanoformulation, when used in combination with copper ions, can inhibit the expression of key glycolytic enzymes, PDK1, GLUT1, HKII, and LDHA, in pancreatic cancer cells. Experimental results show that this combination significantly reduces the expression levels of these key glycolytic enzymes, thereby cutting off the energy supply to tumor cells.

[0062] In some embodiments of the present invention, the nanoformulation combined with copper ions can reduce lactate levels, glucose uptake, and ATP production in pancreatic cancer cells, while increasing ROS levels. These metabolic changes help inhibit tumor cell proliferation and viability.

[0063] In some embodiments of the present invention, the nanoformulation, when used in combination with copper ions, can inhibit the expression of the stemness markers Nanog, OCT-4, and SOX-2 in pancreatic cancer cells. Stemness is closely associated with tumor recurrence and metastasis, and inhibiting the expression of these markers is expected to reduce the risk of pancreatic cancer recurrence.

[0064] In some embodiments of the present invention, the nanoformulation combined with copper ions can inhibit the expression of the multidrug resistance protein MRP1 in pancreatic cancer cells. MRP1 is an important member of the ATP transporter family, and its inhibition helps enhance the sensitivity of tumor cells to chemotherapy drugs.

[0065] The experimental results showed that the combination of HPC-NPs prepared by the above method and copper ions had an IC of 1.547 and 1.571 for PANC-1 and HS766T pancreatic cancer cells. 50 The values were 0.13μM and 0.28μM, respectively, significantly lower than the 27.98μM and 21.36μM of the commonly used clinical drug gemcitabine (GEM). In a nude mouse subcutaneous tumor model, the tumor inhibition rate of the HPC-NP combined with copper ions group reached 66.10%, significantly better than the 45.95% of the GEM group, and did not cause adverse reactions such as bone marrow suppression, demonstrating good safety and anti-tumor efficacy.

[0066] Compared with the prior art, the advantages of the present invention include:

[0067] The hyaluronic acid-modified CPD12C15-loaded PLGA nanoformulation (HPC-NP) provided by the present invention and its overall technical solution of combined use with copper ions have many beneficial effects and advantages compared with the existing technology.

[0068] The present invention successfully improved the water solubility problem of CPD12C15 by designing a core-shell structure in which CPD12C15 is encapsulated in a PLGA core and surface-modified with hyaluronic acid. The results of in vitro release experiments showed that the cumulative release rate of the nanoformulation reached 50.69% in 0.5 hours under pH 5.5 conditions, and the drug can be continuously released in different pH environments, effectively solving the problem of poor water solubility of DSF drugs. At the same time, hyaluronic acid modification enables the nanoparticles to bind to the CD44 receptor expressed on pancreatic cancer cells, thereby achieving targeted delivery to pancreatic cancer. In vivo live imaging experiments showed that the accumulation of HPC-NP in the tumor site was significantly higher than that of unmodified nanoparticles and free drugs, reaching a peak fluorescence intensity in 8 hours, and a significant signal could still be observed after 24 hours.

[0069] In terms of anti-tumor effect, the technical solution of the present invention exhibits excellent performance. In vitro experiments have shown that the IC50 values of HPC-NP combined with copper ions for PANC-1 and HS766T pancreatic cancer cells were 0.13μM and 0.28μM, respectively, which are significantly lower than the 27.98μM and 21.36μM of the commonly used clinical drug GEM. Cell scratch experiments showed that the scratch healing rates of the HPC-NP / Cu group on PANC-1 and HS766T cells were approximately 13.83% and 6.00%, respectively, while the scratch healing rates of the Control group were 40.70% and 34.65%, respectively, proving that it effectively inhibited cell migration ability. In the nude mouse subcutaneous tumor model, the tumor inhibition rate of the group using HPC-NP combined with copper ions reached 66.10%, which was significantly better than the 45.95% of the GEM group. The tumor mass weight was only 0.19±0.09g, while that of the Model group was 0.62±0.11g.

[0070] Safety evaluation shows that the technical solution of the present invention has obvious advantages. Hemolysis experiments show that the hemolysis rate is less than 5% in the concentration range of 0.5-40μg / mL, indicating good blood compatibility. In animal experiments, unlike the GEM group, the group using HPC-NP in combination with copper ions did not cause bone marrow suppression, and the levels of neutrophils and platelets were not significantly different from those in the Model group. The number of nucleated cells in the bone marrow remained at a normal level, and the levels of TPO and EPO in the serum did not show a significant decrease. The liver and kidney function indicators ALT, AST, BUN and Cr were all maintained within the normal range, further demonstrating the good safety of this technical solution.

[0071] Mechanism of action studies have shown that the technical solution of the present invention exerts its effects through multiple pathways. Western Blot and RT-PCR experiments have shown that the combined use of HPC-NP and copper ions significantly inhibited the expression of key glycolysis enzymes such as GLUT1, PDK1, HKⅡ and LDHA. Functional experiments have shown that this technical solution can reduce the lactate content, glucose uptake and ATP production levels in pancreatic cancer cells, while increasing the ROS content, successfully inhibiting the aerobic glycolysis pathway. In addition, experiments have also shown that the combined use of HPC-NP and copper ions can significantly reduce the expression of cell stemness markers such as Nanog, OCT-4 and SOX-2, as well as the MRP1 multidrug resistance protein, effectively inhibiting cell stemness and drug resistance.

[0072] In summary, the technical solution provided by the present invention as a whole provides a new and effective strategy for the treatment of pancreatic cancer by improving drug water solubility, enhancing targeted delivery, improving anti-tumor activity, reducing toxic side effects, and achieving multi-target synergy, with obvious technological advancement and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] The drawings constituting part of the present application are provided to provide a further understanding of the present application. The illustrative embodiments and their descriptions of the present application are provided to explain the present application and do not constitute an undue limitation on the present application. The following describes the implementation scheme of the present application in detail in conjunction with the drawings, wherein:

[0074] Figure 1 : Schematic diagram of the preparation of HPC-NPs.

[0075] Figure 2 : Single factor investigation on the effect of drug loading capacity and encapsulation efficiency of PC-NP.

[0076] Figure 3 : Particle size distribution and zeta potential distribution of HPC-NPs.

[0077] Figure 4 : Transmission electron microscopy (TEM) image of HPC-NPs, scale bar 0.3 μm.

[0078] Figure 5 : Hemolysis test results of HPC-NPs, including (a) hemolysis photos of HPC-NPs at different concentrations; (b) hemolysis rates of HPC-NPs at different concentrations.

[0079] Figure 6 : In vitro release results of HPC-NPs under different pH conditions.

[0080] Figure 7 : Fluorescence distribution images at different time points after tail vein injection of HPD-NP (a) and DiR (b) in nude mice.

[0081] Figure 8 : Qualitative analysis results of PANC-1 cell uptake (scale bar = 200 μm).

[0082] Figure 9 : Qualitative analysis results of HS766T cell uptake (scale bar = 200 μm).

[0083] Figure 10 : Flow cytometric analysis results of PANC-1 and HS766T cell uptake.

[0084] Figure 11 : Effects of each group on the proliferation of pancreatic cancer PANC-1 and HS766T cells (n=3). (a) Effects of CuCl2·2H2O and Free-NP on the proliferation of PANC-1 and HS766T cells; (b) Effects of C15 and its nanoparticle preparation combined with copper on the proliferation of PANC-1 and HS766T cells.

[0085] Figure 12: Effects of different concentrations of GEM on the proliferation ability of pancreatic cancer PANC-1 and HS766T cells (n=3).

[0086] Figure 13 : Cell cloning experiments investigated the effects of drug intervention on the spheroid formation ability of pancreatic cancer PANC-1 (a) and HS766T (b) cells (n=3). ***P<0.001, indicating significant differences compared with the HPC-NP / Cu group.

[0087] Figure 14 : Experimental results of inhibition of PANC-1 (a) and HS766T (b) cell migration by C15 / Cu, PC-NP / Cu, HPC-NP / Cu, and GEM (Scale bar = 500 μm). n = 3, ***P < 0.001, indicating significant difference compared with the HPC-NP / Cu group.

[0088] Figure 15 : Experimental results of inhibition of PANC-1 (a) and HS766T (b) cell invasion by C15 / Cu, PC-NP / Cu, HPC-NP / Cu, and GEM (scale bar = 500 μm). n = 3, ***P < 0.001, indicating significant difference compared with the HPC-NP / Cu group.

[0089] Figure 16 : Flow cytometry analysis of PANC-1 (a) and HS766T (b) cell apoptosis (48 h). n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, indicating significant differences compared with the HPC-NP / Cu group.

[0090] Figure 17 RT-PCR experiments investigated the mRNA expression of key glycolytic enzymes GLUT1, HKⅡ, PDK1, and LDHA in pancreatic cancer PANC-1 (a) and HS766T (b) cells after drug intervention. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, indicating significant differences compared with the HPC-NP / Cu group.

[0091] Figure 18 Western blot analysis revealed changes in protein expression of key glycolytic enzymes, GLUT1, HKⅡ, PDK1, and LDHA, in pancreatic cancer cells PANC-1 (a) and HS766T (b) following drug intervention. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, indicating significant differences compared with the HPC-NP / Cu group.

[0092] Figure 19: Effects of each drug group on lactate content in pancreatic cancer PANC-1 and HS766T cells. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, indicating significant differences compared with the HPC-NP / Cu group.

[0093] Figure 20 : Effects of each drug group on glucose uptake in pancreatic cancer PANC-1 and HS766T cells. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 indicate significant differences compared with the HPC-NP / Cu group.

[0094] Figure 21 Effects of C15 / Cu, PC-NP / Cu, and HPC-NP / Cu on ATP production in pancreatic cancer PANC-1 and HS766T cells. n = 3, *P < 0.05, ***P < 0.001, indicating significant differences compared with the HPC-NP / Cu group.

[0095] Figure 22 Effects of C15 / Cu, PC-NP / Cu, and HPC-NP / Cu on ROS levels in pancreatic cancer PANC-1 (a) and HS766T (b) cells. n = 3, ***P < 0.001, indicating significant differences compared with the HPC-NP / Cu group.

[0096] Figure 23 RT-PCR experiments examined the mRNA expression changes of the stemness markers OCT-4, Nanog, and SOX-2 in pancreatic cancer cells PANC-1 (a) and HS766T (b) after drug intervention. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, indicating significant differences compared with the HPC-NP / Cu group.

[0097] Figure 24 Western blot analysis revealed changes in the expression of stemness markers OCT-4, Nanog, and SOX-2 in pancreatic cancer PANC-1 (a) and HS766T (b) cells after drug treatment. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 indicates significant differences compared with the HPC-NP / Cu group.

[0098] Figure 25 Effects of each drug-treated group on the mRNA and protein expression of MRP1 in pancreatic cancer cells PANC-1 (a) and HS766T (b) cells. n = 3, *P < 0.05, **P < 0.01, indicating significant differences compared with the HPC-NP / Cu group.

[0099] Figure 26: Schematic diagram of the mechanism of action of HPC-NP.

[0100] Figure 27 :Body weight and tumor mass changes in nude mice (n=5)

[0101] Figure 28 : H&E staining results of tumor tissue in a nude mouse subcutaneous tumor model of pancreatic cancer, scale 100×.

[0102] Figure 29 : TUNEL apoptosis results of pancreatic cancer subcutaneous tumor in nude mice model, scale 100×.

[0103] Figure 30 Immunohistochemical results of tumor tissue from a nude mouse subcutaneous pancreatic cancer model. n = 3, ***P < 0.001, indicating significant differences compared to the HPC-NP / Cu group. (a) Immunohistochemical image of tumor tissue from a nude mouse subcutaneous pancreatic cancer model (scale 100×); (b) Quantitative expression of immunohistochemical expression in tumor tissue from a nude mouse subcutaneous pancreatic cancer model.

[0104] Figure 31 : H&E staining results of different groups (scale 100×).

[0105] Figure 32 : Mouse body weight curve. n=5, ***P<0.001, indicating significant difference compared with the Model group.

[0106] Figure 33 : The number of neutrophils, platelets, and bone marrow nucleated cells in nude mice after drug treatment. n = 5, **P < 0.01, ***P < 0.001, indicating significant differences compared with the Model group.

[0107] Figure 34 : Serum TPO and EPO levels in different groups. n=5, *P<0.05, **P<0.01, indicating significant differences compared with the Model group.

[0108] Figure 35 : Results of the test of serum biochemical indicators ALT, AST, BUN, and Cr in mice. n = 5, *P < 0.05, ***P < 0.001, indicating significant differences compared with the Model group. DETAILED DESCRIPTION

[0109] The present application will be further described in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope thereof. Experimental methods for which specific conditions are not specified in the examples are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0110] Unless otherwise defined, all technical terms and scientific terms used in this application should have the meaning familiar to those skilled in the art. Unless otherwise specified, the reagents or raw materials used in this application can be obtained by conventional means and used in accordance with conventional methods or product specifications in this area. In addition, any content similar to or equivalent to the methods or materials described can be applied to the methods of this application. The preferred embodiments and materials described in this application are for illustrative purposes only.

[0111] CPD12C15 was prepared according to the method disclosed in Chinese patent CN116444408A with a purity of >95%. Unless otherwise specified, C15 mentioned in the examples and figures is the abbreviation of CPD12C15.

[0112] Example 1 Preparation and Characterization of Hyaluronic Acid-Modified PLGA Nanoformulations Loaded with CPD12C15 (HPC-NPs)

[0113] 1. Preparation method and process optimization

[0114] 1.1 Basic preparation method:

[0115] HPC-NPs were prepared using an emulsification solvent evaporation method and electrostatic adsorption. PLGA and CPD12C15 were weighed into a 10 mL centrifuge tube, and an organic solvent was added. Dissolved by ultrasonication and thoroughly mixed. A cationic surfactant was weighed and dissolved in ultrapure water. The organic phase was slowly added dropwise to the aqueous phase under ultrasonic conditions, and ultrasonication was performed for 3 minutes. The mixture was stirred at room temperature for 5 hours. After the organic solvent evaporated, PC-NPs (PLGA nanoparticles) were formed and filtered through a 0.22 μm microporous filter membrane.

[0116] Then, at room temperature, the prepared PC-NP (PLGA nanoparticles) solution was added dropwise to the HA aqueous solution, and stirring was ensured while adding, and then stirring was continued for 1 hour. After filtering through a 0.45 μm microporous filter membrane, HPC-NP was obtained. The specific preparation process is as follows Figure 1 shown.

[0117] 1.2 Nanoformulation Process Optimization

[0118] PC-NP and HPC-NP were prepared according to the aforementioned preparation methods, as shown in Preparation Examples 1-24 and Tables 1 and 2.

[0119] Table 1 Preparation Examples 1-18 (Preparation of PC-NP)

[0120]

[0121] Table 2 Preparation Examples 19-24 (Preparation of HPC-NPs)

[0122]

[0123] 1.3 Preparation process optimization and evaluation methods

[0124] The nanoformulations obtained in the above preparation examples were tested for drug loading, encapsulation efficiency, zeta potential, and particle size. Drug loading, encapsulation efficiency, and CPD12C15 content were determined using high-performance liquid chromatography. Particle size and zeta potential were measured using a Malvern nanometer particle size analyzer. Samples were diluted with ultrapure water before testing, and three measurements were taken for each sample, averaging the results.

[0125] Method for measuring the content of CPD12C15:

[0126] Preparation of stock solution: Accurately weigh 6.25 mg of C15 powder synthesized in our laboratory and place it in a 25 mL volumetric flask. Dissolve the solution in a small amount of acetonitrile, then add more acetonitrile to the mark. Mix thoroughly to obtain a C15 stock solution with a final concentration of 240 μg / mL. Refrigerate at 4°C until needed.

[0127] Determination of the ultraviolet absorption wavelength of CPD12C15: Take an appropriate amount of the prepared C15 stock solution, dilute it appropriately with acetonitrile, and use pure acetonitrile solution as a blank control. Use an ultraviolet spectrophotometer to scan the C15 solution at all wavelengths (scanning range: 190-600nm), and record the maximum absorption wavelength as the detection wavelength.

[0128] The HPLC chromatographic conditions were as follows: chromatographic column: Phenomenex Luna 5u C18 column (5 μm, 250×4.6 mm); mobile phase: 0.2% formic acid water-acetonitrile (40:60, v / v); injection volume: 10 μL; column temperature: 30°C; flow rate: 1 mL / min; detection wavelength: 284 nm.

[0129] Establish a standard curve and determine linearity: Use a precise pipette to dispense 0.625, 1.25, 2.5, 5, 7.5, and 10 mL of a 240 μg / mL C15 stock solution into a 10 mL volumetric flask. Add acetonitrile to the volume and mix thoroughly to obtain a series of C15 standard solutions with final concentrations of 15, 30, 60, 120, 180, and 240 μg / mL. Filter all samples through a 0.22 μm filter membrane and analyze by HPLC. Peak areas are recorded. Repeat this experiment three times. Plot a C15 standard curve using C15 concentration and the average of the measured peak areas as the horizontal and vertical coordinates, respectively, and analyze its linearity.

[0130] Determination of drug loading and encapsulation efficiency: The drug loading and encapsulation efficiency of the product were determined using ultracentrifugation. A certain volume of drug-loaded nanoparticles was placed in an ultracentrifuge tube and centrifuged at 5000 rpm for 20 minutes. An appropriate volume of the ultrafiltrate was filtered through a 0.22 μm microporous membrane and analyzed by HPLC to determine the free C15 content. 100 μL of the drug-loaded nanoparticles was ultrasonically demulsified with acetonitrile and filtered through a 0.22 μm microporous membrane. HPLC analysis was performed to determine the total C15 content in the formulation and to calculate the encapsulation efficiency and drug loading. Drug loading (%) = (mass of CPD12C15 in the product / total mass of the product) × 100%; Encapsulation efficiency (%) = [(total amount of CPD12C15 added - amount of free CPD12C15 in the supernatant) / total amount of CPD12C15 added] × 100%.

[0131] 1.4 Preparation process optimization results

[0132] (1) Effect of surfactant types on PC-NP particle size, drug loading, and encapsulation efficiency: In Preparation Examples 1 and 2, PLGA nanoparticles (i.e., PC-NPs) without HA modification were prepared. The particle size, drug loading, and encapsulation efficiency of PLGA nanoparticles prepared with surfactants CTAB and DTAB were investigated. The results are shown in Table 3.

[0133] Table 3 Effects of different surfactants on drug loading, encapsulation efficiency and particle size of PC-NPs (x±sd, n=3)

[0134] Surfactant type Drug loading (%) Encapsulation efficiency (%) Particle size (nm) DTAB (Preparation Example 1) 4.63±0.19 72.52±1.17 67.15±1.64 CTAB (Preparation Example 2) 5.00±0.01 78.05±0.98 184.2±3.18

[0135] The results show that when CATB was used as the surfactant, the drug loading capacity, encapsulation efficiency, and particle size were 5.00%, 78.05%, and 184.2 nm, respectively. When DATB was used as the surfactant, the average drug loading capacity, encapsulation efficiency, and particle size were 4.71%, 70.32%, and 67.15 nm, respectively. Although the drug loading capacity and encapsulation efficiency of nanoparticles prepared using CATB were slightly higher than those of DTAB, the particle size was significantly larger than that of DTAB. Considering the subsequent modification of HA and the particle size for in vivo administration, DTAB was the preferred surfactant.

[0136] (2) The effects of organic solvent types (preparation examples 1, 3-5) on drug loading and encapsulation efficiency of PC-NPs: Figure 2 In (a), when different organic solvents are used, the drug loading and encapsulation efficiency of PC-NPs vary to varying degrees. When the organic solvent is dichloromethane (Preparation Example 1), the drug loading and encapsulation efficiency are both optimal, approximately 72% and 4.6%, respectively. Therefore, dichloromethane is the preferred organic solvent for preparing PC-NPs.

[0137] (3) The effect of the volume ratio of oil phase to water phase (preparation examples 1, 10-12) on drug loading and encapsulation efficiency of PC-NPs: Figure 2 In (b), when the volume ratio of the oil phase to the aqueous phase increases from 1:2 to 1:5, the encapsulation efficiency increases slightly, while the drug loading decreases. Taking all factors into consideration, the optimal volume ratio of the oil phase to the aqueous phase for preparing PC-NPs, i.e., 1:2, is selected to achieve the highest drug loading.

[0138] (4) The effect of the CPD12C15 to PLGA ratio (preparation examples 1, 6-9) on the drug loading and encapsulation efficiency of PC-NPs: Figure 2 In (c), when the CPD12C15:PLGA ratio increased from 1:2 to 1:6, the encapsulation efficiency increased slightly, while the drug loading decreased significantly. Taking all factors into consideration, a 1:2 ratio (highest drug loading) was selected as the optimal condition for preparing PC-NPs.

[0139] (5) The effect of PLGA concentration (preparation examples 1, 13-14) on drug loading and encapsulation efficiency of PC-NPs: Figure 2 In (d), the drug loading capacity was lowest at a PLGA concentration of 10 mg / mL, and did not change significantly when the PLGA concentration increased from 15 mg / mL to 20 mg / mL. The encapsulation efficiency was highest at a PLGA concentration of 15 mg / mL and lowest at 20 mg / mL. Therefore, 15 mg / mL was selected as the optimal PLGA concentration for preparing PC-NPs.

[0140] (6) The effect of surfactant concentration (preparation examples 1, 15-18) on drug loading and encapsulation efficiency of PC-NPs: Figure 2 In (e), the encapsulation efficiency remained essentially unchanged when the surfactant concentration increased from 0.5% to 2.5%. The highest drug loading was achieved at a surfactant concentration of 0.5%. Furthermore, considering the toxicity of cationic surfactants, a lower surfactant concentration of 0.5% was selected as the optimal surfactant concentration for preparing PC-NPs.

[0141] (7) Effect of HA Molecular Weight (Preparation Examples 22-24) on HPC-NP Particle Size and Potential

[0142] The results, as shown in Table 4, show that as the molecular weight of HA increases, its particle size increases significantly. The smallest particle size is achieved at a molecular weight of 6,000. Furthermore, at a molecular weight of 240,000, flocculent precipitation is observed in the nanoparticle solution. Therefore, the smaller molecular weight of 6,000 is selected as the optimal HA molecular weight for preparing HPC-NPs.

[0143] Table 4 Effect of HA molecular weight on nanoparticle size and potential (x ± sd, n = 3)

[0144]

[0145] (8) Effect of HA Concentration (Preparation Examples 19-22) on the Particle Size and Potential of HPC-NPs

[0146] The results are shown in Table 5. As the HA concentration gradually increases, its particle size increases, with a more significant effect on the nanoparticle size. The smallest particle size is achieved at a HA concentration of 0.1 mg / mL. At a HA concentration of 1.5 mg / mL, the nanoparticle solution exhibits a slight flocculent precipitation. Therefore, a HA concentration of 0.1 mg / mL is selected as the optimal concentration.

[0147] Table 5 Effect of HA concentration on nanoparticle size and potential (x±s, n=3)

[0148]

[0149] (9) Determination and verification of the optimal preparation process

[0150] Based on the aforementioned tests, the optimal formulation for HPC-NPs was determined as follows: dichloromethane as the organic solvent, a CPD12C15 to PLGA ratio of 1:2, a V:V ratio of 1:2, a PLGA concentration of 15 mg / mL, a DTAB concentration of 0.5%, a HA molecular weight of 6,000, and a HA concentration of 0.1 mg / mL. Three batches of HPC-NPs were prepared using this optimal formulation and evaluated for drug loading, encapsulation efficiency, particle size, and zeta potential. As shown in Table 6, the HPC-NPs prepared using the optimal formulation had an average particle size of 122.3 ± 4.15 nm, an average zeta potential of -16.90 ± 1.15 mV, an average encapsulation efficiency of 70.01% ± 0.30, and an average drug loading of 17.07% ± 0.17.

[0151] Table 6 Verification of the optimal formulation process for three batches of HPC-NP (n=3)

[0152]

[0153] 2. Comprehensive characterization and quality evaluation of the optimal formulation HPC-NP

[0154] The HPC-NPs (batches shown in Table 6 ) prepared based on the optimal formulation conditions determined in Section 1.4 were subjected to the following comprehensive characterization and quality evaluation.

[0155] (1) Determination of particle size and zeta potential: A small amount of HPC-NP prepared from the optimal formula was diluted with ultrapure water to obtain an HPC-NP solution, and its particle size and zeta potential were measured using a Malvern nanoparticle size potential analyzer. The results are as follows: Figure 3 As shown, Figure 3 (a) shows that the HPC-NPs have a small and uniform particle size distribution, and (b) shows that the HPC-NPs have a negative surface charge. The measured particle size distribution is consistent with the average particle size of 122.3 ± 4.15 nm shown in Table 6.

[0156] (2) Determination of morphological characteristics: A small amount of HPC-NP prepared by the optimal formula was diluted with ultrapure water to obtain an HPC-NP solution, which was then dropped onto a carbon film copper mesh. After the solution was naturally evaporated, the morphological characteristics of the HPC-NP were observed under TEM. The morphological characteristics of the HPC-NP were characterized using a transmission electron microscope. The results are shown in Figure 2. Figure 4 As shown in the figure, HPC-NPs are spherical and have a particle size of about 130-150 nm, which is basically consistent with the aforementioned particle size distribution results. The difference may be due to the different principles of the two measurement methods.

[0157] (3) Stability Study: An appropriate amount of prepared HPC-NPs was taken and stored at 4°C. The particle size and zeta potential were measured using a Malvern nanoparticle size analyzer on days 0, 1, 7, 14, and 28. The drug loading and encapsulation efficiency were also measured. The results are shown in Table 7. There were no significant changes in particle size, zeta potential, drug loading, or encapsulation efficiency, indicating good storage stability.

[0158] Table 7 Stability test results of HPC-NP (x±sd, n=3)

[0159]

[0160] (5) Hemolysis test

[0161] Place an appropriate amount of SD rat whole blood in a heparinized 10mL centrifuge tube and add an equal volume of normal saline. Mix thoroughly and centrifuge at 3000 rpm for 10 minutes. Discard the supernatant and add an equal volume of normal saline. Repeat this process until the supernatant becomes clear. The precipitate is the red blood cells. Place 1mL of red blood cells in a 50mL volumetric flask and add an appropriate volume of normal saline to obtain a 2% red blood cell suspension.

[0162] Take an appropriate amount of HPC-NP with a concentration of 0.5μg / mL-40μg / mL, add an equal volume of 2% red blood cell suspension, and mix well. At the same time, set up negative and positive control groups, that is, treat 2% red blood cell suspension with physiological saline and ultrapure water respectively. After oscillating each group of solutions in a 37°C water bath for 3 hours, centrifuge at 3000r / min for 10 minutes, take 200μL of the supernatant after centrifugation in a 96-well plate, measure the OD value at a specific wavelength, and calculate the hemolysis rate of each group. The calculation formula of the hemolysis rate is as follows:

[0163]

[0164] After the biomaterial enters the human body through intravenous injection, it will exert its medicinal effect through the blood circulation. If the biomaterial comes into contact with red blood cells and produces hemolysis, it will cause hemolytic toxicity and have a significant adverse effect on the human body. Therefore, the hemolysis experiment can be used to explore the compatibility of the material of the drug preparation with the blood. Considering that the subsequent animal experiments will be administered by tail vein injection, the biosafety of HPC-NP is explored through hemolysis experiments. The results of the red blood cell suspension after treatment with various concentrations of nanoparticle solution, ultrapure water (positive control) and normal saline (negative control) are as follows Figure 5 As shown in (a). The results show that the positive control group after ultrapure water treatment showed significant hemolysis, while the groups treated with HPC-NP solutions of various concentrations and the normal saline group did not show hemolysis. Figure 5 The quantitative analysis results in (b) show that the hemolysis rate of HPC-NP solutions at all concentrations is less than 5%, further indicating that no hemolysis occurs within the range of 0.5-40 μg / mL. This hemolysis experiment result demonstrates that HPC-NP has good blood compatibility and biosafety.

[0165] (6) In vitro release experiment

[0166] The in vitro release behavior of HPC-NPs was detected by dynamic dialysis. Three different pH conditions were selected: pH 5.5 (simulating the pH conditions of the intracellular environment of tumor cells), pH 6.8 (simulating the pH conditions of the extracellular microenvironment of tumor cells), and pH 7.4 (simulating the pH conditions of normal body fluid environments such as blood). The dialysis bag had a molecular weight cutoff of 3500 and needed to be activated before use.

[0167] The corresponding HPC-NPs (containing 2.00 mg of CPD12C15) were precisely calculated and then filled to 1 mL with ultrapure water. This solution was then placed in a pre-activated dialysis bag, tightly tied at both ends with string to prevent leakage. 30 mL of release medium was added to a 50 mL centrifuge tube, and the dialysis bag was placed inside. The 50 mL centrifuge tube was then placed in a thermostatic oscillator maintained at 37°C and 100 rpm. At 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours, 500 μL of release medium was removed from the centrifuge tube and simultaneously added to the corresponding 500 μL of fresh, isothermal release medium. The release medium solution removed at different time points was filtered through a 0.22 μm filter membrane and entered into HPLC for detection. The corresponding peak area was recorded, and the content of CPD12C15 was calculated (the method was the same as described in Section 1.3). At the same time, the cumulative release rate of HPC-NP under different pH conditions was calculated, and the corresponding cumulative release curve was drawn.

[0168] According to the above method, the release characteristics of HPC-NP in three release media of pH 5.5, pH 6.8 and pH 7.4 were investigated respectively. The in vitro release results of HPC-NP under different pH conditions are shown in Figure 2. Figure 6 shown.

[0169] The experimental results show that CPD12C15 releases rapidly at pH 7.4, 6.8, and 5.5. At pH 5.5, the cumulative release rate of CPD12C15 reaches 50.69% within 0.5 hours. HPC-NPs can effectively improve the release performance of C15, enhancing its sustained release and achieving more complete release. Furthermore, the release of HPC-NPs at pH 5.5 is more rapid than that at pH 7.4 and 6.8, indicating that HPC-NPs can achieve rapid and efficient drug release within tumor cells to a certain extent. These results demonstrate that HPC-NPs can improve the sustained release of CPD12C15 compared to other pH conditions, with the most rapid and complete drug release at pH 5.5, demonstrating excellent in vitro release properties. The results revealed that HPC-NPs exhibit pH-responsive release properties, which have significant implications for the functionality of drug delivery systems. HPC-NPs successfully improved the release behavior of CPD12C15, transforming the "rapid release" of the free drug into a controlled, sustained-release pattern. This helps prolong the drug's duration of action in vivo and reduce dosing frequency. Importantly, HPC-NPs exhibited a distinct pH-dependent release profile. At pH 5.5 (simulating the intracellular environment and lysosome / endosome environments of tumor cells), the drug release rate was significantly faster than at pH 7.4 (simulating the blood environment) and pH 6.8 (simulating the extracellular microenvironment of tumor cells). While the nanoparticles circulate in the blood (pH 7.4), drug release is slower, helping to minimize drug leakage and systemic toxicity. However, upon reaching the tumor site and being internalized by the cells (pH 5.5), the nanoparticles rapidly and efficiently release the drug, increasing local drug concentrations. This pH-sensitive release pattern enables the HPC-NPs to remain stable in normal tissues while rapidly releasing the drug within tumor cells.

[0170] (7) In vivo targeting evaluation in mice

[0171] In vivo imaging technology can be used to observe biological processes such as tumor growth and metastasis in living animals in real time. DiR is a commonly used lipophilic dialkyl dye that is often used for in vivo fluorescence tracing. DiR was used instead of CPD12C15 to encapsulate hyaluronic acid nanoparticles (HA-PLGA-DiR nanoparticles, HPD-NPs). Nude mice with subcutaneous tumors were selected as the experimental model. Free DiR and HPD-NPs were administered via the tail vein, respectively. Small animal in vivo imaging technology was used to monitor the distribution of fluorescence in the mice at different time points to investigate the ability of the formulation to actively target tumors in vivo.

[0172] 1) Establishment of a PANC-1 tumor nude mouse model. PANC-1 cells in the logarithmic growth phase and in good growth condition were prepared to a cell density of 2×10 7 The cell suspension was prepared at a concentration of 100 μL / mL and placed on ice for later use. First, the skin of the nude mouse armpit was disinfected with alcohol. 100 μL of the prepared cell suspension was slowly injected into the subcutaneous part of the nude mouse armpit using a 1 mL syringe to obtain a nude mouse PANC-1 subcutaneous transplant tumor model. When the tumor grew to 500 mm 3 In vivo imaging experiments can begin at this time.

[0173] 2) HPD-NPs were prepared according to the optimized formulation. Free DiR solution and HPD-NP solution were injected into the tail vein at a DiR dose of 1.5 mg / kg per mouse. At 4, 8, 12, and 24 hours after administration, mice were subjected to gas anesthesia and imaged using a small animal live imaging system. Twenty-four hours later, mice in each group were sacrificed by cervical dislocation, and their hearts, livers, spleens, lungs, kidneys, and tumor masses were removed. After careful washing with saline and drying, the samples were collected and photographed using a small animal live imaging system to investigate the distribution of HPD-NPs within the tumor mass and various tissues and organs.

[0174] 3) Results: The in vivo fluorescence distribution experimental results of the DiR solution group and the HPD-NP group at different time points are shown in Figure 3. Figure 7 As shown in (a). From the experimental results, it can be seen that the fluorescence signal can be observed in the tumor site of the HPD-NP group at 4 hours, while the fluorescence signal in the tumor site of the DiR solution group is very weak, and the accumulation in the liver and spleen is obvious. As time goes on, the fluorescence intensity of the tumor site in the HPD-NP group can continue to accumulate, and the fluorescence intensity is the strongest at 8 hours; at 24 hours, the fluorescence intensity of the tumor site begins to slowly weaken. As time goes on, the DiR solution group also mainly accumulates in the liver and spleen, and less in the tumor site. After 24 hours, the mice in each group were dissected, and the tumor masses and major organs (heart, liver, spleen, lungs and kidneys) were removed for 24-hour in vitro imaging. The experimental results are shown as follows. Figure 7As shown in (b), the fluorescence intensity at the tumor site of the HPD-NP group was significantly stronger than that of the DiR solution group, and the fluorescence intensity in liver tissue was also weaker than that of the DiR solution group, indicating that HPD-NPs have good tumor targeting while reducing their accumulation in liver tissue and reducing in vivo toxicity. These results fully demonstrate that HA-modified CPD12C15-loaded PLGA nanoparticles can accumulate well in the subcutaneous tumor site of nude mice, demonstrating excellent tumor targeting.

[0175] (8) Study on the cellular uptake and intracellular mechanism of HPC-NPs

[0176] Coumarin 6 (C6) is one of the commonly used liposoluble fluorescent dyes. It was replaced by CPD12C15 to prepare PLGA nanoparticles (PLGA-C6 nanoparticles, PC6-NP) and HA-modified nanoparticles (HA-PLGA-C6 nanoparticles, HPC6-NP) to investigate the drug uptake ability of pancreatic cancer PANC-1 and HS766T cells.

[0177] Pancreatic cancer PANC-1 and HS766T cells in the logarithmic growth phase were seeded in 6-well plates at appropriate cell densities. The experiment was divided into four groups: C6 group, PC6-NP group, HPC6-NP group, and HA-Blocked group pretreated with HA in advance, and the C6 concentration in each group was ensured to be 20 ng / mL. Among them, the HA-Blocked group was pretreated by adding HA solution (concentration of 0.1%), cultured for 1 hour, and then the corresponding HPC6-NP was added for incubation. After 1 and 4 hours respectively, the culture medium was discarded, the cells were washed with PBS solution 3 times, and then 4% paraformaldehyde was added to fix the cells at room temperature for 15 minutes. After washing with PBS solution 3 times, DAPI solution was added to mark the cell nucleus at room temperature for 10 minutes, and PBS solution was added to wash 3 times. After being completely dried, the cells were photographed under a fluorescence microscope and the cell uptake at each time point in each group was observed for qualitative analysis.

[0178] For quantitative analysis, the culture medium was discarded after 1 and 4 h, respectively, and the cells were washed three times with PBS solution, digested with trypsin, centrifuged, and prepared into a concentration of 1 × 10 6 / mL single cell suspension, and flow cytometry was used to detect the intensity of each group at each time point.

[0179] Results: The qualitative results of the cellular uptake experiments of PANC-1 and HS766T were as follows: Figure 8 and Figure 9As shown, similar trends were observed in PANC-1 and HS766T cells. Compared with the Control group, the uptake of PC6-NP and HPC6-NP by PANC-1 and HS766T cells increased significantly, and the uptake of HPC6-NP by cells was greater than that of PC6-NP and showed a time-dependence. This indicates that after the nanoparticles are modified with HA, their ability to be taken up by cells can be greatly increased. However, when HA solution is added for pretreatment in advance, the uptake of HPC6-NP is reduced. This may be because the HA and HPC6-NP added in advance competitively inhibit the HA receptor CD44 in the cells, thereby hindering the entry of nanoparticles into the cells and reducing their cellular uptake ability. The quantitative experimental results are shown in Figure 10 As shown, the results are consistent with the qualitative results. Therefore, these results suggest, to a certain extent, that HPC-NPs can enter cells via endocytosis mediated by the CD44 receptor on the cell surface. HA-modified nanoparticles exhibit excellent in vivo tumor targeting while reducing accumulation in liver tissue, thereby reducing in vivo toxicity. Experiments exploring the cellular uptake and intracellular entry mechanisms demonstrated that HPC-NPs can utilize HA to specifically bind to its receptor, enter cells via CD44-mediated endocytosis, achieve drug accumulation at the tumor site, and enhance cellular uptake.

[0180] Example 2 In vitro anti-pancreatic cancer efficacy of HPC-NPs

[0181] In this example, human PANC-1 and HS766T pancreatic cancer cells were selected as model cells, and gemcitabine (GEM), a first-line chemotherapy drug for the clinical treatment of pancreatic cancer, was used as a positive control drug. In vitro pharmacodynamic studies used MTT assays to determine the effects of C15 / Cu, PC-NP / Cu, HPC-NP / Cu, and GEM on PANC-1 and HS766T cell proliferation and to determine the subsequent in vitro dosing concentrations. Cell cloning, wound healing, invasion, and apoptosis experiments were performed to investigate the effects of C15 and its nanoparticle preparations combined with copper on the migration, invasion, proliferation, and apoptosis-inducing abilities of pancreatic cancer cells.

[0182] 1. Materials

[0183] Experimental cells: Human pancreatic cancer cells PANC-1 and HS766T were obtained from the School of Basic Medical Sciences, Shandong University.

[0184] Preparation of experimental reagents:

[0185] DMEM medium: Place a certain amount of DMEM medium in a 50mL centrifuge tube and add DMEM:FBS:antibody in a volume ratio of 10:1:0.1. Shake up and down to mix thoroughly to obtain complete DMEM medium. Store at 4°C and preheat in a 37°C water bath before use.

[0186] Cell freezing solution: Prepare according to the ratio of 70% basal culture medium: 20% fetal bovine serum: 10% DMSO (biological grade). Prepare 1 mL of cell freezing solution for each cryovial and label it.

[0187] CPD12C15 Solution: Prepare a 2 mM CPD12C15 stock solution by accurately weighing 1.36 mg of CPD12C15 and dissolving it in 2 mL of DMSO. Sonicate to dissolve the CPD12C15 solution. Sterilize the solution in a clean hood with UV light for 30 minutes, then filter the solution through a 0.22 μm microporous filter to sterilize. Aliquot and transfer to 1.5 mL centrifuge tubes. Storage is possible at -20°C.

[0188] CuCl2·2H2O Solution: Prepare a 2 mM CuCl2·2H2O stock solution by precisely weighing 0.68 mg of CuCl2·2H2O powder and dissolving it in 2 mL of ultrapure water. After UV sterilization for 30 minutes in a laminar flow hood, filter the solution through a 0.22 μm microporous filter membrane to sterilize. Aliquot and transfer to 1.5 mL centrifuge tubes for long-term storage at -20°C.

[0189] GEM solution: Prepare 10 mM GEM solution as the mother solution: accurately weigh 1.05 mg of GEM powder before the experiment, irradiate it with UV light for 30 min in a cell laminar flow hood, then dissolve it in 2 mL of culture medium, filter the solution through a microporous filter membrane (0.22 μm) and set aside for use. Prepare it before use.

[0190] MTT solution: Accurately weigh an appropriate amount of MTT powder and dissolve it in sterile PBS to a final concentration of 5 mg / mL. Filter through a 0.22 μm filter under aseptic conditions, away from light, to sterilize. Aliquot into 10 mL centrifuge tubes and wrap with tin foil. For short-term use at 4°C, aliquots can be stored at -20°C for long-term storage. Protect from light during use.

[0191] PC-NP solution: PC-NP was prepared according to the process conditions of Preparation Example 19 in Example 1. The CPD12C15 content was determined by HPLC. The solution was stored at 4°C and sonicated for 1 minute before use to ensure uniform dispersion.

[0192] HPC-NP solution: HPC-NP was prepared according to the process conditions of Preparation Example 19 in Example 1. The CPD12C15 content was determined by HPLC. The solution was stored at 4°C and sonicated for 1 minute before use to ensure uniform dispersion.

[0193] PC-NP / Cu Solution: Based on the CPD12C15 content in PC-NPs determined by HPLC, take an appropriate amount of PC-NP suspension and add the required amount of 2 mM CuCl2·2H2O solution to maintain a 1:1 molar ratio of CPD12C15 to Cu. Dilute with complete DMEM medium to the desired working concentration. Sonicate for 1 minute before use to ensure uniform dispersion.

[0194] HPC-NP / Cu solution: Based on the CPD12C15 content in HPC-NPs determined by HPLC, take an appropriate amount of HPC-NP suspension and add the required amount of 2mM CuCl2·2H2O solution to maintain a 1:1 molar ratio of CPD12C15 to Cu. Dilute with DMEM complete medium to the desired working concentration. Sonicate for 1 minute before use to ensure uniform dispersion. Table 8 Primer sequences

[0195]

[0196] 2. Experimental methods

[0197] 2.1 Cell Culture: Both pancreatic cancer PANC-1 and HS766T cells are adherent cells. Culture cells using complete DMEM medium and passage them approximately every 2-3 days in a cell culture incubator maintained at 37°C and 5% CO2. Cells were handled in a laminar flow hood, and all materials used in cell culture were sterilized.

[0198] 2.2 Cell recovery

[0199] (1) First, turn on the UV lamp to irradiate the sterile clean bench for 30 minutes, preheat in advance, adjust the constant temperature water bath to 37℃ and put the DMEM complete culture medium into it to preheat and set aside. After 30 minutes, in the cell clean bench, use tweezers to pick up an appropriate amount of alcohol cotton balls to wipe and disinfect the table surface and light the alcohol lamp. The whole process should be carried out in a sterile environment. (2) After removing the target cells from the liquid nitrogen tank, quickly place them in a preheated 37℃ constant temperature water bath. Gently shake the cryovial to convert the frozen cells from solid to liquid; when shaking, avoid contact between the cryovial mouth and water to avoid contaminating the cells. When there are still a few ice cubes, remove the cryovial directly. (3) In a clean bench, transfer the target cell suspension to a sterilized 10 mL centrifuge tube, add 1 mL of DMEM complete medium, and centrifuge at 1000 r / min for 3 minutes; (4) After the centrifugation, remove the centrifuge tube and discard the supernatant solution in the clean bench. First, resuspend the precipitated cells at the bottom with an appropriate amount of culture medium, then transfer them to a culture dish of the corresponding specifications, add complete medium to a volume of 4 mL, shake the culture dish thoroughly to evenly disperse the cells; then place it in a cell culture incubator for further culture. (5) After the experiment, clean the clean bench.

[0200] 2.3 Cell passaging

[0201] (1) After cell recovery, check the cells at irregular intervals. When the cells have grown to 80%-90% by adhering to the wall, they can be passaged. First, turn on the ultraviolet sterilization of the cell clean bench for more than 30 minutes, turn on the constant temperature water bath, take out the DMEM complete medium and trypsin, preheat them, and set them aside. (2) In the sterile clean bench, use tweezers to pick up an appropriate amount of alcohol cotton balls to wipe and disinfect the table surface and light the alcohol lamp to ensure that the operating environment is sterile. Discard the upper solution of the culture dish and wash it three times with sterilized PBS solution to remove dead cells and culture medium. (3) Use a pipette to discard the PBS and add 1mL of trypsin to digest the cells. After digesting them in the cell culture incubator for 2 minutes, observe the cell status under a microscope. When the intercellular space becomes larger, the cytoplasm shrinks, and the cells become round and bright, move the culture dish to the clean bench and add 1mL of DMEM complete medium to stop the digestion. Slowly blow the cells to evenly disperse them, then transfer them to a 10mL centrifuge tube and centrifuge them at 1000r / min for 3 minutes. (4) After centrifugation, remove the supernatant medium and add an appropriate amount of DMEM complete medium to thoroughly mix the cells. Transfer the cells to a new culture dish according to the appropriate subculture ratio and add DMEM complete medium to a final volume of 8 mL. Place the cells in a cell culture incubator for culture. (5) After the experiment, clean the clean bench.

[0202] 2.4 Cell cryopreservation

[0203] (1) When the cells are growing well and the cell density has grown to 80%-90%, they can be frozen. First, turn on the ultraviolet sterilization of the cell clean bench for more than 30 minutes, and turn on the constant temperature water bath, take out the DMEM basal medium, trypsin, serum and DMSO and preheat them in advance for use. (2) According to the operation under "2.3", after digesting the cells and centrifuging them, discard the supernatant solution, resuspend the cells with cell freezing solution and transfer them to the corresponding cryopreservation tube. (3) Place the cryopreservation tube in a cell freezing box, freeze it in a -80℃ refrigerator overnight, and then place it in a liquid nitrogen tank for long-term storage and keep storage records in time.

[0204] 2.5 Cell Counting

[0205] (1) Clean the blood cell counting plate and coverslip required for cell counting and place them in the clean bench. (2) Follow the operation under "2.3" to digest the cells with trypsin, centrifuge, and resuspend the cells with an appropriate amount of culture medium. Use a pipette to draw an appropriate amount of cell suspension and slowly inject it from one side of the coverslip along the edge at a uniform speed so that the cover slip is filled with suspension and the bubbles are discharged. (3) Place the blood cell counting plate under a microscope for observation. When the counting grid is observed, count the cells in the four large grids at the corners of the counting plate. Calculate the total number of cells in the grid as a, and follow the counting principle of "counting the top but not the bottom, and counting the left but not the right" to count the cells. (4) The cell density is (a / 4)×10 4 pieces / mL.

[0206] 3. MTT assay to detect pancreatic cancer cell viability

[0207] 3.1 Principle: The MTT assay is one of the most commonly used methods for assessing cell viability. MTT is reduced to formazan by succinate dehydrogenase in the mitochondria of living cells, forming blue-purple crystals. These crystals are insoluble in water but soluble in DMSO, and precipitate in living cells. However, MTT does not react with dead cells. Within a certain cell population range, the OD value measured at a specific wavelength is directly proportional to the cell number. Therefore, to a certain extent, MTT can indirectly reflect the viability of target cells.

[0208] 3.2 Experimental steps: Human pancreatic cancer PANC-1 and HS766T cells that are in good growth and in the logarithmic growth phase were digested with trypsin according to the procedures in "2.3", centrifuged and resuspended in appropriate amount of culture medium. The density was 8×10 cells per well. 3 Cells were evenly plated in a 96-well plate, with 100 μL of cell suspension per well, and incubated overnight in a cell culture incubator. The next day, when the cells grew to 70%-80% and adhered to the wall, drugs were added and allowed to act for 48 hours.

[0209] Experimental Design: The experiment was divided into four treatment groups: C15 combined with copper (hereinafter referred to as the C15 / Cu group), PC-NP combined with copper (hereinafter referred to as the PC-NP / Cu group), HPC-NP combined with copper (hereinafter referred to as the HPC-NP / Cu group), and the positive drug gemcitabine (hereinafter referred to as the GEM group). In addition, a blank group (containing only culture medium without cells) and a control group (containing cells and culture medium without drug) were set up as controls.

[0210] In the C15 / Cu group, a C15 solution was mixed with an equimolar CuCl2·2H2O solution (C15 to Cu molar ratio of 1:1). The resulting mixed solution was diluted with complete DMEM medium to prepare treatment solutions with different concentration gradients. The concentration gradients were 0.025, 0.050, 0.100, 0.200, 0.400, and 0.800 μM (based on the final C15 concentration).

[0211] In the GEM group, a 10 mM gemcitabine (GEM) stock solution was first prepared and diluted with DMEM complete medium to set the final concentrations to 0.5, 5, 25, 50, 100, and 500 μM.

[0212] PC-NP / Cu group: The PC-NP suspension was mixed with CuCl2·2H2O solution (the molar ratio of C15 to Cu in PC-NP was 1:1) to obtain a PC-NP / Cu mixed solution, which was diluted with DMEM complete medium. 100 μL of the prepared drug solution was added to each well to make the final C15 concentrations of 0.025, 0.050, 0.100, 0.200, 0.400, and 0.800 μM in both cell lines.

[0213] HPC-NP / Cu group: The HPC-NP suspension was mixed with CuCl2·2H2O solution (the molar ratio of C15 to Cu in HPC-NP was 1:1) to obtain the HPC-NP / Cu mixed solution, which was diluted with DMEM complete medium. 100 μL of the prepared drug solution was added to each well to make the final C15 concentrations of 0.025, 0.050, 0.100, 0.200, 0.400, and 0.800 μM in both cell lines.

[0214] Six replicate wells were set up for each concentration in all treatment groups, with 100 μL of drug solution added to each well. After 48 hours of treatment, 20 μL of MTT solution was added to each well under sterile, dark-protected conditions and incubated in a 37°C incubator for 4 hours. Subsequently, 150 μL of DMSO solution was added to each well, and the 96-well plate was gently shaken on a shaker at room temperature for 15 minutes. Finally, the OD values of PANC-1 and HS766T cells were measured at 490 nm using a microplate reader. This experiment was repeated three times. Cell viability was calculated using the formula shown below.

[0215]

[0216] As: OD value of the experimental group, Ab: OD value of the blank group, Ac: OD value of the control group.

[0217] To ensure consistency in experimental design, subsequent in vitro experiments, including cell cloning, wound healing, invasion, and apoptosis, used the same grouping and drug concentration treatment conditions as the MTT assay. Specifically, the experimental groups were C15 / Cu, PC-NP / Cu, HPC-NP / Cu, and GEM. Drug solutions were diluted in complete DMEM medium, and the final concentrations were set based on the MTT assay screening concentrations. Testing was performed after 48 hours of treatment.

[0218] 4. Cell scratch test

[0219] 4.1 Experimental principle: The cell scratch test is an experimental method that can be used to explore the migration ability and repair degree of cells, which can simulate the cell migration process in vivo to a certain extent. The adherent cells in good growth state and in the logarithmic growth phase are cultured in the cell culture plate until they are confluent. A "cross" line is drawn in the middle area with a micropipette tip, and the cells in the middle area are washed with an appropriate amount of sterile PBS and discarded, that is, a blank "scratch" is artificially created; then the cells are cultured until the given time of the experiment. The cells in the control group can gradually grow towards the "scratch" area due to their own migration and repair abilities; however, the experimental group will induce or inhibit their migration and repair abilities to a certain extent due to the addition of different drug interventions. Finally, the final migration area is counted by the data processing software Image J to obtain the scratch healing rate. The scratch healing rate can be used to characterize the migration and repair abilities of cells. The scratch healing rate is shown in the following formula:

[0220]

[0221] 4.2 Experimental Procedure: Healthy, logarithmically growing human pancreatic cancer PANC-1 and HS766T cells were digested with trypsin according to the procedures described in "2.3." After centrifugation, the cells were resuspended in an appropriate amount of culture medium and evenly plated into 6-well plates at a high cell density. The plates were incubated in a cell culture incubator. When the cell density reached 90% or higher, a pre-sterilized 200 μL pipette tip was used to create a "cross" pattern vertically across the bottom of each well. The cells were then washed three times with PBS and observed and photographed under an inverted microscope. The scratch area for each group at 0 h was recorded, and the position of the scratch was marked. After photographing, the PBS solution was discarded, and the cells were divided into five groups: control, C15 / Cu, PC-NP / Cu, HPC-NP / Cu, and GEM. 2 mL of serum-free culture medium or the corresponding serum-free drug-containing culture medium was added to each well, and the cells were placed in the incubator for further incubation. After 48 hours, the 6-well plates were removed, the culture medium discarded, and the cells were washed three times with PBS. Images were then taken using an inverted microscope at the same location marked at 0 hours, and the cell scratch area at 48 hours was recorded. Data were analyzed using Image J software, and the changes in scratch area between 0 and 48 hours were statistically analyzed for each group. The scratch healing rate was also calculated for each group.

[0222] 5. Cell invasion assay

[0223] 5.1 Experimental Principle: The polycarbonate membrane at the bottom of a Transwell chamber separates the upper and lower culture medium layers. Placing this membrane in a corresponding culture plate separates the serum-free upper layer of culture medium from the complete lower layer of culture medium. Cells are seeded at an appropriate density within the chamber. To obtain more nutrients, they will migrate through the polycarbonate membrane. Matrigel, pre-coated on the membrane, simulates the extracellular matrix. Therefore, counting the number of cells that migrate to the lower layer of the chamber can be used to investigate cell invasion.

[0224] 5.2 Experimental steps: Take out the matrigel stored at -20℃ in advance and thaw it in a 4℃ refrigerator. Use serum-free culture medium to dilute the liquid matrigel at a ratio of 1:8; then take out 100μL of matrigel and add it to the bottom of the Transwell chamber and place it in the corresponding position of the 24-well plate, and transfer it to the incubator to wait for the matrigel to solidify. Human pancreatic cancer PANC-1 and HS766T cells that are in good growth condition and in the logarithmic growth phase are digested with trypsin and centrifuged according to the operation under "2.3". Resuspend them in an appropriate amount of culture medium. After counting the cells and diluting them to 5×10 5 / mL cell suspension, add 100 μL into each chamber.

[0225] The experiment was divided into five groups: control, C15 / Cu, PC-NP / Cu, HPC-NP / Cu, and GEM. To each group, 100 μL of the corresponding serum-free drug-containing culture medium was added to the designated concentration. Simultaneously, 800 μL of complete culture medium was added to the interior of the 24-well plate below the chamber and cultured for 48 hours. After incubation, the cells were washed three times with sterile PBS and fixed with 4% paraformaldehyde for 30 minutes at room temperature. Each well was then washed three times with PBS and stained with crystal violet solution for 30 minutes. After three washes with PBS, any cells that did not penetrate the chamber were gently wiped off with a cotton swab. After complete drying, the cells were observed under an inverted microscope and photographed. The number of cells that penetrated the chamber below was counted using Image J software, and the cell invasion rate was calculated.

[0226] 5.3 Apoptosis Assay: Annexin V / PI double staining was used to examine the ability of different drugs to induce apoptosis in pancreatic cancer PANC-1 and HS766T cells. Well-grown human pancreatic cancer PANC-1 and HS766T cells in the logarithmic growth phase were digested with trypsin according to the procedures described in "2.3." Cells were centrifuged and resuspended in an appropriate amount of culture medium. Cells were seeded at an appropriate density in 6-well plates and incubated overnight in an incubator. When the cell density reached 60%-70%, the experimental group was divided into five groups: control, C15 / Cu, PC-NP / Cu, HPC-NP / Cu, and GEM. The corresponding drug concentration was added to each group until the target concentration was reached. Cells were then transferred to an incubator for continued culture.

[0227] After 48 hours, the supernatant culture medium from each group was collected and washed three times with PBS. EDTA-free trypsin was then added to each well to digest the cells, and the cells were centrifuged at 1000 rpm for 3 minutes. After discarding the supernatant, the cells were washed again three times with PBS. Then, 100 μL of 1× Binding Buffer was added and the cells were pipetted to mix evenly. 5 μL of Annexin V and 5 μL of PI dye were added, mixed, and incubated in the dark for 10 minutes. Finally, 400 μL of 1× Binding Buffer was added, the cells were thoroughly pipetted, and then transferred to flow cytometry tubes. The apoptosis rate of each group was measured using flow cytometry (within 1 hour). Blank and single-staining groups were set up in this experiment, i.e., no dye was added and only PI dye or Annexin V dye was added.

[0228] 6. Cell cloning experiment

[0229] 6.1 Experimental Principle: Cell survival rate reflects the number of cells that adhere after inoculation. However, not all adherent cells will proliferate and form clones. However, the size and number of cell clones formed can be used to assess the cell's ability to adhere and proliferate. Therefore, cell cloning assays are a powerful tool for studying tumor cell proliferation. The plate-based cloning assay is suitable for adherent cells, is simple to perform, and offers a high cloning rate.

[0230] 6.2 Experimental steps: Human pancreatic cancer PANC-1 and HS766T cells that are in good growth condition and in the logarithmic growth phase were digested with trypsin and centrifuged according to the operation under "2.3", and then resuspended with an appropriate amount of culture medium. Cells were seeded in 6-well plates at a density of 2000 cells per well and placed in an incubator overnight. The experiment was divided into four groups: Control group, C15 / Cu group, PC-NP / Cu group and HPC-NP / Cu group. Drug intervention of corresponding concentrations was added to each group for 48 hours, and then replaced with DMEM complete culture medium. The 6-well plates were transferred to an incubator and cultured for 2 weeks, during which the cell culture medium was replaced approximately every 2 days. The culture was terminated when it was observed that the number of most monoclonal cells was greater than 50. After discarding the culture medium, the cells were washed three times with PBS solution and fixed with 4% paraformaldehyde at room temperature for 30 minutes; then washed again with PBS solution three times; crystal violet solution was added for staining for 30 minutes and then washed three times with PBS solution. The cells were left to dry naturally at room temperature, and finally photographed and counted for the number of cell clones in each group.

[0231] 7. Lactate Determination: Healthy, logarithmic-phase human pancreatic cancer PANC-1 and HS766T cells were digested with trypsin according to the procedures described in "2.3." Cells were centrifuged and resuspended in an appropriate amount of culture medium. After cell count, the cells were seeded at a density of 2,000 cells per well in a 6-well plate and transferred to an incubator for continued culture. After cell attachment, the cells were divided into four groups: control, C15 / Cu, PC-NP / Cu, and HPC-NP / Cu. The corresponding drug concentration was added to each group. After incubation for 48 hours, the supernatant was discarded, the cells were washed three times with PBS, and then digested with trypsin and collected from each group for testing. The samples were then processed according to the instructions in the lactate assay kit. The OD value of each group was measured at 530 nm, and the lactate content in each group was calculated.

[0232] 8. Determination of glucose uptake: Human pancreatic cancer PANC-1 and HS766T cells that are in good growth condition and in the logarithmic growth phase were digested and centrifuged according to the operation under "2.3", and then resuspended with an appropriate amount of culture medium. After counting the cells and seeding them in a 6-well plate at an appropriate density, they were transferred to an incubator for continued culture. After the cells adhered to the wall, the experiment was divided into four groups: Control group, C15 / Cu group, PC-NP / Cu group, and HPC-NP / Cu group. The corresponding concentration of drugs was added to each group, and after continuing to culture for 48 hours, the cells were washed three times with PBS solution, digested with trypsin, and collected from each group as the sample to be tested. Then, according to the relevant steps on the instructions of the glucose test kit, the sample to be tested was operated, and finally the OD value of each group was measured at 505nm and the glucose content in each group was calculated.

[0233] 9. Determination of ROS Content: Following the procedures described in "2.3," well-grown human pancreatic cancer PANC-1 and HS766T cells in the logarithmic growth phase were digested and centrifuged, resuspended in an appropriate amount of culture medium, and seeded into 6-well plates at an appropriate density. The cells were then transferred to an incubator for continued culture. After cell attachment, the cells were divided into four groups: control, C15 / Cu, PC-NP / Cu, and HPC-NP / Cu. The indicated concentrations of drug were added to each group and cultured in an incubator. After 48 hours, the supernatant was discarded and the cells were washed three times with PBS. 500 μL of DCFH-DA working solution, previously diluted to a 10 μM concentration in serum-free culture medium, was added to each well. The 6-well plates were then placed in a 37°C cell culture incubator protected from light for 30 minutes. After discarding the supernatant, the cells were washed three times with serum-free medium. The digested cells were collected, thoroughly mixed with an appropriate amount of PBS solution, and transferred to a flow cytometer. The fluorescence intensity of DCFH-DA was detected by flow cytometry, and the ROS content in each group was quantitatively analyzed.

[0234] 10. Determination of ATP content: Human pancreatic cancer PANC-1 and HS766T cells in good growth state and in the logarithmic growth phase were digested and centrifuged according to the operation under "2.3" and resuspended in appropriate amount of culture medium. 2×10 5The cells were seeded in a 6-well plate and then transferred to an incubator for further culture. After the cells adhered to the wall, the experiment was divided into four groups, namely the Control group, the C15 / Cu group, the PC-NP / Cu group and the HPC-NP / Cu group. The corresponding concentration of drugs was added to each group, and the intervention was continued for 48 hours. After washing with PBS solution 3 times, the cells in each group were digested with trypsin and collected as the samples to be tested. Then, according to the relevant steps on the instructions of the ATP test kit, the samples to be tested were operated, and finally the fluorescence intensity of each group was measured at a specific wavelength using a chemiluminescence instrument, and the RLU (Relative Light Unit) value was calculated.

[0235] 11. PCR experiments to determine the expression of related genes

[0236] 11.1 Extraction of Total Cellular RNA: Healthy, logarithmic-phase human pancreatic cancer PANC-1 and HS766T cells were plated at an appropriate density in 6-well plates according to the protocol described in "2.3" and cultured in an incubator. After the cells adhered, they were divided into four groups: control, C15 / Cu, PC-NP / Cu, and HPC-NP / Cu. The given concentration of drugs was added to each group for a continuous intervention of 48 h, and the cells were washed three times with sterile PBS solution. Subsequently, 1 mL of Trizol solution was added to each well to thoroughly blow the cells, and the cells were transferred to a 1.5 mL centrifuge tube. After adding 200 μL of chloroform solution, the cells were slowly shaken up and down for about 10 times, and then allowed to stand at room temperature for 5 min and centrifuged at 12000 g for 15 min at 4°C. 400 μL of supernatant solution was taken from each tube, and then 400 μL of isopropanol solution was added to recover RNA, and the cells were slowly shaken up and down for about 10 times, and allowed to stand at room temperature for 5 min and centrifuged at 12000 g for 15 min at 4°C. The supernatant solution was discarded, and the centrifuge tube was turned upside down at room temperature and naturally dried. Then, 1 mL of 75% ethanol solution (prepared with DEPC water) was added, and the RNA precipitate was gently blown with a pipette to remove the isopropanol. The cells were centrifuged at 12000 g for 10 min at 4°C. The supernatant solution was discarded, and the cells were allowed to stand at room temperature until the ethanol evaporated naturally. 20 μL of After the RNA was completely dissolved in DEPC water and thoroughly mixed, the concentration of RNA in the sample was measured and recorded using a nucleic acid quantitative analyzer.

[0237] 11.2 cDNA Synthesis: Prepare the reaction system as shown in Table 9, referring to the instructions in the reverse transcription kit. After setting up the reverse transcription program, mix the system thoroughly and load it onto the instrument to obtain cDNA, which can be stored at -20°C.

[0238] Table 9 Reverse transcription system

[0239]

[0240] 11.3 RT-PCR reaction: Take out the cDNA stored at -20℃ and dilute it 10 times with DEPC water. Place the sample on ice until use. Prepare the RT-PCR reaction system according to Table 10. Add the prepared reaction system to the eight strips and place them in the PCR instrument. Set the reaction conditions according to the program in Table 11. After the RT-PCR program is completed, record the Ct value and calculate 2 -ΔΔCt The mRNA expression was evaluated.

[0241] Table 10 RT-PCR reaction system

[0242]

[0243] Table 11 RT-PCR reaction conditions

[0244]

[0245] 12. Western Blot assay to determine the expression of related proteins

[0246] 12.1 Total Cell Protein Extraction: Following the procedures described in "2.3," pancreatic cancer PANC-1 and HS766T cells in logarithmic growth phase were digested with trypsin, centrifuged, and resuspended in an appropriate amount of DMEM complete medium. The cells were seeded at an appropriate cell density in 6-well plates and transferred to an incubator for overnight culture. After cell attachment, the cells were divided into four groups: control, C15 / Cu, PC-NP / Cu, and HPC-NP / Cu. The corresponding concentration of drugs was added to each group for a continuous intervention of 48 h, and then the cells were washed three times with pre-cooled PBS solution. Subsequently, 150 μL of protein lysis buffer was added to each well, and all the cells were scraped off and collected with a micropipette tip, and placed on ice for 30 min for full lysis. The supernatant solution was collected after centrifugation at 12000 g for 30 min at 4°C, and an appropriate amount of buffer was added to each centrifuge tube according to the volume of the supernatant solution and 5× loading buffer at a ratio of 1:4. After thorough mixing, the cells were heated for denaturation at 98°C for 10 min, and stored in a -20°C refrigerator after cooling.

[0247] 12.2 BCA protein quantification: Prepare BCA working solution according to the required amount for the experiment. After diluting the protein standard with PBS, add 0, 1, 2, 4, 8, 12, 16, and 20 μL to a 96-well plate, respectively. Then add PBS to each well until the total system is 20 μL. After appropriately diluting the sample to be tested, add 20 μL to each well of the 96-well plate. After adding 200 μL of BCA working solution to all the liquid addition wells, place them on a shaker and incubate at 37°C at low speed for 30 minutes. Use a microplate reader to measure the absorbance of the sample at a specific wavelength. Draw a standard curve based on the concentration and absorbance of the protein standard solution. Then, use the standard curve and the absorbance of the sample to be tested to calculate the protein concentration of the sample. Calculate the sample volume based on the protein concentration.

[0248] 12.3 SDS-PAGE gel electrophoresis: (1) Electrophoresis: Wash the glass plate required for the experiment with distilled water, clamp it with a plate clamp, and add an appropriate amount of ultrapure water to check for leaks. After the leak test is completed, pour out the ultrapure water from one side and absorb it with filter paper. Select a separation gel of appropriate concentration according to the molecular weight of the required protein, first prepare the lower layer of gel according to the preparation method in the instructions, then add anhydrous ethanol to flatten the gel surface and let it stand at room temperature. After the lower layer of gel is completely solidified, pour out the anhydrous ethanol. Then continue to prepare the upper layer of gel according to the preparation method in the instructions, add it to the upper edge of the glass plate; then insert a comb from the upper end of the upper layer of gel, and then let it stand at room temperature until it solidifies. Place the glass plate in the electrophoresis tank and fill the tank with electrophoresis fluid. After checking for leaks, slowly pull out the comb; add 2.5μL of protein pre-stained marker to the appropriate position of the gel hole to mark the protein molecular weight, and then add the sample to the appropriate position of the gel hole. Adjust the voltage to 80V and the electrophoresis time to 30min. After the samples have run smoothly, adjust the voltage to 120V and the electrophoresis time to 50min. Stop the electrophoresis when the samples have reached the bottom of the glass plate. (2) Transfer: Cut the PVDF membrane into appropriate sizes according to the experimental requirements in advance and mark its lower left corner. Then activate it in methanol solution in advance. After the electrophoresis is completed, remove the gel and place the required materials flatly on the transfer clamp according to the principle of "black gel and white film" (be careful to avoid bubbles in the PVDF membrane and gel). Then clamp the transfer clamp and place it in the transfer tank. Fill it with transfer solution and adjust the current to 200mA. The transfer time is 90min. (3) Blocking: Prepare 5% skim milk powder in advance. After the transfer is completed, remove the PVDF membrane and completely immerse it in the milk powder. Block it with low speed shaking at room temperature for 2h. After blocking, wash the PVDF membrane with TBST solution 3 times, each time for 10min. (4) Antibody incubation: Prepare the corresponding primary antibody in an appropriate ratio in advance, place the washed PVDF membrane after blocking in the corresponding primary antibody according to the corresponding molecular weight, and incubate it overnight at 4°C with low speed shaking. After the primary antibody incubation, wash the PVDF membrane with TBST solution three times, each time for 10 minutes; then immerse it in the corresponding secondary antibody. Incubate it with slow shaking at room temperature for 1 hour; then wash the PVDF membrane with TBST solution three times. (5) Development: According to the required amount, mix the A solution and B solution in the ECL developer solution in a ratio of 1:1, place the PVDF membrane flat on the designated position in the chemiluminescence imaging analysis system, use a pipette to absorb an appropriate amount of developer and evenly drop it on the PVDF membrane, then develop, take pictures and record. Finally, quantitatively analyze the protein bands using Lane 1D software.

[0249] 13. Results

[0250] 13.1 HPC-NPs inhibit the proliferation of pancreatic cancer cells

[0251] Human pancreatic cancer cells PANC-1 and HS766T cells were selected as experimental cell lines. Both are commonly used human pancreatic cancer cell lines and can highly express the hyaluronic acid receptor CD44. First, the MTT experiment was used to detect the cell survival rate of the two pancreatic cancer cell lines after drug administration. The MTT test results of PANC-1 and HS766T cells in each group were as follows: Figure 11 (a) and Figure 11 As shown in (b); the MTT test results of GEM on PANC-1 and HS766T cells are shown in Figure 12 shown.

[0252] As the drug concentration increased, when blank hyaluronic acid nanoparticles (Free-NP) and CuCl2·2H2O (hereinafter referred to as Cu) were used alone, the cell survival rate did not change significantly, and the survival rate of both cell lines could reach more than 85%, indicating good safety. However, after intervention with C15 / Cu, PC-NP / Cu, HPC-NP / Cu and GEM, the cell survival rate of each group showed a downward trend and showed a concentration-dependent characteristic. 50 The value can reflect the ability of the drug to inhibit tumor cell proliferation; therefore, Graphpad Prism was used to calculate the IC values of each group for the two pancreatic cancer cell lines. 50 value,

[0253] The experimental results in Table 12 show that the HPC-NP / Cu group has the best ability to inhibit the proliferation of pancreatic cancer cells. Its IC 50 The values were 0.29 μM and 0.13 μM, respectively, while the IC values of the GEM group for PANC-1 and HS766T cells were 50 The values were 28.0μM and 21.4μM, respectively, which fully demonstrated that HPC-NP / Cu had good anti-tumor activity and its in vitro anti-pancreatic cancer activity was better than that of the positive drug GEM. In addition, compared with C15 / Cu, the IC 50 The values increased by about 2 times, indicating that the anti-tumor activity of C15 can be further improved after it is prepared into hyaluronic acid nanoparticles.

[0254] Table 12 IC values of C15 / Cu, PC-NP / Cu, HPC-NP / Cu and GEM on pancreatic cancer PANC-1 and HS766T cells, respectively 50 Value (n=3)

[0255]

[0256] According to the IC of HPC-NP / Cu on two pancreatic cancer cells 50The appropriate dosage concentrations for subsequent cell experiments were selected based on the values obtained. For PANC-1 cells, the effective concentrations for the C15 / Cu, PC-NP / Cu, and HPC-NP / Cu groups were 0.13 μM (based on C15), and the effective concentration for GEM was 25 μM. For HS766T cells, the effective concentrations for the C15 / Cu, PC-NP / Cu, and HPC-NP / Cu groups were 0.28 μM (based on C15), and the effective concentration for GEM was 20 μM.

[0257] To further verify the effect of C15 and its nanoparticle preparation combined with copper on the proliferation of pancreatic cancer cells, cell clone formation experiments were performed to detect the effect of C15 and its nanoparticle preparation combined with copper on the spheroid formation ability of pancreatic cancer cells PANC-1 and HS766T. Figure 13 As shown in (a), compared with the Control group, each drug-treated group could significantly reduce the spheroidization ability of the two pancreatic cancer cell lines. The HPC-NP / Cu group reduced the spheroidization ability of PANC-1 cells by about 2.24 times that of the C15 / Cu group. Figure 13 The HPC-NP / Cu group shown in (b) reduced the spheroidization ability of HS766T cells by about 2.71 times that of the C15 / Cu group, indicating that the HPC-NP / Cu group has the best ability to inhibit the spheroidization of pancreatic cancer cells.

[0258] HPC-NPs inhibit pancreatic cancer cell migration and invasion and induce apoptosis

[0259] The cell scratch test can be used to determine the migration of cells before and after drug administration. The ability of drugs to inhibit cell migration is evaluated by calculating the scratch healing rate. The results of the cell migration experiments on PANC-1 and HS766T cells in each drug group are shown as follows: Figure 14 As shown in (a) and (b). Compared with the control group, C15 and its nanoparticle preparation combined with copper group and GEM showed the ability to inhibit pancreatic cancer cell migration to varying degrees. In PANC-1 and HS766T cells, the scratch healing rates of the control group were 40.70% and 34.65%, the scratch healing rates of the C15 / Cu group were 31.91% and 22.05%, the scratch healing rates of the PC-NP / Cu group were 26.87% and 13.14%, the scratch healing rates of the HPC-NP / Cu group were 13.83% and 6.00%, and the scratch healing rates of the GEM group were 29.05% and 17.19%. The above experimental results show that after C15 is prepared into HPC-NP, it can better inhibit the migration of two pancreatic cancer cells. Its effect of inhibiting the migration of PANC-1 and HS766T cells is approximately 2.3 times and 3.6 times that of the C15 / Cu group, 1.9 times and 2.2 times that of the PC-NP / Cu group, and 2.1 times and 2.9 times that of the GEM group, respectively.

[0260] The cell invasion assay can be used to evaluate the cell invasion before and after drug administration. The ability of the drug to inhibit cell invasion is evaluated by counting the number of cells that pass through the polycarbonate membrane and reach the bottom of the chamber. Figure 15 As shown in the figure, compared with the control group, the number of cells that passed through the polycarbonate membrane and reached the bottom of the chamber was significantly reduced after intervention with C15 and its nanoparticle preparation combined with copper group and GEM, indicating that C15 and its nanoparticle preparation combined with copper group and GEM all showed the ability to inhibit pancreatic cancer cell invasion to varying degrees. Figure 15 As shown in (a), the numbers of invasive cells in the Control group, C15 / Cu group, PC-NP / Cu group, HPC-NP / Cu group, and GEM group of PANC-1 cells were 396.9, 311, 255.7, 108.6, and 228.6, respectively. Figure 15 As shown in (b), the numbers of invasive cells in the HS766T cells in the control, C15 / Cu, PC-NP / Cu, HPC-NP / Cu, and GEM groups were 239.7, 171.6, 154.9, 83.8, and 142.0, respectively. In summary, the HPC-NP / Cu group had the best inhibitory effect on cell invasion, with relative invasion rates on PANC-1 and HS766T cells approximately 2.86- and 2.05-fold higher in the C15 / Cu group, 2.36- and 1.85-fold higher in the PC-NP / Cu group, and 2.10- and 1.70-fold higher in the GEM group, respectively.

[0261] In order to detect the effect of drugs on the ability to induce apoptosis of PANC-1 and HS766T cells, flow cytometry was used to quantitatively detect cell apoptosis after 48 hours of treatment with different drugs. Figure 16 The results in (a) show that compared with the control group, the apoptosis rates of C15 and its nanoparticle preparation combined with copper and GEM groups in PANC-1 cells increased, showing the ability to induce apoptosis of pancreatic cancer cells to varying degrees. The apoptosis rates of the control group, C15 / Cu group, PC-NP / Cu group, HPC-NP / Cu group, and GEM group were 5.28%, 10.58%, 17.16%, 18.20%, and 13.95%, respectively. Figure 16 Results in (b) show that in HS766T cells, the apoptosis rates of the control, C15 / Cu, PC-NP / Cu, HPC-NP / Cu, and GEM groups were 3.45%, 8.34%, 14.26%, 19.06%, and 16.86%, respectively. In summary, C15 and its nanoparticle formulation combined with copper treatment can effectively induce apoptosis in pancreatic cancer cells in vitro, with the HPC-NP / Cu group showing the best effect, and its effect is superior to that of the positive drug GEM.

[0262] 13.3HPC-NPs as PDK inhibitors inhibit aerobic glycolysis in pancreatic cancer cells

[0263] 13.3.1 HPC-NPs Inhibit the Expression of Key Glycolytic Enzymes in Pancreatic Cancer Cells

[0264] Studies have shown that pancreatic cancer cells display high levels of glycolysis, and the activity of key glycolytic enzymes is crucial for aerobic glycolysis in tumor cells. Therefore, measuring the expression of key enzymes in the glycolytic pathway before and after drug administration can reflect the extent of drug inhibition of glycolysis. To further explore the mechanism by which C15 combined with copper inhibits pancreatic cancer in vitro, the expression of key enzymes involved in the aerobic glycolytic pathway in pancreatic cancer cells before and after drug administration was examined. First, RT-PCR and Western Blot experiments were used to investigate whether C15 and its nanoparticle formulation combined with copper could inhibit the expression of key glycolytic enzymes in pancreatic cancer PANC-1 and HS766T cells.

[0265] Figure 17 (a) and (b) show that C15 and its nanoparticle preparation combined with copper as PDK inhibitors can inhibit the expression of PDK1, as well as the key enzymes GLUT1, HKⅡ and LDH mRNA in the aerobic glycolysis pathway in PANC-1 and HS766T cells respectively; Figure 18 Figures (a) and (b) show that C15 and its nanoparticle preparation combined with copper as PDK inhibitors can inhibit the expression of PDK1, as well as the key enzymes GLUT1, HKⅡ, and LDHA proteins in the aerobic glycolysis pathway in PANC-1 and HS766T cells, respectively. The inhibitory effect was most significant in the HPC-NP / Cu group. These results further demonstrate that C15 and its nanoparticle preparation combined with copper can act as PDK inhibitors to inhibit the aerobic glycolysis pathway in pancreatic cancer cells, thereby inhibiting tumor growth and proliferation.

[0266] 13.3.2 HPC-NPs Can Reduce Lactic Acid Content in Pancreatic Cancer Cells

[0267] Due to the "Warburg" effect, tumor cells are more inclined to metabolize energy through aerobic glycolysis than normal cells, that is, pyruvate undergoes glycolysis in the cytoplasm and is then metabolized into lactate by lactate dehydrogenase. Previous results have shown that C15 and its nanoparticle preparations combined with copper can act as PDK inhibitors to inhibit the aerobic glycolysis pathway, converting the energy metabolism of tumor cells to oxidative phosphorylation and reducing the production of lactate. Therefore, the ability of C15 and its nanoparticle preparations combined with copper to inhibit glycolysis was evaluated by exploring the changes in lactate content in pancreatic cancer PANC-1 and HS766T cells before and after drug administration. The experimental results are as follows Figure 19As shown in the data, compared with the Control group, each drug-treated group could reduce the lactate content in PANC-1 and HS766T cells, respectively. Among them, the HPC-NP / Cu group had the most significant effect on reducing the lactate content of the two pancreatic cancer cells, and its inhibition level of lactate production was approximately 6.02 times and 2.00 times that of the C15 / Cu group, respectively.

[0268] 13.3.3 HPC-NPs Can Reduce Glucose Uptake in Pancreatic Cancer Cells

[0269] Due to the active glycolysis in tumor cells, glucose can be consumed in large quantities to meet the raw material needs of biomacromolecule synthesis in tumor cells. C15 and its nanoparticle preparation combined with copper, as a PDK inhibitor, can inhibit the aerobic glycolysis pathway in tumor cells, thereby reducing the cells' ability to uptake glucose. Therefore, the ability of C15 and its nanoparticle preparation combined with copper to inhibit glycolysis was evaluated by investigating changes in glucose uptake in pancreatic cancer PANC-1 and HS766T cells before and after treatment.

[0270] The experimental results are as follows Figure 20 As shown in the results, compared with the Control group, each drug-treated group significantly reduced the glucose uptake in pancreatic cancer PANC-1 and HS766T cells, respectively. Among them, the HPC-NP / Cu group had the strongest ability to reduce glucose uptake in both PANC-1 and HS766T pancreatic cancer cells, with relative glucose uptake of 5.5% and 9.8% relative to the Control group, respectively. Compared with the C15 / Cu group, its inhibitory ability was approximately 6.65 times and 2.49 times, respectively. The above results indicate that C15 and its nanoparticle preparation combined with copper as a PDK inhibitor can reduce the glucose uptake capacity of pancreatic cancer cells by inhibiting the glycolysis pathway, thereby inhibiting tumor growth. Moreover, after C15 was prepared into HPC-NP, its effect was significantly enhanced, thereby improving its anti-tumor efficacy.

[0271] 13.3.4 HPC-NPs Can Reduce ATP Production in Pancreatic Cancer Cells

[0272] Aerobic glycolysis provides the large amounts of energy required for rapid tumor cell proliferation. C15 and its nanoparticle formulation combined with copper as a PDK inhibitor can shift tumor cells' energy metabolism from aerobic glycolysis to oxidative phosphorylation, thereby reducing ATP production. Therefore, the ability of C15 and its nanoparticle formulation combined with copper as a PDK inhibitor to inhibit glycolysis was evaluated by investigating ATP production in pancreatic cancer PANC-1 and HS766T cells before and after treatment.

[0273] The experimental results are as follows Figure 21As shown. Compared with the Control group, each drug-treated group can inhibit the level of ATP production in pancreatic cancer PANC-1 and HS766T cells. Among them, the ability of PANC-1 and HS766T pancreatic cancer cells to generate ATP was the weakest after intervention with the HPC-NP / Cu group, with their ATP production levels being 44.31% and 33.7% of the Control group, respectively, and the reduction in ATP production was approximately 1.83 and 1.98 times that of the C15 / Cu group, respectively. The above results indicate that C155 and its nanoparticle preparation combined with copper as a PDK inhibitor can reduce the level of ATP production in pancreatic cancer cells by inhibiting the aerobic glycolysis pathway, thereby inhibiting tumor growth, and the effect is significantly enhanced after C15 is prepared into HPC-NP.

[0274] 13.3.5 HPC-NPs Increase ROS Levels in Pancreatic Cancer Cells

[0275] Numerous studies in recent years have demonstrated that the level of glycolysis is closely correlated with intracellular ROS levels. During aerobic glycolysis in tumor cells, a large amount of pyruvate remains unoxidized, thereby reducing ROS production via the mitochondrial electron transport chain. This leads to reduced ROS production. Long-term maintenance of low ROS levels promotes tumor growth and creates a hypoxic tumor microenvironment. C15 and its nanoparticle formulation combined with copper as a PDK inhibitor can shift tumor cells' energy metabolism from glycolysis to oxidative phosphorylation, thereby increasing ROS levels. Flow cytometry was used to investigate the effects of these drugs on ROS production by measuring the mean fluorescence intensity of DCF-DA in pancreatic cancer PANC-1 and HS766T cells before and after treatment. This study evaluated the ability of C15 and its nanoparticle formulation combined with copper as a PDK inhibitor to inhibit glycolysis.

[0276] The quantitative results of ROS in PANC-1 and HS766T cells were analyzed as follows Figure 22 As shown in (a) and (b) above, compared with the control group, all groups increased ROS levels in pancreatic cancer PANC-1 and HS766T cells. HPC-NP / Cu treatment significantly increased ROS levels in PANC-1 and HS766T cells, with levels in these two groups increasing 2.67 and 4.37 times higher than those in the control group, respectively.

[0277] This result shows that C15 and its nanoparticle preparation combined with copper as a PDK inhibitor can inhibit the aerobic glycolysis pathway, thereby increasing the ROS level in pancreatic cancer cells to inhibit tumor growth, and the effect is best when C15 is prepared into HPC-NP.

[0278] 13.4HPC-NPs can inhibit the stemness of pancreatic cancer cells

[0279] The strength of cell stemness can, to a certain extent, reflect the degree of malignant events such as tumorigenesis, proliferation, metastasis, and drug resistance, as well as the potential for adverse events such as metastasis and drug resistance. RT-PCR and Western Blot experiments were used to examine changes in OCT-4, Nanog, and SOX-2 gene and protein expression in pancreatic cancer PANC-1 and HS766T cells following treatment with C15 and its nanoparticle preparation combined with copper to investigate whether C15 and its nanoparticle preparation combined with copper could inhibit the stemness characteristics of pancreatic cancer cells.

[0280] The RT-PCR results of PANC-1 and HS766T cells were shown in Figure 2 Figure 23 As shown in (a) and (b), compared with the control group, the expression of OCT-4, Nanog, and SOX-2 genes in both PANC-1 and HS766T pancreatic cancer cell lines decreased after treatment with C15 and its nanoparticle preparation combined with copper. The HPC-NP / Cu group showed the most significant reduction in the expression of OCT-4, Nanog, and SOX-2 genes in both pancreatic cancer cell lines.

[0281] Western Blot results of PANC-1 and HS766T cells are shown in Figure 2. Figure 24 As shown in (a) and (b) of Figure 3 . The experimental results show that compared with the control group, C15 and its nanoparticle preparation combined with copper intervention showed a downward trend in the expression of OCT-4, Nanog, and SOX-2 proteins in both PANC-1 and HS766T pancreatic cancer cell lines. The HPC-NP / Cu group showed the most significant reduction in the expression of OCT-4, Nanog, and SOX-2 genes in both pancreatic cancer cell lines. These results are consistent with the results of the RT-PCR experiment. These results indicate that C15 and its nanoparticle preparation combined with copper can simultaneously inhibit the expression of cell stemness.

[0282] 13.5HPC-NPs can inhibit the drug resistance of pancreatic cancer cells

[0283] Although GEM is a first-line clinical treatment for pancreatic cancer, patients are prone to developing drug resistance during GEM treatment, leading to poor efficacy and prognosis. Numerous studies have demonstrated that the level of aerobic glycolysis in various tumor cells is positively correlated with their drug resistance. On the one hand, through glycolysis, tumor cells can rapidly obtain ATP and the carbon source required for rapid proliferation and growth. Furthermore, high levels of glycolysis provide favorable conditions and environments, promoting tumor growth and metastasis. For example, the accumulation of large amounts of lactic acid creates a slightly acidic tumor microenvironment, which degrades and destroys the extracellular matrix, promoting metastasis and drug resistance. Furthermore, upregulation of glycolysis can promote the expression of members of the intracellular ATP transporter family, thereby facilitating drug efflux. Among the various regulatory factors that promote multidrug resistance, the high affinity of multidrug resistance protein 1 (MRP1) for GEM may be a key factor in pancreatic cancer cell resistance to GEM. Therefore, RT-PCR and Western Blot experiments were performed to explore the changes in MRP1 gene and protein after the intervention of C15 and its nanoparticle preparations combined with copper on pancreatic cancer PANC-1 and HS766T to further explore whether C15 and its nanoparticle preparations combined with copper as PDK inhibitors can also inhibit the expression of MRP1 in pancreatic cancer cells to inhibit the expression of drug resistance. The experimental results are as follows Figure 25 Compared with the control group, the expression of MRP1 gene and protein in PANC-1 and HS766T cells was reduced after intervention with C15 and its nanoparticle preparation combined with copper, among which HPC-NP had the most significant inhibitory effect.

[0284] In summary, C15 and its nanoparticle preparation combined with copper can act as PDK inhibitors to inhibit the aerobic glycolysis pathway, and at the same time inhibit the expression of cell stemness markers OCT-4, Nanog, SOX-2 and ATP transporter family member MRP1 to inhibit the cell stemness and drug resistance of pancreatic cancer cells ( Figure 26 ); and after C15 is prepared into hyaluronic acid-modified nanoparticles, its efficacy can be further exerted and its anti-tumor effect can be enhanced.

[0285] 13.6 Conclusion

[0286] This example first determined the in vitro dosing concentration through an MTT assay; then, through cell cloning assays, scratch assays, invasion assays, and cell apoptosis assays, it was demonstrated that HPC-NP / Cu can significantly enhance the ability of C15 to inhibit the proliferation, migration, and invasion of pancreatic cancer PANC-1 and HS766T cells, and induce cell apoptosis, and its effect is superior to that of the positive drug GEM. HPC-NP / Cu can inhibit the expression of key glycolytic enzyme genes and proteins in pancreatic cancer PANC-1 and HS766T cells; at the same time, it can reduce lactate production, glucose uptake capacity, and ATP generation levels and increase ROS content, thereby demonstrating that HPC-NP / Cu can act as a PDK inhibitor to block the aerobic glycolysis pathway of tumor cells to exert an anti-tumor effect. The level of aerobic glycolysis is positively correlated with the acquisition of cell stemness and drug resistance; and the strength of tumor cell stemness can also, to a certain extent, reflect the strength of its drug resistance. HPC-NP / Cu can inhibit the expression of pancreatic cancer PANC-1 and HS766T cell stemness markers Nanog, OCT-4 and SOX-2, as well as the multidrug resistance protein MRP1, indicating that HPC-NP / Cu can inhibit pancreatic cancer cell stemness and drug resistance.

[0287] Example 3 In vivo efficacy of HPC-NPs against pancreatic cancer

[0288] 1. Experimental Materials

[0289] Experimental cell line: Human pancreatic cancer PANC-1 cells were obtained from the School of Basic Medical Sciences, Shandong University.

[0290] Experimental animals: 30 female Balb / c nude mice, 4-5 weeks old, purchased from Jinan Pengyue Co., Ltd. Six mice per cage were housed in individually ventilated cages at a temperature of 25-27°C and a 12-h day-night cycle. Formal experiments were performed after one week of stable housing.

[0291] 2. Experimental methods

[0292] 2.1 Establishment of nude mouse subcutaneous tumor model: PANC-1 cells required for the experiment were prepared to a cell density of 2×10 7 Prepare a single-cell suspension of 100 μL / mL and store on ice until ready for use. Disinfect the axillary skin of nude mice with alcohol. Use a 1 mL syringe to draw up 100 μL of the prepared cell suspension and slowly inject it into the subcutaneous area of the axilla of the nude mouse to establish a nude mouse PANC-1 subcutaneous tumor model.

[0293] 2.2 Animal Grouping and Dosing: After the model was established, mice were randomly divided into five groups, each consisting of six mice: (1) Model group; (2) C15 / Cu group; (3) PC-NP / Cu group; (4) HPC-NP / Cu group; and (5) GEM group. The specific dosing schedule is shown in Table 13. During the experiment, the body weight and tumor size of the mice were recorded every two days. After 14 days, blood was collected from the right eyeball of five mice randomly selected from each group, and the mice were then sacrificed by cervical dislocation.

[0294] Table 13 Animal Dosing Regimen

[0295]

[0296] Copper gluconate solution: Accurately weigh 1.00 mg of copper gluconate powder and dissolve it in 1 mL of normal saline. Ultrasonicate the solution to obtain a 1 mg / mL copper gluconate aqueous solution.

[0297] C15 solution: Accurately weigh 2.00 mg of C15 powder and dissolve it in 1 mL of normal saline. Ultrasonicate the solution to obtain a 2 mg / mL C15 aqueous solution.

[0298] PC-NP solution: Accurately measure 667 μL of the PC-NP solution prepared in Example 1 (3.0 mg / mL, calculated as C15), add physiological saline to 1 mL, and sonicate to fully dissolve it to obtain a PC-NP aqueous solution with a concentration of 2 mg / mL (calculated as C15).

[0299] HPC-NP solution: Accurately measure 800 μL of the prepared HPC-NP solution (2.5 mg / mL, calculated as C15), add normal saline to 1 mL, and sonicate to fully dissolve it to obtain a 2 mg / mL HPC-NP aqueous solution (calculated as C15).

[0300] GEM solution: Accurately weigh 5.00 mg of gemcitabine powder and dissolve it in 1 mL of normal saline. Ultrasonicate to fully dissolve it to obtain a 5 mg / mL copper gluconate aqueous solution.

[0301] 2.3 H&E Staining: Tumor masses and major organs of mice were stained with H&E to assess pathological changes. The procedure was as follows: Tumor masses and major tissues were carefully cleaned with saline and then fixed in 4% paraformaldehyde for at least 24 hours. The fixed tumor masses and tissues were dehydrated with graded ethanol and placed in xylene until transparent before embedding in paraffin. Paraffin blocks were sectioned at 3-5 μm thickness, stretched with hot water, and flattened. After placement on glass slides, the sections were placed at 60°C for 1 hour. Dewaxed with xylene, hydrated with graded ethanol, and rinsed with ultrapure water. The sections were stained with hematoxylin, 1% ethanolic hydrochloric acid, 1% ammonia, and eosin, followed by color separation, bluing, and staining. The sections were then rinsed with ultrapure water. The slides were dehydrated with graded ethanol and placed in xylene until transparent. After the sections were sealed with neutral gum, the slides were placed under a microscope and photographed and recorded.

[0302] 2.4 TUNEL method to detect cell apoptosis: The TUNEL staining method was used to detect the apoptosis of cells in the tumor masses of each group of mice. The specific experimental procedures were as follows: the paraffin-embedded tumor sections were dewaxed and placed in 3% H2O2 solution. After incubation at room temperature for 20 minutes, they were washed with PBS solution three times, and 20 μg / mL proteinase K working solution was added and continued to incubate at 37°C; after 30 minutes, they were washed with PBS solution three times and immersed in blocking solution for 10 minutes; then they were washed again with PBS solution three times, 100 μL of enzyme reaction solution was evenly dropped onto the sections, and incubated at 37°C in the dark for 1 hour; then an appropriate amount of Conventer-POD reagent was added to the sections and incubated at 37°C in the dark for 30 minutes, and finally 50 μL of DAB color development solution was added to the section samples. After staining, they were counterstained and dehydrated and mounted. The processed sections were placed under a microscope to photograph and record.

[0303] 2.5 Immunohistochemistry: Immunohistochemistry (IHC) was used to examine protein expression in tumor masses from each group of mice. The following experimental procedures were performed: Deparaffinized, paraffin-embedded tumor sections were placed in a pH 6.0 citrate buffer and microwaved for 3 minutes on high heat, then on medium heat for 15 minutes. After cooling to room temperature, the sections were washed three times with PBS. The sections were then immersed in serum blocking solution and blocked for 30 minutes at room temperature. After blocking, the blocking solution was discarded, and the primary antibody was applied to the corresponding sections, incubated overnight at 4°C. Secondary antibody was then applied to the PBS-washed sections and incubated for 30 minutes at room temperature. The secondary antibody was removed and recovered, and the sections were washed three times with PBS for 10 minutes each. Fifty microliters of DAB colorimetric solution was then applied to the sections. After staining, the sections were counterstained, dehydrated, and mounted. The sections were photographed and recorded under a microscope.

[0304] 2.6 Serum Biochemical Index Assay: After the last dose, mice were fasted but not watered for 24 hours. An appropriate amount of blood from the right eyeball of each group was collected in a 1.5 mL centrifuge tube and centrifuged at 3000 rpm for 10 minutes at 4°C. The supernatant was then collected and stored at -20°C for testing. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) in the serum of each group of mice were assayed according to the methods described in the respective kit instructions.

[0305] 2.7 Myelosuppression Experiment

[0306] 2.7.1 Extraction and Counting of Peripheral Blood and Bone Marrow Nucleated Cells: After the last dose, mice were fasted but not watered for 24 hours. 0.2 mL of blood was collected from the right eyeball of each group and placed in an anticoagulant tube. Neutrophil and platelet counts were determined using a Mindray CAL 7000 hematology analyzer. The remaining blood was centrifuged and serum collected. The right femur of each mouse was carefully removed, and the muscle and tissue were carefully removed. The femur was then cut open at both ends. The bone marrow cavity was repeatedly flushed with an appropriate amount of PBS solution until the bone marrow cavity gradually changed from light red to white. Bone marrow cells were collected to prepare a single-cell suspension. The suspension was centrifuged at 1000 rpm for 5 minutes, the supernatant discarded, and 1 mL of red blood cell lysis buffer was added. The suspension was allowed to stand at room temperature for 5 minutes. The suspension was centrifuged at 1000 rpm for 5 minutes, the supernatant discarded, and the suspension was washed three times with PBS. The cells were resuspended and the bone marrow nucleated cells were counted under a microscope.

[0307] 2.7.2 Bone Marrow Histopathological Examination: The left femurs of the mice were removed, muscle and tissue removed, carefully wiped with anhydrous ethanol, and fixed in 4% paraformaldehyde for at least 24 hours. Fixation and decalcification were performed as described in "2.3" of this Example. Paraffin sections were stained with H&E, and the morphological changes of the femoral bone marrow tissue of each group of mice were observed under a microscope.

[0308] 2.7.3 ELISA Assay for Serum TPO and EPO Contents: After the final dose, mice were fasted for 24 hours. An appropriate amount of blood was collected from the right eyeball of each group and placed in a 1.5 mL centrifuge tube. The tube was centrifuged at 3000 rpm for 10 minutes at 4°C. The supernatant was then stored at -20°C for testing. The serum levels of thrombopoietin (TPO) and erythropoietin (EPO) in each group of mice were determined using a double-antibody one-step sandwich ELISA.

[0309] 3. Experimental results

[0310] 3.1 HPC-NPs can inhibit the growth of pancreatic cancer cells in vivo

[0311] After PANC-1 cells were injected subcutaneously into the right side of nude mice, a clear tumor mass appeared in the right armpit about a week later. When the tumor mass reached 80-100 mm 3 The mice were then randomly divided into five groups and drug administration began. Changes in mouse body weight and tumor volume were recorded every two days during drug administration. After two weeks of continuous drug administration, eye blood was collected and the nude mice were sacrificed by cervical dislocation. The tumors were then removed and photographed.

[0312] Depend on Figure 27 In (a), it can be seen that the average tumor volume of the HPC-NP / Cu group is the smallest, and its effect is better than that of the positive drug GEM. In (b), it can be seen that the tumor mass in the Model group grows the fastest, and the tumor mass in the HPC-NP / Cu group grows the slowest, indicating that it can effectively inhibit tumor growth. Figure 27 In (c), the average tumor mass weight of the Model group was 0.62±0.11g, the average tumor mass weight of the C15 / Cu group was 0.34±0.14g, the average tumor mass weight of the PC-NP / Cu group was 0.26±0.09g, the average tumor mass weight of the HPC-NP / Cu group was 0.19±0.09g, and the tumor mass weight of the GEM group was 0.33±0.12g. The tumor inhibition rate of each group was also calculated: 48.72% for the C15 / Cu group, 58.09% for the PC-NP / Cu group, 66.10% for the HPC-NP / Cu group, and 45.95% for the GEM group. This indicates that the HPC-NP / Cu group had the highest relative tumor inhibition rate and the best in vivo pancreatic cancer growth inhibition effect, approximately 1.44 times that of the GEM group.

[0313] The comprehensive analysis of the experimental results showed that the tumor mass in the HPC-NP / Cu group was the smallest, the lightest and had the highest relative tumor inhibition rate, indicating that this group had the best effect in inhibiting tumor growth and was superior to the positive drug GEM. In addition, the peeled tumor mass was stained with H&E, and the results are shown in Figure 28 The experimental results show that the cells in the Model group are densely arranged and grow more vigorously; after treatment with C15 and its nanoparticle preparation combined with copper and GEM, the cell density decreased to varying degrees, the cell nucleus shrank, and vacuoles and necrosis appeared between cells, indicating that each drug-treated group can inhibit the proliferation and spread of pancreatic cancer in vivo, among which the HPC-NP / Cu group had the best effect and was better than the positive drug GEM group.

[0314] The above results collectively indicate that C15 and its nanoparticle preparation can effectively inhibit the growth of pancreatic cancer in mice, and HPC-NP / Cu has the best efficacy and is superior to the positive drug GEM.

[0315] 3.2HPC-NPs induce apoptosis of pancreatic cancer cells in nude mice

[0316] The TUNEL apoptosis method was used to detect the apoptosis of cells in subcutaneous tumors of pancreatic cancer nude mice. Figure 29 As shown, almost all cells in the Model group appeared blue (negative expression), with only a few appearing yellow-brown (positive expression). After treatment with C15 / Cu, PC-NP / Cu, HPC-NP / Cu, and the positive drug GEM, positive expression increased. Among them, the increase in positive expression in the HPC-NP / Cu group was the most significant, and the effect was better than that of the positive drug GEM, indicating that HPC-NP / Cu can accumulate in the tumor site and effectively induce apoptosis of pancreatic cancer cells in nude mice.

[0317] 3.3 Mechanism of HPC-NPs in Inhibiting Pancreatic Cancer Cell Growth in Vivo

[0318] C15 and its nanoparticle preparation combined with copper as PDK inhibitors can inhibit the aerobic glycolysis pathway in tumor cells by inhibiting the activity of key enzymes in the glycolysis pathway, while also inhibiting cell stemness and drug resistance. Therefore, to explore the mechanism of action of C15 and its nanoparticle preparation combined with copper in inhibiting pancreatic cancer growth in vivo, immunohistochemistry (IHC) was used to detect the expression of key glycolytic enzymes GLUT1, PDK1, and LDHA, key cell stemness factors Nanog and OCT-4, and multidrug resistance protein MRP1 in tumor masses of mice in the Model group, C15 / Cu group, PC-NP / Cu group, and HC-NP / Cu group.

[0319] IHC qualitative and quantitative results were as follows Figure 30 As shown in (a) and (b). Compared with the Model group, after treatment with C15 and its nanoparticle preparation combined with copper, the protein expression of key enzymes in the aerobic glycolysis pathway, such as GLUT1, PDK1, and LDHA, showed a downward trend, indicating that it can inhibit the aerobic glycolysis process of tumor cells. At the same time, it was also observed that the protein expression of key cell stemness factors Nanog, OCT-4, and multidrug resistance protein MRP1 also decreased, indicating that C15 and its nanoparticle preparation combined with copper as a PDK inhibitor can inhibit the aerobic glycolysis pathway and inhibit the expression of cell stemness and drug resistance.

[0320] In summary, the results of immunohistochemistry experiments show that C15 and its nanoparticle preparation combined with copper as a PDK inhibitor can block the aerobic glycolysis pathway of pancreatic cancer in vivo; at the same time, it inhibits the expression of cell stemness and drug resistance-related proteins, thereby inhibiting the growth of pancreatic cancer, which is consistent with the research results obtained in vitro experiments.

[0321] 3.4 Analysis of in vivo toxicity and bone marrow suppression

[0322] Although GEM is a first-line drug for the clinical treatment of pancreatic cancer, it has severe adverse reactions such as myelosuppression, mainly manifested as leukopenia and thrombocytopenia. Therefore, the degree of myelosuppression of mice by C15 and its nanoparticle preparation combined with copper and GEM was compared through mouse body weight analysis and H&E staining, peripheral blood and bone marrow nucleated cell count measurement, EPO and TPO content determination, and liver and kidney function tests to explore whether C15 and its nanoparticle preparation can better perform than GEM in terms of adverse reactions such as myelosuppression.

[0323] 3.4.1 Analysis of mouse body weight and H&E staining results

[0324] The main organs and right femur bone marrow of each group of mice were taken for H&E staining, and the results of the sections were as follows: Figure 31 As shown, compared with the Model group, no major organ changes were observed in any of the groups. In bone marrow tissue sections, there were similarities between the Model, C15 / Cu, PC-NP / Cu, and HPC-NP / Cu groups. Their bone marrow hematopoietic tissue was relatively intact, with a high bone marrow content within the bone marrow cavity, abundant sinusoids, and normal morphology. However, in the GEM group, mice exhibited bone marrow destruction, bone marrow shedding, a significant decrease in bone marrow content within the bone marrow cavity, a significant decrease in sinusoids, a significantly disorganized structure, a reduced proportion of nucleated bone marrow cells, and numerous vacuoles within the bone marrow tissue. This suggests that compared with the Model group, mice treated with GEM exhibited more severe bone marrow suppression. However, treatment with C15 and its preparations combined with copper did not exhibit bone marrow suppression, demonstrating its superior in vivo safety profile compared to GEM.

[0325] Depend on Figure 32 It can be seen that, except for the GEM group, there was no statistical difference in the changes in mouse body weight in the other groups compared with the Model group, which to a certain extent shows that HPC-NP / Cu has good safety and low toxicity in vivo; while the weight of mice in the GEM group decreased significantly, indicating that GEM has a greater impact on the weight of mice and has certain toxicity.

[0326] 3.4.2 Analysis of the number of nucleated cells in peripheral blood and bone marrow

[0327] White blood cells (leukocytes) phagocytose foreign matter such as viruses and bacteria and serve as a crucial line of defense for the human body. Platelets, on the other hand, are crucial coagulants in the body, their primary function being to stop bleeding and accelerate coagulation. Bone marrow suppression can cause a decrease in white blood cells, which can rapidly progress to the more severe neutropenia, ultimately leading to a decrease in platelets. Bone marrow suppression also reduces the number of bone marrow cells and inhibits the bone marrow microenvironment. Therefore, counting white blood cells, platelets, and nucleated bone marrow cells in mouse peripheral blood is an important criterion for assessing the degree of bone marrow suppression.

[0328] Eyeball blood was collected from mice in each group, and the neutrophil and platelet counts in the blood were measured using a Mindray CAL 7000 blood cell analyzer. Figure 33 It can be seen that the neutrophil and platelet levels of C15 and its nanoparticle preparation combined with copper did not change significantly compared with the Model group; however, the neutrophil and platelet levels of the GEM group decreased to a greater extent than those of the Model group, indicating that a certain degree of bone marrow suppression occurred after treatment with GEM.

[0329] After blood was collected from the eyeballs, the mice were killed by cervical dislocation. The bone marrow nucleated cells were collected from the right femur and counted under an inverted microscope. The number of bone marrow nucleated cells in each group was calculated. 6 The GEM group showed the most significant decrease in bone marrow nucleated cell count, which was 253±25.05 (10 6 / femur), indicating that GEM treatment significantly reduced the number of bone marrow nucleated cells in mice (***P<0.001), indicating severe myelosuppression. However, HPC-NP / Cu treatment showed no significant change in bone marrow nucleated cell count, indicating no myelosuppression and good in vivo safety. In summary, C15 and its nanoparticle formulation combined with copper treatment did not cause myelosuppression, whereas GEM treatment exhibited severe myelosuppression, suggesting, to some extent, that its in vivo safety is superior to GEM.

[0330] 3.4.3 Analysis of serum TPO and EPO levels

[0331] EPO and TPO are crucial in the proliferation of hematopoietic cells. Erythropoietin (EPO) is one of the most important regulatory factors that promotes red blood cell production in the body. It plays an important role in promoting the division, differentiation and maturation of bone marrow erythroid cells. If the EPO content is reduced, it indicates that anemia may occur. The main function of thrombopoietin (TPO) is to regulate the production of platelets. Hematopoietic stem cells can differentiate into megakaryocytes under the stimulation of TPO. In addition, TPO can also promote the proliferation and maturation of megakaryocytes, and can produce and release platelets. Figure 34Compared with the Model group, the GEM group showed varying degrees of decrease in TPO (**P<0.01) and EPO levels (*P<0.05), indicating that GEM treatment resulted in myelosuppression in mice. In contrast, the serum TPO and EPO levels in the remaining groups did not change significantly compared to the Model group, resulting in statistically insignificant differences. These results suggest that C15 and its nanoparticle formulation combined with copper treatment do not exhibit the myelosuppression characteristic of GEM, demonstrating a superior in vivo safety profile compared to GEM.

[0332] 3.4.4 Liver and kidney function index testing

[0333] The serum of mice in each group was taken to test the routine liver and kidney functions. The experimental results are as follows: Figure 35 Compared with the model group, mice treated with GEM showed slight increases in ALT and AST levels, indicating mild liver damage. Furthermore, GEM-treated mice also showed increases in BUN and Cr levels, suggesting a certain degree of kidney damage. In contrast, mice treated with C15 and its nanoparticle formulation combined with copper showed no significant differences in ALT, AST, BUN, or Cr levels compared with the model group, further demonstrating the good in vivo safety of C15 and its nanoparticle formulation combined with copper.

[0334] 3.5 Conclusion

[0335] Tail vein injection of C15 and its nanoparticle formulation combined with copper administration inhibited the growth of subcutaneous pancreatic cancer tumors in nude mice and induced apoptosis. HA modification of the nanoparticles enhanced their tumor-targeting ability, resulting in superior antitumor efficacy compared to the positive-drug GEM. HPC-NP / Cu, as a PDK inhibitor, inhibited the growth of subcutaneous pancreatic cancer tumors in nude mice by inhibiting the expression of key glycolytic enzymes GLUT1, PDK1, and LDHA, as well as the stemness markers Nanog and OCT-4, and the multidrug resistance protein MRP1, exerting anti-pancreatic cancer effects. Compared with the model group, H&E staining, blood routine tests, bone marrow nucleated cell counts, serum TPO and EPO levels, and liver and renal function tests demonstrated a favorable in vivo safety profile for HPC-NP / Cu. In contrast, the positive-drug GEM group exhibited adverse reactions such as myelosuppression. This suggests that HPC-NP / Cu not only enhances antitumor efficacy but also exhibits a superior in vivo safety profile compared to GEM.

[0336] The above description is merely a preferred embodiment of the present application and is not intended to limit the scope of the present application. Although the present application has been described in detail with reference to the above embodiments, those skilled in the art may, after reading this description, make various modifications to the technical solution or replace some of the technical features with equivalents. Any modification, equivalent replacement, or improvement made within the spirit and principles of the present application shall be deemed to fall within the scope of protection of the present application.

Claims

1. A hyaluronic acid-modified PLGA nanoformulation loaded with CPD12C15, characterized in that: The nanoformulation comprises PLGA, CPD12C15 and hyaluronic acid, wherein PLGA forms a core layer structure, CPD12C15 is encapsulated in the PLGA core layer structure, and hyaluronic acid forms a modified layer covering the surface of the core layer structure through electrostatic adsorption, wherein the structure of CPD12C15 is:

2. The nanoformulation according to claim 1, characterized in that The mass ratio of CPD12C15 to PLGA is 1:2 to 1:6, preferably 1:

2.

3. The nanoformulation according to claim 1 or 2, characterized in that The hyaluronic acid is bound to the surface of the nanoparticles by forming electrostatic interactions with the positively charged groups on the surface of PLGA; Preferably, the molecular weight of the hyaluronic acid is 5,000-250,000 Daltons, preferably 6,000-240,000 Daltons, more preferably 6,000 Daltons.

4. The nanoformulation according to claim 1 or 2, characterized in that The nanoformulation has a particle size of 50-200 nm, a zeta potential of -5 to -30 mV, an encapsulation efficiency of 50%-85%, and a drug loading of 10%-25%; Preferably, the nanoformulation has a particle size of 100-150 nm, a Zeta potential of -15 to -20 mV, an encapsulation efficiency of 60%-80%, and a drug loading of 16%-18%.

5. A method for preparing the hyaluronic acid-modified CPD12C15-loaded PLGA nanoformulation according to any one of claims 1 to 4, characterized in that: include: CPD12C15 and PLGA were dissolved in an organic solvent to form an organic phase; Dissolving the cationic surfactant in the aqueous phase and mixing it with the organic phase to form an emulsion; The organic solvent is removed to form PLGA nanoparticles; The PLGA nanoparticles are brought into contact with a hyaluronic acid aqueous solution, so that the hyaluronic acid is modified on the surface of the PLGA nanoparticles by electrostatic adsorption, thereby obtaining a hyaluronic acid-modified PLGA nanoformulation loaded with CPD12C15.

6. The method according to claim 5, characterized in that The organic solvent is selected from one or a mixture of dichloromethane, chloroform, ethyl acetate or acetone; Preferably, the cationic surfactant is selected from dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide or a combination thereof; Preferably, the mass ratio of CPD12C15 to PLGA is 1:2 to 1:6; Preferably, the volume ratio of the organic phase to the aqueous phase is 1:2 to 1:5; Preferably, the concentration of PLGA is 5-25 mg / mL, preferably 10-20 mg / mL; Preferably, the concentration of the cationic surfactant is 0.5%-2.5%, preferably 0.5%-1.5%; Preferably, the molecular weight of the hyaluronic acid is 5,000-250,000 Daltons, preferably 6,000-240,000 Daltons; Preferably, the concentration of the hyaluronic acid is 0.05-2 mg / mL, preferably 0.1-1.5 mg / mL; Preferably, the organic phase and the aqueous phase are mixed by one or more of ultrasound, high-speed stirring or high-pressure homogenization.

7. A pharmaceutical composition comprising the nanoformulation according to any one of claims 1 to 4; Preferably, it further comprises a pharmaceutically acceptable copper salt; Preferably, the copper salt is selected from one or more of copper gluconate, copper chloride, and copper sulfate, preferably copper gluconate; Preferably, the molar ratio of the nanoformulation to copper ions is 0.5:1 to 1.5:1, preferably 1:

1.

8. A medical kit comprising: A first container containing the nanoformulation according to any one of claims 1 to 4; a second container containing a pharmaceutically acceptable copper salt solution; The two containers are designed for simultaneous, separate or sequential administration.

9. Use of the nanoformulation according to any one of claims 1 to 4 in the preparation of a drug for treating pancreatic cancer.

10. The use according to claim 9, characterized in that The drug is used to treat pancreatic cancer that is resistant to gemcitabine; Preferably, the nanoformulation is used in combination with copper ions, wherein the copper ions are divalent copper ions; Preferably, the divalent copper ion is a copper ion provided by copper chloride, copper sulfate or copper gluconate; Preferably, the molar ratio of the nanoformulation to copper ions is 0.5:1 to 1.5:1, preferably 1:1; Preferably, the nanoformulation is administered intravenously at a dose of 5-15 mg / kg, calculated as CPD12C15; the copper ion is administered orally in the form of copper gluconate at a dose of 1-5 mg / kg, calculated as copper gluconate.

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