Polylysine nanomaterial for oxaliplatin delivery and preparation method thereof

Abstract

The application provides a polylysine nanomaterial for oxaliplatin transmission and a preparation method thereof, and relates to the technical field of biopharmacy. The synthesis method has clear synthesis route and strong controllability. Through modular design, alkynyl hyaluronic acid, azide polylysine-vitamin E succinate and MMP enzyme sensitive peptide segment connector are prepared in sequence, and finally, click chemistry is used for efficient coupling. The product has clear structure, high purity and good batch reproducibility, and overcomes the problems of low coupling efficiency and many by-products in traditional methods. The prepared drug has the abilities of active targeting and double response drug release. The hyaluronic acid can specifically recognize the CD44 receptor highly expressed on the surface of tumor cells to realize active targeting. The MMP enzyme sensitive peptide segment can be cleaved by the overexpressed MMP-9 enzyme in the tumor microenvironment to realize enzyme response drug release. Meanwhile, the disulfide bond structure introduced in the carrier can be broken in the high concentration glutathione environment to realize reduction response drug release.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a polylysine nanomaterial for oxaliplatin delivery and its preparation method. Background Technology

[0002] Oxaliplatin, a third-generation platinum-based antitumor drug, is a core component of chemotherapy regimens for colorectal cancer. It inhibits tumor cell proliferation by inducing interstrand cross-linking of DNA. However, the clinical application of oxaliplatin faces multiple challenges: firstly, its lack of tumor tissue-specific distribution leads to severe dose-limiting toxicities, such as peripheral neurotoxicity and myelosuppression; secondly, the complex microenvironment within solid tumors (such as high pressure and hypoxia) hinders effective drug penetration and accumulation; and thirdly, tumor cells readily develop resistance to oxaliplatin, reducing treatment response.

[0003] To improve efficacy and safety, nanomedicine delivery systems have been extensively studied. Current technologies mostly employ polyethylene glycol (PEG)-modified nanocarriers, utilizing the enhanced penetration and retention (EPR) effect to achieve passive targeting. However, the EPR effect varies significantly among different patients and tumor types, and PEGylation may lead to an "accelerated blood clearance" phenomenon. Furthermore, most delivery systems are single-stimuli responsive (e.g., sensitive only to glutathione), lacking sufficient intelligence in drug release, making it difficult to achieve precise localization and rapid release of drugs at the lesion site.

[0004] In recent years, nanocarriers based on active targeting ligands (such as hyaluronic acid) and multiple response mechanisms have shown great potential. However, how to efficiently and controllably integrate these functional modules into a single carrier system while ensuring carrier stability, high drug loading capacity, and biocompatibility remains a critical technical challenge. Existing synthetic methods are often cumbersome, have low coupling efficiency, or produce numerous byproducts, making it difficult to guarantee the uniformity and reproducibility of the final product structure, thus limiting their clinical translation prospects.

[0005] Therefore, developing a novel nanodelivery system with a well-defined synthetic route, active targeting and multiple-response release capabilities, and synergistic effects with oxaliplatin is of great significance for improving the treatment efficacy of colorectal cancer.

[0006] Therefore, this invention is proposed. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a polylysine nanomaterial for oxaliplatin delivery and its preparation method. This preparation method has a clear synthetic route, possesses both active targeting and multiple response release capabilities, and can synergistically enhance the effects of oxaliplatin, providing a novel solution for the treatment of rectal cancer.

[0008] In order to achieve the objective of this invention, the following technical solution is adopted: This invention provides a method for preparing polylysine nanomaterials for oxaliplatin delivery, comprising the following steps: S1. Hyaluronic acid was dissolved in PBS buffer, and 4-pentyneic acid, EDC and NSH were added to react. After the reaction was completed, the mixture was dialyzed and lyophilized to obtain synthesized alkynylated hyaluronic acid (HA-Alk). S2. Poly-L-lysine was dissolved in DMSO, and vitamin E succinate, EDC and NSH were added to react. Then, it was reacted with 3-azidopropylamine under the action of EDC and NHS, dialyzed and lyophilized to obtain azidolated polylysine-vitamin E succinate (PLLS-TOS-N3). S3. Modify the C-terminus of the MMP-9 sensitive sequence with an alkynyl group and the N-terminus with an azide group to obtain the MMP enzyme-sensitive peptide-linker. S4. The alkydized hyaluronic acid, azidolated polylysine-vitamin E succinate and MMP enzyme-sensitive peptide linker were dissolved in PBS, and CuSO4 and sodium ascorbate were added. The mixture was reacted at room temperature, purified by dialysis, and lyophilized to obtain polylysine nanomaterials (HA-Peptide-PLLS-TOS).

[0009] Furthermore, in S1, the pH of the reaction is 6.0, and the reaction temperature is 4°C.

[0010] Furthermore, in S1, dialysis is performed using a dialysis bag with a molecular weight cutoff of 3.5 kDa; The specific steps are as follows: Dialyze with 0.1M NaCl solution at 4℃ for 6 hours, followed by dialysis with ultrapure water for 48 hours. When dialysis with ultrapure water, change the water every 8 hours.

[0011] Furthermore, the MMP-9 sensitive sequence is shown in SEQ ID NO.1.

[0012] SEQ ID NO.1: GPLGVRGK.

[0013] The present invention also provides polylysine nanomaterials for oxaliplatin transport prepared by the above preparation method.

[0014] This invention also provides a method for preparing an oxaliplatin-loaded drug, using the above-mentioned polylysine nanomaterials for oxaliplatin delivery, comprising the following steps: Oxaliplatin and polylysine nanomaterials were co-dissolved in DMSO, and the organic solvent was removed after ultrasonic emulsification to obtain oxaliplatin-loaded drugs (OXA@HA-PLLS-TOS NPs).

[0015] Furthermore, the mass ratio of oxaliplatin to polylysine nanomaterials is 1:(3-4).

[0016] Furthermore, the mass ratio of oxaliplatin to polylysine nanomaterials is 1:3.3.

[0017] The present invention also provides a method for preparing the above-mentioned oxaliplatin-loaded drug to obtain an oxaliplatin-loaded drug.

[0018] This invention also provides the use of oxaliplatin-loaded drugs in the preparation of cancer treatment drugs.

[0019] Furthermore, the cancer in question is colorectal cancer.

[0020] The present invention has the following technical effects: (1) The synthetic route is well-defined and highly controllable. Through modular design, alkynylated hyaluronic acid, azidolated polylysine-vitamin E succinate and MMP enzyme-sensitive peptide linker were prepared sequentially. Finally, click chemistry was used for efficient coupling. The product structure was well-defined, the purity was high, and the batch-to-batch reproducibility was good, which overcame the problems of low coupling efficiency and many by-products in traditional methods.

[0021] (2) The prepared drug possesses both active targeting and dual-response drug release capabilities. Hyaluronic acid can specifically recognize the CD44 receptor highly expressed on the surface of tumor cells, achieving active targeting; the MMP enzyme-sensitive peptide can be cleaved by the overexpressed MMP-9 enzyme in the tumor microenvironment, achieving enzyme-responsive drug release; simultaneously, the disulfide bond structure introduced in the carrier can be cleaved in a high-concentration glutathione environment, achieving reduction-responsive drug release. The synergistic effect of these two factors significantly enhances the accumulation and precise release of the drug at the tumor site.

[0022] (3) The carrier has both therapeutic synergy and safety. Vitamin E succinate not only serves as a hydrophobic core for drug encapsulation, but also has anticancer activity itself, which can synergistically enhance the effect of oxaliplatin; the hydrophilic shell of hyaluronic acid can prolong the in vivo circulation time of nanoparticles, reduce immune clearance, and improve bioavailability.

[0023] (4) The drug delivery system has excellent performance. The nanoparticles prepared by this method have uniform particle size and good stability, high drug loading capacity and good encapsulation efficiency for oxaliplatin, and the in vitro release experiment shows that its release behavior is significantly enhanced in the simulated tumor microenvironment, and it has good sustained-release and intelligent drug release characteristics.

[0024] This invention provides an efficient, intelligent, and safe nanocarrier system for the targeted delivery of oxaliplatin, which has important application value in improving the treatment effect of colorectal cancer and reducing toxic side effects. Attached Figure Description

[0025] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0026] Figure 1 TEM image of polylysine nanomaterials transported by oxaliplatin. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0028] In a first aspect, the present invention provides a method for preparing polylysine nanomaterials for oxaliplatin delivery, comprising the following steps: S1. Hyaluronic acid was dissolved in PBS buffer, and 4-pentyneic acid, EDC and NSH were added to react. After the reaction was completed, the mixture was dialyzed and lyophilized to obtain synthesized alkynylated hyaluronic acid (HA-Alk). S2. Poly-L-lysine was dissolved in DMSO, and vitamin E succinate, EDC and NSH were added to react. Then, it was reacted with 3-azidopropylamine under the action of EDC and NHS, dialyzed and lyophilized to obtain azidolated polylysine-vitamin E succinate (PLLS-TOS-N3). S3. Modify the C-terminus of the MMP-9 sensitive sequence with an alkynyl group and the N-terminus with an azide group to obtain the MMP enzyme-sensitive peptide-linker. S4. The alkydized hyaluronic acid, azidolated polylysine-vitamin E succinate and MMP enzyme-sensitive peptide linker were dissolved in PBS, and CuSO4 and sodium ascorbate were added. The mixture was reacted at room temperature, purified by dialysis, and lyophilized to obtain polylysine nanomaterials (HA-Peptide-PLLS-TOS).

[0029] This application adopts a modular functional construction approach. First, three functionally defined modules are constructed: one that provides active targeting (HA), one that constitutes a hydrophobic core and synergistically treats (PLLS-TOS), and one that acts as a tumor microenvironment response switch (MMP-sensitive peptide).

[0030] The three modules were efficiently and specifically covalently linked using the Huisgen cycloaddition reaction of azido-alkynyl groups (click chemistry). This reaction has the advantages of mild conditions, high yield, few byproducts, and high tolerance to functional groups, ensuring the accuracy and uniformity of the structure of the final product (HA-Peptide-PLLS-TOS).

[0031] Compared to the potential for multi-site random linkages that can result from traditional amide coupling, click chemistry offers a site-specific and quantitative coupling method, ensuring consistent molecular structures across batches of products, which is beneficial for industrial scale-up and quality control. This method successfully integrates three major functions—active targeting (HA), enzyme-responsive release (MMP peptides), and hydrophobic drug delivery / synergistic therapy (PLLS-TOS)—into a single nanocarrier. The constructed carrier provides the physicochemical basis for subsequent active tumor site recognition, microenvironment-triggered drug release, and synergistic therapy.

[0032] In some embodiments, in S1, the pH of the reaction is 6.0 and the temperature is 4°C.

[0033] The activation and coupling of HA carboxyl groups were performed in MES buffer (pH=6.0). This pH environment is conducive to the efficient activation of carboxyl groups by EDC / NHS, forming a reactive intermediate, while minimizing the hydrolytic degradation of hyaluronic acid under strong acid or strong alkaline conditions, thus maintaining its molecular integrity and biological activity. Low-temperature reaction further inhibits side reactions such as hydrolysis and thermal degradation of the HA chain, ensuring the structural stability and functional integrity of the product (HA-Alk). By suppressing side reactions, the selectivity and efficiency of the target reaction (alkynylation) are improved, ensuring a relatively uniform number of alkynyl groups grafted onto each HA molecule.

[0034] The mild reaction conditions preserve the natural structure of hyaluronic acid to the greatest extent possible, which is crucial for its subsequent specific binding (targeting) to the CD44 receptor on the surface of tumor cells.

[0035] In some embodiments, in step S1, dialysis is performed using a dialysis bag with a molecular weight cutoff of 3.5 kDa; The specific steps are as follows: Dialyze with 0.1M NaCl solution at 4℃ for 6 hours, followed by dialysis with ultrapure water for 48 hours. When dialysis with ultrapure water, change the water every 8 hours.

[0036] First, dialyze with 0.1M NaCl solution. This utilizes the salt effect and concentration gradient to effectively remove unreacted ionic small molecule reagents (such as EDC, NHS, and their byproducts) from the reaction system. Then, dialyze with ultrapure water for an extended period to thoroughly remove salts and other small molecule impurities, yielding high-purity HA-Alk.

[0037] Stepwise dialysis can efficiently and thoroughly purify products, avoiding the influence of impurities on subsequent click chemistry reactions and the biosafety of the final nanomaterials. Specific purification parameters (dialysis bag molecular weight cutoff, time, and fluid exchange frequency) ensure the reproducibility and stability of the purification process.

[0038] In some embodiments, the MMP-9 sensitive sequence is shown in SEQ ID NO.1.

[0039] SEQ ID NO.1: GPLGVRGK.

[0040] The sequence GPLGVRGK is a highly specific substrate for matrix metalloproteinase-9 (MMP-9). MMP-9 is overexpressed in a variety of tumor microenvironments.

[0041] The peptide was introduced into the vector as a linker. When the vector was enriched at the tumor site, MMP-9 could specifically cleave the peptide.

[0042] The cleavage process causes the hydrophilic shell (HA) to dissociate from the hydrophobic core (PLLS-TOS), disrupting the nanostructure and rapidly releasing the loaded oxaliplatin. This results in a "burst" release of the drug at the lesion site, increasing local drug concentration. This release mechanism is spatially specific, being triggered only in MMP-9-rich tumor tissue, further reducing toxicity to normal tissues.

[0043] Secondly, the present invention also provides polylysine nanomaterials for oxaliplatin transport prepared by the above preparation method.

[0044] Thirdly, the present invention also provides a method for preparing an oxaliplatin-loaded drug, using the above-mentioned polylysine nanomaterials for oxaliplatin delivery, comprising the following steps: Oxaliplatin and polylysine nanomaterials were co-dissolved in DMSO, and the organic solvent was removed after ultrasonic emulsification to obtain oxaliplatin-loaded drugs (OXA@HA-PLLS-TOS NPs).

[0045] The hydrophobic drug oxaliplatin is co-dissolved with an amphiphilic polymer carrier in an organic phase (DMSO). Nanoscale droplets are formed through ultrasonic emulsification. Subsequently, during dialysis to remove the organic solvent, the carrier molecules self-assemble in the aqueous phase to form stable nanoparticles, simultaneously encapsulating the drug within its hydrophobic core (PLLS-TOS region). This method utilizes the self-assembly properties of the carrier to effectively encapsulate the hydrophobic drug within its hydrophobic core, typically achieving high drug encapsulation efficiency and loading. It avoids conditions such as high temperatures and vigorous stirring that could damage the activity of the drug or carrier, making it suitable for water-sensitive drugs such as oxaliplatin.

[0046] In some embodiments, the mass ratio of oxaliplatin to polylysine nanomaterials is 1:(3-4).

[0047] In some embodiments, the mass ratio of oxaliplatin to polylysine nanomaterials is 1:3.3.

[0048] At this ratio, a sufficiently high oxaliplatin drug loading capacity can be ensured to meet therapeutic needs, while also ensuring that the carrier molecules form structurally stable and uniformly sized nanoparticles, preventing nanoparticle aggregation or leakage due to excessive drug loading. Optimized drug-loaded nanoparticles possess suitable particle size (facilitating EPR effect and cellular uptake) and stable structure (facilitating long-term circulation in vivo), thereby optimizing their overall in vivo pharmacodynamics and pharmacokinetic behavior, maximizing efficacy and minimizing toxicity.

[0049] From the underlying chemical synthesis (modular assembly via click chemistry) to key process control (reaction conditions, purification, drug loading ratio), and finally to the final product and application, a complete and meticulous patent protection system is in place. Its core innovation lies in using click chemistry as a bridge to efficiently integrate the three major functions of hyaluronic acid targeting, MMP-9 enzyme response, and vitamin E succinate synergistic therapy with polylysine nanocarriers for the first time. This creates a novel oxaliplatin delivery system with controllable structure and intelligent function, aiming to systematically solve key problems in the clinical application of oxaliplatin, such as poor targeting, high toxicity, and easy drug resistance.

[0050] Fourthly, the present invention also provides a drug loaded with oxaliplatin prepared by the above-mentioned method for preparing the drug loaded with oxaliplatin.

[0051] Fifthly, the present invention also provides the use of oxaliplatin-loaded drugs in the preparation of cancer treatment drugs.

[0052] In some embodiments, the cancer is colorectal cancer.

[0053] The following is a detailed explanation using specific embodiments: Example 1: Preparation of polylysine nanomaterials S1. Synthesize acetylated hyaluronic acid (HA-Alk) Dissolve 1.0 g of hyaluronic acid (MW=10kDa) in PBS buffer, add 0.5 g of 4-pentyneic acid, 0.75 g of EDC and 0.45 g of NHS, and react at 4 °C for 24 h.

[0054] After the reaction was completed, the solution was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed with 0.1 M NaCl solution at 4 °C for 6 hours, followed by dialyzed with ultrapure water for 48 hours. When using ultrapure water for dialyzed analysis, the water was changed every 8 hours. The solution was then freeze-dried to obtain HA-Alk.

[0055] S2, Synthesize azidolated polylysine-vitamin E succinate (PLLS-TOS-N3) 1.0 g of poly-L-lysine (PLLS, MW=15kDa) was dissolved in DMSO, and vitamin E succinate (TOS, 0.8 g), EDC (0.6 g) and NHS (0.36 g) were added. The mixture was reacted at 40 °C for 24 h to obtain PLLS-TOS.

[0056] PLLS-TOS was reacted with 3-azidopropylamine (0.3 g) under EDC and NHS for 12 h, and then lyophilized by dialyzing to obtain PLLS-TOS-N3.

[0057] S3. Synthesize MMP-sensitive peptide-linker. The peptide GPLGVRGK (MMP-9 sensitive sequence) was designed and synthesized, with an alkynyl group modified at the C-terminus and an azide group modified at the N-terminus.

[0058] Synthesized using solid-phase peptide synthesis method, with a purity >95%.

[0059] S4, Click chemical assembly HA-Peptide-PLLS-TOS HA-Alk (0.5g), Peptide-Linker (0.2g) and PLLS-TOS-N3 (0.5g) were dissolved in PBS, and CuSO4 (5mM) and sodium ascorbate (10mM) were added. The mixture was reacted at room temperature for 12h.

[0060] The reaction solution was purified by dialysis (MWCO=10kDa) and lyophilized to obtain the final product HA-PLLS-TOS.

[0061] Example 2: Preparation of oxaliplatin (OXA)-loaded drugs 10 mg of HA-PLLS-TOS and 3 mg of OXA were co-dissolved in a DMSO / PBS mixed solution, ultrasonically emulsified, and then dialyzed (MWCO=3.5kDa) to remove the organic solvent, yielding OXA@HA-PLLS-TOS nanoparticles.

[0062] Comparative Example 1: Preparation method of conventional PEG-PLLS-TOS@OXA NPs The traditional method for preparing PEG-PLLS-TOS@OXA NPs is as follows: PLLS-TOS Synthesis: PLLS and TOS were dissolved in anhydrous DMSO at a specific molar ratio. Condensing agents EDC and NHS were added, and the reaction was carried out under inert gas protection at 40-50°C for 24-48 hours. After the reaction was complete, the mixture was transferred to a dialysis bag (MWCO 3.5 kDa), dialyzed against DMSO, then against an ethanol / water mixture and pure water, and finally lyophilized to obtain the PLLS-TOS intermediate.

[0063] Synthesis of mPEG-PLLS-TOS: The above PLLS-TOS intermediate was subjected to an amidation reaction with mPEG-COOH again via EDC / NHS catalysis, under conditions similar to the first step. After the reaction, the mixture was dialyzed (MWCO 7-10 kDa) and lyophilized to obtain the final amphiphilic copolymer mPEG-PLLS-TOS.

[0064] The mPEG-PLLS-TOS polymer and OXA are dissolved together in an organic solvent (such as DMSO or ethanol) at a certain mass ratio (1:5).

[0065] Under magnetic stirring, the above organic phase is slowly added dropwise or injected into a certain volume of aqueous phase (ultrapure water is used in this method).

[0066] Immediately use a probe sonicator to sonicate the mixture (ice bath, 200-300W, intermittent sonication for several minutes) to form a uniform nanoemulsion.

[0067] The resulting emulsion was transferred to a dialysis bag (MWCO 3.5 kDa) and dialyzed against a large volume of ultrapure water for 12–24 hours to completely remove organic solvents and unencapsulated free OXA.

[0068] Collect the liquid from the dialysis bag and sterilize it by filtration through a 0.45 or 0.22 µm microporous membrane to obtain a PEG-PLLS-TOS@OXA NPs colloidal solution, which can be stored at 4 °C or lyophilized.

[0069] Experimental Example 1: Characterization of the product prepared in Example 1 The drug prepared in Example 1 was characterized, and the experimental results are as follows: Figure 1 As shown in the TEM image, the mPssPC-OXA NPs are spherical with a uniform particle size distribution. The size of the drug-loaded nanoparticles in the field of view is around 200 nm.

[0070] Experimental Example 2: Stability Verification of Materials and Drugs Objective: To evaluate the physical stability of colloidal solutions of nanoparticles under different conditions, and the chemical stability of the drug after loading with oxaliplatin (OXA).

[0071] 2.1 Experimental Procedure Sample preparation: Three batches of OXA@HA-PLLS-TOS NPs colloidal solutions (1 mg / mL, based on carrier) were prepared and labeled as P1, P2 and P3, respectively; And as a control, unloaded blank nanoparticles HA-PLLS-TOS NPs; Traditional PEGylated control nanoparticles PEG-PLLS-TOS@OXA NPs.

[0072] Accelerated stability testing: 4℃ storage: Place the sample in a 4℃ refrigerator and take samples on days 0, 1, 3, 7, 14 and 30.

[0073] Storage at 25℃: Place the sample in a 25℃ constant temperature chamber, and take samples at the same time points as above.

[0074] Repeated freeze-thaw cycle: The sample is frozen at -20°C and then thawed at 25°C, which constitutes one cycle. Three cycles are performed, and samples are taken after each cycle to calculate the average value.

[0075] Detection indicators: The hydrated particle size (DLS), polydispersity index (PDI), and zeta potential of each sample were measured using a dynamic light scattering instrument. Simultaneously, the presence of precipitation or flocculation in the solution was observed.

[0076] Freeze-drying and reconstitution experiment: A batch of nanoparticle colloidal solution was freeze-dried to obtain a solid powder. On day 0 and day 30, it was reconstituted with ultrapure water to the original concentration, and the above-mentioned physical properties were immediately tested.

[0077] The experimental results are shown in Table 1.

[0078] Table 1: Results of stability validation of materials and drugs The nanoparticles (OXA@HA-PLLS-TOS NPs) in this scheme exhibited minimal changes in particle size, PDI (polydispersity index), and Zeta potential after 30 days of storage at 4°C, and remained clear and transparent, demonstrating excellent long-term stability.

[0079] At 25°C or after repeated freeze-thaw cycles, the stability decreased slightly (particle size increased slightly, PDI increased, and slight turbidity occurred), but the basic structure was still maintained, indicating that it can withstand certain fluctuations in transportation or storage conditions.

[0080] The blank vector (HA-PLLS-TOS NPs) showed the best stability, indicating that the drug loading process has a controllable impact on the vector structure.

[0081] Traditional PEGylated nanoparticles (PEG-PLLS-TOS@OXA NPs) showed more significant particle size growth, higher PDI, and precipitation after 30 days under the same 4℃ conditions, highlighting the stability advantage of this scheme in terms of structural design.

[0082] 2.2 Chemical stability test of the drug (OXA) step: Plasma stability: Free OXA solution and OXA@HA-PLLS-TOS NPs (with the same OXA concentration) were mixed with 50% (v / v) fresh rat plasma and incubated at 37°C.

[0083] Sampling and Detection: Samples were taken at 0, 1, 2, 4, 8, and 24 hours. Proteins were immediately precipitated with acetonitrile and centrifuged. The content of intact OXA in the supernatant was determined using high-performance liquid chromatography (HPLC). Chromatographic conditions: C18 column, mobile phase: methanol / water (containing 0.1% formic acid), detection wavelength: 210 nm.

[0084] Data analysis: Calculate the percentage of remaining OXA at ​​each time point.

[0085] The experimental results are shown in Table 2.

[0086] Table 2: Chemical stability test of drug (OXA) After 24 hours of incubation in plasma, free oxaliplatin (OXA) was largely degraded (65% remaining), while the OXA encapsulated in the nanoparticles of this method remained highly intact (92% remaining). This demonstrates that the nanocarrier can effectively protect the drug and prevent it from prematurely degrading in the bloodstream, which is beneficial for targeted drug delivery.

[0087] Experimental Example 3: Study on in vitro drug release behavior Objective: To verify the dual-response (GSH and MMP enzyme) drug release characteristics of drug-loaded nanoparticles in a simulated tumor microenvironment.

[0088] step: Release medium settings: Four different release media are set to simulate different physiological environments: Group A (normal physiological environment): PBS (pH=7.4); Group B (high reducing environment): PBS (pH=7.4) + 10 mM GSH; Group C (enzyme response environment): PBS (pH=7.4) + 100 ng / mL MMP-9; Group D (tumor microenvironment simulation): PBS (pH=7.4) + 10 mM GSH + 100 ng / mL MMP-9; Experimental procedure: Take an equal amount of OXA@HA-PLLS-TOS NPs (containing approximately 1 mg of OXA) and place it in a dialysis bag with a molecular weight cutoff (MWCO) of 12-14 kDa. Immerse the bag in 50 mL of the above release medium and shake at 37°C and 100 rpm.

[0089] Sampling and detection: At 0.5, 1, 2, 4, 8, 12, 24, and 48 hours, 1 mL of the external release medium was collected (and 1 mL of fresh medium was added). The concentration of OXA in the collected medium was determined by HPLC.

[0090] Data processing: The cumulative release rate was calculated. The release of free OXA in PBS (pH=7.4) was used as a control.

[0091] The experimental results are shown in Table 3.

[0092] Table 3: Results of in vitro drug release In normal physiological environment (PBS), the cumulative release rate over 24 hours was only 30%, indicating that the nanoparticles have good sustained-release properties and can reduce systemic exposure.

[0093] When high concentrations of glutathione (GSH, mimicking a tumor-reducing environment) or MMP-9 enzyme (mimicking a tumor-overexpressing enzyme) are present alone, the release is significantly accelerated (up to 60% and 55%, respectively), confirming its intelligent drug release capabilities in both reduction and enzyme responses.

[0094] Under the "tumor microenvironment simulation" conditions where GSH and MMP-9 coexist, the release rate was the highest (85%), confirming that the dual response mechanism has a synergistic effect and can achieve efficient and rapid "explosive" drug release at the tumor site, thereby increasing the local drug concentration.

[0095] Experiment Example 4: Evaluation of the inhibitory effect on colon cancer cells 4.1 Cellular uptake and targeting validation Fluorescent labeling: Synthesized FITC-labeled HA-PLLS-TOS-FITC NPs and PEG-PLLS-TOS-FITC NPs (without HA targeting).

[0096] Cell culture: Select human colon cancer cells HCT-116 with high CD44 expression and cells with low CD44 expression (such as LoVo or SW480).

[0097] Competitive inhibition assay: HCT-116 cells were pre-incubated with excess free HA (1 mg / mL) for 1 hour to block the CD44 receptor, and the mean fluorescence intensity (MFI) of the cells was analyzed. The experimental results are shown in Table 4.

[0098] Table 4: Cellular uptake and targeting validation HA-modified nanoparticles exhibited extremely high uptake in CD44-overexpressing HCT-116 cells (MFI = 8500), and this uptake was significantly blocked by free HA (a competitive inhibitor) (MFI decreased to 2200). Conversely, uptake was very low in CD44-underexpressing LoVo cells (MFI = 1800). HA-deficient PEGylated nanoparticles showed moderate uptake (MFI = 2500). This strongly demonstrates hyaluronic acid (HA)-mediated CD44 receptor targeting, which is key to achieving active targeting in this protocol.

[0099] 4.2 Cytotoxicity (CCK-8 assay) and synergistic effects Grouping and treatment: HCT-116 cells were seeded in 96-well plates and the following treatment groups were set up: Blank control, HA-PLLS-TOS NPs (blank vector); Free OXA; Free TOS; Free OXA + Free TOS (physical mixture); PEG-PLLS-TOS@OXA NPs (without targeted drug delivery); OXA@HA-PLLS-TOS NPs (this solution); Incubation and detection: 48 hours after drug administration, CCK-8 reagent was added, absorbance at 450 nm was measured, and cell viability was calculated.

[0100] Data analysis: The half-maximal inhibitory concentration (IC50) of each group was calculated. The interaction between OXA and TOS in OXA@HA-PLLS-TOS NPs was analyzed using the Chou-Talalay combined index method.

[0101] The experimental results are shown in Table 5.

[0102] Table 5: Cytotoxicity (CCK-8 assay) and synergistic effects A combined index of <1 indicates that OXA and TOS have a synergistic effect.

[0103] The nanoparticles in this scheme (OXA@HA-PLLS-TOS NPs) had the lowest IC50 value (3.8 μg / mL), and their anti-tumor effect was significantly better than that of free OXA (8.5 μg / mL) and traditional non-targeted nanoparticles (7.0 μg / mL).

[0104] The combined index (CI) was 0.62 (<1), which clearly confirmed that vitamin E succinate (TOS) in the carrier and oxaliplatin (OXA) had a synergistic effect, enhancing the overall efficacy.

[0105] The experimental results above demonstrate that this application has the following advantages: Stable and controllable structure: The synthesis of nanomaterials is reproducible and exhibits excellent physicochemical stability.

[0106] Dual intelligent drug delivery: It can respond to the tumor microenvironment (high GSH, high MMP-9) to achieve efficient and precise drug release at the lesion site.

[0107] Active targeted delivery: By binding to HA-CD44, the accumulation of nanoparticles in tumor cells is significantly enhanced.

[0108] Synergistic therapeutic effect: The carrier itself (TOS) and the loaded drug (OXA) have a synergistic effect, which greatly enhances anti-tumor activity.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing polylysine nanomaterials for oxaliplatin delivery, characterized in that, Includes the following steps: S1. Hyaluronic acid was dissolved in PBS buffer, and 4-pentyneic acid, EDC and NSH were added to react. After the reaction was completed, the mixture was dialyzed and lyophilized to obtain synthesized alkynylated hyaluronic acid. S2. Poly-L-lysine was dissolved in DMSO, and vitamin E succinate, EDC and NSH were added to react. Then, it was reacted with 3-azidopropylamine under the action of EDC and NHS, dialyzed and lyophilized to obtain azidolated polylysine-vitamin E succinate. S3. Modify the C-terminus of the MMP-9 sensitive sequence with an alkynyl group and the N-terminus with an azide group to obtain the MMP enzyme-sensitive peptide linker. S4. The alkydized hyaluronic acid, azidolated polylysine-vitamin E succinate and MMP enzyme-sensitive peptide linker were dissolved in PBS, and CuSO4 and sodium ascorbate were added. The mixture was reacted at room temperature, purified by dialysis, and lyophilized to obtain polylysine nanomaterials.

2. The method for preparing polylysine nanomaterials for oxaliplatin transport according to claim 1, characterized in that, In S1, the pH of the reaction is 6.0, and the reaction temperature is 4°C.

3. The method for preparing polylysine nanomaterials for oxaliplatin transport according to claim 1, characterized in that, In step S1, dialysis is performed using a dialysis bag with a molecular weight cutoff of 3.5 kDa. The specific steps are as follows: Dialyze with 0.1M NaCl solution at 4℃ for 6 hours, followed by dialysis with ultrapure water for 48 hours. When dialysis with ultrapure water, change the water every 8 hours.

4. The method for preparing polylysine nanomaterials for oxaliplatin transport according to claim 1, characterized in that, The MMP-9 sensitive sequence is shown in SEQ ID NO.

1.

5. A polylysine nanomaterial for oxaliplatin transport prepared by the preparation method according to any one of claims 1-4.

6. A method for preparing a drug loaded with oxaliplatin, characterized in that, Using the polylysine nanomaterial as described in claim 5 includes the following steps: Oxaliplatin and polylysine nanomaterials were co-dissolved in DMSO, and the organic solvent was removed after ultrasonic emulsification to obtain a drug loaded with oxaliplatin.

7. The method for preparing the oxaliplatin-loaded drug according to claim 6, characterized in that, The mass ratio of oxaliplatin to polylysine nanomaterials is 1:(3-4).

8. A oxaliplatin-loaded drug prepared by the method for preparing an oxaliplatin-loaded drug as described in any one of claims 6-7.

9. The use of the oxaliplatin-loaded medicament as described in claim 8 in the preparation of a cancer treatment medicament.

10. The use of the oxaliplatin-loaded drug according to claim 9 in the preparation of a cancer treatment drug, characterized in that, The cancer in question is colorectal cancer.

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