Biological orthogonal catalytic microneedle based on cytotoxic polyethyleneimine matrix as well as preparation method and application of biological orthogonal catalytic microneedle

Abstract

The invention belongs to the technical field of biomedical materials and tumor treatment, and discloses a biological orthogonal catalytic microneedle based on a cytotoxic polyethyleneimine matrix as well as a preparation method and application of the biological orthogonal catalytic microneedle. The microneedle patch comprises a backing layer and a microneedle array, wherein the microneedle array is formed by mixing polyvinyl alcohol (PVA) loaded with metal nanoparticles and polyethyleneimine (PEI) with a hydrogel matrix. According to the invention, the inherent cytotoxicity (membrane destroying ability) of PEI is creatively combined with the catalytic activity of a metal catalyst (such as palladium) to construct a synergistic treatment system which turns toxins into treasures. The PEI matrix has dual functions in the microneedle: on one hand, the PEI matrix is used as a potent ligand to anchor and stabilize the metal nanoparticles and prevent the metal nanoparticles from leaking; on the other hand, as a therapeutic agent, tumor cell membranes are destroyed through electrostatic interaction. The microneedle patch is activated by catalyzing a prodrug (such as doxorubicin protected by propargyloxycarbonyl) in situ, meanwhile, the physical membrane destruction effect of PEI is utilized, the synergistic anti-tumor effect of physical destruction and chemical killing is achieved, and the treatment efficiency of superficial tumors such as melanoma is remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and drug delivery, specifically to a microneedle patch for loading a metal catalyst using the cytotoxicity of polyethyleneimine (PEI), and its application in the synergistic treatment of superficial tumors (such as melanoma) through bioorthogonal catalytic prodrug activation and physical membrane disruption. Background Technology

[0002] Cancer treatment still faces severe challenges such as the high systemic toxicity and poor targeting of chemotherapy drugs. In recent years, bioorthogonal catalysis (BOC) has attracted widespread attention as an emerging therapeutic strategy. This strategy utilizes transition metal catalysts (such as Pd, Ru, Au, etc.) to activate non-toxic or low-toxic prodrugs into highly toxic therapeutic drugs in situ within the body, thereby achieving high-concentration delivery to the tumor site and significantly reducing systemic side effects.

[0003] In existing bioorthogonal catalysis systems, to improve the stability and biocompatibility of catalysts in vivo, researchers typically support metal catalysts on bioinert supports, such as mesoporous silica, polystyrene microspheres, MOF materials, or bioinert resins. The primary function of these supports is to immobilize the catalyst, preventing its aggregation or poisoning by biomolecules; the supports themselves do not possess the ability to kill tumor cells. This "single-function" support design limits the overall efficiency of the therapeutic system.

[0004] Polyethyleneimine (PEI) is a cationic polymer with a high density of amino groups, often used as a gene delivery vector due to its excellent "proton sponge effect." However, the high positive charge of PEI readily interacts strongly with the negatively charged cell membrane, leading to cell membrane rupture and cell death. This inherent cytotoxicity has long been considered a major obstacle to the clinical application of PEI, and researchers typically use chemical modifications such as acetylation and PEGylation to shield its positive charge and reduce toxicity.

[0005] Current microneedle drug delivery systems often prioritize absolute biocompatibility of materials, failing to fully utilize the inherent bioactivity of these materials. However, if we can think in reverse and transform the "toxicity" (i.e., membrane-damaging ability) of PEI into a "weapon" for treating tumors, and utilize its abundant amino groups as excellent ligands for metal catalysts, we could construct a novel and highly efficient tumor treatment system with a dual mechanism of "physical disruption + chemical killing." Currently, there are no reports on constructing bioorthogonal catalytic microneedles using the dual functions of the PEI matrix (stabilizing catalyst + physical killing). Summary of the Invention

[0006] Objective of the Invention: The objective of this invention is to overcome the shortcomings of existing bioorthogonal catalytic carriers, such as limited functionality and lack of active therapeutic activity, and to provide a bioorthogonal catalytic microneedle based on a cytotoxic PEI matrix. This microneedle utilizes the PEI matrix to stabilize palladium nanoparticles and directly disrupt cancer cell membranes, thereby producing a synergistic anti-tumor effect with catalytically activated chemotherapeutic drugs.

[0007] Technical Solution: To solve the above-mentioned technical problems, in a first aspect, the present invention provides a bioorthogonal catalytic microneedle based on a cytotoxic polyethyleneimine matrix, comprising a substrate and a microneedle array disposed on the substrate; the microneedle array is prepared from a hydrogel material containing polyvinyl alcohol (PVA), polyethyleneimine (PEI) and metal nanoparticles dispersed therein. The polyethyleneimine (PEI) plays a dual role in the microneedle: (1) as a ligand, it stabilizes the metal nanoparticles and prevents their leakage in the physiological environment by coordinating with the surface of the metal nanoparticles through its amino groups; (2) as an active therapeutic component, it interacts with the tumor cell membrane using its cationic properties, thereby disrupting the cell membrane integrity.

[0008] Preferably, the metal nanoparticles are palladium (Pd) nanoparticles with a particle size range of 5-20 nm. Preferably, the mass ratio of PVA to PEI in the microneedle matrix is ​​(2-6):1, more preferably 4:1. Preferably, the molecular weight of the PEI is 600 Da - 70 kDa, more preferably 25 kDa or higher to ensure sufficient membrane disruption capability. Preferably, the bioorthogonal catalytic microneedles can catalyze the deprotection reaction of N-propyneoxycarbonyl-protected prodrugs (such as P-DOX) to release the active drug (such as DOX).

[0009] Secondly, the present invention also provides a method for preparing the above-mentioned bioorthogonal catalytic microneedles, comprising the following steps: (1) preparing a mixed aqueous solution containing PVA and PEI; (2) adding a metal salt precursor (such as sodium chloropalladium) to the mixed solution and stirring to coordinate the metal ions with PEI; (3) adding a reducing agent (such as hydrazine hydrate or sodium borohydride) to reduce the metal ions in situ to metal nanoparticles, thereby obtaining a metal-loaded PVA / PEI mixed gel solution; (4) injecting the mixed gel solution into a microneedle mold, centrifuging to fill, and drying and solidifying; (5) peeling off from the mold to obtain the bioorthogonal catalytic microneedles.

[0010] Thirdly, the present invention also provides the application of the above-mentioned microneedles in the preparation of drugs or medical devices for treating superficial tumors (especially melanoma).

[0011] Beneficial effects: Compared with the prior art, the present invention has the following significant features: 1. This invention pioneers a synergistic model of "physical membrane disruption + in-situ chemotherapy". The PEI matrix physically disrupts tumor cell membranes through electrostatic interaction, which not only directly kills cells but also increases cell membrane permeability, promoting the entry of in-situ generated chemotherapy drugs (DOX) into cells, significantly improving efficacy; 2. This invention cleverly utilizes the cytotoxicity of PEI, which is usually avoided. Studies have found that the coordination between Pd nanoparticles and PEI consumes some of the free amino groups, appropriately reducing the excessive toxicity of PEI and placing it at a balance point where it can kill cancer cells while remaining relatively safe for normal tissues; 3. The high-density amino groups of PEI involved in this invention tightly anchor Pd nanoparticles. Experiments show that the leakage of Pd is extremely low (<1.5%) during immersion for up to 48 hours, effectively solving the risk of heavy metal toxicity caused by leakage of metal catalysts in the body. 4. The microneedle patch provided by this invention allows for self-administration of medication, is painless and minimally invasive, and avoids the pain of frequent injections. Attached Figure Description

[0012] Figure 1 Characterization diagrams of the PVA / PEI@Pd nanocomposite material prepared in this invention are shown. (a) is a schematic diagram of the preparation of the PVA / PEI@Pd nanocomposite material; (b) is a transmission electron microscope (TEM) image showing uniform dispersion of Pd nanoparticles; and (c) is a statistical diagram of the particle size distribution of Pd nanoparticles. Figure 2 The morphology and mechanical properties of the PPPd-MNs microneedle patch of the present invention are characterized. Among them, (a) is an optical photograph of the microneedle array; (b) is a scanning electron microscope (SEM) image of the microneedle, scale bar 1 mm; (c) is an elemental distribution image of the microneedle cross section, scale bar 500 μm; (d) is the force-displacement curve of the mechanical property test of the microneedle of the present invention. Figure 3 This invention relates to a study of microneedle-mediated prodrug activation and cytotoxicity. Specifically, (a) is a liquid phase analysis of P-DOX release mediated by PPPd-MNs microneedles; (b) is a quantitative analysis of the conversion rate of P-DOX to DOX in (a); (c) is the survival rate of B16-F10 cells, 4T1 cells, and HUVEC cells treated with PPPd-MNs microneedles; (d) is the survival rate of B16-F10 cells treated with DOX, P-DOX, and a combination of P-DOX / PPPd-MNs at different drug concentrations; and (e) is the survival rate of 4T1 cells treated with DOX, P-DOX, and a combination of P-DOX / PPPd-MNs at different drug concentrations. Figure 4 In the image, (a) is a schematic diagram of apoptosis detected by flow cytometry using the Annexin V-FITC / PI double staining method; (b) is a staining diagram of live and dead cells. Figure 5 The images show the therapeutic effect of the microneedles of this invention in a mouse melanoma model. (a) is a photograph of the tumor after treatment; (b) is a curve showing the tumor volume change during treatment; (c) is the mass of the tumor tissue after treatment; and (d) is an H&E stained section of the tumor tissue after treatment. Detailed Implementation

[0013] The present invention will now be described in detail with reference to specific embodiments. Those skilled in the art should understand that these embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, the materials, reagents, etc., used in the following embodiments are commercially available. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer. The PDMS silicon mold was purchased from Taizhou Microchip Medical Technology Co., Ltd. Example 1

[0014] A method for preparing a PVA / PEI@Pd catalytic hydrogel material includes the following steps: Weigh polyvinyl alcohol (PVA, Mw 89000~98000 kDa, degree of hydrolysis 99%) and dissolve it in deionized water at 95°C with stirring until completely dissolved. Add polyethyleneimine (PEI, Mw ~25 kDa) and stir at 60°C until completely dissolved. To ensure the film-forming properties and functionality of the matrix, control the mass ratio of PVA to PEI to be 4:1, with a total polymer concentration of approximately 25 wt%. After cooling to room temperature, add an equal volume of 20 mM sodium chloropalladium (Na₂PdCl₄) aqueous solution to the above PVA / PEI mixed solution. Stir the mixture for 2 hours to allow the PdCl₄ to react. 2- Ions were fully adsorbed and coordinated to the amino groups of the PEI chain. The mixed solution was poured onto a glass plate to form a film, which was then dried at room temperature. The prepared polymer film was placed in a 10% hydrazine hydrate solution (hydrazine to Pd molar ratio in excess, e.g., 10:1). The color of the reaction system rapidly changed from pale yellow to black, indicating the formation of Pd(0) nanoparticles. The reaction was continued to be stirred for 2 hours to ensure complete reduction and stable dispersion of the nanoparticles. The resulting black gel was washed three times with deionized water, each time for 1 hour, and then dried to obtain PVA / PEI@Pd.

[0015] Material characterization: PVA / PEI@Pd was redissolved in pure water and dropped onto a copper grid for TEM imaging and mapping analysis. Results showed (corresponding to...) Figure 1 The Pd nanoparticles are spherical and uniformly dispersed in the polymer matrix without obvious agglomeration. Statistical analysis shows that the average particle size is approximately 11.6 ± 2.3 nm.

[0016] A method for preparing PPPd-MNs bioorthogonal catalytic microneedle patches includes the following steps: A polydimethylsiloxane (PDMS) microneedle mold was selected, with a 15×15 array, a single needle height of 1500 μm, a hole spacing of 800 μm, and each needle hole being conical with a base diameter of 500 μm. The substrate size was 10 mm × 10 mm. The stock solution (15 wt%) of the PVA / PEI@Pd hydrogel prepared in Example 1 at 95°C was carefully drop-dropped onto the mold surface, avoiding the formation of air bubbles. After the needle tips had slightly dried, a layer of pure PVA solution or PVA / PEI solution could be added on top as a backing layer to provide better mechanical support (optional step). The filled mold was allowed to air dry at room temperature for 48 hours. After complete curing, the microneedle patch was carefully peeled off from the mold.

[0017] Morphological characterization: Scanning electron microscopy (SEM) showed that the microneedles were sharp, morphologically intact, and neatly arranged (corresponding to...). Figure 2 b). The elemental distribution image of the microneedle cross-section shows that Pd is uniformly distributed in all parts of the microneedle (corresponding to...). Figure 2 c).

[0018] The mechanical properties of the microneedles obtained in Example 2 were tested using the following specific steps: A universal testing machine was used to perform compression tests on a single microneedle. For example... Figure 2 The results showed that the single-needle yield force of PPPd-MNs was approximately 0.83 N, which is much higher than the minimum force required to pierce the skin (approximately 0.1 N), demonstrating that it has sufficient strength to penetrate the stratum corneum.

[0019] The bioorthogonal catalytic microparticles in Example 2 catalyze the release of doxorubicin prodrug in vitro. The specific steps are as follows: (1) Synthesis of N-propynoxycarbonyl-protected doxorubicin (P-DOX). This prodrug has extremely low toxicity in its unactivated state. The bioorthogonal catalytic microneedles from Example 2 were placed in 1 mL of reaction solution containing PBS buffer (pH 7.4) and P-DOX (100 μL, 1 mMDMSO) and incubated at 37°C. At a specified time point, 10 μL of the reaction solution was taken out and mixed with 90 μL of methanol for HPLC analysis. HPLC analysis was performed using a Shimadzu LC-20A system equipped with a WondaSil C18 Superb reversed-phase column (5 μm particle size, 4.6 × 150 mm), a flow rate of 1.0 mL / min, and a column temperature of 30°C. The mobile phase gradient of the P-DOX reaction solution was as follows: 0.00–5.30 min, water / acetonitrile (70:30); 5.31–17.00 min, water / acetonitrile (58:42); 17.00–20.00 min, water / acetonitrile (70:30). The aqueous phase contained 0.1% trifluoroacetic acid. All chromatograms were detected at a wavelength of 254 nm. Prodrug conversion and drug release rates were calculated using standard curves. Figure 3 Results a and b show that P-DOX gradually decreases over time, while the active DOX peak gradually increases. After 8 hours of reaction, the conversion rate can reach over 90%.

[0020] The bioorthogonal catalytic microneedles in Example 2 can synergistically enhance the toxicity of the material and the release of P-DOX in vitro to achieve toxicity to B16-F10 cells. The specific steps are as follows: (1) The 48-hour cytotoxicity of the bioorthogonal catalytic microneedles in Example 2 was determined using the CCK8 assay. Figure 3 As shown in Figure c, the PEI-containing bioorthogonal catalytic microneedles exhibited cytotoxicity against B16-F10 cells, 4T1 cells, and HUVEC cells, with significantly greater cytotoxicity against both cancer cell types than against normal cells. This demonstrates that the microneedle material itself possesses cytotoxicity and a degree of selectivity.

[0021] (2) The 48-hour cytotoxicity of the prodrug P-DOX catalyzed by the bioorthogonal catalytic microneedles in Example 2 was determined using the CCK8 assay. Figure 3 As shown in d, data points represent mean ± standard deviation (n = 3), and the maximum half-maximal inhibitory concentration (IC50) of P-DOX combined with PPPd-MNs for B16-F10 cells. 50 The value was 0.10 ± 0.02 μM, much smaller than that of P-DOX (0.97 ± 0.12 μM) and comparable to that of DOX (0.09 ± 0.02 μM). Similarly, in 4T1 cells, the maximum inhibitory concentration (IC50) of cells treated with the combined use of P-DOX and PPPd-MNs was significantly higher. 50The value was 0.4 ± 0.08 μM, much smaller than that of P-DOX (2.55 ± 0.3 μM) and similar to that of DOX (0.45 ± 0.1 μM). Figure 3 e). All of the above results demonstrate that microneedles can catalyze the release of prodrugs in an in vitro environment.

[0022] (3) B16-F10 cells were treated with P-DOX and PPPd-MNs for 24 hours. The treated cells were then stained for apoptosis using the Annexin V-FITC / PI kit. Flow cytometry showed that the treated cells exhibited significant apoptosis. Figure 4 a).

[0023] (4) Live and dead cells were stained with Calcein-AM / PI, and confocal microscopy images showed that the treated cells exhibited significant cell death. Figure 4 b, Scale bar 100 μm), Calcein-AM penetrates the living cell membrane, and after esterase hydrolysis, it emits green fluorescence inside the cell. PI staining shows nucleic acids from necrotic and apoptotic cells with damaged membranes.

[0024] Comparative Example 1 The remaining steps of this comparative example are the same as those in Example 5, except that: only orthogonal catalytic microneedles PPPd-MNs are introduced; no PPPd-MNs are introduced for treatment, and doxorubicin prodrug is used directly; and doxorubicin is used directly. In the results of the comparative examples, the pure P-DOX group showed high cell survival (non-toxic); the pure PPPd-MNs group exhibited dose-dependent cytotoxicity (IC50). 50 (approximately tens of μg / mL), confirming the killing ability of the PEI matrix itself; the combination therapy group had the lowest cell survival rate, significantly lower than that of single therapy.

[0025] The doxorubicin prodrug P-DOX was combined with bioorthogonal catalytic microneedles (PPPd-MNs) to treat melanoma in mice. The specific procedure is as follows: Select 6-8 week old female C57BL / 6 mice and subcutaneously inoculate them with 1×10⁻⁶ mol / L on their backs. 6 B16-F10 cells were collected. After one week of tumor growth, mice were randomly divided into groups of 5 mice each using a random number table. The treatment group (F group) received an intraperitoneal injection of doxorubicin prodrug (50 mg / kg), and PPPd-MNs were cut to the appropriate size for the tumor and attached to it. The microneedle patch was removed after 24 hours. Treatment was repeated twice, once every 3 days, with tumor size and mouse weight monitored during the treatment period. On day 12, mouse tumors were collected for HE staining.

[0026] Comparative Example 2 The remaining steps of this comparative example are the same as those in Example 6, except that: A: PBS group was injected intraperitoneally; B: P-DOX (50 mg / kg) was injected intraperitoneally only; C: DOX (2.5 mg / kg) was injected intraperitoneally only; D: Blank microneedle PP-MNs were introduced only for treatment; E: PPPd-MNs were introduced only for treatment.

[0027] The results are as follows: Depend on Figure 5 As shown, tumor growth in the treatment group mice was significantly inhibited. Figure 5 A visual representation of the tumor size at the end of treatment. Figure 5 Data points b and 5c represent mean ± standard deviation (n = 5). In group C (microneedle only), tumor growth was partially inhibited, demonstrating the physical therapy activity of the PEI matrix in vivo. Group E (synergistic therapy group) had the smallest tumor volume and a tumor inhibition rate as high as 98%, significantly superior to group D (direct chemotherapy group), indicating the highly effective tumor-killing ability of this therapy. Tumor sections were stained with hematoxylin and eosin to observe pathological features. Figure 5 (d) Significant tumor necrosis can be observed in the treatment group. Scale bar, 100 μm. This further confirms the effectiveness of the microneedle-mediated physiochemical synergistic therapy strategy.

Claims

1. A bioorthogonal catalytic microneedle based on a cytotoxic polyethyleneimine matrix, characterized in that, The invention includes a substrate and a microneedle array disposed on the substrate; the microneedle array is prepared from a hydrogel material comprising polyvinyl alcohol (PVA), polyethyleneimine (PEI) and metal nanoparticles dispersed therein; the polyethyleneimine (PEI) serves as a ligand stabilizing the metal nanoparticles and also as an active ingredient that exerts antitumor effects by disrupting cell membranes.

2. The bioorthogonal catalytic microneedles according to claim 1, characterized in that, The metal nanoparticles are palladium (Pd) nanoparticles with a particle size range of 5-20 nm; preferably, the content of the metal nanoparticles in the microneedle material is about 0.5wt%-5.0wt%.

3. The bioorthogonal catalytic microneedles according to claim 1, characterized in that, The polyvinyl alcohol (PVA) and polyethyleneimine (PEI) form a physically cross-linked interpenetrating network structure with a mass ratio of (2~6):1; the microneedles can absorb body fluid and swell to form a porous hydrogel structure after being inserted into the tissue, allowing small molecule prodrugs to penetrate.

4. A method for preparing bioorthogonal catalytic microneedles according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Polyvinyl alcohol (PVA) and polyethyleneimine (PEI) are dissolved and mixed to obtain a polymer mixed solution; (2) A metal salt precursor solution is added to the mixed solution, and the metal ions are coordinated by the amino groups of PEI. (3) Add a reducing agent to reduce metal ions in situ to metal nanoparticles to obtain a PVA / PEI mixed gel liquid loaded with metal nanoparticles (PVA / PEI@Metal); (4) Inject the mixed gel liquid into a microneedle mold, dry and solidify it and then demold to obtain the bioorthogonal catalytic microneedles.

5. The preparation method according to claim 4, characterized in that, The metal salt precursor mentioned in step (2) is sodium chloropalladium (Na2PdCl4) or potassium chloropalladium (K2PdCl4), and the reducing agent mentioned in step (3) is hydrazine hydrate or sodium borohydride.

6. A tumor treatment system, characterized in that, include: (1) The bioorthogonal catalytic microneedles according to any one of claims 1-3; (2) A bioorthogonal prodrug, wherein the prodrug contains a protecting group that can be catalytically removed by the metal nanoparticles.

7. The tumor treatment system according to claim 6, characterized in that, The bioorthogonal prodrug is N-propargyloxycarbonyl-protected doxorubicin (P-DOX); the protecting group is propargyloxycarbonyl.

8. The use of the bioorthogonal catalytic microneedles according to any one of claims 1-3 in the preparation of drugs or medical devices for treating melanoma.

9. The application according to claim 8, characterized in that, The application utilizes polyethyleneimine (PEI) in microneedles to disrupt tumor cell membranes and microneedles to catalyze and activate pro-chemotherapeutic drugs, resulting in a synergistic therapeutic effect.

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