Preparation method and use of nanogel with intelligent cross-linking density regulation

Abstract

The application discloses a preparation method and application of a nanogel with intelligent cross-linking density regulation, and the nanogel is formed by adding a tumor microenvironment sensitive cross-linking agent into a polyvinyl alcohol-acetal-acrylate derivative nanogel cross-linked by ultraviolet light to form a nanogel system with a double cross-linking structure. Research shows that the requirements of the mechanical properties of the nanoparticles are different and even opposite in different stages of in-vivo delivery, the nanogel in the design can respond to the tumor site to partially de-cross-link the nanoparticles, the hardness of the nanoparticles can be intelligently regulated in different stages in vivo, the range of the intelligent drug delivery system is expanded, and the nanogel provides a useful reference value and a new idea for designing a multifunctional drug delivery carrier.

Description

Technical Field

[0001] This invention relates to methods for preparing polymer materials and their applications, and particularly to a method for preparing and using a smart responsive double cross-linked nanogel. Background Technology

[0002] Nanogels are considered promising drug delivery carriers due to their excellent biocompatibility, unique cross-linked network structure, and functionality. The unique cross-linked network structure endows nanogels with high stability, effectively improving their pharmacokinetic parameters. By introducing functional groups or segments into the nanogel structure, ligand-modified nanogels can be obtained for active targeted drug delivery to tumors; stimulus-responsive nanogels can achieve tumor-site-responsive drug release; charge-conversion nanogels can enhance tumor uptake; size-scalable nanogels can achieve deep tumor penetration; and other multifunctional nanogels are also possible. Furthermore, the mechanical strength of nanogels can be adjusted by changing the cross-linking density, endowing them with structural and functional diversity. Meanwhile, the three-dimensional network structure of nanogels, with its high water content, good chemical and mechanical properties, and large multivalent bioconjugated specific surface area, enables them to stabilize bioactive compounds such as drugs, peptides / proteins, and DNA / RNA within polymer networks.

[0003] Currently, an increasing number of researchers are studying how the physical properties of nanoparticles, such as size, surface properties, and shape, affect their biological functions. Many studies have elucidated the impact of particle hardness on in vivo circulation and endocytosis. For example, Mitragotri et al. (ACS Nano 2015, 9, 3, 3169-3177.) prepared hard (3000 kPa) nanogels, which showed that they entered J774 macrophages more easily than soft (10 kPa) nanogels, resulting in a better long-term in vivo circulation effect for soft particles. PengGuo et al. (Nat. Commun., 2018, 9, 130.) found that breast cancer cells MCF-7 took up significantly more soft particles (47.7 MPa) than hard particles (233.4 MPa), because soft particles enter cells via membrane fusion endocytosis, which consumes less energy and is more efficient.

[0004] In fact, the impact of hardness on particle transport in vivo is highly controversial, and even contradictory results have emerged. Furthermore, the mechanical properties required for nanoparticles differ or even contradict each other at different stages of in vivo delivery. For example, in the nanogel system prepared by Mitragotri et al., not only macrophages showed a tendency to take up hard particles, but 4T1 tumor cells also exhibited this tendency. This means that it is impossible to simultaneously achieve long-term circulation of the drug delivery system (soft particles preferred) and enhance the ability of tumor cells to take up particles (hard particles preferred) by selecting a certain hardness. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a method for preparing nano-hydrogels with intelligently adjustable crosslinking density.

[0006] Another object of the present invention is to provide the use of the nanohydrogel with intelligently adjustable crosslinking density.

[0007] Technical Solution: The present invention describes a method for preparing a smartly responsive nanogel with adjustable crosslinking density: Polyvinyl alcohol-acetal-acrylate derivative (PVA-VEA) undergoes two chemical crosslinking processes to form a nanogel with a double crosslinking structure. First, under ultraviolet light, the PVA-VEA double bonds undergo a free radical reaction crosslinking to form a stable primary structure. Subsequently, a tumor microenvironment-sensitive crosslinking agent bonds with the hydroxyl groups between polyvinyl alcohol molecules to obtain a smart, responsive nanogel with a double crosslinking structure.

[0008] Furthermore, the molecular weight of the polyvinyl alcohol is 5–120 kDa.

[0009] Furthermore, the polyvinyl alcohol-grafted vinyl ether acrylate is selected from compounds with the following structures: R is selected from H or CH3.

[0010] Furthermore, the proportion of the crosslinking group vinyl ether propionate group in the single crosslinked nanogel is 0.5% to 8%.

[0011] Furthermore, the tumor microenvironment-sensitive crosslinking agent is a reactive oxygen species-sensitive crosslinking agent.

[0012] Furthermore, the tumor microenvironment-sensitive crosslinking agent is a reactive oxygen species-sensitive crosslinking agent and a matrix metalloproteinase (MMP)-sensitive crosslinking agent, respectively, and consists of compounds with the following structures:

[0013]

[0014] Furthermore, the proportion of the added tumor microenvironment-sensitive cross-linking agent is 1% to 15%.

[0015] The method for preparing the above-mentioned smart responsive nanogels includes the following steps:

[0016] (1) Polyvinyl alcohol with a certain proportion of vinyl ether acrylate is dissolved in water at low temperature, then slowly dispersed in acetone, and after adding a photoinitiator, a free radical reaction of the double bond occurs under ultraviolet light to obtain a nanogel with a primary structure.

[0017] (2) The primary structure nanogel obtained in step (1) is redispersed in water, acetone is removed by dialysis, and a certain proportion of tumor microenvironment sensitive crosslinking agent solution is dropped into the nanogel aqueous solution for further crosslinking to obtain intelligent responsive double crosslinked nanogel.

[0018] The prepared smart responsive nanogel, due to its unique double-crosslinked three-dimensional network structure, can be used as a carrier for a variety of drugs.

[0019] The free radical reaction is a carbon-carbon double bond reaction that occurs between the propylene double bonds provided by the vinyl ether acrylate attached to polyvinyl alcohol under the action of ultraviolet light and a photoinitiator.

[0020] Smart responsive nanogels with a dual cross-linking structure refer to nanogel particles with two cross-linking structures, which are constructed by adding a tumor microenvironment-sensitive cross-linking agent to the hydroxyl groups of polyvinyl alcohol molecules on the basis of ultraviolet cross-linked polyvinyl alcohol-acetal-acrylate derivative nanogels.

[0021] The intelligent responsive nanogel of this invention utilizes the free radical reaction of carbon-carbon double bonds and the action of a tumor microenvironment-sensitive crosslinking agent to prepare a polyvinyl alcohol-acetal-acrylate nanogel with a double crosslinking structure. Because the particles contain tumor microenvironment-sensitive bonds, partial breakage of the crosslinking bonds occurs when the particles reach the tumor site, causing the particles to transform from a double crosslinking structure to a primary structure. Under the influence of stable carbon-carbon bonds, the nanogel can still maintain its original shape and size, ultimately achieving intelligent responsiveness to the crosslinking density of the particles.

[0022] This invention prepares a nanogel with intelligently tunable mechanical strength to achieve high stiffness during in vivo circulation and low stiffness during tumor uptake. The nanogel possesses two types of cross-linking structures, increasing the internal complexity of the particles without altering other physical properties, thus reducing macrophage uptake during in vivo circulation; however, when in the tumor microenvironment, the nanogel undergoes partial cross-linking bond breakage and alteration of mechanical properties, thereby facilitating tumor cell uptake. The particles can achieve intelligent regulation of mechanical properties in vivo, simultaneously enhancing the carrier's long-term circulation and cellular uptake capabilities, effectively solving the long-standing dilemma of prioritizing one aspect over the other. Due to its unique double-crosslinked three-dimensional network structure, this intelligent responsive nanogel can be used as a carrier for anti-tumor drugs and other drugs. This invention is significant for studying the relationship between drug release from nanoparticles and the degree of cross-linking / mechanical properties, as well as the impact of the internal complexity of nanoparticles on different stages of in vivo delivery.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0024] The intelligent responsive double-crosslinked nanogel of this invention undergoes the breakage of sensitive groups under responsive conditions, thereby altering the crosslinking density and internal complexity of the nanogel, while its surface properties and size remain unchanged. This enables intelligent regulation of its mechanical properties at different stages in vivo. This characteristic can be used to study the relationship between drug release from nanoparticles and crosslinking density, as well as the impact of the internal complexity of nanoparticles on their long-term circulation, cellular internalization, and pharmacokinetics in vivo. The diversity of sensitive crosslinking agents used in the second crosslinking stage of this invention also allows for applications in the treatment of other diseases. This double-crosslinked nanogel system has great application potential in the fields of intelligent responsiveness and controlled drug release. Attached Figure Description

[0025] Figure 1 The hydrogen nuclear magnetic resonance spectrum of the polymer polyethylene glycol-acetal-acrylate (PVA-VEA) in Example 1 is shown below.

[0026] Figure 2 The hydrogen nuclear magnetic resonance spectrum of TSPBA, the reactive oxygen species-sensitive crosslinking agent in Example 2;

[0027] Figure 3 The hydrogen nuclear magnetic resonance spectrum of the PVA-VEA-TSPBA nanogel with intelligently tunable mechanical properties in Example 4;

[0028] Figure 4 The figure shows the particle size variation of the PVA-VEA-TSPBA nanogel with intelligently adjustable mechanical properties in Example 5 under 50 μM H2O2 conditions.

[0029] Figure 5 This is a graph showing the cytotoxicity results of the nanogel obtained in Example 7 on RAW264.7 macrophage cells. Detailed Implementation

[0030] Example 1: Synthesis of PVA-VEA with different molecular weights

[0031] (1) Synthesis of PVA-VEA (5kDa)

[0032]

[0033] Under nitrogen protection, 2.00 g of 5 kDa polyvinyl alcohol (PVA), containing 33.8 mmol of hydroxyl groups, was dissolved in 50 mL of anhydrous dimethyl sulfoxide. 1.43 mL of vinyl ethyl ether acrylate (VEA), containing 30% hydroxyl groups and 10 mmol, was added, along with a catalytic amount of PTSA. The reaction was carried out at room temperature for 6 h. After the reaction was complete, 300 μL of triethylamine (TEA) was added to terminate the reaction. The reaction solution was dialyzed against methanol, concentrated under reduced pressure, and precipitated three times with ice-cold diethyl ether to obtain the product. Its proton NMR spectrum is shown below. Figure 1As shown.

[0034] (2) Synthesis of PVA-VEA (15kDa)

[0035] Under nitrogen protection, 2.00 g of 15 kDa polyvinyl alcohol (PVA), containing 33.8 mmol of hydroxyl groups, was dissolved in 50 mL of anhydrous dimethyl sulfoxide (DMSO). 1.43 mL of vinyl ethyl ether acrylate (VEA), containing 30% hydroxyl groups and 10 mmol, was added, along with a catalytic amount of PTSA. The reaction was carried out at room temperature for 6 h. After the reaction was complete, 300 μL of triethylamine (TEA) was added to terminate the reaction. The reaction solution was dialyzed against methanol, concentrated by vacuum evaporation, and washed three times with ice-cold diethyl ether precipitate to obtain the product.

[0036] (3) Synthesis of PVA-VEA (50kDa)

[0037] Under nitrogen protection, 2.00 g of 50 kDa polyvinyl alcohol (PVA), containing 33.8 mmol of hydroxyl groups, was dissolved in 50 mL of anhydrous dimethyl sulfoxide. 1.43 mL of vinyl ethyl ether acrylate (VEA) (10 mmol) with 30% hydroxyl content was added, along with a catalytic amount of PTSA. The reaction was carried out at room temperature for 6 h. After the reaction was completed, 300 μL of triethylamine (TEA) was added to terminate the reaction. The reaction solution was dialyzed against methanol, concentrated by vacuum evaporation, and washed three times with ice-cold diethyl ether precipitate to obtain the product.

[0038] Example 2: Synthesis of reactive oxygen species-sensitive crosslinking agent TSPBA

[0039]

[0040] 4-(bromomethyl)phenylboronic acid (500 mg, 2.3 mmol) was dispersed in 10 mL of acetone and dissolved by stirring at 50 °C. N2 was then added. 1 N 1 N 3 N 3 -Tetramethylpropane-1,3-diamine (0.1 g, 0.75 mmol) was reacted with stirring at 50 °C for 12 h. After the reaction was complete, the precipitate was added to 100 mL of tetrahydrofuran and washed three times. After vacuum drying, a white solid TSPBA was obtained. The proton NMR spectrum is shown below. Figure 2 As shown.

[0041] Example 3: Preparation of single cross-linked nanogels (PVA-VEA)

[0042] PVA-VEA monocrosslinked nanogels were prepared using a reverse nanoprecipitation method. Under nitrogen protection, 200 μL of PVA-VEA (10 mg / mL) aqueous solution was slowly added dropwise to 10 mL of rapidly stirred acetone at low temperature. Then, 5% (polymer weight) of photoinitiator I2959 was added. After irradiation with UV light for 30 minutes, 4 mL of high-purity water was added. Following rotary evaporation and dialysis, a 0.5 mg / mL PVA-VEA nanogel aqueous solution was obtained. Dynamic light scattering analysis revealed that the average particle size of the monocrosslinked nanogels was 166.67 nm, the particle size distribution index was 0.12, and the average Zeta potential was -3.5 mV.

[0043] Example 4: Preparation of nanogels (PVA-VEA-TSPBA) with intelligently tunable mechanical properties

[0044] A 2 mg / mL aqueous solution of TSPBA was prepared. Under stirring, a 5% molar amount of TSPBA solution (containing hydroxyl groups) was slowly added to the PVA-VEA nanogel solution. After reacting for 1 hour, excess reactants were removed by dialysis. Dynamic light scattering analysis revealed that the average particle size of the double-crosslinked nanogel was 156.80 nm, the particle size distribution index was 0.09, and the average Zeta potential was -2.55 mV. Figure 3 Integrating the peak around 8 ppm, the crosslinking efficiency of the secondary crosslinking can be calculated to be over 80%. The appearance of the characteristic aryl hydrogen peak in the NMR spectrum of the double-crosslinked gel (…) Figure 3 The fact that there was no significant difference in particle size and charge before and after the addition of the sensitive crosslinking agent (Table 1) indicates that the secondary crosslinking of the particles only changed the internal structure of the nanogel and had almost no effect on other physical properties, thus achieving single control of mechanical properties.

[0045] Table 1. Characterization of double-crosslinked nanogels a

[0046]

[0047] a The final concentration of the nanogel was 0.5 mg / mL.

[0048] b The average particle size (nm) and particle size distribution were determined by dynamic light scattering at 25°C and pH 7.4.

[0049] Example 5: Responsiveness Experiment of Smart, Tunable Mechanical Properties Nanogel (PVA-VEA-TSPBA) in H2O2 Solution

[0050] Add 10 μL (5 mmol / L) H₂O₂ solution to 1 mL of PVA-VEA-TSPBA nanogel solution, stir thoroughly, and let stand for 2 h. Dynamic light scattering was used to detect the change in particle size in the solution before and after hydrogen peroxide solution treatment. Figure 4 Before hydrogen peroxide solution treatment, the average particle size of the double-crosslinked gel was 156.80 nm, with a particle size distribution of 0.09. After hydrogen peroxide solution treatment, two particle size peaks appeared. The smaller peak corresponds to the particle size of small molecules generated by the breakage of crosslinking bonds, with an average particle size of 5.76 nm. The larger peak corresponds to the particle size of single-crosslinked gel particles formed after the particles respond to reactive oxygen species, with an average particle size of 160.12 nm. The change in particle size compared to the previous double-crosslinked structure was not significant. Figure 4 This indicates that the breakage of oxidation-sensitive groups under the action of H2O2 weakens the complex internal structure without changing the size of the nanogel. This further proves that the nanogel system can achieve single intelligent regulation of particle mechanical properties. This property can be applied to the intelligent response of nanoparticle mechanical properties under the high reactive oxygen species conditions of tumors.

[0051] Example 6: Cytotoxicity test of RAW 264.7 cells using a nanogel with intelligently tunable mechanical properties (PVA-VEA-TSPBA).

[0052] The toxicity of the nanogel (PVA-VEA-TSPBA) in RAW264.7 cells was determined by the MTT assay. First, 100 μL of DMEM suspension containing 10% fetal bovine serum, 100 IU / mL penicillin, and 100 μg / mL streptomycin was spread into 96-well plates and cultured at 37°C with 5% CO2 for 12 h to achieve a monolayer cell coverage of 70–80%. Then, 10 μL of aqueous solutions of different concentrations of the nanogel were added to each well. After culturing for another 24 h, 10 μL of 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) in PBS (5 mg / mL) was added to each well, and the plates were incubated for another 4 h to allow the MTT to interact with the live cells. The culture medium containing MTT was then removed, and 150 μL of DMSO was added to each well to dissolve the purple formazan crystals produced by the live cells and MTT. The absorbance at 570 nm for each well was measured using a ELISA reader (SpectraMax i3x). The relative cell viability was obtained by comparing the absorbance at 570 nm with that of the control well containing only blank cells. All experimental data were performed in triplicate.

[0053] Cell viability (%) = (OD570 sample / OD570 control) × 100%

[0054] Figure 5The image shows the cytotoxicity results of the intelligently tunable mechanically modulated nanogel (PVA-VEA-TSPBA) on RAW264.7 cells. The results indicate that both the primary structure and the double-crosslinked nanogel showed almost no cytotoxicity to the cells at the set concentrations, demonstrating the good biocompatibility of the designed nanogel and its suitability for use as a drug delivery system.

Claims

1. A method for preparing a nanogel with intelligently controllable crosslinking density, characterized in that: A nanogel formed by two chemical crosslinking processes of polyvinyl alcohol grafted with vinyl ether acrylate (PVA-VEA) involves: first, under UV irradiation, the double bonds of PVA-VEA undergo a free radical reaction to form a stable mono-crosslinked nanogel; then, an additional reactive oxygen species-sensitive crosslinking agent is added to further react and form a bi-crosslinked smart responsive nanogel; wherein the PVA-VEA has the structure shown in the following formula: ； The reactive oxygen species-sensitive crosslinking agent is a compound with the following structure: 。 2. The method for preparing the intelligently controllable crosslinking density nanogel according to claim 1, characterized in that: The polyvinyl alcohol has a molecular weight of 5 to 120 kDa.

3. The method for preparing the intelligently controllable crosslinking density nanogel according to claim 1, characterized in that: The proportion of the crosslinking group vinyl ether propionate group in the single crosslinked nanogel is 0.5% to 8%.

4. The method for preparing the intelligently controllable crosslinking density nanogel according to claim 1, characterized in that: The proportion of reactive oxygen species-sensitive crosslinking agent added is 1% to 15%.

5. The application of a nanogel with intelligently controllable crosslinking density obtained by any one of claims 1-4 in the preparation of a drug carrier.

6. The application of a nanogel with intelligently tunable crosslinking density obtained by any one of claims 1-4 in the preparation of antitumor drugs.

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