Method for preparing ultralong polypeptide polymer brushes by using a two-phase polymerization system and application thereof

Abstract

This invention relates to a method for preparing ultralong polypeptide polymer brushes using a biphase polymerization system, belonging to the field of functional nanomaterials. In the reaction system of this invention, an aminated magnetic nanostirring rod is used as the core, proposing a universally applicable biphase polymerization system that successfully prepares nanorods modified with ultralong polypeptide polymer brushes. This invention employs a dichloromethane / water biphase polymerization system, utilizing the in-situ purification effect of the aqueous phase and the synergistic dipole-covalent polymerization between polypeptide chains to achieve grafting of ultrathick polypeptide polymer brushes with a single-sided shell thickness exceeding 358 nm (up to 490 nm) and a polymer molecular weight of 455.6 kDa, breaking the thickness limit of traditional surface-initiated polymerization. Addressing the problem that traditional polypeptide deprotecting agents easily corrode magnetic nanomaterials, this invention uses anhydrous aluminum chloride as the deprotecting agent, successfully achieving non-destructive deprotection. The ultralong polypeptide polymer brushes possess abundant drug binding sites; the thicker the polypeptide shell on the nanorod surface, the greater the drug adsorption capacity.

Description

Technical Field

[0001] This invention relates to a method for preparing ultralong polypeptide polymer brushes using a biphase polymerization system, belonging to the field of functional nanomaterials and their biochemical synthesis. Background Technology

[0002] Peptide-inorganic nanohybrids prepared by surface-initiated ring-opening polymerization of α-amino acids and N-carboxylic anhydrides (NCA-ROP) combine the excellent biocompatibility, secondary structure responsiveness, and side-chain functional groups of peptide polymers with the inherent characteristics of inorganic nanomaterials, showing broad application prospects in biomedical fields such as antibacterial and antifouling, drug delivery, tissue regeneration engineering, and biosensing. One-dimensional magnetic nanostirring rods (MNS), due to their high aspect ratio, high coercivity, and low cost, can generate high-intensity local shear forces under the drive of a rotating magnetic field, demonstrating unique value in scenarios such as thrombus mechanical destruction and targeted drug mixing. However, MNS with only a shell encapsulation face many problems in vivo, such as a lack of sufficient repulsive force to prevent non-specific protein adsorption and aggregation; limited functionality, providing only basic magnetomechanical effects; and drug loading relying on silica pore adsorption, resulting in low loading capacity and difficulty in release, failing to meet the needs of magnetic-drug synergistic diagnosis and treatment.

[0003] Grafting biomimetic peptide polymer brushes onto the surface of nanomaterials is a more promising approach to solving the aforementioned problems. Peptide polymer brushes not only endow nanomaterials with a dense physical barrier, a stable hydration layer, and electrostatic repulsion, improving the colloidal stability of nanomaterials, but more importantly, their core properties are significantly positively correlated with shell thickness: the thicker the shell, the stronger the steric barrier formed, and the better the anti-protein adsorption and anti-aggregation effects; simultaneously, the thicker the shell, the more linearly the number of carboxyl active sites on the side chains that can load drugs, enabling ultra-high drug loading.

[0004] However, traditional peptide-modified nanomaterial systems have long been limited by chain termination side reactions, mass transfer barriers, and insufficient kinetic efficiency caused by trace acidic impurities in the monomers. This results in poor shell uniformity and a difficult-to-break upper limit on polymerization thickness (typically difficult to exceed 100 nm). Furthermore, conventional strong acidic peptide deprotection agents (such as hydrobromic acid / trifluoroacetic acid) readily and severely corrode and damage the magnetic substrate. Therefore, there is an urgent need to develop a novel polymerization strategy and deprotection process to overcome this technological bottleneck. Summary of the Invention

[0005] Based on the secondary structure-mediated autocatalytic effect during ribosomal enzymatic protein synthesis in nature, this invention provides a method for preparing ultralong polypeptide polymer brushes using a biphase polymerization system. By constructing an water / oil biphase heterogeneous polymerization system, this invention aims to completely solve the core problems of slow kinetics, limited mass transfer, and easy substrate corrosion by traditional homogeneous polymerization systems, achieving rapid synthesis of ultrathick polypeptide shells and efficient targeted drug loading.

[0006] To solve the technical problem of this invention, the specific technical solution proposed by this invention is: a method for preparing ultralong polypeptide polymer brushes using a biphase polymerization system, comprising the following steps:

[0007] In a water / oil biphase heterogeneous interfacial polymerization system, an amino-modified inorganic nanomaterial is used as an initiator to initiate the ring-opening polymerization of α-amino acid-N-carboxyl anhydride NCA monomers that have a tendency to form α-helical secondary structures. By using aqueous phase extraction to remove monomer impurities and synergistic dipole covalent polymerization generated by the spontaneous folding of polypeptide chains into α-helices, ultralong polypeptide polymer brushes are generated by in-situ grafting onto the surface of inorganic nanomaterials.

[0008] Preferably, the inorganic nanomaterial is a one-dimensional magnetic nanostirring rod (MNS) with a silica shell on its surface, having a length of 4-9 µm and a width of 150-200 nm; the surface amination modification is achieved by pre-hydrolyzing 3-aminopropyltriethoxysilane (APTES) and protonating it under weakly acidic conditions to introduce primary amine initiating groups onto the surface of the inorganic nanomaterial; in the biphase system, the oil phase is a low-polarity organic solvent such as dichloromethane (DCM) or chloroform, and the aqueous phase is PBS phosphate buffer at pH 7.0; the volume ratio of the oil phase to the aqueous phase is constant at 10:1; the mass ratio of the NCA monomer to the initiator of the amination inorganic nanomaterial is controlled within the range of 2:1 to 131.5:1.

[0009] A method for preparing ultralong polypeptide polymer brushes using a biphase polymerization system, the specific steps of which are as follows:

[0010] (1) Preparation of MNS: Magnetic particles were prepared by high-temperature thermal decomposition and magnetic nano-stirring rods (length: 4~8 µm, width: 150~200 nm) were assembled by external magnetic field assembly.

[0011] (2) Amination modification of MNS surface: APTES was used to pre-hydrolyze and add acetic acid to protonate the amino group, and a primary amine initiating group was introduced into the MNS surface. After multiple purifications, it was vacuum dried.

[0012] (3) Synthesis of ultrathick polypeptide polymer brush in biphase system: MNS-NH2 dispersed in the aqueous phase of PBS was mixed with BLG-NCA monomer in the oil phase, and after sonication to form an emulsion, the mixture was allowed to stand for reaction. After multiple purifications, a magnetic nanostirring rod (MNS-PBLG) modified with poly(γ-benzyl-L-glutamic acid) was obtained.

[0013] (4) Deprotection with aluminum chloride: The peptide-modified magnetic nanorods were dispersed in anhydrous dichloromethane, and 9.0 eq of anhydrous AlCl3 with the same molar NCA were added. The mixture was allowed to stand for 24 h to obtain poly(L-glutamic acid) modified magnetic nanostirring rods (MNS-PLGA).

[0014] Preferably, the synthesis steps of the magnetic nanorods modified with a surface polypeptide shell thickness of 358 nm are as follows:

[0015] Step (1), Preparation of MNS: Fe3O4 nanoparticles of approximately 59 nm were prepared using a modified high-temperature thermal decomposition method. After ligand exchange and directional assembly under an external magnetic field, the silica shell was coated by TEOS hydrolysis to obtain MNS with an average length of 8.5 µm. Multiple batches of synthesized MNS were concentrated in 10 mL of anhydrous ethanol. The solution was then calibrated by mass concentration. 1 mL of the solution was transferred to 200 µL centrifuge tubes in several batches. After removing the waste liquid using magnetic adsorption, the tubes were dried in a vacuum oven. The mass change of the centrifuge tubes before and after drying was measured. The mass difference was calculated as the actual mass of MNS in 1 mL of MNS dispersion. Multiple parallel measurements were performed, and the average value was taken as the final calibration result.

[0016] Step (2), MNS surface amination: First, APTES (4 µL) was added to 1 mL of ethanol / water mixed solvent (95:5 v / v), stirred at 800 rpm for 1 h to ensure complete hydrolysis, and the pH was adjusted to 5 with acetic acid; then 1 mL of MNS solution (0.1 mg / mL) was added, and the mixture was allowed to stand at room temperature for 2 h. The reaction product was collected by centrifugation (3000 g, 10 min), washed repeatedly with anhydrous ethanol to remove free APTES, and finally dried under vacuum.

[0017] Step (3), standard synthesis group of the biphasic system: Disperse the weighed NCA monomer in 0.5 mL DCM solution, then add 50 µL of MNS-NH2 in PBS solution, V DCM / V 水 =10:1, m BLG-NCA / m MNS The ratio of the two components was 65.75:1. After ultrasonic mixing to form an emulsion, the mixture was allowed to stand for reaction. Once the reaction was complete, the mixture was centrifuged at 3000 g for 10 min and washed multiple times with DCM, DMF, and ethanol to remove free peptides. The final product was dispersed and stored in DMF.

[0018] Step (4), Deprotection: The peptide-modified MNS was dispersed in 4 mL of DCM, and Lewis acid deprotecting agent (9.0 eq of NCA molar) weighed in the glove box was added. After mixing evenly, the mixture was allowed to stand for 24 h. After magnetic separation to remove waste liquid, the product was washed with 1 mL of saturated sodium bicarbonate to precipitate aluminum ions. Then, 1 mL of HCl (1 M) was added to dissolve the precipitate. The mixture was washed repeatedly until no precipitate was formed upon the addition of sodium bicarbonate. The product was then washed once with 1 mL of EDTA (0.1 M) solution, and finally washed with water to obtain a clean product.

[0019] Preferably, the NCA monomers used include γ-benzyl-L-glutamic acid-N-carboxylic anhydride (BLG-NCA), γ-benzyl-D-glutamic acid-N-carboxylic anhydride (BDG-NCA), and Nε-benzyloxycarbonyl-L-lysine-N-carboxylic anhydride (ZLL-NCA).

[0020] Preferably, the Lewis acid deprotecting agent used is anhydrous AlCl3.

[0021] The application of the inorganic nano-hybrid material modified with the ultralong polypeptide polymer brush in the preparation of drug adsorption carriers or magnetically controlled targeted drug delivery systems.

[0022] The specific application involves the adsorption and targeted loading of methylene blue, which mimics anticancer or antitumor drugs. Utilizing the massive number of active sites exposed after the deprotection of the ultra-long, loose polypeptide polymer shell, drug molecules are massively loaded through electrostatic adsorption or covalent coupling. The amount of drug adsorbed per unit volume of the nanocarrier increases significantly and proportionally with the increase of the polypeptide shell thickness.

[0023] The specific application involves the adsorption and targeted loading of anticancer or antitumor drug molecules, including doxorubicin or imatinib. Utilizing the massive active sites exposed after deprotection of the ultralong, loose polypeptide polymer shell, drug molecules are massively loaded via electrostatic adsorption or covalent coupling. The amount of drug adsorbed per unit volume of the nanocarrier increases significantly and proportionally with the increase in the thickness of the polypeptide shell. Under the drive of an alternating magnetic field, the inorganic nanohybrid material can construct multifunctional magnetically controlled nanorobots, achieving dual-modal synergistic therapy of in-situ magnetomechanical destruction of tumors and microenvironment-responsive drug release.

[0024] The beneficial effects of this invention are:

[0025] 1. Breakthrough in the upper limit of peptide polymer brush shell thickness. Traditional surface-initiated NCA-ROP technology typically struggles to achieve shell thicknesses exceeding 100 nm due to chain termination side reactions and mass transfer limitations. This invention, by constructing a DCM / water biphase polymerization system, successfully grafted ultra-thick peptide shells with single-sided thicknesses reaching 358 nm and a maximum of 490 nm onto the surface of nanorods, achieving a significant breakthrough over the previously reported upper limit of thickness.

[0026] 2. Significantly improved polymerization kinetic efficiency, enabling rapid and controllable synthesis. The biphase polymerization system employed in this invention achieves a monomer conversion rate of 99% within 2 hours, which is several times higher than the traditional homogeneous dichloromethane system, greatly shortening the production cycle. The thickness of the peptide shell on the surface of the magnetic nanostirring rod can be controlled by adjusting the mass ratio of NCA monomer to MNS.

[0027] 3. A unique "dense-loose" double-shell topology was constructed, enhancing material properties. The polypeptide polymer brush prepared in this invention possesses a unique asymmetric double-shell structure: a dense inner shell (approximately 45 nm) closely adhering to the substrate provides strong protection, while the outwardly extending loose outer shell exhibits a radial "brush" conformation. This structure not only provides an extremely high polypeptide grafting capacity (up to 50.6% by mass), but also offers an extremely large and flexible three-dimensional space for the subsequent massive loading of anticancer drugs.

[0028] 4. Non-destructive deprotection treatment of peptide-modified magnetic nanorods. This invention uses anhydrous aluminum chloride as the deprotection agent, avoiding corrosion of the magnetic nanomaterials and degradation of the peptide backbone, thus successfully achieving non-destructive deprotection of peptide-modified magnetic nanorods.

[0029] 5. Imparting ultra-high drug loading capacity to nanocarriers. After removing the benzyl group, the porous polypeptide shell, hundreds of nanometers thick, can achieve a maximum drug loading capacity of 0.448 mg / mg, overcoming the limitations of traditional mesoporous inorganic materials that rely solely on surface pores, resulting in extremely low adsorption capacity. This higher drug loading capacity means that, with the same clinical therapeutic effect, the total dose of carrier material injected into the patient can be reduced by nearly 80%, demonstrating significant commercial application potential in translational medicine fields such as high-load drug delivery to deep solid tumors. Attached Figure Description

[0030] The present invention will be further described below with reference to the accompanying drawings.

[0031] Figure 1 This is the NCA preparation process.

[0032] Figure 2 The MNS preparation process is as follows: (a) Transmission electron microscope image of Fe3O4 particles prepared by high-temperature pyrolysis, and (b) Scanning electron microscope image of MNS prepared by assembly under an external magnetic field.

[0033] Figure 3 This is a flowchart of the synthesis of ultra-long polypeptide polymers using a biphase polymerization system.

[0034] Figure 4 The images are (a) in-situ infrared spectra of BLG-NCA polymerization initiated by MNS-NH2 in a two-phase system, (b) the relationship between NCA monomer consumption and polymerization time in different systems, (c) transmission electron microscopy images of polymerization products in a single-phase DCM system, and (d) transmission electron microscopy images of polymerization products in a two-phase system.

[0035] Figure 5 (a) Infrared spectra of samples MNS, MNS-NH2, BLG and MNS-PBLG, (b) Gel chromatography characterization of samples under standard synthesis, and (c) Thermogravimetric standard chromatograms of MNS, MNS-NH2 and MNS-PBLG.

[0036] Figure 6 The polymerization products under different DCM / water volume ratios are: (a) the relationship between NCA monomer consumption and polymerization time, (b) the change in unilateral shell thickness, transmission electron micrographs: (c) 400:1, (d) 200:1, (e) 100:1, (f) 50:1.

[0037] Figure 7 The following are transmission electron micrographs of the polymer products at different NCA / MNS mass ratios: (a) 2:1, (b) 5:1, (c) 13.2:1, (d) 26.3:1, (e) 65.75:1, (f) 132:1.

[0038] Figure 8 This is the relationship between the thickness of the single-sided shell of the polymer product MNS-PBLG and the amount of NCA added.

[0039] Figure 9 The following are verifications of the covalent polymerization: (a) MNS-PBDG was synthesized using levorotatory BDG-NCA, and (b) MNS-PBDLG was synthesized using racemic BDLG-NCA.

[0040] Figure 10 The general applicability was verified by: (a) synthesis of MNS-PZLL using ZLL-NCA, and (b) block polymerization using BLG-NCA and ZLL-NCA.

[0041] Figure 11 This is the infrared spectrum of MNS-PLGA after deprotection with aluminum chloride.

[0042] Figure 12(a) UV spectrum of MNS-PLGA adsorbed with methylene blue after deprotection, and (b) standard curve of methylene blue. Detailed Implementation

[0043] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0044] Example 1: Preparation of the core monomer BLG-NCA (Fuchs-farthing method)

[0045] Referring to Nat Commun 12, 5810 (2021), the NCA monomer synthesis process is as follows: Figure 1 As shown, anhydrous reagents were used throughout the experiment. The Fuchs-farthing method was employed, where triphosgene activated amino acids to generate carbamoyl chloride intermediates, which then underwent a cyclization reaction to form the five-membered heterocyclic NCA structure. After the reaction, the mixture was cooled and purified with cold pure water to quench excess triphosgene. The mixed solution was washed and extracted with saturated brine, then dried over anhydrous magnesium sulfate and filtered. The filtrate was evaporated to remove the solvent, and the crude product was purified by recrystallization three times using a hexane / THF (6:1 v / v) mixture. Vacuum drying yielded white needle-like crystals of the BLG-NCA monomer. (The preparation of other monomers, such as ZLL-NCA and BDG-NCA, followed the same process and purification standards.)

[0046] Example 2: Preparation and surface amination of MNS (MNS-NH2)

[0047] Referring to Angewandte Chemie International Edition, 2013, 52(33): 8570-8573; Nanoscale, 2020, 12(36): 18640-18645; ACS Nano, 2019, 13(7): 7716-7728, the MNS synthesis procedure is as follows: Figure 2 As shown. Fe3O4 nanoparticles of approximately 59 nm were prepared using an improved high-temperature thermal decomposition method. Figure 2 a) After ligand exchange and assembly under the direction of an applied magnetic field, MNS with an average length of 8.5 µm was obtained by TEOS hydrolysis and coating of a silica shell. Figure 2 b).

[0048] MNS surface amination: First, APTES (4 µL) was added to 1 mL of ethanol / water mixture (95:5 v / v) and stirred at 800 rpm for 1 h to ensure complete hydrolysis. The pH was then adjusted to 5 with acetic acid. Subsequently, 1 mL of MNS solution (0.1 mg / mL) was added, and the mixture was allowed to stand at room temperature for 2 h. The reaction product was collected by centrifugation (3000 g, 10 min), washed repeatedly with anhydrous ethanol to remove free APTES, and finally dried under vacuum.

[0049] Example 3: Standard synthesis group of two-phase system (MNS-PBLG) and synthesis of single-phase system

[0050] Standard synthesis group for two-phase systems: Synthesis process as follows Figure 3 As shown, BLG-NCA prepared in Example 1 was dispersed and dissolved in 0.5 mL of anhydrous dichloromethane (DCM), and then 50 µL of PBS aqueous buffer solution of MNS-NH2 synthesized in Example 2 was added to it. DCM / V 水 =10:1, m BLG-NCA / m MNS The ratio of 65.75:1 was used to form an emulsion by ultrasonic mixing, followed by static reaction. After the reaction was completed, the mixture was centrifuged at 3000 g for 10 min and washed multiple times with DCM, DMF, and ethanol to remove free peptides. The final product, MNS-PBLG, was dispersed and stored in DMF.

[0051] Single-phase homogeneous comparison group: The MNS-NH2 synthesized in Example 2 was dispersed in 0.5 mL of anhydrous DCM or chloroform solution and mixed with the anhydrous DCM or chloroform solution of BLG-NCA prepared in Example 1. BLG-NCA / m MNS =65.75:1, allowed to stand for reaction. After the reaction was complete, centrifuged at 3000 g for 10 min, and washed repeatedly with DCM, DMF and ethanol to remove free peptides. The final product MNS-PBLG was dispersed and stored in DMF.

[0052] In-situ infrared spectroscopy monitoring of polymerization kinetics showed that in the pure DCM single-phase system, due to the quenching effect of trace impurities remaining in the monomer, the monomer conversion rate only increased by 12% within 2 hours, and ultimately only a 6 nm ultrathin polypeptide layer grew on the MNS surface. Figure 4 (b, c) Even with chloroform, a solvent with lower polarity, the polymerization reaction still requires 12 hours to achieve a 99% monomer conversion rate, and the peptide shell on the MNS surface is only 13 nm.

[0053] In the biphase system constructed in this invention, the monomer conversion rate of NCA ring-opening polymerization initiated by MNS-NH2 reaches 99% in only 2 hours. Figure 4a, b). MNS-PBLG prepared using a two-phase polymerization system exhibits an ultra-thick polymer shell structure. Under phosphotungstic acid negative staining, the peptide shell shows an asymmetric double-shell structure with contrasting electronic contrast: a uniformly thick, brightly contrasting ring closely adhering to the MNS surface, where the dye has difficulty penetrating and may even remain unstained; while the outermost region exhibits an ultra-thick outer shell that the dye can penetrate. Figure 4 d). By measuring the shell thickness, it was found that the average thickness of the MNS-PBLG shell on one side reached about 358 nm, which far exceeds the peptide shell thickness reported by conventional grafting strategies in existing literature, achieving a breakthrough in shell thickness in this field.

[0054] Infrared spectroscopy confirmed the structure of the ultrathick MNS-PBLG. Figure 5 a). MNS-PBLG, while retaining the characteristic absorption bands of the substrate, exhibits a stretching vibration peak (1730 cm⁻¹) of the benzyl carbonyl (C=O) group of the polypeptide side chain. -1 ), belonging to the amide I band of the polypeptide backbone (1650 cm). -1 (C=O stretching) and amide II band (1550 cm) -1 The characteristic peaks (NH bending and CN stretching coupling) confirmed the grafted ultrathick double-shell structure and α-helical structure. Gel chromatography showed that the molecular weight distribution of this structure exhibited a single-peak distribution, indicating that the ultrathick polypeptide shell originated from the in-situ ring-opening polymerization of monomers on the MNS surface. Figure 5 b). The molecular weight of the peptide polymer on the MNS surface reached 455.6 kDa, with a polydispersity index of 1.14. This type of nanomaterial modified with ultra-high peptide polymer molecular weight has never been reported before. Thermogravimetric characterization showed that the mass of the peptide grafted onto the MNS surface in the biphase system accounted for 50.6% of the total volume. Figure 5 c).

[0055] Example 4: Regulation of peptide shell by different DCM / water volume ratios in standard synthesis systems ( Figure 6 )

[0056] Based on the biphase system (m) in Example 3 BLG-NCA / m MNS Under a constant ratio of 65.75:1, we investigated the effect of the size of the water-provided interface on polymerization kinetics and the final shell structure, by systematically changing the volume ratio of DCM / water in the standard system (i.e., setting VC). DCM / V 水 The gradients are 10:1, 50:1, 100:1, 200:1, and 400:1.

[0057] Figure 6The results show that when the proportion of the aqueous phase in the system decreases, i.e., the interfacial area for polymerization decreases, the polymerization kinetics slow down significantly, especially in V. DCM / V 水 The polymerization kinetics differ significantly around a water-to-water ratio of 100:1, but the differences are smaller at low aqueous phase ratios (200:1, 400:1). Even with a water ratio of 400:1 (volume ratio of soluble water) added to pure DCM, the polymerization kinetics are significantly faster than in the single-phase pure DCM system. However, the variation in PBLG shell thickness, characterized by TEM, shows little difference, only at V... DCM / V 水 When the ratio is 50:1, the grafted shell thickness is 291 ± 14 nm, while the others remain around 350 nm, showing no obvious linear trend.

[0058] During polymerization, water forms an emulsion with the oil phase, allowing MNS-NH2 to anchor at the interface for polymerization. The proportion of the water phase reflects the contact area of ​​MNS polymerization at the interface; a larger interface allows for more NCA polymerization, resulting in faster polymerization kinetics. However, analysis of the peptide shell thickness under different DCM / water volume ratios showed that changing the water phase proportion did not alter the shell thickness. This demonstrates that the polymerization reaction does not primarily occur at the interface, but the presence of water accelerates peptide deposition onto the MNS surface.

[0059] Example 5: Regulation of peptide shell by different NCA / MNS mass ratios in standard synthesis system ( Figure 7 )

[0060] While maintaining the basic biphase system of Example 3 (V) DCM / V 水 Under the operation of constant ratio 10:1, the system changes the mass ratio of monomers fed into the system (i.e., setting m). BLG-NCA / m MNS The gradients are 2:1, 5:1, 13.15:1, 26.3:1, 65.75:1, and 131.5:1.

[0061] Figure 7 The results showed that the thickness of the one-sided peptide shell on the MNS surface exhibited a linear increasing trend with the increase of monomer supply. When the NCA / MNS mass ratio was 2:1, the system lacked monomers, and only a shell with a thickness of 17 ± 4 nm was formed on the MNS surface. Figure 7 a). The formation of a relatively thick peptide shell at this low NCA monomer addition level is also unprecedented. With the mass ratio increased to 5:1, MNS-PBLG exhibited a "dense-loose" double-shell structure, with an inner shell thickness of 39 ± 11 nm and an overall shell thickness of 147 ± 25 nm. Figure 7b). When the mass ratio was further increased to 131.5:1 and above, all samples exhibited a clear and stable double-shell structure. The overall shell thickness gradually increased with the increase of NCA monomer content, reaching 252 ± 26, 331 ± 19, 358 ± 24 and 490 ± 41 nm, respectively. Figure 7 (cf). This ultra-thick peptide shell thickness-modified nanomaterial breaks the record for shell thickness in this field. We further analyzed the following four groups of samples with high mass ratios. Although the overall shell thickness varied significantly, the thickness of their dense inner shell remained stable within the range of approximately 45 ± 5 nm. Figure 8 ).

[0062] Example 6: Confirmation and generalization of the α-helical chain-to-chain synergistic covalent accelerated polymerization mechanism

[0063] While maintaining the basic biphase system of Example 3 (V) DCM / V 水 =10:1, m BLG-NCA / m MNS Under constant operation (65.75), ultra-long PBDG and PBDLG polymer brushes were grown using levorotatory BDG-NCA monomers and racemic BDLG-NCA monomers (BLG-NCA and BDG-NCA were mixed in a 1:1 mass ratio).

[0064] Figure 9 The results showed that the MNS-PBDG shell maintained an ultra-thick double-shell structure, with an inner shell of 48 nm and an overall shell of 326 nm; while the MNS-PBDLG shell only exhibited an ultra-thick double-shell structure in a few instances, with most existing as a thinner shell (12.25 nm). The polypeptide chains formed by the polymerization of BDLG-NCA monomers mainly exhibited a random coil conformation, lacking the dipolar synergistic acceleration effect between α-helical chains, which reduced the polymerization kinetics. However, water still played a role in the two-phase system, promoting the ring-opening polymerization of NCA monomers, resulting in a large amount of NCA being degraded by water rather than deposited on the MNS surface.

[0065] While maintaining the basic biphase system of Example 3 (V) DCM / V 水 =10:1, m BLG-NCA / m MNS Under constant conditions (65.75), ultra-long PZLL polymer brushes were grown using ZLL-NCA, and block-type ultra-long P(BLG-ZLL) polymer brushes were grown using a 1:1 mass mixture of BLG-NCA and ZLL-NCA monomers. Figure 10The polymerization effect diagrams of MNS-PZLL and MNS-P (BLG-ZLL) are shown. The polypeptide chains grown by ZLL-NCA and BLG-NCA both have α-helical structures, and both have grown ultra-thick polypeptide shells on the surface of MNS, proving that the polymerization system of the present invention has the universality of NCA monomers that tend to form α-helical secondary structures.

[0066] Example 7: Non-destructive deprotection of magnetic substrates using anhydrous aluminum chloride (MNS-PLGA)

[0067] The MNS-PLGA obtained in Example 3 was dispersed in 4 mL of DCM, and anhydrous AlCl3 (9.0 eq of NCA molars) weighed from a glove box was added. After mixing thoroughly, the mixture was allowed to stand for 24 h. After magnetic separation to remove waste liquid, the product was washed with 1 mL of saturated sodium bicarbonate to precipitate aluminum ions. Then, 1 mL of HCl (1 M) was added to dissolve the precipitate, and the mixture was washed repeatedly until no precipitate was formed upon the addition of sodium bicarbonate. The product was then washed once with 1 mL of EDTA (0.1 M) solution, and finally washed with water to obtain clean MNS-PLGA. The infrared spectrum of the deprotected MNS-PLGA is shown below. Figure 11 a), where it is located at 1730 cm -1 The characteristic peak of the benzyl carbonyl group disappeared after deprotection, indicating that the protecting group was completely removed. Transmission electron microscopy showed that the MNS-PLGA structure was intact, avoiding the problems of commonly used deprotecting agents corroding magnetic particles and degrading the polypeptide backbone. Figure 11 b).

[0068] Example 8: Evaluation of "massive model drug" loading as a targeted anti-tumor drug carrier ( Figure 12 )

[0069] To verify the potential adsorption / sustained-release performance of the extended "ultra-thick loose peptide brush coating" of the MNS-PLGA structure modified with an ultra-long peptide polymer brush in clinical targeted medicine, Example 8 specifically selected methylene blue (MB) cationic molecule as an evaluation mimic for "antitumor / anticancer active drugs." Because MB possesses a planar aromatic heterocyclic structure, molecular size, and cationic charge characteristics highly similar to typical clinical anticancer chemotherapy drugs (such as doxorubicin and imatinib), its electrostatic adsorption and intermolecular interaction behavior in the peptide polymer brush can realistically reflect the loading potential of actual anticancer drugs.

[0070] The MNS-PLGA products with different thickness gradients obtained according to the method in Example 7 were co-incubated with aqueous solutions of simulated drug molecules at a concentration of 0.5 mg / mL at room temperature. The simulated drug molecules were loaded into the loose peptide brush through electrostatic attraction or hydrogen bonding / covalent coupling effects. The adsorption amount of the products was determined by ultraviolet absorption spectroscopy (peak at 664 nm) after magnetic separation.

[0071] Figure 12 a shows the UV spectrum of MNS-PLGA after adsorption of methylene blue, all exhibiting an absorption peak of methylene blue near 664 nm, and the absorption peak intensity increases with the increase of the peptide shell thickness of MNS-PLGA. According to the standard curve ( Figure 12 (b) The adsorption capacity of MNS-PLGA synthesized with NCA / MNS mass ratios of 2:1, 5:1, 13.15:1, 26.3:1 and 65.75:1 (mg / mg) for methylene blue was 0.096, 0.124, 0.132, 0.244 and 0.448 mg / mg, respectively.

[0072] Experimental data strongly demonstrate that the polypeptide polymer brush carrier, which breaks through the thermodynamic thickness limit and possesses a unique double-shell structure, overcomes the limitations of traditional solid inorganic materials with their shallow surface physical pores, resulting in low drug loading capacity, achieving a leap in drug loading capacity several times over. When it is used to actually load anticancer drugs such as doxorubicin and imatinib in the future, combined with the physical penetration properties driven by an external rotating magnetic field, it is expected to construct a multifunctional magnetically controlled nanorobot integrating "in-situ magnetomechanical destruction" and "microenvironment-responsive drug release," providing a highly efficient new material basis for targeted therapy of deep and complex solid tumors in vivo.

Claims

1. A method for preparing ultralong polypeptide polymer brushes using a biphase polymerization system, characterized in that, Includes the following steps: In a water / oil biphase heterogeneous interfacial polymerization system, an amino-modified inorganic nanomaterial is used as an initiator to initiate the ring-opening polymerization of α-amino acid-N-carboxyl anhydride NCA monomers that have a tendency to form α-helical secondary structures. By using aqueous phase extraction to remove monomer impurities and synergistic dipole covalent polymerization generated by the spontaneous folding of polypeptide chains into α-helices, ultralong polypeptide polymer brushes are generated by in-situ grafting onto the surface of inorganic nanomaterials.

2. The method for preparing ultralong polypeptide polymer brushes using a biphase polymerization system according to claim 1, characterized in that: The inorganic nanomaterial is a one-dimensional magnetic nanostirring rod (MNS) with a silica shell on its surface, with a length of 4-9 µm and a width of 150-200 nm. The surface amination modification is achieved by pre-hydrolyzing 3-aminopropyltriethoxysilane (APTES) and protonating it under weakly acidic conditions to introduce primary amine initiating groups onto the surface of the inorganic nanomaterial. In the biphase system, the oil phase is a low-polarity organic solvent such as dichloromethane (DCM) or chloroform, and the aqueous phase is PBS phosphate buffer at pH 7.

0. The volume ratio of the oil phase to the aqueous phase is constant at 10:

1. The mass ratio of the NCA monomer to the initiator of the amination inorganic nanomaterial is controlled within the range of 2:1 to 131.5:

1.

3. The method for preparing ultralong polypeptide polymer brushes using the biphase polymerization system according to claim 1, characterized in that: The specific steps of the method for preparing ultralong polypeptide polymer brushes using the biphase polymerization system are as follows: (1) Preparation of MNS: Magnetic particles were prepared by high-temperature thermal decomposition and magnetic nano-stirring rods were assembled by external magnetic field assembly method. The length was 4~8 µm and the width was 150~200 nm. (2) MNS surface amination modification: APTES was used to pre-hydrolyze and add acetic acid to protonate the amino group, and a primary amine initiating group was introduced into the MNS surface. After multiple purifications, the surface was vacuum dried. (3) Synthesis of ultra-thick polypeptide polymer brush in biphase system: MNS-NH2 dispersed in the aqueous phase of PBS was mixed with BLG-NCA monomer in the oil phase, and after ultrasonication to form an emulsion, the mixture was allowed to stand for reaction; after multiple purifications, a magnetic nanostirring rod MNS-PBLG modified with poly(γ-benzyl-L-glutamic acid) was obtained. (4) Deprotection with aluminum chloride: The peptide-modified magnetic nanorods were dispersed in anhydrous dichloromethane, and 9.0 eq of anhydrous AlCl3 with the same molar NCA were added. The mixture was allowed to stand for 24 h to obtain poly(L-glutamic acid) modified magnetic nanostirring rods MNS-PLGA.

4. The method for preparing ultralong polypeptide polymer brushes using a biphase polymerization system according to claim 1, characterized in that: The NCA monomer with a tendency to form an α-helical secondary structure is selected from γ-benzyl-D-glutamic acid-N-carboxylic anhydride BDG-NCA, γ-benzyl-L-glutamic acid-N-carboxylic anhydride BLG-NCA, Nε-benzyloxycarbonyl-L-lysine-N-carboxylic anhydride ZLL-NCA, or a block copolymer mixture of the two; wherein, the preparation method of the NCA monomer includes: using triphosgene and the corresponding amino acid derivative, propylene oxide as raw materials, cyclization reaction in an anhydrous organic solvent system for synthesis, and purification by crystallization in a hexane / tetrahydrofuran system; The generated ultralong polypeptide polymer brush exhibits an asymmetric "dense-loose" double-shell topology; the thickness of the dense inner shell closely attached to the inorganic material substrate is maintained at a constant threshold of 45 ± 5 nm, while the loose outer shell extends outward in a radially radiating mushroom shape; the thickness of the overall polypeptide shell on one side ranges from 147 nm to 490 nm, and the molecular weight (Mn) of the grafted polypeptide ranges from 280 kDa to 460 kDa.

5. The method for preparing ultralong polypeptide polymer brushes using a biphase polymerization system according to claim 1, characterized in that, After the reaction is complete, a non-destructive deprotection step of the peptide side chains is also included: Nanomaterials modified with ultralong polypeptide polymer brushes were dispersed in an anhydrous organic solvent. After reacting with anhydrous aluminum chloride (AlCl3) as a deprotection reagent, the nanomaterials were washed and purified sequentially with sodium bicarbonate, hydrochloric acid, and EDTA solution. In the non-destructive deprotection step, the molar amount of anhydrous aluminum chloride added was 9.0 equivalents of the initial molar amount of NCA monomer. This reagent is used to specifically cleave ester bonds and remove benzyl protecting groups on the side chains of polypeptides at specific sites to expose a large number of highly active free carboxyl or amino sites. Moreover, this deprotection step does not corrode the internal magnetic inorganic nanosubstrate.

6. The method for preparing ultralong polypeptide polymer brushes using a biphase polymerization system according to claim 1, characterized in that: The specific steps are as follows: The Fuchs-farthing method was used to activate amino acids with triphosgene to generate carbamoyl chloride intermediates, which then underwent a cyclization reaction to form a five-membered heterocyclic NCA structure. After the reaction was completed, the mixture was cooled and cooled pure water was added to quench excess triphosgene. The mixed solution was washed with saturated brine and extracted. Finally, it was dried over anhydrous magnesium sulfate and filtered. The filtrate was subjected to rotary evaporation to remove the solvent. The crude product was purified by recrystallization three times using a hexane / THF mixed solvent. After vacuum drying, white needle-like crystals of γ-benzyl-L-glutamic acid-N-carboxylic anhydride BLG-NCA monomer were obtained. 59 nm Fe3O4 nanoparticles were prepared by an improved high-temperature thermal decomposition method. After ligand exchange and directional induction by an external magnetic field, they were assembled and coated with a silica shell by TEOS hydrolysis to obtain a magnetic nanostirring rod MNS with an average length of 8.5 µm. MNS surface amination: First, 4 µL of APTES was added to 1 mL of ethanol / water mixed solvent (95 / 5 v / v), and stirred at 800 rpm for 1 h to fully hydrolyze the solution. The pH was then adjusted to 5 with acetic acid. Subsequently, 1 mL of MNS solution (0.1 mg / mL) was added, and the reaction was allowed to stand at room temperature for 2 h. The reaction product was collected by centrifugation and washed repeatedly with anhydrous ethanol to remove free APTES. Finally, the product was dried under vacuum to obtain MNS-NH2. Preparation of 358 nm ultralong polypeptide polymer brush-modified magnetic nanorods: MNS-NH2 dispersed in PBS solution was mixed with the prepared BLG-NCA DCM solution, V DCM / V 水 =10:1, m BLG-NCA / m MNS =65.75:1, after ultrasonic mixing, the mixture was allowed to stand for reaction; after the reaction was completed, it was washed multiple times to remove free peptides, and the final product, poly(γ-benzyl-L-glutamic acid) modified magnetic nanostirring rod MNS-PBLG, was dispersed and stored in DMF. The deprotection method for MNS-PBLG is as follows: MNS-PBLG is dispersed in DCM, and anhydrous AlCl3 weighed in a glove box is added, along with 9.0 eq of NCA. The mixture is allowed to stand for 24 h. After magnetic separation to remove waste liquid, the mixture is washed and purified, and finally washed with water to obtain clean poly(L-glutamic acid) modified magnetic nanostirring rods MNS-PLGA.

7. An inorganic nano-hybrid material modified with an ultralong polypeptide polymer brush, prepared by the method according to any one of claims 1-6.

8. The application of the inorganic nano-hybrid material modified with the ultralong polypeptide polymer brush as described in claim 7 in the preparation of drug adsorption carriers or magnetically controlled targeted drug delivery systems.

9. The application according to claim 8, characterized in that: The specific application involves the adsorption and targeted loading of methylene blue, which mimics anticancer or antitumor drugs. Utilizing the massive number of active sites exposed after the deprotection of the ultra-long, loose polypeptide polymer shell, drug molecules are massively loaded through electrostatic adsorption or covalent coupling. The amount of drug adsorbed per unit volume of the nanocarrier increases significantly and proportionally with the increase of the polypeptide shell thickness.

10. The application according to claim 8, characterized in that: The specific application involves the adsorption and targeted loading of anticancer or antitumor drug molecules, including doxorubicin or imatinib. Utilizing the massive active sites exposed after deprotection of the ultralong, loose polypeptide polymer shell, drug molecules are massively loaded via electrostatic adsorption or covalent coupling. The amount of drug adsorbed per unit volume of the nanocarrier increases significantly and proportionally with the increase in the thickness of the polypeptide shell. Under the drive of an alternating magnetic field, the inorganic nanohybrid material can construct multifunctional magnetically controlled nanorobots, achieving dual-modal synergistic therapy of in-situ magnetomechanical destruction of tumors and microenvironment-responsive drug release.

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