Biomimetic mineralized nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses biomimetic mineralized nanoparticles as well as a preparation method and application thereof. The surface of the biomimetic mineralized nanoparticle is a mineralized virus shell, virus infection can be simulated, cell uptake can be enhanced, a specific antibody for recognizing target protein can be effectively encapsulated, and meanwhile, a virus membrane component can induce expression of TRIM family protein in cells. After the biomimetic mineralized nanoparticles are taken into cells, the antibody released by the biomimetic mineralized nanoparticles can bind to the target protein and bind to the TRIM family protein to mediate degradation of the target protein. In this process, although the TRIM family protein is continuously consumed, the TRIM family protein can be compensated by the TRIM family protein endogenous expression induced by the particle of the present invention, thereby maintaining efficient degradation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of targeted protein degradation, in particular to biomimetic mineralized nanoparticles and methods of making and uses thereof. BACKGROUND

[0002] Targeted Protein Degradation (TPD) is a technology that achieves the precise elimination of target proteins by utilizing the natural degradation system in cells, such as the ubiquitin-proteasome system, lysosomal pathway, autophagy pathway, etc. Among various emerging TPD technologies, antibody-mediated TPD has received extensive attention due to its high affinity, which significantly improves the selectivity and targeting accuracy of TPD. However, this strategy still faces many challenges, including the limited cell membrane penetration ability of antibodies and the depletion of E3 ubiquitin ligase during the degradation process, which can lead to decreased degradation efficiency.

[0003] The information in the background section merely serves to explain the general background of the application and should not be considered as admitting or in any way suggesting that this information forms prior art to the application. SUMMARY

[0004] To solve at least some of the technical problems in the prior art, the present application provides biomimetic mineralized nanoparticles and methods of making and uses thereof. Specifically, the present application includes the following content.

[0005] In a first aspect of the present application, a biomimetic mineralized nanoparticle is provided, which comprises a core structure and a shell structure, the core structure comprising an antibody, and the shell structure coating the core structure and comprising a mineral and a membrane component.

[0006] In certain embodiments, the biomimetic mineralized nanoparticle according to the present application, wherein the membrane component comprises a component derived from a virus, such as a viral envelope or a viral membrane structure or a component thereof. The type of virus is not particularly limited.

[0007] In certain embodiments, the biomimetic mineralized nanoparticle according to the present application, wherein the surface main elements of the biomimetic mineralized nanoparticle comprise Na, C, Ca, N and P when analyzed using X-ray photoelectron spectroscopy.

[0008] In certain embodiments, the biomimetic mineralized nanoparticle according to the present application, wherein the particle size of the biomimetic mineralized nanoparticle is in the range of 50-500 nm, such as 80 nm, 90 nm, 100 nm, or 150-500 nm, 150-400 nm, 150-300 nm, 150-250 nm, 150-200 nm.

[0009] In the present invention, the kind of antibody is not particularly limited, and an antibody comprising an Fc fragment is preferred, and any neutralizing antibody or any therapeutic antibody is also preferred. In certain embodiments, the antibody is a neutralizing antibody.

[0010] In a second aspect of the present invention, a pharmaceutical composition is provided, comprising the biomimetic mineralized nanoparticle described above and a pharmaceutically acceptable carrier.

[0011] In certain embodiments, the pharmaceutical composition according to the present invention, wherein the pharmaceutical composition further comprises a TRIM activator. The TRIM activator can be any exogenously added component to further compensate for the continuous consumption of TRIM itself in addition to the induction of endogenous expression of TRIM family proteins by the biomimetic mineralized nanoparticle viral membrane component. Examples of the TRIM activator include, but are not limited to, IFN-α, a recombinant plasmid expressing or overexpressing TRIM. When an exogenously added component is used, the concentration of the additive can be 100-1000 U / mL, for example, less than 500-1000 U / mL, to achieve stable expression of TRIM family proteins.

[0012] In a third aspect of the present invention, a method for preparing a biomimetic mineralized nanoparticle is provided, comprising the step of incubating an antibody-containing particle in a mineralization solution, wherein the mineralization solution comprises divalent metal ions and a solvent.

[0013] In a specific embodiment, the method for preparing a biomimetic mineralized nanoparticle comprises:

[0014] (1) preparing an antibody-containing particle, comprising: lysing virus-infected cells to obtain a lysate, the lysate is first subjected to low-speed centrifugation to collect the supernatant, and then the supernatant is subjected to high-speed centrifugation to collect the precipitate, the precipitate is washed with a buffer, and then ultrasonic treatment is performed in serum-free DMEM, to obtain a viral membrane solution, an antibody is added to the viral membrane solution, and the mixed solution is extruded through an extruder to generate an antibody-containing particle;

[0015] (2) biomimetic mineralization, comprising: dissolving the particle obtained in step (1) in serum-free DMEM, adding a mineralization solution, and reacting under sealed conditions, vortex mixing, and then centrifuging to obtain a biomimetic mineralized nanoparticle.

[0016] In step (1) of the above method, the virus-infected cells are lysed in a lysis buffer, and the cells are broken by ultrasonication. The components of the lysis buffer are not particularly limited, and any cell lysis solution known in the art can be used, for example, the components thereof can include 1-50 mM Tris-HCl, 1-50 mM KCl, 0.1-50 mM MgCl2. The lysis solution is first subjected to low-speed centrifugation to collect the supernatant, and the low-speed centrifugation conditions are specifically at a speed of less than 5000 x g, for example, less than 4000 x g, 3000 x g, 2000 x g, and the centrifugation time is 1-10 minutes, for example, 1-5 minutes. Subsequently, the supernatant is subjected to high-speed centrifugation and the precipitate is collected, and the high-speed centrifugation conditions are specifically at a speed of not less than 15000 x g, for example, not less than 16000 x g, 17000 x g, 18000 x g, 19000 x g, 20000 x g, and the centrifugation time is 15-45 minutes, for example, 25-35 minutes. The precipitate is washed with a buffer solution, and examples of the buffer solution include, but are not limited to, PBS buffer.

[0017] In step (1) of the above method, the concentration of the antibody in the virus membrane solution is 1-50 mg / mL, for example, 1-40 mg / mL, 1-30 mg / mL, 1-20 mg / mL, 1-10 mg / mL, 1-5 mg / mL, such as 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL. The mixed solution is extruded using an extruder through a polycarbonate membrane with a pore size of 200 nm.

[0018] In step (2) of the above method, the mineralization solution contains at least one divalent metal ion selected from Ca, Mg, Zn, Fe, Sr, and Ba. Preferably, the mineralization solution contains a soluble metal ion salt, in particular a halogen salt (such as a chloride salt, a fluoride salt, an iodide salt, etc.), a nitrate salt, a sulfate salt, etc. The mineralization solution can be any solution of the above-mentioned divalent metal ions, for example, 0.1-5 M, such as 0.5-4, 0.5-3, 0.5-2 M CaCl2, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 M CaCl2. The reaction temperature is 25-45°C, for example, 30-40°C, and the reaction time is 15-60 minutes, for example, 20-50 minutes, 20-40 minutes, 25-35 minutes.

[0019] In a fourth aspect of the present application, a method for degrading a target protein in a cell is provided, which comprises the step of contacting the above-mentioned biomimetic mineralized nanoparticles with the cell, and optionally further comprises the step of evaluating the level of protein degradation. The method can be an in vivo method or an in vitro method.

[0020] In a fifth aspect, the present application provides use of the biomimetic mineralized nanoparticles in drug development and scientific research.

[0021] In a sixth aspect, the present application provides use of the biomimetic mineralized nanoparticles in preparation of a target protein degradation drug.

[0022] The biomimetic mineralized nanoparticles (hereinafter sometimes referred to as ViTPD) constructed by the present application can mimic viral infection and target protein degradation. In exemplary embodiments, the mineralized viral coat of ViTPD with a positively charged surface can enhance cellular uptake, enabling efficient encapsulation of specific antibodies recognizing target proteins (POIs), while the introduced viral membrane components can induce expression of TRIM21 in cells. After being taken into cells, the antibodies released from ViTPD can bind to POIs and bind to TRIM21 to mediate degradation of POIs. With degradation, TRIM21 is continuously consumed, but it can be compensated by the endogenous expression of TRIM21 induced by the ViTPD system, thereby maintaining efficient degradation.

[0023] In certain embodiments, the present application exemplarily verifies the enhanced cellular uptake ability and the ability to up-regulate TRIM21 expression level and the efficient protein degradation activity of ViTPD, which has applicability in target protein degradation in various cells. Therefore, as a new TPD platform, it has broad potential and application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Preparation process of ViTPD nanoparticles and protein degradation process under the mechanism of simulating viral infection. (A) Construction of ViTPD nanoparticles: ViTPD nanoparticles are formed by antibodies coated with mineralized viral membranes. (B) Typical characteristics of the viral infection process: ① Viruses enhance their ability to enter cells through mineralization; ② Viruses bound by neutralizing antibodies can be recognized by TRIM21 in cells and degraded under the mediation of TRIM21; ③ Viral infection is often accompanied by activation of the anti-viral immune response, inducing up-regulation of IFN-α / β and TRIM21 expression, while exogenous IFN-α can also directly promote the expression of TRIM21. (C) ViTPD-mediated POI degradation process: By simulating the key mechanisms of the above viral infection, ViTPD achieves efficient degradation of POIs, including: (i) efficient delivery of antibodies into cells through the mineralized shell structure; (ii) up-regulation of TRIM21 expression through viral membrane components; (iii) recruitment of TRIM21 and mediation of POI degradation.

[0025] Figure 2Characterization of ViTPD nanoparticles. (A) Western blot analysis of viral membrane and VSV-G expression in VSV infected HeLa cells. (B) ViTPD-M@IgG production at different Ca2+ concentrations after 30 min of mineralization. The lower panel is a top view, and the white dotted circle is the bottom of the ep tube, with the white material being the product. (C) ViTPD-M@IgG production at different mineralization times. The lower panel is a top view, and the white dotted circle is the bottom of the ep tube, with the white material being the product. (D) Transmission electron microscopy images of viral membrane, non-mineralized ViTPD-M@IgG, and ViTPD-M@IgG (scale bar = 200 nm). (E) Dynamic light scattering analysis of viral membrane (peak at 251.92 nm), non-mineralized ViTPD-M@IgG (monodisperse peak at 184.28 nm), and ViTPD-M@IgG nanoparticles (peak at 220.74 nm). (F) Zeta potential measurements of IgG, non-mineralized ViTPD@IgG, ViTPD@IgG without viral membrane, and intact ViTPD@IgG. (G) Dynamic light scattering (DLS) analysis showing the size distribution of ViTPD@IgG without viral membrane, non-mineralized ViTPD@IgG, and intact ViTPD@IgG (n = 3).

[0026] Figure 3 Composition analysis of ViTPD nanoparticles. (A) Infrared spectroscopy analysis of ViTPD-M@IgG, CaP, and viral membrane, with characteristic absorption peaks at 3406.74 cm -1 (N-H bending vibration), 2918.28 cm-1(C-H stretching vibration), 1649.01 cm -1 (C=O stretching vibration), 1426.65 cm -1 (carboxyl stretching vibration), 1053.73 cm -1 (phosphate stretching vibration), and 870.31 cm -1(P-O bending vibration). (B) X-ray photoelectron spectroscopy (XPS) analysis of ViTPD@IgG, showing the surface elemental composition, including Na, C, Ca, N and P. (C) Scanning electron microscopy-energy dispersive X-ray (SEM-EDX) analysis image, showing the spatial distribution of C, O, N, Ca and P elements in ViTPD@IgG (scale bar = 100 nm). (D) Quantitative analysis results of ViTPD@IgG based on the image of (C). (E) Confocal fluorescence microscopy image: green for DIO-labeled viral membrane, blue for AMCA-labeled IgG, showing that IgG was successfully encapsulated in the viral membrane (scale bar = 50 pm). (F) Protein encapsulation and release verification of ViTPD@IgG (Coomassie brilliant blue staining): lanes are free IgG, ViTPD@IgG nanoparticles and ViTPD@IgG treated with PBS at pH 6.0, respectively, showing that the antibody can be released under pH conditions. (G) X-ray diffraction analysis of ViTPD-M@IgG and CaP. (H) Cell viability detected by CCK-8 method. HeLa cells were treated with ViTPD-M@IgG or free IgG at different concentrations of IgG for 24 hours (n = 3), "ns" represents no significant difference.

[0027] Figure 4ViTPD promotes POI degradation by activating intracellular TRIM21 expression. (A) ViTPD@FAK without viral membrane mediates FAK degradation accompanied by downregulation of TRIM21 expression. HeLa cells were treated with free FAK antibody or ViTPD@FAK without viral membrane for 6 hours, and then the FAK protein expression level was detected by WB, and the WB band intensity was quantified by normalizing to GAPDH (mean ± standard deviation, n = 3). (B) Transient overexpression of TRIM21 can enhance the degradation effect of ViTPD@IKKa without viral membrane. HeLa cells were transiently transfected with mCherry-TRIM21 and treated with ViTPD@IKKa without viral membrane for 6 hours, and then the IKKa protein expression level was detected by WB, and the WB band intensity was quantified by normalizing to GAPDH (mean ± standard deviation, n = 3). (C) VSV infection can upregulate the expression of TRIM21 in THP-1 cells. THP-1 cells were treated with VSV for different times, and the TRIM21 protein expression level was analyzed by WB, and the WB band intensity was quantified by normalizing to GAPDH (mean ± standard deviation, n = 3). (D, E) VSV membrane can significantly upregulate the mRNA levels of TRIM21 and IFN-a. THP-1 cells were treated with VSV membrane or uninfected cell membrane for different times, and the TRIM21 or IFN-a mRNA levels were analyzed by real-time fluorescent quantitative PCR, and the results were normalized to HPRT1 (mean ± standard deviation, n = 3). (F) ViTPD@TREX1 can effectively degrade TREX1 protein in THP-1 cells. THP-1 cells were treated with ViTPD@TREX1 or ViTPD@TREX1 without biomineralization for different times, and the TREX1 protein expression level was analyzed by WB, and the WB band intensity was quantified by normalizing to GAPDH (mean ± standard deviation, n = 3). DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present application will now be described in detail with reference to the drawings. The detailed description is not to be considered to limit the application in any way, but rather to explain certain aspects, features, and embodiments of the application.

[0029] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, for a range of values of a parameter, unless otherwise indicated, each intervening value by each intervening value, as well as each individual value of the recited range, is also contemplated. In each instance, any value can be combined with any other value, unless otherwise indicated. These and other modifications, as well as numerous further details of the present application, are described in the following detailed description.

[0030] Unless otherwise indicated, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application.

[0031] Herein, the term "biomimicry" refers to the simulation of a living organism or its activities, in particular the simulation of the process of viral infection. The biomimicry of the present application includes at least one of the following scenarios: (1) the mineralized virus membrane-coated antibody forms nanoparticles, and the mineralized virus membrane shell can enhance the cellular uptake efficiency through clathrin-mediated endocytosis; (2) when the antibody released by the particles of the present application binds to the target protein, the Fc fragment of the antibody is bound by the key enzyme TRIM21 for protein degradation, achieving protein degradation, just like the neutralizing antibody binding virus entering the cell; (3) the virus infection-related components in the particles of the present application induce the up-regulation of the expression of TRIM21 on cells, thereby enhancing the efficiency of antibody-mediated protein degradation.

[0032] Herein, the term "mineralization" or "biomineralization" refers to the process of generating inorganic minerals through the regulation of biological macromolecules. Unlike general mineralization, the mineralization of the present application involves the participation of biological macromolecules, organism metabolism or organic matrix, and under certain physicochemical conditions, the ions in the solution are converted into solid-phase minerals under the control or influence of biological organic matter.

[0033] Viruses have high entry ability and efficient protein degradation pathways in host cells during the infection process. During the process of viral infection of mammals, neutralizing antibodies are induced, which can bind to viruses and promote their entry into cells. After entering the cells, the Fc end of the antibody can be recognized and bound by the E3 ligase TRIM21 (viral infection or IFN-α / β stimulation can induce the expression of TRIM21), and after forming a virus-antibody-TRIM21 ternary complex, the cell can rapidly degrade the virus particles, and in this process, the TRIM21 itself is continuously consumed. In addition, it is worth noting that certain influenza viruses can be mineralized in the high-concentration calcium ion intestine of birds, improving their infectivity and cellular uptake ability. Inspired by the above-mentioned viral infection mechanism, the present application develops a biomimetic mineralized nanoparticle capable of targeting and degrading target proteins, as shown in the following examples.

[0034] Examples

[0035] I. Preparation of ViTPD

[0036] 1. Preparation of virus membrane-coated antibody

[0037] HeLa cells were infected with VSV virus (enveloped virus) overnight, then harvested, counted and suspended in hypotonic lysis buffer (20 mM Tris-HCl, 10 mM KCl, 2 mM MgCl2, with protease inhibitor cocktail). Then, the cells were disrupted by sonication for 5 minutes in ice bath. The lysate was treated with DNase and RNase (Invitrogen) for 20 minutes on ice. Next, the lysate was centrifuged at 2000 x g for 5 minutes, the supernatant was collected and further centrifuged at 18,000 x g for 30 minutes. The pellet was washed with pre-chilled PBS (with protease inhibitor) for 3 times. Next, serum-free DMEM (0.5 mL / 10 7 cells) was added, and the viral membrane solution (cell membrane containing viral components) was obtained by sonication for 5 minutes in ice bath. Then, 1 mg of antibody was added to 0.5 mL of viral membrane solution, and the mixed solution was extruded through a polycarbonate membrane with a pore size of 200 nm using an extruder (Avanti Polar Lipids). Finally, the volume of the extruded solution was adjusted to 1 mL to generate a solution of viral membrane-coated antibody nanoparticles (1 mg / mL).

[0038] 2. Biomineralization process

[0039] An appropriate amount of viral membrane-coated antibody was dissolved in 1 mL of serum-free DMEM, and 10 μL of CaCl2(1 M) was added and reacted for 30 minutes at 37°C. The whole process was carried out under sealed conditions with brief vortex mixing. Then, the ViTPD was collected by centrifugation (12,000 rpm, 10 minutes) and resuspended in serum-free DMEM. The nanoparticles were stored at 4°C for later use.

[0040] II. Characterization of ViTPD

[0041] In the construction of ViTPD nanoparticles, the antibody was first coated on the viral membrane extracted from HeLa cells infected with vesicular stomatitis virus (VSV), then subjected to biomineralization treatment, and finally formed ViTPD nanoparticles Figure 1 A). Western Blot (WB) detection results showed that VSV glycoprotein (VSV-G) was expressed in both viral membrane and VSV-infected HeLa cells Figure 2 A).

[0042] In the process of biomimetic mineralization, DMEM medium was used to simulate the mineralization environment. The concentration of commercial antibody product is generally 1 mg / mL or below, so the two key parameters, calcium ion concentration and mineralization time, were optimized in the preparation of ViTPD. First, the calcium ion concentration for mineralization was screened, and 100 μL of 1 mg / mL IgG and 30 min of mineralization time were fixed. Different concentrations of calcium ions were used for mineralization, and the mineralized products were finally centrifuged at the bottom of a 1.5 mL ep tube. The results are as follows Figure 2 B. There was no obvious product when the calcium ion concentration was less than 10 μmol / L, and the amount of product was basically the same at 10 μmol / L and 30 μmol / L, so 10 μmol / L was selected as the calcium ion concentration for subsequent mineralization experiments. Next, the IgG concentration and calcium ion concentration were fixed (1 mg / mL IgG, 10 μmol / L Ca 2+ ), and the mineralization time was adjusted (0.5-60 min). The results are as follows Figure 2 C. When the mineralization time was less than 30 min, there was no obvious precipitated product. When the mineralization time reached 30 min, there was obvious product, and the yield did not increase significantly with the extension of time, so 30 min was selected as the mineralization time for subsequent experiments.

[0043] Transmission electron microscopy showed that ViTPD@IgG without biomimetic mineralization showed a clear lipid bilayer structure, while the mineralized ViTPD showed a core-shell structure, and the lipid bilayer still existed but was not as clear as before Figure 2 C). Dynamic light scattering (DLS) analysis showed that there were differences in the particle size distribution of virus membrane, unmineralized ViTPD-M@IgG and complete ViTPD-M@IgG Figure 2 E). The characteristic peak of the virus membrane was at 251.92 nm, unmineralized ViTPD-M@IgG showed a monodisperse peak (184.28 nm), and the particle size distribution peak of complete ViTPD-M@IgG was 220.74 nm. Zeta potential test results showed that the biomimetic mineralization process affected the surface charge of the particles: the potential of free IgG was -3.4 mV, the potential of unmineralized ViTPD@IgG was -11.86 mV, and the potential of mineralized ViTPD@IgG jumped to +18.60 mV Figure 2 F). At the same time, the particle size increased from about 230 nm to about 250 nm Figure 2 G).

[0044] To further analyze the composition of ViTPD, various detection methods were used. Fourier transform infrared (FTIR) spectroscopy analysis confirmed the presence of calcium phosphate (CaP), virus membrane and protein components in ViTPD Figure 3A). Characteristic peaks show: 3406.74 cm -1 (N-H bending vibration) and 1649.01 cm -1 (C=O stretching vibration) are amide groups; 1426.65 cm -1 (carboxyl stretching vibration) and 870.31 cm -1 (PO43- vibration) confirm the presence of CaP; 2918.28 cm -1 (C-H stretching vibration) indicates the presence of lipid groups. X-ray photoelectron spectroscopy (XPS) and energy dispersive spectroscopy (EDS) further verify the elemental composition of ViTPD@IgG, detecting Ca, P, C, N and O as its main elements Figure 3 B-D). Confocal fluorescence microscopy imaging shows that the antibody encapsulated in the viral membrane is successfully labeled: AMCA-stained IgG and DIO-stained viral membrane fluorescence are highly co-localized, verifying the effectiveness of viral membrane wrapping Figure 3 E). Polyacrylamide gel electrophoresis (SDS-PAGE) experiments further confirm that the antibody is effectively wrapped: ViTPD@IgG treated with pH 6.0 PBS releases similar IgG bands to free IgG, while untreated ViTPD@IgG shows no bands Figure 3 F). X-ray diffraction (XRD) analysis results show that the formed CaP shell layer is amorphous structure Figure 3 G).

[0045] Finally, CCK-8 cell viability detection shows that ViTPD has no significant cytotoxicity at various concentrations, and its cell activity is comparable to that of the free IgG treatment group, showing good biocompatibility Figure 3 H). Based on the above results, the successful construction of ViTPD nanoparticles and their excellent biocompatibility are fully verified.

[0046] III. ViTPD enhances POI degradation efficiency by activating intracellular TRIM21 expression

[0047] In previous studies, Western blot (WB) analysis found that the ViTPD system without viral membrane wrapping, although it can induce POI degradation to some extent, is accompanied by a significant decrease in TRIM21 protein levels Figure 4 A). To further verify the key role of TRIM21 in the degradation process, mCherry-TRIM21 was transiently transfected into cells in this embodiment, and treated with ViTPD@IKKα without viral membrane, the results show that the TRIM21 expression level is restored stable and the POI degradation efficiency is significantly improved Figure 4 B) (antibody+) (positive control).

[0048] Notably, TRIM21 expression can also be activated endogenously under certain physiological conditions, such as during viral infection. WB results showed that the expression level of TRIM21 in THP-1 cells gradually increased within 4 to 16 hours after VSV infection Figure 4 C). In addition, the level of TRIM21 mRNA in cells after viral membrane treatment increased nearly 2-fold within 12 hours Figure 4 D), while the level of interferon alpha (IFN-a) mRNA also showed an upward trend, although the up-regulation was lower than that of the VSV infection group Figure 4 E). These results indicate that whether it is a complete viral particle, a viral membrane component, or a partial factor during infection, it can induce the up-regulation of TRIM21 expression in cells.

[0049] Based on the above mechanism, the present embodiment introduces a viral membrane wrapping strategy in the construction of ViTPD to simulate the endogenous immune response induced by viral infection, thereby increasing the expression of TRIM21 and enhancing the degradation effect of target proteins.

[0050] The present embodiment further constructs ViTPD@TREX1 and uses it to treat THP-1 cells. WB results show that ViTPD@TREX1 has a clear advantage in degradation efficiency compared to ViTPD@TREX1 without a mineralized shell layer - the former can achieve about 70% degradation of TREX1 protein within 6 hours of treatment, while the latter requires 12 hours to achieve a 50% degradation rate Figure 4 F). Both groups showed a decrease in TRIM21 protein levels in the early degradation stage (6 hours), but TRIM21 gradually recovered after 12 hours, suggesting that although ViTPD can induce TRIM21 expression, the initial rapid degradation process may cause temporary depletion of TRIM21, and the expression recovery lags behind.

[0051] In summary, the above results preliminarily verify that ViTPD can mediate the intracellular degradation of POI through antibody targeting, and significantly enhance the degradation efficiency through the up-regulation of TRIM21 expression, showing a good application prospect as a new targeted protein degradation platform.

[0052] This example successfully developed a general antibody-guided targeted protein degradation (TPD) platform that can efficiently and specifically degrade a variety of intracellular proteins. This is the first time that a virus infection biomimetic strategy has been used to design a TPD system. Although a variety of virus-like particles have been designed as nanocarriers before, these carriers mainly enhance the delivery of small molecules or nucleic acids by mimicking the morphology and structure similar to viruses, without specifically considering the interactions between viruses and host cells. In contrast, the biomimicry of ViTPD is carried out in an integrated manner, aiming to comprehensively mimic the characteristics of virus internalization, virus-neutralizing antibodies, and the activation of host protein degradation pathways by host responses during viral infection.

[0053] Enzymes related to protein degradation play a key role in the design of TPD systems. In traditional TPD strategies, such as PROTAC, the depletion of resources or the possible “hook effect” that occurs during protein degradation can lead to a decrease in intracellular E3 ubiquitin ligases, which is detrimental to the subsequent efficiency of protein degradation. In contrast, the ViTPD method promotes the expression of TRIM21 by mimicking viral infection, thereby maintaining the steady-state level of this E3 ubiquitin ligase and promoting protein degradation, ensuring that the effect of protein degradation does not decrease over time. In addition, the stable expression of TRIM21 not only affects the degradation efficiency of ViTPD, but also plays an important physiological function in cells. The consistent expression of TRIM21 during degradation helps to reduce potential side effects associated with ViTPD.

[0054] It is important to note that TRIM family proteins are an important class of E3 ubiquitin ligases. Most members contain a conserved RING domain that can bind to ubiquitin-conjugating enzyme E2 and promote the transfer of ubiquitin to the target protein (POI). These proteins are widely involved in various biological processes, including protein degradation, signal transduction, immune response, and antiviral defense. For example, TRIM27 regulates the NF-κB signaling pathway by ubiquitinating and degrading the transcription factor p65 / RelA, while TRIM63 (MuRF1) promotes muscle atrophy by degrading muscle-specific proteins such as actin and myosin. The wide-ranging effects of the TRIM family provide important insights into expanding the E3 ligase library and offer valuable perspectives for the study of protein degradation.

[0055] The selection of ligands that bind POIs is also crucial for TPD design. In this work, antibodies were chosen as the core component of the ViTPD platform. First, antibodies have a strong compatibility with TRIM21. TRIM21 can recognize and bind multiple types of antibodies, such as IgG, IgM, and IgA, ensuring the versatility of the ViTPD platform. Second, the wide availability of antibodies makes it easier to select appropriate antibodies to recognize target proteins. There are now a variety of validated commercial antibodies available, and an increasing number of recombinant nanobodies are emerging as a source of target protein recognition units in ViTPD. By specifying POIs, it is easy to construct new ViTPD, a process much simpler than designing the synthetic route of PROTAC. This work successfully constructed multiple different types of ViTPD, each capable of degrading proteins involved in cellular material transport, cell migration, immune pathways, and fluorescently labeled proteins. In addition, compared to existing PROTACs, ViTPD antibodies have stronger binding affinity and specificity to target proteins. Notably, many protein targets lack high-affinity small molecule compounds, but antibodies against these proteins are often readily available. Therefore, ViTPD is particularly suitable for degrading “undruggable” proteins. This work successfully utilized ViTPD@COPZ1 to degrade the COPZ1 protein, which lacks small molecule inhibitors.

[0056] ViTPD is a mineralized particle prepared by mimicking the biomineralization process of viral particles, effectively solving the problem of antibody transmembrane entry. ViTPD enters cells in large quantities after six hours of incubation with cells, due to its positively charged biomineralized shell, which easily adsorbs to the negatively charged cell membrane surface. Biomineralization also helps ViTPD escape the acidic environment of lysosomes, allowing antibodies to enter the cytoplasm for protein degradation. In addition, this work shows that ViTPD internalization depends on multiple pathways, indicating that a non-specific internalization strategy may help alleviate the problem of drug resistance. Once inside the cell, the released antibodies retain the antigen recognition ability of the Fab region and the function of the Fc region to bind to TRIM21, mainly benefiting from the fact that the mild mineralization conditions maintain the structure and function of the antibodies. Therefore, ViTPD exhibits good biocompatibility, with no significant toxicity observed in 24-hour cytotoxicity experiments. In addition, the reported biomineralized materials often have various stimulus-responsive properties (e.g., temperature, light), which can help design ViTPD with enhanced functionality.

[0057] In summary, by mimicking three features of viral infection, the present embodiment constructs a virus-mimicking mineralized particle ViTPD. Through effective mineralized particle internalization and upregulation of E3 ligase (TRIM21) levels on the viral membrane components, the effective degradation of multiple intracellular proteins is achieved. ViTPD not only provides a new means for efficient degradation of multiple intracellular target proteins, but also provides a new idea for the design of future innovative functional protein degradation systems.

[0058] While the application has been described with reference to example embodiments, it will be understood by those skilled in the art that various changes can be made and equivalents can be substituted for elements thereof without departing from the scope or spirit of the application. In addition, many modifications can be made to the example embodiments of the disclosure in light of the teachings below. The scope of the claims should be accorded the broadest interpretation so as to encompass all such modifications and equivalent structures and functions.

Claims

1. A biomimetic mineralized nanoparticle, characterized in that, It includes a core structure and a shell structure, the core structure including an antibody, the shell structure enclosing the core structure and containing minerals and membrane components.

2. The biomimetic mineralized nanoparticles according to claim 1, characterized in that, The membrane components include those derived from viruses.

3. The biomimetic mineralized nanoparticles according to claim 1, characterized in that, X-ray photoelectron spectroscopy analysis revealed that the main surface elements of the biomimetic mineralized nanoparticles include Na, C, Ca, N, and P.

4. The biomimetic mineralized nanoparticles according to claim 1, characterized in that, The particle size of the biomimetic mineralized nanoparticles is in the range of 50-500 nm.

5. The biomimetic mineralized nanoparticles according to claim 1, characterized in that, The antibody is a neutralizing antibody.

6. A pharmaceutical composition, characterized in that, Includes biomimetic mineralized nanoparticles according to any one of claims 1-5 and pharmaceutically acceptable carriers.

7. The pharmaceutical composition according to claim 6, characterized in that, Further includes TRIM activators.

8. A method for preparing biomimetic mineralized nanoparticles, characterized in that, The method includes the step of incubating antibody-containing particles in a mineralization solution, wherein the mineralization solution contains divalent metal ions and a solvent.

9. A method for degrading target proteins within cells, characterized in that, The step includes contacting the biomimetic mineralized nanoparticles according to any one of claims 1-5 with cells, and optionally, further includes the step of evaluating the level of protein degradation.

10. Use of the biomimetic mineralized nanoparticles according to any one of claims 1-5 in drug development and scientific research.

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