A protein corona delivery system, and methods of making and using the same

Abstract

This invention belongs to the field of biomedicine and nanodelivery technology, specifically relating to a protein crown delivery system, its preparation method, and its applications. The protein crown delivery system provided by this invention utilizes a specific bifunctional polypeptide to modify the surface of gold nanoparticles, enabling efficient and stable binding of various active proteins to form a protein crown, thereby achieving intracellular delivery of active proteins. Specifically, this invention utilizes the abundant side chain groups of the bifunctional polypeptide molecule to solve the problem of limited binding sites between the protein crown delivery system and protein molecules, allowing them to bind through non-covalent interactions, assembling into a uniform and stable protein crown structure. This improves the binding efficiency between the carrier and the protein, thus constructing a universal, efficient, and safe intracellular protein nanoparticle delivery carrier. The protein crown delivery system provided by this invention can couple with various types of proteins while maintaining the protein's own biological activity and function, thereby enabling intracellular delivery of proteins with different activities.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and nanodelivery technology, specifically relating to a protein crown delivery system, its preparation method, and its application. Background Technology

[0002] Compared to small molecule drugs, protein drugs possess unique biological advantages, such as excellent targeting ability, better therapeutic effects, and lower toxicity, leading to their widespread application in biomedicine and clinical trials. However, due to the large molecular weight and volume of proteins, they are difficult to enter cells on their own and require specific methods for intracellular delivery. There are three main methods for intracellular protein delivery: physical methods, protein modification methods, and carrier methods. Among these, carrier methods utilize larger structures or components to increase the protein's ability to be taken up by cells, escape from endosomes, and release into the cell, resulting in high intracellular delivery efficiency and significant advantages.

[0003] Nanoparticles are often used as protein carriers because their surfaces are easily modified. However, nanoparticles exhibit strong interactions with cells, tissues, and their biological environment. For example, after being ingested, nanoparticles are rapidly coated with a large amount of proteins and other biomolecules, forming a "protein corona," a nanoparticle / protein coupling structure known as a protein crown. On one hand, protein coronas can endow nanoparticles with new biological properties by altering their size distribution, surface charge, aggregation behavior, and interfacial properties, or by decorating their surfaces with specific proteins and epitope materials, enabling them to interact with and be recognized by specific cell receptors. On the other hand, excessive free proteins in solution can also affect nanoparticle entry into cells because proteins interact with cell receptors, thus competing with nanoparticles. Therefore, while the protein crown formed by nanoparticles and proteins is significant in some respects, its non-specific formation makes it difficult for nanoparticles to specifically deliver protein drugs into the body.

[0004] In summary, how to enable nanoparticles to efficiently deliver protein drugs intracellularly remains a challenge for those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide a protein crown delivery system, its preparation method, and its application. The protein crown delivery system provided by this invention can efficiently deliver protein drugs intracellularly.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a protein crown delivery system, comprising gold nanoparticles and a bifunctional polypeptide modified on the surface of the gold nanoparticles; the sequence of the bifunctional polypeptide is shown in any one of SEQ ID NO. 1-2:

[0008] SEQ ID NO. 1: Nap-FF-GPLGLAGCRRRCRRRCRRRC;

[0009] SEQ ID NO. 2: Nap-FF-GPLGLAGCKKKCKKKCKKKC.

[0010] Preferably, the gold nanoparticles have a particle size of 15.97~20 nm.

[0011] Preferably, the particle size of the protein crown delivery system is 100~200 nm.

[0012] Preferably, the protein crown delivery system further includes structural domains for enhancing the stability and dispersibility of the gold nanoparticles.

[0013] Preferably, the protein crown delivery system further includes one or more of the following: a domain for providing a specific non-covalent interaction with the target protein, a domain for promoting cellular uptake, and a domain for releasing the target protein in response to the intracellular environment.

[0014] The present invention also provides a method for preparing the protein crown delivery system described above, comprising the following steps:

[0015] The protein crown delivery system was obtained by assembling a colloidal solution of gold nanoparticles and a bifunctional peptide.

[0016] The present invention also provides the application of the protein crown delivery system described in the above-described scheme or the protein crown delivery system prepared by the above-described scheme in intracellular protein delivery.

[0017] Preferably, the method of application includes the following steps: mixing the protein crown delivery system and the target protein and incubating them.

[0018] The present invention also provides a protein delivery system, comprising a protein crown delivery system and a target protein bound to the protein crown delivery system via non-covalent interactions, wherein the target protein and the protein crown delivery system form a protein crown; the protein crown delivery system is the protein crown delivery system described in the above-described scheme or the protein crown delivery system obtained by the preparation method described in the above-described scheme.

[0019] Preferably, the target protein includes non-gene-edited proteins and gene-edited proteins; the non-gene-edited proteins include one or more of bovine serum albumin, β-galactosidase, superoxide dismutase, horseradish peroxidase, ribonuclease A, cytochrome C, and trypsin; the gene-edited protein is Cas9 protein.

[0020] This invention provides a protein crown delivery system. The system utilizes a specific bifunctional peptide to modify the surface of gold nanoparticles (AuNPs). Based on the bifunctional peptide mediation, it can efficiently and stably bind to various active proteins to form functional protein crowns, thereby achieving intracellular delivery of active proteins. Specifically, this invention utilizes the abundant side chain groups of the bifunctional peptide molecule to solve the problem of limited binding sites between the protein crown delivery system and protein molecules. This allows the two to bind through non-covalent interactions (including hydrophobic interactions, hydrogen bonds, π-π stacking, van der Waals forces, and electrostatic interactions), assembling into a uniform and stable protein crown structure. This improves the binding efficiency between the carrier and the protein, thus constructing a universal, efficient, and safe intracellular protein nanoparticle delivery carrier. The protein crown delivery system provided by this invention has strong versatility, capable of coupling with various types of proteins (e.g., proteins with different relative molecular weights and isoelectric points) while maintaining the protein's own biological activity and function, thereby enabling intracellular delivery of proteins with different activities.

[0021] This invention also provides a method for preparing the protein crown delivery system described above. This invention uses affinity peptide molecules as ligands and, through ligand exchange reactions, obtains bifunctional peptide-modified gold nanoparticles. The bifunctional peptides on the surface of the gold nanoparticles have specific amino acid sequences and functions, providing abundant side chain groups for binding target protein molecules through non-covalent interactions to form a stable protein crown structure. Experimental results show that the protein crown delivery system prepared by this invention has the following advantages: The protein crown delivery system provided by this invention can efficiently adsorb various protein molecules and assemble them on its surface to form a uniform and stable protein molecule layer; the protein crown delivery system combines the membrane-penetrating properties of peptide molecules with the good biocompatibility of gold nanoparticles, not only effectively solving the biotoxicity problem of the carrier, but also making the delivery process independent of classical endocytosis, with the advantage of efficiently carrying protein molecules, even Cas9 / sgRNA ribonucleoproteins, directly across the cell membrane barrier and exerting corresponding functions; furthermore, no modification to the protein molecules is involved in the entire process, and the biological activity of the protein molecules can be efficiently preserved during delivery.

[0022] This invention also provides the application of the protein crown delivery system described in the above-described scheme or the protein crown delivery system prepared by the above-described scheme in intracellular protein delivery. The protein crown delivery system provided by this invention can efficiently overcome the cell membrane barrier, promote endosome escape, and maintain the biological activity of the protein, delivering the protein into the cell efficiently, safely, and stably, thus providing a foundation for functional in vivo applications such as intracellular delivery of protein drugs, intracellular catalytic reactions, and gene editing. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Fourier transform infrared spectrum of the protein crown delivery system prepared in Example 1;

[0025] Figure 2 Electron microscopy images of the protein crown delivery system prepared in Example 1, where a1 is an electron micrograph of AuNPs, b1 is an electron micrograph of (CR3)3C-AuNPs, c1 is an electron micrograph of (CK3)3C-AuNPs, a2 is a particle size distribution map of AuNPs, b2 is a particle size distribution map of (CR3)3C-AuNPs, and c2 is a particle size distribution map of (CK3)3C-AuNPs.

[0026] Figure 3 The ultraviolet spectrum of the protein crown delivery system prepared in Example 1;

[0027] Figure 4 Dynamic light scattering diagram of the protein crown delivery system prepared in Example 1;

[0028] Figure 5 The image shows an electron microscope image of the protein delivery system prepared in Example 7, where a is β-Gal, b is SOD, c is HRP, d is Cyt C, e is RNase A, and f is Trypsin.

[0029] Figure 6 The graph shows the intracellular delivery efficiency of the protein delivery system prepared in Example 6.

[0030] Figure 7 The cellular uptake mechanism of bovine serum albumin by the protein crown delivery system prepared in Example 1 is shown, where a is the average fluorescence intensity of the cells and b is the percentage of fluorescent positive cells.

[0031] Figure 8This image shows the effect of the protein crown delivery system prepared in Example 1 delivering β-galactosidase into HepG2 cells.

[0032] Figure 9 This image shows the effect of the protein crown delivery system prepared in Example 1 delivering horseradish peroxidase into HepG2 cells.

[0033] Figure 10 Cytotoxicity diagram of ribonuclease A delivered into HepG2 cells using the protein crown delivery system prepared in Example 1;

[0034] Figure 11 The image shows the effect of the protein crown delivery system prepared in Example 1 delivering superoxide dismutase into HepG2 cells;

[0035] Figure 12 The image shows the gene editing effect of delivering Cas9 protein into Hela-EGFP cells using the protein crown delivery system prepared in Example 1. Detailed Implementation

[0036] This invention provides a protein crown delivery system, comprising gold nanoparticles and a bifunctional polypeptide modified on the surface of the gold nanoparticles; the sequence of the bifunctional polypeptide is shown in any one of SEQ ID NO. 1-2:

[0037] SEQ ID NO. 1: Nap-FF-GPLGLAGCRRRCRRRCRRRC;

[0038] SEQ ID NO. 2: Nap-FF-GPLGLAGCKKKCKKKCKKKC.

[0039] In this invention, the particle size of the gold nanoparticles is preferably 15.97~20 nm, more preferably 16~17 nm.

[0040] In this invention, the molar ratio of the gold nanoparticles to the bifunctional polypeptide is preferably 1:50000.

[0041] In this invention, the particle size of the protein crown delivery system is preferably 100-200 nm, more preferably 140-170 nm.

[0042] In this invention, the protein crown delivery system preferably further includes structural domains for enhancing the stability and dispersibility of the gold nanoparticles.

[0043] In this invention, the protein crown delivery system preferably further includes a domain for providing specific non-covalent interactions with the target protein.

[0044] In this invention, the protein crown delivery system preferably further includes a domain for promoting cellular uptake.

[0045] In this invention, the protein crown delivery system preferably further includes a domain for releasing the target protein in response to the intracellular environment.

[0046] The present invention also provides a method for preparing the protein crown delivery system described above, comprising the following steps:

[0047] The protein crown delivery system is obtained by assembling a colloidal solution of gold nanoparticles and a bifunctional peptide (referred to as the first mixture).

[0048] In this invention, the concentration of the gold nanoparticle colloidal solution is preferably 0.3~2.0 μM, more preferably 2.0 μM.

[0049] In this invention, the volume ratio of the gold nanoparticle colloidal solution to the mass ratio of the bifunctional polypeptide is preferably (3~7) mL:(8~12) mg, more preferably 5 mL:10 mg.

[0050] In this invention, the first mixing is preferably agitated mixing; the agitated mixing equipment is preferably a rotary mixer; the agitated mixing time is preferably 8 to 24 hours, more preferably 12 hours.

[0051] In this invention, the assembly process preferably includes centrifuging the obtained product, discarding the supernatant, and resuspending or freeze-drying the precipitate for preservation; the centrifugation speed is preferably 12000 rpm, and the time is preferably 30 minutes; the resuspension is preferably resuspended in ultrapure water; the freeze-drying temperature is preferably -60℃ to -80℃, and the holding time is preferably 24h to 48h.

[0052] The present invention also provides the application of the protein crown delivery system described in the above-described scheme or the protein crown delivery system prepared by the above-described scheme in intracellular protein delivery.

[0053] In this invention, the method of application preferably includes the following steps: mixing the protein crown delivery system and the target protein and incubating them.

[0054] In this invention, the molar ratio of the protein crown delivery system to the target protein is preferably 5:1, 15:1, 30:1 and 45:1, more preferably 30:1.

[0055] In this invention, the incubation temperature is preferably room temperature, and the incubation time is preferably 30 minutes. Through the above incubation, this invention enables the target protein to interact non-covalently with the protein crown delivery system via side chain groups provided by the bifunctional polypeptide, forming a stable protein crown on the surface of the protein crown delivery system.

[0056] The present invention also provides a protein delivery system, comprising a protein crown delivery system and a target protein bound to the protein crown delivery system via non-covalent interactions, wherein the target protein and the protein crown delivery system form a protein crown; the protein crown delivery system is the protein crown delivery system described in the above-described scheme or the protein crown delivery system obtained by the preparation method described in the above-described scheme.

[0057] In this invention, the target protein preferably includes non-gene-edited proteins and gene-edited proteins; the non-gene-edited protein preferably includes one or more of bovine serum albumin (BSA), β-galactosidase (β-Gal), superoxide dismutase (SOD), horseradish peroxidase (HRP), ribonuclease A (RNase A), cytochrome C (Cyt C), and trypsin; the gene-edited protein is preferably Cas9 protein.

[0058] In this invention, the molar ratio of the protein crown delivery system to the target protein is preferably 5:1 to 90:1, more preferably 45:1 to 90:1.

[0059] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. The scope of protection of the present invention is not limited to the following embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention and are protected by the appended claims. The processes, conditions, reagents, experimental methods, etc., for implementing the present invention, except as specifically mentioned below, are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0060] Example 1

[0061] This embodiment prepared a protein crown delivery system. An excess of bifunctional peptide was used in the preparation of the protein crown delivery system. The specific steps are as follows:

[0062] 10 mg of (CR3)3C (SEQ ID NO. 1) peptide and 10 mg of (CK3)3C (SEQ ID NO. 2) peptide were weighed into clean centrifuge tubes, and 5 mL of ultrapure water was added to each to obtain bifunctional peptide solutions with a concentration of 2 mg / mL. Subsequently, 5 mL of gold nanoparticle colloidal solution (2 μM) was added to each of the two bifunctional peptide solutions to obtain two mixtures. To ensure complete reaction, the two mixtures were stirred overnight (12 h) on a rotary mixer, centrifuged at 12000 rpm for 30 min, and the supernatant was discarded. The precipitates were resuspended in ultrapure water or lyophilized to obtain two protein crown delivery systems—(CR3)3C-AuNPs and (CK3)3C-AuNPs complexes.

[0063] Example 2: Fourier transform infrared spectroscopy analysis:

[0064] The infrared spectra of AuNPs, (CR3)3C peptides, (CK3)3C peptides, (CR3)3C-AuNPs, and (CK3)3C-AuNPs were characterized using a potassium bromide (KBr) pellet method on a Fourier transform infrared spectrometer (VERTEX 70, Bruker). The specific steps were as follows: Dry sample powder and KBr powder were mixed at a mass ratio of 1:100 in an agate mortar and repeatedly ground until homogeneous. Then, 1–2 mg of the homogeneous powder was collected into a clean pelleting mold, which was assembled sequentially to form a 1 mm thick transparent disc. Finally, the infrared absorption spectra were measured using a Fourier transform infrared spectrometer. The results are shown below. Figure 1 As shown.

[0065] according to Figure 1 It can be seen that, compared with AuNPs, (CR3)3C-AuNPs exhibit better performance in the amide I band at 1670 cm⁻¹. -1 Amide II band 1550 cm -1 Amide III band 1200 cm -1 and 3350 cm -1 The presence of new amide bonds nearby confirms the successful binding of the bifunctional peptide to the surface of the gold nanoparticles. Compared to (CR3)3C-AuNPs, (CK3)3C-AuNPs only showed weak amide I and amide II bands characteristic peaks of peptide molecules, while other characteristic peaks disappeared. Furthermore, the SS bond (~658 cm⁻¹) was also observed. -1 ) and Au-S bond (~760 cm) -1 The signal-to-noise ratio in the region where the characteristic peak signal is located is low, and the characteristic peak signal is weak, so it is impossible to accurately determine whether there is the disappearance of disulfide bonds and the formation of Au-S bonds.

[0066] Example 3: Electron microscopy characterization:

[0067] A JEOL JEM-1400plus transmission electron microscope was used with an electron accelerating voltage of 200 kV to characterize AuNPs, (CR3)3C-AuNPs, and (CK3)3C-AuNPs. The specific steps were as follows: First, after cleaning the tweezers used for TEM sample preparation, the carbon support film was carefully removed and placed on the sealing film, ensuring the carbon support film side was facing upwards. Then, 10 μL of sample solution was added to the carbon support film. After 5 minutes, excess sample solution was blotted off with filter paper. Since the samples contained polypeptide molecules, further negative staining was required. Specifically, 10 μL of uranyl acetate was added to the pre-treated carbon support film, and after 5 minutes, excess sample solution was blotted off with filter paper. Finally, the copper mesh containing the sample was placed under a transmission electron microscope for microscopic morphology characterization. Images were acquired using a Gatan CCD, and particle size was statistically analyzed using Image J. The results are shown below. Figure 2 As shown.

[0068] according to Figure 2 As can be seen, AuNPs, (CR3)3C-AuNPs, and (CK3)3C-AuNPs are spherical, uniformly distributed, and well-dispersed, with average sizes of 15.97 ± 3, 17.1 ± 3, and 18.5 ± 3 nm, respectively, according to Image J statistics. Compared with AuNPs, the sizes of (CR3)3C-AuNPs and (CK3)3C-AuNPs are slightly increased. Upon magnification of the TEM images, a shadow was clearly observed on the surface of the bifunctional peptide-modified gold nanoparticles. This is due to the bifunctional peptide molecules replacing citrate on the AuNP surface through ligand exchange reactions. Notably, the shadow range on the surface of (CK3)3C-AuNPs is significantly smaller than that of (CR3)3C-AuNPs.

[0069] Example 4: Ultraviolet Spectroscopy Analysis

[0070] Ultraviolet (UV) spectroscopic analysis was performed on (CR3)3C-AuNPs and (CK3)3C-AuNPs. The specific steps were as follows: a quartz cuvette with a path length of 1 cm and a width of 0.4 cm was used; the absorbance of the samples was scanned within the range of 200–800 nm; and the absorbance of the sample solution was measured using a UV-2450 spectrophotometer (Pharma Spec, Shimadzu). The results are as follows: Figure 3 As shown.

[0071] according to Figure 3It can be seen that the UV absorption peaks of (CR3)3C-AuNPs and (CK3)3C-AuNPs red-shifted from 519 nm to 524 nm and 531 nm, respectively, and the solution color changed from pink to purplish-red and purple. This is due to the increase in size of the bifunctional peptides after binding to AuNPs. Furthermore, the characteristic peaks of the coupled (CR3)3C-AuNPs did not broaden significantly, indicating that the interparticle distance was greater than the particle radius, suggesting that (CR3)3C did not aggregate significantly after binding to the AuNPs surface. However, the characteristic peaks of (CK3)3C-AuNPs were much broader than those of AuNPs and shifted to larger wavelengths. This may be because the isoelectric point of lysine is lower than that of arginine, therefore the synthesized (CK3)3C-AuNPs tended to red-shift and aggregate, exhibiting poor stability.

[0072] Example 5: Dynamic Light Scattering Analysis

[0073] Dynamic light scattering analysis was performed on AuNPs, (CR3)3C-AuNPs, and (CK3)3C-AuNPs. The specific steps were as follows: the sample solution was diluted to a concentration of 100 μM, then transferred to a Malvern potential sample cell (DTS 1070). The size distribution and zeta potential of the samples were measured at room temperature using a nanoparticle size analyzer (ZEN3600, Malvern Instruments). Each sample was tested repeatedly to ensure data accuracy. The results are as follows: Figure 4 As shown.

[0074] according to Figure 4 It can be seen that since the DLS measurement results are the particle hydration radius, the data results are larger than those of TEM, which are 18.2 nm, 37.8 nm and 58.5 nm respectively.

[0075] Example 6

[0076] This embodiment prepared a protein delivery system, and the specific steps are as follows:

[0077] Using bovine serum albumin (BSA) as the model protein, the protein molecule was fixed at a concentration of 3 μM, and the carrier concentration was varied for testing. Specifically, the protein crown delivery system stock solution ((CR3)3C-AuNPs) was diluted to 3 μM, 15 μM, 45 μM, 90 μM, and 135 μM respectively with BSA solution at molar ratios of 1:1, 5:1, 15:1, 30:1, and 45:1 to obtain the protein crown delivery system solution. Then, an equal volume of BSA solution was added to the protein crown delivery system solution, and the mixture was gently pipetted to mix thoroughly. The mixture was incubated at room temperature for 30 min to obtain the protein delivery system—(CR3)3C-AuNPs / BSA.

[0078] Example 7 Electron Microscopy Characterization:

[0079] Following the preparation method in Example 6, protein delivery systems for β-galactosidase (β-Gal), superoxide dismutase (SOD), horseradish peroxidase (HRP), ribonuclease A (RNase A), cytochrome C (Cyt C), and trypsin were prepared. The six different types of protein delivery systems were characterized by electron microscopy, and the results are as follows: Figure 5 As shown.

[0080] according to Figure 5 It can be seen that all six different types of proteins can couple with (CR3)3C-AuNPs to some extent, and their surfaces all exhibit crown-like shadows of varying sizes due to the uniform coating of protein molecules. Among them, β-Gal and SOD showed the best coupling effect, with shadow diameters of 10 nm and 6 nm, respectively; HRP and Cyt C were next, with a diameter of 3 nm; RNase A followed closely, with a diameter of 2 nm; and Trypsin showed the worst coupling effect.

[0081] Example 8: Characterization of intracellular delivery efficiency of bovine serum albumin:

[0082] The intracellular delivery efficiency of the protein delivery system prepared in Example 6 was characterized. The specific steps were as follows: First, HepG2 cells were loaded with the protein at a rate of 1×10⁻⁶ cells / cells. 5 Cells were seeded at a density of 1 / mL in 24-well plates and cultured overnight. Once the cell density reached 80%, the old culture medium was removed. A premixed solution containing 50 μL of (CR3)3C-AuNPs / BSA at different molar ratios (1:1, 5:1, 15:1, 30:1, and 45:1) and 400 μL of serum-free medium was added to each well for 20 min. Cells treated with BSA-FITC alone served as a control group. Each group was run in triplicate to minimize experimental error. The plates were incubated at 37°C (5% CO2) for 4 h. Cells were washed three times with PBS and collected in centrifuge tubes. Flow cytometry was used to quantitatively detect the mean fluorescence intensity and the percentage of fluorescent positive cells. Specifically, 200 μL of trypsin was added to each well to digest the cells from the plate, followed by the addition of 600 μL of complete culture medium to terminate the digestion process. Cells were collected in centrifuge tubes and centrifuged at 800 rpm for 5 min. The supernatant was then discarded, and the cells were resuspended in 800 μL PBS to obtain the flow cytometry sample. Results are as follows: Figure 6 As shown.

[0083] according to Figure 6As can be seen, the protein delivery efficiency significantly increases with the increase of the molar ratio of the protein crown delivery system to the target protein. When the molar ratio is 30:1, the fluorescence intensity of HepG2 cells is 20 times that of the control group, and the number of fluorescent cells detected reaches 100%, indicating that (CR3)3C-AuNPs deliver BSA-FITC into all HepG2 cells. Therefore, this test can prove that the synthesized conjugate has the ability to deliver proteins efficiently and is an excellent intracellular protein delivery carrier. It is worth noting that when the molar ratio continues to increase to 45:1, the detected fluorescence intensity decreases. It can be considered that the protein crown delivery system at this molar ratio has poor stability due to excessively high concentration, resulting in precipitation and thus reduced protein delivery efficiency.

[0084] Example 9: Characterization of cellular uptake mechanism:

[0085] The cellular uptake mechanism of bovine serum albumin by the protein crown delivery system prepared in Example 1 was characterized. The specific steps were as follows: 100 μL of each of the four endocytosis pathway inhibitors—cytochalasin D (10 μM), chlorpromazine (20 μM), genistein (700 μM), and methyl-β-cyclodextrin (MβCD (10 mM)—was added to each well of a 24-well plate containing overnight cultured HepG2 cells. After pretreatment for 2 h, all four inhibitors were completely removed, and the plate was washed three times with PBS. Then, a mixture containing 50 μL of (CR3)3C-AuNPs / BSA at a molar ratio of 30:1 and 400 μL of serum-free culture medium was added for 20 min, maintaining a BSA-FITC concentration of 3 μM. A control group was established using only (CR3)3C-AuNPs / BSA-FITC treatment for 4 h, without treatment with endocytosis pathway inhibitors. Three replicates were performed in each group to minimize experimental error. After incubating the wells at 37°C (5% CO2) for 4 h, the cells were washed three times with PBS and collected in centrifuge tubes. Flow cytometry was used to quantitatively detect the mean fluorescence intensity and the percentage of fluorescent positive cells. The results are as follows: Figure 7 As shown.

[0086] according to Figure 7 As can be seen, compared with the control group, the fluorescence intensity in HepG2 cells treated with various endocytosis inhibitors did not decrease significantly. This indicates that the protein crown delivery system does not rely on traditional endocytosis for cell entry, but rather directly crosses the cell membrane barrier through the interaction between a positively charged amphiphilic peptide and a negatively charged cell membrane. Therefore, it can be considered that after the protein molecule complexes with (CR3)3C-AuNPs, it does not cover the surface of the protein crown delivery system, but is embedded in the gaps between the bifunctional polypeptide molecules on the surface of the protein crown delivery system.

[0087] Example 10: Analysis of the intracellular delivery effect of β-galactosidase:

[0088] The efficacy of the protein crown delivery system prepared in Example 1 in delivering β-galactosidase into HepG2 cells was tested. The specific steps were as follows: The cell sample solution was replaced with 50 μL of a mixture of (CR3)3C-AuNPs / β-Gal at different concentrations (molar ratios of the protein crown delivery system to the target protein were 30:1, 45:1, 60:1, 90:1, 120:1, 150:1, and 200:1) and 400 μL of serum-free culture medium. Three parallel experiments were set up for each group to reduce experimental error, with the β-Gal-treated experimental group serving as a control. After co-incubation for 4 h, the cell culture medium was aspirated, the cells were washed three times with PBS, and 250 μL of β-Gal staining fixative was added. After fixation at room temperature for 10 min, the cells were washed three times with PBS. 250 μL of staining working solution containing 5% X-gal was added to the treated cells, and the wells were incubated at 37°C without CO2 for 2 h. Finally, the results were observed under an inverted fluorescence microscope. Figure 8 As shown.

[0089] according to Figure 8 As can be seen, compared with the control group that only added β-Gal, all experimental groups using (CR3)3C-AuNPs for β-Gal delivery exhibited varying degrees of blue color. Since the blue substrate directly reflects the intracellular β-Gal uptake, it can be assumed that (CR3)3C-AuNPs can deliver biologically active β-Gal into cells. Individual β-Gal is relatively large and therefore cannot easily cross the cell membrane barrier on its own. Furthermore, as the concentration of (CR3)3C-AuNPs increased, the blue color in the field of view deepened continuously. The blue color reached its maximum and remained constant when the molar ratio exceeded 90:1. This may indicate that at this β-Gal concentration, its binding to the corona delivery system has reached saturation. Therefore, a high concentration of the corona delivery system is expected to load more β-Gal, thereby delivering more active proteins into the cell.

[0090] Example 11: Analysis of the intracellular delivery effect of horseradish peroxidase:

[0091] The efficacy of the protein crown delivery system prepared in Example 1 in delivering horseradish peroxidase (HRP) into HepG2 cells was tested. The specific steps were as follows: In the presence of hydrogen peroxide, HRP catalyzes the non-fluorescent substrate Amplex Red to generate the fluorescent product Resorufin. During the experiment, the HRP concentration was fixed at 3 μM, and HepG2 cells were treated for 4 h at a molar ratio of 30:1 between the protein crown delivery system and the target protein. Cells were washed three times with PBS and incubated at room temperature for 30 min in PBS containing 50 μM Amplex Red and 500 μM hydrogen peroxide. Subsequently, the treated cells were washed three times with PBS, and fluorescence was observed by inverting the cells. The HRP-treated experimental group served as a control. The results are as follows: Figure 9 As shown.

[0092] according to Figure 9 As can be seen, after Amplex red treatment, no red fluorescence was observed in HepG2 cells delivered with HRP alone, indicating that HRP could not enter the cell interior at this time. However, uniformly distributed red fluorescence was observed in HepG2 cells treated with (CR3)3C-AuNPs / HRP, indicating that (CR3)3C-AuNPs can efficiently deliver HRP into the cell interior.

[0093] Example 12 Cytotoxicity Analysis:

[0094] The cytotoxicity of the protein crown delivery system prepared in Example 1 in delivering ribonuclease A into HepG2 cells was tested. The specific steps were as follows: HepG2 cells were injected with 1×10 5 Cells were seeded at a density of 1 / mL in 96-well plates and incubated overnight at 37°C to allow cell adhesion. Cells were then washed with PBS, and 50 μL of a mixture of (CR3)3C, (CR3)3C-AuNPs, (CR3)3C-AuNPs / RNase A, and 100 μL of serum-free medium were added to each well according to different concentrations of RNase A (0 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM). Five replicates were set up for each group to minimize experimental error. After 6 hours of incubation at 37°C, the medium was aspirated, washed with PBS, and replaced with 100 μL of fresh DMEM complete medium. After another 24 hours of incubation, 20 μL of 5 mg / mL MTT solution was added to each well in the dark, and the cells were incubated in the dark for 4 hours. After completely removing the supernatant, 150 μL of DMSO was added, and the mixture was shaken for 10 min to completely dissolve the purple crystals. Finally, the absorbance of the solution at 570 nm was measured using a microplate reader to calculate cell viability. The formula for calculating cell viability is: Cell viability = (A... t -A b) × 100% / (A c -A b ), where A t For the absorbance of the experimental group, A c The absorbance of the control group, A b The absorbance of the blank is shown in the following figures. Figure 10 As shown.

[0095] according to Figure 10 It can be seen that, for the RNase A control group not combined with the protein crown delivery system, even with the protease concentration increased to 100 μM, no toxicity was observed in HepG2 cells, indicating that RNase A cannot enter cells on its own. However, for cells treated with (CR3)3C-AuNPs, the cell viability decreased significantly with increasing RNase A concentration, indicating that (CR3)3C-AuNPs effectively delivered RNase A into the cells, and that the delivered RNase A still possessed the ability to catalyze intracellular nucleic acid degradation. Therefore, (CR3)3C-AuNPs can effectively deliver toxic proteins into cells, efficiently maintain the activity and function of proteases, regulate protein expression levels, and play a role in killing cancer cells and treating cancer.

[0096] Example 13: Analysis of Superoxide Dismutase Delivery Efficiency:

[0097] The effect of the protein crown delivery system prepared in Example 1 on the delivery of superoxide dismutase into HepG2 cells was analyzed. The specific steps were as follows: During the experiment, the concentration of SOD-FITC was fixed at 3 μM. (CR3)3C-AuNPs were diluted to appropriate concentrations at molar ratios of 5:1, 15:1, 30:1, 45:1, and 60:1. Subsequently, adherent cells in each well were treated with different molar ratios of (CR3)3C-AuNPs / SOD-FITC according to the above experimental steps. After incubation for 4 h, the cells were washed three times with PBS buffer. Finally, the samples treated according to the aforementioned experimental steps were used for flow cytometry analysis to quantitatively analyze the intracellular uptake of SOD-FITC mediated by (CR3)3C-AuNPs.

[0098] The intracellular delivery activity of SOD protein was monitored using the pyrogallol oxidation method. Cell treatment was the same as described above: after washing three times with PBS buffer, 100 μL of RIPA cell lysis buffer was added to each well, and the cells were incubated at 37°C for 1 h for lysis. Finally, the inhibitory effect of each experimental group on the pyrogallol autoxidation rate was detected using the modified Marklund method described above, and the absorbance at 325 nm was measured. The results are as follows: Figure 11 As shown.

[0099] according to Figure 11It can be seen that normal cells naturally contain a certain level of ROS, while the ROS level of cells in an inflammatory state is significantly enhanced compared to normal cells. When cells were treated with (CR3)3C-AuNPs / SOD, although they underwent inflammatory stimulation, the fluorescence intensity was no different from that of normal cells, indicating that intracellular ROS decreased to the level of normal cells, restoring the inflammatory cells to a normal state. Simultaneously, to avoid the influence of individual components in the carrier on intracellular ROS, the fluorescence intensity of AuNPs, (CR3)3C, and (CR3)3C-AuNPs after Rosup treatment was measured. The results showed that the fluorescence signals of the three components were not significantly different from those of the Rosup-positive group, indicating that the components of the protein crown delivery system did not interfere with the accuracy of the experimental results. The confocal microscopy results were consistent with the flow cytometry results; the intracellular fluorescence intensity of the cells in the experimental group delivered with SOD enzyme via the protein crown delivery system decreased to the level of normal cells.

[0100] Example 14: Analysis of the effect of Cas9 protein gene editing:

[0101] The gene editing effect of the protein crown delivery system prepared in Example 1 on the delivery of Cas9 protein into HeLa-EGFP cells was analyzed. The specific steps were as follows: EGFP-p65 HeLa cells were seeded in 24-well cell culture plates. After cell adhesion, (CR3)3C-AuNPs / RNPs were added. The molar ratio of (CR3)3C-AuNPs to RNPs was 30:1, and each well contained 80 nM Cas9, 80 nM sgRNA, and 2.4 μM (CR3)3C-AuNPs. After co-culturing for 4 h, the sample solution was discarded, and the cells were refreshed with fresh culture medium and cultured for another 48 h. Finally, the samples were prepared for flow cytometry analysis for quantitative fluorescence analysis. The results are as follows: Figure 12 As shown.

[0102] according to Figure 12 As can be seen from the flow cytometry analysis of the green fluorescence expression of EGFP-p65 HeLa cells 48 h after editing, compared with the control group, the intracellular fluorescence intensity of the experimental group treated with (CR3)3C-AuNPs / RNP decreased from 430 to 270 after editing, and the number of positive cells decreased from 79.4% to 66.1%, with an editing efficiency of approximately 30%.

[0103] As can be seen from the above embodiments, the protein crown delivery system provided by the present invention can efficiently overcome the cell membrane barrier, promote the escape of endosomes, and maintain the biological activity of proteins. It can efficiently, safely, and stably deliver proteins into cells, providing a foundation for functional in vivo applications such as intracellular delivery of protein drugs, intracellular catalytic reactions, and gene editing.

[0104] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of a protein crown delivery system in intracellular protein delivery, wherein the application is not for the purpose of disease diagnosis and / or treatment, characterized in that, The protein crown delivery system and the target protein are mixed and co-incubated with cells to achieve intracellular protein delivery; The protein crown delivery system includes gold nanoparticles and a bifunctional polypeptide modified on the surface of the gold nanoparticles; the particle size of the gold nanoparticles is 15.97~20 nm. The sequence of the bifunctional polypeptide is shown in SEQ ID NO.1: SEQ ID NO. 1: Nap-FF-GPLGLAGCRRRCRRRCRRRC.

2. The application according to claim 1, characterized in that, The protein crown delivery system also includes structural domains for enhancing the stability and dispersibility of the gold nanoparticles.

3. The application according to claim 1, characterized in that, The protein crown delivery system further includes one or more of the following: a domain for providing a specific non-covalent interaction with the target protein, a domain for promoting cellular uptake, and a domain for releasing the target protein in response to the intracellular environment.

4. The application according to claim 1, characterized in that, The preparation method of the protein crown delivery system includes the following steps: mixing and assembling a colloidal solution of gold nanoparticles and a bifunctional peptide to obtain the protein crown delivery system.

5. The application according to claim 1, characterized in that, The protein crown delivery system binds to the target protein through non-covalent interactions, and the target protein and the protein crown delivery system form a protein crown.

6. The application according to claim 1, characterized in that, The target proteins include non-gene-edited proteins and gene-edited proteins; the non-gene-edited proteins include one or more of bovine serum albumin, β-galactosidase, superoxide dismutase, horseradish peroxidase, ribonuclease A, cytochrome C, and trypsin. The gene-editing protein is the Cas9 protein.

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