Polypeptide delivery compositions

Abstract

The present invention relates to a polypeptide delivery composition comprising porous artificial partner particles having an average pore size of 1-100 nm, thereby pinning a polypeptide, thereby fixedly changing the randomly coiled three-dimensional structure of the polypeptide to a stable alpha-helical structure, so that the stability and efficacy of the polypeptide can be significantly improved.

Description

[0001] This application is a divisional application of Patent Application No. 201980049415.9 (International Application No. PCT / KR2019 / 009564) filed on July 31, 2019, entitled "Polypeptide Delivery Composition". TECHNICAL FIELD

[0002] The present application relates to a composition for delivering a polypeptide. BACKGROUND

[0003] In order to maintain life activities, various proteins in the body interact with other biopolymers, such as protein-protein interactions, protein-RNA interactions, and protein-DNA interactions, and play an important role in signal transduction systems in the body. It is often found that the interaction between specific proteins is abnormally activated, leading to the occurrence of a specific disease and maintaining the corresponding disease state. In many cases, the interaction between these polymers is carried out through specific secondary structures of proteins such as α-helices and β-strands.

[0004] The α-helix structure is one of the most well-known secondary structures of proteins, accounting for more than 30% of the secondary structures found in globular proteins, and these α-helix structures play a crucial role in interactions with biopolymers. In addition, since the peptides constituting the proteins have a short length, it is easy to chemically synthesize polypeptides and manipulate them. In addition, since polypeptides have specific activities depending on the structure and morphology, interest in self-assembled nanostructures based on these characteristics is increasing. Therefore, it can be considered that short sequence peptides having the above-described secondary structure have a great potential as a useful research tool for biological mechanism research or as a drug for treating diseases.

[0005] Peptides are stable in proteins, but when isolated from proteins and exist in the form of peptides, peptides are unstable in thermodynamics, so that the secondary structure such as a helical structure is hardly maintained and has a disordered form. As a result, it is difficult to achieve high affinity and selectivity to a target. Therefore, in order to solve the above-described problems and be applied as a drug that can control or inhibit various interactions of organisms, there is an urgent need to prepare a solution that can maintain the activated form and the folded form of a peptide, regardless of changes in the environment, and stabilize the self-assembled peptide nanostructure. SUMMARY

[0006] Problem to be solved by the invention

[0007] An object of the present application is to provide a composition for delivering a polypeptide, which can significantly improve stability and efficacy by stapling a polypeptide.

[0008] The present invention aims to provide a composition for delivering a polypeptide, which can significantly improve stability and efficacy by tacking a polypeptide.

[0009] Solution for solving the problem

[0010] 1. A composition for delivering a polypeptide, comprising porous artificial chaperone particles having an average pore diameter of 1 to 100 nm.

[0011] 2. The composition according to the above 1, wherein the particles are used to tack a polypeptide loaded inside the pores thereof.

[0012] 3. The composition according to the above 1, wherein the porous artificial chaperone particles have an average diameter of 150 to 1000 nm.

[0013] 4. The composition according to the above 1, wherein the porous artificial chaperone particles have a BET surface area of 200 to 700 m 2 / g, and a volume per gram of pores of 0.7 to 2.2 ml.

[0014] 5. The composition according to the above 1, wherein the porous artificial chaperone particles are prepared by reacting silica particles having pores with a pore diameter of less than 5 nm with a swelling agent at 120 to 180°C for 24 to 96 hours to swell the pores with a pore diameter of less than 5 nm; and calcining the silica particles having the swollen pores at a temperature of 400°C or higher for at least 3 hours.

[0015] 6. The composition according to the above 1, wherein the porous artificial chaperone particles are characterized in that, when the absorbance ratio in the following Formula 1 reaches 1 / 2, t is 24 or more.

[0016] [Formula 1]

[0017] A t / A0

[0018] (wherein A0 is the absorbance of the porous artificial chaperone particles measured by placing 5 ml of a suspension containing 1 mg / ml of the porous artificial chaperone particles in a cylindrical osmotic membrane having pores with a pore diameter of 50 kDa,

[0019] 15 ml of the same solvent as the suspension is brought into contact with the outside of the osmotic membrane, and the inside and outside of the osmotic membrane are stirred horizontally at 60 rpm at 37°C,

[0020] the pH of the suspension is 7.4, and

[0021] A t represents the absorbance of the porous artificial chaperone particles measured after t hours from the measurement of A0).

[0022] 7. The composition according to 1 above, wherein the porous artificial chaperone particle has a hydrophilic substituent on its outer surface and a hydrophobic substituent inside the pores.

[0023] 8. The composition according to 1 above, wherein the polypeptide is an alpha helix polypeptide or an enzyme polypeptide.

[0024] 9. The composition according to 8 above, wherein the alpha helix polypeptide is at least one selected from the group consisting of a Bcl-2 family protein inhibitor, an MDM2 / MDMX inhibitor, an HIV envelope protein gp41 inhibitor, an epidermal growth factor receptor inhibitor, a Kirsten rat sarcoma viral oncogene homolog (KRAS) inhibitor, a Rab (Ras-related in brain) inhibitor, and a myoA (malaria invasion motor myosin) tail interacting protein (MTIP) inhibitor.

[0025] 10. The composition according to 8 above, wherein the enzyme polypeptide is at least one selected from the group consisting of carbonic anhydrase, lysozyme, insulin, BSA, lipase, amylase, chymotrypsin, citrate synthase, formate dehydrogenase, GFP, and mitochondrial malate dehydrogenase (mMDH).

[0026] 11. The composition according to 1 above, wherein the polypeptide is p53.

[0027] 12. A composition for delivering a polypeptide, comprising: a porous artificial chaperone particle having an average pore diameter of 1 to 100 nm; and a polypeptide loaded and pinned inside the pores of the particle.

[0028] 13. The composition according to 12 above, wherein the porous artificial chaperone particle has an average diameter of 150 to 1000 nm.

[0029] 14. The composition according to 12 above, wherein the porous artificial chaperone particle has a BET surface area of 200 to 700 m 2 / g and a volume per gram of pores of 0.7 to 2.2 ml.

[0030] 15. The composition according to 12 above, wherein the porous artificial chaperone particle is prepared by reacting a silica particle having pores with a pore diameter of less than 5 nm with a swelling agent at 120 to 180°C for 24 to 96 hours to swell the pores with a pore diameter of less than 5 nm; and calcining the silica particle having the swollen pores at a temperature of 400°C or higher for at least 3 hours.

[0031] 16. The composition according to 12 above, wherein the porous artificial partner particle is characterized in that t is 24 or more when the absorbance ratio in the following Formula 1 reaches 1 / 2.

[0032] [Formula 1]

[0033] A t / A0

[0034] (wherein A0 is the absorbance of the porous artificial partner particle measured by placing 5 ml of a suspension containing 1 mg / ml of the porous artificial partner particle in a cylindrical permeable membrane having pores with a pore diameter of 50 kDa,

[0035] 15 ml of the same solvent as the suspension is brought into contact with the outside of the permeable membrane, and the inside and outside of the permeable membrane are stirred horizontally at 37°C at 60 rpm,

[0036] the pH of the suspension is 7.4, and

[0037] A t represents the absorbance of the porous artificial partner particle measured t hours after the measurement of A0).

[0038] 17. The composition according to 12 above, wherein the porous artificial partner particle has a hydrophilic substituent on its outer surface and a hydrophobic substituent inside the pores.

[0039] 18. The composition according to 12 above, wherein the polypeptide has an a-helix structure inside the pores.

[0040] 19. The composition according to 12 above, wherein the polypeptide is an a-helix polypeptide or an enzyme polypeptide.

[0041] 20. The composition according to 19 above, wherein the a-helix polypeptide is at least one selected from the group consisting of a Bcl-2 family protein inhibitor, an MDM2 / MDMX inhibitor, an HIV envelope protein gp41 inhibitor, an epidermal growth factor receptor inhibitor, a Kirsten rat sarcoma viral oncogene homolog (KRAS) inhibitor, a Rab (Ras-related in brain) inhibitor, and a myoA (malaria invasion motor myosin) tail interacting protein (MTIP) inhibitor.

[0042] 21. The composition according to 19 above, wherein the enzyme polypeptide is at least one selected from the group consisting of carbonic anhydrase, lysozyme, insulin, BSA, lipase, amylase, chymotrypsin, citrate synthase, formate dehydrogenase, GFP, and mitochondrial malate dehydrogenase (mMDH).

[0043] 22. The composition according to 12 above, wherein the polypeptide is p53.

[0044] 23. A method for tacking a polypeptide, comprising: mixing a porous artificial chaperone particle having an average pore diameter of 1 to 100 nm and a polypeptide to load the polypeptide inside the pores; and tacking the polypeptide.

[0045] Effects of the invention

[0046] The present application can tack a polypeptide to fix a random coil tertiary structure of the polypeptide as a stable α-helix structure, thereby significantly improving stability and efficacy of the polypeptide. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 A photomicrograph of a porous artificial chaperone (ACPN; hereinafter also referred to as "porous artificial chaperone particle") according to one embodiment of the present application.

[0048] Figure 2 A photomicrograph of a porous artificial chaperone particle according to one embodiment of the present application.

[0049] Figure 3 A photomicrograph of a small pore particle during the manufacturing process of a porous artificial chaperone particle according to one embodiment of the present application.

[0050] Figure 4 A photomicrograph of a small pore particle according to one embodiment of the present application.

[0051] Figure 5 A photomicrograph of a porous artificial chaperone particle according to one embodiment of the present application. Degradable Delivery Vehicle (DDV) is a particle according to the embodiment, wherein the number in the parenthesis means the diameter of the particle and the number of the subscript means the pore diameter. For example, DDV(200) 10 means a particle according to the embodiment having a particle diameter (i.e., particle size) of 200 nm and a pore diameter of 10 nm.

[0052] Figure 6 A photomicrograph for identifying biodegradability of a porous artificial chaperone particle according to one embodiment of the present application.

[0053] Figure 7 A view to show a tube having a cylindrical permeation membrane according to one illustrative example.

[0054] Figure 8 A graph to show the results of absorbance decrease over time of a porous artificial chaperone particle according to one embodiment of the present application.

[0055] Figure 9 A graph to show the results of absorbance reduction of each pore size of porous artificial chaperone particles over time according to an embodiment of the present application.

[0056] Figure 10 A graph to show the results of absorbance reduction of each pore size of porous artificial chaperone particles over time according to an embodiment of the present application.

[0057] Figure 11 A graph to show the results of absorbance reduction of each pore size of porous artificial chaperone particles over time according to an embodiment of the present application.

[0058] Figure 12 A graph to show the results of absorbance reduction of each pore size of porous artificial chaperone particles over time according to an embodiment of the present application.

[0059] Figure 13 A graph to show the results of absorbance reduction of each pore size of porous artificial chaperone particles over time according to an embodiment of the present application.

[0060] Figure 14 A graph to show the design and properties of porous artificial chaperone particles according to an embodiment of the present application. Specifically, (a) is a design diagram of porous artificial chaperone particles. A short polyethylene glycol moiety is conjugated to the outer surface. Hydroxyl, propyl and octyl groups are introduced to the inner surface. (b) is a fluorescence spectrum of Nile red encapsulated in porous artificial chaperone particles (λex= 510 nm). (c) shows TEM images of ACPN-C8 and ACPN-C8@p53 (scale bar, 200 nm). (d) shows DLS data of ACPN-C8 and ACPN-C8@p53.

[0061] Figure 15 A table to show the alpha helix value (helicity) when p53 is loaded on porous artificial chaperone particles according to an embodiment of the present application. This is calculated based on CD spectra using CONTIN program method.

[0062] Figure 16 A graph to show the results of cell experiments and cell death when p53 is loaded on porous artificial chaperone particles according to an embodiment of the present application.

[0063] Specifically, (a) are bright field images and fluorescence images showing absorption of clicked p53 itself and ACPN-C8@clicked p53 into Hela cells (bar = 25 μm). (b) shows cytotoxicity of various concentrations of ACPN-C8@clicked p53 and a comparative group against HeLa cells. (c) shows cytotoxicity of various concentrations of ACPN@clicked p53 against HeLa cells. EC50 values of ACPN@clicked p53 were collected by a log curve fitting method. (d) shows relative caspase-3 activity of ACPN-C8@clicked p53.

[0064] Figure 17 A graph showing results of an in vivo experiment when p53 was loaded on a porous artificial chaperone particle according to one embodiment of the present application. Specifically, (a) shows time-dependent relative tumor volume of tumor xenograft mice and a comparative group introduced with TAMRA-labeled ACPN-C8@clicked p53 at 5-day intervals (N = 4). (b) shows time-dependent whole body fluorescence images of tumor xenograft mice and a comparative group introduced with TAMRA-labeled ACPN-C8@clicked p53 (N = 4) (green: clicked p53, red: ACPN-C8). (c) shows time-dependent relative FAM fluorescence intensity of clicked p53, wherein the intensity was obtained by ROI analysis based on whole body fluorescence images of tumor xenograft mice.

[0065] Figure 18 A graph showing results of polypeptide release evaluation of ACPN-C8@clicked p53. DETAILED DESCRIPTION

[0066] Hereinafter, the present application will be described in detail.

[0067] The present application provides a composition for delivering a polypeptide comprising a porous artificial chaperone particle having an average pore diameter of 1 to 100 nm.

[0068] The porous artificial chaperone particle of the present application has pores of nanometer size, and can carry a polypeptide on the outer surface of the particle and / or inside the pores.

[0069] The polypeptide (protein) includes not only a chemically synthesized polypeptide but also a naturally synthesized polypeptide encoded by a gene of a cultured cell, and a recombinant polypeptide secreted by a cell. The recombinant polypeptide can refer to a peptide, a polypeptide, an oligopeptide, and / or a fusion polypeptide, and any polypeptide or a biologically active portion thereof (e.g., a portion retaining the biological activity of the entire polypeptide) expressed from a recombinant genetic material encoding an amino acid. The recombinant polypeptide product can include a therapeutic product, a prophylactic product, or a diagnostic product.

[0070] In the present application, polypeptide stapling means changing a random coil structure to an alpha helix and maintaining the structure.

[0071] When the structural disorder of a polypeptide as a drug is high, the three-dimensional structure is not fixed. Therefore, a protein to be bound thereto has difficulty in recognizing the three-dimensional structure, thereby reducing the performance of the protein as a drug.

[0072] For the composition of the present application, nanoparticles function as a chaperone particle to stabilize the tertiary structure of a polypeptide, and also participate in click-stapling of a peptide to irreversibly maintain an alpha-helix of the polypeptide. In addition, a plurality of pores can carry a target peptide to be delivered, thereby having a stable alpha-helix structure from a random coil tertiary structure. Therefore, the efficacy of the polypeptide can be maximized.

[0073] In the present application, polypeptides can be stapled, and can be applied without limitation as long as the three-dimensional structure of the polypeptide can be unstable, and specific examples of the polypeptide can include, for example, an alpha helix polypeptide or an enzyme polypeptide.

[0074] The alpha helix polypeptide means a polypeptide having an alpha-helix motif formed by binding an alpha-helix motif to a hydrophobic pocket through several polypeptide-polypeptide interactions in vivo. The alpha helix polypeptide of the present application can have such a motif or mimic a motif. For example, the above-described alpha helix polypeptide can include a Bcl-2 family protein inhibitor, an MDM2 / MDMX inhibitor, an HIV envelope protein gp41 inhibitor, an epidermal growth factor receptor inhibitor, a Kirsten rat sarcoma viral oncogene homolog (KRAS) inhibitor, a Rab (Ras-related in brain) inhibitor, and a myoA (malaria invasion motor myosin) tail interacting protein (MTIP) inhibitor, and more specifically, p53, but is not limited thereto.

[0075] The enzyme polypeptide can be stapled into a natural protein by carrying a non-natural protein, and can include, for example, carbonic anhydrase, lysozyme, insulin, BSA, lipase, amylase, chymotrypsin, citrate synthase, formate dehydrogenase, GFP, and mitochondrial malate dehydrogenase (mMDH), but is not limited thereto.

[0076] The composition of the present application can be used to deliver the loaded polypeptide to an individual, i.e., a subject. In this regard, the polypeptide used herein can include any one as long as it has a sequence derived from the same species as the subject to be delivered, or even if the polypeptide is derived from other species but is applicable to the corresponding subject.

[0077] The porous artificial companion particle of the present application is a biodegradable particle. In such a case, when the companion particle carries a polypeptide and is administered into the body, the companion particle is biodegraded to release the polypeptide. The porous artificial companion particle of the present application can be slowly degraded in the body to release the loaded polypeptide in a sustained manner. For example, when the absorbance ratio in the following Formula 1 reaches 1 / 2, t is 24 or more.

[0078] [Formula 1]

[0079] A t / A0

[0080] (wherein A0 is the absorbance of the porous artificial companion particle measured by placing 5 ml of a suspension containing 1 mg / ml of the porous artificial companion particle in a cylindrical permeable membrane having pores with a pore diameter of 50 kDa,

[0081] 15 ml of the same solvent as the suspension is brought into contact with the outside of the permeable membrane, and the inside and outside of the permeable membrane are stirred horizontally at 60 rpm at 37°C,

[0082] the pH of the suspension is 7.4, and

[0083] A t represents the absorbance of the porous artificial companion particle measured t hours after the measurement of A0).

[0084] The above Formula 1 means the rate at which the porous artificial companion particle is degraded in an environment similar to that in the body.

[0085] As Figure 7 indicated in the above Formula 1, for example, the absorbances A0 and A can be measured after the porous artificial companion particle and the suspension are placed in the cylindrical permeable membrane and the same suspension is also placed outside the permeable membrane. t .

[0086] The porous artificial companion particle of the present application is biodegradable, and can be slowly degraded in the suspension. A diameter of 50 kDa corresponds to about 5 nm, which allows the biodegradable porous artificial companion particle to pass through the permeable membrane with a diameter of 50 kDa, and the cylindrical permeable membrane is stirred horizontally at 60 rpm to uniformly mix the suspension, so that the degraded porous artificial companion particle can come out of the permeable membrane.

[0087] The absorbance in Equation 1 above can be measured, for example, in an environment in which a new suspension is replaced with a suspension outside of a permeable membrane. The suspension can be replaced continuously or every period, wherein the period is regular or irregular. For example, the suspension can be replaced at 1 hour intervals, 2 hour intervals, 3 hour intervals, 6 hour intervals, 12 hour intervals, 24 hour intervals, 2 day intervals, 3 day intervals, 4 day intervals, 7 day intervals, etc., but is not limited thereto, in a range of 1 hour to 1 week.

[0088] An absorbance ratio of 1 / 2 means that the absorbance is half of the initial absorbance after t hours, i.e., means that about half of the porous artificial companion particles are degraded.

[0089] The suspension can be a buffer solution, for example, at least one selected from the group consisting of phosphate buffered saline (PBS) and simulated body fluid (SBF), and more particularly PBS.

[0090] At the time when the absorbance ratio reaches 1 / 2, t in Equation 1 of the present application can be 24 or more, for example, t can be 24 to 120. That is, t can be 24 to 96, 24 to 72, 30 to 70, 40 to 70, 50 to 65, etc., in the above range, but is not limited thereto.

[0091] For the porous artificial companion particles of the present application, at the time when the absorbance ratio in Equation 1 above reaches 1 / 5, t can be 70 to 140. For example, t can be 80 to 140, 80 to 120, 80 to 110, 70 to 140, 70 to 120, 70 to 110, etc., in the above range, but is not limited thereto.

[0092] For the porous artificial companion particles of the present application, at the time when the absorbance ratio in Equation 1 above reaches 1 / 20, t can be 130 to 220. For example, t can be 130 to 200, 140 to 200, 140 to 180, 150 to 180, etc., in the above range, but is not limited thereto.

[0093] For the porous artificial companion particles of the present application, at the time when the absorbance ratio in Equation 1 above reaches 0.01 or less, t can be 250 or more. For example, t can be 300 or more, 350 or more, 400 or more, 500 or more, 1000 or more, etc., and the upper limit can be 2000, but is not limited thereto.

[0094] For the porous artificial companion particles of the present application, the absorbance ratio in Equation 1 above has a high positive correlation with t. For example, the Pearson correlation coefficient can be 0.8 or more, for example, 0.9 or more and 0.95 or more.

[0095] t in the above formula 1 means the speed at which the porous artificial chaperone particle degrades in an environment similar to that in vivo. That is, t can be adjusted by adjusting, for example, the surface area of the porous artificial chaperone particle, the particle diameter, the pore diameter, the substituents on the surface of the particle and / or inside the pores, and the compactness of the surface, and the like.

[0096] For example, the surface area of the particle can be increased to decrease t, or the surface area can be decreased to increase t. The surface area can be adjusted by adjusting the particle diameter and the pore diameter of the particle. Furthermore, t can be increased if the direct exposure of the porous artificial chaperone particle to the environment (e.g., a solvent) is reduced by having the substituents on the surface of the particle and / or inside the pores. Furthermore, when the porous artificial chaperone particle is loaded or carries a polypeptide and the affinity between the polypeptide and the porous artificial chaperone particle is increased, the direct exposure of the porous artificial chaperone particle to the environment can be reduced, thereby increasing t. Furthermore, t can be increased by preparing a particle having a more compact surface. As described above, various examples of adjusting t in the above formula 1 have been described, but are not limited thereto.

[0097] The porous artificial chaperone particle of the present application can have a spherical shape, but is not limited thereto.

[0098] The average diameter of the porous artificial chaperone particle of the present application can be, for example, 150 to 1000 nm. For example, the average diameter can be 150 to 800 nm, 150 to 500 nm, 150 to 400 nm, 150 to 300 nm, and 150 to 200 nm, and the like within the above range, but is not limited thereto.

[0099] The average pore diameter of the porous artificial chaperone particle of the present application can be, for example, 1 to 100 nm. For example, the average diameter can be 5 to 100 nm, 7 to 100 nm, 7 to 50 nm, 10 to 50 nm, 10 to 30 nm, 7 to 30 nm, and the like within the above range, but is not limited thereto. The chaperone particle having a larger diameter as described above can carry a large amount of polypeptide, and can also carry a polypeptide of a large size.

[0100] The BET surface area of the porous artificial chaperone particle of the present application can be, for example, 200 to 700 m 2 / g. For example, the BET surface area can be 200 to 700 m 2 / g, 200 to 650 m 2 / g, 250 to 650 m 2 / g, 300 to 700 m 2 / g, 300 to 650 m 2 / g, 300 to 600 m 2 / g, 300 to 550 m 2 / g, 300 to 500 m 2 / g, 300 to 450 m 2 / g, etc., but is not limited thereto.

[0101] The volume per gram of the porous silica nanoparticles of the present application can be, for example, 0.7 to 2.2 ml. For example, the volume can be 0.7 to 2.0 ml, 0.8 to 2.2 ml, 0.8 to 2.0 ml, 0.9 to 2.0 ml, 1.0 to 2.0 ml, etc., within the above range, but is not limited thereto. If the volume per gram is too small, the degradation rate can be too high. In addition, it is difficult to manufacture particles that are too large or have a complete shape.

[0102] The porous artificial chaperone particles of the present application can have a hydrophilic substituent and / or a hydrophobic substituent on the outer surface thereof and / or inside the pores. For example, only a hydrophilic substituent or only a hydrophobic substituent can be present both on the surface of the particles and inside the pores, a hydrophilic substituent or a hydrophobic substituent can be present on the surface of the particles or inside the pores, or a hydrophilic substituent can be present on the surface of the particles and a hydrophobic substituent can be present inside the pores, or vice versa.

[0103] The release of the polypeptide loaded on the porous artificial chaperone particles according to the present application is mainly performed by degradation of the nanoparticles. Specifically, the interaction of the porous artificial chaperone particles with the release environment of the polypeptide is adjusted to regulate the degradation rate of the nanoparticles, so that the release rate of the polypeptide can be regulated. In addition, the polypeptide can diffuse and be released from the nanoparticles, in which the adjustment of the substituent can regulate the binding force of the polypeptide to the nanoparticles, thereby controlling the release of the polypeptide.

[0104] In addition, in order to increase the binding force of the porous artificial chaperone particles to the poorly soluble (hydrophobic) polypeptide, a hydrophobic substituent can be present inside the pores of the particles. In addition, in terms of easy use and formulation, the surface of the particles can also be treated to have a hydrophilic substituent.

[0105] Specifically, the porous artificial chaperone particles can have a hydrophilic substituent on the surface of the particles and a hydrophobic substituent inside the pores. Such porous artificial chaperone particles are preferred from the viewpoint of excellent dispersibility and the polypeptide carried.

[0106] The hydrophilic substituent can include, for example, a hydroxyl group, a carboxyl group, an amino group, a carbonyl group, a mercapto group, a phosphoric acid group, a thiol group, an ammonium group, an ester group, an imide group, a thioimide group, a ketone group, an ether group, an indene group, a sulfonyl group, and a polyethylene glycol group, etc. In addition, the hydrophobic substituent can include, for example, a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, a substituted or unsubstituted C2 to C30 heteroaryl group, a halogen group, a C1 to C30 ester group, and a halogen-containing group, etc.

[0107] In addition, the porous artificial chaperone particles of the present application can be positively charged and / or negatively charged on the outer surface of the particles and / or inside the pores. For example, both the surface of the particles and the inside of the pores can be positively charged or negatively charged, and only the surface of the particles or the inside of the pores can be positively charged or negatively charged. Alternatively, the surface of the particles can be positively charged and the inside of the pores can be negatively charged, or vice versa.

[0108] Charging can be performed, for example, by the presence of a cationic substituent or an anionic substituent.

[0109] The cationic substance can include, for example, an amino group or any other nitrogen-containing group. In addition, the anionic substituent can include, for example, a carboxyl group (-COOH), a sulfonic acid group (-SO3H), a thiol group (-SH), etc. as an acidic group, but is not limited thereto.

[0110] Likewise, when the interaction of the porous artificial chaperone particles with the release environment of the polypeptide is adjusted by adjusting the substituent by charging, the degradation rate of the nanoparticles can be adjusted to control the release rate of the polypeptide. In addition, the polypeptide can diffuse and be released from the nanoparticles. In this regard, adjusting the substituent can adjust the binding force of the polypeptide to the nanoparticles, thereby controlling the release of the polypeptide.

[0111] In addition, the porous artificial chaperone particles of the present application can include a substituent for the purpose of loading a polypeptide on the surface of the particles and / or inside the pores; delivering the polypeptide into a target cell; loading other substances for other purposes; or binding other substituents. In addition, the porous artificial chaperone particles can further include an antibody, a ligand, a cell permeable peptide, or an aptamer bound thereto.

[0112] The above-mentioned substituents, charges, binders, etc. on the surface of the particles and / or inside the pores can be added, for example, by surface modification.

[0113] Surface modification can be performed, for example, by reacting a compound having a substituent to be introduced with the particles, where the compound can be, for example, an alkoxysilane having a C1 to C10 alkoxy group, but is not limited thereto. The alkoxysilane has one or more alkoxy groups, for example, 1 to 3 alkoxy groups. In addition, there can be a substituent to be introduced to a site not bonded with an alkoxy group, or a substituent substituted with an alkoxy group.

[0114] The porous artificial chaperone particles of the present application can be manufactured, for example, by a small-pore particle preparation and pore expansion process, and, if necessary, can be further manufactured by a calcination or surface modification process, etc. If both the calcination and surface modification processes are performed, the surface modification can be performed on the particles after calcination.

[0115] The small-pore particles can be, for example, particles having an average pore diameter of 1 to 5 nm.

[0116] The small-pore particles can be harvested by adding a surfactant and a silica precursor in a solvent, followed by stirring and homogenization.

[0117] The solvent can be water and / or an organic solvent, and the organic solvent can include, for example: ethers such as 1,4-dioxane (particularly cyclic ethers); halogenated hydrocarbons such as chloroform, dichloromethane, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride, 1,2-dibromoethane, and the like; ketones such as acetone, methyl isobutyl ketone, γ-butyrolactone, 1,3-dimethyl-imidazolidinone, methyl ethyl ketone, cyclohexanone, cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, and the like; aromatic carbon-based materials such as benzene, toluene, xylene, tetramethylbenzene, and the like; alkyl amides such as N,N-dimethylformamide, N,N-dibutylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, and the like; alcohols such as methanol, ethanol, propanol, butanol, and the like; glycol ethers (cellosolves) such as ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, diethylene glycol monomethyl ether, diethylene glycol monobutyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether, dipropylene glycol diethyl ether, triethylene glycol monoethyl ether, and the like; and others such as dimethylacetamide (DMAc), N,N-diethylacetamide, dimethylformamide (DMF), diethylformamide (DEF), N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), N-ethylpyrrolidone (NEP), 1,3-dimethyl-2-imidazolidinone, N,N-dimethylmethoxyacetamide, dimethyl sulfoxide, pyridine, dimethyl sulfone, hexamethyl phosphoramide, tetramethyl urea, N-methylcaprolactam, tetrahydrofuran, m-dioxane, p-dioxane, 1,2-dimethoxyethane, and the like. Specifically, an alcohol, more specifically methanol, can be used, but is not limited thereto.

[0118] When a mixed solvent of water and an organic solvent is used, the relative ratio of water to the organic solvent can be, for example, 1:0.7 to 1.5 by volume, for example, 1:0.8 to 1.3, but is not limited thereto.

[0119] The surfactant can include, for example, cetyltrimethylammonium bromide (CTAB), hexadecyltrimethylammonium bromide (TMABr), hexadecyltrimethylpyridinium chloride (TMPrCl), tetramethylammonium chloride (TMACl), and the like, and specifically, CTAB can be used.

[0120] The surfactant can be added, for example, in an amount of 1 to 10 g, for example, 1 to 8 g, 2 to 8 g, or 3 to 8 g, per 1 liter of the solvent, but is not limited thereto.

[0121] In a case where the surfactant is added to the solvent, the silica precursor can be added after stirring. The silica precursor can be, for example, tetramethyl orthosilicate (TMOS), but is not limited thereto.

[0122] The stirring can be performed for, for example, 10 to 30 minutes, but is not limited thereto.

[0123] The silica precursor can be added in an amount of 0.5 to 5 ml per 1 liter of the solvent, for example, 0.5 to 4 ml, 0.5 to 3 ml, 0.5 to 2 ml, 1 to 2 ml, etc., within the above range, but is not limited thereto.

[0124] If necessary, sodium hydroxide can be further used as a catalyst, specifically, can be added after the surfactant is added to the solvent and before the silica precursor is added to the solvent, with stirring.

[0125] The sodium hydroxide can be added in an amount of 0.5 to 8 ml per 1 liter of the solvent, for example, 0.5 to 5 ml, 0.5 to 4 ml, 1 to 4 ml, 1 to 3 ml, 2 to 3 ml, etc., within the above range, based on 1M aqueous sodium hydroxide solution, but is not limited thereto.

[0126] After the silica precursor is added, the solution can be allowed to react with stirring. The stirring can be performed for 2 to 15 hours, for example, 3 to 15 hours, 4 to 15 hours, 4 to 13 hours, 5 to 12 hours, 6 to 12 hours, 6 to 10 hours, etc., within the above range, but is not limited thereto. If the stirring time (reaction time) is too short, nucleation can be insufficient.

[0127] After the stirring, the solution can be allowed to age. The aging can be performed for 8 to 24 hours, for example, 8 to 20 hours, 8 to 18 hours, 8 to 16 hours, 8 to 14 hours, 10 to 16 hours, 10 to 14 hours, etc., within the above range, but is not limited thereto.

[0128] Thereafter, the reaction product can be washed and dried to harvest the porous artificial mate particles, and, if necessary, separation of unreacted materials can be performed before washing.

[0129] The separation of unreacted materials can be performed by, for example, separating the supernatant by centrifugation. For example, the centrifugation can be performed at 6,000 to 10,000 rpm, and the centrifugation time can be 3 to 60 minutes, for example, 3 to 30 minutes, 3 to 30 minutes, 5 to 30 minutes, etc., within the above range, but is not limited thereto.

[0130] The washing can be performed with water and / or an organic solvent. Specifically, since different substances are dissolved in different solvents, water and an organic solvent can be used alternately one or several times, or the washing can be performed with water or an organic solvent alone one or several times. The above several times can be 2 or more and 10 or less, for example, 3 or more and 10 or less, 4 or more and 8 or less, 4 or more and 6 or less.

[0131] The organic solvent can include, for example, ethers such as 1,4-dioxane (particularly, a cyclic ether); halogenated hydrocarbons such as chloroform, dichloromethane, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride, 1,2-dibromoethane, and the like; ketones such as acetone, methyl isobutyl ketone, γ-butyrolactone, 1,3-dimethyl-imidazolidinone, methyl ethyl ketone, cyclohexanone, cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, and the like; aromatic carbon-based materials such as benzene, toluene, xylene, tetramethylbenzene, and the like; alkyl amides such as N,N-dimethylformamide, N,N-dibutylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, and the like; alcohols such as methanol, ethanol, propanol, butanol, and the like; glycol ethers (cellosolves) such as ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, diethylene glycol monomethyl ether, diethylene glycol monobutyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether, dipropylene glycol diethyl ether, triethylene glycol monoethyl ether, and the like; and others such as dimethylacetamide (DMAc), N,N-diethylacetamide, dimethylformamide (DMF), diethylformamide (DEF), N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), N-ethylpyrrolidone (NEP), 1,3-dimethyl-2-imidazolidinone, N,N-dimethylmethoxyacetamide, dimethyl sulfoxide, pyridine, dimethyl sulfone, hexamethyl phosphoramide, tetramethyl urea, N-methylcaprolactam, tetrahydrofuran, m-dioxane, p-dioxane, 1,2-dimethoxyethane, and the like. Specifically, an alcohol, more specifically methanol, can be used, but is not limited thereto.

[0132] The washing can be performed under centrifugation, for example, at 6,000 to 10,000 rpm, and the centrifugation time can be 3 to 60 minutes, for example, 3 to 30 minutes, 3 to 30 minutes, 5 to 30 minutes, and the like within the above range, but is not limited thereto.

[0133] Alternatively, the washing can be performed without centrifugation by passing the particles through a filter to be filtered out. The filter can have pores having a size smaller than or equal to the diameter of the porous artificial partner particles. When the reaction solution is filtered with such a filter as described above, only the particles are retained on the filter, which can be washed by pouring water and / or an organic solvent on the filter.

[0134] In the cleaning, water and the organic solvent can be used alternately once or several times, or the cleaning can be performed using water or the organic solvent alone once or several times. The above several times can be 2 or more and 10 or less, for example, 3 or more and 10 or less, 4 or more and 8 or less, 4 or more and 6 or less.

[0135] The drying can be performed, for example, at 20 to 100°C, but is not limited thereto, and can also be performed in a vacuum state.

[0136] Thereafter, the pores of the harvested porous artificial partner particles can be expanded, and such pore expansion can be performed using a pore expander.

[0137] The pore expander can include, for example, trimethylbenzene, triethylbenzene, tripropylbenzene, tributylbenzene, tripentylbenzene, trihexylbenzene, toluene, benzene, or the like, and specifically, trimethylbenzene can be used, but is not limited thereto.

[0138] Further, the pore expander used herein can be, for example, N,N-dimethylhexadecylamine (DMHA), but is not limited thereto.

[0139] The pore expansion can be performed, for example, by mixing the porous artificial partner particles with the pore expander in a solvent and heating the mixture to induce a reaction.

[0140] The solvent can be water and / or an organic solvent, and the organic solvent can include, for example, ethers such as 1,4-dioxane (particularly, cyclic ethers); halogenated hydrocarbons such as chloroform, dichloromethane, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride, 1,2-dibromoethane, or the like; ketones such as acetone, methyl isobutyl ketone, cyclohexanone, or the like; aromatic carbon-based materials such as benzene, toluene, xylene, or the like; alkyl amides such as N,N-dimethylformamide, N,N-dibutylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, or the like; alcohols such as methanol, ethanol, propanol, and butanol, or the like. Specifically, an alcohol, more specifically, methanol, can be used, but is not limited thereto.

[0141] The porous artificial partner particles can be added in a proportion of 10 to 200 g per 1 liter of the solvent, for example, 10 to 150 g, 10 to 100 g, 30 to 100 g, 40 to 100 g, 50 to 100 g, 50 to 80 g, 60 to 80 g, or the like within the above range, but is not limited thereto.

[0142] The porous artificial partner particles can be uniformly dispersed in the solvent. For example, the porous artificial partner particles can be added to the solvent and ultrasonically dispersed. In the case of using a mixed solvent, the porous artificial partner particles can be dispersed in a first solvent, and then a second solvent can be added thereto.

[0143] The pore expander can be added in a proportion of 10 to 200 parts by volume, for example, 10 to 150 parts by volume, 10 to 100 parts by volume, 10 to 80 parts by volume, 30 to 80 parts by volume, 30 to 70 parts by volume, or the like, relative to 100 parts by volume of the solvent, but is not limited thereto.

[0144] The reaction can be performed at 120 to 190°C, for example, 120 to 190°C, 120 to 180°C, 120 to 170°C, 130 to 170°C, 130 to 160°C, 130 to 150°C, 130 to 140°C, or the like, within the above range, but is not limited thereto.

[0145] The reaction can be performed for 6 to 96 hours, for example, 30 to 96 hours, 30 to 96 hours, 30 to 80 hours, 30 to 72 hours, 24 to 80 hours, 24 to 72 hours, 36 to 96 hours, 36 to 80 hours, 36 to 72 hours, 36 to 66 hours, 36 to 60 hours, 48 to 96 hours, 48 to 88 hours, 48 to 80 hours, 48 to 72 hours, 6 to 96 hours, 7 to 96 hours, 8 to 80 hours, 9 to 72 hours, 9 to 80 hours, 6 to 72 hours, 9 to 96 hours, 10 to 80 hours, 10 to 72 hours, 12 to 66 hours, 13 to 60 hours, 14 to 96 hours, 15 to 88 hours, 16 to 80 hours, 17 to 72 hours, or the like, within the above range, but is not limited thereto.

[0146] The time and temperature can be adjusted as desired within the range of the above examples, so that the reaction can be performed sufficiently but not excessively. For example, when the reaction temperature is lowered, the reaction time can be increased, and when the reaction temperature is increased, the reaction time can be shortened. If the reaction is not performed sufficiently, the pore expansion can be insufficient. On the other hand, if the reaction is performed excessively, the particles can collapse due to excessive expansion of the pores.

[0147] The reaction can be performed, for example, by gradually increasing the temperature. Specifically, the reaction can be performed by gradually increasing the temperature from room temperature to the temperature defined above at a rate of 0.5 to 15°C / min. For example, the temperature can be increased at a rate of 1 to 15°C / min, 3 to 15°C / min, 3 to 12°C / min, 3 to 10°C / min, or the like, but is not limited thereto.

[0148] The reaction can be performed with stirring. For example, the stirring can be performed at a speed of 100 rpm or more, specifically, at a speed of 100 to 1000 rpm, but is not limited thereto.

[0149] After the reaction, the reaction solution can be slowly cooled, for example, by gradually lowering the temperature. Specifically, the reaction can be performed by gradually lowering the temperature from the above-defined temperature to room temperature at a rate of 0.5 to 20°C / min. For example, the temperature can be lowered at a rate of 1 to 20°C / min, 3 to 20°C / min, 3 to 12°C / min, 3 to 10°C / min, or the like within the above range, but is not limited thereto.

[0150] After the cooling, the reaction product can be washed and dried to harvest the porous artificial mate particle having expanded pores. If necessary, the unreacted material can be first separated before the washing.

[0151] The separation of the unreacted material can be performed by, for example, separating the supernatant by centrifugation. Herein, the centrifugation can be performed at 6,000 to 10,000 rpm, for example, and the centrifugation time can be 3 minutes to 60 minutes. For example, the centrifugation can be performed for 3 to 30 minutes, 3 to 30 minutes, 5 to 30 minutes, or the like within the above range, but is not limited thereto.

[0152] The washing can be performed with water and / or an organic solvent. Specifically, since different substances are dissolved in different solvents, water and an organic solvent can be used alternately one or several times, or the washing can be performed with water or an organic solvent alone one or several times. The above several times can be 2 or more and 10 or less, for example, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, or the like.

[0153] The organic solvent can include, for example: ethers such as 1,4-dioxane (particularly, a cyclic ether); halogenated hydrocarbons such as chloroform, dichloromethane, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride, 1,2-dibromoethane, or the like; ketones such as acetone, methyl isobutyl ketone, cyclohexanone, or the like; aromatic carbon-based materials such as benzene, toluene, xylene, or the like; alkyl amides such as N,N-dimethylformamide, N,N-dibutylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, or the like; alcohols such as methanol, ethanol, propanol, and butanol, or the like. Specifically, an alcohol, more specifically, methanol, can be used, but is not limited thereto.

[0154] The washing can be performed under centrifugation performed at 6,000 to 10,000 rpm, for example, and the centrifugation time can be 3 to 60 minutes, for example, 3 to 30 minutes, 3 to 30 minutes, 5 to 30 minutes, or the like within the above range, but is not limited thereto.

[0155] Alternatively, the washing can be performed by filtering the particles through a filter without centrifugation. The filter can have pores having a size smaller than or equal to the diameter of the porous artificial partner particles. When the reaction solution is filtered with such a filter as described above, only the particles are retained on the filter, which can be washed by pouring water and / or an organic solvent on the filter.

[0156] In the washing, water and an organic solvent can be used alternately one or several times, or the washing can be performed one or several times with water or an organic solvent alone. The above-mentioned several times can be 2 or more and 10 or less, for example, 3 or more and 10 or less, 4 or more and 8 or less, 4 or more and 6 or less.

[0157] The drying can be performed, for example, at 20 to 100°C, but is not limited thereto, and can also be performed under a vacuum state.

[0158] Thereafter, the harvested particles can be calcined, which is a process of heating the particles to remove silanol groups present on the surface of the particles and inside the pores to thereby reduce the reactivity of the particles, provide a denser structure, and remove organic substances filling the pores. For example, the particles can be heated to a temperature of 400°C or higher. The upper limit of the temperature is not particularly limited, but can be 1000°C, 900°C, 800°C, 700°C, or the like. The heating can be performed, for example, for 3 hours or more. The upper limit of the heating time is not particularly limited, and can be 24 hours, 12 hours, 10 hours, 8 hours, 6 hours, or the like. More specifically, the heating can be performed at 400 to 700°C for 3 to 8 hours or at 500 to 600°C for 4 to 5 hours, but is not limited thereto.

[0159] The removal of the organic substances filling the pores can prevent some problems of cytotoxicity or bubbling caused by the residual organic substances.

[0160] Then, the harvested porous artificial partner particles can be surface-modified, and the surface modification can be performed on the surface of the particles and / or inside the pores. Both the surface of the particles and the inside of the pores can be surface-modified in the same manner, or can be surface-modified in different manners.

[0161] The particles can be charged or have hydrophilicity and / or hydrophobicity by the surface modification.

[0162] More specifically, in order to efficiently load a polypeptide, the surface modification of the porous artificial partner particles can be performed by having at least one substituent selected from the group consisting of an amino group, an aminoalkyl group, an alkylamino group, a heterocyclic aromatic compound group containing a nitrogen atom, a cyano group, and a guanidino group.

[0163] The surface modification can be performed, for example, by reacting the porous artificial partner particle with a compound having a hydrophilic substituent, a hydrophobic substituent, a cationic substituent, or an anionic substituent to be introduced, where the compound can be, for example, an alkoxysilane having a C1 to C10 alkoxy group, but is not limited thereto.

[0164] The alkoxysilane has one or more alkoxy groups, for example, 1 to 3 alkoxy groups. In addition, there can be a substituent to be introduced to a site not bonded with an alkoxy group, or a substituent substituted with an alkoxy group.

[0165] When the alkoxysilane is reacted with the porous artificial partner particle, a covalent bond is formed between the silicon atom and the oxygen atom so that the alkoxysilane can be bonded to the surface and / or the inside of the pores of the porous artificial partner particle. Since the alkoxysilane has a substituent to be introduced, the corresponding substituent can be introduced to the surface and / or the inside of the pores of the porous artificial partner particle.

[0166] The reaction can be performed by reacting the porous artificial partner particle dispersed in a solvent with the alkoxysilane.

[0167] The solvent can be water and / or an organic solvent, and the organic solvent can include, for example: ethers, such as 1,4-dioxane (particularly, cyclic ethers); halogenated hydrocarbons, such as chloroform, dichloromethane, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride, 1,2-dibromoethane, and the like; ketones, such as acetone, methyl isobutyl ketone, γ-butyrolactone, 1,3-dimethyl-imidazolidinone, methyl ethyl ketone, cyclohexanone, cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, and the like; aromatic carbon-based materials, such as benzene, toluene, xylene, tetramethylbenzene, and the like; alkyl amides, such as N,N-dimethylformamide, N,N-dibutylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, and the like; alcohols, such as methanol, ethanol, propanol, butanol, and the like; glycol ethers (cellosolves), such as ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, diethylene glycol monomethyl ether, diethylene glycol monobutyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether, dipropylene glycol diethyl ether, triethylene glycol monoethyl ether, and the like; and others, such as dimethylacetamide (DMAc), N,N-diethylacetamide, dimethylformamide (DMF), diethylformamide (DEF), N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), N-ethylpyrrolidone (NEP), 1,3-dimethyl-2-imidazolidinone, dimethylmethoxyacetamide, dimethyl sulfoxide, pyridine, dimethyl sulfone, hexamethyl phosphoramide, tetramethyl urea, N-methylcaprolactam, tetrahydrofuran, m-dioxane, p-dioxane, and 1,2-dimethoxyethane, and the like. Specifically, an alcohol, more specifically, methanol, can be used, but is not limited thereto.

[0168] Positive charge can be imparted by reacting the porous artificial partner particle with an alkoxysilane having a basic group such as a nitrogen-containing group (e.g., an amino group or an aminoalkyl group). Specifically, N-[3-(trimethoxysilyl)propyl]ethylenediamine, N1-(3-trimethoxysilylpropyl)diethylenetriamine, (3- aminopropyl)trimethoxysilane, N-[3-(trimethoxysilyl)propyl]aniline, trimethoxy[3- (methylamino)propyl]silane, 3-(2-aminoethylamino)propyldimethoxymethylsilane, and the like can be used, but are not limited thereto.

[0169] Negative charge can be imparted by reacting the porous artificial partner particle with an alkoxysilane having an acidic group such as a carboxyl group, a sulfonic acid group, a thiol group, and the like. Specifically, (3-mercaptopropyl)trimethoxysilane can be used, but is not limited thereto.

[0170] Hydrophilicity can be imparted by reacting the porous artificial partner particle with an alkoxysilane having a hydrophilic group such as a hydroxyl group, a carboxyl group, an amino group, a carbonyl group, a mercapto group, a phosphoric acid group, a thiol group, an ammonium group, an ester group, an imide group, a thioimide group, a ketone group, an ether group, an indenyl group, a sulfonyl group, and a polyethylene glycol group, and the like. Specifically, N-[3-(trimethoxysilyl)propyl]ethylenediamine, N1-(3-trimethoxysilylpropyl)diethylenetriamine, (3- aminopropyl)trimethoxysilane, (3-mercaptopropyl)trimethoxysilane, trimethoxy[3- (methylamino)propyl]silane, 3-(2-aminoethylamino)propyldimethoxymethylsilane, and the like can be used, but are not limited thereto.

[0171] Hydrophobicity can be imparted by reacting the porous artificial partner particle with an alkoxysilane having a hydrophobic substituent such as a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, a substituted or unsubstituted C2 to C30 heteroaryl group, a halogen group, a C1 to C30 ester group, and a halogen-containing group, and the like. Specifically, trimethoxy(octadecyl)silane, trimethoxy-n-octylsilane, trimethoxy(propyl)silane, isobutyl(trimethoxy)silane, trimethoxy(7-octen-1-yl)silane, trimethoxy(3,3,3-trifluoropropyl)silane, trimethoxy(2-phenylethyl)silane, vinyltrimethoxysilane, cyanomethyl, 3-(trimethoxysilyl)propyl]trithiocarbonate, (3-bromopropyl)trimethoxysilane, and the like can be used, but are not limited thereto.

[0172] Further, in order to increase the binding force of the porous artificial partner particle to a poorly soluble (hydrophobic) polypeptide, a hydrophobic substituent can be present inside the pores of the particle. Further, in terms of ease of use and formulation, the surface of the particle can also be treated to have a hydrophilic substituent.

[0173] Further, the surface modification can be performed in combination. For example, the surface modification can be performed more than twice on the outer surface of the particles or inside the pores. As a specific example, a compound containing a carboxyl group can be bound to a silica particle into which an amino group is introduced through an amide bond, thereby changing the positively charged particles to have different surface properties, but is not limited thereto.

[0174] The reaction of the porous artificial companion particles with the alkoxysilane can be performed, for example, under heating. The heating can be performed at 80 to 180°C, for example, 80 to 160°C, 80 to 150°C, 100 to 160°C, 100 to 150°C, 110 to 150°C, etc., within the above range, but is not limited thereto.

[0175] The reaction of the porous artificial companion particles with the alkoxysilane can be performed for 4 to 20 hours, for example, 4 to 18 hours, 4 to 16 hours, 6 to 18 hours, 6 to 16 hours, 8 to 18 hours, 8 to 16 hours, 8 to 14 hours, 10 to 14 hours, etc., within the above range, but is not limited thereto.

[0176] The reaction temperature, time, and the amount of the compound for surface modification can be selected as desired according to the degree of surface modification. In other words, the reaction conditions will vary depending on the hydrophilicity, hydrophobicity, and charge level of the polypeptide. Specifically, by controlling the hydrophilicity, hydrophobicity, and / or charge level of the porous artificial companion particles, the release rate of the polypeptide can be controlled. For example, if the polypeptide has a strong negative charge at neutral pH, the reaction temperature can be increased, the reaction time can be extended, or the amount of the treated compound can be increased, thereby making the porous artificial companion particles have a strong positive charge, but is not limited thereto.

[0177] Further, the porous artificial companion particles of the present application can be manufactured by, for example, preparation of small-pore particles, pore expansion, surface modification, and interior pore modification.

[0178] The preparation of small-pore particles and the pore expansion can be performed by the above-described procedures, and, after the preparation of small-pore particles and after the pore expansion, a washing procedure and a drying procedure can be performed.

[0179] If necessary, the unreacted material can be separated before washing, and the separation of the unreacted material can be performed by separating the supernatant by centrifugation.

[0180] The centrifugation can be performed at 6,000 to 10,000 rpm, and the centrifugation time can be 3 to 60 minutes, for example, 3 to 30 minutes, 3 to 30 minutes, 5 to 30 minutes, etc., within the above range, but is not limited thereto.

[0181] The washing after the preparation of the small-pore particles can be performed by the method / conditions within the above-described range, but is not limited thereto.

[0182] The washing after the pore expansion can be performed under conditions looser than the above-described embodiments. For example, the washing can be performed three times or less, but is not limited thereto.

[0183] The surface modification and the pore interior modification can be performed by the above-described procedures, respectively. Herein, the surface modification and the subsequent pore interior modification can be performed sequentially, and a washing procedure can be further performed between the two procedures.

[0184] When the washing is performed under looser conditions after the preparation of the small-pore particles and the pore expansion, the reaction solution such as a surfactant used for the particle preparation and the pore expansion is filled in the pores, so that the interior of the pores is not modified during the surface modification, but only the surface of the particles can be modified. Thereafter, the reaction solution in the interior of the pores can be washed away and removed.

[0185] The particle washing between the surface modification procedure and the pore interior modification procedure can be performed using water and / or an organic solvent. Specifically, since different substances are dissolved in different solvents, the water and the organic solvent can be used alternately one or several times, or the washing can be performed one or several times using water or the organic solvent alone. The above-described several times can be 2 times or more and 10 times or less, for example, 3 times or more and 10 times or less, 4 times or more and 8 times or less, 4 times or more and 6 times or less.

[0186] The washing can be performed under centrifugation, for example, at 6,000 to 10,000 rpm, and the centrifugation time can be 3 to 60 minutes, for example, 3 to 30 minutes, 3 to 30 minutes, 5 to 30 minutes, etc. within the above-described range, but is not limited thereto.

[0187] Alternatively, the washing can be performed by passing the particles through a filter without centrifugation. The filter can have pores having a size smaller than or equal to the diameter of the porous artificial partner particles. When the reaction solution is filtered with such a filter as described above, only the particles are retained on the filter, which can be washed by pouring water and / or an organic solvent on the filter.

[0188] In the washing, the water and the organic solvent can be used alternately one or several times, or the washing can be performed one or several times using water or the organic solvent alone. The above-described several times can be 2 times or more and 10 times or less, for example, 3 times or more and 10 times or less, 4 times or more and 8 times or less, 4 times or more and 6 times or less.

[0189] The drying can be performed, for example, at 20 to 100°C, but is not limited thereto, and can also be performed under a vacuum state.

[0190] The polypeptide can be loaded on the surface of the porous artificial partner particle and / or inside the pores. In this context, the loading can be performed, for example, by mixing the porous artificial partner particle with the polypeptide in a solvent.

[0191] The solvent can be water and / or an organic solvent, and the solvent can include, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); halocarbons such as chloroform, dichloromethane, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride, 1,2-dibromoethane, and the like; ketones such as acetone, methyl isobutyl ketone, cyclohexanone, and the like; aromatic carbon-based materials such as benzene, toluene, xylene, and the like; alkyl amides such as N,N-dimethylformamide, N,N-dibutylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, and the like; alcohols such as methanol, ethanol, propanol, and butanol, and the like. Specifically, an alcohol, more specifically methanol, can be used, but is not limited thereto.

[0192] Further, PBS (phosphate buffered saline solution), SBF (simulated body fluid), borate buffered saline, tris buffered saline can be used as the solvent.

[0193] The polypeptide loaded on the porous artificial partner particle can be gradually released over an extended period of time. Such sustained release can be continuous or discontinuous, or linear or non-linear. Further, the release can vary depending on the properties of the porous artificial partner particle and / or the interaction between the porous artificial partner particle and the polypeptide.

[0194] The polypeptide loaded on the porous artificial partner particle is released when the porous artificial partner particle is biodegraded. Specifically, the porous artificial partner particle according to the present application is slowly degraded, thereby allowing the loaded polypeptide to be released in a sustained manner. Such release can be controlled, for example, by adjusting the surface area, particle diameter, pore diameter, substituents on the surface of the particle and / or inside the pores, surface density, and the like of the porous artificial partner particle, but is not limited thereto.

[0195] The polypeptide loaded on the porous artificial partner particle can be released while being separated from and diffusing from the porous artificial partner particle. Since the release is affected by the relationship between the porous artificial partner particle, the polypeptide, and the environment of the release, adjusting the relationship can control the release of the polypeptide. For example, the release of the polypeptide can be controlled by enhancing or weakening the binding force of the porous artificial partner particle to the polypeptide by means of surface modification.

[0196] More specifically, in the case where the loaded polypeptide is poorly soluble (hydrophobic), the surface of the particle and / or the inside of the pores have a hydrophobic substituent, thereby increasing the binding force of the porous artificial chaperone particle to the polypeptide, whereby the polypeptide can be released in a sustained manner. For example, the porous artificial chaperone particle can be surface-modified with an alkoxysilane having a hydrophobic substituent.

[0197] As used herein, the term "poorly soluble" means insoluble, almost insoluble, or only slightly soluble (in water), which is a term defined in "Pharmaceutical Science" 18th edition (published by U.S.P., Remington and Mack Publishing Company).

[0198] The poorly soluble substance can have, for example, a water solubility of less than 10 g / L, specifically less than 5 g / L, and more specifically less than 1 g / L at 1 atm and 25°C, but is not limited thereto.

[0199] When the loaded polypeptide is water-soluble (hydrophilic), the surface of the particle and / or the inside of the pores have a hydrophilic substituent, thereby increasing the binding force of the porous artificial chaperone particle to the polypeptide, whereby the polypeptide can be released in a sustained manner. For example, the porous artificial chaperone particle can be surface-modified with an alkoxysilane having a hydrophilic substituent.

[0200] For example, the water-soluble substance can have a water solubility of 10 g / L or more at 1 atm and 25°C, but is not limited thereto.

[0201] In the case where the loaded polypeptide is charged, the surface of the particle and / or the inside of the pores have an opposite charge, thereby increasing the binding force of the porous artificial chaperone particle to the polypeptide, whereby the polypeptide can be released in a sustained manner. For example, the porous artificial chaperone particle can be surface-modified with an alkoxysilane having an acidic group or a basic group.

[0202] Specifically, if the polypeptide is positively charged at neutral pH, the surface of the particle and / or the inside of the pores can be negatively charged at neutral pH, thereby increasing the binding force of the porous artificial chaperone particle to the polypeptide, whereby the polypeptide can be released in a sustained manner. For example, the porous artificial chaperone particle can be surface-modified with an alkoxysilane having an acidic group such as a carboxyl group (-COOH), a sulfonic acid group (-SO3H), or the like.

[0203] Further, if the polypeptide is negatively charged at neutral pH, the surface of the particle and / or the interior of the pores can be positively charged at neutral pH, thereby increasing the binding force of the porous artificial chaperone particle to the polypeptide, whereby the polypeptide can be released in a sustained manner. For example, the porous artificial chaperone particle can be surface modified with alkoxysilanes having basic groups such as amino groups or any other nitrogen-containing groups.

[0204] The polypeptide can be released for a period of, for example, 7 days to more than 1 year, depending on the type of treatment desired, the release environment, and the type of porous artificial chaperone particle to be used.

[0205] Further, if the porous artificial chaperone particle of the present application is biodegradable, it can be 100% degraded. Thus, the polypeptide loaded thereon can be 100% released.

[0206] The pharmaceutical composition of the present application can further comprise a pharmaceutically acceptable carrier, and can be formulated with such a carrier. As used herein, the term "pharmaceutically acceptable carrier" means a carrier or diluent that does not stimulate biological organisms and does not inhibit the biological activity and properties of the administered compound. In a composition formulated as a liquid solution, the acceptable pharmaceutical carrier is sterile and biocompatible, and can include saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components. In addition, if necessary, other usual additives such as antioxidants, buffers, and bacteriostatic agents can be added. Diluents, dispersants, surfactants, binders, and lubricants can also be added to formulate the pharmaceutical composition into injectable preparations such as aqueous solutions, suspensions, and emulsions, etc., pills, capsules, granules, or tablets.

[0207] The pharmaceutical composition of the present application is suitable for any formulation form, and can be prepared in oral or parenteral preparations. The pharmaceutical preparation of the present application can include a form suitable for oral administration, rectal administration, nasal administration, topical (including buccal and sublingual) administration, subcutaneous administration, vaginal administration, or parenteral (intramuscular, subcutaneous) administration. Alternatively, a form suitable for administration by inhalation or insufflation can also be included.

[0208] The pharmaceutical composition of the present application is administered in a pharmaceutically effective amount. The effective dose level can be determined depending on the type, severity of the patient's disease, activity of the drug, sensitivity to the drug, time of administration, route of administration and release rate, duration of treatment, factors including the simultaneous presence of other drugs, and other factors well known in the medical field. The pharmaceutical composition of the present application can be administered as a single therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered in a single dose or multiple doses. It is important to administer the pharmaceutical composition in an amount that can achieve the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art, taking into account all the above factors.

[0209] The dose of the pharmaceutical composition according to the present application can vary greatly depending on the weight, age, sex, health condition or diet of the patient, the time of administration, the method of administration, the excretion rate, and the severity of the disease, and the appropriate dose varies depending on, for example, the amount of drug accumulated in the patient's body and / or the specific efficacy of the carrier of the present application used. In general, the amount can be calculated based on the EC50 generally determined to be effective in vivo animal models and in vitro, for example, 0.01 pg to 1 g per kg of body weight. In addition, the pharmaceutical composition of the present application can be administered once or several times per unit time during a unit time period such as every day, every week, every month, or every year, or can be continuously administered for a long time using an infusion pump. The number of repeated administrations is determined taking into account the retention time of the drug in the body, the drug concentration in the body, etc. According to the course of treatment of the disease, the composition can be further administered to prevent recurrence, i.e., relapse of the disease, even after treatment.

[0210] In addition, the pharmaceutical composition of the present application can be formulated using any method known in the art to allow rapid release, sustained release, or delayed release of the active ingredient after administration to a mammal. The preparation can be produced in the form of a powder, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injection solutions, sterile powders.

[0211] Hereinafter, the present application will be described in detail with reference to the following examples.

[0212] Embodiment

[0213] Example 1. Preparation of artificial chaperone (ACPN) based on porous nanoparticles

[0214] 1. Preparation of porous artificial chaperone particles

[0215] (1) Preparation of porous artificial chaperone particles

[0216] 1) Preparation of small pore particles

[0217] Put 960 ml of distilled water (DW) and 810 ml of MeOH in a 2 L round bottom flask. Add 7.88 g of CTAB to the flask, and then rapidly add 4.52 ml of 1M NaOH under stirring. After adding the uniform mixture while stirring for 10 minutes, further add 2.6 ml of TMOS. After stirring for 6 hours to uniformly mix, the reaction solution is aged for 24 hours.

[0218] Then, the reaction solution is centrifuged at 8000 rpm and 25℃ for 10 minutes to remove the supernatant, centrifuged at 8000 rpm and 25℃ for 10 minutes, and washed with ethanol and distilled water alternately five times.

[0219] Thereafter, the resulting product is dried in an oven at 70℃ to harvest 1.5 g of powdery small-pore porous artificial mate particles (pore average diameter of 2 nm and particle size of 200 nm).

[0220] 2) Pore expansion

[0221] Add 1.5 g of small-pore porous artificial mate particle powder to 10 ml of ethanol and perform ultrasonic dispersion, and further add 10 ml of water and 10 ml of TMB (trimethylbenzene), and then perform ultrasonic dispersion.

[0222] Thereafter, the dispersion liquid is put in an autoclave, and reacted at 160℃ for 48 hours.

[0223] The reaction is started at 25℃ and proceeds while increasing the temperature at a rate of 10℃ / min, and then slowly cools at a rate of 1 to 10℃ / min in an autoclave.

[0224] Centrifuge the cooled reaction solution at 8000 rpm at 25℃ for 10 minutes to remove the supernatant, centrifuge at 8000 rpm at 25℃ for 10 minutes, and wash with ethanol and distilled water alternately five times.

[0225] Then, the product is dried in an oven at 70℃ to harvest powdery porous artificial mate particles (pore size of 10 to 15 nm, and particle size of 200 nm).

[0226] 3) Calcination

[0227] Put the porous artificial mate particles prepared in 2) in a glass vial, heat at 550℃ for 5 hours, and slowly cool to room temperature after the reaction is completed to prepare particles.

[0228] (2) Preparation of porous artificial mate particles

[0229] Porous artificial mate particles were prepared by the same method as in Example 1-1-(1) except that the reaction conditions at the time of pore expansion were changed to 140°C and 72 hours.

[0230] (3) Preparation of porous artificial mate particles (10 L scale)

[0231] Porous artificial mate particles were prepared by the same method as in Example 1-1-(1) except that a 5 times larger vessel was used and each substance was used in 5 times the amount.

[0232] (4) Preparation of porous artificial mate particles (particle size of 300 nm)

[0233] Porous artificial mate particles were prepared by the same method as in Example 1-1-(1) except that 920 ml of distilled water and 850 ml of methanol were used to prepare the small pore particles.

[0234] (5) Preparation of porous artificial mate particles (particle size of 500 nm)

[0235] Porous artificial mate particles were prepared by the same method as in Example 1-1-(1) except that 800 ml of distilled water, 1010 ml of methanol, and 10.6 g of CTAB were used to prepare the small pore particles.

[0236] (6) Preparation of porous artificial mate particles (particle size of 1000 nm)

[0237] Porous artificial mate particles were prepared by the same method as in Example 1-1-(1) except that 620 ml of distilled water, 1380 ml of methanol, and 7.88 g of CTAB were used to prepare the small pore particles.

[0238] (7) Preparation of porous artificial mate particles (pore size of 4 nm)

[0239] Porous artificial mate particles were prepared by the same method as in Example 1-1-(1) except that 2.5 ml of TMB was used for pore expansion.

[0240] (8) Preparation of porous artificial mate particles (pore size of 7 nm)

[0241] Porous artificial mate particles were prepared by the same method as in Example 1-1-(1) except that 4.5 ml of TMB was used for pore expansion.

[0242] (9) Preparation of porous artificial mate particles (pore size of 17 nm)

[0243] Porous artificial mate particles were prepared by the same method as in Example 1-1-(1) except that 11 ml of TMB was used for pore expansion.

[0244] (10) Preparation of porous artificial mate particles (pore diameter: 23 nm)

[0245] Porous artificial mate particles were prepared by the same method as in Example 1-1-(1) except that 12.5 ml of TMB was used for pore expansion.

[0246] (11) Preparation of porous artificial mate particles (double modification)

[0247] 1) Preparation of small-pore particles

[0248] Small-pore particles were prepared by the same method as in Example 1-1-(1)-1).

[0249] 2) Pore expansion

[0250] The small-pore particles were reacted with TMB, cooled, and centrifuged to remove the supernatant by the same method as in Example 1-1-(1)-2). Thereafter, the remaining solution was centrifuged under the same conditions as in Example 1-1-(1)-2), washed with ethanol and distilled water alternately three times, and then dried under the same conditions as in Example 1-1-(1)-2), thereby harvesting powder-like porous artificial mate particles (pore diameter: 10 to 15 nm, and particle diameter: 200 nm).

[0251] 3) Surface modification

[0252] After 0.8 g to 1 g of porous artificial mate particles having expanded pores were dispersed in 50 ml of toluene, 5 ml of (3-aminopropyl)triethoxysilane was added thereto, and then reflux-heated at 120°C for 12 hours. This process was followed by the washing and drying processes described above, and then 1 ml of triethylene glycol (PEG3, 2-[2-(2-methoxyethoxy)ethoxy]acetic acid), 100 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), and 200 mg of N-hydroxysuccinimide (NHS) were dispersed in 30 ml of PBS and allowed to react under stirring at room temperature for 12 hours. The product was then washed and dried.

[0253] Since the reaction solution of the previous step remains inside the pores, the inside of the pores is not modified.

[0254] 4) Washing the inside of the pores

[0255] 800 mg of the surface-modified particle powder was dissolved in 40 ml of 2M HCl / ethanol and refluxed for 12 hours under vigorous stirring.

[0256] Thereafter, the cooled reaction solution was centrifuged at 8000 rpm for 10 minutes to remove the supernatant, centrifuged at 8000 rpm and 25°C for 10 minutes, and washed with ethanol and distilled water alternately five times.

[0257] Thereafter, the product was dried in an oven at 70°C, thereby harvesting the powdered porous artificial companion particles.

[0258] 5) Modification of the inside of the pores

[0259] ① Propyl was introduced into the pores by the same method as in Example 1-2-(2)-1).

[0260] ② Octyl was introduced into the pores by the same method as in Example 1-2-(2)-2).

[0261] 2. Surface modification of the porous artificial companion particles

[0262] (1) Positively charged

[0263] 1) Particles with a particle size of 300 nm

[0264] The porous artificial companion particles of Example 1-1-(4) were reacted with (3- aminopropyl)triethoxysilane (APTES) to make them positively charged.

[0265] Specifically, 100 mg of the porous artificial companion particles were dispersed in 10 ml of toluene in a 100-ml round-bottom flask using a bath sonicator. Then, 1 ml of APTES was added and stirred at 400 rpm and 130°C for 12 hours.

[0266] After the reaction, the product was slowly cooled to room temperature and centrifuged at 8000 rpm for 10 minutes to remove the supernatant, further centrifuged at 8000 rpm and 25°C for 10 minutes, and then washed with ethanol and distilled water alternately five times.

[0267] Thereafter, the product was dried in an oven at 70°C to harvest the powdered porous artificial companion particles having an amino group on the surface of the particles and inside the pores.

[0268] 2) Particles with a particle size of 200 nm

[0269] ① The particles were made positively charged by reacting the porous artificial companion particles of Example 1-1-(1) with (3-aminopropyl)triethoxysilane (APTES), and the modification was performed in the same manner as in Example 1-2-(1)-1) except that 0.4 ml of APTES was added and the reaction time was 3 hours.

[0270] ② The porous artificial companion particles of Example 1-1-(9) were positively charged by reacting them with (3-aminopropyl)triethoxysilane (APTES) and modified in the same manner as in Example 1-2-(1)-1).

[0271] ③ The porous artificial companion particles of Example 1-1-(10) were positively charged by reacting them with (3-aminopropyl)triethoxysilane (APTES) and modified in the same manner as in Example 1-2-(1)-1).

[0272] (2) Introduction of hydrophobic groups

[0273] 1) Propyl

[0274] The porous artificial companion particles of Example 1-1-(1) were reacted with trimethoxy(propyl)silane to introduce propyl groups into the surface of the particles and the interior of the pores, and were modified in the same manner as in Example 1-2-(1), except that 0.35 ml of trimethoxy(propyl)silane was added instead of APTES and the reaction was carried out for 12 hours.

[0275] 2) Sinki

[0276] The porous artificial companion particles of Example 1-1-(1) were reacted with trimethoxy-n-octylsilane to introduce octyl groups into the surface of the particles and the interior of the pores. The modification was carried out in the same manner as in Example 1-2-(1), except that 0.5 ml of trimethoxy-n-octylsilane was added instead of APTES and the reaction was carried out for 12 hours.

[0277] (3) Carrying a negative charge

[0278] 1) Carboxyl group

[0279] The porous artificial companion particles of Example 1-1-(1) were negatively charged by reacting them with succinic anhydride. Furthermore, the charged particles were modified in the same manner as in Example 1-2-(1)-1), except that DMSO (dimethyl sulfoxide) was used instead of toluene, 80 mg of succinic anhydride was added instead of APTES to allow the reaction to proceed at room temperature with stirring for 24 hours, and DMSO was used instead of distilled water during washing.

[0280] 2) Thiol group

[0281] Except that 1.1 ml of MPTES was used instead of APTES, the particles were modified in the same manner as in Example 1-2-(1)-1).

[0282] 3) Sulfonic acid group

[0283] The porous silica nanoparticles of 100 mg of Example 1-2-(3)-2) were dispersed in 1 ml of 1 M aqueous sulfuric acid solution and 20 ml of 30% hydrogen peroxide solution, and stirred at room temperature to initiate the oxidation reaction to oxidize the thiol group to a sulfonic acid group. Thereafter, the product was washed and dried in the same manner as in Example 1-2-(1)-1).

[0284] 3. Identification of particle formation and pore expansion

[0285] The small-pore particles and the porous artificial partner particles prepared in Experimental Example 1-1-(1) to (3) were observed under a microscope to determine whether the small-pore particles were uniformly formed or the pores were sufficiently expanded to uniformly form the porous artificial partner particles. Figures 1 to 4 ).

[0286] Figure 1 are photographs of the porous artificial partner particles in Example 1-1-(1), and Figure 2 are photographs of the porous artificial partner particles in Example 1-1-(2), and it can be seen from these photographs that the spherical porous artificial partner particles having pores sufficiently expanded were uniformly formed.

[0287] Figure 3 are photographs of the small-pore particles in Example 1-1-(1), and Figure 4 are comparative photographs of the small-pore particles in Example 1-1-(1) and 1-1-(3), and it can be seen from these photographs that the spherical small-pore particles were uniformly formed.

[0288] 4. Calculation of BET surface area and pore volume

[0289] The surface area and pore volume of the small-pore particles in Example 1-1-(1) and the porous artificial partner particles of Example 1-1-(1), (7), (8), and (10) were calculated. The surface area was calculated by the Brunauer-Emmett-Teller (BET) method, and the pore size distribution was calculated by the Barrett-Joyner-Halenda (BJH) method.

[0290] Microphotographs of the particles are shown in Figure 5 , and the calculation results are shown in Table 1 below.

[0291] [Table 1]

[0292]

[0293] 5. Identification of biodegradability

[0294] To identify the biodegradability of the porous artificial companion particles in Example 1-1-(1), the biodegradability in SBF (pH 7.4) at 37°C was observed under a microscope at 0 hours, 120 hours, and 360 hours, and the results thereof are shown in FIG. 1. Figure 6

[0295] Referring to Figure 6 , it can be seen that the porous artificial companion particles are biodegraded and almost completely degraded after 360 hours.

[0296] 6. Measurement of absorbance ratio

[0297] The absorbance ratio over time was measured according to the following Equation 1.

[0298] [Equation 1]

[0299] A t / A0

[0300] (where A0 is the absorbance of the porous artificial companion particles measured by placing 5 ml of a suspension containing 1 mg / ml of the porous artificial companion particles in a cylindrical permeable membrane having pores with a pore diameter of 50 kDa,

[0301] 15 ml of the same solvent as the suspension was brought into contact with the outside of the permeable membrane, and the inside and outside of the permeable membrane were stirred horizontally at 60 rpm and 37°C, and

[0302] A t represents the absorbance of the porous artificial companion particles measured after t hours from the measurement of A0).

[0303] Specifically, 5 mg of the porous artificial companion particle powder was dissolved in 5 ml of SBF (pH 7.4). Thereafter, 5 ml of the porous artificial companion particle solution was placed in a permeable membrane having pores with a pore diameter of 50 kDa, shown in FIG. 2. 15 ml of SBF was added to the outside membrane, and the SBF of the outside membrane was replaced every 12 hours. The degradation of the porous artificial companion particles was performed at 37°C under horizontal stirring at 60 rpm. Figure 7

[0304] Then, the absorbance was measured by UV-visible spectroscopy and analyzed at λ = 640 nm.

[0305] (1) Measurement of absorbance ratio

[0306] The absorbance ratio of the porous artificial companion particles in Example 1-1-(1) was measured according to the above-described method, and the results thereof are shown in FIG. 1. Figure 8

[0307] Referring to Figure 8 ​​​It can be seen that t is about 58 hours at the time when the absorbance ratio reaches 1 / 2, proving very slow degradation.

[0308] (2) Particle diameter

[0309] The absorbance of the porous artificial partner particles in Examples 1-1-(1), (5), and (6) was measured according to the above Formula 1, and the results thereof are shown in Figure 9 (SBF was used as the suspension and solvent).

[0310] Referring to Figure 9 It can be seen that t decreases as the particle diameter increases.

[0311] (3) Average pore diameter

[0312] The absorbance of the porous artificial partner particles in Examples 1-1-(1) and (9) and the small-pore porous artificial partner particles in Example 1-1-(1) as a control was measured according to the above Formula 1, and the results thereof are shown in Figure 10 (SBF was used as the suspension and solvent).

[0313] Referring to Figure 10 It can be seen that the porous artificial partner particles of the present application embodiment have a significantly greater t than the control.

[0314] (4) pH

[0315] The absorbance of the porous artificial partner particles in Example 1-1-(4) was measured for each pH. The absorbance was measured in SBF and Tris at pH 2, 5, and 7.4, and the results thereof are shown in Figure 11 .

[0316] Referring to Figure 11 It can be seen that, although there are differences in t for each pH, t is 24 or more at the time when the absorbance ratio reaches 1 / 2 for all.

[0317] (5) Charge

[0318] The absorbance of the porous artificial partner particles in Example 1-2-(1)-1) was measured, and the results thereof are shown in Figure 12 (Tris (pH 7.4) was used as the suspension and solvent).

[0319] Referring to Figure 12 It can be seen that t is 24 or more at the time when the absorbance ratio of the positively charged particles reaches 1 / 2.

[0320] Example 2. Tethering of polypeptides

[0321] 1. Design of porous artificial partner particles and evaluation of properties

[0322] As porous artificial companion particles, the porous artificial companion particles of Example 1-1-1) which were not surface-modified, the particles of Example 1-2-2)-1) in which propyl groups were introduced, and the particles of Example 1-2-2)-2) in which octyl groups were introduced were used.

[0323] Each ACPN (10 mg) was suspended in 100 μl of Nile Red solution (1 mg / ml methanol solution) and stirred for 1 hour. The complexes were collected by centrifugation and washed several times with methanol and water alternately to remove Nile Red adsorbed to the surface of the particles. Each ACPN-Nile Red complex solution was prepared to have a constant Nile Red content using UV / vis absorption measurement. The fluorescence emission spectrum of Nile Red encapsulated in the pores inside the particles (λex= 510 nm) was measured and the relative emission intensity of each complex solution was compared.

[0324] It was confirmed by TEM images and dynamic light scattering (DLS) that there was no significant change in the particle size and size distribution before and after loading the p53 peptide (Formula 1) to the particles. Figure 14

[0325] [Formula 1]

[0326] FAM-GGQSQQTF*KNLWRLL**KQN-NH2

[0327] (*K: pentynoic acid conjugated with d-lysine, **K: 2-amino-5-azido-pentanoic acid)

[0328] 2. Evaluation of the degree of pegylation of the polypeptide according to the difference in surface modification

[0329] A CD spectrum was measured using a J-810 spectropolarimeter (JASCO, Japan). The spectrum was recorded at a wavelength ranging from 250 to 190 nm using a cuvette with a path length of 1 cm. The test was repeated five times and the average value was taken. The ACPN suspension concentration was 0.2 mg / ml (p53 concentration: 20 μΜ). All sample suspensions were allowed to stabilize at room temperature for at least 2 days before CD measurement. The spectrum was scanned at intervals of 10°C in the temperature range of 25 to 75°C, thereby performing a CD experiment with respect to temperature. The α-helix degree of the peptide was calculated using the CONTIN-LL algorithm.

[0330] When substituted with an octyl group, the highest α-helix level stability was shown. Figure 15

[0331] 3. Cell experiment

[0332] ​​Human hepatocellular carcinoma line HepG2 and human cervical carcinoma line HeLa were grown in DMEM containing 10% FBS and 1% penicillin / streptomycin under a humid atmosphere of 37°C and 5% CO2. After incubating each cell in a 4-well glass plate for 24 hours, ACPN (0.02 mg / ml) containing p53 and free clicked p53 (2 μM) were added to each well containing serum-free medium, and then incubated for 12 hours. Cells were carefully washed with 1×PBS and the medium was replaced with serum-containing medium. After staining the cell nuclei with Hoechst 33342, bright-field and fluorescence images were obtained using a DeltaVision Elite microscope system (GE Healthcare, USA). Cell nuclei were treated with ACPNP-C8 conjugated with TAMRA and containing clicked p53 (0.25 mg / ml), and the mander overlap coefficient was calculated using the JACoP plugin in ImageJ to confirm co-location of fluorescence signals over time. HepG2 and HeLa cells (1 × 10⁻⁶) were used. 4 Cells (10 cells / well) were incubated in 96-well plates for 24 hours, followed by the addition of various concentrations of ACPN and ACPN complex. After incubation, cells were carefully washed with 1×PBS. Cell viability was calculated using CCK-8 assay. EC50 values ​​were determined using logarithmic curve fitting from Origin (OriginLab, USA).

[0333] Caspase-3 activity assays were performed to identify the role of ACPNP-C8@clicked p53 in inducing apoptosis. HepG2 cells were treated with ACPNP-C8, ACPNP-C8@clicked p53 (12.5 μM, 25 μM), and PBS for 4 h each, followed by incubation in fresh culture medium for 20 h. Cells were washed, treated with lysis buffer (Promega, USA), and centrifuged at 4 °C for 10 min. The collected lysates were incubated in a humidified incubator at 37 °C with caspase-3 substrate (Ac-DEVD-pNA, 100 μM) for 2 h. Fluorescence intensity was then measured (λex = 400 nm, λem = 505 nm).

[0334] Reference Figure 16 It can be seen that the p53 of ACPNP-C8@click has significantly superior activity.

[0335] 4. In vivo experiments

[0336] Five-week-old male Balb / c nude mice were subcutaneously injected with HepG2 cells (6 × 10⁻⁶). 6suspension of cells, and the tumor-bearing mice were raised to a tumor volume of about 50 mm 3 To investigate the tumor growth inhibition efficacy, ACPN-C8@clicked p53, ACPN-C8 and clicked p53 (50 mg ACPN-C8 / kg, 5 μmol clicked p53 / kg) were injected directly into the tumor (IT injection) in 100 μl of 1 x PBS solution, respectively. An equal volume of 1 x PBS solution was also injected directly into the tumor as a control. The intratumoral injection was performed at 5-day intervals, and the changes in tumor volume and body weight were monitored for 12 days. The tumor volume was calculated relative to the initial tumor volume. For the imaging experiment, TAMRA-labeled ACPN-C8 instead of ACPN-C8 was processed in the same manner as described above. Fluorescent images of the mice were obtained over time in 3 days using an in vivo imaging system, FoBI (Neoscience, Korea).

[0337] Referring to Figure 17 It can be seen that ACPNP-C8@clicked p53 has a significantly superior activity.

[0338] 5. Polypeptide release

[0339] The experiment for releasing the clicked p53 peptide from ACPN-C8 was performed in PBS buffer and PBS buffer containing 10% FBS, respectively. ACPN-C8 (1 mg / ml) loaded with clicked p53 was each dispersed in PBS buffer solution and PBS buffer solution containing 10% FBS, and then incubated at 37°C under stirring (N=3). At each time point, the supernatant was collected by centrifugation, and the fluorescence intensity of FAM-labeled clicked p53 from the supernatant (λex=480 nm, λem=520 nm) was measured to calculate the concentration of the released peptide.

[0340] Referring to Figure 18 It can be seen that the release rate of the polypeptide is significantly slower in the buffer solution containing no FBS.

Claims

1. A method for stabilizing polypeptides in vitro, comprising: The step of mixing porous artificial chaperone particles with an average pore size of 5 nm to 100 nm with a peptide including an α-helical motif to load the peptide inside the particles. The porous artificial companion particles are silica particles. The porous artificial companion particles have an average diameter of 150 nm to 1000 nm. The porous artificial companion particles have hydrophobic substituents inside the pores.

2. The method for stabilizing polypeptides in vitro according to claim 1, wherein, The method further includes the step of stapled with the loaded polypeptide.

3. The method for stabilizing polypeptides in vitro according to claim 1, wherein, To stabilize the loaded polypeptide, the α-helix structure is formed or maintained.

4. The method for stabilizing polypeptides in vitro according to claim 1, wherein, The porous artificial companion particles have a BET surface area of ​​200 m². 2 / g to 700m 2 / g, and the volume of each pore is 0.7ml to 2.2ml.

5. The method for stabilizing polypeptides in vitro according to claim 1, wherein, The porous artificial companion particles are prepared by a method comprising the following steps: reacting silica particles having pores smaller than 5 nm with an expander at 120°C to 180°C for 24 to 96 hours to expand the pores smaller than 5 nm; and calcining the expanded silica particles at a temperature above 400°C for 3 hours or more.

6. The method for stabilizing polypeptides in vitro according to claim 1, wherein, When the absorbance ratio of the porous artificial companion particles in Formula 1 reaches 1 / 2, t is 24 or higher: [Formula 1] TO t / A0 (In the formula, A0 is the absorbance of the porous artificial companion particles, measured by placing 5 ml of a suspension containing 1 mg / ml of the porous artificial companion particles in a cylindrical permeation membrane with a pore size of 50 kDa.) 15 ml of the same solvent as the suspension is located outside and in contact with the permeation membrane, and the inside and outside of the permeation membrane are horizontally stirred at 60 rpm at 37°C. The pH of the suspension is 7.

4. A t This represents the absorbance of the porous artificial companion particle measured after "t" hours from the start of measurement A0.

7. The method for stabilizing polypeptides in vitro according to claim 1, wherein, The porous artificial companion particles have hydrophilic substituents on their outer surface.

8. The method for stabilizing polypeptides in vitro according to claim 1, wherein, The polypeptide including the α-helical motif is selected from at least one of the following groups: Bcl-2 family protein inhibitors, MDM2 / MDMX inhibitors, HIV envelope protein gp41 inhibitors, epidermal growth factor receptor inhibitors, Kirsten rat sarcoma virus oncogene homolog (KRAS) inhibitors, Rab (brain Ras-associated) inhibitors, and myoA (malaria invasion motor myosin) tail interacting protein (MTIP) inhibitors.

9. The method for stabilizing polypeptides in vitro according to claim 1, wherein, The loading is a randomly coiled polypeptide.

10. The method for stabilizing polypeptides in vitro according to claim 1, wherein, The polypeptide has a length of 10 to 18 amino acids.

11. The method for stabilizing polypeptides in vitro according to claim 1, wherein, The polypeptide is p53.

12. The method for stabilizing polypeptides in vitro according to claim 11, wherein, The p53 is defined by the following formula: FAM-GGQSQQTF*KNLWRLL**KQN-NH2 Wherein, *K: pentynic acid conjugated with d-lysine, and **K: 2-amino-5-azido-pentanoic acid.

13. A method for stapled polypeptides performed in vitro, comprising: The step of mixing porous artificial chaperone particles with an average pore size of 5 nm to 100 nm with a peptide including an α-helical motif to load the peptide inside the pores; and The step of pinning the loaded peptide via a click reaction. The porous artificial companion particles are silica particles. The porous artificial companion particles have an average diameter of 150 nm to 1000 nm. The porous artificial companion particles have hydrophobic substituents inside the pores.

14. The method for anchoring polypeptides in vitro according to claim 13, wherein, The polypeptide has a length of 10 to 18 amino acids.

15. The method for anchoring polypeptides in vitro according to claim 13, wherein, The polypeptide is p53.

16. The method for anchoring polypeptides in vitro according to claim 15, wherein, The p53 is defined by the following formula: FAM-GGQSQQTF*KNLWRLL**KQN-NH2 Wherein, *K: pentynic acid conjugated with d-lysine, and **K: 2-amino-5-azido-pentanoic acid.

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