Drug delivery system comprising mesoporous silica nanoparticles

Abstract

The present invention relates to a drug carrier comprising mesoporous silica nanoparticles and, more specifically, to a carrier comprising mesoporous silica nanoparticles and a bioactive component, wherein the mesoporous silica nanoparticles have a predetermined diameter and surface zeta potential, and the bioactive component is a peptide containing a fatty acid chain. The carrier comprising mesoporous silica nanoparticles of the present invention effectively encapsulates peptides containing fatty-acid chains, and exhibits excellent intracellular delivery and release efficiency of the bioactive ingredient. Accordingly, effective drug delivery can be achieved with a small amount of the carrier.

Description

Drug delivery system comprising mesoporous silica nanoparticles

[0001] The present invention relates to a drug delivery system comprising mesoporous silica nanoparticles.

[0002] Mesoporous silica nanoparticles (MSNs) are nanomaterials with high surface area and a porous structure, enabling them to effectively carry various bioactive substances. MSNs are widely used as carriers in drug delivery systems (DDSs). Their biocompatibility, high physicochemical stability, and surface multifunctionality have led to their application in various drug delivery routes, including oral, intravenous, subcutaneous, and topical administration.

[0003] Peptides are bioactive ingredients with excellent biological activity. However, most peptide-based drugs, such as GLP-1 receptor agonists (GLP-1RAs) commercially available, are administered as injections, limiting patient convenience. In particular, oral administration of peptides is easily altered or degraded by saliva in the mouth or the harsh environment of the gastrointestinal tract (pH, enzymes, etc.), and absorption in the body is severely limited due to the intestinal wall's permeability barrier. Furthermore, the structural characteristics of peptides often result in low encapsulation efficiency within delivery systems, reducing bioavailability. This necessitates frequent administration of large doses, which can lead to side effects such as vomiting. To address this issue, gastrointestinal mucosal penetrants are sometimes used in combination. However, these adjuvants themselves can cause side effects, and the high doses required for peptide administration increase manufacturing costs.

[0004] In addition, peptides are sometimes designed to be combined with fatty acids for the purpose of improving bioavailability and extending half-life. However, in such cases, encapsulation into porous silica nanoparticles is difficult due to the three-dimensional structural characteristics of the peptide or increased molecular weight, which ultimately limits the use of porous nanoparticles as drug delivery vehicles.

[0005] Therefore, a delivery system designed to not only effectively encapsulate peptides but also efficiently uptake them into cells and release them in a therapeutically optimized manner is needed. The present inventors have discovered a novel drug delivery system capable of simultaneously achieving high encapsulation efficiency, intracellular uptake, and controlled release of peptides, thereby completing the present invention.

[0006] The purpose of the present invention is to provide a carrier capable of delivering a drug effectively even in a small amount by having a high encapsulation efficiency of a bioactive ingredient.

[0007] The purpose of the present invention is to provide a delivery vehicle in which a bioactive ingredient is encapsulated with high efficiency, while at the same time effectively delivering the bioactive ingredient into cells and releasing the bioactive ingredient.

[0008] 1. A carrier comprising mesoporous silica nanoparticles and a bioactive ingredient, wherein the mesoporous silica nanoparticles have a diameter of 20 to 300 nm and a zeta potential of -15 to 60 mV, and the bioactive ingredient is a peptide comprising a fatty acid chain.

[0009] 2. In the above 1, the peptide containing a fatty acid chain is any one selected from the group consisting of semaglutide, tirzepatide, retatrutide, survodutide, liraglutide, cotadutide, cagrilintide, insulin detemir, tesamorelin, daptomycin, polymyxin B, insulin degludec, somapacitan and MEDI0382.

[0010] 3. A carrier in the above 1, wherein the diameter of the mesoporous silica nanoparticles is 30 to 150 nm.

[0011] 4. A carrier having a zeta potential of mesoporous silica nanoparticles of the above 1, which is -13 to 55 mV.

[0012] 5. In the above 1, the mesoporous silica nanoparticles are spherical, carriers.

[0013] 6. In the above 1, the mesoporous silica nanoparticles are spherical in shape with protrusions formed on the surface, and are a carrier.

[0014] 7. A carrier having an encapsulation efficiency of a bioactive ingredient within the carrier of 1 above of 40% or more.

[0015] 8. A carrier having a release rate of bioactive ingredients of 40% or more in the above 1.

[0016] 9. In the above 1, a carrier in which the bioactive ingredient is encapsulated in the pores of mesoporous silica nanoparticles.

[0017] 10. In the above 1, the weight ratio of the bioactive ingredient and the mesoporous silica nanoparticles is 1:1 to 10, the carrier.

[0018] 11. A carrier comprising mesoporous silica nanoparticles containing lysine and a bioactive ingredient, wherein the mesoporous silica nanoparticles have a diameter of 20 to 300 nm and a zeta potential of -15 to 60 mV, and the bioactive ingredient is a peptide containing a fatty acid chain.

[0019] 12. In the above 11, the peptide containing a fatty acid chain is any one selected from the group consisting of semaglutide, tirzepatide, retatrutide, survodutide, liraglutide, cotadutide, cagrilintide, insulin detemir, tesamorelin, daptomycin, polymyxin B, insulin degludec, somapacitan and MEDI0382.

[0020] 13. A carrier according to 11 above, wherein the diameter of the mesoporous silica nanoparticles is 30 to 150 nm.

[0021] 14. A carrier according to the above 11, wherein the diameter of the mesoporous silica nanoparticles is 30 to 80 nm.

[0022] 15. In the above 11, a carrier having a zeta potential of mesoporous silica nanoparticles of -5 to 55 mV.

[0023] 16. A carrier having a zeta potential of mesoporous silica nanoparticles of 11 above, of 5 to 55 mV.

[0024] 17. In the above 11, the mesoporous silica nanoparticles are carriers having a diameter of 30 to 70 nm and a zeta potential of 5 to 55 mV.

[0025] 18. A carrier having an encapsulation efficiency of a bioactive ingredient within the carrier of 11 above of 40% or more.

[0026] 19. A carrier having a release rate of bioactive ingredients of 40% or more in the above 11.

[0027] 20. In the above 11, the mesoporous silica nanoparticles are spherical, carriers.

[0028] 21. In the above 11, the mesoporous silica nanoparticles are spherical in shape with protrusions formed on the surface, and are a carrier.

[0029] 22. In the above 11, a carrier in which the bioactive ingredient is encapsulated in the pores of mesoporous silica nanoparticles.

[0030] 23. In the above 11, the weight ratio of the bioactive ingredient and the mesoporous silica nanoparticles is 1:1 to 10, the carrier.

[0031] The delivery system of the present invention encapsulates a bioactive ingredient with high efficiency, enabling effective drug delivery even in small amounts.

[0032] The delivery system of the present invention is effectively delivered into cells, and the release of bioactive ingredients is also smoothly achieved.

[0033] The delivery system of the present invention can maximize drug delivery efficiency by encapsulating a high content of bioactive ingredients while simultaneously exhibiting excellent release efficiency at a target site.

[0034] Figures 1a to 1n are electron microscope photographs of mesoporous silica nanoparticles according to a manufacturing example of the present invention.

[0035] Figure 2 shows the results of a fluorescent microscope analysis to confirm whether mesoporous silica nanoparticles according to the manufacturing example of the present invention are delivered into cells.

[0036] Figure 3 shows the results of encapsulating various peptides into a carrier according to the present invention.

[0037] Figures 4a to 4h compare the encapsulation results of various peptides depending on whether the mesoporous silica nanoparticles of the present invention contain lysine.

[0038] Figures 5a and 5b show the results of encapsulating semaglutide into a delivery vehicle according to the present invention.

[0039] Figures 6a and 6b show the results of encapsulating servo-dutide into a carrier according to the present invention.

[0040] Figures 7a and 7b show the results of encapsulating letatrutide into a carrier according to the present invention.

[0041] Figures 8a and 8b show the results of encapsulating tert-butyl ...

[0042] Figure 9 shows the results of analysis of the release of a bioactive component using a carrier according to the present invention.

[0043] Figures 10a and 10b show the results of confirming whether the mesoporous silica nanoparticles of the present invention are delivered into the body.

[0044] The present invention provides a drug delivery system comprising mesoporous silica nanoparticles.

[0045] The present invention relates to a drug delivery system comprising mesoporous silica nanoparticles, and more particularly, to a delivery system comprising mesoporous silica nanoparticles and a bioactive ingredient, wherein the mesoporous silica nanoparticles have a predetermined diameter and surface zeta potential, and the bioactive ingredient is a peptide comprising a fatty acid chain. The delivery system comprising mesoporous silica nanoparticles and a bioactive ingredient of the present invention effectively encapsulates a peptide comprising a fatty acid chain, and has excellent efficiency in delivery into cells and release of the bioactive ingredient.

[0046] The present invention provides a carrier comprising mesoporous silica nanoparticles and a bioactive ingredient.

[0047] “Mesoporous Silica Nanoparticles (MSN)” are nano-carriers based on silica particles, characterized by a high surface area and porous structure. The term “mesoporous” refers to a large number of pores with a diameter of 2 to 50 nm. Mesoporous silica nanoparticles have a core-shell structure, with the core portion being made of high-density silica to provide stability, and the shell portion having a porous structure to support or encapsulate substances.

[0048] The mesoporous silica nanoparticles of the present specification have a predetermined size and surface charge, and depending on the size and charge of the particles, the interaction with the bioactive ingredient encapsulated in the pores of the nanoparticles is affected, thereby changing the encapsulation rate and release rate of the bioactive ingredient.

[0049] The mesoporous silica nanoparticles may be round, or may have a basically spherical shape but may have star-shaped protrusions or spikes formed on a portion of the surface. A structure with such protrusions formed on the surface may be referred to as a “virus-like shape.” The shape of the mesoporous silica nanoparticles can change the encapsulation rate of the bioactive ingredient loaded on the particle by controlling the surface area of ​​the particle.

[0050] As used herein, the term “bioactive ingredient” means a substance intended to be delivered into the body by being encapsulated in the pores of the delivery vehicle of the present invention, and the bioactive ingredient of the present invention is a peptide containing a fatty acid chain.

[0051] The peptide containing the above fatty acid chain refers to a molecule having a structure in which a fatty acid is bonded to a peptide chain, and the fatty acid chain is a carbon chain structure having a carboxyl group at the terminal, and may include a single fatty acid, a fatty diacid, or more fatty acids. The fatty acid may be introduced into the peptide through various methods such as acylation and PEGylation, but is not limited to these methods.

[0052] The peptide containing the fatty acid chain may be, for example, at least one selected from the group consisting of Semaglutide, Tirzepatide, Retatrutide, Survodutide, Liraglutide, Cotadutide, Cagrilintide, Insulin detemir, Tesamorelin, Daptomycin, Polymyxin B, Insulin Degludec, Somapacitan, and MEDI0382.

[0053] The bioactive ingredient of the present invention may be encapsulated in the pores of mesoporous silica nanoparticles.

[0054] The peptide containing the above fatty acid chain can be effectively encapsulated into the pores of mesoporous silica nanoparticles using electrostatic interactions.

[0055] Mesoporous silica nanoparticles generally have a negative charge, but the charge may vary depending on the environment in which the carrier containing the mesoporous silica nanoparticles exists, and the charge may also be affected by the amount of peptide encapsulated in the mesoporous silica nanoparticles or the size of the nanoparticles. Therefore, in order to effectively encapsulate, deliver, and release the bioactive ingredient of the present invention, the size and surface charge of the mesoporous silica nanoparticles must be controlled within an appropriate range.

[0056] The mesoporous silica nanoparticles of the present invention may have a diameter of 20 to 300 nm. Particles with a diameter smaller than 20 nm may have a small surface area, which may reduce the amount of peptide loaded, and may shorten the retention time in the body, which may reduce the delivery efficiency of the bioactive ingredient. In addition, if the diameter of the nanoparticles is small, the surface curvature increases, which changes the spatial arrangement and ionization degree of the surface functional groups, making it difficult to stably implement a charge.

[0057] Nanoparticles with a diameter greater than 300 nm have limited cellular uptake and may exhibit reduced delivery efficiency of bioactive ingredients relative to their internal volume. Furthermore, larger nanoparticle diameters result in a relatively low functional group density per unit surface area, which reduces surface modification efficiency and makes it difficult to ensure uniformity and reproducibility of surface charge. Therefore, nanoparticle size must be appropriately controlled to ensure a desired charge range.

[0058] The diameter of the mesoporous silica nanoparticles of the present invention may be 20 to 300 nm, for example, 20 to 300 nm, 23 to 300 nm, 23 to 250 nm, 25 to 250 nm, 30 to 250 nm, 30 to 230 nm, 30 to 200 nm, 30 to 170 nm, 30 to 150 nm, 30 to 130 nm, 30 to 100 nm, 30 to 80 nm, 30 to 70 nm, 30 to 60 nm, 50 to 250 nm, 70 to 250 nm, 100 to 250 nm, 100 to 230 nm, 100 to 200 nm or 130 to 200 nm, the ranges including the end values ​​of each range.

[0059] In one embodiment, a carrier comprising mesoporous silica nanoparticles having a diameter of 143 nm encapsulated more than 50% of the bioactive ingredient.

[0060] In one embodiment, a carrier comprising mesoporous silica nanoparticles having a diameter of 50 nm encapsulated more than 90% of the bioactive ingredient.

[0061] The mesoporous silica nanoparticles of the present invention may have a surface zeta potential of -15 to 60 mV.

[0062] If the surface potential of a nanoparticle is too negative, it may bind excessively to the encapsulated bioactive ingredient, slowing or impeding release. This can result in reduced delivery rates even if the ingredient is encapsulated. Alternatively, the encapsulation efficiency itself may be reduced due to repulsion between the nanoparticle and the bioactive ingredient.

[0063] If the surface potential of the nanoparticle is too large and positive, it may interact strongly with the negatively charged cell membrane, causing cytotoxicity. When encapsulating a positively charged bioactive ingredient, the encapsulation efficiency may be reduced due to electrical repulsion, or the release of the encapsulated bioactive ingredient may be inhibited due to strong binding with a negatively charged bioactive ingredient.

[0064] The zeta potential of the mesoporous silica nanoparticles of the present invention may be -15 to 60 mV, for example -15 to 60 mV, -15 to 55 mV, -13 to 55 mV, -10 to 55 mV, -5 to 55 mV, 0 to 55 mV, 5 to 55 mV, 5 to 50 mV, 5 to 40 mV, 5 to 35 mV, or 5 to 30 mV, the ranges including the end values ​​of each range.

[0065] In one embodiment, a carrier comprising mesoporous silica nanoparticles having a zeta potential of -10.9 mV and a diameter of 34.3 nm encapsulated more than 50% of the bioactive ingredient.

[0066] As previously mentioned, the most appropriate charge range may vary depending on the size (diameter) of the mesoporous silica nanoparticles. Furthermore, the size and potential of mesoporous silica nanoparticles are not independent variables in drug delivery systems, but rather are interrelated and require optimization. Furthermore, the surface potential and size characteristics of the nanoparticles may vary depending on the type and nature of the bioactive ingredient being encapsulated, necessitating adjustments to physicochemical conditions accordingly.

[0067] In one embodiment, mesoporous silica nanoparticles having a diameter of 20 to 30 nm, nanoparticles having a potential of about 40 mV encapsulated twice as much semaglutide as nanoparticles having a potential of -20 mV.

[0068] The carrier according to the present invention has excellent encapsulation efficiency of a bioactive ingredient within the carrier, and the encapsulation efficiency of the bioactive ingredient of the present invention within the carrier may be 40% or more.

[0069] In this specification, the encapsulation efficiency may refer to a value indicating how much of a bioactive ingredient is actually encapsulated in mesoporous silica nanoparticles. The encapsulation efficiency may refer to loading efficiency (%), i.e., the ratio of the bioactive ingredient actually encapsulated in the carrier among the total bioactive ingredient (API, Active Pharmaceutical Ingredient) injected.

[0070] In one embodiment, the encapsulation efficiency was confirmed by setting the mass ratio of API and mesoporous silica nanoparticles to 1:5.

[0071] Encapsulation of the bioactive ingredient into the carrier is mostly completed within 1 hour, but the encapsulation efficiency was confirmed after the reaction was carried out for 2 to 4 hours for stable encapsulation and testing under saturated conditions.

[0072] The encapsulation efficiency of the bioactive component in the delivery vehicle according to the present invention may be 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[0073] In one embodiment, the carrier according to the present invention encapsulates at least 50% of a peptide comprising a fatty acid chain.

[0074] The delivery system according to the present invention exhibits high encapsulation efficiency of bioactive ingredients, thereby reducing the total amount of silica (mesoporous silica nanoparticles) required to deliver the same drug dose. This reduces the volume or dosage unit of the final formulation, ultimately improving patient convenience and enhancing medication compliance when administered as a film, oral, or injectable formulation.

[0075] The carrier according to the present invention has excellent release of a bioactive ingredient from the carrier, and the release rate of the bioactive ingredient of the present invention from the carrier can be 40% or more.

[0076] The release rate of the bioactive ingredient of the present invention from the carrier may be 40% or more, 45% or more, 50% or more, 55% or more, or 60% or more.

[0077] In this specification, “release” may mean a process in which a bioactive ingredient encapsulated in mesoporous silica nanoparticles is separated from the nanoparticles over time in a release solution that mimics a living organism or moves into the body environment or is delivered to a target tissue due to external stimuli such as changes in the environment, such as pH or enzymes.

[0078] In this specification, “release rate” refers to the ratio of the bioactive ingredient released outside the carrier within a certain period of time based on the total amount of the bioactive ingredient actually encapsulated in the nanoparticle during the release process.

[0079] In one embodiment, the release characteristics of the bioactive ingredient were evaluated under conditions simulating the in vivo environment. Preferably, the carrier of the present invention was suspended in a solution having a pH of 6 to 8, reacted for 12 hours, and then the supernatant was collected to quantify the amount of the released bioactive ingredient.

[0080] The weight ratio of the bioactive ingredient and the mesoporous silica nanoparticles may be 1:1 to 10, for example, 1:1 to 10, 1:2 to 9, 1:3 to 8, 1:3 to 7, 1:4 to 7 or 1:4 to 6.

[0081] Since the carrier according to the present invention has a high bioactive ingredient encapsulation efficiency, a smaller amount of the carrier can be used to deliver the same amount of bioactive ingredient.

[0082] The weight ratio of the above bioactive ingredient and the above mesoporous silica nanoparticles may vary depending on the type of the bioactive ingredient.

[0083]

[0084] In addition, the present invention provides a drug delivery system comprising mesoporous silica nanoparticles containing lysine and a bioactive ingredient.

[0085] “Mesoporous Silica Nanoparticles (MSN)” are nano-carriers based on silica particles, characterized by a high surface area and porous structure. The term “mesoporous” refers to a large number of pores with a diameter of 2 to 50 nm. Mesoporous silica nanoparticles have a core-shell structure, with the core portion being made of high-density silica to provide stability, and the shell portion having a porous structure to support or encapsulate substances.

[0086] The mesoporous silica nanoparticles of the present specification have a predetermined size and surface charge, and depending on the size and charge of the particles, the interaction with the bioactive ingredient encapsulated in the pores of the nanoparticles is affected, thereby changing the encapsulation rate and release rate of the bioactive ingredient.

[0087] The mesoporous silica nanoparticles may be round, or may have a basically spherical shape but may have star-shaped protrusions or spikes formed on a portion of the surface. A structure with such protrusions formed on the surface may be referred to as a “virus-like shape.” The shape of the mesoporous silica nanoparticles can change the encapsulation rate of the bioactive ingredient loaded on the particle by controlling the surface area of ​​the particle.

[0088] As used herein, the term “bioactive ingredient” means a substance intended to be delivered into the body by being encapsulated in the pores of the delivery vehicle of the present invention, and the bioactive ingredient of the present invention is a peptide containing a fatty acid chain.

[0089] The peptide containing the above fatty acid chain refers to a molecule having a structure in which a fatty acid is bonded to a peptide chain, and the fatty acid chain is a carbon chain structure having a carboxyl group at the terminal, and may include a single fatty acid, a fatty diacid, or more fatty acids. The fatty acid may be introduced into the peptide through various methods such as acylation and PEGylation, but is not limited to these methods.

[0090] The peptide containing the fatty acid chain may be, for example, at least one selected from the group consisting of Semaglutide, Tirzepatide, Retatrutide, Survodutide, Liraglutide, Cotadutide, Cagrilintide, Insulin detemir, Tesamorelin, Daptomycin, Polymyxin B, Insulin Degludec, Somapacitan, and MEDI0382.

[0091] The bioactive ingredient of the present invention may be encapsulated in the pores of mesoporous silica nanoparticles.

[0092] The above mesoporous silica nanoparticles may contain lysine.

[0093] In the present specification, lysine may be added during the manufacturing process of mesoporous silica nanoparticles to be used as a charge control agent or stabilizer, or may be added for surface modification. The lysine may be bound to the mesoporous silica nanoparticles or may be contained within the mesoporous silica nanoparticles themselves, but is not limited thereto.

[0094] Mesoporous silica nanoparticles containing more lysine can have wider pores, and the negative charge of the mesoporous nanoparticles themselves can be converted to positive charge or increased in some positive charge direction.

[0095] Meanwhile, when a fatty acid chain as a bioactive component is conjugated to a peptide, the fatty acid can be bound to the lysine residue of the peptide through acylation or pegylation, etc., thereby extending the half-life of the peptide or increasing its structural stability, thereby improving its bioactivity. When a peptide containing a fatty acid is encapsulated in mesoporous silica nanoparticles containing lysine, the encapsulation of the peptide can be promoted by the competitive action between the lysine of the mesoporous silica nanoparticles and the lysine residues present in the peptide, and the peptide encapsulation can be further controlled by, but is not limited to, pH adjustment, use of organic solvents such as Dimethyl sulfoxide (DMSO) or N,N-Dimethylformamide (DMF), or use of amine coupling agents such as 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-Hydroxysuccinimide (NHS), or sulfo-NHS.

[0096] Mesoporous silica nanoparticles containing lysine can further enhance the encapsulation efficiency of bioactive ingredients. Meanwhile, even if lysine is included in mesoporous silica nanoparticles, the size and surface potential of the mesoporous silica nanoparticles should preferably be maintained within a certain range to ensure stable encapsulation and effective release of the bioactive ingredient simultaneously.

[0097] In one embodiment, mesoporous silica nanoparticles comprising lysine encapsulated a greater amount of bioactive components than mesoporous silica nanoparticles not comprising lysine.

[0098] The mesoporous silica nanoparticles of the present invention may have a diameter of 20 to 300 nm. Particles with a diameter smaller than 20 nm may have a small surface area, which may reduce the amount of peptide loaded, and may shorten the retention time in the body, which may reduce the delivery efficiency of the bioactive ingredient. In addition, if the diameter of the nanoparticles is small, the surface curvature increases, which changes the spatial arrangement and ionization degree of the surface functional groups, making it difficult to stably implement a charge.

[0099] Nanoparticles with a diameter greater than 300 nm have limited cellular uptake and may exhibit reduced delivery efficiency of bioactive ingredients relative to their internal volume. Furthermore, larger nanoparticle diameters result in a relatively low functional group density per unit surface area, which reduces surface modification efficiency and makes it difficult to ensure uniformity and reproducibility of surface charge. Therefore, nanoparticle size must be appropriately controlled to ensure a desired charge range.

[0100] The diameter of the mesoporous silica nanoparticles of the present invention may be 20 to 300 nm, for example, 20 to 300 nm, 23 to 300 nm, 23 to 250 nm, 25 to 250 nm, 30 to 250 nm, 30 to 230 nm, 30 to 200 nm, 30 to 170 nm, 30 to 150 nm, 30 to 130 nm, 30 to 100 nm, 30 to 80 nm, 30 to 70 nm, 30 to 60 nm, 50 to 250 nm, 70 to 250 nm, 100 to 250 nm, 100 to 230 nm, 100 to 200 nm or 130 to 200 nm, the ranges including the end values ​​of each range.

[0101] The mesoporous silica nanoparticles of the present invention may have a surface zeta potential of -15 to 60 mV.

[0102] If the surface potential of a nanoparticle is too negative, it may bind excessively to the encapsulated bioactive ingredient, slowing or impeding release. This can result in reduced delivery rates even if the ingredient is encapsulated. Alternatively, the encapsulation efficiency itself may be reduced due to repulsion between the nanoparticle and the bioactive ingredient.

[0103] If the surface potential of the nanoparticle is too large and positive, it may interact strongly with the negatively charged cell membrane, causing cytotoxicity. When encapsulating a positively charged bioactive ingredient, the encapsulation efficiency may be reduced due to electrical repulsion, or the release of the encapsulated bioactive ingredient may be inhibited due to strong binding with a negatively charged bioactive ingredient.

[0104] The zeta potential of the mesoporous silica nanoparticles of the present invention may be -15 to 60 mV, for example -15 to 60 mV, -15 to 55 mV, -13 to 55 mV, -10 to 55 mV, -5 to 55 mV, 0 to 55 mV, 5 to 55 mV, 5 to 50 mV, 5 to 40 mV, 5 to 35 mV, or 5 to 30 mV, the ranges including the end values ​​of each range.

[0105] As mentioned above, the most appropriate charge range may vary depending on the diameter of the mesoporous silica nanoparticles, and the size and potential of the mesoporous silica nanoparticles do not act independently in a drug delivery system. In addition, the lysine of the mesoporous silica nanoparticles can increase the charge of the particles, so it is desirable to optimize the diameter and potential of the nanoparticles by taking these variables into consideration. In addition, since the surface potential and size characteristics of the nanoparticles may change depending on the type and nature of the bioactive ingredient to be encapsulated, the physicochemical conditions must be adjusted accordingly.

[0106] In one embodiment, the diameter of the mesoporous silica nanoparticles of the carrier may be 30 to 150 nm and the zeta potential of the mesoporous silica nanoparticles may be -5 to 55 mV. In a preferred example, the diameter of the mesoporous silica nanoparticles of the carrier may be 30 to 80 nm and the zeta potential of the mesoporous silica nanoparticles may be 0 to 55 mV. In a more preferred example, the diameter of the mesoporous silica nanoparticles of the carrier may be 30 to 60 nm and the zeta potential of the mesoporous silica nanoparticles may be 5 to 55 mV.

[0107] The carrier according to the present invention has excellent encapsulation efficiency of a bioactive ingredient within the carrier, and the encapsulation efficiency of the bioactive ingredient of the present invention within the carrier may be 40% or more.

[0108] In this specification, the encapsulation efficiency may refer to a value indicating how much of a bioactive ingredient is actually encapsulated in mesoporous silica nanoparticles. The encapsulation efficiency may refer to loading efficiency (%), i.e., the ratio of the bioactive ingredient actually encapsulated in the carrier among the total bioactive ingredient (API, Active Pharmaceutical Ingredient) injected.

[0109] In one embodiment, the encapsulation efficiency was confirmed by setting the mass ratio of API and mesoporous silica nanoparticles to 1:5.

[0110] Encapsulation of the bioactive ingredient into the carrier is mostly completed within 1 hour, but the encapsulation efficiency was confirmed after the reaction was carried out for 2 to 4 hours for stable encapsulation and testing under saturated conditions.

[0111] The encapsulation efficiency of the bioactive component in the delivery vehicle according to the present invention may be 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[0112] In one embodiment, the carrier comprising the mesoporous silica nanoparticles of the present invention encapsulates at least 50% of the peptide comprising a fatty acid chain, but in a more preferred example, the carrier comprising the mesoporous silica nanoparticles comprising lysine encapsulates at least 80% of the peptide comprising a fatty acid chain.

[0113] The delivery system according to the present invention exhibits high encapsulation efficiency of bioactive ingredients, thereby reducing the total amount of silica (mesoporous silica nanoparticles) required to deliver the same drug dose. This reduces the volume or dosage unit of the final formulation, ultimately improving patient convenience and enhancing medication compliance when administered as a film, oral, or injectable formulation.

[0114] The carrier according to the present invention has excellent release of a bioactive ingredient from the carrier, and the release rate of the bioactive ingredient of the present invention from the carrier can be 40% or more.

[0115] The release rate of the bioactive ingredient of the present invention from the carrier may be 40% or more, 45% or more, 50% or more, 55% or more, or 60% or more.

[0116] In this specification, “release” may mean a process in which a bioactive ingredient encapsulated in mesoporous silica nanoparticles is separated from the nanoparticles over time in a release solution that mimics a living organism or moves into the body environment or is delivered to a target tissue due to external stimuli such as changes in the environment, such as pH or enzymes.

[0117] In this specification, “release rate” refers to the ratio of the bioactive ingredient released outside the carrier within a certain period of time based on the total amount of the bioactive ingredient actually encapsulated in the nanoparticle during the release process.

[0118] In one embodiment, the release characteristics of the bioactive ingredient were evaluated under conditions simulating the in vivo environment. Preferably, the carrier of the present invention was suspended in a solution having a pH of 6 to 8, reacted for 12 hours, and then the supernatant was collected to quantify the amount of the released bioactive ingredient.

[0119] The weight ratio of the bioactive ingredient and the mesoporous silica nanoparticles may be 1:1 to 10, for example, 1:1 to 10, 1:2 to 9, 1:3 to 8, 1:3 to 7, 1:4 to 7 or 1:4 to 6.

[0120] Since the carrier according to the present invention has a high bioactive ingredient encapsulation efficiency, a smaller amount of the carrier can be used to deliver the same amount of bioactive ingredient.

[0121] The weight ratio of the bioactive ingredient and the mesoporous silica nanoparticles may vary depending on the type of the bioactive ingredient. In addition, the present invention relates to a pharmaceutical composition comprising the carrier according to the present invention as an active ingredient.

[0122] In addition to the above-mentioned effective ingredient, the composition according to the present invention may further include additional ingredients, i.e., pharmaceutically acceptable or nutritionally acceptable carriers, excipients, diluents or auxiliary ingredients, depending on the formulation, method of use and purpose of use.

[0123] In addition to the above-mentioned effective ingredients, the above-mentioned composition may further contain nutrients, vitamins, electrolytes, flavoring agents, coloring agents, fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0124] The delivery system according to the present invention or the pharmaceutical composition comprising the same may be administered via various routes, either oral or parenteral, as long as it can reach the target tissue. For example, the composition of the present invention may be administered intravenously, intraperitoneally, intramuscularly, intraarterially, orally, intracardiacly, intramedullary, intrathecally, transdermally, enterally, subcutaneously, sublingually, or topically, but is not limited thereto.

[0125] The therapeutically effective dosage of the pharmaceutical composition of the present invention varies depending on the species, body weight, age, and individual condition of the subject, the disorder or disease being treated, or the severity thereof. The daily dosage of the composition of the present invention is 0.0001 to 1000 mg / kg, preferably 0.001 to 100 mg / kg, based on the amount of the carrier of the present invention, and may be administered once a week, 1 to 5 times a day, but the above dosage does not limit the scope of the present invention in any sense.

[0126]

[0127] Hereinafter, the present invention will be described in more detail with examples.

[0128]

[0129] <Manufacturing Example>

[0130] Manufacturing Example A. Manufacturing of mesoporous silica nanoparticles

[0131] To prepare mesoporous silica nanoparticles, first, (1) 3 mM to 100 mM CTAB (cetrimonium bromide) as a surfactant was dissolved in purified water, and 0.2 mM to 3.5 mM of 0.1 M sodium hydroxide (NaOH) aqueous solution was added to prepare a reaction solution. (2) The solution in step (1) was stirred at 60 to 70°C to form CTAB micelle structures. Then, (3) a mixture of TEOS, a silica precursor, dissolved in cyclohexane was prepared, and then added drop by drop to the solution in step (2). (4) The reaction was maintained at 50 to 70°C for 24 to 96 hours under stirring, and (5) cyclohexane or purified water, or APTES (0.5 mM to 50 mM) was added to some experimental groups. (6) After the reaction was completed, the resulting mesoporous silica nanoparticles were washed and centrifuged to obtain the particles. Depending on the embodiment, the particles obtained after washing and centrifugation were used as is, or (7) a freeze-drying process was additionally performed for more stable storage and subsequent experiments. In this case, the sample was freeze-dried in a freeze dryer for 24 hours to obtain a powder form.

[0132] The composition ratio of precursor, surfactant, and solvent can be controlled under various conditions, and accordingly, the average particle size, pore size, and surface charge can be changed.

[0133]

[0134] Manufacturing Example B: Manufacturing of mesoporous silica nanoparticles containing lysine

[0135] Nanoparticles were manufactured using the same method as in Manufacturing Example A, but 6.8 mM to 250 mM lysine was added to the reaction solution in step (1) of Manufacturing Example A to additionally manufacture mesoporous silica nanoparticles.

[0136]

[0137] Manufacturing Example C. Manufacturing of drug delivery vehicle loaded with peptide

[0138] A drug delivery system was manufactured by encapsulating a peptide into the nanoparticles of the examples and comparative examples manufactured according to the above manufacturing example A.

[0139] Both peptides and mesoporous silica nanoparticles (MSNs) were prepared at a concentration of 10 mg / ml, diluted in PBS (Phosphate Buffered Saline, pH 7.2) according to the ratio of each API (Active Pharmaceutical Ingredient, bioactive ingredient or effective ingredient) to MSN, and reacted at 200 rpm at room temperature for 2 hours.

[0140]

[0141] <Evaluation example>

[0142] Evaluation Example 1. Characteristics of mesoporous silica nanoparticles

[0143] The characteristics (size, charge) of mesoporous silica nanoparticles manufactured according to the above manufacturing examples A to C were confirmed.

[0144] Transmission electron microscopy (TEM) was used to confirm the size and shape of nanoparticles. Specifically, nanoparticles according to the above manufacturing example A were prepared in purified water at a concentration of 1 mg / ml, 100 μl was placed on a TEM grid, and then dried at room temperature for 2 hours. Afterwards, the microstructure of MSNs was measured using a field emission transmission electron microscope (FE-TEM, Tecnai G2 F30).

[0145] Additionally, dynamic light scattering (DLS) analysis was used to measure the zeta potential of nanoparticles. Specifically, a Zetasizer Nano ZS90 instrument from Malvern Panalytical was utilized, and the sample was prepared at 1 mg / ml in purified water, loaded onto the Zeta cell, and the zeta potential value was measured (3 times, 10-15 sec / each).

[0146] As a result, it was confirmed that nanoparticles having a spherical or virus-like shape according to Manufacturing Example A of the present invention were successfully manufactured as shown in Fig. 1. The names (exemplary or comparative examples) and specific sizes and surface charge characteristics of the nanoparticles of Manufacturing Example A of the present invention are as shown in Table 1 below.

[0147] ClassificationType of MSNMSN size (nm)Zeta Potential (mV)APTESLysineComparative Example 1Round type29.7-21.9××Example 1Round type2540.5O×Example 2virus / star36.418.5OOExample 3virus / star3054OOExample 4virus / star50-1.1O×Example 5Round type566.6OOExample 6Round type6036.5OOExample 7virus / star655.7O×Example 8virus / star6539.4O×Example 9virus / star780.02OOExample 10virus / star100-12.7×OComparative Example 2Round type200-34.8××Comparative Example 3Round type10001.89O×Comparative Example 4Round type30002.4× ×

[0148]

[0149] Evaluation Example 2. Evaluation of intracellular delivery efficiency of mesoporous silica nanoparticles (cellular uptake)

[0150] In order to confirm whether the porous nanoparticles according to the manufacturing example of the present invention were delivered into cells, the fluorescently labeled particles were treated to the cells, and the degree of cellular uptake was confirmed at 24 hours using a flow cytometer and a fluorescent microscope. Specifically, CaCo2 cells were pre-cultured for 48 hours and then treated with each mesoporous silica nanoparticle loaded with 1 mg / ml of mIgG-FITC. At 24 hours, the proportion (population) of cells into which FITC had penetrated into the cells was measured using a Cytek Aurora Flow cytometer and High-resolution Fluorescence Microscopes from Carl Zeiss. As a result, it was confirmed that the nanoparticles could be effectively absorbed into the cells, as shown in Table 2 below. In particular, the flow cytometry results showed that the nanoparticles according to the examples tended to show a higher cellular uptake rate overall than the comparative examples within the same particle size range. Under most conditions, the nanoparticles of the examples showed higher intracellular fluorescence signal intensity than the comparative examples, or a fluorescence signal above a certain level was clearly confirmed within the cells, confirming that the nanoparticles of the present invention have excellent cellular absorption properties (Fig. 2).

[0151] Classification % Population (Flow cytometer) Fluorescence intensity (Fluorescent microscope) Comparative Example 115.44++++Example 110.13+Example 221.83++++Example 359.94++++Example 462.35++Example 547.29+++Example 627.77++Example 743.13++Example 824.21++Example 930.33++++Example 1053.36++Comparative Example 255.71++Comparative Example 377.10+++Comparative Example 446.30+++

[0152]

[0153] Evaluation Example 3. Peptide Loading (Encapsulation) Analysis

[0154] As described in the above Manufacturing Example C, after encapsulating various peptides into nanoparticles according to Manufacturing Examples A and B, the encapsulation efficiency of each was evaluated to compare the encapsulation characteristics according to the type of peptide.

[0155] The results of encapsulating various peptides in nanoparticles according to Manufacturing Example A are shown in Fig. 4. The encapsulation efficiency differed depending on the type of nanoparticle and the type of peptide, and the correlation between particle size and surface charge and encapsulation efficiency was analyzed for each combination (correlation coefficient).

[0156] As a result of the analysis, it was observed that the sign of the correlation coefficient tended to change depending on the presence or absence of a fatty acid chain in the peptide (Table 3 below), indicating that the presence or absence of a fatty acid chain in the peptide can significantly affect the encapsulation efficiency in nanoparticles.

[0157] Correlation coefficientMSN Size (nm)Zeta potentialSemaglutide-0.5880.342Tirzepatide-0.4100.197Survodutide-0.4660.416Retatrutide-0 .4530.562Exenatide-0.0440.174Leuprolide0.268-0.544Octreotide-0.261-0.345Mhexa0.427-0.542

[0158]

[0159] As a result of specifically analyzing the encapsulation efficiency of each peptide according to the structural / physicochemical characteristics of the nanoparticles, the nanoparticles according to the examples of the present invention showed excellent encapsulation efficiency for peptides containing fatty acid chains (Figs. 3 to 8).

[0160] Specifically, when semaglutide, tirzepatide, survodutide, and retatrutide, which have fatty acid chains, were encapsulated, generally more than 40% of the peptides were encapsulated, and for some nanoparticles, more than 80% or 90% of the peptides were encapsulated. On the other hand, exenatide, which does not have a fatty acid chain, generally showed an encapsulation rate of approximately 20%.

[0161] In addition, nanoparticles with added lysine showed significantly higher encapsulation efficiency for encapsulating peptides containing fatty acid chains compared to nanoparticles without added lysine, suggesting that encapsulation performance may vary depending on the surface charge or lysine modification of the nanoparticles even within the same size range (Figures 4a to 4h).

[0162] In addition, for the nanoparticles having a positive charge, semaglutide, tirzepatide, survodutide, and retatrutide, which are peptides having fatty acid chains, the nanoparticles according to the examples of the present invention generally showed results corresponding to an encapsulation efficiency (% EE) of 50% or more when the API (active ingredient, i.e., peptide): MSN (nanoparticle) ratio was 5, confirming that the carrier of the present invention has excellent encapsulation performance.

[0163] In particular, this efficiency was more prominent when nanoparticles with very weak negative or positive charges were used as carriers, and it was confirmed that the nanoparticles of the present example showed higher encapsulation efficiency compared to particles with strong negative charges or large diameters (Comparative Example 2 or 3). Accordingly, it was confirmed that the examples of the present example can effectively encapsulate peptides even with a smaller peptide input compared to nanoparticles, especially when delivering peptides containing fatty acid chains.

[0164] Meanwhile, in the case of peptides that do not contain a fatty acid chain (exenatide, leuprorelin, octreotide, m-hexa), when encapsulated in nanoparticles according to the above manufacturing example, relatively low encapsulation efficiency was observed, and it was confirmed that a sufficient level of encapsulation was not achieved under some conditions.

[0165] This suggests that whether or not the peptide contains fatty acids is a factor that significantly affects the interaction with the nanoparticles, and indicates that the nanoparticles of the present invention have a structure more appropriately designed for peptides containing fatty acids.

[0166]

[0167] Evaluation Example 4. In vitro release test

[0168] To confirm the peptide release characteristics of the carrier according to the present invention, an in vitro release test was performed. MSNs loaded with semaglutide and terzepatide were diluted to 1 mg / ml in PBS (pH 6.8), incubated at 50 rpm and 37°C for a specified period of time, and the amount of peptide released in the supernatant was measured by centrifugation.

[0169] As a result of a peptide release test of a nanoparticle complex encapsulating terzepatide or semaglutide in a nanoparticle according to a manufacturing example of the present invention, the nanoparticle according to the example released more than 50% of terzepatide within 12 hours, indicating that the peptide can be effectively released from the nanoparticle (Fig. 9). In addition, it was confirmed that it generally showed excellent release efficiency for semaglutide as well.

[0170] When comparing examples containing nanoparticles of the same size and charge range, the nanoparticles containing lysine (Example 3) showed a release rate nearly twice that of the nanoparticles without lysine (Example 7).

[0171] This indicates that mesoporous silica nanoparticles containing lysine possess structural characteristics that enable effective release of peptides, along with peptide encapsulation. Therefore, it was confirmed that the nanoparticles of the present invention can be utilized as a delivery system that simultaneously satisfies controlled drug release and delivery efficiency.

[0172]

[0173] Evaluation Example 4. Confirmation of in vivo delivery of mesoporous silica nanoparticles

[0174] In order to confirm the in vivo delivery of the mesoporous silica nanoparticles (diameter: 110 nm, standard charge: -5 mV, pore size: 3 nm) according to the present invention, a pharmacokinetic evaluation was conducted in rats (SD rats) at 10 mg / kg of the semaglutide-encapsulated carrier itself. Specifically, the semaglutide-encapsulated carrier itself was administered per os (PO) at a concentration of 10 mg / kg using oral gavage (n=5), and plasma was collected at a set time. The concentration of semaglutide in the plasma was then measured using LC-MS. As a result, the carrier according to the present invention showed an in vivo delivery rate about 2.5 times higher than that of commercially available oral semaglutide, confirming that the carrier of the present invention has excellent bioavailability (Fig. 10).

Claims

1. A carrier comprising mesoporous silica nanoparticles and a bioactive ingredient, The above mesoporous silica nanoparticles have a diameter of 20 to 300 nm and a zeta potential of -15 to 60 mV, The above bioactive ingredient is a carrier which is a peptide containing a fatty acid chain.

2. In claim 1, the peptide containing the fatty acid chain is a carrier selected from the group consisting of semaglutide, tirzepatide, retatrutide, survodutide, liraglutide, cotadutide, cagrilintide, insulin detemir, tesamorelin, daptomycin, polymyxin B, insulin degludec, somapacitan, and MEDI0382.

3. A carrier according to claim 1, wherein the diameter of the mesoporous silica nanoparticles is 30 to 150 nm.

4. A carrier according to claim 1, wherein the zeta potential of the mesoporous silica nanoparticles is -13 to 55 mV.

5. In claim 1, the mesoporous silica nanoparticles are spherical, a carrier.

6. In claim 1, the mesoporous silica nanoparticle is a spherical carrier having protrusions formed on the surface.

7. A carrier according to claim 1, wherein the encapsulation efficiency of the bioactive ingredient within the carrier is 40% or more.

8. A carrier according to claim 1, wherein the release rate of the bioactive ingredient is 40% or more.

9. A carrier according to claim 1, wherein the bioactive ingredient is encapsulated in the pores of the mesoporous silica nanoparticles.

10. A carrier according to claim 1, wherein the weight ratio of the bioactive ingredient and the mesoporous silica nanoparticles is 1:1 to 10.

11. A carrier comprising mesoporous silica nanoparticles containing lysine and a bioactive ingredient, The above mesoporous silica nanoparticles have a diameter of 20 to 300 nm and a zeta potential of -15 to 60 mV, The above bioactive ingredient is a carrier which is a peptide containing a fatty acid chain.

12. In claim 11, the peptide containing the fatty acid chain is any one selected from the group consisting of semaglutide, tirzepatide, retatrutide, survodutide, liraglutide, cotadutide, cagrilintide, insulin detemir, tesamorelin, daptomycin, polymyxin B, insulin degludec, somapacitan, and MEDI0382.

13. A carrier according to claim 11, wherein the diameter of the mesoporous silica nanoparticles is 30 to 150 nm.

14. A carrier according to claim 11, wherein the diameter of the mesoporous silica nanoparticles is 30 to 80 nm.

15. A carrier according to claim 11, wherein the zeta potential of the mesoporous silica nanoparticles is -5 to 55 mV.

16. A carrier according to claim 11, wherein the zeta potential of the mesoporous silica nanoparticles is 5 to 55 mV.

17. A carrier according to claim 11, wherein the mesoporous silica nanoparticles have a diameter of 30 to 70 nm and a zeta potential of 5 to 55 mV.

18. A carrier according to claim 11, wherein the encapsulation efficiency of the bioactive ingredient within the carrier is 40% or more.

19. A carrier according to claim 11, wherein the release rate of the bioactive ingredient is 40% or more.

20. In claim 11, the mesoporous silica nanoparticles are spherical, a carrier.

21. In claim 11, the mesoporous silica nanoparticles are spherical in shape with protrusions formed on the surface, a carrier.

22. A carrier according to claim 11, wherein the bioactive ingredient is encapsulated in the pores of the mesoporous silica nanoparticles.

23. A carrier according to claim 11, wherein the weight ratio of the bioactive component and the mesoporous silica nanoparticles is 1:1 to 10.

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