A sustained anticancer drug delivery system based on a mixture of diatomaceous earth and lipid nanoparticles

Abstract

The present invention relates to a drug delivery system comprising a drug polymer containing a drug supported on lipid nanoparticles, a drug delivery vehicle containing biosilica purified from diatomite and transporting the drug polymer, wherein the drug polymer is positively charged and electrostatically bound to the drug polymer.

Description

Technology Field

[0001] The present invention relates to a continuous anticancer drug delivery system based on a mixture of diatomite and lipid nanoparticles.

[0002] More specifically, the invention relates to a drug delivery system and a method for manufacturing the same, which can eliminate toxicity and ensure stability and mass production in a drug delivery system comprising a lipid nanoparticle drug polymer bound to biosilica. Background Technology

[0004] Many new drugs, particularly anticancer agents, possess strong hydrophobicity (or low solubility) to enhance receptor specificity for pharmacological activity, but this results in low bioavailability within the body, which is composed primarily of water.

[0005] To address the side effects caused by these hydrophobic limitations, technologies are currently emerging to improve the solubility and retention time of hydrophobic drugs or to deliver drugs through various drug delivery systems (DDS).

[0006] These various drug delivery system (DDS) platforms must possess specialized targeting capabilities, such as enhanced permeability and retention (EPR) effects. Drug delivery via various DDSs can primarily be achieved by coating drugs with specialized functional materials through physicochemical reactions or by synthesizing specialized formulations by loading drugs onto carriers.

[0007] Among conventional drug delivery system (DDS) platforms, lipid nanoparticles (LNPs) with high biocompatibility are currently emerging as an important solution for hydrophobic drug delivery. Lipid nanoparticles (LNPs) are micelle structures synthesized based on lipids and surfactants, capable of efficiently capturing hydrophobic drugs and transporting them into cells through a hydrophobic environment composed of lipids. In particular, nanostructured lipid carriers (NLCs) are attracting attention as advanced drug delivery carriers that demonstrate high capture efficiency and storage stability for both hydrophobic and hydrophilic drugs compared to conventional lipid nanoparticles (LNPs) and solid lipid nanoparticles (SLNs). The aforementioned cationic lipid nanoparticles (cationic nanostructured lipid carriers) are synthesized using cationic lipids (or surfactants) and can enhance drug delivery efficiency by improving cell adhesion through increased electrical attraction with the cell membrane.

[0008] However, conventional cationic lipid nanoparticles (LNPs), such as cationic nanostructured lipid carriers, have had limitations in that they cannot be directly applied in the medical field outside of laboratory conditions because they exhibit cytotoxic symptoms, such as inflammatory responses induced by cationic surfactants, when treated at high concentrations.

[0009] Furthermore, conventional lipid nanoparticle (LNP)-based drug delivery systems (DDS) had problems that caused adverse effects, such as side effects and non-specific absorption, due to the difficulty in controlling blood drug concentrations caused by an initial drug burst.

[0010] Furthermore, conventional drug delivery systems had a fundamental limitation in that they could not be directly utilized in industrial sectors such as pharmaceuticals, as they only offered the possibility of producing small quantities of drug carriers carrying drug polymers under experimental conditions and could not be manufactured to the extent that mass production could be carried out while guaranteeing stability and uniformity.

[0011] Recently, technology has also emerged that uses nanostructures containing diatomaceous earth for antibacterial purposes (see Korean Patent Publication No. 10-2022-0145441). However, since the purpose of this technology is to amplify the antibacterial properties of nanostructures containing diatomaceous earth that exhibit toxicity, it had the problem of retaining the toxicity issues of existing drug delivery systems.

[0012] In addition, there has been a technology that excludes toxicity in the past as a composition related to skin regeneration containing diatomite (see Korean Registered Patent Publication No. 10-2253894). However, this technology had a fundamental limitation in that it could not be applied to the field of pharmaceuticals where diatomite transports the active extract to target cells, as it only provided a simple physical mixture state rather than an electrochemical bonding between the diatomite and the active extract.

[0013] Finally, a technology utilizing diatom-based biosilica as a drug delivery system has also emerged as an academic paper. (See KSBB Journal, Recent Researches for Diatom as Inorganic and Bioenvironmental Materia.) However, this technology also had limitations in that it only provided the possibility that biosilica could be used as a drug delivery system, but did not disclose at all the specific mechanism by which it could be combined with and transported with a drug polymer, thus failing to suggest its potential for industrial use. The problem to be solved

[0015] The present invention aims to solve the problem of providing a drug delivery system capable of supplying a drug while stabilizing the blood drug concentration.

[0016] The present invention aims to solve the problem of providing a drug delivery system capable of sufficiently loading even hydrophilic drugs onto lipid nanoparticles.

[0017] The present invention aims to solve the problem of providing a drug delivery system capable of achieving in vivo stability and mass production by applying a diatomite-based biosilica as a drug delivery vehicle that guides and transports drug polymers to target cells.

[0018] The present invention aims to solve the problem of providing a drug delivery system that can ensure in vivo stability by excluding chemical bonds and chemicals during the process of combining a drug polymer and a drug delivery system. means of solving the problem

[0020] To solve the above-mentioned problem, the present invention provides a drug delivery system comprising a drug polymer containing a drug supported on lipid nanoparticles, a drug delivery vehicle containing biosilica purified from diatomite and transporting the drug polymer, wherein the drug polymer is positively charged and electrostatically bound to the drug polymer.

[0021] The above drug contains hydrophilic doxorubicin (DOX) and can be hydrophobized and loaded onto the lipid nanoparticles.

[0022] The above drug polymer can be coated with chitosan and charged with a positive charge.

[0023] To solve the above-mentioned problem, the present invention provides a method for manufacturing a drug delivery system comprising: a purification step of obtaining a drug delivery vehicle by purifying biosilica from diatomite; a manufacturing step of manufacturing a drug polymer by loading a drug onto lipid nanoparticles; and a production step of manufacturing a drug complex by combining the drug delivery vehicle and the drug polymer, wherein the manufacturing step includes coating the surface of the drug polymer with chitosan to charge it with a positive charge, and the production step is characterized by electrostatically combining the positively charged drug polymer with the negatively charged drug delivery vehicle.

[0024] The above manufacturing step may include hydrophobizing a hydrophilic drug.

[0025] The above drug may include doxorubicin (DOX). Effects of the invention

[0026] The present invention has the effect of being able to supply drugs while stabilizing the blood drug concentration.

[0027] The present invention has the effect of sufficiently loading even hydrophilic drugs onto lipid nanoparticles.

[0028] The present invention has the effect of enabling in vivo stability and mass production by applying a drug delivery system that guides and transports drug polymers to target cells using diatomite-based biosilica.

[0029] The present invention has the effect of ensuring in vivo stability by excluding chemical bonds and chemicals during the process of combining a drug polymer and a drug delivery system. Brief explanation of the drawing

[0031] FIG. 1 illustrates a method for manufacturing the drug delivery system of the present invention described above. FIG. 2 illustrates the drug delivery system of the present invention. Figure 3 verifies the manufacturing steps of the drug delivery system of the present invention. Figure 4 illustrates the process of determining the optimal size of the nanostructured lipid carrier of the present invention. Figure 5 illustrates the chitosan attachment effect of the drug polymer. FIG. 6 illustrates the purification step of the drug delivery system of the present invention. FIG. 7 illustrates the steps for manufacturing the drug delivery system of the present invention. Figure 8 illustrates the verification of the manufacturing steps in a drug delivery system manufacturing method in a different way. FIG. 9 illustrates the efficacy of the drug delivery system of the present invention. Specific details for implementing the invention

[0032] Hereinafter, embodiments disclosed in this specification will be described in detail with reference to the attached drawings. In this specification, identical or similar reference numbers are assigned to identical or similar components even if they are different embodiments, and the description thereof is replaced by the first description. Singular expressions used in this specification include plural expressions unless the context clearly indicates otherwise. Furthermore, in describing the embodiments disclosed in this specification, detailed descriptions of related prior art are omitted if it is determined that such detailed descriptions may obscure the essence of the embodiments disclosed in this specification. Additionally, it should be noted that the attached drawings are intended only to facilitate understanding of the embodiments disclosed in this specification, and should not be interpreted as limiting the technical concept disclosed in this specification.

[0033] Generally, the drug (110) can be defined as a series of chemical substances that are delivered to target cells and produce a therapeutic effect according to a set pharmacological mechanism. In order for the drug (110) to be delivered to the target cells, a method of direct administration to the target cells may be applied, or a method of indirect administration to the target cells through the digestive system via ingestion may be applied.

[0034] Regardless of the method, in order for the above drug (110) to be delivered to target cells and produce the intended therapeutic effect, it is necessary to ensure a certain dosage and concentration delivered to the target cells. However, since the above drug (110) diffuses or is diluted within the body during the process of delivery to the target cells when administered directly into the body, the drug delivery system (10) of the present invention may additionally require a carrier (120) to deliver the above drug (110) directly to the target cells.

[0035] The above carrier (120) can perform the role of transporting the drug (110) to target cells. By being contained in the above carrier (120), the drug (110) can be prevented from arbitrarily spreading or being diluted within the body.

[0036] When the carrier (120) reaches the target cell, it needs to bind to the target cell to administer the drug (110) to the target cell. Generally, since the wall of the target cell is composed of lipids, the carrier (120) may also be composed of lipids and may be provided in nanometer units to effectively deliver a large amount of the drug (110) to the target cell. That is, the carrier (120) may be provided as a nano-lipid carrier (NLC).

[0037] The drug (110) can be transported together by being loaded onto a nano-lipid carrier (120) to form a drug polymer (100).

[0038] In order for the above drug polymer (100) to be formed, it is presupposed that the drug (110) is contained or bound inside the nano lipid carrier (120). However, since the nano lipid carrier (120) is composed of lipids and is fundamentally hydrophobic, if the drug (110) is hydrophilic, it may be difficult to load the drug (110) onto the nano lipid carrier (120).

[0039] Accordingly, in order to manufacture the drug delivery system (10) of the present invention, a process of hydrophobizing the hydrophilic drug (110) may be required. By doing so, the hydrophilic drug (110) can also be hydrophobized and easily loaded onto the nano-lipid carrier (120). For example, the drug (110) may be composed of or contain doxorubicin (DOX), which is a representative hydrophilic anticancer agent. In this case, the drug polymer (100) may be an anticancer agent, and the drug delivery system (10) of the present invention may correspond to an anticancer drug delivery system.

[0040] Meanwhile, when the above-mentioned nano-lipid carrier (120) reaches a target cell through the digestive system, if it is decomposed by various enzymes, the drug (110) may also be digested, diluted, or diffused and may not reach the target cell.

[0041] To this end, the drug delivery system (10) of the present invention may further include a drug delivery vehicle (200) capable of stably transporting the nano-lipid carrier (120) to the target cell without it being degraded by the digestive system, etc.

[0042] The above drug delivery system (200) may be made of silica (210). The silica (210) can protect the drug polymer (100) from digestive enzymes, etc. due to the silica-based matrix.

[0043] In addition, silica (210) has a negative charge due to the structure of silanol or the surface, so it has the advantage that the surface is easily bonded to other objects chemically and electrically.

[0044] At this time, the silica (210) may be biosilica obtained in large quantities through diatomite, which is an algae such as diatoms and a fossil of the algae. Since diatomite can be collected in large quantities in a freshwater environment and can also be purified in a stable and eco-friendly manner, it can be a major material for manufacturing biosilica.

[0045] In addition, the biosilica (210) can prevent a sudden increase in blood drug concentration, such as a drug explosion, because it separates and releases the drug polymer (100) to target cells at a stable rate according to the silica surface matrix structure.

[0046] Accordingly, the drug delivery system (10) of the present invention can obtain biosilica (210) based on such diatomite and use it as the drug delivery body (200).

[0047] The drug delivery system (10) of the present invention may apply an electrostatic method to bind a drug polymer (100) to such biosilica (210). In other words, by binding the drug polymer (100) and the drug delivery agent (200) in an electrostatic manner, the drug delivery system (10) of the present invention may eliminate the need for separate chemical substances and chemical bonding to bind the drug polymer (100) and the drug delivery agent (200).

[0048] Thus, the drug delivery system (10) of the present invention can maintain characteristics that are harmless to the human body, so it may be suitable for application to pharmaceuticals.

[0049] The drug delivery system (10) of the present invention needs to charge the surface of the drug polymer (100) with a positive charge in order to electrically bond the drug polymer (100) to a negatively charged drug delivery body (200).

[0050] To this end, when the drug polymer (100) is charged with a positive charge using a surfactant or the like as in the conventional method, or is charged with a positive charge while being treated at a high concentration, the drug polymer (100) itself may exhibit cytotoxic symptoms.

[0051] Accordingly, the drug delivery system (10) of the present invention may be coated with chitosan (130), which is a biosafe material, to charge the surface of the drug polymer (100) with a positive charge. By doing so, cytotoxic symptoms can be prevented even if the drug polymer (100) is condensed or processed at a high concentration.

[0052] FIG. 1 illustrates a method for manufacturing the drug delivery system of the present invention described above.

[0053] The drug delivery system (10) of the present invention may include a drug polymer (100) comprising a drug (110) loaded on lipid nanoparticles (210), and a drug delivery vehicle (200) comprising biosilica (210) purified from diatomite and transporting the drug polymer (100).

[0054] The drug delivery system (10) of the present invention may include the drug polymer (100) being charged with a positive charge and electrostatically bonded to the drug polymer (200).

[0055] The above drug (110) may include a hydrophilic anticancer agent such as doxorubicin (DOX). The above drug (110) may be hydrophobized and loaded onto the lipid nanoparticle (210) to form the drug polymer (110).

[0056] To this end, the method for manufacturing a drug delivery system of the present invention may include a purification step (S1) of obtaining a drug delivery body (200) by purifying biosilica (210) from diatomite, a manufacturing step (S2) of manufacturing a drug polymer by loading a drug onto lipid nanoparticles, and a production step (S3) of manufacturing a drug complex by combining the drug delivery body (200) and the drug polymer (100).

[0057] The above manufacturing step (S2) may include coating the surface of the drug polymer (100) with chitosan (130) (130) to charge it with a positive charge.

[0058] The above manufacturing step (S3) may include electrostatically bonding the positively charged drug polymer (100) to the negatively charged drug delivery system (200).

[0059] In addition, the above manufacturing step (S2) may include hydrophobizing a hydrophilic drug.

[0060] The drug delivery system of the present invention and the method for manufacturing the same will be explained below through specific examples.

[0061] The drug delivery system of the present invention may combine a drug polymer (100) containing cationic lipid nanoparticles (120) with a drug delivery system (200), such as natural / synthetic silica and hydrogel, to control initial drug release, cytotoxicity, and instability caused by excessive positive charge.

[0062] The drug delivery system (10) of the present invention may utilize diatomite biosilica (210) as a drug delivery body (200).

[0063] Diatomous biosilica (210) is a natural porous silica particle with a size of tens of micrometers, which is a fossil form of diatoms widely found in underwater environments, and has a hierarchical structure of uniform porous nano-patterns on its surface.

[0064] In addition, diatomaceous earth biosilica (210) has the advantage of being able to protect drugs (110) from various enzymes due to the silica-based matrix, and having a negative charge due to the silanol (-SiOH) structure on the surface, making it easy to chemically functionalize the surface.

[0065] FIG. 2 illustrates the drug delivery system of the present invention.

[0066] The present invention can provide a hybrid drug delivery system (10) that integrates a diatomite biosilica (210) with a strong negative charge and a cation nanostructure lipid carrier (120) through electrical interaction to solve the limitations of conventional cation nanostructure lipid carriers.

[0067] The drug (110) that can be applied to the drug delivery system (10) of the present invention may include doxorubicin (DOX), a representative hydrophobic anticancer agent. In the following description, the drug (110) is provided as doxorubicin (DOX). However, this is merely an example to explain the effect of the drug delivery system (10) of the present invention and does not exclude the application of other drugs.

[0068] The above drug (110) can be loaded onto a nanostructured lipid carrier (120) and mixed with biosilica (210) based on electrical interactions. The synthesis of the above drug delivery system (10) can be verified through FT-IR analysis and various imaging techniques (e.g., SEM, TEM, AFM). The enhanced sustained-release capability of the nanostructured lipid carrier (120) combined with biosilica (210) can be confirmed through analysis of the in vitro drug release profile in PBS and extreme environments.

[0069] The cytotoxicity and drug delivery efficiency of the manufactured formulation can be measured through in vitro cytotoxicity tests using normal (L929) and cancer (SK-BR-3) cells.

[0070] The drug delivery system (10) of the present invention, produced through the above analysis, has the effect of increasing the bioavailability and sustained release of the hydrophobic drug (110).

[0071] The drug manufacturing system (10) of the present invention comprises doxorubicin-HCl, dimethyl sulfoxide (DMSO), diatomite, hydrogen peroxide (30%), polycarbonate track etching (PCTE) membrane, anhydrous ethyl alcohol, surfactant Tween 80 and poloxamer 188 (Pluronic F-127). Co-surfactant dimethyldioctadecylammonium bromide (D18DAB), Precirol® ATO 5 (solid lipid) and CAPRYOL 90 (liquid lipid), centrifugal filtration device (Amicon Ultra-4 mL, 100 kDa) and Dulbecco's Phosphate Buffered Saline (PBS, Cat. No. 40-067753-03), dialysis membrane Spectra / Por® 6 MWCO 1000, chitosan (130), Roswell Park Memorial Institute (RPMI) 1640 medium Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), antibiotics, trypsin, WST-8 reagent used for cell viability analysis, 96-well cell culture plates, acetic acid, chitosan (130) oligosaccharide, lauryl aldehyde, sodium hydroxide (NaOH), It can be manufactured using sodium borohydride (NaBH4).

[0072] Figure 3 verifies the manufacturing steps of the drug delivery system of the present invention.

[0073] The manufacturing step (S1) of the drug delivery system of the present invention may include loading a drug (110) onto the drug delivery body (120).

[0074] The above drug (110) can be hydrophobized and encapsulated in a drug delivery system (120) equipped with hydrophobic lipid nanoparticles.

[0075] Referring to FIG. 3, the loading of doxorubicin (DOX, 110) in the nanolipid carrier (120) can be verified through FT-IR and fluorescence analysis.

[0076] First, by comparing the FT-IR spectrum of the nano lipid carrier (120) and the drug polymer (100) as the nano lipid carrier (120) loaded with the drug (110), the loading of the drug (110) onto the nano lipid carrier (120) can be confirmed through the appearance of the same peak.

[0077] The absence of peak shift when loading the drug (110) indicates that the amount of drug (110) not accommodated in the nanolipid carrier (120) is negligible, and indicates that there is no chemical bonding between the drug (110) and the nanolipid carrier (120).

[0078] Specifically, the manufacturing step (S1) of the drug delivery system (10) of the present invention can be performed through ultrasonic nanoemulsification.

[0079] It is more difficult to incorporate a hydrophilic drug into the nucleus of a nanostructured lipid carrier (120) than a hydrophobic drug, and the tendency for early release may be higher. Therefore, DOX-HCl, a hydrophilic universal anticancer drug (110), can be hydrophobized and loaded into a nanostructured lipid carrier (120).

[0080] To do this, DOX-HCl (10 mg) can be dissolved in 1 mL of dimethyl sulfoxide (DMSO) and stirred for 5 minutes. Next, 100 μL of triethylamine (TEA) and 2 μL of NaOH can be added and stirred overnight to obtain hydrophobic DOX.

[0081] The lipid matrix of the nanostructured lipid carrier (120) can be obtained by mixing solid lipids and liquid lipids in a 10:1 ratio and heating at 75°C, which is higher than the melting point of the two materials. Dimethyldioctadecylammonium bromide (D18DAB) can be used in this process.

[0082] A certain amount of surfactant may be added for uniform emulsification of the aqueous layer containing D18DAB and hydrophilic chitosan (130).

[0083] After the two phases are completely dissolved, a lipid matrix is ​​added and sonicated at 70% amplitude for 10 minutes using an ultrasonic processor, and the resulting emulsion is cooled at room temperature for at least 5 hours to produce a drug polymer (100) corresponding to a nano-lipid carrier (120) loaded with a drug (110).

[0084] Then, to maintain the stability of the drug polymer (100), it can be stored at 4°C.

[0085] The characteristics of the above drug polymer (100), such as hydrodynamic particle size, polydispersity index (PDI), and zeta potential, can be analyzed using ELS-Z-200ZS.

[0086] To this end, for particle size measurement, the sample can be diluted 100-fold with deionized water to minimize multiple scattering effects and achieve a uniform distribution. Measurements can be performed at 25°C with a scattering angle of 165°.

[0087] Similarly, zeta potential measurements can be performed at 25°C with a scattering angle of 165°, and the sample can be diluted 10-fold with deionized water to achieve a concentration suitable for analysis. All measurements can be repeated three times to ensure the reliability and reproducibility of the data.

[0088] Using an atomic force microscope (AFM), the morphology of the drug polymer (100, cationic nanostructured lipid carrier) can be characterized.

[0089] In summary, 2 μL of a diluted cation nanostructured lipid carrier sample can be placed on a silicon wafer and dried. AFM imaging can be performed in non-contact mode using a PPP-NCHR canilever and a silicon tip.

[0090] For example, the resonant frequency of the tip was 330 kHz, and the spring constant could be 42 N / m. The scan speed was set to 0.50 Hz, and the resolution could be 512 pixels. The generated AFM image can be acquired using Park XEI software, etc.

[0091] Meanwhile, scanning electron microscope (SEM) analysis can be performed to analyze the surface characteristics of the diatomite, diatomite biosilica (210), and drug delivery system (10).

[0092] Each sample can be prepared by fixing it to aluminum foil, dispensing the sample solution, and drying it at room temperature.

[0093] SEM images can be acquired using a field emission scanning electron microscope operating at an acceleration voltage of 10.0 kV.

[0094] Additionally, transmission electron microscopy (TEM) analysis can be performed using the JEM-2100F in high-resolution TEM (HRTEM) mode with an acceleration voltage of 200 kV. TEM samples can be prepared by dispersing a solution of appropriate concentration on a copper grid and drying it.

[0095] The efficiency (EE %) of encapsulating the drug (110) in the nanolipid carrier (120) and the release profile of the capsule can be evaluated through the intrinsic fluorescence of the drug (110).

[0096] Fluorescence measurements can be performed using a multimode reader, and the excitation and emission results can be measured at 485 nm and 580 nm accordingly.

[0097] After manufacturing the drug polymer (110), the drug polymer (110) and deionized water are mixed in a 1:1 ratio, and then the drug (110) is not loaded, and then the separation of the drug polymer (110) or the support can be performed using a centrifugal filtration device.

[0098] After the fluorescence for the conversion of the unloaded drug (110) is completely processed, the EE (%) of the DOX loaded in the drug polymer (110) can be calculated by taking the fluorescence into account.

[0099] The formula for calculating the EE(%) of DOX using fluorescence is as follows:

[0100] EE(%) = 100 - (FL - F) / (F0 - F) Х 100,

[0101] Here, FL is the fluorescence of the expected layer after separation, F is the fluorescence of the deionized water, and F0 is the fluorescence corresponding to the loading of doxorubicin (DOX).

[0102] To check the degree of drug release in the body environment of the drug delivery system (10) of the present invention, the release period can be checked under dark conditions for 48 hours at 37°C.

[0103] The release period can be recorded in phosphate-buffered saline (PBS) and under the above environmental simulation conditions.

[0104] The results can be compared between the drug delivery system (10) and the drug polymer (100).

[0105] Each sample can be dialyzed in 5 mL of dialysis tubing and a test kit, and 45 mL of buffer can be used.

[0106] The above simulation solution can be prepared using pepsin and NaCl by referring to previous studies. The pH can be maintained at approximately 2.

[0107] After extracting 100 μL per hour, the fluorescence can be measured, and the solution can be replaced with a new buffer to maintain the dose gradient.

[0108] The formula for checking the frame output is as follows:

[0109] Release(%) = ((FRelease - F) / (F0 - F)) Х 100,

[0110] Here, FRelease represents the requirement for the fluorescence of the buffer over time, F0 represents the fluorescence at 100% emission, and F represents the fluorescence of the new buffer.

[0111] FT-IR analysis can be performed in attenuated total reflection mode using a ZnSe crystal. The frequency range can be set from 650 to 4000 cm⁻¹ using an FT-IR spectrometer. All spectra can be acquired by scanning 32 times at a resolution of 4 cm⁻¹.

[0112] Meanwhile, to verify the effect of the drug delivery system (10) of the present invention, mouse fibroblast cell line L929 and human breast cancer cell line SK-BR-3 can be used as target cells (300).

[0113] SK-BR-3 cells were maintained in RPMI 1640 medium, and L929 cells can be cultured in DMEM medium.

[0114] Both media can be supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics.

[0115] Mouse fibroblast cell line L929 was incubated in a VS-2180 C incubator at 37°C and 5% CO₂ It can be cultured in a humidified environment.

[0116] Before subculture, cells can be washed with Dulbecco's phosphate-buffered saline (DPBS) and isolated using 0.25% (w / v) trypsin-EDTA.

[0117] SK-BR-3 cells were subcultured at a 1:3 ratio, and L929 cells can be subcultured at a 1:4 ratio.

[0118] The cytotoxicity of the drug delivery system (10) of the present invention can be confirmed using WST-8 analysis.

[0119] Mouse fibroblast cell line L929 and breast cancer cell line SK-BR-3 can be used as target cells (300).

[0120] Cell viability can be evaluated through concentration gradient experiments on diatomite biosilica (10) and nanostructured lipid carriers (120).

[0121] Diatomous earth biosilica (210) was tested within a concentration range of 0 - 1,000 μg / mL, and the cytotoxicity of the nanostructured lipid carrier (110) was evaluated based on lipid concentrations of 0 - 10 mg / mL.

[0122] L929 and SK-BR-3 cells were placed in 96-well plates at 5 x 10³ cells / well and 1 x It can be inoculated at a cell / well density and cultured for 48 hours.

[0123] The drug (110) can be diluted in DMEM medium to a specific concentration and administered to cells, after which they can be cultured for 24 hours.

[0124] After treatment, 10 μL of WST-8 solution can be added to each well, and the plate can be incubated at 37°C for an additional 4 hours.

[0125] Absorbance can be measured at 450 nm and 650 nm using a multimode reader (Synergy LX, BioTek, USA).

[0126] Cell viability can be calculated based on the following formula.

[0127] Cell viability (%) = OD treat (450 nm-650 nm) - OD blank (450 nm -650 nm) / OD nontreat (450 nm -650 nm) - OD blank (450 nm -650 nm) x 100

[0128] The efficacy of the drug delivery system (10) of the present invention can be confirmed using WST-8 analysis.

[0129] Target cells (300) can be cultured under the same conditions applied to the cytotoxicity evaluation of diatomite biosilica (210) and cationic nanostructured lipid carriers (120).

[0130] To investigate the effect of the concentration gradient, drug concentrations in the range of 0 to 2.5 μM can be prepared.

[0131] The drug (110), drug polymer (100), and drug delivery system (10) may be included as an experimental group to evaluate the improvement of cell permeability of the nanolipid carrier (120) and the gradual drug release by the biosilica (110).

[0132] Each drug was diluted to a specified concentration in DMEM and administered after 24 hours of incubation. Subsequently, 10 μL of WST-8 solution was added to each well and incubated at 37°C for 4 hours. Absorbance was measured at 450 nm and 650 nm using a microplate reader. Cell viability can be calculated based on the following equation (Eq. 2).

[0133] Cell viability (%) = (OD drug treat (450 nm-650 nm) - OD blank (450 nm -650 nm)) / (OD nontreat (450 nm -650 nm) - OD blank (450 nm -650 nm)) Х 100 All experimental data described below were obtained from at least three independent experiments, and the mean ± standard error (SE) was used.

[0134] For cytotoxicity analysis, the standard error and balance error can be calculated using SPSS software.

[0135] Statistical significance can be evaluated by cross-testing using one-way analysis of variance (ANOVA) with Tukey and LSD post-hoc tests under the assumption of equal variances.

[0136] Statistical significance can be determined to identify significant differences at *p < 0.05, **p < 0.01, and ***p < 0.001.

[0137] Figure 4 illustrates the process of determining the optimal size of the nanostructured lipid carrier of the present invention.

[0138] Referring to FIG. 4(a), the particle size of the nanostructured lipid carrier (120) of the present invention was confirmed to be +79.3 ± 2.43 nm, and the PDI is 0.205 ± 0.014.

[0139] Referring to FIG. 4(b), the surface charge of the nanostructured lipid carrier (120) was found to be greater than +20 mV in the recommended surfactant concentration range (1% to 3% (w / v)), and the average zeta potential of the optimized-size cationic nanostructured lipid carrier was +26.46 ± 1.67 mV. It is considered that when the absolute value of the zeta potential of the nanoparticle is greater than 30 mV, particle aggregation is significantly inhibited, which has a negative effect on particle stability.

[0140] In addition, since the stability of lipid nanoparticles (120) is greatly affected by environmental factors such as temperature, humidity, and ultraviolet rays, optimizing storage conditions can maintain stability even at low zeta potential values.

[0141] In the case of the optimized cationic nanostructured lipid carrier (120), it can be seen that the particle size is appropriate and the zeta potential is close. Therefore, in the drug delivery system (10) of the present invention, the nano lipid carrier (120) can be used as a stable drug formulation.

[0142] Referring to FIG. 4(c), the change in surface roughness of the drug polymer (100) and the drug polymer (100) with hydrophilic chitosan (130) attached can be analyzed using AFM.

[0143] When hydrophilic chitosan (130) is coated on the surface of the drug polymer (100), the final drug polymer (100) exhibits a spherical shape of about 200 nm in size.

[0144] Referring to FIG. 4(d), it can be seen that the change in surface roughness of the final drug polymer (100) due to modification of hydrophilic chitosan (130) is that the average roughness (Ra) increased from 39.406 nm to 63.281 nm and the average mean squares (RMS) (Rq) increased from 42.994 nm to 69.852 nm.

[0145] Figure 5 illustrates the chitosan attachment effect of the drug polymer.

[0146] Referring to FIG. 5, the successful synthesis and attachment of chitosan (130) to the nano-lipid carrier (120) can be confirmed.

[0147] FIG. 6 illustrates the purification step of the drug delivery system of the present invention.

[0148] The purification step (S2) of the drug delivery system (10) of the present invention may include a series of heat and chemical treatments to extract biosilica (210) from diatomite.

[0149] For example, 1 g of diatomite can be suspended in 100 mL of a 30% hydrogen peroxide (H2O2) solution to perform an initial treatment. The mixture can be ultrasonically treated for 10 minutes to partially desorb organic material attached to the inner and outer walls of the diatomite.

[0150] After ultrasonic treatment, a mixture of diatomite and hydrogen peroxide can be reacted at 1.5 atmospheres and 120°C for 15 minutes. This step may correspond to a heat treatment for desorbing and removing residual organic material at high temperature and high pressure.

[0151] After that, the reacted biosilica (210) dispersion can be filtered using PCTE (pore size 8 μm). The biosilica (210) powder filtered through the filter can be redispersed in ethanol (EtOH) and stirred for 30 minutes to remove residual material.

[0152] After filtering the ethanol dispersion again using PCTE, it can be completely dehydrated by drying it in a 75°C oven for 12 hours and stored in a sealed container at room temperature for use.

[0153] Referring to Fig. 6(a), for biological applications, diatomite is coated with foreign substances containing various organic materials, which can cause potential cytotoxicity, so a purification process is required.

[0154] Referring to FIG. 6(b), the present invention can purify diatomite to obtain diatomite biosilica (210) free of organic materials and other impurities through hydrogen peroxide treatment at high temperature and high pressure.

[0155] Referring to FIG. 6(c), the structural characteristics of the obtained diatomite biosilica (210) can be investigated through scanning electron microscope-energy dispersive X-ray spectroscopy (SEM-EDX) and transmission electron microscope (TEM) analysis. SEM-EDX analysis of the purified DB shows peaks of Si, O, and Al.

[0156] The peaks of Si and O are the main components of the diatomite biosilica (210), and Al is a component of the aluminum foil (Al2O3) used for sample preparation and fixation.

[0157] This suggests that the hydrogen peroxide-based autoclave used is suitable for purifying biosilica (210).

[0158] Referring to Fig. 6(d), the unique porous layered structure of the diatomite biosilica (210) was confirmed through TEM imaging analysis, and it can be seen that this can affect continuous drug release.

[0159] Referring to FIG. 6(e), it can be seen that the diatomaceous earth biosilica (210) has an amorphous structure as no specific lattice pattern is observed in the Fast Fourier Transform (FFT) analysis.

[0160] These characteristics show that diatomite biosilica (210) can have excellent biodegradability and biosensor properties for in vivo applications.

[0161] FIG. 7 illustrates the steps for manufacturing the drug delivery system of the present invention.

[0162] Referring to FIG. 7(a), fluorescence analysis using the intrinsic fluorescence of DOX was performed to quantify the drug loading efficiency of the drug polymer (100) in the drug delivery system of the present invention, and it was confirmed that the EE% was 93.07 ± 3.96%.

[0163] The method (S3) for manufacturing a drug delivery system of the present invention may include a step of electrostatically binding the drug delivery system (200) to a drug polymer (100) on which the drug (110) is loaded by coating the nano-lipid carrier (120) with the chitosan (130) and the drug (110) is loaded, through physical contact such as stirring.

[0164] FIG. 7(b) shows the manufacturing step (S3) for forming the drug delivery system (10) verified by SEM imaging, and FIG. 7(c) shows the manufacturing step (S3) for forming the drug delivery system (10) further verified through EDX analysis.

[0165] As observed in the SEM image, the attachment of the drug polymer (100) was demonstrated by the presence of a lipid layer on the surface of the biosilica (210). Additionally, SEM-EDX analysis of the drug delivery system (10) revealed the presence of a C peak derived from the nanolipid carrier (120) and chitosan (130), in addition to the Si and O peaks which are characteristics of the purified biosilica (210).

[0166] These results indicate that the drug delivery system (200) and the drug polymer (100) were successfully mixed through electrical interaction.

[0167] Referring to FIG. 7(d), the manufacturing step (S3) of this drug delivery system (10) can be verified through zeta potential measurement. It can be seen that the surface charge of the biosilica (210) has shifted from -27.43 ± 2.05 mV to +4.03 ± 2.61 mV of the drug delivery system (10).

[0168] Figure 8 illustrates the verification of the manufacturing steps in a drug delivery system manufacturing method in a different way.

[0169] The adhesion ratio of the drug delivery system (200) and the drug polymer (100) can be optimized through fluorescent labeling using coumarin-6, and a lipid to biosilica (210) ratio of 1:30 based on the lipid content of the drug polymer (100) can be identified as the most suitable composition.

[0170] In summary, as a result of this, it can be seen that the drug delivery system (200) containing diatomite biosilica (210) and the drug polymer (100) containing a cationic nanostructure lipid carrier (120) are successfully combined through the manufacturing step (S3) of the drug delivery system of the present invention.

[0171] The performance of the drug delivery system of the present invention manufactured by the method of manufacturing the drug delivery system of the present invention is described below.

[0172] Diatomous biosilica (210) has a high silica content, which provides strong resistance to various environmental stresses. In addition, its unique nanoscale porous layered structure effectively controls the sustained release of drugs.

[0173] FIG. 9 illustrates the efficacy of the drug delivery system of the present invention.

[0174] To evaluate whether the sustained release characteristics of the drug delivery system (10) can be improved by mixing the nanostructured lipid carrier (120) loaded with a hydrophobic drug (110) with diatomite biosilica (210), the drug release profiles of the nanostructured lipid carrier (120) and the drug delivery system (10) can be compared and analyzed in a 1X PBS and gastric fluid simulation environment.

[0175] Referring to FIG. 9(a), the drug release rates of the nanostructured lipid carrier (120) and the drug delivery system (10) in a 1X PBS environment are 78.43% and 38.54%, respectively.

[0176] Referring to Fig. 9(b), in a gastric fluid simulation environment, the values ​​are 52.25% and 39.59%, respectively.

[0177] These results indicate that the porous layered structure of the diatomite biosilica (210) in the drug delivery system (10) of the present invention induced continuous drug release from the mixed nanostructured lipid carrier (120) even under conditions similar to saline solution and gastric fluid. In other words, it can be seen that the drug delivery system (10) of the present invention can achieve gradual drug release under in vivo conditions.

[0178] The sustained-release characteristics of the drug delivery system (10) of the present invention can be attributed to two main mechanisms.

[0179] First, the multilayer structure of the diatomite biosilica (210) acts as a physical barrier that controls drug diffusion and inhibits release.

[0180] Second, the electrical interaction between lipid nanoparticles (120) and biosilica (210) contributes to the chemical inhibition of drug release.

[0181] These results show that the drug delivery system (10) of the present invention can maintain stable drug release even in complex biological environments.

[0182] Before evaluating the drug delivery efficiency of the drug delivery system (10) of the present invention, the cytotoxicity of the diatomite biosilica (210) and the nanostructured lipid carrier (120) can be evaluated using WST-8 analysis to verify their potential as drug carriers.

[0183] Since high concentrations of biosilica (120) can cause acute inflammatory reactions or tissue damage upon administration, it may be important to determine the upper limit of cytotoxicity in vitro.

[0184] In addition, the cationic surfactant constituting the outer membrane of the nanostructured lipid carrier (120) can cause local irritation, membrane damage, and cell death at high concentrations.

[0185] Similarly, excessive lipid concentrations can lead to side effects such as vascular occlusion and metabolic disorders.

[0186] Therefore, since all three substances constituting the drug delivery system (10) of the present invention exhibit concentration-dependent cytotoxicity, it is important to determine the non-cytotoxic concentration range. To this end, an in vitro cytotoxicity analysis can be performed using mouse fibroblast cell line L929, which is a standard model for the initial evaluation of biocompatibility.

[0187] Referring to Fig. 9(c), the results of the cytotoxicity test showed that in the case of diatomite biosilica (DB), the cell viability exceeded 90% in SK-BR-3 and L929 cells at concentrations of 20 μg / mL and 10 μg / mL or less, respectively.

[0188] Biosilica (210) can be degraded by intravenous silica-degrading enzymes. In particular, amorphous silica such as diatomite biosilica (210) exhibits a superior biodegradation rate compared to conventional silica.

[0189] In the case of the nanostructured lipid carrier (120), SK-BR-3 and L929 cells exhibit negligible cytotoxicity at lipid concentrations of 0.1 mg / mL and 0.05 mg / mL or less, respectively.

[0190] Referring to FIG. 9(d), the concentration of the cationic surfactant (< 0.05 mg / mL) used in the drug delivery system (10) of the present invention does not cause significant cytotoxicity.

[0191] As a result, it can be seen that the drug delivery system (10) of the present invention is harmless to the human body and does not provide toxins to normal cells or the internal environment.

[0192] The drug delivery efficiency of the nanostructured lipid carrier (120) and the drug delivery system (10) can be measured by WST-8 analysis using DOX as a model anticancer agent.

[0193] The concentration of the material used to synthesize each carrier can be determined based on the results of cytotoxicity tests.

[0194] To evaluate the performance of the nanostructured lipid carrier (120), the drug efficacy of DOX (110) and the DOX-cation nanostructured lipid carrier (100) was first investigated. In L929 and SK-BR-3 cell lines, the drug polymer (100) showed higher cytotoxicity than the drug (110) itself, and it was found that there was about a twofold difference in IC 50 values.

[0195] These results mean that the drug (110) is delivered more efficiently through the drug polymer (100) than when it moves independently.

[0196] This improvement in delivery efficiency can be understood as the positive charge characteristics of the nanostructured lipid carrier (120) promoting attachment to the target cell (300) and the hydrophilic chitosan (130) coating improving the endosome escape efficiency.

[0197] To investigate whether the drug delivery performance is affected when the drug delivery agent (200) is combined with the drug polymer (100), the drug polymer (100) and the final drug delivery system (10) of the present invention can be compared.

[0198] The in vitro drug release profile can be seen to confirm that the presence of diatomaceous earth biosilica (210) reduced the initial explosive release typically observed in conventional lipid nanoparticles (120). This reduction in explosive release means minimizing unintended cytotoxic reactions caused by excessive initial drug release.

[0199] Referring to FIGS. 9(e) and FIGS. 9(f), it can be seen that the drug delivery system (10) of the present invention exhibits a higher cell viability than the drug polymer (100) itself.

[0200] These results indicate that the drug delivery system (10) of the present invention has significant potential as a sustained-release formulation for delivering hydrophobic drugs (110). Thus, the drug delivery system of the present invention exhibits excellent drug loading efficiency, controlled release characteristics, and enhanced cell permeability, and can be seen to have potential as a strategy for delivering hydrophobic therapeutic agents.

[0201] In summary, the drug delivery system (10) of the present invention provides a pharmaceutical system that integrates diatomite biosilica (210) and a cationic nanostructured lipid carrier (120) to improve the bioavailability of a hydrophobic anticancer drug (10).

[0202] The method for manufacturing the drug delivery system of the present invention optimizes the particle size and surface charge of the cationic nanostructured lipid carrier (120) as the main drug carrier by adjusting the surfactant concentration during synthesis, which means promoting the efficient delivery of DOX, a model hydrophobic anticancer drug (110). The method for manufacturing the drug delivery system of the present invention further improves the cell adhesion and permeability of the cationic nanostructured lipid carrier (120) by modifying the surface with amphiphilic chitosan (130). Subsequently, the method for manufacturing the drug delivery system of the present invention can provide a drug delivery system that constructs a hybrid drug delivery system platform (lipid carrier complex (DBNC)) by electrically combining diatomite biosilica (120) exhibiting a strong negative charge and the cationic nanostructured lipid carrier (100).

[0203] This provides enhanced sustained-release characteristics and a stable pharmacokinetic profile.

[0204] The drug delivery system (10) of the present invention can verify structural and functional properties using electron microscopy, atomic force microscopy (AFM), and Fourier transform infrared (FT-IR) spectroscopy.

[0205] The drug release profile under biological conditions shows that a mixture of diatomite biosilica (210) and drug polymer (100) significantly extends the release of the drug (110).

[0206] Additionally, in cell-based analysis, the drug polymer (100) shows similar therapeutic efficacy at a lower concentration compared to the drug (110) itself, demonstrating improved drug efficiency.

[0207] The drug delivery system (10) of the present invention can provide the potential of this hybrid drug delivery system platform as a multifunctional drug delivery system that overcomes the limitations of existing single drug carriers. This approach will provide a promising strategy for the delivery of hydrophobic drugs in various therapeutic applications as well as cancer treatment. These results are expected to contribute significantly to future research and clinical development of drug delivery systems.

[0208] The present invention may be modified and implemented in various forms, and its scope of rights is not limited to the embodiments described above. Therefore, if a modified embodiment includes the components of the claims of the present invention, it should be considered to fall within the scope of the present invention. Industrial applicability

[0210] This invention was carried out as a research and development project (task) of the National Nakdong River Biological Resources Center as follows.

[0211] Base Year: 2025

[0212] Project ID: NNIBR20253104

[0213] Project Name: Development of Utilization Technology

[0214] Sub-project number: None

[0215] Project Title: Development of Biomaterials for Drug Delivery Utilizing Porous Ultrastructures of Diatoms

[0216] Principal Investigator: Kwon Dae-ryul

[0217] Project Management Agency Name: National Nakdong River Biological Resources Center

[0218] Project Executing Institution: National Nakdong River Biological Resources Center

[0219] Current year project period: 20250101 ~ 20251231

[0220] No content

[0221] No content Explanation of the symbols

[0222] 10 Drug Delivery Systems 100 drug polymers 110 drugs 120 nanolipid carriers 130 Chitosan 200 drug delivery systems 210 Diatomite Biosilica 300 target cells

Claims

Claim 1 A drug delivery system comprising: a drug polymer containing a drug supported on lipid nanoparticles; a drug delivery vehicle containing biosilica purified from diatomaceous earth and transporting said drug polymer; wherein said drug polymer is positively charged and electrostatically bound to said drug polymer. Claim 2 A drug delivery system according to claim 1, characterized in that the drug comprises hydrophilic doxorubicin (DOX) and is hydrophobized and loaded onto lipid nanoparticles. Claim 3 A drug delivery system according to claim 1, characterized in that the drug polymer is coated with chitosan and charged with a positive charge. Claim 4 A method for manufacturing a drug delivery system comprising: a purification step of obtaining a drug delivery vehicle by purifying biosilica from diatomaceous earth; a manufacturing step of manufacturing a drug polymer by loading a drug onto lipid nanoparticles; and a production step of manufacturing a drug complex by combining the drug delivery vehicle and the drug polymer; wherein the manufacturing step includes coating the surface of the drug polymer with chitosan to charge it with a positive charge, and the production step is characterized by electrostatically binding the positively charged drug polymer to the negatively charged drug delivery vehicle. Claim 5 A method for manufacturing a drug delivery system according to claim 4, wherein the manufacturing step comprises hydrophobizing a hydrophilic drug. Claim 6 A method for manufacturing a drug delivery system according to claim 5, characterized in that the drug comprises doxorubicin (DOX).

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