Nanoparticles

Abstract

The present invention relates to nanoparticles which comprise: a liposome core; a chitosan coating layer on the surface thereof; and bromelain bound to at least a part of chitosan of the chitosan coating layer, thereby facilitating mucus attachment and penetration and having excellent stability.

Description

nanoparticles

[0001] The present invention relates to nanoparticles.

[0002] Nanoparticles are a groundbreaking research topic in life sciences and medicine, attracting particular attention for their potential applications in drug delivery systems. Existing drug delivery systems have limitations in various aspects, including drug stability, biodistribution, and therapeutic efficacy. For example, many drugs are rapidly degraded in the body or eliminated from the body before reaching the desired target site. To overcome these limitations, drug delivery systems utilizing nanoparticles are actively being researched. Nanoparticles offer the advantages of improved drug solubility, stability, and biodistribution through their microscopic size and large surface area, thereby maximizing therapeutic efficacy.

[0003] Liposomes are a widely used nanoparticle in drug delivery systems, spherical structures composed of a phospholipid bilayer. Liposomes possess the ability to entrap water-soluble drugs or transport fat-soluble drugs by inserting them into the bilayer. However, extensive research is needed to improve the inherent stability of liposomes and their target delivery efficiency in the body. For example, liposomes can be rapidly eliminated from the body, and their delivery to the target site can be inefficient. To improve this, surface modification of liposomes is crucial for enhancing their persistence in the body and biocompatibility.

[0004] The present invention aims to provide nanoparticles.

[0005] 1. A nanoparticle comprising a liposome core, a chitosan coating layer on the surface thereof, and bromelain bound to at least a portion of the chitosan of the chitosan coating layer.

[0006] 2. In the above 1, the liposome is a nanoparticle having a particle size of 10 to 500 nm.

[0007] 3. In the above 1, the resoform is a nanoparticle containing a physiologically active substance between the inner core or phospholipids.

[0008] 4. In the above 1, the nanoparticles are covalently bonded to the chitosan.

[0009] 5. A method for producing nanoparticles, comprising: a step of mixing a liposome solution and a chitosan solution to form a chitosan-coated liposome; and a step of binding bromelain to chitosan of the chitosan-coated liposome.

[0010] 6. A method for producing nanoparticles in the above 5, wherein the mixing is performed by adding a stirred liposome solution to a chitosan solution.

[0011] 7. In the above 5, the method for producing nanoparticles is performed by mixing a chitosan-coated liposome solution and a bromelain solution.

[0012] The nanoparticles of the present invention are easily attached to mucus and penetrate the mucosa.

[0013] The nanoparticles of the present invention have excellent stability.

[0014] The nanoparticles of the present invention are easily internalized into cell membranes and can be used as excellent drug delivery vehicles.

[0015] Figure 1. (A) Z-average diameter, PDI, and zeta potential of chitosan-coated and uncoated liposomes according to chitosan concentration. All values ​​were measured by DLS analysis. (B) TEM images of uncoated liposomes and 0.1% (w / v) chitosan-coated liposomes (C1-Lip).

[0016] Figure 2. (A) Apparent microviscosity (η) and membrane fluidity (1 / P) of chitosan-coated liposomes according to the concentration of the chitosan coating solution. (B) Emission spectra of FITC-labeled chitosan-coated liposomes (FITC-CS-Lip) and dual fluorescently labeled liposomes (Nile Red-labeled liposomes coated with FITC-chitosan, FITC-CS-Nile Red-Lip) excited at 450 nm. Insets E and R represent the FRET efficiency and the distance between the donor and acceptor, respectively. C1-Lip and C3-Lip represent uncoated liposomes and chitosan-coated liposomes containing 0.1% and 0.3% (w / v) chitosan, respectively.

[0017] Figure 3. Quantification of liposomes internalized in cells and mucus layers. Caco-2 monocultures and Caco-2 / HT29 (9:1) coculture models were cultured for 12 days. Lip, C1-Lip, and C3-Lip represent uncoated liposomes and chitosan-coated liposomes containing 0.1% and 0.3% (w / v) chitosan, respectively.

[0018] Figure 4. (A) Schematic diagram of the fabrication process of chitosan-coated liposomes decorated with bromelain. Purple represents bromelain, red and blue vesicles represent liposomes, and green represents chitosan. (B) Emission spectra of FITC-labeled chitosan-coated liposomes (C1-FITC-Lip) and Alexa Fluor594-labeled bromelain-decorated FITC-labeled chitosan-coated liposomes (Bro10-AF594-C1-FITC-Lip, Bro05-AF594-C1-FITC-Lip) excited at 450 nm.

[0019] Figure 5. Cumulative transport across the porcine intestinal mucosa of unmodified (Lip), chitosan-coated (C1-Lip, C3-Lip), and bromelain-decorated chitosan-coated (Bro05-C1-Lip, Bro10-C1-Lip, Bro05-C3-Lip, Bro05-C3-Lip) liposomes. Values ​​shown are means ± SD of three experiments.

[0020] Figure 6. Cumulative transport of chitosan-coated (C1-Lip and C3-Lip) and bromelain-decorated chitosan-coated (Bro05-C1-Lip, Bro10-C1-Lip, Bro05-C3-Lip, and Bro05-C3-Lip) liposomes in a Caco-2 cell model. Liposomes were labeled with Nile Red to quantify the amount permeated in paracellular permeability studies. Values ​​shown are the means ± SD of three experiments.

[0021] Figure 7. Confocal fluorescence images (blue, DAPI; red, Nile Red) and corresponding Z-stack images of HT29 cells after incubation with (A) chitosan-coated liposomes (C1-Lip) and (B) bromelain-decorated chitosan-coated liposomes (Bro10-C1-Lip). The yellow line indicates the position of the upper image from the bottom of the cell layer.

[0022] Figure 8. TEM image of 0.5% (w / v) chitosan-coated liposomes.

[0023] The present invention is described in detail below.

[0024]

[0025] The present invention relates to nanoparticles.

[0026] The nanoparticle of the present invention comprises a liposome core, a chitosan coating layer on the surface thereof, and bromelain bound to the chitosan coating layer.

[0027] The nanoparticle of the present invention is a particle with a core-shell structure, with a liposome forming the core.

[0028] The liposome core may be a conventional liposome comprising a phospholipid bilayer.

[0029] Liposomes may contain conventional phospholipids, the type of which is not limited. For example, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 2-dihexanoyl-sn-glycero-3-phosphocholine, 1,2-diheptanoyl-sn-glycero-3-phosphocholine, 1,2-dioctanoyl-sn-glycero-3-phosphocholine, 1,2-dinonanoyl-sn-glycero-3-phosphocholine, 1,2-didecanoyl-sn-glycero-3-phosphocholine, 1,2-diundecanoyl-sn-glycero-3-phosphocholine, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-ditridecanoyl-sn-glycero-3-phosphocholine, 1,2-Dipentadecanoyl-sn-glycero-3-phosphocholine, 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Diheptadecanoyl-sn-glycero-3-phosphocholine, 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-Dinonadecanoyl-sn-glycero-3-phosphocholine, 1,2-Diarachidoyl-sn-glycero-3-phosphocholine, 1,2-Dihenalachidoyl-sn-glycero-3-phosphocholine, 1,2-Dibehenoyl-sn-glycero-3-phosphocholine, 1,2-Ditricosanoyl-sn-glycero-3-phosphocholine, 1,2-Dilignoceroyl-sn-glycero-3-phosphocholine, hydrogenated phosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), L-α-phosphatidylcholine (HSPC), 1-myristoyl-2-steroyl-sn-glycero-3-phosphocholine (MSPC), 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC), soybean phosphatidylcholine (SPC), etc.

[0030] Additionally, the liposomes may contain pegylated phospholipids, such as PEG-linked DMPC, PEG-linked 1,2-dihexanoyl-sn-glycero-3-phosphocholine, PEG-linked 1,2-diheptanoyl-sn-glycero-3-phosphocholine, PEG-linked 1,2-dioctanoyl-sn-glycero-3-phosphocholine, PEG-linked 1,2-dinonanoyl-sn-glycero-3-phosphocholine, PEG-linked 1,2-didecanoyl-sn-glycero-3-phosphocholine, PEG-linked 1,2-diundecanoyl-sn-glycero-3-phosphocholine, PEG-linked DLPC, PEG-linked 1,2-ditridecanoyl-sn-glycero-3-phosphocholine, PEG-linked 1,2-Dipentadecanoyl-sn-glycero-3-phosphocholine, DPPC with PEG, 1,2-Diheptadecanoyl-sn-glycero-3-phosphocholine, DSPC with PEG, DSPE with PEG, 1,2-Dinonadecanoyl-sn-glycero-3-phosphocholine with PEG, 1,2-Diarachidoyl-sn-glycero-3-phosphocholine with PEG, 1,2-Dihenarachidoyl-sn-glycero-3-phosphocholine with PEG, 1,2-Dibehenoyl-sn-glycero-3-phosphocholine with PEG, 1,2-Ditricosanoyl-sn-glycero-3-phosphocholine with PEG It can be 1,2-dilignoceroyl-sn-glycero-3-phosphocholine, PEG-linked hydrogenated phosphatidylcholine, PEG-linked DOPC, PEG-linked HSPC, PEG-linked MSPC, PEG-linked MPPC, PEG-linked SPC, etc.

[0031] When the liposome comprises non-PEGylated phospholipids and PEGylated phospholipids, the ratio is not limited. For example, the non-PEGylated phospholipids and PEGylated phospholipids may be comprised in a weight ratio of 1:0.01 to 2, 1:0.05 to 1.5, 1:0.1 to 1, 1:0.1 to 0.9, 1:0.1 to 0.8, 1:0.1 to 0.7, 1:0.1 to 0.6, 1:0.1 to 0.5, 1:0.1 to 0.4, 1:0.1 to 0.3, or 1:0.1 to 0.2.

[0032] The liposomes may further comprise cholesterol or an analogue thereof. Examples thereof include cholesterol, phytosterol, ergosterol, stigmasterol, campesterol, brassicasterol, sitosterol, stigmasterol, beta-sitosterol, desmosterol, crypsterol, cycloartenol, gamma-sitosterol, campesterol, avemasterol, and the like.

[0033] Liposomes may have particle sizes of, for example, 10 to 500 nm, 20 to 300 nm, or 30 to 300 nm.

[0034] Liposomes can encapsulate bioactive substances in their inner core or between phospholipids.

[0035] It may be a therapeutically active agent capable of providing direct or indirect therapeutic, physiological and / or pharmacological effects to a human or animal organism.

[0036] The therapeutically active agent may be, for example, a general medicine, a drug, a prodrug or targeting agent, or a drug or prodrug comprising a targeting agent.

[0037] The therapeutically active agent may be, for example, a natural product, a peptide, a protein, a compound, a nucleic acid, etc.

[0038] Liposomes can be prepared by methods known in the art, such as thin film hydration, sonication, and emulsification.

[0039] The liposome core has a chitosan coating layer on its surface.

[0040] The chitosan coating layer enhances the mucus adhesion of nanoparticles and facilitates their penetration into the mucosa. It also improves the stability of liposomes.

[0041] The chitosan coating layer is a layer located on the outer surface of the liposome core and contains chitosan.

[0042] Chitosan can have a deacetylation rate of, for example, 85 to 95%.

[0043] Chitosan may have a molecular weight of, for example, 50 to 500 kDa, 100 to 400 kDa, etc.

[0044] The method for forming the chitosan coating layer is not particularly limited, and for example, it can be formed by mixing a liposome solution and a chitosan solution.

[0045] At least a portion of the chitosan in the chitosan coating layer is bound to bromelain.

[0046] Bromelain is an enzyme that breaks down proteins. It is found in foods like pineapple.

[0047] Bromelain promotes cellular uptake and improves intercellular permeability through the mucus layer.

[0048] Bromelain is bound to at least a portion of chitosan, and the type of bond is not limited and may include electrical interaction, covalent bond, non-covalent bond, etc. For example, it may be bound by an EDC-NHS coupling reaction involving an amino group of chitosan and a carboxyl group of bromelain.

[0049] The above at least some means one or more chitosan molecules of the chitosan layer, the upper limit of which may be, but is not limited to, the entire chitosan molecule.

[0050] As described above, the liposome core can carry a bioactive substance, and such nanoparticles can be utilized as a bioactive substance delivery vehicle.

[0051]

[0052] In addition, the present invention relates to a method for producing the nanoparticles.

[0053] The method for manufacturing nanoparticles of the present invention includes a step of mixing a liposome solution and a chitosan solution to form chitosan-coated liposomes; and a step of binding bromelain to chitosan of the chitosan-coated liposomes.

[0054] The solvent of the liposome solution can be water.

[0055] Liposomes may contain any of the ingredients exemplified above.

[0056] Liposomes may have particle sizes as exemplified above.

[0057] The concentration of the liposome solution may be, for example, 0.2 to 2 (w / v)%.

[0058] The solvent for the chitosan solution may be water. The solvent for the chitosan may include an acid. The acid may be, for example, acetic acid.

[0059] The concentration of the chitosan solution may be, for example, 0.025 to 1 (w / v)%, 0.025 to 0.8 (w / v%), or 0.025 to 0.5 (w / v)%.

[0060] Mixing the liposome solution with the chitosan solution forms a chitosan layer on the liposome surface. This can be accomplished, for example, by gradually adding the liposome solution to the chitosan solution. For example, the stirred liposome solution can be added dropwise to the chitosan solution.

[0061] The mixing ratio of liposomes and chitosan is not particularly limited, and may be, for example, a concentration (w / v) ratio of 1: 0.01 to 0.2, 1: 0.01 to 0.1.

[0062] Bromelain is then conjugated to at least a portion of the chitosan in the chitosan-coated liposomes.

[0063] This can be done by mixing a chitosan coated liposome solution with bromelain.

[0064] During mixing, ingredients for binding bromelain may also be mixed. These may be, for example, EDC, sulfo-NHS, etc.

[0065] The solution concentration, mixing ratio, etc. can be appropriately selected to ensure sufficient reaction. To obtain a sufficient bromelain density, an excess of bromelain relative to chitosan can be processed. For example, a bromelain solution can be processed at a concentration of 0.2 to 0.6 mg / ml for a 0.1 (w / v)% chitosan solution.

[0066]

[0067] The present invention is described in detail with reference to the following examples.

[0068]

[0069] Example

[0070] Materials and Methods

[0071] ingredient

[0072] Chitosan (CS, deacetylated 87.6–92.5%, 150–300 kDa) was purchased from High-Tech Grunderfonds (Berlin, Germany). Soy phospholipids (Soy PC) and hydrogenated phosphatidylcholine (HSPC) were obtained from Lipoid® (Ludwigshafen, Germany). Bromelain extracted from pineapple stem (≥3 units / mg protein), cholesterol (≥99%), N-(3-domethylaminopropyl)-N'-ethyl carbodiimide hydrochloride (EDC, ≥98.0%), N-hydroxysulfosuccinimide sodium salt (NHS, ≥98.0%), 2-ethylsulfonic acid (MES, ≥99.0%), fluorescein isothiocyanate (FITC), acetate buffer (pH 5.2), diphenhydramine (DPH), Nile Red, Triton X-100, and Alcian blue were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Folin-Ciocalteu phenol reagent (FCP reagent) was purchased from Deoksan Chemical (Seoul, Korea). Alexa Fluor 594 C5 Malemide was purchased from Thermo Fisher (Waltham, MA, USA).

[0073]

[0074] Preparation of chitosan-coated liposomes

[0075] Liposomes were produced using the thin-film method, which involves evaporating a lipid mixture dissolved in chloroform. The resulting thin film was hydrated in 100 mM acetate buffer at pH 5.2. The final composition of the liposome solution included 0.5% (w / v) HSPC, 0.5% (w / v) soybean PC, 0.3% (w / v) Tween 80, and 0.3% (w / v) cholesterol. The solution was then sonicated using a sonicator (VC 505, Vibracell Sonics, Newton, USA) to miniaturize and homogenize the solution.

[0076] For liposome coating using CS, a 0.5% (w / v) CS solution dissolved in 1% (v / v) acetic acid was prepared. To determine an appropriate coating concentration, the CS solution was further diluted to concentrations of 0.025, 0.05, 0.1, and 0.3% (w / v), respectively. The liposome solutions were then added dropwise to each CS solution at a 1:1 volume ratio with continuous stirring at 120 rpm for 1 h at room temperature.

[0077]

[0078] Particle size and shape of liposomes

[0079] The average hydrodynamic size (Z-average), polydispersity index (PDI), and zeta potential of the liposomes were measured using dynamic light scattering (DLS) analysis using a Zetasizer Nano ZS90 (Malvern instrument, UK). The morphology of each sample was observed by transmission electron microscopy (TEM). The liposome suspension (0.25%, w / v) was applied to a carbon grid and then stained with uranyl acetate (1%, w / v). TEM images were obtained using a JEM 1010 electron microscope (Jeol LTD, Japan).

[0080]

[0081] Membrane microviscosity evaluation using DPH

[0082] To evaluate the membrane fluidity of CS-coated liposomes, a DPH fluorescent probe was used. In a simple procedure, DPH in methanol was added to the liposome solution at a final molar ratio of 1:200 (DPH: lipid). After incubation for 30 min, the solution was stimulated with vertically polarized light at 360 nm, and the emission intensity was measured perpendicular to the excitation light at 430 nm (I 0,90 ) and parallel (I 0,0 ) were all measured. Polarization (P) was determined by the following mathematical formula:

[0083]

[0084]

[0085] The instrument correction factor (G) is vertical (I) for horizontally polarized light at 360 nm. 90,0 ) and parallel (I 90,90 ) was calculated using the emission intensity measured for all membranes. Since the mobility is inversely proportional to the polarization, the membrane mobility is expressed as 1 / P.

[0086] The surface microviscosity (η) was calculated using the steady-state fluorescence anisotropy (r) of DPH:

[0087]

[0088]

[0089] Fluorescence resonance energy transfer analysis

[0090] To verify the success of CS coating, the distance between the polymer and liposome membranes was measured using fluorescence resonance energy transfer (FRET) analysis. FITC-labeled CS (FITC-CS) served as the donor (Ex / Em: 495 nm / 519 nm), and Nile Red-labeled liposomes (NR-Lip) served as the acceptor (Ex / Em: 554 nm / 638 nm). Dual fluorescently labeled liposomes (FITC-CS-NR-Lip) and FITC-CS-coated liposomes (FTIC-CS-NR-Lip) were excited at 450 nm, and their emission spectra were recorded from 490 to 800 nm. The FRET efficiency (E) and the distance between the donor and acceptor (R) were calculated using the formulas:

[0091]

[0092] Above F DA and F D represents the fluorescence intensity in the presence and absence of the receptor, respectively. R o represents the Fφrster distance at 50% transfer efficiency. In the FITC-Nile Red FRET pair, R o is 58.8 Å.

[0093] The binding of bromelain to the CS-coated liposome surface was also confirmed by FRET analysis. In this experimental setup, Alexa Fluor 594-labeled bromelain and FITC-labeled CS were used. FRET efficiency was evaluated by measuring the emission spectrum upon excitation at 519 nm. R for the FITC-Alexa Fluor 594 FRET pair o The value is 58.7 Å.

[0094]

[0095] Mucus adhesion test

[0096] The mucoadhesiveness of CS-Lip was confirmed through cell adhesion studies using both Caco-2 monoculture and Caco-2 / HT29 coculture under mild conditions in a fixed environment. Cells were initially cultured at a density of 3.3 × 10 4 Cells were seeded in 24-well plates at a concentration of 10 cells / mL and cultured for 11 days. After culture, the cells were washed with HBSS buffer and treated with FITC-labeled liposome solution at a concentration of 0.02% (w / v) at 37°C. After 3 h of incubation, the cells were rinsed three times with ice-cold PBS (pH 6.8) to remove free liposomes. Subsequently, the cells were lysed with 4% (v / v) Triton X-100. Cell adhesion was quantified as μg of liposomes per ng of protein. The amount of protein was determined by the Bradford assay.

[0097]

[0098] Preparation of bromelain-conjugated chitosan-coated liposomes

[0099] CS-Lips were prepared using the method described above, denoted C1-Lip and C3-Lip, at CS concentrations of 0.1% and 0.3% (w / v), respectively. To prepare Bro-Lips, the primary amine of CS located on the surface of CS-Lips was conjugated to the carboxyl group of bromelain via an EDC / NHS coupling reaction. To form an intermediate amine-reactive ester from the carboxyl group, 0.3 mg of EDC and 1.1 mg of sulfo-NHS were added per 1 mg of bromelain. The reaction was carried out in MES buffer (0.1 M, pH 6.0) containing 0.5 M NaCl at room temperature for 15 min. Excess EDC was quenched with BME to a final concentration of 20 mM. The reactant solution was then mixed with CS-Lips in a 1:1 volume ratio and incubated at room temperature for 2 h with constant stirring at 120 rpm. To adjust the enzyme density on the liposome surface, two concentrations of bromelain solution (0.1 and 0.2 mg / mL) were used in the conjugation process, designated Bro05-CS-Lip and Bro10-CS-Lip, respectively. Depending on the coating concentration of CS, Bro-CS-Lip is also designated Bro-C1-Lip or Bro-C3-Lip. After the reaction, sulfo-NHS was quenched with Tris salt to a final concentration of 50 mM, and free bromelain was removed using a 100 kDa cutoff size dialysis kit. The conjugation efficiency was determined by measuring the residual protein concentration after dialysis.

[0100]

[0101] Proteolytic activity assay

[0102] A protease assay using casein as a substrate was performed to evaluate the residual activity of bromelain after conjugation. The assay was performed in 50 mM PBS buffer (pH 7.2) containing 0.22% (w / v) casein at 37°C for 10 min with continuous shaking at 150 rpm. The reaction was stopped by adding TCA to a final concentration of 27.5 mM, and the amount of tyrosine produced in the reaction solution was measured using FCP reagent. One unit (U) of bromelain activity was determined as the amount of tyrosine (μmol) produced per unit time (min) at 37°C. The specific enzyme activity (U / mg) was calculated using the following equation:

[0103]

[0104]

[0105] Mucus permeability analysis

[0106] The mucus permeability of the nanoparticles was assessed using a transwell plate assay featuring a dual-compartment chamber consisting of a basolateral and an apical side separated by a microporous membrane. Both sides were filled with 1x PBS buffer at pH 6.8, and the upper side of the membrane was coated with 50 mg of native mucin (10%, w / v) extracted from porcine small intestine. A 24-well plate with a membrane with a 0.4 μm pore size was used (CLS3513, Corning).

[0107] Liposome suspensions with a lipid concentration of 0.1% (w / v) were loaded onto the apical side, and the penetration of liposomes through the mucus layer was quantified by sampling 100 μl from the basal compartment at 1-h intervals and supplementing the same volume of PBS each time. For quantification, all liposome samples were labeled with FITC at a concentration of 0.001% (w / v) and measured (λ) using a microplate reader (Varioskan LUX, Thermo Scientific). ex = 485 nm, λ emThe fluorescence of the recovered sample was measured at 520 nm. The analysis was performed in a shaking incubator at 37°C for 4 hours with constant stirring at 120 rpm.

[0108]

[0109] Intercellular permeability assay

[0110] To assess paracellular permeability, transwell plate assays were performed using Caco-2 and Caco-2 / HT29 (7:3 ratio) models. Caco-2 cells (passage 30-36, KCLB 30037.1) and HT29 cells (passage 35-40, KCLB 30038) were obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were cultured at a concentration of 1X10 5 cells / cm 2 Cells were seeded on each microporous membrane at a density of 150–250 Ω cm and cultured in DMEM medium with 5% CO2, 10% (v / v) FBS, 100 μg / mL streptomycin, and 100 U / mL penicillin at 37°C for 21 days. Throughout the culture period, the culture medium was changed daily. The density and integrity of the cell layer were confirmed by measuring the transepithelial electrical resistance (TEER) with an EVOM epithelial volt-ohm meter (World Precision Instruments, Sarasota, FL, USA). 150–250 Ω cm 2 Cell monolayers exhibiting TEER values ​​within the range were selectively used.

[0111]

[0112] CLSM z-stack analysis

[0113] To determine the mucus permeability of liposomes, Z-stack images representing the cross-sections of mucus and cell layers were acquired using a confocal laser scanning microscope (CLSM) (FV3000, Olympus, Japan). For this analysis, HT29 cells were cultured at a density of 1.25X10 5 cells / cm 2Cells were seeded in confocal dishes at a density of 10 μg / mL and cultured for 12 days. Nile red was incorporated into liposomes, and cell nuclei were stained with DAPI. For C1-Lip, CS-labeled FITC was used. Cells were cultured with a 0.02% (w / v) liposome suspension, fixed with 70% ethanol containing PBS, and z-stack images were scanned.

[0114]

[0115] result

[0116] Physical properties of chitosan-coated liposomes

[0117] Size, zeta potential and morphological characteristics

[0118] The effect of CS coating on liposome membrane properties varies depending on the application range. Membrane fluidity decreases until the polymer chains saturate the surface, but exceeding the maximum application range leads to an increase in fluidity. Since liposome membrane stability improves with fluidity, CS coating at the saturation point demonstrated the highest stability. The CS coating solution is a critical factor affecting coating efficiency. Consequently, liposomes were coated at various concentrations and nanoparticle size, surface charge, and membrane fluidity were evaluated to identify the optimal formulation for CS-coated liposomes (CS-Lip).

[0119] CS coating on liposomes was confirmed by DLS and TEM analyses with increasing CS concentration (Fig. 1). The average particle size increased, except at the lowest concentration. Upon CS coating, the negative zeta potential of uncoated liposomes shifted to a positive value, indicating electrostatic interactions between the cationic polymer and the anionic liposome surface. These observed changes in size and zeta potential suggest the formation of a CS layer, with an increasing degree of coating as the CS concentration increased.

[0120] The CS layer on the liposome surface was visually confirmed by TEM images (Fig. 1D), which displayed a distinct dark gray border compared to bare liposomes (Fig. 1C). Furthermore, the PDI significantly increased with increasing CS concentration. This increase in PDI may be due to the increased viscosity of the surrounding medium, which interferes with liposome dispersion and can lead to visible aggregation. This observation is confirmed by TEM images (Fig. 8), which show significant aggregation of large clumps.

[0121]

[0122] Membrane microviscosity and chitosan coverage on the liposome surface

[0123] To evaluate the effect of CS coating on liposome membrane properties, we utilized DPH, a widely used lipophilic probe known to investigate structural and dynamic aspects of lipid bilayers. DPH fluorescence serves as a measure of microviscosity within anisotropic lipid bilayers, with higher values ​​indicating greater stiffness and stability against liposome aggregation. Figure 2A shows that membrane microviscosity increased after coating with 0.05–0.5% (w / v) CS solutions compared to bare liposomes, reaching a peak at a 0.1% CS concentration. These results suggest that CS coating enhances liposome stability.

[0124] Conversely, membrane fluidity, as assessed by DPH fluorescence polarization, decreased compared to bare liposomes at all CS concentrations except the lowest. This decrease is attributed to the efficient adsorption of polymer chains onto the liposome membrane surface, enhancing membrane rigidity and stability. Interactions between CS and the liposome membrane, including both hydrophobic and electrostatic interactions, contribute to this effect.

[0125] Confirmation of CS coating formation was achieved through FRET analysis using FITC-labeled CS and Nile Red-labeled liposomes (Fig. 2B). Compared to single-probe labeling, the decrease in emission intensity at 519 nm and increase at 628 nm for the dual-fluorescently labeled CS-lip indicated energy transfer between FITC-labeled CS (donor) and Nile Red-labeled liposomes (acceptor). In this context, C1-Lip and C3-Lip represent CS-coated liposomes with 0.1% and 0.3% CS, respectively.

[0126] FRET results provided important insights into the CS coating of liposomes. For C1-Lip, the FRET efficiency was determined to be 59.64%, and the average distance between interaction partners was calculated to be 55.12 Å. These results confirmed the successful CS coating on the liposome surface. Notably, the FRET efficiency of C1-Lip exceeded that of C3-Lip (22.88%), indicating a greater interaction between CS molecules and the liposome surface at a concentration of 0.1%. This difference highlights that the optimal coating efficiency was achieved at a CS concentration of 0.1%.

[0127]

[0128] Mucoadhesiveness of chitosan-coated liposomes

[0129] The mucoadhesive properties of liposomes were evaluated using both Caco-2 monocultures and Caco-2 / HT29 coculture models. Given the mucus-secreting properties of HT29 cells, coculture cell layers were characterized by the presence of a mucus layer. Cell layers were then incubated with FITC-labeled liposomes for quantification purposes.

[0130] As shown in Figure 3, the uptake of CS-Lips was higher in Caco-2 / HT29 cells compared to Caco-2 cells. The increased binding of CS-Lips to Caco-2 / HT29 cells suggests that CS confers mucoadhesive properties to liposomes. Conversely, no significant imbalance in the uptake of bare liposomes was observed between the two cell layers, indicating a lack of mucoadhesion in the absence of CS.

[0131] Additionally, the mucosal adhesion of C1-Lip was superior to that of C3-Lip. This result emphasizes the importance of optimal formulation, as C1-Lip exhibited the highest membrane stiffness and CS coverage among the tested formulations.

[0132]

[0133] Preparation of Bro-CS-Lip: Enzyme Density and Conjugation Efficiency

[0134] Bromelain was bound to the CS-Lip surface via a coupling reaction involving the primary amine group of CS and the carboxyl group of the protein, forming an amide bond promoted by EDC / sulfo-NHS. A schematic diagram of this preparation process and the resulting product, termed Bro-CS-Lip, is shown in Figure 4A. C1-Lip was functionalized with two different concentrations of bromelain, yielding Bro05-C1-Lip and Bro10-C1-Lip, representing the bound bromelain at concentrations of 0.5 mg / mL and 1.0 mg / mL, respectively.

[0135] Confirmation of conjugation between bromelain and CS was achieved through FRET analysis using FITC-labeled CS and Alexa Flour 594-labeled bromelain. Figure 4B shows the emission spectra of liposome solutions excited at 495 nm. Compared to CS-liposomes labeled with a single fluorescent probe, a decrease in emission intensity at 519 nm coupled with an increase at 625 nm was observed for dual-labeled Bro-CS-liposomes. Furthermore, the emission spectrum of Alexa Flour 594-labeled bromelain alone showed minimal interference excited at 495 nm (data not shown), further confirming the specificity of the FRET signal indicating energy transfer from FITC-labeled CS to Alexa Flour 594-labeled bromelain.

[0136] The FRET efficiencies of Bro05-C1-Lip and Bro10-C1-Lip were confirmed to be 47.26% and 76.10%, respectively, indicating a higher density of conjugated bromelain on the surface of Bro10-C1-Lip. The formation of Bro-CS-Lip was further verified by calculating the R value, which represents the average distance between bromelain and CS, using the FRET efficiency. The R values ​​of Bro05-C1-Lip and Bro10-C1-Lip were measured to be 58.73 Å and 48.28 Å, respectively, confirming successful conjugation.

[0137]

[0138] Physicochemical properties of chitosan-coated liposomes decorated with bromelain

[0139] Four formulations of Bro-CS-Lip were prepared based on various concentrations of CS and bromelain: Bro05-C3-Lip and Bro10-C3-Lip represent C3-Lip conjugated with 0.5 mg / mL and 1.0 mg / mL bromelain solutions, respectively. The bromelain density, conjugation efficiency, size, zeta potential, and enzyme activity on the liposome surface of each liposome are summarized in Table 1. The conjugation efficiency exceeded 50% for all formulations. Notably, the formulation with a higher bromelain concentration achieved approximately twice the bromelain density compared to the formulation with a lower concentration, which is consistent with the observed FRET efficiency.

[0140] Bro-C1-Lip and Bro-C3-Lip exhibited particle sizes of approximately 220 nm and 260 nm, respectively, which were slightly increased compared to CS-Lip (Table 1). The observed PDI values ​​were also consistent with the trends observed in the C1-Lip and C3-Lip formulations. These results suggest that the original properties of CS-Lip were consistently maintained after bromelain conjugation in all groups, indicating that the degree of conjugation did not significantly affect these properties and that liposomes could maintain their unique characteristics.

[0141] The residual proteolytic activity of the complex bromelain decreased within the range of 25.5% to 40.0%. This decrease in activity may be due to structural changes in the active site and the formation of an amide bond with CS during the conjugation process. The total activity per unit volume of a 0.1% (w / v) liposome solution was found to be proportional to the enzyme density.

[0142]

[0143]

[0144] Physicochemical properties of bromelain-decorated chitosan-coated liposomes 1) Enzyme payload per unit lipid weight of liposomes 2) Ratio of fixed bromelain content to initial amount 3) Remaining enzyme activity after conjugation 4) Total enzyme activity of 0.1% (w / v) liposomes

[0145]

[0146] Mucus permeability of chitosan-coated liposomes decorated with bromelain

[0147] To evaluate the effects of mucoadhesiveness and mucolytic activity of liposomal carriers on mucus permeability, a transwell plate assay was performed using natural porcine intestinal mucin (Figure 5). CS-Lips, characterized by single-character mucoadhesiveness, exhibited reduced permeability compared to bare liposomes. This reduced transport suggests that the mucoadhesive nature of the nanocarriers may hinder their ability to penetrate mucus due to strong interactions with mucin proteins.

[0148] However, Bro-CS-Lips, which possess the dual properties of mucoadhesiveness and mucolytic activity, showed significantly enhanced mucus penetration compared to bare liposomes and CS-Lip. This suggests that the proteolytic activity of bromelain on the mucin matrix enhances the mucus penetration of CS-Lip.

[0149]

[0150] Transcellular permeability of chitosan-coated liposomes decorated with bromelain

[0151] The paracellular transport of CS-Lips and Bro-CS-Lips across Caco-2 and Caco-2 / HT29 cell layers is shown in Figure 6. Both CS-Lips and Bro-CS-Lips showed increased permeability toward Caco-2 / HT29 compared to Caco-2 cell layers. This finding suggests that mucus-adhesive liposomal vehicles have the advantage of being retained in the mucus layer, thereby increasing their potential for transport across cell layers. Furthermore, the presence of bromelain on the liposome surface enhances permeability through enzymatic mucolytic activity. Bro10-C1-Lip showed significantly higher permeability, particularly toward the Caco-2 / HT29 coculture layer.

[0152] Apparent permeability coefficients (P) of each liposome formulation for both Caco-2 and Caco-2 / HT29 cell models app ) was determined from the results of paracellular permeability (Table 2). In both Caco-2 and Caco-2 / HT29, all formulations of Bro-CS-Lip showed higher (P app ) values ​​were consistently observed. This indicates that the presence of bromelain enhances the cellular uptake and mucus permeation of liposomes. Bro10-C1-Lip demonstrated significantly enhanced permeability, especially in both cell layers, and P app The values ​​increased 6.50-fold and 2.53-fold for Caco-2 and Caco-2 / HT29, respectively.

[0153] The permeability of C1-Lip was found to be lower than that of Bro10-C1-Lip. To gain further insight into the behavior of these liposomes within the mucus layer, both C1-Lip and Bro10-C1-Lip were exposed to HT29 cell layers. For visualization using CLSM, all liposome formulations were labeled with Nile Red, while C1-Lip was additionally formulated with FITC-labeled CS.

[0154] As shown in Figure 7, strong red and weak green signals were detected in the mucus layer, indicating that C1-Lip was entrapped within the mucus matrix. In contrast, when cells were treated with Bro-C1-Lip, red fluorescence was primarily observed within the cell layer. This suggests that liposomes successfully penetrated the mucus layer and were internalized into the cell membrane. Conversely, a significant portion of C1-Lip remained outside the cell membrane, remaining intact within the upper mucus layer.

[0155] These findings highlight that while strong mucoadhesion facilitates adhesion to the mucosa, it can also hinder mucosal penetration. Therefore, surface modification with mucolytic agents could potentially overcome these limitations and enhance mucus permeability.

[0156]

[0157]

[0158] Permeability values ​​for chitosan-coated liposomes (CS-Lip) and bromelain-decorated chitosan-coated liposomes (Bro-CS-Lip) 1) Enhanced permeation of Bro-CS-Lips compared to CS-Lips

Claims

1. A nanoparticle comprising a liposome core, a chitosan coating layer on its surface, and bromelain bound to at least a portion of chitosan of the chitosan coating layer.

2. In claim 1, the liposome is a nanoparticle having a particle size of 10 to 500 nm.

3. In claim 1, the resoform is a nanoparticle containing a physiologically active substance between the inner core or phospholipids.

4. A nanoparticle according to claim 1, wherein the bromelain is covalently bonded to the chitosan.

5. A method for producing nanoparticles, comprising: a step of mixing a liposome solution and a chitosan solution to form a chitosan-coated liposome; and a step of binding bromelain to chitosan of the chitosan-coated liposome.

6. A method for producing nanoparticles according to claim 5, wherein the mixing is performed by adding a stirred liposome solution to a chitosan solution.

7. A method for producing nanoparticles according to claim 5, wherein the binding of bromelain is performed by mixing a chitosan-coated liposome solution and a bromelain solution.

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