Iodine-based nanoparticles manufactured by electron beam irradiation, method for manufacturing same, and medical use thereof

Abstract

The present invention relates to a novel synthesis method for iodine-based nanoparticles and, more specifically, to iodine-based nanoparticles manufactured by electron beam irradiation, a method for manufacturing same, and a medical use thereof. Unlike conventional chemical synthesis methods, the method for manufacturing iodine-based nanoparticles, according to the present invention, can rapidly mass-produce small- and uniform-sized nanoparticles with a simple synthesis method using electron beam irradiation, and the iodine-based nanoparticles manufactured thereby are loaded with melittin and conjugated with transferrin, and thus can be used as a contrast agent for diagnostic imaging, as a drug delivery platform targeting a transferrin receptor, and as a therapeutic agent capable of treating vascular diseases by delivering drugs such as melittin.

Description

Iodine-based nanoparticles produced by electron beam irradiation, method of production thereof, and medical uses thereof

[0001] The present invention relates to a novel synthesis method for iodine-based nanoparticles, and more specifically, to iodine-based nanoparticles produced using electron beam irradiation, a method for producing the same, and medical applications thereof.

[0002] Research on iodine contrast agents has evolved from high osmolarity (ionic monomers) to low osmolarity (nonionic monomers, ionic dimers) and isosmolarity (nonionic monomers) to ensure more comfortable and safer patient administration. However, a disadvantage of these commercial iodine X-ray contrast agents is their rapid renal clearance. Consequently, there are limitations in imaging the biodistribution of blood vessels and tissues. Furthermore, due to this rapid renal clearance, these iodine contrast agents are used at high concentrations to obtain high-quality X-ray images, leading to nephrotoxicity.

[0003] Poly(ethylene glycol) (PEG) is the most widely used polymer in the field of polymer-based drug delivery and is the gold standard for stealth polymers because it can evade immune recognition, thereby extending circulation time in the body and providing an opportunity to reach targets. Modifying nanoparticles with polymeric materials such as PEG is an efficient method to reduce interactions with phagocytes and extend circulation time. This improves the stability of formulations during storage and use. Changes in drug pharmacokinetics extend blood circulation, allowing the drug to reach the desired location before being detected as a foreign substance and eliminated from the body, thereby enhancing therapeutic efficacy. To date, only PEG-functionalized products have been available on the market as polymer-based stealth drug delivery systems.

[0004] Nanotechnology plays a crucial role in diagnosing and treating diseases such as solid tumors, atherosclerosis, and infections. Among various nanoparticle synthesis methods, radiation treatment offers a clean and additive-free approach for manufacturing novel materials based on non-toxic and biodegradable natural polymers. Furthermore, the use of radiation offers numerous advantages: for instance, the manufacturing process is easy to execute with high reliability, generates no waste, and requires short processing times. Due to its high production speed and productivity, this method is commonly used in industrial applications. Since bacteria and viruses can be killed at low radiation doses, it can be utilized for sterilization purposes. Additionally, it is widely used to modify the physical, chemical, and biological properties of materials. Compared to chemical methods, radiation treatment offers advantages such as eco-friendly synthesis, rapid processing, high purity, and low toxicity resulting from the use of low-toxicity chemicals. Consequently, it enables a wide range of applications, including crosslinking, curing, grafting, sterilization, polymer degradation, and nanoparticle formation. For example, inorganic nanoparticles can be formed by irradiating an aqueous solution containing suitable precursors, such as Fe3O4 nanoparticles, silver nanoparticles, and gold nanoparticles, with gamma rays or accelerated electrons at room temperature. The size of the prepared particles can be controlled depending on the dose, dose rate, and composition of the irradiated solution.

[0005] The currently accepted procedures for synthesizing iodine-based nanoparticles involve using chemicals for oxidation, polymerization, and crosslinking, but there is still a need for synthesis technologies that can produce small, homogeneous nanoparticles for use in industrial and medical environments more easily, quickly, and simply.

[0006] The objective of the present invention is to provide a new method for synthesizing iodine-based nanoparticles.

[0007] Another objective of the present invention is to provide iodine-based nanoparticles prepared by the above method and their medical uses.

[0008] To achieve the above objective, the present invention provides a method for producing iodine-based nanoparticles, comprising the steps of: preparing a mixed solution by mixing iopamidol, an iodine-based contrast agent, and polyethylene glycol (PEG), a biocompatible polymer, in water at a concentration ratio of 1: (0.1 to 10); and obtaining iodine-polymer nanoparticles by irradiating the prepared mixed solution with an electron beam, wherein the step of obtaining the nanoparticles is performed by irradiating the electron beam with a beam energy of 1 to 3 MeV, a beam current of 3 to 5 mA, a radiation dose of 5 to 10 kGy, and a velocity of 5 to 15 m / min to obtain nanoparticles having an average particle size of 1 to 20 nm.

[0009] The above manufacturing method may further include the step of conjugating melittin to the obtained iodine-polymer nanoparticles to obtain melittin-loaded iodine-polymer nanoparticles, and may further include the step of conjugating transferrin to the obtained melittin-loaded iodine-polymer nanoparticles to obtain transferrin-bound melittin-loaded iodine-polymer nanoparticles.

[0010] The present invention provides an iodine-based nanoparticle prepared according to the above-described manufacturing method, wherein the carboxyl group of the iodine-polymer nanoparticle formed from iopamidol and polyethylene glycol is joined with the amine group of the melittin to load the iodine-polymer nanoparticle, and the amine group of the melittin is joined with the carboxyl group of the transferrin to the iodine-polymer nanoparticle loaded with the melittin to further bind the transferrin to the melittin, and the nanoparticle is spherical in shape with an average diameter of 1 to 20 nm and a negative surface charge.

[0011] The above iodine-based nanoparticles are non-cytotoxic and bio-stable, and can target macrophages and vascular endothelial cells.

[0012] The present invention provides a contrast agent for imaging diagnosis comprising the above-mentioned nanoparticles and characterized by targeting the transferrin receptor of the cell.

[0013] The present invention provides a pharmaceutical composition for the prevention or treatment of vascular disease comprising the above-mentioned nanoparticles, wherein the nanoparticles target transferrin receptors of the cells, and the vascular disease is one or more cerebrovascular diseases selected from the group consisting of Alzheimer's disease, atherosclerosis, stroke, cerebral hemorrhage, cerebral infarction, and cerebral aneurysm.

[0014] In addition, the present invention provides a drug delivery composition comprising the above-mentioned nanoparticles and characterized by targeting the transferrin receptor of the cell to promote drug delivery.

[0015] The method for manufacturing iodine-based nanoparticles according to the present invention is an efficient and sustainable green synthesis method that, unlike conventional chemical synthesis methods relying on oxidation, polymerization, and crosslinking agent reagents, can rapidly mass-produce nanoparticles of small and uniform size using only water as a solvent and a simple synthesis method of electron beam irradiation without a purification process to remove organic solvents.

[0016] The iodine-based nanoparticles produced by the manufacturing method according to the present invention are loaded with melittin and bound to transferrin, and can be utilized as a contrast agent for imaging diagnostics such as X-rays or CT, as a drug delivery platform targeting transferrin receptors, and as a therapeutic agent capable of treating vascular diseases by delivering drugs such as melittin.

[0017] Figure 1 shows the results of dynamic light scattering (DLS) analysis of iopamidol (IOP)-NH2PEGCOOH 2 kDa nanoparticles prepared according to one embodiment of the present invention, said nanoparticles were prepared by irradiating a mixture of iopamidol 0.25% and NH2PEGCOOH 2 kDa (0.25, 0.5, 1%) (AC) aqueous solutions with an electron beam of 5 kGy and 1 MeV.

[0018] Figure 2 shows the DLS analysis results of iopamidol-NH2PEGCOOH 2 kDa nanoparticles prepared by irradiating a mixture of iopamidol 0.25% and NH2PEGCOOH 2 kDa (0.25, 0.5, 1%) (AC) aqueous solutions with an electron beam of 10 kGy and 1 MeV.

[0019] Figure 3 shows the DLS analysis results of iopamidol-NH2PEGCOOH 2 kDa nanoparticles prepared by irradiating a mixture of iopamidol 0.25% and NH2PEGCOOH 2 kDa (0.25, 0.5, 1%) (AC) aqueous solutions with an electron beam of 50 kGy and 1 MeV.

[0020] Figure 4 shows the DLS analysis results of iopamidol-NH2PEGCOOH 2 kDa nanoparticles prepared by irradiating a mixture of 0.25% iopamidol and 1% NH2PEGCOOH 2 kDa aqueous solutions with an electron beam of 100 kGy and 1 MeV.

[0021] Figure 5 shows the DLS analysis results (A) and transmission electron microscope (TEM) images (B, C) of iopamidol-NH2PEGCOOH 2 kDa nanoparticles prepared by irradiating a mixture of 0.25% iopamidol and 1% NH2PEGCOOH 2 kDa aqueous solutions with an electron beam of 5 kGy and 2.5 MeV.

[0022] Figure 6 shows the DLS analysis results (A), TEM image (B), and zeta potential (C) of iopamidol-NH2PEGCOOH 2 kDa nanoparticles prepared by irradiating a mixture of 0.25% iopamidol and 1% NH2PEGCOOH 2 kDa aqueous solutions with an electron beam of 10 kGy and 2.5 MeV.

[0023] Figure 7 shows iopamidol-NH2PEGCOOH 2 kDa nanoparticles through elemental mapping.

[0024] Figure 8 shows the FT-IR analysis results of iopamidol (IOP) (A), NH2PEGCOOH (B), and iopamidol-NH2PEGCOOH nanoparticles (IOP-NH2PEGCOOH NPs).

[0025] Figure 9 shows the DLS analysis results of iopamidol-PEG 8 kDa nanoparticles prepared by irradiating an aqueous solution of iopamidol 0.25% and PEG 8 kDa (0.1, 0.25, 0.5, 1%) (AD) at various concentrations with an electron beam of 5 kGy and 1 MeV.

[0026] Figure 10 shows the DLS analysis results of iopamidol-PEG 8 kDa nanoparticles prepared by irradiating an aqueous solution of iopamidol 0.25% and PEG 8 kDa (0.1, 0.25, 0.5, 1%) (AD) at various concentrations with an electron beam of 10 kGy and 1 MeV.

[0027] Figure 11 shows the DLS analysis results of iopamidol-PEG 8 kDa nanoparticles prepared by irradiating an aqueous solution of iopamidol 0.25% and PEG 8 kDa (0.1, 0.25, 0.5, 1%) (AD) at various concentrations with an electron beam of 50 kGy and 1 MeV.

[0028] Figure 12 shows the DLS analysis results (A) and TEM image (B) of iopamidol-PEG 8 kDa nanoparticles prepared by irradiating an aqueous solution mixed with 0.25% iopamidol and 1% PEG 8 kDa with an electron beam of 5 kGy and 1 MeV.

[0029] Figure 13 shows the DLS analysis results (A) and TEM image (B) of iopamidol-PEG 8 kDa nanoparticles prepared by irradiating an aqueous solution of 0.25% iopamidol and 1% PEG 8 kDa with an electron beam of 10 kGy and 1 MeV.

[0030] Figure 14 shows the DLS analysis results (A) and TEM image (B) of iopamidol-PEG 8 kDa nanoparticles prepared by irradiating an aqueous solution mixed with 0.25% iopamidol and 1% PEG 8 kDa with an electron beam of 5 kGy and 2.5 MeV.

[0031] Figure 15 shows the characteristics of melittin-loaded iodine nanoparticles (MeLiN), where DLS analysis shows the hydrodynamic size (7 nm) (A) and zeta potential (+3.75 mV) (B) of MeLiN, TEM shows well-dispersed spherical nanoparticles (size, 5-10 nm) (C), and MALDI-TOF demonstrated that melittin was successfully loaded onto the iodine nanoparticles via the EDC coupling method (D).

[0032] Figure 16 shows the characteristics of transferrin-bound melittin-loaded iodine nanoparticles (Tf-MeliN), where DLS analysis shows the hydrodynamic size (15 nm) (A) and zeta potential (-2.88 mV) (B) of Tf-MeLiN, TEM shows well-dispersed spherical nanoparticles (size, 5-10 nm) (C), and MALDI-TOF demonstrated successful conjugation between transferrin and melittin-loaded iodine via the EDC coupling method (D).

[0033] Figure 17 shows the biosafety profile of Tf-MeLiN, in which blood samples were evaluated at 48 hours, 1 week, and 1 month after a single dose injection of 0.1 mL Tf-MeLiN (0.3 mg / mL melittin, 0.9 mg I / mL) into healthy mice. Figures 17(A) to (C) show the results for aspartate aminotransferase (AST), alanine aminotransferase (ALT), and blood urea nitrogen (BUN) levels in each experimental group, and Figures 17(D) to (K) show the total blood cell counts in each experimental group; data are presented as mean ± SEM (n=3). After using one-way ANOVA, p-values ​​were calculated using Dunnett's post-hoc comparison (ns, not significant).

[0034] Figure 18 shows the cell viability of RAW 264.7 (A) and C166 (B) cells treated with Tf-MeLiN and free melittin for 24 hours, and (C) shows the change in in vitro melittin drug release from Tf-MeLiN at 37°C and 4°C for up to 72 hours, with data presented as mean ± SEM.

[0035] Figure 19 confirms the phagocytic activity and transferrin receptor targeting efficacy of Tf-MeLiN. The top image is an immunofluorescence image showing the phagocytic activity of Tf-MeLiN by RAW 264.7 cells (A) and C166 cells (B) after 24 hours of treatment. Cells were treated with Tf-MeLiN and FITC-conjugated Tf-MeLiN and compared to a control group. The bottom image is a representative immunofluorescence image of transferrin receptor staining in RAW 264.7 cells (C) and C166 cells (D) upon Tf-MeLiN treatment, where 4',6-diamidino-2-phenylindole (DAPI) was used to stain the cell nuclei (blue). Fluorescein isothiocyanate (FITC) indicates the presence of Tf-MeLiN in the samples (green). The transferrin receptor (Tf-Receptor) was stained with a red fluorescent antibody. Differential interference contrast (DIC) was used to enhance the contrast of unstained clear samples (scale bar = 50 μm).

[0036] The present invention will be described in detail below.

[0037]

[0038] The inventors prepared iodine-based nanoparticles using a novel synthesis method utilizing electron beam irradiation, and completed the present invention by confirming drug delivery efficacy through the combination of melittin, which has therapeutic potential, and transferrin, which can target and pass through the blood-brain barrier (BBB).

[0039]

[0040] The present invention provides a novel method for synthesizing iodine-based nanoparticles using electron beam irradiation.

[0041] More specifically, a method for producing iodine-based nanoparticles according to the present invention may include the step of preparing a mixed solution of an iodine-based contrast agent and a biocompatible polymer; and the step of obtaining iodine-polymer nanoparticles by irradiating the prepared mixed aqueous solution with an electron beam.

[0042] In the manufacturing method according to the present invention, the step of preparing the mixed solution can be performed by adding the iodine-based contrast agent and the biocompatible polymer to water and mixing them.

[0043] The above iodine-based contrast agent may be iopamidol, but is not limited thereto.

[0044] The above biocompatible polymer may be selected from the group consisting of polyethylene glycol (PEG) and derivatives thereof, and preferably may be polyethylene glycol with an average molecular weight of 8 kDa or less, but is not limited thereto.

[0045] The above iodine-based contrast agent and the above biocompatible polymer may be mixed in a concentration ratio of 1: (0.1 to 10), preferably in a concentration ratio of 1: (0.4 to 4), but are not limited thereto.

[0046] In the manufacturing method according to the present invention, the step of obtaining the iodine-polymer nanoparticles can be performed by irradiating the prepared mixed solution with an electron beam, wherein the electron beam irradiation can be performed with a beam energy of 1 to 3 MeV, a beam current of 3 to 5 mA, a dose of 5 to 10 kGy, and a velocity of 5 to 15 m / min.

[0047] With the molecular weight of the polymer, the concentration ratio of the mixed solution, and the electron beam irradiation conditions as described above, the iodine-polymer nanoparticles can be produced into small particles with an average particle size of 1 to 20 nm.

[0048]

[0049] The manufacturing method according to the present invention may further include the step of conjugating melittin to the obtained iodine-polymer nanoparticles to obtain melittin-loaded iodine-polymer nanoparticles.

[0050] The above-mentioned melittin is a positively charged cell-lytic peptide present in bee venom, and has remarkable therapeutic potential such as anti-inflammatory, antioxidant, and antibacterial activity, promotion of angiogenesis, and smooth muscle relaxation.

[0051] More specifically, the step of obtaining the melittin-loaded iodine-polymer nanoparticles may include: dispersing the obtained iodine-polymer nanoparticles in a buffer solution; adding N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride [N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, EDC] and N-hydroxysuccinimide (N-Hydroxysuccinimide, NHS) to the buffer solution in which the nanoparticles are dispersed and stirring; and adding melittin to the stirred solution.

[0052] The above buffer solution may be a 2-(N-morpholino)ethanesulfonic acid [(2-(N-morpholino)ethanesulfonic acid, MES] buffer, but is not limited thereto.

[0053] That is, the step of obtaining the above-mentioned melittin-loaded iodine-polymer nanoparticles can be performed by activating the carboxyl groups of the iodine-polymer nanoparticles through the coupling reaction of EDC / NHS and binding them to the amine groups of melittin.

[0054]

[0055] In addition, the manufacturing method according to the present invention may further include the step of conjugating transferrin to the melittin-loaded iodine-polymer nanoparticles to obtain transferrin-bound melittin-loaded iodine-polymer nanoparticles.

[0056] The above transferrin is a glycoprotein found in vertebrates that binds to iron (Fe) in the blood and mediates the transport of iron.

[0057] More specifically, the step of obtaining the transferrin-bound melittin-loaded iodine-polymer nanoparticles may include: dispersing the obtained melittin-loaded iodine-polymer nanoparticles in a buffer solution; adding N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride [N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, EDC] and N-hydroxysuccinimide (N-Hydroxysuccinimide, NHS) to the buffer solution in which the nanoparticles are dispersed and stirring; and adding transferrin to the stirred solution.

[0058] The above buffer solution may be NaHCO3 buffer, but is not limited thereto.

[0059] That is, the step of obtaining the transferrin-bound melittin-loading iodine-polymer nanoparticles can be performed by activating the carboxyl group of transferrin through the coupling reaction of EDC / NHS to bind to the amine group of melittin of the melittin-loaded iodine-polymer nanoparticles.

[0060]

[0061] The present invention provides iodine-based nanoparticles produced by the above-described manufacturing method.

[0062] The above nanoparticles may be spherical in shape and have an average particle size of 1 to 20 nm.

[0063]

[0064] The present invention provides iopamidol-PEG nanoparticles prepared by the aforementioned electron beam irradiation method, preferably iopamidol as an iodine-based contrast agent and polyethylene glycol (PEG) as a biocompatible polymer. The nanoparticles may be transferrin-bound melittin-loading iodine-polymer (transferrin-militin-iopamidol-PEG) nanoparticles, wherein melittin is loaded and transferrin is further bound to the melittin.

[0065] The above nanoparticles can reduce interaction with phagocytes and extend circulation time in the body by using polyethylene glycol, have biological stability due to non-cytotoxicity, release melittin—a therapeutic substance—even after 4 hours at 37°C, and can target transferrin receptors; thus, these multifunctional nanoparticles can be utilized as contrast agents for imaging diagnostics, disease treatments characterized by the overexpression of transferrin receptors, and drug delivery platforms.

[0066]

[0067] Accordingly, the present invention provides a contrast agent for imaging diagnosis comprising the above-mentioned nanoparticles.

[0068] The above contrast agent can be used in imaging diagnostic examinations and procedures for X-ray or computed tomography (CT).

[0069]

[0070] The present invention provides a pharmaceutical composition for the prevention or treatment of vascular diseases comprising the above-mentioned nanoparticles.

[0071] The above-mentioned vascular disease is a disease characterized by the overexpression of transferrin receptors, and refers collectively to a condition in which blood circulation is not smooth due to narrowing of blood vessels, such as cerebral blood vessels, caused by some reason. Preferably, it may be a cerebrovascular disease such as Alzheimer's disease, atherosclerosis, stroke, cerebral hemorrhage, cerebral infarction, or cerebral aneurysm, but is not limited thereto.

[0072]

[0073] The pharmaceutical composition according to the present invention can be prepared according to conventional methods in the pharmaceutical field. The pharmaceutical composition may be combined with a suitable pharmaceutically acceptable carrier depending on the formulation, and may be prepared by further including excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. as needed. The suitable carrier, etc., does not impair the activity and properties of the nanoparticles according to the present invention and may be selected differently depending on the dosage form and formulation.

[0074] The above pharmaceutical composition can be applied in any dosage form, and more specifically, can be formulated and used in oral dosage forms and parenteral dosage forms such as topical preparations, transdermal patches, suppositories, and injections according to conventional methods.

[0075] Among the above oral formulations, solid formulations may be in the form of tablets, pills, powders, granules, capsules, etc., and may be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc., and may also include lubricants such as magnesium stearate and talc in addition to simple excipients. Furthermore, in the case of capsule formulations, in addition to the substances mentioned above, liquid carriers such as fatty oils may be further included. Among the above oral formulations, liquid formulations may include suspensions, liquid formulations, emulsions, syrups, etc., and may include various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.

[0076] The parenteral formulations described above may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, witepsol, macrogol, Tween 61, cacao oil, laurin oil, glycerogelatin, etc., may be used. However, they are not limited thereto, and any suitable formulation known in the art may be used.

[0077] The pharmaceutical composition according to the present invention can be administered in a pharmaceutically effective amount.

[0078] In this specification, "pharmaceuticalally effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause adverse effects.

[0079] The effective dose level of the above pharmaceutical composition may be determined differently depending on factors including the purpose of use, the patient's age, gender, weight and health status, type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. For example, although not constant, it may generally be administered at a dose of 0.001 to 1000 mg / kg, preferably 0.01 to 100 mg / kg, once or several times daily. The above dosage does not limit the scope of the present invention in any way.

[0080] The above pharmaceutical composition may be administered to any animal that may develop vascular disease, and said animal may include, for example, humans and primates, as well as livestock such as cattle, pigs, horses, and dogs.

[0081] The above pharmaceutical composition may be administered via a suitable route of administration depending on the formulation form, and may be administered via various oral or parenteral routes as long as it reaches the target tissue. The method of administration may be administered by conventional methods, such as oral, transdermal, subcutaneous, rectal or intravenous, intramuscular, topical application, respiratory inhalation, intradural or intracerebroventricular injection, without needing to be particularly limited.

[0082] The above pharmaceutical composition may be used alone for the prevention or treatment of vascular diseases, or may be used in combination with surgery or other drug treatments.

[0083]

[0084] In addition, the present invention provides a drug delivery composition comprising the above-mentioned nanoparticles.

[0085] The above composition can target transferrin receptors and pass through the blood-brain barrier (BBB) ​​to deliver drugs, and thus can be used to help treat related diseases characterized by transferrin overexpression more effectively.

[0086] Preferably, the drug is a brain delivery drug, and the related disease may be a cerebrovascular disease, but is not limited thereto.

[0087] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0088]

[0089] <Preparation Example 1> Experimental Materials and Methods

[0090] All compounds were commercially available and used without further purification. All solvents were reagent grade and used as received.

[0091] Polyethylene glycol (PEG) polymers with various molecular weights, such as NH2PEGCOOH 2 kDa (Biochempeg Scientific, USA) and PEG 8 kDa (Code 89510, Sigma-Aldrich), were used. In addition, iopamidol was purchased from MedChemExpress (MCE) (Code HY-B0684), and water (HPLC grade) was purchased from Duksan. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride [N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, EDC] (E7750), N-hydroxysuccinimide (NHS) (Code 8045180100), melittin (M2272), and holo-Transferrin human (T0665) were purchased from Sigma-Aldrich. Amicon Ultra-0.5 centrifugal filter units at 3 kDa (Code UFC 5003) and 50 kDa were used. Fourier transform infrared (FT-IR) spectra were prepared using a PerkinElmer instrument (Frontier) at 400–4000 cm⁻¹. -1It was acquired at the wavelength. Dynamic light scattering (DLS, Zetasizer nano Malvern) measured hydrodynamic magnitude and zeta potential. Mass was analyzed using MALDI-TOF MS (Matrix-assisted laser desorption / ionization time-of-flight mass spectrometer; AB Sciex TOF / TOF™ Series Explorer™ 72110; AB Sciex LLC; Old Connecticut Path Framingham, MA, USA).

[0092]

[0093] <Example 1> Synthesis of Iopamidol (IOP)-Polyethylene Glycol (PEG) (I-PEG) Nanoparticles

[0094] 1-1. Method for Synthesizing Iopamidol-Polyethylene Glycol (I-PEG) Nanoparticles

[0095] Iopamidol solutions with various molecular weights and PEG concentrations were prepared in water (HPLC grade). Subsequently, 650 μL of each solution was added to 12-well culture plates. The plates were arranged in large trays. Then, to prevent dust contamination, the trays were wrapped in a thin plastic film and placed on a conveyor belt. The conveyor moved the plates to the irradiation zone, where an electron beam (EB) was irradiated at a 90° angle. Various radiation doses were obtained by varying the beam current, velocity, and reaction time. The doses were calibrated using a cellulose triacetate thin film. Detailed information regarding the electron beam irradiation conditions is as follows:

[0096] - Beam energy 1 MeV, beam current 3.5 mA, dose 5, 10, 50, 100, 200 kGy, velocity 10 m / min.

[0097] - Beam energy 2.5 MeV, beam current 4.1 mA, dose 5, 10, 50, 100, 200 kGy, velocity 10 m / min

[0098]

[0099] 1-2. Optimization and Characterization

[0100] Referring to Figure 1, it can be seen that when the concentration of NH2PEGCOOH 2 kDa is increased from 0.25 to 1% at the same concentration of iopamidol (0.25%) and the same dose (5 kGy), small nanoparticles around 2 nm have an intensity of 50%, 74%, and 64%, respectively.

[0101] Referring to Figure 2, it can be seen that when the concentration of NH2PEGCOOH 2 kDa is increased from 0.25 to 1% at the same concentration of iopamidol (0.25%) and the same dose (10 kGy), small nanoparticles around 3 nm have an intensity of 24%, 33%, and 55%, respectively.

[0102] Referring to Figure 3, when the concentration of NH2PEGCOOH 2 kDa was increased from 0.25 to 1% at the same concentration of iopamidol (0.25%) and the same dose (50 kGy), no small nanoparticles were formed.

[0103] That is, screening was performed according to the concentration and dose of NH2PEGCOOH 2 kDa, and the following trends in nanoparticle formation were observed: at doses higher than 10 kGy, iopamidol was unstable, released free I2 (purple iodine vapor), and no small nanoparticles were observed (Figs. 3 and 4). The PEG concentration increases support for nanoparticle formation, and PEG acts as a crosslinking agent.

[0104]

[0105] However, since this pilot screening did not show high yield nanoparticle formation, a higher beam energy of 2.5 MeV with better penetration was applied to 1% NH2PEGCOOH 2 kDa.

[0106] Referring to Fig. 5, the distribution of small-sized nanoparticles increased at 5 kGy compared to 1 MeV (A), and hydrodynamic size 4 nm (B) and transmission electron microscope (TEM) size (8-20 nm) exhibited spherical shape in terms of morphology and aggregation (C).

[0107] Referring to Figures 6A and 6B, agreement between DLS and TEM measurements at 10 kGy was achieved, revealing a hydrodynamic size of 3 nm and a morphology characterized by sphericity and excellent dispersion. The hydrodynamic size and distribution intensity were less than 5 nm and 76%, respectively. The zeta potential indicates that the nanoparticles carry a negative charge of 5.15 mV (Figure 6C).

[0108]

[0109] Referring to Fig. 7, the formation of iopamidol-NH2PEGCOOH 2 kDa nanoparticles can be confirmed through elemental mapping that identifies the iodine presented within the nanoparticles.

[0110] Referring to Figure 8, FT-IR analysis of iopamidol (IOP), NH2PEGCOOH, and iopamidol-NH2PEGCOOH nanoparticles (IOP-NH2PEGCOOH NPs) showed that the 2 kDa functional groups of iopamidol and NH2PEGCOOH were still intact on the nanoparticles, which can be further used for conjugation with drugs or antibodies to expand the application of these nanoparticles.

[0111] Specifically, 3220-3450 cm -1 The very strong broadband of the range is attributed to the OH and NH bonds observed in both IOP and IOP-NH2PEGCOOH NPs. 1740 cm⁻¹ -1C=O stretching of the carboxyl group (COOH-), 2881 cm -1 At CH height, 1466 and 1342 cm -1 At CH bending, 1279 and 1097 cm -1 OH and COH elongation was obtained in both NH2PEGCOOH and IOP-NH2PEGCOOH NPs. Therefore, the successful synthesis of IOP-NH2PEGCOOH NPs without change in the major functional group was confirmed.

[0112]

[0113] To investigate the effect of molecular weight of PEG on nanoparticle formation, a higher molecular weight PEG of 8 kDa was applied. High molecular weight PEG can extend blood circulation time.

[0114] Referring to Fig. 9, it can be seen that when the concentration of PEG 8 kDa is increased from 0.1% to 1% at the same concentration of iopamidol (0.25%) and the same dose (5 kGy), the distribution of small nanoparticles around 5 nm increases from 31.6% to 91.8%, respectively.

[0115] Referring to Fig. 10, it can be seen that when the concentration of PEG 8 kDa is increased from 0.1 to 1% at the same concentration of iopamidol (0.25%) and the same dose (10 kGy), the distribution of small nanoparticles around 5 nm increases up to 90.2%.

[0116] Referring to Fig. 11, it can be seen that when the concentration of PEG 8 kDa is increased from 0.1 to 1% at the same concentration of iopamidol (0.25%) and the same dose (50 kGy), small nanoparticles are not formed.

[0117] In other words, screening results according to the concentration and dose of PEG 8 kDa showed the following trend of nanoparticle formation: at doses higher than 10 kGy, iopamidol was unstable and released free I2 (purple iodine vapor). The PEG concentration increased support for nanoparticle formation, and PEG acts as a crosslinking agent.

[0118]

[0119] Afterwards, bulk synthesis was performed under optimal conditions and characterized by transmission electron microscopy (TEM). Referring to Fig. 12, consistent results were obtained between DLS and TEM. Referring to the TEM image in Fig. 13, nanoparticle aggregation was observed at 10 kGy.

[0120] Next, the nanoparticle formation efficiency was verified by gradually varying only the beam energy at the same iopamidol concentration of 0.25%, PEG 8 kDa 1%, and dose of 5 kGy. Referring to Figure 14, as confirmed by DLS and TEM analysis, it can be seen that using a beam energy of 2.5 MeV produces larger nanoparticles (13 nm) with reduced intensity (80%).

[0121]

[0122] <Example 2> Synthesis of Melittin-Loaded Iopamidol-PEG (MeLiN) Nanoparticles

[0123] According to Example 1 above, after electron beam irradiation, the sample solution was freeze-dried to obtain a nanoparticle solid to be used for melittin loading. To this end, an EDC (4 mg) / NHS (6 mg) mixture was added to 880 μL of 50 mM 2-(N-morpholino)ethanesulfonic acid [MES] buffer pH 5.5 containing 1.5 mg I-PEG and stirred for 0.5 hours to activate the carboxyl groups of I-PEG for conjugation with the amine groups of melittin (0.6 mg / 120 μL). The conjugation reaction was carried out at 4°C for 48 hours. Finally, MeLiN was purified using an Amicon Ultra-4 centrifugation filter (3 kDa) and redispersed in 1 mL of water. The successful preparation of MeLiN was characterized by DLS, TEM, zeta potential, and MALDI-TOF MS to measure the mass of the conjugated MeLiN.

[0124]

[0125] Referring to Figures 15(A) and (B), DLS analysis revealed that MeLiN exhibited a hydrodynamic size of 7 nm and a surface charge of 3.75 mV. Notably, these results confirm that melittin-positively charged cell lysis peptides were successfully loaded onto I-PEG NPs by exhibiting larger size and a shift from negative to positive charge compared to iopamidol-PEG nanoparticles (I-PEG NPs). Furthermore, as shown in Figure 15(C), TEM measurements revealed a spherical shape and excellent dispersion at sizes smaller than 10 nm.

[0126] Melittin, a 2840 Da cationic peptide composed of 26 amino acids, exhibits hydrophilic and lipophilic amphiphilic properties due to the uneven distribution of polar and non-polar residues with 4 positive charges at the N-terminus and 2 positive charges at the C-terminus.

[0127] Referring to Fig. 15(D), MALDI-TOF mass spectrometry analysis revealed that the molecular weight of MeLiN was 5,919 Da, which contrasts with the disclosed molecular weight of 2,840 Da. The difference of 3,079 Da observed between MeLiN and pure melittin implies a mass contribution from I-PEG. This calculated mass is consistent with the theoretical mass of I-PEG, proving that melittin was successfully conjugated to I-PEG.

[0128]

[0129] <Example 3> Synthesis of Transferrin-Conjugated Melittin-Loaded Iopamidol-PEG (Tf-MeLiN) Nanoparticles

[0130] To prepare Tf-MeLiN, 5 mg of EDC and 2.5 mg of NHS were conjugated with 500 μg / 500 μL of transferrin in 1 M NaHCO3 buffer (100 μL) at pH 8 and stirred for 0.5 hours to activate the carboxyl groups of transferrin. This promoted conjugation with the amine groups of MeLiN (0.3 mg / 500 μL). The conjugation reaction was carried out at 4°C for 49 hours. Subsequently, Tf-MeLiN was purified using an Amicon Ultra-4 centrifugation filter (50 kDa) and redispersed in 1 mL of water. The successful synthesis of Tf-MeLiN was characterized by DLS, TEM, zeta potential, and MALDI-TOF MS to measure the mass of the conjugated Tf-MeLiN.

[0131]

[0132] Referring to Figures 16(A) and (B), DLS measurements revealed a hydrodynamic size of 15 nm and a surface charge of -2.88 mV. This indicates an increase in size and a transition from positive to negative charge compared to MeLiN nanoparticles, confirming that transferrin is effectively conjugated to MeLiN nanoparticles. Furthermore, as shown in Figure 16(C), TEM analysis revealed a spherical shape of the nanoparticles, demonstrating excellent dispersion with a size of less than 10 nm.

[0133] Transferrin, a 79.57 kDa monomeric glycoprotein, consists of 679 amino acid residues along with an asparagine-linked glycan residue. Referring to Fig. 16(D), MALDI-TOF mass spectrometry analysis revealed that the molecular weight of Tf-MeLiN is 84,954 Da, which differs from the recorded molecular weight of pure transferrin, 79,570 Da. The identified difference of 5,384 Da between Tf-MeLiN and pure transferrin represents the mass imparted by MeLiN. This calculated mass closely matches the mass of MeLiN (5,919 Da), confirming the effective binding of transferrin to MeLiN.

[0134]

[0135] <Experimental Example 1> Confirmation of Biostability of Tf-MeLiN in Healthy Mice

[0136] The biosafety profile of Tf-MeLiN was evaluated by analyzing blood samples obtained from healthy mice at various time points after administration. Blood samples were collected at 48 hours (n=3), 1 week (n=3), and 1 month (n=3) after a single intravenous administration.

[0137] Liver and renal function were evaluated by analyzing serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and blood urea nitrogen (BUN). Hematological abnormalities were evaluated via total blood count.

[0138]

[0139] As a result of comparing the control group with various time intervals investigated, as shown in Figure 17, there were no significant differences in the levels of AST, ALT, BUN, red blood cells (RBC), white blood cells (WBC), platelets, hemoglobin, hematocrit, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and mean corpuscular volume (MCV) between the control group and various time intervals.

[0140] Specifically, the AST value of the control sample (125.7±47.3) showed no significant difference from the AST values ​​at 48 hours (117.3±11.5), 1 week (111.3±45.5), and 1 month (53.3±0.3) after administration. Similarly, the ALT value of the control sample (32.0±2.0) showed no significant difference from the ALT values ​​at 48 hours (32.0±2.1), 1 week (27.7±3.0), and 1 month (34.7±2.2) after administration. The BUN value of the control sample (32.0±2.0) showed no significant difference from the BUN values ​​at 48 hours (32.0±2.1), 1 week (27.7±3.0), and 1 month (34.7±2.2) after administration.

[0141] Similar trends were observed for RBC, WBC, platelets, hemoglobin, hematocrit, MCH, MCHC, and MCV values ​​at all investigated time points. The RBC value of the control sample (9.4±0.2) was similar to the values ​​at 48 hours, 1 week, and 1 month (9.1±0.1, 9.7±0.2, and 9.6±0.1, respectively). The WBC value of the control sample (1.0±0.2) was similar to the values ​​at 48 hours, 1 week, and 1 month (1.0±0.4, 1.7±0.2, and 1.5±0.1, respectively). The platelet value (609.7±73.4) of the control sample was similar to the values ​​at 48 hours, 1 week, and 1 month (653.0±89.2, 508.3±30.8, and 584.7±64.6, respectively). The hemoglobin value (13.9±0.1) of the control sample was similar to the values ​​at 48 hours, 1 week, and 1 month (13.9±0.1, 14.1±0.3, and 13.6±0.2, respectively). The hematocrit value (49.9±0.8) of the control sample was similar to the values ​​at 48 hours, 1 week, and 1 month (47.2±0.3, 51.4±0.8, and 51.4±1.5, respectively). The MCH value of the control sample (14.8±0.2) was similar to the values ​​at 48 hours, 1 week, and 1 month (15.3±0.1, 14.6±0.1, and 14.1±0.1, respectively). The MCHC value of the control sample (27.9±0.6) was similar to the values ​​at 48 hours, 1 week, and 1 month (29.4±0.2, 27.5±0.3, and 26.6±0.4, respectively). The MCV value of the control sample (53.2±1.2) was similar to the values ​​at 48 hours, 1 week, and 1 month (52.1±0.4, 53.1±0.4, and 53.4±0.8, respectively).

[0142]

[0143] <Experimental Example 2> In vitro cytotoxicity test of Tf-MeLiN

[0144] The cytotoxicity of Tf-MeLiN was evaluated on macrophage RAW 264.7 cells and endothelial C166 cells using the Cell Counting Kit-8 assay (CCK-8; Dojindo Molecular Technologies, Rockville, MD, USA) according to the manufacturer's protocol.

[0145] Cells were cultured in HyClone Dulbecco’s Modified Eagle Medium complete medium (DMEM; Cytiva, Marlborough, MA, USA) supplemented with high-concentration glucose. The DMEM complete medium contains 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin (10,000 U / mL, Thermo Fisher Scientific, Waltham, MA, USA). 2 × 10⁶ cells per well in 100 μL of DMEM complete medium 4 Cells were seeded into 96-well culture plates at cell densities and allowed to attach overnight at 37°C. After incubation for 24 hours in a humidified atmosphere containing 5% CO2, the cells were exposed to various concentrations of free melittin (1, 5, 10, 20, 50 μg / mL) and Tf-MeLiN (equivalent doses) for 24 hours at 37°C in a humidified atmosphere containing 5% CO2. After treatment, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated for an additional 1 hour at 37°C.

[0146] Cell viability was measured by absorbance at 450 nm using a microplate reader (Molecular Devices, San Jose, CA, USA). Each experiment was performed three times, and relative cell viability (%) was calculated by normalizing the absorbance of the test sample to the absorbance of the control sample as shown in Equation 1 below:

[0147] <Equation 1>

[0148] Cell viability (%) = [(Abs_sample -Abs_blank) / (Abs_control-Abs_blank )]

[0149] Here, Abs_sample, Abs_control, and Abs_blank are the absorbances of the test, control, and blank samples, respectively.

[0150]

[0151] As shown in Figures 18(A) and (B), CCK-8 analysis results showed an identifiable pattern of dose-dependent cell viability when exposed to free melittin in both RAW264.7 and C166 cell lines.

[0152] In RAW264.7 cells, viability decreased from 1 μg / mL, 97.99% ± 3.50 to 50 μg / mL, 17.12% ± 0.71, whereas in C166 cells, viability decreased from 1 μg / mL, 180.93% ± 24.10 to 50 μg / mL, 35.66% ± 0.39.

[0153] In particular, Tf-MeLiN showed no signs of cytotoxicity at any tested concentration, even at high doses, in contrast to the apparent cytotoxic effects observed in free melittin-treated cells. This suggests that Tf-MeLiN may not penetrate the cell membrane in the same manner as free melittin. Rather, it appears to function as a distinct nanoparticle entity with unique properties, going beyond a simple conjugation of transferrin, melittin, and iodine nanoparticles.

[0154]

[0155] <Experimental Example 3> Drug Release Test

[0156] The methodology for performing the drug release analysis is the same as previously described. Briefly, Tf-MeLiN was dissolved in PBS, and the supernatant was collected at specific intervals (4, 24, 48, and 72 hours) under various temperature conditions (37°C and 4°C). To isolate melittin, the solution was centrifuged at 10,000×g for 10 minutes using an Amicon Ultra-4 centrifuge filter tube (50 kDa). The concentration of released melittin was quantified at 595 nm using a microplate reader (Molecular Devices) relative to a standard calibration curve of bovine serum albumin (BSA) via the Bradford assay kit.

[0157]

[0158] As a result of confirming the behavior of melittin in the Tf-MeLiN formulation through in vitro drug release experiments, referring to Fig. 18(C), a notable initial burst release was observed at a physiological temperature of 37°C, and approximately 19.11% of melittin was released within the first 4 hours. Subsequently, the release rate decreased to approximately 14% over 24 and 48 hours, indicating sustained release due to the development of a diffusion barrier surrounding the drug reservoir.

[0159] In contrast, under low temperature conditions (4°C), a contrasting trend was observed in which drug release increased from 12.7% to 27.4% between the initial 4 and 48 hours. Notably, a deceleration of drug release was observed at the 72-hour mark, which was a decrease of approximately 4.5% and 5.1% for 37°C and 4°C, respectively.

[0160] These findings highlight the efficacy of Tf-MeLiN in mitigating melittin-induced cytotoxicity while simultaneously tuning a finely controlled drug release profile. The controlled release profile, combined with the absence of cell membrane disruption, suggests a potential delivery mechanism for MeLiN targeting specific lesions through various nanoparticle uptake mechanisms by monocytes or macrophages. Confocal microscopy images of Tf-MeLiN-treated macrophage cultures support these findings.

[0161]

[0162] <Experimental Example 4> Confirmation of Phagocytosis by Tf-MeLiN and Receptor Targeting by Transferrin

[0163] Immunofluorescence staining was performed to evaluate the phagocytic activity and transferrin receptor targeting efficacy of Tf-MeLiN in both RAW 264.7 and C166 cell lines.

[0164] After fixing with 4% paraformaldehyde dissolved in PBS (pH 7.4) for 10 minutes, cells were incubated with 0.1% Triton X-100 dissolved in PBS for 10 minutes to induce permeability, followed by washing with PBS three times for 5 minutes each. To prevent non-specific antibody binding, a blocking step was performed, which included incubating with 10% donkey serum dissolved in PBST (PBS containing 0.05% Tween-20) at room temperature for 1 hour. Subsequently, cells were exposed for 2 hours to a primary antibody targeting the transferrin receptor (ab214039, rabbit) dissolved in PBST containing 10% donkey serum in a humidified chamber at room temperature. After washing with PBS three times for 5 minutes each, the cells were treated with a secondary antibody (anti-rabbit, Alexa Fluor 555) dissolved in PBST for 1 hour in a dark room at room temperature. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) for 10 minutes and rinsed with PBS. Coverslips were mounted using a special medium and secured with nail polish to ensure stability during microscopic examination. Samples were stored in a dark room at -20°C or +4°C until imaging.

[0165]

[0166] Referring to Figures 19(A) and (B), immunofluorescence imaging showed phagocytosis of Tf-MeLiN by both RAW 264.7 and C166 cells after 24 hours of exposure. The uptake of Tf-MeLiN was depicted by green fluorescence emanating from fluorescein isothiocyanate (FITC)-bound Tf-MeLiN, which was primarily localized within the cytoplasm. Notably, phagocytosis was more pronounced in RAW 264.7 cells compared to C166 cells.

[0167] Evaluation of the transferrin receptor targeting efficacy was performed using red fluorescent staining. Referring to Figures 19(C) and (D), co-localization of Tf-MeLiN (green) and the transferrin receptor (red) was evident in both cell lines, indicating successful targeting by Tf-MeLiN.

[0168] These findings highlight the potential of Tf-MeLiN as a promising candidate for the treatment of vascular diseases, particularly by targeting transferrin receptors and overcoming the blood-brain barrier (BBB) ​​to facilitate brain-mediated drug delivery.

[0169]

[0170] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A step of preparing a mixed solution by mixing iopamidol, an iodine-based contrast agent, and polyethylene glycol (PEG), a biocompatible polymer, in water at a concentration ratio of 1: (0.1 to 10); and The method includes the step of obtaining iodine-polymer nanoparticles by irradiating the above-prepared mixed solution with an electron beam. A method for producing iodine-based nanoparticles, characterized in that the step of obtaining the nanoparticles is performed by irradiating an electron beam with a beam energy of 1 to 3 MeV, a beam current of 3 to 5 mA, a radiation dose of 5 to 10 kGy, and a velocity of 5 to 15 m / min to obtain nanoparticles having an average particle size of 1 to 20 nm.

2. In Paragraph 1, The above manufacturing method is, A manufacturing method characterized by further including the step of conjugating melittin to the iodine-polymer nanoparticles obtained above to obtain melittin-loaded iodine-polymer nanoparticles.

3. In Paragraph 2, The above manufacturing method is, A manufacturing method characterized by further including the step of conjugating transferrin to the obtained melittin-loaded iodine-polymer nanoparticles to obtain transferrin-bound melittin-loaded iodine-polymer nanoparticles.

4. With iodine-based nanoparticles prepared according to Claim 3, The carboxyl group of the iodine-polymer nanoparticle formed from the iopamidol and polyethylene glycol is joined with the amine group of the melittin, thereby loading the melittin onto the iodine-polymer nanoparticle, and The amine group of the melittin and the carboxyl group of the transferrin are conjugated to the iodine-polymer nanoparticle loaded with the melittin, so that the transferrin is further bound to the melittin, Iodine-based nanoparticles characterized by being spherical nanoparticles with an average diameter of 1 to 20 nm and having a negative surface charge.

5. In Paragraph 4, The above iodine-based nanoparticles are, Iodine-based nanoparticles characterized by having no cytotoxicity and biological stability, and targeting macrophages and vascular endothelial cells.

6. Comprising nanoparticles according to claim 5, A contrast agent for imaging diagnostics characterized by targeting the transferrin receptor of the above-mentioned cell.

7. A pharmaceutical composition for the prevention or treatment of vascular diseases comprising nanoparticles according to claim 5, The above nanoparticles target the transferrin receptor of the cell, and A pharmaceutical composition characterized in that the above-mentioned vascular disease is one or more cerebrovascular diseases selected from the group consisting of Alzheimer's disease, atherosclerosis, stroke, cerebral hemorrhage, cerebral infarction, and cerebral aneurysm.

8. Comprising nanoparticles according to claim 5, A drug delivery composition characterized by promoting drug delivery by targeting the transferrin receptor of the above-mentioned cell.

Images