Patent Application
20230173098
This application claims priority to and the benefit of Korean Patent Application No. 10-2021-0173100, filed on Dec. 6, 2021, and Korean Patent Application No. 10-2022-0074985, filed on Jun. 20, 2022, the disclosures of which are incorporated herein by reference in its entirety.
The present invention relates to a nanoparticle complex for oral administration, a pharmaceutical composition for oral administration for treating brain tumors, including the same, a pharmaceutical composition for oral administration for photothermal therapy (PTT) or photodynamic therapy (PDT), and a method of treating brain tumors using the pharmaceutical composition.
Korea has the fastest aging population in the world, and the demand for drugs for degenerative brain diseases and geriatric diseases in this regard is rapidly increasing. In particular, cancer is the leading cause of death among people aged 65 and over, accounting for 865.4 deaths per 100,000 people, and cerebrovascular disease (410.7), heart disease (332.6), and diabetes (146.6) follow. In particular, according to data published by the Korea Central Cancer Registry in 2013, there were 1,679 cases of brain tumors for both men and women, and by age group, those in their 50s accounted for the most with 17.6%, those in their 60s accounted for 16.5% and followed by those in their 70s with 14.9%, showing a high incidence rate in the older age groups. Frequent administration of intravenous injections with a short half-life to elderly patients may not only induce stress in the patient, but may also make it impossible to expect an effective therapeutic effect. Accordingly, there is an urgent need for developing a brain tumor therapeutic agent for oral absorption, which can provide convenience to patients through oral administration with a long half-life and can prevent stress induced by drug administration.
Meanwhile, there are three major methods for treating brain tumors, the first being a surgical operation, the second being radiation therapy, and the third being chemotherapy, and the like. Currently, drugs such as Avastin, temozolomide, and doxorubicin, which are anticancer agents used in clinical practice, are widely used to treat brain tumors, but they are known to have severe side effects such as nausea and vomiting, stomatitis, diarrhea, abdominal pain, alopecia, and infections (pneumonia and urinary tract infection). As other therapeutic methods, gene therapy, immunotherapy, photothermal therapy (PTT), photodynamic therapy (PDT), and the like are currently being studied.
However, nanoparticles used in photothermal therapy (PTT) have a technical problem in reaching brain tumor tissue with high efficiency through the blood-brain barrier (BBB). Further, photosensitizers used in photodynamic therapy (PDT) have a problem in that they have difficulties in reaching and accumulating in brain tumor tissue with high efficiency due to their low water solubility. In addition, since the nanoparticles or photosensitizers have a disadvantage of having a very low oral absorption rate due to the low pH in the gastrointestinal tract even in therapeutic methods including gold nanoparticles, there is a limitation in utilizing them as therapeutic agents.
Therefore, there is a need for developing a complex capable of being orally administered while including gold nanoparticles suitable for photothermal therapy (PTT) and photodynamic therapy (PDT).
Objects for solving the aforementioned problems are as follows.
An object is to provide a nanoparticle complex for oral administration, in which gold nanoparticles coated with glutathione; a photosensitizer bonded to the gold nanoparticles; and lactoferrin bonded to the gold nanoparticles are bonded.
Further, another object is to provide a pharmaceutical composition for oral administration or a pharmaceutical composition for oral administration for photothermal therapy (PTT) or photodynamic therapy (PDT), including the nanoparticle complex for oral administration.
In addition, another object is to provide a method of treating brain tumors using the pharmaceutical composition for oral administration.
The nanoparticle complex for oral administration according to an aspect of the present invention includes: gold nanoparticles coated with glutathione; a photosensitizer bonded to the gold nanoparticles; and lactoferrin bonded to the gold nanoparticles.
The photosensitizer may have a substituted thiourea group to be disulfide-bonded to gold nanoparticles.
The photosensitizer may be one or more selected from the group consisting of a chlorin-based compound, a porphyrin-based compound, a bacteriochlorin-based compound, a phthalocyanine-based compound, a naphthalocyanine-based compound, and a 5-aminoevuline ester-based compound.
The lactoferrin may be surface-modified with a biocompatible polymer.
The biocompatible polymer may be one or more selected from the group consisting of polyethylene glycol, polycaprolactone, polylactic acid, polyglycolic acid, polylactate-co-glycolic acid, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(L-lactide-co-caprolactone), and poly(L-lactide-co-D-lactide).
The biocompatible polymer may be substituted with a thiol group (—SH) to be disulfide-bonded to gold nanoparticles.
The nanoparticle complex for oral administration may have an average diameter of 5 nm to 20 nm.
The weight ratio of the gold nanoparticles:the photosensitizer:the lactoferrin may be 19.7 to 20.0:90.0 to 88.0:1.
The pharmaceutical composition according to another aspect of the present invention is characterized by including the nanoparticle complex for oral administration, an isomer thereof, or a pharmaceutically acceptable salt thereof.
The pharmaceutical composition for oral administration may be for treating brain tumors.
The pharmaceutical composition for oral administration for photothermal therapy (PTT) or photodynamic therapy (PDT) according to still another aspect of the present invention is characterized by including the nanoparticle complex for oral administration.
The pharmaceutical composition for oral administration for photothermal therapy (PTT) or photodynamic therapy (PDT) may be targeted to brain tumor tissue.
The method of treating brain tumors according to yet another aspect of the present invention is characterized by including: orally administering the pharmaceutical composition for photothermal therapy (PTT) or photodynamic therapy (PDT); forming a region to be treated by accumulating the pharmaceutical composition in brain tumor tissue; irradiating the region to be treated with output light for photodynamic therapy (PDT); and irradiating the region to be treated with output light for photothermal therapy (PTT).
The nanoparticle complex for oral administration of the present invention has not only excellent metal enhanced fluorescence (MFF), but also metal-enhanced reactive oxygen generation (MERos) due to a surface plasmon resonance effect by being bonded to a photosensitizer while having an excellent oral absorption rate by being bonded to lactoferrin.
Further, a pharmaceutical composition including a nanoparticle complex for oral administration can effectively permeate the small intestinal epithelium and the blood-brain barrier without toxicity and can be effectively accumulated in brain tumor tissue to have an excellent photothermal therapy (PTT) or photodynamic therapy (PDT) effect in brain tumor tissue.
In addition, there is an advantage in that brain tumors can be effectively treated by adjusting the order of photothermal therapy (PTT) and photodynamic therapy (PDT), and the like after administering a pharmaceutical composition including a nanoparticle complex for oral administration.
The above and other objects, features and advantages of the present invention will become more apparent to those of ordinary skill in the art by describing in detail exemplary embodiments thereof with reference to the accompanying drawings, in which:
The above objects, other objects, characteristics, and advantages will be easily understood through the following preferred embodiments related to the accompanying drawings. However, the present invention is not limited to the embodiments described herein, and may be implemented in various different forms. Rather, the embodiments introduced herein are provided to make the disclosed content thorough and complete, and sufficiently convey the technical spirit to those skilled in the art.
In the description of each drawing, like reference numerals are used for like constituent elements. In the accompanying drawings, the dimensions of the structures are illustrated while being enlarged compared with actual dimensions for clarity of the present invention. Terms such as first and second may be used to describe various constituent elements, but the constituent elements are not limited by the terms. The terms are used only to distinguish one constituent element from another constituent element. For example, without departing from the scope of the invention, a first constituent element may be called a second constituent element, and similarly, the second constituent element may be called the first constituent element. Singular expressions include plural expressions unless the context clearly indicates otherwise.
In the present specification, it will be appreciated that the term “include” or “have” is intended to designate the presence of characteristics, numbers, steps, operations, constituent elements, and parts described in the specification or combinations thereof, and does not exclude in advance the possibility of the presence or addition of one or more other characteristics, numbers, steps, operations, constituent elements, and components, or combinations thereof. Furthermore, a case where a part such as a layer, a film, a region, and a plate is present “on” another part includes not only a case where the part is present “directly on” another part, but also a case where still another part is present therebetween. Conversely, a case where a part such as a layer, a film, a region, and a plate is present “under” another part includes not only a case where the part is present “directly below” another part, but also a case where still another part is present therebetween.
Unless otherwise specifically described, all numbers, values, and/or expressions for expressing quantities of ingredients, reaction conditions, polymer compositions, and mixtures, which are used in the specification, are to be understood as modified in all instances by the term “about” because these numbers are essentially approximations that are reflective of, among other things, various uncertainties of measurement encountered in obtaining such values. In addition, when a numerical range is disclosed in the present description, the numerical range is continuous, and includes, unless otherwise indicated, every value from a minimum value to a maximum value of the numerical range. Furthermore, when the numerical range refers to integers, unless otherwise indicated, the integers include every integer from a minimum value to a maximum value of the numerical range.
It will be appreciated that throughout the present specification, when a range is described for a variable, the variable includes all the values in the described range including the end points described in the range. It will be appreciated that for example, a range of “5 to 10” includes not only values of 5, 6, 7, 8, 9, and 10, but also any sub-range of 6 to 10, 7 to 10, 6 to 9, 7 to 9, and the like, and also includes any value between appropriate integers within the described range, such as 5.5, 6.5, 7.5, 5.5 to 8.5, and 6.5 to 9. Further, it will be appreciated that for example, a range of “10% to 30%” includes not only all the integers including values of 10%, 11%, 12%, 13%, and the like and up to 30%, but also any sub-range of 10% to 15%, 12% to 18%, 20% to 30%, and the like, and also includes any value between appropriate integers within the described range, such as 10.5%, 15.5%, and 25.5%.
Nanoparticles used in the photothermal therapy (PTT) in the related art have a technical problem in reaching brain tumor tissue with high efficiency through the blood-brain barrier (BBB), and photosensitizers used in the phototherapy (PDT) have a problem in that they have difficulties in reaching and accumulating in brain tumor tissue with high efficiency due to their low water solubility. In addition, since the nanoparticles or photosensitizers have a disadvantage of having a very low oral absorption rate due to the low pH in the gastrointestinal tract even in therapeutic methods including gold nanoparticles, there is a limitation in utilizing them as therapeutic agents.
Accordingly, as a result of intensive studies to solve the above problems, the present inventors found that a nanoparticle complex, in which a photosensitizer and lactoferrin are disulfide-bonded to the surface of glutathione-coated gold nanoparticles has not only excellent metal-enhanced fluorescence (MEF) but also excellent metal-enhanced reactive oxygen generation (MERos) due to a surface plasma resonance effect through a bond with the photosensitizer while having excellent oral absorption rate through a bond with lactoferrin, and found that when a pharmaceutical composition including the nanoparticle complex is administered, the pharmaceutical composition can effectively permeate the small intestinal epithelium and the blood-brain barrier without toxicity and can be effectively accumulated in brain tumor tissue to have an excellent photothermal therapy (PTT) or photodynamic therapy (PDT) effect in brain tumor tissue, thereby completing the present invention.
The nanoparticle complex for oral administration according to an exemplary embodiment is characterized by including: gold nanoparticles coated with glutathione; a photosensitizer bonded to the gold nanoparticles; and lactoferrin bonded to the gold nanoparticles.
In the present invention, gold (Au) nanoparticles are configured to generate heat by surface plasmon resonance upon irradiation with electromagnetic waves, and serve to form the core of the nanoparticle complex. Although iron oxide nanoparticles, silver nanoparticles, or silica nanoparticles may be applied instead of the gold nanoparticles, if necessary, gold nanoparticles having absorbance in the near-infrared region are preferred.
Since metal nanoparticles in the related art have a very low oral absorption rate due to low pH in the gastrointestinal tract, making it difficult for the metal nanoparticles to be utilized as a therapeutic agent, the metal nanoparticles may be bonded to or coated with glutathione such that the biocompatibility of the metal particles may be improved and the stability thereof may be maintained even at low pH. According to an embodiment, the glutathione may form a disulfide bond with the surface of metal nanoparticles to coat the surface of the metal nanoparticles.
According to an embodiment, the mass ratio of the gold nanoparticles:the glutathione may be 1:1 to 3. When the content of glutathione is too low and outside the above range, there is a disadvantage in that it is difficult to maintain the stability of the nanoparticles due to the antioxidant effect of glutathione in the oral absorption route under acidic conditions, and when the content of glutathione is too high and outside the above range, there is a disadvantage in that the treatment of tumors may be adversely affected by enhancing the activity of the antioxidant 7-glutamylcysteine synthetase which suppresses tumor cell apoptosis after tumor cell endocytosis.
In the present invention, the photosensitizer may be bonded to the gold nanoparticles and irradiated with light of a specific wavelength to be energetically excited, and in this case, may cause apoptosis or necrosis of surrounding tumor cells while generating fluorescence signals or producing reactive oxygen species (such as singlet oxygen, oxygen radicals, superoxide and peroxide) by transferring the excited energy to the surrounding substrate or oxygen.
In an exemplary embodiment, the photosensitizer may include one or more selected from the group consisting of a phorphyrin-based compound, a chlorin-based compound, a bacteriochlorin-based compound, a phthalocyanine-based compound, a naphthalocyanine-based compound, and a 5-aminoevuline ester-based compound.
According to an embodiment, chlorin e6 (Ce6) may be used as the photosensitizer. The chlorin e6 may be bonded to gold nanoparticles, preferably, glutathione on the surface of gold nanoparticles.
According to an embodiment, the photosensitizer may have a substituted thiourea group to be disulfide-bonded to gold nanoparticles. As the photosensitizer has a substituted thiourea group, there is an advantage in that a highly reactive disulfide bond reaction can be performed without an additional catalyst, and since the thiourea group bonded to the end of the photosensitizer is bonded to gold nanoparticles through a disulfide bond with glutathione on the surface of the gold nanoparticles, there is an advantage in that a highly efficient photodynamic therapy can be achieved by a metal-enhanced reactive oxygen generation (MERos) phenomenon generated from the gold nanoparticles.
According to an embodiment, the mass ratio of the gold nanoparticles:the photosensitizer may be 1:1 to 5. When the content of the photosensitizer is too low and outside the above range, there is a disadvantage in that the MERos effect cannot be sufficiently exhibited, and when the content of the photosensitizer is too high and outside the above range, there is a disadvantage in drug treatment due to an increase in hydrophobic properties.
As used herein, lactoferrin refers to a material that binds to iron in the human body and changes into a strong antioxidant, and exhibits activities such as inhibition of bacterial growth in the body, and is abundant in milk and contained in the highest content in colostrum among types of milk, and since lactoferrin is a ligand that can bind to a low-density lipoprotein receptor-related protein (LRP), which is one of the cell membrane proteins, and many lactoferrin receptors are expressed in the small intestinal epithelium, lactoferrin may be used as a composition for targeting brain tumors and improving the bioavailability, particularly, oral absorption rate, of metal nanoparticles.
In the present invention, lactoferrin may be surface-modified with a biocompatible polymer to minimize modification of lactoferrin in the gastrointestinal tract and blood circulation processes. By such a configuration, the oral absorption rate of the nanoparticle complex may be improved, and ultimately, the targeting to brain tissue may be enhanced. In addition, there is an advantage of being able to prevent an aggregation phenomenon between nanoparticles and ultimately contribute to the enhancement of a photothermal or photodynamic therapeutic effect by maintaining a certain distance between nanoparticle complexes by the repulsive force between biocompatible polymers bonded to lactoferrin.
According to an exemplary embodiment, the biocompatible polymer may be a polysaccharide monomer or polymer which preferably has biocompatible characteristics, and preferably, the polymer may be polyethylene glycol, polycaprolactone, polylactic acid, polyglycolic acid, polylactate-co-glycolic acid, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(L-lactide-co-caprolactone), and poly(L-lactide-co-D-lactide).
According to an embodiment, polyethylene glycol, which is used as the biocompatible polymer, has an advantage in that by preventing the adsorption of proteases present in vivo onto nanoparticles, the degradation of lactoferrin is prevented and the duration of the nanoparticles in the bloodstream is increased.
According to an embodiment, the mass ratio of lactoferrin:polyethylene glycol may be 1:10 to 100, preferably 1:10 to 50. When the content of polyethylene glycol is too low and outside the above range, there is a disadvantage in that it is not possible to prevent in vivo proteases from being adsorbed onto nanoparticles, and when the content of polyethylene glycol is too high and outside the above range, there is a disadvantage in that lactoferrin may interfere with a binding domain capable of binding to a lactoferrin acceptor.
According to an embodiment, the weight ratio of the gold nanoparticles:the photosensitizer:the lactoferrin may be 19.7 to 20.0:90.0 to 88.0:1. The disadvantages of a content outside the above range are as described above.
According to an embodiment, a gold nanoparticle complex was prepared by reacting polyethylene glycol-bound lactoferrin and a photosensitizer with gold nanoparticles coated with glutathione, and it could be confirmed that the nanoparticle complex maintained the inherent physical characteristics of gold nanoparticles, that is, a significant absorbance peak and exothermic effect in the near-infrared wavelength range.
In another embodiment, it could be confirmed that stability was maintained even under strongly acidic conditions and there is an excellent oral absorption rate-enhancing effect due to low cytotoxicity to small intestine endothelial cells and vascular endothelial cells.
According to the present invention, the nanoparticle complex may be used as a nanoparticle complex for oral administration.
In another embodiment, since it could be confirmed that the nanoparticle complex has an excellent brain tumor tissue targeting effect and an excellent photothermal therapy (PTT) or photodynamic therapy (PDT) effect in a brain tumor animal model, it could be seen that the nanoparticle complex may be utilized for the treatment of brain tumors.
In the present invention, the pharmaceutical composition for oral administration may include a nanoparticle complex for oral administration, an isomer thereof, or a pharmaceutically acceptable salt thereof.
In the present invention, the nanoparticle complex may be used for brain tumor treatment, photothermal therapy, and photodynamic therapy, and may be used for oral administration because the nanoparticle complex exhibits high stability in the gastrointestinal tract in terms of administration method.
As used herein, the term “treatment” refers to all actions that ameliorate or beneficially change symptoms caused by brain tumors by administering the pharmaceutical composition according to the present invention.
As used herein, the “individual” refers to a subject in need of treatment for a brain tumor, and more specifically, refers to a mammal such as a human or a non-human primate, a mouse, a dog, a cat, a horse, and a cow.
“Tumors,” which are a disease to be treated in the present invention, refer to a group of diseases characterized by excessive cell proliferation and infiltration into surrounding tissues when the normal apoptotic balance is disrupted, and in consideration of the brain tissue targeting effect of the nanoparticle complex, the tumors may be preferably brain cancer (brain tumors), more specifically glioblastoma multiforme (GBM), but are not limited thereto.
In the present invention, the pharmaceutical composition may further include a pharmaceutically acceptable carrier in addition to an active ingredient. In this case, the pharmaceutically acceptable carrier is one typically used during formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but is not limited thereto. Furthermore, the pharmaceutically acceptable carrier may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like in addition to the above components.
In the present invention, the pharmaceutical composition may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or applied topically) according to the desired method, and most preferably administered orally. The dose varies depending on the patient's condition and body weight, severity of the disease, drug form, administration route, and duration, but may be suitably selected by those skilled in the art.
In the present invention, the pharmaceutical composition is administered in a pharmaceutically effective amount. The term “pharmaceutically effective amount” as used herein refers to an amount sufficient to treat diseases at a reasonable benefit/risk ratio applicable to medical treatment, and an effective dosage level may be determined according to factors including type of diseases of patients, the severity of disease, the activity of drugs, sensitivity to drugs, administration time, administration route, excretion rate, treatment period, and simultaneously used drugs, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with therapeutic agents in the related art, and may be administered in a single dose or multiple doses. It is important to administer the composition in a minimum amount that can obtain the maximum effect without any side effects in consideration of all the aforementioned factors, and this amount may be easily determined by a person skilled in the art.
In the present invention, the pharmaceutical composition for oral administration may be used as a composition for photothermal therapy using the gold nanoparticle property of generating heat when irradiated with electromagnetic waves. The photothermal therapy refers to a treatment method in which light energy is converted into heat energy when a light beam (light) of a certain wavelength is applied, and the heat energy emitted from the light burns cancer cells. Upon irradiation with a laser, heat is generated by the surface plasma resonance (SPR) phenomenon, and is emitted to the surrounding region to affect the external region (for example, tumor cells). As an example, cancer cells may be killed.
In the present invention, the pharmaceutical composition for oral administration may be used as a composition for photodynamic therapy using a photosensitizer and a light beam. The photodynamic therapy may be a treatment method including the procedure of treating a subject in a pathological state with a photosensitizer and irradiating the photosensitizer with a light beam in order to obtain a therapeutic effect by activating the photosensitizer.
In the present invention, the energy amount of the light beam for generating heat energy or activating the photosensitizer may be appropriately selected and used according to the use environment and purpose. For example, a suitable wavelength, power, power density, energy density, application period proportional to the photosensitizer treatment time, and the like may be appropriately selected and adjusted. As the wavelength of the light beam, any wavelength that may be absorbed by the gold nanoparticles or the photosensitizer can be used, and any wavelength capable of producing a desired biological response in the target cells may be included without limitation.
In the present invention, as a light source that produces the light beam, any light source that supplies necessary light energy to generate heat energy or produce a wavelength capable of activating the photosensitizer can be used. Examples of the light source include a laser, a lamp, an optoelectric magnetic device, a diode, a diode-laser, or the like.
In an embodiment, as a result of administering a nanoparticle complex for oral administration to brain tumor cell tissue or a brain tumor animal model, and then applying treatment by varying the order of photodynamic therapy (PDT) or photothermal therapy (PTT), it could be confirmed that upon photothermal therapy (PTT) after photodynamic therapy (PDT), brain tumor cells were relatively efficiently reduced.
In the present invention, the method of treating brain tumors may include: orally administering a pharmaceutical composition for oral administration; forming a region to be treated by accumulating the pharmaceutical composition in brain tumor tissue; irradiating the region to be treated with output light for photodynamic therapy (PDT); and irradiating the region to be treated with output light for photothermal therapy (PTT).
Hereinafter, the present invention will be described in more detail through Examples. However, these Examples are provided only for exemplarily describing the present invention, and the scope of the present invention is not limited by these Examples.
[Materials, etc.]
Materials
Gold (III) chloride trihydrate (HAuCl4), glutathione (GSH), lactoferrin (human lactoferrin (hLf)), sodium hydroxide (NaOH), a thiourea solution (H2NCSNH2), sodium borohydride (NaBH4), 100-kDa and 10-kDa MWCO dialysis tubing cellulose membranes, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), n-hydroxysuccinimide (NHS), osmium tetroxide (OsO4), pepsin, a Spurr low-viscosity embedding kit, paraformaldehyde, cresyl violet-acetate, acetic acid, octanol, CHIR-99021, retinoic acid (RA), collagen IV, fibronectin, chlorpromazine hydrochloride and sodium acetate were purchased from Sigma-Aldrich, Mo., USA. A photosensitizer (Chlorin e6 (Ce6, C34H36N4O6, MFCD08669566)) was purchased from Frontier Scientific, USA. A Centricon centrifugal filter (MWCO; 3 kDa, UFC9003) was purchased from Millipore, Germany. Bifunctional poly(ethylene glycol) (SH-PEG-COOH) was purchased from Quanta BioDesign, Plain City, USA. JEM-301 HR-TEM grids were purchased from Nanolab Technologies, N.Y., USA. InstantBlue™ was purchased from Expedeon, UK. A BCA protein assay kit was purchased from Pierce Biotechnology, Rockford, Ill., USA. A Transwell insert was purchased from Coming, Inc., Corning, N.Y., USA. Cell cytotoxicity assay EZ-Cytox was purchased from DoGenBio, Seoul, Korea. 3% isoflurane was purchased from HanaPharm, Seoul, Korea. A DeadEnd Fluorometric Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) System kit was purchased from Promega, USA. Singlet oxygen sensor green (SOSG, S36002), 1×B-27 Supplement, DMEM/F12, Knockout™ Serum Replacement, Non-Essential Amino Acids (100×), β-mercaptoethanol, human Endothelial SFM and a GlutaMAX™ supplement were purchased from Thermo Fisher Scientific, USA. FITC-Dextran 3 kDa was purchased from Invitrogen, USA. An Annexin V-DY-634/PI apoptosis stain (ab214484), anti-Ki67 antibodies (ab15580), anti-HMGB1 antibodies (ab18256), goat anti-rabbit IgG-H&L Alexa Fluor 488 (ab150077) and goat anti rabbit IgG H&L Alexa Fluor 647 (ab150079) were purchased from Abcam, UK. A DAPI mounting kit was purchased from Vector Laboratories, Inc., Burlingame, Calif., USA. ELISAs for IL6 (KET9007), IFN-γ (KET7017) and TNFα (KET9007) were purchased from Abbkine, Wuhan, China.
Experimental Cell Lines and Animals
Experiments were performed using a human umbilical vein endothelial cell line (HUVEC; LONZA, N.J., USA), a human epithelial colorectal cell line (Caco-2; Korean Cell Line Bank, Seoul, Korea), a C6 rat glioma cell line (Rockville, Md., USA), a U87MG human glioblastoma cell line (Korean Cell Line Bank, Seoul, Korea), human brain microvascular endothelial cells (BMVECs) generated from human induced pluripotent stem cells (IMR90-4 iPSCs; WiCell Research Institute, Madison, Wis., USA). HUVECs (passage numbers 4 to 6) were cultured using an endothelial growth medium (EGM-2 bullet kit; LONZA, N.J., USA).
Caco-2, U87MG and C6 cells were cultured using Dulbecco's modified Eagle's medium (DMEM; GenDEPOT, Tex., USA) containing 10% fetal bovine serum (FBS; GenDEPOT, Tex., USA) and 1% penicillin-streptomycin under standard culture conditions at 37° C. and 5% CO2.
For the culture protocol for BMVECs derived from IMR90-4 iPSCs, reference was made to that described in the methodology section of human blood-brain barrier culture and formation of a cell monolayer.
In vivo experiments were carried out using five- to seven-week-old male Balb/c nude mice, Balb/c mice (Nara-Bio Company, Seoul, Korea), and seven-week-old male Sprague Dawley (SD) rats (DBL, Incheon, Korea).
All animals were housed under specific pathogen-free conditions and maintained under guidelines of the Institutional Animal Care and Use Committee (IACUC: 2020-0081) of Hanyang University.
A solution of gold (III) chloride trihydrate (HAuCl4) (11.1 mM), glutathione (GSH) (37.8 mM) and sodium hydroxide (NaOH) (178 mM) was dissolved in methanol/water (1.3:1 v/v, 20 mL).
Next, this solution was diluted to a final Au3+ concentration (0.48 mM) by adding methanol (104 mL) and water (294 mL) thereto. In this case, Au3+ was reduced by adding sodium borohydride (NaBH4) (0.25 M, 4 mL) thereto. The reduction of Au was allowed to proceed for 24 hours at 100° C. with constant stirring.
The gold nanoparticles (AuNPs) produced by the reduction were precipitated by adding NaCl (68 mM) to methanol (200 mL), and then gold nanoparticles (AuNPs) coated with GSH could be finally obtained by centrifugation (3200 rpm, 5 min).
The GSH-coated gold nanoparticles (AuNPs) obtained by the centrifugation were reconstituted in distilled water (DW).
After a photosensitizer (Chlorin e6; Ce6) was diluted with a sodium hydroxide (0.1 M) solution according to the manufacturer's instructions, methanol was added thereto to a final concentration of Ce6 (5 mM), and a photosensitizer solution (Ce6 solution) was prepared by adjusting the pH to 6.2.
Next, the Ce6 solution (108 μL) was mixed with Sulfo-NHS (990 μL, 40.7 mg/mL) and EDC (900 μL, 16 mg/mL) in PBS (10 mM, pH 6.2), and the mixed solution was mixed every 5 minutes for 30 minutes.
Next, a thiourea solution (3006 μL, 4 mM) was added and the solution was occasionally stirred for 120 minutes. Next, sodium hydroxide (42 μL, 0.1 M) was added to quench the reaction.
Next, a Ce6-thiol solution was prepared by removing excess unreacted reagents using a thiourea-conjugated Ce6 (Ce6-thiol) purification process.
Next, after hydrochloric acid (3.3 μL) was added to the Ce6-thiol solution (1 mL), the resulting solution was homogenized and centrifuged at 15,000 rpm for 2 minutes, and a photosensitizer (Ce6-thiol) with a substituted thiourea group could be finally obtained by discarding the supernatant.
In this case, a pellet including the photosensitizer (Ce6-thiol) with the substituted thiourea group was resuspended in DW (600 μL), and this process was repeated twice. Additionally, the pellet was resuspended once more in DW (200 L), and then stored at 4° C.
The GSH-coated gold nanoparticles (AuNPs) prepared according to Preparation Example 1 and the thiourea group-substituted photosensitizer (Ce6-thiol) solution (23 μM) prepared according to Preparation Example 2 were mixed at a feed molar ratio of 1:1, allowed to react and stirred for 24 hours.
In this case, the molar extinction coefficients of Ce6 and AuNP were 45,000 cm−1/M and 55,000 cm−1/M at 532 nm and 671.0 nm, respectively.
Molar concentrations of Ce6 and AuNP were calculated by the following Equation 1 by UV-vis spectroscopy according to the Beer-Lambert law.
* A (λ) is the measured wavelength-dependent absorbance, I0 is the incident light intensity, I is the transmitted light intensity, ε is the wavelength-dependent extinction coefficient of the substrate, c is the substrate concentration, and l is the path length (0.1 cm) of the quartz cuvette
After stirring for 24 hours, the unconjugated-Ce6 was removed using a Centricon centrifugal filter having a membrane pore size of 3 kDa NMWCO, and the final product photosensitizer-bonded nanoparticle complex (Ce6-AuNPs) was reconstituted with DW (1 mL).
Lactoferrin was surface-modified with a biocompatible polymer as follows.
Specifically, lactoferrin-conjugated poly(ethylene glycol) was synthesized by EDC/NHS amide bond conjugation. That is, a PEG mixed solution was prepared by dissolving EDC (250 mM) and NHS (500 mM) in PBS containing PEG (12.5 mM) with constant stirring. After 15 minutes, lactoferrin surface-modified with a biocompatible polymer polyethylene glycol (Lf-PEG) was obtained by adding the PEG mixed solution (12.5 mM) to a PEG mixed solution containing lactoferrin (0.125 mM) at 4° C. with constant stirring for 24 hours.
Next, polyethylene glycol surface-modified lactoferrin (Lf-PEG) was collected by dialysis using a 10-kDa-pore membrane at 4° C. and lyophilized.
Next, a nanoparticle complex (Ce6-AuNPs-Lf) to which lactoferrin and a photosensitizer are bonded was prepared by mixing the solution (70 μM) of the nanoparticle complex (Ce6-AuNPs) bonded to the photosensitizer prepared in Preparation Example 3 with the Lf-PEG solution (5 mL) dissolved in PBS at 4° C. with constant stirring for 24 hours. Next, a nanoparticle complex (Ce6-AuNPs-Lf) bonded to lactoferrin and the photosensitizer was collected by dialysis using a 100-kDa-pore membrane at 4° C. and lyophilized.
1-1. Confirmation of Structure of Nanoparticle Complex for Oral Administration
Through FT-IR spectra, it was intended to identify thiourea binding to the photosensitizer (Ce6) through amide formation
Specifically, Ce6-thiol is a Ce6 solution modified with thiourea (Example 4 solution), and Ce6+thiourea is a Ce6 solution simply mixed with thiourea (Comparative Example).
Referring to
In addition, observation was made using a UV-visible spectrophotometer (NanoDrop 2000; Thermo Scientific, Wilmington, Del., USA) in order to confirm that the thiourea group-substituted photosensitizer (Ce6-thiol) is bonded to the AuNP surface through a disulfide bond.
Referring to
Furthermore, as a result of calculating the binding ratio between Ce6 and AuNP in the Ce6-AuNP using the absorbance measured at each wavelength and the molar extinction coefficients of AuNP and Ce6, it could be confirmed that an average of 4.5 Ce6 were bonded to one AuNP.
1-2. Examination of Physicochemical Properties of Nanoparticle Complex for Oral Administration
Measurements were made using a Zetasizer Nano ZS (Malvern, UK) to confirm the surface charge of a simple photosensitizer, the gold nanoparticles bonded to the photosensitizer (Ce6-AuNP) of Preparation Example 3, and the nanoparticle complex for oral administration (Ce6-AuNP-Lf) of Preparation Example 4, respectively.
Referring to
Meanwhile, TEM images were confirmed in order to confirm physical properties such as size, dispersion and aggregation of the gold nanoparticles bonded to the photosensitizer (Ce6-AuNP) of Preparation Example 3 and the nanoparticle complex for oral administration (Ce6-AuNP-Lf) of Preparation Example 4.
In this case, the elemental mapping and sizes of the Ce6-AuNP and Ce6-AuNP-Lf were measured by high-resolution TEM (HR-TEM). Specifically, HR-TEM grids were prepared by placing each sample on a carbon film-covered copper mesh grid for 1 minute, and the grids were allowed to air dry before being imaged by TEM.
Referring to
Referring to
Through this, it could be confirmed that cations present in the solution were bonded to the carboxylic acid of glutathione coated on the AuNP surface to neutralize the surface charge, thereby inducing irreversible aggregation of AuNPs into large structures
On the other hand, it could be confirmed that the aggregated diameters of Ce6-AuNP-Lf were found to be within 7.8±2.6 nm, 15.9±6.2 nm, and 81.1±15.4 nm with hydrodynamic size percentages of 64.8%, 20.2%, and 15.1%, respectively.
It could be confirmed that the steric stabilization mediated by PEGylation of Ce6-AuNP-Lf prevented particle aggregation to facilitate dispersion.
In particular, it is desirable to maintain Ce6-AuNP-Lf at an average diameter from 5 nm to 20 nm for the nanoparticle complex for oral administration according to an exemplary embodiment because the surface plasmon resonance (SPR) of AuNP irradiated with PTT laser (532 nm wavelength) is highly dependent on NP size.
Meanwhile, it was intended to evaluate the PTT efficiency and photothermal stability of the nanoparticle complex for oral administration (Ce6-AuNP-Lf).
Specifically, a PTT laser (LRS-0532 DPSS Laser System, 532 nm; laser glow Part Number: R5310B1FL, Toronto, ON, Canada) was applied to vials containing Ce6-AuNP (gold equivalent concentration of 10 μM) or Ce6-AuNP-Lf (gold equivalent concentration of 10 μM) for 240 seconds, the heating profiles of Ce6-AuNP and Ce6-AuNP-Lf were measured using a thermal imaging camera (FLIR C2, Wilsonville, Oreg., USA) and quantified by SigmaPlot 10.0 (System Software, Calif., USA), and the results are shown in
* Tmax=maximum temperature; T0=initial temperature
Referring to Table 1 and
That is, it could be confirmed that Ce6-AuNP-Lf showed the highest PTT efficiency despite bonding of AuNP with the Lf protein and Ce6 photosensitizer. Further, PEGylated Lf-conjugated AuNP (AuNP-Lf) also showed higher PTT efficiency, confirming that Lf protein conjugation did not affect the PTT efficiency of AuNP itself. That is, it could be confirmed that steric stabilization by PEGylation of AuNP has a significant effect on PTT efficiency.
1-3. Examination of Hydrophobicity of Nanoparticle Complex for Oral Administration
It was intended to examine the hydrophobicity of a simple photosensitizer (Ce6), the gold nanoparticles bonded to the photosensitizer (Ce6-AuNP) of Preparation Example 3, and the nanoparticle complex for oral administration (Ce6-AuNP-Lf) of Preparation Example 4.
Specifically, absorbance at 400 nm and 671 nm was measured when Ce6, Ce6-AuNP, and Ce6-AuNP-Lf were dissolved in DW (600 μL) and octanol, which has strong hydrophobicity, was added to cause a phase transition, and the results are illustrated in
Referring to
Therefore, it could be confirmed that Ce6-AuNP-Lf is expected to be a promising Ce6 delivery carrier to enhance the accumulation of hydrophobic Ce6 in glioblastoma multiforme (GBM), which is a brain tumor tissue, through oral absorption.
2-1. Examination of Metal-Enhanced Reactive Oxygen Generation (MERos) of Nanoparticle Complex for Oral Administration
Photobleaching was measured to examine the metal-enhanced reactive oxygen generation (MERos) of the nanoparticle complex for oral administration.
Specifically, the fluorescence decays of a simple photosensitizer (Ce6), the photosensitizer (Ce6-AuNP) of Preparation Example 3, and the nanoparticle complex for oral administration (Ce6-AuNP-Lf) of Preparation Example 4 according to laser irradiation were measured using fluorescence spectroscopy (Thermo Scientific™ VLBL00D0, USA).
In this case, each sample (Ce6 equivalent concentration of 2.5 μM) was dissolved in ethanol and irradiated with a PDT laser (MRL-III-671, 671±1 nm, China) for 10 minutes, and then the photobleaching of Ce6 fluorescence (λ excitation/λ emission=400 nm/671 nm) of each sample was measured according to laser irradiation time, and the results are illustrated in
Specifically,
Referring to
It could be confirmed that this is due to metal enhanced fluorescence (MEF) by plasmon coupling between AuNP and Ce6, and it could be confirmed that this improves fluorescence intensity but induces photobleaching.
Through this process, since it could be confirmed that this phenomenon is caused by MEF rather than FRET of the distance-dependent energy transfer process between two fluorophores, and AuNPs do not act as fluorophores, unlike Ce6, it could be confirmed that metal nanoparticles such as Au, Ag, Cu, and Pt are more suitable as MEFs that increase the fluorescence intensity of fluorophores.
Referring to
In addition, in order to examine the relationship between the generated ROS and the decrease of Ce6 fluorescence, mixtures of the SOSG reagent (0.5 μM) and Ce6, Ce6-AuNP, and Ce6-AuNP-Lf (Ce6 equivalent concentration of 2.5 μM) were applied, and then the mixtures were irradiated with a PDT laser for 5 minutes, SOSG assay for detecting the generation of ROS was performed by measuring the production of singlet oxygen (1O2) at the fluorescence intensity (λ excitation/λ emission=488 nm/525 nm), and then the results are illustrated in
Referring to
That is, it seems that the proximity of Ce6 to the gold ions on the AuNP surfaces enhanced spin-orbit coupling due to the external heavy atom effect, increasing triplet formation, so that the mechanism of the relationship between MEF and MERos could be expected to be due to inter-system crossover between AuNP and PS, which promotes the triplet state of the photosensitizer (PS).
2-2. Examination of Metal-Enhanced Reactive Oxygen Generation (MERos) of Nanoparticle Complex for Oral Administration According to PDT and PTT Application Order
To examine the metal-enhanced reactive oxygen generation (MERos) of the nanoparticle complex for oral administration according to the PDT and PTT application order, PDT and PTT to mixtures of SOSG reagent (0.5 μM) along with Ce6, Ce6-AuNP, and Ce6-AuNP-Lf (Ce6 equivalent concentration of 2.5 μM) were applied, and then single laser groups (PTT or PDT) were irradiated for 5 minutes, and groups of a combination of lasers (PTT+PDT or PDT+PTT) were irradiated for 2.5 minutes for each laser to determine the generation of singlet oxygen (1O2) at the fluorescence intensity (λ excitation/λ emission=488 nm/525 nm), thereby performing SOSG assay for detecting the generation of ROS, and then the results are illustrated in
Referring to 13A, it could be confirmed that Ce6-AuNP and Ce6-AuNP-Lf showed a significant difference in ROS generation ability according to the PDT and PTT application order, and as a result, it could be confirmed that PTT+PDT (application of PTT after application of PDT) generated the lowest ROS, whereas single PDT and PDT+PTT (application of PTT after application of PDT) were rather effective.
Referring to
It was confirmed whether the nanoparticle complex for oral administration is stable even in the oral absorption environment.
Specifically, in order to evaluate the colloidal stability of the nanoparticle complex for oral administration (Ce6-AuNP-Lf) in the oral absorption process, the stability of the nanoparticle complex for oral administration was evaluated after the pH environments of the stomach and intestinal system were mimicked.
Referring to
Referring to
Further, referring to
Through this, it could be confirmed that Ce6-AuNP-Lf maintained the colloidal stability of AuNP and undesired release of Ce6 was not detected at the pH conditions of the GI tract.
Referring to
Based on these findings, it could be confirmed that Ce6-AuNP-Lf is suitable as an oral formulation.
4-1. Evaluation of Cytotoxicity of Nanoparticle Complex for Oral Administration
The nanoparticle complex for oral administration (Ce6-AuNP-Lf) targets a lactoferrin receptor present in the small intestine and enters the blood through the small intestine endothelial cells, and then, Ce6-AuNP-Lf present in the blood is targeted to brain tumor tissue and accumulated in brain tissue or blood vessels. Accordingly, in the present example, cytotoxicity experiments were performed on small intestine endothelial cells and vascular endothelial cells
Specifically, human umbilical vein endothelial cells (HUVECs) and small intestine endothelial cells (Caco-2) were seeded in 96-well plates at a seeding density of 5×103 cells for each well and incubated for 24 hours in a CO2 incubator. Next, AuNP, Ce6-AuNP and Lf-PEG-AuNP (gold equivalent concentration of 10 μM) were applied for 24 hours. Next, after washing with PBS buffer, wells were treated with a culture medium containing EZ-Cytox at 37° C. and 5% CO2 for 4 hours. In this case, the absorbance of the medium was measured with a micro-well plate reader at a wavelength of 450 nm, and the results are illustrated in
Referring to
4-2. Measurement of Small Intestine Endothelial Cell Permeability of Nanoparticle Complex for Oral Administration
In the present example, Caco-2 cell permeability assay was performed to confirm the oral absorption rate of the nanoparticle complex for oral administration in small intestine endothelial cells in vitro.
Specifically, small intestine endothelial cells (Caco-2) were inoculated onto a Transwell insert with a diameter of 6.5 mm and a pore size of 0.4 m (seeding density: 2×104 cells/insert; Corning, Inc., Corning, N.Y., USA).
After the Caco-2 were incubated for approximately 2 to 3 weeks in a CO2 incubator, trans epithelial electrical resistance (TEER) was measured using a voltmeter (EVOM2; World Precision Instruments, Sarasota, Fla., USA) to confirm tight junctions. In this case, the TEER value >3,700 Ω·cm2 was used for the assay. Caco-2 cells were treated with Ce6, Ce6-AuNP, and Ce6-AuNP-Lf (gold equivalent concentration of 5 μM and Ce6 equivalent concentration of 2.5 μM). For competitive binding, free-Lf (5 μM) was pretreated for 2 hours, followed by Ce6-AuNP-Lf (gold equivalent concentration of 5 μM). During the treatment, TEER values were measured at each designated time, and the results are illustrated in
Referring to
Through this, transmittance of hydrophobic Ce6 (about 47.6%) may be interpreted as the result of intercellular diffusion into the cellular plasma membrane. Meanwhile, it could be confirmed that hydrophilic Ce6-AuNP and Ce6-AuNP-Lf were also transported across the barrier in amounts of 37.3% and 51.1%, respectively. Through this, it could be confirmed that Ce6-AuNP and Ce6-AuNP-Lf were able to cross the Caco-2 cell barrier through the paracellular pathway in the tight junctions.
Referring to
That is, since intestinal epithelial cells including Caco-2 cells express lactoferrin receptors (LfRs) on their membranes, it could be confirmed that the permeability of Ce6-AuNP-Lf in the gastrointestinal tract was increased by the lactoferrin receptors (LfRs).
4-3. Examination of Passive Transport of Nanoparticle Complex for Oral Administration to Small Intestine Endothelial Cells
Pretreated-Lf/Ce6-AuNP-Lf was used to examine passive transport excluding lactoferrin receptor (LfR)-mediated transport.
Referring to this, Lf pretreatment (pretreated-Lf) was performed to saturate LfR expressed in the Caco-2 cell monolayer, and two hours after Lf pretreatment (pretreated-Lf), the medium was exchanged with a Ce6-AuNP-Lf-containing medium. Thereafter, when the results are examined, since it can be confirmed that Ce6-AuNP-Lf could not be subjected to transcytosis through LfR due to pretreated-Lf, it could be confirmed that that Ce6-AuNP-Lf penetrated the Caco-2 cell monolayer only through passive transport between tight junctions, and it could be confirmed even through
5-1. Measurement of Blood-Brain Barrier Permeability of Nanoparticle Complex for Oral Administration
In the present example, a human BBB Transwell model was used to confirm the oral absorption rate of the nanoparticle complex for oral administration in vitro at the blood-brain barrier (BBB).
Specifically, human BMVECs were generated from human iPSCs as previously described by modification of oxygen conditions to mimic the hypoxic microenvironment of the developing brain.
A human iPSC line IMR90-4 (WiCell Research Institute) was maintained according to the WiCell Feeder Independent Pluripotent Stem Cell Protocol provided by the WiCell Research Institute.
IMR90-4 iPSCs were singularized using Accutase™ and seeded on a 6-well plate coated with Corning Matrigel® at a density of 1.7×104 cells per well in the presence of Y27632 (10 M, Tocris Bioscience).
Next, cells were cultured with TeSR™-E8™ (STEMCELL Technology) for 3 days until the cell density reached 3×105 cells per well (DO-D3).
To initiate differentiation into endothelial cells and neural progenitor cells, IMR90-4 iPSCs were switched from TeSR™-E8™ to unconditioned media (UM). In this case, the UM consisted of DMEM/F12 (78.5 mL), Knockout™ serum replacement (20 mL), non-essential amino acids (1 mL, 100×), GlutaMAX™ supplement (0.5 ml), and β-mercaptoethanol (182 μL) supplemented with CHIR-99021 (6 μM).
On the next day, the cell culture media were switched to UM supplemented with 1×B-27 supplement without CHIR-99021 and were changed daily for 5 days (D4-D9).
For the next 2 days (D9-D11), the endothelial cells were selectively expanded by changing to endothelial cell media (EC).
The endothelial cell media were human endothelial SFM supplemented with 20 ng/mL of basic fibroblast growth factor, 1×B-27 supplement, and retinoic acid (RA, 10 μM).
On day 11, cells were harvested from the 6-well plates using Accutase™ and seeded on a 0.4 m pore-sized 24-well Transwell insert chamber coated with collagen IV (400 μg/mL) and fibronectin (100 μg/mL) at a density of 3.3×104 cells per insert.
Then, to recapitulate the phenotypic features of BBB, BMVECs inoculated onto the insert chamber were cultured with a mixture of human primary astrocytes (ScienCell) and pericytes (ScienCell) in the basal chamber.
On day 12, media were switched to EC without bFGF and RA and changed daily to maintain the BBB culture.
From D6-D12, cells were cultured in a flushed hypoxic incubator (EppendorfGalaxy® 48R) with a 5% O2-5% CO2—N2 balance and transferred to a regular CO2 incubator.
Referring to the drawing, it could be confirmed that the BBB Transwell model was prepared by culturing human brain microvascular endothelial cells on the apical side of the insert connected with primary human pericytes and astrocytes.
The BBB Transwell model was prepared and a TEER value, which is an indicator of development of tight junctions, was measured to determine the blocking function of BBB.
Specifically, as a result of measuring the impedance values for the BBB using a TEER measurement machine (EVOM2, World Precision Instruments), the BBB endothelium showed a physiological level of the TEER value (average 4079 Ω·cm2), confirming that the BBB model can provide very limited paracellular transport.
The amount of AuNPs that crossed the BBB was measured using the prepared human BBB Transwell model.
Specifically, the human BBB model prepared above with TEER values >3,700 Ω·cm2 was used for the assay, and FITC-Dextran 3 kDa was used to monitor the barrier integrity of the BBB during the assay.
The media were changed to human endothelial SFM media at 2 hours before the assay, and Ce6-AuNP and Ce6-AuNP-Lf (gold equivalent concentration of 5 μM) were applied to the apical side of an in vitro BBB system in the absence of FITC-Dextran 3 kDa (250 μg/mL).
The Transwell plates were incubated at 37° C. with stirring, and samples (200 μL) from the basal chamber were collected every 30 minutes for 2 hours while adding the same volume of human endothelial SFM media to the basal chamber.
The quantification of nanoparticles in the basal chamber was performed using an inductively coupled plasma mass spectrometer (ICP-MS, iCAP RQ; Thermo Fisher Scientific, USA) and the fluorescent intensities of the samples at excitation wavelength of 495 nm and emission wavelength of 519 nm were measured. The apparent permeability (Papp) was analyzed using a micro-plate reader (Thermo Scientific™ VLBL00D0, USA).
In this case, the Papp of NP and FITC-Dextran was calculated as follows.
*Vb is the volume of basolateral chamber, Ct is the change in concentration, Δt is change in time at steady state, A is the growth area (0.33 cm2 in the 24-well Transwell), and C0 is the initial concentration in the apical chamber.
Further, to determine the endocytosis mechanism of Ce6-AuNP-Lf, the change in the Papp of Ce6-AuNP-Lf was determined in the presence of chlorpromazine hydrochloride, which is a clathrin-mediated endocytosis blocker.
After pre-treatment with chlorpromazine hydrochloride (50 μM) for 2 hours, Ce6-AuNP-Lf (5 μM) was added to the apical chamber of the in vitro BBB system in the presence of an inhibitor.
In addition, to identify the receptor specificity of Ce6-AuNP-Lf, the in vitro BBB system was pre-treated with Lf (5 μM) for 2 hours.
After pre-treatment with Lf for 2 hours, Ce6-AuNP-Lf (5 μM) was added to the apical chamber of the in vitro BBB system in the presence of pre-treated Lf.
Referring to
Furthermore, referring to
Specifically, for bio-TEM images of the Transwell-cultured human BBB model, the samples were fixed after 2 hours of exposure according to the experimental conditions for each group (Ce6-AuNP, Ce6-AuNP-Lf, pretreated-Lf/Ce6-AuNP-Lf, and Clathrin inhibitor/Ce6-AuNP-Lf). In this case, reagent setup and the procedure followed the reported method. Next, a 4% PFA solution was added to the apical and basolateral chambers of the Transwell filters (0.5 and 1.5 mL, respectively) for 1 hour. Sorensen's phosphate buffer, which consists of solutions A and B (A is 0.2 M Na2HPO4→2H2O, B is 0.2 M NaH2PO4→H2O), was added for 10 minutes to rinse off the fixative. Next, 1% osmium tetroxide (OsO4) was added to stain the cell monolayer for 1 hour. Next, Sorensen's phosphate buffer was used to wash the remaining OsO4 for 10 minutes. Dehydration of cell monolayers on both sides of the filters was performed with different concentrations of ethanol as follows: 30% for 10 minutes, 50% for 10 minutes, 70% for 10 minutes, 90% for 10 minutes, and finally 100% for 20 minutes three times. The method of forming an epoxy resin block with a Low Viscosity Embedded Media Spurr's Kit method was applied to the entire Transwell containing the fixed cell monolayer. The epoxy resin block was cut to 4 mm, and the surrounding plastic and resin were removed by additional cutting to reveal the cubic alignment including plastic, epoxy resin, filter membrane, epoxy resin and the like from left to right. The specimens were cut into 80 nm-thick sections using a microtome and the obtained sections were air-dried for at least 1 hour. Copper grids were mounted in 2% uranyl acetate for 20 minutes, briefly washed with DW and mounted in lead citrate (0.4%) for staining for 10 minutes. Next, the section placed on the grid was observed using an 80-kV transmission electron microscope.
Referring to
5-2. Examination of Brain Tumor Targeting Enhancement Effect of Nanoparticle Complex for Oral Administration
In the present example, the absorption rate of the nanoparticle complex for oral administration to brain tumor cells (U87MG) was confirmed in vitro.
Specifically, brain tumor (U87MG) cells (seeding density: 5×104 cells/well) in a 4 well Nunc™ Lab-Tek™ II Chamber Slide™ System (Thermo Scientific™ 154526PK, USA) were treated with Ce6, Ce6-AuNP, or Ce6-AuNP-Lf (Ce6 equivalent concentration of 2.5 μM) for 18 hours, and then after washing three times with PBS, the cells were fixed with 4% PFA for 15 minutes. Next, DAPI mounting medium (Vector Laboratories, Inc., Burlingame, Calif., USA) was used and the intracellular Ce6 fluorescence was observed under a confocal microscope (TCS SP5, Leica, Germany).
Referring to the drawing, it could be confirmed that the intracellular fluorescence was significantly increased in the Ce6-AuNP and Ce6-AuNP-Lf groups compared to that of free-Ce6.
Meanwhile, flow cytometry (FACS Calibur™; BD Biosciences, Franklin Lakes, N.J.) was used to quantify the intracellular Ce6 fluorescence. Next, brain tumor (U87MG) cells (80% confluency in 100 π culture dish) were treated with Ce6, Ce6-AuNP, or Ce6-AuNP-Lf (Ce6 equivalent concentration of 2.5 μM) for 18 hours. After washing three times with PBS, the cells were detached with trypsin/EDTA, centrifuged for 3 minutes at 1000 rpm, and then analyzed with FACS.
As a result, it could be confirmed that the amounts of Ce6, Ce6-AuNP and Ce6-AuNP-Lf uptake into the cells were 43.6±2.2, 61.3±1.6 and 82.1±2.1, respectively.
That is, it could be confirmed that the cellular uptake of Ce6-AuNP-Lf was statistically increased compared to that of Ce6-AuNP, which lacks Lf as a targeting ligand, confirming that brain tumor cell (U87MG) uptake for targeted conjugates was enhanced compared to non-targeted conjugates.
5-3. Examination of Photothermal Therapeutic Effect of Nanoparticle Complex for Oral Administration
The photothermal therapeutic effect of the nanoparticle complex for oral administration was confirmed through cell cytotoxicity assay.
Specifically, brain tumor (U87MG) cells (seeding density: 5×103 cells/well) were treated with Ce6, Ce6-AuNP, or Ce6-AuNP-Lf (5 μM of gold and Ce6 equivalent concentration of 2.5 μM) for 12 hours. Next, after washing three times with PBS, the cells were irradiated with a single laser (PTT or PDT) for 5 minutes or a combination of lasers (PTT+PDT or PDT+PTT) for 2.5 minutes each. Cell viability was measured with an EZ-Cytox kit.
Meanwhile, the intracellular ROS generation by laser irradiation was measured using the fluorescent probe DCFH-DA according to the manufacturer's instructions. Annexin V-DY-634/PI apoptosis was used to evaluate apoptosis. That is, brain tumor (U87MG) cells were suspended in 400 μL binding buffer, and then stained with Annexin V-DY-634 (5 μL) at 2 to 8° C. for 15 minutes. Next, PI (5 μL) was added and the cells were incubated for 5 min. In this case, brain tumor (U87MG) cells were analyzed using FACS.
Referring to the drawing, it could be confirmed that the apoptotic cell populations (Annexin-V+/PI+) of Ce6-AuNP-Lf, Ce6-AuNP and Ce6 groups to which PDT+PTT was applied were 45.9%, 26.5%, and 22.0% of the total, respectively, confirming that PDT+PTT (application of PTT after application of PDT) showed an excellent therapeutic effect compared to other treatment methods such as PTT+PDT (application of PDT after application of PTT) and single PDT or PTT.
Referring to
Referring to
That is, it could be confirmed that when PTT was first applied, Ce6-AuNP and Ce6-AuNP-Lf, in which Ce6 is bonded to AuNP, lost the MERos effect, resulting in decreased ROS generation, confirming once again that the therapeutic effect of PTT+PDT (application of PDT after application of PTT) is lower than that of PDT+PTT (application of PTT after application of PDT) and PDT alone.
In the present example, it was intended to examine improved oral availability through an in vivo pharmacokinetics (PK) study.
Specifically, Balb/c mice were administered Ce6-AuNP-Lf at 30 mg/kg, 60 mg/kg, and 5 mg/kg via subcutaneous (SC), oral, and intravenous (IV) administration, respectively. Blood samples (500 μl) were collected by intra-cardiac puncture at each time point of 10 minutes, 20 minutes, 30 minutes, 60 minutes, 2 hours, 6 hours, and 12 hours after administration of Ce6-AuNP-Lf. In this case, the exact weights of blood samples were measured in a borosilicate glass tube.
Next, 70% nitric acid (800 μl) was added to each glass tube and samples were heated in a hot water bath at 60° C. for 3 hours. Next, HCl (37%) was added to each glass tube, and samples were heated under the same conditions. Next, the blood samples were transferred into 50 mL tubes and the pH was adjusted with 2% nitric acid and 0.5% HCl in DW. Next, the adjusted blood samples were filtered (0.22 μm pore-size) and analyzed by ICP-MS. The Au concentration was calculated and adjusted for sample weight. In this case, the standard curve of Au (0.0001 to 0.05 μg/mL) was linear, and the limit of detection was 0.0005 μg/mL. Background gold concentration, which is a measured pre-dose, was subtracted from measured values to derive a gold concentration attributed to the dose. In this case, the gold concentration in each sample was determined from the mean of six replicate measurements. Further, the bioavailability (BA) value was calculated from the following Equation 3.
* AUCoral or SC is the area under curve in the case of oral or SC administration, respectively, AUCIV is the area under curve in the case of IV administration, Doseoral or SC is the dosage of drug injected orally or via SC administration, and DoseIV is the dose of drug injected via IV administration.
Referring to
Further, SC injection is used as a sustained diffusible method among various drug injection routes, which is due to the large amount of capillaries in the fatty layer of subcutaneous tissue just beneath the skin. However, the mean residence time (MRT) of orally administered Ce6-AuNP-Lf was better than that of SC. In addition, it could be confirmed that the percentages of bioavailability (Fabs) were similar at 8.6±1.2% and 7.3±0.6% in the SC group and the oral group, respectively.
Furthermore, fluorescence signals were used to confirm whether orally administered Ce6-AuNP-Lf can be delivered to brain tumor tissue via systemic circulation.
Specifically, for the fluorescence tracer image, Balb/c mice were administered Ce6-AuNP-Lf at 60 mg/kg, and after 2, 6, 12, and 24 hours, the experimental mice were sacrificed, and the organs were extracted. In this case, the fluorescence signals of Ce6-AuNP-Lf in organs were imaged using an in vivo imaging system (FOBI, CELLGENTEK, Korea), and the exposure time was fixed to 550 seconds for analyzing fluorescence signals from tissues.
In this case, intensity unit indicates the intensity/min/gain, and a red dashed line indicates the detected fluorescence signal in the brain.
Referring to
7-1. Examination of Brain Tumor Targeting Effect Using Brain Tumor Animal Model
In the present example, it was intended to confirm the brain tumor targeting effect of the nanoparticle complex for oral administration using a brain tumor animal model.
Specifically, PBS (containing 0.8×106 C6 glioma cells, 100 μL) was inoculated subcutaneously in the right flanks of athymic Balb/c nude mice. Next, five days after cell inoculation, the mice were randomly divided into 3 groups (n=4). A CON group received saline administration. Ce6-AuNP-Lf was administered via oral (gold equivalent concentration of 0.07 μM) and intravenous (IV) (gold equivalent concentration of 0.012 μM) methods, respectively.
Referring to
Referring to
7-2. In Vivo Experiment Using Subcutaneous GBM Xenograft Mouse Model
To additionally confirm the presence of GBM targeting and the non-specific drug distribution of Ce6-AuNP-Lf, in vivo experiments using a subcutaneous GBM xenograft mouse model were performed.
Specifically, PBS (containing 0.8×106 C6 glioma cells, 100 μL) was inoculated subcutaneously in the right flanks of athymic Balb/c nude mice. Next, five days after cell inoculation, the mice were randomly divided into 3 groups (n=4). A CON group received saline administration. Ce6-AuNP-Lf was administered via oral (gold equivalent concentration of 0.07 μM) and intravenous (IV) (gold equivalent concentration of 0.012 μM) methods, respectively. Next, 24 hours after administration, the mice were irradiated sequentially with PDT and PTT lasers for 5 minutes. This treatment cycle was repeated three times and the survival rate, body weight, and tumor size were monitored until day 18. Tumor size was determined using calipers to measure the length a and width b of tumors and was calculated as 4/3π×α2×b2 (a: smaller radius; b: larger radius). On day 18, the mice were sacrificed for further analysis.
Referring to
In order to clearly evaluate whether the transmitted PDT laser can have an energy level capable of generating ROS from AuNPs and Ce6-AuNP-Lf, the outside of mouse skin tissue (1 cm thickness) was irradiated with a PDT laser (671 nm).
Specifically, after 1 cm-thick biological tissue was extracted from mouse skin, 10 μL of a DPBF solution (10 mg/mL in DMSO) was added to Ce6, Ce6-AuNP, and Ce6-AuNP-Lf (Ce6 equivalent concentration of 10 μM) diluted with 1.5 mL of water. Next, the mouse skin tissue was irradiated with an NIR laser (671 nm) for 1, 3, and 5 minutes with or without the insertion of the biological tissue.
Referring to
8-1. Preparation of Orthotopic GMB Mouse Model
An orthotopic GBM mouse model for glioblastoma multiforme (GBM), which is a brain tumor tissue, was prepared as follows. Specifically, GBM cells (U87MG cells) were intracranially injected into seven-week-old male nude mice. That is, male nude mice were anesthetized with isoflurane (3%) and fixed by ear bars in a stereotaxic instrument (Stoelting Co., Ill., USA). Once each mouse was anesthetized, the scalp at the surgical position was removed and a small hole located at 2 mm right lateral and 2 mm posterior to bregma was drilled under sterile conditions. Thereafter, PBS (containing 1×106U87MG cells, 8 μL) was loaded into a 26-G Hamilton syringe (Hamilton Company, Nev., USA), and then the syringe was placed on the stereotaxic apparatus. Next, after the needle of the syringe was positioned at a depth of 3 mm, cells were injected at an injection rate of 1 μL/min, followed by a waiting time of 3 minutes to prevent overflow. Next, after injection, the hole was sealed with bone wax and the scalp was closed by suturing. After this procedure, the mice were maintained for 3 weeks until the injected cells reached an appropriate size of GBM tissue. Since previous histology studies have verified that GBM exhibits a spherical shape with a diameter of about 2 mm at the site of cell injection, the boundary between GBM and normal brain tissue was established from these shapes. To evaluate GBM targeting efficacy, Ce6-AuNP and Ce6-AuNP-Lf were administered by oral (gold equivalent concentration of 0.07 μM) and IV (gold equivalent concentration of 0.012 μM) methods, respectively. 24 hours after administration, GBM and normal brain tissues were excised according to the boundary criterion and analyzed by ICP-MS and TEM. To evaluate the phototherapeutic efficacy of Ce6-AuNP-Lf in an orthotopic GBM mice model, the groups were randomized into nine groups (n=5): Con, oral No laser, IV No laser, oral PDT+PTT, oral PTT, oral PDT, IV PDT+PTT, IV PTT, and IV PDT. Then, Ce6-AuNP-Lf was administered by oral (gold equivalent concentration of 0.07 μM) and IV (gold equivalent concentration of 0.012 M) methods, respectively. 24 hours after Ce6-AuNP-Lf administration, GBM was irradiated with a single laser (PTT or PDT, 10 minutes) or a combination of lasers (PDT+PTT, for 5 minutes each). The survival rate and body weight were monitored from day 1 to day 17 after the beginning of treatment. On day 17, the brain in the mice was excised for histology analysis.
8-2. Histological Examination in Orthotopic GMB Mouse Model
Meanwhile, in vivo histological examination of the orthotopic GBM (U87MG) mouse model was performed as follows.
Specifically, tissues were immobilized in 4% paraformaldehyde for 2 days and then placed in a Leica TP1020 Semi-enclosed Benchtop Tissue Processor (Wetzlar, Germany) for washing, dehydration, clearing and paraffin infiltration of the tissue samples, followed by embedding in paraffin blocks. The paraffin blocks were cut into 6 m-thick cross sections using a Leica RM2145 Microtome (Wetzlar, Germany).
For Nissl staining, the brain slides were stained with a staining solution prepared by dissolving cresyl violet-acetate (0.2 g) in distilled water (150 mL) and a buffer solution containing acetic acid (0.1 M) and sodium acetate (0.1 M). H&E staining and TUNEL assay were used to detect necrosis and apoptosis, respectively, in the GBM regions according to the manufacturer's instruction.
Meanwhile, 24 hours after Ce6-AuNP-Lf administration, the GBM mouse model was irradiated with a single laser (PTT or PDT, 10 minutes) or a combination of lasers (PDT+PTT, for 5 minutes each). Next, on the day after laser irradiation, blood was collected from the lateral saphenous vein using a 23-gauge needle. The collected blood was allowed to clot for 30 min at room temperature and then centrifuged at 2,200×g for 10 minutes to obtain serum. Next, the obtained serum samples were divided into aliquots (1.0 mL) in polypropylene tubes and stored at −80° C. for further experiments.
8-3. Examination of Brain Tumor Therapeutic Effect in Orthotopic GMB Mouse Model
In the present example, in vivo GBM targeting of the nanoparticle complex for oral administration was evaluated using an orthotopic GBM (U87MG) mouse model, which is a brain tumor animal model, and then the therapeutic photothermal effect (PTT) and photodynamic effect (PDT) of Ce6-AuNP-Lf accumulated in GBM were evaluated.
Referring to this drawing, laser irradiation was applied to the GBM-induced region of the orthotopic GBM mouse model 24 hours after administration, and the treatment cycle including Ce6-AuNP-Lf administration and laser irradiation was repeated three times. Next, the survival rates and weight changes were measured until sacrifice.
Referring to
Furthermore, in order to histologically evaluate the photothermal effect (PTT) and photodynamic effect (PDT) of the orthotopic GBM mouse model, all of the mouse models were sacrificed after the treatment.
Referring to
Through this, it could be confirmed that the PDT+PTT combination treatment, which is the most potent treatment strategy by MERos of Ce6-AuNP, is also consistent in the orthotopic GBM mouse model.
Furthermore, the therapeutic effect of Ce6-AuNP-Lf with PDT+PTT combination treatment was evaluated by immunohistologically analyzing the extracted brain tissues.
Referring to
9-1. Preparation of Orthotopic GMB Rat Model
An orthotopic GBM rat model for glioblastoma multiforme (GBM), which is a brain tumor tissue, was developed from seven-week-old male Sprague Dawley rats. The rats were anesthetized using 5% isoflurane in 70% N2O and 30% O2, and a 2-mm hole was made in the skull. In this case, the injection point was 2.0 mm lateral to bregma and was carefully drilled using a saline (0.89% NaCl) drip (coordinates to bregma: anteroposterior, 0 mm; lateral, 2.0 mm; ventral, 4.0 mm). Then, C6 cells (1×105 cells/10 μL) were injected into the cerebral cortex using a 26-gauge Hamilton syringe. Next, ten days after tumor implantation, the rats were randomly divided into 9 groups (n=5) [Con, oral No laser, intravenous (IV) No laser, oral PDT+PTT, oral PTT, oral PDT, intravenous (IV) PDT+PTT, intravenous (IV) PTT, and intravenous (IV) PDT]. In this case, Ce6-AuNP-Lf was administered by oral (gold equivalent concentration of 0.07 μM) and IV (gold equivalent concentration of 0.012 μM) methods, respectively.
24 hours after Ce6-AuNP-Lf administration, the GBM rat model was irradiated with a single laser (PTT or PDT, 10 minutes) or a combination of lasers (PDT+PTT, for 5 minutes each). Next, on the day after laser irradiation, blood was collected from the lateral saphenous vein using a 23-gauge needle. The collected blood was allowed to clot for 30 min at room temperature and then centrifuged at 2,200×g for 10 minutes to obtain serum. Next, the obtained serum samples were divided into aliquots (1.0 mL) in polypropylene tubes and stored at −80° C. for further experiments. In this case, ELISAs for IL6, IFN-7, and TNFα were performed with rat serum from each group according to the manufacturer's manual. After three repeated treatment cycles, the rats were sacrificed by perfusion and the brains were harvested and fixed with 4% paraformaldehyde for further analysis.
9-2. Histological Examination in Orthotopic GMB Rat Model
Meanwhile, in vivo histological examination of the orthotopic GBM (U87MG) rat model was performed as follows.
Specifically, tissues were immobilized in 4% paraformaldehyde for 2 days and then placed in a Leica TP1020 Semi-enclosed Benchtop Tissue Processor (Wetzlar, Germany) for washing, dehydration, clearing and paraffin infiltration of the tissue samples, followed by embedding in paraffin blocks. The paraffin blocks were cut into 6 m-thick cross sections using a Leica RM2145 Microtome (Wetzlar, Germany).
For Nissl staining, the brain slides were stained with a staining solution prepared by dissolving cresyl violet-acetate (0.2 g) in distilled water (150 mL) and a buffer solution containing acetic acid (0.1 M) and sodium acetate (0.1 M). H&E staining and TUNEL assay were used to detect necrosis and apoptosis, respectively, in the GBM regions according to the manufacturer's instruction.
Meanwhile, 24 hours after Ce6-AuNP-Lf administration, the GBM rat model was irradiated with a single laser (PTT or PDT, 10 minutes) or a combination of lasers (PDT+PTT, for 5 minutes each). Next, on the day after laser irradiation, blood was collected from the lateral saphenous vein using a 23-gauge needle. The collected blood was allowed to clot for 30 min at room temperature and then centrifuged at 2,200×g for 10 minutes to obtain serum. Next, the obtained serum samples were divided into aliquots (1.0 mL) in polypropylene tubes and stored at −80° C. for further experiments. Meanwhile, ELISAs for IL6, IFN-7, and TNFα were performed with rat serum from each group according to the manufacturer's manual. After three repeated treatment cycles, the rats were sacrificed by perfusion and the brains were harvested and fixed with 4% paraformaldehyde for further analysis. Furthermore, for immunofluorescence (IF) staining, GBM tissue slides were stained with an anti-Ki67 antibody and an anti-HMGB1 antibody and, diluted 1:100 in a mixture of phosphate buffered saline with Tween-20 (PBST) and goat serum. Next, goat anti-rabbit IgG-H&L Alexa Fluor 488 and goat anti rabbit IgG H&L Alexa Fluor 647 were used as secondary antibodies, followed by DAPI mounting.
9-3. Examination of Immune Response of Orthotopic GMB Rat Model
In the present example, in vivo GBM targeting of the nanoparticle complex for oral administration was evaluated using an orthotopic GBM (U87MG) rat model, which is a brain tumor animal model, and then the therapeutic photothermal effect (PTT) and photodynamic effect (PDT) of Ce6-AuNP-Lf accumulated in GBM were evaluated.
Referring to
Referring to
That is, the phototherapy (PDT and PTT)-induced cell apoptosis generated a strong and acute local inflammatory response at treated sites to attack tumor cells, and it could be confirmed that this immune system involves the expression of the NF-κB transcription factor which induces the release of cytokines. Therefore, it can be inferred that pro-inflammatory cytokines (IL-6, IFN-γ, and TNFα) during the treatment cycle are upregulated because PDT+PTT of Ce6-AuNP-Lf subsequently destroys GBM due to an immune response.
Furthermore, the therapeutic effect of Ce6-AuNP-Lf with PDT+PTT combination treatment was evaluated by immunohistologically analyzing the extracted brain tissues.
Referring to
That is, as a result of destruction of tumor blood vessels by ROS due to PDT, as the PDT-containing group showed a significant reduction in tumor volume, since ROS generated by PDT causes irreversible damage in endothelial cells and the vascular membrane and tumor growth is related to vasculature function due to oxygen and nutrient supply, it could be confirmed that microvasculature destruction by PDT damages tumor blood vessels, causes hemorrhaging, and destroy tumors.
Further, Ki67, tumor cell proliferation and angiogenesis markers were examined from the GBM tissue of the orthotopic GBM rat model.
Referring to
In addition, HMGB1 expression in GBM tissue from an orthotopic GBM rat model was investigated. High-mobility-group box 1 (HMGB1), which is an alarmin protein released from tumor cells, is considered a DAMP in cancer therapy, and acts as an endogenous ligand for toll-like receptor (TLR)-2, TLR-4, and TLR-9. Upon receptor binding, HMGB1 induces the activation of signaling pathways and immune responses, and depending on the tissue type, HMGB1 may suppress tumor growth or promote tumorigenesis. The release of DAMP may activate the immune system by inducing the maturation of dendritic cells (DCs) that eventually migrate to the lymph nodes.
Referring to
In summary, with respect to the high expression of Ki67 and HMGB1 in malignant GBM, since the expression was reduced by PDT and PTT treatment, it could be confirmed that the PDT+PTT of Ce6-AuNP-Lf targeting GBM by oral administration or IV injection significantly destroy tumors.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2021-0173100 | Dec 2021 | KR | national |
| 10-2022-0074985 | Jun 2022 | KR | national |
