Drug delivery via pore-modified mesoporous silica nanoparticles

Abstract

To provide mesoporous silica nanoparticles having modifications on the surface of the mesopores, which can be further loaded with one or more types of bioactive ingredients within the mesopores, processes of preparing the same, and applications of the same.SOLUTION: A mesoporous silica nanoparticle comprises organic modification on the surface of its pores and has a particle size of 100 nm or less and a hydrodynamic size in a medium measured by dynamic light scattering of 150 nm or less, where the organic modification comprises at least one terminal hydrocarbyl moiety, and the medium is biologically similar or equivalent to phosphate buffered saline.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to mesoporous silica nanoparticles having modifications on the surface of the (enlarged) mesopores, which can be further loaded with one or more bioactive components within the (enlarged) mesopores, and to a process for their preparation and their applications. [Background technology]

[0002] Combination drug therapy is the most widely used treatment for the most dreaded diseases, including cancer and infectious diseases. The primary goals of using drug combinations are to achieve synergistic therapeutic effects, reduce dosage and adverse effects, and minimize the induction of drug resistance. Therefore, the simultaneous administration of two or more drugs to patients usually demonstrates greater therapeutic efficacy than each drug treatment alone. In cancer treatment, combination therapy is standard clinical practice to overcome drug resistance and improve treatment outcomes. The synergistic effect of two (or more) drugs with different mechanisms of action (targeting different cell cycle checkpoints, genes, or cancer metabolic pathways) increases the chances of eliminating cancer. However, differences in the physicochemical and pharmacokinetic properties of drugs lead to many challenges when developing combination drug therapies. The main issues involved in developing combination drug therapies are (1) identifying appropriate drug combinations and drug ratios, (2) correlating in vitro studies with in vivo behavior, and (3) whether drugs can reach the same tumor cells at effective doses and ratios (drug pharmacokinetics). Nanoparticle multidrug co-delivery offers a potential route to resolving these challenges in combination drug therapy.

[0003] Nanoparticle formulations offer several advantages for multidrug delivery, including: (1) nanoparticles can simultaneously deliver hydrophobic and hydrophilic drugs and maintain an optimized synergistic drug ratio in a single nanoparticle; (2) nanoparticles can extend circulation time and enhance the tumor targeting ability of drugs; and (3) drug-encapsulated nanoparticles can simultaneously deliver multiple drugs into target cells and normalize pharmacokinetic differences between drugs. Mesoporous silica nanoparticles (MSNs) have been considered to have great potential as drug delivery systems due to their unique physical and chemical properties, such as large pore volume, chemical and thermal stability, high loading capacity, tunable surface properties, and excellent biocompatibility. The outer or pore surfaces of MSNs can be easily modified individually with various functional groups. However, encapsulation of hydrophobic and hydrophilic drugs in the same particle usually affects the monodispersity of the particles in solution, and therefore, this delivery system still presents challenges and needs improvement. In the present invention, small-sized MSNs (<100 nm) with enlarged pore size (optionally) and pore surface modification with specific functional groups and ratios can encapsulate two drugs (one hydrophobic and another hydrophilic in one embodiment) in the same particle, exhibiting synergistic therapeutic effects against multidrug-resistant cancer cells. The co-delivery of multidrugs by the particles mentioned in the present invention can also be used to treat various diseases, such as cancer, multidrug-resistant cancer, brain cancer, metastatic brain cancer, and central nervous system diseases. Summary of the Invention

[0004] The present invention provides mesoporous silica nanoparticles having modifications on the surface of the mesopores. Mesoporous silica nanoparticles (MSNs) have pore sizes less than 50 nm, and can be referred to as "exMSNs" when the pore size is greater than 3 nm (i.e., the pores are "expanded").

[0005] The present invention also provides pore-modified MSNs and exMSNs loaded with one or more bioactive components within the (expanded) mesopores. The present application also provides methods for producing pore-modified MSNs and exMSNs with or without loading with bioactive components.

[0006] In one aspect, the present invention provides mesoporous silica nanoparticles whose pore surfaces are modified with functional groups for stably entrapping or encapsulating one or more bioactive components on the surfaces of the pores.

[0007] In one embodiment, the mesoporous silica nanoparticles have a pore size of less than 50 nm. In one embodiment, the functional groups are hydrophobic or hydrophilic or both.

[0008] In one embodiment, the surfaces of the pores of the mesoporous silica nanoparticles are modified with functional groups having terminal hydrocarbyl moieties, which in one embodiment comprise terminal aromatic moieties, terminal (cyclo)aliphatic moieties, or a combination thereof.

[0009] In some embodiments, the terminal aromatic moiety is substituted with lower alkyl or halogen. In further embodiments, the terminal aromatic moiety is derived from trimethoxyphenylsilane (TMPS). In some embodiments, the terminal (cyclo)aliphatic moiety comprises a (cyclo)alkyl, a (cyclo)alkenyl, or a combination thereof, which may optionally be substituted with lower alkyl or halogen.

[0010] In some embodiments, the bioactive ingredient is hydrophilic, hydrophobic, or lipophilic. In some embodiments, the bioactive moiety is a small molecule, a chemical drug, an enzyme, a protein drug, an antibody, a vaccine, an antibiotic, or a nucleotide drug.

[0011] In some embodiments, at least two bioactive components are loaded within the pores, e.g., on the surface of the pores. In one embodiment, mesoporous silica nanoparticles are loaded with one or more hydrophilic bioactive components and one or more hydrophobic bioactive components within the pores, e.g., on the surface of the pores. In one embodiment, mesoporous silica nanoparticles are loaded with one or more hydrophobic bioactive components within the pores, e.g., on the surface of the pores.

[0012] In another aspect, the present disclosure provides a method for delivering a bioactive ingredient to a subject, comprising administering to the subject the mesoporous silica nanoparticles of the present invention.

[0013] In one embodiment, the method is capable of delivering mesoporous silica nanoparticles of the present disclosure to penetrate the blood-brain barrier and / or the blood-eye barrier. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1 shows the synergistic effect of JM17 and Dox codelivery by exMSNs against MCF-7 / ADR cancer cells. [Figure 2] FIG. 2 shows the anti-MCF-7 / ADR tumor efficacy of JM17 and Dox co-delivery by NTT2_131 nanoparticles. DETAILED DESCRIPTION OF THE INVENTION

[0015] To facilitate understanding of this disclosure, terms used herein are defined below. In the context of the specification and claims, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated. Unless otherwise indicated, any and all examples or exemplary language (e.g., "such as") provided herein do not limit the scope of the invention, but are merely used to better illustrate the invention.

[0016] It should be understood that any numerical range recited herein is intended to include all subranges encompassed therein. For example, the range "50 to 70°C" includes all subranges and specific values between the specified minimum value of 50°C and the specified maximum value of 70°C (e.g., 58 to 67°C, 53 to 62°C, 60°C, or 68°C). Because the disclosed numerical ranges are continuous, they include each value between the minimum and maximum values. Unless otherwise specified, the various numerical ranges set forth herein are approximate.

[0017] In the present invention, the term "about" refers to an acceptable deviation of a given value as measured by one of ordinary skill in the art, which depends in part on how the value is measured or determined.

[0018] For purposes of the present invention, unless otherwise specified, the prefix "nano-" as used herein means a size of about 300 nm or less, and unless otherwise specified, the prefix "meso-" as used herein means a size of less than about 50 nm.

[0019] In the present invention, the term "silane" as used herein refers to a derivative of SiH4. Typically, at least one of the four hydrogen atoms is replaced with a substituent such as alkyl, alkoxyl, or amino, as described below. The term "alkoxysilane" as used herein refers to a silane having at least one alkoxyl substituent directly bonded to the silicon atom. The term "organoalkoxysilane" as used herein refers to a silane having at least one alkoxyl substituent and at least one hydrocarbyl substituent directly bonded to the silicon atom. The term "silica source" as used herein refers to a substance that can be considered to be in the form of a salt or ester of orthosilicic acid, such as sodium orthosilicate, sodium metasilicate, tetraethylorthosilicate (tetraethoxysilane, TEOS), tetramethylorthosilicate, or tetrapropylorthosilicate. Optionally, the hydrocarbyl substituent may be further substituted or interrupted by a heteroatom.

[0020] In the present invention, the term "hydrocarbyl" as used herein refers to a monovalent radical derived from a hydrocarbon. As used herein, the term "hydrocarbon" refers to a molecule consisting solely of carbon and hydrogen atoms. Examples of hydrocarbons include, but are not limited to, (cyclo)alkanes, (cyclo)alkenes, alkadienes, aromatic compounds, etc. When the hydrocarbyl is further substituted as described above, the substituents may be halogens, amino groups, hydroxyl groups, thiol groups, etc. When the hydrocarbyl is interrupted by a heteroatom as described above, the heteroatom may be S, O, or N; in the present invention, the hydrocarbyl preferably contains 1 to 30 C atoms.

[0021] In the present invention, the term "alkyl" refers to saturated, straight-chain or branched alkyls preferably containing 1 to 30 carbon atoms, more preferably 1 to 20 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, 2-ethylbutyl, n-pentyl, isopentyl, 1-methylpentyl, 1,3-dimethylbutyl, n-hexyl, 1-methylhexyl, n-heptyl, isoheptyl, 1,1,3,3-tetramethylbutyl, 1-methylheptyl, 3-methylheptyl, n-octyl, 2-ethylhexyl, 1,1,3-trimethylhexyl, 1,1,3,3-tetramethylpentyl, nonyl, decyl, undecyl, 1-methylundecyl, dodecyl, 1,1,3,3,5,5-hexamethylhexyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, and the like.

[0022] In the present invention, the term "alkoxyl" or "alkoxy" as used herein means a group having the formula "-O-alkyl", wherein the definition of "alkyl" has the meaning of "alkyl" above.

[0023] In the present invention, the term "cycloalkyl" as used herein means a saturated or partially unsaturated cyclic carbocyclic radical containing 3 to 10 ring carbon atoms, and more preferably 3 to 8 ring carbon atoms, and optionally alkyl substituents on the ring. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclopropenyl, cyclobutyl, cyclopentyl, cyclohexyl, 2-cyclohexen-1-yl, and the like. As used herein, the term "halogen" or "halo" means fluorine, chlorine, bromine or iodine.

[0024] For purposes of the present invention, the term "amino" as used herein means a functional group of formula -NR1R2, wherein R1 and R2 each independently represent hydrogen or a hydrocarbyl group as defined above.

[0025] In the present invention, the term "aqueous phase" as used herein means a phase that is substantially miscible with water. Examples of aqueous phases include, but are not limited to, water itself, aqueous buffer solutions, aqueous dimethyl sulfoxide (DMSO) solutions, aqueous alkanol solutions, etc. The aqueous phase can be adjusted to be acidic, neutral, or alkaline based on the requirements of the synthesis and / or the stability of the substances present in the aqueous phase.

[0026] In the present invention, the term "oil phase" as used herein means a phase that is substantially immiscible with the aqueous phase as described above. Examples of oil phases include, but are not limited to, liquid, substituted or unsubstituted (cyclo)alkanes (e.g., hexane, decane, octane, dodecane, cyclohexane, etc.); substituted or unsubstituted aromatic solvents (e.g., benzene, toluene, xylene, etc.).

[0027] In the present invention, the term "bioactive ingredient" as used herein refers to a substance that has activity in living organisms. Examples of bioactive ingredients include, but are not limited to, small molecules, chemical drugs, enzymes, protein drugs, antibodies, vaccines, antigens, antibiotics, or nucleotide drugs.

[0028] Pore modified MSN Nanoparticle formulations offer several advantages for multidrug co-delivery compared with free drug combinations: (1) Nanoparticles can simultaneously deliver hydrophobic and hydrophilic drugs, maintaining an optimized synergistic drug ratio in a single nanoparticle. (2) Nanoparticles can extend circulation time and enhance the tumor-targeting ability of drugs. (3) Drug-encapsulated nanoparticles can simultaneously deliver multiple drugs into target cells and normalize pharmacokinetic differences between drugs. Mesoporous silica nanoparticles (MSNs) have been considered to have great potential as drug delivery systems due to their unique physical and chemical properties, such as large pore volume, chemical and thermal stability, high loading capacity, tunable surface properties, and excellent biocompatibility. The outer or pore surfaces of MSNs can be easily modified individually with various functional groups. However, although encapsulation of hydrophobic and hydrophilic drugs in the same particle did not affect the monodispersity of the particles in solution, it remains a challenge and requires improvement. In the present invention, small-sized MSNs (<100 nm) with enlarged pore size (optionally) and pore surface modified with specific functional groups and ratios can encapsulate two drugs (one hydrophobic and another hydrophilic in one embodiment) in the same particle, exhibiting synergistic therapeutic effects in anti-multidrug resistant cancer cells. The co-delivery of multiple drugs by the particles mentioned in the present invention can also be used to treat various indications, such as cancer, multidrug resistant cancer, brain-related cancer, metastatic brain cancer, and central nervous system diseases.

[0029] The pore-modified MSNs and exMSNs of the present invention have appropriate apparent size, dynamic light scattering size, and pore size so that they can be sufficiently loaded with bioactive components and transported through the circulatory system of a subject. In certain embodiments, the pore-modified MSNs and exMSNs can even penetrate the blood-brain barrier (BBB) and / or the blood-ocular barrier.

[0030] The pore size of MSNs and exMSNs can affect the loading capacity and / or efficiency of bioactive components. If the pore size is too small, the loading capacity of larger molecules may be insufficient. If the pore size is too large, the loading efficiency may be low and the loaded drug may easily leak through the pores. In one embodiment, the pore size of MSNs and exMSNs is 50 nm or less, preferably 1 to 20 nm, more preferably 3 to 10 nm, 1 to 10 nm, or 1 to 5 nm. The pore size can be appropriately controlled by appropriate synthesis procedures and / or materials based on requirements, for example, based on the size of the bioactive component to be loaded into the pores.

[0031] The size of MSNs and exMSNs can play an important role in enhancing their transport in the circulatory system. If the size is too large, the nanoparticles may be quickly identified and removed by the immune system. If the size is too small, the nanoparticles may be quickly and easily removed from the body by renal filtration. In one embodiment, the size of MSNs and exMSNs is 100 nm or less, preferably 80 nm or less, 65 nm or less, 60 nm or less, or 50 nm or less, more preferably 40 nm or less. In one embodiment, the size of MSNs and exMSNs is at least 60 nm, preferably at least 40 nm, more preferably at least 30 nm. In one embodiment, the size of MSNs and exMSNs varies from any of the endpoints of the range disclosed herein, such as a feasible range consisting of 30 nm to 100 nm.

[0032] Dynamic light scattering (DLS) size of MSNs and exMSNs is used to evaluate their suspension in different solutions. If the DLS size is too large, they may easily aggregate in stock solutions or under physiological conditions, which can be disadvantageous in producing pharmaceutically stable compositions and providing stable processes. Furthermore, the compositions may not be usable for clinical applications due to insufficient blood circulation and a high risk of vascular occlusion. To better evaluate the potential of MSNs and exMSNs for application in living subjects, DLS size is preferably measured both in water and in a medium biologically similar or equivalent to phosphate-buffered saline (PBS). In one embodiment, the dynamic light scattering size of MSNs and exMSNs is at least 60 nm, more preferably at least 30 nm. In one embodiment, the dynamic light scattering size of MSNs and exMSNs is 150 nm or less, preferably 100 nm or less, more preferably 60 nm or less, or 30 nm or less. In one embodiment, the dynamic light scattering size of the MSNs and exMSNs ranges from any of the endpoints of the numerical ranges disclosed herein, such as a feasible numerical range consisting of 30 nm to 150 nm. Without being bound by theory, nanoparticles with a DLS size greater than 150 nm may easily aggregate or be removed by the immune system, and therefore may not be able to adequately deliver bioactive ingredients.

[0033] The surfaces of the pores of MSNs and exMSNs are modified with functional groups, hereinafter referred to as "internal modification." Internal modification can enable bioactive components to be more stably trapped or encapsulated within the pores. Internal modification can also affect the dispersibility of MSNs and exMSNs. The functional groups for internal modification are typically hydrophobic, particularly aromatic. In one embodiment, the surfaces of the pores of MSNs and exMSNs are modified with functional groups having terminal hydrocarbyl moieties. In one embodiment, the hydrocarbyl moieties are aromatic moieties selected from benzene (phenyl), naphthalene, anthracene, phenanthrene, diphenyl ether, ellagic acid, carbazolyl, quercetin, and the like. In one embodiment, the aromatic moieties are substituted with lower alkyl, alkenyl, alkoxyl, or halogen. In one embodiment, the aromatic moieties can be derived from aromatic siloxanes, such as trimethoxyphenylsilane (TMPS). The amount (e.g., content) of the terminal aromatic moieties can be measured to ensure that the terminal aromatic moieties are sufficient. In one embodiment, the terminal hydrocarbyl moieties are (cyclo)aliphatic moieties selected from (cyclo)pantanyl, (cyclo)hexanyl, (cyclo)heptanyl, (cyclo)octanyl, (cyclo)nonanyl, (cyclo)decanyl, (cyclo)undecanyl, (cyclo)dodecanyl, etc. In one embodiment, the terminal hydrocarbyl moieties comprise aromatic moieties, (cyclo)aliphatic moieties, or a combination thereof. Without being bound by theory, if the amount of terminal hydrocarbyl moieties on the interior surface is too high, the dispersibility of MSNs and exMSNs may be insufficient, and if the amount (proportion) of terminal moieties is too low, the loading capacity / efficiency of hydrophobic bioactive ingredients may be insufficient.

[0034] The "external" surface of MSNs and exMSNs, i.e., the particle surface, can be modified with functional groups, which also alter the properties of the MSNs and thereby their bioapplication performance. For example, poly(alkoxyl glycol) (PAG)-type group modification can improve particle suspension in culture media, reduce immunogenicity, and extend circulation time in the body. Although MSNs do not have any external surface modifications, they typically have a negative charge on their surface. Therefore, modification with polyethyleneimine (PEI), alkoxysilane-terminated (poly)alkylene (poly)amine, or amine-containing organic alkoxysilanes can be applied to give particles a positive or weakly negative surface charge, or to make the outer surface electrically neutral. On the other hand, modification with carboxyl, phosphoryl, or sulfonate-containing organic alkoxysilanes can give particles a strong negative charge. Furthermore, the combination of functional groups on the particle surface can provide a variety of surface properties.

[0035] EPR effect of MSN and exMSN In general, EPR-mediated passive targeting relies heavily on extending the circulation time of nanocarriers. Tumor targeting based on the permeability and retention (EPR) enhancement effect can be addressed by (1) high-density PEGylation; (2) spatial control of surface functional groups; (3) fabrication of small MSNs and exMSNs; and (4) control of protein corona formation. Two particularly important parameters are particle size and surface properties, which are expected to play a key role in the circulatory half-life, pharmacokinetics, and biodistribution of nanocarriers. Typically, injected materials are recognized by serum opsonins, rapidly bound, subsequently phagocytosed, and substantially accumulated in both the liver and spleen (also known as the mononuclear phagocyte system). Furthermore, comprehensive studies have highlighted protein corona neutrality as a critical design factor in the development of targeted nanomaterial delivery and demonstrated that even slight differences in surface heterogeneity can have the opportunity to result in significantly different interactions with cells and tissues. Therefore, controlling and understanding protein corona composition may be crucial for developing successful EPR-targeted nanomedicines.

[0036] BBB penetration effect The blood-brain barrier (BBB) restricts the delivery of most therapeutic drugs to the brain. Nanomedicines can increase BBB penetration by manipulating nanoparticle size, shape, surface charge, and conjugated ligands. Nanoparticles conjugated with targeting ligands that bind to receptors on endothelial cells, such as transferrin, lactoferrin, glutathione, and low-density lipoprotein receptors, can also promote BBB penetration. However, modification of the outer nanosurface with targeting ligands can also affect the nanosurface's suspension and circulation in the blood, accelerating its blood clearance. We increased the BBB penetration ability of PEGylated MSNs and exMSNs by varying and controlling their size, surface composition, and zeta potential. These modifications, spatial arrangement, and charge allow MSNs and exMSNs to exhibit properties including minimal nonspecific binding, adequate circulation duration in physiological environments, and their transport from the blood to the brain.

[0037] Blood-ocular barrier penetration effect Major causes of visual impairment and blindness are posterior segment-related diseases, including age-related macular degeneration, diabetic macular edema, glaucoma, and endophthalmitis. However, like the blood-brain barrier, the blood-ocular barrier prevents most therapeutic drugs from being delivered to the eye, especially the posterior segment. To overcome this barrier, the properties of MSNs and exMSNs, such as nanoparticle size, surface charge, and configuration, can be adjusted to increase penetration of the ocular static and dynamic barriers, thereby improving ocular bioavailability. Therefore, MSNs and exMSNs are potential ocular drug delivery carriers for treating ocular diseases. The administration routes of MSNs and exMSNs for such applications can be topical (eye drops), intravitreal, subconjunctival, subretinal, peribulbar, posterior scleral, retrochoroidal, intracameral, subtenon, and systemic injection.

[0038] bioactive ingredients The bioactive ingredient used herein may be hydrophilic, hydrophobic, or lipophilic. In one embodiment, the bioactive ingredient may be hydrophilic or modified to be hydrophilic, and may be selected from those that are water-soluble or have surface modifications that allow them to be dispersed or dissolved in an aqueous phase. In one embodiment, the bioactive ingredient is an enzyme, a protein drug, an antibody, a vaccine, an antibiotic, or a nucleotide drug. Examples of enzymes include, but are not limited to, agalsidase, imiglucerase, taliglucerase, velaglucerase, alglucerase, sebelipase, laronidase, idursulfase, elosulfase, galsulfase, alglucosidase, asparaginase, glutaminase, arginine deminase, arginase, methioninase, cysteinase, homocysteinase, phenylalanine hydroxylase, phenylalanine ammonia-lyase, urate oxidase, catalase, horseradish peroxidase, superoxide dismutase, or glutathione peroxidase.

[0039] In one embodiment, the bioactive ingredient can be appropriately selected based on its hydrophilicity, hydrophobicity, or amphiphilicity and the associated disorder / disease. Examples of bioactive ingredients include, but are not limited to, everolimus, trabectedin, Abraxane, TLK286, AV-299, DN-I01, pazopanib, GSK690693, RTA744, ON 0910.Na, AZD6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, FLT-3 inhibitors, VEGFR inhibitors, EGFR inhibitors, TK inhibitors, Aurora kinase inhibitors, PIK-1 modulators, Bcl-2 inhibitors, HDAC inhibitors, c-MET inhibitors, PARP inhibitors, Cdk inhibitors, EGFR TK inhibitors, IGFR-TK inhibitors, anti-HGF antibodies, PI kinase inhibitors, AKT inhibitors, JAK / STAT inhibitors, checkpoint 1 or 2 inhibitors, focal adhesion kinase inhibitors, MAP kinase kinase (mek) inhibitors, VEGF trap antibodies, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171, vatabulin, ofatumumab, zanolimumab, edotecarin, tetrandrine, rubitecan, tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490, cilengitide, gimatecan, IL 13-PE38QQR, INO 1001, IPdR1 KRX-0402, Lucanton, LY 317615, Neuradiab, Baytespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 311, romidepsin, ADS-I00380, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, liposomal doxorubicin, 5´-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709, seliciclib, PD0325901,AZD-6244, capecitabine, L-glutamic acid, N-[4-[2-[2-amino-4,7-dihydro-4-oxo-1-H-pyrrolo[2,3-d]pyrimidin-5-yl]ethyl]benzoyl]-disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, DES (diethylstilbestrol), estradiol, estrogen, conjugated estrogens, bevacizumab, IMC-1C11, CHIR-258, 3-[5-(methyl (methyl)indolyl)quinolone, vatalanib, AG-013736, AVE-0005, goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megstol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megstol acetate, CP-724714; TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody, Erbitux, EKB-569, PKI-166 , GW-572016, ionafarnib, BMS-214662, tipifamib; amifostine, NVP-LAQ824, hydroxamic acid suberoylanalide, valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951, aminoglutethimide, amsacrain, anagrelide, L-asparaginase, Bacillus Calmette-Guerin (BCG) vaccine, bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, cyprotease ron, cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol, epirubicin, fludarabine, etc., fludrocortisone, fluoxymesterone, flutamide, gemcitabine, hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole, lomustine, mechlorestamine, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin, pamidronate, pentostatin,Plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, teniposide, testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine, 13-cis-retinoic acid, phenylalanine mustard, uracil mustard, estramustine, altretamine, floxuridine, 5-deoxyuridine, cytosine arabinoside, 6-mercaptopurine, deoxycodone Lumycin, calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine, topotecan, razoxine, marimastat, COL-3, neovastatin, BMS-275291, squalamine, endostatin, SU5416, SU6668, EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene, idoxifene, spironolactone, finasteride Cimitidine, trastuzumab, denileukin diftitox, gefitinib, bortezimib, paclitaxel, cremophos-free paclitaxel, docetaxel, ixabepilone, epithilone B, BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR-3339, ZK186619, topotecan, PTK787 / ZK222584, VX-745, PD 184352, rapamycin, 40-O-(2-hydroxyethyl)rapamycin, tensirolimus, AP-23573, RAD001, ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, Wortmermin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin, erythropoietin granulocyte colony-stimulating factor, zoledronic acid, prednisone, cetuximab, granulocyte-macrophage colony-stimulating factor, histrelin, pegylated interferon alpha-2a, interferon alpha-2a, pegylated interferon alpha-2b, interferon alpha-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab, hydrocortisone,Interleukin-11, dexrazoxane, alemtuzumab, all-trans retinoic acid, ketoconazole, interleukin-2, megestrol, immunoglobulin, nitrogen mustard, methylprednisolone, ibriggumomab tiuxetan, androgen, decitabine, hexamethylmelamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, liposomal daunorubicin, asparaginase, strontium-89, casopitan acetaminophen, netupitant, NK-1 receptor antagonists, palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, dolasetron, tropisetron, pegfilgrastim, erythropoietin, epoetin alfa, curcumin, ALZ001, JM17, and darbepoetin alfa.

[0040] The bioactive ingredients can be in various forms so that they can be appropriately loaded into or onto the MSNs and exMSNs, including, but not limited to, aqueous forms, dispersed forms, ionized forms in solution or dispersion, hydrated or solvated forms in solution or dispersion, etc.

[0041] Bioactive ingredient-loaded MSNs and exMSNs MSNs and exMSNs can be loaded with bioactive components within the pores. Indeed, the present invention can provide MSNs or exMSNs loaded with at least one bioactive component. In particular, the present invention provides MSNs or exMSNs loaded with at least two bioactive components. The at least two bioactive components may have similar or different hydrophobicities. In one embodiment, the MSNs or exMSNs are loaded with a hydrophilic bioactive component and a hydrophobic bioactive component. In one embodiment, the MSNs or exMSNs are loaded with at least two bioactive components, at least one of which is hydrophilic and the rest is hydrophobic. In one embodiment, the MSNs or exMSNs are loaded with at least two bioactive components, at least one of which is hydrophobic and the rest is hydrophilic. In one embodiment, the MSNs or exMSNs are loaded with at least two bioactive components, all of which are hydrophilic. In one embodiment, the MSNs or exMSNs are loaded with at least two bioactive components, all of which are hydrophobic. In one embodiment, two or more bioactive components provide a synergistic effect. The ratio of the bioactive ingredients can be adjusted to meet the requirements of the intended purpose.

[0042] Methods for producing pore-modified MSNs or exMSNs and nanoparticles loaded with bioactive ingredients The present application also provides a method for producing MSNs and exMSNs with internal organic modifications on the pores. In one embodiment, the method comprises the following steps: (a) providing an alkaline solution containing a surfactant to form micelles; (b) adding to the solution a first silica source and a second silica source providing terminal hydrocarbyl moieties, wherein the molar ratio of the first silica source to the second silica source is 5:1 or greater; (c) subjecting the solution to a hydrothermal treatment; and (d) Extracting and optionally purifying the MSNs from the solution.

[0043] In a further embodiment, the method further comprises at least one of the following steps: (e) after step (a) and before step (b), introducing an oil phase into the solution for pore expansion; (f) adding an additional silica source after step (b); and (g) performing a surface modification of the outer surface of the MSNs, the surface modification being performed after step (b) or, if step (f) is performed, after step (f).

[0044] The ratio of the first silica source providing the terminal hydrocarbyl moiety to the second silica source can be adjusted to improve the bioactive component loading capacity and / or efficiency of the MSNs thus prepared, particularly by 5:1 or more. In one embodiment, the molar ratio of the silica source to the terminal aromatic moiety-providing reagent ranges from 29:1 to 4:1, preferably from 26:1 to 5:1, and more preferably from 21:1 to 11:1. Furthermore, the ratio may be an important factor affecting the DLS size of MSNs and exMSNs. Without being bound by theory, the applicants postulate that if the ratio of the first silica source to the second silica source is too low, the outer surface will have organic modifications such that the DLS size dramatically expands beyond 150 nm, hindering their application in delivering bioactive components to living subjects.

[0045] The modifiers for interior surface modification provide terminal hydrocarbyl moieties on the surfaces of the pores of MSNs and exMSNs. Examples of the modifying agent include trimethoxyphenylsilane (TMPS), triethoxyphenylsilane, diphenyldiethoxysilane, 1-naphthyltrimethoxysilane, 2-hydroxy-4-(3-triethoxysilylpropoxy)diphenylketone, O-4-methylcoumarinyl-N-[3(triethoxysilyl)propyl]carbamate, 7-triethoxysilylpropoxy-5-hydroxyflavone, 3-carbazolylpropyltriethoxysilane, bis(2-diphenylphosphinoethyl)methylsilylethyltriethoxysilane, 2-(diphenylphosphino)ethyltriethoxysilane, propyltriethoxysilane, n-butyltriethoxysilane, pentyltriethoxysilane, hexyltriethoxysilane, heptyltriethoxysilane, octyltriethoxysilane, nonyltriethoxysilane, and decyltriethoxysilane. , undecyltriethoxysilane, dodecyltriethoxysilane, cyclopropyltriethoxysilane, cyclobutyltriethoxysilane, cyclopentyltriethoxysilane, cyclohexyltriethoxysilane, cycloheptyltriethoxysilane, cyclooctyltriethoxysilane, propyltrimethoxysilane, n-butyltrimethoxysilane, pentyltrimethoxysilane, hexyltrimethoxysilane, heptyltrimethoxysilane, octyltrimethoxysilane, nonyltrimethoxysilane, decyltrimethoxysilane, undecyltrimethoxysilane, dodecyltrimethoxysilane, cyclopropyltrimethoxysilane, cyclobutyltrimethoxysilane, cyclopentyltrimethoxysilane, cyclohexyltrimethoxysilane, cycloheptyltrimethoxysilane, and cyclooctyltrimethoxysilane, etc.

[0046] The outer surface of MSNs and exMSNs can be modified de novo or post-treatment. Examples of modifications include hydrophilic modifications such as polyethylene glycol (PEG), polyethylenimine (PEI), 3-(trihydroxysilyl)propylmethylphosphonate (THPMP), N-(trimethoxysilylpropyl)ethylenediaminetriacetic acid (EDTAS)-N-[3-(trimethoxysilyl)propyl]ethylenediamine, N-[3-(trimethoxysilyl)propyl]-N,N,N-trimethylammonium (TA-trimethoxysilane), (3-mercatopropyl)trimethoxysilane (MPTMS), and zwitterionic silanes; specific modifications such as biomarker modifications, e.g., antibody modifications, linker modifications, and tumor-targeting ligand modifications; or modifications of the shell surface properties, e.g., modifications of nonspecific activity, e.g., charge type and distribution modifications.

[0047] The present subject matter also provides methods for producing MSNs and exMSNs loaded with bioactive ingredients. In one embodiment, the methods include the following steps: (a) providing MSNs or exMSNs prepared by any of the methods described above; (b) loading the MSNs or exMSNs with a first bioactive component by contacting the MSNs or exMSNs with the first bioactive component; (c) loading a second bioactive component by contacting the first bioactive component-loaded MSNs or exMSNs; (d) Optionally, loading additional bioactive ingredients by repeating step (c) and independently and sequentially contacting the bioactive ingredient-loaded MSNs or exMSNs with the additional bioactive ingredients.

[0048] In one embodiment, the first and second bioactive components have similar or the same hydrophobicity. In one embodiment, the first and second bioactive components differ in terms of hydrophobicity. In a specific embodiment, the first bioactive component is hydrophilic and the second bioactive component is hydrophobic. In another specific embodiment, the first bioactive component is hydrophobic and the second bioactive component is hydrophilic. In one embodiment, the additional bioactive components are independently hydrophilic or hydrophobic.

[0049] Applications of bioactive ingredient-loaded MSNs and exMSNs Mesoporous silica nanoparticles (MSNs) have been considered to have great potential as drug delivery systems due to their unique physical and chemical properties, such as large pore volume, chemical and thermal stability, high loading capacity, tunable surface properties, and excellent biocompatibility. In particular, the MSNs and exMSNs of the present subject application may have the ability to penetrate the blood-brain barrier (BBB) and / or the blood-ocular barrier, making them suitable for certain applications. [Example]

[0050] The following examples are provided to help those skilled in the art to better understand the present invention, but are not intended to limit the scope of the present invention. Materials, methods and test models

[0051] Transmission electron microscope (TEM) The appearance of silica nanoparticles was directly examined and verified using transmission electron microscopy (TEM). TEM images were taken with a Hitachi H-7100 transmission electron microscope operating at an accelerating voltage of 75–100 kV. Samples dispersed in ethanol were dropped onto a carbon-coated copper grid and air-dried for TEM observation.

[0052] Dynamic Light Scattering (DLS) Size measurements of silica nanoparticles in different solution environments were performed using dynamic light scattering (DLS) on a Malvern Zetasizer Nano ZS (Malvern, UK). The size of the (solvated) particles formed in various solutions was analyzed: HO, Dulbecco's modified Eagle's medium (DMEM) with 10% FBS, PBS buffer (pH 7.4), and 5% glucose at room temperature.

[0053] elemental analysis The mass percentages of carbon, nitrogen, oxygen and hydrogen in the silica nanoparticles were measured by an elemental analyzer (elementar Vario EL cube type for NCSH, German). Nanoparticle Tracking Analysis (NTA) The particle concentration (number / mg) of the sample liquid is measured by Nanosight NS300.

[0054] Quantification of drugs in nanoparticles To 5 μL of drug-loaded nanoparticle suspension (from stock, 200 mg / mL), 45 μL of HO was added for a 10-fold dilution, and then 19.6 μL of the diluted solution (20 mg / mL) was taken to mix with 32.4 μL of DMSO and 78 μL of ACN (containing 0.5% HF). After shaking at 4 °C for 10 min, additional bare silica nanoparticles were added to the solution to deplete HF residues. The solution was then centrifuged at 14,000 rpm for 10 min, and 120 μL of the supernatant was taken for HPLC analysis.

[0055] In vivo imaging system for detecting the biodistribution and EPR effect of nanoparticles In vivo biodistribution images of nanoparticles were obtained using an IVIS imaging system (Lumina). Balb / c mice (4 weeks old) were purchased from BioLASCO. Tumor-bearing mice were established by subcutaneous injection of 4T1 (ATCC® CRL-2539™) tumor cells for heterotopic transplantation. 4T1 cells were allowed to grow for 2–3 weeks, after which samples in PBS were injected intravenously. 24 h after injection, major organs (heart, lungs, spleen, liver, and kidneys) as well as tumors, urine, and blood were carefully collected, and fluorescence images and intensity of the collected samples were acquired using the IVIS imaging system.

[0056] Two-photon fluorescence microscopy for detecting nanoparticles in cerebral blood vessels Healthy ICR mice (27-30 g) were intravenously injected with 200 mg / kg of nanoparticles, and dynamic imaging of the mouse earlobes was performed using multiphoton microscopy (Olympus FVMPE-RS) with tunable excitation wavelengths (800-1000 nm). After the nanoparticles no longer circulated in the cerebral blood vessels, the mice were anesthetized and then underwent skull removal (craniotomy). In this study, normal saline was used instead of placing a glass cover on the brain surface for short-term observation. To image the blood vasculature, 0.6 mL of 2.5% (w / V) fluorescein isothiocyanate dextran (FITC-dextran, Mw: 70 kDa) dissolved in sterile saline was intravenously injected into the mice. Depth profile imaging of nanoparticles in the mouse cerebrum was collected from 0-300 μm below the brain surface (axial intervals of 1 μm).

[0057] Determination of drug combination dosage ratios by in vitro antitumor synergy testing The synergistic cytotoxic effect of JM17 (a curcumin analog) and doxorubicin (Dox) was evaluated by Alamar Blue assay (Invitrogen). For proliferation assays, 5 × 10 cells were cultured per well. 4MCF7 / ADR cells (Dox-resistant breast cancer cells) were seeded in 24-well plates. Various concentrations of JM17, various concentrations of Dox, and various combination dose ratios of JM17 and Dox were incubated with the cells. After 48 hours of incubation, the cells were washed twice with culture medium and then incubated with Alamar Blue reagent at 37°C for 2 hours. The fluorescent signal from the Alamar Blue assay was proportional to the number of live cells, and the signal (Ex / Em = 560 / 590) was measured using a microplate reader (Bio-Rad, Model 68). The Zheng-Jun Jin method (Q method) or the Chou-Talalay combination index (CI) theorem was used to analyze the synergistic effects of drug combinations.

[0058] MCF7 / ADR cancer animal model Female BALB / c nude mice (5-6 weeks old) were purchased from the National Laboratory Animal Center. Estrogen was continuously administered orally through the drinking water of the mice during the experiment. 5 × 10 estrogen-containing mice were cultured in 25 μL of PBS and 25 μL of Matrigel matrix. 6 MCF-7 / ADR cells were xenografted subcutaneously into the left flank of BALB / c nude mice. Tumor growth was monitored for approximately 1 month (size ~50 mm). 3 ) and then intravenously injected into all groups on days 0, 4, and 8. Mouse weights and tumor volumes were measured twice weekly.

[0059] Synthesis Example 1 Pore-expanded MSN-PEG+TA (exMSN-PEG-TA) synthesis Expanded mesoporous silica nanoparticles (exMSNs) have a well-defined structure, large pores, and a high density of surface silanol groups, and can be modified with a wide range of organic functional groups. First, 0.386 g of CTAB was dissolved in 160 g of ammonium hydroxide solution (0.22 M) in a sealed beaker at the desired temperature (50 °C). After 10 min, 16.2 mL of a diluted decane-alcohol solution (7.4% v / v) was added, and the mixture was continuously stirred for at least 8 h (decane was added as an oil phase to expand the pore size). The sealed lid was then removed, and 660 μL of TEOS in 2.64 mL of ethanol was added to the mixture with stirring for an additional 1 h. Next, 550 μL of 2-[methoxy(polyethyleneoxy)propyl]trimethoxysilane (PEG) and 300 μL of N-[3-(trimethoxysilyl)propyl]-N,N,N-trimethylammonium chloride (TA) in 3 mL of ethanol were introduced into the reaction system. After stirring the mixture for 30 min, the solution was aged overnight at the desired temperature (50 °C) without stirring. Next, the solution was filtered through 0.45 μm and 0.22 μm filters to remove membrane by-products. The filtrate was then sealed and placed in an oven at 80 °C for 24 h for hydrothermal treatment. The as-synthesized sample was washed and collected by cross-flow filtration. To remove the surfactant in the nanoparticle pores, the as-synthesized sample was incubated in 50 mL of acidified ethanol containing 848 μL (first) and 50 μL (second) of 37% hydrochloric acid at 60 °C for 1 h, respectively. The product was washed and recovered by cross-flow filtration and then stored in 90% ethanol.

[0060] NH2+COOH-exMSN-PEG synthesis First, 0.386 g of CTAB was dissolved in 160 g of 0.22 M ammonium hydroxide solution in a sealed beaker at the desired temperature (50 °C). After 10 min, 16.2 mL of diluted decane alcohol solution (7.4% v / v) was added, and the mixture was continuously stirred for at least 8 h. The sealed lid was then removed, and 700 μL of TEOS, 50 μL of 3-(2-aminoethylamino)propyltrimethoxysilane (AAS), and 5 μL of N-(trimethoxysilylpropyl)ethylenediaminetriacetic acid trisodium salt (EDTAS) in 3.35 mL of ethanol were introduced into the mixture with stirring for 1 h. Next, 550 μL of PEG in 3 mL of ethanol was introduced into the reaction system. After stirring the mixture for 30 min, the solution was aged overnight at the desired temperature (50 °C) without stirring. The solution was then filtered through 0.45 μm and 0.22 μm filters to remove membrane by-products. The filtrate was then sealed and placed in an oven at 80 °C for 24 hours for hydrothermal treatment. The as-synthesized sample was washed and collected by cross-flow filtration. To remove the surfactant in the nanoparticle pores, the as-synthesized sample was incubated in 50 mL of acidified ethanol containing 848 μL (first) and 50 μL (second) of 37% hydrochloric acid at 60 °C for 1 hour, respectively. The product was washed and collected by cross-flow filtration and then stored in 90% ethanol.

[0061] Synthesis of BisTESB-exMSN-PEG+TA (with different TEOS / BisTESB ratios) First, 0.386 g of CTAB was dissolved in 160 g of ammonium hydroxide solution (0.22 M) in a sealed beaker at the desired temperature (50 °C). After 10 min, 16.2 mL of diluted decane alcohol solution (7.4% v / v) was added, and the mixture was continuously stirred for at least 8 h. The sealed lid was then removed, and 583.3 μL of TEOS / 200 μL of 1,4-bis(triethoxysilyl)benzene (BisTESB) (4:1), 636.4 μL of TEOS / 109 μL of BisTESB (9:1), or 656.3 μL of TEOS / 75 μL of BisTESB (14:1) in 2.8 mL of ethanol was added to the mixture with stirring for 1 h. Next, 550 μL of PEG and 300 μL of TA in 3 mL of ethanol were added to the reaction system. After stirring the mixture for 30 minutes, the solution was aged overnight at the desired temperature (50 °C) without stirring. The solution was then filtered through 0.45 μm and 0.22 μm filters to remove membrane by-products. The filtrate was then sealed and placed in an oven at 80 °C for 24 hours for hydrothermal treatment. The as-synthesized sample was washed and collected by cross-flow filtration. To remove the surfactant in the nanoparticle pores, the as-synthesized sample was incubated in 50 mL of acidified ethanol containing 848 μL (first) and 50 μL (second) of 37% hydrochloric acid at 60 °C for 1 hour, respectively. The product was washed and collected by cross-flow filtration and then stored in 90% ethanol.

[0062] Synthesis of phenyl-exMSN-PEG+TA (with different TEOS / phenylsilane ratios) The exMSN structure is formed by the co-condensation of inorganic tetraethoxysilane (TEOS) and organic trimethoxyphenylsilane (TMPS), which imparts hydrophobicity to the exMSN pores and can improve hydrophobic drug loading efficiency. Monodisperse phenyl-exMSN-PEG+TA with enlarged pores was prepared by further introducing decane into a highly diluted surfactant-containing ammonia solution. First, 0.386 g of CTAB was dissolved in 160 g of ammonium hydroxide solution (0.22 M or 0.35 M) in a sealed beaker at the desired temperature (50 °C). After 10 min, 16.2 mL of diluted decane-alcohol solution (7.4% v / v) was added, and the mixture was continuously stirred for at least 8 h. Then, the sealed lid was removed, and 2.5 mL of diluted APTMS alcohol solution (10.7 mM) and 350 μL–700 μL of TEOS were added, and 37 μL, 47 μL, 56 μL, and 112 μL of TMPS mixed in 1.9 mL–3.3 mL of ethanol (TEOS / TMPS molar ratios = 16:1, 13:1, 11:1, and 5:1; varying the TEOS:TMPS ratio can adjust the hydrophobicity of the pore surface for various applications or loaded drugs) were added to the solution under vigorously stirring. After 3–60 min, 140 μL–350 μL of ethanolic TEOS in 0.56 mL–1.4 mL of EtOH was added under several conditions (TEOS / TMPS = 16:1, 13:1, and 11:1). After 1–2 h of reaction, 550 μL–1000 μL of PEG and 155.8 μL–450 μL of TA mixed in 2 mL–3 mL of EtOH were introduced into the reaction. After stirring the mixture for 60 min, the mixture was aged overnight at the desired temperature (50 °C) without stirring. Next, the solution was filtered through 0.45 μm and 0.22 μm filters to remove membrane by-products. The filtrate was then sealed and placed in an oven at 80 °C for 24 h for hydrothermal treatment. The as-synthesized sample was washed and collected by cross-flow filtration. To remove the surfactant in the pores of phenyl-exMSN-PEG+TA, the as-synthesized samples were incubated in 50 mL of acidic ethanol containing 848 μL (first time) and 50 μL (second time) of hydrochloric acid (37%) at 60 °C for 1 h, respectively.The product was washed, collected by cross-flow filtration, and then stored in 90% ethanol. The particle sizes of phenyl-exMSN-PEG+TA with various internal modifications measured by dynamic light scattering (DLS) in different solution environments are shown in Table 1. DLS results showed that all MSNs dispersed well within the range of approximately 40 nm to 70 nm in HO, but the dispersibility of highly phenyl-incorporated particles (TEOS:TMPS = 5:1) was affected in PBS buffer. This suggests that partial phenyl groups may be modified on the surface of highly phenyl-incorporated particles, affecting their dispersibility.

[0063] [Table 1]

[0064] Synthesis Example 2 Synthesis of phenyl-MSN-PEG+TA (with different TEOS / phenylsilane ratios) Phenyl-MSN-PEG+TA was prepared using an ammonia base catalyzed method under highly diluted, low-surfactant conditions. The amount of phenyl groups in phenyl-MSN-PEG+TA was adjusted by introducing various amounts of TEOS and TMPS (TEOS / TMPS molar ratios of 29:1, 26:1, 21:1, 20:1, 15:1, and 11:1) into the reaction. Typically, 0.29 g of CTAB was dissolved in 150 mL of ammonium hydroxide solution (0.171 M or 0.205 M) at the desired temperature (50 °C). After stirring for 15 min, the sealing membrane was removed, and then 250 μL of ethanolic TEOS containing 16 μL, 20 μL, 24 μL, 32 μL, or 40 μL of TMPS mixed in 1 mL of EtOH was added to the solution with vigorous stirring. After 10–30 min, 250 μL or 300 μL of ethanolic TEOS in 1 mL or 1.2 mL of EtOH was added. After 1–2.5 h of reaction, 50 μL of ethanolic TEOS dissolved in 200 μL of EtOH was added for 10 min, followed by the addition of 550 μL–825 μL of PEG and 300 μL–450 μL of TA mixed in 2 mL–2.6 mL of EtOH. After stirring the mixture for 1 h, the mixture was aged at the desired temperature (50 °C) without stirring for at least 12 h. The solution was then sealed and hydrothermally treated in a 70 °C oven for 24 h. The as-synthesized samples were washed and collected by cross-flow filtration. To remove the surfactant in the pores of phenyl-MSN-PEG+TA, the as-synthesized samples were extracted and recovered in 40 mL of acidic ethanol containing 678 μL (first) and 40 μL (second) of 37% hydrochloric acid at 60 °C for 1 h. The products were washed, collected by cross-flow filtration, and then stored in 90% ethanol. Table 2 shows the particle sizes of phenyl-MSN-PEG+TA with various internal modifications measured by dynamic light scattering (DLS) in different solution environments. DLS results showed that all MSNs dispersed well within the range of approximately 30 nm to 50 nm in HO, but the dispersibility of highly phenyl-incorporated particles (TEOS:TMPS = 11:1 and 15:1) was affected in PBS buffer. This suggests that partial phenyl groups may be modified on the surface of highly phenyl-incorporated particles, affecting their dispersibility.

[0065] [Table 2]

[0066] Synthesis Example 3 Synthesis of C8-MSN-PEG+TA (with different TEOS / C8-silane ratios) C8-MSN-PEG+TA was prepared using an ammonia base catalysis method using a highly diluted and low surfactant solution. The octyl group content in C8-MSN-PEG+TA was adjusted by adjusting the total TEOS and C8-reactant amounts (the TEOS / C8-reactant molar ratio varied from 20:1, 15:1, 10:1, and 5:1). Typically, 0.29 g of CTAB was dissolved in 150 ml of ammonium hydroxide solution (0.205 M) at the desired temperature (50 °C) in a sealed beaker. After stirring for 15 min, the sealing membrane was removed, and then 250 μL of ethanolic TEOS containing 39.2 μL, 52.2 μL, 78.4 μL, or 156.8 μL of C8-silane mixed in 1 mL of EtOH was added to the solution with vigorous stirring. Another addition of 300 μL of ethanolic TEOS in 1.2 mL of EtOH was introduced after 1 h. After 3 hours of reaction, 825 μL of PEG and 450 μL of TA mixed in 2.6 mL of ethanol were introduced for further reaction. After stirring for 1 hour, the mixture was aged at the desired temperature (50 °C) without stirring for at least 12 hours. The resulting solution was then sealed and hydrothermally treated in a 70 °C oven for 24 hours. Finally, the as-synthesized sample was washed, and the product was collected by a cross-flow system. To remove the surfactant in the pores of C8-MSN-PEG+TA, the as-synthesized product was introduced into 40 mL of acidic ethanol containing 678 μL (first) and 40 μL (second) of hydrochloric acid (37%) for two 1-hour extractions at 60 °C. The product was washed and collected by a cross-flow system to obtain the final product, which was finally stored in 90% ethanol. The particle sizes of C8-MSN-PEG+TA with various internal modifications measured by dynamic light scattering (DLS) in various environments are listed in Table 3. All MSNs dispersed well, with sizes of approximately 25–40 nm in HO; however, the dispersibility of highly C8-loaded particles (TEOS:C8-silane = 10:1 and 5:1) in PBS buffer was affected. The results indicate that when a higher amount of C8-silane was used, some of the alkyl groups attached to the outer surface of the C8-loaded particles, thereby reducing their dispersibility.

[0067] [Table 3]

[0068] Example 4 Doxorubicin and JM17 co-loading in pore-modified MSNs and exMSNs 50 mg of exMSNs were first immersed in a NaHCO3 solution (pH 9.95) for 30 minutes, and then the solution was concentrated using a Vivaspin® Turbo 15 to obtain a concentrate. The concentrate was then diluted with deionized water, and the diluted solution was then concentrated again. Doxorubicin (Dox) solution was added to the concentrated solution, and the mixture was shaken at 4°C for 30 minutes. The mixed solution (containing Dox and exMSNs) was then slowly added dropwise to a JM17 solution (in 100% DMSO) and shaken continuously for 2 hours at 4°C. Next, DI water was added to the mixture while vigorously shaking to reduce the DMSO concentration to the maximum DMSO tolerance level of the Vivaspin membrane. Prior to the concentration and washing process, the solution was centrifuged at low speed (3500 g) for 10 minutes to separate any partial JM17 precipitate (if necessary). Finally, the product was stored in DI water. The loading amounts of JM17 and Dox in exMSNs can be adjusted by using different concentrations of JM17 and Dox solutions during the drug loading process.

[0069] All types of exMSNs were loaded with JM17 and Dox using the above method. It was noted that JM17 and Dox could not be simultaneously loaded into exMSN-PEG+TA and NH2+COOH-exMSN-PEG, implying that simply enlarging the pore size of MSNs or modifying the pore surface (positively charged, negatively charged, H-bond donor, or H-bond acceptor) alone does not allow MSNs to simultaneously adsorb a hydrophobic drug (JM17) and a hydrophilic drug (Dox). Although BisTESB-exMSN-PEG+TA nanoparticles were constructed with hydrophobic phenyl groups in their structure, they still could not encapsulate both drugs within the particles. In contrast, phenyl-exMSN-PEG+TA had the ability to simultaneously encapsulate JM17 and Dox, implying that the phenyl groups were modified on the pore surface, thereby enhancing the drug loading capacity. The hydrophobic phenyl group derived from TMPS in phenyl-exMSN-PEG+TA plays an important role in improving hydrophobic drug loading efficiency. Therefore, the TMPS / TEOS ratio for phenyl-exMSN-PEG+TA synthesis is optimized to achieve higher drug loading capacity without affecting the material's dispersibility in solution. Therefore, TEOS / TMPS ratios of 5:1, 11:1, 13:1, and 16:1 were selected for exMSN synthesis to evaluate their effectiveness. The results demonstrate that nanoparticles synthesized with a TEOS / TMPS ratio of 11:1 exhibited the best drug loading capacity (DOX: 1-5%; JM17: 1-5%) and still maintained excellent dispersibility in PBS (DLS size / PDI: 44.4 nm / 0.095). On the other hand, nanoparticles synthesized with a lower TEOS / TMPS ratio, such as 5:1, may severely aggregate during the drug loading process. This aggregation may be caused by the phenyl groups attached to the particle surface, which allows the hydrophobic drug to be attached to the particle surface rather than within the pore space. Aggregated nanoparticles (>200 nm in size even in H2O) cause low drug loading rates, making them unusable for in vivo experiments due to poor circulation and the risk of vascular occlusion.Furthermore, although nanoparticles with a low TEOS / TMPS ratio (16:1) exhibit good suspension in solution, they do not adsorb JM17 efficiently; the loading capacity is too low to be of any use.

[0070] Phenyl-MSN-PEG+TA can also encapsulate drugs with higher drug loading capacities without affecting the dispersibility of the material. Six TEOS / TMPS ratios, 29:1, 26:1, 21:1, 20:1, 15:1, and 11:1, were selected for the synthesis of pore-modified MSNs. Nanoparticles synthesized at TEOS / TMPS ratios ranging from 29:1 to 20:1 exhibited high drug loading capacities (>3% loading mass%) and still maintained excellent dispersibility in PBS (DLS size <60 nm). On the other hand, when synthesized at TEOS / TMPS ratios of 15:1 or 11:1, the nanoparticles severely aggregated after the loading step, and the DLS size of the aggregated nanoparticles exceeded approximately 200 nm in HO.

[0071] Phenyl-exMSN-PEG+TA, phenyl-MSN-PEG+TA, and C8-MSN-PEG+TA can also encapsulate various hydrophobic drugs, such as ixabepilone, paclitaxel, and irinotecan. Test results show that by modifying the specific surface area of the pores and adjusting the ratio of functional groups, MSNs and exMSNs can encapsulate multiple drugs with different physicochemical properties in the same particle.

[0072] Example 5 Phenyl group concentration of phenyl-exMSN In the synthesis of phenyl-MSN-PEG+TA and phenyl-exMSN-PEG+TA, different molar ratios of TEOS and TMPS were used in the reaction to adjust the surface modification of the pores. To quantify the phenyl functional groups on the inner surface of MSN and exMSN nanoparticles, the elemental composition of the phenyl-MSN particles was measured by an elemental analyzer. The number of phenyl groups per mg of particle was derived from the mass percent of carbon in the phenyl-MSN and phenyl-exMSN particles. The number of phenyl groups in phenyl (29:1), (21:1), and (11:1)-MSN obtained from elemental analysis was approximately 6.68 × 10, respectively. 17 , 7.9×10 17 , 1.03×10 18 (molecules / mg), and the number of phenyl groups in phenyl (16:1), (11:1), and (5:1) exMSNs obtained from elemental analysis was approximately 8.08 × 10 17 , 1.04c10 18 , 1.62×10 18 The particle concentrations (number / mg) of the sample solutions were measured by nanoparticle tracking analysis. The concentrations of phenyl (29:1), (21:1), and (11:1)-MSN were 2.29 x 10 12 ,2.27×10 12 ,3.02×10 12 (particles / mg), and the concentrations of phenyl (16:1), (11:1), and (5:1)-exMSN were 2.98 × 10 12 ,3.97×10 12 ,4.44×10 12 (particles / mg). The results show that the amount of phenyl groups on the nanoparticles of phenyl (29:1), (21:1), and (11:1)-MSN is 2.92 × 10 5 , 2.85×10 5 , 3.41 × 10 5 (number of phenyls / particle), and the amount of phenyl groups on each nanoparticle of phenyl(16:1), (11:1), and (5:1)-exMSN was 2.71 × 10 5 , 2.61 × 10 5 , 3.64 × 10 5 (number of phenyls / particle).

[0073] Example 6 In-vitro synergistic effect of JM17 and Dox codelivery by exMSNs According to the method for determining the drug combination dosage ratio, a range of JM17:Dox ratios (1:0.4–1:0.6) showed a higher synergistic effect on the inhibition of Dox-resistant cancer cells (MCF-7 / ADR). Therefore, specific JM17:Dox dose ratios encapsulated by exMSNs were synthesized to evaluate the synergistic cytotoxicity effect of the particles. For the proliferation assay, 5 × 10 cells per well were used. 4 MCF-7 / ADR cells were seeded in a 24-well plate. Four groups containing various concentrations of (1) phenyl-exMSN-PEG + TA (NTT2_131), (2) DOX@phenyl-exMSN-PEG + TA (Dox@NTT2_131), (3) JM17@phenyl-exMSN-PEG + TA (JM17@NTT2_131), and (4) JM17 + DOX@phenyl-exMSN-PEG + TA (JM17 / Dox@NTT2_131) were incubated with the cells. The JM17 / Dox@NTT2_131 treatment group showed efficient inhibition of MCF-7 / ADR cancer cells; it was revealed that co-delivery of JM17 and Dox by nanoparticles can exhibit good synergistic effects in vitro. The results are shown in Figure 1.

[0074] Example 7 In vivo anti-MCF7 / ADR tumor efficacy Female BALB / c nude mice were randomly assigned to six groups (n = 5–6): (1) control, (2) DOX, (3) JM17 + DOX (solution form), (4) JM17 + DOX@NTT2_131, (5) JM17 + DOX@NTT2_131 (half dose), and (6) NTT2_131 (particles only). Groups (1)–(4) received the same doses of Dox and JM17. 5 × 10 cells / ml were added to 25 μL of PBS and 25 μL of Matrigel matrix. 6MCF-7 / ADR cells (Dox-resistant breast cancer cells) were subcutaneously xenografted into the left flank of BALB / c nude mice. After tumor growth continued for approximately one month (size ~50 mm), all groups were intravenously injected on days 0, 4, and 8, and the half-dose group was injected three more times on days 12, 16, and 20. Mouse weights and tumor volumes were measured twice weekly. Results showed that co-delivery of JM17 and Dox demonstrated synergistic antitumor effects. The JM17 / Dox@NTT2_131 (half-dose) group exhibited comparable antitumor efficacy and lower toxicity to the JM17+Dox (solution form) group; furthermore, the JM17 / Dox@NTT2_131 group (standard dose) exhibited the highest antitumor efficacy (Figure 2). The results demonstrated that nanoparticles offer several advantages for multidrug co-delivery compared with free drug (solution) combinations: (1) nanoparticles can simultaneously deliver hydrophobic and hydrophilic drugs and maintain an optimized synergistic drug ratio in a single nanoparticle; (2) nanoparticles can prolong the circulation time and enhance the tumor-targeting ability of drugs; and (3) by simultaneously delivering multiple drugs into target cells, drug-encapsulated nanoparticles can normalize the pharmacokinetic differences between drugs. These advantages of nanoparticle formulations may enhance the synergistic effect of combined drugs on antitumor efficacy.

[0075] Example 8 Blood-brain barrier penetration ability of phenyl-exMSN-PEG+TA particles Delivering therapeutic drugs to the brain remains a major challenge due to the blood-brain barrier (BBB). Nanoparticles smaller than 100 nm offer the advantage of improving drug transport across the BBB. 30 nm phenyl-exMSNs modified with positively charged TA molecules on their surface may have the potential to cross the BBB. To understand the BBB penetration ability of nanoparticles in vivo, we used two-photon fluorescence spectroscopy to monitor the distribution of nanoparticles in the blood vessels of mouse brains. Fluorescently labeled nanoparticles were administered through the tail vein. Two days after injection, we detected cerebral blood vessels located within a depth of 0–300 μm from the surface. Meanwhile, FITC-dextran was injected intravenously to map vasodilation and reveal the boundaries of the vascular wall. When the fluorescent signal from the nanoparticles overlapped with the FITC-dextran signal (green signal), it appeared as a yellow signal (sometimes red), indicating that the nanoparticles were not present in the blood vessels but may have crossed the BBB and entered brain tissue. 3D images of cerebral blood vessels showed numerous red signals from phenyl-exMSN-PEG+TA nanoparticles distributed in the vessel walls and brain tissue regions. The results indicated that 30 nm phenyl-exMSN-PEG+TA exhibited the potential to cross the BBB into brain tissue.

[0076] It should be understood that those skilled in the art of the present invention can make variations and modifications to the teachings and disclosures of the present invention without departing from the spirit and scope of the present application. Based on the above, it is intended that the present application covers all changes and modifications thereof, provided that the changes or modifications fall within the scope defined in the appended claims or their equivalents.

Claims

1. 1. A mesoporous silica nanoparticle comprising an organic modification on the surface of its pores, the mesoporous silica nanoparticle having an apparent particle size of 100 nm or less and a hydrodynamic size in a medium measured by dynamic light scattering (DLS) of 150 nm or less, the organic modification comprising at least one terminal hydrocarbyl moiety, the medium being biosimilar or bioequivalent to phosphate buffered saline (PBS), and the terminal hydrocarbyl moiety comprising a terminal aromatic moiety, a terminal aliphatic moiety having 4 to 30 carbon atoms, or a combination thereof.

2. 2. The mesoporous silica nanoparticles according to claim 1, wherein the pore size of the mesoporous silica nanoparticles is 50 nm or less.

3. 2. The mesoporous silica nanoparticles according to claim 1, wherein the hydrodynamic size of the mesoporous silica nanoparticles is 100 nm or less.

4. 2. The mesoporous silica nanoparticles of claim 1, wherein the terminal aromatic moieties are derived from a silane source selected from the group consisting of trimethoxyphenylsilane (TMPS), triethoxyphenylsilane, diphenyldiethoxysilane, 1-naphthyltrimethoxysilane, 2-hydroxy-4-(3-triethoxysilylpropoxy)diphenylketone, O-4-methylcoumarinyl-N-[3(triethoxysilyl)propyl]carbamate, 7-triethoxysilylpropoxy-5-hydroxyflavone, 3-carbazolylpropyltriethoxysilane, bis(2-diphenylphosphinoethyl)methylsilylethyltriethoxysilane, and 2-(diphenylphosphino)ethyltriethoxysilane.

5. The terminal aliphatic moiety is n-butyltriethoxysilane, pentyltriethoxysilane, hexyltriethoxysilane, heptyltriethoxysilane, octyltriethoxysilane, nonyltriethoxysilane, decyltriethoxysilane, undecyltriethoxysilane, dodecyltriethoxysilane, cyclopropyltriethoxysilane, cyclobutyltriethoxysilane, cyclopentyltriethoxysilane, cyclohexyltriethoxysilane, cycloheptyltriethoxysilane, cyclooctyltriethoxysilane, n-butyltrimethoxysilane, pentaerythritol, pentyltriethoxysilane, hexyltriethoxysilane, heptyltriethoxysilane, octyltriethoxysilane, n-butyltriethoxysilane, pentaerythritol, pentyltriethoxysilane, octyltriethoxysilane ...

2. The mesoporous silica nanoparticles of claim 1, derived from a silane source selected from the group consisting of cyclohexyltrimethoxysilane, hexyltrimethoxysilane, heptyltrimethoxysilane, octyltrimethoxysilane, nonyltrimethoxysilane, decyltrimethoxysilane, undecyltrimethoxysilane, dodecyltrimethoxysilane, cyclopropyltrimethoxysilane, cyclobutyltrimethoxysilane, cyclopentyltrimethoxysilane, cyclohexyltrimethoxysilane, cycloheptyltrimethoxysilane, and cyclooctyltrimethoxysilane.

6. The amount of terminal hydrocarbyl moieties per particle is 1 x 10 6 The mesoporous silica nanoparticles of claim 1 which are sub-molecular / sub-particle.

7. 10. The mesoporous silica nanoparticles of claim 1, further comprising at least one hydrophobic bioactive component or at least one hydrophilic bioactive component loaded within the pores.

8. The mesoporous silica nanoparticles of claim 7, wherein the hydrophobic bioactive component is a small molecule, a chemical drug, an enzyme, a protein drug, an antibody, a vaccine, an antibiotic, a nucleotide drug, or a combination thereof.

9. 8. Mesoporous silica nanoparticles according to claim 7, further comprising at least one hydrophilic or hydrophobic bioactive component loaded within the pores.

10. The mesoporous silica nanoparticles according to claim 9, wherein the bioactive components have a synergistic effect.

11. A delivery agent for use in a method for delivering a bioactive ingredient to a subject, comprising the mesoporous silica nanoparticles of any one of claims 1 to 10, wherein the method (1) loading a bioactive ingredient into the pores of the mesoporous silica nanoparticles according to any one of claims 1 to 10; and (2) A delivery agent, comprising administering the mesoporous silica nanoparticles according to (1) to a subject.

12. The delivery agent of claim 11, wherein the mesoporous silica nanoparticles are delivered so as to penetrate the blood-brain barrier.

13. The delivery agent of claim 11 , wherein the mesoporous silica nanoparticles are delivered so as to penetrate the blood-ocular barrier.

14. A method for producing mesoporous silica nanoparticles (MSNs) according to any one of claims 1 to 10, comprising: (a) providing an alkaline solution containing a surfactant to form micelles; (b) adding to the solution a first silica source and a second silica source providing a terminal hydrocarbyl moiety, wherein the terminal hydrocarbyl moiety comprises a terminal aromatic moiety, a terminal aliphatic moiety having from 4 to 30 carbon atoms, or a combination thereof, and wherein the molar ratio of the first silica source to the second silica source is 5:1 or greater; (c) subjecting the solution to a hydrothermal treatment; and (d) extracting and optionally purifying MSNs from said solution; A method comprising:

15. 15. The method of claim 14, wherein the method further comprises at least one of the following steps: (e) after step (a) and before step (b), introducing an oil phase into the solution for pore expansion; (f) adding an additional silica source after step (b); and (g) performing a surface modification of the outer surface of the MSN, the surface modification being performed after step (b) or, if step (f) is performed, after step (f).

16. 15. The method of claim 14, wherein the surfactant is a cationic surfactant, an anionic surfactant, a nonionic surfactant, or any combination thereof.

17. 15. The method of claim 14, wherein the first silane source comprises tetraethoxysilane (TEOS), tetramethoxysilane (TMOS), sodium silicate, or a mixture thereof.

18. the second silane source is selected from the group consisting of trimethoxyphenylsilane (TMPS), triethoxyphenylsilane (TEPS), diphenyldiethoxysilane, 1-naphthyltrimethoxysilane, 2-hydroxy-4-(3-triethoxysilylpropoxy)diphenylketone, O-4-methylcoumarinyl-N-[3(triethoxysilyl)propyl]carbamate, 7-triethoxysilylpropoxy-5-hydroxyflavone, 3-carbazolylpropyltriethoxysilane, bis(2-diphenylphosphinoethyl)methylsilylethyltriethoxysilane, 2-(diphenylphosphino)ethyltriethoxysilane, n-butyltriethoxysilane, pentyltriethoxysilane, hexyltriethoxysilane, heptyltriethoxysilane, octyltriethoxysilane, nonyltriethoxysilane, decyltriethoxysilane, undecyltriethoxysilane, 15. The method of claim 14, wherein the silane is selected from the group consisting of ethoxysilane, dodecyltriethoxysilane, cyclopropyltriethoxysilane, cyclobutyltriethoxysilane, cyclopentyltriethoxysilane, cyclohexyltriethoxysilane, cycloheptyltriethoxysilane, cyclooctyltriethoxysilane, n-butyltrimethoxysilane, pentyltrimethoxysilane, hexyltrimethoxysilane, heptyltrimethoxysilane, octyltrimethoxysilane, nonyltrimethoxysilane, decyltrimethoxysilane, undecyltrimethoxysilane, dodecyltrimethoxysilane, cyclopropyltrimethoxysilane, cyclobutyltrimethoxysilane, cyclopentyltrimethoxysilane, cyclohexyltrimethoxysilane, cycloheptyltrimethoxysilane, and cyclooctyltrimethoxysilane.

19. 15. The method of claim 14, wherein the oil phase described in step (e) comprises a substituted or unsubstituted (cyclo)alkane, a substituted or unsubstituted aromatic solvent, or a combination thereof.

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