Method of treating cancer and / or cancer metastasis

Abstract

To provide a method of treating cancer and / or cancer metastasis.SOLUTION: The present disclosure relates to a method of treating cancer and / or cancer metastasis in a subject, comprising administering to the subject irinotecan filled into mesoporous silica nanoparticles. The present disclosure also provides a conjugate comprising a drug filled into mesoporous silica nanoparticles (MSNs) defining at least one pore and having at least one functional group on a sidewall of the at least one pore.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to cancer treatment. In particular, the present disclosure provides a cancer treatment using mesoporous silica nanoparticles (MSNs). [Background technology]

[0002] Cancer is known as a mass of undifferentiated cells that grow indefinitely, ignoring the necessary conditions in tissues, and is also called a tumor. These cancer cells with the ability to grow indefinitely can invade the surrounding tissues and in more severe cases, metastasize to other organs in the body, causing severe pain and ultimately leading to death.

[0003] Recently, some targeted drugs have been used to treat certain cancers, but so far, surgery, radiation therapy, and chemotherapy using chemotherapeutic agents that inhibit cell proliferation have been the main methods. However, because chemotherapeutic agents are not targeted drugs, the biggest problem with existing chemotherapeutic agents is side effects due to cytotoxicity and drug resistance, which are the main reasons why treatment ultimately fails despite initial success with anticancer drugs. Therefore, in order to overcome the limitations of such chemotherapeutic agents, it is necessary to continuously develop targeted drugs with clear anticancer action mechanisms. Summary of the Invention [Problem to be solved by the invention]

[0004] Accordingly, the present disclosure relates to methods of treating cancer and / or cancer metastasis in a subject. [Means for solving the problem]

[0005] Accordingly, the present disclosure provides a method of treating a subject having or at risk of having cancer and / or metastasis of cancer, the method comprising administering to the subject irinotecan (IRI) loaded in mesoporous silica nanoparticles (MSN) as a conjugate and optionally a pharma- ceutically acceptable carrier.

[0006] In some embodiments of the present disclosure, the cancer is a metastatic cancer. In some embodiments, the cancer is a solid cancer. Examples of cancer include, but are not limited to, squamous cell carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, multiple myeloma, B-cell lymphoma, brain cancer, head and neck cancer, or metastatic cancer related thereto.

[0007] In some embodiments, the method is for inhibiting angiogenesis.

[0008] In some embodiments, the method is for inhibiting tube formation.

[0009] The present disclosure also provides a method of inhibiting focal adhesion turnover and / or cell migration in a cell of a subject in need thereof, the method comprising administering to the subject MSNs or a conjugate and optionally a pharma- ceutically acceptable carrier.

[0010] In some embodiments, the cell is a cancer cell. Examples of cancer cells include, but are not limited to, squamous cell carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatic cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, multiple myeloma, B-cell lymphoma, brain cancer, head and neck cancer, or metastatic cancers related thereto.

[0011] In some embodiments, the MSNs define at least one pore and have at least one functional group on at least one sidewall, and the pK a The value is less than or equal to 4.5.

[0012] In one embodiment, at least one functional group on the sidewall of at least one pore is an acidic group. In another embodiment, at least one functional group comprises a sulfonic acid group, a sulfate ester group, a carboxylic acid group, a phosphonic acid group, a phosphinic acid group, a phosphate ester group, or a phosphite ester group. In a further embodiment, at least one functional group is an alkylsulfonic acid group.

[0013] In one embodiment, the molar ratio of the amount of silane to the amount of functionalized silane is in the range of 60:1 to 5:1, 50:1 to 5:1, 40:1 to 5:1, 30:1 to 5:1, 20:1 to 5:1, 10:1 to 5:1, 9:1 to 7:1, 50:1 to 10:1, 40:1 to 20:1, 35:1 to 25:1, or 30:1 to 27:1.

[0014] In one embodiment, the MSNs further have external surface modifications with organic molecules, oligomers, or polymers, and / or positively charged molecules, oligomers, or polymers.

[0015] Examples of organic molecules, oligomers, or polymers include, but are not limited to, poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), or PEG-PPG copolymers, or combinations thereof.

[0016] Examples of positively charged molecules, oligomers, or polymers include, but are not limited to, (N-[3-(trimethoxysilyl)propyl]-N,N,N-trimethylammonium chloride), N-[3-(trimethoxysilyl)propyl]ethylenediamine, polyethyleneimine (PEI); alkoxylsilane terminated (poly)alkylene(poly)amine, or organoalkoxysilane with amino groups, or combinations thereof.

[0017] In some embodiments, the average particle size of the MSNs is less than 100 nm as measured by transmission electron microscopy (TEM), and in other aspects, the average hydrodynamic diameter of the MSNs is less than 100 nm as measured in PBS medium by dynamic light scattering (DLS).

[0018] In some embodiments, the zeta potential of the MSNs ranges from -30 mV to +30 mV.

[0019] The present disclosure also provides a conjugate comprising an agent loaded into a mesoporous silica nanoparticle defining at least one pore and having at least one functional group on a sidewall of the at least one pore, the at least one functional group having a pK a The value is less than or equal to 4.5.

[0020] In some embodiments of the present disclosure, the drug is loaded into at least one pore.

[0021] In some embodiments of the present disclosure, the pK a The pK value is the pK of one of the functional groups of MSN. a and / or the drug has a pK value equal to or greater than the pK value of any one of the functional groups of the MSN. a It exhibits a change in properties (e.g., active / inactive conversion, charge state, etc.) at pH values ​​equal to or greater than the pH value.

[0022] In some embodiments of the present disclosure, the drug is positively charged and / or has a pK of any one of the functional groups on the sidewall of at least one pore of the MSNs. a Examples of drugs include, but are not limited to, irinotecan (IRI), amantadine, atenolol, amiodarone, axitinib, barbital, clindamycin, clozapine, chlorambucil, camptothecin, chloroquine, chlorpromazine, clomiphene, cetirizine, doxorubicin, daunorubicin, diphenoxylate, epirubicin, ephedrine, epinephrine, ethionamide, etoposide, 5-fluorouracil, idarubicin, lidocaine, mitoxantrone, mechlorethamine, papaverine, propranolol, promethazine, quinacrine, ranitidine, sertraline, sunitinib, topotecan, trimipramine, toremifene, terconazole, triparanol, vincristine, vinblastine, or venlafaxine.

[0023] The present disclosure also relates to a method of preparing the MSNs disclosed herein, the method comprising: (a) providing an alkaline solution containing a surfactant at a concentration sufficient to form micelles; (b) introducing a silane source and a silane source that functions as a pore sidewall into the alkaline solution; the silane source that functions as a pore sidewall contains a functional group or a precursor group of the functional group, and the pK a a value of 4.5 or less; and (c) performing a hydrothermal treatment with the alkaline solution of step (b) to obtain MSNs.

[0024] In some embodiments, the silane source of step (b) and the silane source that functions as the pore sidewalls are introduced sequentially or simultaneously.

[0025] In some embodiments, the method further comprises introducing a second silane source to the alkaline solution of step (b).

[0026] In some embodiments, the method further comprises performing a surface modification of the exterior surface of the MSNs, in some embodiments of the present disclosure, the surface modification is performed after step (b) or after the step of introducing a second silane source to the alkaline solution of step (b).

[0027] In some embodiments, the method further comprises removing the surfactant from the alkaline solution of step (c).

[0028] In some embodiments, the method further comprises purifying or washing the MSNs.

[0029] In one embodiment, the functional group is an alkylsulfonic acid group.

[0030] In one embodiment, the precursor group to the functional group is an alkylthiol group.

[0031] In one embodiment, the molar ratio of the amount of silane source used in step (b) to the amount of silane source used that functions as a pore side wall is in the range of 60:1 to 5:1, 50:1 to 5:1, 40:1 to 5:1, 30:1 to 5:1, 20:1 to 5:1, 10:1 to 5:1, 9:1 to 7:1, 50:1 to 10:1, 40:1 to 20:1, 35:1 to 25:1, or 30:1 to 27:1.

[0032] The present disclosure provides a method of treating a disease and / or prolonging survival in a subject in need thereof, the method comprising administering to the subject a conjugate and optionally a pharma- ceutically acceptable carrier.

[0033] The present disclosure also provides a method of inhibiting angiogenesis in a subject in need thereof, comprising administering to the subject a conjugate and optionally a pharma- ceutically acceptable carrier. [Brief description of the drawings]

[0034] [Figure 1] Figure 1 shows a TEM image of 30 nm (SO3-)-MSNs (NTT2_186k).

[0035] [Diagram 2] FIG. 2 shows the anti-pancreatic cancer efficacy and toxicity of irinotecan, irinotecan@(SO3 −)-MSN (IRI@NTT2_185), and combination treatment of irinotecan and gemcitabine.

[0036] [Diagram 3] FIG. 3 shows the anti-colorectal cancer effects of irinotecan and irinotecan@(SO 3 − )-MSN (IRI@NTT2_186k).

[0037] [Figure 4A-4B] Figures 4A and 4B show the results of bioluminescence imaging to evaluate the effect of IRI@NTT2_186k in inhibiting tumor growth and metastasis in a metastatic colorectal cancer mouse model (Figure 4A), as well as the quantitative bioluminescence intensity in tumors and overall survival results (Figure 4B).

[0038] [Diagram 5] Figure 5 shows the results of a tube formation assay to evaluate the effect of MSNs in inhibiting tube formation.

[0039] [Figure 6] FIG. 6 shows the results of the chorioallantoic membrane (CAM) assay to evaluate the effect of MSNs on angiogenesis.

[0040] [Figure 7] FIG. 7 shows the anti-metastasis and overall survival improvement of MSNs (combined treatment of MSNs with antitumor drugs).

[0041] [Figure 8A-8B] 8A and 8B show protein expression (FIG. 8A) and protein markers (FIG. 8B) associated with inhibition of metastasis of MSNs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] To facilitate understanding of this disclosure, terms used herein are defined here.

[0043] "Optional" or "optionally or optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where said event or circumstance occurs and cases where it does not occur. For example, the phrase "optionally including a drug" means that the drug may or may not be present.

[0044] In the context of this specification and the claims, the singular forms "a," "an," and "the" include plural referents unless specifically indicated otherwise. Unless otherwise specified, any and all examples or exemplary words (e.g., "such as") provided herein are not intended to limit the scope of the present invention, but are merely used to better describe the present invention.

[0045] It is to be understood that any numerical range recited herein is intended to include all subranges contained therein. For example, the range "50-70°C" includes all subranges and specific values ​​between the stated minimum value of 50°C and the stated maximum value of 70°C, including, for example, 58°C to 67°C, and 53°C to 62°C, 60°C, or 68°C. The disclosed numerical ranges are continuous, and therefore include each value between the minimum and maximum values ​​in the disclosed numerical range. Unless otherwise indicated, the various numerical ranges set forth herein are approximate.

[0046] The terms "treatment," "treating," and "treat" generally refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that a disease, disorder, or symptom thereof is completely or partially prevented, and may be therapeutic, in that a disease, disorder, and / or symptom resulting therefrom is partially or completely cured. As used herein, "treatment" encompasses any treatment of a disease in a mammal, preferably a human, and includes (1) inhibiting the onset of the disease, disorder, or symptom thereof in a subject, or (2) alleviating or ameliorating the disease, disorder, or symptom thereof in a subject.

[0047] In this disclosure, the term "therapeutic agent" or "drug" as used herein refers to a substance that has a therapeutic effect in an organism. Examples of therapeutic moieties include, but are not limited to, small molecule drugs, peptides, enzymes, protein drugs, antibodies, vaccines, antibiotics, or nucleotide drugs.

[0048] The term "subject" as used herein refers to any animal that can benefit from administration of a compound or composition disclosed herein. In some embodiments, the subject is a mammal, such as a human, a primate, a dog, a cat, a horse, a cow, a pig, or a rodent, such as a rat or a mouse. Typically, the mammal is a human.

[0049] The term "effective amount" as described herein means a sufficient amount of a component to provide the desired regulation of a desired function. As noted below, the exact amount required will vary from subject to subject, depending on the disease state, physical condition, age, sex, species, and weight of the subject, the specific identity and formulation of the composition, and the like. The administration schedule can be adjusted to induce optimal therapeutic effects. For example, several divided doses may be administered daily, or the dose may be relatively reduced as indicated by the exigencies of the therapeutic situation. Therefore, it is not possible to specify an exact "effective amount". However, an appropriate effective amount can be determined by one skilled in the art using only routine experimentation.

[0050] The term "pharmacologically acceptable" as used herein refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of a subject (either a human or non-human animal) without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio that is within the scope of sound medical judgment. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts.

[0051] In this disclosure, unless otherwise specified, the prefix "nano-" as used herein means a size of about 300 nm or less. Unless otherwise specified, the prefix "meso-" as used herein means a size of about 50 nm or less.

[0052] In this disclosure, the term "silane" as used herein refers to derivatives of SiH4. Typically, at least one of the four hydrogens is substituted with a substituent such as an alkyl, alkoxyl, or amino, as described below. The term "alkoxysilane" as used herein refers to a silane having at least one alkoxyl substituent bonded directly to the silicon atom. The term "organoalkoxysilane" as used herein refers to a silane having at least one alkoxyl and at least one hydrocarbyl substituent bonded directly to the silicon atom. The term "silicic acid source" as used herein refers to a material that can be considered to be a salt or ester form of orthosilicic acid, such as sodium orthosilicate, sodium metasilicate, tetraethyl orthosilicate (tetraethoxysilane, TEOS), tetramethyl orthosilicate, or tetrapropyl orthosilicate. Optionally, the hydrocarbyl substituents can be further substituted or interrupted with heteroatoms.

[0053] In this disclosure, the term "alkyl" refers to saturated, straight-chain, or branched-chain alkyl, 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, or octadecyl.

[0054] In the present disclosure, the term "alkoxyl" or "alkoxy" as used herein means a group having the formula "-O-alkyl", where "alkyl" has the meaning of "alkyl" as defined above.

[0055] The term "carrier" or "excipient" as used herein refers to any substance that is not itself a therapeutic agent, but is used as a carrier and / or diluent and / or adjuvant, or vehicle for delivery of a therapeutic agent to a subject, or is added to a formulation to improve its handling or storage characteristics, or to allow or facilitate the formation of a dosage unit composition into a separate article, such as a liquid solution, suspension, emulsion, granule, ampoule, injection, implant, insert, infusion, kit, ointment, lotion, liniment, cream, gel, spray, drop, aerosol, or combination thereof for topical administration. Suitable carriers or excipients are well known to those skilled in the art of manufacturing pharmaceutical formulations or food products. Carriers or excipients may include, by way of example and not limitation, buffers, diluents, disintegrants, binders, adhesives, wetting agents, polymers, lubricants, glidants, substances added to mask or neutralize unpleasant tastes or odors, flavorants, dyes, fragrances, and substances added to improve the appearance of the composition. Acceptable carriers or excipients include citrate buffer, phosphate buffer, acetate buffer, bicarbonate buffer, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphate and sulfate, magnesium carbonate, talc, gelatin, gum acacia, sodium alginate, pectin, dextrin, mannitol, sorbitol, lactose, sucrose, starch, gelatin, cellulosic materials (such as cellulose esters of alkanoic acids and cellulose alkyl esters), the low melting wax cocoa butter, amino acids, urea, alcohol, ascorbic acid, phospholipids, proteins (e.g., serum albumin), ethylenediaminetetraacetic acid (EDTA), dimethylsulfoxide (DMSO), sodium chloride or other salts, liposomes, mannitol, sorbitol, glycerol or powder, polymers (such as polyvinylpyrrolidone, polyvinyl alcohol, and polyethylene glycol), and other pharma- ceutically acceptable materials. The carrier should not destroy the pharmacological activity of the therapeutic agent and should be non-toxic when administered in dosages sufficient to deliver a therapeutic amount of the agent.

[0056] For example, the MSNs and fillers can be specially formulated for administration in solid or liquid form, including for: (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), troches, dragees, capsules, pills, tablets (e.g., intended for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension, or sustained release preparation; (3) topical application, e.g., as a cream, lotion, gel, ointment, or controlled release patch or spray applied to the skin; (4) vaginal or rectal administration, e.g., as a pessary, cream, suppository, or foam; (5) sublingual administration; (6) ocular administration; (7) transdermal administration; (8) transmucosal administration; or (9) nasal administration.

[0057] Methods for Treating Cancer and / or Cancer Metastasis

[0058] The present disclosure provides a method of treating a subject having or at risk of having cancer and / or metastasis of cancer, the method comprising administering to the subject irinotecan (IRI) loaded in mesoporous silica nanoparticles (MSN) as a conjugate and optionally a pharma- ceutically acceptable carrier.

[0059] The present disclosure provides a method of treating a disease and / or prolonging survival in a subject in need thereof, the method comprising administering to the subject a conjugate and optionally a pharma- ceutically acceptable carrier.

[0060] The present disclosure also provides a method of inhibiting angiogenesis in a subject in need thereof, comprising administering to the subject a conjugate and optionally a pharma- ceutically acceptable carrier.

[0061] In some embodiments of the present disclosure, the disease is cancer; in particular, the disease is metastatic cancer. In some embodiments, the disease is solid cancer. Examples of diseases include, but are not limited to, squamous cell carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, multiple myeloma, B-cell lymphoma, brain cancer, head and neck cancer, or metastatic cancer related thereto.

[0062] In one embodiment of the present disclosure, MSNs and conjugates are effective in treating colorectal cancer, particularly metastatic colorectal cancer. Colorectal cancer (CRC) is the third most commonly diagnosed cancer in men, the second most common cancer in women, and the second most common cause of cancer death worldwide. Five-year survival ranges from 90% in localized stages of CRC (stages I-III) to 10% in distant metastatic patients (stage IV). Metastasis is the leading cause of CRC-related deaths. 22% of CRCs are metastatic at the time of initial diagnosis, and approximately 70% of patients eventually develop metastatic recurrence. Colorectal cancer has a tendency to metastasize, spreading to adjacent lymph nodes, liver, lungs, bone, brain, or spinal cord. There is a dire need for effective therapies that can also inhibit the growth and metastasis of primary colorectal tumors.

[0063] Metastasis of cancer cells to the brain is also problematic and can lead to high mortality. Once tumors metastasize to the brain, both surgery and radiation therapy are less effective, and systemic chemotherapy has limited efficacy due to the BBB. The BBB is an important physiological barrier in the central nervous system, controlling the movement of ions and molecules from the circulating blood to the brain and protecting the brain from invading pathogens and toxins. However, the BBB also blocks the movement of most drugs to the brain for treating brain diseases. The inventors have found that MSNs with specific inner surface modification of pores and surface modification of particles exhibit excellent loading ability of therapeutic agents, especially irinotecan, good dispersibility in buffer or physiological conditions, and good stability in storage or physiological conditions, which can be used for tumor targeting, blood-brain tumor barrier (BBTB) crossing, BBB crossing, brain tumor, and brain metastasis treatment. Furthermore, MSNs exhibit anti-cell migration activity to prevent, inhibit, or suppress cancer metastasis based on in vitro and in vivo models, and may reduce the incidence of cancer metastasis. Therapeutic drug-loaded MSNs offer a new approach for the treatment of cancers with a high propensity to metastasize.

[0064] Mesoporous silica nanoparticles are believed to have great potential as drug delivery systems due to their unique physical / chemical properties such as large pore volume, chemical / thermal stability, high loading capacity, tunable surface properties, and excellent biocompatibility. MSNs offer the combined advantages of both silica and mesoporous materials. The versatility of silica chemistry allows for easy integration with other materials, including metal nanoparticles, fluorescent molecules, and rare earth elements. Mesoporous materials offer large surface areas, high pore volumes, and uniform pore size distribution. The combination of the aforementioned advantages of both bulk and nanosized materials provides properties that can be used in a variety of applications.

[0065] Irinotecan is approved for colorectal and pancreatic cancer (first-line treatment in combination with different drugs). Irinotecan is an important chemotherapy agent for such cancers, but it has a high incidence of bone marrow and gastrointestinal (GI) toxicity. Furthermore, liposomal irinotecan (Onivyde) is approved for pancreatic cancer (second-line treatment in combination with different drugs), but the liposome does not have any anti-metastatic effect. The longer circulation period (several days) helps Onivyde accumulate in the tumor area, but it may remain in the body as well, resulting in undesirable side effects. Onivyde's GI toxicity remains a concern for its use in colorectal cancer.

[0066] In one embodiment of the present disclosure, the conjugates containing irinotecan in MSN significantly improve the anti-cancer effect against colorectal cancer. Moreover, MSN has been shown to block cancer metastasis based on in vitro and in vivo models, and may reduce the incidence of metastasis in colorectal cancer, which is very common. The conjugates containing irinotecan@MSN provide additional clinical benefits compared to irinotecan and liposomal irinotecan, especially in cancers with a high propensity to metastasize.

[0067] In one embodiment of the present disclosure, a conjugate containing irinotecan in MSN significantly improves the anti-cancer effect against pancreatic cancer. The anti-cancer effect of Irinotecan@NTT_MSN is superior to irinotecan alone and causes less weight loss compared to conventional irinotecan + gemcitabine combination therapy.

[0068] In one embodiment of the present disclosure, a conjugate comprising irinotecan in MSN can inhibit primary tumor growth and metastasis and prolong survival, whereas irinotecan alone exhibits inefficient tumor suppression against primary tumor growth and metastasis.

[0069] In some embodiments of the present disclosure, MSNs alone inhibit tube formation of vascular epithelial cells and inhibit angiogenesis around tumors.

[0070] The present disclosure also provides a method of inhibiting focal adhesion turnover and / or cell migration in a subject in need thereof, comprising administering MSN or a conjugate and an optional pharma- ceutically acceptable carrier to the subject. In some embodiments of the present disclosure, MSN inhibits the migration ability of endothelial cells, resulting in irregular growth of blood vessels. In some embodiments, MSN inhibits phosphorylation of ERK, paxillin (PXN), and FAK proteins, and phosphorylation of these proteins has been shown to be associated with focal adhesion turnover. In some embodiments, MSN does not affect actin and microtubule cytoskeleton.

[0071] In some embodiments, the cell is a cancer cell. Examples of cancer cells include, but are not limited to, squamous cell carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatic cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, multiple myeloma, B-cell lymphoma, brain cancer, head and neck cancer, or metastatic cancers related thereto.

[0072] Mesoporous silica nanoparticles with functional groups on the pore surface

[0073] MSNs can be used as carriers for delivering drugs. Usually, the surface of silica nanoparticles can have a negative charge due to dissociation of hydrogen from -SiOH groups. Therefore, a person skilled in the art would consider loading MSNs with drugs that have a positive charge, which can be utilized by electrostatic interactions. However, the surface charge of MSNs is believed to be slightly negative in most situations. Without being bound by theory, it is believed that the silanol groups on silica nanoparticles have a pK of about 4.5-5.5 or 8.5-9.9. a This means that MSNs in a neutral or slightly acidic environment have only a small amount of negative charge on their surface. That is, the drug loaded on the MSNs may have a pK value equal to or lower than that of the silanol groups on the MSNs. aIf the drug exhibits a high or low electrostatic value or changes in properties (e.g., active / inactive conversion, charge state, etc.), it may be difficult to efficiently load the drug due to low or repulsive electrostatic interactions. Therefore, it may be necessary to modify or optimize the surface properties of MSNs. In addition, it may be preferable to load the drug in the pores rather than on the "external" surface of the particles, because this avoids potential problems such as easy drug leakage from the particles, particle aggregation, or affecting the behavior of MSNs in vivo.

[0074] To achieve these goals, functional groups have been introduced onto the surface of MSNs, especially onto the side walls of the pores, and these functional groups have a pK below 4.5. a The (pore) surfaces of MSNs may have sufficient negative charge at lower pH values, e.g., below 7, 6, or 5.5, to have a pK value of 0.01, where the present disclosure provides mesoporous silica nanoparticles defining at least one pore and having at least one functional group on the sidewall of the at least one pore, and the pK value of the at least one functional group is a The value is less than or equal to 4.5.

[0075] In one embodiment, the functional group can be an acidic group such as a sulfonic acid group, a sulfate group, a carboxylic acid group, a phosphonic acid group, a phosphinic acid group, a phosphate group, or a phosphite group. In one embodiment, the precursor group can be converted to a functional group and can be a thiol, a sultone, an acyl chloride, a nitrile, anhydride, an amide and a carboxylate ester, phosphorus oxychloride, phosphorus trichloride.

[0076] In one embodiment, the molar ratio of the amount of silane to the amount of functionalized silane is in the range of 60:1 to 5:1, 50:1 to 5:1, 40:1 to 5:1, 30:1 to 5:1, 20:1 to 5:1, 10:1 to 5:1, 9:1 to 7:1, 50:1 to 10:1, 40:1 to 20:1, 35:1 to 25:1, or 30:1 to 27:1.

[0077] In one embodiment, the MSNs further have an external surface modification with an organic molecule, oligomer, or polymer, and / or a positively charged molecule, oligomer, or polymer.

[0078] Examples of organic molecules, oligomers, or polymers include, but are not limited to, poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), or PEG-PPG copolymers, or combinations thereof.

[0079] Examples of positively charged molecules, oligomers, or polymers include, but are not limited to, (N-[3-(trimethoxysilyl)propyl]-N,N,N-trimethylammonium chloride), N-[3-(trimethoxysilyl)propyl]ethylenediamine, polyethyleneimine (PEI); alkoxylsilane terminated (poly)alkylene(poly)amine, or organoalkoxysilane with amino groups, or combinations thereof.

[0080] Features of MSN

[0081] In the present disclosure, the MSNs preferably have an average diameter (particle size) of less than 100 nm as measured by transmission electron microscopy (TEM). In one embodiment, the mesoporous silica nanoparticles of the present disclosure have an average particle size of 100 nm or less, 80 nm or less, 65 nm or less, 50 nm or less, 40 nm or less, 30 nm or less, or 20 nm or less as measured by TEM. In one embodiment, the mesoporous silica nanoparticles of the present disclosure have a pore size of 20 nm or less, 10 nm or less, 5 nm or less, 3 nm or less, or 2 nm or less. In one embodiment, the mesoporous silica nanoparticles of the present disclosure have an average hydrodynamic diameter of 100 nm or less, 80 nm or less, 60 nm or less, 50 nm or less, 40 nm or less, or 30 nm or less as measured in phosphate buffered saline (PBS) by dynamic light scattering.

[0082] In certain embodiments, the zeta potential of the MSNs (under pH 7.4 conditions) can be in the range of -30 to +30 mV, -28 to +25 mV, -23 to +22 mV, -20 to +20 mV, -15 to +15 mV, or -10 to +10 mV, or any reasonable numerical range within the endpoints mentioned herein, such as -15 to +20 mV, -10 to +25 mV, -15 to +10 mV, etc. In one embodiment, the mesoporous silica nanoparticles are 2 / g or less, 750m 2 / g or less, or 500m 2 / g or less BET surface area.

[0083] Method for preparing pore-modified MSNs

[0084] In one embodiment, MSNs can be prepared by the following steps: (a) providing an alkaline solution containing a surfactant at a concentration sufficient to form micelles; (b-1) introducing a silane source into the alkaline solution, and sequentially introducing a silane source that functions as a pore sidewall having a desired functional group or a precursor group thereof (hereinafter referred to as a "silane that functions as a pore sidewall") into the alkaline solution, or (b-2) introducing a silane source and a silane that functions as a pore sidewall into the solution; optionally introducing a second silane source and introducing an external surface modifier; and (c) performing a hydrothermal treatment with the alkaline solution; optionally recovering the product; optionally removing residual surfactant from the product; and optionally purifying or washing the product.

[0085] In one embodiment, 0.2-0.5 g of surfactant is dissolved in 100-250 mL of aqueous alkaline solution (e.g., ammonium hydroxide solution (0.05-1.2 M)) at the desired temperature (e.g., 45-65 °C) in a sealed beaker. After stirring for 10-30 minutes, 200-550 μL of silane source and 10-100 μL of silane acting as an internal surface in 300-2500 μL of solvent (e.g., alcohol such as ethanol) are added and stirred for 30-120 minutes. Then, an additional 50-200 μL of silane source in 0.6-2 mL of solvent (e.g., alcohol such as ethanol) is introduced and stirred for 1.5-3 hours. A surface modifier, for example 650-1700 μL of PEG silane and 30-1000 μL of silane with optional positively charged groups, in 2-6 mL of solvent (for example alcohol such as ethanol) is introduced and stirred for 0.5-1.5 hours. The mixture is aged at the desired temperature (for example 40-80 ° C) with or without stirring for at least 15 hours. Finally, the solution is sealed and placed in an oven at 60-100 ° C for hydrothermal treatment for 12-48 hours. The as-synthesized product is collected by washing by centrifugation or crossflow. The as-synthesized product is finally treated for surfactant removal and purification and collected by washing by centrifugation or crossflow. If the silane source has a precursor group of the desired functional group, further treatment such as oxidation or hydrolysis, including optional further purification, is carried out to convert the precursor group to the desired functional group. The final product is then stored, preferably in water or ethanol 85% or more.

[0086] Examples of surfactants suitable for use in step (a) include, but are not limited to, cationic surfactants, anionic surfactants, and nonionic surfactants.Suitable surfactants are selected based on reaction conditions such as pH value, ionic strength, temperature, reactants, and products.Examples of cationic surfactants include, but are not limited to, pH-dependent primary, secondary, or tertiary amines with long-chain hydrocarbyl groups (terminal amine groups have a positive charge when below a certain pH value, and primary and secondary amines become positively charged at pH<10), such as octenidine dihydrochloride, and permanently charged quaternary ammonium salts, such as cetrimonium bromide (CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyldioctadecylammonium chloride, and dioctadecyldimethylammonium bromide (DODAB). Examples of anionic surfactants include, but are not limited to, sulfates, sulfonates, and phosphates or esters, such as ammonium lauryl sulfate, sodium lauryl sulfate (sodium dodecyl sulfate, SLS, or SDS) and related alkyl ether sulfates, sodium laureth sulfate (sodium lauryl ether sulfate or SLES), and sodium myreth sulfate, docusate (dioctyl sodium sulfosuccinate), perfluorooctane sulfonate (PFOS), perfluorobutane sulfonate, alkyl-aryl ether phosphates, alkyl ether phosphates, etc. Examples of nonionic surfactants include, but are not limited to, poly(oxyethylene) nonylphenyl ether, polyoxyethylene glycol sorbitan alkyl esters, polyethylene glycol alkyl ethers, glucoside alkyl ethers, polyethylene glycol octylphenyl ethers, polyethylene glycol alkylphenyl ethers, glycerol alkyl esters, polypropylene glycol alkyl ethers, block copolymers, poloxamers, cocamide MEA, cocamide DEA, lauryl dimethylamine oxide, or polyethoxylated tallow amine.

[0087] In one embodiment, the silane source used in steps (b-1), (b-2) comprises tetraethoxysilane (TEOS), tetramethoxysilane (TMOS), sodium silicate, or a mixture thereof.

[0088] In one embodiment, a silane that functions as a pore sidewall can be used in step (b-1) or (b-2). Examples of silanes that function as the pore sidewalls include, but are not limited to, (mercaptoalkyl)trialkoxysilanes such as (3-mercaptopropyl)trimethoxysilane (MPTMS), (3-mercaptopropyl)triethoxysilane, (11-mercaptoundecyl)trimethoxysilane, (trihydroxysilyl)alkanesulfonic acids such as 3-(trihydroxysilyl)propane-1-sulfonic acid (TPS), 3-(trihydroxysilyl)propylmethylphosphonate, (3-triethoxysilyl)propylsuccinic anhydride, bis(3-triethoxysilylpropyl)carbonate, carboxyethylsilanetriol, 2-(4-chlorosulfonylphenyl)ethyltrimethoxysilane, triethoxysilylpropylmaleamic acid, N-(trimethoxysilylpropyl)ethylenediaminetriacetate, and diethylphosphatoethyltriethoxysilane.

[0089] In one embodiment, surface modifiers can be used to tune the properties of MSNs. In one embodiment, (organic) modifiers include, but are not limited to, propyltriethoxysilane, butyltrimethoxysilane, octyltrimethoxysilane, diphenyldiethoxysilane, n-octyltriethoxysilane, chloromethyltrimethoxysilane, isobutyltriethoxysilane, ethyltrimethoxystyrenesilane, methyltriethoxysilane, phenyltriethoxysilane (PTEOS), phenyltrimethoxysilane (PTMOS), methyltrimethoxysilane (MTMOS), ethyltriacetoxysilane (ETAS), N -(trimethoxysilylpropyl)ethylenediaminetriacetic acid (EDTAS), (3-trihydroxysilyl)propyl methylphosphonate (THPMP), methyltriacetoxysilane (MTAS), zwitterionic silane, (N-[3-(trimethoxysilyl)propyl]-N,N,N-trimethylammonium chloride), N-[3-(trimethoxysilyl)propyl]ethylenediamine, polyethyleneimine (PEI); alkoxyl silanes, terminated (poly)alkylene(poly)amines, or organoalkoxysilanes with amino groups.

[0090] In one embodiment, the molar ratio of the silane sources (total, i.e., used in steps (b-1) / (b-2)) to the silane functioning as the pore sidewalls can be in the range of 60:1 to 5:1, 50:1 to 5:1, 40:1 to 5:1, 30:1 to 5:1, 20:1 to 5:1, 10:1 to 5:1, 9:1 to 7:1, 50:1 to 10:1, 40:1 to 20:1, 35:1 to 25:1, or 30:1 to 27:1, or any reasonable numerical range consisting of end points.

[0091] Conjugates containing drugs loaded into pore-modified MSNs

[0092] The inventors have demonstrated that the specific pore sidewall modification allows MSNs to have pK values ​​equal to or greater than that of any one of their functional groups. a and / or the pK value of any one of the functional groups of MSN. aWe speculated that MSNs may have the potential to provide desirable pharmacological effects and superior loading efficiency or stability for drugs that exhibit a change in properties (e.g., active / inactive conversion, charge state, etc.) at pH values ​​equal to or greater than the pK value of any functional group on the sidewall of at least one pore of the MSNs. In addition, drugs may be positively charged and / or have a pK value equal to or greater than the pK value of any functional group on the sidewall of at least one pore of the MSNs. a It exhibits its active form at a pH value equal to or higher than this value.

[0093] One specific example of a drug is irinotecan (IRI). In particular, IRI will be in lactone form (considered therapeutically active) in acidic environments, especially pH<5.5, and in carboxylate form (considered inactive) in slightly acidic, neutral, or basic environments, especially pH>6. Therefore, IRI needs to be loaded in active form, i.e., acidic environment, but the dissociation level of silanol groups on MSNs cannot provide sufficient attractive force for IRI, resulting in low loading efficiency and / or long-term stability of IRI. Examples of these agents include, but are not limited to, amantadine, atenolol, amiodarone, axitinib, barbital, clindamycin, clozapine, chlorambucil, camptothecin, chloroquine, chlorpromazine, clomiphene, cetirizine, doxorubicin, daunorubicin, diphenoxylate, epirubicin, ephedrine, epinephrine, ethionamide, etoposide, 5-fluorouracil, idarubicin, lidocaine, mitoxantrone, mechlorethamine, papaverine, propranolol, promethazine, quinacrine, ranitidine, sertraline, sunitinib, topotecan, trimipramine, toremifene, terconazole, triparanol, vincristine, vinblastine, or venlafaxine.

[0094] In one embodiment, the drug is loaded into the nanoparticles. Encapsulation of the drug into MSNs does not significantly affect the degree of dispersion and hydrodynamic size in a medium such as a medium that is biosimilar or equivalent to phosphate buffered saline (PBS).

[0095] For the treatment of diseases, additional bioactive ingredients can be loaded onto and / or within the MSNs, e.g., distributed within the spaces of the MSNs, on the surfaces of the MSNs, etc. The bioactive ingredients can be appropriately selected based on their size and the associated disorder / disease. Examples of bioactive moieties include, but are not limited to, everolimus, trabectedin, abraxane, TLK286, AV-299, DN-101, pazopanib, GSK690693, RTA744, ON0910.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 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, PI3 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, butabulin, ofatumumab, zanolimumab, edotecarin, tetrandrine, rubitecan, tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol, Bio111, 131-I-TM-601, ALT-110, BIO140, CC8490, cilengitide, dimatecan, IL13-PE38QQR, INO1001, IPdR1 KRX-0402, Lucanton, LY317615, Neurajiab, Vitespan, Rta744, Sdx 102, Talampanel, Atrasentan, Xr311, Romidepsin, ADS-100380, Sunitinib, 5-fluorouracil, Vorinostat, Etoposide, Gemcitabine, Doxorubicin liposomal, 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-(methylsulfonyl) nylpiperazine methyl)-indolyl]-quinolones, vatalanib, AG-013736, AVE-0005, goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714; TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibodies, Erbitux, EKB-569, PKI-166, GW-572016, Io Nafarnib, BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoylanalide hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951, aminoglutethimide, amsacrine, anagrelide, L-asparaginase, Bacillus Calmette-Guerin (BCG) vaccine, bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, cyproterone, cytarabine, dacarbazine, dactinostatin mycin, daunorubicin, diethylstilbestrol, epirubicin, fludarabine, etc., fludrocortisone, fluoxymesterone, flutamide, gemcitabine, hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole, lomustine, mechlorethamine, 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-mecaptopurine, deoxycoformycin, calcitriol, barbicine, mithramycin, vinblastine, vinorelbine, topotecan, razoxine, marimastat, COL-3, neovast , BMS-275291, squalamine, endostatin, SU5416, SU6668, EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene, idoxifene, spironolactone, finasteride, cimitidine, trastuzumab, denileukin diftitox, gefitinib, bortezimib, paclitaxel, cremophor free paclitaxel, 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, PD184352, rapamycin, 40-O-(2-hydroxyethyl)rapamycin, temsirolimus, AP-23573, RAD001, ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmarmin min), ZM336372, L-779,450, PEG-filgrastim, darbepoetin, erythropoietin, granulocyte colony-stimulating factor, zoledronate, prednisone, cetuximab, granulocyte-macrophage colony-stimulating factor, histrelin, pegylated interferon α-2a, interferon α-2a, pegylated interferon α-2b, interferon α-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab,All-trans retinoic acid, ketoconazole, interleukin-2, megestrol, immunoglobulin, nitrogen mustard, methylprednisolone, ibritumomab tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, liposomal daunorubicin, edwina-asparaginase, strontium-89, casopitant, netupitant, NK-1 receptor antagonist agonists, palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, dolasetron, tropisetron, pegfilgrastim, erythropoietin, epoetin alfa and darbepoetin alfa, docetaxel, cabazitaxel, curcumin, and curcumin analogs.

[0096] The following examples are provided to facilitate understanding of the present invention to those skilled in the art to which the present invention pertains, but are not intended to limit the scope of the present invention. EXAMPLES

[0097] Materials, methods, and test models

[0098] Transmission electron microscope (TEM)

[0099] Transmission electron microscopy (TEM) is used to directly examine and verify the appearance of silica nanoparticles. TEM images were taken with a Hitachi H-7100 transmission electron microscope operated at an accelerating voltage of 100 kV. Samples dispersed in ethanol were dropped onto a carbon-coated copper grid, dried in air, and observed under TEM.

[0100] Dynamic Light Scattering (DLS) and Zeta Potential

[0101] The size of silica nanoparticles in different solution environments was measured using dynamic light scattering (DLS) on a Malvern Zetasizer Nano ZS (Malvern, UK). The (solvated) particle size formed in different solutions was analyzed in H2O and PBS buffer solutions (pH 7.4) at room temperature. The surface charge (zeta potential) of silica nanoparticles in PBS (0.01x, pH 7.4) at a particle concentration of 0.1 mg / mL was measured by a Malvern Zetasizer Nano ZS.

[0102] elemental analysis

[0103] The mass percentages of carbon, nitrogen, oxygen, sulfur, and hydrogen in the silica nanoparticles were determined by an elemental analyzer (elementar Vario EL cube type for NCSH, German).

[0104] Example 1

[0105] Preparation of mesoporous silica nanoparticles with modified outer surface of the particles but not the pore sidewalls

[0106] MSNs were prepared using an ammonia base catalysis method under highly diluted low-surfactant conditions. The particle size was controlled by adjusting the ammonia concentration, the amount of TEOS added, and the reaction temperature. Typically, 0.29 g of CTAB was dissolved in 150 mL of ammonium hydroxide solution (0.128 M to 0.17 M) at 60 °C in a sealed beaker. After 15 min of stirring, the sealed lid was removed, and then 390 μL of tetraethoxysilane (TEOS) and 5 μL of 3-aminopropyltrimethoxysilane (APTMS) in 4.105 mL of ethanol were added to the solution with vigorous stirring. After 1 h of stirring, 550 μL of PEG and 85.7 to 300 μL of TA (optionally added) in 2 mL of ethanol were introduced into the reaction. The mixture was stirred for 30 min and then aged overnight at 60 °C without stirring until the reaction volume was reduced to 50 mL. The solution was then filtered through a 0.22 μm filter and placed in an oven at 70 °C for hydrothermal treatment for 24 h. The as-synthesized samples were washed and collected by cross-flow. To remove the surfactant in the pores of the MSNs, the as-synthesized samples were collected in 50 mL of ethanol containing 848 μL of HCl (36.5–38%) for the first 1 h acid extraction and 50 μL of HCl for the second 1 h acid extraction at 60 °C. The products were washed and collected by cross-flow and finally stored in HO or organic solvent. The particle size of the MSNs (25 nm and 50 nm) was controlled by adjusting the concentrations of TEOS and NH4OH (25 nm MSNs: MSN-PEG25, MSN-PEG / TA25; 50 nm MSNs: MSN-PEG50, MSN-PEG / TA50).

[0107] Example 2

[0108] Mesoporous silica nanoparticles with sulfonic acid functional groups on the pore sidewalls ((SO3 - Preparation of ZnS-MSNs

[0109] 0.2-0.5 g of CTAB was dissolved in 100-250 mL of aqueous ammonium hydroxide solution (approximately 0.05-1.2 M) at 45-65 °C in a sealed beaker. After stirring for 10-30 min, 200-550 μL of tetraethoxysilane (TEOS) and 10-100 μL of (3-mercaptopropyl)trimethoxysilane (MPTMS) in 900-2500 μL of ethanol (molar ratios of TEOS / MPTMS = 50:1, 40:1, 25:1, 20:1, 10:1, 8.3:1, and 5:1, the TEOS:MPTMS ratio can be changed to adjust the amount of functional groups on the pore surface for different applications or drugs to be loaded) were added and stirred for 30-90 min. Afterwards, an additional 50-130 μL of tetraethoxysilane (TEOS) in 0.8-2 mL of ethanol was introduced and stirred for 1.5-3 h. Then, 650-1700 μL of 2-[methoxy(polyethyleneoxy)propyl]-trimethoxysilane (PEG-silane) and 100-260 μL of N-[3-(trimethoxysilyl)propyl]-N,N,N-trimethylammonium chloride (TA-silane) in 2000-5400 μL of ethanol were introduced and stirred for 0.5-1.5 h. The mixture was then aged at 40-60 °C for at least 15 h. Finally, the solution was sealed and placed in an oven at 65-75 °C for hydrothermal treatment for 15-24 h. The intermediate product was washed and collected by centrifugation or crossflow. The intermediate product was finally treated for surfactant removal and purification and was washed and collected by centrifugation or crossflow. To convert the mercapto (thiol) group to a sulfonic acid group, an oxidizing agent was introduced into the intermediate to carry out oxidation, and the final product was further purified. The product was stored in water, an organic solvent, or a buffer solution.

[0110] (SO3 - The hydrodynamic diameter of the (SO3 -The results show that )-MSNs are well dispersed within the range of about 30 nm to 50 nm, but the degree of dispersion of particles with a large amount of sulfonated groups (TEOS:MPTMS=5:1) may be affected (hydrodynamic diameter of about 60 nm in water and PBS buffer). The synthesis conditions for NTT2_185 and NTT2_186k are TEOS / MPTMS (8.3:1) and PEG-silane / TA-silane (7:1). Characterization of NTT2_185 and NTT2_186k includes particle size of about 20.6 nm and 25.1 nm measured by TEM; DLS particle size of about 33.5 nm and 39.7 nm measured in PBS; and zeta potential of about -25 mV and -25.3 mV. Characterization of NTT2_186k is similar to NTT2_185, but the surface modification and manufacturing process of NTT2_186k were optimized.

[0111] Has a neutral surface charge (zeta potential) (SO3 - Preparation of )-MSNs

[0112] 0.2–0.5 g of CTAB was dissolved in 100–250 mL of aqueous ammonium hydroxide solution (approximately 0.05–1.2 M) at 55–65 °C in a sealed beaker. After stirring for 10–30 min, 25–70 µL of 3-(trihydroxysilyl)propane-1-sulfonic acid (TPS) in 250–700 µL of water, 200–550 µL of tetraethoxysilane (TEOS) in 900–2500 µL of ethanol, and an additional 50–130 µL of tetraethoxysilane (TEOS) in 800–2000 µL of ethanol were introduced separately, with stirring at 55–65 °C for 5 min–2.5 h after each introduction. Then, 650-1700 μL of 2-[methoxy(polyethyleneoxy)propyl]-trimethoxysilane (PEG-silane) and 30-90 μL of N-[3-(trimethoxysilyl)propyl]ethylenediamine (EDPTMS) in 2000-5400 μL of ethanol were introduced and stirred for 0.5-1.5 h. The mixture was then aged at 45-65 °C for at least 15 h. Finally, the solution was sealed and placed in an oven at 60-90 °C for hydrothermal treatment for 18-30 h to obtain the final product (SO3 - )-MSN(NTT2_202) was obtained.

[0113] Characterization of NTT2_202 includes particle size of approximately 28.2 nm as measured by TEM; DLS particle size of approximately 52.9 nm as measured in PBS; and zeta potential of approximately +1.41 mV.

[0114] Example 3

[0115] Irinotecan (IRI) was loaded into MSNs.

[0116] 300 mg of MSNs in 16.785 mL of H2O were mixed with 495 μL of NaHCO3 (0.1 M, pH 9.96) or acetate buffer (0.1 M, pH 5.5). After shaking for 5 min at room temperature, IRI solution was introduced into the solution and stirred for 30 min at room temperature. The mixture was then purified by washing with NaHCO3 (2.5 mM, pH 8.5) or acetate buffer (2.5 mM, pH 5.5) by Vivaspin or crossflow. Finally, the product was stored in NaHCO3 or acetate buffer.

[0117] Irinotecan (IRI) (SO3 - )-MSN (NTT2_185 and NTT2_186k)

[0118] 600 g of particles in 6 mL of acetate buffer (2.5 M, H.5) was mixed with IRI solution and stirred at room temperature for 30 min. The mixture was then purified by washing with acetate buffer by Vivaspin or crossflow. Finally, the product was stored in acetate buffer.

[0119] Irinotecan (IRI) (SO3 - )-MSN(NTT2_202) was filled

[0120] For alkaline treatment, 150 mg of NTT2_202 in 1.5 mL of H2O was mixed with 375 μL of NaHCO3 (93 mM, pH 11.5). IRI solution was then introduced and stirred at room temperature for 10 min. The mixture was then washed first with H2O and then with acetate buffer (2.5 mM, pH 5.5) by Vivaspin. IRI@NTT2_202 was then immersed in 7.5 mL of acetate buffer at 4 °C for at least 15 h. The mixture was then further washed with acetate buffer and the product was stored in acetate buffer.

[0121] IRI to MSN or (SO3 - ) - The driving force for loading into MSNs is electrostatic interaction. Positively charged IRI is adsorbed into the pores of MSNs, which have negatively charged silanol groups or functional groups on the inner surface of the pores. Irinotecan (pka of about 10.9) was positively charged in an environment below pH 10.9, and the conformation of IRI changed with pH. IRI would be in lactone form (considered therapeutically active) in acidic environment (pH<5.5) and in carboxylate form (considered inactive) in slightly acidic, neutral, or basic environment (pH>6). According to the properties of IRI, the appropriate condition for the IRI loading process was a pH below 5.5, which can make IRI positively charged and active. Various loading strategies were tested with MSNs, NTT2_186k, and NTT2_202. The loading of IRI into MSNs under acidic conditions (pH ≦ 5.5) showed low loading capacity (3.93%) and loading efficiency (33%), as well as low long-term stability of IRI@MSNs. The reason is that the silanol groups on the inner surface of the pores are acidic. This is because IRI exhibits a weak negative charge under alkaline conditions, resulting in insufficient electrostatic interaction between IRI and the silanol groups on the pore sidewalls. Carrying out the IRI loading process under alkaline conditions promoted negative charging in the pores of MSNs, and could improve the loading capacity by 10%-15%. However, the ratio of lactone type IRI in IRI@MSN was about 63% and decreased with the storage time. In contrast, (SO3 -It could be shown that the (SO3)-MSNs had sulfonic acid functional groups (pKa of about -7) on the pore sidewalls and were strongly negatively charged even under acidic conditions. The IRI loading capacity (11.43%) and loading efficiency (>80%) of NTT2_186k were much higher than those of MSNs that did not have sulfonic acid functional groups on the inner surface of the pores. More importantly, almost all of the IRI molecules were in lactone form (>99%). Another (SO3 - )-MSN(NTT2_202) exhibited a neutral zeta potential. To load IRI into NTT2_202, the loading process is adjusted slightly. First, to reduce the electrostatic repulsion between the positively charged amine groups and IRI, the NTT2_202 particles were treated with alkaline NaHCO3 buffer to deprotonate the amine groups on the surface of NTT2_202. IRI molecules could be loaded into the pores via the electrostatic interaction between the negatively charged sulfone groups on the sidewalls of the pores and the positively charged IRI. After the loading process, IRI@NTT2_202 was washed with acetate buffer and stored in acidic conditions for long-term storage. The ratio of lactone form of IRI in IRI@NTT2_202 could be achieved to be about 96% or more. Although the loading capacity of NTT2_202 is lower than that of NTT2_186k, NTT2_202 provides an approach to synthesize IRI@MSN particles with neutral surface charge (zeta potential), which can still exhibit good loading capacity, loading efficiency, and lactone-type ratio of IRI. In conclusion, sulfonic acid functional groups (functional groups with pka ≦ 4.5) were modified on the inner pore surface of MSNs, which was important for loading IRI into MSNs, especially the loading process carried out under acidic conditions. The characterization of IRI-loaded MSNs is shown in Table 1. [Table 1]

[0122] Example 4

[0123] Irinotecan in MSN nanoformulations for the treatment of pancreatic cancer

[0124] In vivo IRI@(SO3- To evaluate the anti-pancreatic tumor effect of )-MSNs, human pancreatic cancer cells (5 × 10 6 PANC-1 cells were subcutaneously implanted into the left flank of NOD-SCID mice to serve as a PANC-1 xenograft mouse model. The first injection was performed at a tumor volume of 100 mm 3 The treatment was started when the tumor reached a tumor size of 100 mg / kg. Tumor-bearing mice were administered irinotecan (IRI), combination treatment of IRI and gemcitabine GEM (an approved drug for pancreatic treatment), and IRI@NTT2_185 with 20 mg / kg IRI once a week for a total of four doses. Tumor size and body weight were observed throughout the study period. The results showed that the efficacy of IRI@NTT2_185 and (IRI+GEM) combination therapy in tumor growth inhibition was superior to IRI. The anticancer effect of IRI@NTT2_185 was similar to IRI+GEM, but IRI@NTT2_185 showed lower toxicity (less body weight loss) compared to (IRI+GEM) combination therapy (Figure 2). IRI@(SO3 - The anti-pancreatic cancer effect of )-MSNs was also evaluated in another subcutaneous allograft tumor model. 6 KPC cells were implanted subcutaneously into the left flank of B6 mice. The first injection was performed when the tumor volume was 100 mm 3 Tumor-bearing mice were administered 20 mg / kg IRI and IRI@(SO3 - )-MSN was administered twice a week for a total of four times. - )-MSNs showed significant tumor growth inhibition compared to the IRI group. Furthermore, IRI@(SO3 - )-MSNs exhibited dose-dependent anticancer effects. In conclusion, irinotecan in MSN nanoformulations had better tumor targeting ability (based on the EPR effect of MSNs), leading to reduced drug-related toxicity and improved tumor inhibition. MSN nanoformulations of IRI proposed a potential therapeutic approach for unmet medical needs in pancreatic cancer treatment.

[0125] Example 5

[0126] Irinotecan in MSN nanoformulations for the treatment of colorectal cancer

[0127] In vivo IRI@(SO3 - To evaluate the anti-colorectal tumor effect of IRI@NTT2_186k-MSNs, 5 × 10 human colorectal cancer cells were cultured in vitro. 6 HCT-116 cells were subcutaneously implanted into the left flank of NOD-SCID mice to serve as HCT-116 heterotopic xenograft mouse models. Tumor-bearing mice were administered IRI and IRI@NTT2_186k at dose levels of 20 mg / kg and 40 mg / kg twice a week for a total of six doses. Tumor size and body weight were observed over the study period. Irinotecan in MSN nanoformulation showed more effective tumor inhibition than irinotecan alone. Results showed that IRI@NTT2_186k exhibited a dose-dependent anticancer effect. In the 40 mg / kg IRI@NTT2_186k group, tumor growth was significantly inhibited and no weight loss was observed during administration. After treatment, the tumors in mice treated with 40 mg / kg IRI@MSN almost disappeared. Furthermore, IRI@NTT2_186k can achieve similar efficacy at two-fold lower doses than IRI alone (Figure 3).

[0128] Example 6

[0129] Irinotecan in MSN nanoformulation can inhibit primary tumor growth and metastasis and extend survival in mice

[0130] Colorectal cancer is highly metastatic and there is a dire need for effective therapies that can also suppress the growth and metastasis of primary colorectal tumors. The orthotopic metastatic colorectal cancer model is an excellent model for testing the efficacy of therapeutic approaches. 6Luciferase-expressing HCT-116 cells were directly injected into the cecal wall via surgery to establish an orthotopic colorectal cancer metastasis model. In this model, highly metastatic tumors naturally develop in adjacent intestinal tissues, peritoneum, and distant metastases (spleen, kidney, liver, and diaphragm). Tumor-bearing mice were administered IRI and IRI@NTT2_186k at a dose level of 40 mg / kg twice a week for a total of eight doses. Tumor growth and metastasis were observed once a week by the IVIS system, and body weight was monitored over the study period. To assess overall survival, animals were monitored until the time of natural death or approaching moribundity. IVIS images and quantitative bioluminescence intensity results showed fast tumor growth and significant peritoneal metastasis in the control group, which promoted the death of the mice. Mice treated with IRI alone showed slower tumor growth rates compared to the control group, but IRI showed inefficient inhibition of tumor growth and metastasis. Thus, when treatment was discontinued, tumor recurrence and tumor metastasis were observed. IRI@NTT2_186k significantly inhibited primary tumor growth and cancer metastasis, and the bioluminescence signal of cancer cells was not detectable after treatment. All mice survived until the end of the study, and the survival time of the IRI@NTT2_186k-treated group was much longer than that of the IRI-treated and control groups (Figures 4A and 4B). Another metastatic mouse model was also used for efficacy testing. A tumor mass (2-3 mm x 2-3 mm mass) of HCT-116-Luc cells was fixed to the cecal wall and used as an orthotopic colorectal cancer metastasis model. Similar results were observed, and the IRI@NTT2_186k-treated group showed significant inhibition of primary tumor growth and cancer metastasis, and prolonged survival. In conclusion, irinotecan in MSN nanoformulation offered a superior advantage over irinotecan, inhibiting primary tumor growth and cancer metastasis while simultaneously prolonging survival. Anti-neoplastic drugs in MSN nanoformulations offered a potential treatment for highly metastatic cancers.

[0131] Example 7

[0132] Tube formation assay

[0133] Cancer cell migration and tumor angiogenesis were important factors in cancer metastasis. During the tumor angiogenesis process, endothelial cells originated from existing blood vessels and formed new blood vessels near the tumor site to supply nutrients for the rapid growth of tumors. A tube formation assay was used to investigate the effect of MSNs on cell migration and angiogenesis. The tube formation assay was established by seeding HUVEC cells on Matrigel. After 6 h, the formation of tube structures was observed and the vascular junctions were calculated using ImageJ. The control and SSN (solid silica nanoparticles) groups showed a complete network of tube structures. However, in the four MSN groups, the formation of tube structures was inhibited. The results show that MSNs can inhibit the migration of HUVECs and prevent the formation of tube structures (Figure 5). Example 8

[0134] Chorioallantoic membrane (CAM) analysis for angiogenesis

[0135] To evaluate the effect of MSNs on tumor angiogenesis, a chick embryo chorioallantoic membrane (CAM) tumor model was used. Fertilized eggs were incubated in an incubator for 10 days, then a small window was opened in the eggshell and cancer cells were implanted in the CAM membrane near the aorta. MSNs or doxorubicin (DOX) were administered on the 12th day after tumor formation, and angiogenesis near the tumor was observed on the 14th and 16th days.

[0136] In the DOX-treated group, blood vessels showed collapse and decreased density due to DOX inhibiting blood vessel growth. MSNs did not reduce blood vessel density as DOX did, but they caused irregular growth of blood vessels compared to the blood vessels in the control group, blocking angiogenesis in the tumor area. In the MSN group, the number of branched microvessels increased, but the length of the blood vessels was much shorter (Figure 6). MSNs inhibited the migration of endothelial cells and tumor cells, so the tumor-induced proliferation of vascular endothelial cells could not spread outward and only accumulated at their location. In conclusion, in the MSN-treated group, the density of blood vessels did not decrease, but the migration ability of endothelial cells was affected, causing irregular growth of blood vessels.

[0137] Example 9

[0138] MSNs inhibit metastasis and extend overall survival in in vivo studies (combined treatment with Lipo-Dox and MSNs)

[0139] A spontaneous cancer metastasis mouse model was used to evaluate the effect of MSNs in inhibiting metastasis on survival. 6 Luciferase-expressing 4T1 cancer cells were subcutaneously implanted into the left flank of BALB / c mice and used as an ectopic allograft mouse model. Tumor-bearing mice were administered Lipo-Dox (IV injection, 4 mg / kg) with or without MSN (IV or IT injection) for a total of three doses at 3-day intervals. Tumor growth and overall survival were monitored after tumor injection. Bioluminescence images were taken by IVIS system on days 22, 26, 29, and 34 to track cancer metastasis. In the control group of mice, it was observed that the primary tumor grew rapidly and the cancer cells metastasized to other parts of the body. In the Lipo-Dox-treated group, the growth of the primary tumor was inhibited, but cancer metastasis was still observed. Severe metastasis in the control and Lipo-Dox-treated groups caused the death of mice, resulting in similar survival times in both groups. In contrast, combined treatment of Lipo-Dox with MSNs (MSNs injected via intravenous or intratumoral injection) significantly inhibited metastasis and extended survival time (Figure 7). The results showed that MSNs significantly inhibited cancer metastasis but had no effect on body weight and primary tumor size. Metastasis is the main factor causing death, and combined treatment of antineoplastic drugs (chemotherapeutic agents, proteins, and nucleic acid drugs) or antineoplastic drugs with MSNs in MSN nanoformulations could simultaneously inhibit primary tumor growth and cancer metastasis and extend survival time. MSNs could provide additional clinical benefits and reduce the incidence of metastasis, especially for cancers with a high propensity to metastasize.

[0140] Example 10 Mechanism of metastasis inhibition

[0141] To investigate the mechanism by which MSNs inhibit cell migration, angiogenesis, and cancer metastasis, the protein expression levels of cancer cells (4T1) treated with MSNs were evaluated by Western blot. Western blot results showed that the protein expression levels of p-ERK, p-paxillin, and p-FAK in cells treated with MSN-PEG / TA25 were lower than those in the control group, indicating that MSNs inhibited the phosphorylation of ERK, paxillin, and FAK (Figure 8A). It has been shown that the phosphorylation of these proteins (ERK, paxillin, and FAK) is associated with focal adhesion turnover, which is related to cell migration. The results showed that MSNs could disrupt the balance of focal adhesion turnover, and consequently inhibit cell migration, angiogenesis, and cancer metastasis. Furthermore, in the in vivo test of anti-metastasis, tumor-bearing mice treated with MSNs were sacrificed, and tumors were harvested and sectioned for immunohistochemical staining. The green signals from p-FAK and p-EPR in tumors (from MSN-treated mice) were significantly reduced compared to the control group (Figure 8B). Analysis of protein expression in cells and immunofluorescence of protein markers in tumors indicated that the mechanism by which MSNs inhibit metastasis is related to the imbalance of focal adhesion turnover.

[0142] Another mechanism by which nanoparticles limit cell movement has been presented in the literature, and this mechanism was the severe inhibition of intracellular microtubule polymerization via nanoparticles. To evaluate the effect of MSNs on the cytoskeleton of cells, protein expression related to the cytoskeleton (G-actin and F-actin) and changes in cytoskeleton morphology (F-actin and α-tubulin) of MSN-treated cells were detected by Western blot and immunofluorescence assay. The data of Western blot and immunofluorescence assay showed no difference between the MSN-treated group and the control group; as a result, MSNs had no effect on actin and microtubule cytoskeleton. The inhibition of MSN migration was associated with the disturbance of the balance of focal adhesion turnover, rather than the interference of the intracellular microtubule cytoskeleton.

[0143] The components, reaction conditions, and parameters illustrated in the examples are for illustrative purposes only and are not intended to be limitations on the materials or preparation methods.

[0144] It will be appreciated that those skilled in the art of the present invention may make changes 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, the present application intends to cover any changes and modifications, provided that the changes or modifications are within the scope defined in the appended claims or their equivalents.

Claims

1. 1. A pharmaceutical composition for use in a method for treating a subject having or at risk of having cancer and / or metastasis of said cancer, the pharmaceutical composition comprising irinotecan (IRI) loaded into mesoporous silica nanoparticles (MSNs) and optionally a pharmaceutically acceptable carrier, the method comprising administering the pharmaceutical composition to the subject.

2. 2. The pharmaceutical composition of claim 1, wherein the cancer is squamous cell carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, multiple myeloma, B-cell lymphoma, brain tumor, or head and neck cancer.

3. The pharmaceutical composition of claim 1, wherein the method is for inhibiting angiogenesis, inhibiting tube formation, or inhibiting focal adhesion turnover and / or cell migration of cells of the cancer.

4. The MSNs define at least one pore and have at least one functional group on a sidewall of the at least one pore, and the pK ａ The pharmaceutical composition of claim 1, wherein the value is 4.5 or less.

5. 5. The pharmaceutical composition of claim 4, wherein the mean hydrodynamic diameter of the MSNs measured in PBS medium is less than 100 nm.

6. 5. The pharmaceutical composition of claim 4, wherein the at least one functional group comprises a sulfonic acid group, a sulfate ester group, a carboxylic acid group, a phosphonic acid group, a phosphinic acid group, a phosphate ester group, or a phosphite ester group.

7. 5. The pharmaceutical composition of claim 4, wherein the molar ratio of the amount of silane to the amount of said functionalized silane ranges from 60:1 to 5:

1.

8. The pharmaceutical composition of claim 4 , wherein the MSNs further have an external surface modification with an organic molecule, oligomer, or polymer, and / or a positively charged molecule, oligomer, or polymer.

9. 9. The pharmaceutical composition of claim 8, wherein the organic molecule, oligomer, or polymer is selected from poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), or a PEG-PPG copolymer, or a combination thereof.

10. 9. The pharmaceutical composition of claim 8, wherein the positively charged molecule, oligomer, or polymer is selected from (N-[3-(trimethoxysilyl)propyl]-N,N,N-trimethylammonium chloride), N-[3-(trimethoxysilyl)propyl]ethylenediamine, polyethyleneimine (PEI); alkoxylsilane-terminated (poly)alkylene(poly)amine, or organoalkoxysilane with amino groups, or a combination thereof.

11. A conjugate comprising a drug loaded in mesoporous silica nanoparticles (MSNs) defining at least one pore and having at least one functional group on the sidewall of said at least one pore, wherein the pK ａ A conjugate having a value of 4.5 or less.

12. The conjugate of claim 11, wherein the mean hydrodynamic diameter of the MSNs measured in PBS medium is less than 100 nm.

13. 12. The conjugate of claim 11, wherein the at least one functional group comprises a sulfonic acid group, a sulfate ester group, a carboxylic acid group, a phosphonic acid group, a phosphinic acid group, a phosphate ester group, or a phosphite ester group.

14. 12. The conjugate of claim 11, wherein the molar ratio of the amount of silane to the amount of said functionalized silane ranges from 60:1 to 5:

1.

15. 12. The conjugate of claim 11, further comprising an external surface modification with an organic molecule, oligomer, or polymer, and / or a positively charged molecule, oligomer, or polymer.

16. 16. The conjugate of claim 15, wherein the organic molecule, oligomer, or polymer is selected from poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), or a PEG-PPG copolymer, or a combination thereof.

17. 16. The conjugate of claim 15, wherein the positively charged molecule, oligomer, or polymer is selected from (N-[3-(trimethoxysilyl)propyl]-N,N,N-trimethylammonium chloride), N-[3-(trimethoxysilyl)propyl]ethylenediamine, polyethyleneimine (PEI); alkoxylsilane-terminated (poly)alkylene(poly)amine, or organoalkoxysilane with amino groups, or a combination thereof.

18. The conjugate of claim 11 , wherein the drug is loaded into the at least one pore.

19. pK of the drug ａ value is equal to or greater than the pK value of the silanol groups of the MSNs, and / or the drug has a pK ａ or the drug is positively charged and / or exhibits a change in properties at pH values ​​equal to or greater than the pK of any one functional group on the broad wall of the MSN. ａ 12. The conjugate of claim 11, which exhibits an active form at a pH value equal to or greater than that of the conjugate of claim 11.

20. The conjugate of claim 11, wherein the drug is irinotecan (IRI).

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