WO2016204896A1 - Inorganic controlled release particles with fast drug loading - Google Patents

Abstract

Disclosed herein are nanoparticles comprising an inorganic material having pores that can quickly absorb pharmaceutical moieties and then release such moieites in a controlled fashion for an extended period of time. The nanoparticles can be aggregated in order to form microaggregates that provide the same advantages of quick absorption and controlled release of pharmaceutical moieties. Quick absorption permits the preparation of drug-loaded nanoparticles and microaggregates essentially contemporaneously with the time of treatment, such as during a surgical process. Methods of preparing such nanoparticles and microaggregates are also provided, as are methods for administering a pharmaceutical moiety using the nanoparticles and microaggregates.

Description

INORGANIC CONTROLLED RELEASE PARTICLES WITH FAST DRUG LOADING

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional App. No. 62/180,345, filed June 16, 2015, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

[0002] The present disclosure concerns mesoporous nanoparticles that can be used to deliver pharmaceutical agents, as well as materials that are formed from such nanoparticles.

BACKGROUND

[0003] Silica gel obtained by sol-gel processing is an inorganic polymer produced synthetically by controlled hydrolysis and condensation of alkoxysilanes. This material, originally developed for engineering applications, is currently also being studied as a material for the entrapment and conversely the sustained release of bioactive compounds in laboratories worldwide. Important benefits associated with sol gel processing of bioactive ceramics and glasses are the excellent biocompatibility, as demonstrated in vivo (Radin, S., G. El-Bassyouni, E.J. Vresilovic, E. Schepers, and P. Ducheyne, In vivo tissue response to resorbable silica xerogels as contr oiled-release materials. Biomaterials, (2005) 26 1043-52), and the extensive control of release kinetics {Radin, S. and P. Ducheyne, Nanostructural control of implantable xerogels for the controlled release of biomolecules , in Learning from nature to design new implantable biomaterials, R. Reis, Editor., Kluwer Academic Publishers: Netherland. (2004) p. 59-74).

[0004] Since 1991, when Mobil Oil Corporation synthesized the MCM-41 mesoporous silica material, highly ordered mesoporous materials have attracted the attention of the scientific community, mainly due to their potential technological applications (S. Bhattacharyya, G.

Lelong, M-L. Saboungi, Recent progress in the Synthesis and Applications of MCM-41 based

Mesoporous materials- a short review. J. Exp. Nanosci. (2006) 1 375-395). Mesoporous materials are characterized by their large surface area, pore volume and pore size, and their narrow pore size distribution. For this reason, applications in the fields of catalysis, lasers, sensors, solar cells and so forth have been proposed and/or developed (S. Bhattacharyya, H.

Wang, P. Ducheyne, Polymer coated mesoporous silica controlled release nanoparticles for macromolecules , Acta Biomater. 8(9), 3429-35, (2012)). [0005] Recent breakthroughs in the synthesis of mesoporous silica nanoparticles (MSNs) and microparticles (e.g., diameter > 1000 nm) (MSMPs) with high surface areas (e.g., >700 m²/g) and tunable pore diameter (e.g., 2-10 nm) have led to the development of a series of new delivery systems, where various guest molecules, such as pharmaceutical drugs, fluorescent imaging agents, and other molecules, could be adsorbed into the mesopores and later released into various solutions (see, e.g., Balas, F, et al, Confinement and Controlled Release of Bisphosphonates on Ordered Mesoporous Silica-Based Materials. J. Am. Chem. Soc. (2006) 128, 8116-8117; Vallet-Regi M, et al, Medical applications of organic-inorganic hybrid materials within the field of silica-based biocer amies. Chem. Soc. Rev., 2011, 40, 596-607; Manzano M, et al., New developments in ordered mesoporous materials for drug delivery. J. Mater. Chem., 2010, 20, 5593-5604).

[0006] Furthermore, recent reports have published concerning the design of functional mesoporous silica nanoparticles by decorating the pore surfaces with organic or inorganic moieties that serve as gating devices to regulate the release of guest molecules and that are under the control of several different external stimuli, such as chemicals (Giri, S, et al., Magnetic Nanoparticle-Capped Mesoporous Silica Nanorod-Based Stimuli-Responsive Controlled Release Delivery System. Angew. Chem., Int. Ed. (2005) 44, 5038-5044; Lai, C.-Y., et al, A Mesoporous Silica Nanosphere-Based Carrier System with Chemically Removable CdS Nanoparticle Caps for Stimuli-Responsive Controlled Release of Neurotransmitters and Drug Molecules. J. Am. Chem. Soc. (2003) 125, 4451-4459; Radu, D. R., et al, A Polyamidoamine Dendrimer -Capped Mesoporous Silica Nanosphere-Based Gene Transfection Reagent. J. Am. Chem. Soc. (2004) 126, 13216-13217; Leung, K. C. F., et al, Supramolecular Nanovalves Controlled by Proton Abstraction and Competitive Binding. Chem. Mater. (2006) 18, 5919-5928), temperature (Fu Q, et al., A reversible molecular valve. Adv. Mater. (2003) 15, 1262-1266), redox reactions (Hernandez, R, et al., An Operational Supramolecular Nanovalve. J. Am. Chem. Soc. (2004) 126, 3370-3371; Nguyen, T. D., et al., A reversible molecular valve. Proc. Natl. Acad. Sci. U.S.A. (2005) 102, 10029-10034), and photo-irradiation (Mai, N. K, et al, Photocontr oiled reversible release of guest molecules from coumarin-modified mesoporous silica. Nature (London) (2003), 421, 350-353; Mai, N. K., et al, Photo-Switched Storage and Release of Guest Molecules in the Pore Void of Coumarin-Modifled MCM-41. Chem.Mater. (2003) 15, 3385- 3394; Knezevic NZ, et al., Functionalized mesoporous silica nanoparticle-based visible light responsive controlled release delivery system. Chem. Commun., 2011, 47, 2817-2819) have highlighted the potential of utilizing this kind of nanodevice for many controlled release applications. Recently, it has been demonstrated that mesoporous silica nanoparticles can be internalized efficiently into cells, are nontoxic without affecting cell viability, growth, and differentiation, can escape from endo-lysosomal vesicles, and resist lysosomal degradation {Mai, N. K., et al ). It has also been demonstrated that these particles are capable of intracellular delivery of DNA {Zhao YN, et al., Capped mesoporous silica nanoparticles as stimuli-responsive controlled release systems for intracellular drug/gene delivery. Expert Opinion on Drug Delivery 7(9) 1013-1029 2010). Surface functionalization of mesoporous silica nanoparticles and its biomedical applications are also being studied {Cauda V, et al, Impact of different PEGylation patterns on the long-term bio-stability of colloidal mesoporous silica nanoparticles. Journal Of Materials Chemistry 20(39) 8693-8699 2010; Cauda V, et al, Bio-degradation study of colloidal mesoporous silica nanoparticles: Effect of surface functionalization with organo- silanes and poly (ethylene glycol) Microporous And Mesoporous Materials, 132(1-2) 60-71 2010; He QJ, et al., The effect of PEGylation of mesoporous silica nanoparticles on nonspecific binding of serum proteins and cellular responses. Biomaterials 31 (6) 1085-1092 2010; S.

Bhattacharyya, et al, Polymer coated mesoporous silica controlled release nanoparticles for macromolecules , Acta Biomater. 8(9), 3429-35, (2012)).

[0007] However, none of the existing systems are capable of fast absorption of single or multiple drug molecules, while providing long-term controlled release immediately after absorption. Moreover, none of the above mentioned systems avoid initial burst release if the drug solution is hand mixed with MSNs or MSMPs and put in a release solution after only a short period of mixing. Most existing systems are, in fact, developed for intracellular delivery of therapeutics, where controlled release is not necessary once the mesoporous silica nanoparticle penetrates into the cell.

[0008] New systems for fast absorption and controlled release of therapeutic agents using mesoporous nanoparticles would have wide ranging benefits in the drug delivery field.

SUMMARY

[0009] Disclosed herein are nanoparticles comprising an inorganic material comprising a plurality of pores of which at least 90% have a diameter of 2 to 50 nm, the nanoparticle being surface-functionalized with a plurality of aldehyde groups that are present in an amount of 0.1 to 1.0 mmol/g.

[0010] Also disclosed are microaggregates comprising a plurality of nanoparticles aggregated together, each of the nanoparticles comprising an inorganic material comprising a plurality of pores of which at least 90% have a diameter of 2 to 50 nm; and, the nanoparticles being surface-functionalized with a plurality of aldehyde groups that are present in an amount of 0.1 to 1.0 mmol/g.

[0011] The present disclosure also provides methods for making an aldehyde surface- functionalized mesoporous nanoparticle comprising synthesizing the nanoparticle using a single pot reaction procedure, wherein said procedure results in the nanoparticle being surface- functionalized with a plurality of aldehyde groups that are present in an amount of 0.1 to 1.0 mmol/g.

[0012] Also provided are methods for loading a microaggregate with a pharmaceutical agent comprising contacting a solution comprising the pharmaceutical agent with the microaggregate for a duration of time that is sufficient to load the microaggregate with the pharmaceutical agent, the microaggregate comprising a plurality of nanoparticles aggregated together, each of the nanoparticles comprising an inorganic material comprising a plurality of pores of which at least 90% have a diameter of 2 to 50 nm; and, the nanoparticles being surface- functionalized with a plurality of aldehyde groups that are present in an amount of 0.1 to 1.0 mmol/g, wherein less than about 30 minutes of the contacting are required in order to load at least some of the pores of the nanoparticles with the pharmaceutical agent.

[0013] The present disclosure also relates to methods for administering a

pharmaceutical agent to a subject comprising contacting the subject with a microaggregate, the microaggregate comprising a plurality of nanoparticles aggregated together, each of the nanoparticles comprising an inorganic material comprising a plurality of pores of which at least 90% have a diameter of 2 to 50 nm, at least some of the pores of the nanoparticle being loaded with the pharmaceutical agent; and, the nanoparticles being surface-functionalized with a plurality of aldehyde groups that are present in an amount of 0.1 to 1.0 mmol/g.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 is a scanning electron micrograph of microaggregates according to the present disclosure.

[0015] FIG. 2A and FIG. 2B depict individual mean cumulative release of vancomycin and rifampin, respectively, from inventive microaggregates as a function of immersion time in PBS.

[0016] FIG. 3 illustrates the simultaneous release of vancomycin and rifampin from a single microaggregate as a function of immersion time in PBS. [0017] FIG. 4A shows the bactericidal activity of microaggregates that are loaded 10% vancomycin, are loaded with both 10% vancomycin and 10% rifampin, or do not contain any drugs, on the first day of exposure to a solution containing 10⁴ CFU/mL of MRS A. FIG. 4B shows the bacterial activity of the microaggregates on days 2-4.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0018] The present inventions may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures and examples, which form a part of this disclosure. It is to be understood that these inventions are not limited to the specific products, methods, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed inventions.

[0019] The entire disclosures of each patent, patent application, and publication cited or described in this document are hereby incorporated herein by reference.

[0020] As employed above and throughout the disclosure, the following terms and abbreviations, unless otherwise indicated, shall be understood to have the following meanings.

[0021] In the present disclosure the singular forms "a," "an," and "the" include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to "a particle" is a reference to one or more of such particles and equivalents thereof known to those skilled in the art, and so forth. Furthermore, when indicating that a certain element "may be" X, Y, or Z, it is not intended by such usage to exclude in all instances other choices for the element.

[0022] When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. As used herein, "about X" (where X is a numerical value) preferably refers to ±10% of the recited value, inclusive. For example, the phrase "about 8" refers to a value of 7.2 to 8.8, inclusive; as another example, the phrase "about 8%" refers to a value of 7.2% to 8.8%, inclusive. Where present, all ranges are inclusive and combinable. For example, when a range of "1 to 5" is recited, the recited range should be construed as including ranges "1 to 4", "1 to 3", "1-2", "1-2 & 4-5", "1-3 & 5", and the like. In addition, when a list of alternatives is positively provided, such a listing can also include embodiments where any of the alternatives may be excluded. For example, when a range of "1 to 5" is described, such a description can support situations whereby any of 1, 2, 3, 4, or 5 are excluded; thus, a recitation of "1 to 5" may support "1 and 3-5, but not 2", or simply

"wherein 2 is not included."

[0023] As used herein, the terms "treatment" or "therapy" (as well as different word forms thereof) includes preventative (e.g., prophylactic), curative, or palliative treatment. Such preventative, curative, or palliative treatment may be full or partial. For example, complete elimination of unwanted symptoms, or partial elimination of one or more unwanted symptoms would represent "treatment" as contemplated herein.

[0024] As employed above and throughout the disclosure the term "effective amount" refers to an amount effective, at dosages, and for periods of time necessary, to achieve the desired result with respect to the treatment of the relevant disorder, condition, or side effect. It will be appreciated that the effective amount of components of the present invention will vary from patient to patient not only with the particular compound, component or composition selected, the route of administration, and the ability of the components to elicit a desired response in the individual, but also with factors such as the disease state or severity of the condition to be alleviated, hormone levels, age, sex, weight of the individual, the state of being of the patient, and the severity of the condition being treated, concurrent medication or special diets then being followed by the particular patient, and other factors which those skilled in the art will recognize, with the appropriate dosage ultimately being at the discretion of the attendant physician. Dosage regimens may be adjusted to provide the improved therapeutic response. An effective amount is also one in which any toxic or detrimental effects of the components are outweighed by the therapeutically beneficial effects. As an example, the materials useful in the methods of the present invention are administered at a dosage and for a time such that the level of the undesired pathogen or symptom is reduced as compared to the level before the start of treatment.

[0025] "Pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio.

[0026] The present disclosure relates to inorganic mesoporous materials that can quickly absorb pharmaceutical moieties and provide controlled release of such moieties over time. [0027] Although previous work has included functionalization of the surface of mesoporous silica nanoparticles in order to regulate the release of drug molecules from the pores of the nanoparticles, the time required to introduce the drug molecules into the pores roughly correlated to the amount of time over which release occurred. That is, although such materials could provide extended release of a therapeutic molecule, loading of these materials with the therapeutic molecule required exposure of the latter to the former for a correspondingly extended period of time. This requirement meant that the drug loading process typically occurred well prior to the time of intended use of the loaded particles. Thus, it was necessary for those who wished to use drug-loaded nanoparticles to have already selected and acquired the desired drug / particle combination prior to the time of intended use. This limited the opportunity for the user (such as a surgeon) to be able to make contemporaneous decisions as to the type of drug to be used. Circumstances during acute medical treatment, such as surgery, can change rapidly and without warning, and medical practitioners benefit from the ability to select or change the nature of treatment as needed.

[0028] In contrast with previously known materials, the presently disclosed

nanoparticles and microparticles provide the unprecedented benefit of rapid, complete absorption of one or more drug moieties, coupled with subsequent controlled release. These characteristics mean that a medical practitioner can choose one or more pharmaceutical materials essentially contemporaneously with time of intended use, can load the inventive particles with the chosen pharmaceutical materials at the facility where treatment is to take place, and can deliver the drug- loaded particles to the treatment site, all within a very brief time frame. This characteristic greatly facilitates, for example, surgical planning and intraoperative decision-making, by permitting the surgeon to select a drug regimen on an intraoperative basis, and thereby at such time to load the chosen pharmaceutical or biological material and administer the loaded particles to the surgical site. Furthermore, despite the rapidity with which the instant particles absorb a desired drug, the particles provide long term, controlled release. These and other advantageous characteristics of the inventive particles are described more fully herein.

[0029] Disclosed herein are nanoparticles comprising an inorganic material comprising a plurality of pores, the nanoparticle being surface-functionalized with a plurality of aldehyde groups that are present in an amount of 0.1 to 1.0 mmol/g. The inorganic material may be, for example, silica, calcium phosphate, hydroxyapatite, alumina, titanium dioxide, or any combination thereof. [0030] As used herein, the term "nanoparticle" preferably refers to a particle that does not include any physical dimension that exceeds 1000 nm. For example, the nanoparticles may have at least one dimension that is 100-1000 nm, at least one dimension that is 100-500 nm, or at least one dimension that is 200-500 nm. The nanoparticles may therefore have at least one dimension that is about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, about 800 nm, about 850 nm, about 900 nm, about 950 nm or about 1000 nm.

[0031] The pores of a given nanoparticle will fall within a certain size distribution. In some embodiments of the present nanoparticles, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the pores have a diameter of 2 to 50 nm. In a preferred embodiment, the nanoparticles comprise a plurality of pores of which at least 90% have a diameter of 2 to 50 nm, and the nanoparticles are surface-functionalized with a plurality of aldehyde groups that are present in an amount of 0.1 to 1.0 mmol/g.

[0032] The present nanoparticles may be characterized as having a large surface area. For example, the surface area of an inventive nanoparticle may be at least or about 500 m²/g, at least or about 600 m²/g, at least or about 700 m²/g, at least or about 800 m²/g, at least or about 900 m²/g, at least or about 1000 m²/g, at least or about 1100 m²/g, at least or about 1200 m²/g, at least or about 1300 m²/g, at least or about 1400 m²/g, or at least or about 1500 m²/g.

[0033] The nanoparticles are surface-functionalized with a plurality of aldehyde groups (-CHO) that are present in an amount of 0.1 to 1.0 mmol/g. In a preferred embodiment, the aldehyde groups are bound directly to the inorganic material of which the nanoparticles are made. Previous mesoporous materials were functionalized with aldehyde groups in order to enable covalent attachment to other molecules, such as polymer (e.g., poly(methyl methacrylate), polyethylene glycol), metallic particles, or enzymes (e.g., lysozyme, penicillin G acylase). For example, a recent publication describes the preparation of trimethoxysilylpropanal- functionalized mesoporous cellular foams as an intermediate step pursuant to the attachment of penicillin G acylase to such foams (see Gao WZ, et al, Journal of Molecular Catalysis B:

Enzymatic 105 (2014) 111-117). Trimethoxysilylpropanal (TMSP) bears a terminal aldehyde group. Gao, et al. report that mesoporous foams "with high content of aldehyde groups were successfully prepared by post-synthetical functionalization of [the foams] with TMSP" in order to improve the "operational stability" of the final product, i. e., mesoporous foams with penicillin G acylase immobilized on the surface thereof. Thus, Gao, et al, as well as other reports of surface functionalization with aldehyde groups in order to enable covalent attachment to other molecules, were focused on maximizing the amount of aldehyde surface functional groups in order to increase the basis for covalent attachment of secondary molecules.

[0034] The present inventors have surprisingly discovered that using a comparatively low concentration of aldehyde surface functionalization on mesoporous nanoparticles enables quick absorption of molecules into the pores of such particles, and, at the same time, enables controlled release of such materials from the pores over time. The conditions that are reported in the prior art is unsuitable for preparing quick absorbing, controlled release particles, because the higher concentration of surface aldehyde groups sterically interferes with the absorption and release processes. At the same time, the low concentration of aldehyde surface functionalization disclosed herein is unsuitable for the purpose of covalently attaching secondary molecules (such as polymer, metallic particles, enzymes, and the like), because it is insufficient for supporting the desired concentration of secondary material.

[0035] The instant nanoparticles are surface-functionalized with a plurality of aldehyde groups that are present in an amount of 0.1 to 1.0 mmol/g, 0.1 to 0.8 mmol/g, 0.1 to 0.7 mmol/g, 0.1 to 0.5 mmol/g, 0.1 to 0.4 mmol/g, or 0.1 to 0.3 mmol/g, such as about 0.1 mmol/g, about 0.2 mmol/g, about 0.3 mmol/g, about 0.4 mmol/g, about 0.5 mmol/g, about 0.6 mmol/g, about 0.7 mmol/g, about 0.8 mmol/g, about 0.9 mmol/g, or about 1.0 mmol/g.

[0036] The instant surface aldehyde functionalized nanoparticles may be prepared using a "one pot" reaction procedure. In other words, a single reaction procedure may be used to accomplish both the synthesis of the nanoparticles and the inclusion of aldehyde groups on the surface of the nanoparticles. This single reaction procedure may be carried out by adding reagents to and appropriately manipulating the conditions within a single vessel. Typically, a one pot reaction procedure is characterized as involving a single chemical reaction. This is to be contrasted with a multiple reaction procedure for producing surface functionalized nanoparticles, which is characterized by the formation of nanoparticles in one reaction, and often in a first vessel, followed by the surface functionalization of the preformed nanoparticles in a second reaction, and often in a second vessel, or in the first vessel following formation and optional isolation of the nanoparticles. One-pot reaction procedures that may be used to form the instant nanoparticles are described more fully infra in connection with the presently disclosed methods for making aldehyde surface-functionalized mesoporous nanoparticles.

[0037] In preferred embodiments, the aldehyde groups are distributed substantially homogeneously (substantially evenly distributed) on the surface of a nanoparticle according to the present disclosure. It has been reported that when mesoporous materials are prepared in a procedure that is separate from the surface functionalization of the materials (i. e., are preformed prior to the functionalization reaction), the resulting product features functional groups that are comparatively non-homogeneously distributed on the surface of the mesoporous materials. See, e.g., Lim MH, et al, Chem. Mater. 1999, 11, 3285-3295. Therefore, one advantage of the presently disclosed one-pot reaction procedure is that there is substantially homogeneous distribution of the aldehyde groups on the surface of the nanoparticles.

[0038] The pores of the nanoparticles may be loaded with one or more desired molecules, and the present nanoparticles are capable of absorbing a molecule having a molecular weight that is, for example, about 10 Da, about 20 Da, about 40 Da, about 50 Da, about 60 Da, about 70 Da, about 80 Da, about 90 Da, about 100 Da, about 200 Da, about 300 Da, about 400 Da, about 500 Da, about 600 Da, about 700 Da, about 800 Da, about 900 Da, about 1 kDa, about 1.1 kDa, about 1.2 kDa, about 1.3 kDa, about 1.4 kDa, about 1.5 kDa, about 1.6 kDa, about 1.7 kDa, about 1.8 kDa, about 1.9 kDa, about 2 kDa, about 2.1 kDa, about 2.2 kDa, about 2.3 kDa, about 2.4 kDa, about 2.5 kDa, about 2.6 kDa, about 2.7 kDa, about 2.8 kDa, about 2.9 kDa, about 3 kDa, about 3.2 kDa, about 3.5 kDa, about 3.7 kDa, about 3.8 kDa, or about 4 kDa.

[0039] The nanoparticles may optionally be loaded with two or more different desired atomic or molecular moieties (see, for example, Figure 3 and 4 and corresponding examples). In one example, some of the pores of a given nanoparticle are loaded with a first molecule, while some of the other pores of the nanoparticle are loaded with a second, different molecule. In another example, a given pore of a nanoparticle is loaded with two or more different molecules. Procedures for loading the pores of the instant nanoparticles are described more fully infra.

[0040] Moieties with which the pores of the instant nanoparticles may be loaded include any therapeutic or otherwise pharmaceutically active moiety that meets the presently disclosed size requirements. Exemplary "large molecules" that may be loaded into the pores of the present nanoparticles include nucleic acids, proteins, DNA, RNA, polysaccharides, enzymes, carbohydrates, and lipids. Classes of moieties with which the pores of the nanoparticles may be loaded include analgesics, antibiotics, antifungals, antiviral agents, antioxidants, antineoplastics, antiangiogenics, antithrombogenics, anti-inflammatories, steroids, cytokines, monoclonal antibodies, genetically modified biological molecules that have therapeutic effects, and growth factors. Specific examples of drugs that may be loaded into the pores of the present

nanoparticles include vancomycin, rifampin, gentamycin, tobramycin, bupivacaine, mepivacaine, ibuprofen, insulin and its analogues, or any other desired drug agent. No limitation as to the type of pharmaceutical moiety that can be loaded into the pores of the instant nanoparticles is intended by the preceding list of exemplary drugs.

[0041] As disclosed herein, the present inventors have unexpectedly found that the inventive nanoparticles avoid the problem of initial burst release and are in fact capable of providing controlled release of moieties that has been loaded within at least some of the nanoparticle pores. For example, the delivery of moieties may be characterized as substantially zero order release kinetics. The delivery of moieties at a controlled rate may be, for example, for about three days, about four days, about 5 days, about 6 days, about one week, about ten days, about two weeks, about three weeks, about four weeks, about five weeks, about six weeks, about seven weeks, about eight weights, about nine weeks, about ten weeks, about 11 weeks, or about 12 weeks. The delivery of the moieties over any such period of time may, in some embodiments, be characterized as substantially zero order release kinetics.

[0042] The present disclosure also provides methods for making an aldehyde surface- functionalized mesoporous nanoparticle comprising synthesizing the nanoparticle using a one pot reaction procedure, wherein the procedure results in the nanoparticle being surface- functionalized with a plurality of aldehyde groups that are present in an amount of 0.1 to 1.0 mmol/g. In certain embodiments, the one pot reaction procedure may proceed in accordance with the following reaction: source of inorganic material

source of aldehyde functional groups

> inorganic nanoparticles with structure directing agent

alkaline pH conditions surface aldehyde functional

Structure directing agents are used for the synthesis of mesoporous silica materials and are responsible for creating the mesostructure, i.e. , the mesopores. The structure directing agent may be, for example, a cationic surfactant, such as cetyltrimethylammonium bromide (C_xTMABr), wherein "x" refers to the length of the carbon chain and can be, for example, 8 (Cg) to 18 (Cig); an anionic surfactant, such as cetyltrimethylammonium chloride (C_XTMAC1) with length of carbon chain "x" being from 8 (Cg) to 18 (Cig); a nonionic surfactant, such as the poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer family known as

Pluronic®; or, any combination of these surfactants. In the instant reaction procedure, the structure directing agent may be combined with a solvent, such as an alcohol or water. [0043] The source of inorganic material may be a source of silica, calcium phosphate, hydroxyapatite, alumina, titanium dioxide, or any combination thereof. When the inorganic material is silica, the source of such material may be, for example, tetraethyl orthosilicate (TEOS) or tetramethyl orthosilicate (TMOS).

[0044] The source of aldehyde functional groups may be, for example, 2- (triethoxysilyl)butyraldehyde, 4-(triethoxysilyl)butyraldehyde, 3- (triethoxysilyl)propionaldehyde, or 4-(trimethoxysilyl)butyraldehyde.

[0045] The source of inorganic material and the source of aldehyde functional groups are preferably added to the reaction medium with the structure directing agent already present. The source of inorganic material and the source of aldehyde functional groups may be added sequentially or at the same time.

[0046] The reaction medium in which the present nanoparticles are formed is characterized by alkaline pH conditions. For example, the pH of the reaction medium may be raised above neutral following introduction of the structure directing agent, following introduction of the source of inorganic material, or following introduction of the source of aldehyde functional groups. Any means for raising the pH above neutral may be used, for example, by adding sodium hydroxide. The alkaline pH conditions of the reaction medium may be about pH 10-13, about pH 12 being preferred.

[0047] The reaction medium is optionally heated above room temperature. When the structure directing agent is combined with a solvent, the solvent may be above room temperature, and thereafter the reaction medium may be maintained above room temperature. The temperature to which the reaction medium is raised and maintained may be about 30°C to about 100°C. For example, at any given point during the reaction procedure, the temperature may be, about 30°C, about 40°C, about 50°C, about 60°C, about 70°C, about 80°C, about 90°C, or about 100°C. Following combination of the structure directing agent, the source of inorganic material, and the source of aldehyde functional groups, the reaction medium may be maintained at the heated temperature for a desired period of time, for example, for about 30 min, about 45 min, about 1 hr, about 2 hr, about 3 hr, about 4 hr, about 5 hr, about 6 hr, about 8 hr, about 10 hr, or about 12 hr.

[0048] At a certain point following the combination of the structure directing agent, the source of inorganic material, and the source of aldehyde functional groups, the reaction medium will form a precipitate, and this precipitate may be separated, and optionally filtered, washed, and/or dried in order to yield the instant nanoparticles. [0049] In one example, the instant methods for making an aldehyde surface- functionalized mesoporous nanoparticle may comprise forming a solution of

octadecyltrimethylammonium bromide in a solvent, increasing the pH of the solution, adding a source of silica to the solution, adding a source of aldehyde functional groups to the solution, heating the solution for a time that is sufficient to form a precipitate, and drying the precipitate to obtain the nanoparticle. The solvent may be water. The pH of the solution may be raised to about pH 12. The source of silica may be tetraethyl orthosilicate. The source of aldehyde functional groups may be triethoxysilylbutyraldehyde. The source of silica and the source of aldehyde functional groups may be added to the solution separately or substantially

simultaneously. The solution to which each of these ingredients has been added may be heated at about 80°C, and the time of heating may be about two hours. The resulting white precipitate may be dried for about 1-2 days in order to yield the instant nanoparticles.

[0050] Following preparation of aldehyde-functionalized nanoparticles according to the present disclosure, the nanoparticles may be subjected to a pore-expansion procedure in order to increase the proportion of mesopores in the nanoparticles. Those of ordinary skill in the art are familiar with procedures for pore expansion in order to yield mesopores. For example, the aldehyde-functionalized nanoparticles may be combined with a surfactant in order to form a suspension of the nanoparticles. The emulsion may then be heated, preferably under pressurized conditions. Following heating, the mixture may be filtered and washed in order to remove unreacted surfactant, and then dried in order to yield the pore-expanded nanoparticles. The pore expansion procedure may alternatively be carried out with respect to the instantly described microaggregates.

[0051] The inventive microaggregates comprise a plurality of nanoparticles aggregated together, each of the nanoparticles comprising an inorganic material comprising a plurality of pores of which at least 90% have a diameter of 2 to 50 nm; and, the nanoparticles being surface- functionalized with a plurality of aldehyde groups that are present in an amount of 0.1 to 1.0 mmol/g. The nanoparticles that are aggregated together in order to form the instant

microaggregates may have any of the characteristics described above with respect to the inventive nanoparticles. Accordingly, the preceding description of the inventive nanoparticles is fully applicable to the nanoparticles that are aggregated in order to form the instant

microaggregates.

[0052] For example, at least 90% of the pores of at least some of the nanoparticles of the instant aggregates may have a diameter of 2 to 40 microns, 2 to 30 microns, 2 to 25 microns, 2 to 20 microns, 2 to 10 microns, or 2 to 5 microns. In another example, the nanoparticles of the instant microaggregates may have at least one dimension that is 100-1000 nm, at least one dimension that is 100-500 nm, or at least one dimension that is 200-500 nm. In a further example, the nanoparticles of the instant microaggregates may be surface-functionalized with a plurality of aldehyde groups that are present in an amount of 0.1 to 1.0 mmol/g, 0.1 to 0.8 mmol/g, 0.1 to 0.7 mmol/g, 0.1 to 0.5 mmol/g, 0.1 to 0.4 mmol/g, or 0.1 to 0.3 mmol/g.

Likewise, because the nanoparticles of the instant microaggregates may have any of the characteristics described above with respect to the inventive nanoparticles, the nanoparticles of the instant microaggregates may also be prepared using a one-pot reaction procedure, for example, of the type described above.

[0053] The instant microaggregates may comprise an aggregation of two or more different types of nanoparticles, each of which types of nanoparticles is consistent with any of the characteristics described above with respect to the presently disclosed nanoparticles.

Respective "types" of nanoparticles may differ from one another, for example, in terms of one or more of dimensions, pore sizes, surface area, type of inorganic material, type of moiety loaded into the pores, and amount of aldehyde functional groups. A desired treatment and/or release profile can be optimized by permitting aggregation of different types of nanoparticles into a given microaggregate, and using the resulting microaggregate.

[0054] Although the present disclosure is not meant to be limited to any particular theory of operation, it is believed that the nanoparticles form the instant microaggregates as a result of attractive van der Waals forces among the respective particles. For example, the absence of electronegative hydroxyl groups on the nanoparticle surfaces and their replacement by less electronegative aldehyde groups could reduce the repulsive force between individual particles and facilitate aggregation. Regardless of the precise mechanism, we have found that there is no aggregation of nanoparticles to microparticles without aldehyde functionalization such as we disclosed here.

[0055] The present microaggregates comprise a plurality of aggregated nanoparticles. The number of nanoparticles in the aggregate is preferably sufficient to confer at least one micron-scale dimension on the resulting aggregate. For example, the microaggregates may have at least one dimension that is from 0.5 microns to 15 microns, 0.5 microns to 12 microns, 1 micron to 15 microns, 1 micron to 12 microns, 1 micron to 10 microns, 2 microns to 12 microns, 2 microns to 10 microns, 3 microns to 12 microns, 4 microns to 12 microns, or 4 microns to 10 microns. In other embodiments, the microaggregate has at least one dimension that is about 0.5 microns, about 1 micron, about 2 microns, about 3 microns, about 4 microns, about 5 microns, about 6 microns, about 7 microns, about 8 microns, about 9 microns, about 10 microns, about 11 microns about 12 microns, about 13 microns, about 14 microns, or about 15 microns. The number of nanoparticles in a microaggregate may be about 5, about 7, about 10, about 12, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 80, about 100, about 200, about 300, about 500, about 600, about 800, or about 1000.

[0056] Also provided are methods for loading a microaggregate with a pharmaceutical agent comprising contacting a solution comprising the pharmaceutical agent with the

microaggregate for a duration of time that is sufficient to load the microaggregate with the pharmaceutical agent, the microaggregate comprising a plurality of nanoparticles aggregated together, each of the nanoparticles comprising an inorganic material comprising a plurality of pores of which at least 90% have a diameter of 2 to 50 nm; and, the nanoparticles being surface- functionalized with a plurality of aldehyde groups that are present in an amount of 0.1 to 1.0 mmol/g, wherein less than about 30 minutes of the contacting are required in order to load at least some of the pores of the nanoparticles with the pharmaceutical agent.

[0057] As noted supra, the present inventors have surprisingly discovered that using a low concentration of aldehyde surface functionalization on mesoporous nanoparticles enables quick absorption of molecules into the pores of such particles. As compared with previous materials, which required exposure to a drug agent for an extended period of time in order to absorb the agent, the instantly described nanoparticles (which form the inventive

microaggregates) are capable of absorbing moieties into their pores after only about one hour or less of exposure to the moieties. For example, less than about 1 hour, less than about 45 minutes, less than about 30 minutes, less than about 25 minutes, less than about 20 minutes, less than about 15 minutes, or less than about 10 minutes of the contacting may be required in order to load at least some of the pores of the instant nanoparticles with a desired moiety. The described time periods are sufficient to load the instant nanoparticles with a pharmaceutically relevant quantity of the desired pharmaceutical moiety.

[0058] The pharmaceutical agent may be any therapeutic or otherwise pharmaceutically active moiety that meets the size requirements previously described in connection with the inventive nanoparticles. Thus, the previously provided listing of classes of pharmaceutical agents and specific examples of pharmaceutical agents is applicable with respect to the instant methods. The instant nanoparticles and microaggregates can be loaded with one pharmaceutical agent, or with two or more pharmaceutical agents. In other words, a single nanoparticle or a single microaggregate may be loaded with more than one pharmaceutical agent, as desired.

[0059] The pharmaceutical agent is in a solution, mixture, suspension, slurry, or other like form when it is contacted with the microaggregate. When the microaggregate is to be loaded with more than one pharmaceutical agent, the solution may contain the desired two or more pharmaceutical agents. The solution may comprise the pharmaceutical agent or agents and an appropriate solvent, carrier, excipient, or diluent. The concentration of the pharmaceutical agent or agents may be optimized in accordance with the particular requirements for loading a therapeutically effective amount of pharmaceutical agent within the microaggregate, and can readily be determined by those of ordinary skill in the art.

[0060] The step of contacting the solution with the microaggregate may be a simple matter of mixing the microaggregate into the solution comprising the pharmaceutical agent. In certain instances, the microaggregate may be suspended in a carrier substance, such as a liquid, such that the resulting suspension can be contacted with the solution comprising the

pharmaceutical agent. As described above, because of the unexpectedly beneficial properties of the instant nanoparticles and microaggregates, the duration of the step of contacting the microaggregate with the solution containing the pharmaceutical agent may be considerably shorter than that which was required to load previous materials with a drug moiety. As a result, medical practitioners can prepare the drug-loaded microaggregates essentially

contemporaneously with the time of treatment, such as during a surgical process.

[0061] Following the step of contacting the solution comprising the pharmaceutical agent with the microaggregate, the mixture may be at least partially dried. For example, the mixture may be air-dried. The duration of the drying period may be, for example, about 5 minutes, about 7 minutes, about 10 minutes, about 12 minutes, about 15 minutes, about 20 minutes, about 25 minutes, or about 30 minutes. In other instances, it will not be necessary to dry the mixture at all, and the mixture can be used immediately after the contacting step.

[0062] The present disclosure also relates to methods for administering a

pharmaceutical agent to a subject comprising contacting the subject with a microaggregate, the microaggregate comprising a plurality of nanoparticles aggregated together, each of the nanoparticles comprising an inorganic material comprising a plurality of pores of which at least 90% have a diameter of 2 to 50 nm, at least some of the pores of the nanoparticle being loaded with the pharmaceutical agent; and, the nanoparticles being surface-functionalized with a plurality of aldehyde groups that are present in an amount of 0.1 to 1.0 mmol/g. Pursuant to the present methods, the respective characteristics of the microaggregate and the nanoparticles may be in accordance with any aspect of the preceding disclosure pertaining to the inventive microaggregages and nanoparticles, respectively.

[0063] The step of contacting the subject with the microaggregate refers to creating physical contact between the subject and the microaggregate so that the pharmaceutical agent may, over time, release from the pores of the nanoparticles and effect the desired treatment. A site of treatment or a subject may be contacted with the microaggregate in any manner that is effective to yield this result. For example, the microaggregate may be injected into the subject. In another embodiment, when the microaggregate is part of a population of like microaggregates, the population including the microaggregate may be injected, poured, spread, or sprinkled on to the site of interest on or in the subject. The microaggregate may be incorporated into or attached to a medical device or other item that is in turn contacted with the subject. Examples include a bandage, a cast, an implant, or a prosthesis. Thus, the present application also pertains to an article comprising a nanoparticle that comprises an inorganic material comprising a plurality of pores of which at least 90% have a diameter of 2 to 50 nm, and, the nanoparticle being surface- functionalized with a plurality of aldehyde groups that are present in an amount of 0.1 to 1.0 mmol/g, or a microaggregate that comprises an aggregation of a plurality of such nanoparticles. As suggested above, the article may be, for example, a bandage, a cast, an implant, or a prosthesis.

[0064] Because the instant nanoparticles and microaggregates are biocompatible, and in most instances can be cleared by the subject's own biological processes, there is no need to remove them from the subject following the step of contacting the subject with the

microaggregate, or following the useful lifetime of the microaggregate (i.e., after the period of release of the pharmaceutical agent from the microaggrgegate has terminated).

Examples

[0065] The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the nanoparticles and microaggregates claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for.

Example 1 - Preparation of Nanoparticles and Microaggregates [0066] Tetra-ethylorthosilicate (TEOS), octadecyltrimethylammoniumbromide (CigTAB), sodium hydroxide pellets, poly(ethyleneglycol)-bis-amine (PEG-amine) (molecular weight 3 kDa), dimethylhexadecylamine (DMH) and trimethyloctadecylammonium bromide, were obtained from Sigma Aldrich (St. Louis, MO), triethoxysilylbutyraldehyde was purchased from Gelest Inc (Philadelphia, PA) and used without further purification. Rifampin and vancomycin were separately obtained.

[0067] Aldehyde functionalized mesoporous silica microparticles (MSMPs) were synthesized as follows: 1.00 g of CigTAB was dissolved in 240 ml of distilled water at 80°C. The solution was made basic (pH -12) with the addition of 3.5 ml of 2.00 M sodium hydroxide. After the addition of the NaOH, 5 ml of TEOS was added. Also, 1 ml of

triethoxysilylbutyraldehyde was added to aldehyde-functionalize the surface of the MSMps. The reaction temperature was maintained at 80 °C for 2 h, resulting in a white precipitate. The mixture was filtered and washed several times with deionized water until the supernatant reached neutral pH (~7). The samples were then dried in an oven at 80°C for 2 days. The aldehyde functionalized mesoporous silica nanoparticles (MSNs) were synthesized using the same procedure, except that 480 ml distilled water was used to dissolve the CigTAB.

[0068] Pore expansion was carried out by adding 1.6 g of dried aldehyde functionalized MSMPs or MSNs to an aqueous emulsion of 1.0 g of DMH in 60 ml of water and stirring for 20 min. The emulsion was transferred to a Teflon®-coated pressure vessel and kept in an oven at 110°C for 3 days. Then, the mixture was filtered and washed several times with deionized water to remove unreacted DMH. The samples were dried at 80°C for 1 day.

[0069] The surfactants (CigTAB and DMH) were subsequently removed as follows. 0.8 g of pore-expanded MSNs or MSMPs were refluxed in 100 ml of methanol containing 1 ml of concentrated hydrochloric acid for 24 hr. Following refluxing, the sample was washed several times with ethanol, methanol, and water. The cleaned sample was then dried in an oven at 80°C for 2 days.

[0070] FIG. 1 shows a scanning electron micrograph of the MSMPs. The average sizes of MSMPs were in the range of 4-10 μιτι, and were formed through the aggregation of MSNs. Example 2 - Loading of MSMPs with Pharmaceutical Moieties

[0071] Dual loading of 20 mg vancomycin and 20 mg rifampin was carried out by dissolving the vancomycin and rifampin in separate 125 μΐ aliquots of distilled water, and mixing the solution with 100 mg of MSMPs in a glass crucible with the help of a spatula, followed by air drying for 20 min before examining the release characteristics of the loaded MSMPs. For loading of single drug, the same amount of drug was dissolved in 250 μΐ of distilled water and mixed with 100 mg of MSMPs using the same procedure as described above for dual loading. The loading period prior to the air drying step had a duration of less than 30 minutes.

Example 3 - Drug Release Study

[0072] Release characteristics of drug-loaded MSMPs that were prepared according to Examples 1 and 2 were measured in triplicate in phosphate buffer solution at pH 7.4. Each of three 20 mg samples of drug loaded MSMPs were placed in respective 15 ml centrifuge tubes containing 5 ml of phosphate buffer solution (PBS) at pH 7.4. The samples were then incubated at 37°C on a platform shaker. On the first day following incubation, samples were centrifuged and supernatants were collected at intervals of 1, 3, and 6 h. Following the first day, supernatants were collected at intervals of 24 h. After each interval, the PBS was replaced by fresh buffer. The procedure was employed until the release of both rifampicin and vancomycin was zero. Released rifampicin and vancomycin concentrations were determined by optical absorption at 337 nm and 281 nm respectively.

[0073] The individual mean cumulative vancomycin and rifampin release from MSMPs as a function of immersion time in PBS is shown in FIGS. 2A and 2B, respectively. It follows from the figures that the release of vancomycin and rifampin was time dependent. In both instances, drugs were released for about 3 weeks in controlled fashion.

[0074] The initial vancomycin and rifampin release concentrations were 54.4 g/mL and 65.6 μg/mL. These amounts of vancomycin or rifampin released at day one were well above the minimum inhibitory concentration (MIC) values for MSSA and MRSA, and therefore suffice to prevent these microbes. In this context, it should be noted that MIC values are given at time zero, while thin sol-gel films continue to deliver drugs in a controlled manner over longer periods of time. The in vitro study demonstrated a long term, time dependent release of vancomycin or rifampin from MSMPs.

[0075] FIG. 3 shows the simultaneous release of vancomycin and rifampin from the instant MSMPs. It shows that vancomycin and rifampin can be released simultaneously, in time dependent and controlled fashion. Initial release of both vancomycin (56.7 μg/mL) and rifampin (38.8 μg/mL) at day 1 exceeds the MIC level for individual drugs for both MSSA and MRSA. It was noted that the amount of release for both of the drugs could be increased by increasing the MSMPs dose.

Discussion [0076] Controlled release of vancomycin or rifampin for 3 weeks without any burst release was observed. The release rate of rifampin was slightly higher than that of vancomycin, likely due to their molecular size differences: vancomycin (1.5 kDa) is almost twice of the size of rifampin (0.82 kDa), and each released slowly when they are absorbed on MSMPs having same pore size.

[0077] It is believed that during the absorption of vancomycin or rifampin on the aldehyde functionalized MSMPs, the electrophilic carbon atoms of aldehyde groups were targets of nucleophilic attack by amino groups present in the drugs, and imine bonds were rapidly formed as a result, as shown in Schematic 1 :

O R

_D.^C. + RNH₂ + H₂0

R R R ^

Schiff base (i rae)

Schematic 1

These imine bonds slowly degrade back to aldehyde functionalized microparticles and the respective drug molecules by hydrolysis in the presence of excess water in the tested release solution. Mechanistically, this is simply the reverse of imine formation, as shown in Schematic 2:

Schematic 2

[0078] As noted previously, the drug-loaded MSMPs provided release of an amount of drug that was greater than the respective MICs (2 μ^ητΐ for vancomycin and 0.5 μ^ητΐ for rifampin against MRSA) for almost 3 weeks. The amount of drug release can be increased by increasing the dose of antibiotic loaded MSMPs.

[0079] Antibiotic combination therapy, as compared with monotherapy, can provide a broader spectrum of antibacterial effect, possible synergistic effects, and reduced risk for the emergence of resistance during therapy. The efficacy of combination therapy can be enhanced using a system that can deliver combinations of antibiotics locally and in controlled fashion. Such systems can minimize the adverse effects of combination therapies at are presently administered via intravenous injection or oral delivery.

Example 4 - In-vitro bactericidal study

[0080] Bactericidal studies were performed with methicillin-resistant

Staphylococcus aureus (MRSA) subspecies Aureus Rosenback (ATTC strain 33591).

Methicillin-resistant Staphylococcus aureus subspecies were purchased from ATTC (Manassas, VA). All experiments were run with at least three samples per data point, and measurements were made in triplicate.

[0081] Using the working bacterial plate, one well-formed colony was collected using a sterile inoculating loop and aseptically transferred into a test tube containing 5 mL sterile TSB. This bacterial solution was kept on a shaker in a 37°C incubator for 18 hours. This solution was then diluted in 5 mL TSB to 10⁸ CFU/mL. 10⁸ CFU/mL was confirmed using a

spectrophotometer (Ultrospec Plus Spectrophotometer; Pharmacia LKB, Piscataway, NJ). The absorbance reading for a 0.5 McFarland standard (BD, Franklin Lakes, NJ) at 625nm was 0.08. A 0.5 McFarland standard corresponds to the same turbidity achieved of a bacterial solution of 10^s CFU/mL; therefore, the 10^s CFU/mL solution of S. aureus was created to match an absorbance of 0.08 ± 10%.

[0082] The 10⁸ CFU/mL bacterial solution was serially diluted in 4.5 mL TSB to 10⁴ CFU/mL and this inoculum was used to inoculate the MSMPs.

[0083] For the bacterial inoculation, three MSMPs samples were used: MSMPs without any drug loaded, MSMPs loaded with 10% vancomycin, and MSMPs loaded with 10% vancomycin and 10% rifampin. For this study, 15 mg of each sample was used, and each test was performed in triplicate.

[0084] Fifteen mg of each sample was placed in sterile 10 mL test tubes. Five mL of 10⁴ CFU/mL MRSA bacterial solution was added to each tube. The tubes were sealed and maintained in a 37°C incubator for 18-24 hours.

[0085] After the incubation, each sample was vortexed, and 2 mL of the solution was pipetted out and centrifuged @ 13000 rpm to precipitate the remaining bacteria in the solution. The bacterial pellet was separated from culture medium TSB and suspended in 1 mL PBS.

These solutions were then serially diluted by a factor 10 to arrive at dilutions ranging from 10 to 10⁸ in 1 mL PBS. These diluted solutions were plated on individual petrifilms (3M, St. Paul, MN), which were placed in an incubator at 37°C for 24 hours. They were then removed and the bacterial colonies per samples were determined.

[0086] In order to determine whether these samples can provide the bactericidal activity up to 4 days, the samples were immersed in PBS for 1, 2, or 3 days, and recovered by centrifugation and subjected to bactericidal experiment.

Bactericidal Effect

[0087] MSMPs loaded with 10% vancomycin, or 10% vancomycin + 10% rifampin, and MSMPs without any drugs were challenged with 10⁴ CFU/ml of MRS A. FIG. 4A shows the bactericidal activity on day 1. For the control sample MSMPs (without any drug), bacteria increased from 10⁴ CFU/ml to 10⁵ CFU/mL, whereas MSMPs with vancomycin alone were able to reduce the bacteria counts by 2.5 order of magnitude. MSMPs with dual antibiotics vancomycin and rifampin were able to completely kill the MRSA colonies.

[0088] FIG. 4B shows the bactericidal effect of MSMPs, MSMPs with vancomycin and MSMPs with both vancomycin and rifampin after releasing drugs for 2, 3, and 4 days. The samples were challenged each day with fresh MRSA solution having concentration of 10⁴ CFU/mL. It can be observed from these results that MSMPs with vancomycin alone and with vancomycin and rifampin retained their antimicrobial activity even at day 4. For MSMPs with vancomycin, there were two orders of magnitude reductions in bacteria growth, and with respect to MSMPs with dual antibiotics a complete inhibition of MRSA was observed even at day 4. These results demonstrate a synergistic effect of releasing vancomycin and rifampin in a controlled fashion from the instant MSMPs.

Claims

What is Claimed:

1. A nanoparticle comprising:

an inorganic material comprising a plurality of pores of which at least 90% have a diameter of 2 to 50 nm; and,

the nanoparticle being surface-functionalized with a plurality of aldehyde groups that are present in an amount of 0.1 to 1.0 mmol/g.

2. The nanoparticle according to claim 1 wherein at least 90% of said pores have a diameter of 2 to 10 nm.

3. The nanoparticle according to claim 1 wherein at least 90% of said pores have a diameter of 2 to 5 nm.

4. The nanoparticle according to claim 1 wherein the nanoparticle has at least one dimension of 100 to 500 nm.

5. The nanoparticle according to claim 1 wherein said aldehyde groups are present in an amount of 0.1 to 0.8 mmol/g.

6. The nanoparticle according to claim 1 wherein said aldehyde groups are present in an amount of 0.1 to 0.5 mmol/g.

7. The nanoparticle according to claim 1, wherein said nanoparticle is surface- functionalized with said aldehyde groups using a one-pot reaction procedure.

8. The nanoparticle according to claim 1 wherein said inorganic material is silica.

9. A microaggregate comprising a plurality of nanoparticles aggregated together,

each of said nanoparticles comprising

an inorganic material comprising a plurality of pores of which at least 90% have a diameter of 2 to 50 nm;

and, the nanoparticles being surface-functionalized with a plurality of aldehyde groups that are present in an amount of 0.1 to 1.0 mmol/g.

10. The microaggregate according to claim 8 having at least one dimension of 0.5 to 12 μηι.

11. The microaggregate according to claim 8 having at least one dimension of 2 to 10 μηι.

12. The microaggregate according to claim 9 wherein at least 90% of said pores of at least some of said nanoparticles have a diameter of 2 to 10 nm.

13. The microaggregate according to claim 9 wherein at least 90% of said pores of at least some of said nanoparticles have a diameter of 2 to 5 nm.

14. The microaggregate according to claim 9 wherein at least some of the nanoparticles have at least one dimension that is 100 to 500 nm.

15. The microaggregate according to claim 9 wherein said aldehyde groups on said nanoparticles are present in an average amount of 0.1 to 0.8 mmol/g.

16. The microaggregate according to claim 9 wherein said aldehyde groups on said nanoparticles are present in an average amount of 0.1 to 0.5 mmol/g.

17. The microaggregate according to claim 9 wherein said nanoparticles are surface- functionalized with said aldehyde groups using a one-pot reaction procedure.

18. The microaggregate according to claim 9 wherein said inorganic material is silica.

19. A method for making an aldehyde surface-functionalized mesoporous nanoparticle comprising synthesizing said nanoparticle using a one-pot reaction procedure, wherein said procedure results in the nanoparticle being surface-functionalized with a plurality of aldehyde groups that are present in an amount of 0.1 to 1.0 mmol/g.

The method according to claim 19 comprising the steps of forming a solution of octadecyltrimethylammonium bromide in a solvent; increasing the pH of the solution;

adding a source of silica to the solution;

adding a source of aldehyde functional groups to the solution;

heating said solution for a time that is sufficient to form a precipitate;

drying said precipitate to obtain said nanoparticle.

21. The method according to claim 20 wherein said source of silica is tetraethyl orthosilicate.

22. The method according to claim 20 wherein said source of aldehyde functional groups is triethoxysilylbutyraldehyde.

23. A method for loading a microaggregate with a pharmaceutical agent comprising:

contacting a solution comprising the pharmaceutical agent with the microaggregate for a duration of time that is sufficient to load said microaggregate with said pharmaceutical agent, the microaggregate comprising a plurality of nanoparticles aggregated together, each of said nanoparticles comprising

an inorganic material comprising a plurality of pores of which at least 90% have a diameter of 2 to 50 nm;

and,

the nanoparticles being surface-functionalized with a plurality of aldehyde groups that are present in an amount of 0.1 to 1.0 mmol/g; wherein less than about 30 minutes of said contacting are required in order to load at least some of the pores of said nanoparticles with the pharmaceutical agent.

24. The method according to claim 23 wherein the solution comprising the pharmaceutical agent is contacted with the microaggregate by mixing the solution with a suspension containing the microaggregate.

25. A method for administering a pharmaceutical agent to a subject comprising contacting the subject with a microaggregate,

the microaggregate comprising a plurality of nanoparticles aggregated together, each of said nanoparticles comprising an inorganic material comprising a plurality of pores of which at least 90% have a diameter of 2 to 50 nm, at least some of the pores of the nanoparticle being loaded with the pharmaceutical agent; and,

the nanoparticles being surface-functionalized with a plurality of aldehyde groups that are present in an amount of 0.1 to 1.0 mmol/g.