WO2009094580A2

WO2009094580A2 - Nano-devices having releasable seals for controlled release of molecules

Info

Publication number: WO2009094580A2
Application number: PCT/US2009/031891
Authority: WO
Inventors: Jeffrey I. Zink; Jie Lu; Fuyuhiko Tamanoi; Andre E. Nel; Fraser Stoddart; Kaushik Patel; William Dichtel; Sarah Angelos
Original assignee: The Regents Of The University Of California
Priority date: 2008-01-23
Filing date: 2009-01-23
Publication date: 2009-07-30
Also published as: WO2009094580A3

Abstract

A nanodevice has a containment vessel defining a storage chamber therein and defining at least one port to provide access to and from said storage chamber, and a stopper assembly attached to the containment vessel. The stopper assembly has a blocking unit arranged proximate the at least one port and has a structure suitable to substantially prevent material after being loaded into the storage chamber from being released while the blocking unit is arranged in a blocking configuration. The stopper assembly is responsive to the presence of a predetermined stimulus such that the blocking unit is released in the presence of the predetermined stimulus to allow the material to be released from the storage chamber. The predetermined stimulus is a predetermined catalytic activity that is suitable to at least one of cleave, hydrolyze, oxidize, or reduce a portion of the stopper assembly, and the nanodevice has a maximum dimension of about 1 µm.

Description

NANO-BEVICES HAVING RELEASABLE SEALS FOR CONTROLLED RELEASE OF

MOLECULES

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 61/006,599 filed January 23, 2008, the entire contents of which are hereby incorporated by reference,

[0002] The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require that the patent owner license others on reasonable terms as provided for by the terms of Grant Nos. CHE 0507929 and DMR 0346601 , awarded by the National Science Foundation, and of Grant No. 32737, awarded by NIH.

BACKGROUND

1. Field of Invention

[0003] The current invention relates to nano-devices, and more specifically to nano- nano-devices that have releasable seals for controlled release of molecules contained therein.

2. Discussion of Related Art

[0004] Control of molecular transport in, through, and out of mesopores has important potential applications in nanoscience including fluidies and drug delivery. Surfaciant-tempiated silica (Kresge, C. T.; Leonowicz., M. E,; Roth, W₁ J,; Vartuli, J. C; Beck, J. S. Nature 1992, 359, 710-712) is a versatile material in which ordered arrays of mesopores can be easily synthesized, providing a convenient platform for attaching molecules that undergo large amplitude motions to control transport. Mesostructiired silica is transparent (for photoeontrol and spectroscopic monitoring), and can be fabricated into useful morphologies (thin films (Lu, Y , F.; GanguJi, R.; Drewiert, C, A,; Anderson, M, T.; Brinker, C. J.; Gong, W, L.: Guo, Y. X,; Soyez, H.; Dunn, B.; Huang. M, H.; Zink, J. I. Nature 1997, 389, 364-368), particles (Kresge, C. ^'T.; Leonowicz, M, E.; Roth, W. J.; Vartuli. J, C; Beck, J. S, Nature 1992, 359, 710-712; Huh, S.; Wiench, J. W.; Yoo. J. C: Pruski, M.: Lin, V. S. Y. Chem. Mater. 2003, 15, 4247-425o)) with designed pure sizes and structures,

[0005] Ylesoporous silica nanoparticles coated with molecular valves hold the promise to encapsulate a pay load of therapeutic compounds, to transport them to specific locations in the body, and to release them m response to either external or cellular stimuli. Sequestering drug molecules serves the dual purpose of protecting the payload from enzymatic degradation, while reducing the undesired side-effects associated with many drugs, Although these benefits are common to pro-drug strategies {(a) Hirano, 1 .; Klesse, W.: Ringsdorf, II. Makromol. Chem. 1979, ISO, I 125, (b) Kataoka, K.; Harαda, A.; Nagasaki, Y. Λάv Drug Delivery Rev 2001, 47, 1 13. Cc) Padilia De Jesus, O. L.; Ihre H. R.; Gagne, L.; Frcchet, J, M. J.; Szoka, F. C. Jr. Bwcύnjug Chem. 2002, 13, 453. (d) Denny, W. A. Cancer Invest. 2004, 22, 604. (e) Lee, C. C; MacKay, J. A,; Frechet, J. M. J., et ai. Nat Biotechnol. 2005, 23, 1517. (f) Duncan, R.; Ringsdorf, H.; Satchi-Fainaro, R. / Drug larger. 2006, 14, 337, (g) ϊ ietze, L. F.; Major, F.; Schuherth, I. Augew Chem. Int. Ed. 2006, 45, 6574), the nanoparticle-supported nanovalve system does not require covalent modification of the therapeutic compounds and allows for the release of many drug molecules upon each stimulus event ((a) Duncan, R.; Vicent, M. J,; Greco, F., et al. Endocr-Relat. Cancer 2005, 12, S 189. (b) Pantos, A.; Tsiourvas, D ; Nounesis, G.; Paleos, C, M. Lmgnmir 2005, 21, 7483, (c) Dhanikula, R. S.; Hildgen, P. Bioconjug Chem. 2006, 17. 29, (ά) Darbre. T., Reymond, J.-l.. Ace Chem Res 2006, 39, 925. (e) Gopin, A.; Ebner, S ; Λttaii, B.; Shαbat, 1). Bioi nnjug Chem. 2006, i ⁷, 1432). Recently, it was demonstrated that πicsoporous silica nanopariicles, not modified with molecular machinery, can deliver the water-insoluble drug camptothecin into human pancreatic cancer cells with very high efficiency (Lu, J. Liong, M.; /ink, J.I ; famanoi, F₁ Small 2007, 3, 134] ), For more sophisticated drug delivery applications, the ability to functionalize ((a) Hernandez, R.; I\eng, R-R₁; Wong, J. W.; Stoddart, J. F.; Zink, J. I, J. Am Chem. Soc, 2004, /26, 3370. (b> Nguyen, 1 . D.; Tseng, H-R,; Ceiesire. F. C; Flood, A. H.; Liu, Y,; Stoddart, J. F.; Zink, J. I. Proc Nail Acad Sa U SA. 2005, 102. 10029. ^c) Nguyen, T. D.; ϊ eung. k. C-P.; Liong, M.; Pentecost, C. D.; Stoddart. J. F.; /ink, J. I. (Vg. Lett. 2006, δ, 3363. (d) Leung, K, C -F.; Nguyen, T. D.; Stoddart, J. F.; /ink. J. I. Chem Mater 2006. IS. 5919, (e) Nguyen, T. D.; Liu, Y ; Saha. S., Leung, K, C-F.; Stoddart, J. P ; Zink, J ) J Am Chem. Soc 2007, 129, 62o. (f) Nguyen, T. D., [ eung, K, C. F.; Liong, M., Liu, Y.: Stoddar!. j. F ; Zink, 3 I AJv b und Mater 2007, i ⁷, 2101. Cg) Saha, S.: Leung, K. C F.; Ngisven, f D., Moddart, J. F.: Zink. J. l. Adv Fund Maicr 2007, 17, 685. (h) Angelos, S.; Johansson, E.: Stoddaπ, J. F.: Zink, J. I. Adv. Fund Mater. 2007, ASAP article) nanoparticles with nanovalves and other controlled-release mechanisms has become an area of widespread interest ((a) MaI, N. K.., Fujiwara, M.; Tanaka, Y.; Nature 2003, 421, 350. (b) Giri, S.; Trewyn, B. G.; Steelmaker, M. P.; Lin, V. S. Y. Angen Chem Im Fd. 2005, 44, 5038. (c) Kocer, A,; Walko. M., Meijberg, W.; Feringa, B. I . Sc ience 2005, 309, 755.

(d) Angelos, S.; Choi, E.; Vogtle, F.; De Cola, I .; Zink. J. I. J Phys. Chem C 2007, 111, 6589.

(e) Slowing, I.; Trewyn, B. G.; Giri, S.; I in, V. !>. Y. Adv. Fund Mater. 2007, 17, 1225). Previously, we have demonstrated the operation of molecular and supramυlecular valves in non- biologically relevant contexts using redox (I kmande?, R.; Tseng, H, -R.; Wong, J. W.; Stoddart, J. F.; Zink. J. I. J. Am Chem. Soc. 2004, 126, 3370. Nguyen, T 1).; Tseng, H.-R ; Cclestre, P. C; Flood, A. H.; Liu, Y.; Stoddart, J. F.; Zink, J. I. Proc. Natl Acad. Sci. USA 2005, 102, 10029. Nguyen, T. D.; Liu, Y.; Saha. S.; Leung, k. C-F.; Stoddart, J. F.; Zink, J. I. J. Am. Chem Soc. 2007, J 29. 626.), pH (Nguyen, T. D.; Leung, K. C-F.: 1 iong, M; Pentecost, C. D.; Stoddart, J. F.; Zink, L I. Org Lett. 2006, S, 3363,), competitive binding (Leung, K. C-F.: Nguyen, T. D.; Stoddart, J. F.: Zink. J. 3. Chem Mater 2006, /S, 5919.), and light (Nguyen, T. D.; Leung, K. C. F,; Liong, M.; Liu, Y,; Stoddart, J. F.: /ink, j. I. Adv. Fund. Mater. 2007, 17, 2101.) as actuators, Other controlled release systems include photoresponsive a/oben/ene-bascd nanoimpellers (Angelos, S,; Choi, E.: Vogtle, F.; De Cola, L.; Zink, J. I. J. Phys. Chem C 2007, 111, 6589.), chemically removable CdS nanoparticle caps (Giri, S.; Trewyn, B. G.; Steϊlmaker, M. P.; Lin, V. S. Y. Angew. Chem. Int. Ed. 2005, 44, 5038. Slowing, L; Trewyn, B. G.; Giri, S.; Lin, V, S. Y, Adv. Fund. Mater. 2007, 17, 1225.), and reversible photo-dimerization of tethered coumarins (MaI, N. k.; Fujiwara, M.: Tanaka, Y,; Nature 20©3, 421, 350,).

[0006] Although there has been substantial research activity in this field, there still remains a need for suitable nano-devices that can selectively release molecules from a containment vessel and that can also keep the molecules substantially contained within the containment vessel when not being selectively released There further remains a need for such nano-deviees that can be useful for bioiogical and biomedical applications. SUMMARY

[0007] A rsanodevicc according to some embodiments of the current invention has a containment vessel defining a storage chamber therein and defining at least one port to provide access to and from said storage chamber, and a stopper assembly attached to the containment vessel. The stopper assembly has a blocking unit arranged proximate the at least one port and has a structure suitable to substantially prevent material after being loaded into the storage chamber from being released while the blocking unit is arranged in a blocking configuration. The stopper assembly is responsive to the presence of a predetermined stimulus such that the blocking unit is released in the presence of the predetermined stimulus to allow the material to be released from the storage chamber. The predetermined stimulus is a predetermined catalytic activity that is suitable to at least one of cleave, hydrolyze, oxidize, or reduce a portion of the stopper assembly, and the nanodevice has a maximum dimension of about 1 μm.

[0008] A composition of matter according to some embodiments of the current invention has a plurality of nanopartieles, each defining a storage chamber therein; and a guest material contained within the storage chambers defined by the plurality of nanoparticies. The guest material is substantially chemically non-reactive with the nanoparticles The plurality of nanoparticles are operable to cause the guest material contained within the storage chambers to be released in a presence υf a predetermined stimulus, and each nanoparticle of the plurality of nanoparticiεs has a maximum dimension of about 1 μm.

[0009] Λ method of administering at least one υf a biologically active substance or a diagnostic substance according to some embodiments of the current invention includes administering a composition to at least one of a person, animal, or organism, the composition comprising nanoparticles therein, wherein the nanoparticles contain the at least one of a biologically active substance or ars imaging/tracking substance therein; and at least one of directing or allowing the nanoparticles of the administered composition to come into contact with a predetermined catalytic activity that is suitable to at least one of cleave, hydrolyze, ovidt/e, or reduce a portion of the nanoparticles to release the biologically active substance or the imaging/tracking substance from the nanoparticles. [0010] Further objectives and advantages will become apparent from a consideration of the description, drawings, and examples.

[0011] Figure IA is a .schematic illustration of a nano-device according to an embodiment of the current invention.

[0012] Figure IB is a schematic illustration of a nano-device, and methods of production, that can serve as a precursor according to .some embodiments of the current invention.

[0013] Figure 2 is schematic illustration to help explain additional embodiments of the current invention.

JΘ014J Figure 3 is a schematic illustration of two embodiments of the current invention that have different stoppers.

[0015] Figure 4 shows, emission intensity plot

514 nm) of HHFhS buffer solutions

(50 m\L pl l - 7.5) containing ester (green) or amide (blue) stoppered snap-tops corresponding to Figure 3. The response of the ester system to the deactivated enzyme (red) is also shown.

[0016] Figure 5 illustrates an example of a mechanism for chemically attaching stoppers to nanodevices according to some embodiments of the current invention.

[0017] Figure 6 summarizes some examples of stoppers according to some embodiments of the current invention.

DF I AILED DESCRIPTION

[0018] Some embodiments of the current invention are discussed in detail below Sn describing embodiments, specific terminology is employed for the sake of clarity. However, the invention rs not intended to be limited to the specific terminology so selected, A person skilled in the relevant art vv ii! recognize that other equivalent components can be employed and other methods developed without departing from the broad concepts of the current invention. All references cited herein are incorporated by reference as if each had been individually incorporated,

Figure I A is a schematic illustration of a nanodevice 100 according to an embodiment of the current invention. The nanodevice 100 has a containment vessel 102 defining a storage chamber 104 therein and defining at ieavt one port 106 to provide access for the transfer of material 108 into and/or out of the storage chamber 104, The containment vessel 102 can be a mcsoporous silica nanoparticle in some embodiments of the current invention. The material 108 can be molecules which are sometimes also referred to as guest molecules herein However, the material 108 does not always have to be in the form of molecules in some embodiments of the current invention, The material 108 is also referred to as cargo herein since it can be loaded into the nanodevice 100. As is indicated in Figure IA, the nanodevice can be referred to as a Snap-l op Covered Silica Nanocomainεr (SCSN) in some embodiments of the current invention. The nanodevice 100 also has a stopper assembly 1 10 attached to said containment vessel 102, The stopper assembly 1 10 has a blocking unit 1 12 arranged proximate the at least one port 106 and has a structure suitable to substantially prevent materia! 108 after being loaded into said storage chamber 104 from being released while the blocking unit 1 12 is arranged in a blocking configuration. The stopper assembly 1 10 is responsive to the presence of a predetermined stimulus such that the blocking unit 1 12 is released in the presence of the predetermined stimulus to allow the material 108 to be released from the storage chamber 106. rhe predetermined stimulus can be a predetermined catalytic activity, for example, that is suitable to at least one of cleave, hydrolyze, oxidize, or reduce a portion of the stopper assembly 1 10.

[0020] The nanodevice 100 has a maximum dimension of less than about 1 μm and greater than about 50 πm in some embodiments, bar some embodiments, the nanodevice 100 has a maximum dimension of less than about 400 nm and greater than about 50 nm. When the nanodevice 100 is greater than about 400 nm, it becomes too large to enter into biological cells. On the other hand, when the nanodevice J 00 is less than about 50 nm, it becomes less able Io contain a useful number of molecules therein. Furthermore, when the nanodevioes are less than about 300 nm, they become more useful in some applications to biological systems. For some embodiments of the current invention, nanodevsces having a maximum dimension in the iarsee of about 50 nm to about 150 nm are suitable. T he containment vessel can be, but is not limited to, a rnesoporous silica nanoparticje according to some embodiments of the current invention.

[0021] In some embodiments of the current invention, the stopper assembly 1 10 can include a thread 1 14 onto which the blocking unit 1 12 can bε threaded as is illustrated schematically in Figure I A The thread 1 14 has a longitudinal length that is long relative to a transverse length and ts suitable to be attached at one longitudinal end to the containment vessel 102. The stopper assembly 1 10 can also have a stopper 1 16 attached to a .second longitudinal end of the thread 1 14 in some embodiments of the current invention. According to some aspects of the current invention, the stopper 1 16 can be selected among a wide range of possible stoppers bάsaά on the type of environment for which the material 108 will be released.

[0022] In operation, the blocking unit 1 12 of the stopper assembly 1 10 is held in place at the port 106 by the thread 1 14 and the stopper 1 16 according to some embodiments of the current invention. The stopper 1 16 is selected to respond to a stimulus so that it allows the blocking unit 1 12 to move away from the port 106. The stimulus can be an environmental condition such as a local chemical environment or can be an applied condition such as illumination with light, etc. The stopper 1 16 can be cleaved, for example, from the thread 1 14 by an environmental condition according to some embodiments of the current invention, Once the blocking unit 1 12 is released to move away from the port 106, the material 108 can then escape from the storage chamber 104.

[0023] According to some embodiments of the current invention, a synthetic strategy can involve the use of a snap-top "precursor'^*. The nanodevice 100 with the stopper 1 16 can serve as a precursor according to some embodiments of the current invention. The assembly of the snap-top precursors can be performed step-wise from the silica nanopartiele surfaces outward according to an embodiment of the current invention, a? illustrated in Figure I B. However, the general concepts of the current invention are not limited to only the materials used in the example of F igure I B. First, the silica nanoparticles are treated with dminupropyltrietboxysiiane iy\PTES) to achieve an amine-modifled nanoparticle surface. An a/ϊdεterminated tπ(eth> lεnε)giycol thread is attached to the armne-nioditϊed nanoparticles, and the pores are then loaded by soaking in a concentrated cargo solution and allowing the cargo to diffuse into the empty pores I he precursor is completed through the addition of α-cyclodextπn as the blocking unit at 5⁰C, which complexes vvuh the threads at the low temperature 1 he precursor can enable the preparation of many different systems based on a common general structure in which different stoppers can be attached depending on the specific desired application according to some embodiments of the current invention.

[0024] The material or molecules of interest to be stored in and released from the containment vessels 102 can include, but are not limited to, biologically active substances, The term "biologically active substance^"' as used herein is intended to include all compositions of matter that can cause a desired effect on biological material oi a biological system and may include in situ and in vivo biological materials and systems. The biologically active substance may be selected from such substances that have molecular sizes such that they can be loaded into the nanodevices, and can also be selected from such substances that don^'t react with the nanodevices. Λ biological system may include a person, animal υr plant, fur example,

[0025] Biologically active substances may include, but are not limited to, the following:

(1) Small molecule drugs for anticancer treatment such as camptothecin, paclitaxel and doxorubicin;

(2) Ophthalmic drugs such as flurbiprofen, levobbunolol and neomycin;

(3) Nucleic acid reagents such as siRNA and DNAzymes;

(4) Small molecule antioxidants such as n-acetylcysteine, sulfurophane, vitamin E, vitamin C, etc.;

(5 ) Small molecule drugs for immune suppression such as rapamycin, FKSOό, cyclosporin; and

(6) Any pharmacological compound that can fit into the nanodevice, e.g., analgesics, NSAiDS, steroids, hormones, anti-epileptics, anti-arrythmics, anti-hypenten_Lsives, antibiotics, antiviral agents, anticoagulants, platelet drugs, cardiostimulants, cholesterol lowering agents, etc. [0026] Molecules of interest can also include imaging and'Or tracking substances.

Imaging and_'Or tracking substances ma\ include, but are not limited to, dye rnoiecules such as propidium iodide, fluorescein, rhodarmne, green fluorescent protein and derivatives thereof.

[0027] Figure 2 is a schematic illustration to facilitate the explanation of additional embodiments of the current invention. For the sake of clarity, Figure 2 does not show storage chambers, such as a plurality of pores of a mcsoporous silica nanoparticie, and does not show stopper assemblies. However, it should be understood that they can be present in addition to the features illustrated in Figure 2. According to some embodiments of the current invention, the nanodevices, such as nanodevice 100, can include a plurality of anionic molecules attached to the surface of the nanodevice as is illustrated schematically in Figure 2. For example the anionic molecules can be phosphonate moieties attached to the outer surface of the nanodevice to effectively provide a phosphonate coating on the nanodevice. For example, the anionic molecules can be trihydroxysilylpropyl methylphosphonate molecules according to an embodiment of the current invention.

[0028] A phosphonate coating on the containment vessel, such as containment vessel

102, can provide an important role in some biological applications according to some embodiments of the current invention. This phosphonate coating can provide a negative ?eta potential that is responsible for electrostatic repulsion to keep such subrnicron structures dispersed in an aqueous tissue culture medium, for example. This dispersion can also be important for keeping the particle size limited to a size scale that allows endocytic uptake (i.e., hinders clumping). In addition to size considerations, the negative ?eta potential may play a role in the formation of a protein corona on the particle surface that can further assist cellular uptake in some applications. It is possible that this could include molecules such as albumin, transferrin or other serum proteins that could participate in receptor-mediated uptake. In addition to the role of the phosphonate coating for drug delivery, u can also provide beneficial effects for molecule loading according to some embodiments of the current invention. (Set- co-pending application number PCT/US08/13476, co-owned by the assignee of the current application, the entire contents of which are incorporated by reference herein.)

[0029] l hc nanodevice 100 can also be functional i/ed with molecules in additional to anionic molecules according to some embodiments of the current invention. For example, a pltjfdlitv υf folate Uganda can be attached to the outei surface of the containment vessel KC according to some embodiments of the current invention, as is illustrated schematically in Figure 2 (stopper assemblies are not shown for clarity).

[0030] In some embodiments of the current invention, the nanodevice 100 can also include fluorescent molecules contained in or attached to the containment vessel 102. For example, fluorescent molecules may be attached inside the pores of mesoporous silica nanoparticles according to some embodiments of the current invention. For example, the fluorescent molecules can be an amine-reactive fluorescent dye attached by being conjugated with an amine-functionalized silane according to some embodiments of the current invention. Examples of some fluorescent molecules, without limitation, can include fluorescein isothiocyanate, NHS-fluorescein, rhodamine B isothiocyanate, tetramethylrhodamine B isothiocyanate, and/or Cy5.5 NHS ester.

[0031] In further embodiments of the current invention, the nanodevices 100 may further comprise one or more nanoparlicle of magnetic material formed within the containment vessel 102, as is illustrated schematically in Figure 2 for one particular embodiment. For example, the nanoparticies of magnetic materia! can be iron oxide nanoparticles according to an embodiment of the current invention. However, the broad concepts of the current invention are not limited to only iron oxide materials for the magnetic nanoparticles. Such nanoparticles of magnetic material incorporated in the subinicron structures can permit them to be tracked by magnetic resonance imaging (MRI) systems and/or manipulated magnetically, for example.

[0032] In further embodiments of the current invention, the nanodevices 100 may further comprise one or more nanoparticle of a material that is optically dense to x-rays. For example, gold nanoparticles may be formed within the containment vessel 102 of the nanodevice 100 according to some embodiments of the current invention,

EXAMPLES

[0033] In the following example, we describe the design, synthesis, and operation of a novel, biocompatible controlled release motif we cail snap-top covered silica nanocontainers (SCSNs), based on an embodiment of the current invention. This is an example of a nanodevice according to an embodiment of the current invention in which the "snap-top" assembly corresponds to a stopper assembly. Silica nanoparticles (-400 nni in diameter) that contain hexagonaiiy arranged pores {-2 nm diameter) function as both the snap-top supports and as containers for guest molecules, The porous mesostruciure ((a) Kresge, C. T.; Leonowicz, M. E,; Roth, W. J.; Vartuli, J. C; Beck, J. S. Nature 1992, 359, 710, (b) ) Lu, Y. F.; Ganguli, R.; Drewien, (7, A.; Anderson, M, T.; Brinker, C. J.; Gong, W. L,; Gυo, Y. X.; Soyez, H,; Dunn, B.; Huang, M. H.; Zink, J. I. Nature 1997, 389, 364. (c) Huang, M. H.; Dunn, B, S.; Soyez, H.; Zink, J. I. Langmuir 1998, 14, 7331) is templated by cetyltrimethylammonium bromide (CTΛB) surfactants, and particle synthesis is accomplished using a base-catalyzed soi-gel procedure (Huh, S.; Wiench, J. W.; Yoo, J. C; Pruski, M; Lin, V. S. Y. Chem. Mater. 2003, 15, 4247). Methods for derivatiziπg silica are well-known {(a) Hernandez, R.; Franville, A. C; Minoofar, P.; Dunn, B.: Zink, J. I. J, Am. Chem. Soc, 2001, 123, 1248, (b) Minoofar, P, N.; Hernandez, R.; Chia, S.; Dunn, B.; Zink, J. I.; Franville, A. C. J. Am. Chem. Soc. 2002, 124, 14388. (c) Minoofar, P. N.; Dunn, B. S.; Zink, J. I. J. Am. Chem. Soc. 2005, 127, 2656) and are used here to functionalize the nanoparticle surfaces with the snap-top machinery. In general, a snap-top consists of a [2]rotaxane tethered to the surface of a nanoparticle in which an α-cyclodextrin (α- CD) tori encircles a polyethylene glycol thread and is held in place by a cleavable stopper. When closed, the snap-iop contains guest molecules stored within the pores, but releases the guests following cleavage of the stopper and dethreading of the tori. Based on the design of the stopper, we conceive that a multitude of stimuli could be exploited to activate snap-top systems. The specific snap-top system we describe here in this example releases encapsulated cargo molecules following enzyme-mediated hydrolysis.

[0034] We have taken divergent approaches in the design and synthesis of SCSNs in which the use of a single versatile snap-top precursor that can enable the preparation of multiple systems that are ultimately highly specific and differentiated in their function. In the divergent design, a snap-top precursor having an unstoppered [2]pseudorotaxanes serves as a foundation from which various snap-top systems can be created depending on the specific stopper that is attached. The synthesis of the snap-top precursor is carried out in a step-wise fashion from the nanoparticle surface outward (Figure I). The mesoporous silica is first treated with aminopropyltriethoxysilane to achieve an amine-rnαdifiεd surface. The amine-fυnctionalizεd materia! is then alkylated with a triethyienεglycol monoazide monotosylate unit to give an azide- terminated surface. Cargo molecules are ioadεd into the nanopores by diffusion, and the loaded, a/.tde-modified particles are then incubated with α-CD at 5'^JC for 24 h. The α-CD tori thread onto the trielhyienegSyeo! chains at low temperature effectively blocking the nanopores, while the azide function serves as a handle to attach a stoppering group. The stoppers are chemically attached to the snap-top precursors using the Cu(I)-catalyzed azide-alkyne cycloaddition ((a) Rostovtsev, V. V,; Green, L. G.; Fokin, V, V.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 2596, (b) ϊ ornoe, C. W.; Christensen, C; Meldal, M. J. Org. Chem, 2002, 67, 3057), a transformation chosen because of its remarkable functional group tolerance and high efficiency as well as our recent success in utilizing it for the preparation of interlocked molecules ((a) Dichtel, W. R.; Miljanic, O. S,; Spruell, J. M.; Heath, J. R.; Stoddart, J. F. J. Am. Chem Soc. 2006, 128, 10388. (b) Miljanic, O. S.; Dichtel, W. R.; Mortezaei. S.; Stoddart. J. F. Org. Lett. 2006, 8, 4835. (c) Aprahamian, I.; Dichtel, W. R,; ϊkeda, T.: Heath, J. R.; Stoddart, J. F. Org. LeU. 2007, 9, 1287. (d) Braunschweig, A. B.; Dichtel, W. R.; Miljanic, O. S.; Olson, M. A.; Spruell, J. M.; Khan, S. L; Heath, J. R.; Stoddart, J. F. Chem. Asian J. 2007, 2, 634).

[0035] To test the viability of an enzyme-responsive snap-top motif, a system activated by Porcine Liver Esterase (PLE) (Woodroofe, C. C; Lippard, S. J. J. Am. Chem. Soc. 2003, 125, 1 1458) was designed (Figure 3). To prepare a PLE-responsive SCSN, a precursor loaded with luminescent cargo molecules (Rhodamine B) was capped with the ester-linked adamantyl stopper 2a. in this snap-top system, PLE catalyzes the hydrolysis of the adamantyl ester stopper, resulting in dethreading of the ex-CD, and release of the cargo molecules from the pores. As a control, an SCSN was also prepared using the adamantyl amide analog 2b, which does not undergo hydrolysis by PLE. After the stoppering reactions, the dye-loaded silica particles were filtered and washed to remove non-specifically adsorbed small contaminants.

[0036] The successful functions ϋzation of the nanoparticle surface was confirmed by

FF-IR spectroscopy at various stages of loading and release. For the azide-modified nanoparticles, the peak at 3450 cm^"1 Ls indicative oi^" an N-H stretch while a strong absorption between 1050 cm ' and 1 300 cm ' indicates the presence of different kinds of C-N bonds. The control amide snap-top system shows two distinctive absorption peaks for the amide C-O group at 1650 cm^"' and 1600 cm^"'. The εster-functionalized snap-top system shows instead the expected ester C=O stretch at J 731 cm ' with pronounced C-H absorptions arising from the adamantyl group. In the spectra of the nanoparticles after guest release, the region around 3000 era^" is broad, a feature which is characteristic of the new carboxyHc acid functionality while the C-O peak is still evident at 173 1 errf¹ indicating some remaining ester functionalities on the surface of the nanoparticles. [0037] The enzyme-triggered release of cargo molecules was monitored using luminescence spectroscopy. The dye-loaded, stoppered particles (15 mg) were placed into the corner of a cuvette before carefully adding HEPES buffer (50 mM, 32 mL, pl l - 7.5). To open the snap-tops, a solution of PLE [0.12 mL, 10 mg/mL in 3.2 M (^"N I L_J)₂SO ,] was carefully added while the solution was stirred The emission of Rhodamine B in the solution above the particles was measured as a function of lirne using a 514 nm probe beam (15 mW), both before and after addition of PLE (Figure 4).

[0038] Prior to the addition of PLE, the emission intensity of Rhodamine B is essentially constant, indicating that, the dye remains trapped in the pore^ of the silica particles, The emission intensity begins to increase almost immediately following addition of PLt, l he emission intensity asymptotically approaches its maximum value with a half-life of ~5 min. By contrast, no such increase in emission was observed for the amide-stoppered snap-top system. In order to further demonstrate that the enzyme is responsible for the release, it was dcnatuied by heating at 5O⁰C for 30 mins before addition to the ester-stoppered snap-tops, No release of dye was observed. Taken together, these results are consistent with the specific opening of the snap-tops as a result of the enzyme-mediated hydrolysis of the adamantyl ester stoppers.

[0039] In order to estimate the paySυad of molecules that are released by the snap-top system, the absorbancc of the solution above the particles was measured before and after release, Using these data, it was calculated that for 15 mg of particles, 0.45 μmol (1.4 wt %} of Rhodamine B is released,

[0040] Described herein is a versatile system that is capable of entrapment and controlled release of cargo molecules. Ws have used one snap-top precursor to prepare two different snap-top systems, one with an ester-linked stopper, and the other with an amide-linked stopper. I King luminescence spectroscopy, we have demonstrated the ability of Pl E to selectively activate the ester-linked snap-top system while the amide-linked system is left intact. The can provide a biocompatible controlled release system that exploits en/ymatic specificity according to some embodiments of the current invention. Because of the wide range of stoppering units that cυuid be attached to the SCSN precursor, a multitude of snap-top systems with differentiated modes of activation coυkl be prepared with relative ease. In the future, the divergent synthetic approach that we have described will allow the snap-top motif to be very Further Snap Top Embodiments

[0041] l he reactivity of a given snap-top system can be determined by the specific stopper that is attached (Figure 5) to the snap-top precursor. ! he a/ide function of the precursors can serve as a handle to attach a stoppering group. Stoppers can be attached through Cu(I)-catalyzed azide-alkyne cycloaddition ('Click' Chemistry). Figure 6 shows three different stoppers according to some embodiments of the current invention that respond to enzymes, pH, and redox stimulation. However, the broad concepts of the current invention arc not limited to only these specific examples. T here arc a wide range of possible stoppers thai may be selected according to the particular application.

[0042] In describing embodiments of the invention, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. The above-described embodiments of the invention may be modified or varied, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.

Claims

WE CLAIM:

1 . A nanodevice, comprising: a containment vessel defining a storage chamber therein and defining at least one port to provide access to and from said storage chamber; and a stopper assembly attached to said containment vessel, said stopper assembly comprising a blocking unit arranged proximate said at least one port and having a structure suitable to substantially prevent material after being loaded into said storage chamber from being released while said blocking unit is arranged in a blocking configuration, wherein said stopper assembly is responsive to the presence of a predetermined stimulus such that said blocking unit is released in the presence of said predetermined stimulus to allow said material to be released from said storage chamber, wherein said predetermined stimulus is a predetermined catalytic activity that is suitable to at least one of cleave, hydrolyze, oxidize, or reduce a portion of said stopper assembly, and wherein said nanodevice has a maximum dimension of about 1 μm.

2. A nanodevice according to claim 1, wherein said nanodevice has a maximum dimension of less than about 400 nm and greater than about 50 nm.

3. A nanodevice according to claim 1 , wherein said nanodevice has a maximum dimension of less than about 300 nm and greater than about 50 nm.

4. A nanodevice according to claim S ₅ wherein said nanodevice has a maximum dimension of less than about 150 nm and greater than about 50 nm.

5. A nanodev ice according to any one of claims 1 -4, further comprising a thread attached to said containment vessel proximate said port, said blocking unit having a structure so that it can become threaded over said thread,

6. Λ nanodevice according to claim 5, further comprising a stopper attached to said thread such that said stopper at least assists in holding said blocking unit in said blocking configuration.

7. A nanodevice according to any one of claims. 1-6, wherein said nanodεvice is operable in an aqueous environment.

8. A nanodevice according to any one of claims 1 -7, wherein said nanodevice consists essentially of biocompatible materials in a composition thereof.

9. A nanodevice according to any one of claims 1 -8, wherein said containment vessel comprises silica in a material thereof.

10. A nanodevice according to claim 9, wherein said containment vessel is a mesoporous sibea nanoparticle defining a plurality of substantially parallel pores therein, said storage chamber being one of said plurality of substantially parallel pores.

1 1. A nanodevice according to any one of claims 1 -10, wherein said stopper assembly comprises at least one oi a [2]rotaxane or a [2 jpseudorotaxane macrυmolecule.

12. A nanodevice according to claim 1 1 , wherein said blocking unit of said stopper assembly is an α-cyclodextrin toroidal molecule.

13. A nanodevice according to claim 12, wherein said stopper assembly comprises a polyethylene thread attached to said containment vessel.

14. A nanodevice according to claim 13, wherein said stopper assembly further comprises a stopper attached to said polyethylene thread, said stopper being responsive to said predetermined stimulus to release said blocking unit, wherein said stopper is suitable to hold said blocking unit in said blocking conliguration prior to being exposed to said predetermined stimulus.

S 5, A nanodevice according Io any one of claims 1-14, further comprising a plurality of anionic or electrostatic molecules attached to an outer surface of said containment vessel, wherein said anionic or electrostatic molecules provide hydrophiiicily or aqueous dispersabihty to said nanodevice and are suitable to provide repulsion between other similar nanodevices.

16. A nanodevice according to claim 15, wherein said anionic molecules comprise a phosphonalε moiety.

17. A nanodevice according to claim 15, wherein said plurality of anionic molecules are trihydroxysilylpropyl methylphosphonate.

18. Λ nanodevice according to any one of claims 1-17, further comprising folate ligands attached Io said containment vessel,

19. Λ nanodevice according to any om of claims 1 -18, further comprising a nanoparticle of magnetic materia! formed within said containment vessel of said nanodevice.

20. A nanodevice according to claim 19, wherein said naπoparϋcle of magnetic material is an iron oxide nanoparticle.

21. A nanodevice according to any one of claims 1-20, further comprising a nanoparticle of gold formed within said containment vessel of said nanodevice.

22. A composition of matter, comprising: a plurality of nanoparticles, each defining a storage chamber therein; and a guest material contained within said storage chambers defined by said plurality of nanoparticles, said guest material being substantially chemically non-reactive with said nanoparticles, wherein said plurality of nanoparticles arc operable to cause said guest material contained within said storage chambers to be released in a presence of a predetermined stimulus, and wherein each rsanopartieie of said plurality of nanopartkies has a maximum dimension of about 1 μm.

23. A composition of matter according to claim 22. wherein said release in the presence of saκl predetermined stimulus comprises a predetermined enzyme cleaving a portion of a stopper assembly to release a stopper.

24. A composition according to claim 22 or 23, wherein said plurality of nanoparticles arc each mesoporous silica nanoparticies. each defining a plurality of substantially parallel pores therein, said storage chambers each being a respective one of said plurality of substantially parallel pores.

25. A composition according to any one of claims 22-24, wherein said stopper assembly comprises at least one of a [2]rotaxane or a [2]pseudorotaxane macromolecule.

26 A composition according to any one of claims 22-25, wherein said blocking unit is an α- cyclodextrin toroidal molecule.

27. A composition according to any one of claims 22-26, wherein said stopper assembly comprises a polyethylene thread attached to said containment vessel.

28. A composition according to any one of claims 22-27, further comprising a h> drophilic sHane.

29. Λ composition according to any one of claims 22-28, further comprising folate.

30. Λ composition according to any one of claims 22-29, further comprising a iigand for targeting a specific cell, a specific tissue, specific organ or specific biological component.

3 1. A method of administering at least one of a biologically active substance, a therapeutic substance, a neutraceuticai substance, a cosmetic substance or a diagnostic substance, comprising: administering a composition to at least one of a person, animal, plant or organism, said composition comprising nanopartseies therein, wherein said nanoparticies contain said at least ons of a biologically active substance or an imaging/tracking substance therein; and at least one of directing or allowing said nanoparticles of said administered composition to come into contact with a predetermined catalytic activity that is suitable to at least one of cleave, bydro3y/e, oxidi/ε, or reduce a portion of said nanoparticles to release said .substance from said nanoparticles.