(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
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`International Bureau
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`(19) World Intellectual Property Organization if
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`“11111)
`I .
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`(43) International Publication Date
`WO 2010/009146 A1
`PCT
`21 January 2010 (21.01.2010)
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`(10) International Publication Number
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`(51)
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`(21)
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`International Patent Classification:
`A61K 9/26 (2006.01)
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`(81)
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`International Application Number:
`PCT/US2009/050565
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`(22)
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`International Filing Date:
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`(25)
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`(26)
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`(30)
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`(71)
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`(72)
`(75)
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`Filing Language:
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`Publication Language:
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`14 July 2009 (14.07.2009)
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`English
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`English
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`Priority Data:
`61/081,034
`61/081,037
`
`15 July 2008 (15.07.2008)
`16 July 2008 (16.07.2008)
`
`US
`US
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`Applicant 0’or all designated States except US): UNI-
`VERSITY OF KANSAS [US/US]; 2385 Irving Hill
`Road, Youngbcrg Hall, Lawrence, KS 66045 (US).
`
`Inventors; and
`(for US only): BERKLAND,
`Inventors/Applicants
`Cory. J. [US/US]; 18329 Northwind Drive, Lawrence,
`KS 66044 (US). BAILEY, Mark [US/US]; 6612 Tenth
`Street, B2, Alexandria, VA 22307 (US). EL GENDY,
`Nashwa [EG/EG]; 29 El-Galaa Street, #5, El-Naam,
`Helmiate El-Zaiyoun, Cairo
`PLUMLEY, Carl
`[US/US]; 11717 Elmridge Circle, Little Rock, AR 72211
`(US).
`
`(74)
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`Agent: LECOINTE, Michelle, M.; Baker Botts L.L.P.,
`98 San Jacinto Blvd., 1500 San Jacinto Center, Austin,
`TX 78701 -4039 (US).
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ,
`CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO,
`DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI,
`NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD,
`SE, SG, SK, SL, SM, ST, SV, SY, TJ, TM, TN, TR, TT,
`TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(84)
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`Designated States (unless otherwise indicated, for every
`kind ofregional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ,
`TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE,
`ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`ML, MR, NE, SN, TD, TG).
`Declarations under Rule 4.17 :
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`as to applicant’s entitlement to apply for and be granted
`apatent (Rule 4.17(ii))
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`as to the applicant’s entitlement to claim the priority of
`the earlier application (Rule 4.1 7(iii))
`Published:
`
`with international search report (Art. 21(3))
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`(54) Title: NANOCLUSTERS FOR DELIVERY OF POORLY WATER SOLUBLE DRUG NANOPARTICLES
`
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`(57) Abstract: The present invention discloses compositions and methods for preparing a nanocluster that includes a plurality of
`nanoparticles that comprise a drug substance. Also disclosed are methods for preventing or treating a disease in a subject by ad-
`ministering a therapeutically effective amount of a composition comprising the nanoclusters of the present invention.
`
`Abraxis EX20‘I‘I
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`Actavis LLC v. Abraxis Bioscience, LLC
`|PR2017-O‘| ‘IOO
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`W02010/009146A1|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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`WO 2010/009146
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`PCT/U52009/050565
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`NANOCLUSTERS FOR DELIVERY OF POORLY WATER SOLUBLE DRUG
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`NANOPARTICLES
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`CROSS-REFERENCE TO RELATED APPLICATIONS
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`This application claims the benefit of US. Provisional Patent Application Serial No.
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`61/081,034, filcd July 15, 2008 and 61/081,037, filed July 16, 2008, the entire disclosure ofwhich
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`is hereby incorporated by reference.
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`FIELD OF THE INVENTION
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`The present invention relates generally to delivery vehicles that can be used to transport
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`active ingredients to a subject. In certain aspects, the delivery vehicles can be nanoclusters that
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`can be used in preventative or therapeutic applications.
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`BACKGROUND
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`Millions of people worldwide suffer from a wide variety of diseases or conditions that
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`would benefit from the effective delivery of therapeutic and or preventative agents. Examples of
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`these diseases or conditions include pulmonary diseases, circulatory diseases, muscular diseases,
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`bone diseases, cancers, etc.
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`The use of nanoparticles as drug delivery vehicles has been employed for a variety of
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`indications (John 2003). Nanoparticles, for example, have been shown to improve the dissolution
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`of poorly water-soluble drugs and enhance the transport of drugs both intra— and paracellularly. In
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`addition, literature indicates that plasmid DNA can be effectively delivered by polycantionic
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`polymers that form nanoparticles when mixed with DNA resulting in enhanced gene expression
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`(Kumar 2003). Research efforts on nanoparticle-mediated gene therapy also address treating
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`genetic disorders such as Cystic Fibrosis (Griesenbach 2004).
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`Most nanoparticle formulations are designed for action at the cellular level. This assumes
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`the efficient delivery of the nanoparticle to the appropriate cellular target. However, current
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`nanoparticle treatment options are limited in the ability to access the cellular target. For example,
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`two research groups are currently investigating microencapsulated nanoparticles as a mode of
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`nanoparticle delivery to the pulmonary epithelium (Sham 2004, Grenha 2005). These efforts are
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`hindered by the common inability to control microparticle size, distribution, and difficulty in
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`delivering a large payload of therapeutic nanoparticles.
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`SUMMARY OF THE INVENTION
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`The present invention overcomes the deficiencies in the art by providing effective drug
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`delivery systems that can: (1) formulate nanoparticles as a nanocluster to facilitate handling,
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`administering, or targeting, for example; and (2) maintain the cluster or disperse the nanoparticles
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`at the targeted site.
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`In one aspect of the present invention, there is disclosed a nanocluster comprising a
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`plurality of nanoparticles. In certain non—limiting aspects, the nanocluster is maintained at the
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`targeted site (e.g., the nanocluster does not disperse into separate nanoparticles). In other aspects,
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`the nanoparticles disperse in response to an environmental cue. The nanocluster, in certain
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`non-limiting embodiments, can have a size of about 1 to about 200 microns. In certain aspects, the
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`nanocluster size is 1, 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,l6,17,18, 19, 20, 21, 22, 23, 24,
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`25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
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`100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 microns. In other aspects, the size ofthe
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`nanocluster can be greater than 200 microns (e.g., 210, 220, 230, 240, 250, 300, 350, 400, 450,
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`500, 600, 700, or more microns in size.) The nanocluster of the present invention can also have a
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`variety of shapes (e.g., spherical and non-spherical shapes). In certain embodiments, the
`nanocluster can be solid or hollow. A person of ordinary skill in the art will recognize that a solid
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`nanocluster can be completely solid throughout or can have spaces, such as pores or a hollow core,
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`that are created by the packing of the nanoparticles within the nanocluster. The size of these
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`packing spaces can be from about 1 nm to about 1000 nm, in non—limiting aspects.
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`In certain
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`aspects, the size ofthe packing spaces can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,
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`80 , 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,
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`1000 or more nanometers, in non—limiting aSpects. Hollow nanoclusters can have an empty space
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`or cavity. The size of the cavity can vary, for example, from about 50 m to about 20 pm, in non
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`limiting aspects. The size of the cavity, for example, can be 50, 100, 150, 200, 250, 300, 3500,
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`400, 450, 500, 550, 600, 650, 700, 750, 800 .
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`.
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`. 20 um, and any range derivable therein.
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`The nanoparticles that are included in the nanocluster, in some embodiments, are not held
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`(e.g., adhered or chemically bound (e.g., covalent bond, non—covalent bond, van der waals forces»
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`together by a functional group on the nanoparticles. The nanoparticles can be in direct contact
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`with one another in some aspects. In other aspects, the nanoparticles are not in direct contact with
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`one another. In certain embodiments of the present invention, the nanoparticles are not
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`encapsulated. In other embodiments, the nanoparticles do not include a functional group. In other
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`aspects, however, the nanoparticles can include a functional group such as, for example, a
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`carboxyl, sulhydryl, hydroxyl, or amino group. All types of functional groups that can be used to
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`bind other nanoparticles together, active ingredients to the surface of nanoparticles, or other
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`compounds are contemplated as being useful with the present invention.
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`In certain embodiments, the nanocluster can include an active ingredient. Non-limiting
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`examples of active ingredients that are contemplated as being useful in the context of the present
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`invention include those known to a person of ordinary skill and those described throughout this
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`specification. By way of example only, active ingredients can include medical pharmaceuticals
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`and specialties such as preventive agents, for example vaccines, diagnostic agents, for example
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`tracers of various types and imaging enhancers, therapeutic agents, for example small molecules
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`(e.g., nucleic acids, proteins, peptides, polypeptides, etc.), drugs, peptides, and radiation,
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`immuno—modulators, vaccine and virus vectors, and combinations of these classes. The
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`nanoparticles can include particular embodiments, respirable non—medical specialties such as
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`physiochemical agents, for example gas antidotes, biophysical modulators, for example
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`paramagnetics, emitters, for example electromagnetic wave emitters, and imaging enhancers. The
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`active ingredients, in certain embodiments, can be associated with the nanoparticles. For example,
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`the active ingredients can be entangled, embedded, incorporated, encapsulated, bound to the
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`surface (e.g., covalently or non—covalently bonded), or otherwise associated with the nanoparticle.
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`In certain preferred aspects, the active ingredient is the nanoparticle. In other aspects, the
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`nanoparticles can include a polymer material (including, for example, biodegradable and
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`non—biodegradable polymers). Non—limiting examples of polymer materials that can be used
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`include those known to a person of ordinary skill and those described throughout this
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`specification. In certain embodiments, the nanoparticles can include a mixture of a polymer and
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`an active ingredient.
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`In other non-limiting embodiments, the nanocluster or nanoparticles, or both, can include
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`at least one, two, three, four, five, six, seven, or more different active ingredients.
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`In a preferred
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`embodiment, the nanocluster or nanoparticles include a first drug on its surface, and a second
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`active ingredient encapsulated within the nanocluster or nanoparticles or other incorporated into
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`the nanocluster or nanoparticle material. It is contemplated that a nanocluster can release the
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`active ingredients in a given environment, or after a given period of time in a controlled manner.
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`For example, a nanocluster having at least one active ingredient can be released in response to an
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`environmental cue or after a pre-determined amount of time. Also by way of example only, a
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`nanocluster having at least two different active ingredients can be released in response to different
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`environmental cues or after pre-determined periods of time. For example, active ingredient 1 can
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`be released first and then active ingredient 2 can be released second. In certain non—limiting
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`aspects, the release of the first active ingredient can improve the performance of the second active
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`ingredient.
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`In other particular aspects, the nanoclusters of the present invention can include a
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`dispersing material that holds the plurality of nanoparticles together and/or disperses the
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`nanoparticles in response to an environmental cue. The dispersing materials that can be used with
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`the present invention include those materials that are known to a person of skill in the art and those
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`that are disclosed throughout this specification. Non—limiting examples of dispersing material
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`include liquid sensitive materials (e.g., water-soluble materials (e.g., polymers», biodegradable
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`polymers, polyelectrolytes, metals, surfactants, polymeric cross-linkers, small molecule
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`cross-linkers, pH sensitive materials, pressure sensitive materials, enzymatic sensitive materials,
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`and temperature sensitive materials. Non-limiting examples of environmental cues that can be
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`used with the present invention include liquid (e.g., water, blood, mucous, solvent, etc), a selected
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`pH range, a selected temperature range, an electric current, a selected ionic strength, pressure, the
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`presence of a selected enzyme, protein, chemical, electromagnetic wavelength range (e.g., visible
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`light, UV light, infrared, ultraviolet light, microwaves, X-rays, and gamma-rays), or the presence
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`of an external force (e.g., vibration, shearing, shaking, etc.).
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`In certain aspects, the dispersing
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`material can be coated onto the surface of the nanoparticles before or after nanocluster formation.
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`In certain embodiments, the dispersing material can be between the nanoparticles or link the
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`nanoparticles together (e.g., covalently or non—covalently couple a first nanoparticle to a second
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`nanoparticle). The dispersing material can be adhered to or covalently or non—covalently coupled
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`to the nanoparticles.
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`In particular embodiments of the present invention, the nanocluster can include from about
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`1% to about 99% by weight or volume of the nanoparticles or dispersing materials. The
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`nanocluster can also be completely made up of nanoparticles (i.e., 100%). In preferred
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`embodiments, the nanocluster includes from about 10% to about 90%, 15% to about 80%, 20% to
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`about 70%, 30% to about 60%, and about 40% to about 50% of nanoparticles or dispersing
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`materials. In certain embodiments, the nanocluster includes at least 50% of the nanoparticles or
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`dispersing material.
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`Another embodiment to the present invention is a composition comprising a nanocluster of
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`the present invention. The composition in certain non-limiting aspects can have a plurality (e.g,, at
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`least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 , 60, 70, 80, 90, 100, 200, 300, 400, 500, or more
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`nanoclusters. The composition can fiirther include an active ingredient. As discussed throughout
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`this specification, the composition can be formulated into a dry powder, an aerosol, a spray, a
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`tablet, or a liquid. The compositions of the present invention can include at least about 1%, 2%,
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`3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
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`65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nanoclusters of the present invention.
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`In
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`certain aspects, the compositions of the present invention can include a plurality of identical or
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`similar nanoclusters. In other aspects, the compositions of the present invention can include at
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`least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nanoclusters that have different characteristics (e.g., different
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`active ingredients attached, different shapes, hollow or solid, etc.). The compositions of the
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`present invention can be formulated into a pharmaceutically acceptable carrier.
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`In another embodiment, there is disclosed a method of preventing or treating a disease or
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`condition in a subject comprising administering a therapeutically effective amount of a
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`composition comprising a nanocluster of the present invention to a subject (e.g., human, pigs,
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`horses, cows, dogs, cats, mouse, rat, rabbit, or any other mammal and non-mammals) in need of
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`the composition. The method can further include a method for determining whether a subject is in
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`need of the prevention or treatment. The disease or condition can include all types of diseases or
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`conditions known to a person of skill in the art and discussed throughout this specification.
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`In
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`certain preferred aspects, the disease or condition can be a pulmonary associated disease or
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`condition (e.g., common cold, flu, cystic fibrosis, emphysema, asthma, tuberculosis, severe acute
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`respiratory syndrome, pneumonia, lung cancer, etc.), a circulatory disease or condition, a muscular
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`disease or condition, a bone disease or condition, an infection, a cancer, etc. In certain
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`embodiments, the method can include the administration of a second therapy used to treat or
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`prevent the disease (e.g., combination therapy).
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`In preferred embodiments, the compositions of
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`the present invention are administered nasally. Other modes of administration known to those of
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`skill in the art or discussed in this specification are also contemplated.
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`In particular aspects, the
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`nanoclusters within the composition are delivered to the deep lung (e.g., bronchiole or alveolar
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`regions ofthe lung).
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`In certain preferred aspects of the present invention, the nanoclusters of the present
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`invention can be used to deliver vaccines or components of vaccines. For instance, cells of the
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`immune system, especially macrophages and dendrocytes, are targets for immunization. These
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`"professional" antigen-presenting cells (APCs) can elicit a desired T—cell response to vaccine
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`components. APCs are typically capable of phagocytosis of particles in the range of 1 to 10 pm.
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`By generating in this size range nanoclusters or nanoparticles containing vaccine components, one
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`can passively target delivery of the vaccine to APCs. US. Pat. No. 6,669,961, for example,
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`provides a non-limiting explanation of this process.
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`The nanoclusters of the present invention can also have a particular mass density. In
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`certain preferred embodiments, for example, the mass density can be greater than, equal to, or less
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`than 0.1 g/cm3. In particular embodiments, the mass density of the nanoclusters of the present
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`invention can be about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
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`0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 g/cm3, or greater.
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`Also disclosed is a method of preparing a nanocluster comprising: (i) obtaining a plurality
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`of nanoparticles; (ii) obtaining a dispersion material (when desired); and (iii) admixing (i) and (ii),
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`wherein the admixture is formulated into a nanocluster. In certain aspects, obtaining a plurality of
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`nanoparticles comprises: (i) obtaining an aqueous suspension of nanoparticles; (ii) emulsifying the
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`suspension into a non-aqueous phase; (iii) allowing water in the aqueous suspension to absorb into
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`the non-aqueous phase; (iv) allowing the nanoparticles to aggregate together; and (v) retrieving
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`the aggregated nanoparticles. In other non—limiting embodiments, obtaining a plurality of
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`nanoparticles includes: (i) obtaining a non-aqueous suspension of nanoparticles; (ii) emulsifying
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`the suspension into an aqueous phase; (iii) allowing liquid in the non-aqueous suspension to
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`absorb into the aqueous phase; (iv) allowing the nanoparticles to aggregate together; and (v)
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`retrieving the aggregated nano particles. The disclosed method represents a non—limiting method
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`with other methods being evident by one skilled in the art (e.g. Emulsion/solvent evaporation,
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`extraction, spray-drying, spray freeze—drying, self-assembly in solution, etc.).
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`In certain aspects, it
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`is contemplated that the nanoclusters can be prepared in a solution without using spray and/0r
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`freeze dry techniques.
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`It is also contemplated that the nanoclusters can be recovered from the
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`solution by using freeze dry or spray dry techniques that are known to those of skill in the art. As
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`noted throughout this specification, the nanocluster can be included within a composition. The
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`composition can be formulated into a liquid, a spray, an aerosol, or a dry powder in non—limiting
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`embodiments.
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`Also disclosed is a method of delivering an active ingredient to a subject in need
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`comprising obtaining composition comprising a nanocluster ofthe present invention and an active
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`ingredient and administering the composition to the subject. In non-limiting aspects, the active
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`ingredient is encapsulated in the nanoparticle, incorporated within the nanoparticle material,
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`conjugated to the nanoparticle, absorbed or coupled to the nanoparticle.
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`In yet another embodiment of the present invention, there is disclosed a method of
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`preparing a nanocluster comprising: (i) obtaining a first nanoparticle and a second nanoparticle;
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`and (ii) admixing the first and second nanoparticles, wherein the nanoparticles self assemble to
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`form a nanocluster. The first and second nanoparticles, for example, can have hydrophobic
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`properties, hydrophilic properties, or a mixture of both.
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`In other aspects, the first or second
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`nanoparticles can have an electrostatic charge. For example, the first nanoparticle can be
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`positively charged and the second nanoparticle negatively charged, and vice versa. The
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`self—assembly, in particular embodiments can be based on an electrostatic interaction between the
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`first and second nanoparticles. In other non-limiting aspects, the self—assembly can be based on a
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`hydrophobic or hydrophilic interaction between the first and second nanoparticles. The first and
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`second nanoparticles can self assemble in solution to form the nanocluster in certain embodiments.
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`In particular aspects, preparation ofthe nanoclusters does not require the use of spray and/or freeze
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`dry techniques; rather nanocluster formation can occur in solution. The nanoclusters can be
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`recovered from the solution by using freeze dry or spray dry techniques that are known to those of
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`skill in the art. In other aspects, the method of preparing the nanocluster can further comprise
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`obtaining a dispersion material and admixing the dispersion material with the first and second
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`nanoparticles.
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`As disclosed is a method of storing nanoparticles comprising forming the nanoparticles
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`into a nanocluster. The nanoparticles, for instance, can be stored as a liquid, a spray, and aerosol,
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`or a dry powder. The method of storing the nanoparticles can further comprise returning the
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`nanocluster to nanoparticles.
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`In certain aspects, retuming the nanocluster to nanoparticles can
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`include subjecting the nanocluster to an environmental cue. As noted above and throughout this
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`specification, non—limiting examples of environmental cues include water, a selected pI-l, a
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`selected temperature, a selected enzyme, a selected chemical, a selected electromagnetic
`
`wavelength range, vibration, or shearing. In certain particular aspects, the nanocluster can include
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`a dispersing material that holds the nanoparticles together and/or disperses the nanoparticles in
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`response to an environmental cue. Non—limiting examples of dispersing materials include a water
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`soluble polymer, a biodegradable polymer, a polyelectrolyte, a metal, a polymeric cross-linker, a
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`small molecule cross—linker, a pH sensitive material, a surfactant, or a temperature sensitive
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`material.
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`It is contemplated that any embodiment discussed in this specification can be implemented
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`with respect to any method or composition of the invention, and vice versa. Furthermore,
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`compositions of the invention can be used to achieve methods of the invention.
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`The terms "inhibiting," "reducing," or "prevention," or any variation of these terms, when
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`used in the claims and/or the specification includes any measurable decrease or complete
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`inhibition to achieve a desired result.
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`The term "effective," as that term is used in the specification and/or claims, means
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`adequate to accomplish a desired, expected, or intended result.
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`The use ofthe word "a" or "an" when used in conjunction with the term "comprising" in the
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`claims and/or the specification may mean "one," but it is also consistent with the meaning of "one
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`or more," "at least one," and "one or more than one."
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`The term "about" or "approximately" are defined as being close to as understood by one of
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`ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%,
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`preferably within 5%, more preferably within 1%, and most preferably within 0.5%.
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`The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated
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`to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure
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`supports a definition that refers to only alternatives and "and/or."
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`As used in this specification and claim(s), the words "comprising" (and any form of
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`comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as
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`"have" and "has"), "including" (and any form of including, such as "includes" and "include") or
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`"containing" (and any form of containing, such as "contains" and "contain") are inclusive or
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`open-ended and do not exclude additional, unrecited elements or method Steps.
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`Other objects, features and advantages of the present invention will become apparent from
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`the following detailed description. It should be understood, however, that the detailed description
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`and the examples, while indicating specific embodiments of the invention, are given by way of
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`illustration only. Additionally, it is contemplated that changes and modifications within the spirit
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`and scope of the invention will become apparent to those skilled in the art from this detailed
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`description.
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`DRAWINGS
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`Illustrative embodiments of the invention are illustrated in the drawings, in which:
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`FIG. 1. Therapeutic nanoparticles are organized into a nanocluster having a defined (and
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`tunable) diameter. Upon contact with an environmental cue, the dispersive material triggers
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`dispersion of the nanoparticles.
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`FIG. 2. Electron micrographs of (A) ~100 nm silica particles that compose the (B) ~6 um
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`nanocluster. (C) Represents typical nanocluster distribution. Scale bar in (C) represents 10 um.
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`FIG. 3. Nanoclusters can be fabricated with a broad or narrow size distribution (left top
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`and bottom). Adjusting fabrication conditions and/or dispersing material used allows for the
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`formation of a solid (top right) or hollow (bottom right) clusters.
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`FIG. 4. Uniform (~75 um) nanoclusters composed of polystyrene nanoparticles.
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`FIG. 5. Electron micrographs of (A) 225 nm silica nanoparticles coated with a dispersion
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`material (light gray corona) and (B) a 9 pm nanocluster of the silica nanoparticles coated with
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`dispersion material.
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`FIG. 6. The dispersion ofnanoclusters over time composed of nanoparticles coated with a
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`hydrolysable polymer was a function of pH as determined by (A) absorption of light at 480 nm
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`and (B) visual inspection. (C) Size analysis of the dispersion shows polydisperse agglomerates
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`are liberated from the nanoclusters, which then break down into monodisperse nanoparticles.
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`FIG. 7A, FIG. 7B, FIG. 7C. The (FIG. 7A) geometric and (FIG. 7B) aerodynamic size
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`distributions of PLGA nanoclusters produced by increasing the concentration of nanoparticles
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`(black=0.68 mg/ml, red=l .36 mg/ml, green=2.l6 mg/ml, blue:2.72 mg/ml). FIG. 7C Scanning
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`electron micrograph of nanocluster structure. Scale bar=5 um.
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`FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG. 8E, FIG. 8F. Laser scanning confocal
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`micrographs of PLGA nanoparticle nanoclusters. FITC—labeled PVAm—coated nanoparticles
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`(FIG. 8A and FIG. 8D) and rhodamine—labeled PEMA—coated nanoparticles (FIG. 8B and FIG.
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`8B) are both identified within the nanocluster structure. FIG. 8C and FIG. 8F Overlays of the
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`micrographs reveal the diffuse structure of the nanoclusters. Scale bar=5 um.
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`FIG. 9. Scanning electron microscope (SEM) image of a population of nifedipine
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`nanoparticles.
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`FIG. 10. SEM image of nifedipine nanOparticle clusters.
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`FIG. 1 1. Illustration of the geometric diameter of the nanoclusters comprising
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`DOTAP/PLGA nanoparticles and ovalbumin.
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`FIG. 12. SEM images of the nanoclusters comprising DOTAP/PLGA nanoparticles and
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`ovalbumin.
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`FIG. 13. The particle size distributions of paclitaxel nanoparticle agglomerates in
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`suspension after flocculation and resuspended after lyophilization.
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`FIG. 14. Aerodynamic size distributions of paclitaxel nanoparticle agglomerates after
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`lyophilization.
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`FIG. 15. The distribution of Paclitaxel powder as received and nanoparticle agglomerate
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`formulations deposited on the stages of a cascade impactor at a flow rate of ~30 L/min.
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`FIG. 16. ln—vitro dissolution profiles of paclitaxel in PBS (pH 7.4) from pure paclitaxel
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`powder and two different nanoparticle (NP) and nanoparticle agglomerate formulations (NA).
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`FIG 17. Viability of A549 cells in the presence of formulation components as determined
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`by an MTS assay,
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`FIG. 18. The particle size distributions ofbudesonide nanoparticle agglomerates in
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`suspension after flocculation and resuspended afier lyophilization.
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`FIG. 19. Aerodynamic size distributions of budesonide nanoparticle agglomerates after
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`lyophilization.
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`FIG. 20. The distribution of budesonide nanoparticle agglomerate formulations (A) F1,
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`(B) F2, and (C) F3 deposited on the stages of a cascade impactor. (D) Formulations were
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`compared with stock budesonide at a flow rate of ~30 L/min.
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`FIG. 21. Transmission electron micrographs ofA) F1 nanoparticles and B) F1
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`nanoparticle agglomerates.
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`FIG. 22. 13C CP/MAS spectra of budesonide, excipients, and budesonide formulations.
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`The nanoparticle agglomerates spectrum was expanded 8 times vertically to produce the
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`nanoparticle agglomerates x8 spectrum to aid the interpretation of the budesonide peaks.
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`FIG. 23. Structure of budesonide with carbon numbering.
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`FIG. 24. 13C CP/MAS spectra from spectral editing experiment.
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`All = all carbon types are shown, C+CH3 = only unprotonated and methyl carbons are
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`shown, C = only unprotonated carbons are shown, CH = only methine carbons are shown, and
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`CH2 = only methylene carbons are shown.
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`FIG. 25.
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`ln-vitro dissolution profiles of budesonide in PBS (pH 7.4) from budesonide
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`stock and three different nanoparticle (NP) and nanoparticle agglomerate formulations (NA).
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`FIG. 26. Viability of A549 cells in the presence of formulation components as determined
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`by an MTS assay.
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`FIG. 27. Percent volume as a function of particle diameter for a flocculated solution of
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`NIF/SA nanoparticles in water (421 .7 +/— 26.2 nm, —32. 16 +/— 3.75 mV) after addition ofNaCl to
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`0.1M. Also shown is the same solution after homogenization at 25000 RPM for 30 seconds.
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`FIG. 28. Aerodynamic Diameter size distribution for the sample of nanoparticle
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`flocculates shown in Figure 1.
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`FIG. 29. A collection of SEM images for nanoparticles directly after sonication (A), newly
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`prepared flocculates (B), flocculate powders after residing under room conditions and devoid of
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`light for 1 month (C), and pure nifedipine crystals as received (D).
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`FIG. 30. DSC outputs for the optimal formulation of nanoparticles, pure nifedipine, and
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`flocculated nanoparticles.
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`FIG. 3 1. Percent drug dissolution vs. Time as deduced via HPLC UV spectroscopy for the
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`nifedipine/stearic acid nanoparticles, flocculates, and the drug in pure crystalline form.
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`FIG. 32. Cascade impactor readings for nifedipine/stearic acid nanoparticles, flocculates,
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`and drug as received in pure form.
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`FIG. 33. Particle size distributions for a flocculate sample and portions of the sample after
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`three homogenization regimes. A nanoparticle solution (Before) was allowed to flocculate to
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`completion without homogenization for 4 hours. Portions of the sample were then subject to
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`increasingly powerful homogenization regimes (Low, Mid, High) from 5, 15, and 25 kRPM for
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`30 seconds, respectively.
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`FIG. 34. Particle size distributions for the flocculation of a nanoparticle suspension ( 336.1
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`+/— 5.9 n