WO2013112215A1 - Synthesis, dye functionalization and bioimaging of polymeric nanocapsules - Google Patents

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit, of priority of U.S. Provisional Application No. 61/5.90,137, filed January 24, 2012.

Any foregoing applications, and all documents cited therein or during their prosecution ("application cited documents") and all documents cited or referenced in the application cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herei by reference, and may be employed in the practice of the invention.

FIELD OF THE INVENTION

The present invention relates to functionaiized polymeric nanocapsuies, synthesis of said polymeric nanocapsuies, methods of making thereof and use thereof.

BACKGROUND OF THE INVENTION

Progress in nanoscience based imaging technologies along with advancements in molecular biology, genoniics, to name but a few can result in viable solutions to cun-ent diagnostic and therapeutic challenges. See Hood et al., "Systems biology and new technologies enable predictive and preventative medicine," Science 2004; 306(5696); 640-3; see also Michalet et al, "Quantum dots for live cells, in vivo imaging, and diagnostics," Science 2005; 307 (5709): 538-44. Among various imaging techniques, fluorescence based imaging platforms are poised to make the clinical translation. See Frangioni JV. "In vivo near-infrared fluorescence imaging," Curr Opin Che Biol. 2003; 7(5): 626-34.

Recent innovations in fluorescence microscopy have enabled the visualization of microscopic structures in three dimensions. See Lichtman et al,, ^Fluorescence microscopy," Nat Methods, 2005; 2(12): 910-9. Fluorescence imaging probes can. be classified as conventional organic dyes (Sevick-Muraca et al., "Fluorescence-enhanced, near infrared diagnostic imaging with contrast agents," Curr Opin Chem Biol. 2002; 6(5): 642-50), quantum dots (Michalet, supra), or hybrid architectures (Bums et al., "Fluorescent core-shell silica nanoparticles: towards 'Lab on a Particle' architectures for nanobioiech.no! ogy^"" Chem Soc Rev. 2006; 35(1 1 ): 1028-42), where the fluorophores are incorporated into an inert matrix.

The low absorption of the near infrared (NIK) light (700 - 1000 nm) by the tissue chroxnophores such as oxygenated and deoxygenated hemoglobin, the development of highly fluorescent NIR emitting fluorophores, and the recent advances in tomography have enabled imaging of tissues deeper than 1 cm. See Lichtman, supra; see also Sevick-Muraca, supra. Ideally for biomedical imaging, the fluorophores or hybrid architectures must be

biocompatible, inert, and should be efficiently excreted from the body; and satisfying all of these criteria still remains a challenge. See Bums et al., "Fluorescent Silica Nanoparticles with Efficient Urinary Excretion for Nanomedicine," Nana Lett. 2009; 9(1): 442-8.

Currently, dyes such as indigo carmine (Gustilo-Ashby at al., "The incidence of ureteral obstruction and the value of intraoperati ve cystoscopy during vaginal surgery for pelvic organ prolapse," Am J Obstet Gynecol. 2006; 194(5): 1478-85), methylene blue (Matsui et al., "Real-time, near-infrared, fluorescence-guided identification of the ureters using methylene bine," Surgery 2010; 148(1 ): 78-86), and most recently a near-infrared (NIR) fiuorophore, indocyanine green (Tanaka et al., "Real-time intraoperative ureteral guidance using invisible near-infrared fluorescence," J Urol 2007; 178(5): 2197-202) are preferred for visualizing ureters. See Jabs at al.. "The role of intraoperative cystoscopy in prolapse and incontinence surgery," Am J Obstet Gynecol. 2001 ; 185(6): 1368-71.

However, their stability (Saxena at al., "Degradation kinetics of indocyanine green in aqueous solution," J P harm Set. 2003: 92(10): 2090-7), interaction with plasma, components and subsequent alteration (Yu et al., "Self-Assembly Synthesis, Tumor Cell Targeting, and Photothermal Capabilities of Antibody-Coated Indocyanine Green Nanocapsuies," J Am Chem Soc, 20.10; 132(6): 1929-38), safety concerns (Grazlano at al, "Life-threatening reaction to indigo carmine - A sulfa allergy?" Int Urogynecol J. 2005; 16(5): 418-9), and rapid clearance (El-Desoky et a!.. "Experimental study of liver dysfunction evaluated by direct indocyanine green clearance using near infrared spectroscopy," Br J Surg. 1999; 86.(8):

7 .1005-11) pose restrictions. Hence, there is a need to develop novel hybrid dye architectures for visualization of ureters during laparoscopic or robotic procedures.

U.S. Food and Drug Administration (FDA) requires that nanocarriers used for bioimaging and other purposes be completely cleared from the human body in a reasonable period of time. See Choi et al, "Renal clearance of quantum dots," Nat Biotechnol 2007; 25(10): 1 165-70. This prerequisite is one of the challenges that limit the clinical translation of various nanotechnological platforms. Another major obstacle is the lack of biocompatibility. See Liu et al, "Self-assembled hollow nanocapsule from amphiphatic carboxymethyl- hexanoyl chitosan as drug carrier," Macro ohcules 2008; 41(17): 651 1 -6.

Nanocarriers are usually Injected intravenously for rapid presentation through the entire body.. Removal can be achieved through renal, filtration, biliary excretion or

metabolism. See Fox et al., "Soluble Polymer Carriers for the Treatment of Cancer: The Importance of Molecular Architecture," Accounts Chem Res. 2009; 42(8): 1 141.-51. Due to structural limitations (Venkatachalam at al., "Structural and Molecular Basis of Glomerular Filtration," CircRes. 1978; 43(3): 337-47). uch as dimensions of the fenestrae (50 - 100 ran), the renal filtration is thought to be limited to nanoparticies < 14 ran. See Fox, supra.

However, it. is worth noting that other reports suggest that nanoparticies with as large as - 45 nm dimensions can also be excreted by the renal filtration route. See He et at, "In Vivo Study of Biodistribution and Urinary Excretion of Surface-Modified Silica

Nanoparticies/' Anal Chem. 2008; 80(24): 9597-603. Notably, mutli-walled and single- walled nanotubes with lengths > 200 nm and thickness ~ 35 - 40 nm have been excreted intact in urine. See Singh et al, "Tissue biodistribution. and blood clearance rates of

Intravenously administered carbon nanotube radiotracers/' Proc Natl Acad Set U SA. 2006; 103(9): 3357-62.

Nanocapsules and nanocontainer molecules, developed, to mimic the

compartrnentalization exhibited by nature, have attracted enormous attention in recent years. See Meier W., "Polymer nanocapsules/' Chem Soc Rev. 2000; 29(5): 295-303; see also Lou et al., "Hollow Micro-/Nanostructures: Synthesis and Applications," Adv Mater. 2008;

20(21): 3987-4019. They can be made of organic (see Meier, supra), inorganic (see Low, supra), or biological materials (see Yu et al., Nano Lett. 2008; 8(10): 3510-5). The techniques currently employed for the fabrication of polymeric or organic nanocapsules and other related nanocontainer architectures require one of the following; self- assembly, sacrificial templates, or surfactants. See Meier, supra; see.. also Lensen et al., "Polymeric Microcapsules for Synthetic Applications," Macromol Biosci. 2008; 8(1 1 ): 991 - 1005. A variety of self-assembled nanocapsules mimicking biological systems such as liposomes, polymersomes, etc. have been investigated. See Lensen, supra. Typical ly in a template based approach, an organic/polymeric shell is either grown or self-assembled around the template by a variety of means, following which the sacrificial template is selectively removed, resulting in the formation of nanocapsules. See Marinakos et al, "Gold particles as templates for the synthesis of hollow polymer capsules. Control of capsule dimensions and guest encapsulation," J Am. Chem Soc. 1999: 121(37): 8518 -22.

A number of templates including gold (see Marinakos, supra) and silica nanoparticles (see Lou, supra see also Caruso F, "Hollow capsule processing through colloidal templating and self-assembly," Chem-Eur J. 2000; 6(3): 413-9), dendrimers (see Wend!and et aL "Synthesis of cored dendrimers," J Am Chem oc. 1999; 121(6): 1389-90) and self-assembled amphophilic block copolymers (see levins et al, "Synthesis of hollow responsive functional nanocages using a meial-ligand complexation strategy,^1' Macromolectdes 2008; 41 ( 10): 3571- 8) have been employed for the synthesis of nanocapsules. A hybrid approach termed polymeric multilayer capsules (PMLCs), which involves the layer-by- layer (LbL)

electrostatic deposition of oppositely charged polymers on a sacrificial template, and subsequent removal of the template leading to the formation of capsules, has been developed and investigated over the years. See Caruso, supra; see also De Cock et ah, "Polymeric Multilayer Capsules in Drug Delivery," Angew Chew.. Int. Ed. , 2010; 49(39): 6954-73; see also del Mercato et al., "LbL multilayer capsules: recent progress and future outlook for their use in life sciences," Nanoscale 2010; 2(4): 458-67.

Conventional emulsion, mini-emulsion ( see Lu at al. "A facile route to synthesize highly uniform nanocapsules: Use of amphophilic poly(acryiic acidVblock-po!ystyrene RAFT agents to interfacialiy confine miniemuision polymerization,'' Macromol Rapid Coinmi . 2007; 28(7): 868-74) and interfacial polymerizations have also been employed for the fabrication of nanocapsules. See Li et al "Dual -Reactive Surfactant Used for Synthesis of Functional Nanocapsules in Miniemuision," J Am Chem Soc. 2010; 132(23): 7823. However, the current strategies for nanocapsuie formation have certain intrinsic limitations such as incomplete removal of sacrificial templates or surfactants, and the tedious procedures involved in the removal process, lack of nanocapsuie robustness, low efficiency etc. See Lou, supra; see also Li. et aL "Reactive Surfactants for Polymeric Nanocapsuies via Interfacially Confined iniemulsion ATRP," Macromolecuks 2009; 42(21): 8228-33.

These na.nosiz.ed container molecules are capable of encapsulating a variety of species and have been used as imaging agents {see del Mercato, supra), drug delivery vehicles {see De Cock, supra), nanoreactors ( see Shchukin et al, "Nanoparlicle synthesis in engineered organic nanoscale reactors," Adv Mater. 2004; 16(8):671-82.}, and in catalysis, See Meier, supra. Drug molecules such as cisplatin {see Chupin et al, "Molecular Architecture of Nanocapsuies, Bi!ayer-Enclosed Solid Particles of Cisplatin," J^m Chem Soc. 2004;

126(42): 13816-21) and doxorubicin {see Wang et al, "Templated synthesis of single- component polymer capsules and their application in drug delivery," Nana Lett. 2008; 8(6): 1741-5) encapsulated in nanocapsuies have been evaluated in tumor therapy. Substantial changes in H, ionic strength, solvent polarity, temperature, etc. are often required to move guest molecules into the capsules. See De Cock, supra.

Given the porous nature of these capsules, smaller guest molecules (< 5 kDa) can easily permeate them; and their encapsulation is usually accomplished by changes in their solubility or their crystallization upon encapsulation. Id. The current guest loading techniques suffer from low encapsulation efficiencies, and require the capsules to be physically robust to endure repeated centrifugation and solven exchange processes. Id. Self- assembled structures held together by weak iniermolecular attractions have limited stability, and hence covaienily linked polymeric nanocapsuies which offer greater mechanical stability are needed for a variety of imaging and drug-delivery applications. See Meier, supra. In this context, (here exists a strong need to develop new, simpler and direct routes to prepare robust nanocapsuies with well-defined morphology, surface composition and mechanical properties for biological applications.

SUMMARY OF THE INVENTION

The present invention provides functionaiized polymeric resorcinarene nanocapsules. In certain embodiments, the functionalized polymeric resoreinarene nanocapsules comprise functional groups that improve the water solubility of the nanocapsules, In other embodiments, the polymeric resoreinarene nanocapsules of this invention comprise fluorescent moieties, which for example, may be useful for

bioimaging. in certain embodiments, the present invention provides polymeric resoreinarene nanocapsules, which are capable of being cleared from the human body by renal filtration.

The present invention relates to functionalized polymers of resorcinarenes which are formed by the thi rmula (I):

(I)

wherein;

X¹, X², X³ and X⁴ are independently -(CH₂)„- ;

n is from 0 - 8;

ll R², R^~ and R⁴ are each independently an alkerie;

wherein fo [lowing photopolymerization residual alkenes and/or thiols of the polymer nanocapsules are covalentiy modfied with one or more functional groups that impro ve the water solubility of the nanocapsules; and

wherein residual alkenes and/or thiols of the polymers are optionally covalentiy modfied with one or more functional groups that contain a fluorescent moiety.

In specific embodiments, residual alkenes are functionalized with with one or more functional groups that improve the water solubility of the polymer. In specific embodiments, residual thiols are functionalized with one or more functional groups that improve the water solubility of the polymer.

In specific embodiments, residual alkenes are functionalized with with one or more functional groups thai comprise one or more fluorescent moieties, in other embodiments, residual thiols are functionalized with one or more functional groups that comprise one or more fluorescent moieties.

In specific embodiments, the functionalized polymers of resorcinarenes described herein may be functionalized by a chemical group that is suitable for bioimaging. In a particular embodiment, the functional group suitable for bioimaging may contai a f!ttorophore, for example, a fluorescent dye.

In an embodiment of the present invention, upon administration to a human or an animal, the functionalized polymers of resorcinarene nanocapsules of the present

Invention may be cleared via the renal filtration route,

In certain embodiments, the nanocapsules of the present invention may he hollow, la other embodiments, the nanocapsules may encapsulate a substance. Examples of substances that may be encapsulated within the nanocapsuie are chemical compounds, biological compounds and metals. The chemical compounds, include, but are not United to active pharmaceutical ingredients (APIs), proteins, peptides, polypeptides,

organometaiiic compounds, solvents, and salts.

Another embodiment of the present invention is a process of making the functionalized polymer resorcinarene nanocapsules of the invention comprising photopolymerization of the thiol arid alkene moieties of a compound of formula (I), wherein the residual alkenes and thiols of the polymer resorcinarenes are further co.valently modified by moeities containing functional groups.

Another embodiment of the present invention is a process of functionaliz ng the nanocapsules described herein, by covaiently modifying residual thiols or alkenes, so that the -resulting nanocapsules comprise moieities that improve the water solubility of the nanocapsules and/or fluorophores. in certain embodiments, one or more of the functionalization steps is followed by the ionization of one or more functional groups.

In some embodiments, the present invention provides: (a) a direct, template-free fabrication of resorcinarene nanocapsules by thioi-ene photopolymerization; (b) generation of rich polymeric architectures by varying the photopolymerization reaction medium; and (c) a one-pot synthesis and functionalization of resorcinarene nanocapsules by covalently attaching moieities that impro ve the water solubility of the nanocapsules and/or have fluorescent properties.

The present invention also relates methods of using the fimctionaHzed nanocapsules. In particular, functionafized nanocapsules of the present invention may be administered to a human or animal subject. For example, the nanocapsules may encapsulate a substance for treating and/or preventing a disease and/or condition. In. this fashion, the nanocapsules may serve as a delivery system for the substance that is being administered to treat and/or prevent a disease and/or condition.

In another embodiment, the nanocapsules of the present invention may be

administered for bio-imaging applications. For example, the nanocapsules may themselves contain covalently attached moieties that allow for bio-imaging, and/or they may encapsulate substances that are capable of being used for bio-imaging.

In an embodiment, the present invention provides a method of tracing the presence of a nanocapsules administered to a human or animal in vivo. For example, the distribution of the nanocapsules within the body may be traced by bio-imaging techniques, such as, fluorescence. In another embodiment, the present invention includes a method of administering nanocapsules, wherein the nanocapsules may be eliminated via renal clearance.

It is further noted that the invention does not intend to encompass within the scope of the invention any previously disclosed product, process of making the product or method of using the product, which meets the written description and enablement requirements of the USPTO (35 U.S.C. 1 12, first paragraph) or the EPO (Article 83 of the EPC), such that applicants) reserve the right and hereby disclose a disclaimer of any previously described product, method of making the product or process of using the product.

It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised", "comprising^"' and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "'includes", "included", "including", and the like; and that terms such as "consisting essentially of ^' and "consists essentially of have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude dements that axe found in the prior art or that affect a basic or novel characteristic of the invention.

These and other embodiments are disclosed or are apparent from and encompassed by, the following Detailed Description.

BRIEF PESCR1 PTION OF THE DRAWINGS

FIG. 1 A. An example of a resorcinarene tetra alkene tetra thiol (RTATT) monomer

FIG. IB, Transmission electron microscopy (TEM ) of RTATT nanocapsules obtained. FIG. iC, Scanning electron microscopy (SEM) of RTATT nanocapsules obtained.

FIG. 113. Dynamic light scattering (DLS) size data of RTATT nanocapsules obtained, FIG. 2. One pot synthesis and surface functionaiization of nanocapsules under

photopolymerization conditions.

FIG. 3, Fourier transform infrared (FTIR) spectra of a) nanocapsules b) vinyl acetic acid functionalized nanocapsules and c) mercapto propionic acid functionalized nanocapsules. Samples b) and c) were subjected to multiple water washes to remove unbound vinyl acetic acid or mercapto propionic acid,

FIG. 4. Atomic force microscop (AFM) characterization of water soluble nanocapsules before and after Alexa Fluor functionaiization.

FIG, 5, Synthesis Alexa Fluor 750 functionalized, water-soluble resorcinarene nanocapsules. FIG. 6. FTIR spectra of RTATT nanocapsules before and after MPA functionaiization.

FIG. 7, DLS (left), absorption (right, solid fine) and emission spectra (right, broken line, with an excitation wavelength of 750 nm) of Alexa Fluor 750 functionalized nanocapsules, AF-NC-A ( i) and AF-NC-B (H). Absorbance and emission spectra (iii) of 3-niercapto- i- propanol functionalized Alexa Fluor 750 (control dye) are also included. TEM images were obtained from Os0 stained samples.

FIG. 8. Plots of fluorescence intensities in distinct locations (bladder, upper body and abdomen) over time in mice injected with (a) AF-NC-A (b) AF-NC-B and (c) control dye. FIG. 9. Whole body fluorescence imaging of mice (ventral aspect ) injected with nanocapsules AF-NC-A (row A), AF-NC-B (row B) and control dye (row C) over a period - 5.5 h. Images were obtained in epi -illumination mode with an excitation wavelength of 745 nm and an emission wavelength of 800 nm, DESCRIPTION OF THE INVENTION

The present invention relates to functionalized polymeric resorcinarene nanocapsules. in particular, the nanocapsules described herein may comprise moeities that that improve the water solubility of the nanocapsul es as compared to

unfunctionalized nanocapsules.

Another aspect of this invention, is thai the functionalized polymeric resorcinarene nanocapsules of this invention may undergo renal filtration and excretion. In particular, the nanocapsules of the invention may be cleared from the body in a reasonable period of time in accordance with FDA guidelines.

in yet another aspect of this invention, the functionalized polymeric resorcinarene nanocapsules comprise covaleniiy bound groups comprising fluorescent nioities. In particular, the fluorescent moieties may be suitable for bioimaging and/or tracing the presence of nanocapsules within humans and/or animals.

In an embodiment, the functionalized polymeric resorcinarene nanocapsules comprise one or more functional groups covalently bound to the surface of the

nanocapsule. In an embodiment, the functional groups improve the water solubility of the nanocapsules when compared to their unfunctionaiized state.

In other embodiments, the functionalized polymeric resorcinarene nanocapsules comprise functional groups covalently bound to the surface of the nanoparticle, where the covalently bound functional groups comprise a fluorescent moiety. For example, the fluorescent moiety .may be a fluorophore for bioimaging. In a particular embodiment, said fluorophore is a fluorophore that is generally recognized as safe (GRAS) for human administration by FDA.

In certain embodiments, the functionalized polymeric resorcinarene nanocapsules of the present invention comprise one or more functional groups for improving the water solubility of the nanocapsule and a another functional group with a fluorescent moiety. i another embodiment, the polymeric nanocapsules are functionalized with charged functional groups. In another embodiment, the polymeric nanocapsules are functionalized with polar hydrophilic functional groups.

wherein:

X¹, X\ X³ and X are independently ~(CH₂)«- ;

n is from 0 - 8;

R¹, R², R³ and R⁴ are independently each an alkene; and

wherein following photopolymerizalion residual thiols and/or alkenes are further functionalized- with a group that improves water solubilit of the nanocapsuies and/or a group that contains a fluorescent moiety.

In one aspect of the resorcinarene polymer nanocapsule, for the compound of formula (I):

X², X³ and X⁴ are independently -(CI¾V ;

n is from 0 ··- 8:

R¹, R², R^J and R^'÷ are each independently a C₂-C30 alkene; and wherein following photopolymerizati on residual thiols and/or alkenes are further

^■functionalized with a group that improves water solubility of the nanocapsuies and/or a group that contains a fluorescent moiety,

in another aspect of the resorcinarene polymer nanocapsuies, for the compound of formula (1): n is 0;

R^*, R^*", R and R^" are each independently a C₁₂-C₂₂ aikene; and

wherein following photopolymerizaiion residua! thiols and/or alkenes are further funct onalized with a group that improves water solubility of the nanocapsules and/or a group that, contains a fluorescent moiety.

In another aspect of the resorcinarene polymer nanocapsules, for the compound of formula (!):

n is 0;

R^!, R^z, R³ and R⁴ are independently a C4-C₁₂ alkene; and

Wherein following photopolymerizati on residual thiols and/or alkenes are further functionalized with a group that improves water soiubility of the nanocapsules and/or a group that contains a fluorescent moiety,

in another aspect of the resorcinarene polymer nanocapsules, for the compound of formula (I):

-R = -C₈H ₆CH=CH₂ ana

wherein following photopolymerizaiion residual thiols and/or alkenes are further functionalized with a group that improves water soiubility of the nanocapsules and/or a group that contains a fluorescent moiety.

In certain embodiments, the functionalized nanocapsules of the present invention are insoluble in water in their non-ionized form, but soluble in their ionized form.

in certain embodiments, the functional group n oieities are covalentiy bound to the surface of the nanocapsule in a random manner. In one embodiment, the random arrangement of functional groups prevents the nanocapsules from crystallizing and/or precipitating. In a particular embodiment, the funciionalization allows the minimization or elimination of self-quenching of fluorescent groups.

The nanocapsules described herein may be eliminated via renai clearance. This is particularly noteworthy, because in some embodiments the nanocapsules of the present invention have hydrodynamic diaineters that are larger than typically associated with compounds tha can undergo renal clearance. In one embodiment, the nanocapsules have a hydrodynamic diameter of about 40 to about 150 nm, 50 to about 140 nm, or about 60 to about 130 nm. in other embodiments the nanocapsules may have hydrodynamic diameters of about 70 to about 90 nro, about 90 to about 1 10 nm, about 1 10 to about 130 nm, or about 130 to about 150 nm.

In certain embodiments, the nanocapsules of the present invention may be hollow. In other embodiments, the nanocapsules may encapsulate a substance. Examples of substances that may be encapsulated within the nanocapsule are chemical/biological compounds, chemical elements and metals. For the purpose of illustration the chemical compounds may incude include, but are not limted to, antibodies, deoxyribonucleic acid (D A), ribonucleic acid (RNA), pharmaceutical ingredients (APIs), proteins, peptides, polypeptides, organomeiallic compounds, solvates and/or salts thereof

The present invention also relates nanocapsules. which encapsulate metal nanoparticies. The present invention also relates to compositions which comprise the metal nanoparticies that are stabilized and encapsulated in the nanocapsule polymers of resorc arenes.

The metal of the metal nanoparticies is selected from the group consisting of a metal from Groups 4-12 of the Periodic Table. (Groups 4-12 metals refers to the nomenclature recognized by the international Union of Pure and Applied Chemistry (IUPAC) as of 21 January 201 1 may also be recognized under CAS as being Group !VB- VOB, VII , IB and ΙΪΒ). The metal nanoparticies may be obtained by methods known in the art, e.g. reduction of metal salts to form metal nanoparticies.

In other embodiments, the metal of the metal nanoparticies is selected from the group consisting of a metal Au, Ag, Cd, Hg, Os, Pb, Pt, Pd, R.h₅ Ru and Zn. In another aspect of the invention, the metal of the meta l nanopariieles is selected from the gro up consisting of Au, Ag, Pt, Pd, Rh, and Ru,

in another embodiment, the metal nanoparticles are gold (Au) nanoparlicles.

Definitions

The terms used herein are intended to have their customary meaning in the art, unless otherwise explicitly indicated.

The term "functional group" may refer to an atom or atoms within a molecule, which affects that chemical behvior or reactivity of the molecule. A functional group of the present invention may be neutral, charged or zwitterionic. A functional group of the present invention may be in salt form.

For illustration purposes non-limiting examples of functional groups of interest axe halides, alcohols, ethers, amines, nitrites, nitro groups, sulfide groups, sulfoxide groups, suSfone groups, thiol groups, aldehydes, ketones, carboxylic acids, esters, amides, carboxylic acid chlorides, carboxylic acid anhydrydes, amino acids, phosphonic acid, sulph.on.ic acid, and corresponding ions or salts thereof.

in an embodiment, the functionalized polymers of resorcinarenes are

functionalized with charged or ionized functional groups, The charged iunciional groups may have a positive or negative charge. For example, the functional groups may be present in ionized form accompanied by a counterion.

in an embodiment, the charged functional groups have a negative charge. In another embodiment, the charged functional groups have a positive charge. The functionalized polymers of resorcinarenes described herein, may be in salt form.

In an embodiment, residual thiols or alkenes of this invention may be

functionalized with a compound selected from the group consisting of po3y(ethylene glycol) methacrylate (PEGMA), 3 -mercapto ropionic acid (MPA), 3-butenoic acid and 3- mercaptopropanoi. in another embodiment, the functional moities of the present invention may comprise -S(Cll2)m~C(0)OH_> -S(CH2)_m-C(0)NH2, or any ion or salt thereof, wherein m is from 0-8.

in an embodiment, the functional moiety is a -S(Cil₂)_m-C(0)0^"M^'!', wherein M is a pharmaceutically acceptable salt. In an embodiment, the water soluble functional group is -S(CH₂)2-C(0)0^'M⁺. In particua!ar embodiment M is a sodium. In a specific embodiment, water soluble functional group is -S(CH₂)₂-C(0)0^"Na⁺

in another aspect of the invention, residual thiols and/or alkenes have been functionalized with either R⁵-CH~CH₂ or R⁶~SH, wherein:

R⁵ and R⁶ are independently -CrGj-alkyl-OH, -Ci-C₄-alkyl-C(0)OH_f -Ci-C - alkyl-C(0)NH₂ or -Ci-C₄-alkyl-C(0)OR⁷,

wherein

R⁷ is H, a ~Cj-C₄-aikyl or -(C_s -C_halky l-0},-,H wherein n is 0-4,

in an embodiment, R⁶ is -CH₂-CH₂-C(0)OH.

"Fluorescent groups," "fluorescent moieities" or "fluorophores," refer to chemical gropus that can he chosen to absorb and emit light in the visible spectrum or outside the visible spectrum, such as in the ultraviolet, or infrared ranges. In a specific embodiment, f!uorophores of the present invention may be a near infrared (NIR) emitting fluorophores.

Suitable fluorophores of the present invention include, but are not limited to, Alexa Fluor dyes (Invitrogen), eoumarin, fluorescein (e.g., 5-carboxyfluorescein (5- FAM), 6-carboxyfluorescem (6-FAM), 2',4', 1 ,4,-tetrachlorofluorescein (TET),

2'_>4',5^I,7', 1 ,4~hexach!oroiluorescein (HEX), ana 2\7^l-dimethoxy-4\5'-dichlorx>-6- carboxyfluorescein (JOE), Lucifer yellow, rhodaxnine (e.g., tetrameth l-6- carboxyrhodamrae (TAMRA), and tetrapropano-6-carboxyrbodamine (ROX)), 4,4- difiuoro-5,7~dim.ethyi-4-bora-3a,4a-diaza-s-indacene (BODIPY), DABSYL, DABCYL, cyanine (e.g., Cy3, Cy5, and Cy7), cosine, Texas red, ROX, quantum dots, a thraquinone, nitrothiazole, and nitroimidazole compounds, Quasar and Cal-fluor dyes, and dansyl derivatives, Combination fluorophores such as fluorescein-rhodamine dinners are also suitable.

The terms "residual alkene" and "residual ene" may refer to alkenes or carbon - carbon double bonds that are on the surface of the nanocapsule after following

pbotopolymri zation. Thes residual groups must be capable of undergoing a reaction with one or more chemical compounds, for example a chemical compound with one or more functional groups, to form a covalent bond. In particular embodiments, residual alkenes or enes may undergo such reactions under photopolymerization conditions.

The term "residual thiols" refers to thiol (-SB) groups of the nanocapsule that remain following photopolymrization to form the polymeric nanocapsules of the invention, in certain embodiments, residual thiol groups may undergo reactions with chemical compounds containing functional groups and/or fluorophores.

"Salts" of the compounds of this invention include those derived from

pharmaceutical ly acceptable inorganic and organic acids and bases. In certain

embodiments, salts of the present invention may be found on FDA's GRAS salts.

Non-limiting examples of.suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, besylate, bisulfate, bromide, butyrate, citrate, camphorate, camphorsulfonate, chloride, cyciopentanepropionate, digiuconate, dodecyls^'ulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycol ate, hemisulfate, heptanoate, hexanoaie, hydrochloride, hydrobromide, hydroiodide. 2-hydroxyethanes lfonate, iodide, lactate,, maleate, malonaie, methanesulfonate, 2- napfathalenesulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate, 3 - phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate.

Salts derived from appropriate bases include alkali metal (e.g., sodium and potassium), alkaline earth metal (e.g., magnesium and calcium) salts, ammonium salts and N⁺¾(CM a3kyi)_y, wherein x is 0-3 and y is 1 -4, provided that x+y-4, salts. This invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil -soluble or dispersible products may be obtained by such uaternization.

Whenever a range is referred to herein, the range includes independently and separately ever member of the range. As a non-limiting example, the term "Cj-Cio alkyi" is considered to include, independently, each member of the group, such that, for example, Cs -C;o alkyi includes straight, branched and where appropriate cyclic d, C₂, C3, C₄, Cs, C₆, C₇, Cg, C and C ; Q alkyi functionalities. Similarly, as another non-limiting example, 1 - 10% includes independently, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 0%, as well as ranges in between such as 1 -2%. 2-3%, etc.

Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the i? and S configurations for each

asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the invention.

Processes of Making Res £iaare¾e Polymer

The present invention also relates to processes of making polymers of

resorcinarenes via phoiopolymerization. In particular, the present invention includes processes of synthesizing functionalized resorcinarene polymer nanocapsules.

in certain embodiments, the process for the synthesis of functional! zed resorcinarene polymer nanocapsules comprises:

(a) a phoiopolymerization step wherein monomers of formula (I);

wherein:

X\ X\ X³ and X⁴ are independently -(CH₂)„- ;

n is from 0 - 8;

R\ Rl R³ and R⁴ are independently an alkene; and

are irradiated to form resorcinarene polymer nanocapsules:

(b) a first fuiictlonalization step, wherein residual thiol or alkene groups of the resorcinarene polymer nanocapsules are covalently functionalized with, moieties that improve water solubility of the nanocapsules; and

(c) a second functionalization step, wherein residual thiol or alkene groups of the resorcinarene polymer nanocapsules are covalently functionalized with fluorescent moieties. In certai n embodiments of the process, steps (a) arid (b) are done in the same reaction vessel.

In certain embodiments of the process, the photopo lymerization reaction of step (a) take place o ver a time period of greater than 1 minute and less than 6 hours. In other embodiments, the photopolymerization reaction of step (a) take place over a time period of greater than 10 minutes and less than 3 hours, m other embodiments, the

photopolymerization reaction of step (a) takes place over a time period of 1 to 60 minutes, 5 to 45 minutes, or 10 minutes to 30 minutes.

The wavelength of the photopolymerization reaction may be in the UV range. in another aspect of the process, residual thiols and/or alkenes are functionalized in ther first funciionaiization step (b) with either R^;~CH=^:CH? or R⁶-SH respectively, wherein:

R⁵ and R⁶ are independently -Ci-C₄-alkyl-OH, -Cj-C₄-alkyl-C(0)OH or -C_{-C₄ alkyl-C(0)OR⁷,

wherein

R⁷ is H, a -Cj ~C₄-alkyl or ~(C] -C₄-alkyI-0)_?JH wherein n is 0-4,

In another aspect of the process, residual thiols or alkenes may be functionalized with a compound selected from the group consisting of poly(ethylene glycol}

methacryiate (PEGMA), 3 -mercaptopropionic acid (MP A), 3-butenoic acid and 3- mercaptopropanol .

in certain embodiments, the first funciionaiization step (b) further comprises the ionization of the functional group,

The present invention also relates to a process of stabilizing and encapsulating metal nanoparticles by adding nanoeapsules formed by polymers of resorcinarenes to the metal nanoparticles.

.In a specific embodiment, a process of making resorcinarene polymer

nanoeapsules comprises:

a) photopolymerization of compounds of formula (I);

b) functional ization of residual alkeiie or thiol groups; and

c) an optional second fnnctionalizontion of residual alkene or thiol groups.

In certain embodiments the photopolymerization and funciionaiization steps are completed in a single pot.

in some embodiments, the first funtionalization step (b) is completed by a photochemical reaction. In some embodiments, some residual aikenes remain

unfunctionalized after a functional ization step, In some embodiments, some residual thiols remain unfunctionalized after a functional ization step. Ln other embodiments., step (c) is not an optional step of the process,

In some embodiments of the process, step (b) comprises the addition of reagents with one or more water soluble moieities. In some embodiments, the reagent with one or more water soluble moieities may be reacted with the product of step (a), such that the ratio of reagent to monomer of formula (I) is from about 0.5: 1 to about 20: 1 , about 1 : 1 to about 1: 1 0, about 2:1 to about 15: 1, about 4: 1 to about 12: 1 , or about 6: 1 to about 10: 1.

In some embodiments, the processes described herein comprise an ionization step. For example, functionalized nanocapsules may be further reacted with an acid or base to form ions or zwitterions. As another embodiment, processes of the invention include steps wherein the tunctionalized nanocapsul es are converted to salt form.

An embodiment of the present invention is a process or forming water soluble functionalized nanocapsules, comprising the step of ionizing a functional ized nanocapsule that is insoluble in water in its non-ionized state, whereby the functionalized nanocapsule becomes water soluble foi lowing the ionization reaction.

(a) Nanocapsule formation

The following description is meant to provide a description of the process of forming water soluble resorcinarene polymer nanocapsules, and is considerd non-lirniting. A solution of RTATT in solvent, for example chloroform, is filtered through a nylon membrane and transferred into a quartz tube.

The RTATT solution is degassed with an inert gas, for example argon, sealed, and irradiated in a photoreaetor, such as a Srmivasan-Griffin Rayo.net photoreaetor equipped with 4 x 254 nm (35 watts) and 4 x 300 run (21 watts) lamps.

(b) Nanocapsule fuKctionalizatson with a water soluble group

Following irradiation, the nanocapsules are allowed to stand in dark for another 15 min, after which a thiol compound for functionalization, such as a .mercapto-carboxyiic acid compound, is added to the solution in excess. In certain embodiments, the

I compound for functionalizauon is 3-mercapfo-propionic acid (MPA) in a ratio of 8 equivalents with respect to RT^'ATT monomer.

The reaction mixture is degassed with an inert gas such as Ar for approximately 30 sec, sealed and UV irradiation is continued for an additional 30 min. The solution of MPA functional! zed nanocapsules in chloroform thus obtained is concentrated to dryness,

(c) Functionalized anoea suSe ionization

The following step is an ionization step, where the residue is dispersed with a base in an amount sufficient to ionize the newly added functional moeity. in a certain embodiments, the base is an equal volume of aqueous NaOH (0,08 M) solution. This may result in the solubilization of the nanocapsules in water.

At this stage, insoluble material present, if any, may be removed by filtration, for example via 0.45 μ*η nylon syringe filter. The water soluble nanocapsules are then dialyzed against water for a period of time. For example, the functionalized nanocapsules may be transferred into a spectra por membrane (25,000 molecular weight cut off) and dialyzed against water for at least 3 days with several water changes.

(d) Nanocapsule fimctionalization with a bioiin aging group, such as a group with a fluorescent moiet

Water soluble functionalized nanocapsules may be further functionalized with imaging groups, such as chemical groups containing fluorescent moieities. In a specific embodiment, the nanocapsules are Alexa Fluor 750 functionalized nanocapsules. in a typical fimctionalization procedure, degassed solutions of dialyzed functionalized resorcinarene nanocapsules in water, NC-A (3,4 mL, 0,45 mg of poiymer/mL) and NG-B (5.6 mL, 0.71 mg of RTATT/rnL for NC-B), axe reacted with maleimide functionalized Alexa Fluor 750 (~ 0.3 mg in 0.3 mL) for 2 hours under Ar atmosphere in dark. Alexa Fluor 750 functionalized nanocapsules, labeled AF-NC-A and AF-NC-B from NC-A and NC-B respectively, are transferred into a spectra por membrane (25,000 molecular weight cut off) and dialyzed against water exhaustively to remove unreached dye. Alexa Fluor functionalized nanocapsules are protected from light during all these operations.

Another aspect of the process, comprises an additional encapsulation step, wherein one or more substances are encapsulated by the nanocapusuies of the invention.

In another aspect of this process, the metal of the metal nanoparticles is selected from the group consisting of a metai from Groups 4-12 of the Periodic Table. (Groups 4- 12 metals refers to the nomenclature recognized by the international Union of Pure and Applied Chemistry (TUPAC) as of 21 January 201 1 ; may also be recognized under CAS as being Group IVB-VOB, VII], IB and IIB). The metal nanoparticles may be obtained by methods known in the art, e.g. reduction of metal salts to form metal nanoparticles.

In another aspect of this process, the metal, of the metal nanoparticles is selected from the group consisting of a metal Au, Ag, Cd, Hg. Os, Pb, Pt, Pd, Rh, Ru and Zn. In another aspect of the invention, the metal of the metal nanoparticles is selected from the group consisting o Au, Ag, Pt, Pd, Rh, and Ru.

In another aspect of the process, the metal nanoparticles are gold (Au) nanoparticles.

it is further noted that the direct, template -free synthesis and in situ

functionalization method for nanopcapsules, substantially reduces the number of synthetic operations typically employed.

Described herein is a direct, template- and emulsion- free method for the synthesis of hollow polymeric nanocapsules. See Balasubramaman et a!,, "Solvent dependent

morphologies in thiol-ene photopolymerization: A facile route to the synthesis of

resorcinarene nanocapsules," J Mater Chem. 2010; 20(31): 6539-43. The

photopolymerization of resorcinarene tetraaikene tetrathioi (RTATT, FIG. 1), a single- component multi-thiol and muiti-ene monomer, resulted in. the formation of hollow polymeric nanocapsules. Id

Given the surface engineering via the inventive one pot synthesis and

functionalization approach and their inherent guest encapsulation capability, the nanocapsules and .nanoparticle-nanocapsule composites of the invention are ideal platforms for "theranostic nanomedicine", (The integration of diagnostic and therapeutic functions is labeled as

"theranostic nanomedicine").

Therefore, it is an aspect of this invention to provide methods for the use and administration of the resorcinarene nanocapsules described herein. In particular,

nanocapsules of the present invention may be admini stered to a human or animal subject. For example, the nanocapsules may encapsulate a substance that is suitable for treating and/or preventing a disease or condition. In this fashion, the nanocapsules may serve as a delivery system.

An embodiment of the present invention is a method of administering

functional ized resorcinarene polymer nanocapsule, wherein the resorcinarene polymer nanocapsule is formed by the ihiol-ene photopolyraerization of a compound of formula

(I):

(!)

wherein:

X¹, X², X^'' and X⁴ are independently -(CH₂)_n- ;

n is from 0 - 8:

R⁵ , R% R^! and R⁴ are independently an alkene; and

wherein residual alkenes of the photopolymerization product are covalentiy modfied with one or more functional groups thai improve the water solubility of the polymer;

wherein residual thiols are covalentiy modified with one or more functional groups containing a fluorescent moiety; and

wherein the functional ized resorcinarene polymer nanocapsuie are capable o f being excreted through renal clearance.

In. another embodiment, the nanocapsules of the present invention ma be

administered for bio-imaging. For example, the nanocapsules may themselves contain cova!entiy attached moieties that allow for bioimaging, and/or they may encapsulate substances that are capable of being used for bio-imaging,

in an embodiment, the present invention provides a method of tracing the presence of a nanocapsules administered to a human or animal, In another embodiment, the method includes the tracing of nanocapsules comprising one or more therapeutic agents and bio- imaging agents.

In another embodiment, the present invention includes a method of administering nanocapsules, wherein the nanocapsules may be eliminated via renal clearance. In one embodiment, the clearance of nanocapsules described herein begins to appear in the bladder within 0.01 to 1 hours, 1 to 3 hours, 3 to 6 hours, 7 to 12, 12 to 18 hours, or 1 to 36 hours post-administration,

In another embodiment;, nanocapsules that have been administered to a human or animal are completely cleared within 1 to 72 hours, I to 48 hours, 1 to 36 hours, 1 to 24 hours, 1 to 12 hours, 1 to 6 hours, or I to 3 hours. In other embodiments, the nanocapsules are cleared within I to 3 hours, 3 to 6 hours, 6 to 12 hours, 12 to 24 hours, 24 to 48 hours, 48 to 72 hours, 72 to 96 hours, or 96 to 120 hours.

Compositions for administration to humans or animals comprising functional ized resorcinarene polymer nanocapsules of the invention are formulated to be compatible with their intended routes of administration, e.g., parenteral, subcutaneous, injectable, intravenous, oral, intradermal, subcutaneous, transdermal, (topical), transmucosai, and rectal

administration.

EXAMPLES

in the examples described herein, the fol lowing techniques and instrumentation were used in the characterization, animal studies and bioimaging of nanocapsules.

Characterization: AFM images were obtained in a Veeco di Nanoscope 3D using Tap 300-G taps (300 kHz resonant frequency and 40 N/rn force constant) supplied by

BudgetSensors under tapping mode conditions.

Samples for AFM analysis were prepared by drop-casting and drying nanocapsule dispersions on a freshly cleaned glass slide, TEM was carried out in a JEOL JEM-2100F field emission microscope operating at 200 kV equipped with a Gatars SC I 000 ORIUS CCD camera (1 1 megapixel).

Nanocapsules dispersions were stained by mixing them with an equal volume of 0.1% aqueous Os0₄ solution for at least 45 mm, prior to their deposition on a carbon coated copper grid and drying.

The dimensions of the nanocapsules were manually measured using ImageJ software. The size analysis by dynamic light scattering was performed on a Zetasizer nano (model: ZEN 3200) supplied by Malvern Instruments. UV-vis and Fluorescence spectra were recorded on a Gary 5000 UV-Vis-NIR and Gary Eclipse Fluorescence spectrophotometer respectively.

Fluorescence spectra were recorded with an excitation and emission slit width of 5 nm. FTIR spectra were recorded on a Thermo Electron Nicolet 370 DTGS spectrophotometer operating in transmission mode.

Animal studies and biosm aging. Hairless female mice (S H-1) were obtained from Charles River laboratories and maintained according to the protocol (number # 1 1 -01 1 ) approved by Old Dominion University's lACUC, Prior to the administration of nanocapsules and throughout the imaging time, mice were maintained under gas anesthesia (1.2 -^■ 1.5 % isof rane with 1 L/.m.in O2 flow) and supported by a heated pad during the experiment. AF- NC--A, AF-NC-B and control dye (50 ,uL) were diluted with an equal amount of 2x saline solution, filtered with a 0.45 μτη nylon fi lter to prevent any inadvertent introduction of larger or aggregated material, and intravenously injected into mice via the lateral tail vein.

Biodistribution of the nanocapsules was followed by whole body imaging using an IVIS .spectrum imaging system supplied by Caliper Lifesciences located in Old Dominion University's Center for Bioelectrics. The fluorescence imaging was carried out in epi- illumination mode in two different channels with an excitation and emission wavelength of 710 and 780 nm in channel 1. and 745 and 800 nm in channel 2. Fluorescence images obtained with arbitrary color scales were modified to make a valid comparison of the biodistribution. Unless otherwise mentioned, all images shown in this application have a mini-mum radiant efficiency of 4.0e7 and the maximum value was set at 8.0e9 for nanocapsules and 3.0e9 for the control compound, Example 1 : Synthesis and characterization of nanocapsules

Nanocapsuie synthesis was accomplished by irradiating a solution of RTATT monomer in chloroform (1.5 mM) for 3 h in a Rayonet reactor equipped with 4 x 254 nm and 4 x 300 nm lamps. The polymerization reaction was initiated by the generation of a tliiyl (RS¹) radical (eqn 1), which adds to the double bond (eqn 2); leading to the formal addition of the thiol across the double bond (eqn 3). See Hoyle at ah, "Thiol-enes: Chemistry of the past with promise for the future," J Poly m Sci Pol Chern. 2004; 42(21 ): 5301 -38; see also Kade et al, "The Power of Thiol-ene Chemistry," J Pofym Sci Pol Ghent 2010; 48(4): 743-50.

The fact that thiol-ene photopolymerization indeed took place was established by FR and NMR spectroscopies. The hollow nature of the nanocapsuie was established by

Transmission electron microscopy (TEM) analysis (FIG. IB), which showed the formation of a darker rim with a lighter core. Further, energy dispersive spectroscopic (EDS) analysis conducted on the rim and center of a nanocapsuie under identical conditions showed enhanced S content on the rim, when compared to the center of the nanocapsuie, confirming its hollow nature. The nanocapsuie dimension of 106 ± .18 nrn obtained by the size analysis of scanning electron microscopy (SEM) images (FIG. 1 C) agreed well with the ~ 90 nm size obtained from dynamic light scattering (DLS) data (FIG. I D).

Example 2; Synthesis of water soluble nanoeaps les RTATT nanocapsules, such as those described in Example 1 , contain residual thiol and ene groups even after UV irradiation for several hours. Id These functional groups are amenable for further reaction with other thiol or ene molecules under photopoiymerization conditions. This may be accomplished by a simple one-pot two-stage photoreaction for the fabrication and functionalization of nanocapsules as shown in FIG, 2.

In this example, RTATT nanocapsules were functionalized with representative thiol. 3-mercaptopropionic acid (MPA) according .to the protocol described below (FIG. 2). See Balasuhramanian et aL "Amphiphiles: Molecular Assembl and Applications," ACS Symposium Series, Vol, 1070, Chapter 16, pp 263-276.

RTATT monomer was phoiopolymerized for 15 minutes; as such the resulting nanocapsules had a higher amount of residual thiol and ene functional groups permitting maximum functionalization. In a typical synthesis and surface functional! zation experiment, RTATT solution (1.5 m!Vf) was irradiated for 15 min to generate nanocapsules, to which MPA (8 equiv) was added and UV irradiation continued for an additional 30 min. Unreacted MPA was removed by aqueous workup.

Atomic force microscopic (AFM) analysis of the parent nanocapsules, i.e., before MPA functionalization, showed a. nanocapsule diameter of 98.0 ± 28.4 nm (n ^~ 51), agreeing well with the dimension s of the resorcinarene nanocapsules obtained after 3 h of

photopolymerization. FTIR spectra of the functionalized nanocapsules clearly showed the presence of carbonyl. (~ 1700 cm^*1) and COOH (-3000 cm^"!) groups, confirming the incorporation of MPA. on nanocapsules (FIG. 6).

After evaporation of chloroform from the MPA functionalized nanocapsules (labeled NC--A), the residue obtained could be solubilized in water in the presence of excess aOH. The water soluble nanocapsules thus obtained were transferred into a spectr por membrane (25,000 molecular weight cut off) and dialyzed against water for at least 3 days with several water changes. The dialyzed MPA functionalized nanocapsules had an average dimension of 49,9 ± 7.5 nm (n - 84) and were significantly smaller than the parent nanocapsules.

Consistent with literature reports (See, e.g. , Kim E et aL, "Solvent-responsive polymer nanocapsules with controlled permeability: encapsulation and release of a

.fluorescent dye by swelling and desweiling," Chem Commun. 2009(12): 1472-4) occasionally we observed noticeable batch -dependent size variations in nanocapsules. Preliminary evidence suggests that such size variations could perhaps be related to the rate of Ar bubbling (degassing) prior to photopolymerization. Smaller nanocapsules were obtained when the rate of degassing was arbitrarily slo wer, Though oxygen does not fully inhibit thiol-ene photopolymerization, it can reduce the overall rate. See Ho ie et a!., Chem. Soc. Rev, 2010, 39, 1355,

In the case of photo polymerization reactions done with a slower degassing rate there may be a slight reduction in the rate of photopolymerization due to the presence of some residual oxygen, leading to a reduction in nanocapsule dimensions. Relatively smaller and more monodispersed parent, nanocapsules with dimensions of 71.5 ± 14.6 nm (n ^ 66) have also been produced. As before, such relatively smaller parent nanocapsules were also functional ized with MPA (labeled NC-B) and could be dispersed in water in the presence of excess base. Such nanocapsules were dialyzed and their AFM analysis gave an average dimension of 38.1 ± 6.2 nm (n 63).

Consistent with the functionalization of Aiexa Fluor dyes there was a slight increase in the dimensions of the nanocapsules as noticed in AFM and DLS (FIG. 5). The presence of larger entities in DLS perhaps suggests the aggregation of some of these nanocapsules.

Note that these AFM analyses described in the examples were done on dried samples drop-cast from either chloroform or water. In principle, such a size reduction could be due to either the selective solubilization of smaller nanocapsules in water or the shrinking of nanocap-sules in water.

To probe the origin of size reduction, nanocapsules dispersed in water were precipitated by the addition of a few drops of concentrated hydrochloric acid and the precipitate obtained was redispersed in chloroform. The AFM analysis of such

redispersed nanocapsules (with COOH functional groups) drop-cast from chloroform showed a much larger size when compared to the dimensions of the nanocapsules (with COONa functional groups) drop-cast from water. This ruled out the selective

solubilization of smaller nanocapsules in water and indicated thai the apparent size reduction observed by AFM for dialyzed MPA functioualized nanocapsules may be due to the shrinking of nanocapsules in water.

Further characterization of dye-funct onal ized water soluble nanocapsules (vide infra) indicated tha the apparent shrinking observed here could be due to drying effects rather than actual shrinking of nanocapsuies.

Exam le 3: Alternative synthesis of functionaHzed nanocapsuies

This one-pot synthesis and surface functionalization approach can also be

accomplished by addition of alkenes. RTATT nanocapsuies have been successfully fonctionalized with PEG A and other olefins.

Note that the normalized FTIR spectra (FIG, 3) of mercapto propionic acid functional ized nanocapsuies (c) showed more C-0 group intensity (around 1718 cm^"'} when compared to vinyl acetic acid fonctionalized nanocapsuies (b). Similar results were obtained with the alcohol counterparts as shown by NMR spectroscopy (results not shown). This approach can readily be applied for the functionalization of nanocapsuies with a variety of groups.

Example 4: Dye functionaHzaiion of nanocapsuies

Encapsulation and functional zat on of dyes in these nanocapsuies was accomplished by three distinct, approaches; in situ encapsulation (See, e.g., Kim D et a!., "Direct synthesis of polymer nanocapsuies with a noncova!ently tailorabie surface," Angew. Chem, Int. Ed, 2007; 46(19): 347.1 -4). post-synthetic encapsulation (See, e.g. , Kim E et aL "Solvent- responsive polymer nanocapsuies with controlled permeability: encapsulation and release of a fluorescent dye by swelling and desweliing," Chem Commun. 2009(12): 1472-4) and dye functi onalization .

This example focuses on the dye functionalization approach, in this approach, MPA- functionalized water soluble nanocapsuies. produced in accordance with Example 2 (2b in FIG. 2), were reacted with Alexa Fluor 750 - ma!eiraide in water as shown in FIG. 5. The nanocapsuies were dialyzed with a Spectra Por membrane (MW cutoff- 25,000) for 2 days with several water changes in dark.

Maieimide fonctionalized Alexa Fluor 750 was cova!ent!y attached to the nanocapsule surfaces through the residual thiol groups present on them (FIG. 5). These Alexa Fluor 750 functional-zed nanocapsuies, AF-NC-A and AF-NC-B, were marginally larger than their corresponding precursors NC-A and NC-B respectively. AFM analysis of AF-NC-A (FIG.7) gave an average dimension of 56.1 ± 10.8 nra (n 50). while AF-NC-B (FIG. 7) had a size of 43.1 ·ι· 6.7 nm (n - 97). These dimensions were further confirmed by TEM analysis. TEM also confirmed the hollow nature of the Alexa Fluor 750 functionalized resorcinarene nanocapsules. However, dynamic light scattering (DLS) experiments gave a hydrodynamic diameter of 122 nm and 68 nm for AF-NC-A and AF-NC-B respectively (FIG. 7).

Note that these dimensions from DLS measurements agree reasonably well with the dimensions of the corresponding parent nanocapsules of NC-A (98,0 ± 28.4 nm) and NC-B (71.5 ± 14.6 nm) obtained by AFM analysis.

Previously (See Balas bramanian et al . Mater. Chern. 2010, 20, 6539), we have shown a good correlation between the dimensions of the nanocapsules dispersed in chloroform from analytical techniques such as TEM. SEM and DLS. Based on these observations, it is inferred that the apparent reduction in dimensions of water dropcast nanocapsules observed in AFM could be due to drying effects rather than the shrinking of the nanocapsules in water.

The pliotophysicai characterization of Aiexa Fluor 750 functionalized resorcinarene nanocapsules, AF-NC-A and AF-NC-B, and 3-mercapio~1 -propanol functionalized Alexa Fluor 750 (as control dye) are provided in FIG. 7. Consistent with the specifications of the manufacturer for the parent dye. the absorption spectra of Aiexa Fluor 750 containing nanocapsules and control dye had absorption maxima at 754 ± 3 nm. When excited at 750 nm. they showed emission maxima ~ 778 ± 3 nm.

Based on the absorbances obtained from UV-vis spectra and die extinction coefficients of RTATT and Alexa Fluor 750, we estimated the amount of RTATT monomer and Alexa Fluor 750 as 0.37 mg/ L and 0.04 mg/roL in AF-NC-A, and 0.71 mg/mL and 0.03 rag mL in AF-NC-B. Assuming nanocapsules as perfect spheres 41 and a surface area of 1 nm² per resorcinarene monomer, the above estimate from the UV-vis data suggests the covalent incorporation of 2918 Alexa Fluor 750 molecules/ AF-NC-A nanocapsuie and 567 Alexa Fluor 750 moleeules/AF-NC-B nanocapsuie,

The average extinction coefficient of Alexa Fluor 750 is 270,000 M^"!cm^"'_? A!exa Fluor 750 functionalized nanocapsules, AF-NC-A and AF-NC-B, have extinction coefficients of 8.46 x 108 M^"'cm^"1 and 1.64 x 108 M^"!cm^"' respectively. These values are impressive, as they are at least 2 - 4 orders of magnitude higher than the extinction coefficient observed for quantum dots.

Example 5: Biodistribution of fluorescent functionalized nanocapsules in mice.

Alexa Fluor functionalized nanocapsules, produced as described in Example 4, were injected into mice. Their biodistribution and clearance was followed using IVIS imaging systems in the Center for Bioelectrics.

Prior to administration of nanocapsules, mice were sedated with Ketamine-HCl (lOOmg/kg) and Xylazine HQ (10mg/kg) IP. Mice were maintained under gas anesthesia (1 ,2-1.5% isolflurane with 1 L/min ⁽¾ flow) throughout the imaging time. Prior to injection, the nanocapsules were mixed with 2x brine and subsequently filtered through a 0.45 micron filter to avoid any inadvertent introduction of larger or aggregated material into the mouse.

The detailed time evolution of Alexa Fluor functionalized dyes in mice was analyzed, as shown in FIG. 9. Note that there was a slight delay (around 5 minutes or so) between the injection and the time imaging started. Within the first 5 minutes, the nanocapsules injected in the tail vein started to accumulate in the bladder. After 30 minutes it was dominant!}' present in the bladder. After 3 hours, nanocapsules were primarily located in the bladder and adjacent area.

After 6 hours of imaging, the mouse was sacrificed by cervical dislocation and the contents of the bladder removed through a syringe. The mouse was dissected to varying degrees and imaging studies confirmed the absence of nanocapsules. No te that the presence of nanocapsules in the dissected mouse was limited to the bladder (remaining nanocapsules) and near the tail region where the nanocapsules were initially injected. The microscopic (AFM and TEM) analysis of the recovered fluid contents from the bladder (after the imaging) confirmed the presence of nanocapsules.

The clearance of nanocapsules through the kidney in their intact form is noteworthy and opens up ne opportunities in biomedical imaging applications,

Example 6: Biohnstging with Alexa Fluor 750 functionalized uan capsaies

To evaluate the utility of Alexa Fluor 750 functionalized nanocapsules in biomedical imaging, we evaluated their biodistribution and clearance in hairless female mice (SKH- l) via whole body fluorescence imaging. Hairless mice were chosen because this minimizes aiitofluorescence from for. Given the negatively charged surfaces of these nanocapsules,_. they were expected to evade the mononuclear phagocyte system. Mice were anesthetized with isoflurane prior to the injection of AF-NC-A., AF-NC-B and the control dye in the lateral tail vein. Mice injected with, various fluorescent materials were placed in a ventral position in the rVIS spectrum imaging system and maintained under gas anesthesia during the entire imaging.

Fluorescence imaging was carried out in epi-illumination mode in 2 different channels with an excitation and emission wave-length of 710 urn and 780 ran i channel 1 and 745 run and 800 nm in channel 2. AH data shown in this article are from channel 2, as they showed higher fluorescence intensity when compared to channel 1 , The imaging data shown in this article were obtained by overlaying the fluorescence images on simultaneous mouse photographs. They show regions with higher NIR fluorescence intensity in brighter yellow colors and regions with lower NIR fluorescence intensity in darker colors.

FIG, 9 summarizes the biodistribution and clearance of AF-NC-A, AF-NC-B and control dye in mice over a period of ~ 5.5 h. Immediately after the injection of fluorescent material measurable fluorescence intensity was observed all over the mouse in ail three cases. Within the next few minutes, we observed an increase in the fluorescence intensity in the bladder. Note that NIR fluorescence could be observed even from the dorsal view. While active urination is blocked by gas anesthesia, occasionally we observed some minimal urination of fluorescent material by mice at the later stages of imaging.

At the end of the imaging, nanocapsules AF-NC-A and AF-NC-B and the control dye were primarily located in the bladder (FIG. 9). The observation of somewhat reduced fluorescence intensity in areas surrounding the bladder could be due to depth-dependent fluorescence scattering of fluorescent material in the bladder and not necessarily due to the presence of fluorescent material in those areas. Simulations using diffusion approximation of light transport under epi-illurnination conditions have shown that fluorescence intensity from deeply buried fiuorophores can result in blurred images.

There were significant differences in the clearance and the rate of clearance of fluorescent material from various regions of the mouse, depending on the nature of the injected fluorescent material. As seen in row A of FIG. 9, AF-NC-A, the nanocap-sules with larger dimensions, remained in circulation for the longest period of time and started clearing from the system at ~ 3 h. On the other hand, AF-NC-B, the relatively smaller nanocapsules were mostly localized in the bladder and the adja-cent area within 2,5 h (row B, FIG. 9). Not surprisingly, the Alexa Fluor control dye cleared in around 2 (row C, FIG . 9).

A semi -quantitative region of interest ( OI) analysis was carried out to compare the clearance profiles of fluorescent nanocapstiles and control dye. At least three distinct, non- overlapping ROI locati ons were chosen in the upper body of the mouse (near the bead and snout) and in the abdomen. Two overlapping ROFs were chosen in the bladder region. Ail these ROFs were circular regions of 5 mm width. The plot of the average fluorescence intensities from various ROFs in distinct locations at various time periods during the imaging are summarized in FIG. 8. In all three cases, the intensity in the bladder increased

significantly during the early stages and then eventually remained constant.

Interestingly, during the early stages of imaging with AF-NC-A there was a significant increase in the fluorescence Intensity in all three regions examined, i.e., in the upper body, abdomen and bladder (FIG. 8). The fluorescence intensity in the upper body and abdomen started reducing after ~ 60 min or so, although some residual fluorescence remained in the upper abdomen region even after several hours. At the end of the imaging, the ROI analysis showed that even in the most intense fluorescent spot in the abdomen region from AF-NC-A injection, the fluorescence intensity was only - 1 % of the maximum fluorescence intensity observed in the bladder. In contrast, AF-NC-B (FIG. 8) and control dye (FIG, 8) behaved differently. In these cases, reduction in the fluorescence intensity in the upper body and abdomen started right from the beginning. While a miniscule amount of fluorescent material was retained in the abdomen of the mouse injected with AF-NC-B, the mouse injected with control dye did not sho any noticeable fluorescence intensity in the abdomen at the end of imaging. The reduced presence of nanocapstiles in various locations such as brain, bone, blood and spleen, when compared to the bladder, suggests their lack of specific affinity to tissues.

After the imaging, the anesthetized mice were euthanized by cervical dislocation and the contents of the bladder removed through a syringe. M ice were dissected and imaged, which confirmed that the NIR fluorescence was primarily limited to the bladder (from residual nanocapsules), tail (the site where the nanocapsules were Initially injected) and the abdomen. The TEM analysis of the bladder contents (urine) of the mice injected with AF- NC-A and AF- C-B unambiguously revealed the presence of intact hollow nanoeapsules.

Note that these nanoeapsules appear somewhat elongated when compared to the nanoeapsules. Initial necropsy and histological analysis of the internal organs of the mice injected with nanoeapsules revealed no evidence of pathological effects. Note that this is based on a 6 h maximum observation window, and any effect withi that brief period would require a very strong biochemical effect induced by the nanoeapsules.

Overall, the whole body imaging studies and the presence of intact nanoeapsules in urine conclusively proves that these nanoeapsules are being cleared via the renal filtration route. The renal clearance of these nanoeapsules is impressive as several other nanoparticles including liposomes are known to dorainantiy accumulate in the liver and spleen. Also, the relatively rapid clearance of these nanoeapsules is noteworthy as polymer vesicles have extended circulation time. Though the specific mechanistic details of the glomerular filtration of these nanoeapsules are yet to be unraveled, it could be related to the urinary excretion of multi- and single- walled nanotubes. The apparent paradoxical filtration of intact nanotubes with lengths > 200 run. and thickness ~ 35 - 40 ran in urine was explained in terms of their rotational diffusivity by modeling studies.

Though we have studied the in vivo behavior of negatively charged PA

runctionalized nanoeapsules, the one-pot synthesis and functionalization approach reported here can be readily extended for the fabrication of resorcmare e nanoeapsules with other functional groups and surface charges.

Having thus described in detail various embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

Claims

1. A functionalized resorcinarene polymer nanocapsule, wherein the resorcinarene polymer nanocapsule is formed by the thiol-ene photopolymerization of a compound, of formula (I):

(I)

wherein:

X X², X³ and X⁴ are independently -(CH₂)_n- ;

n is from 0 - 8;

R\ R², R³ and R⁴ are independently an afkene; and

wherein residual alkenes or thiols of the photopolymerization product are covalently raodfied with one or more functional groups.

2. The functionalized resorcinarene polymer nanocapsule of Claim 1 , wherein residual alkenes of the photopolymerization product are co valently modfied with one or more functional groups.

3. The functionalized resorcinarene polymer nanocapsule of Claim 3, wherein the functional group modification result in improving the water solubility of the nanocapsule when compared to its unmodified state.

4. The functionalized resorcinarene polymer nanocapsule of Claim .1 , wherein residual thiols of the photopolymerization product are covalently modfied with one or more functional groups.

5. The functionalized resorcinarene polymer nanocapsule of Claim 2, wherein residual thiols of the photopolymerization product are covalently modfied with one or more functional groups that comprise a fluorescent moiety.

6. The fuacrionalized resorcinarene polymer nanocapsule of Claim 2, wherein the water soluble functional group comprises a functional group selected from halides, alcohols, ethers, amines, nitriles, nitro groups, sulfide groups, sulfoxide groups, sulfone groups, th ol groups, aldehydes, ketones, carboxylic acids, esters, amides, carboxylic acid chlorides, carboxylic acid anhydrydes, amino acids, phosphonic aeicl, sulphonic acid, and corresponding ions or salts thereof.

7. The functionalized resorcinarene polymer nanocapsule of Claim 6, wherein the water soluble functional group comprises a carboxylic acid, or corresponding ion or salt thereof.

8. The functionalized resorcinarene polymer nanocapsule of Claim 7, wherein the water soluble functional group comprises a carboxylate ion or salt,

9. The functionalized .resorcinarene polymer nanocapsule of Claim 7, wherein the water soluble functional group is -S(CH₂)_m-C(0)0^" a^t, wherein m is 0 to 8.

10. The functionalized resorcinarene polymer nanocapsule of Claim 9, wherein the water soluble functional group is -S(CH₂)₂-C(0)0^"Na^†.

1 1. The functionalized resorcinarene polymer nanocapsule of Claim 6, 7, 8, 9, or 10, further comprising residual thiols covalently modfied by a chemical group comprising a fluorescent moiety.

12. The functionalized resorcinarene polymer nanocapsuie of Claim 1 1 , wherein the fluorescent moiety is an Alexa Fluor.

13. A process for the synthesis of functionalized resorcinarene polymer nanocapsules comprising:

(a) a photopolymerization step wherein monomers of of formula (I):

CD

wherein:

X X², X³ and X⁴ are independently -(CH₂)„- ;

n is from 0 - 8;

R¹, R², R³ and R^"1 are independently an alkene; and

are irradiated to form resorcinarene polymer nanocapsules;

(b) a first functionalization step, wherein residual thiol or alkene groups of the resorcinarene polymer nanocapsules are covalently functionalized with moieties that improve the water solubility of the nanocapsules; and

(c) a second fanciionalization step, wherein residual thiol or alkene groups of the resorcinarene polymer nanocapsules are covalently functionalized with fluorescent moieties.

14. The process of claim 1.3, wherein the first functionalization step further comprises the ionization of the functional group.

15, The process of Claim 13, wherein steps (a) and (b) are done in the same reaction vessel

16, The process of Claim 12; wherein wherein the photopolymerization reaction of step (a) take place over a time period of greater than 10 minutes and less than 3 ho urs,

17, A method for bio-imaging comprising:

the administration of a functlonalized resoreinarene polymer nanocapsule, wherein the resoreinarene polymer nanocapsule is formed by the thiol-ene

photopolymerization of a compound of formula (I):

10

wherein:

X¹, X^?\ X^J and X⁴ are independently (C bj_n- ;

n is from 0 - 8;

R¹, R², R³ and R^-* are independently an a!kene; and

wherein residual alkenes of the photopolymerization product are covaiently roodfied with one or more functional groups that improve the water solubility of the polymer;

wherein residual thiols are covaiently modified with one or more functional groups containing a bio-imaging moiety; and wherein the functionalized resorcinarene polymer nanocapsiile are capable of being excreted through renal clearance.

18. The method of Claim 1 7, wherein the water soluble functional group comprises a carboxylic acid moiety, or a corresponding ion or salt thereof.

19. The method of Claim 18, wherein the water soluble functional group is

-S(CH₂)_m~C(0)0^"Na⁺, wherein m is 0 to 8.

20. The method of Claim 19, wherein the water soluble functional group is -S(CH₂)2~ C(0)(XNa⁺.

21. The method of Claim 17, wherein the bioimaging moiety is a fluorescent moiety.

22. The method of Claim 17, wherein the functional group for bioimaging is an Alexa Fluor,