WO2019108838A1 - Cross-linked fluorescent supramolecular nanoparticles and method of making - Google Patents

Abstract

A cross-linked fluorescent supramolecular nanoparticle (cFSNP) having a supramolecular nanoparticle (SNP), the SNP including a plurality of binding components, each having at least one binding region; a plurality of core components suitable to at least provide some mechanical structure to the supramolecular nanoparticle (SNP), the plurality of core components comprising each having at least one core binding element adapted to bind to the binding region to form a first inclusion complex; a plurality of terminating components, each having at least one terminating binding element adapted to bind to the binding regions to form a second inclusion complex, wherein the plurality of terminating components are present in a sufficient quantity relative to the binding region of the binding components to terminate further binding; a fluorescent conjugated polymer encapsulated within the SNP to form a fluorescent supramolecular nanoparticle (FSNP); and a cross-linking agent, and methods of manufacture and use are described.

Description

CROSS-LINKED FLUORESCENT SUPRAMOLECULAR

NANOPARTICLES AND METHOD OF MAKING

CROSS-REFERENCE OF RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No.

62/592,174 filed November 29, 2017, the entire contents of which are hereby incorporated by reference.

[0002] This invention was made with Government support under EB016270, awarded by the National Institutes of Health (NIH). The Government has certain rights in this invention.

BACKGROUND

1. Technical Field

[0003] The field of the currently claimed embodiments of this invention relates to supramolecular nanoparticles, and more particularly cross-linked fluorescent supramolecular nanoparticles.

2. Discussion of Related Art

[0004] Tattooing has been utilized by the medical community for precisely demarcating anatomic landmarks. This practice is especially important for identifying biopsy sites of non-melanoma skin cancer (NMSC) after the long interval (i.e., up to three months) between the initial diagnostic biopsy and surgical treatment. Commercially available tattoo pigments possess several issues, which include causing poor cosmesis, being mistaken for a melanocytic lesion, requiring additional removal procedures when no longer desired, and potentially inducing inflammatory responses. Therefore, there is a need for an ideal tattoo pigment for labeling of skin biopsy sites for NMSC that is (i) invisible under ambient light, (ii) detectable under a selective light source, (iii) retain a finite intradermal retention time (ca. three months), and (iv) biocompatible.

INCORPORATION BY REFERENCE [0005] All publications and patent applications identified herein are incorporated by reference in their entirety and to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

SUMMARY

[0006] An embodiment relates to a cross-linked fluorescent supramolecular nanoparticle (cFSNP) having a supramolecular nanoparticle (SNP). The SNP including a plurality of binding components, each having at least one binding region; a plurality of core components suitable to at least provide some mechanical structure to the supramolecular nanoparticle (SNP), the plurality of core components comprising each having at least one core binding element adapted to bind to the binding region to form a first inclusion complex; a plurality of terminating components, each having at least one terminating binding element adapted to bind to the binding regions to form a second inclusion complex, wherein the plurality of terminating components are present in a sufficient quantity relative to the binding region of the binding components to terminate further binding; a fluorescent conjugated polymer encapsulated within the SNP to form a fluorescent supramolecular nanoparticle (FSNP); and a cross-linking agent.

[0007] An embodiment relates to a method for delivering a fluorescent agent to a subject including the steps of: penetrating an epidermis tissue layer of the subject such that an accession point to an underlying dermis layer in the subject is created; and delivering a plurality of cross-linked fluorescent supramolecular nanoparticles (cFSNPs) to the underlying dermis layer in the subject through the accession point. In such an embodiment, each of the plurality of cross-linked fluorescent supramolecular nanoparticle (cFSNP) comprises a supramolecular nanoparticle (SNP) has: a plurality of binding components, each having at least one binding region; a plurality of core components suitable to at least provide some mechanical structure to the supramolecular nanoparticle (SNP), the plurality of core components comprising each having at least one core binding element adapted to bind to the binding region to form a first inclusion complex; a plurality of terminating components, each having at least one terminating binding element adapted to bind to the binding regions to form a second inclusion complex, wherein the plurality of terminating components are present in a sufficient quantity relative to the binding region of the binding components to terminate further binding; and a fluorescent conjugated polymer encapsulated within the SNP to form a fluorescent supramolecular nanoparticle (FSNP). The plurality of cross-linked fluorescent supramolecular nanoparticles (cFSNPs) are configured to retain the fluorescent conjugated polymer for a predetermined retention time, and the plurality of cross-linked fluorescent supramolecular nanoparticles are visible under a selective light source.

[0008] An embodiment relates to a method of making a cross-linked fluorescent supramolecular nanoparticle (cFSNP) including the steps of: synthesizing a plurality of fluorescent supramolecular nanoparticles (FSNPs) including: providing a first solution comprising a plurality of binding components, each having at least one binding region;

providing a second solution comprising a plurality of core components, each having at least one core binding element adapted to bind to the binding region to form a first inclusion complex; providing a third solution comprising a plurality of terminating components, each having at least one terminating binding element adapted to bind to the binding region to form a second inclusion complex, wherein the plurality of terminating components are present in a sufficient quantity relative to the binding region of the binding components to terminate further binding; providing a fourth solution comprising a fluorescent conjugate polymer; mixing the first solution, the second solution, the third solution, and the fourth solution, such that mixing brings into contact the plurality of binding components and the plurality of core components such that the plurality of binding components and the plurality of core components self-assemble to form the plurality of fluorescent supramolecular nanoparticles (FSNPs), and such that the fluorescent conjugate polymer is encapsulated within each of the plurality of fluorescent supramolecular nanoparticles (FSNPs); and cross-linking the plurality of fluorescent supramolecular nanoparticles (FSNP) using a cross-linking agent.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Further objectives and advantages will become apparent from a consideration of the description, drawings, and examples.

[0010] FIGS 1A-1C are schematics and images showing generation and use of the cross-linked fluorescent supramolecular nanoparticles (cFSNPs).

[0011] FIGS 2A-2B show a schematic of cFSNP assembly and graphs showing the results of fluorescent profile testing. [0012] FIGS 3A-3F are graphs showing results of characterization of fluroscence profiles of various cFSNPs.

[0013] FIGS 4A-4E are graphs and images of cFSNPs.

[0014] FIGS 5A-5E are images and graphs showing the results of a size-dependent intradermal retention study of c-FSNPs.

DETAILED DESCRIPTION

[0015] Some embodiments of the current invention are discussed in detail below. In describing embodiments, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. A person skilled in the relevant art will recognize that other equivalent components can be employed and other methods developed without departing from the broad concepts of the current invention. All references cited anywhere in this specification, including the Background and Detailed Description sections, are incorporated by reference as if each had been individually incorporated. In particular, U.S. Patent No. 9845237 and U.S. Patent Application No.

20160000918 are hereby incorporated by reference.

[0016] An embodiment relates to a cross-linked fluorescent supramolecular nanoparticle (cFSNP) having a supramolecular nanoparticle (SNP). The SNP including a plurality of binding components, each having at least one binding region; a plurality of core components suitable to at least provide some mechanical structure to the supramolecular nanoparticle (SNP), the plurality of core components comprising each having at least one core binding element adapted to bind to the binding region to form a first inclusion complex; a plurality of terminating components, each having at least one terminating binding element adapted to bind to the binding regions to form a second inclusion complex, wherein the plurality of terminating components are present in a sufficient quantity relative to the binding region of the binding components to terminate further binding; a fluorescent conjugated polymer encapsulated within the SNP to form a fluorescent supramolecular nanoparticle (FSNP); and a cross-linking agent.

[0017] An embodiment relates to the cFSNP above, where the fluorescent conjugated polymer and the binding components are present in a ratio (w/w) between about 0.001 : 1 to 0.2: 1. [0018] An embodiment relates to the cFSNP above, where the fluorescent conjugated polymer and the binding components are present in a ratio (w/w) between about 0.005: 1 to 0.125: 1.

[0019] An embodiment relates to the cFSNP above, where the core components and the binding components are present in a ratio (w/w) between about from about 0.1 : 1 to 2.5: 1.

[0020] An embodiment relates to the cFSNP above, where the core components and the binding components are present in a ratio (w/w) between about 0.15: 1 to 2.2: 1.

[0021] An embodiment relates to the cFSNP above, where the core components and the binding components are present in a ratio (w/w) between about 2.0: 1.

[0022] An embodiment relates to the cFSNP above, where the FSNP has a predetermined size of at least about 100 nm and less than about 1000 nm.

[0023] An embodiment relates to the cFSNP above, where the FSNP has a predetermined size of at least about 200 nm and less than about 700 nm.

[0024] An embodiment relates to the cFSNP above, where the FSNP has a predetermined size of about 670 nm.

[0025] An embodiment relates to the cFSNP above, where the fluorescent conjugated polymer is poly[5-methoxy-2-(3-sulfopropoxy)-l, 4-phenyl enevinylene] (MPS-PPV).

[0026] An embodiment relates to the cFSNP above, where the cross-linking agent is a cross-linker.

[0027] An embodiment relates to the cFSNP above, where the cross-linker is bis(sulfosuccinimidyl)suberate.

[0028] An embodiment relates to the cFSNP above, where the plurality of binding components comprises polythylenimine, po!y(L-lysine), or poly( -amino ester).

[0029] An embodiment relates to the cFSNP above, where the at least one binding region comprises beta-cyclodextrin, alpha-cyclodextrin, gamma-cyclodextrin, cucurbituril or calixarene.

[0030] An embodiment relates to the cFSNP above, where the plurality of core components comprise at least one of a dendrimer, branched polyethyleneimide, linear polyethyleneimide, polylysine, polylactide, polylactide-co-glycolide, polyanhydrides, poly-e- caprolactones, polymethyl methacrylate, poly(N-isopropyl acrylamide), polypeptides, polyamidoamine dendrimers, poly(prophylenimine) (PPI) dendrimer, triazine dendrimer, carbosilane dendrimer, poly(ether imine) (PETIM) dendrimer or phosphorus dendrimer.

[0031] An embodiment relates to the cFSNP above, where the at least one core binding element comprises adamantanamine, azobenzene, ferrocene or anthracene.

[0032] An embodiment relates to the cFSNP above, where the plurality of terminating components comprises at least one of polyethylene glycol, polymer, polypeptide, ligosaccharide, or polypropylene glycol) (PGG).

[0033] An embodiment relates to the cFSNP above, where the at least terminating binding element comprises adamantanamine, azobenzene, ferrocene or anthracene.

[0034] An embodiment relates to the cFSNP above, where the at least one binding region binds to the at least one core binding element or the at least one terminating binding element to form a molecular recognition pair selected from the group consisting of antibody- antigen; protein-substrate; protein-inhibitor; protein-protein; a pair of complementary oligonucleotides; and an inclusion complex.

[0035] An embodiment relates to the cFSNP above, where at least one of the first inclusion complex and the second inclusion complex is ada antane-P-cycl odextri n or azobenzene-a-cyclodextrin.

[0036] An embodiment relates to the cFSNP above, where the cross-linked fluorescent supramolecular nanoparticle (cFSNP) is cross-linked to one or more cross-linked fluorescent supramolecular nanoparticles (cFSNP) to form a fluorescent supramolecular nanoparticles complex or molecule.

[0037] An embodiment relates to the cFSNP above, where the fluorescent supramolecular nanoparticles complex or molecule has a predetermined size of at least about 300 nm and less than about 2,000 nm.

[0038] An embodiment relates to the cFSNP above, where the fluorescent supramolecular nanoparticles complex or molecule has a predetermined size of at least about 600 nm and less than about 1,600 nm.

[0039] An embodiment relates to the cFSNP above, where the fluorescent supramolecular nanoparticles complex or molecule has a predetermined size of at about 1,500 nm. [0040] An embodiment relates to a method for delivering a fluorescent agent to a subject including the steps of: penetrating an epidermis tissue layer of the subject such that an accession point to an underlying dermis layer in the subject is created; and delivering a plurality of cross-linked fluorescent supramolecular nanoparticles (cFSNPs) to the underlying dermis layer in the subject through the accession point. In such an embodiment, each of the plurality of cross-linked fluorescent supramolecular nanoparticle (cFSNP) comprises a supramolecular nanoparticle (SNP) has: a plurality of binding components, each having at least one binding region; a plurality of core components suitable to at least provide some mechanical structure to the supramolecular nanoparticle (SNP), the plurality of core components comprising each having at least one core binding element adapted to bind to the binding region to form a first inclusion complex; a plurality of terminating components, each having at least one terminating binding element adapted to bind to the binding regions to form a second inclusion complex, wherein the plurality of terminating components are present in a sufficient quantity relative to the binding region of the binding components to terminate further binding; and a fluorescent conjugated polymer encapsulated within the SNP to form a fluorescent supramolecular nanoparticle (FSNP). The plurality of cross-linked fluorescent supramolecular nanoparticles (cFSNPs) are configured to retain the fluorescent conjugated polymer for a predetermined retention time, and the plurality of cross-linked fluorescent supramolecular nanoparticles are visible under a selective light source.

[0041] An embodiment relates to the method above, where the predetermined retention time up to 100 days.

[0042] An embodiment relates to the method above, where the selective light source is ultraviolet light.

[0043] An embodiment relates to a method of making a cross-linked fluorescent supramolecular nanoparticle (cFSNP) including the steps of: synthesizing a plurality of fluorescent supramolecular nanoparticles (FSNPs) including: providing a first solution comprising a plurality of binding components, each having at least one binding region; providing a second solution comprising a plurality of core components, each having at least one core binding element adapted to bind to the binding region to form a first inclusion complex; providing a third solution comprising a plurality of terminating components, each having at least one terminating binding element adapted to bind to the binding region to form a second inclusion complex, wherein the plurality of terminating components are present in a sufficient quantity relative to the binding region of the binding components to terminate further binding; providing a fourth solution comprising a fluorescent conjugate polymer; mixing the first solution, the second solution, the third solution, and the fourth solution, such that mixing brings into contact the plurality of binding components and the plurality of core components such that the plurality of binding components and the plurality of core components self-assemble to form the plurality of fluorescent supramolecular nanoparticles (FSNPs), and such that the fluorescent conjugate polymer is encapsulated within each of the plurality of fluorescent supramolecular nanoparticles (FSNPs); and cross-linking the plurality of fluorescent supramolecular nanoparticles (FSNP) using a cross-linking agent.

[0044] An embodiment of the current invention provides a cross-linked fluorescent supramolecular nanoparticle (cFSNP), which is invisible under ambient light, but visible under selective light source for a predetermined amount of time. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) according to the present invention includes a supramolecular nanoparticle (SNP) having a plurality of binding components, each having at least one host-binding region; a plurality of core components, each having at least one core guest-binding element adapted to bind to the host-binding region to form a first inclusion complex; a plurality of terminating components, each having at least one terminating guest- binding element adapted to bind to the host-binding region to form a second inclusion complex, wherein the plurality of terminating components are present in a sufficient quantity relative to the host-binding region of the binding components to terminate further binding; a fluorescent conjugated polymer encapsulated within the SNP to form a fluorescent supramolecular nanoparticle (FSNP); and a cross-linking agent.

[0045] In one embodiment, the fluorescent conjugated polymer is poly[5-methoxy-2-

(3-sulfopropoxy)-l,4-phenylenevinylene] (MPS-PPV). And in one embodiment, the cross- linking agent is a cross-linker and the cross-linker is bis(sulfosuccinimidyl)suberate.

[0046] The covalent cross-linking of FSNPs with cross-linker results in pm-sized c-

FSNPs, which exhibit a size-dependent intradermal retention. It has been found that l456-nm sized c-FSNPs display an ideal intradermal retention time (ca. three months) for NMSC lesion labeling, as observed in an in vivo tattoo study. Advantageously, unlike conventional carbon graphite and India ink, the c-FSNPs of the present invention do not induce any inflammatory responses after tattooing. Hence, the newly developed c-FSNPs may be served as a new type of“finite tattoo” pigment to label potential malignant NMSC lesions.

[0047] Another aspect of the invention provides a method of making a cross-linked fluorescent supramolecular nanoparticle (cFSNP). A supramolecular synthetic approach was used to prepare a fluorescent supramolecular nanoparticle (FSNP) by encapsulating poly[5- methoxy-2-(3-sulfopropoxy)-l, 4-phenyl enevinylene] (MPS-PPV) into a supramolecular nanoparticle (SNP) using three molecular building blocks. The ratio between the building blocks and the fluorescent conjugated polymer was determined to yield an optimal fluorescent performance.

[0048] An embodiment of the invention relates to self-assembling supramolecular nanoparticles (SNPs). A detailed discussion regarding how such SNPs are made is described in U.S. Patent No. 9845237 and U.S. Patent Application No. 20160000918, which are hereby incorporated by reference. Briefly, in such embodiments, the self-assembling SNPs include a plurality of binding components, each having a plurality of binding regions; a plurality of core components that are suitable to at least provide some mechanical structure to the plurality of self-assembled supramolecular nanoparticles (SNPs), the plurality of cores having at least one core binding element adapted to bind to the binding regions to form a first inclusion complex; and a plurality of terminating components, each having a single terminating binding element that binds to remaining binding regions of one of the plurality of binding components by forming a second inclusion complex. In such an embodiment, the plurality of binding components and the plurality of core components self-assemble when brought into contact to form the plurality of self-assembled supramolecular nanoparticles (SNPs). The plurality of terminating components act to occupy the remaining binding regions of the plurality of binding components, and the plurality of terminating components are present in a sufficient quantity relative to the plurality of binding regions of the plurality of binding components to terminate further binding, thereby forming a discrete particle.

[0049] In an embodiment, the self-assembling SNPs are further configured to include a reporting agent. In some embodiments, the reporting agent is a fluorescent agent, however, one of ordinary skill in the art can envision that any reporting agent can be included within the SNPs. In an embodiment, the reporting agent fluoresces in response to specific stimuli. In an embodiment, an example of such a stimuli is exposure to a specific wavelength of light. In an embodiment, the reporter agent is mostly invisible or not visible under ambient conditions or when not exposed to the stimuli. The reporter agent becomes activated and visible following or during exposure to the stimuli. In an embodiment, the reporter agent is encapsulated within the SNPs for a predetermined period of time or for a predetermined retention time. In such an embodiment, the duration of the retention time is determined at least in part by manipulation of one or more of: the ratio of the individual components making up the SNPs, the ratio of the reporter agent to the SNPs and/or to the individual components making up the SNPs, the nature of the interaction between the reporter agent and the SNPs, the size of the SNPs, or the size of larger complexes formed from the joining of two or more SNPs.

[0050] In an embodiment, two or more individual SNPs are joined to form larger complexes. One of ordinary skill in the art can readily envision various methods for joining individual SNPs; a non-limiting example of such a method would be cross-linking. In an embodiment, two or more SNPs are chemically cross-linked to one another via the use of a crosslinking agent.

[0051] In an embodiment, individual SNPs carrying a reporter agent and/or complexes made up of joined SNPs carrying a reporter agent are delivered to or deposited into tissue sites for finite labeling of the tissue site. In such an embodiment, the term finite means that the reporter agent remains associated with and/or encapsulated within the SNPs and/or complexes made up of joined SNPs for a predetermined period of time or for a predetermined retention time. Such an embodiment is useful for a variety of applications where it is desirable to label a specific tissue or a region of tissue for a finite period of time. The finite period of time which the tissue is labeled for is correlated with the predetermined period of time or the predetermined retention time which the reporter agent is associated with and/or encapsulated within the SNPs. A non-limiting example of such an application would be to label tissues at or proximate to the site of a previous or pending surgical procedure. An example of such a surgical procedure includes a biopsy or any other surgical procedure involving the removal of tissue.

EXAMPLES [0052] The following examples help explain some concepts of the current invention.

However, the general concepts of the current invention are not limited to the particular examples.

[0053] In the recent decades of increased sun exposure, over 3.3 million cases of nonmelanoma skin cancers (NMSC) are diagnosed each year, rising at a faster rate than breast, prostate, lung and colon cancers combined.¹ In routine clinical practice, a suspected NMSC lesion is diagnosed via histologic characterization of biopsied skin tissues. Once the biopsy is confirmed to be a NMSC, the patient is then scheduled for a definitive treatment, which includes electrodessication, cryotherapy, topical chemotherapy, immune-modulator, surgical excision, or Mohs micrographic surgery. Since the time interval between the initial diagnostic biopsy and the scheduled surgical treatment can last up to three months, depending on availability of treatment,² the biopsy site can recover remarkably well and thus, become nearly invisible for a physician to identify. Besides photographic and pictorial documentation,^{3 5} labeling a potential NMSC lesion with a“tattoo”, a form of dermal pigmentation, is an approach that can effectively identify the biopsy site of NMSC lesion for the treatment.^{6 8} There are two major types of tattoo pigments in clinical use but with significant limitations: (i) conventional carbon graphite and India ink,⁶ which are

cosmetically unappealing and may be mistaken for a melanocytic lesion, and (ii) fluorescent particles ( e.g ., ZnO or dye-doped PMMA particles),^{7 8} which can only be visualized under UV light. To permanently remove these long-lasting fluorescent tattoo marks,^{9 10} laser or surgical treatments are often needed. In addition, both tattoo pigments may cause local inflammatory responses (e.g., dermatitis), leading to discomfort at the tattoo sites.^{11 12} Ideal tattoo pigments for short-term labeling of potential NMSC lesions require (i) invisibility under ambient visible light, (ii) fluorescent properties for detection under a given light source, (iii) a finite intradermal retention time of approximately three months, and (iv) biocompatibility to prevent any irritation at or around the tattoo sites.

[0054] In this example, a new class of fluorescent tattoo pigment is introduced: cross- linked fluorescent supramolecular nanoparticles (c-FSNPs). It is demonstrated that these c- FSNPs exhibit unique photo-physical properties, a finite intradermal retention, and biocompatibility, making them a promising candidate as an ideal tattoo pigment for short- term labeling of potential NMSC lesions. To prepare c-FSNPs with desired photo-physical and in vivo properties, fluorescent supramolecular nanoparticles (FSNPs), with enhanced fluorescent properties, were synthesized by encapsulating a fluorescent conjugated polymer, poly[5-methoxy-2-(3-sulfopropoxy)-l,4-phenylenevinylene] (MPS-PPV), into the intra- particular space of supramolecular nanoparticles (SNP) via a supramolecular synthetic approach^{13 25} (Figure la). By altering the synthetic parameters {i.e., ratios among MPS-PPV and the molecular building blocks), differential photo-physical properties can be

programmed into a small combinatorial library of FSNPs. 670-nm sized FSNPs were found to exhibit an optimal fluorescent performance with lO-fold enhancement compared to free MPS-PPV. Furthermore, a cross-linking reaction on 670-nm sized FSNPs was conducted to generate pm-sized c-FSNPs, which have a size-dependent and finite intradermal retention time (Figure lb-c). In this study, short-term labeling using c-FSNPs, which are invisible under ambient light but retain their fluorescent signal up to ca. three months (Figure lc) was successfully evaluated. Furthermore, an in vivo study reveals that the newly developed c- FSNPs do not activate any inflammatory responses, which are commonly triggered by commercially available tattoo pigments.

[0055] Fig. 1 A shows a two-step synthetic process employed for the preparation of c-

FSNPs as“finite tattoo” pigments (i) Step I: Supramolecular assembly of MPS-PPV and three molecular building blocks {i.e., Ad-PAMAM, Ad-PEG, and CD-PEI) for the combinatorial formulation of different FSNPs, and (ii) Step II: Cross-linking of FSNPs for the generation of pm-sized c-FSNPs. Fig. 1B is a schematic illustration of the tattooing procedure by which c-FSNPs are deposited in the skin of a nu/nu mouse: (i) wound generation in the dermis layer of the mouse skin through poking with needle, (ii) the deposition of c-FSNPs, (iii) wound healing, and (iv) the clearance of tattooed c-FSNPs with a finite intradermal retention time. Fig. 1C shows imaged showing that the tattooed sites cannot be visualized (i) under ambient light irradiation and their fluorescent signals can be detected (ii) under 465 nm light irradiation (ii-iv) These tattooed c-FSNPs exhibited size- dependent fluorescent signals with finite intradermal retention times.

[0056] Results and Discussion

[0057] A supramolecular synthetic approach^{13 25} was employed to prepare FSNPs, which encapsulate MPS-PPV (0.005 - 0.15 mg/mL) into a SNP core, from three molecular building blocks, i.e., cationic adamantane (Ad)-grafted polyamidoamine dendrimer (Ad- PAMAM; 0.15 - 1.6 mg/mL), cationic b-cyclodextrin (CD)-grafted branched polyethylenimine (CD-PEI; 0.8 mg/mL), and Ad-grafted polyethylene glycol (Ad-PEG;

1.836 mg/mL) (Figure 2a). Given the fact that the intra-particular space of SNPs is composed of a cationic Ad-PAMAM/CD-PEI hydrogel network, it is conceivable that FSNPs can encapsulate anionic MPS-PPV via electrostatic interactions.^{14 15, 26 27} FSNPs with optimal fluorescent performance were identified through a combinatorial screening process. A small combinatorial library composed of 27 different formulations of FSNPs was prepared by changing the ratios among the molecular building blocks: (i) MPS-PPV/CD-PEI ratios (w/w; 0.006: 1, 0.016: 1, 0.031 : 1, 0.125: 1 and 0.186: 1) and (ii) Ad-PAMAM/CD-PEI ratios (w/w; 0.19: 1, 0.25: 1, 0.5: 1, 1.0: 1, 1.5: 1, 1.7: 1, and 2.0: 1). The fluorescent intensities of the resulting FSNPs were measured with a fluorometer and normalized to the observed intensities of free MPS-PPV at corresponding concentrations for each FSNP formulation. A three-dimensional (3D) profile summarizes the normalized fluorescent intensities of the resulting FSNPs (Figure 2b). The FSNPs exhibiting optimal fluorescent performance C^), where FSNPs possess the highest normalized fluorescent intensities with the largest MPS- PPV loading in the core, were obtained with a specific formulation of the mixing ratio among the molecular building blocks, i.e. , MPS-PPV/CD-PEI ratio (w/w) = 0.125 : 1 and Ad- PAMAM/CDPEI ratio (w/w) = 2.0: 1.

[0058] FSNPs with the optimal fluorescent performance, identified from the combinatorial library (Figure 2b; Ά^"), were then subjected to (i) characterization of their structural and photo-physical properties and (ii) cluster formation studies via a cross-linking process.

[0059] Fig. 2A shows a combinatorial library of FSNPs with 27 different formulations is achieved by performing supramolecular assembly of MPS-PPV and three molecular building blocks {i.e., Ad-PAMAM, Ad-PEG, and CD-PEI) in different mixing ratios. Fig. 2B shows a 3D profile of FSNPs’ fluorescent intensities with variation (27 data points) of (i) MPS-PPV/CD-PEI ratio (w/w) and (ii) Ad-PAMAM/CD-PEI ratio (w/w). The fluorescent intensities of FSNPs are normalized to the observed intensities of free MPS-PPV of corresponding concentration for each FSNP formulation. The XZ and YZ planes across the optimal performance present the normalized fluorescent intensity variation of FSNPs depending on MPS-PPV/CD-PEI ratio (w/w) and Ad-PAMAM/CD-PEI (w/w), respectively. The FSNPs exhibiting optimal fluorescent performance f^) are obtained with a specific formulation of the mixing ratio among the molecular building blocks, i.e., MPS-PPV/CD-PEI ratio (w/w) = 0.125 : 1 and Ad-PAMAM/CDPEI ratio (w/w) = 2.0: 1.

[0060] As shown in Figure 3 a, the hydrodynamic size, obtained by dynamic light scattering (DLS), of the FSNPs prepared by the optimal condition as described above was 670 ± 43 nm, and their morphology observed in transmission electron microscopic (TEM) image reveals homogeneous spherical nanoparticles (Figure 3b). The size of FSNPs in TEM images was measured as 498 ± 37 nm, which is smaller than the hydrodynamic size obtained by DLS due to dehydration of FSNPs during the sample preparation for TEM imaging.

[0061] Tinder TiV light irradiation, the 670-nm sized FSNPs displayed dramatically enhanced fluorescence compared to the free MPS-PPV aqueous solution. The difference in their fluorescent performance was so dramatic that it was easily distinguished with the naked eye (Figure 3c). Comparison of the fluorescence spectra confirmed that the emission of 670- nm sized FSNP was ca. lO-fold greater than that of free MPS-PPV (Figure 3d). FSNPs possessed a broad absorption band (maximum absorption peak at 440 nm), where FSNPs exhibit ca. 6.8 times higher absorption than free MPS-PPV (Figure 3e). Analysis of the absorption and fluorescence spectra of 670-nm sized FSNPs indicated an absorption cross- section of 1.17x 1 O ¹⁶ cm ² and quantum yield (QY) of 4%. In contrast, free MPS-PPV showed a smaller absorption cross-section of 1.72^c 10 ¹⁷ cm ² and a lower QY of 0.45% (Figure 3f). These improved photo-physical properties of 670-nm sized FSNPs resulted in their enhanced fluorescent performance.^{28 29}

[0062] Fig. 3 A shows DLS data and Fig. 3B shows a TEM image of the FSNPs selected from the combinatorial library (Ά^', Figure 2b). The hydrodynamic size of the FSNPs, obtained by DLS, is 670 ± 43 nm and TEM image reveals that FSNPs are

homogeneous spherical nanoparticles. Fig. 3C shows a photograph of free MPS-PPV and the 670-nm sized FSNP solution with UV light irradiation (excitation: 365 nm). Comparison of Fig. 3D emission and Fig. 3E absorption spectrum of free MPS-PPV and the 670-nm sized FSNP. Fig. 3F shows photo-physical properties of free MPS-PPV and the 670-nm sized FSNP. All properties were calculated based on the repeating unit of MPS-PPV. Dramatically enhanced fluorescence is observed with the 670-nm sized FSNPs which exhibits ca. lO-fold higher emission and ca. 6.8-fold higher absorption than free MPS-PPV. [0063] In previous studies, conjugated polymer-based nanoparticles have generally required multiple optimization cycles of design and modification of the molecular structures of the core conjugated polymer^{28 31} to reach the best optical performance. In contrast, FSNPs with optimal fluorescent properties can be selected from a combinatorial library in which different formulations of FSNPs were readily prepared by changing the ratios among MPS- PPV and the three molecular building blocks via our flexible and convenient supramolecular synthetic approach. Distinct photo-physical properties can be programmed into individual FSNPs in the combinatorial library. The supramolecular synthetic approach is capable of rapid and parallel programming of a combinatorial library of FSNPs and provides a new developmental pathway to overcome the limitation of the conventional time- and cost- consuming optimization process in search of new fluorescent nanoparticles.

[0064] After identification of the 670-nm sized FSNPs with optimal fluorescent performance, the correlation between size and the intradermal retention time of FSNPs was explored. The commercially available fluorescent tattoo pigments ( e.g ., ZnO, fluorescent dye doped PMMA, and India ink), which possess a permanent intradermal retention time, have a size of one to several pm, as observed from TEM studies. Based on the dimensions of these commercially available tattoo pigments, the intradermal retention time of FSNPs with sizes ranging from hundreds of nm to a few pm was tested. A collection of FSNPs with controllable sizes up to 670 nm was obtained by using the supramolecular synthetic approach. In order to construct pm-sized particles, a covalent cross-linker

bis(sulfosuccinimidyl)suberate (BS3, 10-30 pg/mL) was used to“glue” several 670-nm FSNPs to form pm-sized c-FSNPs (Figure 4a). BS3 reacts with the free amine groups in the FSNPs, leading to both intra- and inter-FSNP cross-linking reactions. Inter-FSNP cross- linking resulted in the formation of various sized c-FSNPs, whose sizes depended on the concentration of BS3. By controlling the quantity of the cross-linker, c-FSNPs with sizes up to 1,456 nm were successfully produced. The hydrodynamic size of c-FSNPs increased from 679 ± 51 nm to 884 ± 62 and 1,456 ± 110 nm when 10, 20, and 30 pg/mL of BS3 were introduced to the 670-nm sized FSNP solution, respectively (Figure 4b-e). TEM analysis confirmed that c-FSNP is composed of 670-nm sized FSNPs, which are cross-linked inter- particularly, maintaining their size and morphology. [0065] Fig. 4A shows a cross-linker, bis(sulfosuccinimidyl)suberate (BS3, 10-30 pg/rnL) is introduced to the 670-nm sized FSNP solution to form pm-sized c-FSNPs. Fig. 4B shows DLS data and TEM images of 670-nm sized FSNPs and Figs. 4C-E show c-FSNPs under the BS3 treatment with various concentrations (c. 10 pg/rnL, d. 20 pg/mL, and e. 30 pg/mL) Hydrodynamic size of the resulting c-FSNPs measured as 679 ± 51, 884 ± 62 and 1456 ± 110 nm when 10, 20, and 30 pg/mL of BS3 were introduced to the 670-nm sized FSNP solution, respectively. TEM images reveal that the resulting c-FSNPs are composed of clusters of 670-nm sized FSNPs maintaining their size and morphology.

[0066] The intradermal retention times of 679-nm, 884-nm, and l,456-nm sized c-

FSNPs along with 240-nm, 4l0-nm, and 670-nm sized FSNPs were explored. These c- FSNPs and FSNPs were tattooed at six different locations on the back of nu/nu mice (// = 3) (Figure 5a). Following a typical tattooing protocol, FSNPs and c-FSNPs, each containing 0.75 pg of MPS-PPV, were deposited in the dermis through wounds generated by poking with a needle (Figure lb). The deposited FSNPs and c-FSNPs stayed in the dermis after the wounds healed. After tattooing, no visible signal from FSNPs and c-FSNPs was observed under ambient light irradiation, as shown in Figure 5b. The fluorescent signals from these tattooed FSNPs and c-FSNPs were obtained up to 94 days using an in vivo optical imaging system (excitation/emission: 465/520 nm, exposure time: 2 s; Figure 5c). The time-dependent fluorescent signals of FSNPs and c-FSNPs are summarized in Figure 5d, where fluorescent signals are normalized to the initial fluorescent signal intensity at day 0. The tattooed FSNPs and c-FSNPs exhibit a size-dependent intradermal retention time (Figure 5c and d). Longer intradermal retention times can be obtained with the larger sized particles. As a result, 1456- nm sized c-FSNPs (white arrow in Figure 5c) show the slowest fluorescent signal decay and maintain their fluorescent signal up to 84 days (12 weeks, ca. 3 months). At day 94, all fluorescent signals from the tattooed FSNPs and c-FSNPs diminished to undetectable levels. In contrast, the commercially available tattoo pigment (z.e., ZnO, ca. 5 pm) exhibited a consistent fluorescent intensity over 94 days (Figure 5d).

[0067] The size of c-FSNPs plays an important role in modulating their intradermal retention time. Moreover, it is noteworthy that 670-nm sized FSNPs exhibit a fast fluorescent signal drop in a week while 679-nm sized c-FSNPs with a similar size show a gradual fluorescent signal decrease over eight weeks (56 days). It is clear that the covalent intra- FSNP cross-linking plays an important role to delay the dynamic disassembly of c-FSNPs by tightening the structure of FSNPs. The dynamic nature of the Ad-CD self-assembly motifs,^{14, 32} employed in the preparation of our FSNPs, leads to disassembly of FSNPs under physiological condition. This disassembly rate is too fast to satisfy the current needs for the “finite tattoo,” in the animal study with the 670-nm sized FSNPs. The additional intra- particular cross-linking in c-FSNPs is thus necessary to delay this disassembly process so that the intradermal retention time of 679-nm sized c-FSNPs is elongated compared to 670- nm sized FSNPs. Overall, the intradermal retention time of c-FSNPs is governed by both their size and cross-linking.

[0068] A pathological study of animal skin was conducted at two days after tattooing to validate the biocompatibility of c-FSNPs. In the H & E (hematoxilin (nucleus staining) and eosin (cytoplasm staining)) stained tissue sections, no obvious inflammatory cells were observed after c-FSNP tattooing compared to normal skin (Figure 5e (i) and (ii)). In contrast, commercially available tattoo pigments {i.e., ZnO and PMMA) attract a large population of inflammation cells, such as lymphocytes, after tattooing (Figure 5e (iii)). Because c-FSNPs are composed of molecular building blocks ( e.g ., CD, PEG) which are known to be non- immunogenic^{33 35}, they exhibit biocompatibility without inducing any inflammatory reactions at or around the tattoo sites.

[0069] Fig. 5A shows three different sized c-FSNPs (i.e., 679 nm, 884 nm, and 1456 nm) along with three different sized FSNPs (i.e., 240 nm, 410 nm, and 670 nm), each containing 0.75 pg of MPS-PPV, are tattooed at six different locations on the back of the nu/nu mice (n = 3). Fig. 5B shows a photograph of a mouse tattooed with various sized FSNPs and c-FSNPs under ambient light irradiation. Fig. 5C shows fluorescent images of the tattooed mouse obtained using the in vivo optical imaging system (excitation/emission: 465/520 nm; exposure time: 2 s), for 94 days. Fig. 5D shows time-dependent fluorescent signal of tattoo pigments, including a commercially available pigment {i.e., ZnO), FSNPs, and c-FSNPs. The fluorescent signals are normalized to the initial fluorescent signal intensity at day 0. In a long-term fluorescent signal observation up to 94 days, both FSNPs and c- FSNPs display a size-dependent fluorescent signal with a finite intradermal retention time. Among various FSNPs and c-FSNPs, l456-nm sized c-FSNPs (arrow in Figure 5c) show a fluorescent signal up to 84 days with the slowest fluorescent signal decay. The commercially available tattoo pigment, ZnO, exhibits a consistent fluorescent intensity over 94 days (e) H&E stained skin sections from a nu/nu mouse tattooed with l456-nm sized c-FSNPs after 2 d post tattooing (magnification: 100^c). Compared to (i) normal skin without tattooing, (ii) no obvious inflammation cells were observed in the skin of nu/nu mouse after c-FSNP tattooing compared (iii) In contrast, commercially available tattoo pigments (i.e., ZnO) attract a large population of inflammation cells (black circles).

[0070] Conclusions

[0071] A new type of tattoo pigment, c-FSNPs, with strong fluorescent properties, a controllable lifetime, and biocompatibility to achieve successful in vivo finite tattooing is described above. The 670-nm sized FSNPs with optimal fluorescent properties selected via a combinatorial screening process were cross-linked to form pm-sized c-FSNPs. c-FSNPs provide a fluorescent signal in the skin under 465 -nm light excitation without being cosmetically unappealing under ambient light irradiation. Unlike commercially available tattoo pigments with permanent intradermal retention, c-FSNPs are clearable but, with an appropriate intradermal retention time in the skin. The retention time of c-FSNPs can be modulated by controlling the size and cross-linking chemistry. c-FSNPs with a l,456-nm hydrodynamic size show an intradermal retention time of up to 84 days (12 weeks, 3 months), which matches the typical period between biopsy and treatment in the clinic. In addition, c-FSNPs are biocompatible and do not induce any dermal inflammatory reactions. The described c-FSNPs can serve as a new type of“finite tattoo” pigment to label potential malignant NMSC lesions. The translation of c-FSNPs into NMSC patients is underway with the hope of providing the correct information of the biopsied potential malignant sites.

[0072] Materials

[0073] Reagents and solvents were used as received without further purification otherwise noted. Branched polyethyl enimine (PEI, MW = 10 kD) was purchased from Polysciences Inc (Washington, PA). Polymers contain primary, secondary and tertiary amine groups in approximately 25/50/25 ratio. l^st-generation polyamidoamine dendrimer

(PAMAM) with 1, 4-diaminobutane core and amine terminals in 20% wt methanol solution was purchased from Dendritic Nanotechnologies, Inc (Mount pleasant, MI). 1- Adamantanamine (Ad) hydrochloride and b-cyclodextrin (b-CD) were purchased from TCI America (San Francisco, CA). N-hydroxysuccinimide (SCM) and maleimido (MAL) hetero- functionalized polyethylene glycol (SCM-PEG-MAL, MW = 5 kD) was obtained from NANOCS Inc (New York, NY). 6-Mono-tosyl-P-cyclodextrin (6-OTs-P-CD) was prepared according to the literature reported method.³⁶ Octa- Ad-grafted polyamidoamine dendrimer (Ad-PAMAM), CD-grafted branched polyethylenimine (CD-PEI) and Ad-grafted

polyethylene glycol (Ad-PEG) were prepared by the method we previously reported.¹³ Poly[5-methoxy-2-(3-sulfopropoxy)-l,4-phenylenevinylene] potassium salt (MPS-PPV) was purchased from Aldrich (St. Louis, MO). The hydrodynamic size of FSNPs and c-FSNPs was measured on a Zetasizer Nano instrument (Malvern Instruments Ltd., United Kingdom). Transmission electron microscope (TEM) images of FSNPs and c-FSNPs were taken with a Philips CM 120 electron microscope operating with an acceleration voltage of 120 kV. PL measurements were conducted with a HORIBA Jobin Yvon system (Horiba, Japan) and the UV-Vis absorption spectrum was obtained with the Hitachi UV-Vis system (Hitachi U-4100 spectrophotometer, Hitachi, Japan). In vivo fluorescent images were obtained with the IVIS- 200 optical imaging system (PerkinElmer, Waltham, MA, USA). Pathological images of tissue sample after tattooing were obtained with the Aperio ScanScope AT microscope (Leica biosystem, USA).

[0074] Preparation of FSNPs

[0075] To a solution of Ad-PEG (1.836 mg/mL) in 485-pL of PBS buffer, CD-PEI

(0.8 mg/mL) was injected under vigorous stirring. MPS-PPV (0.005 - 0.15 mg/mL) was then added sequentially and the mixture solution was stirred vigorously for 2 min. A 5-pL aliquot of DMSO containing Ad-PAMAM (0.15 - 1.6 mg/mL) was added into the mixture solution to obtain FSNPs.

[0076] Characterization methods and settings

[0077] Dynamic light scattering (DLS) : The hydrodynamic sizes of FSNPs and c-

FSNPs were measured with a Zetasizer Nano instrument (Malvern Instruments Ltd., United Kingdom) equipped with a lO-mW helium-neon laser (l = 632.8 nm) and a thermoelectric temperature controller. Measurements were taken at a 90° scattering angle. The

hydrodynamic sizes of FSNPs and c-FSNPs were obtained by averaging the values of three or more measurements.

[0078] Transmission electron microscope (TEM) The morphology and sizes of

FSNPs and c-FSNPs were examined using a TEM. The studies were carried out on a Philips CM 120 electron microscope, operating at an acceleration voltage of 120 kV. TEM samples were prepared by drop-coating 2 pL of sample suspension solutions onto carbon-coated copper grids. Excess amounts of solution were removed by filter papers after 45 s.

Subsequently, the samples were negatively stained with 2% uranyl acetate for 45 s before TEM studies.

[0079] Photoluminescence (PL): Steady-state photoluminescence of free MPS-PPV and FSNPs was measured using a custom-made PL system (HORIBA Jobin Yvon system, Horiba, Japan). The solutions of FSNPs and MPS-PPV were excited by a Xenon lamp which filtered its wavelength using a monochromator with a wavelength of 450 nm.

[0080] UV-Vis absorption: The ETV-Vis absorption of free MPS-PPV and FSNPs was obtained with a Hitachi ETV-Vis system (Hitachi EG-4100 spectrophotometer, Hitachi, Japan). The absorption of free MPS-PPV and FSNPs was obtained from 350 nm to 800 nm with a scan speed of 10 nm /sec with 1 nm intervals.

[0081] Fluorescent lifetime measurement: Time-Correlated Single Photon Counting

(TCSPC) is used to determine the lifetime of free MPS-PPV and FSNPs. In TCSPC, each sample was measured with time between sample excitation by a pulse of laser and the arrival of the emitted photons to the detector. The measured lifetime spectrum was then fitted by an exponential fit time scan to get the fitting curve and lifetime data. All the data were obtained for three times on an Edinburgh FLS920 time-correlated single-photon-counting instrument. Samples were excited at 475 nm from a picosecond diode laser with 90 ps pulse width transmitted through a Czemy-Tumer design monochromator. The emission wavelength can be detected at 90° via a second Czemy-Tumer design monochromator onto a MCP-PMT with a response time down to 25 ps. The instrument response function was profiled using a scatter solution and subsequently deconvoluted from the emission data to yield an undisturbed decay. Nonlinear least-square fitting of the decay curves was performed with the Levenberg- Marquardt algorithm and implemented by the Edinburgh Instruments FAST software.

[0082] Calculation of absorption cross-section

[0083] The absorption cross-section of free MPS-PPV and FSNPs was calculated from the mass absorption coefficient³⁷ using: 2.303(1000) s „

s = - (cm⁴ /po y er i st)

[0084] ¾

[0085] where:

[0086] e is the stated molar extinction coefficient (M 'em ')

[0087] N_A is Avogadro’s number.

[0088] The factor of 1000 originates from a conversion between dm³ and cm³ and l/log e = 2.303.

[0089] Calculation of fluorescent quantum yield (QY)

[0090] The relative QY of free MPS-PPV and FSNPs was determined by comparison with a fluorophore (Cy3) of known QY with the same experimental parameters as free MPS- PPV and FSNPs.

[0091] The QY was calculated by:

[0093] where F is the QY, Int is the area under the emission peak, A is absorbance at the excitation wavelength, and n is the refractive index of the solvent. The subscript R denotes the respective values of the reference substance, Cy3 (Lumiphore, Hallandale Beach, FL, USA; QY = 0.31).

[0094] Preparation of c-FSNPs

[0095] The 670-nm sized FSNPs were mixed with various concentrations of BS3 from 10 to 20 and 30 pg/mL at RT with vigorous stirring. After 15 min, Tris buffer (1 x) was added to the reaction solution in order to stop the cross-linking reaction of BS3.

[0096] Examination on intradermal retention time of c-FSNPs

[0097] All animal manipulations were performed with sterile technique and were approved by the Institutional Animal Care and Use Committee of University of Southern California. Female athymic nude mice (about 6-8 weeks old, with a body weight of 20-25 g) were purchased from Envigo (Livermore, CA, USA). The FSNPs and c-FSNPs were tattooed at 6 different locations on the back of nu/nu mice {n = 3), following the typical tattooing protocol. After the mice were anesthetized with 2% isoflurane in a heated (37°C) induction chamber, mouse skin was poked with a 25 G needle to make wounds to dermis layer. FSNP or c-FSNP solution was dropped on the wounded mouse skin (nu/nu) to depositing FSNP or c-FSNP into the dermis layer through the wounds. The FSNP or c-FSNP solution was washed away with saline several times. After tattooing, the signals of FSNP and c-FSNP were measured with the in vivo optical imaging system (IVIS-200, PerkinElmer, Waltham, MA, USA) for 94 days.

[0098] Pathological studies of skin tissues tattooed with c-FSNPs

[0099] Another group of mice, treated the same as described above in the in vivo study, had their skin tissues taken 2 days after tattooing for pathological studies. Skin tissues were fixed with 10% formalin and blocked with paraffin, following conventional laboratory methods. Slices of skin tissue were stained with H&E (Hematoxylin and eosin) solution for pathological study. Tissues were then examined using an Aperio ScanScope AT microscope (Leica biosystem, USA). Each H&E stained tissue slide was evaluated by two independent pathologists.

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[00101] The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art how to make and use the invention. In describing embodiments of the invention, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. The above-described embodiments of the invention may be modified or varied, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.

Claims

WE CLAIM:

1. A cross-linked fluorescent supramolecular nanoparticle (cFSNP) comprising:

a supramolecular nanoparticle (SNP) comprising:

a plurality of binding components, each having at least one binding region; a plurality of core components suitable to at least provide some mechanical structure to the supramolecular nanoparticle (SNP), the plurality of core components comprising each having at least one core binding element adapted to bind to the binding region to form a first inclusion complex;

a plurality of terminating components, each having at least one terminating binding element adapted to bind to the binding regions to form a second inclusion complex, wherein the plurality of terminating components are present in a sufficient quantity relative to the binding region of the binding components to terminate further binding;

a fluorescent conjugated polymer encapsulated within the SNP to form a fluorescent supramolecular nanoparticle (FSNP); and

a cross-linking agent.

2. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 1, wherein the fluorescent conjugated polymer and the binding components are present in a ratio (w/w) between about 0.001 : 1 to 0.2: 1.

3. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 2, wherein the fluorescent conjugated polymer and the binding components are present in a ratio (w/w) between about 0.005: 1 to 0.125: 1.

4. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 1, wherein the core components and the binding components are present in a ratio (w/w) between about from about 0.1 :1 to 2.5: 1.

5. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 4, wherein the core components and the binding components are present in a ratio (w/w) between about 0.15: 1 to 2.2: 1.

6. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 1, wherein the core components and the binding components are present in a ratio (w/w) between about 2.0: 1.

7. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 1, wherein the FSNP has a predetermined size of at least about 100 nm and less than about 1000 nm.

8. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 7, wherein the FSNP has a predetermined size of at least about 200 nm and less than about 700 nm.

9. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 7, wherein the FSNP has a predetermined size of about 670 nm.

10. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 1, wherein the fluorescent conjugated polymer is poly[5-methoxy-2-(3-sulfopropoxy)-l,4- phenylenevinylene] (MPS-PPV).

11. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 1, wherein the cross-linking agent is a cross-linker.

12. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 11, wherein the cross-linker is bis(sulfosuccinimidyl)suberate.

13. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 1, wherein the plurality of binding components comprises polythylenimine, poly(L-iysine), or polyp-amino ester).

14. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 1, wherein the at least one binding region comprises beta-cyclodextrin, alpha-cyclodextrin, gamma-cyclodextrin, cucurbituril or calixarene.

15. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 1, wherein the plurality of core components comprise at least one of a dendrimer, branched polyethyleneimide, linear polyethyleneimide, polylysine, polylactide, polylactide-co- glycolide, polyanhydrides, poly-e-caprolactones, polymethyl methacrylate, poly(N-isopropyl acrylamide), polypeptides, polyamidoamine dendrimers, poly(prophylenimine) (PPI) dendrimer, triazine dendrimer, carbosilane dendrimer, poly(ether imine) (PETIM) dendrimer or phosphorus dendrimer.

16. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 1, wherein the at least one core binding element comprises adamantanamine, azobenzene, ferrocene or anthracene.

17. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 1, wherein the plurality of terminating components comprises at least one of polyethylene glycol, polymer, polypeptide, ligosaccharide, or polypropylene glycol) (PGG).

18. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 1, wherein the at least one terminating binding element comprises adamantanamine,

azobenzene, ferrocene or anthracene.

19. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 1, wherein the at least one binding region binds to the at least one core binding element or the at least one terminating binding element to form a molecular recognition pair selected from the group consisting of antibody-antigen; protein-substrate; protein-inhibitor; protein-protein; a pair of complementary oligonucleotides; and an inclusion complex.

20. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 1, wherein at least one of the first inclusion complex and the second inclusion complex is adamantane-P-cyclodextrin or azobenzene-a-cyclodextrin.

21. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 1, wherein the cross-linked fluorescent supramolecular nanoparticle (cFSNP) is cross-linked to one or more cross-linked fluorescent supramolecular nanoparticles (cFSNP) to form a fluorescent supramolecular nanoparticles complex.

22. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 21, wherein the fluorescent supramolecular nanoparticles complex has a predetermined size of at least about 300 nm and less than about 2,000 nm.

23. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 21, wherein the fluorescent supramolecular nanoparticles complex has a predetermined size of at least about 600 nm and less than about 1,600 nm.

24. The cross-linked fluorescent supramolecular nanoparticle (cFSNP) of claim 21, wherein the fluorescent supramolecular nanoparticles complex has a predetermined size of at about 1,500 nm.

25. A method for delivering a fluorescent agent to a subject comprising:

penetrating an epidermis tissue layer of the subject such that an accession point to an underlying dermis layer in the subject is created; and

delivering a plurality of cross-linked fluorescent supramolecular nanoparticles

(cFSNPs) to the underlying dermis layer in the subject through the accession point, wherein each of the plurality of cross-linked fluorescent supramolecular nanoparticle (cFSNP) comprises a supramolecular nanoparticle (SNP) comprising:

a plurality of binding components, each having at least one binding region; a plurality of core components suitable to at least provide some mechanical structure to the supramolecular nanoparticle (SNP), the plurality of core components comprising each having at least one core binding element adapted to bind to the binding region to form a first inclusion complex;

a plurality of terminating components, each having at least one terminating binding element adapted to bind to the binding regions to form a second inclusion complex, wherein the plurality of terminating components are present in a sufficient quantity relative to the binding region of the binding components to terminate further binding; and a fluorescent conjugated polymer encapsulated within the SNP to form a fluorescent supramolecular nanoparticle (FSNP);

wherein the plurality of cross-linked fluorescent supramolecular nanoparticles (cFSNPs) are configured to retain the fluorescent conjugated polymer for a predetermined retention time, and

wherein the plurality of cross-linked fluorescent supramolecular nanoparticles are visible under a selective light source.

26. The method of claim 25, wherein the predetermined retention time up to 100 days.

27. The method of claim 25, wherein the selective light source is ultraviolet light.

28. A method of making a cross-linked fluorescent supramolecular nanoparticle (cFSNP) comprising:

synthesizing a plurality of fluorescent supramolecular nanoparticles (FSNPs)

comprising:

providing a first solution comprising a plurality of binding components, each having at least one binding region; providing a second solution comprising a plurality of core components, each having at least one core binding element adapted to bind to the binding region to form a first inclusion complex;

providing a third solution comprising a plurality of terminating components, each having at least one terminating binding element adapted to bind to the binding region to form a second inclusion complex, wherein the plurality of terminating components are present in a sufficient quantity relative to the binding region of the binding components to terminate further binding;

providing a fourth solution comprising a fluorescent conjugate polymer;

mixing the first solution, the second solution, the third solution, and the fourth solution, wherein the mixing brings into contact the plurality of binding components and the plurality of core components such that the plurality of binding components and the plurality of core components self-assemble to form the plurality of fluorescent supramolecular nanoparticles (FSNPs), and such that the fluorescent conjugate polymer is encapsulated within each of the plurality of fluorescent supramolecular nanoparticles (FSNPs); and

cross-linking the plurality of fluorescent supramolecular nanoparticles (FSNP) using a cross-linking agent.