[0001] The present invention generally relates to the field of imaging, sensing and therapeutics delivery. More specifically, the present invention relates to a dynamic host-guest interactive system that possesses unique characteristics and finds utility in multitude of areas, specifically, in imaging, sensing and site directed drug delivery.

BACKGROUND

[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] In supramolecular chemistry, host-guest chemistry describes complexes that are composed of two or more molecules or ions that are held together in unique structural relationships by non-covalent forces. The non-covalent forces can be hydrogen bonds, ionic bonds, van-der-Waals forces, and hydrophobic interactions. The tailoring of host molecules or compounds with other agents can be of great importance and recent advancement seek to incorporate utilization of host-guest chemistry in a variety of biological applications.

[0004] Super-resolution fluorescence microscopy provides in-situ visualization of molecular organization and their interactions beyond the classical diffraction limit of light. In general, the super-resolution imaging techniques rely on switching molecules between fluorescent ON and OFF states to consecutively localize individual molecules at nanometer precision and eventually obtaining a super-resolved image. In most of traditional techniques, switching between ON and OFF states is usually achieved externally, either through a targeted mechanism, for example, Stimulated Emission Depletion Microscopy (STED), or through a stochastic approach, for example, Stochastic Optical Reconstruction Microscopy (STORM) (Huang, B., Jones, S. A., Brandenburg, B. & Zhuang, X. Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nat. Methods, 5, 1047-1052 (2008); Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642-1645 (2006); Hell, S.W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated emission-depletion fluorescence microscopy. Opt. Lett. 19, 780-782 (1994); Hell, S.W. Far-field optical nanoscopy. Science 316, 1153-1158 (2007)). Although these techniques provide improved resolution, they suffer from several shortcomings. Often times they are difficult for the standard laboratories to apply due their complex requirements. For example, the instrumentation for STED microscopy is complicated and cost intensive. In STORM microscopy, dyes with photo-switchable property are essential and specialized buffer conditions (e.g. buffer with reducing/oxidizing agents) have to be employed to get optimal results. Additionally, initial exposure of the specimen to high-power laser illumination is necessary for converting molecules to fluorescence OFF-states. The specialized buffer conditions along with the requirement of high-power laser illumination limit their compatibility with live cell imaging experiments.

[0005] PAINT (points accumulation for imaging in nanoscale topography) represents a conceptually different and easy-to-implement strategy to perform super-resolution microscopy (Sharonov et al, Proc. Natl. Acad. Sci. USA 2006, 103, 18911-18916; Jungmann et al, Nano Lett. 2010, 10, 4756-4761; Agasti et al, Chem. Sci. 2017, 8, 3080- 3091). In this strategy, imaging is carried out by using an influx of diffusing fluorescent molecules that interact transiently with the sample. This strategy benefits by their use of conventional fluorophores and importantly, the implementation of PAINT does not require any specialized equipment or experimental conditions to switch fluorophores between ON- and OFF-states. Initially, PAINT strategy was applied to image lipid bilayers and double stranded DNA by using dye molecules (Kuo et al, J. Am. Chem. Soc. 2011, 133, 4664-4667; Schoen et al, Nano Lett. 2011, 11, 4008-4011). However, lack of specificity hindered the use of these PAINT-compatible dyes for imaging other biomolecules of interest. Recently, DNA and protein fragment based PAINT probes (i.e. DNA-PAINT and IRIS) were introduced to achieve specificity. However, the DNA and protein probes are membrane- impermeable. Further, digestion by nucleases and proteases compromises the stability of these probes under intracellular environment. Therefore, despite being simple and easy-to- implement, the application of PAINT based strategies in live cell microscopy remains challenging.

[0006] Another significant challenge in biology is to deliver relatively membrane impermeable materials to live cell to achieve therapeutic benefit or to visualize specific biomolecules inside living cells through targeted labeling. For example, cytoskeletal proteins perform a variety of cellular activities and thus imaging of cytoskeleton proteins in living cells leads to the understanding of various intracellular dynamics. Researchers have attempted to image actin, an important cytoskeletal protein by labeling actin utilizing various complicated techniques such as microinjection or electroporation due to the serious issue of membrane impermeability of the targeting agents (e.g. phalloidin) or the organic fluorophores. Researchers also followed alternative strategy of fusing GFP with actin filaments following the transfection method. However, these methods are laborious and time consuming which might alter the fate of the live cells.

[0007] There is, therefore, a need in the art to develop a dynamic host-guest interactive system that possesses unique characteristics and which can find utility in a multitude of areas, specifically, in imaging and site directed drug delivery. The present disclosure satisfies the existing needs, as well as others and alleviates the shortcomings of the traditional imaging and drug delivery techniques.

OBJECTS OF THE INVENTION

[0008] An object of the present disclosure is to provide a dynamic host-guest interactive system that can overcome the deficiencies associated with the prior art.

[0009] Another object of the present disclosure is to provide a dynamic host-guest interactive system for single molecule fluorescence ON and OFF blinking.

[0010] Another object of the present disclosure is to provide a dynamic host-guest interactive system for single molecule based super-resolution imaging.

[0011] Another object of the present disclosure is to provide a dynamic host-guest interactive system for live cell super-resolution imaging.

[0012] Another object of the present disclosure is to provide a dynamic host-guest interactive system for carrying membrane impermeable materials into the intracellular environment.

[0013] Another object of the present disclosure is to provide a dynamic host-guest interactive system for drug delivery.

[0014] Another object of the present disclosure is to provide an imaging and a drug delivery system that can utilize supramolecular host-guest interaction.

[0015] Another object of the present disclosure is to provide an imaging and a drug delivery system that exhibits desired specificity.

[0016] Another object of the present disclosure is to provide an imaging and a drug delivery system that gives image of the biological system without utilization of any of an external stimuli or an additive.

[0017] Another object of the present disclosure is to provide an imaging and a drug delivery system that offers improved resolution without any complex instrumentation or specialized experimental conditions. [0018] Another object of the present disclosure is to provide an imaging and a delivery system that is easy to implement.

[0019] Another object of the present disclosure is to provide an imaging and a delivery system that is cost-effective.

SUMMARY

[0020] The present invention generally relates to the field of imaging and drug delivery. More specifically, the present invention relates to a dynamic host-guest interactive system that possesses unique characteristics and finds utility in multitude of areas, specifically, in imaging and site directed drug delivery.

[0021] An aspect of the present disclosure provides a dynamic host-guest interactive system including at least one host molecule; and at least one guest molecule wherein the at least one host molecule interacts with the at least one guest molecule through non-covalent forces, and wherein the at least one host molecule is associated with any or a combination of at least one targeting moiety and at least one therapeutic agent with proviso that when the host molecule is associated with the at least one targeting moiety, the guest molecule is associated with at least one imager, and when the host molecule is associated with the at least one therapeutic agent, the guest molecule is associated with at least one targeting moiety, and when the host molecule is associated with a combination of the at least one therapeutic agent and the at least one targeting moiety, the guest molecule is associated with at least one imager.

[0022] In an embodiment, the at least one host molecule is selected from any or a combination of cucurbituril, cyclodextrins, calixarenes, pillararenes, porphyrins, metallacrowns, crown ethers, cyclotriveratrylenes, cryptophanes, cyclophanes and carcerands. In an embodiment, the at least one guest molecule is selected from any or a combination of propylamine, hexamethylenediamine and adamantylamine. In an embodiment, the cucurbituril is CB[7], and the association of CB[7] with the at least one therapeutic agent increases solubility of said at least one therapeutic agent. In an embodiment, the association of CB[7] with the at least one therapeutic agent affords protection to said at least one therapeutic agent from degradation in a biological medium. In an embodiment, the system affords site specific delivery of the at least one therapeutic agent by targeting tissue and organelles. In an embodiment, the system affords a convenient route to any or a combination of cell surface engineering and cell surface modification. In an embodiment, the host molecule associated with said at least one therapeutic agent exhibits capability to bind with proteins to afford protection to pharmacokinetics of the at least one therapeutic agent. In an embodiment, the system affords delivery of any or a combination of DNA, RNA, siRNA and protein by non-covalent interaction thereof with the host molecule.

[0023] In an embodiment, the at least one guest molecule is associated with the at least one imager through a first linking moiety and wherein the at least one host molecule is associated with the at least one targeting moiety through a second linking moiety, and wherein the first linking moiety and the second linking moiety is selected independently from any or a combination of trara-cyclooctene (TCO), 1,2,4,5-tetrazine (TZ) and N- hydroxysuccinimide (NHS). In an embodiment, the interaction of the at least one guest molecule associated with the imager with the at least one host molecule through the non- covalent forces effects ON-state of the imager associated with the guest molecule when subjected to points accumulation for imaging in nanoscale topography (PAINT).

[0024] In an embodiment, the imager associated with the at least one guest molecule exhibits OFF-state in an event of absence of interaction between the at least one guest molecule and the at least one host molecule through the non-covalent forces when subjected to points accumulation for imaging in nanoscale topography (PAINT). In an embodiment, the ON-state and OFF-state of the imager associated with the at least one guest molecule is effected without utilization of any of an external stimuli and an additive.

[0025] Another aspect of the present disclosure provides a dynamic host-guest interactive system including at least one host molecule; and at least one guest molecule wherein the at least one host molecule is associated with at least one targeting moiety, and wherein the at least one guest molecule is associated with at least one imager, and wherein the at least one imager associated with the at least one guest molecule exhibits OFF-state in an event of absence of non-covalent forces operative between the at least one guest molecule and the at least one host molecule, and wherein non-covalent interaction of the at least one guest molecule associated with the imager with the at least one host molecule effects ON-state of the imager associated with the guest molecule, when subjected to points accumulation for imaging in nanoscale topography (PAF T). In an embodiment, the at least one host molecule is selected from any or a combination of cucurbiturils, cyclodextrins, calixarenes, pillararenes, porphyrins, metallacrowns, crown ethers, cyclotriveratrylenes, cryptophanes, cyclophanes and carcerands. In an embodiment, the at least one guest molecule is selected from any or a combination of propylamine, hexamethylenediamine and adamantylamine. In an embodiment, the at least one imager is selected from any or a combination of Alexa Fluor, Cy5, Atto 655 and Rhodamine. In an embodiment, the at least one targeting moiety is selected from any or a combination of antibodies and small molecules.

[0026] Still further aspect of the present disclosure provides a dynamic host-guest interactive system including at least one host molecule; and at least one guest molecule wherein the at least one host molecule is associated with at least one therapeutic agent, and wherein said at least one guest molecule is associated with at least one targeting moiety, and wherein said at least one guest molecule is associated with said at least one targeting moiety through covalent bond. In an embodiment, the at least one host molecule is selected from any or a combination of cucurbiturils, cyclodextrins, calixarenes, pillararenes, porphyrins, metallacrowns, crown ethers, cyclotriveratrylenes, cryptophanes, cyclophanes and carcerands. In an embodiment, the at least one guest molecule is selected from any or a combination of propylamine, hexamethylenediamine and adamantylamine. In an embodiment, the at least one targeting moiety is selected from any or a combination of antibodies and small molecules.

[0027] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings are included to provide a further understanding of the present disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0029] FIG. 1A illustrates an exemplary snippet depicting the general principle behind utilization of the dynamic host-guest interactive system, realized in accordance with embodiments of the present disclosure, in imaging.

[0030] FIG. IB illustrates an exemplary snippet depicting a series of images, wherein in each frame only a fraction of the molecules are stochastically in ON-state, in accordance with embodiments of the present disclosure.

[0031] FIG. 1C illustrates exemplary snippets depicting accuracy of the imaging technique, realized in accordance with embodiments of the present disclosure, in comparison to traditional imaging technique. [0032] FIG. 2 illustrates an exemplary snippet depicting the general principle behind utilization of the dynamic host-guest interactive system, realized in accordance with embodiments of the present disclosure, in drug delivery.

[0033] FIG. 3A illustrate an exemplary image depicting F-actin fibers in MEF cells stained with CB[7] conjugated phalloidin (scale bar 1 μπι) utilizing super-resolution fluorescence microscopy, in accordance with an embodiment of the present disclosure.

[0034] FIG. 3B illustrate an exemplary image (zoomed-in view, scale bar 500 nm) depicting F-actin fibers in MEF cells utilizing traditional imaging technique (diffraction limited image).

[0035] FIG. 3C illustrate an exemplary image (zoomed-in view of image illustrated in FIG. 3A, scale bar 500 nm) depicting F-actin fibers in MEF cells, in accordance with an embodiment of the present disclosure.

[0036] FIG. 3D illustrates an exemplary graph depicting comparison of super-resolution fluorescence microscopy of F-actin, in accordance with an embodiment of the present disclosure, with the results obtained from traditional imaging technique.

[0037] FIG. 4A illustrate an exemplary image depicting super-resolution fluorescence microscopy of phalloidin targeted F-actin, in accordance with an embodiment of the present disclosure.

[0038] FIG. 4B illustrate an exemplary image of phalloidin targeted F-actin utilizing traditional imaging technique.

[0039] FIG. 4C illustrates an exemplary graph depicting comparison of super-resolution fluorescence microscopy of phalloidin targeted F-actin, in accordance with an embodiment of the present disclosure, with the results obtained from traditional imaging technique.

DETAILED DESCRIPTION

[0040] The following is a detailed description of embodiments of the disclosure depicted in the accompanying drawings. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0041] Each of the appended claims defines a separate invention, which for infringement purposes is recognized as including equivalents to the various elements or limitations specified in the claims. Depending on the context, all references below to the "invention" may in some cases refer to certain specific embodiments only. In other cases it will be recognized that references to the "invention" will refer to subject matter recited in one or more, but not necessarily all, of the claims.

[0042] As used in the description herein and throughout the claims that follow, the meaning of "a," "an," and "the" includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise.

[0043] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0044] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

[0045] The present invention generally relates to the field of imaging and drug delivery. More specifically, the present invention relates to a dynamic host-guest interactive system that possesses unique characteristics and finds utility in multitude of areas, specifically, in imaging and site directed drug delivery.

[0046] An aspect of the present disclosure provides a dynamic host-guest interactive system including at least one host molecule; and at least one guest molecule, wherein the at least one host molecule interacts with the at least one guest molecule through non-covalent forces, and wherein the at least one host molecule is associated with any or a combination of at least one targeting moiety and at least one therapeutic agent with proviso that when the host molecule is associated with the at least one targeting moiety, the guest molecule is associated with at least one imager, and when the host molecule is associated with the at least one therapeutic agent, the guest molecule is associated with at least one targeting moiety, and when the host molecule is associated with a combination of the at least one therapeutic agent and the at least one targeting moiety, the guest molecule is associated with at least one imager. [0047] In an embodiment, the at least one host molecule is selected from any or a combination of cucurbituril, cyclodextrins, calixarenes, pillararenes, porphyrins, metallacrowns, crown ethers, cyclotriveratrylenes, cryptophanes, cyclophanes and carcerands. In an embodiment, the at least one host molecule is cucurbituril. In an embodiment, the at least one host molecule is cucurbit[n]uril, wherein n ranges from 5 to 10. In an embodiment, the at least one host molecule is cucurbit[7]uril (hereinafter referred to as CB[7]). However, any other host molecule, as known to or appreciated by a person skilled in the art, can be utilized to serve its intended purpose, as laid down in the present disclosure, without departing from the scope and spirit of the present invention.

[0048] In an embodiment, the at least one guest molecule is selected from any or a combination of propylamine, hexamethylenediamine (HMD) and adamantylamine. However, any other guest molecule, as known to or appreciated by a person skilled in the art, can be utilized to serve its intended purpose, as laid down in the present disclosure, without departing from the scope and spirit of the present invention.

[0049] In an embodiment, the at least one guest molecule is associated with the at least one imager through a first linking moiety and the at least one host molecule is associated with the at least one targeting moiety through a second linking moiety. In an embodiment, the first linking moiety and the second linking moiety is selected independently from any or a combination of tram'-cyclooctene (TCO), 1,2,4,5-tetrazine (TZ) and N-hydroxysuccinimide (NHS). However, any other linking moiety, as known to or appreciated by a person skilled in the art, can be utilized to serve its intended purpose, as laid down in the present disclosure, without departing from the scope and spirit of the present invention.

[0050] In an embodiment, the at least one imager is selected from any or a combination of fluorophores like Alexa Fluor, Cy5, Atto 655 and Rhodamine, but not limited thereto. However, any other imager, as known to or appreciated by a person skilled in the art, can be utilized to serve its intended purpose as laid down in embodiments of the present disclosure without departing from the scope and the spirit of the present invention.

[0051] In an embodiment, the at least one targeting moiety is selected from any or a combination of antibodies, small molecules and nanoparticles, but not limited thereto. Any other targeting moiety, as known to or appreciated by a person skilled in the art, can be utilized to serve its intended purpose as laid down in embodiments of the present disclosure without departing from the scope and the spirit of the present invention.

[0052] In an embodiment, any one or a plurality of therapeutic agents can be utilized for the present invention to effect advantages realized herein in accordance with embodiments of the present disclosure. The term "therapeutic agent" and "drug" used throughout the present disclosure, interchangeably and synonymously, includes within its scope, any natural, semisynthetic or synthetic molecule that may bear therapeutic efficacy or which may aid in diagnosis, prognosis, prevention or cure of any ailments or disease or disorder in a mammal. In an exemplary embodiment, the therapeutic agent(s) can be any of anti-cancer agents to effect site-directed delivery thereof, in accordance with embodiments of the present disclosure. In an exemplary embodiment, the therapeutic agent(s) can be a diagnostic agent. In an exemplary embodiment, the therapeutic agent(s) can be a contrast media.

[0053] In an embodiment, the cucurbituril is CB[7], and the association of CB[7] with the at least one therapeutic agent increases solubility of said at least one therapeutic agent. In an embodiment, the association of CB[7] with the at least one therapeutic agent affords protection to said at least one therapeutic agent from degradation in a biological medium. In an embodiment, the system affords site specific delivery of the at least one therapeutic agent by targeting tissue and organelles. In an embodiment, the system affords a convenient route to any or a combination of cell surface engineering and cell surface modification. In an embodiment, the host molecule associated with said at least one therapeutic agent exhibits capability to bind with proteins to afford protection to pharmacokinetics of the at least one therapeutic agent. In an embodiment, the system affords delivery of any or a combination of DNA, RNA, siRNA and protein by non-covalent interaction thereof with the host molecule.

[0054] In an embodiment, the interaction of the at least one guest molecule associated with the imager with the at least one host molecule through the non-covalent forces effects ON-state of the imager associated with the guest molecule when subjected to points accumulation for imaging in nanoscale topography (PAINT). In an embodiment, the imager associated with the at least one guest molecule exhibits OFF-state in an event of absence of interaction between the at least one guest molecule and the at least one host molecule through the non-covalent forces when subjected to points accumulation for imaging in nanoscale topography (PAINT). In an embodiment, the ON-state and OFF-state of the imager associated with the at least one guest molecule is effected without utilization of any of an external stimuli and an additive.

[0055] FIG. 1A illustrates an exemplary snippet depicting the general principle behind utilization of the dynamic host-guest interactive system, realized in accordance with embodiments of the present disclosure, in imaging. As can be seen from FIG. 1A, a host molecule (102) is targeted to the biomolecule of interest (106) through the use of a targeting agent/moiety (104). Once a guest molecule (108), associated with a fluorophore/imager (110) is introduced into the medium, it interacts transiently with the host through non-covalent forces operative therebetween. In the unbound state only background fluorescence (OFF state) is observed (shown as 112), whereas a bright fluorescence emission (ON state) is detected upon transient immobilization of the imager through specific binding with host (shown as 114). This autonomous fluorescence blinking provides the foundation for the super-resolution imaging. As illustrated in FIG. IB, the imaging process includes recording a series of images where in each frame only a fraction of the molecules are stochastically in ON state.

[0056] A person skilled in the art would immediately realize that the imaging technique, realized in accordance with embodiments of the present disclosure, utilizes the dynamic interaction between a macrocyclic host (or similar supramolecular system) and fluorescently labeled guest molecule to obtain autonomous ON/OFF-switching of fluorescence under physiological condition without using any additives. Surprisingly, through coupling of macrocyclic host with antibodies or small molecule based targeting platforms, the inventors of the present disclosure for the first time establish dynamic host-guest interactive system mediated autonomous single molecule blinking with prescribed brightness and frequency, enabling two-dimensional (2D) and three-dimensional (3D) super-resolution imaging of biomolecules in cells. Further, the inventors of the present disclosure for the first time establish that cell permeable nature of host-guest interactive system along with their interaction specificity in biological environment allow the instant technique to be applicable in live-cell super-resolution microscopy. Supramolecular interaction between the host-guest system enables super-resolution fluorescence imaging of molecules inside living cells, fixed cells and tissue sample. The dynamic host-guest interactive system, realized in accordance with embodiments of the present disclosure, represents the first of its kind of synthetic PAINT labels that are small, membrane-permeable, provide interaction specificity, and can be applied to live-cell super-resolution imaging.

[0057] Accordingly, another aspect of the present disclosure provides a dynamic host- guest interactive system including at least one host molecule; and at least one guest molecule wherein the at least one host molecule is associated with at least one targeting moiety, and wherein the at least one guest molecule is associated with at least one imager, and wherein the at least one imager associated with the at least one guest molecule exhibits OFF-state in an event of absence of non-covalent forces operative between the at least one guest molecule and the at least one host molecule, and wherein non-covalent interaction of the at least one guest molecule associated with the imager with the at least one host molecule effects ON- state of the imager associated with the guest molecule, when subjected to points accumulation for imaging in nanoscale topography (PAINT). FIG. 1C illustrates exemplary snippets depicting accuracy of the imaging technique, realized in accordance with embodiments of the present disclosure, in comparison to traditional imaging technique (diffraction limited image).

[0058] In an embodiment, the at least one host molecule is selected from any or a combination of cucurbiturils, cyclodextrins, calixarenes, pillararenes, porphyrins, metallacrowns, crown ethers, cyclotriveratrylenes, cryptophanes, cyclophanes and carcerands. However, any other host molecule as known to or appreciated by a person skilled in the art can be utilized to serve its intended purpose as laid down in the present disclosure without departing from the scope and spirit of the invention.

[0059] In an embodiment, the at least one guest molecule is selected from any or a combination of propylamine, hexamethylenediamine and adamantylamine. However, any other guest molecule as known to or appreciated by a person skilled in the art can be utilized to serve its intended purpose as laid down in the present disclosure without departing from the scope and spirit of the invention.

[0060] In an embodiment, the at least one imager is selected from any or a combination of Alexa Fluor, Cy5, Atto 655, Rhodamine and the like fluorophores. However, any other imager as known to or appreciated by a person skilled in the art can be utilized to serve its intended purpose as laid down in the present disclosure without departing from the scope and spirit of the invention.

[0061] In an embodiment, the at least one targeting moiety is selected from any or a combination of antibodies and small molecules. However, any other targeting moiety/agent as known to or appreciated by a person skilled in the art can be utilized to serve its intended purpose as laid down in the present disclosure without departing from the scope and spirit of the invention.

[0062] Further, the inventors of the present disclosure, for the first time, establish dynamic host-guest interactive system mediated site directed drug delivery, optionally, along with imaging of the same. Interestingly and surprisingly, non-covalent host-guest interactions provide efficient and novel means to deliver phalloidin or like therapeutic agents of interest to the site of interest. Further, it is established by the inventors of the present disclosure that utilization of non-covalent interaction between guest molecule(s) associated with targeting moiety(ies) such as nanoparticles and the likes and host molecule(s) associated with therapeutic agent can allow therapeutic agent(s) with relatively less cell membrane permeability to be delivered to the site of interest (e.g. cytosolic delivery). [0063] Accordingly, another aspect of the present disclosure provides a dynamic host- guest interactive system including at least one host molecule; and at least one guest molecule wherein the at least one host molecule is associated with at least one therapeutic agent, and wherein the at least one guest molecule is associated with at least one targeting moiety, and wherein the at least one guest molecule is associated with the at least one targeting moiety through covalent bond. FIG. 2 illustrates an exemplary snippet depicting the general principle behind utilization of the dynamic host-guest interactive system, realized in accordance with embodiments of the present disclosure, in drug delivery. As can be seen from FIG. 2, a dynamic host-guest interactive system including at least one host molecule (206); and at least one guest molecule (208) wherein the at least one host molecule (206) is associated with at least one therapeutic agent (204), and wherein the at least one guest molecule (208) is associated with at least one targeting moiety (210), and wherein the at least one guest molecule (208) is associated with the at least one targeting moiety (210) through a covalent bond.

[0064] In an embodiment, the at least one host molecule is selected from any or a combination of cucurbiturils, cyclodextrins, calixarenes, pillararenes, porphyrins, metallacrowns, crown ethers, cyclotriveratrylenes, cryptophanes, cyclophanes and carcerands, but not limited thereto. In an embodiment, the at least one guest molecule is selected from any or a combination of propylamine, hexamethylenediamine and adamantylamine, but not limited thereto. In an embodiment, the at least one targeting moiety is selected from any or a combination of antibodies, small molecules and nanoparticles, but not limited thereto.

EXAMPLES

[0065] EXAMPLE 1 : Imaging of F-actin fibers in MEF cells utilizing a dynamic host-guest interactive system including CB[7] as a host molecule conjugated with Phalloidin (a targeting moiety), and hexamethylene diamine (a guest molecule) conjugated with Cy5 (an imager)

[0066] Preparation of CB[7] conjugated with Phalloidin and Hexamethylene Diamine (HMD) conjugated with Cy5

[0067] As illustrated in Scheme 1 below, CB[7] was attached with tram'-cyclooctene (TCO) through functional modification of CB[7], and the 1,2,4,5-tetrazine (Tz) was anchored onto Phalloidin using its amine functionality. CB[7] conjugated Phalloidin was prepared via strain promoted cyclo-addition reaction between TCO and Tz.

Scheme 1: Synthesis of CB[7] conjugated with a targeting moiety

[0068] Synthesis of CB[7]-OH: CB[7] (250 mg, 0.215 mmol) was taken in a 50 ml quartz tube. It was dissolved in 40 ml solution of miliQ water and 10 M HC1 solution (1/1 vol. %) under argon atmosphere. Sample was sonicated for a brief period to ensure that all the CB[7] was solubilized before the addition of a solution of hydrogen peroxide (49 μΐ from 30% solution, 0.215 mmol). Then the solution was subjected to UV irradiation at wavelength of 250-400 nm (450 W) under vigorous stirring condition for 7.5 h. After that, the acidic solution was evaporated using rotary evaporator upto 2 ml and 250 ml methanol was added to it. The resulted precipitate was allowed to settle down for overnight. Next, the supernatant was decanted and remaining volume was centrifuged at 5500 rpm to get a light-yellow colored precipitate. The precipitate was washed with 50 ml of methanol for 3 times and finally dried in vacuum oven for overnight.

CB[7] CB[7]-OH

[0069] Synthesis of CB[7]-0-allyl: NaH (64.8 mg, 60% dispersion in mineral oil, 1.62 mmol) was added to 6 ml anhydrous DMSO solution of CB[7]-OH (191 mg, 0.162 mmol) under dry argon atmosphere. The reaction was stirred for 1 h at room temperature. Allyl bromide (138.86 μΐ, 1.62 mmol) was then added to this reaction mixture maintaining the temperature of reaction mixture at 0°C during addition. The reaction was further stirred at room temperature for 18 h. After that the reaction mixture was poured into 200 ml diethyl ether, sonicated and allowed to stand for overnight. Then the supernatant was decanted and the remaining volume was centrifuged at 5500 rpm to get white precipitate. Then it was washed with 200 ml diethyl ether for 3 times and finally dried under high vacuum.

CB[7]-0-allyl

[0070] Synthesis of CB[7]-NH2: CB[7]-0 Allyl (124 mg, 0.1017 mmol) was taken in a quartz cuvette and dissolved in 3 ml miliQ water. Cystamine hydrochloride (115.53 mg, 1.017 mmol) was added to it and the reaction mixture was purged with N2 gas followed by subjected to UV irradiation at wavelength of 254-400 nm (450 W) for 12.5 h. After completion of the reaction, 50 ml ethanol was added to get white precipitate. The precipitate was allowed to settle down for overnight. Then supernatant was decanted and remaining volume was centrifuged at 5500 rpm to get solid precipitate. Next, it was washed with 50 ml ethanol and dried under high vacuum.

[0071] Synthesis of CB[7]-PEG-TCO: CB[7]- H2 (1.7 mg, 1.297 μιηοΐ) was taken in 3 ml glass vial and dissolved in 200 μΐ dry DMF. Et3N (1.8 ul, 12.97 μιηοΐ) was added to it. TCO-PEG- HS ester (1 mg, 1.945 μιηοΐ) was dissolved in 100 μΐ DMF in another vial and added to the reaction mixture. The reaction was stirred at room temperature for 28 h. After completion of reaction, 12 ml MeOH was added to the reaction mixture to get a white precipitate. It was allowed to settle down for 15 min. The precipitate was collected after centrifugation at 7200 rpm for 5 min. The above step was repeated twice to remove all the unreacted starting materials.

CB[7]-PEG-TCO

[0072] Synthesis of CB[7] conjugated phalloidin: Amino phalloidin (120 μg, 60 μΐ from 2 mg ml ^-1 stock solution in dry DMF, 0.1523 μιηοΐ) was taken in 1.5 ml microcentrifuge tube and 5 μΐ DMF solution containing 0.106 μΐ Et3N was added to it. Afterward, 30.34 μΐ Tetrazine- HS ester (from a stock solution of 10 mg ml-1 in DMF, 0.7615 μιηοΐ) was added the above reaction mixture and stirred at room temperature for 3 h to obtain Phalloidin-tetrazine conjugate and purified later by using HPLC. Purified Phalloidin- tetrazine conjugate (30 μg, 0.038 μιηοΐ) in 50 μΐ water was taken in 1.5 ml microcentrifuge tube and CB[7]-PEG-TCO (130 μg, 39.1 μΐ from 3.33 mg ml-1 stock solution in H20, 0.076 μιηοΐ) was added to it and stirred at room temperature for overnight to prepare CB[7] conjugated phalloidin (Scheme 2).

Phalloidine-NH2 Tetrazine-NHS (Tz-NHS) Phalloidin-Tetrazine

CB[71 conjugated phalloidin Scheme 2: Synthesis of CB[7] conjugated phalloidin

[0073] Hexamethylene diamine conjugated with Cy5

[0074] Synthesis of thiol derivative of Hexamethylene diamine (HMD)

0°C→-RT Compound 4

Compound 3 Compound 4

Compound 6 Compound 5

Scheme 3: Synthesis of thiol derivative of HMD

[0075] Synthesis of Compound 1: Tetra ethylene glycol (TEG, 10 g, 51.22 mmol) was taken in 250 ml RB Flask. 130 ml acetonitrile and triethyl amine (7.13 ml, 51.22 mmol) were added to it. Tosyl chloride (9.76 g, 51.22 mmol), dissolved in 20 ml acetonitrile was added drop wise from dropping funnel over 1 h keeping the reaction mixture at 0°C. After the addition, the reaction mixture was stirred at room temperature for 14 h. During the reaction, a white precipitate of triethyl ammonium hydrochloride was formed. After completion of reaction, the precipitate was filtered and washed with acetonitrile. The acetonitrile was evaporated and the compound was purified using flash silica (230-400 mesh) column (eluent: ethyl acetate (EtOAc)/hexane, gradient elution from 0 to 80%). [0076] Synthesis of Compound 2: A solution of NaOH (0.864 g, 21.6 mmol) in 8 ml water was added to a solution of triphenylmethanethiol (3.98 g, 14.402 mmol) in a mixture of toluene/ethanol (EtOH) (1: 1 v/v, 50 ml). Compound 1 (5.019 g, 14.402 mmol) was dissolved in a second solution of toluene/EtOH (1: 1 v/v, 50 ml), which was then added to the triphenylmethanethiol solution in one portion. The reaction mixture was stirred at room temperature for 18 h. After completion of reaction (monitored by TLC) the reaction mixture was poured into 20 ml saturated NaHC03 solution and extracted with diethyl ether (Et20) (3 x 40 ml). The combined organic layers were washed with brine (3 x 40 ml), dried over Na2S04 and solvent was removed under vacuum to give a pale-yellow oil. The crude product was purified by silica column (60-120 mesh) chromatography (eluent: EtOAc/hexane, 1 :2→3 : 1) to yield compound 2 as a pale-yellow oil.

[0077] Synthesis of Compound 3: A solution of compound 2 (5.43 g, 12.0 mmol) and triethylamine (2.5 ml, 18 mmol) in 60 ml dry dichloromethane (DCM) was stirred below 5°C under argon atmosphere. Methanelsulfonyl chloride (1.68 ml, 24.0 mmol) was added in dropwise fashion while maintaining the temperature below 5°C. The reaction mixture was stirred for 30 min below 5°C and then allowed to stir at room temperature for another 12 h. After completion of reaction (monitored by TLC), the resulting residue was diluted to 100 ml with DCM and washed with 0.1 M HC1 (2 x 25 ml), saturated NaHC03 solution (2 x 25 ml), and brine solution (1 x 25 ml). The organic layer was dried over Na2S04 and concentrated under reduced pressure. The crude product was purified by flash silica column (eluent: EtOAc/hexane 1: 1) to yield compound 3 as a pale-yellow oil.

[0078] Synthesis of Compound 4: Compound 4 was synthesized by following a literature reported preocedure6. Hexamethylenediamine (HMD, 13.3 g, 114.5 mmol) was taken in 120 ml chloroform. Di-tert-butyldicarbonate (2.5 g, 11.45 mmol) dissolved in 30 ml chloroform was added to the reaction mixture over two h using dropping funnel keeping the reaction mixture below 5°C. Then the reaction mixture was stirred at room temperature for 12 h. During the reaction a white precipitate was formed which was filtered. The filtrate was concentrated to 50 ml using rotary evaporator, washed with 0.1N HC1 (2 x 50 ml), water (4 x 50 ml) and brine solution (2 x 30 ml). The organic layer was filtered through Na2S04 to give compound 4.

[0079] Synthesis of Compound 5: Compound 4 (812.1 mg, 3.7545 mmol) was first dissolved in 2 ml dry DCM/mehanol (MeOH) (1: 1 v/v) and placed in a 10 ml round bottom (RB) flask. Compound 3 (400 mg, 0.7509 mmol) was dissolved in 2 ml of dry DCM/MeOH (1:1 v/v) in a vial and added to it. The reaction mixture was heated to 50°C and stirred for 48 h. After completion of reaction (monitored by TLC), the reaction mixture was concentrated under reduced pressure, diluted with DCM and directly charged on a flash silica column.

[0080] Synthesis of Compound 6: Compound 5 (300 mg, 0.4613 mmol) was dissolved in 4.5 ml Dry DCM in a 10 ml RB flask. The solution was purged with nitrogen and an excess of trifluoro acetic acid (TFA, 710.8 μΐ, 9.226 mmol) was added. During the addition period of TFA, the color of the solution turned to yellow. Subsequently, triisopropylsilane (TIPS, 472.52 μΐ, 2.3065 mmol) was added to the reaction mixture and the color of mixture slowly changed to colorless. Then the reaction mixture was allowed to stir at room temperature for 6 h under nitrogen atmosphere. The volatile components (DCM, TFA and TIPS) were then removed under reduced pressure. The pale-yellow residue was purified by washing with hexane (3 x 10 ml) and diethyl ether (3 x 10 ml). A viscous colorless liquid was obtained after drying under high vacuum.

[0081] Synthesis of HMD-Cy5: Cy5 maleimide (66.67 μg, 50 μΐ from 1.33 mg ml-1 stock solution in PBS (pH 7.4), 0.0876 μιηοΐ) was taken in 1.5 ml microcentrifuge tube. A stock solution of Compound 6 (16.4 μg, 50 μΐ from 0.82 mg ml-1 stock solution in PBS (pH 7.4), 0.1314 μιηοΐ) was added to the Cy5-maleimide solution and stirred for overnight at room temperature. After that the reaction mixture was directly injected to reverse phase HPLC for purification to yield HMD-Cy5 (90% from HPLC chromatogram).

[0082] Imaging of F-actin using phalloidin as small molecule targeting moiety

[0083] For labeling F-actin, fixed (4% PFA) and permeabilized cells were incubated with 2 μΜ solution of CB[7] conjugated phalloidin for 12 h at 4°C. Excess phalloidin was removed and imaging was performed after washing the cells once with PBS. The fluorescence images were acquired using an inverted Zeiss ELYRA PS 1 microscope, capable of illuminating the sample with TIRF illumination mode. The microcope was fitted with a definite focus system to eliminate z drift. Two lasers have been used for excitation: 561 nm (200 mW) and 642 nm (150 mW). Imaging was performed using a Zeiss oil-immersion TIRF objective (alpha Plan-apochromat DIC lOOx/1.46 Oil DIC M27, numerical aperture (NA) 1.46 oil). Fluorescence light was spectrally filtered with emission filters (MBS-561+EF BP 570-650/LP 750 for laser line 561 and MBS-642+EF LP 655 for laser line 642) and imaged on an electron-multiplying charge-coupled device (EMCCD) camera (Andor iXon DU897, quantum yield >90%, 512 x 512 pixels). In vitro single molecule data was then analysed by using a custom-programmed software package named 'Picasso' . Super-resolution 2D and 3D images were reconstructed using PALM analysis package for Zen software (Zeiss). The drift correction was implemented using fiducial based method. Additional softwares have been used for color adjustment (ImageJ) and data analysis (Origin 9.0).

[0084] FIG. 3A illustrates an exemplary super-resolution image of F-actin fibers in MEF cells stained with CB[7] conjugated phalloidin (scale bar 1 μιη). FIG. 3B illustrate an exemplary image (zoomed-in view, scale bar 500 nm) depicting F-actin fibers in MEF cells utilizing traditional imaging technique (diffraction limited image). FIG. 3D illustrates an exemplary graph depicting comparison of super-resolution fluorescence microscopy of F- actin, cross-section profile of a single filament dl in FIG. 3C, that shows a Gaussion fit with FWHM of 29.87+2.91 nm) in accordance with an embodiment of the present disclosure, with the results obtained from traditional imaging technique.

[0085] EXAMPLE 2

[0086] Site directed delivery (cytosolic delivery) of membrane impermeable Phalloidin derivative into the HeLa cells utilizing a dynamic host-guest interactive system including CB[7] as a host molecule conjugated with Phalloidin (therapeutic agent), and hexamethylene diamine (a guest molecule) conjugated with gold nanoparticles (a targeting moiety)

[0087] Synthesis of 2 nm Gold nanoparticles (AuNPs): Pentane thiol capped gold nanoparticles were synthesised following Brust Schiffrin method as reported in "preparation of 2nm gold nanoparticles for in vivo and in vitro applications, Methods Mol. Biol. 2013, 1025, 3-8".

[0088] Synthesis of hexamethylene diamine (HMD) functionalized Gold nanoparticles: 5 mg of 2 nm pentane thiol capped AuNPs were taken in 1 ml dry DCM (DCM purged with N2) to vial containing magnetic stirring bar. 30 mg of Compound 6 thiolated ligand was weighed, immediately transferred and dissolved in 1 ml of Dry DCM. Mix them and stirred at room temperature for 60 h. Solvents were removed in a rotary evaporator. Solid residue was purified by hexane followed by DCM by the support of sonication and centrifugation, redispersed in Milli-Q water and dialysed against water for 2 days. The dialysed nanoparticle was concentrated using Amicon lOKDa MWCO filter. Portion was dissolved in D20 and characterized.

[0089] Preparation of live cell: HeLa cells (epithelial, human cervical cancer cell line) were used for the live-cell experiment. The cells were cultured in a humidified atmosphere (5% C02) at 37°C and grown in Dulbecco's Modified Eagle's Medium (DMEM, high glucose) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 2 mM Glutamax (Invitrogen, USA) and 1% antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin) (Gibco, USA). At -80% confluence, the cells were washed 3 times with DPBS (Gibco, USA), trypsinized and suspended in culture medium. Afterward, cells were then counted and then in a typical experiment, -25,000 cells/well were plated in 8- well chamber slide system (Eppendorf). The cells were then maintained again in a humidified atmosphere (37°C, 5% C02) for 24 h to reach -60% confluence. Thereafter, cells were used for imaging experiment.

[0090] Live cell delivery of phalloidin-CB[7] conjugate: Culture media was removed from the chamber wells and cells were washed twice with DPBS (pH 7.4). 2 μΜ concentration of Phalloidin-CB[7] conjugate was assembled with HMD associated with gold nanoparticles at a concentration ratio of 10: 1 and added to the cells. Cells were then maintained at 37°C with 5% C02 in a humidified atmosphere for 3 hours. After 3 hours of incubation, cells were washed twice with DPBS.

[0091] EXAMPLE 3

[0092] Site directed delivery (cytosolic delivery) of membrane impermeable Phalloidin derivative into the HeLa cells utilizing a dynamic host-guest interactive system including CB[7] as a host molecule conjugated with Phalloidin (therapeutic agent), and hexamethylene diamine (a guest molecule) conjugated with gold nanoparticles (a targeting moiety) with imaging thereof.

[0093] HMD functionalized Gold nanoparticles and CB[7] conjugated with Phalloidin were prepared in accordance with the procedure provided in Example 2 above. Further, HMD conjugated Rhodamine was prepared to effect imaging of F-actin fibers in MEF cells.

[0094] Synthesis of HMD-Rhodamine: Rhodamine maleimide (37.62 μg from 1.33 mg ml-1 stock solution in PBS (pH 7.4), 0.068 μιηοΐ) was taken in 1.5 ml microcentrifuge tube. A stock solution of Compound 6 (63 μΐ from 1 mg ml-1 stock solution in PBS (pH 7.4), 0.2042 μιηοΐ) was added to the Rhodamine maleimide solution and stirred for overnight at room temperature. After that the reaction mixture was directly injected to HPLC for purification to yield HMD-Rhodamine.

[0095] Imaging with super-resolution fluorescence microscopy technique: After nanoparticle mediated delivery of Phalloidin-CB[7] conjugate utilizing methodology as provided in Example 2 hereinabove, cells were immediately incubated with 10 nM PBS solution of silicon Rhodamine conjuagted HMD imager. The imaging was started immediately after the imager incubation. The images were captured using an inverted Zeiss ELYRA PS 1 microscope, capable to illuminating the sample with TIRF illumination mode. The microscope was fitted with a definite focus system to eliminate z drift. 642 nm (150 mW) laser has been used for excitation of Alexa 647 dye. Imaging was performed using a Zeiss oil-immersion TIRF objective (alpha Plan-apochromat DIC lOOx/1.46 Oil DIC M27, numerical aperture (NA) 1.46 oil). Fluorescence light was spectrally filtered with emission filter (MBS-642+EF LP 655 for laser line 642) and imaged on an electron -multiplying charge-coupled device (EMCCD) camera (Andor iXon DU897, quantum yield >90%, 512 x 512 pixels). Camera readout rate was 1 MHz at 16 bit. The recorded image dimension was 51.10 μιη x 51.10 μιη. EMCCD camera gain was kept at 200 during image acquisition. Laser power density of ~ 138 W/cm2 was used to excite the fluorophore molecules. Power denisty was estimated from the field of view and the laser power at the back focal plane of the objective. Super-resolution images were reconstructed using PALM analysis package for Zen software (Zeiss). FIG. 4A illustrate an exemplary image depicting super-resolution fluorescence microscopy of phalloidin targeted F-actin (scale bar: 5 μιη), in accordance with an embodiment of the present disclosure. Non-covalent interaction mediated delivery of phalloidin allowed the access to the CB[7] functionality for imaging via super-resolution fluorescence microscopy technique. After target binding, incubation of the cells with silicon rhodamine labeled HMD imager resulted in spontaneous blinking. FIG. 4B illustrate an exemplary image of phalloidin targeted F-actin (scale bar: 5 μιη) utilizing traditional imaging technique. As can be seen from FIG. 4A and FIG. 4B, a significant improvement of resolution and much sharper image from live cell could be obtained utilizing the technique in accordance with embodiments of the present disclosure. FIG. 4C illustrates an exemplary graph depicting comparison of super-resolution fluorescence microscopy of phalloidin targeted F-actin (cross-section profile of an actin filament - region marked with a square in FIG. 4A and FIG. 4B), in accordance with an embodiment of the present disclosure, with the results obtained from traditional imaging technique. FIG. 4C shows a Gaussion fit with full width at half maximum (FWHM) of 59.39+1.86 nm for image obtained in accordance with embodiment of the present disclosure as compare to a FWHM of 358.27+8 nm for the diffraction limited image.

[0096] While the foregoing describes various embodiments of the invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art. ADVANTAGES OF THE INVENTION

[0097] The present disclosure provides a dynamic host-guest interactive system that can overcomes the deficiencies associated with the prior art.

[0098] The present disclosure provides a dynamic host-guest interactive system for imaging.

[0099] The present disclosure provides a dynamic host-guest interactive system for drug delivery.

[00100] The present disclosure provides an imaging and a drug delivery system that can utilize supramolecular host-guest interaction.

[00101] The present disclosure provides an imaging and a drug delivery system that exhibits desired specificity.

[00102] The present disclosure provides an imaging and a drug delivery system that gives image of the biological system without utilization of any of an external stimuli or an additive.

[00103] The present disclosure provides an imaging and a drug delivery system that offers improved resolution without any complex instrumentation or specialized experimental conditions.

[00104] The present disclosure provides an imaging and a delivery system that is easy to implement.

[00105] The present disclosure provides an imaging and a delivery system that is cost- effective.