Near infrared dyes and conjugates for targeting tumors

Abstract

Comprising the design, synthesis and functionalization of conjugates of tumor targeting near infrared (NIR) dyes and therapeutic and / or diagnostic agents, whereby the NIR dyes can act to target the therapeutic and / or diagnostic agents to tumor cells.

Description

[0001] This International Patent Cooperation Treaty patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 344,972, filed on May 23, 2022, which is incorporated herein by reference. Background Art

[0002] There remains a need for targeted cancer therapeutics that have maximum efficacy in treating or killing tumor cells and minimal toxicity to non-tumor or healthy cells. Additionally, there remains a need for targeted cancer diagnostics. Summary of the invention

[0003] In general, the present invention details the use of tumor-targeting near-infrared (NIR) fluorescent dyes in conjunction with various therapeutic and / or diagnostic agents.

[0004] Next, a broad object of specific embodiments of the present invention may be to provide a conjugate comprising a tumor targeting NIR dye and a therapeutic agent, whereby the dye functions to target and / or deliver the therapeutic agent to tumor cells.

[0005] Another broad object of specific embodiments of the present invention may be to provide a conjugate comprising a tumor targeting NIR dye and a diagnostic agent, whereby the dye acts to target and / or deliver the diagnostic agent to tumor cells.

[0006] Another broad object of specific embodiments of the present invention may be to provide a method of killing tumor cells by targeting a conjugate comprising a tumor-targeting NIR dye and a therapeutic agent to the tumor cells.

[0007] Another broad object of specific embodiments of the present invention may be to provide methods of diagnosing the presence of tumor cells by targeting a conjugate comprising a tumor-targeting NIR dye and a diagnostic agent to the tumor cells, if present.

[0008] Naturally, further objects of the invention are disclosed throughout other areas of the specification, drawings and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 is a graphical representation of the change in the mean NIR intensity per well over time for a lung cancer cell line (A549) and four normal cell lines (colon cells, kidney cells, hepatocytes, and HUVEC) after a 20 μM dose of the dye represented by Formula V.

[0010] Figure 2 Illustrative examples of zwitterions that can be used in the present invention when attached to tumor targeting NIR dyes and / or nanoparticles are shown.

[0011] Figure 3The hydration ability of zwitterions useful in the present invention, which have positively charged groups and negatively charged groups that can strongly associate with polar water molecules through ion-dipole interactions, is shown.

[0012] FIG. 4 shows an exemplary molecule comprising a negatively charged sulfonate group and a positively charged indole group, whereby the positive charge of the nitrogen is part of a resonance structure that delocalizes the positive charge.

[0013] Figure 5A Shown are the experimental heating temperature profiles of gold nanorods irradiated with a NIR laser with a wavelength of 808 nm and different power densities.

[0014] Figure 5B The experimental heating temperature curves of the gold nanorod conjugate irradiated with NIR laser of wavelength 808 nm and different power densities are shown.

[0015] Figure 5C Theoretical heating temperature curves of gold nanorods irradiated with NIR lasers with a wavelength of 808 nm and different power densities are shown.

[0016] Figure 5D The theoretical heating temperature curves of the gold nanorod conjugate irradiated with NIR laser at a wavelength of 808 nm and different power densities are shown.

[0017] Figure 6 The experimental heating temperature curves of gold nanorods and gold nanorod conjugates irradiated with LED light source are shown.

[0018] Figure 7 Shown are the UV-vis extinction spectra of gold nanorods (GNRs, blue line) and gold nanorod conjugates with the dye of Formula II (GNRCs, red line).

[0019] Figure 8 The UV-vis extinction spectrum of the dye of formula II is shown.

[0020] Fig.9A Dynamic light scattering (DLS) size distribution of gold nanorods is shown.

[0021] Fig. 9B The dynamic light scattering (DLS) size distribution of the gold nanorod conjugate with the dye of Formula II is shown.

[0022] Fig. 10A Specific examples of conjugates of the invention are presented, comprising a tumor-targeting NIR dye attached to zwitterionic nanoparticles.

[0023] Fig. 10BSpecific examples of conjugates of the invention are presented, comprising zwitterionic tumor-targeting NIR dyes attached to zwitterionic nanoparticles.

[0024] Fig.11 Specific examples of conjugates of the invention are shown, comprising DOX conjugated to a tumor-targeting NIR dye via a pH-sensitive cleavable linker having an acid-labile hydrazone bond.

[0025] Fig. 12A Specific examples of conjugates of the invention comprising a PEG linker and SN-38 as a chemotherapeutic agent are shown.

[0026] Fig. 12B Specific examples of conjugates of the invention comprising a PEG linker and SN-38 as a chemotherapeutic agent are shown.

[0027] Fig. 12C Specific examples of conjugates of the invention comprising a PEG linker and DOX as a chemotherapeutic agent are shown.

[0028] Fig.12D Specific examples of conjugates of the invention comprising a PEG linker and DOX as a chemotherapeutic agent are shown.

[0029] Fig.13 Specific examples of conjugates of the invention are shown, which have a linker comprising a VC dipeptide that can be cleaved by cathepsin B to release a conjugate portion comprising a chemotherapeutic agent (SN-38) and a self-cleavable PABC spacer. Subsequently, PABC can undergo spontaneous 1,6-elimination in an acidic environment to release carbon dioxide, p-azaquinone methide, and SN-38.

[0030] Fig.14A Specific examples of conjugates of the invention prepared using click chemistry and including SN-38 as a chemotherapeutic agent are shown.

[0031] Fig. 14B Specific examples of conjugates of the invention prepared using click chemistry and including SN-38 as a chemotherapeutic agent are shown.

[0032] Fig. 14C Specific examples of conjugates of the invention prepared using click chemistry and including DOX as chemotherapeutic agent are shown.

[0033] Fig.14D Specific examples of conjugates of the invention prepared using click chemistry and including DOX as chemotherapeutic agent are shown. DETAILED DESCRIPTION

[0034] The present invention can provide an effective and versatile delivery method that combines tumor-targeting near-infrared (NIR) fluorescent dyes with various cancer therapeutics and / or cancer diagnostics to produce conjugates for: (i) targeting tumor cells and / or (ii) killing tumor cells and / or (iii) imaging tumor cells and / or (iv) theranostics, whereby the NIR dye acts to direct the conjugate to a target tumor cell population; this may be in contrast to the non-specific delivery of conventional anticancer agents, which may cause significant adverse side effects. In addition to targeting tumor cells, the present NIR dyes can also absorb NIR light and emit it in the same area with appreciable brightness, which can be extremely desirable given the non-invasive nature of NIR light and its tissue penetration, which can be several orders of magnitude greater than that of ultraviolet or visible light.

[0035] As to a specific embodiment, the tumor targeting NIR dye may be a cyanine dye, whereby chemically, the cyanine dye comprises two nitrogen atoms connected by a polymethine chain.

[0036] As for a specific embodiment, the tumor-targeting NIR dye may be a heptamethine cyanine dye.

[0037] As for specific embodiments, the tumor-targeted NIR dye may have the following formula I:

[0038]

[0039] in:

[0040] R1 to R8 may each independently be an unsubstituted or substituted C1 to C10 alkyl group, an unsubstituted or substituted aryl group, an unsubstituted or substituted C1 to C10 alkoxy group, an alkyl sulfonate group, an alkyl carboxylic acid group or an alkylamino group;

[0041] X can be Br - 、Cl-、I-、Perchlorate ion (ClO4 - ), toluenesulfonate ion PTS - or absent if another covalently linked anionic moiety is present in the dye molecule;

[0042] Y may be Cl, substituted C, O, S or N; and

[0043] n can be 0 or 1.

[0044] As for specific embodiments, one or more of R1 to R8 can be functionalized with zwitterions, small molecule chemotherapeutic drugs, photodynamic therapy (PDT) agents, or hyperthermia agents (such as gold nanoparticles, iron oxide nanoparticles, etc.) through various linkers.

[0045] As for specific embodiments, regarding Y, substituted C, O, S or N can be functionalized with zwitterions, small molecule chemotherapeutic drugs, photodynamic therapy (PDT) agents or hyperthermia agents (such as gold nanoparticles, iron oxide nanoparticles, etc.) through various linkers.

[0046] As to specific embodiments, the linker can be heterobifunctional and thus react with both the tumor targeting NIR dye and the therapeutic or diagnostic agent to form a conjugate, whereby as just a few illustrative examples, the linker can be polyethylene glycol (PEG), a polymer, a peptide, DNA, a silica nanoparticle, a shell structure (such as iron oxide or silica), an amide bond, an ester bond, an amine, an alcohol, a phenol, sulfur, a phenyl group or its derivatives, etc.

[0047] As to specific embodiments, the linker may be cleavable.

[0048] The tumor-targeting NIR dyes of the present invention may be symmetrical or asymmetrical, whereby exemplary dyes are shown in Formulas II, III, IV and V.

[0049]

[0050]

[0051] To illustrate the tumor targeting capability of a tumor targeting NIR dye, and specifically a specific embodiment of the dye shown in Formula V, 20 μM of the dye (absorbance is about 780 nm, emission is about 808 nm) was administered to cells, and Figure 1 The change in the mean NIR intensity per well over time associated with dye uptake for a lung cancer cell line (A549) versus four normal cell lines (colon cells, kidney cells, hepatocytes, and human umbilical vein endothelial cells (HUVEC)) is shown, whereby significant dye uptake is only evident in the cancer cell line.

[0052] As for specific embodiments, tumor-targeted NIR dyes can be modified to have zwitterionic functionality, whereby such modification can include attaching one or more zwitterions to the dye, each of which acts as a polyelectrolyte or dipole ion having both positively charged groups and negatively charged groups, but can also be overall neutral in charge. Additionally, zwitterions can have a high hydration capacity and can accordingly be highly hydrated through electrostatic interactions of their positively charged groups and negatively charged groups with polar water molecules, whereby the resulting tightly bound water layer forms a dense and stable hydration shell or solvation shell that can provide a physical and energy barrier that can prevent undesirable adsorption (such as but not limited to protein adsorption); therefore, zwitterions can have effective antifouling properties.

[0053] As for specific embodiments, illustrative examples of zwitterions that can be used in the present invention when attached to tumor targeting NIR dyes include sulfobetaines, carboxybetaines, phosphorylcholine, and Figure 2 The zwitterion shown in . According to the above, Figure 2 The zwitterions shown in can be overall neutrally charged and have significant hydration capabilities achieved through strong ion-dipole interactions of their positively and negatively charged groups with polar water molecules, such as Figure 3 shown.

[0054] In order to impart zwitterionic functionality to tumor-targeted NIR dyes, the zwitterions can be covalently or electrostatically linked or bound to the dye through a central ring (e.g., a central cyclopentyl or cyclohexyl ring), through the nitrogen of the indole moiety, through a covalent position, or through an aromatic ring. As for specific embodiments, one or more of R1 to R8 or Y as shown in Formula I can be used as a linking site to link or bind the zwitterion to the tumor-targeted NIR dye through various linkers to form the conjugate of the present invention.

[0055] Significantly, while some molecules may be polyelectrolytes with both positively charged groups and negatively charged groups and have an overall neutral charge, these molecules may not have the high hydration capacity required for the present invention because one or more of the charged atoms are part of a resonance structure. In detail, if the charged atoms are part of a resonance structure, the charge is delocalized or diffused over multiple atoms rather than associated with a single atom. This "diluted" charge then loses its hydration capacity through electrostatic interactions with polar water molecules and, accordingly, may not be used for the present invention. As just one illustrative example, Figure 4A The molecule shown in includes a negatively charged sulfonate group and a positively charged indole group; the positive charge of the nitrogen is part of a resonance structure involving a total of eleven atoms. Accordingly, the positive charge is delocalized and therefore does not have a high hydration capacity achieved by electrostatic interaction with polar water molecules. Subsequently, such a molecule may not be used in the present invention.

[0056] As another illustrative example, Figure 4B The molecule shown in also includes a negatively charged sulfonate group and a positively charged indole group. Similar to the above, the positive charge of nitrogen is part of a resonance structure involving a total of eleven atoms. Accordingly, the positive charge is delocalized and therefore does not have a high hydration capacity achieved by electrostatic interaction with polar water molecules. Next, this molecule may not be used in the present invention.

[0057] Similarly, the charged groups in the two NIR dyes shown in Formulas IV and V above may not have high hydration capabilities through electrostatic interactions with polar water molecules due to their resonance structures.

[0058] In view of the above, as to specific embodiments, zwitterions useful in the present invention may include charged groups in which the charge is associated with a single atom rather than being delocalized over multiple atoms, as this property imparts a desirable high hydration capacity to the zwitterion through electrostatic interactions with polar water molecules to form a hydration shell that can provide a physical and energetic barrier to prevent undesired adsorption.

[0059] As for specific embodiments, the zwitterionic tumor targeting NIR dye may have the following formula VI:

[0060]

[0061] As for specific embodiments, the zwitterionic tumor targeting NIR dye may have the following formula VII:

[0062]

[0063] As for specific embodiments, the zwitterionic tumor targeting NIR dye may have the following formula VIII:

[0064]

[0065] As for specific embodiments, the zwitterionic tumor targeting NIR dye may have the following formula IX:

[0066]

[0067] As for specific embodiments, the zwitterionic tumor targeting NIR dye may have the following formula X:

[0068]

[0069] As described above, the present invention can combine tumor-targeted NIR fluorescent dyes with various agents to produce conjugates for the treatment of different cancers, whereby these conjugates can be delivered directly to target tumor cell colonies. In order to form such conjugates, therapeutic agents or diagnostic agents can be covalently or electrostatically connected or bound to tumor-targeted NIR dyes, for example, through a central ring (such as a central cyclopentyl or cyclohexyl ring), through the nitrogen of the indole moiety, through a co-position or through an aromatic ring. As for specific embodiments, one or more of R1 to R8 or Y as shown in Formula I can be used as a connection site, to connect or bind a therapeutic agent or diagnostic agent to a tumor-targeted NIR dye to form a conjugate of the present invention through various joints.

[0070] As to specific embodiments, the conjugate comprising a tumor targeting NIR dye and a therapeutic or diagnostic agent may have the following formula XI:

[0071]

[0072] Wherein X can be NH or O.

[0073] As for specific embodiments, the conjugate comprising a tumor-targeting NIR dye and a therapeutic or diagnostic agent may have the following Formula XII:

[0074]

[0075] As to specific embodiments, the conjugate comprising a zwitterionic tumor targeting NIR dye and a therapeutic or diagnostic agent may have the following Formula XIII:

[0076]

[0077] Wherein X can be NH or O.

[0078] As to specific embodiments, the conjugate comprising a zwitterionic tumor targeting NIR dye and a therapeutic or diagnostic agent may have the following Formula XIV:

[0079]

[0080] With respect to therapeutic agents, as to specific embodiments, tumor-targeted NIR dyes can be coupled (e.g., by binding) to thermotherapy agents through the use of various linkers for use in hyperthermia (also known as thermal therapy or thermotherapy), a type of cancer treatment, in which body tissue can be exposed to high temperatures (heat) to damage and / or kill cancer cells or to make cancer cells more sensitive to the effects of radiation and / or certain anticancer drugs. Next, the thermotherapy agent can generate heat when exposed to energy such as radio waves, microwaves, infrared light, alternating magnetic fields, or ultrasound. As to specific embodiments, the thermotherapy agent can be a photothermal therapy agent, which can induce cancer cell death or sensitization by heat generated in tumor tissue exposed to NIR light.

[0081] With respect to specific embodiments in which the conjugate of the invention comprises a hyperthermia agent, NIR dyes can be used to direct the conjugate comprising the hyperthermia agent to a targeted tumor cell population; this can be in contrast to the non-specific delivery of conventional hyperthermia agents, which can cause significant adverse side effects when the recipient is exposed to the corresponding radiation.

[0082] As an illustrative example, a hyperthermia agent may comprise metal or semiconductor nanoparticles (eg, gold, silver, iron oxide, carbon, etc.) that can be heated in an electromagnetic field (ie, a radio frequency (RF) or NIR field).

[0083] As for specific embodiments, the hyperthermia agent may include metal nanoparticles configured as gold nanoparticles (AuNPs), such as gold nanospheres, gold nanorods, gold nanoshells, gold nanomatryoshka, gold nanoboxes, gold nanocages, gold nanostars, silica-coated gold nanorods, gold nanodimers, etc.

[0084] As for a specific embodiment, the conjugate comprising a tumor-targeting NIR dye and gold nanoparticles that can be used in the present invention can have the following formula XV:

[0085]

[0086] Notably, tumor-targeting NIR dyes with -SH functionality can be directly conjugated to gold nanoparticles via relatively strong S-AuNP bonds (as shown in Formula XV).

[0087] As described above, conjugates comprising gold nanoparticles can be heated in an electromagnetic field (ie, RF or NIR field) and act as hyperthermia agents.

[0088] Next, as to specific embodiments, the conjugate can be administered to a patient (e.g., by injection) using a method comprising the following conjugates of the invention: (i) a tumor-targeted NIR dye attached to gold nanoparticles or (ii) a zwitterionic tumor-targeted NIR dye attached to gold nanoparticles or (iii) a tumor-targeted NIR dye attached to zwitterionic coated gold nanoparticles or (iv) a zwitterionic tumor-targeted NIR dye attached to zwitterionic coated gold nanoparticles, whereby upon administration, the NIR dye can direct the conjugate to a target tumor cell population. Subsequently, an RF field can be generated, whereby exposure to the RF field can heat the gold nanoparticles and subsequently induce local hyperthermia in the tumor cells.

[0089] As for other specific embodiments, the conjugate can be administered to a patient (e.g., by injection) using a method comprising the following conjugates of the invention: (i) a tumor-targeted NIR dye attached to gold nanoparticles or (ii) a zwitterionic tumor-targeted NIR dye attached to gold nanoparticles or (iii) a tumor-targeted NIR dye attached to zwitterionic coated gold nanoparticles or (iv) a zwitterionic tumor-targeted NIR dye attached to zwitterionic coated gold nanoparticles, whereby upon administration, the NIR dye can direct the conjugate to a target tumor cell population. Subsequently, a NIR field can be generated (e.g., by laser irradiation or light emitting diode (LED) irradiation), whereby upon exposure to the NIR field, the gold nanoparticles can convert the absorbed light into heat and induce local hyperthermia in the tumor cells with minimal damage to intervening and surrounding normal tissues. Specifically, gold nanoparticles and specifically gold nanorods can act as strong NIR light absorbers, whereby such therapy can exploit the defects of naturally occurring NIR absorbing chromophores in most tissues, thereby allowing NIR light to be transmitted through the tissue with scattering-limited attenuation and minimal heating. Light in the NIR spectral region has been shown to penetrate tissue with high spatial precision at depths exceeding 1 cm without observable damage to intervening tissues, thus allowing the non-invasive delivery of heat to tissue volumes by using in vitro low-power irradiation to selectively induce hyperthermia in tumor cells targeted with gold nanoparticles / gold nanorods.

[0090] Notably, the conjugates of the present invention comprising: (i) a tumor-targeting NIR dye attached to gold nanoparticles (and specifically gold nanorods) or (ii) a zwitterionic tumor-targeting NIR dye attached to gold nanoparticles (and specifically gold nanorods) or (iii) a tumor-targeting NIR dye attached to zwitterionic-coated gold nanoparticles (and specifically gold nanorods) or (iv) a zwitterionic tumor-targeting NIR dye attached to zwitterionic-coated gold nanoparticles (and specifically gold nanorods) can produce synergistic heating of the conjugate when irradiated (e.g., by a laser or LED) in the wavelength range of about 700 nm to about 1,500 nm, which means that when irradiated with NIR light, the conjugate can generate more heat than the tumor-targeting NIR dye or the gold nanorods alone or a mixture of the two (non-covalently bound together); therefore, the conjugate can have greater photothermal transduction efficiency.

[0091] Figures 5A to 5D The experimental and theoretical heating temperature curves of gold nanorods and gold nanorod combinations irradiated with NIR lasers at a wavelength of 808 nm and different power densities (fluxes) are shown. Specifically, Figure 5A and 5C The experimental and theoretical temperature curves of the gold nanorods over time are shown, respectively, and Figure 5Band 5D The experimental and theoretical temperature distributions of the gold nanorod conjugate over time are shown, respectively.

[0092] About 5.1W / cm 2 The maximum temperature (T max ), Experimental T of gold nanorods max is 25.4°C, so the experimental T max is 48.3℃, the latter is 1.9 times larger than the former.

[0093] About 2.5W / cm 2 T max , Experimental T of gold nanorods max is 158°C, so the experimental T max is 22.1℃, the latter is 1.4 times larger than the former.

[0094] About 1.2W / cm 2 T max , Experimental T of gold nanorods max is 7.7°C, so the experimental T max It is 7.7℃.

[0095] About 0.3W / cm 2 T max , Experimental T of gold nanorods max is 1.7°C, so the experimental T of the gold nanorod conjugate max is 4.8℃, the latter is 4.7 times larger than the former.

[0096] For all power densities (5.1 W / cm 2 , 2.5W / cm 2 , 1.2W / cm 2 and 0.3W / cm 2 ), T of the gold nanorod conjugate generated by theoretical calculation max is the T of gold nanorods max 1.8 times.

[0097] Figure 6 The experimental heating temperature curves of gold nanorods and gold nanorod conjugates under the LED light source are shown, whereby, like the NIR laser, the T of the gold nanorod conjugate is lower than that of the gold nanorods. max and the rate of temperature increase is greater.

[0098] With respect to gold nanorods, such nanoparticles can exhibit strong extinction of light in the NIR spectrum caused by longitudinal and transverse surface plasmon resonances. These coherent resonant oscillations of conduction electrons can produce enhanced light extinction and heat generation through electron-electron and electron-phonon relaxation processes, whereby this effect can be used for photothermal therapy.

[0099] Illustratively, Figure 7 As shown in, the UV-vis extinction spectra of gold nanorods (GNR, blue line) and gold nanorod conjugates (GNRC, red line) having tumor targeting NIR dyes of formula II bound to gold nanorods have two peaks corresponding to longitudinal and transverse surface plasmon resonance modes. The shift of the extinction peak of the gold nanorod conjugate compared to the gold nanorods is caused by the increase in the local dielectric constant of the gold nanorod conjugate.

[0100] Figure 8 The UV-vis extinction spectrum of the tumor targeting NIR dye of Formula II is shown in FIG.

[0101] Fig.9A and 9B The hydrodynamic sizes of gold nanorods and gold nanorod conjugates with tumor targeting NIR dyes of formula II respectively bound to the gold nanorods are shown, whereby both show a bimodal distribution with maxima of about 3 nm and 80 nm for gold nanorods (GNRs) and maxima of about 10 nm and 100 nm for gold nanorod conjugates (GNRCs). The larger size distribution of the gold nanorod conjugates may be caused by the reduced aspect ratio resulting from the addition of tumor targeting NIR dyes to the surface of the gold nanorods.

[0102] Now with respect to cancer diagnostic agents, the conjugates of the present invention comprising (i) a tumor-targeted NIR dye attached to gold nanoparticles (and specifically gold nanorods) or (ii) a zwitterionic tumor-targeted NIR dye attached to gold nanoparticles (and specifically gold nanorods) or (iii) a tumor-targeted NIR dye attached to zwitterionic-coated gold nanoparticles (and specifically gold nanorods) or (iv) a zwitterionic tumor-targeted NIR dye attached to zwitterionic-coated gold nanoparticles (and specifically gold nanorods) may also be useful, as the NIR dye can emit NIR light to enable corresponding detection and imaging / visualization of the tumor location.

[0103] With respect to the conjugates of the present invention comprising (i) a tumor-targeted NIR dye attached to gold nanoparticles (and in particular gold nanorods) or (ii) a zwitterionic tumor-targeted NIR dye attached to gold nanoparticles (and in particular gold nanorods) or (iii) a tumor-targeted NIR dye attached to zwitterionic coated gold nanoparticles (and in particular gold nanorods) or (iv) a zwitterionic tumor-targeted NIR dye attached to zwitterionic coated gold nanoparticles (and in particular gold nanorods), depending on the distance between the dye molecule and the surface of the nanorod, there may be either radiative decay or non-radiative decay of the emission of the excited dipole in the molecule. For imaging purposes, an enhancement of the radiative decay rate may be desirable because it may result in an enhancement of fluorescence by the dye molecule. In addition to radiative decay, there is also a non-radiative decay channel for excited dye molecules, which can be used for hyperthermia applications, because the non-radiative decay of the dye molecule can allow the desired temperature increase to be achieved with a lower concentration of nanoconjugate and / or less irradiation.

[0104] It is contemplated herein that the properties of silver nanorods may be the same or similar to the gold nanorods used in the present invention.

[0105] Now, again with respect to cancer therapeutic agents, as another illustrative example, a hyperthermia agent may comprise magnetic nanoparticles, which may include iron oxide nanoparticle (IONP) based materials such as Fe3O4 or materials that can be heated in an alternating magnetic field such as FeCo, FePt, or Fe 1-x Si x ).

[0106] As for a specific embodiment, the combination comprising a tumor-targeted NIR dye and iron oxide nanoparticles that can be used in the present invention can have the following formula XVI:

[0107]

[0108] Notably, tumor-targeting NIR dyes with catechol functionality can be directly conjugated to iron oxide nanoparticles (such as the conjugate shown in Formula XVI) via relatively strong catechol-TONP bonds.

[0109] As described above, the conjugate containing iron oxide nanoparticles can be heated in an alternating magnetic field and act as a hyperthermia agent.

[0110] Next, as to specific embodiments, the conjugate can be administered to a patient (e.g., by injection) using a method comprising the following conjugates of the invention: (i) a tumor-targeted NIR dye attached to iron oxide nanoparticles or (ii) a zwitterionic tumor-targeted NIR dye attached to iron oxide nanoparticles or (iii) a tumor-targeted NIR dye attached to zwitterionic coated iron oxide nanoparticles or (iv) a zwitterionic tumor-targeted NIR dye attached to zwitterionic coated iron oxide nanoparticles, whereby upon administration, the NIR dye can direct the conjugate to a target tumor cell population. Subsequently, an alternating magnetic field can be generated, whereby exposure to the alternating magnetic field can heat the iron oxide nanoparticles and subsequently induce local hyperthermia in the tumor cells.

[0111] As for other specific embodiments, the conjugate can be administered to a patient (e.g., by injection) using a method comprising the conjugate of the invention comprising: (i) a tumor-targeted NIR dye attached to iron oxide nanoparticles or (ii) a zwitterionic tumor-targeted NIR dye attached to iron oxide nanoparticles or (iii) a tumor-targeted NIR dye attached to zwitterionic coated iron oxide nanoparticles or (iv) a zwitterionic tumor-targeted NIR dye attached to zwitterionic coated iron oxide nanoparticles, whereby upon administration, the NIR dye can direct the conjugate to a target tumor cell population. Subsequently, a NIR field can be generated (e.g., by laser irradiation or light emitting diode (LED) irradiation), whereby upon exposure to the NIR field, the iron oxide nanoparticles can convert the absorbed light into heat and induce local hyperthermia in the tumor cells with minimal damage to intervening and surrounding normal tissues.

[0112] In addition, the conjugates of the present invention comprising: (i) a tumor-targeted NIR dye attached to iron oxide nanoparticles or (ii) a zwitterionic tumor-targeted NIR dye attached to iron oxide nanoparticles or (iii) a tumor-targeted NIR dye attached to zwitterionic-coated iron oxide nanoparticles or (iv) a zwitterionic tumor-targeted NIR dye attached to zwitterionic-coated iron oxide nanoparticles can be used as diagnostic agents because (i) the NIR dye can emit NIR light and / or (ii) the iron oxide nanoparticles can act as magnetic resonance imaging (MRI) contrast agents to achieve corresponding detection and imaging / visualization of the tumor location.

[0113] As for specific embodiments, carbon nanoparticles (such as carbon nanospheres, carbon nanoshells, carbon nanotubes, other so-called "lossy dielectric" materials, etc.) can also act as strong NIR light absorbers and can therefore be used in the present invention. It is worth noting that carbon nanotubes can be targeted to specific cells by directly covalently functionalizing to targeting moieties (such as tumor-targeted NIR dyes) or by non-covalently encapsulating targeting moieties (such as tumor-targeted NIR dyes).

[0114] Now with respect to nanoparticles (such as gold nanoparticles or iron oxide nanoparticles), an obstacle to their medical application and translation to the clinic (e.g., as a hyperthermia agent) may be the tendency of such particles to accumulate in the liver and spleen due to opsonization and clearance by the mononuclear phagocyte system. Specifically, nanoparticles may associate very easily with proteins, lipids and other biomolecules, resulting in the formation of a dynamic "biomolecular corona", which makes it difficult to predict and / or control their behavior in vivo. Binding of serum proteins, followed by recognition and engulfment by phagocytes may be a particularly common fate for many nanoparticles, resulting in rapid clearance from the bloodstream. Zwitterionic coatings may address this problem and make nanoparticles more biocompatible, for example by reducing the rate and / or extent of nonspecific adsorption of proteins and lipids to the nanoparticle surface, thereby inhibiting the generation of biomolecular coronas. As described above, zwitterions may obtain their antifouling properties through strong electrostatic interactions of their positively and negatively charged groups with polar water molecules, thereby producing a dense and stable hydration shell that may provide a physical and energy barrier that may prevent undesired adsorption. Additionally, because the zwitterionic coating may be generally neutrally charged, ion pair formation with charged adsorbents may be less likely.

[0115] Fig. 10A Specific examples of conjugates of the invention are presented, comprising a tumor-targeting NIR dye attached to zwitterionic nanoparticles.

[0116] Fig. 10B Specific examples of conjugates of the invention are presented, comprising zwitterionic tumor-targeting NIR dyes attached to zwitterionic nanoparticles.

[0117] As for specific embodiments, illustrative examples of zwitterions that can be used in the present invention when attached to nanoparticles include sulfobetaines, carboxybetaines, phosphorylcholine, and Figure 2 The zwitterion shown in .

[0118] As for specific embodiments, with respect to attachment, a 5:1 ratio of zwitterion:tumor targeting NIR dye or zwitterion:zwitterion tumor targeting NIR dye may be used in the present invention.

[0119] As to a specific embodiment, the conjugate comprising a tumor targeting NIR dye attached to a zwitterionic nanoparticle (such as a gold nanoparticle, for example, a gold nanorod) may have the following formula XVII:

[0120]

[0121] As to a specific embodiment, the conjugate comprising a tumor targeting NIR dye attached to a zwitterionic nanoparticle (such as a gold nanoparticle, for example, a gold nanorod) may have the following formula XVIII:

[0122]

[0123] As to a specific embodiment, the conjugate comprising a tumor targeting NIR dye attached to a zwitterionic nanoparticle (such as a gold nanoparticle, for example, a gold nanorod) may have the following formula XIX:

[0124]

[0125] As to a specific embodiment, the conjugate comprising a tumor targeting NIR dye attached to a zwitterionic nanoparticle (such as a gold nanoparticle, for example a gold nanorod) may have the following formula XX:

[0126]

[0127] Now again with respect to therapeutic agents, as to specific embodiments, according to the present invention, tumor-targeted NIR dyes or zwitterionic tumor-targeted NIR dyes can be coupled (e.g., by binding) to chemotherapeutic agents through various linkers for use in chemotherapy. For example, some conventional chemotherapeutic agents that can be bound to tumor-targeted NIR dyes according to the present invention can include actinomycin, all-trans retinoic acid, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, gemcitabine), hydroxyurea, idarubicin, imatinib, irinotecan, dichloromethyl diethylamine, methotrexate, mitoxantrone, nitrogen mustard, oxaliplatin, paclitaxel, pemetrexed, teniposide, tioguanine, topotecan, valrubicin, vemurafenib, vinblastine, vincristine, vindesine, etc.

[0128] For use with the present invention, the chemotherapeutic agent can be fully pharmacologically active after conjugation, or alternatively, the chemotherapeutic agent can be a prodrug, which means an inactive or less active derivative of the chemotherapeutic agent that can be converted by metabolism (e.g., by chemical and / or enzymatic mechanisms) to its pharmacologically active form, whereby the pharmacologically active form can effectively kill tumor cells.

[0129] In contrast to administration in its pharmacologically active form, administration of a chemotherapeutic agent in its prodrug form can improve the absorption, distribution, metabolism and / or excretion (ADME) of the chemotherapeutic agent. For example, a prodrug form can have improved bioavailability relative to its pharmacologically active form, which may be beneficial for chemotherapeutic agents that are poorly absorbed through the gastrointestinal tract. Additionally, a prodrug form can facilitate how a chemotherapeutic agent selectively interacts with cells or processes that are not its intended target, which can reduce unexpected, undesirable and / or adverse side effects of a chemotherapeutic agent.

[0130] Because many thermotherapy agents (such as gold nanoparticles, iron oxide nanoparticles, etc.) can be inherently non-toxic to cells, the prodrug forms of these thermotherapy agents used to form the conjugates of the present invention may not be as necessary as the prodrug forms of the chemotherapeutic agents used in the conjugates of the present invention. For example, doxorubicin (DOX), a commonly used clinical anticancer agent, can be non-specific, and therefore, DOX can be toxic to normal cells (especially cells of the heart, liver, and kidney). Next, it is considered herein that the adverse side effects of DOX can be reduced, minimized, or eliminated by incorporating it into the conjugates of the present invention, wherein the conjugate targets DOX to tumor sites through the NIR dyes or zwitterionic NIR dyes of the present invention, whereby the specific linker of DOX bound to the dye can contribute to the reduction of the adverse side effects of the DOX, because the linker may affect the pharmacokinetics, pharmacodynamics, stability, toxicity, etc. of the chemotherapeutic agents incorporated into the conjugates. The preferred linker can ensure sufficient stability of the prodrug form of the chemotherapeutic agent in the circulation, effectively prevent premature release of the chemotherapeutic agent near the unintended target, and timely promote the release and / or pharmacological activation of the chemotherapeutic agent only near the targeted tumor cells.

[0131] The linkers that can be used to combine the tumor-targeted NIR dyes of the present invention and therapeutic agents (such as chemotherapeutic agents) can be classified as cleavable linkers and non-cleavable linkers. Non-cleavable linkers can change the activity of active metabolites, which may affect efficacy, toxicity and transportation, thereby showing significant limitations. Next, cleavable linkers may be more desirable for use in the conjugates of the present invention and can significantly contribute to their success in effectively killing target tumor cell populations.

[0132] In particular, cleavable linkers that are stable in circulation for extended periods of time and exploit the intrinsic properties of tumor cells to selectively release the chemotherapeutic agent from the conjugate may be particularly desirable.

[0133] As for specific embodiments, a cleavable linker with glutathione (GSH) sensitivity (redox-promoted) can be used for the conjugate of the present invention to connect tumor-targeted NIR dyes and chemotherapeutic agents. Certain cancer cells have a higher intracellular GSH concentration than in plasma, and thus the elevated GSH level can reduce disulfide bonds to maintain intracellular redox balance. The disulfide bond can be used as a cleavable linker, which can remain stable in the blood system and release active payloads uniquely in tumor cells with elevated GSH levels. Two illustrative examples of cleavable links with GSH sensitivity are shown in formula XXI (faster release / lower stability) and XXII (lower release / better stability).

[0134]

[0135] As for other specific embodiments, a cleavable linker with pH sensitivity (acid-induced) can be used for the conjugate of the present invention to connect tumor-targeted NIR dyes and chemotherapeutic agents. Acid-sensitive cleavable linkers can use the lower pH of endosomes (pH = 5-6) and lysosomes (pH = 4.8) relative to the cytoplasm (pH = 7.4) to trigger the hydrolysis of acid-labile groups in the linker. The three bond types shown in formula XXIII (hydrazone-based bond), XXIV (carbonate-based bond) and XXV (silyl ether-based bond) can be used with a cleavable linker with pH sensitivity:

[0136]

[0137] As an illustrative example only, Fig.11 Specific embodiments of conjugates of the invention are shown, comprising DOX conjugated to a tumor-targeted NIR dye via a pH-sensitive cleavable linker having an acid-labile hydrazone bond that may be cleaved once the conjugate enters the more acidic environment of the tumor site / cell, thereby releasing DOX at the targeted location.

[0138] As for other specific embodiments, a cleavable linker with protease sensitivity (enzyme catalysis) can be used in the present conjugates to connect tumor-targeted NIR dyes and chemotherapeutic agents. A protease-sensitive cleavable linker can employ a protease found in the lysosomes of tumor cells that recognizes and cleaves a specific peptide sequence in the linker. Dubowchik and Firestone et al. were the first to discover the valine-citrulline (VC) dipeptide (shown in Formula XXVI below) as an intracellular cleavage mechanism for cathepsin B, which can be overexpressed in tumor cells, thereby allowing the linked chemotherapeutic agent to be precisely released near the tumor site. Similarly, some tumor cells overexpress the MMP-2 protease, which can recognize and cleave a specific peptide sequence in the linker, such as a synthetic octapeptide (GPLGIAGQ) (shown in Formula XXVII below). In addition, due to the presence of protease inhibitors in the blood, a cleavable linker with protease sensitivity may be stable in the systemic circulation.

[0139]

[0140] Protease-sensitive cleavable peptide linkers have been developed as key components of antibody-drug conjugates (ADCs), whereby linkers can play a vital role in the overall success of ADC drugs due to their excellent plasma stability and controlled payload release mechanism; nine out of fourteen approved ADC drugs use protease-sensitive cleavable peptide linkers. For example, brentuximab vedotin A valine-citrulline linker was used.

[0141] Instead of using antibodies as tumor targeting agents, specific embodiments of the conjugates of the present invention employ (i) tumor targeting NIR dyes to selectively and efficiently deliver chemotherapeutic prodrugs to targeted tumor cells, and (ii) protease-sensitive cleavable peptide linkers to release and / or pharmacologically activate chemotherapeutic agents only in the vicinity of targeted tumor sites. Formulas XXVIII, XXIX, and XXX show exemplary tumor targeting NIR dyes conjugated to various chemotherapeutic agents via protease-sensitive cleavable peptide linkers.

[0142]

[0143] As for specific embodiments, the linker of the present invention may include polyethylene glycol (PEG) (e.g. Fig. 12A and 12B , which includes SN-38 as a chemotherapeutic agent, and as Fig. 12C and 12D As shown, it includes DOX as a chemotherapeutic agent), whereby PEG can increase solubility and provide the desired drug metabolism and pharmacokinetics (DMPK) of the delivered chemotherapeutic agent.

[0144] As for specific embodiments, the conjugate of the present invention may include two linkers, a first linker connecting a tumor-targeted NIR dye and a protease-sensitive cleavable peptide, and a second linker connecting a protease-sensitive cleavable peptide and a chemotherapeutic agent. The first linker must be stable (e.g., in circulation) but not necessarily cleavable, and once the conjugate is delivered to the tumor site, the second linker must be stable (e.g., in circulation) and cleavable, so that the chemotherapeutic agent can be released and / or pharmacologically activated near the targeted tumor site. Accordingly, a self-degradable spacer can be inserted between the protease-sensitive cleavable peptide and the chemotherapeutic agent to promote the effective release of the latter.

[0145] As an illustrative example, cathepsin B can cleave a linker comprising a VC dipeptide to release a binding moiety comprising a chemotherapeutic agent (e.g., SN-38) and a self-degradable p-aminobenzylcarbamate (PABC) spacer. Subsequently, PABC can undergo spontaneous 1,6-elimination in an acidic environment to release carbon dioxide, p-azaquinone methide, and SN-38, such as Fig.13 shown.

[0146] In addition to linkers comprising the VC dipeptide, other exemplary linkers with protease-sensitive peptide sequences that can be used in the conjugates of the invention include linkers shown in Formulas XXXI (valine-alanine), XXXII (valine-glycine), XXXIII (glycine-glycine), XXXIV (alanine-alanine-asparagine), and XXXV (phenylalanine-lysine).

[0147]

[0148] Formulas XXXVI to XXXXI show exemplary conjugates of the present invention, comprising a tumor-targeted NIR dye having a zwitterionic functional group, a chemotherapeutic prodrug, a cathepsin B-cleavable linker, and a self-cleavable spacer that promotes efficient release of the chemotherapeutic prodrug. Due to the zwitterionic functionality of the tumor-targeted NIR dye and the resulting high hydration, PEG can be omitted from these conjugates.

[0149]

[0150]

[0151]

[0152] As for specific embodiments, click chemistry can be used to link tumor-targeted NIR dyes and cleavable peptide sequences. As an illustrative example only, dibenzocyclooctyne (DBCO) reagents include a highly reactive DBCO group that can react with azide-labeled molecules or biomolecules via copper-free click chemistry. DBCO click chemistry can be run in aqueous buffer or organic solvent, depending on the properties of the substrate molecule. Fig.14A and 14B (which includes SN-38 as a chemotherapeutic agent) and the formula Fig. 14C and 14D (which includes DOX as a chemotherapeutic agent) Exemplary conjugates of the invention prepared using click chemistry are shown.

[0153] As for specific embodiments, according to the present invention, tumor-targeted NIR dyes or zwitterionic tumor-targeted NIR dyes can be coupled to (e.g., by binding to) nitroxide free radicals. Since cancer cells can produce free radicals at higher levels than normal cells, due to the active metabolism of the cancer cells being associated with the disorder of various cellular events, these cells can be under constant oxidative stress. Although excessive production of high levels of free radicals can lead to harmful cell damage and ultimately cell death, moderate levels of free radicals can promote cancer cell survival and promote tumor growth. Next, cancer cells may rely heavily on antioxidant enzymes and other adaptive antioxidant defenses to maintain the intracellular level of reactive oxidative species (ROS) within an acceptable threshold and therefore protect these cells from damage.

[0154] Accordingly, nitroxides may be suitable cancer therapeutics because these compounds, alone or when combined with another therapy (such as a chemotherapeutic agent), can enhance oxidative stress in cancer cells, which may lead to cell death.

[0155] As for specific embodiments, according to the present invention, tumor-targeted NIR dyes or zwitterionic tumor-targeted NIR dyes can be coupled to (e.g., by binding to) photodynamic therapy (PDT) agents. Typically, PDT includes administering a photosensitizer, such as by injection into the bloodstream or applied to the affected skin area. After the photosensitizer is activated by light, one or more processes that may cause tumor cell death can be initiated. However, the accumulation of conventional photosensitizers in tumor cells may be low, whereby an example of such a PDT agent may be chlorin-e6 PS. It is contemplated herein that the present invention may increase the accumulation of PDT agents in tumor cells.

[0156] From the foregoing it can be easily understood that the basic concept of the present invention can be embodied in a variety of ways.The present invention relates to many and different embodiments of tumor-targeted NIR dyes and related conjugates and methods for making and using such dyes and related conjugates.

[0157] Therefore, the specific embodiments or elements of the present invention disclosed by the specification or shown in the drawings or tables attached to the present application are not intended to be limiting, but rather to illustrate the many different embodiments generally covered by the present invention or the equivalents covered with respect to any specific element thereof. In addition, the specific description of a single embodiment or element of the present invention may not explicitly describe all possible embodiments or elements; many alternatives are implicitly disclosed by the specification and drawings.

[0158] It should be understood that each element of the device or each step of the method can be described in device terms or method terms. Such terms can be replaced in the case where it is desired to make the implicit broad scope to which the present invention is entitled clear. As just one example, it should be understood that all steps of the method can be disclosed as actions, means for taking the actions, or elements that cause the actions. Similarly, each element of the device can be disclosed as a physical element or an action promoted by the physical element. As just one example, the disclosure of "therapeutic agent" should be understood to cover the disclosure of the behavior of "providing therapy" - whether or not it is explicitly discussed, and conversely, if the behavior of "providing therapy" is effectively disclosed, such disclosure should be understood to cover the disclosure of "therapeutic agent" and even "means for providing therapy". Such alternative terms for each element or step should be understood to be explicitly included in the specification.

[0159] In addition, with respect to each term used, it should be understood that unless its usage in this application is inconsistent with such interpretation, the ordinary dictionary definition should be understood to be included in the description of each term contained in Random House Webster's Unabridged Dictionary, Second Edition, each definition is hereby incorporated by reference.

[0160] Whether or not explicitly stated, it is assumed that all numerical values ​​herein are modified by the term "about". For purposes of the present invention, a range may be expressed as "about" one specific value to "about" another specific value. When such a range is expressed, another embodiment includes one specific value to another specific value. The description of a numerical range by an endpoint includes all numerical values ​​incorporated within the range. Numerical ranges one to five include, for example, numerical values ​​1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc. It will be further understood that the endpoints of each range in the range are important relative to the other endpoint and independently of the other endpoint. When a value is expressed as an approximation by using the antecedent "about", it should be understood that the specific value forms another embodiment. The term "about" generally refers to a series of numerical values ​​that a person skilled in the art considers to be equivalent to the stated numerical value or to have the same function or result. Similarly, the antecedent "substantially" means the same form, manner or degree to a large extent but not entirely, and a particular element will have a series of configurations that a person of ordinary skill in the art considers to have the same function or result. When particular elements are expressed as approximations by using the antecedent "substantially," it should be understood that the particular elements form another embodiment.

[0161] In addition, for the purposes of the present invention, the term "a" or "an" entity refers to one or more of the entity, unless otherwise limited. Therefore, the terms "a" or "an", "one or more" and "at least one" can be used interchangeably herein.

[0162] Applicants should therefore be understood to claim at least: i) each of the NIR dyes and related combinations disclosed and described herein; ii) the related methods disclosed and described; iii) similar, equivalent and even implicit variations of each of these devices and methods; iv) those alternative embodiments that implement each of the functions shown, disclosed or described; v) those alternative designs and methods that implement each of the functions shown that are implicit in implementing the functions disclosed and described; vi) each feature, component and step shown as a separate and independent invention; vii) applications enhanced by the various systems or components disclosed; viii) the resulting products produced by such systems or components; ix) methods and apparatus substantially as described above and with reference to any accompanying examples; x) various combinations and arrangements of each of the aforementioned elements disclosed.

[0163] The background section of this patent application, if any, provides a statement of the field of endeavor to which the invention pertains. This section may also incorporate or contain explanations of certain U.S. patents, patent applications, publications, or subject matter of the claimed invention that may be used to describe information, problems, or concerns about the state of the art involved in the invention. Any U.S. patent, patent application, publication, statement, or other information cited or incorporated herein is not intended to be construed, understood, or considered to be prior art to the present invention.

[0164] The claims set forth in this specification, if any, are hereby incorporated by reference as part of the description of the invention, and applicants expressly reserve the right to use all or part of such incorporated content of such claims as additional description to support any or all claims, or any elements or components thereof, and applicants further expressly reserve the right to transfer any part or all of such incorporated content of such claims from the specification to the claims, or vice versa as necessary, to define the matter sought to be protected by this application or any subsequent or continuation, divisional or continuation-in-part application thereof, or to obtain any benefit of, obtain fee reductions relating thereto, or comply with the patent laws, rules or regulations of any country or treaty, and such content incorporated by reference will continue in effect throughout the pendency of this application, including any subsequent continuation, divisional or continuation-in-part application thereof, or any reissue or extension thereof.

[0165] Additionally, the claims set forth in this specification, if any, are further intended to describe the scope and limits of a limited number of preferred embodiments of the invention and should not be construed as the broadest embodiment of the invention or a complete list of embodiments of the invention that may be claimed. Applicants do not waive any right to generate additional claims based on the description set forth above as part of any continuation, divisional, or part continuation application or similar application.

Claims

1. A composition comprising: A combination, comprising: Tumor-targeted near-infrared dyes; and Therapeutic agents; wherein the near infrared dye targets the therapeutic agent to tumor cells.

2. The composition of claim 1, wherein the near infrared dye comprises a zwitterion.

3. The composition of claim 2, wherein the zwitterion comprises both positively charged groups and negatively charged groups.

4. The composition of claim 3, wherein the zwitterion comprises an overall neutral charge. The composition according to claim 3 , wherein the zwitterion has a high hydration capacity.

6. The composition according to claim 5, wherein the zwitterion can be highly hydrated through electrostatic interactions of the positively and negatively charged groups with polar water molecules.

7. The composition of claim 6, wherein the interaction results in the formation of a hydration shell.

8. The composition of claim 7, wherein the hydration shell provides a physical and energetic barrier to prevent undesired adsorption.

9. The composition of claim 7, wherein the hydration shell provides a physical and energetic barrier to prevent undesired protein adsorption.

10. The composition of claim 8, wherein the charge of the positively charged group is associated with a single atom.

11. The composition of claim 10, wherein the charge of the positively charged group is not delocalized over multiple atoms.

12. The composition of claim 10, wherein the charged atoms of the positively charged group are not part of a resonant structure.

13. The composition of claim 8, wherein the charge of the negatively charged group is associated with a single atom.

14. The composition of claim 13, wherein the charge of the negatively charged group is not delocalized over multiple atoms.

15. The composition of claim 13, wherein the charged atoms of the negatively charged group are not part of a resonant structure.

16. The composition of claim 8, wherein the zwitterion comprises a sulfobetaine.

17. The composition of claim 8, wherein the zwitterion comprises carboxybetaine.

18. The composition of claim 8, wherein the zwitterion comprises phosphorylcholine.

19. The composition of claim 8, wherein the zwitterion comprises the molecule shown in Figure 2.

20. The composition of claim 8, wherein the near infrared dye comprises a cyanine dye.

21. The composition of claim 20, wherein the cyanine dye comprises a heptamethine cyanine dye.

22. The composition of claim 21, wherein the heptamethine cyanine dye has formula I.

23. The composition of claim 22, wherein the zwitterion is bound to the near infrared dye via its central ring.

24. The composition of claim 22, wherein the zwitterion is bound to the near infrared dye via the nitrogen of its indole moiety.

25. The composition of claim 22, wherein the zwitterion is geminally bound to the near infrared dye.

26. The composition of claim 22, wherein the zwitterion is bound to the near infrared dye through an aromatic ring.

27. The composition of claim 8, wherein the near infrared dye comprising the zwitterion has Formula VI.

28. The composition of claim 8, wherein the near infrared dye comprising the zwitterion has Formula VII.

29. The composition of claim 8, wherein the near infrared dye comprising the zwitterion is of Formula VIII.

30. The composition of claim 8, wherein the near infrared dye comprising the zwitterion is of Formula IX.

31. The composition of claim 8, wherein the near infrared dye comprising the zwitterion has formula X.

32. The composition of claim 1 or claim 8, wherein the therapeutic agent comprises a hyperthermia agent.

33. The composition of claim 8, wherein the therapeutic agent comprises a photothermal agent.

34. The composition of claim 32, wherein the hyperthermia agent comprises metal or semiconductor nanoparticles capable of heating in an electromagnetic field.

35. The composition of claim 34, wherein the nanoparticles are capable of being heated in a radio frequency field.

36. The composition of claim 34, wherein the nanoparticles are capable of heating in a near infrared field.

37. The composition of claim 34, wherein the nanoparticles comprise gold nanoparticles.

38. The composition of claim 37, wherein the conjugate has one of Formula XV, Formula XVII, Formula XVIII, Formula XIX or Formula XX.

39. The composition of claim 37, wherein the gold nanoparticles are zwitterionic.

40. The composition of claim 37, wherein the gold nanoparticles comprise gold nanorods.

41. The composition of claim 40, wherein the gold nanorods are capable of heating in the near infrared field.

42. The composition of claim 40, wherein the gold nanorods are zwitterionic.

43. The composition of claim 42, wherein the zwitterionic gold nanorods are capable of heating in the near infrared field.

44. The composition of claim 32, wherein the hyperthermia agent comprises magnetic nanoparticles capable of heating in an alternating magnetic field.

45. The composition of claim 44, wherein the nanoparticles comprise iron oxide nanoparticles.

46. ​​The composition of claim 45, wherein the conjugate is of Formula XVI.

47. The composition of claim 45, wherein the iron oxide nanoparticles are zwitterionic.

48. The composition of claim 1 or claim 8, wherein the therapeutic agent comprises a chemotherapeutic agent.

49. The composition of claim 48, wherein the conjugate comprises a pharmacologically active form of the chemotherapeutic agent.

50. The composition of claim 48, wherein the conjugate comprises a prodrug form of the chemotherapeutic agent.

51. The composition of claim 48, wherein the near infrared dye and the chemotherapeutic agent are covalently bound via a linker.

52. The composition of claim 51, wherein the linker is a cleavable linker.

53. The composition of claim 52, wherein the cleavable linker comprises glutathione sensitivity.

54. The composition of claim 53, wherein the cleavable linker comprises one or more disulfide bonds.

55. The composition of claim 52, wherein the cleavable linker comprises pH sensitivity.

56. The composition of claim 55, wherein the cleavable linker comprises one or more acid-labile groups.

57. The composition of claim 55, wherein the cleavable linker comprises a hydrazone-based bond.

58. The composition of claim 55, wherein the cleavable linker comprises a carbonate-based bond.

59. The composition of claim 55, wherein the cleavable linker comprises a silyl ether-based bond.

60. The composition of claim 52, wherein the cleavable linker comprises protease sensitivity.

61. The composition of claim 60, wherein the cleavable linker comprises a peptide sequence capable of being cleaved by the protease.

62. The composition of claim 61, wherein the cleavable linker comprises a valine-citrulline dipeptide.

63. The composition of claim 62, wherein the valine-citrulline dipeptide is capable of being cleaved by cathepsin B.

64. The composition of claim 61, wherein the cleavable linker comprises an octapeptide (GPLGIAGQ).

65. The composition of claim 64, wherein the octapeptide is capable of being cleaved by MMP-2.

66. The composition of claim 51, wherein the linker comprises polyethylene glycol.

67. A method of killing tumor cells, the method comprising: administering to a subject a conjugate comprising: Tumor-targeted near-infrared dyes; and Therapeutic agents; wherein the near infrared dye targets the therapeutic agent to the tumor cells.

68. The method of claim 67, wherein the tumor targeting component of the conjugate consists of the near infrared dye.

69. The method of claim 67, wherein the near infrared dye comprises a zwitterion.

70. The method of claim 69, wherein the therapeutic agent comprises a hyperthermia agent.

71. The method of claim 70, further comprising inducing hyperthermia in the tumor cells by generating an electromagnetic field.

72. The method of claim 70, further comprising inducing hyperthermia in the tumor cells by generating an alternating magnetic field.

73. The method of claim 67 or claim 69, wherein the therapeutic agent comprises a chemotherapeutic agent.

74. The method of claim 73, wherein the conjugate comprises a prodrug form of the chemotherapeutic agent.

75. The method of claim 73, wherein the near infrared dye and the chemotherapeutic agent are covalently bound via a linker.

76. The method of claim 75, wherein the linker is a cleavable linker.

77. A method of preparing a composition, the method comprising: Combining tumor-targeting near-infrared dyes with therapeutic agents; wherein the near infrared dye targets the therapeutic agent to tumor cells.

78. The method of claim 77, wherein the near infrared dye comprises a zwitterion.

79. The method of claim 77 or claim 78, wherein the therapeutic agent comprises a chemotherapeutic agent.

80. The method of claim 79, wherein the near infrared dye and the chemotherapeutic agent are covalently bound via a cleavable linker.

81. A composition comprising: A combination, comprising: Tumor-targeted near-infrared dyes; and Diagnostic agents; wherein the near infrared dye targets the diagnostic agent to tumor cells.

82. A method for imaging tumor cells, the method comprising: administering to a subject a conjugate comprising: Tumor-targeted near-infrared dyes; and Diagnostic agents; wherein the near infrared dye targets the diagnostic agent to the tumor cells.

Images