Near-infrared dyes and conjugates for targeting tumors

Abstract

Design, synthesis, and functionalization of conjugates comprising a tumor-targeted near-infrared (NIR) dye and a therapeutic agent and / or a diagnostic agent, wherein the NIR dye is capable of functioning to target the therapeutic agent and / or the diagnostic agent to tumor cells. A broad object of certain embodiments of the present invention can be to provide a conjugate comprising a tumor-targeted NIR dye and a therapeutic agent, wherein the dye functions to target the therapeutic agent to tumor cells and / or deliver the therapeutic agent to tumor cells. Another broad object of certain embodiments of the present invention can be to provide a conjugate comprising a tumor-targeted NIR dye and a diagnostic agent, wherein the dye functions to target the diagnostic agent to tumor cells and / or deliver the diagnostic agent to tumor cells.

Description

Technical Field

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

Background Art

[0002] I. Background There is still a need for targeted cancer therapeutics that are most effective in treating or killing tumor cells and have minimal toxicity to non-tumor or healthy cells. Additionally, there is a need for targeted cancer diagnostic agents.

Summary of the Invention

Means for Solving the Problems

[0003] II. Disclosure of the Invention Generally, the present invention details the use of near-infrared (NIR) fluorescent dyes that target tumors in combination with various therapeutic and / or diagnostic agents.

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

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

[0006] A broad object of certain 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 objective of certain embodiments of the present invention may be to provide a method for diagnosing the presence of tumor cells by targeting a conjugate comprising a tumor-targeted NIR dye and a diagnostic agent to the tumor cells, if present.

[0008] Of course, further objectives of the present invention are disclosed throughout the remainder of the specification, drawings, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] III. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1

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Figure 12D

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Figure 14B

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Figure 14C

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Figure 14D

Embodiments for Carrying Out the Invention

[0034] IV. Embodiments for Carrying Out the Invention The present invention can provide an effective and versatile delivery method by combining a tumor-targeting near-infrared (NIR) fluorescent dye for creating conjugates for (i) targeting tumor cells and / or (ii) killing tumor cells and / or (iii) imaging tumor cells and / or (iv) seranostics with various cancer therapeutics and / or cancer diagnostics; the NIR dye functions to direct the conjugate towards the target tumor cell population; this can be in contrast to the non-specific delivery of conventional anti-cancer agents which can cause significantly harmful side effects. In addition to targeting tumor cells, this NIR dye can absorb NIR light and emit with significant brightness in the same region, and such properties can be highly desirable considering the non-invasive nature of NIR light and its tissue penetration which can be significantly different compared to ultraviolet or visible light.

[0035] For certain embodiments, the tumor-targeting NIR dye can be a cyanine dye, and chemically, the cyanine dye contains two nitrogen atoms joined by a polymethine chain.

[0036] For certain embodiments, the tumor-targeting NIR dye can be a heptamethine cyanine dye.

[0037] For certain embodiments, the tumor-targeting NIR dye can have the following formula I:

Chemical formula

[0038] For certain embodiments, one or more of R 1 to R 8 can be functionalized with zwitterion(s), small molecule chemotherapeutic agent(s), photodynamic therapy (PDT) agent(s), or hyperthermia agent(s) such as gold nanoparticles, iron oxide nanoparticles, etc. via various linkers.

[0039] For certain embodiments, with respect to Y, substituted C, O, S, or N can be functionalized with zwitterion(s), small molecule chemotherapeutic agent(s), photodynamic therapy (PDT) agent(s), or hyperthermia agent(s) such as gold nanoparticles, iron oxide nanoparticles, etc. via various linkers.

[0040] For certain embodiments, the linker is hetero-bifunctional and thus reacts with both the tumor-targeting NIR dye and the therapeutic or diagnostic agent to form a conjugate. Exemplary examples include that the linker can be polyethylene glycol (PEG), polymer, peptide, DNA, silica nanoparticles, shell structures (such as iron oxide or silica), amide bond, ester bond, amine, alcohol, phenol, thio, phenyl or their derivatives, etc.

[0041] For certain embodiments, the linker can be cleavable.

[0042] The tumor-targeting NIR dyes of the present invention can be symmetric or asymmetric, and exemplary dyes are shown in Formulas II, III, IV, and V.

Chemical formula

Chemical formula

[0043] To illustrate the tumor targeting ability of the tumor-targeting NIR dyes of certain embodiments, specifically the dyes represented by Formula V, cells were dosed with 20 μM of the dye (absorbance at about 780 nm, emission at about 808 nm), and the change over time in the average value of the NIR intensity per well correlated with dye uptake is shown in FIG. 1 for a lung cancer cell line (A549) and four normal cell lines (colon cells, kidney cells, liver cells, and human umbilical vein endothelial cells (HUVEC)). Significant dye uptake was observed only in the cancer cell lines.

[0044] For certain embodiments, the tumor-targeting NIR dyes can be modified to have an amphoteric ion function, and such modifications can include the attachment of one or more amphoteric ions to the dye. Each of said amphoteric ions functions as a polyelectrolyte or zwitterion having both positively and negatively charged groups, but can have an overall neutral charge. Furthermore, the amphoteric ion has a high hydration capacity, and accordingly, is highly hydrated by the electrostatic interaction between its positively and negatively charged groups and polar water molecules. The resulting strongly bound water layer forms a dense and stable hydration shell or solvation shell, and can provide physical and energetic barriers that can prevent unwanted adsorption such as protein adsorption. Thus, the amphoteric ion can have effective anti-fouling properties.

[0045] For certain embodiments, exemplary examples of zwitterions that may be useful in the present invention when attached to a tumor-targeted NIR dye include sulfobetaine, carboxybetaine, phosphorylcholine, and those shown in Figure 2. In accordance with the above, the zwitterions shown in Figure 2 can be overall neutrally charged and, as shown in Figure 3, have significant hydration ability through strong ion-dipole interactions between their positively and negatively charged groups and polar water molecules.

[0046] To confer a zwitterionic functionality on a tumor-targeted NIR dye, the zwitterion can be covalently or electrostatically attached or bound to the dye via a central ring (such as a central cyclopentyl or cyclohexyl ring), via the nitrogen of the indole moiety, via a geminal position, or via an aromatic ring. For certain embodiments, one or more of R 1 from R 8 or Y can be used as a linking site for attaching or binding a zwitterion to a tumor-targeted NIR dye via various linkers to form the conjugate.

[0047] Significantly, some molecules can be polyelectrolytes with groups bearing both positive and negative charges and have a neutral charge overall, but because one or more charged atoms are part of the resonance structure, these molecules may not have the high hydration ability required for the present invention. More specifically, when a charged atom is part of the resonance structure, in contrast to the charge being associated with a single atom, the charge is delocalized or extended over multiple atoms. Next, such a "diluted" charge loses its hydration ability through electrostatic interaction with polar water molecules and correspondingly may not be useful in the present invention. As one illustrative example, the molecule shown in Figure 4A contains a negatively charged sulfonate group and a positively charged indole group; the positive charge on nitrogen is part of a resonance structure containing a total of 11 atoms. Correspondingly, this positive charge is delocalized and thus does not have a high hydration ability through electrostatic interaction with polar water molecules. Next, such a molecule cannot be useful in the present invention.

[0048] As another illustrative example, the molecule shown in Figure 4B also contains a negatively charged sulfonate group and a positively charged indole group. Similar to the above, the positive charge on nitrogen is part of a resonance structure containing a total of 11 atoms. Correspondingly, this positive charge is delocalized and thus does not have a high hydration ability through electrostatic interaction with polar water molecules. Next, such a molecule cannot be useful in the present invention.

[0049] Similarly, the charged groups in the two NIR dyes shown in Formulas IV and V above may not have a high hydration ability through electrostatic interaction with polar water molecules as a result of their resonance structures.

[0050] In view of the above, for certain embodiments, zwitterions useful in the present invention can include a group bearing a charge that is associated with a single atom, as opposed to the charge being delocalized over multiple atoms, and at the same time, this property can provide a hydration shell that confers a desirable high hydration ability of the zwitterion through electrostatic interaction with polar water molecules and provides physical and energetic barriers that prevent undesirable adsorption.

[0051] For certain embodiments, the zwitterionic tumor-targeting NIR dye can have the following formula VI:

Chemical formula

[0052] For certain embodiments, the zwitterionic tumor-targeting NIR dye can have the following formula VII:

Chemical formula

[0053] For certain embodiments, the zwitterionic tumor-targeting NIR dye can have the following formula VIII:

Chemical formula

[0054] For certain embodiments, the zwitterionic tumor-targeting NIR dye can have the following formula IX:

Chemical formula

[0055] For certain embodiments, the zwitterionic tumor-targeting NIR dye can have the following formula X:

Chemical formula

[0056] As described above, the present invention can produce conjugates for the treatment of different cancers by combining tumor-targeted NIR fluorescent dyes with various agents, and these conjugates can be directly delivered to the target tumor cell population. To form such conjugates, a therapeutic or diagnostic agent can be covalently or electrostatically attached or bound to the tumor-targeted NIR dye via, for example, a central ring (such as a central cyclopentyl or cyclohexyl ring), via the nitrogen of the indole moiety, via the geminal position, or via an aromatic ring. For certain embodiments, as shown in Formula I, one or more of R 1 to R 8 or Y can be used as a linking site for attaching or binding a therapeutic or diagnostic agent to the tumor-targeted NIR dye via various linkers to form the conjugate.

[0057] For certain embodiments, a conjugate comprising a tumor-targeted NIR dye and a therapeutic or diagnostic agent can have the following Formula XI:

Chemical formula

[0058] For certain embodiments, a conjugate comprising a tumor-targeted NIR dye and a therapeutic or diagnostic agent can have the following Formula XII:

Chemical formula

[0059] For certain embodiments, a conjugate comprising an amphoteric ionic tumor-targeted NIR dye and a therapeutic or diagnostic agent can have the following Formula XIII:

Chemical formula

[0060] For certain embodiments, a conjugate comprising a zwitterionic tumor-targeting NIR dye and a therapeutic or diagnostic agent can have the following formula XIV: [Chemical Formula]

[0061] With respect to the therapeutic agent, for certain embodiments, the tumor-targeting NIR dye can be linked (e.g., via conjugation) to a hyperthermia agent through various linkers for use in hyperthermia (also referred to as thermal therapy or thermotherapy), a type of cancer treatment that can expose body tissue to high temperatures (heat) to damage and / or kill cancer cells or make them more sensitive to the effects of radiation and / or certain anticancer drugs. Next, the hyperthermia agent can generate heat when exposed to energy such as radio waves, microwaves, infrared light, alternating magnetic fields, or ultrasound. For certain embodiments, the hyperthermia agent can be a photothermal therapy agent, which can induce cancer cell death or sensitization by the heat generated in tumor tissue exposed to NIR light.

[0062] For certain embodiments where the conjugate comprises a hyperthermia agent, the NIR dye can function to direct the conjugate comprising the hyperthermia agent towards the target tumor cell population; this can be in contrast to the non-specific delivery of conventional hyperthermia agents that can cause significantly harmful side effects when the recipient is exposed to the corresponding irradiation.

[0063] As an illustrative example, the hyperthermia agent can comprise an electromagnetic field, i.e., a metal or semiconductor nanoparticle (e.g., gold, silver, iron oxide, carbon, etc.) that can be heated in the radio frequency (RF) or NIR region.

[0064] For certain embodiments, the thermotherapeutic agent can include metal nanoparticles configured as gold nanoparticles (AuNP) such as, for example, gold nanospheres, gold nanorods, gold nanoshells, gold nanomatryoshkas, gold nanoboxes, gold nanocages, gold nanostars, silica-coated gold nanorods, gold nanodimers, and the like.

[0065] For certain embodiments, a conjugate comprising a tumor-targeting NIR dye and gold nanoparticles that can be useful in the present invention can have the following formula XV:

Chemical formula

[0066] Notably, a tumor-targeting NIR dye having a -SH functionality can be directly conjugated to gold nanoparticles via a relatively strong S-AuNP bond (such as the conjugate shown in formula XV).

[0067] As described above, a conjugate comprising gold nanoparticles can be heated in an electromagnetic field (i.e., the RF or NIR region) and function as a thermotherapeutic agent.

[0068] Next, for certain embodiments, a method of using such a conjugate comprising (i) a tumor-targeting NIR dye attached to gold nanoparticles, or (ii) an amphoteric tumor-targeting NIR dye attached to gold nanoparticles, or (iii) a tumor-targeting NIR dye attached to gold nanoparticles coated with an ampholyte, or (iv) an amphoteric tumor-targeting NIR dye attached to gold nanoparticles coated with an ampholyte can be such that the conjugate can be administered to a patient (such as via injection), and after administration, the NIR dye can direct the conjugate towards a target tumor cell population. Thereafter, an RF region can be generated, and exposure to the RF region can result in heating of the gold nanoparticles and induction of resultant local thermotherapy in the tumor cells.

[0069] For other specific embodiments, methods of using a conjugate comprising (i) a tumor-targeted NIR dye attached to gold nanoparticles, or (ii) an amphoteric tumor-targeted NIR dye attached to gold nanoparticles, or (iii) a tumor-targeted NIR dye attached to amphoteric-coated gold nanoparticles, or (iv) an amphoteric tumor-targeted NIR dye attached to amphoteric-coated gold nanoparticles, can administer the conjugate to a patient (e.g., via injection), and after administration, the NIR dye can direct the conjugate towards the target tumor cell population. Thereafter, the NIR region can be generated (e.g., via laser irradiation or light-emitting diode (LED) irradiation), and by exposing to the NIR region, the gold nanoparticles can convert the absorbed light into heat and induce local hyperthermia treatment in tumor cells with minimal damage to intervening and surrounding normal tissues. In particular, gold nanoparticles, and specifically gold nanorods, can act as strong NIR light absorbers, and such therapies can take advantage of the naturally occurring lack of NIR-absorbing chromophores in most tissues, thereby allowing transmission of NIR light through the tissue and minimal heating with scattering-limited attenuation. Light in the NIR spectral region has been shown to penetrate tissue with high spatial accuracy at depths exceeding 1 cm without observable damage to the intervening tissue, and as a result, non-invasive delivery of heat to tissue volumes is enabled by using low-power irradiation outside the body, selectively inducing hyperthermia treatment in tumor cells targeted with gold nanoparticles / gold nanorods.

[0070] Notably, the conjugate comprising (i) a tumor-targeting NIR dye attached to gold nanoparticles (and in particular, gold nanorods), or (ii) an amphoteric tumor-targeting NIR dye attached to gold nanoparticles (and in particular, gold nanorods), or (iii) a tumor-targeting NIR dye attached to zwitterion-coated gold nanoparticles (and in particular, gold nanorods), or (iv) an amphoteric tumor-targeting NIR dye attached to zwitterion-coated gold nanoparticles (and in particular, gold nanorods) can produce synergistic heating of the conjugate upon irradiation (e.g., via a laser or an LED) within a wavelength range of about 700 nanometers to about 1500 nanometers, which means that when irradiated with NIR light, the conjugate can produce more heat than either the tumor-targeting NIR dye or the gold nanorods alone, or a mixture of the two (not covalently bound together). Thus, the conjugate can have a greater light-to-heat conversion efficiency.

[0071] Figures 5A through 5D show the experimental and theoretical heating temperature profiles of gold nanorods and gold nanorod conjugates irradiated with an NIR laser having a wavelength of 808 nm and different output densities (fluence). Specifically, Figures 5A and 5C show the experimental and theoretical temperature profiles over time for the gold nanorods, respectively, and Figures 5B and 5D show the experimental and theoretical temperature profiles over time for the gold nanorod conjugates, respectively.

[0072] 5.1 W / cm 2 Regarding the maximum temperature (T max ), the experimental T max of the gold nanorods is 25.4 °C, and the experimental T max of the gold nanorod conjugate is 48.3 °C, and the latter is 1.9 times higher relative to the former.

[0073] 2.5 W / cm 2 Regarding T max at, the experimental T max of the gold nanorods is 15.8 °C, and the experimental T of the gold nanorod conjugatemax is 22.1 °C, and the latter is 1.4 times higher relative to the former.

[0074] 1.2 W / cm 2 of T max Regarding the experimental T of the gold nanorods max is 7.7 °C, and the experimental T of the gold nanorod conjugate max is 7.7 °C.

[0075] 0.3 W / cm 2 of T max Regarding the experimental T of the gold nanorods max is 1.7 °C, and the experimental T of the gold nanorod conjugate max is 4.8 °C, and the latter is 4.7 times higher relative to the former.

[0076] For all output densities (5.1 W / cm 2 , 2.5 W / cm 2 , 1.2 W / cm 2 , and 0.3 W / cm 2 ), the theoretical calculation yields a T of the gold nanorod conjugate that is 1.8 times higher than that of the gold nanorod. max max

[0077] Figure 6 shows the experimental heating temperature profiles of the gold nanorods and gold nanorod conjugates using an LED light source. Similar to the NIR laser, the T max and the rate of temperature increase are high for the gold nanorod conjugate when compared to the gold nanorod.

[0078] Regarding the 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 vibrations of the conduction electrons can lead to enhanced light extinction and heat generation through electron–electron and electron–phonon relaxation processes, and such effects should be useful for photothermal therapy.

[0079] Exemplarily, as shown in FIG. 7, the UV-vis extinction spectra of gold nanorods (GNR, blue line) and gold nanorod conjugates (GNRC, red line) having a tumor-targeting NIR dye of Formula II conjugated to the gold nanorods have two peaks corresponding to longitudinal and transverse surface plasmon resonance modes. The shift in the extinction peak of the gold nanorod conjugate compared to the gold nanorod is caused by an increase in the local dielectric permittivity of the gold nanorod conjugate.

[0080] The UV-vis extinction spectrum of the tumor-targeting NIR dye of Formula II is shown in FIG. 8.

[0081] FIGS. 9A and 9B show the hydrodynamic sizes of gold nanorods and gold nanorod conjugates having a tumor-targeting NIR dye of Formula II conjugated to the gold nanorods, respectively, both showing a bimodal distribution with maxima at about 3 nm and 80 nm for the gold nanorods (GNR) and 10 nm and 100 nm for the gold nanorod conjugates (GNRC). The larger size distribution of the gold nanorod conjugate should be due to the decreased aspect ratio caused by the addition of the tumor-targeting NIR dye to the surface of the gold nanorod.

[0082] Now, with regard to cancer diagnostic agents, such conjugates comprising (i) a tumor-targeting NIR dye attached to gold nanoparticles (and in particular, gold nanorods), or (ii) an amphoteric tumor-targeting NIR dye attached to gold nanoparticles (and in particular, gold nanorods), or (iii) a tumor-targeting NIR dye attached to gold nanoparticles (and in particular, gold nanorods) coated with an ampholyte, or (iv) an amphoteric tumor-targeting NIR dye attached to gold nanoparticles (and in particular, gold nanorods) coated with an ampholyte may be useful as such because the NIR dye can emit NIR light for the corresponding detection and imaging / visualization of tumor location.

[0083] (i) tumor-targeted NIR dyes attached to gold nanoparticles (and in particular, gold nanorods), or (ii) zwitterionic tumor-targeted NIR dyes attached to gold nanoparticles (and in particular, gold nanorods), or (iii) tumor-targeted NIR dyes attached to zwitterion-coated gold nanoparticles (and in particular, gold nanorods), or (iv) zwitterionic tumor-targeted NIR dyes attached to zwitterion-coated gold nanoparticles (and in particular, gold nanorods), for the conjugate, depending on the distance between the dye molecule and the nanorod surface, there can be either radiative or non-radiative decay of the emission from the excited dipole within the molecule. For imaging purposes, an enhanced radiative decay rate can be desirable in that it can result in enhanced fluorescence by the dye molecule. In addition to radiative decay, there is also a non-radiative decay pathway for the excited dye molecule, which can be useful for hyperthermia treatment applications, but the non-radiative decay of the dye molecule is because it can achieve a desirable temperature increase with a lower nanoconjugate concentration and / or less irradiation.

[0084] It is contemplated herein that silver nanorods can function equivalently or similarly to gold nanorods for use in the present invention.

[0085] Now turning again to cancer therapeutics, as another exemplary example, the hyperthermia therapeutic agent can include magnetic nanoparticles, and the hyperthermia therapeutic agent can be a material based on iron oxide nanoparticles (IONP) such as Fe 3 O 4 or materials including FeCo, FePt, or Fe 1-x Si x and the like.

[0086] For certain embodiments, the conjugate comprising a tumor-targeted NIR dye and iron oxide nanoparticles that can be useful in the present invention can have Formula XVI as follows.

Chemical Formula

[0087] Notably, tumor-targeting NIR dyes having a catechol function can be directly conjugated to iron oxide nanoparticles via a relatively strong catechol-IONP bond (such as the conjugate shown in Formula XVI).

[0088] As described above, the conjugate containing iron oxide nanoparticles can be heated in an alternating magnetic field and function as a hyperthermia agent.

[0089] Next, for a particular embodiment, a method of using such a conjugate comprising (i) a tumor-targeting NIR dye attached to iron oxide nanoparticles, or (ii) an amphoteric tumor-targeting NIR dye attached to iron oxide nanoparticles, or (iii) a tumor-targeting NIR dye attached to iron oxide nanoparticles coated with an ampholyte, or (iv) an amphoteric tumor-targeting NIR dye attached to iron oxide nanoparticles coated with an ampholyte, the conjugate can be administered to a patient (such as via injection), and after administration, the NIR dye can direct the conjugate towards the target tumor cell population. Thereafter, an alternating magnetic field can be generated, and exposure to the alternating magnetic field can result in heating of the iron oxide nanoparticles and induction of local hyperthermia in the resulting tumor cells.

[0090] For other specific embodiments, methods of using the conjugate comprising (i) a tumor-targeted NIR dye attached to iron oxide nanoparticles, or (ii) an amphoteric ion tumor-targeted NIR dye attached to iron oxide nanoparticles, or (iii) a tumor-targeted NIR dye attached to amphoteric ion-coated iron oxide nanoparticles, or (iv) an amphoteric ion tumor-targeted NIR dye attached to amphoteric ion-coated iron oxide nanoparticles, the conjugate can be administered to a patient (e.g., via injection), and after administration, the NIR dye can direct the conjugate towards the target tumor cell population. Then, the NIR region can be generated (e.g., via laser irradiation or light-emitting diode (LED) irradiation), and by exposing to the NIR region, the iron oxide nanoparticles can convert the absorbed light into heat, inducing local hyperthermia treatment in tumor cells with minimal damage to intervening and surrounding normal tissues.

[0091] Furthermore, the conjugate comprising (i) a tumor-targeted NIR dye attached to iron oxide nanoparticles, or (ii) an amphoteric ion tumor-targeted NIR dye attached to iron oxide nanoparticles, or (iii) a tumor-targeted NIR dye attached to amphoteric ion-coated iron oxide nanoparticles, or (iv) an amphoteric ion tumor-targeted NIR dye attached to amphoteric ion-coated iron oxide nanoparticles may be useful as a diagnostic agent because (i) the NIR dye can emit NIR light and / or (ii) the iron oxide nanoparticles can function as a magnetic resonance imaging (MRI) contrast agent for detection and imaging / visualization of the corresponding tumor location.

[0092] For certain embodiments, for example, carbon nanoparticles such as carbon nanospheres, carbon nanoshells, carbon nanotubes, and other so-called "lossy dielectric" materials can also function as strong NIR light absorbers and can thus be useful in the present invention. Notably, carbon nanotubes can be targeted to specific cells through either direct covalent functionalization to a target moiety (such as a tumor-targeting NIR dye) or non-covalent wrapping of a target moiety (such as a tumor-targeting NIR dye).

[0093] Now, with respect to nanoparticles (such as gold nanoparticles or iron oxide nanoparticles), for example, as hyperthermia agents, an obstacle to their medical applications and translation to the clinic can be the tendency of such particles to accumulate in the liver and spleen as a result of opsonization and capture by the mononuclear phagocyte system. Specifically, nanoparticles can be highly prone to association with proteins, lipids, and other biomolecules, leading to the formation of a dynamic "biomolecular corona" that can make it difficult to predict and / or control their behavior in vivo. Recognition and phagocytosis by phagocytes following binding of serum proteins can be a fate for many nanoparticles in particular, resulting in rapid clearance from the bloodstream. Zwitterionic coatings can counter this problem and make nanoparticles more biocompatible, for example, by inhibiting the production of the biomolecular corona by reducing the rate and / or extent of non-specific adsorption of proteins and lipids to the nanoparticle surface. As described above, zwitterions can acquire their anti-fouling properties through strong electrostatic interactions between their positively and negatively charged groups and polar water molecules, providing a dense and stable hydration shell that can create physical and energetic barriers to prevent unwanted adsorption. Furthermore, since zwitterionic coatings can be overall neutrally charged, the formation of ion pairs with charged adsorbents is less likely to occur.

[0094] Figure 10A shows a particular embodiment of this conjugate comprising a tumor-targeting NIR dye attached to a zwitterionic nanoparticle.

[0095] Figure 10B shows a particular embodiment of the present conjugate comprising zwitterionic tumor-targeting NIR dyes attached to zwitterionic nanoparticles.

[0096] For particular embodiments, exemplary examples of zwitterions that may be useful in the present invention when attached to nanoparticles include sulfobetaine, carboxybetaine, phosphorylcholine, and those shown in Figure 2.

[0097] For particular embodiments, with regard to attachment, a ratio of 5:1 of zwitterion:tumor-targeting NIR dye or zwitterion:zwitterionic tumor-targeting NIR dye may be useful in the present invention.

[0098] For particular embodiments, a conjugate comprising a tumor-targeting NIR dye attached to a zwitterionic nanoparticle (such as a gold nanoparticle, for example, a gold nanorod) can have the following formula XVII.

Chemical formula

[0099] For particular embodiments, a conjugate comprising a tumor-targeting NIR dye attached to a zwitterionic nanoparticle (such as a gold nanoparticle, for example, a gold nanorod) can have the following formula XVIII.

Chemical formula

[0100] For particular embodiments, a conjugate comprising a tumor-targeting NIR dye attached to a zwitterionic nanoparticle (such as a gold nanoparticle, for example, a gold nanorod) can have the following formula XIX.

Chemical formula

[0101] For certain embodiments, a conjugate comprising a tumor-targeting NIR dye attached to zwitterionic nanoparticles (such as gold nanoparticles, e.g., gold nanorods) can have the following formula XX.

Chemical Formula

[0102] Here again with respect to the therapeutic agent, for certain embodiments, as in the present invention, a tumor-targeting NIR dye or a zwitterionic tumor-targeting NIR dye can be linked (e.g., by conjugation) to a chemotherapeutic agent via various linkers for use in chemotherapy. For example, as in the present invention, among a few conventional chemotherapeutic agents that can be conjugated to a tumor-targeting NIR dye, there can be mentioned actinomycin, all-trans retinoic acid, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, mechlorethamine, methotrexate, mitoxantrone, nitrogen mustard, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vemurafenib, vinblastine, vincristine, and vindesine.

[0103] For use in 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 a chemotherapeutic agent that can be transformed into its pharmacologically active form via metabolism such as by chemical and / or enzymatic mechanisms, and its pharmacologically active form can be effective in killing tumor cells.

[0104] 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, the prodrug form may have improved bioavailability compared to its pharmacologically active form, which can be beneficial for chemotherapeutic agents that are poorly absorbed through the gastrointestinal tract. Further, the prodrug form can facilitate how selectively the chemotherapeutic agent interacts with cells or processes that are its unintended targets, which can reduce unintended, undesirable, and / or harmful side effects of the chemotherapeutic agent.

[0105] Since many hyperthermia agents, such as gold nanoparticles, iron oxide nanoparticles, etc., may be essentially non-toxic to cells, the prodrug form of these hyperthermia agents that form the conjugate may not be as necessary as the prodrug form of the chemotherapeutic agent for the conjugate. For example, doxorubicin (DOX) is a commonly used clinical anti-cancer agent and may be non-specific, so DOX can be toxic to normal cells, especially cells of the heart, liver, and kidneys. Subsequently, incorporation of DOX into the conjugate that targets the tumor site via the present NIR dye or zwitterionic NIR dye can reduce, minimize, or eliminate the harmful side effects of DOX, whereby the linker can act on the pharmacokinetics, pharmacodynamics, stability, toxicity, etc. of the chemotherapeutic agent incorporated into the conjugate, so it is contemplated herein that a specific linker that conjugates DOX to the dye can contribute to the reduction in the harmful side effects of DOX. Preferred linkers can ensure appropriate stability of the prodrug form of the chemotherapeutic agent in circulation, effectively prevent premature release of the chemotherapeutic agent near unintended targets, and timely promote the release and / or pharmacological activation of the chemotherapeutic agent only near the targeted tumor cells.

[0106] Linkers that may be useful for conjugating the present tumor-targeted NIR dyes with therapeutic agents such as chemotherapeutic agents can be classified as cleavable linkers and non-cleavable linkers. Non-cleavable linkers can alter the activity of the active metabolite, which can affect efficacy, toxicity, and transport, thereby causing significant limitations. Next, cleavable linkers may be more desirable for use in the present conjugate and can significantly contribute to its success in effectively killing the target tumor cell population.

[0107] In particular, cleavable linkers that are stable during long-term blood circulation and selectively release chemotherapeutic agents from the conjugate by taking advantage of the unique properties of tumor cells may be particularly desirable.

[0108] For certain embodiments, cleavable linkers having glutathione (GSH) sensitivity (facilitated by oxidation-reduction) can be used in the present conjugate that connects the tumor-targeted NIR dye and the chemotherapeutic agent. Certain cancer cells have a higher intracellular GSH concentration than in plasma, and the elevated GSH levels can reduce disulfide bonds and maintain the intracellular redox balance. This disulfide bond can remain stable in the blood system and can be used as a cleavable linker that releases the active payload only in tumor cells having elevated GSH levels. Exemplary examples of two cleavable linkers having GSH sensitivity are shown in Formulas XXI (faster release / lower stability) and XXII (lower release / better stability).

Chem.

Chem.

[0109] For other specific embodiments, a cleavable linker having pH sensitivity (induced by an acid) can be used in this conjugate that connects a tumor-targeting NIR dye and a chemotherapeutic agent. The acid-sensitive cleavable linker can utilize the lower pH of endosomes (pH = 5 - 6) and lysosomes (pH = 4.8) compared to the cytoplasm (pH = 7.4) to cause hydrolysis of the acid-labile group(s) within the linker. The three types of bonds shown in Formulae XXIII (hydrazone-based bond), XXIV (carbonate-based bond), and XXV (silyl ether-based bond) can be used in a cleavable linker having pH sensitivity.

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[0110] However, as one exemplary example, FIG. 11 shows a specific embodiment of this conjugate that contains DOX conjugated to a tumor-targeting NIR dye via a pH-sensitive cleavable linker having an acid-labile hydrazone bond that can be cleaved when the conjugate enters the more acidic environment of the tumor site / cells, thus releasing DOX at the targeted location.

[0111] For other specific embodiments, a cleavable linker having protease sensitivity (catalyzed by an enzyme) can be used in this conjugate that connects the tumor-targeting NIR dye and the chemotherapeutic agent. The protease-sensitive cleavable linker is found in the lysosomes of tumor cells, and a protease that recognizes and cleaves a specific peptide sequence in the linker can be used. Dubowchik and Firestone et al. pioneered the discovery of the valine-citrulline (VC) dipeptide (as shown in XXVI below), which can be overexpressed in tumor cells as an intracellular cleavage mechanism by cathepsin B, and thus enables the connected chemotherapeutic agent to be released precisely in the immediate vicinity of the tumor site. Similarly, some tumor cells overexpress MMP-2 protease, which can recognize and cleave specific peptide sequences in the linker, such as the synthetic octapeptide (GPLGIAGQ) (as shown in formula XXVII below). Furthermore, the cleavable linker with protease sensitivity can be stable in systemic circulation due to the presence of protease inhibitors in the blood.

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[0112] Protease-sensitive cleavable peptide linkers have been developed as extremely important components of antibody-drug conjugates (ADCs). As a result of their excellent plasma stability and controlled payload release mechanism, the linker can play an indispensable role in the overall success of ADC drugs; 9 out of the 14 approved ADC drugs use protease-sensitive cleavable peptide linkers. For example, brentuximab vedotin (ADCETRIS®) uses a valine-citrulline linker.

[0113] Instead of using an antibody as a tumor targeting agent, certain embodiments of the present conjugate use (i) a tumor targeting NIR dye for the selective and effective delivery of a chemotherapeutic prodrug to target tumor cells, and (ii) a protease-sensitive cleavable peptide linker for releasing and / or pharmacologically activating the chemotherapeutic agent only in the vicinity of the target tumor site. Formulas XXVIII, XXIX, and XXX show exemplary tumor targeting NIR dyes conjugated to various chemotherapeutic agents via protease-sensitive cleavable peptide linkers.

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[0114] For certain embodiments, the linker can include polyethylene glycol (PEG) (as shown in FIGS. 12A and 12B including SN-38 as a chemotherapeutic agent, and FIGS. 12C and 12D including DOX as a chemotherapeutic agent), and PEG can increase solubility and provide desirable drug metabolism and pharmacokinetics (DMPK) of the chemotherapeutic agent being delivered.

[0115] For certain embodiments, the conjugate can include two linkers, a first linker that connects a tumor-targeting NIR dye and a protease-sensitive cleavable peptide, and a second linker that connects the protease-sensitive cleavable peptide and a chemotherapeutic agent. The first linker should be stable (e.g., in blood circulation), but does not necessarily have to be cleavable. The second linker should be stable (e.g., in blood circulation) and must be cleavable when the conjugate is delivered to the tumor site such that the chemotherapeutic agent can be released and / or pharmacologically activated in the vicinity of the target tumor site. Accordingly, a self-degradable spacer can be inserted between the protease-sensitive cleavable peptide and the chemotherapeutic agent to facilitate the effective release of the latter.

[0116] As an illustrative example, cathepsin B can cleave a linker containing a VC dipeptide to release a conjugate moiety containing a chemotherapeutic agent, e.g., SN-38, and a self-degradable para-aminobenzyl carbamate (PABC) spacer. Subsequently, as shown in Figure 13, PABC can undergo spontaneous 1,6-elimination in an acidic environment to release carbon dioxide, para-azidomethide, and SN-38.

[0117] In addition to the linker containing the VC dipeptide, other exemplary linkers having protease-sensitive peptide sequences that may be useful in the conjugate include those shown in formulae XXXI (valine-alanine), XXXII (valine-glycine), XXXIII (glycine-glycine), XXXIV (alanine-alanine-asparagine), and XXXV (phenylalanine-lysine).

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[0118] Formulas XXXVI to XXXXI show exemplary conjugates of the present invention comprising a tumor-targeted NIR dye having zwitterionic functional group(s), a chemotherapeutic agent prodrug, a cathepsin B-cleavable linker, and a self-degradable spacer that promotes effective release of the chemotherapeutic agent prodrug. Due to the zwitterionic function of the tumor-targeted NIR dye and the resulting high hydration, PEG can be excluded from these conjugates.

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[0119] For certain embodiments, click chemistry can be used to connect a tumor-targeting NIR dye and a cleavable peptide sequence. While just one exemplary example, dibenzocyclooctyne (DBCO) reagents contain highly reactive DBCO groups that can react with azide-labeled molecules or biomolecules via copper-free click chemistry. The DBCO click chemistry can be carried out in an aqueous buffer or in an organic solvent depending on the properties of the substrate molecule. Figures 14A and 14B (containing SN-38 as a chemotherapeutic agent) and schematic diagrams 14C and 14D (containing DOX as a chemotherapeutic agent) show exemplary conjugates of the invention made using click chemistry.

[0120] With respect to certain embodiments, as in the present invention, a tumor-targeting NIR dye or zwitterionic tumor-targeting NIR dye can be linked to a nitroxide radical (e.g., by conjugation). Cancer cells may generate higher levels of free radicals than normal cells due to their active metabolism associated with dysregulation of various cellular events, so these cells may be under a certain level of oxidative stress. Overproduction of high levels of free radicals can lead to harmful cell damage and ultimately cell death, but at moderate levels it may facilitate 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 thus protect these cells from damage.

[0121] Correspondingly, nitroxide radicals can be suitable cancer therapeutic agents because these compounds may enhance the oxidative stress of cancer cells and lead to cell death when used alone or in combination with another therapy (such as chemotherapy).

[0122] With respect to certain embodiments, as in the present invention, a tumor-targeting NIR dye or zwitterionic tumor-targeting NIR dye can be linked to a photodynamic therapy (PDT) agent (e.g., by conjugation). Generally, PDT involves administration of a photosensitizer, for example, by injection into the bloodstream or application to the affected area of the skin. When the photosensitizer is activated by light, one or more processes that can result in tumor cell death can be initiated. However, the accumulation of conventional photosensitizers in tumor cells can be low, and an example of such a PDT agent can be chlorin-e6 PS. It is contemplated herein that the present invention can increase the accumulation of PDT agents in tumor cells.

[0123] As can be readily understood from the foregoing, the basic concepts of the present invention can be embodied in a variety of ways. The present invention includes numerous variant embodiments of tumor-targeting NIR dyes and related conjugates, as well as methods for making and using such NIR dyes and related conjugates.

[0124] Accordingly, the specific embodiments or elements of the present invention disclosed herein or shown in the accompanying drawings or tables of this application are not intended to be limiting, but are intended to be examples of equivalents encompassed by the present invention with respect to numerous variant embodiments or specific elements generally included in the present invention. Moreover, a 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 herein and in the figures.

[0125] It is of course understood that each element of the device or each step of the method can be described in terms of the device or in terms of the method. Such terms can be replaced if necessary to clarify the implicitly broad scope to which the present invention is entitled. As an example, it should be understood that all steps of a method can be disclosed as an action, as means for performing that action, or as an element that causes that action. Similarly, each element of a device can be disclosed as a physical element or as an action facilitated by that physical element. As an example, the disclosure of "treatment" should be understood to include the disclosure of the act of "providing treatment", whether or not explicitly discussed, and conversely, if the act of "providing treatment" is effectively disclosed, such disclosure should be understood to include the disclosure of "treatment" and even "means for providing treatment". Such alternative terms for each element or step should be understood to be explicitly included in this specification.

[0126] Also, with respect to each term used, unless the use in this application is inconsistent with such interpretation, the general dictionary definition should be understood to be included in the description of each term contained in Random House Webster’s Unabridged Dictionary, 2nd Edition, and each definition is incorporated herein by reference.

[0127] All numerical values in this specification are assumed to be modified by the term "about" whether explicitly indicated or not. For the purposes of the present invention, a range can be expressed as from a particular value modified by "about" to another particular value modified by "about". When such a range is expressed, another embodiment includes from a particular value to another particular value. The recitation of a numerical range by endpoints includes all numerical values included within that range. A numerical range of 1 to 5 includes, for example, 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, and the like. It is further understood that each endpoint of each range is significant both in relation to the other endpoint and independently of the other endpoint. When a value is expressed as an approximation by use of the antecedent "about", it will be understood that the particular value forms another embodiment. The term "about" generally refers to a range of numerical values that one of ordinary skill in the art would consider equivalent to the recited numerical value or having the same function or result. Similarly, the antecedent "substantially" means mostly the same form, manner or degree but not completely, and a particular element will have a configuration within a range that one of ordinary skill in the art would consider having the same function or result. When a particular element is expressed as an approximation by use of the antecedent "substantially", it will be understood that the particular element forms another embodiment.

[0128] Furthermore, for the purposes of the present invention, an entity modified by the term "a" or "an" refers to one or more of that entity unless otherwise specifically limited. Accordingly, the terms "a" or "an", "one or more" and "at least one" can be used synonymously herein.

[0129] Thus, it should be understood that one or more applicants claim at least the following: i) each of the NIR dyes and related conjugates 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) alternative embodiments that achieve each of the functions shown, disclosed, or described, v) alternative designs and methods that achieve each of the functions implicitly shown to achieve the functions disclosed and described, vi) each of the functions, components, and steps shown as separate and independent inventions, vii) applications enhanced by the various systems or components disclosed, viii) products obtained as a result of the results produced by such systems or components, ix) methods and apparatuses substantially as described above in the present specification and with reference to any of the appended examples, x) various combinations and permutations of each of the elements prior to disclosure.

[0130] The Background section of this patent application, if any, provides a description of the field of endeavor to which the invention pertains. This section may incorporate or include specific U.S. patents, patent applications, publications, or paraphrases of the claimed invention's subject matter that are useful in relating the information, problems, or concerns regarding the state of the art to which the invention pertains. It is not intended that any U.S. patents, patent applications, publications, statements, or other information cited or incorporated herein be construed, understood, or admitted as prior art with respect to the present invention.

[0131] The claims set forth in this specification are hereby incorporated by reference as part of this description of the invention, if any, and the applicant expressly reserves the right to use all or part of the incorporated content of such claims, or additional description supporting its elements or components, as part of or in addition to the claims. Further, the applicant reserves the right, if necessary to define the subject matter sought to be protected by this application or any subsequent application, continuation, divisional, or partial continuation thereof, or to obtain or comply with any benefits, fee reductions, or requirements of the patent laws, rules, or regulations of any country or treaty, to move all or part of the incorporated description of such claims, or any of its elements or components, from the specification into the claims or vice versa. Additionally, such content incorporated by reference shall survive the entire pendency of this application, including any subsequent continuation, divisional, or partial continuation application, or any reissue or extension thereof.

[0132] Furthermore, the claims set forth in this specification are further intended, if any, to describe the boundaries and limits of a limited number of preferred embodiments of the invention, and should not be construed as the broadest embodiment of the invention that can be claimed or as a complete list of embodiments of the invention. The applicant does not waive the right to make further claims based on the foregoing description as part of any continuation, divisional, partial continuation, or similar application.

Claims

[Claim 1] The invention described herein.

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