Carborane and borohydride salts

Abstract

A salt for administration to a mammalian subject in treatment of neoplasms or dysplasias includes an ion and a counterion. The ion may be a cation and the counterion may be an anion. The anion includes a transition metal free carborane or borohydride cluster. The salt is configured to lead to targeted cell death of the neoplasms or dysplasias when subjected to an external stimulus including light, neutrons, or a combination thereof.

Description

CARBORANE AND BOROHYDRIDE SALTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 626,1 12, filed on January 29, 2024. The entire disclosure of the above applications is incorporated herein by reference.GOVERNMENT SUPPORT

[0002] This invention was made with government support under R01 CA270136 awarded by the National Cancer Institute of the National Institutes of Health and under the CAREER Grant CBET 1845006 by the National Science Foundation. The government has certain rights in the invention.

[0003] All publications cited in this application are incorporated by reference herein.FIELD

[0004] The present disclosure relates to carborane and borohydride salts.BACKGROUND

[0005] This section provides background information related to the present disclosure which is not necessarily prior art.

[0006] Since its initial application for the treatment of bladder cancer in 1976, photodynamic therapy (PDT) has emerged as a clinically approved, noninvasive therapeutic approach against a range of diseases including basal cell carcinoma, actinic keratosis, advanced cutaneous T-cell lymphoma, and non-healing ulcers. Generally, PDT relies on three components: a photosensitizer (PS), oxygen, and light. The PS is activated by light of a specific wavelength that initiates a series of photochemical reactions leading to localized cell death in cancer cells. While many PSs have been used for PDT, agents with higher phototoxicity (toxicity with light irradiation), reduced or minimal cytotoxicity (toxicity without light irradiation), and improved tumor targeting are desired to improve efficacy while reducing side effects.

[0007] Cyanine dyes offer several advantages for PDT applications, including strong absorption near the bandgap (E > 105M-1cm-1) that can be tuned to the near-infrared (NIR) region for deep tissue penetration and efficient activation of photosensitization. Utilization of indocyanine green (ICG) in clinical cancer studies for sentinel lymph node mapping has demonstrated their potential in diagnostics. However, the inherent challenges of poor chemical stability, nonspecific binding, and off-target toxicity associated with cyanine-based agents have constrained their medical applications. This challenge persists in cancer therapy despite implementing advanced strategies such as nanoparticle formulation and antibody conjugation. Additionally, current PDT agents used clinically, photofrin and temoporfin (Foscan), may cause prolonged light sensitivity in patients, who should avoid bright light (even indoors) for up to 8 weeks due to delayed clearance from organs.SUMMARY

[0008] This section provides a general summary of the disclosure, and is not a comprehensive disclosure of its full scope or all of its features.

[0009] Photodynamic therapy (PDT) has emerged as a promising targeted treatment for cancer. However, current PDT is limited by low tissue penetration, insufficient phototoxicity (toxicity with light irradiation), and undesirable cytotoxicity (toxicity without light irradiation).

[0010] At least one example embodiment relates to cyanine-carborane salts as potent photosensitizers (PSs). In at least one example embodiment, the salts harness the near-infrared (NIR) absorbing [cyanine+] with the inertness of [carborane ]. The implementation of [cyanine+][carborane ] salts dramatically enhances the cancer targeting of the PSs and decreases cytotoxicity. At least one example embodiment, characterizes the cellular uptake of the cyanine-carborane PSs, organelle localization, generation of reactive oxygen species (ROS) with the ability to cogenerate multiple ROS species, suppression of pro-metastatic pathways, and activation of apoptotic pathways. At least one example embodiment further demonstrates the ability of improved PSs to eliminate tumors in vivo using an orthotopic mouse model of breast cancer. In at least one example embodiment, these newly developed salt PSs introduce a potent therapeutic approach against aggressive cancer while decreasing side effects.

[0011] At least one example embodiment relates to a salt for administration to a mammalian subject in treatment of neoplasms or dysplasias.

[0012] In at least one example embodiment, the salt includes a cation and an anion. The anion includes a transition metal free carborane or borohydride cluster. Thesalt is configured to lead to targeted cell death of the neoplasms or dysplasias when subjected to an external stimulus including light, neutrons, or a combination thereof.

[0013] In at least one example embodiment, the cation is a photoactive cation. The photoactive cation is configured to become excited and generate reactive oxygen species (ROS) when the salt is subjected to the external stimulus including the light.

[0014] In at least one example embodiment, the light has a wavelength ranging from 700 nm to 1200 nm.

[0015] In at least one example embodiment, the wavelength ranges from 750 nm to 950 nm.

[0016] In at least one example embodiment, the ROS include superoxide, peroxide, singlet oxygen, or any combination thereof.

[0017] In at least one example embodiment, the ROS include at least two of superoxide, peroxide, and singlet oxygen.

[0018] In at least one example embodiment, the ROS include singlet oxygen.

[0019] In at least one example embodiment, the neoplasms or dysplasias are in a tumor.

[0020] 9 In at least one example embodiment, the salt is configured to accumulate inside the tumor while an exo-tumor residue is excreted from the mammalian subject.

[0021] In at least one example embodiment, the exo-tumor residue is configured to be excreted from the mammalian subject by a liver of the mammalian subject, a kidney of the mammalian subject, a spleen of the mammalian subject, or any combination thereof.

[0022] In at least one example embodiment, the salt is water insoluble.

[0023] In at least one example embodiment, a quantity of atoms in the anion ranges from 20 to 1000.

[0024] In at least one example embodiment, the quantity of atoms in the anion ranges from 20 to 54.

[0025] In at least one example embodiment, the quantity of atoms in the anion ranges from 20 to 30.

[0026] In at least one example embodiment, a molecular weight of the anion ranges from 100 g / mol to 3000 g / mol.

[0027] In at least one example embodiment, the molecular weight of the anion ranges from 200 g / mol to 1500 g / mol.

[0028] In at least one example embodiment, the salt has an absolute highest occupied molecular orbital (HOMO) energy ranging from 4 eV to 6eV.

[0029] In at least one example embodiment, the absolute HOMO energy ranges from 4.9 eV to 5.4 eV.

[0030] In at least one example embodiment, the salt has an absolute lowest unoccupied molecular orbital (LUMO) energy ranging from 3.5 eV to 4.5 eV.

[0031] In at least one example embodiment, the salt has a bandgap ranging from 1 eV to 1 .75 eV.

[0032] In at least one example embodiment, the salt has an ICso under the external stimulus of less than or equal to 5 micromolar (pM).

[0033] In at least one example embodiment, the ICso under the external stimulus is less than or equal to 2 pM.

[0034] In at least one example embodiment, the salt has an ICso absent external stimulus of greater than or equal to 2 micromolar (pM).

[0035] In at least one example embodiment, the salt has a zeta potential ranging from -30 millivolts (mV) to -10 mV.

[0036] In at least one example embodiment, the anion is a dianion.

[0037] In at least one example embodiment, the anion includes one or more halogens.

[0038] In at least one example embodiment, the cation and the anion are chemically linked such that the salt is an inner salt or zwitterion.

[0039] In at least one example embodiment, the salt is in the form of a nanoparticle.

[0040] In at least one example embodiment, the nanoparticle is free of a coating.

[0041] In at least one example embodiment, the nanoparticle has a diameter ranging from 5 nm to 40 nm.

[0042] In at least one example embodiment, the diameter of the nanoparticle ranges from 22 nm to 38 nm.

[0043] In at least one example embodiment, the anion is the transition metal free carborane. The transition metal free carborane is one of CBgRi(R2)4(R3)4R4N’, as shown in FIG. 2B, or CBII RI(R2)S(R3)SR4N’, as shown in FIG. 2C. Ri, R2, R3, and R4 are independently selected from the group consisting of: H, F, Cl, Br, I, alkyl, functionalized alkyl, aryl, functionalized aryl, OH, O-alkyl, O-aryl, NH2, NR2, NR3, SH, SR, SR2,haloalkyl, perfluoroaryl, acetyl, ester, ether, formyl, amide, formamide, carboxyl, alkanoate, and carbamate. N is an integer. R is any functional group.

[0044] In at least one example embodiment, the anion is the transition metal free carborane. The transition metal free carborane is one of CB9H10", as shown in FIG. 2F, CBuHeCle", as shown in FIG. 2G, CB9HCI9", as shown in FIG. 2H, CioBnH32Si", as shown in FIG. 2I, or C5B22N2H25", as shown in FIG. 2J.

[0045] In at least one example embodiment, the anion is the borohydride cluster. The borohydride cluster is one of BioRioN-, as shown in FIG. 2D, or Bi2Ri2N-, as shown in FIG. 2E. R is selected from the group consisting of: H, F, Cl, Br, I, alkyl, functionalized alkyl, aryl, functionalized aryl, OH, O-alkyl, O-aryl, NH2, N(RI)2, N(RI)3, SH, SR1, S(RI)2, haloalkyl, perfluoroaryl, acetyl, ester, ether, formyl, amide, formamide, carboxyl, alkanoate, and carbamate. N is an integer. R1 is any functional group.

[0046] In at least one example embodiment, the cation is an organic cation.

[0047] In at least one example embodiment, the organic cation is a cyanine, a polymethine, or a combination thereof.

[0048] In at least one example embodiment, the organic cation includes the cyanine. The cyanine has the structure as shown in FIG. 2A.

[0049] At least one example embodiment relates to a salt for administration to a mammalian subject in treatment of neoplasms or dysplasias.

[0050] In at least one example embodiment, the salt includes an ion and a counterion. The counterion includes a transition metal free carborane or borohydride cluster. The salt is configured to lead to targeted cell death of the neoplasms or dysplasias when subjected to an external stimulus including light, neutrons, or a combination thereof.In at least one example embodiment, the counterion is the transition metal free carborane. The transition metal free carborane is one of CBgRi(R2)4(R3)4R4N+, as shown in FIG. 2B, or CBn Ri(R2)5(R3)sR4N+, as shown in FIG. 2C. R1, R2, R3, and R4 are independently selected from the group consisting of: ammonium, quarternized ammonium, phosphonium, stibonium, bismuthonium, NR2, NR3, SR, SR2, functionalized aryl, functionalized alkyl. N is an integer. R is any functional group.

[0051] In at least one example embodiment, the counterion is the borohydride cluster. The borohydride cluster is one of B RioN+, as shown in FIG. 2D, or Bi2Ri2N+, as shown in FIG. 2E. R is selected from the group consisting of: ammonium, quarternizedammonium, phosphonium, stibonium, bismuthonium, N(RI)2, N(Ri)s, SRi, S(RI)2, functionalized aryl, functionalized alkyl. N is an integer. Ri is any functional group.

[0052] At least one example embodiment relates to a salt for administration to a mammalian subject in treatment of neoplasms or dysplasias.

[0053] In at least one example embodiment, the salt includes an organic cation and a dianion including a carborane or borohydride cluster. The salt is configured to lead to targeted cell death of the neoplasms or dysplasias when subjected to an external stimulus including light, neutrons, or a combination thereof. The salt has a IC50 under the external stimulus of less than or equal to 2 pM.

[0054] At least one example embodiment relates to a salt nanoparticle for administration to a mammalian subject in treatment of neoplasms or dysplasias.

[0055] In at least one example embodiment, the salt includes an organic cation and a dianion. The dianion includes a carborane or borohydride cluster. The salt nanoparticle is configured to lead to targeted cell death of the neoplasms or dysplasias when subjected to an external stimulus including light, neutrons, or a combination thereof. The salt nanoparticle has a diameter ranging from 5 nm to 40 nm.

[0056] At least one example embodiment relates to a salt for use in photodynamic therapy.In at least one example embodiment, the salt includes a photoactive organic cation and an anion. The photoactive organic cation includes a cyanine having the shown in FIG. 2A. The anion includes one of a first transition metal free carborane, a second transition metal free carborane, a first borohydride cluster, or a second borohydride cluster. The first transition metal free carborane has the formula CBgRi(R2)4(R3)4R4N; as shown in FIG. 2B, where N is an integer and R1, R2, R3, and R4 are independently selected from the group consisting of: H, F, Cl, Br, I, alkyl, functionalized alkyl, aryl, functionalized aryl, OH, O-alkyl, O-aryl, NH2, NR2, NR3, SH, SR, SR2, haloalkyl, perfluoroaryl, acetyl, ester, ether, formyl, amide, formamide, carboxyl, alkanoate, and carbamate, and where R is any functional group. The second transition metal free carborane has the formula CB11 R1 (R2)5(R3)SR4N’, as shown in FIG. 2C, where N is an integer and R1, R2, R3, and R4 are independently selected from the group consisting of: H, F, Cl, Br, I, alkyl, functionalized alkyl, aryl, functionalized aryl, OH, O-alkyl, O-aryl, NH2, NR2, NR3, SH, SR, SR2, haloalkyl, perfluoroaryl, acetyl, ester, ether, formyl, amide, formamide, carboxyl, alkanoate, and carbamate, and where R is any functional group. The first borohydride cluster has the formula BioRioN-, as shown in FIG. 2D, where N is an integer and R isselected from the group consisting of: H, F, Cl, Br, I, alkyl, functionalized alkyl, aryl, functionalized aryl, OH, O-alkyl, O-aryl, NH2, N(RI)2, N(Ri)s, SH, SR1, S(RI)2, haloalkyl, perfluoroaryl, acetyl, ester, ether, formyl, amide, formamide, carboxyl, alkanoate, and carbamate, and where R1 is any functional group. The second borohydride cluster has the formula Bi2Ri2N-, as shown in FIG. 2E, where N is an integer and R is selected from the group consisting of: H, F, Cl, Br, I, alkyl, functionalized alkyl, aryl, functionalized aryl, OH, O-alkyl, O-aryl, NH2, N(RI)2, N(Ri)s, SH, SR1, S(RI)2, haloalkyl, perfluoroaryl, acetyl, ester, ether, formyl, amide, formamide, carboxyl, alkanoate, and carbamate, and where R1 is any functional group. The salt is configured to become excited and generate reactive oxygen species (ROS) when subjected to an external stimulus including light having a wavelength ranging from 700 nm to 1200 nm, the ROS including singlet oxygen.

[0057] At least one example embodiment relates to a method of exciting an salt in a cell of a subject.

[0058] In at least one example embodiemnt, the method includes exciting the salt in the cell. The salt includes an organic cation and an anion. The organic cation includes a cyanine or a polymethine. The anion includes a transition metal free carborane or a borohydride nanocluster.

[0059] In at least one example embodiment, the method further includes, prior to the exciting, administering a composition including the salt to the subject such that the cell takes up the salt.

[0060] In at least one example embodiment, during the exciting, the salt is in the form of a nanoparticle.

[0061] In at least one example embodiment, the exciting includes exposing the salt to light. The light has a wavelength ranging from 700 nm to 1200 nm.

[0062] In at least one example embodiment, the exciting causes the salt generate reactive oxygen species (ROS).

[0063] In at least one example embodiment, the ROS include singlet oxygen.

[0064] In at least one example embodiment, the exciting includes exposing the salt to a neutron beam.

[0065] In at least one example embodiment, the anion includes the transition metal free carborane or the borohydride nanocluster. Greater than 40% of boron atoms in the transition metal free carborane or the borohydride nanocluster are10B.

[0066] In at least one example embodiment, the cell is a cancer cell. The cancer cell includes brain cancer cell, head and / or neck cancer cell, lung cancer cell, breastcancer cell, gastrointestinal cancer cell, pancreatic cancer cell, prostate cancer cell, liver cancer cell, skin cancer cell, ovarian cancer cell, cervical cancer cell, bladder cancer cell, renal cancer cell, soft tissue sarcoma cell, thyroid cancer cell, endometrial cancer cell, or any combination thereof.

[0067] In at least one example embodiment, the salt is taken up by mitochondria, endoplasmic reticulum, Golgi apparatus, cytoplasm, or any combination thereof.

[0068] In at least one example embodiment, the salt is taken up by the mitochondria, the endoplasmic reticulum, and the Golgi apparatus.

[0069] Further areas of applicability will become apparent from the description provided herein. The description and specific examples in this summary are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0070] The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations, and are not intended to limit the scope of the present disclosure.

[0071] FIGS. 1 A-1 B illustrate example embodiments of salt configurations. FIG. 1 A is a schematic illustration of a salt including an ion (e.g., a cation) and a counterion (e.g., an anion). FIG. 1 B is a schematic illustration of aggregated particles or nanoparticles of a salt. FIG. 1 C is a schematic illustration of salt being activated by an external stimulus.

[0072] FIGS. 2A-2J relate to a cation and anions that can be paired to form salts according to at least one example embodiment. FIG. 2A illustrates the structure of the cyanine cation (Cy+) according to at least one example embodiment. FIG. 2B illustrates the general structure of a carborane ion according to at least one example embodiment. FIG. 2C illustrates the general structure of another carborane ion according to at least one example embodiment. FIG. 2D illustrates the general structure of a borohydride cluster according to at least one example embodiment. FIG. 2E illustrates the general structure of another borohydride cluster according to at least one example embodiment. FIG. 2F illustrates the structure of a first carborane anion, CB9H10", according to at least one example embodiment. FIG. 2G illustrates the structure of a second carborane anion, CBuHeCle", according to at least one example embodiment. FIG. 2H illustrates the structure of a third carborane anion, CB9HCI9", according to at least one exampleembodiment. FIG. 21 illustrates the structure of a fourth carborane anion, CioBnH32Si", according to at least one example embodiment. FIG. 2J illustrates the structure of a fifth carborane anion C5B22N2H25", according to at least one example embodiment.

[0073] FIG. 3 is a schematic illustrating multifaceted assessment of cyaninecarboranes on tumor growth in PDT.

[0074] FIGS. 4A-4B relate to cyanine-carborane PSs that form stable nanocluster formation in 10% DMSO solution. PSs were either diluted in 100% DMSO (monomer solution) or 10% DMSO (nanoparticle solution) and tested for the formation of nanoparticles using UV-VIS spectroscopy. The spectral broadening of the NIR peaks was observed when the cyanine-carboranes were dissolved in 10% DMSO, with the full width at half maximum increasing from 600 nm to 1000 nm, compared to their narrower peaks in 100% DMSO. FIG. 4A is a graph illustrating normalized absorbance as a function of wavelength for CyCBuHeCle. Hour 1 is shown at 400; Hour 3 is shown at 402; Hour 6 is shown at 404; Day 2 is shown 406; Day 3 is shown at 408; Day 4 is shown at 410; Day 7 is shown at 412; Day 8 is shown at 414; Day 15 is shown at 416; and Day 22 is shown 418. FIG. 4B is a graph illustrating normalized absorbance as a function of wavelength for CyCBgHClg. Hour 1 is shown at 440; Hour 3 is shown at 442; Hour 6 is shown at 444; Day 2 is shown 446; Day 3 is shown at 448; Day 4 is shown at 450; Day 7 is shown at 452; Day 8 is shown at 454; Day 15 is shown at 456; and Day 22 is shown 458.

[0075] FIGS. 5A-5D relate to measurement of particle size of cyanine-carboranes using Scanning Electron Microscopy (SEM). FIGS. 5A-5D are representative SEM images of CyCBuHeCle and CyCBgHClg nanoparticles displaying their relative sizes at different magnifications. The relative sizes of nanoparticles were normalized to the Au reference sample (30 ± 5 nm). FIG. 5A shows CyCBuHeCle at 200 x magnification. FIG. 5B shows CyCBuHeCle at 400x magnification. FIG. 5C shows CyCBgHClg at 200x magnification. FIG. 5D shows CyCBgHClg at 400x magnification.

[0076] FIG. 6 is a chart illustrating frontier energy levels of cyanine-carboranes determined by ultraviolet photoelectron spectroscopy (UPS).

[0077] FIGS. 7A-7N relate to structural characterization of cyanine-carborane photosensitizers using solid-state NMR and high-resolution mass spectrometry (HR-MS). FIGS. 7A-7C are portions of a stacked13C{1H} CP MAS SSNMR spectrum of cyanine [CBuHeCle] recorded at 8 kHz (top) and 10 kHz (bottom). HR-MS spectra of cyanine [CBuHeCle] in both positive and negative ion modes. FIGS. 7D-7F are portions of a stacked13C{1H} CP MAS SSNMR spectrum of cyanine [HCBgClg] recorded at 8 kHz (top)and 10 kHz (bottom). HR-MS spectra of cyanine [HCB9CI9] in both positive and negative ion modes. FIG. 7G is an1H NMR spectrum of [CBuHeCle] in de-Acetone. FIG. 7H is an1H NMR spectrum of [HCB9CI9] in de-Acetone. FIG. 7I is an1H NMR spectrum of cyanine [CBuHeCle] in de-Acetonitrile. FIG. 7J is an1H NMR spectrum of cyanine [HCB9CI9] in de- Acetonitrile. FIG. 7K is a13C NMR spectrum of [CBuHeCle] in de-Acetone. FIG. 7L is a13C NMR spectrum of [HCB9CI9] in de-Acetone. FIG. 7M is a13C{1H} NMR spectrum of cyanine [CBuHeCle] in de-Acetone. FIG. 7N is a13C{1H} NMR spectrum of cyanine [HCB9CI9] in de-Acetone.

[0078] FIGS. 8A-8B relate to cellular viability of 4T 1 mouse mammary cancer cells treated with indicated concentrations of CyCBuHeCle with or without 30 min of daily 850 nm light irradiation over a 5-day duration. FIG. 8A shows crystal violet staining, which was used to visualize the cellular confluency on day 5. Scale bars: 300 pm. FIG. 8B is a graph illustrating number of viable cells, quantified using trypan blue and presented as Iog2 fold changes. Data are presented as the mean ± S.D. (n = 3, *p < 0.05, **p< 0.01 ,m*p < 0.001 ).

[0079] FIGS. 9A-9B relate to cellular viability of 4T 1 mouse mammary cancer cells treated with indicated concentrations of CyCBgHClg with or without 30 min of daily 850 nm light irradiation over a 5-day duration. FIG. 9A shows crystal violet staining, which was used to visualize the cellular confluency on day 5. Scale bars: 300 pm. FIG. 9B is a graph illustrating number of viable cells, quantified using trypan blue and presented as Iog2 fold changes. Data are presented as the mean ± S.D. (n = 3, *p < 0.05, **p< 0.01 ,m*p < 0.001 ).

[0080] FIGS. 10A-10B relate to cyanine-carboranes that inhibit proliferation of breast cancer cells. Viability of 6DT 1 mouse mammary cancer cells treated with indicated concentrations of CyCBuHeCle with or without 30 min of daily 850 nm light irradiation over a 5-day duration. FIG. 10A shows crystal violet staining, which was used to visualize the cellular confluency on day 5. Scale bars: 300 pm. FIG. 10B is a graph illustrating number of viable cells was quantified using trypan blue and presented as Iog2 fold changes. Data are presented as the mean ± S.D. n = 3, *P < 0.05, **P < 0.01 , ***P < 0.001 ).

[0081] FIGS. 1 1 A-1 1 B relate to cyanine-carboranes that inhibit proliferation of breast cancer cells. Viability of 6DT 1 mouse mammary cancer cells treated with indicated concentrations of CyCBgHClg with or without 30 min of daily 850 nm light irradiation over a 5-day duration. FIG. 1 1 A shows crystal violet staining, which was used to visualize the cellular confluency on day 5. Scale bars: 300 pm. FIG. 1 1 B is a graph illustrating numberof viable cells, which was quantified using trypan blue and presented as Iog2 fold changes. Data are presented as the mean ± S.D. n = 3, *P < 0.05, **P < 0.01 , ***P < 0.001 ).

[0082] FIG. 12 is a graph illustrating assessment of cellular viability in 4T1 cells exposed to high fluence 850 nm light. 4T1 cells were exposed to 850 nm light for varying time points and then incubated for an additional 24 hours. After the incubation period, the number of viable cells was quantified using trypan blue staining. Data are presented as mean ± S.D.

[0083] FIGS. 13A-13B relate to assessment of potential cyanine-carboranes for use in PDT. Cellular viability of 4T1 cancer cells treated with indicated concentrations of CyCBgHio with or without 30 min of daily 850 nm light irradiation over a 5-day duration. FIG. 13A shows staining used to visualize cellular confluency. FIG. 13B is a graph illustrating number of viable cells, which was quantified using trypan blue and presented as Iog2 fold changes.

[0084] FIGS. 14A-14B relate to assessment of potential cyanine-carboranes for use in PDT. Cellular viability of 4T1 cancer cells treated with indicated concentrations of CyCioBnH32Si with or without 30 min of daily 850 nm light irradiation over a 5-day duration. FIG. 14A shows staining used to visualize cellular confluency. FIG. 14B is a graph illustrating number of viable cells, which was quantified using trypan blue and presented as Iog2 fold changes.

[0085] FIGS. 15A-15B relate to assessment of potential cyanine-carboranes for use in PDT. Cellular viability of 4T1 cancer cells treated with indicated concentrations of CyCsB22N2H25 with or without 30 min of daily 850 nm light irradiation over a 5-day duration. FIG. 15A shows staining used to visualize cellular confluency. FIG. 15B is a graph illustrating number of viable cells, which was quantified using trypan blue and presented as Iog2 fold changes.

[0086] FIGS. 16A-16B relate to counterions tuning of toxicity of [cyanine+] in 4T1 breast cancer cells. The toxicity of the photoactive cation heptamethine cyanine (Cy+) is modulated by varying anionic pairings. 4T1 breast cancer cells were incubated with different concentrations of Cy+combined with a Cyl (Iodide; cytotoxic) anion, with or without NIR (850 nm) excitation. FIG. 16A illustrates the structure Cyl. FIG. 16B is a graph illustrating cell viability, which was assessed on day 4 using trypan blue staining and cell counting. All data are presented as mean ± S.D. (n = 3; *P< 0.05, ***P< 0.001 ).

[0087] FIGS. 17A-17B relate to counterions tuning of toxicity of [cyanine+] in 4T1 breast cancer cells. The toxicity of the photoactive cation heptamethine cyanine (Cy+) is modulated by varying anionic pairings. 4T1 breast cancer cells were incubated with different concentrations of Cy+combined with a CyFPhB (tetrakis 4-fluorophenyl borate; phototoxic) anion, with or without NIR (850 nm) excitation. FIG. 17A illustrates the structure CyFPhB. FIG. 17B is a graph illustrating cell viability, which was assessed on day 4 using trypan blue staining and cell counting. All data are presented as mean ± S.D. (n = 3; *P < 0.05, ***P< 0.001 ).

[0088] FIGS. 18A-18B relate to counterions tuning of toxicity of [cyanine+] in 4T1 breast cancer cells. The toxicity of the photoactive cation heptamethine cyanine (Cy+) is modulated by varying anionic pairings. 4T1 breast cancer cells were incubated with different concentrations of Cy+combined with a CyTPFB (tetrakis pentafluoropheny borate; nontoxic) anion, with or without NIR (850 nm) excitation. FIG. 18A illustrates the structure CyTPFB. FIG. 18B is a graph illustrating cell viability, which was assessed on day 4 using trypan blue staining and cell counting. All data are presented as mean ± S.D. (n = 3; *P < 0.05, ***P< 0.001 ).

[0089] FIGS. 19A-19B relate to cyanine-carboranes that exhibit non-cytotoxic effects on non-tumorigenic cells. Cellular viability of L-929 mouse non-tumorigenic cells treated with indicated concentrations of CyCBuHeCle with or without 30 min of daily 850 nm light irradiation over a 5-day duration. FIG. 19A illustrates crystal violet staining, which was used to visualize the cellular confluency on day 5. Scale bars: 300 pm. FIG. 19B is a graph illustrating number of viable cells, which was quantified using trypan blue and presented as Iog2 fold changes.

[0090] FIGS. 20A-20B relate to cyanine-carboranes that exhibit non-cytotoxic effects on non-tumorigenic cells. Cellular viability of L-929 mouse non-tumorigenic cells treated with indicated concentrations of CyCBgHClg with or without 30 min of daily 850 nm light irradiation over a 5-day duration. FIG. 20A illustrates crystal violet staining, which was used to visualize the cellular confluency on day 5. Scale bars: 300 pm. FIG. 20B is a graph illustrating number of viable cells, which was quantified using trypan blue and presented as Iog2 fold changes.

[0091] FIGS. 21 A-21 D are images related to cyanine-carboranes localizing to mitochondria. Cyanine-carboranes cyanine-carboranes preferentially localize to the mitochondria and their uptake relies on organic-anion transporting polypeptides. Each of FIGS. 21 A-21 D shows PSs on the left column (originally in red) and respective targettracker in the middle column (originally in green) distribution in 4T1 cells. The rvalue corresponds to the Pearson correlation coefficient, reflecting the level of colocalization between cyanine-carboranes and their respective target tracker. Scale bars: 100 pm. FIG. 21 A is a first set of representative microscope images. FIG. 21 B is a second set of representative microscope images. FIG. 21 C is a third set of representative microscope images. FIG. 21 D is a fourth set of representative microscope images.

[0092] FIGS. 22A-22D are microscope images related to the effective update of cyanine-carboranes by human breast cancer cells. FIGS. 22A show the PSs in the left column (originally in red) and respective target trackers in the middle column (originally in green) distribution in 4T1 cells. The rvalue corresponds to the Pearson correlation coefficient, reflecting the level of colocalization between cyanine-carboranes and their respective target tracker. Scale bars: 100 pm. FIG. 22A is a first set of representative microscope images. FIG. 22B is a second set of representative microscope images. FIG. 22C is a third set of representative microscope images. FIG. 22D is a fourth set of representative microscope images.

[0093] FIGS. 23A-23B are fluorescent images of targeted endocytosis inhibitors showing the nucleus in the left column (Hoechst; originally shown in blue) and PS in the middle column (originally shown in red) distribution in 4T1 cells. Scale bars: 100 pm. Control cells (vehicle; Veh) were treated with 0.1 % DMSO. FIG. 23A is a first set of representative fluorescent images. FIG. 23B is a second set of representative fluorescent images.

[0094] FIGS. 24A-24B relate to inhibition of cyanine-carboranes uptake by OATP1 B1 and OATP1 B3 inhibitors. FIG. 24A is a first set of representative fluorescent images of Bromocresol inhibitors showing the PS in the left column (originally shown in red) distribution in 4T1 cells. Scale bars: 100 pm. Control cells (0 pM) were treated with 0.1 % DMSO. FIG. 24B is a first set of representative fluorescent images of Rifampicin inhibitors showing the PS in the left column (originally shown in red) distribution in 4T1 cells. Scale bars: 100 pm. Control cells (0 pM) were treated with 0.1 % DMSO.

[0095] FIGS. 25A-25F relate to photoactivation of cyanine-carboranes to generate simultaneous mitochondrial and cytoplasmic reactive oxygen species in breast cancer cells. After 24 h incubation of 4T1 cells with or without PSs (CyCBuHeCle and CyCBgHClg), cells were briefly washed with PBS and irradiated with 850 nm or remained in dark conditions (Veh). Following light irradiation, cells were immediately incubated with three ROS-sensing dyes: a general cytoplasmic ROS-sensing dye, a singlet O2 ROS-sensing dye, and a mitochondrial ROS-sensing dye. Scale bars: 100 pm. The white arrows in the phase contrast indicate early cell death (apoptosis). The ROS levels were measured in relative fluorescence units and normalized to the mean ROS level of control cells (Veh) at the 30-min mark. Data are presented as the mean ± S.D. (n = 3, *p < 0.05, **p < 0.01 , ***p < 0.001 ). ns; not significant. FIG. 25A is a collection of images of cells intubated in the general cytoplasmic ROS-sensing dye. FIG. 25B is a graph illustrating ROS levels in the general cytoplasmic ROS-sensing dye. FIG. 25C is a collection of images of cells intubated in the singlet O2 ROS-sensing dye. FIG. 25D is a graph illustrating ROS levels in the singlet O2 ROS-sensing dye. FIG. 25E is a collection of images of cells intubated in the mitochondrial ROS-sensing dye. FIG. 25F is a graph illustrating ROS levels in the mitochondrial ROS-sensing dye.

[0096] FIGS. 26A-26D relate to detection of reactive oxygen species (ROS) generated by cyanine-carboranes in solution using specific ROS sensors. Relative fluorescence units of ROS were measured after 30 minutes of incubation of CyCBuHeCle and CyCBgHClg with a general cytoplasmic ROS sensor (CM-H2DCFDA) and a singlet oxygen sensor in culture media, under dark conditions or 850 nm light exposure. ROS levels were normalized to the mean ROS level of the control group (media + sensor only). Data are presented as mean ± S.D. (n = 3). Statistical significance is indicated as follows: *P < 0.05, **P < 0.01 , ***P < 0.001 ; ns, not significant. FIG. 26A is a graph illustrating ROS levels of CyCBuHeCle with the general cytoplasmic ROS sensor (CM-H2DCFDA). FIG. 26B is a graph illustrating ROS levels of CyCBgHClg with the general cytoplasmic ROS sensor (CM-H2DCFDA). FIG. 26C is a graph illustrating ROS levels of CyCBuHeCle with the singlet oxygen sensor. FIG. 26D is a graph illustrating ROS levels of CyCBgHClg with the singlet oxygen sensor.

[0097] FIGS. 27A-27B relate to photoactivation of cyanine-carboranes to suppress metastatic capacity of breast cancer cells. FIG. 27A is a collection of representative microscopic images after 24 h of wound closure in cells treated with or without PSs under 850 nm light exposure or dark conditions. FIG. 27B is a graph illustrating real-time wound closure of the cells for 24 h immediately following irradiation with 850 nm light.

[0098] FIGS. 28A-28E relate to cyanine-carborane photosensitizers that inhibit cellular migration of highly metastatic breast cancer cells. FIG. 28A is a schematic illustration of the experimental procedure using a transwell culture system. After a 2-hour incubation of 4T1 cells with CyCBuHeCle or CyCBgHClg the cells were irradiated with 850nm light or kept in dark conditions (Veh). Following light irradiation, the cells that migrated to the bottom of the transwell inserts with 8 pm and 12 pm pore sizes were fixed with 4% PFA, stained with Hoechst (a nuclear dye). FIG. 28B is a collection of images of cells intubation with CyCBuHeCle and stained with Hoechst, as described above. FIG. 28C is a graph illustrating total numbers of cells counted using fluorescence microscopy. Scale bars: 100 pm. The total number of cells (intubated with CyCBuHeCle) from 5 random images per group were analyzed using Imaged and plotted. The cells were suspended in a serum-free medium to encourage invasion toward the chemoattractant in the lower chamber, which contained complete medium with 15% FBS. FIG. 28D is a collection of images of cells intubation with CyCBgHClg and stained with Hoechst, as described above. FIG. 28E is a graph illustrating total numbers of cells (intubated with CyCBgHClg) counted using fluorescence microscopy. Scale bars: 100 pm. The total number of cells from 5 random images per group were analyzed using Imaged and plotted. The cells were suspended in a serum-free medium to encourage invasion toward the chemoattractant in the lower chamber, which contained complete medium with 15% FBS.

[0099] FIG. 29 is a Western blot analysis of metastatic markers E-cadherin, N- cadherin, Vimentin, and Snail expression levels in 4T1 cells treated with or without PSs under 850 nm light exposure (30 min) or dark conditions harvested at 6 h time-point.

[0100] FIGS. 30A-30B relate to quantitative RT-PCR was performed to assess the mRNA levels of MMP3 and MMP9. Data were normalized to the expression of the [3- actin reference gene. All data are presented as mean ± S.D. (n = 3; *P< 0.05, **P< 0.01 , ***P < 0.001 , ****p < 0.0001 ). FIG. 30A is a graph illustrating mRNA levels of MMP3. FIG. 30B is a graph illustrating mRNA levels of MMP9.

[0101] FIG. 31 A is a collection of representative microscopic images of F-actin filaments (rhodamine-phalloidin) and nucleus (Hoechst) in 4T1 cells treated with or without PSs under 850 nm light exposure (30 min) or dark conditions at 6 h time-point. Scale bars: 400 pm. FIG. 31 B is a graph illustrating total GTP / ATP ratio of cells was quantified using liquid chromatography-mass spectrometry at 6 h time-point. All data are shown as the mean ± S.D. (n = 3, *p< 0.05). ns; not significant.

[0102] FIGS. 32A-32F relate to photoactivation of cyanine-carboranes that induces early apoptosis and disrupts mitochondrial function in breast cancer cells. Cells were stained with annexin V conjugated to fluorescein isothiocyanate (FITC) and propidium iodide (PI) and analyzed by flow cytometry with or without PSs under 850 nm light exposure (30 min) or in dark conditions at 6 h time-point. FIG. 32A is a graph forcells with no treatment in dark conditions. FIG. 32B is a graph illustrating cells for cells with no treatment under 850 nm light. FIG. 32C is a graph for cells with CyCBuHeCle treatment in dark conditions. FIG. 32D is a graph illustrating cells for cells with CyCBuHeCle treatment under 850 nm light. FIG. 32E is a graph for cells with CyCBgHClg treatment in dark conditions. FIG. 32F is a graph illustrating cells for cells with CyCBgHClg treatment under 850 nm light.

[0103] FIGS. 33A-33C relate to quantitative RT-PCR that was employed to assess the messenger ribonucleic acid (mRNA) levels of Bax, Bak, and Bid. Data were normalized to the [3-actin reference gene expression. FIG. 5A is a graph illustrating mRNA levels of Bax. FIG. 5B is a graph illustrating mRNA levels of Bak. FIG. 33C is a graph illustrating mRNA levels of Bid.

[0104] FIG. 34 is a Western blot analysis of p-ERK 1 / 2, T-ERK 1 / 2, p-MLKL, PARP, Cytochrome c, and Bax expression levels in 4T1 cells at 6 h time-point.

[0105] FIGS. 35A-35H are Western blots of EMT and apoptosis signature proteins in 4T1 cells. Scheme for membrane cuts and western blot images of target protein expression in six different membranes with consideration of the molecular weight. The protein name and size marker were indicated in the top and right side of each blot, respectively. FIG. 35A illustrates original blots. FIG. 35B shows the blots of FIG. 35A cropped. FIG. 35C illustrates original blots. FIG. 35D shows the blots of FIG. 35C cropped. FIG. 35E illustrates original blots. FIG. 35F shows the blots of FIG. 35E cropped. FIG. 35G illustrates original blots. FIG. 35H shows the blots of FIG. 35G cropped.

[0106] FIG. 36 illustrates representative fluorescence images of mitochondrial membrane potentials (AMJm) in 4T1 cells. N.I: Normalized intensity; intensities of mitochondrial membrane potential were normalized against the no-treatment cell conditions in dark or under 850 nm exposure. Data are presented as the mean ± S.D. (n = 3, *p < 0.05, **p < 0.01 , ***p < 0.001 ).

[0107] FIGS. 37A-37G relate to cyanine-carborane localization in tumors of a mouse model of breast cancer. FIG. 37A is a schematic illustrating timeline. 10,000 6DT 1 cells were implanted into the fourth mammary fat pad of female FVB mice, and tumor growth was monitored until day 8. On day 9, mice were intravenously administered a fluorescent organic salt at a dose of 3 pmol / kg, followed by exposure to 850 nm light for 20 minutes. The PDT treatment was subsequently repeated every 48 hours for a total duration of 20 days. FIG. 37B is a collection of representative microscopic imagesshowing the precise localization of PSs within breast tumor cells. FIG. 37C illustrates the fluorescence intensity of CyCBuHeCle uptake within the tumor site was monitored over a period of 5 days following the administration of the PSs. Representative microscopic images showing the precise localization of PSs within breast tumor cells. The dot-shaped circle indicates specific breast tumors in the fourth mammary fat pad region. FIG. 37D illustrates the fluorescence intensity of CyCBgHClg uptake within the tumor site was monitored over a period of 5 days following the administration of the PSs. Representative microscopic images showing the precise localization of PSs within breast tumor cells. The dot-shaped circle indicates specific breast tumors in the fourth mammary fat pad region. FIG. 37E is ex vivo fluorescence imaging of PSs in major organs of an FVB mouse model. Representative fluorescence images of CyCBuHeCle were captured 20 days after PS administration, showing their distribution in the tumor site and major organs including the liver, kidneys, and spleen. FIG. 37F is ex vivo fluorescence imaging of PSs in major organs of an FVB mouse model. Representative fluorescence images of CyCBgHClg were captured 20 days after PS administration, showing their distribution in the tumor site and major organs including the liver, kidneys, and spleen. FIG. 37G is a graph illustrating the fluorescence intensity of cyanine-carboranes uptake within the tumor site, which was monitored over a period of 5 days following the administration of the PSs.

[0108] FIG. 38A is a graph illustrating tumor volume measured every other day following the initiation of PDT in mice. Data are presented as the mean ± S.D. (n = 5, ****p < 0.001 ). FIG. 38B is a graph illustrating that PDT with cyanine-carboranes does not affect the weight of mice. The well-being of the animals and their weight were closely monitored by recording mouse weights every other day before the initiation of PDT. Data are presented as the mean ± S.D. (n = 5).

[0109] FIGS. 39A-39C relate to cyanine-carboranes that inhibit tumor growth in rechallenged FVB mouse model. FIG. 39A is a schematic illustration of experimental design to assess the potential involvement of a rejection antigen in the PDT effect. FIG. 39B is a collection of representative microscopic images showing the precise localization of PSs within breast tumor cells. FIG. 39C is a graph illustrating the tumor volume, which was measured every other day following the initiation of PDT in mice. Data are presented as the mean

[0110] FIGS. 40A-40C relate to cyanine-carboranes that inhibit tumor growth in immunodeficient NSG mouse model. FIG. 40A is a schematic illustration of experimental design. FIG. 40B is a collection of representative microscopic images showing theprecise localization of PSs within breast tumor cells. FIG. 40C is a graph illustrating the tumor volume, which was measured every other day following the initiation of PDT in mice. Data are presented as the mean ± S.D. (n = 5,0.001 ).

[0111] FIGS. 41 A-41 G relate to CyFPhB that exclusively produces mitochondrial reactive oxygen species following NIR irradiation in breast cancer cells. FIG. 41 A illustrates a photoactive heptamethine cyanine cation [Cy+] is tuned with tetrakis 4- fluorophenyl borate [FPhB ] to modulate toxicity. After 24 h incubation of 4T1 cells with or without CyFPhB cells were briefly washed with PBS and irradiated with 850 nm or remained in dark conditions (Veh). Following light irradiation, cells were immediately incubated with three ROS-sensing dyes: a general cytoplasmic ROS-sensing dye, a singlet O2 ROS-sensing dye, and a mitochondrial ROS-sensing dye. Scale bars: 100 pm. FIG. 41 B is a collection of images illustrating cells intubated with the general cytoplasmic ROS-sensing dye. FIG. 41 C is a graph illustrating relative fluorescence of the cells intubated with the general cytoplasmic ROS-sensing dye. FIG. 41 D is a collection of images illustrating cells intubated with the mitochondrial ROS-sensing dye. FIG. 41 E is a graph illustrating relative fluorescence of the cells intubated with the mitochondrial ROS-sensing dye. FIG. 41 F is a collection of images illustrating cells intubated with the singlet O2 ROS-sensing dye. FIG. 41 G is a graph illustrating relative fluorescence of the cells intubated with the singlet O2 ROS-sensing dye.

[0112] FIG. 42 is a schematic illustration of the mechanism of action of cyaninecarborane PSs in breast cancer cells.

[0113] Corresponding reference numerals indicate corresponding parts throughout the several views of the drawings.DETAILED DESCRIPTION

[0114] Example embodiments will now be described more fully with reference to the accompanying drawings.

[0115] Example embodiments are provided so that this disclosure will be thorough, and will fully convey the scope to those who are skilled in the art. Numerous specific details are set forth such as examples of specific components, devices, and methods, to provide a thorough understanding of embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be employed, that example embodiments may be embodied in many different forms and that neither should be construed to limit the scope of the disclosure. In some example embodiments, well-known processes, well-known device structures, and well-known technologies are not described in detail.

[0116] The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” may be intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms “comprises,” “comprising,” “including,” and “having,” are inclusive and therefore specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. The method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed.

[0117] When an element or layer is referred to as being “on,” “engaged to,” “connected to,” or “coupled to” another element or layer, it may be directly on, engaged, connected or coupled to the other element or layer, or intervening elements or layers may be present. In contrast, when an element is referred to as being “directly on,” “directly engaged to,” “directly connected to,” or “directly coupled to” another element or layer, there may be no intervening elements or layers present. Other words used to describe the relationship between elements should be interpreted in a like fashion (e.g., “between” versus “directly between,” “adjacent” versus “directly adjacent,” etc.). As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0118] Although the terms first, second, third, etc. may be used herein to describe various elements, components, regions, layers and / or sections, these elements, components, regions, layers and / or sections should not be limited by these terms. These terms may be only used to distinguish one element, component, region, layer or section from another region, layer or section. Terms such as “first,” “second,” and other numerical terms when used herein do not imply a sequence or order unless clearly indicated by the context. Thus, a first element, component, region, layer or section discussed below could be termed a second element, component, region, layer or section without departing from the teachings of the example embodiments.

[0119] Spatially relative terms, such as “inner,” “outer,” “beneath,” “below,” “lower,” “above,” “upper,” and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. Spatially relative terms may be intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, elements described as “below” or “beneath” other elements or features would then be oriented “above” the other elements or features. Thus, the example term “below” can encompass both an orientation of above and below. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.CompositionsUse of Compositions for Treatment of Neoplasms and / or Dysplasias

[0120] At least one example embodiment relates to compositions for treatment of neoplasms and / or dysplasias in a mammalian subject. The compositions are suitable for use in photodynamic therapy (PDT), boron neutron capture therapy (BNCT), and / or cell / tissue imaging. The compositions include a salt. The salt is configured to facilitate targeted cell uptake and death of the neoplasms and / or dysplasias when subjected to an external stimulus including light, neutrons, or a combination thereof.

[0121] In at least one example embodiment, the neoplasms and / or dysplasias are tumors, such as cancer. In at least one example embodiment, the compositions are suitable for treatment of brain cancer, head and / or neck cancer, lung cancer, breast cancer, gastrointestinal cancer, pancreatic cancer, prostate cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, bladder cancer, renal cancer, soft tissue sarcoma, thyroid cancer, endometrial cancer, or any combination thereof. In at least one example embodiment, PDT and BNCT may be used to treat cancers as shown in Table 1 below.Table 1

[0122] In at least one example embodiment, the neoplasms and / or dysplasias are in a tumor. The salt may be configured to accumulate inside the tumor while an exotumor residue (i.e., the remaining salt not uptaken in the tumor) is excreted from the mammalian subject. That is, the exo-tumor residue is cleared from a body of the mammalian subject as the salt accumulates in the tumor. In at least one example embodiment, the exo-tumor residue is configured to be excreted from the mammalian subject by a liver of the mammalian subject, a kidney of the mammalian subject, a spleen of the mammalian subject, or any combination thereof. This clearance can occur over a time period of hours, days, or several weeks. In at least one example embodiment, greater than or equal to about 50% by mass of the exo-tumor residue (e.g., greater than or equal to about 55%, greater than or equal to about 60%, greater than or equal to about 65%, greater than or equal to about 70%, greater than or equal to about 75%, greaterthan or equal to about 80%, greater than or equal to about 85%, or greater than or equal to about 90%) is cleared from the body of the mammalian subject after a time period. The period of time may be greater than or equal to about 5 hours (e.g., greater than or equal to about 10 hours, greater than or equal to about 20 hours, greater than or equal to about 40 hours, greater than or equal to about 100 hours, greater than or equal to about 150 hours, greater than or equal to about 200 hours, greater than or equal to about 400 hours, greater than or equal to about 1000 hours). The time period may be less than or equal to about 2000 hours (e.g., less than or equal to about 1000 hours, less than or equal to about 400 hours, less than or equal to about 200 hours, less than or equal to about 150 hours, less than or equal to about 100 hours, less than or equal to about 40 hours, less than or equal to about 20 hours, or less than or equal to about 10 hours).Salt Characteristics

[0123] In at least one example embodiment, the salt is an ion-counterion pair (a “pair”; FIG. 1 A), or an aggregate or nanoparticle (at least two pairs; FIG. 1 B). In at least one example embodiment, the photoactive ion is a cation and the counterion is an anion. In at least one other example embodiment, the photoactive ion is an anion and the counterion is a cation. In at least one example embodiment, the salt is in the form of a pair, an aggregate or a nanoparticle when it is located in a cell (i.e., in cytosol), in an extracellular environment or matrix, or in a cell culture medium. The salt is configured to become activated or excited in response to an external stimulus (e.g., light and / or neutrons), as shown in FIG. 1 C and discussed in greater detail below.

[0124] In at least one example embodiment, the salt is in the form of a nanoparticle having an average diameter of greater than or equal to about 5 nm (e.g., greater than or equal to about 6 nm, greater than or equal to about 8 nm, greater than or equal to about 10 nm, greater than or equal to about 12 nm, greater than or equal to about 14 nm, greater than or equal to about 15 nm, greater than or equal to about 16 nm, greater than or equal to about 18 nm, greater than or equal to about 20 nm, greater than or equal to about 22 nm, greater than or equal to about 24 nm, greater than or equal to about 25 nm, greater than or equal to about 26 nm, greater than or equal to about 28 nm, greater than or equal to about 30 nm, greater than or equal to about 32 nm, greater than or equal to about 34 nm, greater than or equal to about 35 nm, greater than or equal to about 36 nm, greater than or equal to about 38 nm, greater than or equal to about 40 nm, greater than or equal to about 45 nm, greater than or equal to about 50 nm, or greater than or equalto about 55 nm). The average diameter of the salt nanoparticle may be less than or equal to about 60 nm (e.g., less than or equal to about 55 nm, less than or equal to about 50 nm, less than or equal to about 45 nm, less than or equal to about 40 nm, less than or equal to about 38 nm, less than or equal to about 36 nm, less than or equal to about 35 nm, less than or equal to about 34 nm, less than or equal to about 32 nm, less than or equal to about 30 nm, less than or equal to about 28 nm, less than or equal to about 26 nm, less than or equal to about 25 nm, less than or equal to about 24 nm, less than or equal to about 22 nm, less than or equal to about 20 nm, less than or equal to about 18 nm, less than or equal to about 16 nm, less than or equal to about 15 nm, less than or equal to about 14 nm, less than or equal to about 12 nm, less than or equal to about 10 nm, less than or equal to about 8 nm, or less than or equal to about 6 nm).

[0125] In at least one example embodiment, the salt has a bandgap of greater than or equal to about 1 eV (e.g., greater than or equal to about 1 .05 eV, greater than or equal to about 1.1 eV, greater than or equal to about 1.15 eV, greater than or equal to about 1 .2 eV, greater than or equal to about 1 .25 eV, greater than or equal to about 1 .3 eV, greater than or equal to about 1 .35 eV, greater than or equal to about 1 .4 eV, greater than or equal to about 1 .45 eV, greater than or equal to about 1 .5 eV, greater than or equal to about 1 .55 eV, greater than or equal to about 1 .6 eV, greater than or equal to about 1 .65 eV, or greater than or equal to about 1 .7 eV). The bandgap of the salt may be less than or equal to about 1 .75 eV (e.g., less than or equal to about 1 .7 eV, less than or equal to about 1 .65 eV, less than or equal to about 1 .6 eV, less than or equal to about 1 .55 eV, less than or equal to about 1 .5 eV, less than or equal to about 1 .45 eV, less than or equal to about 1 .4 eV, less than or equal to about 1 .35 eV, less than or equal to about 1 .3 eV, less than or equal to about 1 .25 eV, less than or equal to about 1 .2 eV, less than or equal to about 1 .15 eV, less than or equal to about 1 .1 eV, or less than or equal to about 1 .05 eV).

[0126] As used herein, “zeta potential” means the electrical potential at the slipping plane of a nanoparticle in suspension that is a measure of the electrostatic attraction / repulsion of the particles. In at least one example embodiment, the salt has a zeta potential of greater than or equal to about -30 millivolts (mV) (e.g., greater than or equal to about -27.5 mV, greater than or equal to about -25 mV, greater than or equal to about -22.5 mV, greater than or equal to about -20 mV, greater than or equal to about - 17.5 mV, greater than or equal to about -15 mV, or greater than or equal to about -12.5 mV). The zeta potential of the salt may be less than or equal to about -10 mV (e.g., lessthan or equal to about -12.5 mV, less than or equal to about -15 mV, less than or equal to about -17.5 mV, less than or equal to about -20 mV, less than or equal to about -22.5 mV, less than or equal to about -25 mV, or less than or equal to about 27.5 mV).

[0127] In at least one example embodiment, the salt is insoluble in water. Thus, the salt is insoluble when it is located within a cell or organelle or in an extracellular environment or matrix, such as in a mammalian subject.Ion

[0128] In at least one example embodiment, the ion (e.g., cation) is a photoactive ion. The ion may be organic or inorganic. The ion may be photoactive or non-photoactive. In at least one example embodiment, the ion is a cation (e.g., having a charge of +1 , +2, +3, or +4). In at least one other example embodiment, the ion is an anion (e.g., having a charge of -1 , -2, -3, or -4). In at least one example embodiment, the ion is a photoactive ion including a cyanine, a polymethine, or a combination thereof. In at least one example embodiment, the ion is an organic photoactive cyanine cation having the structure shown in FIG. 2A. In at least one example embodiment, the salt is an “inner” salt (also known as a zwitterion) so that the cation (e.g., having a charge of +1 , +2, +3, or +4) and anion (e.g., having a charge of -1 , -2, -3, or -4) are chemically linked and the overall molecule is neutral with a net charge of zero.

[0129] In at least one example embodiment, the photoactive ion is a cationic or anionic form of a photoactive molecule selected from the group consisting of: porphyrins, rhodamines, cyanines, polymethines, heptamethines, phthalocyanines, squaraines, perylenes, quinines, xanthenes, naphthalenes, coumarins, oxadiazoles, oxazines, acridines, arylmethines, tetrapyrroles, indocarbocyanines, oxacarbocyanines, thiacarbocyanines, merocyanines, porfimers, derivatives thereof, oligomers thereof, or any combination thereof. As used herein, “derivatives” of any of the ions described herein refer to or include ions that resemble a base ion, but that contain minor changes, variations, or substitutions, such as in, for example, solubilizing groups with varying alkyl chain lengths or substitution with other solubilizing groups, which do not substantially change the bandgap or electronic properties of the organic ion, as well as substitutions at a central methane position with various halides, halogens, and / or ligands. As used herein, a “substantial change” to the bandgap or electronic properties is a change of greater than about 5% or greater than about 10%. In regard to oligomers, in certain aspects of the current technology, the photoactive ion is an oligomer of one photoactivemolecule or an oligomer of a plurality of photoactive molecules, formed by ether and ester linkages. Further, the oligomer can be a mixture of oligomers.

[0130] In at least one example embodiment, the photoactive ion includes 1 -Butyl- 2-(2-[3-[2-( 1 -butyl- 1 H-benzo[cd]indol-2-ylidene)-ethylidene]-2-phenyl-cyclopent-1 -enyl]- vinyl)-benzo[cd]indolium, 1 -Butyl-2-(2-[3-[2-( 1 -butyl-1 H-benzo[cd]indol-2-ylidene)- ethylidene]-2-chloro-cyclohex-1 -enyl]-vinyl)-benzo[cd]indolium , 1 -Butyl-2- (2-[3-[2- (1 - butyl-1 H-benzo[cd]indol-2-ylidene)-ethylidene]-2-phenyl-cyclohex-1 -enyl]-vinyl)- benzo[cd]indolium, 1 -Butyl-2- (2-[3-[2- (1 -butyl-1 H-benzo[cd]indol-2-ylidene)-ethylidene]- 2-diphenylamino-cyclopent-1 -enyl]-vinyl)-benzo[cd]indolium, 1 -Butyl-2-[7- (1 -butyl-1 H- benzo[cd]indol-2-ylidene)-hepta-1 ,3,5-trienyl]-benzo[cd]indolium, 2-[2-[2-chloro-3-[2- (1 ,3-dihydro-3,3-dimethyl-1 -ethyl-2H-benz[e]indol-2-ylidene)ethylidene]-1 -cylohexen-1 - yl]-ethenyl]-3,3-dimethyl- 1 -ethyl- 1 H-benz[e]indolium (“Cy”), N,N,N',N'-Tetrakis-(p-di-n- butylaminophenyl)-p-benzochinon-bis-immonium, 4-[2-[2-Chloro-3-[(2,6-diphenyl-4H- thiopyran-4-ylidene)ethylidene]-1 -cyclohexen-1 -yl]ethenyl]-2 ,6-diphenylthiopyryliu m , 1 - Butyl-2-[2-[3-[(1 -butyl-6-chlorobenz[cd]indol-2(1 H)-ylidene)ethylidene]-2-chloro-5- methyl-1 -cyclohexen-1 -yl]ethenyl]-6-chlorobenz[cd]indolium, 1 - Butyl-2-[2-[3-[( 1 -butyl-6- chlorobenz[cd]indol-2(1 H)-ylidene)ethylidene]-2-chloro-1 -cyclohexen-1 -yl]ethenyl]-6- chlorobenz[cd]indolium, Dimethyl{4-[1 ,7,7-tris(4-dimethylaminophenyl)-2,4,6- heptatrienylidene]-2,5-cyclohexadien-1 -ylidenejammonium, 5,5'-Dichloro-1 1 - diphenylamino-3,3'-diethyl-10,12-ethylenethiatricarbocyanine, 2-[2-[2-Chloro-3-[2-( 1 ,3- dihydro-1 ,1 ,3-trimethyl-2H-benzo[e]-indol-2-ylidene)-ethylidene]-1 -cyclohexen-1 -yl]- ethenyl]- 1 ,1 ,3-trimethyl- 1 H-benzo[e]indolium, 2-[2-[2-Chloro-3-[2-(1 ,3-dihydro-1 ,3,3- trimethyl-2H-indol-2-ylidene)-ethylidene]-1 -cyclopenten-1 -yl]-ethenyl]- 1 ,3,3-trimethyl- 3H-indolium, 2-[2-[3-[(1 ,3-Dihydro-3,3-dimethyl-1 -propyl-2H-indol-2-ylidene)ethylidene]- 2-(phenylthio)-1 -cyclohexen-1 -yl]ethenyl]-3,3-dimethyl- 1 -propylindolium, 1 ,1 ',3 ,3 ,3',3'-4,4',5,5'-di-benzo-2,2'-indotricarbocyanine perchlorate, 2-[2-[2-Chloro-3-[2-( 1 ,3-dihydro- 1 ,3,3-trimethyl-2H-indol-2-ylidene)-ethylidene]-1 -cyclohexen-1 -yl]-ethenyl]-1 ,3,3- trimethyl-3H-indolium, 3,3'-Diethylthiatricarbocyanine, 2-[[2-[2-[4-(dimethylamino)phenyl]ethenyl]-6-methyl-4H-pyran-4-ylidene]methyl]-3-ethyl, 2-[7-(1 ,3- Dihydro-1 ,3,3-trimethyl-2H-indol-2-ylidene)-1 ,3,5-heptatrienyl]- 1 ,3,3-trimethyl-3H- indolium, 2-[2-[2-Chloro-3-[[1 ,3-dihydro-1 ,1 -dimethyl-3-(4-sulfobutyl)-2H-benzo[e]indol- 2-ylidene]-ethylidene]-1 -cyclohexen-1 -yl]-ethenyl]-1 ,1 -dimethyl-3-(4-sulfobutyl)-1 H- benzo[e]indolium hydroxide, 2-[7-[1 ,3-Dihydro-1 ,1 -dimethyl-3-(4-sulfobutyl)-2H- benz[e]indol-2-ylidene]-1 ,3,5-heptatrien-1 -yl]-1 ,1 -dimethyl-3-(4-sulfobutyl)-1 H-benz[e]indolium, cyanine3 (Cy3), cyanine3.5 (Cy3.5), cyanine5 (Cy5), cyanine5.5 (Cy5.5), cyanine? (Cy7), cyanine?.5 (Cy7.5), derivatives thereof, and combinations thereof.

[0131] The photoactive ion is a photoactive organic cation or a photoactive organic anion. In at least one example embodiment, a photoactive cation can be converted into a corresponding photoactive anion and a photoactive anion be converted into a corresponding photoactive cation. For example, a photoactive cation can be converted into a photoactive anion by adding groups to the photoactive cation that have a negative charge. Examples of groups having a negative charge include sulfate (SC2), sulfonate (SO3 ), nitrate (NO3 ), nitrite (NO2 ), phosphate (PO43), and phosphonate (PO3 ). Similarly, a photoactive anion can be converted into a photoactive cation by adding groups to the photoactive anion that have a positive charge. Examples of groups having a positive charge include ammonium (NH4+), quarternized ammonium (F N+), carbenium (R3C+), carbonium (RsC+), arsonium (R4As+), phosphonium (R4P+), stibonium (R4Sb+), and bismuthonium (R4BF) where R is any group (same or different), cyclic or not.Counterion

[0132] The counterion (e.g., anion) which modulates dark and light cytotoxicity of the photoactive ion, may be a weakly coordinating counterion or a non-coordinating counterion. As used herein, a “weakly coordinating counterion” and “non-coordinating counterion” are counterions that weakly interact with ions and metal centers, and that can reduce or eliminate ligand coordination, oxidation, and abstraction. Weakly coordinating ions can replace strong electrostatic interactions by a variety of weak interactions via greater charge delocalization. Such weak interactions are often influenced by coverage with poorly polarizable halogen atoms, such as, for example, fluorine, that can also increase thermodynamic stability. Bulky and weakly coordinating ions do not include PnXe", X , XO4-, XO3 , XO2 , or XO , where X is a halogen and Pn is a pnictogen, which may be considered strongly coordinating ions. Examples of counterions that are not suitable include the anions F-, Cl , Br, I , BF4-, PFe, SbFe", AsFe", SOsF-, and CIO4-; and the cations Li+, LiH+, BeH+, GeH3+, BeO+, Na+, K+, Rb+, Cs+, Be2+, Mg2+, Ca2+, Sr2+, Ba2+.

[0133] In at least one example embodiment, the counterion (e.g., anion) has a volume averaged spherical radius of greater than or equal to about 0.25 nm (e.g., greaterthan or equal to about 0.275 nm, greater than or equal to about 0.3 nm, greater than or equal to about 0.325 nm, greater than or equal to about 0.35 nm, or greater than or equal to about 0.375 nm). The volume averaged spherical radius of the counterion may be less than or equal to about 0.4 nm (e.g., less than or equal to about 0.375 nm, less than or equal to about 0.35 nm, less than or equal to about 0.325 nm, less than or equal to about 0.3 nm, or less than or equal to about 0.275 nm).

[0134] In at least one example embodiment, the counterion is a carborane, a borohydride cluster, or a combination thereof. In at least one example embodiment, the carborane is a transition metal free carborane. In at least one example embodiment, the counterion includes one or more halogens. In at least one example embodiment, the counterion is an anion. The anion may have a net charge of less than or equal to -1 . In at least one example embodiment, the anion has a net charge of -1. In at least one example embodiment, the anion has a net charge of -2 such that it is a dianion.

[0135] In at least one example embodiment, the anion is a carborane having the formula CBgRi(R2)4(R3)4R4[N- or N+land the structure shown in FIG. 2B. N is an integer (e.g., ranging from 1-10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10). R-i, R2, R3, and R4 are independently selected from the group consisting of: H, F, Cl, Br, I, alkyl, functionalized alkyl, aryl, functionalized aryl, OH, O-alkyl, O-aryl, NH2, NR2, NR3, SH, SR, SR2, haloalkyl, perfluoroaryl, acetyl, ester, ether, formyl, amide, formamide, carboxyl, alkanoate, carbamate, ammonium, quarternized ammonium, phosphonium, stibonium, bismuthonium, NR2, NR3, SR, and SR2, where R is any functional group. In at least one example embodiment, R1, R2, R3, and R4 are independently selected from the group consisting of: R1, R2, R3, and R4 are independently selected from the group consisting of: H, F, Cl, Br, I, alkyl, functionalized alkyl, aryl, functionalized aryl, OH, O-alkyl, O-aryl, NH2, NR2, NR3, SH, SR, SR2, haloalkyl, perfluoroaryl, acetyl, ester, ether, formyl, amide, formamide, carboxyl, alkanoate, and carbamate, where R is any functional group, and a salt including the carborane has the formula [CBgRi(R2)4(R3)4R4N][NC+], where C+is a cation. In at least one other example embodiment, R1, R2, R3, and R4 are independently selected from the group consisting of: ammonium, quarternized ammonium, phosphonium, stibonium, bismuthonium, NR2, NR3, SR, SR2, functionalized aryl, and functionalized alkyl, where R is any functional group, and a salt including the carborane has the formula [CBgRi(R2)4(R3)4R4N+][NA ], where A- is an anion.

[0136] In at least one example embodiment, the anion is a carborane having the formula CBH RI (R2)5(R3)5R4[N- or N+land the structure shown in FIG. 2C. N is an integer(e.g., ranging from 1-10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10). R-i, R2, R3, and R4 are independently selected from the group consisting of: H, F, Cl, Br, I, alkyl, functionalized alkyl, aryl, functionalized aryl, OH, O-alkyl, O-aryl, NH2, N(RI)2, N(RI)3, SH, SR1, S(RI)2, haloalkyl, perfluoroaryl, acetyl, ester, ether, formyl, amide, formamide, carboxyl, alkanoate, carbamate, ammonium, quarternized ammonium, phosphonium, stibonium, bismuthonium, NR2, NR3, SR, and SR2, where R is any functional group. In at least one example embodiment, R1, R2, R3, and R4 are independently selected from the group consisting of: H, F, Cl, Br, I, alkyl, aryl, OH, O-alkyl, O-aryl, NH2, NR2, SH, SR, haloalkyl, perfluoroaryl, acetyl, ester, ether, formyl, amide, formamide, carboxyl, alkanoate, and carbamate, and a salt including the carborane has the formula [CBH RI(R2)5(R3)5R4N-][NC+], where C+is a cation. In at least one other example embodiment, R1, R2, R3, and R4 are independently selected from the group consisting of: ammonium, quarternized ammonium, phosphonium, stibonium, bismuthonium, NR2, NR3, SR, SR2, functionalized aryl, and functionalized alkyl, wherein R is any functional group, and a salt including the carborane has the formula [CBH RI(R2)5(R3)5R4N+][NA ], where A- is an anion.

[0137] In at least one example embodiment the anion is a borohydride cluster having the formula BioRio[N or N+land the structure shown in FIG. 2D. N is an integer (e.g., ranging from 1-10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10). R is selected from the group consisting of: H, F, Cl, Br, I, alkyl, functionalized alkyl, aryl, functionalized aryl, OH, O- alkyl, O-aryl, NH2, N(RI)2, N(RI)3, SH, SR1, S(RI)2, haloalkyl, perfluoroaryl, acetyl, ester, ether, formyl, amide, formamide, carboxyl, alkanoate, carbamate, ammonium, quarternized ammonium, phosphonium, stibonium, bismuthonium, N(RI)2, N(RI)3, SR1, and S(RI)2, where R1 is any functional group. In at least one example embodiment, as shown in FIG. 2D, all of the R groups are the same. In at least one other example embodiment, at least a portion of the R groups are different from other R groups. In at least one example embodiment, R is selected from the group consisting of: H, F, Cl, Br, I, alkyl, functionalized alkyl, aryl, functionalized aryl, OH, O-alkyl, O-aryl, NH2, N(RI)2, N(RI)3, SH, SR1, S(RI)2, haloalkyl, perfluoroaryl, acetyl, ester, ether, formyl, amide, formamide, carboxyl, alkanoate, and carbamate, wherein R1 is any functional group, and a salt including the borohydride cluster has the formula [BioRioN][NC+], where C+is a cation. In at least one other example embodiment, R is selected from the group consisting of: ammonium, quarternized ammonium, phosphonium, stibonium, bismuthonium, N(RI)2, N(RI)3, SR1, S(RI)2, functionalized aryl, and functionalized alkyl wherein R1 is anyfunctional group, and a salt including the borohydride cluster has the formula [BIORION+][NA ], where A- is an anion.

[0138] In at least one example embodiment, the anion is a borohydride cluster having the formula Bi2Ri2[N or N+land the structure shown in FIG. 2E. N is an integer (e.g., ranging from 1-10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10). R is selected from the group consisting of H, F, Cl, Br, I, alkyl, functionalized alkyl, aryl, functionalized aryl, OH, O- alkyl, O-aryl, NH2, N(RI)2, N(Ri)s, SH, SR1, S(RI)2, haloalkyl, perfluoroaryl, acetyl, ester, ether, formyl, amide, formamide, carboxyl, alkanoate, carbamate, ammonium, quarternized ammonium, phosphonium, stibonium, bismuthonium, N(RI)2, N(Ri)s, SR1, and S(RI)2, where R1 is any functional group. In at least one example embodiment, as shown in FIG. 2E, all of the R groups are the same. In at least one other example embodiment, at least a portion of the R groups are different from other R groups. In at least one example embodiment, R is selected from the group consisting of: H, F, Cl, Br, I, alkyl, functionalized alkyl, aryl, functionalized aryl, OH, O-alkyl, O-aryl, NH2, N(RI)2, N(RI)3, SH, SR1, S(RI)2, haloalkyl, perfluoroaryl, acetyl, ester, ether, formyl, amide, formamide, carboxyl, alkanoate, and carbamate, where R1 is any functional group, and a salt including the borohydride cluster has the formula [Bi2Ri2N][NC+], where C+is a cation. In at least one other example embodiment, R is selected from the group consisting of: ammonium, quarternized ammonium, phosphonium, stibonium, bismuthonium, N(RI)2, N(RI)3, SR1, S(RI)2, functionalized aryl, and functionalized alkyl, wherein R1 is any functional group, and a salt including the borohydride cluster has the formula [BI2RI2N+][NA ], where A- is an anion.

[0139] In at least one example embodiment, the anion is a carborane having the formula CB9H10" and the structure shown in FIG. 2F. In at least one example embodiment, the anion is a carborane having the formula CBuHeCle" and the structure shown in FIG. 2G. In at least one example embodiment, the anion is a carborane having the formula CB9HCI9" and the structure shown in FIG. 2H. In at least one example embodiment, the anion is a carborane having the formula CioBnH32Si" and the structure shown in FIG. 2I. In at least one example embodiment, the anion is a carborane having the formula C5B22N2H25" and the structure shown in FIG. 2J.

[0140] In embodiments where the ion (e.g., photoactive ion) is a cation, the counterion is an anion. In embodiments where the ion (e.g., photoactive ion) is an anion, the counterion is a cation. Similar to the ion, a cationic counterion can be converted into a corresponding anionic counterion and an anionic counterion can be converted into acorresponding cationic counterion. For example, a cationic counterion can be converted into an anionic cation by adding groups to the photoactive cation that have a negative charge. Examples of groups having a negative charge include sulfate (SO42), sulfonate (SO3 ), nitrate (NO3 ), nitrite (NO2 ), phosphate (PO43), and phosphonate (PO3 ). Similarly, an anionic counterion can be converted into a cationic counterion by adding groups to the photoactive anion that have a positive charge. Examples of groups having a positive charge include ammonium (NH4+), quarternized ammonium (F N+), phosphonium (R4P+), carbenium (R3O), carbonium (RsC+), arsonium (R4As+), stibonium (R4Sb+), and bismuthonium (R4BF) where R is any group (same or different), cyclic or not.

[0141] In at least one example embodiment, the counterion is non-spherical. By “non-spherical,” it is meant that the counterion is either non-elemental or not of the form AX4-, AXe where A is, for example, B, P, S, Sb, or Cl, and X is, for example, F, Cl, Br, I, H, or O.

[0142] In at least one example embodiment the counterion (e.g., anion) has a molecular weight of greater than or equal to about 100 g / mol (e.g., greater than or equal to about 125 g / mol, greater than or equal to about 150 g / mol, greater than or equal to about 175 g / mol, greater than or equal to about 200 g / mol, greater than or equal to about 225 g / mol, greater than or equal to about 250 g / mol, greater than or equal to about 275 g / mol, greater than or equal to about 300 g / mol, greater than or equal to about 325 g / mol, greater than or equal to about 350 g / mol, greater than or equal to about 400 g / mol, greater than or equal to about 425 g / mol, greater than or equal to about 450 g / mol, greater than or equal to about 475 g / mol, greater than or equal to about 500 g / mol, greater than or equal to about 600 g / mol, greater than or equal to about 700 g / mol, greater than or equal to about 800 g / mol, greater than or equal to about 900 g / mol, greater than or equal to about 1000 g / mol, greater than or equal to about 1 100 g / mol, greater than or equal to about 1200 g / mol, greater than or equal to about 1300 g / mol, greater than or equal to about 1400 g / mol, greater than or equal to about 1500 g / mol, greater than or equal to about 1750 g / mol, greater than or equal to about 2000 g / mol, greater than or equal to about 2250 g / mol, greater than or equal to about 2500 g / mol, or greater than or equal to about 2750 g / mol). The molecular weight of the counterion may be less than or equal to about 3000 g / mol (e.g., greater than or equal to about 2750 g / mol, greater than or equal to about 2500 g / mol, greater than or equal to about 2250 g / mol, greater than or equal to about 2000 g / mol, greater than or equal to about 1750g / mol, greater than or equal to about 1500 g / mol, greater than or equal to about 1400 g / mol, greater than or equal to about 1300 g / mol, greater than or equal to about 1200 g / mol, greater than or equal to about 1 100 g / mol, greater than or equal to about 1000 g / mol, greater than or equal to about 900 g / mol, greater than or equal to about 800 g / mol, greater than or equal to about 700 g / mol, greater than or equal to about 600 g / mol greater than or equal to about 500 g / mol, greater than or equal to about 475 g / mol greater than or equal to about 450 g / mol, greater than or equal to about 425 g / mol greater than or equal to about 400 g / mol, greater than or equal to about 375 g / mol greater than or equal to about 350 g / mol, greater than or equal to about 325 g / mol greater than or equal to about 300 g / mol, greater than or equal to about 275 g / mol greater than or equal to about 250 g / mol, greater than or equal to about 225 g / mol greater than or equal to about 200 g / mol, greater than or equal to about 175 g / mol greater than or equal to about 150 g / mol, or greater than or equal to about 125 g / mol).

[0143] In at least one example embodiment, the counterion (e.g., anion) includes a quantity of atoms that is greater than or equal to about 20 (e.g., greater than or equal to about 22, greater than or equal to about 24, greater than or equal to about 26, greater than or equal to about 28, greater than or equal to about 30, greater than or equal to about 32, greater than or equal to about 34, greater than or equal to about 36, greater than or equal to about 38, greater than or equal to about 40, greater than or equal to about 42, greater than or equal to about 44, greater than or equal to about 46, greater than or equal to about 48, greater than or equal to about 50, greater than or equal to about 52, greater than or equal to about 54, greater than or equal to about 56, greater than or equal to about 58, greater than or equal to about 60, greater than or equal to about 70, greater than or equal to about 80, greater than or equal to about 100, greater than or equal to about 150, greater than or equal to about 200, greater than or equal to about 250, greater than or equal to about 300, greater than or equal to about 350, greater than or equal to about 400, greater than or equal to about 450, greater than or equal to about 500, greater than or equal to about 550, greater than or equal to about 600, greater than or equal to about 650, greater than or equal to about 700, greater than or equal to about 750, greater than or equal to about 800, greater than or equal to about 850, greater than or equal to about 900, or greater than or equal to about 950). The quantity of atoms in the counterion may be less than or equal to about 1000 (e.g., less than or equal to about 900, less than or equal to about 800, less than or equal to about 700, less than or equal to about 600, less than or equal to about 500, less than or equal to about 400, lessthan or equal to about 300, less than or equal to about 200, less than or equal to about 100, less than or equal to about 80, less than or equal to about 70, less than or equal to about 60, less than or equal to about 58, less than or equal to about 56, less than or equal to about 54, less than or equal to about 52, less than or equal to about 50, less than or equal to about 48, less than or equal to about 46, less than or equal to about 44, less than or equal to about 42, less than or equal to about 40, less than or equal to about 38, less than or equal to about 36, less than or equal to about 34, less than or equal to about 32, less than or equal to about 30, less than or equal to about 28, less than or equal to about 26, less than or equal to about 24, or less than or equal to about 22).Counterion Substitution

[0144] Whether a counterion will make a salt suitable for PDT, combined PDT and BNCT, or imaging (or both as a theranostic) depends on its absolute highest occupied molecular orbital (HOMO) energy and its lowest unoccupied molecular orbital (LUMO) energy. In at least one example embodiment, the salt has an absolute HOMO energy of greater than or equal to about 4 eV (e.g., greater than or equal to about 4.1 eV, greater than or equal to about 4.2 eV, greater than or equal to about 4.3 eV, greater than or equal to about 4.4 eV, greater than or equal to about 4.5 eV, greater than or equal to about 4.6 eV, greater than or equal to about 4.7 eV, greater than or equal to about 4.8 eV, greater than or equal to about 4.9 eV, greater than or equal to about 5 eV, greater than or equal to about 5.1 eV, greater than or equal to about 5.2 eV, greater than or equal to about 5.3 eV, greater than or equal to about 5.4 eV, greater than or equal to about 5.5 eV, greater than or equal to about 5.6 eV, greater than or equal to about 5.7 eV, greater than or equal to about 5.8 eV, or greater than or equal to about 5.9 eV). The absolute HOMO energy of the salt may be less than or equal to about 6 eV (e.g., less than or equal to about 5.9 eV, less than or equal to about 5.8 eV, less than or equal to about 5.7 eV, less than or equal to about 5.6 eV, less than or equal to about 5.5 eV, less than or equal to about 5.4 eV, less than or equal to about 5.3 eV, less than or equal to about 5.2 eV, less than or equal to about 5.1 eV, less than or equal to about 5 eV, less than or equal to about 4.9 eV, less than or equal to about 4.8 eV, less than or equal to about 4.7 eV, less than or equal to about 4.6 eV, less than or equal to about 4.5 eV, less than or equal to about 4.4 eV, less than or equal to about 4.3 eV, less than or equal to about 4.2 eV, or less than or equal to about 4.1 eV). In at least one example embodiment, the salt has an absolute LUMO energy of greater than or equal to about 3.5 eV (e.g., greater than or equal toabout 3.6 eV, greater than or equal to about 3.7 eV, greater than or equal to about 3.8 eV, greater than or equal to about 3.9 eV, greater than or equal to about 4 eV, greater than or equal to about 4.1 eV, greater than or equal to about 4.2 eV, greater than or equal to about 4.3 eV, or greater than or equal to about 4.4 eV). The absolute LUMO energy of the salt may be less than or equal to about 4.5 eV (e.g., less than or equal to about 4.4 eV, less than or equal to about 4.3 eV, less than or equal to about 4.2 eV, less than or equal to about 4.1 eV, less than or equal to about 4 eV, less than or equal to about 3.9 eV, less than or equal to about 3.8 eV, less than or equal to about 3.7 eV, or less than or equal to about 3.6 eV).

[0145] “Cytotoxicity” refers a compound’s (e.g., an ion’s or salt’s) quality of being toxic to cells, regardless of presence or absence of an external stimulus (e.g., light or neutrons). “Dark cytotoxicity” refers to toxicity of the salt to cells when it is not an excited state, i.e., in the absence of external stimulus that excites an electron from a ground state to an excited state (“in the dark”) or excited by neutron capture. “Cytotoxicity under external stimulus” is cellular toxicity that is induced from the salt when it becomes “excited” by light or neutrons. Similarly, “phototoxicity” refers to toxicity that results from a salt becoming excited due to the presence of illumination, wherein the salt is not toxic to cells in the dark. For example, a salt that is phototoxic is not toxic to cells in the dark, but is toxic to cells in the light.

[0146] “Activated” salts are salts that are excited and the excitation causes cellular toxicity. Accordingly, salts that are toxic to cells in the dark can become “deactivated,” i.e., made to be non-toxic to cells in the dark, by substituting the counterion with a second counterion that renders the salt nontoxic in the dark only (i.e. , phototoxic) or non-toxic in both the dark and under external stimulus (e.g., light).

[0147] The cellular toxicity of the salt can be described by a half maximal inhibitory concentration (IC50), where the IC50 is concentration of the salt at which 50% of a cell population dies. The term “dark IC50” refers to the IC50 of a salt in the dark. The Term “IC50 under external stimulus” refers to the IC50 under external stimulus, such as light or neutrons. The term “light IC50” refers to the IC50 of a salt in the light.

[0148] In at least one example embodiment, a dark ICso of the salt (i.e., in a cell) is greater than or equal to about 1 pM (e.g., greater than or equal to about 1 .2 pM, greater than or equal to about 1 .4 pM, greater than or equal to about 1 .6 pM, greater than or equal to about 1 .8 pM, greater than or equal to about 2 pM, greater than or equal to about 2.2 pM, greater than or equal to about 2.4 pM, greater than or equal to about 2.5 pM,greater than or equal to about 2.6 pM, greater than or equal to about 2.8 pM, greater than or equal to about 3 pM, greater than or equal to about 3.2 pM, greater than or equal to about 3.4 pM, greater than or equal to about 3.5 pM, greater than or equal to about 3.6 pM, greater than or equal to about 3.8 pM, greater than or equal to about 4 pM, greater than or equal to about 4.5 pM, greater than or equal to about 5 pM, greater than or equal to about 6 pM, greater than or equal to about 7 pM, greater than or equal to about 8 pM, greater than or equal to about 9 pM, greater than or equal to about 10 pM, greater than or equal to about 15 pM, or greater than or equal to about 20 pM). The dark IC50 of the salt is less than or equal to about 25 pM (e.g., less than or equal to about 20 pM, less than or equal to about 15 pM, less than or equal to about 10 pM, less than or equal to about 9 pM, less than or equal to about 8 pM, less than or equal to about 7 pM, less than or equal to about 6 pM, less than or equal to about 5 pM, less than or equal to about 4.5 pM, less than or equal to about 4 pM, less than or equal to about 3.8 pM, less than or equal to about 3.6 pM, less than or equal to about 3.5 pM, less than or equal to about3.4 pM, less than or equal to about 3.2 pM, less than or equal to about 3 pM, less than or equal to about 2.8 pM, less than or equal to about 2.6 pM, less than or equal to about2.5 pM, less than or equal to about 2.4 pM, less than or equal to about 2.2 pM, less than or equal to about 2 pM, less than or equal to about 1 .8 pM, less than or equal to about1 .6 pM, less than or equal to about 1 .4 pM, or less than or equal to about 1 .2 pM).

[0149] In at least one example embodiment, an IC50 under external stimulus (e.g., light, neutrons) of the salt (i.e., in a cell) is greater than or equal to about 1 pM (e.g., greater than or equal to about 1 .2 pM, greater than or equal to about 1 .4 pM, greater than or equal to about 1 .5 pM, greater than or equal to about 1 .6 pM, greater than or equal to about 1 .8 pM, greater than or equal to about 2 pM, greater than or equal to about 2.2 pM, greater than or equal to about 2.4 pM, greater than or equal to about 2.5 pM, greater than or equal to about 2.6 pM, greater than or equal to about 2.8 pM, greater than or equal to about 3 pM, greater than or equal to about 3.5 pM, greater than or equal to about 4 pM, greater than or equal to about 4.5 pM, or greater than or equal to about 5 pM). The IC50 under external stimulus of the salt is less than or equal to about 10 pM (e.g., less than or equal to about 5 pM, less than or equal to about 4.5 pM, less than or equal to about 4 pM, less than or equal to about 3.5 pM, less than or equal to about 3 pM, less than or equal to about 2.8 pM, less than or equal to about 2.6 pM, less than or equal to about 2.5 pM, less than or equal to about 2.4 pM, less than or equal to about 2.2 pM, less than or equal to about 2 pM, less than or equal to about 1.8 pM, less than or equal to about1 .6 pM, less than or equal to about 1 .5 pM, less than or equal to about 1 .4 pM, or less than or equal to about 1 .2 pM).Compositions for PDT

[0150] In at least one example embodiment, a first composition for PDT includes a first salt. The first salt includes a first photoactive ion and a first counterion. The first photoactive ion is an organic photoactive ion (e.g., an organic photoactive cation), such as any of those described herein. The first counterion is any of the counterions described herein (e.g., the counterions of FIGS. 2B-2J).

[0151] As discussed above, the first ion is a photoactive ion. As used herein, the term “photoactive” refers to a molecule or compound, in particular, an ion, capable of chemical or physical change in response to illumination. In particular, photoactive ions absorb light at particular wavelengths, which excites an electron and induces the chemical or physical change. At the same time, the energy of the excited electron and a hole is modulated by the counterion.

[0152] In at least one example embodiment, the first salt includes the first photoactive ion that absorbs light having a particular wavelength becomes excited. As used herein, “exciting” a salt refers to promoting an electron from a lower-energy occupied orbital to a higher-energy empty orbital resulting in an excited state salt. Accordingly, an “excited salt” is a salt comprising a photoactive ion with at least one electron in an excited state as a result of light absorption. In at least one example embodiment, the first photoactive ion is excited when it absorbs near-infrared (NIR) or infrared (IR) light. In some embodiments, the excited ion luminesces (more specifically, fluoresces), i.e., emits light. As used herein “visible light” is light having a wavelength ranging from about 400 nm to about 700 nm and “IR light” is light having a wavelength of ranging from about 700 nm to about 1.2 mm. As used herein, “IR” light includes nearinfrared (NIR) light.

[0153] In at least one example embodiment, the first salt is configured to become excited upon exposure to NIR light. In at least one example embodiment, the first salt is configured to become excited upon exposure to light having a wavelength of greater than or equal to about 700 nm (e.g., greater than or equal to about 725 nm, greater than or equal to about 750 nm, greater than or equal to about 775 nm, greater than or equal to about 800 nm, greater than or equal to about 825 nm, greater than or equal to about 850 nm, greater than or equal to about 875 nm, greater than or equal to about 900 nm, greaterthan or equal to about 925 nm, greater than or equal to about 950 nm, greater than or equal to about 1000 nm, greater than or equal to about 1050 nm, greater than or equal to about 1 100 nm, or greater than or equal to about 1150 nm). The light may have a wavelength of less than or equal to about 1200 nm (e.g., less than or equal to about 1 150 nm, less than or equal to about 1 100 nm, less than or equal to about 1050 nm, less than or equal to about 1000 nm, less than or equal to about 950 nm, less than or equal to about 925 nm, less than or equal to about 900 nm, less than or equal to about 875 nm, less than or equal to about 850 nm, less than or equal to about 825 nm, less than or equal to about 800 nm, or less than or equal to about 775 nm).

[0154] In at least one example embodiment, the first salt (e.g., the photoactive cation) is configured to generate reactive oxygen species (ROS) upon becoming excited. In at least one example embodiment, the ROS include superoxide, peroxide, singlet oxygen, or any combination thereof. In at least one example embodiment, the ROS include at least two of superoxide, peroxide, and singlet oxygen. In at least one example embodiment, the ROS include singlet oxygen.Compositions for BNCT

[0155] In at least one example embodiment, a second composition for BNCT includes a second salt. The second salt includes a second ion and a second counterion. The second ion (e.g., cation) may be organic or inorganic. The second ion may be any of those described herein or any other suitable ion. The second counterion (e.g., anion) includes boron, as described in herein and in greater detail below. One of the significant challenges hindering BNCT is the ability to achieve both boron uptake and selective boron uptake into cancer cells. Nanoparticle salt formulations enable selective uptake without the need for linkage to antibodies (or other targeting moieties) to target cancer cells.

[0156] The boron in the second counterion may be natural abundance11B / 10B isotopes or enriched up to 99.999%10B. The boron in the second counterion may be a mixed composition of stable B isotopes of10B and11B.10B has the highest neutron capture rate. In at least one example embodiment, the boron in the second counterion is greater than 0%10B (e.g., greater than or equal to about 10%10B, greater than or equal to about 20%10B, greater than or equal to about 30%10B, greater than or equal to about 40%10B, greater than or equal to about 50%10B, greater than or equal to about 60%10B, greater than or equal to about 70%10B, greater than or equal to about 80%10B, greaterthan or equal to about 9010B, or greater than or equal to about 95%10B). In at least one example embodiment with an increased neutron capture radius, the boron in the second counterion is greater than or equal to about 50%10B (e.g., greater than or equal to about 60%10B, greater than or equal to about 70%10B, greater than or equal to about 80%10B, greater than or equal to about 9010B, greater than or equal to about 95%10B, greater than or equal to about 96%10B, greater than or equal to about 97%10B, greater than or equal to about 98%10B, greater than or equal to about 99%10B, greater than or equal to about 99.9%10B, or greater than or equal to about 99.99%10B). In at least one example embodiment, the boron in the second counterion is substantially all10B.

[0157] In at least one example embodiment, the second salt is configured to generate alpha particles and / or high energy lithium 7 nuclei upon becoming excited by an external stimulus including neutrons. Neutrons can have a range of energies range from about 0.5 eV to about 10 keV for suitable depth of penetration. Neutrons with energies of less than about 0.5 eV are less preferable due to low penetration. Upon exposure of the salt to the external stimulus of neutrons,10B forms11B* that decays to form high energy alpha particles and high energy Li7, both of which produce closely spaced ionization that destroy cells.Compositions for Combined PDT and BNCT

[0158] In at least one example embodiment, a single salt, such as the second salt described above, may be used for combined PDT and BNCT. In at least one other example embodiment, a mixture of salts, such as the first salt and the second salt described above, may be used for combined PDT and BNCT.Other Elements of Composition

[0159] In at least one example embodiment, the salt is substantially free of a coating or substantially free of a non-bioresorbable coating when it is located within a cell or organelle or in an extracellular environment or matrix. As used herein, “substantially free” of a coating means that less than or equal to about 50% (e.g., less than or equal to about 40%, less than or equal to about 30%, less than or equal to about 20%, less than or equal to about 15%, less than or equal to about 10%, less than or equal to about 5%, less than or equal to about 2.5%, or less than or equal to about 1 %) of the surface area of the salt is covered with a coating, such that the salt is free to interact with a target cell and / or become endocytosed.

[0160] In at least one example embodiment, the salt is a coated when administered to a subject, but is substantially free of the coating when the salt is in an extracellular environment or matrix or within a cell, i.e., in the cytosol or organelle. In other words, when present during administering to a subject, the coating can be absorbed, digested, or otherwise removed in at least one of the digestive tract, circulation, and intracellular environment of the subject. Accordingly, the coating can be removed before or after the salt enters a cell. In at least one other example embodiment, the salt is administered without a coating.

[0161] In at least one example embodiment, the salt comprises a polymeric or non- polymeric bioresorbable coating. As used herein, a “bioresorbable coating” is a coating that is at least partially bioresorbed in the bloodstream, digestive system, or in a cell after intravenous injection or in the gut after oral administration, such that the salt becomes substantially free of the coating as it interacts with or enters a target cell. Bioresorbable non-polymeric coatings include, as examples, MgF2, calcium phosphate, apatite, calcium carbonate, calcium fluoride, and mixtures thereof. Bioresorbable polymeric coatings comprise, as examples examples, polysaccharides, polylactides (PLA); PLGA (poly(lactide-co-glycolide)); polyanhydrides; polyorthoesters; poly(N-(2-hydroxypropyl) methacrylamide); DLPLA — poly(dl-lactide) ; LPLA — poly(1 -lactide); PGA-polyglycolide; PDO — poly(dioxanone); PGA-TMC — poly(glycolide-co-trimethylene carbonate); PGA- LPLA — poly( 1 -lactide-co-glycolide); PGA-DLPLA — poly(dl-lactide-co-glycolide); LPLA- DLPLA — poly(1 -lactide-co-dl-lactide); and PDO-PGA-TMC -poly(glycolide-co- trimethylene carbonate-co-dioxanone), and combinations, copolymers, and derivatives thereof. In at least one example embodiment, the bioresorbable coating comprises pores having an opening diameter of greater than or equal to about 1 nm to less than or equal to about 1 pm and a porosity, i.e., ratio of void volume to total volume ranging from about 30% to about 75%.

[0162] In at least one example embodiment, the salt is coupled to an adjunct material. The adjunct material is a biomolecule; a sugar, such as a sugar molecule or a polysaccharide; a protein; a peptide; an intracellular organelle localization peptides / signals; an outer surface of a nanoparticle; a lipid carrier, such as an outer surface of a micelle or liposome; a nucleic acid; such as a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA); an antibody (monoclonal or polyclonal), including an antibody fragment (such as, for example Fab, Fab', Fab2, Fab'2, Fd, Fd', scFv, scFv2, dAb, or combinations thereof), and an antibody fragment fusion molecule; a targeting agent, or acombination thereof. Targeting agents include agents that direct the salt to a specific tissue type or cell type. In at least one example embodiment, the targeting agent is an antibody that recognizes a molecule on a specific cell or a ligand that binds to a receptor expressed by a specific cell. Examples of tumor-targeting peptides are provided by Liu et aL, Advanced Drug Delivery Reviews 1 10-1 1 1 (2017) 13-37, which is incorporated herein by reference in its entirety. Intracellular organelle localization peptides / signals include peptides and nucleic acids that localize the salt at or near an organelle, such as a mitochondrion, a nucleus, a Golgi apparatus, or an endoplasmic reticulum. In at least one example embodiment, the salt is additionally or alternatively taken up by cytoplasm.

[0163] In at least one example embodiment, the composition further comprises a pharmaceutically acceptable carrier, diluent, stabilizing agent, or a combination thereof. Examples of suitable carriers include water, phosphate buffer, phosphate buffer saline (PBS), isotonic saline, dextrose solution (greater than or equal to about 1% dextrose to less than or equal to about 10% dextrose), sodium chloride solution (greater than or equal to about 0.1 % sodium chloride to less than or equal to about 1.5% sodium chloride), dimethyl sulfoxide (DMSO), isotonic saline, other aqueous solutions, or any combination thereof. Various non-aqueous solutions may be suitable as well. Examples of suitable diluents include starch, cellulose derivatives, magnesium stearate, and combinations thereof. Examples of suitable stabilizing agents include cellulose, calcium carbonate, dicalcium phosphate, carboxymethyl cellulose, starch, mannitol, and mixtures thereof. In at least one example embodiment, the salt has a concentration in the composition of greater than or equal to about 0.5 mg / mL to less than or equal to about 10 mg / mL (e.g., greater than or equal to about 1 mg / mL to less than or equal to about 7.5 mg / mL, or greater than or equal to about 1 .5 mg / mL to less than or equal to about 5 mg / mL). In at least one other example embodiment, the composition consists essentially of the salt, or consists of the salt.

[0164] In at least one example embodiment, the composition further comprises a secondary agent. The secondary agent is a therapeutic molecule, a drug for treating a tumor / cancer, an antimicrobial agent, or any combination thereof.Methods of Making and Using the Composition

[0165] At least one example embodiment relates to a salt, as described above, for use in a method of treating cancer, wherein the salt is substantially free of a coating when located within a cancer cell, comprises an ion and a counterion, and is non-toxic to cellsin the dark. At least one example embodiment relates to a salt, as described above, for use in a method of treating cancer, wherein the salt comprises an ion, a counterion, and a bioresorbable coating, and is non-toxic to cells in the dark. At least one example embodiment relates to a salt, as described above, for use in the treatment of cancer; and a salt, as described above, for use as a medicament for the treatment of cancer. At least one example embodiment relates to the use of a salt, as described above, for the manufacture of a medicament for the treatment of cancer.Method Of Tuning the Cytotoxicity of Ions

[0166] At least one example embodiment provides methods of tuning the cellular toxicity of photoactive ions. More specifically, the cellular toxicity can be dark cellular toxicity or light cellular toxicity. For example, if a salt comprising an ion and an unsuitable counter ion is cytotoxic in the dark at a desired concentration, the dark cellular toxicity can be deactivated, that is, the salt can be made nontoxic to cells, by removing the unsuitable counterion and pairing the photoactive ion with a second different acceptable counterion.

[0167] Accordingly, at least one example embodiment provides a method of deactivating dark cellular toxicity of a salt to a cell. The method includes obtaining a first salt that is cytotoxic to the cell in the dark, wherein the first salt comprises an ion and a first (unsuitable) counterion. The method also comprises selecting a second (suitable) counterion, and substituting the first counterion with the second counterion to generate a deactivated second salt, wherein the deactivated second salt has a lower cellular toxicity to the cell than the first salt prior to the substituting. In at least one example embodiment, the substituting is performed by ion exchange or ion synthesis. This can be done by “exchange” or during an initial reaction, or via a subsequent reaction. Moreover, the second counterion is a suitable counterion as described above. In at least one example embodiment, the salt becomes activated or excited in response to external stimulus (e.g., light). In at least one example embodiment, the deactivated salt luminesces when exposed to light having a particular wavelength (e.g., NIR light).

[0168] Even though a salt may be nontoxic to cells in the dark, in at least one example embodiment, cellular toxicity is activated when the photoactive ion is excited. This phototoxicity is desired during, for example, PDT and / or BNCT, but may not be desired during, for example, imaging. Therefore, phototoxicity is tunable by substituting counterions.

[0169] Accordingly, at least one example embodiment relates to a method of deactivating or activating phototoxicity of a salt to a cell. The method comprises obtaining a first salt that is either phototoxic or non-phototoxic, wherein the first salt comprises a photoactive ion and a first counterion that provides either cellular toxicity in the external stimulus or no cellular toxicity in the external stimulus.

[0170] In at least one example embodiment, an electron of the photoactive ion become excited when the photoactive ion absorbs light of a certain wavelength, the first counterion modulates the energy of the electron and a hole. The method also comprises selecting a second counterion, which modulates the energy of the electron and the hole to a different extent than the first counterion, and substituting the first counterion with the second counterion to generate a second light activated or light deactivated salt, wherein the light activated salt has a phototoxicity that is higher than that of the first salt and the light deactivated salt has a phototoxicity that is lower than that of the first salt. In at least one example embodiment, the substituting is performed by ion exchange or ion synthesis. Moreover, the second counterion is a suitable counterion as described above. In other aspects of the current technology, the deactivated or activated second salt luminesces when exposed to light. In example embodiments where the first salt is toxic to cells in the dark, the second counterion may be chosen to deactivate dark cellular toxicity and to be either phototoxic or non-phototoxic.Method Of Exciting a Salt in a Cell

[0171] At least one example embodiment relates to a method of exciting a salt in a cell. The method includes contacting the cell with a composition including the salt, the salt including a photoactive ion and a counterion, wherein the cell uptakes the salt such that the salt is substantially free of a coating within the cell and the salt is non-toxic to the cell in the dark. The method also includes exposing the cell to light having a first wavelength or neutrons to become excited (e.g., a photoactive ion of the salt absorbs the light to become excited).

[0172] As discussed above, the light having the first wavelength may be NIR light. In at least one example embodiment, the excited salt emits light having a second wavelength. In at least one example embodiment, the excited salt is non-toxic to the cell (in the light), such that the excited salt does not cause cell death within a desired (or alternatively, predetermined) concentration range. In at least one example embodiment, the excited salt is activated to generate reactive oxygen species (ROS) predominantlywithin the mitochondria, where, for example, it disrupts mitochondrial electron transport chains, leading to cell death. Additionally, minor localization in the endoplasmic reticulum (ER), Golgi apparatus, and cytoplasm may contribute to the overall cellular stress and cellular death.

[0173] The method may also include, prior to the contacting, tuning the salt to have a desired toxicity after the exposing the cell to external stimulus by pairing the photoactive ion with the counterion, wherein the counterion induces the desired toxicity of the excited photoactive ion. Tuning the salt can be performed by the methods described above.

[0174] The cell can be a prokaryotic cell, such as a bacterium, or a eukaryotic cell, such as from a tissue. In at least one example embodiment, the cell is a cancer cell or a tumor cell. For example, the cell may be located in a tumor of a subject having cancer. The cancer can be brain cancer, head and / or neck cancer, lung cancer, breast cancer, gastrointestinal cancer, pancreatic cancer, prostate cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, bladder cancer, renal cancer, soft tissue sarcoma, thyroid cancer, endometrial cancer, or any combination thereof. In at least one example embodiment, when the cell is a tumor cell, the excited salt may be activated to be toxic and to kill the tumor cell by generating ROS, disrupting electron transport chains, generating alpha particles, or generating high energy lithium 7 nuclei, or the salt may be non-toxic, but luminescent, such that the tumor cell can be visualized optically if the luminescence has a wavelength in the visible range or with a detector if the luminescence has a wavelength in the IR range. The exposing the cell to external stimulus (e.g., light) may comprise exposing a tissue comprising the cell to external stimulus.

[0175] When the tissue is in a subject, the method may also include, prior to the contacting, administering a safe and effective amount of the composition including a salt to the subject, wherein the salt contacts the cell in vivo. The administering can be performed intravenously, orally, or topically, depending on the location of the tissue and the exposing the cell to external stimulus (e.g., light having a desired wavelength) includes exposing the tissue comprising the cell to external stimulus, which may be performed greater than or equal to about 1 hour after the administering, greater than or equal to about 10 hours after the administering, greater than or equal to about 24 hours after the administering, or greater than or equal to about 48 hours after the administering.

[0176] Similarly, at least one example embodiment relates to a method of exciting a salt in a tissue of a subject. The method includes exposing the tissue in the subject to external stimulus, wherein, prior to the exposing a composition comprising a salt wasadministered to the subject, the salt comprising an ion (e.g., photoactive ion) and a counterion, wherein cells of the tissue took up the salt and the salt is substantially free of a coating within the cells. The exposing the tissue to external stimulus excites the salt. The excited salt is at least one of activated to be toxic to the tissue or luminescent, such that the tissue can be visualized. When the excited salt is luminescent, it emits light having a second wavelength and the method can further comprise visualizing the light having the second wavelength in real time, and resecting the tissue. In at least one example embodiment, the salt was administered to the subject greater than or equal to about 1 hour before the exposing, greater than or equal to about 10 hours before the exposing, greater than or equal to about 24 hours before the exposing, or greater than or equal to about 48 hours before the exposing.Method Of Treating a Subject Having a Tumor

[0177] At least one example embodiment relates to a method of treating a subject having a tumor. The method includes exposing the tumor in the subject to external stimulus (e.g., having a first wavelength), wherein the tumor comprises tumor cells containing a salt being substantially free of a coating and comprising an ion (e.g., photoactive ion) and a counterion, wherein the external stimulus excites the salt.

[0178] In at least one example embodiment, prior to the exposing, the method includes obtaining the subject, wherein the subject was previously administered a composition comprising the salt. The composition could be any composition described above, including a composition wherein the salt is coated with a bioresorbable coating. However, the bioresorbable coating is resorbed by the subject after the administering, such that the salt is substantially free of the coating as it contacts or enters cells of the tumor.

[0179] In at least one example embodiment, the excited salt emits light having a second wavelength and the method further comprises visualizing the light having the second wavelength in real time, and resecting the tumor from the subject. Because the dye is selectively accumulated in tumor cells, the tumor can be identified and removed while leaving healthy, non-cancerous tissue substantially intact, i.e., minimizing damage to healthy tissue.

[0180] In at least one example embodiment, the excited salt generates ROS within the tumor cells and / or disrupts electron transport chains in mitochondria. Additionally or alternatively, the salt may localize and contribute to cellular stress and / or death in theendoplasmic reticulum, Golgi apparatus, and / or cytoplasm of the tumor cells. Because the organic dye is selectively accumulated in tumor cells, the tumor cells are selectively killed while leaving healthy, non-cancerous tissue substantially intact, i.e., minimizing damage to healthy tissue.

[0181] In at least one example embodiment, the method further includes administering a safe and effective amount of a conventional drug for treating the tumor to the subject.Method Of Treating a Subject Having a Tumor Using Photodynamic Therapy

[0182] At least one example embodiment relates to a method of treating a subject having a tumor. The subject may have, for example, brain cancer, head and / or neck cancer, lung cancer, breast cancer, gastrointestinal cancer, pancreatic cancer, prostate cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, bladder cancer, renal cancer, soft tissue sarcoma, thyroid cancer, and / or endometrial cancer. The method includes obtaining the subject, wherein the subject received a pretreatment comprising administering a composition comprising a salt to the subject, the salt comprising a photoactive ion and a counterion, wherein the salt selectively accumulates in cells of the tumor and is substantially free of a coating within the cells of the tumor. In at least one example embodiment, prior to the pretreatment, the salt was deactivated by substituting a first counterion with counterion by ion exchange or ion synthesis.

[0183] The method further includes inducing the salt to become toxic to the tumor cells. The inducing comprises exposing the tumor in the subject to light having a first wavelength, wherein the light having the first wavelength excites and activates the salt, the activated salt being toxic to the tumor cells. Additionally or alternatively, the salt may localize and contribute to cellular stress and / or death in the endoplasmic reticulum, Golgi apparatus, and / or cytoplasm of the tumor cells. Because the salt is selectively accumulated in tumor cells, the tumor cells are selectively killed while leaving healthy, non-cancerous tissue substantially intact, i.e., minimizing damage to healthy tissue.

[0184] Embodiments of the present technology are further illustrated through the following non-limiting examples.Example 1

[0185] Other examples reported a counterion-pairing strategy to manipulate the cyto- / photo-toxicity profile of cyanine-based PSs for PDT. This example observed that specific counterions decreased the undesirable cytotoxicity of cyanine-based PSs. Thisexample hypothesized that a novel, inert counterion could be discovered to target the cancer cells in a highly specific manner. This example reports that when cyanine cations are paired with suitable carborane anions, improved selectivity is observed in the potent targeting of highly metastatic breast cancer cell lines. Equally important, mouse studies reveal utilizing improved [cyanine+][carborane-] salts eliminate highly aggressive primary breast tumors while also suppressing metastatic capacity of breast cancer cells. FIG. 3 is a schematic illustrating multifaceted assessment of cyanine-carboranes on tumor growth in PDT.

[0186] Carborane anions are a subclass of borohydride clusters that have inherently weakly coordinating properties, as bestowed by their delocalized charge and 3-dimensional aromaticity. Icosahedral and bicapped square antiprism borohydride clusters and their isoelectronic carborane cousins possess high intrinsic thermal, chemical, and photochemical stabilities and have wide applications in molecular chemistry and materials science. Examples have shown that such clusters can exhibit limited toxicity in rats at a level similar to NaCI (7.5 g / Kg) and their chlorinated derivatives have recently demonstrated similar nontoxicity. The weak coordination has been shown to help stabilize reactive or labile cations as well as shift the frontier energy levels of the cation by several eV. In contrast to other weakly coordinating anions like phenyl borates, carboranes are the most inert and least nucleophilic anions; thus, carboranes make a suitable anion pairing for cyanine PSs with proper energy level tuning and improved stability. Indeed, unlike all other known weakly coordinating anions, carboranes are the most synthetically modular, and their properties can be fine-tuned via cluster surface modifications. Additionally, many carborane anions exhibit no absorption in the NIR and visible regions of the electromagnetic spectrum. While carboranes have been employed in boron neutron capture therapy (BNCT), where their unique properties enable selective cell damage through the absorption of low-energy thermal neutrons by10B atoms, the use of halogenated carboranes in PDT has not been reported. This example demonstrates implementation of utilizing carboranes as counterions to cyanine cations facilitate capitalizing on the toxicity tuning and cancer-targeting ability of these photosensitizers.

[0187] This example screens and improves cyanine carborane-based PSs for PDT in primary breast tumors as well as metastatic breast cancer cells. To create a clinically relevant orthotopic mouse model of metastatic breast cancer, 6DT1 cells derived from an MMTV-Myc breast tumor were injected into the fourth mammary fat pad of syngeneicFVB mice. By using this model, counterion-tuned PSs were tested in a physiologically relevant tumor microenvironment in immunocompetent mice, which is important in studying tumor metastasis and PDT mechanism. This example tested PDT using Cy+ paired with five distinct carborane anions in vitro in both mouse (4T1 and 6DT1 ) and human (MDA-MB-231 ) cell lines, as well as in vivo using an orthotopic mouse model of breast cancer. This example demonstrates the ability of improved PSs to eliminate reduce or eliminate tumors in vivo, indicating a potent therapeutic approach against aggressive breast cancer while reducing side effects in healthy cells.

[0188] This example reports a multifaceted assessment of novel cyaninecarborane salts as potent photosensitizers for photodynamic therapy (PDT). These compounds exhibit efficient tumor targeting through uptake by organic anion-transporting polypeptides (OATP). Upon near-infrared (NIR) light irradiation, cyanine-carborane salts generate reactive oxygen species (ROS), triggering apoptosis and significantly reducing or suppressing tumor cell proliferation and migration. In vivo examples demonstrate antitumor activity with reduced or minimal systemic toxicity, highlighting their potential as next-generation PDT therapeutics.

[0189] I. Results

[0190] A. Cyanine-carborane photosensitizers impede breast cancer cell proliferation.

[0191] This example synthesized and tested nanoparticle formulations of various cyanine-carborane pairs including CyCBgH , CyCBuHeCle, CyCBgHClg, CyCioBnH32Si, and CyCsB22N2H25. FIGS. 2A-2F relate to cyanine-carborane photosensitizers that are potent inhibitors of breast cancer cellular viability. A photoactive heptamethine cyanine cation (Cy+) may be paired with carborane anions CBgHio", CBuHeCle", CBgHClg", CioBiiH32Si", and / or C5B22N2H25" to tune the energy level of organic salt PSs. FIG. 2A illustrates the structure of the cyanine cation (Cy+). FIG. 2F illustrates the structure of a first carborane anions, CBgHio". FIG. 2G illustrates the structure of a second carborane anion, CBuHeCle". FIG. 2H illustrates the structure of a third carborane anion, CBgHClg" FIG. 21 illustrates the structure of a fourth carborane anion, CioBnH32Si". FIG. 2J illustrates the structure of a fifth carborane anion C5B22N2H25".

[0192] Initially, the PSs was dissolved in pure dimethyl sulfoxide (DMSO) and assessed the ultraviolet-visible (UV-VIS) transmittance spectrum of the resulting solution. Subsequently, a portion of this solution was dispersed in water at a volumetric ratio of 10% DMSO to 90% water and the UV-VIS transmittance spectrum was then examined.The results showed significant spectral broadening of the NIR peaks with a blue shift of the 764 nm shoulder peak and a bathochromic shift of the 834 nm peak, resulting in a full width at half maximum of ~68 nm for the cyanine-carboranes in 100% DMSO increasing to ~290 nm when the cyanine-carborane nanoparticles were dissolved in 10% DMSO and 90% water. This peak broadening stems from both H- and J- aggregation during the formation of stable “flash precipitated” nanoparticles (FIGS. 4A-4B). The absorption profiles of CyCBgHio and CyCBuHeCle exhibited a broadening of the absorption profile over 22 days (FIGS. 4A-4B), indicating that these PSs rapidly aggregate and remain stable in their aggregated state without significant dissociation into [Cy+] and [CBuHeCle ]. SEM analysis further revealed that the particle sizes of PSs ranged from 23 ± 4 nm to 38 ± 5 nm (FIGS. 5A-5D). The incorporation of different combinations of cyanine-carboranes resulted in significant shifts in the energy levels of the highest occupied molecular orbital (HOMO) (FIG. 6 and Table 2, below), while maintaining essentially the same bandgap (absorption edge) in the solid-state, consistent with earlier examples. Notably, the HOMO of CyCBuHeCle and CyCBgHClg are both energetically resonant around 5 eV, while the other carboranes that are energetically off- resonant are less selectively photoactive. The important compounds CyCBuHeCle and CyCBgHClg were further characterized using13C,11B, and1H solid-state nuclear magnetic resonance (SSNMR) spectroscopy, confirming the formation of [cyanine+] [carborane ] salts (FIGS. 7A-7N). The solution-state13C and1H spectra in de-acetone were consistent with an intact cyanine cation and distinct carborane C-H protons.11B NMR showed that the carborane clusters retained their Csv or C4v symmetry without degradation. High-resolution mass spectrometry further confirmed the expected masses of each component in the nanoparticle formulations.

[0193] Table 2, below, illustrates highest occupied molecular orbital energy levels of example cyanine-carboranes.Table 2

[0194] To investigate the potential effects of PSs on cellular proliferation, this example exposed two highly metastatic breast cancer cell lines, 4T1 and 6DT1 , to cyanine-carborane nanoparticles at various concentrations (0.2, 0.5, 1 , 3, and 10 pM) under dark or 850 nm light exposure conditions. Following 5 days of daily 30-min exposure to 850 nm light, cells were stained with crystal violet to visualize the surviving cell population, which was subsequently quantified using trypan blue. In the absence of 850 nm light (dark), exposure of cancer cells to CyCBuHeCle and CyCBgHClg cyaninecarboranes within the concentration range of 0.2 to 1 pM did not yield significant alterations in cellular viability (FIGS. 8A-11 B), suggesting that cyanine-carborane at the highest concentration of 1 pM does not induce cytotoxicity. Upon exposure to 850 nm light, breast cancer cells exhibited distinct cluster formations in cellular morphology accompanied by a substantial reduction in cancer cell proliferation by ~3-fold at a concentration of 1 pM (FIGS. 8A-9B), indicating pronounced phototoxic effects. Cellular viability of 4T 1 cells exposed to 850 nm light for up to 1 hour (765 J / cm2) did not decrease, indicating this fluence is not cytotoxic (FIG. 12). The phototoxicity response of 4T1 cells to other cyanine-carboranes including CyCBgHio, CyCioBnH32Si, and CyCsB22N2H25 demonstrated a higher ICso (2.5 pM) compared to the lower ICso value (1 .6 pM; Table 3, below) observed with CyCBuHeCle and CyCBgHClg cyanine-carboranes (FIGS. 13A- 15B); thus, this led to focus on CyCBuHeCle and CyCBgHClg for subsequent PDT investigations. To explore how counterions influence the toxicity of [cyanine+], additional control experiments were conducted using [cyanine+] paired with different counterions, [I ], [FPhB ], and [TPFB ], in 4T1 cells (FIGS. 16A-18B). The results confirmed that counterions modulate the toxicity of [cyanine+] in 4T1 breast cancer cells: [cyanine+] is tuned to become cytotoxic by [I ], phototoxic by [FPhB ], and nontoxic by [TPFB ].

[0195] Table 3 illustrates half maximal inhibitory concentrations (ICso) of cyanine- carboranes with and without NIR irradiation in 4T1 cells. ICso values were generated by nonlinear regression analysis using Graph Pad Prism. Error is displayed as a 95% confidence interval.Table 3Dark ICso NIR ICsoCompoundsCyCBuHeCle 2 3 1 .3-4.3 k6 0.9-2.8CyCBgHClg 2.4 1.4-4.5 1.6 0.9-2.8CyCBgHio 2.7 2.5-3.6 1.7 0.6-4.4CyCioBnH32Si 2.9 2.4-3.7 1.9 1.1-3.8CyC5B22N2H25 3.1 2.5-3.6 2.1 1 .2^.1

[0196] This Example further examined the effect of PSs on non-tumorigenic cells (L-929) to demonstrate the cancer-selective phototoxicity of cyanine-carboranes (FIGS. 19A-20B). Under dark conditions, no changes in cellular proliferation were observed. However, exposure to 850 nm light induced pronounced cellular phototoxicity, resulting in a 2.5-fold reduction in cellular viability. These results imply that the CyCBuHeCle and CyCBgHClg cyanine-carboranes do not have detrimental impacts on normal cells (i.e., non-tumorigenic cells) under dark conditions, while their activation with 850 nm light renders them highly promising for PDT due to their phototoxic properties that could impede the proliferation of highly metastatic cancer cells. -

[0197] B. Subcellular localization and mechanism of uptake in breast cancer cells.

[0198] To track the translocation of cyanine-carboranes, this example conducted fluorescent staining experiments targeting specific cellular organelles: mitochondria (Mito-tracker), endoplasmic reticulum (ER-tracker), Golgi apparatus (Golgi-tracker), and lysosomes (Lyso-tracker) in live cells (FIGS. 21 A-21 D). Following a 24 h exposure of cyanine-carboranes to 4T1 cells, the intracellular distribution of PSs and various trackers were monitored using fluorescence microscopy (FIGS. 21 A-21 D). The results revealed predominant localization of CyCBuHeCle and CyCBgHClg within the mitochondria of the cancer cells (with Pearson's correlation coefficients of r = 0.78 and 0.69, respectively) (FIGS. 21 A-21 D). A minor fraction of the salts was detectable within the ER (r = 0.53 and 0.63) and the Golgi (r = 0.49 and 0.52). In contrast, only a marginal portion of the cyanine-carboranes exhibited detectability within the lysosomes (r = 0.43 and 0.15), suggesting that cellular lysosomes may not be the primary sites of action or interaction for the PSs. Subsequently, this example conducted a parallel subcellular localization analysis on human breast cancer cells (MDA-MB-231 ) using CyCBuHeCle and CyCBgHClg (FIGS. 22A-22D), which revealed the highest localization rate within the mitochondria. This consistent intracellular distribution pattern of cyanine-carboranes across both human and murine breast cancer cell lines emphasizes their targeted accumulation within mitochondria, offering valuable insights into their potential mechanisms of action.

[0199] To explore the uptake mechanism of cyanine-carboranes, cells were treated with three distinct inhibitors targeting specific endocytosis pathways: clathrin- dependent (Dynasore), caveolae-dependent (Methyl-[3-cyclodextrin), and organic anion transporting polypeptide (OATP)-mediated transport (Bromsulphthalein; BSP) (FIGS. 23A-23B). Cells were initially pretreated with different inhibitors and subsequently exposed to CyCBuHeCle and CyCBgHClg cyanine-carboranes in the presence of respective endocytosis inhibitors for 24 h. Remarkably, inhibiting clathrin-dependent or caveolae-dependent endocytosis pathways in cancer cells yielded no alterations in the uptake of PSs. However, a significant reduction in the internalization of cyanine- carboranes was evident upon the introduction of OATP inhibitor (BSP), indicating that their uptake predominantly relies on the OATP-mediated transport mechanism. Further supporting the role of OATPs in the uptake of these PSs, bromocresol and rifampicin, which inhibit both OATP1 B1 and OATP1 B3, significantly blocked PS uptake compared to the control (FIGS. 24A-24B).

[0200] C. Coupling cyanine-carborane treatment with NIR generates reactive oxygen species (ROS) in malignant cells.

[0201] ROS play a pivotal role in the mechanism of PDT wherein the activation of a photosensitizer triggers ROS generation, leading to phototoxic effects and targeted cellular damage in cancer cells. To elucidate the impacts of PDT on breast cancer cells, this example used three types of ROS sensors capable of quantifying ROS levels in live cells: mitochondrial ROS, cytoplasmic ROS, and singlet O2. 4T1 cells were exposed to CyCBuHeCle and CyCBgHClg PSs for 24 h and the ROS generation was visualized immediately after a 30 min-exposure of 850 nm light. The untreated cells (Veh) and the negative control cells (cyanine-carboranes exposed) were both maintained under dark conditions. Upon exposure of the cells to CyCBuHeCle under 850 nm light, both CyCBuHeCle and CyCBgHClg PSs induced substantial mitochondrial ROS generation (FIGS. 25A-25B). The levels of cytoplasmic ROS and singlet O2 were increased by 10.3 and 5.4-fold (FIGS. 25C-25F and FIGS. 26A-26D), respectively, in comparison with the control group cells (dark). This increase in ROS levels was accompanied by developing cellular blebbing phenotypes (indicated by white arrows) observed in bright-field images, suggesting an early phase of apoptosis. In contrast, exposure to CyCBgHClg resulted in a comparatively lower cytoplasmic (6.7-fold) and singlet O2 ROS generation generated in comparison with the cells under dark conditions. Collectively, these results demonstrate that the combined cyanine-carboranes treatment with 850 nm lightcogenerates the cytoplasmic ROS, singlet O2, and mitochondrial ROS, resulting in the effective death of breast cancer cells.

[0202] D. NIR PDT with cyanine-carboranes disrupts EMT signaling in breast cancer progression.

[0203] To explore the in vitro anti-metastatic effects of PSs, this example examined how PDT affects cancer cell migratory capacity and the modulation of epithelial-mesenchymal transition (EMT), an evolutionarily conserved developmental program implicated in cancer development. EMT enables cancer cells to acquire metastatic traits, including enhanced mobility, invasiveness, and resistance to apoptosis. 4T1 cells were grown in a confluent cell monolayer and treated with CyCBuHeCle and CyCBgHClg PSs for 24 h, while the control cells (Veh) were treated with DMSO. Following a 30-min exposure to 850 nm light, this example created a consistent wound width and monitored the real-time scratch closure for 24 h (FIG. 27 A). Under dark conditions, the control (Veh) and cyanine-carborane treated cells were able to migrate faster and completely close the gap (FIG. 27B), suggesting the highly metastatic potential of 4T1 cells. The combined treatment of CyCBuHeCle and CyCBgHClg with 850 nm light significantly reduced wound closure by 74.0% ± 2.6% and 87.0% ± 4.1 %, respectively. Additionally, CyCBuHeCle and CyCBgHClg under 850 nm light displayed significantly reduced invasion compared to the dark control group (FIGS. 28A-28E). The western blotting analysis of EMT signaling markers revealed a notable decrease in the protein expression levels of N-cadherin, Vimentin, and Snail in cells co-exposed to cyaninecarboranes and 850 nm light as compared to cells treated with PSs in the absence of light (FIGS. 28A-28B, 29, and 30A-30B). This result suggests that the combined treatment of cyanine-carboranes with 850 nm light may be associated with the inhibition or reversal of the EMT process.

[0204] To determine the cause of reduced cellular migration during PDT, this example investigated the cellular cytoskeleton changes including the reorganization of F-actin filaments - an integral component regulating cell migration across various cellular processes. After exposure to cyanine-carboranes under either dark or light conditions, cells were fixed and stained for actin filaments at the 6 h time-point (FIG. 31 A). Cytochalasin D was used as a positive control specifically targeting and depolymerizing F-actin filaments. This example found no qualitative changes in cytoplasmic and peripheral F-actin filaments when the cells were exposed to CyCBuHeCle and CyCBgHClg under dark conditions. However, a profound reduction of cytoplasmic F-actinfilaments was noted upon exposure to PSs with 850 nm light as compared to cells under dark conditions. The dynamic assembly and disassembly of F-actin filaments are regulated by the cycling between guanosine triphosphate (GTP)-bound and guanosine diphosphate (GDP)-bound states of actin monomers, while adenosine triphosphate (ATP) serves as a vital cofactor in regulating actin filament stability and turnover. The GTP / ATP ratio is crucial as it reflects the balance between filament assembly and stability, with higher GTP promoting polymerization and ATP stabilizing the filaments. To further understand the cellular response, this example measured the GTP / ATP ratio of 4T1 cells using liquid chromatography-mass spectrometry (LC-MS) when exposed to PSs. Interestingly, there were no statistically significant changes when cells were exposed to CyCBgHClg under both dark and light conditions (FIG. 31 B). In contrast, treatment with CyCBuHeCle resulted in a substantial reduction in the GTP / ATP ratio under 850 nm light conditions. These results suggest that the combination of cyaninecarboranes with NIR treatment may disrupt cancer cell migration by destabilizing F-actin filaments and reducing the expression of EMT proteins. This, in turn, may suppress the migration behavior of highly metastatic cancer cells.

[0205] E. NIR PDT with cyanine-carboranes triggers apoptosis signaling in breast cancer cells.

[0206] To investigate the effects of the combination of PSs and NIR light on apoptosis, cells were treated with cyanine-carboranes for 30 min either with or without light. Annexin-V / PI double staining was used to determine apoptosis in 4T1 cells at 6 h post-exposure time-point. Under dark conditions, cells treated with CyCBuHeCle and CyCBgHClg showed no significant changes in early apoptotic cell proportion (Q3), i.e., 6.8% and 6.1 %, respectively (FIGS. 32A-32F), compared with the control cells (4.7%). However, when these cells were treated with CyCBuHeCle and CyCBgHClg, the early apoptotic cell proportion increased to 21.5% and 15.7% under light conditions, respectively. Moreover, the action of cyanine-carboranes on apoptosis signaling was validated by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Bax, Bak, and Bid genes were upregulated in cells treated with CyCBuHeCle and CyCBgHClg compared with those of the control and cells under dark conditions (FIGS. 33A-33C). Following NIR light exposure, protein levels of Poly (ADP-ribose) polymerase (PARP) and its cleaved form (cleaved PARP) increased in cells treated with CyCBuHeCle and CyCBgHClg (FIGS. 34-35H). However, when cells were treated with cyanine-carborane without NIR light, there were no observed changes in their proteinexpressions (FIG. 34). Notably, increased PARP levels in cells treated with cyaninecarboranes and irradiated with NIR light led to the downregulation of Cytochrome c, indicating impact on mitochondrial function in breast cancer cells.

[0207] To further explore the impact of PSs on mitochondria, this example assessed the magnitude of mitochondrial membrane potential (AMJm) in 4T1 cells (FIG. 36). The fluorescence level of the voltage-sensitive dye tetramethylrhodamine methyl ester (TMRM) is positively correlated with mitochondrial metabolic activity. A significant decrease in mitochondrial membrane potential was observed for both CyCBuHeCle and CyCBgHClg treated under light conditions, 0.4 and 0.6, respectively, indicating that PSs activated by light have a detrimental effect on mitochondria. Notably, CyCBuHeCle and CyCBgHClg treatment under dark conditions had no significant effect on mitochondrial activity (FIG. 36). Altogether, these results indicate that activated PSs by NIR light disrupts mitochondrial activity, leading to early apoptosis in cancer cells.

[0208] F. NIR PDT with cyanine-carboranes impedes tumor growth in vivo.

[0209] To validate trends observed in vitro and demonstrate potential clinical applications of cyanine-carboranes, this example further performed in vivo experiments. 10,000 syngeneic 6DT1 mammary cancer cells were orthotopically injected into the right fourth mammary fat pad of FVB mice (FIG. 37A). After the formation of a palpable tumor (2 x 2 mm3), mice were given an intravenous injection of either CyCBuHeCle or CyCBgHClg through the tail vein followed by PDT treatment every 48 h. A fluorescent stereomicroscope was used to track the biodistribution of cyanine-carboranes in tumor cells located on the right mammary fat pad and their clearance from normal tissues (FIGS. 37B-37F). The results show that CyCBuHeCle was rapidly uptaken with initial localization in the liver for the first 24 h as compared with the slower uptake of CyCBgHClg (FIG. 37G). Both CyCBuHeCle and CyCBgHClg demonstrated remarkable tumor-specific uptake after 24 h, achieving a 7- to 9-fold increase in fluorescence signal over 48 to 72 h and stably retained for more than 100 h, suggesting promising therapeutic potential for PDT.

[0210] To further explore the potential effects of fluorescent cyanine-carboranes as potent PSs for PDT, tumor-bearing mice were dosed with 2 pmol / kg of PSs. Subsequently, they were irradiated with 150 J / cm2of 850 nm light every 48 h following the administration of cyanine-carboranes, repeated a total of six times. Under dark conditions, a significant increase in tumor volume in mice groups dosed with CyCBuHeCle and CyCBgHClg was measured (FIG. 38A), i.e., 580 ± 30 and 390 ± 43 mm3,respectively. Under NIR light treatment, tumor volume shrank significantly until they were no longer detectable (FIG. 38A). No alterations in mice weights were observed over the entire experiment for all groups under either dark or light conditions (FIG. 38B). Collectively, these in vivo biodistribution results indicate the potential clinical applications and therapeutic efficacy of these cyanine-carboranes in PDT.

[0211] Building on our in vitro findings, this example investigated whether the elevated ROS levels from our PSs upon NIR exposure contribute to antitumor effects in vivo, independent of the immune response. Experiments in both immunocompetent FVB mice and immunodeficient NSG mice revealed that only NIR-exposed tumors showed significant reduction, while non-exposed tumors grew (FIGS. 39A-40C). The consistency of this effect in both mouse models indicates a localized action not mediated by systemic immunity. These findings confirm that ROS generation upon NIR exposure is the primary driver of the therapeutic effect of our cyanine-carborane PSs, independent of the immune system.

[0212] II. Discussion.

[0213] A major challenge in treatment of metastatic breast cancer is the lack of targeted therapies. Thus, there is a desire to create targeted therapies with reduced side effects. PDT offers advantages by exerting localized effects without the systemic side effects seen in other treatments. PDT can also be combined with other clinical interventions such as surgery, radiotherapy, or chemotherapy. Considering the benefits of PDT, this example developed and investigated cyanine-carborane photosensitizers in a physiologically relevant tumor microenvironment within an immunocompetent mouse model. Our findings reveal that these newly developed PSs significantly reduce cancer cell metastatic potential, altering ATP / GTP ratios, and affecting mitochondrial membrane potential, ultimately leading to tumor elimination in vivo.

[0214] A suitable PS should exhibit low cytotoxicity while demonstrating a strong phototoxic effect and specificity on cancer cells. This example showed that CyBuHeCle and CyCBgHClg at a concentration of 1 pM were not cytotoxic for both non-tumorigenic mouse fibroblast (L-929) (FIGS. 19A-20D) and metastatic breast cancer (4T 1 and 6DT 1 ) cell lines (FIGS. 8A-8B & 10A-1 1 B). Moreover, when cyanine-carboranes were administered to FVB mice at 2 pmol / kg, there were no significant changes in their weight after 20 days, suggesting no cellular cytotoxicity in vivo. These findings are consistent with our previous examples in lung cancer, melanoma, and 6DT 1 breast cancer cell lines, demonstrating that the choice of counterion can finely tune the biological effects of[cyanine+]. Examples have demonstrated that steric effects introduced by different counterions, combined with their impact on the HOMO energy levels and zeta potentials, are likely responsible for the varying phototoxicity and cytotoxicity observed in cancer cells.

[0215] The in vivo results of this example indicate that while the liver initially absorbed the majority of cyanine-carboranes, ex vivo fluorescence imaging confirm they were effectively cleared from most tissues including the tumor, spleen, and liver, within 20 days post-PDT treatment (FIGS. 37B & 37E-37F). Notably, these salts persisted in breast tumors for up to 5 days, indicating their selective affinity and stability in tumor cells. In contrast, ICG has rapid metabolism and limited tumor specificity, and using ICG alone for PDT is challenging. Therefore, various carriers have been developed to deliver ICG to tumors; however, these carriers retain ICG in normal organs for a longer duration. Many negatively charged anti-cancer drugs such as methotrexate and etoposide are efficiently transported by OATPs, which facilitate the uptake of organic anions, and their bidirectional transport capabilities make them essential in cellular transport processes. Blocking OATPs with the inhibitors BSP, bromocresol, and rifampacin prevented the uptake of cyanine-carboranes (FIGS. 24A-24B), whereas inhibitors of other endocytic mechanisms such as clathrin and caveolae had no significant effect on their uptake (FIGS. 23A-23B). These findings suggest that OATPs are important players in the cellular uptake of these photosensitizers. Further studies are desired to determine which specific OATP subtypes are responsible.

[0216] The enhanced uptake of PSs by cancer cell mitochondria can be attributed to their inherent negative charge. Given the substantially negative membrane potential within mitochondria, typically ranging from -160 to -180 mV, specific anion channels on the outer membrane as voltage-dependent anion channel may facilitate this process. These channels serve as conduits for the entry of negatively charged molecules, including salt nanoparticle PSs, into the mitochondria. This phenomenon is driven by the electrostatic interactions between the negatively charged salt PSs and the mitochondrial anion channels, allowing for efficient and higher transport into the organelle. However, our experiments using OATP inhibitors suggest that this is not the only mechanism driving the uptake of our PSs. This example observed a significant reduction in PS accumulation in cells when OATPs were inhibited (FIGS. 21A-21 D & 23A-23B), indicating that OATP-mediated transport is essential for their cellular uptake. This implies that our PSs may rely on a more complex mechanism involving both their chemical natureand transport via OATPs. The interplay between their potential lipophilic properties and the involvement of OATPs may collectively contribute to their mitochondrial targeting. The negative zeta potential of our particles further supports this mechanism (Table 4, below), as it enhances the attraction between the negatively charged PSs and the mitochondrial anion channels, improving their uptake and localization within mitochondria. This example reports higher localization in mitochondria than other organelles using both mouse (4T1 ) and human (MDA-MB-231 ) cell lines (FIGS. 21 A- 22D). PDT-targeted mitochondria are good candidates for cancer therapy; PDT using mitochondria-targeting PSs reduces mitochondrial membrane potential and inhibits the activity of complexes I to IV. Consistently, mitochondria-targeting CyCBuHeCle and CyCBgHClg effectively lower mitochondrial membrane potential and Cytochrome c protein expression (FIGS. 34 & 36).

[0217] Table 4 illustrates zeta potential changes as a function of counterion pairing. The Zeta potential of photosensitizers was calculated from electrophoretic mobility using a Malvern Zetasizer NS. The anion shifts the zeta potential, similar to what is observed in the solid-state using ultraviolet photoemission spectroscopy (UPS).Table 4Compounds Zeta Potential(mV)CyCB9Hio -21.7 + 0.3CyCBnH6Cl6-26.4 + 0.9CyCB9HCl9-14.0 + 0.8CyC5B22N2H25 3.54 + 2.28

[0218] Reactive oxygen species (ROS) generation plays a crucial role in therapeutic and effectiveness of PSs in cancer therapy. In our prior generation of PSs, this example observed that tetrakis (4-fluorophenyl) borate (FPhB- or C24HieBF4“) induced ROS generation within the mitochondria of 4T1 cells when exposed to 850 nm light (FIGS. 41 A-41 G). Notably, this ROS generation was limited to mitochondria only and did not extend to the cytoplasm or result in singlet oxygen production. In contrast, CyCBuHeCle and CyCBgHClg generate ROS within the mitochondria as well as the cytoplasm, leading to the additional formation of singlet oxygen (FIGS. 25A-25F). Changes in the HOMO energy level can significantly impact the efficiency of these processes, thereby influencing the overall photodynamic efficacy of the photosensitizer. A lower HOMO energy level may stabilize the excited state, leading to enhanced ROSgeneration, which is crucial for effective PDT. In contrast, a higher HOMO energy level may cause the molecule to undergo non-radiative decay, reducing ROS production. ROS are intricately associated with tumor metastasis, affecting processes such as tumor cell migration, invasion, and angiogenesis. Elevated ROS levels can suppress E-cadherin expression, an important molecule for maintaining epithelial characteristics, thereby inhibiting the transition to a mesenchymal state. In contrast, high ROS levels may induce a mesenchymal-to-epithelial transition (MET), reducing the invasive and metastatic potential of cancer cells. This example shows that exposure of cells to CyCBuHeCle and CyCBgHClg substantially diminished the metastatic behavior of 4T1 cells (FIGS. 27A- 27D), accompanied by a significant decrease in the expression of Snail and Vimentin proteins (FIG. 29). Consistently, analysis by transwell invasion assay revealed that the invasive potential of 4T1 cells was markedly diminished following treatment with CyCBuHeCle and CyCBgHClg under 850 nm light exposure. This suggests that these PSs inhibit the invasive properties of 4T1 cells, likely by interfering with matrix metalloproteinases (MMPs) involved in ECM degradation. qPCR results show that exposure of 4T1 cells to 850 nm light with PSs led to a 1 .1 -fold and 0.5-fold increase in MMP3 and MMP9 expression, respectively (FIGS. 30A-30B), both of which are known to degrade the ECM and disrupt the stability of F-actin filaments. Elevated ROS can oxidize signaling proteins that govern the intricate balance between actin polymerization and depolymerization that is crucial for the maintenance of the actin cytoskeleton's structural integrity and cancer metastasis. This example indicates qualitative reduction of cytoplasmic F-actin filaments along with reduced GTP / ATP ratio when the cells were exposed to CyCBuHeCle (FIG. 31 A). Therefore, elevated levels of ROS may initiate the oxidation of F-actin filaments and potentially reducing cancer metastasis.

[0219] Our findings suggest a dual inhibitory mechanism of cyanine-carboranes in PDT (FIG. 42): it suppresses EMT signaling by downregulating markers such as Vimentin and Snail, while concurrently enhancing the expression of cleaved PARP protein. This dual action induces early apoptosis in metastatic cancer cells. Moreover, the generation of ROS levels leads to a substantial decrease in mitochondrial membrane potential (AMJm) and a reduction in Cytochrome c protein levels within cells. The simultaneous generation of mitochondrial ROS, cytoplasmic ROS, and singlet O2 results in a reduction in the GTP / ATP ratio that could perturb the polymerization of F-actin filaments. This coordinated sequence of events, inducing early apoptosis and the disorganization of F-actin filaments, inhibits migration of metastatic breast cancer cells and arrests the growth of breast tumors in vivo.

[0220] III. Conclusion.

[0221] This example demonstrated the power of cyanine-carboranes for nextgeneration PDT of metastatic breast cancer cells. Carborane anions provide distinct advantages through their intrinsic lack of toxicity, outstanding thermal and chemical stability, coupled with the three-dimensional aromaticity. Their remarkable inertness and synthetic flexibility become particularly advantageous when paired with cyanine PSs, allowing for precise energy level adjustment and enhanced stability in the context of PDT applications. PDT can also be combined with other clinical interventions such as surgery, radiotherapy, or chemotherapy. Our findings reveal that the mechanism of action in these systems involves altering ATP / GTP ratios and affecting mitochondrial membrane potential to ultimately lead to tumor elimination in vivo. Equally important is the apparent non-toxicity, coupled with greater and more diverse ROS generation of these cyanine carborane nanoparticles in cancer cells.

[0222] IV. Experimental Section.

[0223] A. Materials.

[0224] All reagent-grade chemicals, including Mitomycin C, Cytochalasin D, ethanol, Triton X-100 and formaldehyde were purchased from Sigma-Aldrich (Louis, MO, USA) unless otherwise mentioned. Hoechst 33342, a nucleic acid stain dye, was obtained from Invitrogen (Waltham, MA, USA). CsCBgHw, CSC5B22N2H25, CsCioBnH32Si, and CsCBuHeCle were prepared according to literature procedures.

[0225] B. Synthesis of Cs[CBgHCIg].

[0226] CsCBgH (2.0 g, 7.9 mmol) was dried in vacuo at room temperature overnight. An oven-dried Teflon-lined pressure vessel was then charged with the dried CsCBgHw and cooled to room temperature under a stream of argon. Once cooled, 20 mL of iodine monochloride was added to the reactor followed by 20 mL of Trifluoromethane sulfonic acid. The Argon stream was removed and the reactor was sealed immediately. The reactor was slowly brought up to 200°C and was left reacting behind a blast shield for 3 days. After 3 days, the reaction was cooled to room temperature and monitored using a combination of11B NMR and mass spectrometry for complete conversion to the CBgHClg- cluster. If the reaction was incomplete, more iodine monochloride was added and the reaction was allowed to continue heating. Once complete, the cooled reaction was transferred to a dry, cool round bottom flask rinsingthe reactor with portions of dichloromethane (-125 mL total) and set up for trap-to-trap distillation under inert atmosphere. After removal of the liquids, the distillation was cooled to room temperature and subsequently opened to atmosphere. 50 mL of deionized water was added followed by a sodium sulfite solution until all the halogens were quenched. After filtration, the solution underwent extraction with Ethyl Acetate (4 x 50 mL). The organic extracts were combined and the solvent was evaporated. Subsequently, 200 mL of water was introduced and the mixture was hot-filtered. Two grams of Cesium chloride was incorporated into the filtrate and recrystallized from hot water. CsCBgHClg was collected in two batches of crystal crops resulting in an 87% yield (3.9 g, 6.9 mmol). The compound was characterized by mass spectroscopy and a combination of1H,11B and13C NMR and was consistent with previously reported data. The photosensitizers were characterized by NMR and high-resolution mass spectrometry (FIGS. 7A-7N).

[0227] C. Synthesis and purification of cyanine-carboranes.

[0228] 2-[2-[2-chloro-3-[2-(1 ,3-dihydro-3,3-dimethyl-1 -ethyl-2H-benz[e]indol-2- ylidene)ethylidene]-1 -cylohexen-1 -yl]-ethenyl]-3,3-dimethyl- 1 -ethyl- 1 H-benz[e]indolium iodide (Cyl, American Dye Source), the parent cyanine salt, was dissolved in methanol at a concentration of 10 mg / mL and stirred at room temperature in air. An equimolar amount of carborane precursor (CsCBuHeCle, CsCBgHClg, CsCBgHio, CsCioBnH32Si, and CSC5B22N2H25) was added to the stirring Cyl solution. The cyanine-carborane salt formed solid precipitates after approximately 5 min, and the stirring was stopped after 10 min. The resulting cyanine-carboranes were collected via vacuum filtration and were washed several times with methanol to remove excess precursor and unreacted CyCarborane. The salts were then redissolved in dichloromethane (DCM) and purified through a silica gel plug with DCM as the eluent. The resulting colored solution was then dried in a rotary evaporator. The organic salt purity was verified using a Waters Xevo G2- XS QToF, a high mass resolution time-of-flight mass spectrometer paired with ultra high- performance liquid chromatography system (UHPLC-MS), in both positive and negative modes to verify the presence of the Cy+cation and the carboranes, respectively. Reaction and purification protocol follow the methods in the previous report.

[0229] D. Cell Culture.

[0230] Mouse mammary cancer cells (4T1 , 6DT1 ) and non-metastatic murine fibroblast originating from connective tissue lineage (L-929) were cultured in an incubator at 37°C and 5% CO2 in growth Dulbecco’s Modified Eagle’s Medium (Cat. No. 10-017CM, Corning, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (Cat. No.F0392, Sigma, St. Louis, MO, USA), 2 mM glutamine, 100 U / mL penicillin, and 100 mg / mL streptomycin (Cat. No. 15323671 , Corning, NY, USA).

[0231] E. Cyanine-carboranes nanoparticle formation.

[0232] Fluorescent cyanine-carboranes were dissolved to 2 mM in dimethyl sulfoxide (Cat. No. D4540, Millipore Sigma, St. Louis, MO, USA), and then further diluted in Dulbecco’s Modified Eagle’s Medium to form aggregated nanoparticles (confirmed by peak broadening in optical spectroscopy) for various experiments.

[0233] F. Particle Size Analysis.

[0234] 0.5 pM nanoparticle solutions were prepared in water and 50 pL of each solution was spin-coated onto a silicon wafer at 2000 rpm for 30 seconds. Prior to spin coating, the silicon wafer was meticulously cleaned by sonication in deionized water, acetone, and isopropanol for 10 minutes each, followed by oxygen plasma treatment for 10 minutes. A 2.5 nm iridium film was deposited onto the SEM samples to enhance contrast and reduce charging during imaging. SEM imaging was carried out using a JEOL JSM-7000F microscope at an accelerating voltage of 5 kV and a working distance of 5- 15 mm. Particle dimensions were then measured using Imaged software, ensuring a representative number of particles were analyzed for accurate size distribution.

[0235] G. Cell Viability.

[0236] 4T1 cells were seeded at a density of 40,000 cells per well in 6-well tissue culture plates with or without different concentrations of CyCBuHeCle and CyCBgHClg. After 24 h of incubation, culture media with treated cyanine-carboranes were aspirated and replaced with fresh media without PSs. Each well was irradiated with an 850 nm LED lamp with an irradiation flux of 425 mW / cm2for 30 min in the incubator, and control cells were left in a dark incubator without irradiation. Following irradiation treatment, the media was changed to fresh media containing PSs and allowed to incubate for another 24 h. The same procedure was repeated at 48 and 72 h; however, the cells received no further PSs dissolved in culture media at 72 h. Viable cell number was determined at 96 h using 4% trypan blue and counted by a cell counter (Cellometer Auto T4, Nexcelom Bioscience, MA, USA). All assays were performed with three biological replicates.

[0237] H. Crystal Violet Staining.

[0238] 4T1 cells were fixed using ice-cold methanol for 10 min on ice and further stained with 1 % crystal violet (Cat. No. C581 , Thermo Scientific, Rockford, IL, USA) for 30 min at 25°C. Cells were further washed three times with deionized water and an optical microscope was then used to observe the number of viable cells.

[0239] I. Colocalization analysis.

[0240] The cells were grown on 20 mm glass bottom dish (Cat. No. 801001 , Nest Biotechnology) containing culture media for 24 h. Next, the cells were incubated either with 1 pM of CyCBuHeCle or CyCBgHClg and allowed to incubate for 24 h. The cells were further independently incubated with mitochondrial staining Rhodamine123 (Cat. No. R302, Thermo Scientific), LysoTracker Green (Cat. No. L7526, Thermo Scientific), ER- Tracker Green (Cat. No. E34251 , Thermo Scientific), or Golgi (Cat. No. D7450, Thermo Scientific) according to the manufacturer's instructions. After incubation, the cells were washed three times with phosphate-buffered saline (PBS) and the images were taken with a 63x oil-immersion objective lens. The organelle trackers were excited and detected as recommended by the supplier. The nanoclusters were detected under a customized mounted set of lenses (Aex=350 / 50x nm, Aem=650 Ip nm) manufactured by Chroma Technology Corp attached to an empty filter cube, size P (1 mm) compatible with the DMi8 microscope manufactured by Leica-microsystems.

[0241] J. Endocytosis inhibition.

[0242] To investigate the mechanism of uptake of cyanine-carboranes, the cells were pre-treated with the clathrin-mediated endocytosis inhibitor Dynasore (200 pM) for 20 min, the clathrin-caveolae mediated endocytosis inhibitor Methyl-[3-cyclodextrin (1 mM) for 1 h, and the organic anion-transporting polypeptides inhibitor Bromsulphthalein (250 pM) for 30 min. The control cells (Veh) were treated with 0.1 % DMSO dissolved in the culture media. Subsequently, the cells were incubated with either 1 pM of CyCBuHeCle or CyCBgHClg and allowed to incubate for 24 h in the presence of the inhibitors. Next, the cells were washed three times with PBS and the uptake of PSs into the cells was immediately captured using live cell imaging with the DMi8 microscope manufactured by Leica-microsystems.

[0243] K. Migration assays.

[0244] 4T1 cells were washed with PBS and pretreated with 10 pg / mL MitomycinC to inhibit cellular proliferation for the wound healing assay. Following 2 h of incubation, cells were then trypsinized and seeded at 50,000 cells into the 96-well IncuCyte plates with or without 1 pM of CyCBuHeCle or CyCBgHClg. Cells were further incubated for at least 12 h in an incubator at 37°C and 5% CO2 in a growth culture medium to reach confluence. The cells treated with cyanine-carboranes were irradiated with an 850 nm LED lamp for 30 min in the incubator, and control cells were left in a dark incubator without irradiation. The IncuCyte wound maker tool (Essen Bioscience) was used tocreate a consistent wound width for 96 well-plate. Subsequently, the plate was placed in the IncuCyte system and the scratch closure was monitored in real-time for 24 h. The data of wound confluence over time was exported as an average of three biological replicates. All data are shown as the mean ± S.D.

[0245] L. Liquid chromatography-mass spectrometry (LC-MS).

[0246] To detect the total intracellular ATP and GTP levels, 4T 1 cells were seeded in six-well plates at a density of 150,000 cells / mL in the presence of 1 pM of CyCBuHeCle or CyCBgHClg. After 24 h of incubation, the cells treated with PSs were irradiated with an 850 nm LED lamp for 30 min inside the incubator, while the control cells were kept in a dark incubator without irradiation. After a 6 h incubation period, cells were washed with room-temperature saline and quenched by the addition of cold methanol. Subsequently, methanol-water-chloroform extraction (5:3:5 ratio) was added to the samples and the mixture vortexed for 10 min at 4°C followed by centrifuging at 16,000 xg for 10 min to achieve phase separation. The methanol-water phase, which contains polar metabolites, was separated and dried using a freeze-dryer benchtop for 24 h (Labconco, MO, USA). The remaining protein from the extraction was dissolved in 0.2 mM KOH overnight and then quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific). Dried metabolite extracts were resuspended in HPLC-grade water containing 1 pM piperazine- N, N'-bis (PIPES) added as an internal standard and transferred to HPLC vials for analysis. LC-MS / MS analysis was performed with ion-pairing reverse phase chromatography using an Ascentis Express column (C18, 5 cm x 2.1 mm, 2.7 pm) and a Waters Xevo TQ-S triple quadrupole mass spectrometer. Mass spectra were acquired using negative mode electrospray ionization operating in multiple reaction monitoring (MRM) mode. Peak processing was performed in MAVEN, and data for each sample was normalized to PIPES intensity. Statistical tests were performed in R using the t-test and p-value functions for Student’s t-test and Benjamini-Hochberg false discovery rate analysis, respectively.

[0247] M. Actin filaments staining.

[0248] 4T1 cells cultured in the 20 mm glass bottom dish were fixed in 4% formaldehyde solution for 15 min at 25°C and washed three times with PBS (pH 7.4). Permeabilization was accomplished using 0.2% Triton X-100 (Cat. No. T8787, Sigma- Aldrich, St. Louis, MO, USA) in PBS for 15 min at 25°C. The samples were further incubated in blocking solution using 1 % bovine serum albumin (BSA) for 1 h at 25°C. Next, rhodamine phalloidin dye (actin filaments staining probe) (Cat. No. R415,Invitrogen, Waltham, MA, USA) was diluted in 3% BSA and then cells were stained for 30 min at 25°C. Finally, the cells were mounted using Gold antifade reagent with DAPI, a nucleic acid stain dye (Cat. No. P6931 , Invitrogen, Waltham, MA, USA).

[0249] N. Measuring reactive oxygen species (ROS).

[0250] The cells were grown on a 20 mm glass bottom dish containing culture media with or without 1 pM of CyCBuHeCle or CyCBgHClg. After 24 h, cells treated with PSs were irradiated with an 850 nm LED lamp for 30 min in the incubator while the control cells were left in a dark incubator without irradiation. Immediately after irradiation, cells were stained with chloromethyl-2', 7'-dichlorodihydrofuorescein diacetate (CM- H2DCFDA) (Cat. No. C6827, Invitrogen), MitoSOX (Cat. No. M36006, Invitrogen) or Singlet Oxygen Sensor Green (Cat. No. S36002, Invitrogen) for 20 min. This staining period allowed for the detection of ROS generated by both the photochemistry of the photosensitizer and the mitochondrial response to PDT-induced damage. Live cell imaging was then performed immediately after staining to capture ROS levels. This protocol was specifically designed to measure total ROS production, including secondary ROS generated by mitochondria following PDT, providing a comprehensive assessment of the photodynamic effect on mitochondrial ROS generation.

[0251] O. Quantitative polymerase chain reaction (q-PCR) analysis.

[0252] Total RNA was extracted from the 4T1 cells at 6 h post-treatment of PSs using RNeasy Plus Micro kit (Cat. No. 74034, Qiagen, MD, USA). Next, 2 pg of RNA was used for cDNA synthesis using 2X RT master mix (Cat. No. 4368814, Thermo Scientific) according to the manufacturer’s protocol. Quantitative PCR (q-PCR) was performed using qPCR Luna 2X master mix (Cat. No. M3003S, New England BioLabs) in real-time PCR system (Agilent, USA). The base sequences of mice gene primers are mentioned in Table 5.

[0253] Table 5 illustrates sequences of primers used for real-time quantitative PCR.Table 5Gene Primer sequenceSense: 5’- AG G CCfCCT CTCCTACTTCG-3' R YAntisense: 5’-GCCTCAGCCCATCTTCTTCC-3’Sense: 5 -TCAGGTGACAAGTGACGGTG-3BakAntisense: 5 -CTGCTGTTCAGAAGGGGACG-Sense: ^^YTGCCTTQC C(3TTC-3-BidAntisense: 5’-CGTTGCTGACCTCAGAGTCC-3’Sense: 5 -TACAATGAGCTG CGTGTG GCCC-3'P-actinAntisense: 5’-

[0254] P. Western blotting.

[0255] Equal amounts of 50 pg total protein were separated on either 10% or 12% sodium dodecyl sulfate-polyacrylamide gel and electrotransferred onto nitrocellulose membranes. The membranes were washed with Tris-buffered saline (10 mM Tris HCI, 150 mM NaCI, pH 7.5) supplemented with 0.05% (v / v) Tween 20 (TBST) followed by blocking using TBST containing 5% (w / v) skimmed milk. The membranes were incubated overnight with specific primary antibodies to Snail (Cat. No. 3879, Cell Signaling Technology, 1 :500), PARP (Cat. No. 9542, Cell Signaling Technology, 1 :1 ,000), p- ERK1 / 2 (Cat. No. 4370, Cell Signaling Technology, 1 :1 ,000), ERK1 / 2 (Cat. No. 4695, Cell Signaling Technology, 1 :1 ,000), Bax (Cat. No. 14796, Cell Signaling Technology, 1 :500) N-cadherin (Cat. No. 610920, BD Bioscience, 1 :500), E-cadherin (Cat. No. 610181 , BD Bioscience, 1 :500), p-MLKL (Cat. No. 37333, Cell Signaling Technology, 1 :1 ,000), Cytochrome c (Cat. No. 11940, Cell Signaling Technology, 1 :1 ,000), and GAPDH (Cat. No. 5174, Cell Signaling Technology, 1 :1 ,000) at 4°C. The membranes were exposed to secondary antibodies conjugated to horseradish peroxidase for 1 h at 25°C and treated with ECL reagents. Chemiluminescence signals were visualized using the luminescent image analyzer BioRad (Hercules, CA, USA).

[0256] Q. Flow cytometry analysis.

[0257] 2 x 105cells were cultured in the culture medium containing 1 mM ofCyCBuHeCle or CyCBgHClg. The cells treated with cyanine-carboranes were irradiated with an 850 nm LED lamp for 30 min in the incubator while the control cells were kept in a dark incubator without irradiation. After 6 h, cells were washed two times with PBS (pH 7.4) and harvested with 0.05% trypsin / EDTA. Cell pellets were then resuspended in 500 mL Annexin binding buffer with FITC-conjugated Annexin V and PI (Cat. No. K101 -25, BioVision, Milpitas, CA, USA) according to the manufacturer’s instructions. The flow cytometry was performed using an Attune CytPix benchtop analyzer (ThermoFisher). Annexin-FITC fluorescence was collected through a 525 / 40 bandpass filter, whereas PI fluorescence was collected through a 610 / 20 bandpass filter. Data acquisition (2 x 104events per sample) was performed using the CytExpert 1 .2 software (BeckmanCoulter Life Sciences). Flow cytometry data acquisition and analysis were performed using FCS Express 7 software (De Novo Software, USA). The waiting time between sample preparation and measurement was kept constant, at a minimum of 30 min, to ensure complete stability of the live samples.

[0258] R. Assessment of mitochondrial membrane potentials.

[0259] Mitochondrial membrane potential was measured with a fluorescent dye, tetramethylrhodamine methyl ester (TMRM) (Cat. No. T668, Molecular Probes, Eugene, OR, USA). The mitochondrial membrane potential was quantified according to the intensity of intracellular fluorescence that was averaged over three different regions of interest for each experiment.

[0260] S. Orthotopic cancer model.

[0261] All animal protocols were approved and performed in accordance with guidelines set by the Institute of Animal Care and Use Committee (IACUC) of Michigan State University (license number: 201900200). Ethical approval for the animal experiments was obtained from the IACUC, ensuring compliance with all relevant guidelines. 6DT1 cells were harvested for tumor implantation at 80% confluence while in the logarithmic phase of growth. 10,000 6DT1 cells in 50 pL of sterile saline were inoculated into the right fourth mammary fat pad of 6-8 week-old syngeneic FVB / NJ female mice (purchased from Jackson Laboratories, Bar Harbor, ME, USA). Tumor growth was monitored every other day with external caliper measurements to determine tumor length and width to calculate volume, V = L x W2 / 2. Animal well-being was also monitored by recording mouse weight every other day and watching for potential skin irritation at the tumor site. Mice were euthanized at a 28-day endpoint when the majority of control mice exhibit excessive morbidity due to tumor burden.

[0262] T. Highest occupied molecular orbital (HOMO) level estimation.

[0263] Previous examples with Cy+organic salts paired with tetrakis (4- fluorophenyl) borate (C24H16BF4 ), cobalticarborane (C4B18C0 ), and tetrakis (pentafluorophenyl) borate (C24BF20 ) yielded open-circuit voltages ( Voc) from photovoltaic (PV) devices and experimentally determined HOMO energy levels from ultraviolet photoelectron spectroscopy measurements. This data was fitted with a linear model to produce a relationship between measured Voc and measured HOMO levels. PVs were fabricated with the carborane-based organic salts as described previously. The Voc was measured and used with the Voc -HOMO level model to estimate the HOMO level of the cyanine-carborane organic salts.

[0264] U. Ultraviolet-visible (UV-VIS) NIR spectroscopy.

[0265] To confirm the formation of the CyCBuHeCleand CyCBgHClg nanoparticles, monomer solutions were prepared in DMSO. The DMSO solutions were mixed with water at a ratio of 10% DMSO and 90% water to form nanoparticles. Both DMSO (monomer) and 10% DMSO (nanoparticle) solutions were analyzed in a Perkin Elmer Lambda 900 UV / VIS / NIR Spectrometer subtracting the blank reference solution in transmittance (7) mode. The resulting transmittance spectra were then converted to absorbance (A) as 1(A) = 100 - T(A).

[0266] V. Photodynamic therapy (PDT).

[0267] Nine days after orthotopic injection, mice bearing tumors were assigned randomly to one of four treatment groups: 1. vehicle injection (Veh), 2. organic salt injection, 3. vehicle injection with near-infrared (NIR) light irradiation (Veh + 850 nm), and 4. organic salt injection with NIR light irradiation. For organic salt injection treatment groups (2 and 4), mice were given a 2 pmol / kg intravenous injection of a fluorescent organic salt dissolved in 10% DMSO and 0.03% Tween 20 in 100 pL of sterile saline prior to injection through the lateral tail vein. Vehicle groups (1 and 3) received a tail vein injection of 10% DMSO and 0.03% Tween 20 in 100 pL of sterile saline. Leica software was used to quantitate relative brightness and localization within tumor tissue relative to normal tissue at various time points throughout the experimental example. At 24 h post IV injections of the organic salt, NIR light irradiation groups (3 and 4) were anesthetized with 2.5% isoflurane, placed on a heated pad, and underwent tumor irradiation with an 850 nm LED. Mice received a 150 J / cm2dose over 20 min. The PDT treatment was repeated every 48 h for a period of 20 days.

[0268] W. In vivo imaging.

[0269] For biodistribution examples, at 1 1 days post orthotopic injection tumorbearing mice were dosed with 2 pmol / kg of CyCBuHeCle or CyCBgHClg via intravenous tail vein injection. For imaging purposes, the mice were subjected to anesthesia with 2.5% isoflurane, and subsequently, brightfield and NIR fluorescent images were captured at specified time intervals. This was achieved by employing a Leica M165FC stereoscope equipped with a 740 nm PE4000 LED light source, and an InGaAs NIR DFC9000GT camera. The Leica software was employed to analyze the fluorescence intensity and identify specific regions of interest (ROIs) encompassing the tumor in the right fourth mammary fat pad, the liver, and the left fourth mammary fat pad. The measured fluorescence values were then normalized relative to a vehicle-injected mouse.

[0270] X. Statistical analyses.

[0271] Statistical analyses were performed using an unpaired student’s t-test and all error bars are representative of standard deviation, except where otherwise noted. All displayed data has a minimum of three biological replicates. Curve fittings were done using GraphPad Prism software. P-values < 0.05 are reported as statistically significant (*)■

[0272] The foregoing description of the embodiments has been provided for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosure. Individual elements or features of a particular embodiment are generally not limited to that particular embodiment, but, where applicable, are interchangeable and can be used in a selected embodiment, even if not specifically shown or described. The same may also be varied in many ways. Such variations are not to be regarded as a departure from the disclosure, and all such modifications are intended to be included within the scope of the disclosure.

Claims

CLAIMSWhat is claimed is:1 . A salt for administration to a mammalian subject in treatment of neoplasms or dysplasias, the salt comprising: a cation; and an anion including a transition metal free carborane or borohydride cluster, the salt configured to lead to targeted cell death of the neoplasms or dysplasias when subjected to an external stimulus including light, neutrons, or a combination thereof.

2. The salt of claim 1 , wherein the cation is a photoactive cation, and the photoactive cation is configured to become excited and generate reactive oxygen species (ROS) when the salt is subjected to the external stimulus including the light.

3. The salt of claim 2, wherein the light has a wavelength ranging from 700 nm to 1200 nm.

4. The salt of claim 3, wherein the wavelength ranges from 750 nm to 950 nm.

5. The salt of claim 2, wherein the ROS include superoxide, peroxide, singlet oxygen, or any combination thereof.

6. The salt of claim 5, wherein the ROS include at least two of superoxide, peroxide, and singlet oxygen.

7. The salt of claim 4, wherein the ROS include singlet oxygen.

8. The salt of claim 1 , wherein the neoplasms or dysplasias are in a tumor.

9. The salt of claim 8, wherein the salt is configured to accumulate inside the tumor while an exo-tumor residue is excreted from the mammalian subject.

10. The salt of claim 9, wherein the exo-tumor residue is configured to be excreted from the mammalian subject by a liver of the mammalian subject, a kidney of the mammalian subject, a spleen of the mammalian subject, or any combination thereof.11 . The salt of claim 1 , wherein the salt is water insoluble.

12. The salt of claim 1 , wherein a quantity of atoms in the anion ranges from 20 to 1000.

13. The salt of claim 12, wherein the quantity of atoms in the anion ranges from 20 to 54.

14. The salt of claim 13, wherein the quantity of atoms in the anion ranges from 20 to 30.

15. The salt of claim 1 , wherein a molecular weight of the anion ranges from 100 g / mol to 3000 g / mol.

16. The salt of claim 15, wherein the molecular weight of the anion ranges from 200 g / mol to 1500 g / mol.

17. The salt of claim 1 , the salt has an absolute highest occupied molecular orbital (HOMO) energy ranging from 4 eV to 6eV.

18. The salt of claim 17, wherein the absolute HOMO energy ranges from 4.9 eV to 5.4 eV.

19. The salt of claim 1 , wherein the salt has an absolute lowest unoccupied molecular orbital (LUMO) energy ranging from 3.5 eV to 4.5 eV.

20. The salt of claim 1 , wherein the salt has a bandgap ranging from 1 eV to 1 .75 eV.21 . The salt of claim 1 , wherein the salt has an IC50 under the external stimulus of less than or equal to 5 micromolar (pM).

22. The salt of claim 21 , wherein the IC50 under the external stimulus is less than or equal to 2 pM.

23. The salt of claim 1 , wherein the salt has an IC50 absent external stimulus of greater than or equal to 2 micromolar (pM).

24. The salt of claim 1 , wherein the salt has a zeta potential ranging from -30 millivolts (mV) to -10 mV.

25. The salt of claim 1 , wherein the anion is a dianion.

26. The salt of claim 1 , wherein the anion includes one or more halogens.

27. The salt of claim 1 , wherein the cation and the anion are chemically linked such that the salt is an inner salt or zwitterion.

28. The salt of claim 1 , wherein the salt is in the form of a nanoparticle.

29. The salt of claim 28, wherein the nanoparticle is free of a coating.

30. The salt of claim 28, wherein the nanoparticle has a diameter ranging from 5 nm to 40 nm.31 . The salt of claim 30, wherein the diameter of the nanoparticle ranges from 22 nm to 38 nm.

32. The salt of claim 1 , wherein the anion is the transition metal free carborane, the transition metal free carborane is one of:CB9R1 (R2)4(R3)4R4N’, Or CBllRl(R2)5(R3)5R4N-, andN is an integer, andRi, R2, R3, and R4 are independently selected from the group consisting of: H, F, Cl, Br, I, alkyl, functionalized alkyl, aryl, functionalized aryl, OH, O-alkyl, O-aryl, NH2, NR2, NR3, SH, SR, SR2, haloalkyl, perfluoroaryl, acetyl, ester, ether, formyl, amide, formamide, carboxyl, alkanoate, and carbamate, where R is any functional group.

33. The salt of claim 1 , wherein the anion is the transition metal free carborane, and the transition metal free carborane is one of:CB9H10 , or CBuHeCle , orCB9HCI9 , or CioBiiH32Si”, or34. The salt of claim 1 , wherein the anion is the borohydride cluster, the borohydride cluster is one of:BioRioN-, or Bi2Ri2NN is an integer, andR is selected from the group consisting of: H, F, Cl, Br, I, alkyl, functionalized alkyl, aryl, functionalized aryl, OH, O-alkyl, O-aryl, NH2, N(RI)2, N(Ri)s, SH, SR1, S(RI)2, haloalkyl, perfluoroaryl, acetyl, ester, ether, formyl, amide, formamide, carboxyl, alkanoate, and carbamate, where R1 is any functional group.

35. The salt of claim 1 , wherein the cation is an organic cation.

36. The salt of claim 35, wherein the organic cation is a cyanine, a polymethine, or a combination thereof.

37. The salt of claim 36, wherein the organic cation includes the cyanine, and the cyanine includes:

38. A salt for administration to a mammalian subject in treatment of neoplasms or dysplasias, the salt comprising: an ion; and a counterion including a transition metal free carborane or borohydride cluster, the salt configured to lead to targeted cell death of the neoplasms or dysplasias when subjected to an external stimulus including light, neutrons, or a combination thereof.

39. The salt of claim 38, wherein the counterion is the transition metal free carborane, the transition metal free carborane is one of:CB9RI(R2)4(R3)4R4N+, or CB11 R1 (R2)5(R3)5R4N+N is an integer, andR1, R2, R3, and R4 are independently selected from the group consisting of: ammonium, quarternized ammonium, phosphonium, stibonium, bismuthonium, NR2, NR3, SR, SR2, functionalized aryl, and functionalized alkyl, where R is any functional group.

40. The salt of claim 39, wherein the counterion is the borohydride cluster, the borohydride cluster is one of:B RioN+, or Bi2Ri2N+, andN is an integer, andR is selected from the group consisting of: ammonium, quarternized ammonium, phosphonium, stibonium, bismuthonium, N(RI)2, N(Ri)s, SRi, S(RI)2, functionalized aryl, and functionalized alkyl, where Ri is any functional group.41 . A salt for administration to a mammalian subject in treatment of neoplasms or dysplasias, the salt comprising: an organic cation; and a dianion including a carborane or borohydride cluster, the salt configured to lead to targeted cell death of the neoplasms or dysplasias when subjected to an external stimulus including light, neutrons, or a combination thereof, and the salt having a IC50 under the external stimulus of less than or equal to 2 pM.

42. A salt nanoparticle for administration to a mammalian subject in treatment of neoplasms or dysplasias, the salt nanoparticle comprising:An organic cation; and a dianion including a carborane or borohydride cluster, the salt nanoparticle configured to lead to targeted cell death of the neoplasms or dysplasias when subjected to an external stimulus including light, neutrons, or a combination thereof, and the salt nanoparticle having a diameter ranging from 5 nm to 40 nm.

43. A salt for use in photodynamic therapy, the salt comprising: a photoactive organic cation including a cyanine having the structure:an anion including one of:(i) a first transition metal free carborane having the formula CBgRi(R2)4(R3)4R4N; where N is an integer and R-i, R2, R3, and R4 are independently selected from the group consisting of: H, F, Cl, Br, I, alkyl, functionalized alkyl, aryl, functionalized aryl, OH, O-alkyl, O-aryl, NH2, NR2, NR3, SH, SR, SR2, haloalkyl, perfluoroaryl, acetyl, ester, ether, formyl, amide, formamide, carboxyl, alkanoate, and carbamate, and where R is any functional group, the first transition metal free carborane having the structure:, or(ii) a second transition metal free carborane having the formula CB11 R1 (R2)5(R3)SR4N’, where N is an integer and R1, R2, R3, and R4 are independently selected from the group consisting of: H, F, Cl, Br, I, alkyl, functionalized alkyl, aryl, functionalized aryl, OH, O-alkyl, O-aryl, NH2, NR2, NR3, SH, SR, SR2, haloalkyl, perfluoroaryl, acetyl, ester, ether, formyl, amide, formamide, carboxyl, alkanoate, and carbamate, and where R is any functional group, the second transition metal free carborane having the structure:(iii) a first borohydride cluster having the formula BioRioN-, where N is an integer and R is selected from the group consisting of: H, F, Cl, Br, I, alkyl, functionalized alkyl, aryl, functionalized aryl, OH, O-alkyl, O-aryl, NH2, N(RI)2, N(Ri)s, SH, SR1, S(RI)2, haloalkyl, perfluoroaryl, acetyl, ester, ether, formyl, amide, formamide, carboxyl, alkanoate, and carbamate, and where R1 is any functional group, the first borohydride cluster having the structure:(iv) a second borohydride cluster having the formula Bi2Ri2N-, where N is an integer and R is selected from the group consisting of: H, F, Cl, Br, I, alkyl, functionalized alkyl, aryl, functionalized aryl, OH, O-alkyl, O-aryl, NH2, N(RI)2, N(Ri)s, SH, SR1, S(RI)2, haloalkyl, perfluoroaryl, acetyl, ester, ether, formyl, amide, formamide, carboxyl, alkanoate, and carbamate, and where R1 is any functional group, the second borohydride cluster having the structure:the salt configured to become excited and generate reactive oxygen species (ROS) when subjected to an external stimulus including light having a wavelength ranging from 700 nm to 1200 nm, the ROS including singlet oxygen.

44. A method of exciting an salt in a cell of a subject, the method comprising: exciting the salt in the cell, the salt including an organic cation and an anion, the organic cation including a cyanine or a polymethine, and the anion including a transition metal free carborane or a borohydride nanocluster.

45. The method of claim 44, further comprising: prior to the exciting, administering a composition including the salt to the subject such that the cell takes up the salt.

46. The method of claim 44, wherein, during the exciting, the salt is in the form of a nanoparticle.

47. The method of claim 44, wherein the exciting includes exposing the salt to light, and the light has a wavelength ranging from 700 nm to 1200 nm.

48. The method of claim 47, wherein the exciting causes the salt generate reactive oxygen species (ROS).

49. The method of claim 48, wherein the ROS include singlet oxygen.

50. The method of claim 44, wherein the exciting includes exposing the salt to a neutron beam.51 . The method of claim 50, wherein the anion includes the transition metal free carborane or the borohydride nanocluster, and greater than 40% of boron atoms in the transition metal free carborane or the borohydride nanocluster are10B.

52. The method of claim 44, wherein the cell is a cancer cell, and the cancer cell includes brain cancer cell, head and / or neck cancer cell, lung cancer cell, breast cancer cell, gastrointestinal cancer cell, pancreatic cancer cell, prostate cancer cell, liver cancer cell, skin cancer cell, ovarian cancer cell, cervical cancer cell, bladder cancer cell, renal cancer cell, soft tissue sarcoma cell, thyroid cancer cell, endometrial cancer cell, or any combination thereof.

53. The method of claim 52, wherein the salt is taken up by mitochondria, endoplasmic reticulum, Golgi apparatus, cytoplasm, or any combination thereof.

54. The method of claim 53, wherein the salt is taken up by the mitochondria, the endoplasmic reticulum, and the Golgi apparatus.

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