Red blood cell-derived nanoparticle system

Abstract

The disclosure herein relates to an engineered, nano-sized platform, derived from erythrocyte ghosts (EGs), with dual near infrared (NIR) fluorescence and magnetic resonance (MR) characteristics. In some embodiments, the EGs comprise co-encapsulation of a brominated carbocyanine compound and gadobenate dimeglumine, respectively. Some embodiments of the present disclosure relate to usage of the EGs, as well as methodology for screening of cancer and tumor tissues.

Description

RED BLOOD CELL-DERIVED NANOPARTICLE SYSTEMCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 620,077, entitled “RED BLOOD CELL-DERIVED NANOPARTICLE SYSTEM,” which was filed January 11, 2024, and to U.S. Provisional Application No. 63 / 644,270, entitled “RED BLOOD CELL-DERIVED NANOPARTICLE SYSTEM ,” which was filed May 8. 2024. The entire disclosure of both of which are expressly incorporated herein by reference as if fully set forth herein.STATEMENT REGARDING FEDERALLY SPONSORED R&D

[0002] The findings of the present disclosure were made with government support under funds awarded by The National Science Foundation, Grant No. 1940965. The government retains certain rights.BACKGROUND DESCRIPTION

[0003] The present disclosure relates broadly to biomedical imaging, cancer imaging, drug delivery, laser therapy, and pharmaceuticals. It has applications to medical imaging, and to treatment of specific types of cancers.

[0004] A major problem related to cancer diagnosis is late diagnosis. For example, ovarian cancer is the deadliest gynecological cancer since only about 15% of patients are diagnosed at stage I where the tumor is localized to the ovaries. Most individuals with ovarian cancer are diagnosed with regional (20%) or distant disease (65%) with 5-year survival rates of 72% and 27%, respectively. Nearly all patients undergo surgery to remove the tumors. The current surgical standard is to remove implants >1 cm. However, even these lesions are difficult to detect by current pre-operative imaging methods, or visually in the open abdomen during surgery’. Therefore, clinically impactful methods are needed to identify ovarian tumors early, and remove all of them entirely.SUMMARY OF SOME EXAMPLE EMBODIMENTS

[0005] Some embodiments of the present disclosure relate to a liposome, micelle, vesicle-based nanoparticle, cell membrane, or RBC-derived nano-sized particle.In some embodiments, the nanoparticle is a derived from a red blood cell, such as a hemoglobin-depleted, or '’ghost" red blood cell. In some embodiments, the nanoparticle has a dual imaging modality. In some embodiments, the nanoparticle comprises a paramagnetic gadolinium ion chelated with benzyloxypropionictetra-acetate gadolinium (Gd-BOPTA), and NIR halogenated carbocyanine dyes containing indoline, and indolenine heterocycles. In some embodiments, the nanoparticle comprises at least two imaging agents. In some embodiments, the two imaging agents result in an enhanced fluorescence emission. Also disclosed is a use of the nanoparticle platform and its resulting high imaging sensitivities for both early detection and localization of small ovarian tumors prior to surgery by MRI, andNIRFI at surgery to aide staging and guide complete surgical removal of all tumors which cannot otherwise be visualized.

[0006] Some embodiments of the present disclosure relate to a vesicle-based nanoparticle system with therapeutic applications towards a tumor and / or a cancer. In some embodiments, the vesicle-based nanoparticle system comprises membrane, a magnetic resonance imaging agent and a fluorescence imaging agent, wherein the vesicle-based nanoparticle system is formulated for magnetic resonance imaging and fluorescence imaging. In some embodiments, the membrane is a plasma membrane and / or bilayer. In some embodiments, the membrane comprises a phospholipid. In some embodiments, the fluorescence imaging is Near-IR fluorescence imaging (NIRFI), fluorescence endoscopy, and / or fluorescence laparoscopy. In some embodiments, the magnetic resonance imaging agent is incorporated inside the nanoparticle. In some embodiments, the magnetic resonance imaging agent is incorporated on the surface of the nanoparticle. In some embodiments, the magnetic resonance imaging agent is incorporated on the surface and inside of the nanoparticle. In some embodiments, the fluorescence imaging agent is incorporated inside the nanoparticle. In some embodiments, the fluorescence imaging agent is incorporated inside and on the surface of the nanoparticle. In some embodiments, the nanoparticle is a liposome, micelle, vesicle, cell, or a cell-derived particle. In some embodiments, the cell is mammalian and / or human. In some embodiments, the cell is an erythrocyte. In some embodiments, the erythrocyte is a hemoglobin-depleted, ‘"ghost” erythrocyte. In some embodiments, the magnetic resonance imaging agent comprises one or more of: gadolinium, gadopiclenol, gadodiamide, gadoversetamide, gadoxetate, Gadofosveset, gadobenate, gadopentetate, Gadoteridol. Gadobutrol. Gadoterate, Iron Oxide, Manganese, or any combination thereof. In some embodiments, the magnetic resonance imaging agent comprises gadolinium. In some embodiments, the magneticresonance imaging agent comprises one or more of: gadopiclenol, Gadopentetate dimeglumine (Gd-DTPA). gadobenate dimeglumine. Gd-BOPTA. gadoxetate disodium, Gadofosveset trisodium, Gd-DTPA-BMA, Gd-DTPA-BMEA, Gd-EOB-DTPA, Gd-HP- D03A, Gd-BT-DO3A, Gd-DOTA, Ferumoxides, Ferumoxtran-10, Ferumoxsil, Mangafodipir trisodium, Gadoterate meglumine, or any combination thereof. In some embodiments, the magnetic resonance imaging agent comprises paramagnetic gadolinium ion chelated with benzyloxypropionictetra-acetate gadolinium (Gd-BOPTA). In some embodiments, the fluorescence imaging agent comprises cyanine, indoline, indolenine, or any combination thereof. In some embodiments, the fluorescence imaging agent comprises a halogenated carbocyanine dye, a brominated cyanine dye, an indolenine heterocycle, a closed chain cyanine, a hemi cyanine, a streptocyanine, a cyanine with a hetrocyclic moiety (such as indole, benzothiazole), or any combination thereof. In some embodiments, the fluorescence imaging agent comprises BrCylO6, BrCyl l l, BrCyl l2, IR700, IR800, indocyanine green, or any combination thereof.

[0007] Some embodiments of the present disclosure relate to a cell comprising a magnetic resonance imaging agent and a fluorescence imaging agent. In some embodiments, the fluorescence imaging is Near-IR fluorescence imaging (NIRFI), fluorescence endoscopy, and / or fluorescence laparoscopy. In some embodiments, the magnetic resonance imaging agent is present inside the cell. In some embodiments, the magnetic resonance imaging agent is present on the surface of the cell. In some embodiments, the magnetic resonance imaging agent is present inside and on the surface of the cell. In some embodiments, the fluorescence imaging agent is present inside the cell. In some embodiments, the fluorescence imaging agent is present on the surface of the cell. In some embodiments, the cell is mammalian and / or human. In some embodiments, the cell is an erythrocyte. In some embodiments, the erythrocyte is a hemoglobin-depleted, ‘'ghost” erythrocyte. In some embodiments, the magnetic resonance imaging agent comprises one or more of: gadolinium, gadodiamide, gadoversetamide, gadoxetate, Gadofosveset, gadobenate, gadopiclenol, gadopentetate, Gadoteridol, Gadobutrol, Gadoterate, Iron Oxide, Manganese, or any combination thereof. In some embodiments, the magnetic resonance imaging agent comprises gadolinium. In some embodiments, the magnetic resonance imaging agent comprises one or more of: gadopiclenol, Gadopentetate dimeglumine (Gd-DTPA), gadobenate dimeglumine, Gd-BOPTA. gadoxetate disodium, Gadofosveset trisodium. Gd-DTPA-BMA, Gd-DTPA-BMEA. Gd-EOB-DTPA, Gd-HP- DO3A, Gd-BT-DO3A, Gd-DOTA, Ferumoxides, Ferumoxtran-10, Ferumoxsil,Mangafodipir trisodium. Gadoterate meglumine, or any combination thereof. In some embodiments, the magnetic resonance imaging agent comprises paramagnetic gadolinium ion chelated with benzyloxypropionictetra-acetate gadolinium (Gd-BOPTA). In some embodiments, the fluorescence imaging agent comprises cyanine, indoline, indolenine, or any combination thereof. In some embodiments, the fluorescence imaging agent comprises a halogenated carbocyanine dye, a brominated cyanine dye, an indolenine heterocycle, a closed chain cyanine, a hemi cyanine, a streptocyamne, a cyanine with a hetrocyclic moiety (such as indole, benzothiazole), or any combination thereof. In some embodiments, the fluorescence imaging agent comprises BrCylO6, BrCyl l l, BrCyl l2, IR700, IR800, indocyanine green, or any combination thereof.

[0008] Some embodiments of the present disclosure relate to a composition. In some embodiments, the composition comprises the nanoparticle of any one the embodiments of the present disclosure. In some embodiments, the composition comprises the cell of any one of the embodiments of the present disclosure. In some embodiments, the composition is formulated for administration to a subject.

[0009] Some embodiments of the present disclosure relate to a medicament. In some embodiments, the medicament comprises the nanoparticle of any one the embodiments of the present disclosure. In some embodiments, the medicament comprises the cell of any one of the embodiments of the present disclosure. In some embodiments, the medicament is formulated for administration to a subject.

[0010] Some embodiments of the present disclosure relate to a kit. In some embodiments, the kit comprises the nanoparticle of any one of the embodiments of the present disclosure. In some embodiments, the kit comprises the cell of any one of the embodiments of the present disclosure. In some embodiments, the kit is formulated for administration to a subject.

[0011] Some embodiments of the present disclosure relate to a use for the kit, medicament, and / or composition of any embodiment herein in detecting and / or the localization of a cancer and / or a tumor. Some embodiments of the present disclosure relate to a use for the kit, medicament, and / or composition of any embodiment herein in screening for the presence of a cancer and / or tumor. In some embodiments, the cancer and / or tumor is a solid tumor. In some embodiments, the cancer and / or tumor is a human cancer and / or tumor. In some embodiments, the cancer and / or tumor is present in an ovary, lymph node, pancreas, colorectal tissue, and / or intraperitoneal tissue. In some embodiments, the detection and / or localization is carried out in a subject.

[0012] Some embodiments of the present disclosure relate to a use for the kit, medicament, and / or composition of any embodiment herein in detecting and / or the localization of lymph nodes. In some embodiments, the lymph node is a sentinel lymph node. In some embodiments, the lymph nodes contain cancer or tumor that is a human cancer or tumor. In some embodiments, the lymph nodes contain cancer or tumor that is an ovarian cancer or tumor. In some embodiments, the detection and / or localization is carried out in a subject.

[0013] Some embodiments of the present disclosure relate to a use for the kit, medicament, and / or composition of any embodiment herein in diagnosing a subject as having a cancer and / or a tumor. In some embodiments, the cancer or tumor is a human cancer or tumor. In some embodiments, the subject is mammalian and / or human. In some embodiments, the cancer or tumor is an ovarian cancer or tumor.

[0014] Some embodiments of the present disclosure relate to a method for diagnosing a subject as having a cancer and / or a tumor. In some embodiments, the method comprises administering to the subject the kit. medicament, and / or composition of any embodiment herein, and imaging the subject using MRI and a fluorescence-based scan, wherein positive labeling of a cell or tissue indicates that the subject has the cancer and / or tumor. In some embodiments, the subject is mammalian and / or human. In some embodiments, the cancer and / or tumor is ovarian. In some embodiments, the fluorescencebased scan is NIRFI. In some embodiments, the fluorescence scan is fluorescence endoscopy. In some embodiments, the fluorescence scan is fluorescence laparoscopy.

[0015] Some embodiments of the present disclosure relate to a method for detecting the presence of lymph node(s) and / or of cancer and / or tumor in a subject. In some embodiments, the method comprises administering to the subject the kit, medicament, and / or composition of any embodiment herein, and imaging the subject using MRI and / or a fluorescence-based scan, wherein the localization of the magnetic resonance imaging agent and the fluorescence imaging agent within a subject is indicative of the cancer and / or tumor position in that subject. In some embodiments, the subject is mammalian and / or human. In some embodiments, the cancer and / or tumor is ovarian. In some embodiments, the fluorescence-based scan is NIRFI, fluorescence endoscopy, and / or fluorescence laparoscopy.

[0016] Some embodiments of the present disclosure relate to a method for treating a cancer and / or tumor in a subject. In some embodiments, the method comprises administering to the subject the kit, medicament, and / or composition of any embodimentherein, imaging the subject using MRI to detect the localization of the cancer and / or tumor within the subject’s body, and / or conducting surgery on the subject while simultaneously imaging the subject with a fluorescence-based scan, wherein the surgery removes the cells or tissues that are positively labeled by the magnetic resonance imaging agent and / or the fluorescence imaging agent. In some embodiments, the subject is mammalian and / or human. In some embodiments, the cancer and / or tumor is ovarian. In some embodiments, the fluorescence-based scan is NIRFI, fluorescence endoscopy, and / or fluorescence laparoscopy. In some embodiments, the method further comprises administering to the subject radiation, chemotherapy, and / or an effective dose of a pharmaceutical molecule for treating the cancer and / or tumor.

[0017] Some embodiments of the present disclosure relate to a method for removing a lymph node(s) and / or cancer and / or tumor from a subject. In some embodiments, the method comprises administering to the subject a vesicle-based nanoparticle system comprising a magnetic resonance imaging agent and a fluorescence imaging agent, imaging the subject using MRI to detect the localization of the cancer and / or tumor within the subject’s body, and / or conducting surgery on the subject while simultaneously imaging the subject with a fluorescence-based scan, wherein the surgery removes the cells or tissues that are positively labeled by the magnetic resonance imaging agent and / or the fluorescence imaging agent. In some embodiments, the cancer and / or tumor is completely removed from the subject. In some embodiments, the subject is mammalian and / or human. In some embodiments, the cancer and / or tumor is ovarian. In some embodiments, the fluorescence-based scan is NIRFI, fluorescence endoscopy, and / or fluorescence laparoscopy. In some embodiments, the method further comprises administering to the subject radiation, chemotherapy, and / or an effective dose of a pharmaceutical molecule for treating the cancer and / or tumor. In some embodiments, the magnetic resonance imaging agent is incorporated inside the nanoparticle. In some embodiments, the magnetic resonance imaging agent is incorporated on the surface of the nanoparticle and / or within the nanoparticle. In some embodiments, the fluorescence imaging agent is incorporated inside the nanoparticle. In some embodiments, the fluorescence imaging agent is incorporated on the surface of the nanoparticle. In some embodiments, the nanoparticle is a liposome, micelle, vesicle, cell, or cell-derived particle. In some embodiments, the cell is mammalian and / or human. In some embodiments, the cell is an erythrocyte. In some embodiments, the erythrocyte is a hemoglobin-depleted, "‘ghost” erythrocyte. In some embodiments, the magnetic resonance imaging agent comprises oneor more of: gadolinium, gadodiamide, gadoversetamide, gadoxetate, Gadofosveset, gadobenate, gadopiclenol, gadopentetate, Gadoteridol, Gadobutrol. Gadoterate, Iron Oxide, Manganese, or any combination thereof. In some embodiments, the magnetic resonance imaging agent comprises gadolinium. In some embodiments, the magnetic resonance imaging agent comprises one or more of: gadopiclenol, Gadopentetate dimeglumine (Gd-DTPA). gadobenate dimeglumine. Gd-BOPTA. gadoxetate disodium, Gadofosveset trisodium. Gd-DTPA-BMA, Gd-DTPA-BMEA. Gd-EOB-DTPA, Gd-HP- D03A, Gd-BT-DO3A, Gd-DOTA, Ferumoxides, Ferumoxtran-10, Ferumoxsil, Mangafodipir trisodium, Gadoterate meglumine, or any combination thereof. In some embodiments, the magnetic resonance imaging agent comprises paramagnetic gadolinium ion chelated with benzyloxypropionictetra-acetate gadolinium (Gd-BOPTA). In some embodiments, the fluorescence imaging agent comprises cyanine, indoline, indolenine, or any combination thereof. In some embodiments, the fluorescence imaging agent comprises a halogenated carbocyanine dye, a brominated cyanine dye, an indolenine heterocycle, a closed chain cyanine, a hemi cyanine, a streptocyanine, a cyanine with a hetrocyclic moiety (such as indole, benzothiazole), or any combination thereof. In some embodiments, the fluorescence imaging agent comprises BrCylO6, BrCyl l l, BrCyl l2, IR700, IR800, indocyanine green, or any combination thereof.

[0018] Some embodiments of the present disclosure relate to a nanoparticle vesicle for magnetic resonance imaging and fluorescence imaging, comprising: a membrane-bound vesicle; a magnetic resonance imaging agent within the membranebound vesicle; and a fluorescence imaging agent within the membrane-bound vesicle. In some embodiments, the vesicle is a liposome, micelle, cell membrane, or a cell-derived nanoparticle. In some embodiments, the membrane is a plasma membrane and / or bilayer. In some embodiments, the plasma membrane and / or bilayer comprises a phospholipid. In some embodiments, the nanoparticle vesicle is configured for imaging in a Near-IR fluorescence imaging (NIRFI) system, in a fluorescence endoscopy system, and / or in a fluorescence laparoscopy system. In some embodiments, the magnetic resonance imaging agent is incorporated inside the nanoparticle. In some embodiments, the magnetic resonance imaging agent is incorporated on the surface of the nanoparticle. In some embodiments, the magnetic resonance imaging agent is incorporated on the surface of the nanoparticle and inside the nanoparticle. In some embodiments, the vesicle is a hemoglobin-depleted, "‘ghost” erythrocyte. In some embodiments, the magnetic resonance imaging agent comprises one or more of: gadolinium, gadodiamide, gadoversetamide,gadoxetate. Gadofosveset, gadobenate. gadopiclenol, gadopentetate, Gadoteridol, Gadobutrol, Gadoterate. Iron Oxide, Manganese, or any combination thereof. In some embodiments, the magnetic resonance imaging agent comprises one or more of: gadopiclenol, Gadopentetate dimeglumine (Gd-DTPA), gadobenate dimeglumine, Gd- BOPTA, gadoxetate disodium, Gadofosveset trisodium, Gd-DTPA-BMA, Gd-DTPA- BMEA, Gd-EOB-DTPA, Gd-HP-DO3A, Gd-BT-DO3A, Gd-DOTA, Ferumoxides, Ferumoxtran-10. Ferumoxsil, Mangafodipir trisodium, Gadoterate meglumine, or any combination thereof. In some embodiments, the fluorescence imaging agent comprises a halogenated fluorophore, cyanine, indoline, indolenine, or any combination thereof. In some embodiments, the fluorescence imaging agent comprises BrCylO6, BrCyl l l, BrCy 112, IR700. IR800. indocyanine green, or any combination thereof.

[0019] Also disclosed herein is a method of detecting the presence of a cancer and / or a tumor in a subject. In some embodiments, the method comprises administering to the subject the nanoparticle vesicle of any one of the previous claims; and imaging the subject using magnetic resonance imaging and / or a fluorescence-based scan, wherein the localization of the magnetic resonance imaging agent and the fluorescence imaging agent within a subject is indicative of the cancer and / or tumor position. In some embodiments, the subject is mammalian and / or human. In some embodiments, the cancer and / or tumor is a solid tumor. In some embodiments, the cancer and / or tumor is present in an ovary, lymph node, pancreas, colorectal tissue, intraperitoneal tissue, brain, eyes. head. neck, alimentary canal, lungs, mediastinum, heart, liver, spleen, gallbladder, adrenal glands, bladder, uterus, prostate, cervix, vagina, bones, muscles, skin, or any combination thereof. In some embodiments, the fluorescence-based scan is infrared, visual, NIRFI, fluorescence endoscopy, and / or fluorescence laparoscopy. In some embodiments, the method further comprises administering to the subject an effective amount of a molecule or therapy for treating the cancer and / or the tumor.

[0020] Also disclosed herein is a method of preparing a subj ect for dual imaging analysis, the method comprising administering to the subject the nanoparticle vesicle of any one of the embodiments of the present disclosure. In some embodiments, the dual imaging analysis comprises fluorescence and / or MR imaging. In some embodiments, the nanoparticle vesicle is administered through inhalation / spray, or through intravenous, intraperitoneal, intramuscular, or subcutaneous injection.

[0021] Also disclosed herein is a nanoparticle vesicle for magnetic resonance imaging and fluorescence imaging, comprising: a membrane-bound vesicle; a magneticresonance imaging agent Gd-BOPTA, gadobenate dimeglumine, or a chemical derivative thereof within the membrane-bound vesicle; and a fluorescence imaging agent comprising BrCyl l l, BrCyl l2, indocyanine green, or a chemical derivative thereof, within the membrane-bound vesicle.

[0022] Also disclosed herein is a method of conducting magnetic resonance imaging and fluorescence imaging in a subject in need thereof, the method comprising: administering to the subject a nanoparticle vesicle, wherein the nanoparticle vesicle comprises: an outer membrane; a magnetic resonance imaging agent comprising Gd- BOPTA, and / or gadobenate dimeglumine; and a fluorescence imaging agent comprising BrCyl l l, BrCyl l2. and / or indocyanine green, wherein the nanoparticle vesicle is administered through inhalation / spray, or through intravenous, intraperitoneal, intramuscular, or subcutaneous injection.

[0023] Also disclosed herein is a nanoparticle vesicle for magnetic resonance imaging and fluorescence imaging, comprising an at least one brominated carbocyanine (BrCy) dye, and a magnetic resonance imaging agent.

[0024] Also disclosed herein is a cell-derived nanoparticle vesicle comprising a magnetic resonance imaging agent. In some embodiments, the cell-derived nanoparticle vesicle is derived from a red blood cell.

[0025] Some embodiments of the present disclosure are also as noted in the below numbered arrangements:1. A nanoparticle vesicle for magnetic resonance imaging and fluorescence imaging, comprising: a membrane-bound vesicle; a magnetic resonance imaging agent within the membrane-bound vesicle; and a fluorescence imaging agent within the membrane-bound vesicle.2. The nanoparticle vesicle of arrangement 1, wherein the vesicle is a liposome, micelle, cell membrane, or cell-derived particle.3. The nanoparticle vesicle of arrangement 1 or 2, wherein the membrane is a plasma membrane and / or a lipid bilayer.4. The nanoparticle vesicle of arrangement 3, wherein the plasma membrane and / or the lipid bilayer comprises a phospholipid.5. The nanoparticle vesicle of any one of arrangements 1-4, wherein the fluorescence imaging is Near-IR fluorescence imaging (NIRFI), fluorescence endoscopy, and / or fluorescence laparoscopy.6. The nanoparticle vesicle of arrangement 5, wherein the fluorescence imaging is NIRFI.7. The nanoparticle vesicle of any one of arrangements 1 -6, wherein the magnetic resonance imaging agent is incorporated inside the nanoparticle.8. The nanoparticle vesicle of any one of arrangements 1-7, wherein the magnetic resonance imaging agent is incorporated on the surface or through the membrane (i.e. transmembrane) of the nanoparticle.9. The nanoparticle vesicle of any one of arrangements 1-8, wherein the fluorescence imaging agent is incorporated inside the nanoparticle.10. The nanoparticle vesicle of any one of arrangements 1-9, wherein the fluorescence imaging agent is incorporated on the surface or through the membrane of the nanoparticle.11. The nanoparticle vesicle of any one of arrangements 1-10, wherein the vesicle is derived from a mammalian and / or human cell.12. The nanoparticle vesicle of arrangement 11, wherein the vesicle is derived from an erythrocyte.13. The nanoparticle vesicle of arrangement 12, wherein the vesicle is a hemoglobin-depleted, “ghost” ery throcyte.14. The nanoparticle vesicle of any one of arrangements 1-13, wherein the magnetic resonance imaging agent comprises one or more of: gadolinium, gadodiamide, gadoversetamide, gadoxetate, Gadofosveset, gadobenate, gadopiclenol, gadopentetate, Gadoteridol, Gadobutrol, Gadoterate, Iron Oxide, Manganese, or any combination thereof.15. The nanoparticle vesicle of arrangement 14, wherein the magnetic resonance imaging agent comprises gadolinium.16. The nanoparticle vesicle of arrangement 14 or 15, wherein the magnetic resonance imaging agent comprises one or more of: gadopiclenol, Gadopentetate dimeglumine (Gd-DTPA), gadobenate dimeglumine, Gd-BOPTA, gadoxetate disodium, Gadofosveset trisodium, Gd-DTPA-BMA, Gd-DTPA-BMEA. Gd-EOB-DTPA, Gd-HP- DO3A, Gd-BT-DO3A, Gd-DOTA. Ferumoxides, Ferumoxtran-10, FerumoxsiL Mangafodipir trisodium, Gadoterate meglumine, or any combination thereof.17. The nanoparticle vesicle of arrangement 16, wherein the magnetic resonance imaging agent comprises paramagnetic gadolinium ion chelated with benzyloxypropionictetra-acetate gadolinium (Gd-BOPTA).ls. The nanoparticle vesicle of any one of arrangements 1-17, wherein the fluorescence imaging agent comprises cyanine, indoline, indolenine, or any combination thereof.19. The nanoparticle vesicle of arrangement 18, wherein the fluorescence imaging agent comprises a halogenated carbocyanine dye, abrominated cyanine dye, an indolenine heterocycle, a closed chain cyanine, a hemicyanine, a streptocyanine, a cyanine with a hetrocyclic moiety (such as indole, benzothiazole), or any combination thereof.20. The nanoparticle vesicle of any one of arrangements 18 or 19, wherein the fluorescence imaging agent comprises BrCylO6, BrCyl l l, BrCyl l2, IR700, IR800, indocyanine green, or any combination thereof.21. A composition comprising the nanoparticle vesicle of any one of arrangements 1-20, formulated for administration to a subject.22. The composition of arrangement 21, wherein the subject is mammalian and / or human.23. The composition of arrangement 21 or 22, wherein the composition further comprises Phosphate Buffered Sahne (PBS).24. A kit comprising the vesicle-based nanoparticle system of arrangements 1-20.25. The composition of arrangements 21-23 and / or the kit of arrangement 24 for use in the screening of a tissue, organ, and / or system in a subject.26. The use of arrangement 25, wherein the tissue, organ, and / or system is an ovary, lymph node, pancreas, colorectal tissue, or intraperitoneal tissue.27. The use of arrangement 26, wherein the lymph node is a sentinel lymph node.28. The use of any one of arrangements 25-27, wherein the subject is mammalian and / or human.29. The composition of arrangements 21-23 and / or the kit of arrangement 24 for use in the detection and / or localization of a cancer and / or tumor.30. The use of arrangement 25, wherein the cancer and / or tumor is a solid tumor.31. The use of arrangement 28 or 29, wherein the cancer and / or tumor is a human cancer and / or tumor.32. The use of any one of arrangements 29-31, , wherein the cancer and / or tumor is present in an ovary, lymph node, pancreas, colorectal tissue, and / or intraperitoneal tissue.33. The use of any one of arrangements 29-32, wherein the detection and / or localization is carried out in a subject.-I l34. The use of arrangement 33, wherein the subject is mammalian and / or human.35. The composition of arrangements 21-23 and / or the kit of arrangement 24 for use in diagnosing a subject as having a cancer and / or tumor.36. The use of arrangement 35, wherein the cancer and / or tumor is a solid tumor.37. The use of arrangement 35 or 36, wherein the cancer and / or tumor is present in an ovary, lymph node, pancreas, colorectal tissue, and / or intraperitoneal tissue.38. The use of any one of arrangements 35-37, wherein the subject is mammalian and / or human.39. A method for diagnosing a subject as having a cancer and / or tumor, the method comprising: administering to the subject the composition of arrangements 21-23 and / or the kit of arrangement 24; and imaging the subject using MRI and / or a fluorescence-based scan, wherein positive labeling of a cell or tissue indicates that the subject has a cancer and / or tumor.40. The method of arrangement 39. wherein the subject is mammalian and / or human.41. The method of arrangement 39 or 40, wherein the cancer and / or tumor is a solid tumor.42. The method of any one of arrangements 39-41, wherein the cancer and / or tumor is present in an ovary, lymph node, pancreas, colorectal tissue, and / or intraperitoneal tissue.43. The method of any one of arrangements 39-42, wherein the fluorescence-based scan is NIRFI, fluorescence endoscopy, and / or fluorescence laparoscopy.44. A method of detecting the presence of a cancer and / or tumor in a subject, the method comprising: administering to the subject the composition of arrangements 21-23 and / or the kit of arrangement 24; and imaging the subject using MRI and / or a fluorescence-based scan, wherein the localization of the magnetic resonance imaging agent and the fluorescence imaging agent within a subject is indicative of the cancer and / or tumor position.45. The method of arrangement 44, wherein the subject is mammalian and / or human.46. The method of arrangement 44 or 45, wherein the cancer and / or tumor is a solid tumor.47. The method of any one of arrangements 44-46, wherein the cancer and / or tumor is present in an ovary, lymph node, pancreas, colorectal tissue, and / or intraperitoneal tissue.48. The method of any one of arrangements 44-47, wherein the fluorescence-based scan is NIRFI. fluorescence endoscopy, and / or fluorescence laparoscopy.49. A method of treating a cancer and / or tumor in a subject, the method comprising: administering to the subject the composition of arrangements 21-23 and / or the kit of arrangement 24; imaging the subject using MRI to detect the localization of the cancer and / or tumor within the subject’s body; and / or conducting surgery on the subject while simultaneously imaging the subject with a fluorescence-based scan, wherein the surgery removes the cells or tissues that are positively labeled by the magnetic resonance imaging agent and / or the fluorescence imaging agent.50. The method of arrangement 49, wherein the subject is mammalian and / or human.51. The method of arrangement 49 or 50, wherein the cancer and / or tumor is a solid tumor.52. The method of any one of arrangements 49-51, wherein the cancer and / or tumor is present in an ovary, lymph node, pancreas, colorectal tissue, and / or intraperitoneal tissue.53. The method of any one of arrangements 49-52, wherein the fluorescence-based scan is NIRFI, fluorescence endoscopy, and / or fluorescence laparoscopy .54. The method of any one of arrangements 49-53, wherein the method further comprises administering to the subject an effective amount of a molecule or therapy for treating the cancer and / or tumor.55. A method of removing a cancer and / or tumor from a subject, the method comprising: administering to the subject the composition of arrangements 21-23 and / or the kit of arrangement 24;imaging the subj ect using MRI to detect the localization of the cancer and / or tumor within the subject’s body; and / or conducting surgery on the subject while simultaneously imaging the subject with a fluorescence-based scan, wherein the surgery removes the cells or tissues that are positively labeled by the magnetic resonance imaging agent and / or the fluorescence imaging agent.56. The method of arrangement 55, wherein the subject is mammalian and / or human.57. The method of arrangement 55 or 56, wherein the cancer and / or tumor is a solid tumor.58. The method of any one of arrangements 55-57, wherein the cancer and / or tumor is present in an ovary, lymph node, pancreas, colorectal tissue, and / or intraperitoneal tissue.59. The method of any one of arrangements 55-58, wherein the fluorescence-based scan is NIRFI. fluorescence endoscopy, and / or fluorescence laparoscopy.60. The method of any one of arrangements 55-59, wherein the method further comprises administering to the subject an effective amount of a molecule or therapy for treating the cancer and / or tumor.61. The vesicle-based nanoparticle system of any one of arrangements 1-20, further comprising an effective amount of a molecule for treating a cancer and / or tumor.62. A method of preparing a subject for dual imaging analysis, the method comprising administering the composition of arrangements 21-23 and / or the kit of arrangement 24 to the subject.63. The method of arrangement 62, wherein the dual imaging analysis comprises fluorescence and / or MR imaging.64. A nanoparticle vesicle for magnetic resonance imaging and fluorescence imaging, comprising: a membrane-bound vesicle; a magnetic resonance imaging agent Gd-BOPTA, gadobenate dimeglumine, or a chemical derivative thereof within the membrane-bound vesicle; and a fluorescence imaging agent comprising BrCy l ll, BrCyl l2, indocyanine green, or a chemical derivative thereof, within the membrane-bound vesicle.65. A method of conducting magnetic resonance imaging and fluorescence imaging in a subject in need thereof, the method comprising: administering to the subject a nanoparticle vesicle, wherein the nanoparticle vesicle comprises: an outer membrane; a magnetic resonance imaging agent comprising Gd-BOPTA, and / or gadobenate dimeglumine; and a fluorescence imaging agent comprising BrCyl l l, BrCyl l2, and / or indocyanine green, wherein the nanoparticle vesicle is administered through inhalation / spray, or through intravenous, intraperitoneal, intramuscular, or subcutaneous injection.66. A method of acquiring an image of a portion of a subject, the method comprising: administering to the subject a nanoparticle vesicle; and imaging the portion of interest in the subject through magnetic resonance imaging and / or fluorescence imaging, wherein the nanoparticle vesicle comprises: an outer membrane; a magnetic resonance imaging agent comprising Gd-BOPTA, and / or gadobenate dimeglumine; and a fluorescence imaging agent comprising BrCy 1 1 1 , BrCy 1 12, and / or indocyanine green, wherein the nanoparticle vesicle is administered through inhalation / spray, or through intravenous, intraperitoneal, intramuscular, or subcutaneous injection.67. The method of arrangement 66, further comprising acquiring a second image of a portion of a patient using magnetic resonance imaging and / or fluorescence imaging.68. The method of any one of arrangements 39-63, or 65-67, wherein the vesicle is a liposome, micelle, or cell membrane.69. The method of any one of arrangements 39-63, or 65-68, wherein the membrane is a plasma membrane and / or a lipid bilayer.70. The method of arrangement 69, wherein the plasma membrane and / or the lipid bilayer comprises a phospholipid.71. The method of any one of arrangements 39-63, or 65-70, wherein the fluorescence imaging is Near-IR fluorescence imaging (NIRFI), fluorescence endoscopy, and / or fluorescence laparoscopy.72. The method of arrangement 71, wherein the fluorescence imaging is NIRFI.73. The method of any one of arrangements 39-63, or 65-72, wherein the magnetic resonance imaging agent is incorporated inside the nanoparticle.74. The method of any one of arrangements 39-63, or 65-73, wherein the magnetic resonance imaging agent is incorporated on the surface or through the membrane (i.e. transmembrane) of the nanoparticle.75. The method of any one of arrangements 39-63, or 65-74, wherein the fluorescence imaging agent is incorporated inside the nanoparticle.76. The method of any one of arrangements 39-63, or 65-75, wherein the fluorescence imaging agent is incorporated on the surface or through the membrane of the nanoparticle.77. The method of any one of arrangements 39-63, or 65-76, wherein the vesicle is derived from a mammalian and / or human cell.78. The method of arrangement 77, wherein the vesicle is derived from an erythrocyte.79. The method of arrangement 78, wherein the vesicle is a hemoglobin-depleted, "ghosf ’ erythrocyte.80. The method of any one of arrangements 39-63, or 65-79, wherein the magnetic resonance imaging agent comprises one or more of: gadolinium, gadodiamide, gadoversetamide, gadoxetate, Gadofosveset, gadobenate, gadopiclenol, gadopentetate, Gadoteridol, Gadobutrol, Gadoterate, Iron Oxide. Manganese, or any combination thereof.81. The method of arrangement 80, wherein the magnetic resonance imaging agent comprises gadolinium.82. The method of arrangement 80 or 81, wherein the magnetic resonance imaging agent comprises one or more of: gadopiclenol, Gadopentetate dimeglumine (Gd-DTPA), gadobenate dimeglumine, Gd-BOPTA, gadoxetate disodium, Gadofosveset trisodium. Gd-DTPA-BMA, Gd-DTPA-BMEA, Gd-EOB-DTPA, Gd-HP-DO3A, Gd-BT-DO3A, Gd-DOTA, Ferumoxides, Ferumoxtran-10, Ferumoxsil, Mangafodipir trisodium, Gadoterate meglumine, or any combination thereof.83. The method of arrangement 82, wherein the magnetic resonance imaging agent comprises paramagnetic gadolinium ion chelated with benzyloxypropionictetra-acetate gadolinium (Gd-BOPTA).84. The method of any one of arrangements 39-63, or 65-83, wherein the fluorescence imaging agent comprises cyanine, indoline, indolenine, or any combination thereof.85. The method of arrangement 84. wherein the fluorescence imaging agent comprises a halogenated carbocyanine dye, a brominated cyanine dye, an indolenine heterocycle, a closed chain cyanine, a hemicyanine, a streptocyanine, a cyanine with a hetrocyclic moiety (such as indole, benzothiazole), or any combination thereof.86. The method of any one of arrangements 84 or 85, wherein the fluorescence imaging agent comprises BrCylO6, BrCyl l l, BrCyl l2, IR700, IR800, indocyanine green, or any combination thereof.87. The method any one of arrangements 39-63 or 65-86, wherein the fluorescent imaging agent is present at a concentration that is at least about 1, 5, or 10 uM.88. The method of arrangement 87. wherein the fluorescent imaging agent is present at about 10 uM to about 10 mM; preferably wherein the fluorescent imaging agent is present at about 10 uM to about 5 mM.89. The method of any one of arrangements 39-63 or 65-88, wherein the magnetic resonance imaging agent is present at a concentration that is at least about 0. 1. 0.5, 1, or 5 mM.90. The method of arrangement 89, wherein the magnetic resonance imaging agent is present at about 1 mM to about 10 M; preferably wherein the magnetic resonance imaging agent is present at about 1 mM to about 1 M.91. The nanoparticle vesicle of any one of arrangements 1-20, wherein the fluorescent imaging agent is present at a concentration that is at least about 1, 5, or 10 uM.92. The nanoparticle vesicle of arrangement 91, wherein the fluorescent imaging agent is present at about 10 uM to about 10 mM; preferably wherein the fluorescent imaging agent is present at about 10 uM to about 5 mM.93. The nanoparticle vesicle of any one of arrangements 1-20 or 91-92, wherein the magnetic resonance imaging agent is present at a concentration that is at least about 0.1, 0.5, 1, or 5 mM.94. The nanoparticle vesicle of arrangement 93, wherein the magnetic resonance imaging agent is present at about 1 mM to about 10 M; preferably wherein the magnetic resonance imaging agent is present at about 1 mM to about 1 M.95. The nanoparticle vesicle of any one of arrangements 1-20 or 91-94, wherein the nanoparticle vesicle comprises at least two magnetic resonance imaging agents, and / or at least two fluorescent imaging agents.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1A shows the mean loading efficiency (%) of various BrCy dyes and ICG into nano-sized EGs. Error bars represent the standard deviation for three independent measurements. Single asterisk (*) denotes a statistically significant differences (p < 0.05) between the indicated pair. Triple asterisks (***) for BrCyl 11-nEGs fabricated at 750 and 1,000 pM) indicate that those mean zeta-potential values were significantly different (p < 0.001) as compared to the values for other nanoparticles

[0027] FIG. IB shows the mean zeta-potentials for RBCs, nEGs, and BrCy- and ICG-encapsulating nano-sized EGs suspended in IX PBS. Error bars represent the standard deviation for three independent measurements. Single asterisk (*) denotes a statistically significant differences (p < 0.05) between the indicated pair. Triple asterisks (***) for BrCyl l l-nEGs fabricated at 750 and 1,000 pM) indicate that those mean zeta-potential values were significantly different (p < 0.001) as compared to the values for other nanoparticles.

[0028] FIGs. 2A-2D show the hydrodynamic diameter distributions for nEGs loaded with various BrCy dyes or ICG measured in IX PBS by DLS. The average (circles) are presented with SD values (error bars) based on three independent measurements per samples. FIG. 2A is BrCy 106-nEGs, FIG. 2B is BrCyl 11-nEGs, FIG. 2C is BrCyl 12- nEGs, and FIG. 2D is ICG-nEGs.

[0029] FIGs. 3A-3D show the absorption spectra of the RBC-NPs: (FIG. 3 A) BrCy 106-nEGs, (FIG. 3B) BrCy 111 -nEGs, (FIG. 3C) BrCyl 12-nEGs, and (FIG. 3D) ICG- nEGs suspended in IX PBS. Dye concentrations used to fabricate the RBC-NPs were 100, 750, and 1,000 pM.

[0030] FIGs. 4A-4C show the fluorescence emission spectra of the RBC-NPs: (FIG. 4 A) BrCy 106-nEGs, (FIG. 4B) BrCyl 11-nEGs, and (FIG. 4C) BrCyl 12-nEGs in response to photoexcitation at 750 ± 2.5 nm.

[0031] FIG. 4D shows the fluorescence emission spectra of ICG-nEGs in response of photoexcitation at 780 ± 2.5 nm.

[0032] FIG. 4E shows the normalized spectrally integrated fluorescence values (ct) in the range of 795-900 nm for various BrCy- nEGs and 795-900 nm for ICG-nEGs. Each bar is an average of three independent measurements with SD values (error bar).

[0033] FIG. 4F shows the relative fluorescence quantum yield (<|)) for various RBC-NPs, fabricated at different concentrations of the dyes, as compared to the value for free ICG IX PBS.

[0034] FIG. 5A shows the DLS-based Hydrodynamic diameters of nEGs and Gd-BOPTA-BrCyl 12-nEGs collected at day 0, 2. and 7.

[0035] FIG. 5B shows the mean zeta-potentials of RBC, nEGs, and Gd- BOPTA-BrCyl 12-nEGs collected at day 0 (immediately after fabrication), 2, and 7. The average and SD values (error bars) of three measurements per sample are presented.

[0036] FIGs. 6A-6F show the stability results of Gd-BOPTA-BrCyl 12-nEGs up to seven days post-fabrication. FIG. 6A shows the time-dependent absorption spectra of Gd-BOPTA-BrCyl 12-nEGs and their corresponding supernatants (Sup) immediately after fabrication (day 0), and at 2 and 7 days of storage at 4 °C. FIG. 6B show s the fluorescence emission spectra, and FIG. 6C shows the normalized spectrally-integrated fluorescence values (s) over the 765-900 nm band in response to photoexcitation at 750 ± 2.5 nm. FIG. 6D shows the relative fluorescence quantum yield (4>) for Gd-BOPTA-BrCyl l2-nEGs at day 0 (immediately after fabrication), day 2, and day 7, as compared to the value for free ICG IX PBS. FIG. 6E shows the MR images of Gd-BOPTA standards at different concentrations, and Gd-BOPTA-BrCyl 12-nEGs at days 0 (immediately after fabrication), day2, and day7. Samples suspensions were in IrnL vials when acquiring the MR images. Images of the standards at 100 X dilution, and Gd-BOPTA-BrCyl 12-nEGs samples without dilution and at 100 X dilutions are presented. FIG. 6F shows the T1 relaxation rate (Rl) of Gd-BOPTA-BrCyl 12-nEGs. Each data point is an average of three independent measurements with error bars representing the SD values.

[0037] FIGs. 7A-7D show the concentration-dependent absorption spectra of the RBC-NPs: (FIG. 7A) BrCylO6-nEGs, (FIG. 7B) BrCyl 11-nEGs, (FIG. 7C) BrCyl 12- nEGs, and (FIG. 7D) ICG-nEGs suspended in IX PBS.

[0038] FIGs. 8A-8F show the fluorescence emission spectra of free BrCylO6 (FIG. 8A), BrCyl l l (FIG. 8B), BrCyl 12 (FIG. 8C) and ICG (FIG. 8D) in IX PBS inresponse to photoexcitation wavelengths of 750 ± 2.5 nm (for BrCy dyes) and 780 ± 2.5 nm (for ICG). FIG. 8E shows the normalized spectrally-integrated fluorescence (a) over the 765-900 spectral band for free BrCy dyes (excited at 750 nm) and 795-900 spectral band for ICG (excited at 780 nm). FIG. 8F shows the relative fluorescence quantum yield (<|)) for the three free BrCy dyes as compared to free ICG at different concentrations of the dyes in IX PBS.

[0039] FIGs. 9A-8B show the quantification of MR signal intensity (FIG. 9A) and CNR (“contrast-to-noise ratio”) (FIG. 9B), normalized by protein content, of nEG (nano-sized erythrocyte ghosts), nEG with encapsulated gadolinium-chelates (EC, Gd- BOPTA), nEG with surface Gd-chelates (SC, Gd-DTPA). and nEG with both encapsulated and surface Gd-chelates (EC+SC), measured using a 3T MRI with a T1 FSPGR sequence. * P < 0.05; n=3.

[0040] FIGs. 10A-10C show the MR signal intensity (FIG. 10A), CNR (FIG. 10B). and fluorescence (FL) intensities (FIG. 10C) of nEG, nEG + BrCy 112, nEG + BrCyl I2 + SC (Gd-DTPA), and nEG + BrCyl l2 +SC + EC (Gd-BOPTA), respectively. MR intensity was acquired using a 3T MRI with a T1 FSPGR sequence, with MR signal intensity and CNR values normalized by protein content. FL intensity was measured using a fluorometer with 750 nm excitation and integrated emission between 765 nm and 900 nm. The integrated FL intensity- was normalized to the protein content and the loading efficiency of BrCy 112 into the nEG particles. * P < 0.05; n=3.

[0041] FIGs. 11A-1 IB show a non-limiting example schematic of the chemical structures for indocyanine green (ICG) (FIG. 11A) and BrCy 112 (FIG. 1 IB).

[0042] FIGs. 12A-12B show the aborption (FIG. 12A) and fluorescence spectra (FIG. 12B) of 15.6 pM ICG or BrCyl 12 in DI water. The absorption maxima for ICG and BrCyl l2 are observed at 780 nm and 742 nm, respectively. The peak maxima of the fluorescence spectra are located at 775 nm for ICG and 820 nm for BrCy 112. Fluorescence measurements were conducted using 730 nm excitation for ICG and 680 nm excitation for BrCy 112. The integrated fluorescence intensity of BrCyl 12 within the NIR-I window (up to 1000 nm) is approximately 2.5 times greater than that of ICG at an equivalent concentration of 15.6 pM.

[0043] FIGs. 13A-13B show the optimization of ICG and BrCyl 12 loading concentrations for liposomal formations. The optimal loading concentrations were determined as shown in FIG. 13 A, resulting in the brightest fluorescence intensity withinthe liposomal formations, are 150 pM for ICG and 25 pM for BrCyl l2. All fluorescence measurements were performed using 745 nm excitation, with emission filters ranging from 800 to 860 nm. Fluorescence quantum yields of dual-mode-dual-gadolinium (DMDG) were quantified at the optimal loading concentrations (FIG. 13B), specifically DMDG-ICG at 150 pM and DMDG-BrCy 112 at 25 pM.

[0044] FIG. 14A shows the size measurement as measured by DLS; the sizes (PDI values) are 151 nm (0.096). 146 nm (0.083), and 173 nm (0.117) for DMDG-ICG. DMDG-BrCy 112, and vehicle (without MR and FL agents), respectively.

[0045] FIG. 14B shows the T1 relaxivity measurement of DMDG-ICG and DMDG-BrCy 112 at 3 Tesla MRI. T1 relaxivity are calculated from linear regression and show similar slopes for R1 relaxivity ( / 2values) at 15.55 mM'1(0.998) for DMDG-ICG and 16.00 mM‘ls'1(0.988) for DMDG-BrCyl 12, respectively.

[0046] FIGs. 14C-14D show the absorption (FIG. 14C) and fluorescence spectra (FIG. 14D) of DMDG-ICG and DMDG-BrCy 112 in Histidine buffer are shown. The absorption peaks of DMDG-ICG and DMDG-BrCy 112 are observed at 800 nm and 750 nm, respectively. The fluorescence intensity peak of DMDG-BrCyl 12 is approximately 5 times higher than that of DMDG-ICG. Fluorescence measurements were performed using 730 nm excitation for DMDG-ICG and 680 nm excitation for DMDG- BrCy 112.

[0047] FIG. 15 shows non-limiting representative axial 2D-FSPGR MR images of mice abdomen pre- and 2 days-post IV injection with DMDG-BrCyl 12, DMDG-ICG, vehicle, free-BrCyl l2, and free-ICG, respectively. Tumor enhancement is observed with DMDG-BrCyl l2 and DMDG-ICG, as indicated by the yellow arrow. The yellow region indicates the tumor area. All MR images are displayed with the same window level and width.

[0048] FIG. 16A shows non-limiting representative open abdomen fluorescence images of nude mice with HeyA8 tumor after two days post- injection of various imaging agents in HeyA8 tumors. The excitation (emission) windows were 710 ± 15 nm (800 ± 10 nm) for BrCyl l2, 745 ± 15 (820 ± 10 nm) for ICG and their average for vehicle, respectively. The yellow region and arrow indicate the tumor area. The expected background signal is seen in RES organs liver and spleen.

[0049] FIG. 16B shows non-limiting representative fluorescence images of various imaging agents by intraperitoneal HeyA8 tumors.

[0050] FIG. 16C shows the semi-quantitative evaluation of fluorescence after two days of injected imaging agents: DMDG-BrCyl 12, DMDG-ICG, vehicle, free- BrCyl l2, and free-ICG. (P < 0.05, n=6).

[0051] FIG. 17A shows non-limiting representative open abdomen fluorescence images in color of nude mice with HeyA8 tumor after two days post- injection of various imaging agents in HeyA8 tumors. The excitation (emission) windows were 710 ± 15 nm (800 ± 10 nm) for BrCyl 12. 745 ± 15 (820 ± 10 nm) for ICG and their average for vehicle, respectively. The yellow region and arrow indicate the tumor area. The expected background signal is seen in RES organs liver and spleen.

[0052] FIG. 17B shows non-limiting representative fluorescence images in color of various imaging agents by intraperitoneal HeyA8 tumors.

[0053] FIG. 17C shows the semi-quantitative evaluation of fluorescence in color after two days of injected imaging agents: DMDG-BrCy 112, DMDG-ICG, vehicle, free-BrCyll2, and free-ICG. (P < 0.05, n=6).DETAILED DESCRIPTION

[0054] The present inventors have engineered a nano-sized platform, derived from erythrocyte ghosts (EGs), with dual near infrared (NIR) fluorescence and magnetic resonance (MR) characteristics by co-encapsulation of a brominated carbocyanine compound and gadobenate dimeglumine, respectively. In some embodiments, magnetic resonance has use for percutaneous imaging enabling tumor detection and pre-surgical planning, enabling the surgeon to know where to expect to find the tumor nodules. Near infrared fluorescence imaging has use in, as a non-limiting example, visualizing of the nodules at surgery, and complement MR imaging at surgical staging and resection. Some aspects of the present disclosure therefore relate to a dual imaging method based on magnetic resonance and fluorescence for early detection, staging, and guided-resection of tumors, particularly ovarian intraperitoneal tumors.

[0055] In particular, the use of three brominated carbocyanine dyes (referred to as BrCyl 06. BrCyl 11. BrCyl 12) have been investigated with various degrees of bromination, structural symmetry and acidic modifications for encapsulation by nano-sized EGs (nEGs), and compared the resulting optical characteristics with nEGs doped with the FDA-approved indocyanine green (ICG). It was found that an unsymmetric dye (BrCy 106) with only one dibromobenzene ring, and acidic groups attached to all aromatic rings reduce dye aggregation and enhance the fluorescence emission characteristics of the dye in bothits non-encapsulated and encapsulated forms. Specifically, the mean value of the relative fluorescence quantum yield ( / i) for nEGs fabricated using 1 mM of BrCylO6 is about 54- fold, 61-fold, and 1.3-fold higher than nEGs fabricated using the same concentrations of ICG, BrCyl 11, and BrCyl 12, respectively. The dual mode nEGs containing BrCy 112 and gadobenate dimeglumine show a nearly twofold increase in their <f> as compared to their single mode counterpart, and retain their fluorescence and T1 relaxation rate characteristics for at least seven days when stored at 4 °C. These dual mode nano-constructs may prove useful in various biomedical imaging applications such as image-guided tumor surgery7where MR imaging can be used for tumor staging and mapping, and fluorescence imaging can help visualize small tumor nodules for resection.

[0056] The dual agent particles disclosed herein have been fabricated and characterized for their fluorescence and MR characteristics. The disclosed particles have several advantages over other imaging particles in the literature, including an enhanced fluorescence emission of the particles when co-loaded with Gd-BOPTA. which may lead to more effective surgical removal of tumors and improved survival of the patients.Terms

[0057] In the present disclosure, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in this disclosure, including the drawings and claims, are not meant to be limiting. Some embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0058] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which the disclosed subject matter belongs when read in light of the current disclosure.

[0059] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0060] The articles “a” and “an” are used herein to refer to one or to more than one (for example, at least one) of the grammatical object of the article, unless the contextdictates otherwise. By way of example, “an element” means one element or more than one element.

[0061] By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is approximately the recited value. Where it is not clear from the context what is encompassed by “about,” it will mean the value recited + / - 10%.

[0062] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity' or action of the listed elements.

[0063] The terms “individual”, “subject”, or “patient” as used herein have their plain and ordinary meaning as understood in light of the specification, and mean a human or a non-human mammal, e g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate, or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate. The term “mammal” is used in its usual biological sense. Thus, it includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys) and humans, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, or the like.

[0064] As used herein, the term “isolated” has its plain and ordinary meaning as understood in light of the specification, and refers to a substance and / or entity' that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from equal to, about, at least, at least about, not more than, or not more than about, 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%. about 80%, about 90%, about 95%. about 98%, about 99%, substantially 100%, or 100% of the other components with which they were initially associated (or rangesincluding and / or spanning the aforementioned values). In some embodiments, isolated agents are, are about, are at least, are at least about, are not more than, or are not more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure (or ranges including and / or spanning the aforementioned values). As used herein, a substance that is "‘isolated” may be “pure” (e g., substantially free of other components). As used herein, the term “isolated cell” may refer to a cell not contained in a multi-cellular organism or tissue.

[0065] As used herein, “in vivo” has its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method inside living organisms, usually animals, mammals, including humans, and plants, as opposed to a tissue extract or dead organism.

[0066] As used herein, “ex vivo” has its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method outside a living organism with little alteration of natural conditions.

[0067] As used herein, “in vitro” has its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method outside of biological conditions, e.g., in a petri dish or test tube.

[0068] The term “purity” of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and refers to the actual abundance of the substance, compound, or material relative to the expected abundance. For example, the substance, compound, or material may be at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between. Purity may be affected by unwanted impurities, including but not limited to nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membrane, cell debris, small molecules, degradation products, solvent, carrier, vehicle, or contaminants, or any combination thereof. In some embodiments, the substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes. host cell culture components, process related components, mycoplasma, pyrogens, bacterial endotoxins, and adventitious agents. Purity can be measured using technologies including but not limited to electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, UV-visible spectrometry, infraredspectrometry, mass spectrometry, nuclear magnetic resonance, gravimetry, or titration, or any combination thereof.

[0069] The term ‘'yield’’ of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and refers to the actual overall amount of the substance, compound, or material relative to the expected overall amount. For example, the yield of the substance, compound, or material is, is about, is at least, is at least about, is not more than, or is not more than about. 80. 85. 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected overall amount, including all decimals in between. Yield may be affected by the efficiency of a reaction or process, unwanted side reactions, degradation, quality of the input substances, compounds, or materials, or loss of the desired substance, compound, or material during any step of the production.

[0070] The terms “function” and “functional” as used herein have their plain and ordinary meaning as understood in light of the specification, and refer to a biological, enzymatic, or therapeutic function.

[0071] As used herein, the terms '‘treating” or “treatment” have their plain and ordinary meaning as understood in light of the specification, and refer to an approach for obtaining beneficial or desired results in a subject's condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (e.g., not worsening) the state of disease, prevention of a disease's transmission or spread, delaying or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the recurrence of disease, and remission, whether partial or total and whether detectable or undetectable. “Treating” and “treatment” as used herein also include prophylactic treatment. Treatment methods include administering to a subject a therapeutically effective amount of an active agent. The administering step may include a single administration or may include a series of administrations. The compositions are administered to the subject in an amount and for a duration sufficient to treat the subject. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the age and genetic profile of the subject, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and becomeapparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required.

[0072] The term '‘administering’’ includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intra-tumoral, intrathecal, intranasal, or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g.. buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, ortransdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intra-tumoral, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By “coadminister” it is meant that a first compound described herein is administered at the same time, just prior to, or just after the administration of a second compound described herein.

[0073] As used herein, “pharmaceutically acceptable” has its plain and ordinary meaning as understood in light of the specification and refers to earners, excipients, and / or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed or that have an acceptable level of toxicity. A “pharmaceutically acceptable” “diluent,” “excipient,” and / or “carrier” as used herein have their plain and ordinary meaning as understood in light of the specification and are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans, cats, dogs, or other vertebrate hosts. Typically, a pharmaceutically acceptable diluent, excipient, and / or carrier is a diluent, excipient, and / or carrier approved by a regulatory agency of a Federal, a state government, or other regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans as well as non-human mammals, such as cats and dogs. The term diluent, excipient, and / or carrier can refer to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical formulation is administered. Such pharmaceutical diluent, excipient, and / or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and / or excipients include sugars, starch, glucose, fructose, lactose, sucrose, maltose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, salts, sodium chloride, dried skim milk,glycerol, propylene, glycol, water, ethanol and the like. A non-limiting example of a physiologically acceptable earner is an aqueous pH buffered solution. The physiologically acceptable carrier may also include one or more of the following: antioxidants, such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids, carbohydrates such as glucose, mannose, or dextrins, chelating agents such as EDTA. sugar alcohols such as glycerol, erythritol, threitol. arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, isomalt, maltitol, orlactitol, salt-forming counterions such as sodium, and nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The formulation, if desired, can also contain minor amounts of wetting, bulking, emulsifying agents, or pH buffering agents. These formulations can take the form of solutions, suspensions, emulsion, sustained release formulations and the like. The formulation should suit the mode of administration.

[0074] The term ‘‘pharmaceutically acceptable salts'’ has its plain and ordinary meaning as understood in light of the specification and includes relatively non-toxic, inorganic and organic acid, or base addition salts of compositions or excipients, including without limitation, analgesic agents, therapeutic agents, other materials, and the like. Examples of pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. Examples of suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, the class of such organic bases may include but are not limited to mono-, di-, and trialkylamines, including methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines including mono-, di-, and triethanolamine; amino acids, including glycine, arginine and lysine; guanidine; N-methylglucosamine; N-methylglucamine; L- glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; trihydroxymethyl aminoethane.

[0075] The term “% w / w” or “% wt / wf ’ as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a percentage expressed in terms of the weight of the ingredient or agent over the total weight of the composition multiplied by 100. The term “% v / v” or “% vol / vol” as used herein has its plain and ordinarymeaning as understood in the light of the specification and refers to a percentage expressed in terms of the liquid volume of the compound, substance, ingredient, or agent over the total liquid volume of the composition multiplied by 100.

[0076] The terms “first,” “second,” and “third” used in combination with substances are intended to designate distinguishable features to similar substances and do not imply any particular order unless otherwise specified.

[0077] The term “nanoparticle” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a small particle. In some embodiments, a “nanoparticle” can refer to a micelle, liposome, vesicle, cell, or any combination thereof. In some embodiments, the cell has been emptied of at least part of its intracellular contents. For example, a ghost erythrocyte refers to a red blood cell whose intracellular hemoglobin has been significantly depleted.

[0078] The term “membrane” as used herein has its plain and ordinary' meaning as understood in light of the specification and refers to a biological or synthetic membrane. In some embodiments, the membrane comprises a lipid and / or a phospholipid. In some embodiments, the membrane is a single layered membrane. In some embodiments, the membrane is a bilayer membrane.

[0079] The term “vesicle” as used herein has its plain and ordinary' meaning as understood in light of the specification and refers to a spherical nanoparticle that has a center liquid portion that is surrounded by a membrane. In some embodiments, the membrane is a bilayer. In some embodiments, the membrane comprises a plasma membrane, lipid, phospholipid, steroid (for example, a cholesterol), or any combination thereof. In some embodiments, the liquid portion inside the vesicle comprises a salt. In some embodiments, the liquid portion inside the vesicle is isotonic with the plasma of a human subject.

[0080] The term “chemical derivative” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a modification to a molecule that does not significantly alter its function. For example, a fluorescence imaging agent is any agent that has use in fluorescence imaging. A chemical derivative of a fluorescence imaging agent is any modified form of the fluorescence imaging agent, wherein the agent still has use in fluorescence imaging. In some embodiments, the chemical derivation does not alter the use of the chemical. In some embodiments, the chemical derivation does alter the use of the chemical.

[0081] Although the present disclosure has been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of embodiments of the disclosure as defined in the appended claims.Nanoparticle synthesis

[0082] The following describes a non-limiting embodiment to synthesize the nanoparticles. To synthesize the nanoparticles, RBCs are isolated from whole blood by centrifugation and re-suspended in 320 mOsm (1 x) isotonic phosphate buffer saline (PBS). Isolated RBCs are then incubated in hypotonic PBS (0.5X, pH=8, ~20 min, 4°C), and subsequently centrifuged. This process is repeated until an opaque pellet that contains RBC ghosts is obtained. RBC ghosts then undergo sequential mechanical extrusion through polycarbonate porous membranes to form nano-sized erythrocyte ghosts (nEGs). These nano ghosts are then incubated with Sorensen’s phosphate buffer (Na2HPO4 / NaH2PO4, ~140 mOsm. pH ~ 8) containing both agents (Gd-BOPTA (concentrations in the range of 100-1000 mM) and the halogenated dye (concentrations in the range of 100-1000 pM) in a 1 : 1 : 1 : 1 volume ratio for 30 min at 4°C, followed by washing in IX PBS twice. The resulting pellets are resuspended in IX PBS.

[0083] Fluorescent organic dyes provide an enabling platform for image-guided surgery and other clinical applications. In particular, when activated by near infrared (NIR) light (700-2,500 nm), such materials mediate fluorescence imaging on the order of about 1 cm (depending on wavelength and tissue type), and enhance the image contrast as a result of the lower autofluorescence in the NIR spectral band. The FDA-approved indocyanine green (ICG) (molecular weight (MW) = 775 Da) remains as the principal NIR dye used in specific clinical applications such as ophthalmic angiography and liver function assessment, and has been investigated for its utility in intraoperative cancer imaging. It is a dye with two tricarbocyanine systems connected by a polyene bridge, and terminal sulfonate groups attached to each of the nitrogen-containing heterocycles (Scheme 1A). The delocalization of the electrons across the bridge gives rise to NIR absorption and fluorescence properties of ICG and other carbocyanine dyes. Despite its long clinical usage dating back to 1950, drawbacks of ICG include weak fluorescence emission (e.g., relative fluorescence quantum yield of- 2.58% in water when photoexcited at 780 nm at 6.45 pM), and rapid clearance from bloodstream in a biexponential manner with half-life of less than 10 minutes. Previous studies indicate that once within the vasculature, ICG binds toalbumin and lipoproteins, and is transported to the liver where it is then excreted via the hepatobiliary system.

[0084] To address the weak emission of ICG, new NIR fluorophores have been under development and investigation. One class of organic fluorophores are carbocyanine dyes with added heavy halogen elements such as bromine, chlorine, fluorine, or iodine. In particular, specific brominated carbocyanine dyes containing indoline and indolenine heterocycles are the subject of this study: (1) BrCylO6 (in its acid form) (MW = 934.73 Da), an unsymmetric dye with: one dibromobenzene ring, carboxyl and sulfonic acid attached to the indolenine group, and further modified with the addition of sulfonic acid and sulfonate (conjugate base of sulfonic acid) to the indoline and indolenine groups, respectively (Scheme IB); (2) BrCyl l l (MW = 940.40 Da), a more symmetric dye with two dibromobenzene rings, an indoline heterocycle with an attached sulfonic acid group, and sulfonic acid and sulfonate attached to the indoline and indolenine groups, respectively (Scheme 1C); and (3) BrCyl l2 (MW = 754.55 Da), another unsymmetric dye with one dibromobenzene ring, and sulfonic acid and sulfonate attached to the indoline group (Scheme ID).

[0085] When dissolved in an aqueous buffer containing NaAc and MgAc, at the same concentration of 20 pg / ml, the peak emission intensity7of the BrCy 106 dye containing a N-hydroxy succinimide (NHS-ester) group (instead of the carboxyl group) emission at 780 nm (when photoexcited at 720 nm) was ~ 65 times higher than that of ICG emission at 775 nm (in response to 680 nm excitation). Under these conditions, the spectrally- integrated emission (a measure of the total fluorescence over all emitted wavelengths) of BrCy 106 with the attached NHS-ester group was nearly 57 times higher than that of ICG. The superior emission characteristics of the brominated carbocyanine dye, attributed to the presence of the bromine elements that reduce the non-radiative relaxation pathways involve C — H vibrations modes. The inventors had also previously encapsulated this BrCylO6 by capsid proteins obtained from the plant-infecting brome mosaic virus and used the resulting nano-constructs for NIR fluorescence imaging of intraperitoneal ovarian tumors in mice. Encouraged by these results, herein, the inventors of the present disclosure expand their studies to other brominated carbocyanine dyes shown in Scheme 1.Scheme 1. Molecular Structures of (A) ICG, (B) BrCylO6, (B) BrCyl l l, (C) BrCyl l2, and (D) BrCyl l2.

[0086] To shield ICG from binding to plasma proteins and extend its circulation, it has been encapsulated into various nano-sized constructs including those composed of micelles, vesicles, liposomes, polymers, and viral capsid proteins. The inventors of the present disclosure reported the first demonstration of encapsulating ICG within nano-constructs derived from red blood cells (RBCs), and have shown their utility in mediating NIR fluorescence imaging and photo-destruction of tumors in mice. Use of RBCs as delivery systems has also been reported by other investigators.

[0087] A particular feature of RBCs is their naturally long circulation time (~ 90-120 days), attributed to the presence of specific membrane proteins, including CD47 which impedes phagocytosis by macrophages, CD55 which protects against cell lysis by the complement system, and CD59 which blocks the assembly of membrane-attackcomplexes. The proteomics analysis, based on tandem mass spectroscopy, indicate that CD47, CD55, and CD59 are retained on the surface of ICG-containing nano-constructs fabricated from RBCs. This built-in immune-inhabiting machinery, absent on synthetic nano-constructs such as liposomes, but available on RBC-derived nanoparticles (RBC- NPs), is a key distinguishing characteristic needed for prolonged circulation of the particles. For example, it has been reported that the circulation half-life of ICG-containing liposomes with mean diameter of ~ 200 nm was only about 7.5 minutes in immunodeficient Balb / C mice incapable of producing T cells. In a previous study, the inventors of the present disclosure found that the half-life of RBC-NPs containing ICG in bloodstream of healthy Swiss Webster (hSW) mice was nearly two hours, and about 11% of these constructs were still within the vasculature 48 hours post-tail vein injection. Another advantage of RBC- NPs is that as materials fabricated autologously, they present personalized constructs; expected to be highly biocompatible, non-immunogenic, and non-toxic. The inventors have shown that the histological sections of various organs of hSW mice injected with RBC- NPs, as well as the hematological panels and serum biochemistry assaying for liver and kidney functions were similar to those for mice injected with phosphate buffered saline (PBS) control. Previous acute immunogenic studies based on measurements of specific cytokines following intravascular administration of RBC-NPs in hSW mice have shown that the mean values of interleukin (IL)-6, IL-10, and monocyte chemoattractant protein-1 in blood serum at up to 6 hours post-injection were not significantly different than those in response to PBS administration.

[0088] Herein, for the first time, the inventors report on the encapsulation of three brominated carbocyanine fluorophores (BrCylO6, BryCylll, and BrCyl l2) into RBC-NPs, and compare the optical characteristics of these particles with ICG-containing RBC-NPs. Another innovation reported in this paper is the co-encapsulation of a brominated carbocyanine dye (BrCyl l2) with an inorganic magnetic resonance (MR) contrast agent to enable dual optical and MR imaging (MRI) by the RBC-NPs.

[0089] Gadolinium (a rare earth metal element) as an MR contrast agent and ICG have been assembled into hybrid nanoparticles, or encapsulated into liposomes or silicon dioxide matrices. Iron oxide nanoparticles have been encapsulated into RBCs and nano-sized RBC-derived vesicles. However, co-encapsulation of a brominated NIR fluorophore with an MR contrast agent into RBC-NPs, presented in this study, is new'. Specifically, the inventors of the present disclosure have co-encapsulated gadobenate dimeglumine (Gd-BOPTA, MW=1058.17) (Multihance™), which consists of theparamagnetic gadolinium ion chelated with benzyloxypropionictetra-acetate (BOPTA). Gd-BOPTA is an FDA-approved agent for use in MRI of the central nervous system in adults and pediatric patients, and MR angiography of renal and aorto-iliofemoral occlusive vascular disease. The RBC-NPs with dual MR and NIR fluorescence imaging capabilities can be useful in surgical applications where the MRI component can provide deep tissue imaging on the order of tens of centimeters at millimeters resolution, and the NIR fluorescence component would allow imaging of the shallow and superficial layers. As an example, for image-guided resection of epithelial ovarian tumors, which account for more than 85% of ovarian tumors, MRI can be used for pre-surgical planning to stage and map the tumors distribution, and fluorescence imaging would enable intraoperative imaging to visualize small tumor nodules (e.g., < 1 mm) that may not otherwise be detectable for resection.Magnetic Resonance (MR) Contrast Agents

[0090] Non-limiting example contrast agents for MR include: (1) Gadolinium Agents (Gadopentetate dimeglumine (Gd-DTPA), gadopiclenol, Gadodiamide (Gd-DTPA- BMA), Gadoversetamide (Gd-DTPA-BMEA), Gadoxetate disodioum (Gd-EOB-DTPA), Gadofosveset trisodium, Gadoteridol (Gd-HP-DO3A), Gadobutrol (Gd-BT-DO3A), Gadoterate meglumine (Gd-DOTA)), (2) Iron Oxide Nanoparticles (Ferumoxides, Ferumoxtran-10. Ferumoxsil). and (3) Manganese-Based Contrast Agents (Mangafodipir trisodium).Magnetic Resonance Agents in Combination with Near Infrared (NIR) Agents

[0091] Ovarian cancer is the most fatal gynecologic malignancy. The five-year survival rate is 94% if detected as stage I, localized disease, but only 28% for metastatic cases. It is diagnosed with tumors already within the peritoneal cavity (intraperitoneal disease) in -75% of cases. Treatment of ovarian cancer necessitates precise tumor localization for staging and preoperative planning. Methods for imaging tumor at surgery are needed. Current imaging modalities, such as computed tomography (CT) and magnetic resonance (MR) with traditional contrast agents, suffer from limited sensitivity and transient tumor enhancement. Complete tumor removal is crucial for successful surgical outcomes but is hindered by the challenge of detecting small lesions dispersed within the large peritoneal space. Thus, developing a multimodal contrast agent with long residencetime in tumor for both MR for staging and presurgical planning as well as optical imaging during surgery are needed for managing ovarian cancer.

[0092] MRI presents notable advantages in soft-tissue contrast when compared to CT and does so without ionizing radiation. Traditional intravenous (IV) contrast agents used are primarily extravascular-extracellular in tumors and provide transient enhancement lasting only minutes. The dual-Gd platform, incorporating both surface-bound and encapsulated gadolinium (Gd) chelates, demonstrates superior relaxivity / nanoparticle compared to single surface or encapsulated presentations alone, resulting in approximately 10,000-fold higher relaxivity per particle compared to clinically used chelates. This platform has exhibited enhanced signal-to-noise ratio (SNR) such as for vascular imaging. The nanoparticle's diameter range within 100 to 250 nm makes it amenable to enhanced permeability and retention (EPR) effect that is observed in tumors due to abnormal angiogenic vessels. It is PEGylated to decrease background reticuloendothelial system (RES) uptake. For tumor resection, optical imaging has potential to provide real-time surgical guidance. Compared to the visible spectrum, near-infrared (NIR) imaging offers distinct advantages such as reduced background signal and improved penetration depth. This real-time optical-imaging modality has been proposed for intraoperative applications. Incorporating indocyanine green (ICG) into the dual Gd platform allows for both MR and NIR imaging of ovarian tumors two days after IV nanoparticle injection.

[0093] Among NIR agents, ICG stands as an FDA-approved fluorophore for specific clinical indication, primarily in eye angiography. Free (non-encapsulated) ICG exhibits a brief plasma half-life of approximately 2-4 minutes. Encapsulation in various nanoparticle formulations including liposome, micelles, and polymeric nanoparticles, prolongs circulation time and can enhance in vivo stability as well as signal intensity. However, ICG has limited fluorescence (FL) quantum yield and readily aggregates in aqueous solution, impacting its optical properties. Moreover, rapid decomposition under light exposure can be limiting and potentially compromise sensitivity in optical imaging.

[0094] New NIR fluorophores have been developed and investigated for their high brightness and stability in aqueous solutions. One class of these organic fluorophores is carbocyanine dyes that incorporate heavy halogen elements such as bromine, chlorine, fluorine, or iodine. Notably, BrCyl l2 (FIG. 11B) incorporates into the aromatic structure found in traditional cyanine dyes heavier bromine atoms to reduce non-radiative relaxation pathway and act as electron donors. Increasing the electron density of the donor units for enhanced fluorescent performance has proven to be an effective strategy. NIR fluorophores,such as indocyanine green (ICG) (FIG. 11 A), are widely utilized; however, there is significant potential to enhance their optical properties, for example, by altering the donor and acceptor groups and their position in the molecule. The introduction of heavy bromine atoms can increase the FL quantum yield and stability’ of cyanine dyes.

[0095] Clinically, in addition to MR agents for staging, bright NIR agents for identifying tumors to resect are wanting. In clinical practice, there is a demand for imaging agents capable of identifying and charactenzing peritoneal metastases.

[0096] The present disclosure will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the disclosure in any way.EXAMPLESExample 1: Fabrication of RBC-NPs containing ICG or various brominated cyanine dyes.

[0097] Erythrocytes were isolated from whole human blood (BioIVT, Westbury, NY) and washed three times in isotonic (-320 mOsm) PBS (Fisher Scientific, Hampton, NH), referred to as IX PBS, at 1000g for 10 mins at 4°C. Isolated ery throcytes were then incubated with hypotonic PBS (-80 mOsm; 0.25X PBS) at 4°C for 1 hour, followed by centrifugation at 20,000g for 20 mins at 4°C. The resulting micro-sized erythrocyte ghosts (pEGs) were resuspended in IX PBS. To obtain nano-sized EGs (nEGs), pEGs were diluted 1 : 10 in I X PBS prior to the extrusion process. The diluted pEGs were extruded three times sequentially through 800, 400, and 200 nm polycarbonate porous filters (Sterlitech Corp., Kent, WA) using a 10 rnL automatic LIP EX® extruder (TRANSFERRA Nanosciences Inc., Burnaby, B.C., Canada). The resulting nEGs were then centrifuged at 100,000g for 1 hour at 4°C, resuspended in IX PBS, and concentrated back by 10 times.

[0098] Three brominated cyanine dyes, BrCylO6, BrCyl l l and BrCyl l2 (NanoQuantum Sciences, Bellevue, WA), and ICG (MP Biochemicals, Santa Ana. CA) were used to form variants of RBC-NPs. The inventors of the present disclosure incubated the nEGs solution with Sorensen’s phosphate buffer (Na2HPO4 / NaH2PO4, ~140 mOsm, pH ~ 8), and solutions of each of the dyes, previously dissolved in water, in a 1 : 1 : 1 volume ratio for 30 mins at 4°C to form the RBC-NP variants BrCylO6-nEGs, BrCyl l l-nEGs, BrCy l l2-nEG and ICG-nEGs. The resulting pellets were then washed twice in IX PBS and centrifuged at 100,000g for 1 hour at 4°C to remove excess unencapsulated dyes. Theinventors of the present disclosure experimented with dye concentrations of 100, 750, and 1000 pM in the loading solution. Triplicate RBC-NP samples loaded with each of the dyes were prepared.Example 2: Fabrication of RBC-NPs containing Gd-BOPTA and BrCyl l2 (Gd-BOPTA- BrCyl 12-nEGs).

[0099] To load the nEGs with Gd-BOPTA (Bracco Diagnostics Inc., Princeton. NJ) and BrCy 112, the inventors of the present disclosure incubated the nEGs solution with Sorensen’s phosphate buffer (Na2HPO4 / NaH2PO4, -140 mOsm, pH ~ 8) containing both agents (Gd-BOPTA (final concentration of 125 mM) and BrCy 112 (final concentration of 100 pM) in a 1 : 1 : 1 : 1 volume ratio for 30 min at 4°C, followed by washing in IX PBS twice. The resulting pellets were resuspended in IX PBS. The inventors of the present disclosure refer to these RBC-NPs as Gd-BOPTA-BrCyl 12-nEGs.Example 3: Characterizations of non-encapsulated dyes and RBC-NPs.

[0100] Absorption spectra of free (non-encapsulated) BrCylO6, BrCyl l l, BrCy 112, ICG, and the various RBC-NPs in IX PBS were obtained using a spectrophotometer (Jaco V-670 UV-vis spectrophotometer, JASCO) with an optical path length of 1 cm. Free dyes were initially dissolved in IX PBS at concentrations of 100, 750, and 1,000 pM. For spectrometric recordings, the free dye solutions were diluted by 20 times, hence resulting in concentrations of 5, 37.5, and 50 pM in PBS. The suspensions of RBC-NPs in IX PBS were diluted by 10 times prior to spectral recordings.

[0101] Fluorescence emission spectra of 1 OX-diluted RBC-NPs and 20X- diluted free dyes in response to photoexcitation at 750 ± 2.5 nm (for brominated dyes, and BrCy-loaded nEGs) and 780 ± 2.5 nm (for free ICG, and ICG-loaded nEGs) filtered from a 450 W xenon lamp were acquired using a fluorometer (Fluorolog-3 spectrofluorometer, Edison). The inventors of the present disclosure obtained the normalized spectrally- integrated fluorescence emission cr as:

[0102] where F(2) is the recorded fluorescence emission intensity in response to the given excitation wavelength (Aex), and A (Aex) is the absorbance of the sample at the excitation wavelength. Fluorescence emission was integrated over the range of 765-900 nm for BrCy-nEGs, and 795-900 nm for ICG-nEGs.

[0103] The inventors of the present disclosure determined the relative fluorescence quantum yield / > for each of the free BrCy dyes and RBC-NPs variants in response to 750 (for BrCy-encapsulating NPs) or 780 (for ICG-encapsulating NPs) nm excitation wavelength as:

[0104] where <pICG^ex= 780 nni) is the fluorescence quantum yield (~2.58%)16 of free ICG in water (6.45 pM) at -780 nm excitation wavelength, <Jsampieis the normalized spectrally-integrated fluorescence emission of free BrCy dyes or RBC-NPs in response to a given excitation wavelength, AICGis the absorbance value of free ICG at 780 nm, and the integral represents the spectrally-integrated fluorescence emission of free ICG in response to 780 nm excitation wavelength.

[0105] The inventors of the present disclosure define the loading efficiency of each dye in RBC-NPs as:Loading Efficiency = 1 — mm'' inuipticar(3) l

[0106] where mlnltialis the amount of the dye introduced into the loading buffer, and msuperis the amount of the dye present in the supernatant upon completing the fabrication of the RBC-NPs. To determine the amount of dye in the supernatant, the inventors of the present disclosure first measured the absorbance value of the supernatant solution, and then compared the peak absorbance value of the supernatant to a calibration curve that related peak absorbance value at the same wavelength to various known concentrations of ICG or the BrCy dye in the same supernatant buffer.

[0107] The hydrodynamic diameter distributions for all RBC-NPs suspended in IX PBS were measured by dynamic light scattering (DLS) (Zetasizer nanoseries, NanoZS90. Malvern. UK). Three individual measurements were collected for each sample, averaged, and standard deviation (SD) determined for each measured diameter. The zeta potentials (Zetasizer nanoseries, NanoZS90, Malvern, UK) of the RBC-NPs suspended in folded capillary cells were measured in IX PBS. Five measurements were collected for each sample and averaged to determine the mean ± SD values of the zeta potential for each sample type.Example 4: Magnetic resonance imaging.

[0108] MRI was performed using a 3T Siemens Prisma scanner. The Tl-values of the contrast agent (Gd-BOPTA) were measured with a spin echo based inversion recovery experiment (repetition time = 5000 ms; echo time = 15 ms, field of view=210 mm x 72 mm, matrix size = 256 x 88). Inversion times (TI) were 30, 100, 400, 1,000, 2000, 3,000, 4,000. and 4,900 ms. Tl values were calculated voxelwise by fitting the resulting signal intensity (S) curve with an exponential function:S(t) = A - Bexp — TI / Tl') (4)Example 5: Quantification of Gd-BOPTA and fluorochromes loading efficiency into nEGs.

[0109] To quantify the loading efficiency of Gd-BOPTA, the inventors of the present disclosure used a similar methodology as described above (Equation 3). The inventors of the present disclosure obtained the MR images of the particles and the supernatants collected upon completing the fabrication of Gd-BOPTA-BrCyl l2-nEGs. To quantify the amount of Gd-BOPTA in the supernatant, the inventors of the present disclosure prepared various concentrations of the Gd-BOPTA (10, 30, 60, 125, 180, and 250 mM) in the water, and measured the Tl relaxation rate (1 / T1=R1) of these suspensions diluted by 100 times to generate a standard curve of Tl value vs concentration. By comparing to the standard curve, the inventors of the present disclosure obtained the concentrations of non-encapsulated Gd-BOPTA in the supernatant after fabrication of Gd- BOPTA-BrCyl l2-nEGs.Example 6: Assessment of BrCyl 12 and Gd-BOPTA leakage from Gd-BOPTA-BrCyll2- nEGs.

[0110] Upon fabricating the Gd-BOPTA-BrCyl l2-nEGs, the suspension containing the particles was stored at 4°C. At specific times (0, 2, 7 days), approximately 1.3 mL of particle suspension was centrifuged, and the pellet was resuspended in original volume of IX PBS. The inventors of the present disclosure recorded the absorption and the fluorescence emission spectra of the resuspended pellet and supernatant. To determine BrCyl 12 leakage, the inventors of the present disclosure compared the absorption and fluorescence emission spectra, and their spectrally-integrated emission from day 2 and 7 to day 0. The inventors of the present disclosure also measured the Tl-values of non-diluted and 100 time diluted Gd-BOPTA-BrCy 112-nEGs at these time points to determine the Gd- BOPTA leakage by comparing their corresponding R1 values at days 0, 2, and 7. Threemeasurements were obtained for each time point and averaged to determine the mean R1 value.Example 7: Absorption and fluorescence characteristics of free dyes

[0111] Absorption spectra of free BrCy and ICG fluorophores dissolved in IX PBS at three different concentrations (5, 37.5, and 50 pM) are shown in FIGs. 7A-7D. Spectra of free BrCylO6 showed a narrow distinct spectral peak at ~ 750 nm (FIG. 7A). which are attributed to the monomeric form of the dye. As the concentration of BrCyl06 increased from 5 to 37.5 and 50 pM, a more visible shoulder in the range of 671-700 nm emerged, which can be associated with an H-like aggregate form of the dye. In such aggregates, fluorophores are stacked in a sandwich-like structure. In accordance with the exciton theory, aggregates split the excited electronic state of the monomer into two where one level is higher, and the other lower than the monomer electronic state. In H-like aggregates, transition only to the upper excited states is allowed; and hence, the absorption spectrum includes a band (671-700 nm in the case of BrCy 106) which is blue-shifted (i.e., a hypsochromic shift) with respect to the monomer peak at 750 nm.

[0112] As the concentration of free BrCy 111 in IX PBS was increased to 37.5 and 50 pM, bimodal spectra with respective primary and secondary peaks at 850 and 643 nm, and a notably broadened spectrum without a distinct monomeric peak emerged (FIG. 7B). The inventors of the present disclosure attribute the bathochromic (red) shift to 850 nm to J-like aggregates of the dye. In such aggregates, the dye molecules are arranged in a head-to-tail manner, and transition only to the lower level of the split excited state is allowed.

[0113] In comparison to BrCy 106 and BrCy 112. BrCyl l l has two dibromobenzene rings (Scheme 1). The fact that BrCy 111 is more extensively brominated combined with the chemical composition of PBS can make BrCyl l l more prone to aggregation. Specifically, with increased bromination, the role of hydrophobic driving forces, including London dispersion forces (induced dipole-induced dipole interactions), for aggregation increases. The increased hydrophobicity can be the resultant of enhanced electron-withdrawing effects by the additional bromine atoms, that due to their electronegativity, can draw electrons from the carbon atoms to which they are bonded, and create partially positive charges around them. Such partially positive charged portions increase the affinity of the dye for less polar and non-polar environments, leading toincreased hydrophobicity. Furthermore, electron-withdrawing effects allow the energy levels of the dye to be adjusted over a broad range.

[0114] The symmetry of BrCy 111 can also make it more prone to aggregation. Since fundamentally formation of aggregates is driven by intermol ecul ar electrostatic interactions, the symmetry can promote stronger intermolecular interactions, leading to stackings (e.g., 7t-7t stacking), particularly resulting from the additional symmetric bromination in the case of BrCyl l l, dipole-dipole interactions, and London forces that ultimately alter the electronic energy7levels of the dye.

[0115] The ionic strength of the solvent is also a contributing factor to the propensity of the dye molecules to aggregate. For example, the presence of salts (NaCl, KC1, Na2HPO4, and KH2PO4) in PBS contributes to the formation of J-aggregates. The salt ions reduce the electrostatic repulsions between the dye molecules, whereby, accelerating the formation of aggregates. In summary7, the inventors of the present disclosure attribute the observed absorption spectra of free BrCyl l l to the formation of dye aggregates, resulting from the additional bromination which increases the hydrophobic forces among the dye molecules, the symmetry of the dye, combined with the effects of the salt ions in PBS which further substantiate the strength of aggregation among the dye molecules.

[0116] As the concentration of free BrCyll l in IX PBS was increased to 37.5 and 50 pM, bimodal spectra with respective primary and secondary peaks at 850 and 643 nm, and a notably broadened spectrum (FIG. 7B). The bathochromic (red) shift to 850 nm was attributed to J-like aggregates of the dye. In such aggregates, the dye molecules are arranged in a head-to-tail manner, and transition only to the lower level of the split excited state is allowed.

[0117] In comparison to BrCy l06 and BrCyl l2. BrCy l l l has two dibromobenzene rings (Scheme 1). The fact that BrCy 111 is more extensively brominated combined with the chemical composition of PBS can make BrCyl l l more prone to aggregation. Specifically, with increased bromination, the role of hydrophobic driving forces, including London dispersion forces (induced dipole-induced dipole interactions) for aggregation increases. The increased hydrophobicity can be the resultant of enhanced electron-withdrawing effects by the additional bromine atoms, that due to their electronegativity, can draw electrons from the carbon atoms to which they are bonded, and create partially positive charges around them. Such partially positive charged portions increase the affinity of the dye for less polar and non-polar environments, leading toincreased hydrophobicity. Furthermore, electron-withdrawing effects allow the energy levels of the dye to be adjusted over a broad range.

[0118] The symmetry of BrCy 111 can also make it more prone to aggregation. Since fundamentally formation of aggregates is driven by intermol ecul ar electrostatic interactions, the symmetry can promote stronger intermolecular interactions, leading to stackings (e.g., 7t-7t stacking), particularly resulting from the additional symmetric bromination in the case of BrCyl l l, dipole-dipole interactions, and London forces that ultimately alter the electronic energy7levels of the dye.

[0119] The ionic strength of the solvent is also a contributing factor to the propensity of the dye molecules to aggregate. For example, the presence of salts (NaCl, KC1, Na2HPO4, and KH2PO4) in PBS contributes to the formation of J-aggregates. The salt ions reduce the electrostatic repulsions between the dye molecules, whereby, accelerating the formation of aggregates. In summary7, the observed absorption spectra of free BrCyl l l was attributed to the formation of dye aggregates, resulting from the additional bromination which increases the hydrophobic forces among the dye molecules, the symmetry' of the dye, combined wi th the effects of the salt ions in PBS which further enhance the strength of aggregation among the dye molecules.

[0120] As the concentration of free BrCyl 12 was increased to 37.5 and 50 pM in IX PBS (FIG. 7C), the absorption spectra showed a distinct spectral peak at 744 nm, corresponding to the monomer form of the dye, and shoulders in the range of 668-697 nm, and 790-825 nm, suggesting the simultaneous presence of both the H-like and J-like aggregates, respectively. For free ICG dissolved in IX PBS (FIG. 2D), the peak at 780 nm corresponds to its monomeric form. Increasing the ICG concentrations to 37.5 and 50 pM were associated with the emergence of another peak at 706 nm, attributed to the H-like aggregate form of the dye.

[0121] In response to photoexcitation of free BrCy dyes at 750 ± 2.5 nm and ICG at 780 ± 2.5 nm, the peaks values of the fluorescence emission intensities for free BrCy 106 and BrCy 112 (FIGs. 8A. 8C) were considerably greater than those for free BrCyl l l and ICG (FIGs. 8B, 8D). For example, for BrCyl06 and BrCyl l2 at 37.5 pM, the peak emission intensities were, respectively, 38 and 26 times higher as compared to free ICG. Similarly, the values of the normalized spectrally-integrated fluorescence (s), were considerably greater for free BrCylO6 and 112 (FIG. 8E). Both free BrCy 106 and BrCyl l2 had the highest values of cr at 37.5 pM. At this concentration, value of rassociated with free BrCy 106 was about 33 times higher as compared with the value for free ICG prepared at the same concentration (FIG. 8E). Increasing the concentrations of free BrCyl l l and ICG from 5 to 37.5 and 50 pM were associated with progressively lowered values of s, suggestive of dye aggregation-induced weakening of fluorescence.

[0122] At each of the experimented concentrations, free BrCy 106 produced the highest value of the relative fluorescence quantum yield ( (FIG. 8F). While ^ for free BrCy 106 nearly doubled when increasing the concentration from 5 to 37.5 pM, there was not much further increase in its (p with increased concentration to 50 pM. The lower values of ^ for free BrCy 112 as compared to BrCy 106 is suggestive of the greater aggregated forms of free BrCy 112, evidenced by both hypsochromic and bathochromic shifts in its absorption spectrum (FIG. 7C). The minimal values of (p for free BrCy 111 can be attributed to aggregation-induced fluorescence quenching, indicating that the excited state energy is mostly dissipated through non-radiative relaxation pathways.Example 8: Characterizations of BrCy- and ICG-encapsulating nano-sized EGs.

[0123] At a given concentration used to fabricate the particles, BrCyl l l had the highest loading efficiency (in the range of ~ 79-95%) (FIG. 1A). The high loading efficiency of BrCy I l l is suggestive of its favorable interactions (e.g. , stronger hydrophobic interactions) with the membrane of the EGs. However, despite its relatively high loading efficiency, BrCyl l l-nEGs exhibited inferior fluorescence characteristics as compared to the nEGs loaded with BrCy 106 or BrCy 112 (FIGs. 4A-4F). For BrCy 106. its mean loading efficiency was increased by ~ 48% when its concentration in the loading buffer was increased from 750 to 1,000 pM, whereas for BrCyl l l and BrCyl l2, this change in concentration was not accompanied by an increase in loading efficiency. In the case of ICG, its loading efficiency remained at about 54% independent of its concentration in the loading buffer.

[0124] Except for BrCy 111 -nEGs, which had the respective mean zeta potential values of -22.34 and -25.25 mV when fabricated using 750 and 1,000 pM of BrCyl l 1, the remaining RBC-NPs had mean zeta potentials in the range of -12.79 to -14.43 mV (FIG. IB). This range was similar to the mean values for nEGs (-13.68 mV) and untreated RBCs (-13.7 mV). The more negative zeta potential for BrCy 111-nEGs may be a resultant of the presence of BrCy 111 -nEGs aggregates when using the higher concentrations of 750 pM and 1,000 pM in fabricating the particles. Given the larger surface area of the aggregates,there could be increased number of exposed negatively charged species (e.g., sulfonates) to cause a more negative value of the zeta potential. Further evidence in support of the formation of BrCy 111 -nEGs aggregates is shown in FIG. 2B.

[0125] Illustrative profiles of the diameters of RBC-NPs loaded with the various dyes, acquired by DLS, are shown in FIGs. 2A-2D. For a given RBC-NP, there were minimal variations in the diameter distribution and the mean peak diameter as a function of the dye concentration used in fabricating the RBC-NP (FIGs. 2A, 2C, 2D), with the exception of BrCyl 11 -nEGs fabricated at 750 and 1,000 pM, which exhibited bimodal distributions (FIG. 2B). For these particular sets of particles, the inventors of the present disclosure attribute the right-shifted distributions (population distributions with larger diameters) to the aggregates of these RBC-NPs. Such aggregates can be formed as a result of attractive forces (e.g., van der Waals forces) between the BrCyl 11-nEGs if the BrCyl l l molecules, which themselves are aggregated (as supported by the absorption spectra shown in FIG. 7B), are not fully encapsulated. For example, the exposed portions of the nonencapsulated of BrCyl 11 molecules may consist of the negatively charged sulfonates that would have electrostatic interactions with the positively charged hydrogens of the sulfonic acid groups protruding out from BrCyl l l molecules on other the BrCyl 11-nEGs. The remaining RBC-NPs had mean peak diameters in the range of 84-137 nm.

[0126] Absorption spectra of the RBC-NPs doped with the various dyes are shown in FIG. 5. Absorbance at 280 nm originates from the aromatic amino acids of the RBC proteins such as tyrosine and tryptophan. The absorbance values at 280 nm for all RBC-NPs was ~1, indicating that the constructs had approximately the same amount of membrane materials. For BrCylO6-nEGs, as the dye concentration used in fabricating the particles was increased to 1,000 pM. the spectral peak at 750 nm. associated with the monomer form of BrCyl 06, emerged (FIG. 3 A). As the BrCyl l l concentration used in fabricating the BrCyl 11-nEGs was increased from 100 to 750 and 1,000 pM, the monomer peak at 854 nm was no longer distinct (FIG. 3B). Hypsochromic and bathochromic shifts with peaks at 652 and 854 nm. respectively, and a broadened spectrum, similar to the features for free BrCy l 11 (FIG. 7B) were observed, suggesting that the dye was mostly in the forms of H-like and J-like aggregates when encapsulated. The spikes associated with the spectra of BrCyl 06-nEGs fabricated using the higher concentrations of the dye (750 and 1,000 pM) are possibly due to instrumentation artifacts or the sample impurities.

[0127] The absorption spectra of BrCyl 12-nEGs (FIG. 3C) were also similar to those for free BrCyl 12 (FIG. 1C) with a distinct peak at 744 nm, and shoulders in the rangeof 661-697 nm and 797-850 nm, which can be atributed to the monomer form, H-like, and the J-like aggregates of the dye, respectively. The ICG-nEGs exhibited a primary peak at 804 nm, and a shoulder in the range of 727-758 nm, associated with the monomer and the H-like aggregate forms of the dye, respectively (FIG. 3D). Consistent with previous results, the monomeric peak had a bathochromic shift from 780 nm as compared to free ICG, indicative of altered electronic states of ICG when nano-encapsulated. As the ICG concentration used in fabricating the ICG-nEGs was increased to 750 and 1.000 pM. another peak at 919 nm emerged, suggestive of the formation of J-like aggregates.

[0128] In response to photoexcitation at 750 ± 2.5 nm for BrCy-nEGs and 780 ± 2.5 nm for ICG-nEGs, BrCylO6-nEGs and BrCyl l2-nEGs in general produced the highest peak emission intensities (FIGs. 4A. 4C) as compared to BrCyl 11-nEGs and ICG- nEGs (FIGs. 4C, 4D). Increasing the concentration of BrCylO6 from 750 to 1,000 pM was associated with nearly a five-fold increase in the peak emission intensity of BrCyl 06-nEGs (FIG. 4A), consistent with the increased loading efficiency of the dye with this change in concentration (FIG. 1A). Increasing the concentration of BrCyl l l from 100 pM was associated with a decrease in the peak emission intensity of the BrCyl 11-nEGs (FIG. 4B). consistent with aggregation-induced fluorescence quenching. In agreement with loading efficiency of BrCyl l2 and ICG (FIG. 1A), there were minimal changes in the emission spectra of BrCyl 12-nEGs (FIG. 4C) and ICG-nEGs (FIG. 4D) with increasing concentrations of the dyes used to fabricate these RBC-NPs. However, depending on the concentration, the peak emission intensity of BrCyl 12-nEGs was at least 55 times higher than ICG-nEGs.

[0129] Consistent with the trends in the fluorescence emission spectra (FIGs. 4A-4D), values of <7 for BrCylO6-nEGs and BrCyl 12-nEGs were higher than those for BrCy 111-nEGs and ICG-nEGs, except when using 100 pM of BrCyl l l, which produced a similar value to BrCyl 06-nEGs fabricated at 100 pM (FIG. 4E). Since the value of <7 for BrCyl 12-nEGs fabricated using 100 pm of the dye was only about 16% lower than the highest <7, which was associated with BrCylO6-nEGs fabricated at 10 times higher concentration, the inventors of the present disclosure chose to proceed with BrCyl 12-nEGs as the test construct for co-encapsulation with Gd-BOPTA.

[0130] The highest value of < / > (- 32.6%) was associated with BrCyl06-nEGs fabricated using 1,000 pM of the dye whereas ICG-nEGs had the lowest values of ( / > (-0.6%) (FIG. 4F). For example, when fabricated using 1,000 pM of the respective dye,value of ( / > was nearly 54 times higher for BrCy 106-nEGs as compared to ICG-nEGs. While the highest value of for RBC-NPs fabricated using 100 pM of a given dye w as associated with BrCyl l2-nEGs (-16.6%), the rate of increase in < / > with increased concentration beyond 100 pM was associated with BrCyl 06-nEGs.Example 9: Dual -encapsulation of NIR fluorescent and MR materials.

[0131] To combine the strengths of NIR fluorescence imaging and MRI into a single nano-construct, BrCyl 12 and Gd-BOPTA were into RBC-NPs. Illustrative DLS- based estimates of nEGs and Gd-BOPTA-BrCyl l2-nEGs diameters show a unimodal distribution, suggesting that the particles were not aggregated (FIG. 5A). The mean peak hydrodynamic diameters for nEGs and Gd-BrCy 112-nEGs immediately after fabrication were -129 and 131 nm, respectively. There were minimal changes in the diameter distributions of BOPTA-BrCy 112-nEGs for up to seven days post-fabrication, suggesting that the particles remained physically stable.

[0132] The inventors of the present disclosure estimated the loading efficiency of Gd-BOPTA into the particles as approximately 33.6%. The mean zeta potential of Gd- BrCy 112-nEGs (-16.03 mV) was similar to the values for nEGs (-14.53 mV) and RBCs (- 15.17) (FIG. 5B), with minimal variation for up to seven days. These results suggest that sialoglycoproteins, the primary negatively charged component of the RBC membrane, and other charged structures, were retained after encapsulating BrCyl 12 and Gd-BOPTA into nEGs.

[0133] Illustrative absorption spectra of Gd-BOPTA-BrCy 112-nEGs pellets suspended in IX PBS, and stored for up to seven days at 4 °C, along with their corresponding supernatants are shown in FIG. 6A. The spectra of the Gd-BOPTA- BrCyl 12-nEGs pellets were similar to those for BrCyl 12-nEGs (FIG. 3C) featuring spectral peak at -745 nm with shoulders in the range of 668-697 and 816-853 nm, suggesting that in these RBC-NPs loaded with both optical and MR materials, BrCyl 12 molecules were still present as monomers, and H-like and J-like aggregates. There was only about 12% reduction in the monomer absorbance value at seven days post-fabrication, concomitant by a similar increase (~ 13%) in the value for the supernatant, indicating minimal leakage or degradation of the over this time interval.

[0134] Similarly, fluorescence emission spectra (FIG. 6B), in response to 750 nm excitation, and their corresponding values of cr (FIG. 6C) remained nearly unchangedover seven days, further confirming the fluorescence stability of these RBC-NPs. Interestingly, the peak emission intensity, and the mean value of cr at day 0 for the dual mode particles (Gd-BOPTA-BrCyl 12-nEGs), fabricated using 125 mM and 100 pM of Gd-BOPTA and BrCyl l2, respectively, were about 2.6 and twofold higher, respectively, as compared to the values for the single mode particles (BrCyl 12-nEGs) fabricated using 100 pM of BrCyl 12 (FIGs. 4C, 4E). This trend was maintained for the Gd-BOPTA- BrCyl 12-nEGs particles at Days 2 and 7 post-fabrication. Similarly, the mean value of <j) for the dual mode particles at day 0 (FIG. 8D) was about twofold higher than the value for single mode particles (FIG. 4F),and remained higher up to seven days post-fabrication. These enhanced fluorescence characteristics resemble a metal-induced-like enhancement in fluorescence when a fluorophore is within a near-field distance of a metal. Photoexcitation of the subwavelength Gd-BOPTA molecules creates an enhanced local electric field. In accordance with plasmons-fluorophore coupling, the presence of BrCy 112 molecules in the vicinity’ of the intense electric field enhances the intrinsic fluorescence emission of the dye.

[0135] MR images of Gd-BOPTA-BrCyl 12-nEGs suspensions in IX PBS obtained immediately after fabrication, and at two and seven days of storage at 4 °C are shown in FIG. 8E. There were minimal variations between the samples collected at day 0 and 7. Values of R1 remained nearly constant for up to seven days following fabrication (FIG. 8F), indicating that there was minimal leakage (<1%) of Gd-BOPTA over this time interval.Conclusion

[0136] The inventors of the present disclosure have engineered a nano-sized platform, derived from erythrocyte ghosts, with dual NIR fluorescence and MR characteristics by co-encapsulation of a brominated-indolenine carbocyanine dye containing an indoline heterocycle, and gadobenate dimeglumine, respectively. The inventors of the present disclosure find that the degree of bromination, structural symmetry, and acidic modifications to the heterocycles have a considerable influence on the fluorescence emission characteristics of the nano-constructs. For example, nano ghosts fabricated using 1.000 pM of BrCyl 06, an unsymmetric dye with brominated indoline heterocycle, and acidic groups attached to both indoline and indolenine heterocycles, demonstrate a nearly 54-fold increase in their relative fluorescence quantum yield ascompared to those fabricated using the FDA-approved ICG. The dual mode nano ghosts containing BrCy 112 and gadobenate dimeglumine show a nearly twofold increase in their relative fluorescence quantum yield as compared to the single mode nano-constructs, and retain their fluorescence and T1 relaxation rate characteristics for at least seven days when stored at 4 °C.Example 10: MR intensity when for encapsulated and surface materials.

[0137] Next, the magnetic resonance intensity was quantified when the MR agent was both encapsulated and on the surface. nEG (nano-sized erythrocyte ghosts), nEG with encapsulated gadolinium-chelates (EC, Gd-BOPTA), nEG with surface Gd-chelates (SC, Gd-DTPA). and nEG with both encapsulated and surface Gd-chelates (EC+SC). were measured for MR signal intensity and contrast-to-noise ratio (CNR) (FIGs. 9A-9B). It was found that surface conjugation increases MR signal intensity and CNR compared to the encapsulation, and these effects are further increased with encapsulation + surfaceconjugation. Next, each cell was exposed to BrCyl l2 fluorescent dye (FIGs. 10A-10C). Not only was MR signal maintained in the presence of BrCyl 12; the BrCy 112 fluorescence signal increased in the presence of MR agents.Example 11: Near-Infrared Imaging of Intraperitoneal Ovarian Cancer Enhanced by BrCy 112 in a Dual Mode (MR and NIR), Dual-Gd nanoparticle

[0138] It was hypothesized that adding BrCyl 12 to DMDG would increase DMDG NIR FL and increase tumor FL. It was evaluated whether a DMDG nanoparticle incorporating BrCy 112 enables MR imaging and NIR imaging of intraperitoneal ovarian tumors in mice, two days after IV injection.Methods:

[0139] DMDG nanoparticles were equipped with either of two different nearinfrared (NIR) fluorophores (ICG or BrCyl 12) along with positive contrast magnetic resonance (MR) imaging agents. The optical properties of the NIR fluorophores were evaluated in vitro using absorption and fluorescence (FL) spectroscopy. For liposomal formulation, the loading concentrations of the NIR fluorophores were optimized. In vitro, MR relaxivity and optical properties including FL were characterized. Subsequently, two days after nanoparticle injection. MR and NIR FL imaging was performed in mice implanted with human intraperitoneal ovarian cancer xenografts.Liposomal formation.

[0140] l,2-Dipalmitoyl-sn-Glycero-3-Phosphatidylcholine (DPPC), cholesterol (Choi), and diethylenetriaminepentaacetic acid-bis (stearylamide) gadolinium salt (Gd-DTPA-BS) were purchased from Avanti Polar lipids (Alabaster, AL, USA). N- (carbonyl-methoxy polyethylene glycol 2000)-1.2-Distearoyl-sn-Glycero-3 phosphatidylethanolamine (mPEG-2000-DSPE) was purchased from Corden Pharma (Switzerland LLC). Indocyanine green (ICG) was obtained from Sigma-Aldrich (St. Louis, MO, USA). BrCyl l2 was purchased in NanoQuantum Science (Georgetown, TX, USA). Whatman Nucl epore polycarbonate track-etch membranes of 100 and 400 nm pore sizes were purchased from Fisher Scientific (Waltham, MA, USA).

[0141] Dual-Gd liposomes consisting of encapsulated and surface Gd with a nominal 150 mM lipid content and encapsulating BrCyl 12 and ICG were made as follows. Lipids consisting of 30:40:5:25 mole percent of DPPC: Choi: DSPE-mPEG-2000-DSPE: Gd-DTPA-BS were dissolved in ethanol at 65°C. A 1 mg / mL stock solution (approximately 1.3 mM) of BrCyl 12 or ICG was made in DI water and used to make DMDGwith different concentration of fluorophores. For example, the solution was used to make a 25 pM solution of BrCyl l2 or 150 pM solution of ICG in gadobenate dimeglumine (505 mg / ml, Bracco Diagnostics Inc., Princeton, NJ, USA), respectively. The lipids dissolved in ethanol were then hydrated with this solution to encapsulate the fluorophore and gadobenate dimeglumine using constant stirring for 30 minutes at 65 °C. The liposomes were then extruded 5 times through 400 nm followed by 6 extrusions through 100 nm Nucleopore membranes using a high-pressure extruder (Northern Lipids, Vancouver. BC, Canada). Extrusions were processed at 65 °C.

[0142] The liposomal suspensions were diafiltered using a MicroKros module (Spectrum Laboratories Inc., CA, USA) of 500 kDa molecular weight cutoff (MWCO) membrane tubes to remove unencapsulated fluorophores (BrCyl 12 or ICG) and Gd- complex. 10 mM histidine with 140 mM saline (pH~7.4) was used as the replacement buffer. Liposome particle size was determined using a Dynamic Light Scattering instrument (Malvern Panalytical Inc. Zetasizer ZS, Westborough, MA, USA). The correlation function was fitted by analyzing a cumulant method. The measurement was repeated on separate samples (N = 3) of each batch. The Z-average (mean) diameter and polydispersity index were recorded for each measurement. The correlation function was also inverted using the CONTIN algorithm, and the intensity-weighted distribution ofparticle sizes that resulted was converted to a volume-weighted distribution using the Mie coefficients at the mid-point of each size interval in the histogram. Cumulative size distributions were calculated using the volume-weighted distributions.Fluorescence intensity measurements.

[0143] FL intensity was measured using a wide-field microscope (DMi-8, Leica) with a objective (HC PL FLUOTAR, 4* / NA 0.13, Leica). Excitation was provided by a 745 nm LED light source, and emission (780-850 nm) was collected with an sCMOS camera (K5, Leica). Samples (2 pL) were placed in a Take3 microvolume plate, and FL images were acquired with a 200 ms exposure for all samples. FL intensity was calculated across the entire image area.Absorption and fluorescence spectra.

[0144] Details of the absorption and FL spectra have previously been reported elsewhere. The absorption spectra were acquired on a multi-mode multiplate reader (Synergy HTX, Agilent, Savage, MD, USA) using the Take3 microvolume plate with 0.5 mm thickness. FL spectra were obtained on a spectrofluorometer (Nanolog, Horiba, NJ, USA) with an InGaAs detector (Sy mphony II, Horiba, NJ) for all near-infrared regions. All sample solutions were measured using a 2 mm x 2 mm quartz cuvette. The excitation wavelength was selected from the Xe lamp centered at 680 nm or 730 nm with a 3 nm bandwidth in a monochromator. The emission spectrum was acquired five times in 1 s each. The FL spectra presented were derived from the average of five individual spectra and were subsequently corrected by subtracting the background spectrum, considering the dark count of the InGaAs detector.Quantum yield measurements.

[0145] The quantum yield of a fluorophore is a fundamental photophysical parameter defined as the ratio of the number of the emitted to the number of the absorbed photons. The relative quantum yield (S) of the samples was calculated using the following equation:

[0146] In this equation, the subscripts S and R denote the sample and reference, respectively. <PRis the known quantum yield of the reference standard. I represents the integrated FL intensity. A is the absorbance of the solution at the excitation wavelength at 745 nm, and n is the refractive index of the solvent (1.33 in water). In this study, the FL quantum yield of ICG in water used as the reference, was 2.9%.T1 relaxivitv measurement.

[0147] T1 relaxation rate measurements of liposomes such as DMDG-BrCy 112 and DMDG-ICG were performed prior to animal experiments. Dilutions were imaged in a 3T MR (Biospec 3T, Bruker, Billerica, MA). Quantitative T1 relaxation measurements were performed using a fast spin-echo saturation-recovery sequence (TR = 25-4000 ms [25 ms, 50 ms, 75 ms, 100 ms, 150 ms, 250 ms, 400 ms, 600 ms, 800 ms, 1000 ms, 2000 ms, 4000 ms]; TE = 50 ms; echo train = 8; field of view (FOV) = 4 cm x 3 cm; Image matrix = 128 x 128; number of signal averages = 3). In all images, a region of interest of constant size was positioned in the center of the cross-sectional view of each tube. Tl-relaxivity is defined as the slope of the linear correlation between relaxation rate (1 / T1) and the concentration of DMDG containing Gadolinium-based contrast agents for MR imaging. ParaVision 3.6 v2.0 was used to calculate the T1 relaxation values fit by mono-exponential function from saturation recovery as a function of TR (recovery’ time, ms). The relaxation rates of the diluted samples were obtained using the inverse of these values.Animals

[0148] Female nude mice aged (7-8 weeks were purchased from Charles-River Laboratory (Wilmington, MA, USA). All mice were housed in specific pathogen-free conditions. All experiments were conducted in accordance with the animal care guidelines at the University of Maryland School of Medicine approved by the Institutional Animal Care and Use Committee.

[0149] Tumors were established by intraperitoneal injection of 1 x io7HeyA8- Luc cells using a syringe with a 28-gauge needle. At weekly intervals after injection, the mice were monitored for tumor growth. After 4-5 weeks, mice (n = 6 per group) were randomly assigned to control groups or experimental groups. Then, the mice were injected intravenously with 200 pL of DMDG-ICG, or DMDG-BrCy 112. As controls, mice (n = 6) bearing intraperitoneal HeyA8 tumors were injected intravenously with free BrCy 112, freeICG, or vehicle (liposome without MR or fluorophore), for robustness, delivered at the same dose delivered with DMDG. Two days later, MR and NIR imaging was performed.Magnetic Resonance Imaging.

[0150] All animal MR studies were performed using a 3 T scanner (Bruker Biospec 3T, Billerica, MA) with a 60 mm gradient insert and a volume resonator with a 35 mm inner diameter. Animals were anesthetized with isoflurane and placed headfirst and prone on a positioning sled. Orthogonal 3-plane scout scans were initially acquired to confirm animal positioning. Animal placement and tumor location were confirmed using images from a respiratory gated axial T2-weighted fast spin echo (FSE) sequence (repetition time = 3813 ms. effective echo time (TE) = 57 ms, echo train = 12; field of view = 4 x 3 x 4 cm, slice thickness = 1 mm, image matrix = 256 x 192, number of signal averages = 3, flip angle = 180°). For T1 -weighted imaging, axial 2D fast spoiled gradient echo (2D-FSPGR) sequence (TR = 120.54 ms; TE = 1.50 ms; echo train = 1; field of view = 3 x 3 x 4 cm; slice thickness = 1 mm; image matrix = 128 x 128; number of signal averages = 20, flip angle = 90°) was acquired. The axial 2D-FSPGR sequence was used to measure the signal intensity of the tumor tissue.Near Infrared-Fluorescent Imaging.

[0151] After MR imaging, the animals were euthanized, and immediate NIR imaging was performed of the open abdomen. Subsequently, tumors and organs, such as the heart, lung, liver, kidney, spleen, stomach, and intestine, were dissected and removed for NIR imaging of the excised tissues. NIR imaging w as performed using a Xenogen 2022 IVIS spectrum optical imaging system (Perkin Elmer Inc., Waltham, MA, USA). Total photon radiant efficiency (p / sec / cm2 / sr / pW) was quantified utilizing excitation / emission filters centered (with bandwidth) at 710(30) / 800(20) for BrCyl 12 and 745(30)7820(20) for ICG, respectively. Regions of interest (ROI) were drawn for each organ / tumor using Living Image (64-bit) software, and total radiant efficiencies were subsequently measured and for excised tumors divided by the tumor weight.Statistical Analysis

[0152] Absorption and emission spectra were analyzed using Microsoft Excel (version 2403, Microsoft Office, Redmond. WA. USA) software. Student's one-tailed t-tests were performed to compare groups, utilizing spreadsheet software. A significant level of p-value < 0.05 was considered statistically significant.Results:

[0153] To address the low quantum yield of ICG (MW ~ 774.96 Da), various investigators have pursued development of alternative dyes. Here a new NIR cyanine dye, brominated cyanine 112 (MW « 754.55 Da) (developed and manufactured by NanoQuantum Sciences, Bellevue, WA) is utilized. The chemical structure of ICG and BrCy 112 are show n in FIGs. 11 A- 11 B. Briefly, the strategy7in the development of BrCy 112 is based on altering the aromatic structure in cyanine dyes including asymmetrically with brominated nitrogen containing heterocyclic compound, specifically the indolenine group.Optical characterization of ICG and BrCy 112

[0154] The optical properties of BrCy 112 was compared to the standard ICG. Deionized (DI) water was used as the aqueous medium to assess the photophysical properties of the dyes, as both ICG and BrCy 112 exhibit amphiphilic solubility7. FIG. 12A displays the absorption spectra of 15.6 pM ICG and BrCy 112 in DI water. The absorption maximum of ICG in DI w ater was observed at 780 nm, while that of BrCy 112 was at 742 nm, indicating an approximately 40 nm blue shift. The absorbance values in BrCy 112 (0.390) and ICG (0.347) at the same concentration, 15.6 pM, were consistent at their respective maxima, and a similar absorption was observed for both. For both BrCy 112 and ICG, a second absorption peak was observed around 680 nm and 700 nm, corresponding to H-type aggregate forms of each fluorophore.

[0155] In order to characterize the fluorescent properties in the NIR region. FL spectra of ICG and BrCy 112 w ere conducted using a sample cell with a thickness of 2 mm and a spectrometer with InGaAs detector to minimize spectral artifacts and optimize detection efficiency in the NIR region. FIG. 12B illustrates the FL spectra of ICG and BrCy 112 at the same concentration in DI water Excitation wavelengths of 680 nm for BrCy 112 and 730 nm for ICG were selected to investigate the spectral features and avoid scattering of excitation light. The fluorescent maxima of BrCy 112 and ICG were observed at 780 nm and 820 nm, respectively, indicating a 40 nm FL blue shift of BrCy 112 compared to ICG, consistent with the amount of absorption peak shifts observed between both compounds. Notably, the FL intensity7of BrCy 112 was approximately 2.5 times greaterthan that of ICG, suggesting that BrCy 112 represents a promising candidate as a bright NIR fluorophore surpassing ICG FL intensity.Optimization of loading fluorophore concentrations in liposomal formation

[0156] The fluorophore concentration was optimized, and the fluorescent properties in the NIR region was monitored (FIGs. 13A-13B). All liposomal formulations maintained consistent ratios of materials, including phospholipid, cholesterol, and Gd- complex, except for the loading fluorophore concentration. Thus, only the fluorophores within the liposomes were altered in otherwise identical liposomal formulations. Integrated FL intensities were measured within the emission range of 770 nm to 860 nm at 745 nm excitation. Low FL intensity was observed at lower concentrations, while higher concentrations exhibited reduced FL intensity suggesting intermolecular quenching resulting from self-aggregation. Peak FL intensity suggesting optimal loading concentrations for the liposomal formulations were 25 pM for BrCyl l2 and 150 pM for ICG (FIG. 13A). Under these optimal conditions, the FL quantum yield (QY) of DMDG with 25 pM BrCyl 12 was approximately five times higher than that of DMDG with 1 0 pM ICG, even though the BrCyl 12 liposome used less amount of fluorophore (FIG. 13B). Therefore, liposomal formulations were prepared with loading concentrations of 25 pM for BrCy 112 and 150 pM for ICG as these were expected to maximize FL intensity; and, these nanoparticles were further characterized.Characterization of liposomal formations; DMDG-BrCyl l2 and DMDG-ICG

[0157] DMDG containing ICG or BrCy 112 at optimal concentrations along with negative control liposomes lacking any Gd-complex or fluorophore was prepared. The size distribution and polydispersity index (PDI) values depicted in FIG. 14A indicate that all liposomes had similar size distributions of approximately 150 nm with PDI values around 0.1. To assess the magnetic properties of the Gd-complex for MR imaging, T1 relaxivity measurements were conducted using a 3 T MRI scanner. FIG. 14B illustrates the T1 relaxivity measurement and shows a similar slope for the T1 relaxivity of the two DMDG samples. The slopes of T1 rate vs liposome-concentration were similar and no significant difference in T1 relaxivity was noted, as expected, with relaxivity of 15.55 mM' 's'1for DMDG-BrCyl l2 and 16.00 mM''s'' for DMDG-ICG. These values are twice as high as the 7.52 mM 's'1observed in conventional liposome formulations based solely on external Gd imaging agents, such as Gd(III)-DTPA-BSA.

[0158] To characterize the optical properties of the liposomes, absorption and FL spectra were measured for DMDG samples optimized in terms of fluorophore loading concentration for FL. Absorption peak of DMDG-ICG was -800 nm, whereas the peak for DMDG-BrCyl l2 was -750 nm (FIG. 14C). The absorption peaks of the liposomes with Gd-based contrast agents were approximately 10 nm red-shifted compared to free fluorophores of ICG or BrCy 112. Based on optimal loading concentration of the liposomal formation, the absorption values of 150 pM DMDG-ICG was approximately 6 times greater than that of 25 pM DMDG-BrCy 1 12. The FL spectra of the nanoparticles are presented in FIG. 14D. The FL peak of DMDG-ICG was observed at -870 nm, accompanied by a shoulder peak at -960 nm. In comparison, the peak maximum of DMDG-BrCy 112 was seen at -760 nm. with a shoulder peak at -880 nm. Within the NIR-I region, extending up to 1,000 nm, the integrated FL intensity of DMDG-BrCy 1 12 was approximately five times greater than that of DMDG-ICG. These findings are consistent with previous observations of FL intensity of free fluorophores and quantum yield measurements of nanoparticles depicted in FIGs. 12B and 13B, respectively. Thus. DMDG-BrCy 112 emerged as a promising candidate for NIR imaging when compared to DMDG-ICG.Imaging IP Ovarian Cancers by MR

[0159] Intraperitoneal tumors were established using human ovarian cancer HeyA8 cells. Two days after injection, HeyA8 tumors from mice injected with DMDG- ICG and DMDG-BrCyl l2 demonstrated enhancement on MR but not before injection. This was not seen with vehicles (liposomes without MR or NIR agent) nor with free dye (FIG. 15). Thus, MR imaging was enabled 2 days post injection by DMDG containing either BrCyl 12 or ICG, as indicated by the yellow arrow.Imaging IP Ovarian Cancers by NIR

[0160] Following MR, mice were sacrificed and immediately open abdomen optical NIR imaging followed by imaging of excised tumors was conducted. FIGs. 16A and 17A depict the merged representative coronal necropsy and coronal open abdomen NIR FL images of nude mice. Of note, excitations and emission specific for each fluorophore was used. Two days after injection, HeyA8 tumors from mice injected with DMDG-ICG and DMDG-BrCy 112 demonstrated NIR signal, but this w as not seen with vehicle (liposomes without MR or NIR agent) nor with free dye. The NIR signal in the tumor region, highlighted in yellow; appeared to be greater with DMDG-BrCyl l2 thanDMDG-ICG. Expected background signal was seen in reticuloendothelial system (RES) organs liver and spleen involved in scavenging liposomes. The tumors were removed and subsequent excised tumor analysis again revealed that HeyA8 tumors from mice injected with either DMDG-ICG or DMDG-BrCyl l2 exhibited enhanced signals compared to tumors from control mice injected with free ICG, free BrCyl l2, or vehicles (FIGs. 16B and 17B). Again, the NIR signal appeared to be greater with DMDG-BrCyl l2 than DMDG-ICG. Semi-quantitative evaluation of FL intensity of intraperitoneal HeyA8 tumors showed significantly greater FL signal / g tumor in the DMDG-BrCyl l2 and DMDG-ICG groups compared to the control groups (p < 0.05, n = 6) and the FL signal / g tumor was greater with DMDG-BrCyl 12 than DMDG-ICG (P< 05, n=6, FIGs. 16C and 17C). Thus, there was greater NIR signal in the intraperitoneal tumors in mice two days after both DMDG-BrCyl 12 or DMDG-ICG IV injection than controls and greater NIR signal in the intraperitoneal tumors in mice two days after DMDG-BrCyl 12 than DMDG-ICG IV injection. In particular, the FL tumor enhancement of DMDG-BrCyl 12 was ~ twice greater than that of DMDG-ICG.Conclusion and Discussion:

[0161] In DI water, the FL intensity of BrCyl l2 was ~2.5 times greater than that of ICG (p<0.05, n=5). In liposomal formulations with optimized loading concentrations. DMDG-BrCyl 12 exhibited FL intensity in the NIR region approximately three times greater than DMDG-ICG (p<0.05, n=5). Intraperitoneal ovarian tumors (HeyA8) in mice showed enhanced MR signals two days after intravenous injection of DMDG-BrCyl l2 and DMDG-ICG compared to controls. Open abdomen and excised tumor analysis revealed animals injected with DMDG-BrCyl 12 had twice the FL intensity (radiant efficiency ) / g tumor compared to those injected with DMDG-ICG (p < 0.05, n = 6).

[0162] BrCyl l2 has greater fluorescence intensity than ICG and in a nanoparticle (DMDG) can be used days after a single injection for MR and NIR imaging of intraperitoneal ovarian tumors in a mouse model suggesting clinical potential for pre- surgical planning and image-guided resection of intraperitoneal ovarian cancer.

[0163] In the DMDG platform, it was found that BrCyl l2 at a lower concentration for peak FL led to ~3 fold greater FL in the nanoparticle than ICG; and, 2 days after IV injection in an IP ovarian cancer model, approximately 2-fold greater NIR FL per gram tumor. Both DMDG-BrCyl 12 and DMDG-ICG enabled MR imaging. Thisimplies that DMDG-BrCyl 12 enables MR imaging and greater fluorescence intensity NIR imaging, thus, may enable ovarian cancer staging and pre-surgical planning as well as more sensitive (brighter tumors) real-time guidance for surgical resection.

[0164] The dual-mode imaging of tumors was demonstrated, incorporating both MR and NIR modalities, utilizing a single positive MR contrast nanoparticle in a human peritoneal ovarian cancer model. This nanoparticle exhibits prolonged residence with imaging performed two days after nanoparticle administration I.V. allowing for both MR and optical NIR imaging. The inventors foresee utilizing such an agent using MR for tumor staging and pre-surgical planning as well as using NIR for image-guided surgery. The dualmode design integrates external and internal Gd-chelate for MR imaging, BrCy 112 or ICG for NIR imaging, and PEGylation to decrease uptake by the reticuloendothelial system. This design harnesses the EPR effect for tumor localization, enhances MR and optical intensity within tumors, and prolongs residence time to facilitate pre-surgical planning and surgery. This approach may potentially enhance clinical workflow' efficiency for MR imaging by providing an extended window between nanoparticle injection and both MR and NIR imaging.

[0165] Dual-mode imaging offers significant advantages over single-mode imaging. MR imaging excels in displaying anatomy with exceptional clarity, facilitating the interpretation of nanoparticle signals w ithin the context of anatomic structures. In vivo percutaneous imaging with MRI is typically not depth-limited and provides clinical resolution on the order of millimeters in patients and -100 pm in mice. However, MRI machines are not utilized during abdominal surgery'. Given the potential limitations in signal detection with MRI, amplification at the nanoparticle level, as provided by the Dual- Gd approach, enables lesion visualization. Optical imaging, on the other hand, should enable dynamic real-time imaging during surgery, facilitating lesion detection and confirmation of resection. Optical imaging systems can be deployed in the operating room environment, and FL of the tumor should enable resection and aid avoiding eloquent structures complementing white light imaging of lesions and surrounding anatomy. Additionally, optical imaging offers superior spatial resolution theoretically down to submicron levels. Among wavelength of the light, near-infrared (NIR) imaging provides better depth of penetration (-10 mm) compared to visible light imaging to enable surgical guidance, although it remains insufficient for clinical percutaneous imaging for tumor staging and treatment planning enabled by MR.0166] MRI is a crucial modality in clinical diagnostic radiology, providing tunable soft-tissue contrast, high spatial and temporal resolution, and the benefit of no ionizing radiation exposure. The use of gadolinium (III) based contrast agents significantly enhances anatomical detail and improves diagnostic accuracy. Additionally, liposomes containing MR imaging agents have been investigated as nanocarriers for non-invasive MRI. These contrast agents, such as Gd-DTPA derivatives, incorporate an amphiphilic component into the liposome membrane, allowing for the encapsulation of therapeutic molecules within the lumen. This lumen, which accommodates both hydrophilic core and hydrophobic shell components within the liposome, can be utilized for drug delivery' or enhancing the imaging modality'. The benefits of liposomes include high agent loading efficiency, excellent stability, and controllable responses, providing superior performance compared to many other carriers. Furthermore, the dual Gd-nanoparticles feature both internal and external Gd imaging agents, facilitating increased T1 relaxation than single presentation and the liposomal formulation results in tumor localization due to the EPR effect. Liposomes with a similar lipid composition and size to DMDG have demonstrated the ability to localize to and have prolonged residence time in tumors for over five days. In practice, this extended duration should allow for sufficient time to conduct pre-surgical MR imaging and subsequent optical imaging-guided surgery'.

[0167] For optical imaging, ICG is the FDA approved fluorescent agent in the NIR region. Nevertheless, its detection is hindered by a modest quantum yield, a tendency to aggregate in aqueous solutions, and degradation of FL intensity' over imaging time, limiting its application in NIR optical imaging. These can be improved by encapsulation, which can prolong circulation time, increase in vivo stability', and increase signal intensity', but further improvements are needed. To improve NIR FL, BrCyl 12 was investigated for the first time as a potential candidate for the NIR fluorophore. It is in the same class as ICG, however, aromatic rings are modified including by an asymmetric brominated indolenine group at one end of the molecule. This resulted in greater FL intensity' compared to the conventional ICG fluorophore as the free dye. Upon encapsulation, less amount was needed for greater FL. resulting in improved quantum yield. Moreover, greater FL signal per gram of IP fluorescent ovarian tumor was noted with the DMDG-BrCyl l2 NP formulation.

[0168] BrCyl 12 is approximately 40 nm blue-shifted than ICG towards the visible spectrum. This is compatible with Si-based cameras recognized for their high efficiency, cost-effectiveness, and low background noise but w hose efficiency substantiallydecreases beyond 800 nm. However, this blue shift may compromise the benefits associated with conventional NIR imaging, potentially leading to reduced tissue penetration depth and increased interference from auto-background signals. Nonetheless, findings with the DMDG-BrCyl l2 formulation demonstrate intraperitoneal tumor imaging in animals with improved signal / g tumor compared to the nanoparticle formulation with ICG.

[0169] Most current nanoparticle research has focused on single optical imaging modalities, for example using quantum dots in extracellular vesicles or using polymers such as PLGA. The former can have binding proteins that target the tissue from which they originated but can be difficult to produce in large quantities. The latter can have several areas to bind imaging agents but have been used primarily for single modality imaging. Liposomes can incorporate materials internally such as in aqueous medium like BrCyl l2 and MR agents, in the hydrophilic membrane, as well as display moi eties externally such as an MR agent enabling interaction with surrounding water for signal generation; and encapsulation can increase fluorophore signal.

[0170] DMDG-ICG (or -BrCy 112) was designed to serve as a positive contrast agent for MR and NIR optical imaging after a single injection. In this study, DMDG- BrCyl l2 (or -ICG) detected intraperitoneal tumors in a peritoneal human ovarian cancer model by two different imaging modalities, both MR and NIR, after a single injection. The current findings suggest the potential for using a single injection of a single DMDG- BrCy 112 (or -ICG) to localize ovarian tumors by MR for pre-surgical planning and by NIR at the time of surgery to guide resection.

[0171] In summary, dual-mode imaging of tumors was conducted using both complimentary MR and NIR modalities with a single injection of MR positive contrast nanoparticle in human peritoneal ovarian cancer models using BrCy 112 in the nanoparticle formulation. Using BrCyl 12 at lower concentration within the DMDG nanoparticle than ICG resulted in ~3-fold increase in FL, consistent with greater quantum yield, and ~2-fold increase in FL intensity per gram intraperitoneal ovarian tumor compared to the ICG nanoparticle formulation. The DMDG nanoparticle remains within ovarian tumors for days; thus, there is strong potential for both presurgical planning with excellent anatomic context by MR and surgical guidance after a single injection providing real-time tumor imaging by NIR.

[0172] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein alsoencompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as "up to," "at least," and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1 -3 markers refers to groups having 1 , 2, or 3 markers . Similarly, a group having 1-5 markers refers to groups having 1, 2, 3. 4, or 5 markers, and so forth.

[0173] All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. Any feature, step, element, embodiment, or aspect disclosed herein can be used in combination with any other unless specifically indicated otherwise.

[0174] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g.. the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases ”at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a“ or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as ”a“ or ”an“ (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited,those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a formulation having at least one of A, B, and C” would include but not be limited to formulations that have A alone, B alone. C alone. A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g.. “ a formulation having at least one of A, B. or C” would include but not be limited to formulations that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0175] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0176] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. For example, “about 5”, shall include the number 5. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a grouphaving 1-3 values refers to groups having 1, 2, or 3 values. Similarly, a group having 1-5 values refers to groups having 1. 2, 3, 4, or 5 values, and so forth.

[0177] It would be understood that the various sizes, materials, configurations and arrangements disclosed herein may be combined and constructed in any way that is feasible to create a hybrid for any particular end use. Accordingly, all suitable modifications and equivalents may be resorted to falling within the scope of the appended claims. Unless defined otherwise, all technical and scientific terms used herein have same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Also, as used herein and in the appended claims, the singular form “a”, “and"’, and “the” include plural referents unless the context clearly dictates otherwise.

[0178] It is to be understood that the present invention is not to be limited to the exact description and embodiments as illustrated and described herein. To those of ordinary skill in the art, one or more variations and modifications will be understood to be contemplated from the present disclosure. Accordingly, all expedient modifications readily attainable by one of ordinary skill in the art from the disclosure set forth herein, or by routine experimentation therefrom, are deemed to be within the true spirit and scope of the invention as defined by the appended claims.

[0179] From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A nanoparticle vesicle for magnetic resonance imaging and fluorescence imaging, comprising: a membrane-bound vesicle; a magnetic resonance imaging agent within the membrane-bound vesicle; and a fluorescence imaging agent within the membrane-bound vesicle.

2. The nanoparticle vesicle of claim 1. wherein the vesicle is a liposome, micelle, cell membrane, or a cell-derived nanoparticle.

3. The nanoparticle vesicle of any of the previous claims, wherein the membrane is a plasma membrane and / or bilayer.

4. The vesicle-based nanoparticle system of claim 3. wherein the plasma membrane and / or bilayer comprises a phospholipid.

5. The nanoparticle vesicle of claim any of the previous claims, wherein the nanoparticle vesicle is configured for imaging in a Near-IR fluorescence imaging (NIRFI) system, in a fluorescence endoscopy system, and / or in a fluorescence laparoscopy system.

6. The nanoparticle vesicle of any one of the previous claims, wherein the magnetic resonance imaging agent is incorporated inside the nanoparticle.

7. The nanoparticle vesicle of any one of the previous claims, wherein the magnetic resonance imaging agent is incorporated on the surface of the nanoparticle.

8. The nanoparticle vesicle of any one of the previous claims, wherein the magnetic resonance imaging agent is incorporated on the surface of the nanoparticle and inside the nanoparticle.

9. The nanoparticle vesicle of any one of the previous claims, wherein the vesicle is a hemoglobin-depleted, ’'ghost” erythrocyte.

10. The nanoparticle vesicle of any one of the previous claims, wherein the magnetic resonance imaging agent comprises one or more of: gadolinium, gadodiamide, gadoversetamide, gadoxetate, Gadofosveset, gadobenate, gadopiclenol, gadopentetate, Gadoteridol, Gadobutrol, Gadoterate, Iron Oxide, Manganese, or any combination thereof.

11. The nanoparticle vesicle of any one of the previous claims, wherein the magnetic resonance imaging agent comprises one or more of: gadopiclenol, Gadopentetate dimeglumine (Gd-DTPA), gadobenate dimeglumine, Gd-BOPTA, gadoxetate disodium, Gadofosveset trisodium, Gd-DTPA-BMA, Gd-DTPA-BMEA. Gd-EOB-DTPA, Gd-HP- DO3A, Gd-BT-DO3A, Gd-DOTA, Ferumoxides, Ferumoxtran-10, Ferumoxsil, Mangafodipir trisodium, Gadoterate meglumine, or any combination thereof.

12. The nanoparticle vesicle of any one of the previous claims, wherein the fluorescence imaging agent comprises a halogenated fluorophore, cyanine, indoline, indolenine, or any combination thereof.

13. The nanoparticle vesicle of any one of the previous claims, wherein the fluorescence imaging agent comprises BrCylO6, BrCyll l, BrCyl l2, IR700, IR800, indocyanine green, or any combination thereof.

14. A method of detecting the presence of a cancer and / or a tumor in a subject, the method comprising: administering to the subject the nanoparticle vesicle of any one of the previous claims; and imaging the subject using magnetic resonance imaging and / or a fluorescence-based scan, wherein the localization of the magnetic resonance imaging agent and the fluorescence imaging agent within a subject is indicative of the cancer and / or tumor position.

15. The method of claim 14, wherein the subject is mammalian and / or human.

16. The method of claim 14 or 15, wherein the cancer and / or tumor is a solid tumor.

17. The method of any one of claims 14-1 , wherein the cancer and / or tumor is present in an ovary, lymph node, pancreas, colorectal tissue, intraperitoneal tissue, brain, eyes, head, neck, alimentary canal, lungs, mediastinum, heart, liver, spleen, gallbladder, adrenal glands, bladder, uterus, prostate, cervix, vagina, bones, muscles, skin, or any combination thereof.

18. The method of any one of claims 14-17, wherein the fluorescence-based scan is infrared, visual. NIRFI. fluorescence endoscopy, and / or fluorescence laparoscopy.

19. The method of any one of claims 14-18, wherein the method further comprises administering to the subject an effective amount of a molecule or therapy for treating the cancer and / or the tumor.

20. A method of preparing a subject for dual imaging analysis, the method comprising administering to the subject the nanoparticle vesicle of any one of claims 1- 13.

21. The method of claim 20, wherein the dual imaging analysis comprises fluorescence and / or MR imaging.

22. The method of claim 20 or 21, wherein the nanoparticle vesicle is administered through inhalation / spray, or through intravenous, intraperitoneal, intramuscular, or subcutaneous injection.

23. A nanoparticle vesicle for magnetic resonance imaging and fluorescence imaging, comprising: a membrane-bound vesicle; a magnetic resonance imaging agent Gd-BOPTA, gadobenate dimeglumine, or a chemical derivative thereof within the membrane-bound vesicle; and a fluorescence imaging agent comprising BrCylll, BrCyl l2, indocyanine green, or a chemical derivative thereof, within the membrane-bound vesicle.

24. A method of conducting magnetic resonance imaging and fluorescence imaging in a subject in need thereof, the method comprising: administering to the subject a nanoparticle vesicle, wherein the nanoparticle vesicle comprises: an outer membrane; a magnetic resonance imaging agent comprising Gd-BOPTA, and / or gadobenate dimeglumine; and a fluorescence imaging agent comprising BrCyl l l, BrCyl l2, and / or indocyanine green, wherein the nanoparticle vesicle is administered through inhalation / spray, or through intravenous, intraperitoneal, intramuscular, or subcutaneous injection.

25. A nanoparticle vesicle for magnetic resonance imaging and fluorescence imaging, comprising an at least one brominated carbocyanine (BrCy) dye, and a magnetic resonance imaging agent.

26. A cell-derived nanoparticle vesicle comprising a magnetic resonance imaging agent.

27. The cell-derived nanoparticle vesicle of claim 26, wherein the cell-derived nanoparticle vesicle is derived from a red blood cell.