MODIFIED J-AGGREGATES AND CONJUGATES THEREOF
20230118682 · 2023-04-20
Assignee
Inventors
Cpc classification
A61B5/0059
HUMAN NECESSITIES
C09B23/04
CHEMISTRY; METALLURGY
A61K49/0091
HUMAN NECESSITIES
A61K49/0093
HUMAN NECESSITIES
International classification
Abstract
Provided is a modified J-aggregate (MJ). In some embodiments, the MJ is nanoscale, sub-micronscale, or microscale. Also provided is an MJ conjugate. Also provided are processes for making an MJ or an MJ conjugate. Photoacoustic imaging methods employing the MJ or MJ conjugate are also provided.
Claims
1. A modified J-aggregate (MJ).
2. The MJ of claim 1, wherein said MJ comprises a J-aggregate forming dye.
3. The MJ of claim 1, wherein said MJ comprises a J-aggregate forming dye that has been functionalized.
4. The MJ of claim 1, wherein said MJ comprises a J-aggregate forming dye and a J-aggregate forming dye that has been functionalized.
5. The MJ of claim 3, wherein said J-aggregate forming dye has been functionalized with an azide group.
6. The MJ of claim 4, wherein said J-aggregate forming dye has been functionalized with an azide group.
7. The MJ of claim 2, wherein said J-aggregate forming dye is a cyanine dye.
8. The MJ of claim 2, wherein said J-aggregate forming dye is selected from the group consisting of indocyanine green (ICG), ICG-NHS, ICG-NH.sub.2, pseudoisocyanine, merocyanine, bis(2,4,6-trihydroxyphenyl)squaraine, tetrtakis(4-sulfonatophenyl)-porphyrin, antimony(III)-phthalocyanine, copper phthalocyanine, perylene bismide, hypericin, subphtalocyanine, and combinations thereof.
9. An MJ produced by the process of mixing a J-aggregate forming dye and a J-aggregate forming dye that has been functionalized.
10. The MJ of claim 9, wherein the mixing a J-aggregate forming dye and a J-aggregate forming dye that has been functionalized occurs in the presence of a salt.
11. The MJ of claim 10, wherein said salt is selected from the group consisting of KCl, NaCl, MgCl.sub.2, CaCl.sub.2), NiCl.sub.2, MnCl.sub.2, EuCl.sub.3, TbCl.sub.3, and a salt from a rare earth element.
12. The MJ of claim 1, wherein the MJ is nanoscale, sub-micronscale, or microscale.
13. The MJ of claim 12, wherein the MJ is nanoscale.
14. An MJ conjugate of Formula I MJ—E—D Formula I wherein MJ is a modified J-aggregate; E is a linker or a bond; D is a functional moiety.
15. The MJ conjugate of claim 14, wherein said MJ comprises a J-aggregate forming dye.
16. The MJ conjugate of claim 14, wherein said MJ comprises a J-aggregate forming dye that has been functionalized.
17. The MJ conjugate of claim 14, wherein said MJ comprises a J-aggregate forming dye and a J-aggregate forming dye that has been functionalized.
18. The MJ conjugate of claim 16, wherein said J-aggregate forming dye has been functionalized with an azide group.
19. The MJ conjugate of claim 17, wherein said J-aggregate forming dye has been functionalized with an azide group.
20. The MJ conjugate of claim 14, wherein D has been attached to MJ by employing copper-free click chemistry with said azide group.
21. The MJ conjugate of claim 15, wherein said J-aggregate forming dye is a cyanine dye.
22. The MJ conjugate of claim 15, wherein said dye is selected from the group consisting of ICG, ICG-NHS, ICG-NH.sub.2, pseudoisocyanine, merocyanine, bis(2,4,6-trihydroxyphenyl)squaraine, tetrtakis(4-sulfonatophenyl)-porphyrin, antimony(III)-phthalocyanine, copper phthalocyanine, perylene bismide, hypericin, subphtalocyanine, and combinations thereof.
23. The MJ conjugate of claim 14, wherein said MJ was produced by the process of mixing a J-aggregate forming dye and a J-aggregate forming dye that has been functionalized.
24. The MJ conjugate of claim 23, wherein the mixing a J-aggregate forming dye and a J-aggregate forming dye that has been functionalized occurs in the presence of a salt.
25. The MJ conjugate of claim 24, wherein said salt is selected from the group consisting of KCl, NaCl, MgCl.sub.2, CaCl.sub.2), NiCl.sub.2, MnCl.sub.2, EuCl.sub.3, TbCl.sub.3, NaOH, K.sub.2PO.sub.4, Mn(NO.sub.3).sub.2, Ni(NO.sub.3).sub.2 and a salt from a rare earth element.
26. The MJ conjugate of claim 14, wherein E is a linker comprising a copper-free click chemistry group.
27. The MJ conjugate of claim 14, wherein E is a linker selected from the group consisting of DBCO-PEG-NHS, DBCO-PEG-Biotin, DBCO-PEG-Thiol, DBCO-PEG-NH.sub.2, DBCO-PEG-Mal, Thiol-PEG-NHS, Thiol-PEG-Biotin, Thiol-PEG-NH.sub.2, Mal-PEG-NHS, Mal-PEG-NH.sub.2, Mal-PEG-Acrylate, Thiol-PEG-Acrylate, DBCO-PEG-Folate, and combinations thereof.
28. The MJ conjugate of claim 14, wherein D comprises streptavidin, biotin, RGD, or a derivative thereof.
29. The MJ conjugate of claim 14, wherein D comprises a drug, a protein, a targeting protein, or an antibody.
30. A method of photoacoustic imaging at a target site comprising providing the MJ of claim 1 at the target site; and monitoring absorbance at the target site.
31. A method of photoacoustic imaging at a target site comprising providing the MJ conjugate of claim 14 at the target site; and monitoring absorbance at the target site.
Description
DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION
[0082] While the general inventive concepts are susceptible of embodiment in many forms, there are shown in the drawings, and will be described herein in detail, specific embodiments thereof with the understanding that the present disclosure is to be considered an exemplification of the principles of the general inventive concepts. Accordingly, the general inventive concepts are not intended to be limited to the specific embodiments illustrated herein.
[0083] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
[0084] The articles “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “a cell” means one cell or more than one cell.
[0085] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±5%, preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
[0086] As used herein, the term “nanoscale” means particles having a size range between approximately 1 and 100 nanometers (nm).
[0087] As used herein, the term “nanometer” means 1/1,000,000,000 meter (m).
[0088] As used herein, the term “sub-micronscale” means particles having a size less than a micron (μm) and larger than 100 nm.
[0089] As used herein, the term “micron” means 1/1,000,000 meter (m).
[0090] As used herein, the term “microscale” means having a size of approximately 1 to 5 microns (μm).
[0091] As used herein, the term “RGD” means a common peptide responsible for cell adhesion comprising arginine, glycine and aspartic acid, or a derivative of the peptide, for example a derivative comprising biotin. RGD examples include, but are not limited to, Cyclo-Arg-Gly-Asp-D-Phe-Lys(Biotin) (c[RGDfK(Biotin)]) (SEQ ID NO: 1), which is commercially available from Vivitide (Louisville, Ky., USA), Sigma Aldrich (St. Louis, Mo., USA), Anaspec (Fremont, Calif., USA), or other vendors.
[0092] As used herein, the term “antibody” and “antibodies” are meant in a broad sense and include immunoglobulin molecules including polyclonal antibodies, monoclonal antibodies including murine, human, human-adapted, humanized and chimeric monoclonal antibodies, antibody fragments (e.g. F(ab).sub.2, Fab, Fv, or a domain antibody (dAb) fragment), bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, diabodies, nobodies, and single chain antibodies (e.g. scFv).
[0093] In some embodiments of any of the compositions or methods described herein, a range is intended to comprise every integer or fraction or value within the range.
[0094] Embodiments described herein as “comprising” one or more features may also be considered as disclosure of the corresponding embodiments “consisting of and/or “consisting essentially of such features.
[0095] Thus, there still exists a critical need to assemble J-aggregates whose size can be easily tailored for imaging applications without using complicated synthesis methods or nanocarriers. Therefore, developing a facile method for direct functionalization of ICG J-aggregates with targeting molecules would avoid the complexities and limitations of nanocarrier encapsulation and enable high-throughput and cost-efficient synthesis of a targeted PAI contrast agent for use in the NIR regime.
[0096] Modified J-aggregates (MJs) described herein are designed to provide improved photoacoustic probes of various sizes, including nanoscale. Functional moieties, including but not limited to a drug, a protein, an antibody or a payload may be attached to the MJs either directly or via a linker to form MJ conjugates. Also provided herewith are methods of producing photoacoustic probes, as well as methods of using photoacoustic probes.
[0097] Modified J-Aggregates
[0098] Provided is a modified J-aggregate (MJ). In some embodiments, the MJ comprises a J-aggregate forming dye. In some embodiments, the MJ comprises a J-aggregate forming dye that has been functionalized. In some embodiments, the MJ comprises a J-aggregate forming dye and a J-aggregate forming dye that has been functionalized.
[0099] In some embodiments, the J-aggregate forming dye has been functionalized with an azide group.
[0100] In some embodiments, the J-aggregate forming dye is a cyanine dye. In further embodiments, the J-aggregate forming dye is selected from the group consisting of indocyanine green (ICG), ICG-NHS, ICG-NH.sub.2, pseudoisocyanine, merocyanine, bis(2,4,6-trihydroxyphenyl)squaraine, tetrtakis(4-sulfonatophenyl)-porphyrin, antimony(III)-phthalocyanine, copper phthalocyanine, perylene bismide, hypericin, subphtalocyanine, and combinations thereof.
[0101] Provided is an MJ produced by the process of mixing a J-aggregate forming dye and a J-aggregate forming dye that has been functionalized. The J-aggregate forming dye and the J-aggregate forming dye that has been functionalized may be mixed at various molar ratios.
[0102] In some embodiments, the mixing a J-aggregate forming dye and a J-aggregate forming dye that has been functionalized occurs in the presence of a salt. In further embodiments, the salt is selected from the group consisting of KCl, NaCl, MgCl.sub.2, CaCl.sub.2, NiCl.sub.2, MnCl.sub.2, EuCl.sub.3, TbCl.sub.3, and a salt from a rare earth element.
[0103] Nanoscale MJ Conjugates
[0104] For certain applications, it may be advantageous to have nanoscale size MJs. Examples of such applications include applications that require the contrast agents to pass through biological barriers, for example, applications such as contrast-enhanced imaging of tumor lesions require nanosized particles. For such applications, the synthesis protocoal may be adapted to enable control over the size of MJ particles.
[0105] In some embodiments, the MJ is nanoscale, sub-micronscale, or microscale. In further embodiments, the MJ is nanoscale. In some embodiments, the MJ is 100 nm to 400 nm in size. In further embodiments, the MJ is 100 to 300 nm in size. In yet further embodiments, the MJ is 150 nm to 250 nm in size. In yet further embodiments, the MJ is 180 nm to 220 nm in size, for example about 200 nm in size.
[0106] Functionalization of MJs
[0107] Provided is an MJ conjugate of Formula I
[0108] MJ—E—D
[0109] Formula I
[0110] wherein
[0111] MJ is a modified J-aggregate;
[0112] E is a linker or a bond;
[0113] D is a functional moiety.
[0114] In some embodiments, the MJ comprises a J-aggregate forming dye.
[0115] In some embodiments, the MJ comprises a J-aggregate forming dye that has been functionalized.
[0116] In some embodiments, the MJ comprises a J-aggregate forming dye and a J-aggregate forming dye that has been functionalized.
[0117] In some embodiments, the J-aggregate forming dye has been functionalized with an azide group. In further embodiments, D has been attached to MJ by employing copper-free click chemistry with said azide group.
[0118] A schematic representation of how copper free click chemistry is carried out in an embodiment is shown below, wherein R is ICG:
##STR00001##
[0119] In some embodiments, the J-aggregate forming dye is a cyanine dye. In further embodiments, the J-aggregate forming dye is selected from the group consisting of indocyanine green (ICG), ICG-NHS, ICG-NH.sub.2, pseudoisocyanine, merocyanine, bis(2,4,6-trihydroxyphenyl)squaraine, tetrtakis(4-sulfonatophenyl)-porphyrin, antimony(III)-phthalocyanine, copper phthalocyanine, perylene bismide, hypericin, subphtalocyanine, and combinations thereof.
[0120] In some embodiments, E is a linker comprising a copper-free click chemistry group. In further embodiments, E is a linker selected from the group consisting of DBCO-PEG-NHS, DBCO-PEG-Biotin, DBCO-PEG-Thiol, DBCO-PEG-NH.sub.2, DBCO-PEG-Mal, Thiol-PEG-NHS, Thiol-PEG-Biotin, Thiol-PEG-NH.sub.2, Mal-PEG-NHS, Mal-PEG-NH.sub.2, Mal-PEG-Acrylate, Thiol-PEG-Acrylate, DBCO-PEG-Folate, and combinations thereof.
[0121] In some embodiments, D comprises streptavidin, biotin, RGD, or a derivative thereof. In some embodiments, D comprises a drug, a protein, a targeting protein, or an antibody. In further embodiments, the drug, protein, targeting protein or antibody is connected to E via a biotin-streptavidin interaction. In some embodiment, E comprises biotin and D comprises streptavidin. In some embodiments, E comprises streptavidin and D comprises biotin.
[0122] In some embodiments, the drug is a chemotherapeutic (e.g., Doxorubicin, Daunorobicin). In some embodiments, the drug is a steroid, a chemokine, a cytokine, a hormone, a therapeutic protein, an antimicrobial peptide, or a nucleic acid. In some embodiments, the nucleic acid is mRNA or siRNA. In some embodiments, the drug is bradykinin (BK).
[0123] In some embodiments, the protein is avidin, streptavidin, neutravidin, an enzyme, a signal peptide, a marker, a growth factor, a neurotransmitter, an antibody, an interferon, thrombin, an anticoagulant protein, a targeting peptide, or a cell penentrating peptide. In further embodiments, the antibody is a broad neutralizing antibody. In further embodiments, the anticoagulant protein is Protein C, Protein S or antithrombin.
[0124] In some embodiments, the targeting protein is a receptor, for example a chemokine receptor, a cytokine receptor, a hormone receptor, a growth factor receptor, a neurotransmitter receptor, or a cell adhesion receptor.
[0125] In some embodiments, the antibody is an antibody to a target tissue or an antibody to a target deep tissue. In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, or a single domain antibody. In some embodiments, the antibody is anti-CTL4-4, anti-VCAM 1, anti-asialoglycoprotein receptor, or anti-VEGFR. In some embodiments, the monoclonal antibody is anti-CTL4-4, for example for targeting T lymphocytes. In some embodiments, the polyclonal antibody is anti-VCAM 1, for example for targeting kidney tissues. In some embodiments, the single domain antibody is anti-asialoglycoprotein receptor, for example for targeting liver tissues. In some embodiments, the single domain antibody is anti-VEGFR, for example a nanobody anti-VEGFR.
[0126] In some embodiments, the MJ conjugate is Bio-JAAZ, Strep-JAAZ or RGD-JAAZ, as further described in the Examples.
[0127] In some embodiments, the MJ conjugate is nanoscale, sub-micronscale, or microscale. In further embodiments, the MJ conjugate is nanoscale. In some embodiments, the MJ conjugate is 100 nm to 400 nm in size. In further embodiments, the MJ conjugate is 100 to 300 nm in size. In yet further embodiments, the MJ conjugate is 150 nm to 250 nm in size. In yet further embodiments, the MJ conjugate is 180 nm to 220 nm in size, for example about 200 nm in size.
[0128] Photoacoustic Imaging Using MJs or MJ Conjugates
[0129] Provided is a method of photoacoustic imaging at a target site comprising providing the MJ of any one of the embodiments described herein at the target site; and monitoring absorbance at the target site.
[0130] In some embodiments, the MJ absorbs in the near-infrared I (NIR-I) region (750-950 nm). In some embodiments, the MJ absorbs in the region of 450 to 950 nm. In further embodiments, the MJ absorbs in the near-infrared II (NIR-II) region of 1000 to 1700 nm.
[0131] In some embodiments, the MJ absorb incident light in the region of 450 to 950 nm and fluoresce to emit light in the near-infrared II (NIR-II) region of 1000 to 1700 nm.
[0132] In some embodiments, the target site is subjected to laser pulses at 1 to 100 pulses/s. In some embodiments, the target site is subjected to laser pulses at 1 to 2000 pulses/s, for example 10 pulses/s. In further embodiments, the target site is subjected to light pulses from light-emitting diodes at 1 to 2000 pulses/s.
[0133] In some embodiments, the target site is a tissue. Tissues include, but are not limited to, vascular tissue, placental vasculature, skin, brain, breast, ovarian, testicular, prostate, lung, colon, liver and all the hepatocellular system, spleen, uterine cervix, and kidney.
[0134] In further embodiments, the target site is a deep tissue. Deep tissues include, but are not limited to, lymph node, gastrointestinal tract, bladder, pancreas, colon, kidney, liver, and brain.
[0135] Provided is a method of photoacoustic imaging at a target site comprising providing the MJ conjugate of any one of the embodiments described herein at the target site; and monitoring absorbance at the target site.
[0136] In some embodiments, the MJ conjugate is Bio-JAAZ, Strep-JAAZ or RGD-JAAZ, as further described in the Examples.
[0137] In some embodiments, the MJ conjugate absorbs in the near-infrared I (NIR-I) region (620-950 nm). In some embodiments, the MJ absorbs in the region of 450 to 950 nm. In further embodiments, the MJ absorbs in the near-infrared II (NIR-II) region of 1000 to 1700 nm.
[0138] In some embodiments, the MJ conjugate absorbs incident light in the region of 450 to 950 nm and fluoresce to emit light in the near-infrared II (NIR-II) region of 1000 to 1700 nm.
[0139] In some embodiments, the target site is subjected to laser pulses at 1 to 100 pulses/s. In some embodiments, the target site is subjected to laser pulses at 1 to 2000 pulses/s, for example 10 pulses/s. In further embodiments, the target site is subjected to light pulses from light-emitting diodes at 1 to 2000 pulses/s.
[0140] In some embodiments, the target site is a tissue. Tissues include, but are not limited to, vascular tissue, placental vasculature, skin, brain, breast, ovarian, testicular, prostate, lung, colon, liver and all the hepatocellular system, spleen, uterine cervix, and kidney.
[0141] In further embodiments, the target site is a deep tissue. Deep tissues include, but are not limited to, lymph node, gastrointestinal tract, bladder, pancreas, colon, kidney, liver, and brain.
[0142] Use of the MJ or MJ conjugates described herein may reduce the rate of secondary tumor resection surgeries. It may allow conservation of more healthy tissue. This may improve patient outcome.
[0143] Tumor Margin Staining Using MJs or MJ Conjugates
[0144] In some embodiments, MJ or MJ conjugate is formulated in a water-based sprayable form (solvents can be water, PBS or other water-based physiological solution) that can be used for staining tumor margin directly after surgical resection of the tumor from the patient. The MJ or MJ conjugate may be micro or nanoscale.
[0145] In some embodiments, the MJ or MJ conjugate helps detect the presence of tumor cells near to the surface of the resected tumors via specific targeting moieties (adapted to the tumor targeted) that enable tumor-cell specific labelling.
[0146] Using photoacoustic imaging, presence of tumor cells close to the margin of the resected tumor is evaluated.
[0147] Near-Infrared II (NIR-II) Imaging
[0148] NIR-II is the second NIR optical window that encompasses wavelengths ranging from 1000-1700 nm, respectively. Using these longer wavelengths to perform fluorescence imaging reduces the light scattering and autofluorescence typically observed in biological tissues and allows for deeper imaging with improved signal-to-background ratio. MJs and MJ conjugates have a strong and characteristic fluorescence emission signal detectable in the NIR-II windows when excited at wavelengths of 808 (that correspond to ICG absorption peak) and 890 nm (that correspond to ICG aggregates absorption peak).
[0149] In some embodiments, MJ or MJ conjugate is used to perform targeted deep in vivo imaging using dual imaging modality, namely photoacoustic and fluorescence imaging.
[0150] Photothermal Therapy
[0151] MJ or MJ conjugate is used to perform photothermal therapy that can be used to kill cancer cells locally. In some embodiments, the MJ or MJ conjugate is free. In some embodiments, the MJ or MJ conjugate is encapsulated in a liposome. In some embodiments, the MJ or MJ conjugate performs photothermal therapy using NIR excitation (e.g., 808 nm laser-irradiation for 100 to 300 s). By including tumor targeting moieties in the MJ or MJ conjugate, photothermal destruction of tumors may be conducted.
EXAMPLES
[0152] Materials and Methods
[0153] Materials:
[0154] Indocyanine Green (ICG) and ICG-Azide were purchased from AAT Bioquest (Pleasanton, Calif., USA). Dibenzylcyclooctyne-PEG4-Biotin (DBCO-PEG4-Biotin) was acquired from Jena Biosciences (Jena, Germany). Streptavidin and Cyclo-Arg-Gly-Asp-D-Phe-Lys(Biotin) (c[RGDfK(Biotin)]) (SEQ ID NO: 1) were purchased from Vivitide (Louisville, Ky., USA). Filtration unit Amicon Ultra-0.5 centrifugal filter, potassium chloride (KCl) and Bicinchoninic Acid (BCA) Kit for protein determination were acquired from Sigma-Aldrich (St. Louis, Mo., USA). Triton X-100, Tween-20, Dimethylsulfoxide (DMSO) and glycerol were purchased from Fischer Scientific (Waltham, Mass., USA). Phosphate-Buffered Saline (PBS, 10X solution at pH 7.4) was acquired from ThermoScientific (Waltham, Mass., USA). Agarose, high strength (1200 g/cm.sup.2) was purchased from Agarose Unlimited (Alachua, Fla., USA). Agarose, low melt (>500 g/cm.sup.2) was purchased from IBI Scientific (Dubuque, Iowa, USA). Molecular biology grade water was purchased from Quality Biological (Gaithersburg, Md., USA). Low-volume quartz glass cuvettes (ZEN2112) and DTS-1070 folded capillary zeta cells were purchased from Malvern Analytical (Malvern, U.K.), 1X Dulbecco's Modified Eagle's Medium was purchased from Corning Inc (Corning, N.Y., USA) and Fetal Bovine Serum (FBS) were purchased from VWR Life Sciences (Radnor, Pa., USA). India Ink and whole sheep blood containing anticoagulant were purchased from Hardy Diagnostics (Santa Maria, Calif., USA). Eagle's Minimum Essential Media (EMEM) and Ethylenediaminetetraacetic acid (EDTA) were purchased from ATCC (Manassas, Va., USA). Penicillin was purchased from Millipore Sigma (Burlington, Mass., USA).
[0155] Methods:
[0156] Preparation of ICG-JA and JAAZ particles: ICG stock solutions were prepared at a concentration of 5 mg/ml in molecular biology grade water and stored at −20° C. ICG-azide stock solution was made at 10 mg/ml in DMSO and stored at −20° C. To assemble the ICG-JA, ICG dye was prepared at 1 mM in molecular biology grade water complemented with 20 mM of KCl and incubated at 60° C. for 20 hours. For each molar ratio of ICG-azide:ICG solution, 1:20, 1:10, 1:5 and 1:3, a solution of the corresponding molarity of ICG and ICG-azide was prepared. For JAAZ microparticles, the different solutions of ICG and the corresponding molarity of ICG-azide was incubated in 20 mM KCl for 20 hours at 60° C. to a final concentration of 1 mM total dye. Unless otherwise specified, JAAZ microparticle samples were prepared with 1:10 ICG-azide:ICG molar ratio to a final concentration of 1 mM total dye.
[0157] Preparation of N-JAAZ particles: For JAAZ particles in the 600-700 nm range, the reaction solution of 1:10 ICG-azide:ICG molar ratio was incubated in 1 mM KCl for 20 hours at the same temperature as mentioned above to a final concentration of 1 mM total dye. To make nanosized JAAZ particles of size ˜200 nm, 250 μM total dye solution was incubated in 0.1 mM KCl for 14 hours at 60° C. A complete summary of the total dye concentration at 1:10 ICG-azide:ICG molar ratio, KCl molarity and time required for the formation of JAAZ particles is given in
[0158] Filtration of JA: Unless otherwise specified, prepared ICG-JA and JAAZ samples were filtered using 0.5 ml Amicon centrifugal filters (100 kDa cutoff) to remove the non-aggregated free monomers or dimers of ICG dye, if present. Filtration was carried out by centrifugation at 4000 g for 3 minutes at room temperature. Three cycles of filtration in molecular biology water at a total volume of 0.5 ml were used to ensure complete removal of unreacted dye. The percentage of samples recovered after filtration was calculated by:
% recovery=[(A894.sub.(BF)−A894.sub.(AF)/A894.sub.(BF)]*100
where A894(BF) is the absorbance intensity at 894 nm before filtration (BF) and, A894.sub.(AF) is the 459 absorbance intensity after filtration.
[0159] Characterization of JA
[0160] Standard Curve: The amount of free dye in all J-aggregate samples was determined by incubating the formed J-aggregate particles in 1% Triton X-100 for 10 minutes at 37° C. and scanning the samples from 400-990 nm. A standard curve of a 1:10 molar solution of ICG-azide:ICG dye was created to determine the free dye concentration in the dissociated J-aggregate samples.
[0161] Absorbance measurements: To confirm the presence of J-aggregates, the samples were scanned from 440 nm to 990 nm with 2 nm step size using a PowerWavex microplate spectrophotometer by Bio-Tek Instruments Inc. The excitation and emission wavelengths of free ICG dye are 780 and 800 nm respectively. The emission wavelength of ICG-JA was determined at 895 nm. Unless specified, all readings were taken at a free dye concentration of 25 μM determined using the standard curve.
[0162] Fluorescence measurements: Fluorescence intensity of different concentrations of free ICG dye and J-aggregates was recorded using a Tecan Safire.sup.2™ (Mannendorf, Switzerland) fluorescence microplate reader. Both free ICG dye and J-aggregate particles were excited at 720 nm with an emission at 770 nm. The fluorescence emission spectrum was recorded from 770 to 850 nm with a step size of 2 nm.
[0163] Dynamic Light Scattering (DLS) measurements: The hydrodynamic diameter of the formed ICG-JA and JAAZ particles was measured using a Malvern Zetasizer Nano ZS™ (Malvern, United Kingdom) instrument. The size measurements were taken using low-volume quartz glass cuvettes at 25 μM of free dye concentration. The zeta potential (surface charge) of all samples was determined using the Malvern Zetasizer Nano ZS™ using DTS-1070 folded capillary zeta cells. The measurements for Zeta potential were taken at a dye concentration of 15 μM and a volume of 700 μl in 0.1X PBS at pH 7.4.
[0164] Gel Electrophoresis: A 2% (w/v, in MilliQ water) agarose gel was prepared using high strength agarose. The agarose gel was casted in a square gel caster. A square window of 6×6 cm was cut in the 2% gel using a blade. Low melting agarose of 0.05% (w/v, in MilliQ water) was poured in the cut window containing well-combs and the gel polymerized at 4° C. overnight. 30 μl of 500 μM JAAZ and N-JAAZ samples were mixed with an equal volume of glycerol and loaded into the wells. The gel was run at 120 V for 10 minutes.
[0165] Scanning Electron Microscopy: 5 μl of the JAAZ samples prepared at dye concentration of 1 mM with 20 mM KCl or 5 μl of the N-JAAZ (183 nm) samples prepared at dye concentration of 250 μM with 0.1 mM KCl incubated for 20 or 14 hours at 60° C., respectively, were deposited on a clean mica surface and vacuum dried overnight. SEM images were collected using Jeol JSM-IT500HR InTouchScope™ (Tokyo, Japan) at an accelerating voltage of 15 kV and a working distance of 10 mm. EDX spectra were collected using Octane Elect EDS System at an accelerating voltage of 15 kV and a resolution of 127 eV. ImageJ (63) was used to determine the size distribution of the particles in the obtained SEM images.
[0166] Streptavidin functionalization of JAAZ particles: 500 μM of JAAZ or N-JAAZ particles with 1:10 ICG-azide:ICG molar ratio were labelled with DBCO-PEG4-Biotin at a concentration 10 times greater than the estimated number of azide groups available (Table 2).
TABLE-US-00001 TABLE 2 Samples and their attached functional groups/biomolecules Composition and Functional Groups Attached ICG- DBCO- Strepta- RGD- Sample ICG N.sub.3 Biotin vidin Biotin Name (mM) (mM) (Molar ratio with respect to ICG-N.sub.3) JA 1 — — — — JAAZ 1 0.1 — — — Bio-JAAZ 1 0.1 10X — — Strep-JAAZ 1 0.1 10X 10X — RGD-JAAZ 1 0.1 10X 10X 10X
[0167] The azide group on the J-aggregates reacts with the DBCO end of DBCO-PEG4-Biotin in an aqueous buffer free of metal catalysts (water or 1X PBS) and this reaction is known as copper-free click chemistry. The samples were incubated and mixed in an incubator shaker overnight at room temperature protected from light. To remove excess DBCO-Biotin, the samples were filtered using the same procedure mentioned in the filtration section. These biotin functionalized samples (Bio-JAAZ) were diluted to 100 μM of free dye concentration and treated with 10 mg/ml streptavidin at 5X concentration of the available biotin sites (Strep-JAAZ). The samples were kept at 4° C. for two hours to ensure attachment of streptavidin to the available biotin sites. Excess streptavidin was removed by filtration using 100 kDa mini-centrifugal filters. The samples were filtered by centrifugation at 4000 g for three minutes. Four cycles of filtration were carried out to ensure complete removal of free streptavidin. The absorbance spectra of all samples were tabulated. The Strep-JAAZ samples were labelled with c[RGDfK(Biotin)] at 10X molar concentration and incubated for two hours at 4° C. Excess c[RGDfK(Biotin)] was removed by filtration using the same procedure as mentioned before. The labelled samples were named as RGD-JAAZ. All Bio-JAAZ, Strep-JAAZ and RGD-JAAZ samples were characterized using the same procedure as mentioned in the characterization section.
[0168] Determination of amount of streptavidin attached: All Strep-JAAZ samples after filtration were diluted to 5 μM of free dye concentration. BCA working reagent was made following the instructions provide by the supplier. 25 μl of 5 μM samples are incubated in 200 μl of BCA working solution and the amount of streptavidin attached is calculated using a BSA standard curve. Bio-JAAZ samples are used as control in every experiment.
[0169] Photoacoustic Measurement: The 96-well plate was secured to the bottom of a plastic water reservoir. The reservoir was filled with deionized water, completely submerging the sample. For comparison of smaller volumes, 2-inch segments of 24-gauge PTFE tubing (˜100 μL) was filled with various samples. The tubes were placed in a clear acrylic chamber submerging the samples in deionized water. A single element piezoelectric 35 MHz or 5 MHz focused transducer (V326, Olympus, Mass., USA) was fixed at 12 mm or 45 mm above each sample with their functional element submerged in deionized water. Optimization of positioning and photoacoustic signal from each sample was done though movement of a fine x-y axis stage, moving above each sample with a fixed transducer and pulsed laser. Excitation of the sample was done obliquely by a wavelength-tunable (690-950 nm) pulsed laser designed for photoacoustic imaging (Phocus Mobile, Opotek) at the peak absorption wavelength for the JAAZ (895 nm) for magnitude comparison. The photoacoustic signal recorded from the transducer is sent to a 20/40db amplifier (HVA-200M, Femto) from there the signal is recorded through a lock-in amplifier (Zurich Instruments, Zurich, Switzerland) triggered on each laser pulse using a photodiode placed near the laser source. For each measurement, a 10 second recording (100 waveforms) was taken for each sample (or at each wavelength, for PA spectrum generation). The absolute amplitude of each waveform was calculated, averaged, and normalized to the signal recorded from India ink or whole sheep's blood containing anticoagulant.
[0170] Photoacoustic Imaging
[0171] Cell culture: Hela cells were cultured in EMEM augmented with 10% Fetal Bovine Serum, and 1% Penicillin and kept in an incubator at 37° C. and 5% CO.sub.2. HeLa cells were seeded using 0.25% (w/v) Trypsin-0.53 mM EDTA solution to glass slides at a concentration of 1.0×10.sup.5 cells/mL.
[0172] Cell and Phantom Imaging: Plain glass slides cultured with Hela cells were stained with 10 μM JAAZ or RGD-JAAZ for 20 minutes in an incubator at 37° C. and 5% CO.sub.2. Short segments of 24-gauge PTFE tubing (˜100 μL) were filled with 10 μM RGD-JAAZ and arranged on a glass slide. The slides were then placed under a single element piezoelectric 35 MHz (or 15 MHz) focused transducer fixed at 12 mm (or 2 inches) above the surface of the slide. A wavelength-tunable (690-950 nm) pulsed laser designed for photoacoustic imaging (Phocus Mobile, Opotek) was obliquely shown over the slide. The peak absorption wavelength for the JAAZ (895 nm) was used to excite the sample and the photoacoustic signal recorded from the transducer was sent to a 20/40 dB amplifier (HVA-200M, Femto) from there the signal was recorded through a lock-in amplifier (Zurich Instruments, Zurich, Switzerland) triggered on each laser pulse using a photodiode placed near the laser source. The sample was then raster scanned through movement of the stage at 100 μm steps (with fixed laser source and transducer) mounted on motorized translation stages (Thorlabs). Absolute signal magnitude was then plotted in 2D space to create the image. A simple Matlab image gaussian filter was then used to smooth the image.
[0173] Statistical Analysis: All bar graphs are expressed as the mean±standard deviation. Statistical analyses were performed using the unpaired, two-tailed Student's t-test and One-Way Analysis of Variance (ANOVA) with a Tukey post-hoc test using the software GraphPad Prism 9.0.2. Data was deemed statistically significant if the p value was <0.05. Absolute PA signal amplitude is defined as the sum of the absolute minimum and maximum peak PA signal.
Example 1: Formation of Azide-Modified J-Aggregates (JAAZ)
[0174] To assemble functionalized ICG-JA that could be further modified with targeting moieties using simple and robust copper-free click chemistry, J-aggregates were prepared using a combination of ICG and ICG-azide dyes (
[0175] The same protocol was applied to assemble JAAZ particles. Mixtures of ICG dye and azide-modified ICG dye were incubated at different molar ratios of ICG-azide to ICG. After incubation for 20 and 40 hours at 60° C. in water, the typical J-aggregate peak was not observed at 895 nm (
Example 2: Characterization and Stability of JAAZ Microparticles
[0176] After confirming formation of JAAZ particles, the presence and accessibility of the azide group was examined using measurements of size and surface charge (zeta potential). The surface zeta (ζ) potential of both ICG-JA and JAAZ was first measured, knowing that the difference in charge between the ICG-azide and the regular ICG dye should modify the overall surface charge of the JAAZ particles. The average ζ potential of the JAAZ particles was measured at −50.32 mV, as opposed to the ζ potential of ICG-JA at −60.11 mV (
[0177] Importantly, the synthesized JAAZ microparticles could be lyophilized and resuspended in water with no significant change in the absorption spectra (
TABLE-US-00002 TABLE 1 Size and charge (zeta potential) of JAAZ and N-JAAZ before and after vacuum drying and resuspension in water. Size (nm) Zeta Potential (mV) Sample Before Drying After Drying Before Drying After Drying JAAZ 1,083 ± 52 1,030 ± 256 −52.1 ± 2 −55.0 ± 5 Nanosized 281.8 ± 37 360.9 ± 38 −54.7 ± 1 −57.1 ± 3 JAAZ Sample size is n = 3 for DLS measurement; p = 0.8470 = ns for JAAZ; p = 0.4622 = ns for N-JAAZ (Statistics are for DLS measurement); n = 2 for zeta potential measurement.
[0178] Before further modification of JAAZ particles, the free dye concentration of ICG-201 JA and JAAZ particles was determined, to quantify the incorporation of dye into these J-aggregates and correlate the absorbance with the total concentration of dye. The filtered JAAZ particles were dissolved into free dye molecules using 1% Triton X-100 (TX-100) and the concentration of free dye was determined from a standard curve of ICG dye dispersed in the same TX-100 concentration (
Example 3: Functionalization of JAAZ Microparticles
[0179] To enable conjugation of JAAZ with streptavidin, copper-free click chemistry was performed to graft biotin linkers onto the azide groups on the surface of the JAAZ particles. The biotin-streptavidin complex has been abundantly used in biotechnological applications due to its biocompatibility, strong binding affinity and availability of different biotinylated functional modalities for targeting or imaging. Copper free click chemistry was performed with the azide groups of JAAZ using a DBCO-PEG-biotin linker to create a biotinylated version of the JAAZ particles (Bio-JAAZ,
[0180] Attachment of biotinylated targeting molecules such as RGD was also explored (
Example 4: Synthesis, Characterization, and Functionalization of Nanoscale JAAZ (N-JAAZ)
[0181] The micrometer scale of the JAAZ particles might restrict their use in applications that require the contrast agents to pass through biological barriers, for example, applications such as contrast-enhanced imaging of tumor lesions require nanosized particles. Therefore, to meet the requirements for such applications of J-aggregates, the synthesis protocol was adapted to enable control over the size of JAAZ particles. J-aggregate formation is dependent on time, salt and dye concentration; hence, the impact of these three parameters on the size of JAAZ particles was explored. Solutions of 250, 500, 750 and 1000 μM total dye with 1:10 ICG-azide:ICG molar ratio were incubated at 60° C. for 20 hours under KCl concentrations of 0.1, 1 and 20 mM. At 0.1 mM KCl, the 895 nm absorption peak appeared after incubation for 6 hours for 1000 and 750 μM of total dye (
[0182] Following the same biomolecule functionalization strategy used for the microparticles (
Example 5: PA Properties of Different JAAZ Particles and Cellular Targeting Capabilities of RGD-JAAZ
[0183] To quantify their viability for PAI applications, the in-vitro photoacoustic properties of micro and nanoscale JAAZ along with RGD functionalized JAAZ were characterized.
[0184] To explore the possibility of using JAAZ in vivo, the photoacoustic signal was compared to whole blood. Short segments of 24-gauge PTFE tubing (containing approximately100 μL, of sample) were filled with 20 μM of ICG dye, JAAZ, N-JAAZ, RGD-JAAZ, or whole sheep's blood. PA signals were recorded from each sample and the signal amplitude was normalized to the whole-blood PA-signal amplitude (
Example 6: Theranostic Applications of MJ Conjugates
[0185] An MJ can be further conjugated with a combination of a targeting moiety and a therapeutic cargo such as a chemotherapeutic drug (e.g., Doxorubicin, Daunorobicin), an antibody, a therapeutic protein, and a peptide. The cargo may be conjugated using a chemical linker such as that which was used for attachment of the targeting moiety.
[0186] These MJ conjugates are used as theranostic particles. They have targeted delivery capability to accumulate to any specific area of the body or specific diseased tissue using specific targeting moieties. They allow for imaging and diagnosis, and can be used to release drug on demand and with high spatial precision to treat diseased tissues. Ultrasound, light or a combination of both can be used to trigger dissolution of the MJ conjugate and the subsequent release of the drug in the body area of interest. The light can be pulsed or continuous.
[0187] Tables of P-Values for Indicated Figures
TABLE-US-00003 TABLE 3 P-values for FIG. 2A-2B Row JA JAAZ FIG. 2A JA Na 0.0001 JAAZ 0.0001 Na FIG. 2B JA Na 0.0161 JAAZ 0.0161 Na
TABLE-US-00004 TABLE 4 P-values for FIG. 3C FIG. 3C Row JAAZ Bio-JAAZ Strep-JAAZ RGD-JAAZ JAAZ Na 0.9058 0.0013 0.0019 Bio-JAAZ 0.9058 Na <0.0001 <0.0001 Strep-JAAZ 0.0013 <0.0001 Na 0.9011 RGD-JAAZ 0.0019 <0.0001 0.9011 Na
TABLE-US-00005 TABLE 5 P-values for FIGS. 4B, 4E and 4F FIG. 4B Row 0.1 mM KCl 1 mM KCl 20 mM KCl 0.1 mM KCl Na 0.0007 <0.0001 1 mM KCl 0.0007 Na <0.0001 20 mM KCl <0.0001 <0.0001 Na Row N-JAAZ N-Bio-JAAZ N-Strep-JAAZ N-RGD-JAAZ FIG. 4E N-JAAZ Na 0.3627 0.3634 0.0690 N-Bio-JAAZ 0.3627 Na 0.0645 0.0882 N-Strep-JAAZ 0.3634 0.0645 Na 0.0919 N-RGD-JAAZ 0.0690 0.0882 0.0919 Na FIG. 4F N-JAAZ Na 0.7620 0.0007 0.0004 N-Bio-JAAZ 0.7620 Na 0.0004 0.0002 N-Strep-JAAZ 0.0007 0.0004 Na 0.4006 N-RGD-JAAZ 0.0004 0.0002 0.4006 Na
TABLE-US-00006 TABLE 6 P-values for FIGS. 5B, 5D FIG. 5B, 25 μM Row JAAZ N-JAAZ JAAZ Na <0.0001 N-JAAZ <0.0001 Na Row JAAZ N-JAAZ RGD-JAAZ FIG. 5B, 12 μM JAAZ Na <0.0001 <0.0001 N-JAAZ <0.0001 Na <0.0001 RGD-JAAZ <0.0001 <0.0001 Na FIG. 5B, 5 μM JAAZ Na <0.0001 <0.0001 N-JAAZ <0.0001 Na <0.0001 RGD-JAAZ <0.0001 <0.0001 <0.0001 FIG. 5D, 25 μM Row ICG JAAZ N-JAAZ RGD-JAAZ ICG Na <0.0001 <0.0001 <0.0001 JAAZ <0.0001 Na <0.0001 <0.0001 N-JAAZ <0.0001 <0.0001 Na <0.0001 RGD-JAAZ <0.0001 <0.0001 <0.0001 Na
TABLE-US-00007 TABLE 7 P-values for FIG. 15B FIG. 15B Row Bio-JAAZ Strep-JAAZ RGD-JAAZ Bio-JAAZ Na 0.4386 0.0358 Strep-JAAZ 0.4386 Na 0.0477 RGD-JAAZ 0.0358 0.0477 Na
TABLE-US-00008 TABLE 8 P-values for FIGS. 16A-16B Row JAAZ Bio-JAAZ Strep-JAAZ RGD-JAAZ FIG. 16A JAAZ Na 0.5494 0.8409 0.0940 Bio-JAAZ 0.5494 Na 0.5175 0.1036 Strep-JAAZ 0.8409 0.5175 Na 0.2148 RGD-JAAZ 0.0940 0.1036 0.2148 Na FIG. 16B JAAZ Na 0.9729 0.6851 0.3637 Bio-JAAZ 0.9729 Na 0.7482 0.4650 Strep-JAAZ 0.7482 0.7482 Na 0.7118 RGD-JAAZ 0.3637 0.4650 0.7118 Na
TABLE-US-00009 TABLE 9 P-values for FIG. 17B FIG. 17B Row 1:20 1:10 1:5 1:3 1:20 Na 0.2901 0.7246 0.2769 1:10 0.2901 Na 0.4064 0.7123 1:5 0.7246 0.4064 Na 0.4275 1:3 0.2749 0.7123 0.4275 Na
TABLE-US-00010 TABLE 10 P-values for FIGS. 22A-22D Row 0.1 mM 1 mM 20 mM FIG. 22A, 1000 μM, 6 hrs 0.1 mM Na 0.2833 0.0007 1 mM 0.2833 Na 0.0006 20 mM 0.0007 0.0006 Na FIG. 22A, 1000 μM, 8 hrs 0.1 mM Na 0.0157 0.0165 1 mM 0.0157 Na 0.0010 20 mM 0.0165 0.0010 Na FIG. 22A, 1000 μM, 14 hrs 0.1 mM Na 0.2834 0.6409 1 mM 0.2834 Na 0.2570 20 mM 0.6409 0.2570 Na FIG. 22A, 1000 μM, 20 hrs 0.1 mM Na 0.7945 0.0462 1 mM 0.7945 Na 0.0296 20 mM 0.0462 0.0296 Na FIG. 22B, 750 μM, 6 hrs 0.1 mM Na 0.4137 0.0506 1 mM 0.4137 Na 0.0502 20 mM 0.0506 0.0502 Na FIG. 22B, 750 μM, 8 hrs 0.1 mM Na 0.0428 0.00001 1 mM 0.0428 Na 0.0502 20 mM 0.00002 0.00001 Na FIG. 22B, 750 μM, 14 hrs 0.1 mM Na 0.9776 0.2496 1 mM 0.0428 Na 0.3148 20 mM 0.2496 0.3148 Na FIG. 22B, 750 μM, 20 hrs 0.1 mM Na 0.9776 0.0421 1 mM 0.9776 Na 0.0089 20 mM 0.0421 0.0089 Na FIG. 22C, 500 μM, 4 hrs Row 1 mM 20 mM 1 mM Na 0.0005 20 mM 0.0005 Na Row 0.1 mM 1 mM 20 mM FIG. 22C, 500 μM, 6 hrs 0.1 mM Na 0.00003 0.00007 1 mM 0.00003 Na 0.00002 20 mM 0.00007 0.00002 Na FIG. 22C, 500 μM, 8 hrs 0.1 mM Na 0.00007 <0.00001 1 mM 0.00007 Na 0.00002 20 mM <0.00001 0.00002 Na FIG. 22C, 500 μM, 14 hrs 0.1 mM Na 0.0419 0.0030 1 mM 0.0419 Na 0.0022 20 mM 0.0030 0.0022 Na FIG. 22C, 500 μM, 20 hrs 0.1 mM Na 0.0785 0.0055 1 mM 0.0785 Na 0.0013 20 mM 0.0055 0.0013 Na Row 1 mM 20 mM FIG. 22D, 250 μM, 4 hrs 1 mM Na 0.0013 20 mM 0.0013 Na FIG. 22D, 250 μM, 6 hrs 1 mM Na 0.0009 20 mM 0.0009 Na FIG. 22D, 250 μM, 8 hrs 1 mM Na 0.00003 20 mM 0.00003 Na Row 0.1 mM 1 mM 20 mM FIG. 22D, 250 μM, 14 hrs 0.1 mM Na 0.1494 0.0322 1 mM 0.1494 Na 0.0627 20 mM 0.0322 0.0627 Na FIG. 22D, 250 μM, 20 hrs 0.1 mM Na 0.0149 0.0186 1 mM 0.0149 Na 0.0530 20 mM 0.0186 0.0530 Na
Example 7: In Vivo Imaging of Two Mice Using RGD Functionalized J-Aggregates
[0188] To further demonstrate the clinical translation potential of the JAAZ particles, an in vivo pilot study was conducted on the commercially available small animal PAI system TriTom from PhotoSound Technologies (Houston, Tex., USA) (
TABLE-US-00011 TABLE 11 Contrast-to-noise ratio and circulation half-life of ICG and RGD-JAAZ particles. Injection Contrast-to-noise ratio Circulation half-life (mins) ICG 1.59 1.19 ± 0.45 RGD-JAAZ 2.42 10.26 ± 1.24 n = 2. Data is represented as mean ± SD.
Example 8: Further Synthesis, Characterization, and Functionalization of Nanoscale JAAZ (N-JAAZ)
[0189] Stock solution of ICG dye was prepared at 5 mg/ml (6.5 mM) in water and stock of ICG-azide dye was prepared at 10 mg/ml (12 mM) in DMSO. Different molar ratio N-JAAZ particles, namely, 1:10, 1:5, 1:3, 2:5 and 1:1 was prepared by mixing the corresponding ratios of ICG and ICG-azide dye in different concentrations of potassium chloride. The final concentration of total dye (ICG and ICG-azide) was 350 μM for every sample. Unless stated otherwise, all samples were incubated for 18 hours at 60° C. In order to synthesize N-JAAZ particles of size ˜100 nm, a 350 μM total dye reaction solution of 1:1 ICG-azide:ICG molar ratio was incubated with 5 mM potassium chloride at the same temperature and time as specified previously. A summary of the different ratios of ICG-azide:ICG molar (1:10,1:5,1:3,2:5,1:1) and the different concentrations of potassium chloride (1,2,3,4.5 mM) used is given in
[0190] For the formation of N-JAAZ particles, different molar ratio solutions of ICG dye and azide modified ICG dye (ICG-azide) were incubated with different KCl concentrations at 60° C. (
TABLE-US-00012 TABLE 12 Polydispersity Index (PDI) of different molar ratio ICG-azid:ICG samples Molar ratio Molarity of KCl [mM] ICG-azide:ICG 1 2 3 4 5 1:10 0.462 ± 0.066 0.423 ± 0.033 0.533 ± 0.077 0.533 ± 0.077 0.581 ± 0.095 1:5 0.413 ± 0.093 0.478 ± 0.098 0.727 ± 0.075 0.721 ± 0.082 0.691 ± 0.194 1:3 0.168 ± 0.022 0.163 ± 0.02 0.175 ± 0.016 0.202 ± 0.012 0.210 ± 0.024 2:5 0.124 ± 0.023 0.143 ± 0.023 0.151 ± 0.024 0.158 ± 0.013 0.143 ± 0.019 1:1 0.079 ± 0.011 0.087 ± 0.026 0.116 ± 0.019 0.127 ± 0.013 0.109 ± 0.016 n = 3 distinct samples; data is represented as mean ± SD.
[0191] On increasing the molar ratio of ICG-azide:ICG to 1:3 the size of the J-aggregates decreased to 171±21.7 nm, 187±8.5 nm, 181±5.3 nm and 149±20.8 nm at 1, 2, 3 and 4 mM KCl respectively (
[0192] Along with molar ratio of the ICG and ICG-azide dye and concentration of KCl, the influence of the time of incubation was also studied for N-JAAZ formation. The absorbance spectra of 1:3, 2:5 and 1:1 molar ratio solution demonstrate the appearance of the J-aggregate peak at 895 nm at 6 hours (
[0193] Hence, to demonstrate and summarize the effective control over the size of the nanosized J-aggregates (N-JAAZ), the above-mentioned ratios of ICG-azide:ICG (1:10, 1:5, 1:3, 2:5 and 1:1) were incubated at 60° C. in 5 mM KCl. All ratios demonstrated a maximum optical absorption at 895 nm (
[0194] The in-vitro stability of N-JAAZ particles was tested in physiological media such as 1X PBS at pH 7.4, media supplemented with 10% serum and 100% serum at 37° C. N-JAAZ particles were stable in 1×PBS and media with 10% serum for 48 hours with no decrease in the intensity of the absorbance peak at 895 nm (
[0195] The incorporated azide groups in the synthesized N-JAAZ particles can be used for the attachment of targeting moieties by using click-chemistry groups. As described elsewhere herein, biomolecules such as biotin and streptavidin were functionalized onto the surface of micron sized JAAZ particles. Following the same protocol, DBCO-biotin followed by streptavidin was functionalized onto N-JAAZ particles. Zeta potential of the bare N-JAAZ particles was −56.6±4.2 mV. The addition of DBCO-biotin changed the zeta potential value to −45.3±5.4 mV. The zeta potential value changed to −17.4±2.4 mV on the coating of streptavidin onto the available biotin groups (
[0196] The versatility of N-JAAZ particles was further demonstrated by functionalizing the azide groups available with DBCO-nitriloaceticacid (DBCO-NTA). The NTA group interacts with histidine (His) and can be used for the attachment of His tagged proteins or peptides. Functionalization with more positively charged DBCO-NTA caused a significant change in the value of the zeta potential of 1:10, 1:5, 1:3, 2:5 and 1:1 molar ratio sample (
Example 9: PA Properties of N-JAAZ Particles
[0197] The in-vitro photoacoustic (PA) properties of the N-JAAZ particles were further evaluated. The PA signal magnitude of the particles showed a dependence on the equivalent concentration of free dye, as seen with absorbance. With increased free dye concentration (
Example 10: Formation of J-Aggregates with Modified ICG Dyes
[0198] Other modified dyes with N-Hydroxysuccinimide (NHS) and amine (NH.sub.2) functional groups were incorporated in a J-aggregate preparation to demonstrate that the method can work for dyes other than ICG-azide. Incorporating different functional groups also allowed the liberty to perform different functionalization chemistry onto the aggregates. The same method as described elsewhere herein (varying salt concentration) was used to prepare these aggregates. Results are shown in
Example 11: Tumor Margin Staining
[0199] MJ or MJ conjugate is formulated in a water-based sprayable form (solvents can be water, PBS or other water-based physiological solution) that can be used for staining tumor margin directly after surgical resection of the tumor from the patient. The MJ or MJ conjugate may be micro or nanoscale. The MJ or MJ conjugate helps detect the presence of tumor cells near to the surface of the resected tumors via specific targeting moieties (adapted to the tumor targeted) that will enable tumor-cell specific labelling. The MJ or MJ conjugate is sprayed directly onto the tumor resects and the resects are washed with physiological solution after 5 to 10 minutes of staining. Only the tumor cells are labelled and non-tumor cells are not labelled. Using photoacoustic imaging, the surgeons and the pathologists are able to evaluate the presence of tumor cells close to the margin of the resected tumor and decide if there is a need to remove more tissues. (
Example 12: Near-Infrared II (NIR-II) Imaging
[0200] NIR-II is the second NIR optical window that encompasses wavelengths ranging from 1000-1700 nm, respectively. Using these longer wavelengths to perform fluorescence imaging reduces the light scattering and autofluorescence typically observed in biological tissues and allows for deeper imaging with improved signal-to-background ratio. MJs and MJ conjugates have a strong and characteristic fluorescence emission signal detectable in the NIR-II windows when excited at wavelengths of 808 (that correspond to ICG absorption peak) and 890 nm (that correspond to ICG aggregates absorption peak).
Results
[0201] Free ICG (monomeric ICG) in PBS were compared with MJ in PBS at a same dye concentration (˜100 μM) placed in capillary tubes of 20 μL. A higher emission signal was shown from the MJs than from the free ICG in NIR-II (
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[0265] All publications and patents referred to herein are incorporated by reference. Various modifications and variations of the described subject matter will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to these embodiments. Indeed, various modifications for carrying out the invention are obvious to those skilled in the art and are intended to be within the scope of the following claims.