WO2019126565A1

WO2019126565A1 - Lipid derivatives for in vitro or in vivo delivery

Info

Publication number: WO2019126565A1
Application number: PCT/US2018/066914
Authority: WO
Inventors: Dmitri Simberg; Weston J. SMITH
Original assignee: The Regents Of The University Of Colorado, A Body Corporate
Priority date: 2017-12-20
Filing date: 2018-12-20
Publication date: 2019-06-27

Abstract

The present invention relates to novel constructs that allow for delivery of biologically active agents into cells. In certain embodiments, the constructs comprises conjugates of a lipophilic membrane dye, or a derivative or analogue thereof, with the biologically active agent.

Description

TITLE OF THE INVENTION

Lipid Derivatives for In Vitro or In Vivo Delivery

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/608,346, filed Dec 20, 2017, which application is hereby incorporated by reference in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

This invention was made with government support under grant number CA194058 awarded by National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Improving the pharmacokinetics (prolonging circulation time and delaying hepatic and renal clearance) of biologies and drugs is of great importance to the field of drug delivery. To meet this end, several strategies have been developed, including

nanoformulations, pegylation, fusion with polypeptides, and physical drug reservoirs. An alternative approach is to covalently link molecules onto biological structures that exhibit inherent prolonged circulation, such as albumin and blood cells.

Due to biocompatibility, red blood cells (RBCs) have been used for delivery of genes, chemotherapy, contrast agents, and enzymes. The bulk of research on RBCs as drug delivery vehicles has been focused on hypotonic loading of erythrocytes to contain bioactive cargo, and a lesser taken approach has been to bind molecules to the surface of erythrocytes covalently, with high affinity protein-protein interactions, and with lipids. Unfortunately, covalent modifications to red blood cells alter the membrane non-selectively, leading to damage and clearance. Further, affinity tags involving antibodies and peptides are expensive and synthetically cumbersome.

Thus, there is a need for novel constructs that allow delivery of biologically active agents, such as genes, chemotherapy, contrast agents, and/or enzymes, into cells in vitro or in vivo. The present invention addresses this unmet need.

BRIEF SUMMARY OF THE INVENTON

In one aspect, the invention provides a construct comprising a lipophilic membrane dye which is covalently linked through a linker to a cargo. In another aspect, the invention provides a composition comprising the construct of the invention and at least one cell, wherein at least a fraction of the construct is incorporated into the membrane of the cell. In yet another aspect, the invention provides a method of delivering a cargo to a cell. In yet another aspect, the invention provides a method of delivering a cargo to a subject in vivo. In yet another aspect, the invention provides a lipophilic membrane dye), or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof. In yet another aspect, the invention includes a compound comprising a linker (L) and a lipophilic membrane dye. In yet another aspect, the invention provides a method of solubilizing a compound in an aqueous solution. In yet another aspect, the invention comprises a method of enhancing the endothelial membrane crossing of a cargo in a subject.

In certain embodiments, the cargo is selected from the group consisting of a therapeutic drug, nucleic acid, polypeptide, enzyme, antibody, ligand, biologically active lipid, dye (or chromophore), fluorophore, bioluminescent label, biosensor, contrast agent, radioisotope, hydrophilic polymer, hydrophilic copolymer, and chemiluminescent label. In other embodiments, the lipophilic membrane dye comprises the compound of formula (I) or (II), or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof:

wherein: R¹ is selected from the group consisting of C₁₄-C₂₂ alkyl, C₁₄-C₂₂ alkenyl, C₁₄-C₂₂ alkynyl, and C₁₄-C₂₈ acyl; R² is selected from the group consisting of C₁₄-C₂₂ alkyl, C₁₄-C₂₂ alkenyl, C₁₄-C₂₂ alkynyl, and C₁₄-C₂₈ acyl; R³¹, R^3u, R³¹¹¹, and R^3lv are each independently selected from the group consisting of H and Ci-C₆ alkyl; each occurrence of R⁴ is independently selected from the group consisting of Ci-C₆ alkyl, C₃-C₈ cycloalkyl, halo, - N<¾, -CN, -OR^4a, -NR^4aR^4a, -NR^4a-C(=0)R^4a, -NR^4a-S0₂R^4a, -C(=0)0R^4a, -C(=0)NR^4aR^4a, - SR^4a, and -S(=0)i-₂(Ci-C₆ alkyl), wherein each occurrence of R^4a is independently selected from the group consisting of H and Ci-C₆ alkyl; each occurrence of R⁵ is independently selected from the group consisting of Ci-C₆ alkyl, C₃-C₈ cycloalkyl, halo, -NO₂, -CN, -OR^5a, -NR^5aR^5a, -NR^5a-C(=0)R^5a, -NR^5a-S0₂R^5a, -C(=0)0R^5a, -C(=0)NR^5aR^5a, -SR^5a, and -S(=0)i. ₂(Ci-C₆ alkyl), wherein each occurrence of R^5a is independently selected from the group consisting of H and Ci-C₆ alkyl; R⁶ is selected from the group consisting of CH₃ C₁₄-C₂₈ acyl, C₁₅-C₂₂ alkyl, C₁₅-C₂₂ alkenyl, C₁₅-C₂₂ alkynyl, and phospholipid; R⁷ is selected from the group consisting of CH₃ C₁₄-C₂₈ acyl, C₁₅-C₂₂ alkyl, C₁₅-C₂₂ alkenyl, C₁₅-C₂₂ alkynyl, and phospholipid; R⁸ is selected from the group consisting of Ci-C₆ alkyl and -NHR^8a, wherein R^8a is selected from the group consisting of H, C₁₆-C₂₂ alkyl, C₁₆-C₂₂ alkenyl, and C₁₆-C₂₂ alkynyl; R⁹ is selected from the group consisting of H and S0₃H; R¹⁰ is selected from the group consisting of -NR^10aR^10a and -NR^10a-C(=O)R^10a, wherein each occurrence of R^10a is independently selected from the group consisting of H and Ci-C₆ alkyl; m is 0, 1, 2, 3, 4, 5, or 6; n is 0, 1, or 2; p is 0, 1, or 2; wherein each occurrence of alkyl, cycloalkyl, or acyl is optionally substituted.

In certain embodiments, R¹ and R² are independently selected from the group consisting of Cie acyl, C₂₀ acyl, C₂₂ acyl, C₂₄ acyl, Cie alkyl, C₂₀ alkyl, C₂₂ alkyl, C₂₄ alkyl, Ci₈ alkenyl, C₂₀ alkenyl, C₂₂ alkenyl, C₂₄ alkenyl, Cie alkynyl, C₂₀ alkynyl, C₂₂ alkynyl, and C₂₄ alkynyl. In certain embodiments, m is 0 or 1. In certain embodiments, R³¹, R^3u, R^3m, and R^3lv are each methyl.

In certain embodiments, the phospholipid comprises

In certain embodiments, in the construct having formula (I), the linker is attached directly to the phenyl ring of at least one indolinyl group, to a R⁴ group, and/or to a R⁵ group, and/or the linker is attached to R¹ and/or R².

In certain embodiments, in the construct having formula (II), the linker is attached directly to the phenyl ring of at least one indolinyl group, to a R⁹ group, and/or to a R¹⁰ group, and/or the linker is attached to R⁶ and/or R⁷.

In certain embodiments, the compound of formula (I) comprises the compound of formula (III), or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof:

wherein:

S-, -S(C=0)-, or -CRR-; and Y comprises the linker conjugated to the cargo.

In certain embodiments, the compound of formula (II) comprises the compound of formula (IV), or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof:

In certain embodiments, the compound of formula (II) comprises the compound of formula (V), or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof:

wherein A comprises the linker conjugated to the cargo.

In certain embodiments, the linker comprises a disulfide linker. In certain embodiments, the disulfide linker comprises

In certain embodiments, the linker comprises at least one -OCH₂CH₂- group. In certain embodiments, the linker comprises from 1 to about 5,000 -OCH₂CH₂- groups. In certain embodiments, the linker comprises formula (A), (B) or (C): *-(CH₂)_mi-Xi-(CH₂-CH₂- X₂)_m2-(CH₂)_m3-C(X₃)- (A); *-(CH₂)_ml-0-(CH₂-CH₂-0)_m2-(CH₂)rn₃-C(0)- (B); *-

(CHR')_mi-0-(CHR'-CHR'-0)_m2-(CHR')_m3-C(0)- (C); wherein: * indicates the bond between the linker and the compound of formula (I) or (II); each ml, m2, and m3 is independently an integer ranging from 0-5000; each X_l X₂, and X₃ is independently absent (a bond), O, or N-R'; each R' is independently selected from the group consisting of hydrogen, optionally substituted Ci-C₆ alkyl, optionally substituted C₃-Cg cycloalkyl, and optionally substituted C3-C8 cycloheteroalkyl.

In certain embodiments, the linker is conjugated to the cargo through a 3-thio- succinimido group.

In certain embodiments, the cargo is further covalently linked through an

independently selected additional linker to at least one other independently selected lipophilic membrane dye of formula (I) or (II), or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof.

In certain embodiments, the additional linker comprises a disulfide linker. In certain embodiments, the additional linker comprises formula (A), (B) or (C): *-(CH₂)_mi-Xi-(CH₂- CH₂-X₂)_m2-(CH₂)_m3-C(X₃)- (A); *-(CH₂)_ml-0-(CH₂-CH₂-0)_m2-(CH₂)rn₃-C(0)- (B);

*-(CHR')_mi-0-(CHR'-CHR'-0)_m2-(CHR')_m3-C(0)- (C); wherein: * indicates the bond between the linker and the at least one other independently selected lipophilic membrane dye of formula (I) or (II); each ml, m2, and m3 is independently an integer ranging from 0-5000; each Xi, X₂, and X₃ is independently absent (a bond), O, or N-R'; each R' is independently selected from the group consisting of hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C₃-Cg cycloalkyl, and optionally substituted C₃-Cg cycloheteroalkyl.

In certain embodiments, the cargo is enzymatically cleavable, whereby upon cleavage of the cargo the at least two lipophilic membrane dyes are not covalently connected with each other.

In certain embodiments, the hydrophilic polymer is at least one selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone, polyethylenimine, polymethacrylate, and polyvinyl alcohol. In certain embodiments, the hydrophilic copolymer comprises at least one polymer selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone, polyethylenimine, polymethacrylate, and polyvinyl alcohol, or any copolymer thereof.

In certain embodiments, at least a fraction of the lipophilic membrane dye of the construct inserts into the membrane of the cell. In certain embodiments, the cell is a red blood cell. In certain embodiments, the cell does not undergo significant lysis upon incorporation of the construct into the membrane of the cell.

In certain embodiments, the composition further comprises at least one

pharmaceutically acceptable carrier.

In certain embodiments, the composition is formulated for administration by a route selected from the group consisting of oral, parenteral, transdermal, transmucosal, intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical.

In certain embodiments, the method comprises contacting the construct of the invention with a cell under conditions whereby at least a fraction of the lipophilic membrane dye of the construct inserts into the membrane of the cell.

In certain embodiments, the cell is a red blood cell. In certain embodiments, the cell does not undergo significant lysis upon insertion of the lipophilic membrane dye of the construct into the membrane of the cell.

In certain embodiments, the cargo is selected from the group consisting of a therapeutic drug, nucleic acid, polypeptide, enzyme, antibody, ligand, biologically active lipid, dye (or chromophore), fluorophore, bioluminescent label, biosensor, contrast agent, radioisotope, hydrophilic polymer, hydrophilic copolymer, and chemiluminescent label.

In certain embodiments, the method comprises administering to the subject a composition comprising the construct of the invention and at least one cell, wherein at least a fraction of the construct is incorporated into the membrane of the cell.

In certain embodiments, the administering is by a route selected from the group consisting of oral, parenteral, transdermal, transmucosal, intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra arterial, intravenous, intrabronchial, inhalation, and topical.

In certain embodiments, the cargo has a higher circulating half-life in the subject as compared to when the cargo, which is not part of the construct, is administered to the subject. In certain embodiments, the cargo comprises a therapeutically effective agent, thereby treating or preventing a disease or disorder in the subject.

In certain embodiments, the cargo is covalently linked through independently selected linkers to at least two independently selected lipophilic membrane dyes of formula (I) or formula (II), or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof. In certain embodiments, the cargo is enzymatically cleavable in vivo, whereby upon cleavage of the cargo the at least two lipophilic membrane dyes are not covalently connected with each other. In certain embodiments, the subject is further administered at least one additional therapeutically effective agent.

In certain embodiments, the subject is a mammal. In certain embodiments, the mammal is a human.

In certain embodiments, the lipophilic membrane dye comprises the compound of formula (IIA)

wherein: R is selected from the group consisting of H, CH₃, and -CH₂R¹⁰; R⁶ is selected from the group consisting of CH₃ C₁₄-C₂₈ acyl, C₁₅-C₂₂ alkyl, C₁₅-C₂₂ alkenyl, C₁₅-C₂₂ alkynyl, and phospholipid; R⁷ is selected from the group consisting of CH₃ C₁₄-C₂₈ acyl, C₁₅-C₂₂ alkyl, C₁₅-C₂₂ alkenyl, C₁₅-C₂₂ alkynyl, and phospholipid; R⁸ is selected from the group consisting of Ci-C₆ alkyl and -NHR^8a, wherein R^8a is selected from the group consisting of H, C₁₆-C₂₂ alkyl, C₁₆-C₂₂ alkenyl, and C₁₆-C₂₂ alkynyl; R⁹ is selected from the group consisting of H and S0₃H; R¹⁰ is selected from the group consisting of -NR^10aR^10a and -NR^10a-C(=O)R^10a, wherein each occurrence of R^10a is independently selected from the group consisting of H and Ci-C₆ alkyl; wherein each occurrence of alkyl, cycloalkyl, or acyl is optionally substituted.

In certain embodiments, the lipophilic membrane dye comprises the compound of formula (I) or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof:

wherein in (I): R¹ is selected from the group consisting of C₁₄-C₂₂ alkyl, C₁₄-C₂₂ alkenyl, C₁₄-C₂₂ alkynyl, and C₁₄-C₂₈ acyl; R² is selected from the group consisting of C₁₄-C₂₂ alkyl, C₁₄-C₂₂ alkenyl, C₁₄-C₂₂ alkynyl, and C₁₄-C₂₈ acyl; R³¹, R^3u, R³¹¹¹, and R^3lv are each independently selected from the group consisting of H and Ci-C₆ alkyl; each occurrence of R⁴ is independently selected from the group consisting of Ci-C₆ alkyl, C₃-C's cycloalkyl, halo, -NO₂, -CN, -OR^4a, -NR^4aR^4a, -NR^4a-C(=0)R^4a, -NR^4a-S0₂R^4a, -C(=0)0R^4a, -C(=0)NR^4aR^4a, - SR^4a, and -S(=0)i_-2(Ci-C₆ alkyl), wherein each occurrence of R^4a is independently selected from the group consisting of H and Ci-C₆ alkyl; each occurrence of R⁵ is independently selected from the group consisting of Ci-C₆ alkyl, C₃-C's cycloalkyl, halo, -N0₂, -CN, -OR^5a, -NR^5aR^5a, -NR^5a-C(=0)R^5a, -NR^5a-S0₂R^5a, -C(=0)0R^5a, -C(=0)NR^5aR^5a, -SR^5a, and -S(=0)i. ₂(Ci-C₆ alkyl), wherein each occurrence of R^5a is independently selected from the group consisting of H and Ci-C₆ alkyl; m is 0, 1, 2, 3, 4, 5, or 6; n is 0, 1, or 2; p is 0, 1, or 2.

In certain embodiments, each occurrence of alkyl, cycloalkyl, or acyl is optionally substituted.

In certain embodiments, L is attached directly to the phenyl ring of at least one indolinyl group, to a R⁴ group, and/or to a R⁵ group. In other embodiments, L is attached to R¹ and/or R².

In certain embodiments, the linker comprises a disulfide linker.

In certain embodiments, the linker comprises formula (A), (B) or (C): *-(CH₂)_ml-X₁- (CH₂-CH₂-X₂)_m2-(CH₂)_m3-C(X₃)- (A); *-(CH₂)_ml-0-(CH₂-CH₂-0)_m2-(CH₂)rn₃-C(0)-

(B); *-(CHR')_ml-0-(CHR'-CHR'-0)_m2-(CHR')_m3-C(0)- (C); wherein: * indicates the bond between the linker and the lipophilic membrane dye of formula (I); each ml, m2, and m3 is independently an integer ranging from 0-5000; each Xi, X₂, and X₃ is independently absent (a bond), O, or N-R'; each R' is independently selected from the group consisting of hydrogen, optionally substituted Ci-C₆ alkyl, optionally substituted C₃-Cg cycloalkyl, and optionally substituted C₃-Cg cycloheteroalkyl.

In certain embodiments, the lipophilic membrane dye comprises the compound of formula (II) or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof:

wherein in (II): R⁶ is selected from the group consisting of CH₃ Ci4-C₂₈ acyl, Cis-C₂₂ alkyl, Ci5-C₂₂ alkenyl, Cis-C₂₂ alkynyl, and phospholipid; R⁷ is selected from the group consisting of CH₃ CI4-C₂₈ acyl, Cis-C₂₂ alkyl, Cis-C₂₂ alkenyl, Cis-C₂₂ alkynyl, and phospholipid; R⁸ is selected from the group consisting of Ci-C₆ alkyl and -NHR^8a, wherein R^8a is selected from the group consisting of H, Ci₆-C₂₂ alkyl, Ci₆-C₂₂ alkenyl, and Ci₆-C₂₂ alkynyl; R⁹ is selected from the group consisting of H and S0₃H; R¹⁰ is selected from the group consisting of - NR^10aR^10a and -NR^10a-C(=O)R^10a, wherein each occurrence of R^10a is independently selected from the group consisting of H and Ci-C₆ alkyl; m is 0, 1, 2, 3, 4, 5, or 6; n is 0, 1, or 2; p is 0, 1, or 2.

In certain embodiments, L is attached directly to the phenyl ring of at least one indolinyl group, to a R⁹ group, and/or to a R¹⁰ group. In other embodiments, L is attached to R⁶ and/or R⁷.

wherein each occurrence of alkyl, cycloalkyl, or acyl is optionally substituted.

In certain embodiments, the linker comprises a disulfide linker.

In certain embodiments, the linker comprises formula (A), (B) or (C): *-(CH₂)_mi-Xi-(CH₂- CH₂-X₂)_m2-(CH₂)_m3-C(X₃)- (A); *-(CH₂)_ml-0-(CH₂-CH₂-0)_m2-(CH₂)m₃-C(0)- (B);

*-(CHR')_mi-0-(CHR'-CHR'-0)_m2-(CHR')_m3-C(0)- (C); wherein: * indicates the bond between the linker and the lipophilic membrane dye of formula (II); each ml, m2, and m3 is independently an integer ranging from 0-5000; each X_l X₂, and X₃ is independently absent (a bond), O, or N-R'; each R' is independently selected from the group consisting of hydrogen, optionally substituted Ci-C₆ alkyl, optionally substituted C₃-C₈ cycloalkyl, and optionally substituted C₃-C₈ cycloheteroalkyl.

In certain embodiments, the linker comprises a hydrophilic polymer or copolymer selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone, polyethylenimine, polymethacrylate, polyvinyl alcohol.

In certain embodiments, the method comprises contacting the compound with at least one agent selected from the group consisting of the construct of the invention and the compound of the invention, wherein at least one applies: (a) the cargo of the construct of the invention comprises a hydrophilic polymer or copolymer, (b) the linker in the construct of the invention and/or the compound of the invention comprises a hydrophilic polymer or copolymer, wherein the compound is solubilized in aqueous solution.

In certain embodiments, the hydrophilic polymer or copolymer is selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone, polyethylenimine, polymethacrylate, polyvinyl alcohol, or any copolymers thereof.

In certain embodiments, the linker further comprises a disulfide linkage.

In certain embodiments, the method comprises administering to the subject a composition comprising a construct of the invention, wherein the construct comprises the cargo.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of certain embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, specific embodiments are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

FIG. 1 comprises a non-limiting illustration of lipid painting of cell membrane with Dil (distearyl indocyanine dye) constructs (or conjugates). The constructs (three of which are illustrated in the bottom of the figure) are incorporated into the membrane either by transfer of individual lipid molecules or by fusion with the membrane. RBCs are shown for illustrative reasons, but other cells can be painted with this principle.

FIGs. 2A-2B illustrate certain aspects of stability of PEG-Dil construct painted RBCs in vivo. FIG. 2A: Structure of the GST-PEG-Dil construct. FIG. 2B: Erythrocytes were painted with the GST-PEG34oo-DiI construct and detected with fluorescence microscopy in the Cy3 channel. FIG. 2C: Painted RBCs were injected into a BALB/c mouse. Flow cytometry analysis shows painted RBCs in peripheral blood. FIG. 2D: Percentage of labeled cells in the blood stream was measured at 1, 2, and 7 days post injection shows stability of RBCs.

FIGs 3A-3D illustrate stability of cetuximab-PEG-Dil painted RBCs in vivo. FIG.

3 A: IgG was labeled with IRDye800 and thiolated Traut’s reagent. The modified antibody was conjugated to mal-PEG-Dil as described elsewhere herein. FIG. 3B: Reducing SDS PAGE was used to characterize the antibody constructs. Heavy and light chains of cetuximab are shown with arrow. Antibody-Dil constructs were compared to native antibody. The additional mass of the PEG-Dil adducts resulted in shifted bands (left side arrows). The heavy chain appears to be far more reactive than the light chain. FIG. 3C: RBCs were painted with fluorescent cetuximab, and the loading efficiency of IgG was determined by measuring the antibody recovered from washed red blood cells after the painting process.

The loading efficiency was similar for all concentrations tested (-17%). Inset shows microscopic images of IgG-PEG-Dil painted RBCs. FIG. 3D: Painted RBCs were injected intravenously. Mice were bled at different time points, and NIR fluorescence in blood spots was determined by dot blot analysis as described elsewhere herein. The negatively charged NIR molecules on IgG (9 IRDye800/Ab) leads to poor circulation of free antibody which was cleared within 1 day, whereas the labeled IgG painted on RBCs shows prolonged circulation (20% of the initial concentration at 48 hours). N=3 per group.

FIGs. 4A-4F illustrate stability of superoxide dismutase (SOD)-PEG-Dil painted RBCs in vivo. FIG. 4A: SOD was thiolated with Traut’s reagent and conjugated to mal- PEG-Dil. FIG. 4B: Reduced SDS PAGE was used to characterize the constructs. The different bands (on left side) correspond to the Dil linker modified SOD. FIG. 4C: RBCs were painted with Cy3-SOD-PEG-DiI and the loading efficiency of the enzyme was determined as described in Methods. The painting efficiency was similar for all

concentrations (-10%). Inset shows microscopic images of RBCs painted with 10 mM Cy3- SOD-PEG-Dil. FIG. 4D: Painted RBCs were injected into a BALB/c mouse. Flow cytometry analysis shows that the painted RBCs are still in circulation after 24h post injection. FIG. 4E: Percentage of labeled cells in the blood stream was measured at 30 min, lh, 2h, and 24h post injection. Numbers remain relatively stable for the first two hours and drop to 68% of initial abundance after 24 h. FIG. 4F: RBCs were painted with IRDye800- SOD-PEG-Dil and injected intravenously. Control mice were injected with IRDye800-SOD. Mice were bled at different time points and NIR fluorescence in blood spots was determined by dot blot analysis as described elsewhere herein. Painting SOD on RBCs resulted in higher SOD levels in blood and much higher AUC (Table 1) compared to free SOD. Thus, the majority (93%) of non-anchored enzyme had been cleared by 30 min, whereas 23% of the initial concentration of the anchored enzyme remained in circulation after four days. N=3 for each group.

FIGs. 5A-5D illustrate stability of DyLight800-DiI painted RBCs in vivo. FIG. 5 A: Dy Light 800-DiI construct was synthesized as described elsewhere herein. FIG. 5B: RBCs were painted with DyLight800-DiI, and the loading efficiency of the dye was determined as described elsewhere herein. Loading efficiency was over 60% for all concentrations tested. Image next to the graph shows microscopic image of double-labeled RBCs. FIG. 5C: RBCs were painted with Dy Light 800-DiI and injected intravenously. Control mice were injected with free Dy Light 800. Free dye was cleared within 1 hour whereas the dye still circulated bound to RBCs. AUC of painted dye was much higher than free dye (Table 1). FIG. 5D: Following injection, mice were sacrificed and the organs were scanned with Li-COR at 800nm. Fluorescent images of organs of mice sacrificed 72h post-injection show significant organ deposition of the dye in painted RBC mouse group. Free dye did not show any deposition probably due to high clearance rate. In painted RBC group, in addition to the liver and the spleen (main clearance organs), there was a signal in the lungs and the femur.

Representative images are shown. N=3. Fluorescence intensity was determined with ImageJ software.

FIGs. 6A-6B illustrate hemolysis and complement activation of the painted RBCs in mouse serum. FIG. 6A: Control and painted RBCs were incubated in serum for lh as described elsewhere herein, and the level of hemoglobin was measured in the supernatant. There was minimal release of hemoglobin compared to the control RBCs. FIG. 6B:

Complement C3 opsonization of RBCs in mouse serum shows some increase over control RBCs (6% increase for SOD RBCs and 51% increase for cetuximab RBCs). Without wishing to be limited by any theory, this increase in C3 opsonization did not cause excessive lysis, but potentially could lead to accelerated clearance of RBCs.

FIG. 7 illustrates discrete (top) vs. continuous (bottom) change in the signal. The discrete clustered pixels on the RBC surface provide a sensitive parameter for quantification of changes. N is the number of clusters. Integrated intensity change in gray value within the gray boundary is 2-fold for both the clustered and non-clustered signal.

FIGs. 8A-8C illustrate RBCs as sensors. FIG. 8A: RBCs were painted with anti- FITC IgG-PEG-DSPE construct and incubated with FITC-dextran for 30 min. RBCs were washed and probed with AlexaFluor 488 anti-FITC IgG. Phase contrast + fluorescence images show clusters of fluorescence on RBC surface. FIG. 8B: Fluorescence clusters were detectable down to a l-femtomolar concentration of FITC-dextran. FIG. 8C: Number of fluorescent dots per field was quantified and plotted against a FITC-dextran concentration.

FIG. 9 illustrates design of biosensors based on the phenomenon of lateral diffusion of lipid molecules in the RBC membrane.

FIG. 10 illustrates a dialkyl cyanine-modified antibody construct with PEG linker for capturing serum biomarkers.

FIG. 11 A-l 1B illustrate an example of a MMP-2 sensor module. FIG. 11 A shows that lipophilic fluorescent dyes are linked together through MMP-2 cleavable peptide and anchored in the membrane. FIG. 11B illustrates that upon cleavage of peptide linking lipophilic fluorescent dyes, the color components separate due to lateral diffusion causing color separation or change in FRET. Ri and FC are Cl 8, C20, C22, C24 lipid chains.

FIG. 12A-12B illustrate a sensor module where detachable parts are detected ex vivo with antibodies or nanoparticles. FIG. 12A shows a construct based on DiO and Dil, which forms a FRET pair and their cleavage leads to a decrease in FRET or color separation in the membrane that is detected ex vivo by nanoparticles. FIG. 12B shows fluorescence co- localization of uncleaved construct and delocalization of cleaved construct.

FIG. 13 A illustrates a sensor module with double parallel cyclic substrate.

FIG. 13B illustrates that in a sensor module with double parallel cyclic substrate, the cleavage of one bond does not lead to separation, and higher enzyme concentrations are required.

FIG. 14A comprises a graph illustrating concentration-time profiles resulting in different /%

FIG. 14B comprises a graph illustrating that values of £_¾ can be calculated and plotted against fluorescence change on the RBC surface to derive the relationship.

FIGs. 15A-15B illustrate lipid painting of RBC cell membrane with DIR-PEG3400- MTz. Near infrared fluorescence were detected by flow cytometry.

FIG. 16 illustrates experiments demonstrating that DiR-PEG MTZ was conjugated with IgG-transcyclooctene. Methyl tetrazine reacts with transcyclooctene, forming IgG-lipid conjugate. The conjugate was visualized after SDS-PAGE by near infrared scanner.

FIGs. 17A-17B illustrates lipid painting of RBC cell membrane with DIR-PEG3400- IgG. FIG. 17A shows human erythrocytes first painted with DIR-PEG3400-MTz and then conjugated with human IgG labelled with TCO. IgG on human erythrocytes painting were detected with green fluorescence of secondary antibody Alexa 488 Goat anti human IgG.

FIG. 17B shows IgG conjugated with DIR-PEG3400-MTz and then human erythrocytes painted with DIR-PEG3400-IgG. IgG on human erythrocytes painting were detected with green fluorescence of secondary antibody Alexa 488 Goat anti human IgG.

FIGs. 18A-18C shows lipid painting of RBC cell membrane with DIR-PEG3400-IgG. FIG. 18A shows Human Erythrocytes painted with DIR-PEG3400-MTz. FIG. 18B shows Human Erythrocytes were first painted with DIR-PEG3400-MTz and then conjugated with Human IgG labelled with TCO. IgG on Human Erythrocytes painting were detected with green fluorescence of secondary antibody Alexa 488 Goat anti human IgG. FIG. 18C shows IgG conjugated with DIR-PEG3400-MTz and then human Erythrocytes painted with DIR- PEG3400-IgG. IgG on Human Erythrocytes painting were detected with green fluorescence of secondary antibody Alexa 488 Goat anti human IgG.

FIG. 19 shows IgG concentration on painted human erythrocytes. (a)RBC + (DIR- PEG-IgG): Human erythrocytes were first painted with DIR-PEG3400-MTz and then conjugated with Human IgG labelled with TCO. (b) RBC-DIR-PEG-MTz + IgG-TCO: IgG was conjugated with DIR-PEG3400-MTz and then human erythrocytes were painted with DIR-PEG3400-IgG. The number of IgG on human Erythrocytes painting was detected by dot blot analysis of IR fluorescence of secondary antibody goat anti human 680.

FIG. 20 shows reaction between click RBCs and immune cells and leukemic cells.

FIGs. 21A-21H shows that indocyanine lipids exhibit efficient skin accumulation. FIGs. 21A-21B illustrate that liposomes were formulated with lissamine rhodamine PE or Dil. FIGs. 21C-21D illustrate that mouse skin was excised 7 days after injection, placed on a slide and imaged under low magnification with NIR microscope (Hoechst and dextran were preinjected to label nuclei and blood vessels). Dil shows much more efficient extravasation. N=3 mice. FIGs. 21E-21F show that DyLight800-DiI conjugate was synthesized and formulated into long circulating PEGylated liposomes. DyLight-Dil shows longer circulation than free DyLight (l2h vs. 5 min). FIGs. 21G-21H show DyLight accumulation and penetration in freshly excised skin flaps (abdominal and dorsal) measured with Li-COR NIR scanner (FIG. 22G) and NIR microscope (FIG. 22H) 14 days after injection of liposomes. Many liposomes accumulate in the extravascular skin cells. N=3 mice.

FIG. 22 shows that Dil-PEG was formulated with silibinin (free drug). Left vial: Dil formulation, right vial, silibinin only.

FIGs. 23A-23C show a non-limiting synthesis of silibinin-lipid conjugates and formulations. FIG. 23A shows conjugates of NIR indocyanine and silibinin or PEG. FIG. 24B shows the synthetic route for conjugation via self-immolating disulfide linker. FIG. 23C shows different formulations using lipids shown in (FIG. 23A) or combination of free drug (gray hexagons) and lipids.

FIGs 24A-24B show Dexamethasone formulations in PBS buffer. FIG. 24A illustrates that Dexamethasone is solubilized in PBS in the presence of DIR-PEG-3400- MTZ. FIG. 24B shows phase contrast microscopy images illustrating that the free

Dexamethasone as well as DSPE-PEG with Dexamethasone forms aggregates and crystals, whereas the Dexamethasone formulated with DIR-PEG-3400-MTZ shows minimal aggregation.

FIG. 25 illustrates Zeta sizer measurements. Upper graph is for Dexamethasone +DiR-PEG3400-MTZ. Lower graph is for Dexamethasone only.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery of novel constructs that allow for delivery of biologically relevant cargoes into cells. The constructs comprise a lipophilic membrane dye, or a derivative or analogue thereof, conjugated with the biologically relevant cargo(es).

The commonly used lipophilic membrane dye Dil is very efficient at RBC painting and stably retained on the surface of red blood cells for several days in circulation. Some of the stability can be attributed to the highly lipophilic nature of Dil and mild cationic charge on the indocyanine ring, allowing deep embedding into the bilayer. As described herein, an amine derivative of Dil, where the aminomethyl moiety is conjugated to the cyanine chromophore, was prepared as covalently conjugated with various biologically active agents. Using these Dil constructs, the conjugates of biologically active agents such as antibodies, enzymes, and small molecules, showed enhancement of their circulation times. These three non-limiting examples demonstrate the generality of the strategy of membrane retention, as means of altering the pharmacokinetics of bioactive molecules.

In order to use Dil as a membrane anchor for cell membrane painting, a non-limiting methylamine Dil derivative was prepared. This derivative was subsequently reacted with NHS-PEG34oo-mal to afford mal-PEG34oo-DiI (FIG. 2A). To determine the ability of the construct to paint biological membranes (similar to Dil), the maleimide group of mal- PEG3400-D1I was blocked with glutathione and then incubated with murine erythrocytes for 30 min at 37°C in the presence of a small amount of fetal bovine serum. Without wishing to be limited by any theory, the derivative may self-assemble into micelles at high enough concentration, but undergoes exchange with the RBC membrane and becomes stably incorporated due to interaction with negatively charged glycocalix (FIG. 2B). Indeed, RBCs showed efficient painting with the dye as verified by fluorescent microscopy (FIG. 2C). The painted RBCs were then injected intravenously into a BALB/c mouse. Flow cytometry analysis of blood samples taken during 7 days showed excellent stability of painted RBCs (FIG. 2D), with over 60% of the RBCs still present in the circulation at 7 days post-injection (FIG. 2D).

To demonstrate the applicability of Dil as anchor of targeting ligands, mouse IgG was modified with both IRdye800 and Traut’s reagent and coupled it to mal-PEG34oo-DiI (FIGs. 3A-3B). The painting of RBCs with IRdye800-IgG-PEG₃₄oo-DiI showed linear dependency on the concentration in the loading buffer (FIG. 3C), and the loading yield was 16.9 ± 0.9%. Next, the effect of Dil anchor on circulation of IRDye800-modified IgG was determined in vivo. Despite the long-circulating properties of engineered antibodies, heavy modification with small molecules and drugs triggers their accelerated clearance. Thus, accelerated clearance is the primary factor that constrains the number of drugs that can be conjugated to an IgG molecule, limiting the potency of drug antibody conjugates. Anti-EGFR antibody cetuximab was modified with ~9 IRDye800 molecules. In this case, IRDye 800 was used as both a drug mimetic and fluorescent reporter. IRDye800-cetuximab-PEG₃₄oo-DiI was loaded onto RBCs and injected into a cohort of 3 BALB/c mice via the tail vein. IRDye800- Cituximab was injected into another 3 mice, and blood samples were taken at various time points over the course of two days. During that time the RBC-tethered cetuximab still showed over 20% of the initial concentration after 2 days post-injection, whereas free cetuximab was eliminated after 1 day (FIG. 3D). The RBC-anchored cetuximab showed 5.6- fold higher AUC than free cetuximab (Table 1).

To demonstrate the ability of Dil to serve as membrane anchor for proteins, the sulfhydryl reactive mal-PEG34oo-DiI was conjugated to thiolated protein. Superoxide dismutase (SOD) is a naturally occurring enzyme in mammalian cells that breaks down highly reactive superoxide radicals that can lead to cellular damage. SOD has been considered a potentially useful tool as enzyme replacement therapy against off target oxidative damage caused by radiation therapy. Unfortunately, the enzyme has very poor retention in the blood stream and is therefore of limited use in the clinic unless it can be tethered to a more stable platform in vivo. IRDye 800CW was conjugated to the enzyme and used as a reporter to determine SOD concentration in blood. The fluorescent enzyme was then thiolated using Traut’s reagent and coupled to mal-PEG34oo-DiI forming SOD-PEG3400- Dil (FIG. 2A). SDS-PAGE analysis indicated an overall yield of 60% (FIG. 2B). In order to determine the efficiency of RBC loading, various concentrations of SOD-PEG34oo-DiI were titrated in the loading buffer to determine the efficiency of the painting process.

Measurement of infrared fluorescence of washed RBCs revealed that the amount of incorporated enzyme increased linearly with the increase in the incubation concentration. On average, 9.9 ± 0.3% of the SOD-PEG3400-D1I in the loading buffer became stably bound to erythrocyte membranes (FIG. 2C), and at the highest loading concentration, each RBC had over 1.3 million SOD molecules. Microscopy of cells loaded with Cy3-SOD-PEG3400-DiI revealed that the red blood cells remained intact and undamaged after being coated with the lipidated enzyme (FIG. 4C).

The in vivo stability of SOD-painted RBCs was assessed. Cy3-SOD-PEG34oo-DiI was prepared, and mouse RBCs were painted with the construct and injected into BALB/c mice. As shown in FIG. 2D-2E, the percentage of RBCs painted with Cy3-SOD-PEG34oo-DiI remained relatively constant (around 10%) for two hours post-injection and dropped to around 6% after 24h. The effect of membrane tethering on the pharmacokinetics of SOD was determined by tail vein injection of IRDye800-SOD-PEG₃₄oo-DiI painted RBCs into cohort of 3 mice. Separately, 3 mice were injected with an equal amount of IRDye800-SOD. Blood samples were collected at various time points, lysed in SDS buffer and blotted on

nitrocellulose for near infrared fluorescence analysis. Without the anchor, SOD was rapidly cleared from the blood (less than 10% of initial blood concentration, in 30 min), whereas levels of SOD anchored to RBCs remained above 10% for 4 days (FIG. 2F). SOD anchored to RBCs showed 6.5 fold higher blood AUC than free SOD (Table 1).

Further, the effect of Dil anchor on membrane painting and stability of small molecules was investigated. To this end Dy Light 800, a hydrophilic small molecule that has poor circulatory retention, was employed. The dye was directly conjugated to aminomethyl Dil viaNHS ester (FIG. 4A). The painting resulted in RBCs being fluorescent for both Cy3 and DyLight 800 (FIG. 4B). Similar to the previously described constructs, the loading efficiency of DyLight 8OO-PEG3400-D1I was linear (FIG. 4C) and the yield was 60%, which is surprisingly high considering the expected repulsion between the negatively charged construct and the anionic RBC membrane. Mice were injected with both either free DyLight 800 or DyLight 800-DiI bound to red blood cells, and the circulation was measured in vivo. According to FIG. 4D, the free Dylight 800 was completely gone by lh post-injection, whereas RBC bound DyLight 800 was at nearly 60% of its initial concentration. The RBC- anchored dye showed 77.7-fold higher AUC than free dye (Table 1). Four days after injection the 25% of the initial Dil was still in circulation, and there was none of the free dye. At day 4 post injection, the organs of the mice were analyzed for near infrared fluorescence, and it was found that Dy Light 800 was retained in the tissues of the mice that had been injected with RBC bound dye and was nearly undetectable in the organs of the mice that had been injected with free dye (FIG. 4E, insert). The increase in average Dy Light 800 fluorescence was over 100-fold in liver and spleen, 67-fold in the lung, and over 5-fold in the kidney, heart and bone (FIG. 4).

In order to check if painting of RBC with DI triggers hemolysis and complement activation, mouse RBCs were incubated with either IRDye800-SOD-PEG₃₄oo-DiI or

IRDye800-Cituximab-PEG₃₄oo-DiI (5 mM in RBC loading buffer), or with PBS. After washing three times to remove unbound constructs, RBCs were incubated with mouse serum for 1.5 h at 37°C and the absorbance of the supernatants was measured at 550 nm. Painting RBCs with SOD and cituximab resulted in minimal lysis compared to plain RBCs (FIG. 6A). Complement C3 opsonization assay showed no increase in C3 opsonization of SOD-painted RBCs, and small increase of cetuximab painted RBCs, over plain RBCs (FIG. 6B).

The present data demonstrate that lipophilic membrane dye Dil anchor can be used for efficient incorporation of variety of molecules in the RBC membrane. Moreover, the data clearly demonstrate the increased in vivo retention of molecular entities painted onto RBC membranes. Molecules that are rapidly cleared from the blood stream can be retained for days via Dil anchor. In this work RBCs were used as model“cell” with biological membrane. RBCs are also attractive drug and enzyme carriers, and ligand-coated can be used for scavenging of pathogens, toxins and circulating cells, therefore the Dil anchor can advance of these applications. In certain embodiments, the same painting strategy can be used to incorporate enzymes, small molecules and antibodies into membrane of other cells, for example immune cells or stem cells. There are several advantages in the lipid painting strategy over chemical linkage, including fast kinetics, scalability and minimal damage. The constructs of the invention showed low level of hemolysis and complement activation.

Moreover, proteins conjugated to the surface of red blood cells show to be resistant to the adaptive immune response and can trigger anergy to entities that have already triggered an immune reaction.

In certain embodiments, the linkers within the constructs of the invention have at least one labile linkage, thus allowing for release drugs slowly over time from these RBC-bound reserves. Non-limiting examples of such linkages include oximes, carbonates, hydrazones, disulfide bonds, esters, and the like. Other non-limiting examples include peptide linkages that remain stable until exposed to certain proteases associated with disease (Singh, et al, 2008, Curr. Med. Chem. 15(18): 1802—1826; Yang, et al, 2011, Acta Pharm. Sin. B

1(3): 143—159; Wang, et al, 2016, Drug Delivery : Principles and Applications, Wiley, 2nd Edition). In certain embodiments, such labile linkages allow for slow release of biologically active agent from RBCs or other storage cells, maintaining the agent at therapeutic levels in the body while minimizing adverse side effects.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.

As used herein, each of the following terms has the meaning associated with it in this section.

The articles“a” and“an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

The term“abnormal,” when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the“normal”

(expected) respective characteristic. Characteristics that are normal or expected for one cell or tissue type might be abnormal for a different cell or tissue type.

“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 ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

A disease or disorder is“alleviated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.

As used herein, the term“composition” or“pharmaceutical composition” refers to a mixture of at least one compound useful within the invention with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, pulmonary and topical administration.

A“disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.

In contrast, a“disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

The terms“patient,”“subject,” or“individual” are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In a non-limiting embodiment, the patient, subject or individual is a human.

As used herein, the term“pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material can be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

As used herein, the term“pharmaceutically acceptable carrier” means a

pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the patient such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil;

glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid;

pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein,“pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The“pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the invention.

Other additional ingredients that can be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

As used herein, the language“pharmaceutically acceptable salt” refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic acids or bases, organic acids or bases, solvates, hydrates, or clathrates thereof. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present invention, such as for example utility in process of synthesis, purification or formulation of compounds useful within the methods of the invention. Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include sulfate, hydrogen sulfate, hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate). Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2- hydroxyethanesulfonic, p-toluenesulfonic, sulfanibc, cyclohexylaminosulfonic, stearic, alginic, b-hydroxybutyric, salicylic, galactaric, galacturonic acid, glycerophosphonic acids and saccharin (e.g., saccharinate, saccharate). Suitable pharmaceutically acceptable base addition salts of compounds of the invention include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N’- dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.

As used herein, the terms“pharmaceutically effective amount” and“effective amount” and“therapeutically effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

A“therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.

As used herein, the term“treatment” or“treating” is defined as the application or administration of a therapeutic agent, /. e.. a compound of the invention (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a condition contemplated herein, a symptom of a condition contemplated herein or the potential to develop a condition contemplated herein, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect a condition contemplated herein, the symptoms of a condition contemplated herein or the potential to develop a condition contemplated herein. Such treatments can be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.

As used herein, the term“alkyl,” by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e.

means one to six carbon atoms) and including straight, branched chain, or cyclic substituent groups. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and cyclopropylmethyl. A non-limiting example is (C_|-G,)alkyl. particularly ethyl, methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropylmethyl.

As used herein, the term“substituted alkyl” means alkyl as defined above, substituted by one, two or three substituents selected from the group consisting of halogen, -OH, alkoxy, -NH₂, -N(CH₃)₂, -C(=0)0H, trifluoromethyl, -CºN, -C(=0)0(Ci-C₄)alkyl, -C(=0)NH₂, - S0₂NH₂, -C(=NH)NH₂, and -N0₂, preferably containing one or two substituents selected from halogen, -OH, alkoxy, -NH₂, trifluoromethyl, -N(CH₃)₂, and -C(=0)0H, more preferably selected from halogen, alkoxy and -OH. Examples of substituted alkyls include, but are not limited to, 2,2-difluoropropyl, 2-carboxy cyclopentyl and 3-chloropropyl.

As used herein, the term“alkoxy” employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined above, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, l-propoxy, 2-propoxy (isopropoxy) and the higher homologs and isomers. A non-limiting example is (Ci-C₃) alkoxy, particularly ethoxy and methoxy.

As used herein, the term“halo” or“halogen” alone or as part of another substituent means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine, more preferably, fluorine or chlorine.

As used herein, the term“cycloalkyl” refers to a mono cyclic or polycyclic non aromatic radical, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom. In certain embodiments, the cycloalkyl group is saturated or partially unsaturated. In other embodiments, the cycloalkyl group is fused with an aromatic ring. Cycloalkyl groups include groups having from 3 to 10 ring atoms. Illustrative examples of cycloalkyl groups include, but are not limited to, the following moieties:

Monocyclic cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Dicyclic cycloalkyls include, but are not limited to, tetrahydronaphthyl, indanyl, and tetrahydropentalene. Polycyclic cycloalkyls include adamantine and norbomane. The term cycloalkyl includes“unsaturated nonaromatic carbocyclyl” or“nonaromatic unsaturated carbocyclyl” groups, both of which refer to a nonaromatic carbocycle as defined herein, which contains at least one carbon carbon double bond or one carbon carbon triple bond.

As used herein, the term“substituted” means that an atom or group of atoms has replaced hydrogen as the substituent attached to another group. The term“substituted” further refers to any level of substitution, namely mono-, di-, tri-, tetra-, or penta-substitution, where such substitution is permitted. The substituents are independently selected, and substitution can be at any chemically accessible position. In certain embodiments, the substituents vary in number between one and four. In other embodiments, the substituents vary in number between one and three. In yet other embodiments, the substituents vary in number between one and two.

As used herein, the term“optionally substituted” means that the referenced group can be substituted or unsubstituted. In certain embodiments, the referenced group is optionally substituted with zero substituents, /. e.. the referenced group is unsubstituted. In other embodiments, the referenced group is optionally substituted with one or more additional group(s) individually and independently selected from groups described herein.

In certain embodiments, the substituents are independently selected from the group consisting of oxo, halogen, -CN, -NH₂, -OH, -NH(CH₃), -N(CH₃)₂, alkyl (including straight chain, branched and/or unsaturated alkyl), substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, fluoro alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, fluoroalkoxy, -S-alkyl, S(=0)₂alkyl, - C(=0)NH [substituted or unsubstituted alkyl], -C(=0)NH[substituted or unsubstituted phenyl], -C(=0)N[H or alkyl]₂, -OC(=0)N[substituted or unsubstituted alkyl]₂,

-NHC(=0)NH[substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl], - NHC(=0)alkyl, -N[substituted or unsubstituted alkyl]C(=0)[substituted or unsubstituted alkyl], -NHC(=0) [substituted or unsubstituted alkyl], -C(OH)[substituted or unsubstituted alkyl]₂, and -C(NH₂)[substituted or unsubstituted alkyl]₂. In other embodiments, by way of example, an optional substituent is selected from oxo, fluorine, chlorine, bromine, iodine, - CN, -NH₂, -OH, -NH(CH₃), -N(CH₃)₂, -CH₃, -CH₂CH₃, -CH(CH₃)₂, -CF₃, -CH₂CF₃, -OCH₃, - OCH₂CH₃, -OCH(CH₃)₂, -OCF₃, - OCH₂CF₃, -S(=0)₂-CH₃, -C(=0)NH₂, -C(=0)-NHCH₃, - NHC(=0)NHCH₃, -C(=0)CH₃, and -C(=0)0H. In yet one embodiment, the substituents are independently selected from the group consisting of Ci_₆ alkyl, -OH, Ci_₆ alkoxy, halo, amino, acetamido, oxo and nitro. In yet other embodiments, the substituents are independently selected from the group consisting of Ci_₆ alkyl, Ci_₆ alkoxy, halo, acetamido, and nitro. As used herein, where a substituent is an alkyl or alkoxy group, the carbon chain can be branched, straight or cyclic.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Compounds and Compositions

The compounds of the present invention can be synthesized using techniques well- known in the art of organic synthesis. The starting materials and intermediates required for the synthesis can be obtained from commercial sources or synthesized according to methods known to those skilled in the art.

The invention provides a construct comprising a lipophilic membrane dye. In certain embodiments, the lipophilic membrane dye is covalently linked through a linker to a cargo selected from the group consisting of a therapeutic drug, nucleic acid, polypeptide, enzyme, antibody, ligand, biologically active lipid, dye (or chromophore), fluorophore, bioluminescent label, biosensor, contrast agent, radioisotope, hydrophilic polymer, hydrophilic copolymer, and chemiluminescent label. In other embodiments, the lipophilic membrane dye comprises the compound of formula (I), or formula (II), or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof:

wherein:

R¹ is selected from the group consisting of C₁₄-C₂₂ alkyl, C₁₄-C₂₂ alkenyl, C₁₄-C₂₂ alkynyl, and C₁₄-C₂₈ acyl;

R² is selected from the group consisting of C₁₄-C₂₂ alkyl, C₁₄-C₂₂ alkenyl, C₁₄-C₂₂ alkynyl, and C₁₄-C₂₈ acyl;

R³ⁱ, R³ⁱⁱ, R³ⁱⁱⁱ, and R^3iv are each independently selected from the group consisting of H and Ci-C₆ alkyl;

each occurrence of R⁴ is independently selected from the group consisting of Ci-C₆ alkyl, C₃-C₈ cycloalkyl, halo, -N0₂, -CN, -OR^4a, -NR^4aR^4a, -NR^4a-C(=0)R^4a, -NR^4a-S0₂R^4a, - C(=0)0R^4a, -C(=0)NR^4aR^4a, -SR^4a, and -S(=0)i-₂(Ci-C₆ alkyl), wherein each occurrence of R^4a is independently selected from the group consisting of H and Ci-C₆ alkyl;

each occurrence of R⁵ is independently selected from the group consisting of Ci-C₆ alkyl, C₃-C₈ cycloalkyl, halo, -N0₂, -CN, -OR^5a, -NR^5aR^5a, -NR^5a-C(=0)R^5a, -NR^5a-S0₂R^5a, - C(=0)0R^5a, -C(=0)NR^5aR^5a, -SR^5a, and -S(=0)i-₂(Ci-C₆ alkyl), wherein each occurrence of R^5a is independently selected from the group consisting of H and Ci-C₆ alkyl;

R⁶ is selected from the group consisting of CH₃ C₁₄-C₂₈ acyl, C₁₅-C₂₂ alkyl, C₁₅-C₂₂ alkenyl, C₁₅-C₂₂ alkynyl, and phospholipid;

R⁷ is selected from the group consisting of CH₃ C₁₄-C₂₈ acyl, C₁₅-C₂₂ alkyl, C₁₅-C₂₂ alkenyl, C₁₅-C₂₂ alkynyl, and phospholipid;

R⁸ is selected from the group consisting of Ci-C₆ alkyl and -NHR^8a, wherein R^8a is selected from the group consisting ofH, C₁₆-C₂₂ alkyl, C₁₆-C₂₂ alkenyl, and C₁₆-C₂₂ alkynyl;

R⁹ is selected from the group consisting ofH and S0₃H;

R¹⁰ is selected from the group consisting of -NR^10aR^10a and -NR^10a-C(=O)R^10a, wherein each occurrence of R^10a is independently selected from the group consisting of H and Ci-C₆ alkyl;

m is 0, 1, 2, 3, 4, 5, or 6;

n is 0, 1, or 2;

p is 0, 1, or 2; wherein each occurrence of alkyl, cycloalkyl, or acyl is optionally substituted.

In certain embodiments, R¹ and R² are independently selected from the group consisting of Ci₈ acyl, C₂o acyl, C₂₂ acyl, C₂₄ acyl, Ci₈ alkyl, C₂o alkyl, C₂₂ alkyl, C₂₄ alkyl, Cis alkenyl, C₂o alkenyl, C₂₂ alkenyl, C₂₄ alkenyl, C₄₈ alkynyl, C₂o alkynyl, C₂₂ alkynyl, and C₂₄ alkynyl.

In certain embodiments, m is 0 or 1.

In certain embodiments, R³¹, R^3u, R^3m, and R^3lv are each methyl.

In certain embodiments, in the construct having formula (II), the linker is attached directly to the phenyl ring of at least one indolinyl group, to a R⁹ group, and/or to a R¹⁰ group, and/or

the linker is attached to R⁶ and/or R⁷

In certain embodiments, the phospholipid comprises glycerol-3-phosphate. In other embodiments, the phospholipid comprises l,2-0-diacyl-glycerol-3-phosphate, wherein the acyl groups are independently selected. In yet other embodiments, the phospholipid

comprises

wherein:

p is 1 or 2;

CRR-; and

Y comprises the linker conjugated to the cargo.

In certain embodiment, the compound of formula (II) comprises the compound of formula (IV), or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof:

embodiments, the compound of formula (II) comprises the compound of formula (V), or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof:

wherein A comprises a linker conjugated to the cargo. In other embodiments, the linker is a disulfide linker.

In certain embodiments, the disulfide linker comprises

. In certain embodiments, the cargo is a therapeutic drug, such as but not limited to a chemotherapeutic drug, such as but not limited to doxorubicin, auristatin, cytarabine, and/or camptothecin.

In certain embodiments, the cargo is a nucleic acid, such as but not limited to a siRNA, mRNA, miRNA, DNA, oligodeoxynucleotide, and so forth.

In certain embodiments, the cargo is a polypeptide.

In certain embodiments, the cargo is a ligand, such as but not limited to folate, RGD, VEGF, Sialyl-Lewis molecule, and so forth. In certain embodiments, the cargo is an enzyme, such as but not limited to superoxide dismutase (SOD), asparaginase, protease, catalase, and so forth.

In certain embodiments the cargo comprises tetrazine, such as but not limited to 4- methyl-tetrazine.

In certain embodiments, the cargo is an antibody, such as but not limited to cetuximab, trastuzumab, ramucirimab, and so forth.

In certain embodiments, the cargo is a bioactive or biologically active lipid.

In certain embodiments, the cargo is a dye or chromophore, such as but not limited to a near infrared dye (Cy7, IRDye800, Cy5.5, and so forth).

In certain embodiments, the cargo is a fluorophore.

In certain embodiments, the cargo is a bioluminescent label.

In certain embodiments, the cargo is a chemiluminescent label.

In certain embodiments, the cargo is a biosensor, such as but not limited to an enzyme sensor, pH sensor, hypoxia sensor, metabolite sensor, and so forth.

In certain embodiments, the cargo is a contrast agent, such as a chelator that can complex gadolinium, iron oxide, perfluorocarbon bubble, iodine, and so forth.

In certain embodiments, the cargo is a radioisotope, such as but not limited to chelated Actinium 225, chelated Tc99m, chelated Lutecium-l77, chelated Cu-64, F-18, and so forth.

In certain embodiments, the linker comprises a Ci-C₂o hydrocarbon chain. In other embodiments, the linker comprises 1-20 amino acids. In yet other embodiments, the linker comprises at least one -OCH₂CH₂- group. In yet other embodiments, the linker comprises from 1 to about 5,000 -OCH₂CH₂- groups. In yet other embodiments, the linker is conjugated to the cargo through a 3-thio-succinimido group.

In certain embodiments, the linker comprises formula (A), (B) or (C):

*-(CH₂)_ml-X₁-(CH₂-CH₂-X₂)_m2-(CH₂)_m3-C(X₃)- (A)

*-(CH₂)_ml-0-(CH₂-CH₂-0)_m2-(CH₂)m₃-C(0)- (B)

*-(CHR')_mi-0-(CHR'-CHR'-0)_m2.(CHR')_m3-C(0)- (C)

wherein:

* indicates the bond between the linker and the compound of formula (I) or (II); each ml, m2, and m3 is independently an integer ranging from 0-5000;

each Xi, X₂, and X₃ is independently absent (a bond), O, or N-R';

each R' is independently selected from the group consisting of hydrogen, optionally substituted Ci-C₆ alkyl, optionally substituted C₃-C₈ cycloalkyl, and optionally substituted C₃-C₈ cycloheteroalkyl. In certain embodiments, the cargo is further covalently linked through an

independently selected additional linker to at least one other independently selected lipophilic membrane dye of formula (I) or (II), or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof

In certain embodiments, the additional linker comprises a disulfide linker.

In certain embodiments, the additional linker comprises formula (A), (B) or (C):

*-(CH₂)_ml-X₁-(CH₂-CH₂-X₂)_m2-(CH₂)_m3-C(X₃)- (A)

*-(CH₂)_ml-0-(CH₂-CH₂-0)_m2-(CH₂)m₃-C(0)- (B)

*-(CHR')_mi-0-(CHR'-CHR'-0)_m2.(CHR')_m3-C(0)- (C)

wherein:

* indicates the bond between the linker and the at least one other independently selected lipophilic membrane dye of formula (I) or (II);

each ml, m2, and m3 is independently an integer ranging from 0-5000;

each Xi, X₂, and X₃ is independently absent (a bond), O, or N-R';

each R' is independently selected from the group consisting of hydrogen, optionally substituted Ci-C₆ alkyl, optionally substituted C₃-C₈ cycloalkyl, and optionally substituted C₃-C₈ cycloheteroalkyl.

In other embodiments, the cargo is enzymatically cleavable, whereby upon cleavage of the cargo the at least two lipophilic membrane dyes are not covalently connected with each other.

In certain embodiments, the hydrophilic polymer is at least one selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone, polyethylenimine, polymethacrylate, and polyvinyl alcohol.

In certain embodiments, the hydrophilic copolymer comprises at least one polymer selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone,

polyethylenimine, polymethacrylate, and polyvinyl alcohol, or any copolymer thereof.

The invention further provides a composition comprising the construct of the invention and at least one cell, wherein at least a fraction of the construct is incorporated into the membrane of the cell. In certain embodiments, at least a fraction of the lipophilic membrane dye of the construct inserts into the membrane of the cell. In other embodiments, the cell is a red blood cell. In yet other embodiments, the cell does not undergo significant lysis upon incorporation of the construct into the membrane of the cell. In yet other embodiments, the composition further comprises at least one pharmaceutically acceptable carrier. In yet other embodiments, the composition is formulated for administration by a route selected from the group consisting of oral, parenteral, transdermal, transmucosal, intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical.

The invention further provides a lipophilic membrane dye of formula (IIA), or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof:

wherein:

R is selected from the group consisting of H, -CH₃, and -CH₂R¹⁰;

R⁶ is selected from the group consisting of CH₃ Ci -C₂₈ acyl, Ci₅-C₂₂ alkyl, Ci₅-C₂₂ alkenyl, Cis-C₂₂ alkynyl, and phospholipid;

R⁷ is selected from the group consisting of CH₃ Ci -C₂₈ acyl, Ci₅-C₂₂ alkyl, Ci₅-C₂₂ alkenyl, Cis-C₂₂ alkynyl, and phospholipid;

R is selected from the group consisting of Ci-C₆ alkyl and -NHR , wherein R is selected from the group consisting ofH, Ci₆-C₂₂ alkyl, Ci₆-C₂₂ alkenyl, and Ci₆-C₂₂ alkynyl;

R⁹ is selected from the group consisting of H and S0₃H;

In another aspect, the invention provides a compound comprising a linker (L) and a lipophilic membrane dye of formula (I) or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof;

wherein in (I):

R¹ is selected from the group consisting of Ci₄-C₂₂ alkyl, Ci₄-C₂₂ alkenyl, Ci₄-C₂₂ alkynyl, and Ci₄-C₂₈ acyl; R² is selected from the group consisting of C14-C22 alkyl, C14-C22 alkenyl, C14-C22 alkynyl, and C14-C28 acyl;

m is 0, 1, 2, 3, 4, 5, or 6;

n is 0, 1, or 2;

p is 0, 1, or 2; and

wherein L is:

(a) attached directly to the phenyl ring of at least one indolinyl group, to a R⁴ group, and/or to a R⁵ group, and/or

(b) attached to R¹ and/or R²;

In certain embodiments, the linker comprises a disulfide linker.

In certain embodiments, the linker comprises formula (A), (B) or (C)

*-(CH₂)_ml-X_l-(CH₂-CH₂-X₂)_m2-(CH₂)_m3-C(X₃)- (A)

*-(CH₂)_mi-0-(CH₂-CH₂-0)_m2-(CH₂)m₃-C(0)- (B)

*-(CHR')_mi-0-(CHR'-CHR'-0)_m2-(CHR')_m3-C(0)- (C)

wherein:

* indicates the bond between the linker and the lipophilic membrane dye of formula

(i);

each ml, m2, and m3 is independently an integer ranging from 0-5000;

each Xi, X₂, and X₃ is independently absent (a bond), O, or N-R';

each R' is independently selected from the group consisting of hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C₃-C₈ cycloalkyl, and optionally substituted C₃-C₈ cycloheteroalkyl.

In yet another aspect, the invention provides a compound comprising a linker (L) and a lipophilic membrane dye of formula (II) or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof;

wherein in (II):

R⁶ is selected from the group consisting of CH₃ C14-C28 acyl, C15-C22 alkyl, C15-C22 alkenyl, C15-C22 alkynyl, and phospholipid;

R⁹ is selected from the group consisting ofH and S0₃H;

m is 0, 1, 2, 3, 4, 5, or 6;

n is 0, 1, or 2;

p is 0, 1, or 2; and

wherein L is:

(a) attached directly to the phenyl ring of at least one indolinyl group, to a R⁹ group, and/or to a R¹⁰ group, and/or

(b) attached to R⁶ and/or R⁷;

In certain embodiments, the linker comprises a disulfide linker.

In certain embodiments, the linker comprises formula (A), (B) or (C)

*-(CH₂)_ml-X_l-(CH₂-CH₂-X₂)_m2-(CH₂)_m3-C(X₃)- (A)

*-(CH₂)_mi-0-(CH₂-CH₂-0)_m2-(CH₂)rn₃-C(0)- (B)

*-(CHR')_mi-0-(CHR'-CHR'-0)_m2-(CHR')_m3-C(0)- (C)

wherein:

(II); each ml, m2, and m3 is independently an integer ranging from 0-5000;

each Xi, X₂, and X₃ is independently absent (a bond), O, or N-R';

each R' is independently selected from the group consisting of hydrogen, optionally substituted Ci-C₆ alkyl, optionally substituted C3-C8 cycloalkyl, and optionally substituted C3-C8 cycloheteroalkyl.

In certain embodiment, the linker comprises a hydrophilic polymer or copolymer selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone, polyethylenimine, polymethacrylate, polyvinyl alcohol.

The compounds of the invention can possess one or more stereocenters, and each stereocenter can exist independently in either the (R) or (S) configuration. In certain embodiments, compounds described herein are present in optically active or racemic forms.

It is to be understood that the compounds described herein encompass racemic, optically - active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein. Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. In certain embodiments, a mixture of one or more isomer is utilized as the therapeutic compound described herein. In other embodiments, compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including

stereoselective synthesis, enantioselective synthesis and/or separation of a mixture of enantiomers and/ or diastereomers. Resolution of compounds and isomers thereof is achieved by any means including, by way of non-limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography.

The methods and formulations described herein include the use of N-oxides (if appropriate), crystalline forms (also known as polymorphs), solvates, amorphous phases, and/or pharmaceutically acceptable salts of compounds having the structure of any compound of the invention, as well as metabolites and active metabolites of these compounds having the same type of activity. Solvates include water, ether (e.g., tetrahydrofuran, methyl tert-butyl ether) or alcohol (e.g., ethanol) solvates, acetates and the like. In certain embodiments, the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, and ethanol. In other embodiments, the compounds described herein exist in unsolvated form.

In certain embodiments, the compounds of the invention may exist as tautomers. All tautomers are included within the scope of the compounds presented herein.

In certain embodiments, compounds described herein are prepared as prodrugs. A “prodrug” refers to an agent that is converted into the parent drug in vivo. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound. In other embodiments, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the compound.

In certain embodiments, sites on, for example, the aromatic ring portion of compounds of the invention are susceptible to various metabolic reactions. Incorporation of appropriate substituents on the aromatic ring structures may reduce, minimize or eliminate this metabolic pathway. In certain embodiments, the appropriate substituent to decrease or eliminate the susceptibility of the aromatic ring to metabolic reactions is, by way of example only, a deuterium, a halogen, or an alkyl group.

Compounds described herein also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.

Examples of isotopes suitable for inclusion in the compounds described herein include and are not limited to ²H, ³H, ⁿC, ¹³C, ¹⁴C, ³⁶Cl, ¹⁸F, ¹²³I, ¹²⁵I, ¹³N, ¹⁵N, ¹⁵0, ¹⁷0, ¹⁸0, ³²P, and ³⁵S. In certain embodiments, isotopically-labeled compounds are useful in drug and/or substrate tissue distribution studies. In other embodiments, substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements). In yet other embodiments, substitution with positron emitting isotopes, such as C, F, O and N, is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed.

In certain embodiments, the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.

The compounds described herein, and other related compounds having different substituents are synthesized using techniques and materials described herein and as described, for example, in Fieser & Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989), March, Advanced Organic Chemistry 4^th Ed., (Wiley 1992); Carey & Sundberg, Advanced Organic Chemistry 4th Ed., Vols. A and B (Plenum 2000, 2001), and Green & Wuts, Protective Groups in Organic Synthesis 3rd Ed., (Wiley 1999) (all of which are incorporated by reference for such disclosure). General methods for the preparation of compound as described herein are modified by the use of appropriate reagents and conditions, for the introduction of the various moieties found in the formula as provided herein.

In certain embodiments, reactive functional groups, such as hydroxyl, amino, imino, thio or carboxy groups, are protected in order to avoid their unwanted participation in reactions. Protecting groups are used to block some or all of the reactive moieties and prevent such groups from participating in chemical reactions until the protective group is removed. In other embodiments, each protective group is removable by a different means. Protective groups that are cleaved under totally disparate reaction conditions fulfill the requirement of differential removal. In certain embodiments, protective groups are removed by acid, base, reducing conditions (such as, for example, hydrogenolysis), and/or oxidative conditions.

Methods

The invention provides a method of delivering a cargo to a cell. In certain

embodiments, the method comprises contacting the construct of the invention with the cell under conditions whereby at least a fraction of the lipophilic membrane dye of the construct inserts into the membrane of the cell. In other embodiments, the cell is a red blood cell. In yet other embodiments, the cell does not undergo significant lysis upon insertion of the lipophilic membrane dye of the construct into the membrane of the cell.

The invention further provides a method of delivering a cargo to a subject in vivo. In certain embodiments, the cargo is selected from the group consisting of a therapeutic drug, nucleic acid, polypeptide, enzyme, antibody, ligand, biologically active lipid, dye (or chromophore), fluorophore, bioluminescent label, biosensor, contrast agent, radioisotope, hydrophilic polymer, hydrophilic copolymer, and chemiluminescent label.

In certain embodiments, the method comprises administering to the subject a composition comprising the construct of the invention and at least one cell, wherein at least a fraction of the construct is incorporated into the membrane of the cell. In other embodiments, the administering is by a route selected from the group consisting of oral, parenteral, transdermal, transmucosal, intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous,

intrabronchial, inhalation, and topical. In yet other embodiments, the cargo has a higher circulating half-life in the subject as compared to when the cargo, which is not part of the construct, is administered to the subject. In yet other embodiments, the cargo comprises a therapeutically effective agent, thereby treating or preventing a disease or disorder in the subject. In yet other embodiments, the cargo is covalently linked through independently selected linkers to at least two independently selected lipophilic membrane dyes of the invention, or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof. In yet other embodiments, the cargo is enzymatically cleavable in vivo, whereby upon cleavage of the cargo the at least two lipophilic membrane dyes are not covalently connected with each other. In yet other embodiments, the subject is further administered at least one additional therapeutically effective agent.

The invention further provide a method of solubilizing a compound in an aqueous solution, wherein the method comprises contacting the compound with at least one agent selected from the group consisting of the construct of the invention and the compound of the invention, wherein at least one applies: (a) the cargo of the construct of the invention comprises a hydrophilic polymer or copolymer, (b) the linker in the construct of the invention and/or the compound of the invention comprises a hydrophilic polymer or copolymer..

In certain embodiment, the linker further comprises a disulfide linkage.

The invention further provides a method of enhancing the endothelial membrane crossing of a cargo in a subject, the method comprising administering to the subject a composition comprising the construct of the invention, wherein the construct comprises the cargo.

In certain embodiments, the subject is a mammal. In other embodiments, the mammal is a human.

Combination Therapies

The compounds useful within the methods of the invention can be used in

combination with one or more additional agents useful for treating or preventing diseases or disorders contemplated herein. These additional agents can comprise compounds that are commercially available or synthetically accessible to those skilled in the art. These additional agents are known to treat, prevent, or reduce the symptoms of diseases or disorders contemplated herein.

A synergistic effect can be calculated, for example, using suitable methods such as, for example, the Sigrnoid-E_max equation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv.

Enzyme Regul. 22:27-55). Each equation referred to above can be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the

concentration-effect curve, isobologram curve and combination index curve, respectively.

Administration/Dosage/Formulations

The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations can be administered to the subject either prior to or after the onset of diseases or disorders contemplated herein. Further, several divided dosages, as well as staggered dosages can be administered daily or sequentially, or the dose can be continuously infused, or can be a bolus injection. Further, the dosages of the therapeutic formulations can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

Administration of the compositions of the present invention to a patient, preferably a mammal, more preferably a human, can be carried out using known procedures, at dosages and for periods of time effective to treat, ameliorate, or prevent diseases or disorders contemplated herein. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound to treat, ameliorate, or prevent diseases or disorders contemplated herein. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the invention is from about 1 and 5,000 mg/kg of body weight/per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention can be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

In particular, the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.

A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of heart failure in a patient.

In certain embodiments, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of a compound of the invention and a pharmaceutically acceptable carrier.

The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.

In certain embodiments, the compositions of the invention are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions of the invention are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the invention varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the ahending physical taking all other factors about the patient into account.

Compounds of the invention for administration can be in the range of from about 1 pg to about 10,000 mg, about 20 pg to about 9,500 mg, about 40 pg to about 9,000 mg, about 75 pg to about 8,500 mg, about 150 pg to about 7,500 mg, about 200 pg to about 7,000 mg, about 350 pg to about 6,000 mg, about 500 pg to about 5,000 mg, about 750 pg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments there between.

In certain embodiments, the present invention is directed to a packaged

pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the invention, alone or in combination with a second

pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of diseases or disorders contemplated herein.

Formulations can be employed in admixtures with conventional excipients, /. e..

pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wehing agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.

Routes of administration of any of the compositions of the invention include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds for use in the invention can be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.

Oral Administration

For oral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. The compositions intended for oral use can be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets can be uncoated or they can be coated by known techniques for elegance or to delay the release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.

Parenteral Administration

For parenteral administration, the compounds of the invention can be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and/or dispersing agents can be used.

Additional Administration Forms

Additional dosage forms of this invention include dosage forms as described in U.S. Patents Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790.

Additional dosage forms of this invention also include dosage forms as described in U.S. Patent Applications Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms of this invention also include dosage forms as described in PCT Applications Nos. WO 03/35041; WO 03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO 02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO 98/11879; WO 97/47285; WO 93/18755; and WO 90/11757.

Controlled Release Formulations and Drug Delivery Systems

In certain embodiments, the formulations of the present invention can be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.

The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time can be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.

For sustained release, the compounds can be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use the method of the invention can be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.

In certain embodiments of the invention, the compounds of the invention are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.

The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.

The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.

As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration. As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.

Dosing

The therapeutically effective amount or dose of a compound of the present invention depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of heart failure in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.

A suitable dose of a compound of the present invention can be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day.

The dose can be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage can be the same or different. For example, a dose of 1 mg per day can be administered as two 0.5 mg doses, with about a l2-hour interval between doses.

It is understood that the amount of compound dosed per day can be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose can be initiated on Monday with a first subsequent 5 mg per day dose administered on

Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.

In the case wherein the patient's status does improve, upon the doctor's discretion the administration of the inhibitor of the invention is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e.. a“drug holiday”). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days,

5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%- 100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the viral load, to a level at which the improved disease is retained. In certain embodiments, patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and/or infection.

The compounds for use in the method of the invention can be formulated in unit dosage form. The term“unit dosage form” refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form can be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form can be the same or different for each dose.

Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD₅₀ and ED₅₀. The data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this invention and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents, with art- recognized alternatives and using no more than routine experimentation, are within the scope of the present application.

It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application.

The following examples further illustrate aspects of the present invention. However, they are in no way a limitation of the teachings or disclosure of the present invention as set forth herein.

EXAMPLES

The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Example 1: Synthesis of Aminomethyl-Dil (Scheme 1)

Step 1: 2 mL of 2,3,3-Trimethylindolenine 1 (1.984 g, 0.013 mol, 1 eq.) and 8.19 g of l-Iodooctadecane (0.022 mol, 1.75 eq.) were dissolved in 4.2 mL of chloroform and heated at reflux for 96 hours. The solvent was then removed under reduced pressure, and the resulting dark red residue was washed multiple times with hexanes and ether to give 5.0712 g of indolium 2 as a dusty reddish brown solid.

Step 2: 3 mL of 2,3,3-Trimethylindolenine 1 (2.98 g, 18.7 mmol, 1.1 eq.) and 3.01 g of N-(Hydroxymethyl)phthalimide 3 (16.93 mmol, 1 eq.) were added to 14.7 mL of concentrated sulfuric acid and stirred at room temperature for 6 days. The reaction was then poured over ice and made basic with concentrated aqueous ammonium hydroxide. After crystallizing at -8 °C overnight, the resulting precipitate was filtered, rinsed with ice cold water, and dried under vacuum to give indolenine 4 as a light yellow shiny crystalline solid.

Step 3: 5.5 g of indolenine 4 (17.3 mmol, 1 eq.) and 11.5 g of 1 -Iodooctadecane (30.2 mmol, 1.75 eq.) were dissolved in 5.5 mL of chloroform and stirred at reflux for 5 days. The solvent was then removed under reduced pressure, and the residue was washed, filtered, and rinsed with hexanes and diethyl ether to give 5 as a dark red solid. The crude product was used in the next step without further purification.

Step 4: 0.98 g of 5 (1.71 mmol, 1 eq.), 0.70 g ofN,N’-Diphenylformamidine 6 (3.42 mmol, 2 eq.) and 0.4 mL of triethyl orthoformate (0.356 g, 2.14 mmol, 1.2 eq.) were dissolved in 5.1 mL absolute ethanol and stirred at reflux. The reaction was monitored by TLC using 10% methanol in DCM as solvent, and was complete after 22 hours. The solvent was then removed under reduced pressure and the resulting red residue was purified by preparatory HPLC using a gradient of 65-100% acetonitrile in water (+ 0.1% TFA) with detector absorbance set to 411 nm. Hemi cyanine 7 was isolated as 565 mg of a bright yellow solid (0.84 mmol, 49% yield). Step 5: 1.13 g of hemicyanine 7 (1.68 mmol, 1 eq.), 1.39 g of indolium 2 (3.36 mmol, 2 eq.), and 1.6 mL acetic anhydride (1.73 g, 16.8 mmol, 10 eq.) were dissolved in 8.4 mL of pyridine and heated at 60°C. The reaction was monitored by TLC (10% methanol in DCM) and was complete after 2 days. The solvent was azeotroped with toluene and removed under reduced pressure to give cyanine 8 as a dark purple residue. For characterization, a small amount of this material was purified by semi-preparatory HPLC using a gradient of 65- 100% acetonitrile in water (+0.1% TFA). The rest of the material was used crude in the next step.

Step 6: 500 mg of cyanine 8 (0.503 mmol, 1 eq.) was dissolved in 2 mL of methanol, and then 1.5 mL of anhydrous hydrazine (1.53 g, 47.8 mmol, 95 eq.) was added slowly. The reaction was stirred at room temperature for 2 days and monitored by TLC (10% methanol in DCM). The solvent was then evaporated under reduced pressure, and the resulting dark pink residue was purified by prep HPLC using a gradient of 85-100% acetonitrile in water (+0.1% TFA) to give 289 mg of pure cyanine 9 as a bright pink solid (0.33 mmol, 67% yield).

Sfhpmp 1.

Example 2: Synthesis of Mal-PEG3400-DiI 15 mihoΐ (10 nm) of aminomethyl-Dil 9 was combined with 37.5 mihoΐ (135 mg, 2.5 eq) NHS-PEG34oo-Maleimide in DMSO with 5 eq (with respect to amino-Dil, 10 pL) TEA and allowed to react for 1.5 h at room temperature. The product 10 was purified on an Isolera purification system (Biotage, LLC, Charlotte, NC, USA) using a Biotage SNAP Ultra C18 30 g column with a 30-95% methanol-water gradient (0.1% TFA). The product was dried under vacuum with an overall yield of 10%. The product was verified by mass spectrometry.

Example 3: Synthesis of Superoxide Dismutase (SOD)-PEG₃₄oo-DiI

SOD (58 mM, 50 pL) in PBS (pH 7.4) was reacted with 25 pg either IR Dye 800 CW NHS or sulfo-cy3 NHS. After 2 h incubation at room temperature, excess dye was removed by size exclusion chromatography (7 kD cutoff Zebra column, Thermo). Thiolation was achieved with a 30-fold excess of Traut’s reagent along with 2.5 mM EDTA in PBS. Excess Traut’s reagent was removed by size exclusion chromatography. A 2.5 fold excess of Mal- PEG3400-D1I in DMSO (final concentration less than 5%) was added, and the mixture was allowed to react for 48 h at 4°C. The synthesis was confirmed by SDS PAGE analysis on a 4- 20% acrylamide gel (Bio-Rad; FIGs. 4A-4B). Densitometry revealed an overall yield of 60%.

Example 4: Synthesis of IgG-PEG₃₄oo-DiI

Mouse IgG (10 mg/mL, 50 pL) in PBS (pH 7.4) was reacted with 25 pg

IRDye800CW NHS. After 2 h incubation at room temperature, excess dye was removed by Zebra column. Thiolation was achieved with a 30-fold excess of Traut’s reagent along with 2.5 mM EDTA in PBS. Excess Traut’s reagent was removed by size exclusion

chromatography. A 2.5 fold excess of mal-PEG34oo-DiI (DMSO) was added and the mixture was allowed to react for 48 h at 4°C. The synthesis was confirmed by SDS PAGE analysis on a 4-20% acrylamide gel (FIGs. 3A-3B). Densitometry revealed an overall reaction yield of 60%.

For cetuximab-PEG34oo-DiI synthesis, ERBITUX (2 mg/mL, 100 pL) was solvent exchanged into PBS (pH 7.4) using a Zebra spin column and reacted with 75 pg

IRDye800CW NHS. After 2 h incubation at room temperature, excess dye was removed by size exclusion chromatography. UV-Vis spectral analysis revealed an average of 9 dyes conjugated per antibody. Thiolation was achieved with a 30-fold excess of Traut’s reagent along with 2.5 mM EDTA in PBS. Excess Trauf s reagent was removed by size exclusion chromatography. A 2.5 fold excess of mal-PEG34oo-DiI (DMSO) was added and the mixture was allowed to react for 48 h at 4°C. The synthesis was confirmed by SDS PAGE analysis on a 4-20% acrylamide gel. Densitometry revealed an overall reaction yield of 17%.

Example 5: Synthesis of Dy Light 800-DiI

952 nmol (1 mg) of Dy Light 800 NHS ester was combined with 750 nmol aminomethyl-Dil 9 in 100 pL dry DMSO with a 5 fold excess of triethylamine. The solution was maintained at room temperature in the dark for 5 h. Excess Dy Light 800 was removed by 5x dichloromethane : PBS extraction (1 : 10), keeping the organic layer. The structure 11 was confirmed by MALDI-TOF high resolution mass spectrometry.

Example 6: Erythrocyte loading

Whole blood was obtained from adult B ALB/c mice. The blood was washed three times in PBS by centrifugation at 1500 g being careful to remove the huffy coat. The erythrocyte pellet was combined with various concentrations of Dil constructs and 2% FBS in PBS at a volume ratio of 2: 1 : 1. The mixture was incubated at 37 C for 30 min, and RBCs were washed 3 times in PBS. To determine loading efficiency, RBCs were bath sonicated and loaded in lpL triplicates on a 0.22 pm nitrocellulose membrane (Bio-Rad). The membrane was scanned at 800 nm using Li-COR Odyssey (for near infrared labeled constructs) or at 565 nm using Bio-Rad gel camera (for Dil). The spot integrated density of a 16-bit TIFF image was measured with ImageJ and plotted as a function of time with Prism (GraphPad, San Diego CA). Concentrations were calculated using a standard curve for the respective constructs. Labeled RBCs were imaged with Zeiss Axio Observer 5 epifluorescent microscope equipped with X-Cite 200DC light source and Axiocam 506 monochromatic camera. Near infrared labeled RBCs were imaged with Cy7 filter set, catalog number 49007, Chroma Corporation (McHenry, IL, USA).

Example 7 : Monitoring of RBCs in circulation

To monitor levels of painted RBCs at different times post-injection, flow cytometry was used as described in Staples, 2010, Wiley Interdiscip. Rev. Nanomedicine

Nanobiotechnology 2(4):400-4l7. PEG3400-D1I (200 mM, 100 pL in PBS) was treated with 1 mM glutathione for 1 h. After this time a shift of around 300 Daltons was observed in Gaussian distribution of the mass spectrum of PEG3400-D1I by MALDI, confirming the maleimide had been inactivated. Fresh washed RBCs (40 pL) were painted using 50 pM thiolated PEG3400-D1I in the loading buffer. The pellet was washed and then injected into a B ALB/c mouse, which was bled at 30 min, 1 d, 2 d, and 7d post injection. Blood samples were diluted in PBS (1 mL), and analyzed on a flow cytometer (FACScan, BD or Galios) in FL-2 channel. The percentage of Dil-positive RBCs was determined. The same protocol was used for monitoring of SOD painted RBCs.

For monitoring the circulation of NIR-labeled molecules, RBCs were painted as previously described. RBCs or controls (NIR-labeled molecules without Dil anchor) were injected into the tail vein of BALB/c mice, and blood was collected at various time points via retro orbital bleeding. After blood was diluted two-fold with lysis buffer (0.1% SDS in H₂0), samples were dotted in triplicate on a nitrocellulose membrane (2 pL) and the fluorescence intensity was measured with a LiCOR infrared scanner at 800nm wavelength.

Example 8: Hemolysis and complement C3 opsonization

Blood was withdrawn from a BALB/c mouse and washed three times to obtain a RBC pellet. 10 pL of pellet was either loaded with 5 pM IRDye800-SOD-PEG₃₄oo-DiI or

IRDye800-Cituximab-PEG₃₄oo-DiI as previously described. After painting, lOpL RBCs were mixed with 30pL mouse serum and incubated at 37 C for 1.5 h. Then, the RBCs were briefly pelleted, and the absorbance in the supernatant was measured by UV-Vis spectrometry at 550 nm. The absorbance of hemoglobin in control supernatant (non-loaded RBCs incubated in the same conditions) was considered 100%, and the hemolysis of painted RBCs was expressed as percentage of control (FIGs. 6A-6B).

Example 9: Injectable in vivo biosensors

In cancer, it is extremely challenging to detect the indolent disease before the appearance of clinical signs. The established approach to measuring disease biomarkers is based on sampling accessible fluids in the body, usually blood. At late stages many cancers have elevated biomarker levels. However, the biomarkers are difficult to detect in peripheral blood in the early stages of the disease. This is because diagnostic sampling provides only a snapshot at the time of analysis, whereas the levels of biomarkers could be below the detectable level at the time of measurement. Moreover, the microenvironment of diseased tissue is not readily accessible with peripheral sampling and the released biomarkers are diluted and/or eliminated.

The present invention contemplates optically-coded injectable biosensors that circulate in the blood and collect signals over time. In certain embodiments, a more sensitive detection of rare cues from the tumor microenvironment can be achieved with these sensors. In other embodiments, the intravenously injected RBC biosensors can circulate in the blood from days to weeks, pass through diseased tissue, and become modified by high local concentration of proteases or biomarkers in the tumor. The sensor“switch” is proportional to the integrated concentration of biomarker/enzyme over time (area under curve). The switch can be quantified in a peripheral blood sample and correlated with the disease status.

In certain non-limiting embodiments, the invention contemplates the following procedure: a patient comes to an outpatient clinic, where a nurse collects 0.1-1 ml blood; the RBCs are purified, modified with optical sensors in a sterile automated setup and reinjected back into the patient; the patient then receives a personal home kit for finger blood sampling (similar to a blood glucose test). Each day after the injection, the patient collects microliters of blood on a special membrane or into microcapillaries. The samples are then shipped to a central lab where they are analyzed according to their optical signature.

In certain embodiments, the constructs of the invention identify and/or quantitate protease activity and/or soluble biomarkers for cancer diagnostics. Matrix metalloproteinases (MMPs) and vascular endothelial growth factor-A (VEGF-A) are critical biomarkers of cancer progression, angiogenesis, and metastasis. These and many other biomarkers have higher concentration in tumors but are harder to detect at early stages in peripheral blood. In certain embodiments, exposure to biomarkers over time (concentration versus time) cannot be determined using a standard blood test; long-circulating biosensors that continuously collect information in the body are best suited for this type of monitoring. In other embodiments, direct access to the tissue increase the chance of detecting rare biomarkers as compared to peripheral blood tests.

Use of autologous RBCs is recommended due to availability and biocompatibility. Moreover, lipid-anchored molecules undergo lateral diffusion and clustering in the RBC membrane. Discrete clusters of fluorescence improve the sensitivity of detection of changes on the RBC surface. The concept of continuous vs. discrete change of signal of the same magnitude (2 -fold) is shown in FIG. 7. Membrane-anchored molecules undergo lateral diffusion and nano-clustering, thereby greatly improving detection of molecules on the RBC surface. In one experiment (FIGs. 8A-8C), anti-fluorescein (FITC) antibody -painted RBCs captured FITC-labeled dextran, which was then detected with Alexa 488-anti-FITC antibody (ELISA on RBCs). Due to the clustering of dextran FITC on the RBC surface by the antibody, the nano-sized dots were detectable at a l-fM concentration of FITC-dextran.

Libraries of sensor modules can be quickly and controllably inserted into the RBC membrane via lipid anchors. The modified RBC biosensors circulate for prolonged periods in the body and collect cues from the disease. The following sensors can be designed and tested: (a) protease biosensor; (b) soluble biomarker biosensor.

A protease biosensor (FIG. 9, top) responds to tumor-specific proteases. The sensor unit consist of non-exchangeable fluorescent lipophilic anchors with“donor” and“acceptor” fluorescence that are be bridged via a cleavable protease substrate. The cleavage by tumor- specific protease induces lateral separation of the fluorophores in the membrane, which is detected ex vivo due to change in fluorescence resonance energy transfer (FRET). In certain embodiments, the cleaved components are detected ex vivo with fluorescent antibodies or fluorescent nanoparticles. In other embodiments, ex vivo detection can improve sensor sensitivity due to clustering of molecules in the membrane. To increase the specificity (tumor vs. healthy tissues), multiple protease-cleavable linkers can be used in parallel, which would require an above-threshold concentration of the enzyme to cleave the bond between color components. As a model protease, MMPs can be used.

A biomarker biosensor (FIG. 9, bottom) uses ELISA-on-RBCs. Fluorescent lipid constructs with biomarker-specific and control antibodies (orange) are incorporated into RBCs. The RBCs are stained ex vivo with the secondary antibody against the biomarker, causing preferential clustering of the fluorophore associated with the specific antibody but not with the control antibody. As a model biomarker, the vascular endothelial growth factor VEGF-A, which is the main angiogenesis factor involved in cancer progression and metastasis, can be used.

Sensor synthesis:

Sensor modules are based on lipid anchors to allow incorporation into the RBC membrane. For biomarker sensor, antibodies are conjugated to lipophilic cyanine dye derivatives of different colors, such as the antibody -lipid construct in FIG. 10. For protease sensor, the lipophilic cyanine dyes of different colors are joined together through a MMP-2/9 cleavable sequence (Olson, et al, 2009, Integr. Biol. (Camb) 1(5-6): 382-393), such as in FIG. 11 A. When the dyes are in close proximity, FRET or quenching of fluorescence are activated. The cleavage will result in a physical separation of color components in the membrane due to lateral diffusion, as shown in FIG. 11B.

In order to improve detection of separated fluorescent components, each half of the sensor contains a small molecule“handle”, e.g., fluorescein (FITC) and biotin (FIG. 12A). After blood collection, the RBCs are stained with AlexaFluor 488 anti-FITC antibody and Alexa-Fluor 555-labeled streptavidin. Detection of an antibody causes clustering of fluorescent molecules in the membrane so that the non-cleaved sensor shows a cluster with both colors colocalized, whereas cleaved sensors show separated clusters of different colors (FIG.12B).

In order to increase the stringency of protease detection, sensor module is such that a cyclic double MMP-2/9 substrate is placed in parallel (FIG. 13 A). Cleavage of only one bond does not lead to color separation, and above-threshold concentrations of enzyme are needed to induce a complete cleavage (FIG. 13B). The same scheme can be used to place two different substrates in parallel, for example MMP-2 and thrombin.

Correlation between optical switch and biomarker exposure

As opposed to the far more limited static biomarker/enzyme concentration parameter measured in a single time point blood test, circulating sensors can continuously respond to and measure“biomarker exposure” E_t, which is the integrated parameter that takes into consideration the biomarker/enzyme concentration over time (FIG. 14A). Technically, £_¾ is

equal to the area under curve AUC“concentration over time”, or , where

Cb(t) is the concentration of the biomarker/enzyme as a function of time. In order to measure the response of biosensors to different L) values in vitro, RBCs are incubated with different concentrations of biomarkers/enzymes for different times, and the optical change on the RBC surface is quantified and plotted as a function of £_¾ values (FIG. 14B). The optical changes are determined by comparing a selected optical parameter before and after

biomarker/protease exposure. The measurable optical change can be the fluorescence colocabzation or FRET, or number of fluorescent clusters per RBC/field.

In vivo testing

A transgenic adenocarcinoma mouse prostate model (TRAMP) and transgenic mouse breast cancer model MMTV-PyMT can be used, as these models most closely represent human disease. Tumors at different stages of growth are used.

a) Protease sensor: Correlation between MMP-2/9 levels in the tumor and blood with the optical change are studied. RBCs are injected intravenously and the optical changes are quantified in peripheral blood at different times post injection. In parallel, levels of MMP in plasma are measured several times a day with a commercial ELISA kit. In addition, the tumor interstitial fluid is sampld daily with a 28G-needle and MMP levels are measured with western blotting. £_¾ values for MMP are calculated and statistically correlated with the optical switch.

b) Biomarker sensor: Correlation between VEGF-A levels in blood and tumor and the optical change are studied. A VEGF-A biosensor is injected into tumor-bearing mice at different stages of growth. Levels of VEGF-A are measured in blood 3-4 times a day with an ELISA kit. RBCs are stained daily with a fluorescent anti-VEGF-A antibody and the changes in fluorescence are used to calculate £_¾ based on the correlation obtained in vitro. E_b values obtained in vivo are statistically compared with E_b based on measured VEGF-A levels in vitro.

Example 10: Synthesis of DIR-methylamine (Scheme 2), DIR-MTz (Scheme 3), DIR- PEG-MTz (Scheme4), and DIR-PEG-NH2 (Scheme 5)

DIR-methylamine were synthesized with modification of previously reported DIR synthesis (Konig & Kramer, 2017, Chem. Eur. J. 23:9306-9312; Salon, et al., 2005, J.

Heterocyclic Chem. 42:959-961; Smith, et al, 2018, Biomaterials 161 :57-68).

Synthesis of 2,3,3-trimethyl-l-octadecyl-3i/-indol-l-ium (2) involved reaction of commercially available 2,3,3-Trimethylindolenine (1) and 1 -iodooctadecane.

For the synthesis of 5-(( 1.3-dio\oisoindolin-2->/)methyl)-2.3.3-trimethyl- l -octadecyl- 3//-indol- 1 -ium (5), 2,3,3-Trimethylindolenine (1) was converted to 2-((2.3.3-trimethyl-3//- indol-5-yl)methyl)isoindoline-l,3-dione (4) upon reaction with (Hydroxymethyl)phthalimide (3), then uponn reaction with 1 -iodooctadecane. Compound dianil hydrochloride (8) was synthesized from /V-formyl-/V-methylaniline (7), l-methylcyclohexene, aniline, and phosphoous oxychloride (POCI3) in

dimethylformamide (DMF).

Compound 9 was synthesized by reacting compound 2.3.3-tri methyl- 1 -octadecy 1-3//- indol-l-ium (2) with 5-((l,3- dioxoisoindolin-2-y/)methyl)-2.3.3-tri methyl- 1 -octadecyl-3//- indol-l-ium (5) and dianilhydrochloride (8) in presence of acetic anhydride. The amine protecting group, phthalimide, was removed with hydrazine to obtain compound DIR- methylamine (10)(Scheme 2).

DIR-PEG-MTz, DIR-MTz and DIR-PEG-NH2 were synthesized from DIR methylamine upon reaction with methyl tetrazine NHS ester, MTz-PEG-COOH, and NHBoc- PEG-COOH under EDC/NHS coupling conditions (Scheme 3-5).

Scheme 2.

Scheme 5.

10.1. Synthesis of2,3,3-trimethyl-l-octadecyl-3H-indol-l-ium (2): A stirred mixture of compound 2,3,3-Trimethylindolenine (1) (1.0 g 6.3 mmol, 1 eq.) and 1- Iodooctadecane (3.61 g, 9.5 mmol, 1.5 eq.) in chloroform (5 mL) were heated at reflux temperature for 96 hours. The solvent was then removed under reduced pressure and the resulting dark red residue was washed multiple times with hexanes and ether to give 2. lg of indolium 2 as a dusty reddish brown solid. Yield 80.9%, ^XH NMR (400 MHz, CDCI₃): d 7.57 (m, 4 H, Ar-H); 4.68 (t, J= 7.8 Hz, 2H, CH₂); 3.12 (s, 3H, CH3); 1.87-1.97 (m, 2H,

CH₂); 1.67 (s, 6H,CH₃); 1.42-1.51 (m, 2H, CH₂); 1.32-1.41 (m, 2H, CH₂); 1.19-1.32 (m, 26H, CH₂); 0.88 (t ,J= 6.8 Hz, 3H, CH₃).

10.2. Synthesis of 2-((2,3,3-trimethyl-3H-indol-S-yl)methyl)isoindoline-l ,3-dione (4):

To a mixture of compound 2,3,3-Trimethylindolenine (1) (2.0 g, 12.5 mmol, 1.1 eq.) and A (Hydroxymethyl)phthalimide (3) (2.02 g 11.4 mmol, 1 eq.) were added 14.7 mL of concentrated sulfuric acid and the system stirred at room temperature for 6 days. The reaction was then poured over ice and made basic with concentrated aqueous ammonium hydroxide. After crystallizing at -20 °C overnight, the resulting precipitate was filtered, rinsed with ice cold water, and dried under vacuum to give indolenine 4 as a light yellow shiny crystalline solid. Yield 88.3%. ^XH NMR (400 MHz, CDCl₃); d 7.82-7.87 (m, 2H, Ar- H); 7.67-7.72 (m, 2H, Ar-H); 7.44-7.48 (m, 1H, Ar-H); 7.35-7.42 (m, 2H, Ar-H); 4.87 (s, 2H,CH₂); 2.25 (s, 3H, CH₃) 1.28 (s, 6H, CH₃).

10.3. Synthesis of 5-((l,3-dioxoisoindolin-2-yl)methyl)-2,3,3-trimethyl-l-octadecyl-3H- indol-l-ium (5):

A stirred mixture of compound 4 (1.0 g, 3.1 mmol, 1 eq.) and 1 -Iodooctadecane (2.09 g, 5.5 mmol, 1.75 eq.) in chloroform (5 mL) was heated at reflux temperature for 5 days. The solvent was then removed under reduced pressure and the residue was washed, filtered, and rinsed with hexanes and diethyl ether to give 5 as a dark red solid. Yield 83.6%. ' H NMR (400 MHz, CDC1₃): d 7.85-7.89 (m, 2H, Ar-H); 7.73-7.76 (m, 2H, Ar-H); 7.69 (d, J= 8.4 Hz, 1H, Ar-H); 7.64-7.67 (m, 1H, Ar-H); 7.52 (d, =8.3 Hz, 1H, Ar-H); 4.93 (s, 2H, CH₂); 4.64 (t, J= 7.7 Hz, 2H, CH₂); 3.08 (s, 3H, CH₃);l.81-1.93 (m, 2H, CH₂); 1.64 (s, 6H, CH₃); 1.38- 1.48 (m, 2H, CH₂); 1.31-1.37 (m, 2H,CH₂); 1.21-1.29 (m, 26H, CH₂); 0.87 (t, J= 6.8 Hz, 3H, CH₃).

10.4. Synthesis of N-((E)-(2-niethyl-3-((E)-(phenyliniino)niethyl)cyclohex-2-en-l- ylidene)methyl) aniline hydrogen chloride (8): A stirred solution o G N- formy 1 -A- methy 1 an i 1 i ne(7 ) ( 2.11 g, 15.6 mmol) in chloroform (2 mL) at -5 °C was treated with phosphorous oxychloride (1.5 mL, 15.6 mmol) dropwise, and stirred for 1 h at 10 °C. l-methylcyclohexene (0.6 mL, 5.2 mmol) was added dropwise and the solution stirred at 45°C for 20 h. The reaction mixture was poured into a beaker containing vigorously stirred water (20 mL). Solid potassium carbonate (2 g, 14.5 mmol) was added carefully added. A solution of aniline hydrochloride salt (1.52 g, 11.7 mmol) in water (3 mL) was added and the mixture stirred at ambient temperature for 30 min. At this time, potassium carbonate (2 g, 14.5 mmol) was added portionwise and the resulting solution cooled to give a precipitate that was filtered, washed several times with cold water, and stirred vigorously with acetone (23 mL) filtered and dried in vacuum to afford 8 dark red crystal. Yield 37 %; ^XH NMR (400 MHz, DMSO-d₆) d 10.57 (bs, 2H, NH); 8.44 (s, 2H, =CH); 7.59 (d, J= 7.8 Hz, 4H, Ar-H); 7.43 (t, .7=8.0 Hz, 4H, Ar-H); 7.22 (t, J= 7.4 Hz, 2H, Ar-H); 2.56 (s, 3H, CH₃); 2.50-2.54 (m, 4H, CH₂); 1.75-1.84 (m, 2H, CH₂).

10.5. Synthesis of 2-((E)-2-((E)-3-(2-((E)-3,3-dimethyl-l-octadecylindolin-2- ylidene)ethylidene)-2-methylcyclohex-l-en-l-yl)vinyl)-5-((l,3-dioxoisoindolin-2-yl)methyl)- 3, 3-dimethyl- 1 -octadecyl-3H-indol-l -ium ( 9) :

A stirred mixture of compound 5 (200 mg, 0.35 mmol, 1 eq.), dianil hydrochloride (8) (142 mg, 0.42 mmol, 1.1 eq.),indolium (2) (144 mg, 0.35 mmol, 1 eq.), and sodium acetate (95 mg, 0.70 mmol, 2 eq.) were dissolved in a mixture of acetic acid (5 mL) and acetic anhydride (5 mL) under N₂ was heated to 100 °C for 4 h, cooled down to 25 °C, and agitated into a heterogeneous mixture by addition of ethyl acetate (20 mL). The reaction was monitored by TLC using 10% methanol m DCM as solvent. The reaction mixture was azeotroped with toluene and removed under reduced pressure, extracted with chloroform, washed with sat. NaHCCL solution to give cyanine 9 as a dark green residue used as crude for the next step. MALDI-TOF (DHB matrix) Calculated M/z = 1098.8749, Found M/z =

1098.7701,1099.7740 [M+H], 1100.7786 [M+2H]

10.6. Synthesis of 5-(aminomethyl)-2-((E)-2-((E)-3-(2-((E)-3,3-dimethyl-l- octadecylindolin-2-ylidene)ethylidene)-2-methylcyclohex- 1 -en- 1 -yI)vinyI)-3, 3-dimethyl- 1- octadecyl-3Hindol-l-ium (10):

A cyanine compound 9 (100 mg, 0.091 mmol, 1 eq.) was dissolved in 5 mL of methanol, and then anhydrous hydrazine (271 pl, 8.65 mmol, 95 eq.) was added slowly. The reaction was stirred at room temperature for 48h and monitored by TLC (10% methanol in DCM). The solvent was then evaporated under reduced pressure, and the resulting dark pink residue was purified by using prep HPLC and eluted with 90% to 95% methanol, to obtain 17 mg of pure cyanine 10 as a dark green solid. Yield 19%. MALDI-TOF (DHB matrix) Calculated M/z = 968.8694, Found M/z = 969.0248, 970.0417 [M+H], 971.0408 [M+2H]

10.7. Synthesis of DIR-MTz .

A mixture of compound DiR-methyl amine(lO) (5 mg 0.0052 mmol, 1 eq.), Methyl tetrazine NHS (2.53 mg, 0.0077 mmol, 1.5 eq.) and DIEA (2.8 mΐ, 0.015 mmol, 3 eq.) were stirred in DMF at room temperature for 4 hours. The solvent was then evaporated under reduced pressure and the resulting dark pink residue was purified by using prep HPLC and eluted with 80% to 90% methanol, to obtain DIR-MTz as a dark green solid.

10.8 Synthesis of MTzNH-PEG-COOH (3400):

A mixture of B-PEG-VA (50 mg, 0.015 mmol, 1 eq.), Methyl tetrazine NHS (7.21 mg 0.022 mmol, 1.5 eq.), and DIEA (8 mΐ, 0.044 mmol, 3 eq.) were stirred in DMF at room temperature for 4 hours. The solvent was then evaporated under reduced pressure and the resulting dark pink residue was purified by using prep HPLC and eluted with 40% to 50% methanol, to obtain MTzNH-PEG-COOH (3400) as a pink solid. Yield 68.3%.

10.9. Synthesis of DIR-PEG-MTz (3400):

A mixture of DIR-methyl amine (10) (5 mg, 0.0052 mmol, 1 eq.), MTz-PEG-COOH (3400) (17.5 mg, 0.0052 mmol, 1 eq.), HBTU (3 mg, 0.0077 mmol, 1.5 eq.) and DIEA (2.8 mΐ, 0.015 mmol, 3 eq.) were stirred in DMF at room temperature for l2h. The solvent was then evaporated under reduced pressure, and the resulting dark pink residue was purified by using prep HPLC and eluted with 50% to 60% methanol, to obtain DIR-PEG-MTz (3400) as a dark green solid.

10.10. Synthesis ofNHBoc-PEG-COOH (2000):

A mixture of NHB0C-PEG-NH2 (50 mg, 0.015 mmol, 1 eq.), succinic anhydride (7.21 mg 0.022 mmol, 2.0 eq.), and DIEA (8 mΐ, 0.044 mmol, 3 eq.) were stirred in dichloromethane at room temperature for 2 hour. The solvent was then evaporated under reduced pressure and the resulting residue was purified by lkd dialysis membrane to give NHBoc-PEG-COOH (2000) as a white solid. 10.11. Synthesis of DIR-PEG-NH2 (2000):

A mixture of DIR-methyl amine (10) (4 mg, 0.0041 mmol, 1 eq.), MTz-PEG-COOH (3400) (13 mg, 0.0061 mmol, 1.5 eq.), HBTU (2.8 mg, 0.0082 mmol, 2 eq.), and DIEA (2.2 pl, 0.012 mmol, 3 eq.) were stirred in dimethylformamide at room temperature for l2h. The solvent was then evaporated under reduced pressure and the resulting dark pink residue was purified by using prep HPLC and eluted with 50% to 65% methanol, to obtain DIR-PEG- NHBoc as a dark green residue. The residue was stirred in 25% TFA/DCM for 30 min to give DIR-PEG-NH2 (2000) as a dark green solid. Example 11: Synthesis of DIR Cytarabine (Scheme 6) and Daunorubicin (Scheme 7) conjugates using disulfide linker.

For the synthesis of disulfide linker, intermediates 1 and 3 were synthesized from g- valerolactone and dipyridyldisulfide. Cytarabine disulfide acid (5) was synthesized from the cytarabine upon reaction with intermediate 3 and then with intermediate 1. DIR-SS- Cytarabine (6) was prepared by reacting Cytarabine disulfide acid (5) with DIRmethylamine using HBTU as a coupling reagent (Scheme 6).

Similarly, Daunorubicin disulfide acid (8) was synthesized from Daunorubicin upon reaction with intermediate 3 and then with intermediate 1. DIR-SS-Daunorubicin (9) was made by reacting daunorubicin disulfide acid (8) and DIR-methylamine using HBTU as a coupling regent (Scheme 7).

Scheme 6.

Scheme 7.

Methods

77.7. Synthesis of 4-Sulfhydrylpentanoic acid (1):

g-valerolactone (2.0 g, 2lmmol) was refluxed with HBr (8.4 g) at 70 °C. After reflux was established, thiourea (8.0 g) was added to the mixture and solution was further refluxed for 24 hours. After 24 hours of reflux, the clear solution was diluted with ice-water and washed with three allotments of methylene chloride and ether. The aqueous layer was then treated with 10 N NaOH to adjust the pH to 10. The mixture was refluxed for 24 hours. After 24 h, the reaction mixture was allowed to cool to room temperature. The pH was adjusted to 1 with 1 N HC1. The aqueous layer was extracted with methylene chloride. The combined organic layers were washed with brine and H₂0. The organic layer was dried under MgSCfl and concentrated in vacuum to give compound 1 as a yellow oil with strong stench (Yield 39%). ^XH NMR (400 MHz, CDCl₃): d 10.7 (bs, 1H, OH), 2.92-3.05 (m, 1H, CH), 2.47-2.65 (m, 2 H, CH₂), 1.94-2.04 (m, 1H, CH₂), 1.73-1.86 (m, 1H, CH₂), 1.48 (d, J= 7.1 Hz, 1 H, SH), 1.40 (d, J= 6.8 Hz, 3H, CH₃). 77.2. Synthesis of 2-(pyridin-2-yldisulfaneyl)ethan-l-ol (2):

To the mixture of 2,2'- Dipyridyldisulfide (2.17 g, 9.87 mmol, 1.5 eq. ) in EtOH (10 mL), a solution of 2-mercaptoethanol (461 pL, 6.6 mmol, 1 eq.) in EtOH (5 mL) was added dropwise at room temperature over a 30-min period. Then, the reaction mixture was stirred at room temperature overnight. Subsequent evaporation of the solvent afforded the crude product as a yellow oil, which was then purified by silica gel column chromatography (EtO Ac/Hexane = 1:9 1: 1, v/v) to give the product as a white solid (Yield, 90%). ' H NMR (400 MHz, CDCl₃): d 8.5l(d, J= 5.0 Hz, 1H, Ar-H), 7.55-7.61 (m, 2H, Ar-H), 7.40 (d, J= 8.1 Hz, 1H, Ar-H), 7.12-7.18 (m, 2H, Ar-H), 5.59 (bs, 1H, OH), 3.81 (t, .7=5.2 Hz 2H, CH₂), 2.80 (t, J= 5.2 Hz, 2H, CH₂).

11.3. Synthesis of 2-(pyridin-2-yldisulfaneyl)ethyl IH- 1,2,1-triazole- 1-carboxylate (3):

To a solution of compound 2 (500 mg, 2.7 mmol, leq.) in methylene chloride was added carbonyl ditriazole (CDT) (658 mg, 4.0 mmol, 1.5 eq.) at room temperature. Then, the reaction mixture were stirred at room temperature for 2 hour. The methylene chloride solution were washed with sat. NaHC0₃ and brine. The organic layer was dried under MgSCf and concentrated in vacuo to give compound 1 as a yellow oil, which was then purified by silica gel column chromatography (EtO Ac/Hexane = 2:8 7:3, v/v) to give the product as a white solid (Yield, 73%). 1H NMR (400 MHz, CDCl₃): d 8.81 (s, 1H, Ar-H), 8.47 (d, J= 4.6 Hz, lH,Ar-H), 8.08 (s, 1H, Ar-H), 7.52-7.67 (m, 2H, Ar-H), 7.11 (t, J= 5.6 Hz, 1H, Ar-H), 4.79 (t, J= 6.5 Hz 2H, CH₂), 3.24 (t, J= 6.5 Hz, 2H, CH₂).

11.4. Synthesis of 2-(pyridin-2-yldisulfaneyl)ethyl ( l-((3S,4S)-3,4-dihydroxy-5 - (hydroxyniethyl)tetrahydrofuran-2-yl)-2-oxo-I,2-dihydropyriinidin-4-yl)carhaniate (4):

A mixture of cytarabine hydrochloride (10 mg, 0.036 mmol, 1 eq.), compound 3 (15.1 mg,

5.4 mmol, 1.5 eq.) and DIEA (19 pL, 0.11 mmol, 3 eq.) were dissolved in methylene chloride (5mL) and stirred at room temperature for 2 hour. The solvent was removed in vacuum and the crude product was purified by using prep HPLC and eluted with 30% to 90% methanol, to obtain compound 4 as white solid (Yield 63%). 'H NMR (400 MHz, DMSO-d₆): d 9.5l(s,

1H, N-H), 8.84 (s, 1H, Ar-H), 8.46 (d, J= 5.6 Hz, 1H, Ar-H), 7.79-7.87 (m, 2H, Ar-H), 7.75 (d, J= 8.1 Hz, 1H, Ar-H), 7.21-7.29 (m, 1H, Ar-H), 6.01-6.10 (m, 2H, CH), 4.25 (m, 5H,

CH, OH), 4.00-4.09 (m, 2H, CH), 3.15 (t, J= 6.0 Hz, 2H,CH₂), 2.54 (s, 1H, OH).

11.5. Synthesis of 4-((2-(((l-((3S,4S)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2- yl)- 2-oxo-l,2-dihydropyrimidin-4-yl)carbamoyl)oxy)ethyl)disulfaneyl)pentanoic acid (5): To a solution of 4 (5 mg, 0.011 mmol) and 4-mercaptopentanoic acid 1 (2 pL, 0.012 mmol) in MeOH (5 mL) was added acetic acid (10 pL). The reaction mixture was stirred at room temperature for l6h. The solvent was removed in vacuum and the crude product was purified by using prep HPLC and eluted with 30% to 90% methanol, to obtain compound 4 as white solid (Yield

9.32(s, 1H, N-H), 8.55 (s, 1H, Ar-H), 7.82 (d, J= 7.8 Hz, 1H, Ar-H), 6.0-6.08 (m, 2H, CH), 5.75 (bs, 1H, OH), 4.28-4.37 (m, 4H, CH, OH), 4.00-4.08 (m, 2H, CH₂), 3.59 (s, 1H, CH), 2.89-3.00 (m, 2H, CH₂), 2.54 (s, 1H, CH), 2.42 (t, J= 7.4 Hz, 1H, CH₂), 2.33 (t, J= 7.6 Hz, 1H, CH₂), 1.66-

I.90 (m, 2H, CH₂), 1.25 (d, J = 6.7 Hz, 3H, CH₃).

II.6. Synthesis of 5-((4-((2-(((l-((3S,4S)-3,4-dihydroxy-5-(hydroxymethyl)

tetrahydrofuran-2-yl)-2-oxo-l,2-dihydropyrimidin-4-yl)carbamoyl)oxy)ethyl)disulfaneyl) pentanamido)methyl)-2-((E)-2-((E)-3-(2-((E)-3,3-dimethyl-l-octadecylindolin-2-ylidene) ethyUdene)-2-methylcyclohex- I-en- 1-yl) vinyl)-3, 3-dimethyl- 1 -octadecyl-3 H-indol- 1 -ium (6):

A mixture of DIR-methyl amine (10) (5mg, 0.0052 mmol, 1 eq.), Compound 5 (5 mg, 0.0077 mmol, 1.5 eq.), HBTU (5.2 mg, 0.015 mmol, 3 eq.) and DIEA (4.6 mΐ, 0.015 mmol, 3 eq.) were stirred in DMF at room temperature for l2h. The solvent was then evaporated under reduced pressure and the resulting dark pink residue was purified by using prep HPLC and eluted with 30% to 100% methanol, to obtain DIR-SS-cytarabine (6) as a dark green solid (Yield 43%). MALDI-TOF (DHB matrix) Calculated M/z = 1429.9621, Found M/z = 1430.2083.

11.7. Synthesis of 2-(pyridin-2-yldisulfaneyl)ethyl (( 2S,3S,4S,6R)-6-(((lS,3S)-3-acetyl - 3,5, 12-trihydroxy- 10-niethoxy-6, 11-dioxo- 1 ,2,3,4,6, 11 -liexahydrotetracen- 1 -yi)oxy)-3- hydroxy-2-methyltetrahydro-2H-pyran-4-yl)carbamate (7):

A mixture of daunorubicin hydrochloride (10 mg, 0.036 mmol, 1 eq.), compound 3 (15.1 mg, 5.4 mmol, 1.5 eq.) and DIEA (19 pL, 0.11 mmol, 3 eq.) were dissolved in methylene chloride (5 mL) and stirred at room temperature for 2 hour. The solvent was removed in vacuum and the crude product was purified by using prep HPLC and eluted with 30% to 90% methanol, to obtain compound 4 as white solid (yield 63%). ^XH NMR (400 MHz, CDCl₃): d 13.98 (s, 1H, OH), 13.27 (s,lH, OH), 8.63 (s, IH, NH), 8.03 (d, = 7.6 Hz, 1H, Ar-H), 9.94 (d, J= 4.4 Hz, 2H, Ar-H), 7.78 (t, J= 8.1 Hz, 1H, Ar-H), 7.32-7.42 (m, 3H, Ar-H), 5.55 (m, 1H), 5.28 (m, lH),4.97 (m, 1H), 4.13-4.32 (m, 3H),4.08 (s, 3H, CH₃), 3.97-4.05 (m, 1H), 3.18-3.27 (m,

1H), 3.14 (t, J= 5.6 Hz, 2H, CH₂), 2.97-3.04 (m, 1H), 2.96 (s, 1H), 2.91 (s, 1H), 2.76 (s,lH), 2.41 (s, 3H, CH₃), 2.26-2.34 (m, 1H), 2.10-2.18 (m, 1H), 1.81-1.90 (m, 1H), 1.22 (t, J= 6.4 Hz, 3H, CH₃). ESI-MS Calculated M/z = 740.10, Found M/z = 74l. l[M+H] 11.8. Synthesis of 4-((2-((((2S,3S,4S,6R)-6-(((lS,3S)-3-acetyl-3,5,12-trihydroxy-10- methoxy-6,11-dioxo- 1,2, 3, 4, 6,11-hexahydrotetracen- l-yl)oxy)-3-hydroxy-2-methyl tetrahydro-2H-pyran-4-yl)carbamoyl)oxy)ethyl)disulfaneyl)pentanoic acid (8):

To a solution of 7 (5mg, 0.011 mmol) and 4-mercaptopentanoic acid 1 (2 pL, 0.012 mmol) in MeOH (5 mL) was added acetic acid (10 pL). The reaction mixture was stirred at room temperature for l6h. The solvent was removed in vacuum and the crude product was purified by using prep HPLC and eluted with 30% to 90% methanol, to obtain compound 4 as white solid (yield 51%).

11.9. Synthesis of 5-((4-((2-((((2S,3S,4S,6R)-6-(((lS,3S)-3-acetyl-3,5,12-trihydroxy-10- niethoxy-6, 11-dioxo- 1 ,2, 3, 4, 6, 11-hexahydrotetracen- 1 -yl)oxy)-3-hydroxy- 2- methyltetrahydro-2H-pyran-4-yl)carbamoyl)oxy)ethyl)disulfaneyl)pentanamido)methyl)-2- ((E)-2-((E)-3-(2-((E)-3,3-dimethyl-l-octadecylindolin-2-ylidene)ethylidene)-2- niethylcyclohex- 1-en-l-yl) vinyl)-3, 3-dimethyl- 1 -octadecyl-3 H-indol- 1 -ium (DIR-SS- Daun oruhicin ) (9 ):

A mixture of DIR-methyl amine (10) (5 mg, 0.0052 mmol, 1 eq.), Compound 8 (5 mg,

0.0077 mmol, 1.5 eq.), HBTU (5.2 mg, 0.015 mmol, 3 eq.) and DIEA (4.6 pl, 0.015 mmol, 3 eq.) were stirred in DMF at room temperature for l2h. The solvent was then evaporated under reduced pressure and the resulting dark pink residue was purified by using prep HPLC and eluted with 30% to 100% methanol, to obtain DIR-SS-daunorubicin (9) as a dark green solid.

Example 12: Indocyanine lipids exhibit highly efficient extravasation and can be used for drug delivery

In order to compare extravasation efficiency of indocyanine lipids and phospholipids, 2mol% indocyanine lipid Dil or 2% phospholipid lissamine rhodamine-PE (FIG. 21A) were formulated into serum-stable hydrogenated soy PC (HSPC)/Chol/DSPE-PEG2ooo liposomes (similar composition to DOXIL). Both liposomes had the same hydrodynamic diameter (~90nm) and negative zeta potential (-25mV) as DOXIL (FIG. 21B). Seven days post injection, mouse capillaries showed accumulation of both types of liposomes. However, in the case of Dil there was 8 times more extravasation and penetration into the tissue (FIGs. 21C-21D)

Further, to test whether indocyanine lipids can be used as carriers, amino methyl derivative of Dil were prepared and covalently conjugated to a NIR dye DyLight800 (FIG. 21E, Smith et al, 2018, Biomaterials 161 :57-68). This conjugate was formulated into stealth HSPC/CI10I/DSPE-PEG2000 liposomes. Liposomal DyLight-Dil showed 25h half-life versus <5 min as free molecules (FIG. 21F). There was a highly efficient accumulation of liposomal DyLight-Dil (FIG. 21 G), as well as efficient extravasation, as detected in freshly excised skin flaps (FIG. 21H).

Example 13: Silibinin formulation with Indocyanine lipids

In order to test whether silibinin can be formulated with indocyanine lipids (as a free drug), DiI-PEG34oo derivative of amino methyl Dil was synthesized (FIG. 23). DiI-PEG34oo was mixed with silibinin (weight ratio 1: 1; molar ratio 1 : 8.8) in ethanol and then diluted in PBS to 5% ethanol (final silibinin concentration 2 mg/mL). The Dil-PEG formulation with silibinin formed colloidally stable clear solution, whereas silibinin alone formed visibly turbid aggregation (FIG. 22). These data demonstrate that indocyanine lipids can formulate silibinin with up to 50% loading capacity. For comparison, DOXIL has a weight ratio of doxorubicin: total lipid 1 :8, or 12.5% doxorubicin loading capacity.

Further, a non-limiting synthesis of silibinin-lipid conjugates and formulations is shown in FIG. 23B. Same scheme can be applied to other drug compounds, as would be contemplated by one skilled in the art.

Example 14: Dexamethasone formulation with Indocyanine lipids in PBS

In order to test whether Dexamethasone can be formulated with indocyanine (DiR) lipids, Dexamethasone was dissolved in DMSO to final concentration of 20 mg/ml. 10 ul Dexamethasone dissolved in DMSO was then mixed with 10 ul of lOmM DIR-PEG-MTZ in DMSO. To the solution of dexamethasone and DIR-PEG-MTZ , 100 ul PBS was added. Free Dexamethasone as well as formulation with DSPE-PEG were used as controls. Free

Dexamethasone as well as formulation with DSPE-PEG precipitated from the solution, whereas Dexamethasone formulation with DiR-PEG-MTZ remained suspended and clear FIG 24 A. Both free Dexamethasone and Dexamethasone formulated with DSPE- PEG showed aggregates and crystals in phase contrast microscopy. Dexamethasone formulated with DiR-PEG-MTZ showed minimal aggregation as shown in FIG. 24B.

Table 1. Area under the curve (AUC) of different cargoes (in arbitrary units)

Values were calculated using corresponding blood elimination profiles (FIGs. 3D, 4F, 5C) using Prism software.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention can be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

What is claimed is:

1 A construct comprising a lipophilic membrane dye,

wherein the lipophilic membrane dye is covalently linked through a linker to a cargo selected from the group consisting of a therapeutic drug, nucleic acid, polypeptide, enzyme, antibody, ligand, biologically active lipid, dye (or chromophore), fluorophore, bioluminescent label, biosensor, contrast agent, radioisotope, hydrophilic polymer, hydrophilic copolymer, and chemiluminescent label,

wherein the lipophilic membrane dye comprises the compound of formula (I) or formula (II), or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof:

wherein:

each occurrence of R⁵ is independently selected from the group consisting of Ci-C₆ alkyl, C₃-C₈ cycloalkyl, halo, -N0₂, -CN, -OR^5a, -NR^5aR^5a, -NR^5a-C(=0)R^5a, -NR^5a-S0₂R^5a, - C(=0)0R^5a, -C(=0)NR^5aR^5a, -SR^5a, and -S(=0)i_-2(Ci-C₆ alkyl), wherein each occurrence of R^5a is independently selected from the group consisting of H and Ci-C₆ alkyl;

R⁹ is selected from the group consisting ofH and S0₃H;

m is 0, 1, 2, 3, 4, 5, or 6;

n is 0, 1, or 2;

p is 0, 1, or 2;

2. The construct of claim 1, wherein R¹ and R² are independently selected from the group consisting of C_l8 acyl, C₂₀ acyl, C₂₂ acyl, C₂₄ acyl, C_l8 alkyl, C₂₀ alkyl, C₂₂ alkyl, C₂₄ alkyl, Ci₈ alkenyl, C₂o alkenyl, C₂₂ alkenyl, C₂₄ alkenyl, Ci₈ alkynyl, C₂o alkynyl, C₂₂ alkynyl, and C₂₄ alkynyl.

3. The construct of claim 1, wherein the phospholipid comprises

4. The construct of claim 1, wherein m is 0 or 1.

5. The construct of claim 1, wherein R³¹, R^3u, R^3m, and R^3lv are each methyl.

6 The construct of claim 1, wherein in (I): (a) the linker is attached directly to the phenyl ring of at least one indolinyl group, to a R⁴ group, and/or to a R⁵ group, and/or

(b) the linker is attached to R¹ and/or R².

7. The construct of claim 1, wherein in (II):

(a) the linker is attached directly to the phenyl ring of at least one indolinyl group, to a R⁹ group, and/or to a R¹⁰ group, and/or

(b) the linker is attached to R⁶ and/or R⁷.

8. The construct of claim 1, wherein the compound of formula (I) comprises the compound of formula (III), or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof:

wherein

p is 1 or 2;

or -CRR-; and

Y comprises the linker conjugated to the cargo.

9. The construct of claim 1, wherein the compound of formula (II) comprises the compound of formula (IV), or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof:

10. The construct of claim 1, wherein the compound of formula (II) comprises the compound of formula (V), or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof:

wherein A comprises the linker conjugated to the cargo.

11. The construct of claim 1, wherein the linker comprises a disulfide linker.

12 The construct of claim 11, wherein the disulfide linker comprises

13. The construct of claim 1, wherein the linker comprises at least one -OCH₂CH₂- group.

14. The construct of claim 13, wherein the linker comprises from 1 to about 5,000 - OCH₂CH₂- groups.

15. The construct of claim 1, wherein the linker comprises formula (A), (B) or (C):

*-(CH₂)_ml-X₁-(CH₂-CH₂-X₂)_m2-(CH₂)_m3-C(X₃)-

(A)

*-(CH₂)_ml-0-(CH₂-CH₂-0)_m2-(CH₂)m₃-C(0)-

(B)

*-(CHR')_mi-0-(CHR'-CHR'-0)_m2-(CHR')_m3-C(0)- (C)

wherein:

each Xi, X₂, and X₃ is independently absent (a bond), O, or N-R';

each R' is independently selected from the group consisting of hydrogen, optionally substituted Ci-C₆ alkyl, optionally substituted C₃-C₈ cycloalkyl, and optionally substituted C3-C8 cycloheteroalkyl.

16. The construct of claim 1, wherein the linker is conjugated to the cargo through a 3- thio-succinimido group.

17. The construct of claim 1, wherein the cargo is further covalently linked through an independently selected additional linker to at least one other independently selected lipophilic membrane dye of formula (I) or (II), or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof.

18. The construct of claim 17, wherein the additional linker comprises a disulfide linker.

19. The construct of claim 17, wherein the additional linker comprises formula (A), (B) or (C):

*-(CH₂)_ml-X₁-(CH₂-CH₂-X₂)_m2-(CH₂)_m3-C(X3)-

(A)

*-(CH₂)_ml-0-(CH₂-CH₂-0)_m2-(CH₂)m₃-C(0)-

(B)

*-(CHR')_mi-0-(CHR'-CHR'-0)_m2-(CHR')_m3-C(0)-

(C)

wherein:

each ml, m2, and m3 is independently an integer ranging from 0-5000;

each Xi, X₂, and X₃ is independently absent (a bond), O, or N-R';

each R' is independently selected from the group consisting of hydrogen, optionally substituted CVG, alkyl, optionally substituted C3-C8 cycloalkyl, and optionally substituted C3-C8 cycloheteroalkyl.

20. The construct of claim 17, wherein the cargo is enzymatically cleavable, whereby upon cleavage of the cargo the at least two lipophilic membrane dyes are not covalently connected with each other.

21. The construct of claim 1, wherein the hydrophilic polymer is at least one selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone, polyethylenimine, polymethacrylate, and polyvinyl alcohol.

22. The construct of claim 1, wherein the hydrophilic copolymer comprises at least one polymer selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone, polyethylenimine, polymethacrylate, and polyvinyl alcohol, or any copolymer thereof.

23. A composition comprising the construct of claim 1 and at least one cell, wherein at least a fraction of the construct is incorporated into the membrane of the cell.

24. The composition of claim 23, wherein at least a fraction of the lipophilic membrane dye of the construct inserts into the membrane of the cell.

25. The composition of claim 23, wherein the cell is a red blood cell.

26. The composition of claim 23, wherein the cell does not undergo significant lysis upon incorporation of the construct into the membrane of the cell.

27. The composition of claim 23, further comprising at least one pharmaceutically acceptable carrier.

28. The composition of claim 23, which is formulated for administration by a route selected from the group consisting of oral, parenteral, transdermal, transmucosal, intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical.

29. A method of delivering a cargo to a cell, the method comprising contacting the construct of claim 1 with a cell under conditions whereby at least a fraction of the lipophilic membrane dye of the construct inserts into the membrane of the cell.

30. The method of claim 29, wherein the cell is a red blood cell.

31. The method of claim 29, wherein the cell does not undergo significant lysis upon insertion of the lipophilic membrane dye of the construct into the membrane of the cell.

32. A method of delivering a cargo to a subject in vivo, wherein the cargo is selected from the group consisting of a therapeutic drug, nucleic acid, polypeptide, enzyme, antibody, ligand, biologically active lipid, dye (or chromophore), fluorophore, bioluminescent label, biosensor, contrast agent, radioisotope, hydrophilic polymer, hydrophilic copolymer, and chemiluminescent label, wherein the method comprises administering to the subject a composition comprising the construct of claim 1 and at least one cell, wherein at least a fraction of the construct is incorporated into the membrane of the cell.

33. The method of claim 32, wherein the administering is by a route selected from the group consisting of oral, parenteral, transdermal, transmucosal, intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra arterial, intravenous, intrabronchial, inhalation, and topical.

34. The method of claim 32, wherein the cargo has a higher circulating half-life in the subject as compared to when the cargo, which is not part of the construct, is administered to the subject.

35. The method of claim 32, wherein the cargo comprises a therapeutically effective agent, thereby treating or preventing a disease or disorder in the subject.

36. The method of claim 32, wherein the cargo is covalently linked through

independently selected linkers to at least two independently selected lipophilic membrane dyes of formula (I) or formula (II), or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof.

37. The method of claim 36, wherein the cargo is enzymatically cleavable in vivo, whereby upon cleavage of the cargo the at least two lipophilic membrane dyes are not covalently connected with each other.

38. The method of claim 32, wherein the subject is further administered at least one additional therapeutically effective agent.

39. The method of claim 32, wherein the subject is a mammal.

40. The method of claim 39, wherein the mammal is a human.

41. A lipophilic membrane dye of formula (IIA), or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof:

wherein:

R is selected from the group consisting of H, -CH₃, and -CH₂R¹⁰;

R⁹ is selected from the group consisting ofH and S0₃H;

42. A compound comprising a linker (L) and a lipophilic membrane dye of formula (I) or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof;

wherein in (I):

R¹ is selected from the group consisting of C14-C22 alkyl, C14-C22 alkenyl, C14-C22 alkynyl, and C14-C28 acyl;

R² is selected from the group consisting of C14-C22 alkyl, C14-C22 alkenyl, C14-C22 alkynyl, and C14-C28 acyl;

m is 0, 1, 2, 3, 4, 5, or 6;

n is 0, 1, or 2;

p is 0, 1, or 2; and

wherein L is:

(b) attached to R¹ and/or R²;

43. The compound of claim 42, wherein the linker comprises a disulfide linker.

44. The compound of claim 41, wherein the linker comprises formula (A), (B) or (C)

*-(CH₂)_ml-X_l-(CH₂-CH₂-X₂)_m2-(CH₂)_m3-C(X₃)- (A)

*-(CH₂)_ml-0-(CH₂-CH₂-0)_m2-(CH₂)rn₃-C(0)-

(B)

*-(CHR')_mi-0-(CHR'-CHR'-0)_m2-(CHR')_m3-C(0)-

(C)

wherein:

* indicates the bond between the linker and the lipophilic membrane dye of formula each ml, m2, and m3 is independently an integer ranging from 0-5000;

each Xi, X₂, and X₃ is independently absent (a bond), O, or N-R';

45. A compound comprising a linker (L) and a lipophilic membrane dye of formula (II) or a salt, solvate, enantiomer, diastereoisomer, tautomer, or geometric isomer thereof;

wherein in (II):

R⁹ is selected from the group consisting ofH and S0₃H;

m is 0, 1, 2, 3, 4, 5, or 6;

n is 0, 1, or 2;

p is 0, 1, or 2; and wherein L is:

(a) atached directly to the phenyl ring of at least one indolinyl group, to a R⁹ group, and/or to a R¹⁰ group, and/or

(b) atached to R⁶ and/or R⁷;

46. The compound of claim 45, wherein the linker comprises a disulfide linker.

47. The compound of claim 45, wherein the linker comprises formula (A), (B) or (C)

*-(CH₂)_ml-X₁-(CH₂-CH₂-X₂)_m2-(CH₂)_m3-C(X₃)-

(A)

*-(CH₂)_mi-0-(CH₂-CH₂-0)_m2-(CH₂)m₃-C(0)-

(B)

*-(CHR')_mi-0-(CHR'-CHR'-0)_m2.(CHR')_m3-C(0)-

(C)

wherein:

* indicates the bond between the linker and the lipophilic membrane dye of formula (II);

each ml, m2, and m3 is independently an integer ranging from 0-5000;

each Xi, X₂, and X₃ is independently absent (a bond), O, or N-R';

48. The compound of claim 45, wherein the linker comprises a hydrophilic polymer or copolymer selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone, polyethylenimine, polymethacrylate, polyvinyl alcohol.

49. A method of solubilizing a compound in an aqueous solution, wherein the method comprises contacting the compound with at least one agent selected from the group consisting of the construct of claim 1, the compound of claim 42, and the compound of claim 45, wherein at least one applies: (a) the cargo of the construct of claim 1 comprises a hydrophilic polymer or copolymer, (b) the linker in the construct of claim 1, the compound of claim 42, or the compound of claim 45 comprises a hydrophilic polymer or copolymer.

50. The method of claim 49, wherein the hydrophilic polymer or copolymer is selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone, polyethylenimine, polymethacrylate, polyvinyl alcohol, or any copolymers thereof.

51. The method of claim 49, wherein the linker further comprises a disulfide linkage.

52. A method of enhancing the endothelial membrane crossing of a cargo in a subject, the method comprising administering to the subject a composition comprising the construct of claim 1, wherein the construct comprises the cargo.