# US 9,872,926 B2 · Multicolored pH-activatable fluorescence nanoplatform

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- Number: US 9,872,926 B2
- Document ID: US9872926B2
- Status: Granted
- Kind: B2
- Application number: 15/297,862
- Filed: 2016-10-19
- Published: 2017-02-23
- Granted: 2018-01-23
- Earliest claimed: 2012-04-05
- Assignees: Board of Regents, The University of Texas System
- Applicants: The Board of Regents of the University of Texas System
- Inventors: Jinming Gao; Kejin Zhou; Baran D. Sumer
- Web page: https://patentgrep.com/patent/US9872926B2

## CPC

- A61K 49/0054: {Macromolecular compounds, i.e. oligomers, polymers, dendrimers}
- A61K 49/0032: {Methine dyes, e.g. cyanine dyes}
- A61K 49/0082: {micelle, e.g. phospholipidic micelle and polymeric micelle}
- G01N 21/6486: {Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence)}
- G01N 33/502: {for testing non-proliferative effects}
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present invention relates to pH-tunable, highly activatable multicolored fluorescent nanoplatforms and methods of using the nanoplatforms in a variety of applications including, but not limited to, investigating fundamental cell physiological processes such as pH regulation in endocytic vesicles, endosome/lysosome maturation, and effect of pH on receptor cycling and trafficking of subcellular organelles.

## Claims

### Claim 1

A method of monitoring vesicular trafficking comprising:
(i) contacting a cell with a composition comprising at least a first and second micelle population, wherein each micelle population comprises a block copolymer and a fluorescent dye, and further wherein the first micelle population has a first pH transition point and a first fluorescent emission spectra, the second micelle population has a second pH transition point and a second fluorescent emission spectra, and further wherein the first and second pH transition points are not identical and the first and second fluorescent dyes are not identical, under conditions whereby the cell takes up the composition by endocytosis;
wherein the first micelle population and the second micelle population are each independently a copolymer comprising
a) a hydrophilic polymer segment according to formula:
b) a hydrophobic polymer segment according to formula A1; and
c) a polymer segment according to formula A2;
or a pharmaceutically acceptable salt thereof; and stereoisomers, isotopic variants and tautomers thereof;
wherein
each A1 and A2 is independently selected from
each R1a is independently H, substituted or unsubstituted alkyl, or
each R2a, R2b, R3a, R3b, R4a, R4b, R4c, R4d, and R5 is independently H or substituted or unsubstituted alkyl;
each R6a , and R6b is independently substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R6a, and R6b taken together with the N atom they are attached to form heterocycle;
R7 is a moiety comprising a dye selected from the group consisting of rhodamine, BODIPY, coumarin, and cyanine;
the subscript n is an integer between 10 to 200;
and wherein the copolymer is a random copolymer;
(ii) detecting in the cell a first and/or second fluorescent signal corresponding to the first, and/or second fluorescent emission spectra, wherein the detection of first and/or second fluorescent signals indicates that each micelle population comprising a corresponding fluorescent dye has reached its pH transition point and that the micelle population has disassociated; and
(iii) determining what compartment the endocytosed micelle population was in when it dissociated based on the pH transition point of the micelle.

### Claim 2 (depends on 1)

The method of claim 1, wherein the A1 has the formula:

### Claim 3 (depends on 1)

The method of claim 1, wherein R7 is a moiety comprising a cyanine dye.

### Claim 4 (depends on 1)

The method of claim 1, wherein the A2 has the formula:

### Claim 5

A method of monitoring vesicular trafficking comprising:
(i) contacting a cell with a composition comprising at least a first, second, and third micelle population, wherein each micelle population comprises a block copolymer and a fluorescent dye, and further wherein the first micelle population has a pH transition point between about pH 6.3-7.4 and a first fluorescent emission spectra, the second micelle population has a pH transition point between about pH 5.9-6.2 and a second fluorescent emission spectra, and the third micelle population has a pH transition point between about pH 5.0-5.8 and a third fluorescent emission spectra, under conditions whereby the cell takes up the composition by endocytosis;
wherein the first micelle population, the second micelle population, and the third micelle population are each independently a copolymer comprising
a) a hydrophilic polymer segment according to formula:
b) a hydrophobic polymer segment according to formula A1; and
c) a polymer segment according to formula A2;
or a pharmaceutically acceptable salt thereof; and stereoisomers, isotopic variants and tautomers thereof;
wherein
each A1 and A2 is independently selected from
each R1a is independently H, substituted or unsubstituted alkyl, or
each R2a, R2b, R3a, R3b, R4a, R4b, R4c, R4d, and R5 is independently H or substituted or unsubstituted alkyl;
each R6a, and R6b is independently substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R6a, and R6b taken together with the N atom they are attached to form heterocycle;
R7 is a moiety comprising a dye selected from the group consisting of rhodamine, BODIPY, coumarin, and cyanine;
the subscript n is an integer between 10 to 200;
and wherein the copolymer is a random copolymer;
(ii) detecting in the cell a first, second, and third fluorescent signal corresponding to the first, second, and third fluorescent emission spectra, wherein the detection of first, second, and third fluorescent signals indicates that each micelle population comprising a corresponding fluorescent dye has reached its pH transition point and that the micelle population has disassociated; and
(iii) determining that a vesicle is an early endosome when the first fluorescent signal is detected, determining that a vesicle is a late endosome/lysosome when the second fluorescent signal is detected, and determining that a vesicle is a lysosome when the third fluorescent signal is detected.

### Claim 6 (depends on 5)

The method of claim 5, wherein the A1 has the formula:

### Claim 7 (depends on 5)

The method of claim 5, wherein R7 is a moiety comprising a cyanine dye.

### Claim 8 (depends on 5)

The method of claim 5, wherein the A2 has the formula:

### Claim 9

A method of monitoring endosome pH comprising:
(i) contacting an endosome with a composition comprising at least a first and second micelle population, wherein each micelle population comprises a block copolymer and a fluorescent dye, and further wherein the first micelle population has a pH transition point between about pH 5.7-6.3 and a first fluorescent emission spectra, the second micelle population has a pH transition point between about pH 5.7-6.3 and a second fluorescent emission spectra, wherein the first and second micelle populations have different pH transition points and different fluorescent emission spectra, under conditions whereby the endosome takes up the composition;
wherein the first micelle population and the second micelle population are each independently a copolymer comprising
a) a hydrophilic polymer segment according to formula:
b) a hydrophobic polymer segment according to formula A1; and
c) a polymer segment according to formula A2;
or a pharmaceutically acceptable salt thereof; and stereoisomers, isotopic variants and tautomers thereof;
wherein
each A1 and A2 is independently selected from
each R1a is independently H, substituted or unsubstituted alkyl, or
each R2a, R2b, R3a, R3b, R4a, R4b,R4c, R4d, and R5 is independently H or substituted or unsubstituted alkyl;
each R6a , and R6b is independently substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R6a, and R6b taken together with the N atom they are attached to form heterocycle;
R7 is a moiety comprising a dye selected from the group consisting of rhodamine, BODIPY, coumarin, and cyanine;
the subscript n is an integer between 10 to 200;
and wherein the copolymer is a random copolymer;
(ii) detecting in the endosome at least one of a first and second fluorescent signal corresponding to the first and second fluorescent emission spectra, wherein the detection of first and/or second fluorescent signals indicates that each micelle population comprising a corresponding fluorescent dye has reached its pH transition point and that the micelle population has disassociated; and
(iii) determining a pH or pH range for the endosome.

### Claim 10 (depends on 9)

The method of claim 9, wherein the A1 has the formula:

### Claim 11 (depends on 9)

The method of claim 9, wherein R7 is a moiety comprising a cyanine dye.

### Claim 12 (depends on 9)

The method of claim 9, wherein the A2 has the formula: