Selective delivery molecules and methods of use
Disclosed herein is a selective delivery molecule comprising: (a) an acidic sequence (portion A) which is effective to inhibit or prevent the uptake into cells or tissue retention, (b) a molecular transport or retention sequence (portion B), and (c) a linker between portion A and portion B, and (d) at least one cargo moiety.
1. A method of preparing a selective delivery molecule comprising:
a) incubating a peptide according to Peptide P-3 in a solution comprising a first fluorescent moiety for a first time period sufficient to generate a first intermediate, wherein Peptide P-3 has the structure:
b) to the first intermediate in the solution, adding a second fluorescent moiety and incubating the second fluorescent moiety for a second time period sufficient to generate a second intermediate;
c) processing the second intermediate by a chromatographic means to generate an isolated second intermediate; and
d) incubating the isolated second intermediate with a PEG polymer for a third time period sufficient to generate the selective delivery molecule.
2. The method of claim 1 , wherein the first fluorescent moiety is a donor fluorescent moiety.
3. The method of claim 2 , wherein the second fluorescent moiety is an acceptor fluorescent moiety.
4. The method of claim 3 , wherein the acceptor and donor fluorescent moieties that are capable of undergoing Försters/fluorescence resonance energy transfer with the other.
5. The method of claim 3 , wherein the acceptor and donor fluorescent moieties are selected from: Cy5 and Cy7, Cy5 and IRDye750, Cy5 and IRDye800, and Cy5 and ICG.
6. The method of claim 2 , wherein the donor fluorescent moiety is Cy5.
7. The method of claim 3 , wherein the acceptor fluorescent moiety is Cy7.
8. The method of claim 1 , wherein the PEG polymer has an average molecular weight of about 500 Da, 2 kDa, 5 kDa, or 10 kDa.
9. The method of claim 1 , wherein the PEG polymer has an average molecular weight of about 2 kDa.
10. The method of claim 1 , wherein the PEG polymer is in a solution comprising glycine buffer.
11. The method of claim 10 , wherein the glycine buffer is at pH 3.
12. The method of claim 10 , wherein the glycine buffer is at 0.1M final concentration.
13. The method of claim 10 , wherein the solution further comprises aniline.
14. The method of claim 13 , wherein the aniline is at 20 mM final concentration.
15. The method of claim 1 , wherein the first intermediate has the structure:
16. The method of claim 1 , wherein the second intermediate has the structure:
17. The method of claim 1 , wherein each of the incubating steps is carried out at room temperature.
18. The method of claim 1 , wherein each of the incubating steps is carried out in the dark.
19. The method of claim 1 , wherein the first intermediate is purified prior to incubating with the second fluorescent moiety.
20. The method of claim 1 , wherein the chromatographic means is a reverse phase chromatography.