Synthesis and Characterization of POSH-KKP2 Micelles

Example 1

POSH, TAT-POSH, and POSH-KKP2 Synthesis

POSH and TAT-POSH peptides were synthesized by Fmoc solid phase synthesis and purified by high pressure liquid chromatography. An ε-lysyllysine residue was added to the C-terminus of POSH through which two hydrophobic aliphatic palmitic acid moieties were conjugated, yielding the amphiphile POSH-KKP₂.

Micelle Formation and Characterization

POSH-KKP2 was solubilized in water and micelle formation and morphology were assessed by previously established characterization techniques. The capacity for POSH-KKP2 to self-assemble into micelles was evaluated by a critical micelle concentration (CMC) assay where 1,6-diphenylthexatriene greatly increases in fluorescence intensity when trapped within the micelle core. POSH-KKP₂ amphiphiles readily formed micelles as indicated by a very low CMC of 0.18 μM as shown in FIG. 2.

Transition electron microscopy (TEM) revealed that POSH-KKP₂ self-assembled into micelles (FIG. 3) of which most were spheres (diameter—9.7±1.9 nm) though some were short cylinders (length—21.0±6.2 nm). As shown in FIG. 3, POSH-KKP2 amphiphile solutions (20 μM) were incubated on copper grids and the solution rapidly wicked away. Grids were negatively stained with an aqueous Nano-Tungsten solution and then observed using a transmission electron microscope (TEM). Micrographs were taken at 15,000× (left) and 45,000× (right) magnification and have scale bars of 100 nm and 20 nm, respectively, which showed the presence of mostly spherical micelles and some short cylindrical micelles. TAT-POSH and POSH-KKP₂ were incubated at 37° C. for 24 hours with different leukemic cells (LM-138, Mec-1, and Mec-2) at a variety of doses (1.25, 2.5, 5, 10, and 20 μM) after which therapeutic cytotoxicity was evaluated by flow cytometry. The results are shown in FIG. 4.

Results

When leukemic cells (LM-138) were exposed to TAT-POSH or POSH-KKP₂, dose dependent cytotoxicity was observed for both products (FIG. 4) though POSH-KKP₂ outperformed TAT-POSH at every dose. LM138 mouse proB leukemic cells transduced to overexpress BCR-ABL were cultured and 10⁶ cells were incubated with varying concentrations of TAT-POSH or POSH-KKP₂ micelles at 37° C. for 24 hours. Cell death was assessed by flow cytometry and experimental groups were standardized against cells exposed to no stimulus (Control).

The capacity for POSH-KKP₂ micelles to enhance cytotoxicity over a gold-standard intracellular delivery system like TAT-POSH is an exciting and surprising discovery. Even more remarkable is that POSH-KKP₂ micelles induced similar cell death (˜37% v. ˜39%) at a sixteenth of the dose of TAT-POSH (1.5 μM v. 2.0 μM). Additionally, the highly hydrophilic and charged nature of the POSH peptide facilitated the formation of small POSH-KKP₂ micelles which will allow for their future intravenous delivery and in vivo evaluation.

Example 2

The results of Example 1 are consistent with results generated with human B cell leukemia Mec-1 and Mec-2 cells as discussed herein. When peptide amphiphiles comprised of hydrophilic CD19-aptamer-Tat-POSH peptide and a hydrophobic moiety were exposed to water they self-assembled into spherical or cylindrical micelles by hiding their hydrophobic moieties in their core and displaying their hydrophilic aptamer-peptides on their surface. As shown in FIG. 3, an electron micrograph of the POSH-KKP2 micelles confirmed their spherical shape. 2× serial dilutions of POSH-KKP2 micelles (20 μM starting concentration) were incubated with murine Bcr/Abl+ BCP-ALL LM138, Human B-CLL Mec1 and Mec2 cells for 24 hours in vitro. Percent survival was measured by flow cytometry (7AAD, active caspase-3). Curves (n=3 for each) were analyzed by non-linear regression. F test analyses of Tat-POSH vs. of POSH-KKP₂. p<0.001 (Prism by GraphPad). The results are shown in FIG. 4 (P2K means POSH-KKP2).

From the foregoing it will be seen that this invention is one well adapted to attain all ends and objectives herein-above set forth, together with the other advantages which are obvious and which are inherent to the invention.

Since many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matters herein set forth or shown in the accompanying drawings are to be interpreted as illustrative, and not in a limiting sense.

While specific embodiments have been shown and discussed, various modifications may of course be made, and the invention is not limited to the specific forms or arrangement of parts and steps described herein, except insofar as such limitations are included in the following claims. Further, it will be understood that certain features and subcombinations are of utility and may be employed without reference to other features and subcombinations. This is contemplated by and is within the scope of the claims.