Systemicin vivodistribution of activatable cell penetrating peptides is superior to that of cell penetrating peptides

Abstract

Cell penetrating peptides (CPPs) have been developed as vehicles for payload delivery into cells in culture and in animals. However several biologic features limit their usefulness in living animals. Activatable cell penetrating peptides (ACPPs) are polycationic CPPs whose adsorption and cellular uptake is minimized by a covalently attached polyanionic inhibitory domain. Cleavage of the linker connecting the polyanionic and polycationic domains by specific proteases (tumor associated matrix metalloproteases discussed herein) dissociates the polyanion and enables the cleaved ACPP to enter cells. In contrast to CPP counterpart, ACPPs are relatively nonadherent and distributed uniformly to normal tissues. While nonaarginine (r 9 ) CPP administered intravenously into mice initially bind to the local vasculature and redistribute to the liver, where > 90% of the injected dose accumulates 30 min after injection. Regardless of the presence of the polyanionic inhibitory domain, confocal imaging of live tissues reveals that the majority of the ACPP and CPP remain in punctate organelles, presumably endosomes. Therefore further improvements in the efficiency of delivery to the cytosol and nucleus are necessary. In addition to improved target specificity, a major advantage of ACPPs over CPPs for potential clinical applications is reduced toxicity. Systemically administered r 9 CPP causes acute toxicity in mice at a dose 4 fold lower than the MMP cleavable ACPP, a complication not observed with an uncleavable ACPP presumably because the polycationic charge remains masked systemically. These data suggest that ACPPs have greater potential than CPPs for systemic delivery of imaging and therapeutic agents.

Surgery with molecular fluorescence imaging using activatable cell-penetrating peptides decreases residual cancer and improves survival

“…To test whether ACPPs are taken up differentially between tumor and normal tissue, we examined four tumor models: isografts derived from spontaneous mammary adenocarcinoma tumors in transgenic mice (line 8119, MMTV-PyMT) (11,12), the murine melanoma cell line B16F10 transplanted into immune-competent mice, and human cancer cell lines (MDA-MB 435 melanoma and HT1080 fibrosarcoma) xenografted into nude mice. Both unadorned ACPPs not conjugated to macromolecules [free ACPPs (12)] and ACPPs conjugated to a generation 5 poly(amidoamine) dendrimer [ACPPs conjugated to dendrimers (ACPPDs) (13)] were separately injected i.v.…”

“…Standardized uptake values (SUV) were measured as described in Aguilera et al (11) and Olson et al (12,13). Briefly, 30-mg tissues were homogenized and heated in SDS buffer, frozen, and then imaged by fluorescence.…”

Gelatinase activity imaged by activatable cell-penetrating peptides in cell-based and in vivo models of stroke

“…[16][17][18] ACPPs and ucACPPs consist of polycationic CPPs (arginine 9 ) labeled with Cy5 and fused using cleavable (-PLGC(Me)AG-) or resistant (-plgc(Me)ag-) linkers, respectively, to matching polyanions (succinyl-glutamate 8 ). This strategy reduces the net charge towards zero and inhibits adhesion and uptake into cells (Scheme 1), allowing improved tissue distribution.…”

Enhancing cellular uptake of activable cell-penetrating peptide–doxorubicin conjugate by enzymatic cleavage

“…[16][17][18][19][20] The ACPPs are inactive in the circulation since the cell-penetrating function of CPP is efficiently blocked by intramolecular electrostatic interactions with a polyanionic peptide. Proteolysis of a cleavable enzyme-sensitive substrate present between the CPP and polyanionic peptide by specific enzymes overexpressed on the disease site affords dissociation of both domains and enables the activated CPP to enter cells.…”