Harnessing the power of cell-penetrating peptides: activatable carriers for targeting systemic delivery of cancer therapeutics and imaging agents

Abstract

Targeted delivery of cancer therapeutics and imaging agents aims to enhance the accumulation of these molecules in a solid tumor while avoiding uptake in healthy tissues. Tumor-specific accumulation has been pursued with passive targeting by the enhanced permeability and retention effect, as well as with active targeting strategies. Active targeting is achieved by functionalization of carriers to allow specific interactions between the carrier and the tumor environment. Functionalization of carriers with ligands that specifically interact with overexpressed receptors on cancer cells represents a classic approach to active tumor targeting. Cell-penetrating peptides (CPPs) provide a non-specific and receptor-independent mechanism to enhance cellular uptake that offers an exciting alternative to traditional active targeting approaches. While the non-specificity of CPP-mediated internalization has the intriguing potential to make this approach applicable to a wide range of tumor types, their promiscuity is, however, a significant barrier to their clinical utility for systemically administered applications. Many approaches have been investigated to selectively turn on the function of systemically delivered CPP-functionalized carriers specifically in tumors to achieve targeted delivery of cancer therapeutics and imaging agents.

Figure 1

MMP-2 cleavage of PEG controlled cellular uptake of quantum dots. Streptavidin-coated quantum dots (QD-strep) (red – top row) achieved little internalization by GFP-expressing MDA-MB-435 melanoma cells (green – bottom row, merged with top row). Functionalization of the quantum dot surface with biotin-CPP increased cellular uptake, while addition of removable PEG (biotin-substrate-PEG) decreased the internalization by blocking interactions of the CPP with the cell surface. Cellular uptake was recovered in the presence of MMP-2 with cleavage of the substrate-PEG linker, as the CPPs could interact with the cell surface following the removal of PEG. Reprinted with permission from Ref Copyright 2009 American Chemical Society.

Figure 2

Cytotoxicty was controlled by pH-mediated removal of PEG. Protective PEG was added to TAT-functionalized micelles by ionic interactions between the cationic TAT and PEG-conjugated anionic polysulfonamide. Neutralization of polysulfonamide charge in acidic conditions caused the dissociation of PEG that allowed interaction of TAT with the cell surface. Doxorubicin-loaded PEG-shielded TAT-micelles (white) demonstrated selective cytotoxicity, with increased cell death occurring only at pH 7.0 and below. No pH-dependence in cytotoxicity was shown with controls including TAT-micelles (dark grey), free doxorubicin (light grey), or untreated cells (black). Reprinted from Ref with kind permission from Springer Science and Business Media.

Figure 3

Tumor accumulation was enhanced with removal of PEG from tumor-localized stealth liposomes (SL). TAT-functionalized liposomes with cleavable disulfide-linked PEG (C-TAT-SL) accumulated in C26 colon cancer tumors via the EPR effect prior to the intravenous administration of thiol-reactive cysteine (Cys). Local cleavage and release of the PEG increased the cellular uptake in the tumor by 56% in cysteine administered animals, compared to the PBS administered controls, as measured by flow cytometry of excised tumor cells. This tumor uptake was 130% greater than that achieved with controls including non-functionalized stealth liposomes (SL), non-cleavable PEG coated TAT-liposomes (N-TAT-SL), and cleavable PEG coated non-functionalized liposomes (C-SL). Reprinted with permission from Ref . Copyright 2011 American Chemical Society.

Figure 4

Triggered display of TAT in response to the acidic tumor environment improved tumor regression in drug resistant ovarian tumor xenografts. Three intravenous doses of doxorubicin-loaded pH-sensitive activatable TAT-functionalized micelles (PHSM^pop-upTAT - circle) achieved superior tumor regression in comparison to controls including a non-functionalized pH-sensitive micelle (PHSM - square), a pH-sensitive micelle that displayed TAT at all pH (PHSM^TAT - triangle), and free doxorubicin (DOX – inverted triangle). The in vitro cytotoxicity of these constructs at physiological (7.4) and acidic (6.8) pH demonstrated the controlled cytotoxicity of the activatable PHSM^pop-upTAT (inset graph). Reprinted from Ref . Copyright 2008 with permission from Elsevier.

Figure 5

Dendrimeric nanoparticles functionalized with MMP-2 activatable CPPs permitted the targeted tumor delivery of Cy5 fluorophore and MR contrast agent, gadolinium. The dual imaging properties of these particles permitted surgical planning with MRI, real-time fluorescence imaging for intraoperative assistance of tumor resection, and post-operative evaluation with MRI. Accumulation of nanoparticles provided MR contrast to identify a fibrosarcoma xenograft tumor (arrow - A). Intraoperative visualization of the tumor was achieved with fluorescence imaging prior to resection (B). Post-operative MRI revealed residual tumor tissue (box – C) whose cancerous character was confirmed with histology following removal with a second surgery (D). Similar surgical assistance was provided for melanoma xenografts. The tumor was identified with MRI (arrow – E) and fluorescence provided intraoperative visualization (F). Surgical resection was performed such that no visible fluorescence remained at the surgical site (G) and post-operative MRI confirmed the successful removal of all cancer cells (H). Tumor resection assistance with guidance by activatable CPP-nanoparticles accumulated in tumor tissue lead to prolonged tumor-free survival after removal of murine melanoma and mammary adenocarcinoma tumors. Reprinted with permission from Ref .

Figure 6

Cellular uptake of CPP-functionalized HPMA was controlled by the dissociation of neutralizing inhibitory domains in response to UV illumination. Charge-neutralizing groups conjugated to the CPP by UV-labile linkers prevented internalization of FITC labeled polymer (green) in PC-3 prostate cancer cells kept in the dark (bottom). Exposure to UV light for 10 minutes was sufficient to remove the neutralizing groups and allow significant increase in cellular uptake (top). Lysosomes were labeled with LysoTracker Red and colocalized, in part, with the internalized HPMA. The delivery to lysosomes provided the acidic conditions necessary to allow intracellular drug release from the carrier using a pH-sensitive hydrazine linker. Reprinted from Ref . Copyright 2011 with permission from Elsevier.

Figure 7

Cellular uptake of a transportan-derived CPP was controlled by the pH-triggered ionization of histidine residues. Peptides whose cationic lysine residues were replaced with histidine residues (TH) exhibited low levels of cellular uptake, as measured by flow cytometry, in HeLa cervical cancer cells at pH 7.4 due to the neutral charge of the histidines at this pH. At pH 6.0 the ionization of the histidine residues conferred positive charge to the CPP and increased cellular uptake. This pH-triggered cellular uptake was in contrast to the original lysine-containing peptide (TK), which demonstrated equivalent cellular uptake at both pH 7.4 and 6.0. Reprinted with permission from Ref . Copyright 2011 American Chemical Society.

Figure 8

Temperature-triggered micelle assembly controlled cellular uptake by modulation of arginine density. Arginine₅-functionalized elastin-like polypeptide diblock copolymers (Arg₅-ELP_BC) achieved cellular uptake in HeLa cervical cancer cells, as quantified by flow cytometry, comparable to controls at 37 °C in their soluble unimer state, with arginine content below the threshold required to achieve significant cellular internalization. At 42 °C the micelle assembly of Arg₅-ELP_BC enhanced the local arginine density on the micelle corona, which increased cellular uptake 8-fold over that achieved at 37 °C. Non-functionalized diblock copolymers capable of temperature-triggered micelle assembly (ELP_BC) and soluble temperature-insensitive arginine-functionalized unimers (Arg₅-ELP) demonstrated negligible change in cellular uptake between 37 and 42 °C. Data adapted with permission from Ref Copyright 2012 American Chemical Society.