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Selective Uptake Into Drug Resistant Mammalian Cancer By Cell Penetrating Peptide-Mediated Delivery Kate J. F. Carnevale, Megan E. Muroski, Parth Nalin Vakil, Megan E. Foley, Geoffry Laufersky, Rachael Kenworthy, Diego A.R. Zorio, Thomas J. Morgan, Cathy W. Levenson, and Geoffrey F. Strouse Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00429 • Publication Date (Web): 21 Sep 2018 Downloaded from http://pubs.acs.org on September 22, 2018
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Bioconjugate Chemistry
Selective Uptake Into Drug Resistant Mammalian Cancer By Cell Penetrating Peptide-Mediated Delivery Kate J. F. Carnevale,† Megan E. Muroski,†⸸ Parth N. Vakil,† Megan E. Foley,†♦ Geoffry Laufersky,†⸹ Rachael Kenworthy,†⸷ Diego A. R. Zorio,‡ Thomas J. Morgan Jr.,‡ Cathy W. Levenson‡ & Geoffrey F. Strouse*† †
Dept. of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32304 College of Medicine, Florida State University, Tallahassee, FL 32304
‡
ABSTRACT
Research over the past decade has identified several of the key limiting features in multidrug resistance (MDR) cancer therapy applications, such as evolving glycoprotein receptors at the surface of the cell that limit therapeutic uptake, metabolic changes that lead to protection from multidrug resistant mediators which enhance degradation or efflux of therapeutics, and difficulty ensuring retention of intact and functional drugs once endocytosed. Nanoparticles have been demonstrated to be effective delivery vehicles for a plethora of therapeutic agents and, in the case of nucleic acid based agents, they provide protective advantages. Functionalizing cell penetrating peptides (CPPs), also known as protein transduction domains, onto the surface of a fluorescent quantum dots creates a labelled delivery package to investigate the nuances and difficulties of drug transport in MDR cancer cells for potential future clinical applications of diverse nanoparticle therapeutic delivery strategies. In this study, eight distinct cell penetrating peptides were used (CAAKA, VP22, HIV-TAT, HIV-gp41, Ku-70, and hCT(9-32), integrin-β3, and K-FGF) to examine the different cellular uptake profiles in cancer versus drug resistant melanoma (A375 & A375-R), mesothelioma (MSTO & MSTO-R), and glioma (rat 9L & 9L-R, and human U87 & LN18) cell lines. The results of this study demonstrate that cell penetrating peptide uptake varies with the amount of drug resistance and cell type, likely due to changes in cell surface markers. This study provides insight to developing functional nanoplatform delivery systems in drug resistant cancer models.
KEYWORDS: multi-shelled quantum dot, QD, cell penetrating peptide, CPP, multidrug resistant, MDR, cancer, cell uptake
INTRODUCTION Non-viral approaches to overcoming drug resistant pathways include the use of cell penetrating peptides (CPP) coupled to nanoparticles to effectively act as a transfection carrier of chemotherapeutics.1 CPPs are derived from small sequence fragments of the protein transduction domain of viral sheath proteins, or human carrier proteins. Packaging of CPPs onto the surface of nanoparticles is an important focus area for packaging of therapeutics into non-viral transfection methodologies.2–5 When the nanoparticle – loaded with a therapeutic agent – is combined with a CPP, cellular uptake and delivery of the therapeutic agent can be enhanced. Nano-therapeutic delivery agents have also been shown to enhance the survivability and therefore effectiveness of the delivered nucleic acid agent due to reduced nuclease activity, continuous agent release, and pH clamping of the endosomal package.6,7 There are reports suggesting that CPPs exhibit selectivity for different cell types.8–13 Thus, we have hypothesized that CPPs may also exhibit specificity for chemoresistance in different cancer cells and have designed an approach to screen CPPs for nanocarrier uptake ashelled CdTe@CdSe@CdS@ZnS quantum dots (QDs) (Figure 1) and the effective enhancement of cellular internalization in a series of cancer cell lines of human melanoma (A375), human mesothelioma (MSTO), and human (LN18, U87) and rat glioma (9L), along with drug resistant sub-clones. QDs are used for this study as they are ideal optical probes for molecular tracking in cell uptake studies owing to their brightness and resistance to photo-bleaching, and for their comparability to other clinically relevant, nonemissive, nanomaterials. Although the heavy metals that comprise QDs and allow for the unique optical properties, such as cadmium and tellurium, are potentially cytotoxic and therefore not applicable to potential clinical treatments, the use of a non-therapeutic photoluminescent multi-shelled quantum dot (QD), composed of CdTe@CdSe@CdS@ZnS, allows for quantification of nano-package cellular uptake through fluorescence microscopy, and provides clinically transferrable insights. The studied CPPs are appended to the QD surface via an N-terminal cysteine which allows for bidentate coordination using the thiol and amine of cysteine to bind onto the ZnS outermost QD shell. A biphasic ligand exchange allows the displacement of the weakly coordinated organic passivating ligands from the as-synthesized QDs, and replacement by the aqueous CPPs.
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The study provides a method that shows promise for selectively targeting multi-drug resistant cancer that could offer new therapies that increase drug delivery to tumors, while circumventing drug efflux and other mechanisms responsible for chemoresistance. Despite improvements in therapeutic approaches for many types of cancer, the development of multidrug resistance (MDR) remains a significant barrier to improving survival time in patients with many types of aggressive cancers. Developing new potentially therapeutic strategies for highly aggressive, metastatic cancers with poor 5-year survival rates (melanoma (
