Poster X1 - Exploring the Loading of Cell Penetrating Peptides into Extracellular Vesicles for Therapeutic Applications

Saturday, October 26, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Neona M. Lowe (she/her/hers)

Ph.D. Candidate
University of California, Davis, United States

Co-Author(s)

Alyssa Panitch, PhD (she/her/hers)

Professor/ Chair
Georgia Institute of Technology and Emory University, Georgia, United States

Last Author(s)

Randy Carney, PhD

Associate Professor
University of California, Davis, United States

Introduction: Cell penetrating peptides (CPPs) are small ( < 30 amino acid residues) biomolecules that are capable of passively crossing biological membranes. There is interest in exploiting CPPs for the development of functional therapeutics. However, a limitation of CPP-based therapeutics is that most CPPs do not have targeting capabilities. We therefore propose using extracellular vesicles (EVs) as a drug delivery vehicle for CPP-based therapeutics. EVs are heterogenous lipid-bilayer wrapper nanoparticles release by all cells and have high potential as next generation drug delivery vehicles. As there is very little research previously done on CPP loading into EVs, here we first investigated how physiochemical properties of CPP subclasses affect their interaction and loading into EVs.

Materials and

Methods: Representative CPPs from the three major categories with different physicochemical properties were synthesized: TP10 (primary amphiphilic CPP), penetratin (secondary amphiphilic CPP), and TAT (cationic CPP). Additionally, YARA and MK2i were also synthesized. YARA is a modified version of TAT to increase transduction potential. MK2i is YARA conjugated to peptide based therapeutic cargo that functions as a MK2 inhibitor. All synthesized CPPs were tagged with fluorescein isothiocyanate (FITC). EVs were collected from HEK293T cells via ultracentrifugation and non-specifically labeled with cell tracker far red (CTFR). EVs were incubated with various concentrations of CPPs for 24 hours at room temperature and isolated from free peptides via size exclusion chromatography. Samples were then analyzed by single particle flow cytometry to determine the location of CPP on the EV (i.e., EV lumen or outer membrane), by assessing fluorescence labeling with or without trypan blue, fluorescent quencher of FITC.

Results, Conclusions, and Discussions: All CPPs were observed to show dose dependent loading into the HEK293T EVs with more loading with increased CPP concentration. TP10 (primary amphiphilic CPP) and penetratin (secondary amphiphilic CPP) were shown to have low loading into the EV lumen. On the other hand, majority of the loaded TAT (cationic CPP), YARA (modified TAT), and MK2i (YARA with therapeutic cargo) were observed to be in the EV lumen. Of the loaded EVs, the CPP loading per EV volume was investigated by calculating the median FITC intensity per median EV volume. For TP10 and penetratin, the value decreased with increased CPP concentration. Meanwhile for TAT, YARA, and MK2i, the amount of CPP loaded per EV volume did not change significantly across all tested CPP concentrations. Finally, EV integrity was investigated by looking at total event number on the cytometer. The event number decreases drastically with increase concentration when EVs are incubated with TP10, penetratin, and TAT. However, minimal decrease is overserved with YARA and moderate decrease with MK2i. Overall, we have demonstrated the ability of CPPs to be dose dependently loaded into EVs, as well as the ability of CPPs to load therapeutic cargo into EVs. Additionally, we have demonstrated that the physicochemical properties of the CPPs affect loading location, amount of CPP loaded per EV volume, and EV integrity.

Acknowledgements (Optional):