Preparation of modified peptide solutions: ‒ Preheated (80⁰C) for 20min, kept at 37⁰C.

Cell culture: Mouse fibroblast (NIH 3T3) cells were seeded and incubated with several peptides [15uM, preheated] for 1 hr in different temperature. Flow cytometer (Cell Lab Quanta SC MPL, Beckman Coulter): Cells were washed, trypsinized and analyzed. Circular dichromism (CD) spectroscopy: CD provided conformational data of peptides at different temperatures. Confocal microscopy (Olympus Fluoview 1000): Confocal imaging was used to investigate peptide internalization.

Peptide Internalization Triggered by Temperature Myungeun Oh, Chloe Hu, Katarzyna Slowinska, Ph.D.

Department of Chemistry and Biochemistry, California State University Long Beach

Cell penetrating peptides (CPPs) are known for their ability to carry molecules across cell membrane. CPPs are efficient carriers of molecular cargo in micro-molar concentration and penetrate cell membrane without damage to the cell. Yet, CPP carriers are not selective and are not capable of targeted delivery unless specific modification is added into CPP structure. This modification is usually attachment of small molecule or peptide sequence targeting specific molecular receptor. Here we report the attempt to design a protocol for selective uptake of CPP that is based on peptide conformation. This protocol opens a possibility of targeting cells without molecular targets. Here we modified CPPs with collagen-like domain that allows peptide to fold into triple helix conformation. This folding process is reversible and temperature dependent. With confocal microscopy and flow cytometry we show the controlled internalization of CPP, dependent on peptide folding that is controlled with temperature. This type of controlled delivery may open the possibility of active targeting cells that does not have known molecular targets.

Peptide Sequence Tm (⁰C)

V2R FITC-Aβ-GG- GPPOOGPGGGPOOPGOOPGGOOPP- R6 -

FL8V1 FITC-Aβ-GG-(POG)8- (RRG)2 48.8

FL6V1 FITC-Aβ-GG-(POG)6- (RRG)2 15.33

FL7V1 FlTC-Aβ-GG-(POG)7-(RRG)2 16.29

CPPs are well known for their ability to penetrate cell membrane and their potential application as drug carriers. Yet their indiscrimination of cellular membrane penetration hinders their potential clinical use. Here, we showed the controlled localization of modified peptides using their conformational change triggered by temperature. Result consistently indicated that the peptides were internalized at temperature lower than their Tm. We concluded that the cellular localization of peptides can be manipulated by temperature.

1. Bakota, E. , Sensoy, O. , Ozgur, B. , Sayar, M. , & Hartgerink, J. (2013). Self-assembling multidomain peptide fibers with aromatic cores.Biomacromolecules, 14(5), 1370-1378.

2. Fretz, M. , Penning, N. , Al-Taei, S. , Futaki, S. , Takeuchi, T. , et al. (2007). Temperature-, concentration- and cholesterol-dependent translocation of l- and d-octa-arginine across the plasma and nuclear membrane of cd34+ leukaemia cells. The Biochemical Journal, 403(2), 335-342

3. Madani, F. , Lindberg, S. , Langel, U. , Futaki, S. , & Gräslund, A. (2011). Mechanisms of cellular uptake of cell-penetrating peptides. Journal of Biophysics (Hindawi Publishing Corporation : Online), 2011, 414729

4. Shinde, A., Feher, K.M., Hu, C., Slowinska, K. (2014).Folding short arginine-rice sequences into triple helix enables efficient internalization. [submitted]

Introduction

Conclusion

Result

Methods and Materials

References

4⁰C 1 hr

Both Brightfield FITC

37⁰C 1 hr

4⁰C 1 hr

Both Brightfield FITC

37⁰C 1 hr

4⁰C 1 hr

Both Brightfield FITC

37⁰C 1 hr

Flow

4⁰C 1 hr

Both Brightfield FITC

37⁰C 1 hr

Flow

A B

Figure 2. Diagram of methods. (A) Procedure for taking confocal image with cell-seeded MatTek dish. (B) Setup for dynamic temperature experiment.

We used confocal imaging and flow cytometer to investigate the internalization of peptides by conformational change due to temperature difference. Resulting data (Fig. 3) showed peptide internalization at temperature lower than Tm for the peptides that have collagen mimetic sequence. No internalization was observed for peptide at temperature higher than Tm. In order to look at peptide internalization controlled by temperature, apparatus was designed which involved temperature gradient. This tubing apparatus (Fig. 4) was set up inside the incubator to keep the cells at body temperature (37⁰C). Fountain pump was used to circulate cold water through the tube. One end of the coverslip with cells was put on top of the tube to give low temperature (8 ⁰C -13 ⁰C). The other end of the coverslip was put on top of a piece of tube to level out. This created the temperature gradient on the coverslip which ranged from 8 ⁰C to 25 ⁰C. Using this temperature gradient, dynamic peptide internalization can be investigated.

Contacts: Chloe Hu Chloe.hu@student.csulb.edu

Katarzyna Slowinska Katarzyna.slowinska@csulb.edu Website: http://slowinskalab.weebly.com/research.html

Merlyn Arostegui Merlyn.arostegui@gmail.com

Myungeun Oh Myungeun.oh@student.csulb.edu

This project was supported by National Institutes of Health. Special thanks to Dr. Slowinska and all the members of Slowinska lab .

Acknowledgements:

Figure 1. Diagram of methods. (A) Structure of modified peptide. (B) CD curve showing transition temperature (Tm) and peptide conformation of FL7V1 peptide.

Table 1. Modified peptides information

Figure 3. Images from confocal microscopy (Both, Brightfield, and FITC) and flow cytometer. All flow data represent peptides at 4⁰C and 37⁰C except for FL6V1 (15 ⁰C and 37⁰C).

A

Flow

Flow

Figure 4. Experiment looking at dynamic internalization of peptide. (A) Control coverslip sitting inside incubator shelf. Edge of the coverslip is marked with red tape. (B) Coverslip with warm end (bottom) and cold end (top, water tube running underneath). Edge of the coverslip is drawn on screen with black line.

B

Related posters: ORGM 414: Circular dichroism studies of hybrid cell penetrating–collagen peptides Author: Chloe Hu [8:00-10:00pm Mar 24, Hall C – Colorado Convention Center] BIOT 256: Feasibility of targeting cells without unique molecular targets Author: Katarzyna Slowinska [6:00-9:00pm Mar 24, Imperial Ballroom – Grand Hyatt Denver]