Supporting Information
PTD-Modified ATTEMPTS for Enhanced Toxin-based Cancer Therapy: An
In Vivo Proof-of-Concept Study
Meong Cheol Shina,b, Jian Zhangc, Kyoung Ah Minb, Huining Hea, Allan E. Davidd, Yongzhuo
Huange and Victor C. Yanga,b,f*
a
Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and
Diagnosis, School of Pharmacy, Tianjin Medical University, Tianjin 300070, China
b
Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, 428
Church St., Ann Arbor, MI 48109, USA
c
Biomedical Polymers Laboratory, and Jiangsu Key Laboratory of Advanced Functional
Polymer Design and Application, Department of Polymer Science and Engineering, College of
Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou
215123, China
d
Department of Chemical Engineering, Auburn University, Auburn, AL 36849, USA
e
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Hai-ke Rd, Shanghai
201203, China
f
Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of
Convergence Science and Technology, and College of Medicine or College of Pharmacy,
Seoul National University, Seoul, 151-742, Republic of Korea
* Author to whom correspondence should be addressed:
Victor C. Yang, Ph.D.
Albert B Prescott Professor
Department of Pharmaceutical Sciences
The University of Michigan
Ann Arbor, Michigan 48109-1065
Tel: 01-734-764-4273; Fax: 01-734-763-9772
Email: vcyang@umich.edu
Internalized TAT-Gel (%)
100
w/o Protamine
with Protamine
80
60
40
**
**
**
**
20
0
0
1
2
3
4
5
6
Incubation Time (h)
Figure S1. Quantification of cell internalized TAT-Gel fractions with or without addition of
protamine to the TAT-Gel/T84.66-Hep pre-treated cells. LS174T cells were seeded on 24-well
plates at a density of 2×104 cells per well and, after overnight incubation, TAT-Gel (TRITClabeled)/T84.66-Hep (TAT-Gel: final 1 µM concentration; TAT : heparin = 1:3) were added to
the wells and incubated at 37ºC for 2 h. After washing the unbound TAT-Gel/T84.66-Hep with
PBS, the cells were further incubated at 37ºC up to 6 h with or without addition of protamine
(final concentration: 3 µM). At intended time points (0, 0.5, 1, 2, 6h), the cell internalized TATGel amount was quantified by following the identical procedures used for quantification of the
cell internalized T84.66-Hep (please see ‘MATERIALS AND METHODS’ section). The
internalized TAT-Gel fraction (%) was calculated by dividing the mean fluorescence intensity
(M.F.I.) of internalized TAT-Gel by the M.F.I. of total cell associated TAT-Gel (at time point
“0”), and then multiplying by 100. The results showed that with addition of protamine, the cell
internalization of TAT-Gel significantly increased (at 0.5 h, from 9.8% to 26% and, at 6h, 13.9%
to 39.2%). **P < 0.01 by Student’s t-test. (TAT-Gel: recombinant TAT-gelonin fusion chimera,
T84.66-Hep: T84.66-heparin chemical conjugate)