Form 1

2017 Report Form for Collaboration withResearch Center for Biomedical Engineering

2018/3/9

Year/month/date2069

Numberdate:

Date /Month/Year
9/March/2018

To Chairman, Board of Directors, Research Center for Biomedical Engineering

ApplicantAffiliation ; Jahangirnagar University
Title: Lecturer
Name: Dr. Md. Zahidul Islam
Address : Department of Biotechnology and Genetic Engineering,
Jahangirnagar University, Savar, Dhaka-1342, Bangladesh.
Phone : +88-01787-086965
Fax
E-mail : zahidul_ku@yahoo.com; zahidul.islam@bgeju.edu.bd
Report Form for Collaboration Research
Effect of membrane potential on entry of cell-penetrating peptide Transportan10

Research Theme

Research Area

Research Period

into single vesicles.
1. Biomaterials
2. Bioengineering√3. Functional molecules
4. Chemistry/Electrical Engineering/Mechanical Engineering/Materials Science

From:Date/month/Year
01/06/2017

To: Date/month/Year
31/03/2018

Applicant Organization
Name
Md. Zahidul Islam

Department
Department of Biotechnology and Genetic
Engineering, Jahangirnagar University, Ban
gladeshTitle
Lecturer

Role
Experiments andanalysis

Md. Mizanur Rahman
MoghalIntegrated Bioscience Section, Graduate
School of Science and Technology, Shizuoka
Univ.

Ph. D
student

ExperimentsMasahito Yamazaki

Research Institute of Electronics, Shizuoka
Univ.

Professor

Collaboration Partners
Research CenterintheMasahito Yamazaki

courseAnalysis

Research Results (Including Purpose, Results, Figures, etc.)

1200
800
40000100200
Time (s)300400Lumen intensity of CF-TP101600

Normalized FI

entered the GUV lumen by translocating across the GUV membrane and (B)1.2
then bound to the LUVs before pore formation and that CF-TP10
1.00.8concentration in the lumen increased with time.
0.6
The above results clearly indicate that the new method using single GUVs0.4containing LUVs can detect continuously the entry of CF-TP10 into single
0.20.0
GUVs without pore formation and the values of the fraction of entry ofCF-TP10 increased with CF-TP10 concentration. This method also enabled us

Fraction of Entry of CF-TP10

Normalized FI

Lumen intensity of CF-TP10

Plasma membrane or biomembrane of biological cells prevents the entry of pharmacologically active
biomacromolecules (polypeptides and oligonucleotides) from outside of the cells. In contrast, cell-penetrating
peptides (CPPs) such as transportan 10 (TP10) can translocate across the plasma membrane of living eukaryotic
cells to enter their cytoplasms, and thus can be used for the intracellular delivery of biological cargo such asproteins and oligonucleotides. However, the elementary processes of the entry of CPPs into cells, the mechanisms
underlying its translocation, and the delivery of large cargo remain unclear. On the other hand, all biological cells,
irrespective of eukaryote or prokaryote, have membrane potential in their plasma membranes, which play
important physiological roles. The purpose of this project is to reveal the effect of membrane potential ontranslocation of CF-TP10 into single giant unilamellar vesicles (GUVs) using the single GUV method.
First, we have developed a new method for the continuous, quantitative detection of the entry of CPPs intoGUVs, where we investigate the interaction of fluorescent probe-labeled CPPs with single GUVs containing large
unilamellar vesicles (LUVs) with a diameter of 100 nm and water-soluble fluorescent probes in their lumens using
confocal laser scanning microscopy (CLSM). Using thisFigure 1
method, we investigated the interaction of (A)carboxyfluorescein (CF)-labeled TP10 (CF-TP10)
with single GUVs comprised of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine(DOPC) containing LUVs of the same membrane andAlexa Fluor 647 hydrazide (AF647) in their lumens. (B)(C)
1.0
At low concentrations of CF-TP10 (such as 1.0 µM
1.01600
0.8CF-TP10), first the fluorescence intensity (FI) of the0.8
1200
0.60.6
GUV membrane due to the binding of CF-TP10
8000.40.4
increased with time (green square in Fig. 1B), and then
0.20.2400
after some lag time the FI of the GUV lumen due to the0.00.0020.510100200300
400
binding of CF-TP10 with the LUVs increased
CF-TP10 Conc. (µM)
Time (s)continuously with time (Fig. 1A (2) and black square inFig. 1B), whereas FI of the GUV lumen due to AF647 did not change (Fig. 1A and red circle in Fig. 1B),
indicating no pore formation in the GUV membrane. As a measure of the rate of entry of CPPs into GUV lumen,
we can use the fraction of GUVs which CPPs entered before a specific time t with respect to total examined
GUVs, P entry (t), because the entry of CPPs occurred stochastically. Here we can determine the values of P entry (t)based on the FI of the GUV lumen due to CF-TP10 at a specific time t. The values of the I lumen (6 min) for all
examined GUVs (n = 20) interacting with CF-TP10 were larger than 50, indicating that the fraction of GUVs
which CPPs entered before 6 min without pore formation among all examined GUVs, P entry (6 min), was 1.0 for
1.0 µM CF-TP10. Figure 1C (red circle) shows the dependence of P entry (6 min) on CF-TP10 concentration; at andless than 0.50 µM CF-TP10, P entry (6 min) = 0, and above 0.50 µM, P entry (6 min) increased with an increase in
CF-TP10 concentration, indicating that the rate of entry of CF-TP10 into GUV lumen increased with CF-TP10concentration.
Figure 2
At higher concentrations of CF-TP10 (such as 2.0 (A)µM CF-TP10), after the FI of the GUV lumen due toCF-TP10 (black square in Fig. 2B) increasedsignificantly, leakage of AF647 started (Fig. 2A and red
circle in Fig. 2B). These results indicate that CF-TP10

to follow the time course of CF-TP10 concentration in the GUV lumen continuously and quantitatively. Moreover,in this method, the I lumen greatly increased after entry of CF-TP10 into GUV lumen, and therefore otherexperimental methods such as flow cytometry can detect the entry of CF-TP10. We can reasonably expect that thismethod can be applied to the interaction of other CPPs with single GUVs.
Next, to elucidate the mechanism of the translocation of TP10 across lipid bilayers, we investigated the effectof the membrane potential on the entry of CF-TP10 into single DOPG/DOPC-GUVs using the single GUV
method. Various membrane potential were applied to GUV membranes using the K+ concentration gradient, which
was confirmed by the potential sensitive-fluorescence probe. The rate of entry of CF-TP10 into the GUV lumen
before leakage of AF647, i.e., pore formation, increased with an increase in membrane potential. There are several
elementary processes involved in the entry of CF-TP10 into GUV lumen. It is thus important to elucidate whichelementary process is affected greatly by the membrane potential. We have to investigate more this point nextyear.

List of Publications Related to the Collaboration Research(1) Md. Mizanur Rahman Moghal, Md. Zahidul Islam, Sabrina Sharmin, Victor Levadnyy, Md. Moniruzzaman,
and Masahito Yamazaki, Continuous detection of entry of cell-penetrating peptide transportan 10 into single
vesicles, Chem. Phys. Lipids, 212, 120-129, 2018.
(2) Md. Zahidul Islam, Sabrina Sharmin, Md. Moniruzzaman, and Masahito Yamazaki
Elementary Processes for the Entry of Cell-Penetrating Peptides into Lipid Bilayer Vesicles and BacterialCells, Appl. Microbiol. Biotechnol. in press, 2018
List of Presentations ( Conference, Meeting, etc)
(1) Md. Mizanur Moghal, Md. Zahidul Islam, Sabrina Sharmin, Masahito Yamazaki, Effect of Membrane
Potential on the Translocation of Cell-Penetrating Peptide Transportan 10 (TP10) across Lipid Bilayers, The
55th Annual Meeting of Biophysical Society of Japan, 19−21 Sept., 2017, Kumamoto Univ., Kumamoto

List of Awards

Research plan for the next year (from April 1, 2018 to March 31, 2019), if the collaboration research is continued.
Prior consent from the collaboration partner in the Research Center is necessary.
As described in the above section, in the last year’s project, we found that the rate of entry of CF-TP10 into theGUV lumen before pore formation increased with an increase in membrane potential applied to the GUV
membrane. However, there are several elementary processes involved in the entry of CF-TP10 into GUV lumen.
It is thus important to elucidate which elementary process is affected greatly by the membrane potential. For thispurpose, we examine the effect of membrane potential on the rate constants of binding of CF-TP10 to GUV
membrane from aqueous solution and that of unbinding of CF-TP10 from GUV membrane to aqueous solution
using the method developed by us (1). Briefly, we investigate the interaction of CF-TP10 with single
DOPG/DOPC-GUVs (not containing vesicles) under the membrane potential using confocal laser scanningmicroscopy measure, and analyze the time course of the FI of the GUV membrane due to CF-TP10 to obtain theserate constants. Then we compare the effect of membrane potential on the rate constants, and determine which
process is greatly affected by the membrane potential.
So far we investigated the negative membrane potential. To increase our understanding of the effect ofmembrane potential on the entry of CF-TP10, we also want to investigate the effect of the positive membranepotential on the entry of CF-TP10 into GUV lumen and also on the rate constant of each elementary process. Onthe basis of the results of the effects of both negative membrane potential and positive one on the entry ofCF-TP10 and its elementary processes, we want to improve the mechanism of the entry of CF-TP10 proposed by
us recently (2).
Next, to generalize the concept of the effect of the membrane potential on the entry of CPPs, we investigate theeffect of membrane potential on the entry of other CPPs such as CF-R 9 (3) and Rh-LfcinB (4-9) (4) and its
elementary processes.
The results of the effect of membrane potential on the entry of CPPs and the mechanism of the translocation ofCPPs across the membranes are very helpful to develop novel vectors for the delivery of drugs, chemicals,oligonucleotides, and proteins into cells for curing various diseases.
<References>(1) Islam MZ, Ariyama H, Alam JM, Yamazaki M (2014) Entry of cell-penetrating peptide transportan 10 into asingle vesicle by translocating across lipid membrane and its induced pores. Biochemistry 53:386-396.
(2) Islam MZ, Sharmin S, Levadnyy V, Shibly SUA, Yamazaki M (2017) Effects of Mechanical Properties ofLipid Bilayers on Entry of Cell-Penetrating Peptides into Single Vesicles. Langmuir 33:2433-2443.
(3) Sharmin S, Islam MZ, Karal MAS, Shibly SUA, Dohra H, Yamazaki M (2016) Effects of lipid composition on
the entry of cell-penetrating peptide oligoarginine into single vesicles. Biochemistry55:4154-4165.
(4) Moniruzzaman M, Islam MZ, Sharmin S, Dohra H, Yamazaki M (2017) Entry of a Six-Residue Antimicrobial
Peptide Derived from Lactoferricin B into Single Vesicles and Escherichia coli Cells without Damaging theirMembranes. Biochemistry 56:4419-4431.