l i v e r c r 2 - peptide therapeutics...

Longer acyl chains induce more stable nanoparticle formation

Transfection efficiency improves with longer acyl chains bothin vitro and in vivo

Self-association of the acylated peptides is dependent on protonation

Principles of in vivo nucleic acid deliverywith acylated cell-penetrating peptidesTõnis Lehto,1 Kadi-Liis Veiman,2 Mattias Hällbrink,1 Ülo Langel1,3

1 Department of Neurochemistry, The Svante Arrhenius Laboratories for Natural Sciences, Stockholm University, 10691 Stockholm, Sweden2 Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia

Introduction

N-terminal acylation of cell-penetrating peptides (CPPs) with fattyacids is a well-known method to improve their ability to deliver non-covalently bound nucleic acid cargoes inside cells.The aim of this study was to systematically describe the role of theN-terminal acyl chain length in translation of plasmid delivery fromin vitro to in vivo.We took a previously studied set of acylated CPPs with varying N-terminal acyl chain length (2 to 22 carbons) as the basis1. To evaluatethe delivery efficiency, the peptides were non-covalently complexedwith plasmid DNA and tested for gene induction both in cell culturesand in mice after systemic administration.

Tõnis LehtoDepartment of Neurochemistry, Stockholm UniversityPhone: + 46 (0)8 16 4264E-mail: tonis.lehto@gmail.com

Toxicity of the complexes in vivo is related to the freefraction of peptide and acyl chain length

Self-association of peptides wasstudied by DLS. 10 μM peptide wasdiluted either in Milli-Q (MQ) or in 10mM phosphate buffers at differentpH.

Transfection efficiency of complexeswas evaluated in CHO cell line at 0.1 µgper well pGL3 plasmid dose after 24h.

Formation of non-covalent peptide/nucleic acid complexes

C0-1

4

C2-1

4

C4-1

4

C6-1

4

C8-1

4

C10-1

4

C12-1

4

C14-1

4

C16-1

4

C18-1

4

C20-1

4

C22-1

4

0 .1

1

1 0

1 0 0

1 0 0 0

1 0 0 0 0

Nu

mb

er m

ea

n s

ize

(n

m)

p H 4 .0

p H 6 .0

p H 8 .0

MQ

Conclusions

• Modifying CPPs with longer acyl chains improves the transfectionefficiency of plasmid DNA both in cell cultures and in liver aftersystemic administration in vivo.

• CPPs with longer acyl chains form more stable nanoparticles uponnon-covalent complexation of nucleic acids.

• Although free peptide fraction in the peptide/nucleic acidcomplexes is known to cause hemolysis and toxicity in vitro1,2,these side-effects can be overcome in vivo by minimizing theamount of free CPP fraction in the complexes.

Complexes Free fraction of peptideMixing

Complexes are formed in Milli-Q at diferent [peptide:nucleic acid] charge ratios(CR).

!

0 5 1 0 1 5 2 0 2 5 3 0

0

2 0

4 0

6 0

8 0

1 0 0

t im e (m in )

% o

f re

lea

se

d p

DN

A

C 0 -1 4

C 2 -1 4

C 4 -1 4

C 6 -1 4

C 8 -1 4

C 1 0 -1 4

C 1 2 -1 4

C 1 4 -1 4

C 1 6 -1 4

C 1 8 -1 4

C 2 0 -1 4

C 2 2 -1 4

C0-1

4

C2-1

4

C4-1

4

C6-1

4

C8-1

4

C10-1

4

C12-1

4

C14-1

4

C16-1

4

C18-1

4

C20-1

4

C22-1

4

1 0 1

1 0 2

1 0 3

1 0 4

1 0 5

1 0 6

1 0 7

Lu

cif

era

se

ex

pre

ss

ion

(RL

U)

C R 2

In vitro transfection In vivo gene induction

Gene induction in liver 24h afterintravenous administration of 50 µg ofpLuc2 plasmid complexed at CR2.(n=2-6 animals per group).

L iv e r

C6-1

4

C10-1

4

C14-1

4

C18-1

4

C22-1

4

1

2

4

8

1 6

3 2

6 4

Fo

ld i

nc

re

as

e o

f R

LU

/mg

(ov

er p

DN

A t

re

ate

d)

References1. Lehto, T., Vasconcelos, L., Margus, H., Figueroa, R., Pooga, M., Hällbrink, M., and Langel, Ü. (2017)

Saturated Fatty Acid Analogues of Cell-Penetrating Peptide PepFect14: Role of Fatty Acid Modification inComplexation and Delivery of Splice-Correcting Oligonucleotides. Bioconjugate Chemistry 28, 782–792.

2. Vasconcelos, L., Lehto, T., Madani, F., Radoi, V., Hällbrink, M., Vukojević, V., Langel, Ü. Simultaneousmembrane interaction of amphipathic peptide monomers, self-aggregates and cargo complexes detectedby Fluorescence Correlation Spectroscopy. Biochim. Biophys. Acta – Biomembranes (In press).doi:10.1016/J.BBAMEM.2017.09.024

C0-1

4

C2-1

4

C4-1

4

C6-1

4

C8-1

4

C10-1

4

C12-1

4

C14-1

4

C16-1

4

C18-1

4

C20-1

4

C22-1

4

1 0

1 0 0

1 0 0 0

1 0 0 0 0

-1 0

0

1 0

2 0

3 0

4 0

5 0

Nu

mb

er m

ea

n s

ize

(n

m)

Ze

ta P

ote

ntia

l (mV

)

S iz e

Z e ta P o te n tia l

The size and zeta potential of the complexes at CR2 (A) and stability tocompeptitive polyanion displacement (B) were studied by DLS and PicoGreenassay respectively.

Stability ↑

Number and survival of BALB/c mice in treatment groups 24h after i.v.administration of complexes at diferent charge ratios. All animal experiments andprocedures were approved by the Estonian Laboratory Animal Ethics Committee(approvals no. 69 and 70, dated Feb 9, 2011).

CR2 50µg CR4 20µg

Peptide

No of animals in group

Live after 24h

Survival Rate (%)

No of animals in group

Live after 24h

Survival Rate (%)

C6-14 2 2 100 5 5 100

C10-14 4 3 75 7 1 14

C14-14 4 2 50 7 3 43

C18-14 6 6 100 9 7 78

C22-14 4 4 100 7 2 29

A B

Nucleic acid

Nucleic acid