Cationic oligopeptides modified with lipophilic fragments: Use for DNA delivery to cells

Article abstract of DOI:10.1007/s11171-005-0002-z

Cationic oligopeptides, including the amphipathic α-helical peptides, are applied to the targeted delivery of DNA to eukaryotic cells due to their DNA-compacting properties and the ability to destabilize the cell lipid bilayer in some cases. We synthesize

Full text of DOI:10.1007/s11171-005-0002-z

Russian Journal of Bioorganic Chemistry, Vol. 31, No. 1, 2005, pp. 18–26. Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 22–30.
Original Russian Text Copyright © 2005 by Guryanov, Vlasov, Lesina, Kiselev, Baranov, Avdeeva, Vorob’ev.
Cationic Oligopeptides Modified with Lipophilic Fragments:
Use for DNA Delivery to Cells
1
I. A. Guryanov*, G. P. Vlasov*, E. A. Lesina**, A. V. Kiselev**, V. S. Baranov**,
E. V. Avdeeva***, and V. I. Vorob’ev***
* Institute of Macromolecular Compounds, Russian Academy of Sciences, Bol’shoi pr. 31, St. Petersburg, 119004 Russia
** Ott Institute of Obstetrics and Gynecology, Russian Academy of Medical Sciences, St. Petersburg, 199034 Russia
*** Institute of Cytology, Russian Academy of Sciences, Tikhoretskii pr. 4, St. Petersburg, 194064 Russia
Received August 1, 2003; in final form, January 27, 2004
Abstract—Cationic oligopeptides, including the amphipathic α-helical peptides, are applied to the targeted
delivery of DNA to eukaryotic cells due to their DNA-compacting properties and the ability to destabilize the
cell lipid bilayer in some cases. We synthesized the peptides differing in the number and location of residues of
decanoic acid covalently attached to Lys residues in order to combine the DNA-binding and the membrane
activities in a single molecule. We chose peptide structures that assisted in the formation of α helices. The DNA-
binding ability of the peptides and the membrane activity of their complexes with DNA were shown to depend
on the structure. The study of erythrocyte hemolysis by complexes with DNA of the pCMV LacZ plasmid and
the peculiarities of transfection of these complexes revealed a correlation between the hemolytic activity and
the expression level of the lacZ gene in cells.
Key words: cationic α-helical oligopeptides, hemolysis, nonviral DNA carriers, transduction, transfection
1
INTRODUCTION
of the transforming DNA or RNA, to be the specifically
bound by the cell surface, to penetrate trough cell mem-
brane structures, and to have a distinct signal of nuclear
localization for the active transport into nucleus [7–10].
The search for effective methods of systemic deliv-
ery of genes to eukaryotic cells is one of the most
important areas of development of gene therapy [1–4].
2
The membrane-active fragments of viral proteins
often are α-helical and amphipatic [11, 12]. In this
study, we describe the synthesis of a number of pep-
tides with various contents and locations of hydropho-
bic amino acid residues and Lys residues, including the
Lys residues acylated with the residues of decanoic
acid. These peptides combined the complex-forming
and membrane-active properties in a single molecule.
We found that the hemolytic activity and the helicity
degree of the synthesized peptides depended on their
decreased hydrophobicity. We also demonstrated the
ability of the peptides to form the complexes with
DNA, to protect DNA from the cleavage by nucleases,
and to facilitate the DNA transport into cell.
Three approaches are mainly used for the insertion of
marker or “therapeutic” genes into cDNA: (1) applica-
tion of recombinant viral vectors; (2) physical transfec-
tion methods (such as electroporation, DNA injections,
“gene gun” method, and others); and (3) the application
of nonviral systems for the DNA delivery to cells [5, 6].
The carriers on the basis of recombinant viruses (e.g.,
adenovirus ADV), retroviruses (RV), and herpes sim-
plex type 1 virus (HSV-1) have a number of advantages
over the other delivery methods due to their high spec-
ificity to certain types of tissues and their ability to be
effectively transfected. However, this method has some
serious drawbacks: potential pathogenicity, possibility
of a high immune response, and a limited quantity of
DNA that can be packed into small particles. Therefore,
a search for nonviral vector systems devoid of these
drawbacks and displaying some advantages of viral
carriers is an urgent problem. The modeling of the virus
penetration into cells is one of the approaches to the
search for nonviral transfection systems. The choice of
a model depends on its ability to form compact particles
RESULTS AND DISCUSSION
The introduction of alien genes into cell nucleus is
one of the important problems of gene therapy. A high
efficiency of the viral gene delivery is still not achieved
despite all the efforts in the field of search for the viral
systems transporting gene material into cells. An artifi-
cial DNA carrier should meet a number of demands, in
particular, it should be able to compact DNA and trans-
fer it trough cell membranes. This problem is often
overcome by the use of liposomes (lipoplex) [13]. The
1
Corresponding author; phone: +7 (812) 323-1050; e-mail: igury-
anov@mail.ru
2
Abbreviations: Acm, acetamidomethyl; Ahx, residue of ε-amino-
caproic acid; DIC, diisopropylcarbodiimide; TFA, trifluoroacetic
acid; and TFE, trifluoroethanol.
1068-1620/05/3101-0018 © 2005 Pleiades Publishing, Inc.

CATIONIC OLIGOPEPTIDES MODIFIED WITH LIPOPHILIC FRAGMENTS
Table 1. Structure of the synthesized peptides synthesized
19
Peptide
Structure*
C(Acm)–Ahx–YKAKKKKKKKWK–NH2
Mexp
FP
1877.1
2161.6
3063.0
FP1
C(Acm)–[K(C10)]–Ahx–YKAKKKKKKKWK–NH2
FP4
C(Acm)–Ahx–WK[K(C10)]KK[K(C10)]KKK[K(C10)]KYK[K(C10)]KK–NH2
ε
* K(C10) is N -decanoyllysined.
use of membrane-active compacting peptides can sig- sion of positive charges of the ε-amino groups of Lys
nificantly increase the transduction efficiency. Such residues. On the other hand, the neutralization of nega-
peptides very frequently have an amphipatic α-helical tive charges of Glu residues of JTS-1 in acidic medium
structure, with one of the helix side being hydrophilic led to an increase in the content of α-helices. The max-
and another being hydrophobic [14–16]. Such struc- imum helicity of the peptides was observed in 80% tri-
tures are formed by the alternating hydrophilic and fluoroethanol, because its dielectric properties are sim-
hydrophobic amino acid residues (positions i
i + ilar to cell membranes and it is often used for the con-
3/4) [17–19]. One of the examples of such peptides is formational studies of peptides in hydrophobic
JTS-1 (GLFEALLELLESLWELLLEA) whose intro- environment. Thus, we could expect a significant mem-
duction into the complex of the K-8 peptide brane activity for the peptides modified with the resi-
(YKAK8WK) with DNA sharply increases (by dues of decanoic acid owing to a high content of α-heli-
ces in them and their hydrophobicity.
6000 times) the expression level of luciferase gene in
the HepG2 cells [6, 20]. However, JTS itself cannot
bind DNA owing to the absence of positively charged
groups in its molecule. In this study, we synthesized a
number of peptides with various contents and locations
of unnatural hydrophobic residues (Table 1), in order to
find the structure that could form complexes with DNA
and, simultaneously, increase the interaction of such
carrier with the cell membrane [21]. Also, we plan fur-
ther to modify the peptides through sulfhydryl group
and, therefore, attached the N-terminal Cys residues
linked to the basic sequence via an Ahx residue.
Hemolytic activity of the peptides. Endosomolytic
capacity (i.e., the ability to penetrate through the endo-
some membranes into the cell cytoplasm) is an impor-
tant factor in the penetration of DNA with the carrier
into a cell. Erythrocyte membranes were chosen as a
model of biological membranes. The hemolytic activity
depended on the peptide hydrophobicity and the
medium pH (Fig. 4). The hemolytic activity increased
along with the increase in hydrophobicity: FP4 ≈ JTS-1 >
FP1 > FP. The FP peptide had no effect on the erythro-
cyte membranes. The FP4 and the JTS-1 peptides
exerted the maximum activity that was exhibited at the
pH values causing no aggregation due to the decrease in
the number of charges on the Glu and Lys residues.
Unlike the case of FP4, the hemolytic activity of JTS-1
increased with the decrease in its concentration and,
then, sharply decreased [6]. At the same time, the value
of medium pH did not affect the FP1 activity. Thus, a
correlation between the hemolytic activity and the con-
tent of α-helical conformation was observed in the pep-
tides. The peptides adopt this conformation in hydro-
phobic environment, probably including the erythro-
cyte membrane. One can expect that the hydrophobic
peptides would more effectively interact with cell
Ohmori et al. demonstrated by the example of mas-
toparan [17] that the introduction of one hydrophobic
residue into a peptide chain did not prevent interaction
of the peptide with nucleic acid and can also contribute
to the appearance of the peptide ability to form a com-
pact structure. The introduction of the capric (decanoic)
acid residue proved to be optimal in this case. Further
increase in the hydrophobic radical length did not lead
to a significant increase in the binding ability. In addi-
tion, one can expect a decrease in the solubility of such
a compound in water along with a further increase in
the length of hydrophobic radical. Therefore, it was
decanoic acid that we here chose for the modification of
ε-amino groups of Lys residues in cationic peptides.
The purity of the peptides synthesized was deter-
mined by HPLC, and their structures were confirmed
by mass spectrometry (see Figs. 1, 2).
Table 2. Percentage of the α-helical regions in the peptide
structure in various media
Peptide 0.01 N NaOH 0.01 N HCl
80% TFE
The study of the peptide structures by CD. The
structural characteristics of the peptides synthesized
were determined from their CD spectra under various
conditions (Fig. 3). One can see from the results
(Table 2) that the helicity of the peptides increased with
the increase in their hydrophobicity; however, the struc-
ture became unordered in acidic medium due to repul-
FP
8
25
27
15
Unordered
Unordered
Unordered
45
25
70
63
70
FP1
FP4
JTS-1
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 1 2005

20
GURYANOV et al.
membranes and intracellular structures than the peptide protection of the plasmid DNA by the FP carrier from
not modified with decanoic acid residues. The pH-
dependent membrane activity of the FP4 peptide that
was similar to the membrane activity of JTS-1 should
probably facilitate the release of the complex from the
cell endosomes.
the enzyme began with the charge ratio of 1 : 1.4. We
can see in the gel the fragments of cleaved DNA along
with the conformation forms characteristic of the intact
plasmid at a charge ratio of 1 : 0.7, which suggested that
the plasmid was partially cleaved at this charge ratio. A
sufficiently effectively protection of DNA by the FP1
and FP4 peptides began starting from the ratio of
1 : 0.7.
The formation of DNA–peptide complexes. The
ability of the peptides to the binding to DNA was exam-
ined by gel electrophoresis of DNA–peptide complexes
with different molar ratio of the components (method
of gel retardation) [22]. The electrophoretic mobility of
the complexes gradually decreased with the increase in
the peptide amount in complex (Fig. 5b). The complete
retardation of the plasmid mobility took place even at
the charge ratio 1 : 1 for DNA–FP peptide and 1 : 0.5
for DNA–FP1 peptide. In the case of FP1, an increase
in the complex density was observed for along with the
increase in the carrier content in complex. This fact was
proved by the practically complete disappearance of
The efficiency of cell transformation by the
DNA–FP, DNA–FP1, and DNA–FP4 complexes. The
cell transformation by the complexes of peptides FP,
FP1, and FP4 with the marker LacZ gene (plasmid
pCMVLacZ) was studied in each case at the DNA–car-
rier ratios close to those corresponding to the maximum
DNA protection from the action of DNase I (Fig. 7). We
found that the transformation efficiency that was pro-
vided by the FP peptide–carrier was less than 0.01%
fluorescence of the ethidium bromide bound to DNA in and was reliably the same as that of the native DNA.
the gel wells. The complete DNA retardation proceeded
at the charge ratio of DNA–FP4 of 1 : 0.7.
The maximum transformation efficiency when using
the FP1 peptide was achieved at the DNA–carrier
charge ratio of 1 : 2.8 and was approximately 0.34
0.05% of the cells. The most effective DNA–FP4
charge ratio was 1 : 1.4; it provided the expression of
3.61 0.7% of the cells. Thus, the introduction of four
residues of decanoic acid significantly increased the
expression level of the plasmid. This result can be
explained by a rather high rate of release of the complex
from the cell endosomes due to the membrane activity
Protection of DNA within the complex with
amphipatic peptides from DNase I. The complete
protection of plasmid from the action of hydrolytic
enzymes requires a higher content of the peptides syn-
thesized in complex than that necessary for the forma-
tion of completely compact DNA conformation. This
was proved by studying the stability of plasmid DNA
within the DNA–FP, DNA–FP1, and DNA–FP4 com-
plexes to the action of DNAase I (Fig. 6). The effective by the peptide itself.
A230
(a)
(b)
(c)
0
10 20 30 40 50 min
0
10 20 30 40 50 min
0
10 20 30 40 50 min
Fig. 1. Analytical HPLC of the (a) FP, (b) FP1, and (c) FP4 synthesized peptides on the Nucleosil C18 column (4.6 × 150 mm)
eluted with a gradient of acetonitrile in 0.1% TFA [(a, b) from 10 to 40% and (c) from 30 to 60% of acetonitrile within for 25 min]
at a flow rate of 1 ml/min; detection at 230 nm.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 1 2005

CATIONIC OLIGOPEPTIDES MODIFIED WITH LIPOPHILIC FRAGMENTS
21
(a)
(b)
6000
6000
4000
2000
4000
2000
0
0
1500
2000
m/z
2000
2500
3000 m/z
(c)
6000
4000
2000
0
1500
2000
2500
3000
m/z
Fig. 2. MALDI TOF mass spectra of the (a) FP, (b) FP1, and (c) FP4 peptides in water (pH 6.4).
The endosomolytic properties of the FP1 and FP4 ment of cells by chloroquine provides the most com-
peptides were investigated after a number of transfor- plete exhibition of the intrinsic membrane-active prop-
mations of the cells in vitro by the DNA–peptide com- erties of the carrier. These experiments demonstrated
plexes in the presence of chloroquine that disturbs the that the presence of chloroquine increased the effi-
process of endosome acidification and, by such a way, ciency of transfection by the DNA–FP1 complex from
prevents the action of hydrolytic enzymes. The treat- 0.08 0.02% to 5.15 0.31%. On the other hand, chlo-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 1 2005

22
GURYANOV et al.
(b)
6
4
40
20
0
(a)
2
210 220 230 240 250 260 270 280 290 300
0
205 215 225 235 245 255 265 275 285 295 305 315
–20
–40
–60
–80
–2
–4
–6
–8
–10
1
2
3
4
1
2
3
4
–100
(c)
5
0
(d)
5
0
205215 225 235 245 255 265 275 285 295 305 315
205 215 225 235 245 255 265 275 285 295 305 315
–5
–5
–10
–15
–20
–25
–30
–35
–10
–15
–20
–25
–30
–35
–40
–45
1
2
3
4
1
2
3
4
λ, nm
Fig. 3. CD spectra of the peptides (a) FP, (b) FP1, (c) FP4, and (d) JTS-1 in (1) water (pH 6.4), (2) HCl (pH 3.0), (3) NaOH (pH
10.0), and (4) 80% TFE.
roquine exerted little effect on the efficiency of trans-
fection by the DNA–FP4 complex: the content of trans-
fected cells remained practically the same.
H, %
100
On the basis of these results, we hypothesized that
the carrier FP4 peptide itself had endosomolytic prop-
erties, which are much more pronounced than those of
the FP1 peptide. This result agrees well with the exper-
iments of erythrocyte hemolysis. The similarity of
hemolytic properties of peptides FP4 and JTS-1 sug-
gests that their endosomolytic properties are similar.
However, the FP4 peptide can compact DNA and pro-
tect it from the action of hydrolytic enzymes present in
cell endosomes and lysosomes, which differs it from
the JTS-1 peptide.
80
60
40
20
0
1
2
3
4
5
6
CONCLUSIONS
0
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45
mM
We synthesized and studied a number of peptides
with different content and location of the residues of
hydrophobic amino acids and lysine, including the Lys
residues modified by decanoic acid, and demonstrated
that the ability of the studied peptides to transfect cells
Fig. 4. Hemolytic activity of the peptides (1, 2) FP1, (3, 4)
FP4, and (5, 6) JTS-1 at (1, 3, 5) pH 5.0 and (2, 4, 6) pH 7.8.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 1 2005

CATIONIC OLIGOPEPTIDES MODIFIED WITH LIPOPHILIC FRAGMENTS
(‡) (b) (c)
23
1
2
3
4
5
*
*
1
2
3
4
5
1
2
3
4
5
*
Fig. 5. Evaluation of the efficiency of binding of the pCMVlacZ plasmid with the peptides by the electrophoresis of DNA complexes
with (a) FP, (b) FP1, and (c) FP4 in 0.8% agarose gel containing ethidium bromide (0.5 µg/ml) at the DNA–peptide charge ratio of
(1) 1 : 0.1, (2) 1 : 0.3, (3) 1 : 0.5, (4) 1 : 0.7, and (5) 1 : 1. The intact DNA is marked with an asterisk.
with DNA increased with the increase in the number of gue). The products were further purified by the reversed
decanoic acid residues in these peptides. We consider phase HPLC on a Waters 600E chromatograph (Waters,
the FP4 peptide to be currently the most promising United States) equipped with a Vydac C-18 analytical
compound that exhibits a pronounced endosomolytic column (4.6 × 150 mm, Supelco, United States) and
activity among all the studied peptides. A further mod- Vydac C-18 preparative column (22 × 250 mm,
ification of the FP4 peptide (introduction of the signal Supelco, United States). The amino acid analysis was
of nuclear localization and the ligand for the directed performed on an AAA T339 M analyzer (Microtechna,
transport to the desired cell types) should enhance its Prague, Czech Republic). TLC was carried out on Silu-
endosomolytic and transfection properties and could fol plates (Kavalier, Czech Republic).
significantly increase the gene expression level.
The MALDI TOF mass spectrometry was per-
formed on a Voyager-DE spectrometer (United States)
using α-cyano-4-hydroxycinnamic acid (State Techni-
cal University, St. Petersburg). CD spectra were
recorded on a Mark V dichrograph (Gobin Ivan,
France). Cells were counted in the transformation
experiments using a light microscope (Karl Zeiss, Ger-
many).
EXPERIMENTAL
Derivatives of L-amino acids, DIC, TFA, trifluo-
romethanesulfonic acid, thioanisole, ethanedithiol,
1-hydroxybenzotriazole, (Fluka, Germany), 5-bromo-
4-chloro-3-indolyl β-D-galactopyranoside (X-gal,
Sigma, United States), DNase I (Promega, United
States), chloroquine, proteinase K (Sigma, United
States), and Escort TM (Promega, United States) were
used in this study. The solvents were from OAO Vecton
(St. Petersburg) and purified before use. The transfec-
tion experiments in vitro were carried out on cell cul-
tures of the HeLa human epithelial carcinoma (Institute
of Cytology, Russian Academy of Sciences, St. Peters-
burg). The cells were cultured on the RPMI 1640
medium (OOO Biolot, St. Petersburg), containing calf
blood serum (OOO Biolot, St. Petersburg) and genta-
mycin (OAO Dal’khimfarm, Khabarovsk).
The pCMVLacZ plasmid DNA (kindly presented by
Dr. B. Scholte from Erasmus University, Rotterdam)
was used in the experiments on DNA compacting,
DNA protection from enzymatic cleavage, and the plas-
mid transformation. The complex formation was stud-
ied by CD using DNA from calf thymus with molecular
mass of 8.6 MDa (Serva, Germany).
Peptide synthesis. Ne-Decanoyl-Na-tert-butyloxy-
carbonyllysine. A solution of N,N'-dicyclohexylcarbo-
diimide (2.3 g, 0.011 mol) in chloroform (15 ml) was
added to a solution of decanoic acid (1.7 g, 0.01 mol)
and pentafluorophenol (2 g, 0.011 mol) in chloroform
(25 ml) at cooling with ice. After 3 h, acetic acid (sev-
eral drops) was added to the reaction mixture, the pre-
cipitated N,N'-dicyclohexylurea was filtered off, and
the filtrate was evaporated in a vacuum. Triethylamine
(2 ml) and α-tert-butyloxycarbonyllysine (2.5 g,
0.01 mol) were added to the solution of pentafluo-
rophenyl ester of decanoic acid in DMF (25 ml). After
24 h, the reaction mixture was diluted with 1 N sulfuric
acid (200 ml), and the product was extracted with ethyl
acetate. The ethyl acetate solution was two times
washed with water, dried over anhydrous sodium sul-
fate, and evaporated to get the title product; yield 3.7 g;
Rf 0.62 (85 : 10 : 5 chloroform–methanol–acetic acid)
and 0.4 (1 : 3 petroleum ether–ethyl acetate).
Equipment. The peptides were synthesized on an
NPS 4000 semi-automatic peptide synthesizer (Neo-
Solid phase peptide synthesis was performed
system Laboratories, France) on a p-methylbenzhydryl- according to the Boc-strategy using p-methylbenzhy-
amine resin (Neosystem Laboratories, France). The drylamine resin as a polymer support, DIC/HOBt as a
peptides were purified by the gel exclusion chromatog- coupling reagent, and trifluoroacetic acid as a depro-
raphy on a Bio-Gel P2 (BioRad Laboratories, United tecting reagent. The following protocol was used when
States) with the detection on an Uvicord S 2138 (Pra- calculating for 1 g of resin with the capacity of
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 1 2005

24
(‡)
GURYANOV et al.
% of the LacZ+ cells
7
6
5
4
3
2
1
0
Without chloroquine
With chloroquine
6
5
4
3
2
1
0
1:0.7 1: 1.4 1 : 2.8
1:0.7 1:1.4 1:2.8
Fig. 7. The dependence of the efficiency of transfection of
the HeLa cells on the charge ratios between DNA and the
FP1 peptide and between the DNA and the FP4 peptide.
1
2
3
4
5
6
K–
K+
(b)
if the ninhydrin test was positive (incomplete cou-
pling). The complete deprotection and the cleavage of
the peptide from the polymer were achieved by the
treatment with trifluoromethanesulfonic acid (1 ml) in
TFA (10 ml) in the presence of thioanisole (1 ml) and
ethanedithiol (0.5 ml) for 1 h at cooling with ice and for
1.5 h without cooling. The reaction mixture was diluted
with ether (100 ml), and the precipitate was filtered.
The resulting peptide was dissolved in TFA (30 ml), fil-
tered for the removal of resin, and precipitated with
anhydrous ether (200 ml). The nonadecapeptide JTS-1
was prepared according to the procedure [20]. The pep-
tides were preliminarily purified by gel chromatogra-
phy on a column (600 × 25 mm) with BioGel P-2 (Phar-
macia, United States) eluted with 6% aqueous solution
of acetic acid with detection at 226 nm.
1
2
3
4
5
6
K– K+
(c)
The further purification of peptides was carried out
by HPLC using 0.1% aqueous TFA as eluent A and
mixtures of acetonitrile solution in eluent A as eluent B
at a flow rate of 1 ml/min (for the analytical HPLC) and
10 ml/min (for the preparative HPLC). The purity of
peptides was >95%. The structures of the synthesized
peptides were confirmed by mass spectrometry and
amino acid analysis.
1
2
3
4
5
6
K–
K+
Fig. 6. The efficiency of protection of the plasmid DNA
from DNAase I by (a) FP, (b) FP1, and (c) FP4 at the charge
DNA–peptide ratio of (1) 1 : 0.2, (2) 1 : 0.4, (3) 1 : 0.5, (4)
1 : 0.7, (5) 1 : 1.4, and (6) 1 : 2.8 compared with (K–) the
action of DNaase I without the peptides and with (K+) the
intact DNA.
Determination of hemolytic activity was done as
described in [6]. Human erythrocytes were isolated
from the fresh blood by centrifugation at 1500 rpm on
a Janetzki centrifuge (Germany) in the presence of
3.8% sodium citrate and thrice washed with 0.9% solu-
tion of sodium chloride. In each experiment, a solution
of peptide at the chosen concentration was prepared in
a buffer (0.15 ml) containing 0.01 M Na2HPO4, 1.6 mM
NaH2PO4, and 0.15 M NaCl (pH 5.0 or 7.8). Then, the
erythrocyte suspension (10 µl) was added. The mixture
was incubated for 1 h at 37°ë at the occasional shaking.
The hemolysis degree was determined according to the
change in absorption of the supernatant at λ 492 nm
after the removal of cells by centrifugation. We
assumed the absorption values corresponding to 0% of
hemolysis and to 100% hemolysis observed after the
0.5 mmol/g: (1) twice deprotection with 65% TFA (2 ×
1 min), and washing with dichloromethane (2 × 10 ml,
15 min); (2) washing with dichloromethane (3 × 10 ml)
and DMF (2 × 10 ml); (3) deprotonation with 10% TEA
for 1 min and washing with DMF (2 × 10 ml) for 2 min;
(4) washing with DMF (2 × 10 ml); (5) coupling with
the corresponding Boc-amino acid (1.5 mmol) using
DIC (1.5 mmol) and 1 M solution of HOBt in DMF
(1.5 ml); and (6) washing with DMF (2 × 10 ml) and
dichloromethane (2 × 10 ml); monitoring with ninhy-
drin test. The protocol was repeated starting from stage 3 incubation of erythrocytes in the buffer solution and in
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CATIONIC OLIGOPEPTIDES MODIFIED WITH LIPOPHILIC FRAGMENTS
25
water, respectively. The given data are the average culture medium (1 ml per well) was added. The plasmid
results of three independent experiments.
DNA compacted with the EscortTM commercial car-
rier and the native DNA were used as a positive and a
negative control, respectively.
CD spectroscopy. CD spectra were recorded at
room temperature. The measurements were made at the
range of wavelength from 195 to 320 nm. The working
peptide concentration was 30 µg/ml. Cuvettes with the
lengths of optical path of 1 cm and 0.2 cm were used.
The conformational states of peptides were estimated
using the CDNNV2.1 program (Gerald Bohm, Institute
für Biotechnologie, Wittenberg, Germany).
The activity of β-galactosidase was measured 48 h
after the introduction of complexes in the following
way. The cells were fixed on the cultural plates by the
treatment with 0.5% glutaraldehyde in PBS (1.7 mM
KH2PO4, 5.2 mM Na2HPO4, and 150 mM NaCl, pH
7.5); washed with PBS containing 2 mM MgCl2 and
with the detergent solution (0.01% sodium deoxycho-
late, 0.02% Nonidet P-40, and 2 mM MgCl2); and incu-
bated for 16 h in a mixture of X-gal (2 mg/ml),
ä3Fe(CN)6 (5 mM), K4Fe(CN)6 (5 mM), and MgCl2
(2 mM) in PBS. The plates were washed with PBS and
inspected under the Zeiss phase-contrast microscope
(Germany). The presence of β-galactosidase in the
nuclei of transfected cells was determined according to
the specific violet coloring. The portion of cells
expressing the bacterial β-galactosidase was the crite-
rion of the transfection efficiency of the peptide com-
plexes. The data on the activity of β-galactosidase are
the average result of three independent experiments.
Preparation of the complexes of peptides with
DNA of the pCMVLacZ plasmid and studies of their
stability by the method of gel retardation. A solution
(20 µl) containing various amounts of peptide was
gradually added to an aqueous solution (20 µl) of plas-
mid DNA (1 µg). After 1-h incubation, the resulting
complexes were analyzed by the method of gel retarda-
tion (according to decrease in the electrophoretic
mobility of DNA in 0.8% agarose gel containing 2.7 µl
of ethidium bromide in 400 g of dry agarose). The
results presented in Fig. 5 are average from two inde-
pendent experiments.
An analysis of stability of the plasmid DNA in the
peptide complexes to the hydrolysis by nucleases.
The efficiency of protection of the pCMVLacZ plasmid
construct from the hydrolysis by DNase I was studied
in the complexes with each synthesized peptide at dif-
ferent DNA–peptide charge ratio. DNase I (0.1 activity
U) was added to the solutions of complexes and incu-
bated for 1 h at 37°C. DNase was then inactivated by
heating to 70°C for 15 min. The complex was treated
with proteinase K and then subjected to the phenol–
chloroform extraction [23]. The DNA state was evalu-
ated by gel electrophoresis. The incompact DNA
treated with DNase I and the native plasmid DNA were
used as a negative (K–) and a positive (K+) controls,
respectively. The average results of two independent
experiments are given.
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