Arch Pharm Res Vol 30, No 8, 1027-1040, 2007

http://apr.psk.or.kr

Nucleic Acid-Phospholipid Recognition: Fourier Transform

Infrared Spectrometric Characterization of Ternary Phospho-
lipid-Inorganic Cation-DNA Complex and its Relevance to
Chemicopharmaceutical Design of Nanometric Liposome
Based Gene Delivery Formulations
Yasemin Manavbasi and Erhan Süleymanoglu 1,2

Dept. of Pharmacology, Faculty of Pharmacy, Gazi University, 1Dept. of Pharmaceutical Chemistry Faculty of Phar-
macy, Gazi University, and 2The Central Laboratory, Faculty of Pharmacy, Gazi University, Hipodrom, 06330-
Ankara, Turkey

(Received December 28, 2006)

The present work is a continuation of our previous microscopic, spectroscopic and microcalori-
metric measurements of liposomes and poly(ribo)nucleotides and their ternary complexes with
inorganic cations as an alternative formulation employing zwitterionic phospholipids instead of
cytotoxic cationic lipids. Current report describes Fourier transform infrared spectrometric
study as employed to follow structural transitions of newly proposed ternary solid neutral lipid-
Mg2+-DNA complexes as promising gene delivery formulation. Spectra of the unbound compo-
nents are compared with those obtained after their complexation as binary and ternary mix-
tures. Results are described at the levels of carbonyl, phosphate, choline and CH groups and
discussed as effects of nucleic acid and phosphatidylcholine moiety on each other in the
absence and in the presence of Mg2+. The infrared spectra of DNA-lipid phases are dominated
by the lipid specific absorption bands, with a very little contribution of DNA. Data suggest that
upon recognition of DNA with lipids, the DNA undergoes helical transition. Mg2+ effects are
interpreted as dehydrations of phosphates and H-bonding inducing effects on carbonyl groups.
The role of residual and surface water on these associations, as well as on chain packing is
also discussed followed by possible implications of the ternary complex formation for further
gene transfer designs.
Key words: Phospholipid-nucleic acid recognition, FTIR, Hydration forces, Mg , Vibrational
2+

bands

INTRODUCTION delivery vehicles in human gene therapy (Templeton,

Besides general interest of depicting and visualizing
membrane-nucleic acid self-assemblies in terms of cell
biological phenomena such as transcription, translation
and cell division, research efforts on determining interations
between lipids and nucleic acids has arisen recently after
realization of their potential for employment as gene
Correspondence to: Faculty of Pharmacy, The Central Laboratory
and Dept. of Pharmaceutical Chemistry, Erhan Süleymanoglu,
Gazi University, Gazi Mahallesi, Polatli Caddesi, No:115/5, Yeni-
mahalle, 06560-Ankara, Turkey
Tel: 90-312-211-1947; Fax: 90-312-223-5018
E-mail: esuleymanoglu@gazi.edu.tr
2001, 2002, 2003; Ulrich, 2002). Currently employed lipid
based gene transfer designs utilize lipofection mostly
undertaken by cationic and helper zwitterionic lipids
(Ulrich, 2002; Templeton, 2003). Having considered the
cytotoxicity of cationic lipids (Dass, 2002), complexes
formed between neutral phospholipids and nucleic acids
in a variety of conformations and sizes attract research
interest as a possible alternative therapeutic gene delivery
formulation. Gene transfection assays rely on structural
information on partcipating macromolecules in overall
supramolecular arrangement of lipoplexes. Such data
concerning physicochemical properties of lipid-nucleic
acid complexes of various structures required for efficient

1027
1028
transfection is incomplete.
Despite regular reports on nucleic acid (both DNA and
RNA) aggregation with liposomes of different lipid com-
position, the colloidal factors and intermolecular forces
governing their complex formation remain to be determined.
Prerequisites to be met regarding their size homogeneity,
serum stability, ability to keep the entrapped poly(ribo)
nucleotide sequences in desired concentration and re-
producible manufacturing issues additionally render the
development of such gene delivery tools difficult. In this
context, following secondary structural transitions of both
DNA and phospholipids upon their interaction becomes
essential. Using optical microscopic, thermodynamic,
hydrodynamic, spectroscopic, as well as other methods of
structure determination, multilamellar, liquid crystalline
phases of alternating lipid-DNA arrays have been proposed
as the resulting structure (Choosakoonkriang, 2001;
Safinya, 2001). Attempts to monitor changes in secondary
structure of DNA after binding with lipids employing
circular dichroism (Choosakoonkriang, 2001; Brown, Kueltzo
and Middaugh, 2001) has encountered certain hurdles
due to formation of large fluctuations in peak position and
intensity from the induction of chiral supramolecular struc-
tures, as well as problems arising from scattering and
absorption flattering artifacts (Choosakoonkriang, 2001).
Fourier Transform Infrared (FTIR) spectroscopy and
Raman scattering can be potentially used to obtain useful
information about the fine structure of both lipids and
nucleic acids upon their association (Choosakoonkriang,
2001) without the use of bulky extrinsic probes which
could perturb the membrane structure, as employed by
fluorescent, NMR, electron spin resonance (ESR), and
other techniques.
Particularly, IR spectroscopy have been extensively
applied for studying nucleic acid structural changes
(Taillandier and Liquier, 2002). Thus, vibrational studies
have been performed on single-, double-, and triple-strand-
ed nucleic acid conformations, as well as on different RNA
structural elements, backbone conformational substances
and on various structural transitions, in the study of
nucleic acid hydration and its interaction with ions and
drugs, and informative review on infrared bands of nucleic
acids in solution was recently published (Banyay, Sarkar
and Gräslund, 2003). On the other hand, FTIR spectroscopy
has been used in the field of lipid chemistry, as well-for
structural studies on a submolecular level (Le Bihan and
Pézolet, 1998; Selle and Pohle, 1998; Silvestro and Axelsen,
1998; Ruthven, Lewis and Lewis et al., 2001; Lewis .,
et al
2001). Following this approach, important features con-
cerning polar headgroup, nonpolar CH residues and the
glycerol backbone have been clarified (Lewis and
McElhaney, 1996; Selle, Pohle and Fritzsche, 1999; Gauger
et al., 2001; Pohleet al ., 2001).
Y. Manavbasi and E. Süleymanoglu
The versatility of FTIR comes from the fact, that it can
be employed in a wide range of environments, such as
following membrane binding with ions, polymers, drugs,
and other biomolecules (Lewis and McElhaney, 1996;
Garidel, Blume and Hübner, 2000; Taillandier and Liquier,
2002), at the air-water interface (Hübner and Blume,
1998), in organic solvents, or in dehydrated state (Zundel,
1983; Johnston, 1991; Forato, Bernardes-Filho and Colnago,
1998; van der Weert et al ., 2001; Günzler and Gremlich,
2002). Since FTIR spectroscopy operates with diminished
effects of light scattering and can be applied to heteroge-
neous systems providing the researcher with informative
bands concerning participating molecules, the potential of
this method for characterization of complex formed
between various lipids and nucleic acids of different con-
formations appears to be greater than other spectrometric
techniques.
While large amount of data on DNA, lipids and their
assemblies have been collected (Templeton, 2001, 2002,
2003; Ulrich, 2002; Dass, 2002), mainly using hydrated
phospholipids (Choosakoonkriang et al., 2001; Pohle et
al ., 2000), data reporting their behaviour in dry or solid
state, which could be of comparative value, is insufficient.
Therefore, we have used FTIR spectroscopy to characterize
the recognition and complexation of a synthetic model
phospholipid with DNA, to compare these results with
previous reports on similar supramolecular assemblies in
solution. Since, development of lipid based delivery vehicles
relies on physicochemical information on their structure-
activity relationships, compairing their physical properties
in different states would improve further their designs as
pharmaceutical formulations. In this context, we present
here a comparative study of complexation between L-α-
phosphatidylcholine, Mg2+ and DNA in the dry state. We
have used FTIR spectroscopy to investigate their recogni-
tion at the levels of carbonyl, the phosphate, the choline
group, and CH groups.
MATERIALS AND METHODS
Materials
Calf thymus DNA (D-4764; 5 units) of approximate size
>13 kb was obtained from Sigma-Aldrich Chemie GmbH,
89552 Steinheim, Germany and stored at 4oC. L-α-phos-
phatidylcholine (P3556), FT-IR-grade KBr (22.186) and
MgCl2.2H2O (M8266) was obtained from the same supplier.
All reagents were of highest analytical grade and were
used without further purification.
Methods
Preparation of ternary DNA-Mg2+-phospholipid com-
plexes
FTIR spectra were taken for each single component
Nucleic Acid-Phospholipid Recognition
engaged in DNA-lipid complex formation for three combina-
tions of binary mixtures (Mg2+-DNA, DNA-phosphatidyl-
choline, phosphatidylcholine-Mg2+) and for the ternary
mixture (DNA-Mg2+-phosphaidylcholine) in equimolar ratios
for comparison of our recent calorimetric studies on this
ternary complex (Süleymanoglu, 2005).
Fourier transform infrared spectroscopy
KBr pellet method was employed as FTIR sampling
technique. The samples were prepared by admixing ca.
2-3 mg of their content with ca. 300 mg of spectropscopy-
grade KBr (KBr/DNA=75:1 by mass) and pressing the
mixtures with a die press, as described previously (van
der Weert et al ., 2001; Pevsner and Diem, 2003). For
comparison, transmission spectra of aqueous solutions of
nucleic acids, phosphatidylcholine and their complexes in
the presence of Mg2+ were measured on KBr windows.
Similarly to the mentioned reports, our criterion for sub-
traction of water from the spectra of aquoeous solutions of
nucleic acids was a straight baseline between 1750 cm-1
and 1990 cm-1, while that for lipids was the CH2 stretching
vibration signal seen as bands in the region from ~2800-
3100 cm-1 (Hauser, 1991; Pohle et al ., 2000; Binder and
Zschörnig, 2002). Spectra were measured on Vectra 22
FTIR spectrometer (Bruker Analytische Messtechnik GmbH,
Karlsruhe, Germany), equipped with He-Ne laser. Data
were collected at 2 cm-1 resolution, converted to a convini-
ent format showing authomatically the vibrational frequen-
cies of the major functional groups using the original
software OPUS 3.0, provided by the manufacturer.
RESULTS AND DISCUSSION
The objective of this study was to follow structural
transitions of DNA, phospholipids and their complexes for
comparative reasons, which can be further used in bio-
pharmaceutical reasoning and physicochemical profiling
and design of promising lipid-based gene delivery vehicles,
as well as for analytical purposes-in sequence-specific
and non-specific double stranded and single stranded
nucleic acid chromatography.
FTIR has been widely used to determine biomolecular
structures in unbound form and in lipid environment as an
alternative to the other spectroscopic methods (Zundel,
1983; Johnston, 1991; Lewis and McElhaney, 1996; Hübner
and Blume, 1998; Forato, Bernardes-Filho and Colnago,
1998; Garidel, Blume and Hübner, 2000; van der Weert et
al., 2001; Taillandier and Liquier, 2002; Günzler and
Gremlich, 2002). In the majority of cases, the vibrational
spectra are recorded using samples prepared in water,
D2O, organic solvent, as well as in membraneous sur-
roundings (Le Bihan and Pézolet, 1998; Selle and Pohle,
1029
1998; Silvestro and Axelsen, 1998; Pohle et al., 2000;
Ruthven, Lewis and Lewis et al., 2001; Lewis et al., 2001;
Choosakoonkriang et al., 2001). Under these conditions,
the FTIR spectrum is obtained only after the subtraction of
the solvent spectrum, which can introduce artificial
spectral shifts due to solvent concentration effects. To
correct these distortions, certain precautions have to be
met (Forato, Bernardes-Filho and Colnago, 1998). One
way to reduce these complications is to use dried sample
preparations-films or powder dispersions, or KBr pellets.
All these procedures themselves have to be employed
carefully, since frequently the sample has to be lyophilized
and submitted to pressure (KBr pellet), which can cause
sample denaturation. However, since FTIR spectra show
large fluctuations in solution and in solid state, performing
these experiments in parallel-both with hydrated and
dehydrated samples provides with useful information
regarding structural transitions of free molecules and after
their complex formations with a wide variety of ligands.
These differences were mainly assigned either to changes
in secondary structures, or to removal of water (Forato,
Bernardes-Filho and Colnago, 1998; van der Weert .,
et al
2001).
To address the relevance of this reasoning to DNA-lipid
assemblies, we have carried out studies on their binary
and ternary complexes with Mg2+, comparing their struc-
tures in the solid and solution states. Mg2+ was chosen as
agent trigerring DNA-phospholipid complex formation in
terms of its role in regulating cellular functions. Having
considered its high intracellular concentration, its function
in energy metabolic pathway reactions, with further impli-
cations in enzymatic interconversions utilizing phosphate
group transfers, makes it preferred naturally occuring
inorganic cation, as compared to currently employed
synthetic polymers as DNA compaction agents. Mg2+ has
attracted scientific interest in terms of its stabilizing role on
nucleic acid structure, in cellular inorganic chemistry,
cytolasmic macromolecular crowding, as well as in DNA
packaging. Thus, DNA-Mg2+-lipid ternary system has
gained importance both as a model for transcription-
translation, and more recently as a promising gene delivery
formulation. However, despite that its structure determination
and thermotropic phase behaviour have been followed
using spectroscopic (Süleymanoglu, 2004) and thermody-
namic (Süleymanoglu, 2005) approaches, its study by
infrared spectrometry becomes complicated due to
interfering vibrations of water at 1645 cm-1 and D2O at
1450 cm-1 and 1210 cm-1 (Banyay, Sarkar and Gräslund,
2003). Therefore, we have undertaken this FTIR study of
this ternary complex using samples prepared by KBr
pellet method.
1030 Y. Manavbasi and E. Süleymanoglu

Overall IR absorption pattern of phospholipid- Apparently, despite water removal, the spectral region
metal ions-DNA ternary complex and its individ- arround 3420 cm-1 and below 850 cm-1 are due to O-H
ual components bending vibrations of water (Choosakoonkriang ., et al

Fig. 1 is a FTIR absorption spectra of calf thymus DNA-
phosphatdylcholine complex and its components. The
results of these molecules are grouped together because
they share major functional group frequency bands.
These are also shown in Table I. The major bands
showing structural transitions of DNA and lipid as binary
and ternary complexes with metal cation, as well as in
free form and the effects they insert in each other are
given in following figures. The infrared absorption spectra
of calf thymus DNA is based on PO2- groups' vibrational
modes, which are also sensitve to hydration (Fig. 1-A). In
this respect, the figure demonstrates the responce of DNA
to dehydration. Approximately 10 well seen DNA absorption
bands occur in the region between 530-3425 cm-1. The
majority of hese are given also in Table I, where the OPO
group stretching motions, ribose ring vibration, furanose
CO stretch, existance of particular type of helices and
bond conformations, as well as C=N stretchings are
considered as the most informative. Interestingly, the IR
absoption peak at 1068.7 cm-1 is an indication for possible
formation of C-form of DNA (Pohle ., 2000), despite
et al
the presence of stronger bands indicating the predominating
false Z-helices formed possibly due to KBr effects (Table
I). The region encompassing 530-800 cm-1 represents out
of plane base vibrations (Banyay, Sarkar and Gräslund,
2003). The band seen at 3425 cm-1 is ascribed to water.
2001). The region between 1450-1750 cm-1 concerns
C=O, C=C and C=N stretchings and is sensitive to H-
bonding. Water substraction allows an increased possibility
for base-base H-bonding. In comparison with DNA samples
in the form of thin films, the KBr pellet sample spectra
yields peaks shifted to higher wavenumbers (Pevsner and
Diem, 2003). The intensities of symmetric stretching vi-
bration of the group (νsPO2−) at 1068.7 cm-1 and antisym-
metric (νasPO2−) drop after dehydration (data not shown
for brevity). The effects appear to be the result of the
water content. Fig. 1B. is absorption spectrum of DNA-
Mg2+ binary mixture, where major structural transitions are
observed in the region 500-1750 cm-1. Changes of native
DNA spectrum are apparent in the shifts of the character-
istic absorpton bands of the phosphate groups and bases
and in alterations of their intensities. Some sort of out-of-
plane and wagging vibrations of C-NH2 bond takes place
at 470 and 616 cm-1, respectively following metal cation
binding. Furanose CO stretching vibration of pure DNA
shifts to 1051.8 cm-1 upon recognition by Mg2+. This cation
binding also distorts Z-helices of free DNA (Fig. 1A), as
well as the C=N ring vibration of guanines in double-
stranded chain, indicating changes of base stacking and
base pairing, giving further evidence that ligand binding to
specific bases takes place. The major surface water band
at arround 3400 cm-1 of native DNA appears as two

Fig. 1. IR absorption spectrum of unbound DNA and lipid structures and their equimolar binary and ternary complexes: A. calf thymus DNA; B.
DNA-Mg ; C. L-α-phosphatidylcholine; D. DNA-L-α-phosphatidylcholine; E. L-α-phosphatidylcholine-Mg ; F. ternary DNA-Mg -L-α-phosphatidyl-
2+ 2+ 2+

choline complex.
Nucleic Acid-Phospholipid Recognition
Fig. 2. Effect of phospholipid on DNA structure employing vibrational
frequency region 850-2000 cm , where the most characteristic nucleic
-1
acid bands are usually reported. Three different IR absorption spectra
show unbound DNA, its binary complex with L-α-phosphatidylcholine
and their ternary complex with Mg from top to the bottom,
2+
respectively.
distinguishible bands upon Mg2+ binding seen at 3405.7
cm-1 signal and a smaller one preceding it at 3240.4 cm-1.
This is apparently due to formation of water molecules in
an effective H-bonded structure, referred to as ‘lattice
water'’(Binder and Zschörnig, 2002). Similarly to Mg2+, L-α-
phophatidylcholine affects DNA in a more profound way,
as seen by its structural transitions (Fig. 1C vs. Fig. 1D).
Obviously, this is a phospholipid dominated absorbance
spectrum with a very little conribution from DNA. Interest-
ingly, however, the surface water bands are diminished,
indicating a Mg2+-driven hydration-dehydration effects.
This result confirms the recent FTIR spectroscopic study
of cationic lipid-DNA complexes, leading to interesting
suggestion to use the structural data of the lipid part in this
1031
complex without significant disturbance by DNA absorption
(Pohle et al ., 2000). Fig. 1C represents a phospholipid
spectrum and is given for referative and quantitative
purposes with all important vibrational band frequencies
also separately (Fig. 4). Similarly, Fig. 1E is an important
case of phospholipid-metal ion interaction with significant
biological implications and thus is also given as a separate
figure in three different IR absorption regions (Fig. 6).
FTIR analysis of the triple complexes formed between
calf thymus DNA-Mg2+-phosholipid was performed ac-
cording to previously reported reasoning (Bruni et .,
al
2001; Süleymanoglu, 2004; 2005). The evaluation of the
parallel effects of divalent metal cation and L-α-phosphati-
dylcholine on the nucleic acid absorption spectra, or
alternatively of metal ion and DNA on lipid is emphasized.
The comparisons were performed in those IR regions,
where the bands of DNA and L-α-phosphatidylcholine do
not intefere (phosphate, carbonyl and CH vibrations).
Thus, the ternary complex is compared with two controls
of binary mixtures trying to obtain synergic effects. The
complete IR absorption spectrum is shown in Fig. 1F and
is depicted in different wavenumber regions corresonding
to characteristic bands of phosphate, carbonyl and CH
stretching vibrations in Fig. 2, 3 and Table I. The last
spectrum in the bottom of Fig. 1 indicates that major
structural changes occur in ternary mixture, as compared
with unbound lipid, as well as with DNA-phospholipid
mixture. As oppose to phospholipid dominated spectrum
(Fig. 1D and Fig. 4), ternary complex formation leads to
alterations of stretching motions of the OPO group, as
well as rocking vibrations (500-750 cm-1). Mg2+-induced
effects are clearly seen in Fig. 1B, 1E and 1F. These are
appearance of new CO band (~1735 cm-1), new H-
bonding effect in ~2254 cm-1, as well as the surface water
effect at ~3400 cm-1. The carbonyl bands appear in the
ternary complex spectrum with a decreased intensity,
demonstrating the competition of Mg2+ with surface water
molecules for ligand binding. The new CO band at ~1735
cm-1 is a typical marker for H-bond formation between the
carbonyls and water (Binder and Zschörnig, 2002). Ap-
parently, carbonyl groups become more accessible to the
water molecules because of their involvement in the
hydration shell of ions. Mg2+ induces less effects on
phosphate moieties and the spectrum bears the profile of
lipid-metal cation binary mixture in this region (Fig. 1E vs.
1F). Having considered that the interaction with the polar
lipid headgroups results in alterations in the acyl chains,
the retention of the hexagonal packing in the triple com-
plex with Mg2+ is highly probable. The characteristic bands
of ion binding are diminished, as compared with DNA-
phospholipid binary complex, indicating the formation of
increased fraction of gauche conformers. Effect of bound
water formation, also discussed below as a significant part
1032
Fig. 3.Effect of nucleic acid recognition on phospholipid spectrum. Three different IR absorption pattern characteristic for unbound lipid, its nucleic
acid bound binary form and their ternary complex with Mg , respectively are shown in three different wavenumber regions for better comparison of
2+
DNA and Mg effects on phosphate, carbonyl and CH stretching modes.
2+
of lipid-metal cation complex, is obvious (3000-4000 cm-1).
This IR absorption is ascribed to that peculiar water, which
creates an additive effect to that present in the DNA-metal
cation complex and its lipid-bound form (Pohle ., 2000). et al
The appearance of two water bands are not observed for
the OH bands in neither DNA (Fig. 1A), nor in lipid (Fig.
n to highly immobilized water molecules engaged in long-
1C) and lipid-DNA complex (Fig. 1D). The effect of this
residual water in the lipid sample was confirmed by
differential scanning calorimetric thermogram of the L-α-
phosphatidylcholine (Fig. 5), which otherwise could be
thought and misinterpreted as an antisymmetric stretching
of C-NH2 bond vibration (Table I). The thermogram clearly
shows shift to much lower melting temperatures (Tm) of
~31oC, instead of 39oC, as usually reported for this lipid in
LIPIDAT database for lipid phase transitions (http://www.
lipidat.chemistry.ohio-state.edu), the effect being acsribed
to the presence of residual water in the commercially sold
lipid sample (http://www.sigma-aldrich.com). Interestingly,
its glass-transition temperature (Tg) remains unaffected.
Extrapolations from solid dispersions in FTIR experiments
to structural aggregations in aqueous state should be
done with precautions. The broad water band at ~3400
cm-1 is a result of superposition of several underlying single
bands forming a number of coexisting subpopulations of
Y. Manavbasi and E. Süleymanoglu
water aggregates with different size and binding states.
The two resolved OH sub-bands seen in DNA-Mg2+,
n
phospholipid-Mg2+, and their ternary complex are probably
due to the formation of different types of hydration water
(Pohle ., 2000). The stronger band appears to be due
et al
lasting strong H-bonds as a part of bound water in the
interfacial region between DNA and lipid contact zone.
The preceding smaller shoulder is ascribed to the formation
of second type of weakly H-bonded water, situated in
interstitial space between adjacent lipid structure and
DNA, the formation of which is determined by overall
geometry of the complex keeping it away from possible
thermodynamically unfavourable structure formations.
IR absorption of DNA structural transitions
Since the vibrational spectra of DNA contains a high
degree of masking and overlapping bands, it is useful to
examine the structural changes occuring with the nucleic
acid employing the 800-1800 cm-1. Fig. 2 depicts structural
transitions of nucleic acids within this wavenumber region,
in unbound form, after recognition with phospholipid as
binary mixture and as ternary complex upon association
with phosphatidylcholine moiety, trigerred by Mg2+, shown
Nucleic Acid-Phospholipid Recognition 1033

Table The wavenumbers and spectral assignments of major strong IR bands in DNA-Mg2+-phosphatidylcholine complex and its
I.

components.
Moiety Band position (cm )
-1
Assignment References
835.0 Stretching motions from OPO group [10]
917.5 Ribose ring vibration
1068.7 Furanose CO stretch of backbone; possible formation of C-type DNA [10,27]
DNA a) 1265-1264 GC helices [10]
1275 dT (CN3H bond) [10]
1337.8 N-type of dA, dT in 3'-endo/anti- [10]
1594.3 C=N ring vibrations of G-ds [10]
3425.0 Water band; in other circumstances antisymmetric stretching of C-NH bond 2
[10,32,33]
470.0 Out-of-plane vibration [10]
616 Wagging of planar C-NH bond 2
[10]
1051.8 Vibration of ribose backbone [10]
DNA-Mg 2+ b)
1634.4 In-plane ring vibration of T/ss; C=N, C=C ring vibration of A/ss [10]
2253.7
3240.4 Residual water [10]
3405.7 Residual water; antisymmetric stretching of C-NH bond 2
[10]
925.7 Shifted to higher wavnumbers ribose ring vibration [10]
970.3 CC stretch frequency of the B-form DNA backbone (when seen as a singlet peak) This study
1090.6 →
Symmetric PO − stretching-insensitive to B A transition of DNA backbone
2
This study
1175-1188 Indication of A-form of helix. Sugar-phosphate backbone vibration with a high This study
contribution from the sugar moiety in C3'-endo/anti-type of puckering
DNA- 1244.6 Main marker for antisymmetric PO stretching-depicting A form of DNA
-

2
This study
phospholipid c)
1339.5 N-type dA, dT in in C3’-endo/anti-type conformation This study
1409.3 N-type C3’-endodeoxyribose in A-form helices This study
1467.2 Indication of A-form helix (1453-1457) This study
1594.2 C=N ring vibration of double stranded G-strand (when sen as a weak peak between This study
1575-1590)
1737.1 νC=O stretch signal This study
3424.9 Water band [39-41]
2922 ν s[(CH ) ] [28,35,36,37,38]
νs[(CH ) ]
3 4

2852.2 [28,35-38]
νs[C=O]
3 4

1736.6 [28,34-37]
700 – 1500 δ[C-H] [36-38]
1467.4 scissoring [(CH ) ] δ [28,37,38]
Phospholipid 1378.1 δ[CH ]
2 n

[28]
1242.6 ν 2

as[PO ]
-
[28,34,35]
ν [PO ]
2

1091.5 2
-
[28,37]
970.4 asymm. [ N-(CH ) ] stretching region
+
[35]
ν [C-C-N]
3 3

927.0 [37]
826.9 ν [P(-O-C) ] [37]
rock. ν[(CH ) ]
n

721.8 2 n
[37]
975.7 ν [C-C-N] [27,39-41]
1095.4 ν [PO −] [27,39-41]
1467.6
2

scissoring [(CH ) ] δ 2 n
[27,39-41]
1635.3 H-unbound CO [39-41]
Phospholipid- 1734.5 H-bonded CO [27,39-41]
Mg 2+
2261.5 CH stretching [27,39-41]
2852.9 CH stretching
2
[27,39-41]
2923.3 CH stretching
2
[27,39-41]
3241.4 Interstitial water [39-41]
3405.5 Bound water [39-41]
975.1 Shifted CC stretch of B-from backbone of DNA [10]
1095.8 Diminished and shifted to higher wavenumbers symmetric PO − stretching-insensitive This study
→
to B A transition of DNA backbone
2

DNA-Mg - 2+
1255.8 Diminished and shifted to higher wavenumbers antisymmetric PO − stretch-indicative
2
This study
Phospholipid of A-helix formation
1467.2 Formation of A-helix [27],This study
1633.2 phospholipid overlap [27],This study
1732.0 nC=O stretch signal [27],This study
Calf thymus DNA
a)

L-α-phosphatidylcholine
b)

MgCl .2H 0
c)
2 2

Abbreviations: G: guanine; A: adenine; T: thymine; ds: double stranded; d: deoxyribose

1034 Y. Manavbasi and E. Süleymanoglu

Fig. 4. Complete IR absorption spectrum of L-α-phosphatidylcholine.

Fig. 5. Thermotropic phase behaviour of L-α-phosphatidylcholine. Differential scanning calorimetric scan was performed with Diamond -DSC
®

(Perkin-Elmer-UK) employing directly phosphatidylcholine derived signals for better approaching the FTIR sample preparation conditions.

from top to bottom, respectively. Stretching motions of the
OPO group (800 to 850 cm-1), ribose ring vibrations (917.5
cm-1) and the CO stretching of the furanose backbone
(1068.7 cm-1) are clearly distinguishible. Vibrations origi-
nating in the region from 1200 to 1500 cm-1 attributed to
the base and base-sugar entities give rise to marker band
sensitive to glycosidic bond rotation, backbone conformation
and sugar puckering modes. These can be used to obtain
information about nucleotide-specific interactions and
conformations. Frequency region spanning the wavenum-
Nucleic Acid-Phospholipid Recognition
Fig. 6. FTIR spectra of L-α-phosphatidylcholine- Mg binary complex.
2+
Three different wavenumber regions are given from top to the bottom
for better visualization of the divalent metal cation effects on phos-
phate, carbonyl and CH stretching vibrations, as well as the effects of
surface and bound water.
bers from 1500 to 1800 cm-1 is attributed to infrared bands
originating from in-plane double bond vibrations (C=C,
C=N and C=O stretches) leading to nucleobase vibrations,
which are highly sensitive markers for base stackings and
pairings (Table I).
Upon recognition with phospholipid moiety, the ribose
ring vibration shifts to higher wavenumbers. A new band
occurs as a singlet at 970.3 cm-1, corresponding to CC
stretch of the DNA backbone and depicts a switch from Z-
to B-form helix (Banyay, Sarkar and Gräslund, 2003).
Similarly, another new bands appear at 1065 cm-1 and at
1090.6 cm-1. The latter serves as a marker for insensitive
B-to-A transition and symmetric PO2− stretching of the
backbone (Table I). An indication for occurrence of the A-
1035
type of helix comes from the minor peak seen between
the range of 1175-1188 cm-1, which is sugar phosphate
backbone vibration with a high contribution from the sugar
moiety in C3’-endo/anti-conformational type of puckering.
The next clue for A-form of helix is obtained from the next
new and unique band, at 1244.6 cm-1, which is a main
marker of antisymmetric PO2− stretching (Banyay, Sarkar
and Gräslund, 2003). The following two bands at 1339.5
and 1409.3 cm-1, which are diminished peaks of previously
recorded infrared spectrum of the free DNA, correspond-
ing to N-type deoxyadenosine and deoxythymine riboses
in A-form helices and a new band seen at 1467.2 cm-1
indicate the occurrence of A-form duplex (Table I). Thus,
upon association with phosphatidylcholine entity, calf
thymus DNA undergoes Z- → B- → A-helix structural
transitions. Major changes are observed within the region
1500-1800 cm-1. Newly formed weak and wide band at
1594.2 cm-1 and the the sharper peak at 1737.1 cm-1 indi-
cate the role of C=N group of double stranded G-strand
and of C=O strands in associating with DNA, respectively
(Table I).
Ternary phospholipid-Mg2+-DNA complex forma-
tion
The third bottom part of Fig. 2 is a depiction of IR spec-
trum of ternary DNA-Mg2+-phosphatidylcholine complex
formation. The singlet of CC stretch of B- form DNA, the
frequency of B → A-helical transition (1095.8 cm-1), as
well as the two bands correponding to A-helical form of
calf thymus DNA (1255.8 and 1467.2 cm-1, respectively)
are diminished and shifted to higher wavenumbers. Ap-
parently, the A-helical form of the nucleic acid predominates
in this ternary complex. The last two new unique bands
seen at 1663.2 and 1732.0 cm-1 (ν C=O) are overlaps of
phospholipid signals. This finding is worth comparing with
previous FTIR studies depicting the occurrence of mainly
B-form (Choosakoonkriang ., 2001; Bruni
et al et al., 2001),
or partly C- and A-forms of DNA (Pohle et al., 2000) in
DNA-liposome complexes. DNA is polymorphic and can
adopt different forms, depending on the degree of hydra-
tion, method of preparation, as well as other factors. The
observation of coexistance of various conformations with
intermediate structural characteristics seems to be a
common feature, both in aqueous and solid forms, as also
reported previously (Pohle et al ., 2000).
Phospholipid-nucleic acid recognition
Fig. 3 is given mainly for qualitative purposes for con-
vinience, to follow the structural transitions of lipid imposed
by DNA at three informative frequency regions, while the
quantitative parameters are shown in Table I. The left-
hand-side of the figure depicts the changes of major
phospholipid bands induced by nucleic acid recognition.
1036
Comparison of the first two of them from top to bottom
shows again a phospholipid dominated IR absorption
spectrum. The trend is also maintained in the adjacent
regions of 1000-2400 cm-1 and 2400-4000 cm-1, respectively.
Intensive sorption of water, shown in middle part of the
figure give rise to a typical shift of a couple of stretching
vibratons of the phosphate group. This effect is attributed
to Mg2+ influence on IR absorption bands of the phosphate
group moiety of L-α-phosphatidylcholine and is observed
by the modes of antisymmetric and symmetric PO2−
stretching bands near 1240 and 1090 cm-1, respectively.
Mg2+ increases the centre of gravity (νasPO2−), partially
dehydrating the phosphate groups (Hauser, 1991; Binder
and Zschörnig, 2002). The CO stretching vibration also
diminishes. The middle column of the figure from top to
the bottom shows effects in the CH2 scissoring region and
on the wagging progression of uncomplexed phosphati-
dylcholine (Fig. 4), as compared to its binary complex
formed with DNA and as ternary DNA-Mg2+-phospholipid
complex, respectively. The frequencies of the scissoring
vibrations of the methylene and methyl groups are located
in the region ~1350-1500 cm-1. The region is depicted
here mainly as a CH2 scissoring mode, seen as a band at
1467.4 cm-1 indicating a hexagonal packing of all- trans
methylene chains and orientational disorder of the hydro-
carbon chains. Compared with the unbound phospholipid,
its recognition by DNA leads to splitting of scissoring band
into two separate bands with lowered intensity, showing
the appearance of antisymmetric deformation of methyl
groups. The effect is somehow reversed to a much lesser
extend upon formation of a ternary lipid-metal cation-DNA
complex, shown at the middle bottom part of the figure.
The emergence of CO vibrations with low frequencies at
~1710 cm-1 are typical for crystalline Mg2+-phosphatidylcho-
line complexes and are explained by strong H-bonding of
trapped and immobilized water molecules (Binder and
Zschörnig, 2002). Comparison with the case of free phos-
pholipid (Fig. 4) depicts the reversal of the intensities
belonging to H-bonded and unbonded CO stretching
vibrations upon Mg2+ recognition leading to the ternary
complex formation (Fig. 3). The right-hand-side of Fig. 3
shows the CH2 stretching vibration seen as bands in the
region from 2800-3100 cm-1 with the CH2 antisymmetric
and symmetric stretching modes situated at 2922.4 and
2852.2 cm-1, respectively. These frequencies are conforma-
tion sensitive and respond to alterations of the trans-
gauche ratio of C-C bond in the acyl chains. A slight shift
of both CH2 to higher frequncies (~2 cm-1) indicates an
increased proportion of gauche conformers in the acyl
chain. In this respect, this is a different situation when
compared to lipid-metal cation complex formation in the
liquid crystalline phase, where small amount of gauche
conformers exist (Binder and Zschörnig, 2002). The bottom
Y. Manavbasi and E. Süleymanoglu
row of the figure depicts major conformational transitions,
induced by Mg2+ recognition of both nucleic acid and phos-
phatidylcholine, as seen from left to the right. The metal
cation decreases both PO2- and CO stretching vibrations.
Mg2+ recognition leads to appearance of new bands at
~1635 and 1736 cm-1, attributed to the formation of H-
bonded effects on carbonyl group moiety, rather than to
possible conformational changes of this group. Mg2+
affects the CH2 vibrations, seen at the right bottom of Fig.
3 resulting in second shoulder in the water spectrum
depicting the formation of another shell of water and
possibly the additive effect of bound water molecules, as
well as the decreased in intensity bands of CH2 stretching
vibrations. The latter effect obviously shows the occurrence
of higher proportion of gauche andgauche/trans/gauche
conformers next to the carbonyl end of the sn-1 acyl chain
(Binder and Zschörnig, 2002). Apparently, Mg2+ effects
starts with a partial dehydration of phosphate groups of
lipid and possibly ends up with their complete dehydration
in crystalline samples.
Phospholipid-metal ion binary mixture
Fig. 6 represents an important case of lipid-metal
binding and is given in three IR spectral regions for better
following the Mg2+-effects on phosphatidylcholine moiety-
namely, on phosphate, carbonyl stretching, as well as CH2
stretching and water bands. The first two are quantified
usually between 850-1850 cm-1, 1000-2500 cm-1, the latter
two-at 2800-3600 cm-1, respectively. This binary complex
is depicted separately, having considered its huge biological
role in variety of cellular phenomena, such as membrane
transport and/or maintanance of ionic fluxes on cell surface
(Saenger, 1987; Bloomfield, Crothers and Tinoco, 2000;
Hackl et al ., 1997), as well as in lipid mediated signal
transduction events. The top and middle part of the figure
represent the first two regions mentioned, where little
effect is seen to be inserted on the phosphate moiety
(1000-1175 cm-1), while H-bonded and unbonded carbonyl
group is affected to a greater extent (1635.3 and 1734.5
cm-1). The proposed effect on carbonyl stretching could
be due the reduction of H-bond in responce to lowering
the interaction of the carbonyl groups with water mole-
cules. A new band following the phosphate vibrations is
observed at 1467.6 cm-1, which could be related to the
effect of Mg2+ of diminishing the intensity of the higher
frequency bond showing the transition to the hexagonal
packing. The bottom part of the figure depicts only slight
shift of lipid CH2 signals and gives almost no clue on
possible effects on lipid chain order, although the similar
unaltered position of the CH2 rocking at 720 cm-1 somehow
confirms previous data in favour of a lowering in motional
states and increase in chain order (Binder and Zschörnig,
2002). The expected effect of Mg2+ of increasing νasPO2−
Nucleic Acid-Phospholipid Recognition
in the gel phase, by partially dehydrating the phosphate
groups is not observed here. Apparently, the employed
hydration conditions are not sufficient enough for such a
conformational change, as evidenced from the lack of
vibrational bands at arround 1115 and 1137 cm-1 (Hauser,
1991; Binder and Zschörnig, 2002). Interesting effects are
seen in the IR absorption pattern of the carbonyl group of
the phosphatidylcholine (Fig. 4 vs Fig. 6). The peak
observed at 1736.6 cm-1 is an indication of H-bonded
carbonyls and the water. Mg2+ ions cause a shift of C=On
towards smaller wavenumbers. This effect is due to increase
of the accessability of the carbonyl groups to water
because of their engagement in the hydration shell of the
ions. Alternatively, the effect could also be due to confor-
mational changes in the carbonyl region, which also has
the potential to affect the structure and phase behaviour
of lipid structures due to their location near the polar
interface (Binder and Zschörnig, 2002). However, to relate
the latter possibility to a specific Mg2+ binding to phos-
phatidylcholine via hydration forces is rather difficult. The
salt used is a crystalline metal chloride hydrate, which can
give rise to a characteristic IR signal with a typical fine
structure, probably caused by crystal water of free metal
chloride. Mg2+ ions recognize the lipid headgroups (Hauser,
1991; Binder and Zschörnig, 2002). The two water bands
describe also for DNA-metal ion absorption spectrum (Fig.
1B) appearing also here at 3405.5 and that preceding it at
3241.4 cm-1 represents the additional water effect in the
hydration shell of lipids acting separately to hydration of
ions. Mg2+ phase separation, due to specific distributions
of H-bonds with various strength, resulting in increased
binding sites, degree of inter- and intramolecular vibrational
coupling within the water structure. Apparently, a non-
random distribution of ions among the lipids resulting in
insertion of Mg2+ into the polar region of the phospholipid
structre can be suggested. The larger band can be ascrib-
ed to a mode of out of phase symmetric stretches of
neighbouring water molecules, whereas the smaller left-
hand-side band is probably due to a complex mixture of
different modes with major contributions from antisymmetric
water stretches (Binder and Zschörnig, 2002).
The biologically relevant structure of the result-
ing nucleic acid-Mg2+-phospholipid ternary com-
plex
Even though neutral lipids undertaken also in the
current study are thought to form similar to cationic lipids
structures with DNA, the mechanisms of lipoplex forma-
tion appear to be different. Thus, as oppose to cationic
lipids, the complexation between various zwitterionic
phospholipids and DNA is governed by divalent metal
catons acting as bridges between the phosphate groups
of nucleic acid and the uncharged lipid headgroups (Bruni
1037
et al., 2001). The formation of a particular ternary structure
depends on charge and degree of saturation of lipids
employed, as well as on type and topology (linear or
plasmid form) of the nucleic acid, as well as on the
laboratory protocol. Since, the divalent metal cations also
lead to nucleic acid compaction prior to interaction with
phospholipids, the advantage of their use is realized having
considered the fact that the probably formed supercoiled
form of DNA is the transfection competent conformation
and therefore the preferred structure in lipofection. DNA,
metal cations and phosphatidylcholine molecules form a
ternary self-assembly, consising of DNA molecules sand-
wiched between the lipid bilayers (Bruni et ., 2001;
al
Pisani et al ., 2006). The formation of the structure is
trigerred by metal ions and is particularly relevant in liquid-
crystalline state. Our previous thermodynamic measure-
ments depicted that ternary complex coexist with the
unbound phospholipid phase and phosphatidylcholine-
metal cations binary mixture (Süleymanoglu, 2005).
A note on biopharmaceutical production issues
The instability of lipid-based pharmaceutical formulations
is a major problem to bypass before their commercial
application in gene therapy. Thus, manufacturing of stable
and easy to handle lipoplex preparations is an important
item. For this reason, it is useful to examine them at
different physical states. The described features of phos-
phatidylcholine-divalent metal cation-DNA ternary com-
plexes can be applied in biopharmaceutical field in several
ways. The expected huge potential of non-viral gene
therapy vectors can be realized provied that vehicles with
controllable physicochemical properties are manufactured
on an industrial scale. Currently, non-viral gene delivery
formulations are made in small batches employing
laboratory protocols based on using either simple or
peristaltic pump-controlled continuous mixing of plasmid
DNA or other type of nucleic acid with liposome suspension
at various ratios, followed by the lyophilisation of the
resulting lipoplexes or genosomes. Although this approach
already permits the production of large quantities of lipid-
DNA particles, the reported parameters are often specu-
lative and contradictory to each other, since the pro-
cedures are highly dependent on the nucleic acid and lipid
vesicles' sizes and compositions, instruments used, personal
training, storage conditions and expermetntal designs.
This especially holds true reagarding DNA/lipid ratio and
how it influences size distribution of resulting lipoplexes
and subsequently transfection efficiency. Moreover, following
mixing of DNA and liposomes often aggregate formations
are observed leading to heterogeneous structures that are
characterized by various charge ratios and dimensions.
Frequently, the particle size, z potential and other colloidal,
interfacial and biophysical features affecting gene delivery
1038
efficacy themselves are governed by many factors, such
as mixing protocol, buffer components, ionic strength and
pH, temperature, as well as complexation time. Therefore,
one of the important factors concerning clinical application
of lipoplexes and polyplexes is the necessity of their fresh
preparation prior to administration, to avoid the aggrega-
tion tendency or possible lipid fusion resulting in difficult to
employ multilamellar structures in liquid formulations.
Decrease of tranfection efficiency following storage of
lipoplexes in liquid form was also reported (Lai and van
Zanten, 2002). On the other hand, dried preparations offer
the potential for extended physical stability at room tem-
perature and can be ready for use after a simple rehydra-
tion step (Anchordoquy et al., 2001). The reasoning on
employment of dried multicomponent systems such as
lipoplexes were overviwed recently (Anchordoquy et al.,
2001; Clement et al., 2005). It is worth pointing out that it
is encouraging that lyophilization studies to date have
reported stabilization of polymer-, lipid- and viral-based
vectors. Such transfection samples can offer suitability for
production of pharmaceutical products in terms of desired
transfection efficiencies, decreased cytotoxicities, as well
as GMP-conformity (Clement ., 2005). The motivation
et al
for studying dried formulations has come from relevant
report on increased rates of gene transfer to lung using
such preparation (Sorgi and Huang, 1995). Therefore, we
focused on studying the nucleic acid-lipid recognition in
water free samples in attempt to mimic the case of
removing water from the liquid formulations, e.g. during
lyophilization. This is imporatant also in terms of still
unknown mechanisms by which excipients stabilize non-
viral vectors during the latter process. The macromolecular
integrity is perturbed in aqueous formulations by removing
water from the surrounding and thereby creating closer
contacts between interacting moieties in the dry state.
This could beneficially minimize associations between
particles and thus diminish the unwanted aggregation
(Anchordoquy et al., 2001). In our opinion, the ternary
complex described formed between nucleic acids,
divalent metal cations and phospholipid structures can be
reasonably linked to aqueous lipoplex designs. In the light
of our recent emphasis on the divalent cation driven
nucleic acid-lipid recognition, membrane dsetabilization,
lipid fusion and internalization of the therapeutic DNA
(Süleymanoglu, 2004), it is worth noting that in general,
membrane fusion involves several events such as vesicle
aggregation, interfacial dehydration and membrane desta-
bilization, the importance of which is also underlined in the
current IR study. Since hydration forces are detrimental in
polar headgroups approaching, as well as repulsing each
other, one of the suggested mechanisms of fusion involves
an increase in membrane dehydration leading to a strong
adhesion of two opposing membrane surfaces, membrane
Y. Manavbasi and E. Süleymanoglu
deformation and destabilization (Binder and Zschörnig,
2002). Divalent metal cations such as Ca2+ and Mg2+ can
compete with water molecules for binding to lipid head-
groups. Such a complex formed between ions and
phosphates of the polyanions (DNA and cellular lipids)
can increase membrane surface hydrophobicity, reducing
the repulsive hydration forces and enhancing fusion
(Süleymanoglu, 2004). The described ternary system can
also be employed as affinity matrix design in immobilized
liposome chromatography for sequence specific and se-
quence non-spcific nucleic acid separation and purification
(Manavbasi and Süleymanoglu, 2006). Maintaining phy-
sicochemical stability of lipid matrix-associated nucleic
acids for prolonged time enabling their further analyses
remains the major objective.
CONCLUSIONS
The principal conclusion of this study that under the
conditions employed Fourier Transform Infrared Spectros-
copy (FTIR) gives valuable information about the structural
transitions of nucleic acids, phospholipids and metal ions
in free and bound form, which may be employed as an
analytical tool for predicting physicochemical behaviour of
lipoplexes prior to gene transfer trials. Based on hydration
profiles of the engaged biomolecules (nucleic acid vs. lipid
hydration), the biologically relevant structures of the
resulting nucleic acid-Mg2+-phospholipid ternary complex
can be deduced. The infrared spectra of DNA-lipid binary
complexes is dominated by the lipid phase. Nucleic acid-
phospholipid recognition is followed by the helical transi-
tions of DNA. Inorganic cation effects are seen as dehy-
drations of phosphates and H-bonding effects on carbonyls.
Although the pharmacodynamical features of the zwitterionic
lipid-metal cation-DNA nanocondensates in fluid form
remain to be tested in further gene transfection experiments,
at least from physicochemical viewpoint, their preliminary
surface recognition parameters are encouraging to
consider them as a promising metal-based nucleic acid
nanopharmaceuticals. Our work with fluorescent microscopy
of fluid liposome-nucleic acid complexes is in progress
and will be reported separately.
ACKNOWLEDGEMENTS
This work was supported by The Scientific and
Technological Research Council of Turkey (TÜBITAK) in
the frame of Project No: 105S151 (SBAG-HD-42). The
technical assistance of Mrs. Yasemin Akkoç is greatly
acknowledged. We also thank Dr. Necati Özkan from
Central Laboratory of Middle East Technical University for
his help with DSC scans of lipids.
Nucleic Acid-Phospholipid Recognition
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