α-Tocopherol-ascorbic acid hybrid antioxidant based cationic amphiphile for gene delivery: Design, synthesis and transfection

Article abstract of DOI:10.1016/j.bioorg.2018.02.025

Natural antioxidants and vitamins have potential to protect biological systems from peroxidative damage induced by peroxyl radicals, α-tocopherol (Vitamin E, lipid soluble) and ascorbic acid (vitamin C, water soluble), well known natural antioxidant molecules. In the present study we described the synthesis and biological evaluation of hybrid of these two natural antioxidants with each other via ammonium di-ethylether linker, Toc-As in gene delivery. Two control cationic lipids N14-As and Toc-NOH are designed in such a way that one is with ascorbic acid moiety and no tocopherol moiety; another is with tocopherol moiety and no ascorbic acid moiety respectively. All the three cationic lipids can form self-assembled aggregates. The antioxidant efficiencies of the three lipids were compared with free ascorbic acid. The cationic lipids (Toc-As, N14-As and Toc-NOH) were formulated individually with a well-known fusogenic co-lipid DOPE and characterization studies such as DNA binding, heparin displacement, size, charge, circular dichroism were performed. The biological characterization studies such as cell viability assay and in vitro transfection studies were carried out with the above formulations in HepG2, Neuro-2a, CHO andHEK-293T cell lines. The three formulations showed their transfection efficiencies with highest in Toc-As, moderate inN14-As and least in Toc-NOH. Interestingly, the transfection efficiency observed with the antioxidant based conjugated lipid Toc-As is found to be approximately two and half fold higher than the commercially available lipofectamine 2000 at 4:1 charge ratio in Hep G2 cell lines. In the other cell lines studied the efficiency of Toc-As is found to be either higher or similarly active compared to lipofectamine 2000. The physicochemical characterization results show that Toc-As lipid is showing maximum antioxidant potency, strong binding with pDNA, least size and optimal zeta potential. It is also found to be least toxic in all the cell lines studied especially in Neuro-2a cell lines when compared to other two lipids. In summary, the designed antioxidant lipid can be exploited as a delivering system for treating ROS related diseases such as malignancy, brain stroke, etc.

Full text of DOI:10.1016/j.bioorg.2018.02.025

Accepted Manuscript
α-Tocopherol-ascorbic acid hybrid antioxidant based cationic amphiphile for
gene delivery: Design, Synthesis and transfection
Venkanna Muripiti, Lohchania Brijesh, Hari Krishnareddy Rachamalla, Srujan
Kumar Marepally, Rajkumar Banerjee, Srilakshmi V. Patri
PII:
S0045-2068(18)30062-2
DOI:
https://doi.org/10.1016/j.bioorg.2018.02.025
YBIOO 2285
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
24 January 2018
23 February 2018
23 February 2018
Please cite this article as: V. Muripiti, L. Brijesh, H. Krishnareddy Rachamalla, S. Kumar Marepally, R. Banerjee,
S.V. Patri, α-Tocopherol-ascorbic acid hybrid antioxidant based cationic amphiphile for gene delivery: Design,
Synthesis and transfection, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.02.025
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α-Tocopherol-ascorbic acid hybrid antioxidant based cationic amphiphile
for gene delivery: Design, Synthesis and transfection
Venkanna Muripiti1, Lohchania Brijesh2, Hari KrishnareddyRachamalla3, Srujan Kumar
Marepally2, Rajkumar Banerjee3, Srilakshmi.V Patri1*
3
1National Institute of Technology, Warangal-506004, Telangana, India, Division of Lipid
Science and Technology, Indian Institute of Chemical Technology, Hyderabad-500607,
Telangana, India, 2Centre for Stem Cell Research, Vellore-632002, Tamil Nadu, India.
Email: patrisrilakshmi@gmail.com
ABSTRACT:
Natural antioxidants and vitamins have potential to protect biological systems from
peroxidative damage induced by peroxyl radicals, α-tocopherol (Vitamin E, lipid soluble) and
ascorbic acid (vitamin C, water soluble), well known natural antioxidant molecules. In the
present study we described the synthesis and biological evaluation of hybrid of these two
natural antioxidants with each other via ammonium di-ethylether linker, Toc-As in gene
delivery. Two control cationic lipids N14-As and Toc-NOH are designed in such a way that
one is with ascorbic acid moiety and no tocopherol moiety; another is with tocopherol moiety
and no ascorbic acid moiety respectively. All the three cationic lipids can form self-
assembled aggregates. The antioxidant efficiencies of the three lipids were compared with
free ascorbic acid. The cationic lipids (Toc-As, N14-As and Toc-NOH) were formulated
individually with a well-known fusogenic co-lipid DOPE and characterization studies such as
DNA binding, heparin displacement, size, charge, circular dichroism were performed. The
biological characterization studies such as cell viability assay and in vitro transfection studies
were carried out with the above formulations in HepG2, Neuro-2a, CHO andHEK-293T cell
1

lines. The three formulations showed their transfection efficiencies with highest in Toc-As,
moderate inN14-As and least in Toc-NOH. Interestingly, the transfection efficiency observed
with the antioxidant based conjugated lipid Toc-As is found to be approximately two and half
fold higher than the commercially available lipofectamine 2000 at 4:1 charge ratio in Hep G2
cell lines. In the other cell lines studied the efficiency of Toc-As is found to be either higher
or similarly active compared to lipofectamine 2000. The physicochemical characterization
results show that Toc-As lipid is showing maximum antioxidant potency, strong binding with
pDNA, least size and optimal zeta potential. It is also found to be least toxic in all the cell
lines studied especially in Neuro-2a cell lines when compared to other two lipids. In
summary, the designed antioxidant lipid can be exploited as a delivering system for treating
ROS related diseases such as malignancy, brain stroke etc.
Keywords: α-tocopherol, Ascorbic acid, Transfection, Gene delivery, Cationic liposomes,
Antioxidant.
Introduction
Basically, gene therapy is a potential strategy to deliver foreign DNA into a target cell
nucleus, resulting in the modulation of hosts malfunctioning gene thereby helps in disease
treatment[1]. Gene therapy plays an important role in the prevention of acquired diseases,
such as cardiovascular diseases, cancer, AIDS, etc [2],[3],[4]. But, the ideal gene-carriers are
needed to deliver and protect the genetic material that has to be entered in to the host cell,
against to the enzymatic degradation inside the cell milieu[5], [6],[7]. Though the viral vectors
are one of the widely using DNA-carriers, they are proven to be imperfect in the past decades
due to their less efficiency in gene transfer due to the existence of maximum
2

immunogenicity, mutagenicity, and sometimes fatal toxicity[8],[9]. However, synthetic DNA-
delivering vehicles (non-viral vectors) were emerged as promising alternatives, because of
their extensive characterization, structural and synthetic simplicity, as well as synthetic ease
of modification of their size, functionality[10]. Synthetic cationic lipids were having been in
use since 1987 for gene delivery. In general, non-viral vectors are categorized into cationic
lipids, polymers, and peptides. The cationic lipids are considered to be one of the most
efficient non-viral over viral approaches and it already has been using for in vitro transfection
of a mammalian cell[11]. The cationic lipid-DNA complexes (lipoplexes) are one of the
highly efficient systems for easy trafficking of gene of interest into host nucleus via an
endocytosis pathway[12]. The transfection efficacy of liposomes depends up on the
formulation of cationic lipids and the architecture of the gene-expression
system.[13],[14],[15] However, toxicity is one of the barrier limiting the clinical applications
of cationic liposomes[16],[17]. Hence, regulating this cytotoxicity should facilitate the
development of safe cationic liposomes for use as non-viral vectors and there by clarifying
the mechanism of cytotoxicity of cationic liposomes[18]. Recently it is demonstrated that the
cytotoxicity of cationic liposomes is a result of apoptosis [19],[20], and that cationic
liposome-induced apoptosis exhibited due to the following features: generation of reactive
oxygen species (ROS)[21],[22]; the activation of p38 mitogen-activated protein kinase
(MAPK)[23],[24]; the activation of caspase-8[20-23],[24] the cleavage of Bid and its
translocation to the mitochondria and the release of cytochrome c [23], [24]; and the
activation of caspase-3[25]. However, it remains unclear how cationic liposomes lead to ROS
generation. Particularly neurons are vulnerable to increases in ROS levels[22],[26], because
these cells have a reduced capacity to detoxify ROS[24],[27]. Hence, the delivery systems
possessing radical scavenging ability along with transfection potency, may be helpful in
treating ROS (reactive oxygen species)[28] related diseases such as brain stroke/ischemia and
3

malignancy. Antioxidant cationic lipids display their obvious role as transporters of foreign
DNA into the nucleolus of host cell, with-out showing any toxicity[29], [30],[31],[32, 33].
Herein, we designed and synthesized a new cationic lipid hybrid (Toc-As) by conjugating
theα-tocopherol and ascorbic acid together by ammonium di-ether linker and allowed it to
form the self-assembled amphiphilic molecule. The rational for a synthesis of this conjugated
lipid is that these two vitamins (E and C) are potent natural antioxidants and can form an
amphiphilic surfactant when conjugated. The main role of α-tocopherol as antioxidant is
quenching of peroxyl radicals and per hydroxyl radicals and produce “tocopheroxyl radicals”.
Ascorbic acid (vitamin C) scavenges oxygen radicals in the aqueous phase and it converts
tocopheroxyl radical into α- tocopherol, thereby permitting α-tocopherol to act as a free
radical chain-breaking antioxidant and maintain biological systems. It is now apparent that
ascorbic acid and tocopherol function together can protect membrane lipids from oxidative
damage. To observe the individual role of each antioxidant in transfection, we have
developed two control lipids one is with ascorbic acid moiety without tocopherol moiety
(N14-As) and another is with tocopherol moiety without ascorbic acid moiety (Toc-NOH).
4

Figure.1. Chemical Structures of Cationic Lipids
The nano structure of individual Toc-As, Toc-NOH and N14-As cationic lipids characterized
in terms of size, antioxidant activity, stability, and morphology. The lipoplexes of Toc-As,
N14-As and Toc-NOH were evaluated for theirs in vitro activities in multiple cell lines. The
cytotoxicity of the lipids is also studied using MTT assay in multiple cell lines.
RESULTS AND DISCUSSION
Chemistry
The titled cationic lipids Toc-As, N14-As and Toc-NOH were synthesized as described in
the Schemes 1, 2 and 3. The cationic lipid Toc-As possess α-tocopherol as the hydrophobic
group and ascorbic acid as hydrophilic group linked through N,N-diehtylether ammonium
linker, whereas N14-As lipid contains N,N dialkyl chain as hydrophobic group and ascorbic
5

acid as hydrophilic group linked through ethyl ether linker. The lipids Toc-NOH possess α-
tocopherol as the hydrophobic and hydroxyethyl group as hydrophilic moiety linked via ethyl
ether linker. The precursor intermediate O-aminoethyl-[N-hydroxy ethyl, N-methyl]--
tocopherol (1D, Scheme 1)in the synthesis of both the final lipids Toc-NOH and Toc-As is
prepared conventionally as shown in the Scheme1. Briefly, O-alkylation of α-tocopherol
using ethylbromo acetate in presence of 50% potassium hydroxide gave O-acetic acid-α-
tocopherol, 1A which upon reduction with Lithium aluminium hydride gave intermediate O-
hydroxyethyl-α -tocopherol1B (Scheme 1). The intermediate 1D was obtained by O-
tosylation of intermediate 1B followed by N-alkylation of intermediate 1C. Subsequently the
lipid Toc-NOH is obtained by the quaternization of 1D using 1N HCl in methanol. Toc-As is
obtained by treating the intermediate 1D with5,6 isopropylidine ascorbic acid in DCM in
presence of N,N dimethylamine followed by deprotection of acetonide group using dry
hydrochloric acid in methanol (Scheme 3). The intermediate N-(2-chloroethyl)-N-tetradecyl
tetradecane-1-amine, 2C for synthesis of N14-Aslipid is prepared conventionally as
mentioned in the scheme 2. The intermediate 2C upon treatment with 5,6 isopropyledine
ascorbic acid as described above for Toc-As, followed by the deprotection using dry
hydrochloric acid gave lipid N14-As (Scheme 2). Chemical structures of all the synthetic
1
intermediates lipids were confirmed by H-NMR, ESI-MASS and final lipids shown in
1
Schemes 1-3 are confirmed by H NMR,13C NMR and molecular ion peaks in their ESI–
HRMS mass spectra. The purity of final lipids was characterized by RP-HPLC, as described
in the Experimental Section.
6

Scheme 1
Synthesis of Toc-NOH lipid
Reagentsand condition: i) Ethyl bromo acetate, Dry DMF, 50% KOH solution, 12 h RT ii)

Dry THF, LiAlH4, 6h RT, 0-5 C iii) p-Toulene sulfonyl chloride , DMAP, Dry DCM iv) N-
methyl ethanol amine, Methanol v) 1N HCl in Methanol, 12 h RT
Scheme 2
Synthesis of N14-As lipid
7

Reagents and conditions: i) Dry acetone, 2,2 Dimethoxy propane, Sncl2, ii) Anhydrous
K2CO3, ethyl acetate, 48 h reflux iii) SOCl2, dry chloroform, 6h reflux iv) 5,6 isopropylidine
ascorbic acid, dry DMSO, anhydrous K2CO3, refluxed for 18h v) 6N HCl in Methanol, 6h
RT.
Scheme 3
Synthesis of Toc-As lipid
Reagents and conditions: i) p-Toluene sulfonylchloride, Triethyl amine, DMAP, dry DCM
ii) Diethylisopropylethylamine, 5,6 isopropylidine ascorbic acid, DMAP, dry DCM iii) 1N
HCl in methanol, 12 h RT.
8

Size and charge of liposomes and lipoplexes
Physicochemical characteristics viz., particle size and surface potential of liposomes and
lipoplexes of cationic lipids (Toc-As, N14-As and Toc-NOH) and DOPE as co-lipid were
examined using DLS technique (Figure 2). These measurements were made across the
lipid:DNA charge ratios 1:1 to 8:1 in presence of Dulbecco’s Modified Eagle’s medium
(DMEM). It is observed that the particle size of liposomes of Toc-As and N14-As are found
to be 350 nm 398 nm respectively and slightly increased upon complexation with pDNA at
1:1. As we increase the charge ratio up 4:1 the particle size observed to be decreased upto
180 nm and 220 nm respectively and further increase in the charge ratio i.e. at 8:1 the particle
size is increased i.e. 380 nm and 350 nm respectively. The surface potential of liposomes
Toc-As and N14-As (+12 mV and +9.5 mV respectively) also observed to be decreasing
with increasing charge ratio upto 2:1 (+6 mV and +3.6 mV respectively)and started
increasing at higher charge ratio’s 4:1 and 8:1 upto +16 mV and +20 mV respectively. The
least size and optimal zeta potential of lipoplexes of Toc-As and N14-As at 4:1 charge ratio
may be responsible for their greater transfection potentials at this charge ratio. It clearly
indicate that when the resulting DNA-liposome complexes exhibit more charge positive or
negative charge, electrostatic repulsive forces prevent extensive aggregation and/or fusion,
thus leading to the formation of smaller complexes. It might also be the size of the nano-
particle can be seen as a vital factor in evaluating lipoplexes suitability for gene delivery,
given the size plays a role in lipoplexes endocytosis and transfection activity[34], [35], [36],.
Moreover, the minimal global charge of lipoplexes benefit reduces the cytotoxicity and serum
stability[37]. Whereas the sizes of lipoplexes of Toc-NOH observed to be increasing with
increase in charge ratio 1:1 to 8:1 from 250 nm to 490 nm and similarly the surface charge
9

also increasing with increase in charge ratios 1:1 to 8:1from + 7.2 mV to 13.7 mV (Figure 2).
This may be because of the aggregation of lipoplexes in presence of DMEM. Such increase in
the sizes of the lipoplexes in presence of DMEM is reported previously[38]. The same
reason may be considered for the increase in the sizes of lipoplexes of Toc-As and N14-As at
8:1 charge ratio.
Figure.2. Physicochemical Characterization of liposomes and Lipoplexes: Particle sizes (A)
and zeta-potentials (B) of liposome of lipids Toc-As, N14-As and Toc-NOH and pDNA
complexes at various N/P ratios (DLS at room temperature). Data represent mean ± SD (n =
3).
Liposome-DNA binding and Heparin displacement
The electrostatic binding interactions between the cationic liposome and pDNA were studied
using conventional agarose gel retardation assay and heparin displacement assay across 1:1-
8:1 charge ratios. The corresponding gel images reveal that) all the three cationic lipids have
similar capabilities in inhibiting the mobility of pDNA (Figure 3A). All the three lipids is
capable of inhibiting 90% of pDNA mobility even at lower charge ratio’s such that 2:1
(Figure 3A). Further, heparin displacement assay is carried out to observe the DNA binding
10

strengths of these liposomal formulations. The results of heparin displacement assay showed
significant difference in the binding strengths of the three liposomal formulations. Liposomal
formulation with Toc-As liposomal formulation completely resisted displacement of DNA
with heparin across the lipid: DNA charge ratio 8:1-1:1 (Figure 3B). While, N14-As
liposomal formulation could survive DNA displacement by heparin only at higher charge
ratios i.e. 4:1 and above. (Figure 3B).Whereas, liposomal formulations of Toc-NOH could
not resist much even at charge ratio 4:1 and could survive heparin displace only at higher
charge ratio 8:1. It therefore appears that Toc-As and N14-As lipids having ascorbic acid
moiety in the head group region are capable of binding pDNA strongly and it may show any
crucial role in the transfection biology. These observations are well in agreement with the
size and surface potential data. The CD spectrum data also supports the binding abilities of
the lipids having ascorbic acid moiety with pDNA.
Figure.3(A) Electrophoretic gel patterns for lipoplex-DNA in gel retardation assay, (B)
Electrophoretic gel patterns for lipid-DNA complexes in Heparin displacement assay for
lipids Toc-NOH, N14-As and Toc-As. The lipid: DNA charge ratios are indicated at the top
of each lane. The details of the treatment are as described in the text.
CD Spectra of pDNA in Lipoplexes
To observe the change in the morphology of pDNA when complexed with the liposomes of
the present lipids, circular dichroism (CD) measurements were carried out. Surprisingly, the
11

formulations of Toc-As and N14-As lipids perturbed the double helix structure of DNA
(Figure 4).Such transformed mode of compaction of pDNA by Toc-As and N14-As might be
responsible for their relatively higher transfection activity, though this may be further
corroborated by other physical methods to provide a comprehensive model and to prove the
role of ascorbic acid derivative of the lipids in gene transfection activity. As ascorbic acid is
generally negatively charged, it cannot neutralize the charges on the negative phosphate
groups of DNA. Toc-As and N14-As lipids having ascorbic acid head group, may interact
with the bases inside the double-helix structure of pDNA and causes distortion in the double-
helix structure. Neault et al[39]. carried out an infrared and Raman spectroscopic studies on
the effect of ascorbic acid on DNA[40]. They found that the OH and C-O groups of ascorbic
acid interact directly with DNA bases[39],[41],[42],[43],[44]. Figure 4 demonstrate that,
Toc-As and N14-As lipoplexes showed linear conformation changes when compared to Toc-
NOH lipoplex and this indicates that morphology change of lipoplexes induced by ascorbic
acid head group contained lipids. These results suggest that torsional stress is generated along
the double-helix DNA in ascorbic acid based lipids and this may be useful for the gene
delivery. To the best of our knowledge, gene delivery efficiency of ascorbic acid head group
(Toc-As and N14-As) lipids has not previously reported. In this study, it has become clear
that ascorbic acid head group lipids have a dramatic effect on the conformation of giant DNA
molecules.
12

Figure 4. Circular dichroism (CD) spectra of lipoplexes. Liposomes were prepared at 1/1
molar ratio of Toc-As, N14-As and Toc-NOH as cationic lipids DOPE taken as a co-lipid.
Complexes were prepared at 4:1 charge ratio of cationic liposome-double helix structure of
DNA, and CD was recorded at a pDNA concentration of 50 µg/mL. The CD spectra have
been compared to the profile for pDNA alone. Spectral profiles of liposomes with dextrose or
of dextrose alone were subtracted as blank.
Antioxidant Activity
As the cytotoxicity of cationic liposomes is majorly because of apoptosis exhibited due to the
generation of reactive oxygen species (ROS) during transfection[45],[16]. Hence, the cationic
lipids having radical scavenging ability along with transfection potency may be helpful in
treating ROS (reactive oxygen species) induced toxicity[16]. So, it is necessary to study the
antioxidant efficiencies of the designed anti-oxidant cationic lipid Toc-As along with the
control lipids N14-As and Toc-NOH. The radical scavenger ability of the synthesised lipids
was evaluated calorimetrically by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. The
DPPH radicals show a deep-violet color in an aqueous solution of ethanol giving a strong
absorption in the visible region (460–560 nm),. The increase in the intensity of DPPH
solution is directly proportional to the concentration of radicals and hence used as a measure
13

of the radical-scavenging ability of Toc-As, N14-As and Toc-NOH. The antioxidant
efficiencies of the cationic lipids Toc-As, N14-As and Toc-NOH were calculated with
respect to the control samples that contain only DPPH. The cationic lipids at different
concentrations (0 –50 μM) were incubated in aqueous ethanol solutions of DPPH (100 μM) at
room temperature in the dark. The difference in the concentration of DPPH radicals upon
treatment with different cationic lipids was calculated by measuring the colorimetric intensity
of the samples at 490 nm using a microplate reader after 30, 60, 90, and 120 min of
incubation (Figure 5). The untreated DPPH sample at every time point used as controls and
the anti-oxidant efficiencies observed were time and concentration dependant. After 30 min
of incubation, low concentrations of the cationic lipids (<5 μM) did not show significant
antioxidant activity (Figure 5). However, the cationic lipids, Toc-As, Toc-NOH and N14-
Asshowed antioxidant activity at concentrations that are above 10 μM. Toc-As showed
higher radical scavenger abilities when compared to control lipids Toc-NOH, N14-As.
Moreover, the anti-oxidant activity of Toc-As was found to be 2 fold higher than the natural
antioxidant ascorbic acid which is used as a positive control (Figure 2). The cationic lipid
Toc-NOH did not show any appreciable antioxidant activity even at higher concentrations
(Figure 5). However, N14-As lipids molecules having ascorbic acid moiety as head group
showed some radical-scavenging effect after 30 min of incubation. The experimentally found
superior radical-scavenging activity of Toc-As is attributed to the ability of the natural
antioxidant properties and neutralization of the DPPH radicals.
14

Figure.5. (A) Antioxidant assay of cationic lipids. Concentration-dependent radical-
scavenging ability of Toc-As, N14-As and Toc-NOH cationic lipids against DPPH free
radicals after 30 min of incubation at room temperature in the dark. The absorbance was
measured at 490 nm. The values shown are mean ± SEM of three independent experiments
carried out in three to five replicates. DPPH = 2, 2-diphenyl-1-picrylhydrazyl radicals, AA =
ascorbic acid.
Cationic liposome preparation
Toc-As/DOPE, N14-As /DOPE and Toc-NOH/DOPE liposomes were prepared at 1:1 mol
ratio with 1.4 × 10-2 M total lipid concentration, as stock solution. The mixture was dissolved
in chloroform (1.5 mL) in an autoclaved glass vial. The solvent was evaporated and dried
under a thin flow of moisture-free nitrogen gas. The resulting thin film is dried farther under
high vacuum for 4h. The dried film was hydrated by the addition of 1 mL of deionized water.
The mixture was allowed to swell overnight. The liposomes were vortexed for 1-2 min to
remove any adhering lipid film and then sonicated in a bath sonicator for 5 min at room
temperature to obtained multilamellar vesicles. The vesicles were then sonicated in an ice
15

bath using a probe sonifier to afford the cationic liposomes. All the liposomes formed were
ο
stable and uniform. Liposomes were stored at 4 C and it was observed that no precipitation
even after 3 months.
Transfection Biology: In vitro transfection studies
The relative transfection potentials of lipoplexes of lipids Toc-As, Toc-NOH and N14-
Aswereestablished against multiple cell lines viz., HepG2, HEK-293T, CHO andNeuro-2a
across charge ratio 1:1 to 8:1.pCMV-SPORT--gal plasmid DNA was used as a reporter gene
and is complexed with cationic liposomes across1:1 to 8:1 lipid:DNA charge ratios. The
transfection efficacies of Toc-As, Toc-NOH and N14-As lipids were compared with that of
commercial formulation, lipofectamine 2000 and the results are summarized in Figure 6. All
the cationic lipids showed their maximum transfection efficacies at the charge ratio 4:1
irrespective of the cell lines. This may be due to the optimal particle sizes and zeta potentials
of the lipoplexes at these charge ratios, which in turn affect the cellular uptake (Figure 6).
The lipoplexes of lipids Toc-As and N14-As showed similar transfection profile in HEK-
293T and CHO cells at charge ratios 2:1 and 4:1 and nearly two folds higher efficient than
Lipofectamine 2000 in HEK-293T cell lines at 4:1 charge ratio. Where as in Neuro-2a cell
lines Toc-As is slightly better active than N14-Asat 4:1 charge ratio and comparable to that
of lipofectamine 2000. The lipoplexes of Toc-As, i.e. lipid having both tocopherol and
ascorbic acid moieties, exhibited much superior transfection efficacy among three lipids and
two and half fold higher transfection than Lipofactamine 2000 against HepG2 cell lines at 4:1
charge ratio (Figure 6). It is apparently, due to the better uptake of tocopherol lipids through
cellular transport pathways mechanism involving cell surface receptors (tocopherol-transfer
protein) present on HepG2 cell lines[46]. At the same time the superior activity of Toc-As
may be attributed because of the least cytotoxicity of the lipid due to its maximum anti-
16

oxidant activity. In contrast the lipoplexes derived from Toc-NOH showed least transfection
against all the tested cell lines, indicating that the ascorbic acid in the head group region in
others played a crucial role in transfection. The higher transfection efficiencies of Toc-As
and N14-As compared to Toc-NOH may also be due to the presence of more number of
hydroxyl groups present in the head group region as reported earlier[38] which is also
confirmed by CD spectral data. The superior activity of Toc-As cationic liposomes also can
be attributed because of the diehtylether ammonium linker, which might have greatly
influenced the process of liposomes formation and lipoplex-membrane fusion thanN14-As
liposomes. In detail, the lipid Toc-As possessed α-tocopherol and chiral ascorbic acid may
show some degree of lateral freedoms to each other because of the diehtylether liker and it
might contributed to the maximum release of DNA from lipoplexes into cytosol and also the
flexibility in the lipid favours the multiple H-bonding interaction of ascorbic acid moiety of
the lipid with negatively charged surface of double helix plasmid DNA to significantly
enlarge the overall strength of interaction between the lipid and the DNA. In summary, Toc-
As lipid is capable of transfecting multiple cell lines effectively along with its higher radical
scavenging ability and low cytotoxicity may be helpful in treating ROS (reactive oxygen
species) related diseases such as brain stroke/ischemia and malignancy.
17

Figure.6. (A-D) Transfection efficiencies of lipids Toc-As, N14-As and Toc-NOH in (A)
HepG2 cells, (B) HEK-293T cells, (C) CHO cells and (D) Neuro-2a cells with DOPE as co-
lipid (each lipid formulated with co-lipid in 1:1 molar ratio respectively).The transfection
efficiencies of the lipids were compared to that of commercial formulation Lf2000.
Transfection experiments were performed as described in the text. All the cationic lipids were
tested on the same day, and the data presented are the average of three experiments
performed on three different days. The error bar represents the standard error. The difference
in the data obtained is statistically significant in all charge ratios (P < 0.002).
EGFP expression
The transfection results revealed that the present lipids Toc-As,N14-As and Toc-NOH
showed their maximum transfection ability at 2:1 & 4:1, which was further supported by the
qualitative examination using eGFP plasmid under epifluorescence microscopy. Towards this
end, lipoplexes were prepared using eGFP plasmid at transfection efficient charge ratios 2:1
and 4:1. The formulations were incubated with HEK-293T and HepG2 cell lines in DMEM +
18

10% FBS for 4 hrs. The green fluorescent images were visually observed under epi-
fluorescent microscope and evaluated in terms of % GFP positive transfected cells (Figure 7
& 8) and geometric mean fluorescence intensities (GMFI) (Figure 7C & 8C). The
representative fluorescent images (Figure 7A & 8A) show that the relative fluorescent gene
expression obtained from HepG2 was relatively higher than that obtained from HEK-293T
cell lines. The results represent that maximum GFP expression was observed at 4:1 charge
ratio irrespective of the cell lines for all the three lipid formulations. It is also observed that
Toc-As lipoplex showed maximum GFP expression when compared to N14-As and Toc-
NOH lipoplexes at both the charge ratios in both the cell lines studied (Figure 7 & 8). These
results were reliable with the transfection results and it reemphasize that tocopherol-ascorbic
acid hydride cationic lipid has superior transfection profile. The lipoplexes of Toc-NOH
showed least expression of eGFP among the three lipids studied (Figure 7 & 8). The order of
cationic lipids according to their intensity of fluorescent expression observed in Figure 7 & 8
can be given as Toc-As>N14-As>Toc-NOH.
19

Figure 7.Transient transfection using the reporter gene, eGFP in HepG2 cells: A)
Representative fluorescence images of eGFP expression at the indicated charge ratios.
Liposomes of all cationic lipids formulated with colipid DOPE (1:1) lipid and were
complexes with negatively charged GFP in the optimized charge ratios 2:1 and 4:1 were
used. The liposome-DNA complexes were incubated for 4 h in presence of 10% serum.
Images were acquired 48 h post transfection. B) Representative graph depicts the transfection
efficiencies in terms of percentage GFP positive cells. C) The graph indicating geometric
mean fluorescent intensity (GMFI) of transfected cells using eGFP as the reporter gene. The
data shown is the mean and standard deviation of three different experiments.
20

Figure 8 .Transient transfection using the reporter gene, eGFP in HEK-293T cells: A)
Representative fluorescence images of eGFP expression at the represents charge ratios.
Liposomes of all cationic lipids formulated with colipid DOPE (1:1) lipid and were
complexes with negatively charged GFP in the optimized charge ratios 2:1 and 4:1 were
used. The liposome-DNA complexes were incubated for 4 h in presence of 10% serum.
Images were acquired 48 h post transfection. B) Representative graph depicts the transfection
efficiencies in terms of percentage GFP positive cells. C) The graph indicating geometric
mean fluorescent intensity (GMFI) of transfected cells using eGFP as the reporter gene. The
data shown is the mean and standard deviation of three different experiments.
Effect of serum on transfection efficiency
Generally the gene transfer efficacies of cationic amphiphiles are evaluated either in absolute
absence of added serum or in the presence of only 10% (v/v) serum as reported in many prior
21

investigations[38],[47],[48].Still the serum-incompatibility is one of the major setbacks
retarding the clinical success of cationic transfection lipids. The high in vitro transfection of
many cationic amphiphiles are often found to be adversely affected in the presence of serum.
The cationic lipids show their serum incompatibility due to adsorption of negatively charged
serum proteins onto the positively charged cationic liposome surfaces which in turn prevents
their efficient interaction with cell surface and/or internalization67-69.Hence, evaluation of
gene transfer efficacies across a range of lipid:DNA charge ratios in multiple cultured cells in
presence of increasing concentrations of added serum is needed for obtaining meaningful
systemic potential of any in vitro efficient cationic transfection lipid. In order to know the
effect of serum on the transfection efficiencies of the present lipids, transfection experiment
was performed by complexing with eGFP plasmid at transfection efficient charge ratio 4:1 in
HEK-293T and CHO cell lines with increasing added serum concentrations from 10-50%.
The in vitro transfection studied with increasing concentrations of added serum results show
that all the three lipids Toc-As, N14-As and Toc-NOH lipids were unaffected even in the
presence of high concentration of serum up to50% of added serum (Figure 9). The results
demonstrate that Toc-As lipid showed slight increase in the transfection with increase in the
concentration of added serum. Toc-As and N14-As exhibited higher transfection potentials
even at higher concentration of added serum compared to Toc-NOH, which is found to be the
least. It clearly indicates that lipoplexes having zeta potential values <10 mV, which greatly
benefit the serum stability of lipoplexes and decrease the cytotoxicity, exhibited serum
compatibility. The maximum serum compatibility of lipids Toc-As and N14-Asexpected to
be due to enhanced surface charge shielding of the liposome-DNA complexes induced by
hydroxyl functionalities of ascorbic acid in the head group region.
22

Figure9.Transfection efficacies of the cationic lipoplexes Toc-As, N14-As and Toc-NOH in
the presence of increasing concentrations of added serum. In vitro transfection efficiencies of
liposome-eGFP complexes prepared using GFP reporter gene at a lipid/DNA charge ratio of
4:1 were evaluated in the presence of enhancing concentrations of added serum in (A) HEK-
293T and (B) CHO types of cells. The error bar indicates that the standard error. The
difference in the data obtained is statistically significant in all charge ratios (P < 0.003).
Cellular uptake
The transfection efficiencies of the non-viral gene delivery systems seem to be linked to the
internalization (endocytosis) of DNA associated complexes. In order to verify the cellular
uptake efficiency of lipoplexes of the present lipids, membrane green fluoresce-labeled CHO
cell lines were incubated with the lipoplexes comprising of Rhodamine-PE labelled
liposomes of lipids (Toc-As, N14-As and Toc-NOH) and pCMV-SPORT--gal plasmid at
4:1 charge ratio for 4 hrs. The internalization of rhodamine labelled lipoplexes was observed
under epifluorescence microscope. The intracellular red fluorescence intensity of the cells
was quantified using microplate fluorescent reader. Figure 10 shows the red and green
fluorescent images of CHO cells after incubation for 4 h. The results of the cellular uptake
experiment show that the % of rhodamine positive cells follows the order: Toc-As>N14-
As>Toc-NOH. The lipoplexes particle size may play a vital role in facilitating the cellular
23

uptake. Hence, it is obvious that Toc-As lipoplexes having the least particle size at 4:1 is one
of the reasons for its high potency for uptake by cell lines. The other reason for the higher
uptake efficiency may be the multiple hydroxyl group functionality in its head group region,
which may involve in the favourable hydrogen bonding interactions with the biological
membrane components. Thus, the higher uptake efficiency of the lipid Toc-As may also an
attribute to its superior in vitro transfection efficiency compared to N14-As and Toc-NOH.
24

Figure.10. Cellular Uptake of rhodamine labelled lipoplexes. Fluorescence images (A) and %
uptake (B) of CHO cells incubated for 4 h with the complexes of rhodamine labeled
liposomes of Toc-As, N14-As, and Toc-NOH and plasmid DNA. The percentage uptake was
calculated using the formula% uptake = 100x (fluorescence intensity of the fluorescence
lipoplex treated cell lysate-background) / (fluorescence intensity of lipoplex added to the
cells-background).The details of the experiments are as described in the text. Data are
showed as mean ± SD (n = 3). * represent sp<0.03.
Cytotoxicity assay
The cytotoxicity is a major limiting factor for the success of gene therapy in clinical
applications. Especially, in liposomal gene delivery, the cytotoxicity of cationic liposomes is
a result of apoptosis[22], [18], [21], [21], [23],[27] and that cationic liposome-induced
apoptosis exhibited due to the generation of reactive oxygen species (ROS) [23],
[24].Particularly neurons are vulnerable to increases in ROS levels, because these cells have a
reduced capacity to detoxify ROS [24], [23].Towards, this end to observe the cytotoxicities of
the lipoplexes of the designed antioxidant lipid Toc-AS and control lipids, N14-As and Toc-
NOH, MTT assay was performed in four different cell lines i.e. HepG2, HEK-293, Neuro-2a,
and CHO across the charge ratios 1:1 to 8:1. The cell viability results are summarised in
Figure 11.The results indicate that the lipoplexes of Toc-As, N14-As and Toc-NOH lipids
found to be non-toxic up to the charge ratios of 4:1- 1:1 in HepG2, HEK-293T and CHO cell
lines and slightly toxic (80%) at 8:1 charge ratio. Whereas in Neuro-2a cell lines the lipid
Toc-NOH showed less viability i.e.83 % at 4:1 charge ratio, the transfection efficient charge
ratio, when compared to lipids (Toc-As and N14-As)containing ascorbic acid moiety in the
head group region. It is clear that the neuro cell line which are vulnerable to ROS related
apoptosis could survive in the presence lipids (Toc-As and N14-As) containing ascorbic acid
moiety in the head group region than in the presence of lipid (Toc-NOH) not having ascorbic
25

acid in the head group region. Hence, the delivery systems consisting of our designed
antioxidant lipid i.e. TOC-AS having high radical scavenging ability along with maximum
transfection efficiency and least cytotoxicity, may be helpful in treating ROS (reactive
oxygen species) related diseases such as brain stroke/ischemia and malignancy.
Figure.11Representative percent of cell viabilities up on treatment of HepG2 (A), HEK-293T
(B), CHO (C), and Neuro-2a (D) cells with lipids Toc-As, N14-As and Toc-NOH using
MTT assay. The absorbance obtained with reduced formazan with cell in the absent of
cationic lipids was taken to be 100. The toxicity assays were evaluated as depicted in the text.
The data obtained is the average values of three independent experiments (n = 3).
26

Conclusions
In summary, the synthesized α-tocopherol-ascorbic acid hybrid cationic amphiphile can
efficiently deliver genes into multiple culture cells. Among the three cationic lipids studied
(Toc-As,N14-As and Toc-NOH),lipid Toc-As with ascorbic acid head group showed
superior transfection activity. This α-tocopherol-ascorbic acid hybrid lipid has significantly
affected the physico-chemical and biological properties of cationic lipids. Hence, ascorbic
acid head group might be attractive in designing new lipids for gene delivery and it might be
useful for drug delivery. Based on the results it is suggested that Toc-As lipid will be a
promising non-viral gene delivery vector with superior transfection efficiency, strong radical
scavenger activity and least cytotoxicity for treating ROS related diseases in brain.
Experimental Section
General procedure and chemicals reagents
Mass spectral data were acquired by using a commercial LCQ ion trap mass spectro-meter
13
(Thermo Finnigan, SanJose, CA, U.S.) equipped with an ESI source.1H NMR and C NMR
spectra were recorded on a Varian FT400 MHz NMR spectrometer. α-Tocopherol was
purchased from Sigma Co. Super negatively charged eGFP plasmid, and rhodamine-PE were
ample gifts from IICT (Indian Institute of Chemical Technology, Hyderabad, India).
Lipofectamine-2000 was purchased from Invitrogen Life Technologies, polyethylene glycol
8000, and o-nitrophenyl-β-D-galactopyranoside was purchased from Sigma (St. Louis, MO,
U.S.). NP-40, antibiotics, and agarose were purchased from Hi-media, India. 1,2-dioleoyl-sn-
glycero-3-phosphoethanolamine (DOPE) was purchased from Fluka (Switzerland). Unless
27

otherwise stated, various organic solvents including α-tocopherol chloroform, acetone,
ethanol, methylene chloride, phosphomolybdic acid spray reagent, ethyl bromo acetate, p-
toluene sulfonylchloride, and N,N-dimethylformamide (DMF) were purchased from Sigma-
Aldrich Co. and were used without further purification. The progressive of the reaction was
monitored by thin-layer chromatography using 0.25 mm silica gel plates. Column
chromatography technique was executed with silica gel (Acme Synthetic Chemicals, India;
finer than 200 and 60-120mesh). Elemental analyses were performed by High Resolution
Mass Spectrometry (HRMS) using QExactive equipment (Thermo Scientific) and purity of
lipids were characterised by HPLC (Shimadzu LC Solution) and showed more than 95%
purity. HepG2, CHO, Neuro-2a and HEK-293T cells were procured from the National Centre

for Cell Sciences (NCCS), Pune, India. Cell were grown at 37 C in Dulbecco’s modified
Eagle’s medium (DMEM) with 10% FBS in humidified atmosphere containing 5% CO2 / 95
% air.
Synthesis of O-acetic acid-α-tocopherol(1A, scheme 1)
A solution of α-tocopherol (0.5 g, 1.16 mmol) in N, N-dimethylformamide (20mL) was
treated with ethyl bromo acetate (3.4 g, 8.3 mmol) and an excess of powdered KOH (1.2 g,
30 mmol). The resulting yellow residue was stirred vigorously for 24 h at room temperature.
The reaction was acidified with 6N HCl and extracted with ethyl acetate (3 × 30 mL). The
combined ethyl acetate layers were washed with H2O (3 × 30 mL) and brine (1 × 30 mL), and
then dried with Na2SO4. The ethyl acetate solution was concentrated to yellow oil and
purified by silica gel chromatography eluting with 20% (v/v) EtOAc and 1% acetic acid in
hexanes. This yielded 1A as yellow color (0.50 g, 88%).
28

1H NMR (400 MHz, CDCl3) δ/ppm 0.8-0.90 (m, 12H), 1.00-1.50 (m, 25H), 1.77-1.88 (m,
2H), 2.08 (s, 3H), 2.13 (s, 3H), 2.17 (s, 3H), 2.55-2.50 (t, 2H), 4.3 (s, 2H), 7.98 (broad,
1H).ESI-MS: Calculated 488; found [M+18] 506
Synthesis of O-ethyl alcohol -α-tocopherol (1B, scheme 1)
To a solution of lithium aluminium hydride (0.08 g, 1.1 mmol) in 10 mL of dry THF, a
solution of 1A (2.0 g, 1 mmol) in 5 mL dry THF was added slowly at 0C with the help of
addition funnel about 10 minutes. The reaction mixture was stirred for 6 h at room
temperature and then the excess LiAlH4 was quenched with ethyl acetate. The ethyl acetate
layer was washed with water (3×50 mL), dried with anhydrous Na2SO4, filtered, and
concentrated under reduced pressure. The crude product was purified by silica gel column
chromatography to give O-ethyl alcohol-α-tocopherol 1B (1.89 g), 96% as a clear oil. Rf =
0.2 (20% EtOAc/hexane, 2:8).
1H NMR (400 MHz, CDCl3) δ/ppm 0.8-0.90 (m, 12H), 1.00-1.5 (m, 25H), 1.77-1.88 (m,
2H), 2.08 (s, 3H), 2.13 (s, 3H), 2.17 (s, 3H), 2.52-2.59 (t, 2H), 3.66 (t, 2H), 3.85 (t, 2H).
).ESI-MS: Calculated 474; found [M+] 474
Synthesis of O-ethyl-O-sulfonyl benzyl -α-tocopherol (1C, Scheme 1)
To a solution of 1B (4.0 g, 9.0 mmol) in 10 mL of dry DCM were added tosyl chloride (2.12
g, 18 mmol), pyridine (1.46 g, 18 mmol), and a catalytic amount of DMAP. The reaction
mixture was stirred at room temperature for about 12h.The solvent was evaporated in vacuum
to dryness. The residue was dissolved in 25 mL of ethyl acetate and washed twice with2 ×30
mL of copper sulphate solution to remove any excess pyridine. The organic layer was dried
on anhydrous sodium sulphate, the solvent was evaporated, and the residue was purified by
29