RESEARCH

Characterization of Zwitterionic Phosphatidylcholine-BasedBilayer Vesicles as Efficient Self-Assembled Virus-Like GeneCarriers

Reihaneh Ramezani • Majid Sadeghizadeh •

Mehrdad Behmanesh • Saman Hosseinkhani

� Springer Science+Business Media New York 2013

Abstract Entrapment of plasmid DNA (pDNA) in an

aqueous compartment separated from the bulk external

aqueous medium by a phospholipid bilayer resembles a

structure similar to a primitive living cell, and interestingly,

this phenomenon occurs completely self-assembled. Being

inspired by such a structure as well as using the dehydra-

tion–rehydration technique, we were able to encapsulate

pDNA without using multivalent cations and with high

efficiency (98 %) into noncationic lipid bilayer vesicles.

These liposomes which were composed of dimyristoyl-sn-

glycero-3-phosphocholine unlike cationic liposomes, were

nontoxic. The obtained liposome structure was able protect

DNA against nuclease and was completely stable, in a way

that even after 6 months, it still kept the pDNA in its

structure, and there was a small change in its size

(100–150 nm) determined by dynamic light scattering. The

purpose of this research is to polarize the researchers’

interest toward utilization of neutral liposomes originating

from the cell membrane as the most efficient carrier for

gene delivery. We indicated that in using such carriers,

which are the most similar synthetic structures to viruses,

their inability in electrostatic interaction with DNA would

not be an obstacle for entrapping nucleic acids.

Keywords Zwitterionic liposome � Bilayer vesicle � Self-

assembly � Entrapment efficiency � Artificial virus

Introduction

Nonviral gene transfer systems for human gene therapy

applications represent one of the widest fields of chemical,

biological, and medical research today. Although viral

carriers have high efficiency in gene delivery, owing to

potential oncogenic activity of these structures and the high

cost of building these carriers in vitro, their use have

become increasingly limited [1]. Hence, artificial struc-

tures, such as polymers, proteins, lipids, and many other

structures have been considered for this purpose [2, 3].

For a long time, scientists have been attempting to create

artificial viruses which are as proficient as natural ones in

delivering materials to cells. Successful artificial viruses

could carry therapeutic agents into human cells to treat a

variety of diseases [4, 5]. It goes without saying that the

more accurate the mimicking in terms of structure and

function of viruses is made, the more promising would be

the results that are achieved. Among all the available

structures, Liposome, due to its having a simple and self-

assembled structure besides being economic, has been of

interest from the beginning [6, 7]. With the introduction of

a new theory in which lipid bilayer vesicles or liposomes

are strongly regarded as the origins of life [8–10], we

expect that in near future, the dream of creating phospho-

lipid-based artificial viruses with promising therapeutic

potential will come true.

Lipid vesicles (liposomes) are tiny bubble structures

which enclose an aqueous compartment, as membranes of

biological cells. During formation, liposomes in a self-

assembly process are able to entrap DNA, RNA, protein,

R. Ramezani � M. Sadeghizadeh � M. Behmanesh �S. Hosseinkhani

Department of Nanobiotechnology, School of Biological

Sciences, Tarbiat Modares University, Tehran, Iran

M. Sadeghizadeh (&) � M. Behmanesh

Department of Genetics, School of Biological Sciences, Tarbiat

Modares University, P.O. Box 14115-175, Tehran, Iran

e-mail: sadeghma@modares.ac.ir

S. Hosseinkhani

Department of Biochemistry, School of Biological Sciences,

Tarbiat Modares University, Tehran, Iran

123

Mol Biotechnol

DOI 10.1007/s12033-013-9663-7

and small molecules like nucleotides and even multivalent

ions within a confined area of a bilayer lipid membrane

[11–13]. This advantage of liposomes facilitates the

delivery of both gene and drug to the target cells. However,

despite all the advantages, why have the neutral liposomes

not yet received wide attention in the context of gene

delivery in vivo as well as drug delivery systems?

Before the introduction of cationic liposomes, neutral

liposomes were used as a model for studying cell membranes

[14–16], but because of the absence of positive charge for

establishing an electrostatic interaction with DNA, these

structures were not considered in gene delivery process [1].

Later on, the advantages of cationic liposomes in gene

delivery were overshadowed because of high cytotoxicity

and their serum instability. On the other hand, neutral lipo-

somes that originate from cell membrane lipids are com-

pletely nontoxic and are stable in blood circulation [1]. These

carriers can be rendered tissue specific following conjuga-

tion with a specific ligand. Neutral liposome-based drugs are

currently used clinically for the treatment of cancers and

infectious diseases. However, as a gene or DNA delivery

vehicle, neutral liposomes suffer from low encapsulation

efficiency because of their poor interaction with nucleic

acids [17]. By increasing the efficiency of DNA encapsula-

tion within these liposomes, not only the problem of DNA

attachment would be solved, but also the protection of DNA

from degrading caused by nuclease enzymes would be

achieved. Among all the methods for entrapping DNA inside

liposomes, the dehydration–rehydration technique seems to

be more efficient [11, 18–21]. The interesting point is that, in

the theory of creation of primitive living cell from liposomes

[9], this dehydration–rehydration method has been consid-

ered—since, without any external force or additional mate-

rial, any material can be entrapped in the liposome. The

assumption is that the primitive bilayer vesicles which had

been formed through self-assembly process after being

adjacent to RNA, DNA, or some enzymes experienced a

draft and after gradual hydration, all these materials were

accidentally encapsulated in liposomes, and the first sign of

life had appeared [22].

Can we repeat such a phenomenon in vitro conditions?

What about the amount of DNA encapsulated in this lipo-

somal structure? In order to find a reasonable answer to this

question, in this study, being inspired from the theory above

and using dehydration–rehydration technique, DNA was

entrapped into neutral liposomes consisting of dimyristoyl-

sn-glycero-3-phosphocholine (DMPC) with great efficiency.

Since DMPC can be found abundantly in membranes of

living cells and liposomes resulting from this phosphati-

dylcholine have been studied extensively as a model for cell

membranes, using them in designing the delivery systems

can help us derive benefit not only from their nontoxicity but

also from the extensive information in previous studies

regarding their structure and performance [12, 23]. Owing to

promising results obtained in this study, it can be expected

that cell membrane-based neutral liposomes would be con-

sidered in the near future as an alternative to cationic carriers

in gene delivery [24]. By mimicking virus functions in fusion

to cell membrane and transferring DNA to mammalian cells,

these neutral liposomes can be introduced as artificial viruses

in gene therapy [4].

Materials and Methods

Materials

DMPC and cholesterol were purchased from Avanti Polar

lipids (Alabaster, AL), Miniextruder system from Avanti

Polar Lipids (Alabaster, AL), RNase-free DNase I from

Fermentas, Sodium cholate and triton X-100 from Sigma

Chemical Co. (St Louis, MO), and Plasmid Maxiprep

purification kit from Qiagen, Inc. (Santa Clarita, CA). All

other reagents were of analytical grade.

Preparation of Zwitterionic Empty Bilayer Vesicles

Vesicles composed of DMPC or DMPC/cholesterol were

prepared using slight modification of the thin film method

employed by Kudsiova et al. [19]. In brief, lipid (typically

5 mg) was dissolved in chloroform, and the chloroform

evaporated to dryness under vacuum to leave a thin film of

lipid which was hydrated with 1 ml of 40 mM Tris–Hcl

buffer solution pH 8 and 100 mM trehalose, at approxi-

mately 10 �C greater than the phase-transition temperature

of the lipid, yielding a final lipid concentration of 5 mg/ml.

It is important to add the solution to lipid film gradually to

get better suspension. Upon being hydrated, the crude

vesicular suspension was briefly vortexed and then soni-

cated using a probe sonicator fitted with a tapered microtip

operating at 50 % of maximum output at 5–7 cycles of

sonication (30 s ‘‘on’’, 30 s ‘‘off’’) at approximately 10 �C

greater than the phase-transition temperature of the lipid to

produce a suspension of small unilamellar vesicles (SUV).

The resulting suspension was centrifuged at 13,000 rpm

(Heraeus PIC6 Biofuge) for 10 min to remove any titanium

particles which might have been shed by the probe during

sonication. Afterward, the vesicular suspensions were

allowed to anneal at 25 �C for at least 1 h prior to use.

Vesicle size remained unaltered after centrifugation.

Entrapment of Plasmid in Liposome by Dehydration–

Rehydration Method

Dehydration–rehydration vesicles (DRV) encapsulating

DNA were prepared using a slight modification of the

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123

method originally proposed by Kirby [18–21] for the

effective entrapment of DNA into liposomes. An aqueous

solution (1 ml) containing a range of DNA concentra-

tions (between 0.05 and 0.5 mg/ml) was slowly added to

2 ml of a 2.5 mg/ml suspension of SUV which was

prepared and diluted in ultrapure water. The resulting

suspension was mixed, and dried in rotary evaporator

overnight. The dried lipid–DNA mixtures were then

rehydrated at 37 �C with the required volume of ultra-

pure water to produce a DRV suspension encapsulating

DNA. Afterward, the lipid suspension containing multi-

lamellar vesicles was pushed through polycarbonate fil-

ters (200-nm pore size) 11 times by using an Avanti

manual extruder. This resulted in LUVs with a well-

defined and homogeneous size.

Some experiments were carried out to identify the

factors that are influential in increasing the encapsulation

efficiency of DNA in liposome. In several previous

studies [25–29], a strong interaction was reported

between neutral liposome and DNA in the presence of

Ca2? and Mg2?. In order to examine the role of cations

in DNA entrapment in liposomes, various concentrations

of Mg2? were used in liposome preparation buffer, and

the results were taken into consideration. While planning

one of the experiments, the sonication level was skipped

to examine the necessity of production of SUV. In this

experiment, after adding buffer to lipid film and the

formation of multilamellar vesicle (MLV) by skipping

the sonication step, DNA was added directly to the

liposome suspension. In another experiment, after the

dehydration process and the formation of the dry lipid

film, upon adding the buffer containing DNA to dry

lipid, liposomes were formed, and the importance of

adding DNA to liposome suspension before the dehy-

dration for increasing the amount of DNA entrapped into

liposomes was investigated.

Determination of Entrapment Efficiency by Agarose

Gel Electrophoresis

Samples of liposome-entrapped DNA were subjected to

agarose gel (0.8 %) electrophoresis to determine the

retention of DNA by the liposomes [30, 31]. In brief,

10 ll (1–2 lg, DNA) of DRV or SUV suspension was

mixed with 4 ll gel loading buffer (bromophenol blue

0.25 % w/v; xylene cyanol 0.25 %; glycerol 30 % w/v;

EDTA 0.1 M, pH 8), the resulting mixture then was

subjected to agarose gel electrophoresis for 35 min at

90 V, and then finally dyed in the presence of ethidium

bromide (0.5 lg/ml). DNA visualization of the gels was

carried out using ultraviolet illumination (UV-tech, Kiel,

Germany).

Determination of Maximum Loading of DNA

in Bilayer Vesicles Using UV Spectroscopy

The liposomes were centrifuged at 35,000 rpm for precip-

itation. Next, the supernatant was separated and transferred

in another tube. UV–vis spectroscopy (Hitachi U-3300

spectrophotometer) was employed to determine the amount

of DNA to be incorporated in the DMPC liposomes (in the

precipitate) by measuring the concentration of free DNA

remaining in the supernatant from the absorbance of the

DNA peak near 260 nm [19]. The concentration of DNA

present in the liposome was determined by comparing the

concentration of DNA in the supernatant with standard

solutions (i.e., solutions of known DNA concentration).

Nuclease Protection Assay

Free or encapsulated DNA in liposome was incubated with

DNase I at 37 �C for 30 min according to manufacturer’s

instructions (Fermantas). Next, all experimental aliquots

were placed in EDTA buffer and were incubated at 65 �C

for 10 min to inactivate the nuclease. For analyzing Plas-

mid on agarose gel electrophoresis, each sample was added

to an equal volume of 1 % SDS (0.5 % final concentration)

to solubilize lipid vesicles.

Stability of DNA Entrapped in Bilayer Vesicles

The stability of the DNA-encapsulating vesicles with

respect to size and DNA leakage was monitored weekly

over a period of 2–45 days after storage at 4 �C [30]. DNA

leakage from vesicles was measured by centrifuging the

sample at 35,000 rpm for 1 h at 4 �C and measuring the

DNA in the supernatant using UV spectroscopy [19]. The

agarose gel electrophoresis was applied for DNA leakage

determination as well.

Vesicle Sizing and Assessment of Zeta Potential Using

Laser Light Scattering

The average particle size and the polydispersity of the

particle size distribution of the liposomes were determined

by dynamic light scattering using photon correlation spec-

troscopy [19, 30, 31]. The measurements were performed at

25 �C using a Zetasizer Nano ZS instrument (Malvern

Instruments Ltd, Malvern, Worcestershire, UK) equipped

with a helium–neon laser and a scattering angle of 173�.

Before light scattering and zeta potential measurement, the

vesicular samples were diluted with ultrapure, filtered water

to yield a concentration of approximately 0.025 mg/ml of

lipid. At these concentrations, the sizes of the vesicles

recorded were independent of concentration.

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Transmission Electron Microscopy

The TEM specimens of DMPC liposomes were prepared

by negative staining method, which effectively vitrified the

structure in the excess water state. A 5-ll drop of the

aqueous suspension was deposited onto a copper grid

bearing a carbon-coated Formvar film. The solution was

allowed to stand for 1 min and was then withdrawn with

the tip of a piece of filter paper until a very thin layer of

fluid was formed on the grid surface. The samples were

immediately stained for 1 min with an aqueous 4 % acidic

uranyl acetate solution. The ultrathin specimens were then

examined by a Zeiss-EM10C transmission electron

microscope operated at 80 kV.

Cell Viability Assay

CHO cells were seeded in 96-well plates, were incubated

for 24 h, and were allowed to adhere overnight. Afterward,

cells were treated with empty as well as DNA-encapsu-

lating liposomes. The MTT assay was performed to

determine cell viability [27]. 50 ll volume of MTT

(0.5 mg/ml in Dulbecco’s phosphate buffered saline) was

added to the medium in each well, and the plates were

incubated for 3 h at 37 �C. The medium was then dis-

carded, and a 200 ml of dimethyl sulfoxide was added to

each well to insure solubilization of formazan crystals.

Absorbance was measured at 540 nm with a Microplate

Reader (ELx800TM, BioTek, Winooski, VT). Viability was

expressed as a percentage of the control. To calculate cell

survival rate, the absorbance of each sample well (Asample),

blank well (Ab), and negative control well (Ac) were

entered in the equation below:

Survival rate %ð Þ ¼ Asample � Ab

� �= Ac � Abð Þ � 100:

Results and Discussion

Incorporation of Plasmid DNA (pDNA) in Liposome

One of the methods for testing DNA entrapment into

liposome is gel electrophoresis and examination of the

behavior of DNA toward gel (gel retardation assay). In case

of entrapment of pDNA in liposome, we expected to see

retardation of DNA movement in agarose gel electropho-

resis. Encapsulated pDNA remained at the site of appli-

cation in gel electrophoresis, and no free pDNA band was

observed (Fig. 1a), which suggested that a large amount of

plasmid had entered into the liposome, which was later

corroborated by UV spectroscopy. In another experiment, it

was indicated that the formation of SUV before adding

pDNA can increase encapsulation efficiency (Fig. 1b).

Adding DNA to MLV liposomes without sonication and

the formation of SUV liposome to some extent can cause a

decline in DNA entrapment.

As was mentioned in DNA encapsulation protocol, no

cation was added to liposome preparation buffer in any of

the phases. This fully proves that cations have nothing to

do with pDNA encapsulation.

Considering previous reports regarding DNA interaction

and neutral liposome, the existence of cations was found

necessary [27–29], which emphasizes the importance of

these findings. In previous reports, DNA interaction with

liposome surface was discussed [29–31], and since neutral

liposomes do not have positive charge on their surface,

they need multivalent cation for interaction with DNA. The

purpose of this study was to encapsulate DNA into lipo-

some structure, without connecting to the liposome surface.

Therefore, by using the dehydration–rehydration method

without the use of cation, DNA was inserted into liposome

with very high efficiency. The advantage of this method

over other ones which used liposome–DNA–Ca2? complex

is that, alongside with DNA, we can deliver other sub-

stances into liposome. This method can be used for dif-

ferent purposes, such as delivering DNA and drug at the

same time, which would also make it possible to entrap all

necessary elements for transcription and translation sys-

tems into liposomes to produce an artificial minimal cell [8,

32].

Previous studies indicate that adding DNA before the

dehydration procedure for increasing the DNA encapsula-

tion in liposome is necessary, and if buffers containing

pDNA are added to film lipid resulting from liposome

dehydration, the amount of encapsulated DNA would be

decreased [19]. Since adding pDNA to SUV liposome

happens when we stir the suspension, DNA molecules

would be dispersed evenly in liposome structures, which

after being dried they would remain in lipid layers.

Therefore, by adding water, obviously a larger percentage

of the DNA will remain within liposome. When dry lipid

film is formed and DNA containing buffer is added to be

encapsulated in the structure of liposomes, DNA cannot be

placed in lipid layers easily, which leaves some part of it to

remain out of the liposome. As indicated in Fig. 1b, adding

DNA before lipid dehydration and their correct placement

in lipid layers can be very effective in entrapment of DNA

in liposome.

After 1 % SDS treatment, it was expected that pDNA

would be released from the liposome, and its structure

appears clearly on gel after gel electrophoresis. As seen in

Fig. 1a, when pDNA was entrapped in liposome, DNA

remained at the site of application and was not able to

migrate toward the cathode as expected, but the interesting

finding was that, after adding SDS to solubilize liposome

and release the pDNA, there was no change in pDNA

behavior on the gel. It was obvious that adding SDS did not

Mol Biotechnol

123

make pDNA to be released from liposome. In order to

examine this phenomenon, we used the SDS, triton

X-100, with different concentrations for liposome solu-

bilization, but in all cases, the same result was achieved,

and pDNA did not enter the gel (Fig. 2). After conducting

a comprehensive literature search, we found that this

phenomenon had also been reported in another study, and

it was regarded as a reason for the entrance of DNA into

liposome structure [31]. In this report, it was mentioned

that if DNA is attached to liposome surface, it can easily

be freed by detergent, but if it enters inside the liposome,

the DNA cannot be easily extracted from the liposome.

Although we were not able to release plasmid from

liposome, we discovered that plasmid maintained its

biological activity by means of transfecting CHO cells by

this system and observing GFP gene expression. The

results of these experiments have not been reported in this

article.

Fig. 1 Agarose gel electrophoresis of pDNA entrapped in bilayer

vesicles. a Determination of pDNA incorporation: lane 1 DMPC

vesicles encapsulating pDNA, lane 2 DMPC:Chol vesicles encapsu-

lating pDNA (molar ratio, 9:1), lane 3 DNA ladder, and lane 4 Std

pDNA. b Determination of effective factors in entrapping pDNA:

lane 1 DNA ladder; lane 2, 3 adding pDNA to multilayer vesicles

without sonication; lane 4 adding pDNA after dehydration of

liposomes; and lane 5 preparation of liposome encapsulating pDNA

without any modification as a control

Fig. 2 Gel electrophoresis of liposomes encapsulating pDNA to

determine the detergent effect on solubilizing of liposomes. a in the

presence of triton X-100: lane 1 without triton treatment, lane 2 with

0.05 % triton, lane 3 0.1 % triton, lane 4 0.5 % triton, lane 5 1 %

triton, lane 6 2 % triton, lane 7 5 % triton, lane 8 10 % triton, lane 9DNA ladder, and lane 10 Std pDNA. b in the presence of SDS, lane 1

without SDS treatment, lane 2 0.05 % SDS, lane 3 DNA ladder, lane4 0.1 % SDS, lane 5 0.5 % SDS, and lane 6 1 % SDS. All test tubes

were incubated at 37 �C for 30 min after adding detergent. The

concentrations of lipid and DNA in liposome suspension were 5 mg/

ml and 100 lg/ml, respectively

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Determination of Maximum Loading of DNA

in Bilayer Vesicles Using UV Spectroscopy

After measuring the concentration of DNA at supernatant

(Cs) resulting from centrifuging liposomes at 35,000 rpm

and comparing it to the amount of DNA that was added to

liposomes at first (Ct), the amount of inserted DNA to

liposomes was estimated.

Plasmid encapsulation %ð Þ ¼ Ct � Cs=Ct½ � � 100:

Either when cholesterol was added to liposome structure or

when liposomes only consisted of DMPC, almost the entire

DNA was placed inside the liposome structure. Despite the

increase in DNA level up to 500 lg for each 5 mg lipid,

the encapsulation efficiency of DNA was measured at

about 98 % (Fig. 3). Based on previously reported results

[20, 21, 33], DNA could enter the liposome with this

efficiency only when cationic lipids were used in its

structure; however, as was mentioned earlier, there are no

cationic lipids in the structure of liposomes prepared in this

study to avoid their toxic effects. We were able to entrap

DNA into liposome not only without employing cationic

lipids but also in the absence of cations for making elec-

trostatic interaction with DNA and facilitating its entrance

into liposome. These promising results reflect that in the

future, neutral liposomes are going to have a significant

role in gene delivery systems.

Nuclease Protection Assay

As shown in Fig. 4, free pDNA was readily degraded by

the DNaseI. In contrast, pDNA formulated in DMPC lipid

Fig. 3 Encapsulation of pDNA in neutral liposomes prepared using

DMPC and DMPC/Chol (molar ratio 9:1) as a function of DNA

concentration. As shown, pDNA was completely encapsulated in

liposome until its concentration reached to 0.5 mg/2.5 mg lipid. At

this concentration, about 35 % of DNA amount could not be

encapsulated and left in supernatant. Results denote mean ± SD

from five measurements

Fig. 4 Nuclease protection assay: lane 1 Std pDNA, lane 2 naked

pDNA in the presence of DNase I, lane 3 DNA–cationic liposome

(lipofectamine) complex, lane 4 DNA–lipofectamine complex in the

presence of 1 % triton X-100, lane 5 DNA–lipofectamine complex in

the presence of 1 % triton and DNaseI, lane 6 DNA encapsulated in

DMPC liposomes in the presence of 1 % triton X-100, lane 7 DNA

encapsulated in DMPC liposomes in the presence of 1 % triton X-100

and DNase I, and lane 8 DNA ladder

Fig. 5 Measurement of cytotoxicity of liposome components. Cells

were seeded at 1.4 9 104 cells/well in a 96-well plate and incubated

at 37 �C. Percentage of cell viability was determined following 24-h

exposure to varying amounts of each liposomes. MTT assay was

employed to calculate the cell survival rates. a Determination of cell

toxicity of various liposome concentrations, liposome components

consist of empty DMPC, empty DMPC/Chol, and DMPC with DNA

inside; b comparison of cytotoxicities of naked DNA, empty liposome

composed of DMPC and DMPC/Chol, liposome encapsulating DNA,

and lipofectamin along with control of untreated cells, in identical cell

population. Lipofectamine (2 ll) was used as per manufacturer’s

instructions. Liposome concentration was 0.2 mg/ml (100 lg/well) in

all experiments. Values represent the average of triplicates ± SD

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vesicles was substantially protected from degradation by

DNaseI, suggesting that pDNA was largely encapsulated

within lipid vesicles. The point which is certain is that the

resistance of the encapsulated pDNA inside the liposome

against nuclease is a strong reason for DNA encapsulation

inside the liposome, and as seen in Fig. 4, Lipofectamine,

as a cationic liposome, was not able to protect DNA against

DNase. This result is a proof that DNA was attached to the

surface in cationic liposome under the test and was not

delivered to the space inside the liposome.

Toxicity Assay

Nowadays, nontoxicity of DNA transfer carriers is of sig-

nificant value in the design of these systems. This is

because regardless of their high efficiency, cationic carriers

used in DNA delivery are highly toxic, and therefore, their

use in delivering drugs and DNA is decreasing every day.

By examining the resulted liposomes, it was found in this

study that they are not toxic for CHO cells (Fig. 5a, b), and

it was predictable as these liposomes were made of phos-

phatidylcholine that is abundant in living cell membranes.

Nowadays, this liposome structure is very commonly used

for drug delivery, and its nontoxicity in vivo has been

approved. In some cases, phosphatidylcholines are injected

to humans for treatment of particular diseases. Recent

studies point to many potential benefits of phosphatidyl-

choline for liver repair [34, 35]. One study shows phos-

phatidylcholine produces healing effect with hepatitis A, B,

and C. Phosphatidylcholines administration for chronic,

active hepatitis resulted in significant reduction of disease

activity in mice [35].

Stability of DNA Entrapped in Bilayer Vesicles

The stability of the resulting liposomes was evaluated by

examining the size changes during a time period, and the

results were extremely satisfactory. As shown in Fig. 6, no

change in liposome size was seen until after 1 month, and

afterward, although the liposome size increased a little,

aggregation in liposomal suspension was, however, not

obvious.

Fig. 6 Measurement of liposome size changes during a month.

Liposome size distribution was determined in a Zetasizer Nano ZS

instrument at 25 �C. Liposomes were prepared by the dehydration–

rehydration procedure and composed of DMPC:Chol at a molar ratio

of 9:1. The size distribution of liposome did not have any significant

changes after 3 weeks, but a minor size increase occurred after

4 weeks of keeping liposomes containing DNA at 4 �C

Fig. 7 Percentange of DNA leakage from liposome encapsulating

pDNA during a specific period of time. Liposomes were composed of

DMPC and DMPC:Chol (molar ratio 9:1). To measure the amount of

DNA released from liposome, they were precipitated, and the amount

of DNA in supernatant were compared to the amount of DNA that

was added to liposomes at first. UV spectroscopy was employed for

determination of all DNA concentration. The amount of DNA added

to lipids was 100 lg/5 mg lipid

Fig. 8 Determination of DNA leakage from liposome by agarose gel

electrophoresis after being stored for a long time at 4 �C: lane 1 after

1 day, lane 2 after 1 week, lane 3 after 1 month, lane 4 after

6 months, lane 5 DNA ladder, lane 6 standard pDNA, lane 7 standard

pDNA stored for 1 month at 4 �C, and lane 8 standard pDNA stored

for 6 months at 4 �C. The amount of DNA added to lipids was

100 lg/5 mg of lipid

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Examining the DNA leakage from liposome yielded

promising results. After keeping liposomes containing

DNA at 4 �C for a period of 6 months, a negligible amount

of DNA leakage was noticed from the liposome. The

results of UV spectroscopy after liposome precipitation

and gel electrophoresis are shown in Figs. 7 and 8,

respectively.

The temperature stability of the created liposomes with

incubation at 4, 37, and 55 �C indicate that the increase in

temperature does not lead to destruction of the structure

and release of pDNA (Fig. 9). All these results corroborate

that the liposome structure thus formed has very high

stability.

Vesicle Sizing and Assessment of Zeta Potential Using

Laser Light Scattering

The results of measuring the size of liposomes (Table 1)

indicate that DNA entry into the structure of liposome has

not led to any change in its size, and since the extrusion

method and poly carbonate filters sized 200 nm were used

for homogenization of liposomes, the size of liposomes

were always similar, and were in the range of 100–150 nm.

Investigating the size of liposomes in different time frames,

it was found that liposomes were quite stable and passage

of time did not change their size (Fig. 6). Even after

keeping liposome containing DNA at 4 �C for 3 weeks, no

notable change was observed in their zeta potential value

(Fig. 10). By examining the zeta potential, it was found

that DNA encapsulation inside the liposome structure has

had no effect on this characteristic of liposome (Table 1),

which itself proves that DNA was encapsulated in this

structure completely without any attachment to the lipo-

some surface.

Transmission Electron Microscopy

The images from TEM (Fig. 11) indicate that these lipids’

bilayer structures have high potency for fusion. It seems

that the transition of this structure into living cells through

cell membrane would not be problematic any more. There

is no doubt that one of the most challenging parts for

designing each delivery system is penetrating the cell

membrane barrier. It would be a great advantage for a

system if a structure can deliver genetic materials to a

living cell without having positive surface charge and only

with the help of the fusion process.

By comparing the images of liposomes before and after

treatment with triton X-100, it was found that these

structures are resistant to detergent treatment. This finding

Fig. 9 Determination of heat stability of liposome encapsulating

pDNA by analysis of pDNA release from liposome on agarose gel

electrophoresis: lane 1, 2, 3, 4 liposome encapsulating DNA (lane 1stored at 4 �C, lane 2 stored at 25 �C, lane 3 stored at 37 �C, lane 4stored at 55 �C); lane 5 DNA ladder; lane 6 Std pDNA stored at 4 �C;

lane 7 Std pDNA stored at 25 �C; lane 8 Std pDNA stored at 37 �C;

and lane 9 Std pDNA stored at 55 �C. Storage time for all

experiments was 48 h. The concentrations of lipid and DNA in

liposome suspension were 5 mg/ml and 100 lg/ml, respectively

Table 1 Particle sizes in diameter (nm) and zeta potential (mv) of

neutral liposome entrapping and not entrapping pDNA

Formulation Sizea ± SD (nm) fb ± SD (mv)

With encapsulated pDNA

DMPC/Chol (8:2) 96 ± 4.8 -10 ± 4.1

DMPC/Chol (9:1) 93 ± 7.5 -13.7 ± 3

DMPC/Chol (9.5:0.5) 120 ± 25.7 -13 ± 2.5

DMPC 149 ± 17.6 -22.2 ± 3

Without pDNA (blank liposome)

DMPC/Chol (9:1) 99.86 ± 5.9 -14.3 ± 2.2

DMPC 135.7 ± 15.4 -19.8 ± 1.5

a Particle diameter (nm)b Zeta potential (mv)

Fig. 10 Measurement of zeta potential change in liposome encapsu-

lating DNA (100 lg) during 20 days. Liposomes were composed of

DMPC:Chol (9:1). Zeta potential was determined in a Zetasizer Nano

ZS instrument at 25 �C

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confirmed the phenomenon that had been observed in gel

electrophoresis assay. Currently, we are investigating the

factors that are responsible for these observations, and the

results will be published in due course of time. It has pre-

viously been found that one of the reasons for the insolu-

bility of lipids in detergent is the way lipid acyl chains are

ordered [36, 37]. Some detergent-resistant membrane

(DRM) fragments can be isolated from cell lysates after

extraction. DRMs are similar in several ways to the liquid-

ordered (lo) phase. The lo phase has properties intermediate

between gel and lc phases. At low temperatures, lipids exist

in a solid, ordered gel phase that is the characteristic of any

lipid, but above melting temperature (Tm), they form the

fluid, disordered lc phase. Since most phospholipids in cell

membranes have low Tm values, they are believed to be in

the lc phase. It has been observed that most saturated chain

phosphatidylcholines can form the lo phase in the presence

of cholesterol, and at this phase, they are detergent resistant

[37–39]. According to our observations and previous stud-

ies by other researchers, we hypothesize that the resulting

bilayer vesicle in this study might have a structure similar to

this phase, which can be the reason why it is resistant to

Fig. 11 The negative-staining TEM images of phospholipid-based

bilayer vesicles containing pDNA prepared by dehydration–rehydra-

tion method. a TEM images before adding triton X-100, b TEM

image after adding 1 % triton X-100. c the resulting images of

transmission electronic microscopy show that these lipids’ bilayer

structures have high potency for fusion. The areas in which this

phenomenon is clearer have been marked by arrow at image

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detergent treatment. These liposomes are composed of

DMPC which is a saturated chain phosphatidylcholine and

according to previous studies, they can easily form the lophase.

Conclusion

Nowadays, researchers believe that to design an efficient

system in gene delivery and overcome the problems

regarding nonviral delivery systems, we have to mimic

viruses in a better and more accurate way [1, 5]. Creating

an artificial virus not for killing humans but for treating

diseases has always seemed like a charming dream for

researchers. Nowadays, thanks to new and astonishing

achievements, particularly regarding the origin of life and

creation of artificial cells [9, 22], this dream does not seem

to be out of reach anymore. The theory of self-assembled

bilayer lipid vesicles that are the primitive living cells is

advocated to a great extent [8, 32]. The simple and self-

assembled structure of liposomes had been interesting to

scientists since a very long time [7, 15]. Another unique

advantage of liposomes is that they are able to entrap

biological macromolecules such as proteins, RNA, DNA,

or small substances like drugs, nucleotides, and even ions

into their structures. They can even entrap these materials

simultaneously. As mentioned in previous reports, it is

possible to place all necessary elements for transcription,

translation, and protein synthesis inside the liposome, a

system known as an artificial cell [12, 32]. This unique

feature completely distinguishes these structures from

other nonviral delivery systems. Using this feature, the

treatment of diseases and delivery of drug and genes can be

conducted simultaneously, which, as a result, can increase

the effectiveness of the treatment to a great extent. In this

study, by means of phospholipid-based bilayer vesicles,

which have complete organic origins, we intended to

demonstrate that it is possible to entrap the DNA in a

closed space. These results also showed that lack of posi-

tive charge on the surface of liposomes, and even the

existence of cations does not have any important effects for

the self-assembly process. By creating and introducing this

system, we intended to show that the structures with

organic origins can be the best candidate for the design of

delivery systems, and are capable of encapsulating the

DNA structure in themselves with very high efficiency

(98 %), and protecting them against nucleases. Neutral

liposomes which originate from living membrane, despite

many advantages, are not able to interact with nucleic

acids, which therefore led to the less interest being shown

by scientists for using them in delivery systems for the past

several years. The main purpose of this study was to

introduce structures that offer the stablest and the safest

gene and drug delivery system to be used by the scientists

as the delivery system for DNA and gene. Although

delivery of these carriers into cells needs further studies,

the days are not very far from now when these neutral

liposomes can be used as the most efficient carriers for

protein, RNA, DNA, and drug delivery into human cells.

Acknowledgments The authors would like to express their grate-

fulness to the Research Council of Tarbiat Modares University for

providing financial support for this study.

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