RESEARCH PAPER

Microemulsion extrusion technique: a new methodto produce lipid nanoparticles

Marcelo Bispo de Jesus • Allan Radaic •

Inge S. Zuhorn • Eneida de Paula

Received: 11 December 2012 / Accepted: 20 August 2013 / Published online: 3 September 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Solid lipid nanoparticles (SLN) and nano-

structured lipid carriers (NLC) have been intensively

investigated for different applications, including their

use as drug and gene delivery systems. Different

techniques have been employed to produce lipid

nanoparticles, of which high pressure homogenization

is the standard technique that is adopted nowadays.

Although this method has a high efficiency, does not

require the use of organic solvents, and allows large-

scale production, some limitations impede its applica-

tion at laboratory scale: the equipment is expensive,

there is a need of huge amounts of surfactants and co-

surfactants during the preparation, and the operating

conditions are energy intensive. Here, we present the

microemulsion extrusion technique as an alternative

method to prepare lipid nanoparticles. The parameters to

produce lipid nanoparticles using microemulsion extru-

sion were established, and the lipid particles produced

(SLN, NLC, and liposomes) were characterized with

regard to size (from 130 to 190 nm), zeta potential, and

drug (mitoxantrone) and gene (pDNA) delivery prop-

erties. In addition, the particles’ in vitro co-delivery

capacity (to carry mitoxantrone plus pDNA encoding

the phosphatase and tensin homologue, PTEN) was

tested in normal (BALB 3T3 fibroblast) and cancer (PC3

prostate and MCF-7 breast) cell lines. The results show

that the microemulsion extrusion technique is fast,

inexpensive, reproducible, free of organic solvents, and

suitable for small volume preparations of lipid nano-

particles. Its application is particularly interesting when

using rare and/or costly drugs or ingredients (e.g.,

cationic lipids for gene delivery or labeled lipids for

nanoparticle tracking/diagnosis).

Keywords Lipid nanoparticles �Microemulsion extrusion � Solid lipid

nanoparticles � Nanostructured lipid carrier �Liposomes

Introduction

Solid lipid nanoparticles (SLN) and their second

generation, called nanostructured lipid carriers

(NLC), have been firstly described in the 1990s

Marcelo Bispo de Jesus and Allan Radaic have contributed

equally to this study.

M. B. de Jesus � A. Radaic � E. de Paula

Department of Biochemistry, Institute of Biology,

University of Campinas—UNICAMP, CP 6109,

Campinas, SP 13083-970, Brazil

M. B. de Jesus � I. S. Zuhorn

Department of Membrane Cell Biology, University

Medical Center, University of Groningen, A. Deusinglaan

1, 9713 AV Groningen, The Netherlands

M. B. de Jesus (&)

Department of Biochemistry, Institute of Biology,

University of Campinas, Rua Monteiro Lobato no 255,

Campinas, SP CEP 13083-862, Brazil

e-mail: dejesusmb@gmail.com

123

J Nanopart Res (2013) 15:1960

DOI 10.1007/s11051-013-1960-3

(Muller et al. 2000). They have no inner aqueous

compartments and their lipid content is higher than that

of liposomes, the most well-known lipid-based drug

delivery system (Alberts et al. 1985; Gasco 2007). SLN

and NLC have been used as drug delivery systems

(Jiang and Liu 2008; Muchow et al. 2008; Puglia and

Bonina 2012; Puri et al. 2009), gene delivery systems

(Bondi and Craparo 2010; del Pozo-Rodrıguez et al.

2010; Vighi et al. 2010; Zhang et al. 2004), and several

of these applications have been subject of patents

(Attama 2011; Mehnert and Mader 2001). The use of

lipid nanoparticles presents advantages over that of

polymeric nanoparticles, since their production is

water based (avoiding use of organic solvents) and

most lipids used in the formulations are biodegradable,

which guarantees an excellent biocompatibility. In

addition, the standard method (high pressure homog-

enization) for SLN and NLC production is already

successfully implemented in pharmaceutical industry

(Anton et al. 2008; Manjunath et al. 2005; Zhang et al.

2009).

Several techniques have been applied to produce

lipid nanoparticles (Justo and Moraes 2011; Manjunath

et al. 2005; Mehnert and Mader 2001; Mukherjee et al.

2009; Souto et al. 2011), and new methods are being

reported, such as coacervation method and Mumper

and Jay method (Corrias and Lai 2011; Kuntsche et al.

2011; Souto et al. 2011). Among those the high

pressure homogenization technique (HPH), described

by Muller and Lucks (Bunjes et al. 2007; Muller et al.

2000), is the standard method to produce lipid nano-

particles. Through the high pressure (100–2,000 bar)

and high shear stress generated by the homogenizer

particle size can be reduced to the desired sub-micron

range (Hinrichs et al. 2006; Liedtke et al. 2000;

Schwarz and Mehnert 1997). HPH is highly efficient, it

does not require the use of organic solvents, and it

allows lipid nanoparticles production at large-scale.

On the other hand, the equipment has a significant

capital cost, there is a need of large amounts of

surfactants/co-surfactants during the nanoparticles

preparation, and the operating conditions are energy

intensive (Hecht et al. 2011). Such pitfalls of HPH are

especially critical when only small sample volumes are

desirable, for example, in preformulation studies or

with rare and/or expensive ingredients.

As an alternative to HPH, we propose the use of the

microemulsion extrusion technique to produce lipid

nanoparticles. The microemulsion extrusion technique

is fast, inexpensive, reproducible, free of organic

solvents, and suitable for small volumes (MacDonald

et al. 1991). This method is particularly interesting

when using rare or costly compounds, such as certain

drugs, targeting molecules, and nanoparticle compo-

nents (e.g., cationic lipids). Since small-volumes

extrusion is an established method to prepare liposo-

mal formulations (MacDonald et al. 1991) we have

included liposomal formulations in our investigation

of SLN and NLC particles for the codelivery of drugs

and genes. First, we have optimized the production of

lipid nanoparticles by extrusion, using a mini-extruder

apparatus. Second, the lipid particles prepared were

characterized by dynamic light scattering (DLS) and

tested for their use as drug (mitoxantrone) and gene

(pDNA-containing the phosphatase and tensin homo-

log, PTEN construct) delivery systems for cancer

therapy. Mitoxantrone inhibits DNA duplication in the

S-phase of the cell cycle (Alberts et al. 1985) while

PTEN is related to tumor suppression and phospho-

inositide-3 kinase (PI3K) signal transduction, being

deleted or dysfunctional in prostate and breast cancer

(Jiang and Liu 2008). The formulations capacity to

affect cell viability in vitro was tested in normal

(BALB/3T3 fibroblasts) versus cancer (PC3 prostate,

and MCF-7 breast) cells.

Materials and methods

Materials

Egg phosphatidylcholine (EPC), Pluronic F68

(PLF68), mitoxantrone, and stearic acid (SA) were

purchased from Sigma-Aldrich (St. Louis, MO). N-[1-

(2,3-dioleoyloxy) propyl]-N,N,N-trimethyl-ammo-

nium chloride (DOTAP) was obtained from Avanti

Polar Lipids (Alabaster, AL) and glyceryl monostea-

rate (GMS) was kindly donated by Croda (Campinas,

Brazil). pTrcHis Topo TA (Invitrogen, Carlsbad, CA)

expression vector containing the sequence of the

PTEN was kindly provided by M. Peppelenbosch,

Erasmus University Rotterdam.

Microemulsion extrusion method

The composition of SLN, NLC, and liposomes is

given in Table 1. To prepare the formulations, we used

an adaption of the microemulsion method followed by

Page 2 of 15 J Nanopart Res (2013) 15:1960

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extrusion through a 100-nm polycarbonate membrane

(Millipore, Darmstadt, Germany) using an Avanti

mini-extruder (Avanti Polar Lipids, Inc., Alabaster,

AL). In brief, a solution of PLF68 (surfactant) and

DOTAP (co-surfactant) (pre-heated above the melting

temperature of the solid lipid used) was added to the

melted lipid followed by homogenization using vig-

orous stirring. For some samples sonication was

necessary to produce a homogeneous suspension

before extrusion.

Then, the hot emulsion was loaded into the donor

syringe and extruded 15 times, unless otherwise stated,

through the double-syringe extruder (Fig. 1), always

finishing at the receiver syringe, to avoid contamina-

tion. A heating block, pre-heated above the melting

temperature of the lipid, warmed the system during the

whole process. Finally, the lipid nanoparticles were

transferred to an ice bath and then stored at 4 �C.

A systematic study was performed to test different

production parameters, such as the number of extru-

sion cycles (0, 5, 10, 15, 20, 25, and 30 times), and

temperature (5, 10, or 15 �C above the melting point of

the solid lipid, being 58–59 �C for GMS and 69.6 �C

for SA, used in the formulation).

Of note, to avoid extruder membrane disruption

10 mM was the maximum lipid concentration used in

our studies. This concentration may vary when other

lipid mixtures are used and also depends on the type of

formulation (liposomes vs. SLN vs. NLC) that is

produced. Second, to guarantee that the complete

system has reached the same temperature, the heating

block, the lipid, and the aqueous phases should be

heated to the desired temperature, at least 15 min

before the extrusion.

Particle size and zeta-potential measurements

The hydrodynamic diameters, polydispersity index

(PDI) and zeta potentials of the lipid nanoparticles and

liposomes were measured using dynamic laser light

scattering on a Malvern Zetasizer Nano system

(Malvern Instruments Ltd., Worcestershire/UK) at

Table 1 Composition and physicochemical characteristics of the lipid carriers (liposomes, SLN, NLC) prepared by the micro-

emulsion extrusion technique

Formulations Stearic

acid (mM)

DOTAP

(mM)

Pluronic

F68 (mM)

GMS

(mM)

EPC

(mM)

Size (nm) Zeta potential

(mV)

PDI

EPC Liposomes – – – – 3 113.3 ± 4.68 2.90 ± 0.43 0.101 ± 0.001

NLC – – 1 5 3 115.7 ± 8.07 1.30 ± 0.42 0.131 ± 0.001

DOTAP Liposomes – 2.5 – – – 189.5 ± 0.76 57.60 ± 3.39 0.127 ± 0.009

SLN 7 2.5 1 – – 135.9 ± 1.31 40.65 ± 0.49 0.164 ± 0.007

Each value represents the mean ± SD (n = 3)

Fig. 1 Microemulsion extrusion method to prepare lipid

nanoparticles. Step 1 the aqueous surfactant–cosurfactant

solution (pre-heated above the melting temperature of the solid

lipid) was added to the melted lipid followed by homogenization

using vigorous stirring and avoiding changes in temperature.

Step 2 the hot emulsion was loaded into the donor syringe and

extruded several times, always finishing at the receiver syringe

to avoid contamination. Extrusion was performed at a temper-

ature above the melting temperature of the lipid, using a heating

block. Step 3 the lipid nanoparticles were transferred to an ice

bath and stored at 4 �C

J Nanopart Res (2013) 15:1960 Page 3 of 15

123

25 �C. One-hundred microliters of the formulations

were diluted in 1-mL deionized water prior to the

measurement.

Lyophilization studies

Lipid nanoparticles were diluted (75 lL of sample in

1.5 mL of deionized water) and freeze dried in the absence

or presence of a cryoprotector (trehalose or sucrose, at a

final concentration of 4 wt/v%). The resulting dispersions

were frozen in liquid nitrogen, and subsequently lyophi-

lized using a Freezone 4.5 (Labconco, USA) for 48 h

under vacuum. Lyophilized samples were reconstituted in

deionized water and their size, zeta potential, and PDI were

measured and compared with freshly prepared (non-

lyophilized) samples.

Morphology study using transmission electron

microscopy (TEM)

The TEM grid was exposed to a droplet (10 lL) of each

formulation for 10 s and let to air-dry. After that, negative

staining was performed to improve the contrast: the TEM

grid was exposed to a droplet (10 lL) of uranyl acetate

solution (1 wt%, pH 4) for 10 s. Then, to avoid over

staining, the grid was placed in a droplet of water for a few

seconds and the excess of fluid was removed with a filter

paper. Subsequently, the sample was analyzed using a

JEOL JEM-2010HC transmission electron microscope

(JEOL Co. Ltd., Tokyo, Japan).

Lipid nanoparticles entrapment efficiency

Lipid nanoparticles were prepared as previously

described with addition of mitoxantrone (MTX

5 mM) in the lipid phase. For liposomal formulations,

an aqueous solution containing MTX was used to

suspend the lipids. The entrapment efficiency (%EE)

of MTX into lipid nanoparticles was determined by

ultrafiltration method using centrifugal filter tubes

with a molecular weight cut-off of 10 kDa (Amicon

Ultra, Millipore, Ireland) at 3,5509g for 5 min. The

concentration of MTX in lipid nanoparticles and in the

filtrate (freeMTX) was analyzed at 672 nm, using a

Cary� 50 UV–Vis spectrophotometer (Varian, USA)

and quantified using equation for developed analytical

curve (absorbance = 0.0975 9 concentration (M) ?

0.1525, r = 0.9982). %EE was calculated according

to:

%EE ¼ totalMTX� freeMTX

totalMTX� 100

Drug-release assay

MTX release was determined using Franz diffusion cells

(1 mL in donor compartment and 4 mL in acceptor

compartment). Membranes of 12–14-kDa (Spectrumlab,

EUA) molecular weight cut off were used and PBS 5 mM

(pH 7.4) was set as the release medium. Sample was

diluted to 5 lmol of MTX (in solution or in lipid

nanoparticles) and placed in the donor compartment

(1 mL). The concentration of MTX in the acceptor

compartment was quantified at 672 nm using a Cary� 50

UV–Vis spectrophotometer (Varian, USA) at specified

time points (1, 2, 3, 4, 5, 6, 7, 8, 24, 48, and 72 h). The

acceptor compartment was homogenized using a mag-

netic stirrer. The drug concentration was quantified using

the equation of a previously determined analytical curve

(absorbance = 0.0975 9 concentration (M) ? 0.1525,

r = 0.9982).

Plasmid purification

The pTrcHis Topo TA (Invitrogen, Carlsbad, CA)

expression vector containing the sequence of the

PTEN gene was transformed into Escherichia coli.

The bacteria were incubated in Luria–Bertani (Bertani

2004) medium (10 g tryptone, 5 g yeast extract and

10 g NaCl was dissolved in 1 L of distilled water and

the pH was adjusted to 7.0 with 1 M NaOH; then the

mixture was autoclaved for 25 min at 120 �C) in the

presence of the selective antibiotic ampicillin. Later,

the plasmid was purified using GenEluteTM HP

Plasmid Maxiprep Kit (Sigma-Aldrich Corporation)

according to manufacturer’s instructions.

DNA-binding assay and DNase I protection assay

For DNA binding, complexes of DNA and lipid

nanoparticles or liposomal formulations were prepared

using 1 lg of pDNA and 2 lL of the carrier (EPC

liposomes, SLN, DOTAP liposomes, and NLC) in

water. After 20 min of incubation at room temperature,

the samples were loaded in 10 % glycerol on a 0.8 %

agarose gel-containing ethidium bromide (0.5 lg/mL),

and immersed in TBE buffer (89 mM Tris, 89 mM

boric acid, 2 mM ethylenediaminetetraacetic acid,

Page 4 of 15 J Nanopart Res (2013) 15:1960

123

EDTA). The samples were run at 50 V for 2 h. Images

were taken using a UV trans illuminator and processed

using ImageJ software (http://imagej.nih.gov/ij/). For

DNase I protection assay complexes of DNA and lipid

carriers were prepared as described above for the DNA-

binding assay. Then, samples were exposed to DNase I

(1 unit DNase/2.5 lg pDNA) at 37 �C for 30 min. After

that the samples were treated with 1 % sodium dodecyl

sulfate to release DNA from the lipoplexes, and sub-

mitted to agarose gel electrophoresis and analyzed, as

described for the DNA-binding assay.

Cell culture

BALB/3T3 and MCF-7 cells were maintained in

Dulbecco’s Modified Eagle’s Medium, DMEM (Sigma

Chemical Co.), while PC3 cells were maintained in

RPMI 1640 culture medium. DMEM and RPMI 1640

culture media were supplemented with 10 % (v/v) fetal

bovine serum and 1 % (v/v) penicillin/streptomycin.

Cells were grown at 37 �C under a humidified atmo-

sphere with 5 % CO2 in air and subcultured every

2 days using trypsin/EDTA. Mitoxantrone-resistant

MCF-7 cells were cultured in medium containing

100 nM of MTX (Zhang et al. 2004).

Cytotoxicity

The cytotoxicity of the lipid nanoparticles formula-

tions was tested on mouse embryonic fibroblasts

(BALB/3T3), prostate cancer cells (PC3), and human

mammary cancer cells (MCF-7). The cells were plated

at a concentration of 104 cells per well in 96-well

plates and incubated (37 �C and 5 % CO2) overnight

for adhesion. Then, the cells were treated for 24 h with

lipid nanoparticles formulations containing the drug

(10 lM of MTX), the plasmid-containing PTEN gene,

or double-loaded with drug and gene, in RPMI 1640

medium without serum. After that, the medium was

replaced with 100 lL of 3-[4,5-dimethylthiazol-2-yl]-

2,5-diphenyl tetrazolium bromide (MTT solution-

0.5 mg/mL) for 4 h incubation at 37 �C; following

removal of the MTT solution, the formazan crystals

formed were dissolved in 100 lL of DMSO. The

solutions were homogenized for 5 min on a plate

shaker, and the absorbance was measured at 570 nm

on a microplate ELX800 spectrophotometer (Biotek,

Winooski/EUA). Cell viability was expressed as

percentage of the untreated control.

Statistical analysis

All the experiments were performed in triplicate and

were repeated independently, at least three times.

Mean and standard deviations were calculated.

ANOVA followed by Tukey’s post-test were per-

formed to test for statistical significance (p \ 0.05)

between the treatment groups.

Results and discussion

Microemulsion extrusion technique as a new

method to produce lipid nanoparticles

Mini-extruder systems are commonplace in bioscience

laboratories and have been used to prepare liposomes

for many years. Here, we put forward the use of this

instrument to prepare also lipid nanoparticles, specif-

ically SLN and NLC. The formulation compositions

tested are shown in Table 1. To establish the micro-

emulsion extrusion as a new method to produce lipid

nanoparticles, we have analyzed changes in two

parameters: extrusion cycles and temperature. We

studied the effect of such parameters on the physico-

chemical characteristics of the SLN and NLC pro-

duced and compared them to similar liposomal

formulations.

First, we determined the minimum number of

extrusion cycles (Fig. 2) required to produce a mono-

disperse and homogeneous population of lipid nano-

particles. The number of extrusion cycles being

defined as the number of passages of the hot emulsion

from the donor syringe to the receiver syringe and

back. As shown in Fig. 2a–c, after ten extrusion cycles

no statistical differences were found among the

particles, regarding size, zeta potential, and PDI, in

comparison to particles produced with 30 extrusion

cycles. Similar results were observed for the liposomal

formulations. Therefore, we determined 15 extrusion

cycles as the minimum number of extrusion cycles to

guarantee homogeneous production of lipid

nanoparticles.

The temperature effect on the production of the

lipid nanoparticles regarding size, zeta potential, and

PDI was determined next. The SLN formulation

(Fig. 2d) showed a decrease in particle size when

prepared at ?15 �C above the melting point of stearic

acid, while the size of the NLC was not affected by

J Nanopart Res (2013) 15:1960 Page 5 of 15

123

Fig. 2 Optimization of process parameters for the production

of lipid nanoparticles by microemulsion extrusion technique.

Left the effect of the number of extrusion cycles (0–30) on the

production of EPC liposomes, NLC, DOTAP liposomes, and

SLN was evaluated regarding a average diameter, b zeta

potential, and c PDI. Each value represents the mean ± SD

(n = 3). The results are expressed as the mean ± SEM of three

independent experiments, letters indicate significant differences

as determined by one-way ANOVA followed by Tukey’s

(p \ 0.05) post-test in comparison to: a the EPC liposomal

formulation after 30 cycles; b the NLC formulation after 30

cycles; c the DOTAP liposomal formulation after 30 cycles;

d the SLN formulation after 30 cycles. Right the effect of

temperature (?5, ?10 and ?15 �C above the lipid-melting

point for SLN and NLC and ?5, ?10, and ?15 �C above room

temperature for liposomes, which is well above the lipid melting

point) used to produce EPC liposomes, NLC, DOTAP

liposomes, and SLN was also evaluated regarding the d average

diameter, e zeta potential, and f PDI. Each value represents the

mean ± SD (n = 3). The results are expressed as the mean ±

SEM of three independent experiments, letters indicate signif-

icant differences as determined by one-way ANOVA followed

by Tukey’s (p \ 0.05) post-test in comparison to: a the EPC

liposomal formulation prepared at Tf ? 5 �C; b the DOTAP

liposomal formulation prepared at Tf ? 5 �C; c the SLN

formulation prepared at Tf ? 5 �C

Page 6 of 15 J Nanopart Res (2013) 15:1960

123

increasing the temperature. Mehnert and Mader

(2001) using HPH, have reported lower particle sizes

in SLN prepared at higher temperatures, as a result of

decreased viscosity in the inner particles phase.

Figure 2e also shows that the zeta potential of both

formulations was not affected by the increase in

temperature. Nevertheless, high temperatures

increased SLN heterogeneity (Fig. 2f). At ?10 and

?15 �C, the PDI values for SLN were higher than 0.3.

Interestingly, the NLC produced at all temperatures

kept the PDI values below 0.2. A PDI value lower than

0.3 represents a homogenous and monodisperse par-

ticle population (Anton et al. 2008; Zhang et al. 2009).

Also for the liposomes increasing the temperature

resulted in larger and more dispersed vesicles. Both

EPC and DOTAP liposomes presented a significant

increase in their average diameter, and their PDI

values were higher than 0.3 when prepared at ?15 �C.

Such instability agrees with previous reports in the

literature (Justo and Moraes 2011) showing that high

temperatures can affect the physicochemical charac-

teristics of lipid vesicles.

Taking into account the effects of temperature on

particle size, zeta potential, and PDI, we have

established ?5 �C above the lipid-melting point as

the optimum temperature during extrusion (15 cycles)

in order to obtain homogeneous lipid nanoparticles.

In conclusion, it is shown that the microemulsion

extrusion technique is a valid method to prepare (small

amounts of) lipid nanoparticles.

Morphological and physicochemical comparison

between lipid nanoparticles and liposomes

produced by the microemulsion extrusion

technique

After optimizing the production process of lipid

nanoparticles, we have proceeded with the character-

ization of the formulations presented in Table 1. We

initiated investigating the morphological differences

between liposomes and lipid nanoparticles. It is known

that the morphology and architecture of nanoparticles

depends on their composition and preparation process

(Kuntsche et al. 2011). Therefore, the morphology of

SLN, NLC, and liposomes was investigated using

electron microscopy. Preparations of EPC and DO-

TAP liposomes (Fig. 3a, c, respectively) reveal quite

homogeneous populations of vesicles, showing the

existence of bilayer structures spaced by an internal

(aqueous core) compartment. In contrast, the NLC and

SLN preparations (Fig. 3b, d, respectively) show

onion-like layered structures, as previously described

for nanoparticles stabilized with saturated phospho-

lipids in the a-conformation (Bunjes et al. 2007).

Thus, while an aqueous core surrounded by a hydro-

phobic lipid bilayer characterizes liposomal structure,

a high-density core characterizes both SLN and NLC

lipid nanoparticles.

Table 1 summarizes the physicochemical proper-

ties of lipid nanoparticles and liposomal formulations

produced. EPC liposomes and NLC showed an

average diameter of *110 nm, neutral zeta potential

(*0 mV) and homogenous particle-size distribution

(PDI * 0.1). DOTAP liposomes and SLN had

positive zeta potentials ([?40 mV) and also pre-

sented a narrow-size distribution (\0.2). DOTAP

liposomes (189.5 ± 0.76 nm) showed slightly bigger

particles than SLN (135.9 ± 1.31 nm). Because

nanoparticle stability is of remarkable importance

for a successful therapeutic application, we investi-

gated the stability of all lipid formulations, regarding

size, zeta potential, and PDI, over 180 days of storage

under refrigeration. Liposomal particles underwent a

significant increase in size after 150 days, whereas

lipid nanoparticles were stable over the 180 days

tested (Fig. 3e). The cationic (DOTAP) liposomes

showed a decrease in the zeta potential after 90 days

(Fig. 3f), which could compromise its ability to bind

genetic material, because it is mediated by electro-

static interactions. In addition, the homogeneity of the

liposomal formulations was compromised after

90 days (Fig. 3g), as demonstrated by the increase in

the PDI. Remarkably, the lipid nanoparticles (SLN

and NLC) size, zeta potential, and PDI values

remained stable over the 180 days of storage.

Subsequently, it was tested whether the lipid

nanoparticles and liposomal formulations could keep

their physicochemical properties after freeze drying.

Lyophilization of nanoparticles brings several phar-

maceutical advantages, including the possibility for

long-time storage, transportation at room temperature,

and increased particle stability (Hinrichs et al. 2006;

Schwarz and Mehnert 1997). Nevertheless, this pro-

cess exposes nanoparticles to freezing and drying

stresses that by itself can cause changes in physical

characteristics, e.g., due to water sublimation, even-

tually leading to particle aggregation. Indeed, as

indicated in Table 2 the formulations that were

J Nanopart Res (2013) 15:1960 Page 7 of 15

123

lyophilized without cryoprotector showed a significant

increase in size compared to freshly prepared lipid

formulations (non-lyophilized), i.e., 30–50-fold for

the neutral formulations (EPC liposomes and NLC)

and 8–10 fold for the cationic formulations (DOTAP

liposomes and SLN), the latter according to the report

Page 8 of 15 J Nanopart Res (2013) 15:1960

123

of Das and Chaudhury (2011) showing that SLN

lyophilization without cryoprotectant resulted in par-

ticle aggregation (Das and Chaudhury 2011; Gasco

2007). To prevent the aggregation of particles cryo-

protectors can be used, which will also improve the

redispersion of the dry product. Although the lipo-

somes kept their original sizes following redispersion

after lyophilization in the presence of cryoprotectant

(trehalose and sucrose), their homogeneity was

severely compromised (PDI [ 0.3). Conversely, lipid

nanoparticles only slightly increased their average

diameter, when freeze dried with sucrose, and kept

their size, zeta potential, and homogeneity, when

lyophilized with trehalose. Therefore, trehalose was

demonstrated to be an effective cryoprotectant to

avoid aggregation during lyophilization and subse-

quent reconstitution of these lipid nanoparticles.

Lipid nanoparticles produced by microemulsion

extrusion as drug delivery system

Next we investigated the lipid nanoparticles produced

as drug delivery system, taking mitoxantrone (MTX)

as a model drug. MTX is a hydrophobic synthetic

anthracenedione chemotherapeutic drug, extensively

used for the treatment of several types of cancer,

including breast cancer, prostate cancer, lymphoma,

and leukemia. Several nanoparticle formulations con-

taining MTX are reported (Lu et al. 2006; Muchow

et al. 2008; Puglia and Bonina 2012; Puri et al. 2009;

Zhang et al. 2011a, b). First, the SLN and NLC

capacity to entrap MTX was measured and compared

to the entrapment efficiency of the liposomal formu-

lations. Liposomal formulations showed low Ta

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Fig. 3 Transmission electron micrographies and long-term

stability of lipid nanoparticles: a–d morphological differences

between the ultrastructure of lipid nanoparticles and liposomal

formulations. a EPC liposomes, b NLC, c DOTAP liposomes,

d SLN (scale bars 100 nm). Samples used here were prepared

using the optimal conditions established: 15 extrusion cycles

and ?5 �C above the solid lipid melting point. The samples

were staining with uranyl acetate to improve the contrast and

investigated using a JEOL JEM-2010HC transmission electron

microscope (JEOL Co. Ltd., Tokyo, Japan). e–g The colloidal

stability of EPC liposomes, NLC, DOTAP liposomes and SLN

stored under refrigeration (4 �C) were followed up to 180 days.

Regarding: e diameter, f zeta potential, and g PDI. Each value

represents the mean ± SD (n = 3). The measurements were

carried using dynamic light scattering on a Malvern Zetasizer

Nano system (Malvern Instruments Ltd., Worcestershire/UK),

at 25 �C in polystyrene cuvettes with a path length of 10 mm

b

J Nanopart Res (2013) 15:1960 Page 9 of 15

123

entrapment efficiency: EPC and DOTAP liposomes

entrapment efficiency was 9.94 (±4.6) and 14.05 %

(±4.1), respectively. In contrast, lipid nanoparticles

showed higher entrapment efficiency: NLC entrap-

ment efficiency was 81.03 % (±2.9) and SLN entrap-

ment efficiency was 63.95 % (±3.8).

Second, the MTX-release profile from the formu-

lations was assessed using a Franz diffusion cell. As

depicted in Fig. 4, liposomal formulations displayed a

release kinetic profile very similar to free MTX.

Within the first 8 h, all the MTX was detected in the

receptor compartment. Presumably, the drug is loaded

into the lipid bilayer in liposomes; therefore fast

equilibrium with the medium could be responsible for

the fast release profile. Conversely, lipid nanoparticles

displayed a significantly different release kinetic

profile from free MTX and liposomes formulations.

Even after 72 h, little MTX was detected in the

receptor compartment (ca. 10.4 % from SLN and

12.7 % from NLC). In lipid nanoparticles, the drug is

loaded in the lipid matrix, which may delay drug

release, resulting in a sustained release system.

Otherwise, in some cases the payload is expelled in

one go from the lipid nanoparticles, this is called

‘‘burst effect’’, and it is a highly undesirable feature

found in some SLN formulations (Battaglia and

Gallarate 2012; Bondi and Craparo 2010; del Pozo-

Rodrıguez et al. 2010; Vighi et al. 2010). From a

therapeutic perspective, it was interesting to note that

lipid nanoparticles prepared here provided a sustained

release of MTX, as previously described for lipid

nanoparticles incorporation MTX (Attama 2011; Lu

et al. 2006).

Lipid nanoparticles produced by microemulsion

extrusion as gene delivery system

Having determined that lipid nanoparticles properly

accommodate MTX and show a sustained release

profile, we assessed whether these particles could also

act as gene delivery system. First, the DNA-binding

capacity of the lipid nanoparticles was evaluated and

compared with that of liposomal formulations

(Fig. 5a). As expected, the formulations containing

the cationic phospholipid DOTAP, i.e. DOTAP lipo-

somes and SLN, were able to efficiently bind pDNA.

On the other hand, formulations without DOTAP, i.e.

EPC liposomes and NLC, presented no capacity to

bind pDNA. The ability of these formulations to

protect pDNA from DNase I action correlated with

their DNA-binding capacities (Fig. 5b). Specifically,

the formulations containing DOTAP completely pro-

tected pDNA from DNase I, while DOTAP-free

formulations could not protect against DNA degrada-

tion. Together, these results support the idea that

cationic lipid nanoparticles (SLN) and cationic (DO-

TAP) liposomes are able to carry pDNA.

Codelivery of drugs and genes by lipid

nanoparticles produced by microemulsion

extrusion

Recently, codelivery systems, which are able to carry

drugs and genes together, have been described as a

new strategy for cancer treatment (Chen et al. 2012;

Manjunath et al. 2005; Yu et al. 2012). Since the lipid

nanoparticles produced here have drug and gene

delivery properties, we tested their codelivery capacity

on cancer cells.

The chemotherapeutic drug MTX and the phos-

phatase and tensin homolog (PTEN) tumor suppressor

gene were chosen for delivery into cancer cells. MTX

intercalates into DNA and binds topoisomerase II,

thereby compromising cellular replication. Neverthe-

less, it provides modest survival and palliative benefits

in advanced stages of cancer (Mahon et al. 2011;

Manjunath et al. 2005; Mehnert and Mader 2001;

Mukherjee et al. 2009; Souto et al. 2011). With that in

Fig. 4 Mitoxantrone release assay. Drug-release profiles of MTX

from EPC liposomes, NLC, DOTAP liposomes, and SLN in

phosphate-buffered saline pH 7.4 (mean ± SD, n = 3). The MTX

release was measured using a Franz diffusion cells (1 mL in donor

compartment and 4 mL in acceptor compartment) at specified time

points (1, 2, 3, 4, 5, 6, 7, 8, 24, 48, and 72 h). Drug concentration was

quantified according to the analytical curve (absorbance =

0.0975 9 concentration (M) ? 0.1525, r = 0.9982)

Page 10 of 15 J Nanopart Res (2013) 15:1960

123

mind, we tested MTX in association with the tumor

suppressor PTEN, aiming for therapeutic benefits to

the treatment of advanced stages of cancer. PTEN is

downregulated in cancer cells, which has been shown

to play an important role in advanced cancer (Corrias

and Lai 2011; Jiang and Liu 2008; Souto et al. 2011),

amongst others by the development of resistance or

reduced sensitivity to radiation and chemotherapy

(Jiang et al. 2007; Muller et al. 2000). The combina-

tory effect of MTX and pDNA-PTEN in advanced

cancer treatment, was tested on PC3 prostate cancer

cells that lack PTEN, and MTX-resistant MCF-7

breast cancer cells (Liedtke et al. 2000; Zhang et al.

2004).

Delivery of MTX and pDNA-PTEN by liposomes

and lipid nanoparticles: effect on cell viability

To first determine the IC50 of free MTX to induce cell

death, the cell viability of BALB/3T3 fibroblasts, PC3

prostate cancer cells, and MCF-7 resistant and non-

resistant breast cancer cells in the presence of different

concentrations of MTX, ranging from 0 to 100 lM,

was assessed (Fig. 6). At a concentration of 10 lM a

cell viability of 50 % was observed for all cell types,

except for the MCF-7 resistant cells, that showed an

IC50 at a MTX concentration of 55 lM.

Subsequently, the BALB/3T3 cell viability follow-

ing treatment with the lipid-based formulations con-

taining 10 lM MTX and/or pDNA-PTEN was

determined (Fig. 7a, b). EPC and DOTAP liposomes

containing MTX or containing MTX and pDNA-

reduced BALB/3T3 cellular viability to a similar

extent. Cell viability was 47.5 ± 10.6 % for cells

treated with EPC lipMTX, 62.7 ± 7.7 % with EPC

lipMTX?pDNA (Fig. 7a), 62.9 ± 9.4 % with DOTAP

lipMTX and 58.8 ± 7.1 % with DOTAP lipMTX?pDNA

(Fig. 7b). Liposomal formulations and MTX in solu-

tion showed similar effect on BALB/3T3 cell viability,

this could be explained by the high percentage of free

MTX in the liposomal formulations.

Free plasmid, empty liposomes, and lipid nanopar-

ticles showed no effect on BALB/3T3 cell survival.

Interestingly, neither lipid nanoparticles containing

Fig. 5 DNA binding and protection by lipid nanoparticles and

liposomal formulations produced by microemulsion extrusion.

DNA-binding assay and DNase I protection assay evaluated the

potential for gene delivery of lipid nanoparticles and liposomal

formulations. a DNA-binding assay for EPC liposomes, NLC,

DOTAP liposomes, and SLN. b DNase I protection assay for

EPC liposomes, NLC, DOTAP liposomes, and SLN. Complexes

of DNA and lipid nanoparticles or liposomal formulations were

prepared using 1 lg of pDNA and 2 lL of carrier in water. After

20 min of incubation at room temperature, the samples were

loaded in 10 % glycerol on a 0.8 % agarose gel containing

0.5 lg/mL ethidium bromide, and immersed in TBE buffer. The

samples were run at 50 V for 2 h and images were taken using a

UV transilluminator. For DNase I protection assay, prior to

submitting the samples to electrophoresis, complexes of DNA

were exposed to DNase I (1 unit DNase/2.5 lg pDNA) for

30 min at 37 �C

J Nanopart Res (2013) 15:1960 Page 11 of 15

123

MTX, nor lipid nanoparticles containing MTX plus

pDNA-reduced BALB/3T3 cellular viability, as

shown in Fig. 7a (NLC) and Fig. 7b (SLN). Presum-

ably, the slow release of MTX from these formulations

prevents a cytotoxic effect on BALB/3T3 cells.

Next, we tested the formulations activity against a

prostate cancer cell line (Fig. 7c, d). PC3 prostate

cancer cells treated with free MTX (10 lM) showed a

reduction in cell viability to 52.3 ± 5.9 %. As

observed for BALB/3T3 cells, EPC liposomal formu-

lations reduced PC3 cellular viability to similar

extents (57.6 ± 10.5 % for EPC lipMTX and

52.7 ± 10.3 %, for EPC lipMTX?pDNA, Fig. 7c), prob-

ably due to the high percentage of free MTX in those

formulations. For EPC lippDNA no effect was noticed

on PC3 cell viability (96.2 ± 8.6 %), most probably

because of the lack of pDNA-PTEN binding to such

liposomes (see Fig. 5a).

Treatment of PC3 cells with NLC did not reduce

cell viability (Fig. 7c), neither with NLCMTX

(103.6 ± 5.4 %) nor with NLCMTX?pDNA (101.6 ±

7.1 %). This result can possibly be explained by the

high entrapment efficiency (81 ± 5.9 %) but slow

release kinetics of NLC, which may prevent release of

sufficient amounts of MTX to reach a therapeutic level

within the cells. Alternatively, the intracellular fate of

this nanoparticle, for instance in degradative lyso-

somal compartments, could prevent its therapeutic

effect (MacDonald et al. 1991; Sahay et al. 2010).

pDNA-PTEN (98.1 ± 5.1 %), EPC Lip.pDNA

(92.2 ± 7.4 %) and NLCpDNA (102.2 ± 5.0 %) were

nontoxic to PC3 cells either, probably because of an

inefficient delivery of pDNA-PTEN (Fig. 7c).

On the other hand, treatment of PC3 cells with

cationic formulations (DOTAP liposomes and SLN)

caused reduction in cell viability (Fig. 7d). Delivery of

pDNA-PTEN with SLN resulted in a more pro-

nounced cytotoxic effect in PC3 cells than with

DOTAP liposomes (40.2 ± 8.5 %), either alone

(SLNpDNA 25.8 ± 5.4 %) or in conjugation with

MTX (SLNMTX?pDNA 25.1 ± 7.1 %). PC3 cells via-

bility was not affected after treatment with SLNMTX

(101.3 ± 7.6 %). Similar to EPC liposomes, DOTAP

LipMTX (54.5 ± 8.6 %) and DOTAP LipMTX?pDNA

(40.7 ± 7.7 %) were as cytotoxic as free MTX to PC3

cells (Fig. 7d).

Altogether, the delivery of PTEN with DOTAP

liposomes resulted in a slightly higher reduction in cell

viability than delivery of MTX with DOTAP lipo-

somes in PC3 cells. Codelivery of PTEN and MTX

with DOTAP liposomes gave a similar reduction in

cell viability as delivery of just PTEN. Therefore,

MTX seems not to have an additional effect over

PTEN in PC3 cells. In addition, SLNs showed an

inefficient delivery of MTX into PC3 cells, while

delivery of PTEN resulted in a greater reduction in cell

viability than delivery of PTEN with DOTAP lipo-

somes. Therefore, gene delivery with SLN seems more

efficient than with DOTAP liposomes, but the SLN

formulation used here is not yet suitable for codelivery

of MTX. The SLN formulation would need to be

improved to obtain better release kinetics for the

cytotoxic drug into PC3 cells.

Finally, the formulations were tested on resistant

breast cancer cells. Resistant MCF-7 breast cancer

cells (see ‘‘Materials and methods’’) treated with free

MTX (10 lM) showed no reduction in cell viability,

as expected from its higher IC50 value (55 lM,

Fig. 6). Similarly, empty EPC liposomes, NLCMTX,

NLCpDNA, and NLCMTX?pDNA did not reduce MCF-7

cellular viability (Fig. 7e). Neither DOTAP-contain-

ing MTX formulations (DOTAP lipMTX = 97.5 ±

8.5 % and SLNMTX = 100.1 ± 4.7 %) were cyto-

toxic to resistant MCF-7 cells (Fig. 7f). Interestingly,

both DOTAP formulations containing pDNA-PTEN

were able to reduce resistant MCF-7 cells viability to

similar extents (DOTAP lippDNA = 60.2 ± 8.5 % and

DOTAP lipMTX?pDNA = 62.7 ± 7.5 %, Fig. 7f),

Fig. 6 In vitro antitumor effect of MTX in 3T3 fibroblasts,

prostate cancer cells, breast cancer cells, and resistant breast

cancer cells. BALB/3T3 cells, PC3 cells, MCF-7 and MCF-7

resistant were treated for 24 h with increasing concentrations of

MTX and cell viability was assessed by MTT assay. The results

are expressed as the mean ± SEM of three independent

experiments

Page 12 of 15 J Nanopart Res (2013) 15:1960

123

Fig. 7 Cytotoxic and in vitro antitumor effect of MTX

codelivered with PTEN gene using lipid nanoparticles and

liposomal formulations. BALB/3T3 cells (a, b), PC3 cells (c, d),

and MCF-7 resistant (e, f) cells were treated for 24 h with empty

carriers, MTX, naked pDNA-PTEN, MTX-containing carriers,

pDNA-PTEN-containing carriers or with carriers containing

both MTX and pDNA-PTEN. Lefts panels (a, c, e) carriers are

EPC liposomes and NLC; Right panels (b, d, f) carriers are

DOTAP liposomes and SLN. Cell viability was assessed by MTT

assay. The results are expressed as the mean ± SEM of three

independent experiments. Different letters indicate significant

differences: a compared to empty EPC liposomes group;

b compared to empty DOTAP liposomes group; c compared to

free pDNA-PTEN group; d compared to empty SLN group;

e compared to DOTAP Lip.MTX group; f compared to DOTAP

Lip.MTX?pDNA-PTEN group and g compared to SLNpDNA-PTEN

group. The significance was determined by one-way ANOVA

followed by Tukey’s post-test (n = 3, p \ 0.05)

J Nanopart Res (2013) 15:1960 Page 13 of 15

123

indicating no synergistic effect between MTX and

pDNA in DOTAP liposomes. On the other hand,

SLNpDNA-reduced MCF-7 viability to 49.6 ± 4.4 %

and SLNMTX? pDNA was even more effective,

decreasing the cell survival to 23.7 ± 5.2 %. This

indicates a synergistic effect for the combination of

MTX and pDNA-PTEN in SLN, resulting in reversion

of mitoxantrone resistance of MCF-7 cells.

Conclusions

Mini-extruder systems are commonplace in bioscience

laboratories. The results presented here provide a

satisfying proof of principle that such extruders can be

used to produce lipid nanoparticles at laboratory scale.

In addition, the lipid nanoparticles produced were

stable and versatile, being applicable to drug and gene

delivery. In fact, the codelivery of the chemothera-

peutic drug MTX and the pDNA-PTEN construct

successfully reduced the viability of PC3 prostate

cancer cells and reverted the resistance of MCF-7

breast cancer cells to mitoxantrone, with no effect on

normal (3T3) cells viability. Therefore, we conclude

that the microemulsion extrusion technique is a very

efficient way to screen for new formulations of lipid-

based carriers, such as SLN, NLC, and liposomes.

Acknowledgments This research has benefited from the

financial support of State of Sao Paulo Research Foundation

(FAPESP). M. B. J. and A. R. were the recipients of fellowships

from FAPESP (Proc. 2012/01038-9 and Proc. 2009/13110-3,

respectively). E. P. received fellowship from The National

Council for Scientific and Technological Development (CNPq).

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