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Lipid nanoparticles have received considerable attention in the field of drug delivery, due their ability to incorporate lipophilic drugs and to allow controlled drug release. Solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), and nanoemulsion (NE) are three different lipid nanostructured systems presenting intrinsically physical properties, which have been widely studied in recent years. Despite the extensive applicability of lipid nanoparticles, the toxicity of these systems has not been sufficiently investigated thus far. It is generally believed that lipids are biocompatible. However, it is known that materials structured in nanoscale might have their intrinsic physicochemical properties modified. Thus, the aim of this study was to evaluate the cytotoxicity of these three nanoparticle systems. To this end, In Vitro and In Vivo toxicity studies were carried out. Our results indicate that nanoparticles containing the solid lipid GMS (SLN and NLC) induced an important cytotoxicity In Vitro, but showed minimal toxicity In Vivo—evidenced by the body weight analysis. The NE did not induce In Vitro toxicity and did not induce body weight alteration. On the contrary, the SLN and NLC possibly induce an inflammatory process In Vivo. All nanoparticle systems induced lipid peroxidation in the animals’ livers, but only SLN and NLC induced a decrease of antioxidant defences indicating that the main mechanism of toxicity is the induction of oxidative stress in liver. The higher toxicity induced by SLN and NLC indicates that the solid lipid GMS could be the responsible for this effect. Nevertheless, this study provides important insights for toxicological studies of different lipid nanoparticles systems.
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Article
Journal of
Nanoscience and Nanotechnology
Vol. 15, 1–10, 2015
www.aspbs.com/jnn
Development and Evaluation of Lipid Nanoparticles for
Drug Delivery: Study of Toxicity In Vitro and In Vivo
Evelyn Winter1, Carine Dal Pizzol1, Claudriana Locatelli2, and Tânia Beatriz Crezkynski-Pasa1
1Departamento de Ciências Farmacêuticas, Universidade Federal de Santa Catarina P.O. Box 476,
Florianópolis, SC, 88040-900, Brazil
2Curso de Farmácia, Universidade do Oeste de Santa Catarina, Videira, SC, 89560-000, Brazil
Lipid nanoparticles have received considerable attention in the field of drug delivery, due their ability
to incorporate lipophilic drugs and to allow controlled drug release. Solid lipid nanoparticles (SLN),
nanostructured lipid carriers (NLC), and nanoemulsion (NE) are three different lipid nanostructured
systems presenting intrinsically physical properties, which have been widely studied in recent years.
Despite the extensive applicability of lipid nanoparticles, the toxicity of these systems has not been
sufficiently investigated thus far. It is generally believed that lipids are biocompatible. However, it is
known that materials structured in nanoscale might have their intrinsic physicochemical properties
modified. Thus, the aim of this study was to evaluate the cytotoxicity of these three nanoparticle
systems. To this end, in vitro and in vivo toxicity studies were carried out. Our results indicate
that nanoparticles containing the solid lipid GMS (SLN and NLC) induced an important cytotoxicity
in vitro, but showed minimal toxicity in vivo—evidenced by the body weight analysis. The NE did
not induce in vitro toxicity and did not induce body weight alteration. On the contrary, the SLN and
NLC possibly induce an inflammatory process in vivo. All nanoparticle systems induced lipid per-
oxidation in the animals’ livers, but only SLN and NLC induced a decrease of antioxidant defences
indicating that the main mechanism of toxicity is the induction of oxidative stress in liver. The higher
toxicity induced by SLN and NLC indicates that the solid lipid GMS could be the responsible for this
effect. Nevertheless, this study provides important insights for toxicological studies of different lipid
nanoparticles systems.
Keywords: Lipid Nanoparticles, Solid Lipid Nanoparticle, Nanostructured Lipid Carriers,
Nanoemulsion, Nanotoxicology, Drug Delivery.
1. INTRODUCTION
Drug loaded nanoparticles have some advantages over free
drugs. An ideal targeting system has a long circulating
time, presents appropriate drug concentration at the target
site, and helps to maintain the drug activity or therapeutic
efficacy while in circulation. Nanotechnology provides the
rescue of numerous chemicals for the treatment of brain
disorders, which without manipulation using such technol-
ogy are not clinically useful because they cannot circum-
vent the blood-brain barrier.12
Lipid-based nanoparticles (LNP) are widely used for
drug delivery, and the nature, shape and charge of the
biomaterials that constitute these LNP affect the interac-
tion with different cell types.34Solid Lipid Nanoparticles
Author to whom correspondence should be addressed.
(SLN) are commonly defined as nano-scaled lipid matri-
ces, which are solid at physiological temperatures and
which exhibit limited drug encapsulation and drug expul-
sion as a result of the high crystallinity of the lipids and
consequently present a polymorphic transition.56Nanos-
tructured lipid carriers (NLC) are systems composed of
an amorphous core containing a solid and a liquid oil,
and which present a higher capacity for encapsulation than
SLN.7Nanoemulsions (NE) are heterogeneous systems
with a great stability, composed of oil droplets dispersed in
aqueous media and stabilized by surfactant molecules.68
Nanostructured lipid carriers are generally composed
of biocompatible lipids, although it should be noted that
lipids might nonetheless exhibit toxicity when given at
high doses. Furthermore, other than the dose, the type of
lipid used is another important factor affecting toxicity.9
J. Nanosci. Nanotechnol. 2015, Vol. 15, No. xx 1533-4880/2015/15/001/010 doi:10.1166/jnn.2015.11667 1
Development and Evaluation of Lipid Nanoparticles for Drug Delivery: Study of Toxicity In Vitro and In Vivo Winter et al.
The positive charges on cationic lipids can promote bind-
ing to circulating blood cells, and the presence of unpro-
tected surface negative charges on lipid molecules serve as
binding sites for plasma opsonin, which favors uptake by
macrophages.1011
The number of publications relating to nanotechnology
has increased and currently stands at about 29,000 in total,
while the number of publications on nanotoxicology has
reached around 1,000, according to the ISI Web of Knowl-
edge database in January of 2015. Among publications
relating to nanotoxicology, the toxicity mainly reported is
related to metals or polymers and not to lipid nanoparti-
cles, because there is a widespread belief that lipids are
generally biocompatible. Furthermore, the most commonly
used strategy to evaluate the toxicity of nanoparticles con-
sist of in vitro assays, with cells lines, and our experience
shows that in vitro and in vivo studies do not always point
toward the same conclusions. Thus, due to the poor avail-
ability of data about toxicity in the literature, the aim of
this study was to investigate the effect of different LNP
systems through in vitro and in vivo assays.
2. MATERIALS AND METHODS
2.1. Materials
For the assembly of nanoparticles, solid lipid glyceryl
monostearate (GMS) was purchased from Galena (Brazil),
liquid oil Miglyol 812 was purchased from Caelo GmbH
(Germany) and the lecithin S75 (Lipoid S75) was pur-
chased from Lipoid (Switzerland). The cell culture media
and fetal bovine serum were purchased from Culti-
lab (Brazil). The antibiotics penicillin/streptomycin and
DCFH-DA (2,7-dichlorofluorescein diacetate) were pur-
chased from Life Technologies (Brazil). Dimethyl sulfox-
ide (DMSO) was purchased from Merck, and all other
reagents were purchased from Sigma-Aldrich.
2.2. Lipid Nanoparticles Preparation
The formulations were obtained by ultrasound method.12
Briefly, the matrix lipid and surfactant lecithin S75 were
heated to about 56–70 C. After that, the aqueous solution
containing the PBS buffer and polysorbate 80—previously
heated to the same temperature as the lipid phase—was
mixed in the oil phase by stirring. Next, the sonication
probe (6 mm diameter) of an ultrasonic processor (Vibra-
cells, USA) was placed in the pre-emulsion and set to
produce an output power with 70% amplitude for 3 min
at 4 C, leading to droplet breakage by acoustic cavitation
and subsequent nanoparticles formation.
The formulations consisted of 20 mg/ml lipid stabi-
lized by 1% (w/v) of surfactant mixture. An overview of
the formulations is given in Table I: SLN (=100% solid
lipid), the NLC with 50% solid lipid and 50% liquid oil
and NE (=100% liquid oil). The pH of all formulation
was 7.4.
Tab l e I . LNPs composition.
Lipids Surfactant
Glyceryl
Miglyol812 Monostearate Lecitin Polysorbate
LNPs (mg/ml) (mg/ml) S75 (%) 80 (%)
SLN – 20 0.2 0.8
NLC 10 10 0.2 0.8
NE 20 0.2 0.8
Notes: SLN—solid lipid nanoparticles; NLC—nanostructured lipid carriers; NE—
nanoemulsion.
2.3. Characterization of Nanoparticles’
Physicochemical Properties
The particle size, polydispersity index (PDI) and elec-
trophoretic mobility (zeta potential) of nanoparticle disper-
sions were measured by dynamic light scattering (DLS) in
a Zetasizer Nano ZS (Malvern Instruments, UK), equipped
with scattering angle of 173. The measurements were
made at 25 C after appropriated sample dilution in ultra-
pure water (Milli-Q, Millipore, USA) or with sodium chlo-
ride solution (0.9%) for zeta potential determination. To
measure zeta potential, nanoparticle samples were placed
in a specific cell in which a potential of 150 mV was
established. The zeta potential values were calculated
from the mean of electrophoretic mobility values using
Smoluchowski’s equation.13
In addition, the particle shape was visualized by field
emission scanning electron microscopy transmission elec-
tron microscopy (FE-SEM) (JEOL JSM-6701F, Tokyo,
Japan).
2.4. Differential Scanning Calorimetry
Thermal analysis was performed to verify the melt-
ing and the crystallinity of the lipids in the nanopar-
ticles. DSC analyses were performed using a DSC-50
(Shimadzu, Japan) instrument. Samples of physical mix-
ture or lyophilized nanoparticles (2–5 mg), were loaded in
aluminum pans and hermetically sealed. An empty stan-
dard aluminum pan was used as reference. Scans were
recorded at a heating rate of 10 C/min from 25 Cto
200 C. For physical mixtures, a second heating cycle was
performed.
2.5. Cell Culture
Monkey kidney epithelial cells (VERO) and acute lym-
phoblastic leukemia cells (L1210) were obtained from
American Type Culture Cell (ATCC). VERO cells were
cultured in DMEM and L1210 cells were cultured in
RPMI, both supplemented with 10% heat-inactivated
fetal bovine serum, 100 U/ml penicillin, 100 g/ml
streptomycin and 10 mM HEPES. The cell culture was
maintained at 37 C and at pH 7.4, in a 5% CO2humid-
ified atmosphere. In all experiments, viable cells were
checked at the beginning of the experiment by Trypan Blue
2J. Nanosci. Nanotechnol. 15, 1–10,2015
Winter et al. Development and Evaluation of Lipid Nanoparticles for Drug Delivery: Study of Toxicity In Vitro and In Vivo
exclusion and only cells with viability higher than 80%
were used.
2.6. Viability Assay
The cytotoxicity of LNP was evaluated by MTT.14 Vero
cells (2 ×104/well) and L1210 cells (1 ×105/well) were
seeded in 96-well culture plates. After overnight incuba-
tion, cells were exposed to lipid nanoparticles for 24 and
48 h with concentrations ranging from 0.05 to 1 mg/ml
of lipid content. After 24 h, a fresh culture medium with
5 mg/ml of MTT was added and incubated for 2 h. The
precipitated formazan formed was dissolved in 100 l
of DMSO, and the absorbance was measured at 540 nm
using a micro-well system reader. The CC50 (cytotoxic
concentration to 50% of the cells) was calculated with the
GraphPad Prism5 (GPW6-242831-RBMZ-03274) using a
sigmoidal curve.
2.7. Erythrocyte Hemolysis
The hemolytic effect of LNP was evaluated according
to Wang et al.15 with modifications. Blood samples
were obtained from healthy donors by venipuncture and
collected into tubes containing sodium citrate (Ethics
Committee from Federal University of Santa Catarina
number 717.557). The erythrocytes were immediately sep-
arated by centrifugation at 200×gfor5minandwashed
three times with four volumes of a normal saline solution.
Immediately thereafter lipid nanoparticles were dissolved
in the erythrocyte suspension (up to 20% of nanopar-
ticles in erythrocyte suspension; v/v). Incubations were
carried out at 37 C with gentle tumbling of the test
tubes. After 1 h of incubation, samples were diluted in
dichloromethane and centrifuged for 5 min at 200×gto
avoid the interference of lipid nanoparticles in absorbance.
The absorbance of the supernatant was measured at
415 nm to determine the percentage of cells undergoing
hemolysis. Hemolysis induced with distilled water was
used as a positive control and was taken as 100% of
hemolysis.
2.8. Animal Care and Treatments
Male Swiss albino mice (6–8 weeks old) were maintained
at 23 ±2C, with relative humidity of 50–60% under a
12:12 h light-dark cycle with food and water ad libitum.
Prior to performing the experimental procedures, mice
were matched for body weight (30 to 35 g). Animals used
in this study were handled in accordance with The Use
on the Principles of Animal Care, previously approved by
the Ethics Committee for Animals. Animals were divided
into four groups (n=5 each): control, which received only
the vehicle (saline); NE-treated; SLN-treated and NLC-
treated, which received the formulation corresponding to
200 mg/Kg in 0.3 ml of formulation. The doses at which
humans may be exposed to nanoparticles are still to be
determined. As there are currently no guidelines and stan-
dard methodologies for the in vivo toxicity assessment
of lipid nanoparticles, we chose to work with the high-
est dose possible to administrate by intraperitoneal route.
The formulations or saline alone were administered via
intraperitoneal (i.p.) injection daily for 14 days, which
characterize a repeated-dose study.
On the final day of the treatment the animals were
killed, their blood was collected using EDTA as antico-
agulant and selected organs (heart, liver, spleen, brain,
stomach, lung and kidney) were removed and weighed.
Blood was used to evaluate hepatic, renal, and hemato-
logic toxicity. The oxidative stress was assayed in the liver
homogenates. Body weight was evaluated every two days
and at the end of treatment.
2.9. Preparation of Homogenates
The livers were rapidly removed and homogenized (liver
1:10 w/v) in a buffer containing 1% triton X-100, 150 mM
NaCl, 20 mM sodium phosphate, pH 7.4. The livers were
homogenized in a tissue homogenizer for 30 seconds on
ice, followed by centrifugation at 10,000×g for 10 min.
Protein content was determined by Lowry’s method.16
Lipid peroxidation, reactive species generation and thiols
contents analyses were performed with freshly prepared
samples. To measure the activity of catalase, glutathione
peroxidase and glutathione reductase, the supernatants
were stored at 80 C until utilization.
2.10. ROS Determination
Intracellular free radical formation was determined using
2,7-dichlorofluorescein diacetate (DCFH-DA), which is
oxidized to dichlorofluorescein (DCF) in the presence
of ROS.17 Liver homogenates (200 g) were incubated
with 0.1 M DCFH-DA in 96-well plates for 30 min at
37 C. The DCF fluorescence signal was measured using
a Perkin–Elmer LS55 spectrofluorimeter.
2.11. Enzyme Assays
Glutathione peroxidase (GPx) was assayed according
to Flohé and Gunzler18 using 200 g of protein, and
NADPH oxidation was monitored spectrophotometrically
at 340 nm. Catalase activity was determined according
to Aebi,19 using 0.1 mg of protein. In this assay, the
disappearance of H2O2was evaluated by measuring the
decrease in absorbance at 240 nm. Glutathione reduc-
tase was assayed according to Carlberg and Mannervick20
using 200 g of protein, and the NADPH oxidation, which
resulted from GSSG reduction by GR, determined spec-
trophotometrically at 340 nm. Glutathione S-transferase
was assayed according to Keen et al.21 using 100 gof
protein. This assay is based on the conjugation of GSH
with CDNB by GST. The conjugate was detected spec-
trophotometrically at 340 nm.
J. Nanosci. Nanotechnol. 15, 1–10, 2015 3
Development and Evaluation of Lipid Nanoparticles for Drug Delivery: Study of Toxicity In Vitro and In Vivo Winter et al.
2.12. Lipid Peroxidation Measurements
Thiobarbituric acid reactive species (TBARS) levels were
determined according to the method already established,
in which malondialdehyde, the major end product of fatty
acid peroxidation, reacts with thiobarbituric acid to form
a colored complex.22 Freshly prepared tissue homogenates
were sequentially mixed in test tubes with equal volumes
of 60 mM Tris-HCl, pH 7.4, 0.1 mM DPTA buffer, 12%
TCA and 0.73% thiobarbituric acid (TBA), stirring after
each addition. The tubes were kept at 100 Cfor1h
and subsequently cooled and centrifuged (10,000×gfor
5 min). The absorbance of the supernatant was measured
at 535 nm. The TBARS concentration in the samples was
calculated using an analytical curve of malondialdehyde
performed in parallel and expressed in nmol/mg protein.
2.13. Non-Protein Thiol Determination
GSH concentration in the samples was determined by the
DTNB method.23 This method is based on the reaction of
GSH with acid 5,5-ditiobis 2-nitrobenzoic (DTNB), gen-
erating a thiolate anion (TNB), whose yellow color is
measured spectrophotometrically at 412 nm. To determine
the GSH content, the samples were added to a reaction
medium containing 20 M DTNB in 200 mM sodium
phosphate buffer, pH 8.0. The formation of thiolate anion
was monitored after 10 min at 405 nm. The GSH concen-
tration was calculated using an analytical curve of GSH
performed in parallel and expressed in mol/mg protein.
2.14. Biochemical Parameters
The blood samples were centrifuged at 200×g for 10 min
at room temperature, the serum was separated to measure
aspartate (AST) and alanine transaminase (ALT) activities,
as well as the contents of total protein and albumin to eval-
uate the hepatic function. Renal function was determined
based on serum urea and creatinine levels. The evalua-
tion of the content of plasma cholesterol and triglycerides
was also conducted to verify whether the lipid nanopar-
ticles could affect the normal lipid levels. Commercially
available kits (Labtest Diagnóstica SA, Lagoa Santa, MG,
Brazil) were used for the biochemical analysis.
2.15. Hematological Evaluation
Hematological parameters such as red blood cell number
(RBC), white blood cell number (WBC), lymphocyte and
neutrophil counts were evaluated according to the method
published elsewhere.24 The content of hemoglobin, hemat-
ocrit, mean corpuscular hemoglobin (MCH), mean corpus-
cular volume (MCV) and mean corpuscular hemoglobin
concentration (MCHC) were evaluated according to Pari
and Murugavel.25
2.16. Statistical Analysis
The results were presented as means ±standard error of
mean (SE) of triplicates from at least three-independent
experiments. Statistical significance was assessed by
ANOVA followed by Dunnett’s test, and a pvalue of less
than 0.05 was considered significant.
3. RESULTS
3.1. Lipid Nanoparticles Characterization
Nanoparticle formulations were produced and analyzed for
their cytotoxicity by in vitro and in vivo assays. Glyceryl
Monostearate (GMS) was used as a core material for the
SLN. For the NLC formulations, the medium chain triglyc-
eride oil, as the liquid oil matrix, was mixed with glyceryl
monostearate in the same proportions, and for the NE for-
mulations only the medium chain triglyceride oil was used
as a core material for the dispersion. Values of the particle
size, polydispersity index (PDI) and zeta potential of the
developed SLN, NLC and NE are shown in Table II. After
production, the mean diameters of the particles were in
the range of 101–157 nm, depending on the lipid loading.
The size of the nanoparticles was confirmed by electronic
microscopy (FE-SEM) (Fig. 1). In general, the particle
sizes of the different LNP formulations decreased in accor-
dance with the order of SLN >NE >NLC. The formula-
tions presented a negative surface charge (zeta potential)
of between 22 and 13 mV, as expected for the types
of mixture of surfactant utilized.
3.2. Differential Scanning Calorimetry Investigation
DSC analysis was performed to investigate the melting
of the solid lipid crystalline material, which was in the
composition of the SLN and NLC. Figure 1 shows an
overview of the melting process of the physical mixture
and lyophilized nanoparticles. A second heating cycle was
performed for all physical mixtures in order to compare
with the nanoparticles DSC analyses, which were first
melted in their synthesis.
The melting point for all analysis is described in
Table III. In the physical mixture of SLN and NLC, the
first melting process of GMS took place with maximum
peak of 57.3 and 56.2 C respectively, according to the
literature.26 However, for GMS-based nanoparticles and
for the second heating cycle of physical mixtures the
melting points decreased, which could be attributed to a
smaller crystallite size of the GMS. Another important
effect observed with the nanoparticles was the decrease in
Table II. Characterization of formulations.
Particle Polydispersity Zeta
Formulation size (nm) index potential (mV)
SLN 157 ±18 0.26 ±0.01 13 ±2.8
NLC 101 ±15 0.14 ±0.01 14 ±0.7
NE 117 ±40.19±0.02 22 ±1.4
Notes: SLN—solid lipid nanoparticles; NLC—nanostructured lipid carriers; NE—
nanoemulsion. (Mean ±S.E., n=3).
4J. Nanosci. Nanotechnol. 15, 1–10,2015
Winter et al. Development and Evaluation of Lipid Nanoparticles for Drug Delivery: Study of Toxicity In Vitro and In Vivo
Figure 1. Heating curves of differential scanning calorimetry (DSC) for lyophilized nanoparticles (solid line) and physical mixture (first and second
heating cycle—dotted line) (Left side). FE-SEM images of the lipid nanoparticles (Right side). SLN: solid lipid nanoparticles; NLC: nanostructured
lipid carriers; NE: nanoemulsion. Scan rate: 10 Cmin
1. Bar scale X-axis: Major unit: 50 C and minor unit: 10 C. Bar scale Y-axis: Major unit:
2 mW and minor unit: 1 mW.
the fusion peak of the GMS in the SLN, and the near dis-
appearance of the fusion peak in the NLC. These results
suggest that the lipid within the SLN and NLC should be
in a less ordered arrangement compared to the isolated
Table III. Melting point (peak maximum) obtained from the DSC
analysis.
Sample Heating cycle Melting point (C)
SLN First 57.3
PM First 57.3
Second 47.9
NLC First 46.2
PM First 56.2
Second 45.1
NE First
PM First
Second –
Notes: PM—Physical mixture; SLN—solid lipid nanoparticles; NLC—nano-
structured lipid carriers; NE—nanoemulsion.
lipid. For the less ordered crystal or amorphous state,
melting the substance required less energy than for the
perfect crystalline substance.
The physical mixture and the NE did not show any
fusion peak as expected for the liquid oil (Mygliol 812),
indicating that its form is completely amorphous and that
there is no contaminant or degradation product in the
nanoparticles.
3.3. In Vitro Cell Toxicity
To determine the cytotoxicity of the formulations, an
adherent cell line (VERO) and a non-adherent cell line
(L1210) were used. Cells were exposed for 24 and 48
hours with increasing concentrations of the formulations
corresponding to 0.05 and 1 mg/ml of lipids, respectively.
The effects of lipid nanoparticles on the cell viability are
presented in Figure 2. In general, all formulations were
more cytotoxic to the non-adherent cell line. The NE did
not show cytotoxicity even after 48 hours of incubation in
J. Nanosci. Nanotechnol. 15, 1–10, 2015 5
Development and Evaluation of Lipid Nanoparticles for Drug Delivery: Study of Toxicity In Vitro and In Vivo Winter et al.
Figure 2. Cytotoxicity of lipid nanoparticles in VERO and L1210 cells. Cells were incubated for 24 h and 48 h with the nanoparticles at concentrations
up to 1 mg/ml of lipids. Cell viability was assayed by MTT and optical density of zero time was taken as 100% cell viability. SLN—solid lipid
nanoparticles; NLC—nanostructured lipid carriers; NE—nanoemulsion.
VERO cells, but presented cytotoxic effects in L1210 cells
after 48 hours of incubation. The NLC showed an interme-
diary toxicity without reaching 50% of cell death, tested
at concentrations up to 1 mg/ml in VERO cells, however
it induced a considerable toxicity in L1210 after 24 hours
and 48 hours of incubation with CC50 =0.5 mg/ml and
0.3 mg/ml respectively. The SLN induced a higher cytoxi-
city then the others formulations, reaching CC50 values of
0.7 and 0.4 mg/ml in VERO cells and 0.5 and 0.3 mg/ml in
L1210 cells, after 24 and 48 hours of incubation, respec-
tively. These results suggest that the use of GMS might
provide a more cytotoxic formulation for the cells than the
caprylic capric triglyceride oil (Miglyol 812).
Figure 3. Hemolysis induced by lipid nanoparticles. Erythrocytes suspension was incubated for 1 h with the nanoparticles at concentrations up to
20% (v/v). The absorbance of the supernatant was measured at 405 nm. The percentages of nanoparticles correspond to 0.2, 1.0, 2.0 and 4.0 mg/ml of
lipids, respectively. (A) Hemolysis induced by water was taken as 100% and was used to calculate the hemolysis percentage induced by nanoparticles.
(B) Data in an expanded scale calculated based on the C+as 100% hemolysis. SLN—solid lipid nanoparticles; NLC—nanostructured lipid carriers;
NE—nanoemulsion.
3.4. Erythrocyte Hemolysis
To evaluate the safety of the nanoparticles per se,the
hemolytic activity was determined. As shown in Figure 3,
all the three formulations induced hemolysis of lower than
5% at concentrations up to 20% (v/v). Thus, all systems
would be tolerable by parenteral administration.
3.5. In Vivo Toxicity
In order to evaluate and compare the cytotoxicity of the
three LNP in vivo, groups of five mice were treated daily
with the nanoparticles (200 mg/Kg) or with saline for
14 days.
6J. Nanosci. Nanotechnol. 15, 1–10,2015
Winter et al. Development and Evaluation of Lipid Nanoparticles for Drug Delivery: Study of Toxicity In Vitro and In Vivo
Tab l e I V. Evaluation of animals’ weight during the LNPs treatment.
Days Control (g) SLN (g) NLC (g) NE (g)
0 33. 0 ±1.3 33.0±1.3 32.6 ±1.6 32.2±3.2
2 33. 6 ±1.1 32.0 ±1.2 32.8 ±1.4 32.0±3.2
4 33. 4 ±1.0 33.0 ±1.6 32.8 ±1.2 32.4±2.5
6 33. 2 ±1.5 34.0 ±1.2 33.8 ±1.3 33.2±2.5
8 32. 8 ±1.4 33.8 ±1.3 33.8 ±0.9 33.4±2.2
10 34.8 ±1.5 33.8 ±1.2 33.8 ±1.0 33.6 ±2.3
12 35.6 ±1.6∗∗ 35.4 ±1.6∗∗ 34. 8 ±0.8 35.2 ±2.2∗∗
14 35.8 ±1.5∗∗ 35.0 ±1.5∗∗ 35. 0 ±0.934.4 ±2.1
Notes:p<005 and ∗∗p<001 using repeated-measures ANOVA followed by
Dunnet’s test when compared with the 0 day. SLN—solid lipid nanoparticles;
NLC—nanostructured lipid carriers; NE—nanoemulsion.
The animals were weighed every two days and the
results are shown in Table IV. For the statistical analy-
sis, the weight recorded each day was compared with the
weight on the first day (day 0). The control animals pre-
sented an increase in body weight (+2.8 g) at 12day,
as did the animals treated with SLN (+2.0 g) and NE
(+2.2 g). Despite the weight increase in all animals, the
nanoparticles induced a lower increase in body weight
when compared with the animals of the control group.
To improve the cytotoxicity analysis, some of the ani-
mals’ important organs were weighed at the end of the
treatment and the results are presented in the Table V.
The nanoparticles did not significantly affect the weight of
the organs after 14 days of treatment.
3.5.1. Biochemical Parameters Investigation
The results of biochemical studies are summarized in
Table VI. Only the treatment with NLC showed a slight
alteration in creatinine, however this change seems to
be unrelated to renal damage. Other parameters did not
present significant alterations.
3.5.2. Hematological Evaluation
To evaluate the toxicity produced by exposure to nanopar-
ticles, hematological profiles of the animals were deter-
mined and compared with respective values obtained
for the control group. Table VII shows the results
obtained from the hematological analysis. The treatment
with all nanoparticles caused changes in hematological
Tab l e V. Evaluation of organs weight after the LNPs treatment.
Organs Control (g) SLN (g) NLC (g) NE (g)
Spleen 0.20 ±0.01 0.28 ±0.06 0.21 ±0.01 0.22 ±0.02
Liver 2.00 ±0.11 2. 27 ±0.18 1.92 ±0.12 2.18 ±0.09
Heart 0. 18 ±0.01 0.22 ±0.02 0.17 ±0.01 0. 19 ±0.02
Lung 0.24 ±0.01 0.23 ±0.02 0.21 ±0.01 0.22 ±0.01
Brain 0.32 ±0. 02 0.33 ±0.02 0.29 ±0.02 0.35 ±0.02
Kidneys 0. 25 ±0.01 0.28 ±0.02 0.27 ±0.03 0.26 ±0.02
Stomach 0.30 ±0.03 0.22 ±0.01 0.27 ±0.01 0.27 ±0.02
Notes: SLN—solid lipid nanoparticles; NLC—nanostructured lipid carriers; NE—
nanoemulsion.
Tab l e V I . Biochemical parameters analysis after 14 days of LNPs
treatment.
Parameters Control SLN NLC NE
ALT (UI/L) 184.0 ±19.0 157.0 ±10.9 145.0 ±14.5 157.0±10.0
AST (UI/L ) 181.0 ±29.0 154.0 ±15.0 140.0 ±13.0 145.0 ±6.0
Albumin (g/dl) 2.0 ±0.1 1.87 ±0.05 2.02 ±0.08 2.02 ±0.08
Total proteins 5.8 ±0.2 6.10 ±0.19 5.71 ±0.16 5.95 ±0.18
(g/dL)
Urea (mg/dL) 61.0 ±5.3 67. 0 ±4.3 59.4 ±5.46 61.0 ±4.6
Creatinine 0.40 ±0.03 0.36 ±0.01 0.33 ±0.020.41 ±0.01
(mg/dL)
Triglycerides 71.0 ±4.0 86.0 ±6.0 86.0 ±10.0 96.0 ±14.0
(mg/dl)
Total Cholesterol 121.0 ±13.0 113.0 ±3.3 118.0 ±4.5 120.0 ±6.5
(mg/dl)
Notes:p<005 using ANOVA followed by Dunnet’s test when compared with the
control group. SLN—solid lipid nanoparticles; NLC—nanostructured lipid carriers;
NE—nanoemulsion.
parameters, with a significant increase in total leukocytes
and mononuclear cells. The treatment with SLN induced
a significant increase of band cell and the treatment with
NLC induced an increase of basophil cells.
3.5.3. Oxidative Stress Evaluation
The results obtained for all parameters of oxidative
stress evaluated in the liver homogenates are shown in
Table VIII. The LNP did not induce ROS generation, but
induced an elevation in lipid peroxidation. The nanopar-
ticles containing the solid lipid (SLN and NLC) induced
a decrease in antioxidant enzyme activities, indicating the
induction of an oxidative stress.
GST is an important member of the phase II detox-
ification enzymes family, which is responsible for the
protection of cellular macromolecules from attack by reac-
tive electrophiles via GSH conjugation.27 The SLN and
NLC induced a strong elevation of GST activity, indicat-
ing that these nanoparticles can activate phase II of the
detoxification system.
Table VII. Hematological parameters analysis after 14 days of LNPs
treatment.
Parameters Control SLN NLC NE
Total RBC count (×105/mm352 ±365±254±468±4
Leucocytes (×102/mm348 ±780±1396±12∗∗ 91 ±3∗∗
MCV (fL) 72 ±865±10 95 ±7∗∗ 81 ±8
MCH (pg) 24±326±331±2∗∗ 26 ±4
MCHC (%) 33 ±332±232±432±3
Hemoglobin (g/dL) 14 ±217±217±318±3
Hematocrit (%) 46 ±352±652±455±5
Neutrophil (×102/mm314 ±121±220±121±2
Band cells (×10/mm35±532±8∗∗ 10 ±10 9 ±9
Mononuclear (×102/mm334 ±154±3∗∗∗ 69±1∗∗∗ 69 ±4∗∗∗
Eosinophil (×10/mm35±58±810±10 9 ±9
Basophil (×10/mm35±532±16 67 ±199±9
Notes:p<005 and ∗∗p<001 using ANOVA followed by Dunnet’s test
when compared with the control group. SLN—solid lipid nanoparticles; NLC—
nanostructured lipid carriers; NE—nanoemulsion.
J. Nanosci. Nanotechnol. 15, 1–10, 2015 7
Development and Evaluation of Lipid Nanoparticles for Drug Delivery: Study of Toxicity In Vitro and In Vivo Winter et al.
Table VIII. Liver oxidative stress analysis after 14 days of LNPs
treatment.
Parameters (%) Control SLN NLC NE
TBARS 100 ±7 131 ±3134 ±5∗∗ 144 ±10∗∗∗
ROS 100 ±16 77 ±792±10 95 ±14
GSH 100±20 99 ±17 108 ±30 118 ±6
Catalase 100 ±15 105 ±990±584±4
GR 100±059±4∗∗ 76 ±9106 ±4
GPx 100 ±5.9 68 ±9∗∗ 87 ±889±3
GST 100±5.6 178±19∗∗ 160 ±25113 ±14
Notes:p<005 and ∗∗p<001 using ANOVA followed by Dunnet’s test
when compared with the control group. SLN—solid lipid nanoparticles; NLC—
nanostructured lipid carriers; NE—nanoemulsion.
4. DISCUSSION
Nanotechnology has been expanding in several fields
such as biotechnology, pharmaceutical technology, med-
ical sciences, diagnostic, among others, but although its
benefits have been widely publicized, discussion about the
effects of their widespread use is just beginning.28 Due
to their small size, nanostructured materials can enter the
circulatory and lymphatic systems, and eventually body
tissues and organs. Some particles, depending on their
composition and size, can cause irreversible damage to
cells by oxidative stress or/and organelle injury.29
Many studies have used the LNP for drug deliv-
ery, and have compared the effects of the encapsu-
lated drugs using the formulations SLN, NLC and NE
as nanocarriers.7153031 However, none of the studies
showed the cytotoxicity of the empty nanoparticles. Few
works have evaluated the cytotoxicity of empty LNP
with different constituents;3432–35 and Doktorovova and
collaborators showed in a good systematic review, the
comparison of published results of in vitro effects of lipid
nanoparticles.4
It is widely accepted that the toxicity of nanoparticles
depends on the physiochemical parameters such as particle
size, shape, surface charge, composition, and subsequent
formulation stability. However, studies with NP in general
are incomparable because of one or more among the fol-
low parameters used in each case are different:
(i) administration routes;
(ii) materials used in the nanoparticles production;
(iii) animal models.36
In this work, we compared the cytotoxicity of three dif-
ferent LNP through in vitro and in vivo methodologies.
The results from in vitro assays showed that the nanopar-
ticles containing GMS induced higher toxicity than NE in
VERO cells with CC50 values similar to other studies.3237
Nanoparticles containing GMS (SLN and NLC) were also
more toxic than NE to L1210 cells, although NE was
also toxic to this cell line when the incubation time was
increased to 48 h. Petersen and collaborators also have
studied LNP and have demonstrated that nanoparticles
containing solid and crystalline matrix induced a higher
toxicity than the ones containing liquid lipid matrix.35
L1210 cells seem to be more sensitive to LNP than
VERO cells, which can be explained by the greater inter-
action of LNP with cells in suspension than with adherent
ones. Furthermore, the higher toxicity of LNP in L1210
cells could be an outcome of the higher expression of lipid
receptors in the membranes of leukemic cells.38–40
To determine the toxicity of the LNP in vivo,animals
were treated with the three formulations for 14 days by
intraperitoneal route. The administration was performed by
i.p. route because:
(i) the nanoparticles composition is mainly lipid and may
be degraded when administered orally;
(ii) this route mimics the i.v. route as this region of the
body is richly vascularized;
(iii) the i.p. route is one of the most effective ways of
dispensing test-drugs into animals under experimentation
in a short term-procedure.
No deaths and few weight differences were observed
in the animals during the treatment. The non-change of
several parameters such as body and organ weight and bio-
chemical parameters indicate that the nanoparticles SLN
and NLC did not induce toxicity, contrarily of the effects
observed in vitro.
The only difference among the three LNP systems is the
lipid content, which indicates that the cell lines are more
sensitive than the animals to the formulations in which
GMS is used, as concluded from the results obtained by
in vitro assays. The zeta potential and size values are very
similar for all the formulations and should therefore not
be involved in different toxicity profiles observed.
The GMS is widely used in cosmetic formulations, in
foods as a surfactant, and in nanoparticles synthesis.41–43
The few studies about the GSM toxicity demonstrated that
it was non-toxic in acute oral studies in rats. However,
when rats were fed with a diet containing 25% of GMS
for two years, they developed renal calcifications. GMS
(100%) also did not present dermal toxicity in rabbits.44
The cytotoxicity of GMS observed in this study could be
a result of the nanoparticulate form, as it is known that
GMS bulk presented no toxicity.44 Doktorovova and col-
laborators have compiled in a table several results of toxi-
city after treatment with nanoparticles containing different
kind of lipids. Nanoparticles containing GMS are in the
first part of the table among nanoparticles that presented
the lowest IC50 values (from 0.17 to 0.45 mg/ml).
The cytotoxicity of metallic nanoparticles such as TiO2,
silica, silver and zinc oxide has been widely related to
oxidative stress.45–48 However, few studies with LNP have
related their effects to oxidative stress.3249 In this work it
is shown that all nanoparticles induced an increase in liver
lipid peroxidation, determined by the thiobarbituric acid
reactive substances (TBARS), and that the SLN and NLC
induced a decrease in GR and GPx activities. These are
further evidences that the presence of solid lipid (GMS)
8J. Nanosci. Nanotechnol. 15, 1–10,2015
Winter et al. Development and Evaluation of Lipid Nanoparticles for Drug Delivery: Study of Toxicity In Vitro and In Vivo
in the formulation could provide conditions of higher tox-
icity than the liquid oil used in the nanoemulsion prepa-
ration. The increase in TBARS amounts associated with
a decrease in antioxidant defenses indicate that the main
mechanism of toxicity induced by SLN and NLC is by the
induction of oxidative stress in liver. This effect, however
can be or not reversed after discontinuation of the treat-
ment. The non-reduction of GSH content could be a con-
sequence of several events as the increase in GST activity,
as well as the alteration of the y-glutamyl cysteine syn-
thase activity, which is the responsible enzyme for GSH
synthesis (not evaluated in this work).
The most significant hematological changes induced
by nanoparticles are associated with an increase in total
leukocyte, mainly mononuclear cells. Mononuclear phago-
cytes are potent phagocytic cells that initiate the innate
immune system.50 These cells can interact with nanopar-
ticles in the blood and induce stimulation or suppres-
sion of the immune system. The immuno-stimulation may
affect therapeutic efficacy as in the case of cancer treat-
ment and in vaccine efficacy. In contrast, an undesirable
immuno-stimulation response can induce cytokine storm,
interferon response and/or lymphocytes activation causing
severe adverse events.51 The increase in band cells induced
by SLN suggests that this formulation could induce a
slight and reversible inflammatory process. In addition, the
increase in basophil cells induced by SLN and NLC indi-
cates that the nanoparticles with a solid lipid core could
induce an allergic response in the animals. Our in vivo
observations are consistent with previous studies, which
showed that different SLN can act as immunomodulators
inducing the activation of IFN-.5253 The i.p. administra-
tion of lactic co-glycolic acid (PLGA) nanoparticles was
also capable of inducing expression of proinflammatory
cytokines (including IL-2, IL-6 and TNF- in plasma and
peritoneal fluid of rats.54 Park and Park55 have also shown
that single treatment with silica nanoparticles (50 mg/Kg,
i.p) activate peritoneal macrophages increase the levels of
IL-1,TNF-and the release of nitric oxide, as well as
the expression of inflammation-related genes.
The FDA recommends that the evaluation of in vitro
hemolysis should be performed for pharmaceutical excip-
ients that will be administrated intravenously, to evaluate
the hemolytic potential.56 According to the “Standard test
method for analysis of hemolytic properties of nanoparti-
cles (E2524-08),” a percentage of hemolysis higher than
5% indicates that the test material will cause damage to
red blood cells.57 As the hemolysis induced by nanoparti-
cles was lower than 5%, the particles studied here could
be safe for intravenous administration. These results are in
agreement with other works, which showed LNP as blood
compatible.5859
Although, these lipid nanoparticles have shown low
toxicity both in vitro and in vivo, it is not possible to
predict the safe use of them without further studies, espe-
cially immunological evaluation as shown in a study of
our group.35 Likewise, other studies have shown that lipid
nanoparticles are able to promote the activation of the
immune response by inducing an inflammatory response
and cell death.52–54
In conclusion, our findings suggest that the lipid com-
position plays an important role in the toxicity of nanopar-
ticles independently of the constituent’s safety, and can
induce conflicting results when assays in vitro and in vivo
are performed. The results of this study extend the knowl-
edge on the safety of LNP in view of their applications
and provides important data for toxicological studies of
LNP in vivo exposure.
Declaration of Interest Section
The authors declare that they have no conflict of interest
related to this work.
Acknowledgments: This study was supported by
grants from CNPq (Conselho Nacional de Desenvolvi-
mento Científico e Tecnológico), CAPES (Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior—Rede de
Nanobiotecnologia) and FAPESC (Fundação de Amparo à
Pesquisa de Santa Catarina). This paper forms part of the
doctoral studies in Pharmacy of Carine Dal Pizzol, who
prepared the formulations, and part of the doctoral studies
in Pharmacy of Evelyn Winter, who carried out the toxic-
ity assays. The group wishes to thank the Laboratório de
Controle de Qualidade, Departamento de Ciências Farma-
cêuticas, UFSC, for the DSC measurements.
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10 J. Nanosci. Nanotechnol. 15, 1–10,2015
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... Solid-lipid nanoparticles are colloidal dispersions consist of solid, biodegradable lipid matrices, ranging in diameter (10-1000 nm) and remain solid at room temperature (58). They can incorporate both lipophilic and hydrophilic drugs providing controlled release and excellent stability and protection of sensitive drugs from the surrounding environment. ...
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The potential role of human low density lipoprotein (LDL) particles as delivery system for lipophilic, cytotoxic drugs critically depends on their structural integrity. In the present study, LDL particles were loaded with antineoplastic prodrugs, i.e. monooleoyl (MOT)- and dioleoyl (DOT)- thymidine esters by different techniques. Using the reconstitution method MOT shows the highest incorporation efficiency with over 80% of the initial drug associated with LDL. In contrast, for the more lipophilic DOT the incorporation efficiency for reconstitution, dry film as well as dimethylsulfoxide method was extremely low. Structural changes upon drug loading were monitored by differential scanning calorimetry (DSC) and small angle X-ray scattering (SAXS). The results show that the influence of MOT and DOT is predominantly confined to the surface monolayer of LDL seen as a destabilisation of the protein moiety and a small increase in particle diameter. The core lipid region of the LDL-drug complexes remains essentially unaffected, as verified by undisturbed core lipid arrangement and core lipid melting behaviour.
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The use of an efficient carrier for nucleic acid-based medicines is considered to be a determinant factor for the successful application of gene therapy. The drawbacks associated with the use of viral vectors, namely those related with safety problems, have prompted investigators to develop alternative methods for gene delivery, cationic lipid-based systems being the most representative. Despite extensive research in the last decade on the use of cationic liposomes as gene transfer vectors and the development of elegant strategies to enhance their biological activity, these systems are still far from being viable alternatives to the use of viral vectors in gene therapy. In this review considerations are made regarding the structure-activity relationships of cationic liposome/DNA complexes and the key formulation parameters influencing the features of lipoplexes are presented and discussed in terms of their effect on biological activity. Particular emphasis is given to the interaction of the lipoplexes with serum components as well as to novel strategies developed to circumvent difficulties that may emerge upon iv administration of the complexes. Finally, since the ability of the lipoplexes to be stored while preserving their transfection activity is a crucial issue for the repeated use of such carriers, approaches reported on the improvement of their physical stability are also reviewed.
  • K V Jaspreet
  • K R Maram
  • D L Vinod
K. V. Jaspreet, K. R. Maram, and D. L. Vinod, Curr. Nanoscience 1, 47 (2005).