European Journal of Pharmaceutics and Biopharmaceutics 87 (2014) 1–18

Contents lists available at ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics

journal homepage: www.elsevier.com/locate/ejpb

Review Article

Nanotoxicology applied to solid lipid nanoparticles and nanostructured

lipid carriers – A systematic review of in vitro data
Slavomira Doktorovova a,b, Eliana B. Souto b,c, Amélia M. Silva a,d,⇑
a
Department of Biology and Environment, School of Life and Environmental Sciences (ECVA, UTAD), University of Trás-os-Montes and Alto Douro, Vila Real, Portugal
b
Institute of Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes and Alto Douro (IBB/CGB-UTAD), Vila-Real, Portugal
c
Department of Pharmaceutical Technology, Fernando Pessoa University, UFP, Porto, Portugal
d
Centre for the Research and Technology of Agro-Environmental and Biological Sciences, University of Trás-os-Montes and Alto Douro (CITAB-UTAD), Vila-Real, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) were developed as alternative to
Received 29 June 2013 other colloidal carriers. They were designed to overcome lipid nanoemulsions and liposomes in stability
Accepted in revised form 4 February 2014 and ability to control the release of an encapsulated substance, and at the same time to be better toler-
Available online 12 February 2014
ated than polymeric nanoparticles. Since the patenting of SLN discovery, large amount of data became
available on the behaviour of these systems in vitro. SLN/NLC have many prerequisites to be a well tol-
Keywords: erated carrier – the currently available data seem to confirm it, but there are also some contradictory
Lipid nanoparticles
results. In this review, we collected the available data from cytotoxicity, oxidative stress and hemocom-
Solid lipid nanoparticles
Nanostructured lipid carriers
patibility studies in vitro and analysed their outcomes. We also provide a summary of the available data
Cytotoxicity in a form of reference table.
Genotoxicity Ó 2014 Elsevier B.V. All rights reserved.
Oxidative stress
Blood compatibility
In vitro studies

1. Introduction
Solid lipid nanoparticles (SLN), nanoparticles prepared from a
lipid matrix that is solid at body and room temperature, stabilised
by suitable surfactants and having size <1 lm, were invented
20 years ago and, since then they have received great and still
increasing attention in pharmaceutical technology research [1,2].
Nanostructured lipid carriers (NLC), i.e. nanoparticles composed
of a mixture of a solid and a liquid lipid which lipid matrix is solid
Abbreviations: Chol, cholesterol; COM, Compritol™ 888 ATO; CP, cetyl palmitate;
CPC, cetylpyrimidium chloride; CPT, camptothecin; CTAB, cetyltrimethylammonium
bromide, hexadecyltrimethylammonium bromide; CysA, Cyclosporine A; DOPE,
dioleoylphosphatidyl ethanolamine; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-
trimethylammonium chloride; DOX, doxorubicin; DTX, docetaxel; EQ1, esterquat1;
EYPS, egg yolk phosphatidyl choline; FA, fatty acid(s); GMS, glyceryl monostearate;
GO, glyceride oleate; MTX, methotrexate; OA, oleic acid; P188, Poloxamer 188; P407,
Poloxamer 407; PEG, polyethyleneglycol; p-gp, p-glycoprotein; PTC, paclitaxel; PVA,
polyvinylalcohol; SA, stearic acid; SDS, sodium dodecyl sulphate; StA, stearylamine;
STC, sodium taurocholate; T80, Tween 80; TL, glyceryl trilaurate, trilaurine; TM,
glyceryl trimyristate, trimyristine; TP, glyceryl tripalmitate, tripalmitine; TS, glyceryl
tristearate, tristearine; +, lipids (or surfactants) used simultaneously in one lipid
matrix; /, lipids (or surfactants) used in the same study in different lipid matrices.
⇑ Corresponding author at: Department of Biology and Environment, School of
Life and Environmental Sciences (ECVA, UTAD), University of Trás-os-Montes and
Alto Douro, Quinta de Prados, P-5001-801 Vila Real, Portugal. Tel.: +351
259350106; fax: +351 259350480.
E-mail address: amsilva@utad.pt (A.M. Silva).
at room and body temperature [3], are 2nd generation of solid li-
pid-based colloidal carriers, with improved stability and drug
encapsulation ability. SLN and NLC have all pre-requisites to enter
the market faster than other colloidal carriers – among the reasons
it is the use of starting materials that are biodegradable and al-
ready in use in pharmaceutical and cosmetic products, many of
them of GRAS status (generally regarded as safe). Several cosmetic
products based on SLN are already marketed [4]. Secondly, provid-
ing an advantage over liposomes or polymeric nanoparticles, prep-
aration methods feasible at industrial scale are available, and these
methods avoid the use of organic solvents [5]. Furthermore, in vitro
tolerability of SLN and NLC appears to be much higher than that of
polymeric nanoparticles (see further).
Toxicology of nanomaterials is becoming an important issue
nowadays, especially with regard to nanomaterials present in envi-
ronment and nanomaterials intended for medical use. The biggest
concern about nanomaterial safety is focused on nanomaterials with
at least one dimension smaller than 100 nm. Although SLN typically
do not reach this size (z-average data are most often reported over
100 nm; it was suggested SLN be called ‘‘submicron particles’’ rather
than nanoparticles [6]), facilitated cellular uptake of lipid nanoparti-
cles themselves or their drug payload have been reported repeatedly.
SLN and NLC have many pre-requisites to be safe nanocarriers.
Indeed during the first 10 years of development very encouraging

http://dx.doi.org/10.1016/j.ejpb.2014.02.005
0939-6411/Ó 2014 Elsevier B.V. All rights reserved.
2 S. Doktorovova et al. / European Journal of Pharmaceutics and Biopharmaceutics 87 (2014) 1–18
results were reported. However, for approval in use in medicinal
products, more evidence will be required. Research reports reflect
this challenge, even despite expected low toxicity or no toxicity at
all. Up to date, considerable amount of data on SLN/NLC behaviour
in cell culture is available. As of December 2013 (accessed on 1st of
December 2013), a Pubmed search by keywords ‘‘solid lipid nano-
particles’’ and ‘‘cytotoxicity’’ returns 128 results; the same search
in ISI Web of Knowledge returns 174 entries. The existing data on
toxicity in cell culture experiments are however still difficult to
compare – they were obtained (i) in various cell lines and cell types,
at different passages, (ii) the methods used have slight variations
among research groups, and, clearly, (iii) different formulations
for various purposes were tested. To the best of our knowledge, a
systematic study with a large series of formulations and performed
in one sole laboratory has not been published up to date.
Among the currently available data, there are a few studies
which main purpose is to address the safety issues of SLN/NLC
for the selected route of administration: Weyenberg and collabora-
tors tested a series of SLN with both positive and negative surface
charge for the purpose of dermal delivery [7], Ridolfi and collabo-
rators [8] also tested a small series of SLN in fibroblasts and kerat-
inocytes. Another group tested two SLN formulations intended for
lung delivery by different cell viability assessment methods and
also reports an ex vivo study in murine lung tissue [9,10]. Tolerabil-
ity of a series of SLN by normal human granulocytes from healthy
volunteers and leukemic HL-60 macrophages was tested by the
group of Müller [11–13]. Petersen and colleagues [14] investigated
the influence of lipid physical state on the toxicity towards cells in
culture and Silva and collaborators [15] reported a study with a
series of SLN in monkey kidney cells.
Cell studies have also been performed for various purposes.
Studies using chemotherapeutic drug(s) loaded in SLN/NLC usually
include a cell viability study to prove that the formulation is
efficient. The main reason of this testing is a proof of efficiency of
the drug loaded formulation. A control using the blank SLN/NLC is
a logical component of such a study to prove that the carrier itself
is not toxic, however, there are reports where this control is omitted
and others that only refer to blank SLN/NLC non-toxicity marginally.
Improvement in chemotherapeutic drug delivery is a hot topic in
current research; SLN/NLC research also follows this trend and there
are up to 100 formulations reported, most of them with cell toxicity
data. These are useful sources of information on SLN/NLC safety.
Another big source of cell viability data are SLN/NLC formulations
intended for gene delivery. Again, the proof of efficiency requires the
use of cell culture and the information of the maximal tolerable con-
centration that can be used in transfection experiments. Furthermore,
lately more and more reports on non-chemotherapeutic drugs stud-
ies include cell viability assays; aiming to show that neither the blank
nor the drug loaded formulation causes cellular damage/toxicity.
In order to compare the published results, we collected the
information on cell lines, methods used, time of exposure, concen-
tration range and the study outcome, together with basic informa-
tion about the formulations that were tested (solid lipid, surfactant,
size – mostly reported as z-average diameter, zeta potential – ZP).
Outcomes of the studies are reported here as given by the authors;
i.e., by % of viability at a particular concentration or as IC50. Concen-
tration of SLN/NLC in the tested dispersion is given as mass solid
lipid per volume, when possible to calculate this information from
the published data. For uniformity, the concentrations are all given
here in mg/ml, i.e., solid lipid content/ml cell culture media.
2. Methods for risk identification in vitro
Basic test for a toxicological study is a cell viability study. The
endpoint is a concentration that causes 50% cell death (IC50). In
order to calculate IC50, several concentrations need to be tested. If
IC50 is not required for further studies, many researchers report a
concentration at which no toxicity is observed (or toxicity at
acceptable levels) and select this concentration for further studies.
This reporting is naturally less precise. Decrease in cell viability by
more than 30% (i.e. cell viability of 70% and less) is to be considered
as a cytotoxic effect [16]. This norm is frequently forgotten when
referring the results, thus it is important to review the actual cell
viability % reported rather than the evaluation made by the authors.
Besides cell viability studies, the advanced toxicological studies
also search for effects on cells that do not necessarily lead to cell
death. These include oxidative stress, i.e. increased production of
reactive oxygen species (ROS); lipid (integral part of cell mem-
branes) peroxidation, imbalance in oxidised/reduced glutathione
pool or DNA damage. An excellent revision of toxicology of nanom-
aterials including various methods was published by Lewinski and
collaborators [17] where more detail on the toxicological assays
can be obtained.
2.1. Cell viability assays
The majority of cell viability studies of SLN/NLC were performed
by MTT assay. MTT is a tetrazolium dye which is converted into
formazan by metabolically active cells [18]. The product is insolu-
ble in water; a solubilisation step by an organic solvent is neces-
sary to dissolve the crystals and enable absorbance reading. The
use of an organic solvent may raise the logical question if the lipids
used for SLN may be solubilised as well. Even though some lipids
such as Precirol ATO 5 (Glycerol distearate, Gattefosse SAS) are sol-
uble in chloroform [19], the extent of solubilisation of the particles
should be low. Furthermore, SLN/NLC in the dispersion themselves
absorb visible light. Absorbance reading of MTT assay is usually
done at 570 nm; at this wavelength the absorbance from SLN/
NLC is very low. To avoid the possibility of lipid interaction with
the assay, some research groups include a washing step before
incubation with organic solvent [7,20,21]. Again, majority of pub-
lished reports do not enclose if the particles had been removed
or not before incubation with MTT.
Apart from MTT, other formazan dye based assays have been
used, including Alamar Blue (AB), WST-1 and WST-8 (Water Solu-
ble Tetrazolium salts) based assays. These dyes are water-soluble;
the absorbance reading can be done without solubilisation step.
When using these assays, the presence of SLN in the cell culture
media is even more critical. The blank measurements should in-
clude also the particles in all concentrations used in the experi-
ment. Fig. 1 shows such blanks and illustrates that over a
Fig. 1. Absorbance at 570 nm of various SLN in DMEM. Absorbance readings were
obtained from Multiskan Ex microplate reader (MTX Labsystems, USA) and are
presented as mean from quadruplicate with standard deviation. (A, B and C)
Represents three distinct SLN formulations, differing among themselves in solid lipid
and/or surfactant composition. Unpublished data (SD and AMS), for information on
formulation composition, please see Doktorovova, et al. 420 (2011) 341–349.
 S. Doktorovova et al. / European Journal of Pharmaceutics and Biopharmaceutics 87 (2014) 1–18
concentration range the blank absorbance shows remarkable dif-
ferences, depending on the SLN composition.
Other type of cell viability assay is based on dye uptake. Neutral
red (NR), Crystal violet or Trypan Blue were used in SLN/NLC tests.
NR assay is based on accumulation of NR in lysosomes of living
cells [22]; Trypan Blue is based on the fact that living cells can ac-
tively exclude the dye, while dead cells accumulate the dye [23].
The evaluation is based on the count of living cells vs. dead cells
marked by the accumulated dye.
Serving for the same purpose (estimation of % of living cells) but
based on different mechanism; these two types of methods are
complementary and were used simultaneously within one study
with the same materials and cell lines [9,15]. From these two re-
ports it can be concluded that the results are comparable: the re-
sults of Silva and collaborators showed excellent correlation of
the results obtained with MTT and NR results in MDCK cells; a
slightly higher IC50 was calculated by NR assay in Vero cells [15].
Nassimi and collaborators [9] reported an IC50 for the tested SLN
of 2.09 mg/ml by NR assay and of 3.09 mg/ml by MTT assay. These
findings are perfectly in agreement with previous comparisons of
various cell viability assays [24].
Lactate dehydrogenase (LDH) release assay can also be taken as
a measure of cell viability. It reflects the damage to cell membrane,
thus gives additional information to mitochondrial activity-based
assays. Some authors, however, reported that LDH assay is less sen-
sitive to cell damage than MTT or WST assays (see further).
The results that we mention here were collected at time points
varying from 4 h through the most common 24 h, 48 h to 72 h
incubation times. This fact causes even further difficulties in com-
paring them. Ma and colleagues showed that MTT metabolism by
HaCaT cells shows a decrease between 4 h and 12 h of incubation.
After 12 h, the MTT conversion gradually increased again [25]. On
the contrary, Zhuang and collaborators [26] observed steady de-
crease in IC50 of three drug-loaded SLN formulations (mitoxan-
throne, methotrexate, paclitaxel; using MCF-7 cell line)
determined at 24 h, 48 h and 72 h.
Box 1 – List of substances used in cell viability assays.
MTT 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyl
tetrazolium bromide
MTS 3-(4,5-dimethyltiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-
dihydro tetrazolium
XTT 2,3-bis-(2-methoxy-4-nitro-5sulfophenyl)-
dihydroxy-terazolium-5-carboxyanilide
WST-1 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-
disulfophenyl)-2H-tetrazolium
WST-8 2-(2-methoxy-4-nitrophenyl)-3-(4-
nitrophenyl)-5-(2,4-disulfophenyl)-
dihydroxy tetrazolium
Alamar Blue 7-hydroxy-3-hydro-phenoxazin-3-one-10-
(AB) oxide), syn. Resazurin
Crystal violet Tris(4-(dimethylamino)phenyl)methylium
chloride, syn. Gentian violet
Neutral Red 3-amino-7-dimethylamino-2-
(NR) methylphenazine hydrochloride, syn.
toluylene red
Propidium 3,8-diamino-5-[3-
Iodide (PI) (diethylmethylammonio)propyl]-6-
phenylphenanthridinium diiodide
Trypan Blue (3Z,30 Z)-3,30 -[(3,30 -dimethylbiphenyl-4,40 -
diyl)di(1Z)hydrazin-2-yl-1-ylidene]bis(5-
amino-4-oxo-3,4-dihydronaphthalene-2,7-
disulfonic acid), syn. Azidine Blue
3
3. Special issues with SLN
By definition, SLN/NLC are formulated in aqueous dispersion;
higher concentration of electrolytes may cause instability and pre-
cipitation of SLN [27]. This is an especially important issue in cell
studies with this kind of carriers, as they need to be performed
in buffers (to maintain physiological pH and osmolarity) or cell cul-
ture media. This fact should immediately raise a concern about
SLN/NLC stability in these media. The vast majority of reports do
not address this issue at all. From the few reports that include
some stability data we can conclude that SLN/NLC can be formu-
lated so that they were stable in these media.
Petersen and collaborators [14] reported no aggregation of their
samples in DMEM at 37 °C in the whole range of concentrations
used in cell studies, Silva and colleagues [28] also reported that
the tested SLN were stable in cell culture media, although at lower
concentrations. Almost no change in size over a large concentra-
tion range (0.9 ng/ml–9 mg/ml) in a buffer was reported by Rivolta
and collaborators [29], How and collaborators [30] reported that
their NLC formulations were stable in RPMI with or without foetal
bovine serum (FBS), showing only few nm difference from the ori-
ginal z-ave value in aqueous dispersion. This change occurred
within 60 min of incubation; up to 5 h no further changes in diam-
eter were observed [30]. On the other hand, Siddiqui and collabo-
rators [31] reported aggregation of cationic SLN in cell culture
media. Positively charged SLN are usually less stable then nega-
tively charged SLN, hence this is an expected result. Some aggrega-
tion that could be prevented by ultrasound treatment was also
mentioned for negatively charged SLN [28,32].
Some authors adjusted their formulations to physiological pH
before all further tests took place [33–36], and others worked with
freeze-dried SLN/NLC that were directly re-dispersed in appropri-
ate buffer solution [37]. In response to the need of stable
SLN/NLC dispersions for in vitro studies, Peters and collaborators
[38] prepared NLC especially with the aim of being stable in buffer
solutions.
4. SLN effect on cells in culture
4.1. Comparison by IC50
The most accurate comparison of SLN/NLC toxicity can be done
by using the IC50 values provided by the authors. Although very
few studies were conducted with enough concentrations to calcu-
late IC50, all of them tested a set of formulations which allows look-
ing at the overall information. Table 1 lists these results from the
most toxic (lowest IC50) to the most well tolerated (highest IC50).
From a brief look it is apparent that the IC50s are found mostly in
the range of 0.1–1.0 mg/ml particles in dispersion. Higher IC50s
than 1 mg/ml were reported by several authors, e.g. Silva and col-
laborators [15] reported an IC50 over 1 mg/ml, Schubert and Mul-
ler-Goymann [39] reported a value of 1.3 mg/ml, or Nassimi and
collaborators [9] 2–3 mg/ml.
There is no clear tendency of neither surfactant nor lipid core
influence on the obtained IC50 values. We can find glyceryl monos-
tearate (GMS)-composed formulations in both ends of the table,
including both GMS-alone and in combination with stearic acid
(SA) and oleic acid (OA). Surfactants are considered to be decisive
in toxicity of nanoparticles [40], from the listed IC50 (Table 1) it
can be concluded that only poly-vinyl alcohol (PVA) and sodium
dodecyl sulphate (SDS) used as surfactants lead to low IC50 values,
which is an expected finding, and that frequently used surfactants
in SLN/NLC formulations like Tween 80 (T80) are to be find in the
bottom of the table.
With exact measure of toxicity as IC50, it is possible to compare
the influence of size and charge. The importance of size on toxicity
4 S. Doktorovova et al. / European Journal of Pharmaceutics and Biopharmaceutics 87 (2014) 1–18

Table 1
Effect of blank SLN and NLC (i.e. without any encapsulated active ingredient) on cell viability expressed as half maximal inhibitory concentration (IC50). The results are ordered
from the most toxic formulations (lowest IC50) to formulations with lowest effect on cell viability (highest IC50). The column ‘‘drug’’ refers to the substance that the formulation
was intended to encapsulate.

Type Lipid matrix composition Surfactant Drug Size; PdI; ZP Cell line Assay Exposure IC50 (mg/ml) Refs.
t (h) [] (mg/ml)
SLN+ CP, DOTAP Oleth20, 522 nm; 0.29; n.a. Du-145 MTT 24 0.005–2.5 0.125 [43]
GO
SLN GMS Chitosan, DOX 83–164 nm; 0.15–0.23; MCF-7 MTT 48 n.a. 0.17456 [37]
PVA 18 to 22 mV
SLN(?) S154 Soy PC, n/a 145 nm, 0.15, 17.3 mV MCF-7 MTT 72 0.037–0.3 0.2223 [45]
octadecanol
SLN TS SDS n/a 116 nm; 0.22; 39 mV Vero MTT 24 0.1–1.0 0.247 ± 0.007.2 [15]
SLN(?) S154 Soy PC, n/a 145 nm, 0.15, 17.3 mV MCF-7 MTT 48 0.037–0.3 0.2621 [45]
octadecanol
SLN(?) S154 Soy PC, n/a 145 nm, 0.15, 17.3 mV MDA-MB- MTT 72 0.037–0.3 0.275 [45]
octadecanol 231
SLN(?) S154 Soy PC, n/a 145 nm, 0.15, 17.3 mV MCF-7 MTT 24 0.037–0.3 0.2875 [45]
octadecanol
SLN(?) S154 Soy PC, n/a 145 nm, 0.15, 17.3 mV MDA-MB- MTT 24; 48 0.037–0.3 0.29 [45]
octadecanol 231
SLN GMS + SA-FA PTC 300–460 nm; n.a.; n.a. A549 MTT 48 n.a. 0.30872 ± 0.04189 [57]
SLN SA P188 PTC 300–460 nm; n.a.; n.a. A549 MTT 48 n.a. 0.33112 ± 0.03412 [57]
SLN+(?) Triolein, Chololeate, Chol, DSPE-PEG (Plasmid) 102 nm; n.a.; n.a. HepG2 WST- 10 n.a. 0.3335 [127]
DC-Chol, DOPE 1
SLN GMS + SA-PEG PTC 300–460 nm; n.a.; n.a. A549 MTT 48 n.a. 0.34789 ± 0.04240 [57]
SLN GMS P188 PTC 300–460 nm; n.a.; n.a. A549 MTT 48 n.a. 0.39682 ± 0.03319 [57]
SLN GTS P188 PTC 185 nm; 0.2; 31.7 mV A549 MTT 48 n.a. 0.40149 ± 0.02768 [57]
SLN COM P188 PTC 300–460 nm; n.a.; n.a. A549 MTT 48 n.a. 0.47148 ± 0.03091 [57]
NLC GMS + OA P188 PTC, DOX 134.4 ± 21.0 nm; MCF-7 MTT 48 n.a. 0.45549 ± 0.02768 [66]
0.361 ± 0.024;
45.5 ± 3.0 mV
NLC GMS + OA P188 PTC, DOX 134.4 ± 21.0 nm; MCF-7/Adr MTT 48 n.a. 0.46974 ± 0.03002 [66]
0.361 ± 0.024;
45.5 ± 3.0 mV
SLN Solid white vaseline USP T80 n/a 128 nm; 0.32; 18 mV Vero MTT 24 0.1–1.0 0.489 ± 0.004.9 [15]
SLN GMS P188 PTC, DOX 273 nm, 0.101, 36.4 mV SKOV-3 MTT 48 n.a. 0.544 [128]
SLN GMS P188 PTC, DOX 273 nm, 0.101, 36.4 mV MCF-7 MTT 48 n.a. 0.549 [128]
SLN GMS P188 PTC, DOX 273 nm, 0.101, 36.4 mV MCF-7/ADR MTT 48 n.a. 0.564 [128]
SLN GMS P188 PTC, DOX 273 nm, 0.101, 36.4 mV Skov-3/ MTT 48 n.a. 0.558 [128]
TR30
SLN(?) S154 + PhospholiponP90G Solutol n/a 98 nm; 0.148; 14.6 mV PCSL WST- 24 n.a. 0.575 [9]
HS15 1
SLN TS SDS n/a 116 nm, 0.22, 39 mV MDCK MTT 24 0.1–1.0 0.586 ± 0.015 [15]
SLN TS SDS n/a 116 nm; 0.22; 39 mV MDCK NR 24 0.1–1.0 0.603 ± 0.0027 [15]
SLN TS SDS n/a 116 nm; 0.22; 39 mV Vero MTT 24 0.1–1.0 0.643 ± 0.0024 [15]
SLN TS T80 n/a 110 nm; 0.22; 26 mV Vero MTT 24 0.1–1.0 0.682 [15]
SLN TS T80 n/a 110 nm; 0.22; 26 mV Vero NR 24 0.1–1.0 0.694 ± 0.0093 [15]
SLN CP P60 CPT <200 nm; <0.25; 20 mV U373 MTT 24 0.005–50 0.716 [129]
SLN CP P60 CPT A172 MTT 24 0.005–50 0.863 [129]
SLN S154 + PhospholiponP90G Solutol n/a 95 nm; 0.182; 9.8 mV HDF(NHDF) 24 0.0–3.0 (0–6%) 1.3 [39]
HS15
SLN CP P80 CPT <200 nm; <0.25; 20 mV U251 MTT 24 0.005–50 1.17025 [129]
SLN CP P80 CPT <200 nm; <0.25; 20 mV A172 MTT 24 0.005–50 2.06 [129]
SLN CP P60 CPT <200 nm; <0.25; 20 mV U251 MTT 24 0.005–50 2.086 [129]
SLN S154 + PhospholiponP90G Solutol n/a 98 nm; 0.148; 14.6 mV A549 MTT 24 0–15.0 2.09 [9]
HS15
SLN CP P80 CPT <200 nm; <0.25; 20 mV U373 MTT 24 0.005–50 2.135 [129]
SLN S154 + PhospholiponP90G Solutol n/a 98 nm; 0.148; 14.6 mV A549 MTT 24 0–15.0 3.09 [9]
HS15
SLN S154 + PhospholiponP90G Solutol n/a 144 nm; 0.225; 12.3 mV/ A549 MTT 24 0–15.0 3.43 (SLN20); 1.253 [10]
HS15 93 nm; 0.157; 22.6 mV (SLN50)
SLN CP P60 or P80 CPT <200 nm; <0.25; 20 mV U87 MTT 24 0.005–50 5.0 [129]
SLN CP P60 or P80 CPT <200 nm; <0.25; 20 mV THP1 MTT 24 0.005–50 5.0 [129]
SLN Plant lipids T80 n/a n.a.; n.a.; n.a. Vero, MTT 24 0.1–1.0 IC50  1 mg/ml (100% [15]
MDCK viability at 0.5 mg/ml)
SLN Solid white vaseline USP T80 n/a 128 nm; 0.32; 18 mV MDCK MTT 24 0.1–1.0 IC50  1 mg/ml (100% [15]
viability at 0.5 mg/ml)
SLN TS T80 n/a 110 nm; 0.22; 26 mV MDCK MTT 24 0.1–1.0 IC50  1 mg/ml (100% [15]
viability at 0.5 mg/ml)
SLN Solid white vaseline USP T80 n/a 128 nm; 0.32; 18 mV Vero NR 24 0.1–1.0 IC50  1 mg/ml (100% [15]
viability at 0.5 mg/ml)
SLN Solid white vaseline USP T80 n/a 128 nm; 0.32; 18 mV MDCK NR 24 0.1–1.0 IC50  1 mg/ml (100% [15]
viability at 0.5 mg/ml)
SLN TS T80 n/a 110 nm; 0.22; 26 mV MDCK NR 24 0.1–1.0 IC50  1 mg/ml (100% [15]
viability at 0.5 mg/ml)

Abbreviations (in this table) n.a. = not available, NR = Neutral Red uptake method, MTT = MTT method; WST-1 = WST-1 method.
 S. Doktorovova et al. / European Journal of Pharmaceutics and Biopharmaceutics 87 (2014) 1–18 5

has been reported for other types of colloidal carriers [41,42]. Here, Box 3 – List and identification of cancer cell lines.
with the few data available, we cannot draw any conclusion yet.
Most of the tested SLN/NLC had size below 200 nm, but among
the formulations with lowest IC50 we can find formulation with A172 human glioma cells
z-average (z-ave) of 522 nm [43]. The size effect reported for other A549 human lung carcinoma cells
colloidal carriers like carbon nanotubes, silica nanoparticles or B16F10 murine melanoma cells
other types of inorganic particles reported on substantially smaller BEL7402 human hepatoma cells
particles, i.e., 20–40 nm [44]. This means that most SLN/NLC effects bEnd.3 murine cerebral cortex endothelioma cells
on cells cannot be explained just on their ‘‘small size’’ (which com- BxPC-3 human pancreatic adenocarcinoma cells
pared to inorganic nanoparticles is nothing but small, actually). Caco-2 human colorectal adenocarcinoma cells
Fig. 2 shows graphically the influence of SLN/NLC size on cell CPH 54A human small-cell lung cancer cells
viability at 24 h time point, collected from both IC50 and cell viabil- CT26 murine colon carcinoma cells
ity data. As can be observed in this figure, no clear tendency of Du-145 human prostate cancer androgen-non-
influence of size can be traced, although the highest values of via- responsive
bility with the highest concentrations tested were observed with HCT116 human colorectal carcinoma
SLN ranging from 100 to 300 nm. HCT-15 human colorectal carcinoma
HDF(NHDF) normal human dermal fibroblasts
Box 2 – List and identification of non-cancer cell lines.
HeLa human cervical adenocarcinoma
HepG2 human hepatocellular carcinoma
293T human kidney epithelial cell line HL-60 human promyelotic (susp) acute
3T3 BALB/ murine embryonic fibroblast promyelocytic leukemia
c HNGC1 human glioma cells
TC-1 lung epithelial cells HT-29 human colorectal adenocarcinoma
A30 human alveolar cells HuH6, HuH7 human hepatoblastoma
COS-1 African green monkey kidney fibroblast-like cell J774 murine monocyte/macrophage macrophage
line (from reticulum cell sarcoma)
COS-7 African green monkey kidney fibroblast-like cells L929 murine aneuploid fibrosarcoma
HaCaT human dermal fibroblast cells K562 Human myelogenous leukemia
MDBK Madin–Darby bovine kidney LAN5 human neuroblastoma
MDCK dog kidney fibroblasts MCF-10A human breast adenocarcinoma
NiH/3T3 murine embryonic fibroblast MCF-7 human breast adenocarcinoma
NHFa human foreskin fibroblast MCF-7/Adr human breast adenocarcinoma, resistant to
NR6-W mouse fibroblast cell lineb Adriamycin
NR8383 rat alveolar macrophages MCF-7/MX human breast adenocarcinoma, multidrug
RAT-1 rat immortalised fibroblasts resistant (Mitoxanthrone)
SDHCEC human corneal epithelia cell MDA-MB-231 human breast adenocarcinoma
TC-1 lung epithelial cells MDA-MB-435 human breast ductal adenocarcinoma
U87MG human brain-microvascular endothelial cells Na 1300 murine neuroblastoma
VK2/E6E7 human vaginal epithelial NCI/ADR-RES human ovarian cancer
Vero African green monkey kidney fibroblasts OCI-AML-2 human acute myeloid leukemia
a
OVCAR-3 human ovarian adenocarcinoma
Also designed as NHDF.
b
Transfected to express the human EGF receptor.
PC-3 human prostate adenocarcinoma
RAW 264.7 murine macrophage, Abelson murine
leukemia virus transformed
SKOV-3 human ovary adenocarcinoma
SKOV-3/TR30 human ovary adenocarcinoma, paclitaxel
resistant
SW-480 human colorectal adenocarcinoma
THP1 human acute monocytic leukemia
(phagocytic)
U87 human glioblastoma; astrocytoma
U118 human glioblastoma
U251 human neuronal glioblastoma
(astrocytoma)b
U373 human glioblastoma cellsa
a
Cell line was withdrawn by ATCC.
b
Identical to U373 [111].

Fig. 2. The effect of SLN/NLC size (z-ave, diameters in nm) on cell viability in vitro.
Cell viability (in % viable cells) vs. concentration of SLN/NLC in cell culture media 4.2. Comparison by cell viability data
reported in references listed in Tables 1–3 were used. From reports that list three
and more different SLN/NLC concentration, the highest and lowest cell viabilities Much more data on SLN/NLC safety are reported as % of viable
were used. Citation, in the respective references, as ‘‘No decrease in cell viability’’
was plotted as 100% viability, as ‘‘no toxicity’’ was plotted as 85% viability (as non-
cells in the culture, or stating that no toxicity was observed at a
toxic could be between 70% and 100% viability) and IC50 data were plotted as 50% certain concentration or concentration range. From these data,
viability. Size not available on the reference denoted by n.a. when possible to gather all necessary data, we summarised
6 S. Doktorovova et al. / European Journal of Pharmaceutics and Biopharmaceutics 87 (2014) 1–18

Tables 2 and 3 assuming that viability >70% is considered as ‘‘no tox- concentrated SLN dispersion is needed in order to achieve higher
icity’’ (Table 2) and in case that viability <70% was reported the result SLN concentration in cell culture so that the amount of cell culture
is considered as ‘‘cytotoxic effects’’ were observed (Table 3). In order media did not need to be reduced [12,14].
to better understand the data, we plotted the reported cell viability Although there are some isolated reports on the lack of toxicity
or IC50 vs. SLN/NLC concentration, for 24 h exposure (Fig. 3). at higher SLN/NLC concentration, the reasonable range to design a
About 100% viability was reported up to 0.1 mg/ml SLN in hu- toxicological study with this type of system seems to be below
man granulocytes [13] or NHF-cells [46], up to 0.5 mg/ml SLN com- 1 mg/ml.
posed by cetyl esters had no influence on 3T3 cells viability [8], up
to 1 mg/ml in NHF cells [47] and 100% viability at 1.32 mg/ml was
4.3. Influence of lipid-core material
reported by Trombino and collaborators [48] in RAT-1 cells. SLN at
1 mg/ml and 2.5 mg/ml without decrease in cell viability (HL-60
In one of the very first in vitro trials with TM and COM-com-
and MCF-7 cells) was reported by Miglietta and colleagues [49].
posed SLN (stabilised by different poloxamers) it was noted that
The only ‘‘100% viability’’ report on NLC discloses 0.9 mg/ml NLC
although overall very well tolerated, COM-SLN had slightly higher
in NHF cells, tested within the same studies as SLN (0.1 mg/ml)
impact on cell viability than TM-SLN. This was attributed to the
[46]. It is interesting to note that all of these results come from
longer time needed for degradation of the longer-chain lipids by li-
studies on cell lines that do not originate from cancer cells. The
pases [13].
material used for the formulation includes common lipids used
In a further study with a series of SLN composed of different lipid
in SLN and NLC preparation – Compritol 888 ATO (COM), trimyri-
core but stabilised with same surfactant, Scholer and collaborators
state (TM), cetyl palmitate (CP), Witepsol E58 and less frequently
[56] reported significant influence of solid lipid. COM, TM, and CP
used Apifil (PEG-8 beeswax) or cetyl esters; the surfactants include
did not cause any cytotoxic effect up to 1 mg/ml, but stearic acid-
poloxamers, polysorbates (Tweens), phospholipids, sodium tauro-
composed SLN caused a drastic decrease (2.2% cell viability) at this
cholate and even butanol.
concentration. Tristearate (TS) and GMS also were cytotoxic to mac-
Over 90% viability was reported for concentrations varying from
rophages at 1.0 mg/ml (around 60% and 70% viability, respectively).
0.015 to 1.5 mg/ml in HL-60 cells (Poloxamer stabilised SLN, while
The reason seems to be attributed to stearic acid, both as lipid-
T80 SLN stabilised reduced viability to 50% at 0.1 mg/ml [13], and
matrix material and component of respective mono- and tri-
all other reports of >90% viability state exposure of 0.1–0.4 mg/ml,
glycerides [56]. Miglietta and colleagues [49] reported however
including both cancer (HeLa [50], Raw 264.7 [51]) and non-cancer
reported no decrease in cell viability when using 1 mg/ml stearic
cell lines (HaCaT [7]). Over 80% viability of various formulations
acid-SLN. The study by Scholer et al. was performed in primary
was reported, again roughly in the same concentration range,
macrophages, the study by Miglietta et al. used HL-60 macrophages
including cationic SLN formulations (stearic acid + stearylamine
[56,49]. Interestingly, primary, ‘‘normal’’ macrophages were more
in COS-1 cells [52] and GMS + LHNH in RAW 264.7 [33]).
susceptible to damage. Stearic acid is a widely used lipid in SLN
Maintaining viability >70% was in fact reported over broad con-
and NLC formulations; however cytotoxicity testing was not per-
centration ranges: 0.4–6.0 mg/ml [53] or up to 15.0 mg/ml [54].
formed in the most of the cases. From the few reports of stearic
The lowest viabilities reported come logically from reports on
acid-based SLN we can only conclude that concentrations about
cationic SLN/NLC. 0.03 mg/ml SLN composed of stearic acid and
0.1–0.2 mg/ml are mostly reported as the highest non-toxic
stearylamine were toxic to HaCaT, 3T3 and J774 cells; viability
amounts [52,57].
<70% was reported also for stearic acid-composed SLN in the same
Ridolfi and collaborators tested a series of wax-based SLN and
study, while Witepsol composed SLN were not toxic [7].
reported different cell viabilities for cetyl esters, cetyl palmitate
From these results, it might seem that cancer cell lines were
and myristyl myristate SLN, showing that cetyl palmitate-based
more susceptible to SLN/NLC than non-cancer cell lines. The same
SLN caused the lowest obtained cell viability values in the tested
tendency was noted by Martins et al. [32] who observed higher
concentration range (up to 0.5 mg/ml, cell viability >70%) [8].
toxicity of the tested SLN towards a series of glioma cells than to-
Other reports with similar sets of SLN/NLC (different lipid, same
wards monocytes. This apparent tendency may still be misleading,
surfactant) did not point out any significant differences [47,57].
as it is not possible to draw a conclusion if this is true from the
How and colleagues tested the components of their NLC formula-
available information. The reason is different endpoints of the
tion alone; in 1% acetone in RPMI mixture. In this work, tripalmi-
studies summarised on Tables 1–3. The data cited for Tables 2
tate (TP) reduced viability of BALB/c 3T3 cells to 30% only, while
and 3 are mostly taken from studies that did not focus on toxicity
trilaurine or hydrogenated palm oil were not toxic [30]. SLN/NLC
in much detail or were designed to prove that the given formula-
may form aggregates in cell culture media or suffer changes to
tion is not toxic at a given concentration. Most of the experiments
its structure (e.g., desorption of surfactants). Testing of the unpro-
with cancer cell lines studied the toxicity in much more detail and
cessed starting material present in the same quantities and ratios
provided IC50 results. The reported IC50 (=50% viability) are mostly
as in the final formulation would therefore clarify if the observed
in the range 0.1–1.0 mg/ml which is exactly the same concentra-
effects are result of that particular combination of materials per
tion range where no cytotoxic effects (>70% viability, Table 2) are
se or of the SLN/NLC in question. Controls like the one reported
reported.
by How and colleagues [30] are rare. One reason is the materials
Fig. 4 correlates the available data on cell viability vs. SLN/NLC
are already considered safe; the other is the practical point of
concentration discriminated by the type of cell line (normal vs.
testing water-insoluble solid lipids in quantities usually used in
cancer cell line). As can be seen, no tendency of toxicity suscepti-
SLN/NLC without damaging the cells with necessary organic
bility can be attributed to normal or cancer cell lines.
solvents (DMSO, acetone, or others).
An interesting explanation of why low toxicity of SLN/NLC is of-
ten reported was given by Liu and colleagues [55] – blanks SLN are
tested in concentrations suitable to deliver small amounts of drug 4.4. Influence of surfactant
– the EU directive recommends testing of concentrations up to
0.5 mg/ml. At typical SLN/NLC loading capacity of around 5–10% Surface properties of nanocarriers have decisive influence on
[5], the used concentrations are too low to cause any effect at all. their interactions with living cells and tissues. It is therefore obvi-
Another limiting factor in SLN/NLC testing in cell culture assays ous that surfactant type, amount and interaction with lipid core
is the highest SLN concentration in the cell culture media. A highly will have effect on SLN/NLC performance in cell cultures.
Table 2
Effect of blank SLN and NLC (i.e. without any encapsulated active ingredient) on cell viability expressed as % of viability reported by the authors of the study. This table includes the results that may be interpreted as not cytotoxic (cell
viability >70%). The column ‘‘drug’’ refers to the substance that the formulation was intended to encapsulate.

Type Lipid matrix Surfactant Active Size; PdI; ZP Cell line Assay Exposure Outcome (cell viability) Refs.
t (h) [] (mg/ml)a
SLN COM Epikuron + STC Ferulic acid LAN5 MTS 24 0.014– 95% to 0.056 mg/ml; 100% to [100]
0.056 other []
SLN COM/TM 0.5 wt% Polox 184/188/235/ n/a 186–280 nm; n.a.; HL-60 MTT 12 1  105– 100% up to 0.01 mg/ml [13]
335/407/T80/SDS n.a.; 1.0
SLN COM/TM 0.5 wt% Polox 184/188/235/ n/a 186–280 nm; n.a.; Granulocytes MTT 12 1  105– 100% up to 0.01 mg/ml [13]
335/407/T80/SDS n.a. 1.0
SLN CP/myristyl myristate (MM)/cetyl P188 n/a 185–197 nm; 3T3 BALB/c MTT 24 0.01–0.5 100% (CE up to 0.5 mg/ml); 85–65% [8]
esters (CE) 0.15–0.23; 30.5 (CP 0.01–0.5 mg/ml), 100–65%

S. Doktorovova et al. / European Journal of Pharmaceutics and Biopharmaceutics 87 (2014) 1–18

to 37.5 mV (MM 0.01–0.5 mg/ml)
SLN. SLN = 1%Apifil, P188 2.5% Alfa Lipoic Acid 103–121 nm; 0.18– NHF MTT 16 0.9 NE & 100% viability [46]
NLC, NLC = 8%Apifil + 1%Miglyol, 0.22, 27.8 to NLC; 0.1
NE NE = 9%Mygliol 36.8 mV SLN
SLN, NE SLN1 = CP, SLN2 = D114, P188 2.5% Gamma Oryzanol 200 nm; n.a.; n.a. NHF(NHDF?) MTT 48 1 100% viability [47]
SLN3 = WitepsolE85
SLN COM PC + T80 Mitoxantrone 108 nm; 0.219; MCF-7 MTT 72 100% viability [26]
26 mV
SLN TM PC, +STC PTC 224 nm; 0.278; n.a. MCF-7 MTT 72 100% viability [26]
SLN TP PC, +STC MTX 192 nm; 0.245; n.a. MCF-7 MTT 72 100% viability [26]
SLN+ Stearylalcohol/CTAB 2:1 or n/a 167 nm; 0.18; NCI/ADR- CellT- ? 0.1– 100% up to 1 lg CTAB, over 90% at [31]
Stearylalcohol/ceramide/CTAB 1:1:1 +39.3 mV or RES Glo 51.16 lg/ 51.16 lg CTAB/ml
75 nm; 0.21; ml CTAB
35.8 mV
SLN Stearylalcohol (ferrulate) T20 + ButOH + STC b-carotene/- 159 nm; 0.11; n.a RAT-1 MTT 24 1.32–13.52 100% up to 1.32 mg/ml, 81.5% at [48]
tocopherol 13.5 mg/ml, 58.1% 50 lM
Stearylalcohol ferrulate
SLN SA T80 + P188 Emodin 29 nm; n.a.; MCF-10A MTT n.a. n.a. 100% (all conc.) [88]
31 mV
SLN TM PC, Galactosylated DOPE DTC 120 nm; 0.2; 12.4 BEL7402 MTT 24 n.a. 100% (all conc.) [114]
to 13.2 mV
SLN CP T80 CPT n.a.; n.a.; n.a. BCECa AB 24 (0.25– 100% [115]
2.5 lM
CPT)
SLN TP T80 CPT n.a.; n.a.; n.a. BCECa AB 24 (0.25 lM 100% [115]
CPT)
SLN(?) Precirol 3 wt% T80 (50 wt%) PTC 114 nm; 0.117; MPMac MTT 2 0.075–7.5 100% [130]
20.1 mV
SLN(?) Precirol 3 wt% T80 (50 wt%) 114 nm; 0.117; MTX-B2 2 0.075–15 100% [130]
20.1 mV
SLN+ SA, stearylamine P188, Chol/PC/Chol + PC n.a. 272–305 nm; n.a.; HepG2 12 0.2 100% [131]
25–28 mV
SLN Precirol T80 Edelfosine 124 nm; 0.26; OCI-AML-2 Annexin- 24; 0.199 100% [132]
28.1 mV V 72
SLN COM P407/Poloxamine 908 n/a 200 nm, n.a.; n.a. Granulocytes MTT 12 1.0–25.0 100% (up to 1 mg/ml), 80–95% (up [12]
to 25 mg/ml)
SLN TP PTC 160 nm; 0.2; HL60 Trypan 48 2.5 No decrease in viability [49]
31 mV Blue
SLN SA DOX 80 nm; 0.2; HL60 Trypan 72 1 No decrease in viability [49]
35 mV Blue
SLN TP PTC 160 nm; 0.2; MCF-7 Trypan 48 2.5 No decrease in viability [49]
31 mV Blue
SLN SA DOX 80 nm; 0.2; MCF-7 Trypan 72 1 No decrease in viability [49]
35 mV Blue

(continued on next page)

7
 8
Table 2 (continued)

Type Lipid matrix Surfactant Active Size; PdI; ZP Cell line Assay Exposure Outcome (cell viability) Refs.
t (h) [] (mg/ml)a
NLC COM (1.5 mg/ml) + Miglyol812 Epikuron (36 mg), STC (chemotherapeutic) 116 nm; 0.24; HuH6, HuH7 MTS 72 ? No cytotoxicity [116]
(0.3 mg/ml) (130.7 mg) 32.22 mV
NLC COM 1.50 mg/ml + Miglyol812 Epikuron (36 mg), STC (chemotherapeutic) 116 nm; 0.24; PC-3 MTS 72 ? No cytotoxicity [116]
0.3 mg/ml) (130.7 mg) 32.22 mV
SLN Hydrogenated Castor Oil 100 mg PVA 2 wt% Tilmicosin 189 nm; n.a.; COS-7 MTT 18 0. 5–4.0 No cytotoxicity [81]
7.8 mV
SLN Hydrogenated Castor Oil 100 mg PVA 2 wt% Tilmicosin 189 nm; n.a.; MDBK MTT 18 0. 5–4.0 No cytotoxicity (4 mg/ml [81]
7.8 mV decreased viability)
SLN(?)+ 5 mg/ml lipids (in wt%: 45 cholesteryl siRNA 117 nm; n.a.; MDAMB435 WST-8 24 0.003– 100% up to 0.048 mg/ml; 78 ± 1% [96]
ester, 3 TG, 14 DOPE, 10 (w/w) chol, 41.76 mV 0.072 at 0.072 mg/ml

S. Doktorovova et al. / European Journal of Pharmaceutics and Biopharmaceutics 87 (2014) 1–18

28 DC-chol)
SLN(?) E.wax, Brij78 5 mM (2 mg/ml lipids) PTC <100 nm; n.a.; n.a. U118 MTS 48, 0.023 No cytotoxicity [64]
72
SLN COM 0.3 wt%, StA 0.075 wt% 0.7 wt% ButOH n/a 100–200 nm; n.a.; CPH 54A Trypan 2 0.094 No toxicity up to 0.07 mg/ml [117]
(3.95 mg/ml) n.a. Blue
SLN COM 0.3 wt%, StA 0.075 wt% 0.7 wt% ButOH n/a 116 nm; 0.24; NR6-WA Trypan 2 0.094 No toxicity up to 0.07 mg/ml [117]
(3.95 mg/ml) 32.22 mV Blue
SLN COM 0.3 wt%, StA 0.075 wt% 0.7 wt% ButOH pDNA 116 nm; 0.24; CPH 54A Trypan 2 0.094 No toxicity up to 0.1 mg/ml [117]
(3.95 mg/ml) 32.22 mV Blue
SLN COM 0.3 wt%, StA 0.075 wt% 0.7 wt% ButOH pDNA 116 nm; 0.24; NR6-WA Trypan 2 0.094 No toxicity up to 0.1 mg/ml [117]
(3.95 mg/ml) 32.22 mV Blue
SLN COM Soy PC + P188 Epirubicin <450 nm; n.a.; n.a. A549 CV-A 24 ? No cytotoxicity [118]
SLN COM P188 + T80 CysA 227 nm; 0.214; n.a. Rabbit NR 2 2 99% [119]
cornea uptake
eptihelium
SLN+ SA, stearylamine P188, Chol/PC/Chol + PC n.a. 272–305 nm; n.a.; COS-1 PI 12 0.2 95% [131]
25–28 mV
SLN+ COM, Samine T80 Doxorubicin 127–151 nm; n.a.; A549 Trypan 48 0.002174 95% [133]
n.a. Blue
5
SLN TM PC PTC 129 nm; n.a.; OVCAR-3 MTT 24 14  10 – 90% highest [], 82.7 ± 7.4% at [20]
38.1 mV 14.0 100 lM Ptc
SLN(?) HSPC:DSPE:Chol:triolein = 1.5:1:1.2:1 P188 Curcumin/ 194 ± 2.89 nm; MCF-7 MTT 3, 6, ? >90% [21]
transferrin 0.15; 12.43 mV 12,
24,
48
SLN+ SA, stearylamine P188, Chol/PC/Chol + PC n.a. 272–305 nm; n.a.; Na1300 PI 12 0.2 90% [131]
25–28 mV
SLN Precirol T80 Edelfosine 124 nm; 0.26; HL60 Annexin- 72 0.199 90% [132]
28.1 mV V
SLN SA + PA PC Rifampicin 830 nm; 0.176; n.a. A549, MTT 24 0.03 90% [134]
NR8383,
pAEC, pAM
SLN Witepsol E85 P60 CPT n.a.; n.a.; n.a. BCECa AB 24 (0.25 lM >90% [115]
CPT)
SLN SA:isopropylmyristate = 3:1 T80 + TPGS + SoyPC + P188 Tripterine 93–321 nm; n.a.; Caco-2 MTT 36 ? >90% [120]
n.a.
SLN+ COM StA + T80 DOX 127–151 nm; A549 Trypan 48 ? >90% [69]
0.27–0.35; 27 mV Blue
SLN TS PC + T80 Ketoconazol 184–225 nm; 0.2; NIH-3T3 MTT 18 (1 mM >90% [121]
19 to 44 mV active)
SLN+ SA + StA P188 + PC + Chol n/a 336 nm; 0.38; HepG2 PI 12 1.0–3.0 >90% [122]
35 mV
SLN+ SA + StA P188 + Chol n/a 272 nm; 0.24; HepG2 PI 12 1.0–3.0 >90% [122]
28 mV
SLN COM/TM PC n/a HL-60 MTT 12 0.015–1.5 >90% to all []; (TM > COM viability) [13]
SLN COM P407/T80 n/a 268/214 nm; n.a.; HL-60 MTT 12 0.015–1.5 >90% Polox; 50% (T80 0.1 mg/ml); [13]
n.a. <40% 1.5 mg/ml
NLC COM + Miglyol 812 NTC Celecoxib 217 nm; 0.2; A549 CV-A 72 ? >90% [90]
27.38 mV
SLN CP/myristyl myristate (MM)/cetyl P188 n/a 185–197 nm; 3T3 BALB/c MTT 24 0.01–0.5 100% up to 0.5 mg/ml CE; 85–65% [8]
esters (CE) 0.15–0.23; 30.5 CP 0.01–0.5 mg/ml; 100–65% MM
to 37.5 mV 0.01–0.5 mg/ml
SLN SA PC + NTC DOX HT-29 72 (172 nM >90% v [65]
DOX)
NLC(?) Cacao butter + OA Pullulan-folate conjugate Pheo-A HeLa MTT 24 ? >90% NLC-Pheo A without access of [123]
light
SLN GMS PC + VitE + T80 DTC, ketoprofen, <100 nm; n.a.; n.a. bEnd.3 MTT 24 ? 97%, both SLN and SLN-folate [67]
folate targeted

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SLN GMS PC + VitE + T80 DTC, ketoprofen, <100 nm; n.a.; n.a. U373 24 ? 97%, both SLN and SLN-folate [67]
folate targeted
SLN SA PC + Myrj 59 BHR 75 nm; n.a.; 293T MTT 48 0.05–0.4 >95% viability [124]
2.5 mV
SLN SA PC + Myrj 59 BHR 75 nm; n.a.; HeLa MTT 48 0.05–0.4 >95% viability [124]
2.5 mV
SLN Witepsol (various) PC/T80 n/a 283–366 nm; n.a.; HaCaT MTT 18 0.15 >93.66% (all) [7]
26 to 32 mV
NLC(?) Chol oleate + Chol + trioleate PC Tan IIA 8 nm, n.a.; n.a.; RAW 264.7 MTT 3 0.03–0.15 95% (HDL-coated NLC), 76–88% [51]
viability
SLN SA PC + Myrj59 PPT d90 < 100 nm; n.a.; 293T MTT 24 0.1–0.4 >90% [50]
n.a.
SLN SA PC + Myrj59 PPT d90 < 100 nm; n.a.; HeLa MTT 24 0.1–0.4 >90% [50]
n.a.
SLN Softisan 100 PAA, PLL, Heparin Tenofovir VK2/E6E7 MTS 48 0.4–6.0 >80% (all conc.) [53]
SLN(?) Cacao butter/SA/CB + SA ButOH + CTAB + SDS + DSPE- DOX 200–250 nm; n.a.; U87MG XTT ? 0.8 88% (SA) – 95 (CB)% [70]
PEG-ca n.a.
SLN TM PC PTC 129 nm; n.a.; MCF-7 MTT 24 14  105– 80% (highest conc.) 70.7 ± 15% (at [20]
38.1 mV 14.0 equal 100 lM PTC)
SLN+ SA, stearylamine P188, Chol/PC/Chol + PC n/a 272–305 nm, n.a.; HepG2 PI 12 0.6 85% [131]
25–28 mV
SLN TP T20 + NTC Oryzalin 128 ± 5 nm; 0.12; THP1 MTT 24 ? >80% [91]
37 ± 4 mV
SLN TP PC + T20 + NTC Oryzalin 101 ± 4 nm; 0.22; THP1 MTT 24 ? >80% [91]
33 ± 2 mV
SLN TM/TL P188/P188 + NTC Ibuprofen 111 nm; n.a.; Caco-2 MTT 24 0.0–15.0 >80% [54]
+0.03 mV/121 nm;
n.a.; +0.02 mV
SLN+ SA + StA P188 + PC + Chol n/a 336 nm; 0.38; Na 1300 PI 12 1.0–3.0 >80% [122]
35 mV
SLN+ SA + StA P188 + PC n/a 305 nm; 0.24; HepG2 PI 12 1.0–3.0 >80% [122]
25 mV
SLN+ SA + StA P188 + PC + Chol n/a 336 nm; 0.38; COS-1 PI 12 1.0–2.0 >80% [122]
35 mV
SLN+ SA + StA P188 + Chol n/a 272 nm; 0.24; COS-1 PI 12 1.0–3.0 >80% [122]
28 mV
SLN CP/myristyl myristate (MM)/cetyl P188 n/a 185–197 nm; HaCaT MTT 24 0.01–0.5 80–70% (CP); 100–80% (MM); 100– [8]
esters (CE) 0.15–0.23; 30.5 85% (CE)
to 37.5 mV
SLN+ SA + CPC/SA + GMS + CPC P407 DOX n.a.; n.a.; 22– B16F10 MTT 24 1.32 80% [125]
40 mV
SLN CP, DA Octyldecanol, soy PC, P188 Etoposide 152 nm; n.a. K562 MTT 48 0.00625 80% [135]
>30 mV
SLN CP, SP Octyldecanol, soy PC, P188 Etoposide 171 nm; n.a.; K562 MTT 48 0.00625 80% [135]
25 mV

(continued on next page)

9
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Table 2 (continued)

Type Lipid matrix Surfactant Active Size; PdI; ZP Cell line Assay Exposure Outcome (cell viability) Refs.
t (h) [] (mg/ml)a
SLN(?) Precirol 3 wt% T80 50 wt% 114 nm; 0.117; MPMac MTT 2 15 80% [135]
20.1 mV
SLN(?) HPO/TL/TP/docosanoic T80/T85/T20 n/a 61–103 nm, 0.39– BALB/c 3T3 MTT Over 80% viability [136]
0.47, 12.93 to
18.3 mV
SLN+ COM, Samine T80, Chol Retinoic acid 104–115 nm; n.a.; MCF-7, HCT- MTT 48 0.00625 80% [137]
n.a. 116 PBMC
NLC TP, OA T80 Simvastatin 149 nm; n.a.; HaCaT 72 0.3924 80% [138]
36 mV
SLN+ SA + StA + Chol P188 + PC + protamine n/a 336 nm; 0.38; COS-1 PI 12 0.1; 0.2; 80% (0.1 and 0.2 mg/ml), 70% at [52]

S. Doktorovova et al. / European Journal of Pharmaceutics and Biopharmaceutics 87 (2014) 1–18

35 mV 0.4 0.3 mg/ml
SLN SA T80 + P188 Emodin 29 nm; n.a.; MDA-MB- MTT 12, (up to >80% all times points and conc. [88]
31 mV 231 24, 30 lg
48 Emodin)
SLN SA T80 + P188 Emodin 29 nm; n.a.; MCF-7 MT 12, (up to >80% all times points and conc. [88]
31 mV 24, 30 lg
48 Emodin)
SLN+ GMS + CTAB/GM + LHNH PC n/a 50 nm; 0.1–0.23; A549 MTT 24, ? 80% CTAB-SLN; 90% LHNH SLN [36]
n.a. 48
SLN+ GMS + CTAB/GM + LHNH PC n/a 50 nm; 0.1–0.23; HeLa MTT 24, ? 80% CTAB-SLN; 90% LHNH SLN [36]
n.a. 48
SLN Witepsol (various) PC/T80 n/a 283–366 nm; n.a.; 3T3 MTT 18 Up to 0.15 >87.8% (all []) [7]
26 to 32 mV
SLN GMS + LHNH PC n/a 53 nm; n.a.; RAW 264.7 MTT 24 0.005–0.2 80–100% [33]
39.5 mV
SLN CP P407 n/a 200 nm; n.a.; n.a. Granulocytes MTT 12 25 80% [12]
SLN SA P188 Ibuprofen n.a.; 115; 0.32 mV Caco-2 MTT 24 0.0–15.0 >70% [54]
SLN+ SA + StA P188 + Chol n/a 272 nm; 0.24; Na 1300 PI 12 1.0–3.0 >70% [122]
28 mV
SLN+ SA + StA P188 + PC n/a 305 nm; 0.24; Na 1300 PI 12 1.0–3.0 >70% [122]
25 mV
SLN+ SA + StA P188 + PC n/a 305 nm; 0.24; COS-1 PI 12 1.0–2.0 >70% [122]
25 mV
SLN TM T80 CPT ? BCECa AB 24 (2.5 lM >70% [115]
CPT)
SLN CP, SA Octyldecanol, soy PC, P188 Etoposide 125 nm; n.a.; K562 MTT 48 0.006025 70% [135]
>40 mV
SLN CP, SP Octyldecanol, soy PC, P188 Etoposide 171 nm; n.a.; K562 MTT 48 0.0121 70% [135]
25 mV
SLN+ COM, Samine T80, Chol Retinoic acid 104–115 nm; n.a.; Jurkat MTT 48 0.007511 70% [137]
n.a.

Abbreviations (for this table): AB, alamar blue assay; BCEC, primary porcine brain capillary endothelial cells; BHR, Benzoic Acid; ButOH, butanol; CellT-Glo, Cell Titer Glo Assay (Luciferase& Luciferyl); CV-A, crystal violet assay;
E.wax, emulsifying wax; 2-Hydroxy-, 2-D-ribofuranosylhydrazide; MPMac, mouse peritoneal macrophages; MTT, MTT assay; NR, neutral red assay; PI, Propidium iodide exclusion assay; PPT, Podophyllotoxin; Tan IIA, Tanshinone
IIA; WST-8, WST-8 (Cell Counting Kit); [], concentration.
a
If not mentioned comes in mg/ml.
 S. Doktorovova et al. / European Journal of Pharmaceutics and Biopharmaceutics 87 (2014) 1–18 11

Table 3
Effect of blank SLN and NLC formulation (i.e. without any encapsulated active ingredient) on cell viability expressed as % of viability reported by the authors of the study. This
table summarises the reported results that may be interpreted as cytotoxic (cell viability <70%) of SLN/NLC. The column ‘‘drug’’ refers to the substance that the formulation was
intended to encapsulate.

Type Lipid matrix Surfactant Active Size; PdI; ZP Cell Assay Exposure Outcome (cell viability) Refs.
line
t Conc.
(h) (mg/ml)a
SLN+ CP 3 wt%, DDAB T80:Span85 = 7:3 Plasmid n.a.; n.a.; n.a. HeLa AB 24 0.1–10.0 100% at 0.1 mg/ml 0.1–15% [126]
viability, above 0.4 mg/ml
SLN E.wax, Brij78 5 mM PTC <100 nm; n.a.; n.a. HCT- MTS 8– 0.023 68.6 ± 13.9% (24 h) [64]
(2 mg/ml lipids) 15 72 64.6 ± 10.5% (72 h)
SLN Witepsol E85 T60 CPT ? BCECa AB 24 (2.5 lM 50% [115]
CPT)
SLN CP, DA Octyldecanol, soy Etoposide 152 nm; n.a.; K562 MTT 48 0.0121 65% [135]
PC, P188 >30 mV
SLN+ SA, stearylamine P188, Chol/PC/ n/a 272–305 nm; n.a.; COS-1 PI 12 0.6 65% [131]
Chol + PC 25–28 mV
SLN CP 5 wt% T60/T80 2 wt% n/a 130–165 nm; <0.3; U251, MTT 24 0.05–5.0 <60% [32]
18 to 23 mV U373
SLN CP, DA Octyldecanol, soy Etoposide 152 nm; n.a.; K562 MTT 48 0.0241 60% [135]
PC, P188 >30 mV
SLN SA 0.33 wt% Lipoid/T80 n/a 283–366 nm; n.a.; 3T3 MTT 18 0.03 (1%) 40–64% [7]
26 to 32 mV
SLN SA 0.33 wt% Lipoid/T80 n/a 283–366 nm; n.a.; HaCaT MTT 18 0.03 (1%) 43–74% [7]
26 to 32 mV
SLN CP, SP Octyldecanol, soy Etoposide 171 nm; n.a.; 25 mV K562 MTT 48 0.0241 45% [135]
PC, P188
SLN CP, SA Octyldecanol, soy Etoposide 125 nm; n.a.; K562 MTT 48 0.0121 40% [135]
PC, P188 >40 mV
SLN(?) Precirol 3 wt% T80 50 wt% n/a 114 nm; 0.117; MPMac MTT 2 37.5 40% [130]
20.1 mV
SLN SA 0.33 wt% Lipoid/T80 n/a 283–366 nm; n.a.; J774 MTT 18 0.03 (1%) 8–18% [7]
26 to 32 mV
SLN(+) SA 0.33 wt%, StA n/a 284–381 nm; n.a.; J774 MTT 18 0.03 (1%) 1.45–2.47% [7]
0.066–0.13 wt% 18.0 to 24.0 mV
SLN COM 10 wt% PC 3 wt%, Risperidone n.a.; n.a.; n.a. Caco-2 MTT 24 40.6– 40–60% [28]
tyloxapol 1 wt% 550.0 lg/
cm2

Abbreviations for this table: BCEC, primary porcine brain capillary endothelial cells; MPMac, mouse peritoneal macrophages.
a
Concentration is given in mg/ml if no other unit is mentioned.

Fig. 3. Cell viability after 24 h exposure (in % viable cells) vs. concentration of SLN/
NLC in cell culture media. Data were extracted from that reported in the references
listed in Tables 1–3. From reports that list 3 and more different SLN/NLC
concentration, the highest and lowest cell viabilities were used. ‘‘No decrease in
cell viability’’ was plotted as 100% viability, ‘‘no toxicity’’ was plotted as 85%
viability (as non-toxic could be between 70% and 100% viability). IC50 data are
plotted as 50% viability.
In a series of SLN composed of same lipid but different Polox-
Fig. 4. The effect of normal vs. cancer cell line on the outcome of cell viability
studies after exposure to SLN. Data were extracted from references that used
normal (non-cancer) cell lines (open squares) and/or cancer cell lines (closed
circles). Cell viability (in % viable cells) vs. concentration of SLN/NLC in cell culture
media reported in references listed in Tables 1–3 were used. From reports that list 3
and more different SLN/NLC concentration, the highest and lowest cell viabilities
were used. ‘‘No decrease in cell viability’’ was plotted as 100% viability, ‘‘no toxicity’’
was plotted as 85% viability (as non-toxic could be between 70% and 100% viability).
IC50 data are plotted as 50% viability.

amer (or Tween 80), slight differences were found among the for-
mulations, but none of them was toxic [13]. The surfactants used,
although non-toxic themselves (as illustrated by exposure to the
same cell line in the same report), could even improve their toler-
ability when bound to SLN [30]. Binding of surfactant to SLN/NLC
surface decreases its availability to interact with cells and thus de-
creases its toxicity [13]. Schubert and Muller-Goymann however
observed a contrary tendency in triglyceride-composed SLN, but
in a smaller series of formulations [39].
The effect of surfactant influence is more pronounced with cat-
ionic SLN/NLC, where the cationic surfactants/lipids alone show
some toxicity towards living cells on the membrane level. Tabatt
and collaborators [58] reported low viability of COS-1 cells after
treatment SLN/NLC with positive surface charge; this was however
still higher than when the same cells were treated with aqueous
solution of the same cationic surfactants. Single alkyl chain con-
12 S. Doktorovova et al. / European Journal of Pharmaceutics and Biopharmaceutics 87 (2014) 1–18
taining surfactants were found to be more toxic than double-
chained surfactants. Erni and collaborators reported that the viabil-
ity after solid lipid microparticles (SLM) treatment was similar to
the treatments with aqueous cationic surfactant solutions [59],
and, furthermore, noted that single-chain cationic surfactants
coated particles had slightly smaller impact on the cells. These re-
sults might seem contradictory, but as different methods were
used to obtain cell viability (Tabbat used LDH release which di-
rectly correlates to cell membrane damage, while Erni used ethi-
dium bromide uptake assay) they are not to be automatically
correlated. Although the paper of Erni and collaborators is entitled
‘‘solid lipid microparticles’’, the composition of the nanoparticles is
relevant for SLN formulation and the resulting size (1.89–2.37 lm
for cationic SLM) was not much higher than the SLN size range,
therefore information from this study is still relevant also for eval-
uation of SLN/NLC with size <1 lm.
4.5. SLN vs. NLC
Nanostructured lipid carriers contain, compared to SLN, addi-
tional oil component. Theoretically, the oil should be firmly incor-
porated in the nanoparticle core and increase the stability of NLC
by limiting the polymorphic phase changes during the storage
[2]. However, Jores and colleagues [60] found out that the oil
may be attached to the solid lipid core surface. Although this
‘‘spoon-like’’ appearance was also observed by Schwarz and col-
leagues [61], it is generally overseen in the literature. The implica-
tions for tolerability in vitro include the availability for cell
interaction of another formulation component on the particle sur-
face. The other question, yet without answer, is whether the oil,
adsorbed on the lipid core surface, stays on the surface in environ-
ment with higher ionic strength or if it suffers desorption as surfac-
tants may suffer [62].
From the collected information, there is no apparent difference
between the toxicity of SLN and NLC. In Tables 1–3, NLC formula-
tions can be found all around the tables, organised according to the
toxicity. Only a few direct comparisons in SLN to NLC exist: 100%
viability (in NHF cells) up to 0.1 mg/ml and 0.9 mg/ml for SLN vs.
NLC, respectively [46], and IC50 of 0.0264 mg/ml for SLN vs. IC50
of 0.0151 mg/ml for NLC in MDA-MB-231 cell line [63]. As these
only two available comparisons do not have concordant conclu-
sions, it is not possible to do a conclusion in favour of one of the
systems. NLC have improved characteristics in terms of drug load-
ing and stability compared to SLN [2]; therefore, more data are to
be expected in near future.
4.6. SLN formulations for delivery of chemotherapeutic drugs
Almost all available reports about SLN/NLC loaded with chemo-
therapeutic drugs state that despite blank formulations were not
toxic to the cell line in use, upon loading with the drug the toxicity
exceeded the toxicity of drug solution alone. There are examples
for paclitaxel [20,49,64–66], docetaxel [55,67,68], doxorubicin
[49,69,70], mitoxantrone [71] and others. Surface modified or
targeted formulations showed even greater toxicity than non-
targeted formulation [21,72]. Increased susceptibility to chemo-
therapeutic drugs administered in SLN/NLC was reported also in
resistant cell lines [64,66]. Blank NLC were shown to be non-toxic
to the cell line in question (MCF-7 vs. MCF-7/MX resistant to mito-
xanthrone). Viability below 70% was reported for paclitaxel resis-
tant HCT-15 cell line [64]. A study by Baek and Cho suggests that
encapsulation of paclitaxel in SLN caused similar improvement in
drug effect as pre-treatment with a p-glycoprotein inhibitor
(verapamil), and that combination of verapamil pre-treatment
and encapsulation could further improve the outcome [73]. The
efficiency of SLN and NLC for delivery of chemotherapeutic drugs
was reviewed in detail elsewhere [74].
There might be various reasons for these observations: the che-
motherapeutic drugs, mostly highly lipophilic, are better soluble in
lipid phase than in aqueous phase and therefore reach the cells in
higher concentrations than from an aqueous solution, also the drug
can be protected by possible degradation by encapsulation [75].
Furthermore, cellular uptake might be improved by encapsulation
into lipid nanoparticles of various types, not limited to SLN/NLC
[57,76–78].
On the contrary to the majority of published results, Ying and
collaborators [37] reported that the IC50 of DOX-SLN was higher
than that of DOX solution (after 48 h exposure). The authors justi-
fied this finding by incomplete release of the drug from SLN. This is
an important remark, once one reasons of encapsulation of drugs
into SLN/NLC is to achieve controlled release. Yet drug release is
mostly tested in different conditions than those found in cell cul-
ture (appropriate buffer solution or solvent mixture). It would be
interesting to see how well the in vitro release data correlate to ob-
served drug effects in cell culture.
The report by Carneiro and collaborators [79] shows that Treti-
noin (here tested for chemotherapeutic effect) loaded SLN had very
similar effect on cell viability of Jurkat, HCT-116, MCF-7 and nor-
mal peripheral blood monocytes compared to blank SLN.
Also Wang and collaborators [80] tested a novel compound
(2-hydroxy-, 2-D-ribofuranosylhydrazide, BHR) with possible anti-
cancer effect in both normal and cancer-derived cell lines. In 293T
cells, encapsulation into SLN could improve cell survival after
treatment, compared to BHR solution. On the contrary, HeLa cells
were more susceptible to BHR-loaded SLN treatment than BHR
solution. Treatment with blank SLN did not influence cell viability
in neither of cell lines [80].
4.7. SLN formulations for delivery of non-chemotherapeutic drugs
Evaluation of cytotoxicity of SLN and NLC formulations of other
then cytotoxic drug is still not common, despite a large number of
existing SLN/NLC formulations. Again, there are also studies that
state the drug-loaded formulation is not toxic, but do not disclose
the data of blank formulation – although in this case it is logical
that a non-toxic drug-loaded formulation is not toxic without the
drug, too. As the drugs approved for human use should be non-
toxic per se, there are even few reports on SLN formulation only,
without testing the drug-loaded formulation – those are the fol-
lowing [53,81,82].
Considering the same fact, some of the reports of non-chemo-
therapeutic drugs which are non-toxic in vitro, are raising some
concern. Alpha-lipoic acid, on its own non-toxic to NHF cells over
the concentration range used (0.01–10.0 lM), caused decrease in
cell viability by 30% when loaded into NLC and by 20% when loaded
into Apifil-composed SLN. The blank formulations were both very
well tolerated (no decrease in cell viability up to 0.1 mg/ml for
SLN and 0.9 mg/ml for NLC) [46]. Similar effect was observed in
the same cell line by the same research group when examining a
gamma-oryzanol SLN formulation. The drug, by itself non-toxic,
and non-toxic when encapsulated in CP-composed SLN, while
when encapsulated into TM-composed SLN, the cell viability
dropped to 60% [47]. A time-dependent and dose dependent toxic-
ity was observed for beta-carotene encapsulated in otherwise very
well tolerated (80% cell viability at 13.5 mg/ml) formulation in
RAT-1 cells [48].
On the other hand, Souza and collaborators [83] reported im-
proved cell viability of HepG2 cells treated with praziquantel-
loaded SLN compared to those treated with praziquantel solution
(in ethanol). The authors raised questions of the observed toxicity
to be caused by the solvent used for preparation of drug solution,
 S. Doktorovova et al. / European Journal of Pharmaceutics and Biopharmaceutics 87 (2014) 1–18
despite it being applied in minimal amounts [75,83]. Similar obser-
vation was reported by Teskac and Kristl with resveratrol-loaded
SLN and tripterin-loaded NLC [84]. Resveratrol-loaded SLN in this
case caused even higher rate of keratinocytes cell growth than that
observed in untreated control and drug-solution treated control
[85].
Li and colleagues [86] observed higher cytotoxicity of their buf-
adienolides-loaded NLC after first 48 h of treatment than free
drugs; at 72 h and 96 h more damage to HGC-27 cells was caused
by free drug than NLC. The authors observed that this behaviour
correlated with the release pattern of the drug, showing a burst re-
lease during the first 8 h and then showed sustained release at
steady rate, releasing 85% of encapsulated drug [86].
Other authors reported absence of toxicity of neither blank nor
drug loaded SLN [53,54,87–91]. Wolf and collaborators confirmed
excellent tolerability of SLN in keratinocytes, skin fibroblasts and
in a reconstructed human epidermis model (Episkin), showing that
the system is harmless to skin cells and can be used for therapy of
skin diseases and wounds [92].
These contradictory results imply the importance of toxicity
testing for each particular formulation. While the effect of in-
creased toxicity upon encapsulation in SLN/NLC can again be ex-
plained by the fact that the drugs are better solubilised and thus
available in higher concentration in the system, on the other hand,
the effect of reduced cytotoxicity may be explained by incomplete
release of the drug from the particles.
4.8. Comparison to other types of colloidal carriers
As evidenced already, in the very first years of development of
SLN and later NLC, the biocompatibility of these systems is remark-
ably better as that of comparable colloidal systems prepared from
polymers. First of all, the lipid material used is biodegradable and
thus does not accumulate in the tissue/organism. Secondly, the
typical average size of SLN/NLC between 100 nm and 1 lm, by
some not even considered as ‘‘nanoscale’’, was until recently con-
sidered too large for cells to internalise [93]. Recent insights how-
ever prove that even non-phagocytic cells are able to effectively
internalise objects as large as 2–3 lm [94].
Direct comparison of SLN to other colloidal drug (or gene) deliv-
ery systems in terms of safety favours lipid nanoparticles. While
0.35 mg/ml of polycyanoacrylate nanoparticles was a dose 100%
lethal for HL-60 macrophages, 1.5 mg/ml SLN could be well toler-
ated (90% cell viability). The lethal dose of PMCA-NP towards HL-
60 in the same study was even smaller – 0.05 mg/ml [13]. IC50 of
poly-L-lysine (22 kDa) and poly-ethyleneimine (PEI, 25 kDa) in
COS-1 cells was 10 lg/ml and 20 lg/ml, while IC50 of cationic
SLN was 0.6–0.7 mg/ml, respectively, depending on the solid lipid
used [95]. PEI was also compared to SLN in MDA-MD-435 cells,
the outcome being IC50 of 0.009 mg/ml for PEI and no toxicity
caused by SLN at 0.048 mg/ml [96].
5. Uptake by cells
Evaluation of all of the effects on living cells in culture has the
pre-requisite of uptake of the particles by the cells, or interaction of
SLN/NLC components with the cells. As documented by many re-
ports, SLN and NLC are promptly internalised by a wide varieties
of cell lines, including cancer cell lines [32,57,76], non-cancer cell
lines [32,85], and both phagocytic and non-phagocytic cells.
Attempts to clarify the uptake mechanism have been published
recently. Coumarin-6 marked SLN could be well internalised into
A30 cells at 37 °C. The uptake was decreased but not completely
stopped at 4 °C incubation, which suggests that energy-dependent
13
processes (endocytosis) might be involved in SLN uptake [29]. By
pre-incubation with specific uptake mechanism inhibitors, two
independent research group could specify that clathrin-mediated
endocytosis might be the preferential uptake mechanism, while
caveolae-mediated endocytosis seems to play only minor role or
not involved at all [32,71]. Martins and collaborators also found
out that macropinocytosis may be involved to smaller extent in
some cell types.
6. Membrane integrity
LDH release assay was used in several reports on SLN/NLC
safety. Many of the authors used it as a measure of cell viability,
most of them alongside with a formazan-dye based assay. These
authors concluded that LDH assay was less sensitive to potential
effects on cell than WST-1 [95] or MTT assay [10,29], from which
result higher IC50 values.
Alukda and colleagues reported no increase in LDH release com-
pared to control in the range of 0.4–6.0 mg/ml SLN in VK2/E6E7
cells after 48 h exposure, while MTS assay revealed a decrease in
cell viability (80% viable cells) [53]. Even greater differences where
quite surprisingly reported by Olbrich and collaborators of cationic
SLN: while IC50 for Esterqaut-containing SLN was 0.6–0.7 mg/ml by
WST-1, LDH assay indicated 3.0–5.0 mg/ml as IC50 [95].
This difference in sensitivity can also be interpreted as higher
susceptibility of mitochondrial enzymes (responsible for formazan
dye metabolism) to SLN/NLC presence than the cellular membrane,
or by decrease in mitochondrial metabolism and other intracellular
pathways occurrence prior cell membrane rupture.
Montenegro and colleagues [35] used LDH-assay to examine the
ability of idebenone loaded SLN to protect the cells (primary astro-
cytes) from oxidative damage induced by 2,20 -azobis-(2-amidino-
propane) dihydrochloride (AAPH). By increasing the dose, the
LDH release values could return to values similar to AAPH
untreated group [35].
7. ROS generation
The presence of nanoparticles and their interaction with biolog-
ical membranes can lead to such disturbances in cell metabolism
that lead to increased reactive oxygen species (ROS) production.
Many reports on increased oxidative stress in cells in culture after
exposure to nanomaterials exist (for example, see [44,97]). In case
of SLN/NLC, this issue has not been addressed yet in a manner
allowing for a general conclusion. As increased ROS may not neces-
sarily lead to cell death, it may occur also in the cells and can be
induced by formulations that have been successfully tested by cell
viability methods.
From the few existing report addressing the oxidative stress is-
sue, most of them used the DCFH-DA method. DCFH-DA (20 ,70 -
Dichlorofluorescein diacetate) is a highly permeable substance that
enters cells easily. In the presence of oxidants, it is metabolised
into fluorescent product DCFH. However, DCFH-DA can react with
a variety of radicals present in the cells, and it is prone to interfer-
ence [98]. Other methods for detection of free radicals and their ef-
fects were reviewed elsewhere [99]. Mulik and collaborators
reported that the increase in ROS content after SLN exposure was
only negligible compared to untreated MCF-7 cells [21]. Picone
and colleagues also reported ROS generation levels near to control
[100]. In addition, ferulic acid loaded SLN could protect the COS-1
cells better than ferulic acid solution. This effect was found dose-
dependent [101]. Idebenone-loaded SLN also showed strong pro-
tective effect against AAPH induced ROS generation [35]. Gökce
and colleagues reported that ROS level in skin fibroblasts was
14 S. Doktorovova et al. / European Journal of Pharmaceutics and Biopharmaceutics 87 (2014) 1–18
lower after treatment with coenzyme Q10-loaded SLN than after
treatment with Q10 alone [102].
In contradiction to other reports [21,100], Silva and collabora-
tors [15] reported that ROS generation associated to SLN exposure
may occur. The cells, when pre-incubated with N-acetylcysteine
(an antioxidant) and/or catalase had significantly higher viability
after SLN exposure [15], meaning that these antioxidants could
protect the cells from SLN induced loss of viability.
Very interesting in vivo results were reported by Kakkar and
Kaur [103] in a murine model of Alzheimer disease. After AlCl3
damage induction of damage, the levels of oxidised lipids were in-
creased, reduced glutathione (GSH) pool decreased and activity of
superoxide dismutase and catalase (which are the enzymes in-
volved in cellular protection against ROS) increased. Treatment
with curcumin-loaded SLN could reverse these changes. Interest-
ingly, a reverse to values close to normal was also reported after
administration of blank SLN only [103].
8. Genotoxicity
One of the sub-lethal effects of nanoparticles, addressed
throughout in the research of inorganic nanoparticles is the dam-
age to DNA. DNA damage can include oxidation of the bases, strand
breaks or disturbances in DNA repair. A fixation of an error in DNA
sequence is the pre-requisite for mutation development. Our group
tested a small set of cationic SLN in two cell lines by comet assay
(for method details see [104,105]. Although we found a small insig-
nificant increase in comet tail intensity (which represent the DNA
strand breaks or the sites with defect or missing bases converted
into strand breaks), the overall result allowed to conclude that
these formulations were not genotoxic.
The material used for SLN/NLC production gives the pre-requi-
site of a well tolerated carrier also in the perspective of genotoxi-
city, however it would be interesting to confirm the absence of
genotoxicity by more studies. As DNA damage is often linked to in-
creased oxidative damage [106], and SLN may have some effect on
ROS generation (increase, as reported by [15]), and many reports
showed SLN reaching perinuclear region of the cells, the genotox-
icity studies might have interesting outcomes.
9. Phagocytic cells, inflammation response
SLN uptake by macrophages which is not desired as it leads to
rapid elimination from the blood circulation was tested in three re-
ports. SLN stabilised by Poloxamer 188 (polyethylene–polypropyl-
ene glycol) or Myrj (polyethylene glycol monostearate) are
expected to have ‘‘stealth’’ characteristics and avoid phagocytosis
for a prolonged time. In in vitro and in vivo studies, murine macro-
phages could internalise such SLN to a smaller extent; the peak
absorption occurred at 120 min in both studies [51,107]. Wang
and collaborators reported that increasing Myrj concentration in
formulation and/or polyoxyethylene chain length of Myrj could re-
duce the uptake [107]. Zhang and colleagues [51] found out that
phagocytosis of SLN stabilised with Poloxamer 188 occurred to
the same extent independently of the presence of serum in the cul-
ture medium, while non-stealth SLN were internalised to higher
extend in serum presence in the culture medium. Phagocytic up-
take of SLN by normal human granulocytes measured by luminal
assay could be highly decreased compared to luminal control. Fur-
thermore, Poloxamine 908 stabilised-SLN was shown more resis-
tant against the uptake than Poloxamer 407 covered SLN [12].
This is interesting information and raises a question, why polox-
amine 908 is only rarely used in SLN formulations intended for
intravenous administration?
10. Blood compatibility (red blood cells)
Important information about safety of formulations intended
for intravenous administration is the compatibility with red blood
cells (RBCs). There are some reports on RBC compatibility testing,
and the results are all very encouraging. Studies by Schubert and
Muller-Goymann [39] and Li and collaborators [86] reported a very
low % of released oxy-haemoglobin – RBCs exposed SLN composed
of Softisan 100 and varying content of phospholipids up to 50% of
lipid matrix, stabilised by Solutol HS15 presented only (1.8 ± 1.6)%
release, which corresponded to about 0.4% of positive control (dis-
tilled water) [39]. Treatment with NLC composed of GMS, OA and
Miglyol 812, stabilised with a combination of P188, phosphatidyl-
choline (PC) and even SDS, lead to less than 5% haemolysis at
1.2 mg/ml SLN, depending on the final SLN amount [86].
Koziara and collaborators showed that, similarly to cell viability
in vitro, the effect of surfactant alone is reduced when used in SLN.
Time dependent and concentration effect was observed after treat-
ment with Brij 78 in solution in the same amount as used in SLN.
While haemolysis ratio increased with time and concentration up
to 80% (6 h, highest concentration), haemolysis after 1 mg/ml SLN
treatment was still below 10% [108]. Further reports exist on actu-
ally lower haemolysis of drug-loaded SLN/NLC than free drugs –
DOX caused higher extent of haemolysis than DOX-SLN, even smal-
ler impact was observed after mannosylated DOX-SLN treatment
[109]. Same tendency was reported for SLN loaded with metoth-
rexate [110]. It is interesting that both SLN formulations in the last
two mentioned reposts had positive surface charge, and their sur-
faced variant (mannosylated, albumin-covered) was also success-
fully tested.
Taken together, there seems to be no reason for concerns about
negative effects on RBSc.
11. Safety in vivo?
Despite large number of published in vivo studies, the large
majority focuses on SLN efficiency rather than their safety. Among
the few in vivo toxicological studies, there are even fewer that al-
low for comparison of the dose used in in vivo experiments with
the observed toxicity in vitro in cell culture. Therefore, from the
available data, it is not possible to establish a link between a ‘‘safe’’
SLN/NLC concentration for in vitro and in vivo.
Tolerability of SLN by airways and lung tissue were examined in
mice and rats. Nassimi and collaborators [9] tested tolerability
both in vitro and in vivo. In vitro assays revealed IC50 in A549 cells
of 2 mg/ml (by NR uptake assay) or 3 mg/ml (by MTT assay). Doses
between 1 and 200 lg, of lung-deposited solids, were tested in fe-
male BALB/c mice. SLN did not trigger inflammation; neither LDH
activity in bronchoalveolar lavage was increased. Microscopic
appearance of the lung parenchyma was found normal; although
some minor aggregates of lipid-filled macrophages were found [9].
Although cytotoxicity of blank NLC was not tested in cell lines
(Bufadienolides-loaded NLC were cytotoxic to HGC-27 and U87-
MG cells, presenting similar IC50 values as Bufadienolides solution
in DMSO), a LD50 in mice was 1.15 higher than LD50 of drug solu-
tion (2.31 mg/kg and 2.95 mg/kg for drug solution and NLC, respec-
tively). Strong presentation of toxic effects in mice administered
with drug solution was observed at 1.32 mg/kg drug, while no
marked toxic effects were observed after receiving bufadieno-
lides-NLC [86]. Fangueiro and colleagues [112] tested the effect
of a SLN formulation on Drosophila melanogaster survival and
reproduction. Although small reduction in number of progeny flies
was detected, this was similar to the effect of bulk lipid (Softisan
145) admixed in the standard Drosphila medium.
 S. Doktorovova et al. / European Journal of Pharmaceutics and Biopharmaceutics 87 (2014) 1–18
A good correlation between in vitro and in vivo results was re-
ported by Olbrich and collaborators [113] with SLN used as vaccine
adjuvants in chicken. A cationic SLN formulation (solid paraf-
fin + esterquat1 (EQ), stabilised by T80 + Span85 mixture) which
was toxic towards RAW 246.7 macrophages in vitro (1 mg/ml), also
caused significant damage in tissue at injection site (dose 7.2 mg/
ml). This damage was still lower when compared to standard
vaccine adjuvant. This formulation caused decrease in viability in
human foreskin fibroblast, but by no more than 20%. On the con-
trary, a negatively charged formulation without EQ, non-toxic
in vitro, was also well tolerated in chicken [113].
12. Conclusions and further perspectives
As shown in this review, great amount of work has been done
and there is large amount of in vitro data available. The basic con-
cept of SLN and NLC as a well tolerated carrier seems to be con-
firmed and well documented. When it comes to more detailed
questions, the currently available data still do not offer straightfor-
ward answers. Some researchers questioned if cancer cell lines are
more susceptible to SLN/NLC than ‘‘normal’’ cell lines, but with cur-
rently available data one should be very careful to draw a conclu-
sion like this. ‘‘Normal’’ cell lines, despite being derived from
healthy tissue, still represent a set of dividing cells that does not
normally occur with mature differentiated cells. To the best of
our knowledge, no study on ‘‘normal’’ cells exists up to date and
it is a questionable if it would to be feasible with nanocarriers that
are not expected to cause any damage. Different situation is with
leukocytes, where normal macrophages and granulocytes from hu-
man healthy volunteers appear much more susceptible to SLN/NLC
than leukaemia-derived HL-60 or virus containing RAW 264.7
macrophages [12,13].
We divided the SLN/NLC in vitro data also by the potential active
substance they might carry by purpose. Except from one report, all
studies on chemotherapeutic drug loaded SLN/NLC report an in-
crease in toxicity towards the target cancer cells. However, the
other group of formulations for non-chemotherapeutic drugs refers
to both increase and decrease in toxicity.
Taken altogether, prediction of action of a drug encapsulated in
SLN/NLC can still be tricky. We think that the contradictory results
are also due to some bias of the studies of chemotherapeutic drugs.
Furthermore, all of these data are only a small hint on how SLN/
NLC will behave in vivo. We still have almost no information on
side effects at site of administration (except from dermal products
which were already tested on human volunteers), and the systemic
toxicity on the organism as whole. More important questions
would arise when considering per oral route of administration, as
– although biodegradable – an increased uptake of lipids will have
an effect on the organism in question.
To summarise all, a large amount of in vitro data about SLN and
NLC is already available, but the overall question if SLN/NLC are
really so well tolerated at any circumstances is still not answered.
We would like to draw the attention to the fact that there are many
contradictory results, and also many results which might suffer for
some influence of the actual purpose of the SLN/NLC formulation.
Animal studies with SLN/NLC are reaching almost the same num-
ber as in vitro studies reviewed here. Once the authors also focus
on screening for possible side effects or their absence apart from
efficiency, bio-distribution and pharmacokinetics, only then it
would be able to confirm that SLN and NLC are safe nanocarriers.
Acknowledgments
Financial support from Portuguese Science and Technology
Foundation (FCT) under reference SFRH/BD/60552/2009 (Doctoral
15
grant to SD) and PEst-OE/EQB/LA0023/2011 is acknowledged.
AMS was supported by European Union Funds (FEDER/COMPETE)
and by national funds (FCT) under the project FCOMP-01-0124-
FEDER-022696 and by a research project grant (PEst-C/AGR/
UI4033/2011). The authors also wish to acknowledge FCT, under
the reference PTDC/SAU-FAR/113100/2009.
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