Solid Lipid Nanoparticles For Targeted Brain Drug Delivery

Advanced Drug Delivery Reviews 59 (2007) 454 477
www.elsevier.com/locate/addr
Solid lipid nanoparticles for targeted brain drug delivery

Paolo Blasi , Stefano Giovagnoli, Aurlie Schoubben, Maurizio Ricci, Carlo Rossi
Department of Chemistry and Technology of Drugs, School of Pharmacy, University of Perugia, Via del Liceo 1, 06123 Perugia, Italy
Received 21 November 2006; accepted 24 April 2007
Available online 22 May 2007
Abstract
The present review discusses the potential use of solid lipid nanoparticles for brain drug targeting purposes. The state of the art on surfactantcoated poly(alkylcyanoacrylate) nanoparticles specifically designed for brain targeting is given by emphasizing the transfer of this technology to
solid lipid matrices. The available literature on solid lipid nanoparticles and related carriers for brain drug targeting is revised as well. The potential
advantages of the use of solid lipid nanoparticles over polymeric nanoparticles are accounted on the bases of a lower cytotoxicity, higher drug
loading capacity, and best production scalability. Solid lipid nanoparticles physicochemical characteristics are also particularly regarded in order to
address the critical issues related to the development of suitable brain targeting formulations. A critical consideration on the potential application
of such technology as related to the current status of brain drug development is also given.
2007 Elsevier B.V. All rights reserved.
Keywords: CNS drug delivery; Brain targeting; Blood-brain barrier (BBB); Solid lipid nanoparticles (SLN); NLC; LDC; Polymeric nanoparticles; Polysorbate 80
(Tween 80)
Contents
1.
2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Blood-brain barrier biology . . . . . . . . . . . . . . . . . . . .
Brain drug delivery using surfactant coated polymeric nanoparticles
Advantages of SLN over PACA NPs . . . . . . . . . . . . . . .
4.1. Preparation and sterilization . . . . . . . . . . . . . . . .
4.2. Toxicity, stability and biocompatibility issues . . . . . . .
5. SLN physicochemical features with respect to brain drug delivery
5.1. SLN structure, drug loading, and stability issues. . . . . . .
5.2. Surfactants and brain targeting features . . . . . . . . . . .
6. SLN for brain drug targeting . . . . . . . . . . . . . . . . . . . .
7. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . .
8. Legend for lipid substances . . . . . . . . . . . . . . . . . . . .
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
This review is part of the Advanced Drug Delivery Reviews theme issue on
Lipid Nanoparticles: Recent Advances.
Corresponding author. Tel.: +39 075 5855133; fax: +39 075 5855163.
E-mail address: kaolino@unipg.it (P. Blasi).
0169-409X/$ - see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2007.04.011
In the last decade, an emerging interest has been growing

towards brain drug targeting where issues have been widely
discussed [18]. The increasing awareness of the lack of rational
P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477
and common efforts among different and complementary research areas has pointed out the need for a deeper understanding
and a closer collaboration among diverse research experts [7,9]
of the field. These issues along with poor knowledge regarding
the physiology of the central nervous system (CNS) have been
the main limiting factors in the development of effective drugs
and appropriate drug delivery systems (DDS) for brain targeting
[1,2,6,7]. In fact, delivering drugs to the CNS is impaired by the
presence of the blood-brain barrier (BBB) that represents the
main obstacle for CNS drug development [2,10].
A worrying updated picture of the worldwide CNS pathology
incidence displayed that, in Europe alone, about 35% of the total
burden of all diseases is caused by brain diseases [1] and about
1.5 billion people worldwide suffer from CNS disorders. Moreover, due to an average lifespan increase to almost 70 years of age
at which about 50% of the population starts revealing evidence of
Alzheimer's disease (AD) [5], an increase of CNS disorder global
burden from the current 11% to 14%, is expected by 2020 [11].
For genetic, age and contingent reasons, most of the degenerative
CNS pathologies, such as Creutzfeldt-Jakob's, Parkinson's (PD),
Alzheimer's, Huntington's disease, multiple sclerosis and
amyotrophic lateral sclerosis, are developing as a consequence
of wrong lifestyle and increased risk factors, such as alcohol,
drugs or dietary abuse as well as stress [1214]. In addition, CNS
involvement may occur because of primary effects of human
immunodeficiency virus (HIV) infection or secondary effects of
immune suppression. About 75% of patients with acquired
immune deficiency syndrome eventually develop some features
of subacute encephalitis and other secondary effects, such as
primary CNS lymphoma and progressive multifocal leukoencephalopathy [12,1520]. This emergency is becoming particularly
serious in developing countries, as a result of a wide and
uncontrolled spread of HIV infection.
In spite of this worrying picture, in the last number of years
the CNS drug market has become the largest of all therapeutic
areas, recording worldwide an amazing + 13% growth in 2003,
reaching 77.2 billion USD [21]. This is also due to the advances
reported in the treatment and understanding of some serious
CNS pathologies [2227]. In this context, the pharmaceutical
technology contribution to this area has been found of paramount importance [5,6,2830]. Several DDS and strategies
have been developed for the sole purpose of an effective drug
deposition to the CNS [3136], while other carriers, fabricated
for different aims, have been turned to a possible brain targeting
application [27,37]. Among others, nanoparticles (NPs) have
been considered as carriers of election to overcome the BBB
issue [36,38,39].
Several systems have been developed and among these, poly
(ethylene glycol) (PEG) stabilized poly(lactide) (PLA) [40] and
chitosan [41] NPs, conjugated with the peptidomimetic
monoclonal antibody OX26, gave good results. NPs obtained
by conjugating poly(lactide-co-glycolide) PLGA to five short
synthetic peptides, to mimic the synthetic opioid peptide MMP2200 [42], and a LectinPEGPLA conjugated system [43]
showed to be equally effective in brain targeting. Moreover, a
significant brain uptake was achieved by using polysorbate 80
(P80) as a coating system [4446].
455
Among other NP formulations, solid lipid nanoparticles

(SLN) have recently found reappraisal as potential DDS for
brain targeting [36,38,44,4749]. The present review will report
the state of the art on surfactant-coated NPs designed for brain
targeting, and the transfer of this technology to SLN and related
carriers will be emphasized on the basis of their cytotoxicity,
drug loading capacity, and production scalability. Besides, SLN
physicochemical characteristics will be discussed in order to
address the issues related to the development of suitable brain
targeting formulations.
2. Blood-brain barrier biology
The brain is one of the most important organs of the human
body, if not the most, and its homeostasis is of primary
importance. In fact, specific interfaces, also referred to as
barriers, tightly regulate the exchange between the peripheral
blood circulation and the cerebrospinal fluid (CSF) circulatory
system. These barriers are represented by the choroid plexus
(CP) epithelium (blood-ventricular CSF), the arachnoid epithelium (blood-subarachnoid CSF), and the BBB (blood-brain
interstitial fluid) [50]. The concentration and clearance of
endogenous and exogenous molecules, essential for the normal
brain functions or dangerous because of their toxicity, are strictly
regulated by the anatomic and physiologic features of such
barriers [51,52].
The presence of the BBB is certainly the most critical issue
encountered in brain drug delivery. Indeed, among the possible
strategies to deliver therapeutic molecules into the brain, namely
intracerebral, intraventricular, and intravascular delivery, the
latest represents the most reliable one because of its potential
efficacy, safety, and compliance. The brain microvasculature, if
not for the BBB, would represent an extraordinary way to access
the brain, with the possibility of virtually distributing therapeutic
molecules to all brain areas [7]. In fact, even though the volume
occupied by capillaries and endothelial cells is about 1% and
0.1% of the brain volume respectively, the brain microvasculature develops a total surface area of 20 m2 and a total length
of 400 miles [6]. This high vascularization confers to the brain
a very intricate network of capillaries with an average distance
between capillaries of 40 m. This means that every single
brain cell is placed virtually at no more than 20 m from a
capillary, thus creating a space that can be permeated (simple
diffusion) by small molecules in a half a second [6]. Unfortunately, the physiological features of the brain microvasculature
restrict enormously the number of drugs that can enter the brain
upon systemic administration. It becomes hence easier to
understand the difficulties in developing new CNS drugs by
considering that more than 98% of the new potential
neurotherapeutic molecules are unable to cross the BBB [1].
Generally, but not always, a molecule has to possess peculiar
physicochemical characteristics to overcome the BBB, such as
high lipophilicity and a molecular weight b 500 Da [6]. This rule
is invalidated by the presence of the transport systems (generally
divided in 3 classes: carrier-mediated transport, active efflux
transport, and receptor-mediated transport) to different regions
of the BBB, conferring to this barrier dynamic permeability
456
characteristics, barely predictable only by considering its

lipophilicity and molecular weight [53,54].
Brain capillaries, differently from the peripheral capillaries,
present no fenestrae, a low amount of pinocytosis vesicles and
particular tight junctions (TJs) also known as zonula occludens
[5557]. TJs are structures that form a narrow and continuous
seal surrounding each endothelial and epithelial cell at the apical
border (Fig. 1) and are aimed at strictly regulating the movement
of molecules through the paracellular pathway [58]. The BBB
endothelial TJs show some differences in morphology, composition, and complexity features from those of both the epithelia
and peripheral endothelia [59]. These structures, together with
the brain endothelial cells, make an almost impermeable barrier
for drugs administered through the peripheral circulation.
A further contribution to the peculiar BBB functions is given
by the periendothelial accessory structures represented by
astrocytes [51,60], pericytes [61,62], and the basal membrane.
(Fig. 1) Astrocytes, generally classified into fibrous and
protoplasmic, represent the major component (90%) of the
brain mass. Fibrous astrocytes have a star-like morphology and
often present many long processes, known as end-feet, that end
on the basal membrane of the BBB (Fig. 1). These cells have a
multitude of functions important for the brain homeostasis
(maintenance of K+ levels, inactivation of neurotransmitters,
regulation and production of growth factors and cytokines), many
of which are related to the production of apolipoprotein E (ApoE)
[60]. Pericytes are spherical cells holding a prominent nucleus and
many primary and secondary lysosomes in the cytoplasm and
seem to be virtually involved in the BBB formation, differentiation processes, and integrity [62].
As previously mentioned, the presence of BBB transport
systems further complicates this scenario. In fact, from a drug
delivery point of view, these transporters may assist or hinder the
delivery of drugs to the brain. The carrier-mediated transport
(e.g. transporter proteins for glucose, essential amino acids,
monocarboxylic acids, and nucleosides) may be able to shuttle
drugs or pro-drugs into the brain in therapeutic concentrations,
mimicking nutrients or endogenous compounds [6365]. The
use of the receptor-mediated transport to gain access to the brain
is another very attractive strategy. Receptors for endogenous
large molecules (e.g. insulin, transferrin, leptin, Apo, and many
others) highly expressed on the BBB luminal side, are
responsible for their shuttling to the brain by transcytosis. The
binding of drugs or drug containers (e.g. liposomes and NPs) to
specific ligands (peptides) or peptidomimetic monoclonal
antibodies makes it possible to ship the construct into the brain
by mimicking an endogenous molecule [6,9,66,67]. Unfortunately, the presence of active efflux transporters to the BBB also
limits the therapeutic efficacy of drugs virtually able to access
the brain.
The P-glycoprotein (P-gp) is an ATP-dependent drug
transport protein present at the apical membranes of different
epithelial cell types including those forming the BBB [6870]. It
was demonstrated, both in vitro and in vivo, that BBB P-gp can
prevent the accumulation of many molecules including a variety
of drugs (e.g. vincristine, anthracyclines, taxanes, cyclosporine
Fig. 1. A representative cross-section of a cerebral capillary of the BBB (adapted from: Expert Reviews in Molecular Medicine 2003 Cambridge University Press,
http://www.expertreviews.org/).
A, digoxin, dexamethasone, and others) in the brain [70]. P-gp

inhibition has been proposed as a possible strategy for enhancing
the brain drug penetration [71,72]. Some of the surfactants
generally employed in pharmaceutical formulations have shown
to inhibit P-gp and some of them are currently undergoing
clinical screening for the treatment of multidrug resistant tumors
[71].
3. Brain drug delivery using surfactant coated polymeric
nanoparticles
Out of the several different approaches previously mentioned,
the use of NPs has been regarded as having great potential for the
delivery of drugs into the CNS. The term NPs refers to welldefined particles ranging in size approximatively from 10 to
1000 nm (1 m) [73] with a core-shell structure (nanocapsules) or
a continuous matrix structure (nanospheres). The active compounds may be entrapped within NPs and/or adsorbed or bound
to their surface. Different kinds of polymers such as poly
(alkylcyanoacrylate) (PACA), chitosan, PLA, poly(glycolide)
(PGA) and their copolymers (PLGA) have been used to prepare
NPs [40,74]. To date, poly(butylcyanoacrylate) (PBCA) has been,
perhaps, the most employed for NP fabrication [75].
The major limiting factor for the systemic use of NPs is their
rapid clearance from the blood circulation by the reticuloendothelial system (RES). The extent of this uptake depends mainly on
particle size, surface charge, and surface properties [76,77]. In
fact, colloidal particles, characterized by variable hydrophobic
surface properties, are efficiently coated by specific plasma
components (opsonins), such as immunoglobulins (IgG), albumin, the elements of the complement system, fibronectin, and
others, and then cleared from the blood stream by the phagocytic
cells within minutes [7880]. Intravenously (i.v.) administered
NPs mainly distribute into liver (6090% of the injected dose),
spleen (210%), lungs (320% and more), and bone marrow
(N 1%) [73]. Additionally, extravasation affects consistently NP
body distribution. In fact, the structure of the peripheral capillary
walls in different organs and tissues displays interendothelial gaps
as large as 150 nm. This passive targeting to the liver strongly
limits the use of NPs in delivering drugs to sites in the body other
than RES, such as the CNS, that, on the contrary, is characterized
by a very low permeable continuous endothelium [58].
Based on the above considerations, a proper modification of
NP surface characteristics and size, can represent a highly
457
effective strategy to alter NP biodistribution, enhancing thus their

blood circulation time and deposition in non-RES organs [81].
In this respect, as early as in the '80s, different approaches
were proposed for the modification of the hydrophobic particle
surface; most of them were based on the physical adsorption of
PEG-containing surfactants [8290]. More recently, surface covalent modification with PEG derivatives has been explored as an
alternative strategy [9195]. All authors showed that this coating
reduced NP liver uptake increasing simultaneously their persistance in the pheripheral circulation as well as their deposition into
other organs, including non-RES organs, for several hours.
Trster et al. first hypothesized that the surfactant contact
angle may be indicative in predicting the body distribution of
coated NPs [96]. Based on this assumption, in a later work [88],
the authors studied the in vivo body distribution of surfactantcoated and non-coated poly(methyl methacrylate) (PMMA)
NPs of 131 30 nm in size. A series of poloxamers (also known
with the proprietary name Pluronic) (poloxamer 188, 407,
184, 338, and poloxamine 908), polysorbates (also known with
the proprietary name Tween) (polysorbates 20, 60, and 80),
and Brij 35, were used as coating material to investigate the
possible correlation between the NP surface properties and their
in vivo biodistribution [88]. The results indicated that: i) no
correlation exists between the body distribution pattern and the
contact angle of the NP coating materials; ii) although all used
surfactants decreased the liver uptake and increased the uptake
in other organs and tissues, poloxamer 338 and poloxamine 908
were the most powerful surfactants in enhancing NP blood
levels and in increasing the spleen uptake while reducing the
liver uptake; iii) P80 and poloxamer 184 yielded the highest
accumulation in the heart, muscles, kidneys, and the brain.
Therefore, it was thought that polysorbates and poloxamers
could be useful for specific targeting to non-RES organs. In
particular, the total NP brain amount was increased with all
surfactants up to 24 h, especially by polysorbates 60, and 80,
poloxamers 407, 184, and 338, and poloxamine 908. However,
as time increased the levels decreased approaching those of the
uncoated PMMA NPs.
In light of these findings, NP surfactant coating was considered
as a new potential approach to improve brain drug uptake.
P80 coated PBCA NPs (Fig. 2) were first investigated in vivo
to enhance the transport across the BBB of the hexapeptide
dalargin, a Leu-enkephalin analogue having an opioid activity
[9799]. The peptide is not effective when administered i.v. but
Fig. 2. Schematic representation of P80-coated PACA NPs (1) and P80 stabilized SLN (2).
458
when it was bound to PBCA NPs (250 nm) coated with P80,
a strong analgesic effect was observed. In fact, all controls,
consisting of a solution of dalargin, a solution of P80 (1% w/v,
200 mg/kg), a suspension of PBCA NPs, a mixture of dalargin
with P80, a mixture of dalargin with NPs, a mixture of dalargin,
P80 and NPs (mixed extemporaneously before injection) as
well as dalargin adsorbed to NPs without P80 coating, resulted
ineffective.
The investigation of the in vivo fate of NPs coated with
different surfactants further confirmed the P80 effectiveness at
enhancing brain drug uptake [100]. The results showed that,
after i.v. injection, only polysorbates 20, 40, 60 and 80 were
able to induce analgesia in mice. Other surfactants, such as
poloxamers 184, 188, 338, 407, poloxamine 908, Cremophor
EZ, Cremophor RH40, and Brij 35, were totally or almost
inactive. P80 enabled the highest induction of analgesia at both
7.5 and 10 mg/kg dalargin dosages.
Accordingly, other drugs that normally do not penetrate the
BBB, such as tubocurarine [101], loperamide [102], 8-chloro-4hydroxy-1-oxo1, 2-dihydropyridazino[4,5]quinoline-5-oxide
choline salt (MRZ 2/576) [103], and doxorubicin [104] showed
higher brain concentrations when associated with P80-coated
NPs. The transport across the BBB of tubocurarine loaded NPs
[101] has been evaluated by in situ perfused rat brain technique
together with simultaneous electroencephalogram recording.
Thirteen to fifteen minutes after the introduction of tubocurarine
loaded P80-coated PBCA NPs into the perfusate, frequent severe
spikes typical of epileptic seizures were observed in the
electroencephalogram. As for dalargin, neither tubocurarine
solution or NP suspension nor NP suspension in tubocurarine
solution or a simple mixture of P80 and tubocurarine influenced
the electroencephalogram in the control experiments.
The results obtained with loperamide [102], a poorly watersoluble opioid agonist unable to cross the BBB, were in
excellent agreement with the earlier results obtained with
dalargin and, similarly, it was concluded that the transport of
P80-coated drug loaded NPs across the BBB may occur through
an endocytosis process by the brain capillary endothelial cells.
Similar results were observed for MRZ 2/576, a novel Nmethyl-D-aspartate receptor antagonist characterized by a potent
but rather short anticonvulsant effect (515 min). This short
activity was assumed to be due to its rapid elimination by efflux
pump-mediated transport processes. In fact, MRZ 2/576 showed a
prolonged anticonvulsive activity up to 150 min after probenecid
pre-treatment (an efflux process inhibitor). Better results were
achieved by i.v. administration of the drug when adsorbed to P80coated PBCA NPs and after previous probenecid treatment [103].
In these cases, the duration of the anticonvulsive activity in mice
rose up to 210 and 270 min respectively. These results demonstrated that P80-coated PBCA NPs can be useful both for
delivering drugs to the brain and for prolonging CNS drug mean
retention time (MRT).
Accordingly, 5 mg/kg of doxorubicin loaded PBCA NPs
coated with P80 administered to healthy rats [104] were able to
elicit a brain drug concentration as high as 6 mg/g by i.v.
administration, versus b 0.1 mg/g of the control injection.
Furthermore, doxorubicin bound to P80-coated NPs produced,
in 20% of the treated rats with intracranial transplanted 101/

8 glioblastomas, long term remission if compared to control
animals or animals treated with other doxorubicin formulations.
Additionally, binding to NPs may reduce doxorubicin systemic
toxicity [105].
Recently, PBCA NPs coated with P80 have been proposed as
suitable carriers for nerve growth factor (NGF) brain delivery
[106]. NGF is essential for the survival of peripheral ganglion
cells as well as central cholinergic neurons of the basal forebrain.
Although its inability to cross the BBB, the NGF action on these
neuronal population and its efficacy in preventing neurodegeneration suggested its potential use in the treatment of
neurological pathologies such as AD, diabetic neuropathies
and Huntington's disease. Systemic administration of NGF
adsorbed on P80-coated PBCA NPs successfully reversed
scopolamine-induced amnesia and improved recognition and
memory in acute amnesia rat model.
Lately, clioquinol (CQ) (5-chloro-7-iodo-8-hydroxyquinoline)
loaded NPs have been proposed for diagnostic and therapeutic
applications in AD [28]. CQ is a highly hydrophobic molecule
able to cross the BBB and it is known to solubilize, in vitro, the
amyloid beta protein (A) (senile plaques or A plaques)
presumably by chelation of the A-associated zinc and copper
[107]. Moreover, after oral treatment, CQ inhibits the A
accumulation in AD transgenic mice model [107,108]. CQ
treatment appears to inhibit successfully A accumulation and
hence CQ may be considered the first credible drug candidate to
take care of AD. In addition, after coating with 1% P80 solution,
the NPs were shown to be able to enhance the drug transport
across the BBB.
Likewise PEG-containing surfactants as surface modifiers,
also PEGylated NPs have been accounted as potentially useful
tool to deliver drugs into the brain. New synthetic methoxypolyethyleneglycol cyanoacrylate-co-hexadecylcyanoacrylate
(MePEGCA-HDCA) amphiphilic block-copolymer have been
fabricated for the preparation of novel PEGylated polymeric NPs
(Fig. 3) [91]. Upon [14C]MePEGCA-HDCA NPs i.v. administration in mice, 30% of the radioactivity was still found in the
blood circulation 6 h after injection, regardless of the PEGylation
degree employed. On the other hand, the non-PEGylated NPs
were cleared from the bloodstream within a few minutes [92]. The
overall biodistribution showed that the hepatic accumulation was
dramatically reduced whereas an increased spleen uptake was
observed as already noticed for PEGylated PLA NPs [93,94].
PEGylated NPs have been hence proposed as a new carrier
for brain drug delivery and a comparison of the biodistribution
profiles, after i.v. administration in mice and rats, of radiolabelled PEGylated NPs, P80- or poloxamine 908-coated poly
(hexadecyl cyanoacrylate) (PHDCA) NPs and the uncoated
PHDCA NPs has been performed [95]. In this case [14C]
Fig. 3. Molecular structure of the MePEGCA-HDCA copolymer.
PEGylated NPs (60 mg/kg) penetrated into the brain to a larger

extent than P80- or poloxamine 908-coated PHDCA NPs, and
uncoated PHDCA NPs, both in mice and rats. On the contrary,
the injection of half of the P80-coated NP dose (30 mg/kg) in rats
produced a significant accumulation enhancement in the brain
that resulted to be the double of that recorded for PEGylated
particles. The authors hypothesized that low amounts of
suspended PHDCA NPs in the P80 solution (1%), increased
the concentration of free surfactant in the blood to a level
sufficient to modify the BBB permeability. However, this
reasonable hypothesis is not supported by experimental data and
is strongly contrasting with what reported in the literature, where
good performances by P80-coated NPs are described but no
theories on the effect on BBB permeability due to P80 are
postulated [95].
Following the evidences that P80-coated NPs could target
therapeutic molecules to the brain, a number of potential
mechanisms have been proposed [81,109,110]. In particular, six
plausible explanations were provided: (i) the increased NP
retention in the brain blood capillaries, and/or their adsorption to
the capillary walls, can create a higher concentration gradient
enhancing the transport across the BBB and leading to brain
drug accumulation; (ii) the solubilization of the endothelial cell
membrane lipids by the surfactant can lead to membrane
fluidization and then an enhanced BBB drug permeability; (iii)
the opening of the tight junctions by NPs can lead to an increased
drug paracellular permeation in the free or NP-bound form; (iv)
the NP endocytosis by the endothelial cells can permit the drug
release within these cells and the following drug diffusion in the
brain parenchyma; (v) the transcytosis of NPs with the bound
drug can release directly into the brain parenchyma; (vi) the
inhibition of the efflux system (especially P-gp) by means of
P80 can prolong the activity of drugs. These mechanisms can
also work in combination. Nevertheless, some of these possible
explanations can be excluded. In fact, it has been demonstrated
that control groups treated with: a mixture of drug and P80,
blank NP suspension, P80-uncoated drug loaded NPs, and an
extemporaneously prepared mixture of the three components
(P80, NPs, and drug), failed to display any pharmacological
effect [97,99,101103]. By reason of these results, the
mechanisms (ii), (iii) and (vi) may be obviously excluded.
Moreover, the fact that NP-coated with other PEG-containing
surfactants (e.g.: poloxamers 184, 188, 338, 407, poloxamine
908, Cremophor EL, Cremophor RH 40 and Brij 35) were
not able to significantly induce antinociceptive activity, ruled out
the possibility of an enhanced BBB drug permeability due to a
general surfactant effect (ii).
The remaining three hypothetic mechanisms are usually
considered the most likely. In fact, evidence of P80-coated NP
adsorption on the brain capillary wall [111] and NP endocytosis,
[97,112114] has been reported both in vitro and in vivo.
Furthermore, transcytosis was observed with different types of
NPs [115,116].
However, none of these mechanisms may be completely
confirmed or rejected. Lately the hypothesis of NP endocytosis
by the BBB endothelial cells, followed by drug release and
diffusion to the brain, has gained more credit [117119]. The
459
P80-coated NP endocytosis is believed to be mediated by protein

adsorption on their surface followed by subsequent interactions
(triggering the endocytosis) with specific BBB surface
receptors.
As previously mentioned, when an exogenous particulate
material (e.g. polymeric NPs and SLN) is exposed to serum or
plasma, proteins readily adsorb (within the first few seconds) on
its surface [120,121]. The type and the relative amount of
adsorbed proteins will decide the fate of particles. This concept
was originally defined as differential protein adsorption in
1989 [121]. Based on this concept, the hypothesis was
postulated that an adsorption of the ApoE or B on P80-coated
NPs could be responsible for the interaction with the BBB and
the subsequent endocytosis (Fig. 4) [81,109,110]. Abundant
evidence of Apo adsorption on P80-coated NPs by different
techniques have been reported [45,109,111,122126].
Fig. 4. Schematic representation of the proposed mechanism of P80-coated NP

brain uptake. 1. Apo adsorption on P80-coated NP surface. 2. NP binding to the
BBB LDL receptors. 3. Drug release from NPs upon endocytosis and transcytosis
through the BBB endothelial cells.
460
It has been shown that Apo (particularly ApoJ, ApoA-I and

ApoC-III) were the dominant proteins adsorbed on cetylpalmitate
SLN, regardless of the surfactant used (polysorbates 20, 40, 60,
80) [45]. All those SLN formulations showed ApoE adsorption,
while typical opsonins (e.g. IgG or complement factors) were
either not at all detected or in very low amounts. Interestingly
enough, there seems to be a relationship between ApoE
adsorption on polysorbate-stabilized SLN and the surfactant
hydrophiliclipophilic balance (HLB) number [45]. This may
explain some of the findings regarding a significant effect of
dalargin when using PBCA NPs coated with polysorbates 20, 40,
60, 80 although the highest effect was achieved using P80 [100].
However, it was not possible to correlate the amount of adsorbed
ApoE with the observed pharmacological effect.
Naturally, the fate of colloidal drug carriers cannot be
determined by the sole amount of a single protein adsorbed on
their surface. More likely, the composition of the entire protein
layer, the type, and the relative quantity of the adsorbed
proteins, will determine the body distribution of the carrier. In
fact, ApoC-I and ApoC-II were found to inhibit the binding of
ApoE-containing lipoproteins to the low-density lipoprotein
receptors [127]. Therefore, a high ApoE/ApoC-II ratio on the
NP surface should be achieved to successfully accomplish brain
targeting. P80 stabilized SLN displayed the highest ApoE/
ApoC-II ratio [45].
In order to attempt to elucidate the mechanism leading to
P80-coated NP brain targeting, by means of systematic
experiments, it has been shown [117,119] that just dalarginor loperamide-loaded NPs, coated with P80 and/or ApoB or
ApoE, were able to significantly induce the antinociceptive
effect. The highest effects were observed when both P80 and
ApoB or ApoE were coating NPs. In ApoE deficient mice, a
reduced effect was observed [117]. Further studies using ApoE
linked to human serum albumin NPs were carried out by means
of the biotinneutravidin strategy. This system loaded with
loperamide was able to induce an analgesic effect in a dosedependent manner. In particular, the variant ApoE3 was found
responsible for this effect [119]. Both results sustained the
specific role of ApoE in mediating the brain targeting of this
type of NPs, supporting the hypothesis of NP endocytosis [128]
and transcytosis (Fig. 4) [129]. In order to further investigate
P80-coated NP uptake mechanism and to establish whether the
main role was played by phagocytosis or pinocytosis, additional
experiments were performed. The preincubation of bovine brain
endothelial cells with cytochalasin, a phagocytosis inhibitor led
to a significantly reduced uptake ( 40%) of labeled P80-coated
NPs, while the pretreatment with colchicine, a pinocytosis
inhibitor, had no effect on NP uptake [112].
Even though these results support the aforementioned
mechanisms, further investigation and other evidence is
required in order to draw an unambiguous conclusion on this
matter. In fact, up to now, none of the studies performed have
demonstrated the presence of P80-PBCA NPs in the vesicular
structures of the brain endothelial cells [130].
Besides, if this hypothesis will turn out to be true, NP size
will be demonstrated to represent a critical parameter for brain
targeting. Indeed, NPs with particle size close to low density
lipoproteins (LDL), in the range from 18 to 25 nm should be

more effectively taken up by the BBB [129]. This hypothesis is
now being supported only by indirect evidence [131].
Methotrexate loaded NPs with various mean sizes (70, 170,
220, 345 nm) were coated with P80, administered by i.v.
injection and the methotrexate concentrations in CSF and brain
tissues were measured. In this experiment, among the tested
NPs, those with a diameter below 100 nm led to the highest drug
level in the brain, even though no statistically significant
differences were observed [131].
Recent preliminary results demonstrated the possibility of
targeting P80-coated NPs to the brain following oral administration as well [132,133]. This opportunity appears most
interesting and should be further investigated as a possible
alternative to the parenteral route due to its higher compliance.
In light of the promising results obtained with P80-coated
polymeric NPs for brain targeting, the transfer of this technology
to novel lipid nanocarriers, such as SLN, nanostructured lipid
carriers (NLC) and lipid drug conjugated (LDC) NPs (Fig. 2)
will be discussed. In fact, SLN, developed almost fifteen years
ago [134136], have been proposed as alternative carrier to
polymeric NPs to overcome some of their common problems.
SLN have been used for various purposes, such as drug
bioavailability enhancement [137,138], controlled release [139]
and drug targeting [140]. Due to the high lipid biocompatibility,
all the existing administration routes are virtually possible and
many of them have been investigated, namely the parenteral
[49], oral [141], transdermal [142], pulmonary [143] and ocular
[144].
4. Advantages of SLN over PACA NPs
4.1. Preparation and sterilization
Many approaches for SLN preparation exist. The most
common methods consist in high pressure homogenization at
elevated or low temperatures, microemulsion, solvent emulsificationevaporation or diffusion, w/o/w double emulsion and
high speed stirring and/or sonication [4749]. High pressure
homogenization at elevated temperatures [145,146] and microemulsion [147149] are among the most versatile techniques
and for this reason have been largely employed for SLN
preparation. Briefly, the high pressure homogenization method
regards the production of a lipid and drug melt, which is added to
an aqueous surfactant solution at the same temperature. Then, a
pre-emulsion is produced by high shear stirring and subsequently processed by a high pressure homogenizer at controlled
temperature for several cycles and finally cooled down. In
contrast, the microemulsion method consists in the formation of
a warm microemulsion containing about 10% melted lipid, 15%
surfactant and up to 10% cosurfactant. The microemulsion is
then poured upon stirring in a large volume of cold water and the
exceeding water is eliminated by ultra-filtration or lyophilization. Both these techniques, avoiding the use of organic solvents,
do not lead to residue contaminations. SLN metallic particle
contamination, that may occur by using sonication, is avoided as
well. Moreover, the elevated temperatures used do not represent
an issue because of the limited time of exposure. Another

fundamental advantage of these two methods is the scale up
feasibility as high pressure homogenization easily allows a
laboratory, pilot or large scale production. In fact, 50 to 150 kg of
SLN dispersions per hour can be obtained by placing two
homogenizers in series [145]. However, the microemulsion
technique is less scalable, even though the production of batches
as large as 1.1 L has been achieved [49].
Differently from SLN, the preparation of PACA NPs can be
performed either by polymerization of alkylcyanoacrylate monomers or by using the formed polymers [150]. The first process
includes two methods of preparation, emulsion [102] or interfacial
polymerization [151]. Emulsion polymerization is the most
widely used method and consists in a very complex medium
where its initiation is carried out by the water hydroxyl ions and
elongation of the polymer occurs by anionic mechanism. The
interfacial polymerization technique has been developed to obtain
nanocapsules containing a water or oil core by using a water-in-oil
or oil-in-water emulsion system respectively. PACA NPs
prepared with block copolymer can be obtained by nanoprecipitation or by emulsion-solvent evaporation [91,92,95]. In the former
case, colloidal particles are obtained adding a non-solvent to a
dilute solution of the polymer. Finally, the solvent is removed
from the suspension. In the latter case, a polymer solution is added
to an aqueous phase upon high pressure homogenization to form a
fine emulsion. The solvent is then evaporated to induce polymer
precipitation and NP formation. The scale up of these preparation
methods is more complicated than for SLN as a result of the
intensive use of solvents needed to solubilize polymers or
monomers. Unfortunately, in the literature there is a consistent
lack of studies concerning the scale up issues in the production of
these systems, although in recent years more attention has been
devoted to the industrial development aspect of batch production.
Sterilization is another fundamental step in the production of
NPs intended for parenteral administration. In the case of SLN,
normal procedures consist in carrying out the SLN preparation in
aseptic conditions and the possible application of common
sterilization processes, such as filtration, -irradiation and
autoclaving [48]. However, these methods can present some
important drawbacks. In particular, sterilization by filtration
requires particle sizes below 0.2 m and therefore cannot be used
in all circumstances. Sterilization by -irradiation generally may
lead to free radical production that could provoke SLN chemical
modification, while heat sterilization can induce modifications
related to their physical stability such as the increase of particle
size due to their aggregation [48]. In fact, it seems that poloxamer
did not provide enough stabilization to avoid particle size increase
upon heating [135,151]. Even though, autoclaving has been
proposed for SLN suspensions containing reduced lipid concentrations (2%), since no important alterations were observed [139].
Beyond the nature of the lipid and the presence of surfactants, the
medium represents an important parameter that may allow
autoclaving without particle size increase. In fact, when SLN
were suspended in a solution of Pluronic F68 prior to
sterilization, no increase in particle size was observed [152]. Of
course, the possible drug loss and lipid or drug degradations have
to be carefully considered as well [151,153].
461
Sterilization of PACA NPs is hardly accounted for in the

literature [154], although general methods, already considered
for SLN and applied to other kind of particle systems [155157]
can be considered useful. However, for both particulate
systems, the preparation performed in sterile conditions still
remains the best choice over all the methods above reported.
4.2. Toxicity, stability and biocompatibility issues
Since these DDS are designed for human application, the
material used for their preparation has to be non-toxic,
biocompatible, and able to be excreted or biodegradable.
PACA NPs are completely excreted only when the molecular
weight of the used polymers is sufficiently low [150]. Hence, it is
fundamental to understand the degradation mechanisms which
can determine the possible PACA NP suitability or limitations.
Three mechanisms of degradation have been described for
PACA NPs [150]. The first, which is believed to be the most
important, is based on the enzymatic degradation by esterases of
the serum, lysosomes and pancreatic juice. This mechanism
gives rise to alkylalcohol and poly(cyanoacrylic acid) byproducts. The second is based on the unzipping polymerization
followed by an instantaneous repolymerization into a lower
molecular weight polymer. The third, that at the early stages of
PACA NP development was believed to be the most important,
is well known under the name of Knoevenagel reaction. The
degradation products are formaldehyde and cyanoacetic ester
and the reaction yields only 5% of degradation after 24 h. These
byproducts are all potentially harmful to cells as they are small or
medium size non-self molecules. In this regard, the knowledge
of the degradation pathway is fundamental as it deeply
influences the kind and the accumulation rate of byproducts in
tissues and organs from which the toxicity depends. For this
reason, toxicity studies have been performed to understand the
implication of PACA biodegradability in the occurrence of
possible side effects.
The investigation of PACA NP in vitro cytotoxicity was
carried out on several cell lines, namely macrophages [158,159],
L929 fibroblasts [160,161], hepatocytes [162,163], cancer cells
DC3F [164], mesenchymal cells [165], Swiss 3T3 cells
[166,167] and murine cerebral microvessels [168]. Since
macrophages are characterized by a high phagocytic propensity,
they represent good models to study the toxicity of PACA NPs
intended for intracellular targeting [158,159]. The results
revealed that polyisohexylcyanoacrylate induced a respiratory
burst in macrophages, and thus macrophage activation [159].
Another study showed that polymethylcyanoacrylate NPs
provoked perforation of the macrophage membrane. In the
same conditions, perforation was not observed with polyisobutylcyanoacrylate NPs. The contrasting results can be explained
by the different degradation rate that is correlated to the alkyl
chain length [158]. The influence of the alkyl chain length on the
PACA NP cytotoxicity was investigated more accurately in a
L929 fibroblast cell model analyzing cell growth inhibition
[161]. Polyethyl-, polyisobutyl-, polymethyl-, and polyisohexylcyanoacrylates were investigated for cell growth at an amount of
20 g/ml. The first two polymers showed a considerably higher
462
toxicity compared to polyisohexyl-cyanoacrylate, while polymethyl-cyanoacrylate is only slightly less toxic. The observed
toxic effects could be due to the combination of two
mechanisms, PACA degradation and adhesion. In the presence
of longer alkyl chains, NPs undergo a slower degradation
favoring cell adhesion likely leading to high local degradation
product concentrations [161]. Besides the length of the alkyl
chain, the particle size influences the toxicity too. In fact, it was
found that smaller particle sizes provoke higher cytotoxicity.
This has been explained by the large surface area that leads to a
faster degradation and a higher degradation product concentration [161].
Another very useful model to study PACA NP toxicity is the
liver [162,163] since it is responsible for a rapid uptake upon
NP i.v. administration. PACA NP i.v. administrations initiate an
inflammation process accompanied by metabolic disorders in
the glucidic metabolism and by massive glycoprotein undersialylation. This process, reversible 14 days after the end of the
treatment, can be correlated to the NP phagocytosis by Kupffer
cells. In fact, in vivo, PACA NPs are supposed to principally
distribute in the Kupffer cells initiating their activation, while
the direct contact with hepatocytes is not likely to occur. For this
reason, PACA degradation products are not mainly responsible
for the low level of reduced glutathione. Indeed, the decrease of
the reduced glutathione level could be due to a phagocytosis
induced burst respiratory effect.
It can be concluded that the PACA NP cytotoxicity is likely
due to the burst release of degradation byproducts, directly
related to the length of the polymer alkyl chains. Even if NPs
made of longer alkyl chain polymers are less toxic, they could
adhere to cells, leading to a high local degradation product
concentration. For brain targeting, less toxic slow degrading
PACA NPs should be more suitable, although their chronic
administration would lead to waste disposal' in brain
endothelial cells and/or other brain structures.
On the other hand, since SLN are prepared from biocompatible
materials, better tolerability with respect to polymeric NPs can be
hypothesized. In addition, triglycerides, fatty acids, or waxes are
known to release natural occurring degradation byproducts. To
date, no SLN formulations for parenteral use are present on the
market. Nevertheless, lipid emulsions composed by fatty acids are
largely used in parenteral nutrition since the '60s [49].
Consequently, the use of glycerides for SLN preparation could
be conceivable without expecting important toxic effects resulting
from their degradation products [47]. Although SLN for
parenteral administration are of great clinical interest, little is
known about their metabolism. Moreover, GRAS (Generally
Recognized As Safe) surfactants must be used for their
preparation to obtain approval and a good in vivo tolerability [49].
The phagocytosis of SLN can be controlled modifying their
surface properties as has been done for liposomes and
polymeric micro- and nanoparticles [169,170]. In this way, it
is possible to target molecules to the brain by limiting RES
uptake. At any rate, it is essential to understand if the interaction
of SLN with cells leads to cytotoxic effects.
The in vivo toxicity on the liver and the spleen of mice after i.v.
injection of two SLN formulations composed of cetylpalmitate
or Compritol has been assessed [171]. Even if cetylpalmitate

is not a physiological wax, no accumulation was observed
since its degradation is very rapid. On the contrary, Compritol
provoked a reversible liver and spleen weight increase.
Moreover, the liver histological analyses revealed the presence
of mononuclear cell infiltration, Kupffer cell hyperplasia and
liver cell necrosis. These effects were almost totally reversible
6 weeks after the injection since neither inflammation nor fresh
infiltrations were observed. Besides, the Compritol SLN
injected dose was remarkably high, in fact, if correlated to the
human body mass, the dose was equivalent to 100 g solid lipid
mass administered 6 times by i.v. injection. In light of these
considerations, the effects observed can be ascribed to the
enormous dose administered, much larger than that theoretically needed for therapy.
Several studies have investigated the influence of the lipid
matrix, surfactants, and even particle size on the SLN
degradation rate. The degradation of the lipid matrix occurs
mainly by lipases while only a small part is degraded by nonenzymatic hydrolytic processes [172]. Another enzyme responsible for SLN degradation is the endogenous alcohol dehydrogenase [173]. The influence of surfactants, namely cholic acid
sodium salt, lecithin, P80 and poloxamer 407, on the degradation
rate of SLN has been evaluated [174]. The last two stabilizers are
PEG-containing surfactants that provide a sterically protective
layer of varying thickness depending on the structure and
number of PEG units. The protective layer more or less hinders
the lipid from the enzyme attack obstructing the anchorage of the
lipase/colipase system. When comparing the same surfactant, a
higher degradation rate was observed for glyceride based SLN
than for wax based SLN (e.g. cetylpalmitate). In fact, waxes are
not an optimal substrate for lipase/colipase that preferentially
metabolizes glycerides. The faster degradation occurred with
cholic acid sodium salt that is recognized as promoting enzyme
anchorage to surfaces [175]. Since lecithin did not promote or
hinder the enzyme anchorage, SLN degradation rate is not
influenced by the presence of this stabilizer. Poloxamer 407 was
able to reduce the SLN degradation rate to a larger extent
compared to P80 that had a limited steric effect. In fact, the
adsorption layer thickness was approximately 20 for P80 and
around 120 for poloxamer 407. A faster degradation rate was
observed for triglycerides containing short fatty acid chains,
while an opposite effect for long fatty acid chain triglycerides
was noticed. Since SLN are degraded by surface erosion, size is
expected to have an influence on the degradation rate [176]. For
SLN possessing the same diameter and polydispersity index,
degradation was faster with cholic acid sodium salt than with
poloxamer 407. Poloxamer 407-coated SLN degradation
seemed to be more sensible to size variations. In fact, small
differences in particle diameter led to a significantly different
degradation behavior. In addition, two poloxamer 407 SLN
possessing two different diameters were characterized by a
similar degradation rate. This phenomenon was explained by an
increased polydispersity of the smaller size SLN batch, due to
the presence of microparticle population fractions. However, in
general small size SLN were characterized by a faster
degradation rate while cholic acid sodium salt-coated SLN
degradation was just slightly sensible to particle size. In fact,

different degradation rates were observed only when sufficiently
different particle populations (300 nm and 800 nm mean size)
were compared. Generally, the influence of particle size on SLN
degradation rate was observed when hindering surfactants were
used in SLN preparation.
Investigation on SLN in vitro cytotoxicity have been carried
out on several cell lines, such as granulocytes, HL 60 and MCF7 cells and murine peritoneal macrophages [149,177180]. In
comparison to Food and Drug Administration (FDA) approved
polymers, such as PLA or PLGA, that show 50% viability at
0.2% of polymeric NPs, SLN (Compritol, cetylpalmitate)
were 20 times less toxic on human granulocytes and HL 60
[177]. Moreover, tripalmitin and stearic acid based SLN, in the
range 0.1 to 0.25%, resulted non-toxic to HL60 or MCF-7 cells
[149]. Comparing Dynasan 114 and Compritol SLN in the
range 0.0151.5%, no significant viability differences on HL 60
cells were observed. These results mean that SLN are very well
tolerated by HL60 cells even if their degradation rate is
significantly different [179]. These two SLN formulations did
not have any effect on murine peritoneal macrophage viability
while an amount of 0.1% Dynasan 118 and Imwitor SLN
provoked a clear viability reduction. Other lipid-based SLN,
such as dimethylstearoyl-ammonium bromide (0.01%), cetylpalmitate and stearic acid (0.1%) resulted in a macrophage
viability of 15.3%, 61.8% and 2.2%, respectively [178,180].
These results show how the lipid nature fundamentally
influenced the cytotoxicity. Since Dynasan 118 and Imwitor
are two lipids containing glycerides of stearic acid, their
cytotoxicity was compared with stearic acid SLN; the latter
resulted much more toxic than the former ones. This higher
cytotoxicity was explained by the presence of larger amounts of
stearic acid produced by SLN biodegradation [180].
SLN size did not lead to macrophage activation, and thus to
cytokine production. Moreover, no cytotoxic effects were
associated to SLN size modifications, confirming the findings
previously reported [181]. In addition to the lipid type, the
surfactant, used to stabilize SLN, could affect cell viability. In
fact, while poloxamer 407 (up to 10%) did not have any effect
on HL 60 cell viability, P80 reduced it markedly when used at a
concentration 0.01% [179]. Moreover, P80 toxicity resulted
higher when used as a free molecule (concentration of 0.001%,
viability of 50%) with respect to P80 bound or incorporated in
SLN. Non-ionic surfactants (poloxamine 908, poloxamer 407
and 188, Solutol HS15 and P80) revealed a slight concentration dependent cytotoxicity on murine peritoneal macrophages,
while cationic surfactants, such as cetylpyridinium chloride,
provoked a clear viability reduction [178,181]. Furthermore, the
same concentration (concentration presented in 0.01 or 0.1%
SLN) of free cetylpyridinium chloride incubated with macrophages led to a lower toxicity if compared with the toxicity
observed when it was incorporated in SLN. The cetylpyridinium chloride antimicrobial activity is based on its interaction
with microorganism membranes [182]. Indeed, the positively
charged surfactant present at the surface of SLN can interact
with the negatively charged cellular membrane resulting in a
cytotoxic effect. The lipid matrix seems to play a role in cell
463
adherence since such toxic effects are not observed with free
cetylpyridinium chloride [181].
The comparison between polyester NPs and SLN cytotoxicity showed that SLN were ten-fold [183] or even twenty-fold
[177] less toxic to human granulocytes if compared to the
polyester particles. In fact, for 0.5% PLGA and PLA NPs,
important cytotoxic effects were detected, while, for 5% SLN, a
7090% granulocyte viability was still observed [184]. SLN
cytotoxicity resulted much lower than that of PACA NPs
[179,185], since the cytotoxic concentration of the less toxic
PACA polymer (polyhexylcyanoacrylate) was 0.005% when
incubated with L929 fibroblasts and 0.05% when incubated
with hepatocytes. Moreover, the exposure of human granulocytes to 0.05% hexylcyanoacrylate NPs killed all cells.
The very low cytotoxicity of SLN makes them very attractive
candidates for brain delivery. It is important to underline that
their toxicity is not only related to the lipid or wax used but also
to the surfactant employed. The most common surfactant
exploited for NP brain targeting is P80 and it has been
demonstrated that free P80 is more toxic than when bounded
[179]. Because P80 is incorporated into the SLN matrix while it
is only adsorbed on PACA NPs [46], its leakage is more likely
to occur from PACA NPs (Fig. 2). That means that even if the
cytotoxicity was similar for both systems, SLN would be safer
for brain targeting since little P80 would be released in the body.
High incorporation efficiencies are desirable, of course, since it
can reduce the amount of particles needed to reach pharmacological levels. In the case of SLN, NLC or LDC NPs, drugs are
located in the core, in the shell or are molecularly dispersed in
the entire matrix according to their lipophilicity and hydrophilicity. SLN are generally able to entrap lipophilic drugs,
yielding high encapsulation efficiencies [49]. On the contrary,
hydrophilic molecules are hardly incorporated into SLN due to
the low affinity with the lipid matrix. However, biotechnological drug loaded SLN have been successfully formulated
[186,187]. Conscious of this issue, Mller et al. developed
P80 stabilized NPs for brain targeting made by lipophilic prodrug, or LDC NPs, of diminazene (diminazene covalently
linked to oleic and stearic acids) [188]. In this fashion, a drug
content, up to 33%, was achieved.
In the case of PACA NPs, drugs can be incorporated by
using the interfacial polymerization method that allows the
entrapment of both hydrophilic and hydrophobic molecules.
However, polymerization can produce a consistent amount
of free radicals that may react with the loaded drug upon
NP formation. Perhaps for this reason, in the preparation of
PACA NPs for brain targeting, drugs are generally adsorbed on
the surface of the carrier made with the preformed polymer
[98,100,101]. It is thus evident that drug loading in PACA
NPs, intended for brain targeting, is much lower than in SLN
and, even under the unlikely assumption of equal toxicity, the
latter are preferable over the former. In fact, larger amounts of
PACA NPs with respect to SLN are needed to carry the same
amount of drug to the brain. Moreover, considering that only
12% of the injected dose generally reaches the brain, drug
loading is an essential issue to address in order to develop an
effective therapy.
464
5. SLN physicochemical features with respect to brain drug

delivery
affect physical and chemical stability and suitability of SLN

formulations such as DDS.
Lipids are rightly being considered as safe and useful

materials for drug delivery [49,135,189193]. Conventional
lipid-based systems, consisting of emulsions and microemulsions, have been widely used to enhance bioavailability of class
II molecules [194], and the absorption of class III molecules
[194]. The stability of such systems is strictly related to particle
size distribution, the lipid content, and the presence of a
surfactant capable of stabilizing the dispersion. Fig. 5 reports a
general ideal diagram for the possible situations that can be
generated according to surfactant, lipid, and aqueous phase
interplay. Of course, the molecular properties of the phases
involved deeply influence lipid organization and assembly.
Stable lipid aggregates form only when the right surfactant
and concentration are being employed. Surfactants are usually
chosen according to their HLB and the Israelachvili's surfactant
packing parameter (P) [195] reported in the following form
(Eq. (1)):
5.1. SLN structure, drug loading, and stability issues
V
AL
where V is the molar volume of the hydrophobic moiety of the

surfactant molecules, A the cross sectional area of the
hydrophilic portion when situated at the interface, and L the
critical length of the hydrophobic chain. HLB and packing
parameter describe the same basic concept, though the latter is
more suitable for microemulsions. According to the geometry of
the surfactant molecules, P values can range from 1 / 3 to 1 and
to values N 1 for conical, cylinders or reverse phase conical
geometries, respectively [194,195]. For lipid aggregates
dispersed in an aqueous phase a value between 1 / 3 and 1 is
considered optimal [194].
These general considerations also apply to peculiar lipid
systems such as SLN. In this context, stable SLN dispersions
have been associated with the use of surfactants possessing
HLB values lower than 12 [194,195]. Furthermore, lipid
molecular characteristics, bulk, and surface properties heavily
Fig. 5. Ternary phase diagram of a generic lipid-based system showing the

possible lipid aggregates (adapted from Ref. [194]).
Recently, some papers have highlighted the need for a wider

comprehensive investigation of SLN properties, which have
been correlated to their composition and nanoscale size
[49,194]. The connection existing between SLN physical
characteristics and their in vitro and in vivo behavior has been
also pointed out [196,197]. Investigations on the possible SLN
inner structure should always be developed since its lack can
bring to possible misinterpretation of the in vivo results [198]. It
has been reported that materials formulated at nanoscale level
show different characteristics compared to the bulk material
[194,199]. These differences can be ascribed to changes in the
physical state and level of interaction as well as in the energies
involved. In fact, nanosize systems always possess peculiar
characteristics not found in the microscale systems, such as large
surface areas, high curvature radius, and high interaction
energies. In this regard, surface properties deeply impact
nanosystems and are correlated to the diameter of the particles.
In fact, when their size is 10100 nm, a large portion of
molecules (10% or more) are located at or near the surface. The
surface molecules will affect the mechanical (strength, flexibility, etc.) and physical behaviors of particles in a way that is not
yet fully understood [199].
For a volume fraction FV of randomly distributed spherical
particles of radius r, the number of particles per unit volume NV
(number density) is given by Eq. (2).
Nv
3Fv
4kr3
This feature is important if one considers that the particle

dose administered is proportional to their loading capacity and
to NV. In this regard, being NV closely related to particle size, for
colloidal systems the number of particles per volume fraction is
several orders of magnitude higher than that for microparticles
and it decreases rapidly as size increases (Fig. 6). This
consideration has to be accounted for when assessing NP in
vivo performances. In addition, the inner structure of the lipid
matrix has been seen to vary from large to small size particles
[200,201]. Since SLN are made of solid lipids, long and short
chain tri-, di-, or monoglycerides, their inner structure can result
quite different compared to the bulk material. Therefore, it is
obvious that the investigation of the solid lipid structure is of
great importance in order to improve the understanding of SLN
properties and to handle possible stability issues.
Several techniques have been applied to outline the SLN
physical and chemical inner organization, such as differential
scanning calorimetry (DSC), nuclear magnetic resonance
(NMR), and small angle and wide angle X-ray scattering
techniques (SAXS, WAXS) [202206]. Likewise, rheological
characterization has also been found useful to assess the SLN
lipid matrix structural characteristics [201,207,208]. In this
regard, Compritol, stearic alcohol and Softisan 138 SLN
465
Fig. 6. Particle size effect on the surface area () and on the number of particles per volume fraction () (Eq. (2)) of lipid-based systems.
revealed high storage modulus G and loss modulus G1,

suggesting the presence of a gel-like organization [209]. In
particular, Compritol and stearic alcohol SLN had a larger G
elastic modulus, whereas Softisan 138 SLN resulted more
viscous (larger G). These features were ascribed to the higher
crystallinity of Compritol and stearic alcohol compared to
Softisan SLN that, in contrast, were depicted as supercooled
melt dispersions. In addition, cetylpalmitate SLN flow properties in comparison with cetylpalmitate microparticles were
investigated as well. Cetylpalmitate SLN have been found to
possess about ten fold increased G and G moduli compared to
cetylpalmitate microparticles [201]. This effect was correlated
with the higher viscoelasticity of the NP inner structure, which
was assumed to resemble a gel-like structure. These results also
demonstrated that size may have a predominant role in
assessing lipid particle physical characteristics.
The formation, in the SLN, of a network-like structure
having a semisolid consistency was correlated to the lipid
molecules assembling themselves into a platelet-like arrangement rather than a conventional spherical particle (Fig. 7). In
fact, platelet structures are being formed mainly according to
size and when highly ordered and compact crystalline
arrangements occur, whereas it is likely that a contingent
entropy increase followed by a size increase, forces the system
to redistribute into a more thermodynamically stable spheroid.
The assumption of a platelet structural assembling of lipid
layers has been confirmed by transmission electron microscopy
(TEM) analysis [207,210]. These non-spherical particles offer a
much larger surface available for particleparticle interactions
thus favoring the formation of a network-like structure. Such
structures are naturally occurring especially for pure triglyceride
SLN. This layer organization has been reported in lipid systems
1
G = Measurement of energy stored during deformation and related to the
solid-like or elastic portion of the material. G = Measurement of energy lost
(usually lost as heat) during deformation and related to the liquid-like or
viscous portion of the material.
such as Compritol SLN characterized by field flow fractionation, photon correlation spectroscopy (PCS), and cryo-TEM
[210]. Platelets are consistent with few lipid layers (two or
three) forming a 1018 nm thick structure. Solid cetylpalmitate
SLN tend to crystallize in a platelet-like structure with a steplike surface. SAXS studies highlighted an orthorhombic
lamellar lattice arrangement for cetylpalmitate layers [211].
Since the conventional preparation methods imply the SLN
formation upon fast cooling from hot lipid melts [49], the solid
colloidal dispersion is the result of lipid crystallization into well
established ordered structures. Crystallization of triglycerides
Fig. 7. Schematic representation of solid lipid nanoparticle shapes (adapted from

Ref. [210]).
466
from the melt occurs in a metastable hexagonal -form which

converts, via an orthorombic -form, into a stable triclinic form upon reheating and storage [212214]. The rate of such
transition is sensitive to size, and the -to- transformation has
been observed to be much faster in colloidal dispersions than in
the bulk material and, likely, via an altered course. In general, it
seems that the lower the lipid melting point, the better the
chance is of -to- transformation during storage. Also particle
shape and morphology were modified as a result of the change
in the intimate physical structure of the lipid matrix. In fact, a
platelet-like shape was observed when the content of -form
was higher (in the case of ionic surfactants), whereas a higher
amount of -form produced particles of different shapes as a
function of size. Larger particles (N200 nm) were mostly
spherical, while smaller particles (b100 nm) had a blocky
isometric layered shape [215]. Another point to be accounted
for is the fact that, as a consequence of the -to- transition, the
particle surface area increases considerably as the platelets due
to the polymorph are being formed (Fig. 8) [213] This is the
result of the building up of a more complex step-like platelet
structure as is reported for cetylpalmitate SLN [211].
Melting and crystallization temperatures can be very
different between bulk materials and SLN, according to lipid
chain length [212].
The particle size dependence of the melting temperature has
been often described by using the Thomson equation modified
for crystalline materials or GibbsThomson equation (Eq. (3))
[216],
T0 T
T
2g Vs
cln sl
T0
T0
rDHfus
where T is the melting temperature of a particle with radius r, T0

is the melting temperature of the bulk material at the same
external pressure, sl is the interfacial tension at the solidliquid
interface, Vs is the specific volume of the solid, and Hfus is the
specific heat of fusion. It has been shown that this equation does
not only apply to spherical (curved) particles but can be used to
describe the behavior of particles with plane surfaces [217] and
it turned out to be useful to explain the melting point depression
of triglyceride NPs compared to their bulk phase [217]. This
effect can represent an important issue for the development of
suitable SLN formulations. In fact, not always does crystallization occur properly and the formation of supercooled melts
has been reported for several lipid systems [196,199]. Tristearin
and tripalmitin SLN are crystalline at room temperature. Under
the same conditions, trimyristin and trilaurin particles remain
liquid for several months upon storage. According to DSC
Fig. 8. Schematic representation of surface area increase upon step-like platelet

SLN formation (adapted from Ref. [213]).
analysis trimyristin particles start to crystallize at 10C while

trilaurin dispersions retain the emulsion state even at 4 C [212].
As already reported, the small size itself can reduce melting
temperatures of several degrees with respect to the bulk
material. The same effect may happen also for the crystallization temperature. This suggests that supercooling may often
occur especially for short chain lipids or lipids that are not of the
purest quality. Since the main advantages of SLN reside in the
particle solid state, low purity along with the presence of
stabilizers can indeed represent a serious issue. Moreover, the
possibility of polymorph coexistence heavily affects SLN
stability. In this regard, trilaurin exists as four different
polymorphic forms: 12 [203]. The melt of trilaurin
SLN was found to crystallize straight into the metastable -form
upon fast cooling.
Another factor strongly affecting SLN characteristics is drug
loading. In spite of the high efficiency achieved, especially for
lipophilic drugs, the amount of entrapped drug into SLN is
limited by their small size. Lipid polymorphic structures often
undergo modification upon drug loading as a result of the
intercalation of the drug between lipid layers [196]. As proof,
blank Compritol SLN showed small amounts of the unstable
-form that disappeared upon heating or by drug molecule
loading [218]. Generally, drug loading was found to decrease
lipid layer organization. In this regard, several examples of
supercooled lipid melts have been described [196,199,212].
Trimyristin SLN and SLN made of a blend of triglycerides with
chain lengths between 12 and 18 carbons and stabilized with a
mix of lecithins and glycerols were evaluated upon loading of
several steroidal drugs [196]. A large depression of melting and
crystallization temperatures was observed in agreement with
lipid chain length, indicating a strong tendency towards
supercooling, even the recrystallization course was different
between bulk material and SLN as shown by X-ray pattern
analysis [196].
Drug loading capacity was higher in the case of glyceride
blends as a result of their lower crystallinity with respect to pure
lipids, and different drug distribution and motility could be
detected by NMR analysis [196]. Drug loading from 1% up to
50% (w/w) caused drastic changes in the lipid structure and
drug expulsion was reported upon storage. The rate of drug
expulsion was different for different lipid matrices and this
feature was correlated to the rate of polymorph transformation.
Lipid annealing was also taken into consideration.
Further studies on tripalmitin SLN loaded with ubidecarenone (coenzyme Q10, CQ10) highlighted again the destabilizing effect because of the entrapped drug as shown by the
different DSC profiles of drug free and up to 70% (w/w) drug
loaded systems [219].
Melting point depletion was observed as the CQ10 concentration was increased and it persisted even after long-term
storage. Such behavior was ascribed to an eutectic formation
between the drug and tripalmitin. Above a 50% CQ10 loading,
SLN remained as a supercooled melt even upon cooling below
room temperature. However, CQ10 was found to be strongly
associated with the NPs and consequently no separation
occurred even with 5070% drug loading.
Characteristic platelet structures were detected by TEM. The

excess drug was seen to remain outside of the particle as liquid
droplets adsorbed to their surface forming thus a two phase
particle (Fig. 7). In contrast, solid state NMR studies performed
on CQ10 (5% w/w) loaded cetylpalmitate SLN showed mainly a
solid state occurrence of the particles [220]. About 60% of the
drug was found homogeneously dispersed in the lipid matrix.
Other studies demonstrated that drugs, such as mifepristone,
incorporated into SLN, can lead to stable formulations with a less
ordered crystalline organization [221]. The advantage is that less
rigid and ordered structures provide a larger space to accommodate drugs. In this situation drug expulsion is also less likely to
occur upon storage. Moreover, SLN showed a more regular
spherical shape and this may be correlated to the lower crystalline
grade. The physical state of particles is thus of paramount
importance both from the technological and biopharmaceutical
viewpoint. Since the actual physical condition of the lipid system
is not known, misinterpretation of in vitro or in vivo behavior can
easily occur as supercooled melts behave mainly as emulsions.
When solubilization of poorly soluble drugs is required while
their modified release does not constitute a major aim, the use of
supercooled melts may be advantageous as they can hold larger
amounts of drug and they possess fewer stability issues. However,
supercooled melts are not thermodynamically stable and
recrystallization over a long period of time cannot be excluded.
Such an issue enforces the possibility of product changes upon
long-term storage; and their pharmaceutical quality, especially
when intended for parenteral administration, cannot be ensured.
In fact, even though some of these systems, such as trimyristin
SLN, can be forced to recrystallize if cooled below their critical
crystallization point, undesired irreversible phenomena, such as
gelling or expulsion of the incorporated drug, can always occur in
an unpredictable manner.
5.2. Surfactants and brain targeting features
The choice of a proper stabilizer is essential to ensure SLN
suitability as brain drug targeting systems. SLN inner crystal
structures have been extensively characterized because of their
importance in determining particle behavior and stability. Parallel
investigations have been carried out to determine the best
surfactant characteristics impacting positively SLN stability and
their in vitro and in vivo behavior [214,222,223]. The surfactant
suitability is being based on the mentioned HLB and packing
parameter. However, the ability to stabilize lipid dispersions
cannot be only related to the surfactant intrinsic properties but is
also influenced by lipid and particle characteristics. Several
studies report the effect of stabilizers on size, polydispersity and
structure of triglyceride SLN [224226].
The most common stabilizers employed are ionic and
nonionic molecules, such as lecithins, polysorbates, poloxamers, derivatized fatty acids, and their combinations [96].
Surfactants are being used to prevent aggregation but they
have been found to change SLN characteristics, such as
crystallinity [227]. In particular, the extent of SLN triglyceride
crystallinity and stability is correlated to their chain length and
the presence of stabilizers, such as lecithin [228].
467
Tripalmitin SLN have been observed to organize into the

stable -form when different surfactants were used for SLN
preparation. However, ionic surfactants caused the retention of a
higher amount of -form compared to nonionic surfactants
upon cooling. This can affect the SLN behavior during storage
as the kinetic pattern of transformation into the -form is
profoundly altered [215].
The different polymorphic behavior related to the hydrophilic surfactants suggests that polymorphic transitions in
triglycerides start mainly from the surface of the particle. As
previously mentioned, SLN degradation rate was modified
according to the surfactant used for their preparation
[174,176,229]. Surfactants, therefore, are fundamental for
developing proper SLN formulations. In fact, even upon
preparation, these molecules have been reported to influence
particle size and aggregation of freshly produced SLN intended
for brain drug delivery. As reported earlier, most of the
preparation methods are based on high pressure homogenization techniques to improve SLN dispersion, their effectiveness
and scale up perspectives [145]. Several studies have been
conducted on homogenization pressure, cycle number, and
equipment effects on SLN size [226,230,231]. Tri-, di-, and
monoglycerides show a similar sensitivity to pressure and
number of homogenization cycles [226]; triglyceride SLN size
increased when using higher pressures, meanwhile di- and
monoglyceride SLN remained almost unchanged. These
contrasting effects of the homogenization pressure have been
elsewhere reported also for Compritol and Softisan 142 SLN
[231]. The explanation may reside in the coupled effect of
cavitation forces and surface area increase [226]. In fact,
although the expected effect at higher pressures and number of
cycles is size reduction, the high shear at which the particles are
being processed and the larger surface area that is generated by
particle breaking down favor aggregation. These effects are
being observed for many SLN preparations and, of course, are
susceptible to lipid and surfactant molecular characteristics. In
addition, the same effects can be obtained by increasing the
processing temperature, which may lead to size reduction as a
consequence of a reduced melt viscosity, but, similarly, the
surface area increase can produce higher tendency to aggregation. In this regard, the formation of microparticle populations is
being generally observed [226,231].
All these features have to be accounted for especially when
developing SLN intended for brain delivery. In fact, as already
stated, size and surface properties are fundamental for particle
brain uptake [110,131]. In this context, the use of a proper
surfactant is even more important because of its role of particle
surface modifier. In particular, polysorbates have been found
potentially predominant over other surfactants for brain delivery
[100]. Pressure and number of cycles upon homogenization
affect similarly the P80-coated Compritol SLN size [231]. P80
concentration had a larger effect on particle size compared to the
number of homogenization cycles (Fig. 9). Moreover, in the
case of Softisan 142, it was observed that concentration values
higher than 2% may be required in order to reduce the size down
to 150 nm [231,232]. The question arising is what kind of effect
P80 exerts on the lipid matrix. Hypothetically, the P80 oleic
468
Fig. 9. Response surface plot of the effect of P80 concentration and number of homogenization cycles on the size intended as mean volume diameter (1.) and Gaussian
distribution width (2.) of P80 stabilized Compritol SLN (adapted from Ref. [231]).
chain may penetrate the lipid matrix and anchor the surfactant
molecule onto the SLN surface (Fig. 2). This may have a large
impact on the lipid physical state. However, DSC investigations
showed a much larger melting point depression for Softisan
142 with respect to Compritol SLN (Fig. 10). This effect could
be due to the diverse impact of the surfactant on the lipid matrix
organization as well as to the different SLN particle size [232].
How these features can affect the in vivo effectiveness of
such preparations is yet to be understood. Moreover, in spite of
the long history of P80 application to NPs for brain delivery, a
comprehensive investigation of the physicochemical characteristics of the coated SLN is still partially missing. Indeed, such
information could be helpful to the development of suitable

SLN for improving brain therapies.
6. SLN for brain drug targeting
In the late '90s SLN were proposed for brain drug targeting
application independently by two research groups [233,234]
even though the first proof of lipid particle transport across the
BBB had already been provided [235].
Studying the pharmacokinetics of two anticancer agents,
namely camptothecin and doxorubicin, drug accumulation into
the brain was observed after both oral and i.v. administration
469
Fig. 10. DSC data of the bulk Softisan 142 and Compritol and their respective P80 stabilized SLN formulations.
when loaded into SLN [233,234]. As previously shown with

PACA NPs, better results in brain targeting were achieved when
SLN surface characteristics were modified by means of PEGderivatives or PEG-containing surfactants [116,233,236,237].
Both stealth and non-stealth stearic acid labeled SLN were
found in rat CSF 20 min after i.v. administration even though low
amount of radioactivity was found in the CSF samples. An
underestimation due to the sampling into the cysterna magna
was then hypothesized [116]. When the same kind of SLN were
loaded with doxorubicin, significantly higher drug concentration was found in the brain of the group treated with stealth SLN
as compared to non-stealth SLN and doxorubicin solution.
Remarkably, 30 min after administration of stealth SLN, the
same doxorubicin concentration ( 10 g/g of tissue) was found
either in the brain, heart, liver, lungs and spleen. The overall
plasma kinetics of stealth and non-stealth SLN provided to be
significantly different from that of the doxorubicin solution
[237].
Poloxamer 188 stabilized stearic acid camptothecin-loaded
SLN targeted to the brain after both oral and i.v. administration
in mice [233,236]. Following SLN i.v. administration, the
maximum concentration (Cmax) increased by 180% if compared
to the Cmax of the drug solution. The area under the curve
(AUC)/dose and the MRT of SLN were 10.4- and 4-fold higher,
respectively [236].
Two new SLN formulations made with biocompatible
materials, such as emulsifying wax and Brij 72, and stabilized
by P80 and Brij 78 were proposed for brain drug targeting
[46,140,238]. The aforementioned particles showed a significant brain uptake, measured during a short term in situ rat brain
perfusion experiment [46]. Brij 78 stabilized wax NPs
containing paclitaxel were able to significantly increase brain
drug distribution as compared to paclitaxel dissolved in a 50:50
v/v mixture of the surfactant Cremophor ELR and dehydrated
ethanol (as the commercially available Taxol formulation). In

this case it was speculated that the employed carrier, masking
paclitaxel characteristics, limited its binding to P-gp leading to
higher brain drug concentration due to the avoided efflux [140].
A very interesting series of experiments, regarding the effect of
these NPs on the baseline BBB parameters were carried out as
well [238]. Both in vitro and in vivo data showed no significant
changes on cerebral perfusion flow, integrity, permeability, and
the facilitated transport of choline [238]. The tight junction
molecular integrity was also confirmed by occludin and
claudin-1 western blot analyses, showing no changes in protein
expression. The authors concluded that the two investigated NP
formulations turned out to have minimal effects on the primary
BBB parameters [238].
Surface charged SLN were also proposed in order to achieve
brain targeting. Positively (+ 5 mV; 391 nm) and negatively
( 47 mV; 362 nm) charged tripalmitin SLN were loaded with
labeled etoposide and their organ biodistribution was compared
to that of free labeled drug in mice after i.v. administration.
Positively charged SLN showed a higher brain accumulation
compared with both negatively charged SLN and free labeled
drug. The etoposide concentration, achieved using positively
charged SLN, was 10- and 14-fold higher with respect to the
negatively charged SLN at 1 and 4 hours after administration,
respectively. Even though the overall concentration was not as
high, some preferential BBB uptake could be hypothesized
[239].
Clozapine loaded tripalmitin SLN, with (+ 23.2 0.9 mV;
163 nm) and without stearylamine (+ 0.2 0.1 mV; 233 nm),
were able to significantly increase drug brain concentration after
mice i.v. administration when compared to clozapine suspension
[240]. Particularly, if compared to the clozapine suspension, the
Cmax increased by 199 to 243%, the AUC by 5.3- and 4.1-fold,
and the MRT by 3.9- and 4.3-fold, for charged and non-charged
470
particles, respectively. It is noteworthy that, for the positively

charged SLN preparation, over the investigated organs (liver,
spleen, heart, kidney, and brain), the highest relative bioavailability was seen in the brain [240]. Significantly higher brain
etoposide concentrations, after mice intraperitoneal administration of negatively charged tripalmitin SLN ( 47 mV; 387 nm),
with respect to etoposide alone were observed [241]. Unfortunately, the strategy of using surface charged SLN, or NPs in
general, to cross the BBB has shown some important drawbacks
limiting its usefulness [242]. Neutral ( 14 mV; 75 53 nm),
negatively ( 60 mV; 127 71 nm) and positively (+ 45 mV; 97
69 nm) charged SLN were prepared and their rat BBB
permeability was studied using the in situ brain perfusion
method. Neutral SLN or low amounts (10 g/mL) of negatively
charged SLN showed no effect on the cortical cerebrovascular
volume, indicating a good BBB integrity. Higher amounts of
negatively charged SLN (20 g/mL) or positively charged SLN
significantly increased the cortical cerebrovascular volume
pointing out a BBB disruption. Significant increases in vascular
volume were noted in all brain regions when perfused with high
doses (20 mg/mL) of either anionic or cationic NPs. Comparing
organ distribution of tobramycin-loaded SLN and tobramycin
solution, it was found that a statistically higher brain
concentration was achieved with i.v. administered SLN if
compared to duodenal administration. Nevertheless, SLN could
reach the brain after duodenal administration in rats [243]. In
other works, biodistribution studies showed that idarubicinloaded SLN were able to cross the BBB after duodenal
administration [244,245].
Superparamagnetic iron oxide loaded SLN (159 nm) were
proposed as a new type of nuclear magnetic resonance contrast
agent [246]. After i.v. injection in rats, SLN were able to cross
the BBB and to accumulate in the brain parenchyma. A high
signal suppression was found in the hypothalamus, even though
the strongest suppression was in the liquoral space, where SLN
were present in high amounts [116]. Different results were

obtained with charged NPs in vitro. This discrepancy was
probably due to the non-adequate BBB in vitro model, having
low permeability to sucrose and inulin but an electrical
resistance of only 500800 cm2 [115].
A lipophilic pro-drug of 5-fluoro-2-deoxyuridine (FUdR),
the 3,5-dioctanoyl-5-fluoro-2-deoxyuridine (DO-FUdR), was
efficiently incorporated into SLN (76 nm) [247]. Drug brain
concentration (intended as FUdR or DO-FUdR) was higher at
each time point for the group treated with the SLN formulations
with respect to FUdR solution. In the same study, DO-FUdR
pharmacokinetics and body distribution were investigated as
well. Unfortunately, statistical data analysis and information
concerning DO-FUdR formulation (solution, suspension, particle size, stabilizer, etc.) are missing [247], impeding further
comparisons between the brain distribution of DO-FUdR alone
and that loaded into SLN. However, the pro-drug approach can
be very useful in the development of SLN for brain drug
targeting. In fact, not all the therapeutic molecules possess
suitable characteristics to be efficiently incorporated into a lipid
matrix. Moreover, as a result of the low particle uptake available
at the BBB surface, SLN drug content may become a critical
issue for the clinical success of this strategy. For this reason
LDC NPs have been developed [188]. Even though preliminary
in vivo studies showed just an accumulation of NPs on the BBB
surface in place of a real particle uptake [111], it is our opinion
that this elegant approach should be more deeply investigated
because of its enormous potentiality. Another possibility to
maximize NP drug loading may be represented, when possible,
by the formulation of the sole drug into nanocrystals,
successively coated with a proper surfactant (e.g. P80)
[126,248]. This technology would permit the delivery of the
therapeutic molecules to the target site, maximizing the amount
delivered and avoiding all possible toxic effects from the carrier
matrix.
Fig. 11. Situation of the CNS drug research progress updated through September 2005.
7. Concluding remarks
The amazing growth in recent years of CNS drugs on the
market has generated enormous research efforts in an attempt to
develop new drugs for brain diseases. However, the main interest
has been focused on the discovery of new therapeutic molecules
rather than developing new approaches and systems to target
actives to the brain. This is a general trend in pharmaceutical
science. Since the importance of drug delivery to improve
therapies was understood more than 30 years ago, it is deeply
disappointing that most efforts are still oriented to the sole
development of drug discovery programs. Such a picture is
exacerbated in the case of CNS drugs, for which virtually no BBB
programs have ever been accounted [1,2]. In fact, between the 40
and 50% of the compounds under development remained at the
early stages of the discovery process while between the 23 and
30% are those divided within the clinical phase Iphase III
(Fig. 11). Less than 5% of the total molecules under screening
have reached the final step before marketing and less than 1% have
been registered over the past three years. In addition, about 15
20% of molecules have reported no development over a period of
18 months [249251]. Of the whole drug development program,
more than 43% is represented by AD and PD therapeutics,
whereas just about 14% are related to epilepsy, Huntington's
disease, and multiple sclerosis. This disproportion on behalf of
AD and PD is likely a consequence of the large incidence of these
diseases in the population as a result of lifespan increase.
The low number of drugs entering the market reflects the
complexity of the CNS drug development, certainly in part due
to the presence of the BBB. It is clear that this failure is
ascribable to a wrong design in the drug discovery and development programs that overlook the fundamental contribution of
a well sustained drug delivery program. Only the integration of
these two complementary steps from the early stages of the
development pathway can lead to more successful achievements
in brain disease therapy. This is even more evident in light of the
fact that most of the potentially available drugs for AD and PD
therapies are large hydrophilic molecules, e.g. peptides, proteins, and oligonucleotides that do not cross the BBB. Among
the several strategies attempted in order to overcome this problem, properly tailored NPs may have a great potential.
The large amount of evidence regarding brain drug delivery
by means of P80-coated NPs cannot be ignored or considered as
single evidence even though its action mechanism is not
completely understood. Lipid NPs, e.g. SLN, NLC, LDC NPs,
may represent, in fact, promising carriers since their prevalence
over other formulations in terms of toxicity, production
feasibility, and scalability is widely documented in the literature.
The use of surfactants is the main controversial point of this
approach [252], but, at the same time, data on P80 toxicity, when
formulated together with NPs, are less conspicuous [95,253]
than those reported by other authors [97100,102,104,106,109]
and not very convincing [254]. Moreover, such a controversial
point is less applicable to lipid NPs by the fact that the use of a
lipid matrix strongly reduces the possibility of P80 desorption
upon administration [46]. Therefore, the use of these carriers
should be more deeply investigated as brain DDS for newer and
471
older neurotherapeutics. Furthermore, an optimal CNS drug

development strategy should consider the design of suitable
carriers for these drugs as soon as they are demonstrated to be
active but unable to reach their molecular target. This would allow
one to find the proper carrier, according to molecule characteristics, for an early preclinical and clinical evaluation of the
final formulation. Such an approach would certainly improve drug
developing strategies by increasing the number of therapeutics
thus far not exploited but that could effectively reach the market.
8. Legend for lipid substances
Brij 35: Polyoxyethylene (35) lauryl ether
Brij 72: Polyoxyethylene 2 stearyl ether
Brij 78: Polyoxyethylene (20) stearyl ether
Compritol 888 ATO: Mixture of mono-, di-, and triglycerides
of behenic acid (referred in the text as Compritol)
Cremophor ELR: Polyoxyethylated castor oil
Cremophor RH40: Polyoxyl 40 hydrogenated castor oil
Cremophor EL: Polyoxyl 35 castor oil
Dynasan 114: Trimyristin
Dynasan 118: Tristearin
Imwitor: Glycerol monostearates
Pluronic F68: Ethylene oxide/propylene oxide block
copolymer
Softisan 138: Hydrogenated coco glycerides
Softisan 142: Hydrogenated coco glycerides
Solutol HS15: Polyethyleneglycol 660 12-hydroxystearate
Acknowledgment
Thanks are due to Miss Maria Vigilante for kindly revising
the English version of the manuscript.
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Solid Lipid Nanoparticles For Targeted Brain Drug Delivery

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Solid Lipid Nanoparticles For Targeted Brain Drug Delivery

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Advanced Drug Delivery Reviews 59 (2007) 454 477

Solid lipid nanoparticles for targeted brain drug delivery

In the last decade, an emerging interest has been growing

P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477

Among other NP formulations, solid lipid nanoparticles

P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477

characteristics, barely predictable only by considering its

P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477

A, digoxin, dexamethasone, and others) in the brain [70]. P-gp

effective strategy to alter NP biodistribution, enhancing thus their

P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477

in 20% of the treated rats with intracranial transplanted 101/

Fig. 3. Molecular structure of the MePEGCA-HDCA copolymer.

P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477

PEGylated NPs (60 mg/kg) penetrated into the brain to a larger

P80-coated NP endocytosis is believed to be mediated by protein

Fig. 4. Schematic representation of the proposed mechanism of P80-coated NP

P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477

It has been shown that Apo (particularly ApoJ, ApoA-I and

lipoproteins (LDL), in the range from 18 to 25 nm should be

P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477

an issue because of the limited time of exposure. Another

Sterilization of PACA NPs is hardly accounted for in the

P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477

or Compritol has been assessed [171]. Even if cetylpalmitate

P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477

degradation was just slightly sensible to particle size. In fact,

P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477

5. SLN physicochemical features with respect to brain drug

affect physical and chemical stability and suitability of SLN

Lipids are rightly being considered as safe and useful

5.1. SLN structure, drug loading, and stability issues

where V is the molar volume of the hydrophobic moiety of the

Fig. 5. Ternary phase diagram of a generic lipid-based system showing the

Recently, some papers have highlighted the need for a wider

This feature is important if one considers that the particle

P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477

revealed high storage modulus G and loss modulus G1,

Fig. 7. Schematic representation of solid lipid nanoparticle shapes (adapted from

P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477

from the melt occurs in a metastable hexagonal -form which

where T is the melting temperature of a particle with radius r, T0

Fig. 8. Schematic representation of surface area increase upon step-like platelet

analysis trimyristin particles start to crystallize at 10C while

P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477

Characteristic platelet structures were detected by TEM. The

Tripalmitin SLN have been observed to organize into the

P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477

information could be helpful to the development of suitable

P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477

when loaded into SLN [233,234]. As previously shown with

ethanol (as the commercially available Taxol formulation). In

P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477

particles, respectively. It is noteworthy that, for the positively

were present in high amounts [116]. Different results were

P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477

older neurotherapeutics. Furthermore, an optimal CNS drug

P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477

[12] A. Croquelois, G. Assal, J.M. Annoni, F. Staub, A. Gronchi, L.

[38] E. Garcia-Garcia, K. Andrieux, S. Gil, P. Couvreur, Colloidal carriers and

P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477

P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477

P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454477