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FEATURE ARTICLE

Cite this: J. Mater. Chem. B, 2013, 1,

5201

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Recent trends in the use of lipidic nanoparticles as

pharmaceutical carriers for cancer therapy and
diagnostics
Samuel V. Mussi and Vladimir P. Torchilin*
Lipidic nanoparticles have recently gained attention in cancer research. In this review we are focused on the
solid lipid nanoparticle (SLN) and nanostructured lipid carrier (NLC). They have signicant advantages
including low toxicity of the lipids and the controlled release of the drugs incorporated into the matrix.
The recent trends described here contain functions added to nanoparticles to improve the therapeutic
ecacy, such as long-circulation, co-loading of drugs, the combination with RNA/DNA, pH stimulus-

Received 17th July 2013

Accepted 29th July 2013

sensitive drug release, incorporation of agents for imaging and the attachment of ligands for active
targeting. By putting it all together, it may be possible to obtain an ideal multifunctional nanocarrier for
cancer therapy. Among the many eorts made so far to obtain one, SLN/NLC should have a place in this

DOI: 10.1039/c3tb20990c

search for a combined therapeutic and diagnostic system with dramatically enhanced ecacy in cancer

www.rsc.org/MaterialsB

therapy.

Introduction

Solid lipid nanoparticles (SLN) that have size of 501000 nm are

derived from parenteral nutrition emulsions by replacement of
the liquid lipid (oil) of the emulsion droplets with a lipid solid at
room temperature. The lipids used to prepare SLN can be fatty
acids, highly puried triglycerides, complex glyceride mixtures
or waxes and they can be stabilized by surfactants or polymers
and their mixtures.1 Usually, the SLN matrix is composed of
physiological lipids and has low potential of toxicity. This is one
of the most important advantages, although the following
features should also be highlighted the greater physical
stability, control of drug release, alteration of the drug dissolution rate and consequently an enhanced bioavailability, sitespecic targeting, an ability to avoid or minimize degradation of
the entrapped drug, ease manufacturing and industrial scaleup, non-use of organic solvents, and the low cost of lipids. Due
to the similarity of the method of preparation to parenteral
emulsions, SLN have gained attention as a promising alternative over the others well-known drug nanocarriers, such as
liposomes and polymeric nanoparticles. The low loading
capacity and drug expulsion aer polymorphic transition of the
lipid from matrix during storage have been described as the
main disadvantages.24
To overcome these limitations a similar formulation referred
as nanostructured lipid carriers (NLC) was proposed. NLC are
made of a mixture of solid and liquid lipids, resulting in special

Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University,

360 Huntington Avenue, 140 The Fenway, Boston, MA 02115, USA. E-mail:
v.torchilin@neu.edu

This journal is The Royal Society of Chemistry 2013

nanostructures that are solid at room and body temperatures.

Both SLN and NLC share all the advantages but NLC may have
further more benets. This nanocarrier has higher drug incorporation capacity and improved properties for drug release. The
lipid matrix has some dierent characteristics from SLN with an
imperfect crystallization of the solid lipid or even showing an
amorphous prole. This fact enables a drug loading as molecular form or aggregates at matrix imperfections that allow
greater incorporation of drugs, leading also to a lower or to no
drug expulsion aer storage.2,3
The literature has demonstrated the potential of SLN and
NLC as promising candidates for delivery of anticancer agents.
The noticeable features of SLN and NLC such as the capability
to carry anticancer compounds with dierent physiochemical
characteristics, higher drug stability, improved pharmacokinetics and drug activity, make them an attractive carrier for
antitumor drug delivery. Several in vitro and in vivo studies have
demonstrated the potential of these lipidic nanoparticles in the
increase of the ecacy of anticancer agents.4
SLN loaded with doxorubicin were more cytotoxic than free
drug against resistant cancer cell lines and these SLN are
promising as an approach to overcome the chemoresistance of
adriamycin-resistant breast cancer.57 The authors of ref. 8
veried an increase in the uptake and cytotoxicity of SLN loaded
with paclitaxel in the human glioblastoma cell line U-118. They
also evaluated the in vitro and in vivo activity against HCT-15.
These SLN overcame drug resistance and increased the ecacy
of the treatment. Other studies investigated the activity of NLC
loaded with paclitaxel and doxorubicin against dierent cell
lines. These NLC overcame the drug resistance of SKOV3-TR30
cells. NLC loaded with paclitaxel were 31 times more cytotoxic,

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Table 1

Feature Article

New trends with lipid nanocarriers for enhanced tumor activity and diagnosis

Formulation

Drug/agent

Added function

Reference(s)

SLN
SLN
SLN
SLN
SLN
NLC
SLN
SLN

Drug-free/stearic acid-PEG 2000

PK-L4/stearic acid-PEG 2000
Paclitaxel/Pluronic F68 and Brij78
Anthracyclines/Pluronic F68
Chemosensitizer GG918 (Elacridar)/doxorubicin
Simvastatin/tocotrienol
Doxorubicin/mitomycin
Doxorubicin/ceramide/oligonucleotide
(MBO-asGCS)
Paclitaxel/siRNA
Doxorubicin/DHA (ionic pair)
Doxorubicin free base
Triamcinolone acetonide/n-glutaryl
phosphatidylethanolamine coating
Superparamagnetic iron oxide
99m
Tc
64
Cu
Paclitaxel/siRNA/quantum dot

PEGylation
PEGylation
PEGylation
PEGylation
Co-loading
Co-loading
Co-loading
Co-loading

15 and 19
21 and 22
23
24
29
30
31 and 32
33

Co-loading/pegylation/gene delivery
Co-loading/pH
1 stimulus-sensitive
pH stimulus-sensitive
pH stimulus-sensitive

34
35
7
38

Imaging diagnostic
Imaging diagnostic
Imaging diagnostic
PEGylation/co-loading/imaging
diagnostic/gene delivery
Active targeting/imaging diagnostic

39
40
42
41

Active targeting
Active targeting
Active targeting
Active targeting/PEGylation/gene delivery
Active targeting/PEGylation/gene delivery
Active targeting/PEGylation

45
46
47
48
49
50

Active targeting/PEGylation

51

Active targeting/PEGylation/co-loading
Active targeting/PEGylation/ph stimulus-sensitive
Active targeting

52
53
59

SLN
SLN
SLN
SLN
SLN
SLN
SLN
SLN
SLN
SLN
SLN
SLN
SLN
SLN
NLC
SLN
SLN
NLC
NLC

Integrin-specic ligand cyclic Arg-Gly-Asp

(cRGD)/quantum dots
Doxorubicin/mannose
Paclitaxel/octaarginine
Doxorubicin/anti-EGFR
Dexamethasone/transferrin/plasmid GFP
Transferrin/mannan/plasmid GFP
Docetaxel/folate-poly(PEG-cyanoacrylateco-cholesteryl cyanoacrylate)
Paclitaxel/folate-poly(ethylene glycol)
(PEG)-phosphatidylethanolamine
Docetaxel/ketoconazole/folate
Docetaxel/cell-penetrating peptide/folate
Paclitaxel/hyaluronic acid

and NLC loaded with doxorubicin were 2.5 more eective than
the respective free drugs.9,10 SLN loaded with docetaxel were
shown to be more eective than the commercial docetaxel,
Taxotere, in vitro against colorectal (C-26) and malignant
melanoma (A-375) cell lines. In in vivo tests with C-26-implanted
BALB/c mice, SLN loaded with docetaxel showed better tumor
inhibitory ecacy and survival than Taxotere.11
SLN carrying dierent antineoplastic agents (doxorubicin,
paclitaxel, cholesteryl butyrate, and anti-VEGF antisense oligonucleotides) were tested in experimental animal models of
cerebral gliomas and induced an eective anti-tumoral therapeutic response.12 An explanation involves the accumulation of
drugs encapsulated in SLN in brain aer intravenous administration. This was attributed to the transport of intact SLN
through the bloodbrain barrier and also to the sustained
release from the lipid matrix.12,13,14 Pharmacokinetics studies
showed a prolonged circulation up to 24 hours, a signicant
increase in t1/2, Cmax and low uptake of SLN by the reticuloendothelial system (RES). A lower concentration of doxorubicin
was found in heart when administered as SLN, indicating the
possibility of lower cardiotoxicity compared to the commercial
doxorubicin solution, which is one of the eects limiting the
usefulness of doxorubicin.15

5202 | J. Mater. Chem. B, 2013, 1, 52015209

31

Thus, SLN and NLC can oer many benets for cancer
therapy. Recent works have described new proposals to further
optimize chemotherapy using these drug delivery systems. They
have been successfully multifunctionalized to capture a payload
of drugs, to target specic cells and to release entrapped drugs
in a controlled manner. The following topics of this review focus
on these new trends, related to the addition of more functions
to these nanoparticles, such as PEGylation, co-encapsulation of
anticancer agents, pH-sensitivity, magnetic nanoparticle
loading and other agents for imaging and for attachment of
ligands on the surface (see Table 1).

2 PEGylated lipid nanoparticles longevity

in the blood
Nanoparticles are rapidly opsonized and removed from the
blood circulation without entering the tumor once they are
identied as foreign particles by the body's defence system
(reticuloendothelial system RES). One important property
aecting the ecacy of a nanocarrier is its blood longevity, and
long-circulating pharmaceuticals currently represent an
important area of research on cancer drug delivery systems. The
long-circulating nanoparticles will slowly accumulate in solid

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Feature Article
tumors due to the defective vasculature, resulting in a passive
targeting. This is a well-established phenomenon, termed the
enhanced permeability and retention (EPR) eect.16,17 Poly(ethylene)glycol (PEG) derivatives and others hydrophilic polymers have been widely employed to modify nanoparticles
surface. This modication results in a steric stabilization of the
carriers and reduction of their uptake by the RES cells. The
steric stabilization is a phenomenon in which the solid
particulates are protected from interactions with dierent
solutes and this term has been used to describe the polymermediated protection.18
Drug-free PEGylated SLN and non-PEGylated SLN were
prepared and injected intravenously in rats to study their tissue
distribution and transport across the bloodbrain barrier.19 The
average size of these lipid nanoparticles was below 100 nm. To
PEGylate the SLN, stearic acid-PEG 2000 at 0.15% w/v was
added. Localization of SLN toward in the brain and in the
cerebrospinal uid (CSF) was higher for both formulations
comparing to the free drug. However, there was a lower uptake
in the liver and lung for PEGylated-SLN than for the nonPEGylated-SLN, conrming the inuence of PEGylation.19
Another study investigated the potential of the same PEGylated
and non-PEGylated SLN for improved parenteral delivery of
doxorubicin.20 The pharmacokinetics and tissue distribution of
doxorubicin in these SLN were compared with a commercial
solution of doxorubicin. There was a signicantly prolonged
circulation, an increase of t1/2 and of Cmax with PEGylated SLN.20
The inuence of stearic acid-PEG 2000 concentration for these
SLN was also evaluated. The AUC of doxorubicin increased as a
function of the amount of PEGylating agent present in the SLN.
Doxorubicin was detected in blood 6 hours post-injection from
both non-PEGylated and PEGylated SLN, while no doxorubicin
was detectable aer intravenous injection of doxorubicin solution. The highest concentration of doxorubicin in the blood was
detected in PEGylated SLN at the highest concentration of
stearic acid-PEG 2000.15
The PEGylation of SLN also increased their cytotoxicity and
uptake for some cancer cell lines. PK-L4 was synthesized as a
potential anticancer compound. IC50 values were signicantly
lower for PK-L4-loaded SLNs with stearic acid-PEG 2000 in the
human lung carcinoma cell line (A549) and a human hepatocellular liver carcinoma cell line (HepG2). This eect was
strongly connected to the SLN incorporation of PEG and was
attributed to the degradation delay using stabilizing surfactants
such as stearic acid-PEG 2000. However, further investigations
about the mechanism need to be done. There was a signicantly
higher uptake of the PK-L4-loaded SLN PEGylated with stearic
acid-PEG 2000 at 10 wt% than with the plain SLN, but the
cytotoxicity was lower and the IC50 of the PEGylated SLN was
higher than with plain SLN.21,22
Long-circulating SLN made with two dierent surfactants,
Pluronic F68 (also known as poloxamer) and Brij78, revealed
marked dierences in pharmacokinetic parameters, especially
in the t1/2 and AUC, in comparison with Cremophor EL
containing a solution of paclitaxel.23 F68 SLN and Brij 78-SLN
were long-circulating with a 7.4-fold and 3.6-fold higher t1/2,
respectively. This was attributed to the reduced uptake of SLN

This journal is The Royal Society of Chemistry 2013

Journal of Materials Chemistry B

by the mononuclear phagocytic system and also to the lower
clearance rate. SLN prepared with F68 had longer circulation
than Brij 78 due to its longer PEG chain length. Thus, these
surfactants used to prepare the SLN are at the same time
PEGylating agents, performing a dual function. Many of the
surfactants used to prepare lipid nanoparticles are PEGylated
and investigations on their use to obtain long circulating
particles continue.23 Another advantage of the PEGylation
using Pluronic block copolymers may be better results in
treatment of drug-resistant tumors. These block copolymers
interact with multidrug-resistant cancer (MDR) tumors,
resulting in drastic sensitization of these tumors to various
anticancer agents, particularly anthracycline antibiotics.
Pluronic aects several distinct drug resistance mechanisms
including inhibition of drug eux transporters.24
Although few other hydrophilic polymers in addition to PEG
have been suggested to prepare long-circulating pharmaceutical
nanocarriers (such as polyvinyl pyrrolidone, polyacryl amide,
polyhydrazones, polymorpholines, etc.), there are still no published studies on the use of non-PEG polymers for SLN or NLC
loaded with anticancer compounds and, probably, studies on
this subject are under way.

3 Combination therapy co-loading of

anticancer agents into lipid nanoparticles
Combination chemotherapy regimens in cancer treatment have
shown improved anticancer activity compared to a single drug,
but these regimens are also more toxic than single agent regimens.25,26 Moreover, the varieties of drug administration
schedule have complicated the management of the pharmacokinetic and pharmacodynamic proles. Thus, obtaining
uniform temporal and spatial co-delivery remains challenging.
The combination of drugs co-delivered in a drug delivery system
(DDS) has emerged as an attractive alternative.27 DDS-based
combination therapy oers many advantages, such as more
synchronized, controlled pharmacokinetics of the drugs,
improved drug solubility and bioavailability and the potential to
bypass mechanisms of multidrug resistance. Overall, these
combinations can result in improved drug ecacy with a single
formulation.27,28
To optimize the eectiveness of a combinational approach
with doxorubicin and a chemosensitizer, GG918 (Elacridar), a
SLN capable of co-delivering both drugs was prepared. The
encapsulation eciency for both drugs was high (more than
80%) and not compromised by the co-encapsulation. In vitro
evaluations against the human MDR breast cancer cell line
MDA435/LCC6/MDR1 demonstrated the greatest doxorubicin
uptake and anticancer activity against these MDR cells for SLN
co-loaded with both drugs. Co-administration of two singleagent loaded PLN was least eective.29
Association of tocotrienols and statins has been proven to
synergistically inhibit the growth of highly malignant +SA
mammary epithelial cells in vitro. The encapsulation of simvastatin and a tocotrienol rich fraction (TRF) in NLC was
proposed to further increase the activity. Studies evaluated
their antiproliferative eect and showed the potency of the

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combined treatment in NLC. The IC50 of the NLC loaded with
simvastatin and TRF was 34-fold lower than alpha-tocopherol
nanoparticles, which was used as reference. The addition of
TRF generated compartments into the solid matrix that
promoted the entrapment of simvastatin. The presence of
these compartments were veried by dierential scanning
calorimetry and X-ray diraction analysis that showed an
absence of endothermic or crystalline peaks for the mixture
of the solid lipid and TRF (liquid). Thus, the combination of
TRF and simvastatin entrapped in NLC emerges as an alternative to support the NLC formation and at the same time to
promote an increase of the activity.30
SLN co-loaded with doxorubicin and mitomycin were eective in killing MDR cells in vitro with up to 30-fold lower doses
than the free drugs. This increase was correlated with coencapsulation of dual agents into a nanoparticle formulation. It
was much more eective than concurrent application of single
agent-containing SLN, demonstrating a requirement for
simultaneous uptake of both drugs by the same cells to enhance
the drug synergy.31 These SLN were evaluated in vivo and
demonstrated enhanced ecacy compared to liposomal doxorubicin with a 3-fold increase in animal life span, a 1020%
tumor cure rate, undetectable normal tissue toxicity and
decreased tumor angiogenesis. Thus, the combination of
doxorubicin and mitomycin in SLN can be used to overcome
multidrug resistance at a signicantly lower dose than the
respective free drugs, exhibiting the potential for enhanced
chemotherapy and reduced therapeutic limitations imposed by
systemic toxicity.32
SLN loaded with doxorubicin, ceramide and the mixed
backbone antisense glucosylceramide synthase oligonucleotide
(MBO-asGCS) were proposed.33 The sphingolipid ceramide was
part of the lipid matrix and at the same time could contribute to
the antitumor activity by triggering apoptosis in cancer cells.
Since the glycosylation of ceramide results in its inactivation,
the combination with MBO-asGCS could promote the increase
of ceramide into the cells and promote apoptosis. Due to the
oligonucleotides anionic charge, it was attached to the cationic
SLN surface by ionic attraction. The NCI/ADR-RES cells were
treated with separate SLN: one loaded with doxorubicin and
another carrying MBO-asGCS. The viability of cells treated with
SLN loaded with MBO-asGCS and doxorubicin was much lower
than that with SLN loaded with doxorubicin. This suggested
enhanced apoptosis due to cell sensitization and the eectiveness of intracellular delivery of MBO-asGCS, ceramide and
doxorubicin by SLN.33
SLN for co-delivery paclitaxel and siRNA have also been
formulated.34 The SLN was PEGylated with methoxypolyethylene glycol 2000-distearoyl phosphatidylethanolamine
(mPEG-DSPE). The positive charge needed to attach the siRNA
was obtained with the incorporation of 3b[N-(N0 ,N0 -dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol). The SLN
increased the cellular uptake and the attachment of the therapeutic siRNA, human MCL1-specic siRNA (siMCL1) and
resulted in reduction of the MCL1 mRNA levels. The siMCL1
complexed to SLN loaded with paclitaxel showed a greater in
vitro anticancer eect against KB cells than SLN loaded only

5204 | J. Mater. Chem. B, 2013, 1, 52015209

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with paclitaxel. In KB cell-xenograed mice, this SLN loaded
with paclitaxel and complexed with siMCL1 signicantly
reduced the growth of tumors. Thus, it veried the potential of
SLN for the co-delivery of lipophilic anticancer drug and therapeutic siRNA.
Our previous work describes a new SLN co-loaded with
doxorubicin and docosahexaenoic acid (DHA), an omega 3
polyunsaturated fatty acid that shows synergic eect with
anthracyclines.35 Since doxorubicin is a water soluble drug, its
encapsulation in a matrix lipid is challenging. We proposed
the formation of an ionic pair between the cationic doxorubicin molecule and the anionic DHA in situ, during the SLN
preparation, to improve drug encapsulation and anticancer
activity. The formulation had a size of about 90 nm, entrapment eciency 100% and drug loading of 31 mg g
1 for
doxorubicin. In vitro evaluation showed higher uptake for
drugs encapsulated in SLN compared to the free drugs as well
as higher cytotoxicity against the human lung cancer cell line,
A549.35

4 pH-sensitive lipid nanoparticles for drug

release at lowered pH
The use of acid-sensitive drug carriers has been one of the most
extensively studied active triggering strategies on cancer
research due to the characteristic excess acidity of tumor
regions. Compared to normal tissues, tumors typically exhibit a
weak acidity with pH of 6.8. Upon cellular uptake, drug-loaded
nanocarriers will be subjected to an intracellular pH of 5.96.2
in early endosomes and pH of 5.05.5 in late endosomes and
lysosomes. Thus, nanocarriers responsive to these pH alterations may provide an alternative type of targeting strategy to
further improve therapeutic ecacy by cancer site-specic
delivery and controlled release.36,37
The SLN loaded with doxorubicin and DHA described in
ref. 35 showed an acid-specic drug release of more than
80% of doxorubicin at a pH of 5.0. The ionic pair strategy
used to increase the encapsulation of doxorubicin is unstable
at acidic pH and the higher drug release was attributed to it
(see Fig. 1). Higher doxorubicin release at pH of 5 for SLN
loaded with doxorubicin free base was also veried.7 They
converted the doxorubicin hydrochloride to doxorubicin free
base to increase the encapsulation into the lipid matrix,
since the free base is more lipophilic. This higher doxorubicin release was explained by the cationization of the
molecule at acidic pH, which increased the hydrophilic
character due to the basic nature of doxorubicin (pKa 8.3).
Thus, the cationic doxorubicin has higher solubility at lower
pH and reduced anity with the SLN matrix. In another
study, SLN were prepared using polysorbate 80 as surfactant
and tripalmitin glyceride and n-glutaryl phosphatidylethanolamine (n-glut) as lipid components. The n-glut is an
acidic pH-sensitive derivative of phosphatidylethanolamine.
The results indicated that the carrier had increased triamcinolone acetonide release under acidic conditions (pH 4
to 6).38

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Fig. 1 Schematic of doxorubicin release from SLN at lowered pH. The high encapsulation of doxorubicin into the lipid matrix was achieved by an in situ ionic pair
formation that resulted in a lipophilic salt [(1) DOX-DHA Ionic pair], as previously described in ref. 35. At low pH of 5, the DHA is protonated (2) and will undo the ionic
interaction with doxorubicin (3). Due to the hydrophilic character of the cationic doxorubicin, its diusion from the lipid matrix would be faster and result in enhanced
drug release (4).

5 Imaging agents based on lipid

nanoparticles
SLN are under investigation as a pharmaceutical tool to stably
carry various types of diagnostic agents, such as superparamagnetic iron oxide,39 technetium-99 (99mTc),40 64Cu42 and
quantum dots.31,41
Superparamagnetic SLN have been proposed for magnetic
resonance imaging (MRI) contrast.39 Iron oxide was encapsulated in SLN and in vitro analysis of the relaxometric properties
showed similar results to the commercial superparamagnetic
iron oxide nanoparticles (Endorem). The authors also evaluated the in vivo MRI of the central nervous system (CNS) with
SLN and veried a slower blood clearance than Endorem. MRI
and uptake by CNS data showed that both contrast agents lasted
up to the end of the experiment (135 min). Thus, these results
showed that the SLN are able to bypass the BBB and suggest the
potential for use as a CNS MRI contrast agent.
Assessment of inhaled radiolabelled SLN biodistribution
was described by ref. 40. SLN were radiolabelled with 99mTc
using the lipophilic chelator D,L-hexamehylpropyleneamine
oxime (HMPAO). Aer inhalation of 99mTc-HMPAO-SLN, it was
seen in the biodistribution studies a high uptake in lymphatics
and high rate of distribution in periaortic, axillar and inguinal
lymph nodes. Gamma scintigraphic images also showed a
strong deposition of 99mTc-HMPAO and 99mTc-HMPAO-SLN on
the lung but 99mTc-HMPAO was rapidly cleared via systemic
circulation. They veried 4 hours aer inhalation a higher

This journal is The Royal Society of Chemistry 2013

accumulation of 99mTc in lung for 99mTc-HMPAO-SLN and it was

attributed to the higher particle retention. Thus, they claim
99m
Tc-HMPAO-SLN as an alternative for targeting colloidal
carriers to the lymphatics and for using as a lymphoscintigraphic agent.
The authors of ref. 42 proposed a SLN radiolabeled with
64
Cu for in vivo positron emission tomography (PET) studies.
A copper specic chelator, 6-[p-(bromoacetamido)benzyl]1,4,8,11-tetraazacyclotetradecane-N,N 0 ,N 00 ,N-tetraacetic
acid
(BAT) conjugated with a synthetic lipid was incorporated into
SLN and successfully radiolabeled with 64Cu. Stability assays
conrmed that copper remains associated with the SLN over a
48 hours time period. They veried a 64Cu circulating in the
blood aer 3 hours of injection and an accumulation in the
liver. This pattern of biodistribution was attributed to the 64Cu
associated with SLN. The results indicate the potential for a
quantitative evaluation and in vivo PET imaging for 64Cu-SLN.
SLN loaded with quantum dots for anticancer theragnostics
and delivery of a paclitaxel-siRNA combination were recently
proposed.41 Quantum dots and paclitaxel were incorporated
into the SLN lipid matrix while the anionic siRNA molecules
were electrostatically complexed with the outer cationic surface.
The SLN loaded with paclitaxel-siRNA were eciently taken up
into human lung carcinoma cells and exhibited synergistic
anticancer activity. The strong uorescence from quantum dots
within SLN enabled in situ visualization of the intracellular
translocation of SLN into cancer cells and suggests that this
formulation could be useful as a multifunctional and optically

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traceable nanocarrier for ecient anticancer theranostics.
However, the study was just an in vitro evaluation, and further
investigations, rst of all, in vivo studies, should be done to
verify the real potential of this formulation as a diagnostic tool.
Indeed, the quantum dots are being successfully used for the
sentinel lymph node mapping and tumor angiogenesis
imaging. The emission color of quantum dots can be precisely
tuned from the ultraviolet to the near-infrared, and they are
extremely bright and photostable.60
An integrin-targeted near infrared light-emitting SLN has
been developed by ref. 31. Tumor cells and tumor vascular
endothelium overexpress a(v) b(3) integrin receptors and thus
are useful targets for imaging, chemotherapy and anti-angiogenic therapy. SLN were conjugated with the a(v) b(3) integrinspecic ligand, cyclic Arg-Gly-Asp (cRGD), and loaded with near
infrared quantum dots. Live animal imaging was performed
using nude mice bearing xenograed orthotopic human breast
tumors or dorsal window chamber breast tumors. The active
tumor targeting with cRGD showed a double-edged sword
eect: specic RGD-SLN accumulation in tumor blood vessels
and increased tumor residence time. Thus, the RGD-SLN may
perhaps be useful for tumor neovascular-specic therapies and
imaging applications.

6 Attachment of targeting ligands to the

surface of lipid nanoparticles
Currently, one of the biggest challenges in cancer therapy is to
obtain a drug or a carrier that are able to reach specically the
tumor area, minimizing the accumulation and toxicity in
healthy tissues and organs. Thus, the success of the treatment
with higher ecacy and lower toxicity depends on the ability of
the therapeutics to achieve their target sites. The attachment of
ligands to the surface of nanoparticles to improve the specicity
for cancer cells has emerged as an attractive tool and it has been
widely described.43 This addition makes them able to selectively
bind in cancer cells and enhance the internalization. The
endocytosis mediated by receptor has been extensively explored
as a way to increase internalization of nanoparticles. These
ligands are thought and designed to bind specically in receptors over-expressed in cancer cells and minimally in healthy
tissues. Among many molecules and structures described as
ligands, there are antibodies, antibody fragments, proteins,
small molecules (sugar, folate, vitamins and others), aptamers
and peptides.44 Furthermore, many works proposed a modication of the nanoparticles surface with PEG derivatives for
enhance the circulation time in the blood and at the same time
for incorporation of ligands. The attachment of ligands at the
end of the PEG spacer arm keeps them out of the PEG brush,
reducing the steric impediment and facilitating the binding to
target receptors.18
A mannose coating of SLN, loaded with doxorubicin was
proposed to target lectin-like receptors overexpressed in cancer
cells.45 The mannose was previously linked to stearylamine by a
chemical reaction and then added to the SLN. The ex vivo
evaluation showed that mannosylated-SLN were less hemolytic
than the uncoated SLN and free doxorubicin. Mannosylated-

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SLN were more cytotoxic and were preferably taken up by A549
tumor cells. Pharmacokinetic studies revealed improved
bioavailability, longer half-life and increased mean residence
time of doxorubicin upon mannose conjugation. They also
veried a higher concentration of doxorubicin in tumors for the
mannosylated-SLN as well as lower damage to renal and hepatic
tissues. Thus, the attachment of the small molecule mannose to
the SLN surface seems to be promising to target cancer cells.
However, studies concerning the higher ecacy in vivo of this
formulation have to be investigated.
SLN loaded with paclitaxel and modied with octaarginine
(R8-PTX-SLN) were proposed by ref. 46. The octaarginine was
attached to the SLN through a linkage with stearic acid. The
results showed a higher cytotoxicity against human lung cancer
cells (A549) and an increase of the cell uptake. The authors of
ref. 47 developed SLN loaded with doxorubicin and the antiepithelial growth factor receptor, anti-EGFR, which was electrostatically attached to the catanionic (cationic and anionic)
surface of the particle. Immunochemical staining revealed that
the crosslinked anti-EGFR on the surface of SLN preserved a
high specicity for EGFR on U87MG cells, and it induced a
growth-inhibition eect. Another study proposed a linkage of
transferrin (Tf) on polyethylene glycol-phosphatidylethanolamine (PEG-PE) to obtain Tf-PEG-PE ligands. This polymer was
used for the surface modication of a cationic SLN for dual gene
(plasmid pEGFP) and dexamethasone delivery. In vitro and in
vivo studies showed increase in transfection due to the attachment of Tf.48
To target cancer cells and macrophages in the liver, SLN were
modied with dual ligands Tf and mannan (M). Tf and M were
linked to polyethylene glycol-phosphatidylethanolamine (PEGPE) and PE separately to get transferrin-PEG-PE (T-PEG-PE) and
mannan-PE (M-PE) ligands for the surface modication of SLN.
The in vivo transfection eciency was veried using uorescence protein plasmid (GFP) that was promoted in the presence
of the dual ligands. The evaluation in tumor-bearing animal
models showed that Tf-PEG-PE and M-PE functioned as excellent ligands to improve the cell targeting ability of the carriers.49
A folate-poly(PEG-cyanoacrylate-co-cholesteryl cyanoacrylate)
(FA-PEG-PCHL) was synthesized to modify docetaxel-loaded
NLC and to obtain a long blood circulating eect and improved
targeting.50 This is an amphiphilic copolymer able to locate at
the nanoparticle interface, exposing the PEG chain to the
aqueous phase with the folate moiety at the end. In vivo studies
indicated signicant dierences in the pharmacokinetic prole
and tissue distribution for FA-PEG-PCHL-modied docetaxelloaded NLC compared to the plain docetaxel-loaded NLC. The
PEGylation contributed to an increase of the AUC, t1/2 and MRT,
and there was a signicant accumulation of docetaxel in tumors
due to the folic acid attachment. The authors of ref. 51 veried
that the modication of paclitaxel-loaded SLN with folatepoly(ethylene glycol) (PEG)-phosphatidylethanolamine exhibited a higher tumor inhibition rate in mice with sarcoma180
ascites tumors. It was proposed the co-loading of docetaxel and
ketoconazole in SLN graed with folic acid, which was incorporated by a stearic acid and folic acid conjugate (SA-FA).52 Both
drugs are metabolized hepatically by the cytochrome P-450

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system, and ketoconazole can inhibit p-gp eux of docetaxel at
the blood brain barrier. Thus, a potential drugdrug interaction
for these agents can be useful. The authors veried that the
combination of drugs in SLN plus attached folic resulted in
increased brain uptake of docetaxel and it was 44 times higher
than the commercial Taxotere, which suggests promise for
brain tumor treatment.
NLC loaded with paclitaxel were modied with tumor
microenvironment-sensitive polypeptides (TMSP) and folate.53
The aim of the ligands combination was to create a more
selective and ecient system to deliver the drug in cancer cells.
The TMSP and folate were conjugated to the distal ends of
DSPE-PEG2000-maleimide and DSPE-PEG5000-amine, respectively (see Fig. 2). The folate was attached to increase the
endocytosis by the higher interaction with folate-receptors,
which are overexpressed in some tumors. TMSP are shielded
in the blood circulation but are activated by matrix

Journal of Materials Chemistry B

metalloprotease-2 and -9 (MMP-2/9) that are enzymes oversecreted in tumor tissues. This activation occurs aer a cleavage
by the MMP-2/9 that enables the TMSP to penetrate into the cell.
The authors conrmed this cleavage of TMSP by MMP-2/9
through an in vitro proteolysis assay. The cellular uptake and
cytotoxic of F/TMSP-DTX-NLC were improved in HT-1080, KB,
MCF-7 and A549 cells, and they were attributed to the attachment of folate and TMSP. The penetration into the interior of
tumor spheroids was deeper for F/TMSP-DTX-NLC. In vivo
evaluations using a nude mice model with KB tumor cells
showed a stronger antitumor ecacy and enhanced tumor cell
apoptosis.53
NLC coated with hyaluronic acid (HA) and paclitaxel-loaded
(PXT-NLC) were prepared. The HA was attached via electrostatic
attraction to the cationic PTX-NLC. In vitro investigation using
B16, CT26 and HCT116 cell lines showed that the cytotoxicity of
HA-PTX loaded NLC (HA-PTX-NLC) against these three cancer

Fig. 2 (A) A schematic of the dual-ligand DTX-NLC simultaneously modied with folate and TMSP (F/TMSP-DTX-NLC). (B) The schematic of the multivalent interactions
of F/TMSP-DTX-NLC with cancer cells. Reprinted from ref. 53, Copyright (2013), with permission from Elsevier (License number 3133680405511).

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Journal of Materials Chemistry B

cell lines was superior to that of Taxol. The in vivo evaluation
in B16-bearing Kunming mice showed an increased antitumor
eect of HA-PTX-NLC compared with Taxol. Furthermore, a
prolonged circulation time for PTX in the blood was obtained
plus an increased accumulation in the tumor.59
As one can see, there are quite a few studies describing
various ligands, such as antibodies, antibody fragments,
proteins, small molecules like folate, and peptides, attached to
SLN or NLC loaded with anticancer compounds (with aptamers
remaining probably the only ligand candidate not tested with
SLN and NLC yet).

7 Prospects for SLN and NLC in cancer

therapy
Much work has been described about multifunctional nanocarriers for improved cancer treatment.18,5458 According to ref.
18, an ideal multifunctional pharmaceutical nanocarrier would
provide highly ecient and specialized system for delivery of
drugs, genes, and diagnostic agents. This system would have
individual functions acting in synchronized way, with a more
optimized temporal, spatial deposition and release pattern of
the anticancer agents.
Such promising multifunctional nanocarriers could possess
many useful functions in the same carrier system such as (1)
long-circulation (PEGylation), (2) co-loading of two or more
drugs, (3) magnetic particles loaded into the carrier together
with the drug to allow for carrier sensitivity towards an external
magnetic eld and its use as a contrast agent for magnetic
resonance imaging, (4) pH sensitivity to release drug in acidic
areas of tumors, (5) ligands attached to the surface for active
targeting (monoclonal antibody, peptides, proteins, small
molecules, aptamers), (6) contrast agents for imaging purposes
(heavy metals 111In, 99mTc, Gd, Mn for gamma- or MR imaging
application), (7) cell-penetrating capacity (cell-penetrating
peptide CPP) for carrier-enhanced uptake, (8) a gene-carrying
nanocarrier such as a lipoplex or polyplex (DNA or RNA complexed by the carrier via the carrier surface positive charge). (see
Fig. 3).18,55
All these functions have been addressed at least somewhat in
this review and are summarized in Table 1. The nanoparticles
described have each been developed with a few of the outlined

Fig. 3 The schematic structure of an idealized multifunctional pharmaceutical

nanocarrier. (1) long-circulation PEGylation; (2) co-loading of two or more
drugs; (3) loading of magnetic particles; (4) pH stimulus-sensitivity; (5) ligands
attached to the surface for active targeting; (6) contrast nanocarrier for imaging
purposes; (7) cell-penetrating peptide; (8) gene-carrying.

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functions mentioned. However, the combination of all functions
would be very challenging. Much eort has been done with
liposomes and polymeric nanoparticles to obtain multifunctional nanocarriers. However, the advantages of SLN and NLC
over the others nanosystems qualify them as promising candidates in the development of combined therapeutic and diagnostic multifunctional pharmaceutical nanocarrier systems.

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