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Cetuximab-oxaliplatin-liposomes for Epidermal Growth Factor receptor targeted chemotherapy of colorectal cancer.


Abstract and Figures

Oxaliplatin (L-OH), a platinum derivative with good tolerability is currently combined with Cetuximab (CTX), a monoclonal antibody (mAb), for the treatment of certain (wild-type KRAS) metastatic colorectal cancer (CRC) expressing epidermal growth factor receptor (EGFR). Improvement of L-OH pharmacokinetics (PK) can be provided by its encapsulation into liposomes, allowing a more selective accumulation and delivery to the tumor. Here, we aim to associate both agents in a novel liposomal targeted therapy by linking CTX to the drug-loaded liposomes. These EGFR-targeted liposomes potentially combine the therapeutic activity and selectivity of CTX with tumor-cell delivery of L-OH in a single therapeutic approach. L-OH liposomes carrying whole CTX or CTX-Fab' fragments on their surface were designed and characterized. Their functionality was tested in vitro using four human CRC cell lines, expressing different levels of EGFR to investigate the role of CTX-EGFR interactions in the cellular binding and uptake of the nanocarriers and encapsulated drug. Next, those formulations were evaluated in vivo in a colorectal cancer xenograft model with regard to tumor drug accumulation, toxicity and therapeutic activity. In EGFR-overexpressing cell lines, intracellular drug delivery by targeted liposomes increased with receptor density reaching up to 3-fold higher levels than with non-targeted liposomes. Receptor specific uptake was demonstrated by competition with free CTX, which reduced internalization to levels similar to non-targeted liposomes. In a CRC xenograft model, drug delivery was strongly enhanced upon treatment with targeted formulations. Liposomes conjugated with monovalent CTX-Fab' fragments showed superior drug accumulation in tumor tissue (2916.0 ± 507.84 ng/g) compared to CTX liposomes (1546.02 ± 362.41 ng/g) or non-targeted liposomes (891.06 ± 155.1 ng/g). Concomitantly, CTX-Fab' targeted L-OH liposomes outperformed CTX-liposomes, which on its turn was still more efficacious than non-targeted liposomes and free drug treatment in CRC bearing mice. These results show that site-directed conjugation of monovalent CTX-Fab' provides targeted L-OH liposomes that display an increased tumor drug delivery and efficacy over a formulation with CTX and non-targeted liposomes. Copyright © 2015. Published by Elsevier B.V.
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Cetuximab-oxaliplatin-liposomes for epidermal growth factor receptor
targeted chemotherapy of colorectal cancer
Sara Zalba
, Ana M. Contreras
, Azadeh Haeri
, Timo L.M. ten Hagen
, Iñigo Navarro
Gerben Koning
, María J. Garrido
Department of Pharmacy and Pharmaceutical Technology, University of Navarra, 31008 Pamplona, Spain
Laboratory Experimental Surgical Oncology, Department of Surgery, Erasmus MC Cancer Institute, Rotterdam 3015 GE, The Netherland
Department of Chemistry and Edaphology, University of Navarra, 31008 Pamplona, Spain
abstractarticle info
Article history:
Received 23 February 2015
Received in revised form 8 May 2015
Accepted 11 May 2015
Available online 19 May 2015
Chemical compounds studied in this article:
Oxaliplatin (PubChem CID: 5310940)
hydroxysuccinimide-ester (PubChem CID:
(PubChem CID: 2734570)
tetramethylindocarbocyanine perchlorate
(PubChem CID: 16212735)
β-Mercaptoethanol (PubChem CID: 1567)
Hydrogenatedphosphatidylcholine (PubChem
CID: 94190)
(polyethylenglycol)-2000] (PubChem CID:
Cholesterol (PubChem CID:5997)
(polyethylene glycol) 2000] (PubChem
Targeted liposomes
Colorectal cancer
Oxaliplatin (L-OH), a platinum derivative with good tolerability is currently combined with Cetuximab (CTX), a
monoclonal antibody (mAb), for the treatment of certain (wild-type KRAS) metastatic colorectal cancer (CRC)
expressing epidermal growth factor receptor (EGFR).
Improvement of L-OH pharmacokinetics (PK) can be provided by its encapsulation into liposomes, allowing a
more selective accumulation and delivery to the tumor. Here, we aim to associate both agents in a novel liposo-
mal targeted therapy by linking CTX to the drug-loaded liposomes. These EGFR-targeted liposomes potentially
combine the therapeutic activity and selectivity of CTX with tumor-cell delivery of L-OH in a single therapeutic
L-OH liposomes carrying whole CTX or CTX-Fabfragments on their surface were designed and characterized.
Their functionality was tested in vitro using four human CRC cell lines, expressing different levels of EGFR to
investigate the role of CTX-EGFR interactions in the cellular binding and uptake of the nanocarriers and encapsu-
lated drug. Next, those formulations were evaluatedin vivo in a colorectalcancer xenograft model with regard to
tumor drug accumulation, toxicity and therapeutic activity.
In EGFR-overexpressing cell lines, intracellular drug delivery by targeted liposomes increased with receptor
density reaching up to 3-fold higher levels than with non-targeted liposomes. Receptor specic uptake was
demonstrated by competition with free CTX, which reduced internalization to levels similar to non-targeted
liposomes.In a CRC xenograft model, drug delivery was strongly enhanced upon treatment with targetedformu-
lations. Liposomes conjugated with monovalent CTX-Fabfragments showed superior drug accumulation in
tumor tissue (2916.0 ± 507.84 ng/g) compared to CTXliposomes (1546.02 ± 362.41 ng/g) or non-targeted lipo-
somes (891.06 ± 155.1 ng/g). Concomitantly, CTX-Fabtargeted L-OH liposomes outperformed CTX-liposomes,
which on its turn was still more efcacious than non-targeted liposomes and free drug treatment in CRC bearing
These results show thatsite-directed conjugationof monovalent CTX-Fabprovides targetedL-OH liposomes that
display an increased tumor drug delivery and efcacy over a formulation with CTXand non-targeted liposomes.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
Platinum derivatives are widely used in cancer chemotherapy. They
are used in the chemotherapeutic treatment of approximately 70% of
solid tumors, including non-small andsmall cell lung, breast, colorectal,
gastric, pancreatic, esophageal, testicular, cervical, ovarian cancers and
Journal of Controlled Release 210 (2015) 2638
Corresponding authors.
E-mail addresses: (G. Koning),
(M.J. Garrido).
0168-3659 2015 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Journal of Controlled Release
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non-Hodgkin's lymphoma [1,2]. L-OH is a third generation platinum
derivative used in rst line treatment of colorectal cancer (CRC) [3].
It is one of the platinum derivatives with lower toxicity in patients
[4]. However, L-OH pharmacokinetics is characterized by a high
volume of distribution due to its rapid and irreversible binding to
erythrocytes and to plasma and tissue proteins. This causes serious
limitations to reach desirable unbound drug concentrations in
the target tissue as tumor and subsequent therapeutic efcacy [4].To
overcome this, novel strategies aim at modifying L-OH biodistribution,
for instance by drug encapsulation in nanocarriers, particularly in
liposomes [5].
Liposomal encapsulation causes a strong reduction in the volume
of distribution of the loaded drug, a decrease in drug toxicity,
prolonged presence of drug in circulation and enhanced tumor accu-
mulation [6].
Two important aspects of liposomes that contribute signicantly to
tumor accumulation of entrapped drugs are their small size (usually
around 100 nm) and surface pegylation. Both contribute to increase
the circulation half-life of this formulation in blood stream delaying
its caption by the reticulo-endothelial system and thereby, increasing
the probability to reach the tumor [7]. The latter is promoted by the
enhanced permeability and retention effect (EPR) in tumors, caused
by an increased vascular permeability in this tissue and a lack of
lymphatic ow [8], allowing preferential nanoparticle extravasation
and retention at the tumor site.
This selective retention of nanoparticles, together with a specic
intracellular drug delivery into tumor cells, can be promoted by cell-
specic targeting [9]. For this approach, one needs the presence of a spe-
cic molecule or ligand in the surface of the liposome able to recognize a
receptor or antigen on the cancer cell, promoting the internalization of
the complex to reach the intracellular targets. Different types of ligands
are in use for nanoparticle targeting, such as vitamins, carbohydrates,
growth factors, peptides, and monoclonal antibodies [10,11].Incurrent
cancer therapy, the use of targeted agents is increasing; for example,
growth factor receptor inhibitors, molecules that regulate cell-signaling
and gene expression or inhibitors of angiogenesis [12,13].
In recent years, L-OH combined with Cetuximab (CTX), a chimeric
human-mouse antibody specic for the epidermal growth factor receptor,
is the chemotherapeutic treatment of certain (wild-type KRAS) metasta-
tic CRC expressing EGFR [14,15].
Binding of CTX to EGFR leads to the blockade of the receptor
followed by a rapid internalization, causing not only a down regula-
tion of receptors at the surface of the cells, but also an inhibition
of EGFR, cell proliferation signals triggering of apoptosis and the
activation of the immune system to destroy tumor cells [16]. This
receptor is over-expressed in many solid tumors including 6570%
of CRC [17,18].
In this study, we aim to combine L-OH chemotherapy and CTX
targeted therapy of CRC by designing a L-OH-loaded liposomal formula-
tion targeted to EGFR on CRC cells. Such an approach combines the L-OH
drug delivery advantages of PEG-liposomal encapsulation, with tumor
cell-specic delivery by CTX on the liposomal surface.
Linking whole mAb usually results in random orientation of this
molecule on the liposomal surface, which limits antigen or receptor
binding site exposure and also yields exposure of Fcdomains. The latter
can promote recognition by Fc-receptors on macrophages and faster
liposome clearance from circulation as well as, induce unwanted
immune reactions [19]. To overcome this, smaller targeting molecules
able to recognize the receptor are coupled but they do not display the
unwanted immune activity [20]. Therefore, CTX-Fabfragments, as a se-
lective ligand for EGFR, were coupled site-specically to the liposome.
CTX-mAb and CTX-FabL-OH-liposomes were prepared and character-
ized. Of these formulations, we investigated binding and uptake by
human CRC cell lines and intracellular drug delivery. Next, we studied
in vivo drug delivery to the tumor and therapeutic activity in CRC
xenograft bearing mice.
2. Materials and methods
2.1. Drugs and chemicals
(SPDP), Tris(2-carboxyethyl)-phosphinehydrochloride (TCEP),1,1-
Dioctadecyl-3,3,3,3-tetramethyl-indocarbocyanine perchlorate (Dil),
Vivaspin tubes (300,000 MWCO) and β-Mercaptoethanol were
purchased from Sigma (Barcelona, Spain). The mAb CTX (Erbitux®)
and L-OH (Eloxatin®) were provided by the University Clinic of
Navarre (Pamplona, Spain). Hydrogenated-phosphatidylcholine
(HSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[Methoxy
(polyethylenglycol)-2000] (DSPE-PEG2000) and 1,2-distearoyl-sn-
glycero-3-phosphoethanolamine-N-[Maleimide (polyethylene glycol)
2000] (DSPE-PEG2000-Mal), werefromAvantiPolarLipidsInc.
(Alabaster, Alabama, USA) and Lipoid (Ludwigshafen, Germany)
and were kindly provided by the Laboratory Experimental Surgical
Oncology from the Erasmus MC in Rotterdam (The Netherlands).
Cholesterol (CH) and bovine serum albumin (BSA) were purchased
from Sigma (Barcelona, Spain). Amicon tubes with membranes
of 10,000, 30,000 and 50,000 MWCO were obtained from Millipore
Corporation (Billerica, MA, USA).
2.2. Oxaliplatin liposomes
Liposomes of L-OH were developed using the lm hydration tech-
nique following the methodology previously described by Zalba et al.
[21]. Briey, the lipids HSPC:CH:DSPE-PEG
-Mal, in
a molar ratio of 1.85:1:0.12:0.03, were dissolved in a solution of
chloroform:methanol [9:1 (v/v)]. The solvent was evaporated using a
rotary evaporator (Büchi-R144, Switzerland) at 65 °C to obtain a lipid
lm, which was further dried under vacuum. The lm was hydrated
with a solution of L-OH (5 mg/ml) in glucose 5%, resulting a nal solu-
tion of 10 mM of lipids. The same procedure was followed to prepare
the non-loaded or blank liposomes but adding glucose 5%. The solution
was extruded through a 100 nm polycarbonate membrane (Mini-
Extruder Set, Avanti Polar Lipids Inc., Alabaster, Alabama,USA) to obtain
a homogeneous liposome population (LP-N).
Non-encapsulated L-OH was removed by ultraltration at 2200 g for
60 min using the Amicon system (10,000 MWCO). Liposomes were
washed three times with Hepes saline solution (pH 6.7) and the nal
formulation was stored at 4 °C until use.
In order to prepare labeled liposomes, the uorescent probe Dil was
added to the lipid mixture [1% (w/w)] and dissolved in the solution of
chloroform:methanol [9:1 (v/v)] together with lipids. The Dil-LP-N
preparation procedure was similar to non-uorescent liposomes.
These liposomes were also used to formulate the EGFR targeted lipo-
somes with CTX and Fabfragment.
2.3. Development of immunoliposomes
2.3.1. Liposomes coupled to Cetuximab
The method followed to develop the immunoliposomes has been
previously described by Songs and coworkers [22].Briey, CTX was
mixed by orbital shaking with SPDP at 1:12 molar ratio for 1 h at
room temperature (RT). Afterwards the excess of SPDP was removed
from the mixture by ultraltration at 2200 g for 30 min using the
Amicon system (50,000 MWCO). The ultraltered solution was incubated
under agitation for 1 h with TCEP (1:625 molar ratio) at RT [23].These
two reactions provided thiolation of CTX, which was next co-incubated
with liposomes overnight at 10 °C. The thiol groups behave as crosslinker
with the maleimide groups of the DSPE-PEG
-Mal lipid, forming a
thioether bond, as is shown in Fig. 1.Thenal formulation was puried
by ultraltration at 2200 g for 60 min using the Vivaspin system
(300,000 MWCO), followed by a washout process with Hepes saline
to remove the non-bound ligand. To prevent the particle aggregation,
27S. Zalba et al. / Journal of Controlled Release 210 (2015) 2638
liposomes were incubated with 1 mM of L-cysteine that quenches the free
maleimide radicals avoiding the formation of disulde bond [24]. The
immunoliposome (LP-CTX) was stored at 4 °C until use.
2.3.2. Liposomes coupled to Fabfragment of Cetuximab
To obtain the Fabfragment from CTX, several steps were followed.
First, the mAb was hydrolized using a pepsin solution (1:20 w/w) pre-
pared in sodium acetate (100 mM, pH 3.7). This process was carried
out at 37 °C for 2 h obtaining the (Fab)
and the crystallizable fragment,
Fc.ThisFcwas removed by ultraltration at 2200 g for 30 min using
the Amicon system (50,000 MWCO). Afterwards the collected (Fab)
fragments were incubated at 37 °C for 30 min with a solution of
15 mM of β-Mercaptoethanol to obtain single Fabfragments, as is
shown in Fig. 2. These molecules were puried by several cycles of ultra -
ltration (30,000 MWCO) and washing. To check the isolation process
of Fabfragments and to verify the presence of the different molecules
in each of the steps mentioned (whole mAb, the non-reduced (Fab)
fragments and the Fabfragment) an 8% gel SDS page was used.
The induced hinge-region free thiol group in the structure of Fab
allowed the conjugation with DSPE-PEG
-Mal of the liposome by
incubation at 10 °C overnight. The elimination of the non-coupled
fragments was carried out by three cycles of ultraltration in Amicon
tubes (50,000 MWCO) and washing with Hepes saline solution. To
prevent the instability of the formulation (LP-Fab), it was incubated
with 1 mM of L-cysteine and stored at 4 °C until use [24].
2.3.3. Optimization of the ligand/lipid ratio
In order to select the most adequate ligand to lipid ratio in eachtype
of formulation, different quantities of ligand per μmol of lipid (5, 10, 20,
30, 40 and 50 μg) were tested for both ligands, CTX and Fab.Inthenal
Thioethe r bond
Fig. 1. Schematic representation of the method used for LP-CTX production.
Thioether bond
β- Mercaptoethanol
Fab’ fragments
Fig. 2. Schematic representation of the method used for LP-Fabproduction.
28 S. Zalba et al. / Journal of Controlled Release 210 (2015) 2638
formulation, the efciency of the coupling was measured using the
MicroBCAkit (Thermo Fisher Scientic Inc, Waltham, MA USA). The
efency was calculated as the relation between the initial and the nal
amount of the ligand incorporated in the liposomes.
In a parallel study, uorescent liposomes (Dil-LP), previously formu-
lated with different ligand/lipid ratios, were assayed for cell uptake in
HCT-116 cell line. This human colorectal cancer cell line was purchased
from ATCC and maintained at 37 °C, 5% CO
in RPMI containing 10% (v/v)
fetal bovine serum and 1% (v/v) of PenicillinStreptomycin.
For this study, cells were harvested by trypsinization, counted
and seeded in 96-well black plates at a density of 15 × 10
One day later, the culture medium was replaced by a fresh medium con-
taining a lipid concentration of 100 μM/well of each type of uorescent
formulations, Dil-LP-CTX and Dil-LP-Fab. After 24 h of treatment, the
plates were washed twice with PBS and the uorescence was measured
(Tecan group Ltd, Maennedorf, Switzerland). The signal corresponding
to the internalization with the different formulations was compared to
the control basal signal.
2.4. Characterization of liposomes
The particle size and zeta potential of the formulations were ana-
lyzed by laser diffractometry using a Zetasizer Nano Series (Malvern
Instruments, UK). Formulations were diluted 1:50 (v/v) in deionized
water in order to ensure a convenient scattered intensity on the
detector. The efciency of L-OH encapsulation was measured by atomic
absorption spectrometry. The concentration of the lipid in each formu-
lation was quantied using the phosphate assay method [25].
The efciency of conjugation for the two ligands was quantied
using the MicroBCAkit following the manufacturing instructions.
2.5. Evaluation of L-OH release from the liposomal formulations
The release rate of L-OH from liposomes was characterized for the
three types of formulations. Aliquots of 1 mL corresponding to 60 μg
of L-OH loaded in liposomes were incubated at 37 °C in a dialysis
tube (12,000 MWCO). The receptor medium consisted of 500 mL of
isoosmotic Hepes saline solution under orbital shaking to maintain sink
conditions during the experiment [26]. Several samples of the receptor
medium were collected at 0, 10, 30, 60 min and 4, 7 and 24 h by triplicate
and the L-OH quantied by atomic absorption spectrometry.
The inuence of serum proteins in the stability of liposomes was
assayed by their incubation in presence of 50% FBS (v/v).
The accumulative release curve was built as the percentage of
release (% R) at each assayed time applying the following formula:
where, Qa represents the amount of L-OH measured in the collected
sample, and Qt, the total amount of the drug at the beginning of the
2.6. In vitro studies
2.6.1. Cytotoxicity study
Four human colon cancer cell lines, HCT-116, HT-29 and SW-480,
SW-620 were purchased from ATCC and routinely maintained at stan-
dard conditions in RPMI and DMEM respectively, containing 10% (v/v)
FBS and a 1% (v/v) of PenicillinStreptomycin.
For this study, cells were detached by trypsinization and seeded in
96 well microtiter plates at a density of 5x10
cells/well. Twenty-four
hours later, the cells were exposed for 4 h at different concentrations
of L-OH, (from 0.1 to 100 μM) free and encapsulated in LP-N, LP-CTX
and LP-Fab. After drug exposure times, cells were rinsed with PBS and
new fresh medium was added. Cell viability was measured at 72 h
post-treatment using the Neutral Red Assay [27].Datawereexpressed
as concentration of L-OH that gives a 50% inhibition of cell growth
compared to untreated or control cells (IC
). Blank liposomes were
also tested under the same conditions.
2.6.2. Inuence of liposomes in the EGFR phosphorylation status
In order to evaluate the inuence of the ligands CTXand Fab,freeor
coupled to liposomes in the EGFR phosphorylation status, all formula-
tions were assayed in HCT-116 and SW-480 cell lines.
Cells at a density of 8 × 10
cells/well were seeded in 6 well plates.
Twenty-four hours later, cells were exposed for 15 min at the following
treatments: 20 ng/ml of EGF, 27 μg/ml of CTX and Fab,and1mMofLP-
N, LP-CTX and LP-Fab. Afterwards cells, washed twice with ice-cold
PBS, were treated with 100 μl of RIPA buffer supplemented with sodium
orthovanadate (1 mM), sodium uoride (10 mM), β-glycerophosphate
(100 mM) and Phosphatase inhibitor cocktail tablets (Roche®,
Indianapolis, USA), harvested with a cell scrapper and incubated at
4 °C for 30 min. Lysates were centrifuged at 12,500 g for 30 min at
4 °C to collect the supernatant. The content of protein in those samples
was quantied using the Micro BCAkit. Aliquots of 40 μg of protein
were loaded on the SDS PAGE (8%) and transferred to a nitrocellulose
membrane by wet blotting. This membrane, incubated overnight at
4 °C in Tris buffer solution (TBS) with 1% Tween-20 and 5% of BSA and
the anti-EGFR [phospho Y1068 from abcam®(Cambridge, UK)], was
washed and incubated for 1 h at RT with the secondary antibody, an
anti-rabbit peroxidase-labeled (Cell-signaling, Danvers, USA). Blots
were developed using the ECL system Super Signal ULTRA kit (Thermo
Fisher Scientic Inc, Waltham, MA USA), and immunoreactive proteins
were visualized on the high-performance chemiluminescence lm
(HyperlmTM, Amersham Bioscience, Piscataway, NJ, USA). β-actin
2.6.3. Cellular uptake of liposomes
Cell uptake in four cell lines using uorescent liposomal formula-
tions LP-N, LP-CTX and LP-Fabwas characterized at two temperatures,
37 and 4 °C respectively.
For this study, cells were seeded in 96 well microtiter black plates at
cells/well. After 24 h, 100 μM lipid of each formu-
lation was added to the culture medium. Cells collected at different time
points, from 0.5 to 24 h, were washed twice with PBS to measure the
incorporated uorescence.
In order to investigate the role of the EGFR in the uptake, a parallel
study following the above protocol was carried out, but here, cells
were pretreated with 100 μg/mL of CTX for 1 h at 37 °C before the expo-
sure to liposome treatment. Additionally, cell uptake with and without
CTX pretreatment was also explored by images captured by uores-
cence microscopy at 40× magnication. In this experiment, 60,000
cells were seeded in culture slides chambers (BD Falcon, Bedford,
USA) and incubated for 24 h with 100 μM lipid of each type of formula-
tion. After treatments, cells were xed in formaldehyde 4% by incubation
for 10 min at RT and preserved at 4 °C until analysis. Cell membrane was
visualized by adding anti-alpha 1 sodium potassium ATPase antibody
(abcam®, Cambridge, UK), and DNA in nucleus was stained with DAPI.
Images were acquired using the Axio Cam MR3 video camera connected
to the Zeiss Imager M1 microscope (Carl Zeiss AG, Oberkochen,
Germany) equipped with epiuorescence optics and Axiovison software
( version).
2.7. In vivo study
Fifty-four female athymic nude mice weighing 2025 g (Harlan,
Barcelona, Spain) were housed in plastic cages under standard and
sterile conditions (25 °C, 50% relative humidity, 12 h dark/light), with
water and food ad libitum. All experiments were performed according
to European animal care regulations and the protocol was approved
by Ethical committee of the University of Navarra (075/07).
29S. Zalba et al. / Journal of Controlled Release 210 (2015) 2638
A subcutaneous tumor was induced by the inoculation of 5 × 10
SW-480 cells in 100 μl of PBS, in the right ank of the mice. When the
tumors reached approximately a volume of 200 mm
, mice were ran-
domly divided into different groups: control, free L-OH, LP-N, LP-Fab,
and LP-CTX. Mice were intravenously (i.v.) injected with 2.5 mg/kg
dose of L-OH every three days during three cycles, as is shown in
Fig. 3. The corresponding empty formulations were also evaluated in
three extra-groups by i.v. administration of the equivalent amount of
Tumor size, measured by an electronic caliper, and the body weight
were recorded every two days. Tumor volume was calculated according
to the following formula: V(mm
) = 4/3π(d
), where d and D are
respectively the smallest and the largest tumor diameters.
At the end of the experiment, day 24, all mice were sacriced to
remove the liver, spleen and the tumor to quantify the L-OH levels
reached in those organs. They were homogenized in 1 mL of a mixture
NaCl (150 mM)/HCl (470 mM) using the Mini-Beadbeater device
(Bioespec Products, Bartlesville, UK), and mixed with 3 mL of HNO
(35%). Samples were heated for 2 h in a water bath at 65 °C and stored
until use. Platinum levels were measured by atomic absorption
spectrometry technique.
2.8. Statistical methods
All data were expressed as the mean and standard deviation (SD).
The statistical analysis was performed using a non-parametric test,
KruskallWallis to compared all treatments followed by the U of
MannWhitney test to compare two by two groups. The signicance
level was set at p b0.05.
3. Results
3.1. Preparation of CTX-Fabfragments
Fig. 4 shows the SDS-PAGE results from the CTX fragmentation
(panel A) and the conjugated liposomal formulations (panel B). The
molecules obtained during the process of antibody digestion and Fab
isolation were identied in the different bands observed in each lane.
In lane 3, full CTX antibody is applied resulting in a typical CTX band
found at 150 kDa. CTX treated with pepsin resulted in (Fab)
observed at around 100 kDa and the protease itself, pepsin, at around
40 kDa (corresponding to lane 2). Successful production of monovalent
Fabfragments after 2 h reduction with β-mercaptoethanol was demon-
strated by theband at 50 kDa, observed in lane 5. Formulations obtained
by covalent fusion of the modied ligands, CTX and CTX-Fab,toprevi-
ous prepared liposomes are represented in lanes 2 and 3 respectively,
in panel B. These bands correspond to liposomes puried, suggesting
the non-presence of unbound ligands in the samples.
These results suggest that, the protocol based on an enzymatic
digestion by pepsin followed by the β-mercaptoethanol treatment to
reduce the disulde bonds in the hinge region, was adequate to obtain
the monovalent Fabfragments suitable for conjugation to liposomes.
3.2. Preparation and characterization of targeted L-OH liposomes
Liposomes containing L-OH were prepared and conjugated to CTX or
Fabfragments using thiol-ether linkage.
The physicochemical characterization of nanoparticles in terms of
particle size, polydispersity index (PDI) and zeta potential of the three
types of liposomes developed in this study are listed in Table 1.
All liposomal formulations (targeted and control or non-targeted)
showed a particle size around 120 nm associated with a very low PDI,
suggesting a homogenous nanoparticle population, with a similar
value for zeta potential. Protein conjugation did not affect the main
particle characteristics as none of the assayed parameters reached a
statistical signicant difference among formulations. Liposomes were
stable at 4 °C for, at least, one month.
The three formulations showed similar efciency of encapsulation
with an average value of 32.07 ± 4.9% and a loading capacity of
65.2 ± 7.2 μg/mg lipid, these values are similar to those previously
reported for non-targeted L-OH liposomes [21]. Therefore, there is no
inuence of the coupling process on the amount of the encapsulated
3.3. Protein coupling efciency at different ligand/lipid ratios and effect on
cell binding
Coupling of protein to the liposomes was evaluated by protein and
phospholipid assays at different ligand/lipid ratios. The coupling ef-
ciency represented in Fig. 5 shows similar outcome for both ligands
CTX and Fab.Thisefciency was calculated as the relationship between
the initial and nal amount of ligands conjugated to the surface of the
liposomes. A linear increase between 5 and 30 μg of protein/μmol of
lipid was followed by a plateau in the range of 3040 μgofprotein/
μmol of lipid.
The highest efciency was found at the lowest protein concentra-
tion, 93.3 ± 4.73% at 5 μg CTX or Fabwhile, at higher concentrations
(30 μg) the efciency was statistically lower (p b0.05), reaching values
Day 0
Tum or
Day 12
Day 18
Day 15
End of
Day 24
Fig. 3. Scheme of the in vivo experimental design.
30 S. Zalba et al. / Journal of Controlled Release 210 (2015) 2638
between 66.66 ± 2.9% for Faband 63.40 ± 1.9% for CTX. Taking
into account this result, further studies were carried out using the
ratio 30 μgofprotein/μmol of lipid. Results in Table 1 correspond to
liposomes formulated with this ratio.
The effectof ligand-density on binding to EGFR positive HCT-116 co-
lorectal cancer cells was investigated in a cell association study with
uorescent liposomes. Fig. 6 indicates that the binding, representing
the sum of binding and uptake, for both formulations increased statisti-
cally (p b0.01) as increasing the ligand density from 10 to 30 μg/μmol,
reaching a plateau at 30 μg/μmol. But, no further increase in binding
was observed at 50 μg/μmol.
3.4. In-vitro L-OH release
Kinetics release of L-OH from LP-N, LP-CTX and LP-Fabin Hepes
saline buffer and 50% serum were investigated during the rst 24 h.
Release of L-OH from liposomal formulations was similar among
them, as is observed in Fig. 7. Release increased up to 5 h to approxi-
mately 2535% and remained constant up to 24 h. In order to study
the inuence of serum proteins on the stability of liposomes, formula-
tions were incubated with 50% (v/v) of FBS in Hepes saline. Presence
of serum caused an increase in L-OH release corresponding to 1020%,
which mainly occurred in the initial phase.
3.5. Effect of ligand binding on EGFR activation
To investigate the effect of ligand binding on receptor activity, EGFR
phosphorylation status was studied in SW-480 and HCT-116 after
15 min of exposure to different treatments. Stimulation of cells with
EGF increased the receptor phosphorylation compared to basal levels
in both cell lines; while treatments with free ligands, CTX and Fab,in-
duced a total inhibition of basal phosphorylation (Fig. 8). Treatments
with LP-N, LP-CTX or LP-Fabin both cell lines did not change the
basal receptor status, indicating that the binding of targeted liposomes
and possible multivalent interactions did not cause EGFR receptor
cross linking and activation.
3.6. Targeted liposomes bind selectively to overexpressed EGFR in CRC cell
Cellular association of targeted liposomes was quantied for several
CRC cell lines with different EGFR expression levels at 37 °C and 4 °C
[2831]. This process was time-dependent in the four cell lines
(Fig. 9). For targeted liposomes, LP-Faband LP-CTX, at 37 °C cellular
association was more rapid and higher in HCT-116 and SW-480 than
in HT-29 and SW-620; being more favorable for LP-Fab, especially at
the rst times of incubation 3 and 5 h. The difference in the signal
among celllines is compatible with the low and moderate EGFR expres-
sion levels described for SW-620 and HT-29 respectively, compared to
the elevated expression in the other two [2831].
Fig. 4. Electrophoresis on polyacrylamide gel (8%) to check: A) the molecules obtained during different steps of CTX fragmentation. Lanes represent: 1, molecular weight size marker; 2,
pepsin; 3, monoclonal antibody, CTX; 4, (Fab)
fragments; 5, monovalent Fabfragment; B) the puried targeted liposomes: lane 2, LP-CTX and 3, LP-Fab.
Table 1
Characterization of the three types of liposomes developed. Datarepresent the averageof
three ind ependent studies and th eir SD.
Size (nm) 119.6 (0.36) 123.0 (1.59) 120.0 (1.19)
PDI 0.065 (0.018) 0. 057 (0.022) 0.053 (0.024)
Zeta potential (mV) 22.4 (1.08) 20.8 (2.19) 24.4 (1.45)
Fig. 5. Relationship between the initial and the nal amount of ligands per μmol of lipid
in the developed formulations. Symbols represent the means and vertical lines thei r
corresponding SD of three independent studies. Lines represent the interpolation of the
experimental data. ** p b0.01. (U of MannWhitney test).
31S. Zalba et al. / Journal of Controlled Release 210 (2015) 2638
Blocking the receptor by CTX, strongly reduced the association of
targeted-liposomes by receptor positive cells, reaching similar levels
to those observed for non-targeted formulations. The role of the recep-
tor in the specic binding and internalization of the targeted liposomes
is endorsed by these results and the fact, that the internalization of
targeted and non-targeted liposomes was comparable in SW-620, a
cell line with low EGFR expression.
On the other hand, the active and specic binding to the receptor
was supported by the results found at 4 °C, where the signal
corresponded to non-specic binding [32]. The relative values of the
uorescence at this temperature were very similar in all cell lines and
formulations. The observed differences in cellular association of
targeted liposomes at 4 °C and 37 °C are suggestive of rapid andefcient
internalization of liposomes and their contents.
In positive EGFR cell lines, the association of LP-Fabwas higher than
LP-CTX, while both are considerably higher than for non-targeted
Increased cell-specic association of targeted liposomes by receptor-
positive cells was conrmed by uorescence microscopy (Fig. 10).
Targeted liposomes (red) bound to SW-480 cells in a specic manner
as proven by competition with free CTX (lower panels) and not to
receptor-negative SW-620. HT-29 and HCT-116 incubations had an
outcome similar to SW-480 (data not shown).
Therefore, these images support that targeted liposome uptake
occurs through the EGFR.
3.7. Targeted L-OH liposomes improve cytotoxicity over the free drug and
non-targeted liposomal L-OH
Cytotoxicity of targeted L-OH liposomes was compared to free and
non-targeted liposomal L-OH in four different colorectal cancer cell
lines. IC
values were calculated from the doseresponse curves. Both
targeted liposomal L-OH formulations demonstrated increased efcacy
toward all tested EGFR receptor-positive colorectal cancer cell lines,
when compared to free and non-targeted liposomal L-OH, as evidenced
by the lower IC
values (Table 2). Remarkably, targeted liposomes de-
creased IC
values 24 fold in HT-29 and SW-480, which demonstrated
signicant resistance to free L-OH, reaching nearly similar efcacy as in
the more sensitive HCT-116 cells.
Efcacy of targeted liposomes and non-targeted liposomes wassim-
ilar in the negative EGFR cell line, supporting again the importance of
receptor-binding for intracellular drug delivery and efcacy No cytotoxic
effect was found for empty targeted liposomes (data not shown).
3.8. Targeting L-OH liposomes improves therapeutic efcacy in colon cancer
bearing mice
Therapeutic activity of the L-OH targeted liposomes was studied in
SW-480 tumor bearing mice then, compared to treatments with the
free and non-targeted liposomes. Tumor growth was individually moni-
tored in all animals over time. Three 2.5 mg/kg i.v. doses of L-OH, free and
encapsulated, were administered on days: 12, 15 and 18 post-cancer cell
Fig. 6. Effect of ligand density on association of uorescent targeted liposomes (100 μM)
with 10, 30 or 50 μgofligand/μmol of lipid with HCT-116 cells. Data,expressed as relative
light units (R.L.U.), represent the means of three independent studies with their SD.
** p b0.01. (U of MannWhitney test).
Fig. 7. Time proles of the accumulated drug release for the three formulations: stealth liposomes (LP-N) and targetedliposomes (LP-Faband LP-CTX) in Hepes buffer (panel A) and in
presence of FBS (panel B). Each value represents the average of three independent studies and their corresponding SD.
12 345 6 7
Fig. 8. Bands represent the phosphorylation of EGFR after different treatments. 1: control;
2: EGF; 3: free CTX; 4: free Fabfragment;5:LP-N;6:LP-CTX;7:LP-Fab.
32 S. Zalba et al. / Journal of Controlled Release 210 (2015) 2638
Tumor growth in the control group increased statistically (p b0.01)
in comparison with treated groups just after second cycle administra-
tion on day 15, as is shown in Fig. 11. In addition, the antitumor effect
for liposomal formulations was found statistically (p b0.01) higher
than for the free drug after third cycle.
Results show that L-OH encapsulated was more efcient inducing a
delay in the tumor growth than the free drug, but LP-Fabwas able to
control that delay even one week after the last dose administration,
reaching a statistical signicant difference (p b0.05) with LP-CTX. The
administration of empty targeted liposomes and free ligands, at the
same concentration that was coupled in the formulations, did not
show any statistically signicant inuence on tumor growth (see
supplementary material). It is worthy to conrm that no changes in
body weight was observer in any of the groups over the duration
of the study supporting then, the lack of toxicity of the dose used for
L-OH (see supplementary material).
3.9. Targeting L-OH liposomes improves tumor drug delivery
To study intra-tumor drug delivery, tumors from all animals were
collected at the end of the in vivo experiment, xed on day 24 post-
cells implantation, to measure platinum levels.
The treatment with targeted liposomes led to a higher tumor accu-
mulation of L-OH compared to non-targeted and the free drug,
as shown in Fig. 12. In addition, the drug concentration in tumor for
LP-Fab, 2916.6 ± 507.84 ng/g of tissue, was almost double than for
LP-CTX, 1546.5 ± 362.41 ng/g of tissue. This result likely explains the
higher delay in tumor growth found in mice treated with L-OH LP-Fab
formulation shown in Fig. 11.
Accumulation of targeted liposomes showed higher afnity to
the liver compared to non-targeted, due to the presence of EGFR in
hepatocytes while in the spleen, LP-N and LP-Fabwere found in higher
concentration than LP-CTX and L-OH. Both results together led to
suggest that whole antibody may induce a more rapid clearance than
4. Discussion
To our knowledge, this is the rst study describing the development
and the in vitro/in vivo evaluation of L-OH loaded EGFR targeted lipo-
somes using two specic ligands, CTX or its Fabfragment. This strategy
represents an advanced step to reach specic delivery of L-OH into
colon cancer cells.
Previously, we have reported the development of a pegylated liposo-
mal formulation of L-OH [21]. This formulation formed the basis to fur-
ther advance L-OH delivery by coupling two specic ligands for EGFR
targeting to those liposomes. The selection of the ligands was based
on the therapeutic combination applied in patients with CRC, CTX plus
L-OH. This strategy seeks the improvement of the pharmacodynamics
of L-OH previously found with non-targeted liposomes [21],andthere-
duction of the adverse effects observed in patients after repeated doses
of the free CTX [33]. Moreover, this novel combination treatment aims
to specically deliver L-OH at high levels into tumor cells. Here, we
could demonstrate that the targeting to EGFR on CRC cells increases
Fig. 9. Relative association rate obtained at several times during the rst 24 h of the treatment with targeted and non-targeted liposomes in different conditions: at 37 °C, with a pretreatment
with free CTX and at 4 °C. Bars represent the average of three replicates with their SD.*p b0.05, (KruskallWallis followed by the U of MannWhitney test).
33S. Zalba et al. / Journal of Controlled Release 210 (2015) 2638
cell binding of liposomes and intracellular drug delivery and thereby,
could reverse L-OH resistance.
Targeted liposomes have been described to deliver higher quantities
of drugs to tumors, which resulted in an enhanced therapeutic efcacy
over free drug and liposomal formulations [1013,22,32,34].For
targeting of liposomes, antibodies have been used extensively [1013].
However, the presence of Fcfragments can induce the uptake of
those antibodies by the mononuclear phagocyte system, leading to a
rapid clearance of liposomes from the circulation and developing in
some cases, anti-drug antibodies (ATA) that may contribute to immu-
nogenic reactions [33]. Although the avidity by the receptors is lower
than for the whole mAb, Fabfragments abolish the uptake by the
phagocytic system and reduce that immunogenicity improving in vivo
efcacy [19,20,32].
In this study, Fabwas obtained by enzymatic fragmentation of CTX
followed by a posterior reduction of (Fab)
with β-mercaptoethanol.
This methodology provided similar results to previous described in
the literature, obtaining monovalent fragments Fabfrom mAb but
here, the purication of Fabwas done by ultraltration using Amicon
systems instead of the sepharose columm used by Sapra et al. [32].
This purication process was simple and rapid, allowing the isolation
and identication of various fragments obtained during CTX fragmenta-
tion, as was observed in the analysis by electrophoresis shown in Fig. 4
The optimization of the ligand/lipid ratio in targeted liposomes, for
both ligands, was explored from two different angles. First, we focused
on obtaining a high ligand density with highly efcient coupling quan-
tied as the relationship between the initial and nal amount of the
Membrane Nuclei Liposomes Merge
LP-CTX LP-Fab’ LP-N Control
Membrane Nuclei Liposomes Merge
LP-CTX LP- Fab LP-N Control
Fig. 10. Fluorescencemicroscopy of the uptake of liposomesafter 24 h of exposure in EGFR-positive, SW-480 and EGFR-negative, SW-620 celllines at 40×. Lower panels represent results
from a CTX pretreatment previous to the liposome exposure. Different dyes have been used to stain the membrane (green), the nuclei (blue) and the liposomes (red).
34 S. Zalba et al. / Journal of Controlled Release 210 (2015) 2638
ligand attached to the liposome. Secondly, we studied the effect of
ligand density on tumor cell targeting, and assessed as cell association
of formulations with different ligand/lipid ratios using a cell line with
a high EGFR expression [2831]. Ligand coupling to the liposomes in-
creased linearly with increasing ligand/lipid ratio and reached a plateau
at 30 μgofprotein/μmol lipid. At this density, coupling efciencies
between 60and 70% were achieved for both CTX and Fab. This percent-
age is in line with data previously published, describing a relatively
wide range in efciencies, as this process depends on the formulation
and the ligand [24,32]. In this case, because of the molecular weight of
both molecules are different, the number of ligands attached to the
surface of liposomes were approximately 10 for CTX vs. 31 for Fab.
Membrane Nuclei Liposomes Merge
LP-CTX LP-Fab LP-N Control
Membrane Nuclei Liposomes Merge
LP-CTX LP-Fab LP-N Control
Fig. 10 (continued).
Table 2
(μM) values measured at 72 h after 4 h of drug exposure. Data represent the average and SD of three independent studies.
HCT-116 HT-29 SW-480 SW-620
L-OH 28.67 (5.17)*
57.87 (7.16) *
79.22 (8.11) *
40.49 (0.28) *
LP-N 22.20 (1.73)*
26.00 (3.91) *
39.30 (4.46) *
31.99 (2.12) *
LP-CTX 16.64 (2.71)*
21.26 (1.26) *
26.01 (2.61) *
30.65 (2.69) *
LP-Fab17.19 (1.12) *
23.82 (0.89) *
28.12 (0.40) *
35.86 (3.51) *
*p b0.05; a, difference amongcell lines; b, difference between free andnon-targeted; c, difference between free and targeted; d, difference between non-targeted and targeted, Kruskall
Wallis followed by the U of MannWhitney test, were applied for statistical signicance.
35S. Zalba et al. / Journal of Controlled Release 210 (2015) 2638
The ligandcoupling did not alter the physicochemical characteristics
of the liposomes such as particle size, PDI and zetapotential. The particle
size, around 120 nm, associated with a very low PDI support a good
standardization of the developed method to formulate the different
types of liposomes, which are small enough to suffer the EPR effect
after their i.v. administration to animals [35].
Additionally, the presence of the ligand did not affect the drug
release rate. Release kinetics suggests a certain depot effect that could
be achieved with these liposomes in an in vivo system. The presence
of serum in the dialysis tube increased slightly the drug release from
liposomes (1020%). This may be due to the opsonization of serum
proteins that affects the stability of the liposome, although in this case
that inuence has a low impact [26]. This effect has been also reported
by Abu-Lilla and coworkers (2009) for L-OH liposomes, with similar
impact on the release rate [36].
Next to optimizing ligand density with respectto coupling efciency,
optimal tumor cell binding is also of major importance. Cell binding to
EGFR overexpressing HCT-116 cells increased with increasing ligand
density up to 30 μg/μmol lipid and remained comparably high, when
further increasing liposomal ligand density (Fig. 6). This is expected as
coupling efciencies do not increase much above 30 μg/μmol (Figs. 5
6). A clear relationship between ligand density and tumor cell binding
has been described before for whole mAb [19] and for antibody
fragments [37].
It is interesting to point out that although the Fabfragment presents
only one binding site compared to the two sites present in the whole an-
tibody, similar cell association level was obtained for liposomes
equipped with either one of the ligands. This may well be explained
by the slightly higher number of actual Fabligands at the liposomal
surface compared to the larger mAb, in combination with the random
orientation of the whole antibody, which for a portion of the mAb will
physically hinder binding to the receptors [38].Apparentlyboth
compensate for the lower number of antigen binding sites per ligand
and suggest that Fabcoupling to liposomes for this ligand restores the
multivalent binding [32]. The association studies carried out in cell
lines with different levels of EGFR expression, showed an increase in
the association of targeted liposomes at 37 °C with increasing receptor
density. This result together with the inhibition of the internalization
by a pretreatment with free mAb, reveal that this process takes place
through selective binding to the receptor. The behavior of the negative
control cell line, SW-620, conrmed those results as no differences in
the internalization between targeted and non-targeted liposomes
were found. The high cell association of targeted liposomes at 37 °C
compared to 4 °C strongly indicates cellular uptake of targeted
liposomes via an active process [32].
Regarding the difference between both ligand-targeted liposomes,
Fabfragment seemed to inuence positively the uptake, probably due
to the random orientation of the whole antibody coupled in the lipo-
some as has been commented above. This limitation has also led to
some authors to use the Fabfragment, as a selective ligand for coupling
to nanoparticles, in order to enhance the cell uptake and improve the
in vivo efcacy [24,32,34].
Cellular association was conrmed by uorescence microscopy. The
highest uorescence intensity associated with SW-480 cells was found
for LP-Fab, in comparison with other cell lines and LP-CTX, which
supports the relationship between EGFR expression and the degree of
internalization and the higher receptor binding capacity of LP-Fab.
Cytotoxicity studies conrmed efcacious intracellular L-OH delivery
by targeted liposomes. Targeted liposomal L-OH outperformed liposo-
mal and free L-OH in all EGFR overexpressing cell lines, and was even
able to reverse L-OH resistance in the two cell lines which displayed
decreased L-OH sensitivity. This increase in the drug sensitivity may
be supported by a modication in the mechanisms involved in this cel-
lular uptake. Thus, L-OH resistant mechanisms are related to the copper
receptor which mediates the cell entry of the free L-OH and the efux
pumps that decrease the cytoplasmic drug concentration [39,40]. The
advantage of the drug encapsulation in targeted liposomes lies in the
selectivity of the uptake by a receptor up-regulated enhancing thus,
the intracellular drug concentration. This phenomenon has been de-
scribed even for non-targeted formulations like Lipodox, which reduces
the exchange of doxorubicin by the P-glycoprotein [41].
Differences in cellular uptake observed between LP-Faband LP-CTX
did not markedly affect IC
values in the cytotoxicity studies. Whereas
differences in binding were observed at shorter incubations, in this
Fig. 11. Time prole of the relative tumor growth for the different treatments. Each point
represents the average of six mice and the bars are the SD. Arrows represent the dose
administration at days 12, 15 and 18 after tumor inoculation.* p b0.05, KruskallWallis
followed by the U of MannWhitney.
Fig. 12. L-OH levels measured in spleen and liver (panel A) and tumor (panel B), 144 h
after last dose administration following the different tr eatments. Bars represent the
average of six mice, and the vertical lines their corresponding SD (*, p b0.05; **, p b0.01
provided by the comparison between targeted and non-targeted liposomes using U of
36 S. Zalba et al. / Journal of Controlled Release 210 (2015) 2638
study the IC
was measured at 72 h after 4 h of drug exposure. Other
studies report similar observations [42,43]. Iden and Allen found that
of CD19-targeted Dox liposomes decreased with increasing drug
exposure time or the time to measure the cytotoxicity after treatment
[42]. Importantly, as shown in Table 2, a targeted liposomal L-OH
increased sensitivity of colorectal cancer cell lines more than three
times compared to the free or liposomal L-OH.
The in vivo efcacy study was carried out using a very low dose of
L-OH every three days [44]. This regimen was chosen in order to iden-
tify the importance of targeted delivery during several cycles. L-OH
(2.5 mg/kg i.v. dose) was able to inhibit the growth of the tumor in
mice after the second dose. Liposomal encapsulation together with
the targeting strategy clearly beneted tumor response. One week
after the last cycle, at day 24 post cell implantation, the reduction of
tumor growth for liposomes was much higher than for the free drug
when compared to the control group. These differences, expressed as
% inhibition was ranged from 52%, for LP-N, to 66% for LP-Fab, while
for free L-OH that effect was 34%.
These results show that the antitumor activity of L-OH liposomal
encapsulation was more effective than the free drug and even more
importantly, that targeting using the Fabligand was more efcacious
than CTX. Although the dose of L-OH is lower than the dose used by
other authors, the aim of this work was to elucidate the effectiveness
of the targeted liposomes. Yang et al. [45] found an inhibition of 26%
after several doses of 5 mg/kg administered every four days of non-
targeted L-OH pegylated liposomes, which is slightly lower than the
inhibition found for LP-N in this study. A washout of 72 h was chosen
because very low levels of liposomes were detected by Yang and
coworkers in tumor at that time after liposome administration and be-
cause, similar washout has also been reported by Abu-Lila et al. [44],
which represents approximately ve times the half-life of L-OH in
nude mice [45]. However, we found relatively high levels of drug in
tumor 144 h after the third administration, especially in the case of
targeted liposomes, as shown in Fig. 10. This observation supports a se-
lective accumulation of targeted formulations that correlate to tumor
growth inhibition. LP-Fabpresented the higher L-OH accumulation in
tumor tissue followed by LP-CTX and LP-N. This result is according to
data previously reported by several authors, where a higher efciency
in the selective targeting by Fabligands than for the whole antibody,
was described [32,46]. This may well be explained by the presence of
Fcfragment on whole mAb carrying liposomes, which can be recog-
nized by macrophages leading to a more rapid removal from circulation
than the Fabfragment [19,20,32], in combination with a lower ability to
bind to receptor due to the orientation of the attached antibodies
In addition, data from Fig. 10 can also be supported by previous re-
sults from a pilot study carried out with similar protocol but for only
two doses. L-OH levels increased from 4 until 24 h post-dosing for the
formulations, especially for LP-Fab, and decreased for the free drug, as
is shown in supplementary material (Fig. 1S). These results correlated
with L-OH plasma levels at 24 and 72 h listed in table 1S, where those
levels were maintained for LP-Fabcompared to LP-CTX and LP-N.
Note that animals from the in vivo study did not show signicant
toxicity, loss body weight or cachexia (Fig. 3S), even in groups treated
with targeted formulation, where L-OH tissue accumulation was not
associated to an enhancement of toxicity.
5. Conclusion
In conclusion, we demonstrated that EGFR targeted L-OH liposomes
were more efcient in tumor drug accumulation than free L-OH or non-
targeted liposomes, which improved efcacy in mice inoculated with a
CRC cell line overexpressing this receptor. In vitro results supported
these results by proving that targeted liposomes, particularly LP-Fab
that induced cell-specic binding, uptake, intracellular drug delivery
and subsequent cytotoxicity to EGFR-positive CRC cells. Remarkably,
targeted delivery was also effective in L-OH resistant cell lines, suggest-
ing that this approach could reverse drug resistance by inducing active
cellular drug uptake through the EGFR. Therefore, EGFR targeted L-OH-
liposomes seem to be a promising novel nanomedicine to improve CRC
Funding for this project came from the Spanish Government
(Instituto de Salud Carlos III, ref: PS09/02512-FISS), Government of
Navarra (ref: IIQ14334.RI1) and from the University of Navarra. Sara
Zalba was supported by a grant from the Government of Navarra. The
authors want to thank the Imaging Unit from CIMA (Pamplona, Spain)
for their support to this work.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
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... It has repeatedly been shown that less than 100 nm PEGylated nanoparticles interact less with blood components and less liver filtration. These tiny, PEGylated NPs also improve tumor therapy, have high tumor-targeting effectiveness, and lengthen blood circulation time (Tseng et al., 2009;Wang et al., 2014a;Zalba et al., 2015). ...
Platinum nanoparticles (Pt NPs) have numerous applications in various sectors, including pharmacology, nanomedicine, cancer therapy, radiotherapy, biotechnology and environment mitigation like removal of toxic metals from wastewater, photocatalytic degradation toxic compounds, adsorption, and water splitting. The multifaceted applications of Pt NPs because of their ultrafine structures, large surface area, tuned porosity, coordination-binding, and excellent physiochemical properties. The various types of nanohybrids (NHs) of Pt NPs can be fabricated by doping with different metal/metal oxide/polymer-based materials. There are several methods to synthesize platinum-based NHs, but biological processes are admirable because of green, economical, sustainable, and non-toxic. Due to the robust physicochemical and biological characteristics of platinum NPs, they are widely employed as nanocatalyst, antioxidant, antipathogenic, and anticancer agents. Indeed, Pt-based NHs are the subject of keen interest and substantial research area for biomedical and clinical applications. Hence, this review systematically studies antimicrobial, biological, and environmental applications of platinum and platinum-based NHs, predominantly for treating cancer and photo-thermal therapy. Applications of Pt NPs in nanomedicine and nano-diagnosis are also highlighted. Pt NPs-related nanotoxicity and the potential and opportunity for future nano-therapeutics based on Pt NPs are also discussed.
... Nowadays, active targeting has become the mainstay of cancer therapy, promoting preferential drug accumulation at the tumor sites while sparing healthy tissues, leading to enhanced efficacy and minimizing unwanted toxicities. Therefore, liposomes are often functionalized with various tumor-specific ligands or antibodies, including folic acid [32], hyaluronic acid [33], antibodies [34], and aptamers [35], to achieve active tumor targeting. Liposomes and other nanocarriers can also be constructed with specific materials to facilitate chemotherapeutics release in response to TME. ...
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Simple Summary The advancement of nanotechnology over the last three decades has given new hope to cancer management. The first FDA-approved nanomedicine (DOXIL) was made available in the market in 1995. Since then, numerous nanocarriers have been synthesized and extensively evaluated for antitumor efficacy to establish them as premier therapeutic tools. Even though nanomedicine is one of the most promising breakthroughs in the modern era of medicine, several challenges are still faced during scaling up from a laboratory setup to a clinical arrangement. In this review, we describe and compare various types of nanoparticles and their role in advancing cancer treatment. Moreover, we highlight various nanomedicines currently available for cancer therapy and nanoformulations that are through various stages of clinical testing. Abstract Cancer is one of the most prevalent diseases globally and is the second major cause of death in the United States. Despite the continuous efforts to understand tumor mechanisms and various approaches taken for treatment over decades, no significant improvements have been observed in cancer therapy. Lack of tumor specificity, dose-related toxicity, low bioavailability, and lack of stability of chemotherapeutics are major hindrances to cancer treatment. Nanomedicine has drawn the attention of many researchers due to its potential for tumor-specific delivery while minimizing unwanted side effects. The application of these nanoparticles is not limited to just therapeutic uses; some of them have shown to have extremely promising diagnostic potential. In this review, we describe and compare various types of nanoparticles and their role in advancing cancer treatment. We further highlight various nanoformulations currently approved for cancer therapy as well as under different phases of clinical trials. Finally, we discuss the prospect of nanomedicine in cancer management.
HER2-targeted immunoliposomes with gold payloads exhibit greater accumulation than non-targeted liposomes and free gold compounds and localize in the mitochondria and endoplasmic reticulum leading to cell death at lower nanomolar drug concentrations.
Chemotherapy-induced peripheral neuropathy (CIPN) is an important adverse effect of treatment with oxaliplatin (OXA). We have developed PEGylated nanoliposomal oxaliplatin (OXA-LIP) and tested its activity in an animal model of CIPN. OXA-LIPs were prepared using a combination of egg yolk lecithin, cholesterol, and DSPE-mPEG2000 (at ratios 400, 80, and 27 mg). These liposomes were characterized using several different methods (e.g., polydispersity index (PDI), and zeta potential, FESEM). The in vivo study was performed in 15 male rats comprising three groups: a negative control (normal saline) OXA, and OXA-LIP. These were injected intraperitoneally at a concentration of 4 mg/kg on two consecutive days every week, for 4 weeks. After that, CIPN was assessed using the hotplate and acetonedropmethods. Oxidative stress biomarkers such as SOD, catalase, MDA, and TTG were measured in the serum samples. The functional disturbances of the liver and kidney were assessed by measuring the serum levels of ALT, AST, creatinine, urea, and bilirubin. Furthermore, hematological parameters were determined in the three groups. The OXA-LIP had an average particle size, PDI, and zeta potential of 111.2 ± 1.35 nm, 0.15 ± 0.045, and -52.4 ± 17 mV, respectively. The encapsulation efficiency of OXA-LIP was 52% with low leakage rates at 25 °C.Thermal hyperalgesia changes showed OXA has significant effects in the induction of neuropathy on days 7, 14, and 21 compared to the control group. OXA had a significantly greater sensitivity than the OXA-LIP and control groups in the thermal allodynia test (P < 0.001). OXA-LIP administration did not show significant effects on the changes of oxidative stress, biochemical factors, and cell count. Our findings provide a proof of concept on the potential application of oxaliplatin encapsulated with PEGylated nanoliposome to ameliorate the severity of neuropathy, supporting further studies in clinical phases to explore the value of this agent for Chemotherapy-induced peripheral neuropathy.
Targeted therapies using nanoparticles have been proposed to address some drawbacks of conventional chemotherapy. In the last two decades, surface modification of nanoparticles with antibody ligands has been extensively explored for the safe and effective delivery of chemotherapeutic drugs. By capitalizing on receptors overexpressed on cancer cells or angiogenic endothelial cells, antibody-functionalized nanoparticles are promising candidates to improve selectivity for target cells and, thus, therapeutic efficacy. This work offers a brief overview of nanotechnology applied to targeted cancer therapies and highlights the use of antibodies as targeting ligands. After presenting a summary of the most common methods for nanoparticle functionalization with antibodies, the authors discuss different targeting strategies divided into angiogenesis-associated targeting, uncontrolled cell proliferation targeting, and tumor cell targeting. Special emphasis is given to antibody-nanoparticle conjugates that have entered the clinical testing phase. Some explanations for the discrepancy between preclinical and clinical observations are also provided.
Colorectal cancer (CRC) is among the most rampant diseases globally and still has a high fatality and relapse rate despite the significant developments in conventional treatment modalities. In the realm of effective cancer treatments, nanotechnology-based drug delivery systems have attracted great interest. The most researched drug delivery system for successful cancer therapy is liposomes because of their unique qualities, such as biocompatibility, improved entrapment efficiency, and scalability as well as cost-effective process. While on the contrary, polymeric nanoparticles possess good stability, controlled release, and the capability for several chemical alterations. However, lipid peroxidation, burst release, restricted surface changes, and polymer toxicity limit their applications. Thus, lipid-polymer hybrid nanoparticles (LPHNPs) have been identified as promising nanosystems with respect to a number of biomedical applications. These particles are a prospective delivery system made up of combinatorial properties of liposomes and polymeric nanoparticles. A deeper understanding of LPHNPs types, synthesis aspects with their controllable parameters, release mechanisms, and surface functionalization are all elements of the current review. This review article emphasizes the significant uses of LPHNPs relating to the delivery of drugs and their targeting as well as the obstacles hampering their clinical translation with the potential for utilizing these nanoplatforms to treat colorectal cancer.
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Nanotechnology takes the lead in providing new therapeutic options for cancer patients. In the last decades, lipid-based nanoparticles—solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), liposomes, and lipid–polymer hybrid nanoparticles—have received particular interest in anticancer drug delivery to solid tumors. To improve selectivity for target cells and, thus, therapeutic efficacy, lipid nanoparticles have been functionalized with antibodies that bind to receptors overexpressed in angiogenic endothelial cells or cancer cells. Most papers dealing with the preclinical results of antibody-conjugated nanoparticles claim low systemic toxicity and effective tumor inhibition, which have not been successfully translated into clinical use yet. This review aims to summarize the current “state-of-the-art” in anticancer drug delivery using antibody-functionalized lipid-based nanoparticles. It includes an update on promising candidates that entered clinical trials and some explanations for low translation success.
Bioactive lipids are now widely accepted players in the field of cancer biology. Targeting components like enzymes, bioactive lipids and receptors are important for maintaining lipid homeostasis, metabolism, and signaling so that cell proliferation and metastasis can be reduced. This can be achieved through various means like modifying functions of enzymes involved in lipid biosynthesis, altering the structure and composition of bioactive lipids, disruption of lipid mediated tumor microenvironment, and by promoting apoptosis. These strategies can encourage the treatment and cure of cancer. This book is divided into three parts: chemistry, formulation, and mechanism of bioactive lipids in cancer. In the chemistry portion, Mal et al. stated that ceramides act as second messengers for apoptosis. There are several pieces of evidence to support the use of exogenous C2 and C6 ceramides and ceramide modulators like FTY720, PDMP, NOE, Tamoxifen, etc., with different chemotherapeutic agents to fight cancer in a better way. Another chapter by Rati Kailash Prasad Tripathi states that lipoxins and their epimers, viz. epi-lipoxins, are involved in the performance of crucial functions causing attenuation of the cancer-associated inflammation, which portrays a synergic rationale that integrates the anti-inflammatory hallmarks and raises anti-tumor immunity. Das et al. states that resolvins have the potential for the growth of further anticancer agents. Malik et al. states that Sphingosine-1 phosphate (SIP) functions as both the first and second messenger and act both extracellularly and intracellularly. Although the cell proliferating activity of SIP is good in different disease conditions, it does not affect cancer very well. Another part of this book is based upon formulation of bioactive lipids in cancer. Basu et al. states that lipid synthesis and its signaling are the commonly recognized players in the genetics of cancer. Jain and Pillai state that exosomes have lipids as one of their contents and serve as their natural transporters carrying them to distant cells. So, exosomes can be explored for their utility as prognostic/diagnostic marker and future pharmacological targets. The next part is based on the mechanism of bioactive lipids in cancer. In this category, Garg et al. state that computational techniques are helpful in the identification of bioactive lipid drugs and their targets. However, it is essential to use these techniques correctly way to get reliable information. Singh et al. state that hypoxic cancer cells remain in high demand of lipids, and the increasing demand cannot be accomplished only through glucose metabolism. Therefore, HIF-1α acts at the genetic level to enhance lipid synthesis in cancer cells. The utilization of acetate, lactate, glutamate along with glucose for fatty acid synthesis makes them a crucial metabolite both in the diagnosis and treatment of solid hypoxic tumors. Alongside, enzymes involved in these pathways could be fascinating targets for newer drugs. Sahu et al. state that targeted therapies are used to treat advanced colorectal cancer (CRC) induced by mutated genes. The various novel drug deliveries have been developed and demonstrated significant inhibition of cancerous cells in CRC. Pal and Raj, state that avocado can prevent or diagnose or treat various kinds of human cancer cells (HCCs). If more research work continues, then in the near future, it becomes an excellent therapeutic agent in cancer therapy. Mitra and Banerjee state that lipopolysaccharide (LPS) is a potent inducer of carcinogenesis upon overexposure. The same LPS can be anti-carcinogenic at optimum levels or if modified. Future studies can focus on selective immune activation by targeting specific intermediate adaptor proteins of LPS/TLR-4 interaction to initiate possible immuno-therapy against different cancer types. Pal and Saha state that LA is a glycerophospholipid and worked via G-protein-coupled receptors with six subtypes LA1 –LA6 . Among them, LA2 directly affects the cell survival rate of GRI-977143 (agonist of LA2 ) against lung cancer cell lines. Banerjee et al. state that several clinical pieces of evidence confirm the anticancer potential of ω-PUFAs. Experimental data indicates the beneficial effect of these acids in CRC and advanced metastatic disease, from the primary to tertiary prevention. So, this complete book summarizes the benefits of bioactive lipids for the treatment and prevention of cancer.
The term “liposomes” was given by Weismann and coworkers to the spontaneously formed closed structures of phospholipid bilayers, when they were hydrated in water. Gregoriadis established the concept that the drugs could be encapsulated into liposomes and used as drug‐delivery vehicles. The manufacturing of liposomes has advanced significantly, beginning with the thin lipid film hydration and progressing through reverse‐phase evaporation, freeze‐drying and scalable ethanol injection methods. Stealth technology has been extensively investigated in developing a drug‐delivery system making these liposomes difficult to detect by the mononuclear phagocyte system. Liposome‐based products are complex formulations; hence, small changes in the formulation may significantly affect the clinical outcomes. Targeted drug delivery is a strategy that preferentially and selectively delivers the active therapeutics to the site of action, that is, target site while concurrently minimizing the access and exposure to the nontarget site.
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To identify better regimens in currently available chemotherapy would be beneficial to KRAS mutant metastatic colorectal cancer (mCRC) patients because they have fewer treatment options than KRAS wild-type mCRC patients. Clinicopathologic features and overall survival (OS) of KRAS mutant and wild-type mCRC patients who had used oxaliplatin-based, irinotecan-based, bevacizumab-based, as well as cetuximab-based regimens were compared to those who had never-used oxaliplatin-based, irinotecan-based, bevacizumab-based, as well as cetuximab-based regimens respectively. Between 2007 and 2012, a total of 394 mCRC patients, in whom 169 KRAS mutant and 225 KRAS wild-type, were enrolled. In KRAS mutant patients who had used oxaliplatin-based regimens (N = 131), the OS was significantly longer than that in KRAS mutant patients who had never-used oxaliplatin-based regimens (N = 38). The OS was 28.8 months [95% confidence interval (CI): 23.2-34.4] in KRAS mutant patients who had used oxaliplatin-based regimens versus 17.8 months [95% CI: 6.5-29.1] in KRAS mutant patients who had never-used oxaliplatin-based regimens (P = 0.026). Notably, OS in KRAS wild-type mCRC patients who had used oxaliplatin-based regimens (N = 185) was not significantly longer than that in KRAS wild-type mCRC patients who had never-used oxaliplatin-based regimens (N = 40) (P = 0.25). Furthermore, the OS in KRAS mutant patients who had used either irinotecan-based, bevacizumab-based or cetuximab-based regimens was not significantly different than that in KRAS mutant patients who had never-used either irinotecan-based, bevacizumab-based or cetuximab-based regimens respectively. In multivariate analyses, patients who had used oxaliplatin-based regimens remains an independent prognostic factor for longer OS in KRAS mutant mCRC patients. In conclusion, oxaliplatin-based regimens are more beneficial in KRAS mutant than in KRAS wild-type mCRC patients.
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Fifteen monoclonal antibodies (mAbs) are currently registered and approved for the treatment of a range of different cancers. These mAbs are specific for a limited number of targets (9 in all). Four of these molecules are indeed directed against the B-lymphocyte antigen CD20; 3 against human epidermal growth factor receptor 2 (HER2 or ErbB2), 2 against the epidermal growth factor receptor (EGFR), and 1 each against epithelial cell adhesion molecule (EpCAM), CD30, CD52, vascular endothelial growth factor (VEGF), tumor necrosis factor (ligand) superfamily, member 11 (TNFSF11, best known as RANKL), and cytotoxic T lymphocyte-associated protein 4 (CTLA4). Collectively, the mAbs provoke a wide variety of systemic and cutaneous adverse events including the full range of true hypersensitivities: Type I immediate reactions (anaphylaxis, urticaria); Type II reactions (immune thrombocytopenia, neutopenia, hemolytic anemia); Type III responses (vasculitis, serum sickness; some pulmonary adverse events); and Type IV delayed mucocutaneous reactions as well as infusion reactions/cytokine release syndrome (IRs/CRS), tumor lysis syndrome (TLS), progressive multifocal leukoencephalopathy (PML) and cardiac events. Although the term "hypersensitivity" is widely used, no common definition has been adopted within and between disciplines and the requirement of an immunological basis for a true hypersensitivity reaction is sometimes overlooked. Consequently, some drug-induced adverse events are sometimes incorrectly described as "hypersensitivities" while others that should be described are not.
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Platinum-based chemotherapy, such as cisplatin, oxaliplatin, and carboplatin, is one of the most widely utilized classes of cancer therapeutics. While highly effective, the clinical applications of platinum-based drugs are limited by their toxicity profiles as well as suboptimal pharmacokinetic properties. Therefore, one of the key research areas in oncology has been to develop novel platinum analog drugs and engineer new platinum drug formulations to improve the therapeutic ratio further. Such efforts have led to the development of platinum analogs including nedaplatin, heptaplatin, and lobaplatin. Moreover, reformulating platinum drugs using liposomes has resulted in the development of L-NDPP (Aroplatin™), SPI-77, Lipoplatin™, Lipoxal™, and LiPlaCis®. Liposomes possess several attractive biological activities, including biocompatibility, high drug loading, and improved pharmacokinetics, that are well suited for platinum drug delivery. In this review, we discuss the various platinum drugs and their delivery using liposome-based drug delivery vehicles. We compare and contrast the different liposome platforms as well as speculate on the future of platinum drug delivery research.
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One method for improving cancer treatment is the use of nanoparticle drugs functionalized with targeting ligands that recognize receptors expressed selectively by tumor cells. In theory such targeting ligands should specifically deliver the nanoparticle drug to the tumor, increasing drug concentration in the tumor and delivering the drug to its site of action within the tumor tissue. However, the leaky vasculature of tumors combined with a poor lymphatic system allows the passive accumulation, and subsequent retention, of nanosized materials in tumors. Furthermore, a large nanoparticle size may impede tumor penetration. As such, the role of active targeting in nanoparticle delivery is controversial, and it is difficult to predict how a targeted nanoparticle drug will behave in vivo. Here we report in vivo studies for αvβ6-specific H2009.1 peptide targeted liposomal doxorubicin, which increased liposomal delivery and toxicity to lung cancer cells in vitro. We systematically varied ligand affinity, ligand density, ligand stability, liposome dosage, and tumor models to assess the role of active targeting of liposomes to αvβ6. In direct contrast to the in vitro results, we demonstrate no difference in in vivo targeting or efficacy for H2009.1 tetrameric peptide liposomal doxorubicin, compared to control peptide and no peptide liposomes. Examining liposome accumulation and distribution within the tumor demonstrates that the liposome, and not the H2009.1 peptide, drives tumor accumulation, and that both targeted H2009.1 and untargeted liposomes remain in perivascular regions, with little tumor penetration. Thus H2009.1 targeted liposomes fail to improve drug efficacy because the liposome drug platform prevents the H2009.1 peptide from both actively targeting the tumor and binding to tumor cells throughout the tumor tissue. Therefore, using a high affinity and high specificity ligand targeting an over-expressed tumor biomarker does not guarantee enhanced efficacy of a liposomal drug. These results highlight the complexity of in vivo targeting.
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Introduction: Liposomes represent a versatile system for drug delivery in various pathologies. Platinum derivatives have been demonstrated to have therapeutic efficacy against several solid tumors. But their use is limited due to their side effects. Since liposomal formulations are known to reduce the toxicity of some conventional chemotherapeutic drugs, the encapsulation of platinum derivatives in these systems may be useful in reducing toxicity and maintaining an adequate therapeutic response. Areas covered: This review describes the strategies applied to platinum derivatives in order to improve their therapeutic activity, while reducing the incidence of side effects. It also reviews the results found in the literature for the different platinum-drugs liposomal formulations and their current status. Expert opinion: The design of liposomes to achieve effectiveness in antitumor treatment is a goal for platinum derivatives. Liposomes can change the pharmacokinetic parameters of these encapsulated drugs, reducing their side effects. However, few liposomal formulations have demonstrated a significant advantage in therapeutic terms. Lipoplatin, a cisplatin formulation in Phase III, combines a reduction in the toxicity associated with an antitumor activity similar to the free drug. Thermosensitive or targeted liposomes for tumor therapy are also included in this review. Few articles about this strategy applied to platinum drugs can be found in the literature.
In this study, single-walled carbon nanotubes (SWNTs) conjugated with antibody C225 were used to achieve targeted therapy against EGFR over-expressed colorectal cancer cells. In addition, the control release of the chemotherapeutic drug, 7-Ethyl-10-hydroxy-camptothecin (SN38), was studied. We used three different colorectal cancer cell lines, HCT116, HT29, and SW620, listed in the order of decreasing expression levels of EGFR. Our results showed that SWNT could use C225 to specifically bind to EGFR-expressed cells. The cellular uptakes of SWNT of EGFR over-expressed cells (HCT116 and HT29) were much higher than that of the negative control (SW620). We, next, demonstrated that receptor-mediated endocytosis was the primary cell entry route for SWNT. As a consequence, abundant amount of SN38 was released and EGFR over-expressed cells were killed. The drug control release process was studied by utilizing human carboxylesterase enzyme (hCE) that would break the bond linking SN38 and SWNT-carrier in cytoplasm. The intracellular SN38 release observed by confocal microscopy showed that SN38 actually dissociated from the SWNT-carrier first. SN38's entry to nucleus was then followed while the SWNT-carrier still remained in the cytoplasm. Overall, all these data suggested that SWNT could be a good carrier for targeting controlled release therapy.
Separation of polar lipids by two-dimensional thin layer chromatography providing resolution of all the lipid classes commonly encountered in animal cells and a sensitive, rapid, reproducible procedure for determination of phospholipids by phosphorus analysis of spots are described. Values obtained for brain and mitochondrial inner membrane phospholipids are presented.