SUPPLEMENTARY DATA
Mono and Dually Decorated Nanoliposomes for Brain Targeting, in vitro and in vivo
studies
E. Markoutsa,1, a K. Papadia,1, a A.D. Giannou,2 M. Spella,2 A. Cagnotto,3 M. Salmona,3 G.
T. Stathopoulos,2 and S. G. Antimisiaris,1,4 ,*
1
Laboratory of Pharmaceutical Technology, Department of Pharmacy, University of Patras, Rio 26510,
Greece
2
Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine,
University of Patras, Rio 26510, Greece
3
Department of Biochemistry and Molecular Pharmacology, IRCCS-Istituto di Ricerche Farmacologiche
"Mario Negri", Milan, Italy;
4
Foundation for Research and Technology Hellas, Institute of Chemical Engineering Sciences,
FORTH/ICES, Rio 26504, Greece
a
Equal contribution of two first authors
S1. Methodology for preparation of APOe peptide-decorated LIPs
For preparation of APOe-LIP, pre-formed LIPs with surface Maleimide group anchors on their
surface, consisting of DSPC/Chol/PEG-lipid/PEG-Mal at 20:10:0.8:0.01(mole/mole) [low PEG
(equal to 2.6 mol%-of total lipid-)] or 20:10:1.6:0.01(mole/mole) [high PEG equal to 5.1
mol%-of total lipid-) ], and control liposomes (without PEG-Mal) were prepared, as described
in the Methods section. For conjugation of APOe by formation of a thioether bond between the
cysteine of the peptide and Mal-groups immobilized on the pre-formed LIPs, sample (with Mal
groups) or control LIPs were incubated overnight with the peptide (at 1.1 molar excess of
peptide) at 250C. After incubation, the fluorescence intensity (FI) was measured (EX-280nm,
EM-350nm), and non-attached peptides were removed by extensive dialysis (using Spectrapore
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dialysis tubing, MW-cutoff 10000). Following dialysis, sample FI was re-measured and the
peptide attachment yield (%) was calculated from initial (before dialysis) and final (after
dialysis) FI values (compared as: FI of sample/moles of lipid in sample). Non-specific binding
was estimated from control LIPs, with the same method.
When low PEG-lipid amount (2.6 mol%) was used for initial formation of LIPs, the peptide
attachment yield was very low (approx. 20%) and practically the same for sample and control
formulations (Table S1), indicating that the binding of the peptide on liposomes was “nonspecific“ and was not taking place via the functional MAL moiety of Mal-PEG-lipid. This
inability of the peptide to approach MAL-groups and covalently bind to them is most possibly
attributed to the fact that the MAL-group is hidden in the PEG chain (being in “mushroom”
conformation) as reported previously for other types of interacting moieties [Kaasgaard T,
Mouritsen OG, Jørgensen K. Receptor mediated binding of avidin to polymer covered liposomes.
J Liposome Res 2001;11 (1) : 31-42]. On the contrary, when high PEG-lipid amounts were
incorporated in LIPs (> 5 mol% was found tobe sufficient in the current case), ensuring that
PEG-chains are in brush conformation, the attachment yield of the peptide increased to
practically 100 percent. Results in Table S1 are those measured fort he mpnpmer APOe peptide.
Similar values were also measured when the dimer-peptide was used (differences were always
less than 8%).
Table S1. Attachment yield of APOe (monomer), calculated as percent of initial APOe
incubated with the LIP samples (as described above), for low-PEG and high-PEG, control
(without PEG-Mal) and sample (with PEG-Mal) LIP preparations.
LIP-type
Control
Sample
Control
Sample
Lipid Composition
DSPC/Chol/PEG-lipid/PEG-Mal
20 :10 : 0.81 : 0
20: 10 : 0.80 : 0.01
20 :10 : 1.61 : 0
20 :10 : 1.60 : 0.01
PEG
amount
low PEG
high PEG
Attachment
Yield (%)
18.7 ± 4.3
20.3 ± 2.9
21.7 ± 3.3
108.7 ± 7.1
2
The peptide coupling procedure was carried out at three different pH values (6.50, 7.40 and
8.00), in order to identify optimum pH for thioether bond formation. As observed from Fig. S1, it
**
120
**
**
100
80
40
non-specific
60
non-specific
non-specific
Peptide attachement Yield (%)
is verified that the coupling yield (as also the non-specific binding) is not affected by pH.
20
0
pH
6.50
7.40
8.00
Figure S1. Peptide attachment yield of ApoE (monomer) on DSPC/ Chol / PEG-lipid/ PEG-Mal
(20:10:1.6:0.01 mole/mole) [sample] –patterned bars-, or DSPC/ Chol / PEG-lipid/ PEG-Mal
(20:10:1.61:0 mole/mole) [control] -solid bars- LIPs, when the peptide/ (pre-formed) LIP
incubations (see above for details) is carried out in buffer at different pH values.
S2. Preparation of dual-decorated nanoliposomes (DUAL-LIP)
Initially two different ligation methodologies were investigated, for preparation of DUAL-LIPs.
Pre-formed-LIPs with both, -Mal and -Biotin surface group anchors consisting of
DSPC/Chol/PEG-lipid/PEG-Mal/PEG-Biotin at 20:10:1.6:0.01:0.01 (mole ratios) were prepared,
as described in Methods section of the manuscript. Two different procedure sequences were
tested for attachment of the two ligands (which were performed with the methods described in
detail in the Methods section), and the best one was finally selected, based on the attachment
yields for both, peptide and MAb.
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In procedure A: The ApoE peptides were attached on the pre-formed-LIPs, before attaching
STREP molecules (for subsequent biotin-MAb coupling); while in procedure B: The STREP
molecules were attached before performing the incubation with the peptides. As seen from
results (Table S2), although the peptide attachment yield was not affected by the procedure used
(always >90 %), on the contrary the MAb attachment was significantly affected. Indeed MAb
attachment yield was higher when procedure B was followed; implying that perhaps STREP
coupling to vesicle biotin-groups was disturbed by the presence of the peptide on the vesicle
surface.
Table S2. Attachment yield of MAb and APOe peptides on pre-formed-LIP consisted of
DSPC/Chol/PEG-lipid/PEG-Mal/PEG-Biotin at 20:10:1.6:0.01:0.01 (mole ratios) (with –biotin
and –Mal groups on their surface), when two different procedures (sequences) were used for
attachment of the ligands (described in detail above).
Procedure (sequence)
MAb attachment Yield
APOe attachment Yield
(%)
(%)
38.8 ± 9.4
97.8 ± 2.1
Procedure A
65 ± 10
96.5 ± 3.5
Procedure B
S3. Cytotoxicity studies
hCMEC/D3 cells were grown on 24-well plates until confluent. Medium was replaced and the
various LIP types (at concentrations used in the cell interaction studies) were incubated with
cells for 24 h at 37 0C, (5% CO2 /saturated humidity). After incubation the medium was removed
and the cells were washed with PBS. Fresh medium containing 0.5 mg/ml 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added. Cells were incubated
for 2.5 h, the medium was removed and DMSO was added (at 37 0C for 30 min) to dissolve the
formazan crystals that formed. Alive cells (%) were calculated based on the formula (A570sampleA570background) / (A570control-A570background) x 100, where A570control is the OD-570 nm of
untreated cells, and A570background the OD-570 nm of MTT without cells (Fig S2).
4
80
(% of Control)
h-CMEC/D3 cell viability
100
PEG-LIP (control)
APOe-LIP
MAb-LIP
DUAL-LIP
60
40
20
0
6
9
12
4
Liposome concentration (nmoles lipid / 3x10 cells)
Figure S2. hCMEC/D3 cell viability, as measured by the MTT method (details above) in
presence of the various LIP-types at similar conditions (liposomal lipid/ cells) with those used
for the LIP uptake studies. The positive control Triton X-100 (0.1% v/v) resulted in <5% viability
(not shown).
S4. Verification of expression low-density lipoprotein receptor related protein (LPR) by
hCMEC/D3 cells
For western blot analysis, hCMEC/D3 cells and MDA-MB 231 cell lines (used as control cell
lines) were grown in T-25 flasks to 90% confluence. Cells were lysed with RIPA assay buffer
[PBS, 1% NP-40 (Thermo Fisher Scientific, Illkirch Cedex, France), 0.5% sodium deoxycholate,
and 0.1% SDS] and the lysates were centrifuged at 12,000 × g for 15 minutes. 100-150 μg of
extracts were separated by SDS-PAGE gel 12% and transferred onto nitrocellulose membrane.
Membranes were incubated in blocking Tris-buffered saline buffer (20 mM Tris, 50 mM NaCl)
containing 5% (w/v) low-fat milk and 0.1% (v/v) Tween 20 and then were incubated with anti
LDL-R (SC18823) (1:200) overnight at 4°C. Subsequently, blots were washed and incubated
with the anti-IgG-peroxidase-linked secondary antibody for 1 hour at room temperature before
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detection using a chemiluminescent detection kit (NEN Life Science, Paris). Results show that
hCMEC/D3 cells express the LPR receptor (Fig. S3)
Figure S3. Western blot micrographs which confirm the presence of LPR receptor on
hCMEC/D3 cells
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