SUPPORTING INFORMATION
SUPPORTING METHODS
1. Estimation of total phospholipid content by Stewart method
Principle:
Phospholipids may be measured calorimetrically, without conventional acid digestion and
color development procedures, by forming a complex with ammonium ferrothiocyanate (25).
The red inorganic compound ammonium ferrothiocyanate is insoluble in chloroform, but
forms a complex with phospholipids which is freely soluble in chloroform. When a solution
of chloroform containing phospholipid is mixed intimately with ammonium ferrothiocyanate
at room temperature, a coloured complex (Amax= 472 nm) is formed which partitions in the
chloroform phase.
Preparation of ammonium ferrothiocyanate solution:
Throughout the work a standard solution of ammonium ferrothiocyanate was used. It was
prepared by dissolving 27.03g ferric chloride hexahydrate (FeCl3·6H20) and 30.4g
ammonium thiocyanate (NH4SCN) in deionized distilled water and the volume was made up
to 1 liter.
Calibration curve of lipid mixture:
All glassware’s used in the study were washed with chromic acid solution (5g sodium
dichromate was dissolved in 5 mL deionised water in a 250 mL beaker and to this solution
100mL of concentrated sulphuric acid was slowly added with constant stirring and allowed
the mixture to attain room temperature) to avoid the possible contamination from surface
active cleansing agents. A stock solution containing 90 mg of lipids mixture (HSPC:35 mg;
DPPC:35 mg; DPPG: 8.6 mg; DSPE-mPEG2000: 11.4 mg) was prepared in 100 mL
chloroform (900µg/mL). The stock solution also contains cholesterol of 13.5 mg as one of the
component of the final formulation. The 2.5 mL of the above stock solution was diluted up to
25mL with chloroform to obtain a concentration of 90 µg/mL solution. From the 90 µg/mL
stock solution 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 2mL were pipetted off (9µg to
180µg) and added to 3.0mL ammonium ferrothiocyanate solution in a test tube. Then the
enough chloroform was added to the test tubes to make the final chloroform volume 3.0 mL.
The biphasic system was then vigorously mixed for 3min. The lower chloroform phase was
separated with a syringe and the optical density of the chloroform phase was read at λmax 472
nm against chloroform as a blank and the average optical density was plotted against
concentration.
2. In vitro serum protein adsorption (opsonisation study)
In vitro serum/liposome incubation:
To 300 μL of non-PEGylated and PEGylated liposomal suspensions in a 2 mL polypropylene
micro test tubes (Eppendorf tubes), 1000 μL of 100% foetal bovine serum was added and the
liposome/serum mixture (76.9% final serum concentration) was incubated in a shaker
incubator (Scigenic ORBITEK, Germany) for 1hr at 37 °C. The incubation mixture was
immediately cooled using an ice/water bath for 5 min to stop the reaction.
Isolation of liposomes from the incubation mixture:
To separate liposomes from serum proteins, the incubation mixture (1300 μL) prepared above
was loaded onto a Sepharose CL-4B column (10mL pipette loaded with Sepharose CL-4B
gel) and eluted with double distilled water. Fraction of 600 μL was collected and replaced
with distilled water every time. To determine the liposome concentration in the fractions,
each fraction (600 μL/fraction) was analyzed for phospholipid content using Stewart method
with slight modifications (25). The fractions with the highest lipid content, typically fractions
7, 8, 9 and 10 from each column, were pooled and analysed for mean particle size, zeta
potential, lipid content, and adsorbed amount of serum proteins. The samples were stored at 70 °C until use (26).
Determination of % recovery of liposomes:
The % recovery of the liposomal suspension after separation from the column was
determined by measuring the phospholipid content of the liposome before incubation with
foetal serum and after separation through the column using Stewart method (25) with slight
modifications. Before Incubation with serum: To 0.1 mL liposomal dispersions in 2 mL
polypropylene micro test tubes, 1 ml of distilled water was added (1.1mL). To 0.2mL of
above liposomal dispersions in test tubes, added 3.0mL ammonium ferrothiocyanate solution
and 3.0mL chloroform. The biphasic system was then vigorously vortexed for 3 minutes.
After Recovery from serum: To 0.2 mL of the recovered liposomal suspension (from pooled
2.4 mL) in test tube, added 3.0mL ammonium ferrothiocyanate solution and 3.0mL of
chloroform. The biphasic system was then vigorously vortexed for 3 min.
On separating the lower chloroform phase from both the test tubes with a syringe the optical
density of the chloroform phase was read at λmax 472 nm against chloroform as a blank and
the amount of lipid present was estimated by calibration curve generated.
The percentage of liposomes recovered after separation of liposome serum mixture through
Sepharose CL-4B column was calculated using below mentioned equation.
% Liposome Recovered =
Total amount of lipid in recovered liposome
x 100
Total amount of lipid in liposomal dispersion incubated
3. Estimation of total serum protein associated with recovered liposomes
Quantification of the serum proteins in the pooled liposomal fractions was performed using
the BCA Protein Assay Kit (Bangalore Genei, Bangalore). The lipid in the sample did not
interfere with the protein assay under our experimental conditions. The known amounts of
bovine serum albumin were used to generate a standard curve. The protein concentrations in
test samples and standards were measured at a wavelength of 562 nm using
spectrophotometer. The protein binding index (PB: grams of total protein/mol of total lipid),
was calculated as described earlier (27). To 0.1 mL of recovered liposomal dispersions in 2
mL polypropylene micro test tubes, 1mL of methanol was added in order to extract the lipids.
The methanol was evaporated on a water bath maintained at 50 0C. To the residue, added 1
mL of distilled water and centrifuged at 6000 rpm for 15 min in order to settle down the
lipids. To 40 µL of supernatants in test tubes, added 160µL sodium azide solution (0.05 %
w/v) to make up the volume to 200 µL. To these test tubes 2 mL of BCA working reagent
was added and analysis was carried out at enhanced protocol (60°C for 30 min). After
incubation, cooled all tubes to room temperature and measured the absorbance at 562 nm (A562) of each tube against water as reference.
SUPPORTING TABLES
STI: The % liposomes recovered after separation through Sepharose CL 4B column
Amount of lipid
Amount of lipid in
Used in study
recovered
(µg)
liposomes (2.4 mL) (µg)
CLs-1
888.539
583.86±38.38
65.70±4.32
PLs-1 mol%
916.778
599.26±24.89
65.32±2.71
PLs-3 mol%
965.189
569.19±67.42
58.97±6.98
PLs-5 mol%
999.480
702.68±13.48
70.3±1.34
% Liposomes
Formulation
Recovered
STII: The amount of serum proteins associated with recovered anionic liposomes
lipid in
Amount of serum
Formulations
Protein binding (PB;
recovered
protein (µg)
µg/µM of lipid)
liposomes (µM)
CLs
355.31±18.56
0.718±0.046
494.417±22
PL-1 mol%
228.31±11.49
0.737±0.03
325.899±15
PL-3 mol%
219.06±21.21
0.7001±0.083
297.194±25
PL-5 mol%
209.12±11.93
0.863±0.015
242.085±15
Values are Mean ± SD, n=3. The CLs showed significantly high serum protein
binding than all PEGylated liposomes (***p<0.0001). The PEGylated liposomes with
5 mol% DSPE-mPEG2000 showed significantly less serum protein adsorption than 3
mol% DSPE-mPEG2000 (*p<0.05).
STIII: Cumulative % DTX released from different formulations
Cumulative % DTX released
Time
(hr)
Taxotere®
HSPC Alone
DPPC Alone
HSPC:DPPC
0.5
1.666±0.174
1.562±0.037
1.386±0.079
1.241±0.358
1
2.683±0.160
2.011±0.168
1.726±0.351
1.386±1.972
2
3.874±0.616
2.964±0.063
2.98±0.575
2.235±0.144
4
7.425±1.148
4.962±0.196
4.391±0.835
3.982±0.410
6
9.786±2.282
7.448±0.508
6.705±0.605
5.829±0.537
10
18.439±3.289 11.313±0.850 10.793±0.644
9.479±0.831
24
32.837±3.96
48
49.957±4.223 31.976±1.503 26.454±0.341 19.898±0.507
20.531±0.58
19.911±0.807 13.275±1.311
SUPPORTING FIGURES
SF1: Effect of PEGylation on serum protein binding (PB) of anionic liposomes
(a). After 24hr
(b). After 48hr
(c). After 72hr
SF2: The % A549 cell viability after (a). 24hr, (b). 48hr and (c). 72hr of treatment with TXT,
CLs, PLs and BLs
(a). After 24hr
(b). After 48hr
(c). after 72hr
SF3: The % B16F10 cell viability after (a). 24hr, (b). 48hr and (c). 72hr of treatment with
TXT, CLs, PLs and BLs
SF4: A549 cell uptake of 6-coumarin loaded CLs, and PLs
SF5: B16F10 cell uptake of 6-coumarin loaded CLs, and PLs
SF6: K9 cell uptake of 6-coumarin loaded CLs, and PLs
References:
25.
Stewart
JC.
Colorimetric
determination
of
phospholipids
with
ammonium
ferrothiocyanate. Anal Biochem 1980;104(1):10-4.
26. Ishida T, Ichihara M, Wang X, Yamamoto K, Kimura J, Majima E, et al. Injection of
PEGylated liposomes in rats elicits PEG-specific IgM, which is responsible for rapid
elimination of a second dose of PEGylated liposomes. J Control Release 2006;112(1):1525.
27. Chonn A, Semple SC, Cullis PR. Association of blood proteins with large unilamellar
liposomes in vivo. Relation to circulation lifetimes. J Biol Chem 1992;267(26):18759-65.