lipid digestion using Fourier transform infrared (FTIR) spectroscopy. Lipid was quantitated using perdeuterated nonadecane as an internal standard. Thin films ...
ANALYTICAL
BIOCHEMISTRY
l&31,28-32
(1989)
Fourier Transform Infrared Assay of Liposomal Lipids Purdue
Charles
Pidgeon,’ G. Apostol, and R. Markovich University School of Pharmacy, West Lafayette, Indiana 47907
Received
December
19,1988
MATERIALS Quantitating the lipid content in organic lipid solutions and extracted membrane preparations is described. Fourier transform infrared analysis of thin films using perdeuterated nonadecane as an internal standard permitted quantitation with >95% accuracy. 0 1999 Academic Press, Inc.
Liposomes have been used for in vitro and in vivo gene transfer experiments (l-5) and also as a drug delivery system to macrophages in vitro and in vivo (6-8). In addition, empty liposomes without encapsulated DNA stimulate polyethylene glycol-mediated transfection of protoplasts (9). Our lab is encapsulating potential antiAIDS2 oligonucleotides in liposomes with the intent of enhancing oligonucleotide delivery into cells. Using liposomes for biological experiments, as described above, requires accurate determination of the amount of membrane lipids in the final vesicle preparation. Typically radioactive lipids or a phosphate assay (10) is used for lipid quantitation. We developed a nonradioactive, sensitive, convenient method for quantitating lipid in membrane preparations without phospholipid digestion using Fourier transform infrared (FTIR) spectroscopy. Lipid was quantitated using perdeuterated nonadecane as an internal standard. Thin films prepared from organic extracts of membrane lipids were analyzed by FTIR spectroscopy. Lipid quantitation utilized the methylene symmetric and asymmetric stretches in both membrane lipids and the deuterated internal standard. An FTIR data station facilitated calculations of lipid concentrations from the integrated band intensities associated with the CH and CD stretches. I To whom correspondence should be addressed. ’ Abbreviations used: AIDS, acquired immunodeficiency syndrome; FTIR, Fourier transform infrared; PBS, phosphate-buffered saline; &D4,,, perdeuterated nonadecane; CHC13, chloroform; MEOH, methanol; DMPC, dimyristoylphosphatidylcholine egg PC, egg phosphatidylcholine; CH, cholesterol.
AND
METHODS
Chemicals All lipids were purchased from Avanti Polar Lipids (Birmingham, AL). Perdeuterated nonadecane (C19D4,,) 98.7% atom D was purchased from MSD Canada (Point C-Claire Dorval, Quebec). Perdeuterated nonadecane is solid at room temperature (mp 32-34°C). Stock solutions of perdeuterated nonadecane (1.5 mg/ml) were prepared in HPLC-grade CHCl, obtained from Aldrich Chemical Co. (St. Louis, MO). HPLC-grade methanol (MEOH) used for lipid extraction was purchased from Fisher Scientific. All other chemicals were analytical grade and were used as received. Dulbecco’s phosphatebuffered saline (PBS) 10X obtained from GIBCO (Grand Island, NY) was diluted with filtered water free of trace organics. This PBS was used to disperse lipids into liposomes. Sample Preparation Liposomes were formed by hydrating 50-100 mg of lipid with 1 ml of PBS in a 25- or 50-ml round-bottom flask. Liposome stock suspensions were prepared by quantitatively transferring the liposome suspension to sealed test tubes by carefully washing the round-bottom flask used to prepare the vesicles 2X with PBS. Standard curves were prepared from aliquots of the liposome stock suspension. Liposome sample aliquots, 5-25 ~1, measured with a Hamilton syringe were transferred to 13 X loo-mm glass culture tubes and diluted to 0.5 ml with double-distilled water. Each aliquot was extracted by adding 0.5 ml MEOH, then adding 0.5 ml CHC& containing perdeuterated nonadecane, and vortexing for 30 s3.Both CHC& and MEOH were pipetted using repi3 This extraction cannot be done in plastic Eppendorf tubes. The CHCl, used to extract the lipid leaches hydrocarbon from plastic tubes. Polymer leaching causes large errors in the FTIR measurements by increasing the hydrocarbon content in the CHCl, extract. Because of polymer leaching, plastic pipet tips should not be used to transfer organic solvents during the lipid extraction procedure.
28
0003-2697/89
Copyright All
rights
0 1989 by Academic
of reproduction
in any form
$3.00
Press, Inc. reserved.
FOURIER
TRANSFORM v
3 !To-
2870
Z&O
2410
INFRARED
CD,pYrn
2i80
liJ50
WAVENUMBER
FIG. 1. Typical FTIR spectra of lipids. Peak positions are given in Table 1. The internal standard, perdeuterated nonadecane 98.7% atom
D shows
small
amounts
of CH stretch
(middle
spectrum).
pet dispensers purchased from Fisher Scientific. After centrifugation (2000 rpm, 1 min) to phase separate the organic aqueous mixture, the upper aqueous phase was discarded just prior to obtaining spectra, and the CHCls phase containing extracted membrane lipids was used to prepare thin films for FTIR analysis. Standard curves used six lipid concentrations. Initial experiments showed triplicate determination to be extremely reproducible. Triplicate determination had a coefficient of variation < 4.7. Consequently, triplicate determinations are unnecessary and we routinely use single-point determination for the assay. FTIR spectral measurements for both standard curves and unknowns were single-point determinations. Thin films for analysis were prepared in a manner similar to applying samples to TLC plates, i.e., dropwise, allowing the drops to evaporate before applying the next drop. Thus, a 25~1 Hamilton syringe was used to care-
TABLE Peak Positions
Asym” Sym b Note.
Spectral
’ Asymmetric * Symmetric
LIPOSOMAL
29
LIPIDS
fully deposit in a dropwise manner 5-15 ~1 of the CHCl, extract onto the center of a 25 X 4-mm CaFz disk purchased from McCarthy Scientific (Fullerton, CA). Care was taken to avoid contaminating the thin film with residual aqueous phase. Aqueous phase contaminants may generate variability in the standard curves. Tilting the test tube allows residual water to move aside, permitting the needle of the Hamilton syringe to obtain a clean CHC& sample. CaF, has a cutoff of 1110 cm-’ and is stable to aqueous and organic solvents. CaF, disks were cleaned with CHC& between samples and the same disk was used for each sample. The ir spectrum of CHC& contain a CH stretch at 3019.5 cm-l and the total evaporation of CHC& was monitored by this peak. FTIR spectra used for lipid quantitation did not contain a peak at 3019.5 cm-‘. Thin films prepared from 5-15 ~1 of CHC& had small diameters, -10 mm wide. The positioning of the thin film in the light beam was critical for accurate determination of lipid. The thin film was positioned into the path of the ir beam using the laser beam of the instrument. The CaF, plate was positioned such that diffraction of the laser light occurred on the CaF, plate itself. When this occurred, the thin film was in the light path of the spectrometer. Infrared Spectroscopy A Nicolet 2Osxc FTIR spectrometer equipped with a triglycine sulfate detector was used for all infrared spectra. Infrared spectra were obtained from double-sided interferograms taken at 4 cm-l resolution, utilizing a Happ-Genzel apodization function before Fourier transformation. The correct positioning of the sample in the light path was verified by obtaining preliminary FTIR spectra using 32 scans. This preliminary spectrum (not used for determining lipid content) was visually inspected to ensure evaporation of residual CHC& and complete purge of the sample compartment. After a satisfactory preliminary spectrum was obtained, the sample was rescanned to quantitate lipid. All thin film spectra
1
XH,
XD,
(cm-‘)
(cm-‘)
GCHs (cm-‘)
;CDB (cm-‘)
2918.7
2195.2 2093.7
2956.6
2216.3
2872.3
2069.4
deconvolution stretch. stretch.
OF
after Spectral Deconvolution of the Methylene Symmetric and Asymmetric Stretch of Dimyristoylphosphatidylcholine and Perdeuterated Nonadecane
2850.3
= 1.5.
ASSAY
(11) used a Bessel
function
and half-width
half-height
;CH1/;CDP
Vibrations
XHJ;CD,
1.33 1.36 of 20 cm-’
and a resolution
1.33 1.39 enhancement
factor
of
K
30
PIDGEON,
APOSTOL,
AND
MARKOVICH
ware was written (Appendix) to automatically integrate the area under the bands of interest. Deconvolution was used for band assignments of perdeuterated nonadecane. Parameters are given in the note to Table 1.
Irg Egg PC pi CHCl3
0.633
RESULTS
0.667
Initial attempts to assay membrane lipids using FTIR spectra of KBr pellets obtained from lyophilized liposome suspensions were unsuccessful. Consequently, membrane lipids were extracted and thin films were prepared for FTIR analysis. Similar to KBr pellets, thin films vary in thickness and quantitation requires an internal standard for Beer’s law plots. Perdeuterated nonadecane was chosen as the internal standard because intense bands in a clear region of the ir spectra are observed. Figure 1 compares the ir spectra of the internal standard and dimyristoylphosphatidylcholine (DMPC) in the region of interest. The methylene symmetric and asymmetric bands are clearly different between membrane lipids and the deuterated internal standard. Table 1 gives peak assignments of Fig. 1 based on (12) for DMPC and (13) for perdeuterated nonadecane. The methylene symmetric and asymmetric stretches of deuterated nonadecane are close to the theoretically expected band positions based on XH/XD = (2)1’2 = 1.4. This is shown in Table 1. In Fig. 1 the hydrocarbon region used to quantitate membrane lipids is apparent. The area under the lipid hydrocarbon region between 2992 and 2764 cm-l was used for quantitation. This area was ratioed to the 22802000 cm-* region of the internal standard. Standard
0.500 0.333 0.167 0
3ioo
2670
2690 2410 WVENUMBER
2i80
1450
FIG. 2. Typical spectra used to obtain standard curves for egg PC liposomes. The spectra were scaled to peak heights of the internal standard, perdeuterated nonadecane. The concentrations of egg PC in the CHCls extract used to obtain the spectra are given.
for lipid quantitation were obtained from 128 scans (approximately 0.91 s/scan) and an air reference was used as background. A separate background spectrum was collected for each sample. The background spectrum was collected immediately after the single-beam spectrum of the sample was obtained. Spectral data handling was minimal; water subtraction, baseline correction, and smoothing were unnecessary. FTIR spectra for standard curves were stored in the computer as a consecutive block of files. A computer program utilizing Nicolet soft-
AND
x Egg .9
DISCUSSION
PC:CH(66:34)
y=O.a615x
+ 0.1232
r-0.9947
X
.a
JDMPC
/ I
r-o.9977
J:
I
0
0.167
0.333 pg
FIG.
3.
Standard
curves
obtained
0.500
Lipid/p1
from
0.667
0.833
CHC13extfact
extracting
liposome
suspensions
of known
lipid
concentration.
FOURIER
TRANSFORM
INFRARED
ASSAY
TABLE
OF
LIPOSOMAL
31
LIPIDS
2
Accuracy of FTIR Analysis of Extracted Lipids Egg 1-2 Unknown
Lipid (/e/d)
1 2 3 4 5 6 7 8 9 10
0.692 0.708 0.198 0.358 0.36 0.602 0.138 0.440 0.516 0.388
Egg PC:CH
(66:34)
DMPC
Lipid a
% error
Lipid
% error
(/.%/wl) a
-1.1 t1.1 -1.0 -2.2 0.0 to.3 t3.0 t1.4 +18.gb -3.0
0.030 0.218 0.338 0.321 0.205 0.494 0.079 0.363 0.126 0.372
a This is the concentration of total lipid in the CHCl, extract. Approximately FTIR analysis of lipids (see Materials and Methods). ’ After extraction, a cloudy interface between the aqueous and organic layer ’ Below the sensitivity limit of the assay.
curves were obtained by plotting the ratio given by Eq. [l] vs lipid concentration in the CHCls extract:
Figure 2 shows a typical set of spectra from different concentrations of extracted liposomal lipids. In Fig. 2, a small CO2 band near 2350 cm-’ is apparent. Although occasionally large, subtraction of this COz band is unnecessary. Figure 3 gives typical standard curves for three different lipid mixtures used to prepare the vesicles. Regression analysis of standard curves always gave correlation coefficients > 0.98 for all lipid mixtures tested. The intercept is slightly positive, partially because nonadecane 98.7% atom D elicits CH absorbance in the lipid region used for integration. Table 2 shows the accuracy of the assay using 10 unknowns for each lipid mixture. Accuracy is typically greater than 95%. In the assay of unknowns, Abs(CH)/ ABS(CD) was always kept within the range of standards. Optimum results are obtained when standard curves are prepared between 0.2 and 1.0 pg lipidjyg CHC& extract. The sensitivity of the assay is -0.1 pg lipid/PI CHC&. As shown in Table 1, unknown lipid concentrations near 0.1 pg lipid/pg CHCl, were accurately determined. Thus, organic extracts containing less than 0.1 kg lipid/p1 CHCl, should be concentrated before measuring lipid content by the FTIR assay. Lipids are difficult to assay because they are poor chromophores and do not fluoresce. Unknown mem-
-46.7’ +4.4 +1.9 -3.6 -2.5 -1.0 -5.5 -0.9 -5.4 +1.4 515 caused
0.539 0.158 0.290 0.538 0.778 0.379 0.287 0.226 0.588 0.445
~1 of the CHCl, a large error
% error
(dd)”
extract
-1.9 +5.7 -0.1 -2.3 +1.5 +3.4 -0.9 -3.3 -2.0 +2.8
was deposited
in the assay of this
data
as a thin
film
for
point.
brane extracts are particularly difficult to assay because of the complex nature of membrane lipids. However, artificial membrane systems usually have defined lipid mixtures. Thus for artificial membrane systems, like liposomes, the lipid type is known and stock solutions are available. The FTIR assay described above permits rapid determination of membrane lipids in reconstituted systems. This assay allows quantitating lipid recovery after liposomes are separated or purified from unentrapped aqueous solutes by gel filtration, centrifugation, floatation on Ficoll gradients, and other standard techniques. The assay was developed for unknown lipid concentrations where the lipid mixture is known and the preparation of a standard curve is possible. Alternatively for quantitating the percentage lipid recovered after liposome purification, standard curves do not have to be measured. A single-point determination of the percentage lipid recovered in liposome preparations can be obtained using Eq. [2] if suitable corrections for liposomal volumes are made: % recovery
of lipids after liposome purification x 100. before liposome
[2]
purification
We note that using Eq. [ 21 for lipid recovery is similar to using phosphate content before and after liposome purification to calculate lipid recovered in the final liposome population. Using Eq. [2] does not require a stan-
32
PIDGEON,
APOSTOL,
dard curve and does not require knowledge of the lipid mixtures. However, using Eq. [2] will require that the membrane lipid composition is the same before and after liposome/vesicle purification.
AND
MARKOVICH
STD PRN FCD DFN=DFN+l TEM=3
APPENDIX
NPR
This computer program, denoted as MMM, was written for Nicolet sx software. This program baseline corrects and then integrates the area under a specified band. The program requires the first and last frequency for integration and makes calculations on a block of files. The program activates a digital LA50 printer to allow a hard copy of the integrated band intensities. After execution of the program, the printer is automatically turned off.
OMD xxxxxxxxxxxxxxxxxxx
:LST MMM OMD
END
BEFORE PEAK INTEGRATION, A BASELINE IS DRAWN FROM FXF TO LXF
ACKNOWLEDGMENTS
NXT
III
LXF=400 FXF=4000 TEM=4 NPR
We thank Sharon Armao for help in preparing this manuscript. This work was supported by NIH Grant 1UOlAlCA 25 712-01. This is publication number 2 accessing the Showalter FTIR facility at Purdue University School of Pharmacy and Pharmacal Sciences.
FXF LXF OMD ENTER SRT
FIRST
DATA
FILE
REFERENCES
FOR INTEGRATION
1. Fraley, J. Biol.
OMD
2. R&o,
ENTER
LAST
DATA
FILE
Virol.
FOR INTEGRATION
4. Mannino,
TEM=4 OMD INTEGRATION FXF
A. B. (1983)
J. Gen.
A., Spanjer, H., Londos-GagliG., and Nicolau, C. (1983) Proc. S. (1988)
Biotechniques
6,
FOR III=SRT SMD
BETWEEN
FXF AND
LXF
5. Ohgawara, T., Uchimiya, H., and Harada, H. (1983) Protoplasma 116,145148. 6. Pidgeon, C., Schreiber, R. D., and Schultz, R. M. (1983) J. Zmmunol. 131,311-314. 7. Sone, S., and Fidler, I. J. (1981) Cell. Zmmunol. 57.42. 8. Poste, G., Kirsh, R., Fogler, Res. 37,881. 9. Rodicio, M. R., and Chater,
PRN LXF BAS=YS TIL QIT
W. E., and Fidler, K. F. (1982)
I. J. (1979)
Cancer
151, 107%
J. Bacterial.
1085. 10. Bartlet,
PRN DFN
11. Griffiths, 209-215.
TEM=3
12. Casal,
MTD
R. J., and Gould-Fogeriti,
(1980)
682-690.
NPR
NPR
P., and Papahadjopoulos
J. D., and Mukherjee,
P., Dijkstra, J., Legrand, arbi, D., Roerdink, F., Scherphof, Natl. Acad. Sci. USA 80,7128-7131.
DFN=SRT
PRN
W. B., Schulman, 64,911-919.
3. Soriano,
QIT
PEAK
R., Subramani, S., Berg, Chem. 255,10431-10435.
G. R. (1959)
J. Biol.
P. R., and Parienti, H. L., and Mantsch,
Chem.
234,466-468.
G. C. (1986)
Trends
H. H. (1984)
Biochim.
Anal.
Chem.
Biophys.
5, Acta
779,381-401. 13. Mendelsohn, Dluhy,
R., Brauner, J. W., Faines, L., Mantsch, R. A. (1984) Biochim. Biophys. Acta 774,237-246.
H. H., and
