Low amounts of sucrose are sufficient to depress the phase transition temperature of dry phosphatidylcholine, but not for lyoprotection of liposomes

Abstract

Disaccharides such as sucrose and trehalose play an important role in stabilizing cellular structures during dehydration. In fact, most organisms that are able to survive desiccation accumulate high concentrations of sugars in their cells. The mechanisms involved in the stabilization of cellular membranes in the dry state have been investigated using model membranes, such as phosphatidylcholine liposomes. It has been proposed that the lyoprotection of liposomes depends on the depression of the gel to liquid-crystalline phase transition temperature (T(m)) of the dry membranes below ambient and on the prevention of membrane fusion by sugar glass formation, because both lead to leakage of soluble content from the liposomes. Since fusion is prevented at lower sugar/lipid mass ratios than leakage, it has been assumed that more sugar is needed to depress T(m) than to prevent fusion. Here, we show that this is not the case. In air-dried egg phosphatidylcholine liposomes, T(m) is depressed by >60 degrees C at sucrose/lipid mass ratios 10-fold lower than those needed to depress fusion to below 20%. In fact, T(m) is significantly reduced at mass ratios where no bulk sugar glass phase is detectable by Fourier transform infrared spectroscopy or differential scanning calorimetry. A detailed analysis of the interactions of sucrose with the P=O, C=O, and choline groups of the lipid and a comparison to published data on water binding to phospholipids suggests that T(m) is reduced by sucrose through a "water replacement" mechanism. However, the sucrose/lipid mass ratios necessary to prevent leakage exceed those necessary to prevent both phase transitions and membrane fusion. We hypothesize that kinetic phenomena during dehydration and rehydration may be responsible for this discrepancy.

FIGURE 1

Lipid melting curves of dry EPC liposomes as determined by FTIR spectroscopy. The wavenumber of the symmetric CH₂ stretching band (νCH₂s) is plotted as a function of the sample temperature. The samples contained either only EPC liposomes or Suc and liposomes at the different mass ratios indicated.

FIGURE 2

DSC heating thermograms of dry EPC liposomes in the presence of different amounts of Suc. The resulting Suc/EPC mass ratios are indicated to the right of each trace. Thermograms correspond to the second heating scan after cooling the samples from 100 to −60°C and subsequent equilibration at this temperature for 5 min.

FIGURE 3

DSC heating thermograms of dry EPC liposomes in the presence of Suc at a Suc/EPC mass ratio of 0.1 in the temperature range −40–60°C. Solid line, first heating scan; dashed line, second heating scan. See Fig. 2 for experimental details.

FIGURE 4

Infrared spectra in the Suc OH stretching vibration (νOH) region of dry samples containing Suc and EPC liposomes at the indicated mass ratios. The peak wavenumbers for each spectrum are indicated above the respective curves.

FIGURE 5

Wavenumber of the asymmetric P=O stretching vibrational mode (νP=Oas) as a function of the Suc/EPC mass ratio at 50°C (solid symbols) and 100°C (open symbols).

FIGURE 6

Infrared spectra in the carbonyl stretching region of (A) pure dry EPC liposomes and (B) dry EPC liposomes and Suc at a Suc/EPC mass ratio of 2.0. The peaks were deconvoluted and fitted into two band components corresponding to νC=O_free (short dashes, upfield peak, no H-bonding) and νC=O_bond (long dashes, lowfield peak, H-bonded). The solid curve comprises both the measured and the fitted absorbance curves. The correlation coefficients for the fitted curves were always higher than 0.999. Peak wavenumbers are indicated in panel A.

FIGURE 7

Ratio A_C=Obond/A_C=Ofree (fitted peak areas of the two νC=O band components identified in Fig. 6), (A) as a function of the Suc/EPC mass ratio of dry samples at 50°C (solid symbols) and 100°C (open symbols), or (B) as a function of temperature for the different Suc/EPC mass ratios indicated.

FIGURE 8

Infrared spectra in the asymmetric stretching region of the choline methyl group (νCN(C-H₃)₃as) of dry EPC liposomes in presence of sucrose at different Suc/EPC mass ratios. All spectra were recorded at 50°C.

FIGURE 9

Wavenumber of the asymmetric stretching of the choline C-N bond vibrational mode (νC-N(CH₃)₃as) (A) as a function of the Suc/EPC mass ratio of dry samples at 50°C (solid symbols) and 100°C (open symbols); (B) as a function of temperature for the different Suc/EPC mass ratios indicated.

FIGURE 10

Wavenumber of the asymmetric P=O stretching vibration as a function of the Suc/EPC mass ratio for dry EPC liposomes (solid symbols) or as function of the H₂0/DPPC mass ratio for DPPC liposomes (open symbols). Published data (35,36) for the hydrated DPPC samples were used.

FIGURE 11

Correlation between the position of the νP=Oas peak and T_m for dry samples with different Suc/EPC mass ratios. T_m was determined from the FTIR lipid melting curves (Fig. 1). The wavenumber of νP=Oas was determined from spectra recorded at 50°C. An exponential curve was fitted to the data with a correlation coefficient r = 0.9946.

FIGURE 12

Protection of large unilamellar EPC liposomes from damage during drying by different Suc/EPC mass ratios. Leakage of CF from the vesicles and membrane fusion were determined after air drying and rehydration. T_m was determined from the FTIR lipid melting curves (Fig. 1).