sugars have the ability to stabilize biological cells during desiccation ( see crowe et al . the effects of sugars on the stability of liposomes , which are a convenient model of biological membranes , during drying have been extensively investigated ( see hincha et al . much of this research has focused on the disaccharides suc and trehalose , while other potential protectants , such as oligosaccharides , have received less attention ( see oliver et al . it has , however , been shown that manno - oligosaccharides ( linear oligomers of 1 - 4 linked man ; man mann ; with n  =  number of monosaccharide units ) ( cacela and hincha 2006b ) and malto - oligosaccharides ( linear oligomers of 1 - 4 linked glc ; glc  glcn ) ( cacela and hincha 2006b ; hincha et al . 2002 ; suzuki et al . 1996 ) decrease in their effectiveness to protect liposomes from leakage of a soluble marker during drying with increasing dp . rfo ( linear oligomers of gal 1 - 6 linked with a capping suc ; glc  fru  galn ) ( hincha et al . 2003 ) and inulins ( linear oligomers of fru 2 - 1 linked with a capping glc ; glc  frun ) ( cacela and hincha 2006b ; hincha et al . recently , it has been shown that the branched fructans from cereals such as oat and rye show a more complex dependence of membrane stabilization on dp , with optima at intermediate chain lengths ( hincha et al . the differences between the different linear oligosaccharides have been related to their ability to interact with the lipid headgroups in the dry state . in particular , differences in the extent of h - bonding of different oligo - saccharides of the same dp to the p  =  o groups ( cacela and hincha 2006a , 2002 , 2003 ) have been related to the differential protection of liposomes from leakage during drying . similar differences have been reported for interactions with the more exposed choline group of epc , but not for the more buried c  =  o group ( cacela and hincha 2006b ) . such h - bonding interactions of sugar oh - groups with the lipid headgroups are thought to replace hydration water of the lipids during drying ( oliver et al . 1998a ) and are closely related to the ability of different sugars to depress the lipid liquid crystalline to gel phase transition temperature in the dry state , similar to the effect of water ( cacela and hincha 2006a ) . another important determinant of membrane stability during drying is the prevention of membrane fusion between liposomes , which can also lead to solute leakage . it is generally accepted that sugars prevent liposome fusion during drying by forming a glass ( vitrification ) and thereby preventing the close approach of vesicles necessary for fusion , without the need for specific interactions with the membranes ( see buitink and leprince 2004 ; crowe et al . the ability of a substance to vitrify can be experimentally characterized by its tg . in oligo- and polymers , tg generally increases with molecular weight ( slade and levine 1991 ) and an increase in tg with dp has been reported for rfo ( buitink et al . 1989 ) . however , while rfo and malto - oligosaccharides induced a decrease in membrane fusion with increasing dp ( hincha et al . 2002 , 2003 ) , fusion increased with dp in the presence of inulins ( hincha et al . these effects on fusion are notably different from the effects on leakage and the ability of the oligosaccharides to h - bond to lipid headgroups , indicating that different properties of the sugars may be responsible for their effects on leakage and fusion . in the present paper we report a systematic study of the effect of dp on the ability of inulins , malto - oligosaccharides and manno - oligosaccharides to prevent liposome fusion during drying and compare these data with the tg values of the respective sugars . surprisingly , our analysis shows that while tg increased with dp in all three oligosaccharide families , membrane fusion decreased only in the presence of malto - oligosaccharides up to dp5 . in the presence of inulins , manno - oligosaccharides and higher epc was purchased from lipid products ( south nutfield , surrey , uk ) . nbd and rh - pe were obtained from molecular probes ( leiden , the netherlands ) . suc and malto - oligosaccharides were obtained from sigma , inulins and manno - oligosaccharides were purchased from megazyme ( wicklow , ireland ) . the purity of the sugars was at least 95% , as determined by analytical hplc , as described in detail in previous publications ( hincha et al . lipids were dried from chloroform under a stream of n2 and stored under vacuum overnight to remove traces of solvent . liposomes were prepared from lipids hydrated in ten buffer ( 10  mm tes , 0.1  mm edta ( ph 7.4 ) , 50  mm nacl ) , using a hand - held extruder with two layers of polycarbonate membranes with 100  nm pores ( macdonald et al . liposome fusion after drying and rehydration was determined using fluorescence resonance energy transfer ( struck et al . briefly , two epc liposome samples were prepared : one sample was labeled with 1  mol% each of the fluorescent probe pair nbd - pe and rh - pe , while the other sample was unlabeled . the two samples were combined after extrusion in ten buffer in a 1:9 ( labeled : unlabeled ) ratio , resulting in a final lipid concentration of 10  mg / ml . liposomes ( 40  l ) were mixed with an equal volume of concentrated solutions of sugars in ten and 20  l aliquots were filled into the caps of microcentrifuge tubes . the samples were dried in desiccators at 28c and > 1% rh for 24  h in the dark ( hincha et al . fusion was measured after rehydration in ten buffer by fluorescence resonance energy transfer ( struck et al . 1981 ) with a kontron sfm 25 fluorometer ( kontron instruments , neufahrn , germany ) at excitation and emission wavelengths of 450 and 530  nm , respectively . due to a slight quenching effect of triton - x 100 on nbd fluorescence , fusion values above 100% 1protection of large unilamellar liposomes from damage during drying by inulins , malto - oligosaccharides and manno - oligosaccharides of different degrees of polymerization ( dp ) . fusion of the vesicles after air - drying and rehydration is shown as a function of the concentration of the sugars in the suspending medium . ( 2002 ) protection of large unilamellar liposomes from damage during drying by inulins , malto - oligosaccharides and manno - oligosaccharides of different degrees of polymerization ( dp ) . fusion of the vesicles after air - drying and rehydration is shown as a function of the concentration of the sugars in the suspending medium . ( 2002 ) glass transitions ( tg ) were measured using a perkin elmer dsc-7 ( norwalk , ct , usa ) or a mettler toledo dsc822 differential scanning calorimeter ( columbus , oh , usa ) , both equipped for subambient operation . sugar solutions were dissolved in 18  m water at 200500  mg / ml and air dried into differential scanning calorimetry ( dsc ) pans in a sealed box that was continuously purged with dry air of less than 3% rh over a period of 23  days . finally , samples were incubated for 1  h at elevated temperature ( 6080c ) to remove residual water and pressure - sealed in the dry box . for the state diagram , samples were transferred to chambers maintained at specific rh over saturated salt solutions ( weast 1984 ) for 124  h before sealing . glass transitions were analyzed using the perkin elmer or mettler software , which determine tg as the stable midpoint of the baseline inflection . every experiment was performed twice , with at least three heating runs for each experiment . water contents were determined gravimetrically using a cahn c-33 electrobalance , as previously described ( oliver et al . epc was purchased from lipid products ( south nutfield , surrey , uk ) . nbd and rh - pe were obtained from molecular probes ( leiden , the netherlands ) . suc and malto - oligosaccharides were obtained from sigma , inulins and manno - oligosaccharides were purchased from megazyme ( wicklow , ireland ) . the purity of the sugars was at least 95% , as determined by analytical hplc , as described in detail in previous publications ( hincha et al . lipids were dried from chloroform under a stream of n2 and stored under vacuum overnight to remove traces of solvent . liposomes were prepared from lipids hydrated in ten buffer ( 10  mm tes , 0.1  mm edta ( ph 7.4 ) , 50  mm nacl ) , using a hand - held extruder with two layers of polycarbonate membranes with 100  nm pores ( macdonald et al . liposome fusion after drying and rehydration was determined using fluorescence resonance energy transfer ( struck et al . briefly , two epc liposome samples were prepared : one sample was labeled with 1  mol% each of the fluorescent probe pair nbd - pe and rh - pe , while the other sample was unlabeled . the two samples were combined after extrusion in ten buffer in a 1:9 ( labeled : unlabeled ) ratio , resulting in a final lipid concentration of 10  mg / ml . liposomes ( 40  l ) were mixed with an equal volume of concentrated solutions of sugars in ten and 20  l aliquots were filled into the caps of microcentrifuge tubes . the samples were dried in desiccators at 28c and > 1% rh for 24  h in the dark ( hincha et al . fusion was measured after rehydration in ten buffer by fluorescence resonance energy transfer ( struck et al . 1981 ) with a kontron sfm 25 fluorometer ( kontron instruments , neufahrn , germany ) at excitation and emission wavelengths of 450 and 530  nm , respectively . due to a slight quenching effect of triton - x 100 on nbd fluorescence , fusion values above 100% 1protection of large unilamellar liposomes from damage during drying by inulins , malto - oligosaccharides and manno - oligosaccharides of different degrees of polymerization ( dp ) . fusion of the vesicles after air - drying and rehydration is shown as a function of the concentration of the sugars in the suspending medium . ( 2002 ) protection of large unilamellar liposomes from damage during drying by inulins , malto - oligosaccharides and manno - oligosaccharides of different degrees of polymerization ( dp ) . fusion of the vesicles after air - drying and rehydration is shown as a function of the concentration of the sugars in the suspending medium . glass transitions ( tg ) were measured using a perkin elmer dsc-7 ( norwalk , ct , usa ) or a mettler toledo dsc822 differential scanning calorimeter ( columbus , oh , usa ) , both equipped for subambient operation . sugar solutions were dissolved in 18  m water at 200500  mg / ml and air dried into differential scanning calorimetry ( dsc ) pans in a sealed box that was continuously purged with dry air of less than 3% rh over a period of 23  days . finally , samples were incubated for 1  h at elevated temperature ( 6080c ) to remove residual water and pressure - sealed in the dry box . for the state diagram , samples were transferred to chambers maintained at specific rh over saturated salt solutions ( weast 1984 ) for 124  h before sealing . glass transitions were analyzed using the perkin elmer or mettler software , which determine tg as the stable midpoint of the baseline inflection . every experiment was performed twice , with at least three heating runs for each experiment . water contents were determined gravimetrically using a cahn c-33 electrobalance , as previously described ( oliver et al . we have investigated the effects of three structural families of oligosaccharides , inulins , malto - oligosaccharides , and manno - oligosaccharides , on membrane fusion in the dry state . while fusion data have been reported previously for the first two families ( hincha et al . we used commercially available sugars from dp2 to dp5 for inulins and manno - oligosaccharides , and dp2 to dp7 for malto - oligosaccharides . figure  1 shows the effects of oligosaccharides up to dp5 on liposome fusion after drying and rehydration . while the malto - oligosaccharides showed increased protection up to dp5 , the members of the other two families showed a linear decrease in protection with increasing dp ( figs .  1 , 2 ) . however , at higher dp ( dp6 and 7 ) , the effectiveness of the malto - oligosaccharides also decreased ( fig .  2 ) . these experiments have identified , in addition to data reported before for inulins and malto - oligosaccharides ( hincha et al . 2002 ) , manno - oligosaccharides as another family of sugars that shows less inhibition of fusion with increasing dp . the effect of dp was not as strong as for the inulins , but clearly in the opposite direction than for the malto - oligosaccharides up to dp5 ( figs .  1 , 2 ) . 
 fig . 2percentage of liposome fusion after air - drying and rehydration as a function of the dp of inulins , malto - oligosaccharides and manno - oligosaccharides . all samples contained 2  g sugar / g lipid ( compare fig .  1 ) . the straight lines for samples containing inulins and manno - oligosaccharides were fitted by least - squares linear regression . the regression coefficients ( r ) the line connecting the data from the malto - oligosaccharide containing samples is only meant as a visual aid . ( 2002 ) percentage of liposome fusion after air - drying and rehydration as a function of the dp of inulins , malto - oligosaccharides and manno - oligosaccharides . the straight lines for samples containing inulins and manno - oligosaccharides were fitted by least - squares linear regression . the regression coefficients ( r ) the line connecting the data from the malto - oligosaccharide containing samples is only meant as a visual aid . ( 2002 ) the basic assumption about the role of sugars in preventing membrane fusion during drying is that it is due to the formation of a glass phase ( see crowe et al . in addition , it has been shown that heating samples containing suc above the sugar glass transition temperature leads to a rapid increase in membrane damage ( sun et al . therefore , we compared the vitrification behavior of the oligosaccharides by dsc to elucidate the interdependence of membrane fusion , dp , and tg . figure  3 shows state diagrams for the glass transitions of manno - oligosaccharides , malto - oligosaccharides and inulins of dp5 . in common with other sugar glasses ( buitink and leprince 2004 ; crowe et al . 1998 ) , water acted as a plasticizer and led to a progressive decrease in tg with increasing water content . the degree of reduction in tg , however , was different for the different oligosaccharides , in accordance with findings for other oligo- and polysaccharides ( sun et al . it was not possible to obtain a tg value for extremely dry ( below 0.13  g water / g dry weight ) dp5 manno - oligosaccharide , because the sample decomposed during heating before tg was reached . however , the data suggest that at a given water content the dp5 inulin showed the lowest and the corresponding manno - oligosaccharide the highest tg , with the malto - oligosaccharide intermediate . 
 fig . 3state diagram of dp5 sugars of different oligosaccharide families as a function of water content in the samples state diagram of dp5 sugars of different oligosaccharide families as a function of water content in the samples for all three families of oligosaccharides , tg increased with dp , as expected ( fig . the increase was highest for the malto - oligosaccharides and lowest for the inulins , with the manno - oligosaccharides intermediate . the caveat in the case of the manno - oligosaccharides is ; however , that in the dp5 sample , water content was higher than in all other samples ( compare also fig . 3 ) , so that tg may be underestimated in this case . similarly , tg was slightly higher for the dp2 than for the dp3 manno - oligosaccharide , indicating that its water content was lower . unfortunately , the sugar in this sample decomposed during gravimetric analysis , so that no exact water content data are available . however , a sample of dp2 manno - oligosaccharide with a higher - water content ( 0.26  g water / g dry weight ) showed the expected lower tg ( 27.4c ) . 4b ) yielded the expected straight lines for all oligosaccharide families ( orford et al . 1989 ) , indicating that the data reflect the relative vitrification behavior of the sugars reasonably well . 4glass transition temperatures of sugars from the different oligosaccharide families as a function of dp(a ) . panel ( b ) shows a double reciprocal plot of the data in a. the water content of the samples was below 0.01  g water / g dry weight for all inulins and malto - oligosaccharides , but is indicated in the figure for the manno - oligosaccharides of dp3dp5 ( see text for further details ) glass transition temperatures of sugars from the different oligosaccharide families as a function of dp(a ) . panel ( b ) shows a double reciprocal plot of the data in a. the water content of the samples was below 0.01  g water / g dry weight for all inulins and malto - oligosaccharides , but is indicated in the figure for the manno - oligosaccharides of dp3dp5 ( see text for further details ) a plot of membrane fusion as a function of the tg of the different sugars presents a surprising picture ( fig .  5 ) . only malto - oligosaccharides up to dp5 show the expected behavior , i.e. a reduction in fusion with increasing dp . the higher dp malto - oligosaccharides and all other oligosaccharides , on the contrary , show an increase in membrane fusion with increasing tg . on the other hand , it has been shown before that sorbitol is a much better protectant against membrane fusion during drying than would be expected from its tg ( hincha and hagemann 2004 ) . it has been suggested previously that tc , the critical temperature where a glass changes from the solid - like to the liquid - like state , may be a better parameter for the functional characterization of sugar glasses ( buitink et al . tc for sorbitol , for example , is approximately 40c above tg ( tsujimi et al . 1999a , b ) , while it is only 18c above tg for sucrose ( buitink et al . unfortunately , no tc data for the oligosaccharides investigated in our study have been published . 5percentage of liposome fusion after air - drying and rehydration as a function of the glass transition temperature of the respective oligosaccharides of different dp . the fusion data are taken from samples that contained 2  g sugar / g lipid ( compare figs .  1 , 2 ) . the straight lines for samples containing inulins and manno - oligosaccharides were fitted by least - squares linear regression . the regression coefficients ( r ) the line connecting the data from the malto - oligosaccharide containing samples is only meant as a visual aid percentage of liposome fusion after air - drying and rehydration as a function of the glass transition temperature of the respective oligosaccharides of different dp . the fusion data are taken from samples that contained 2  g sugar / g lipid ( compare figs .  1 , 2 ) . the straight lines for samples containing inulins and manno - oligosaccharides were fitted by least - squares linear regression . the regression coefficients ( r ) the line connecting the data from the malto - oligosaccharide containing samples is only meant as a visual aid in addition , it has been shown in recent spectroscopic investigations that there is spatial and temporal heterogeneity in sugar glasses , even at temperatures considerably below tg and that this heterogeneity is different for different sugars and sugar alcohols ( shirke and ludescher 2005a , b ) . while there is little doubt that vitrification of the sugar matrix during drying is necessary to prevent membrane fusion ( buitink and leprince 2004 ; crowe et al . 1998 ) and that fusion is increased when samples are heated above tg ( sun et al . 1996 ) , the present study shows that measurements of tg are not a good predictor of membrane protection against fusion for different sugars . this is even true for oligosaccharides of the same dp , so that effects of size differences can be largely excluded . therefore , our data indicate that structural differences between different sugars play an important role not only in the ability of sugars to interact with lipid headgroups ( cacela and hincha 2006b ) , but also in their ability to form a glass that effectively prevents membrane fusion . further investigations will be necessary to relate structural differences between different sugars to functional differences , to understand the structural basis of the protective abilities of different oligosaccharides .
sugars play an important role in the desiccation tolerance of most anhydrobiotic organisms . 
 it has been shown in previous studies that different structural families of oligosaccharides have different efficacies to interact with phospholipid headgroups and protect membranes from solute leakage during drying . here , 
 we have compared three families of linear oligosaccharides ( fructans ( inulins ) , malto - oligosaccharides , manno - oligosaccharides ) for their chain - length dependent protection of egg phosphatidylcholine liposomes against membrane fusion . 
 we found increased protection with chain length up to a degree of polymerization ( dp ) of 5 for malto - oligosaccharides , and a decrease for inulins and manno - oligosaccharides . 
 differential scanning calorimetry measurements showed that for all sugars the glass transition temperature ( tg ) increased with dp , although to different degrees for the different oligosaccharide families . 
 higher tg values resulted in reduced membrane fusion only for malto - oligosaccharides below dp5 . 
 contrary to expectation , for inulins , manno - oligosaccharides and malto - oligosaccharides of a dp above five , fusion increased with increasing tg , indicating that other physical parameters are more important in determining the ability of different sugars to protect membranes against fusion during drying . 
 further research will be necessary to experimentally define such parameters .