2.2 Methods
3.1.1 Interaction in sodium chloride solution
3.1.1.4 Discussion
HA are quite small (especially for 50 ◦C) and special care has to be taken when interpreting the electron density profiles. As the quality of the data is not very high and the used model restricts the shape of the added layer there are some uncertainties in the electron density profile are existing, which is reflected in the high relative errors for the HA layer (see tab. 3.3).
mechanisms for high molecular weight HA differ slightly from those of HA with a small molecular weight. However it can be excluded that HA interacts considerably with the alkyl chains of the lipids. Molecules that are present in this region, like for example cholesterol [91], cause a change of the main transition temperature and an increase of the width of the main transition [90]. Further it is known that even small amounts of contamination lead to a disappearance of the pretransition [92].
DLS measurements showed the existence of two different populations of vesicles, as two intensity peaks at two different positions were found. One at around 1µmand one at around 100 nm. It seems reasonable that the peak at ≈ 1 µm stems from aggregated vesicles. As a membrane with a pore size of 200 µm was used for the extrusion of the vesicles it can be assumed that the peak at around 100nmbelongs to non aggregated vesicles. Two different trends could be observed, after adding HA of different molecular weight to the solution: i) an increase of the hydrody-namic radius of the vesicles as a function of the molecular weight ii) a decrease of the amount of non aggregated vesicles with a stronger decrease for HA of low molecular weight HA.
An increased hydrodynamic radius for DPPC vesicles with HA has already been re-ported by Wang et al. 2013 [28], who used HA with a molecular weight of 620kDa.
The HA induced change of the hydrodynamic radius is comparable with the here presented results. From the increase of the hydrodynamic radius it could be con-cluded that HA adsorbs to the DPPC vesicles, which fits to the here presented results obtained from SAXS where an additional layer at the outside of the vesicles was supposed. The increase of the hydrodynamic radius as a function of the HA can be explained by the different conformations of HA with a low and highMW. At very low molecular weights (e.g. 10kDa) HA can be regarded as a stiff rod, while high molecular weight HA in aqueous solution has a random coil conformation with a radius of gyration, that increases as a function of the molecular weight [93]. A HA coil with a larger radius of gyration adsorbing to a DPPC vesicle will lead to large increase of the hydrodynamic radius.
The second trend implied that HA promoted the formation of aggregates, which can be deduced from the decreased intensity of the peak at around 100nmfor sam-ples with HA. This hints at formation of supramolecular HA-DPPC structures, as illustrated in figure 3.6i. Such a formation has already been claimed by Crescenzi et al. 2004 [94], who visualized the aggregates with electron microscopy. The effect got weaker for HA with a higher molecular weight, which hints at an increased interaction strength of HA with low molecular weight. High and low molecular weight HA have the chemical structure and the number of charged carboxyl groups
(i)
(ii)
(iii)
Figure 3.6: Sketch illustrating the possible structure of DPPC/HA aggregates (i) and possible structures of single DPPC vesicles enveloped by HA (ii) or with attached HA chains (iii).
in the solution is the same as the same mass concentration was used for all molec-ular weights of HA. Thus, the reason for the increased interaction is most likely the changed conformation (rod vs. random coil). HA of a higher molecular weight, has, due to its coiled structure, a high amount of mass that is excluded from an interaction with the vesicles since it is situated within the coil. Like the results from DSC, DLS measurements imply an interaction of DPPC vesicles with HA.
For the SAXS measurements DPPC vesicles were mixed with HA (MW = 250kDa) and measured at three different temperatures (25◦C, 37◦C and 50 ◦C). The tem-peratures correspond to the three bilayer phases: gel phase (Lβ0), rippled phase (Pβ0) and fluid phase (Lα) [30]. This was also confirmed by the DSC measure-ments presented here. As the different phases go along with different structural conformations of the lipids the structure of the bilayer changes, while the lipids undergo a phase transition [30]. This is most obvious for the changed d-spacings and head-to-head distances, which were higher in the Pβ0 and Lβ0 phase than in the Lα phase. The head-to-head distance of 4.3 nmand 3.8nm for the Lβ0 Lα, fit well to the values reported by Nagle et al. 1996 [95]. In contrast to that the results for the d-spacing do not match those reported in literature [95]. A reason for this might be the different preparation methods. The vesicles used for the studies in literature are large multilamellar vesicles, while the here presented results stem from small vesicles with a low number of lamellar. They are called pauci-lamellar vesicles and are well known for PC vesicles prepared by extrusion with membranes of a pore size of 200 nm [96]. The smaller vesicle size induces higher stresses due to the high curvature, which could have a strong effect on the d-spacing. Another effect that is observed during the phase transition is the changed electron density ρr. This effect can be explained by an increased volume per DPPC molecule, which
could be the result of higher degree of disorder, due the molten alkyl chains after the phase transitions. An increased volume per molecule for theLα and Pβ0 phase compared to the Lβ0 phase has already been reported [31].The addition of HA did not change the scattering profiles considerably, which shows that the bilayer struc-ture did not change much. It could be observed that the d-spacing for the Pβ0 and the Lα was increased by HA. A possible explanation could be that HA moved between the bilayers and thereby increases the d-spacing as it was suggested in a study by Kreuzer et al. 2012 [97]. The question arises, how HA should enter the space between two lamellae, as vesicles consist of closed shells of lipids and the presented results do not imply a strong interaction between the HA and DPPC.
Thus, an induced disruption of the DPPC bilayers seems very unlikely. Also a penetration through the bilayers is not very likely as the electron density profiles and especially the DSC measurements rule this out. Also Kreuzer et al 2012 [97]
did not find an evidence for such kind of interaction. In general some parts of the multilamellar structure could arise from aggregated vesicles, where it is easily possible for the HA to accumulate. The electron density profiles showed a diffuse layer of HA with low electron density. At 37 ◦C the layer seemed to be most com-pact. At this temperature the bilayer was in the Pβ0 phase which should give the HA chains a better possibility to interact with DPPC molecules, due to its rippled structure. However, it is difficult to give a detailed information about the formed structures as the shape of the layer in the used model is restricted to a Gaussian form and the limited q-range restricts the resolution of the density profile.
The combination of the three methods could confirm that HA and DPPC vesicles do interact with each other, although the interaction is not very strong. In general the interaction of HA and DPPC could be facilitated by two different mechanisms.
First, by an electrostatic interaction of the negatively charged carboxyl groups of HA with the positively charged part of the zwitterionic headgroup of DPPC (as indicated by the sketch in fig. 3.7a) and, second, the hydrophobic patches of HA might enable the interaction with the alkyl chains in the inner part of bilayer. The here presented SAXS measurements showed only a diffuse layer on top of the bi-layer and no changes of the inner bibi-layer (tail group region) structure. Further, the results from DSC measurements do not hint at an interaction of HA with the alkyl chains. Thus an adsorption of HA at the headgroup water interface seems very likely. It can be presumed that the positive charge of the NH+ group, which is located directly at the vesicle interface (see fig. 3.7b), binds to the negative car-boxyl group of HA, i.e. the interaction is dominated by electrostatic forces. This is in agreement with result from recent other works that claimed that HA adsorbs
+ -+ -+
-+
-+
-+
-+
-+
-+
-+ -+ -+
-+
-+ -+
-+
-+
-+
-+
-+
-+
-+
-+
-+
-+
-+
-+
-+
-+
-+
-+ -- + +
--
--
-+
-+
-+
-+
-Inside Outside
(a)
O O O O
OP
O O−ON+
+
-(b)
Figure 3.7: (a) Sketch of a DPPC vesicle in HA solution. (b) Sketch illustrating the charge distribution in the headgroup of a lipid.
to the headgroup region [97, 98]. However DSC results hint also at a low amount of HA at the glycerol - headgroup interface. The sketch in figure 3.7a summarizes the results, where HA binds to the interface of the vesicles via its charged groups.
The measurements with different HA show that the molecular weight has a sig-nificant influence on the interaction of the two components. It is already known from several other studies that the molecular weight plays an important role in the synovial joint. It has been claimed that high molecular weight HA shows a better wear resistance [23] and that the molecular weight plays an important role in arthritis [22, 49]. While it is difficult to interpret the result from the DSC mea-surements which show a somehow stronger effect of the high molecular weight HA on the phase behavior of DPPC, the results from DLS hint at a stronger interac-tion of low molecular weight HA with DPPC. Also in the literature the important question, if high or low molecular weight HA has a different effect on the bilayer is a matter of discussion [98, 99]. Since, up to now, there are no studies, investigat-ing the effect of the molecular weight on the lubrication properties of DPPC/HA mixtures it is hard to conclude from the presented results if a stronger interaction between HA and DPPC is beneficial or not. Only indirect information about the effect of the molecular weight is available as the molecular weight of HA in the synovial fluid of arthritic joints was found to be lower compared to healthy joints [21, 22].