Morphological transition of amphiphilic block copolymer/PEGylated phospholipid complexes induced by the dynamic subtle balance interactions in the self-assembled aggregates

2.1 Materials

1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE–PEG2k, DSPE–PEG1k, DSPE–PEG5k) was purchased from Shenzhen Xingjiafeng Technology Co. Ltd (China). Monomethoxy poly(ethylene glycol)-block-poly(d,l-lactide) (PDLLA10k–MPEG2k) was obtained from Jinan Dai-gang Biological Technology Company (Jinan, China). Doxorubicin hydrochloride (DOX·HCl) was obtained from Beijing Hua-feng United Technology Company (Beijing, China). Triethylamine (TEA) and dimethyl sulfoxide (DMSO) were obtained from Sinopharm Chemical Reagent Company (Shanghai, China). Dialysis tubes (molecular weight cutoff = 8,000–12,000 Da) were purchased from Shanghai Yuanye Biological Technology Company (Shanghai, China). All solvents and reagents were of analytical grade and used as such without further purification.

2.2 Methods

3.1 Characterization of DSPE–PEG/PDLLA–MPEG complex aggregates formed at different DSPE–PEG additions

The effect of DSPE–PEG on the morphology and size of DSPE–PEG/PDLLA–MPEG complex aggregates was studied by TEM. Figure 1 shows the representative TEM images of DSPE–PEG2k/PDLLA–MPEG complex aggregates at different mass ratios of DSPE–PEG2k/PDLLA–MPEG. As expected, the morphological structure of DSPE–PEG2k/PDLLA–MPEG complex aggregates could be changed by increasing the amount of DSPE–PEG addition. For the pure MPEG–PDLLA molecules, a structure of spherical micelles with good dispersity was formed and the calculated average size of micelles was about 231 nm, as shown in Figure 1a. This can be ascribed to their amphiphilic property which makes them spontaneously self-assemble into micelles in the aqueous solution. When the mass ratio of DSPE–PEG2k to PDLLA–MPEG was 0.0005:1, the DSPE–PEG2k/PDLLA–MPEG complex aggregates showed irregular spherical micelles (Figure 1b) with an average size of 283 nm. When the mass ratio of DSPE–PEG2k to PDLLA–MPEG was increased to 0.001:1, the morphology of complex aggregates had a significant transition, where a structure of vesicles with diameters ranging from 2 to 4 µm was obtained, as shown in Figure 1c. As the mass ratio of DSPE–PEG2k/PDLLA–MPEG reached 0.005:1, some destroyed vesicles were found in the TEM images, and it could be observed that some integral vesicles coexisted with the partially ruptured ones, as indicated in Figure 1d. This phenomenon indicated that the stability of vesicles at this mass ratio becomes poor. Then, with the further increase in mass ratio of DSPE–PEG2k/PDLLA–MPEG to 0.01:1, almost no intact vesicles were found, and the morphology of complex aggregates showed semi-vesicle structure (Figure 1e). The semi-vesicular morphological structure has already been mentioned in the literature as a transition state in the structural transition between vesicles and micelles of polymers (35). However, it is interesting to point out that it is the first time to find the existence of this morphological structure experimentally. At an even higher mass ratio of DSPE–PEG2k/PDLLA–MPEG (0.02:1), it was surprised to find that a structure of irregular spherical micelles, similar to that of the mass ratio at 0.0005:1, occurred again. Although the morphology of DSPE–PEG2k/PDLLA–MPEG complex aggregates obtained under these two ratios is similar, the difference in composition ratios of these two complex aggregates is remarkably large and in turn the respective micelle properties may be completely different.

Figure 1

TEM images of aggregates formed from DSPE–PEG2k/MPEG–PDLLA mixtures with different mass ratios of DSPE–PEG2k/PDLLA–MPEG: (a) 0:1, (b) 0.0005:1, (c) 0.001:1, (d) 0.005:1, (e) 0.01:1 and (f) 0.02:1.

It is evident that the addition of DSPE–PEG2k leads to the morphological transition of PDLLA–MPEG aggregates. To further verify the effect of DSPE–PEG2k additions on the morphology of complex aggregates, the DLS test was used to determine the size distribution of DSPE–PEG2k/PDLLA–MPEG complex aggregates as indicated in Figure 2. The DLS results showed that PDLLA–MPEG micelles have an average size of 280 nm at 25°C. When DSPE–PEG2k was added in the 0.0005:1 mass ratio, the average size of DSPE–PEG2k/PDLLA–MPEG complex aggregates increased to 351 nm. Then, with the further increase in DSPE–PEG2k content, the DSPE–PEG2k/PDLLA–MPEG complex aggregates exhibited a dual size distribution, with a dominant size in the nanometer range and the other one in the micrometer range. Consistent with the TEM results in Figure 1, the size distribution changes in DLS data are attributed to the evolution of the complex aggregates from micelles to vesicles and then semi-vesicles. At a higher mass ratio of 0.02:1, the DLS data of DSPE–PEG2k/PDLLA–MPEG complex aggregates just exhibit a single distribution with an average size of 296 nm. This result means that the complex aggregates completely changed into micelle structure again, as indicated in Figure 1f. Consequently, TEM images and DLS data both showed that the addition of DSPE–PEG2k is an effective way to regulate the morphological transition of PDLLA–MPEG self-assembled structures.

Figure 2

Size distributions of DSPE–PEG2k/PDLLA–MPEG complex aggregates prepared from the DSPE–PEG2k/PDLLA–MPEG mixed solutions at different mass ratios of DSPE–PEG2k/PDLLA–MPEG.

The aforementioned results obviously indicated that the gradual increase in DSPE–PEG2k additions resulted in the morphological transition of DSPE–PEG2k/PDLLA–MPEG complex aggregates from micelles to vesicles, semi-vesicles and then to mixed micelles again. To investigate how the DSPE–PEG2k molecules affect the self-assembly structure of DSPE–PEG2k/PDLLA–MPEG mixtures and further explore the interaction between DSPE–PEG2k and PDLLA–MPEG polymers in solutions, the dynamic mixing process of DOX-loaded PDLLA–MPEG micelle solutions with DSPE–PEG2k molecules was monitored by TEM. In this process, MPEG–PDLLA/DOX mixed solution was first dialyzed against aqueous solution to form micelles and then DSPE–PEG2k was added to the PDLLA–MPEG micelle solutions and mixed for different time periods. Figures 3 and 4 show the TEM images of DSPE–PEG2k and PDLLA–MPEG micelles mixed at different time periods with the mass ratio of 0.001:1 and 0.02:1, respectively. As shown in Figures 3a and 4a, only evenly dispersed spherical micelles could be observed at 0 h, i.e., no DSPE–PEG2k was added into the amphiphilic block polymer micelles. With the increase in mixing time to 1 h, the aggregates were still spherical micelles but there was adhesion between the spherical micelles as shown in Figure 3b (the mass ratio of DSPE–PEG2k/PDLLA–MPEG was 0.001:1); a similar phenomenon was also observed at this mixing time in the case where the mass ratio of DSPE–PEG2k/MPEG–PDLLA was 0.02:1 (Figure 4b). Both the vesicles began to be observed in TEM images after the addition of DSPE–PEG2k to PDLLA–MPEG micelle solutions for 6 h at these two different mass ratios (Figures 3c and 4c). However, as the mixing time increased further, the morphological changes of aggregates formed from the DSPE–PEG2k molecules and PDLLA–MPEG micelle mixed solutions showed different evolution at different mixed mass ratios. When the mass ratio of DSPE–PEG2k/PDLLA–MPEG was 0.001:1, the number of vesicles observed in TEM images increased with the further increase in mixed time and finally showed a similar TEM image to that of Figure 1c. In contrast, in the case where the mass ratio of DSPE–PEG2k/PDLLA–MPEG was 0.02:1, the number of vesicles observed in TEM images did not only increase with increasing mixed time but also the vesicles observed in TEM images gradually ruptured and disappeared, and eventually changed into micelles again. The time-dependent morphological evolution of aggregates formed by mixing PDLLA–MPEG micelle solutions and DSPE–PEG2k molecules demonstrated that the DSPE–PEG2k can gradually penetrate into the PDLLA–MPEG micelles which leads micelles to re-assemble into different new aggregates according to the different amounts of DSPE–PEG penetration. The hydrophobic DOX encapsulation in the micelles may enlarge the inner cores and cause more chains to be looped that may facilitate the DSPE–PEG2k to penetrate into the PDLLA–MPEG more easily (36). But during the mixing of PDLLA–MPEG micelles and DSPE–PEG2k, the morphological transition of aggregates formed from the PE–PEG and PDLLA–MPEG micelles mixed solutions, from micelles to vesicles or finally to mixed micelles, should be the result of the interaction between DSPE–PEG and PDLLA–MPEG, not the one due to DOX release. It is because that the released DOX cannot shift the relative volume ratio of hydrophobic block/hydrophilic block to a high value, while the increasing hydrophobic block/hydrophilic block value is a key step for the transition of copolymers from micelles to vesicles. Therefore, these obtained results further confirmed that the addition of DSPE–PEG2k can effectively adjust the morphological transition of PDLLA–MPEG self-assembled structure and form various thermodynamically stable supramolecular structures.

Figure 3

TEM images of aggregates formed from DSPE–PEG2k and PDLLA–MPEG micelle (at a mass ratio of 0.001:1) mixed solutions at different mixing time: (a) 0 h, (b) 1 h, (c) 6 h, (d) 12 h and (e) 24 h.

Figure 4

TEM images of aggregates formed from DSPE–PEG2k and PDLLA–MPEG micelle (at a mass ratio of 0.02:1) mixed solutions at different mixing time: (a) 0 h, (b) 1 h, (c) 6 h, (d) 12 h and (e) 24 h.

3.2 Proposed possible mechanism of morphological transition of DSPE–PEG2k/PDLLA–MPEG complex aggregates

Interactions between two copolymers in a binary mixture of amphiphilic block copolymers mainly involve two ways. One is the occurrence of intrachain cross-linking of the hydrophilic segments of two copolymers through the noncovalent interactions, such as hydrogen bonding or electrostatic interactions. In this case, the hydrophilic/hydrophobic balance of amphiphilic block copolymers changes, and this can be used for convenient regulation of the morphological transition of the obtained polymer aggregates (19,20,21,22). Other interaction between two amphiphilic block copolymers is that the hydrophobic segments of mixtures are packed together into hydrophobic aggregates by hydrophobic interactions. In the latter, co-micellization of two amphiphilic block copolymers is generally the most evident appearance. The structure of co-micellization of binary mixed amphiphilic block copolymers can be mixed micelles or demixed ones which can be easily and precisely regulated by the block length, block chemistry and the stoichiometry of the binary mixture (37,38). Moreover, mixed micelles formed from two amphiphilic block copolymers with obviously distinct hydrophobic lengths usually present ellipsoidal structures in which the inner cores are mainly occupied by the longer hydrophobic blocks while the shorter blocks straddle the core and corona interface by their hydrophobic block inserted in the inner core through the hydrophobic interaction (37). The difference between the two hydrophobic blocks also may cause the microphase separation of the inner core to occur, which induces the morphological change of mixed micelles. However, almost no obvious controllable morphological transition appears for such binary mixtures, mainly because of the relatively weak strength of the hydrophobic interaction and the poor selectivity between two hydrophobic blocks, which hinder the formation of a regular complex from a mixture of amphiphilic block copolymers.

If the assembled aggregates from a mixture of DSPE–PEG/PDLLA–MPEG also prefer to present mixed micelle structures with the DSPE–PEG concentrating in the core and corona interface as that usually observed in a binary mixture of amphiphilic block copolymers (37), the aggregates thus obtained will not be the ones with stable energy due to the structural incompatibility between two-hydrophobic tailed DSPE–PEG and linear PDLLA–MPEG. As it is well-known, due to their two-hydrophobic tailed structure, the phospholipids usually prefer to form bilayer structures by interdigitating between them. DSPE–PEG have a long hydrophilic PEG segment that improves their solubility in water and greatly decreases the hydrophobic interaction between DSPE–PEG themselves. But the interdigitating hydrophobic interaction ability of the two hydrophobic tails of DSPE–PEG should be retained when they interact with the linear PDLLA–MPEG. Consequently, the DSPE segment of DSPE–PEG will be preferentially interdigitated with the end of linear hydrophobic PDLLA segment of PDLLA–MPEG through the hydrophobic interaction, when DSPE–PEG/PDLLA–MPEG mixtures are dialyzed against water. In this interaction way, the two fatty acid tails of DSPE–PEG can interact with three PDLLA segments of PDLLA–MPEG, so that it can obtain as much hydrophobic interaction as possible between the hydrophobic blocks of mixtures and decrease the structural inconsistencies of the hydrophobic aggregation region formed by the DSPE and PDLLA segments together. As such, interaction between the DSPE and PDLLA segments also facilitate the DSPE–PEG and PDLLA–MPEG to form “ABA” temporary supra-amphiphiles. This new building block may cooperatively assemble with PDLLA–MPEG into energetically stable bilayer supramolecular structures in which all the hydrophobic chains are fully contacted while the PEG segments of PDLLA–MPEG form the outer hydrophilic layer and the PEG segments of DSPE–PEG form the inner hydrophilic layer to provide steric repulsion to stabilize the hydrophobic aggregates. Consequently, the unique interaction between DSPE–PEG and PDLLA–MPEG makes them tend to form “ABA” temporary supra-amphiphiles and self-assemble into bilayer aggregates, which is a key step to transit from micelles to vesicles. Therefore, the addition of DSPE–PEG played an active role in the morphological evolution of DSPE–PEG2k/PDLLA–MPEG complex aggregates from micelles to vesicles. It is interesting to point out that the as-formed vesicle from DSPE–PEG2k/PDLLA–MPEG mixtures has an asymmetric structure in which the hydrophilic segments of DSPE–PEG are located in the inner chamber. However, it should be noted that the formation of hydrophilic layer from the PEG segments of DSPE–PEG is necessary to form bilayer structure aggregates, while the repulsive force derived from them in turn may affect the stability of “ABA” temporary supra-amphiphiles. Therefore, when only a small amount of DSPE–PEG is added to PDLLA–MPEG, the PEG segments of DSPE–PEG cannot form a hydrophilic layer with sufficient repulsive force to maintain the formation of the bilayer structured aggregates from the DSPE–PEG/PDLLA–MPEG mixtures. At this time, mixed micelles were formed from the DSPE–PEG/PDLLA–MPEG mixtures by inserting the two fatty acid tails of DSPE–PEG in the inner core. Presumably, the obvious difference in the hydrophobic block structures between DSPE–PEG and PDLLA–MPEG can lead the inner core of micelle to deform and present an irregular sphere structure as indicated in Figure 1b. When the addition of DSPE–PEG is increased by a certain amount, the number of “ABA” temporary supra-amphiphiles formed is sufficient to form aggregates with a bilayer structure in which the PEG segments of PDLLA–MPEG form the outer hydrophilic layer and the PEG segments of DSPE–PEG form the inner hydrophilic layer to provide steric repulsion and stabilize the formed aggregates. In this case, the morphology of DSPE–PEG2k/PDLLA–MPEG complex aggregates presents vesicle structures as indicated in Figure 1c and d. On further increasing the amounts of DSPE–PEG addition, more “ABA” temporary supra-amphiphiles will form to facilitate the DSPE–PEG/PDLLA–MPEG mixtures assemble into vesicles, while the mutual repulsive force between the PEG segments of DSPE–PEG located in the inner chamber of vesicles also increases. It should be noted that the driving force for the formation of “ABA” temporary supra-amphiphiles from DSPE–PEG and PDLLA–MPEG mixtures is the weak van der Waals force. In addition, the interaction strength between the DSPE–PEG2k and PDLLA–MPEG is also weak due to the short hydrophobic tail of DSPE–PEG. Therefore, the increasing repulsive force in the inner chamber of vesicles with increasing DSPE–PEG2k additions would cause “ABA” temporary supra-amphiphiles to be unstable or even to be destroyed. The destruction of “ABA” temporary supra-amphiphiles will, in turn, lead the vesicles to be unstable or even disappear. Therefore, a continuous increase in the DSPE–PEG2k addition can further change the morphology of DSPE–PEG2k/PDLLA–MPEG complex aggregates. To avoid the conflict between the presence of stable “ABA” temporary supra-amphiphiles and the increasing repulsive force caused by the increasing DSPE–PEG2k in a closed chamber, DSPE–PEG2k/PDLLA–MPEG complex aggregates would change from vesicle structures to semi-vesicle structures as indicated in Figure 1e. The semi-vesicles that are nearly disks in structure can allow more “ABA” temporary supra-amphiphiles to exist in the aggregates because the repulsive force between the DSPE–PEG2k molecules in the as-formed structure is less than that in a vesicle structure. This decreased repulsive force between the PEG segments of DSPE–PEG in semi-vesicles is due to the fact that these PEG segments are not subject to the repulsive force from the opposite half of the sphere as that exists in vesicle structures. Semi-vesicles are present as a transition state in the structural transformation between vesicles and micelles of polymers and are important intermediate structures instead of bilayer membranes (35). Generally, semi-vesicles are in a thermodynamically unstable state, and these structures have not been observed in experiment previously. Interestingly, the stable semi-vesicles can be formed at a suitable ratio of DSPE–PEG2k/PDLLA–MPEG since this structure can reconcile the conflicts between the number of stable “temporary shape amphiphiles” formed and the increasing repulsive force coming from the DSPE–PEG2k. However, if DSPE–PEG/PDLLA–MPEG mixtures still preferentially form “ABA” temporary supra-amphiphiles under a high ratio of DSPE–PEG2k/PDLLA–MPEG, then, in the subsequently forming supramolecular structure, the repulsive force generated by the PEG segment of DSPE–PEG will be large enough to cause the structure unstable. Under such conditions, the geometric molecular structure of DSPE–PEG does not play a key role in determining the hydrophobic interaction site between the DSPE–PEG and PDLLA–PEG. And the DSPE–PEG and PDLLA–PEG will be self-assembled into mixed micelles in which the hydrophilic segments of DSPE–PEG and PDLLA–PEG together form a hydrophilic layer while the hydrophobic tails of DSPE–PEG are inserted into the hydrophobic cores via the hydrophobic interaction. In such mixed micelles, although the hydrophobic interaction between DSPE–PEG and PDLLA–PEG is not the strongest, the effect of repulsive force resulting from the PEG segment of DSPE–PEG on their interaction is eliminated and thus allows the formation of a related more stable supramolecular structure. Thereby, DSPE–PEG2k/PDLLA–MPEG complex aggregates would further change from semi-vesicle structures to mixed micelle structures under a high ratio of DSPE–PEG2k/PDLLA–MPEG, as indicated in Figure 1f.

According to the above discussion, a schematic diagram of how the addition of DSPE–PEG regulates the morphological transition of PDLLA–MPEG self-assembled structures is outlined in Figure 5.

Figure 5

Schematic representation of the possible aggregate structures formed from DSPE–PEG2k/PDLLA–MPEG mixtures with increasing mass ratio: (a) pure micelles, (b) vesicles, (c) semi-vesicles and (d) mixed micelles.

This model describes that the geometric molecular structure of DSPE–PEG makes DSPE–PEGA and PDLLA–PEG preferentially form “ABA” temporary supra-amphiphiles; however, subsequently, the repulsive force between the PEG segments of DSPE–PEG resulting from the formation of a bilayer supramolecular structure by the “ABA” temporary supra-amphiphiles and PDLLA–PEG in turn affects the stability of the “ABA” temporary supra-amphiphiles. The dynamic subtle balance between the number of “ABA” temporary supra-amphiphiles formed and the repulsive force strength between the PEG segments of DSPE–PEG under different DSPE–PEG2k additions causes the morphological structure of DSPE–PEG2k/PDLLA–MPEG complex aggregates to transit from micelles to vesicles, semi-vesicles and then to mixed micelles with increasing ratios of DSPE–PEG2k/PDLLA–MPEG. The time-dependent morphological evolution of aggregates formed by directly mixing PDLLA–MPEG micelle solutions and DSPE–PEG2k molecules at different ratios also provides evidence for the plausibility of this proposed model as outlined in Figure 5 (cf. Figures 3 and 4).

To further demonstrate the proposed model for the morphological transition of DSPE–PEG2k/PDLLA–MPEG complex aggregates, two other DSPE–PEG molecules with different PEG molecular weights (DSPE–PEG1k and DSPE–PEG5k) were used to investigate how the DSPE–PEG affect the self-assembly process of DSPE–PEG/PDLLA–MPEG mixtures. By only changing the molecular weight of PEG segments of DSPE–PEG, the hydrophobic interaction strength between the two fatty acid tails of DSPE–PEG and the three PDLLA segments of PDLLA–PEG does not change, while in the bilayer supramolecular structures formed, the repulsive force generated by the PEG segment of DSPE–PEG become stronger or weaker based upon a longer PEG or a shorter PEG segment of DSPE–PEG. TEM images of DSPE–PEG/PDLLA–MPEG complex aggregates prepared from the different mass ratios of DSPE–PEG5k and DSPE–PEG1k to the PDLLA–MPEG are shown in Figures 6 and 7, respectively. The obtained TEM images showed that the changing trends toward morphology of DSPE–PEG/PDLLA–MPEG complex aggregates with increasing addition of DSPE–PEG5k (Figure 6) or DSPE–PEG1k (Figure 7) are both similar to that of DSPE–PEG2k effect on the morphology of DSPE–PEG2k/PDLLA–MPEG complex aggregates (Figure 1). However, it should be noted in this context that less amounts of DSPE–PEG5k addition could effectively lead to a similar change of DSPE–PEG5k/PDLLA–MPEG morphology to that of DSPE–PEG2k/PDLLA–MPEG, while for DSPE–PEG1k, it needs more amounts of DSPE–PEG1k addition to gain the similar effect on the morphological transition. The intrinsic reason behind this phenomenon was due to the fact that the repulsive force between PEG segments of DSPE–PEG in the bilayer supramolecular structures formed by “ABA” temporary supra-amphiphiles and PDLLA–PEG increases with increasing PEG segment length of DSPE–PEG in the condition with the same number of “ABA” temporary supra-amphiphiles. As a result, the longer PEG segment of DSPE–PEG5k has a stronger repulsive force that can either maintain the existence of bilayer structure or destroy the structure of “ABA” temporary supra-amphiphiles at low PEG5k addition. In contrast, for DSPE–PEG1k/PDLLA–MPEG mixtures, the occurrence of morphological transition needs a higher DSPE–PEG1k addition because the shorter PEG segment has a less repulsive force. These obtained data further confirmed that the dynamic subtle balance between the number of “ABA” temporary supra-amphiphiles formed and the repulsive force strength between the PEG segments of DSPE–PEG under different DSPE–PEG additions induces the morphological transition of DSPE–PEG/PDLLA–MPEG complex aggregates.

Figure 6

TEM images of aggregates formed from DSPE–PEG5k/PDLLA–MPEG mixtures with the mass ratio of DSPE–PEG5k to MPEG–PDLLA: (a) 0:1, (b) 0.0001:1, (c) 0.0005:1, (d) 0.001:1, (e) 0.005:1 and (f) 0.01:1.

Figure 7

TEM images of aggregates formed from DSPE–PEG1k/MPEG–PDLLA mixtures with the mass ratio of DSPE–PEG1k to MPEG–PDLLA: (a) 0:1, (b) 0.001:1, (c) 0.005:1, (d) 0.01:1, (e) 0.02:1 and (f) 0.03:1.