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November 2015
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Fig. 2: ZP reversal during LbL-assembly.
on ZP value along with a more gradual plateau onset was verified, following an exponential fitting model (R2 > 0.96, Fig. 1a). DS originated a clear plateau, approaching to a sigmoid fitting model (R2 > 0.95, Fig. 1b). This difference could be explained to the difference in charge density of PEs. Only the two first titrations of the LbL shell were depic- ted, however the procedure was simi- lar for the followed PE layers ensuring no PEs excesses. Values of ZP magni- tudes during the process of adsorption are present in Fig. 2. After adsorption of PAH to RSV nanocores, drug NPs were recharged to high positive surface charge (+26.2 ± 1.0 mV), conferring high physical stability to nanocores. The addition of DS promoted the reversion of the surface charge to negative values (-26.7 ± 2.2 mV). The strongly charged LbL-coated NPs repulsed, maintaining colloidal stability 2. The LbL proceeded by consecutively alternating PE addi- tions. Given the higher ZP magnitude of PAH layers comparing to DS layers, PAH was chosen for
the outermost shell layer coating. Thus, a LbL self-assem- bly technique cou- pled with a washless approach was deve- loped and aqueous RSV nanocolloids with different num- ber of PAH/DS bilayers were per- formed, namely with 2.5 (RSV-(PAH/ DS)2.5), 5.5 (RSV- (PAH/DS)5.5) and 7.5 (IBF-(PAH/DS)7.5) bilayers. These col-
loids showed homogenous particle size populations at the desired nanoscale interval (150-250 nm). LbL 7.5-bilayers coated NPs (the most complex formu- lation) showed 219 ± 1 nm and 0,17 of PI; high electrical surface ZP of +31 ± 0.5 mV; and a high drug content of 92 ± 2%.
In vitro release studies with RSV crys- tals, RSV nanocores and LbL NPs with 2.5, 5.5 and 7.5 bilayers of PAH/DS were investigated in simulated gastric followed by intestinal fluids without en- zymes (Fig. 3). 2.5-bilayered coated NPs and RSV nanocores showed a higher dissolution rate in simulated gastric pH in relation to RSV crystals and 5.5 and 7.5- bilayered coated NPs. RSV crys- tals, in turn, showed higher dissolution rate in relation to the most complex LbL formulations. This indicates an effect of shell wall thickness on RSV delayed dissolution. Besides these differences, after 2 hours of in vitro simulated gas- tric incubation, most RSV remained associated to LbL NPs (> 80%), indica- ting that these systems promoted good
gastric resistance, namely for 5.5- and 7.5-bilayered coated NPs, emphasi- sing the important role of LbL shell on RSV protection. Following a pH change to 6.8, a biphasic release pattern was observed, characterized by an initial rapid release during the first 1.5 hours followed by a delayed release up to 6 h. These results showed a very good fit with the exponential kinetic model (R2 > 0.99), suggesting an apparent first- order behaviour. No differences were detected between 2.5-bilayered coated NPs and RSV nanocores. Both formula- tions led to slightly faster release than non-encapsulated RSV crystals and 5.5- and 7.5-bilayered NPs, due to the low complexity of LbL shells and also because of the NPs small size compa- red to micrometer size of RSV crystals (like it happened previously in simula- ted gastric medium). Surface area was higher between nanocores and 2.5-bi- layered coated NPs and the release medium in comparison to RSV crystals. Moreover, according to Noyes-Whitney equation, an enhancement of satura- tion concentration and a decrease in particle size into the nanoscale caused an increase in the dissolution rate (No- khodchi 2010).
An increasing effect of the number of coating bilayers on delayed release of RSV was observed.
This effect was probably due to the enhancement of shell wall thickness, which conducts to increased diffusio- nal path for RSV and, thus, for a RSV delay from the core to the LbL shell 2. For example, at 4 hours, only 51% of RSV in 7.5-bilayers coated NPs was released as compared with of 60% the 5.5-bilayers and 91% of the 2.5-bilayers samples. Non-complete drug release from LbL PE bilayered shells was re- ported before and it is related to the complexity of the LbL shell 2. Compa- ring to our previous results 2, DS pre- vented significant premature release of RSV by providing higher retention capa- city than polystyrenesulfonate (PSS) at gastric pH. This predicts higher availa- bility of RSV for absorption in the intes- tine. Alongside, LbL technique allowed for the control of RSV release rate from PAH/DS-stabilized NPs depending on the number of coating bilayers in the
Fig. 3: In vitro RSV release from studied formulations in gas- tric and intestinal simulated media.
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