1 POLYARGININE NANOCAPSULES: A VERSATILE NANOCARRIER WITH POTENTIAL IN TRANSMUCOSAL DRUG DELIVERY Ana Gonzalez-Paredes a,b,1 ,DoloresTorres a , María José Alonso a,b,* a Dept. Pharmacy and Pharmaceutical Technology, University of Santiago de Compostela, Spain b Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela, Spain AUTHOR INFORMATION Corresponding Author *Maria José Alonso- CIMUS Research Institute Avda. Barcelona s/n, University of Santiago de Compostela, 15706- Santiago de Compostela (Spain) E-mail: [email protected]Telephone number: +34 881815454 ABSTRACT The objective of this work was to investigate the potential utility of nanocapsules composed of an oily core decorated with a single polyarginine (PARG), or double PARG/ polyacrylic acid (PAA) layer as oral peptide delivery carrier. A step-by-step formulation optimization process 1 Present address: Nanovector Ltd. Via Livorno 60, 10144- Turin (Italy)
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POLYARGININE NANOCAPSULES: A VERSATILE NANOCARRIER WITH
POTENTIAL IN TRANSMUCOSAL DRUG DELIVERY
Ana Gonzalez-Paredesa,b,1,DoloresTorresa, María José Alonsoa,b,*
aDept. Pharmacy and Pharmaceutical Technology, University of Santiago de Compostela, Spain
bCenter for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of
Santiago de Compostela, Spain
AUTHOR INFORMATION
Corresponding Author
*Maria José Alonso- CIMUS Research Institute
Avda. Barcelona s/n, University of Santiago de Compostela,
Finally, considering that the ionic interaction was the main force for sCT encapsulation, it was
hypothesized that the incorporation of sCT into the NCs in the form of a freeze-dried
sCT/lecithin solid dispersion, might promote the association of both components, thereby
contributing to the association of sCT to the oily core. However, the impact of this variable on
the sCT association efficiency was minor (45% ± 4 vs 41 ± 4).
Overall, these data highlight the fact that it is possible to efficiently associate the cationic
peptide sCT to lipid nanostructures, and that this process is mainly driven by ionic interactive
forces between the anionic lipids and the cationic peptide. However, while designing these
formulations, it should be taken into account that the formulation parameters that are important
for achieving the adequate drug loading might interfere with the achievement of the desired
physicochemical/stability properties of the nanocarrier. Therefore, a specific formulation
optimization process is required for each peptide in order to develop a product with the adequate
profile.
4.4.In vitro sCT release study in simulated intestinal fluid
Based on the results of the sCT association efficiency, we selected the formulations made with
Maisine® 35-1 or oleic acid core, MMw or HMw PARG (PARG concentration= 0.5 or 1.5
mg/mL respectively), and lecithin (concentration= 4 mg/mL) as they provided the best drug
loading results, for further evaluation of its in vitro release profile. In this study we chose to
analyze the impact of the oil and also of the way sCT was incorporated into the system (as an
aqueous solution or as a freeze-dried powder)on the release profile (Figure 5).
The prototypes containing Maisine® 35-1 showed an initial burst release of 40% followed by a
slow release. The release rate in this second phase was affected by the way sCT was incorporated
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during the nanocapsules formation process (as a solution or as a freeze-dried powder). This was
attributed to the strong interaction between sCT and lecithin, as previously described (Rao and
Shao, 2008). On the other hand, in the oleic acid-core formulation, where sCT was incorporated
as an aqueous solution, the burst release effect was highly reduced and almost no further release
was observed until the end of the study. In this case, it was hypothesized that a strong ionic
interaction between oleic acid and sCT occurred which hampered the release of sCT. Therefore,
in this situation it could be expected that the release of sCT would occur upon degradation of the
oily core.
4.5.Studies in the Caco-2 model cell monolayer
As a final study, and in order to assess the hypothesis about the favorable interaction of PARG
nanocapsules with the intestinal epithelium, we investigated their toxicity and capacity to affect
the TEER of the Caco-2 model monolayer.
4.5.1. Cytotoxicity studies
The cytotoxicity of selected HMw PARG nanocapsules formulations containing a Maisine®
35-1core was evaluated by the MTT assay. The selected prototypes were those containing
lecithin, lecithin-lysophospholipids or Tween® 80 as surfactants, Maisine® 35-1 as an oily core
and HMw PARG as a polymer shell. The characteristics of these formulations are described in
Section 4.1.1, Table 3. In addition, we have included in the study the free polymers in solution
(LMw PARG and HMw PARG) as controls. Prior to the Caco-2 study, the stability of the
prototypes in DMEM was assessed. With the exception of the formulation containing a Tween®
80 core, all the prototypes suffered an increase in their size (about a200 nm increase) after
dilution in DMEM, which was maintained until the end of the study (3h). This size increase,
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which was attributed to the ionic strength of the cell culture medium (Pavlin and Bregar, 2012),
should be taken into account for the interpretation of the data.
As shown in Figure 6A, the formulations containing lecithin (with or without
lysophospholipids) did not compromise the cell viability at the tested concentrations (4 to 8
mg/mL, corresponding to 1.2 and 2.4 µg/cm2, respectively), whereas those containing Tween®
80, led to an important dose-dependent reduction in the cell viability. On the other hand, as
displayed in Figure 6B, the HMw PARG aqueous solution showed cytotoxicity at concentrations
equal or higher than 0.1 mg/mL, while the LMw PARG solution did not exhibit a significant
toxicity within the same concentration range.
Taking into consideration the effective amount of PARG attached to the nanocapsules, we can
deduce that the highest concentration of nanocapsules tested (8 mg/mL) correspond to a PARG
concentration of 0.1 mg/mL, a result that leads to the conclusion that the toxicity profile of
PARG was improved as a consequence of its attachment to the nanocapsules surface. An
additional conclusion from this study was that the toxicity of PARG nanocapsules was very low
as compared to that of other polycationic nanocapsules, i.e. chitosan nanocapsules, previously
assayed in our laboratory. In that case, doses between 1 and 2 mg/mL caused an important
reduction of the cell viability(Prego et al., 2005, Prego et al., 2006a).
4.5.2. Transepithelial electrical resistance (TEER) study
As indicated in the introduction, PARG has shown the ability to promote the paracellular
transport of hydrophilic macromolecules through the nasal epithelium (Ohtake et al., 2003).
Besides, Yamaki et al. have recently proposed a mechanism by which PARG causes an increase
of the Caco-2 cells permeability, which involved the alteration of the tight junctions (Yamaki et
al., 2014).We therefore considered important to investigate if this property was maintained after
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the incorporation of PARG to the nanocarriers, as it was demonstrated for other polymers by
measuring the TEER of the Caco-2 monolayers. For that purpose, three formulations containing
a Maisine® 35-1 core were tested, which were those containing lecithin, prepared with MMw and
HMw PARG, and one containing lecithin and dodecylphosphocholine, prepared with HMw
PARG. The concentration of nanocapsules was 6 mg/mL, which was previously found to be non-
toxic. The results showed in Figure 7 indicate that, irrespective of their composition, the tested
formulations caused a reduction in the TEER values after 30 min of contact with the monolayer,
reaching a maximum reduction of 43% compared to the control after 2 h. Interestingly, the initial
TEER values were almost recovered 24 h after the removal of the nanocapsules, which indicated
that the perturbation of the monolayer was transitory and disappeared after removing the
nanosystems. Therefore, the conclusion was that irrespective of the PARG Mw, the nanocapsules
increased the permeability of the Caco-2 monolayer and that the presence of
lysophosphatidilcholine did not contribute to this effect. These results are promising and
highlight the potential advantages of PARG vs. other cationic polymers, i.e. chitosan previously
investigated in our laboratory, mainly from the cytotoxicity point of view (Garcia-Fuentes et al.,
2005, Prego et al., 2005, Prego et al., 2006a).The results presented here also showed the fact that
the inherent penetration enhancing properties of PARG were preserved upon association to the
nanocapsules and that this effect was independent of the Mw of PARG.
4.6.Stability of PARG nanocapsules during storage
Among other parameters, the temperature is known to play a crucial role in the stability of
colloidal lipid carriers (Heurtault et al., 2003). For that reason, the stability of selected blank
nanocapsules under refrigerated conditions (4ºC), at room (25ºC) and at physiological
temperature (37ºC) was evaluated by monitoring their size and zeta potential upon their storage
29
during 6 months. The selected prototypes were those prepared with Maisine®35-1, lecithin and
MMw PARG. In addition, the potential contribution of the second layer of PAA to the stability
of the nanocapsules was investigated. As shown in Figure 8, at 4ºC both PARG and PARG:PAA
nanocapsules were stable and their size and zeta potential remained unchanged during a long
period of storage. An increase in size was only observed after 60 days storage at 25 or 37ºC,
which was also accompanied by a modification in the zeta potential values. However, the
presence of the PAA coating helped preventing the aggregation at 25 ºC. In conclusion, the
nanocapsules presented here, could be administered orally, or by any other modality of
administration, in the form of a suspension and be stored at RT for up to 2 months, especially
those PAA-coated. Alternatively, a freeze-dried powder could be developed in order to produce a
solid dosage form.
5. Conclusions
This work describes a number of formulation strategies for the association of hydrosoluble
cationic peptides to lipophilic nanocarriers and highlight the potential to modulate the interaction
of these peptides with anionic lipids. Beyond this, the data reported here underlines the
possibility to design lipophilic nanocarriers that can withstand the harsh conditions of the
intestinal tract and simultaneously permeabilize the intestinal epithelium. In particular, the
selection of the adequate surfactants and the formation of an external PAA layer was found to
have a positive effect. Overall, the message is that by adjusting the formulation variables it is
possible to engineer PARG nanocapsules with a potential for oral peptide delivery.
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ACKNOWLEDGMENTS
This research work was financed by the Xunta de Galicia (Competitive Reference Groups-
FEDER Funds Ref 2014/043).
Conflict of interest
The authors declare no conflict of interest in the present work
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Figure captions
Figure 1. TEM images of HMw PARG nanocapsules containing a Maisine®35-1/lecithin oily
core
Figure 2. Physicochemical characteristics of the double layer MMw PARG/PAA nanocapsules
containing a Maisine® 35-1/lecithin core as a function of the PARG:PAA polymer ratios; (Mean
± SD; n=3) (PI: polydispersity index).
Figure 3. Evolution of the size of MMw PARG nanocapsules after incubation in simulated
intestinal fluid (SIF); MMw PARG nanocapsules were prepared with different oily cores
(Mygliol ® 812 or Maisine® 35-1) and aqueous phase surfactants (Pluronic® F68, Pluronic® F127,
or PEG stearate), or coated with polyacrylic acid (PAA) (Mean ±SD; n=3)
Figure 4. Evolution of the size (solid lines) and zeta potential (dashed lines) of MWw PARG
nanocapsules containing a Maisine®35-1 core, uncoated or coated with polyacrylic acid (PAA),
after incubation in simulated intestinal fluid (SIF) with pancreatin (Mean ±SD; n=3)
Figure 5. In vitro release profiles of sCT from PARG nanocapsules in simulated intestinal
fluid:Maisine® 35-1 core has sCT incorporated i) as a solution or ii) as a freeze-dried powder
with lecithin; oleic acid core has sCT incorporated i) as a solution (Mean ±SD; n=4).
Figure6. Cytotoxicity profiles of Caco-2 cellsafter 2 h of contact with:(A) HMw PARG
nanocapsules containing a Maisine® 35-1core in combination with other surfactants: lecithin
alone or mixed with lysophospholipids, or Tween® 80;and B) HMw and LMw PARG solutions