1

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,

15706- Santiago de Compostela (Spain)

E-mail: mariaj.alonso@usc.es

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

1Present address: Nanovector Ltd. Via Livorno 60, 10144- Turin (Italy)

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was designed, which involved the study of the influence of the surfactants, oils and polymer

shells (PARG of different molecular weight and PAA) on the nanocapsules physicochemical

properties, peptide loading efficiency, stability in simulated intestinal fluids (SIF) and capacity to

enhance the permeability of the intestinal epithelium. Despite the lipophilic nature of the

nanocapsules, it was possible to achieve a moderate loading of the hydrophilic model peptide

salmon calcitonin and control its release in SIF, by adjusting the formulation conditions. Finally,

studies in the Caco-2 epithelial cell line showed the capacity of the nanocapsules to reduce the

transepithelial electric resistance of the monolayer, without compromising their viability.

Overall, these properties suggest the capacity of polyarginine nanocapsules for enhancing the

transport of peptides across epithelia.

KEYWORDS: Polyarginine, nanocapsules, peptides, transmucosal delivery, stability in intestinal

fluids

1. Introduction

In the last decade, an important number of peptides medicines have been approved by the

FDA, currently being more than 60 on the market and this is expected to increase significantly in

the coming years (Fosgerau and Hoffmann, 2015). Although the oral route would be the most

convenient way for the administration of therapeutic peptides with a systemic action,

unfortunately, with a few exceptions (Aguirre et al., 2016), this is not feasible nowadays. This is

due to the harsh gastro-intestinal environment with a high concentration of specialized enzymes,

and the highly restricted transport of these macromolecules across the mucus and the underlying

epithelium (Smart et al., 2014). A number of strategies have been investigated so far in order to

overcome the above indicated biological barriers. These include the modification of the peptide

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chemistry, and also formulation strategies that go from simple physical mixtures of the

therapeutic peptide with protease inhibitors and penetration enhancers, to the use of

nanoparticulate drug delivery systems (Griffin et al., 2016, Lakkireddy et al., 2016, Niu et al.,

2016). These strategies, alone or in combination, have led to a number of oral peptide

formulations currently in clinical trials (Aguirre et al., 2016, Lakkireddy et al., 2016).

The use of cell penetrating peptides (CPPs) has shown some promising results in terms of

increasing the transport of peptides across epithelia (Deshayes et al., 2005, Chugh et al., 2010).

In particular, D-octoarginine, L-penetratin or L-pVEC (peptide derived from murine vascular

cadherin) were shown to significantly increase the bioavailability of insulin in an in situ loop

absorption model. The enhancement of the peptide transport was attributed to the in situ

electrostatic complexation of insulin and the CPP followed by interaction with the epithelial cells

and endocytosis of the nanocomplexes (Kamei et al., 2008, Kamei et al., 2013). Other studies

have shown that the penetration promoting capacity of arginine-rich CPPs relies on the presence

of guanidinium moieities in their backbone, with the capacity to interact with anionic groups on

the cellular membrane not only through ionic interactions, but also through hydrophobic forces

and hydrogen bonds (Reuter et al., 2009, Schmidt et al., 2010, Takechi et al., 2012). On the other

hand, the importance of the number of arginine residues has been a subject of debate. For

example, a few in vitro (Mitchell et al., 2000, Futaki et al., 2001) and in vivo (Morishita et al.,

2007) studies have highlighted that CPPs with 8 arginine aminoacids are those exhibiting the

highest penetration capacity. In contrast, other in vivo studies have underlined that high

molecular weight polyarginine (PARG) is more effective that R8 in terms of enhancing the

absorption of a hydrophilic macromolecular model drug through the nasal mucosa (Natsume et

al., 1999, Miyamoto et al., 2001, Ohtake et al., 2002).

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Recent studies have shown that the inherent penetration enhancing capacity of arginine-rich

molecules is maintained when these molecules are exposed on the surface of nanoparticles. For

example, the attachment of R8 to the surface of PLGA or solid lipid nanoparticles, was reported

to have a positive impact in the insulin absorption (Zhang et al., 2012, Liu et al., 2013).

Taking this background information into consideration, our group developed a novel

nanocarrier named as PARG nanocapsules, consisting of oily nanodroplets surrounded by a

PARG shell (Lozano et al., 2013). More recently, we disclosed the possibility to associate an

amphiphilic peptide to these nanocapsules as well as their capacity to interact with the intestinal

epithelium (Lollo et al., 2017). As a further step in this line, the objective of this work was

conveniently engineer PARG nanocapsules for the oral delivery of hydrophilic peptide drugs.

For this, we investigated a number of formulation variables that could potentially influence the

association of a hydrophilic peptide to the nanocapsules and performed an exploratory study with

regard to their capacity to overcome specific barriers associated to the oral modality of

administration, notably their stability in intestinal fluids and their interaction with the intestinal

epithelium (Caco-2 model).

2. Materials

The surfactant Epikuron® 145V, a phosphatidylcholine-enriched fraction of soybean lecithin

with 45% content in phosphatidylcholine, was donated by Cargill (Spain). Different oils were

used: Miglyol® 812, a mixture of capric and caprylic acid triglycerides, which was offered by

Sasol (Germany); Maisine®35-1 and Peceol®, glycerylmonolinoleate and monooleate

respectively, which were kindly donated by Gattefosse (Spain), and oleic acid, which was

purchased from Sigma-Aldrich (Spain). Simulsol® M52 (PEG-40 stearate) was obtained from

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Seppic (France). Pluronic® F68 and F127 (poloxamer 188 and 407), as well as poylacrylic acid

(PAA; Mw 450 KDa) and Tween® 80 were purchased from Sigma-Aldrich (Spain).

Polyarginine (PARG) of three different molecular weights was used: low (5 KDa; LMw) and

high molecular weight (26.3KDa; HMw) were a gift from PTS (Spain), whereas a medium

molecular weight polymer (5-15KDa; MMw) was purchased from Sigma-Aldrich (Spain).

Palmytoil-lysophosphatidylcholine (PLPC) and dodecylphosphocholine (DPC) were obtained

from Avanti Polar Lipids (Spain). Salmon calcitonin (sCT) was obtained from Polypeptide

Group ES(Sweden). The solvents, ethanol and acetone, were analytical grade and obtained

from Fisher Chemical.

3. Methods

3.1.Preparation of PARG and PARG/PAA nanocapsules

PARG nanocapsules were prepared by either a single step or a two-step procedure based on the

previously described solvent displacement technique (Calvo et al., 1997, Prego et al., 2006a).

The two-step procedure starts with the preparation of a nanoemulsion by the solvent

displacement technique followed by its incubation with PARG. The nanoemulsion was prepared

as follows: 20 mg of lecithin (Epikuron® 145V) were dissolved in 0.25 mL of warm

ethanol(37ºC) and this solution was mixed with 62.5 µL of oil (Miglyol® 812,Maisine®35-1,

Peceol® or oleic acid) and 4.75 mL of acetone. This organic phase (5 mL) was poured over an

external aqueous phase (10 mL) containing the surfactant Pluronic®F68 or F127 (0.25% w/v).

The mixture turned milky immediately because of the formation of the nanoemulsion. Finally,

the organic solvents were removed under vacuum to a final volume of 5 mL. PARG

nanocapsules were prepared by the incubation of the nanoemulsion with a PARG aqueous

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solution (0.25-1.5 mg/mL) of 3 different molecular weights: low (5 KDa; LMw), medium (5-

15KDa; MMw) and high (26.3KDa; HMw), in a 4:1 (v/v) ratio under magnetic stirring. These

experimental conditions were maintained for the single-step procedure, with the exception of that

PARG was dissolved in the external aqueous phase above described (10 mL) containing the

surfactant Pluronic®F68 or F127 (0.25% w/v) or the surfactants mix Pluronic®F68/PEG-stearate

(0.25/0.24 % w/v) (Lozano et al., 2013). Therefore, in this situation, the formation of the oily

nanodroplets occurs simultaneously to the deposition of the PARG coating.

3.2.Incorporation of lysophospholipids in the lipid core of PARG nanocapsules

A specific PARG nanocapsules formulation based on a lecithin-Maisine®35-1 oily core was

selected in order to study the potential benefit of incorporating two types of lysophospholipids,

palmytoil-lysophosphatidylcholine (PLPC) or dodecylphosphocholine (DPC), in the penetration

enhancing properties of the nanocapsules. These phospholipids were added to the ethanol phase

containing lecithin. The total amount of phospholipids (lecithin plus lysophospholipid) was 20

mg, the lysophospholipids representing the 25% of this weight. The final concentration of

phospholipids in the organic phase was 4 mg/mL. A phospholipid-free formulation based on

Tween® 80 (20 mg, 4mg/mL) was prepared as a control. All the formulations were prepared by

the two-step procedure, which involved the mixing of the nanoemulsion with a PARG aqueous

solution (0.5-0.75 mg/mL) in a 4:1 (v/v) ratio under magnetic stirring.

3.3.Formation of a second polyacrylic acid (PAA) layer around PARG nanocapsules

A second polymer coating consisting of PAA was formed onto MMw PARG nanocapsules

having a lecithin-Maisine®35-1 oily core. This was achieved by adding 1 mL of PAA solution

(pH ≈3.5) to 4 mL of PARG nanocapsules, mixing and leaving the mixture under magnetic

stirring for 10 min. Taking into account the theoretical initial concentration of PARG (0.4

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mg/mL), several PARG:PAA ratios were tested ranging from 1:0.1 to 1:2 (w/w), using for that

PAA solutions at different concentrations ranging from 0.04 to 0.8 mg/mL.

3.4.Physicochemical characterization of PARG and PARG/PAA nanocapsules

Particle size and polydispersity index were determined by photon correlation spectroscopy

after nanocapsules dilution in water (1:33) (Zetasizer Nano ZS, software version 5.10, Malvern

Instruments, Malvern, U.K.). The zeta potential values were calculated from the mean

electrophoretic mobility values, which were determined by Laser Doppler Anemometry after

nanocapsules dilution in KCl 1mM (1:33).

The morphological analysis of the nanocapsules was performed by transmission electron

microscopy (TEM, CM12 Philips, Netherlands). For TEM imaging, samples were diluted with

bidistilled water (1:33), placed on a copper grid with Formvar® films and stained with 2% w/v

phosphotungstic acid solution for 1 minute. After that, the grid was washed twice with bidistilled

water and dried at 40ºC for 10 minutes.

3.5.Determination of PARG associated to the nanocapsules

The influence of PARG molecular weight on its association to the oily core was investigated.

For this, a specific PARG nanocapsules formulation contaning lecithin-Maisine® 35-1 in the oily

core and Pluronic® F68 in the aqueous phase, was selected. This formulation was prepared by

either the one-step or the two step procedure. Two different PARG molecular weights were used,

medium molecular weight (5-15 KDa; MMw) and high molecular weight PARG (26.3 KDa;

HMw).

The amount of PARG associated to the oily core was determined indirectly by quantification

of the free PARG after isolation of the nanocapsules. The isolation was achieved by

centrifugation in a Beckman Coulter Optima 90K Ultracentrifuge at 61690xg, 1h, 15ºC. The

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subnatant, containing unattached PARG, was analyzed for PARG quantification by Ultra

Performance Liquid Chromatography (Acquity UPLC, Waters, Spain) using a column Acquity

UPLC BEH C18 1.4 µm 2.1x50mm, at 210 nm. The chromatography conditions were the

following: 0 min: 90% A; 3.5 min: 90% B, at a flow rate of 0.2 mL/min, where A was 0.1%

trifluoroacetic acid in water and B was 0.1% trifluoroacetic acid in acetonitrile. The column was

heated at 35ºC.

PARG association was calculated according with the following equation:

% ‐

100

3.6.Stability of blank PARG nanocapsules in simulated intestinal fluid

Blank MMw PARG nanocapsules prepared with a Mygliol® 812 or a Maisine® 35-1 core by

the one-step method, were selected for studying their stability in pancreatin-free simulated

intestinal fluid. For the formulation based on Mygliol® 812, the influence of the type of

surfactant in the aqueous phase (Pluronic® F68, Pluronic® F127), or the surfactants

mixture(Pluronic® F68/PEG-stearate), on their stability was investigated. Finally, the stability of

the formulation containing Maisine® 35-1 core and Pluronic® F68 was studied with and without

a second PAA layer (PARG:PAA ratio=1:1). Nanocapsules were diluted (1:33) and incubated in

pancreatin-free simulated intestinal fluid (European Pharmacopoeia, 2011) at 37ºC under

horizontal shaking. The stability of the nanocapsules was evaluated by measuring the size and

the polydispersity index at different times during 2 h.

In a complementary study, the effect of the presence of enzymes in the simulated intestinal

fluid was followed upon incubation of the formulations containing a Maisine® 35-1 core and

Pluronic® F68, and coated or not with PAA, for 2 h in pancreatin-supplemented medium. The

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medium was prepared by adding 1% (w/v) of pancreatin to the simulated intestinal fluid. In these

case, the size, polydispersity index and Z potential of the formulations were evaluated.

3.7.Encapsulation of sCT in PARG nanocapsules

Several formulation variables have been evaluated in terms of their influence in the

encapsulation of the model peptide sCT. Among them, regarding the organic phase, the variables

studied were the core oil type (Mygliol 812®, Maisine® 35-1 and oleic acid) and the lecithin

concentration (3-4 mg/mL), whereas those regarding the aqueous phase were the type of

Pluronic® (F68 and F127) and the PARG final concentration (0.25-0.5-1 mg/mL). The influence

of the presence of the PARG coating was also evaluated in the formulations containing the

different oily cores by comparing them with the uncoated nanoemulsions.

The encapsulation of sCT in the nanosystems was achieved in two ways. The first one was the

incorporation of 50 µL of a concentrated sCT aqueous solution (10 mg/mL) into the organic

phase. The second way consisted in mixing the sCT aqueous solution with an aqueous dispersion

of lecithin. Then, this mixture was freeze-dried, and the resulting solid dispersion of sCT in

lecithin was dissolved in ethanol in order to prepare the organic phase as previously described.

All the formulations were prepared by the two-step procedure, using MMw or HMw PARG.

The amount of sCT encapsulated was indirectly calculated by determining the free sCT in the

subnatant after the isolation of the nanocapsules. The isolation was achieved, as previously

described, by centrifugation in a Beckman Coulter Optima 90K Ultracentrifuge at 61690xg, 1h,

15ºC. Then, the subnatant was diluted (1:2) with phosphate buffer (pH=4) and analyzed for sCT

content by Ultra Performance Liquid Chromatography (Acquity UPLC, Waters, Spain) using a

column Acquity UPLC BEH C18 1.4 µm 2.1x50mm, at 210 nm. The chromatography conditions

were the following: 0 min: 70% A; 2.5 min: 60% B, at a flow rate of 0.3 mL/min, where A was

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0.1% trifluoroacetic acid in water and B was 0.1% trifluoroacetic acid in acetonitrile. The

column was heated at 35ºC.

The encapsulation efficiency was calculated according with the equation:

% ‐

100

3.8.In vitro sCT release from PARG nanocapsules

The influence of the core composition in the sCT release was evaluated using two different

oily cores: Maisine® 35-1 and oleic acid. They were prepared using a lecithin concentration of 4

mg/mL, and a PARG concentration of 0.5 mg/mL (MMw PARG) and 1.5 mg/mL (HMw

PARG), respectively. We also tested in the release studies the influence of the way of

incorporation of the sCT in the nanocapsules: (1) as an aqueous solution or (2) as a freeze-dried

powder in the organic phase. The release of sCT from the PARG nanocapsules prototypes was

performed by dilution (1:2) and incubation of the loaded nanocapsules in pancreatin-free

simulated intestinal fluid. The release samples were placed in vials in an incubator at 37ºC with

horizontal shaking. At different time points, an individual sample was ultra-centrifuged

(61690xg, 1h, 15ºC). The amount of sCT released was calculated directly by UPLC analysis of

the subnatant obtained after ultracentrifugation, as described above.

3.9.Cellular studies with blank PARG nanocapsules using the Caco-2 model

In vitro studies were conducted in human colon adenocarcinoma cells (Caco-2 cells). Cells

were cultivated on 80 cm2 flasks (Nunc, Denmark) using DMEM supplemented with 10% fetal

bovine serum , L-glutamine (2mM) and 1% non-essential amino acids (all obtained from Sigma-

Aldrich). Cells were maintained at 37ºC in a humidified atmosphere with 5% CO2. The culture

medium was changed every two days during 5-6 days and when approximately 80-90%

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confluence was reached cells were trypsinized, subcultured and seeded in plates with a cell

density which was dependent on the experiment.

3.9.1. Stability of blank nanocapsules in culture medium

The stability of blank nanocapsules in Dulbecco’s modified Eagle medium-high glucose

(DMEM) was evaluated in order to elucidate the behavior of the nanosystems during the studies

in Caco-2 cell line. Selected blank PARG nanocapsules formulations were isolated as described

before, and then resuspended in ultrapure water, diluted (1:10), and incubated in serum-free

DMEM at 37ºC under horizontal shaking. Their stability was evaluated by measuring the size

and the polydispersity index at different times during 3 h.

3.9.2. Cytotoxicity studies

Cells were cultivated on 96-well plates (Nunc, Denmark) with a cell density of 1.4 x 104

cells/well. Cells were incubated during two days, in order to obtain a homogeneous cell

monolayer. Then, the medium was replaced by several prototypes of blank HMw PARG

nanocapsules containing a Maisine® 35-1 core in combination with different surfactants: lecithin

alone or mixed with lysophospholipids, or Tween® 80. The formulations were isolated as

described before, and then resuspended in ultrapure water, diluted in the cell culture medium

(1:10) and incubated at concentrations 4, 6 and 8 mg/mL, which corresponds to a theoretical

PARG concentration of 0.1, 0.15 and 0.2 mg/mL respectively. Solutions of the free polymers of

two different molecular weights, low molecular weight (5KDa; LMw) and high molecular

weight (26.3 KDa, HMw), were also tested but in wider range of concentrations, between 0.0125

mg/mL and 0.2 mg/mL, which corresponds to 3.8 µg/cm2 and 60 µg/cm2 respectively. DMEM

was used as a positive control.

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Cells monolayers were kept in contact with the samples for 2 hours, at 37ºC and 5% CO2.

After this time, the samples were removed and the cell viability was determined by the MTT

assay. The absorbance (λ=515 nm) of the samples was measured with a Teca Ultra Evolution

spectrophotometer after the addition of DMSO (100 µL), and corrected for the background

absorbance (λ=630 nm). The percentage of cell viability was calculated by comparing the

absorbance of the samples with that of the control.

3.9.3. Measurement of the transepithelial electrical resistance

The transepithelial electrical resistance (TEER) was measured in Caco-2 cells which were

seeded on 6-well Transwell cell culture chambers (Costar, USA) using a Millicell®-ERS system

(Millipore, Spain). Cells were seeded onto the apical compartment of a Transwell at a cell

density of 2.5x105 cell/well. The culture medium (supplemented DMEM) was changed every

other day and the cells were kept for 21 days, in order to obtain a tight cell monolayer.

The TEER of the cell monolayers was measured just before adding the samples. Then, cell

medium in the apical chambers was changed by some selected PARG nanocapsules

formulations, which were previously isolated and resuspended in ultrapure water. The

formulations, based on a Maisine®35-1/lecithin oily core, with Pluronic® F68 as surfactant, were

diluted in culture medium (1:10) at a concentration of 6 mg/mL, which led to a final PARG

theoretical dose per surface of 45 µg/cm2 (0.15 mg/mL). Two different molecular weights of

PARG (MMw and HMw) were tested, as well as a formulation including dodecylphosphocholine

in the oily core. Fresh cell culture medium was used as a control. The samples were incubated

for 2 h and TEER values were recorded at 0.5, 1 and 2 h. After this time, the tested samples were

removed and replaced by fresh medium in order to check the TEER values at 24 h after exposure

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to the nanocapsules. Each TEER value was calculated as a percentage of the initial TEER value

(≥330Ω cm2).

3.10. Stability of blank PARG nanocapsules during storage

The stability of blank MMw PARG and PARG:PAA (1:1 polymers ratio) nanocapsules having

a lecithin-Maisine® 35-1 oily core and Pluronic® F68 as surfactant, was evaluated under storage

conditions. At determined time points a sample of the nanocarriers stored at 4, 25 or 37º C was

taken and analyzed for size and zeta potential, as previously described, during a period of at least

6 months. These parameters can be used as a tool to predict and control the stability of colloidal

suspensions or emulsions.

4. Results and Discussion

Recently, our group reported for the first time the design and production of PARG

nanocapsules composed of a lecithin/Mygliol®812 core, together with their capacity for the

delivery of hydrophobic, i.e. docetaxel (Lozano et al., 2013), or amphiphilic, i.e. plitidepsin

(Lollo et al., 2017) drugs. The aim of the present work was to explore the versatility of these

nanocapsules in terms of: (i) the possibility to modulate their physicochemical properties and

composition, (ii)their potential to load macromolecular hydrophilic drugs and (iii) their capacity

to promote their transport across well-organized epithelia, such as the intestinal epithelium.

Hence, here we describe how the adequate selection of the oil, the surfactants, and the molecular

weight of PARG may lead to nanocapsules with appropriate characteristics for overcoming

important intestinal barriers, i.e. stability and interaction with the intestinal epithelium. We also

report the influence of these variables on the encapsulation of the model peptide sCT.

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4.1. Modulation of the physicochemical characteristics of PARG nanocapsules

PARG nanocapsules were prepared according to the solvent displacement technique, adopting

either a one- or a two-step procedure (Calvo et al., 1997, Prego et al., 2006a). A nanocapsules

composition previously described (Table 1) was adopted in order to adapt it for the purpose of

oral peptide delivery. In a first set of experiments, the influence of specific formulation variables,

i.e. type of oil, type of surfactant, and PARG molecular weight, on the physicochemical

properties of the nanocapsules was investigated. Table 2 shows the result of the characterization

of these nanocapsules prepared by a one-step procedure.

Table 1. Formulation components concentration of PARG nanocapsules in the final suspension (mg/mL)

Oil Lecithin(Epikuron®145) Pluronic® F68 or 127 PARG

12 4 5 0.5

Overall, the results indicated that the nature of oil was the main parameter determining the size

and the polydispersity index of the nanocapsules. The smallest nanocapsules were those with a

Mygliol® 812 (159-205 nm) or a Maisine® 35-1 core (180 nm), and the largest ones were those

with a Peceol® core (336 nm). Interestingly, although Peceol® and Maisine® 35-1, glycerol

monooleate and monolinoleate respectively, are both monoglycerides of C18 fatty acids, they led

to the formation of nanocapsules of significantly different size (180 nm for Maisine® 35-1 and

336 nmfor Peceol®). This can only be attributed to the number and position of double bonds,

being one in the case of Peceol®(monooleate) and two in the case of Maisine®35-1

(monolinoleate), which may influence chain splay and, thus, the interfacial curvature of the oily

droplets and the size of the resulting nanocapsules (Kulkarni et al., 2010).

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Table2. Formulation variables investigated and physicochemical properties of PARG

nanocapsules prepared by the one-step method (Mean ±SD; n=3)(PI: polydispersity index; Z-

Pot: zeta potential)

Formulation variables investigated Properties of the nanocarriers

Oil

Surfactants

(aqueous phase)

PARG Mw

Size (nm) PI Z-Pot (mV)

PARG association

(%)

Mygliol® 812

----

MMwb

171± 01 0.1 +44 ± 1

ND

Pluronic® F68 205 ± 07 0.1 +46 ± 7

Pluronic® F68/ PEG-stearatea

159 ± 02 0.1 +32 ± 3

Pluronic® F127

171± 02 0.1 +27 ± 2

Peceol® Pluronic® F68 MMwc 336 ± 27 0.2 +23 ± 7 ND

Maisine®35-1 Pluronic® F68 MMwb 180 ± 16 0.1 +31 ± 1 31 ± 6

HMwb 182 ± 03 0.1 +29 ± 3 51 ± 2

Oleic acid Pluronic® F68 HMwd 244 ± 10 0.2 +03 ± 1 42 ± 4

aPEG-stearate concentration: 9.6 mg/mL PARG concentration in the final suspension: b0.5 mg/mL; c1 mg/mL; d1.5 mg/mL MMw: Medium Molecular weight PARG; HMw: High Molecular weight PARG ND: notdetermined.

On the other hand, with regard to the influence of the surfactants incorporated in the external

aqueous phase, the results in Table 2 indicate that, in the case of prototypes prepared with

Mygliol®812, the presence of Pluronic®127 did not affect the size of the nanocapsules. However,

the addition of Pluronic®F68 led to a slight increase in the size; an increase that could be reduced

by the simultaneous incorporation of PEG-stearate. This effect could be explained by the

different HLB values of both Pluronics®, being Pluronic® F127 slightly more hydrophobic

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(HLB=22) than Pluronic® F68 (HLB=29). This greater hydrophobicity, which is associated to

the longer polypropilene oxide chain in its structure, could have facilitated its entanglement

around the oily droplets. These results are in agreement with those found by Krishna et al., who

observed that the combination of Pluronic® F127 and lecithin was more appropriate that the

combination of Pluronic® F68 and lecithin with regard to their emulsification properties (Krishna

et al., 1998).

Finally, within the range of conditions investigated in this study, the MW of PARG (5-15 KDa

and 26.3 KDa) did not affect the size of the nanocapsules. The size of this kind of lipidic

nanocarriers may vary or not according to the polymer structure and molecular weight, which

will influence its arrangement around the oily droplets (Quemeneur et al., 2010). In fact, the lack

of effect of molecular weight in nanocapsules size has been also described for different

polyaminoacids nanocapsules, such as polyglutamic acid and polyasparagine nanocapsules

(Abellan-Pose et al., 2016).Our hypothesis is that, due to the ability of PARG to establish

hydrophobic bond, it is coating the nanoemulsion by means of electrostatic and hydrophobic

interactions with lecithin, with a tight flat absorption of the polymer around the oily

nanodroplets, regardless of molecular weight.

Another physicochemical property that may influence the stability of the nanocapsules in the

intestinal fluids as well as their interaction with the intestinal mucosa, is the zeta potential. The

results in Table 2 show that all formulations exhibit a positive surface charge, which is attributed

to the attachment of PARG around the oily nanodroplets. However, the intensity of the positive

charge varied depending on the nature of the oil and the surfactants used. For example, in the

case of Mygliol®812-containing nanocapsules, the zeta potential ranged from +44 mV (without

Pluronic®) to +27 mV, which indicates that surfactants with long hydrophilic chains, such as

17

PEG stearate and Pluronic® F127, shield partially the positive charge on the surface. For

example, in the case of the nanocapsules containing oleic acid, the zeta potential was low (+ 3

mV) and the amount of PARG needed to obtain this positive charge was 3 times higher than the

one required for the rest of the formulations. This could be related to the important negative

charge of oleic acid and its affinity for the positively-charged arginine residues (Xie et al., 2013).

On the other hand, the results in Table 2 indicate that the surface charge of the nanocapsules was

not significantly influenced by the molecular weight of PARG. However, the association

efficiency of PARG to the oily core was dependent on this parameter. Indeed, in the case of

nanocapsules containing Maisine®35-1, the MMw PARG (5-15 KDa) was attached to the oily

core in a 31%, whereas for the HMw PARG (26.3 KDa) this value increased up to a 51%. This

effect is probably a consequence of the number of arginine units present in the polymer,

providing the highest molecular weight more points for the attachment to the oily core surface. In

fact, the number of PARG molecules attached to the nanocores was similar irrespective of their

Mw.

Finally, as expected, we also observed that the method used for the preparation of the

nanocapsules significantly influenced the PARG association to the oily core. In fact, the one-step

procedure, in which the polymer is incorporated at the beginning of the process, was twice more

effective in terms of favoring the polymer deposition around the oily droplets, in comparison

with the two-step procedure (results not shown). This could be due to the fact that in the one-step

method the deposition of the polymer and surfactants around the oily droplets occurs

simultaneously, whereas in the two-step procedure the polymer has to be accommodate after the

organization of the surfactants on the surface, leaving less freedom for its spatial arrangement,

results that are in agreement with those found for other cationic polypeptides, where the nature of

18

the surfactant highly influenced the polymer attachment to the nanocapsules surface (González-

Aramundiz et al., 2017).

Overall, from this initial screening, we concluded that the nature of the oil was the main factor

influencing the size and zeta potential of the nanocapsules, and that the attachment of PARG to

the oily core was effective and influenced by its molecular weight. Taking into account their

small size and the high association of PARG, the formulation containing Maisine®35-1 was

selected for further experiments.

HMw PARG nanocapsules containing Maisine® 35-1 in their core were observed by TEM.

The micrographs in Figure 1 show that the nanocapsules have a round shape and a size around

100 nm, smaller than that observed by laser light scattering. This phenomenon had been

previously observed and attributed to the dehydration of the nanostructure and compaction of the

polymer shell.

4.1.1. Incorporation of lysophospholipids as penetration enhancers in the PARG

nanocapsules

A subsequent study was planned in order to explore the effect of including specific

lysophospholipids, known as potential penetration enhancers, into the Maisine® 35-1 core of the

nanocapsules (Brandhonneur et al., 2011, Kotake-Nara and Nagao, 2012).

These lysophospholipids were palmytoil-lysophosphatidylcholine (PLPC) or

dodecylphosphocholine (DPC). The NCs containing different amounts of these

lysophospholipids were prepared by the two-step procedure using LMw (5 KDa) and HMw (26.3

KDa) PARG. The results showed that the maximum amount of these phospholipids that could be

incorporated into the selected nanocapsules without compromising their physicochemical

properties and stability was a 25% with respect to the total amount of phospholipids (20 mg).

19

The physicochemical characterization of the resulting prototypes, in comparison with a control

containing Tween® 80 (20 mg) instead of phospholipids, is shown in Table 3.

Table 3. Formulation variables investigated and physicochemical characteristics of PARG

nanocapsules containing a Maisine® 35-1 core; (Mean ±SD; n=3) (PI: polydispersity index).

Surfactant/s(oily phase) PARG Mw Size (nm) PI Zeta potential (mV)

Lecithin LMwa 150 ± 02 0.1 +22 ± 1

HMwb 154 ± 05 0.1 +36 ± 1

Lecithin/PLPC LMwa 188 ± 26 0.1 +16 ± 3

HMwb 158 ± 10 0.1 +38 ± 1

Lecithin/DPC LMw a 185 ± 12 0.1 +14 ± 3

HMwb 170 ± 04 0.1 +37 ± 2

Tween® 80 LMwa 187 ± 03 0.1 +0.1 ± 0.1

HMwb 194 ± 15 0.1 +12 ± 2

PLPC: palmytoil-lysophosphatidylcholine; DPC:dodecylphosphocholine; LMw: Low Molecular weight PARG; HMw: High Molecular weight PARG aPARG concentration: 0.75 mg/mL bPARG concentration : 0.5 mg/mL

This last formulation was selected because of the known penetration enhancing properties of

Tween®80 (Maher et al., 2016). The results in Table 3 indicate that the size of both LMw and

HMw PARG nanocapsules only was slightly affected by the nature of the surfactants

incorporated in the formulation. On the contrary, the zeta potential was significantly affected by

the nature of the surfactants and the PARG Mw. Indeed, the formulations containing Tween® 80

showed the lowest zeta potential values, a fact that suggests that, under the current experimental

conditions, this surfactant impairs the attachment of PARG. Moreover, all the formulations

20

prepared with LMw PARG required a higher amount of PARG (1.5 times more), as compared to

those prepared with LMw, in order to obtain stable systems.

4.1.2. Development of double layer PARG/ polyacrylic acid (PAA) nanocapsules

Although the positive charge of PARG nanocapsules was supposed to have a positive effect in

terms of their interaction with the intestinal epithelium, it was also presumed that this polymer

layer might facilitate the interaction of the nanocapsules with the negatively charged enzymes,

which are present in the intestinal fluids, i.e. pancreatin, with their subsequent degradation and/or

aggregation. To avoid this potential problem our idea was to form a second polymer layer around

the nanocapsules and, for this, we chose PAA. In addition to providing a negative charge to the

nanocapsules, the selection of PAA was based on its capacity to open cell tight junctions (Di

Colo et al., 2008, Vllasaliu et al., 2012) and inhibit trypsin and other peptidases in the gut

(Luessen et al., 1995, Bai et al., 1996). Based on the results of the physicochemical

characterization, the formulation selected for the formation of a second PAA layer was the one

containing a Maisine® 35-1/lecithin core, and Pluronic® F68. The second PAA layer was formed

by mixing the PARG nanocapsules suspension with an aqueous solution of PAA, based on the

assumption that the driving force for the formation of the double layer was the ionic interaction

between PARG and PAA. To monitor the efficiency of the enveloping process, we chose

different ratios of PARG:PAA ranging from 1:0.1 to 1:2 (w/w). As shown in Figure 2, the

nanocapsules formed with PARG:PAA ratios 1:1 and 1:2, exhibited adequate size and zeta

potential values, whereas a lesser amount of PAA led to bigger sizes, and high polydispersity

indexes. The ratio 1:1 shifted the nanocapsules mean size from 180 to 260 nm, being confirmed

the formation of the PAA coating by the inversion of the zeta potential value (from +31 mV to

21

-38 mV). The 1:2 ratio conferred very similar characteristics to the nanocapsules, thus we

discarded a further increase in the PAA final content.

4.2.Stability of blank PARG nanocapsules in simulated intestinal fluid (SIF)

As in the case of the physicochemical characterization, we evaluated the influence of the

nanocapsules composition (different oils, surfactants and polymer layers) in the stability of the

nanocapsules in SIF. As shown in Figure 3, the stability of the nanocapsules was particularly

compromised in the case of the formulations containing Pluronic® F68. The use of Maisine® 35-

1, instead of Mygliol® 812, contributed to a slight stabilization of the Pluronic® F68-containing

nanocapsules. The improved stability of nanocapsules containing Pluronic® F127, as compared

to those containing Pluronic® F68, was attributed to the presence of longer polypropylene oxide

block chains and lower HLB of Pluronic® F127 (HLB: 22 vs. 29 for Pluronic® F68) and their

favorable attachment to the oily cores. On the other hand, the results also showed that use of

PEG stearate or the formation of a second PAA coating reverted the deleterious effect of

Pluronic® F68 and led to the formation of stable nanocapsules. The size increase observed for

nanocapsules prepared with Mygliol® 812 was accompanied of a big increase in polydispersity

index (PI), evolving from 0.1 to 0.7 at the end of the study, whereas for the rest of formulations

the PI value was between 0.1 and 0.2.

In a different study we also monitored the stability of selected formulations, in particular those

containing Maisine®35-1 and Pluronic® F68, with and without PAA, in the presence of SIF with

pancreatin. Surprisingly, the results in Figure 4 indicate that, in terms of mean size, the stability

of both formulations, with and without PAA, was similar. However, the polydispersion of the

nanocapsules without a PAA coating increased up to 0.5. To explain these data we hypothesized

that a negatively charged protein corona was formed around the PARG nanocapsules protecting

22

them from aggregation. This hypothesis was confirmed by the inversion of the zeta potential

values of this prototype of PARG nanocapsules (from +30 mV to -30 mV). Therefore, in

conclusion, the presence of a PAA protective layer around PARG nanocapsules was found to

have a protective role against enzyme attachment.

4.3.Association of salmon calcitonin (sCT) to PARG nanocapsules

Once verified that PARG nanocapsules were stable upon incubation in intestinal fluids, our

next step was to assess their capacity for the association of hydrophilic peptides and, with this

idea in mind, we chose salmon calcitonin (sCT) as a model peptide. In the formulation process of

sCT, we selected as a constant parameter the presence of lecithin and investigated a number of

technological variables associated to the two-step nanocapsules fabrication technique. As

indicated in Table 4, these variables were: the type of oil (Mygliol® 812, Maisine® 35-1 or oleic

acid, all of them in a constant concentration of 12 mg/mL), the type of Pluronic® (PF68 and

PF127, both of them in a constant concentration of 5 mg/mL), and the PARG concentration in

the final suspension (0.25, 0.5 and 1 mg/mL). In addition, for some of these formulations we also

investigated the influence of the amount of lecithin (3 and 4 mg/mL) on the properties of the

formulations. The physicochemical characterization and sCT entrapment capacity of the

nanocapsules are reported in Table 4. From the physicochemical point of view, the first

observation is that sCT-loaded PARG nanocapsules showed similar mean particle size and zeta

potential than the blank nanocapsules (Section 4.1, Table 2), thus evidencing no interference of

the peptide in the nanocapsules formation process. However, as shown in Table 4, the

encapsulation efficiency of sCT was significantly influenced by nanocapsule’s composition. First

of all, the presence of the PARG shell led to an important decrease in the capacity of association

of the peptide to the nanocapsules. In fact, when compared the encapsulation efficiency values to

23

those of uncoated nanoemulsions, a decrease of at least a 50% could be appreciated. Similarly,

when using Mygliol®812 as an oily core, the use of increasing concentrations of PARG led to a

drastic reduction in the entrapment of the peptide. Our interpretation of these results was that,

because of its hydrophilic nature, the cationic peptide was mainly associated to the surface of the

oily core and the addition of a PARG layer around this core led to the displacement of the

peptide. In fact, without the PARG coating, the encapsulation efficiency was around 80% and

this value dropped down to 8% for highest PARG concentration used.

These results are in agreement with those previously reported by Prego et al. for chitosan

nanocapsules (Prego et al., 2006a, b), where values of sCT association efficiency around 90% for

the nanoemulsion decreased dramatically after incubation with a chitosan solution. This

competition between the peptide and PARG might also explain the fact that the nanocapsules

prepared with HMw PARG, with aMaisine®35-1 core and a PARG concentration of 0.5 mg/mL,

showed a lower sCT encapsulation (26% ± 7) compared to the same prototype prepared with

MMw PARG (41% ± 4). This effect could be easily understood if we consider that HMw PARG

was more efficiently attached to the nanocapsules surface, and thus a greater displacement of

sCT by the polymer should be expected.

On the other hand, despite the hydrophilic nature of the peptide, the type of oil was found to

slightly influence the sCT encapsulation. Indeed, the use of either more hydrophilic oils, i.e.

Maisine® 35-1 (HLB=1) or more acidic oils, i.e. oleic acid, facilitated the association of sCT,

probably because of the greater facility of these oils to accommodate the hydrophilic cationic

peptide.

With regard to the influence of the Pluronic® type, it was observed that in the case of the

nanocapsules prepared with Maisine® 35-1, the use of Pluronic® F127, which efficiently

24

improved the stability of the system, caused a drastic reduction in the peptide entrapment

efficiency. In fact, in this case and also when PEG stearate was added to the Pluronic® F68 (data

not shown), the long PEG and polypropylene oxide chains probably hindered the sCT binding.

Table 4. Formulation variables investigated, physicochemical properties and encapsulation

efficiency of several sCT-loaded PARG nanocapsules (PI: polydispersity index) (Mean ± S.D.;

n=3-6).

Formulation variables

Properties of the nanocarriers

Size

(nm) PI

Zeta potential

(mV)

sCT encapsulation

efficiency (%)

Oil typea

(Uncoated nanoemulsions)

Miglyol ® 812 189± 04 0.1 -52± 05 79 ± 5

Maisine® 35-1 179 ± 04 0.1 -46 ± 02 81 ± 2

Oleic acid 165± 08 0.1 -42± 03 80 ± 4

Oil typea

Miglyol ® 812c 212 ± 13 0.1 +51 ± 07 32 ± 6

Maisine® 35-1c 177 ± 16 0.2 +41 ± 04 41 ± 4

Oleic acidd 173 ± 12 0.2 +7 ± 01 45 ± 8

PARG concentrationb, e

(mg/mL)

0.25 192 ± 05 0.1 +41 ± 01 18 ± 1

0.5 192 ± 08 0.1 +57 ± 02 22 ± 8

1 179 ± 04 0.1 +55 ± 10 8 ± 7

Pluronic®typea, c, f

Pluronic® free 191 ± 03 0.1 +46 ± 01 48 ± 4

P F68 177 ± 16 0.2 +41 ± 04 41 ± 4

P F127 181 ± 10 0.1 +20± 01 8 ± 3

Constant parameters: Theoretical sCT loading: 0.8%. Final concentration: 0.1 mg/mL Pluronic® F68/127 concentration: 5 mg/mL.

Variable parameters investigated for specific formulations: Lecithin concentration: a4 mg/mL; b3 mg/mL

25

PARG concentration: c0.5 mg/mL; d1.5 mg/mL Oil type: eMygliol ®812; f Maisine®35-1

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

26

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,

27

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

28

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.

30

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

(Mean± SD; n=2)PLPC=palmytoil-lysophosphatidylcholine; DPC= dodecylphosphocholine

Figure 7. Transepithelial electrical resistance (TEER) of Caco-2 monolayer after incubation with

MMw and HMw PARG nanocapsules containing a Maisine® 35-1/lecithin core with or without

dodecylphosphocholine (DPC); control: DMEM (Mean± SD; n=3)*Statistically significant

differences between the 3 formulations and the control (p<0.05)

Figure 8. Evolution of the size (nm) and zeta potential (mV) of: (A) MMw PARG and (B) MMw

PARG-PAA nanocapsules over the time in storage at different temperatures (4º, 25º and 37ºC)

Figure 1.

Figure 2.

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Figure 3.

Figure 4.

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Figure 5.

Figure 6.

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Figure 7.

Figure 8.

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Graphical abstract

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