Accepted Manuscript

Research paper

In situ mucoadhesive-thermosensitive liposomal gel as a novel vehicle for nasal

extended delivery of opiorphin

Paola Mura, Natascia Mennini, Cristina Nativi, Barbara Richichi

PII: S0939-6411(17)30702-6
DOI: https://doi.org/10.1016/j.ejpb.2017.10.008
Reference: EJPB 12613

To appear in: European Journal of Pharmaceutics and Biophar-

maceutics

Received Date: 8 June 2017

Revised Date: 7 October 2017
Accepted Date: 10 October 2017

Please cite this article as: P. Mura, N. Mennini, C. Nativi, B. Richichi, In situ mucoadhesive-thermosensitive
liposomal gel as a novel vehicle for nasal extended delivery of opiorphin, European Journal of Pharmaceutics and
Biopharmaceutics (2017), doi: https://doi.org/10.1016/j.ejpb.2017.10.008

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In situ mucoadhesive-thermosensitive liposomal gel as a novel
vehicle for nasal extended delivery of opiorphin

Paola Mura*, Natascia Mennini, Cristina Nativi, Barbara Richichi

School of Human Health Sciences, Department of Chemistry, University of Florence, via

Ugo Schiff 6, Sesto Fiorentino, 50019 Florence, Italy

*Corresponding author
tel +390553672; e-mail:paola.mura@unifi.it

1
Abstract

Previous studies proved the effectiveness of an intravenous PEGylated liposomal

formulation of opiorphin (1 mg/mL) in protecting the drug from enzymatic degradation,
and improving intensity and duration of its painkilling effect. Therefore, considering the
advantages of nasal administration, the aim of this work was the development of a
liposomal mucoadhesive thermo-sensitive in situ gel for the extended nasal delivery of
opiorphin. With this purpose, the potential of a series of combinations of different
polymers (i.e. chitosan, hydroxypropylmethylcellulose, Poloxamer, Carbopol) in forming
solutions able to rapidly gel at the nasal cavity temperature (34 °C) has been investigated.
The best formulations were further characterized for gel strength and mucoadhesion
properties. The selected formulation, composed by Poloxamer 407 (26.5 %) and
Carbopol 934P (1%), showed short gelation time at 34 °C (10 s) and suitable
mucoadhesion duration (5.5 h) and strength (27 g/cm2). Due to the low volume
administrable via the nasal route, a concentrated liposomal formulation of the peptide
(16.5 mg/mL) was developed and loaded in the selected in situ gel formulation. Ex-vivo
permeation studies, by excised nasal porcine mucosa, showed that the liposomal
hydrogel formulation enabled a sustained and controlled delivery of opiorphin over more
than 5 h, and highlighted the role of the liposomal carrier in enhancing up to 6 times
permeability coefficient and permeation rate of the peptide through the lipophilic nasal
mucosa compared to a free peptide-loaded gel.

Keywords: opiorphin; in situ nasal delivery; PEGylated liposomes; thermosensitive

hydrogel; mucoadhesion; ex vivo permeation.

Chemical compounds studied in this article:

Opiorphin (PubChem CID: 25195667); sodium glycerophosphate hydrate (PubChem
CID: 22251426); Chitosan (PubChem CID: 21896651); hydroxypropyl-
methylcellulose (PubChem CID: 57503849); Poloxamer (PubChem CID:24751);
Carbopol (PubChem CID: 4068533)

2
1. Introduction
Opiorphin is a natural peptide recently isolated from human saliva [1], that shows a
strong analgesic effect, even superior than that induced by morphine [2]. It appears as an
interesting and promising therapeutic agent in the treatment of acute and chronic pain,
also considering that many of the side effects given by morphine and morphine-like
drugs should be absent. In fact opiorphin does not activate directly the opioid receptors,
but it mainly acts by stopping the normal breakdown of enkephalins [1,3]. It has been
shown that repeated treatments with opiorphin did not produce significant abuse liability
and opioid dependence and failed to give rise to tolerance or morphine-cross-tolerance
phenomena [4,5].
However, despite its strong pain-killing activity, the short duration of action after
intravenous administration, probably caused by its rapid degradation by the peptidases in
the bloodstream [2] may seriously hinder the successful use of opiorphin into clinical
practice. In order to overcome this problem, we recently developed a PEGylated-
liposomal formulation of opiorphin, which proved to be able to protect the drug and
significantly enhance extent and duration of its analgesic effect after intravenous
administration to rats, with respect not only to the simple peptide aqueous solution but
also to the peptide loaded in conventional liposomes [6]
Nasal route has gained an increasing interest over recent decades as a potential
alternative to the parenteral administration of a number of therapeutic agents, including
peptides [7,8]. In fact, in addition to its non-invasiveness and painlessness, intranasal
administration presents a series of important advantages such as the relatively large
surface area available for drug absorption, the highly vascularized epithelium, the
avoidance of hepatic first-pass metabolism of drugs and the possibility of delivery drugs
to the brain [9]. In this latter respect, a review about the possible routes and mechanisms
likely involved in intranasal delivery of drugs to the central nervous system (CNS) has
been published [10]. Furthermore, the easy accessibility of the nasal cavity allows a
quick self-administration, thus further improving patient compliance.
Therefore, intranasal delivery of opiorphin, as liposomal formulation, could
represent a valid alternative to its intravenous administration. However, in spite of the
high potential of nasal drug delivery, some important limitations on its adoption have to
be considered, such as eventual presence of pathological conditions (such as colds,
rhinitis, allergies), potential local tissue irritation by formulation components, possible
enzymatic degradation. Moreover, the mucociliary clearance can reduce the in situ

3
residence time of drugs and their absorption by transporting the drug to the nasopharynx
and then to the gastrointe1tinal tract [11]. One of the most attracting and effective
approaches to overcome this critic drawback is the addition in the formulation of suitable
mucoadhesive polymers, owing to their ability to interact with the mucus layer, thus
hampering the clearance of the delivery system and favouring the drug absorption
[12,13]. In particular, it has been shown that mucoahesive polymeric gels are able to
establish an intimate contact with the nasal mucosa surface and thus prolong the drug in
situ residence time [14,15]. However, due to their high viscosity, gels are difficult to
apply and the dose of administered drug cannot be accurately determined. In situ forming
hydrogels have been developed that can allow to overcome both these problems [16].
Among the possible mechanisms leading to in situ gel formation, the thermo-sensitive
approach is considered particularly advantageous [17,18]. Mucoadhesive thermosensitive
polymeric systems are fluid-like before nasal administration, and can be administered as
drops, allowing high accuracy of drug dosage and ease of administration; however they
undergo a fast sol-gel transition at the temperature of the deposition site, so that the
increased viscosity of the resulting mucoadhesive gel system gives rise to an extended in
situ residence time.
On the basis of these premises, the aim of this research work was the development of
mucoadhesive, thermosensitive in situ gels for nasal delivery of opiorphin. With this
purpose, the potential of a series of combinations of different mucoadhesive polymers
(i.e. chitosan, hydroxypropylmethylcellulose, Poloxamer, Carbopol) in forming solutions
able to rapidly gel at the temperature of the nasal cavity has been investigated. The best
formulations were further characterized for gel strength and mucoadhesion properties.
Moreover, it was considered necessary to modify the previously developed
opiorphin liposomal formulation [6], in order to increase the loaded drug amount and
obtain a more concentrated formulation, suitable to the lower volume administrable via
the nasal route. The new opiorphin liposomal formulation, characterized for vesicle size,
homogeneity, Zeta potential and entrapment efficiency, was loaded into the selected in
situ gel formulation and tested ex-vivo for drug permeation properties, using excised
nasal porcine mucosa, compared to a free peptide-loaded gel.

2. Materials and methods

2.1. Materials

4
 Cross-linked lipid polyethylene glycol 2000–distearoyl-phosphatidylethanolamine
(PEG (2000)–DSPE) and cholesterol were from Avanti Polar Lipids Inc) (Alabaster, AL,
USA). Egg L--phosphatidylcholine was from Fluka (Neu-Ulm, Germany), stearylamine
from Merck GmBH (Germany), chitosan hydrochloride from Protasan (Germany),
hydroxypropylmethylcellulose K100M (HPMC) from Colorcon GmBH (Germany),
Poloxamer 407 (MW 40000) (P407) and Poloxamer 188 (MW 1800) (P188) from BASF
(Ludwigshafen, Germany), beta-glycerophosphate disodium salt pentahydrate (GP)
from Sigma Aldrich (Milan, Italy), Carbopol 934P from Lubrizol (Cleveland, Ohio
USA). The amino acid building blocks, resin and reagents used for opiorphin (OPI)
synthesis were from Bachem AG (Switzerland). All other chemicals and solvents were of
analytical grade.

2.2. Synthesis of opiorphin (OPI)

Opiorphin (OPI), a five-amino acid polypeptide (Glutamine-Arginine-

Phenylalanine-Serine-Arginine), was synthesized by using Nα-Fmoc solid-phase

methodology on preloaded H-Arg(Pbf)-2-chlorotrityl resin (200–400 mesh, loading 0.37
mmol/g, 1 eq, 1 g) according to the method previously developed [6]. The identity and
purity of the synthesized peptide was determined by HPLC-DAD-ESI MS analysis, using
an HP 1100 Liquid chromatograph (Agilent Technologies, CA, USA) endowed with a
HP1040 DAD and connected to a mass spectrometer endowed with API-ES
(Atmosphere-Pressure-Ionization-ElectroSpray) (Agilent Technologies, CA, USA). The
analyses demonstrated that the synthetic procedure utilized enabled the obtainment of the
peptide with a high purity, around 98%. The final yield was about 60%.

2.3. Preparation and characterization of opiorphin-loaded liposomes

PEGylated liposomes were prepared by the thin layer evaporation method, according
to the previously developed procedure [6], by keeping constant the types and the
respective molar ratios of the lipid phase components, but varying their total amounts.
Briefly, the desired amounts of phosphatidylcholine, cholesterol, and stearylamine were
dissolved in chloroform in a round bottom flask and added with PEG (2000)–DSPE. The
solvent was then removed under reduced pressure in a rotary evaporator. The dry lipid
film obtained was hydrated, under stirring, by adding 5 mL of NaCl 0.9% w/v solution
containing the drug (16.5 mg/mL). The system was then subjected to 3 cycles of
vortexing (1 min at 30 Hz) and heating (5 min at 58 °C) and finally sonicated (3 min at

5
30 W) in an ice bath. All samples were stored at 4°C in sealed containers under light
protection.
The liposomal formulations were characterized for mean vesicle size, polydispersity
index (PDI) and Zeta potential by Photon Correlation Spectroscopy (Malvern ZetaSizer
Nano ZS90, Malvern, UK) at 25 ±0.1°C. Before analysis, each liposomal dispersion was
suitably diluted with NaCl 0.9% w/v, to avoid multiscattering phenomena. Six
independent samples were taken from each batch and measured for both vesicle size and
Zeta potential.
The drug entrapment efficiency (EE%) was determined by a direct method, after
separation, by size exclusion chromatography, of loaded liposomes from free OPI.
Purified liposomes were then destroyed by methanol addition; after 1 min sonication, the
sample was centrifuged and the OPI concentration in the supernatant assayed by HPLC
analysis, as described below. The entrapment efficiency (EE%) was calculated by the
following equation:
EE%=(WL/WT) x100.
where WL is the amount of OPI actually loaded into the vesicle, and WT the OPI amount
initially added during the batch preparation
Each result is the mean of three separate experiments.

2.4. HPLC assay of opiorphin (OPI)

OPI was assayed by HPLC (Merck Hitachi Elite LaChrom chromatograph,
Darmstadt, Germany, equipped with UV-vis detector L-2400), according to a previously
developed method [6]. The stationary phase was a Thermo-ScientificTM Hypersil BDS
C18 column (100×4.6 mm, 2.4 μm). The mobile phase was: (A) water added with 0.05%
v/v trifluoroacetic acid; (B) CH3CN added with 0.05% v/v trifluoroacetic acid. The

analysis was carried out under gradient: 0-9 min, A% (v/v) 95–85; B% (v/v) 5–15. The
injection volume was 20 μL, the flow rate 1 mL/min. In these conditions The OPI
retention time was 7.6±0.2 min. The method was validated for linearity (r2=0.999), LOQ
(1.14 μg/mL) and LOD (0.34 μg/mL). Adequate controls have been performed to verify
the absence of interference of the components of the liposomal carrier.

2.5. Storage stability studies of liposomal formulations

Stability of OPI-loaded PEGylated liposomal dispersions, as such or added with the
components of the gel, was checked for 6 months. The colloidal dispersions were kept at

6
4 °C in sealed containers under light protection. At given time intervals, aliquots were
withdrawn and examined for mean size, PDI, Zeta potential and EE%.

2.6. Preparation of nasal thermosensitive gels

Temperature-sensitive in situ gelling systems were prepared using the cold
procedure, by combining various mucoadhesive polymers in different percentages and
under different experimental conditions, depending on the polymers used.
Method 1: chitosan.HCl (1% w/v) and HPMC (0-0.7 % w/v) were dissolved under
stirring in a 0.5 % w/v NaCl solution placed in a ice-bath at 4 °C; a saline ß-GP solution
(final concentration 0.282-0.420 M) was then added drop-wise under continuous stirring
to the resulting solution (see Table 1 for formulations composition).
Method 2: P407 (15-30 % w/v) was dissolved under stirring in water at 4 °C; HPMC
(1 % w/v) or Carbopol 934P (0.5-1% w/v), in the presence or not of an additional third
component, were then slowly added at different concentrations, under continuous
stirring, to the resulting solution (see Tables 2 and 3, respectively, for formulations
composition).

2.7. Characterization of thermosensitive gels

2.7.1. Determination of gelation temperature

Gelation temperature was determined by pouring 5 mL of each formulation in a 20-
mL beaker containing a magnetic bar and placing it on a hot plate. A thermometer was
immersed in the liquid formulation, which was heated at a rate of 1 °C/min with constant
stirring at 30 rpm. When the bar stopped moving due to gelation, the temperature was
recorded as the gelation temperature [19]. The results presented are the mean of 4
separate measurements.

2.7.2. Determination of gelation time

The gelation time was determined according to the method of Yuan et al. [20],
slightly modified. Briefly, a 20 mL vial containing a magnetic bar and 5 mL of each
formulation was immersed in a thermostated bath at 34 °C, with a constant stirring at 20
rpm. The time at which the magnetic bar stopped moving due to gelation was recorded as
the gelation time. The results presented are the mean of 4 separate measurements.

2.7.3. Gel strength determination

7
 The gel strength was evaluated according to the technique described by Yong et al.
[21]. Briefly, a sample (50 g) of each formulation was transferred into a 100-mL
graduated cylinder and allowed to jellify in a thermostated water bath at 34 °C. A weight
of 35 g was then positioned on the gelled solution. The gel strength, indicative of the gel
viscosity at the nasal physiological temperature, was measured as the time taken by the
weight to penetrate 5 cm deep into the gel. The results presented are the mean of 4
separate measurements.

2.7.4. Evaluation of gel mucoadhesive strength

Formulations with thermo-gelling properties considered suitable for the purpose of
the work were further characterized for mucoadhesive properties using a tensile test
based on a slightly modified version of the two-arms balance method [22]. Nasal porcine
mucosa, obtained from a local slaughterhouse, was used as a model mucoadhesive tissue.
Sections of freshly excised nasal porcine mucosa (area 4 cm2) were mounted on proper
tissue holders with the mucosal side up. A fixed amount of each formulation, previously
gelled by temperature increase, was interposed between two separate tissue holders. One
holder side was attached to one of the two-arms balance; the other one was connected to
a mobile support, which was carefully lifted up to counterbalance the weight of a
container attached to the balance second-arm. After a pre-set contact time (2 min), water
was added into the container at a constant rate of 50 mg/s by a peristaltic pump. The
water amount needed to detach the two surfaces, expressed as g/cm2, was recorded as
mucoadhesion strength. The results presented are the mean of 4 separate measurements.

2.7.5. Determination of mucoadhesion time

The time of mucoadhesion of the gel formulation to the nasal porcine mucosa was
determined according to the method of Khan et al. [23], slightly modified. Briefly, a
sample (2 g) of each examined formulation, added with 0.1% methylene blue, was left to
gel in a thermostated water bath at 34 °C and then put, by applying with the fingertip a
light pressure for 30 s, on a section of porcine nasal mucosa (obtained from a local
slaughterhouse), previously attached over a plastic plate. This set was placed at an angle
of 40° in a chamber thermostated at 34 °C and subjected to a continuous flux (5 mL/min)
of pH 6.3 simulated nasal fluid (SNF) (aqueous solution containing 8.77 mg/mL NaCl,
2.98 mg/mL KCl and 0.59 mg/mL CaCl2) [24], in order to mimic the physiological
conditions at the nasal cavity. The time required for complete washing of the

8
formulation, detected on the basis of the presence of colour, was measured as the
mucoadhesion time. The test was repeated 4 times for each formulation.

2.8. Preparation of nasal thermosensitive gels containing OPI-loaded liposomes

The method was the same as for blank gel preparation. In this case the polymers
(P407 and Carbopol 934P) were slowly added in sequence, under continuous stirring, to
the drug-loaded liposomal dispersion, kept at 4 °C. A gel containing a free-OPI aqueous
solution was also prepared for comparison purposes. The formulations were stored at 4°C
until use.

2.9. Ex vivo permeation studies

Ex vivo permeation studies were carried out through excised porcine nasal mucosa,
by using vertical Franz diffusion cells (Rofarma, Gaggiano, Italy). Porcine snouts were
obtained from a local slaughterhouse within 10 min of the animal killing. The snouts
were stored on ice during transportation to the laboratory, where they were opened and
the septum wall was fully exposed by a longitudinal incision through the lateral wall of
the nose. Then, using forceps and scalpels, the mucosa was carefully removed, cut into
appropriately sized pieces and mounted in Franz diffusion cells with the anterior surface
faced towards the donor compartment (effective diffusion area 1.27 cm2). A fixed
amount (100 L) of gel loaded with plain OPI or liposomal OPI was placed in the donor
compartment. The acceptor medium, maintained at 37 °C and kept under gentle agitation
with a magnetic bar at 50 rpm, consisted of 7 mL of pH 7.4 phosphate buffer. At
predetermined time intervals, 0.5 mL samples were withdrawn from the receiving
chamber and the drug concentration was assayed by HPLC, as described in section 2.4. A
correction for the cumulative dilution due to the sample replacement with an equal
volume of fresh medium was calculated. Experiments were carried out in sextuple for
each formulation. The cumulative amount of drug permeated into the receptor
compartment was determined and the results were averaged. The area under the drug
permeation curve (AUC) was calculated using the trapezoidal rule. The drug apparent
permeability coefficient was calculated according to the equation:
dQ_
Papp =
dtACo

where dQ/dt is the cumulative amount of drug permeated vs. time per unit of area, A the
effective surface area and Co the OPI initial concentration in the donor compartment.

9
2.10. Statistical analysis.
Statistical comparisons were made by one-way analysis of variance (ANOVA)
followed by the Student-Neuwman Keuls multiple comparison post-test (GraphPad
Prism, version 6). A minimum level of significance of p < 0.05 was used.

3. Results and discussion

3.1. Preparation and evaluation of thermo-sensitive gels

The first step of this research work was the preparation of thermo-responsive gels
having a sol-gel transition temperature around 32-34°C (the temperature of the nasal
cavity), and capable of a very rapid in situ gelation upon contact with the nasal mucosa.
With this aim we tested a series of mucoadhesive polymers in different combinations.
Chitosan, a cationic polysaccharide composed of randomly distributed (1→4) linked D-
glucosamine and N-acetyl-D-glucosamine units, has attracted great interest in the
pharmaceutical field, owing to its several beneficial biological properties, such as
biocompatibility, biodegradability, mucoadhesivity, absence of toxicity and low-
immunogenic power [25]. In particular, chitosan hydrogels proved to be able to improve
the intranasal drug absorption owing to their excellent mucoadhesive properties and their
ability to reversibly open the tight junctions and thus improve drug permeation through
biological membranes [7,26]. Interestingly, cationic chitosan solutions can be
transformed into thermo-sensitive gel-forming systems by addition of β-GP [18]. The
pharmaceutical and biomedical applications of chitosan/glycerophosphate-based
hydrogel formulations, which are liquid at room temperature and turn into semi-solid
hydrogels at the body temperature, have been recently reviewed [27,28]. The gelation
mechanism may involve multiple interactions, such as electrostatic forces between
chitosan ammonium groups and β-GP phosphate group, enhanced hydrophobic
interactions and increased hydrogen bonding between polymer chains, owing to the
reduced electrostatic repulsion after chitosan solution neutralization by β-GP [28].
A first series of gel formulations was then investigated, based on chitosan.HCl-β-GP
combinations, in the presence or not of HPMC. This cellulose derivative was selected not
only for its desirable mucoadhesive properties, which can further concur to prolong the
drug residence time in the nasal cavity and sustain its release, but also for its ability to
interact, via hydrogen bond formation, with the chitosan chains, acting as a cross-linking
agent, thus favoring the gelling process [23]. The composition of the different tested

10
formulations, and the corresponding gelation times at 34 °C, are reported in Table 1.
All the formulations appeared as clear solutions and exhibited gelation temperature
values within the range 32-34 °C, suitable for thermo-reversible nasal gel formulations.
However, the gelation time in the absence of HPMC (G1) was very long, reaching 30
min, thus proving the important role played by HPMC for a fast and efficient gelling
process. In fact, the addition of 0.5 % w/v HPMC (G2) enabled to strengthen the gel
network, and the gelation time fell sharply to 5 min. Nevertheless, a new increase of
HPMC concentration up to 0.7 % w/v, (G3) did not further reduce the gelation time. An
additional increase of HPMC amount was considered not appropriate, due to its very high
viscosity.
The gelation time decreased with increasing ß-GP concentration, as observed also by
other authors [29]. The G6 formulation, containing the highest ß-GP amount consistent
with its solubility in the medium and 0.7% w/v HPMC, gave the best result, exhibiting a
gelation time of 1.5 min. However, also this gelation time was considered still too long
for an effective in situ gel nasal formulation, since gelation times around 10 s are
generally considered as optimal [30].
A new series of gel formulations was then investigated, based on Poloxamer 407
(P407) as principal component. Poloxamers are amphiphilic block copolymers of ABA
type, composed by PEO (A) and PPO (B) units [31]. They have thermo-reversible
properties resulting of particular interest for the design of drug delivery systems to
mucous membranes. In fact, due to their low critical temperature solution, they display a
sol-gel transition at body temperature, causing in situ gelation at the site of interest [7].
This behavior is explained by the PPO blocks dehydration, occurring as the temperature
increases, which causes aggregation of the copolymer molecules into spherical micelles,
with the PPO blocks oriented towards the core and the PEO chains arranged in the outer
shell; above a critical concentration, micelles come into contact forming multi-molecular
aggregates, thus leading to gel formation [32]. Among the different Poloxamers available
on the market, P407 is of particular interest, owing to its bioadhesion properties, good
tolerability, low toxicity, almost complete absence of irritation or sensitivity phenomena
towards skin and mucosa, which make it an useful and safe excipient in topical, rectal,
ocular and nasal formulations [33].
Combinations of P407 and mucoadhesive polymers have been found to be more
advantageous than the use of P407 alone, since such combinations tend to reduce the
gelation temperature of P407, so that the polymer mixture can gel within the temperature

11
range (32-34 °C) observed in the nasal mucosa [34].
A first series of formulations was investigated using P407 in combination with
HPMC, in the presence or not of a third polymer, such as P188 or Carbopol 934P, to
modulate the gelation time. The composition of the different formulations and the related
gelation times at 34 °C are reported in Table 2.
The binary combination of P407 with 1% HPMC resulted in a very viscous solution
which gelled after 60 s at 34 °C. The addition of 1% Carbopol further increased the
viscosity of the system, which, however, did not gel even after 30 min at 34 °C. To
explain this finding, it can be hypothesized that the combined presence of HPMC and
Carbopol gave rise to an entangled network where the interactions among their polymeric
chains hindered the movement and packing of P407 micelles, thus compromising the
gelation process. On the contrary, the addition of P188 (at 10 or 5 % w/v) markedly
reduced the gelation temperature, giving rise to an almost immediate gelation, even at
room temperature.
Considering the unsatisfactory results obtained, as well as the high viscosity of the
systems containing HPMC, which may hamper their nasal administrations, no further
formulations based on P407-HPMC mixtures were considered. Therefore, a new series of
formulations based on P407-Carbopol combinations, was examined (Table 3).
Polyacrylates, including Carbopol, have been widely investigated as components of
many drug delivery systems intended for different administration routes, including the
nasal one [15], because of their excellent mucoadhesive and gel-forming properties. They
may also be able to temporarily open the tight junctions between epithelial cells, during
the swelling process in the nasal cavity, and thus improve the paracellular absorption of
drugs [35]. Moreover, the use of Carbopol as component of the OPI intranasal gel
formulation could be of special interest, considering its ability to enhance the intranasal
bioavailability of hydrophilic macromolecular drugs and to increase the stability of
peptidic drugs by inhibition of proteolytic enzymes [12].
As can be seen in Table 3, the series of P407-Carbopol binary combinations
exhibited a progressive decrease in gelation time with increasing the P407 content in the
formulation, going down from 60 s (G10) up to 0 s (G15 and G16). No variations of
gelation time were instead observed with increasing the Carbopol concentration from 0.5
to 1% w/v, by keeping the P407 amount constant (G13 vs G14, or G15 vs G16).
Considering the interesting results provided by the series of P407-Carbopol binary
combinations in terms of gelation time, as well as the possibility for an ease modulation

12
of such parameter by suitably varying the P407 percentage, more in depth investigations
were performed for their better characterization. The results of these studies are
summarized in Fig. 1.
Values of gelation temperature were in the range 31-35 °C, which can be considered
appropriate for thermo-reversible nasal gel formulations, as the temperature of the nasal
cavity is approximately 32-34°C [34]. Gelation temperatures above 35 °C can result in
loss or drainage of drug from the nasal cavity, while temperatures lower than 30 °C could
cause problems of early gelling before or during administration, leading to difficulty in
handling, and hampering an ease administration of the correct drug dose.
Mucoadhesion strength is a fundamental parameter for the development of a
successful in situ gelling nasal formulation: the stronger the mucoadhesion, the longer
the residence time of the formulation in the nasal cavity. However, too strong
mucoadhesion phenomena can damage or irritate the mucosal membrane. In case of
P407-Carbopol gels, the mucoadhesion strength increased with increasing P407 and,
even more, Carbopol concentration. The series of G13-G16 formulations showed
satisfying mucoadhesion strength, ranging from 22 to 30 g/cm2.
The duration of mucoadhesion increased with increasing the content of both
polymers, but, also in this case, the contribution of Carbopol was clearly more evident.
For example an increase of 0.5 % Carbopol, being the P407 amount kept constant (G13
vs G14), gave rise to a 28 % increase of mucoadhesion duration. This effect could be
attributed to the cross-linked nature of Carbopol polymer, whose very high number of
carboxylic groups can interact, via hydrogen bonding, with the hydroxyl groups of the
oligosaccharide chains present on the mucosal surface, leading to an increase in
mucoadhesion strength and duration [19]. G13-G16 formulations showed adequate
duration of mucoadhesion, ranging from 4.3 to 6 h.
Gel strength is another important parameter to consider in the development of
mucoadhesive nasal formulation. In fact, optimal in situ nasal gel formulations should
have adequate strength to be easily administered and retained in situ, limiting post nasal
drip or anterior leakage phenomena. Formulations with gel strength values lower than 25
s could not maintain their integrity for sufficient time and may rapidly erode; on the
contrary, those with strength values greater than 50 s are too stiff and may cause
discomfort to the mucosal surface and patient non-compliance [21]. As observed for both
mucoadhesive strength and duration, also the gel strength increased with increasing
concentration of gelling and bioadhesive polymers. Anyway, all the examined

13
formulations (excepted G10 and G16) exhibited acceptable gel strength values ranged
between 29-44 s.
Formulations G13 and G14 showed overall the best properties in terms of proper
gelation time (10 s), adequate mucoadhesive and gel strength (22 and 27 g/cm2, and 38
and 44 s, respectively) and mucoadhesion duration (4.3 and 5.5 h, respectively).
The G14 formulation was finally selected, in virtue of its higher Carbopol content
(1% w/v), considering the enhancer properties, and inhibition activity against proteolytic
enzymes of this polymer [12], which could be useful to increase the stability and
bioavailability of the peptidic drug.

3.2. Preparation and characterization of opiorphin-loaded liposomes

Previous studies showed that formulation of OPI in PEGylated liposomes allowed to
enhance the stability of the drug and to improve intensity and duration of its pain killing
effect after intravenous administration in rats, compared not only to a free drug solution,
but also to a classic OPI liposomal formulation [6]. Moreover, it has been shown that
liposomal formulations can be effective in enhancing the nasal absorption of peptides by
increasing their membrane penetration [36,37]. This has been attributed to both increased
nasal retention [36] and protection of the entrapped peptides from enzymatic degradation
[38].
Accordingly, in the second step of this study we developed an OPI PEGylated-
liposomal formulation suitable to be nasally administered by means of the selected in situ
thermoresponsive gel G14. Considering the lower volume administrable via the nasal
route (150 µL for human subjects [7]) than the intravenous one, it was necessary to
modify the previously developed OPI liposomal formulation (containing 1 mg/mL of
drug), in order to obtain a more concentrated formulation.
It has been reported that the liposome entrapment efficiency towards hydrophilic
drugs generally increases with increasing the lipid concentration [39,40]. This has been
attributed to the larger number of vesicles present per unit of volume, and, consequently,
the larger total internal volume available for dug encapsulation.
Therefore, to evaluate the effect of liposome lipid concentration on the OPI
entrapment efficiency, four different liposomal formulations were prepared, by keeping
the types of components (phosphatidylcholine, cholesterol and stearylamine) and their
respective molar ratios (69:13:18) the same as in the previous study [6], but gradually

14
increasing the total lipid amount, and using a fixed drug concentration in the aqueous
hydrating phase of 16.5 mg/mL.
As shown in Fig. 2, an increase in total lipid concentration from 20.6 to 68.3 mM
gave rise to a significant increase in the drug EE%, which raised from 38% up to about
60 %. Nevertheless, a saturation of this effect was observed for lipid concentrations
higher than 70 mM. This might be explained by the increased viscosity of the medium,
which could hamper the free movement of the drug molecules into the inner cavity of
liposomes [40,41].
On the other hand, as observed by other authors [39,42,43], the increase in drug
concentration (from 1 to 16.5 mg/mL) resulted in a decrease in the EE% with respect to
the previous liposomal formulation [6], even though the amount of entrapped drug
increased up to about 11 times.
Based on these results, the formulation with total lipid concentration of 68.3 mM and
entrapment efficiency around 60% (corresponding to a peptide loading of about 18 %
w/w) was chosen for incorporation in the thermosensitive gel G14, and further
characterized in terms of vesicle size, homogeneity, Zeta potential, and stability under
storage.
As can be seen in Table 4, the optimized PEGylated liposomal OPI formulation
showed nanometric mean size, suitable for nasal delivery. In fact the importance of the
nano-dimensions of the particles in the effectiveness of nasal delivery of drugs, has been
proved [44, 45]. Moreover, the vesicles exhibited high homogeneity, as proved by the
low value of polydispersity index, and good physico-chemical stability in time. In fact,
despite their low Z-potential, the vesicles maintained almost unchanged values (P<0.05)
of mean dimensions and PDI during 6 month storage at 4 °C, probably in virtue of the
steric hindrance effect of the PEG chains which hindered aggregation phenomena.
Moreover, the almost unchanged EE% values (P<0.05) also proved the chemical stability
of the peptide, indicating the absence of degradation phenomena. Furthermore, as shown
in the same Table 4, addition of the P407-Carbopol mixture (G14) to the OPI-loaded
optimized PEGylated liposomal formulation did not significantly modify (P<0.05) the
properties of the vesicles in terms of mean size, PDI, Zeta potential and entrapment
efficiency of the vesicles. Furthermore, no significant variations (P<0.05) of all these
parameters was observed at the end of the 6 month storage period at 4 °C, indicating that
the gel formulation did not affect the stability of the liposomal dispersion.

15
 On the other hand, the presence of the liposomal dispersion did not influence the
overall behavior of the thermosensitive system. In fact, the liposomal OPI-loaded gel
exhibited gelation time and temperature similar to those of the blank gel, and only a
small decrease in gel strength (40 rather than 44 s), mucoadhesive strength (24 rather
than 27 g/cm2) and mucoadhesion duration (5.0 rather than 5.5 h) was observed.
The liposomal OPI-loaded gel G14) was then subjected to ex-vivo permeation
studies through excised nasal porcine mucosa, in comparison with the same gel
formulation containing the plain drug. The results of these studies are collected in Table
5, in terms of AUC, apparent permeability coefficient and total permeated amount of
OPI, while the drug permeation profiles are shown in Fig 3. As can be seen, in both cases
an almost linear permeation profile of OPI was observed, indicating that the developed
gel formulation was actually able to allow a controlled and prolonged delivery of the
drug for more than 5 h. However, interestingly, the gel loaded with the plain drug
presented an initial lag time of about 2 h, which can be attributed to the poor tendency of
the hydrophilic drug to escape from the hydrogel and permeate through the lipophilic
nasal mucosa. On the contrary this effect was not observed in the case of the gel
containing liposomal OPI. Evidently, the entrapment of the hydrophilic drug into the
amphiphilic vesicles strongly improved its permeation through the lipophilic membrane.
In fact, as shown in Table 5, the apparent permeability coefficient of liposomal OPI was
about 6 times higher than that from the gel formulation containing the free drug aqueous
solution. The greater permeability of liposomal OPI reflected in an around 60 times AUC
increase and in a greater permeation rate through the mucosal membrane, reaching about
60 % of diffused drug after 5 h, vs the about 10 % obtained with the gel loaded with the
plain drug. The ability of liposomes to enhance nasal absorption of peptides such as
calcitonin [46] and insulin [47] has been previously described and mainly explained with
enhanced membrane penetration, increased nasal retention and protection of the
entrapped peptides from enzymatic degradation. In this case, the improvement in OPI
permeability obtained with its PEGylated liposomal formulation could be also reasonably
attributed to possible mucopenetrative properties of PEGylated liposomes as well as to
the nanometric size of the vesicles. In fact, this is in agreement with the reported greater
ability of PEG-coated PLA nanoparticles, than the non-coated ones, in significantly
improving drug transport through nasal epithelia [44], and with the inverse relation found
between absorption extent of the encapsulated drug and size of the particles [45].

16
Moreover, the high stability of PEGylated liposomes against aggregation phenomena can
further contribute to this result, assuring the maintenance of nano-sized vesicles.
Therefore, the overall results highlighted that in addition to the important role in
protecting the peptide from enzymatic degradation, thus increasing is therapeutic efficacy
after i.v. administration, as emerged in the previous study [6], the PEGylated liposomal
carrier was also able to strongly enhance OPI permeation properties through the nasal
mucosa.

4. Conclusions
In the present study a nasal delivery system of OPI was successfully developed in
the form of a liposomal thermosensitive mucoadhesive in situ-gelling hydrogel.
The optimized hydrogel formulation was based on a P407 (26.5%)-Carbopol (1%)
combination, which presented the best compromise in terms of properly short gelation
time at 34 °C, i.e. the nasal cavity temperature (10 s), suitable gelation temperature (33.7
°C), right gel strength, good mucoadhesive properties and long mucoadhesion duration,
so that to enable an easy drug administration as liquid formulation and, at the same time,
assure a prolonged in situ residence, as a consequence of the rapid gelation process, thus
favoring the drug absorption.
OPI was formulated as PEGylated liposomal dispersion to improve its stability and
reduce problems of rapid enzymatic degradation [6]. The concentrated formulation of
liposomal OPI, purposely developed considering the small liquid volume administrable
via nasal route, showed high entrapment efficiency (near to 60%), nanometric vesicle
size (around 140-150 nm), high homogeneity and good stability after 6 month storage at
4 °C. These properties remained almost unchanged after addition of the components of
the thermo-sensitive gel to obtain the final formulation.
Ex vivo permeation experiments through excised porcine nasal mucosa showed that
the developed hydrogel formulation was actually able to provide a sustained and
controlled delivery of the drug over more than 5 h, and highlighted the fundamental role
of the liposomal carrier in enhancing up to 6 times the permeability coefficient and the
permeation rate of the peptide through the lipophilic nasal mucosa.
Further studies will be necessary to elucidate the possible pathways and mechanism
involved in brain delivery via the intranasal route of OPI as PEGylated liposomal
dispersion, i.e. a direct nose-to-brain-delivery through the olfactory epithelium,
bypassing the blood-brain barrier (BBB), and/or a systemic delivery across the BBB,

17
through the nasal epithelium. This last route, which would appear more likely, based on
the results of the permeation tests using excised nasal mucosa, should be equally suitable,
considering the proved ability of OPI to cross the BBB [48].

References

[1] A. Wisner, E. Dufour, M. Messaoudi, A. Nejdi, A. Marcel, M.N. Ungeheuer, C.

Rougeot, Human Opiorphin, a natural antinociceptive modulator of opioid-
dependent pathways, Proc. Natl. Acad. Sci. USA 103 (2006) 17979-17984.
[2] X. Tian, J. Chen, W. Xiong, T. He, Q. Chen, Effects and underlying mechanisms of
human opiorphin on colonic motility and nociception in mice, Peptides 30 (2009)
1348-1355.
[3] C. Rougeot, Opiorphin peptide derivatives as potent inhibitors of enkephalin-
degrading ectopeptidases. Patent WO (2009) 124948A1.
[4] C. Rougeot, F. Robert, L. Menz, J.F. Bisson, M. Messaoudi, Systemically active
human opiorphin is a potent yet non-addictive analgesic without drug tolerance
effects, J. Physiol. Pharmacol. 61 (2010) 483-490.
[5] P. Popik, E. Kamysz, J. Kreczko, M. Wròbel, Human opiorphin: the lack of
physiological dependence, tolerance to antinociceptive effects and abuse liability in
laboratory mice, Behav. Brain Res. 213 (2010) 88-93.
[6] N. Mennini, P. Mura, C. Nativi, B. Richichi, L. Di Cesare Mannelli, C. Ghelardini,
Injectable liposomal formulations of opiorphin as a new therapeutic strategy in pain
management, Future Sci OA, 1 (3) (2015) DOI 10.4155.
[7] L. Illum, Nasal drug delivery: possibility, problems and solutions, J. Control.
Release 87 (2003) 187-198. 

[8] C. Campbell, B.H. Morimoto, D. Nenciu, A.W. Fox, Drug development of
intranasally delivered peptides, Ther. Delivery 3 (2012) 557-568.
[9] M.U. Ghori, M.H. Mahdi, A.M. Smith, B.R. Conway, Nasal Drug Delivery
systems: an overview, Amer. J. Pharmacol. Sci. 3 (2015) 110-119.
[10] J.J. Lochhead, R.G. Thorne, Intranasal delivery of biologics to the central nervous
system, Adv. Drug Deliv. Rev. 64 (2012) 614-628.
[11] F.W. Merkus, J.C. Verhoef, N.G. Schipper, E. Marttin, Nasal mucociliary
clearance as a factor in nasal drug delivery, Adv. Drug Deliv. Rev. 29 (1998) 13-
38.
[12] M.I. Ugwoke, R.U. Agu, N. Verbeke, R. Kinget, Nasal mucoadhesive drug
delivery: background, applications, trends and future perspectives, Adv. Drug
Deliv. Rev. 57 (2005) 1640-1665.
[13] L. Jiang, L. Gao, L. Tang, J. Ma, The application of mucoadhesive polymers in
nasal drug delivery, Drug Dev. Ind. Pharm. 36 (2010) 323-336.
[14] C. Callens, J.P. Remon, Evaluation of starch-maltodextrin-Carbopol 974 P
mixtures for the nasal delivery of insulin in rabbits, J. Control. Release 66 (2000)
215–220. 

[15] C. Tas, C.K. Ozkan, A. Savaser, Y. Ozkan, U. Tasdemir, H. Altunay, Nasal
absorption of metoclopramide from different Carbopol 981 based formulations: In
vitro, ex vivo and in vivo evaluation, Eur. J. Pharm. Biopharm. 64 (2006) 246-254.

18
[16] U.C. Galgatte, A.B. Kumbhar, P.D. Chaudari, Development of in situ gel for nasal
delivery: design, optimization, in vitro and in vivo evaluation, Drug Deliv. 21
(2014) 62-73. 

[17] B. Jeong, S.W. Kim, Y.H. Bae, Thermosensitive sol-gel reversible hydrogels, Adv.
Drug Deliv. Rev. 54 (2002) 37-51.
[18] E. Ruel-Gariépy, J.C. Léroux, In situ-forming hydrogels - review of temperature-
sensitive systems, Eur. J. Pharm. Biopharm. 58 (2004) 409-426.
[19] H.K. Choi, J.H. Jung, J.M. Ryu, S.J. Yoon, Y.K. Oh, C.K. Kim, Development of
in-situ gelling and mucoadhesive acetaminophen liquid suppository, Int. J. Pharm.
165 (1998) 33-44.
[20] Y. Yuan, C. Ying, Z. Li, Z. Hui-ping, G. Yi-Sha, Z. Bo, H. Xia, Z. Ling, W. Xiao-
hui, C. Li, Thermosensitive and mucoadhesive in situ gel based on poloxamer as
new carrier for rectal administration of nimesulide, Int. J. Pharm. 430 (2012) 114-
118.
[21] C.S. Yong, J.S. Choi, Q.Z. Quan, J.D. Rhee, C.K. Kim, S.J. Lim, K.M Kim., P.S.
Oh, H.G. Choi, Effect of sodium chloride on gelation temperature, gel strength and
bioadhesive force of poloxamer gels containing diclofenac sodium, Int. J. Pharm.
226 (2001) 195-205. 

[22] P. Mura, N. Mennini, I. Kosalec, S. Furlanetto, S. Orlandini, M. Jug, Amidated
pectin-based wafers for econazole buccal delivery: Formulation optimization and
antimicrobial efficacy estimation, Carbohydr. Polym. 121 (2015) 231-240.
[23] S. Khan, K. Patil, N. Bobade, P. Yeole, R. Gaikwad, Formulation of intranasal
mucoadhesive temperature-mediated in situ gel containing ropinirole and
evaluation of brain targeting efficiency in rats, J. Drug Target. 18 (2010) 223-234.
[24] A. Martinac, J. Filipovic-Grcic, D. Voinovich, B. Perissutti, E. Franceschinis,
Development and bioadhesive properties of chitosan-ethylcellulose microspheres
for nasal delivery, Int. J. Pharm. 291 (2005) 69-77.
[25] M.N. Kumar, R.A. Muzzarelli, C. Muzzarelli, H. Sashiwa, A.J. Domb, Chitosan
chemistry and pharmaceutical perspectives, Chem. Rev. 104 (2004) 6017–6084.
[26] B. Luppi, F. Bigucci, T. Cerchiara, V. Zecchi, Chitosan-based hydrogels for nasal
drug delivery: from inserts to nanoparticles, Expert Opin. Drug Deliv. 7 (2010)
811-827
[27] S. Supper, N. Anton, N. Seidel, M. Riemenschnitter, C. Curdy, T. Vandamme,
Thermosensitive chitosan/glycerophosphate-based hydrogel and its derivatives in
pharmaceutical and biomedical applications, Expert Opin. Drug Deliv. 11 (2014)
249-267.
[28] H.Y. Zhou, L.J. Jiang, P.P. Cao, J.B. Li, X.G. Chen, Glycerophosphate-based
chitosan thermosensitive hydrogels and their biomedical applications, Carbohydr.
Polym. 117 (2015) 524-536.
[29] M.J. Ganji, A. Abdekhodaie, S.A. Ramazani, Gelation time and degradation rate of
chitosan-based injectable hydrogel, J. Solgel Sci. Technol. 42 (2007) 47-53.
[30] S.P. Sherafudeen, P.V. Vasantha, Development and evaluation of in situ nasal gel
formulations of loratadine, Res. Pharm. Sci. 10 (2015) 466-476.
[31] A.V. Kabanov, V.Y. Alakhov, Pluronic block copolymers in drug delivery: from
micellar nanocontainers to biological response modifiers. Crit. Rev. Ther. Drug
Carrier Syst. 19 (2002) 1-72.
[32] A. Cabana, A. Ait-Kadi, J. Juhasz, Study of the gelation process of polyethylene
oxide-polypropylene oxide-polyethylene oxide copolymer (poloxamer 407)
aqueous solutions, J. Colloid Interface Sci. 190 (1997) 307-312.
[33] G. Dumortier, J.L. Grossiord, F. Agnely, J.C. Chaumeil, A review of poloxamer
407 pharmaceutical and pharmacological characteristics, Pharm. Res. 23 (2006)

19
 2709-2728.
[34] N. Sharma, A. Sharma, P.K. Sharma, G. Garg, G.T. Kulkarni, Mucoadhesive
thermoreversible nasal delivery system, J. Pharm. Res. 3 (2010) 991-997.
[35] A. Shahiwala, A. Misra, Nasal delivery of levonorgestrel for contraception: An
experimental study in rats, Fertil. Steril. 81 (2004) 893-898.
[36] S.L. Law, K.J. Huang, V.H.Y. Chou, J.Y. Cherng, Enhancement of nasal
absorption of calcitonin loaded in liposomes, J. Lipos. Res. 11 (2001) 164-174.
[37] A.K. Jain, K.B. Chalasani, R.K. Khar, F.J. Ahmed, 
 P.V. Diwan, Muco-adhesive
multivesicular liposomes as an effective carrier for transmucosal insulin delivery, J.
Drug Target. 15 (2007) 417-427.
[38] Y. Kato, T. Hosokawa, E. Hayakawa, K. Ito, Influence of liposomes on tryptic
digestion of insulin, Biol. Pharm. Bull. 16 (1993) 457-461.
[39] X. Xu, M.A. Khan, D.J. Burgess, A quality by design (QbD) case study on
liposomes containing hydrophilic API: I. Formulation, processing design and risk
assessment, Int. J. Pharm. 419 (2011) 52-59.
[40] S. Shariat, A. Badiee, M.R. Jaafar, S.A. Mortazavi, Optimization of a method to
prepare liposomes containing HER2/Neu- derived peptide as a vaccine delivery
system for breast cancer, Iran. J. Pharm. Res. 13 (2014) 15-25.
[41] E. Ducate, M. Brion, F. Lecomte, B. Evrard, G. Piel, The experimental design as a
practical approach to develop and optimize a formulation of peptide-loaded
liposomes, AAPS Pharm. Sci. Tech. 11 (2010) 966- 975.
[42] T. Schneider, A. Sachse, G. Rössling, M. Brandl, Generation of contrast-carrying
liposomes of defined size with a new continuous high pressure extrusion method,
Int. J. Pharm. 117 (1995) 1-12.
[43] X. Xu, M.A. Khan, D.J. Burgess, A quality by design (QbD) case study on
liposomes containing hydrophilic API: II. Screening of critical variables, and
establishment of design space at laboratory scale, Int. J. Pharm. 423 (2012) 543-
553.
[44] A. Vila, H. Gill, O. McCallion, M.J. Alonso Transport of PLA-PEG particles
across the nasal mucosa: effect of particle size and PEG coating density, J. Control.
Release 98 (2004) 231-244.
[45] A. Vila, A. Sanchez, C. Evora, I. Soriano, O. McCallion, M.J. Alonso, PLA-PEG
particles as nasal vaccine carriers: influence of the particle size, Int. J. Pharm 292
(2005) 43-52.
[46] S.L. Law, K.J. Huang, V.H.Y. Chou, J.Y. Cherng, Enhancement of nasal
absorption of calcitonin loaded in liposomes, J. Liposome Res. 11 (2001) 164-117
[47] A.K. Jain, K.B. Chalasani, R.K. Khar, F.J. Ahmed, P.V. Diwan, Muco-adhesive
multivesicular liposomes as an effective carrier for transmucosal insulin delivery, J.
Drug Target. 15 (2007) 417-427. 

[48] A. Bocsik, Z. Darula, G. Toth, M.A. Deli, M. Wollemann, Transfer of opiorphin
through a blood-brain barrier culture model, Arch. Med. Res. 46 (2015) 502-506.

20
Legend for Figures

Fig. 1. Gelation temperature, mucoadhesive strength, gel strength and mucoadhesion

duration of various thermosensitive mucoadhesive gel formulations (see Table 3 for gel
compositions). Data represent mean values ± s.d. (n=4).

Fig. 2. Effect of total lipid concentration variations on the entrapment efficiency (EE%)
of liposomes towards opiorphin (OPI). Data represent mean values ± s.d. (n=3).

Fig. 3. Ex vivo permeation profiles through excised nasal porcine mucosa of opiorphin
(OPI) from the selected thermosensitive mucoadhesive gel formulation G14 (see Table 3
for gel composition) loaded with liposomal () or free () peptide. Data represent mean
values ± s.d. (n=6).

21
22
23
24
25
Table 1
Composition and gelation time at 34 °C of formulations based on mixtures of
chitosan.HCl with β-glycerophosphate (β-GP) and HPMC
Batch Chitosan HCl β-GP HPMC gelation time (min)
code (%w/v) (%w/v) (%w/v) at 34 °C
G1 1 8.88 --- 30±1.0
G2 1 8.88 0.5 5.0±0.4
G3 1 8.88 0.7 5.0±0.5
G4 1 10.5 0.5 4.0±0.3
G5 1 13.2 0.5 2.0±0.2
G6 1 13.2 0.7 1.5±0.2

26
Table 2
Composition and gelation time at 34 °C of formulations based on mixtures of
P407 and HPMC, in the presence or not of an additional component.
Batch P407 HPMC other component gelation time (s)
code % w/v % w/v % w/v at 34 °C
G7 18 1 ---- 60±5
G8 18 1 Carbopol 1% no gel
G9 18 1 P188 5 % gel at R.T.

27
 Table 3
Composition and gelation time at 34 °C of
formulations based on mixtures of P407 and
Carbopol 934P.
Batch P407 Carbopol 934P gelation time
code % w/v % w/v (s) at 34 °C
G10 15 0.5% 60±4
G11 18 0.5% 30±3
G12 22 0.5% 20±2
G13 26.5 0.5 % 10±1
G14 26.5 1% 10±1
G15 30 0.5 % instantaneous
G16 30 1% instantaneous

28
Table 4
Properties of the optimized PEGylated liposomal opiorphin (OPI) formulation, as such
or loaded in the thermosensitive mucoadhesive gel formulation G14 (see Table 3 for gel
composition), freshly prepared or after 6 month storage at 4 °C.
OPI formulation
Mean size PDI -potential EE%
(nm) (mV)
freshly prepared 141 4 0.184 0.024 -0.62 0.18 59 3
liposomes in
solution after 6 months at 4°C 159 9 0.144 0.020 -0.83 0.23 57 3

liposomes in freshly prepared 143 3 0.169 ±0.018 -0.52 0.16 58 3

gel
after 6 months at 4°C 151 7 0.175 0.022 -0.59 0.19 56 3

29
Table 5
Area under curve (AUC0-5h), apparent permeability coefficient
(Papp) and total permeated amount across excised nasal porcine
mucosa of opiorphin (OPI) from the selected thermosensitive
mucoadhesive gel G14 (see Table 3 for gel composition) loaded
with free or liposomal peptide.
Sample AUC 0-5h Papp total permeated
(cm/min) amount (µg)
gel with free OPI 5.96 2.5.10-4 23±1
gel with liposomal OPI 353.3 14.3.10-4 127±6

30
Graphical abstract

31