http://informahealthcare.com/lprISSN: 0898-2104 (print), 1532-2394 (electronic)

J Liposome Res, Early Online: 1–9! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/08982104.2014.911315

RESEARCH ARTICLE

Liposomal lidocaine gel for topical use at the oral mucosa:characterization, in vitro assays and in vivo anesthetic efficacyin humans

Michelle Franz-Montan1, Daniela Baroni1, Giovana Brunetto2, Viviane Roberta Vieira Sobral2,Camila Morais Goncalves da Silva2, Paulo Venancio1, Patricia Wiziack Zago1, Cintia Maria Saia Cereda2,Maria Cristina Volpato1, Daniele Ribeiro de Araujo3, Eneida de Paula2, and Francisco Carlos Groppo1

1Department of Physiological Sciences, Piracicaba Dental School, University of Campinas – UNICAMP, Piracicaba, Sao Paulo, Brazil, 2Department of

Biochemistry, Institute of Biology, University of Campinas – UNICAMP, Campinas, Sao Paulo, Brazil, and 3Human and Natural Sciences Centre,

Federal University of ABC, Sao Paulo, Brazil

Abstract

Objective: To characterize liposomal-lidocaine formulations for topical use on oral mucosa andto compare their in vitro permeation and in vivo anesthetic efficacy with commercially availablelidocaine formulations.Materials and methods: Large unilamellar liposomes (400 nm) containing lidocaine wereprepared using phosphatidylcholine, cholesterol, and a-tocoferol (4:3:0.07, w:w:w) and werecharacterized in terms of membrane/water partition coefficient, encapsulation efficiency, size,polydispersity, zeta potential, and in vitro release. In vitro permeation across pig palatal mucosaand in vivo topical anesthetic efficacy on the palatal mucosa in healthy volunteers (double-blinded cross-over, placebo controlled study) were performed. The following formulations weretested: liposome-encapsulated 5% lidocaine (Liposome-Lido5); liposome-encapsulated 2.5%lidocaine (Liposome-Lido2.5); 5% lidocaine ointment (Xylocaina�), and eutectic mixture oflidocaine and prilocaine 2.5% (EMLA�).Results: The Liposome-Lido5 and EMLA showed the best in vitro permeation parameters(flux and permeability coefficient) in comparison with Xylocaina and placebo groups, as well asthe best in vivo topical anesthetic efficacy.Conclusion: We successfully developed and characterized a liposome encapsulated 5%lidocaine gel. It could be considered an option to other topical anesthetic agents for oralmucosa.

Keywords

Lidocaine, liposome, oral mucosa, topicalanesthesia

History

Received 14 January 2014Revised 27 March 2014Accepted 30 March 2014Published online 7 May 2014

Introduction

Anxiety and fear induced by pain are still associated with

dental treatment, and one of the most feared procedures is

local anesthesia (Armfield & Milgrom, 2011). The pain

during this procedure leads to the lack of cooperation by

patients, prolonged dental attendance time, unsuccessful/

repeated attempts, and additional pain (Taddio et al., 2005).

In long term, repeated painful procedures contribute to

conditioned anxiety responses and increased pain perception

(Meechan, 2002). In order to alleviate pain from needle

insertion and anesthetic injection, and reduce anxiety, topical

anesthesia is recommended in dental practice (Meechan,

2000, 2002).

Lidocaine (LDC) is an amine–amide local anesthetic with

moderate action and fast onset widely used in topical and

local anesthesia in Dentistry (Meechan, 2000, 2002).

Nevertheless, its efficacy in topical anesthesia is questionable

and the results are conflicting. It was demonstrated a high

incidence of inefficacy in reducing pain during: anesthetic

injection in the palatal mucosa (Bhalla et al., 2009; Hutchins

et al., 1997), intraligamentary injection (Meechan &

Thomason, 1999), and gingival probing (Donaldson &

Meechan, 1995).

The palatal mucosa is known for its difficulty in acquiring

adequate topical anesthesia (Meechan et al., 2005). This site

has a firmly attached keratinized tissue (which reduces tissue

distension), poor drug penetration through this highly

keratinized tissue, and a decreased tissue buffering capacity

(Primosch & Robinson, 1996). Therefore, a topical anes-

thetic able to eliminate pain from needle insertion and

Address for correspondence: Michelle Franz-Montan, Faculdade deOdontologia de Piracicaba, Universidade Estadual de Campinas, Av.Limeira, 901, Bairro Areiao, CEP 13414-903, Piracicaba – SP – Brazil.Tel/Fax: +55 19 2106 5306. E-mail: franzmontan@hotmail.com

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anesthetic injection in the palatal mucosa would be a benefit

in dentistry.

The encapsulation of drugs into liposomes represents an

alternative method of drug delivery system for local anes-

thetics because it increases the duration of analgesia, and

decreases both central nervous and cardiac toxicity

(Boogaerts et al., 1993, 1994). Several studies have demon-

strated that local anesthetics, including lidocaine, provide

efficient topical anesthesia of the skin when encapsulated into

these lipid vesicles (Eidelman et al., 2005; Taddio et al.,

2005).

Regarding the oral mucosa, significant topical anesthesia

was induced by liposome-encapsulated local anesthetics

(Franz-Montan et al., 2007, 2010; Paphangkorakit et al.,

2012). However, it was recently demonstrated that liposome

encapsulated ropivacaine at 1 or 2% was not different than a

placebo gel in reducing pain during needle insertion in the

palatal mucosa when compared with the eutectic mixture of

2.5% lidocaine/2.5% prilocaine (EMLA�) (Franz-Montan

et al., 2012).

The aim of the present study was to characterize a

liposomal-lidocaine formulation aimed for topical use at the

oral mucosa, to evaluate its in vitro permeation across pig

palatal mucosa, and to measure its in vivo topical anesthetic

efficacy in reducing the pain caused by needle insertion and

local anesthetic injection in the palatal mucosa of healthy

volunteers.

Materials and methods

Materials

Lidocaine hydrochloride was donated by Cristalia Prod.

Quim. Farm. Ltda (Itapira, SP, Brazil). Egg phosphatidylcho-

line (EPC), cholesterol (Ch), and a-tocopherol (a-T) were

purchased from Sigma Chemical Company (St Louis, MO).

All other reagents used were analytical grade.

The commercial topical formulations used in this study

were eutectic mixture of 2.5% lidocaine and 2.5% prilocaine

(EMLA�, Astra-Zeneca, Cotia, SP, Brazil – batch no. 26099)

and 5% lidocaine ointment (Xylocaina� ointment –

AstraZeneca, Cotia, SP, Brazil – batch no. 26438).

Liposome preparation

The liposomes (large unilamellar vesicles with 400 nm

diameters) were prepared according to previously described

methodology (Cereda et al., 2008; de Araujo et al., 2008).

EPC-Ch-a-T (4:3:0.07, molar ratio) films were obtained by

evaporating stock chloroform solutions under a stream of wet

nitrogen, followed by vacuum for 2 h. Films were suspended

in 20 mM HEPES buffer (pH 7.4, containing 154 mM NaCl),

and multilamellar vesicles were obtained after vortexing at

ambient temperature (5 min, 25 �C). Large unilamellar ves-

icles were prepared by extrusion (15 cycles) of the multi-

lamellar vesicles within 400 nm membrane filters (25 �C),

using a Lipex Biomembranes Inc. (Vancouver, Canada)

extruder.

Lidocaine encapsulation was performed by adding the

local anesthetic directly to the liposomes after extrusion at the

final concentration of 2.5% or 5%. The suspensions were then

sterilized by autoclaving (121 �C, 1 atm during 15 min)

(Cereda et al., 2008).

Liposome characterization

Determination of encapsulation efficiency (EE%) and

partition coefficient (P)

The encapsulation efficiency (EE%) and partition coefficient

(P) values of lidocaine into the liposomes was optically

determined, in triplicates. The liposomal suspension (4 mM)

containing LDC (2 mM) was submitted to ultracentrifugation

(120 000 g for 2 h at 10 �C) (de Araujo et al., 2008) and the

supernatant was analyzed at 260 nm for LDC concentration.

The EE% was calculated by subtracting the supernatant

concentration from the total LDC concentration, measured

previously to phase mixing. The partition coefficient (P) was

calculated by using equation 1 (de Paula & Schreier, 1995).

P ¼ ðnm=VmÞðnm=VwÞ ð1Þ

where n is the number of moles of lidocaine, V denotes the

volume (L), m and w refer to the membrane and aqueous

phase, respectively.

Determination of liposome size and polydispersity

The average particle size (hydrodynamic diameter in nm) and

polydispersity index (PDI) of the extruded liposomes (diluted

to 1 mM) were determined by dynamic light scattering, in a

ZS-90 particle analyzer (Zetasizer, Malvern Instruments,

Malvern, UK). All parameters were analyzed 24 h after

liposomal preparation, in triplicate, in three different days, at

a fixed angle (90�) and at 25 �C.

Determination of liposomes morphology by transmission

electron microscopy (TEM)

Morphology of liposomes containing LDC was performed by

TEM (EM-900; Carl Zeiss, Jena, Germany) according to a

previously described method (Franz-Montan et al., 2013).

A drop of the liposomal suspension was placed on copper

grids of 200 meshes for 15 min. Excess of the formulation was

removed with a filter paper. A 2% uranyl acetate solution was

dropped onto the grid, and the excess of this staining solution

was removed with a filter paper. The grid was examined 2 min

after staining under TEM at 80 kV.

In vitro release study

The release of LDC from liposome vesicles was evaluated

using a two-compartment system consisting of a donor

compartment (2 mL) and an acceptor compartment (250 mL

HEPES buffer, pH 7.4) separated by a cellulose membrane

with 14 000 Da molecular exclusion pores, kept under light

magnetic stirring at 37 �C (Paavola et al., 1995). Aliquots of

2 mL were withdrawn from the acceptor compartment at

intervals of 15, 30, and 60 min, during a total period of

300 min, and quantified by UV spectroscopy (260 nm).

The acceptor compartment was refilled with buffer after

removal of each aliquot to maintain a constant volume of

liquid. Absorbance measures obtained were converted into

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percentages of released drug, using a standard solution of free

lidocaine.

Higuchi and zero-order theoretical models were used to

analyze the release profiles of lidocaine from plain and

liposomal formulation. Zero-order kinetics describes that drug

release rate is independent of the concentration of the drug.

In contrast, the Higuchi model follows Fick’s law and

determines that the mechanism of drug release is dependent

on to the square root time, as described by the following

equations, respectively:

Qt ¼ Q0 þ K0 � t ð2Þ

where Qt is the cumulative amount of drug released at time t,

Q0 is the initial amount of drug, K0 is the zero-order release

constant, and t is the time

Q ¼ K � t12 ð3Þ

where Q is the amount of drug released at a time t and K is the

release kinetics constant.

Topical formulations

The liposome and placebo gel formulations were prepared by

the same operator (not involved in the application or

anesthetic efficacy evaluation) and consisted of identical

color, taste, smell and fluidity, based on a patented

method (Silva et al., 2008), using the components according

to Table 1.

The resulting gel (placebo gel) was stored in the dark in

capped glass containers at 4 �C, until the preparation of

liposomal based gels, which were obtained by mixing

carbopol base gel with liposome suspension with or without

the local anesthetic (50:50, v/v) at the final desired drug

concentration (2.5% w/w or 5% w/w). The following gels

were prepared: liposome-encapsulated 5% lidocaine

(Liposome-Lido5), liposome-encapsulated 2.5% lidocaine

(Liposome-Lido2.5), liposomal placebo gel, and plain pla-

cebo gel.

The gel formulations were placed into coded flasks to

ensure blindness of the volunteers and the investigator

involved in the application and evaluation of the topical

anesthetics.

In vitro permeation of lidocaine through pig palatalmucosa

Lidocaine analysis

Lidocaine was quantified by high performance liquid

chromatography (HPLC – Varian ProStar HPLC,

Biodirect Inc, Taunton, MA, a PS 325 UV–Vis detector, a

PS 210 solvent delivery module, and an automatic injector).

The local anesthetic lidocaine was separated on a C18

reversed-phase column (5 mm, 150� 4.60 mm, Phenomenex)

at 40 �C. The mobile phase consisted of the mixture

methanol:buffer (4.35 mM NH4H2PO4, pH 7.0, adjusted

with triethylamine) at the volume ratio 60:40, pumped at

1.5 mL/min and an injection volume of 40 mL. Lidocaine

detection was monitored at 220 nm.

A calibration curve was constructed from a stock solution,

prepared by dissolving lidocaine in 0.9% NaCl (0.2 mg/mL)

followed by dilution into six working solutions by using 0.9%

NaCl (from 0.2 to 0.04 mg/mL). For each concentration, three

samples were injected in order to obtain calibration curves,

which were analyzed by linear regression analysis of the peak

area versus the concentration (r2¼ 0.9991). The limit of

detection was 0.34 mg/mL, and the limit of quantification was

1.13 mg/mL.

This method was validated according to the ‘‘International

Conference on the Harmonization of Technical Requirements

for the Registration of Pharmaceuticals for Human Use’’

and Resolution 899/2003 of the Brazilian National

Agency for Health Monitoring (Agencia Nacional de

Vigilancia Sanitaria or ANVISA) (ANVISA, 2003; ICH,

1996).

Tissue preparation for permeation study

Pig maxilla was obtained from a local slaughterhouse and

transported to the laboratory in isotonic phosphate buffer pH

7.4. The tissue preparation was adapted from a previous

described methodology (Franz-Montan et al., 2013). Briefly,

the palatal mucosa was removed from the palatal bone and

rinsed with saline. Pieces of palatal tissue were cut out and

immersed into deionized water at 65 �C for 60 s, to allow

separation of the epithelia from the connective tissue.

Samples were quickly rinsed in deionized water, drained on

a cellulose filter, and frozen at �20 �C until use. All

experiments were conducted using tissue from at least two

animals, with eight replicates.

Permeation experiments

Permeation studies were carried out in Franz diffusion cells

(Disa, Magenta Milano, Italy), with 0.6 cm2 of permeation

area and a receiver compartment of 4.2 mL in volume. The

mucosa was placed over a 0.45 mm cellulose filter (connect-

ive side of tissue facing the membrane filter) due to its

fragility, avoiding any damage that could alter permeation

parameters without altering lidocaine transport. Gel formula-

tions were applied in infinite dose conditions (1.66 g/cm2) in

the donor compartment. The receptor chambers were filled

with degassed isotonic saline solution magnetically stirred at

37 �C. Permeation experiments were performed in non-

occlusive conditions during 5 h. Samples (300 mL) were

periodically withdrawn from the receptor phase and analyzed

by HPLC, being replaced with fresh receptor solution in equal

volumes.

The flux of drug was calculated from the slope of the linear

portion of the curve (cumulative amounts of lidocaine

transported across the mucosa per unit of area� time). The

lag time was obtained from the interception to the time axis.

Table 1. Components of the base gel, according to Silva et al. (2008)(Patent # WO/2008/138089).

Component Function

Carbopol (2%) Used as a gelling agentPropylene glycol (5%) Acts as a solvent and wettingMethylparaben (0.2%) PreservativeGlycerin (8%) Wetting and emollient agentDeionized water SolventTriethanolamine Alkalinizing agent pH¼ 7.0

DOI: 10.3109/08982104.2014.911315 Liposomal lidocaine gel for topical use 3

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The permeability coefficient was calculated according to the

following equation (de Araujo et al., 2010):

J ¼ P� Cd ð4Þ

where J (mg cm�2 h�1) is the lidocaine flux across the skin,

P (cm h�1) is the permeability coefficient and Cd is the LDC

concentration in the donor compartment (mg cm�3).

In vivo anesthetic efficacy evaluation in healthyvolunteers

Subjects

The Ethical Committee of Piracicaba Dental School,

University of Campinas Piracicaba, Brazil, approved this

research (Protocol # 112/2007), according to the requirements

of the International Conference on Harmonization Guidelines

for Good Clinical Practice and the Declaration of Helsinki.

The ClinicalTrials.gov trial registration number of this study

is NCT01425840.

A power calculation indicated that a sample size of 40

subjects would provide 95% power to detect a difference of

10 mm in VAS scores between two groups, assuming a

significance level of 5% (two-tailed).

Forty volunteers (20 women) aging 18–29 years-old

(20.3 ± 2.3 years) were selected based on a satisfactory

medical history evaluation and their agreement to provide

informed consent. All the subjects were undergraduate or

graduate students at Piracicaba Dental School and were in

good health. Exclusion criteria included history of allergy to

any of the local anesthetics used, intake of drugs that would

alter pain perception, pregnancy, and presence of lesion in the

site of topical application.

The study was conducted at Piracicaba Dental School/

University of Campinas, Sao Paulo, Brazil, at the ambulatory

office of the Pharmacology, Anesthesiology and Therapeutics

Area.

Anesthetic procedure

The volunteers randomly received six different topical

formulations in a double-blinded, placebo-controlled, cross-

over, three-period treatment design. Each volunteer received

bilaterally 100 mg of two of the following topical anesthetics

applied by the same operator: liposome-encapsulated 5%

lidocaine (Liposome-Lido5), liposome-encapsulated 2.5%

lidocaine (Liposome-Lido2.5), commercial 5% lidocaine

ointment (Xylocaina�), eutectic mixture of 2.5% lidocaine

and 2.5% prilocaine (EMLA�), placebo gel, and liposomal

placebo gel, in three different appointments spaced at least

1 week apart. The subjects served as their own controls.

A researcher not involved in anesthetic application or

anesthetic parameters evaluation selected the volunteers and

performed the randomization. Site (right or left side) and

order of application were predetermined by a random number

table, with no restriction. To allow blindness of the experi-

ment, neither the dentist nor the patient knew which

formulation was applied.

Before topical application, the palatal mucosa of both

right- and left-upper canines was dried using the sterile gauze.

The topical anesthetic agents were previously weighed and

applied on the dried sites, approximately 1.0 cm away from

the canines’ gingival margin, by using a cotton swab. Each

topical anesthetic was kept on the mucosal surface for 2 min.

After topical application, the mucosa was gently wiped with

sterile gauze followed by a water rinse.

According to a previous described procedure (Franz-

Montan et al., 2012), after the removal of the topical

formulations, a second operator inserted a 30-gauge dental

needle attached to an aspirating syringe in the same region of

topical application, until periosteum contact. Immediately

after, the volunteer was informed that the anesthetic solution

would be injected, and 0.3 mL of 2% lidocaine with 1:100.000

epinephrine cartridge (Alphacaine� – DFL Ind. Com. Ltd,

Rio de Janeiro, RJ, Brazil) was injected at a rate of 1 mL/min.

Pain perception was accessed in two different moments,

after needle insertion and after local anesthetic injection, on

two separated visual analogue scales (VAS). VAS consisted of

a 10-cm non-graded line showing ‘‘no pain’’ and ‘‘unbearable

pain’’ at the left and right ends, respectively. Subjects were

asked to mark a point on the line according to their level of

perceived pain. A ruler was used to measure the distance from

the left end-point to the mark made by the volunteer.

In addition, at the end of all sessions, the volunteers were

asked to choose the most efficient topical anesthetic for the

palatal mucosa.

Statistical analysis

Characterization and in vitro permeation data were expressed

as percentage or mean (±SD) and analyzed by a one-way

analysis of variance (ANOVA) and Tukey–Kramer’s post-hoc

test or unpaired t-test. In vivo anesthetic efficacy data were

compared by the Friedman test. Correlation between in vivo

efficacy and in vitro studies was performed by Pearson’s

correlation test. All tests were performed by using GraphPad

Instat (GraphPad Software, Inc., La Jolla, CA) with signifi-

cance level set at 5%.

Results and discussion

Characterization of liposomes containing lidocaine

Table 2 shows particle size, polydispersity index (PDI),

encapsulation efficiency (EE%), and partition coefficient (P)

of liposomal LDC. The encapsulation of LDC did not affect

vesicles size and homogeneity of the system, since there was

no statistically significant difference in size and polydisper-

sity index between empty and lidocaine-containing liposomes

(p40.05). The low polydispersity values indicate a good

stability of the system (Ntimenou et al., 2012).

Few studies were reported in the literature regarding the

development of a liposomal lidocaine formulation for oral

mucosa use. Paphangkorakit et al. observed an improved

Table 2. Encapsulation efficiency (EE%), partition coefficient (P),particle size, and polydispersity index (PI) of empty liposomes andliposome-encapsulated LDC.

Samples EE% ± SD P ± SD Size (nm ± SD) PI ± SD

Liposomes – – 376.9 ± 48.1 0.16 ± 0.01Liposomes

with LDC21.63 ± 2.6 114.5 ± 16.8 392.8 ± 39.3 0.20 ± 0.03

4 M. Franz-Montan et al. J Liposome Res, Early Online: 1–9

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topical anesthetic efficacy in the palatal mucosa of a

liposome-encapsulated 2% lidocaine with epinephrine

1:100 000. The liposomes consisted of cholesterol and egg

phospholipid (1:1, w:w, prepared by the sonication method –

ultrasonic dental scaler). The authors did not study particle

size or encapsulation efficiency (Paphangkorakit et al., 2012).

In the present study, the partition coefficient and encap-

sulation efficiency were similar to the results obtained by

others with different local anesthetics encapsulated into

liposomes with the same composition (Cereda et al., 2006;

de Araujo et al., 2008).

TEM images confirmed the particle size and vesicles

morphology (Franz-Montan et al., 2013). The morphological

analysis of liposomes images demonstrated the existence of

spherical-shaped vesicles (Figure 1) around 400 nm in

diameter, which is in accordance to the DLS results in

Table 2.

The LDC release profile was significantly reduced after

encapsulation in liposomes (1.5–1.2 times, p50.001, after 15

and 60 min, respectively) when compared with the free drug

(Figure 2). The time required for total release (100%) of

LDC+liposome formulation was achieved after 180 min.

Theoretical model analysis revealed that the release rate

from liposomes follows the Higuchi model, as observed by

high correlation coefficient value when compared with zero-

order kinetics analysis (Table 3), suggesting that the release of

LDC from liposomes is a process dependent on diffusion.

In vitro permeation of lidocaine through pig palatalmucosa

Lidocaine permeation profiles across pig palatal mucosa from

different topical formulations and in infinite dose condition

are observed in Figure 3. Permeation parameters are

described in Table 4.

Porcine buccal or esophageal mucosae are frequently used

for in vitro drug permeation studies due to their similar

structure and permeability with human tissues (Diaz Del

Consuelo et al., 2005a,b). In the present study, palatal mucosa

was chosen due to its keratinized layer, allowing a more

efficient permeability barrier characteristic (Ganem-

Quintanar et al., 1998) and it represents the same application

site used in clinical studies.

The Liposome-Lido5 formulation presented the highest

flux when compared with the other formulations (Table 3;

p50.001). This result was unexpected, since in this formu-

lation, 21.6% of lidocaine was encapsulated into the lipo-

somes and, therefore, less free drug was available to cross the

barrier in comparison with the commercial 5% lidocaine

formulation (Xylocaina�). The results observed in the present

study differ from others that demonstrated free drug present-

ing higher flux when compared with drug encapsulated into

Figure 1. Transmission electron micrographs of a typical EPC-Ch-a-T (4:3:0.07, mole%) liposome without (a) and with (b) lidocaine. Magnification100 000�.

0 40 80 120 160 200 240 280 3200

20

40

60

80

100

LDCLUV

LDC

Time (min)

LD

C r

elea

se (

%)

Figure 2. In vitro release profile of lidocaine (plain and encapsulated inliposomes) (mean values ± SD).

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lipid carriers or liposomes (Maestrelli et al., 2009; Puglia

et al., 2011).

Beyond the use of propylene glycol as a solvent in topical

formulations (Herkenne et al., 2008; Kang et al., 2007), the

literature also demonstrated its ability to increase drug

absorption after topical application (Bailey, 1992; Gee

et al., 2014; Herkenne et al., 2008; Melero et al., 2008;

Merino et al., 2008). The presence of propylene glycol at the

liposomal gel could explain why this formulation presented

the highest flux. Curiously, despite having the lowest

lidocaine concentration, EMLA� presented a higher flux

(p50.001) when compared with Xylocaina�. A possible

explanation for its higher flux could be the well-known

physical–chemical properties. Since EMLA is a eutectic

mixture, its melting point is lower than that of the isolated

compounds (EMLA¼ 18 �C; lidocaine¼ 67 �C). Therefore, at

the body temperature (37 �C), the same condition used in the

in vitro experiment, lidocaine was in the liquid form and

could present a faster permeation.

In addition, such altered fluxes could be possibly

associated to the diverse composition and nature of the

formulations, influencing drug solubility and partitioning,

leading to different drug transports across the barrier

(de Araujo et al., 2010). It is worth notice that both

commercial formulations, EMLA� and Xylocaina�, are not

gel-based formulations, but a cream and an ointment,

respectively.

The lidocaine concentration also varies: EMLA� contains

2.5% and Xylocaina� contains 5%. In the present study,

these two different lidocaine concentrations were used in

the liposomal formulations in order to compare them

with the same concentrations of the two commercial

formulations.

In relation to permeability coefficient Xylocaina� pre-

sented the lowest value (p50.001); Liposome-Lido5 did not

differ from EMLA� and Liposome-Lido2.5 (p40.05) and

EMLA� present higher permeability coefficient than

Liposome-Lido2.5 (p50.05). These differences could also

be explained based on the factors previously discussed,

formulation composition, concentration of local anesthetic,

and physical–chemical properties.

In vivo anesthetic efficacy evaluation in healthyvolunteers

Table 5 shows formulation preference considering the anes-

thetic effect and VAS concerning pain during needle insertion

and anesthetic injection. Liposome-Lido5 was equivalent to

EMLA� (positive control) in reducing pain during needle

insertion and anesthetic injection (p40.05). However,

EMLA� was preferred by most of the volunteers, followed

by Liposome-Lido5. Both formulations showed superior

anesthetic efficacy (p50.05) when compared with

Xylocaina� and placebo groups, and probably this result has

influenced the preference reported by the volunteers.

In the present study, the non-encapsulated lidocaine was

not excluded from the formulation. Moreover, the 79% of non-

encapsulated lidocaine also contribute for its efficacy.

Similarly, our research group has previously demonstrated

an increase of 26.1% in the intensity of total anesthetic effect

induced by 2% liposomal lidocaine after infra-orbital nerve

block in rats, when compared with plain lidocaine formulation

(Cereda et al., 2006). In addition, the formulation good

performance can also be attributed to the presence of

propylene glycol, which can act as a permeation enhancer

(Bailey, 1992; Gee et al., 2014; Herkenne et al., 2008; Melero

et al., 2008; Merino et al., 2008), increasing lidocaine

solubility in the palatal mucosa, and leading to a better

efficacy.

Topical anesthesia is widely used in dentistry to reduce

pain from both needle insertion and injection of a local

anesthetic solution. However, an efficient topical anesthetic is

not yet available, especially concerning the palatal mucosa,

and even after topical anesthesia, most patients still feel pain

(Meechan, 2000, 2002).

Adequate topical anesthesia on palatal mucosa is usually

not achieved, especially in the anterior region (Meechan,

2000). Harker (1997) attributed the pain during injection to

the dislocation of the palatine muco-periosteum during local

anesthetic administration. However, in most of the studies

involving topical anesthetics efficacy, pain is evaluated solely

by the needle insertion, with no local anesthetic injection into

the tissues (Meechan, 2000, 2002). In the present study, the

efficacy of the liposomal topical anesthetics was evaluated

considering their ability to reduce pain during injection of an

anesthetic solution in the anterior portion of the palatal

mucosa.

Since 1985, several authors have been evaluating the

anesthetic efficacy of EMLA� on oral mucosa, and most of

them demonstrated its superiority when compared with

other commercial available formulations in dentistry

00,2

5 0,5 0,75 1 1,5 2 2,5 3 4 5

0.0

0.5

1.0

1.5

2.0

2.5 Liposome-Lido5

Liposome-Lido2.5

EMLA®Xylocaína®

Time (h)

Lid

ocai

ne p

erm

eate

d (m

g/cm

²)

Figure 3. Permeation profiles of lidocaine from liposomal andcommercial formulations applied in infinite dose condition (meanvalues ± SD).

Table 3. In vitro release kinetics of lidocaine (free or encapsulated inliposomes) according to Higuchi and zero-order theoretical models.

Release kinetics

FormulationZero-order

modelHiguchimodel

R2

(zero-ordermodel)

R2

(Higuchimodel)

LDC 0.70 ± 0.15% h�1 72.1 ± 5.1% h�1/2 0.8525 0.955LDCLUV 0.65 ± 0.16% h�1 66.8 ± 0.16% h�1 0.8724 0.988

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(Al-Melh & Andersson, 2007; Holst & Evers, 1985). The

reported efficacy of EMLA� in inducing local anesthesia in

the palatal mucosa (Al-Melh & Andersson, 2007; Holst &

Evers, 1985) was the main reason to its choice as a positive

control in the present study.

The efficacy of lidocaine formulations on the oral mucosa

was previously compared with EMLA�. It was concluded that

a 2-min application of EMLA� was better than 2% lidocaine

gel on tongue mucosa and anterior buccal gingiva (Svensson

et al., 1992), and better than 5% lidocaine on the buccal

mucosa (McMillan et al., 2000). However, EMLA� was not

previously compared with a liposome encapsulated-lidocaine

in topical anesthesia in dentistry.

The potential of liposomal local anesthetic formulations to

provide topical anesthesia was demonstrated in the literature

as 4% and 5% lidocaine liposomal formulations provided the

same anesthetic efficacy as EMLA� on intact skin (Friedman

et al., 1999). Similarly, in the present study, the liposomal

encapsulated 5% lidocaine gel was equivalent to EMLA� in

reducing pain from needle insertion and anesthetic injection

at palatal mucosa.

Concerning oral mucosa, another local anesthetic encap-

sulated into liposomes (1% ropivacaine) was equivalent

to EMLA� in reducing pain during needle insertion

(no anesthetic solution was injected) in the maxillary buccal

fold after a 2-min application. Even in a different application

site, the present study also concluded that liposome

encapsulated 5% lidocaine gel was equivalent to EMLA� in

reducing pain during needle insertion.

Nevertheless, Franz-Montan et al. (2012) were not able to

show the effectiveness of a liposomal formulation on the

palatal mucosa, considering pain during needle insertion

using the same methodology of the present study.

These authors also evaluated pain during anesthetic injection

and even the positive control (EMLA�) was not able to

reduce pain during anesthetic injection. Contrary to those

results, in the present study, both liposomal lidocaine (5%)

and EMLA� were effective in reducing pain during injection.

In agreement to the present study, Paphangkorakit et al.

demonstrated that the liposomal lidocaine encapsulation was

able to improve topical anesthetic efficacy in reducing pain

during anesthetic injection in the palatal mucosa, in com-

parison with a commercial formulation. In such study, a

different liposomal formulation (cholesterol and egg phospho-

lipid 1:1, w:w, prepared by the sonication method – ultrasonic

dental scaler) was used (Paphangkorakit et al., 2012).

Correlation between in vitro permeation and in vivoanesthetic efficacy

Figure 4 shows the relationship between flux of lidocaine

across pig palatal mucosa (in vitro) and in vivo efficacy of

lidocaine formulations in reducing pain during needle inser-

tion (Figure 4a) and local anesthetic injection (Figure 4b) in

volunteers. A moderate (rPearson40.3, p40.05) correlation

was found between flux and VAS-Insertion.

Even though a high permeation rate and flux were not

expected for Liposome-Lido5 (Maestrelli et al., 2009; Puglia

et al., 2011), the moderate correlation between flux and

VAS-insertion indicates that the highest in vitro flux deter-

mines a better performance in oral topical anesthesia in vivo.

Similarly, a high correlation between flux of benzocaine

across pig esophageal mucosa and pain scores during needle

insertion in the canine’s maxillary buccal fold was recently

demonstrated (Franz-Montan et al., 2013). Therefore, the

anesthetic flux seems to be a valuable parameter to predict

Table 4. Permeation parameters (mean ± SD) of lidocaine through pig palatal mucosa from liposomal and commercialformulations applied in infinite dose condition (n¼ 6–7).

Lidocaine formulation Flux (mg cm�2 h�1) Permeability coefficient (cm h�1)�10�3 Lag time (h)

Liposome-Lido5 (0.5–5 h) 0.44 ± 0.04a,b,c*** 8.80 ± 0.85c*** –Liposome-Lido2.5 (0.5–4 h) 0.16 ± 0.04 6.51 ± 1.58f*** –EMLA� (0.5–5 h) 0.24 ± 0.05d*** 9.44 ± 2.14d***,e* –Xylocaına� (2–5 h) 0.09 ± 0.05 1.73 ± 1.21 1.46 ± 0.14c,d,f***

aLiposome-Lido5 versus Liposome-Lido2.5.bLiposome-Lido5 versus EMLA�.cLiposome-Lido5 versus Xylocaına�.dEMLA� versus Xylocaına�.eEMLA� versus Liposome-Lido2.5.fLiposome-Lido2.5 versus Xylocaına�.***p50.001 and *p50.05.

Table 5. VAS median (first and third quartiles) and preference considering the anesthetic effect rated by volunteers after needle insertion and anestheticinjection.

Anesthetic agentNeedle insertion VAS median

(first and third quartiles)Anesthetic injection VAS median

(first and third quartiles)Preference reported by

the volunteers in %

Liposome-Lido5 0.70 (0.34–2.75) 0.90 (0.34–2.43) 25.6Liposome-Lido2.5 1.25 (0.60–2.45) 1.60 (0.78–3.79) 7.7EMLA� 0.60 (0.10–1.20) 0.73 (0.24–1.70) 41.0Xylocaina� 2.00 (0.89–2.45) 2.10 (0.90–3.00) 20.5Placebo 2.10 (1.08–3.98) 2.48 (1.10–3.53) 2.6Liposomal placebo 1.40 (1.10–3.45) 2.30 (1.20–3.83) 2.6

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anesthetic efficacy during the preclinical phase, as previously

observed by Mura et al. (2008).

Figure 5 shows the correlation between permeability

coefficient of lidocaine across pig palatal mucosa (in vitro)

and in vivo efficacy of lidocaine formulations in reducing

pain during needle insertion (Figure 5a) and local anesthetic

injection (Figure 5b) in volunteers. A high (rPearson40.7,

p40.05) correlation was verified between P and

VAS-injection; and a moderate (rPearson40.3, p40.05)

correlation between P and VAS-insertion.

The permeability coefficient demonstrated to be another

worthy in vitro permeation parameter to predict topical

anesthetic efficacy in the oral mucosa as a high correlation

was found between P and VAS-injection; and a moderate

between P and VAS-insertion.

The correlation between in vitro parameters and in vivo

topical anesthetic efficacy can be explained by the hypothesis

previously suggested: a drug with a higher flux and perme-

ability coefficient presents a greater penetration into the

epithelium, resulting in a more-intense superficial analgesia

(Franz-Montan et al., 2013).

Conclusion

In conclusion, we successfully developed and characterized a

liposomal lidocaine formulation. The liposome encapsulated

5% lidocaine gel presented both in vitro and in vivo

performances similar to the gold standard commercial

formulation EMLA� in terms of permeation profile and in

reducing pain during needle insertion and anesthetic injection.

Therefore, it should be considered an efficient topical

anesthetic in dentistry.

In addition, the correlation between in vitro and in vivo

parameters suggests that in vitro studies could be helpful to

predict the effect of topical anesthetic agents.

Declaration of interest

No external funding and no competing interests declared. The

authors alone are responsible for the content and writing of

this paper. This study was financially supported by Sao Paulo

Research Foundation – FAPESP (grant # 2006/00121-9).

Daniela Belisario Baroni and Michelle Franz-Montan

acknowledge the scholarship provided by FAPESP (Grants #

2007/05734-1 and # 2009/08860-3, respectively).

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0 5 10 150

1

2

3

P(cm.h-1).103

VA

S I

nse

rtio

n (

in c

m)

0 5 10 150

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2

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