Journal of Liposome Research, 18:329–339, 2008Copyright © Informa UK, Ltd.ISSN: 0898-2104 print / 1532-2394 onlineDOI: 10.1080/08923970802500067

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LLPR0898-21041532-2394Journal of Liposome Research, Vol. 99, No. 99, October 2008: pp. 1–68Journal of Liposome Research

Stability and Local Toxicity Evaluation of a Liposomal Prilocaine Formulation

Liposomal Prilocaine: Stability and local toxicityCereda et al. CINTIA M. SAIA CEREDA,1 GIOVANA R. TÓFOLI,1,2 RUI B. DE BRITO JUNIOR,3 MARCELO B. DE JESUS,1 LEONARDO F. FRACETO,1,4 FRANCISCO C. GROPPO,2 DANIELE R. DE ARAUJO,1 AND ENEIDA DE PAULA1

1Department of Biochemistry, Institute of Biology, State University of Campinas,Campinas, São Paulo, Brazil2Department of Physiological Sciences, Piracicaba Dental School, StateUniversity of Campinas, Piracicaba, São Paulo, Brazil3Department of Molecular Biology, São Leopoldo Mandic Dental ResearchInstitute, Campinas, São Paulo, Brazil4Department of Environmental Engineering, State University of São Paulo,Sorocaba, São Paulo, Brazil

This study reports a physicochemical stability evaluation of a previously reported lipo-somal prilocaine (PLCLUV) formulation (Cereda et al. J. Pharm. Pharmaceut. Sci.7:235, 2004) before and after steam sterilization as well as its local toxicity evaluation.Prilocaine (PLC) was encapsulated into extruded unilamellar liposomes (LUVs) com-posed by egg phosphatidylcholine:cholesterol:alfa-tocopherol (4:3:0.07, mole %).Laser light-scattering analysis (p > 0.05) and thiobarbituric acid reaction (p > 0.05)were used to evaluate the liposomes physical (size) and chemical (oxidation) stability,respectively. The prilocaine chemical stability was followed by 1H-nuclear magneticresonance. These tests detected no differences on the physicochemical stability of PLCor PLCLUV, sterilized or not, up to 30 days after preparation (p > 0.05). Finally, thepaw edema test and histological analysis of rat oral mucosa were used to assessthe possible inflammatory effects of PLCLUV. PLCLUV did not evoke rat paw edema(p > 0.05), and no significant differences were found in histological analysis, whencompared to the control groups (p > 0.05). The present work shows that PLCLUV is sta-ble for a 30-day period and did not induce significant inflammatory effects both in thepaw edema test and in histological analysis, giving supporting evidence for its safetyand possible clinical use in dentistry.

Keywords local anesthetic, prilocaine, liposome, drug delivery

Address correspondence to Eneida de Paula, Departamento de Bioquímica, Instituto de Biologia,UNICAMP, C. P. 6109, CEP 13083-970, Campinas, SP, Brazil; Fax: +55 19 3521 6129; E-mail:depaula@unicamp.br

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330 Cereda et al.

Introduction

Prilocaine (PLC) is an aminoamide local anesthetic widely used in dentistry. Its rapidmetabolism and tissue uptake lead to a quick fall of plasma levels. These features increasethe safety profile of the drug, making PLC a popular choice in regional anesthesia proce-dures (McLure and Rubin, 2005). Liposomes have been extensively described in the liter-ature as drug-delivery systems (Torchilin, 2005). They have been used to prolong theanalgesic effect and to decrease the toxic effects of several local anesthetics, such asbupivacaine (Grant et al., 1997, 2001, 2004; Boogaerts et al., 1993a,b, 1994; Malinovskyet al., 1997; Yu et al., 1998, 2002), mepivacaine, and lidocaine (Araujo et al., 2004;Mashimo et al., 1992; Taddio et al., 2005; Cereda et al., 2006).

In a former study, the PLCLUV formulation prolonged the duration of analgesia, whencompared to the plain PLC (without a vasoconstrictor). In addition, the anesthetic effectinduced by the liposomal PLC formulation on an animal model was similar to that ofvasoconstrictor-containing PLC in an infraorbital nerve blockade model, pointing it out asa new alternative to the use of vasoconstrictor-containing local anesthetics (Cereda et al.,2004). Herein, we have intended to evaluate the PLCLUV physicochemical stability beforeand after sterilization, as well as its local toxicity, in order to consider its possible clinicalapplication. The average particle size and size distribution were used to evaluate the physicalstability of the liposomes (Grit and Crommelin, 1993), whereas the chemical stability oflipids was assessed through the measurement of phospholipid oxidation, using the thiobar-bituric acid (TBA) reaction (Ohkawa et al., 1979; New, 1990). The PLC chemical stabilitywas evaluated by 1H-nuclear magnetic resonance (1H-NMR) spectroscopy, a convenientand informative method for structural analysis of organic compounds (Hanna, 2000). Thepaw edema test, an assay widely used to evaluate anti- and proinflammatory activity(Campos and Calixto, 1995; Hayashi et al., 2001; Fernandes et al., 2003; Duhgaonkaret al., 2006; Ye4ilada and Küpeli, 2007), was the test of choice to investigate the in vivolocal toxicity of this novel liposomal formulation. The tissue reaction induced in the oralmucosa of rats after PLCLUV administration was evaluated by histological analysis.

Methods

Materials and Animal Model

PLC hydrochloride form and thiopental (Thiopentax®) were obtained from CristáliaProdutos Químicos e Farmacêuticos Ltda (SP, Brazil). Egg phosphatidylcholine, choles-terol, α-tocopherol, and carrageenan were purchased from Sigma Chemical Co. (St. Louis,Missouri, USA). All other reagents were of analytical grade.

Male Wistar rats, 250–350 g, were obtained from CEMIB UNICAMP (Centro deBioterismo–State University of Campinas; UNICAMP) and were given free access to waterand food throughout the study. The experiment was approved by the Institutional Committeefor Ethics in Animal Research of UNICAMP (Protocols 816–1 and 871–1), which followsthe recommendations of the Guide for the Care and Use of Laboratory Animals.

Liposomal PLC Preparation and Sterilization

Liposomal PLC formulation was prepared as previously described (Cereda et al., 2004).Briefly, a dry lipid film, containing egg phosphatidylcholine, cholesterol, andα-tocopherol at a 4:3:0.07 molar ratio, was prepared by solvent evaporation under nitrogen

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Liposomal Prilocaine: Stability and Local Toxicity 331

flow. Multilamellar liposomes were obtained by adding 20 mM HEPES buffer, pH 7.4(containing 154 mM NaCl) to the dry lipid film and vortexing the mixture. Unilamellar lipo-somes (LUVs) were prepared by extrusion (Extruder Lipex Biomembranes®, Vancouver,British Columbia, Canada) of the multilamellar vesicles (12 cycles, using a 400-nm poly-carbonate membrane, at 25°C). The total lipid concentration in the LUV was 5 mM, andPLC was added directly to the liposomes after extrusion, up to a concentration of 3%(Cereda et al., 2004). This formulation was sterilized by autoclaving (121°C, 1 atm during15 minutes). Afterward, the sterility was evaluated by microbiological test with Brain-HeartInfusion (BHI) medium (BD-Becton, Dickinson and Company, Franklin Lakes, NJ, USA)(Tortora et al., 2002), and apyrogenicity was assessed by the Endosafe® Limulus amoeb-ocyte Lysate Test (USDHHS, 1987). The liposomal formulations were stored at 4°C,under light shelter (Lichtenberg and Barenholz, 1988).

Liposomal PLC Stability

Chemical Stability. Lipid oxidation was assessed by the TBA colorimetric method(Asakawa and Matsushita, 1980). The results were expressed in terms of the presence of asecondary product of lipid peroxidation, malondialdehyde (MDA).

The chemical stability of the PLC molecule was evaluated by one-dimensional1H-NMR spectra recorded with a 500-MHz Varian INOVA 500 spectrometer (Varian Inc.,Palo Alto, CA, EUA), at 20°C, before and after sterilization. Samples were suspended inD2O (5 mM) and degassed by bubbling N2 directly into the NMR tubes. The spectra werereferenced to the residual water signal (4.81 ppm).

Physical Stability of the Liposomes. The mean diameter and size distribution of theliposomal prilocaine formulation were analyzed by laser light scattering (Dathe et al.,1990; Barth and Flippen, 1995), using the Malvern Autosizer 4700 equipment (MalvernInstruments Ltd., Malvern, Worcestershire, UK). The measurements were made from a90-degree angle at room temperature (25°C) at the initial time and at 30-day intervals.

In Vivo Local Toxicity Evaluation

The paw edema test (Winter et al., 1962; Campos and Calixto, 1995; Hayashi et al.,2001; Fernandes et al., 2003; Duhgaonkar et al., 2006; Ye4ilada and Küpeli, 2007) wasperformed in order to assess the in vivo local toxicity of PLCLUV. Carrageenan, a muco-polysacharide derived from Irish Sea mosses (Chondrus), has been the agent of choice to ver-ify inflammatory responses, since this material stimulates an inflammatory process withoutsystemic effects and with a high degree of reproducibility (Winter et al., 1962).

Rats were distributed in seven groups (n = 6 per group), according to the treatment: group1, PLC-free liposomes (LUVs); group 2, 3% prilocaine in 154 mM NaCl (PLC); group 3, com-mercially available felypressin containing 3% prilocaine (PLCFELYPRESSIN); group 4, liposomalprilocaine 3% (PLCLUV); group 5, 1% carrageenan (positive control); group 6, saline (negativecontrol); and group 7, HEPES buffer (negative control). The animals were anesthetized with40 mg/kg of sodium thiopental solution by the intraperitoneal (i.p.) route, and the paw delimi-tation was made. First, the baseline volume of the animal paw was measured, and after that,intraplantar injections (0.1 mL) of the test and control solutions were given into the paw. Therat-paw volume was analyzed with a plethysmometer (Ugo-Basile, Varese, Italy) at 60, 120,and 180 minutes after injection, and it was expressed in milliliters, as the difference betweenthe baseline and the injected paw volume (Nantel et al., 1999; Posadas et al., 2004).

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332 Cereda et al.

Surgical Procedure to Histological Analysis

The rats were divided into four groups: group 1, LUV; group 2, 3% PLC; group 3,3% PLCFELYPRESSIN; and group 4, 3% PLCLUV. Slightly general anesthesia was inducedby an i.p. injection of sodium thiopental (40 mg/kg) before the administration of the localanesthetics formulations.

The animals received one of the formulations (0.1 mL) in the oral mucosa of theright-upper first molar (Figure 1). The same amount of saline solution (NaCl 0.9%) wasadministered in the left side as control. Six animals (n = 6) from each group were sacri-ficed after 6 hours, 24 hours, and 4 days of administration, and the maxillary bones, alongwith soft tissues, were removed.

The samples were fixed with Bowin solution during 24 hours and, after that, with10% formalin solution for 48 hours. Afterward, the samples were decalcified with ethyl-enediaminetetraacetic acid (EDTA) titriplex (Merck KGaA, Darmstadt, Germany). Fivecross-sections 6 μm thick and with 40 μm of depth were obtained from each sample. Thecross-sections were stained with hematoxylin and eosin.

The sections were submitted to qualitative analysis in order to evaluate the intensityof leucocitary infiltration and/or the presence of any possible necrosis area. The soft tis-sue and the bone near the injection site were analyzed. The scores were defined basedon the following descriptions: (1) minimal infiltrate; (2) mild infiltrate; (3) moderateinfiltrate; (4) severe infiltrate; and (5) severe infiltrate with necrosis areas (Shipperet al., 2005).

Statistical Analysis

Size distribution of liposomes, thiobarbituric acid reaction, and paw edema tests werecompared by one-way analysis of variance (ANOVA) with the post-hoc Tukey Kramertest. Statistical significance was defined as p < 0.05 (Zar, 1999). The oral mucosa tissuereaction was analyzed by Kruskal-Wallis test (intergroups) and Wilcoxon paired test(intragroups).

Figure 1. Pattern region for histological analysis and site of injection (dashed arrow).

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Liposomal Prilocaine: Stability and Local Toxicity 333

Results

The results obtained by the TBA reaction test revealed that the chemical stability of lipidswas maintained up to 60 days after preparation with nonsignificant differences (p > 0.05)between sterilized and nonsterilized samples (Figure 2).

The laser light-scattering analysis showed that the size of PLCLUV, either sterilized ornot, was stable up to 30 days after preparation (Table 1). After a 60-day period the size ofthe liposomes increased in both cases of nonsterilized (2.4-fold; p < 0.001) and sterilized(4.5-fold; p < 0.001) samples.

Figure 2. Malondialdehyde (MDA) concentration in sterilized and nonsterilized prilocaine liposomalformulation (n = 6), as followed in 30-day intervals, up to 120 days after preparation. *p < 0.05; **p< 0.01; ***p < 0.001 (ANOVA/TukeyKramer).

Table 1Physical stability of liposomes: vesicles size distribution (nm) and proportion (%)

of liposomal prilocaine, before and after sterilization; immediately, 30, and 60 days after preparation. Data are expressed as mean ± SD (n = 6)

Non-sterilized Sterilized

Initial 341.71 ± 48 (100%) a(ns),b*** 342.8 ± 56 (100%) c(ns),d***30 days 354.01 ± 61 (100%) 363.3 ± 57 (100%)60 days 812.9 ± 359 (94.1%) 1561.8 ± 954 (83.4%)

Note: Statistical analysis: ANOVA/TukeyKramer, p < 0.001 (***), p < 0.01 (**), p < 0.05 (*),p > 0.05 (ns); ns, nonsignificant.

aNonsterilized samples (30 days) vs. nonsterilized samples (initial time).bNonsterilized samples (60 days) vs. nonsterilized samples (initial time).cSterilized samples (30 days) vs. sterilized samples (initial time).dSterilized samples (60 days) vs. sterilized samples (initial time).

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334 Cereda et al.

In order to check the effect of the sterilization process on the chemical stability ofPLC, 1H-NMR was used. Figure 3 shows the assignment of the 1H spectrum of PLC afterautoclaving. No difference was detected in the spectra before (not shown) and after steril-ization. The peaks assignment is in agreement with the literature, in which the spectrawere referenced to chloroform (Hanna, 2000).

Inflammatory activity assay showed that PLCLUV did not evoke edema in rats, whencompared to the other groups (p > 0.05): PLCFELYPRESSIN, PLC, LUV, saline, and HEPESbuffer. On the other hand, significant difference on inflammatory activity was observedwhen prilocaine liposomal formulation was compared to the 1% carrageenan-treatedgroup (p < 0.001, positive control) (Figure 4).

The histological analysis of the oral mucosa samples is presented in Table 2. To illus-trate the score values used, some histological aspects of tissue reaction caused by theinjection of the formulations are shown in Figure 5.

The intensity score values of the inflammatory infiltrate obtained with the histologicalevaluation of the tested groups, after a 6-hour period, are shown in Table 2a.

A comparative analysis among the different groups showed that the score valuesobtained after PLCLUV administration were significantly smaller than the onesobtained after PLCFELYPRESSIN injection (p < 0.01). On the other hand, comparisonsamong the PLCLUV and PLC or LUV groups did not reveal any significant difference(p > 0.05).

Also, regarding to the 6-hour period after the treatment, the maxilla sides of eachanimal of the group were compared (intragroup analysis) as follows: the right side thatreceived the test solution and the opposite side that received the saline solution as control.This comparison revealed that the PLCFELYPRESSIN and PLC groups showed increasedscore values relative to their respective controls (Table 2a) (p < 0.05).

Figure 3. 1H-nuclear magnetic resonance (1H-NMR) spectra of prilocaine solution after sterilizationwith the assignment of the prilocaine hydrogens. [Prilocaine] = 5 mM, 20°C, 500 MHz.

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Liposomal Prilocaine: Stability and Local Toxicity 335

Twenty-four hours after the treatment, non significant differences wereobserved (Table 2b) (p > 0.05 in the intergroups analysis). In the intragroup analysis, onlyPLCFELYPRESSIN showed increased score values, when compared to its respective control(Table 2b) (p < 0.05).

The group score values obtained 4 days after the treatment did not differ statistically(p > 0.05) from each other. On the other hand, in the intragroup analysis, the PLCFELYPRESSINand PLC treatments exhibited score values greater than the ones of their respectivecontrols (p < 0.05) (Table 2c).

Discussion

Previous works showed the liposomal PLC formulation effectiveness in the increase ofanalgesia in rats (Cereda et al., 2004, 2006). Stability and local toxicity evaluation were thefurther steps in the formulation development, looking forward to its clinical application.

PLCLUV was sterilized by autoclaving and its compound stability and the liposomalvesicle size distribution were checked before and after sterilization. NMR results indicatedthat PLC hydrochloride form resists the sterilization process, which is what was expected,taking into account its melting point (167–168°C) (Merck Index, 1996).

The TBA method was used to evaluate the lipid chemical stability. The oxidation ofphospholipids fatty acids, in the absence of specific oxidants, can occur via a free radicalchain mechanism (New, 1990). Lipid peroxidation is favored in unsaturated fatty acylchains, such as those of egg phosphatidylcholine, and the resultant free radical reactionshave been related to decreases in membrane fluidity and cell lyses (McCall and Frei,1999). The TBA results showed that the chemical stability of lipids was maintained up to60 days after preparation.

Nevertheless, the laser light-scattering analysis revealed that the size of the liposomalvesicles, sterilized or not, was stable just up to 30 days after preparation, being a limitingparameter to the formulation stability.

Figure 4. The graph shows no significant differences (p > 0.05) between PLCLUV and the groups:PLCFELYPRESSIN, PLC, LUV, Saline, and HEPES buffer. On the other hand, when compared to thepositive control, the carrageenan group, the prilocaine liposomal formulation shows a very signifi-cant difference (p < 0.001) (ANOVA/TukeyKramer).

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Finally, in order to test the local toxicity of the PLCLUV formulation, its in vivo inflam-matory response was evaluated in rats and the tissue reaction of rat oral mucosa afterPLCLUV injection was observed by histological analysis. In the paw edema test, the safetyof PLCLUV was cross-checked by comparison with the carrageenan group to which thePLCLUV formulation showed a significant difference (p < 0.001; Figure 4). The results inFigure 4 also show that PLCLUV, its components (PLC, LUV, saline, and HEPES buffer),

Table 2Median (minimum–maximum limits) of the inflammatory scores for all

groups and their respective controls: A: 6 hours after the treatment; B: 24 hours after the treatment; and C: 4 days after the treatment

A) Test Saline

PLCLUV 2.0 (2.0–2.2)a** 2.4 (2.2–3.6)PLCFELYPRESSIN 3.8 (3.2–4.4) 2.1 (2.0–2.8)b*PLC 2.6 (2.2–4.0) 2.0 (2.0–2.4)c*LUV 2.3 (2.0–2.8) 2.4 (2.0–3.4)

B) Test Saline

PLCLUV 2.0 (2.0–2.6) 2.3 (2.0–2.4)PLCFELYPRESSIN 2.8 (2.4–3.0) 2.0 (2.0–2.8)b*PLC 2.1 (2.0–3.0) 2.0 (2.0–2.2)LUV 2.1 (2.0–2.4) 2.0 (2.0–2.8)

C) Test Saline

PLCLUV 1.6 (1.2–1.8) 1.7 (1.4–1.8)PLCFELYPRESSIN 2.3 (1.8–3.0) 1.6 (1.4–2.0)b*PLC 1.8 (1.6–2.2) 1.6 (1.2–1.8)c*LUV 1.9 (1.6–2.4) 1.9 (1.4–2.2)

Note: Statistical analysis: p < 0.001 (***), p < 0.01 (**), p < 0.05 (*), p > 0.05(non significant).

Intergroups (Kruskal-Wallis test) Intragroups (Wilcoxon paired test)aPLCFELYPRESSIN vs. PLCLUV. bPLCFELYPRESSIN vs. control (saline solution).

cPLC vs. control (saline solution).

Figure 5. Photomicrographs showing histological aspects of tissue reaction caused by the injection oftested formulations as examples of the used score values (H&E, ×100). A:Saline (4 days)—score 1;B:LUV (24 hours)—score 2; C:PLCLUV (6 hours)—score 3; D:PLC (6 hours)—score 4; E:PLCFELY-

PRESSIN (6 hours)—score 5.

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Liposomal Prilocaine: Stability and Local Toxicity 337

and PLCFELYPRESSIN were not able to evoke rat-paw edema. This result is in agreement with theliterature, which describes PLC as one of the less allergenic local anesthetics (Ball, 1999).Besides, it shows that encapsulation into liposomes did not elicit local toxic effects, asexpected from the well-known biocompatibility and low toxicity of liposomes (Grant andBansinath, 2001), attributed to their composition, that resemble those of biological membranes(Malinovsky et al., 1997). Moreover, in histological analysis of rats oral mucosa, nonsignificant differences were found between PLCLUV and its control group (p > 0.05). On theother hand, PLCLUV presented a lower intensity of inflammatory reaction than that ofPLCFELYPRESSIN (p < 0.01). This result can be explained by the PLCFELYPRESSIN vasocon-striction action, which is an important cause of cell injury (Cotran et al., 1999).

Conclusion

In this framework, the results presented here show that PLCLUV did not induce inflamma-tory effects on the rat paw and induced less inflammatory reaction in the rat oral mucosathan that of the commercial vasoconstrictor-containing formulation, PLCFELYPRESSIN.These findings may be promising for the future application of liposomal local anestheticsformulations with enhanced safety. Further, according to stability assays, PLCLUV is stableup to a 30-day period. These results are important for upcoming scale-up experiments,since in a previous study (Cereda et al., 2004), this formulation was considered a goodchoice to replace vasoconstrictor-containing local anesthetic formulations when the vaso-active compound is contraindicated.

Acknowledgements

The authors thank PhD Dr. Maria Silvia Viccari Gatti for her contribution in the microbiologic tests;Mr. Márcio Paschoal and Ms. Daniela Ceratti for their technical assistance; and Cristália Prod.Quim. Farmac. Ltda for the PLC samples and for the pyrogen tests. C.M.S.C. and E.P. received fel-lowships from CAPES (Comissão de Aperfeiçoamento de Pessoal do Ensino Superior) and CNPq(Conselho Nacional de Desenvolvimento Cientifico e Technológico), respectively. This work wassupported by FAPESP (Proc. 06/00121–9).

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