Development of novel topical tranexamic acid liposome formulations

A. Manosroi et al. / International Journal of Pharmaceutics 235 (2002) 61–70 65and derivatized, following the procedures as describedin the analytical method section, prior toassay by spectrophotometer. The loading of TAin liposomes was also calculated. Each samplewas prepared in two lots and the experimentswere performed in duplicates.2.5. Analytical method for the determination ofTA contentThe TA content in liposome formulations wasdetermined spectrophotometrically at 415 nm(Milton Roy Spectronic 1001 Plus, Rochester,NY), following derivatization with 2,4,6-trinitrobenzosulfonicacid (Atmaca, 1989). The colorreagent used was 1.68% (w/v) 2,4,6-trinitrobenzosulfonicacid solution in DI water, freshly preparedand protected from light, prior to use. Each0.1 ml of working standard solutions or sampleswas spiked with 0.25 ml of 0.025 M disodiumtetraborate solution (pH 10) and 0.25 ml of colorreagent, prior to standing at 25 °C for 30 min.The solution was then diluted to 5.0 ml with 0.1M potassium dihydrogen phosphate solution (pH4.5). The solution mixture in the absence of TAwas used as a blank. A calibration graph wasconstructed, by plotting the concentrations of TAagainst the absorbance values. The linearity ofassay was determined from five working standardsolutions of TA in DI water (concentrations: 4.0–20.0 g/ml), prepared in triplicates. The correlation(r 2 ), intercept and slope of the calibrationgraph were calculated. The absorbance values ofsamples were observed and compared with thoseobtained from the calibration graph, to determinethe amount of TA in samples. This experimentwas performed in duplicates of three lots ofsamples.3. Results3.1. Physical appearances and pH of liposomesThe appearances and pH of various liposomeformulations, DI water, 5 and 10% TA solutionsin DI water are shown in Table 2. The neutral 7:2,7:2(5%TA) and 7:2(10%TA) liposomes showedsedimentation, following storage at 4 °C for 24 h(Table 2). Thus, the charged liposomes {7:2:1(+),7:2:1(5%TA, +), 7:2:1(10%TA, +), 7:2:1(−),7:2:1(5%TA, −) and 7:2:1(10%TA, −)} demonstratingfavorable physical appearances (no sedimentation)were selected for further experiments(Table 2). The pH values of both positively andnegatively charged liposome formulations withthe entrapped TA (pH 6.9–8.0) were higher thanTable 2Physical appearances and pH of various liposome formulations,DI water, 5 and 10% TA solutions in DI waterFormulations Appearances pH a7:2 Translucent, white ND b7:2(5%TA)7:2(10%TA)dispersion withsedimentationTranslucent, whitedispersion withsedimentationTranslucent, whiteND bND b7:2:1(+)7:2:1(5%TA, +)dispersion withsedimentationTranslucent, whitedispersion, nosedimentationTranslucent, white7.590.067.920.027:2:1(10%TA, +)dispersion, nosedimentationTranslucent, white 7.860.037:2:1(−)dispersion, nosedimentationTranslucent, white 3.40.1dispersion, nosedimentation7:2:1(5%TA, −)7:2:1(10%TA, −)DI waterTranslucent, whitedispersion, nosedimentationTranslucent, whitedispersion, nosedimentationClear solution6.900.097.120.0066.300.065% TA in DI water Clear solution 7.510.0310% TA in DI water Clear solution 7.570.01Physical appearances of samples were visually observed, followingstorage at 4 °C for 24 h. pH was measured immediatelyafter sample preparation.aExperimental data represent the meanSD of three determinations.b ND, not determined.

66A. Manosroi et al. / International Journal of Pharmaceutics 235 (2002) 61–70Table 3Particle sizes of six liposome formulationsFormulationsParticle size (m) a7:2:1(+) 17.90.57:2:1(5%TA,+)17.50.57:2:1(10%TA,+)35.80.57:2:1(−)24.780.047:2:1(5%TA, −)2.760.027:2:1(10%TA, −)2.050.03Particle sizes of liposome formulations were measured 10 daysafter sample preparation (kept at 4 °C).aExperimental data represent the meanSD of six determinations.that of DI water (pH 6.3), except the blank negativeliposome {7:2:1(−)} (pH 3.4, Table 2). Thus,the incorporation of TA in liposomes may increasepH of the system.3.2. Liposome size, morphology and lamellarity ofliposomesThe particle sizes of six liposome formulationsare presented in Table 3. The 7:2:1(+), 7:2:1(−),7:2:1(5%TA, +) and 7:2:1(10%TA, +) liposomesdemonstrated bimodal distribution of particlesize and larger particle size (10 times) thanthe negatively charged liposomes with the entrappeddrug. The smallest size of 2.05 m wasobserved in the 7:2:1(10%TA, −) liposome. Allliposome formulations demonstrated log-normaldistribution of particle size, ranging between 2.0and 35.8 m (Table 3). The transmission electronmicrographs of the blank 7:2:1(+), 7:2:1(5%TA,+) and 7:2:1(5%TA, −) liposomes, showing themorphology and lamellarity of liposomes areshown in Fig. 1. The lamellarity of about 8–15bilayers (multilamellar vesicles) was observed inall liposome formulations (Fig. 1).3.3. Glass transition temperature, transitiontemperature and enthalpy of transitionThe glass transition temperatures of HSC,CHL, SA, DCP, TA, 7:2:1(10%TA, +) and7:2:1(10%TA, −) liposome formulations are presentedin Table 4. The results indicated that allsamples were decomposed at high temperature(200 °C), except SA which decomposed atlower than 200 °C. The T c and H values ofHSC, CHL, SA, DCP, TA, 7:2:1(+),7:2:1(5%TA, +), 7:2:1(10%TA, +), 7:2:1(−),7:2:1(5%TA, −) and 7:2:1(10%TA, −) liposomeformulations are summarized in Table 5. Underthe experimental conditions, there was no peak ofTA observed in the DSC thermogram.3.4. Tranexamic acid entrapped in liposomesThe calibration graph of TA solution in DIwater was shown to be linear (r 2 =0.9937), overthe concentration range 4.0–20.0 g/ml. The regressionequation was as follows: y=20.7903x−0.1942, where y is the concentration of TA(g/ml) and x is the absorbance of the derivativeof TA formed with 2,4,6-trinitrobenzosulfonicacid (mAU*s). The percentages of TA recoveredfrom the liposome formulations with the entrappeddrug, using spectrophotometric assayvaried between 91.4 and 104.7%. The other componentsin liposome formulations neither reactedwith the color reagent nor demonstrated significantabsorption at 415 nm. The percentages ofTA entrapped in liposomes, the free drug and theloading of TA in liposome formulations areshown in Table 6.4. DiscussionThe charged liposomes with and without theentrapped TA, showing no sedimentation followingstorage at 4 °C for 1 day, may indicate betterphysical stability than the neutral liposomes(Table 2). This may be associated with the effectsof charges on the surface of liposomes. TA is asynthetic amino acid, which has amino and carboxylicgroups. When TA dissolves in water, itwill ionize in equilibrium Eq. (1):C 8 H 15 NO 2 +H 2 O C 8 H 16 NO 2+ +OH − (1)The pH of samples increased when TA was incorporatedinto the liposome systems (Table 2). Thismay be due to the effect of concentration of

A. Manosroi et al. / International Journal of Pharmaceutics 235 (2002) 61–70 67hydroxyl ion (Eq. (1)). TA in DI water appearedto have weak positive charges (Eq. (1)). The positivelycharged liposomes with the entrapped TAdemonstrated higher pH values than those withoutthe entrapped drug (Table 2). The entrappedTA may provide more hydroxyl ions to the liposomedispersion sample (Eq. (1)). Similarly, thenegatively charged liposomes with the entrappedTA showed higher pH values than the blanknegative liposome (Table 2).SA and DCP are amphiphilic compounds, andthey may ionize when incorporated into the liposomesand aqueous environments, as shown in thefollowing equations:C 18 H 39 N+H 2 O C 18 H 40 N + +OH − (2)C 32 H 67 O 4 P+H 2 O C 32 H 66 O 4 P − +H 3 O + (3)The positively charged liposomes received hydroxylion from SA (Eq. (2)), whereas the negativelycharged liposomes received hydronium ionfrom DCP (Eq. (3)). Thus, all positively chargedliposomes (pH 7.6–7.9) exhibited higher pH valuesthan all negatively charged ones (pH 3.4–7.1,Table 2). Hydronium ion from DCP (Eq. (3)) maybe neutralized by hydroxyl ion, resulting from theionization of TA (Eq. (1)).SA and DCP incorporated into the liposomalbilayer membranes render the surface electricallyFig. 1. Transmission electron micrographs of the: (a) blank 7:2:1(+) liposomes (6000×); (b) 7:2:1(5%TA, +) liposomes (5000×);(c) 7:2:1(5%TA, −) liposomes (6000×).

68A. Manosroi et al. / International Journal of Pharmaceutics 235 (2002) 61–70Table 4Glass transition temperatures of HSC, CHL, SA, DCP, TA,7:2:1(10%TA, +) and 7:2:1(10%TA, −) liposome formulationsFormulationsHSC257.77CHL246.66SA 162.22DCP218.47TA223.637:2:1(10%TA, +) 222.227:2:1(10%TA, −) 229.63Glass transition temperature (°C)like charges. This results in a repulsion and hence,an increase in the distance between different bilayers(Benita et al., 1984). Similar charges of lipidbilayers to the entrapped TA can increase thedistance between bilayers, as observed in the positivelycharged liposomes with large particle size(17.5–35.8 m, Table 2). In contrast, the negativecharges of lipid bilayers, opposite to the chargesof entrapped TA will decrease the distance (liposomesize: 2.0–2.8 m), due to the neutralizationof charges (Table 2). Thus, the smallest particlesize was observed in the negative liposome withthe entrapped TA concentration of 10% (Table 2).Table 5Transition temperatures and enthalpy of transition of HSC,CHL, SA, DCP, TA, 7:2:1(+), 7:2:1(5%TA, +),7:2:1(10%TA, +), 7:2:1(−), 7:2:1(5%TA, −) and7:2:1(10%TA, −) liposome formulationsFormulationsTransitiontemperature ( °C)Enthalpy oftransition (J/g)HSC61.20.4 38.59.8CHL149.70.2 66.61.0SA57.10.6 293.753.0DCP79.30.2 187.01.87:2:1(+) 77.80.4 50.07.37:2:1(5%TA, +) 76.20.4 11.80.37:2:1(10%TA, +) 77.11.18.32.47:2:1(−)70.00.5 45.410.67:2:1(5%TA, −) 71.30.5 14.72.87:2:1(10%TA, −) 70.80.56.33.2There was no peak of TA observed in the DSC thermogram.Experimental data represent the meanSD of six determinations.The T c values of the positively and negativelycharged liposome formulations with and withoutthe entrapped TA were broadly comparable(70.8–77.1 °C vs. 70.0–77.8 °C). However, theH values of blank positively and negativelycharged liposome formulations were higher(45.4–50.0 J/g) than those of the respective formulationswith the entrapped drug (6.3–14.7 J/g,Table 5). This may indicate that TA was entrappedin the aqueous layer of the charged liposomes.TA may interfere with the formation oflipid bilayers, thereby reducing the H values ofliposome formulations. The higher the entrappedTA concentration in liposomes, the more markedreduction of H was observed (Table 5). TA didnot exhibit T c and H, when scanned in the rangeof 20–200 °C, indicating high thermal stability ofthis drug.All liposome formulations with and without theentrapped TA exhibited an endothermic peak,with an average maximum peak of T c (70.0–77.8 °C) higher than HSC (61.2 °C, Table 5).This may indicate that cholesterol incorporatedinto the liposomal membrane had its hydroxylgroup oriented towards the aqueous layer, withthe aliphatic chain aligned parallel to the acylchains in the center of lipid bilayers. The presenceof rigid steroid nuclei along side the first 10 or soof carbons of the phosphatidylcholine chain ofHSC may reduce the freedom of movement. Thismay result in an enhanced stability of the liposomalmembrane, and hence the high T c value ofliposome formulations.Reaction between TA and 2,4,6-trinitrobenzosulfonicacid in alkaline condition forms a yellowcolor compound, with the maximum absorptionspectrum of 415 nm (Atmaca, 1989). The optimumcondition for the reaction to occur is asfollows: pH 10 at 25 °C for 30 min. The 0.1 Mpotassium dihydrogen phosphate buffer solution(pH 4.5) was added to terminate the reaction. Theintensity of color was relatively stable in the reactionmedium, for at least 3 h when protected fromlight.The percentages of TA entrapped in all liposomeformulations varied between 13.2 and 15.6%(Table 6). There were no significant differences inthe percentages of entrapment of TA between

A. Manosroi et al. / International Journal of Pharmaceutics 235 (2002) 61–70 69Table 6Percentages of entrapment of TA, free TA, and the amount of TA per total lipid in liposome formulationsFormulationsForm of drug% EntrapmentLoading of TA (g/mg lipid) a7:2:1(5%TA, +)Total drug 100.0–Entrapped drug14.70.6275.213.9Free drug85.30.2 –7:2:1(10%TA, +) Total drug100.0 –Entrapped drug15.00.4627.010.8Free drug85.00.9 –7:2:1(5%TA, −) Total drug100.0–Entrapped drug 15.60.7 290.45.7Free drug84.52.6 –7:2:1(10%TA, −) Total drug 100.0 –Entrapped drug 13.20.3 547.313.6Free drug86.80.3 –Experimental data represent the meanSD of four determinations.a Total lipid in 1 ml of liposome dispersion sample was 25 mg.different formulations (Table 6), as analyzed byANOVA (P0.05). This may indicate that thecharges and TA concentrations did not affect thepercentages of TA entrapped in liposome formulations.The negatively charged liposome whichhad opposite charged to TA (weak positive) mayimprove the entrapment of drug in the liposomes.On the other hand, the positively charged liposomeswith the entrapped drug demonstratedlarger particle size, and hence larger volume forthe entrapment of drug than the negative liposomes(Benita et al., 1984). These phenomena mayexplain why the percentages of entrapment of TAin the positive and negative liposomes werebroadly comparable (14.7–15.0% vs. 13.2–15.6%,Table 6).The loading of TA in the charged liposomesincreased, as the initial concentration of drugincreased, from 275 g/mg lipid {7:2:1(5%TA,+)} to 627 g/mg lipid {7:2:1(10%TA, +)}, andfrom 290 g/mg lipid {7:2:1(5%TA, −)} to 547g/mg lipid {7:2:1(10%TA, −)} (Table 6, Foldvariet al., 1993).In conclusion, the liposomes composed ofHSC/CHL/charged lipids at molar ratios of7:2:1(+) and 7:2:1(−) with the entrapped TA (5and 10% solutions in DI water) were demonstratedas multilamellar vesicles. Thus, TA wasadvantageous when entrapped in liposome, sincethis drug delivery system can potentitally reducethe skin irritation and improve moisturizing effect.The particle sizes of all liposome formulations,with and without the entrapped TA were inthe range of 2.05–35.8 m, with the smallest sizewas observed in the negative liposome with theentrapped TA concentration of 10%. The chargedliposomes with the entrapped TA demonstratedhigh physical stability. The concentrations of TAappeared to affect the pH, particle size and enthalpyof transition of liposome formulations,whereas charges seemed to affect the physicalstability, pH and particle size of liposomes. Thebest formulation was concluded to be the negativelycharged 7:2:1(10%TA, −) liposome, whichexhibited high physical stability, small particle sizeand relatively high percentage of drug entrapment.This formulation will be further evaluatedfor release study. Overall, it has been demonstratedthat TA which is a hydrophilic drug canbe favorably entrapped in liposomes.AcknowledgementsThe authors thank Dr Kuncoro Foe for hisassistance in the preparation of the manuscript.We also acknowledge JJ-Degussa (T) Ltd., Thailandfor the gifts of Emulmetik 950 ® , and Thistle