n - methyl-2-pyrrolidone ( nmp ) ( fluka , new jersey , usa ) was used as received .
sabouraud dextrose agar ( sda ) , sabouraud dextrose broth ( sdb ) , tryptic soy agar ( tsa ) and tryptic soy broth ( tsb ) ( difco , usa ) were used as media for antimicrobial test .
the 20% w / w lf127 solutions containing 10 , 20 , 30 , 40 , 50 , 60 and 80% w / w nmp were prepared using a cold method . briefly , lf127 was dissolved in distilled water at 4 until a homogeneous solution was obtained and nmp was subsequently added to with the polymer solution .
the ph values of the systems were measured using a ph meter ( professional meter pp-15 sartorius , goettingen , germany ) .
the test tube inverting method as described previously was employed to roughly determine the phase boundary .
gelation and gel melting temperatures were assessed using a modification of the previously reported technique .
five millilitre aliquot of gel was transferred to a test tube ( internal diameter of 1.8 cm ) and sealed before immersing in a thermostat water bath at 4. the temperature was controlled at the heating rate of 0.2/min , and the transition temperature was monitored at an accuracy of 1. the samples were then examined for gelation , which was said to have occurred when the meniscus would no longer move upon tilting through 90 ( n=3 ) .
the gel melting temperature , the temperature at which a gel started flowing upon tilting through 90 , was recorded ( n=3 ) .
the rheological behavior of prepared systems was investigated by recording their shear stress ( f ) as a function of shear rate ( g ) with a brookfield dv - iii ultra programmable rheometer ( brookfield engineering laboratories .
the measurements were conducted at three different temperatures ( 4 , 28 and 35 ) ( n=3 ) .
these three conditions were selected to represent the temperature in refrigerator , room temperature and temperature of human body , respectively .
the continuous check for viscosity change with temperature was rather difficult to perform since there was notably different apparent viscosity of system at different temperature to measure with the same cone of the rheometer .
rheological behavior of the gels is expressed as n value ( flowing parameter ) of the martin 's equation given by , log g = nlog f  log ' ...
(1 ) , where  ' is apparent viscosity . for n value of 1 or close to 1 ,
a rheological behavior is newtonian and for n value not equal to 1 , a rheological behavior is non - newtonian but are dilatant ( if n>1 ) and pseudoplastics ( if n<1 ) , or plastics if yield value was able to be determined .
the standard microbes used in this study were staphylococcus aureus ( atcc 6538p ) , escherichia coli ( atcc 10536 ) and candida albicans ( atcc 17110 ) .
. the actively growing broth culture of microbes was prepared and the turbidity was adjusted to contain approximately 10 cells / ml .
then , the swab was spread onto the agar plate in three directions to cover whole plate .
the samples were filled into the cup and incubated at 37 for 24 h. antimicrobial activities were determined by measuring diameters ( cm ) of clear zones of growth inhibition .
the 20% w / w lf127 solutions containing 10 , 20 , 30 , 40 , 50 , 60 and 80% w / w nmp were prepared using a cold method . briefly , lf127 was dissolved in distilled water at 4 until a homogeneous solution was obtained and nmp was subsequently added to with the polymer solution .
the ph values of the systems were measured using a ph meter ( professional meter pp-15 sartorius , goettingen , germany ) .
the test tube inverting method as described previously was employed to roughly determine the phase boundary .
gelation and gel melting temperatures were assessed using a modification of the previously reported technique .
five millilitre aliquot of gel was transferred to a test tube ( internal diameter of 1.8 cm ) and sealed before immersing in a thermostat water bath at 4. the temperature was controlled at the heating rate of 0.2/min , and the transition temperature was monitored at an accuracy of 1. the samples were then examined for gelation , which was said to have occurred when the meniscus would no longer move upon tilting through 90 ( n=3 ) .
the gel melting temperature , the temperature at which a gel started flowing upon tilting through 90 , was recorded ( n=3 ) .
the rheological behavior of prepared systems was investigated by recording their shear stress ( f ) as a function of shear rate ( g ) with a brookfield dv - iii ultra programmable rheometer ( brookfield engineering laboratories .
the measurements were conducted at three different temperatures ( 4 , 28 and 35 ) ( n=3 ) .
these three conditions were selected to represent the temperature in refrigerator , room temperature and temperature of human body , respectively .
the continuous check for viscosity change with temperature was rather difficult to perform since there was notably different apparent viscosity of system at different temperature to measure with the same cone of the rheometer .
rheological behavior of the gels is expressed as n value ( flowing parameter ) of the martin 's equation given by , log g = nlog f  log ' ...
(1 ) , where  ' is apparent viscosity . for n value of 1 or close to 1 ,
a rheological behavior is newtonian and for n value not equal to 1 , a rheological behavior is non - newtonian but are dilatant ( if n>1 ) and pseudoplastics ( if n<1 ) , or plastics if yield value was able to be determined .
the standard microbes used in this study were staphylococcus aureus ( atcc 6538p ) , escherichia coli ( atcc 10536 ) and candida albicans ( atcc 17110 ) .
the actively growing broth culture of microbes was prepared and the turbidity was adjusted to contain approximately 10 cells / ml .
then , the swab was spread onto the agar plate in three directions to cover whole plate .
the samples were filled into the cup and incubated at 37 for 24 h. antimicrobial activities were determined by measuring diameters ( cm ) of clear zones of growth inhibition .
the 20% w / w lf127 gel base was transparent liquid at 4 whereas the transparent semisolid gel was formed at 27 and 37. the 20%w / w lf127 systems containing < 40% w / w nmp were transparent liquid at 4 and the preparations containing > 50% nmp exhibited as cloudy gel . on the other hand , at 27 and 37 , the prepared systems containing < 40% w / w nmp exhibited as transparent gel and the systems containing more than 50% w / w nmp exhibited as transparent solution .
these results indicated that the appearance of lf127 systems was affected by the amount of nmp .
the phase behavior and microstructure of poloxamer block copolymers was also affected by various organic solvents such as glycerol , propylene glycol and ethanol .
it was shown that although polar and completely miscible with water , the peo - resembling and the ppo - resembling solvents such as nmp could affect the curvature of the self - assembled structure in opposite directions .
the peo - resembling solvents are located in the polar peo - rich domains far from the interface and increase the curvature .
the ppo - resembling solvents are preferably located in the relatively apolar ppo rich domains and at the interface affecting the curvature constant .
the ph values of gel preparations comprising nmp were in the range of 5.16 + 0.03 to 5.270.02 .
these results indicated that the nmp amount slightly changed ph value of gel preparations because the ph value of nmp was near to that of the gel base .
as desired for any topical and other preparations , the ph of gel was close to the skin ph which is approximately 5.32 .
effect of nmp on sol - gel and gel - sol transitions is shown in fig .
gelation temperature is a temperature that system was changed from sol to gel state , whereas gel melting temperature is a temperature that system was changed to sol state .
sol - gel transition temperature of 20% w / w lf127 gel without nmp was 23.70.6c .
the addition of nmp influenced the sol - gel and gel - sol transitions of gels .
lf127 system with lower nmp ( 30% w / w ) could shift the sol - gel transition to a lower temperature but the gel - sol transition was shifted to a higher temperature .
lf127 system with higher nmp ( 40% w / w ) could shift both sol - gel and gel - sol transitions to a lower temperature .
thus , the gelation range of systems containing < 30% w / w nmp was broader than that of the systems containing > 50% w / w nmp .
the sol - gel transition temperature of gel comprising 40% w / w nmp was not found because this system formed gel even at low temperature at 0. these results suggested nmp decreased the gelation temperature of the system .
the prior studies reported that the gelation of thermosensitive gel was affected by various factors , such as temperature and concentrations of polymer , active ingredients / excipients and electrolytes . the gel formation related to micellar packing and volume fraction .
the type of structures obtained in the presence of selective solvent appeared to be a function of the volume fraction of the polar / nonpolar components . the effects could be viewed in terms of a reduction of water activity leading to an increase in the active concentration of polymer in systems .
the decreased gelation temperature was owing to the interaction between the hydrophobic portions of the polymer molecules , which could disrupt the micellar structure and increase the entanglement of micelles .
most of flavors increased the viscosity of the poloxamer aqueous solution and decreased the gelling point of poloxamer 407 aqueous solution in proportion to amount added .
the flavors might bind to the hydrophilic end chains of poloxamer , promoting dehydration resulting in a decrease in the gelation temperature of polymer solution .
this is in agreement with previous findings on the effects of hydrophobic molecules on the gel formation of peo - ppo - peo block copolymer systems .
for example , t - butylbenzene could reduce the gelation temperature of lf127 while benzoic acid and p - hydroxybenzoate esters can cause a decrease in the gelation temperature of the block copolymer .
thus , the more hydrophobic the solute the greater the decrease in gelation temperature . there was a decreased gelation point of lf127 system with increasing amount of lidocaine and prilocaine .
this effect was owing to the presence of hydrophobic component inducing micellization resulting in the micellar growth .
the ambiguous sol - gel transition temperature of gel comprising 40% w / w nmp might be due to the equilibrium of suitable amount nmp for dehydration of the lf127 molecule , therefore the gel was formed at low temperature .
flow curves of the systems comprising 20% w / w lf127 and different concentrations of nmp at different temperatures , 4 , 27 and 37 , are shown in figs . 2 , 3 and 4 , respectively .
the incorporated nmp caused an apparent change in the flow curve of the lf127 system .
the flow curve shifted from low to high shear stress along the temperature increased when the concentration of nmp was less than 20% .
the unique flow curve with initially increased and subsequently rapidly decreased shear stresses was evident for the systems containing 50 and 60% nmp at 4 because their initial gel strength was destroyed after shear rate was enhanced .
as the concentration of nmp was higher than 50% , the flow curve shifted from high to low shear stress when the temperature increased .
the change of temperature slightly affected the flow of systems containing 30 - 40% w / w nmp .
the flow curve of system was the non - newtonian behavior that the up curve did not coincide with the down curve indicating the presence of thixotropy , with a hysteresis loop .
but the area of the hysteresis loop of all prepared systems did not increase as the nmp concentration increased .
however , the flow curve moved to a higher shear stress value , indicating the higher gel compactness .
the flow parameters of lf127 systems containing different concentrations of nmp at 4 , 27 and 37 are shown in table 1 .
the n values of the l systems containing 0 - 20% nmp increased with increasing temperature .
the n values of 0 - 20% nmp systems at 4 were close to 1 indicating a newtonian flow characteristic , while these values at higher temperature were>1 indicating a non - newtonian flow characteristic .
the n values of the prepared gels containing 30 - 40% nmp were>1 , which were not affected by the temperature . the increased nmp concentration from 50 to
the n values of the prepared gels containing 50 - 80% nmp at 4 were more than one , indicating the non - newtonian flow .
moreover , the n values of 50 - 80% nmp gels at 37 close to 1 indicating the newtonian flow characteristic .
the viscosity coefficients (  ) of systems containing 0 - 20% nmp increased whereas that of 30 - 40% nmp slightly increased with temperature . on the other hand ,
the viscosity coefficient of systems containing 50 - 80% w / w nmp decreased when the temperature increased corresponding with above obtained flow curves .
flow parameters of the aqueous l systems containing different concentrations of nmp at different temperatures ( n=3 ) antimicrobial activity of gels containing different concentrations of nmp against s. aureus , e. coli and c. albicans ( fig .
antibacterial activity against s. aureus and e. coli was increased as the amount of nmp was increased from 20 - 40% w / w while antifungal activity against c. albicans was enhanced by increasing the nmp amount .
the inhibition zones of pure nmp against s. aureus , e. coli and c. albicans were 1.90.1 , 2.00.2 and 3.50.1 cm , respectively ( fig .
therefore , nmp could solubilize the lipid in cell membrane and promote the leakage of microbial cell membranes .
inhibition zones of nmp against three microbes ( s. aureus , e. coli and c. albicans ) using agar diffusion method ( n= 3 ) .
lower nmp amount ( 30% w / w ) could shift the sol - gel transition to a lower temperature but the gel - sol transition was shifted to a higher temperature .
higher nmp amount ( 40% w / w ) could shift both sol - gel and gel - sol transitions of that system to a lower temperature .
the amount of nmp > 60% could reverse the phase of the lf127 system to non - newtonian flow at 4 and the newtonian flow at high temperature .
aqueous lf127 system containing nmp exhibited antimicrobial activities against s. aureus , e. coli and c. albicans in a dose - dependent manner .
 the purpose of this study is to investigate the effects of n - methyl-2-pyrrolidone on the thermosensitive properties of aqueous ethylene oxide - propylene oxide block copolymer ( lutrol f127 ) system . due to the aqueous solubility enhancement and biocompatibility 
 , n - methyl-2-pyrrolidone is an interesting solubilizer for the poorly water soluble drugs to be incorporated in the lutrol f127 system . 
 effect of n - methyl-2-pyrrolidone on physicochemical properties of lutrol f127 system was investigated using appearance , ph , gelation , gel melting temperature and rheology . 
 the antimicrobial activity of the thermosensitive n - methyl-2-pyrrolidone gel was also tested . 
 lower n - methyl-2-pyrrolidone amount ( 30%w / w ) could shift the sol - gel transition to a lower temperature but the gel - sol transition was shifted to a higher temperature . 
 higher n - methyl-2-pyrrolidone ( 40%w / w ) could shift both sol - gel and gel - sol transitions of the system to a lower temperature . 
 the amount of n - methyl-2-pyrrolidone > 60% w / w could reverse the phase of the lutrol f127 system to non - newtonian flow at 4 and newtonian flow at high temperature . 
 aqueous lutrol f127 system containing n - methyl-2-pyrrolidone exhibited antimicrobial activities against staphylococcus aureus , escherichia coli and candida albicans with the n - methyl-2-pyrrolidone in a dose - dependent manner . 
MATERIALS AND METHODS
Preparation of Lutrol
Evaluation of physical properties of gel:
Gelation and gel melting temperature:
Rheological behavior studies:
Antimicrobial studies:
RESULTS AND DISCUSSION