molecules

Communication -Based Thermoreversible Gel for Topical Delivery of Emodin: Influence of P407 and P188 on Solubility of Emodin and Its Application in Cellular Activity Screening

Eunmi Ban, Mijung Park, Seonghee Jeong, Taekhyun Kwon, Eun-Hee Kim, Kiwon Jung and Aeri Kim * College of Pharmacy, CHA University, 521 CHA Bio Complex, 335 Pangyo-ro, Bundang-gu, Seongnam 463-400, Korea; [email protected] (E.B.); [email protected] (M.P.); [email protected] (S.J.); [email protected] (T.K.); [email protected] (E.-H.K.); [email protected] (K.J.) * Correspondence: [email protected]; Tel.: +82-31-881-7166; Fax: +82-31-881-7219

Academic Editor: Guy Van den Mooter Received: 27 December 2016; Accepted: 2 February 2017; Published: 7 February 2017

Abstract: Emodin is a component in a Chinese herb, Rheum officinale Baill, traditionally used for diabetes and anticancer. Its poor solubility is one of the major challenges to pharmaceutical scientists. We previously reported on thermoreversible gel formulations based on poloxamer for the topical delivery of emodin. The present study was to understand the effect of poloxamer type on emodin solubility and its application in cellular activity screening. Various gel formulations composed of poloxamer 407 (P407), poloxamer 188 (P188) and PEG400 were prepared and evaluated. Major evaluation parameters were the gelation temperature (Tgel) and solubility of emodin. The emodin solubility increased with increasing poloxamer concentration and the Tgel was modulated by the proper combination of P407. In particular, this study showed that the amount of P407 in thermoreversible poloxamer gel (PG) was the dominant factor in enhancing solubility and P188 was effective at fixing gelation temperature in the desired range. A thermoreversible emodin PG was selected as the proper composition with the liquid state at room temperature and gel state at body temperature. The gel showed the solubility enhancement of emodin at least 100-fold compared to 10% ethanol or . The thermoreversible formulation was applied for in vitro cellular activity screening in the human dermal fibroblast cell line and DLD-1 colon cancer cell line after dilution with cell culture media. The thermoreversible gel formulation remained as a clear solution in the microplate, which allowed reliable cellular activity screening. In contrast, emodin solution in ethanol or DMSO showed precipitation at the corresponding emodin concentration, complicating data interpretation. In conclusion, the gel formulation is proposed as a useful prototype topical formulation for testing emodin in vivo as well as in vitro.

Keywords: thermoreversible; poloxamer; emodin; solubility; in vitro cellular activity screening

1. Introduction Emodin, 1,3,8-trihydroxy-6-methyl-anthraquinone (Figure1), is an anthraquinone derivative from the roots of rhubarb (Rheum officinale Baill) [1]. Emodin reportedly possesses a number of anti-inflammatory, wound healing and anti-tumor activities [2,3]. Oral bioavailability of emodin is very low because of extensive glucuronidation in and intestine [4]. Therefore, the topical application of emodin will be an attractive alternative to oral route for improved bioavailability with less effect by liver or intestinal metabolism. Emodin is known to be soluble in DMSO, ethanol, or alkali solution but insoluble in water. Low aqueous solubility creates obstacles to conducting and

Molecules 2017, 22, 246; doi:10.3390/molecules22020246 www.mdpi.com/journal/molecules Molecules 2017, 22, 246 2 of 10 Molecules 2017, 22, 246 2 of 10 interpretingvivo and in invitro vivo studiesand in vitrofor thestudies evaluation for the of evaluation toxicity or of toxicitypharmacological or pharmacological activity of activitybioactive of bioactivemolecules, molecules, including including new drug new candidates drug candidates that thathave have demonstrated demonstrated clinical clinical effects effects by by several mechanisms. Specifically,Specifically, during duringin vitroin vitrostudies studie conducteds conducted in cell in culture cell mediaculture to media evaluate to biologicalevaluate propertiesbiological suchproperties as efficacy, such andas genotoxicity,efficacy, and poorlygenotoxicity, soluble drugpoorly candidates soluble drug might candidates precipitate might in the medium,precipitate leading in the to medium, inaccurate leading information to inaccurate regarding information the drug’s propertiesregarding [5the]. Solubilitydrug’s properties is also a very [5]. importantSolubility factoris also for a very performing important a functional factor for and performing pharmacological a functional study and of natural pharmacological compounds study because of natural compoundscompounds suchbecause as emodin natural show compounds activity such at concentration as emodin show of a few activity tens ofat µconcentrationM level. Actually, of a pharmacologicalfew tens of μM level. research Actually, of emodin pharmacological has many research limitations of emodin due to thehas poormany water limitations solubility due to of the emodin poor althoughwater solubility emodin of has emodin potential although for the emodin treatment has ofpotential various for conditions. the treatmen Therefore,t of various improvement conditions. of emodinTherefore, solubility improvement is desirable of emodin for the evaluationsolubility is of de itssirable pharmacological for the evaluation and functional of its pharmacological activity. To date, variousand functional approaches activity. to improving To date, various solubility approaches have been to improving investigated, solubility including have physical been investigated, or chemical modifications,including physical and or the chemical use of solubilizers modifications, or and the use [5 –of8 ].solubilizers Among these or surfactants approaches, [5–8]. the useAmong of a solubilizerthese approaches, or a the use of is a solubilizer simple and or direct a surfactant method is for a simple improving and direct drug solubility.method for , improving composeddrug solubility. of various Poloxamers, sizes of composed polymer poly(ethylene of various sizes oxide) of polymer (PEO) and poly(ethylene poly(propylene oxide) oxide) (PEO) (PPO) and blockspoly(propylene are frequently oxide) used (PPO) with blocks poorly are water-soluble frequently compounds,used with poorly as they water-soluble are effective surfactants,compounds, and as lackthey knownare effective toxicity. surfactants, Poloxamers and have lack also known been used toxicity. in the Poloxamers development have of thermoreversible also been used gelsin the for topicaldevelopment drug deliveryof thermoreversible [9]. Poloxamer gels 407for (P407)topical anddrug poloxamer delivery [9]. 188 Poloxamer (P188) are 407 the (P407) polymers and poloxamer frequently used188 (P188) for preparation are the polymers of thermoreversible frequently used gels for and prepar enhancementation of thermoreversible of drug solubility, gels respectively.and enhancement Topical of formulationsdrug solubility, of respectively. poloxamer-based Topical thermoreversible formulations of poloxamer-based gels are being modifiedthermoreversible to achieve gels improved are being compliancemodified to achieve and bioavailability improved comp [10].liance Poloxamer and bioavailability gels (PG) exist [10]. as Poloxamer low viscosity gels solutions(PG) exist at as room low temperature,viscosity solutions but exist at room as gel temperature, at body temperature, but exist makingas gel at them body ideal temperature, thermosensitive making polymers them ideal for topicalthermosensitive delivery. polymers Therefore, for poloxamer-based topical delivery. thermoreversibleTherefore, poloxamer-based gel is a very thermoreversible attractive formulation gel is a forvery topical attractive delivery formulation through for the topical body delivery surface toth reducerough the metabolism body surface and to improve reduce themetabolism solubility and of emodin.improve the Many solubility researchers of emodin. have studiedMany researcher the effects ofhave varying studied P407/P188 the effect ratios of varying on properties P407/P188 of thermoreversibleratios on properties gels of including thermoreversible gelation temperature gels including [11 –gelation14]. Ban temperature et al. [14] reported [11–14]. the Ban optimization et al. [14] ofreported emodin the thermoreversible optimization of emodin PG for properthermoreversible Tgel and emodin PG for proper solubility Tgel by and quality emodin by design.solubility In by a previousquality by study, design. we In observed a previous that study, poloxamers we observ are capableed that of poloxamers enhancing are the watercapable solubility of enhancing of poorly the solublewater solubility drugs and of P407 poorly is especially soluble moredrugs effective and P407 than is P188especially on the more enhancement effective of than emodin P188 solubility. on the Inenhancement the present study,of emodin the influence solubility. of P407In the and presen P188t on study, thermoreversible the influence PG of properties P407 and and P188 on theon solubilitythermoreversible of a poorly PG water-solubleproperties and drug on wasthe solubi furtherlity examined of a poorly using water-soluble emodin because drug less was is knownfurther aboutexamined the relativeusing emodin effects because of P407 less and is P188 known on drugabout solubility. the relative Furthermore, effects of P407 the and thermoreversible P188 on drug PGsolubility. formulation Furthermore, was applied the thermoreversible for in vitro cellular PG formulation activity screening was applied in the for human in vitro dermal cellular fibroblast activity (HDF)screening cell in line the and human DLD-1 dermal colon cancerfibroblast cell line.(HDF) In cell our preparationsline and DLD-1 of PG, colon emodin cancer concentration cell line. In wasour constrainedpreparations atof aPG, minimum emodin concentration of 0.5 mg/mL, was because constrained previous at a topicalminimum administration of 0.5 mg/mL, studies because of previous emodin heldtopical its administration concentration studies at approximately of emodin held 400 µ itsg/mL concentration [15]. at approximately 400 μg/mL [15].

Figure 1. Chemical structure of emodin.

2. Results and Discussion

2.1. Comparison of P407 and P188 for the Solubilization of Emodin In this study, thermoreversiblethermoreversible PGPG containingcontaining emodin was prepared using P407 and P188, to determine the individual effects of P188 and P407 on emodin solubility. We measured the solubility of emodin in increasing concentrations of P188 and P407. As shown in Figure2 2,, emodinemodin solubilitysolubility increased as the concentrations of both P407 and P188 increased. This result demonstrated that both P407 and P188 have solubilizing properties, and could be used to increase the water solubility of

Molecules 2017, 22, 246 3 of 10 increased as the concentrations of both P407 and P188 increased. This result demonstrated that both P407 and P188 have solubilizing properties, and could be used to increase the water solubility of drugs. Of the two, P407 was more effective, particularly when the poloxamer concentration was higher than 10%, due to its higher hydrophobicity and lower critical micelle concentration than P188 (Figure2). When P407 concentration was 20%, the solubility of emodin was 0.42 mg/mL, which was an appropriate concentration for this study. However, in the case of P188, 40% of P188 was needed to reach 0.5 mg/mL of emodin solubility [14]. Based on these results, we conclude that 20% P407 or 40% P188 was required to solubilize 0.5 mg/mL emodin.

Figure 2. Effect of P407 and 188 concentration on emodin solubility.

2.2. Effect of P407 and P188 on Emodin Solubility and Gelation Temperature in Thermoreversible PG Thermoreversible PG for the topical delivery of emodin should be gel at skin and body temperature (32–36 ◦C), while existing as a solution at room temperature. However, PG composed of 20% P407 or 40% P188 solutions did not meet these criteria. The P407 solution gelled below 25 ◦C, and the 40% P188 solution failed to gel below 50 ◦C. In this study, two approaches were attempted to obtain PG with the proper gelation temperature and emodin solubility. The first approach was to add P188 to 20% of P407 to increase the gelation temperature with the total poloxamer concentration not greater than 40% (Table1). In our second approach, varying concentrations of P407 were added to P188 ranging between 20% and 35% with the total poloxamer concentration fixed at 40% (Table2). It was not feasible to test the total poloxamer concentration greater than 40% because the sample became too viscous to measure emodin solubility. In the first approach, as we expected, the solubility of emodin increased with the addition of P188 to 20% P407 (Figure3A). The gelation temperature of the PG solution increased from 25 ◦C to 33 ◦C with the addition of 5% P188 to the 20% P407 solution. However, when P188 was added to the 20% P407 solution higher than 5%, gelation temperature decreased (Figure3B), which might be due to shifting of the critical micelle concentration and PEO/PPO ratio at a high concentration of P188. When a larger amount of P188 is added to P407, micellization of P188 begins to predominate [13]. Our results show that PG composed of 20% P407 and 5%–15% P188 had a gelation temperature in the physiological range. Precipitation of emodin in PG during Molecules 2017, 22, 246 4 of 10 storage at low temperatures, probably due to the temperature-dependent shift in the critical micelle concentrationMolecules 2017 of, 22 poloxamer,, 246 has been observed. To preserve emodin solubility at low temperatures4 of 10 and retain the desired gelation temperature of PG, various concentrations of PEG400 were added were added according to the previous study. As shown in Figure 4, addition of PEG400 (5%–10%) to according to the previous study. As shown in Figure4, addition of PEG400 (5%–10%) to PG resulted in PG resulted in increased solubility but decrease in gelation temperature below 32 °C. Based on these increased solubility but decrease in gelation temperature below 32 ◦C. Based on these observations, observations, thermosensitive PG composed of P407/P188/PEG400 (20/10/0) was selected considering thermosensitiveits gelation temperature PG composed in the of desired P407/P188/PEG400 range (32–36 °C) (20/10/0) for the topical was selecteddelivery of considering emodin (Table its gelation1). temperature in the desired range (32–36 ◦C) for the topical delivery of emodin (Table1). Table 1. Composition and gelation temperature and the emodin solubility of thermoreversible Tablepoloxamer 1. Composition gel (PG) based and gelationon the poloxamer temperature 407 system. and the emodin solubility of thermoreversible poloxamer gel (PG) based on the poloxamer 407 system. Poloxamer (%, w/w) PEG400 (%, w/w) Gelation Temperature (°C) Solubility (mg/mL) P407 P188 Poloxamer (%, w/w) 20 0 PEG400 0 (%, w/w) Gelation 23.67 Temperature ± 1.15 (◦C) Solubility 0.42 ± 0.05 (mg/mL) P40720 P188 5 0 33.33 ± 0.58 0.47 ± 0.00 2020 0 5 5 0 31.00 23.67 ± ±0.001.15 0.50 0.42 ± 0.03± 0.05 2020 5 5 10 0 27.00 33.33 ± ±1.000.58 0.74 0.47 ± 0.01± 0.00 2020 5 10 0 5 33.33 31.00 ± ±0.580.00 0.61 0.50 ± 0.01± 0.03 2020 5 10 510 30.67 27.00 ± ±1.151.00 0.66 0.74 ± 0.01± 0.01 20 10 0 33.33 ± 0.58 0.61 ± 0.01 20 10 0 28.00 ± 1.73 0.85 ± 0.01 20 10 5 30.67 ± 1.15 0.66 ± 0.01 20 15 0 30.67 ± 1.15 0.63 ± 0.01 20 10 0 28.00 ± 1.73 0.85 ± 0.01 2020 15 15 5 0 26.33 30.67 ± ±0.581.15 0.85 0.63 ± 0.07± 0.01 2020 15 15 20 5 24.33 26.33 ± ±1.150.58 0.96 0.85 ± 0.00± 0.07 2020 15 20 020 25.67 24.33 ± ±0.581.15 0.90 0.96 ± 0.00± 0.00 20 20 0 25.67 ± 0.58 0.90 ± 0.00 Table 2. Composition and gelation temperature and the emodin solubility of thermoreversible PG Tablebased 2. Composition on the poloxamer and gelation188 system. temperature and the emodin solubility of thermoreversible PG based onPoloxamer the poloxamer (%, w 188/w) system. PEG400 (%, w/w)Gelation Temperature (°C)Solubility (mg/mL) P407 P188 Poloxamer (%, w/w) 10 30 PEG400 (%,0 w/w) Gelation36.5 Temperature ± 0.6 (◦C) 0.54Solubility ± 0.06 (mg/mL) P40710 P188 30 5 34.5 ± 0.5 0.56 ± 0.07 1010 3030 10 0 <25.0 36.5 ± 0.60.69 ± 0.05 0.54 ± 0.06 105 3035 50 38.7 34.5 ± 0.4± 0.50.50 ± 0.05 0.56 ± 0.07 105 3035 105 35.5 ± <25.0 0.9 0.52 ± 0.06 0.69 ± 0.05 55 3535 10 0 30.5 38.7 ± 0.4± 0.40.61 ± 0.06 0.50 ± 0.05 5 35 5 35.5 ± 0.9 0.52 ± 0.06 5 35 10 30.5 ± 0.4 0.61 ± 0.06

1.2

0.8

0.4 Solubility (mg/mL)

0 0 5 10 15 20 P188 concentration (%)

(A)

Figure 3. Cont.

Molecules 2017, 22, 246 5 of 10

Molecules 2017, 22, 246 5 of 10 Molecules 2017, 22, 246 36 5 of 10 )

36℃ 32 ) ℃ 32 28 28 24 24 Gelation temperature ( Gelation temperature

Gelation temperature ( Gelation temperature 20 20 0 5 10 15 20 0 5P188 10 concentration 15 (%) 20 P188 concentration (%)

(B) (B) Figure 3. Effect of addition of P188 to the 20% P407 solution on (A) the solubility of emodin and (B) FigureFigure 3. Effect 3. Effect of additionof addition of of P188 P188 toto the 20% 20% P407 P407 solution solution on ( onA) the (A) solubility the solubility of emodin of emodin and (B) and gelationgelation temperature. temperature. (B) gelation temperature.

(A)(A) 1.6 1.6

1.2 1.2

0.8 P188 0.15 0.8 P188 0.15 P188 0.1 0.4 P188 0.1 0.4 P188 0.05 P188 0.05 Solubility (mg/mL) 0 Solubility (mg/mL) 05100

0510PEG concentration (%)

PEG concentration (%) (B)

)

(B) ℃ 36 ) ℃ 36 32

32 28 P188 0.15

P188 0.1 24 28 P188 0.15 P188 0.05 P188 0.1

Gelation temperature ( Gelation temperature 20 24 P188 0.05 0510 PEG concentration (%) Gelation temperature ( Gelation temperature 20 0510 Figure 4. Effect of addition of PEG400PEG to concentration P188 and P407 mixture (%) at 20% P407 on (A) the solubility of emodin and (B) gelation temperature. Figure 4. Effect of addition of PEG400 to P188 and P407 mixture at 20% P407 on (A) the solubility of emodin andFigure (B) 4. gelation Effect of temperature. addition of PEG400 to P188 and P407 mixture at 20% P407 on (A) the solubility of emodin and (B) gelation temperature.

Molecules 2017, 22, 246 6 of 10 Molecules 2017, 22, 246 6 of 10

In our second approach, P407 was added to P188 solutionsolution to bring the gelation temperature of PG to within the range near the skin and body body temperature temperature (32–36 °C)◦C) as as in in the the above above study. study. Varying Varying concentrations of P407 were added to P188 while maintaining a total poloxamer concentration of 40%, and the gelation temperatures were measured atat various P407/P188 ratios ratios.. In In these these experiments, experiments, emodin solubility solubility increased increased and and gelation gelation temperatures temperatures decrease decreasedd with with increasing increasing P407 P407 concentration. concentration. PG composedPG composed of 5%–10% of 5%–10% P407 P407 and 30%–35% and 30%–35% P188 P188had gela hadtion gelation temperatures temperatures in the inphysiological the physiological range (Tablerange (Table2). As2 in). Asthe infirst the approach, first approach, PEG400 PEG400 was added was added to PG toto PGimprove to improve emodin emodin solubility solubility and adjust and theadjust gelation the gelation temperature temperature (Table (Table 2). The2). The result result showed showed that that thermoreversible thermoreversible PG PG composed ofof P407/P188/PEG400 (10/30/5) (10/30/5) yielded yielded proper proper gelation gelation temperature temperature for for the the topical topical delivery delivery of of emodin in the desired range (32–36 ◦°C).C). TheThe thermoreversiblethermoreversible PG with thethe selectedselected composition enhanced emodin solubility solubility by by nearly nearly 100-fold 100-fold compared compared to 10% to 10% ethanol ethanol or water or water (Figure (Figure 5). In5 ).addition, In addition, of the twoof the thermoreversible two thermoreversible PGs, the PGs, formulation the formulation composed composed of 20% P407 of 20% showed P407 higher showed emodin higher solubility emodin thansolubility the other than although the other althoughits total poloxamer its total poloxamer amount was amount lower was and lower PEG and was PEG not wascontained not contained as a co- solvent.as a co-solvent. These results These demonstrate results demonstrate that the thermoreve that the thermoreversiblersible PG is a useful PG is formulation a useful formulation for solubility for enhancementsolubility enhancement and P407 is and a more P407 effective is a more polymer effective than polymer P188 thanin terms P188 of in solubility. terms of solubility.

Figure 5. Enhancement of emodin solubility in poloxamer gel.

2.3. In Vitro Cell Activity Screen Screeninging of Emodin Thermoreversible PG In vitrovitro cellcell experimentsexperiments werewere performedperformed usingusing thermoreversiblethermoreversible PGPG composed of P407/P188/PEG400 (10/30/5) (10/30/5) in inorder order to toobserve observe the the effect effect of of the the enhanced enhanced solubility solubility of of emodin in thermoreversible PG PG in vitro cellcell activityactivity screening.screening. When ethanol solution of emodin was diluted with cell culture media to a concentration of 50 µμg/mL,g/mL, an an effective effective emodin emodin concentration concentration known for various cellular activities, emodin was aggregated and precipitated (arrows in Figure Figure 66A).A). InIn contrast, emodin in in thermoreversible thermoreversible PG PG remained remained as as a aclear clear solution solution after after dilution dilution with with cell cell culture culture media, media, as shownas shown in inFigure Figure 6B.6B. Application Application of of poloxamer poloxamer to to inhibit inhibit precipitation precipitation was previously reported byby Dai et et al. al. [16]. [16]. Emodin Emodin in inethanol ethanol solution solution of PG of di PGluted diluted with the with culture the culture media mediawas treated was treatedwith HDF with or DLD-1HDF or cells DLD-1 to observe cells to effect observe of emodin effect of on emodin the cell on pr theoliferation cell proliferation or cell viability. or cell As viability. shown in As Figure shown 7A, in theFigure precipitation7A, the precipitation of emodin of was emodin visually was observed visually observed and there and was there no wassignificant no significant difference difference in cell morphologyin cell morphology between between emodin emodin and the and vehicle the vehicle contro controll when whenemodin emodin in ethanol in ethanol was treated was treated with HDF with cellsHDF after cells afterdilution dilution with with the thecell cellculture culture media. media. However, However, when when the the emodin emodin thermoreversible thermoreversible PG diluted with the cell culture media was treated with HDF cells, the change of HDF cells’ morphology was observed and no precipitation or aggregation of emodin was observed (Figure 77B).B). TheThe reasonreason that no effect of emodin on HDFHDF waswas observedobserved afterafter treatmenttreatment with emodin ethanol solution might be that the amount of cellular uptake of emodin was extremely low because of emodin precipitation and consequently emodin was ineffectiveineffective at cellular level. In contra contrast,st, effective cellular uptake of emodin must have occurred after treatment with emodin in thermoreversible PG due to the enhanced solubility of emodin. We have conducted another in vitro cell study in DLD-1 cells. In this study,

Molecules 2017, 22, 246 7 of 10 emodin must have occurred after treatment with emodin in thermoreversible PG due to the enhanced solubilityMolecules of2017 emodin., 22, 246 We have conducted another in vitro cell study in DLD-1 cells. In this7 of 10 study, emodinMolecules was 2017 prepared, 22, 246 in DMSO and thermoreversible gel, respectively and treated with DLD-17 of 10 cells afteremodin dilution was to prepared various concentrationsin DMSO and thermoreversible (5, 25, 50, 100 µ M)gel, with respectively the cell and culture treated media. with The DLD-1 cell cells viability changedemodinafter dilution dose was independentlyprepared to various in concentrations DMSO when andemodin thermoreversible (5, 25, solution50, 100 μM) gel, prepared with respectively the incell DMSO culture and treated wasmedia. treated with The DLD-1cell with viability cells the cells. Theafterchanged higher dilution thedose emodinto independently various concentration concentrations when emodin treated,(5, 25, 50,solution the 100 more μ M)prepared with the emodinthe in cell DMSO culture precipitation was media. treated Thein with thecell theviability microplates cells. The higher the emodin concentration treated, the more the emodin precipitation in the microplates waschanged observed. dose These independently results could when be emodin due to solution the precipitation prepared in of DMSO emodin was upon treated dilution with the of DMSOcells. in Thewas higherobserved. the Theseemodin results concentration could be treated,due to the the prec moreipitation the emodin of emodin precipitation upon dilution in the ofmicroplates DMSO in the culture media. In contrast, the change of cell viability was observed dose-dependently without wasthe cultureobserved. media. These In results contrast, could the bechange due to of the cell prec viabilityipitation was of observed emodin upondose-dependently dilution of DMSO without in any precipitation when emodin was prepared in thermoreversible PG (data not shown). These results theany culture precipitation media. when In contrast, emodin the was change prepared of cellin ther viabilitymoreversible was observed PG (data dose-dependently not shown). These without results areanyare consistent consistentprecipitation with with when those those emodin observed observed was with with prepared HDFHDF inand ther impl implymoreversibley that that the the thermoreversiblePG thermoreversible (data not shown). PG PG isThese an is effective an results effective formulationareformulation consistent for for studyingwith studying those emodin observedemodin in in with cell cell cultureHDFculture and and impl its itsy efficacy efficacythat the in thermoreversible in vitro. vitro. PG is an effective formulation for studying emodin in cell culture and its efficacy in vitro.

Figure 6. Optical images of clearness of emodin solution diluted with cultured media (scale bar, 50 μm). Figure 6. Optical images of clearness of emodin solution diluted with cultured media (scale bar, 50 µm). Figure(A) Emodin 6. Optical in ethanol images dilutedof clearness with of cultured emodin mediasolution and diluted (B) emodin with cultured in thermoreversible media (scale bar, PG 50diluted μm). (A) Emodin in ethanol diluted with cultured media and (B) emodin in thermoreversible PG diluted (withA) Emodin cultured in media.ethanol Arrows diluted indicate with cultured precipitates. media and (B) emodin in thermoreversible PG diluted with cultured media. Arrows indicate precipitates. with cultured media. Arrows indicate precipitates.

Figure 7. Morphology of human dermal fibroblasts (HDF) cells after treatment of emodin diluted with cultured media (scale bar, 50 μm). (A) Morphology of HDF cells after treatment of ethanol (control) FigureFigure 7. Morphology7. Morphology of of human human dermaldermal fibroblasts fibroblasts (HDF) (HDF) cells cells after after treatment treatment of emodin of emodin diluted diluted with with and emodin in ethanol diluted with cultured media and (B) Morphology of HDF cells after treatment culturedcultured media media (scale (scale bar, bar, 50 50µ μm).m). ((AA)) MorphologyMorphology of of HDF HDF cells cells after after treatment treatment of ethanol of ethanol (control) (control) of thermoreversible PG without emodin (vehicle control) and emodin in thermoreversible PG diluted andand emodin emodin in in ethanol ethanol diluted diluted with with culturedcultured media media and and (B (B) Morphology) Morphology of HDF of HDF cells cells after after treatment treatment ofwith thermoreversible cultured media. PG Arrow without bars emodin indicate (vehicle cells with control) morphology and emodin changes. in thermoreversible PG diluted of thermoreversible PG without emodin (vehicle control) and emodin in thermoreversible PG diluted with cultured media. Arrow bars indicate cells with morphology changes. with cultured media. Arrow bars indicate cells with morphology changes.

Molecules 2017, 22, 246 8 of 10

3. Materials and Methods

3.1. Materials Poloxamer 407 (P407) and 188 (P188) were obtained from BASF Co. (Ludwigshafen, Germany). Acetonitrile, ethanol and methanol were purchased from Honeywell Burdick & Jackson (Muskegon, MI, USA). Polyethylenglycol 400 (PEG400), emodin, and phosphoric acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of reagent grade and used without further purification.

3.2. Preparation of Emodin Loaded Thermoreversible Poloxamer Gel Poloxamer gel was prepared according to the previous study [14]. Briefly, P188 and P407 was first dissolved in cold water at room temperature under continuous slow agitation. After the completion of dissolution, emodin dissolved in ethanol was added to the poloxamer solution and then stirred. To make emodin-loaded thermoreversible poloxamer gel, PEG400 was finally added to the previously prepared mixture and gently stirred. Emodin loaded thermoreversible poloxamer gel was maintained at 4 ◦C prior to use.

3.3. Measurement of Gelation Temperature The gelation temperature (Tg) of emodin loaded thermoreversible PG was measured using the tube invert method as described in the our previous study [14]. Vials containing 1 g of were incubated in an ice water bath for at least 20 min, in order to allow the samples to equilibrate thermally to the bath temperature. Vials were transferred from an ice-water bath to a water bath. The bath temperature was then increased stepwise (1 ◦C/5 min) from 10 ◦C to 50 ◦C. At each step, the sample vials were removed from the water bath and were inverted immediately to observe the flow of the sample in the vials. The sample remained at the bottom of the vial after inversion at a certain bath temperature, which was recorded as the gelation temperature. All experiments were performed in triplicate.

3.4. Measurement of Solubility Excess emodin was added to the glass vials containing 1 g of thermoreversible PG and the vials were stirred for 24 h in a cold room (3 ± 2 ◦C). Each sample was centrifuged at 15,000 rpm, 4 ◦C for 30 min to spin down the undissolved drug (Labogen 1730R, Labogen, Seoul, South Korea). The supernatant was separated and diluted with ethanol and the mobile phase of high-performance liquid chromatography (HPLC). After filtration through a 0.22 µm filter, the emodin concentration was measured by the Shimadzu UFLC HPLC system coupled with a SPD-M20A PDA detector (Shimadzu, Kyoto, Japan). Chromatographic separation was performed using a RP C8 column (4.6 × 150 mm, 5.0 µm, Waters, New York, NY, USA). The mobile phase consisted of 0.1% phosphoric acid in water and methanol (20:80, v/v) with the flow rate of 1.0 mL/min and injection volume of 20 µL. The PDA detector was set at 254 nm. The data were analyzed using LC solution software (Shimadzu, Japan). All experiments were performed in triplicate.

3.5. Cell Culture Experiments The preciptaion of two emodin formulations—one in thermoreversible PG and the other in organic solvents—was compared in order to confirm the usefulness of emodin thermoreversible PG for in vitro cell culture assays. The culture medium consisted of Dulbecco Modified Eagle Medium (DMEM) (Invitrogen, Carlsbad, CA, USA), 1% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA), and 1% penicillin/streptomycin. The first experiment was to test precipitaion after dilution with the culture media. The two formulations were diluted in DMEM to the final emodin concentration of 50 µg/mL and 100 µL of each diluted sample was transferred into a 24-microwell plate. Samples Molecules 2017, 22, 246 9 of 10 were incubated at 37 ◦C for 6 h and observed under the light microscope (Nikon Eclipse TS100, Tokyo, Japan). In order to demonstrate the importance of compound solubility for in vitro assay, cells were plated in 24 well plates at a density of 5 × 103 cells/well with DMEM medium and grown for ◦ ◦ 24 h at 37 C with 5% CO2 and 95% air. Two formulations were diluted with warm DMEM (37 C) and the diluted samples were treated with the cells at the emodin concentration of 50 µg/mL and incubated for 6 h. Cells were photographed under the microscope (Nikon Eclipse TS100, Tokyo, Japan). For the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay, DLD-1 cells (human epithelial colon cancer cell lines, ATCC CCL 221) were cultured in 96-well plates in RPMI 1640 medium supplemented with 10% FBS. After dilution of cold emodin thermoreversible PG and emodin in DMSO with warm media, the diluted emodin samples were treated with the cells (5 × 103) at various emodin concentrations. After incubation for 24 h, cells were washed in phosphate-buffered saline (PBS, pH 7.4), added to 50 µL of the 1 mg/mL MTT solution in sterile PBS, and incubated for 4 h at 37 ◦C. Then, the plate was centrifugated at 3000 rpm for 10 min, supernatant was carefully removed, and remaining adhered cells were dissolved in 50 µL DMSO. The plate was read at 570 nm using an ELISA reader (VERSAMax, Molecular Devices, VWR, Philadelphia, PA, USA).

4. Concluding Remarks In this study, we have prepared thermoreversible PG containing emodin with high emodin solubility and the gelation temperature suited to physiological use. The emodin thermoreversible PG was applied for in vitro cellular activity screening in the HDF and DLD-1 colon cancer cell line to investigate the vehicle effect. The prepared thermoreversible PG was composed of P407, P188, and PEG400, and was a liquid state at room temperature and a gel state at body temperature. The liquid property of thermoreversible PG at room temperature allows convenient handling and administration for topical delivery. The emodin solubility of thermoreversible PG with an optimized ratio of P407, P188, and PEG400 was improved 100-fold as compared to 10% ethanol or water. This study showed that the amount of P407 in thermoreversible PG was the dominant factor in enhancing the emodin solubility and P188 was effective at fixing gelation temperature in the desired range. In addition, the change of the cell viability and morphology was observed without any emodin precipitation or aggregation after treatment of the cells with emodin in thermoreversible PG, unlike in ethanol or DMSO solution. These results demonstrate that drug solubility and vehicle are very important to in vitro cellular study. Therefore, we suggest that the thermoreversible PG reported in the present study, can be effectively applied to the in vitro cellular study of poorly soluble compounds including emodin.

Acknowledgments: This research was supported by a grant (16172MFDS172) from Ministry of Food and Drug Safety in 2016. Author Contributions: E.B. and A.K. conceived and designed the experiments; M.P., S.-H.J., T.K. and E.-H.K. performed the experiment, HDF proliferation, gel preparation and gelation temperature measurements, solubility test, and DLD-1 cell experiments, respectively; K.J. provided information on various compounds from natural products including emodin and contributed to the design of the experiments; E.B., E.-H.K. and A.K. analyzed the data; E.B. wrote the paper. Conflicts of Interest: The authors declare no conflicts of interest.

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Sample Availability: Samples are not available.

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