Physicochemical Characterization and In Vitro Activity of Poly(ε-Caprolactone)/Mycophenolic Acid Amorphous Solid Dispersions
Abstract
This research was funded by Spanish Ministry of Science and Innovation MICINN (PID2019-106236GB-I00) and the Basque Government Department of Education, Culture and Language Policy (IT1766-22).
Full text
Citation: Sánchez-Aguinagalde, O.; Sanchez-Rexach, E.; Polo, Y.; Larrañaga, A.; Lejardi, A.; Meaurio, E.; Sarasua, J.-R. Physicochemical Characterization and In Vitro Activity of Poly(ε-Caprolactone)/ Mycophenolic Acid Amorphous Solid Dispersions. Polymers 2024,16, 1088. https://doi.org/10.3390/ polym16081088 Academic Editors: Stefano Farris and Masoud Ghaani Received: 10 November 2023 Revised: 27 March 2024 Accepted: 3 April 2024 Published: 13 April 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). polymers Article Physicochemical Characterization and In Vitro Activity of Poly(ε-Caprolactone)/Mycophenolic Acid Amorphous Solid Dispersions Oroitz Sánchez-Aguinagalde 1, Eva Sanchez-Rexach 1, Yurena Polo 2, Aitor Larrañaga 1, Ainhoa Lejardi 1,*, Emilio Meaurio 1and Jose-Ramon Sarasua 1 1Department of Mining-Metallurgy Engineering and Materials Science, POLYMAT, Bilbao School of Engineering, University of the Basque Country (UPV/EHU), Plaza Ingeniero Torres Quevedo 1, 48013 Bilbao, Spain; [email protected] (O.S.-A.); [email protected] (E.S.-R.); aitor[email protected] (A.L.); [email protected] (E.M.); jr[email protected] (J.-R.S.) 2Polimerbio SL, Paseo Miramon 170, 20014 Donostia-San Sebastian, Spain; [email protected] *Correspondence: ainhoa.lejar[email protected] Abstract: The obtention of amorphous solid dispersions (ASDs) of mycophenolic acid (MPA) in poly( ε -caprolactone) (PCL) is reported in this paper. An improvement in the bioavailability of the drug is possible thanks to the favorable specific interactions occurring in this system. Differential scanning calorimetry (DSC) was used to investigate the miscibility of PCL/MPA blends, measuring glass transition temperature (Tg) and analyzing melting point depression to obtain a negative interaction parameter, which indicates the development of favorable inter-association interactions. Fourier transform infrared spectroscopy (FTIR) was used to analyze the specific interaction occurring in the blends. Drug release measurements showed that at least 70% of the drug was released by the third day in vitro in all compositions. Finally, preliminary in vitro cell culture experiments showed a decreased number of cancerous cells over the scaffolds containing MPA, presumably arising from the anti-cancer activity attributable to MPA. Keywords: poly( ε -caprolactone) (PCL); mycophenolic acid (MPA); amorphous solid dispersions (ASDs); miscibility; interactions; drug release; cancer treatment 1. Introduction As new treatments and drugs appear for all kinds of diseases, we are also faced with great challenges to achieve a satisfactory application of these remedies. Although they may be effective in theory, most of the drugs that are being approved are not feasible in terms of their biopharmacological properties. The main causes are low permeability, poor solubility, or rapid elimination from the body. In fact, 90% of the drugs being developed are molecules with low water solubility, in addition to almost 40% of the drugs already approved [1–3]. The dimensions of this problem can be seen, for example, in the case of the oral administration of doses. In order to reach systemic circulation, the drug must be dissolved in the intestinal fluids of the gastrointestinal tract, which is difficult in the case of low solubility [ 4 ]. The cause of this low bioavailability is the different molecular arrangements, where the crystalline compounds are the ones that present the greatest problem [ 5 ]. In order to solve this problem, one of the established strategies is amorphization, which transforms low-energy crystalline substances into high-energy amorphous compounds, giving them greater solubility and bioavailability [ 6 ]. However, these amorphous solids are not thermodynamically stable because of their excess enthalpy, entropy, and free energies, which cause them to tend to form crystals [ 7 ]. For this reason, achieving the stability of these compounds is a great challenge. One of the strategies used for this purpose is developing amorphous solid dispersions (ASDs). In the 1970s, Chiou and Riegelman defined the term solid dispersions as the Polymers 2024,16, 1088. https://doi.org/10.3390/polym16081088 https://www.mdpi.com/journal/polymers
Polymers 2024,16, 1088 2 of 16 dispersion of an active pharmaceutical ingredient (API) in an amorphous carrier in a solid state prepared by solvent, melting, or solvent-melting methods [ 7 ]. In these systems, there is a mixture at the molecular level between a polymer and the drug in an amorphous state, increasing its bioavailability [ 8 – 12 ]. It is known that the low thermodynamic stability due to the high energy of the amorphous state causes relaxation, nucleation, and recrystallization under different variables [ 13 – 16 ]. Thus, the role of the polymeric matrix is to inhibit this process and maintain the mixture in a single homogeneous phase [ 17 , 18 ]. To avoid this crystallization and maintain the mixture in the metastable region of the binary phase diagram, miscibility between the API and polymer is essential [ 19 – 21 ]. The kinetic stability provided by storage below the glass transition temperature (T g ) must also be taken into account. In fact, according to Hancock et al., the stability of the mixture could be ensured for years by storing it at least 50 K below T g [ 22 , 23 ]. One significant challenge in this system is the unpredictable nature of polymer–drug interactions [24]. One interesting drug to test this system is mycophenolic acid (MPA—C 17 H 20 O 6 , 320 g/mol; aqueous solubility: 35.5 mg/L). Mycophenolic acid (Scheme 1) is an antibiotic produced by the Penicillium family and is best known for its use as an immunosuppressive agent to prevent rejection in organ transplants [ 25 , 26 ]. In addition, this drug has more biological properties, such as antifungal or antiviral properties [ 27 ]. It also has the potential to prevent and perhaps treat chronic allograft vasculopathy, as it can inhibit the proliferation of vascular smooth muscle cells (VSMCs), mesangial cells, and myofibroblasts [ 28 ]. However, one of the most striking properties is its ability to act against tumor cells of various types such as leukemia or lymphoma, among others [ 29 ]. This is because MPA is an inhibitor of inosine monophosphate dehydrogenase (IMPDH), which leads to the reduction of xanthine monophosphate (XMP), guanosine-5 ′ -triphosphate (GTP), and deoxyguanosine triphosphate (dGTP), thus inhibiting the proliferation of lympholeukocytes and cancer cells [ 26 , 30 ]. Despite having so many favorable properties, the bioavailability of MPA in vivo is relatively poor due to the high clearance inside a living organism, which limits its possibility of clinical application [ 25 ]. This, in addition to its low aqueous solubility, makes it a perfect candidate for forming amorphous solid dispersions. Polymers2024,16,xFORPEERREVIEW2of17 enthalpy,entropy,andfreeenergies,whichcausethemtotendtoformcrystals[7].For thisreason,achievingthestabilityofthesecompoundsisagreatchallenge. Oneofthestrategiesusedforthispurposeisdevelopingamorphoussolid dispersions(ASDs).Inthe1970s,ChiouandRiegelmandefinedthetermsoliddispersions asthedispersionofanactivepharmaceuticalingredient(API)inanamorphouscarrierin asolidstatepreparedbysolvent,melting,orsolvent-meltingmethods[7].Inthese systems,thereisamixtureatthemolecularlevelbetweenapolymerandthedruginan amorphousstate,increasingitsbioavailability[8–12].Itisknownthatthelow thermodynamicstabilityduetothehighenergyoftheamorphousstatecausesrelaxation, nucleation,andrecrystallizationunderdifferentvariables[13–16].Thus,theroleofthe polymericmatrixistoinhibitthisprocessandmaintainthemixtureinasingle homogeneousphase[17,18].Toavoidthiscrystallizationandmaintainthemixtureinthe metastableregionofthebinaryphasediagram,miscibilitybetweentheAPIandpolymer isessential[19–21].Thekineticstabilityprovidedbystoragebelowtheglasstransition temperature(T g )mustalsobetakenintoaccount.Infact,accordingtoHancocketal.,the stabilityofthemixturecouldbeensuredforyearsbystoringitatleast50KbelowT g [22,23].Onesignificantchallengeinthissystemistheunpredictablenatureofpolymer– druginteractions.[24]. Oneinterestingdrugtotestthissystemismycophenolicacid(MPA—C 17 H 20 O 6 ,320 g/mol;aqueoussolubility:35.5mg/L).Mycophenolicacid(Scheme1)isanantibiotic producedbythePenicilliumfamilyandisbestknownforitsuseasanimmunosuppressive agenttopreventrejectioninorgantransplants[25,26].Inaddition,thisdrughasmore biologicalproperties,suchasantifungalorantiviralproperties[27].Italsohasthe potentialtopreventandperhapstreatchronicallograftvasculopathy,asitcaninhibitthe proliferationofvascularsmoothmusclecells(VSMCs),mesangialcells,and myofibroblasts[28].However,oneofthemoststrikingpropertiesisitsabilitytoact againsttumorcellsofvarioustypessuchasleukemiaorlymphoma,amongothers[29]. ThisisbecauseMPAisaninhibitorofinosinemonophosphatedehydrogenase(IMPDH), whichleadstothereductionofxanthinemonophosphate(XMP),guanosine-5′- triphosphate(GTP),anddeoxyguanosinetriphosphate(dGTP),thusinhibitingthe proliferationoflympholeukocytesandcancercells[26,30].Despitehavingsomany favorableproperties,thebioavailabilityofMPAinvivoisrelativelypoorduetothehigh clearanceinsidealivingorganism,whichlimitsitspossibilityofclinicalapplication[25]. This,inadditiontoitslowaqueoussolubility,makesitaperfectcandidateforforming amorphoussoliddispersions. Poly(ε-caprolactone)Mycophenolicacid Scheme1.ChemicalstructuresofPCLandMPA. Inthiswork,thepolymerselectedasthematrixtodisperseMPAinamorphousform ispoly(ε-caprolactone)(PCL),abiodegradablesemicrystallinepolyester.Itsglass transitiontemperatureisaround−60°Canditsmeltingpointataround60°C.The biodegradationofthispolymerunderphysiologicalconditionshasbeenreportedtolast severalmonthstoyears[31,32],makingitsuitableforlong-termbiomedicalapplications. Inthiswork,miscibilityandinteractionsbetweenPCLandMPAarestudiedtoverifythe suitabilityofthismixturefortheformationofanamorphoussoliddispersion.Inaddition, weseparatelytestedtheinteractionoftheblendscontainingincreasingconcentrationsof Scheme 1. Chemical structures of PCL and MPA. In this work, the polymer selected as the matrix to disperse MPA in amorphous form is poly( ε -caprolactone) (PCL), a biodegradable semicrystalline polyester. Its glass transition temperature is around − 60 ◦ C and its melting point at around 60 ◦ C. The biodegradation of this polymer under physiological conditions has been reported to last several months to years [ 31 , 32 ], making it suitable for long-term biomedical applications. In this work, miscibility and interactions between PCL and MPA are studied to verify the suitability of this mixture for the formation of an amorphous solid dispersion. In addition, we separately tested the interaction of the blends containing increasing concentrations of MPA with both a non-cancerous fibroblast cell line (MRC5), approved by ISO 10993 for cytotoxicity studies [ 33 ], and a widely used immortalized HeLa cell line derived from cervical cancer [34].
Polymers 2024,16, 1088 3 of 16 2. Experimental Section 2.1. Starting Materials Poly( ε -caprolactone) (PURASORB ® PC12 trade name) with an average molecular weight (M w ) of 1.3 × 10 5 g/mol and M w /M n = 1.76 was purchased from Purac Biochem (Gorinchem, The Netherlands). Mycophenolic acid (C 17 H 20 O 6 , M = 320.34 g/mol) was obtained from Fluorochem Ltd. (Gossop, UK), and dichloromethane (DCM) was supplied by Labkem (Dublin, Ireland). 2.2. Blend Preparation Films were prepared by solvent casting from dichloromethane (DCM) solutions containing 2.5 wt% of PCL/MPA blend at room temperature. 2.3. Differential Scanning Calorimetry (DSC) A Modulated DSC Q200 from TA Instruments was used for thermal analyses. All the scans were performed in hermetic aluminum pans under nitrogen atmosphere with sample weights between 5 and 10 mg. Two scans from − 80 ◦ C to 160 ◦ C with a scan rate of 20 ◦ C/min were performed in order to measure glass transition temperatures (T g ) in the second one. 2.4. Melting Point Depression Analysis The melting point depression of MPA was observed in MPA-rich blends containing 0–20 wt% PCL. To obtain the melting temperature of MPA crystals, samples were heated in the DSC with a scan rate of 1 ◦C/min. The samples were weighed again after the DSC scans, and no weight loss was observed during the thermal treatments. 2.5. Fourier Transform Infrared Spectroscopy (FTIR) A Nicolet AVATAR 370 Fourier transform infrared spectrophotometer was used to record FTIR spectra of the blends, with a resolution of 2 cm −1 and averaged over 64 scans in the range of 400–4000 cm −1 . Dichloromethane solutions containing 2 wt% of blends were cast on KBr pellets by evaporation of the solvent at room temperature. The absorbance of the samples was within the range where the Lambert–Beer law is obeyed. 2.6. In Vitro Drug Release In vitro drug release experiments were performed for the PCL/MPA 99.95/0.05, 99.9/0.1, 99.8/0.2, 99.5/0.5, 99/1, and 98/2 blends. Round samples of PCL/MPA of Ø10 mm obtained by solvent casting were immersed in 1 mL of 0.1 M PBS buffer (pH 7.4) at 37 ◦ C. At fixed intervals, samples of 200 µ L were taken and replaced with fresh PBS at 37 ◦ C. The drug concentration in solution was determined using a BioTech Sinergy H1M MicroPlate Reader (Minneapolis, MN, USA) using a calibration curve that was previously obtained measuring the absorbance at a wavelength of 305 nm for solutions of MPA in 0.1 M PBS. The release kinetics of mycophenolic acid were examined by considering four mathematical models as follows: Zero −order : Ct/C∞=k0t(1) First −order : ln (1−Ct/C∞)=−k1t(2) Higuchi : Ct/C∞=kht1 2(3) Korsmeyer–Peppas : Ct/C∞=ktn(4) where C t is the cumulative amount of the drug released at time t,C ∞ is the starting amount of the drug, nis the release exponent, and k 0 ,k 1 ,k h , and kare the kinetic constants. Zeroorder kinetics (Equation (1)) represents a release process that is controlled by the relaxation
Polymers 2024,16, 1088 4 of 16 of polymeric chains, independent of its concentration and with a constant release rate. The first-order kinetics (Equation (2)) model represents a drug release rate that depends on its concentration [ 32 ]. Higuchi (Equation (3)) describes drug release as a diffusion process based on Fick’s law, square root time-dependent. If the release mechanism is not well known or when more than one type of release phenomena could be involved, the Korsmeyer–Peppas (Equation (4)) model is applied. It is possible to define whether the release happens by Fickian diffusion, anomalous transport, Case-II transport, or Super Case-II transport depending on the values obtained for the release exponent, n[35,36]. 2.7. In Vitro Cell Culture Experiments In vitro cell culture experiments were performed on the PCL/MPA 99.5/0.5, 99/1, and 98/2 blends. Circular samples of PCL/MPA of Ø6 mm were obtained, and each side was sterilized for 30 min under UV light. Either the immortalized HeLa cell line (ATCC, Manassas, VA, USA) derived from cervical cancer or the non-cancerous fibroblasts MRC5 (CCL-171, ATCC, Manassas, VA, USA) derived from lung tissue were drop-seeded over the materials at a concentration of 25,000 cells per scaffold. After 1 h, 480 mL of prewarmed DMEM (Fisher Scientific, Madrid, Spain) at 37 ◦ C supplemented with 10% fetal bovine serum (FBS) (Fisher Scientific, Madrid, Spain), 1% L-glutamine (Fisher Scientific, Madrid, Spain), and penicillin/streptomycin (Fisher Scientific, Madrid, Spain) were added. PCL films were used as the negative control, and for the positive control, MPA in dissolution at a concentration of 300 ppm was dissolved on the culture media and filtrated (0.2 µ m). Cells were incubated at 37 ◦C and 5% CO2in a standard cell culture incubator. 2.8. Immunostaining After 1 or 3 days in vitro (DIV), samples were fixed with 4% paraformaldehyde (PFA) (Fisher Scientific, Spain) and permeabilized with 0.3% triton-X100 (Fisher Scientific, Spain) in PBS (Fisher Scientific, Spain) containing 1% Bovine Serum Albumin (BSA) (Sigma Aldrich, Spain). For the staining, rhodamine/phalloidin (Fisher Scientific, Madrid, Spain) and DAPI, 4 ′ ,6-diamidino-2-phenylindole dihydrochloride (Fisher Scientific, Madrid, Spain) were diluted in 1% PBS BSA and incubated for 1.5 h. After washing each sample 2 times in PBS containing 0.1% Tween-20 (Fisher Scientific, Madrid, Spain) and 1 time in PBS, the samples were mounted using mounting medium (Abcam, Waltham, MA, USA). The samples were analyzed in an inverted fluorescence microscope (Nikon Eclipse Ts2). For cell quantification studies, 5 different points were taken. 2.9. Cell Count and Statistical Analysis For cell counts, five aleatory images of 0.1 mm 2 were taken for each of the triplicates in each condition, and nuclear DAPI labeling was used to calculate the total number of cells. The data were subjected to one-way analysis of variance (ANOVA) using Kruskal–Wallis followed by Dunn’s post hoc test. The level of significance was set at p< 0.05. The results were presented as mean ±SD or SEM. 3. Results and Discussion 3.1. Miscibility Analysis by Differential Scanning Calorimetry (DSC) When two components are miscible, a single glass transition temperature (T g ) between the T g of each material, which changes progressively with the composition, is expected [ 37 , 38 ]. On the contrary, the detection of more than one single value would indicate a separation into individual amorphous phases within the system. Different methods have been employed to predict the glass transition temperature of amorphous binary systems, such as the Gordon–Taylor (GT), Couchman–Karasz (CK), and Fox equations (Equation (5)). Considering that the Fox equation was developed to analyze systems formed
Polymers 2024,16, 1088 5 of 16 by components of equal densities, it is appropriate to use it to estimate this intermediate Tg, as the densities of PCL and MPA are 1.14 g/cm3and 1.3 g/cm3, respectively [39]: 1 Tgb =w1 Tg1 +w2 Tg2 (5) where w 1 and w 2 are the weight fractions of components 1 and 2, respectively, T g1 and T g2 are the glass transition temperatures of the pure components, and T gb is the glass transition temperature of the blend. Figure 1shows the first scan DSC traces obtained for the pure components and for different PCL/MPA blends. As can be seen, pure PCL is a semicrystalline polymer displaying a glass transition temperature located at about − 60 ◦ C and a melting endotherm at about 60 ◦ C. On the other hand, MPA is a crystalline compound melting at 145 ◦ C, which can be also supercooled to undergo a glass transition at 11 ◦ C after reheating the quenched melt (see Figure 2). Polymers2024,16,xFORPEERREVIEW5of17 [37,38].Onthecontrary,thedetectionofmorethanonesinglevaluewouldindicateaseparationintoindividualamorphousphaseswithinthesystem.Differentmethodshavebeen employedtopredicttheglasstransitiontemperatureofamorphousbinarysystems,suchas theGordon–Taylor(GT),Couchman–Karasz(CK),andFoxequations(Equation(5)).ConsideringthattheFoxequationwasdevelopedtoanalyzesystemsformedbycomponentsof equaldensities,itisappropriatetouseittoestimatethisintermediateT g ,asthedensitiesof PCLandMPAare1.14g/cm 3 and1.3g/cm 3 ,respectively[39]: 1 𝑇 𝑤 𝑇 𝑤 𝑇(5) wherew 1 andw 2 aretheweightfractionsofcomponents1and2,respectively,T g1 andT g2 aretheglasstransitiontemperaturesofthepurecomponents,andT gb istheglasstransition temperatureoftheblend. Figure1showsthefirstscanDSCtracesobtainedforthepurecomponentsandfor differentPCL/MPAblends.Ascanbeseen,purePCLisasemicrystallinepolymerdisplayingaglasstransitiontemperaturelocatedatabout−60°Candameltingendothermat about60°C.Ontheotherhand,MPAisacrystallinecompoundmeltingat145°C,which canbealsosupercooledtoundergoaglasstransitionat11°Cafterreheatingthequenched melt(seeFigure2). Figure1.FirstscanDSCtracesforPCL,MPA,andPCL/MPAblends. Figure 1. First scan DSC traces for PCL, MPA, and PCL/MPA blends. Polymers2024,16,xFORPEERREVIEW5of17 [37,38].Onthecontrary,thedetectionofmorethanonesinglevaluewouldindicateaseparationintoindividualamorphousphaseswithinthesystem.Differentmethodshavebeen employedtopredicttheglasstransitiontemperatureofamorphousbinarysystems,suchas theGordon–Taylor(GT),Couchman–Karasz(CK),andFoxequations(Equation(5)).ConsideringthattheFoxequationwasdevelopedtoanalyzesystemsformedbycomponentsof equaldensities,itisappropriatetouseittoestimatethisintermediateT g ,asthedensitiesof PCLandMPAare1.14g/cm 3 and1.3g/cm 3 ,respectively[39]: 1 𝑇 𝑤 𝑇 𝑤 𝑇(5) wherew 1 andw 2 aretheweightfractionsofcomponents1and2,respectively,T g1 andT g2 aretheglasstransitiontemperaturesofthepurecomponents,andT gb istheglasstransition temperatureoftheblend. Figure1showsthefirstscanDSCtracesobtainedforthepurecomponentsandfor differentPCL/MPAblends.Ascanbeseen,purePCLisasemicrystallinepolymerdisplayingaglasstransitiontemperaturelocatedatabout−60°Candameltingendothermat about60°C.Ontheotherhand,MPAisacrystallinecompoundmeltingat145°C,which canbealsosupercooledtoundergoaglasstransitionat11°Cafterreheatingthequenched melt(seeFigure2). Figure1.FirstscanDSCtracesforPCL,MPA,andPCL/MPAblends. Figure 2. Second scan DSC traces for PCL, MPA, and PCL/MPA blends. As can be seen in Figure 2, the PCL/MPA blends show composition-dependent single glass transitions located close to the values predicted using the Fox equation (see Table 1and Figure 3). Consequently, it can be concluded that the two components are
Polymers 2024,16, 1088 6 of 16 completely miscible in the amorphous phase. Furthermore, the melting temperature of PCL decreases as the content of MPA increases. Furthermore, the crystallization of PCL is totally suppressed when the drug composition exceeds 50 wt%. Table 1. Thermal properties of PCL/MPA blends. PCL/MPA T g Experimental (◦C) TgTheoretical (Fox) (◦C) TmPCL (◦C) ∆HfPCL (J/g) PCL −60.0 - 57.2 66.4 80/20 −44.1 −48.8 51.7 49.8 60/40 −36.3 −36.3 46.4 25.9 40/60 −25.7 −22.4 - - 20/80 −10.2 −6.8 - - MPA 11.1 - - - Polymers2024,16,xFORPEERREVIEW6of17 Figure2.SecondscanDSCtracesforPCL,MPA,andPCL/MPAblends. AscanbeseeninFigure2,thePCL/MPAblendsshowcomposition-dependentsingle glasstransitionslocatedclosetothevaluespredictedusingtheFoxequation(seeTable1 andFigure3).Consequently,itcanbeconcludedthatthetwocomponentsarecompletely miscibleintheamorphousphase.Furthermore,themeltingtemperatureofPCLdecreases asthecontentofMPAincreases.Furthermore,thecrystallizationofPCListotallysuppressedwhenthedrugcompositionexceeds50wt%. Table1.ThermalpropertiesofPCL/MPAblends. PCL/MPAT g Experimental(°C)T g Theoretical(Fox)(°C)T m PCL(°C)ΔH f PCL(J/g) PCL−60.0-57.266.4 80/20−44.1−48.851.749.8 60/40−36.3−36.346.425.9 40/60−25.7−22.4-- 20/80−10.2−6.8-- MPA11.1--- Figure3.GlasstransitiontemperatureversuscompositionforthePCL/MPAsystem:(■)experimentalvaluesand(■)Foxequation. 3.2.MeltingPointDepressionAnalysis Ifthefreeenergyofthemixingofthetwocomponents(ΔG mix )isnegative,asystem canbeconsideredthermodynamicallymiscible. ∆𝐺 ∆𝐻 𝑇∆𝑆(6) whereΔH mix andΔS mix aretheenthalpyandentropyofmixing,respectively.TΔS mix isalwayspositivesincetheentropyofmixingisaddedtotheentropyofmelting,makingthe entropychangeinamiscibleblendlargerthaninthepurecomponent.Consequently,the signofΔG mix dependsonthevalueofΔH mix .Inordertoavoidphaseseparation,thecohesiveinteractionsneedtobelowerthanthesumofadhesiveinteractions,generatingafavorableenthalpyofmixing.Themiscibilitybetweentwocomponentsintermsofthe changeintheGibbsfreeenergycanbedescribedusingthemeltingpointdepression method,basedonFlory–Hugginstheory.Accordingtothismethod,themeltingpoint Figure 3. Glass transition temperature versus composition for the PCL/MPA system: ( ■ ) experimental values and (■) Fox equation. 3.2. Melting Point Depression Analysis If the free energy of the mixing of the two components ( ∆ G mix ) is negative, a system can be considered thermodynamically miscible. ∆Gmix =∆Hmix −T∆Smix (6) where ∆ H mix and ∆ S mix are the enthalpy and entropy of mixing, respectively. T ∆ S mix is always positive since the entropy of mixing is added to the entropy of melting, making the entropy change in a miscible blend larger than in the pure component. Consequently, the sign of ∆ G mix depends on the value of ∆ H mix . In order to avoid phase separation, the cohesive interactions need to be lower than the sum of adhesive interactions, generating a favorable enthalpy of mixing. The miscibility between two components in terms of the change in the Gibbs free energy can be described using the melting point depression method, based on Flory–Huggins theory. According to this method, the melting point temperature of the drug will decrease as the polymer content in the mixture increases if the cohesive forces in the pure components are weaker than the adhesive forces between the
Polymers 2024,16, 1088 7 of 16 drug and the polymer [ 32 , 40 ]. Flory’s relationship can be used to analyze the depression of the equilibrium melting point: 1 Tm −1 T0 m =−R ∆H2u V2u V1uln ϕ2 m2 +1 m2 −1 m1ϕ1+χ12ϕ2 1(7) where T0 m is the equilibrium melting point of the pure crystallizable component and Tm is the equilibrium melting point of its blends; the subscripts 1 and 2 refer to the amorphous and crystallizable components, respectively. R is the universal gas constant, while ∆H2u is the heat of fusion per mole of crystalline repeat units. Vu is the molar volume of the repeating unit, m is the degree of polymerization, ϕ is the volumen fraction, and χ12 is the interaction parameter. In order to apply Equation (7), the molar volume of MPA ( V2= 246.3 cm 3 /mol) can be considered as the molar volume of the lattice sites, resulting in m2= 1. The same volume can be taken as the molar volume of the polymeric repeat unit V2=V1u . Since m1=Vpol/V1uis large, 1/m1≈0. As a result, Equation (7) simplifies to: 1 Tm −1 T0 m =−R ∆H2ln ϕ2+ϕ1+χϕ2 1(8) The melting points of pure components and different PCL/MPA blends were measured at a low heating rate (1 ◦ C/min). The average melting point of pure MPA is T0 m=140.3 ◦C, and this temperature is decreased by nearly 5 ◦ C when 20 wt% PCL is added to the blend. The data obtained for each blend can be seen in Table 2. These results, with the average melting enthalpy of pure MPA ( ∆HMPA = 114.7 J/g) were used to plot Equation (8) as a function of the square of the volume fraction of the polymer, ϕ2 1 . The slope of this plot, which can be seen in Figure 4, gives an approximation of the interaction parameter of χ=− 1.18. The negative values for the interaction parameter indicate an exothermic reaction, confirming a thermodynamically miscible blend. It is also possible to calculate the interaction energy density, B, at the melting temperature of MPA according to Equation (9): χ=BVr RT (9) where Vris a reference volumen (Vr=V2=246.3 cm3/mol), yielding B=−16.5 J/cm3. Polymers2024,16,xFORPEERREVIEW8of17 Figure4.AnalysisofthemeltingtemperatureofMPAaccordingtoEquation(8)forthePCL/MPA system.Theslopeoftheplotgivestheinteractionparameter𝜒 1.18. Table2.MeltingtemperaturesofMPAobtainedfrom1°Cmin −1 scanrates. MPAwt%T m (°C) Sample1Sample2Sample3 100139.2140.5140.9 95139.6139.9138.3 90138.4138.1137.4 85137.4138.9136.8 80135.8135.7135.9 3.3.FourierTransformInfraredSpectroscopy(FTIR) Theanalysisofthechangesobservedintheinfraredspectrumuponblendingprovidesinformationaboutthechangesinspecificinteractionsandcaneventuallyaidinexplainingtheenergeticcontributionsdrivingthemiscibilityofthesystem.InthePCL/MPA system,boththecarbonylandthehydroxylstretchingregionsareofmaininterestbecause hydrogenbondinginteractionscanbeexpectedforthosegroups.Figure5showsthecarbonylstretchingregionforPCL,MPA,andtheirblends.ThespectrumofpurePCLshows apeakat1725cm −1 attributabletocrystallinePCLandashoulderat1735cm −1 arisingfrom theamorphousphase[32,40].Ontheotherhand,pureMPAshowstwodifferentpeaks locatedat1744and1708cm −1 attributable,respectively,tothelactonecarbonylandthe carboxylicacidcarbonyl.Bothlocationsareatthelowerendofthespectralrangescorrespondingtothosefunctionalgroups[41]becauseofthehydrogenbondinginteractions occurringinpureMPA.Figure6sketchestheseinteractionsasderivedfromXRDstudies [42–44].Asitcanbeseen,inpureMPA,themoleculesarejoinedinthecrystalbycarboxylicacidgroupsformingdimers,alongwithbifurcatedhydrogenbondsbetweenthehydroxylgroupandthecarboxylicacidcarbonyl(absorptionbandat1708cm −1 ).Inaddition, anintramolecularbifurcatedhydrogenbondredshiftstheabsorptionofthelactonecarbonyltothereportedwavenumber(1744cm −1 ). Figure 4. Analysis of the melting temperature of MPA according to Equation (8) for the PCL/MPA system. The slope of the plot gives the interaction parameter χ=−1.18.
Polymers 2024,16, 1088 8 of 16 Table 2. Melting temperatures of MPA obtained from 1 ◦C min−1scan rates. MPA wt% Tm(◦C) Sample 1 Sample 2 Sample 3 100 139.2 140.5 140.9 95 139.6 139.9 138.3 90 138.4 138.1 137.4 85 137.4 138.9 136.8 80 135.8 135.7 135.9 3.3. Fourier Transform Infrared Spectroscopy (FTIR) The analysis of the changes observed in the infrared spectrum upon blending provides information about the changes in specific interactions and can eventually aid in explaining the energetic contributions driving the miscibility of the system. In the PCL/MPA system, both the carbonyl and the hydroxyl stretching regions are of main interest because hydrogen bonding interactions can be expected for those groups. Figure 5shows the carbonyl stretching region for PCL, MPA, and their blends. The spectrum of pure PCL shows a peak at 1725 cm −1 attributable to crystalline PCL and a shoulder at 1735 cm −1 arising from the amorphous phase [ 32 , 40 ]. On the other hand, pure MPA shows two different peaks located at 1744 and 1708 cm −1 attributable, respectively, to the lactone carbonyl and the carboxylic acid carbonyl. Both locations are at the lower end of the spectral ranges corresponding to those functional groups [ 41 ] because of the hydrogen bonding interactions occurring in pure MPA. Figure 6sketches these interactions as derived from XRD studies [ 42 – 44 ]. As it can be seen, in pure MPA, the molecules are joined in the crystal by carboxylic acid groups forming dimers, along with bifurcated hydrogen bonds between the hydroxyl group and the carboxylic acid carbonyl (absorption band at 1708 cm −1 ). In addition, an intramolecular bifurcated hydrogen bond red shifts the absorption of the lactone carbonyl to the reported wavenumber (1744 cm−1). Polymers2024,16,xFORPEERREVIEW9of17 Figure5.CarbonylstretchingregionforpurePCLandMPAandPCL/MPAblendsofdifferentcompositions. Figure6.HydrogenbondingincrystallineMPA(seetext). ThePCL/MPA20/80and40/60blendsshowamajorpeaklocatedatabout1724cm −1 , accompaniedbytwoshouldersathigherwavenumberslocatedatabout1735cm −1 and 1750cm −1 .Atthesecompositions,PCLisalmostinamorphousformaccordingtotheDSC results(hence,thecontributioncorrespondingtocrystallinePCLshouldbenegligible), andtheabsorptionbandscorrespondingtoMPAareexpectedtoprevailoverthoseof Figure 5. Carbonyl stretching region for pure PCL and MPA and PCL/MPA blends of different compositions.
Polymers 2024,16, 1088 9 of 16 Polymers2024,16,xFORPEERREVIEW9of17 Figure5.CarbonylstretchingregionforpurePCLandMPAandPCL/MPAblendsofdifferentcompositions. Figure6.HydrogenbondingincrystallineMPA(seetext). ThePCL/MPA20/80and40/60blendsshowamajorpeaklocatedatabout1724cm −1 , accompaniedbytwoshouldersathigherwavenumberslocatedatabout1735cm −1 and 1750cm −1 .Atthesecompositions,PCLisalmostinamorphousformaccordingtotheDSC results(hence,thecontributioncorrespondingtocrystallinePCLshouldbenegligible), andtheabsorptionbandscorrespondingtoMPAareexpectedtoprevailoverthoseof Figure 6. Hydrogen bonding in crystalline MPA (see text). The PCL/MPA 20/80 and 40/60 blends show a major peak located at about 1724 cm−1, accompanied by two shoulders at higher wavenumbers located at about 1735 cm −1 and 1750 cm −1 . At these compositions, PCL is almost in amorphous form according to the DSC results (hence, the contribution corresponding to crystalline PCL should be negligible), and the absorption bands corresponding to MPA are expected to prevail over those of PCL; hence, the band at 1724 cm −1 is most likely attributable carboxylic acid carbonyls forming dimers in the amorphous phase. This band is probably strongly overlapped with PCL carbonyls hydrogen bonded with hydroxyl groups present in MPA, but unfortunately, these two components are not distinguishable. The shoulder at about 1735 cm −1 can be attributed to free C=O groups in PCL and the one at about 1750 cm −1 to lactone carbonyls in the amorphous phase. Finally, Figure 7shows the hydroxyl stretching region for MPA and its blends with PCL. As can be seen, the OH stretching band in pure MPA is located at about 3416 cm −1 , and blending broadens the band and shifts it to higher wavenumbers. Band broadening is a consequence of the presence of amorphous MPA, while shifting to higher wavenumbers can be attributed to weaker hydrogen bonding interactions in the blends compared with pure MPA. Despite the weaker nature of the interactions, the energetic balance will still render favorable to miscibility as long as the blend achieves a larger number of interactions, arising from the introduction of additional interacting groups (the PCL carbonyls).
Polymers 2024,16, 1088 16 of 16 33. ISO 10993-5:2009; Biological Evaluation of Medical Devices—Part 5: Tests for in vitro Cytotoxicity. ISO Standards: Geneva, Switzerland, 2009. 34. Rashid, F.; Saeed, A.; Iqbal, J. In Vitro Anticancer Effects of Stilbene Derivatives: Mechanistic Studies on HeLa and MCF-7 Cells. Anti-Cancer Agents Med. Chem. 2021,21, 793–802. [CrossRef] [PubMed] 35. Costa, P.; Lobo, J.M.S. Modeling and comparison of dissolution profiles. Eur. J. Pharm. Sci. 2001,13, 123–133. [CrossRef] [PubMed] 36. Sánchez-Aguinagalde, O.; Lejardi, A.; Meaurio, E.; Hernández, R.; Mijangos, C.; Sarasua, J.-R. Novel Hydrogels of Chitosan and Poly(vinyl alcohol) Reinforced with Inorganic Particles of Bioactive Glass. Polymers 2021,13, 691. [CrossRef] [PubMed] 37. Pezzoli, R.; Lyons, J.G.; Gately, N.; Higginbotham, C.L. Investigation of miscibility estimation methods between indomethacin and poly(vinylpyrrolidone-co-vinyl acetate). Int. J. Pharm. 2018,549, 50–57. [CrossRef] 38. Hernandez-Montero, N.; Ugartemendia, J.M.; Amestoy, H.; Sarasua, J.R. Complex phase behavior and state of miscibility in Poly(ethylene glycol)/Poly(l-lactide-co-ε-caprolactone) Blends. J. Polym. Sci. B Polym. Phys. 2014,52, 111–121. [CrossRef] 39. Baird, J.A.; Taylor, L.S. Evaluation of amorphous solid dispersion properties using thermal analysis techniques. Adv. Drug Deliv. Rev. 2012,64, 396–421. [CrossRef] 40. Sánchez-Aguinagalde, O.; Meaurio, E.; Lejardi, A.; Sarasua, J.-R. Amorphous solid dispersions in poly( ε -caprolactone)/xanthohumol bioactive blends: Physicochemical and mechanical characterization. J. Mater. Chem. B 2021,9, 4219–4229. [CrossRef] [PubMed] 41. Colthup, N.B.; Daly, L.H.; Wiberley, S.E. Chapter 9—Carbonyl Compounds, 3rd ed.; Wiberley, E., Ed.; Academic Press: San Diego, CA, USA, 1990; pp. 289–325. [CrossRef] 42. Harrison, W.; Shearer, H.M.M.; Trotter, J. Crystal structure of mycophenolic acid. Journal of the Chemical Society. Perkin Trans. 1972,2, 1542–1544. [CrossRef] 43. Covarrubias, A.; Zúñiga-Villarreal, N.; González-Lucas, A.; Díaz-Domínguez, J.; Espinosa-Pérez, G. Crystal Structure of Mycophenolic Acid: 6-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methyl-hex-4-enoic Acid. Anal. Sci. 2000,16, 783–784. [CrossRef] 44. Zeng, Q.Z.; Ouyang, J.; Zhang, S.; Zhang, L. Structural characterization and dissolution profile of mycophenolic acid cocrystals. Eur. J. Pharm. Sci. 2017,102, 140–146. [CrossRef] 45. Li, J.; Mooney, D.J. Designing hydrogels for controlled drug delivery. Nat. Rev. Mater. 2016,1, 16071. [CrossRef] [PubMed] 46. Baishya, H. Application of Mathematical Models in Drug Release Kinetics of Carbidopa and Levodopa ER Tablets. J. Dev. Drugs 2017,6, 1000171. [CrossRef] 47. Klangjorhor, J.; Chaiyawat, P.; Teeyakasem, P.; Sirikaew, N.; Phanphaisarn, A.; Settakorn, J.; Lirdprapamongkol, K.; Yama, S.; Svasti, J.; Pruksakorn, D. Mycophenolic acid is a drug with the potential to be repurposed for suppressing tumor growth and metastasis in osteosarcoma treatment. Int. J. Cancer 2020,146, 3397–3409. [CrossRef] [PubMed] 48. Dun, B.; Sharma, A.; Teng, Y.; Liu, H.; Purohit, S.; Xu, H.; Zeng, L.; She, J.X. Mycophenolic acid inhibits migration and invasion of gastric cancer cells via multiple molecular pathways. PLoS ONE 2013,8, e81702. [CrossRef] [PubMed] 49. Dun, B.; Sharma, A.; Xu, H.; Liu, H.; Bai, S.; Zeng, L.; She, J.X. Transcriptomic changes induced by mycophenolic acid in gastric cancer cells. Am. J. Transl. Res. 2014,6, 28–42. 50. Dun, B.; Xu, H.; Sharma, A.; Liu, H.; Yu, H.; Yi, B.; Liu, X.; He, M.; Zeng, L.; She, J.X. Delineation of biological and molecular mechanisms underlying the diverse anticancer activities of mycophenolic acid. Int. J. Clin. Exp. Pathol. 2013,6, 2880–2886. [PubMed] 51. Howgate, E.M.; Yeo, K.R.; Proctor, N.J.; Tucker, G.T.; Rostami-Hodjegan, A. Prediction of in vivo drug clearance from in vitro data. I: Impact of inter-individual variability. Xenobiotica 2006,36, 473–497. [CrossRef] [PubMed] 52. Rambhia, K.J.; Ma, P.X. Controlled drug release for tissue engineering. J. Control. Release 2015,219, 119–128. [CrossRef] [PubMed] 53. Hanahan, D.; Weinberg, R.A. The Hallmarks of Cancer. Cell 2000,100, 57–70. [CrossRef] [PubMed] 54. Morath, C.; Reuter, H.; Simon, V.; Krautkramer, E.; Muranyi, W.; Schwenger, V.; Goulimari, P.; Grosse, R.; Hahn, M.; Lichter, P.; et al. Effects of mycophenolic acid on human fibroblast proliferation, migration and adhesion in vitro and in vivo .Am. J. Transplant. 2008,8, 1786–1797. [CrossRef] [PubMed] 55. Chen, K.; Cao, W.; Li, J.; Sprengers, D.; Hernanda, P.Y.; Kong, X.; van der Laan, L.J.; Man, K.; Kwekkeboom, J.; Metselaar, H.J.; et al. Differential sensitivities of fast-and slow-cycling cancer cells to inosine monophosphate dehydrogenase 2 inhibition by mycophenolic acid. Mol. Med. 2015,21, 792–802. [CrossRef] [PubMed] 56. Franklin, T.J.; Jacobs, V.; Bruneau, P.; Ple, P. Glucuronidation by human colorectal adenocarcinoma cells as a mechanism of resistance to mycophenolic acid. Adv. Enzym. Regul. 1995,35, 91–100. [CrossRef] [PubMed] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.