Physicochemical Characterization and In Vitro Activity of Poly(ε-Caprolactone)/Mycophenolic Acid Amorphous Solid Dispersions

Ci a ion: Sánchez-Aguinagalde, O.;
Sanchez-Rexach, E.; Polo, Y.;
La añaga, A.; Leja di, A.; Meau io, E.;
Sa asua, J.-R. Physicochemical
Cha ac e iza ion and In Vi o
Ac i i y o Poly(ε-Cap olac one)/
Mycophenolic Acid Amo phous Solid
Dispe sions. Polyme s 2024,16, 1088.
h ps://doi.o g/10.3390/
polym16081088
Academic Edi o s: S e ano Fa is and
Masoud Ghaani
Recei ed: 10 No embe 2023
Re ised: 27 Ma ch 2024
Accep ed: 3 Ap il 2024
Published: 13 Ap il 2024
Copy igh : © 2024 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
polyme s
A icle
Physicochemical Cha ac e iza ion and In Vi o Ac i i y o
Poly(ε-Cap olac one)/Mycophenolic Acid Amo phous
Solid Dispe sions
O oi z Sánchez-Aguinagalde 1, E a Sanchez-Rexach 1, Yu ena Polo 2, Ai o La añaga 1, Ainhoa Leja di 1,*,
Emilio Meau io 1and Jose-Ramon Sa asua 1
1Depa men o Mining-Me allu gy Enginee ing and Ma e ials Science, POLYMAT, Bilbao School o
Enginee ing, Uni e si y o he Basque Coun y (UPV/EHU), Plaza Ingenie o To es Que edo 1, 48013 Bilbao,
Spain; [email p o ec ed] (O.S.-A.); [email p o ec ed] (E.S.-R.); ai o [email p o ec ed] (A.L.);
[email p o ec ed] (E.M.); j [email p o ec ed] (J.-R.S.)
2Polime bio SL, Paseo Mi amon 170, 20014 Donos ia-San Sebas ian, Spain; [email p o ec ed]
*Co espondence: ainhoa.leja [email p o ec ed]
Abs ac : The ob en ion o amo phous solid dispe sions (ASDs) o mycophenolic acid (MPA) in
poly(
ε
-cap olac one) (PCL) is epo ed in his pape . An imp o emen in he bioa ailabili y o he d ug
is possible hanks o he a o able speci ic in e ac ions occu ing in his sys em. Di e en ial scanning
calo ime y (DSC) was used o in es iga e he miscibili y o PCL/MPA blends, measu ing glass
ansi ion empe a u e (Tg) and analyzing mel ing poin dep ession o ob ain a nega i e in e ac ion
pa ame e , which indica es he de elopmen o a o able in e -associa ion in e ac ions. Fou ie
ans o m in a ed spec oscopy (FTIR) was used o analyze he speci ic in e ac ion occu ing in
he blends. D ug elease measu emen s showed ha a leas 70% o he d ug was eleased by he
hi d day
in i o
in all composi ions. Finally, p elimina y
in i o
cell cul u e expe imen s showed a
dec eased numbe o cance ous cells o e he sca olds con aining MPA, p esumably a ising om he
an i-cance ac i i y a ibu able o MPA.
Keywo ds: poly(
ε
-cap olac one) (PCL); mycophenolic acid (MPA); amo phous solid dispe sions
(ASDs); miscibili y; in e ac ions; d ug elease; cance ea men
1. In oduc ion
As new ea men s and d ugs appea o all kinds o diseases, we a e also aced wi h
g ea challenges o achie e a sa is ac o y applica ion o hese emedies. Al hough hey
may be e ec i e in heo y, mos o he d ugs ha a e being app o ed a e no easible in
e ms o hei biopha macological p ope ies. The main causes a e low pe meabili y, poo
solubili y, o apid elimina ion om he body. In ac , 90% o he d ugs being de eloped
a e molecules wi h low wa e solubili y, in addi ion o almos 40% o he d ugs al eady
app o ed
[1–3].
The dimensions o his p oblem can be seen, o example, in he case o
he o al adminis a ion o doses. In o de o each sys emic ci cula ion, he d ug mus be
dissol ed in he in es inal luids o he gas oin es inal ac , which is di icul in he case o
low solubili y [
4
]. The cause o his low bioa ailabili y is he di e en molecula a ange-
men s, whe e he c ys alline compounds a e he ones ha p esen he g ea es p oblem [
5
].
In o de o sol e his p oblem, one o he es ablished s a egies is amo phiza ion, which
ans o ms low-ene gy c ys alline subs ances in o high-ene gy amo phous compounds,
gi ing hem g ea e solubili y and bioa ailabili y [
6
]. Howe e , hese amo phous solids a e
no he modynamically s able because o hei excess en halpy, en opy, and ee ene gies,
which cause hem o end o o m c ys als [
7
]. Fo his eason, achie ing he s abili y o
hese compounds is a g ea challenge.
One o he s a egies used o his pu pose is de eloping amo phous solid dispe sions
(ASDs). In he 1970s, Chiou and Riegelman de ined he e m solid dispe sions as he
Polyme s 2024,16, 1088. h ps://doi.o g/10.3390/polym16081088 h ps://www.mdpi.com/jou nal/polyme s
Polyme s 2024,16, 1088 2 o 16
dispe sion o an ac i e pha maceu ical ing edien (API) in an amo phous ca ie in a solid
s a e p epa ed by sol en , mel ing, o sol en -mel ing me hods [
7
]. In hese sys ems, he e
is a mix u e a he molecula le el be ween a polyme and he d ug in an amo phous s a e,
inc easing i s bioa ailabili y [
8
–
12
]. I is known ha he low he modynamic s abili y due o
he high ene gy o he amo phous s a e causes elaxa ion, nuclea ion, and ec ys alliza ion
unde di e en a iables [
13
–
16
]. Thus, he ole o he polyme ic ma ix is o inhibi his
p ocess and main ain he mix u e in a single homogeneous phase [
17
,
18
]. To a oid his
c ys alliza ion and main ain he mix u e in he me as able egion o he bina y phase
diag am, miscibili y be ween he API and polyme is essen ial [
19
–
21
]. The kine ic s abili y
p o ided by s o age below he glass ansi ion empe a u e (T
g
) mus also be aken in o
accoun . In ac , acco ding o Hancock e al., he s abili y o he mix u e could be ensu ed
o yea s by s o ing i a leas 50 K below T
g
[
22
,
23
]. One signi ican challenge in his sys em
is he unp edic able na u e o polyme –d ug in e ac ions [24].
One in e es ing d ug o es his sys em is mycophenolic acid (MPA—C
17
H
20
O
6
,
320 g/mol; aqueous solubili y: 35.5 mg/L). Mycophenolic acid (Scheme 1) is an an ibio ic
p oduced by he Penicillium amily and is bes known o i s use as an immunosupp essi e
agen o p e en ejec ion in o gan ansplan s [
25
,
26
]. In addi ion, his d ug has mo e
biological p ope ies, such as an i ungal o an i i al p ope ies [
27
]. I also has he po en ial
o p e en and pe haps ea ch onic allog a asculopa hy, as i can inhibi he p oli e -
a ion o ascula smoo h muscle cells (VSMCs), mesangial cells, and myo ib oblas s [
28
].
Howe e , one o he mos s iking p ope ies is i s abili y o ac agains umo cells o
a ious ypes such as leukemia o lymphoma, among o he s [
29
]. This is because MPA is an
inhibi o o inosine monophospha e dehyd ogenase (IMPDH), which leads o he educ ion
o xan hine monophospha e (XMP), guanosine-5
′
- iphospha e (GTP), and deoxyguanosine
iphospha e (dGTP), hus inhibi ing he p oli e a ion o lympholeukocy es and cance
cells [
26
,
30
]. Despi e ha ing so many a o able p ope ies, he bioa ailabili y o MPA
in i o
is ela i ely poo due o he high clea ance inside a li ing o ganism, which limi s i s
possibili y o clinical applica ion [
25
]. This, in addi ion o i s low aqueous solubili y, makes
i a pe ec candida e o o ming amo phous solid dispe sions.
Polyme s2024,16,xFORPEERREVIEW2o 17


en halpy,en opy,and eeene gies,whichcause hem o end o o mc ys als[7].Fo 
his eason,achie ing hes abili yo  hesecompoundsisag ea challenge.
Oneo  hes a egiesused o  hispu poseisde elopingamo phoussolid
dispe sions(ASDs).In he1970s,ChiouandRiegelmande ined he e msoliddispe sions
as hedispe siono anac i epha maceu icaling edien (API)inanamo phousca ie in
asolids a ep epa edbysol en ,mel ing,o sol en -mel ingme hods[7].In hese
sys ems, he eisamix u ea  hemolecula le elbe weenapolyme and hed uginan
amo phouss a e,inc easingi sbioa ailabili y[8–12].I isknown ha  helow
he modynamics abili ydue o hehighene gyo  heamo phouss a ecauses elaxa ion,
nuclea ion,and ec ys alliza ionunde diffe en  a iables[13–16].Thus, he oleo  he
polyme icma ixis oinhibi  hisp ocessandmain ain hemix u einasingle
homogeneousphase[17,18].Toa oid hisc ys alliza ionandmain ain hemix u ein he
me as able egiono  hebina yphasediag am,miscibili ybe ween heAPIandpolyme 
isessen ial[19–21].Thekine ics abili yp o idedbys o agebelow heglass ansi ion
empe a u e(T
g
)mus alsobe akenin oaccoun .In ac ,acco ding oHancocke al., he
s abili yo  hemix u ecouldbeensu ed o yea sbys o ingi a leas 50KbelowT
g

[22,23].Onesigni ican challengein hissys emis heunp edic ablena u eo polyme –
d ugin e ac ions.[24].
Onein e es ingd ug o es  hissys emismycophenolicacid(MPA—C
17
H
20
O
6
,320
g/mol;aqueoussolubili y:35.5mg/L).Mycophenolicacid(Scheme1)isanan ibio ic
p oducedby hePenicillium amilyandisbes known o i suseasanimmunosupp essi e
agen  op e en  ejec ionino gan ansplan s[25,26].Inaddi ion, hisd ughasmo e
biologicalp ope ies,suchasan i ungalo an i i alp ope ies[27].I alsohas he
po en ial op e en andpe haps ea ch onicallog a  asculopa hy,asi caninhibi  he
p oli e a iono  ascula smoo hmusclecells(VSMCs),mesangialcells,and
myo ib oblas s[28].Howe e ,oneo  hemos s ikingp ope iesisi sabili y oac 
agains  umo cellso  a ious ypessuchasleukemiao lymphoma,amongo he s[29].
ThisisbecauseMPAisaninhibi o o inosinemonophospha edehyd ogenase(IMPDH),
whichleads o he educ iono xan hinemonophospha e(XMP),guanosine-5′-
iphospha e(GTP),anddeoxyguanosine iphospha e(dGTP), husinhibi ing he
p oli e a iono lympholeukocy esandcance cells[26,30].Despi eha ingsomany
a o ablep ope ies, hebioa ailabili yo MPAin i ois ela i elypoo due o hehigh
clea anceinsideali ingo ganism,whichlimi si spossibili yo clinicalapplica ion[25].
This,inaddi ion oi slowaqueoussolubili y,makesi ape ec candida e o  o ming
amo phoussoliddispe sions.

Poly(ε-cap olac one)Mycophenolicacid
Scheme1.Chemicals uc u eso PCLandMPA.
In hiswo k, hepolyme selec edas hema ix odispe seMPAinamo phous o m
ispoly(ε-cap olac one)(PCL),abiodeg adablesemic ys allinepolyes e .I sglass
ansi ion empe a u eisa ound−60°Candi smel ingpoin a a ound60°C.The
biodeg ada iono  hispolyme unde physiologicalcondi ionshasbeen epo ed olas 
se e almon hs oyea s[31,32],makingi sui able o long- e mbiomedicalapplica ions.
In hiswo k,miscibili yandin e ac ionsbe weenPCLandMPAa es udied o e i y he
sui abili yo  hismix u e o  he o ma iono anamo phoussoliddispe sion.Inaddi ion,
wesepa a ely es ed hein e ac iono  heblendscon aininginc easingconcen a ionso 
Scheme 1. Chemical s uc u es o PCL and MPA.
In his wo k, he polyme selec ed as he ma ix o dispe se MPA in amo phous o m
is poly(
ε
-cap olac one) (PCL), a biodeg adable semic ys alline polyes e . I s glass ansi ion
empe a u e is a ound
−
60
◦
C and i s mel ing poin a a ound 60
◦
C. The biodeg ada ion
o his polyme unde physiological condi ions has been epo ed o las se e al mon hs
o yea s [
31
,
32
], making i sui able o long- e m biomedical applica ions. In his wo k,
miscibili y and in e ac ions be ween PCL and MPA a e s udied o e i y he sui abili y
o his mix u e o he o ma ion o an amo phous solid dispe sion. In addi ion, we
sepa a ely es ed he in e ac ion o he blends con aining inc easing concen a ions o
MPA wi h bo h a non-cance ous ib oblas cell line (MRC5), app o ed by ISO 10993 o
cy o oxici y s udies [
33
], and a widely used immo alized HeLa cell line de i ed om
ce ical cance [34].
Polyme s 2024,16, 1088 3 o 16
2. Expe imen al Sec ion
2.1. S a ing Ma e ials
Poly(
ε
-cap olac one) (PURASORB
®
PC12 ade name) wi h an a e age molecula
weigh (M
w
) o 1.3
×
10
5
g/mol and M
w
/M
n
= 1.76 was pu chased om Pu ac Biochem
(Go inchem, The Ne he lands). Mycophenolic acid (C
17
H
20
O
6
, M = 320.34 g/mol) was
ob ained om Fluo ochem L d. (Gossop, UK), and dichlo ome hane (DCM) was supplied
by Labkem (Dublin, I eland).
2.2. Blend P epa a ion
Films we e p epa ed by sol en cas ing om dichlo ome hane (DCM) solu ions con-
aining 2.5 w % o PCL/MPA blend a oom empe a u e.
2.3. Di e en ial Scanning Calo ime y (DSC)
A Modula ed DSC Q200 om TA Ins umen s was used o he mal analyses. All
he scans we e pe o med in he me ic aluminum pans unde ni ogen a mosphe e wi h
sample weigh s be ween 5 and 10 mg. Two scans om
−
80
◦
C o 160
◦
C wi h a scan a e o
20
◦
C/min we e pe o med in o de o measu e glass ansi ion empe a u es (T
g
) in he
second one.
2.4. Mel ing Poin Dep ession Analysis
The mel ing poin dep ession o MPA was obse ed in MPA- ich blends con aining
0–20 w % PCL. To ob ain he mel ing empe a u e o MPA c ys als, samples we e hea ed in
he DSC wi h a scan a e o 1 ◦C/min.
The samples we e weighed again a e he DSC scans, and no weigh loss was obse ed
du ing he he mal ea men s.
2.5. Fou ie T ans o m In a ed Spec oscopy (FTIR)
A Nicole AVATAR 370 Fou ie ans o m in a ed spec opho ome e was used o
eco d FTIR spec a o he blends, wi h a esolu ion o 2 cm
−1
and a e aged o e 64 scans
in he ange o 400–4000 cm
−1
. Dichlo ome hane solu ions con aining 2 w % o blends we e
cas on KB pelle s by e apo a ion o he sol en a oom empe a u e. The abso bance o
he samples was wi hin he ange whe e he Lambe –Bee law is obeyed.
2.6. In Vi o D ug Release
In i o
d ug elease expe imen s we e pe o med o he 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 o PCL/MPA o
Ø10 mm ob ained by sol en cas ing we e imme sed in 1 mL o 0.1 M PBS bu e (pH 7.4)
a 37
◦
C. A ixed in e als, samples o 200
µ
L we e aken and eplaced wi h esh PBS a
37
◦
C. The d ug concen a ion in solu ion was de e mined using a BioTech Sine gy H1M
Mic oPla e Reade (Minneapolis, MN, USA) using a calib a ion cu e ha was p e iously
ob ained measu ing he abso bance a a wa eleng h o 305 nm o solu ions o MPA in
0.1 M PBS.
The elease kine ics o mycophenolic acid we e examined by conside ing ou ma he-
ma ical models as ollows:
Ze o −o de : C /C∞=k0 (1)
Fi s −o de : ln (1−C /C∞)=−k1 (2)
Higuchi : C /C∞=kh 1
2(3)
Ko smeye –Peppas : C /C∞=k n(4)
whe e C
is he cumula i e amoun o he d ug eleased a ime ,C
∞
is he s a ing amoun
o he d ug, nis he elease exponen , and k
0
,k
1
,k
h
, and ka e he kine ic cons an s. Ze o-
o de kine ics (Equa ion (1)) ep esen s a elease p ocess ha is con olled by he elaxa ion
Polyme s 2024,16, 1088 4 o 16
o polyme ic chains, independen o i s concen a ion and wi h a cons an elease a e.
The i s -o de kine ics (Equa ion (2)) model ep esen s a d ug elease a e ha depends
on i s concen a ion [
32
]. Higuchi (Equa ion (3)) desc ibes d ug elease as a di usion
p ocess based on Fick’s law, squa e oo ime-dependen . I he elease mechanism is no
well known o when mo e han one ype o elease phenomena could be in ol ed, he
Ko smeye –Peppas (Equa ion (4)) model is applied. I is possible o de ine whe he he
elease happens by Fickian di usion, anomalous anspo , Case-II anspo , o Supe
Case-II anspo depending on he alues ob ained o he elease exponen , n[35,36].
2.7. In Vi o Cell Cul u e Expe imen s
In i o
cell cul u e expe imen s we e pe o med on he PCL/MPA 99.5/0.5, 99/1,
and 98/2 blends. Ci cula samples o PCL/MPA o Ø6 mm we e ob ained, and each side
was s e ilized o 30 min unde UV ligh . Ei he he immo alized HeLa cell line (ATCC,
Manassas, VA, USA) de i ed om ce ical cance o he non-cance ous ib oblas s MRC5
(CCL-171, ATCC, Manassas, VA, USA) de i ed om lung issue we e d op-seeded o e he
ma e ials a a concen a ion o 25,000 cells pe sca old. A e 1 h, 480 mL o p ewa med
DMEM (Fishe Scien i ic, Mad id, Spain) a 37
◦
C supplemen ed wi h 10% e al bo ine
se um (FBS) (Fishe Scien i ic, Mad id, Spain), 1% L-glu amine (Fishe Scien i ic, Mad id,
Spain), and penicillin/s ep omycin (Fishe Scien i ic, Mad id, Spain) we e added. PCL
ilms we e used as he nega i e con ol, and o he posi i e con ol, MPA in dissolu ion
a a concen a ion o 300 ppm was dissol ed on he cul u e media and il a ed (0.2
µ
m).
Cells we e incuba ed a 37 ◦C and 5% CO2in a s anda d cell cul u e incuba o .
2.8. Immunos aining
A e 1 o 3 days
in i o
(DIV), samples we e ixed wi h 4% pa a o maldehyde (PFA)
(Fishe Scien i ic, Spain) and pe meabilized wi h 0.3% i on-X100 (Fishe Scien i ic, Spain)
in PBS (Fishe Scien i ic, Spain) con aining 1% Bo ine Se um Albumin (BSA) (Sigma Ald ich,
Spain). Fo he s aining, hodamine/phalloidin (Fishe Scien i ic, Mad id, Spain) and DAPI,
4
′
,6-diamidino-2-phenylindole dihyd ochlo ide (Fishe Scien i ic, Mad id, Spain) we e
dilu ed in 1% PBS BSA and incuba ed o 1.5 h. A e washing each sample 2 imes in PBS
con aining 0.1% Tween-20 (Fishe Scien i ic, Mad id, Spain) and 1 ime in PBS, he samples
we e moun ed using moun ing medium (Abcam, Wal ham, MA, USA). The samples we e
analyzed in an in e ed luo escence mic oscope (Nikon Eclipse Ts2). Fo cell quan i ica ion
s udies, 5 di e en poin s we e aken.
2.9. Cell Coun and S a is ical Analysis
Fo cell coun s, i e alea o y images o 0.1 mm
2
we e aken o each o he iplica es in
each condi ion, and nuclea DAPI labeling was used o calcula e he o al numbe o cells.
The da a we e subjec ed o one-way analysis o a iance (ANOVA) using K uskal–Wallis
ollowed by Dunn’s pos hoc es . The le el o signi icance was se a p< 0.05. The esul s
we e p esen ed as mean ±SD o SEM.
3. Resul s and Discussion
3.1. Miscibili y Analysis by Di e en ial Scanning Calo ime y (DSC)
When wo componen s a e miscible, a single glass ansi ion empe a u e (T
g
) be-
ween he T
g
o each ma e ial, which changes p og essi ely wi h he composi ion, is
expec ed [
37
,
38
]. On he con a y, he de ec ion o mo e han one single alue would
indica e a sepa a ion in o indi idual amo phous phases wi hin he sys em. Di e en me h-
ods ha e been employed o p edic he glass ansi ion empe a u e o amo phous bina y
sys ems, such as he Go don–Taylo (GT), Couchman–Ka asz (CK), and Fox equa ions
(Equa ion (5)). Conside ing ha he Fox equa ion was de eloped o analyze sys ems o med
Polyme s 2024,16, 1088 5 o 16
by componen s o equal densi ies, i is app op ia e o use i o es ima e his in e media e
Tg, as he densi ies o PCL and MPA a e 1.14 g/cm3and 1.3 g/cm3, espec i ely [39]:
1
Tgb
=w1
Tg1
+w2
Tg2
(5)
whe e w
1
and w
2
a e he weigh ac ions o componen s 1 and 2, espec i ely, T
g1
and T
g2
a e he glass ansi ion empe a u es o he pu e componen s, and T
gb
is he glass ansi ion
empe a u e o he blend.
Figu e 1shows he i s scan DSC aces ob ained o he pu e componen s and
o di e en PCL/MPA blends. As can be seen, pu e PCL is a semic ys alline polyme
displaying a glass ansi ion empe a u e loca ed a abou
−
60
◦
C and a mel ing endo he m
a abou 60
◦
C. On he o he hand, MPA is a c ys alline compound mel ing a 145
◦
C, which
can be also supe cooled o unde go a glass ansi ion a 11
◦
C a e ehea ing he quenched
mel (see Figu e 2).
Polyme s2024,16,xFORPEERREVIEW5o 17


[37,38].On hecon a y, hede ec iono mo e hanonesingle aluewouldindica easepa-
a ionin oindi idualamo phousphaseswi hin hesys em.Di e en me hodsha ebeen
employed op edic  heglass ansi ion empe a u eo amo phousbina ysys ems,suchas
heGo don–Taylo (GT),Couchman–Ka asz(CK),andFoxequa ions(Equa ion(5)).Con-
side ing ha  heFoxequa ionwasde eloped oanalyzesys ems o medbycomponen so 
equaldensi ies,i isapp op ia e ousei  oes ima e hisin e media eT
g
,as hedensi ieso 
PCLandMPAa e1.14g/cm
3
and1.3g/cm
3
, espec i ely[39]:
1
𝑇 𝑤
𝑇 𝑤
𝑇(5)
whe ew
1
andw
2
a e heweigh  ac ionso componen s1and2, espec i ely,T
g1
andT
g2

a e heglass ansi ion empe a u eso  hepu ecomponen s,andT
gb
is heglass ansi ion
empe a u eo  heblend.
Figu e1shows he i s scanDSC acesob ained o  hepu ecomponen sand o 
diffe en PCL/MPAblends.Ascanbeseen,pu ePCLisasemic ys allinepolyme display-
ingaglass ansi ion empe a u eloca eda abou −60°Candamel ingendo he ma 
abou 60°C.On heo he hand,MPAisac ys allinecompoundmel inga 145°C,which
canbealsosupe cooled ounde goaglass ansi iona 11°Ca e  ehea ing hequenched
mel (seeFigu e2).

Figu e1.Fi s scanDSC aces o PCL,MPA,andPCL/MPAblends.

Figu e 1. Fi s scan DSC aces o PCL, MPA, and PCL/MPA blends.
Polyme s2024,16,xFORPEERREVIEW5o 17


[37,38].On hecon a y, hede ec iono mo e hanonesingle aluewouldindica easepa-
a ionin oindi idualamo phousphaseswi hin hesys em.Di e en me hodsha ebeen
employed op edic  heglass ansi ion empe a u eo amo phousbina ysys ems,suchas
heGo don–Taylo (GT),Couchman–Ka asz(CK),andFoxequa ions(Equa ion(5)).Con-
side ing ha  heFoxequa ionwasde eloped oanalyzesys ems o medbycomponen so 
equaldensi ies,i isapp op ia e ousei  oes ima e hisin e media eT
g
,as hedensi ieso 
PCLandMPAa e1.14g/cm
3
and1.3g/cm
3
, espec i ely[39]:
1
𝑇 𝑤
𝑇 𝑤
𝑇(5)
whe ew
1
andw
2
a e heweigh  ac ionso componen s1and2, espec i ely,T
g1
andT
g2

a e heglass ansi ion empe a u eso  hepu ecomponen s,andT
gb
is heglass ansi ion
empe a u eo  heblend.
Figu e1shows he i s scanDSC acesob ained o  hepu ecomponen sand o 
diffe en PCL/MPAblends.Ascanbeseen,pu ePCLisasemic ys allinepolyme display-
ingaglass ansi ion empe a u eloca eda abou −60°Candamel ingendo he ma 
abou 60°C.On heo he hand,MPAisac ys allinecompoundmel inga 145°C,which
canbealsosupe cooled ounde goaglass ansi iona 11°Ca e  ehea ing hequenched
mel (seeFigu e2).

Figu e1.Fi s scanDSC aces o PCL,MPA,andPCL/MPAblends.

Figu e 2. Second scan DSC aces o PCL, MPA, and PCL/MPA blends.
As can be seen in Figu e 2, he PCL/MPA blends show composi ion-dependen
single glass ansi ions loca ed close o he alues p edic ed using he Fox equa ion (see
Table 1and Figu e 3). Consequen ly, i can be concluded ha he wo componen s a e

Polyme s 2024,16, 1088 6 o 16
comple ely miscible in he amo phous phase. Fu he mo e, he mel ing empe a u e o
PCL dec eases as he con en o MPA inc eases. Fu he mo e, he c ys alliza ion o PCL is
o ally supp essed when he d ug composi ion exceeds 50 w %.
Table 1. The mal p ope ies o PCL/MPA blends.
PCL/MPA
T
g
Expe imen al
(◦C)
TgTheo e ical
(Fox) (◦C) TmPCL (◦C) ∆H PCL (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 - - -
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
Figu e2.SecondscanDSC aces o PCL,MPA,andPCL/MPAblends.
AscanbeseeninFigu e2, hePCL/MPAblendsshowcomposi ion-dependen single
glass ansi ionsloca edclose o he aluesp edic edusing heFoxequa ion(seeTable1
andFigu e3).Consequen ly,i canbeconcluded ha  he wocomponen sa ecomple ely
misciblein heamo phousphase.Fu he mo e, hemel ing empe a u eo PCLdec eases
as hecon en o MPAinc eases.Fu he mo e, hec ys alliza iono PCLis o allysup-
p essedwhen hed ugcomposi ionexceeds50w %.
Table1.The malp ope ieso PCL/MPAblends.
PCL/MPAT
g
Expe imen al(°C)T
g
Theo e ical(Fox)(°C)T
m
PCL(°C)ΔH
PCL(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---

Figu e3.Glass ansi ion empe a u e e suscomposi ion o  hePCL/MPAsys em:(■)expe i-
men al aluesand(■)Foxequa ion.
3.2.Mel ingPoin Dep essionAnalysis
I  he eeene gyo  hemixingo  he wocomponen s(ΔG
mix
)isnega i e,asys em
canbeconside ed he modynamicallymiscible.
∆𝐺  ∆𝐻 𝑇∆𝑆(6)
whe eΔH
mix
andΔS
mix
a e heen halpyanden opyo mixing, espec i ely.TΔS
mix
isal-
waysposi i esince heen opyo mixingisadded o heen opyo mel ing,making he
en opychangeinamiscibleblendla ge  hanin hepu ecomponen .Consequen ly, he
signo ΔG
mix
dependson he alueo ΔH
mix
.Ino de  oa oidphasesepa a ion, hecohe-
si ein e ac ionsneed obelowe  han hesumo adhesi ein e ac ions,gene a inga a-
o ableen halpyo mixing.Themiscibili ybe ween wocomponen sin e mso  he
changein heGibbs eeene gycanbedesc ibedusing hemel ingpoin dep ession
me hod,basedonFlo y–Huggins heo y.Acco ding o hisme hod, hemel ingpoin 
Figu e 3. Glass ansi ion empe a u e e sus composi ion o he PCL/MPA sys em: (
■
) expe imen-
al alues and (■) Fox equa ion.
3.2. Mel ing Poin Dep ession Analysis
I he ee ene gy o he mixing o he wo componen s (
∆
G
mix
) is nega i e, a sys em
can be conside ed he modynamically miscible.
∆Gmix =∆Hmix −T∆Smix (6)
whe e
∆
H
mix
and
∆
S
mix
a e he en halpy and en opy o mixing, espec i ely. T
∆
S
mix
is
always posi i e since he en opy o mixing is added o he en opy o mel ing, making
he en opy change in a miscible blend la ge han in he pu e componen . Consequen ly,
he sign o
∆
G
mix
depends on he alue o
∆
H
mix
. In o de o a oid phase sepa a ion, he
cohesi e in e ac ions need o be lowe han he sum o adhesi e in e ac ions, gene a ing
a a o able en halpy o mixing. The miscibili y be ween wo componen s in e ms o
he change in he Gibbs ee ene gy can be desc ibed using he mel ing poin dep ession
me hod, based on Flo y–Huggins heo y. Acco ding o his me hod, he mel ing poin
empe a u e o he d ug will dec ease as he polyme con en in he mix u e inc eases i he
cohesi e o ces in he pu e componen s a e weake han he adhesi e o ces be ween he
Polyme s 2024,16, 1088 7 o 16
d ug and he polyme [
32
,
40
]. Flo y’s ela ionship can be used o analyze he dep ession o
he equilib ium mel ing poin :
1
Tm
−1
T0
m
=−R
∆H2u
V2u
V1uln ϕ2
m2
+1
m2
−1
m1ϕ1+χ12ϕ2
1(7)
whe e
T0
m
is he equilib ium mel ing poin o he pu e c ys allizable componen and
Tm
is
he equilib ium mel ing poin o i s blends; he subsc ip s 1 and 2 e e o he amo phous
and c ys allizable componen s, espec i ely. R is he uni e sal gas cons an , while
∆H2u
is he hea o usion pe mole o c ys alline epea uni s.
Vu
is he mola olume o he
epea ing uni ,
m
is he deg ee o polyme iza ion,
ϕ
is he olumen ac ion, and
χ12
is he
in e ac ion pa ame e .
In o de o apply Equa ion (7), he mola olume o MPA (
V2=
246.3 cm
3
/mol) can
be conside ed as he mola olume o he la ice si es, esul ing in
m2=
1. The same
olume can be aken as he mola olume o he polyme ic epea uni
V2=V1u
. Since
m1=Vpol/V1uis la ge, 1/m1≈0. As a esul , Equa ion (7) simpli ies o:
1
Tm
−1
T0
m
=−R
∆H2ln ϕ2+ϕ1+χϕ2
1(8)
The mel ing poin s o pu e componen s and di e en PCL/MPA blends we e measu ed
a a low hea ing a e (1
◦
C/min). The a e age mel ing poin o pu e MPA is
T0
m=140.3 ◦C,
and his empe a u e is dec eased by nea ly 5
◦
C when 20 w % PCL is added o he blend.
The da a ob ained o each blend can be seen in Table 2. These esul s, wi h he a e age
mel ing en halpy o pu e MPA (
∆HMPA =
114.7 J/g) we e used o plo Equa ion (8) as a
unc ion o he squa e o he olume ac ion o he polyme ,
ϕ2
1
. The slope o his plo ,
which can be seen in Figu e 4, gi es an app oxima ion o he in e ac ion pa ame e o
χ=−
1.18. The nega i e alues o he in e ac ion pa ame e indica e an exo he mic
eac ion, con i ming a he modynamically miscible blend. I is also possible o calcula e he
in e ac ion ene gy densi y, B, a he mel ing empe a u e o MPA acco ding o Equa ion (9):
χ=BV
RT (9)
whe e V is a e e ence olumen (V =V2=246.3 cm3/mol), yielding B=−16.5 J/cm3.
Polyme s2024,16,xFORPEERREVIEW8o 17
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

Figu e4.Analysiso  hemel ing empe a u eo MPAacco ding oEqua ion(8) o  hePCL/MPA
sys em.Theslopeo  heplo gi es hein e ac ionpa ame e 𝜒  1.18.
Table2.Mel ing empe a u eso MPAob ained om1°Cmin
−1
scan a es.
MPAw %T
m
(°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.Fou ie T ans o mIn a edSpec oscopy(FTIR)
Theanalysiso  hechangesobse edin hein a edspec umuponblendingp o-
idesin o ma ionabou  hechangesinspeci icin e ac ionsandcane en uallyaidinex-
plaining heene ge iccon ibu ionsd i ing hemiscibili yo  hesys em.In hePCL/MPA
sys em,bo h heca bonyland hehyd oxyls e ching egionsa eo mainin e es because
hyd ogenbondingin e ac ionscanbeexpec ed o  hoseg oups.Figu e5shows heca -
bonyls e ching egion o PCL,MPA,and hei blends.Thespec umo pu ePCLshows
apeaka 1725cm
−1
a ibu able oc ys allinePCLandashoulde a 1735cm
−1
a ising om
heamo phousphase[32,40].On heo he hand,pu eMPAshows wodiffe en peaks
loca eda 1744and1708cm
−1
a ibu able, espec i ely, o helac oneca bonyland he
ca boxylicacidca bonyl.Bo hloca ionsa ea  helowe endo  hespec al angesco e-
sponding o hose unc ionalg oups[41]becauseo  hehyd ogenbondingin e ac ions
occu inginpu eMPA.Figu e6ske ches hesein e ac ionsasde i ed omXRDs udies
[42–44].Asi canbeseen,inpu eMPA, hemoleculesa ejoinedin hec ys albyca box-
ylicacidg oups o mingdime s,alongwi hbi u ca edhyd ogenbondsbe ween hehy-
d oxylg oupand heca boxylicacidca bonyl(abso p ionbanda 1708cm
−1
).Inaddi ion,
anin amolecula bi u ca edhyd ogenbond edshi s heabso p iono  helac oneca -
bonyl o he epo edwa enumbe (1744cm
−1
).
Figu e 4. Analysis o he mel ing empe a u e o MPA acco ding o Equa ion (8) o he PCL/MPA
sys em. The slope o he plo gi es he in e ac ion pa ame e χ=−1.18.
Polyme s 2024,16, 1088 8 o 16
Table 2. Mel ing empe a u es o MPA ob ained om 1 ◦C min−1scan a es.
MPA w % 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. Fou ie T ans o m In a ed Spec oscopy (FTIR)
The analysis o he changes obse ed in he in a ed spec um upon blending p o ides
in o ma ion abou he changes in speci ic in e ac ions and can e en ually aid in explaining
he ene ge ic con ibu ions d i ing he miscibili y o he sys em. In he PCL/MPA sys em,
bo h he ca bonyl and he hyd oxyl s e ching egions a e o main in e es because hyd ogen
bonding in e ac ions can be expec ed o hose g oups. Figu e 5shows he ca bonyl
s e ching egion o PCL, MPA, and hei blends. The spec um o pu e PCL shows a peak
a 1725 cm
−1
a ibu able o c ys alline PCL and a shoulde a 1735 cm
−1
a ising om he
amo phous phase [
32
,
40
]. On he o he hand, pu e MPA shows wo di e en peaks loca ed
a 1744 and 1708 cm
−1
a ibu able, espec i ely, o he lac one ca bonyl and he ca boxylic
acid ca bonyl. Bo h loca ions a e a he lowe end o he spec al anges co esponding o
hose unc ional g oups [
41
] because o he hyd ogen bonding in e ac ions occu ing in
pu e MPA. Figu e 6ske ches hese in e ac ions as de i ed om XRD s udies [
42
–
44
]. As i
can be seen, in pu e MPA, he molecules a e joined in he c ys al by ca boxylic acid g oups
o ming dime s, along wi h bi u ca ed hyd ogen bonds be ween he hyd oxyl g oup and
he ca boxylic acid ca bonyl (abso p ion band a 1708 cm
−1
). In addi ion, an in amolecula
bi u ca ed hyd ogen bond ed shi s he abso p ion o he lac one ca bonyl o he epo ed
wa enumbe (1744 cm−1).
Polyme s2024,16,xFORPEERREVIEW9o 17
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
Figu e5.Ca bonyls e ching egion o pu ePCLandMPAandPCL/MPAblendso diffe en com-
posi ions.

Figu e6.Hyd ogenbondinginc ys allineMPA(see ex ).
ThePCL/MPA20/80and40/60blendsshowamajo peakloca eda abou 1724cm
−1
,
accompaniedby woshoulde sa highe wa enumbe sloca eda abou 1735cm
−1
and
1750cm
−1
.A  hesecomposi ions,PCLisalmos inamo phous o macco ding o heDSC
esul s(hence, hecon ibu ionco esponding oc ys allinePCLshouldbenegligible),
and heabso p ionbandsco esponding oMPAa eexpec ed op e ailo e  hoseo 
Figu e 5. Ca bonyl s e ching egion o pu e PCL and MPA and PCL/MPA blends o di e en
composi ions.
Polyme s 2024,16, 1088 9 o 16
Polyme s2024,16,xFORPEERREVIEW9o 17
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

Figu e5.Ca bonyls e ching egion o pu ePCLandMPAandPCL/MPAblendso diffe en com-
posi ions.

Figu e6.Hyd ogenbondinginc ys allineMPA(see ex ).
ThePCL/MPA20/80and40/60blendsshowamajo peakloca eda abou 1724cm
−1
,
accompaniedby woshoulde sa highe wa enumbe sloca eda abou 1735cm
−1
and
1750cm
−1
.A  hesecomposi ions,PCLisalmos inamo phous o macco ding o heDSC
esul s(hence, hecon ibu ionco esponding oc ys allinePCLshouldbenegligible),
and heabso p ionbandsco esponding oMPAa eexpec ed op e ailo e  hoseo 
Figu e 6. Hyd ogen bonding in c ys alline MPA (see ex ).
The PCL/MPA 20/80 and 40/60 blends show a majo peak loca ed a abou
1724 cm−1,
accompanied by wo shoulde s a highe wa enumbe s loca ed a abou 1735 cm
−1
and
1750 cm
−1
. A hese composi ions, PCL is almos in amo phous o m acco ding o he DSC
esul s (hence, he con ibu ion co esponding o c ys alline PCL should be negligible),
and he abso p ion bands co esponding o MPA a e expec ed o p e ail o e hose o
PCL; hence, he band a 1724 cm
−1
is mos likely a ibu able ca boxylic acid ca bonyls
o ming dime s in he amo phous phase. This band is p obably s ongly o e lapped wi h
PCL ca bonyls hyd ogen bonded wi h hyd oxyl g oups p esen in MPA, bu un o una ely,
hese wo componen s a e no dis inguishable. The shoulde a abou 1735 cm
−1
can be
a ibu ed o ee C=O g oups in PCL and he one a abou 1750 cm
−1
o lac one ca bonyls
in he amo phous phase.
Finally, Figu e 7shows he hyd oxyl s e ching egion o MPA and i s blends wi h
PCL. As can be seen, he OH s e ching band in pu e MPA is loca ed a abou 3416 cm
−1
,
and blending b oadens he band and shi s i o highe wa enumbe s. Band b oadening is
a consequence o he p esence o amo phous MPA, while shi ing o highe wa enumbe s
can be a ibu ed o weake hyd ogen bonding in e ac ions in he blends compa ed wi h
pu e MPA. Despi e he weake na u e o he in e ac ions, he ene ge ic balance will s ill
ende a o able o miscibili y as long as he blend achie es a la ge numbe o in e ac ions,
a ising om he in oduc ion o addi ional in e ac ing g oups ( he PCL ca bonyls).
Polyme s 2024,16, 1088 16 o 16
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