nanoma e ials
A icle
Cyclodex in Ca ionic Polyme -Based
Nanoassemblies o Manage In lamma ion by
In a-A icula Deli e y S a egies
Annalau a Co da o 1,2 , Robe o Zagami 1, Milo Malanga 3, Jagadeesh Kuma Venka esan 4,
Ca men Al a ez-Lo enzo 5, Magali Cucchia ini 4, Anna Pipe no 2,* and
An onino Mazzaglia 1,*
1CNR-ISMN, Is i u o pe lo S udio dei Ma e iali Nanos u u a i, V. le F. S agno d’Alcon es 31,
98166 Messina, I aly; annalau a.co da [email p o ec ed].i (A.C.); [email p o ec ed].i (R.Z.)
2
Dipa imen o di Scienze Chimiche, Biologiche, Fa maceu iche ed Ambien ali, Uni e si
à
di Messina, V. le F.
S agno d’Alcon es 31, 98166 Messina, I aly
3CycloLab, Illa os ú 7, H-1097 Budapes , Hunga y; [email p o ec ed]
4Cen e o Expe imen al O hopaedics, Saa land Uni e si y Medical Cen e , Ki be ge s . Bldg 37,
D-66421 Hombu g/Saa
, Ge many; [email p o ec ed] (J.K.V.); [email p o ec ed] (M.C.)
5Depa amen o de Fa macología, Fa macia y Tecnología Fa macéu ica, I+DFa ma (GI-1645), Facul ad de
Fa macia and Heal h Resea ch Ins i u e o San iago de Compos ela (IDIS), Uni e sidade de San iago de
Compos ela, 15872 San iago de Compos ela, Spain; ca men.al a ez.lo [email p o ec ed]
*Co espondence: [email p o ec ed] (A.P.); an onino.mazzaglia@cn .i (A.M.)
Recei ed: 20 July 2020; Accep ed: 27 Augus 2020; Published: 29 Augus 2020
Abs ac :
Injec able nanobiopla o ms capable o locally igh ing he in lamma ion in os eoa icula
diseases, by educing he numbe o adminis a ions and p olonging he he apeu ic e ec is highly
challenging.
β
-Cyclodex in ca ionic polyme s a e p omising ca ilage-pene a ing candida es by
in a-a icula injec ion due o he high biocompa ibili y and abili y o en ap mul iple he apeu ic
and diagnos ic agen s, hus moni o ing and mi iga ing in lamma ion. In his s udy, nanoassemblies
based on poly-
β
-amino-cyclodex in (PolyCD) loaded wi h he non-s e oidal an i-in lamma o y
d ug diclo enac (DCF) and linked by sup amolecula in e ac ions wi h a luo escen p obe
(adaman anyl-Rhodamine conjuga e, Ada-Rhod) we e de eloped o manage in lamma ion in
os eoa icula diseases. PolyCD@Ada-Rhod/DCF sup amolecula nanoassemblies we e cha ac e ized
by complemen a y spec oscopic echniques including UV-Vis, s eady-s a e and ime- esol ed
luo escence, DLS and
ζ
-po en ial measu emen . S abili y and DCF elease kine ics we e in es iga ed
in medium mimicking he physiological condi ions o ensu e con ol o e ime and e icacy. Biological
expe imen s e idenced he e icien cellula in e naliza ion o PolyCD@Ada-Rhod/DCF (wi hin wo
hou s) wi hou signi ican cy o oxici y in p ima y human bone ma ow-de i ed mesenchymal s omal
cells (hMSCs). Finally, polyCD@Ada-Rhod/DCF signi ican ly supp essed IL-1
β
p oduc ion in hMSCs,
e ealing he an i-in lamma o y p ope ies o hese nanoassemblies. Wi h hese p emises, his
s udy migh open no el ou es o exploi o iginal CD-based nanobioma e ials o he ea men o
os eoa icula diseases.
Keywo ds:
polyme ic cyclodex ins; IL-1
β
; human ma ow-de i ed mesenchymal s omal
cells; hodamine
1. In oduc ion
Os eoa h i is (OA) is a p e alen , ch onic and se e e degene a i e disease ha a ec s abou 50%
o he o e -six y popula ion [
1
]. OA is cha ac e ized by al e a ions in he whole join (a icula ca ilage
Nanoma e ials 2020,10, 1712; doi:10.3390/nano10091712 www.mdpi.com/jou nal/nanoma e ials
Nanoma e ials 2020,10, 1712 2 o 19
deg ada ion, bone emodeling and syno ial in lamma ion) ha lead o join ins abili y, ailu e,
in e mi en pain and swelling [
2
]. While a ious su gical and pha macological ea men s a e a ailable
in he clinics o con ol he p og ession o OA, none o hem a e able o ep oduce he o iginal hyaline
a icula ca ilage in a ec ed pa ien s [
3
,
4
]. E en he e icacy and/o sa e y o al eady app o ed d ugs
and o mula ions, such as co icos e oids and hyalu onic acid (HA) dispe sions, a e deba ed o
di e en easons: i) he ime o esidence o ee d ugs in he join upon in a-a icula adminis a ion
is inadequa e because o ad e se pha macokine ics ha beha e wi h apid lympha ic d ainage and
physiological u no e o he syno ial luids and ii) he di usion o d ug ough ca ilage could be
slowe han i s clea ance because o he high densi y o anionic ex acellula ma ix and small po e
size (
15 nm), hus ee d ugs could no achie e he he apeu ic concen a ions in he a ge si e.
To encompass he apid clea ance o ee d ug ( om hou s o ew days o weeks) a ple ho a o enginee ed
bioma e ials we e p oposed [
5
]. Howe e , sus ained in a-a icula deli e y s a egies by means o
d ug conjuga ed o en apped in hyd ogels ha e some limi a ions such as (i) he chemical modi ica ion
could inac i a e he d ug and (ii) he delayed elease o a small d ug is gene ally achie ed by inc easing
he c osslinking o he polyme , which could no low h ough a sy inge and inally no ma ch he
mechanical ea u es o he join [
6
]. To con as he sho he apeu ic ime, dec easing he equency o
adminis a ion, hus op imizing he ca ilage pene a ion, mo e injec able nano o mula ions based on
ca ionic polyelec oly e needed o be p oposed o in a-a icula deli e y [7].
The apeu ic polyme s a e excellen candida es o ge sui able nanosize-d ug deli e y sys ems
wi h he success ul ea u es o OA ea men [
8
]. Cyclodex ins (CDs) a e cyclic oligosaccha ides
capable o encapsula ing gues molecules wi hin hei hyd ophobic ca i y ia non co alen in e ac ions.
The seques a ion o he gues (o pa o i ) inside he ca i y usually imp o es i s physicochemical
p ope ies, meanwhile inc easing i s solubili y and p o ec ing i om he aqueous medium
(deg ada i e enzymes, oxidan s, e c.) [
9
]. CD polyme s show addi ional p ope ies wi h espec o hei
monome ic coun e pa . CD uni s ha e been copolyme ized o di e en unc ionali ies, conjuga ed
on he side chains o c oss-linked in he backbone o polyme s [
10
–
12
] o ming mul i unc ional
nanocons uc s o e ec i e d ug and gene deli e y
in i o
and
in i o
[
13
,
14
]. In pa icula , nanoca ie s
based on nonionic b anched CD polyme s such as CD polyme s sel -assemblies [
15
], CD associa i e
a angemen s [
16
,
17
] and CD nanosponges [
18
,
19
] ha e been widely u ilized as e sa ile ools o
hos ing d ugs (i.e., an icance s, an i ube cula s, an imala ials, pho o he apeu ics, e c.) wi hin hei
ne wo k and modula ing hei elease
in i o
[
20
–
24
]. Anionic b anched CD polyme s ha e been
p oposed as componen s o d ug elu ing sys ems [
25
] o ibe s o s en coa ings [
26
,
27
]. In his
scena io, b anched ca ionic CD polyme s ha e been designed o se e al applica ions. They inc ease he
pe meabili y o d ugs o biological memb anes [
28
], easily sel -assemble in o op imized nanocon aine s
o e icien in acellula deli e y [
29
], o m nanoemulsions o o al deli e y [
30
], o sys ems o
a ge ing an imic obial e ec s in bio ilms [31].
Diclo enac (DCF) is one o he mos widely p esc ibed NSAIDs (non-s e oidal in lamma o y
d ug) o i s analgesic and an i-in lamma o y p ope ies. Un o una ely, simila ly o o he NSAIDs,
DCF use is associa ed wi h some gas oin es inal (GI) side e ec s, including ulce a ion and hemo hage.
In o de o mi iga e such side e ec s, DCF has been success ully adminis e ed by con olled elease
sys ems based on CDs and hei sup amolecula assemblies [
32
,
33
]. The inclusion o DCF inside
he CD ca i y helps educing i s GI mucosal oxici y and imp o es he solubili y o he d ug,
enhancing i s bioa ailabili y in he si e o ac ion and an i-in lamma o y e ec . The in e ac ion
be ween DCF and CD molecules o CD oligome s by o ma ion o inclusion complexes has
al eady been s udied [
34
–
38
]. Howe e , he s abili y o hese complexes could be no su icien o
pa en al/in a-a icula adminis a ion [
39
], and s a egies using CD nanoma e ials migh be equi ed
o inc ease he bioa ailabili y and s abili y o he nanocomplexes [
40
]. In OA ea men DCF is o en
used by o al adminis a ion o elie e pain and in lamma ion, howe e , he opical ou e is p e e ed
due o ewe sys emic side e ec s wi h compa able e icacy [
41
]. Fu he mo e, i is challenging
o ack he e ec i eness o ac ion o he apeu ic nanopa icles in os eoa icula diso de s [
42
].
Nanoma e ials 2020,10, 1712 3 o 19
Among he ongoing esea ch on sup amolecula sel -assemblies based on CD o con olled d ug/gene
deli e y in os eoa icula egene a ion [
43
,
44
], he e we de elop a nanocons uc based on b anched
ca ionic
β
-CD (Poly-
β
-amino-cyclodex in, PolyCD) [
45
] en apping DCF and ancho ing a p obe
luo escen (adaman anyl-Rhodamine conjuga e, Ada-Rhod) [
46
] by a sup amolecula in e ac ion.
Taking ad an age o he high a ini y o adaman ane uni o CD ca i ies and o he lipophilic ea u e o
Ada-Rhod [
47
], he p oposed PolyCD@Ada-Rhod/DCF nanoassembly has he po en iali y o become
he anos ic. In his s udy he p esence o Ada-Rhod as a doping agen ([CD]:[Ada-Rhod]
33:1 mola
a io) was exploi ed o s udy he cellula up ake o he nanoassembly. The biocompa ibili y o he
sys em was assayed on human bone ma ow-de i ed mesenchymal s omal cells (hMSCs) and he
dec ease o in insic le els o IL-1
β
p oduc ion in hMSCs was de ec ed o e alua e he p o ec i e
ac i i ies agains p oin lamma o y esponses.
2. Ma e ials and Me hods
2.1. Ma e ials
Poly-
β
-amino-cyclodex in (PolyCD, A e age MW =25 kDa, CD con en 70%) was syn hesized a
CycloLab(Budapes , Hunga y)by c oss-linking ad-hocde i a ized
β
-CDmonome wi h epichlo ohyd in
as al eady epo ed [
45
]. A colo ime ic Kaise es [
48
,
49
] was pe o med o spec oscopically es ablish
he quan i y o amino g oups p esen in PolyCD, which was es ima ed o be 0.21 mmol/g (see SI
(Supplemen a y In o ma ion)). Adaman anyl-Rhodamine conjuga e (Ada-Rhod, MW =735.5 g/mol)
was syn hesized as p e iously epo ed [
46
]. Diclo enac sodium sal (DCF, MW =318.13 g/mol) and all
he sol en s (analy ical g ade) we e pu chased om Sigma-Ald ich (Milano, I aly). All he dispe sions
used o nanoassemblies p epa a ion and spec oscopic cha ac e iza ions we e p epa ed in ul apu e
wa e (F esenius Kabi I alia) o in 10 mM phospha e bu e con aining NaCl (137 mM) and KCl (2.7 mM)
a pH 7.4 (PBS) a oom empe a u e ( . .
25
◦
C). pH measu emen s we e ob ained using an 827 pH
Lab pHme e —Me ohm.
2.2. Nanoassemblies P epa a ion
2.2.1. P epa a ion o PolyCD@Ada-Rhod
The complex was p epa ed a [CD]:[Ada-Rhod]
33:1 mola a io (0.017
µ
mol o Ada-Rhod/mg o
PolyCD) wi h [CD] equal o a mola concen a ion o epe i i e uni s in PolyCD (see SI). B ie ly, PolyCD
was dissol ed in ul apu e wa e (44 mg/4.4 mL) and sonica ed in an ul asonic ba h (10 min). A hin
Ada-Rhod o ganic ilm (0.6 mg) was p epa ed by slow e apo a ion o a dichlo ome hane (DCM)
solu ion and his la e was hyd a ed wi h he p e iously p epa ed polyme solu ion (hea ed a 50
◦
C),
ollowed by sonica ion in ul asonic ba h (1 h 30 min). The pink aqueous phase was collec ed and
analyzed, whe eas he esidual Ada-Rhod ilm was sepa a ed by sligh cen i uga ion and used o
de e mine Ada-Rhod ac ual loading in o he complex (by di e ence o he weigh ed amoun s o
Ada-Rhod ini ially p esen in o ganic ilm and he esidual ilm a e hyd a ion).
2.2.2. P epa a ion o PolyCD@Ada-Rhod/DCF
An o ganic ilm o DCF (17 mg a [CD]:[DCF] 1:1 mola a io) p e iously p epa ed by
slow e apo a ion o an ace one solu ion was hyd a ed wi h an aqueous solu ion o PolyCD o
PolyCD@Ada-Rhod (92.3 mg/10 mL) and sonica ed o 20 min.
All he samples we e eeze-d ied and hen econs i u ed in aqueous medium. A e eeze-d ying,
eco e y yield was calcula ed conside ing he inal eco e ed amoun o p oduc (mg) s. he ini ial
weighed amoun o each componen .
Nanoma e ials 2020,10, 1712 4 o 19
2.3. Loading and En apmen E iciency
Bo h Ada-Rhod o DCF ac ual loading (AL%), heo e ical loading (TL%) and en apmen e iciency
pe cen ages (EE%) we e e alua ed by UV/Vis spec oscopy using he ollowing Equa ions:
AL (%)=amoun o Ada −Rhod o DCF in o he nanoassembly
weigh o nanoassembly ×100 (1)
TL (%)=amoun o Ada −Rhod o DCF ini ially added o o mula ion
weigh o nanoassembly ×100 (2)
EE (%)=amoun o Ada −Rhod o DCF in nanoassembly
amoun o Ada −Rhod o DCF ini ially added o o mula ion ×100 (3)
The amoun o Ada-Rhod loaded in o he sys em was calcula ed by edissol ing he esidual
ilm om he complexa ion eac ion in DCM and measu ing i s abso p ion in ensi y. A Lambe and
Bee calib a ion cu e o Ada-Rhod in DCM was pe o med in he concen a ion ange 25–200
µ
M
(ε=877.8 ±10 M−1cm−1; see Figu e S1).
DCF ac ual loading inside PolyCD@Ada-Rhod/DCF sys em and EE% we e e alua ed by
UV/Vis by means o di e ence by DCF ini ially added and esidue in o ganic ilm a e hyd a ion.
Calib a ion cu es o ee DCF we e pe o med bo h in ul apu e wa e and PBS: he calcula ed
mola ex inc ion coe icien s we e espec i ely 8130
±
225 M
−1
cm
−1
(DCF ee in wa e ) and
9700 ±767 M−1cm−1(DCF ee in PBS; see Figu e S2).
2.4. UV/Vis and S eady S a e and Time Resol ed Fluo escence Spec oscopy
UV/Vis spec a we e ob ained on a Agilen model 8453 diode a ay spec opho ome e using
1 cm pa h leng h qua z cells a T =25
◦
C by using a he mos a ic ba h. S eady-s a e luo escence
measu emen s we e pe o med on a Jasco model FP-750 spec o luo ime e by using a 0.5 o 1 cm pa h
leng h qua z cells. Time esol ed luo escence emission measu emen s we e pe o med on a Jobin
Y on-Spex Fluo omax 4 spec o luo ime e using ime-co ela ed single-pho on coun ing echnique
and a NanoLED (λ=390 nm) as he exci a ion sou ce, as al eady epo ed [50,51].
2.5. Job Plo and Cha ac e iza ion o he PolyCD/DCF Complex in Solu ion
Job’s plo expe imen s we e pe o med by wo equimola s ock solu ions o PolyCD and DCF
([CD] =[DCF] =1 mM) bo h by mixing hem in ul apu e wa e and main aining he o al olume and
concen a ion cons an ([CD] +[DCF] =100
µ
M) whe e [CD] is he mola concen a ion o epe i i e
uni in PolyCD. Acco dingly, he mola ac ion (
χ
) was changed om 0.1 o 1 and measu ing he
co esponding abso bance by UV/Vis a T =25
◦
C. Plo s show he
χ
s.
∆
A/A
0×
[DCF], whe e
∆
A
is he di e ence be ween abso bance alues a maxima in he p esence (A) and in he absence (A
0
)
o PolyCD espec i ely s.
χDCF
(whe e
χDCF
is he mola ac ion o DCF a he in es iga ed mola
concen a ion [DCF]) [52,53].
The complexa ion o DCF in PolyCD was s udied by UV/Vis i a ion. Di e en aqueous solu ions
o ee DCF and DCF wi h di e en amoun s o PolyCD ([DCF] =100
µ
M and [CD] a ying in he ange
0–150
µ
M) we e p epa ed in sealed ials by adding aliquo s o PolyCD o aqueous solu ions o DCF,
homogenized by sligh sonica ion (10 min) and he mally equilib a ed a T =25
◦
C. The dispe sions
we e analyzed by UV/Vis as desc ibed and he plo o 1/[A
−
A
0
] as a unc ion o 1/[CD] was epo ed,
whe e A and A
0
a e he abso bance o DCF in he p esence and in he absence o PolyCD measu ed a
λmax o complex abso bance.
2.6. Size and ζ-Po en ial Measu emen s
Hyd odynamic diame e (D
H
) o size, wid h o dis ibu ion (polydispe si y index, PDI) and
ζ
-po en ial o he PolyCD-based nanoassemblies we e de e mined by pho on co ela ion spec oscopy
Nanoma e ials 2020,10, 1712 5 o 19
(PCS) by a Ze asize Nano ZS (Mal e n Ins umen , Mal e n, U.K.) a 25
◦
C in ul apu e wa e .
The measu emen s we e ca ied ou a 173
◦
angle s. he inciden beam a 25
±
1
◦
C o each aqueous
dispe sion. The decon olu ion o he co ela ion cu e o an in ensi y size dis ibu ion was ob ained by
using a non-nega i e leas -squa es algo i hm. The
ζ
-po en ial alues we e measu ed using a Ze asize
Nano ZS Mal e n Ins umen equipped wi h a He
−
Ne lase a a powe P =4.0 mW and
λ
=633 nm.
The esul s a e epo ed as he mean o h ee sepa a e measu emen s on h ee di e en ba ches
±
he
s anda d de ia ion (SD).
2.7. S abili y S udies
S abili y s udies we e ca ied ou by dissol ing PolyCD@Ada-Rhod/DCF (0.5 mg/mL) in di e en
biological media: (i) ul apu e wa e , (ii) 0.9 w % NaCl aqueous solu ion, (iii) PBS a pH 7.4. All he
solu ions we e kep unde s i ing (T =25
◦
C) along 14 days and analyzed by UV/Vis and DLS a . . in
iplica e.
ζ
-Po en ial was measu ed along 2 weeks on he dispe sions p epa ed in ul apu e wa e and
s o ed a 25 ◦C.
2.8. Release S udies
Release p o ile o DCF om PolyCD@Ada-Rhod/DCF nanoassembly was e alua ed in PBS a pH
7.4 by a dialysis me hod. PolyCD@Ada-Rhod/DCF (10 mg) in PBS (1 mL) we e pu in o a dialysis ube
(Spec a/Po
®
dialysis bags, MWCO 3.5 kDa) and imme sed in o 10 mL o PBS (sink condi ion) unde
con inuous s i ing (250 pm) a 37
±
0.5
◦
C. A ixed imes, 1 mL o elease medium was wi hd awn
and eplaced wi h an equal olume o esh aqueous solu ion o PBS. The amoun o DCF eleased was
e alua ed by UV/Vis spec oscopy (a
λ
=276 nm) and was exp essed as pe cen age a io be ween he
weigh o eleased DCF and he o al amoun o en apped d ug. The kine ic analysis was ca ied by
h ee models p oposed in he li e a u e such as Higuchi, Bake –Lonsdale and he i s o de p ocess
(see in a and SI) [53,54].
2.9. Biological S udies
2.9.1. Ma e ials
All eagen s we e om Sigma (Munich, Ge many) unless o he wise indica ed. Recombinan FGF-2
( FGF-2) was pu chased a R&D Sys ems (Wiesbaden-No dens ad , Ge many). The Cell P oli e a ion
Reagen WST-1 and he Cy o oxici y De ec ion Ki PLUS (LDH) we e ob ained a Roche Applied
Science (Mannheim, Ge many). The Human IL-1
β
and TNF-
α
enzyme-linked immunoso ben assays
(ELISAs; Human IL-1βQuan ikine ELISA, TNF-αQuan ikine ELISA) we e om R&D Sys ems.
2.9.2. Cell Cul u e
Bone ma ow aspi a es (15 mL) we e ob ained om dis al emu s o pa ien s unde going o al
knee a h oplas y (n=8, age 68–74 yea s). The s udy was app o ed by he E hics Commi ee o he
Saa land Physicians Council. All p ocedu es we e in acco dance wi h he Helsinki Decla a ion and
all pa ien s p o ided in o med consen be o e inclusion in he s udy. Bone ma ow-de i ed human
mesenchymal s omal cells (hMSCs) we e isola ed acco ding o s anda d p o ocols [
55
,
56
] by washing
and cen i uging he aspi a es in Dulbecco’s modi ied Eagle’s medium (DMEM). The cell pelle was
esuspended in ed blood cell lysing bu e (Sigma) and DMEM (1:1). The mix u e was washed,
pelle ed and esuspended in DMEM wi h 10% e al bo ine se um, 100 U/mL penicillin and 100
µ
L/mL
s ep omycin (g ow h medium). The cells we e pla ed in T75 lasks and kep a 37
◦
C unde 5% CO
2
o e nigh . The medium was hen emo ed and eplaced by g ow h medium wi h ecombinan FGF-2
(1 ng/mL), wi h medium exchanged e e y 2–3 days. P oli e a ing cells we e epla ed when eaching an
85% densi y and hMSCs we e u he used a no mo e han passage 1–2. Cell s udies we e ca ied ou
by adding PolyCD-based nanoassemblies (2 mg/mL, [Ada-Rhod] =32 µM, [DCF] =944 µM).
Nanoma e ials 2020,10, 1712 6 o 19
2.9.3. De ec ion o Li e Fluo escence
hMSCs we e seeded in 24-well pla es (2
×
10
4
cells/well) wi h g ow h medium o 12 h a 37
◦
C
unde 5% CO
2
. PolyCD-based nanoassemblies we e hen di ec ly added o he cul u es and li e
luo escence was moni o ed in he samples by luo escen mic oscopy using a hodamine il e se
(568 nm; Olympus CKX41; Hambu g, Ge many) [55,57].
2.9.4. Cell P oli e a ion and Viabili y
hMSCs we e seeded in 24-well pla es (2
×
10
4
cells/well) wi h g ow h medium o 12 h a
37
◦
C unde 5% CO
2
p io o di ec addi ion o he PolyCD-based nanoassemblies o he cul u es.
Cell p oli e a ion was e alua ed using he Cell P oli e a ion Reagen WST-1 acco ding o he
manu ac u e ’s ecommenda ions [
55
–
57
]. Cell iabili y was de e mined wi h he Cy o oxici y
De ec ion Ki PLUS (LDH) in he supe na an s o cul u e by assessing he abso bance a 450 nm on a
GENios spec opho ome e / luo ome e (Tecan, C ailsheim, Ge many). Cy o oxici y was calcula ed as
ollows [57]:
cell iabili y (%) =(expe imen al alue −low con ol)/(high con ol −low con ol) ×100 (4)
2.9.5. In lamma o y Responses
hMSCs we e seeded in 24-well pla es (2
×
10
4
cells/well) wi h g ow h medium o 12 h a
37
◦
C unde 5% CO
2
p io o di ec addi ion o he PolyCD-based nanoassemblies o he cul u es.
In lamma o y esponses we e moni o ed by measu ing he p oduc ion le els o IL-1
β
and TNF-
α
in
he supe na an s o cul u e by espec i e ELISAs on a GENios spec opho ome e / luo ome e .
2.9.6. S a is ical Analysis
All es s we e pe o med in iplica e in h ee independen expe imen s. Da a a e exp essed as
mean
±
s anda d de ia ion (SD) o sepa a e expe imen s. The - es was employed whe e app op ia e,
wi h p<0.05 conside ed s a is ically signi ican .
3. Resul s and Discussion
3.1. Nanoassemblies P epa a ion
Fluo escen ca ionic nanoassemblies en apping DCF (PolyCD@Ada-Rhod/DCF) we e p epa ed
by hyd a ion o Ada-Rhod o ganic ilm and he eco e ed PolyCD@Ada-Rhod was used o he
ollowing hyd a ion o DCF o ganic ilm. Concen a ion o CD epe i i e uni s was used in sligh
excess s. [DCF], hus o achie e a comple e d ug en apmen . Scheme 1summa izes nanoassemblies
o ma ion by molecula componen s.
Nanoassemblies we e ob ained wi h high Ada-Rhod and DCF en apmen e iciency (
92%
and 100%, espec i ely). Ada-Rhod esidual ilm was used o de e mine Ada-Rhod loading (see
expe imen al). No esidual o DCF was ound in he dispe sions o PolyCD@Ada-Rhod/DCF, con i ming
he comple e en apmen . Mo eo e , i was obse ed ha he eco e y yield o all sys ems is 80%,
p obably because o he p esence o a li le wa e pe cen age in he s a ing cyclodex in polyme ,
due o i s highly hyg oscopic na u e. P ope ies o nanoassemblies a e epo ed in Table 1.
DLS analysis o PolyCD and PolyCD@AdaRhod/DCF nanoassemblies (Table 1) shows a size
dis ibu ion cen e ed a a hyd odinamic diame e (D
H
) o abou 250 nm o he main popula ion
and a
ζ
-po en ial o abou +20 mV, due o he posi i e cha ges o he amino g oups o he polyme
ne wo k (see Figu e S3). Su p isingly PolyCD@AdaRhod showed a size ha is abou wo- old s.
he analogue wi h DCF, sugges ing a di e en ea agmen s. he nanossemblies en apping bo h
Ada-Rhod and DCF.
Nanoma e ials 2020,10, 1712 7 o 19
Nanoma e ials 2020, 10, x FOR PEER REVIEW 7 o 19
Scheme 1. Ske ched iew o nanoassemblies p epa a ion (PolyCD@Ada-Rhod/DCF).
Nanoassemblies we e ob ained wi h high Ada-Rhod and DCF en apmen e iciency (≅92% and
100%, espec i ely). Ada-Rhod esidual ilm was used o de e mine Ada-Rhod loading (see
expe imen al). No esidual o DCF was ound in he dispe sions o PolyCD@Ada-Rhod/DCF,
con i ming he comple e en apmen . Mo eo e , i was obse ed ha he eco e y yield o all
sys ems is 80%, p obably because o he p esence o a li le wa e pe cen age in he s a ing
cyclodex in polyme , due o i s highly hyg oscopic na u e. P ope ies o nanoassemblies a e
epo ed in Table 1.
Table 1. O e all p ope ies o PolyCD-based nanoassemblies: mean DH, polidispe si y index (PDI)
and ζ-po en ial alues (ζ), loading and EE% in ul apu e wa e .
Sample Medium
Mean DH (nm
± SD)a,(%)b PDI ζ (mV
± SD)
Theo e ical
Loading (%)
c Ac ual
Loading (%)
d EE
(%)
PolyCD H2O 268 ± 10 (97) 0.07 19 ± 6
PolyCD@Ada-
Rhod H2O 498 ± 54 (85) 0.2 25 ± 5 1.28 (1) 1.18 ± 0.04 (1) 92.0 ±
3.3 (1)
PolyCD@
AdaRhod/DCF H2O 229 ± 35 (85)
29 ± 13 (12) ≤0.3 22 ± 4 1.18 (1) 1.09 ± 0.05 (1) 92.0 ±
3.9 (1)
15.5
(2) 15.5 (2) ∼100 (2)
PBS pH 7.4 230 ± 24 (83)
25 ± 13 (11) ≤0.2
NaCl (0.9
w %)
228 ± 21 (89)
18 ± 13 (12) ≤0.2
a SD was calcula ed on h ee di e en ba ches. b Mean size wi h co esponding in ensi y % dis ibu ion
(only main popula ions). c Ac ual loading is exp essed as he amoun o d ug (mg) encapsula ed pe
100 mg o nanoassembly. d Ra io be ween ac ual and heo e ical loading × 100. (1) Values a e e e ed
o Ada-Rhod; (2) Values a e e e ed o Diclo enac; PolyCD (0.5 mg/mL) and PolyCD@Ada-Rhod/DCF
(0.5 mg/mL, [Ada-Rhod] = 8 μM, [DCF] = 236 µM).
DLS analysis o PolyCD and PolyCD@AdaRhod/DCF nanoassemblies (Table 1) shows a size
dis ibu ion cen e ed a a hyd odinamic diame e (DH) o abou 250 nm o he main popula ion and
a ζ-po en ial o abou + 20 mV, due o he posi i e cha ges o he amino g oups o he polyme
Scheme 1. Ske ched iew o nanoassemblies p epa a ion (PolyCD@Ada-Rhod/DCF).
Table 1.
O e all p ope ies o PolyCD-based nanoassemblies: mean D
H
, polidispe si y index (PDI)
and ζ-po en ial alues (ζ), loading and EE% in ul apu e wa e .
Sample Medium Mean DH(nm ±SD) a,
(%) bPDI ζ(mV ±SD) Theo e ical
Loading (%)
cAc ual
Loading (%)
dEE (%)
PolyCD H2O 268 ±10 (97)
0.07
19 ±6
PolyCD@Ada-Rhod H2O 498 ±54 (85)
0.2
25 ±51.28 (1) 1.18 ±0.04 (1) 92.0 ±3.3 (1)
PolyCD@
AdaRhod/DCF H2O229 ±35 (85)
29 ±13 (12) ≤
0.3
22 ±41.18 (1) 1.09 ±0.05 (1) 92.0 ±3.9 (1)
15.5 (2) 15.5 (2) ~100 (2)
PBS pH 7.4
230 ±24 (83)
25 ±13 (11) ≤
0.2
NaCl (0.9
w %)
228 ±21 (89)
18 ±13 (12) ≤
0.2
a
SD was calcula ed on h ee di e en ba ches.
b
Mean size wi h co esponding in ensi y % dis ibu ion (only main
popula ions).
c
Ac ual loading is exp essed as he amoun o d ug (mg) encapsula ed pe 100 mg o nanoassembly.
d
Ra io be ween ac ual and heo e ical loading
×
100.
(1)
Values a e e e ed o Ada-Rhod;
(2)
Values a e e e ed o
Diclo enac; PolyCD (0.5 mg/mL) and PolyCD@Ada-Rhod/DCF (0.5 mg/mL, [Ada-Rhod] =8
µ
M, [DCF] =236
µ
M).
3.2. In e ac ion S udies and Complexes Fo ma ion
The in e ac ions o PolyCD wi h Ada-Rhod wi hin PolyCD@Ada-Rhod, and wi h bo h Ada-Rhod
and DCF wi hin PolyCD@Ada-Rhod/DCF we e in es iga ed by UV/Vis, s eady-s a e and ime- esol ed
luo escence emission. PolyCD/DCF complex o ma ion was s udied o compa ison. UV/Vis spec a
and luo escence emission o ee Ada-Rhod in DCM s. PolyCD@Ada-Rhod complex a e shown in
Figu e 1. Ada-Rhod’s abso p ion p o ile shows a majo band cen e ed a 558 nm in DCM, which was
ed-shi ed a 561 nm in PolyCD@Ada-Rhod. The appea ance o abso p ion p o ile in wa e was
unambiguous e idence o Ada-Rhod complexa ion since ee Ada-Rhod was no soluble in wa e
(Figu e 1A). S eady s a e emission luo escence (Figu e 1B) o Ada-Rhod in DCM shows a band cen e ed
a 575 nm, whe eas a e he in e ac ion wi h PolyCD in aqueous medium he emission p o ile ac ually
spli in o a band, cen e ed a 542 and a shoulde a ound 576 nm espec i ely.
Nanoma e ials 2020,10, 1712 8 o 19
Nanoma e ials 2020, 10, x FOR PEER REVIEW 8 o 19
ne wo k (see Figu e S3). Su p isingly PolyCD@AdaRhod showed a size ha is abou wo- old s. he
analogue wi h DCF, sugges ing a di e en ea agmen s. he nanossemblies en apping bo h Ada-
Rhod and DCF.
3.2. In e ac ion S udies and Complexes Fo ma ion
The in e ac ions o PolyCD wi h Ada-Rhod wi hin PolyCD@Ada-Rhod, and wi h bo h Ada-
Rhod and DCF wi hin PolyCD@Ada-Rhod/DCF we e in es iga ed by UV/Vis, s eady-s a e and ime-
esol ed luo escence emission. PolyCD/DCF complex o ma ion was s udied o compa ison.
UV/Vis spec a and luo escence emission o ee Ada-Rhod in DCM s. PolyCD@Ada-Rhod complex
a e shown in Figu e 1. Ada-Rhod’s abso p ion p o ile shows a majo band cen e ed a 558 nm in
DCM, which was ed-shi ed a 561 nm in PolyCD@Ada-Rhod. The appea ance o abso p ion p o ile
in wa e was unambiguous e idence o Ada-Rhod complexa ion since ee Ada-Rhod was no soluble
in wa e (Figu e 1A). S eady s a e emission luo escence (Figu e 1B) o Ada-Rhod in DCM shows a
band cen e ed a 575 nm, whe eas a e he in e ac ion wi h PolyCD in aqueous medium he emission
p o ile ac ually spli in o a band, cen e ed a 542 and a shoulde a ound 576 nm espec i ely.
Figu e 1. UV/Vis spec a (A) and s eady s eady s a e emission spec a (B) o ee Ada-Rhod in DCM
( ed ace) and PolyCD@Ada-Rhod (blue cyan ace) in ul apu e wa e , pH = 4. (A): 44 mg/mL, [Ada-
Rhod] = 713 µM, pH = 4, d = 1 cm and sca e ing sub ac ed in he spec um o he PolyCD@Ada-
Rhod. (B): 0.5 mg/mL, [Ada-Rhod] = 8 µM, λexc = 480 nm.
This double band p o ile is ypical o hodamine de i a i es because o he au ome ic
equilib ium ha occu s in wa e , and is s ic ly in luenced e en by sligh pH changes in he aqueous
mic oen i onmen s [58,59].
The in e ac ion o PolyCD wi h DCF was i s ly s udied by UV/Vis spec oscopy. The
complexa ion o he d ug in o PolyCD was ob ained by simple mixing o aqueous solu ions o DCF
and PolyCD as epo ed in expe imen al me hod. The inco po a ion o DCF in o he complex was
e iden om he UV/Vis spec um (Figu e 2A) ha displays a band cen e ed a 276 nm o ee DCF
(black ace) and 278 nm o he complex in wa e (o ange ace). Fu he mo e, a sligh hype ch omic
e ec was obse ed upon complexa ion. Fo compa ison, a UV/Vis spec um was eco ded on
PolyCD/DCF complex ob ained by sol en e apo a ion echnique (hyd a ion o o ganic ilm and
sonica ion) as epo ed in he expe imen al me hod and he same e ec was obse ed, hus
con i ming he in e ac ion (Figu e S4). P obably he e y sligh hype c omici y and shi a e due o
he excellen dispe sibili y o bo h componen s in wa e . The s oichiome y o he complex was
de e mined by he con inuous a ia ion me hod [60]. The shape o Job’s plo (ΔA/A0 × [DCF] s. χ
DCF) was highly symme ical, showing a maximum alue a χ DCF = 0.5 poin ing ou a o ma ion o
complex wi h p e alen 1:1 s oichiome y (Figu e 2B).
Figu e 1.
UV/Vis spec a (
A
) and s eady s eady s a e emission spec a (
B
) o ee Ada-Rhod in DCM ( ed
ace) and PolyCD@Ada-Rhod (blue cyan ace) in ul apu e wa e , pH =4. (
A
): 44 mg/mL, [Ada-Rhod]
=713
µ
M, pH =4, d =1 cm and sca e ing sub ac ed in he spec um o he PolyCD@Ada-Rhod.
(B): 0.5 mg/mL, [Ada-Rhod] =8µM, λexc =480 nm.
This double band p o ile is ypical o hodamine de i a i es because o he au ome ic
equilib ium ha occu s in wa e , and is s ic ly in luenced e en by sligh pH changes in he aqueous
mic oen i onmen s [58,59].
The in e ac ion o PolyCD wi h DCF was i s ly s udied by UV/Vis spec oscopy. The complexa ion
o he d ug in o PolyCD was ob ained by simple mixing o aqueous solu ions o DCF and PolyCD
as epo ed in expe imen al me hod. The inco po a ion o DCF in o he complex was e iden om
he UV/Vis spec um (Figu e 2A) ha displays a band cen e ed a 276 nm o ee DCF (black ace)
and 278 nm o he complex in wa e (o ange ace). Fu he mo e, a sligh hype ch omic e ec was
obse ed upon complexa ion. Fo compa ison, a UV/Vis spec um was eco ded on PolyCD/DCF
complex ob ained by sol en e apo a ion echnique (hyd a ion o o ganic ilm and sonica ion) as
epo ed in he expe imen al me hod and he same e ec was obse ed, hus con i ming he in e ac ion
(Figu e S4). P obably he e y sligh hype c omici y and shi a e due o he excellen dispe sibili y
o bo h componen s in wa e . The s oichiome y o he complex was de e mined by he con inuous
a ia ion me hod [
60
]. The shape o Job’s plo (
∆
A/A
0×
[DCF] s.
χ
DCF) was highly symme ical,
showing a maximum alue a
χ
DCF =0.5 poin ing ou a o ma ion o complex wi h p e alen 1:1
s oichiome y (Figu e 2B).
Nanoma e ials 2020, 10, x FOR PEER REVIEW 9 o 19
Figu e 2. (A) UV/Vis spec a o ee DCF (black ace) and PolyCD/DCF (o ange ace) in wa e , [CD]
= [DCF] = 100 µM; (B) Job’s plo o he complexa ion o DCF wi h PolyCD om UV/Vis
measu emen s in ul apu e wa e (see Expe imen al sec ion); and (C) UV/Vis spec al changes o DCF
s. CD concen a ion (dashed black aces) and plo o (1/ΔA) s. 1/[CD] (inse , o ange ace, R
2
=
0.997) in ul apu e wa e , [DCF] = 100 µM, [CD] = 25–150 µM; d = 1 cm; T = 25 °C.
Fu he mo e, he o ma ion o complex was con i med by inc ease o abso bance measu ed s.
CD concen a ion in he ange 25–150 µM. This plo elea es a bimodal beha iou , wi h a linea
inc ease up o 100 µM and a e wa ds a pseudo pla eau (Figu e 2C). In he i s linea po ion a A
L
ype diag am wi h a slope less o a uni was obse ed. A highe hos concen a ions complexes wi h
a highe o de and unde ined s ochiome y could occu . The appa en binding cons an o he 1:1
complex can be e alua ed by using he Benesi–Hildeb and Equa ion [61].
1
A− A
= 1
K
×A − A×CD 1
A − A (5)
whe e A is he abso bance a maximum o he PolyCD/DCF complex, A
0
is he abso bance o
DCF in he absence o PolyCD, [CD] is he PolyCD concen a ion in CD uni s, A
max
is he abso bance
a [CD]
max
(100 µM) and K
b
is he appa en binding cons an . The appa en binding cons an was
es ima ed om he slope
/A
max
− A
0
(plo o 1/(A − A
0
) s. 1/[CD]) and is ound o be 4.1 × 10
3
M
−1
(log
K
b
≅ 3.60; inse o Figu e 2C). This alue ag ees wi h da a ound o complexa ion o DCF in ca ionic
CD c oss-linked oligome s (log K
b
≅ 3.47) [38].
Fluo escence ime-decays o Ada-Rhod ee and wi hin nanoassemblies we e i ed by one and
h ee exponen ial p o iles espec i ely, es ima ing h ee di e en luo escence li e imes when he
p obe is complexed in o he polyme ic s uc u e (Table 2 and Figu e 3).
Table 2. Fluo escence li e imes (τ) and o a ional co ela ion ime (θ
R
) o PolyCD@Ada-Rhod/DCF s.
Ada-Rhod.
Sample τ
1
± 0.1, ns τ
2
± 0.1, ns τ
3
± 0.1, ns A
1
, % A
2
, % A
3
, % θ
R
± 0.2, ns
Ada-Rhod
a
3.6 -- -- 100 -- -- 0.8
PolyCD@
Ada-Rhod/DCF
a
0.4 2.4 5.8 23 34 43 2.2
a
Fluo escence li e imes we e measu ed a λ
exc
= 390 nm: Ada-Rhod in DCM, λ
em
= 576 nm;
PolyCD@Ada-Rhod/DCF in ul apu e wa e , λ
em
= 576 nm. A is he ampli ude o he in ensi y decay.
Expe imen al condi ions: ee [Ada-Rhod] = 100 µM; PolyCD@Ada-Rhod/DCF (0.5 mg/mL, [Ada-Rhod] =
8 μM, [DCF] = 236 µM).
The ime luo escence decay (Figu e 3) and co esponden luo escen li e imes (Table 2) poin ed
ou ha ee Ada-Rhod in DCM was p esen mos ly as a monome (τ
1
= 3.6 ns) [62–64]. When
analyzing PolyCD@Ada-Rhod/DCF nanoassemblies in ul apu e wa e , h ee li e imes we e
obse ed and one o hese (τ
2
= 2.4 ns) was asc ibable o Ada-Rhod species en apped wi hin he
polyme chains in he monome ic o m [46,65]. This could also explain he o a ional co ela ion ime
a e in e ac ion o Ada-Rhod wi hin PolyCD@Ada-Rhod/DCF (0.8 ns in ee Ada-Rhod s. 2.2 ns in
he nanoassembly), sugges ing ha he p obe is e ec i ely inco po a ed in o he s uc u e, bu s ill
Figu e 2.
(
A
) UV/Vis spec a o ee DCF (black ace) and PolyCD/DCF (o ange ace) in wa e ,
[CD] =[DCF] =100 µM
; (
B
) Job’s plo o he complexa ion o DCF wi h PolyCD om UV/Vis
measu emen s in ul apu e wa e (see Expe imen al sec ion); and (
C
) UV/Vis spec al changes o
DCF s. CD concen a ion (dashed black aces) and plo o (1/
∆
A) s. 1/[CD] (inse , o ange ace,
R2=0.997) in ul apu e wa e , [DCF] =100 µM, [CD] =25–150 µM; d =1 cm; T =25 ◦C.
Fu he mo e, he o ma ion o complex was con i med by inc ease o abso bance measu ed s. CD
concen a ion in he ange 25–150
µ
M. This plo elea es a bimodal beha iou , wi h a linea inc ease up
o 100
µ
M and a e wa ds a pseudo pla eau (Figu e 2C). In he i s linea po ion a A
L
ype diag am
wi h a slope less o a uni was obse ed. A highe hos concen a ions complexes wi h a highe o de
Nanoma e ials 2020,10, 1712 9 o 19
and unde ined s ochiome y could occu . The appa en binding cons an o he 1:1 complex can be
e alua ed by using he Benesi–Hildeb and Equa ion [61].
1
A−A0
=1
Kb×(Amax −A0)×[CD]+1
Amax −A0
(5)
whe e A is he abso bance a maximum o he PolyCD/DCF complex, A
0
is he abso bance o DCF
in he absence o PolyCD, [CD] is he PolyCD concen a ion in CD uni s, A
max
is he abso bance
a [CD]
max
(100
µ
M) and K
b
is he appa en binding cons an . The appa en binding cons an was
es ima ed om he slope /A
max −
A
0
(plo o 1/(A
−
A
0
) s. 1/[CD]) and is ound o be 4.1
×
10
3
M
−1
(log K
b
3.60; inse o Figu e 2C). This alue ag ees wi h da a ound o complexa ion o DCF in
ca ionic CD c oss-linked oligome s (log Kb3.47) [38].
Fluo escence ime-decays o Ada-Rhod ee and wi hin nanoassemblies we e i ed by one and
h ee exponen ial p o iles espec i ely, es ima ing h ee di e en luo escence li e imes when he p obe
is complexed in o he polyme ic s uc u e (Table 2and Figu e 3).
Table 2.
Fluo escence li e imes (
τ
) and o a ional co ela ion ime (
θR
) o PolyCD@Ada-Rhod/DCF
s. Ada-Rhod.
Sample τ1±0.1, ns τ2±0.1, ns τ3±0.1, ns A1, % A2, % A3, % θR±0.2, ns
Ada-Rhod a3.6 – – 100 – – 0.8
PolyCD@
Ada-Rhod/DCF a0.4 2.4 5.8 23 34 43 2.2
a
Fluo escence li e imes we e measu ed a
λexc
=390 nm: Ada-Rhod in DCM,
λem
=576 nm; PolyCD@Ada-Rhod/DCF
in ul apu e wa e ,
λem
=576 nm. A is he ampli ude o he in ensi y decay. Expe imen al condi ions: ee [Ada-Rhod]
=100 µM; PolyCD@Ada-Rhod/DCF (0.5 mg/mL, [Ada-Rhod] =8µM, [DCF] =236 µM).
The ime luo escence decay (Figu e 3) and co esponden luo escen li e imes (Table 2) poin ed ou
ha ee Ada-Rhod in DCM was p esen mos ly as a monome (
τ1
=3.6 ns) [
62
–
64
]. When analyzing
PolyCD@Ada-Rhod/DCF nanoassemblies in ul apu e wa e , h ee li e imes we e obse ed and
one o hese (
τ2
=2.4 ns) was asc ibable o Ada-Rhod species en apped wi hin he polyme
chains in he monome ic o m [
46
,
65
]. This could also explain he o a ional co ela ion ime a e
in e ac ion o Ada-Rhod wi hin PolyCD@Ada-Rhod/DCF (0.8 ns in ee Ada-Rhod s. 2.2 ns in
he nanoassembly), sugges ing ha he p obe is e ec i ely inco po a ed in o he s uc u e, bu s ill
main ains a ce ain eedom o o a e upon i sel [
51
]. Fo wha conce ns he sho e li e imes (0.4 ns in
he PolyCD@Ada-Rhod/DCF) i could be supposedly due o sel -oligome s o ma ion o Ada-Rhod,
likely gene a ed by sel
π
–
π
s acking o o he agg ega ion phenomena ha lead o luo escence
quenching. Finally, he longe ones (
τ3
=5.8 ns) we e en a i ely asc ibed o species o Ada-Rhod
in e ac ing mo e closely o CD ca i ies (i.e., inclusion o Ada po ion).
O e all, ou in es iga ions indica ed ha bo h DCF and Ada-Rhod in e ac wi h CD ca i ies.
Hos –gues complexa ion o Ada-Rhod akes ad an age o he high a ini y o adaman ane po ion o
CD ca i ies (K
b
=5
×
10
4
M
−1
) [
66
], and his in e ac ion is s onge wi h espec o DCF wi h a CD
ca i y: indeed no Ada-Rhod displacemen was obse ed e en i DCF was used in excess s. Ada-Rhod.
The o ma ion o la ge agg ega es in PolyCD@Ada-Rhod a he han PolyCD@Ada-Rhod/DCF is an
expe imen al e idence ha was al eady obse ed in o he s nanoassemblies based on polyme ic sys ems
unc ionalized wi h hodamine [
67
]. We suppose ha upon inclusion o he adaman ane uni in o CD
ca i y, he hodamine esidue o Ada-Rhod is loca ed in a mo e hyd ophilic en i onmen , likely ou side
he CD ca i y and in he p oximi y o he CD ims. Mo eo e , he o ma ion o sup amolecula
sel -oligome s o Ada-Rhod wi h sho e luo escence li e imes (0.4 ns wi h ampli ude o 23%) could
occu . These a angemen s could allow in a e age an inc ease o D
H
(see Table 1) in PolyCD@Ada-Rhod.
On he o he hand, he dec ease o D
H
and
ζ
-po en ial alues in PolyCD@Ada-Rhod/DCF could be
en a i ely asc ibed o he o ma ion o mo e compac ed and smalle nanoassemblies due o an
Nanoma e ials 2020,10, 1712 16 o 19
17.
O hman, M.; Bouchemal, K.; Cou eu , P.; Desmaële, D.; Mo an, E.; Pouge , T.; G e , R. A comp ehensi e
s udy o he spon aneous o ma ion o nanoassemblies in wa e by a “lock-and-key” in e ac ion be ween
wo associa i e polyme s. J. Colloid In e aces Sci. 2011,354, 517–527. [C ossRe ]
18.
T o a, F.; Zane i, M.; Ca alli, R. Cyclodex in-based nanosponges as d ug ca ie s. Beils ein J. O g. Chem.
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