Jou nal o CO2 U iliza ion 81 (2024) 102722
A ailable online 2 Ma ch 2024
2212-9820/© 2024 The Au ho (s). Published by Else ie L d. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/).
G een CO
2
echnology o he p epa a ion o ae ogel d y powde loaded
wi h beclome hasone dip opiona e
Thoa Duong
a
, Cla a L´
opez-Iglesias
a
, Annalisa Bianche a
b
, Ma ia Vi e o-Lopez
a
, In´
es A dao
c
,
Rugge o Be ini
b
, Ca men Al a ez-Lo enzo
a
, Ca los A. Ga cía-Gonz´
alez
a
,
*
a
Ae ogelsLab, I+D Fa ma G oup (GI-1645), Depa men o Pha macology, Pha macy and Pha maceu ical Technology, Facul y o Pha macy, iMATUS 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, San iago de Compos ela E-15782, Spain
b
Food and D ug Depa men , Uni e si y o Pa ma, Pa co A ea delle Scienze 27/A, Pa ma 43124, I aly
c
BioFa ma Resea ch g oup, Depa men o Pha macology, Pha macy and Pha maceu ical Technology, Innopha ma D ug Sc eening and Pha macogenomics Pla o m,
Cen o Singula de In es igaci´
on en Medicina Molecula y En e medades C ´
onicas (CiMUS) 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, San iago de Compos ela E-15782, Spain
ARTICLE INFO
Keywo ds:
G een echnology
Supe c i ical CO
2
D y powde inhale s
Beclome hasone dip opiona e
D ug-loaded ae ogel
ABSTRACT
D y powde inhale s (DPIs) ha e gained inc easing clinical accep ance in he local ea men o lung diseases due
o hei abili y o mee he e ol ing needs o pa ien s while a oiding en i onmen al conce ns. Howe e , some
o mula ions o DPIs s ill encoun e pe o mance limi a ions in e ms o ae odynamic p ope ies and achie e-
men o he apeu ic doses. Inno a i e ae ogel powde o mula ions o DPIs p ocessed using combined supe -
c i ical CO
2
(scCO
2
)-based echnologies can o e come hese limi a ions, especially in he case o poo ly wa e -
soluble d ugs. The loading o hyd ophobic d ugs in o ae ogels can be op imized h ough a ho ough unde -
s anding o he physicochemical p ope ies o he o mula ion and he scCO
2
p ocessing condi ions. In his s udy,
d ug-loaded algina e ae ogel pa icles we e p epa ed by combining gela ion-emulsi ica ion echniques and
scCO
2
-based echnologies. Beclome hasone dip opiona e (BDP), a hyd ophobic co icos e oid, was inco po a ed
in o he ae ogel ma ix by scCO
2
imp egna ion. The in luence o con ac ime, ini ial amoun o d ug, and use o
co-sol en s on he e iciency o scCO
2
imp egna ion we e s udied. The kine ics o he BDP adso p ion p ocess was
modelled o elucida e he ime equi ed o he d ug o a ain equilib ium concen a ion unde speci ic ope a ing
condi ions. Ni ogen adso p ion-deso p ion and helium pycnome y e ealed pa icles wi h la ge su ace a ea
(>200 m
2
/g) and po osi y (ca. 90%). The esul ing ae ogels had excellen ae odynamic p ope ies a ele an
BDP doses, as con i med by in i o lung deposi ion es s. Ex i o pe meabili y es s wi h po cine lung issues
e idenced ha BDP eleased om he inhaled o mula ion could pene a e he b onchial issue.
1. In oduc ion
Pulmona y d ug deli e y is p ima ily used o he local ea men o
lung diseases, as i minimizes sys emic side e ec s and imp o es pa ien
adhe ence [1],[2]. D y powde inhale s (DPIs) ha e gained p ominence
and inc easing ma ke demand as de ices o pulmona y d ug deli e y
due o hei eco- iendliness, ease o use, s abili y o he o mula ion,
and wide po en ial o ea ing espi a o y condi ions [3],[4]. Howe e ,
DPIs encoun e challenges o deli e he apeu ic d ug concen a ions o
he desi ed egions wi hin he lungs, o en due o lung de ense mecha-
nisms, and sys emic abso p ion esul ing om pa icle deposi ion in he
mou h o o opha ynx due o he de icien ae odynamic p ope ies.
Signi ican e o s ha e been di ec ed owa ds no el pa icle enginee ing
o op imizing DPIs pe o mance [5]. Among hem, po ous o mula ions
a e especially p omising since hey p esen e y low pa icle densi y
associa ed wi h a highe su ace a ea ha imp o es hei ae odynamic
beha io . Howe e , only a ew pionee ing powde echnologies, like
Pulmosphe e™, Technosphe e®, and ARCUS echnologies, ha e gained
app o al o comme cial use [5],[6]. Finally, hese challenges o design
DPI o mula ions a e u he ampli ied when dealing wi h d ugs ha
exhibi poo solubili y in wa e [7],[8],[9].
Beclome hasone dip opiona e (BDP) is an ex emely poo ly wa e -
soluble (<1
μ
g/mL in wa e ) an i-in lamma o y p od ug commonly
p esc ibed o as hma pa ien s [9],[10], which is con e ed in he lung
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (C.A. Ga cía-Gonz´
alez).
Con en s lis s a ailable a ScienceDi ec
Jou nal o CO2 U iliza ion
jou nal homepage: www.else ie .com/loca e/jcou
h ps://doi.o g/10.1016/j.jcou.2024.102722
Recei ed 8 Janua y 2024; Recei ed in e ised o m 11 Feb ua y 2024; Accep ed 25 Feb ua y 2024
Jou nal o CO2 U iliza ion 81 (2024) 102722
2
o he ac i e me aboli e beclome hasone 17-monop opiona e by he
ac ion o es e ases [11]. Cu en ly, comme cial p oduc s inco po a ing
BDP a e a ailable in DPIs, such as Easyhale (O ion Pha ma L d., Lon-
don, UK) [12] and Fos ai NEXThale (Chiesi Fa maceu ici S.p.A, Pa ma,
I aly), which con ains a combina ion o BDP and o mo e ol uma a e
(FF) [13]. Ne e heless, he signi ican ly high con en o excipien s in
he inhaled o mula ions, e.g. lac ose in Fos ai NEXThale (100
μ
g o
BDP, 6
μ
g FF, and 9.9 mg o lac ose monohyd a e), migh pose chal-
lenges o pa ien s using inhale s on a daily basis. Simila ly o o he
po en inhaled co icos e oids, such as lu icasone p opiona e and
mome asone u oa e, BDP also has a low nominal dose (less han 1 mg)
and an ex emely poo wa e solubili y [14]. The use o po ous ma ices
can imp o e no only he ae odynamic pe o mance o BDP o mula ions
o DPIs, bu also he dissolu ion a e o BDP, due o hei high su ace
a ea and hei capaci y o inco po a e BDP as solid dispe sions [15].
Supe c i ical luid (SCF) echnologies can be used o p oduce solid
powde s o DPI o mula ions and ha e been ecognized o hei cos -
e ec i eness, non- oxici y, and en i onmen al iendliness [16],[17].
Pa icula ly, CO
2
is a sol en app o ed by he Uni ed S a es Food and
D ug Adminis a ion (FDA) and he mos common SCF, since i is
non- oxic, non- lammable, cos -e ec i e, comes om ecycled sou ces,
and has low su ace ension and mild c i ical poin condi ions (31.1 ◦C,
73.8 ba ) [16],[18],[19]. Namely, he echnological pla o m based on
he use o scCO
2
se s se e al s a egies o o mula e po ous nano-
s uc u ed powde s loaded wi h d ugs o di e ing wa e solubili y [15],
[20],[21]. Among hem, ae ogels a e ma e ials endowed wi h low bulk
densi y, la ge su ace a ea and high po osi y, which a e usually ob ained
by scCO
2
d ying o gels and ha e been p oposed as he nex gene a ion o
DPI o mula ions [22],[20]. scCO
2
d ying p e en s po e collapse o he
s uc u e and p ese es he physicochemical cha ac e is ics o he
polyme ic gels. Ae ogel pa icles wi h he igh g anulome y can
compile he ae odynamic and geome ical equi emen s o pulmona y
d ug deli e y while imp o ing low dispe sibili y [23],[24]. Recen ly,
ae ogels p oduced ia emulsion o he mal inkje p in ing gela ion
echniques ollowed by scCO
2
d ying ha e been s udied ega ding hei
po en ial in pulmona y d ug deli e y [23],[24],[25].
To he bes knowledge o he au ho s, he p esen s udy ma ks he
pionee ing applica ion o scCO
2
-assis ed imp egna ion o in eg a ing
ac i e pha maceu ical ing edien s (APIs) in o nanos uc u ed ae ogels
o DPIs. The use o powde echnology o inhalable o mula ions based
on BDP-loaded ae ogels wi h po en ial o indus ial scale p oduc ion is
he ein s udied. The p ocessing me hod should conside se e al design
aspec s – i mus be able o simul aneously p ese e he physicochemical
p ope ies o he ae ogel ma ix, and o inco po a e poo ly wa e -soluble
BDP in o ae ogels. scCO
2
-soluble d ugs can be loaded in o ae ogel
ma ices by a p ocess known as scCO
2
-assis ed imp egna ion [16],[18],
[19]. In his p ocess, he d ug molecules dissol ed in he supe c i ical
medium e ec i ely pene a e he swollen ae ogel ma ix and a e ap-
ped inside he polyme ic s uc u e upon dep essu iza ion [26],[27].
Howe e , he low solubili y o BDP in scCO
2
p esen s a signi ican
challenge o i s loading by scCO
2
-assis ed imp egna ion. This issue has
been p e iously o e come o o he d ugs by he addi ion o pola
co-sol en s (e.g., e hanol, ace one o me hanol) ha imp o e he solu-
bili y o he d ug in he supe c i ical medium, hus acili a ing he
imp egna ion p ocess [26],[27]. Tuning o o he imp egna ion pa am-
e e s, such as imp egna ion con ac ime o d ug:ca ie weigh a io,
could esul in inc eased loading e iciencies and d ug con en , hence
imp o ing he o e all p oduc i i y o he p ocess as well as i s
cos -e ec i eness [28].
In his s udy, he scCO
2
echnological pla o m was applied wi h a
wo- old in e es : (i) o d y algina e gel pa icles p epa ed h ough an
in e nal gela ion (gela ion-emulsi ica ion) me hod (scCO
2
d ying), and
(ii) o load BDP (scCO
2
imp egna ion p ocess) in o he esul ing ae ogel
pa icles [29],[30]. The ae ogel o mula ions we e hen assessed o
hei d ug loading capaci y and ae odynamic pe o mance in pulmona y
d ug deli e y o po en ial use as DPI o mula ions. The scCO
2
imp egna ion p ocedu e was he ein de eloped and he impac o a ious
pa ame e s (imp egna ion ime, d ug:ca ie a io, co-sol en choice)
was in es iga ed. The ex u al p ope ies and BDP con en o he p o-
duced ae ogels we e de e mined and used o model he imp egna ion
kine ics. In i o d ug elease and deposi ion es s in a compendial
impac o we e pe o med o e alua e he he apeu ic po en ial o he
op imized BDP-loaded o mula ion. Las ly, ex i o pe meabili y es s
using po cine b onchial issue we e ca ied ou o e alua e he pene-
a ion capaci y o he ae ogels in o he b onchial issue.
2. Ma e ials and Me hods
2.1. Ma e ials
Beclome hasone dip opiona e (BDP, >98% pu i y) was p o ided by
Tokyo Chemical Indus y Co., L d (Tokyo, Japan). Alginic acid sodium
sal om b own algae (gulu onic acid/mannu onic acid a io o 70/30,
M
w
=403 kDa) was ob ained om Sigma Ald ich (I ine, UK). Calcium
chlo ide (CaCl
2
) was pu chased om Me ck (Da ms ad , Ge many).
Ca bon dioxide (CO
2
>99.8% pu i y) was supplied by Nippon Gases
(Mad id, Spain). Ace one (100% pu i y) was p o ided by Scha lau
Chemie S.A (Ba celona, Spain). Absolu e e hanol, me hanol (100% pu-
i y), Tween 80, and Span 80 we e om Me ck (Da ms ad , Ge many).
Pa a in oil was pu chased om Ca lo E ba Reagen s (Emmendingen,
Ge many). Ace oni ile was supplied om VWR In e na ional S.A.S
(Rosny-sous-Bois, F ance).
2.2. P epa a ion o BDP-loaded algina e ae ogels
2.2.1. P epa a ion o algina e gel pa icles
Algina e ae ogels we e p epa ed by a gela ion-emulsi ica ion
me hod. B ie ly, 40 mL o 1.5 % w/ algina e solu ions in Milli-Q
wa e we e p epa ed unde magne ic s i ing (600 pm) a oom em-
pe a u e. To p epa e he oil phase, 3 g o a mix u e o Span 80 and
Tween 80 (75 and 25 % w/w, espec i ely) we e dissol ed in o 40 mL o
pa a in oil and homogenized by mechanical s i ing (1000 pm, VOS 60
con ol, VWR) o 10 min. Then, he algina e solu ion was added d op by
d op in o he oil phase and he esul ing wa e -in-oil emulsion (1:1 ol-
ume a io) was mechanically s i ed o 30 min a 1000 pm. The e-
loci y o he mechanical s i ing was dec eased o 600 pm, he
c osslinking solu ion (6 % w/ CaCl
2
) was hen added d opwise, and he
emulsion was le o age 2 h. Subsequen ly, 100 mL o e hanol we e
added, agi a ion was s opped, and he emulsion was le o se le o
15 min o sepa a e he oil and aqueous phase. The oil was emo ed, and
he aqueous phase was ans e ed o a 50 mL-Falcon ube. E hanol was
added (ca. 30 mL) and he mix u e was subsequen ly cen i uged a
3000 pm and 30 ◦C o 10 min o elimina e he emaining oil. This
washing s ep was epea ed h ee imes. The esul ing algina e gel pa -
icles we e le in e hanol un il d ying wi h scCO
2
.
2.2.2. P epa a ion o algina e ae ogel powde
The collec ed algina e alcogel pa icles we e placed in o pape ca -
idges and d ied wi h scCO
2
as ex ac ion medium (Wa e s-Tha P o-
cess, Pi sbu g, PA, USA) a 40 ◦C, 120 ba and a CO
2
low o 7 and 5 g/
mL o 1 and 2.5 h, espec i ely (Fig. 1). The high-p essu e essel u i-
lized had an in e nal olume o 100 mL (heigh : 4.2 cm; diame e :
5.5 cm). The dep essu iza ion a e was 1.0 g/min, and he dep essu -
iza ion du a ion was 66 min.
2.2.3. BDP loading by scCO
2
-assis ed imp egna ion
2.2.3.1. P epa a ion o BDP-loaded algina e ae ogels. Algina e ae ogels
(200 mg) and BDP (20 o 40 mg) we e illed sepa a ely in wo il e
pape ca idges and pu a he bo om o he 100-mL s ainless s eel high-
p essu e essel (Wa e s-Tha P ocess, Pi sbu gh, PA, USA), ensu ing
T. Duong e al.
Jou nal o CO2 U iliza ion 81 (2024) 102722
3
ha he e was no physical con ac be ween ae ogels and he d ug
powde be o e he p ocess. scCO
2
-assis ed imp egna ion was ca ied ou
a 65 ◦C and 215 ba , based on p e iously epo ed solubili y alues
[31]. S i ing was applied du ing he imp egna ion p ocess (500 pm).
Besides a ying he amoun o ini ial BDP, di e en con ac imes (1, 2,
6, 9 and 24 h) and co-sol en s (none, me hanol, e hanol o ace one a
5 ol.%) we e in es iga ed. The dep essu iza ion s ep was con olled
h ough an au oma ic backp essu e (BPR in Fig. 1) a a low o 1 g/min.
T ials we e ca ied ou in iplica e.
2.2.3.2. Quan i ica ion o BDP con en in algina e ae ogels. Samples (ca.
10 mg) o he collec ed o mula ion we e placed in o ials con aining
10 mL o a sol en mix u e comp ised o ace oni ile (ACN) and wa e a
a 65:35 % / . Subsequen ly, he ials unde wen sonica ion o
180 min o ensu e he comple e elease o BDP p esen in he o mula-
ion in o he sol en . BDP con en in he ae ogel pa icles was quan i ied
by HPLC (JASCO, Tokyo, Japan) using a C18 column (Wa e s Symme y
5µm, 3.9 mm ×150 mm) as s a iona y phase. The mobile phase con-
sis ed o ACN:wa e a a 65:35 % / and was pumped a a low a e o
1.4 mL/min. The olume o injec ion was 25
μ
L, and BDP was de ec ed
a 254 nm ( e en ion ime: 2.7 min). BDP concen a ion was calcula ed
using a calib a ion cu e wi h linea i y in he 0.25–80
μ
g/mL concen-
a ion ange (R
2
>0.9999).
2.2.3.3. Modelling o SCF imp egna ion o BDP in o algina e ae ogels. The
expe imen al da a ob ained om SCF-assis ed imp egna ion we e i ed
o pseudo i s -o de and pseudo second-o de kine ic models. By in e-
g a ing Eq. 1, equa ions o pseudo i s -o de (Eq. 2) and pseudo second-
o de (Eq. 3) models we e ob ained:
d(1−q
qe)
d = − k(1−q
qe)n
(1)
ln(qe
qe−q )=k1 (2)
q
=1
k2qe2+
qe
(3)
whe e q
(mmol/g) ep esen s he amoun o d ug ha has been loaded
in o he ae ogel a e a speci ic con ac ime , q
e
(mmol/g) deno es he
adso p ion capaci y a equilib ium, which is he maximum quan i y o
d ug ha can be loaded in o he ae ogel, and k
1
(1/h) and k
2
(g/mmol⋅h)
a e he a e cons an s o pseudo i s -o de and pseudo second-o de
models, espec i ely. Fi ings we e ca ied ou using O iginP o so -
wa e ( . 9.0, O iginlab, No hamp on, MA, USA).
2.3. Physicochemical cha ac e iza ion
Algina e ae ogel pa icle diame e s we e measu ed using a CKC53
op ical mic oscope equipped wi h an EP50 came a and EP iew image
analysis so wa e ( .1.3, Olympus, Tokyo, Japan). The acqui ed images
we e subsequen ly analyzed using Fiji-ImageJ2 (Na ional Ins i u es o
Heal h, NIH, Be hesda, MD, USA) o de e mine he Fe e diame e o he
pa icles (Figu e S1 in Supplemen a y In o ma ion).
Scanning elec on mic oscopy (SEM, EVO LS15, Zeiss, Obe kochen,
Ge many) was used o s udy he mo phology and s uc u e o he ae -
ogels. Spu e coa ing wi h a hin laye (2–20 nm) o i idium was
employed o imp o e he con as o he pa icles. The small g ain size o
i idium, ypically be ween 1 and 2 nm, makes i a a o able coa ing
ma e ial choice o cap u ing p ecise, high-magni ica ion images [32].
Ni ogen adso p ion-deso p ion analysis (ASAP2000, Mic ome i ics
Inc., No c oss, GA, USA) was used o de e mine he speci ic su ace a ea
o BDP-loaded algina e ae ogel pa icles. Be o e he analysis, samples
we e degassed unde acuum (<1 mPa, oom empe a u e, 24 h). The
ex u al p ope ies (speci ic su ace a ea, po e size dis ibu ion and
speci ic po e olume) we e calcula ed using he BET and BJH me hods.
The o e all po osi y (
ε
) o he ma e ial was calcula ed using (Eq. 4)
[23]
ε
=(1−
ρ
bulk
ρ
skel )×100 (4)
whe e
ρ
bulk
ep esen s he bulk densi y and
ρ
skel
ep esen s he skele al
densi y o he algina e pa icles. Bulk densi y was es ima ed based on he
apped densi y (app oxima ely 1.26 imes he apped densi y acco ding
o exis ing li e a u e) [23],[33]. The apped densi y (
ρ
apped
) was
calcula ed using he g adua ed lask me hod in iplica e [23]. Skele al
densi y was measu ed om i e eplica es using helium pycnome y
(Quan ach ome; Boyn on Beach, FL, USA) a a empe a u e o 25 ◦C and
a p essu e o 1.03 ba .
The p esence and po en ial in e ac ion o BDP wi hin he algina e
ae ogel was s udied by A enua ed To al Re lec ance (ATR) spec oscopy
using a Va ian FT-IR 670 spec ome e wi h a Gladi-ATR accesso y (Pike
Technologies, Madison, WI, USA). Pu e BDP, algina e ae ogels, BDP-
loaded algina e ae ogels and BDP-ae ogel physical mix u es we e
examined in he 400–4000 cm
−1
ange using 8 scans a a esolu ion o
2 cm
−1
.
X- ay di ac ion (XRD) pa e ns we e collec ed wi h a Rigaku Min-
iFlex di ac ome e (Rigaku, Neu-Isenbu g, Ge many) ope a ing a
30 kV and a 15 mA, wi h CuK
α
adia ion (1.54056 Å) in he ange om
3 o 60◦(s eps o 0.02◦, scan s ep ime o 2.00 s).
2.4. In i o d ug elease
In i o elease es s o BDP-loaded algina e ae ogels we e ca ied ou
in dialysis bags (MWCO =12,400 Da). Algina e ae ogels (10 mg, con-
aining ca. 450 µg o BDP) we e added in o he dialysis bags con aining
1 mL o PBS solu ion, pH 7.4 (con aining sodium chlo ide, po assium
chlo ide, sodium phospha e and po assium phospha e) wi h 20 ol.%
me hanol o enhance he solubili y o BDP ( he solubili y o BDP in PBS
is 0.15 ±0.04
μ
g/mL [34]). The en i e se up was hen placed in a plas ic
con aine con aining 15 mL o he same medium and pu inside a
shaking incuba o (100 pm, 37 ◦C). Samples (300
μ
L) we e pe iodically
wi hd awn o HPLC analysis, wi h an equi alen olume o esh me-
dium being ein oduced in o he plas ic con aine . Released BDP was
quan i ied ollowing he same HPLC me hod as desc ibed in Sec ion
2.2.3.2.
Modeling o he BDP elease p o ile om he ae ogels was pe o med
by i ing he da a o he i s -o de elease model (Eq. 5) and o wo
simul aneous i s -o de dissolu ion p ocesses (Eq. 6 [[35]]) using
G aphPad P ism 10 so wa e (San Diego, CA, USA).
F=Fmax(1−e−k1 )(5)
D=Dmax,1(1−e−k1 )+Dmax,2(1−e−k2 )(6)
Whe e D is he dosage o BDP eleased a ime (in pe cen age), D
max,i
is
Fig. 1. Se up o con inuous scCO
2
d ying. P1: dual pis on pump, V1-2: needle
al es, HEX12: hea exchange s, BPR: backp essu e egula o (used o main ain
a s able p essu e). Algina e alcogel pa icles we e placed in he high-p essu e
essel. Residual e hanol was ga he ed ia a collec o .
T. Duong e al.
Jou nal o CO2 U iliza ion 81 (2024) 102722
4
he maximum BDP eleased in s age i (in pe cen age), and k
i
is he i s -
o de kine ic coe icien in s age i in h
−1
, being i=1 o 2.
2.5. In i o ae osol deposi ion es s
Ae odynamic deposi ion o BDP-loaded algina e ae ogels was e al-
ua ed wi h a Nex Gene a ion Impac o (NGI) manu ac u ed by Copley
(No ingham, UK) consis ing o se en s ages and a Mic o-O i ice Col-
lec o (MOC). A mou hpiece adap e was a ached o he NGI o simula e
he use o a single-dose DPI de ice wi h low esis ance, meaning ha
h ough he de ice, he p essu e d op is lowe han 5 Mba
0.5
L/min. The
expe imen al se up in ol ed a acuum pump ope a ing a a low a e o
100 L/min o a du a ion o 2.4 s, esul ing in a p essu e d op o 4 kPa
behind he impac o and an ai olume o 4 L.
Inhaled pa icles in each s age a icles we e ga he ed using a sol en
mix u e o ACN: H
2
O 65:35 % / and subjec ed o 180 min o soni-
ca ion. The quan i y o BDP was hen de e mined h ough HPLC analysis
(c . Sec ion 2.2.3.2).
The mass o pa icles ob ained a each s age was es ima ed om he
d ug measu emen s. Wi h hese esul s, he alues o mass median
ae odynamic diame e (MMAD), emi ed ac ion (EF), and ine pa icle
ac ion (FPF) we e calcula ed [23],[36],[37]. The ae odynamic cu -o
diame e o he NGI was se a 6.12
μ
m, 3.42
μ
m, 2.18
μ
m, 1.31
μ
m, 0.72
μ
m, 0.14
μ
m, and 0.24
μ
m o s ages 1–7, co espondingly [38]. Emi ed
dose ac ion (EF) was calcula ed as he pe cen age be ween he emi ed
dose ED (ED is he change in mass be ween he capsule be o e and a e
adminis a ion) and used o al dose [36],[37]. The mass o pa icles wi h
ae odynamic diame e s <5
μ
m we e ma ked as ine pa icle dose (FPD).
Fine pa icle ac ion (FPF) was exp essed as he pe cen age o FPD
e sus ED. Mass median ae odynamic diame e (MMAD) was calcula ed
based on he plo ed g aph o he cumula i e mass pe cen age o he
sample in p obabili y scale e sus he log o he s age cu -o diame e .
All ials we e conduc ed in iplica e (n =3) a oom empe a u e.
2.6. Ex i o b onchial pe meabili y es s
Algina e ae ogels (25 mg) loaded wi h BDP (6.0 w .%) we e sub-
jec ed o b onchial pe meabili y expe imen s alongside an equi alen
amoun o d ug powde (1.5 mg) o compa ison. F eshly pig lungs
( om a local slaugh e house) we e dissec ed, and he b onchial issue
was ca e ully cu , p epa ed, and washed wi h dis illed wa e be o e
being posi ioned in F anz cells o es ing wi h he epi helial wall acing
he dono chambe , and he basemen memb ane in con ac wi h he
ecep o chambe o mimic biological condi ions [39,40]. The s abili y
o he issue in he ecep o medium was con i med by obse ing ha he
issue’s colo and ex u e emained unchanged a e being placed in he
medium o 30 min be o e commencing he pe meabili y es s. The e-
cep o chambe was illed wi h 6 mL o PBS:me hanol (80:20 / ), while
he dono chambe con ained 500 µL o he same sol en mix u e. The
di usion cells we e kep a a cons an empe a u e inside a me hac yla e
ba h con aining wa e wi h magne ic s i ing (37 ◦C, 100 pm). Aliquo s
o 1 mL we e ex ac ed om he ecep o chambe hou ly o e a 1 and
3-hou du a ion, and he wi hd awn olume was eplaced wi h esh
elease medium. The aliquo samples unde wen HPLC analysis as
desc ibed in Sec ion 2.2.3.2.
Following he pe meabili y es s, he o mula ion emaining on he
b onchial issues a e 1 and 3 h was ca e ully emo ed, and he b on-
chial issues we e washed in dis illed wa e and s o ed a −80 ◦C. The
b onchial issues we e la e analyzed using IR-Raman spec oscopy
(WITec, alpha 300 R, Wissenscha liche Ins umen und Technologie
GmbH, Ulm, Ge many) wi h a line scan in he xz plane and iewed
h ough a 50×objec i e lens (Zeiss LD EC Epiplan-Neo lua Dic 50×/
0.55) (exci a ion wa eleng h: 532 nm, lase powe : 1.2 mW, in eg a ion
ime: 1.0 s). Each da a poin was ga he ed using 100 accumula ions. IR-
Raman spec oscopy is commonly employed o he de ec ion o API
wi hin biological ma e ials [41,42]. The s udy ocused on he basemen
memb ane o b onchial issue. The a io be ween he BDP-associa ed
peak a 1666 cm
–1
and he ixed peak a 1345 cm
−1
in lung issue was
compa ed o con i m he p esence o BDP pene a ed h ough b onchial
issue.
2.7. S a is ical analysis
Resul s we e p esen ed as mean alues and hei co esponding
s anda d de ia ions. One-way analysis o a iance (ANOVA) ollowed by
he pos -hoc Tukey’s Hones ly Signi ican Di e ence (HSD) es was
used o analyze he s a is ical signi icance. The g oups exhibi ed a s a-
is ically signi ican di e ence when he p- alue ell below 0.05. All
s a is ical analyses we e conduc ed using G aphPad P ism 9 so wa e
(San Diego, CA, USA).
3. Resul s and discussion
3.1. Tex u al and physicochemical p ope ies o algina e ae ogels
A gela ion-emulsi ica ion me hod was applied o p oduce algina e
ae ogel pa icles. This app oach acili a es he upscaling o algina e
pa icle p oduc ion, ansi ioning om labo a o y-scale o pilo and in-
dus ial scales, owing o i s capaci y o swi ly p oduce a s able emulsion
wi h a consis en a e age d ople size wi hin sho imes ( ypically mi-
nu es) [30]. Wi hin his p ocess, he p ecu so liquid o mula ion is
ans o med in o sphe ical d ople s wi hin an immiscible liquid phase
esul ing in biphasic liquid-liquid sys em. Con inuous s i ing played a
i al ole as a cons an ene gy sou ce, aiding in he dispe sion o he
algina e aqueous solu ion in o he oil phase o ob ain a wa e -in-oil
emulsion. Then, CaCl
2
was in oduced o he emulsion as a
c oss-linking agen o ini ia e gela ion in he d ople s o he dispe sed
phase o he emulsion esul ing in a dispe sion o algina e gel pa icles
wi hin he oil phase. Ae ogel powde was ob ained om he algina e gels
dispe sion a e he emo al o he oil and he ex ac ion o he gel po e
luid by scCO
2
d ying.
The dis inc cha ac e is ics o he algina e ae ogels, including ho-
mogeneous pa icle size dis ibu ion, sphe ici y, high po osi y and
ough ex u e we e obse ed by SEM imaging (Fig. 2(a-d)). These
p ope ies play a c ucial ole in acili a ing he inco po a ion o poo ly
wa e -soluble d ugs and make hese pa icles p omising candida es as
ca ie s in no el DPI o mula ions [16]. Algina e ae ogels had an
a e age Fe e diame e o 2.25 ±1.50
μ
m, wi h an a e age ci cula i y
close o 0.9. The size dis ibu ion o algina e ae ogels was illus a ed in
Fig. 2e. Algina e ae ogels possess a po ous s uc u e, which po en ially
mi iga es di icul ies ega ding mac ophage clea ance and he mo e-
men o pa icles h ough he mac ophage cy oskele on when compa ed
o igid pa icles o he same size [43–45]. Ce ain s udies emphasize he
signi icance o s i ness in in luencing how mac ophages de ec hei
p esence wi hin he lungs. In gene al, sphe ical pa icles lacking po es o
exhibi ing a igid s uc u e a e mo e p one o up ake by mac ophages
han po ous ma e ials. Addi ionally, sphe ical pa icles, as well as o he
ound-like shapes like pollen o cubic pa icles, end o show be e low
p ope ies han o he geome ies (e.g., pla e- and needle-shaped pa i-
cles), which ansla es in o a highe p obabili y o deep lung deposi ion
[15]. Using complemen a y analy ical echniques (ni ogen
adso p ion-deso p ion analysis and helium pycnome y), he excellen
ex u al p ope ies o he ae ogels we e con i med by a high po osi y (ca.
90%), high speci ic su ace a ea (247 ±12 m
2
/g), BJH-speci ic po e
olume (0.8 ±0.04 cm
3
/g) and a e age po e diame e wi hin he
mesopo ous ange (10.3 ±0.5 nm). In his con ex , scCO
2
d ying was
e y e ec i e in p ese ing algina e ae ogel ex u al p ope ies wi h
espec o o he d ying echniques o algina e pa icles [46].
T. Duong e al.
Jou nal o CO2 U iliza ion 81 (2024) 102722
5
3.2. ScCO
2
imp egna ion p ocess o he inco po a ion o BDP in o
ae ogels
3.2.1. E ec o he p ocessing pa ame e s on BDP loading con en s
BDP was loaded in he ae ogels by a scCO
2
imp egna ion p ocess
whe e he empe a u e and p essu e we e se a 65 ◦C and 215 ba ,
espec i ely. The choice o hese alues was aken based on he s abili y
o BDP epo ed wi hin he 64.85–84.85◦C empe a u e ange and he
213–385 ba p essu e ange [31]. Keeping hese pa ame e s ixed, he
e ec s o he con ac ime (i.e., imp egna ion p ocess du a ion a con-
s an p essu e and empe a u e), he ini ial amoun o BDP loaded in he
high-p essu e essel and he use o co-sol en s on d ug loading we e
s udied.
Rega ding con ac ime, he imp egna ion p ocess was ini ially ca -
ied ou using only scCO
2
. The du a ion o he imp egna ion p ocess had
a signi ican in luence on he d ug loadings (Fig. 3a). Fo sho con ac
imes (1 and 2 h), he d ug loading was signi ican ly lowe compa ed o
long imp egna ion pe iods o 6 and 9 h. Howe e , much longe con ac
imes (24 h) esul ed in d ug loading le els simila o hose achie ed a
9 h. The equilib ium s a e o he p ocess seems o be achie ed a e 9 h,
al hough he BDP loadings in he ae ogel we e s ill low (0.19 ±0.02 w .
%). The highly limi ed solubili y o BDP in scCO
2
may explain his
phenomenon as he highes solubili y o BDP in scCO
2
measu ed by
Va ana a e al. was 3.36 ×10
−5
(mole ac ion) a he ha shes p essu e
(385 ba ) and empe a u e (84.85 ◦C) condi ions es ed [31]. scCO
2
exhibi s sol en cha ac e is ics ha closely esemble hose o adi ional
hyd oca bon sol en s [26],[47]. scCO
2
is a good sol en o non-pola
and low molecula weigh solu es, bu i s sol a ion powe is ypically
se e ely es ic ed when i comes o hyd ophilic, pola molecules, o
subs ances wi h a high molecula weigh (>500 g/mol) [27],[47].
Al hough BDP is a hyd ophobic molecule, BDP is also a la ge molecule
ha con ains hyd oxyl, ca bonyl and es e unc ional g oups wi hin i s
s uc u e, which a e pola and could explain he limi ed solubili y o he
molecule in scCO
2
[48].
The e ec o adding small amoun s (5 ol.%) o di e en pola co-
sol en s (ace one, me hanol and e hanol) on BDP loading was es ed
a sho imp egna ion imes (1 h). The addi ion o co-sol en s signi i-
can ly inc eased he BDP loading in he ae ogel; he o al amoun loaded
being dependen on he na u e o he co-sol en (Fig. 3b). The scCO
2
sol en sys em a o ds an augmen a ion in pola i y wi h he use o pola
Fig. 2. Mo phological appea ance o he algina e ae ogel pa icles ob ained by gela ion-emulsi ica ion e alua ed by SEM imaging a di e en magni ica ions: (a,b)
Na ow pa icle size dis ibu ion, (c) images o a ep esen a i e sphe ical pa icle, (d) high mesopo osi y and oughness on he su ace and (e) size dis ibu ion o
algina e ae ogel ca ie s p oduced by gela ion-emulsi ica ion echnique ollowed by scCO
2
d ying.
T. Duong e al.
Jou nal o CO2 U iliza ion 81 (2024) 102722
6
co-sol en s, such as ace one and e hanol, a mino amoun s esul ing in a
supe c i ical mix u e wi h an e ec i e inc ease in he sol a ion powe o
pola compounds [47]. This s a egy no only b oadens he ange o
compounds amenable o scCO
2
ex ac ion and imp egna ion, bu also
showcases he e sa ili y and adap abili y o scCO
2
echnologies in
a ious applica ions, including pha maceu icals, ma e ials science and
en i onmen al chemis y [26],[47],[49].
BDP:ae ogels weigh a io was inc eased om 1:10 o 1:5 in a scCO
2
/
ace one imp egna ion medium o a emp o u he inc ease he BDP-
loading in he ae ogels. Howe e , doubling he amoun o d ug did
no cause a signi ican change in d ug loading (Fig. 3c). In he conduc ed
expe imen , he quan i y o BDP was doubled while keeping cons an
o he pa ame e s, such as empe a u e, p essu e, and dep essu iza ion
a e. I is assumed ha in bo h expe imen al cases, he anspo o he
ae ogel po es’ su ace, he adso p ion o he d ug, and he p ecipi a ion
o he d ug emained unchanged [50]. The simila d ug loading con en s
could be explained by he consis en dissolu ion o he d ug in
CO
2
/ace one supe c i ical medium, likely eaching he sa u a ion in he
said medium in bo h expe imen al cases ega dless o he
d ug- o-ae ogel ca ie weigh a io used.
Ace one was he mos e ec i e co-sol en in inc easing he BDP-
loading in he ae ogel pa icles by supe c i ical imp egna ion. The
es s wi h his co-sol en a inc easing con ac imes om 1 h o an
ex ensi e du a ion (24 h) we e ca ied ou o assess he maximum
loadings o BDP wi h his supe c i ical mix u e (Fig. 3d). The esul s
ollowed a simila pa e n o hose ob ained in pu e scCO
2
bu a much
highe BDP loading alues. Du ing he ini ial s ages o scCO
2
imp eg-
na ion, he d ug loading s ood a ca. 2 w .%. Howe e , a no ewo hy
inc ease in d ug loading (ca. 5 w .%) was obse ed a longe imes o
6 h. The BDP loading signi ican ly inc eased again a e 9 h, achie ing
he highes d ug loading capaci y, which was no imp o ed by ex ending
he imp egna ion du a ion o 24 h. O e all, a mode a ely p olonged
con ac ime coupled wi h he use o ace one as co-sol en eme ges as a
use ul s a egy o enhancing loading o BDP in ae ogel sys ems by
scCO
2
imp egna ion, and may be ex ended o enabling he e icien
loading o o he hyd ophobic compounds.
3.2.2. Adso p ion kine ics o BDP in o algina e ae ogels
The i ing o he expe imen al da a om scCO
2
imp egna ion es s o
kine ic models allows he p edic ion o he loading capaci ies ha can be
Fig. 3. E ec o p ocessing pa ame e s on he d ug loading o BDP in o algina e ae ogels by scCO
2
imp egna ion: (a) Con ac ime using 1:10 BDP:ae ogels weigh
a io wi hou co-sol en . (b) Addi ion o 5 ol.% o di e en co-sol en s in a 1-hou imp egna ion p ocess. (c) BDP:ae ogel weigh a io wi h ace one as co-sol en . As
a esul , (d) inc easing he con ac ime a he op imized condi ions (5 ol.% ace one as co-sol en , 1:10 BDP:ae ogels a io) esul ed in BDP-loadings >6 w %. (n =
3, mean ±s anda d de ia ion), * and ** deno e s a is ical di e ences (p <0.05 and p <0.01, espec i ely).
T. Duong e al.
Jou nal o CO2 U iliza ion 81 (2024) 102722
7
achie ed unde ce ain gi en p ocess condi ions. The expe imen al BDP
imp egna ion da a om scCO
2
and CO
2
/ace one supe c i ical mix u e
ob ained in his wo k (c . Sec ion 3.2.1) we e i ed o he pseudo i s -
o de and pseudo second-o de models (Table 1, and Figu e S2 in Sup-
plemen a y In o ma ion). Pseudo i s -o de sugges s ha su ace
di usion go e ns he a e-con olling s ep, while pseudo-second o de
indica es ha he con olling ac o is he chemical eac ion [51].
Based on he calcula ed R
2
and Akaike in o ma ion c i e ion AIC
alues (Table 1), he pseudo i s -o de kine ic model had a sligh ly
highe R
2
and lowe AIC indica ing a highe le el o accu acy in i ing
he expe imen al da a when compa ed o he pseudo second-o de ki-
ne ic model, e lec ing ha he BDP adso p ion a e-con olling s ep was
he su ace di usion.
The maximum loading o BDP in o algina e ae ogels (q
e
) was
0.004 mmol/g in he case o scCO
2
imp egna ion, while i inc eased
28.5- old wi h he use o ace one as co-sol en . Acco ding o he pseudo
second-o de model, ca. 75 w .% o he maximum d ug quan i y was
adso bed wi hin he ini ial 10 h in bo h expe imen s u ilizing scCO
2
-
assis ed imp egna ion, wi h o wi hou he p esence o he co-sol en
(ace one). Addi ionally, he kine ic coe icien k
1
in scCO
2
-assis ed
imp egna ion wi h co-sol en was highe han scCO
2
-assis ed imp eg-
na ion wi hou co-sol en . This indica ed an enhanced e iciency in he
imp egna ion p ocess when employing a co-sol en .
3.3. Physicochemical p ope ies o BDP-loaded ae ogels
The p esence o BDP in he ae ogels and i s in e ac ion wi h he
algina e ma ix was s udied by ATR-FTIR (Fig. 4). BDP exhibi ed dis inc
conjuga ed and non-conjuga ed C
–
–
O s e ching bands a 1724 cm
−1
and 1658 cm
−1
, espec i ely [36]. Cha ac e is ic C
–
–
C s e ching band
in BDP a 1615 cm
−1
, along wi h C-O bands a 1186 cm
−1
we e also
obse ed [10],[36]. ATR spec a o BDP-loaded algina e and physical
mix u e be ween BDP and algina e ae ogels e ealed a no able C
–
–
O
s e ching band a 1735 cm
−1
(as e isks in Fig. 4). This dis inc i e peak
a 1735 cm
−1
sugges ed he p esence o BDP in he loaded algina e
ae ogels.
The solid-s a e BDP in ae ogel o mula ions con aining BDP was
in es iga ed by XRD (Fig. 5). C ys alline BDP p esen s sha p and in ense
peaks a 11.28, 14.44, and 20.06º 2θ angles [10]. These peaks we e
clea ly isible in he XRD pa e n o a physical mix u e o BDP and
algina e ae ogels, indica ing ha he simple mechanical mixing did no
esul in d ug amo phiza ion.
On he con a y, he pa e n ele an o he imp egna ed ae ogel
indica ed lack o c ys alline d ug being p ac ically supe imposable o
ha o he non-imp egna ed algina e ae ogel. Fo a gi en d ug:ca ie
ma ix se , he selec ed empe a u e, p essu e and dep essu iza ion s eps
a e c i ical pa ame e s o he scCO
2
imp egna ion p ocess o achie e
d ug loadings in he amo phous o m. The e m "adso p i e p ecipi a-
ion" encompassed wo essen ial phenomena ha may ake place du ing
he scCO
2
imp egna ion p ocess: he d ug’s adso p ion on o he su ace
o polyme s, and he d ug’s p ecipi a ion wi hin he polyme ic ma ix.
The high di usi i y and low su ace ension o CO
2
, along wi h he
p esence o a co-sol en , played a signi ican ole in dissol ing he d ug
(BDP) in he supe c i ical luid. Unde con olled empe a u e and
p essu e condi ions, scCO
2
unde wen molecula densi y inc ease, ol-
lowed by a expansion h ough he dep essu iza ion s ep, leading o he
p ecipi a ion o d ug molecules wi hin he po e walls o he ca ie
ma e ial [50],[52]. In his con ex , he apid p ecipi a ion o solidi i-
ca ion o d ug molecules caused he solid s a e o d ug in an amo phous
s uc u e.
3.4. In i o BDP elease o ae ogel ca ie s
BDP elease om he ae ogel ma ix was examined ia he dialysis
bag me hod o a oid di usion o he dissol ing algina e chains o he
ecep o medium. The ae ogels showed a wo-phase elease pa e n,
wi h an ini ial bu s elease in he i s 15 min, ollowed by a sus ained
elease o e he nex hou s (Fig. 6). The e was no signi ican di e ence
in d ug elease be ween 3 and 7 h, sugges ing ha he d ug elease
eached a pla eau a e 3 h.
The modelling o he BDP elease p o ile om he ae ogel un eiled a
complex mechanis ic beha iou (Table 2). The bes i ing was ob ained
o he model conside ing a biexponen ial unc ion (Eq. 6) whe e he
Table 1
Fi ing pa ame e s o he expe imen al da a o BDP-loaded algina e ae ogels ob ained om scCO
2
-assis ed imp egna ion wi h and wi hou co-sol en (ace one) o he
pseudo i s -o de (Eq. (2)) and pseudo second-o de (Eq. (3)) adso p ion kine ic models.
Co-sol en used Pseudo- i s -o de Pseudo-second-o de
q
e
x 10
3
(mmol/g) k
1
(1/h) R
2
AIC q
e
x 10
3
(mmol/g) k
2
(g/mmol⋅h) R
2
AIC
None 4.0±0.42 0.20±0.06 0.92 -50.34 5.0±0.89 39.3±25.86 0.89 -48.62
Ace one 114±7.2 0.27±0.05 0.96 -20.48 135±17.3 2.2±1.17 0.89 -16.35
Fig. 4. ATR-FTIR spec a o he physical mix u e o BDP and algina e compa ed
o BDP-loaded algina e ae ogel. As e isks co espond o he wa enumbe o
1735 cm
−1
, ypical o C
–
–
O s e ching band om BDP molecule. The physical
mix u e exhibi s an equi alen BDP concen a ion in algina e ae ogels,
6.5 w .%.
Fig. 5. X- ay di ac ion pa e ns o algina e ae ogels, BDP-loaded algina e
ae ogels, BDP and physical mix u e o BDP and algina e ae ogels. The physical
mix u e exhibi s an equi alen BDP concen a ion in algina e ae ogels,
6.5 w .%.
T. Duong e al.
Jou nal o CO2 U iliza ion 81 (2024) 102722
8
mass ans e mechanism is mainly go e ned by dissolu ion [35],[53].
This model conside s wo simul aneous i s -o de d ug eleases o wo
di e en BDP ac ions. A i s BDP ac ion (k
1
, D
max,1
) weakly bound o
he su ace o algina e o he ae ogel ma ix wi h a as dissolu ion and
di usion o he elease medium. A second BDP ac ion (k
2
, D
max,2
) wi h
a complex in e ac ion wi h he ae ogel and anspo h ough he
nanopo ous ma ix ha includes he hyd a ion o he ae ogels, PBS
di usion h ough he po es owa ds he in e io pa o he pa icles, and
a combined mechanism o dissolu ion o BDP a he su ace inside he
po es and disassembly o he algina e chains due o ion exchange. The
amoun o d ug eleased unde he second e ec accoun ed o a sig-
ni ican po ion, speci ically 70% o he o e all d ug eleased,
amoun ing o an equi alen o 15 µg.
Toge he wi h he lipophilic na u e o BDP (log P=3.49), he physi-
ological cha ac e is ics o human lung condi ions, such as he p esence
o su ac an s and he lipophilic na u e o he epi helia, did no pe ec ly
mi o he condi ions obse ed in he in i o elease wi h an aqueous
elease medium [14],[54]. Consequen ly, his di e ence accoun ed o
he low amoun o BDP eleased om algina e ae ogels (ca. 22 µg,
equi alen o ca. 4.5 w .% a e 6 h). In ano he wo k, he d ug elease
s udy u ilized a comme cial p oduc o BDP (Clenil®) as a con ol [9].
Ve ical F anz di usion cells we e employed in DPBS wi h a i icial
mucus. Following a 6-hou incuba ion pe iod, he BDP eleased om he
Clenil® o mula ion was close o 1.5 w .%, indica ing a e y low elease
a e, e en hough a i icial mucus was used o mimic he lung
condi ions.
3.5. In i o ae odynamic d ug deposi ion o BDP-loaded algina e
ae ogels
BDP-loaded ae ogel o mula ions ob aining om scCO
2
imp egna-
ion a di e en du a ions (1, 6, 9 and 24 h) wi h 5 ol.% ace one (1:10
BDP:ae ogels weigh a io) we e subjec ed o ae osol pe o mance
es ing using an NGI impac o (Fig. 7 and Table 3). This enabled he
p ecise measu emen and cha ac e iza ion o he amoun o BDP
deposi ed a each s age o he NGI, p o iding aluable insigh s in o he
ae odynamic deposi ion beha io o he ae ogels. In gene al, simila
d ug deposi ion p o iles we e obse ed ega dless o he scCO
2
imp egna ion ime, wi h only changes in he o al amoun o d ug
Fig. 6. In i o elease p o ile o BDP om algina e ae ogels (PBS pH 7.4 wi h
20% me hanol, 37
◦C, 100 pm) (n =3, mean ±s anda d de ia ion). Dashed
line co esponds o he i ing o he da a o he biexponen ial elease model
conside ing wo simul aneous i s -o de dissolu ion p ocesses (Eq. 6).
Table 2
Kine ic i ing pa ame e s o he BDP elease om algina e ae ogel pa icles in
PBS pH 7.4 wi h 20% me hanol medium.
Model Fi s -o de (Eq.5) Double i s -o de (Eq.6)
D
max,1
(%) 4.482 ±0.119 1.43 ±0.19
k
1
(h
−1
) 1.821 ±0.220 17.00 ±5.69
D
max,2
(%) - 3.42 ±0.19
k
2
(h
−1
) - 0.692 ±0.101
R
2
0.938 0.979
Fig. 7. D ug deposi ion o BDP-loaded algina e ae ogels ob ained om scCO
2
imp egna ion wi h di e en con ac imes: (a) 1 h, (b) 6 h, (c) 9 h and (d) 24 h (n =3,
mean ±s anda d de ia ion). No a ion: IP, induc ion po , and MOC, mic o-o i ice collec o .
T. Duong e al.
Jou nal o CO2 U iliza ion 81 (2024) 102722
9
deposi ed due o he di e en loading con en s.
A wide ange o impo an pa ame e s can be assessed when e alu-
a ing d ug ae odynamics in i o, including mass median ae odynamic
diame e (MMAD), emi ed ac ion (EF), and ine pa icle ac ion (FPF)
[23]. MMAD e e s o he ae odynamic diame e a which 50 % o he
o al pa icle mass exhibi s a diame e smalle han he median, while
he o he 50 % exhibi s a diame e la ge han he median. In he case o
he BDP-loaded algina e ae ogels, he mean alue (ca. 1.1 µm) indica ed
ha pa icles we e wi hin he 1–5µm ange wi h a p opensi y o se le
p edominan ly in he b onchioles and al eoli, which cons i u e he
a ge egion o e icien d ug deli e y. EF quan i ies he pe cen age o
d ug eco e ed in he NGI, ep esen ing he o e all dose emi ed du ing
he e alua ion. The ema kable EF (ca. 90 %) o he ae ogel pa icles
indica ed he high lowabili y o he inhaled o mula ion leading o a
high amoun o d ug en e ing o he NGI and o a small esidual amoun
emaining in he capsule. The high lowabili y o inhaled o mula ion
also con i med he success ul emo al o he ace one a e imp egna ion.
A signi ican po ion (ca. 20 %) o he BDP con en in he loaded ae ogel
pa icles was p ima ily deposi ed in he h oa and ini ial s ages. How-
e e , owa ds he inal s ages, he d ug con en app oached o low le els,
endo sing ha a subs an ial amoun o BDP would no be exhaled again
and could be e ec i ely deli e ed o he b onchioles and b onchi wi hin
he espi a o y sys em o a local ea men . Las ly, FPF measu ed he
ac ion o he emi ed dose ha consis ed o pa icles wi h an ae o-
dynamic size below 5
μ
m. The high FPF alue o he algina e ae ogels
(ca. 80 %) indica ed hei sui abili y o localized ea men , as i acil-
i a es a ge ed d ug deli e y o he speci ic egions o in e es . The
indings sugges ed supe io pe o mance o inhaled pa icles u ilizing
algina e ae ogels in compa ison o comme cial p oduc s con aining
BDP, like Fos e ® NEXThale ®, which exhibi ed an FPF o less han 70 %
[55],[56]. O e all, he simila in i o ae odynamic p ope ies o inhaled
o mula ions ob ained om di e en imp egna ion con ac imes indi-
ca ed ha he con ac ime in scCO
2
imp egna ion p ocess did no cause
de imen al e ec s on he ae odynamic pe o mance o he o mula ion
con aining BDP.
3.6. Ex i o pe meabili y s udies
Once he capabili y o he ae ogels o access o lung issue was
demons a ed, ex i o pe meabili y analysis was ca ied ou using
po cine b onchial issue o measu e he capaci y o he d ug o be
eleased om he ca ie and i s pene a ion in o he b onchial issue
(Fig. 8). Po cine lungs we e used in his s udy due o hei simila i y wi h
human lungs in e ms o size and ana omical ea u es [57]. Addi ionally,
po cine lungs exhibi compa able IgE le els, smoo h muscle s uc u e,
and e ec i e yp ase inhibi o s, which a e c ucial ac o s in he s udy o
espi a o y alle gies and as hma.
Acco ding o he indings in Fig. 8a, he p esence o BDP in he e-
cep o chambe indica ed ha BDP was no only eleased om he
inhaled o mula ion bu also pe mea ed h ough he b onchial issue.
The e was no s a is ically signi ican di e ence be ween he amoun o
BDP pe mea ed be ween 1 and 2 h in he ecep o chambe . The
maximum amoun o BDP pe mea ed a he ex i o expe imen was ca.
4µg/cm
2
wi h he a ailable pe mea ion a ea in he F anz cells being
0.785 cm
2
, which co esponded o 3.2 µg o BDP and 0.4 w .% o o al
BDP amoun . This BDP amoun ep esen s only he d ug ha passed
h ough he issues and en e ed o he ecep o chambe , bu he o al
BDP eleased include wha emained in he dono chambe and wha
was e ained wi hin he issue.
The measu ed BDP amoun in he ex i o es was no ably lowe han
he in i o elease (22 µg). This di e ence was ela ed o he smalle
olume o he elease medium in he ecep o chambe in F anz cells o
he ex i o es and he educed pe mea ion a ea o he issue in com-
pa ison o he dialysis bag. Mo eo e , he d ug pe mea ion su ace a ea
o he dialysis bags and o he F anz cells a e 32 cm
2
and 0.785 cm
2
,
espec i ely, which a e much lowe han he o al ai way su ace a ea in
human lungs, spanning om he achea o he b onchioles and al eoli,
wi h alues o app oxima ely 0.24 m
2
and 70 m
2
, espec i ely [58,59].
Highe o al d ug amoun s would be abso bed unde he a ailable su -
ace a ea in eal body condi ions o each he apeu ic doses.
A e 3 h, he quan i ica ion o BDP h ough he HPLC me hod was
no easible, p obably due o: (1) he dilu ion e ec in he ecep o
chambe a e sampling and eplenishmen wi h esh medium, (2) he
incomple e elease o BDP om hei ca ie s and limi ed solubili y o
BDP in he elease medium o PBS:me hanol 80:20 / , (3) he accu-
mula ion o BDP inside he issues o (4) he ins abili y o BDP inside he
b onchial issue a e 2 h. Due o he p esence o es e ases in he lung,
he hyd olysis o BDP p oduces h ee dis inc me aboli es: he ac i e
compound beclome hasone-17-monop opiona e (BMP) and wo inac i e
compounds wi h low binding a ini y o he glucoco icoid ecep o :
beclome hasone-21-monop opiona e (21-BMP), and beclome hasone
(BOH) [11,56]. BDP was epo ed o be ans o med in o inac i e me-
aboli es upon incuba ion wi h lung issue, wi h BMP being he p e-
dominan (a e 2 and 6 h), while BOH was he p ima y me aboli e (a e
24 h).
The p esence o BDP in he b onchial issue was e i ied h ough IR-
Raman spec oscopy (Fig. 8b). A compa ison was made be ween he
b onchial issue a e 1 and 3 h o incuba ion in he F anz cell con aining
he inhaled o mula ion in con as o he con ol b onchial issue. A e
1 h, he ela i e a io ob ained om BDP-loaded algina e ae ogels o he
basemen memb ane o he b onchial issue was highe compa ed o he
non- ea ed b onchial issue. These indings sugges ed ha BDP con-
ained in he inhaled o mula ion was eleased om he algina e ca ie s
and success ully pene a ed he b onchial issue a e 1 h. The e was no
s a is ically signi ican di e ence be ween he signals eco ded o
Table 3
In i o ae odynamic p ope ies ob ained om he in i o deposi ion es s o he
BDP-loaded algina e ae ogels p epa ed by scCO
2
imp egna ion using ace one as
co-sol en a di e en con ac imes.
Imp egna ion ime (h) MMAD (µm) EF (%) FPF (%)
1 1.2 ±0.2 86.8 ±5.4 78 ±3.5
6 1.1 ±0.1 89.6 ±1.0 80 ±1.7
9 1.1 ±0.1 89.6 ±1.1 80 ±1.6
24 1.1 ±0.1 87.3 ±2.9 80 ±3.5
Fig. 8. Ex i o BDP pe mea ion s udies o d ug-loaded ae ogel pa icles: (a)
Cumula i e quan i ies o BDP ha pe mea ed ac oss po cine b onchial issue
a e 1 and 2 h (n =3; mean alues ±s anda d de ia ions). (b) Ra io o Raman
peak in ensi ies o BDP in he basemen memb ane o he issue, ha indica ed
he p esence o BDP. G ay colo was employed o ep esen blank issues, ligh
g een and da k g een was employed o ep esen issues con aining inhaled
o mula ion a e 1 and 3 h con ac , espec i ely.
T. Duong e al.
