molecules
A icle
P epa a ion o Hyd ochlo o hiazide Nanopa icles o
Solubili y Enhancemen †
Eliska Vaculiko a 1, Ane a Ce niko a 2, Daniela Placha 1,3,*, Ma in Pisa cik 4,
Pa lina Peike o a 1,3, Ka e ina Dedko a 1,5, Fe dinand De insky 4and Jose Jampilek 6,*
1Nano echnology Cen e, VSB—Technical Uni e si y o Os a a, 17. lis opadu 15/2172,
708 33 Os a a, Czech Republic; [email p o ec ed] (E.V.); [email p o ec ed] (P.P.);
[email p o ec ed] (K.D.)
2
Depa men o Chemical D ugs, Facul y o Pha macy, Uni e si y o Ve e ina y and Pha maceu ical Sciences,
Palackeho 1/3, 612 42 B no, Czech Republic; [email p o ec ed]
3IT4 Inno a ions Cen um Excellence, VSB—Technical Uni e si y o Os a a, 17. lis opadu 15/2172,
708 33 Os a a, Czech Republic
4Depa men o Chemical Theo y o D ugs, Facul y o Pha macy, Comenius Uni e si y, Kalinciako a 8,
832 32 B a isla a, Slo akia; [email p o ec ed] (M.P.); [email p o ec ed] (F.D.)
5Regional Ma e ials Science and Technology Cen e, VSB—Technical Uni e si y o Os a a,
17. lis opadu 15/2172, 708 33 Os a a, Czech Republic
6Depa men o Pha maceu ical Chemis y, Facul y o Pha macy, Comenius Uni e si y, Odboja o 10,
832 32 B a isla a, Slo akia
*Co espondence: [email p o ec ed] (D.P.); [email p o ec ed] (J.J.);
Tel.: +420-596-991-554 (D.P.); +421-250-117-227 (J.J.)
†
P elimina y Resul s We e P esen ed a The Nine een h Elec onic Con e ence on Syn he ic O ganic Chemis y
(ECSOC-19, h p://sci o um.ne /con e ence/80/pape /3080), 1–30 No embe 2015 (Pape b007).
Academic Edi o s: Eca e ina And onescu and Alexand u Mihai G umezescu
Recei ed: 1 June 2016; Accep ed: 29 July 2016; Published: 2 Augus 2016
Abs ac :
Nanopa icles can be conside ed as a use ul ool o imp o ing p ope ies o poo ly soluble
ac i e ing edien s. Hyd ochlo o hiazide (Class IV o he Biopha maceu ical Classi ica ion Sys em)
was chosen as a model compound. An isol en p ecipi a ion-sol en e apo a ion and emulsion
sol en e apo a ion me hods we e used o p epa a ion o 18 samples con aining hyd ochlo o hiazide
nanopa icles. Wa e solu ions o su ac an s sodium dodecyl sul a e, Tween 80 and ca boxyme hyl
dex an we e used in mass concen a ions o 1%, 3% and 5%. Ace one and dichlo ome hane we e
used as sol en s o he model compound. The pa icle size o he p epa ed samples was measu ed
by dynamic ligh sca e ing. The selec ed sample o hyd ochlo o hiazide nanopa icles s abilized
wi h ca boxyme hyl dex an sodium sal wi h pa icle size 2.6 nm was cha ac e ized addi ionally by
Fou ie ans o m mid-in a ed spec oscopy and scanning elec on mic oscopy. I was ound ha
he solubili y o his sample was 6.5- old highe han ha o bulk hyd ochlo o hiazide.
Keywo ds:
hyd ochlo o hiazide; nanopa icles; dynamic ligh sca e ing; in a ed spec oscopy;
scanning elec on mic oscopy; solubili y
1. In oduc ion
Hyd ochlo o hiazide (HCTZ, Figu e 1), 6-chlo o-3,4-dihyd o-2H-1,2,4-benzo hiadiazine-
7-sul onamide 1,1-dioxide, is a hiazide diu e ic ha is equen ly used in an ihype ensi e he apy
in combina ion wi h an ihype ensi e agen s. HCTZ a ec s he dis al enal ubula mechanism o
elec oly e eabso p ion: i inc eases exc e ion o sodium and chlo ide ions [
1
]. Due o his inc eased
exc e ion, i inc eases diu esis. HCTZ is no a i s choice d ug o hype ension ea men o pa ien s
wi h diabe es melli us because o me abolic side e ec s. HCTZ can cause hype glycaemia; i can
Molecules 2016,21, 1005; doi:10.3390/molecules21081005 www.mdpi.com/jou nal/molecules
Molecules 2016,21, 1005 2 o 8
wo sen glucose ole ance and cause hype choles e olemia [
2
]. I is ques ionable whe he HCTZ
nanopa icles (NPs) would ha e less ad e se e ec s in a ange o me abolic side e ec s. Me abolic side
e ec s o HCTZ a e dose-dependen [
3
–
7
]. I can be s a ed ha he bes balance be ween e ec i eness
and side e ec s is ob ained wi h much smalle doses [
8
]. This ac was connec ed wi h hypokalemia
associa ed wi h high adminis a ed doses o hiazides (up o 150 mg/day o HCTZ) [
8
]. D ug NPs
ha e been p epa ed o p o ide a possibili y o ea men wi h a lowe , bu s ill he apeu ic dose [
9
].
HCTZ belongs o Class IV o he Biopha maceu ical Classi ica ion Sys em (BCS) [
10
]. D ugs o he
men ioned class a e cha ac e ized by poo wa e solubili y and low pe meabili y; his means hey a e
p oblema ic om he poin o iew o o al bioa ailabili y [
11
]. Some nanonized d ug subs ances ha e
been used in human ea men . Doses o nanod ugs can be lowe han doses o bulk d ugs, which
esul s in a lowe pill bu den [6,12].
Molecules 2016, 21, 1005 2 o 8
nanopa icles (NPs) would ha e less ad e se e ec s in a ange o me abolic side e ec s. Me abolic side
e ec s o HCTZ a e dose-dependen [3–7]. I can be s a ed ha he bes balance be ween e ec i eness
and side e ec s is ob ained wi h much smalle doses [8]. This ac was connec ed wi h hypokalemia
associa ed wi h high adminis a ed doses o hiazides (up o 150 mg/day o HCTZ) [8]. D ug NPs ha e
been p epa ed o p o ide a possibili y o ea men wi h a lowe , bu s ill he apeu ic dose [9]. HCTZ
belongs o Class IV o he Biopha maceu ical Classi ica ion Sys em (BCS) [10]. D ugs o he men ioned
class a e cha ac e ized by poo wa e solubili y and low pe meabili y; his means hey a e p oblema ic
om he poin o iew o o al bioa ailabili y [11]. Some nanonized d ug subs ances ha e been used
in human ea men . Doses o nanod ugs can be lowe han doses o bulk d ugs, which esul s in a
lowe pill bu den [6,12].
Figu e 1. S uc u e o hyd ochlo o hiazide.
The aim o his s udy was he p epa a ion o s abilized HCTZ NPs ia an isol en p ecipi a ion
wi h he ollowing solubili y expe imen . I was supposed ha HCTZ NPs would ha e imp o ed
bioa ailabili y (some HCTZ nano o mula ions we e published ecen ly [13–16]). The e a e wo main
app oaches o NPs p epa a ion: bo om-up and op-down. Top-down echniques a e based on milling
o high-p essu e homogeniza ion. The bo om-up app oach is mainly based on p ecipi a ion [12,17,18].
This app oach was used in his s udy.
Pola and nonpola sol en s we e used in his in es iga ion; he e o e, he exac p inciple o he
applied sol en e apo a ion me hod is dependen on he wa e -based sys em, including o no an
aqueous miscible o ganic sol en . Pola ace one and nonpola dichlo ome hane we e chosen as he
mos sui able sol en s o easy dissolu ion o HCTZ; so wo di e en possible mechanisms we e used
o he NP syn hesis. When HCTZ is dissol ed in ace one and hen mixed wi h wa e con aining a
s abilize , NPs a e o med spon aneously and immedia ely upon mixing. This me hod can be called
an isol en p ecipi a ion-sol en e apo a ion, and he p ocedu e is in p inciple simila o he
e apo a i e p ecipi a ion in o aqueous solu ion [19,20] and he liquid an isol en p ecipi a ion [21].
When HCTZ is dissol ed in dichlo ome hane and hen mixed wi h wa e con aining s abilize s, an
emulsion (o/w ype) is o med; HCTZ is clus e ed by he excipien , which esul s in he encapsula ion
o HCTZ in o nano- esicula. This combina ion o emulsi ica ion and sol en e apo a ion in NP
syn hesis is called emulsion sol en e apo a ion [22,23].
2. Resul s and Discussion
Based on he pilo sc eening [24,25], excipien s such as sodium dodecyl sul a e (Se ies 1, 2), Tween
80 (Se ies 3, 4) and ca boxyme hyl dex an sodium sal (Se ies 5, 6) we e chosen om he g oup o
su ac an s. Th ee wa e solu ions o 1%, 3% and 5% mass concen a ion we e p epa ed. HCTZ was
sol ed in dichlo ome hane (Se ies 1, 3, 5) as a non-pola sol en , and ace one (Se ies 2, 4, 6) as a pola
sol en . D ug solu ion was added o he solu ion o excipien unde con inuous s i ing. O ganic
sol en was e apo a ed in an ul asonic ba h ha was used as a sou ce o complemen a y ene gy o
NPs p epa a ion. The combina ion o all excipien s wi h HCTZ p o ided 18 samples, see Table 1. All
p epa ed samples we e measu ed by dynamic ligh sca e ing [26], i.e., he pa icle size and alues o
polydispe si y index we e de e mined (see Table 1). The sui abili y o he used su ac an and he
o ganic sol en was analyzed.
Figu e 1. S uc u e o hyd ochlo o hiazide.
The aim o his s udy was he p epa a ion o s abilized HCTZ NPs ia an isol en p ecipi a ion
wi h he ollowing solubili y expe imen . I was supposed ha HCTZ NPs would ha e imp o ed
bioa ailabili y (some HCTZ nano o mula ions we e published ecen ly [
13
–
16
]). The e a e wo main
app oaches o NPs p epa a ion: bo om-up and op-down. Top-down echniques a e based on milling
o high-p essu e homogeniza ion. The bo om-up app oach is mainly based on p ecipi a ion [
12
,
17
,
18
].
This app oach was used in his s udy.
Pola and nonpola sol en s we e used in his in es iga ion; he e o e, he exac p inciple o
he applied sol en e apo a ion me hod is dependen on he wa e -based sys em, including o no
an aqueous miscible o ganic sol en . Pola ace one and nonpola dichlo ome hane we e chosen
as he mos sui able sol en s o easy dissolu ion o HCTZ; so wo di e en possible mechanisms
we e used o he NP syn hesis. When HCTZ is dissol ed in ace one and hen mixed wi h wa e
con aining a s abilize , NPs a e o med spon aneously and immedia ely upon mixing. This me hod
can be called an isol en p ecipi a ion-sol en e apo a ion, and he p ocedu e is in p inciple simila o
he e apo a i e p ecipi a ion in o aqueous solu ion [
19
,
20
] and he liquid an isol en p ecipi a ion [
21
].
When HCTZ is dissol ed in dichlo ome hane and hen mixed wi h wa e con aining s abilize s,
an emulsion (o/w ype) is o med; HCTZ is clus e ed by he excipien , which esul s in he
encapsula ion o HCTZ in o nano- esicula. This combina ion o emulsi ica ion and sol en e apo a ion
in NP syn hesis is called emulsion sol en e apo a ion [22,23].
2. Resul s and Discussion
Based on he pilo sc eening [
24
,
25
], excipien s such as sodium dodecyl sul a e (Se ies 1, 2),
Tween 80 (Se ies 3, 4) and ca boxyme hyl dex an sodium sal (Se ies 5, 6) we e chosen om he
g oup o su ac an s. Th ee wa e solu ions o 1%, 3% and 5% mass concen a ion we e p epa ed.
HCTZ was sol ed in dichlo ome hane (Se ies 1, 3, 5) as a non-pola sol en , and ace one (Se ies 2, 4, 6)
as a pola sol en . D ug solu ion was added o he solu ion o excipien unde con inuous s i ing.
O ganic sol en was e apo a ed in an ul asonic ba h ha was used as a sou ce o complemen a y
ene gy o NPs p epa a ion. The combina ion o all excipien s wi h HCTZ p o ided 18 samples, see
Table 1. All p epa ed samples we e measu ed by dynamic ligh sca e ing [
26
], i.e., he pa icle size
and alues o polydispe si y index we e de e mined (see Table 1). The sui abili y o he used su ac an
and he o ganic sol en was analyzed.
Molecules 2016,21, 1005 3 o 8
Table 1.
Composi ion o samples (dichlo ome hane Se ies 1, 3, 5; ace one Se ies 2, 4, 6), concen a ion
(%) o indi idual excipien s in wa e samples ela i e o hyd ochlo o hiazide, pa icle size (nm)
and polydispe si y index (PDI) o hyd ochlo o hiazide samples exp essed as mean
±
SD (n= 5
independen measu emen s). (SDS = sodium dodecyl sul a e, TW = Tween 80, DCM = dichlo ome hane,
CMD = ca boxyme hyl dex an sodium sal
, AC = ace one, n.d. = immeasu able due o c ys alliza ion)
Sample Excipien /Concen a ion (%) Pa icle Size (nm) PDI
1a SDS/DCM/1 24.5 ±3.6 0.654 ±0.073
1b SDS/DCM/3 102.2 ±17.6 0.384 ±0.021
1c SDS/DCM/5 21.0 ±1.2 0.356 ±0.011
2a SDS/AC/1 9.6 ±0.4 0.302 ±0.014
2b SDS/AC/3 4.2 ±0.6 0.300 ±0.027
2c SDS/AC/5 7.6 ±1.2 0.271 ±0.012
3a TW/DCM/1 10.5 ±0.5 0.177 ±0.020
3b TW/DCM/3 13.6 ±0.7 0.189 ±0.008
3c TW/DCM/5 n.d. n.d.
4a TW/AC/1 10.2 ±0.1 0.154 ±0.007
4b TW/AC/3 9.3 ±0.3 0.128 ±0.057
4c TW/AC/5 9.9 ±0.1 0.157 ±0.010
5a CMD/DCM/1 107.0 ±51.4 0.396 ±0.025
5b CMD/DCM/3 221.5 ±28.5 0.470 ±0.034
5c CMD/DCM/5 2.6 ±0.2 0.212 ±0.140
6a CMD/AC/1 493.2 ±155.9 0.368 ±0.075
6b CMD/AC/3 510.7 ±33.7 0.396 ±0.055
6c CMD/AC/5 2.8 ±1.2 0.312 ±0.040
The in es iga ed pa icles showed good pa icle size s abili y h oughou he ligh sca e ing
measu emen s, excep o sample
3c
ha could no be measu ed due o c ys alliza ion o HCTZ.
In he cou se o he measu emen s, no signi ican de ia ions om he mean alues o pa icle size,
which could be a esul o possible sample ageing, we e obse ed. In addi ion, a egula isual
check o he samples showed no changes in he sample s uc u e, which was con i med by he
ep oducible da a ob ained by he ligh sca e ing me hod. NPs o size >100 nm we e p epa ed in
he case o sodium dodecyl sul a e and ca boxyme hyl dex an sodium sal in combina ion wi h
dichlo ome hane (
samples 1b,5a,5b
) and ca boxyme hyl dex an sodium sal in combina ion wi h
ace one (
samples 6a,6b
). On he o he hand, ca boxyme hyl dex an sodium sal in 5% concen a ion in
combina ion wi h dichlo ome hane o ace one o med samples wi h he smalles pa icle sizes (2.6 nm
o sample
5c
and 2.8 nm o sample
6c
). Sodium dodecyl sul a e in combina ion wi h dichlo ome hane
p o ided pa icle size nea 20 nm (samples 1a and 1c). O he p epa ed samples con ained NPs in he
size ange om 4.2 o 13.6 nm, mos ly nea 10 nm.
The dispe si y is a measu e/deg ee o he homogenei y/he e ogenei y o sizes o pa icles in a
mix u e/sys em. The uni o mi y o dispe sed sys ems is exp essed as polydispe si y index (PDI), see
Table 1. Low PDI alues demons a e na ow size dis ibu ion and he uni o mi y o samples con a y
o PDI
≈
1, which indica es ha samples ha ha e a e y b oad size dis ibu ion, may con ain la ge
pa icles o agg ega es and a e no sui able o measu emen s [
27
,
28
]. In he p epa ed NPs o HCTZ,
PDI alues anged om 0.128 o 0.396, when he samples
1a
and
5b
(wi h PDI alues 0.654 and 0.470)
we e elimina ed.
Sample
5c
wi h he smalles pa icle size and low PDI, whe e HCZT was s abilized wi h 5%
ca boxyme hyl dex an sodium sal , was chosen o addi ional cha ac e iza ion and solubili y es .
Figu e 2illus a es he Fou ie ans o m mid in a ed (FT-MIR) spec a o he s a ing bulk HCTZ,
ca boxyme hyl dex an sodium sal and HCTZ NPs s abilized wi h he excipien and hus e i ies
he composi ion o sample
5c
. Scanning elec on mic oscopy (SEM) shows bulk HCZT, see Figu e 3A,
while Figu e 3B ep esen s an SEM image showing he su ace s uc u e and he mo phology o
sample 5c. The su ace and s uc u e o nanopa icles we e uni o m wi h no oughness.
Molecules 2016,21, 1005 4 o 8
Molecules 2016, 21, 1005 4 o 8
Figu e 2. FT-MIR spec a o hyd ochlo o hiazide s abilized wi h 5% ca boxyme hyl dex an sodium
sal (sample 5c, A), bulk hyd ochlo o hiazide (B) and ca boxyme hyl dex an sodium sal (C).
Figu e 3. SEM images o bulk hyd ochlo o hiazide a magni ica ion 1600× (A) and hyd ochlo o hiazide
s abilized wi h 5% ca boxyme hyl dex an sodium sal (sample 5c) a magni ica ion 100× (B).
The solubili y o bulk HCTZ (as a con ol sample) and HCTZ NPs (sample 5c) was es ed; he
esul s o his in es iga ion a e p esen ed in Table 2, whe e he concen a ions o HCTZ NPs dissol ed
in wa e wi h pH 6 we e de e mined by HPLC in compa ison wi h bulk HCTZ. The dissol ed amoun
o bulk HCTZ was 16.6 µg/mL, while ha o HCTZ NPs was 107.9 µg/mL, i.e., he applica ion o NPs
led o a signi ican , 6.5- old inc ease o he solubili y o HCTZ.
Table
2
. Resul s o dissolu ion es .
Sample HPLC De e mined Conc. (µg/mL) R
bulk HCTZ (con ol sample) 16.6 –
HCTZ NPs (sample 5c) 107.9 6.5
Wa e soluble, biodeg adable, biocompa ible and non oxic ca boxyme hyl dex an has been
widely used as a s abilize and a nanoca ie o d ug deli e y [29–36]. Based on he expe imen s, i
can be s a ed ha ca boxyme hyl dex an sodium sal can be conside ed as a e sa ile s abilize o a
a ie y o d ugs om classes II–IV o he Biopha maceu ical Classi ica ion Sys em, o example,
lipophilic s e oids [24], highly hyd ophilic ised ona e sodium [18], basic cime idine [37], acid
Figu e 2.
FT-MIR spec a o hyd ochlo o hiazide s abilized wi h 5% ca boxyme hyl dex an sodium
sal (sample 5c,A); bulk hyd ochlo o hiazide (B) and ca boxyme hyl dex an sodium sal (C).
Molecules 2016, 21, 1005 4 o 8
Figu e 2. FT-MIR spec a o hyd ochlo o hiazide s abilized wi h 5% ca boxyme hyl dex an sodium
sal (sample 5c, A), bulk hyd ochlo o hiazide (B) and ca boxyme hyl dex an sodium sal (C).
Figu e 3. SEM images o bulk hyd ochlo o hiazide a magni ica ion 1600× (A) and hyd ochlo o hiazide
s abilized wi h 5% ca boxyme hyl dex an sodium sal (sample 5c) a magni ica ion 100× (B).
The solubili y o bulk HCTZ (as a con ol sample) and HCTZ NPs (sample 5c) was es ed; he
esul s o his in es iga ion a e p esen ed in Table 2, whe e he concen a ions o HCTZ NPs dissol ed
in wa e wi h pH 6 we e de e mined by HPLC in compa ison wi h bulk HCTZ. The dissol ed amoun
o bulk HCTZ was 16.6 µg/mL, while ha o HCTZ NPs was 107.9 µg/mL, i.e., he applica ion o NPs
led o a signi ican , 6.5- old inc ease o he solubili y o HCTZ.
Table
2
. Resul s o dissolu ion es .
Sample HPLC De e mined Conc. (µg/mL) R
bulk HCTZ (con ol sample) 16.6 –
HCTZ NPs (sample 5c) 107.9 6.5
Wa e soluble, biodeg adable, biocompa ible and non oxic ca boxyme hyl dex an has been
widely used as a s abilize and a nanoca ie o d ug deli e y [29–36]. Based on he expe imen s, i
can be s a ed ha ca boxyme hyl dex an sodium sal can be conside ed as a e sa ile s abilize o a
a ie y o d ugs om classes II–IV o he Biopha maceu ical Classi ica ion Sys em, o example,
lipophilic s e oids [24], highly hyd ophilic ised ona e sodium [18], basic cime idine [37], acid
Figu e 3.
SEM images o bulk hyd ochlo o hiazide a magni ica ion 1600
×
(
A
) and hyd ochlo o hiazide
s abilized wi h 5% ca boxyme hyl dex an sodium sal (sample 5c) a magni ica ion 100×(B).
The solubili y o bulk HCTZ (as a con ol sample) and HCTZ NPs (sample
5c
) was es ed;
he esul s o his in es iga ion a e p esen ed in Table 2, whe e he concen a ions o HCTZ NPs
dissol ed in wa e wi h pH 6 we e de e mined by HPLC in compa ison wi h bulk HCTZ. The dissol ed
amoun o bulk HCTZ was 16.6
µ
g/mL, while ha o HCTZ NPs was 107.9
µ
g/mL, i.e., he applica ion
o NPs led o a signi ican , 6.5- old inc ease o he solubili y o HCTZ.
Table 2. Resul s o dissolu ion es .
Sample HPLC De e mined Conc. (µg/mL) R
bulk HCTZ (con ol sample) 16.6 –
HCTZ NPs (sample 5c) 107.9 6.5
Wa e soluble, biodeg adable, biocompa ible and non oxic ca boxyme hyl dex an has been
widely used as a s abilize and a nanoca ie o d ug deli e y [
29
–
36
]. Based on he expe imen s,
i can be s a ed ha ca boxyme hyl dex an sodium sal can be conside ed as a e sa ile s abilize
o a a ie y o d ugs om classes II–IV o he Biopha maceu ical Classi ica ion Sys em, o example,
Molecules 2016,21, 1005 5 o 8
lipophilic s e oids [
24
], highly hyd ophilic ised ona e sodium [
18
], basic cime idine [
37
], acid
candesa an cilexe il and calcium sal o a o as a in [
25
]. In gene al, i p o ided s able NP samples
wi h na ow pa icle size dis ibu ion in concen a ions o 3% and 5% in dichlo ome hane a he
han in ace one [
17
,
24
,
25
,
37
]. This may be connec ed wi h p ope ies o non-pola dichlo ome hane
(wi h dipole momen 1.60 D in con as o pola ap o ic ace one wi h dipole momen 2.88 D [
38
]) and
hyd ophilic cha ged polyme ic ca boxyme hyl dex an sodium sal and hei (non)bonding in e ac ions
wi h used molecules o d ugs [
30
–
34
]. Despi e i s oxici y, dichlo ome hane has been one o he mos
widely used sol en s o d ugs o in insic emulsi ied phase [
29
,
39
] dispe sed in aqueous solu ion o
ca boxyme hyl dex an sodium sal . The di e en mechanisms o gene a ion o NPs in compa ison wi h
he an isol en p ecipi a ion p ocess in he p esence o pola ace one seems o be ano he impo an
pa ame e a ec ing he pa icle size and he homogenei y o he samples. Ne e heless, i can be
s a ed ha he “ne ” o med by 5% concen a ion o ca boxyme hyl dex an sodium sal p e en s
agg ega ion/agglome a ion o d ug subs ance pa icles.
3. Expe imen al Sec ion
3.1. Gene al P ocedu e o P epa a ion o Nanopa icles
The model compound hyd ochlo o hiazide (HCTZ) and he excipien s sodium dodecyl
sul a e, Tween 80 and ca boxyme hyl dex an sodium sal we e pu chased om Sigma-Ald ich
(
S . Louis, MO, USA
). All compounds we e o analy ical g ade. H
2
O-HPLC-Mili-Q G ade was used as
a sol en o he excipien s. Each excipien (0.1, 0.3 o 0.5 g) was dissol ed in wa e (10 mL), and h ee
solu ions wi h mass concen a ions 1%, 3% and 5% we e p epa ed. HCTZ (0.1 g) was dissol ed
in dichlo ome hane o ace one (10 mL), i.e., 1% solu ions we e p epa ed. The solu ion o HCTZ in
dichlo ome hane/ace one was slowly d opped (2 mL/min) o he aqueous solu ions o excipien s, and
he solu ions we e s i ed a 600 pm o 15 min a 25
◦
C, a e which he mix u es we e ans e ed o
he ul asonic ba h in he ume chambe , whe e hey we e mixed again o 20 min o homogeniza ion
o he samples. Finally, he sol en was e apo a ed.
3.2. Pa icle Size Measu emen
The pa icle size was de e mined using a B ookha en dynamic ligh sca e ing sys em BI 9000
(B ookha en Ins umen s Co po a ion, Hol s ille, NY, USA) wi h a goniome e SM-200 and an a gon
gas lase (Lexel 95, wa eleng h 514.5 nm). Sca e ed in ensi y was egis e ed a sca e ing angle 90
◦
and empe a u e 25
◦
C. All he samples we e dispe sed by sonica ion and addi ionally il e ed di ec ly
be o e he measu emen h ough sy inge il e s wi h 0.45
µ
m po e size o emo e mechanical impu i ies.
Fi e independen eco dings o he au oco ela ion unc ion we e done o each in es iga ed excipien
concen a ion. The pa icle size was calcula ed om he ansla ional di usion coe icien using he
S okes-Eins ein o mula. The ansla ional di usion coe icien was ob ained based on he cumulan
expansion o he au oco ela ion unc ion up o he second cumulan . The p esen ed pa icle sizes
a e epo ed as he mean alues aken o he se o i e independen measu emen s. The esul s a e
summa ized in Table 1.
3.3. FT-IR Analysis
Fou ie ans o m mid in a ed (FT-MIR) spec a (Figu e 2) we e measu ed o con i ma ion o he
sample composi ion. The a enua ed o al e lec ance (ATR) echnique was used wi h a diamond c ys al.
FT-MIR spec a we e eco ded by a Nicole 7000 FT-IR spec ome e (The mo Scien i ic, Wes Palm
Beach, FL, USA) in he ange 4000–400 cm−1wi h he esolu ion o 4 cm−1and 32 scans.
3.4. Scanning Elec on Mic oscopy
The mo phology o he sample was s udied using scanning elec on mic oscopy (SEM).
The sample was a ached o a mic oscopic glass, d ied a 25
◦
C and hen spu e -coa ed wi h hin ilm

Molecules 2016,21, 1005 6 o 8
o gold. An elec on mic oscope Quan a 450FEG (FEI, Hillsbo o, OR, USA) was used o imaging o
he sample a he ollowing wo king condi ions: accele a ing ol age 10.0 kV, wo king dis ance 8.0 mm
and p obe cu en 100 mA. S uc u es o he samples a e illus a ed in Figu e 3.
3.5. Solubili y Tes
Two beake s illed wi h 10 mL o phospha e bu e (pH 6) we e placed on magne ic s i e s and
empe ed o 36
◦
C [
40
–
42
]. D y powde s o bulk HCTZ o sample
5c
we e sepa a ely added o beake s
wi h bu e unde con inuous s i ing (250 pm). The beake s we e co e ed by Pa a ilm
®
. When he
u bidi y occu ed, he con en o he beake was s i ed o addi ional 24 h. I he u bidi y did no
disappea , he samples we e cen i uged and analyzed. Analysis o samples was pe o med using
a high pe o mance liquid ch oma og aph Agilen 1200 Se ies (Agilen Technologies, San a Cla a,
CA, USA) equipped wi h a DAD, a qua e pump, and an au oma ic injec o . The HPLC sepa a ion
p ocess was moni o ed and e alua ed by he ChemS a ion so wa e. A ch oma og aphic column
Gemini 5
µ
m C6-Phenyl 110A, 4.6 mm
×
250 mm (Phenomenex
®
, To ance, CA, USA) was used.
Isoc a ic elu ion by a mix u e o MeOH p.a. (35%) and o mic bu e (pH 4.0; 65%) as a mobile phase
was used. The o al low o he column was 1.0 mL/min, injec ion 10
µ
L, column empe a u e 40
◦
C,
and sample empe a u e 22
◦
C. The de ec ion wa eleng h o 273 nm was chosen. The e en ion ime o
HCTZ was 4.19
±
0.05 min, he limi o de ec ion (LOD) was 0.01
µ
g/mL, and he limi o quan i ica ion
(LOQ) o HCTZ was 0.04 µg/mL. The esul s a e p esen ed in Table 2.
4. Conclusions
Eigh een samples o HCTZ NPs we e p epa ed by he an isol en p ecipi a ion-sol en
e apo a ion and he emulsion sol en e apo a ion echniques. Th ee di e en excipien s we e used
as s abilize s in 1%, 3%, and 5% aqueous mass concen a ion. Dynamic ligh sca e ing was used o
pa icle size analysis. The polydispe si y index (PDI) was de e mined. Low PDI alues o he samples
con i med na ow size dis ibu ion and he uni o mi y o he p epa ed sys ems. Sample
5c
o HCTZ
NPs s abilized wi h 5% concen a ion o ca boxyme hyl dex an sodium sal (pa icle size 2.3 nm,
PDI 0.212) was chosen as he sample wi h he smalles pa icle size o ollowing cha ac e iza ion and
analysis. SEM was used o su ace iden i ica ion, and FT-MIR spec oscopy was used o e i ica ion
o he sample composi ion. Finally, he solubili y es con i med he enhanced solubili y o HCTZ NPs
in compa ison wi h he solubili y o bulk HCTZ (6.5- old highe ).
Acknowledgmen s:
This s udy was suppo ed by he Minis y o Educa ion, You h and Spo s o he Czech
Republic om he Na ional P og amme o Sus ainabili y (NPU II) p ojec “IT4Inno a ions Excellence in Science
LQ1602“, by SP2016/85 p ojec , by IGA VFU B no 302/2015/FaF, VEGA 1/0298/16 and by Slo ak Resea ch and
De elopmen Agency G an No. APVV-0516-12.
Au ho Con ibu ions:
Eliska Vaculiko a—p epa a ion o nanopa icles, in e p e a ion o IR and SEM da a,
Ane a Ce niko a—HPLC analysis and solubili y es , Daniela Placha—in e p e a ion o IR and SEM da a, Pa lina
Peike o a—IR analysis, Ka e ina Dedko a—SEM analysis, Ma in Pisa cik, Fe dinand De insky—dynamic ligh
sca e ing expe imen s, Jose Jampilek—design o s udy, ela ionships be ween excipien s, w i ing o he pape .
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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2016 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 p://c ea i ecommons.o g/licenses/by/4.0/).