molecules
A icle
Modeling o he P oduc ion o Lipid Mic opa icles
Using PGSS®Technique
Cla a López-Iglesias 1,* , En ique a R. López 2, Jose a Fe nández 2, Ma iana Landin 1
and Ca los A. Ga cía-González 1,*
1Depa men o Pha macology, Pha macy and Pha maceu ical Technology, I+D Fa ma g oup (GI-1645),
Facul y o Pha macy, Ag upación Es a égica de Ma e iales (AeMAT) 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,
15782 San iago de Compos ela, Spain; [email p o ec ed]
2Labo a o io de P opiedades Te mo ísicas, G upo NaFoMa , Depa amen o de Física Aplicada,
Facul ad de F
í
sica, Ag upaci
ó
n Es a
é
gica de Ma e iales (AeMAT), Uni e sidade de San iago de Compos ela,
15782 San iago de Compos ela, Spain; en ique [email protected] (E.R.L.); jose a. e [email protected] (J.F.)
*Co espondence: [email p o ec ed] (C.L.-I.); [email p o ec ed] (C.A.G.-G.);
Tel.: +34-881-814-882 (C.L.-I. & C.A.G.-G.)
Academic Edi o : Ri a Co esi
Recei ed: 25 Sep embe 2020; Accep ed: 23 Oc obe 2020; Published: 24 Oc obe 2020
Abs ac :
Solid lipid mic opa icles (SLMPs) a e a ac i e ca ie s as deli e y sys ems as hey a e
s able, easy o manu ac u e and can p o ide con olled elease o bioac i e agen s and inc ease hei
e icacy and/o sa e y. Pa icles om Gas-Sa u a ed Solu ions (PGSS
®
) echnique is a sol en - ee
echnology o p oduce SLMPs, which in ol es he use o supe c i ical CO
2
(scCO
2
) a mild p essu es
and empe a u es o he mel ing o lipids and a omiza ion in o pa icles. The de e mina ion o he key
p ocessing a iables is c ucial in PGSS
®
echnique o ob ain eliable and ep oducible mic opa icles,
he e o e he modelling o SLMPs p oduc ion p ocess and a iables con ol a e o g ea in e es o
ob ain quali y he apeu ic sys ems. In his wo k, he mel ing poin dep ession o a comme cial lipid
(glyce yl monos ea a e, GMS) unde comp essed CO
2
was s udied using iew cell expe imen s.
Based on an uncons ained D-op imal design o h ee a iables (nozzle diame e , empe a u e and
p essu e), SLMPs we e p oduced using he PGSS
®
echnique. The yield o p oduc ion was egis e ed
and he pa icles cha ac e ized in e ms o pa icle size dis ibu ion. Va iable modeling was ca ied
ou using a i icial neu al ne wo ks and uzzy logic in eg a ed in o neu o uzzy so wa e. Modeling
esul s highligh he main e ec o empe a u e o une he mean diame e SLMPs, whe eas he
p essu e-nozzle diame e in e ac ion is he main esponsible in he SLMPs size dis ibu ion and in he
PGSS®p oduc ion yield.
Keywo ds: lipid mic opa icles; PGSS®; supe c i ical CO2; modeling; sol en - ee echnology
1. In oduc ion
Pa icula e sys ems like mic opa icles ha e a ac ed in e es in se e al biomedical, ood and
en i onmen al applica ions [
1
–
5
]. Namely, he encapsula ion o bioac i e agen s in hese ca ie s
imp o es hei e icacy and sa e y, since be e con ol o he dosage and elease a e p o ided [
6
,
7
].
Mic opa icles also enhance physicochemical s abili y, p o ec ing he ca go om en i onmen al and
physiological ac o s[
8
]. Thesizeo mic opa icles, be ween 0.1–100
µ
m[
9
], canhampe hei abso p ion
h ough biological memb anes, inc easing hei pe manence in he applica ion si e, hus p o iding
local and sus ained d ug elease and mi iga ing hei oxic e ec s [10].
Lipids a e ad an ageous ma ices o pa icula e d ug deli e y sys ems since hey a e physiological
compounds and he e o e well ole a ed by li ing sys ems [
11
,
12
]. Fo ins ance, a a ie y o lipids
Molecules 2020,25, 4927; doi:10.3390/molecules25214927 www.mdpi.com/jou nal/molecules
Molecules 2020,25, 4927 2 o 14
such as so bi an es e s, phospha idylcholine, and unsa u a ed polyglycolized glyce ides a e widely
used as su ac an s in lipid-based o mula ions [
13
]. Among lipid sys ems, solid lipid mic opa icles
(SLMPs) a e easy o p oduce on a la ge scale and s e ilize, exhibi ing be e s abili y p ope ies han
o he s, such as liposomes [
14
]. Se e al SLMP-based o mula ions ha e been de eloped as d ug deli e y
sys ems o o al, pa en e al, pulmona y and opical applica ions [14,15].
Sol en - ees a egies a eespecially a ac i e o hemanu ac u ing o SLMPs om hep ocessing,
en i onmen al and economical poin s o iew. Namely, supe c i ical CO
2
(scCO
2
) echnology has
been highligh ed as a p ocessing ool o en i onmen ally iendly, sa e and cos -e icien echniques
a mild condi ions—p essu e (P) >73.8 ba and empe a u e (T) >31.1
◦
C) [
16
]. P ocesses based on
supe c i ical luid echnology ( oaming, s e iliza ion) usually a oid o a leas mi iga e he use o o ganic
sol en s hus educing hei ca bon oo p in . The PGSS
®
(Pa icles om Gas-Sa u a ed Solu ions)
echnique is based on he use o comp essed CO
2
o scCO
2
o he p oduc ion o mic opa icles in an
a omiza ion-wise p ocess [
17
–
19
]. PGSS
®
p ocess comp ises wo main s eps: (i) CO
2
so p ion in he
polyme , and (ii) polyme expansion and pa icle o ma ion. In he i s s ep, high amoun s (5–50 w .%)
o CO
2
dissol e in a mol en subs ance a a mode a e p essu e in an ex en depending on he soaking
ime and CO
2
a ini y o he polyme [
20
]. Then a apid expansion o a mosphe ic p essu e o he mel
h ough a nozzle causes an in ense cooling e ec and CO
2
supe sa u a ion wi hin he mel , esul ing
in he p ecipi a ion o solid pa icles [
21
]. scCO
2
used in he PGSS
®
echnique di e s om o he
comp essed luids (e.g., comp essed ai ) used in con en ional a omiza ion p ocesses (sp ay d ying) in
hei chemical in e ac ion wi h he p ocessed polyme s a a molecula le el, as scCO
2
can dec ease he
mel ing empe a u e o he polyme hus con ibu ing o cos s op imiza ion and ene gy consump ion
sa ings [
22
]. PGSS
®
is an adequa e echnique o he p ocessing o polyme ic pa icles inco po a ing
he molabile compounds, al hough i s use is limi ed o polyme ma ices wi h ela i ely low mel ing
empe a u es and wi h an a ini y o CO
2
o he polyme [
23
]. Compa ed o o he p ocesses o pa icle
p oduc ion in ol ing he use o scCO
2
, such as he gas an isol en (GAS), supe c i ical an isol en
(SAS) and supe c i ical luid ex ac ion o an emulsion (SFEE) echniques, he PGSS® echnique does
no use any o ganic sol en s [
16
,
24
]. Mo eo e , he subs ance o be mic onized does no equi e o be
soluble in CO
2
unlike in he apid expansion o supe c i ical luids (RESS) p ocess [
25
,
26
]. O e all,
PGSS
®
eme ges as an appealing and ad an ageous echnique o he p ocessing o SLMPs a educed
mel ing empe a u es and in he absence o o ganic sol en s.
The mo phology and size o he SLMPs p oduced by he PGSS
®
p ocess a e mainly in luenced by
he o mula ion (chemical composi ion and heology o he compounds o be p ecipi a ed), he echnical
de ails o he equipmen used ( olume o he sa u a o , p ecipi a o and collec o , diame e o he nozzle
and leng h o he ubing) and he ope a ing condi ions (p essu e, empe a u e, soaking ime) [
27
,
28
].
The PGSS
®
p ocessing a iables a e nume ous, making i di icul o elucida e hei in luence on he
cha ac e is ics o he mic opa icles using con en ional s a is ical me hods [
29
–
31
]. Despi e PGSS
®
being a simple and e sa ile me hod, he lack o knowledge o he e ec s o he a iables on he esul s
o PGSS
®
echnology may en ail an obs acle owa ds he obus SLMPs p oduc ion and he scaling-up
o he p ocess [
32
]. App oaches based on DoE (design o expe imen s) and mul iple eg ession ha e
been p oposed o manage he numbe o expe imen s, o selec he c i ical a iables and o op imize
he ope a ion condi ions, bu mainly ega ding hei in luence on he dissolu ion p o ile o he d ug
inco po a ed in he pa icles [
33
]. Some ma hema ical models we e also p oposed o simula e he
physicochemical p ocesses aking place du ing he PGSS
®
p ocessing, such as he beha io o a
CO
2
-supe sa u a ed solu ion d op in low-p essu e en i onmen s [
34
,
35
]. In his con ex , a i icial
in elligence echnologies eme ge as ools wi h g ea po en ial o simpli ying he s udy o p ocesses in
which many a iables a e in ol ed, e en when a small numbe o expe imen s a e a ailable. Some o
hem, such as he neu o uzzylogic sys ems, allow mul iple a iables o be modeled and he models
exp essed h ough language, which gene a es in-dep h knowledge abou he p ocess. Neu o uzzylogic
so wa e is a hyb id sys em ha combines a i icial neu al ne wo ks (ANN) and uzzy logic (FL).
ANN a e compu e p og ams ha simula e how he human b ain p ocesses in o ma ion. They de ec
Molecules 2020,25, 4927 3 o 14
pa e ns and ela ionship in da a, and lea n om expe ience, leading o “black-box” ma hema ical
models [
36
]. When combined wi h FL, he models a e exp essed as simple linguis ic IF
. . .
THEN ules
oge he wi h a membe ship deg ee, losing hei black-box cha ac e and being easily unde s andable.
A i icial in elligence ools ha e been p e iously used in he de elopmen and op imiza ion
o mic opa icles [
37
] and polyme ic and lipid nanopa icles [
38
,
39
]. To he bes o ou knowledge,
hese ools a e applied in his wo k o he i s ime o model he p oduc ion o SLMPs by he
PGSS
®
echnology. SLMPs consis o a ma ix o comme cial glyce yl monos ea a e (GMS), a lipid
widely used as an emulsi ie in pha maceu ical p epa a ions due o i s good biocompa ibili y and
sa e y [
40
,
41
]. Fi s , he mel ing poin dep ession o comme cial GMS in con ac wi h scCO
2
was s udied
o es ablish he limi s o he adequa e knowledge space o he p ocessing o PGSS
®
. Subsequen ly,
an uncons ained D-op imal design o h ee a iables (nozzle diame e , p essu e and empe a u e) a 2,
3 and 3 le els, espec i ely, was used o p epa e SLMPs using he PGSS
®
echnique. The mic opa icles
we e cha ac e ized in e ms o size and shape. The gene a ed da abase was modeled h ough
a neu o uzzylogic sys em and he design space was es ablished wi h espec o he mel GMS
p ocessabili y ( ine pa icle p oduc ion yield) and he cha ac e is ics o he pa icles.
2. Resul s and Discussion
2.1. Mel ing Poin Dep ession o GMS in he P esence o CO2
Mel ing p essu e- empe a u e cu e o he comme cial GMS unde comp essed CO
2
was measu ed
o de e mine he easible ope a ing ange o condi ions o he PGSS
®
echnique (Figu e 1). This s ep
is c ucial since i is necessa y o es ablish a se o p essu e- empe a u e condi ions (g ey egion in
Figu e 1) whe e he lipid mix u e is mol en. The mel ing poin o GMS in he p esence o CO
2
has
been p e iously s udied [
42
], bu hese de e mina ions a e essen ial because i is well known ha GMS
can ha e in e -ba ch and in e -manu ac u e a iabili y as i is comme cially p o ided as a mix u e o
componen s (mono- and diglyce ides).
Molecules 2020, 25, x FOR PEER REVIEW 3 o 14
expe ience, leading o “black-box” ma hema ical models [36]. When combined wi h FL, he models
a e exp essed as simple linguis ic IF…THEN ules oge he wi h a membe ship deg ee, losing hei
black-box cha ac e and being easily unde s andable.
A i icial in elligence ools ha e been p e iously used in he de elopmen and op imiza ion o
mic opa icles [37] and polyme ic and lipid nanopa icles [38,39]. To he bes o ou knowledge, hese
ools a e applied in his wo k o he i s ime o model he p oduc ion o SLMPs by he PGSS®
echnology. SLMPs consis o a ma ix o comme cial glyce yl monos ea a e (GMS), a lipid widely
used as an emulsi ie in pha maceu ical p epa a ions due o i s good biocompa ibili y and sa e y
[40,41]. Fi s , he mel ing poin dep ession o comme cial GMS in con ac wi h scCO2 was s udied o
es ablish he limi s o he adequa e knowledge space o he p ocessing o PGSS®. Subsequen ly, an
uncons ained D-op imal design o h ee a iables (nozzle diame e , p essu e and empe a u e) a
2, 3 and 3 le els, espec i ely, was used o p epa e SLMPs using he PGSS® echnique. The
mic opa icles we e cha ac e ized in e ms o size and shape. The gene a ed da abase was modeled
h ough a neu o uzzylogic sys em and he design space was es ablished wi h espec o he mel GMS
p ocessabili y ( ine pa icle p oduc ion yield) and he cha ac e is ics o he pa icles.
2. Resul s and Discussion
2.1. Mel ing Poin Dep ession o GMS in he P esence o CO2
Mel ing p essu e- empe a u e cu e o he comme cial GMS unde comp essed CO2 was
measu ed o de e mine he easible ope a ing ange o condi ions o he PGSS® echnique (Figu e 1).
This s ep is c ucial since i is necessa y o es ablish a se o p essu e- empe a u e condi ions (g ey
egion in Figu e 1) whe e he lipid mix u e is mol en. The mel ing poin o GMS in he p esence o
CO2 has been p e iously s udied [42], bu hese de e mina ions a e essen ial because i is well known
ha GMS can ha e in e -ba ch and in e -manu ac u e a iabili y as i is comme cially p o ided as a
mix u e o componen s (mono- and diglyce ides).
Figu e 1. Glyce yl monos ea a e (GMS) mel ing poin s ob ained a di e en p essu es o CO2 using a
a iable- olume high-p essu e iew cell. G ey a ea ep esen s he p essu e- empe a u e egion a
which GMS will be mol en. The a ea delimi ed by he dashed line ep esen s he ope a ing egion
es ablished o solid lipid mic opa icles (SLMPs) p oduc ion by PGSS® echnique.
The mel ing poin o he comme cial GMS wi hou CO2 was 61 °C a ambien p essu e. CO2 can
ac as a plas icize agen , being able o mel o he subs ances, like lipids o polyme s, below hei
no mal mel ing poin s. Mel ing poin deple ion e ec o GMS in con ac wi h CO2 is highly dependen
on he wo king p essu e and dec eased p opo ionally up o 52 °C as can be seen in Figu e 1. This
e ec was ela ed o he inc ease in he amoun o CO2 dissol ed in he lipid when he p essu e
inc eases [43]. A pla eau in empe a u e was eached a 52 °C and p essu es abo e 120 ba we e no
able o cause an addi ional mel ing poin deple ion. This second e ec was ela ed o he compe ing
mechanism o inc eased CO2 solubili y in he lipid and he hyd os a ic p essu e p omo ing he
52 54 56 58 60 62
0
20
40
60
80
100
120
140
160
180
200
T (ºC)
P (ba )
PGSSâ ope a ing egion
Figu e 1.
Glyce yl monos ea a e (GMS) mel ing poin s ob ained a di e en p essu es o CO
2
using
a a iable- olume high-p essu e iew cell. G ey a ea ep esen s he p essu e- empe a u e egion a
which GMS will be mol en. The a ea delimi ed by he dashed line ep esen s he ope a ing egion
es ablished o solid lipid mic opa icles (SLMPs) p oduc ion by PGSS® echnique.
The mel ing poin o he comme cial GMS wi hou CO
2
was 61
◦
C a ambien p essu e. CO
2
can
ac as a plas icize agen , being able o mel o he subs ances, like lipids o polyme s, below hei
no mal mel ing poin s. Mel ing poin deple ion e ec o GMS in con ac wi h CO
2
is highly dependen
on he wo king p essu e and dec eased p opo ionally up o 52
◦
C as can be seen in Figu e 1. This e ec
was ela ed o he inc ease in he amoun o CO
2
dissol ed in he lipid when he p essu e inc eases [
43
].
A pla eau in empe a u e was eached a 52
◦
C and p essu es abo e 120 ba we e no able o cause an
addi ional mel ing poin deple ion. This second e ec was ela ed o he compe ing mechanism o
inc eased CO
2
solubili y in he lipid and he hyd os a ic p essu e p omo ing he mel ing poin deple ion
Molecules 2020,25, 4927 4 o 14
and inc ease, espec i ely, ha a e coun e ac ing a p essu es abo e 120 ba o GMS [
42
]. The educed
mel ing empe a u e in he p esence o comp essed CO
2
is ad an ageous o he ene gy op imiza ion
o he PGSS®pa icle p ocessing when ans e ing o mula ions om lab o pilo scale [44,45].
2.2. Pa icle Size Dis ibu ion (PSD), Mo phological and Physichochemical Cha ac e iza ion o GMS Pa icles
Based on he mel ing poin alues ob ained in Sec ion 2.1, he ange o alues o p essu e and
empe a u e selec ed o he expe imen al s udy o he PGSS
®
p ocessing o GMS pa icles we e se
a 120–200 ba and 57–67
◦
C, espec i ely. In his wo k, an inc emen o ca. 5
◦
C wi h espec o he
mel ing empe a u e o GMS a a ce ain p essu e in he p esence o comp essed CO
2
was es ablished
as a ule-o - humb (dashed and g ey ec angle in Figu e 1) o ensu e he comple e mel ing and o
a oid clogging o he nozzle du ing he PGSS
®
expansion-sp aying s ep. The selec ion o he nozzle
diame e was based on he echnical possibili ies o he PGSS
®
equipmen , being 4 and 1 mm he
maximum nozzle diame e and he minimum nozzle diame e ha did no cause clogging e en s upon
dep essu iza ion using he es ablished P-T ange in he expe imen al design, espec i ely.
PSDs o he SLMPs showed mean diame e s be ween 100 and 190
µ
m and s anda d de ia ions
be ween 30 and 65
µ
m (Table 1). In gene al, he PSDs i ed well o a no mal dis ibu ion (Figu e 2)
wi h good co ela ion le els (R
2
>0.95) in all cases. The yield o pa icle p oduc ion was de e mined
om he weigh pe cen age o ine pa icles wi h espec o he ini ial GMS (Table 1). The loss o
ma e ial du ing he PGSS
®
p ocessing was due o GMS emaining in he ubing and he sa u a o o
he equipmen , mol en ma e ial ha was no solidi ied in o pa icles and o med a c us in he walls o
he p ecipi a o . Some mass losses we e a ibu ed o small pa icles ha emained suspended in he
ou le gaseous s eam and we e en ed ou du ing he dep essu iza ion s ep along wi h he CO2.
Table 1.
Yield o pa icle p oduc ion, mean diame e and s anda d de ia ion o SLMPs o GMS
p ocessed using PGSS
®
echnique. Pa icles we e deno ed as GMS-x-y-z, whe e x is he nozzle diame e
(mm), y he p ocessing empe a u e (deg ees Celsius) and z he p ocessing p essu e (ba ).
SLMPs Mean Diame e (µm) S anda d De ia ion (µm) % Fine Pa icles
GMS-4-57-120 138.7 47.0 17.4
GMS-4-57-200 182.6 63.3 43.7
GMS-4-62-120 128.0 41.8 12.8
GMS-4-62-200 147.4 48.3 18.3
GMS-4-67-120 103.5 33.1 11.0
GMS-4-67-200 154.3 52.1 27.5
GMS-1-57-120 171.6 56.8 39.5
GMS-1-57-160 172.3 51.6 34.8
GMS-1-57-200 186.2 57.5 25.7
GMS-1-67-120 131.9 44.4 23.5
GMS-1-67-160 130.3 50.0 27.1
GMS-1-67-200 125.4 43.1 34.8
Molecules 2020, 25, x FOR PEER REVIEW 5 o 14
Figu e 2. F equency his og am o GMS-1-67-200 pa icles (mean pa icle diame e = 125.4 ± 43.1 μm).
The no mal dis ibu ion o his his og am is ep esen a i e o all he GMS o mula ions es ed.
The p ocessing using PGSS® echnique led o pa icles wi h educed ci cula i y (60.7 ± 18.2%)
wi h espec o he o iginal GMS ( ound pa icles, Figu e 3A). PGSS®-p ocessed lipid mic opa icles
had a dec eased bulk densi y (0.14 g/cm3) wi h espec o he aw ma e ial (0.53 g/cm3). Howe e ,
skele al densi y was simila (0.995 ± 0.017 g/cm3) o he unp ocessed GMS (0.980 ± 0.003 g/cm3),
sugges ing ha he chemical s uc u e o he GMS was no unal e ed du ing he p ocess, as also
con i med by X- ay di ac ion (XRD) and A enua ed To al Re lec ance/Fou ie T ans o m in a ed
spec oscopy (ATR/FT-IR) (Figu e A1).
Figu e 3. E ec o empe a u e in he PGSS® p ocessing o GMS pa icles: (A) unp ocessed GMS
pa icles and (B) GMS-1-57-200, (C) GMS-1-62-200 and (D) GMS-1-67-200 pa icles.
2.3. Mo phological Cha ac e iza ion and Modeling o GMS Pa icle P oduc ion Using Neu o uzzy Tool
The p ocessing o GMS using he PGSS® echnique esul ed in po ous pa icles o a ied shape
and o lowe pa icle diame e han he o iginal ma e ial (Figu es 3 and 4).
Figu e 4. E ec o p essu e in he PGSS® p ocessing o GMS pa icles: (A) GMS-1-67-120 and (B) GMS-
1-67-200 pa icles.
0-10
10-20
20-30
30-40
40-50
50-60
60-70
70-80
80-90
90-100
100-110
110-120
120-130
130-140
140-150
150-160
160-170
170-180
180-190
190-200
0
2
4
6
8
10
12
Pa icle diame e (mm)
Weigh %
Figu e 2.
F equency his og am o GMS-1-67-200 pa icles (mean pa icle diame e =125.4
±
43.1
µ
m).
The no mal dis ibu ion o his his og am is ep esen a i e o all he GMS o mula ions es ed.
Molecules 2020,25, 4927 5 o 14
The p ocessing using PGSS
®
echnique led o pa icles wi h educed ci cula i y (60.7
±
18.2%) wi h
espec o he o iginal GMS ( ound pa icles, Figu e 3A). PGSS
®
-p ocessed lipid mic opa icles had a
dec eased bulk densi y (0.14 g/cm
3
) wi h espec o he aw ma e ial (0.53 g/cm
3
). Howe e , skele al
densi y was simila (0.995
±
0.017 g/cm
3
) o he unp ocessed GMS (0.980
±
0.003 g/cm
3
), sugges ing
ha he chemical s uc u e o he GMS was no unal e ed du ing he p ocess, as also con i med by
X- ay di ac ion (XRD) and A enua ed To al Re lec ance/Fou ie T ans o m in a ed spec oscopy
(ATR/FT-IR) (Figu e A1).
Molecules 2020, 25, x FOR PEER REVIEW 5 o 14
Figu e 2. F equency his og am o GMS-1-67-200 pa icles (mean pa icle diame e = 125.4 ± 43.1 μm).
The no mal dis ibu ion o his his og am is ep esen a i e o all he GMS o mula ions es ed.
The p ocessing using PGSS® echnique led o pa icles wi h educed ci cula i y (60.7 ± 18.2%)
wi h espec o he o iginal GMS ( ound pa icles, Figu e 3A). PGSS®-p ocessed lipid mic opa icles
had a dec eased bulk densi y (0.14 g/cm3) wi h espec o he aw ma e ial (0.53 g/cm3). Howe e ,
skele al densi y was simila (0.995 ± 0.017 g/cm3) o he unp ocessed GMS (0.980 ± 0.003 g/cm3),
sugges ing ha he chemical s uc u e o he GMS was no unal e ed du ing he p ocess, as also
con i med by X- ay di ac ion (XRD) and A enua ed To al Re lec ance/Fou ie T ans o m in a ed
spec oscopy (ATR/FT-IR) (Figu e A1).
Figu e 3. E ec o empe a u e in he PGSS® p ocessing o GMS pa icles: (A) unp ocessed GMS
pa icles and (B) GMS-1-57-200, (C) GMS-1-62-200 and (D) GMS-1-67-200 pa icles.
2.3. Mo phological Cha ac e iza ion and Modeling o GMS Pa icle P oduc ion Using Neu o uzzy Tool
The p ocessing o GMS using he PGSS® echnique esul ed in po ous pa icles o a ied shape
and o lowe pa icle diame e han he o iginal ma e ial (Figu es 3 and 4).
Figu e 4. E ec o p essu e in he PGSS® p ocessing o GMS pa icles: (A) GMS-1-67-120 and (B) GMS-
1-67-200 pa icles.
0-10
10-20
20-30
30-40
40-50
50-60
60-70
70-80
80-90
90-100
100-110
110-120
120-130
130-140
140-150
150-160
160-170
170-180
180-190
190-200
0
2
4
6
8
10
12
Pa icle diame e (mm)
Weigh %
Figu e 3.
E ec o empe a u e in he PGSS
®
p ocessing o GMS pa icles: (
A
) unp ocessed GMS
pa icles and (B) GMS-1-57-200, (C) GMS-1-62-200 and (D) GMS-1-67-200 pa icles.
2.3. Mo phological Cha ac e iza ion and Modeling o GMS Pa icle P oduc ion Using Neu o uzzy Tool
The p ocessing o GMS using he PGSS
®
echnique esul ed in po ous pa icles o a ied shape
and o lowe pa icle diame e han he o iginal ma e ial (Figu es 3and 4).
Molecules 2020, 25, x FOR PEER REVIEW 5 o 14
Figu e 2. F equency his og am o GMS-1-67-200 pa icles (mean pa icle diame e = 125.4 ± 43.1 μm).
The no mal dis ibu ion o his his og am is ep esen a i e o all he GMS o mula ions es ed.
The p ocessing using PGSS® echnique led o pa icles wi h educed ci cula i y (60.7 ± 18.2%)
wi h espec o he o iginal GMS ( ound pa icles, Figu e 3A). PGSS®-p ocessed lipid mic opa icles
had a dec eased bulk densi y (0.14 g/cm3) wi h espec o he aw ma e ial (0.53 g/cm3). Howe e ,
skele al densi y was simila (0.995 ± 0.017 g/cm3) o he unp ocessed GMS (0.980 ± 0.003 g/cm3),
sugges ing ha he chemical s uc u e o he GMS was no unal e ed du ing he p ocess, as also
con i med by X- ay di ac ion (XRD) and A enua ed To al Re lec ance/Fou ie T ans o m in a ed
spec oscopy (ATR/FT-IR) (Figu e A1).
Figu e 3. E ec o empe a u e in he PGSS® p ocessing o GMS pa icles: (A) unp ocessed GMS
pa icles and (B) GMS-1-57-200, (C) GMS-1-62-200 and (D) GMS-1-67-200 pa icles.
2.3. Mo phological Cha ac e iza ion and Modeling o GMS Pa icle P oduc ion Using Neu o uzzy Tool
The p ocessing o GMS using he PGSS® echnique esul ed in po ous pa icles o a ied shape
and o lowe pa icle diame e han he o iginal ma e ial (Figu es 3 and 4).
Figu e 4. E ec o p essu e in he PGSS® p ocessing o GMS pa icles: (A) GMS-1-67-120 and (B) GMS-
1-67-200 pa icles.
0-10
10-20
20-30
30-40
40-50
50-60
60-70
70-80
80-90
90-100
100-110
110-120
120-130
130-140
140-150
150-160
160-170
170-180
180-190
190-200
0
2
4
6
8
10
12
Pa icle diame e (mm)
Weigh %
Figu e 4.
E ec o p essu e in he PGSS
®
p ocessing o GMS pa icles: (
A
) GMS-1-67-120 and
(B) GMS-1-67-200 pa icles.
Neu o uzzylogic so wa e succeeded in modeling he in luence o he pa ame e s o p essu e,
empe a u eand nozzlediame e (inpu s) on heou pu meandiame e (Table2) wi hhigh p edic abili y
(R
2
>90%) and accu acy (p<0.01). The h ee pa ame e s help o explain he a ia ions in pa icle size,
wi h empe a u e (submodel 1) ha ing he main e ec . An in e ac ion be ween he p essu e and he
nozzle can be also obse ed (submodel 2).
Molecules 2020,25, 4927 6 o 14
Table 2.
Inpu s selec ed by Fo mRules
®
o he di e en ou pu s e alua ed in his wo k, wi h hei
espec i e pa ame e s o e alua e he quali y o each model. The mos ele an submodels a e
highligh ed in bold.
Ou pu Submodel Inpu s
Selec ed R2Deg ees o
F eedom Value C i ical
Value
Mean
diame e
1 T 91.5012 5 and 6 12.92 4.39
2
P
×
Nozzle
S anda d
de ia ion 1
P
×
Nozzle
58.3925 4 and 7 2.46 4.12
% ine
pa icles
1
P
×
Nozzle
75.1098 6 and 5 2.51 4.93
2 T
The p edic abili y is also easonable o he pe cen age o ine pa icles (R
2
>75%), a pa ame e
indica i e o p ocess yield (Table 2). Howe e , adequa e accu acy was no achie ed wi h such a small
numbe o deg ees o eedom. The model shows a main e ec o he in e ac ion p essu e-nozzle,
bu empe a u e also a ec s p ocess yield.
Va iables s udied do no explain su icien ly he a ia ions in he s anda d de ia ion o he pa icle
size dis ibu ion (R
2
<75%). The pa icle size dis ibu ions wi h PGSS
®
echnique a e b oad and
cha ac e ized by high s anda d de ia ions, p obably highe han he a ia ions p omo ed by he
p ocessing pa ame e s ( empe a u e, p essu e and nozzle diame e ) used in his esea ch. The e o e,
he ANN canno de ine a good model o his s anda d de ia ion.
IF
. . .
THEN ules, gene a ed by he neu o uzzylogic so wa e allows acqui ing knowledge in an
easy way (Figu e A1). Acco ding o hese ules, IF he empe a u e is low (up o 62
◦
C) THEN he
mean pa icle size ob ained is high (o e 144.8
µ
m). The inc ease in empe a u e o e 62
◦
C p oduces a
dec ease in pa icle size (Figu e 3).
On he o he hand, (IF) he p essu e inc ease (
. . .
THEN) p omo es a dec ease in he pa icle size
o he mic opa icles (Figu e 4). This ule applies o bo h small and la ge nozzle diame e s, being he
a ia ions in pa icle size wide when he la ge nozzle is used. Figu e 5 ep esen s he p edic ed esul s
by he model o mean pa icle size o he la ge (Figu e 5A) and small (Figu e 5B) nozzle. This e ec
was ela ed o he inc eased solubili y o CO
2
in mol en GMS. A highe p essu es CO
2
solubili y will
inc ease and, upon dep essu iza ion, mo e nuclea ion bubbles will o m due o CO2supe sa u a ion,
b eaking he lipid in o smalle pa icles (Figu e 4) [
42
,
46
]. Using he la ge nozzle diame e , p essu e
a ia ions p oduced a mo e p onounced e ec on he mean pa icle diame e .
Molecules 2020, 25, x FOR PEER REVIEW 6 o 14
Neu o uzzylogic so wa e succeeded in modeling he in luence o he pa ame e s o p essu e,
empe a u e and nozzle diame e (inpu s) on he ou pu mean diame e (Table 2) wi h high
p edic abili y (R2 > 90%) and accu acy (p < 0.01). The h ee pa ame e s help o explain he a ia ions
in pa icle size, wi h empe a u e (submodel 1) ha ing he main e ec . An in e ac ion be ween he
p essu e and he nozzle can be also obse ed (submodel 2).
The p edic abili y is also easonable o he pe cen age o ine pa icles (R2 > 75%), a pa ame e
indica i e o p ocess yield (Table 2). Howe e , adequa e accu acy was no achie ed wi h such a small
numbe o deg ees o eedom. The model shows a main e ec o he in e ac ion p essu e-nozzle,
bu empe a u e also a ec s p ocess yield.
Table 2. Inpu s selec ed by Fo mRules® o he di e en ou pu s e alua ed in his wo k, wi h hei
espec i e pa ame e s o e alua e he quali y o each model. The mos ele an submodels a e
highligh ed in bold.
Ou pu Submodel Inpu s Selec ed R2 Deg ees o F eedom
Value
C i ical
Value
Mean diame e
1
T
91.5012 5 and 6 12.92 4.39
2
P
x Nozzle
S anda d de ia ion
1
P x Nozzle
58.3925
4 and 7
2.4
6
4.12
% ine pa icles
1
P x Nozzle
75.1098 6 and 5 2.51 4.93
2
T
Va iables s udied do no explain su icien ly he a ia ions in he s anda d de ia ion o he
pa icle size dis ibu ion (R2 < 75%). The pa icle size dis ibu ions wi h PGSS® echnique a e b oad
and cha ac e ized by high s anda d de ia ions, p obably highe han he a ia ions p omo ed by he
p ocessing pa ame e s ( empe a u e, p essu e and nozzle diame e ) used in his esea ch. The e o e,
he ANN canno de ine a good model o his s anda d de ia ion.
IF…THEN ules, gene a ed by he neu o uzzylogic so wa e allows acqui ing knowledge in an
easy way (Figu e A1). Acco ding o hese ules, IF he empe a u e is low (up o 62 °C) THEN he
mean pa icle size ob ained is high (o e 144.8 μm). The inc ease in empe a u e o e 62 °C p oduces
a dec ease in pa icle size (Figu e 3).
On he o he hand, (IF) he p essu e inc ease (…THEN) p omo es a dec ease in he pa icle size
o he mic opa icles (Figu e 4). This ule applies o bo h small and la ge nozzle diame e s, being he
a ia ions in pa icle size wide when he la ge nozzle is used. Figu e 5 ep esen s he p edic ed
esul s by he model o mean pa icle size o he la ge (Figu e 5A) and small (Figu e 5B) nozzle.
This e ec was ela ed o he inc eased solubili y o CO2 in mol en GMS. A highe p essu es CO2
solubili y will inc ease and, upon dep essu iza ion, mo e nuclea ion bubbles will o m due o CO2
supe sa u a ion, b eaking he lipid in o smalle pa icles (Figu e 4) [42,46]. Using he la ge nozzle
diame e , p essu e a ia ions p oduced a mo e p onounced e ec on he mean pa icle diame e .
Figu e 5. P edic ed esul s by he model o mean pa icle size o he (A) la ge and (B) small nozzles.
Figu e 5.
P edic ed esul s by he model o mean pa icle size o he (
A
) la ge and (
B
) small nozzles.
Figu e 6shows he p edic ed alues o he pe cen age o ine pa icles as a unc ion o
p essu e and empe a u e. The inc ease in empe a u e leads o a educ ion in he p ocess yield,
Molecules 2020,25, 4927 7 o 14
being especially impo an up o 62
◦
C. In he empe a u e ange o he expe imen al design (57–67
◦
C),
he Joule-Thomson coe icien is e y simila o he 0–200 ba p essu e ange [
47
]. A highe
empe a u es, he posi i e Joule-Thomson e ec con ibu ion may no be enough o solidi y he GMS
when exi ing he nozzle. Unde hese condi ions, a signi ican ac ion o GMS is in a semi-mol en s a e
when i eaches he p ecipi a o and o ms a c us in he walls o he essel ins ead o o ming SLMPs
ha deposi on he collec o .
Molecules 2020, 25, x FOR PEER REVIEW 7 o 14
Figu e 6 shows he p edic ed alues o he pe cen age o ine pa icles as a unc ion o p essu e
and empe a u e. The inc ease in empe a u e leads o a educ ion in he p ocess yield, being
especially impo an up o 62 °C. In he empe a u e ange o he expe imen al design (57–67 °C), he
Joule-Thomson coe icien is e y simila o he 0–200 ba p essu e ange [47]. A highe
empe a u es, he posi i e Joule-Thomson e ec con ibu ion may no be enough o solidi y he GMS
when exi ing he nozzle. Unde hese condi ions, a signi ican ac ion o GMS is in a semi-mol en
s a e when i eaches he p ecipi a o and o ms a c us in he walls o he essel ins ead o o ming
SLMPs ha deposi on he collec o .
Figu e 6. In luence o he pa ame e s p essu e and empe a u e on he yield o ine pa icle o ma ion
using: (A) he la ge nozzle diame e and (B) he smalle nozzle diame e .
The diame e o he nozzle also in luenced he ine pa icle yield p oduc ion. In gene al, he
p ocess pe o ms be e when using he small size nozzle. I has been epo ed ha lowe nozzle
diame e s led o smalle pa icle sizes o o he lipid-based sys ems [48]. Di e ences in he e ec o
p essu e we e also de ec ed depending on he size o he nozzle used. When a nozzle o smalle size
is used, he inc ease in p essu e causes a sligh educ ion in he pe cen age o ines ob ained. This
may be ela ed o he p oduc ion o e en smalle pa icles ha emain suspended in he CO2 and a e
he e o e en ed ou . Howe e , when he nozzle has a la ge diame e , he e ec is he opposi e, and
he p ocess pe o mance is imp o ed wi h inc easing p essu e. The p essu e d op o he lipid-CO2
mel h ough he nozzle is lowe wi h la ge nozzle diame e s, leading o a dec eased Joule-Thomson
cooling e ec . A highe p essu es, his p essu e d op e ec is compensa ed by a highe CO2 con en
in he lipid mel and pa icles a e able o solidi y and each he collec o leading o highe ine pa icle
yield [49].
The expe imen al alues we e compa ed wi h he alues p edic ed wi h he model, showing
high accu acy o he models o ine pa icle ac ion (Figu e 7A) and mean diame e (Figu e 7B).
Figu e 6.
In luence o he pa ame e s p essu e and empe a u e on he yield o ine pa icle o ma ion
using: (A) he la ge nozzle diame e and (B) he smalle nozzle diame e .
The diame e o henozzle alsoin luenced he ine pa icle yieldp oduc ion. Ingene al, he p ocess
pe o ms be e when using he small size nozzle. I has been epo ed ha lowe nozzle diame e s
led o smalle pa icle sizes o o he lipid-based sys ems [
48
]. Di e ences in he e ec o p essu e
we e also de ec ed depending on he size o he nozzle used. When a nozzle o smalle size is used,
he inc ease in p essu e causes a sligh educ ion in he pe cen age o ines ob ained. This may be
ela ed o he p oduc ion o e en smalle pa icles ha emain suspended in he CO
2
and a e he e o e
en ed ou . Howe e , when he nozzle has a la ge diame e , he e ec is he opposi e, and he p ocess
pe o mance is imp o ed wi h inc easing p essu e. The p essu e d op o he lipid-CO
2
mel h ough
he nozzle is lowe wi h la ge nozzle diame e s, leading o a dec eased Joule-Thomson cooling e ec .
A highe p essu es, his p essu e d op e ec is compensa ed by a highe CO
2
con en in he lipid mel
and pa icles a e able o solidi y and each he collec o leading o highe ine pa icle yield [49].
The expe imen al alues we e compa ed wi h he alues p edic ed wi h he model, showing high
accu acy o he models o ine pa icle ac ion (Figu e 7A) and mean diame e (Figu e 7B).
Molecules 2020, 25, x FOR PEER REVIEW 8 o 14
Figu e 7. Pa i y plo s o he p edic ed and expe imen al alues o (A) mean pa icle size and (B) % o
ine pa icles. Con inuous diagonal line is a 45°-slope line; do ed lines co espond o an en elope o
ole ance o 10%.
3. Ma e ials and Me hods
3.1. Ma e ials
Kolliwax® GMS II (glyce ylmonos ea a e 40–55 ype II, powde , Tm = 54–64 °C) was supplied by
BASF GmbH (Ludwigsha en am Rhein, Ge many). CO2 o he PGSS® echnique (pu i y 99.8%) and
o he mel ing poin de e mina ion (pu i y 99.998%) we e pu chased om P axai (Mad id, Spain)
and Ai Liquide (San iago de Compos ela, Spain), espec i ely.
3.2. De e mina ion o he Mel ing Poin o GMS in he P esence o Comp essed CO2 a Di e en P essu es
The mel ing poin o he GMS in he p esence o comp essed CO2 in a 0–200 ba p essu e ange
was de e mined. A sample o GMS (app oxima ely 3.5 mg) on a glass ial was placed inside a a iable
olume high-p essu e cell, consis ing o a ho izon al s ainless-s eel cylinde wi h an in e nal diame e
o 2 cm and a pis on o adjus he olume om 7.9 o 29.5 cm3. The cell was equipped wi h a sapphi e
window (1.6 cm diame e ) ha allowed he de ec ion o phase ansi ions h ough an endoscope
(Olympus 5 se ies, Olympus, Tokyo, Japan) connec ed o a CCD-came a (Mo icam 2000, Mo ic Asia,
Hong Kong, China). In one sidewall o he cylinde , a second sapphi e window (6 mm in diame e )
made i possible o illumina e he in e io o he cell h ough an op ical ibe . A P 100 p obe wi h an
unce ain y o 0.02 °C was used o measu e he empe a u e in he cell wall. The p essu e was
measu ed wi h a Heise model DXD se ies digi al p essu e ansduce , wi h an ope a ing ange 0–500
ba and an unce ain y o 0.02% o he ull scale (FS).
Fo he expe imen al ials, he cell a i s maximum olume was illed wi h CO2 a oom
empe a u e and supply p essu e o 60–65 ba . A e wa ds, he sys em was hea ed o he selec ed
empe a u e ( om 52 o 61 °C) and he p essu e was g adually inc eased mo ing he pis on (i.e.,
educing he olume o he chambe ) un il he solid was comple ely mol en o de e mine he mel ing
poin alue. Thus, he mel ing p essu e o he GMS a he selec ed empe a u e was de e mined.
Subsequen ly, ano he empe a u e was selec ed and he p ocedu e was epea ed o ob ain ano he
alue o he mel ing cu e. Tempe a u e measu emen s we e ca ied ou by iplica e. Resul s we e
exp essed as he mean alue ± s anda d de ia ion (SD). A a ixed empe a u e, his de ice shows
epea abili y o he p essu e lowe han 11.4%. The mel ing poin empe a u e o he GMS a
a mosphe ic p essu e in he same equipmen was also de e mined.
3.3. SLMPs P oduc ion by he PGSS Technique
Fo he pa icle o ma ion p o ocol, 6 g o GMS powde we e placed in o a 250-mL high-p essu e
au ocla e (sa u a o ) (Eu o echnica GmbH, Ba g eheide, Ge many). A e hea ing he sa u a o o he
desi ed empe a u e (T), CO2 en e ed he equipmen a a cons an low o 7 g/min un il he desi ed
80 100 120 140 160 180 200
80
100
120
140
160
180
200
Expe imen al mean diame e (µm)
P edic ed mean diame e (µm)
A
0 10 20 30 40 50
0
10
20
30
40
50
Expe imen al ine pa icle ac ion (%)
P edic ed ine pa icle ac ion (%)
B
Figu e 7.
Pa i y plo s o he p edic ed and expe imen al alues o (
A
) mean pa icle size and (
B
) % o
ine pa icles. Con inuous diagonal line is a 45
◦
-slope line; do ed lines co espond o an en elope o
ole ance o 10%.
Molecules 2020,25, 4927 8 o 14
3. Ma e ials and Me hods
3.1. Ma e ials
Kolliwax
®
GMS II (glyce ylmonos ea a e 40–55 ype II, powde , Tm =54–64
◦
C) was supplied by
BASF GmbH (Ludwigsha en am Rhein, Ge many). CO2 o he PGSS® echnique (pu i y 99.8%) and
o he mel ing poin de e mina ion (pu i y 99.998%) we e pu chased om P axai (Mad id, Spain)
and Ai Liquide (San iago de Compos ela, Spain), espec i ely.
3.2. De e mina ion o he Mel ing Poin o GMS in he P esence o Comp essed CO2a Di e en P essu es
The mel ing poin o he GMS in he p esence o comp essed CO
2
in a 0–200 ba p essu e ange
was de e mined. A sample o GMS (app oxima ely 3.5 mg) on a glass ial was placed inside a a iable
olume high-p essu e cell, consis ing o a ho izon al s ainless-s eel cylinde wi h an in e nal diame e
o 2 cm and a pis on o adjus he olume om 7.9 o 29.5 cm
3
. The cell was equipped wi h a sapphi e
window (1.6 cm diame e ) ha allowed he de ec ion o phase ansi ions h ough an endoscope
(Olympus 5 se ies, Olympus, Tokyo, Japan) connec ed o a CCD-came a (Mo icam 2000, Mo ic Asia,
Hong Kong, China). In one sidewall o he cylinde , a second sapphi e window (6 mm in diame e )
made i possible o illumina e he in e io o he cell h ough an op ical ibe . A P 100 p obe wi h an
unce ain y o 0.02
◦
C was used o measu e he empe a u e in he cell wall. The p essu e was measu ed
wi h a Heise model DXD se ies digi al p essu e ansduce , wi h an ope a ing ange 0–500 ba and an
unce ain y o 0.02% o he ull scale (FS).
Fo heexpe imen al ials, he cella i smaximum olume was illed wi hCO
2
a oom empe a u e
and supply p essu e o 60–65 ba . A e wa ds, he sys em was hea ed o he selec ed empe a u e
( om 52 o 61
◦
C) and he p essu e was g adually inc eased mo ing he pis on (i.e., educing he
olume o he chambe ) un il he solid was comple ely mol en o de e mine he mel ing poin alue.
Thus, he mel ing p essu e o he GMS a he selec ed empe a u e was de e mined. Subsequen ly,
ano he empe a u e was selec ed and he p ocedu e was epea ed o ob ain ano he alue o he
mel ing cu e. Tempe a u e measu emen s we e ca ied ou by iplica e. Resul s we e exp essed as
he mean alue
±
s anda d de ia ion (SD). A a ixed empe a u e, his de ice shows epea abili y o
he p essu e lowe han 11.4%. The mel ing poin empe a u e o he GMS a a mosphe ic p essu e in
he same equipmen was also de e mined.
3.3. SLMPs P oduc ion by he PGSS Technique
Fo he pa icle o ma ion p o ocol, 6 g o GMS powde we e placed in o a 250-mL high-p essu e
au ocla e (sa u a o ) (Eu o echnica GmbH, Ba g eheide, Ge many). A e hea ing he sa u a o o he
desi ed empe a u e (T), CO
2
en e ed he equipmen a a cons an low o 7 g/min un il he desi ed
p essu e (P) was eached. A e 1 h o con ac be ween he mol en lipid and he comp essed CO
2
unde
s i ing a 400 pm, he sys em was dep essu ized by opening he al e placed a he bo om o he
sa u a o . When he mol en lipid lea es he sa u a o h ough a nozzle, apid dep essu iza ion causes
lipid mic opa icles p ecipi a ion wi hin a 2.7 L bo osilica e au ocla e (p ecipi a o ).
Ba ches o GMS pa icles we e p oduced ollowing a D-op imal expe imen al design o h ee
a iables: nozzle diame e (2 le els), ope a ing empe a u e (3 le els) and p essu e (3 le els) (Table 3)
ca ied ou by Da aFo m
®
.3.1 so wa e (In elligensys L d., S okesley, UK). GMS pa icles p ocessed
unde di e en p essu e and empe a u e condi ions we e deno ed as GMS-x-y-z, whe e x is he nozzle
diame e in mm, y he p ocessing empe a u e in deg ees Celsius and z he p ocessing p essu e in ba .
Molecules 2020,25, 4927 9 o 14
Table 3.
Nozzle diame e s and p ocessing empe a u es (T) and p essu es (P) es ed o he p epa a ion
o SLMPs o GMS using he PGSS® echnique.
SLMPs Nozzle (mm) T (◦C) P (ba )
GMS-4-57-120 4 57 120
GMS-4-57-200 4 57 200
GMS-4-62-120 4 62 120
GMS-4-62-200 4 62 200
GMS-4-67-120 4 67 120
GMS-4-67-200 4 67 200
GMS-1-57-120 1 57 120
GMS-1-57-160 1 57 160
GMS-1-57-200 1 57 200
GMS-1-67-120 1 67 120
GMS-1-67-160 1 67 160
GMS-1-67-200 1 67 200
Mic opa icles we e collec ed and weighed o de e mine he p ocess yield acco ding o Equa ion (1):
% ine pa icles =
W
W0
×100 (1)
whe e W
0
is he ini ial weigh o GMS added o he sa u a o and W
is he inal weigh o ine pa icles
collec ed. Also, he amoun o GMS emaining on he walls o he p ecipi a o and he in e io o he
ubing was weighed o e i y all he GMS had le he sa u a o , and wha amoun had no p ecipi a ed
in o SLMPs.
3.4. Mo phological Analysis, Physicochemical Cha ac e iza ion and Pa icle Size Dis ibu ion (PSD)
Fou aliquo s o each ba ch we e cha ac e ized in e ms o pa icle size dis ibu ion by op ical
mic oscopy using a came a (EP50, Olympus, Tokyo, Japan) p o ided wi h he so wa e EP View
(Olympus, Tokyo, Japan). The images we e analyzed using he eewa e ImageJ 1.49 . Calcula ed
pa icle diame e s co espond o he p ojec ed a ea equi alen diame e . The pa icle size dis ibu ions
we e i ed o a no mal dis ibu ion, and mean pa icle size and s anda d de ia ions we e ob ained.
The ci cula i y o he pa icles was also e alua ed by image analysis.
X- ay di ac ion (XRD) and a enua ed o al e lec ance/ ou ie ans o m in a ed spec oscopy
we e used o es possible physicochemical modi ica ions in GMS caused by PGSS
®
p ocessing.
XRD pa e ns we e collec ed (PW-1710, Philips, Eindho en, The Ne he lands) in he 2–50
◦
2
θ
- ange
using a 0.02
◦
s ep and CuK
α1
adia ion. ATR/FT-IR spec a (Gladi-ATR, Pike, Madison, WI, USA) we e
ob ained in he 400–4000 cm−1spec um ange om 32 scans and a a esolu ion o 2 cm−1.
Pa icles we e also analyzed by scanning elec on mic oscopy (SEM Zeiss EVO LS 15; Zeiss,
Obe kochen, Ge many) o e alua e hei mo phology and su ace ex u e. Pa icles we e p e iously
spu e ed-coa ed wi h a laye o 10 nm o i idium o imp o e he con as (Q150 T S/E/ES, Quo um
Technologies, Lewes, UK). Bulk densi y o he pa icles was de e mined by a olume ic me hod and
he skele al densi y was e alua ed using helium pycnome y (MPY-2; Quan ach ome, Del ay Beach,
FL, USA).
3.5. Modeling
The gene a ed da abase (inpu s om Table 3and ou pu s om Table 1) was modeled using
he comme cial so wa e Fo mRules
®
4.03 (In elligensys L d., S okesley, UK) which is a hyb id
sys em ha combines A i icial Neu al Ne wo ks (ANN) and uzzy logic. Nozzle diame e , p essu e
and empe a u e we e in oduced as inpu s, while pe cen age o ine pa icles, mean pa icle size
and s anda d de ia ion we e in oduced as ou pu s. A sepa a e model was de eloped o each
