1529
Review
ISSN 1743-588910.2217/NNM.13.131 © 2013 Future Medicine Ltd Nanomedicine (2013) 8(9), 1529–1548
Liposomal drug products and recent advances in the synthesis of supercritical fluid-mediated liposomes
Bangham et al. first observed that when egg leci-thin dispersed in water, closed bilayer structures formed spontaneously; he subsequently con-firmed these findings by electron microscopy [1]. He referred to these lipid vesicles as ‘smectic mesophases’. Subsequently, colleagues of Bang-ham referred to these vesicles as ‘bangosomes’; the term ‘liposomes’ was first used in 1968 [2]. In the early 1970s, Gregoriadis et al. first estab-lished the concept that liposomes could entrap drugs [3–6]. Several conventional techniques, such as the Bangham, detergent depletion, ether/ethanol injection, reverse-phase evapora-tion and emulsion methods, have been widely used to prepare liposomes with high entrapment efficiency (EE), a narrow particle size distribu-tion and long-term stability [7]. Other alterna-tive advanced methods for liposome prepara-tion, including dense gas and supercritical fluid (SCF) methods, have recently been introduced [8]. We describe the various SCF methods for liposome preparation in ‘SCF-mediated lipo-somes’ later in this review. Liposomes can be classified into three types based on their lamellarity and size, which differ according to preparation method and lipid composition: small unilamellar vesicles, large unilamel-lar vesicles (LUVs) and multilamellar vesicles (MLVs) [9].
Considerable progress was made during the 1970s and 1980s in the field of liposome stabil-ity, resulting in liposomes with prolonged cir-culation times after intravenous administration
and, therefore, improved biodistribution [10]. In the late 1970s, Szoka and Papahadjopoulos developed a preparation method that yields LUVs with a high EE and homogeneity [11]. Furthermore, extrusion of MLVs with polycar-bonate filters was found to reduce their size by up to 100 nm [12]. Similarly, sonication and homogenization processes were used for scal-able production of liposomes in the desired size range [13].
Since the discovery of liposomes, many stud-ies have been published describing the use of liposomes for a wide range of drug and vaccine delivery applications [14]. Despite considerable success at the research level, relatively few lipo-somal products have made it to the market-place. Table 1 shows the liposomal drug products approved by the US FDA and EMA, as well as other agencies [7,15–18,201,202].
Liposomal drug products: features & developmentThere are 15 approved liposome-based drugs that are commercially available. Among these, most have been approved for intravenous application. Many drugs are in the pipeline for commercialization, and are in various stages of clinical trials. Examples of potentially impor-tant drugs are Arikace® (amikacin; Insmed, NJ, USA), Thermodox® (doxorubicin; Cel-sion Corp., NJ, USA) and Lipoplatin™ (cis-platin; Regulon Inc., NY, USA) in Phase III trials, and EndoTAG®-1(paclitaxel; Medigene,
Since the pioneering research of Bangham et al. in 1965, liposomes have attracted a large amount of interest as potential carriers of various bioactive molecules for clinical applications. However, scaling-up conventional methods of liposome preparation has been proven to be challenging. Compared with conventional methods, processes that use supercritical fluid (SCF)-CO2 require a reduced amount of organic solvent, are relatively fast and simple to perform, and yield stable and more uniform liposomes. A number of studies have demonstrated that SCF-CO2 methods might be suitable for industrial-scale manufacturing of liposomes. In this review there are two topics being discussed. We provide an overview of liposomal drug products and aim to describe the physicochemical properties of liposomes prepared using various SCF methods. We review all of the available literature on SCF-CO2-based liposomes and focus on the future applications of these innovative technologies in industrial-scale liposome preparation.
KEYWORDS: conventional method n industrial scale n liposomal drug product n liposome n supercritical fluid CO2
Pankaj Ranjan Karn1, Wonkyung Cho1 & Sung-Joo Hwang*1,2
1Yonsei Institute of Pharmaceutical Sciences, Yonsei University, 162-1 Songdo-dong, Yeonsu-gu, Incheon 406-840, Republic of Korea 2College of Pharmacy, Yonsei University, 162-1 Songdo-dong, Yeonsu-gu, Incheon 406-840, Republic of Korea�*Author for correspondence: Tel.: +82 32 749 4518 Fax: +82 32 749 4105 [email protected]
part of
For reprint orders, please contact: [email protected]
Nanomedicine (2013) 8(9)1530 future science group
Review Karn, Cho & Hwang
Martinsried, Germany), Atragen® (tretinoin; Aronex Pharmaceuticals, TX, USA), SPI-077 (cisplatin; Sequus Pharmaceuticals, Inc., CA, USA), INX-0125 (vinorelbine; Inex Pharma-ceuticals Corp., BC, Canada) and OSI-211 (lurtotecan; OSI Pharmaceuticals, NY, USA) in Phase I/II clinical trials [18]. Marketed prod-ucts, especially AmBisome® (Astellas Pharma, IL, USA) and Doxil® (Janssen Pharmaceuticals, PA, USA), have achieved substantial clinical success, with sales in the hundreds of millions of US$ per year [16]. The composition, char-acteristics and clinical applications of vari-ous liposomal drug products are summarized in Table 2.
Manufacturing of liposomesNumerous conventional techniques to syn-thesize liposomes have been reported, such as the Bangham method [1], detergent-depletion method [19], ether/ethanol injection [20–22], emulsion method [9] and reverse-phase evapo-ration method [11]. The features of these meth-ods along with their drawbacks are summarized in Table 3. The frequent use of organic solvents and complex steps associated with these con-ventional techniques obstruct scaled-up manu-facturing of liposomes [23]. All methods require lipids to be combined by some means with an aqueous phase. Conventional methods gener-ally involve production of a lipid solution using an organic solvent prior to dispersion in the aqueous phase. The use of volatile organic sol-vents may affect the chemical structure of the entrapped chemical and also make the resulting liposome toxic.
SCF-CO2 has been used to produce pharma-
ceutically viable products. We have extensively investigated the applications of SCF-CO
2 and
published many papers detailing how it can be used in nanoparticles [24–27]. We have also explored industrial-scale liposome preparation using SCF-CO
2. Amphotericin B (AmB) and
cyclosporin A (CsA) liposomes are the main drugs that we have studied [28,101]. Liposomes were first prepared using SCF-CO
2 in 1994 [102].
Since then, many groups have used SCF-CO2
to prepare liposomes. Our main aim in the present article is to illus-
trate recent advances in the use of SCF-CO2 for
the preparation of liposomes and to discuss the future of this technology. To ensure that our discussion of SCF-CO
2-mediated liposome syn-
thesis was exhaustive, we reviewed all relevant research papers and patents dealing with the use of SCF-CO
2 for the preparation of liposomes.Ta
ble
1. A
pp
rove
d li
po
som
al d
rug
pro
du
cts
that
are
co
mm
erci
ally
ava
ilab
le.
Act
ive
ph
arm
aceu
tica
l in
gre
die
nt
Pro
du
ctD
osa
ge
form
Typ
eA
pp
rova
l dat
e (D
D/M
M/Y
YY
Y)
Man
ufa
ctu
rer
Ap
pro
val
Dox
orub
icin
hyd
roch
lorid
eD
oxil®
Inje
ctab
le –
iv.
PEG
ylat
ed li
poso
me
17/1
1/19
95Ja
nsse
n Ph
arm
aceu
tical
s (P
A, U
SA)
US
FDA
Lipo
-Dox
®In
ject
able
– iv
.PE
Gyl
ated
lipo
som
eJa
nuar
y 20
01TT
Y B
ioph
arm
(Ta
ipei
, Tai
wan
)D
epar
tmen
t of
Hea
lth T
aiw
an
Cae
lyx®
Inje
ctab
le –
iv.
Lipo
som
e21
/06
/199
6Ja
nsse
n Ph
arm
aceu
tical
sEM
A
Myo
cet®
Inje
ctab
le –
iv.
Lipo
som
e13
/07/
200
0G
P-Ph
arm
(Ba
rcel
ona,
Spa
in)
EMA
Am
phot
eric
in B
Abe
lcet
®In
ject
able
– iv
.Li
pid
com
plex
20/1
1/19
95Si
gma
Tau
(MD
, USA
)FD
A
Am
phot
ec®
Inje
ctab
leLi
pid
com
plex
22/1
1/19
96Be
n V
enue
Lab
orat
orie
s (O
H, U
SA)
FDA
Am
Biso
me®
Inje
ctab
leLi
poso
me
08
/11/
1997
Ast
ella
s Ph
arm
a (IL
, USA
)FD
A
Dau
noru
bici
n ci
trat
e D
auno
Xom
e®In
ject
able
Lipo
som
e0
8/0
4/1
996
Gile
ad S
cien
ces
(CA
, USA
)FD
A
Cyt
arab
ine
Dep
ocyt
®In
ject
able
– s
pina
lLi
poso
me
01/0
4/1
999
Paci
ra P
harm
aceu
tical
s In
c. (
NJ,
USA
)FD
A
Ver
tepo
rfin
Vis
udyn
e®In
ject
able
– iv
.Li
poso
me
12/0
4/2
00
0JH
P Ph
arm
aceu
tical
s (N
J, U
SA)
FDA
Mor
phin
e su
lfate
D
epoD
ur®
Inje
ctab
le –
epi
dura
lLi
poso
me
18/0
5/2
00
4Pa
cira
Pha
rmac
eutic
als
Inc.
FDA
Bupi
vaca
ine
Expa
rel®
Inje
ctab
le –
iv.
Lipo
som
e11
/10
/201
1Pa
cira
Pha
rmac
eutic
als
Inc.
FD
A
Vin
cris
tine
sulfa
teM
arqi
bo® K
itIn
ject
able
– iv
.Li
poso
me
12/0
8/2
012
Talo
n Th
erap
eutic
s (C
A, U
SA)
FDA
Hep
atiti
s A
ant
igen
Ep
axal
®im
.Li
poso
me
NA
Bern
a Bi
otec
h (B
erne
, Sw
itze
rland
)Fi
rst
licen
sed
in S
wit
zerla
nd
Hem
aggl
utin
in
Infle
xal-V
®im
.Li
poso
me
1997
Bern
a Bi
otec
hFi
rst
licen
sed
in S
wit
zerla
nd
im.:
Intr
amus
cula
r; iv
.: In
trav
enou
s; N
A: N
ot a
vaila
ble.
D
ata
take
n fr
om [7
,15–
18,201,202].
www.futuremedicine.com 1531future science group
Liposomal drug products & supercritical fluid-mediated liposomes Review
Tab
le 2
. Co
mp
osi
tio
n a
nd
ch
arac
teri
stic
s o
f lip
oso
mal
dru
g p
rod
uct
s.
Pro
du
ct (
man
ufa
ctu
rer)
Form
ula
tio
n c
om
po
siti
on
(m
ola
r ra
tio
)C
har
acte
rist
ics
of
lipo
som
esTh
erap
euti
c in
dic
atio
n
Ref
.
Dox
il®/C
aely
x® (J
anss
en
Phar
mac
eutic
als,
PA
, USA
)H
SPC
:cho
lest
erol
:PEG
200
0–D
SPE
(56
:39
:5)
Muc
h lo
nger
circ
ulat
ion
time
in b
lood
; bet
ter
safe
ty
profi
le; 1
00
nm; E
E >
90%
M
etas
tatic
ova
rian
canc
er,
AID
S-re
late
d K
apos
i’s s
arco
ma
[7,16,64
]
Lipo
-Dox
® (
TTY
Bio
phar
m,
Taip
ei, T
aiw
an)
DSP
C:c
hole
ster
ol:P
EG20
00
–DSP
E (5
6:3
9:5
)Pr
olon
ged
circ
ulat
ion
time
(65
h)A
dvan
ced
ovar
ian
canc
er,
AID
S-re
late
d K
apos
i’s s
arco
ma
[7,16,65]
Myo
cet®
(G
P-Ph
arm
, Ba
rcel
ona,
Spa
in)
EPC
:cho
lest
erol
(55
:45)
Redu
ced
drug
-rel
ated
tox
icit
y; h
ighe
r ris
k of
st
omat
itis;
19
0 nm
; EE:
95%
In
com
bina
tion
with
cy
clop
hosp
ham
ide
for
met
asta
tic
ovar
ian
canc
er
[7,66,67,202]
Abe
lcet
® (S
igm
a Ta
u,
MD
, USA
)D
MPC
:DM
PG (7
:3)
Shee
t-lik
e sh
ape;
1.6
–11
mm
siz
e; b
ioav
aila
bilit
y lo
wer
th
an F
ungi
zone
® (
Bris
tol-
Mye
rs S
quib
b, N
J, U
SA)
Inva
sive
fun
gal i
nfec
tions
[7,16,68]
Am
Biso
me®
(Ast
ella
s Ph
arm
a, IL
, USA
)H
SPC
:DSP
G:c
hole
ster
ol:A
mB
(2:0
.8:1
:0.4
)SU
V; <
100
nm in
dia
met
er
Serio
us f
unga
l inf
ectio
ns[7,68,115]
Dau
noX
ome®
(G
ilead
Sc
ienc
es, C
A, U
SA)
DSP
C:c
hole
ster
ol (2
:1)
SUV
; 45
nm in
dia
met
er; s
afe
Kap
osi’s
sar
com
a in
AID
S[7,67,69]
Dep
ocyt
® (
Paci
ra
Phar
mac
eutic
als
Inc.
, N
J, U
SA)
Cho
lest
erol
:trio
lein
:DO
PC:D
PPG
(11:
1:7:
1)Sp
heric
al; 2
0 µm
; mul
tilam
ella
rLy
mph
omat
ous
men
ingi
tis[7,203]
Vis
udyn
e® (J
HP
Phar
mac
eutic
als,
NJ,
USA
)EP
G:D
MPC
(3:5
); la
ctos
eLy
ophi
lized
; pho
tose
nsiti
zer
for
phot
odyn
amic
the
rapy
A
MD
, pat
holo
gica
l myo
pia,
ocu
lar
hist
opla
smos
is[7,70,20
2]
Dep
oDur
® (
Paci
ra
Phar
mac
eutic
als
Inc.
)C
hole
ster
ol:t
riole
in:D
OPC
:DPP
G (1
1:1:
7:1)
Mul
tives
icul
ar li
pid
part
icle
s; 1
7–23
µm
; pH
5–8
Trea
tmen
t of
the
pai
n fo
llow
ing
maj
or
surg
ery
[7,204
]
Mar
qibo
® K
it (T
alon
Th
erap
eutic
s, C
A, U
SA)
Cho
lest
erol
:egg
sph
ingo
mye
lin (4
5:55
)C
onve
ntio
nal fi
lm m
etho
d us
ed, f
ollo
wed
by
extr
usio
n; s
tabl
e; im
prov
ed c
ircul
atio
n tim
e; e
nhan
ced
drug
ret
entio
n
Phila
delp
hia
chro
mos
ome-
nega
tive
acut
e ly
mph
obla
stic
leuk
emia
[7,205]
Epax
al® (
Bern
a Bi
otec
h,
Bern
e, S
wit
zerla
nd)
PC a
nd P
ESp
heric
al; 1
50 n
m in
dia
met
er; a
lum
inum
fre
e;
form
alin
-inac
tivat
ed h
epat
itis
A v
irus
atta
ched
to
PL
vesi
cles
Hep
atiti
s A
viru
s in
fect
ions
[71]
Infle
xal-V
® (
Bern
a Bi
otec
h)PC
and
PE
Sphe
rical
; uni
lam
ella
r; 1
50 n
m in
dia
met
er; H
A
glyc
opro
tein
s an
d N
A in
sert
ed in
to P
L m
embr
ane
Influ
enza
pro
phyl
axis
; adj
uvan
t flu
va
ccin
e fo
r al
l age
gro
ups
[72]
Am
B: A
mph
oter
icin
B; A
MD
: Age
-rel
ated
mac
ular
deg
ener
atio
n; D
MPC
: 1-a
-dim
yris
toyl
phos
phat
idyl
cho
line;
DM
PG: 1
-a-d
imyr
isto
yl p
hosp
hatid
ylgl
ycer
ol; D
OPC
: 1,2
-dio
leoy
l-sn
-gly
cero
-3-p
hosp
hoch
olin
e;
DPP
G: D
ipal
mito
ylph
osph
atid
yl g
lyce
rol;
DSP
C: D
iste
aroy
lpho
spha
tidyl
cho
line;
DSP
E: D
iste
aroy
lpho
spha
tidyl
eth
anol
amin
e; D
SPG
: Dis
tear
oylp
hosp
hatid
yl g
lyce
rol;
EE: E
ntra
pmen
t ef
ficie
ncy;
EPC
: Egg
pho
spha
tidyl
ch
olin
e; E
PG: E
gg p
hosp
hatid
yl g
lyce
rol;
HA
: Hem
aggl
utin
in; H
SPC
: Hyd
roge
nate
d so
y ph
osph
atid
yl c
holin
e; N
A: N
eura
min
idas
e; P
C: P
hosp
hatid
yl c
holin
e; P
E: P
hosp
hatid
yl e
than
olam
ine;
PL:
Pho
spho
lipid
; SU
V: S
mal
l un
ilam
ella
r ve
sicl
e.
Nanomedicine (2013) 8(9)1532 future science group
Review Karn, Cho & HwangTa
ble
3. A
dva
nta
ges
an
d d
isad
van
tag
es o
f lip
oso
me
pre
par
atio
n m
eth
od
s.
Met
ho
ds
Ad
van
tag
esD
isad
van
tag
es
Mec
han
ical
dis
per
sio
n
Film
hyd
ratio
nM
LVs;
>1
µm; h
igh
EE; s
impl
est
met
hod
; EE
depe
nds
on t
he t
ype
of li
pid
and
drug
Dif
ficul
ty in
sca
ling-
up; o
rgan
ic s
olve
nt r
esid
ues;
het
erog
eneo
us v
esic
les;
la
rge
amou
nt o
f or
gani
c so
lven
ts
Prol
ipos
omes
MLV
s; s
mal
ler
size
tha
n fil
m h
ydra
tion
met
hod
; hig
h EE
; sui
tabl
e fo
r co
mm
erci
al u
se; l
ong-
term
sto
rage
in d
ry f
orm
is p
ossi
ble;
sta
ble
Part
icul
arly
sui
ted
for
high
mel
ting
poin
t lip
ids;
car
riers
(e.
g., s
orbi
tol)
are
requ
ired
; siz
e co
ntro
l is
not
poss
ible
; lip
ophi
lic d
rugs
are
pre
ferr
ed
Free
ze d
ryin
gM
LVs;
dis
pers
es t
he li
pid
in a
fine
ly d
ivid
ed f
orm
Requ
ires
addi
tiona
l ste
ps; c
ompl
ex m
etho
d; h
igh
cost
; con
sum
es a
larg
e am
ount
of
ener
gy
Mo
difi
cati
on
of
pre
par
ed li
po
som
es
Soni
catio
nSU
Vs;
mos
t w
idel
y us
ed m
etho
d fo
r pr
oduc
ing
SUV
s fr
om M
LVs;
pr
oduc
es li
poso
mes
with
a s
ize
up t
o 15
–50
nmRi
sk o
f de
grad
atio
n of
the
lipi
ds; l
oss
of m
ater
ials
in t
he p
roce
ss; m
uch
low
er E
E; s
uita
ble
for
smal
l vol
umes
of
sam
ple
Mic
roem
ulsi
ficat
ion
(hom
ogen
izat
ion)
Smal
l MLV
s; h
igh
pres
sure
is a
pplie
d to
red
uce
the
size
up
to 0
.1–0
.2 µ
m;
EE u
p to
70%
; sca
labl
e te
chni
que
Hig
h pr
oduc
t lo
ss; r
isk
of d
egra
datio
n du
e to
hig
h pr
essu
re a
nd
tem
pera
ture
; hig
h co
st
Fren
ch p
ress
ure
SUV
s; v
ery
mild
con
ditio
n us
ed, s
impl
e, r
apid
; sui
tabl
e fo
r se
nsiti
ve
mat
eria
ls; s
ize
up t
o 30
–80
nmV
ery
high
cap
ital c
ost
of t
he e
quip
men
t; w
orki
ng v
olum
e is
too
sm
all
(~50
ml);
tem
pera
ture
is d
iffic
ult
to a
chie
ve f
or t
he p
roce
ss
Mem
bran
e ex
trus
ion
SUV
s; h
ighe
r re
duce
d pr
essu
re (<
100
psi)
requ
ired
com
pare
d w
ith o
ther
ex
trus
ion
met
hods
; can
sel
ect
exac
t po
re s
ize
of fi
lter
to p
rodu
ce d
efine
d si
ze o
f lip
osom
es
Hig
h lo
ss o
f aq
ueou
s ve
sicl
e co
nten
ts; r
educ
ed E
E; r
epea
ted
extr
usio
n m
ay
alte
r th
e pr
oper
ties
of li
poso
mes
Drie
d re
cons
titut
ed v
esic
les
SUV
s; e
mpt
y bu
ffer
con
tain
ing
SUV
s an
d re
hydr
ated
with
aqu
eous
so
lutio
n co
ntai
ning
dru
g to
be
entr
appe
d; f
reez
e dr
ied
EE b
elow
40%
; lip
ids
bein
g ly
ophi
lized
mus
t be
in t
he f
orm
of
unila
mel
lar
vesi
cles
bef
ore
dryi
ng; o
smot
ic r
uptu
re; l
oss
of c
onte
nt
Free
ze–t
haw
son
icat
ion
Sim
ple,
rap
id, m
ild p
roce
ss f
or e
ntra
pped
sol
utes
; use
ful f
or s
tudy
ing
mem
bran
e pe
rmea
bilit
yEE
fro
m 2
0–3
0% o
nly;
onl
y su
ited
for
SUV
s; n
ot p
ossi
ble
to p
repa
re n
eutr
al
lipos
omes
Solv
ent
dis
per
sio
n
Etha
nol i
njec
tion
SUV
s; e
xtre
mel
y si
mpl
e m
etho
d an
d a
very
low
ris
k of
deg
rada
tion;
less
th
an 1
00
nmEx
trem
ely
low
EE;
dif
ficul
t to
rem
ove
etha
nol f
rom
lipo
som
es
Ethe
r in
ject
ion
SUV
s or
LU
Vs;
30
–20
0 nm
; lit
tle r
isk
of d
egra
datio
n; c
an r
un f
or lo
ng
perio
ds o
f tim
e; r
elat
ivel
y hi
gh E
ETi
me
cons
umin
g pr
oces
s; c
aref
ul c
ontr
ol n
eede
d; r
equi
re m
echa
nica
lly
oper
ated
infu
sion
pum
p; e
leva
ted
proc
ess
tem
pera
ture
Reve
rse-
phas
e ev
apor
atio
nU
nila
mel
lar
vesi
cle
or O
LV; s
ize
depe
nds
on t
he t
ype
of li
pids
; hig
h aq
ueou
s sp
ace-
to-li
pid
ratio
and
abl
e to
ent
er a
larg
e pe
rcen
tage
of
aque
ous
mat
eria
l
Rem
aini
ng o
rgan
ic s
olve
nts;
brie
f pe
riods
of
soni
catio
n; d
iffic
ulty
in
scal
ing-
up; n
ot e
asy
proc
edur
e
Oth
er m
eth
od
s
Det
erge
nt s
olub
iliza
tion
SUV
s, O
LVs
or M
LVs;
rem
oves
det
erge
nt f
rom
pre
form
ed m
ixed
mic
elle
s co
ntai
ning
pho
spho
lipid
s; p
reci
se c
ontr
ol o
f si
ze; v
ery
high
siz
e ho
mog
enei
ty; p
artic
ular
ly s
uite
d fo
r lip
ophi
lic p
rote
ins
Not
effi
cien
t m
etho
d; v
ery
low
EE;
res
idua
l det
erge
nt c
ause
s in
crea
sed
and
hete
roge
neit
y of
ves
icle
siz
e
EE: E
ntra
pmen
t ef
ficie
ncy;
LU
V: L
arge
uni
lam
ella
r ve
sicl
e; M
LV: M
ultil
amel
lar
vesi
cle;
OLV
: Olig
olam
ella
r ve
sicl
e; S
UV
: Sm
all u
nila
mel
lar
vesi
cle.
D
ata
take
n fr
om [1
1,19].
www.futuremedicine.com 1533future science group
Liposomal drug products & supercritical fluid-mediated liposomes Review
�n SCFs: history, definition & propertiesThe first reported observation of the occurrence of a supercritical phase was made by Baron Cagniard de la Tour in 1822 [29]. He observed that the gas–liquid boundary disappeared when the temperature of certain materials was increased by heating each of them in a closed glass container. Based on these early experiments, the critical points of several substances were first discovered, and this is the first description of a SCF [29]. Hannay and Hogarth published the first report describing a potential process using SCF as a medium for particle production, they reported: “We have then, the phenomenon of a solid with no measurable gaseous pressure, dis-solving in a gas. When the solid is precipitated by suddenly reducing the pressure, it is crystal-line, and may be brought down as snow in the gas, or on the glass as a frost, but it is always easily redissolved by the gas on increasing the pressure” [30].
A SCF is defined by the International Union of Pure and Applied Chemistry as ‘the state of a compound, mixture, or element above its criti-cal pressure and critical temperature’ [31]. The critical pressure is the highest pressure at which a liquid can be converted into a gas by an increase in temperature, while the critical temperature is the highest temperature at which a gas can be converted into a liquid by an increase in pressure. A pure component enters the supercritical phase when both pressure and temperature are above their critical values. In this region, a SCF exists in an intermediate phase between the gas and liquid phases (Figure 1) [31,32]. The physicochemical prop-erties of a SCF are intermediate between liquid and gas. Like a gas, a SCF has lower viscosity and higher diffusivity relative to liquid. These prop-erties facilitate mass transfer phenomena such as matrix extraction or impregnation. Similar to a liquid, SCF has a density high enough to exert a salvation effect. A SCF is dense but highly compressible; thus, any pressure change results in density alteration and, consequently, solvent power variation [25,33].
SCF technology has recently emerged as a green and novel technique for various processes, including liposomal preparation [8]. Simple and convenient SCF-CO
2 methods have been used to
produce liposomes while eliminating the issues involved in conventional preparation. The lev-els of organic solvent required are much lower than that for conventional methods [23], and the amount of residual organic solvent present in the final material is negligible compared with conventional methods [34]. Therefore, SCF-CO
2
can be used for industrial-scale preparation of liposomes, and has attracted a great deal of inter-est because of the numerous advantages that this technology offers over conventional methods.
SCF-mediated liposomesSeveral SCF methods have been used to prepare liposomes. Among these processes, injection and decompression, rapid expansion of super-critical solution (RESS), gas antisolvent (GAS), supercritical antisolvent (SAS), aerosol solvent extraction systems (ASES), and supercritical reverse-phase evaporation (SCRPE) methods are the processes most widely used for liposome preparation [35]. Castor first discovered the SCF-CO
2 injection and decompression method [102].
Indeed, over the past few years, a number of groups have begun investigating the possibility of using a variety of SCF-CO
2 methods to pre-
pare liposomes [14]. These SCF-CO2 methods
are discussed below.
�n Injection & decompression methodThe first SCF-CO
2 liposomes were produced by
Castor in 1994 using the injection and decom-pression method (Figure 2) [102]. In the injection method, a mixture of lipids, organic cosolvents, and supercritical gas is injected through a noz-zle into an aqueous solution. Liposomes form upon injection into the aqueous phase. In the decompression method, a mixture of phospho-lipids, an aqueous phase and a SCF is produced. The mixture is then decompressed to separate the SCF from the phospholipids and aqueous
Pre
ssu
re (
bar
)
Temperature (°C)
Solid
Liquid
Supercritical phase
Triple pointGas
Critical pointTc = 31.3°CPc = 73.3 bar
Figure 1. Pressure–temperature phase diagram of CO2. Pc: Critical pressure; Tc: Critical temperature.
Nanomedicine (2013) 8(9)1534 future science group
Review Karn, Cho & Hwang
media, resulting in liposome formation. The major distinction between these processes is the incorporation of the aqueous solution. In the injection method, the compressed phase is sprayed into water, whereas in the decompres-sion method, the aqueous phase is incorporated into the compressed phase, which is sprayed into the air. The size of the resulting liposomes depends on the rate of decompression and the opening diameter of the nozzle. SCF-CO
2 lipo-
somes with an average size of 478 nm formed when a nozzle diameter of 0.5 mm, pressure of 4000 psig and temperature of 60°C were used [103]. Decreasing the diameter of the nozzle to 0.06 mm decreased the liposome size to 326 nm. The effect of initial critical fluid pressure was also studied. The optimized pressure for solu-bilizing lecithin in SCF-CO
2 was found to be
3000 psig at 60°C for 60 min; under these conditions, mostly liposomes of uniform size (352 nm) were obtained. The authors of these studies claimed that injection and decompres-sion methods are capable of producing sterile,
pharmaceutical-grade liposomes with a suitable size range and narrow particle size distribu-tion that are effectively solvent free. They have reported that the supercritical liposomes exhib-ited good to excellent stability over a period of 6 months [103]. However, for the clinical devel-opment of liposomal drugs, formulations with a shelf-life of more than 12 months are desir-able. In addition, this method is well suited for hydrophobic drug encapsulation.
In 1996, Castor and Chu used the same decompression and injection method for lipo-some preparation to entrap hydrophobic drugs such as taxoids, doxorubicin, michellamine B, vincristine and cisplatin [104]. They reported that their methods could be used for large-scale production of pharmaceutical-grade liposomes that were sterile, of a predetermined size and effectively free of organic solvents. They stud-ied liposome-encapsulated paclitaxel prepared using a SCF-CO
2 injection and decompres-
sion as well as sonication methods. Injection or decompression methods yielded more effective
LipidOrganic cosolvent
MixingMixingchamber
CompressedCO2 + PL mix Compression
Decompression
Ice bath
Aqueousphase
Injection
CO2 storagetank
Nanomedicine © Future Science Group (2013)
Figure 2. Simplified and modified representation of the injection and decompression method explained by Castor et al. [102]. PL mix: Phospholipid mixture.
www.futuremedicine.com 1535future science group
Liposomal drug products & supercritical fluid-mediated liposomes ReviewReview
encapsulation of paclitaxel than sonication. Furthermore, the SCF-CO
2 injection method
produced liposomes with superior physical sta-bility than those formed using conventional sonication methods [104]. The potential disad-vantages of the injection and decompression method are using a nozzle, the high capital cost, and the high process pressure and temperature.
Frederiksen et al. discovered a novel and advantageous process for preparing liposomes for inclusion of water-soluble or hydrophilic sub-stances with the view of reducing the amount of combustible organic solvent used in liposome preparation [105]. They termed this method the supercritical liposome method, which is simi-lar to the injection method described by Castor and Chu [104]. This method has a high-pressure component in which phospholipids and choles-terol are dissolved in SCF-CO
2 using ethanol
as a cosolvent. An increase in pressure at con-stant temperature improved the solubility of the lipids in the supercritical phase. At 60°C and 25 MPa, with a recycling period of 60 min in CO
2 containing 7% ethanol, up to 80% of the
lipids were recovered [36]. The fluid/lipid mix-ture was then rapidly expanded to atmospheric pressure in a manner similar to that described in the RESS process. During the expansion process, an aqueous solution containing the sol-ute to be entrapped was simultaneously added to produce liposomes. Most of the liposomes produced were small unilamellar vesicles, and their EE was reported to be approximately 20% lower than that achieved using conventional methods. However, the consumption of ethanol to produce liposomes was 15-fold less than that consumed by the conventional ethanol injection method [37,38].
In 2005, Castor introduced phospholipid nanosomes. These are small uniform liposomes prepared by methods similar to the injection and decompression methods, which can encap-sulate hydrophilic molecules, such as proteins and nucleic acids. The size and characteristics of phospholipid nanosomes depend on process parameters and material properties, including the size and design of the decompression nozzle, bubble size, pressure, the rate of decompression, interfacial forces, charge distribution and the nature of the compound being encapsulated. The size range of liposomes was reported to be between 100 nm and 4 µm, but the aver-age size was less than 200 nm. Liposomes were found to be stable in terms of their particle size distribution when stored for 6 months at 4°C. In vitro and in vivo data on breast cancer cells
and xenografts in nude mice indicat that pacli-taxel nanosomes were less toxic and much more effective than paclitaxel in Cremophor® EL (BASF Corp., NJ, USA; Taxol®; Bristol–Myers Squibb, NY, USA). A drawback of this method is the high processing temperature; the amount of time and pressure required for this process is relatively high [39].
In 2010, Castor invented an improved pro-cess to coencapsulate hydrophobic and hydro-philic drugs in phospholipid liposomes. In this method, a phospholipid and hydrophobic drug solution are dissolved in SCF with or without a cosolvent or modifier. Afterwards, the phospho-lipid and hydrophobic drug solution is depres-surized, resulting in the formation of uniform liposomes that encapsulate hydrophobic and hydrophilic drugs. This method has been proven to be very useful for combination therapy [106].
Rapid expansion of supercritical solutionsThe RESS process was first patented in the USA in 1986 by Smith and since then has been widely used for the particle precipitation of many active pharmaceutical ingredients [40,107]. In the RESS process, the solute is dissolved in SCF and then the solution is rapidly expanded (decompressed) by passing it through a heated nozzle at super-sonic speed. During the rapid expansion of the supercritical solution, both its density and solvent power decrease dramatically, leading to solute supersaturation that causes very rapid nucleation of the substrate in the form of very small par-ticles that are collected from the gas stream [40,41]. This process yields small particles with a uni-form size. Larger particles can also be obtained by controlling the process parameters [42].
The conventional RESS process cannot be used for liposome formulation because, in the conventional process, solutes must be dissolved in the supercritical solvent. However, phospho-lipids are not soluble in pure SCF-CO
2. Further-
more, phospholipids can only assemble them-selves into liposomes in an aqueous medium. As a result, the conventional RESS process is not applicable for liposome preparation. Wen et al. introduced a modified RESS technique in which phospholipids and essential oils are dissolved in a mixture of SCF-CO
2 and ethanol, and then
the solution is sprayed into a buffer solution to form a liposome suspension. By controlling the pressure and temperature of this process, higher EE and drug loading were achieved. Liposomes appeared as double-layered spheres with a uni-form and narrow size distribution. Therefore, the
Nanomedicine (2013) 8(9)1536 future science group
Review Karn, Cho & Hwang
modified RESS process shows good prospects for the scaled-up production of liposomes [43].
Zhang et al. synthesized sirolimus liposomes based on the RESS process; phospholipids and cholesterol were dissolved in a sirolimus solution containing water and Tween®-80 (Sigma Aldrich Corp., MO, USA), and ethanol was added to the solution. The solution was then placed in a reac-tion vessel, into which SCF-CO
2 gas had been
blown. The researchers studied the effects of temperature, pressure and equilibrium time on the average particle size and envelope rate of lipo-somes. They found that the particles obtained by this process were uniform and medium sized with a narrow size distribution [44].
�n Processes with SCF-CO2 as an antisolvent (GAS/SAS/ASES)Antisolvent precipitation processes, for example, GAS, SAS, ASES and solution-enhanced disper-sion by SCFs, are subdivided based on the specific dispersal and mixing techniques involved. These methods are particularly important method for materials that cannot be easily dissolved in SCF-CO
2 and are, therefore, unsuitable substrates for
the RESS process. For a material to be processed by GAS/SAS/ASES, it must be reasonably insol-uble in antisolvent gases or SCFs, and must be soluble in organic solvents or SCFs. Liposomes prepared using various antisolvent precipitation processes are further described below.
Gas antisolventGallagher et al. first proposed the GAS process [45]. The GAS process was specifically devel-oped to achieve nanosizing of hydrophobic materials that cannot be processed by the RESS technique due to their poor solubility in SCFs. GAS is a batch technique in which the gradual introduction of antisolvent CO
2 expands the
organic solution the solute has been dissolved in. The subsequent reduction in solvent power causes supersaturation of the solution, resulting in precipitation of the substrate [46].
Kadimi et al. introduced AmB-intercalated liposomes by a process based on the GAS prin-ciple. AmB and acidified phosphatidyl choline (PC) were solubilized in an equal volume solu-tion of chloroform and methanol at 65°C. Soya PC and cholesterol were then mixed with the AmB–PC lipohilic complex. Hydrogenated soy PC and cholesterol were also dissolved in a mixture of methanol and chloroform and then mixed with the AmB–PC complex, yielding a translucent orange solution. This complex was then pumped into the reaction vessel, where
SCF-CO2 was introduced into the mix. The
pressure was 150 bar, while the temperature used for this process was high at 60–65°C. The high-pressure vessel was then depressurized and saline solution (0.9%) was introduced to induce the formation of liposomes. These authors compared AmB liposomes prepared using SCF liposomes with those prepared using conventional sonica-tion methods. Both liposome preparations were spherical in shape; however, the size of liposomes obtained by the SCF-CO
2 GAS method was
approximately 0.15–3 µm, whereas liposomes formed by the sonication method ranged in size from 0.15 to 6 µm. The intercalation efficiency of AmB was slightly better when the SCF-CO
2
GAS method was used than the conventional method. SCF liposomes were more stable than liposomes obtained using the conventional sonication method. Kadimi and colleagues concluded that the GAS method is an efficient method to prepare uncontaminated liposomes with improved physicochemical properties [47].
Recently, Ghatnur et al. extended the work of Kadimi et al. by preparing a sea buckthorn (SBK) leaf extract, incorporating liposomes using GAS and conventional Bangham thin-film methods. SBK has been reported to be a medicinal food and it also has hypoglycemic effects. For the SCF-CO
2 GAS method, SBK
leaf extract, PC and cholesterol were dissolved in organic solvent (chloroform and ethanol) and then loaded into a high-pressure reactor vessel, where compressed CO
2 (120 bar) was intro-
duced at a temperature of 65°C. After complete mixing of the loaded materials with SCF-CO
2,
the system was depressurized, the mixture was subsequently hydrated by adding buffer solu-tion and repressurized to the same conditions. Finally, the system was depressurized again to obtain liposomes containing SBK leaf extract. These researchers also prepared liposomes using a thin-film method (Bangham method) and thereafter sonicated them for comparison with liposomes prepared using the SCF-CO
2 GAS
method. The SCF-CO2
GAS method lipo-somes were multilamellar with a mean diam-eter of 930 nm and a narrow size distribution (0.48–1.07 µm), whereas liposomes prepared by the Bangham method were multilamellar with a much larger particle size (3740 nm) and broader size distribution (1.57–6.0 µm). The EE of liposomes prepared using the SCF-CO
2
GAS process was 28.42%; almost double that of the EE (14.6%) of conventional liposomes. The antioxidant effect of the extract encapsulated in the liposomes was superior to that of the pure
www.futuremedicine.com 1537future science group
Liposomal drug products & supercritical fluid-mediated liposomes Review
form. However, further studies are required to draw firm conclusions about the effects of this process [48].
Supercritical antisolventThe SAS technique is quite similar to the GAS process; the only difference is that the polymer is dissolved in a liquid solvent instead of in a gas, and the resulting solution is sprayed in a chamber containing SCF as the antisolvent. The advantage of SAS over GAS is that rapid contact between the two media (antisolvent and polymer solution) can be achieved, which speeds up the process of nucleation and growth, resulting in the formation of smaller particles.
Naik et al. developed a method to prepare and characterize docetaxel-entrapped PEGylated liposomes using the SAS technique that may be suitable for large-scale industrial applica-tion. In this method, docetaxel, phospholipids (hydrogenated soy PC and soy PC) and cho-lesterol are first dissolved in organic solvents (chloroform:methanol) and distearoylphos-phatidyl ethanolamine–PEG is then added to prepare PEGylated liposomes. Liquefied CO
2
is then converted to a supercritical state at an optimized temperature and pressure, and the mixture is sprayed into a high-pressure vessel in which the pressure and temperature of the vessel are at steady state. The SCF-CO
2 in the vessel
allows mixing of the drug–lipid solution and precipitation of proliposomes with entrapped docetaxel. Hydration then converts prolipo-somes into liposomes. The liposomes obtained by this method are small, unilamellar and spher-ical, with a size range of 200–300 nm and an EE of approximately 80%. These formulations were completely free of residual solvents and the lipo-somes were stable for at least 3 months [49]. Very recently, RGD-grafted PEGylated docetaxel lipo-somes were prepared using the same technique; grafting of RGD to the PEGylated docetaxel liposomes significantly improved antiprolifera-tive activity compared with the free drug and PEGylated docetaxel liposomes. Although these RGD-grafted PEGylated docetaxel liposomes are still at an early preclinical stage, they may be a feasible treatment option for breast cancer treatment if future studies demonstrate that they have low toxicity, good bioavailability and high efficacy [50].
Lesoin et al. developed a SAS process for lipo-some preparation, and compared these liposomes with those produced using a conventional Bang-ham method [23]. The SAS process involved the cocurrent spraying of a solution composed of
soy lecithin, cholesterol and ethyl alcohol into a continuous supercritical phase of CO
2. Simulta-
neous dissolution of the SCF in the liquid phase and evaporation of the organic solvent in the supercritical phase resulted in supersaturation of the solute in the liquid phase and then its precipitation. More CO
2 was used to remove
the ethyl alcohol completely. Small samples of microparticles were obtained after depressuriza-tion. All experiments were carried out at 35°C. Similarly, thin films were also obtained by the conventional Bangham method, in which lipids and cholesterol were dissolved in ethyl alcohol, introduced into a rotary evaporator and solvent evaporated at 50°C. Both the micronized phos-pholipid particles (SAS process) and dry phos-pholipid film (conventional method) were fur-ther hydrated at ambient conditions by an aque-ous solution of calcein (encapsulated marker) under stirring to obtain liposomes for further investigation. Lesoin et al., compared the exper-imental parameters, microscopic observations, yield, effect of solvent and EE of both types of liposomes, and reported that the SAS process appeared to be a more efficient and environ-mentally friendly method than the conventional Bangham method [23,34]. They reported that liposomes prepared using the SAS process were more spherical and stable than the Bangham liposomes. Similar results have been reported by several other research groups [28,47]. The stability of liposomes is always an issue when applying liposomes to biological systems [51]. Some stud-ies have demonstrated that the use of dry phos-pholipid microparticles (dry liposomes or proli-posomes) enhances liposome stability and drug encapsulation. However, the micronized parti-cles have to be handled with great precaution before the hydration step. Hence, Lesoin et al. introduced a new method called a continuous antisolvent process in which micronization and hydration are performed in a single step [23,34].
In 2011, Hwang et al. introduced a novel SCF-CO
2 method based on the SAS technique to pre-
pare liposomes in which a dried mixture of lipids, cholesterol and the drug of interest are coated with anhydrous lactose, a water-soluble carrier. The concept underlying this method is the same as that of proliposomes. Hwang and colleagues first studied liposomal AmB and investigated the effect of solvent, process temperature, pressure, surface area of the carrier particle and hydration temperature on an AmB-liposomal preparation by using SCF-CO
2 and the conventional Bang-
ham method. After preparation, liposomes were characterized and the effects of microfluidization
Nanomedicine (2013) 8(9)1538 future science group
Review Karn, Cho & Hwang
and lyophilization on liposomal properties were investigated. AmB liposomes obtained after homogenization were unilamellar and spherical in shape with an average diameter of 100 nm and an EE greater than 80%. SCF-CO
2 liposomes
were found to be more stable than conventional liposomes for a test period of 1 month, leading the authors to propose the possibility of long-term storage of SCF-CO
2 liposomes by lyophilization.
A hemolysis study revealed that liposomal AmB prepared by the SCF-CO
2 method was less hema-
totoxic than a commercial micellar formulation of AmB Fungizone® (Bristol-Myers Squibb, NJ, USA). This method could, therefore, be a supe-rior alternative to the conventional Bangham method, and is well suited to the mass production of liposomes [101].
Xia et al. investigated process parameters for the eff icient preparation of coenzyme Q
10-encapsulating liposomes using the SAS tech-
nique. Like other SAS processes described ear-lier, the mixture of SCF-CO
2 and drug solution
in a high-pressure vessel reached supersaturation, resulting in precipitation of coenzyme Q
10 proli-
posomes in the vessel. The optimal pressure and temperature conditions were found to be 35°C and 8 MPa, respectively [52].
Karn et al. [28] adopted the method invented by Hwang et al. [101] to prepare liposomal CsA. This process is very simple, as illustrated in Figure 3. For preparation, Lipoid S100/E80 (Lipoid GmbH,
Ludwigshafen, Germany), cholesterol and CsA are dissolved in ethanol, and then the mixture is sealed in a reaction vessel with 900 mg of anhy-drous lactose. The optimal pressure and temper-ature were investigated by analyzing the physico-chemical properties of the resulting liposomes; a pressure of 10 MPa and a temperature of 45°C were found to be optimal for the preparation of liposomal CsA [28]. The resulting phospho-lipid–drug mixture is coated on the surface of lactose particles to form a thin film; subsequent hydrolysis generates MLVs. This method is based on the SAS process, but the approach is entirely different from that of other SAS/GAS methods where proliposomes are obtained as fine and aggregated particles before hydration [23,47,49]. As a control in their experiment, liposomes were also prepared using the same composition and conditions with the conventional method, and the differences between liposomes prepared using the SCF-CO
2 and conventional modified
Bangham methods were thoroughly investi-gated. They reported that SCF-CO
2 liposomes
did not show any alteration in size or content after 14 weeks of study. Therefore, liposomes prepared using SCF-CO
2 were physically and
chemically more stable than liposomes prepared using the conventional method. This result was consistent with previous studies that reported that liposomes prepared using the SCF-CO
2
method were more stable [23,34,101]. The stability
Pressure release valve
Pressure indicator
Reactionvessel
H2O
StirrerStirring tip Temperatureindicator
Syringe pump
CO2 storagetank
Figure 3. Experimental apparatus for liposome preparation using a supercritical fluid-CO2 method. Reproduced with permission from [28].
www.futuremedicine.com 1539future science group
Liposomal drug products & supercritical fluid-mediated liposomes Review
of liposomes prepared using SCF-CO2 might be
due to static repulsion of carbonic acids (formed between CO
2 and water) incorporated into the
bilayer membrane [53]. Furthermore, the SCF-CO
2 liposomes were smaller, uniform and more
spherical than the liposomes prepared using the conventional method. Additionally, a nontoxic organic solvent (ethanol) was used, which evap-orated completely before hydration. Although there is strong experimental evidence that SCF-CO
2 methods have several advantages over con-
ventional Bangham methods, these liposomes need to be tested in clinical studies to confirm their effectiveness. As the synthesis of liposomes was easier to scale-up, the technique described above could potentially be used by pharmaceuti-cal companies to develop liposomal products for clinical applications [28].
Aerosol solvent extraction systemsThe ASES process is also used to generate liposomes. This process was first invented by Muller and Fischer in 1989 [54,108]. It utilizes the extraction properties of supercritical gases to produce microparticles [54], and involves spraying or dispersing an organic solution via a nozzle into a flowing or static dense gas [46]. In 2006, Kunastitchai et al. first applied the ASES process to prepare liposomes as dry and reconstitutable vesicles for large-scale produc-tion [55]. This process, essentially similar to the SAS process, uses SCF-CO
2 as an antisolvent.
An antifungal agent, miconazole, was used as the model drug in this study, and liposomes containing miconazole were processed in two steps [56]. First, micro particles were produced by ASES by mixing the drug, phospholipids and cholesterol at different molar ratios, followed by dissolving the mixture in organic solvents (chloroform and methylene chloride). In some experiments, poloxamer 407 was used as a sur-factant. The solution was then injected into a high-pressure vessel containing SCF-CO
2. Par-
ticle formation under the adjusted extraction conditions occurred instantaneously because the organic solvents were miscible with SCF-CO
2.
SCF-CO2 was then discharged from the vessel
and microparticles containing miconazole were obtained in a dry form. In the second step, these dry particles were hydrated with phosphate buf-fer at 55°C and agitated by vortex mixing to produce liposomes. Very little organic solvent residue remains after this process, and solid microparticles/liposomes are produced instan-taneously in a single production step. Aggre-gates of microparticles ranged in size from a few microns to 40 µm. The residual content of methylene chloride and methanol was lower than 30 and 86 ppm, respectively. The percent-age of drug incorporated into the microparticles was increased by increasing the drug concen-tration in the spraying solution. Similarly, EE and the size of the liposomes were increased by increasing the pH of the hydration medium.
Viewthrough eye
Hand pump
Pressure transducer
Reaction vesselor view cell
StirrerStirring tip Temperatureindicator
PL andD-glucosesolution
200 bar, 60°C,40 minCO2 storage
tank
Nanomedicine © Future Science Group (2013)
Figure 4. Simplified and modified representation of the improved supercritical reverse-phase evaporation method explained by Otake et al. [53]. PL: Phospholipid.
Nanomedicine (2013) 8(9)1540 future science group
Review Karn, Cho & HwangTa
ble
4. O
rig
inal
res
earc
h p
aper
s d
escr
ibin
g s
up
ercr
itic
al fl
uid
-med
iate
d li
po
som
e p
rod
uct
ion
.
Stu
dy
(yea
r)Pr
epar
atio
n
met
ho
dD
rug
use
d in
th
e st
ud
yPr
op
erti
es o
f SC
F-C
O2
lipo
som
esA
dva
nta
ges
Ref
.
Fred
erik
sen
et a
l. (1
997)
RE
SSFI
TC, d
extr
an a
nd
TSZn
PcSi
ze: 2
00
± 2
5 nm
/SU
V; h
igh
PDI;
no s
uppo
rt f
or s
hape
; EE
: 20%
Suita
ble
for
hydr
ophi
lic d
rugs
[38]
Ota
ke e
t al
. (20
01)
SCRP
EG
luco
se a
nd
chol
este
rol
100
–20
0 nm
/LU
V; e
llips
oida
l sha
pe; E
E: u
p to
23%
for
hy
drop
hilic
dru
gs, 6
3% f
or li
poph
ilic
mat
eria
lsSi
ngle
ste
p, e
asy
to s
cale
-up
[58]
Imur
a et
al.
(20
02)
SCRP
EG
luco
se
Ellip
soid
al t
o sp
heric
al; L
UV
and
/or
MLV
; siz
e: 0
.1–1
.2 µ
m;
EE: 3
–10%
H
ighe
r co
nten
t of
PC
incr
ease
s EE
[59]
Imur
a et
al.
(20
03)
SCRP
EG
luco
seLU
Vs;
siz
e: v
aria
ble;
EE:
up
to 2
0 (D
PPC
) and
40%
(D
OPC
)M
echa
nism
of
lipos
ome
form
atio
n by
SC
RPE,
co
ntro
llabl
e ph
ysio
chem
ical
pro
pert
ies
[60]
Ota
ke e
t al
. (20
06
)IS
CRP
EG
luco
seU
nila
mel
lar;
loos
ely
pack
ed s
truc
ture
; EE:
up
to 3
5%Ea
sy t
o pe
rfor
m, h
igh
trap
ping
effi
cien
cy, h
ighl
y st
able
[53]
Ota
ke e
t al
. (20
06
) IS
CRP
E, c
hito
san
coat
ing
Glu
cose
LUV
; EE:
up
to 1
7% w
ith o
r w
ithou
t ad
ditio
n of
chi
tosa
nSi
mpl
e m
etho
d to
sca
le-u
p ea
sily
, chi
tosa
n-co
ated
lip
osom
es p
repa
red
in o
ne s
tep
[62]
Brid
son
et a
l. (2
00
6)
Com
pres
sed
CO
2 so
lven
t m
etho
d –
RESS
Cip
roflo
xaci
n hy
droc
hlor
ide
Size
: 50
nm t
o 1
µm; o
ligol
amel
lar;
thr
ee t
o fiv
e bi
laye
rs;
EE: <
20%
for
hyd
roph
ilic
drug
sC
ompl
etel
y fr
ee f
rom
res
idua
l sol
vent
s, p
ossi
ble
alte
rnat
ive
to c
onve
ntio
nal m
etho
ds[14]
Kun
astit
chai
et
al.
(20
06
)A
SES
Mic
onaz
ole
Mic
ropa
rtic
les:
siz
e: 1
–40
µm; y
ield
: 50
–70%
; EE
: 25
–70%
; sph
eric
al. L
ipos
omes
: siz
e: 2
.7–9
.4 µ
m; E
E:
4–1
00%
dep
endi
ng o
n th
e pH
of
the
hydr
atin
g m
ediu
m
Resi
dual
sol
vent
s: e
xtre
mel
y lo
w li
mit
valu
e, d
irect
pr
oduc
tion
of d
ry f
orm
lipo
som
es[55]
Kun
astit
chai
et
al.
(20
07)
ASE
SM
icon
azol
eM
icro
part
icle
s: s
ize:
20
–30
µm; s
pher
ical
but
agg
rega
ted
; D
L: 4
.8–2
6.1%
. Lip
osom
es: s
ize:
3–6
µm
; DL:
3.1
–18.
7M
icro
part
icle
s st
able
at
4°C
for
5 m
onth
s; li
poso
mes
un
stab
le in
hyd
rate
d fo
rm[57]
Kad
imi e
t al
. (20
07)
GA
S A
mph
oter
icin
BSi
ze: 0
.15
–3 µ
m; m
uch
smal
ler
size
lipo
som
es a
re
abun
dant
; sph
eric
al; i
nter
cala
tion
effic
ienc
y: 2
3–3
5%;
high
fun
gici
dal a
ctiv
ity
Stab
le li
poso
mes
; sim
ple,
effi
cien
t pr
epar
atio
n m
etho
d; n
o co
ntam
inat
ion
[47]
Saka
i et
al. (
200
8)
SCRP
EG
luco
seSi
ze: 1
50–6
00
nm; E
E: u
p to
40%
; LU
V; s
pher
ical
Exam
ined
eff
ect
of a
lkyl
gro
up o
n m
embr
ane
prop
ertie
s of
lipo
som
es[63]
Nai
k et
al.
(201
0)
SAS
Doc
etax
elSi
ze: 2
00
–30
0 nm
; z-p
oten
tial:
-27
mV
; EE:
80%
; sm
ooth
su
rfac
e; a
mor
phou
s na
ture
Stab
le li
poso
mes
, fre
e fr
om r
esid
ual s
olve
nts,
can
po
ssib
ly b
e us
ed f
or in
dust
rial a
pplic
atio
ns[49]
Wen
et
al. (
2010
)M
odifi
ed R
ESS
Atr
acty
lode
s m
acro
ceph
ala
Size
: ~20
0 nm
; mon
odis
pers
ed; s
pher
ical
with
dou
ble-
laye
red
lipid
mem
bran
e; n
arro
w p
artic
le s
ize
dist
ribut
ion;
EE
: 78
–85%
; DL:
5–6
%
Phys
icoc
hem
ical
pro
pert
ies
and
stab
ility
mee
t th
e st
anda
rds
of t
he C
hine
se p
harm
acop
oeia
for
fo
rmul
atio
ns
[43]
Leso
in e
t al
. (20
11)
SAS
Cal
cein
(mar
ker)
Size
: 0.1
–10
0 µm
; yie
ld: 7
5–8
5%; E
E: u
p to
20%
Envi
ronm
enta
lly f
riend
ly p
roce
ss, s
tabl
e lip
osom
es[23]
Leso
in e
t al
. (20
11)
CA
SN
o dr
ugSi
ze: 1
0–1
00
µm; m
ultil
amel
lar;
sph
eric
alSi
ngle
ste
p an
d co
ntin
uous
pro
cess
, sca
le-u
p po
ssib
le[34]
ASE
S: A
eros
ol s
olve
nt e
xtra
ctio
n sy
stem
; CA
S: C
ontin
uous
ant
isol
vent
pro
cess
; DL:
Dru
g lo
adin
g; D
OPC
: 1,2
-dio
leoy
l-sn
-gly
cero
-3-p
hosp
hoch
olin
e; D
PPC
: Dip
alm
itoyl
phos
phat
idyl
cho
line;
EE:
Ent
rapm
ent
effic
ienc
y; F
ITC
: Fl
uore
scei
n is
othi
ocya
nate
; GA
S: G
as a
ntis
olve
nt p
roce
ss; I
SCRP
E: Im
prov
ed s
uper
criti
cal r
ever
se-p
hase
eva
pora
tion
proc
ess;
LU
V: L
arge
uni
lam
ella
r ve
sicl
e; M
LV: M
ultil
amel
lar
vesi
cle;
PC
: Pho
spha
tidyl
cho
line;
PD
I: Po
lydi
sper
sity
inde
x; R
ESS:
Rap
id e
xpan
sion
of
supe
rcrit
ical
sol
vent
s; S
AS:
Sup
ercr
itica
l ant
isol
vent
pro
cess
; SC
F: S
uper
criti
cal fl
uid
; SC
RPE:
Sup
ercr
itica
l rev
erse
-pha
se e
vapo
ratio
n pr
oces
s; S
UV
: Sm
all u
nila
mel
lar
vesi
cle;
TSZ
nPc:
Zin
c ph
thal
ocya
nine
tet
rasu
lfoni
c ac
id.
www.futuremedicine.com 1541future science group
Liposomal drug products & supercritical fluid-mediated liposomes Review
Kunastitchai et al. also studied the physical and chemical stability of miconazole liposomes prepared using the ASES process [57]. They used the same method of liposome preparation as Kunastitchai et al [55]. They reported that liposomes in dry form (microparticles) were stable when stored at 4°C, but were significantly degraded after storage at 25°C. Hydrated lipo-somes containing cholesterol aggregated when stored at 4°C tended to decrease in size at pH 7.2. Addition of poloxamer retarded lipo-some degradation. The authors of this study did not explain these findings [57].
�n Supercritical reverse-phase evaporation The SCRPE method was first invented by Otake et al. in 2001 [58]. The basic concept of this method is similar to that of the conventional reverse-phase evaporation method. SCF-CO
2
is used to dissolve phospholipids and organic cosolvents sealed in a view cell at a temperature higher than the phase transition temperature of the phospholipid. An aqueous solution is then introduced into the cell. Liposomes form when the pressure is reduced by the release of the SCF-CO
2.
In 2001, Otake et al. developed a new method called the SCRPE method to prepare liposomes in a single step [58]. This method represents a great advance in liposome technology for practi-cal purposes. In the SCRPE method, liposomes are prepared by introducing water into a homo-geneous mixture of SCF-CO
2, phospholipids
and ethanol. Introduction of additional water results in the formation of a macroemulsion from the water/CO
2 microemulsion based on
reversed lipid micelles. Liposomes then form as CO
2 evaporates from the aqueous phase upon
depressurization. This method permits prepa-ration of liposomes with a high EE for both water-soluble and hydrophobic substances. The SCRPE method yields aqueous dispersions of LUVs with diameters of 0.1–1.2 µm; these lipo-somes are much closer in size to real cells than the liposomes prepared by Frederiksen and col-leagues [38]. The method is physically simpler than the method described by Frederiksen and colleagues. Compared with the Frederiksen method, the SCRPE method requires a smaller amount of CO
2.
In 2002, Imura and colleagues [59] adopted the same method described by Otake et al. [58] to study three different kinds of soybean lecithins varying in their composition of PC, phosphatidyl etha-nolamine, phosphatidyl inositol and phosphatidic Ta
ble
4. O
rig
inal
res
earc
h p
aper
s d
escr
ibin
g s
up
ercr
itic
al fl
uid
-med
iate
d li
po
som
e p
rod
uct
ion
(co
nt.
).
Stu
dy
(yea
r)Pr
epar
atio
n
met
ho
dD
rug
use
d in
th
e st
ud
yPr
op
erti
es o
f SC
F-C
O2
lipo
som
esA
dva
nta
ges
Ref
.
Zhan
g et
al.
(201
2)
RESS
Siro
limus
Mic
ropa
rtic
les
with
a n
arro
w p
artic
le s
ize
dist
ribut
ion
Expl
ains
the
influ
enci
ng f
acto
rs o
f si
rolim
us li
poso
mes
an
d th
e na
rrow
par
ticle
siz
e di
strib
utio
n ob
tain
ed[44]
Xia
et
al. (
2012
) SA
S (p
rolip
osom
e)C
oenz
yme
Q10
Size
: 50
–69
nm; m
onod
ispe
rsed
; SU
V; E
E: 8
2.28
%;
DL:
8.9
2%; r
ecov
ery:
48.
51%
Firs
t re
port
of
coen
zym
e Q
10 p
rolip
osom
es/li
poso
mes
us
ing
SAS
[52]
Gha
tnur
et
al.
(201
2)
GA
SSe
a bu
ckth
orn
leaf
ex
trac
tSi
ze: 9
30–1
425
nm; M
LVs;
sph
eric
al; s
ize
dist
ribut
ion:
0.
48
–1.7
8 µm
Supe
rior
phys
icoc
hem
ical
pro
pert
ies
com
pare
d w
ith
SCF-
CO
2 G
AS
lipos
omes
[48]
Kar
n et
al.
(201
3)
SAS
Cyc
losp
orin
ASi
ze: 1
50–1
40
0 nm
; MU
V; s
pher
ical
; mor
e ho
mog
eneo
us;
EE: >
90%
; no
chan
ges
in s
ize/
cont
ent
afte
r 3
mon
ths
Suita
ble
for
larg
e-sc
ale
use,
eas
y an
d co
nven
ient
m
etho
d, c
ompa
rativ
ely
mor
e st
able
lipo
som
es[28]
ASE
S: A
eros
ol s
olve
nt e
xtra
ctio
n sy
stem
; CA
S: C
ontin
uous
ant
isol
vent
pro
cess
; DL:
Dru
g lo
adin
g; D
OPC
: 1,2
-dio
leoy
l-sn
-gly
cero
-3-p
hosp
hoch
olin
e; D
PPC
: Dip
alm
itoyl
phos
phat
idyl
cho
line;
EE:
Ent
rapm
ent
effic
ienc
y; F
ITC
: Fl
uore
scei
n is
othi
ocya
nate
; GA
S: G
as a
ntis
olve
nt p
roce
ss; I
SCRP
E: Im
prov
ed s
uper
criti
cal r
ever
se-p
hase
eva
pora
tion
proc
ess;
LU
V: L
arge
uni
lam
ella
r ve
sicl
e; M
LV: M
ultil
amel
lar
vesi
cle;
PC
: Pho
spha
tidyl
cho
line;
PD
I: Po
lydi
sper
sity
inde
x; R
ESS:
Rap
id e
xpan
sion
of
supe
rcrit
ical
sol
vent
s; S
AS:
Sup
ercr
itica
l ant
isol
vent
pro
cess
; SC
F: S
uper
criti
cal fl
uid
; SC
RPE:
Sup
ercr
itica
l rev
erse
-pha
se e
vapo
ratio
n pr
oces
s; S
UV
: Sm
all u
nila
mel
lar
vesi
cle;
TSZ
nPc:
Zin
c ph
thal
ocya
nine
tet
rasu
lfoni
c ac
id.
Nanomedicine (2013) 8(9)1542 future science group
Review Karn, Cho & Hwang
acid, in the context of liposome preparation by the SCRPE method. The selected lecithins, namely Lecinol® S-10 EX (Nikko Chemicals Co., Ltd, Tokyo, Japan; 95% PC), Lecinol S-10 (Nikko Chemicals Co., Ltd; 32% hydrogenated PC) and SLP white SP (32% unhydrogenated PC), were selected to evaluate the properties of liposomes. Liposomes with Lecinol S-10 EX were ellipsoidal LUVs with a mean diameter of 0.2–1.2 µm; by contrast, use of the other two lecithins yielded spherical MLVs with a mean diameter of 0.1–0.25 µm. The z-potentials of liposomes prepared using Lecinol S-10 and SLP white SP were higher than that of Lecinol S-10 EX, suggesting that the dispersibility of Lecinol S-10 and SLP white SP was better than that of SLP S-10 EX. Lecithin with a high PC content (Lecinol-S10 EX) was found to be more effec-tive for synthesizing liposomes with a high EE for water-soluble drugs than liposomes synthe-sized with phosphatidyl ethanolamine, phospha-tidyl inositol and phosphatidic acid (negatively charged lipids). They found that the structure of liposomes prepared by the SCRPE method was dependent on the solubility of phospholipids in the SCF-CO
2/ethanol mixture [59].
Imura et al. also studied the mechanism of liposome formation by SCRPE. They reported that a physicochemical mechanism was involved because colloidal structures, such as water/CO
2 or CO
2/water emulsions obtained
before liposome preparation, directly inf lu-enced the physicochemical properties of the liposomes such as their particle size, EE and lamellarity. Importantly, these authors claimed that control of the physicochemical properties of the liposomes could be achieved by chang-ing process parameters slightly (i.e., pressure or amount of ethanol). Liposomes prepared using 1,2-dioleoyl-sn-glycero-3-phosphocholine had a higher EE than dipalmitoylphosphatidyl choline (DPPC) liposomes with the same phospholipid concentration. The reason for this difference is the more loosely packed structure of 1,2-dio-leoyl-sn-glycero-3-phosphocholine bilayers than DPPC bilayers. Owing to the single-step nature of this method, scaled-up liposome production is possible [60].
Liposomes modified with lipopeptides to increase the EE of bovine serum albumin as a model drug were prepared by the SCRPE method [61]. DPPC, cholesterol, lipopeptides and ethanol were sealed in a vessel and CO
2 was introduced.
Temperature and pressure were maintained at 45°C and 20 MPa, respectively, and an aqueous solution of the drug was added into the cell by Ta
ble
5. P
aten
ts r
elat
ed t
o li
po
som
es s
ynth
esiz
ed u
sin
g s
up
ercr
itic
al fl
uid
met
ho
ds.
Pate
nt
Titl
e o
f th
e in
ven
tio
nIn
ven
tor(
s)D
escr
ipti
on
of
pro
cess
es in
volv
edR
ef.
PCT
WO
94/2
7581
(199
4)
Met
hods
and
app
arat
us f
or m
akin
g lip
osom
esC
asto
r In
ject
ion
and
deco
mpr
essi
on[102]
PCT
WO
96/1
5774
(199
6)
Met
hods
and
app
arat
us f
or m
akin
g lip
osom
es f
or h
ydro
phob
ic d
rugs
C
asto
r an
d C
hu
Dec
ompr
essi
on a
nd in
ject
ion
[104
]
US1
997-
570
04
82Pr
oces
s fo
r th
e pr
epar
atio
n of
a li
poso
me
disp
ersi
on u
nder
ele
vate
d pr
essu
re
cont
ents
Fred
erik
sen
et a
l.M
odifi
ed in
ject
ion
and
deco
mpr
essi
on[105]
JP20
03-1
1912
0M
etho
d fo
r pr
oduc
ing
lipos
omes
, cos
met
ics
cont
aini
ng t
he li
poso
me,
and
ski
n ca
re
prep
arat
ion
Abe
et
al.
Mix
ing,
dec
ompr
essi
on a
nd fi
ltrat
ion
[109]
JP20
06
-069
930
Lipo
som
e an
d it
s pr
ecur
sor
emul
sion
mix
ture
Ued
a et
al.
Emul
sion
mix
ture
[111]
JP20
06
-063
052
Ultr
asou
nd im
agin
g ag
ent
cont
aini
ng li
poso
mes
, and
pro
duct
ion
ther
eof
Ued
a M
ixin
g an
d fo
rmin
g m
icel
les
[114]
US2
00
6-0
2399
25 A
1M
etho
d of
man
ufac
turin
g ph
arm
aceu
tical
pre
para
tion
cont
aini
ng li
poso
mes
Wad
a an
d U
eda
Mix
ing,
dec
ompr
essi
on a
nd fi
ltrat
ion
[116]
JP20
06
-063
009
Lipo
som
e co
mpo
sitio
ns f
or c
ance
r th
erap
y, a
nd m
anuf
actu
re t
here
ofA
oki a
nd U
eda
Mix
ing,
dec
ompr
essi
on a
nd fi
ltrat
ion
[113]
JP20
06
-298
842
Met
hod
for
prod
uctio
n of
lipo
som
e co
mpo
sitio
ns b
y us
ing
supe
rcrit
ical
flui
d C
O2
Mot
okui
and
Wad
a M
ixin
g, d
ecom
pres
sion
and
filtr
atio
n[117]
JP20
06
-298
845
Lipo
som
e co
mpo
sitio
ns c
onta
inin
g x-
ray
cont
rast
age
nts,
and
pro
duct
ion
ther
eof
Wad
a an
d M
otok
ui
Mix
ing,
dec
ompr
essi
on a
nd fi
ltrat
ion
[112]
EP-2
007
1776
948
Met
hod
for
prod
ucin
g lip
osom
e-co
ntai
ning
pre
para
tions
Nag
aike
and
M
otok
ui
Mix
ing,
dec
ompr
essi
on a
nd fi
ltrat
ion
[110]
US/
2010
/024
7620
A1
Met
hods
for
coe
ncap
sula
tion
of c
ombi
ned
drug
s an
d co
enca
psul
ated
com
bina
tion
of
drug
pro
duct
sC
asto
r In
ject
ion
and
deco
mpr
essi
on[106
]
KR
2011
-009
6962
New
met
hods
and
app
arat
us f
or p
repa
ring
lipos
omes
Hw
ang
et a
l. M
ixin
g, d
ecom
pres
sion
and
hyd
ratio
n (b
ased
on
SAS)
[101]
EP: E
urop
ean
pate
nt; J
P: J
apan
ese
pate
nt; K
R: R
epub
lic o
f K
orea
pat
ent;
PC
T: P
aten
t co
oper
atio
n tr
eaty
; SA
S: S
uper
criti
cal a
ntis
olve
nt p
roce
ss; U
S: A
mer
ican
pat
ent;
WO
: Wor
ld p
aten
t.
www.futuremedicine.com 1543future science group
Liposomal drug products & supercritical fluid-mediated liposomes Review
a HPLC pump. The vessel was then depressur-ized to obtain a liposome suspension containing the model drug. In all cases, EEs were much higher (up to 70%) than those obtained using the conventional Bangham method, and approx-imately 90% of the entrapped drug was retained for up to 48 h in liposomes produced using the SCRPE method, whereas no drug remained in liposomes produced using the Bangham method after 48 h [61].
Otake et al. reported an improved SCRPE method (ISCRPE) to obtain even higher EE using fewer process steps, which is, therefore, more amenable to scale-up, as illustrated in Figure 4 [53]. This technique does not use any organic solvent, and is very simple compared with the SCRPE method. PC and glucose are sealed in a reactor vessel and SCF-CO
2 is intro-
duced at a temperature of 60°C and pressure of 20 MPa. Liposomes are formed when the vessel is depressurized by releasing CO
2. These
researchers also prepared liposomes using the conventional Bangham method and SCRPE method as controls. The EE of ISCRPE lipo-somes was significantly higher than that of liposomes produced using the other two meth-ods. In comparison with the SCRPE method, the ISCRPE method had a greater mixing effi-ciency, resulting in the formation of a CO
2/water
emulsion with a high trapping efficiency. Phos-pholipids were loosely packed in the ISCRPE liposomes in comparison with their packing in the two other types of liposomes. Differential scanning calorimetry revealed that the ISCRPE liposomes had a smaller transition temperature than the Bangham and SCRPE liposomes. More importantly, liposomes prepared by the ISCRPE method were found to be highly stable, even at room temperature, compared with liposomes produced using the Bangham and SCRPE meth-ods. This method was used to synthesize chi-tosan-coated liposomes; the only modification was that chitosan solution was added to the lipid solution or aqueous glucose solution. These cat-ionic liposomes were stable for 30 days and had an EE of 17%, which is much higher than that of the Bangham method (2%). Owing to the sim-plicity and lack of organic solvent of the ISCRPE method, this technique can easily be scaled-up for mass production of liposomes [53,62].
Earlier studies showed that the EE of water-soluble drugs was much greater when liposomes were synthesized using the SCRPE method than the conventional Bangham method [58–60]. The effect of the length of the alkyl chain and num-ber of unsaturated bonds in the phospholipids
on the properties of liposomes prepared by the SCRPE method was studied by Sakai et al. [63]. Liposomes prepared using unsaturated phospho-lipids had a higher EE than those prepared using saturated phospholipids. The EE of liposomes prepared using the SCRPE method was found to depend on the alkyl chain length of the phos-pholipids. Thus, the EE increased as the number of alkyl groups increased, with the exception of DPPC liposomes.
�n Other methodsSeveral studies have been carried out in Japan using other methods, and patents have been filed [109–116]. The basic principles of these processes are similar to the SCRPE method described by Otake and colleagues [58]. The advantage of these processes is that they use a minimal amount of organic solvents or they do not use any organic solvents. Abe et al. invented liposomes for cos-meceuticals and hydrophilic drugs with a high stability and enclosure ratio [109]. Nagaike and Motokui invented an improved method for manufacturing liposomes with relatively higher hydrophilic drug incorporation and in vivo sta-bility than those produced using the method of Abe and colleagues [110]. In this method, a mixture of phospholipids and liquefied CO
2 is
introduced into a vessel at a suitable temperature (35–50°C) and pressure (100–400 kg/cm2) to create a supercritical state. An aqueous solution of iodine is then added to the mixture, and the pressure within the vessel is decreased and CO
2
is discharged to obtain an aqueous dispersion of liposomes enclosing iodine. Owing to the optimal temperature for liposome preparation being similar to body temperature, the liposomes showed enhanced stability in vivo. An enclosure (drug entrapped within the liposome) percent-age of approximately 20% was reported for these liposomes, which is higher than that reported in previous studies [110].
Ueda et al. used an emulsion process to enca psulate photosensitizers and imaging, anticancer or antifungal agents into liposomes [111]. The mechanism of how water/CO
2 or
CO2/water emulsion mixtures were obtained
before liposome preparation was investigated earlier by Imura et al. [60]. To prepare liposomes containing x-ray contrast agents, a mixture of phospholipids, cholesterol, and phospholipids with low molecular weight PEG were mixed with SCF-CO
2 at 40–65°C and 10–30 MPa.
Contrast-medium solution was injected, and the system was then decompressed and lipo-somes containing contrast-medium agents were
Nanomedicine (2013) 8(9)1544 future science group
Review Karn, Cho & Hwang
obtained as dispersion [112]. Aoki and Ueda introduced liposomes for incorporating anti-cancer drugs, which they termed ‘anti-tumor liposomes’ [113], using the same manufacturing process as Ueda et al. [114].
After reviewing all of the literature available, we have summarized the reviewed studies in the form of Table 4 and Table 5 for published papers and patents, respectively. In addition, the advantages and disadvantages of the preparation methods are listed in Table 6.
Some of the groups have investigated the signif-icance of novel SCF methods for the preparation of liposomes suitable for industrial applications. Liposomes are thermodynamically unstable, and their physicochemical properties directly relate to their preparation methods. In the case of SCF-CO
2 methods, control of physicochemical
properties of liposomes could easily be possible by changing the process parameters. In addition, SCF methods produce sterile, stable and uniform liposomes that are apparently free from residual solvents. These features make SCF-mediated lipo-somes ideal candidates for addressing the barriers of mass-scale production. Despite the remarkable and useful properties of these methods, none of the liposomal drug products approved so far have been produced using supercritical methods. The major reason why SCF-CO
2 methods have not
been applied for industrial-scale production is the lack of fundamental studies accurately describing the phase behavior of multicomponent mixtures and the long-term stability of the resulting lipo-somes, as well as the effects of elevated tempera-tures and pressures on the final drug formula-tions. In addition, obtaining the optimized condi-tions for solubilizing the drug and other materials in a SCF-CO
2 is quite complicated. In addition,
some processes, for example, the injection and decompression methods, RESS and ASES, utilize nozzles where solid components of the mixtures may block the nozzles in supercritical conditions and, thus, impede the efficiency of the process. Production costs reported for the SCF process are reasonable but some of the complex equip-ment requires higher capital cost. However, with continued innovation, some of the problems have already been resolved by introducing simple and improved methods, for example, ISCRPE, GAS and SAS processes. Most of the studies have proved that the liposomes produced by the SCF-CO
2 methods were more stable. However, for
clinical development, 3- or 6-month’s stability evaluations would not be enough. Therefore, long-term stability should be extensively assessed in order to apply these processes for clinical use. Ta
ble
6. A
dva
nta
ges
an
d d
isad
van
tag
es o
f su
per
crit
ical
flu
id-a
ssis
ted
pro
cess
es f
or
lipo
som
e p
rep
arat
ion
.
SCF-
CO
2 m
eth
od
sA
dva
nta
ges
Dis
adva
nta
ges
Inje
ctio
n an
d de
com
pres
sion
Ster
ile a
nd p
harm
aceu
tical
gra
de o
f lip
osom
es; c
ontr
ol o
f si
ze o
f lip
osom
es b
y ch
angi
ng t
he n
ozzl
e di
amet
er; n
arro
w p
artic
le s
ize
dist
ribut
ion;
eff
ectiv
ely
solv
ent
free
Com
plex
equ
ipm
ent;
low
yie
ld; h
igh
pres
sure
and
tem
pera
ture
use
d;
suita
ble
mai
nly
for
hydr
opho
bic
drug
s; n
ozzl
e of
ten
beco
mes
clo
gged
Supe
rcrit
ical
lipo
som
esSc
alab
le m
etho
d; m
uch
low
er c
onsu
mpt
ion
of o
rgan
ic s
olve
nt;
solv
ent
free
Ver
y hi
gh t
empe
ratu
re a
nd p
ress
ure
appl
ied
; ver
y lo
w E
E
RESS
Con
trol
labl
e pa
rtic
le s
ize;
sol
vent
fre
e; s
impl
e an
d ra
pid
Not
suc
cess
ful f
or li
poso
me
prep
arat
ion;
low
yie
ld a
nd E
E
GA
SA
pplic
able
to
varie
ties
of d
rugs
; var
iabl
e si
ze r
ange
s; s
pher
ical
; nar
row
pa
rtic
le s
ize
dist
ribut
ions
; rel
ativ
ely
stab
le; m
oder
ate
pres
sure
and
te
mpe
ratu
re; s
olve
nt f
ree,
unc
onta
min
ated
Batc
h pr
oces
s; u
ses
orga
nic
solv
ents
; sep
arat
ion
of g
as a
nd s
olve
nt
requ
ired
SAS
Low
org
anic
sol
vent
con
sum
ptio
n; c
an p
rodu
ce s
mal
l hom
ogen
eous
lip
osom
es; s
tabl
e lip
osom
es; c
ontr
olla
ble
para
met
ers;
sim
ples
t m
etho
d; m
oder
ate
pres
sure
and
tem
pera
ture
use
d; s
tabl
e lip
osom
es;
scal
able
; sol
vent
fre
e; u
ncon
tam
inat
ed
Use
s or
gani
c so
lven
ts; s
epar
atio
n of
gas
and
sol
vent
req
uire
d; d
iffic
ult
to
achi
eve
optim
ized
con
ditio
ns
ASE
SSu
itabl
e fo
r dr
y lip
osom
es; e
xcep
tiona
lly lo
w o
rgan
ic r
esid
ues;
sin
gle
step
; rap
id; s
cala
ble
For
mic
ropa
rtic
les;
gre
ater
car
e re
quire
d; b
igge
r an
d he
tero
gene
ous
part
icle
s; u
ses
a no
zzle
; lim
ited
stud
ies
cond
ucte
d
SCRP
E/IS
CRP
ELo
w o
r no
org
anic
sol
vent
con
sum
ptio
n; s
impl
e an
d ra
pid
proc
ess;
si
ngle
ste
p; n
o ne
ed f
or n
ozzl
es; s
cale
-up
pote
ntia
l; st
able
lipo
som
esH
igh
pres
sure
use
d; h
igh
cost
; lim
ited
stud
ies
on e
ncap
sula
tion
of d
rugs
co
nduc
ted
ASE
S: A
eros
oliz
ed s
uper
criti
cal e
xtra
ctio
n of
sol
vent
s; E
E: E
ntra
pmen
t ef
ficie
ncy;
GA
S: G
as a
ntis
olve
nt p
roce
ss; I
SCRP
E: Im
prov
ed s
uper
criti
cal r
ever
se-p
hase
eva
pora
tion;
RES
S: R
apid
exp
ansi
on o
f su
perc
ritic
al s
olut
ions
; SA
S: S
uper
criti
cal a
ntis
olve
nt p
roce
ss; S
CF:
Sup
ercr
itica
l flui
d; S
CRP
E: S
uper
criti
cal r
ever
se-p
hase
eva
pora
tion.
www.futuremedicine.com 1545future science group
Liposomal drug products & supercritical fluid-mediated liposomes Review
Executive summary
Major challenges that have to be considered for liposomal formulations � The final products should be completely free from organic residues. Therefore, minimal or no use of organic solvents is preferred in liposome preparation.
� In order to provide stability to the formulation, preventing aggregation and/or fusion of the liposome particles, degradation of entrapped drugs and oxidative degradation of lipids is mandatory.
� The liposomes size should be in the nanoscale in order to take advantage of the enhanced permeability and retention effect, especially for cancer treatment.
� Extended circulation times in human plasma by producing stable liposomal formulations is required in order to achieve therapeutically efficacious passive targeting of liposomes to cancer tissues.
� The method of liposome preparation should allow for scaled-up liposome production.
Liposomal drug products: from Doxil® to Marqibo® Kit � The history of liposomal drugs started with Doxil® (Janssen Pharmaceuticals, PA, USA) when Gabizon and Barenholz initiated the basic research on Doxil in 1979.
� Very recently the US FDA approved vincristine sulfate containing a liposomal drug (Marqibo®; Talon Therapeutics, CA, USA) for the treatment of a rare type of leukemia.
� Most of the liposomal drugs are used for intravenous administration.
� There are 15 approved drugs and many drugs are in the pipeline for commercialization, which are in various stages of clinical trials.
� Marketed products, AmBisome® (Astellas Pharma, IL, USA) and Doxil are the most prominent products, which have achieved substantial clinical success.
Supercritical fluid of CO2 method: an alternative approach for liposome preparation � Supercritical fluid (SCF)-CO2 is green, nontoxic and nonflammable, and the use of SCF-CO2 gives a clean and effective alternative to conventional methods of liposome preparation.
� The solvent properties of SCF can be drastically altered by controlling the temperature and pressure, and, thus, the properties of liposomes can easily be controlled.
� Since the first discovery of SCF-CO2 injection and decompression method by Castor, many studies have been reported to demonstrate the efficient use of SCF-CO2 in preparing liposomes using various methods.
� Injection and decompression, rapid expansion of supercritical solvent (RESS), supercritical antisolvent (SAS), gas antisolvent (GAS), supercritical reverse-phase evaporation (SCRPE) methods are the most commonly used methods for liposome preparation.
SCF-mediated liposomes: advantages of the methods for liposome preparation � Injection & decompression method
– Castor and Chu mainly studied this method in which the control of particle size, reduced use of organic solvent, in situ sterilization and scale-up of the process were easily possible. However, low yield and entrapment efficiency were the drawbacks of this method.
� SCRPE– Otake and his colleagues mainly studied this method and claimed that the SCRPE method is simple, rapid and utilizes very low or no
organic solvents. There was no need for a nozzle and the process was carried out in a single step.
� RESS– Conventional RESS process was not successful for liposome preparation, therefore, there were some modified RESS processes
reported that showed good prospects in the scaled-up production of liposomes.
� GAS– The GAS technique offered an efficient and uncontaminated way of preparing liposomes with improved and controllable
physicochemical properties. Requirement for organic solvents is very low and also final formulations are almost free from solvents. There are a limited number of studies on GAS-based liposomes.
� SAS– At present, this process is well known for liposome preparation. The SAS process provided many successes for incorporating
amphotericin B, cyclosporine A and docetaxel into liposomes, and offered a physically and chemically stable formulation with reduced particle size and narrow size distribution. It is a very simple process and has the potential for scale-up.
In order to prove that the organic residues were present in the final formulations, gas chroma-tography, infrared spectroscopy or other advanced methods should be carefully studied. Similarly, a good number of microbiological studies should be focused on evaluating the sterility effects of SCF-CO
2 in the final liposomal formulations.
Therefore, this field is still emerging, and more research needs to be carried out on SCF liposomes
to confirm their effectiveness before they are approved for clinical use.
Conclusion Although liposomal drugs and SCF liposomes have been reviewed in the past, we have thor-oughly discussed process development and the characteristics of liposomes based on the latest research papers and patents filed worldwide. To
Nanomedicine (2013) 8(9)1546 future science group
Review Karn, Cho & Hwang
date, only conventional methods have been used to prepare commercially available liposomal drug products. However, these processes yield final products that are contaminated to some extent with residual organic solvent, and it is also difficult to maintain scaled-up production. The affordable cost, simple, convenient and mass preparation of liposomes with almost no trace of residual solvents that supercritical techniques offer make these ideal methods for use by pharmaceutical companies. Rapid advances in SCF research have furthered liposome technology for industrial applications and SCF methods are viable alternatives to conventional liposome synthesis approaches.
Future perspectiveThere are few liposomal drugs that have received substantial clinical response mainly for cancer treatment. However, it would be expected that the drugs, which are currently in the pipeline for commercialization, will receive approval in future for use in various clinical applications. There is no doubt that supercritical methods have a bright future in producing for liposomal drug formula-tions, as they allow control of the physicochemical
properties of liposomes with great flexibility. Recently, SCF processes have been the most com-mon alternative approach for scaled-up production of liposomes, but the full potential of this approach in the clinical development of liposomal drug products has not been fully explored. In the future, we believe that the advances and modifications of known SCF methods – injection and decom-pression, RESS, SAS/GAS and ISCRPE – will undoubtedly make the industrial-scale production of liposomes feasible.
Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a finan-cial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
Writing assistance was utilized in the production of this manuscript. The authors would like to acknowledge Yonsei Institute of Pharmaceutical Sciences, Yonsei University, for their writing assistance supported in part by the Yonsei University Research Fund of 2011.
ReferencesPapers of special note have been highlighted as:n of interestnn of considerable interest
1 Bangham A, Standish M, Watkins J. Diffusion of univalent ions across the lamellae of swollen phospholipids. J. Mol. Biol. 13(1), 238–252 (1965).
2 Sessa G, Weissmann G. Phospholipid spherules (liposomes) as a model for biological membranes. J. Lipid Res. 9(3), 310–318 (1968).
3 Gregoriadis G, Ryman B. Liposomes as carriers of enzymes or drugs: a new approach to the treatment of storage diseases. J. Biochem. 124(5), 58P (1971).
4 Gregoriadis G. Drug entrapment in liposomes. FEBS Lett. 36(3), 292–296 (1973).
5 Gregoriadis G. The carrier potential of liposomes in biology and medicine (first of two parts). N. Engl. J. Med. 295(13), 704–710 (1976).
6 Gregoriadis G. The carrier potential of liposomes in biology and medicine (second of two parts). N. Engl. J. Med. 295(14), 765–770 (1976).
7 Chang HI, Yeh MK. Clinical development of liposome-based drugs: formulation, characterization, and therapeutic efficacy. Int. J. Nanomedicine 7, 49–60 (2012).
n� Good overview of the recent developments and advances in liposomal drug products.
8 Meure LA, Foster NR, Dehghani F. Conventional and dense gas techniques for the production of liposomes: a review. AAPS PharmSciTech 9(3), 798–809 (2008).
nn� One of the most comprehensive liposome reviews that describes both the conventional and dense gas liposome preparation methods.
9 New RRC. Introduction. In: Liposomes: a Practical Approach. New RRC (Ed.). Oxford University Press, Oxford, UK, 22–25 (1990).
10 Samad A, Sultana Y, Aqil M. Liposomal drug delivery systems: an update review. Curr. Drug Deliv. 4(4), 297–305 (2007).
11 Szoka F Jr, Papahadjopoulos D. Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation. Proc. Natl Acad. Sci. USA 75(9), 4194–4198 (1978).
12 Hope M, Bally M, Webb G, Cullis P. Production of large unilamellar vesicles by a rapid extrusion procedure. Characterization of size distribution, trapped volume and ability to maintain a membrane potential. Biochim. Biophys. Acta 812(1), 55–65 (1985).
13 Wagner A, Vorauer-Uhl K. Liposome technology for industrial purposes. J. Drug Deliv. 2011, 591325 (2011).
14 Bridson R, Santos R, Al‐Duri B, McAllister S, Robertson J, Alpar H. The preparation of liposomes using compressed carbon dioxide: strategies, important considerations and comparison with conventional techniques. J. Pharm. Pharmacol. 58(6), 775–785 (2006).
15 Patil SD, Burgess DJ. Liposome: design and manufacturing. In: Injectable Dispersed Systems Formulation, Processing, and Performance. Burgess DJ (Ed.). Taylor and Francis, FL, USA, 249–284 (2005).
16 Allen TM, Cullis PR. Liposomal drug delivery systems: from concept to clinical applications. Adv. Drug Deliv. Rev. 65(1), 36–48 (2013).
n� Good overview of liposomal drugs and their clinical applications.
17 Slingerland M, Guchelaar HJ, Gelderblom H. Liposomal drug formulations in cancer therapy: 15 years along the road. Drug Discov. Today 17(3), 160–166 (2012).
18 Song G, Wu H, Yoshino K, Zamboni WC. Factors affecting the pharmacokinetics and pharmacodynamics of liposomal drugs. J. Liposome Res. 22(3), 177–192 (2012).
19 Lasch J, Weissig V, Brandl M. Preparation of liposomes. In: Liposomes: a Practical Approach. Torchilin V, Weissig V (Eds). Oxford University Press, Oxford, UK (2003).
www.futuremedicine.com 1547future science group
Liposomal drug products & supercritical fluid-mediated liposomes Review
www.futuremedicine.com
20 Batzri S, Korn ED. Single bilayer liposomes prepared without sonication. Biochim. Biophys. Acta 298(4), 1015–1019 (1973).
21 Deamer D, Bangham A. Large volume liposomes by an ether vaporization method. Biochim. Biophys. Acta 443(3), 629–634 (1976).
22 Karn PR, Vanic Z, Pepic I, Škalko-Basnet N. Mucoadhesive liposomal delivery systems: the choice of coating material. Drug Dev. Ind. Pharm. 37(4), 482–488 (2011).
23 Lesoin L, Crampon C, Boutin O, Badens E. Preparation of liposomes using the supercritical anti-solvent (SAS) process and comparison with a conventional method. J. Supercrit. Fluids 57(2), 162–174 (2011).
24 Cha KH, Cho KJ, Kim MS et al. Enhancement of the dissolution rate and bioavailability of fenofibrate by a melt-adsorption method using supercritical carbon dioxide. Int. J. Nanomedicine 7, 5565–5575 (2012).
25 Kim MS, Jin SJ, Kim JS et al. Preparation, characterization and in vivo evaluation of amorphous atorvastatin calcium nanoparticles using supercritical antisolvent (SAS) process. Eur. J. Pharm. Biopharm. 69(2), 454–465 (2008).
26 Kim MS, Kim JS, Park HJ, Cho WK, Cha KH, Hwang SJ. Enhanced bioavailability of sirolimus via preparation of solid dispersion nanoparticles using a supercritical antisolvent process. Int. J. Nanomedicine 6, 2997–3009 (2011).
27 Park J, Cho W, Cha KH, Ahn J, Han K, Hwang SJ. Solubilization of the poorly-water soluble drug, telmisartan, using supercritical anti-solvent (SAS) process. Int. J. Pharm. 441(1–2), 50–55 (2013).
28 Karn PR, Cho W, Park HJ, Park JS, Hwang SJ. Characterization and stability studies of a novel liposomal cyclosporin A prepared using the supercritical fluid method: comparison with the modified conventional Bangham method. Int. J. Nanomedicine 8, 365–377 (2013).
nn� Interesting paper that describes a novel and simple approach to preparing liposomes suitable for large-scale production using the supercritical antisolvent process, in which supercritical fluid liposomes were stable compared with liposomes produced with the Bangham method.
29 Taylor L. Supercritical Fluid Extraction. Wiley Interscience Publication, NY, USA, 1–26 (1996).
30 Hannay J, Hogarth J. On the solubility of solids in gases. Proc. Roy. Soc. London 29, 324–327 (1879).
31 Naylor A, Lewis AL, Illum L. Supercritical fluid-mediated methods to encapsulate drugs: recent advances and new opportunities. Ther. Deliv. 2(12), 1551–1565 (2011).
32 Herrero M, Mendiola JA, Cifuentes A, Ibáñez E. Supercritical fluid extraction: recent advances and applications. J. Chromatogr. A 1217(16), 2495–2511 (2010).
33 Byrappa K, Ohara S, Adschiri T. Nanoparticles synthesis using supercritical fluid technology – towards biomedical applications. Adv. Drug Deliv. Rev. 60(3), 299–327 (2008).
34 Lesoin L, Crampon C, Boutin O, Badens E. Development of a continuous dense gas process for the production of liposomes. J. Supercrit. Fluids 60, 51–62 (2011).
35 Zhong J, Dai L. Liposomal preparation by supercritical fluids technology. Afr. J. Biotechnol. 10(73), 16406–16413 (2011).
36 Kompella UB, Koushik K. Preparation of drug delivery systems using supercritical fluid technology. Crit. Rev. Ther. Drug 18(2), 173–199 (2001).
37 Frederiksen L, Anton K, Hoogevest PV, Barratt BJ, Leuenberger H. Use of supercritical carbon dioxide for preparation of pharmaceutical formulations. Presented at: Proceeding of 3rd International Symposium on Supercritical Fluids (Volume 3). Strasbourg, France, 17–19 October 1994.
38 Frederiksen L, Anton K, Hoogevest PV, Keller HR, Leuenberger H. Preparation of liposomes encapsulating water‐soluble compounds using supercritical carbon dioxide. J. Pharm. Sci. 86(8), 921–928 (1997).
39 Castor TP. Phospholipid nanosomes. Curr. Drug Deliv. 2(4), 329–340 (2005).
40 Phillips EM, Stella VJ. Rapid expansion from supercritical solutions: application to pharmaceutical processes. Int. J. Pharm. 94(1–3), 1–10 (1993).
41 Pasquali I, Bettini R. Are pharmaceutics really going supercritical? Int. J. Pharm. 364(2), 176–187 (2008).
42 Girotra P, Singh SK, Nagpal K. Supercritical fluid technology: a promising approach in pharmaceutical research. Pharm. Dev. Technol. 18(1), 22–38 (2013).
43 Wen Z, Liu B, Zheng Z, You X, Pu Y, Li Q. Preparation of liposomes entrapping essential oil from Atractylodes macrocephala Koidz by modified RESS technique. Chem. Eng. Res. Des. 88(8), 1102–1107 (2010).
44 Zhang W, Sun Y, Li Y, Shen R, Ni H, Hu D. Preparation and influencing factors of sirolimus liposome by supercritical fluid. Artif. Cells. Blood Substit. Immobil. Biotechnol. 40(1–2), 62–65 (2012).
45 Gallagher P, Coffey M, Krukonis V, Klasutis N. Gas antisolvent recrystallization: new process to recrystallize compounds insoluble in supercritical fluids. In: Supercritical Fluid Science and Technology (Volume 406). Johnston KP, Penninger JML (Eds). American Chemical Society, DC, USA, 334–354 (1989).
46 Dehghani F, Foster N. Dense gas anti-solvent processes for pharmaceutical formulation. Curr. Opin. Solid State Mater. Sci. 7(4), 363–369 (2003).
47 Kadimi US, Balasubramanian DR, Ganni UR, Balaraman M, Govindarajulu V. In vitro studies on liposomal amphotericin B obtained by supercritical carbon dioxide–mediated process. Nanomedicine 3(4), 273–280 (2007).
48 Ghatnur SM, Sonale RS, Balaraman M, Kadimi US. Engineering liposomes of leaf extract of seabuckthorn (SBT) by supercritical carbon dioxide (SC-CO
2)-mediated process. J. Liposome Res.
22(3), 215–223 (2012).
49 Naik S, Patel D, Surti N, Misra A. Preparation of PEGylated liposomes of docetaxel using supercritical fluid technology. J. Supercrit. Fluids 54(1), 110–119 (2010).
50 Naik S, Patel D, Chuttani K, Mishra AK, Misra A. In vitro mechanistic study of cell death and in vivo performance evaluation of RGD grafted PEGylated docetaxel liposomes in breast cancer. Nanomedicine 8(6), 951–962 (2012).
51 Lasic DD. Novel applications of liposomes. Trends Biotechnol. 16(7), 307–321 (1998).
52 Xia F, Jin H, Zhao Y, Guo X. Preparation of coenzyme Q10 liposomes using supercritical anti-solvent technique. J. Microencapsul. 29(1), 21–29 (2012).
53 Otake K, Shimomura T, Goto T et al. Preparation of liposomes using an improved supercritical reverse phase evaporation method. Langmuir 22(6), 2543–2550 (2006).
nn� One of the most important papers that describes the use of supercritical fluid for the preparation of liposomes.
54 Bleich J, Müller B, Wassmus W. Aerosol solvent extraction system – a new microparticle production technique. Int. J. Pharm. 97(1), 111–117 (1993).
55 Kunastitchai S, Pichert L, Sarisuta N, Müller BW. Application of aerosol solvent extraction system (ASES) process for preparation of liposomes in a dry and reconstitutable form. Int. J. Pharm. 316(1), 93–101 (2006).
56 Bleich J, Müller B. Production of drug loaded microparticles by the use of supercritical gases with the aerosol solvent extraction system
Nanomedicine (2013) 8(9)1548 future science group
Review Karn, Cho & Hwang
(ASES) process. J. Microencapsul. 13(2), 131–139 (1996).
57 Kunastitchai S, Sarisuta N, Panyarachun B, Müller BW. Physical and chemical stability of miconazole liposomes prepared by supercritical aerosol solvent extraction system (ASES) process. Pharm. Dev. Technol. 12(4), 361–370 (2007).
58 Otake K, Imura T, Sakai H, Abe M. Development of a new preparation method of liposomes using supercritical carbon dioxide. Langmuir 17(13), 3898–3901 (2001).
nn� First study to describe the supercritical reverse-phase evaporation method for liposome preparation.
59 Imura T, Gotoh T, Otake K et al. Control of physicochemical properties of liposomes using a supercritical reverse phase evaporation method. Langmuir 19(6), 2021–2025 (2002).
60 Imura T, Otake K, Hashimoto S et al. Preparation and physicochemical properties of various soybean lecithin liposomes using supercritical reverse phase evaporation method. Colloid Surf. B 27(2), 133–140 (2003).
61 Aburai K, Yagi N, Yokoyama Y et al. Preparation of liposomes modified with lipopeptides using a supercritical carbon dioxide reverse-phase evaporation method. J. Oleo Sci. 60(5), 209–215 (2011).
62 Otake K, Shimomura T, Goto T et al. One-step preparation of chitosan-coated cationic liposomes by an improved supercritical reverse-phase evaporation method. Langmuir 22(9), 4054–4059 (2006).
63 Sakai H, Gotoh T, Imura T, Sakai K, Otake K, Abe M. Preparation and properties of liposomes composed of various phospholipids with different hydrophobic chains using a supercritical reverse phase evaporation method. J. Oleo Sci. 57(11), 613–621 (2008).
64 Martin FJ. Case study: doxil, the development of PEGylated liposomal doxorubicin. In: Injectable Dispersed Systems Formulation,
Processing, and Performance. Burgess DJ (Ed.). Taylor and Francis, FL, USA, 427–471 (2005).
65 Chen JR, Yang YC, Chen TC et al. Salvage chemotherapy in recurrent cervical cancer with biweekly PEGylated liposomal doxorubicin (Lipo-Dox). Taiwan J. Obstet. Gynecol. 47(3), 322–326 (2008).
66 Swenson CE, Perkins WR, Roberts P, Janoff AS. Liposome technology and the development of Myocet™ (Liposomal doxorubicin citrate). The Breast 10(Suppl. 2), 1–7 (2001).
67 Allen TM, Martin FJ. Advantages of liposomal delivery systems for anthracyclines. Semin. Oncol. 31(Suppl. 13), 5–15 (2004).
68 Bekersky I, Fielding RM, Buell D, Lawrence I. Lipid-based amphotericin B formulations: from animals to man. Pharm. Sci. Technolol. Today 2(6), 230–236 (1999).
69 Petre CE, Dittmer DP. Liposomal daunorubicin as treatment for Kaposi’s sarcoma. Int. J. Nanomedicine 2(3), 277–288 (2007).
70 Bressler NM, Bressler SB. Photodynamic therapy with verteporfin (Visudyne): impact on ophthalmology and visual science. Invest. Ophthalmol. Vis. Sci. 41(3), 624–628 (2000).
71 Bovier PA. Epaxal®: a virosomal vaccine to prevent hepatitis A infection. Expert Rev. Vaccines 7(8), 1141–1150 (2008).
72 Mischler R, Metcalfe IC. Inflexal®V a trivalent virosome subunit influenza vaccine: production. Vaccine 20(Suppl. 5), B17–B23 (2002).
�n Patents101 Hwang SJ, Park HJ, Cho W et al.:
KR2011-0096962 (2011).
nn� Describes the new method for scaled-up production of liposomal amphotericin B using the supercritical antisolvent method.
102 Castor TP: WO94/27581 (1994).
103 Castor TP: US5554382 (1996).
104 Castor TP, Chu L: WO96/15774 (1996).
105 Frederiksen L, Anton K, Hoogevest PV: US5700482 (1997).
106 Castor TP: US/2010/0247620 A1 (2010).
107 Smith RD: US4582731 (1986).
108 Muller BW, Fischer W: DE3744329 (1989).
109 Abe M, Otake K, Hashimoto S: JP2003-119120 (2003).
110 Nagaike C, Motokui Y: EP-1776948A1 (2007).
111 Ueda E, Nakajima A, Motokui K, Nagaike C: JP2006-069930 (2006).
112 Wada T, Motokui Y: JP2006-298845(2006).
113 Aoki Y, Ueda E: JP2006-063009 (2006).
114 Ueda E: JP2006-063052 (2006).
115 Proffitt RT, Adler-Moore J, Chiang SM: US5965156 (1999).
116 Wada T, Ueda E: US2006-0239925 (2006).
117 Motokui Y, Wada T: JP2006-298842 (2006).
�n Websites201 US FDA. Approved drug products with
therapeutic equivalence evaluations, 33rd edition. www.fda.gov/downloads/Drugs/DevelopmentApprovalProcess/UCM071436.pdf (Accessed 26 March 2013)
202 European Medicine Agency. Search for medicine. www.ema.europa.eu/ema (Accessed 1 March 2013)
203 Depocyt® (Cytarabine liposome injection). www.depocyt.com (Accessed 1 March 2013)
204 DepoDur® (morphine sulfate extended-release liposome injection). www.pacira.com/pdf/depodur-full-information.pdf (Accessed 1 March 2013)
205 US FDA. Marqibo® (vincristine sulfate liposome injection) for intravenous infusion. www.accessdata.fda.gov/drugsatfda_docs/label/2012/202497s000lbl.pdf (Accessed 1 March 2013).