liposomal drug products and recent advances in the synthesis of supercritical fluid-mediated...

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]

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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

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ally

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le.

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ive

ph

arm

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tica

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gre

die

nt

Pro

du

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ge

form

Typ

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rova

l dat

e (D

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rer

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pro

val

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poso

me

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nsse

n Ph

arm

aceu

tical

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A, U

SA)

US

FDA

Lipo

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ject

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ipei

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wan

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som

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arm

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som

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200

0G

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rcel

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in)

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Am

phot

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plex

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plex

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enue

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orat

orie

s (O

H, U

SA)

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Am

Biso

me®

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ctab

leLi

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me

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/11/

1997

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ella

s Ph

arm

a (IL

, USA

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A

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n ci

trat

e D

auno

Xom

e®In

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cien

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, USA

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A

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arab

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Dep

ocyt

®In

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pina

lLi

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me

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ra P

harm

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tical

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c. (

NJ,

USA

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A

Ver

tepo

rfin

Vis

udyn

e®In

ject

able

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poso

me

12/0

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00

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P Ph

arm

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tical

s (N

J, U

SA)

FDA

Mor

phin

e su

lfate

D

epoD

ur®

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ctab

le –

epi

dura

lLi

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me

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rmac

eutic

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Inc.

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vaca

ine

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rel®

Inje

ctab

le –

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som

e11

/10

/201

1Pa

cira

Pha

rmac

eutic

als

Inc.

FD

A

Vin

cris

tine

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arqi

bo® K

itIn

ject

able

– iv

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poso

me

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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

.

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il®/C

aely

x® (J

anss

en

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mac

eutic

als,

PA

, USA

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SPC

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lest

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200

0–D

SPE

(56

:39

:5)

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h lo

nger

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ulat

ion

time

in b

lood

; bet

ter

safe

ty

profi

le; 1

00

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E >

90%

M

etas

tatic

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rian

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er,

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S-re

late

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apos

i’s s

arco

ma

[7,16,64

]

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® (

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Bio

phar

m,

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ei, T

aiw

an)

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C:c

hole

ster

ol:P

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6:3

9:5

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ged

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ion

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dvan

ced

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ian

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late

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ma

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cet®

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rcel

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in)

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:45)

Redu

ced

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-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

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Abe

lcet

® (S

igm

a Ta

u,

MD

, USA

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:3)

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e; b

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y lo

wer

th

an F

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zone

® (

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tol-

Mye

rs S

quib

b, N

J, U

SA)

Inva

sive

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gal i

nfec

tions

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Am

Biso

me®

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ella

s Ph

arm

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)SU

V; <

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dia

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er

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us f

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met

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afe

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com

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S[7,67,69]

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ra

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mac

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A

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2]

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pid

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s; 1

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µm

; pH

5–8

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pai

n fo

llow

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or

surg

ery

[7,204

]

Mar

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® K

it (T

alon

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SA)

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lm m

etho

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n tim

e; e

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cles

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s in

fect

ions

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xal-V

® (

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a Bi

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rical

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A

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d N

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sert

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embr

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Influ

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t flu

va

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e fo

r al

l age

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B: A

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ener

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phos

phat

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line;

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isto

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hosp

hatid

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ol; D

OPC

: 1,2

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leoy

l-sn

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e;

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G: D

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mito

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lyce

rol;

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C: D

iste

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lpho

spha

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cho

line;

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E: D

iste

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lpho

spha

tidyl

eth

anol

amin

e; D

SPG

: Dis

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oylp

hosp

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lyce

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pmen

t ef

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: Egg

pho

spha

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ch

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gg p

hosp

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yl g

lyce

rol;

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: Hem

aggl

utin

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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.


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

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�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.



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.


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



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



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



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].



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.


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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.



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.



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.



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



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.



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



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.

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56 Bleich J, Müller B. Production of drug loaded microparticles by the use of supercritical gases with the aerosol solvent extraction system



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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,

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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).

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72 Mischler R, Metcalfe IC. Inflexal®V a trivalent virosome subunit influenza vaccine: production. Vaccine 20(Suppl. 5), B17–B23 (2002).

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www.fda.gov/downloads/Drugs/DevelopmentApprovalProcess/UCM071436.pdf



www.ema.europa.eu/ema

www.depocyt.com

www.pacira.com/pdf/depodur-full-information.pdf

www.pacira.com/pdf/depodur-full-information.pdf

www.accessdata.fda.gov/drugsatfda_docs/label/2012/202497s000lbl.pdf

www.accessdata.fda.gov/drugsatfda_docs/label/2012/202497s000lbl.pdf

liposomal drug products and recent advances in the synthesis of supercritical fluid-mediated...

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