Review

Therapeutic
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Delivery
Treatment of intermediate stage
hepatocellular carcinoma: a review of
intrahepatic doxorubicin drug-delivery
systems

The biopharmaceutical properties of doxorubicin delivered via two drug-delivery Ilse R Dubbelboer, Elsa
systems (DDSs) for the palliative treatment of unresectable hepatocellular carcinoma Lilienberg, Emelie Ahnfelt,
were reviewed with relation to the associated liver and tumor (patho)physiology. Erik Sjögren, Niklas Axén &
These two DDSs, doxorubicin emulsified with Lipiodol® and doxorubicin loaded into Hans Lennernäs*
Department of Pharmacy, Uppsala
DC Bead® are different regarding tumor delivery, release rate, local bioavailability, if University; Box 580, SE-751 23 Uppsala,
and how they can be given repeatedly, biodegradability, length of embolization and Sweden
safety profile. There have been few direct head-to-head comparisons of these DDSs, *Author for correspondence:
and in-depth investigations into their in vitro and in vivo performance is warranted. Tel.: +46 18 471 4317
Fax: +46 18 471 4223
hans.lennernas@farmaci.uu.se
The global incidence of liver cancer is increas- To date, several DDSs delivering various
ing and this type of cancer has a poor progno- chemotherapeutic drug(s) and pharmaceu-
sis and is ranked as the third most common tical excipients have been developed and
lethal cancer form [1,2] . The recommended clinically evaluated for TACE therapy [5] .
treatment for hepatocellular carcinoma A review of these options of TACE DDSs
(HCC), a primary liver cancer, is dependent is warranted in that it would form the basis
on the stage of the disease [3] . For unresect- for optimizing tumor-targeted therapy for
able, intermediate stage HCC, transarterial HCC treatment, and subsequent develop-
chemoembolization (TACE) is recom- ment of novel, tumor-targeted DDSs and
mended. TACE involves the delivery of the dosing strategies. Two common DDSs used
cytostatic agent(s) in a drug-delivery system for TACE therapy for intermediate stage
(DDS) to the tumor via the hepatic artery (HA) HCC involve cytostatic agents emulsified
[3] . In contrast to normal liver tissue, which in Lipiodol® (LIP) or loaded into drug-
has a dual blood supply from the HA and the eluting beads. LIP is an iodized poppy seed
portal vein [4] , HCC is typically mainly vascu- oil derivative visible on x-ray, and after
larized by the HA. Local drug administration administration, the contrast is preferentially
by the TACE DDS is expected to increase the retained in tumor tissue [6,7] . The combina-
specificity of the tumor response while reduc- tion of the cytostatic agent emulsified in
ing the frequency of side effects and morbidity LIP can be administered with or without
compared with systemic dosed treatments [4] . additional embolizing materials, generat-
Embolization, caused by the DDS, obstructs ing complete or partial embolization of the
the blood flow in the tumor, induces hypoxia, targeted tumor-feeding vessel(s) [3,8] . DC
and results in increased drug concentrations Bead® (DCB) is one of several commercially
and prolonged residence times in the tumor- available drug-eluting embolizing bead sys-
target area [4] . Patients with untreated HCC tems [9] . Positively charged chemotherapeu-
that has not invaded the portal vein or spread tic drugs can be loaded into DCB for sub-
extrahepatically have an expected median sur- sequent local release [9] . The beads have a
vival of approximately 16 months. With cur- double function, serving as both a DDS and
rent palliative TACE strategies, the expected an embolic material, and result in complete,
median survival is prolonged by approximately nonreversible embolization in the targeted
part of
4 months [3] . vessels [9,10] .

10.4155/TDE.14.11 © 2014 IR Dubbelboer, E Lilienberg, Therapeutic Delivery (2014) 5(4), 447–466 ISSN 2041-5990 447
E Ahnfelt, E Sjögren, N Axén & H Lennernäs
Review  Dubbelboer, Lilienberg, Ahnfelt, Sjögren, Axén & Lennernäs
Keyterms
Hepatocellular carcinoma: Has a global incidence of
6% and is the third most common cause of cancer related
death. The primary risk factor is liver cirrhosis, primarily
caused by hepatitis B virus and C virus, or by other diseases
such as alcoholic liver disease, nonalcoholic steatohepatitis,
hemochromatosis, primary biliary cirrhosis and autoimmune
hepatitis.
Lipiodol® : Iodinated and ethylated poppy seed oil,
consisting of ethyl linoleate, ethyl oleate, ethyl palmitate
and ethyl stearate, that contains 37–39% iodine.
DC Bead® : Polyvinyl alcohol-based hydrogel bead system
that can load doxorubicin or irinotecan and is available in
different sizes ranging from 70 up to 700 μm.
Transarterial chemoembolization: An interventional
radiology procedure that involves the injection of
embolizing drug-delivery systems containing a cytostatic
agent into the vicinity of the tumors via the femoral
and hepatic artery. The injection may be followed by an
additional infusion of an embolizing agent to impede the
blood flow locally.
LIPDOX: Drug-delivery system consisting of an aqueous
solution of doxorubicin emulsified with Lipiodol.
DCBDOX: Drug-delivery system with doxorubicin loaded
into DC Bead.
These two DDSs were chosen for review as they
are both clinically used to deliver the chemotherapeu-
tic drug doxorubicin (DOX), a potent agent against
HCC. Recent clinical reports suggest that DOX deliv-
ered via these two DDSs has a similar antitumor effect,
but different adverse event profiles [11–14] . LIP delivery
of DOX was associated with higher incidence of DOX-
related side effects than DCB delivery (such as alope-
cia and myelosuppression), probably related to higher
systemic exposure of DOX and its active metabolite
doxorubicinol (DOXol). However, the total number
of serious adverse events following the two treatments
did not significantly differ [11] . Another study showed
that TACE with drug-eluting beads is associated with
a 6.6-times higher risk of liver/biliary events (i.e.,
dilated bile duct, portal vein narrowing, portal venous
thrombosis and biloma/liver infarct) compared with
TACE and LIP as a DDS [13] . It is obvious that sev-
eral important pharmaceutical and biopharmaceuti-
cal properties of these DDSs differ, with significant
implications for the treatment benefit. The objective
of this review was to examine the pharmaceutical
and biopharmaceutical properties of the two selected
DDSs. The general characteristics of DOX solutions
emulsified in LIP (LIPDOX) and DOX loaded into
DCBs (DCBDOX) are described in detail, and fac-
tors influencing the in vitro release, in vivo release and
liver pharmacokinetics (PK) of DOX are thoroughly
discussed.
448 Therapeutic Delivery (2014) 5(4)
DOX
DOX is the only cytostatic agent that is used in both
LIP emulsion and DCB DDSs and is the dominating
cytotoxic agent for intermediate-stage HCC. Its physi-
cochemical properties are presented in Table 1. Chemi-
cally, DOX is an amphiphilic active pharmaceutical
ingredient often used as its HCl salt in pharmaceuti-
cal formulations. It consists of a daunosamine group
(sugar component) attached to an a glycone by a glyco-
sidic bond (Figure 1) [15] . DOXHCl is sparingly water
soluble (10 mg/ml at unspecified pH) and DOX parti-
tions between octanol and water in a pH-dependent
manner, in accordance with its three pKa values and
alkaline functions (logD7.4 = 2.4; Figure 2 and Table 1).
The polar surface area of 222.9 Å 2 is relatively large
when considering membrane transport properties [16] .
Crystalline DOX is hygroscopic and DOX forms an
orange-red solution in water [17] .
DOX is an anthracycline, antibiotic, antineoplastic
drug, which is indicated for multiple forms of cancer
such as bone sarcomas, Hodgkin’s disease, and ovary,
breast, and lung cancer [23] . In addition, DOX is used
off-label to treat intermediate HCC. The pharmaco-
logical effects of DOX appear to be mediated by at
least three antitumor mechanisms: reversible binding
to topoisomerase I and II, intercalation to DNA base
pairs, and free-radical generation, which causes DNA
damage [15] . The concentration of DOX required for
50% growth inhibition (IC50) in vitro is both time-
and cell-line-dependent [24] . IC50-values decreased
from 3.8–0.029 μM in human HCC Hep-G2 cells
and from 8.8–1.13 μM in human HCC Hep3B cells
over 36–72 h [25–27] . For 13 different HCC cell lines,
the IC50values from 72 h incubations ranged from
0.024–6.0 μM [27–29] .
When DOX is administered as an intravenous (i.v.)
infusion, doses ranging from 60–75 mg/m2 body
surface area (BSA) or 1.2–2.4 mg/kg are repeatedly
administered every 3 or 4 weeks [23] . Systemic exposure
to DOX and its active metabolite DOXol increases the
risk of severe side effects such as cardiomyopathy and
myelosuppression [23] . Suggested risk factors for cardio-
myopathy include: cumulative DOX doses exceeding
500 mg/m2 BSA and high peak blood concentrations
of DOX [30,31] .
In vitro studies in Caco-2 cell lines indicate that
the intestinal epithelium is poorly permeable to DOX
(0.1 × 10 -6 cm2 /s), which is in accordance with its rela-
tively high polar surface area [32,33] . Like many other
drugs, DOX is transported across cell membranes by
both passive diffusion and carrier-mediated (CM)
transport [34] . It has recently been reported that the
solute-carrier influx transporter SLC22A16 is involved
in the influx of DOX into cells [35] . Several CM efflux
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 Treatment of intermediate stage hepatocellular carcinoma: a review of intrahepatic doxorubicin drug-delivery systems  Review

Table 1. The physicochemical properties of doxorubicin.

Parameter Value Ref.
Molecular weight (g/mol) 543.53 [21]

Polar surface area (Å ) 2

222.89 [21]

pKa 7.34, 8.46, 9.46 [22]

LogD    
pH 4.8 -0.91 [19]

pH 5.8 -0.45 [19]

pH 6.8 0.18 [19]

pH 7.2 0.71 [19]

pH 7.5 2.42 ± 0.08 [20]

Solubility† (mg/ml) 10 [21]

Hydrogen bond donor 6 [21]

Hydrogen bond acceptor 12 [21]

†
Solubility is presented for the HCI salt, at an unknown pH.
LogD: Logarithm of the distribution coefficient; pKa: Acid dissociation constant.

proteins, including ATP-binding cassette (ABC)
transporters such as ABCB1, ABCC1, ABCC2 and
ABCG2, and Ral binding proteins such as RALBP1,
have been associated with DOX transport [36] .
The pKa values of DOX indicate that a maximum
50% of the DOX dose is ionized in the physiological
pH range. The level of ionization controls the lipophilic
partitioning (Log Doctanol:buffer) of DOX in both patho-
logical and normal physiological pH ranges (Figure 2)
[19,20] . The pH of intracellular fluid is reported to be
higher than that of extracellular fluid in some tumors,
while the opposite is the case in healthy tissue [18] .
Thus, once DOX has been distributed into the cells in
the tumor tissue, it may bind more extensively to lipid
structures than it would in healthy tissue as the ionized
fraction is greater [15] .
DOX is reported to be extensively bound to the
tissue compartment (e.g., to liver, kidneys, heart and
lungs), which is reflected in its large volume of distri-
bution (22 l/kg) [37,38] . DOX is distributed to healthy
tissues at a higher extent than to tumor tissue, this is
most likely explained by the tumor vasculature, for
example irregular or static blood flow [39] . The PK
of DOX are best described by a three-compartment
model, with a terminal half-life of 35 h [23,37] . DOX is
metabolized into six metabolites; active DOXol is the
ered a minor route of elimination as only 3–4% of an
i.v. DOX dose (60 mg/m2) was excreted within 5 days
of administration in one study [43] .
Lip
The fate of LIP in the hepatic vascular system
LIP (also known as Ethiodol®) is an iodinated and
ethylated poppy seed oil that consists of ethyl linole-
ate (70% w/w), ethyl oleate (15% w/w), ethyl palmi-
tate (10% w/w), ethyl stearate (5% w/w), and contains
37–39% iodine (400 mg/ml) [44,45] . Following injec-
tion into the HA, LIP is accumulated in tumor tis-
sue for at least 3 months, as lipid layers (nonglobular)
extracellularly and as droplets (globular) intracellularly
(Figure 3) [7,46] . This preferential localization in tumor
tissue initiated the pharmaceutical development of an
intrahepatic DDS based on LIP for the treatment of
unresectable HCC [6–7,46–47] . Other important prop-
erties of LIP are a density of 1.280 g/cm3 (at 15°C)
and a viscosity of 25 mPa/s (at 37°C), properties that
are thought to influence the in vivo performance of the
DDS. The density of LIP is an important factor for
H
O H
O H
O O

most abundant of these [40] . The biotransformation to O

H
DOXol occurs mainly in hepatocytes, but also extrahe- O O O O O
H
patically, via the cytosolic enzymes aldoketo-reductase H
H H H
and carbonyl-reductase [41] . The biliary elimination H O
route dominates for both DOX and DOXol. After an N
H H
i.v. dose of DOX, 25% of the dose is excreted into the
bile as the parent drug and 25% as metabolites (mainly
DOXol) within 7 days [42] . Renal excretion is consid- Figure 1. Doxorubicin.

future science group www.future-science.com 449

Review  Dubbelboer, Lilienberg, Ahnfelt, Sjögren, Axén & Lennernäs
pHt,e pHt,i
3 of oil from the liver, increased embolizing effects (as
2 the oil phase after a w/o emulsion than after an oil-
LogD(oct/buffer)
1
0 linoleic acid (main ingredient in LIP) to hypervas-
5 6 7 8
-1
pH pHh,i pHh,e
Figure 2. Experimentally determined distribution
coefficient (logarithm) (LogD; octanol/buffer) values
for doxorubicin. The intracellular pH (pHt,i, dark gray
area) and extracellular pH (pHt,e, light gray area) of
tumor tissue, and intracellular pH (pHh,i, dotted area)
and extracellular pH (pHh,e, striped area) of healthy
tissue are shown [18] .
Data taken from [19,20] .
the stability of the emulsion and the viscosity of LIP
is about five-times higher than that of human blood
(6.1 mP/s in men, 4.5 mPa/s in women), which may
contribute to the chemoembolization effect [48,49] .
The available data indicate that the vascular and
tumor distribution of LIP in vivo are affected by a
dynamic interplay between the size of the oil drop-
lets, the size of the vasculature, and the physiological
hydrodynamics. After intrahepatic infusion of LIP into
healthy animals (mice, rats, rabbits and pigs), either
as a pure oil or emulsified with water, distribution of
LIP into the hepatic-vascular system resulted in tem-
porary embolization, local hypoxia and, if the vessels
were fully embolized, atrophy of the sinusoids [50,51] .
LIP is distributed into the sinusoids and is also shunted
to the portal venules, where it accumulates and slows
the sinusoidal blood flow [50–52] . If the LIP droplets are
smaller than the vessel diameter (25–50 μm in rabbits
and humans) the droplets can pass through the sinu-
soids [50] . Kan et al. demonstrated that the embolizing
effect of LIP is reversible and that the time to complete
restoration of the sinusoidal blood flow is dose-depen-
dent in the clinical dose range of 0.1–0.4 ml/kg. It has
also been suggested that it is possible to control the in
vivo droplet size of LIP by adjusting the injection rate
into the HA: the slower the injection into the HA, the
smaller the generated droplets [51] .
When injected as a water-in-oil (w/o) emulsion, LIP
was observed in the portal venules and sinusoids as a
series of oil droplets containing smaller water drop-
lets [52] . The water droplets in the emulsion eventu-
ally separated from the oil phase and were swept away
450 Therapeutic Delivery (2014) 5(4)
by the perfusing blood. There was a higher content
of oil in the liver tissue, slower clearance (removal)
blood flow was reduced), and more water droplets in
in-water (o/w) emulsion infusion [8,52] . When a w/o
emulsion was infused into the HA in tumor-bearing
rats and mice, the oil phase was preferentially located
in the liver tumor vessels [52] . The accumulation of
cular VX2 tumors in rabbits is shown in Figure 3
[6] . The oil phase of a w/o emulsion contained more
water droplets when administered to hypervascular
tumors than to hypovascular tumors, possibly due to
higher vessel density in hypervascular tumors [52] . A
higher HA blood flow has been shown to increase the
percentage of the LIP dose distributed to the tumor
[53] . The size of the oil droplet has also been corre-
lated to the extent of intra-tumor distribution;oil
droplets less than 1 μm in diameter were distributed
homogeneously in the vascular lumen of the tumor,
while droplets larger than 15 μm were distributed
unevenly [54] . Droplets smaller than 0.1 μm appear
to pass through the hepatic sinusoidal fenestrations
in healthy tissue and are expected to behave simi-
larly in tumor sinusoids [54] . Taken together, these
findings suggest the importance of implementing in
vivo studies and in vitro–in vivo performances early
in the development of pharmaceutical formulations,
especially for parenterals.
The fate of LIP in hepatic extracellular
& intracellular tissue
Having passed the vascular barrier, the lipids accu-
mulate on the surfaces of the tumor cells, as observed
both in vitro and in vivo [7,55–56] . Based on in vitro
studies, membrane-bound droplets of LIP are
thought to enter the cell via pinocytosis (fluid endo-
cytosis) [45,55–58] . After resection of HCC tumors in
patients treated with a LIP-cytostatic agent emulsion,
LIP was found in both the vascular lumen and the
endothelial cells of the tumors [45] . Necrosis in these
resected liver tumors was thought to have occurred
as a result of drug activity and/or of hypoxia related
to embolization. In the same clinical study, resected
untreated tumor lesions were perfused through the
HA with LIP ex vivo and fixed in formalin within
15 min. After fixation, droplets of LIP were observed
in the vascular lumen, in endothelial cells lining the
tumor vessels, and in tumor cells, suggesting that
LIP uptake into both tumor tissue and cells occurs
rapidly (<15 min) [45] . The distribution to, and accu-
mulation of, LIP in tumors (Figure 3) can partly be
explained by the enhanced vascular permeability
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 Treatment of intermediate stage hepatocellular carcinoma: a review of intrahepatic doxorubicin drug-delivery systems  Review

and retention (EPR) effect [59] . The EPR effect is a

500
well-known consequence of abnormal vascularization

Radioactivity (dpm/g × 10-3)

and increased vascular permeability in solid tumors 400 15 min
in combination with impaired lymphatic clearance.
3 days
Reduced lipid metabolism in the diseased liver, seen 300
7 days
as an increase in free fatty acids and a decrease in
(apo-)lipoproteins, probably contributes to further 200
tissue accumulation of LIP [60] . In addition, phago-
cytosis by Kupffer cells is down-regulated in HCC 100
tissue, which slows the elimination of lipids [61,62] .
Although these mechanisms contribute strongly to 0

or

or
or

h
le

ac
the tumor-selective accumulation of LIP, additional

Bi
m
m

om
Tu

tu
tu

to
o
processes may also exist.

St
tt

nt
en

ta
ac

is
rd
dj
Release of DOX from LIPDOX in vitro

ra

ve
ve

Li
The release of DOX from LIPDOX in vitro is affected

by factors such as the partitioning of DOX between the
LIP and aqueous phases, the type and stability of the
LIPDOX emulsion, and the chosen in vitro test method.
Despite extensive use of LIPDOX, the partition-
ing coefficient of DOX between LIP and the aqueous
phase has not yet been established. DOX is relatively
lipophilic at neutral pH (logD7.4 = 2.4), but its distribu-
tion between the oil and aqueous phase is pH-depen-
dent (Figure 2) . The dependency of DOX distribution
in LIPDOX emulsions on pH is an important factor
that needs to be considered in vitro, since it may affect
drug release in vivo.
The emulsion type affects the DOX release and is
determined by the ratio between the aqueous and the
LIP phases. This ratio can be adjusted to form dif-
ferent types of emulsions with different distribution
and release properties. Kan et al. have reported that
aqueous:LIP ratios of 1:2–4 generated LIPDOXw/o
emulsions with a water droplet diameter range of
2–3 μm, and that 2–4:1 aqueous:LIP ratios formed
LIPDOXo/w emulsions, all made with an extempora-
neous syringe mixing technique [52] . The oil droplet
diameters in the LIPDOXo/w emulsions were in the
range of 5–300 μm (mean around 7 μm). As the larger
oil droplets contained smaller water droplets of unspec-
ified size, the emulsion was classified as water-in-oil-in-
water (w/o/w) [52] . The impact of the emulsion type
on in vitro release of DOX is visualized in Figure 4 and
Table 2 : DOX is released faster from LIPDOXo/w emul-
sions than from LIPDOXw/o emulsions. These data
strongly indicate that DOX is preferentially distrib-
uted to the aqueous phase of the emulsion, as adding
a LIPDOXo/w emulsion to the aqueous release medium
resulted in the immediate release of DOX in vitro. If
the DOX had partitioned into the LIP, it would be
expected to be released more slowly. Similarly, when
a LIPDOXw/o emulsion is added to an aqueous-release
medium, the diffusion of DOX through the lipid outer
Li
Figure 3. Distribution of 14C after injection of 0.2
ml [14C]-linoleic acid into the hepatic artery in the
hypervascular VX2 tumor in rabbits. Tissues expressing
less than 5 dpm/g × 10 −3 radioactivity, for example lung,
kidney and heart are not depicted.
Data taken from [6] .
phase will be the rate-limiting step for in vitro release
[63,64] . In conclusion, DOX is released more slowly
from LIPDOXw/o emulsions than from LIPDOXo/w
emulsions in vitro, in accordance with the observed
poor permeation of DOX across lipid bilayers [65] .
The release of DOX from LIPDOXw/o emulsions is
also crucially influenced by the stability of the emul-
sion, since the inner-aqueous phase (containing most
of the DOX) will be in direct contact with the in vitro
release medium if the emulsion breaks, releasing the
DOX instantly. The thermodynamical stability of
LIPDOX emulsions can be improved by the addition
of densifiers (substances that increase the density of the
aqueous phase) and emulsifiers [67] . When the density
of the aqueous phase was increased to bring it closer
to that of LIP (1.28 g/cm3), the stability period for
LIPDOXw/o emulsions was increased from 3–24 h [69] .
Various densifiers, such as 60% diatrizoate sodium
meglumine (Urografin®), iohexol (Omnipaque
300™), or iopamidol, have been used in LIPDOX for-
mulations [52,63,67,69] . Various emulsifiers with different
degrees of hydrophilic–lipophilic balance (HLB) have
also been tested for their ability to improve LIPDOX
emulsion stability. Hydrogenated castor oil 60 (HCO-
60, HLB 14–15) and Pluronic F68 (HLB 29) have
been used for this purpose [63,66–67,70] . As shown in
Figure 4, release of DOX from LIPDOXw/o emulsions
stabilized with emulsifiers and/or densifiers was slower
in vitro than that from the unstabilized LIPDOXw/o
emulsion. In vitro release of DOX from the only
LIPDOXw/o/w emulsion included in Figure 4 (Table 2)
was faster than that from the LIPDOXw/o emulsions,

future science group www.future-science.com 451

Review  Dubbelboer, Lilienberg, Ahnfelt, Sjögren, Axén & Lennernäs
probably because of impaired emulsion stability and
subsequent partitioning of DOX to the outer aque-
ous phase. In general, w/o/w emulsions are unstable
and immediate release from LIPDOXw/o/w emulsions
has been reported [67,71] . However, some published
sustained-release profiles indicate that LIPDOXw/o/w
emulsions can be stabilized [66] .
A third factor influencing the release of DOX from
LIPDOX is the mixing procedure, which influences
both the stability and the type of emulsion formed.
Two main mixing procedures have been described:
homogenization by ultrasonic bath or homogenizer
and an extemporaneous pumping technique where
two connected syringes (one containing an aqueous
solution of DOX and one containing LIP) are used
to emulsify the two phases by pumping the syringes
until an emulsion is formed [52,67] . The preparation
technique chosen will probably influence both the in
vitro and the in vivo release of DOX from the emul-
sion, as the intensity of mixing influences the droplet
size of the inner phase, irrespective of whether the
inner phase is aqueous or LIP. Homogenization cre-
ated emulsions with inner droplet sizes of 10–40 μm,
and the pumping technique created emulsions with
inner droplet sizes of 30–120 μm in one study [8] .
Emulsification via ultrasound, a less used option,
generated smaller droplets and more stable emulsions
than homogenization created emulsions [72] .
100
80
Dose released (%)
60
40
20
0
0 2 4 6 8 20
Time (h)
Figure 4. The in vitro release profiles for aqueous doxorubicin from Lipiodol® emulsions. Details of the
composition of the emulsions are provided in Table 2. Results represented by circles were released using the sample-
and-separate release method, those represented by squares were released using the T-apparatus method, and those
represented by triangles were released using dialysis membrane-based methods. Closed symbols, open symbols, and
framed closed symbols represent water-in-oil, oil-in-water and water-in-oil-in-water emulsions, respectively.
LIPDOX: Lipiodol emulsions.
Data taken from [63,66–68] .
452 Therapeutic Delivery (2014) 5(4)
The release-test method used is also known to
influence the release of DOX from LIPDOX in vitro.
Parameters affecting in vitro release include the agi-
tation speed, the type and rate of flow, and the type
of membrane chosen [73] . The sample-and-separate,
free-flowing, and dialysis membrane-based methods
are commonly used [63,66–67] .
Thus, for prolonged stability and optimal modi-
fied release of DOX from LIPDOX, the emulsion
should be of the w/o type, have an aqueous:LIP ratio
of at least 1:2, be stabilized up to 24 h by the addi-
tion of densifiers and/or emulsifiers, and be prepared
by vigorous mixing prior to administration. Proposed
in vitro DOX release mechanisms from LIPDOX are
shown in Figure 5A .
It has been suggested that lipids may act as perme-
ability enhancers by mechanisms such as increasing
membrane fluidity, inhibiting carrier-mediated efflux
transporters, increasing solubility, and/or decreas-
ing enzymatic hydrolysis both in vitro and in vivo
[74–76] . If so, the intracellular uptake of DOX might
be affected by the presence of LIP. However, most in
vitro models of cellular uptake are directly correlated
with prolonged contact time between the formula-
tion and the cells. As a consequence, it is difficult
to accurately predict the in vivo outcomes and the
relevant mechanisms involved in tumoral uptake of
DOX from LIPDOX formulations based only on
LIPDOX I
LIPDOX II
LIPDOX III
LIPDOX IV
LIPDOX V
LIPDOX VI
LIPDOX VII
40 60 80
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 Treatment of intermediate stage hepatocellular carcinoma: a review of intrahepatic doxorubicin drug-delivery systems  Review

Table 2. The composition of the aqueous doxorubicin from Lipiodol® emulsions reviewed in this
paper and the in vitro method used to study the release of doxorubicin.
Formulation Type of Concentration of Water:oil Emulsifiers Densifiers In vitro Ref.
emulsion DOX in aqueous (:water) (v/v) method
phase (mg/ml)
LIPDOX I w/o 2.6 1:3.33 – – DMB [66]

LIPDOX II NA 5.0 1:1 – – T-app [68]

LIPDOX III w/o 2.6 1:3.33 HCO-60 – DMB [66]

LIPDOX IV w/o 5.0 1:5 HCO-10 – S-S [63]

LIPDOX V w/o 4.0 1:4 HCO-60 Iopamiro DMB [67]

LIPDOX VI w/o/w 0.88 1:3.33:8.33 Pluronic F-68 – DMB [66]

LIPDOX VII o/w 5.0 1:4 †

Kaytwo Urografin ®
S-S [63]
The emulsions were composed of aqueous doxorubicin solution, Lipiodol®, emulsifiers, and/or densifiers. The in vitro release studies were
performed with NaCl 0.9% [66], distilled water [63], or phosphate-buffered saline [67,68].
Kaytwo (10 mg) not included in ratio water:oil.
DMB: Dialysismembrane-based; DOX: Doxorubicin; HCO: Hydrogenated castor oil; Kaytwo: Syrup containing vitamin K; LIPDOX: Aqueous
doxorubicin solutions emulsified in Lipiodol; NA: Not available; o/w: Oil-in-water; S-S: Sample-and-separate; T-app: T-apparatus;
w/o: Water-in-oil; w/o/w: Water-in-oil-in-water.

in vitro models [77] . Instead, a combination of rel-
evant in vivo models and theoretical physiologically
based PK/pharmacodynamics models are required
to better understand and predict the clinical perfor-
mance of a DDS.
In vivo behavior of LIPDOX in the
hepatobiliary system
When LIPDOX is administered in vivo, the droplet
sizes of both the inner and outer phases of the emul-
sion need to be considered. In the blood, the w/o
emulsion droplet size should be smaller than 20 μm
to enable passage through the sinusoids and facilitate
distribution within the tumor [51,54] . It is important
that the aqueous phase of LIPDOX is readily dis-
persed in the oil phase to withstand emulsion break-
age during administration [78] . However, the in vivo
droplet size could potentially be reduced by a slower
infusion speed as suggested by Kan et al. [51] .
In an in vivo comparison of two LIPDOXw/o emul-
sions, one stabilized with HCO-60 and iopamiro
and prepared using homogenization, and one not
stabilized and prepared using the syringe-pumping
technique, in healthy mongrel dogs [67] , the stabilized
emulsion had a longer elimination half-life than the
nonstabilized emulsion. This correlates well with the
slower release from stabilized LIPDOXw/o emulsions
in vitro. In another in vivo study, in VX2 carcinoma-
bearing rabbits, the plasma AUC 0-120min for DOX
was three-times lower for a stabilized LIPDOXw/o
emulsion than for a stabilized LIPDOXo/w emulsion
and, after 24 h, intra-tumoral DOX concentrations
were twice as high for LIPDOXw/o [63] . This could
be explained by the EPR effect in the rabbit tumor
model, which suggests that LIP droplets containing
DOX might accumulate in the tumor. These data
are supported by the higher DOX concentrations in
the tumor than in the surrounding liver tissue after
intra-hepatic LIPDOXw/o infusions in both VX2 rab-
bits and HCC patients [69,79] . Lower DOX concentra-
tions in the liver tissue surrounding the tumor than
in the tumor itself are indicative of a more tumor-
directed anticancer effect and fewer systemic side
effects. Even though co-administration of DOX with
LIP (unknown composition) increased tumor DOX
concentrations, the intra-tumor distribution of the
iodine component of LIP did not predict the intra-
tumor distribution of DOX [80,81] . DOX was mostly
distributed only around the tumor blood vessels 4 h
after intra-arterial administration [82] . Drug penetra-
tion was enhanced with LIP, but adjacent avascular
regions were never reached with DOX [82] .
There is strong in vitro and in vivo evidence indi-
cating that the formulation properties of LIPDOX
have an impact on liver disposition and presum-
ably on the benefit:safety ratio. Surprisingly often,
however, this crucial information on the formula-
tion is not fully described and summarized in clini-
cal reports [12,53,83–86] . When it is reported, ratios
of aqueous:LIP appear to be in the range of 4:1 to
1:3.33, but are mostly 1:1 [87–92] . As suggested by the
in vitro data (Figure 4) , only aqueous:LIP ratios of
1:2–4 will result in w/o emulsions with sustained in
vitro release of DOX. The volume of infused LIP var-
ies widely, with literature reports of 2–20 ml. Because
of the many unknown factors in the preparation of
clinically used LIPDOX emulsions, the published
plasma PK profiles can provide only limited infor-

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Review  Dubbelboer, Lilienberg, Ahnfelt, Sjögren, Axén & Lennernäs
mation on the effectiveness of the emulsion. Hence,
a direct pharmaceutical comparison based on pub-
lished LIPDOX data is virtually impossible, since
information on the crucial formulation properties
with respect to its performance at the site of action
is not available. This apparent lack of consistency in
the clinical use of LIP-based DDSs strongly indicates
a need for development of a more standardized DDS.
In summary, to reach high DOX concentrations
in the tumor and low peak-plasma concentrations,
LIPDOXw/o emulsions should be stabilized and have
a small inner and outer droplet size (<20 μm) to
enable efficient distribution through the permeable,
leaky vascular system of the tumor. Furthermore,
LIPDOX formulations used in both nonclinical and
clinical settings should be described in greater detail
to allow more meaningful comparisons between
studies.
DCBs
DCB (Biocompatibles Ltd, Farnham, UK) is a poly-
vinyl alcohol (PVA)-based hydrogel bead system into
which DOX (DCBDOX) or irinotecan can be loaded.
The beads are available in several size ranges: 70–150
μm, 100–300 μm, 300–500 μm, and 500–700 μm
[93,94] . DCB700–900μm and DCB900–1200μm are no lon-
ger available, but have been extensively investigated
and are therefore included in this review. Few stud-
ies have investigated DCB70–150μm because it has been
on the market for only a short while. DCB is manu-
factured by polymerization of methacyloyl-modified
PVA macromers suspended in an oil phase [95,96] .
The macromers are synthesized by reacting PVA with
N-acrylamido-aminoacetaldehyde, followed by copo-
lymerization with 2-acrylamido-2-methylpropane
sulfonate by free radicals [96] .
DCB is biocompatible, nonbiodegradable and
mechanically flexible. Hence, in the transarterial
treatment of HCC, it provides permanent vascu-
lar embolization, limiting the number of doses that
can be applied to the tumor through the same blood
vessels.
Loading & distribution of DOX in DCB
The loading and release of DOX into and from DCB
has been described as an ion-exchange mechanism.
However, the diffusion of the ions is considered the
rate-limiting step, as ion exchange is considered to
be instantaneous [68,97–99] . DCB is provided in a
sodium phosphate buffer of physiological pH, where
the sodium ions are bound to the sulfonate groups
within the DCB. Prior to DCB loading with DOX,
the excess buffer solution is removed with a needle,
taking care not to remove any DCB and an aque-
454 Therapeutic Delivery (2014) 5(4)
ous solution of DOX is added to DCB directly after
removing the buffer solution. As diffusion and ion
exchange proceed, the positively charged primary
amine of DOX forms an ionic bond with the nega-
tively charged sulfonate groups, thus replacing the
sodium ions [68,99] .
The DCB have a smooth shell containing stabi-
lizer (cellulose acetatebutyrate) and therefore lack
ionic bonding sites [95,98–99] . In the region underlying
the shell, DOX is evenly distributed throughout the
DCB at lower concentrations (up to 0.05 mg DOX/
ml DCB) [98] . However, at higher concentrations
(from 5 mg DOX/ml DCB), two distinct regions are
distinguishable with fluorescent and transmission
microscopy: a bright red outer layer and a black core
region [98,100] . According to the microscopy images
in Biondi et al., the size of the core region appears to
increase and the outer layer to decrease with increas-
ing DOX concentrations [98] . While distribution
throughout the outer layer was uniform at higher
concentrations in this study, the distribution and
concentration of DOX in the black-core region could
not be determined [98] . It is likely that DOX not only
forms ionic bonds with the sulfonate groups, but
also self-aggregates at high concentrations, which
quenches the fluorescent signal [98] . Self-aggrega-
tion is caused by the hydrophobic surface of DOX
and is increased with increasing concentrations of
DOX [101] .
Ion exchange in combination with aggregate for-
mation is thought to form a more hydrophobic envi-
ronment, causing water displacement out from the
DCB, and subsequent increased microsphere density
and shrinkage of the bead size [98,99] . The diameter of
the DCB700–900μm can be reduced by as much as 35%
with increasing DOX concentrations, compared with
the bead size in the standard sodium buffer solution
[95] . DCB size is virtually unchanged (3% decrease)
with increasing concentrations of a saline solution,
suggesting that all negative sulfonate groups are neu-
tralized by sodium ions from the standard sodium
buffer solution, and no self-aggregation of sodium
occurs [68,95,98–99] . The large difference in the reduc-
tion of bead diameter (Na + vs DOX+) is most likely
due to DOX creating a more hydrophobic intra-
particle environment and thus being more efficient
at replacing water. The displacement of water could
also explain the increased resistance of DCB700–900μm
to compressive forces after loading DOX [68] .
The time to load DCB with DOX increases
roughly exponentially with the amount of DOX in
the solution (Table 3) . This has been explained by the
increasing hydrophobicity and reduced water content
within the polymer with increasing DOX concentra-
future science group
 Treatment of intermediate stage hepatocellular carcinoma: a review of intrahepatic doxorubicin drug-delivery systems  Review

A Release medium B Release medium

Unstirred Liquid
water layer film layer

Oil
DC Bead®
phase

Water
phase
D D Deff 1
[ion+] [ion+] [ion+]

[HSO3-]

D Log P D Log P D D Deff

[DOX] [DOX] [DOX] [DOX] [DOX+] [DOX+] [DOX+] 2

Figure 5. Proposed in vitro release mechanisms. (A) Lipiodol® -DOX emulsion and (B) DOX loaded into DC Bead®
(DCBDOX). Release from both Lipiodol-DOX and DCBDOX is controlled by D both within the formulation and
in the unstirred water layer/liquid film layer that forms at the border between the formulation and the release
medium. For Lipiodol-DOX, the partition coefficient between the two phases will affect the release of DOX.
Diffusion within the bulk of DCBDOX is hampered to some degree by DOX–bead interactions, so it is more useful
to describe the bulk transport as the effective diffusion. In addition, the diffusion of counterions is crucial for the
release of DOX from DCBDOX, since ion exchange is needed to trigger its release.
D: Diffusion; Deff: Effective diffusion; DOX: Doxorubicin; LogP: Partition coefficient.

tions in the beads, which reduces the diffusion rate of
DOX through DCB [68,95,100] . The loading time was
also proportional to the decrease in DCB surface area
with increasing bead sizes [68,95] .
For sufficiently long loading times, the maxi-
mum drug load for DCB is approximately 40 mg
of DOX/ml of beads, irrespective of the bead size.
This strongly suggests a correlation with the number
of binding sites available in DCB [68,95] . It was esti-
mated that one single DCB100–300μm contains about
2.54 × 1014 ionic binding sites, which corresponds
to 1.40 × 1019 ionic binding sites/ml of DCB100–300μm
[98,99] . The maximum loadable amount of DOX, 40
mg per ml DCB, corresponds to 1.31 × 1025 mole-
cules, which is more than the number of free bind-
ing sites, however this can be explained by the self-
aggregation of DOX [98] . Associated with the excess
loading, an initial burst release (unknown amount)
has been reported [68] . The burst release is thought
to be the result of a larger concentration gradient
between the DCBDOX and the release medium due
to a higher concentration of DOX in the surround-
ing thin layer, promoting diffusion. The maximum
drug load can be reduced by increased ionic strengths
in the loading solution and by left-over supernatant
(residuals from the excess buffer solution) in the vial,
which indicates competitive binding of DOX and
the counter ion [95] . It is therefore important that
only deionized water should be used to prepare the
DOX-loading solution.
In conclusion, the time for loading DOX into DCB
is dependent on the initial bead size and the concen-
tration of the DOX loading solution. The maximum
drug load for DCBDOX is determined not only by
the number of sulfonate binding sites, but also by the
self-aggregation of DOX within the polymer matrix,
which in its turn contributes to the burst release.
DOX distributes homogeneously within DCB at low
concentrations. At high concentrations, self-quench-
ing of DOX interferes with the analysis of DOX in
the inner region of the DCB and no conclusion on
the distribution of DOX in this region can be drawn.
Future studies need to investigate how pH, tempera-
ture and agitation affect distribution, loading time
and maximum DOX loading in the extemporaneous
preparation of DCBDOX prior to dosing.
Release of DOX from DCBDOX in vitro
The in vitro release of DOX from DCBDOX is
dependent on a number of factors including the
bead size, the ionic strength of the release medium,
the amount of DOX loaded, and the in vitro release
method. The fraction released after 22 h from smaller
beads (DCBDOX100–300μm) was threefold higher than
from larger beads (DCBDOX700–900μm) in one study,
most likely because of the shorter diffusion path-
way within the smaller beads and a larger total-
surface area [68] . A tenfold increase in ionic strength
increased the fraction released in vitro over 24 h by
a factor of three, from DCBDOX900–1200μm loaded

future science group www.future-science.com 455

Review  Dubbelboer, Lilienberg, Ahnfelt, Sjögren, Axén & Lennernäs

Table 3. Time to load DC Bead® with 99% of the loading concentration of doxorubicin in relation to
bead size.
Bead size (μm) Loading concentration of DOX Time to load 99% of loading Ref.
(mg/ml DCB) concentration of DOX (min)
100–300 25.0 20 [68]

300–500 25.0 60 [68]

500–700 5.0 20 [68]

500–700 10.0 45 [68]

500–700 25.0 120 [100]

500–700 25.0 90 [68]

500–700 37.5 360 [68]

500–700 45.0 1440 [68]

DOX: Doxorubicin.

with 25 mg/ml DOX [102] . The influence of differ- T-apparatus for DCBDOX500–700μm loaded with vari-
ent loading doses on the in vitro release of DOX is ous doses (Figure 6A & 6B) [102] . In a sample-and-sepa-
complex and is shown in Figure 6. The maximum rate method, DCB100–300μm released DOX completely
fraction released after 24 h was only about 13% in a within 4 to 24 h, dependent on the loading dose

A 15 B 2.0
6.25 mg/ml 6.25 mg/ml

12.5 mg/ml 12.5 mg/ml

Amount released (mg)
Fraction released (%)

1.5
10 25 mg/ml 25 mg/ml

37.5 mg/ml 37.5 mg/ml

1.0

5
0.5

0 0.0
0 6 12 18 24 0 6 12 18 24
Time (h) Time (h)

C D 15
100 5 mg/ml
10 mg/ml
Amount released (mg)
Fraction released (%)

25 mg/ml
10

50

5 mg/ml 5
10 mg/ml
25 mg/ml
0 0
0 6 12 18 24 0 2 4 6 8
Time (h) Time (h)

Figure 6. The fraction of doxorubicin released and amount released during 24 and 8h after different loading
doses of doxorubicin into DC Bead®. (A & B) Release of doxorubicin from DC Bead doxorubicin500-700μm in phospate
buffered saline in a T-apparatus and (C & D) from DC Bead doxorubicin100–300μm using a phosphate buffered solution
and a sample-and-separate method. Data in (D) were recalculated from (C) assuming that 1 ml doxorubicin loaded
DC Bead was added to the release medium.
Data taken from [99,102] .

456 Therapeutic Delivery (2014) 5(4) future science group

 Table 4. Summary of the influence of DC Bead®, loaded or unloaded with doxorubicin, on hepatic tumor tissue in vivo in humans and hepatic
nontumor tissue in humans and pigs.
Species DCB® Loading (mg Admin. Time post- Subjects Concentrations Proportion of all examined
size (μm) DOX/ml beads) dose treatment (n) cases with specified tissue
(days) lesion surrounding the DCB (%) † 

future science group

            DCB Tissue Non-NT NT CN IFT Viable liver Viable tumor
(mg/ml) (μM) ‡,§  (μM) §,¶  (μM) §  parenchyma
Human # 100–300 37.5 98.3 ± 24.4 8 h 1 – 5.00 ± NA NA 0 NA NA NA
mg DOX 3.45
(75–
150 mg)††
Human # 100–300 37.5 98.3 ± 24.4 9–14 3 – 2.1 ± 2.00 ± 2.30 ± 37 51 8 2
mg DOX 1.70 1.45 1.75
(75–
150 mg)††
Human # 100–300 37.5 98.3 ± 24.4 32–36 2 – 0.65 ± 0.50 ± 0.90 ± 40 60 0 0
mg DOX 0.50 0.40 0.55
(75–
150 mg)††
Pig‡‡ 100–300 0.0 2 ml DCB 28 3 – – – – 0 0 100 –
Pig‡‡ 100–300 0.0 2 ml DCB 90 2 – – – – 0 33 67 –
Pig‡‡ 100–300 37.5 103 mg 28 3 22.5 1.05 0.55 ± 1.45 ± 58 41 1 –
DOX§§ 0.80 1.10
Pig‡‡ 100–300 37.5 103 mg 90 2 4 0.75 0.60 ± 2.30 ± 12 81 7 –
DOX§§ 0.90 1.50
Pig‡‡ 700–900 37.5 103 mg 28 3 15 2.05 1.40 ± 4.80 ± 19 81 0 –
DOX§§ 1.05 2.00
Pig‡‡ 700–900 37.5 103 mg 90 2 7.4 1.25 1.25 ± NA 0 100 0 –
DOX§§ 1.05
†
Results obtained with histology hemateineosin saffron.
‡
Every 20 μm from surface of bead to 600 μm away from bead, independent on tissue lesion.
§
Above or in IC50 range (0.024–6.0 μM; 72 h incubation; 50% cell death) [27–29].
¶
Data taken from [109].
#
Inflammatory fibrotic tissue, viable liver parenchyma and viable tumor.
††
Mean ± standard deviation (minimum–maximum).
‡‡

www.future-science.com
Data taken from [108].
§§
Mean dose for both treated groups combined.
Admin.: Administered; CN: Coagulative necrosis; DCB: DC Bead®; DOX: Doxorubicin; IFT: Inflammatory fibrotic tissue; NA: Not available; NT: Necrotic tissue.
Treatment of intermediate stage hepatocellular carcinoma: a review of intrahepatic doxorubicin drug-delivery systems 

457
Review
Review  Dubbelboer, Lilienberg, Ahnfelt, Sjögren, Axén & Lennernäs

1000
100

Plasma concentration DOX (ng/ml)

Plasma concentration DOX (ng/ml)

Rabbit 100–300 11.25 mg Human 500–700 25 mg
Pig 100–300 50 mg Human 500–700 75 mg

100 Pig 100–300 127.5 mg Human 500–700 100 mg

Pig 700–900 78.7 mg Human 500–700 128 mg

10

10

1 1
0 2 4 6 0 6 12 18 24
Time (h) Time (h)

Figure 7. Plasma concentration-time curves showing the in vivo doxorubicin release from DC Bead® for different
species. (A) The 6 h plasma DOX concentration–time curves showing release from DC Bead® DOX100–300μm (closed
symbols) and DC Bead® DOX 700–900μm (open symbols) after a range of doses in pigs (circles) and rabbits (triangles).
(B) The plasma DOX concentration-–time curves showing release from DC Bead® DOX500–700μm in humans over 24 h.
The lower limit of quantification was 1 ng/ml for Varela et al. and Poon et al., and was unspecified by Lewis et al.
DOX: Doxorubicin.
Data taken from [12,96,104–105,107] .

(Figure 6C–D) . In both DCB100–300μm and DCB500–700μm
the relative release rate (%/h) was faster with lower
concentrations (Figure 6A & 6C) . The absolute release
rate (mg/h), however, is higher with higher loading
doses in DCB500–700μm, while it is similar for differ-
ent loading doses in DCB100–300μm (Figure 6B & D) .
This indicated that the difference in absolute-release
rate (mg/h) might be related to the difference in bead
size, assuming that the ranking order of release rate
will not be affected by the different release methods.
This observation could be explained by the greater
shrinkage of the bigger DCBs when loaded with a
higher dose, thus supporting the previously described
bead size-release rate relationship [68] . It is also
important to consider that these data were regener-
ated using PlotDigitizer (Free Software Foundation
Inc., MA, USA) and the observations could well have
been affected by this process.
Very different release rates have been reported
using different in vitro test methods [68,99,102] . Lewis
et al. reported 17% release of DOX in the T-appara-
tus, whereas Biondi et al. reported complete release
(100%) using a sample-and-separate method [68,99] .
Both studies were performed with 25 mg/ml loaded
DCBDOX100–300μm in PBS over 24 h, but with differ-
ent volumes of DCB and media and different release
methods. As shown by Jordan et al., altering condi-
tions such as flow rate and hydrodynamics will affect
the release rate [100] .
There is a need to improve our understanding
of the mechanism(s) behind the DOX release from
DCBDOX and its rate-limiting steps. Two theoreti-
cal models have been proposed for ion-exchange par-
ticles. One suggests that the rate-limiting process is
diffusion across the film layer (aqueous boundary
layer), while the other proposes that it is diffusion
within the particle (Figure 5B) [97,99,103] . Biondi et al.
proposed that film diffusion was the rate-limiting step
for DCBDOX [99] . However, the change in DCB size,
total surface area and volume during the release of
DOX, and the ionic strength of the release medium,
were not considered when these conclusions were
made. Thus, to achieve a mechanistic understanding
of this highly dynamic DDS, more work is needed in
the development of new in vitro methods and data
analysis models.
In vivo release of & tissue exposure to DOX
from DCBDOX
The in vivo performance of DCBDOX has been
investigated in both nonclinical models and patients
[12,96,104–109] . A study investigating liver tissue from a
single transplanted HCC patient, resected 8 h after
treatment with a transarterial infusion of DCBDOX100–
300μm
(Table 4) , found that DCB was distributed both
within and outside of the tumor, with a range of 13
mm from the tumor surface [109] . In pig liver and kid-
ney, smaller DCBDOX penetrated further into the
tissue from the injection site than larger beads [108,110] .
For instance, DCBDOX700–900μm occluded large ves-
sels in portal spaces whereas DCBDOX100–300μm were
located in the arterial branches closer to the hepatic
lobules and sinusoids. Penetration of DCB deep into
the tumor tissue is desirable, but the risk of passing
through the tumor tissue increased with decreasing
DCB size. So far, the optimal bead size and proper-

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 Treatment of intermediate stage hepatocellular carcinoma: a review of intrahepatic doxorubicin drug-delivery systems  Review

LIPDOX DCBDOX

Blood ECF Hepatocyte ECF Blood

Ion+

DCBDOX

Stasis

Blood Blood
flow flow

LIP LIPDOX w/o emulsion DOX

Figure 8. Proposed mechanisms of in vivo delivery using LIPDOX, (to the left) or DCBDOX, (to the right). LIPDOX
is administered into the liver segment containing the tumor and reaches the tumor feeding vessels via the blood.
In the blood, the LIPDOX is either separated into unloaded LIP and free DOX, after which DOX is transferred into
the extracellular fluid, or delivered through the endothelia into the ECF. In the ECF, LIPDOX releases DOX into the
ECF or into the hepatocytes, or it may be pinocytosed into the hepatocytes where it releases DOX. DCBDOX is
administered adjacent to the tumor via a tumor-feeding vessel. DC Bead® subsequently embolizes the vessel and
slowly releases DOX into the blood in exchange for cations from the blood. The released DOX enters the ECF and
eventually reaches the intracellular spaces in the hepatocytes.
DCBDOX: Doxurubicin loaded into DC Beads® ; DOX: Doxurubicin; ECF: Extracellular fluid; LIPDOX: Aqueous
doxorubicin solution emulsified in Lipiodol® ; w/o: Water in oil.

ties of DCB for tumor-targeted treatment of HCC are
unknown.
Plasma exposure to DOX after administration of
various bead sizes and doses of DCBDOX to rabbits,
pigs and humans is depicted in Figure 7. Increased
plasma exposure to DOX was directly correlated with
higher doses and smaller DCB sizes in vivo, which is
in agreement with the higher in vitro release data for
these beads (Figures 6 & 7) . The systemic concentra-
tions of DOX after DCBDOX injection (various sizes
and doses) were below the limit of quantification, or
very low, after 2 and 7 days in pigs and humans, respec-
tively (Figure 7) [12,104,107] . Plausible explanations for
this include a slow release rate from DCBDOX and/
or high local tissue uptake and accumulation of DOX.
It can also be speculated that the local embolization
retards diffusion of counter ions required for ionic
exchange, thus decreasing the release rate of DOX
from DCBDOX.
Liver concentrations of DOX after DCBDOX
administration have been measured in both HCC
patients and healthy pigs (Table 4) . Concentrations
of DOX in DCBDOX decreased over time in pigs; a
faster decrease was seen for DCBDOX100–300μm than
for DCBDOX700–900μm [108] , which is in good agree-
ment with the higher in vitro release rate seen for this
smaller particle size. The tissue concentrations, mea-
sured up to 0.6 mm from the DCB, tended to decrease
both over distance from DCB and over time for both
species, irrespective of DCB size (from 8 h to 32–36

future science group www.future-science.com 459

Review  Dubbelboer, Lilienberg, Ahnfelt, Sjögren, Axén & Lennernäs

Table 5. Summary of the comparison of the Lipiodol® and DC Bead® drug-delivery systems.
Parameters LIPDOX DCBDOX
Active pharmaceutical ingredient DOX DOX
Vehicle LIP DCB
Type of formulation Emulsion, Dispersion of DOX-loaded beads in aqueous
preferably w/o solution
with higher LIP
volume than
aqueous DOX
volume
Biodegradable Yes, but LIP can No
be accumulated
in tumor tissue
for up to 3
months
Loading mechanism Emulsification Ion-exchange mechanism, where DOX binds to
of aqueous DOX anions in the bead
solution in LIP
Common drug load Variable 37.5 mg DOX/ml DCB
Maximum dose (per treatment) 50 mg 150 mg
Release mechanism Diffusion Ion-exchange and diffusion
Release rate Dependent on Dependent on drug load and bead size
type of emulsion
Technique used for Via image- Via image-guided insertion of catheter into hepatic
administration guided insertion artery via femoral vein
of catheter into
hepatic artery via
femoral vein
Site of administration Whole-liver, Tumor-feeding artery†
lobar, segmental
or subsegmental
Embolization after administration Yes, partial and Yes, full and permanent
temporary‡
In vivo distribution Dependent Dependent on site of injection
on tumor size
and tumor
vasculature
LIPDOX is an emulsion containing an aqueous doxorubicin solution and DCBDOX are embolic drug-eluting beads loaded with DOX.
†
Often described as superselective in clinical research papers.
‡
Embolizing agents, for example a gelatin sponge, can be inserted after administration of the emulsion. These agents cause full permanent
or temporaryembolization.
DCB: DC Bead®; DCBDOX: Doxorubicin loaded into DC Beads; DOX: Doxorubicin; LIP: Lipiodol®; LIPDOX: Aqueous doxorubicin solution
emulsified in Lipiodol; w/o: Water in oil.

days in HCC patients and from 28–90 days in pigs)
[108,109] . However, the concentrations remained in or
above the cytotoxic range (IC50 0.024–6.0 μM). In
agreement with the faster in vitro release from smaller
beads, DCBDOX100–300μm was correlated with lower
tissue concentrations at both time points (28 and 90
days) than DCBDOX700–900μm in healthy pigs [108] .
Necrotic tissue had significantly higher concentrations
of DOX than nonnecrotic tissue in both species, possi-
bly because the DOX tissue concentrations were higher
closer to the DCB and consequently generated more
necrotic tissue. A further explanation to the observa-
tion might be that necrotic tissue cannot eliminate
DOX as effectively as nonnecrotic tissue can.
The tissue surrounding DCBDOX was histologi-
cally classified as necrotic, inflammatory-fibrotic,
healthy liver parenchyma, or healthy tumor (Table 4) .
It appeared that the necrosis caused by DCBDOX in

460 Therapeutic Delivery (2014) 5(4) future science group

 Treatment of intermediate stage hepatocellular carcinoma: a review of intrahepatic doxorubicin drug-delivery systems  Review

healthy pig liver was reversible, while in HCC liver
patients the necrosis remained unaltered [108,109] .
Although differences in the measurement time points
may have been responsible for this trend, the presence
of cirrhosis in the human tissue could have had a major
effect on the difference in outcome [111] . In pig liver,
unloaded DCB100–300μm did not cause necrosis, but the
percentage of inflammatory-fibrotic tissue tended to
increase over time, most likely due to the nondegrad-
able nature of this DDS. This suggests that while embo-
lization in itself does not cause cell death, it does acti-
vate an immune response [112,113] . DCBDOX100–300μm
tended to cause more necrosis in healthy pig liver than
DCBDOX700–900μm [108] , which is again in agreement
with the faster release of DOX from smaller beads.
However, the smaller beads were still surrounded by
some viable liver parenchyma, while the larger beads
had no viable liver parenchyma in their vicinity. In the
clinical group, the amount of viable liver parenchyma
and viable tumor tissue seemed to decrease over time,
suggesting a long-term effect of DCBDOX [109] .
Up to three months after treatment, DCBDOX
was associated with local tissue damage and necro-
sis in both healthy and tumor tissue [108,109] . Smaller
DCBDOX have higher plasma exposure and lower tis-
sue exposure than larger DCBDOX, which correlates
with the faster release rate of DOX. There is, however,
a need for a better description of the distribution of
DOX in the vicinity of the particles and of DCB in
relation to the effects in vivo, and for a definition of
the optimal properties of the particles [107,109–110] .
Comparison of the formulations
The pharmaceutical differences between these DDSs (the
liquid LIP and the solid DCB) are numerous (Table 5).
However, they can both be loaded with DOX, admin-
istered via the same route, and used to treat the same
indication. Although both can be loaded with DOX, the
recommended doses for DCBDOX and LIPDOX are
150 mg and 50 mg, respectively [88,104] . The loading of
DOX into DCB prior to administration is considered to
be more standardized and reproducible than the prepara-
tion of LIPDOX, which can vary greatly between clini-
cal/manufacturing sites [87–91] . Although both the DDSs
are administered as a hepatic TACE infusion, the cathe-
ters are placed with different tissue and tumor specificity.
It is recommended that DCBDOX should be adminis-
tered via the tumor-feeding artery, whereas LIPDOX can
also be administered to (sub)segmental or lobular areas,
or to the whole liver (Figure 8). The liver distribution of
DCBDOX is dependent on the site of injection, and the
size of both beads and microvessels. As these nonbiode-
gradable particles accumulate, the DCBDOX system
provides complete and permanent embolization. Because
instantaneous and irreversible embolization occurs, the
vessels used to administer DCBDOX can only be used
once,although the tumor can be treated repeatedly by
using other vessels [9,10] .
In contrast, repeated administration of LIPDOX
through the same vessel is possible, as LIPDOX causes
only temporary embolization [88] . LIPDOX distribu-
tion is affected by the tumor and droplet size as well as
the vasculature in and around the tumor. These factors
will influence both the local and systemic concentra-
tion–time profiles of DOX and its active metabolite(s).
Both DDSs deliver DOX to the proximity of the
tumor, but penetration of DOX into the tumor is still
diffusion dependent [82,109] .
The in vivo release profiles for DOX from both LIP-
DOX and DCBDOX have been compared in patients
with HCC [12] . A higher plasma exposure (AUC) was
observed with LIPDOX (composition unknown)

Table 6. Summary of the factors affecting the release rate of doxorubicin from drug-delivery
systems comprising an aqueous solution of doxorubicin emulsified with Lipiodol® and doxorubicin
loaded into drug-eluting beads.
Parameters LIPDOX DCBDOX
  In vitro  In vivo  In vitro  In vivo 
Ions Unknown Unknown Y Y
Flow rate Y Y Y Unknown
Membrane used in vitro Y NA Unknown NA
Size of bead NA NA Y Y
Drug load Unknown Unknown Y Y
Type of emulsion Y Y NA NA
Droplet size Unknown Unknown NA NA
Stability of emulsion Y Y NA NA
DCBDOX: Doxorubicin loaded into DC Beads ; DOX: Doxorubicin; LIPDOX: Lipiodol ; NA: Not applicable; Y: Yes.
® ®

future science group www.future-science.com 461

Review  Dubbelboer, Lilienberg, Ahnfelt, Sjögren, Axén & Lennernäs

than with DCBDOX500-700μm (drug load unknown),
although the doses administered were 50–75 mg and
<150 mg, respectively. A direct comparison of the
formulations was also performed in healthy pigs and
in a VX2 rabbit model. The higher systemic plasma
and hepatic intracellular exposure seen with LIPDOX
compared with DCBDOX suggested faster release
from LIPDOX [96,105] . The factors affecting the in vitro
and in vivo release of DOX from both DDSs have been
summarized in Table 6.
This review has demonstrated that LIPDOX
releases DOX faster than DCBDOX in vivo, resulting
in higher local and systemic exposure to both DOX
and its active metabolite(s). The local liver tissue and
tumor tissue concentration–time profiles of DOX have
not been well characterized for either of the DDSs.
However, in vivo release from DCBDOX is thought
to be highly dependent on the intrahepatic environ-
ment of freely diffusible cationic ions. Therefore, it is
still unclear which of the formulations better targets
the cancer cells and provides the most positive clinical
benefit:safety ratio.
Future perspective
As cancer is an increasing health problem in an increas-
ingly elderly population there is a need to develop novel
multifunctional-drug delivery principles to destroy
cancer cells while retaining a minimal effect on non-
malignant cells. The local administration of established
substances as part of innovative modified-release DDSs
has evolved considerably over the last decade as a con-
sequence of multidisciplinary joint ventures. Develop-
ment has been largely driven by novel imaging tools
becoming clinically standard, increased knowledge
of the cellular targets, improvements to catheter tech-
nology, and increased demands from the population.
Although the local DDSs discussed in this paper may
have improved the palliative treatment of intermediate-
stage HCC, this article shows that there is a need for sig-
nificant improvements to release and robustness aspects
of the DDSs, with subsequent improvements to anti-
cancer effects, side effects and associated morbidity. For
instance, there is an ongoing debate regarding the clini-
cal efficacy of, and benefits from, transarterial intrahe-
patic delivery of these formulations [14,114–116] . It seems
likely that, in the foreseeable future, more targeted
DDSs will be developed in multidisciplinary projects.
More specific targeting of malignant cells using multi-
functional nanoparticles could well provide a strategy to
better anticancer effects and lower morbidity associated
with this treatment approach for HCC [117] .
Financial & competing interests disclosure
Financial support to E Ahnfelt was provided by the Swed-
ish Research Council (521-2011-3773). The authors have no
other relevant affiliations or financial involvement with any
organization or entity with a financial interest in or financial
conflict with the subject matter or materials discussed in the
manuscript. This includes employment, consultancies, hono-
raria, stock ownership or options, expert t-estimony, grants
or patents received or pending, or royalties. No writing
assistance was utilized in the production of this manuscript.
Open Access
This work is licensed under the Creative Commons Attribu-
tion-NonCommercial 3.0 Unported License. To view a copy
of this license, visit http://creativecommons.org/licenses/by-
nc-nd/3.0/

Executive summary
Lipiodol® –doxorubicin emulsion
• The optimal aqueous doxorubicin solutions emulsified in Lipiodol (LIPDOX); emulsion is water-in-oil,
thermodynamically stabilized, and prepared by a homogenization technique that will create sufficiently small
(<20 μm) water droplets in the oil phase that efficiently penetrates into tumor tissue.
• Clinical and nonclinical studies should describe the composition as well as the preparation technique for
LIPDOX formulations, to allow comparisons of clinical study results.
Doxorubicin-compatible beads
• Release rate of doxorubicin (DOX) from DOX loaded into DC Bead® (DCBDOX) is dependent on at least bead
size, loading dose and composition and diffusivity of ions in the bead surroundings.
• The in vitro release characteristics of DOX from both investigated drug-delivery systems (DDSs), depend
strongly on the used in vitro release test method and it is therefore necessary to establish the most suitable
method for these DDSs so as to be able to compare in vitro with in vivo situations.
Comparison of the formulations
• LIPDOX and DCBDOX are both mainly passively delivered to hepatocellular carcinoma tissue rather than
nontumor tissue, but act in very different ways.
• LIPDOX releases DOX faster than DCBDOX in vivo, resulting in higher local and systemic exposure to both DOX
and its active metabolite(s).
• More research comparing the in vitro and in vivo performance of these DDSs, in particular regarding their
tumor and systemic exposure-effect relationship, is needed.

462 Therapeutic Delivery (2014) 5(4) future science group

 Treatment of intermediate stage hepatocellular carcinoma: a review of intrahepatic doxorubicin drug-delivery systems  Review
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