Lipid nanoemulsions loaded with doxorubicin-oleic acid ionic complex: Characterization, in vitro and in vivo studies

Article abstract of DOI:10.1691/ph.2011.0379

This study aimed at developing a novel lipid nanoemulsion formulation of doxorubicin (DOX) which is feasible for scale-up production, exhibits good parenteral acceptability, and further improves the therapeutic index of the drug. Oleic acid was used to form ionic complex with DOX in order to enhance its lipophilicity. The lipid nanoemulsions loaded with doxorubicin-oleic acid complex (DOX-OA-LNs) were prepared using a simple high-pressure homogenization method and fully characterized from physicochemical and in vitro release standpoint. Afterwards, the DOX-OA-LNs and free DOX were compared with respect to their in vitro cellular uptake and cytotoxicity, and their in vivo pharmacokinetics and biodistribution behavior in mice were also investigated. The obtained DOX-OA-LNs could achieve high encapsulation efficiency of 93.7±1.2% under optimal conditions. The in vitro release behavior displayed biphasic drug release pattern with rapid release at the initial stage and prolonged release afterwards. The DOX-OA-LNs exhibited higher growth inhibitory effect than free DOX by MTT assay. Flow cytometry and confocal microscopy studies showed that the cellular uptake of free DOX and DOX-OA-LNs were comparable. Pharmacokinetics and in vivo distribution studies in mice showed that DOX-OA-LNs demonstrated significantly higher DOX level in blood and longer circulation time than free DOX. Moreover, DOX-OA-LNs significantly decreased DOX concentration in heart, lung and kidney. These results suggested that DOX-OA-LNs could be a promising formulation for the delivery of DOX in tumor chemotherapy.

Full text of DOI:10.1691/ph.2011.0379

ORIGINAL ARTICLES
Key Laboratory of Drug Targeting and Drug Delivery Systems1, Ministry of Education, West China School of Pharmacy,
Sichuan University, Sichuan, School of Medicine, Tsinghua University2, Beijng, P. R. China
Lipid nanoemulsions loaded with doxorubicin-oleic acid ionic complex:
characterization, in vitro and in vivo studies
Xuanmiao Zhang1, Xun Sun1, Jilong Li1, Xiaoning Zhang2, Tao Gong1, Zhirong Zhang1
Received December 11, 2010, accepted December 29, 2010
Tao Gong, Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School
of Pharmacy, Sichuan University, Sichuan 610041, People’s Republic of China
gongtaoy@126.com
Pharmazie 66: 496–505 (2011)
doi: 10.1691/ph.2011.0379
This study aimed at developing a novel lipid nanoemulsion formulation of doxorubicin (DOX) which is
feasible for scale-up production, exhibits good parenteral acceptability, and further improves the therapeutic
index of the drug. Oleic acid was used to form ionic complex with DOX in order to enhance its lipophilicity.
The lipid nanoemulsions loaded with doxorubicin-oleic acid complex (DOX-OA-LNs) were prepared using
a simple high-pressure homogenization method and fully characterized from physicochemical and in vitro
release standpoint. Afterwards, the DOX-OA-LNs and free DOX were compared with respect to their in
vitro cellular uptake and cytotoxicity, and their in vivo pharmacokinetics and biodistribution behavior in
mice were also investigated. The obtained DOX-OA-LNs could achieve high encapsulation efficiency of
93.7 1.2% under optimal conditions. The in vitro release behavior displayed biphasic drug release pattern
with rapid release at the initial stage and prolonged release afterwards. The DOX-OA-LNs exhibited higher
growth inhibitory effect than free DOX by MTT assay. Flow cytometry and confocal microscopy studies
showed that the cellular uptake of free DOX and DOX-OA-LNs were comparable. Pharmacokinetics and
in vivo distribution studies in mice showed that DOX-OA-LNs demonstrated significantly higher DOX level
in blood and longer circulation time than free DOX. Moreover, DOX-OA-LNs significantly decreased DOX
concentration in heart, lung and kidney. These results suggested that DOX-OA-LNs could be a promising
formulation for the delivery of DOX in tumor chemotherapy.
1. Introduction
ity of the lipids (e.g. stearic acid, hexadecanoic acid) was the
major hurdle for the successful commercialization of SLNs for
parenteral administration and there is no product of SLNs com-
mercially available for i.v. injection on the market so far (Joshi
and Müller 2009). In conclusion, despite vast researches in the
field of colloidal particle delivery systems of DOX, there has
been no colloidal particle delivery system of DOX which could
both satisfy the regulatory acceptance of the excipients for i.v.
administration and the demand on low cost and convenience of
large-scale production. Thus, a need for simple, safe and cheap
colloidal formulations of DOX persists in the clinical trials.
Recently, lipid nanoemulsions (LNs) have emerged as an inter-
esting carrier system for optimized delivery of drugs. LNs, also
frequently known as miniemulsions, ultrafine emulsions, submi-
cron emulsions and so forth (Solans et al. 2005; Gutierrez et al.
2008), were nanometric-scale oil-in-water dispersions, mainly
covering a size range of 20–200 nm and showing narrow size dis-
tributions (Solans et al. 2005; Huynh et al. 2009; Subedi et al.
2009). The basic structure of a nanoemulsion droplet is a neu-
tral lipid core (e.g. liquid triglyceride) stabilized by amphipathic
lipids (e.g. phospholipids). The LNs are appealing to many
administration routes: parenteral (Santos-Magalhaes et al. 2000;
Zhao et al. 2008), transdermal (Sonneville-Auburn et al. 2004;
Yilmaz and Borchert 2005; Mou et al. 2008), oral (Singh et al.
2008; Vyas et al. 2008; Bali et al. 2010) and ocular (Sznitowska
et al. 1999).
Doxorubicin (DOX), an anthracycline antibiotic, is commonly
used in the treatment of malignancy, including leukemias, lym-
phomas, bonesarcoma, andsoon. However, itstherapeuticuseis
limitedbyitscumulativedose-relatedandirreversiblecardiotox-
icity and myelosuppression (Young et al. 1981; Singal et al.
2000). Until now, various colloidal particle delivery systems,
such as liposomes (Working et al. 1994), polymeric nanopar-
ticles (Park et al. 2009; Zhang et al. 2007; Petri et al. 2007)
and solid lipid nanoparticles (SLNs) (Cavalli et al. 1993; Ma
et al. 2009; Subedi et al. 2009) have been developed to mini-
mize the toxic side effect of DOX and enhance its therapeutic
efficacy. However, all of them had their own drawbacks which
limited their clinical use and only the DOX liposomes could be
used clinically. Although the liposomal DOX formulation has
appeared on the pharmaceutical market in the 1990s, complexity
associated with the manufacturing process and enormous cost
of the liposomal formulation were the major barriers in the suc-
cessful commercialization of liposomes (Joshi and Müller 2009;
Wissing et al. 2004). The primary two drawbacks of the poly-
meric nanoparticles which held back their clinical use were the
cytotoxicity of polymers and the lack of a suitable large scale
productionmethod(Mülleretal. 2000). SLNscouldbeproduced
easily by the high pressure homogenization method which was
feasible for scale-up production. But the parenteral acceptabil-
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Intravenously administered LNs are excellent carriers for drug
delivery, particularly for lipophilic drugs. They combine the
advantages of colloidal drug carrier systems like polymeric
nanoparticles and liposomes but at the same time avoid or mini-
mize the drawbacks associated with them. Most importantly, the
components of LNs, such as lecithin and soybean oil (injectable
grade), are clinically available for several decades and are used
in current parenteral formulations.
LNs are useful for the incorporation of lipophilic drugs. Due
to limited solubility of the drug in the lipid matrix and drug
distribution into the aqueous phase during the production pro-
cess, conventionally prepared LNs of hydrophilic drugs will
probably have a poor drug loading and drug encapsulation effi-
ciency. Thus, the difficulty in preparing hydrophilic drug loaded
lipid nanoemulsions formulations combining high drug entrap-
ment efficiency with controlled drug release represents a real
challenge.
The aim of this study was to prepare DOX loaded LNs using bio-
compatiblelipidcompoundswhichareusedincurrentparenteral
formulations. In the present study, we selected oleic acid (OA)
as the lipophilic complexing agent to prepare doxorubicin-oleic
acid ionic complex (DOX-OA) to enhance the lipophilicity of
DOX. Then we prepared doxorubicin-oleic acid complex loaded
lipid nanoemulsions (DOX-OA-LNs) with high encapsulation
efficiency by a simple high pressure homogenization method
and evaluated the physicochemical and in vitro release prop-
erties of DOX-OA-LNs. Afterwards, the DOX-OA-LNs and
free DOX were compared with respect to their in vitro cellu-
lar uptake and cytotoxicity, and their in vivo pharmacokinetics
and biodistribution behaviour in mice were also investigated.
Fig. 1: Structure of the doxorubicin-oleic acid ionic complex
DOX-OA also can be prepared by the codissolve-evaporation
method (Olbrich et al. 2002, 2004). Briefly, DOX-OA was
prepared by dissolving 50 mg of doxorubicin base and 100 mg
of OA in dichloromethane and consequent evaporation of
solvent at 30 ◦C under reduced pressure. Compared with the
codissolve-evaporation method, the stirring method could avoid
the use of toxic organic solvent. Moreover, the process of this
method was more convenient.
The formation of an ionic complex is generally driven by overall
forces such as electrostatic attraction, hydrophobic interaction
and hydrogen bonding (Higashiyama et al. 2007). Although
solvents with low dielectric constant are favored for ion pair for-
mation, the contribution of hydrophobic interaction is relatively
large as well as electrostatic force once the ion pair is formed in
aqueous solution (Quintanar-Guerrero et al. 1997). What men-
tioned above explained why the DOX-OA could be prepared
both in water and dichloromethane. According to the study per-
formed by Kalinkova (2007), the ionic complexes obtained by
interactions between aliphatic amines and carboxylic acids have
a structure type of the ion pair and complex composition of 1:1.
Similarly, it was presumed that DOX-OA was formed through
electrostatic forces derived from the charge neutralization
between the basic amino group of DOX and the acidic carboxyl
group of OA, and the structure of DOX-OA is shown in Fig. 1.
OA was an effective counter-ion for DOX in our LNs formu-
lation. The adding of OA to aqueous solution of doxorubicin
base resulted in immediate formation of ion-pair complex as
red precipitates. More than 99.7% of DOX could ion-pair
with OA during this complexation process, which was almost
complete. Investigations showed that the formed DOX-OA was
very soluble in organic solvents, especially in ethanol, ethyl
acetate, and acetone.
2. Investigations, results and discussion
2.1. Preparation of DOX-OA
Ion pair formation has been used as a method of modifying
the lipophilicity of ionizable drugs by shielding their charge
with an oppositely charged ion without chemical modifications
(Higashiyama et al. 2007). Because DOX has the amino sugar
moiety, daunosamine, which bears the positive electrostatic
charge localized at the protonated amino nitrogen, lipophilic
counterions have been employed to facilitate the entrapment
of DOX in lipid vehicles. The Gasco’s group (Cavalli et al.
1993) showed that both decyl phosphate and hexadecyl phos-
phate could form ion-pairs with DOX, and the resulting ion-pair
complexes increased the lipophilicity of DOX, which facil-
itated the incorporation of DOX into the SLNs. Ma et al.
(2009) used anionic ion-pairing agents, sodium taurodeoxy-
cholate and sodium tetradecyl sulfate, to neutralize the charges
of the cationic DOX and enhance entrapment of DOX in the
SLNs. However, the disadvantage of the ion-pair method used
by the forementioned two research groups is that the ion-pairing
agents they used are potentially toxic and could not be utilized
for i.v. injection. In our study, OA was used as the complex-
ing agent due to the fact that OA is a kind of biodegradable
and physiological long chain fatty acid which shows low tox-
icity. Moreover, it is clinically available for parenteral usage
(Pontes-Arruda 2009).
2.2. Determination of octanol/water partition coefficient
The octanol/water partition coefficient (log PO/W) of doxoru-
bicin hydrochloride, doxorubicin base and DOX-OAwas−1.79,
−1.32 and 1.81, respectively. It was shown that the lipophilicity
of doxorubicin base was a few stronger than that of doxoru-
bicin hydrochloride. However, the lipophilicity of DOX-OA
was much stronger than that of doxorubicin hydrochloride.
These results indicated that the ion pair formed between DOX
and OA could mask the positive charge of DOX, and that the
long alkyl chain of OA increased the lipophilicity of DOX-
OA thereby facilitating the distribution of DOX in the organic
phase. The enhanced lipophilicity of DOX-OA could contribute
to the increased solubility of DOX in lipid matrix and therefore
improved the entrapment efficacy of DOX in LNs.
During the preparation process of DOX-OA, bicarbonate solu-
tion was used to neutralize the hydrochloric acid of doxorubicin
hydrochloride instead of other bases such as sodium hydroxide,
because DOX is instable and decomposes easily in strong alkali-
nous environment. In the preparation process, OA was dissolved
in ethanol and subsequently the solution was added to the aque-
ous solution of doxorubicin base. In this way, OA could disperse
in aqueous solution more easily than adding OA directly into the
DOX solution, so that it could react with DOX more adequately.
2.3. Preparation of DOX-OA-LNs
In this study, we developed an economical, simple and repro-
ducible method, and it was free of toxic organic solvent during
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ORIGINAL ARTICLES
preparation of LNs. Most importantly, the preparation process of
DOX-OA-LNs was feasible for industrial scale-up production.
High pressure homogenization (HPH) has emerged as a reliable
and powerful technique for the preparation of LNs, which is a
technique well established on the large scale since the 1950s and
is already available in the pharmaceutical industry.
The parenteral acceptability of the lipids (e.g. stearic acid,
hexadecanoic acid) is the major hurdle for the successful com-
mercialization of SLNs. It is more feasible to use glycerides of
the fatty acids which are present in the lipid phase of parenteral
fat emulsions (e.g. soybean oil, medium-chain triglyceride) to
fabricate LNs with high biocompatibility (Joshi and Müller
2009; Wolfram et al. 1989). In our study, the components of LNs
were lecithin, OA and soybean oil (injectable grade). They are
biodegradable and physiological lipids which show low toxicity.
Moreover, these materials are accepted by the regulatory author-
ities (e.g. FDA) and are clinically available for i.v. injection
(Pontes-Arruda 2009).
A serious problem related to LNs is the need of large amounts
of surfactants. In most cases, surfactants are added to the formu-
lation of LNs to stabilize the highly increased particle surface
and hinder the coalescence of droplets. Type and amount of sur-
factants involved in the fabrication procedure obviously have
an important effect on physical and pharmaceutical properties
of LNs, such as particle size and zeta potential values being
important for physicochemical stability as well as biopharma-
ceutical properties of the preparation. However, many of these
stabilizers such as Cremophor EL and Tween 80 have a low
biocompatibility in parenteral administration. The safety and
efficacy of the excipients play an important role in case of
parenteral application. It should be kept in mind that GRAS
(Generally Recognized as Safe) surfactants used to prepare LNs
for i.v injection should obtain approval and have a good in vivo
tolerability. In our formulation of DOX-OA-LNs, the only sur-
factant used was lecithin, which is native and well tolerated
in humans. Moreover, it has been widely used in parenteral
nutrition emulsions. Therefore, there was no problem related
to toxicity of surfactants in DOX-OA-LNs.
In order to optimize the formulation of DOX-OA-LNs, the influ-
ence of drug/lipid ratio on the particle size distribution and the
polydispersity index (PDI) was investigated. The size and mor-
phology of LNs were significantly influenced by the content of
phospholipid. When the drug/lipid ratios were more than 1/4
(w/w), LNs showed large particle size and broad size distribu-
tion (data not shown), which could be attributed to insufficient
amount of phospholipid as surfactant. As the drug/lipid ratio
decreased to 1/6 (w/w), the obtained LNs showed the optimal
particle size and polydispersity.
In order to study the conditions of HPH, the influence of
parameters such as applied pressure, homogenization cycles
and temperature on particle size distribution was investigated.
The temperature affected the size distribution of DOX-OA-LNs
slightly due to the fact that the melting point of the lipids used
in DOX-OA-LNs was relatively low, so the homogenization
process could be performed at room temperature to avoid the
degradation of DOX which is sensitive to high temperature. In
our trials, 7 homogenization cycles at an operating pressure of
100 MPa were sufficient to prepare DOX-OA-LNs with optimal
particle size and PDI. Increasing the homogenization pressure
or the number of cycles resulted in an increase of the particle
size due to droplets coalescence which occurred as a result of
the high kinetic energy of the nanoemulsion droplets.
Fig. 2: Transmission electron microscopy micrograph of DOX-OA-LNs aqueous
suspensions. Scale bar: 200 nm
cating that the nanoemulsion droplets population was relatively
homogenousinsize. ThecalculatedmeansizeofDOX-OA-LNs,
based on three separate measurements, was 208.5 26.7 nm
with PDI of 0.136 0.023, which indicated that the DOX-OA-
LNs were uniform and mono-disperse. The mean particle size
and PDI of DOX-OA-LNs were much desirable due to the opti-
mal formulation and preparation parameters of DOX-OA-LNs.
The TEM micrograph of DOX-OA-LNs is shown in Fig. 2, from
which it could be found that the shape of nanoemulsion droplets
was spherical and the particle size approximately ranged from
100 to 200 nm, corresponding to the PCS results.
The zeta potential was −28.5 3 mV, which was as high as
the absolute value of 30 mV to satisfy the stability require-
ment solely deduced from the electrostatic interaction, showing
the good stability of DOX-OA-LNs. Zeta potential is essential
to the storage stability of colloidal dispersion, the presence of
small amounts of phosphatidylserine and phosphatidylglycerol
(2–5%)inLipoidE80resultedinanegativesurfacecharge(Zhao
et al. 2008; Li et al. 2008). In addition, due to an accumulation of
the negatively charged ionized carboxy groups on the interface,
OA could also act as co-emulsifier leading to more negative zeta
potential, which resulted in a higher resistance to coalescence
of the droplets.
2.5. Entrapment efficiency and drug loading
Commonly, it is considered that LNs are mainly applica-
ble to hydrophobic drugs, while they could not achieve high
entrapment efficacy for hydrophilic drugs. The incorporation
efficiency may be altered by several factors such as the physic-
ochemical properties of drugs and the structure of LNs. In our
study, the entrapment efficacy of DOX-OA-LNs based on DOX-
OA was 93.7 1.2%. However, it was found in the preliminary
experiment that the LNs incorporating DOX without the OA
complexation only could reach a low incorporation efficiency
2.4. Particle size, Zeta potential and morphology
The size distribution of DOX-OA-LNs measured by photon
correlation spectroscopy (PCS) showed one narrow peak indi-
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Fig. 3: In vitro release curves of DOX from DOX solution and DOX-OA-LNs in
Fig. 4: Intracellular DOX fluorescence intensity in HepG2 and T2780 cells which
were incubated with DOX solution or DOX-OA-LN at 10 ␮M DOX
concentration for 3 h at 37 ◦C. Data represent mean SD (n = 3)
PBS (0.01 M, pH 7.4) containing 0.2% w/v of Tween 80 (n = 3)
of 30%. The high affinity and incorporation of DOX in the
lipid matrix of DOX-OA-LNs with high incorporation efficiency
were achieved owing to the enhanced lipophilicity of DOX-OA
compared to free DOX.
The drug loading content of DOX-OA-LNs was 7.4%. In this
system, relativelyhighdrugloadingefficiencycouldbeachieved
by DOX-OA-LNs, avoiding the administration of redundant
excipients.
cytometry method. DOX was used directly to measure cellular
uptake without additional markers since DOX itself is fluores-
cent, and its fluorescence intensity is directly proportional to the
amount of DOX internalized (Upadhyay et al. 2010). In addition,
many researchers have utilized flow cytometry for quantitative
determination of DOX uptake in cells (Zheng et al. 2009; Praba-
haran et al. 2009; Zhang et al. 2010).
Fig. 4 shows the intracellular DOX fluorescence intensity after
HepG2 and T2780 cells were incubated with free DOX or
DOX-OA-LNs for 3 h at 37 ◦C. There was little or no statis-
tical difference in the cell associated fluorescence between free
DOX and DOX-OA-LNs within each cell line. For example, the
cell-associated fluorescence intensity of free DOX and DOX-
OA-LNs was 87.6 11 and 107 8 in HepG2, respectively,
and 118 9 and 132 13 in T2780, respectively. The results
shown in Fig. 4 indicate that the DOX also could be successfully
transported into cells by being loaded in DOX-OA-LNs.
The cellular uptake of DOX might be an integrated result of
the nature of drug, the particle size, zeta potential and surface
characteristics of the drug carriers, the type and state of cells,
and so on. As for the liposomal DOX, the intracellular DOX
amount is lower than that of free DOX. Many researchers (Li
et al. 2009; Xiong et al. 2005) reported that the highest amount
of intracellular DOX was found in cells that had been incubated
with free DOX and the intracellular DOX for DOX liposomes in
various cells was much lower than that for free DOX. The rel-
ative enhanced intracellular DOX of DOX-OA-LNs compared
with the liposomal DOX can be ascribed to the presumption
that DOX-OA-LNs were more readily internalized than DOX
liposomes due to the enhanced lipophilicity of DOX by forming
DOX-OA.
2.6. In Vitro release study
The in vitro release behaviors of free DOX solution and DOX-
OA-LNs are shown in Fig. 3, from which it could be found
that the drug release from free DOX solution was much faster
than DOX-OA-LNs. The free DOX solution released 27.8% of
DOX within 0.5 h. In contrast, the cumulative release amount of
DOX-OA-LNs was only 2.74% within 0.5 h, demonstrating that
there was no burst effect for DOX-OA-LNs. This phenomenon
can be attributed to the fact that the hydrophobicity of DOX
has been notably increased by the formation of DOX-OA and
a hydrophobic interaction between drug and matrix lipids may
retain the drug.
More than 80% of drug was released from free DOX solution
into the medium after 8 h, whereas the cumulative amount of
DOX released from DOX-OA-LNs was less than 40% until 12 h,
indicating that the dissolution of DOX from the lipid matrix
was slowly and DOX-OA-LNs were responsible for the delayed
release of DOX.
The drug release profile of DOX-OA-LNs could be best charac-
terized by the exponential model and the following regression
equation was given: F (t) = 15.594·In t + 8.3197, correlation
coefficient (r) = 0.996, where F (t) is the accumulative drug
release (%), and t is time (h). The release profile of DOX-OA-
LNs was biphasic. The initial fast release was observed in the
beginning of 24 h, due to the large surface area of the LNs and
drug enrichment in the outer shell of the droplets. In the latter
stage, drug release was constant and slow. It could be attributed
to the assumption that the lipophilic DOX-OA solubilized or
dispersed in lipid matrix and therefore DOX released mainly by
dissolution and diffusion of drug from the lipid matrix.
In conclusion, the drug release from DOX-OA-LNs was drasti-
cally delayed as a result of the increase in the lipophilicity of the
complex and the incorporation of the drug into the lipid matrix.
2.8. Intracellular distribution of DOX by confocal
microscopy analysis
The intracellular distribution of free DOX and DOX-OA-LNs
was analyzed by confocal microscopy in HepG2 and T2780 cell
lines. Fig. 5 showed the confocal microscopy images of HepG2
and T2780 after incubated with DOX-OA-LNs or free DOX at
37 ◦C for 3 h.
For both the free DOX and the DOX-OA-LNs, the DOX fluo-
rescence was mainly distributed in the nuclei of the cells. After
3 h of incubation with the DOX solution in HepG2 and T2780
cells, strong fluorescence of DOX was observed in cell nuclei in
addition to a very weak fluorescence in the cytoplasm, suggest-
ing rapid intercalation of intracellular DOX to the chromosomal
DNA after passive diffusion into the cells without the release
2.7. Flow cytometry analysis
Uptake of DOX-OA-LNs into HepG2 and T2780 cells in com-
parison to free DOX was quantitatively analyzed by the flow
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ORIGINAL ARTICLES
Fig. 5: Confocal microscopy images of HepG2 and T2780 cells incubated with free DOX solution or DOX-OA-LNs at a DOX concentration of 20 ␮g/mL diluted in culture
medium at 37 ◦C for 3 h. (Magnification 63×)
process. While HepG2 and T2780 cells were incubated with
DOX-OA-LNs, as shown in Fig. 5, intense DOX fluorescence
at the nucleus was also observed in the two cell lines.
Thus, the results illustrated above evidenced that DOX-OA-LNs
nearly did not change the pattern of sub-cellular distribution of
DOX and also could deliver DOX to the nuclear compartment
of the cells successfully, where the chromosomal DNA was the
target site of the DOX.
to 0.41 0.33 ␮g/mL on HepG2, and from 1.11 0.24 to
0.69 0.18 ␮g/mL on T2780, respectively.
The cytotoxicity in vitro might be influenced by many factors
such as the nature of drug, drug release from LNs, the type, char-
acteristics and state of cells, time of contact, and so on (Zhang
et al. 2010). The increase cytotoxicity of DOX incorporated in
LNs may be related to the successful internalization of DOX-
OA-LNs due to the enhanced lipophilicity of DOX by forming
DOX-OA and the successive drug release from DOX-OA-LNs
inside the cells, enhancing the action of DOX.
In addition, blank LNs exhibited low toxicity or even nontoxi-
city under various concentrations, as shown in Fig. 6, indicated
that the excipients in this formulation were little toxic or even
nontoxic under formulation concentrations and the lipid matrix
of LNs were well tolerated.
2.9. InVitro cytotoxicity study
The survival curves of HepG2 and T2780 cells showed a
concentration-dependent manner of action, as shown in Fig. 6.
It showed that DOX-OA-LNs exhibited higher inhibition rates
than those of the free DOX group at the same concentration.
The 50% inhibitory concentration (IC50) of free DOX and
DOX-OA-LNs is shown in Table 1. DOX-OA-LNs exhib-
ited significantly higher inhibition rates than free DOX on
HepG2 and T2780 cells (P< 0.05). Compared with free
DOX, the IC50 of DOX-OA-LNs decreased from 1.44 0.17
2.10. Pharmacokinetics and biodistribution of
DOX-OA-LNs in mice
The plasma concentration-time curves of different DOX formu-
lations are reported in Fig. 7. After injection of DOX-OA-LNs,
DOX was still present in the plasma of mice after 6 h, while
after 3 h the drug could not be detected in the plasma of the
mice injected with the DOX solution. The comparative phar-
macokinetic parameters after i.v. administration of the DOX
formulations are reported in Table 2. The plasma kinetics of
DOX-OA-LNs showed a higher AUC, a lower rate of clearance,
and a smaller volume of distribution in comparison to those of
the DOX solution, as shown in Table 2. This is probably due to
the slower release of DOX from DOX-OA-LNs than from the
free DOX solution.
Table 1: IC50 values of free DOX solution and DOX-OA-LNs
on different cell lines (24 h, n = 3)
Cell lines
IC (␮g/mL)
50
Free DOX solution
DOX-OA-LNs
HepG2
T2780
1.44 0.17
1.11 0.24
0.41 0.33*
0.69 0.18*
However, the circulation time of DOX-OA-LNs was relatively
shorter than that of the pegylated DOX liposomes and SLNs
*
P < 0.05 versus DOX solution
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ORIGINAL ARTICLES
reported in the literature (Working et al. 1994; Fundarò et al.
2000), and this can be attributed to the following two reasons.
Firstly, most of the DOX-OA was located in the oil droplet
of the LNs, but some was located at the interfacial lecithin
layer between the oil droplet and the aqueous phase and might
release rapidly when the DOX-OA-LNs interacted with serum
components in the blood stream. Secondly, the formulation of
DOX-OA-LNs did not include the stealth agent, such as the
pegylated lecithin (e.g. PEG-DSPE). Colloidal drug carriers are
rapidly removed from systemic circulation after i.v. injection due
to their recognition as foreign bodies by the mononuclear phago-
cyte system (MPS) (Moghimi et al. 2001). The elimination of
such colloidal systems is influenced by various parameters such
as: the nature of the components, the size, apparent electrical
charge and hydrophilicity of the colloidal carriers (Vonarbourg
et al. 2006). Coating with hydrophilic polymer chains, such as
PEG and its derivatives, to the surface of the colloidal drug
carriers provides a highly hydrophilic shield for them from elec-
trostatic and hydrophobic interactions with serum components
as well as reduced uptake by MPS. In comparison to conven-
tional nanocarriers without PEG modification, these pegylated
colloidal drug delivery systems showed a prolonged retention
time in blood, primarily due to the reduced recognition and
uptake by phagocytic cells of the MPS located mainly in the
liver and spleen. Thus, they are less readily cleared from cir-
culation and showed prolonged circulation time in the blood
stream (Mosqueira et al. 2001; Moghimi and Szebeni 2003).
Therefore, the DOX loaded pegylated liposomes or SLNs pro-
tected by the stealth agent were more stable while circulating
in the blood stream than DOX-OA-LNs and exhibited relatively
longer circulation time.
Site-specific delivery of drugs and therapeutics can significantly
reduce drug toxicity and increase the therapeutic effect. The
pharmacokinetic properties of the drug in the nanocarriers,
the vesicle size of the carriers and the vascular permeabil-
ity of individual tissues will determine the extravasation and
biodistribution profile of the drug loaded colloidal drug carri-
ers. Biodistribution of DOX in different formulations is shown
in Fig. 8. The area under the DOX concentration-time curves
(AUC) values calculated for 1–48 h of different formulations in
these tissues were listed in Table 3.
Fig. 6: Survival curves of HepG2 and T2780 cells after 24 h of treatment of free
DOX solution, DOX-OA-LNs and blank LNs (n = 3)
In particular, DOX-OA-LNs prevented the accumulation of
DOX in the heart. Fig. 8A shows that DOX concentration in
the heart (a primary target organ for doxorubicin-related car-
diotoxicity) of mice was significantly lower in DOX-OA-LNs
group than in free DOX solution group at all time points. The
lower DOX heart concentration could determine a lower car-
diotoxicity, which was one of the effects limiting the usage of
DOX. Fig. 8B and Fig. 8C show that when DOX-OA-LNs were
i.v. administered, DOX in the lung and kidney was less than
that of DOX solution at each time point, respectively. The lower
DOXconcentrationfoundinheart, lungandkidneyinDOX-OA-
LNs-treated mice and the concomitant higher plasmatic level,
could be related to a slow drug release from DOX-OA-LNs to
the blood or to the targeted organs.
Fig. 7: Plasma concentration-time profiles of DOX following i.v. administration of
DOX solution and DOX-OA-LNs (10 mg/kg). Each value represents the
mean SD (n = 6)
Behavior regarding RES tissues differed between DOX-OA-
LNs and the DOX solution. Figs. 8D and Fig. 8E showed that
when the DOX-OA-LNs were i.v. administered, less DOX was
taken up by the liver and spleen tissue than that of DOX solution
at the initial time (0–3 h), but 6 h later the DOX concentration in
the liver and spleen tissue was higher than that of DOX solution.
The delayed accumulation of the DOX in the DOX-OA-LNs
form in the liver and spleen was in agreement with the slow
plasma clearance rates shown above.
Table 2: Pharmacokinetic parameters of free DOX solution
and DOX-OA-LNs
Parameters
DOX solution
DOX-OA-LNs
T1/2 (h)
AUC (mg/L h)
MRT (h)
Vd (L/kg)
Cl (L/h/kg)
1.554 0.19
1.927 0.31
1.419 0.27
11.284 1.73
4.057 1.32
2.889 0.48*
6.923 0.74*
2.323 0.18*
1.483 0.29*
1.198 0.48*
Overall, the DOX-OA-LNs showed an improved pharmacoki-
netic profile, and altered tissue distribution, which could be
related to the enhanced lipophilicity of DOX by forming the
*
Each value represents the mean SD (n = 6). P < 0.05 versus DOX solution
Pharmazie 66 (2011)
501

ORIGINAL ARTICLES
Fig. 8: Tissue distribution of DOX after intravenous administration of DOX solution and DOX-OA-LNs (10 mg/kg). Each value represents the mean SD (n = 6)
ion-pair and the slow drug release from the DOX-OA-LNs in
the blood or targeted organs.
revealedthattheIC50 valueofDOX-OA-LNswaslowerthanthat
of free DOX. Flow cytometry and confocal microscopy studies
showed that the cellular uptake of free DOX and DOX-OA-LNs
were comparable. Pharmacokinetics and biodistribution stud-
ies in mice showed that DOX-OA-LNs demonstrated higher
DOX levels in blood and longer circulation time than free DOX.
Moreover, DOX-OA-LNs significantly decreased DOX concen-
tration in heart, lung and kidney. Hence, this novel formulation
has a promising potential as an alternative parenteral colloidal
delivery system of DOX for cancer treatment.
In conclusion, the present study demonstrated that a novel
doxorubicin-oleic acid ionic complex was prepared to alter the
solubility of DOX in water and organic solvents and entrap DOX
into lipid nanoemulsions. The method of preparing the ionic
complex with oleic acid opens a new prospective for carrying
positively charged drugs or drugs with basic amino groups into
lipid-based colloidal drug carriers with high entrapment effi-
ciency and controlled release, which needs further research. In
addition, a simple but successful high pressure homogenization
method, feasible for scale-up production, was employed to pre-
pare DOX-OA-LNs. This HPH method allowed instantaneous
and reproducible formation of DOX-OA-LNs, with a diame-
ter value around 200 nm, small PDI (< 0.2), high entrapment
efficiency(>90%)andimprovedreleaseproperties. Mostimpor-
tantly, all the used materials were approved for i.v. injection,
so the preparation has a great potential for clinical application.
The drug release behavior from the lipid nanoemulsions exhib-
ited a biphasic pattern with rapid release at the initial stage and
sustained release afterwards. The in vitro cytotoxicity studies
3. Experimental
3.1. Materials
Doxorubicin hydrochloride was kindly offered by Haizheng Pharmaceutical
CO., Ltd (Taizhou, China). Purified yolk lecithin (Lipoid E80) and oleic acid
were purchased from Lipoid CO., Ltd (Germany). Vitamin E was purchased
from Southwest Synthetic Pharmaceutical Co., Ltd (Chongqing, China).
Soybean oil was provided by Beiya Medical oil CO., Ltd (Tieling, China).
Trypsin and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenltetrazolium bromide
(MTT) were purchased from Sigma (USA). All the other chemical solvents
and reagents were of analytical grade or better.
Table 3: Tissue AUC0–48 (h ␮g/g) values of DOX solution and DOX-OA-LNs in heart, liver, spleen, lung and kidney
Formulations
Heart
Liver
Spleen
Lung
Kidney
DOX solution
DOX-OA-LNs
240.84 57.42
124.68 36.14*
337.68 78.58
577.57 62.17*
591.85 87.32
629.19 93.78
241.47 74.51*
504.38 132.17
295.21 46.25*
821.69 104.46*
*
Each value represents the mean SD (n = 6). P < 0.05 versus DOX solution
502
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ORIGINAL ARTICLES
3.2. Tumor cell lines and cell culture
subsequently removed by a rotary evaporator (Büchi, Switzerland) under
reduced pressure at 45 ◦C until a thin lipid film formed.
HepG2 cell line and T2780 cell line were provided by Shanghai Institutes
for Biological Sciences (SIBS; Shanghai, China). HepG2 cells were grown
in RPMI 1640 medium and T2780 cells were grown in Dulbecco’s modified
Eagle’s medium (DMEM), respectively. Both cells lines were cultured in
medium supplemented with 10% calf serum (Minhai, Gansu, China), 100
IU/mL penicillin and 100 ␮g/mL streptomycin. Cells were maintained at
37 ◦C in a humidified atmosphere containing 5% CO2.
The dried lipid film was rehydrated in 50 mL water, followed by vigorous
vortex. Afterwards, this predispersion was passed through a high-pressure
homogenizer (EmulsiFlex-C5, AVESTIN, Canada) for seven cycles at an
operating pressure of 96.4–115.6 MPa, resulting in the formation of DOX-
OA-LNs solution.
3.7. Characterization of DOX-OA-LNs
3.3. Animals
3.7.1. Measurement of particle size and Zeta potential
Healthy male Kunming mice (15–25 g) were obtained from Laboratory
Animal Center of Sichuan University (Chengdu, China). Prior to the experi-
ments, the mice were housed in a temperature and humidity controlled room
(25 1 ◦C, 55% air humidity) with free access to water and standard mice
chow, and they were acclimated for at least 5 days. All the animal exper-
iment protocols and procedures were approved and supervised by Animal
Ethics Committee of Sichuan University.
The mean particle size and zeta potential of DOX-OA-LNs were determined
by photon correlation spectroscopy (PCS) (Malvern Zetasizer Nano ZS90,
UK) at 25 ◦C.
3.7.2. Transmission Electron Microscopy (TEM)
The morphology of DOX-OA-LNs was examined by transmission electron
microscopy (H-600, Hitachi, Japan). The sample was stained with 2% (w/v)
phosphotungstic acid for 30 s and placed on copper grids with films for
viewing.
3.4. Preparation of DOX-OA
To neutralize the charges of cationic doxorubicin hydrochloride salt form
and facilitate drug entrapment in the oil phase, DOX-OA was prepared
using a co-precipitation method. The amount of DOX used was calcu-
lated as the base form throughout the experiments. Aqueous solution of
doxorubicin base was prepared by addition of 0.2 mL sodium bicarbonate
solution (50 mg/mL) to 10 mL aqueous solution of doxorubicin hydrochlo-
ride (5 mg/mL) in a 25 mL round bottom flask. DOX-OA was prepared by
adding 2 mL ethanol solution of oleic acid (50 mg/mL) to the obtained dox-
orubicin base solution under stirring. Under continuous stirring at room
temperature, a cloudy solution was spontaneously developed as a result
of the complex formation. DOX-OA, a red precipitate, was collected by
centrifuging. The supernatant was diluted with 0.12 M HCl in ethanol and
subsequently quantified (excitation/emission: 490/580 nm) by fluorescence
spectrophotometer (RF-5301 PC, Shimadzu, Kyoto, Japan) to determine the
concentrationofDOXinthesupernatant. ThepercentageofDOXcomplexed
with OA was calculated as follows:
3.7.3. Entrapment efficiency and drug loading
The entrapment efficiency of DOX-OA-LNs was determined by the ultra-
filtration method previously reported (Ma et al. 2009). To separate the free
drug from the LNs suspension, Nanosep® Centrifugal Filtration Devices
(Mw cut-off 300 kDa; PALL Life Science, USA) were used. A fixed vol-
ume (400 ␮L) of the freshly prepared DOX-OA-LNs (1 mg/mL) was added
to the sample reservoir tube and spun at 14000 × g at 25 ◦C for 60 min. The
collected filtrate in the retentate vial was diluted with 0.12 M HCl in ethanol
and analyzed by fluorescence spectrophotometry.
The encapsulation efficiency and drug loading were calculated by the fol-
lowing formulas:
Encapsulation efficiency (%)
(3)
= [ 1 - (amount of drug in filtrate/amount of drug added)] × 100%
Percentage of DOX ion-paired with OA
= [ 1- (weight of drug in supernatant/weight of the drug added)]×100%
Drug loading content (%)
(1)
= (weight of drug added/weight of drug and excipients added) × 100%
(4)
The resultant complex was washed three times with water for injection, and
then it was sealed and kept in a desiccator at room temperature.
3.7.4. In Vitro release study
3.5. Determination of octanol/water partition coefficient
In vitro drug release from DOX-OA-LNs was investigated using a dialysis
bag diffusion technique previously reported (Zhang et al. 2007) with some
modifications. PBS (400 mL, 0.01 M, pH 7.4) containing 0.2% w/v of Tween
80 was used as the release medium at 37 1 ◦C under constant shaking at
100 rpm with a thermostatted shaker bath (Shenzhen worldwide industry,
Co., Ltd., China). An appropriate amount of DOX-OA-LNs was suspended
in the cellulose membrane dialysis bag (Mw cut-off: 8000–12000; Millipore,
USA) and immersed into the release medium. During the whole process the
device was protected from light in order to avoid DOX photodegradation.
At certain time intervals, 1 mL aliquot of the medium was pipetted out, and
each withdrawal was followed by replacement with the same volume of
fresh medium.
Octanol/water partition experiments were performed by the shaking flask
method previously reported (Montero et al. 1997) using the following exper-
imental conditions.
Doxorubicin base was prepared by addition of excess sodium bicarbonate to
an aqueous solution of doxorubicin hydrochloride, followed by extraction
into chloroform and solvent evaporation. Water and octanol were mutually
saturated for 24 h before the experiment.
Firstly, 1 mL aqueous solution of doxorubicin hydrochloride (1 mg/mL)
and 1 mL aqueous solution of doxorubicin base (1 mg/mL) were mixed
with 3 mL octanol respectively, and 1 mL octanol solution of doxorubicin-
oleic acid complex (equivalent to 1 mg/mL doxorubicin base) was mixed
with 3 mL water. Consequently, the two phases were vigorously vortexed
for 10 min and agitated for 24 h in a thermostatted shaker bath (Shenzhen
worldwide industry, Co., Ltd., China) at 25 0.1 ◦C.
The amount of DOX released in the supernatant was determined by flu-
orescence spectrophotometry after the sample solution being diluted with
0.12 M HCl in ethanol. Simultaneously, the same amount of free DOX was
tested as the control. Each experiment was performed in triplicate.
After equilibration, the samples were centrifuged at 18000 × g at 25 ◦C for
15 min, and then both phases were diluted with 0.12 M HCl in ethanol and
subsequently assayed by fluorescence spectrophotometry to determine the
concentration. Each experiment was performed at least in triplicate. The
partition coefficient was calculated by the following equation:
3.8. Cell experiments
3.8.1. Flow cytometry analysis
P = (Co × Vw)/(Cw × Vo)
(2)
HepG2 and T2780celllines wereusedasmodel cancercellsforthe examina-
tion of endocytosis. HepG2 and T2780 cell suspension (50 × 105 cells/well)
was seeded in a six-well tissue culture plate (Corning, NY, USA) and incu-
bated for 24 h at 37 ◦C, respectively. Then DOX-OA-LNs or free DOX
solution was added to designated wells with a concentration of DOX as
10 ␮M. After the incubation period of 3 h, cells were trypsinized and pel-
letedbycentrifugation, thenwashedthreetimeswithcoldPBSandexamined
by flow cytometry using the FACScan AriaTM (Becton Dickinson, San Jose,
CA, USA). The intracellular DOX was excited with an argon laser (488 nm)
and fluorescence was detected at 540 nm. Files were collected of 10000
gated events and analyzed with the FACStation software program.
(Co: concentration of DOX in octanol at equilibrium; Vo: volume of octanol
in sample; Cw: concentration of DOX in water at equilibrium; Vw: volume
of water in sample)
3.6. Preparation of DOX-OA-LNs
DOX-OA-LNs were prepared by a simple lipid film hydration-high pressure
homogenization method. Briefly, 300 mg DOX-OA (DOX/OA, 1/2, w/w),
600 mg lipoid E80, 50 mg vitamin E and 400 mg soybean oil were dissolved
into 200 mL ethanol in a 500 mL round bottom flask. The organic phase was
Pharmazie 66 (2011)
503

ORIGINAL ARTICLES
3.8.2. Confocal microscopy studies
Acknowledgments: The present study was funded by the 973 Program of
Chinese government (No. 2009CB930300) and supported by the National
Science Foundation of People’s Republic of China (No.30430770).
A confocal fluorescent microscopy was used to compare the intracellular
distribution of DOX (excitation/emission: 488/534 nm) between the free
DOX and DOX-OA-LNs. Followed culture of HepG2 and T2780 cells for
24 h on 14-mm2 sterile glass coverslips that were pre-soaked in culture
dishes, free DOX solution and DOX-OA-LNs diluted in culture medium at
a DOX concentration of 20 ␮g/mL were added to each dish and incubated
for another 3 h at 37 ◦C, respectively. The medium was removed and cells
were washed with cold PBS followed by fixing with 4% paraformaldehyde
in PBS for 15 min. Fluorescent images of cells were examined by confocal
microscopy (Carl Zeiss LSM510, Germany).
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