PHARMACEUTICAL NANOTECHNOLOGY
Docetaxel Albumin Conjugates: Preparation, In Vitro
Evaluation and Biodistribution Studies
FARNAZ ESMAEILI,1 RASSOUL DINARVAND,1,2 MOHAMMAD HOSSEIN GHAHREMANI,2,3 MOHSEN AMINI,4
HASTI ROUHANI,4 NIMA SEPEHRI,2 SEYED NASSER OSTAD,3 FATEMEH ATYABI1
1
Novel Drug Delivery Systems Laboratory, Faculty of Pharmacy, Department of Pharmaceutics,
Tehran University of Medical Sciences, PO Box 14155-6451, Tehran, Iran
2
Medical Nanotechnology Research Centre, Tehran University of Medical Sciences, Tehran, Iran
3
Pharmacology Research Laboratory, Faculty of Pharmacy, Department of Toxicology-Pharmacology,
Tehran University of Medical Sciences, Tehran, Iran
4
Faculty of Pharmacy, Department of Medicinal Chemistry, Tehran University of Medical Sciences, Tehran, Iran
Received 19 February 2008; revised 12 June 2008; accepted 26 August 2008
Published online 28 October 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21599
ABSTRACT: Docetaxel (DTX) is one of the most active chemotherapeutic agents for
treating metastatic breast cancer. Its aqueous solubility is very low, hence the available
formulation of DTX for clinical use consists of high concentrations of tween80, which has
been associated with several hypersensitivity reactions. To reduce the systemic toxicity
of DTX as well as to avoid the use of tween80, in this study DTX was chemically
conjugated with human serum albumin via a succinic spacer. A high-performance liquid
chromatography method was developed for the determination of DTX albumin con-
jugate. T47D and SKOV3 cells were used for the evaluation of the in vitro cytotoxicity
of the conjugate by MTT assay. Studies were then done on balb/c mice to elucidate
the tissue distribution of conjugates after intravenous administration. The albumin-
conjugated formulation of DTX with the particle size of 90 110 nm showed enhanced
solubility and in vivo characteristics and significantly higher cytotoxicity against tumor
cells, for example, IC50 of 6.30 0.73 nM for T47D cell line compared to free DTX
with IC50 of 39.4 1.75 nM. Conjugation also maintained DTX plasma level at 16.19% up
to 2 h after injection compared with 2.51% for Taxotere1, hence increasing the chance of
nanoparticles uptake by tumor cells. ß 2008 Wiley-Liss, Inc. and the American Pharmacists
Association J Pharm Sci 98:2718 2730, 2009
Keywords: albumin; drug conjugate; docetaxel; cell culture; nanotechnology;
biodistribution; nanoparticle
INTRODUCTION one of the most active chemotherapeutic agents
for treating patients with metastatic breast
Docetaxel (DTX) is a semisynthetic taxoid derived cancer.1
from the European yew tree, Taxus baccata. It is DTX like other taxanes such as paclitaxel shows
very low water solubility; hence its formulation
Correspondence to: Rassoul Dinarvand (Telephone: þ98-21-
consists of high concentrations of tween80.
66405598; Fax: þ98-21-66959055;
This vehicle has been associated with several
E-mail: [e-mail])
hypersensitivity reactions and has shown in-
Journal of Pharmaceutical Sciences, Vol. 98, 2718 2730 (2009)
compatibility with common PVC intravenous
ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association
2718 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 8, AUGUST 2009
DOSETAXEL-ALBUMIN CONJUGATES NANOPARTICLES 2719
administration sets.2 In order to eliminate the aminopropyl)-N0-ethylcarbodiimide hydrochloride
tween80-based vehicle and in the attempt to and 4-(dimethylamino) pyridine were purchased
increase the drug solubility, alternative dosage from Merck (Hohenbrunn, Germany). N-hydroxy-
forms have been suggested, including polymeric 3-sulfo-succinimide was purchased from Fluka
nanoparticles,3 5 liposomes,6 and cyclodextrins.7 (St. Gallen, Switzerland).
Another approach involves the use of soluble RPMI-1640 modified medium and Penicillin/
conjugates obtained by conjugation to a stable streptomycin solution were obtained from Gibco
macromolecular drug carrier system8 or the Invitrogen (Carlsbad, CA). MTT was obtained
attachment of DTX to water-soluble polymers.9 from Sigma Aldrich (St. Louis, MO). Methanol
It has been shown that macromolecules such as used as the mobile phase in HPLC was purchased
albumin, globulins, and synthetic polymers mark- from Merck (Darmstadt, Germany). Dimethylfor-
edly accumulate in tumor tissues because of mamide (DMF) and dimethylsulfoxide (DMSO)
enhanced tumor vascular permeability and pro- were also obtained from Merck. Deionized water
longed retention time in the tumor interstitium was used throughout the experiment. All other
due to the obstruction of lymphatic drainage.9 chemicals used were of reagent grade.
These findings have led to the development of a
variety of macromolecular drug conjugates using
Synthesis of 2(-Sulfo-NHS-Succinyl-DTX
albumin, dextran, and synthetic molecules as
carriers.10 Another advantage of macromolecular DTX (16 mg) was added to succinic anhydride
conjugates is that they show improved escaping (3.61 mg) in the presence of 4-(dimethylamino)
from the cellular mechanisms underlying drug pyridine (0.22 mg), previously dried under
resistance.11 The choice of albumin for the fabri- vacuum for 2 h, dissolved in 0.2 mL of dry
cation of ligand supportive carriers was guided by pyridine and was stirred for 24 h at room
the high level of albumin biocompatibility, bio- temperature. Ten milliliters of ethyl acetate and
degradability in vivo, and ease of modification.12 10 mL of H2SO4 solution (0.01%, w/v) were added
In 1997 Dosio et al.8 synthesized the conjugate to the mixture and stirred for 30 min. The organic
of the paclitaxel human serum albumin (HSA) phase was then separated and washed with 10 mL
that showed good activity on neoplastic cell lines, of H2SO4 solution (0.01%, w/v) and 10 mL of water,
reduced in vivo cytotoxicity, and increased plasma respectively. The solvent was mixed with sodium
concentration. Their method could avoid the use of sulfate anhydride to remove any remained water
Cremophor EL, a vehicle responsible for signifi- in the solution, and then evaporated under
cant hypersensitivity problems. In 2001, they reduced pressure to obtain a concentrated solu-
prepared the PEG HSA paclitaxel conjugate tion (1 mL). The final solution was purified by
with longer circulation ability.13 chromatography on SiO2 column with ethyl
To eliminate the tween80 from DTX formula- acetate as the mobile phase to separate the
tion, we tried to produce the DTX nanoparticles remained pyridine from 20-succinyl-DTX.
1
using HSA conjugated to DTX via a succinic HNMR (CDCL3) d: 8.1(bs, 2H, H2,6-OBz), 7.66
spacer. A high-performance liquid chromatogra- (t, 1H, J ź 7.1 Hz, H4-OBz), 7.54 (t, 2H, J ź 7.2 Hz,
phy (HPLC) method was developed for the H3,5-OBz), 7.40 7.44 (m,2H, H2,6-Arsidechain), 7.36
determination of DTX HSA conjugate. 7.38 (m, 3H, H4 and H3,5-Arsidechain), 6.2 (bs,
0
T47D and SKOV3 cells were used for in vitro 1H, H13), 5.71 (d, 1H, J ź 2 Hz, H2 ), 5.50 (bs,
0
cell line experiment, and the cytotoxicity of 1H, H3 ), 5.26 (bs, 1H, H10), 4.20 4.40 (m, 3H, H20
the conjugate was investigated by MTT assay. and H7), 3.94 (bs, 1H, H3), 2.8 2.9 (m, 1H, H6),
The conjugate was characterized and studies 2.50 2.7 (m, 4H,-OCOCH2CH2COOH), 2.35 2.45
were carried out on balb/c mice to elucidate the (m, 2H, H14), 1.98 (s, 3H, OAC), 1.87 (t, 1H, J ź 7
tissue distribution of conjugates after intravenous Hz, H6), 1.79 (s, 3H, Me18), 1.38 (s, 9H, t-Bu), 1.29
administration. (s, 3H, Me19), 1.25 (s, 3H, Me16), 1.54 (s, 3H, Me17).
IR (KBR) N: 3440(OH), 2989 and 2919 (CH),
1719 and 1730 (CO), 1494 and 1370 (CH2
MATERIALS AND METHODS
and CH3), 1243,1153 and 1069 (C O), 762 and
698 (mono substituted phenyl).
Materials
To 4.3 mg of 20-succinyl-DTX, 1.8 mg of
DTX as powder was obtained from Cipla Ltd. N-hydroxy-3-sulfo-succinimide and 2 mg of
(Mumbai,India).Succinicanhydride,N-(3-dimethyl- N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide
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2720 ESMAEILI ET AL.
hydrochloride in 1 mL of mixture of DMSO:DMF High-Performance Liquid Chromatography Analysis
(70:30) were added. The mixture was stirred for of DTX and DTX HSA Conjugate
24 h at room temperature. The progress of the
Two milligrams of the dried conjugate was
reaction was maintained by TLC (Rf ź 0.52 in
dissolved in 0.5 mL water and mixed. One
CHCl3:CH3OH, 80:20).
milliliter of methanol containing ZnSO4 (1%, w/v)
was added to precipitate the HSA. The mixture
was then centrifuged for 10 min at 5000g at 48C,
after which 20 mL of the supernatant was
Preparation of Albumin Conjugates With DTX
analyzed.
The 20-sulfo-NHS-succinyl-DTX solution was A reversed-phase C18 column (250 mm 4.6 mm
used directly to derivatize the HSA protein. In a internal diameter, pore size 5 mm; Teknokroma,
general procedure, the HSA (45 53 mM) was Barcelona, Spain) was used. The mobile phase
dissolved in a phosphate-buffered solution (0.1 M, consisted of a mixture of methanol and water
pH 7.3 plus 50 mM NaCl) and was reacted with (80:20, v/v) was delivered at a flow rate of
a suitable molar excess of 20-sulfo-NHS-succinyl- 1.00 mL/min with a pump (WellChrom, K-1001,
DTX ranging from 30 to 40 times. The reaction Knauer, Berlin, Germany). The column effluent
mixture was maintained for 1 h at room tempera- was detected at 230 nm with an ultraviolet
ture, then centrifuged at 4000g for 15 min (Sigma detector (WellChrom, K-2600, Knauer). For dif-
3K30, Ostrode, Germany) and was sterile filtered ferential separation of the DTX HSA conjugate
through the 0.45 mm membrane filter (Sartorius with any nonconjugated DTX, a methanolic
AG, Gottingen, Germany). All conjugate solutions solution of DTX was prepared and injected into
were stored in PBS buffer of pH 7.4 at 48C. For the HPLC column as per the above method.
extended storage, mannitol was added as the Also to confirm the peak related to the
cryoprotectant (2%, w/v) to the conjugate solution conjugate s peak, 2 mg of the dried conjugate
and was lyophilized at 408C for 48 h (Lyotrap was dissolved in 0.5 mL of water and mixed, and
Plus, LTE Scientific Ltd, Oldham, UK) and kept at then 1 mL of ethyl acetate was added into the
208C. mixture and mixed for 2 min. The mixture was
then centrifuged for 10 min at 14,000g at 48C; the
organic phase was separated and evaporated
under nitrogen flow. The dried sample was
reconstituted in HPLC mobile phase, after that
Characterization of the Conjugate
20 mL of the supernatant was analyzed by HPLC.
Gel Permeation Chromatography (GPC)
To primarily identify the DTX HSA conjugate,
GPC was used. To characterize the size of the
Size and Size Distribution
modified protein, the PL Aquagel OH mixed gel-
Particle size and size distribution of the DTX
filtration column (300 mm 7.5 mm internal
HSA conjugate were determined by laser light
diameter, pore size 8 mm) from Agilent Technol-
scattering (Zetasizer ZS, Malvern, UK). The sam-
ogies (Santa Clara, CA) was used. The chromato-
ples were prepared by suspending the freeze-dried
graphic procedures were performed at 238C. All
conjugates in 10 mL deionized water (10 mg/mL)
chromatograms were generated on an Agilent
and were examined to determine the mean
1100 Liquid Chromatographer (Agilent Technol-
diameter.
ogies) and the eluting fractions were monitored
using a RID (refractive index signal detector,
Agilent technologies). Water was used as the
mobile phase at a flow rate of 1.00 mL/min. Fifty
Hydrolysis Studies in Buffer Solutions at pH 5.5,
milligrams of HSA was dissolved in 3 mL phos-
7.4 and Plasma
phate buffer (0.1 M, pH 7.3 plus 50 mM NaCl), and
then 1 mL mixture of DMSO and DMF (70:30, v/v) Two milligrams of the lyophilized conjugate in
was added and analyzed by the GPC system. The 10 mM phosphate buffer, 0.15 M NaCl, pH 7.4, 6,
same process of sample preparation and analysis or 5 was incubated at 378C. At scheduled times,
was performed on the synthesized conjugate of the solutions were sampled and analyzed by
DTX HSA. HPLC according to the protocol reported above.
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DOSETAXEL-ALBUMIN CONJUGATES NANOPARTICLES 2721
For studying the stability of the conjugate in where Ints is the colorimetric intensity of the cells
plasma, 2 mg of the lyophilized conjugate was incubated with the samples and Intcontrol is the
added to 1 mL of rat plasma. The samples were colorimetric intensity of the cells incubated with
kept at 378C, under mild stirring. At scheduled the PBS only (positive control). IC50, the drug
times, 100 mL volume was taken and DTX was concentration at which the inhibition of 50% cell
extracted from plasma by adding ZnSO4 (2%, w/v, growth was calculated by the curve fitting of the
in mixture of 50:50 methanol/water) at a ratio of cell viability data using Prism 4.0 (Graphpad, San
1:2, and analyzed by described HPLC method. Diego, CA).
Standard curves were prepared by the addition of
DTX in plasma following the same process. Each
experiment was repeated in triplicate.14 Body Distribution of DTX HSA Conjugate
In vivo biodistribution studies were performed
using 13-week-old female balb/c mice (provided
Cell Culture
by Animal Care Center, Faculty of Pharmacy,
Human breast cancer cells (T47D) and ovary Medical Sciences/University of Tehran) after a
cancer cells (SKOV3) (American Type Culture 7-day acclimatization period. All mice were ran-
Collection) were cultivated in RPMI-1640 medium domly assigned into two groups (n ź 5 7 mice/
supplemented with 10% fetal bovine serum and group). The mice were fasted overnight to prevent
1% penicillin streptomycin at 378C in a humidi- coprophagia but had free access to water. Each
fied incubator with 5% CO2. Cells were main- group received 2 mg/kg of DTX formulation either
tained in an exponential growth phase by periodic as DTX HSA conjugate or Taxotere1 via tail vein
sub-cultivation. The medium was replenished injection. Both formulations were sterile filtered
every other day. and administered in 13% ethanol in water for
Taxotere1 and in NaCl 0.9% for conjugate. Two
hours after dosing, mice were sacrificed and major
In Vitro Cell Viability organs were collected for analysis. Organ samples,
consisting of lungs, liver, heart, kidneys, intes-
SKOV3 and T47D cells were seeded in 96-well
tine, and spleen were removed, washed with NaCl
plates (Costar, IL) at the density of 1 104 viable
0.9% and accurately weighed. The organ samples
cells/well and incubated for 24 h to allow cell
were then homogenized and centrifuged at
attachment. The cells were incubated with the
21,000g for 10 min. Methanol was added to the
DTX or DTX HSA conjugate suspension at the
supernatant (1:1) to precipitate the unwanted
concentrations of 10 100 nM for 96 h.
proteins and centrifuged (21,000g for 10 min).
For all studies involving cultured tumor cells,
The aliquots were assayed for DTX by the HPLC
we used DMSO instead of ethanolic tween80 as a
to estimate the amount of DTX in each organ.
solvent for DTX-free drug stock solution prepara-
For calculations, standard curves of DTX were
tion, since tween80, which has been used at high
prepared by the addition of DTX solutions in
concentration as surfactant in Taxotere1, has
methanol to tissues following the same treatment
shown to be cytotoxic itself.15 The vehicle group
steps.16 Blood samples were obtained by cardiac
contained the same amount of DMSO used as the
puncture in preweighed heparinized tubes;
vehicle for the DTX solutions preparation.
plasma was separated by centrifugation of the
The diluent for preparing the DTX-free drug
blood samples at 7000g (10 min). DTX was
and DTX HSA conjugate working solution was
extracted from plasma by adding ZnSO4 (2%,
RPMI-1640 culture medium. At designated time
w/v, in the mixture of 50:50 methanol/water) at a
(96 h), 20 mL of MTT (5 mg/mL in PBS) was added
ratio of 1:2, and standard curves were prepared by
to each well and, the culture medium containing
the addition of DTX in plasma following the same
MTT solution was removed after 3 4 h. The
process. Extraction efficiency from plasma and
formazan crystals were dissolved in 100 mL
various tissues was variable (80 95%).
DMSO and read at 570 nm by a microplate
reader. Cell viability was calculated by the follow-
ing equation:
Statistical Analysis
Ints
Results are expressed as mean SD. Statistical
Cell viabilityð%Þ Åº 100
Intcontrol
data analysis was conducted using statistical
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2722 ESMAEILI ET AL.
software program (SPSS1 11.5, Microsoft). After After primarily confirming the formation of
confirmation of normal distribution using the DTX HSA conjugate by GPC analysis, HPLC
Kolmogoroff Smirnoff test, comparison of the analysis was performed to identify and quantify
data was performed using Student s t-test. the DTX HSA conjugate. As it can be seen in
Figure 3, DTX-free drug showed a single peak
(Peak 2) at the retention time of 4.75 min
RESULTS AND DISCUSSION (RT ź 4.75). After the injection of the synthesized
DTX HSA, an additional peak (Peak 1) appeared
We did many efforts to prepare DTX HSA at RT ź 4.25 min, next to the DTX-free drug peak.
conjugates before. Different factors influence the To confirm that this additional peak is related to
conjugation, including incubation time required the DTX HSA conjugate, liquid liquid extraction
for the fabrication of the bonds between DTX and with ethyl acetate was carried out. Injecting the
succinic spacer and DTX HSA, purification and ethyl acetate phase into the HPLC column showed
characterization of the conjugates. In previous only one peak at RT ź 4.75, corresponded to the
works, the radiolabeled drug has been used to peak of the free DTX. In extracting with an
characterize and determine the conjugates in vitro organic solvent such as ethyl acetate, conjugated
and in vivo, such as radiolabeled paclitaxel used or free HSA remained in aqueous phase and only
by Dosio et al.8 In this study instead of radi- nonconjugated HSA was removed by the organic
olabeled DTX, a HPLC method was developed for phase; therefore, one peak related to free DTX
the separation of the DTX HSA conjugate. The appeared in the HPLC chromatogram. Also to
characterization of the conjugate is shown below. confirm this finding, different ratio of the HPLC
mobile phase solvents and flow rates were tested
and the peaks 1 and 2 retention times were
Preparation of 2(-Sulfo-NHS-Succinyl-DTX
changed according to the different conditions
Conjugation of paclitaxel and HSA has been (chromatograms not shown). A good resolution
previously reported by Dosio et al., in which could be observed between peak 1 (DTX HAS
20-succinyl-paclitaxel was used to prepare 20-sulfo- conjugate) and peak 2 (DTX-free drug) and also
NHS-succinyl paclitaxel.8 HSA was covalently nonconjugated HSA peak appeared at the reten-
attached to the paclitaxel through a succinyl tion time of 1.3 2.5, which is far from DTX HSA
spacer group. With the inspiration of Dosio s or DTX-free drug and does not have any interac-
method, in this study, a conjugation between HSA tion in the analysis.
and DTX was achieved via a similar pathway with Comparing the area under the curves of DTX
1
some modification. HNMR spectrum of DTX HSA conjugate and DTX-free drug in HPLC
was well interpreted.17 1HNMR spectra data of chromatograms (Fig. 3) gives an evaluation for
20-succinyl-DTX in comparison with DTX showed the percentage of drug loading. The results
an additional signal at 2.5 2.7 ppm as multiple obtained with this analytical procedure indicated
peaks that could be related to OCOCH2CH2COOH that the ratio of the DTX HSA to free DTX in the
moiety (Fig. 1). This observation is to confirm the final product was about 51%, and the total drug
chemical shift for OCOCH2CH2COOH group loading based on DTX HSA conjugate and DTX-
1
in HNMR spectrum of 20-succinyl-paclitaxel.8 free drug was about 1.3% (w/w).
Finally, 20-sulfo-NHS-succinyl-DTX was used to The resultant conjugate had a mean diameter of
prepare DTX HSA conjugation through a succi- 90 110 nm, approximately one-hundredth of the
nyl spacer group (Scheme 1). size of a single red blood cell, thus eliminating
the need for any solvent. The key factors that
determine the performance of the drug-loaded
Characterization of the DTX HSA Conjugate
nanoparticles include the particle size and surface
In order to evaluate the modified HSA after coating.18 The size is a crucial parameter in the
derivatization, the conjugate was analyzed by design of drug delivery system since it is a main
GPC (Fig. 2). The GPC chromatogram of DTX factor in determining the cellular uptake effi-
HSA conjugate showed additionally a small peak ciency of the particles.18 Moreover, the particle
at 8.8 min missing in the spectrum of HSA. This size is associated with the biological response of
peak illustrated that coupling reaction with the tissue to foreign biomaterials.18
probable formation of bonds between HSA and Stability and drug studies were carried out by
DTX occurred when conjugate was made. incubation of lyophilized DTX HAS conjugate at
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 8, AUGUST 2009 DOI 10.1002/jps
DOSETAXEL-ALBUMIN CONJUGATES NANOPARTICLES 2723
Figure 1. spectra of (A) DTX and (B) 20-succinyl-DTX.
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2724 ESMAEILI ET AL.
Scheme 1. Docetaxel and derivatives.
different pH and plasma. Phosphate buffer at In Vitro Cell Viability of the DTX HSA Conjugate
pH 5.5 and 7.4 was selected to mimic intratumoral
and extracellular pH compartments, respectively. The standard procedure for MTT assay is 48 72 h.
The conjugate degradation profiles at pH 5.5 and We have tested the effect of DTX for up to 168 h
7.4 depicted in Figure 4 confirm that DTX HAS treatment. Our results indicated that 72 or 96 h
conjugate undergoes pH-dependent degradation. incubation of T47D and SKOV3 with DTX
Figure 4 shows that at pH 5.5 the succinyl provided 18% and 12% difference in viability,
derivative undergoes rapid disappearance indi- respectively. However, in the 96 h treatment, the
cating that under these conditions the ester bond experiment had lower deviation compared to 72 h
is quickly hydrolyzed (about 37% of the esteric (with SD of 3% for 72 h and 1.5% for 96 h). Thus,
bond cleavage occurred during the 24-h incuba- 96 h incubation time was used to better report the
tion). The profiles depicted in Figure 4 show that difference. Furthermore, cells were in the pre-
at pH 7.4 the hydrolysis of the ester bond between sence of the drug for 96 h and the medium was not
succinic spacer and DTX degradation is slower as changed when the treatment started.
compared to that at pH 5.5. Figure 5 shows the in vitro cytotoxic effect of
Figure 4 also shows that in plasma the DTX DTX-free drug and DTX HSA conjugate for T47D
HSA conjugate undergoes rapid ester bond (A) and SKOV3 (B) cells (n ź 6), respectively.
hydrolysis and degradation, which are reflected The result indicates that the DTX HSA con-
in the release of about 54% of DTX from the jugate has showed advantages in achieving lower
conjugate in 24 h. The faster degradation in cell viability or equivalently, higher cytotoxicity
plasma as compared to pH 7.4 seems to indicate versus the DTX-free drug. The cell viability
that in plasma the hydrolysis of the succinic ester measured at the 100 nM drug concentration
bond takes place by both chemical and enzymatic was decreased from 20.73% and 25.51% for
mechanisms.14 DTX-free drug to 6.28% and 3.69% for the
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DOSETAXEL-ALBUMIN CONJUGATES NANOPARTICLES 2725
Figure 2. GPC chromatogram of (A) HSA (50 mg/mL) and (B) DTX HSA conjugate;
( ) shows the conjugate peak.
conjugate formulation after 96 h incubation with (39.94 1.75) and has made the DTX HSA more
T47D and SKOV3 cells, respectively. Conjugate toxic to cells in vitro (Tab. 1).
has shown inhibition of proliferation in all cell As in vitro release studies show (Fig. 4), about
lines even at low drug concentration (10 nM) after 20% of the DTX is released from DTX HSA
96 h as seen in Figure 5. The IC50, the dose which conjugate after 24 h at pH 7.4, which is the cell
produces 50% inhibition of cell growth, of DTX- culture medium pH. Thus, the increased cytotoxi-
free drug and DTX HSA conjugate was calculated city of the conjugate formulation is not only via the
and as presented in Table 1, the conjugate DTX released from the conjugate but also it is
formulation has decreased the IC50 value for both probably because of cell uptake of the nanoparti-
cell lines ( p < 0.05). culate conjugate and/or better penetration of the
The IC50 of DTX HSA conjugate is 33% and 25% conjugate into the cells which in either case DTX
lower than that of DTX free in T47D and SKOV3 will be delivered to its site of action.19
cell lines, respectively. The DTX HSA conjugate Transendothelial cell transport of albumin is
has lowered the IC50 of the T47D cells by 50% mediated by the gp60 (albondin) receptor and
to 6.30 0.73 nM compared to DTX-free drug caveolar transport.20 As albumin binding to gp60
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2726 ESMAEILI ET AL.
Figure 3. HPLC chromatogram profiles of ( ) DTX solution in water (10 mg/mL), (- - -)
DTX HSA conjugate solution in BPS (pH 7.4) and ( ) ethyl acetate extract of the
conjugate. (1) shows the peak of the DTX HSA conjugate and (2) represents the DTX
peak.
activates caveolin-1 resulting in the formation of
caveoli, which transports albumin and other
plasma constituents across the endothelial cell
to the interstitial space; the increased antitumor
activity of DTX HSA conjugate may also be
related to the enhanced intratumor delivery of
DTX. The gp60 receptors are specific for albumin
and, once activated, allow for the transport of
albumin complexes across blood vessel wall
barriers into the underlying tumor tissue,21 after
which the conjugate acts as intracellular drug
depots, slowly releasing the attached therapeutic
Figure 4. Stability of the DTX HSA conjugate in Figure 5. Cytotoxic effect of DTX-free drug and
phosphate buffer at pH 5.5 and 7.4 and in rat plasma DTX HSA conjugate incubated with (A) T47D and
(n ź 3). (B) SKOV3 cells (n ź 6).
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DOSETAXEL-ALBUMIN CONJUGATES NANOPARTICLES 2727
Table 1. IC50 of SKOV3 Cells Incubated With sesses a long plasma half-life in vivo, has been
DTX-Free Drug and DTX HSA Conjugate at 96 h
extensively used as a carrier in the preparation of
(n ź 6)
conjugates with antineoplastic27 or antiviral28
properties. The HSA was also very attractive for
IC50 (nM)
the derivatization of DTX, which is almost
completely insoluble in water. 75.57% and
Cell Line DTX-Free Drug DTX HSA Conjugate
59.31% of the original dose after 2 h postinjection
T47D 39.94 1.75 6.30 0.73
was found in blood, spleen, lung, liver, kidney,
SKOV3 55.86 7.79 30.75 2.51
intestine, and heart in DTX HSA conjugate and
Taxotere1 formulation, respectively. Figure 6
shows the result of the biodistribution studies.
agent into the cellular cytoplasm. This results There were significant differences in drug dis-
in the enhancement of therapeutic efficacy for tribution for liver, lung, spleen, kidney, and
drugs like dexamethasone22 and paclitaxel in plasma ( p < 0.05). The amount of DTX and/or
drug-sensitive cells, because cytoplasm is the site its metabolite was six- to sevenfold higher in
of action for these drugs.23 As shown in Figure 4, plasma for DTX HSA conjugate when compared
about 40% of the DTX is released from the to Taxotere1 ( p < 0.05). The amount of the DTX
conjugate at pH 5.5, which is the pH of the tumor in liver and kidneys was lower in DTX HSA
cells, during 24 h incubation. Both cell lines used conjugate group compared to Taxotere1 ( p < 0.05)
in this study were DTX-sensitive and cytoplasm is and there was no significant difference between
the site of action for DTX, and DTX HSA the distribution of the DTX and DTX HSA
nanoparticles with better cytotoxic effects are in conjugate for intestine and heart ( p > 0.05).
agreement with other scientific reports on pacli- The DTX concentration in lungs was also higher
taxel or dexamethasone.23 in DTX HSA conjugate than Taxotere1 ( p < 0.05)
The presence of efflux transporters such as possibly due to the filtration effect of the lung
multi-drug resistance proteins (MRP) or mem- capillary bed that removes larger particles or their
brane bound p-gp in tumor cells also requires the aggregates. Similarly, the relatively high DTX
dosed drug be optimally localized to its desired site concentration in spleen for DTX HSA conjugate
of action within the cell without its exposure to ( p < 0.05) could also be explained by the splenic
these transporters.24 filtration.29
Thus, the lack of tween80-micelle sequestration It is likely that the significant differences in
of DTX and/or increased albumin-mediated trans- DTX biodistribution between DTX HSA conju-
port may explain the increased intratumoral gate and Taxotere1 in kidneys can be explained
accumulation of DTX for DTX HSA conjugate. by the exclusion of the conjugate by kidneys
The ability to give higher doses of DTX in the
absence of tween80 is clinically important. Thus,
the increased antitumor activity of DTX HSA
conjugate, related to enhanced intratumor deliv-
ery of DTX may be regarded as an extra bonus.
Body Distribution of DTX HSA Conjugate in Mice
The goal of the biodistribution studies was to
investigate the differences in organ distribution
of DTX HSA conjugate when compared to
Taxotere1.
In order to reduce the systemic toxicity of DTX
such as cumulative fluid retention25 as well as to
Figure 6. The level of DTX detected in the organs of
avoid the use of toxic excipients such as tween80,
mice after IV administration of DTX HSA conjugate
which causes severe hypersensitive reactions2,26;
and Taxoterte1. The data obtained from lung, liver,
we used HSA chemically conjugated to the drug as
spleen, intestine, heart, kidney, and plasma in 2 h after
a delivery system. This nonspecific carrier, which
injection were calculated as a percent of the initial dose
is easily distributed in tumor tissues and pos- applied/g tissue and presented as mean SD (n ź 6).
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 8, AUGUST 2009
2728 ESMAEILI ET AL.
through filtration process because of the high cells, the antitumor activity of DTX has shown to
molecular weight of the albumin. be greater than that of other recognized agents,
When DTX was conjugated to albumin, the including cisplatin, cyclophosphamide, doxorubi-
plasma level after intravenous administration cin, and etoposide. In a direct comparison with
maintained at a good level (16.19%) for up to 2 h paclitaxel in a large number of freshly explanted
in comparison with DTX formulated in tween80 in tumors (including breast, ovarian, lung, and
which about 2.51% of the initial dose was present colorectal tumors), DTX has been found to have
in plasma up to 2 h. at least equivalent cytotoxicity and incomplete
As the DTX HSA conjugate distributed to the cross-resistance. DTX also had a longer residence
liver, which is the major reticuloendothelial time, accumulated at higher concentrations
system (RES) organ, was less than Taxotere1, within cells, and demonstrated a superior ther-
the RES-evading ability of such conjugate is high. apeutic index,31 besides the DTX being more
Since the size of the colloidal carriers is a key for potent than paclitaxel and also the advantages of
the biological fate of the nanoparticles, and the DTX HSA conjugate prepared in this study
nanoparticles of smaller than 100 nm or sur- such as high circulation life in vivo, the DTX HSA
face-modified nanoparticles (Stealth1) usually conjugate could be more successful than the
bypass the RES macrophages uptake, the DTX paclitaxel HSA conjugate in clinical cancer
HSA particles of 90 110 nm were small enough to therapy.
bypass the RES macrophages30 and this is one
of the advantages of the DTX HSA conjugate
prepared in this study compared to the paclitaxel
CONCLUSIONS
HSA conjugate reported by Dosio et al.8 that there
is no need to prepare the PEG HAS DTX or
In conclusion, an albumin-conjugated formulation
Stealth1 nanoparticles for longer blood circula-
of DTX was developed that was more soluble
tion time.
with enhanced in vivo characteristics and higher
Moreover, it was observed that the presence of
activity against tumor cells. This conjugate ap-
a spacer arm in the drug conjugate resulted in
pears to be a good candidate for further antitumor
slower release of the drug. Incubation of the
tests in animal models. In summary, DTX HSA
conjugate in rat plasma showed that after 24 h,
conjugate is a tween80-free, albumin bound with
about 54% of the DTX was released from the
a size of 90 110 nm form of DTX that showed an
conjugate (Fig. 4). Considering the in vivo features
improved in vitro cytotoxicity and in vivo bio-
of the DTX HSA conjugate, it is possible to
distribution profile.
suggest that after intravenous administration in
mice there may be a depot effect, that is, the
conjugate may remain for a longer time in the
ACKNOWLEDGMENTS
blood and slowly release the pharmacological
active drug over a prolonged period. This finding
The authors would like to thank Medical Nano-
is in agreement with Dosio et al. report on
technology Research Center, Medical Sciences/
paclitaxel HSA conjugate.8
University of Tehran, and The Special Office of
Then as it is obvious, prepared DTX HSA
Nanotechnology Development for their financial
conjugate is able to present a good plasma level
support, and Sobhan Chemotherapeutics Co. for
in vivo in comparison with DTX conventional
providing Taxotere1 vials .The authors would
formulation.
also like to thank Mr. A.R. Kazemi for his tech-
Also as the level of DTX formulated in nano-
nical assistance in animal experiments, and
particles was higher for organs like lung and
Mr. H. Akbari and Mrs. Sh. Tavajjohi for their
spleen in comparison with Taxotere1, and DTX
kind assistance in cell culture experiments.
is a drug of choice in the treatment of lung
carcinoma, it is an added value for conjugation of
DTX with HSA for carcinomas located in these
organs especially lung cancer.
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