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was then filtered using a 0.2 μm syringe filter. The concentration of
cis-[Pt(NH3)2(H2O)2](NO3)2 was measured using inductively coupled
plasma mass spectrometry (ICP-MS) and adjusted to 200 mM.
2.4. Preparation of LPC NPs
The synthesis route of LPC NPs is described in Scheme 2. First, 100 μL
of 200 mM cis-[Pt(NH3)2(H2O)2](NO3)2 was dispersed in a solution
composed of mixture of cyclohexane/Igepal CO-520 (71:29, V:V) and
cyclohexane/triton-X100/hexanol (75:15:10, V:V:V) to form a well-
dispersed, water-in-oil reverse micro-emulsion. Another emulsion con-
taining KCl was prepared by adding 100 μL of 800 mM KCl in water into
a separate 8.0 mL oil phase. One hundred microliters of DOPA (20 mM)
was added to the CDDP precursor phase and the mixture was stirred.
Twenty minutes later, the two emulsions were mixed and the reaction
proceeded for another 30 min. After that, 16.0 mL of ethanol was
added to the micro-emulsion and the mixture was centrifuged at
12,000g for at least 15 min to remove the cyclohexane and surfactants.
After being extensively washed with ethanol 2–3 times, the pellets were
re-dispersed in 3.0 mL of chloroform and stored in a glass vial for fur-
ther modification.
Scheme 1. Classic synthesis route of CDDP.
expected to improve the solubility of platinum based drug candidates
with poor solubility, such as cis-diamminedibromoplatinum(II) and cis-
diamminediiodoplatinum(II). Therefore, we hypothesize that: (1) CDDP
can be encapsulated as a nanoprecipitate in a microemulsion and stabi-
lized in an organic solvent with 1, 2-dioleoyl-sn-glycero-3-phosphate
(DOPA); (2) DOPA-coated CDDP NPs can be further dispersed into aque-
ous solution by adding lipids to form the outer leaflet of the coating bilay-
er; (3) the lipid bilayer-coated CDDP NPs will show anti-cancer activity
in vitro and in vivo.
To prepare the final NPs, 1.0 mL of LPC NPs core, 100 μL of 20 mM
DOTAP/Cholesterol (molar ratio 1:1) and 50 μL of 10 mM DSPE–PEG-
2000 or DSPE–PEG–AA were combined. After evaporating the chloro-
form, the residual lipids were dispersed in 1.0 mL of d-H2O.
2. Materials and methods
2.1. Materials
2.5. Characterization of NPs
Lipids were purchased from Avanti Polar Lipids (Alabaster, AL).
Dulbecco's Modified Eagle Medium (DMEM), L-glutamine, penicil-
lin Gsodium, streptomycin and fetal calf serum were purchased
from Gibco®. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-
N-[anisamide(polyethylene glycol)-2000] (DSPE-PEG-AA) was
synthesized in our laboratory as previously reported [25]. 1-Hexanol
was purchased from Alfa Aesar. Igepal® CO-520, triton™ X-100, cyclo-
hexane, cisplatin and silver nitrate were obtained from Sigma-Aldrich
(St. Louis, MO) without further purification.
The zeta potential and particle size of LPC NPs were determined
using a Malvern ZetaSizer Nano series (Westborough, MA). Transmis-
sion electron microscopy (TEM) images were acquired using a JEOL
100CX II TEM (JEOL, Japan). The LPC NPs were negatively stained with
2% uranyl acetate.
The drug-loading capacity and platinum content were measured
using Varian 820-MS (Palo Alto, CA). Samples were digested in 70%
HNO3 and diluted in water to a final acid content of 2%. Platinum con-
centration was determined using the 195Pt isotope.
2.2. Cell lines
The composition of DOPA-coated CDDP NPs was studied using X-ray
photoelectron spectroscopy (XPS) (Kratos Axis Ultra DLD X-ray Photo-
electron Spectrometer) and NMR (Varain Inova 400 NMR Spectrometer).
1205Lu cells were cultured in DMEM medium supplemented with
10% heat-inactivated fetal bovine serum (FBS), 20 mM of L-glutamine,
100 U/mL of penicillin G sodium, and 100 mg/mL of streptomycin at
37 °C in an atmosphere of 5% CO2 and 95% air.
2.6. Cell toxicity assay
1205Lu cells were seeded in 96-well plates at a density of 2000 cells/
well and incubated in 10% FBS of DMEM containing 100 U/mL penicillin,
and 100 mg/mL streptomycin for 20 h. Then, the medium was removed
and replaced by Opti-MEM containing CDDP or LPC NPs. Forty-eight
hours later, a CellTiter 96 AQueous One Solution Cell Proliferation Assay
(Promega, Madison, WI) kit containing the tetrazolium compound MTS
was used to assay cell viability according to the manufacturer's protocols.
2.3. Synthesis of cis-[Pt(NH3)2(H2O)2](NO3)2 precursor
To a suspension of CDDP (60 mg, 0.20 mmol) in 1.0 mL water,
AgNO3 (66.2 mg, 0.39 mmol) was added. The mixture was heated at
60 °C for 3 h and then stirred overnight in a flask protected from
light with aluminum foil. Afterwards, the mixture was centrifuged at
16,000 rpm for 15 min to remove the AgCl precipitate. The solution
2.7. Cellular uptake
1205Lu cells (2 × 105) were seeded in 35 mm MatTek glass bottom
dishes (MatTek Corporation, MA) 20 h before experiments. The cells
were treated with NBD-PE labeled LPC NPs at a concentration of
100 μM platinum (Pt) at 37 °C for 4 h. The cells were subsequently
washed twice with PBS. The nucleus was stained with Hoechest 33342
(Sigma, St Louis, MO), and lysosomes were stained by lysotracker red
(Invitrogen, Carlsbad, CA). Then, the sample was observed by Olympus
FV 1000-MPE microscope (Olympus, Japan).
To measure the amount of Pt in cells, cells were washed and lysed in
order to determine their uptake of NPs. Samples were digested in 70%
HNO3 and diluted in water to a final acid content of 2%.
Scheme 2. Synthesis of lipid bilayer-coated CDDP NPs using a reverse microemulsion
method.