Y. Liu et al.
Biochemical Pharmacology xxx (xxxx) xxx
0.5% hydrochloride solution in water for 2 days. The Boc-PMet polymer
(compound 4) was then lyophilized.
concentration regions.
The DOX concentration was assayed by high performance liquid
chromatography (HPLC), detected by a Waters 2475 Fluorescence De-
tector with excitation at 490 nm and emission at 590 nm. The drug
encapsulation efficiency (DEE) and drug loading capacity (DLC) were
calculated by the following equations (1–2).
The Boc-PMet polymer (compound 4) was deprotected at room
temperature in DMSO/TFA (1/1, v/v) mixture for 2 h, and then dialyzed
against distilled water for 2 days. The Boc-deprotected PMet product
with 15% free amino group (compound 5) was then lyophilized.
WPTX incorported in micelle
WTotal PTX added in micelle
DEE(%) =
× 100
(1)
2.2.3. Synthesis of PMet-P(cdmPEG2K) polymer
PMet-P(cdmPEG2K) was synthesized by a ring opening reaction of
PEG2K-cdm and pMet polymers. Boc-deprotected PMet (compound 5,
100 mg) and PEG2K-cdm (compound 1, 303 mg) were dissolved in 4 mL
of DMSO and stirred at 37 ◦C for 24 h. The mixture was dialyzed against
DMSO and then distilled water for 2 days. The final product of PMet-P
(cdmPEG2K) polymer was then lyophilized.
WPTX incorported in micelle
DLC(%) =
× 100
(2)
WTotal
micelle
The serum stability of the PMet-P(cdmPEG2K) micelles co-loaded
with pIL-12 and DOX was studied to evaluate the shielding effect of
PEG in preventing aggregation in serum. The pIL-12/PEI25K polyplexes,
blank PMet-P(cdmPEG2K) micelles, pIL-12, DOX and pIL-12/DOX
loaded micelles, were mixed with a BSA solution (1 mg/mL) in HEPES
buffer (10 mM, pH 7.4) under gentle stirring. The average sizes of mi-
celles were monitored over time by a Malvern Zeta Nanosizer.
1H NMR spectrum was analyzed on a Varian-400 FT-NMR spec-
trometer at 400 MHz with DMSO‑d6 and CDCl3 as the solvent.
2.3. Preparation and characterization of DOX and pIL-12 co-loaded
micelles
2.3.4. Tumor extracellular pH (pHe) sensitivity
2.3.1. Preparation of micelles
The sensitivity of pIL-12/DOX co-loaded PMet-P(cdmPEG2K) micel-
leplexes to the acidic pH was evaluated by examining changes in the zeta
potentials at different pHs. The pIL-12 loaded and pIL-12/DOX co-
loaded PMet-P(cdmPEG2K) micelleplexes were incubated in HEPES (10
mM) buffer at pH 7.4 and pH 6.8 at 37 ◦C. Aliquots of the micelleplex
solutions were withdrawn at designated time intervals and the zeta
potential was measured with a Malvern Zeta Nanosizer.
Blank and DOX loaded PMet-P(cdmPEG2K) micelles were prepared
using a thin film hydration method. Briefly, DOX (5 mg/mL in 1:1(v/v)
of DCM/methanol) and PMet-P(cdmPEG2K) (10 mg/mL in DCM) at
designated mass ratios were mixed in a glass tube, and organic solvent
was removed through a gentle stream of nitrogen, followed by drying
under vacuum for 1 h. The obtained thin-film of PMet-P(cdmPEG2K)/
DOX mixture was hydrated in HEPES buffer (10 mM, pH 7.4), forming a
clear solution of DOX-loaded PMet-P(cdmPEG2K) micelles. The blank
micelles were prepared as above in the absence of DOX.
2.3.5. In vitro drug release
In vitro DOX release profile was investigated by the dynamic dialysis
method. Typically, DOX loaded micelles and pIL-12/DOX co-loaded
PMet-P(cdmPEG2K) micelleplexes with 0.5 mg of DOX were transferred
into a dialysis bag (molecular weight cutoff = 3500) and immersed in 80
mL of HEPES (10 mM) buffer at pH 7.4 or pH 6.8, while stirring at 100
rpm and a temperature of 37 ◦C. DOX release from DOX loaded micelles
and pIL-12/DOX co-loaded PMet-P(cdmPEG2K) micelleplexes was
assayed at predetermined times by fluorescence spectrometry with
excitation at 490 nm and emission at 590 nm. The DOX diffusion profile
from the DOX solution was also investigated for comparison.
For preparation of pIL-12 loaded micelleplexes and pIL-12/DOX co-
loaded micelleplexes, equal volumes of pIL-12 and blank micelles or
DOX-loaded PMet-P(cdmPEG2K) micelles were mixed at various N/P
ratios (the ratio of the number of amino groups in PMet-P(cdmPEG2K) to
the number of phosphate groups in pIL-12), and the mixture was further
incubated at room temperature for 20 min.
2.3.2. Gel retardation assay
The pIL-12 binding efficiency of blank and DOX loaded PMet-P
(cdmPEG2K) micelles was analyzed by agarose gel electrophoresis. The
pIL-12 loaded PMet-P(cdmPEG2K) micelleplexes and pIL-12/DOX co-
2.4. Cell culture
loaded PMet-P(cdmPEG2K) micelleplexes, containing 0.5 μg of pIL-12
prepared at various N/P ratios from 1 to 20, and DOX loaded micelles
with equal DOX concentrations as in the counterparts of pIL-12/DOX co-
loadeded micelleplexes, were loaded onto a 0.8% agarose gel and run
under tris–acetate (TAE) buffer at 80 V for 45 min. pIL-12 retardation
was visualized and photographed using a ChemiDoc XRS imaging sys-
tem (Bio-Rad, Hercules, CA, USA).
4T1.2 is a mouse metastatic breast cancer cell line, which was
cultured in DMEM culture medium, containing 10% (v/v) fetal bovine
serum and 100 IU/mL penicillin and 100 μ
g/mL streptomycin at 37 ◦C in
a humidified 5% CO2-95% air atmosphere.
2.5. In vitro cytotoxicity
2.3.3. Characterizations of micelles and micelleplexes
2.5.1. In vitro cytotoxicity of micelleplexes
The size distribution and zeta potential of blank and DOX-loaded
micelles, and pIL-12 loaded and pIL-12/DOX co-loaded micelleplexes
at various N/P ratios, were examined by dynamic light scattering (DLS)
through a Malvern Zeta Nanosizer. The morphology was observed by
transmission electron microscopy (TEM) using a negative staining
method.
In vitro cytotoxicity of PMet-P(cdmPEG2K) micelleplexes with pEGFP
was measured using the MTT method with PEI25K and pEGFP/PEI25K
polyplexes as controls. 4T1.2 cells were seeded onto a 96-well plate at a
density of 3 × 103 cells/well and incubated for 24 h. Culture were
treated with PEI25K, pEGFP/PEI25K polyplex, PMet-P(cdmPEG2K) mi-
celles and pEGFP/PMet-P(cdmPEG2K) micelleplexes of various N/P ra-
tios in FBS-free culture medium (Life Technologies, USA) at pH 7.4 and
pH 6.8 for 4 h. The culture medium was then replaced with fresh com-
plete medium at pH 7.4. After further incubation for 44 h, the MTT assay
was carried out as previously described [35,38]. Relative cell viability
was calculated by the following Eq. (3):
The critical micellar concentration (CMC) of PMet-P(cdmPEG2K) was
measured by fluorescence intensity using nile red as a probe. Briefly, nile
red and differing amounts of PMet-P(cdmPEG2K) dissolved in DCM were
put into tubes, the solvent was removed by nitrogen flow and the formed
thin film was dried under vacuum. HEPES was added to yield a final nile
red concentration of 6.0 × 10ꢀ 7 M, with the PMet-P(cdmPEG2K) micelle
concentrations ranging from 1.0 × 10ꢀ 4 to 5 × 10ꢀ 1 mg/mL. Fluores-
cence was carried out with excitation at 550 nm and emission spectra
recorded from 570 to 720 nm. The CMC value was determined as the
cross-point when extrapolating the intensity at low and high
Asample ꢀ Ablank
Cell viability(%) =
× 100
(3)
Acontrol ꢀ Ablank
Asample and Acontrol are the absorbance in the presence and absence of
4