L. Huang et al. / International Journal of Pharmaceutics 466 (2014) 52–57
53
Scheme 1. The schematic illustration of structure of CP-Folate/CPN = Dox/PTX mixed micelle and the process of the targeted drug delivery.
Sinopharm (China) and used as received. Doxorubicin hydrochlo-
ride (Dox
HCl) and Brij S 100 (average Mn ꢁ4670) were purchased
2.1.4. C18-PEG-Folate (CP-Folate)
18-PEG-CDI (476 mg, 0.1 mmol), Folate-NH2 (241 mg,
ꢀ
C
from Sigma–Aldrich and used as received. Dimethyl sulfoxide
(DMSO) was dried over 4 Å molecular sieve and distilled under
vacuum. Dichloromethane (DCM) was distilled over CaH2 before
use. Methanol was dried over 3 Å molecular sieve and distilled by
rectification. Bovine serum albumin (BSA), Dubelcco’s Modified
Eagle’s Medium (DMEM), penicillin–streptomycin, trypsin, and
phosphate-buffered saline (PBS) were purchased from GIBCO
Invitrogen Corporation. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) and Hochest 33258 were purchased
from Sigma–Aldrich.
0.5 mmol) and triethylamine (0.21 g, 2.1 mmol) were dissolved in
10 mL of DMSO. The mixture was stirring at room temperature for
48 h, and then dialyzed extensively against with DI water for 72 h
(Mw cutoff: 3500 Da). The CP-Folate was obtained by freeze-
drying. Yield: 327 mg (63.5%).
2.1.5. C18-PEG-NH-N = Dox (CPN = Dox)
Dox HCl (87 mg, 0.15 mmol) and C18-PEG-NH-NH2 (476 mg,
ꢀ
0.1 mmol) were dissolved in 15 mL of anhydrous menthol, and
treated with a drop of TFA. The solution was refluxed under dark for
48 and then cooled down to room temperature. The solvent was
evaporated under vacuum and the residue was resolved in 10 mL of
anhydrous DMSO. The solution was dialyzed against with DMSO
for 48 h (Mw cutoff: 3500 Da) and then dialyzed extensively
against with DI water to remove the organic solvent and any other
impurities. The CPN = Dox was obtained by freeze-drying as dark
red solid. Yield: 299 mg (57.2%).
2.1.1. C18-PEG-CDI
Brij S 100 (4.67 g, 1.0 mmol) and CDI (1.62 g, 10.0 mmol) were
dissolved in DCM (50 mL), and then the solution was allowed at
room temperature for 24 h. The most portion of solvent was
evaporated and the residue was poured into excess anhydrous
diethyl ether. The white precipitate was collected by centrifuga-
tion, washed with diethyl ether and dried under vacuum. Yield
4.2 g, (89.9%).1H NMR (300 MHz, CDCl3,
d
ppm): 8.14 (s,1H), 7.47 (d,
2.2. Methods
1H), 7.09 (d, 1H), 4.56 (t, 2H), 3.42–3.88 (m, 400H), 1.57 (m, 2H),
1.25 (m, 30H), 0.88 (t, 3H).
1H NMR spectra were recorded at 300 MHz on a Mercury VX-
300 spectrometer by using tetramethylsilane (TMS) as the internal
reference. A drop of micelle solution was placed onto a copper grid
with carbon film and then stained by phosphotungstic acid. The
TEM images were taken by JEM-2100 (HR) transmission electron
microscope at an acceleration voltage of 200 keV. Size and
distribution of the mixed micelles were measured by Dynamic
Light Satter (Zetasizer, Malvern).
2.1.2. C18-PEG-NH-NH2
C18-PEG-CDI (476 mg, 0.1 mmol), hydrazine (16 mg, 0.5 mmol)
and triethylamine (101 mg, 1 mmol) were dissolved in 10 mL of
DCM. The mixture was concentrated by rotary evaporation and the
residue was poured into excess anhydrous diethyl ether to form
precipitate. The white precipitate was collected by centrifugation,
then washed with diethyl ether and dried under vacuum. Yield:
412 mg, (86.6%). 1H NMR (300 MHz, CDCl3,
d
ppm), 4.27 (m, 2H),
2.2.1. Determination of the content of Dox in CPN = Dox
3.42–3.88 (m, 400H), 1.57 (m, 2H), 1.25 (m, 30H), 0.88 (t, 3H).
The content of Dox in CPN = Dox was determined using
fluorescence spectroscopy. The CPN = Dox was resolved in 1N HCl
solution and kept in dark over night at room temperature. The
content of Dox was calculated based on the fluorescence intensity at
emission wavelength of 560nm, excitation wavelength of 488 nm,
2.1.3. Folate-NH2
The synthesis of Folate-NH2 was according to a published
procedure (Lee and Low, 1995). Folic acid (441 mg, 1 mmol) was
dissolved in 20 mL of DMSO, and then treated with DCC (248 mg,
1.2 mmol) and NHS (230 mg, 2.0 mmol). The mixture was stirring at
50 ꢂC for 6 h. The resulting Folate-NHS was reacted with ethyl-
enediamine (781 mg, 13.0 mmol) and pyridine (500 mg, 6.3 mmol)
at room temperature overnight. The mixture was poured into
excess acetonitrile, and the precipitate was collected and washed
with diethyl ether before drying under vacuum to get yellow
powder. Yield: 297 mg, (61.5%). The obtained Folate-NH2 was
directly used in the next step without further purification.
and slit width of 5 nm calibrated by a standard curve of Dox HCl.
ꢀ
2.2.2. Micelle preparation and drug encapsulation
The mixed micelles (CP-Folate/CPN = Dox or CP-OH/CPN = Dox)
were prepared by dialysis. Typically, 1.0 mg of CPN = Dox and
0.2 mg of CP-Folate were stirring in 200 mL of DMSO, and then
1.8 mL of DI water was added dropwise into the above solution. The
mixture was further stirred for 2 h and then dialyzed (Mw 3500,
cutoff) against with DI water.