Enteric Polymer Based on pH-Responsive Aliphatic Polycarbonate Functionalized with Vitamin E To Facilitate Oral Delivery of Tacrolimus

Article abstract of DOI:10.1021/bm501847u

(Figure Presented) To improve the bioavailability of orally administered drugs, we synthesized a pH-sensitive polymer (poly(ethylene glycol)-poly(2-methyl-2-carboxyl-propylene carbonate)-vitamin E, mPEG-PCC-VE) attempting to integrate the advantages of enteric coating and P-glycoprotein (P-gp) inhibition. The aliphatic polycarbonate chain was functionalized with carboxyl groups and vitamin E via postpolymerization modification. Optimized by comparison and central composite design, mPEG₁₁₃-PCC₃₂-VE₄ exhibited low critical micelle concentration of 1.7 × 10^-6 mg/mL and high drug loading ability for tacrolimus (21.2% ± 2.7%, w/w). The pH-responsive profile was demonstrated by pH-dependent swelling and in vitro drug release. Less than 4.0% tacrolimus was released under simulated gastric fluid after 2.5 h, whereas an immediate release was observed under simulated intestinal fluid. The mPEG₁₁₃-PCC₃₂-VE₄ micelles significantly increased the absorption of P-gp substrate tacrolimus in the whole intestine. The oral bioavailability of tacrolimus micelles was 6-fold higher than that of tacrolimus solution in rats. This enteric polymer therefore has the potential to become a useful nanoscale carrier for oral delivery of drugs.

Full text of DOI:10.1021/bm501847u

Article
pubs.acs.org/Biomac
Enteric Polymer Based on pH-Responsive Aliphatic Polycarbonate
Functionalized with Vitamin E To Facilitate Oral Delivery
of Tacrolimus
Menglin Wang,† Jin Sun,‡ Yinglei Zhai,*,§ He Lian,§ Cong Luo,† Lin Li,† Yuqian Du,† Dong Zhang,†
Wenya Ding,† Shuhong Qiu,† Yuhai Liu,† Longfa Kou,† Xiangfei Han,† Rongwu Xiang,§ Yongjun Wang,†
and Zhonggui He*,†
†Department of Pharmaceutical Sciences, School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road,
Shenyang, 110016, China
‡Municipal Key Laboratory of Biopharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road,
Shenyang, 110016, China
§School of Medical Instrument, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang, 110016, China
S
* Supporting Information
ABSTRACT: To improve the bioavailability of orally administered drugs, we synthesized a pH-sensitive polymer (poly(ethylene
glycol)−poly(2-methyl-2-carboxyl-propylene carbonate)−vitamin E, mPEG−PCC−VE) attempting to integrate the advantages of
enteric coating and P-glycoprotein (P-gp) inhibition. The aliphatic polycarbonate chain was functionalized with carboxyl groups and
vitamin E via postpolymerization modification. Optimized by comparison and central composite design, mPEG113−PCC32−VE4
exhibited low critical micelle concentration of 1.7 × 10−6 mg/mL and high drug loading ability for tacrolimus (21.2% 2.7%, w/w).
The pH-responsive profile was demonstrated by pH-dependent swelling and in vitro drug release. Less than 4.0% tacrolimus was
released under simulated gastric fluid after 2.5 h, whereas an immediate release was observed under simulated intestinal fluid. The
mPEG113−PCC32−VE4 micelles significantly increased the absorption of P-gp substrate tacrolimus in the whole intestine. The
oral bioavailability of tacrolimus micelles was 6-fold higher than that of tacrolimus solution in rats. This enteric polymer therefore
has the potential to become a useful nanoscale carrier for oral delivery of drugs.
for oral drug delivery, since they maintain their structural
stability in the stomach and are disassembled when they transit
into small intestine.6,7
Most pH-sensitive biomedical micelles are related to poly-
mers based on poly(methacrylic acid) (Eudragit, a commercial
formulation of poly(methacrylic acid)),8,9 poly(aspartic acid),10
or poly(glutamic acid)11 and recently reported polycaprolac-
tone.12 These polymers exhibit a pH-dependent ionization/
dissociation profile due to functional carboxyl groups on their
backbones or side chains. However, the pH-sensitive micelles
INTRODUCTION
■
Nanometer-sized particles for drug delivery have been one of
the major advancements in the past decade.1 Poorly water-
soluble drugs historically viewed as highly risky development
candidates have a renaissance in oral delivery due to the new
nanomedicine delivery strategy.2 Abundant research has
revealed superiority of polymeric micelles as nanovehicles for
oral delivery, including small size, easy modification, lower
toxicity, and reduced adverse reactions.3 In contrast to the con-
ventional small-molecule surfactants, the amphiphilic polymers
furnish better kinetic and thermodynamic stability.4 The
hydrophobic cores of polymeric micelles solubilize poorly
soluble drugs and defend them from the aqueous environ-
ment.5 Among these, pH-sensitive polymeric micelles are better
Received: December 19, 2014
Revised: February 25, 2015
© XXXX American Chemical Society
A
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are mostly prepared in acidic media because of the repulsion
among the ionized carboxyl groups.13−15 It is a concern
whether self-assembly could be induced in water by stronger
hydrophobic attraction.
For the last two decades, aliphatic polycarbonates (APCs)
together with other polyesters (polylactides,16 polyglycolide,17
poly(lactide-co-glycolide),18 polydioxanone,19 and polycapro-
lactone20) have been increasingly explored for biomedical
applications as a consequence of their low toxicity, biodegrad-
ability, and biocompatibiliy.18 The leading characteristics of
APCs are low Tm, high susceptibility to hydrolysis, and elas-
ticity. Additionally, degradation products of the conventional
polyesters result in more detrimental acidity to loaded drugs or
surrounding tissues.21 By contrast, those of APCs are less
stimulating, namely CO2 and dihydric alcohol. Functional APCs
have already been applied as an active component of sutures,22
biodegradable elastomers,23,24 and tissue engineering scaf-
folds.25 Apart from applications as biocompatible engineering
materials, APCs have been utilized as more versatile degradable
carriers for drug delivery systems, such as floating micro-
spheres,26 injectable hydrogels,27 and polymeric micelles.28
Efficient postpolymerization modifications of APCs could open
up new ways to fulfill their extended biomedical applications.29
However, efforts have not been made to develop APCs as
stimuli-responsive structures for oral drug delivery.
Figure 1. Schematic drawing of tacrolimus-loaded mPEG−PCC−VE
micelles.
Vitamin E (VE) and its derivatives have shown their poten-
tial as drug carriers in recent years.30 The biocompatibility and
solubilizing capacity for poorly water-soluble drugs make VE a
promising functional group for drug delivery systems.31 As
reported in the literature, VE has an inhibitory effect on cellular
P-gp efflux pumps.32,33 Our previous work showed that the
newly developed amphiphilic VE derivatives (P5KSSLV and
PLV2K) acted as P-gp ATPase reversible inhibitors.34,35
Conjugating VE to APCs could enlarge the hydrophobic core
volume as well as confer a P-gp inhibitory effect.
In this study, aliphatic polycarbonates functionalized with
carboxyl and VE were prepared to exploit obvious pH-
responsive behavior and P-gp inhibitory effect. Tacrolimus
was selected because it is a potent substrate of P-gp and typical
poorly water-soluble drug. As illustrated in Figure 1, the self-
assembled micelles could stay compact and minimize the burst
release of drug in the stomach. When the inner cores of pH-
sensitive units ionized in the intestine, tacrolimus is rapidly
released from the swelling micelles. The pH-responsiveness of
poly(ethylene glycol)−poly(2-methyl-2-carboxyl-propylene car-
bonate)−vitamin E (mPEG113−PCC32−VE4) micelles was
realized by the competition between hydrophobic attraction
and electrostatic repulsions. Further experiments were con-
ducted to test the efficiency of this drug delivery system.
Kermel Chemical Reagent (Tianjin, China). All other reagents were of
analytical grade and used without other purification.
Methods. Synthesis of mPEG−PCC−VE. The 2-methyl-2-benzy-
loxycarbonyl-propylene carbonate (MBC) and poly(ethylene glycol)-
block-poly(2-methyl-2-carboxyl-propylene carbonate) (mPEG−PCC)
were synthesized mainly according to descriptions of Li et al., Danquah
et al., and Guan et al.36−38
Synthesis of 5-Methyl-5-benzyloxycarbonyl-1,3-dioxane-2-one
(BHP). DMPA (100.0 g, 0.738 mol) was dissolved in 370 mL of
DMF in a flask. When DMPA was completely dissolved, 48.72 g
(0.738 mol) of KOH was added into the flask with vigorous stirring at
100 °C. When all of the KOH reacted to form homogeneous potas-
sium salt, 100.0 mL of benzyl chloride (0.790 mol) was dropwise
added into the solution. The reaction was stirred for 12 h with efficient
condensation in a 100 °C oil bath. Upon completion of the reaction,
DMF was removed under vacuum. The residue was dissolved in a
mixture of 480 mL of ethyl acetate, 480 mL of hexanes, and 300 mL of
water. After extraction, the organic layer was washed with 100 mL of
water and then dried with anhydrous sodium sulfate. With solvent
evaporated, the remaining solid was recrystallized from toluene to
produce BHP (121.0 g, yield 73.11%).
Synthesis of 2-Methyl-2-benzyloxycarbonyl-propylene Carbonate
(MBC). BHP (60.0 g, 0.268 mol) and 130.1 mL of ethyl chloroformate
(1.20 mol) was dissolved in 1000 mL of THF, and 123.9 mL of
TEA diluted with 121.1 mL of THF was added dropwise in an ice
bath. The reaction was controlled under 0 °C for 1 h then brought to
room temperature for another 2 h. After filtration of the precipitate
triethylamine hydrochloride, the residue was concentrated under
vacuum and recrystallized from ether/THF (1:1) to give MBC (41.1 g,
yield 61.38%).
Synthesis of Poly(ethylene glycol)-block-poly(2-methyl-2-benzoxy-
carbonyl-propylene carbonate) (mPEG−PBC). mPEG−PBC was syn-
thesized by ring-opening polymerization. Initiator mPEG5000 (18.3 g,
0.00366 mol) and DBU (0.00729 mol) were dissolved in 35 mL of
dichloromethane. MBC (40.0 g, 0.160 mol) ground into fine powder
was added to the above solution. The reaction lasted 20 min under
nitrogen protection at 35 °C. At the end of the reaction, 2 drops of
benzyl alcohol was added to terminate the reaction. Another 20 mL of
dichloromethane was added to dilute the viscous polymer solution.
The polymer was purified by pouring into freezing-cold ether and
then filtered to separate the white solid of mPEG−PBC (52.9 g, yield
90.7%, conversion rate 81.2%). As for synthesis of polymers of
EXPERIMENTAL SECTION
■
Materials. Tacrolimus was obtained from Huifengda Chemical
Corporation (Jinan, China). 2,2-Bis(hydroxoymethyl) propionic acid
(DMPA; AR, 99%), and ethyl chloroformate (AR, 98%) were
purchased from Ai Keda Chemical Technology (Chengdu, China).
Benzyl chloride was procured from Rgent Chemicals (Tianjin, China).
Potassium hydroxide (AR, 85%) was obtained from Hengxing
Chemical Preparation (Tianjin, China). Poly(ethylene glycol) methyl
ether (mPEG5000), average Mn 5000, was purchased from Sigma-
Aldrich. 1,8-Diazabicyclo[5.4.0]undec-7-ene (99%) and Vitamin E
(96%) were bought from Aladdin Industrial Corporation (Shanghai,
China). Pa/C (10%) was obtained from Sinopharm Chemical Reagent
(Beijing, China). Triethylamine (TEA, 99%) was obtained from
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different degrees of polymerization (DP), multiple amounts of
monomer MBC were added into the polymerization reaction.
Synthesis of Poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-
propylene carbonate) (mPEG−PCC). mPEG−PBC (45.4 g) was
dissolved in dichloromethane/THF (1:1). Palladium on charcoal
(2.25 g, 10%, Pd/C) was added into the mixed solution. The flask was
purged with nitrogen twice then exchanged with hydrogen using a
balloon. The reaction was carried out for 24 h with stirring at 40 °C.
At the end of the reaction, Pd/C was completely removed by filtration,
and the liquid was concentrated under vacuum. The remaining mixture
was precipitated with cold ether. The resulting semisolid product was
put under vacuum over a period to give the final mPEG−PCC (31.7 g,
yield 90.7%).
Synthesis of Poly(ethylene glycol)-block-poly(2-methy-2-carboxyl-
propylene carbonate-co-2-methyl-2-carboxyl-propylene carbonate)-
graft-DL-α-tocopherol (mPEG−PCC−VE). mPEG−PCC (30.9 g) was
dissolved in 150 mL of thionyl chloride and a drop of DMF was added
as catalyst. The solvent was refluxed at 50 °C overnight. At the end of
the reaction, the solvent was evaporated under vacuum. Toluene was
used to dissolve the residue, and the excess of thionyl chloride was
completely removed under vacuum. The resultant solid crude acid
chlorides were taken directly to the next reaction. Dichloromethane
(300 mL) was added to the above acid product. The mixture was
stirred and cooled to 0 °C. TEA (14.1 mL, 0.103 mol) and vitamin E
(23.1 g, 0.0515 mol) were dissolved in another 100 mL of
dichloromethane. The mixture was added dropwise to the acid
chorides solution at a slow speed. After 1 h in an ice−water bath, the
solution was allowed to warm to room temperature for another 72 h.
At the end of the reaction, the solvent was evaporated and extracted
with dichloromethane twice. Finally, the organic layer was washed with
1 mol/L HCl three times to fully revive the carboxyl groups from acid
chorides. The organic layer was given one more water wash and dried
with anhydrous sodium sulfate. The final solution was concentrated
under vacuum and purified by pouring into cold ether two times to
obtain mPEG−PCC−VE (20.4 g, yield 55.8%).
300 to 380 nm, with an emission wavelength of 390 nm. At last, the
intensity ratio (I340/I336) versus the logarithm of the polymer con-
centration was plotted. The CMC result is displayed in the Supporting
Information.
Preparation of Polymeric Micelles. Tacrolimus and mPEG−PCC−VE
were codissolved in THF and stirred for 15 min to form a homo-
geneous organic phase. Tacrolimus was incorporated into polymeric
nanomicelles by a simple solvent evaporation method. The organic
phase was injected into distilled water, and the mixture was agitated
on a magnetic stirrer at a speed of 400 rpm for 12 h to evaporate
THF thoroughly. Subsequently, the micelle solution was centrifuged
(13000 rpm, 10 min) to remove the unloaded drug. The formulation
was screened for the optimum by central composite design. The drug
loading property was evaluated by drug loading efficiency and drug
loading content.
Drug Loading Efficiency (DLE) and Drug Loading Content (DLC).
To determine DLE and DLC, 500 μL of drug loaded micelles was
diluted with 1000 μL of methyl cyanide. The solution was centrifuged
(13000 rpm, 10 min) before being transferred to ultra high perfor-
mance liquid chromatography (Waters ACQUITY UPLC). The mobile
phase consisting of acetonitrile and redistilled water (67:33, v/v) is
delivered at a flow rate of 1 mL/min, using a Kromasil-100-5C8 column
(150 mm × 4.6 mm) at a column temperature of 50 °C. The TUV
Detector was kept at 225 nm wavelength. The DLE and DLC were
calculated by the following equations:
MLD
MD
DLE(%) =
× 100%
where MLD stands for the amount of the loaded drug after centrifugation
determined by UPLC and MD is the amount of drug before centrifuga-
tion determined by UPLC.
MFDD
MFDMi
DLC(%) =
× 100%
Nuclear Magnetic Resonance (NMR). 1H NMR spectra were
recorded on a Bruker AVANCE III HD instrument equipped with a
400 MHz magnetic field. The proton signals of mPEG−PCC and
mPEG−PCC−VE were acquired in DMSO-d6 solution; the other
products were measured in CDCl3 solution.
Fourier Transform Infrared Spectroscopy (FT-IR). The chemical
structures and changes of functional groups were examined by FT-IR.
The polymer samples were heated to melt by an infrared generator,
spread in spectroscopic grade KBr matrix by grinding, and then
compressed into disks. MBC was mixed with KBr directly. Spectra
were recorded by Bruker IFS 55 infrared spectrometer in the range of
400 to 4000 cm−1 wavenumber range. FT-IR spectra are displayed in
the Supporting Information.
Gel Permeation Chromatography (GPC). Molecular weight
distribution was measured by gel permeation chromatography using
a Waters GPC system equipped with a GPC column (Waters Styragel
HT 3 THF), THF served as both solvent and eluent at a flow rate
of 1 mL/min. Calibration curve was derived from a series of narrow
polystyrene standards (580−20000 g/mol).
Differential Scanning Calorimetry (DSC). The thermal properties
of synthesized polymers were analyzed by DSC (Mettler Toledo
DSC-1). Accurately weighed samples were sealed in an aluminum pan.
Under nitrogen atmosphere, the pan was cooled to −50 °C then
heated to 100 °C at a 5 °C/min heating rate. For tacrolimus and micelles,
temperature was set from −50 to 200 °C at a rate of 5 °C/min. DSC
results are displayed in the Supporting Information.
where MFDD means the weight of drug in freeze-dried drug loaded
micelles determined by UPLC and MFDMi is the weight of freeze-dried
drug loaded micelles.
Size and Morphology. The particle size and polydispersity index
(PDI) of the resulting micelles were measured by a Malvern Instru-
ments Zetasizer (Nano ZS, Malvern).
Transmission electron microscopy (TEM) measurements were
conducted on a JEM-2100s instrument with the accelerating voltage of
200 kV at up to 30000 magnifications. Micelles were stained by 2.0%
(w/v) phosphotungstic acid for 3 min on a copper grid stabilized with
carbon support film.
Central Composite Design (CCD). The formulation of the
polymeric micelles was optimized by central composite design-
response surface method (CCD-RSM). The aim was to investigate
the influence of the THF and mPEG−PCC−VE on the resultant
micelles. According to the preliminary test, the volume of THF was
designed from 417.2 to 982.8 mL and the amount mPEG−PCC-VE
was from 49.3 mg to 190.7 mg. The three evaluation parameters were
particle size, drug loading efficiency (DLE), and drug loading content
(DLC). Fitting parameters of multivariate linear regression were
obtained by Design and Expert 8.0.6. Experimental data was analyzed
by this software.
X-ray Diffraction (XRD) Analysis. The nanomicelles were lyophilized,
and then samples were sent to XRD and DSC analysis, taking
tacrolimus and mPEG−PCC−VE as controls. The X-ray powder
diffraction patterns were recorded on a DX-2700 X-ray diffraction
instrument (Dandong, Liaoning Province, China) at 40 kV and 30 mA.
XRD results are displayed in the Supporting Information.
Stability Study. The micelles were subjected to the long-term
stability study and physical stability study. In the long term stability
study, micelle solution was kept at room temperature without special
shading or seal handling. Samples were taken for analysis at 0, 15, and
30 days. Physical stability was carried out in a 37 2 °C shaker for
30 h. Changes in particle size, polydispersity, and drug loading content
were chosen to evaluate the stability of micelles. The size and PDI
Critical Micelle Concentration (CMC). The CMC of mPEG−
PCC−VE was determined by using pyrene as a hydrophobic
fluorescent probe. Pyrene in acetone (6.0 × 10−6 M) was added in
a volumetric flask. Acetone was evaporated under nitrogen airflow, and
then 10 mL aliquots of polymer solution in a range of concentrations
(10−2 to 10−9 g/mL) were added into the flask making the final con-
centration of pyrene 6.0 × 10−7 M. The samples are equilibrated in a
shaker in the dark for 24 h. The excitation spectra were scanned on a
Spectra Max M3 (Molecular Devices LLC, USA) at wavelengths from
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a
Scheme 1. Synthesis of mPEG−PCC−VE
a
Conditions: (A) DMF, 100 °C, 12 h; (B) ClCO2C2H5, THF, rt, 3 h; (C) DBU, CH2Cl2, 35 °C, 20 min; (D) Pa/C, THF/CH2Cl2 (1:1), 40 °C,
24 h; (E) SOCl2, DMF, rt, 3 d.
were measured before centrifugation of samples, and DLC was
measured after centrifugation to remove the leakage of drugs. The
results of the stability study are detailed in the Supporting Information.
In Vitro Drug Release. Micelle solution (1 mL) in a dialysis bag
(MWCO = 10000) was suspended in 30 mL of simulated gastric fluid
(SGF, pH = 1.2) or 30 mL of phosphate buffer solution (PBS, pH 6.8
and 7.4). The in vitro release was conducted under the sink condition
(adding 0.1% tween 80). The samples were incubated in a 37 °C
shaker; 3 mL of the release medium was withdrawn and replaced with
3 mL of fresh SGF or PBS at specified intervals. Tacrolimus was anal-
yzed by UPLC as mentioned above. The SGF and PBS in this research
were prepared according to the formulation of Chinese Pharmacopoeia.
pH-Dependent Swelling Profile. Similar to the preparation of
nanomicelles in distilled water, the organic phase was injected into
different pH media (pH 1.2, 8.0) and water. The solvent was
evaporated to observe the self-assembly outcomes.
10 cm at length with glass cannulas attached to tygon tubing system
and secured by sutures. Before the perfusion experiment, the intestinal
segments were gently washed with warm saline solution with a syringe
and then purged with 30 μg/mL fresh tacrolimus-loaded micellar
dispersion (2.25 mL of tacrolimus micellar solution was diluted into
150 mL of K-R buffer, which consisted of 7.8 g of NaCl, 0.35 g of KCl,
1.37 g of NaHCO3, 0.32 g of NaH2PO3, 0.02 g of MgCl2, 1.4 g of
glucose, and 0.32 g of CaCl2 per 1000 mL). Saline-wetted gauzes were
used to prevent the rat’s body from dehydrating. The temperature-
controlled inlet perfusate (37 °C) was furnished at speed of 0.2 mL/min
throughout a peristaltic pump. The outflowing perfusate was collected
at 15 min intervals continuously up to 90 min (n = 6). The difference
in weight between inlet and outlet perfusate was recorded. At the end
of the experiment, the rats were executed. The length and radius of
experimental intestine was measured. The concentration of drug in the
donor and receptor was determined by UPLC. The absorption rate
(Ka) and apparent permeability (Papp) were calculated by the following
equations:
The variation in micelle size versus time (0−31 h) in different PBS
solutions (pH 1.2, 4.5, 6.0, 6.8, 7.8) was studied. After storage at room
temperature for 72 h, the ultimate state of pH-sensitive micelles in
different PBS solutions was determined.
⎛
⎞
Cout
Cin
V
out
v
V
K = 1 −
⎜
⎟
⎠
a
V
⎝
In Situ Single-Pass Intestinal Perfusion. All animals investigated
were executed according to the Guidelines for the Care and Use of
Laboratory Animals Approved by the Ethics Committee of Animal
Experimentation of Shenyang Pharmaceutical University. Sprague−
Dawley rats weighting 230−250 g fasted overnight with free access to
water were anaesthetized with 20% urethane (1 g/kg) by intra-
peritoneal injection. The rats were operated on under a warm infrared
lamp to maintain body temperature. The abdominal area was sterilized
with 75% ethanol, and the intestine was exposed by making a 4 cm
incision along the midline. The isolated segments of duodenum,
jejunum, ileum, and colon were cannulated longitudinally at both the
proximal and distal ends. The intestinal segments measured about
in
Cout
Cin
V
out
V
in
−v ln
(
)
P
=
app
2πrl
where Cout is the concentration of tacrolimus in the outlet perfusate,
Cin is the concentration of tacrolimus in inlet perfusate, Vout is the
volume of perfusate collected in the receptor tube, Vin is the volume of
perfusate pumped from the donor flask, l is the length of the isolated
intestine for perfusion, r is the radius of the intestinal segment, and v is
the flow rate of perfusion.
D
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Figure 3. GPC measurements of mPEG113−PBC32, mPEG113−PCC32,
and mPEG113−PCC32−VE4.
verapamil (100 mg/mL) for 45 min at 37 °C. Afterward, the
incubation media was removed, and 1 mL of a micellar solution of
tacrolimus (100 μg/mL) was added, followed by incubation for 45 min.
The intestinal segments were finally washed and homogenized.
Tacrolimus extracted from the intestinal homogenate was analyzed
by HPLC. The tacrolimus solution group underwent the same process
as described above.
Pharmacokinetic Study. The absorption of tacrolimus was
systematically evaluated for oral administration. Ten rats in fasted
state were randomly divided into two groups. Two groups of rats were
fed with tacrolimus-solution (15 mg/kg) and tacrolimus-nanomicelle
(15 mg/kg); 400 μL of blood was collected at specific time intervals.
The blood concentration during 12 h after administration was
determined by UPLC−MS/MS (Waters ACQUITY UPLC/tandem
quadrupole detector). The blood samples were stored in a −20 °C
freezer until analysis. The gradient elution of UPLC, MS conditions,
and sample processing have been reported in our previous work.39
Briefly, analyses were acquired by an ACQUITY UPLC system
(Waters Co). An ACQUITY UPLC BEH C18 column (50 mm ×
2.1 mm, 1.7 μm) was used, while mobile phase was composed of
acetonitrile and water premixed with 0.1% formic acid. Gradient
elution program began at 15% water for 0.2 min, and then changed
linearly to 10% water (0.2−0.5 min), then to 5% water (0.5−2.8 min),and
at last changed to the initial conditions (2.8−3.0 min). The column
temperature was set at 50 °C with 0.2 mL/min flow rate. The injection
volume was 10 μL.
The ionization source conditions were as follows: source temper-
ature of 120 °C and desolvation temperature of 350 °C; the cone
voltage of 50 V and capillary voltage of 3.2 kV; the cone gas flow rates
of 50 L/h and desolvation gas flow rates of 500 L/h; collision energy
of 34 and 19 V for tacrolimus and glibenclamide (I.S.), respectively.
Quantification was carried out by multiple reaction monitoring with
transitions from m/z 826.3 to 616.5 for tacrolimus and m/z 494.0 to
352.0 for glibenclamide (I.S.). The scan time was set at 0.2 s per
transition.
1
Figure 2. H NMR of the monomer and polymers: (A) MBC; (B)
mPEG113−PBC32; (C) mPEG113−PCC32; (D) mPEG113−PCC32−VE4.
Biodistribution of Coumarin-6 Labeled Nanomicelles in the
Gastrointestinal Tract. Micelles encapsulating coumarin-6 were orally
administrated to Sprague−Dawley rats. After 40 min, the rat was
sacrificed. Isolated by incision and frozen in OTC, the everted
segments of duodenum, jejunum, ileum, and colon were sliced at 7 μm
intervals. The intestinal tissue was incubated with 4% paraformalde-
hyde and washed three times with cold PBS (pH 7.4). Then the tissue
slide was incubated with triton (0.5%) and subsequently washed with
PBS. After staining with DAPI and rhodamine phalloidin for visualiza-
tion of cell nuclei (blue) and cytoskeleton (red), the enterocyte was
observed by confocal laser scanning microscopy (LSM 710 NLO).
Inhibition of P-gp Efflux Tests. P-pg inhibitory effect of mPEG−
PCC−VE was evaluated by the absorption of tacrolimus micelles in the
presence and absence of standard P-gp inhibitor verapamil. The ileum
of SD rats was rinsed with 37 °C KR buffer and everted with a glass
rod. The everted intestine was cut to about 100 mg rings and placed
in a 24-well plate. The intestinal rings were washed with 1 mL of KR
buffer twice and were incubated in KR solution with or without
The mixture of dichloromethane and n-hexane (1:1, 2 mL) and I.S.
solution (100 μL, 20 μg/mL) was added to the 100 μL blood sample.
After vortex for 3 min and centrifugation for 10 min (3500 rpm), the
organic layer was separated and evaporated completely at 35 °C under
Table 1. Molecular Weight Characterization of Polymers in Reactions
polymer
Mn, NMR
Mn, GPC
Mw, GPC
MP, GPC
MZ, GPC
Mw/Mn
yield (%)
mPEG113−PBC32
mPEG113−PCC32
mPEG113−PCC32−VE4
12250
9542
10323
10491
10660
11564
12237
12613
11094
11989
12799
12915
13753
14208
1.12
1.12
1.13
90.8
89.5
44.2
12646
E
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Table 2. Characterizations and Comparison of mPEG113−PCC8−VE2, mPEG113−PCC16−VE4, mPEG113−PCC32−VE4, and
mPEG113−PCC64−VE4
DP,
NMR
VE,
NMR
Mn,
NMR
Mw,
GPC
Mw/
Mn
DLC
(%)
polymers
Mn, GPC
MP, GPC MZ, GPC
Dh (nm)
PDI
DLE (%)
mPEG113−PCC8−VE2
mPEG113−PCC16−VE4
mPEG113−PCC32−VE4
mPEG113−PCC64−VE4
9.3
14.7
35.5
63.5
2.1
3.6
4.6
4.1
7362
8907
9706
8812
11033
11244
12237
18773
9790
9831
12470
13522
13753
19001
1.13
1.20
1.12
1.13
179.1
91.9
0.438
0.623
0.186
0.091
3.0
3.9
0.8
1.0
12646
16933
10491
18550
11989
17333
89.7
81.1
5.9
19.7
1.6
107.2
gentle nitrogen airflow. The residue was dissolved in 100 μL of mobile
phase followed by vortex-mixing and centrifugation (13000 rpm,
10 min). The resultant supernatant was transferred to UPLC-MS for
analysis.
Statistical Analysis. The statistical analysis was accomplished by
student’s t test for experiments, while P < 0.1 was deemed statistical
difference.
all showed a monomodal symmetric distribution with PDI
(Mw/Mn) below 1.13 (listed in Table 1), demonstrating a
well-controlled polymerization and narrow dispersion of
molecular weights. The GPC measurement suggested that Mn
of mPEG113−PCC32 might be higher than that of mPEG113
−
PBC32 contrary to our speculation. This phenomenon might be
caused by the addition of abundant carboxyl, which resulted in
shorter retention in our GPC column. The molecular weights
were almost consistent with the theoretical values. Therefore,
mPEG113−PCC32−VE4 was successfully synthesized and
characterized.
RESULTS
■
Synthesis and Characterization. According to the syn-
thetic route shown in Scheme 1, the carboxyl groups of DMPA
were first protected by benzyl chloride instead of commonly
used benzyl bromide. The yield of BHP turned out to be
73.11%, higher than with protection by the benzyl bromide
method.40 MBC was obtained from ring-closing of BHP, sub-
sequently subjected to organocatalytic ring open polymer-
ization (ROP) initiated by mPEG5000. mPEG−PCC resulted
from deprotection of the benzyl group via hydrogenolysis.
Finally, a portion of reactive pendant carboxyl groups of
Comparison between Different Polymers. The phys-
icochemical properties of a series of polymers and with different
DP are compared in Table 2. These polymers had the same
hydrophilic chains of mPEG5000 but connected with the
different lengths of hydrophobic polycarbonate chains. The
four kinds of the micelles were prepared by the same method,
with identical weight of mPEG−PCC−VE and drug. Small PDI
(Mw/Mn) measured by GPC revealed the narrow dispersion of
mPEG−PCC was conjugated to vitamin E via postpolymeriza-
all the polymers. Both mPEG113−PCC8−VE2 and mPEG113
−
1
tion modification. H NMR spectrum report of mPEG113
PCC32−VE4 is given in Supporting Information (Figure 1).
−
PCC16−VE4 could self-assemble to micelles with PDI > 0.4 and
DLE < 5%. mPEG113−PCC32−VE4 and mPEG113−PCC64−VE4
were able to form homogeneous sized micelles with PDI < 0.2,
but mPEG113−PCC32−VE4 exhibited more than 10 times higher
DLE (81.1%) and DLC (19.7%) than mPEG113−PCC64−VE4.
This implied that the balance between amphiphilic chains
profoundly influenced the formation of micelles. As a result,
mPEG113−PCC32−VE4 was selected to load tacrolimus in
subsequent experiments.
The postpolymerization modification provides two functional
groups and high-yielding polymers. Using thionyl chloride in
conjugation with VE could circumvent the limitation of low
esterification efficiency and result in the maintenance of
carboxyl groups with the hydrolysis of unreacted acyl chlorides.
The thionyl chloride method prohibited the cross-linking
between carboxyl and terminal OH groups and prevented the
carboxyl from connection with conventional condensation
agents (DCC and EDC, for instance) as well. The synthesized
mPEG−PCC−VE exhibited narrow dispersion of molecular
weight.
We explored whether conjugation to vitamin E improved
the drug loading properties of the polymers (Table 3). After
From 1H NMR spectra of mPEG−PBC (Figure 2B), peaks at
δ 4.7 (d, 2H) and 4.2 (d, 2H) of monomer MBC (Figure 2A)
disappeared, and a new peak emerged at δ 4.3, demonstrating
the success of ROP. The repeated CH2 at δ 4.3 in poly-
carbonates was used to estimate the DP and molecular weight
by comparing with CH2 of mPEG at δ 3.7. The polymerization
was well-tailored to generate polymers of different DP (ranging
from 8 to 64) with conversion rate up to 81.2%. The pendant
benzyl protection was completely removed by hydrogenation as
demonstrated by disappearance of the peak at δ 7.3 (phenyl
ring) and appearance of the peak at δ 13.1 (the carboxyl group)
(Figure 2C). The conjugation of vitamin E was confirmed and
quantified by the peak at δ 0.8, belonging to the methyl groups
of the aliphatic chain on vitamin E (Figure 2D).
Table 3. Characterization of Micelles Self-Assembled from
Different Polymers and Comparison of Their Drug Loading
Properties
size
DLE DLC
polymers
mPEG113−PBC32
mPEG113−PCC32
(nm) PDI
ζ
(%)
(%)
100.3 0.23 −1.81
138.9 0.09 −4.37 38.6
111.4 0.23 −4.26 75.0
9.4
4.5
8.9
mPEG113−PCC32-VE4
21.3
drug-loaded mPEG113−PCC32−VE4 126.4 0.14
0.33
attaching five vitamin E groups to the polycarbonate chain,
mPEG113−PCC32−VE4 possessed much higher DLE and DLC
than the others without vitamin E attachment. Therefore, a
vitamin E modified brush-like hydrophobic chain greatly
increased the drug loading efficiency. Moreover, the particle
sizes were ranked as follows: mPEG−PCC > drug loaded
mPEG−PCC−VE > mPEG−PCC−VE > mPEG−PBC. The
size of carboxylic functionalized mPEG−PCC was the largest
among all polymers. Compared with mPEG−PCC, conjugation
to vitamin E led to a decrease in particle size. That is to say,
Molecular weights of polymers were determined by 1H NMR
and GPC as described in Table 1. Mn of mPEG113−PBC32 mea-
sured by 1H NMR decreases after removal of the benzyl groups
(mPEG113−PCC32). Mn of mPEG113−PCC32−VE4 showed an
increasing tendency compared with mPEG113−PCC32 confirm-
ing the incorporation of VE groups. GPC curves (Figure 3) of
mPEG113−PBC32, mPEG113−PCC32, and mPEG113−PCC32−VE4
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Figure 4. Three-dimensional response surface and contour plot spreading on (A) size, (B) DLE, (C) DLC, and (D) desirability.
although the molecular weight of mPEG−PCC−VE was larger
than that of mPEG−PCC, the size of mPEG−PCC−VE
assemblies was smaller. When tacrolimus was loaded, the
increase in drug-loaded micelle volume can be observed in
contrast to that of mPEG−PCC−VE. TEM images of these
different micelles are displayed in Figure 5.
shows the effect of carrier (variable A) and THF (variable B) on
size. Variable B has little effect on the size of micelles. With
variable A amount from 40 to 200 mg, particle size increased
from about 90 to 150 nm. Figure 4B displays the effect of
variables on DLE. The maximum of both A and B led to the
highest DLE. An obvious ridge spread on the surface of DLC
(Figure 4C) along the change of A, indicating that certain range
of A (120−160 mg) resulted in relatively high DLC. Taking the
Central Composite Design. The response surfaces of
CCD are illustrated in Figure 4. The response surface in Figure 4A
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Figure 5. Size and size distribution of drug loaded mPEG113−PCC32−VE4 and morphology of blank mPEG113−PBC32, mPEG113−PCC32,
mPEG113−PCC32−VE4 and tacrolimus loaded mPEG113−PCC32−VE4 micelles.
result of the three responses into account, the desirability versus
A and B was given by Figure 7D. The model was verified by
prediction, indicating a desirable predictive effect and relatively
high reliability. The optimized micelle formulation given by
CCD consisted of 135.9 mg of mPEG113−PCC32−VE4 and
drugs appeared. The detailed description and photograph are
available in Supporting Information.
The pH-dependent profile was further confirmed by in vitro
drug release. Figure 6B showed the in vitro release of tacrolimus
at pH 7.4, 6.8, and 1.2. In pH 7.4 PBS media, 18.9% of
tacrolimus was released from the micelles over a 3 h period
and 96.2% was released after 24 h. At pH 1.2, since pendant
carboxyl groups were protonated, drug release was 3.4% at 3 h
and 52.0% after 24 h, indicating that the pH-sensitive cores
could markedly reduce the release of the loaded cargo in acidic
pH. In addition, mPEG113−PCC32−VE4 micelles efficiently
prolonged the release of drug in a controlled manner in
contrast with the solution group.
Biodistribution of Coumarin-6 Labeled Nanomicelles.
Intestinal segments were imaged with a confocal laser scanning
microscope (CLSM) to determine the epithelial cell disposition
of coumarin-6 loaded micelles in the presence and absence
of mPEG113−PCC32−VE4 (Figure 7). The CLSM micrographs
revealed that the absorbed coumarin-6 micelles (Figure 7A)
were localized intracellularly with stronger fluorescence inten-
sity and deeper permeation in comparison to coumarin-6
solution (Figure 7B). The results provided visual evidence that
the mPEG113−PCC32−VE4 enhances the absorption of loaded
drug in all segments of the intestine. More specifically, colon
was observed to have the most absorption versus duodenum,
jejunum, and ileum.
980.0 μL of THF with size of 101.7
19.4 nm, DLE of
80.2% 7.3% and DLC of 21.2% 2.7% with tacrolimus con-
centration of 7.2 mg/mL. The detailed description is available
in Supporting Information (Tables 1−5).
Characterizations of Micelles. Dynamic light scattering
(DLS) focused on the evaluation of the size and size distribu-
tion of tacrolimus loaded micelles. DLS (Figure 5) results
indicated a size of 101.7 nm and PDI of 0.156 agreeing well
with TEM. TEM images directly visualized the spherical
structures of micelles, indicating that the amphiphilic polymers
yielded spherical micelle.
pH-Sensitive Properties of mPEG113−PCC32−VE4. We
studied the pH-dependent swelling profile of mPEG113
−
PCC32−VE4 (Figure 6A). At pH 6.8 and pH 7.4, the diameter
of micelles increased rapidly and then came to a plateau about
230 nm with the swelling ratio of 2.5. At pH 1.2 and 4.5, the
particle size remained at the initial level for 31 h without
significant variations. Micelles at pH 6.0 were at an intermediate
level, suggesting that pH 6.0 may be around the critical value to
trigger the pH-dependent swelling behavior. Micelles in pH 1.2
and 4.5 shrank to about 70 nm lower than the size of the
control prepared in water. These four micellar solutions were
stored at room temperature for 3 days. Predictable sediment of
tacrolimus caused by the drug leakage in disassembled micelles
was observed in pH 6.0, 6.8, and 7.4, and the supernatant
became completely transparent. By contrast, micelles in pH 1.2
and 4.5 remained their light blue opalescence and no insoluble
In Situ Single-Pass Intestinal Perfusion and Inhibition
of P-gp Efflux Tests. The absorption parameter (Ka) was
introduced to describe the absorption rate of drug; the perme-
ability of the drug was evaluated by the apparent permeability
coefficient (Papp). In situ single-pass perfusion (Figure 8A)
showed that the ileum turned out to have the lowest Ka and Papp
,
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Table 4. Pharmacokinetic Parameters of Tacrolimus in Rats
a
after Oral Administration
tacrolimus-sol
tacrolimus-nanomicelle
dose (mg/kg)
AUC(0−12h)
t1/2 (h)
15.0
15.0
320.8 121.5
5.9 4.4
1926.1 533.0
2.3 1.1
Tmax (h)
0.33 0.17
149.2 71.7
1.70 0.76
467.4 94.8
Cmax (ng/mL)
a
AUC, area under the blood concentration−time curve; T1/2, terminal
half-life; Tmax, time when Cmax is attained; Cmax, maximum blood
concentration; data are given as mean SD (n = 5 for each group).
were 467.4
94.8 ng/mL and 1926.1
533.0 ng/(mL·h)
(6-fold compared with tacrolimus-sol), which is an encouraging
outcome in consideration of the difficulties encountered in the
oral absorption. The pharmacokinetic study demonstrated that
mPEG113−PCC32−VE4 nanomicelles developed in this study
significantly enhanced intestinal absorption and oral bioavail-
ability of tacrolimus.
DISCUSSION
■
Amphiphilic macromolecules form micellar architectures in
aqueous solution, which are capable of loading insoluble
drugs.42 In our study, the hydrophobic core-block length is
varied, and the resultant micellar properties including size,
stability, and loading capacity were measured to help
comprehensive understanding of the relationship between
chemical composition and corresponding physical properties.
To summarize, the optimal polymer structure was composed of
mPEG5000 (Mn), hydrophobic polycarbonate 5000 (Mn) and
2000 (Mn) vitamin E, which spread a brush-shaped hydro-
phobic region.
We chose the central composite design (CCD) for
formulation optimization because of its efficiency in exploring
two-factor interactions and detecting quadratic effects. With
fewer experimental conditions than full factorial designs, CCD
gives output of response surfaces for exploring the significant
amount of formula in micelle preparation.43 CCD herein aided
finding the maximized drug loading formulation. We could
Figure 6. (A) pH-dependent swelling profile of mPEG113−PCC32−VE4
micelles in different pH; size variations were recorded during specific
time intervals. (B) In vitro release of tacrolimus loaded micelle in pH
1.2, pH 6.8, and pH 7.4 PBS with the control of tacrolimus solution.
possibly because the activities of P-gp increased from prox-
imal to the distal intestine segments.41 Colon exhibited the
highest Ka and Papp, in an agreement with the result of bio-
distribution study, because colon was likely to possess a special
mechanism responsible for the high absorption of tacrolimus
micelles.
The presence of verapamil significantly enhanced the
absorption of tacrolimus in ileum (p < 0.1) as illustrated in
Figure 8B, confirming that tacrolimus was a potent P-gp sub-
strate. The absorption of tacrolimus was significantly (p < 0.01)
increased by mPEG113−PCC32−VE4 micelles versus the solu-
tion group, which might be ascribe to two aspects. For one
thing, mPEG113−PCC32−VE4 might have an inhibitory effect
on P-gp. For another, the nanoscale micelles possess more ad-
vantages over solution preparation in oral absorption. Interest-
ingly, adding verapamil made no significant difference to the
micellar group, implying that mPEG113−PCC32−VE4 might act
as verapamil did.
conclude from the response surfaces that the more mPEG113
−
PCC32−VE4 added, the higher DLE can be achieved. However,
DLC did not show an identical trend. When DLE peak value
was attained, adding more mPEG113−PCC32−VE4 remarkably
decreased DLC value. Therefore, a proper ratio of water phase,
organic phase, and surfactant was critical to maximize drug
loading and minimize particle size.
Some pH-sensitive micelles for oral administration were pre-
pared and stored only in acidic pH region. Our newly developed
pH-sensitive polymers were able to remain stable micelle
structures in water for a month (stability study is displayed in
Supporting Information Table 6), which was superior to
previous ones.14,15 These self-assemblies in water are mainly
driven by hydrophobic attractions among vitamin E segments,
suppressing the electrostatic repulsions between charged
carboxyl groups. When pH is increased, the ionized carboxyl
groups favored the dissociation of the structure. As a result,
repulsion-driven forces might dominate the reversal of self-
assembly.
Pharmacokinetic Study. The blood tacrolimus concen-
tration versus time is shown in Figure 9. The related pharma-
cokinetic parameters were displayed in Table 4. At 0.3 h
postadministration, the group orally administrated tacrolimus
solution exhibited rapid absorption (Tmax = 0.33 h), whereas
Tmax of tacrolimus-nanomicelle was 1.7 h. The Cmax and
AUC(0−12h) of the group orally treated with tacrolimus solution
pH-dependent swelling and drug release were affected by
solution variables, such as pH and solution ionic strength.44 For
pH near or above pKa, deprotonation was favored by the
acid−base equilibrium. When repulsion among charged
were 149.2
71.7 ng/mL and 320.8
121.5 ng/(mL·h),
respectively. In contrast, the Cmax and AUC(0−12h) of micelles
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Figure 7. Intestinal segment fluorescence of (A) SD rats treated with nanomicelles encapsulating coumarin-6 (green fluorescence) and (B) SD rats
treated with coumarin-6 solution. DAPI (blue) was used to capture the nucleus of the histological sections. Rhodamine phalloidin (red) was used to
counterstain the cytoskeleton.
Figure 9. Blood tacrolimus level versus time after oral administration of
tacrolimus loaded nanomicelles and solution preparations to SD rats.
carboxyl became sufficiently strong, it caused the hydrophobic
chains to reduce electrostatic interactions by swelling, at the
cost of decreasing the hydrophobic attraction. The formation of
the micelles will therefore be unfavorable. The ionized carboxyl
is surrounded by a charged ionic atmosphere, which partially
screens the carboxyl from protonation. Increasing the ionic
strength by adding salts decreased the formation of protonated
carboxylic acid, and apparently benefitted the swelling of
micelles. The small swelling ratio of the mPEG113−PCC32−VE4
micelles conducted a controlled release.
Oral administration features convenience, painless admin-
istration, and high compliance with patients especially those in
need of chronic therapies. The intravenous-to-oral switch in
chemotherapy is recommended in our recent review due to its
Figure 8. (A) In situ single-pass perfusion, Ka and Papp of duodenum,
jejunum, ileum, and colon (n = 6 for each group). (B) The inhibition
of P-gp efflux test in ileum segment. Verapamil was used as standard
P-gp inhibitor. The groups treated without verapamil were used as
innate patient compliance and mild sustained drug concen-
tration in the circulation.45 At present, more than 60% of
marketed drugs are administered via the oral route,46 but
controls (*p < 0.1; ***p < 0.01).
approximately 70% of new chemical entities exhibit poor
J
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Figure 10. Illustrated mechanism of pH-sensitive oral absorption.
soluble in aqueous solution.47 According to the Biopharma-
ceutics Classification System (BCS), class II drugs are
characterized by poor aqueous solubility and high permeability,
and poor solubility continues to be a therapeutic threshold to
oral absorption.5 Besides, drug efflux by the intestinal epithelial
ATP-binding cassette transporter P-gp plays a determining role
in restricting membrane permeability.48−52
Tacrolimus (FK506) is an immunosuppressant usually used
to prevent graft rejection after organ transplantations, as well as
to treat autoimmune diseases.53,54 Tacrolimus belongs to BCS
class II substances, and it is a typical cosubstrate of P-gp and
cytochrome P450 (CYP) 3A enzymes. The above character-
istics result in poor in vivo absorption of tacrolimus. However,
development of an oral tacrolimus delivery system is of great
significance for chronic treatment to transplant recipients. P-gp
efflux should be responsible for poor oral bioavailability of
tacrolimus to a great extent.55 D-α-Tocopheryl polyethylene
glycol succinate (vitamin E TPGS) was reported to serve as a
P-gp inhibitor for improving the oral bioavailability due to its
functional group vitamin E.56 Similarly, in our study, the
increased absorption of tacrolimus was probably because the
nanocarriers have abundant vitamin E, which functioned as a
P-gp inhibitor.
In this study, anionic polymers with carboxyl and vitamin E
were to maintain the hydrophobic cores at stomach pH but
swell to release drug at intestinal neutral pH. The nanoscale of
micelles might also facilitate adhesion to mucus,57−59 and
prolong residence time in the gut by physical absorption.60,61
In the meantime, the bioadhesion of carriers to mucosa
(pH 5.0−6.5) was augmented, since the swelling micelles
transform from solid to gel enhancing the in vivo absorption.62
The functional vitamin E group might further facilitate the drug
absorption by acting as a P-gp inhibitor to reduce drug efflux.
The possible schematic diagram of nanomicelles across a
pH gradient in GI track is illustrated in Figure 10.
size, DLE, and DLC to desirable values. The resultant micellar
structure exhibited low leakage of drug in pH 1.2 release
media and could overcome the drastic pH environ-
ment in stomach, subsequently conducting the controlled
release of drug in the intestinal pH environment. Experi-
ments were further performed to confirm that this drug
delivery system was adequate to encapsulate drugs efficiently
and transport them in the GI tract with improved oral
bioavailability, as well as inhibiting the drug efflux mediated by
P-gp. The novel anionic pH-responsive polymer was appro-
priate for delivering a variety of drugs, such as preventing
gastric degradation of drug like insulin, improving loading con-
tent of cationic drugs like doxorubicin, improving bioavailability
of a P-gp substrate, and specifically binding drugs with amino,
forming clathrate or chelate with drugs by noncovalent binding.
More work will be done to realize the control and optimization
of this polymer, as well as to explore mechanism of its oral
administration. Therefore, this newly developed pH-responsive
polymer has the possibility to be a useful biomaterial for oral
drug delivery.
ASSOCIATED CONTENT
* Supporting Information
■
S
Two-factor three-level CCD design information, including
details of factors, experimental data of three responses,
quadratic regression models, verification, solution, further
1
description of H NMR spectrum of mPEG113−PCC32−VE4,
FT-IR spectrum, DSC, CMC, XRD, stability study, and pH-
responsive profile. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*Z.H.: tel/fax, +86-24-23986321; e-mail, hezhonggui@vip.163.com.
*Y.Z.: tel/fax, +86-24-23984219; e-mail, zhaiyinglei@aliyun.com.
■
Notes
CONCLUSIONS
The authors declare no competing financial interest.
■
A serial length of hydrophobic polycarbonate chains with
functional carboxyl and VE were successfully synthesized via
postpolymerization modification. We demonstrated that varia-
tions in drug loading capability were attributed to the chain
length and the hydrophobic vitamin E tips. CMC of the optimal
structure was extraordinarily low suggesting that the micelles
were stable against dilution, which was later verified by the
stability test. In the nanomicelle formulation, CCD was used to
build the fitting the second-order models for manipulating
ACKNOWLEDGMENTS
■
We gratefully acknowledge the kind help of Dr. Lianlong
He (Shenyang National Laboratory for Materials Science,
Institute of Metal Research, Chinese Academy of Sciences) in
transmission electron microscopy visualization of our micelle
samples. This work was financially supported by the National
Nature Science Foundation of China (Grant Nos. 81173008,
81273450, and 81373336), the National Basic Research Program
K
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DOI: 10.1021/bm501847u
Biomacromolecules XXXX, XXX, XXX−XXX