Construction of micelles based on biocompatible pseudo-graft polymers via β-cyclodextrin/cholesterol interaction for protein delivery

Article abstract of DOI:10.1039/c4ra07175a

A novel pseudo-graft copolymer was developed for protein delivery, based on the self-assembly of (6-(2-aminoethyl)-amino-6-deoxy)-cyclodextrin (β-CDen)-modified poly(aspartic acid) (PASP-CD) with cholesterol-modified poly(d,l-lactide) (PLA-Chol) by host-g

Full text of DOI:10.1039/c4ra07175a

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PAPER
Construction of micelles based on biocompatible
pseudo-graft polymers via b-cyclodextrin/
cholesterol interaction for protein delivery
Cite this: RSC Adv., 2014, 4, 40882
Hui Han, De-E Liu, Hongguang Lu, Wen-Xing Gu and Hui Gao*
A novel pseudo-graft copolymer was developed for protein delivery, based on the self-assembly of (6-(2-
aminoethyl)-amino-6-deoxy)-cyclodextrin (b-CDen)-modified poly(aspartic acid) (PASP-CD) with
cholesterol-modified poly(D,L-lactide) (PLA-Chol) by host–guest inclusion complexation. The chemical
structures of polymers were confirmed by Fourier transform infrared spectroscopy and proton nuclear
magnetic resonance spectroscopy. These components were then investigated for their ability to form
nanoparticles and encapsulate protein. The diameter of micelles in water ranged from 70 to 200 nm as
determined by dynamic light scattering, and the micelles were spherical in shape as observed by
transmission electron microscopy. A model protein, bovine serum albumin (BSA), was encapsulated into
the pseudo-graft copolymer micelles. The encapsulation efficiency (EE) and loading capacity (LC) of BSA
in the micelles could be well tuned by adjusting the composition of the pseudo-graft copolymers. The
micelles with a lower molar ratio of CD and cholesterol (hydrophilic/hydrophobic) exhibited a higher EE
and LC. While in vitro release studies showed that a shorter chain of hydrophobic segment and higher
hydrophilic/hydrophobic molar ratio could enhance the release rate. Cell viability studies showed that
these materials possessed good cell viability (>95%). These results suggest that the degradable
copolymers with appropriate hydrophilic and hydrophobic composition are able to self-assemble into
micelles that are an effective and biocompatible vehicle for delivering protein, paving a new way for the
application of pseudo-graft copolymers in protein or peptide delivery.
Received 16th July 2014
Accepted 11th August 2014
DOI: 10.1039/c4ra07175a
www.rsc.org/advances
15
or g-CD, respectively. The geometry of CDs gives a hydro-
Introduction
˚
phobic inner cavity having a depth of ca. 7.0 A, and an internal
˚
Noncovalently connected micelles (NCCMs) are arousing the diameter of ca. 4.5, 7.0, and 8.5 A for a-, b-, and g-CD, respec-
interest of many researchers, probably due to the complicated tively. The hydrophobic interior cavity of b-CD can accommo-
1
6–18
polymerization processes of conventional amphiphilic copoly- date a variety of guest molecules, such as adamantane,
1,2
19
20,21
22
mers. Only physical interactions rather than chemical bonds adamantine carboxylic acid, cholesterol,
azobenzene,
23
exist between shell and core in NCCMs, which is different from ferrocene, and so on, to form supramolecular inclusion
In addition, CDs have been chosen as drug
interactions, such as hydrogen bonding interactions, metal– carriers since the 1950s, because of their bioavailability and low
24–26
conventional polymeric micelles. Various types of non-covalent complexes.
3–5
6–8
9–11
27
ligand interactions and host–guest recognition,
have been toxicity. A lot of developments of drug delivery systems based
employed to construct supramolecular block or gra copoly- on CDs have been investigated by us and others in recent years,
12
mers. Compared to conventional polymers, the non-covalent which were for biomedical and pharmaceutical applica-
32–34
2
8–31
nature of supramolecular copolymers endows them with tions,
especially for peptide and protein delivery.
13
enhanced structural versatility, exibility, tunability, robust However, the study of pseudo-gra copolymers based on b-CD/
combination of different building blocks and adaptability to cholesterol inclusion complexes to form nanoparticles as a
14
external stimuli.
Cyclodextrins (CDs), as good candidates to build NCCMs, are
protein delivery system has not been reported.
The supramolecular guest used in this work was poly(D,L-
a series of natural cyclic oligosaccharides composed of 6, 7, or 8 lactide) (PLA) bearing a cholesterol end (PLA-Chol). The
D-(+)-glucose units linked by a-1,4-linkages, and named as a-, b-, cholesterol moiety was chosen because cholesterol is important
in the stability of lipid ras, bearing interaction sites for
35
membrane receptors. PLA was selected as the hydrophobic
core-forming moiety due to its excellent biocompatibility,
having been widely used in the elds of protein delivery and
School of Chemistry and Chemical Engineering, Tianjin Key Laboratory of Organic
Solar Cells and Photochemical Conversion, Tianjin University of Technology, Tianjin
3
2
00384, China. E-mail: ghhigher@hotmail.com; hgao@tjut.edu.cn; Fax: +86-
260214251; Tel: +86-2260214259
32,36,37
tissue engineering.
(6-(2-Aminoethyl)-amino-6-deoxy)-
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Scheme 1 Schematic representation of the formation of pseudo-graft copolymer from PASP-CD and PLA-Chol in water, driven by host–guest
interaction complexation.
cyclodextrin (b-CDen)-graed poly(aspartic acid) (PASP-CD) was
Fourier transform infrared (FT-IR) spectra of CDen, poly-
prepared as the supramolecular host. Recently, PASP as a succinimide (PSI) and PASP-CD were recorded using KBr discs
poly(amino acid) polymer has attracted a great deal of attention with a spectrometer (Bio-Rad 6000, Thermo Electron, USA) at
1
as a biocompatible and water-soluble material as a candidate room temperature. All H NMR spectra were recorded with a
38
drug carrier in biomedical applications. In particular, PASP- NMR spectrometer (AV-400, Bruker, Freemont, CA) at room
3
9,40
41,42
43
based nanoparticles,
micelles,
nanocapsules
and temperature with CDCl , D O and DMSO-d as the solvents,
3 2 6
44
membranes were fabricated for the delivery of drugs, DNA, respectively. The number-average molecular weight (Mn) and
RNA and proteins.
molecular weight distribution (Mw/Mn) of PSI and PLA-Chol
Herein, we present
a
novel protein delivery system were determined with gel permeation chromatography (GPC)
comprising a PASP-CD polymer which forms supramolecular (R1-201H, Shoko Scientic Co. Ltd, Japan), using DMF and
self-assembly micelles with different molecular weights of PLA- tetrahydrofuran (THF) as the mobile phase and polystyrene as
Chol through host–guest inclusion complexation in water calibration standard.
(Scheme 1). Micelles composed of pseudo-gra copolymers
were fabricated using the dialysis method and were used to
encapsulate a model protein, i.e., bovine serum albumin (BSA).
The size, morphology, colloidal stability and cell viability of the
Synthesis of polymers
Synthesis of mono-6-deoxy-6-(p-tolylsulfonyl)-cyclodextrin
micelles were studied, and the encapsulation efficiency and (mono-6-OTs-b-CD). Mono-6-OTs-b-CD was synthesized
45
release proles of BSA-loaded micelles with different PASP-CD according to a previous report. Briey, NaOH solution was
and PLA-Chol molar ratio and different molecular weight of added dropwise to a suspension of b-CD in water. The mixture
PLA-Chol were also investigated.
was stirred in an ice-water bath, while p-toluenesulfonyl chlo-
ride in acetonitrile was added dropwise. Aer 5 h of stirring in
the ice bath, the unreacted p-toluenesulfonyl chloride was
removed by suction ltration, and the pH of the ltrate was
adjusted with HCl solution to be approximately 6, and then
refrigerated overnight. The white crystal product was recovered
Experimental section
Material and techniques
Stannous octoate (Sn(Oct)
2
) was purchased from Sigma-Aldrich by suction ltration and the puried product was dried in a
ꢀ
Co. Ltd (Shanghai, China). D,L-Lactide and 8-anilino-1-naph- vacuum oven at 50 C for 3 days (yield: 20%).
thalene sulfonate (ANS) were purchased from Heowns Biochem
Technologies Co. Ltd (Tianjin, China). Cholesterol was previously reported method. 6.0 g of mono-6-OTs-b-CD was
purchased from Best Reagent Co. Ltd (Chengdu, China). BSA reacted with excess amount of EDA (36 mL) at 75 C for 4.5 h,
Synthesis of b-CDen. b-CDen was synthesized according to a
46
ꢀ
was purchased from Aladdin Reagent Co. Ltd (Shanghai, and then cooled to room temperature. Subsequently, 30 mL of
China). L-Aspartic acid (99%) was purchased from Alfa Aesar Co. cold acetone was added into the mixture, and white precipitate
Ltd (Beijing, China). Tetramethylene sulfone was purchased formed immediately. Aer the precipitate was collected by
from J&K Scientic Co. Ltd (Beijing, China). Mesitylene, etha- suction ltration, the precipitate was repeatedly dissolved in
nediamine (EDA), dimethylformamide (DMF), b-CD (98%), 30 mL of water–methanol mixture and poured into a large
p-toluenesulfonyl chloride and other reagents were purchased amount of cold acetone several times to remove unreacted EDA.
ꢀ
from Tianjin Chemical Reagent Co. Ltd (Tianjin, China). The product was dried at 50 C for 3 days in a vacuum oven
Cholesterol was puried by recrystallization from acetone. (yield: 75%).
p-Toluenesulfonyl chloride was puried by recrystallization
Synthesis of PSI. PSI was synthesized according to a previous
47
from a mixture of n-hexane and benzene. b-CD was used aer report. L-Aspartic acid (25 g, 188 mmol), 85% phosphoric acid
recrystallization from water and drying under vacuum. DMF (2 g, 17.34 mmol) and a mixed solution of mesitylene/sulfolane
and EDA were distilled before use. All other reagents were used (56 g/24 g) were mixed in a three-neck round-bottom ask. The
as received.
mixed solution was reuxed under nitrogen atmosphere at
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ꢀ
ꢁ1
1
80 C, and the water formed in the reaction process was of the micellar solutions (0.1 mg mL ) onto a carbon-coated
removed using the Dean-Stark trap with a reux condenser. The copper grid and dried.
reaction was continued for 4.5 h. Aer the reaction mixture was
cooled down, the solvent was removed. Then DMF was added to
ANS uorescence measurements of PSAP-CD and micellar
dissolve the polymer, and precipitated into excess methanol
twice. Aer ltration, the precipitate was washed several times
with distilled water to remove the phosphoric acid. The product
was nally dried at 80 C in a vacuum for three days to obtain
PSI as white powder (yield: 85%).
solutions
ANS stock solution was added into 3 mL of the micellar solution,
stirred for 2 h, and sonicated for 20 min before measurements.
The nal ANS concentration was set as 0.05 mM. The uores-
cence emissions of these solutions were recorded at an excita-
tion wavelength of 370 nm using a spectrouorophotometer
ꢀ
Synthesis of PASP-CD. PASP-CD was synthesized by the ring-
48
opening reaction of PSI with b-CDen. PSI (1 mmol) was dis-
solved in DMF (10 mL), and the solution was stirred in an ice-
water bath under nitrogen, followed by addition of b-CDen (3 g,
(F-7000, Hitachi, Japan).
Determination of the encapsulation efficiency (EE) and the
loading capacity (LC) of BSA
2
6
.5 mmol) in 30 mL of DMF. The reaction mixture was heated to
0 C and stirred for 3 days. The product was precipitated in
ꢀ
ether. Aer ltration, the precipitate was dissolved in DMF, 5 mL of BSA-loaded micelles were centrifuged at 22 000 rpm
ꢀ
then dialyzed (Mw cut-off ¼ 10–12 kDa) against deionized water (4 C) for 40 min. The EE and LC were determined by measuring
ꢀ
at 0–5 C for 3 days, and then freeze-dried (yield: 80%).
the BSA concentration in the supernatant, which was measured
Synthesis of PLA-Chol. 13.96 g (96.94 mmol) of D,L-lactide according to the Coomassie Blue G250 protein assay. The
49
was polymerized under vacuum using 0.04 g (0.1 mmol) of UV-visible absorbance (Persee, TU-1900, Beijing) was recorded
Sn(Oct)
2
as a catalyst and 1.18 g (3.05 mmol) of cholesterol as an at 595 nm. The LC and EE were calculated from the following
ꢀ
initiator. The reaction was maintained at 150 C for 5 h in bulk. equations:
The solid product was dissolved in acetone, and precipitated in
deionized water. The product was ltered and dried in a vacuum
Initial loading ð%w=wÞ ¼
ꢀ
Mass of initial BSA
oven at 50 C (yield: 55%).
ꢂ 100%
Mass of ðinitial BSA þ polymersÞ
Preparation of micellar solutions and BSA-loaded micellar
solutions
Mass of loaded BSA
LC ð%w=wÞ ¼
ꢂ 100%
Micellar solutions were prepared using the dialysis method
from PLA-Chol of different molecular weights (Mn of PLA-Chol
was 5 kDa, 10 kDa, 15 kDa and named as PLA-Chol1, PLA-Chol2,
PLA-Chol3, respectively) and PASP-CD with molar ratio
Mass of nanoparticles
LC
EE ð%Þ ¼ ꢂ 100%
Initial loading
(cholesterol/CD) of 1 : 1, 1 : 2, 1 : 3, 1 : 4. In a typical example, to
prepare M15K1 (“M” represents micelles, “15K” represents the
molecular weight of PLA-Chol and “1” represents the molar
ratio of CD and cholesterol), 4 mg of PASP-CD with an equiva-
lent molar (1 : 1 cholesterol/CD molar ratio) of PLA-Chol3 (10
Release of BSA from micelles
mg) were dissolved in DMSO (10.0 mL). Aer vigorous stirring 9 mL of BSA-loaded micelles were incubated in a capped
for 5 h, the mixture was added to 5 mL of deionized water or centrifuge tube containing 1 mL of phosphate buffered saline
aqueous solution of BSA (for preparing BSA-loaded micellar (PBS; pH ¼ 7.4). The centrifuge tube was kept in a thermostatic
ꢁ1
solutions) via a syringe pump at a ow rate of 0.5 mL min , and water bath with oscillators (Xinhang Instrument, SHZ-82, Jin-
ꢀ
the dispersion was le stirring for another 5 h at room tan) at 37 C and shaken at 120 rpm. At appropriate intervals, 1
temperature. DMSO was then removed by dialysis (Mw cut-off ¼ mL of the supernatant was sampled and an equal amount of
1
2 kDa) against deionized water for 3 days. Stock solutions with fresh PBS was added to the tube. The samples were centrifuged
a characteristic bluish tinge were obtained.
at 22 000 rpm for 40 min. The BSA concentration in the
supernatant was determined as described above. Each experi-
ment was repeated three times and the result was the mean
value of the three measurements.
Particle size analysis and morphology examination
ꢀ
Micellar particle size was determined in water at 25 C by
dynamic light scattering (DLS) with a Zetasizer Nano (ZS90,
Malvern Instruments, Southborough, MA). Then the variation
Circular dichroism spectra of BSA samples
of particle size at different pH was also measured using DLS. The circular dichroism measurements of free BSA in the release
The pH values of the stock solutions were adjusted with 0.1 M medium and control BSA solutions in water were performed
ꢀ
NaOH or HCl.
with a Jasco-715 spectropolarimeter at 20 C using matched 10
Transmission electron microscopy (TEM) was conducted mm path length quartz cells. Each sample solution was scanned
with a Jeol-1400 instrument (Jeol, Japan) with an accelerating in the range of 190–250 nm. A circular dichroism spectrum was
voltage of 100 kV. Samples were prepared by placing two drops recorded as the average value of three scans.
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Cell viability assays
Results and discussion
Synthesis and characterization of PASP-CD
Cell viability was determined using Cell Counting Kit-8
(
CCK8) assay. Mouse broblast cells (L929) were cultivated in
ꢀ
Scheme 2 shows the synthetic procedure for PSI and PASP-CD.
PASP-CD was synthesized by the ring-opening reaction of PSI
a humidied 5% carbon dioxide atmosphere at 37 C on a 96-
well microplate, with 5000 cells immersed in complete
growth medium per well. The cells with 100 mL of RPMI-1640
per well were allowed to attach for 24 h. Subsequently, the
copolymer suspensions at PASP-CD concentrations of 0.1,
with b-CDen. The molecular weight of PSI measured by GPC was
4
2
.2 ꢂ 10 with polydispersity index (PDI) of 1.47.
1
The H NMR spectra of PSI as well as of b-CDen shown in
ꢁ1
Fig. 1 indicated the successful synthesis of PSI and b-CDen.
0
1
.3, 0.5, 0.7, and 0.9 mg mL were added to 96-well plates at
0 mL per well and incubated for 24 h. The solution was then
1
While the H NMR spectrum of PASP-CD in Fig. 1(III) showed
the disappearance of the peak located at 5.30 ppm, which was
assigned to methine protons (peak “b”) of succinimide units,
indicating that the ring opening of PSI had completed. In
addition, the peak “c” located at 8.0 ppm in Fig. 1(III) assigned
removed and replaced with 100 mL of RPMI-1640. Then, CCK-
solution was added to the 96-well plates at 10 mL per well
8
and incubated for 24 h, and the resulting solution was
analyzed at 450 nm by means of a plate reader with a back-
ground correction using a Bio-Tek FLx800 uorescence
microplate reader. This process was repeated eight times in
parallel. The results are expressed as the relative cell viability
47
to amide groups was observed. Fig. 2 shows the FT-IR spectra
of PSI, b-CDen and PASP-CD. In the FT-IR spectrum of PSI
ꢁ
1
(
Fig. 2(I)), the peak at 1710 cm is attributed to the stretching
ꢁ1
of carbonyl of cyclic imides. The peaks located at 3380 cm and
(
%) with respect to control wells.
ꢁ
1
1
030 cm arise from multihydroxyl groups of b-CDen (Fig. 2(II)
and (III)). Especially, the characteristic stretching of carbonyl
ꢁ
1
groups of cyclic imides located at 1710 cm (Fig. 2(I)) was
ꢁ1
absent in Fig. 2(III), and a new peak located at 1670 cm
appeared, assigned to the stretching vibration of carbonyl in the
amide groups of PASP-CD, indicating that the aminolysis of PSI
had completed.
Synthesis of PLA-Chol
End-functionalized polymers can be synthesized via direct
50,51
polymerization using a functional initiator
or through the
Scheme 2 Synthesis of PSI (I) and PASP-CD (II).
post-modication of polymers with reactive terminal
1
Fig. 1 H NMR spectra of PSI (I) in DMSO-d
6
, and b-CDen (II) and PASP-CD (III) in D
2
O.
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Table 1 Molecular weight of PLA-Chol
Yield
1
a
b
b
Mn (theory) Mn ( H NMR) Mn (GPC) Mw/Mn (%)
PLA-Chol1
PLA-Chol2 10 000
PLA-Chol3 15 000
5000
4800
9900
14 400
5300
9300
17 500
1.21
1.32
1.35
46.3
50.5
55.6
a
1
b
Determined by H NMR. Determined by GPC using polystyrene as
standards.
Fig. 2 FT-IR spectra of PSI (I), CDen (II) and PASP-CDen (III).
51,52
moieties.
The polymerization of lactide in the presence of
hydroxyl functionality-containing compound with Sn(Oct) as
2
53
the catalyst has been reported by several investigators. The
preparation route to PLA-Chol is illustrated in Scheme 3. The
molecular weight of PLA-Chol was adjusted by varying the
D,L-lactide/cholesterol ratio. The polymer composition and
1
molecular weight were characterized by H NMR and GPC
1
(
Table 1). Fig. 3 shows the H NMR spectrum of PLA-Chol. The
1
multiple peaks appearing between 0.67 and 2.33 ppm were Fig. 3 H NMR spectrum of PLA-Chol in CDCl3.
assigned to the –CH , –CH in cholesterol and –CH in D,L-lactic
2
3
3
acid units (e, f, g, h, i). The peak “b” at 5.21 ppm was assigned to
the O]C–CH–O in D,L-lactic acid units. The peak “a” at 5.37
ppm was assigned to the –CH]C in cholesterol, suggesting that
the C]C double bond was not destroyed during the polymeri-
zation. The number-average molecular weight of PLA-Chol was
determined from the integral ratio of peak “b” at 5.21 ppm and
peak “c” at 4.64 ppm (–CH groups in the cholesterol moiety
which neighbor the PLA block) (Table 1). The molecular weights
to induce self-assembly, and subsequent dialysis to remove
DMSO. Aer dialysis, a stable dispersion with a bluish tinge
formed. The micellar solutions exhibited no macroscopic phase
separation upon standing at room temperature for more than
three weeks, suggesting the formation of stable aggregates. The
diameters of the complexes were in the range of 70–200 nm as
determined using DLS (Table 2). The particle size decreased
1
obtained from H NMR and GPC were in good agreement with
the theoretical molecular weights.
Table 2 Characteristics of micelles composed of PASP-CD and PLA-
Chol
Micellar behavior of pseudo-gra copolymer
a
b
b
c
c
Micelles Host–guest Diameter (nm) PDI Diameter (nm) PDI
54
Inspired by the work of Jiang et al., we prepared the pseudo-
gra copolymer in DMSO rst, followed by the addition of water
M
M
M
M
M
M
M
M
15K
15K
15K
15K
10K
10K
10K
10K
1
2
3
4
1
2
3
4
1 : 1
2 : 1
3 : 1
4 : 1
1 : 1
2 : 1
3 : 1
4 : 1
1 : 1
2 : 1
3 : 1
4 : 1
70.5
85.0
102.2
211.3
66.1
0.143
0.107
0.118
0.228
0.379
0.260
0.203
0.193
0.269
0.255
0.205
0.206
64.7
65.3
70.4
92.5
—
0.279
0.197
0.191
0.283
—
72.0
98.0
84.0
—
0.235
—
124.9
119.2
172.4
197.4
219.5
—
—
M
5K
M
5K
M
5K
M
5K
1
—
—
2
3
4
154.0
—
0.219
—
—
—
a
b
The molar ratio of CD in PASP-CD and cholesterol in PLA-Chol. The
c
diameter and PDI of micelles in water. The diameter and PDI of BSA-
loaded micelles in water.
Scheme 3 Synthesis route to PLA-Chol.
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with increased ratio of hydrophobic moiety, or higher molar
ratio of hydrophobic PLA-Chol. It has been reported that the
hydrophobic segment could promote the core compactness of
micelles, and a greater amount of hydrophobic portion could
result in the formation of smaller particles in aqueous solu-
55
tions. DLS measurements were then performed at different pH
Fig. 4). The particles were stable in neutral or alkaline envi-
ronment (pH > 5) while the size became larger rapidly (about
00 nm) in acid environment (pH < 5), and even precipitated at
(
4
pH lower than 3. The solvation effects, as attracting forces
between polymer and solvent molecules, could promote the
dissolution of polymer. The solvation effects of PASP-CD at
different pH inuenced the particle size of the micelles.
Lowering the pH led to the protonation of amino groups in
PASP-CD, and resulted in solvation effects. With the augmen-
tation of the solvation effects, the micelles swelled, causing an
enlarged particle size. Finally, the hydrophilic PASP-CD was
unable to stabilize the hydrophobic core. As a result, hydro-
phobic PLA-Chol was separated from water and precipitated.
M15K3 and M15K1 exhibited a similar trend. The morphology of
these micelles in water was observed using TEM (Fig. 5). The
micelles were spherical in shape with a diameter of approxi-
mately 50 nm. The size observed from TEM was smaller than
that measured by DLS (70 nm) due to the dry nature of the TEM
samples (i.e., polymer solvent swelling is absent).
Fig. 5 TEM images of M15K1 (A) and M10K2 (B).
1
H NMR analysis of the complex
1
Fig. 6 H NMR spectra of PASP-CD (I) and PASP-CD/PLA-Chol
complex (II) in D O (asterisk indicates the water peak).
2
In order to explore the possible interaction of the PASP-CD/PLA-
Chol complex, we compared the H NMR spectra of PASP-CD
1
2
and the inclusion complex in D O (Fig. 6). Owing to its poor
water solubility, the proton signal of cholesterol is invisible in
H NMR analysis under most conditions when D O is used as a
2
1
Table 3 The chemical shifts (d) of PASP-CD and PASP-CD/PLA-Chol
complex
solvent. However, in the presence of PASP-CD, assessment of
1
the complex by H NMR clearly demonstrated the presence of
d (ppm)
the framework protons of the cholesterol molecule. As illus-
trated in Fig. 6, the cholesterol protons displayed chemical
shis at 1.2–1.7 ppm (peak “a”). In addition, aer complexation
PASP-CD/PLA-Chol
complex
CD
PASP-CD
with PLA-Chol, the chemical shis of b-CD (H-1, H-2 protons) in H1
d
dd
dd
dd
4.990
3.561
3.880
3.502
3.798
5.230
3.793
4.073
3.739
4.002
H2
H3
H4
H5, H6
m, dd
PASP-CD exhibited signicant changes (0.19–0.24 ppm) (Fig. 6
and Table 3). It is noteworthy that H-5 protons shied ca. 0.204
ppm, but H-3 protons showed relatively weak shis (0.193 ppm)
in the complex. Both H-3 and H-5 protons are located in the
interior of the b-CD cavity. H-3 protons are near the wide side of
the cavity, while H-5 protons are near the narrow side, indi-
cating that cholesterol probably penetrated into the b-CD cavity
from the narrow side.
ANS uorescence
The loading behavior of the micelles was studied using uo-
Fig. 4 The diameter of M15K1 and M15K3 at different pH.
rescent chromophore 8-anilino-1-naphthalene sulfonate (ANS)
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as a model guest. The uorescent character of ANS largely Table 4 Encapsulation efficiency (EE) and loading capacity (LC) of
micelles
depends on its microenvironment, and an apparent intensity
increase and peak blue shi can be observed when ANS mole-
cules move from water to b-CD cavities. We rst measured the
Micelles
LC (%)
EE (%)
56

uorescence emission of ANS in water containing PASP-CD with M15K
1
2
3
4
2
31.2
30.2
26.9
23.8
24.2
17.1
62.4
60.4
53.8
47.6
48.5
34.2
different concentrations (Fig. 7(I)). The intensity and the blue
shi of the ANS emission increased with the concentration of
the PASP-CD. When the b-CD molar content in the solutions
M
M
M
M
M
15K
15K
15K
10K
ꢁ
5
was equal to that of ANS (5 ꢂ 10 M), the emission intensity
increased 4 times and the peak shied from 522 to 492 nm.
Similar uorescence experiments were performed for solutions
of ANS with the PASP-CD/PLA-Chol micelles. As shown in
Fig. 7(II), compared to the blank solution of ANS in water, all the
micellar solutions of M15K1, M15K2, M15K3 and M15K4 caused an
apparent increase in the emission intensity and a small blue
shi of the emission peak of ANS, which indicated that the
residue CD cavities in the micelles have the ability to accom-
modate the ANS molecules. Furthermore, the effects of the
micelles on the emission behavior of ANS were clearly intensi-
5K
2
M
15K1 > M15K2 > M15K3 > M15K4. The BSA loading was enhanced
as a result of a relatively strong hydrophobic interaction
between polymer and BSA. BSA contains hydrophobic groups,
such as aromatic groups and some large alkyl groups, which
could interact with the hydrophobic moiety of the polymer, and
57
can enhance the EE and LC of BSA. Therefore, the hydrophilic/
hydrophobic balance of the pseudo-gra copolymer inuenced
EE and LC. At the same molar ratio of b-CD/cholesterol of 2, a
longer chain of hydrophobic segment (M15K2 > M10K2 > M5K2) of
the pseudo-gra copolymer facilitates chain coiling, resulting in
the enhanced BSA loading. In addition, as the molar ratio of
cholesterol/b-CD varied from 1 to 4 for M15K, the micelles
composed of more PASP-CD hydrophilic segments exhibited
decreased EE and LC.

ed when the molar ratio of b-CD to cholesterol increased from
to 4 for M15K. More residue b-CD cavities are available with an
1
increase in the molar ratio of b-CD to cholesterol in the micelles
from M15K1 to M15K4. However, no obviously blue shi could be
observed between the micelles. ANS, as a hydrophobic probe,
probably got into the inner hydrophobic core of the micelles.
Consequently, the uorescence intensity of ANS increased due
to the microenvironment change.
In vitro BSA release from the micelles
Fig. 8 shows the release curves of BSA from the micelles in PBS
EE and LC of BSA in the micelles
(pH ¼ 7.4). All micelle formulations can release 45–85% of BSA
The EE and LC of BSA in the micelles were calculated using UV within 10 h, and then reach a plateau. The release rate could be
detection, and the data are listed in Table 4. The LC (17–32%) well tuned by the composition of the pseudo-gra copolymers.
and EE (34–62%) of pseudo-gra copolymer micelles were close The cumulative release rates of the BSA were almost opposite to
to those of conventional nano-carriers. It appeared that the the trends for LC and EE of BSA. The release of protein occurs
ordering of EE and LC was as follows: M15K2 > M10K2 > M5K2; predominantly by its diffusion through aqueous pores
Fig. 7 Fluorescence spectra of ANS in water (black) and in PASP-CD solutions with different concentrations (I), and ANS in M15K1 (red), M15K
2
ꢁ5
(
(
green), M15K3 (blue), and M15K4 (pink) solutions (II). The b-CD molar concentrations (ꢂ10 M) are 1.5 (M15K1), 3.6 (M15K2), 7.0 (M15K3), and 12.6
ꢁ5
M
15K4). The bottom curve is for ANS in water; ANS concentration is 5 ꢂ 10 M. The excitation wavelength is 370 nm, and the emission is
recorded from 450 to 700 nm.
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Fig. 8 In vitro release curves of BSA from micelles in PBS.
Fig. 10 Cell viabilities of polymer complexes against L929 cells. The
cells were treated with increasing concentrations of PASP-CD in
copolymers, incubated for 24 h before analysis by CCK8 assay.
generated in the dosage form, which is associated with gener-
ation of micropores during degradation and enhanced water
5
8
uptake. b-CDs could probably offer channels for the BSA
diffusion. Therefore, the micelles with more residue b-CD (at
lower cholesterol/b-CD ratio) release their cargo faster. Addi-
tionally, the hydrophobicity of the micelles increased with the
molecular weight of PLA-Chol, which contributed to inducing a
compact structure, and thus the cumulative release decreased
with an increase in PLA-Chol molecular weight. The release
results show that the protein can be released effectively and the
copolymer micelles have the ability to be used as a potential
protein carrier.
BSA retained its original structure due to the mild encapsula-
tion condition.
Cell viability assays
The in vitro cytotoxicity of host–guest complexes is an
extremely important factor for their consideration as a safe
protein vector. In this work, the in vitro cytotoxicity of freeze-
dried powders of M15K2, M10K2, and M5K2 was evaluated
using CCK8 assays against L929 cells as a function of
concentration (Fig. 10). All the samples retained high cell
viability (>95%) aer 24 h of incubation at all tested
Circular dichroism spectroscopy is a common method to
analyze the secondary structure of a protein with high reli-
ability. The circular dichroism spectrum of the free BSA in the
supernatant from the release test aer 2 days was measured and
is shown in Fig. 9. Obviously, two extreme valleys at 208 and 222
nm occurred without any signicant difference from the spec-
trum of the native BSA. This result indicated that the released
ꢁ1
concentrations up to 90 mg mL . Some average cell viabil-
ities are slightly larger than 100% (about 120%) in Fig. 10,
probably due to the experimental error of the data. Their low
cytotoxicity further highlighted the advantage of the copoly-
mers as desirable drug carriers.
Conclusions
In summary, well-dened PASP-CD and PLA-Chol of different
molecular weights were synthesized and characterized. A novel
and efficient protein carrier based on the supramolecular
pseudo-gra copolymer has been developed via the self-
assembly of PASP-CD with different molecular weights of PLA-
Chol by host–guest inclusion complexation. The ANS uores-
cence measurement revealed the possible mechanism of
micelle formation. The particle size, EE, LC and release process
can be well tuned by adjusting the pseudo-gra copolymer
composition. A shorter chain of hydrophobic segment and
higher hydrophilic/hydrophobic ratio led to an enlarged
particle size and smaller EE and LC, as well as an enhanced
release rate. Additionally, the released BSA could maintain its
native secondly structure. These complexes, with exible
composition and good biocompatibility, are capable of being
efficient protein delivery vehicles.
Fig. 9 Circular dichroism spectra of the BSA solutions. (a) Native BSA
and (b) BSA released after 2 days from M15K3.
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