Preparation and characterization of amphiphilic calixarene nanoparticles as delivery carriers for paclitaxel

Article abstract of DOI:10.1248/cpb.c14-00699

Two types of amphoteric calix[n]arene carboxylic acid (CnCA) derivative, i.e., calix[6]arene hexa-carboxylic acid (C ₆ HCA) and calix[8]arene octo-carboxylic acid (C ₈OCA), were synthesized by introducing acetoxyls into the hydroxyls of calix[n]arene (n-6, 8). C₆HCA and C₈ OCA nanoparticles (NPs) were prepared successfully using the dialysis method. C nCA NPs had regular spherical shapes with an average diameter of 180-220 nm and possessed negative charges of greater than -30 mV. C₆ HCA and C₈OCA NPs were stable in 4.5% bovine serum albumin solutions and buffers (pH 5-9), with a low critical aggregation concentration value of 5.7 mg?L-^-1 and 4.0 mg?L-^-1, respectively. C₆ HCA and C₈ OCA NPs exhibited good paclitaxel (PTX) loading capacity, with drug loading contents of 7.5% and 8.3%, respectively. The overall in vitro release behavior of PTX from the CnCA NPs was sustained, and C ₈ OCA NPs had a slower release rate compared with C₆ HCA NPs. These favorable properties of CnCA NPs make them promising nanocarriers for tumortargeted drug delivery.

Full text of DOI:10.1248/cpb.c14-00699

1
80
Chem. Pharm. Bull. 63, 180–186 (2015)
Vol. 63, No. 3
Regular Article
Preparation and Characterization of Amphiphilic Calixarene
Nanoparticles as Delivery Carriers for Paclitaxel
a,b
a,b
a,b
a
,a,b
Zi-Ming Zhao, Yu Wang, Jin Han, Hui-Dong Zhu, and Lin An*
a
b
Department of Pharmacy, Xuzhou Medical College; 209 Tongshan Road, Xuzhou 221004, P.R. China: and Jiangsu
Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical College; 209 Tongshan Road,
Xuzhou 221004, P.R. China.
Received October 1, 2014; accepted December 23, 2014
Two types of amphoteric calix[n]arene carboxylic acid (CnCA) derivative, i.e., calix[6]arene hexa-car-
boxylic acid (C HCA) and calix[8]arene octo-carboxylic acid (C OCA), were synthesized by introducing ace-
6
8
toxyls into the hydroxyls of calix[n]arene (nꢀ6, 8). C HCA and C OCA nanoparticles (NPs) were prepared
6
8
successfully using the dialysis method. CnCA NPs had regular spherical shapes with an average diameter of
1
4
80–220nm and possessed negative charges of greater than ꢁ30mV. C HCA and C OCA NPs were stable in
6 8
.5% bovine serum albumin solutions and buffers (pH 5–9), with a low critical aggregation concentration
ꢁ
1
ꢁ1
value of 5.7mg·L and 4.0mg·L , respectively. C HCA and C OCA NPs exhibited good paclitaxel (PTX)
6
8
loading capacity, with drug loading contents of 7.5% and 8.3%, respectively. The overall in vitro release
behavior of PTX from the CnCA NPs was sustained, and C OCA NPs had a slower release rate compared
8
with C HCA NPs. These favorable properties of CnCA NPs make them promising nanocarriers for tumor-
6
targeted drug delivery.
Key words calix[n]arene carboxylic acid; nanoparticle; paclitaxel; tumor therapy
Paclitaxel (PTX) is a well-known anticancer drug that is arenes have attracted enormous interest in biomedical applica-
15)
16)
used primarily to treat lung, breast and ovarian cancers. Simi- tions including ion channel mimics, enzyme mimics, gene
17)
lar to most anticancer drugs, its poor solubility in water limits transfection vectors, and agents for surface recognition of
18)
the clinical applications of PTX. Taxol, a widely used clinical proteins. They were also reported to possess antimicrobial,
formulation of paclitaxel, requires the use of Cremphor-EL as anticancer and anti-human immunodeficiency virus (HIV)
19,20)
a solubilizer, which has been known to cause serious prob- activities.
lems, including hypersensitivity reactions, neurotoxicity, and
However, one major problem limiting the applications of
1,2)
nephrotoxicity. Moreover, the lack of selectivity of PTX re- calixarenes in the pharmaceutical or biomedical fields is their
sults in significant toxicity to noncancerous cells and severely poor water solubility. Consequently, different hydrophilic
impacts patients’ quality of life. Therefore, there is a dire need moieties have been introduced onto the upper or lower rims of
for the development of a novel formulation that improves the calixarenes to obtain water-soluble calixarenes. The tetracar-
solubility of PTX and decreases its side effects.
boxylic acid of p-tert-butylcalix[4]arene introduced by Ungaro
Among various alternatives, nano-sized drug delivery and colleagues was an early example of a water-soluble calix-
21)
system (nano-DDS) is one of the best choices. Nano-DDS arene. Shinkai et al. reported the preparation of p-sulfonato
2
2)
not only greatly enhances the solubility of PTX without side calix[6]arene. Other anionic water-soluble derivatives con-
2
3)
24)
25)
effects, but it also promotes intra-tumoral drug accumula- taining nitro, phosphonic acid, and carboxyl moieties
tion through the enhanced permeability and retention (EPR) emerged, and the first example of a cationic water-soluble
3,4)
26)
effect. Many nano-DDSs of PTX have been reported, and calixarene was reported by Shinkai et al. Other cationic
some of them have been marketed. Abraxane, a nanosuspen- calixarenes contain tetraalkylammonium groups and primary
2
7–29)
sion of paclitaxel encapsulated in human albumin was ap- amines.
Neutral water-soluble calixarenes have been syn-
3
0)
31)
proved by the Food and Drug Administration (FDA) in 2005 thesized with sulfonamides,
sugars,
and polyoxyethyl-
5
)
32)
for the treatment of metastatic breast cancer. A liposome- ene.
based PTX formulation, Lipusu, has been applied for the
In the development of water-soluble calixarenes, some
treatment of ovarian cancer in China. In addition to these ap- hydrophilic-modified calixarenes were found to exhibit am-
6
)
proved drugs, a number of PTX nano-DDSs, such as polymer- phipathy and self-assemble into nano-aggregates in aqueous
7
–9)
10)
11)
ic nanoparticles (NPs),
inorganic NPs, nano-emulsions,
solutions, which were suitable for the delivery of hydropho-
12)
13)
carbon nanotubes, and nanogels, have been reported in bic antitumor drugs. An amphiphilic tetrahexyloxy-tetra-p-
preclinical studies. Although these novel PTX nano-DDSs aminocalix[4]arene (A4C ) was synthesized and used as a
6
33)
exhibit varying levels of success, methods for improving the nanoparticle delivery platform for PTX. PTX-loaded A4C6
safety, stability and specificity of nano-DDS still warrant fur- nanoparticles had an average diameter of 79± 21nm and a
ther research.
drug loading efficiency of approximately 6.5%, and the re-
Calixarenes have been used as an important class of macro- lease of PTX from A4C6 nanoparticles could be sustained
14)
cyclic host molecules in supramolecular chemistry. Because for at least 72h. Amphoteric calix[8]arenes with a negatively
of their intriguing structures and well-defined cavities, high charged sulfonated upper rim and a positively charged qua-
symmetry and stability, and rich modification sites, calix- ternary ammonium lower rim were synthesized and could
*
To whom correspondence should be addressed. e-mail: anlinhx@sina.com
©
2015 The Pharmaceutical Society of Japan

Vol. 63, No. 3 (2015)
Chem. Pharm. Bull.
181
−
1
−
form aggregates of approximately 300nm in the pH range of
7
C OCA: white powder. IR (KBr) cm : 1604.7 (COO ),
8
3
4)
1
–7.6. Hydrophobic drugs, such as ciprofloxacin, could be 1423.4 (Ar-CH -Ar), 1035.7 (Ar-O-C), 767.6 (Ar). H-NMR
2
loaded into amphoteric calix[8]arenes nano-aggregates with a (D O) δ: 6.73 (24H, s, Ar-H), 3.95 (16H, s, Ar-CH -Ar), 3.84
2
2
loading content of 9.8%. The drug release behavior was pH- (16H, s, O-CH -).
2
triggered due to the pH-sensitive disassembly of the ampho-
teric calix[8]arenes.
Preparation of PTX-Loaded CnCA Nanoparticles PTX-
loaded CnCA nanoparticles were prepared by a dialysis
Carboxyls have been conjugated to the lower rim of p-tert- method. PTX (2.0mg) and CnCA (20.0mg) were dissolved in
butylcalix[4]arene or the upper rim of calix[4]arene, and the ethanol (5mL) and sonicated for 30min at room temperature.
resulting calix[4]arene carboxylic acid derivatives have exhib- The ethanol solution was dialyzed (MWCO 1000) overnight
ited enhanced solubilities and outstanding cation binding abili- against several changes of water. The resulting suspension
21,25)
ties.
However, studies on the application of calixarene car- was filtered through a membrane (0.8µm pore) to remove in-
boxylic acid derivatives as drug carriers are not available. The soluble PTX, and the PTX-loaded CnCA nanoparticles disper-
aim of the present work was to develop amphiphilic calixarene sion was obtained. Blank CnCA nanoparticles were prepared
carboxylic acid derivatives that can act as a promising nano- using the same method without adding PTX.
size delivery carrier for PTX. For this purpose, two types of
Characterization of PTX-Loaded CnCA Nanoparticles
calixarene carboxylic acid derivatives, i.e., calix[6]arene hexa- The mean size and zeta potential of the nanoparticles disper-
carboxylic acid (abbreviated as C HCA) and calix[8]arene sions were determined by dynamic light scattering (DLS)
6
octo-carboxylic acid (abbreviated as C OCA), were synthe- technique (Nicomp™380/ZLS, PSS, U.S.A.). The sample con-
8
−1
sized and characterized. These calix[n]arene carboxylic acid centration was kept at 1.0mg·mL .
derivatives (abbreviated as CnCA) formed into nanoparticles
The morphology of the CnCA nanoparticles was examined
using an appropriate method, and their self-assembling ability, under a transmission electron microscope (Tecnai Spirit G2
particle size, stability, drug loading and release behavior were TWIN, FEI, U.S.A.). Samples were placed on carbon-coated
investigated and compared. The focus was to obtain PTX- copper grids and dehydrated at ambient temperature for trans-
loaded nanoparticles based on calixarene carboxylic acid de- mission electron microscope (TEM) observation.
rivatives with the desired size range, acceptable drug loading
The critical aggregation concentration (CAC) of the CnCA
and release profiles for further development as an anti-cancer nanoparticles was determined by fluorescence spectros-
drug delivery system.
copy (F4600, Shimadzu, Japan) with pyrene as a fluorescence
probe. The pyrene solution in methanol (5µL, 6.0×10
M) was added to 5mL volumetric flasks and evaporated to
−4
Experimental
Materials Calix[6]arene, calix[8]arene, and PTX were dryness under a nitrogen atmosphere. CnCA nanoparticles
−4
−1
purchased from TCI (Shanghai, China). Ethyl chloroacetate dispersions at various concentrations (1×10 –0.1mg·mL )
99%), pyrene (99%), Tween 80 (pharmaceutical grade) were were added and then sonicated for 1h before measurement.
(
obtained from Aladdin Reagent (Shanghai, China). Bovine Fluorescence emission spectra were recorded at the excitation
serum albumin (BSA, 98%) was purchased from Santa Cruz wavelength (λ ) of 335nm with a set of 5nm excitation and
ex
(
Dallas, TX, U.S.A.). Acetonitrile (chromatographic grade) 5nm emission slits. Intensity ratio (I373/I384) of the emission
was from Fisher Scientific (Waltham, MA, U.S.A.). Methanol, spectra was plotted against the logarithm of the concentration
ethanol, dichloromethane, tetrahydrofuran (THF), hexane, of CnCA nanoparticles and the CAC values of C HCA and
6
K CO , NaI were all of analytical grade and purchased from C OCA were determined from the inflection point of the plot.
2
3
8
Sinopharm Chemical Reagent (Shanghai, China). All other re-
agents were of analytical grade. Water used in this study was CnCA NPs in buffers at various pH values and in the presence
double distilled water. of serum proteins were investigated by monitoring the varia-
Stability of CnCA Nanoparticles The stabilities of
Synthsis of Calix[n]arene Carboxylic Acids Calix[n]- tion in particle size. The following buffers were used: acetate
arene carboxylic acids were synthesized as previously de- buffer (pH 5, 0.15M), phosphate buffer (pH 6 and pH 7.4,
35)
scribed. To a solution of calix[n]arene (0.5mmol) in anhy- 0.15 M), and borate buffer (pH 9, 0.15M). C HCA and C OCA
6
8
drous acetonitrile (50mL), K CO (5mmol), NaI (5mmol), and NPs were suspended in pH 5, pH 6, pH 7.4, and pH 9 buffers
2
3
ethyl chloroacetate (5mmol) were added, and the solution was at room temperature with gentle agitation for 2h, and the par-
refluxed with stirring under a nitrogen atmosphere for 12h. ticle sizes were measured. To examine the stability of CnCA
The reaction mixture was evaporated in a vacuum, and the NPs over storage time, C HCA and C OCA NPs were stored
6
8
residue was dissolved in dichloromethane (10mL) and filtered. in phosphate buffered saline (PBS) (pH 7.4, 0.15M) at room
The filtrate was dropped into 10-fold hexane. The precipitate temperature for 15d, and the particle sizes were measured at
was filtered and dried under a vacuum to obtain calix[n]arene different times. To investigate the stability of CnCA NPs in
ester. The calix[n]arene ester was dissolved in a 2:1 mixture the presence of serum proteins, C HCA and C OCA NPs were
6
8
of THF and water (12mL), and a solution of NaOH (0.3M, suspended in a 4.5% BSA solution in PBS (pH 7.4, 0.15M).
mL) was added. The reaction was kept at room temperature The suspensions were incubated at 37°C, 100 rpm for 24h and
for 12h and at 4°C for another 12h. The reaction liquid was determined at time intervals.
8
filtered, and the filter cake was dried under a vacuum to ob-
tain calix[n]arene carboxylic acids.
PTX Loading Capacity and Release Studies The drug
loading contents and encapsulation efficiencies were deter-
−1
−
C HCA: white powder. IR (KBr) cm : 1600.8 (COO ), mined by quantifying the paclitaxel content using HPLC
6
1
1
421.4 (Ar-CH -Ar), 1033.8 (Ar-O-C), 769.5 (Ar). H-NMR analysis. Freeze-dried drug-loaded nanoparticles were sonicat-
2
(
D O) δ: 6.79 (18H, s, Ar-H), 3.93 (12H, s, Ar-CH -Ar), 3.87 ed in acetonitrile for 5min and filtered through a membrane
2
2
(
12H, s, O-CH -).
(0.22 µm pore). The concentration of PTX in the supernatant
2

1
82
Chem. Pharm. Bull.
Vol. 63, No. 3 (2015)
was determined by HPLC (LC-20AT, Shimadzu, Japan) using Table 1. Particle Size and Zeta Potential of CnCA NPs and PTX Loaded
CnCA NPs (n=3)
a reverse phase column (Zorbax SB-C , 5µm pore size,
18
4
.6×250mm, Agilent, U.S.A.). The mobile phase was aceto-
Mean diameter
nm)
Zeta potential
(mV)
−1
Samples
PDI
nitrile–water (45:55, v/v), the flow rate was 1mL·min and
(
the detection wavelength was 227nm. Drug loading content
C HCA NPs
222.4± 34.7
179.6± 29.5
285.4± 39.9
236.6± 38.3
0.19
0.08
0.14
0.13
−36.2± 1.5
−31.3± 1.2
−35.8± 1.6
−29.2± 3.2
6
(
LC) was calculated as a percent of the PTX mass over the
C OCA NPs
8
total nanoparticles mass in a lyophilized sample. Drug en-
capsulation efficiency (EE) was calculated as a percent of the
PTX mass loaded in nanoparticles relative to the mass of PTX
used for the formulation.
PTX loaded C HCA NPs
6
PTX loaded C OCA NPs
8
Abbreviation: PDI, polydispersity index.
The in vitro release of PTX from the CnCA nanoparticles
was studied in the presence or absence of serum proteins
using methods similar to those reported in previous stud-
3
6,37)
ies.
PBS (0.15M, pH 7.4) containing 1% (v/v) Tween 80
was used as the medium without serum proteins, and 4.5%
BSA solution in PBS (0.15M, pH 7.4) was used to simulate
blood stream. PTX loaded CnCA nanoparticles suspensions
were mixed with an equal volume of PBS or 4.5% BSA so-
lution. Five milliliters of these mixtures were placed in a
dialysis bag (MWCO 3500) and immersed in the medium at
37°C under constant stirring (100rpm). At predetermined time
intervals, 0.5mL of the liquid outside the bag was collected,
and the same volume of fresh release medium was added.
Into each sample, 0.5mL acetonitrile was added and sonicated
for 5min. After filtration through a 0.22µm membrane, the
samples were measured by HPLC as described previously.
The cumulative amount of released PTX was calculated by
determining the total amount of drug in the release medium
at time points as a percent of the drug loaded in the CnCA
nanoparticles dispersion.
Results and Discussion
Preparation of PTX-Loaded Calixarene Nanoparticles
The solubility of C HCA and C OCA in polar solvents, such
6
8
as ethanol and acetonitrile, was improved greatly. However,
despite being amphipathic, C HCA and C OCA were not easy
6
8
to form nano-aggregates in water by stirring or sonication.
Lee and colleagues prepared aminocalixarene nanoparticles
using the emulsion evaporation method with the help of poly-
33)
vinyl alcohol (PVA). In this study, the dialysis method was
found to be more suitable for the self-assembly of C HCA
6
and C OCA in water. In addition, PTX could be encapsulated
8
into C HCA and C OCA nanoparticles efficiently by dialysis
6
8
without any stabilizing agent or surfactant. Based on their
structures, the phenyl groups formed the inner hydrophobic
core and the (–O–CH –COOH) chains acted as the external
hydrophilic shell.
Characterization of PTX-Loaded Calixarenes Nanopar-
ticles Nanoparticles are able to penetrate into tumor tissues,
2
Fig. 1. TEM Images of C HCA NPs (a) and C OCA NPs (b) in pH 7.4
PBS
6
8
accumulate, and release drugs locally at tumor sites because had an average hydrodynamic diameter of approximately
of the EPR effect. Particle size is an important factor that 180–220nm with mono-disperse size distribution (Table 1).
effects the accumulation and endocytosis of nanoparticles. In The DLS results and TEM observation showed that the size of
general, nanoparticles with the size of less than 200nm are C OCA NPs was smaller than C HCA NPs. The decrease in
8
6
considered to be able to penetrate through the porous blood size might be due to the increasing phenyl moieties that could
vessels in tumors. However, Hobbs et al. reported that the generate stronger hydrophobic interactions between the calix-
pore cutoff size of porous blood vessels in majority tumors arenes molecules, thus making the inner core more compact.
38)
was 380–780nm. Some researchers also confirmed that li- The size difference before and after drug loading showed that
posomes and nanoparticles with larger size, such as 400nm, the incorporation of PTX caused an increase in the size of the
39,40)
could accumulate in tumors.
Therefore, it can be conclud- nanoparticles. It was most likely due to the steric hindrance
ed that particles with a diameter of approximately 50–400nm of the inserted PTX, which disrupted the ordered structure of
41)
effectively concentrate in tumors. In this study, CnCA NPs the hydrohobic core and reduced its compactness. The same

Vol. 63, No. 3 (2015)
Chem. Pharm. Bull.
183
results were observed in other studies using other types of tal pH changed from 9 to 5, there was no significant varia-
42,43)
nanoparticles and drugs.
tion in particle size. When the environmental pH was below
Negative zeta potentials were obtained for the CnCA NPs, 4, the size of C HCA NPs and C OCA NPs both increased
6
8
confirming the presence of carboxyl groups on the NPs’ sur- significantly and precipitation occurred at pH 3 (Fig. 3a).
face. The electrostatic repulsion caused by surface charges is The instability and aggregation of CnCA NPs was due to the
one of the primary factors that maintain the stability of col- deionization of the carboxyls, which caused a rapid decrease
loidal systems. Generally, colloid particles are stable when the in the electrostatic repulsion and the elimination of the hydra-
4
4)
absolute value of the zeta potential exceeds 20mV. The zeta tion layer. When C HCA NPs and C OCA NPs were stored in
6
8
potential of above −30mV suggested that C HCA and C OCA pH 7.4 PBS at room temperature, they both remained stable
6
8
NPs had good dispersion stability in aqueous media. Con- during 2 weeks, which indicated that CnCA NPs had satisfac-
versely, the incorporation of PTX did not significantly change
the zeta potential of CnCA nanoparticles, suggesting that the
PTX molecules had no interaction with the carboxyl groups
on the surface.
As shown in the TEM images in Fig. 1, calixarene nanopar-
ticles exhibited a spherical shape and were moderately uni-
form in size distribution. The particle size, as estimated by
TEM, was smaller than the size measured with DLS. It is
known that DLS measures the hydrodynamic diameter of
the particles core along with the solvation layer attached to
45)
the particles. When CnCA nanoparticles were dispersed
in water, a hydration layer formed outside the nanoparticles.
However, this hydration layer was not present under TEM
4
6)
after sample preparation. Hence, the hydrodynamic diameter
of CnCA nanoparticles was larger than the size estimated by
TEM.
The fluorescence spectrum of pyrene in water exhibits five
predominant peaks, and the ratio of the intensities of the first
peak (I at 373nm) to the third peak (I at 384nm) can reflect
1
3
4
7)
the polarity of the probe’s environment. When nanopar-
ticles form, there is a sudden change in the I373 to I384 ratio.
As shown in Fig. 2, the CAC values of C HCA and C OCA
6
8
−1
−1
were 5.7mg·L and 4.0mg·L , respectively, which meant
that C OCA formed nanoparticles more easily than C HCA.
8
6
This phenomenon could also be contributed to the increase in
phenyl groups, which improved the stability of the nanopar-
ticles by stronger hydrophobic interactions and thus reduced
the CAC values. The low CAC value suggested that CnCA
nanoparticles would provide good stability in solution, even
after extreme dilution by a larger volume of systemic circula-
4
8)
tion in the body.
Stability of CnCA Nanoparticles When the environmen-
Fig. 3. (a) Effect of pH on the Size of C HCA and C OCA NPs; (b)
6
8
Changes in Particle Size of C HCA and C OCA NPs in PBS (0.15M, pH
6
8
7
.4) at Room Temperature; (c) Changes in Particle Size of C HCA and
6
−
1
C OCA NPs in PBS (0.15M, pH 7.4) Containing 45mg·mL BSA at
8
3
7°C, 100rpm
Fig. 2. Plots of Intensity Ratio (I373/I384) from Fluorescence Emission
Spectra of Pyrene versus logC of C HCA and C OCA
Mean diameters of NPs were determined by DLS.
6
8

1
84
Chem. Pharm. Bull.
Vol. 63, No. 3 (2015)
tory stability in media in which they were typically stored
Fig. 3b).
Abraxane has shown more advantages than taxol , such as
(
®
®
higher administration dosage, better tolerability and fewer side
effects. However, the pharmacokinetic properties of Abrax-
®
®
ane are no better than those of the Taxol formulation. This
®
may be due to the poor colloidal stability of Abraxane during
blood circulation. Indeed, upon intravenous (i.v.) injection and
®
subsequent dilution in the large volume of blood, Abraxane
disassembles, allowing PTX to dissociate and resulting in a
49)
nonspecific biodistribution similar to free PTX. NK105, a
PTX incorporating polymeric micellar nanoparticle formula-
tion, has markedly high plasma and tumor area under curve
®
®
(
AUC) as compared with taxol and Abraxane . One principal
reason why NK105 exhibits higher antitumour activity is that
NK105 has good stability in the bloodstream, thereby prolong-
ing its plasma half-life. In order to be effective PTX delivery
carriers, CnCA nanoparticles must remain intact and circulate
in the blood for a sufficiently long time after intravenous
injection. The interactions between CnCA nanoparticles and
serum proteins were evaluated to predict their performance
in vivo.
As a major protein in serum (approximately 60%), albumin
was utilized to evaluated the colloidal stability of nanopar-
5
0,51)
−1
ticles in some studies.
A solution of BSA (45mg·mL )
in 0.15M PBS pH 7.4, which was similar to the concentra-
tion found in plasma, was prepared and mixed with CnCA
37)
nanoparticles.
CnCA nanoparticles remained stable in BSA solution within
4h, suggesting that albumin adsorption is minimal. The
The DLS results showed that the size of
2
isoelectric point of BSA is 4.7, indicating that it is negatively
charged at physiological pH. Since CnCA nanoparticles have
negative zeta potentials at pH 7.4, protein adsorption to the
nanoparticle surface may be limited by electrostatic repulsion.
These results suggested that CnCA nanoparticles had good po-
tential for drug delivery carrier by intravenous administration.
Fig. 4. The in Vitro Release Profiles of PTX-Loaded C HCA NPs and
6
PTX-Loaded C OCA NPs
8
(
a) The release profile in PBS (0.15M, pH 7.4) containing 1% Tween 80 at 37°C,
−1
1
00rpm; (b) The release profile in PBS (0.15M, pH 7.4) containing 45mg·mL
PTX Loading Capacity and Release Studies The drug BSA at 37°C, 100rpm. Values are mean± S.D., n=3.
loading content (LC) and entrapment efficiency (EE) of PTX-
loaded CnCA NPs were determined by HPLC analysis to of PTX from C HCA NPs and C OCA NPs in the absence
6
8
quantify the drug loaded in a known amount of freeze-dried of serum proteins. The release profile of PTX-C HCA NPs
6
nanoparticles. The EE of the C HCA and C OCA NPs were exhibited an apparent burst release of 33.7% within 30min.
6
8
8
1.6% and 90.3%, respectively, which suggested that the dialy- The cumulative percent drug release of C HCA NPs at 4, 8
6
sis method was effective for PTX loading into the inner core and 24h amounted to 57.5%, 73.8% and 96.2%, respectively.
of the CnCA NPs. The C OCA NPs that had a LC of 8.3% The burst release of PTX-C OCA NPs was not as notable as
8
8
exhibited better drug loading ability than did the C HCA NPs that of PTX-C HCA NPs and the cumulative release percent
6
6
that had a LC of 7.5%. The hydrophobic interaction between was only 18.8% at 30min. The overall release rate of PTX-
hydrophobic sites of PTX and phenyl units of calix[n]arene C OCA NPs was 5–15wt% less than PTX-C HCA NPs in
8
6
3
4)
is a key factor that influences LC. Compared with C HCA 12h, and PTX could be completely released from C OCA NPs
6
8
NPs, the density of phenyl units in the hydrophobic region of at 24h. Although the release profiles of PTX loaded CnCA
C OCA NPs was higher, which indicated that there were more NPs showed a burst release within the initial period, the latter
8
drug binding sites and that more PTX could be introduced represented a slow continuous release until 24h, suggesting
into the inner core of nanoparticles. The LC of C OCA NPs that the overall release of PTX from the CnCA NPs was sus-
8
®
was similar to that of Abraxane (approximately 10%), which tained. This could overcome some disadvantages of PTX, such
suggested that the therapeutic concentration of PTX was easy as local toxicity and a short half-life in vivo resulting from a
52)
to achieve using this novel nano-carrier.
The drug release from CnCA nanoparticles was investigated
rapid release.
Drug release from nanoparticles is a complicated process
using a conventional dialysis method at a physiological tem- that can be affected by many factors, including degradation
perature of 37°C. The solubility of PTX in 1% Tween 80 is and erosion of nanoparticles matrix, diffusion process of drug,
−
1
1
4.5 µg·mL , which produces a good sink condition that can crystallinity, and the binding affinity between nanoparticles
53)
solubilize more than 3 times the total amount of PTX in the and drugs. Compared with C HCA NPs, C OCA NPs had
6
8
dialysis bag. Figure 4a shows the accumulative release profiles a minor burst release and a slower release rate, which could

Vol. 63, No. 3 (2015)
Chem. Pharm. Bull.
185
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