Encapsulation of 10-hydroxy camptothecin in supramolecular hydrogel as an injectable drug delivery system

Article abstract of DOI:10.1002/jps.24481

10-Hydroxy camptothecin (HCPT) has been proven to be a cell cycle-specific chemotherapeutic agent, which is a necessary choice to inhibit tumor residue growth and prevent tumor metastasis after surgery. But it suffers from light decomposition, poor solubility, relatively low bioavailability, and some side effects, which are the major obstacles toward its clinical use. Integration of hydrophobic HCPT with hydrophilic hydrogel is a facile approach to change the disadvantageous situation of HCPT. In this study, a novel supramolecular hydrogelator with improved synthetic strategy was triggered by chemical hydrolysis, and then self-assembled to hydrogel. Taking advantage of the high-equilibrium solubility of HCPT in hydrogelator solution, this hydrogel was utilized to load HCPT via encapsulation as an effective carrier. HCPT hydrogels were characterized by several techniques including transmission electronic microscopy, rheology, and UV spectroscopy. In vitro release experiment indicated HCPT hydrogel could maintain long term and sustained release of HCPT at high accumulated rate. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay showed that HCPT hydrogel had an optimized anticancer efficacy. Besides, with prominent physical properties of carrier, HCPT hydrogel possessed satisfactory stability, syringeability, and recoverability, demonstrating itself as a potential localized injectable drug delivery system.

Full text of DOI:10.1002/jps.24481

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Encapsulation of 10-Hydroxy Camptothecin in Supramolecular
Hydrogel as an Injectable Drug Delivery System
RUIXIN LI,1,2 CHANG SHU,1,2 WEI WANG,3 XIAOLIANG WANG,4 HUI LI,3 DANKE XU,3 WENYING ZHONG1,2
1Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, People’s Republic of China
2Key Laboratory of Biomedical Functional Materials, China Pharmaceutical University, Nanjing 210009, People’s Republic of China
3State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University,
Nanjing 210093, People’s Republic of China
4Department of Polymer Science and Engineering, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093,
People’s Republic of China
Received 31 January 2015; revised 26 March 2015; accepted 13 April 2015
Published online 15 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24481
ABSTRACT: 10-Hydroxy camptothecin (HCPT) has been proven to be a cell cycle-specific chemotherapeutic agent, which is a necessary
choice to inhibit tumor residue growth and prevent tumor metastasis after surgery. But it suffers from light decomposition, poor solubility,
relatively low bioavailability, and some side effects, which are the major obstacles toward its clinical use. Integration of hydrophobic HCPT
with hydrophilic hydrogel is a facile approach to change the disadvantageous situation of HCPT. In this study, a novel supramolecular
hydrogelator with improved synthetic strategy was triggered by chemical hydrolysis, and then self-assembled to hydrogel. Taking advantage
of the high-equilibrium solubility of HCPT in hydrogelator solution, this hydrogel was utilized to load HCPT via encapsulation as an
effective carrier. HCPT hydrogels were characterized by several techniques including transmission electronic microscopy, rheology, and
UV spectroscopy. In vitro release experiment indicated HCPT hydrogel could maintain long term and sustained release of HCPT at high
accumulated rate. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay showed that HCPT hydrogel had an optimized
anticancer efficacy. Besides, with prominent physical properties of carrier, HCPT hydrogel possessed satisfactory stability, syringeability,
ꢀC
and recoverability, demonstrating itself as a potential localized injectable drug delivery system.
American Pharmacists Association J Pharm Sci 104:2266–2275, 2015
2015 Wiley Periodicals, Inc. and the
Keywords: 10-hydroxy camptothecin; supramolecular hydrogel; chemical hydrolysis; injectable drug delivery; Drug delivery systems;
Hydrogels; Cancer chemotherapy; Encapsulation; Peptides
INTRODUCTION
was successfully completed by chemical covalent binding, which
might be administrated in the cavity after surgical tumor re-
moval for the long-term release of anticancer drugs to improve
the efficiency, reduce side effects, and overcome drug resistance
during chemotherapy. Recently, Ou et al.20 provided an addi-
tional excellent choice of aromatic capping group, for example,
PTZ-acetic acid, to generate short peptide-based supramolecu-
lar hydrogelators with super gelation ability. This achievement
brought more opportunities for the development of supramolec-
ular hydrogels.
Cancer chemotherapy is essential and to date remains the
best way to eliminate tumor residues and prevent tumor metas-
tasis after surgery, yet widely used anticancer drugs such as
Taxol and cis-platinum-based drugs often have limited water
solubility and result in side effects.19 In order to minimize the
side effects, some research groups have successfully developed
a few supramolecular hydrogels combining chemotherapy with
target. A folic acid–Taxol conjugated molecular hydrogel21 was
formed by the reduction of disulfide bond by glutathione. Com-
pared with intravenous injection of clinically used TaxolꢀR with
four times the dosage, this hydrogel could inhibit tumor growth
more efficiently by a single dose of intratumor administration,
which suggested the big potential of this novel gelation system
of Taxol for cancer therapy. In one of our previous articles,22
a novel synergistic dual-targeting molecular self-assembly hy-
drogel of Taxol was developed by conjugation with estrone and
RGD peptide, which transported drugs, specifically targeted
tumors, and penetrated breast cancer cells. Confocal imaging
Hydrogels, with highly hydrated, porous nature, and alterable
chemical or physical properties, have been applied in fields such
as medical implants, tissue engineering, and drug delivery.1–6
For delivery applications, hydrogels have been used to incor-
porate drug molecules into the gel matrix to create reservoirs
that deliver bioactive agents.7 Although a majority of hydro-
gels are polymeric ones whose networks consist of covalently
cross-linked natural or synthetic polymers, supramolecular hy-
drogels, whose networks consist of nanofibers formed through
the self-assembly of small molecules (i.e., hydrogelators), have
received remarkable scientific interests as promising biomate-
rials in the last decade.2,8–11 Excellent drug delivery systems
have been successfully developed based on hydrogelators of
therapeutic agents.12–15 By introducing a pyrene group, Xing
et al.16 modified vancomycin and turned it into a vancomycin–
pyrene hydrogel, serendipitously enhancing nearly three orders
of magnitude potency. One of these therapeutic agents commer-
cially called “Lanreotide” has been approved by US FDA for
clinical use.17,18 First example of a codelivery hydrogel system
of two complementary anticancer drugs,19 for chemotherapy
Correspondence to: Wenying Zhong (Telephone: +86-025-86185217; Fax: +86-
025-86185517; E-mail: wyzhong@cpu.edu.cn)
This article contains supplementary material available from the authors upon
request or via the Internet at http://onlinelibrary.wiley.com
Journal of Pharmaceutical Sciences, Vol. 104, 2266–2275 (2015)
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
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in vitro and real-time visualization in vivo were carried out to Figure 1, the precursor was turned into hydrogelator (NapFE)
evidence enhanced targeted delivery and anticancer effect of by hydrolysis. The hydrogel, derived from self-assembly of hy-
Taxol.
drogelator, was used to encapsulate HCPT. We believe that the
Except for chemical conjugation, some groups obtained integration of hydrogel with HCPT can not only form a new
promising drug delivery systems by simple noncovalent inter- formulation with better solubility and stability, it also takes
action as well. Soukasene et al.23 used self-assembling peptide advantage of the excellent performance of hydrogel to establish
amphiphile nanofibers to encapsulate camptothecin using sol- a simple yet robust drug delivery system, which can maintain
vent evaporation technique. In vitro and in vivo tests showed a relatively stable and sustained-release HCPT in an aqueous,
the ability of these amphiphilic nanofibers to inhibit tumor physiological medium with a fairly well-accumulated release
growth, demonstrating the potential of these systems to de- rate.
liver hydrophobic drugs.24 Peptide-based hydrogel nanoparti-
The purpose of this article is to demonstrate the suitability
cles also represent a promising alternative to current drug de- of HCPT hydrogel as an injectable drug delivery system. The
livery approaches. Ischakov et al.25 designed and developed a upcoming sections emphatically certified its suitability from
new class of self-assembled aromatic peptide-based HNPs con- the aspects of drug loading, mechanical characteristics, in vitro
sisting of Fmoc-FF core, and vitamin E-TPGS monolayer as drug release, and anticancer efficacy.
an outer shell. Encapsulation of doxorubicin and 5-flourouracil
within the HNPs matrix showed release kinetics of the drugs
depending on their chemical structure, molecular weight, and
hydrophobicity. Results clearly indicated that these HNPs have
MATERIALS AND METHODS
potential to be used as encapsulator and delivery system. In
spite of some significant limitations about supramolecular hy-
drogels, the most important being that injectable liquids can
be cross-linked into hydrogels after injection and then facili-
tate local delivery of drug molecules. For instance, curcumin
hydrogel26 is prepared in situ where curcumin encapsulation
within the hydrogel network is accomplished concurrently with
peptide self-assembly and demonstrates its effectiveness as an
injectable agent for localized curcumin delivery. An injectable
delivery system that can sustainedly release drugs may im-
prove the efficiency and reduce their side effects on other or-
gans, which ameliorate the pains of patients more directly.
Hydrophilicity, biocompatibility, and physiologically benign
processing conditions are also beneficial properties that make
peptide hydrogels attractive candidates as injectable deliv-
ery vehicles for therapeutic agents.27 So hydrogel is abso-
lutely a promising choice to transform hydrophobic drugs into
hydrophilic agents. 10-Hydroxy camptothecin (HCPT), a cell
cycle-specific agent that can inhibit DNA replication and arrest
the cells at S phase, has proven potential for cancer chemother-
apy. Similar to other hydrophobic compounds, it suffers from
light decomposition, poor aqueous solubility, low bioavailabil-
ity, and side effects, which are the major obstacles toward its
medical deployment.
All the Fmoc-amino acid and 2-chlorotrityl chloride resin
were obtained from GL Biochem (Shanghai, China). HCPT
was obtained commercially from Dalian Meilun Biotech
Company, Ltd. (Dalian, China) 1-Hydroxybenzotriazole, N,N-
diisopropylethylamine, O-benzotriazole-N,N,Nꢁ,Nꢁ-tetramethyl
-uroniumhexafluoro-phosphate, trifluoroacetic acid, and 4-
dimethylamino-pyridine were purchased from Aladdin Reagent
Corporation (Shanghai, China). 3-(4, 5-dimethylthiazol-2-yl)-
2, 5-diphenyltetrazolium bromide (MTT) was obtained from
Biosharp Company (Hefei, China). The human breast cancer
stem cells MCF-7 were purchased from American Type Cul-
ture Collection (ATCC, China’s district general agent, Beijing,
China).
Preparation of Precursor Peptide
In this part, we adopted solid phase peptide synthesis method
(SPPS), which is easier and more straightforward than solution
phase synthesis.29 Scheme 1 shows the synthetic route and the
structure of each small molecule.
First, Fmoc–glycinol and succinic anhydride were used to
synthesize the Fmoc–ethanolamine–succinic anhydride (1).
Then, 1 was used to couple with Fmoc–phenylalanine and 2-
naphthylacetic acid by SPPS method. The final peptide (3) was
obtained easily. Such a simple synthetic pathway ensures large-
scale preparation of the compounds for practical use.
As illustrated in Figure 1, upon the action of sodium bicar-
bonate, precursor 3 hydrolyzes to the hydrogelator (2), which
self-assembles into nanofibers and affords a supramolecular
hydrogel.
In order to change the situation, a supramolecular hydrogel
was adopted to load HCPT via noncovalent interaction.
Building a conventional drug delivery system28 usually re-
quires a carrier that loads the therapeutic agents via either
noncovalent interaction (physical loading) or covalent bonds
(chemical loading).18 Zhao et al.29 have successfully developed
a new type of supramolecular hydrogel that induced gelation in
a new way of chemical hydrolysis by replacing a hydroxyl group
with a carboxylate group on the molecular structure of hydro-
gelator. The controlled hydrolysis of a carboxylic ester bond by
a base or an enzyme can act as a simple trigger to initiate
Hydrolysis of 3 and the Formation of Hydrogel of 2
Two milligram of 3 was added to 1 mL weak basic pure water
(pH 7.5). Gradual addition of base (0.1 M Na2
CO3) caused the
self-assembly and form hydrogels, even if the molecule itself is hydrolysis of 3 and induced self-assembly. The solution should
unable to form hydrogels by commonly used methods, such as be oscillated sufficiently after each addition. Till there was no
directly changing pH,30–34 temperature,35,36 or ionic strength.37 more solid and the pH of solution was measured as 8, the total
Besides, this hydrogel is stable over a wide pH range and in-
sensitive to ionic strength.
In this paper, the synthetic method of supramolecular hydro-
gel precursor (NapFES) was thoroughly renewed by combining
with solid phase synthesis, which raised the yield. As shown in
volume of base was 80 :L. A clear solution of 2 at the concen-
tration of 0.2% (w/v) self-assembled into a solid and translucent
hydrogel of 2 in 12 h without a noticeable pH change. The same
protocol was used to prepare 0.3% (w/v) and 0.4% (w/v) hydro-
gels.
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Figure 1. The synthesis route of 3 and schematic of the formation mechanism of HCPT hydrogel.
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Preparation of HCPT Hydrogel
ing peptide concentration, that is, 0.2% (w/v), 0.3% (w/v), and
0.4% (w/v), all at fixed 400 :g/mL drug concentration, were pre-
pared to quantify released HCPT into PBS supernatants. First,
0.2 mL of fresh PBS solution (100 mM, pH 7.4) was added onto
the top of each hydrogel. At each desired time point, the PBS
supernatant was totally taken out for measurement of accumu-
lating release amount of HCPT from hydrogels; then, an equal
volume of fresh PBS buffer was added. The experiment was
conducted in three parallel experiments.
Although HCPT is poorly soluble in organic solvents, it is sol-
uble in weak basic solutions. And the pH of the solution of 2
is just measured as around 8.0. Thus, we took advantage of
the weak basic solution of 2 to load HCPT. HCPT (0.2 mg) was
dissolved and evenly dispersed to 0.5 mL solution of 2. Then,
self-assembly took place and HCPT hydrogel was formed grad-
ually.
Transmission Electron Microscopy of HCPT Hydrogels
Cell Culture and HCPT Hydrogels in Contact with MCF-7 Cell
Several drops of diluted hydrogel of 2, heat-treated hydrogel of
2, HCPT hydrogel at 0.3% (w/v) and 0.8% (w/v) were applied to
a carbon-coated grid and excess water was blotted away with
filter paper. The grids were left to dry in ambient condition for
1 h. Transmission electronic microscopy (TEM) was performed
with JEOL JEM-2100f TEM (Japan) operating at 220 kV and
used to characterize the self-assembled structures in hydrogels.
Michigan Cancer Foundation-7 cells line, MCF-7 cells, were cul-
tured in RPMI-1640 supplemented with 10% fetal bovine serum
and penicillin/streptomycin (complete cell growth medium) in
a humidified, 5% CO2 atmosphere at 37°C.
Five-hundred microliter of 0.1% (w/v), 0.2% (w/v), 0.4% (w/v),
and 0.8% (w/v) HCPT hydrogels was prepared with 0.2 mg and
a control group without HCPT. MCF-7 cells were planted in the
24-well plate (1500 cells/well) and incubated at 37°C overnight
in cell culture medium. One-hundred microliter of each sample
was added into 24-well plate and incubated for another 4 h.
And then, light microscope images were taken by MOTIC AE31
inverted biological microscope (CCIS Ph 10×. Xiamen, China).
Rheology
Rheological measurement with the mode of dynamic time
sweep, dynamic frequency sweep, and dynamic strain sweep
provides useful information on gelation properties of HCPT
hydrogel. Rheology experiments were conducted on a Thermo
RheoStress 600 instrument using a set of 60 mm diameter par-
allel plates with the thickness of 0.3 mm at 37°C. Aged HCPT
hydrogel (0.8%, w/v) was prepared as described previously. Af-
ter aged HCPT hydrogel was subjected to 1000 s−1 shear for
30 s and immediately transferred to the rheometer, the recov-
ery of the storage (Gꢁ) and loss (Gꢁꢁ) modulus as a function of
time (6.28 rad/s frequency, 1% strain) was subsequently moni-
tored for 240 min. Then, dynamic frequency sweep and dynamic
strain sweep were performed in turn. Dynamic frequency sweep
experiment was performed to establish the frequency response
of the samples in the region of 0.1–100 rad/s at the strain of 1%.
Dynamic strain sweep experiment was performed on samples
to establish the linear viscoelastic region at the frequency of
6.28 rad/s and the strain of 0.1%–100%.
Anticancer Efficacy Measurement
A total medium volume of 100 :L MCF-7 cells were seeded in
a 96-well plate with the density of 1500 cells per well. Twenty-
four hour after seeding, the solutions with six geometric con-
centrations of three different compounds (HCPT, hydrogel of
2, and HCPT hydrogel) in 100 :L of medium were added to
each well (three wells for each concentration). Cells in wells
with the treatment of the blank PBS solution were used as the
control. After an extra 48 h culture time, the MTT assays were
performed. Twenty microliter of MTT solution (5 mg/mL) was
added for each well, and then incubated for 4 h in 37°C. All
the spent solution was pipetted out; formazon crystals at the
bottom of each well were dissolved in 150 :L dimethyl sulfox-
ide (DMSO). After oscillation for 15 min at room temperature,
absorbance at wavelength of 490 nm was tested using a mi-
croplate reader (iMark TM, BIO-RAD, Hercules, CA, USA ).19
The experiment was repeated for three times.
UV Spectroscopy
Ultraviolet–visible spectra of HCPT, supernatants of hydrogel
of 2 and HCPT hydrogel at concentration of about 10−5 mol/L
were recorded using Agilent Technologies UV1800 spectropho-
tometer (Shanghai, China). In all experiments, solutions were
taken in quartz cuvette of 0.1 cm path length.
RESULTS AND DISCUSSION
Hydrolysis of 3 and the Formation of Hydrogel of 2
Equilibrium Solubility of HCPT in Solution of 2
Excessive amount of HCPT was added to fresh solutions of 2
at the concentration of 0.2% (w/v), 0.3% (w/v), and 0.4% (w/v),
respectively. After ultrasonic sonication for 6 h to maximize the
solubility of HCPT and centrifugation at 104 r/min for 15 min,
the saturated supernatants were taken out and diluted appro-
priately by phosphate-buffered solution (PBS) solution at pH
7.4. The absorbance of diluted supernatants was determined
and then used to calculate the solubility of HCPT in the solution
of 2 according to the standard curve of HCPT. The experiment
was conducted in three parallel experiments and the results
averaged.
Self-assembly of the peptide is initiated with the generation of
2. The loss of succinic acid from 3 afforded 2 in almost quanti-
tative yield.29 Critical gelation concentration can be reached at
0.1% (w/v). The time of different peptide concentrations needed
for gelation was different: falling within 10 min and approx-
imately 72 h for 1.6% (w/v) and 0.1% (w/v). Higher peptide
concentration resulted in shorter gelation time. Rigidity of hy-
drogel is also proportional to peptide concentration. Increase
in peptide concentration may further strengthen the network
of hydrogel. Interestingly, we found that heat to a boil could
speed up gelation. Once boiled, the formation of hydrogel at
0.3% (w/v) would be observed in about 20 min.
In Vitro Release Profile of HCPT Hydrogels
Hydrogel of 2 is able to remain stable for more than 1 month
In vitro release experiment was studied under physiological at 4°C, which proved that hydrogel of 2 is a stable platform for
temperature (37°C). HCPT (0.5 mL) hydrogels at an increas- loading drugs. Besides, this hydrogel displays shear-thinning
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Figure 2. (a) Optical image of HCPT hydrogel. TEM images of (b) hydrogel fibrils; (c) hydrogel of 2 at 0.8% (w/v); (d) heat-treated hydrogel of
2; HCPT hydrogel at 0.3% (w/v) (e) and 0.8% (w/v) (f).
and recovery properties showing its ability to flow with low vis- HCPT was dissolved in 20 mL 0.9% NaCl solution. The pH of
cosity under shear and soon recover back to a solid hydrogel HCPT hydrogel was measured as approximately 7.5. We took
on cessation of shear. Shear-thinning and recovery properties advantage of the weak basic characteristics of solution of 2 to
enable injection, without syringe clogging, to a specific site and efficiently dissolve and encapsulate HCPT, directly avoiding the
provide site specificity because of solid-like properties of hydro- introduction of toxic substance, such as DMSO, and reaching a
gel after injection.38
preferable effect.
Connecting link in HCPT hydrogel between the drug and
carrier is achieved through noncovalent cross-linking. Struc-
ture of both HCPT and 2 has benzene ring and hydroxide rad-
ical, thus B–B stacking and hydrogen bonding cannot be ig-
nored. Because of the hydroxide radical and lactonic ring in
chemical structure, HCPT has two forms in aqueous solutions:
E-lactone form and carboxylic salt form. In vivo at physiological
Preparation of the HCPT Hydrogel
A clear, stable, bright yellow HCPT hydrogel with 400 :g/mL
drug concentration was obtained after standing 1 day, as shown
in Figure 2a. Its drug concentration is comparable to Hydrox-
ycamptotbecine InjectionꢀR on the market, in which 4–6 mg
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Figure 3. (a) Dynamic frequency sweep (0.1–100 rad/s frequency, 1% strain) and (b) dynamic strain sweep (0.1%–100% strain, 6.28 rad/s
frequency) of 0.8% (w/v) HCPT hydrogel.
pH, HCPT remains a balance of E-lactone form and carboxylic recovered within 4 h at 37°C, indicated by Gꢁ values dom-
salt form. Along with the increasing strength of alkaline so- inating Gꢁꢁ values. The observed modulus directly after the
lution, E-lactone form could transform to carboxylic salt form, shear-thinning treatment was 19.3 and 5.4 Pa, respectively,
so there were likely charge interactions between HCPT and and 304 and 26.2 Pa 240 min after the shear-thinning treat-
peptide.
ment. The growing trend of Gꢁ is much faster than Gꢁꢁ over
time, which is the main characteristics of hydrogel elastic be-
havior, indicating that peptides in the hydrogel system have
successfully formed fibrous network structure by various inter-
molecular forces. Dynamic frequency (0.1–100 rad/s frequency,
1% strain) sweep is shown in Figure 3a. Gꢁ values exhibited
weak frequency dependence at the range of 0.1–100 rad/s, sug-
gesting the presence of high elastic matrixes in hydrogels.26
The result of dynamic strain (0.1%–100% strain, 6.28 rad/s fre-
quency) sweep was Figure 3b. Once applying strain beyond the
critical strain (about 0.5%), Gꢁ and Gꢁꢁ obviously appear declin-
ing, thus HCPT hydrogel system was damaged. As long as the
strain or shear force was removed, HCPT hydrogel system can
recover soon. Rheological experiments not only confirmed the
formation of hydrogel, but also demonstrated that the HCPT
hydrogel indeed has recoverability. The shear-thinning and re-
covery capability of HCPT hydrogel makes it quite suitable for
injecting, laying the foundation as a potential candidate for
injection.
Transmission Electron Microscopy of HCPT Hydrogels and
Nanofiber Morphology
Transmission electronic microscopy image provides useful in-
formation on morphology of self-assembled structures in the
hydrogel. As shown in Figure 2b, nanofibers were fine fil-
aments with a width of about 10 nm and length of sev-
eral microns. These confirmed the self-assembly of nanofibers.
Figure 2c showed the nanofibers of hydrogel of 2 at 0.8% (w/v).
Those nanofibers tightly intertwined together to form three-
dimensional networks to support the hydrogel formation.39 De-
sired stiffness of hydrogel can explain how fiber entanglements
and fiber branching40 account for the mechanical rigidity of the
hydrogel.26 Figure 2d is the TEM image of heat-treated hydro-
gel of 2. We observed uniform rod-like nanofibers with a width
of about 100 nm and length of less than 1 :m stacked with
each other, which are quite different from those in unheated
hydrogel. It seems that heating process could speed the hy-
drolysis of 3 and aggregation of 2, thus forming nanofibers in
different shapes. TEM results of HCPT hydrogel at 0.3% (w/v)
and 0.8% (w/v) are shown in Figures 2e and 2f, respectively.
Nanofibers of 0.8% (w/v) bear similarity to that of 0.3% (w/v),
but it has denser networks. Black dotted HCPT was stuck to
nanofibers or trapped in networks, which distinctly provided
certification for drug encapsulation. Obviously, the quantity of
HCPT in 0.8% (w/v) hydrogel is more than that in 0.3% (w/v)
hydrogel, which proved that the equilibrium solubility of HCPT
may increase with the concentration of solution of 2.
UV Spectroscopy
Concentrations of HCPT in PBS supernatants of samples were
determined by UV. In Figure 4, the absorbance spectra of HCPT
showed ultraviolet absorption peak at 267 and 384 nm. Because
of the benzene structure of 2, absorption peak of pure hydro-
gel of 2 was at about 280 nm, which would apparently affect
the HCPT absorption peak at 267 nm. But, there was no ab-
sorption over 300 nm range for hydrogel of 2. So the ultravio-
let absorption at 384 nm was determined to quantify released
HCPT.
Moreover, TEM was used to investigate whether the fibril-
lar nanostructure was affected by the presence of HCPT. This
result showed that the presence of HCPT does not affect self-
assembly of 2.26 Except for the color from HCPT, HCPT hydro-
gel has the same appearance as neat hydrogel with no discern-
able changes.
Equilibrium Solubility of HCPT in Solution of 2
According to the standard curve of HCPT, the equilibrium sol-
ubility of HCPT in solution of 2 at concentrations of 0.2% (w/v),
0.3% (w/v), and 0.4% (w/v) is shown in Table 1. The equilib-
rium solubility of HCPT in PBS solution at pH 7.4 is deter-
Rheology
As shown in dynamic time sweep (Supplementary Fig. S3), mined as 9.08 :g/mL.41 Compared with that in PBS solution,
HCPT hydrogel at concentrations 0.8% (w/v) spontaneously the solubility of HCPT in solution of 2 increased at least 40
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drogel increased; the volume administrated can be partially
reduced eventually, which is more sustainable for patients.
Furthermore, the increase in equilibrium solubility implied
that the bioavailability of HCPT could be promoted to some
extent.
In Vitro Release Profile of HCPT Hydrogels
Figure 5 shows the release profiles and accumulative percent-
ages of HCPT released from their corresponding hydrogels.
Three curves with different peptide concentrations exhibit sim-
ilar release trend: relatively higher rates during the first 12 h,
followed by lower rates after 60 h. The highest release rate of
1 h was 18%, meeting the required under 40% for the sustained-
and controlled-release preparations.39 For 72 h, approximately
52.6%, 66.3%, and 79.2% of HCPT were released from HCPT
hydrogels at 37°C. Comparison of three accumulative release
rates state clearly that higher peptide concentration has higher
drug release under the prerequisite of the same drug loading.
This result may be associated with the increase of equilibrium
solubility and hydrophilicity of HCPT as peptide concentration
increased. These preliminary results demonstrated the long
term and sustained release of HCPT at high-accumulated re-
lease rate. In addition, either before or after injection, HCPT
hydrogels do not swell when exposed to additional solution or
bodily fluids. Rather, new solutes and aqueous fluids can begin
to diffuse into and within the stable hydrogels as defined by the
porosity of the self-assembled peptide network.26 It means that
HCPT hydrogel is likely to work at a steady state in vivo as an
injectable therapeutic agent.
Figure 4. The ultraviolet absorption spectra of HCPT, pure hydrogel
of 2, and HCPT hydrogel at the concentration of about 10−5 mol/L.
Table 1. The Equilibrium Solubility of HCPT in Solution of 2 at
Concentration of 0.2% (w/v), 0.3% (w/v), and 0.4% (w/v)
Concentration of
Solution of 2
Equilibrium
Solubility (:g/mL)
0.2% (w/v)
0.3% (w/v)
0.4% (w/v)
416
743
863
HCPT Hydrogels in Contact with MCF-7 Cell
Through the microscope (Figure 6), MCF-7 cells showed mor-
phological features of cell death (cell rounding, cell shrinkage,
detachment, or floatation) when incubated with HCPT hydro-
gels, whereas there were few obvious signs of cytotoxicity in
cells cultured with hydrogels lacking HCPT. Cell death ob-
served clearly indicated the release of HCPT from HCPT hydro-
gel network. Comparison of cells cultured with a fixed concen-
tration of HCPT at an increasing peptide concentration, that
is, 0.1% (w/v), 0.2% (w/v), 0.4% (w/v), and 0.8% (w/v), showed
signs of increased cell death. This increasing trend of cell death
is caused by the higher drug release of higher peptide con-
centration. This result is coincident with the previous in vitro
release profile of HCPT hydrogel.
Anticancer Efficacy Measurement
The activity of three compounds was evaluated by treating
MCF-7 cells at a serial of concentrations. Although MCF-
7 cells grew in hydrogel of 2 freely, HCPT hydrogels inhib-
ited their growth (Fig. 7). By calculating cell inhibition rate,
the hydrogel of 2 was nontoxic at gelation concentration,
whereas pure HCPT and HCPT hydrogel possessed IC50 val-
Figure 5. 10-Hydroxy camptothecin accumulative release profiles of
0.2% (w/v) (ꢀ), 0.3% (w/v) (•), and 0.4% w/v (ꢁ) HCPT hydrogel prepared
at fixed drug concentration of 400 :g/mL.
ues of 52.3
2.9 and 23.5
3.5 :g/mL. This result is in
times, which fully demonstrated that a hydrophilic prepara- agreement with the cell state observed in cell imaging. Un-
tion of HCPT have been created. It can be easily concluded that like some other anticancer drug hydrogels,8,19 the anticancer
the equilibrium solubility increases with the concentration of efficacy of HCPT was indeed increased. The most likely rea-
solution of 2, which leads to the increase of maximum drug son is that the solubility and hydrophilicity of HCPT improved
loading in unit volume. The optimization in maximum drug through encapsulation by hydrogel. So hydrophilic HCPT hy-
loading showed that hydrogel of 2 is suitable for loading drug. drogel is much easier than hydrophobic HCPT in entering
Under the same drug dose, drug loading in unit volume of hy- the cells to exert its cytotoxic effect and cause fatal damage
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
2273
Gel of 2
HCPT Gel
0.1% (w/v)
0.2% (w/v)
0.4% (w/v)
0.8% (w/v)
Figure 6. Images of MCF-7 cells affected by 0.1% (w/v), 0.2% (w/v), 0.4% (w/v), and 0.8% (w/v) hydrogel of 2 (left) and HCPT hydrogel (right)
at fixed 400 :g/mL drug concentration. Scale bar represents 100 :m.
to cells. Although HCPT hydrogel showed promising potency, brought HCPT hydrogel a superior drug loading, which indeed
its toxicity toward normal mammalian cells remains to be improved anticancer efficacy simultaneously. Cytotoxicity test
evaluated.
indicated that the hydrogel without drug is nontoxic, ensuring
the safety of carrier. Long term and sustained release of HCPT
enhanced the potential effectiveness of HCPT hydrogel as an
injectable agent for local delivery. We believe that the integra-
tion of hydrophilic hydrogel with hydrophobic drugs is a feasible
approach to formulate hydrophobic drugs into an aqueous form
for improving their clinical applicability. Further studies will
focus on the in vivo stability of HCPT hydrogel, with the hope
that its release rate could withstand the complex and volatile
physiological environment.
CONCLUSION
10-Hydroxy camptothecin hydrogel was successfully developed
as an injectable and hydrophilic anticancer formulation, which
radically turned several drawbacks of HCPT. Excellent phys-
ical properties of carrier, such as stability, shear-thinning,
and recovery properties, enable injection to a special location.
High-equilibrium solubility of HCPT in hydrogelator solution
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
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