CD44-Targeted Facile Enzymatic Activatable Chitosan Nanoparticles for Efficient Antitumor Therapy and Reversal of Multidrug Resistance

Article abstract of DOI:10.1021/acs.biomac.7b01676

Nanoparticles are attractive platforms for the delivery of various anticancer therapeutics. Nevertheless, their applications are still limited by the relatively low drug loading capacity and the occurrence of multidrug resistance (MDR) against chemotherap

Full text of DOI:10.1021/acs.biomac.7b01676

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Article
CD44-targeted Facile Enzymatic Activatable Chitosan Nanoparticles
for Efficient Antitumor Therapy and Reversal of Multidrug Resistance
Xiaodong Zhang, Fan He, Keqi Xiang, Jiajing Zhang, Mingzhi
Xu, Pingping Long, Haijia Su, Zhihua Gan, and Qingsong Yu
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01676 • Publication Date (Web): 05 Feb 2018
Downloaded from http://pubs.acs.org on February 6, 2018
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CD44ꢀtargeted Facile Enzymatic Activatable Chitosan
Nanoparticles for Efficient Antitumor Therapy and
Reversal of Multidrug Resistance
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Xiaodong Zhang‡,⊥, Fan He†,‡,⊥, Keqi Xiang , Jiajing Zhang , Mingzhi Xu ,
,‡
†
∥
†,‡
,
Pingping Long , Haijia Su , Zhihua Gan†,‡,§,*, and Qingsong Yu
†
,‡
‡
† ‡,
*
†The State Key Laboratory of Organicꢀinorganic Composites, Beijing Laboratory of
Biomedical Materials, Beijing University of Chemical Technology, Beijing, 100029,
PR China
‡College of Life Science and Technology, Beijing University of Chemical Technology,
Beijing, 100029, PR China
§Beijing advanced innovation center for soft matter science and engineering, Beijing
University of Chemical Technology, Beijing, 100029, PR China
∥Beijing Hospital & Beijing Institute of Geriatrics, Ministry of Health, Beijing 100730,
PR China
KEYWORDS Active targeting, enzymatic responsiveness, prodrug nanoparticle,
reversal of multidrug resistance, high drug loading capacity
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ABSTRACT
Nanoparticles are attractive platforms for the delivery of various anticancer
therapeutics. Nevertheless, their applications are still limited by the relatively low
drug loading capacity and the occurrence of multidrug resistance (MDR) against
chemotherapeutics. In this study, we report that the integration of Dꢀαꢀtocopherol
succinate (VES) residue with both chitosan and paclitaxel (PTX) led to significant
improvement of drug loading capacity and drug loading efficiency through the
enhancement of drug/carrier interaction. After the incorporation of hyaluronic acid
containing PEG side chains (HAꢀPEG), higher serum stability and more efficient
cellular uptake were obtained. Due to HA coating, VES residues and the enzymatic
responsive drug release property, such facile nanoparticles actively targeted cancer
cells that overexpress CD44 receptor and efficiently reversed the MDR of treated cells,
but caused no significant toxicity to mouse fibroblast (NIHꢀ3T3). More importantly,
with HAꢀPEG coating, longer blood circulation and more effective tumor
accumulation were achieved for prodrug nanoparticles. Finally, superior anticancer
activity and excellent safety profile was demonstrated by HAꢀPEG coated
enzymatically activatable prodrug nanoparticles compared to commercially available
Taxol formulation.
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Graphical Abstract
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1
. INTRODUCTION
Paclitaxel (PTX) is one of the most important chemotherapeutic agents and has
shown clinical efficacy against advanced and refractory ovarian, breast, lung, and
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head/neck cancers. However, two factors severely compromised the therapeutic
efficacy of PTX. One is the high hydrophobicity and crystallinity. The other reason
that limit the usage of PTX is the emergence of multidrug resistance (MDR) during
treatment. Clinically, to overcome the low solubility of PTX, an adjuvant consisting
of Cremophor EL and ethanol has to be used in its administration, which causes
serious sideꢀeffects including hypersensitivity reactions, nephrotoxicity and
2
neurotoxicity. To minimize the adverse effects, various drug delivery systems (DDSs),
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including nanoparticles, polymeric micelles, liposomes as well as PTX conjugates
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have been developed. In this way, PTX could be loaded either by physical
entrapment or by chemical conjugation. Meanwhile, various hydrophobic polymers,
8ꢀ10
including those capable of hydrophobic association and/or πꢀπ stacking
with
11
hydrophobic drugs and those containing hydrotropic groups, have been explored.
However, despite these attempts, the stable and efficient encapsulation of PTX has
still been an important issue that hindered the further application of PTX encapsulated
nanoparticles.
Recent efforts for cancer therapy have focused on nanoparticles with tumor
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2ꢀ14
targetability and siteꢀspecific drug release properties.
In this way, nanoparticles
can selectively accumulate in a tumor tissue following systemic administration via
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enhanced permeability and retention (EPR) effect or by the specific binding of
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receptors, and then release loaded drugs in response to extracellular or intracellular
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stimuli such as lower pH, oxidative or reductive potential, and presence of selected
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enzymes. To enhance the tumor accumulation and endocytosis of nanoparticles,
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various active targeting moieties such as hyaluronic acid, folic acid and peptides
have been explored to bind with specific receptors that are overexpressed on cancer
cells. Hyaluronic acid (HA), also known as hyaluronan has drawn vast attentions due
to its natural origin, biocompatibility, biodegradability and the binding capability to
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CD44, a receptor that is overexpressed on various tumor cells.
Based on HA, a
large number of DDSs have been developed to deliver various agents including
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chemotherapeutics, genes and proteins and some of them have led to efficient
tumor growth inhibition. Despite their active targeting ability, a significant portion of
HA nanoparticles was also found in liver and spleen, possibly owing to the cellular
uptake by phagocytic cells and liver sinusoidal endothelial cells, which overexpress
25, 26
other HA receptors such as HARE and LYVEꢀ1.
To escape from
reticuloendothelial system (RES) and achieve higher tumor accumulation, covalent or
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nonꢀcovalent pegylation
as well as the modification technique with
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poly(Nꢀ2ꢀhydroxylpropyl methacrylamide) have been developed.
As for MDR, several different mechanisms were involved, but the primary
reason has been recognized as the overexpression of ATP binding cassette (ABC)
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transporters, particularly Pꢀglycoprotein (Pꢀgp).
In some cases, receptor mediated
endocytosis of nanoparticles can help to bypass Pꢀgp efflux, but some drugs can still
31, 32
be removed by Pꢀgp transporters before reaching the intracellular target.
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Therefore, other than bypassing Pꢀgp, the capability to suppress Pꢀgp transporters is
also needed for efficient reversal of MDR. To address this issue, a broad range of
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compounds that interact with Pꢀgp and block drug efflux (such as verapamil,
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quinidine, cyclosporine, etc.) have been investigated. However, the inherent
toxicity or the altered pharmacokinetics of cytotoxic drugs have limited their
application. Recently, some safe amphiphilic copolymers such as Dꢀαꢀtocopheryl
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polyethylene glycol 1000 succinate (TPGS) and Pluronics have been identified as
efficient Pꢀgp inhibitors, leading to restored sensitivity to anticancer drugs. The
mechanism study indicated that role of TPGS was related with Pꢀgp ATPase inhibition
and mainly determined by VES residue which prevented anticancer drugs from
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efflux. Except for the ability to reverse MDR, VES modified prodrugs of various
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anticancer agents including paclitaxel (PTX),
doxorubicin
as well as
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camptothecin have shown great potential in improving the anticancer effect and drug
loading efficiency of the parent drugs.
Herein, we hypothesized that similar VES residue in polymer and prodrugs shall
greatly improve its performance in formulating and delivering hydrophobic anticancer
agents. Therefore, to achieve the efficient delivery of PTX and to overcome MDR of
tumor cells, paclitaxelꢀDꢀαꢀtocopherol succinate prodrug (PV) was synthesized and
loaded by αꢀtocopherol succinateꢀmodified chitosan (CV). Since cathepsin B (Ca.B)
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is overexpressed in many cancer cells, GlyꢀPheꢀLeuꢀGly (GFLG) tetra peptide
which could be cleaved by Ca.B, was introduced to realize the responsive release of
loaded drugs. Nanoparticles were then modified with hyaluronic acidꢀPEG (HAꢀPEG)
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conjugate to prolong blood circulation and enhance the receptorꢀmediated endocytosis.
Systemic studies were conducted with respect to the drug loading efficiency and
capacity, stability, cellular uptake, cytotoxicity, receptorꢀmediated endocytosis,
endocytic pathways, in vitro targetability, in vivo pharmacokinetics/biodistribution
and anticancer efficacy of asꢀprepared enzymatically activatable nanoparticles.
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. MATERIALS AND METHODS
.1. Materials
Sodium hyaluronate (HA, MW=9000 Da) was obtained from Frida biological
engineering Co. LTD (Shandong, China). Waterꢀsoluble chitosanꢀoligosaccharide
(Mn=5000 Da) was purchased from heowns biochem technologies, Co., LTD
(
Tianjing, China).
1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl) carbodiimide (EDC), 4ꢀdimethylaminopyridine
DMAP), Nꢀhydroxysuccinimide (NHS) and dicyclohexylcarbodiimide were
mPEG amineꢀ5000, Dꢀαꢀtocopherol succinate (VES),
(
purchased from J&K chemicals (Beijing, China). Paclitaxel (PTX) was purchased
form Huafenginfo CO., LTD (Beijing, China). GFLG tetra peptide was purchased
from
China
peptide
Co.,
LTD.
Ethylꢀ(2S,3S)ꢀ3ꢀ[(S)ꢀ3ꢀmethylꢀ1ꢀ(3ꢀmethylbutylcarbamoyl)ꢀbutylcarbamoyl]ꢀ2ꢀoxiran
ecarboxylate (E64ꢀd) was obtained from Abcam (Shanghai, China).
Bisꢀparaꢀnitrophenylphosphate (BNPP), chlorpromazine hydrochloride, colchicine,
methylꢀβꢀcyclodextran, porcine liver esterase (PLE) and bovine spleen Ca.B were
obtained from SigmaꢀAldrich (Shanghai, China). The NIR dye (Cy7ꢀNHS) was
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purchased from Lumiprobe Co., LTD (Florida, USA). MDAꢀMBꢀ231 (breast cancer
cells), MCFꢀ7 (breast cancer cells) and NIHꢀ3T3 (fibroblasts) were purchased from
the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).
MDAꢀMBꢀ231/PTX (PTX resistant breast cancer cells) was purchased from Cellbio
Inc. (Shanghai, China). Female BALB/c normal mice and nude mice (5ꢀ6 weeks old)
were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.
Cell Counting Kitꢀ8 (CCKꢀ8) was purchased from Dojindo Laboratory (Kumamoto,
Japan). FBS (Fetal bovine serum), DMEM (Dulbecco’s Modified Eagle Medium) and
penicillin/streptomycin solution were obtained from Wisent (Canada). All other
chemicals were obtained from SigmaꢀAldrich (Shanghai, China) and used without
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further purification. Paclitaxelꢀαꢀtocopherol succinate prodrug (PV) and HAꢀPEG
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polymer with 5 PEG side chains per 100 suger residues of HA were synthesized
according to the previously reported methods.
2.2. Synthesis and characterization of VESꢀchitosan (CV) and VESꢀGFLGꢀchitosan
GFLG
(
CV) conjugates
CV polymer was synthesized by the amidation of chitosan and VES. Briefly, VES
1.5 g, 2.8 mmol) was firstly activated with EDC (0.8 g, 4.2 mmol) and NHS (0.483 g,
(
4.2 mmol) in 10 mL DMSO for 2 h. Then chitosan (4.5 g, 0.3 mmol) in 20 mL DMSO
was added to the solution and reacted at ambient temperature for 48 h. The mixture
was added dropwise into 300 mL acetone to precipitate the product and remove the
byproducts. 1 g CV polymer were obtained as pale yellow powder after centrifugation
and vacuum drying. Recovered rate: 34%.
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GFLG
CV conjugate was synthesized by a twoꢀstep reaction. Firstly, VES (0.6 g, 0.99
mmol) was activated with EDC (0.14 g, 1.09 mmol) and NHS (0.086 g, 1.09 mmol) in
mL DMSO for 2h. Then GFLG tetrapeptide (0.51 g, 1.30 mmol) was added to the
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solution and reacted at ambient temperature. After 24 h, the reaction mixture was
added into dilute hydrochloride acid (pH=1) to remove the unreacted GFLG and other
byproducts. The white precipitation was collected and lyophilized (LABCONCO
FreeZone®) to yield 0.7 g Dꢀαꢀtocopherylsuccinylglycylphenylleucylglycine
(
VESꢀGFLG) (yield: 63%). Secondly, VESꢀGFLG was conjugated to the amino
groups of chitosan with the same method as CV polymer. Pale yellow powder could
be obtained after lyophilization, and the recovered rate of this step was ~35%.
GFLG
The fluorescent labelling of RBITC and Cy7 with CV or
CV polymer was
realized by the reaction of RBITC or Cy7ꢀNHS (10 mol% relative to the free amino
group of chitosan) and corresponding polymers with the presence of equal molar
triethylamine. The final product was purified by dialysis in water and lyophilization.
The labelling ratio was maintained the same for different conjugates.
GFLG
The VES content in CV or
CV conjugate, the number of PEG side chains in
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HAꢀPEG polymer and the structure of PV prodrug was determined using H NMR
(
Avance 400, Bruker, Germany). The amount of rhodamine or Cy7 residue in the
conjugates was determined fluorometrically using an Fꢀ4600 fluorescence
spectrometer (Hitachi, Japan).
2
.3. Cell culture and animal models
The human breast cancer cells (MDAꢀMBꢀ231, MDAꢀMBꢀ231/PTX and MCFꢀ7)
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and murine fibroblasts (NIHꢀ3T3) were grown in DMEM medium supplemented with
1% penicillin/streptomycin, 10% fetal bovine serum (FBS). All cells were maintained
at 37 °C in a humidified atmosphere of 5% carbon dioxide and 95% air.
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All animal experiments were performed in accordance with guidelines of Peking
University Health Science Center Animal Care and Use Committee under the
protocols approved by the Institutional Animal Care and Use Committee at Peking
University (Beijing, China). PTX resistant MDAꢀMBꢀ231/PTX murine tumor model
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was established by subcutaneously injecting MDAꢀMBꢀ231/PTX cells (0.1 mL, 1×10
cells) into the right backside of female BALB/c nude mice (18±2 g, 5ꢀ6 weeks old).
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Tumors were allowed to grow up to 50 mm before the subsequent experiments.
The other detailed experimental procedures could be found in the supporting
information.
3. RESULTS AND DISCUSSION
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A
OH
OH
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O
O
x
O
O
EDC, NHS
Chitosan
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y
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HO
NH2
O
O
O
O
O
O
VES
CV
HO
O
NH
EDC, NHS
O
OH
OH
O
HN
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HO
O
O
O
O
O
x
O
O
HN
HO
HO
y
NH
NH2
NH2
NH
O
O
GFLG
NH
HN
O
O
EDC, NHS
Chitosan
O
HN
HN
O
N
O
H
O
O
O
O
HN
O
N
VES-GFLG
O
H
O
O
O
B
GFL
GC
V
H
O
N
O
O
O
HO
O
OH
N
O
H
O
EDC, NHS
O
O
OH
O
VES
H
O
O
O
O
O
O
O
O
OH
O
PV
HO
O
H
O
PTX
O
O
GFLG
Scheme 1. The synthetic routes of CV,
CV polymers and PV prodrug.
Based on our hypothesis, the efficient loading of PTX might be realized by the
similar functionalization of both PTX and carrier materials. To prove this, PV prodrug,
GFLG
CV or
CV polymer containing VES residues were synthesized (Scheme 1). HA
polymer containing PEG side chains was also synthesized to improve the blood
circulation and tumor targeting of drug loaded nanoparticles. The functionalization of
PTX with VES was achieved by esterification between carboxyl of VES and
44
2
`ꢀhydroxyl of PTX. HAꢀPEG copolymer were synthesized with HA and amine
2
2
functionalized PEG (Mn=5000) according to the literature reported method. Since
the molecular weight of HA significantly influence the in vivo fate of HAꢀmodified
45
particles, a relatively low molecular weight HA (9,000 Da) was introduced to
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prevent fast clearance. The degree of substitution (DS) of PEG side chains was set at
~5% for balancing between long blood circulation and efficient cellular uptake. CV or
GFLG
CV polymer was synthesized by the amidation of VES or VESꢀGFLG and
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GFLG
chitosan, respectively. As for the synthesis of
CV, the carboxyl of VES was firstly
activated by EDC and NHS, and then reacted with the amino group of tetrapeptide
linker GFLG to obtain the intermediate VESꢀGFLG. Then the Ca.B responsive
GFLG
CV polymer was obtained by the conjugation of VESꢀGFLG and chitosan in the
GFLG
presence of EDC and NHS. The relatively low yield of both CV and
CV could be
attributed to the precipitating process in acetone. Since VES can be dissolved in
acetone, the conjugates with high VES content would form reverseꢀphase micelles
GFLG
and be difficult to separate. The structures of PV, VESꢀGFLG, CV and
CV and
1
the stability of GFLG tetrapeptide were confirmed by H NMR (Figure S1). The
benzyl group of GFLG tetrapeptide were detected at about δ 7.2 ppm while the signals
of alkyl chain of VES were found between 1.0 and 2.0 ppm, indicating the successful
integration of GFLG with VES. The conjugation of VES or VESꢀGFLG to chitosan
was confirmed by the presence of signals of VES residue between 1.0 and 2.0 ppm
and signals of chitosan between 3.0 and 4.0 ppm. The DS was calculated to be
approximately 5 VES residues on each chitosan chain for both polymers (Table S1).
Critical micelle concentration (CMC) value is a parameter indicative of the
GFLG
micelle’s stability upon dilution. In order to determine the CMC of CV and
CV
polymers, fluorescence measurements were carried out using pyrene as the
fluorescence probe. The ratio between the fluorescence intensity of 384 nm and 375
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nm was used to calculate the CMC (the midꢀpoints of the fluorescent ratio plots were
46
ꢀ3
used to determine IC50 values). The CMC values were calculated to be 5.5×10
ꢀ3
GFLG
mg/mL and 5.2
×
10 mg/mL for CV and
CV, respectively.
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0
Nanoparticles are important tools in improving the solubility and enhancing the
efficacy of hydrophobic drugs. In this work, PTX or PV was loaded in nanoparticles
43
to realize the efficient delivery. Different from the literature, PV alone was able to
selfꢀassemble in water to yield ~100 nm nanoparticles (Figure 1A), but these
nanoparticles tended to form large aggregates and precipitated from the solution after
long storage. Hence, VES modified chitosan was added as a stabilizer to their
formulation. PV loaded chitosan nanoparticles with different drug loading capacity
were prepared via nanoprecipitation method. Then negatively charged HAꢀPEG
polymer was coated on nanoparticle surfaces by electrostatic absorption. The
physicochemical properties of asꢀprepared nanoparticles were characterized with
TEM and DLS. As shown in Figure S2 & S3, compared with free PV, the addition of
GFLG
CV or
with
CV polymers and PV prodrug into water yielded uniform nanoparticles
h
ranged from 10 nm and to 45 nm, indicating the successful encapsulation of
R
prodrug by the polymers. For PV loaded CV nanoparticles, the
h
R increased from 14
nm to 45 nm with increase of the feeding ratio of PV and CV polymer. However, the
GFLG
size of PV loaded
CV nanoparticles decreased from 32 nm to 13 nm under the
same condition. The reason for the different tendency was still unclear, we speculated
that this might be caused by the introduction of GFLG peptide which may change the
stacking structure of polymer and prodrugs. It is noteworthy that the drug loading
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efficiency of prodrugs with various feeding ratio was approximately 100%, and no
precipitation was observed during the fabrication process even for 10mg PV and
10mg polymer. Compared to free PTX loaded nanoparticles whose highest drug
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loading capacity was only 3.56% when 10mg PTX was added to 10mg polymer
solution, the drug loading capacity of prodrug loaded nanoparticles was up to 98.99%
under the same situation as measured by HPLC (Figure S5 & Table S2). Moreover,
our results were much higher than the literature reported values for PTX loaded
chitosanꢀαꢀtocopherol succinate nanoparticles whose drug loading efficiency and drug
4
7
loading capacity were 65.0 ± 4.2% and 7.7 ± 0.7%, respectively. It has been reported
in the literature that VESꢀDOX prodrug can selfꢀassembly with TPGS2k to form stable
48
nanoparticles with lamellar structure and thus high drug loading capacity up to 34%.
Considering the structure of prodrug and polymers, and because hydrophilic chitosan
chain has to distribute on the surface of nanoparticles, we can assume that the driving
GFLG
forces for the selfꢀassembly of the PV and CV or
CV polymers are hydrophobic
43
associations, with PTX interacting with PTX, PTX with VES, or VES with VES.
GFLG
For better investigation of the influence of CV and
CV polymers on the efficacy
of nanoparticles, nanoparticles fabricated with 10 mg CV (CVPV, 24 nm, Figure 1B)
GFLG
GFLG
or
CV (
CVPV, 21 nm, Figure 1D) polymers and 5 mg prodrug were selected
for the subsequent study to ensure uniform size and drug loading capacity.
Even though asꢀprepared chitosan nanoparticles possessed several excellent
performance such as uniform size, high drug loading capacity and high reproducibility,
the in vivo pharmacokinetics were still not satisfactory due to the positive charge
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which induce RES uptake and opsonization. HA coating has been proved to be an
efficient way to enhance the active targeting ability of chitosan nanoparticles towards
37, 50
cancer cells which overexpress CD44.
However, some HA receptors were also
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25, 26
overexpressed in normal organs such as liver or spleen,
causing the rapid
clearance of HA coated nanoparticles by these organs. To overcome these drawbacks,
PEGylation was introduced to modify HA polymers so that it can escape from RES
27, 51
uptake and achieve long blood circulation and efficient tumor accumulation.
Herein, for balancing long circulation and efficient cellular uptake, HA polymer
containing 5% PEG side chains was used to protect prodrug loaded chitosan
51
GFLG
nanoparticles. As shown in Figure 1C & E, the
R
h
of CVPV and
CVPV
nanoparticles increased from 24nm and 21nm to 44nm and 40nm, respectively. The
HA coating could be clearly observed in the TEM images (Figure 1C&E). Before HA
GFLG
modification, CVPV (43.15 mV) and
CVPV (40.25 mV) nanoparticles showed
strong positive charge due to the amino residues on chitosan chain (Figure 1F).
However, after the coating of HAꢀPEG, the zeta potential of corresponding
nanoparticles decreased to 2.17 mV and 5.18 mV, respectively, consistent with the
52
zeta potential of PEG. This result further demonstrated the feasibility of surface
modification via electrostatic absorption. Except for altering the zeta potentials of
nanoparticles, the HAꢀPEG coating also did not influence the drug loading capacity
but increase the serum stability of asꢀprepared nanoparticles (Figure S4). It should be
noted that the HAꢀPEG concentration played important roles in the successful coating,
0.1 mg/mL HAꢀPEG solution yielded successful coating, but higher concentration
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caused the aggregation and precipitation of nanoparticles. The final HAꢀPEG content
on the nanoparticle surface was about 0.08 mg and 0.07 mg HAꢀPEG per 1 mg CV
GFLG
53
and
CV, respectively, according to the Stain All assay of the supernatant after
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removing nanoparticles by centrifugation.
GFLG
Figure 1. DLS and TEM results of PV (A), CVPV (B), CVPV@HA (C),
CVPV (D)
GFLG
and
CV @HA (E) nanoparticles, scale bar represents 100nm. (F) Zeta potential
PV
GFLG
change of CVPV and
CVPV nanoparticles before and after the coating of HAꢀPEG
polymer. ***P<0.001 (n=3).
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GFLG
Figure 2. (A) PV release curves of CVPV and
CVPV nanoparticles under different
GFLG
condition. (B) PTX release curves of CVPV and
CVPV nanoparticles under
GFLG
different condition. (C) PTX release curves of CV @HA and
CV @HA
PV
PV
nanoparticles under different condition.
The release of PV prodrug and free PTX were studied by dialysis method in PBS
containing Ca. B or PLE at pH 7.4 (pH of blood) or pH 5.5 (pH of endosomes),
respectively. The release profiles of different formulations were presented in Figure 2.
The fed PV or PTX in this experiment is soluble in PBS containing 0.1% (v/v) Tween
80, so the sink condition for this experiment was met. From Figure 2A, it could be
GFLG
found that PV prodrugs in
CVPV nanoparticles underwent fastest release in the
presence of Ca.B at pH 5.5 which mimic the endosomal condition. About 80% of PV
GFLG
was released from nanoparticles after 128 h incubation. Compared with
CVPV, the
PV release from CVPV nanoparticles was about 20% under the same condition.
GFLG
Despite PV prodrug released faster from
CVPV than from CVPV nanoparticles in
the presence of Ca.B, the free PTX level in the both condition was similar to each
other, with a release rate lower than 20% when treated with Ca.B or PLE alone. Only
when the nanoparticles were simultaneously treated with Ca.B and PLE, can the
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release rate of free PTX be accelerated, and ~60% of free PTX was released after
GFLG
128h. As for the
CVPV@HA nanoparticle, the efficient release (~60%) of free
PTX only occurred under the condition of pH5.5/Ca.B/PLE plus 0.025 mg/mL HAase.
These results ensured that the loaded drug should remain encapsulated during blood
circulation or outside tumor cells and be released only after the nanoparticles were
endocytosed.
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GFLG
Figure 3. (A) Cellular uptake of CV,
CV, and various nanoparticles by
MDAꢀMBꢀ231/PTX cells at different incubation time. (B) Cellular uptake of
GFLG
GFLG
MDAꢀMBꢀ231/PTX cells after incubation with
CVPV (1),
CVPV+HA
GFLG
GFLG
pretreatment (2),
CVPV@HA (3) and
CVPV@HA+HA pretreatment (4). (C)
GFLG
GFLG
Cellular uptake of MCFꢀ7 cells after incubation with
CVPV (1),
CVPV+HA
GFLG
GFLG
pretreatment (2),
CVPV@HA (3) and
CVPV@HA+HA pretreatment (4). (D)
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GFLG
Cellular uptake of MDAꢀMBꢀ231/PTX cells after incubation with
CVPV (1),
GFLG
GFLG
GFLG
GFLG
CVPV+antiꢀCD44
CVPV@HA+antiꢀCD44 pretreatment (4). (E) Cellular uptake of
CV @HA and CV @HA by MDAꢀMBꢀ231/PTX cells after different
pretreatment
(2),
CVPV@HA
(3) and
GFLG
CVPV, CVPV
,
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PV
PV
pretreatment such as 4 ℃ , NaN
3
, chlorpromazine, colchicine and mβCD. *P<0.05,
*
*P<0.01, ***P<0.001 compared with the control column in each group (n=3).
To study the targeting efficiency of HA coated nanoparticles, the cellular uptake of
rhodamine labeled nanoparticles was quantitatively analyzed by flow cytometry in
5
4
MCFꢀ7 (low CD44 level) and MDAꢀMBꢀ231/PTX cells (high CD44 level). Figure
3A demonstrated the mean fluorescent intensity (MFI) of MDAꢀMBꢀ231 cells treated
with various formulations for different time. The relevant confocal microscopic
images could be found in Figure S6. It could be found that the cellular uptake of all
nanoparticles was timeꢀdependent. CVPV nanoparticles with positive charge exhibited
higher cellular uptake than CVPV@HA nanoparticles with HA coating and slightly
negative charge at the initial 6h. Reversal occurred at 24h when the uptake of HA
coated nanoparticles significantly exceeded the nonꢀtarget ones. This could be
explained by that the electrostatic interaction between nanoparticle surface and cell
membrane played more decisive role in the uptake of nanoparticle at the initial period
of incubation while the targeting ability of HA start to take effect only at longer
incubation time. However, at the initial 6 h, no significant difference was observed for
GFLG
GFLG
CVPV and
CVPV@HA, only at 24 h can the targeting ability of HA appear.
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The reason for this phenomenon was unclear, but we speculated that this might be
GFLG
caused by the different nanoparticle structures between CVPV and
CVPV as shown
by their different size change tendency after loading different amount of prodrug
(Figure S3).
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To elucidate whether the uptake of HAꢀPEG coated nanoparticles was mediated by
HA receptor or CD44, the competitive adsorption experiments with HA and
antiꢀCD44 were carried out and shown in Figure 3. The quantitative analysis of mean
cell associated fluorescence intensity could be found in Figure S7. The flow
GFLG
GFLG
cytometric histograms of
CVPV and
CV @HA incubated with
PV
MDAꢀMBꢀ231/PTX and MCFꢀ7 cells for 3 h with or without HA pretreatment were
shown in Figure 3B & C, respectively. The results showed that HA pretreatment
GFLG
caused significant decrease of
CVPV@HA uptake in MDAꢀMBꢀ231/PTX cells
which overexpress CD44 while caused no obvious change in MCFꢀ7 cells whose
54
GFLG
CD44 level was much lower.
By contrast, the uptake of
CVPV by
MDAꢀMBꢀ231/PTX cells was not influenced by HA pretreatment. Further inhibition
of MDAꢀMBꢀ231/PTX cells with antiꢀCD44 antibody was also carried out and
presented in Figure 3D, it could be found that CD44 antibody can efficiently inhibit
the uptake of HA coated nanoparticles while had no influence on the uptake of
GFLG
nonꢀtarget nanoparticles such as
CV . These results clearly indicated that the
PV
electrostatic coating of HAꢀPEG on the surface of chitosan nanoparticles was an
efficient way to improve its active targeting ability.
The potential endocytic pathways of various nanoparticles were studied with
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MDAꢀMBꢀ231/PTX cells. As shown in Figure 3E, upon preꢀincubation with sodium
azide or low temperature, cellular uptake for all nanoparticles was significantly
reduced to ~70% (P<0.001), demonstrating that the uptake of all nanoparticles was
energyꢀdependent and relied on the membrane mobility. When preꢀtreated with
chlorpromazine, which can disrupt clathrin from the cell membrane and inhibit the
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GFLG
clathrinꢀmediated endocytosis, the uptake of CVPV
,
CVPV, CVPV@HA and
GFLG
CVPV@HA was 82.6, 87.3, 83.9 and 75.6%, respectively. Similarly, when treated
56
with colchicine, a microtubuleꢀdependent endocytosis inhibitor, the uptake of the
aforementioned nanoparticles was reduced to 84.5, 86.3, 82.3 and 84.6%. Moreover,
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the preꢀtreatment of mβCD which influence the lipidꢀraft mediated endocytosis also
caused the decrease of endocytosis to ~80% of control for various nanoparticles.
Therefore, the above results indicated that the endocytosis of HAꢀPEG coated
nanoparticles by MDAꢀMBꢀ231/PTX cells was an energyꢀdependent process and
mediated by the combination of multiple endocytosis pathways.
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Figure 4. The colocalization of nanoparticles (in red) with lysosome (in blue) and
mitochondria (in green).
In addition to the binding to CD44 and the subsequent endocytosis, the intracellular
transportation and endosomal escape were also important factors that influence the
anticancer efficacy. To track the intracellular trafficking of different nanoparticles,
rhodamine labeled nanoparticles were observed with CLSM, and both the lysosomal
and mitochondrial colocalization were studied. As shown in Figure 4, free polymers
GFLG
GFLG
(CV and
CV), drug loaded nanoparticles (CVPV and
CVPV) and HAꢀPEG
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GFLG
coated nanoparticles (CVPV@HA and
CVPV@HA) (in red) all exhibited good
colocalization with lysosome (in blue) as showed by the purple region in the merged
layer, indicating that the lysosome was the major organelle that participated in the
intracellular trafficking of asꢀprepared nanoparticles. Since lysosomes are rich in
various enzymes such as cathepsin B and hyaluronidases, we can assume that HA and
GFLG peptide could be hydrolyzed in this compartment, releasing loaded drugs and
chitosan. Accordingly, the partial colocalization of red fluorescence and mitochondria
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(
in green) could be explained by the adsorption of chitosan on the mitochondrial
membrane, indicating the possible impact on mitochondrial activities. It has been
reported that chitosan nanoparticles can cause the apoptosis of cancer cells through
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possible mitochondrial damage, probably due to the strong positive charge which
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can bind to the negative surface of mitochondria as many antibacterial peptides did.
Figure 5. Cytotoxicity of MDAꢀMBꢀ231 (A), MDAꢀMBꢀ231/PTX (B), MCFꢀ7 (C)
and NIHꢀ3T3 (D) cells after treatment with PTX, PV, and various nanoparticles. (E)
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GFLG
Cytotoxicity of CV and
CV polymers against MDAꢀMBꢀ231/PTX cells. (F) The
GFLG
survival rate of 10 ꢁg/mL PTX, PV, CVPV@HA and
CVPV@HA against
MDAꢀMBꢀ231/PTX cells after pretreatment with 1.0 mM E64ꢀd or BNPP. *P<0.05,
**P<0.01, ***P<0.001 (n=3).
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GFLG
To investigate the in vitro cytotoxicity of PTX, PV prodrug, CV or
CV
polymers and various nanoparticles, MDAꢀMBꢀ231 cells and the corresponding PTX
resistant cell line MDAꢀMBꢀ231/PTX were used in the assay. Meanwhile, to evaluate
the influence of CD44 targeting on the cytotoxicity of prodrug loaded nanoparticles,
nanoparticles with or without HAꢀPEG coating were also incubated with MCFꢀ7 cells.
Mouse fibroblasts (NIHꢀ3T3) were involved in the experiment to evaluate the
cytotoxicity of nanoparticles against normal cells. The IC50 values against different
cells were calculated as the PTX equivalent and presented in Table S3. It could be
GFLG
found that CV and
CV polymers showed a certain toxicity in MDAꢀMBꢀ231/PTX
and NIHꢀ3T3 cells only at higher dose (Figure 5E & Figure S8), indicating the
biocompatibility of the carrier materials. In contrast to this, all nanoparticles and free
drugs exhibited doseꢀdependent toxicity against cancer cells. The PTX resistance of
MDAꢀMBꢀ231/PTX cells could be proved by the much lower toxicity (P<0.001) of
free PTX than in MDAꢀMBꢀ231 cells. Compared with DOXꢀTOS prodrug which
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showed lower toxicity than free DOX, PV prodrug in this work showed higher
toxicity than free PTX in cancer cells. Intriguingly, lower toxicity in NIHꢀ3T3 cells
than in cancer cells could be observed for prodrug nanoparticles, possibly due to the
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low esterase concentration in the former. Such fibroblastꢀexempt nanomedicine may
exert potent anticancer activity with few side effect by escaping from WNT16B
secretion, which stimulates the surviving cancer cells to be drug resistant and
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GFLG
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metastatic.
In cancer cells,
CVPV and
CV @HA nanoparticles showed
PV
higher toxicity than their nonꢀresponsive counterparts, i.e. CVPV and CVPV@HA.
Higher cytotoxicity than free PTX in various cancer cells could also be found for
these enzymatically responsive nanoparticles, corroborating the significance of Ca.B
responsiveness in increasing the intracellular drug release and thus the cytotoxicity.
For both drug sensitive and drug resistant MDAꢀMBꢀ231 cells with CD44
GFLG
GFLG
overexpression,
CVPV@HA showed higher toxicity than
CVPV. By contrast,
for MCFꢀ7 cells (low CD44 level) in which the electrostatic interaction other than HA
targeting played major role in the uptake of nanoparticles, opposite results were
observed, indicating that HA targeting played important role in the action of
nanoparticles in CD44 overexpressed cells.
To investigate the role of Ca.B and esterase, MDAꢀMBꢀ231/PTX cells were
pretreated with E64ꢀd or BNPP in their safe dose, which can inhibit the activity of
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Ca.B and esterase , respectively. The cytotoxicity of free drugs and nanoparticles
was measured with CCKꢀ8 method. As shown in Figure 5F, no obvious influence on
the toxicity of free PTX could be observed for the treatment with both E64ꢀd and
BNPP. Compared with free PTX, the pretreatment with BNPP other than E64ꢀd
caused slight decrease of the cytotoxicity of PV and CVPV@HA, indicating that
esterase might be involved in the activation of PV prodrug, similar as previously
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reported TSꢀSN38 prodrug. The pretreatment of either E64ꢀd or BNPP caused
GFLG
significant decrease of the cytotoxicity of
CVPV@HA (P<0.001), and higher
toxicity could be obtained by the the simultaneous inhibition of Ca.B and esterase
(P<0.05). These results indicated that both Ca.B and esterase played important role in
the effect of enzymatically activatable prodrug nanoparticle. However, even after the
treatment with both inhibitors, cytotoxicity still existed, indicating that the
aforementioned enzymes were not the only factors that determined the intracellular
action of nanoparticles. Though the detailed mechanism is unclear, current results still
revealed the great potential of the asꢀprepared nanoparticles in inhibiting tumor cell
growth, especially for those drug resistant ones.
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Figure 6. (A) Cell cycle analysis of MDAꢀMBꢀ231/PTX cells after treatment with
GFLG
PTX, CV,
CV and various nanoparticles. (B) Apoptosis of MDAꢀMBꢀ231/PTX
GFLG
cells after treatment with PTX, CV,
CV and various nanoparticles, the percentage
of normal cells was normalized based on 100% control.
Paclitaxel enhances the polymerization of tubulin to stable microtubules and also
interacts directly with microtubules, stabilizing them against depolymerization by
cold and calcium, which readily depolymerize normal microtubules. It blocks cells in
the G2/M phase of the cell cycle and such cells are unable to form a normal mitotic
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apparatus. To investigate the mechanism of PV nanoparticles inside cells, the cell
cycle and apoptosis of MDAꢀMBꢀ231/PTX cells incubated with free PTX and various
nanoparticles were analyzed with flow cytometry. As shown in Figure 6A, 64.58% of
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treated cells were trapped in G2/M phase for free PTX. For CVPV and
CVPV
nanoparticles, the percentage of cells in G2/M phase was 51.68% and 68.42%,
respectively. After the coating of HAꢀPEG which enhance the targeting ability and
endocytosis of nanoparticles, the G2/M phase cells increase to 71.23% for CVPV@HA
GFLG
and 81.87% for
CVPV@HA. These results suggested that PV nanoparticles not
only shared similar intracellular mechanism with PTX but also had higher efficiency.
GFLG
Accordingly, the treatment with
CVPV@HA nanoparticles also led to the increase
of the apoptotic ratios of MDAꢀMBꢀ231/PTX cells (Figure 6B). Specifically, the
GFLG
GFLG
normal cell ratios for PTX, CV,
CV, CVPV
,
CVPV, CVPV@HA and
GFLG
CVPV@HA were 69.15%, 79.72%, 84.58%, 72.20%, 64.34%, 67.02% and
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9.45%, respectively. Compared with control group, treated groups exhibited much
GFLG
lower normal cell level. Notably, drug resistant cells treated with
CVPV@HA
showed much lower normal cell ratio than free PTX treated ones, revealing the great
potential of this formulation in treating MDR tumors.
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Figure 7. (A) CLSM images of JCꢀ1 staining of MDAꢀMBꢀ231/PTX cells after
GFLG
treatment with CV,
CV and various nanoparticles. (B) The quantitative analysis of
JCꢀ1 fluorescence intensity of MDAꢀMBꢀ231/PTX cells after treatment with CV,
GFLG
CV and various nanoparticles. (C) The ATP production of MDAꢀMBꢀ231/PTX
GFLG
cells after treatment with PTX, PV, CV,
CV and various nanoparticles. (D) Pꢀgp
GFLG
expression of MDAꢀMBꢀ231/PTX cells after treatment with PTX, PV, CV,
CV
and various nanoparticles. (E) Rhodamine retention capacity of MDAꢀMBꢀ231/PTX
GFLG
cells after treatment with PTX, PV, CV,
*P<0.01, ***P<0.001 (n=3).
CV and various nanoparticles.*P<0.05,
*
It has been proved that the enzymatically responsive nanoparticles can efficiently
overcome the MDR of MDAꢀMBꢀ231/PTX cells, but the mechanism of this was still
unclear. Therefore, we further studied the cellular and molecular mechanism of
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