Rational Design of Multifunctional Polymeric Nanoparticles Based on Poly(l -histidine) and d-α-Vitamin e Succinate for Reversing Tumor Multidrug Resistance

Article abstract of DOI:10.1021/acs.biomac.8b00213

A multifunctional nanoparticulate system composed of methoxy poly(ethylene glycol)-poly(l-histidine)-d-α-vitamin E succinate (MPEG-PLH-VES) copolymers for encapsulation of doxorubicin (DOX) was elaborated with the aim of circumventing the multidrug resist

Full text of DOI:10.1021/acs.biomac.8b00213

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Rational design of multifunctional polymeric
nanoparticles based on poly(L-histidine) and d-#-Vitamin
E Succinate for reversing tumor multidrug resistance
Zhen Li, Qixian Chen, Yan Qi, Zhihao Liu, Tangna Hao, Xiaoxin Sun, Mingxi
Qiao, Xiaodong Ma, Ting Xu, Xiuli Zhao, Chunrong Yang, and Dawei Chen
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00213 • Publication Date (Web): 04 Apr 2018
Downloaded from http://pubs.acs.org on April 5, 2018
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Rational design of multifunctional polymeric nanoparticles
based on poly(Lꢀhistidine) and dꢀαꢀVitamin E Succinate for
reversing tumor multidrug resistance
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a,b,
c
a
a
d
e
b
Zhen Li *, Qixian Chen , Yan Qi , Zhihao Liu , Tangna Hao , Xiaoxin Sun , Mingxi Qiao ,
a
c
b
f,
b,g,
Xiaodong Ma , Ting Xu , Xiuli Zhao , Chunrong Yang *, Dawei Chen
*
a
School of Pharmacy, Dalian Medical University, Dalian, 116044, PR China
b
Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University,
Shenyang, 110016, PR China
c
School of Life Science and Biotechnology, Dalian University of Technology, 116024, PR China
d
Department of Pharmacy, The Second Affiliated Hospital of Dalian Medical University, Dalian,
116011, PR China
e
Institute (College) of Integrative Medicine, Dalian Medical University, Dalian 116044, PR China
f
School of pharmacy, Jiamusi University, Jiamusi 154007, PR China
g
School of Pharmacy, Medical College of Soochow University, Suzhou 215123, PR China
Correspnding author: Zhen Li*
Email: lizhenpharm@126.com
Corresponding author: Chunrong Yang*
Email: jmsyangchunrong@163.com
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Corresponding author: Dawei Chen*
Eꢀmail: chendawei@syphu.edu.cn
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ABSTRACT
A multifunctional nanoparticulate system composed of methoxy poly(ethylene
glycol)ꢀpoly(Lꢀhistidine)ꢀdꢀαꢀVitamin E succinate (MPEGꢀPLHꢀVES) copolymers for
encapsulation of doxorubicin (DOX) was elaborated with the aim of circumventing
the multidrug resistance (MDR) in breast cancer treatment. The MPEGꢀPLHꢀVES
nanoparticles (NPs) were subsequently functionalized with biotin motif for targeted
drug delivery. The MPEGꢀPLHꢀVES copolymer exert no obvious effect on the Pꢀgp
expression level of MCFꢀ7/ADR but exhibited a significant influence on the loss of
mitochondrial membrane potential, the reduction of intracellular ATP level and the
inhibition of Pꢀgp ATPase activity of MCFꢀ7/ADR cells. The constructed
MPEGꢀPLHꢀVES NPs exhibited an acidic pHꢀinduced increase on particle size in
aqueous solution. The DOXꢀencapsulated MPEGꢀPLHꢀVES/biotinꢀPEGꢀVES
(MPEGꢀPLHꢀVES/B) NPs were characterized to possess high drug encapsulation
efficiency of approximate 90%, an average particle size of approximate 130 nm and a
pHꢀresponsive drug release profile in acidic milieu. Confocal laser scanning
microscopy (CLSM) investigations revealed that the DOXꢀloaded NPs resulted in an
effective delivery of DOX into MCFꢀ/ADR cells and a notable carrierꢀfacilitated
escape from endoꢀlysosomal entrapment. Pertaining to the in vitro cytotoxicity
evaluation, the DOXꢀloaded MPEGꢀPLHꢀVES/B NPs resulted in more pronounced
cytotoxicity to MCFꢀ/ADR cells comparing with DOXꢀloaded MPEGꢀPLHꢀVES NPs
and free DOX solution. In vivo imaging study in MCFꢀ7/ADR tumorꢀengrafted mice
exhibited that the MPEGꢀPLHꢀVES/B NPs accumulated at the tumor site more
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effectively than MPEGꢀPLHꢀVES NPs due to the biotinꢀmediated active targeting
effect. In accordance with the in vitro results, DOXꢀloaded MPEGꢀPLHꢀVES/B NPs
showed the strongest inhibitory effect against the MCFꢀ7/ADR xenografted tumors
with negligible systemic toxicity, as evidenced by the histological analysis and change
of body weight. The multifunctional MPEGꢀPLHꢀVES/B nanoparticulate system has
been demonstrated to provide a promising strategy for efficient delivery of DOX into
MCFꢀ7/ADR cancerous cells and reversing MDR.
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KEYWORDS: MPEGꢀPLHꢀVES copolymer; selfꢀassembled nanoparticles;
doxorubicin; multidrug resistance; Pꢀgp function; targeted delivery; antitumor activity
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1
. INTRODUCTION
The failure of chemotherapy is acknowledged to associate with the occurrence of
multidrug resistance (MDR), where the cancerous cells simultaneously display
resistance to a wide range of chemotherapeutics despite single chemotherapeutic drug
was administered. There are several molecular mechanisms underlying the acquired
MDR propensity: (1) increased drug efflux; (2) reduced drug influx; (3) improved
DNA repair activity; (4) changes in drug metabolism; (5) reluctant apoptosis in
accompanied with possession of antiꢀapoptotic defensing mechanisms; (6) genomic
amplification or mutation of relevant proteins and (7) sequestration of drugs within
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cytoplasmic vesicles . Indeed, the acquired MDR of the cancer cells involves a
series of cellular processes. Particularly, multiple mechanisms may simultaneously
conduce to the cellular drug resistance. Therefore, it is an onerous task to design an
adequate drug delivery system to achieve maximal reversal of MDR.
Pꢀglycoprotein (Pꢀgp), characterized with a member of ATPꢀbinding cassette
family, has been documented to be one of the main drug efflux transporters to induce
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effluxꢀmediated MDR . A number of chemotherapeutic drugs (e.g. doxorubicin,
etoposide, vinblastine and paclitaxel) are determined to be Pꢀgpꢀsubstrates. Hence,
Pꢀgp is capable of pumping substrates out of tumor cells using the energy of
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ATPꢀhydrolysis, and thus resulting in reduced intracellular drug accumulation . The
antiꢀcancer drugꢀloaded nanocarriers internalized by endocytosis offer a promising
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therapeutic approach to evade and bypass the Pꢀgp mediated cellular efflux . To this
respect, a string of natural and synthetic polymers has been developed for
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manufacture of nanocarriers with the intention of improving cellular accumulation of
chemotherapeutic drugs. Unfortunately, most of the intracellular drug molecules could
still be pumped out by efflux proteins because the ordinary polymeric nanomaterials
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lack of adequate facilities on inhibiting efflux pump . Hence, it requests the design
of functional polymeric nanomaterials in affording Pꢀgp inhibition functionality so as
to further improve the potency of the chemotherapeutic activity. Some nonionic
surfactants, including Tween 80, Poloxamer, Cremophor EL and DꢀαꢀTocopheryl
polyethylene glycol succinate 1000 (TPGS), have demonstrated the ability in
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inhibiting the Pꢀgp activity . Particularly, TPGS is one of the most promising
surfactants for the reversal of MDR. Vitamin E succinate (VES) exists as the
hydrophobic moiety of TPGS and acts a vital part in the sensitization of MDR cells by
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inhibiting the substrateꢀinduced ATPase activity . TPGS can also be utilized as
emulsifier, solubilizer, absorption enhancer, and a component material of
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nanocarriers . In addition, TPGS has been proved to possess micellar properties, as
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evidenced by formation of stable micelles in aqueous solution .
Despite important characters of TPGS micelles for the drug delivery applications,
considerable drawbacks are imperative to be resolved for their translation into wide in
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vivo availabilities
. One major disadvantage is the relatively small molecular
weight of PEG chain (Mw: 1000 Da) of TPGS, which led to poor capacity in
diminishing the immuneꢀreaction (e.g. adsorption of micelles onto the phagocytes)
and poor persistence in the bloodstream post systemic dosage. To this regard, some
attempts have been made to overcome the aforementioned drawback of TPGS. For
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instance, Mi et al synthesized the hybrid of TPGS 2000 and TPGS3350ꢀFolate
conjugates, followed by selfꢀassembly for construction of micelles to pursue targeted
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docetaxel delivery . TPGS 2000 was characterized to possess a lower CMC value
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(0.0219 mg/ml) relative to that of the original TPGS (0.2 mg/ml). Moreover, the
TPGS 2000 conjugate itself exerted antiꢀcancer activity by mean of substituting
ubiquinone from binding to complex II in the mitochondrial respiratory chain, which
consequently promoted a synergistic interaction with the formulated drug. Besides,
some other nanomedicines based on PEGꢀderivatized VES conjugates have also been
developed to enhance the antiꢀmetastatic performance of the loaded chemotherapeutic
drugs, overcome MDR in chemotherapy, and enhance the antitumor therapeutic
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efficacy
. Therefore, such analogs of TPGS (retaining the structure of VES moiety)
hold promise as an improved nanoparticulate formulation and deserve further
investigations for cancer treatments.
Compared to molecular drug, the polymeric nanocarriers enable improved the
accumulation and delivery of loaded chemotherapeutics agents to the tumors via the
21, 22
enhanced permeability and retention (EPR) effect
. Unfortunately, in many cases
the drug release from polymerꢀbased nanocarriers is characterized to exhibit a
retarded process, thereby becoming difficult to reach an effective therapeutic level at
the target sites. This is the particular obstacle to attain adequate antiꢀtumor efficacy to
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the tumors with abundance composition of drugꢀresistant cancer cells
. This fact
encouraged the pharmaceutic researchers to develop the functional nanoparticulate
systems with triggering drug release profiles responsive to external or internal stimuli
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(
e.g. temperature, light, pH, reductive glutathione (GSH), enzymes and
25ꢀ30
ultrasound)
. Noteworthy was the modality of pHꢀsensitive polymeric nanocarriers,
which has experienced progressive development for the medical and biological
applications. For instance, the extracellular pH in most tumors is estimated to be mild
acidic (pH 6.5ꢀ7.2) in contrast to the physiological milieu (pH 7.4). Moreover,
subcellular compartments of endosomes and lysosomes are characterized to possess
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more acidic conditions (pH 4.0ꢀ6.0) . In addition, MDR cancer cells have the
propensity of creating more acidic organelles (such as recycling endosome, lysosome
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and transꢀGolgi network) comparing with the drugꢀvulnerable cancer cells . Based on
the aforementioned physiological characters, poly(Lꢀhistidine) (PLH) was often
harnessed as an excellent pHꢀsensitive candidate due to its distinct pHꢀresponsive
characters. It is a peptide containing imidazole groups with a pKa value of 6.0ꢀ7.0.
The protonation of PLH would occur when the environmental pH is below the pKa of
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PLH . A string of pHꢀresponsive formulations based on PLH have been design with
3
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the aim of intelligently controlling the drug release at different pH environments
.
The designed nanoparticulate systems were anticipated to maintain the stability in
blood circulation environment (pH 7.4) and suffer structural destabilization in
relatively acidic pH environment (such endosomes and lysosomes) due to the
promoted protonation of PLH. Furthermore, the protonated PLH can interact with
anionic phospholipids (membrane components of the endoꢀlysosomal compartments)
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and result in appreciable endoꢀlysosomal membranes disruption . The postulated
activity is particular vital in facilitating the effective delivery of drugs into the
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cytoplasm to pursue improved potency to the affected tumor cells.
Aiming to facilitate the tumorꢀspecific drug transportation, the surface of the
nanocarriers can be functionalized with various tumorꢀtargeting moieties, including
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peptides, aptamers, saccharides, folate and biotin
. Of note, biotin is a
waterꢀsoluble Bꢀvitamin which acts a critical part in nourishing the healthy hair, nails
42
and skin . Cancer cells requests a markedly larger amount of vitamins (such as folate,
biotin and vitamin B12) to sustain its proliferation, thereby inducing overexpress of
41
wide range of vitamin receptors on the cell surfaces . Hence, the specific interactions
between vitamins and their receptors could be harnessed for pursuit of targeted drug
delivery. In respect to the simply chemical structure, low molecular weight and good
reactivity, biotin has been extensively explored for utilities as the targeting moiety for
tumorꢀtargeted therapeutics. The uptake of biotin could be facilitated by
streptavidinꢀavidin receptors and/or sodiumꢀdependent multivitamin transporter on
43
the surface of cancer cells . In pertinent to nanomedicines, biodegradable polymeric
nanocarriers with functionalization of biotin have been determined to mediate higher
level of the cellular uptake activities in many cancer cell lines (including MCFꢀ7 and
4
4ꢀ48
MCFꢀ7/ADR cells utilized here) than nonꢀfunctionalized nanocarriers
.
Motivated by the abovementioned rationales, we firstly synthesized a novel
amphiphilic polymer–methoxy poly(ethyleneglycol)ꢀpoly(Lꢀhistidine)ꢀdꢀαꢀVitamin E
Succinate (MPEGꢀPLHꢀVES), which was anticipated to encompass the
aforementioned advantages of both VES and PLH. The MPEGꢀPLHꢀVES copolymers
were mixed with the homeꢀmade biotinꢀPEGꢀVES conjugates to form a novel
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nanoparticulate system for targeted and intracellular transportation of DOX (an
effective anticancer agent). The physiological characteristics (e.g. drug encapsulation
efficiency, particle size, zeta potential and in vitro drug release behavior) of the
DOXꢀloaded NPs were performed. The antitumor efficacy and MDR reversal
mechanisms of the DOXꢀloaded NPs were systematically evaluated both in vitro and
in vivo. These investigations validated the rationale of the proposed multifunctional
polymeric NPs, capable of efficiently encapsulating the chemotherapeutic drug of
DOX, promoting the activity of MCFꢀ7/ADR cell internalization and tumor
accumulation, specifically accelerating intracellular release of the encapsulated DOX
responsive to the intracellular acidic subcellular compartment, retrieving DOX from
endoꢀlysosomal entrapment, exerting the MDR reversal effect and minimizing
systemic toxicity of DOX.
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2. MATERIALS AND METHODS
2
.1. Synthesis of MPEG-PLH-VES and biotin-PEG-VES
.1.1. Synthesis of the protected MPEG-PLH
2
α
im
Anhydrous THF (15 mL) containing N ꢀCBZꢀN ꢀDNPꢀLꢀhistidine (3 g, 6.60
mmol) was dropꢀwise supplemented with 5 equivalent thionyl chloride. Subsequently,
the above reaction solution was agitated for 5 h at 25 °C under protection of nitrogen.
The mixture was precipitated in chilled ether, followed by sequential filtration and
im
vacuumꢀdrying. Subsequently, the yielded N ꢀDNPꢀLꢀhistidine NCA·HCl (2 g, 5.77
mmol) was solubilized in anhydrous DMF (40 mL) as the monomer for ringꢀopening
polymerization with the macroꢀinitiator of methoxypolyethylene glycol amine 2000
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(
MPEG2000ꢀNH
2
). The polymerization reaction was performed at 25 °C for 72 h. The
im
yielded MPEGꢀpoly(N ꢀDNPꢀLꢀhistidine) (protected MPEGꢀPLH) was collected by
sequential precipitation in chilled ether and vacuumꢀdrying (yield ca. 64%).
2.1.2. Synthesis of MPEG-PLH-VES
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VES, NHS and EDC·HCl were solubilized in DMF (10 mL) according to a molar
ratio of 1: 1.2: 1.2. The mixture was allowed to stir at room temperature for 4 h,
aiming to preꢀactivate the carboxyl group of VES. Subsequently, the aforementioned
MPEGꢀPLH (1 g, 0.18 mmol) was transferred to the above reaction solution, followed
by stirring at 25 °C for 48 h. Afterwards, the resulting solution was transferred for
precipitation in chilled ether to obtain the protected MPEGꢀPLHꢀVES, followed by
vacuumꢀdrying.
The protected MPEGꢀPLHꢀVES (1 g, 0.16 mmol) and a precalculated amount of
2ꢀmercaptoethanol were solubilized in DMSO, followed by agitating for 24 h.
Subsequently, the above solution was dialyzed against water for 72 h. The end product
(MPEGꢀPLHꢀVES) was harvested by lyophilization (yield ca. 52%).
2
.1.3. Synthesis of biotin-PEG-VES
The synthesis procedure of biotinꢀPEGꢀVES was detailed in the supporting
information.
2
.2. Characterization of the synthesized polymers
The relevant details were introduced in the supporting information.
.3. Acid–base titration of MPEG-PLH-VES
2
The MPEGꢀPLHꢀVES nanoparticulate solution (1 mg/mL) was initially adjusted
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to pH 11 with 1 M NaOH. Furthermore, the diluted solution was titrated with 0.01 M
HCl solution by following stepwise manner to construct the titration curve.
2.4. Preparation and characterization of the polymeric NPs
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The DOXꢀencapsulated MPEGꢀPLHꢀVES NPs and MPEGꢀPLHꢀVES/B NPs were
34
prepared as previously described . The corresponding blank NPs were also provided
in a similar procedure. The experimental procedures of the NPs preparation, particle
size and zeta potential measurement, TEM observation and drug encapsulation
efficiency determination were described in the supporting information.
2.5. In vitro drug release
The release profiles of DOX from nanoparticles were studied utilizing dialysis
method. In brief, 2 mL DOXꢀloaded NPs solution was dialyzed against 25 mL of
release media (pH 7.4 or 5.0) at 37 °C. Periodically, 3 mL aliquots were taken and
replaced by 3 mL blank medium. Finally, the released drug amount was quantified
with UVꢀvis spectrometer at 481 nm.
2.6. Detection of P-gp expression level
MCFꢀ7/ADR cells were treated with TPGS or MEPGꢀPLHꢀVES nanoparticulate
solution (final concentration at 50 ꢁM) for 4 h. Subsequently, the cells were
trypsinized and fixed with 4% paraformaldehyde. Subsequently, the cells were
incubated with rabbit antiꢀhuman Pꢀgp antibody (1:25) for 30 min at 4 °C, and
centrifugated to remove the unbound antibody. After that the cells were treated with
FITCꢀlabeled goat antiꢀrabbit antibody (1:50) for 30 min. eventually, the cells were
rinsed and resuspended in PBS for quantitative fluorescence analysis using a flow
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cytometer.
2.7. Assay of mitochondrial membrane potential
The MCFꢀ7/ADR cells were incubated with TPGS or MEPGꢀPLHꢀVES
nanoparticulate solution (final concentration at 50 ꢁM) for 2 h. The mitochondrial
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membrane potential was determined as previously described .
2.8. Intracellular ATP level assay
The MCFꢀ7/ADR cells were incubated with TPGS or MEPGꢀPLHꢀVES
nanoparticulate solution (final concentration at 50 ꢁM) for 2 h. After that, the ATP
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level was measured as previously described .
2.9. Determination of P-gp ATPase activity
The effect of MPEGꢀPLHꢀVES on Pꢀgp ATPase activity was investigated based
on PꢀgpꢀGlo assay system (Promega, USA). In brief, varied concentrations of the
sample compounds containing verapamil (50 mM) and MPEGꢀPLHꢀVES copolymer
(final concentration at 0, 10, 50 and 100 ꢁM, respectively) or Na VO alone were
3 4
incubated with recombinant Pꢀgp membranes for 5 min at 37 °C. Afterward, the
reaction was stimulated by the MgATP for 40 min. Furthermore, the samples were
mixed with ATP detection reagent to initiate an ATPꢀdependent luminescence reaction
at room temperature. The luminescence signals were determined by multifunctional
microplate reader (Tecan, Austria). The changes of relative light unit (ꢂRLU) were
3 4
acquired by calculating the difference between the Na VO ꢀtreated samples and the
mixture of polymer and verapamilꢀtreated samples. In addition, the ꢂRLU values
were plotted as a function of the reciprocal of verapamil concentrations (25, 40, 50
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and 100 mM) to acquire the linear regression for each concentration of
MPEGꢀPLHꢀVES (10 or 100 mM).
2.10. In vitro cytotoxicity
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The MCFꢀ7 or MCFꢀ7/ADR cells were incubated with free DOX, blank NPs and
DOXꢀencapsulated NPs for 48 h. Afterwards, MTT was added, followed by 4 h
incubation. The generated crystals of purple formazan were solubilized in DMSO,
followed by determination of the absorbance (at 570 nm) with a microplate reader.
The cytotoxicity of blank polymeric NPs on L02 cells (human normal liver cells) was
also evaluated with a similar procedure.
2.11. Cellular uptake and intracellular localization
MCFꢀ7/ADR cells were treated with DOXꢀloaded NPs (10 ꢁg/mL
DOXꢀequivalent) for 1 and 4 h. Furthermore, the cells were rinsed and stained with
100 nM Lysotracker Green (5 min) and 10 ꢁg/mL Hoechst 33342 (10 min), aiming to
observe lysosomes and nuclei, respectively. Subsequently, the cells were subjected to
fixation with 4% paraformaldehyde, followed by being captured utilizing a CLSM
(LSM 710, Carl Zeiss). The cellular uptake activities of various DOX formulations
34
were further quantitatively analyzed using flow cytometry as previous described .
2.12. In vivo imaging
Nonꢀinvasive in vivo imaging systems were employed to gain insight on the
realꢀtime biodistribution activities of DiRꢀlabeled NPs in the female BALB/c nude
7
mice (18ꢀ20 g) engrafted MCFꢀ7/ADR tumors. Briefly, each of 1 × 10 cancerous
cells was inoculated into the abdomen of the mouse. When the tumor volume grew to
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0ꢀ100 mm , the DiRꢀlabeled MPEGꢀPLHꢀVES and MPEGꢀPLHꢀVES/B NPs were
intravenously dosed into the tumorꢀengrafted mice, respectively. The timeꢀdependent
biodistribution was imaged with an IVIS® Lumina II Imaging System at the designed
time points. At last, the tumors and major organs were immediately excised and
transferred for ex vivo imaging.
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.13. In vivo tumor growth inhibition study
Various formulations (including saline, free DOX solution, DOXꢀencapsulated
MPEGꢀPLHꢀVES NPs and DOXꢀencapsulated MPEGꢀPLHꢀVES/B NPs) were
intravenously dosed into the MCFꢀ7/ADR tumorꢀengrafted mice every 3 days at the
dose of 10 mg/kg DOX, respectively. Body weight and tumor volume (length ×
2
width /2, determined with a caliper) were recorded every other day. Finally, the mice
were euthanized to isolate the tumors and hearts. the detailed experimental procedure
for H&E staining was described in the supporting information.
2
.14. Statistical analysis
Data were presented as mean ± SD. Statistical analysis were determined by the
analysis of variance (ANOVA) for the group numbers exceeding 3 or Student’s tꢀtest
between 2 groups. P values less than 0.05 were considered to be statistically
significant.
3
. RESULTS AND DISCUSSIONS
3.1. Characterization of the synthesized polymers
The synthesis routes of MPEGꢀPLHꢀVES and biotinꢀPEGꢀVES were illustrated in
im
Figure 1. The MPEGꢀpoly(N ꢀDNPꢀLꢀhistidine) (protected MPEGꢀPLH) copolymer
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was synthesized via the ringꢀopening polymerization of N ꢀDNPꢀLꢀhistidine
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2
carboxyanhydride using MPEG2000ꢀNH as a macroinitiator (Figure 1A). The
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representative H NMR spectrum of protected MPEGꢀPLH exhibited a series of peaks
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at δ 3.49 ppm (−OCH CH O−), δ 7.79 ppm (−CO−NH−), δ 4.24 ppm (−CH−), δ
a
2
2
b
k
c
8.15 ppm (the proton “c” on the imidazole moiety) and δ
d
8.76 ppm (the proton “d” on
the imidazole moiety). The peaks (δ 9.16 ppm; δ 8.78 ppm; δ
h
i
j
7.93 ppm) belonged to
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various moieties of phenyl group (DNP) (Figure 2A). The representative H NMR
spectrum of MPEGꢀPLHꢀVES exhibited a series of peaks at δ
a
3.58 ppm
(
−OCH CH O−), δ 7.94 and 7.91 ppm (−CO−NH−), δ 8.44 ppm (the proton “c” on
2
2
b
c
the imidazole moiety), δ
2B). The peaks in the aliphatic region (δ
ppm) could be assigned to the protons of vitamin E tail. As opposed to the spectrum in
Fig.2A, the disappearance of the characteristic peaks (δ , δ and δ ) of the phenyl
d
8.87 ppm (the proton “d” on the imidazole moiety) (Figure
e
2.00 and 1.90 ppm; δ 1.75 ppm; δ 0.85
f
g
h
i
j
protons on DNP group indicated that the DNP protection groups were successfully
1
deprotected (Figure 2B). The representative H NMR spectrum of biotinꢀPEGꢀVES
exhibited a series of peaks at δ
a
6.35 and 6.41 ppm (the proton “a” on the biotin
7.83 and 7.91
e 2 2
CH
3.51 ppm (−OCH O−), and the characteristic peaks of VES
moiety), δ 4.13 and 4.30 ppm (the proton “b” on the biotin moiety), δ
b
d
ppm (−CO−NH−), δ
moiety (Figure S1).
The GPC measurement confirmed the peak of MPEGꢀPLHꢀVES at 33.23 min and
biotinꢀPEGꢀVES at 31.04 min, respectively (Figure 3A). All of the synthesized
polymers showed wellꢀdefined unimodal distribution (PDI <1.2) (Figure 3A, table S1).
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Pertaining to the biotinꢀPEGꢀVES conjugate, the molecular weight (M ) measured by
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GPC was relatively higher than M
n
by HꢀNMR measurement (Table S1). Indeed, the
molecular weights based on GPC (sizeꢀexclusion chromatography) reflect the
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molecular size of the tested compounds in solution, which is often influenced by the
34, 50
specific molecular conformation in the solution
. To this respect, the molecular
1
weight of the synthesized polymer calculated from the HꢀNMR spectra was utilized
in this work.
3.2. Acid–base titration of MPEG-PLH-VES
The acid–base titration method was employed to assess the pKa and buffering
capacity of the MPEGꢀPLHꢀVES copolymer (Figure 3B). The copolymer exhibited an
appreciable buffering capacity in physiological pH range from 5.0 to pH 8.0. The
apparent pKa of the MPEGꢀPLHꢀVES polymer was determined to be approximate
6.67 in terms of the firstꢀorder derivation of the titration profile, consistent with
51
previous report . The observed buffering capacity was believed to be attributable to
the PLH segment contained in the MPEGꢀPLHꢀVES copolymer. Along pH gradient
from neutral milieu to acidic milieu, the imidazole rings of PLH segment were
capable of progressively binding protons to induce the change of hydrophobicity of
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the MPEGꢀPLHꢀVES copolymer . Such a pHꢀresponsive amphoteric property of
PLH block is essential for the pHꢀsensitive function of the MPEGꢀPLHꢀVES
copolymer.
3.3. pH-induced particle size and PDI variation
The particle size and PDI variation of the MPEGꢀPLHꢀVES polymeric NPs in
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response to pH gradient was recorded by DLS measurement (Figure 3C). The average
particle size and PDI value remained almost unchanged at pH 8.0 and 7.4. Stepwise
adjustment of the pH of the sample solution (MPEGꢀPLHꢀVES NPs) at pH of 6.5, 6.0
and 5.0, resulted in consistent increase in average size, characterized with a linear
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slope of ꢀ56.9 nm/pH unit (R = 0.9968). Meanwhile, the PDI value of the
MPEGꢀPLHꢀVES also increased (from 0.137 to 0.250) in response to pH gradient
from 7.4 to 5.0. In pertinent to the MPEGꢀPLHꢀVES NPs, the associative
hydrophobic force of VES moieties and deprotonated PLH segments strengthened the
inner core structure under neutral pH milieu. Along pH decrease of the sample
solution, the gradual protonation of the imidazole groups in PLH segments enabled
PLH undergoing a hydrophobic to hydrophilic transition, wherein the electrostatic
repulsive force also progressively generated. This molecular transformation,
eventually accounts for the structural destabilization, evidenced as the swelling or
33
aggregation of NPs .
3.4. Characterization of DOX-encapsulated polymeric NPs
The physical characterizations of the DOXꢀencapsulated polymeric NPs were
summarized in Table 1. The DOXꢀencapsulated NPs were determined to possess
similar DLS size at approximate 130 nm with unimodal distribution of PDI below 0.2
(Figure 3E and Figure S 2B). Biotin decoration appeared to not affect the size and size
distribution of the DOXꢀencapsulated polymeric NPs. TEM morphology
characterization revealed that the drugꢀencapsulated NPs with or without biotin
decoration exhibited nanoscaled spherical formation (Figure 3D and Figure S2A). The
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relative smaller size observed via TEM as compared to DLS measurement may be
ascribed to the shrinkage of the nanoparticles during drying in the TEM sample
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preparation . The zeta potential was measured to be ꢀ5.82 mV for DOXꢀloaded
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MPEGꢀPLHꢀVES NPs and ꢀ2.98 mV for MPEGꢀPLHꢀVES/B NPs, respectively. The
low absolute value of the zeta potential of the polymeric NPs may be attributed to the
existence of the neutrally charged PLH segments near the nanoparticle surface and the
19, 54
spatial charge shielding effect of PEG chains
. In addition, Both DOXꢀloaded
MPEGꢀPLHꢀVES NPs and MPEGꢀPLHꢀVES/B NPs were determined to possess
appreciable drug EE, 90.69 ± 2.63% and 89.53 ± 3.77%, respectively.
3.5. In vitro drug release
The in vitro drug release behaviors of the DOXꢀloaded MPEGꢀPLHꢀVES NPs and
MPEGꢀPLHꢀVES/B NPs were investigated in two dissolution media (pH 5.0 and 7.4)
at 37 °C (Figure 3F). Overall, the DOX release from MPEGꢀPLHꢀVES NPs was not
affected by the biotin decoration. Yet, the DOXꢀloaded polymeric NPs displayed
apparent pHꢀdependent drug release behavior, as evidenced by the faster DOX release
at endoꢀlysosomal pH (~5.0). In pH 7.4 releasing media, both MPEGꢀPLHꢀVES NPs
and MPEGꢀPLHꢀVES/B NPs had a low cumulative drug release around 30% within
72 h. In contrast, as the pH decreased to 5.0, the cumulative DOX release percentage
was increased to 72.1% for MPEGꢀPLHꢀVES NPs and 70.2% for MPEGꢀPLHꢀVES/B
NPs, respectively (P > 0.05). This result may be attributed to the protonation of DOX
and PLH segments under acidic condition. Along pH gradient from pH 7.4 to 5.0, the
protonated PLH segments conduced to the swelling of micellar hydrophobic core due
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to the strengthening of electrostatic repulsion. Moreover, the dissociation of
doxorubicin was also enhanced with the decrease of pH, leading to a higher solubility
in aqueous solution and weaker hydrophobic interaction with the inner core building
blocks of VESꢀPLH. Meanwhile, the electrostatic repulsion between DOX molecules
and PLH segments was also strengthened to further prompt the drug release from the
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3.6. Assay of P-gp expression, mitochondrial membrane potential and ATP level
To study the plausible mechanisms of reversing MDR, the impact of the
MPEGꢀPLHꢀVES or TPGS on the Pꢀgp expression level of the MCFꢀ7/ADR cells
was initially investigated using fluorescently labeled antibody. The TPGS and
MPEGꢀPLHꢀVES did not induce a significant alteration on fluorescence intensity of
MCF7/ADR cells as compared to corresponding mean value in the control group
(Figure 4A). It indicates that the inhibition on the expression level of Pꢀgp is not
52
involved in the reversal of MDR by MPEGꢀPLHꢀVES or TPGS . The impact of the
m
MPEGꢀPLHꢀVES or TPGS on the mitochondrial membrane potential (ꢂΨ ) of the
MCFꢀ7/ADR cells was then investigated based on a potentialꢀdependent accumulation
of JCꢀ1 dye. This dye was characterized to enter selectively into mitochondria and
undergo the redꢀshift in its emission from green (530 nm) to red (590 nm) as a
consequence of the reversible formation of JCꢀ1 aggregates based on membrane
polarization. Hence, a reduced index of red/green fluorescence intensity represents the
depolarizing behavior of the mitochondrial membrane. The index of JCꢀ1 red/green of
MCFꢀ7/ADR cells exposed to MPEGꢀPLHꢀVES was determined to be merely 20.3%
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relative to that of the control cells, lower than that of TPGS group (68.6% of control)
(P<0.01, Figure 4B). It is well acknowledged that mitochondria are responsible for
providing cells with usable energy. Therefore, the intracellular ATP level of
MCFꢀ7/ADR cells was also measured using a luciferin/luciferase assay. The
MCFꢀ7/ADR cells incubated with the blank medium were employed as a control.
TPGS and MPEGꢀPLHꢀVES brought about a significant reduction of intracellular
ATP level in MCF7/ADR cells to 34.4% and 84.4% relative to the control at 2 h post
incubation, respectively (Figure 4C). In consistent with the change in mitochondrial
membrane potential, the MPEGꢀPLHꢀVES also displayed a higher depletion of the
intracellular ATP level as compared to TPGS (p<0.01). Most likely, vitamin E
succinate (VES) and its analogues could induce cell apoptosis by interference of the
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ubiquinoneꢀbinding sites in mitochondrial respiratory complex II . This behavior
would disrupt the electron flux, and consequently induce destabilization onto the
mitochondrial membrane. Meanwhile, the protonated PLH segment of
MPEGꢀPLHꢀVES under acidic condition was believed to interact with the membrane
components of the mitochondrial compartments, which may induce further loss of
mitochondrial membrane potential. Such mitochondrial dysfunction hindered the ATP
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synthesis and resulted in the observed decrease in the intracellular ATP level . Pꢀgp is
an ATPꢀdependent drug efflux transporter. Accordingly, the reduction in mitochondrial
membrane potential and ATP level induced by MPEGꢀPLHꢀVES would exaggerate
the inhibitory potency on Pꢀgp mediated drug efflux, consequently bringing about the
improved drug accumulation in drugꢀresistance tumor cells.
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.7. Inhibition of P-gp ATPase
The Pꢀgp ATPase assay was carried out to explore the mechanism for Pꢀgp
inhibition by the MPEGꢀPLHꢀVES copolymer based on the PꢀgpꢀGlo™ assay system.
Herein, Na VO was selected as a specific inhibitor of Pꢀgp. The samples treated with
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Na VO possess no Pꢀgp ATPase activity. Note that verapamil was selected as a Pꢀgp
substrate. It could stimulate the Pꢀgp ATPase activity, leading to the ATP consumption.
The corresponding assay employed an ATPꢀdependent luminescence reaction. The
difference in luminescent signal (ꢂRLU) between polymer treatedꢀsamples and
Na VO ꢀtreated samples indicates the inhibition for Pꢀgp ATPase activity by the test
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polymer, that is, the lower value of the ꢂRLU, standing for the weaker activity of
Pꢀgp ATPase. Verapamil (50 ꢁM) alone (control group) was capable of stimulating the
Pꢀgp ATPase activity by the specific binding of verapamil in the active center of Pꢀgp,
resulting in a significant ATP consumption (Figure 4D). In contrast, the
MPEGꢀPLHꢀVES and TPGS distinctly inhibited the Verapamilꢀstimulated Pꢀgp
ATPase activity in a concentrationꢀdependent manner. Moreover, the inhibition effect
of verapamilꢀstimulated ATPase activity of Pꢀgp by MPEGꢀPLHꢀVES was better than
that by TPGS at the designed polymer concentrations (10 and 50 ꢁM) (P<0.01, Figure
4D). A plausible reason for this promoted inhibitory activity may be as a result of the
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impact of varying the hydrophobe of TPGS by introducing the PLH segment . In
addition, plots of ꢂRLU against the reciprocal concentration of verapamil exhibited a
parallel trend among a class of MPEGꢀPLHꢀVES concentrations (Figure 4E). It
indicates that MPEGꢀPLHꢀVES may not be a substrate of Pꢀgp and not able to make
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the competitive inhibition of substrate binding. This result is consistent with the
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previous investigations on TPGS and its analogues . Similar to TPGS,
MPEGꢀPLHꢀVES may inhibit ATPase inhibition through blocking substrate binding
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and/or Pꢀgp allosteric modulation
.8. In vitro cytotoxicity
The cytotoxic potency of DOXꢀencapsulated NPs together with the corresponding
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blank NPs was assessed in MCFꢀ7 and MCFꢀ7/ADR cells for 48 h incubation. The
DOX solution was also assessed for comparison. Meanwhile, the cytotoxic profile of
blank NPs was also assessed in L02 cells (human normal liver cells) for 48 h
incubation, with the aim of verifying the selective cytotoxicity of blank NPs to the
cancer cells. The cytotoxic activities of blank NPs (MPEGꢀPLHꢀVES NPs and blank
MPEGꢀPLHꢀVES/B NPs) to MCFꢀ7 cells were markedly higher than that to normal
L02 cells at copolymer concentrations of 100 and 200 ꢁg/mL (P<0.01) (Figure S3),
suggesting that the blank NPs possess selective cytotoxicity to MCFꢀ7 breast cancer
cells. A plausible reason for the acquired anticancer effect could ascribe to the VES
analogs in terms of downꢀregulating the antiapoptotic proteins of cancer cells or/and
activating mitochondriaꢀspecific apoptotic pathways through the production of
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reactive oxygen species (ROS) within tumor cells . In contrast, the normal cells
were envisioned to be less susceptible to the aforementioned ROSꢀinitiated apoptosis
from the VES analogs due to their effective antiꢀoxidant defenses, thereby preventing
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further oxidative damage and cell death . In addition, the cytotoxic impact of the
blank NPs on MCFꢀ7/ADR cells was observed to be lower than that of MCFꢀ7 cells
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Figure 5 and Figure S3). This result was conjectured to be as a result of the
strengthened antioxidant abilities of multidrugꢀresistant cancer cells (e.g.: activation
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of the antioxidant transcription factor Nrf2 or other ROS scavengers) .
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Pertaining to the DOX formulations, free DOX solution exhibited relatively
higher cytotoxic potency to the drugꢀsensitive MCFꢀ7 cancer cells at all designed
DOX concentrations as compared to the NPs of DOXꢀloaded MPEGꢀPLHꢀVES and
MPEGꢀPLHꢀVES/B (Figure 5A). Free DOX solution was calculated to have an IC50
value of 0.3 ꢁg/mL for MCFꢀ7 cells, lower than those obtained for the DOXꢀloaded
nanoparticulate formulations (IC50=2.3 ꢁg/mL for MPEGꢀPLHꢀVES NPs and
IC50=1.7 ꢁg/mL for MPEGꢀPLHꢀVES/B NPs, respectively). The relative low
inhibitory potency of nanoparticulate formulation as compared to molecular DOX was
likely to be as a consequence of the relative retarded release of drug from the
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polymeric nanocarriers . In contrast, free DOX molecules were able to be
expeditiously internalized by sensitive MCFꢀ7 cells with low expression of Pꢀgp and
readily localized inside the cancerous cells, accounting for high cytotoxicity as a
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result of the action of DOX with nucleus DNA in cancer cells
.
As opposed to the drugꢀvulnerable MCFꢀ7 cancer cells, free DOX was observed
to exert no significant cytotoxic potency to MCFꢀ7/ADR cells over a concentration
range of 2.5~40 ꢁg/mL, likely due to the excessive expression of drug resistant Pꢀgp
(Figure 5B). By contrast, the constructed DOXꢀloaded nanoparticle formulations
showed pronounced cytotoxic potency to MCFꢀ7/ADR cells in
a
concentrationꢀdependent manner. The IC50 value was calculated to be 11.1 and 8.6
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ꢁ
g/mL for DOXꢀloaded MPEGꢀPLHꢀVES NPs and MPEGꢀPLHꢀVES/B NPs,
respectively. DOXꢀloaded MPEGꢀPLHꢀVES/B NPs was determined to be
approximate 1.3ꢀtimes as effective as DOXꢀloaded MPEGꢀPLHꢀVES NPs. The
MDRꢀreversing mechanisms of the constructed nanoparticle formulations may
involve the following factors: firstly, the entrapment of DOX molecules in the
polymeric NPs were internalized through endocytosis pathway, thereby evading MDR
efflux pumps; secondly, the MPEGꢀPLHꢀVES, as a TPGS analog, has been validated
in correlation of the inhibition of Verapamilꢀinduced Pꢀgp ATPase activity; thirdly,
the MPEGꢀPLHꢀVES copolymer could induce the loss of the mitochondrial
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membrane potential and ATP level of MCFꢀ7/ADR cells, accounting for further
inhibition of the Pꢀgp mediated drug efflux; lastly, the strategic biotinꢀfunctionalized
formation could augment the intracellular drug concentration by mean of the biotin
receptorꢀmediated endocytosis of the NPs to further exaggerate the cell death to the
affected MCFꢀ7/ADR cells.
3.9. Cellular uptake and intracellular distribution
The cellular uptake activities of DOX solution and diverse DOXꢀloaded NPs in
MCFꢀ7/ADR cells were quantitatively analyzed with flow cytometry. The cellular
uptake of the DOXꢀloaded nanoparticulate formulations exhibited a distinct
timeꢀdependent manner (Figure 6). The fluorescence intensity of the internalized
DOX at 4 h post incubation was higher than that at 1 h post incubation (p < 0.01). Of
note, the intracellular drug fluorescence intensities with the aids of nanoparticle
formulations at 1 and 4 h post incubation were markedly higher than that of DOX
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solution (p < 0.01). Particularly, the DOXꢀencapsulated MPEGꢀPLHꢀVES/B NPs led
to 4.52ꢀ and 4.88ꢀfold higher cellular uptake than the DOX solution, and 1.39ꢀ and
1.52ꢀfold higher cellular uptake than the drugꢀencapsulated MPEGꢀPLHꢀVES NPs at
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1 h and 4 h post incubation, respectively Figure 6C). It suggests the possibility of
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endocytosis of DOXꢀloaded polymeric NPs by evading Pꢀgpꢀmediated efflux of
drugꢀresistance cancerous cells, accounting for increased cellular internalization.
Moreover, the interaction between biotin moiety and its receptors overꢀexpressed on
the surface of the cancerous cells was believed to further improve the cellular uptake
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efficiency of MPEGꢀPLHꢀVES NPs
.
The intracellular distribution of drugꢀloaded NPs in MCFꢀ7/ADR cells was
investigated with CLSM. The endoꢀlysosomes and nuclei were labeled with
LysoTracker DNDꢀ26 (green) and Hoechst 33258 (blue), respectively. In principle,
yellow pixels in the CLSM image stand for the colocalization of DOX (red) and
endoꢀlysosomes (green). As shown in Figure 7, the internalized DOX (sum of yellow
and red pixels) were observed as yellow for both DOXꢀloaded MPEGꢀPLHꢀVES NPs
and MPEGꢀPLHꢀVES/B NPs treated cells at 1 h post incubation. This implied that the
drugꢀencapsulated NPs were internalized into the cancerous cells via the endocytosis
pathway and subsequently localized in the endoꢀlysosome compartments. In contrast,
after 4 h incubation, the population of red pixels was observed to significantly
increase in the cytoplasm as companied with reduced population of yellow pixels,
suggesting an effective endoꢀlysosomal escape of DOX. This result can be possibly
explained by the facilitated protonation of PLH component (proton sponge effect) of
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the MPEGꢀPLHꢀVES copolymer in the acidic endoꢀlysosome compartments,
conducing to sequential endoꢀlysosome osmotic swelling, disruption of the
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endoꢀlysosome membrane and cytoplasmic translocation of DOX . Such facilitated
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endoꢀlysosomal escape by MPEGꢀPLHꢀVES copolymer was believed to be crucial in
inhibiting the DOX exocytosis via the endoꢀlysosomal system or avoiding DOX
degradation in the endoꢀlysosomal compartments, eventually leading to the low drug
elimination of cancerous cells.
3.10. In vivo imaging
A wholeꢀbody NIR imaging system was utilized to visualize the biodistribution of
DiRꢀloaded MPEGꢀPLHꢀVES NPs and MPEGꢀPLHꢀVES/B NPs in the MCFꢀ7/ADR
tumorꢀengrafted nude mice (Figure 8A). Herein, DiR was employed as a nearꢀinfrared
fluorescence probe with respect to its advantageous longꢀwavelength excitation and
66
emission for deep light penetration and low autoꢀfluorescence interference . The dye
loading efficiency and encapsulation efficiency of DiRꢀloaded NPs were approximate
2% and 100%, respectively. The in vitro dye leakage profiles of DiRꢀloaded NPs were
investigated in pH 7.4 releasing media at 37 °C using dialysis method. No leakage of
DiR was detected from polymeric NPs within 48 h, suggesting that the florescence of
DiR could be utilized to evaluate the in vivo distribution activities of
MPEGꢀPLHꢀVES NPs and MPEGꢀPLHꢀVES/B NPs. At 2 h post administration, the
loaded DiR molecules were abundantly localized in the livers and the accumulation in
the tumors was limited for both MPEGꢀPLHꢀVES NPs and MPEGꢀPLHꢀVES/B NPs.
Nevertheless, the fluorescence intensity in the tumors appeared to gradually increase
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over time. Marked fluorescence accumulation in the tumors was validated at 12 h post
administration of both MPEGꢀPLHꢀVES and MPEGꢀPLHꢀVES/B NPs. Moreover, the
MPEGꢀPLHꢀVES/B NPs exhibited more intensive fluorescence signals in tumor
tissue as compared to MPEGꢀPLHꢀVES NPs. Meanwhile, the ex vivo fluorescent
images of the dissected tumors reconfirmed the higher tumor accumulation of
MPEGꢀPLHꢀVES/B NPs compared to MPEGꢀPLHꢀVES NPs (Figure 8B).
Considerable liver and spleen accumulation was observed for the two DiRꢀloaded
NPs, which is believed to be attributable to the nonspecific capture by the
reticuloendothelial system residing in the livers and spleens.
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3.11. In vivo tumor growth suppression
The ability of nanoparticulate formulations to reverse MDR was evaluated in mice
bearing the subcutaneous MCFꢀ7/ADR tumor xenografts. The saline group served as
the control group. DOX solution group exhibited a limited tumor inhibitory activity
(an average 29.4% reduction in tumor volume compared to the saline group) on
account of drug resistant characteristics of MCFꢀ7/ADR cells Figure 9A). By contrast,
(
all DOXꢀloaded nanoparticle formulations exhibited high in vivo antitumor efficacy at
the end of 13 days. Of note, the tumor volume of the mice treated with
MPEGꢀPLHꢀVES/B group was calculated to be 27.4% relative to that of the control
group, lower than that of MPEGꢀPLHꢀVES group (42.2%) (p < 0.05), indicating the
strategic use of MPEGꢀPLHꢀVES and biotinꢀPEGꢀVES for pursuit of systemic tumor
growth suppression (Figure 9A and B). The pathology analysis of the postꢀtreated
tumor tissues was further conducted with H&E staining (Figure 9D). The tumor
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sections collected from DOXꢀloaded nanoparticle formulations treatment groups
(especially for DOXꢀloaded MPEGꢀPLHꢀVES/B) showed extensive hemorrhagic
inflammation and severe spotty necrosis compared with that collected from free DOX
solution treatment group. The improved antitumor efficacy of MPEGꢀPLHꢀVES/B
group might be due to the synergistic outcomes of passive (EPR effectꢀmediated)
tumor targeting and active (biotinꢀmediated) tumor targeting.
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The average body weight of every group was also recorded to assess the overall
systemic toxicity. The saline control group showed a moderate increase in body
weight due to no drug intervention. In contrast, ~30% loss relative to the initial body
weight was confirmed for the mice treated with free DOX solution group at the end of
animal test. By comparision, the body weight change of the DOXꢀloaded nanoparticle
formulations groups was negligible, demonstrating a low degree of systemic toxicity
(Figure 9C). The H&E staining for the crossꢀsection of heart sections further affirmed
the associated cardiotoxicity of the diverse DOX formulations (Figure 9D). Pertaining
to the specimen derived from the free DOX solution, apparent cardiac tissue damages
(such as cytoplasmic vacuolization, myocardial fiber breakage and disarray) were
observed, consistent with the previously documented result of severe cardiotoxicity
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from free DOX solution
. By comparison, no severe cardiac tissue damage was
confirmed for the mice treated with DOXꢀencapsulated nanoparticle formulations,
verifying that nanoparticle formulations of DOX can afford a tempting merit of
reduced systemic cardiotoxicity. Hence, the investigation from DOXꢀbased
therapeutics functionalized with the MPEGꢀPLHꢀVES/B nanocarriers suggests
2
9
ACS Paragon Plus Environment