CD44-Targeted Facile Enzymatic Activatable Chitosan Nanoparticles

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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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Biomacromolecules

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†,‡,* †The

State Key Laboratory of Organic-inorganic 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

ACS Paragon Plus Environment

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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 D-α-tocopherol 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 (HA-PEG), 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 (NIH-3T3). More importantly, with HA-PEG 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 HA-PEG 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 head/neck cancers.1 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 side-effects including hypersensitivity reactions, nephrotoxicity and neurotoxicity.2 To minimize the adverse effects, various drug delivery systems (DDSs), including nanoparticles,3 polymeric micelles,4 liposomes5 as well as PTX conjugates6, 7

have been developed. In this way, PTX could be loaded either by physical

entrapment or by chemical conjugation. Meanwhile, various hydrophobic polymers, including those capable of hydrophobic association and/or π-π stacking

8-10

with

hydrophobic drugs and those containing hydrotropic groups,11 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 targetability and site-specific drug release properties.12-14 In this way, nanoparticles can selectively accumulate in a tumor tissue following systemic administration via enhanced permeability and retention (EPR) effect15 or by the specific binding of


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receptors, and then release loaded drugs in response to extracellular or intracellular stimuli such as lower pH,16 oxidative or reductive potential,17 and presence of selected enzymes.18 To enhance the tumor accumulation and endocytosis of nanoparticles, various active targeting moieties such as hyaluronic acid,19 folic acid20 and peptides13 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 CD44, a receptor that is overexpressed on various tumor cells.21, 22 Based on HA, a large number of DDSs have been developed to deliver various agents including chemotherapeutics,23 genes and proteins24 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 other HA receptors such as HARE and LYVE-1.25,

26

To escape from

reticuloendothelial system (RES) and achieve higher tumor accumulation, covalent or non-covalent pegylation19,

27

as well as the modification technique with

poly(N-2-hydroxylpropyl methacrylamide)28 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) transporters, particularly P-glycoprotein (P-gp).29, 30 In some cases, receptor mediated endocytosis of nanoparticles can help to bypass P-gp efflux, but some drugs can still be removed by P-gp transporters before reaching the intracellular target.31,


32


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Therefore, other than bypassing P-gp, the capability to suppress P-gp transporters is also needed for efficient reversal of MDR. To address this issue, a broad range of compounds that interact with P-gp and block drug efflux (such as verapamil,33 quinidine,34 cyclosporine,35 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 D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS)36 and Pluronics37 have been identified as efficient P-gp inhibitors, leading to restored sensitivity to anticancer drugs. The mechanism study indicated that role of TPGS was related with P-gp ATPase inhibition and mainly determined by VES residue which prevented anticancer drugs from efflux.38 Except for the ability to reverse MDR, VES modified prodrugs of various anticancer agents including paclitaxel (PTX),39 doxorubicin40

as

well as

camptothecin41 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, paclitaxel-D-α-tocopherol succinate prodrug (PV) was synthesized and loaded by α-tocopherol succinate-modified chitosan (CV). Since cathepsin B (Ca.B) is overexpressed in many cancer cells,42 Gly-Phe-Leu-Gly (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 acid-PEG (HA-PEG)


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conjugate to prolong blood circulation and enhance the receptor-mediated endocytosis. Systemic studies were conducted with respect to the drug loading efficiency and capacity, stability, cellular uptake, cytotoxicity, receptor-mediated endocytosis, endocytic pathways, in vitro targetability, in vivo pharmacokinetics/biodistribution and anticancer efficacy of as-prepared enzymatically activatable nanoparticles.

2. MATERIALS AND METHODS 2.1. Materials Sodium hyaluronate (HA, MW=9000 Da) was obtained from Frida biological engineering Co. LTD (Shandong, China). Water-soluble chitosan-oligosaccharide (Mn=5000 Da) was purchased from heowns biochem technologies, Co., LTD (Tianjing,

China).

mPEG

amine-5000,

D-α-tocopherol

succinate

(VES),

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), 4-dimethylaminopyridine (DMAP),

N-hydroxysuccinimide

(NHS)

and

dicyclohexylcarbodiimide

were

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.

Ethyl-(2S,3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)-butylcarbamoyl]-2-oxiran ecarboxylate

(E64-d)

was

obtained

from

Abcam

(Shanghai,

China).

Bis-para-nitrophenylphosphate (BNPP), chlorpromazine hydrochloride, colchicine, methyl-β-cyclodextran, porcine liver esterase (PLE) and bovine spleen Ca.B were obtained from Sigma-Aldrich (Shanghai, China). The NIR dye (Cy7-NHS) was



purchased from Lumiprobe Co., LTD (Florida, USA). MDA-MB-231 (breast cancer cells), MCF-7 (breast cancer cells) and NIH-3T3 (fibroblasts) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). MDA-MB-231/PTX (PTX resistant breast cancer cells) was purchased from Cellbio Inc. (Shanghai, China). Female BALB/c normal mice and nude mice (5-6 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Cell Counting Kit-8 (CCK-8) 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 Sigma-Aldrich (Shanghai, China) and used without further purification. Paclitaxel-α-tocopherol succinate prodrug (PV)43 and HA-PEG polymer with 5 PEG side chains per 100 suger residues of HA22 were synthesized according to the previously reported methods. 2.2. Synthesis and characterization of VES-chitosan (CV) and VES-GFLG-chitosan (GFLGCV) 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 two-step 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 6mL DMSO for 2h. Then GFLG tetrapeptide (0.51 g, 1.30 mmol) was added to the 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

D-α-tocopherylsuccinylglycylphenylleucylglycine

(VES-GFLG) (yield: 63%). Secondly, VES-GFLG 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%. The fluorescent labelling of RBITC and Cy7 with CV or

GFLG

CV polymer was

realized by the reaction of RBITC or Cy7-NHS (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. The VES content in CV or

GFLG

CV conjugate, the number of PEG side chains in

HA-PEG polymer and the structure of PV prodrug was determined using 1H NMR (Avance 400, Bruker, Germany).

The amount of rhodamine or Cy7 residue in the

conjugates was determined fluorometrically using an F-4600 fluorescence spectrometer (Hitachi, Japan). 2.3. Cell culture and animal models The human breast cancer cells (MDA-MB-231, MDA-MB-231/PTX and MCF-7)



and murine fibroblasts (NIH-3T3) 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. 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 MDA-MB-231/PTX murine tumor model was established by subcutaneously injecting MDA-MB-231/PTX cells (0.1 mL, 1×106 cells) into the right backside of female BALB/c nude mice (18±2 g, 5-6 weeks old). Tumors were allowed to grow up to 50 mm3 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 O

O HO

EDC, NHS

O

HO

O NH2

O

O x HO

y O

Chitosan

O

O

NH O

O

O

VES

O

CV

HO O NH

EDC, NHS

O OH

OH

HN HO

O O HN

O HO

NH2

NH

O NH2

O

O x HO

O O

O

O

GFLG

NH

HN

O

O

EDC, NHS

O HN O

y

NH

O

N H

O

HN

Chitosan

O O

O

HN

VES-GFLG

N H

O

B

O O

O

GFL

O O

GC

V

H N O

O

HO O

O OH

N H O

EDC, NHS H O

O

OH O O

VES

O

O O O O

O O

O

OHO

HO H O

PTX O

PV

O

O

Scheme 1. The synthetic routes of CV, GFLGCV 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, CV or

GFLG

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 2`-hydroxyl of PTX.44 HA-PEG copolymer were synthesized with HA and amine functionalized PEG (Mn=5000) according to the literature reported method.22 Since the molecular weight of HA significantly influence the in vivo fate of HA-modified particles,45 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 VES-GFLG and

chitosan, respectively. As for the synthesis of GFLGCV, 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 VES-GFLG. Then the Ca.B responsive GFLG

CV polymer was obtained by the conjugation of VES-GFLG and chitosan in the

presence of EDC and NHS. The relatively low yield of both CV and GFLGCV could be attributed to the precipitating process in acetone. Since VES can be dissolved in acetone, the conjugates with high VES content would form reverse-phase micelles and be difficult to separate. The structures of PV, VES-GFLG, CV and

GFLG

CV and

the stability of GFLG tetrapeptide were confirmed by 1H 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 VES-GFLG 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 micelle’s stability upon dilution. In order to determine the CMC of CV and

GFLG

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 mid-points of the fluorescent ratio plots were used to determine IC50 values).46 The CMC values were calculated to be 5.5×10-3 mg/mL and 5.2×10-3 mg/mL for CV and GFLGCV, respectively. 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 to realize the efficient delivery. Different from the literature,43 PV alone was able to self-assemble 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 HA-PEG polymer was coated on nanoparticle surfaces by electrostatic absorption. The physicochemical properties of as-prepared nanoparticles were characterized with TEM and DLS. As shown in Figure S2 & S3, compared with free PV, the addition of CV or

GFLG

CV polymers and PV prodrug into water yielded uniform nanoparticles

with Rh ranged from 10 nm and to 45 nm, indicating the successful encapsulation of prodrug by the polymers. For PV loaded CV nanoparticles, the Rh increased from 14 nm to 45 nm with increase of the feeding ratio of PV and CV polymer. However, the size of PV loaded

GFLG

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 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 chitosan-α-tocopherol succinate nanoparticles whose drug loading efficiency and drug loading capacity were 65.0 ± 4.2% and 7.7 ± 0.7%, respectively.47 It has been reported in the literature that VES-DOX prodrug can self-assembly with TPGS2k to form stable nanoparticles with lamellar structure and thus high drug loading capacity up to 34%.48 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 forces for the self-assembly of the PV and CV or

GFLG

CV polymers are hydrophobic

associations, with PTX interacting with PTX, PTX with VES, or VES with VES.43 For better investigation of the influence of CV and

GFLG

CV polymers on the efficacy

of nanoparticles, nanoparticles fabricated with 10 mg CV (CVPV, 24 nm, Figure 1B) or

GFLG

CV (GFLGCVPV, 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 as-prepared 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.49 HA coating has been proved to be an efficient way to enhance the active targeting ability of chitosan nanoparticles towards cancer cells which overexpress CD44.37, 50 However, some HA receptors were also overexpressed in normal organs such as liver or spleen,25,

26

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 uptake and achieve long blood circulation and efficient tumor accumulation.27,

51

Herein, for balancing long circulation and efficient cellular uptake, HA polymer containing 5% PEG side chains was used to protect prodrug loaded chitosan nanoparticles.51 As shown in Figure 1C & E, the Rh of CVPV and

GFLG

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 modification, CVPV (43.15 mV) and

GFLG

CVPV (40.25 mV) nanoparticles showed

strong positive charge due to the amino residues on chitosan chain (Figure 1F). However, after the coating of HA-PEG, the zeta potential of corresponding nanoparticles decreased to 2.17 mV and 5.18 mV, respectively, consistent with the zeta potential of PEG.52 This result further demonstrated the feasibility of surface modification via electrostatic absorption. Except for altering the zeta potentials of nanoparticles, the HA-PEG coating also did not influence the drug loading capacity but increase the serum stability of as-prepared nanoparticles (Figure S4). It should be noted that the HA-PEG concentration played important roles in the successful coating, 0.1 mg/mL HA-PEG solution yielded successful coating, but higher concentration



caused the aggregation and precipitation of nanoparticles. The final HA-PEG content on the nanoparticle surface was about 0.08 mg and 0.07 mg HA-PEG per 1 mg CV and

GFLG

CV, respectively, according to the Stain All assay53 of the supernatant after

removing nanoparticles by centrifugation.

Figure 1. DLS and TEM results of PV (A), CVPV (B), CVPV@HA (C), GFLGCVPV (D) and

GFLG

CVPV@HA (E) nanoparticles, scale bar represents 100nm. (F) Zeta potential

change of CVPV and GFLGCVPV nanoparticles before and after the coating of HA-PEG polymer. ***P

CD44-Targeted Facile Enzymatic Activatable Chitosan Nanoparticles

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