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Anisamide-Decorated pH-Sensitive Degradable Chimaeric Polymersomes Mediate Potent and Targeted Protein Delivery to Lung Cancer Cells Ling Lu, Yan Zou, Weijing Yang, Fenghua Meng, Chao Deng, Ru Cheng, and Zhiyuan Zhong Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00193 • Publication Date (Web): 04 May 2015 Downloaded from http://pubs.acs.org on May 10, 2015

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Anisamide-Decorated pH-Sensitive Degradable Chimaeric Polymersomes Mediate Potent and Targeted Protein Delivery to Lung Cancer Cells

Ling Lu, Yan Zou, Weijing Yang, Fenghua Meng*, Chao Deng, Ru Cheng, and Zhiyuan Zhong*

Biomedical Polymers Laboratory, and Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, P. R. China.

*

Corresponding authors. Tel/Fax: +86-512-65882060, Email: [email protected] (F. Meng); [email protected] (Z. Zhong)

Abstract In spite of their high potency and specificity, few protein drugs have advanced to the clinical settings due to lack of safe and efficient delivery vehicles. Here, novel anisamide-decorated pH-sensitive degradable chimaeric polymersomes (Anis-CPs) were designed, prepared and investigated for efficient and targeted delivery of apoptotic protein, granzyme B (GrB), to lung cancer cells. Anis-CPs were readily prepared with varying Anis surface

densities

from

anisamide

end-capped

poly(ethylene

glycol)-b-poly(2,4,6-

trimethoxybenzylidene-1,1,1-tris(hydroxymethyl)ethane methacrylate)-b-poly(acrylic acid) (Anis-PEG-PTTMA-PAA) and PEG-PTTMA-PAA copolymers. Using cytochrome C (CC) as a model protein, Anis-CPs displayed high protein loading efficiencies (40.5-100%) and loading contents (up to 16.8 wt.%). CC-loaded Anis-CPs had narrow distribution (PDI 1

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0.04-0.13) and small sizes ranging from 152 to 171 nm, which increased with increasing CC contents. Notably, the release of proteins from Anis-CPs was accelerated under mildly acidic conditions, due to the hydrolysis of acetal bonds in PTTMA. MTT assays showed that GrB-loaded Anis-CPs (GrB-Anis-CPs) displayed high targetability to sigma receptor over-expressing cancer cells such as H460 and PC-3 cells. For example, GrB-Anis-CPs exhibited increasing antitumor efficacy to H460 cells with increasing Anis contents from 0 to 80%. The antitumor activity of GrB-Anis-CPs was significantly reduced upon pretreating H460 cells with haloperidol (a competitive antagonist). Notably, the half-maximal inhibitory concentrations (IC50) of GrB-Anis70-CPs were determined to be 6.25 nM and 5.94 nM for H460 and PC-3 cells, respectively, which were 2-3 orders of magnitude lower than that of chemotherapeutic drugs such as paclitaxel. Flow cytometry studies demonstrated that GrB-Anis70-CPs induced widespread apoptosis of H460 cells. The confocal laser scanning microscopy (CLSM) experiments using FITC-labeled CC-loaded Anis-CPs confirmed fast internalization and intracellular protein release into H460 cells. GrB-Anis-CPs with high potency and specificity are particularly interesting for targeted therapy of lung cancers. Keywords: Polymersomes; apoptotic proteins; pH-sensitive; lung cancer; tumor targeting

Introduction Many cancers originates from a deficiency or malfunction in somatic proteins participating in cellular homeostasis. Therapies have been developed that treat cancers by silencing abnormal cell signaling patterns using therapeutic proteins as inhibitors.1-4 As compared to chemotherapeutics, protein drugs have unique advantages such as high specificity, superior anticancer efficacy and low side effects. Many apoptotic proteins like TRAIL, TNF-α, cytochrome C (CC), apoptin, caspase 3, Heceptin, ricin, saporin and granzyme B (GrB) have been investigated for cancer therapy.5-10 For instance, GrB is a serine 2


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protease stored in the granules of activated cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, which are the two main types of cytotoxic effector cells of the immune system.11, 12

Upon target cell contact, GrB is secreted by the engaged CTLs and NK cells and

translocated into the cytosol of target cells. Through direct processing of certain caspases which leads to their autoactivation, induction of reactive oxygen species (ROS) or indirectly proteolysis of Bid, a protein that promotes mitochondrial permeabilization and consequent activation of the apoptosome pathway to caspase activation, GrB promotes apoptosis of target cells.13, 14 Meanwhile exogenous GrB was reported to be a highly potent apoptosis mediator in cancer cells.15-17 Nevertheless, to realize high therapeutic efficacy, protein drugs need to overcome several challenges including rapid degradation, poor bioavailability, elimination following i.v. injection, potential immune response, and low cell permeability. Most proteins exert their apoptotic functions in certain cellular compartment, e.g. TRAIL, apoptin and HEMLET proteins taking effect in the cell nuclei18 and CC, saporin and GrB in the cytosol.8 In the past decade, a growing number of innovative strategies have been explored to deliver proteins to the targets.7-10, 19-21 In particular, polymersomes (also referred to as polymer vesicles) with a diameter in the range of virus sizes and a natural watery core to accommodate proteins have appeared as one of the most ideal carriers for protein delivery.5,

22-25

As

compared to liposomes, polymersomes have several advantages including thicker membrane, better stability, and inherent non-fouling properties. However, they usually exhibit slow drug release at the site of action. To improve their drug release, great effort has been directed to the development of smart polymersomes,26-30 which release payloads in response to an internal signal like cytoplasmic glutathione, enzyme and endo/lysosomal pH

31-35

or external stimulus

such as photo and magnetic.36-38 Similar to liposomes, common polymersomes display a very low loading efficiency and loading content toward hydrophilic substances including proteins, which restricts their applications as protein chaperones. In recent years, we have demonstrated

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that chimaeric polymersomes (CPs) self-assembled from asymmetric ABC triblock copolymers could efficiently load hydrophilic drugs like proteins and doxorubicin hydrochloride (DOX·HCl).5,

17

pH-Sensitive degradable CPs based on poly(ethylene

glycol)-b-poly(2,4,6-trimethoxybenzylidene-1,1,1-tris(hydroxymethyl)ethane

methacrylate)

-b-poly(acrylic acid) (PEG-PTTMA-PAA) were shown to effectively load and deliver DOX·HCl into HeLa cells, resulting in high antitumor efficacy.39 Equally important, polymersomes should be equipped with a tumor targeting ligand, such as antibody, peptide, folic acid, aptamers or lactoferrin, to achieve cell-specific and efficient protein delivery.40-48 We recently reported that galactose-decorated reduction-sensitive CPs could efficiently load and chaperone GrB into hepatoma cells resulting in high apoptosis of cancer cells.17 In this study, we designed anisamide-functionalized pH-sensitive degradable chimaeric polymersomes (Anis-CPs) based on Anis-PEG-PTTMA-PAA and PEG-PTTMA-PAA triblock copolymers for efficient loading and delivery of GrB to non-small cell lung cancer (NSCLC) cells (Scheme 1). Lung cancer is among the most lethal malignancies with a high metastasis and recurrence rate. NSCLC accounts for approximately 85 % of all lung cancers. The prognosis of NSCLC metastasis remains poor and chemotherapy provides only minimal gains in overall survival rates, resulting in a 5-year survival rate of < 15%.49, 50 Anisamide (Anis) ligands show high affinity to sigma receptor, a membrane bound protein known to over-express on many human malignancies including lung cancers and prostate cancers, and have been applied for sigma receptor-mediated delivery of doxorubicin, platinum, proteins or siRNAs.19, 51-53 Our results have shown that GrB-loaded Anis-CPs caused superior anti-tumor effects with a particularly low IC50 of 3.75 nM toward H460 cells. Here, the preparation of anis-installed multifunctional polymersomes, loading of proteins, pH-triggered protein release, targetability and in vitro anti-tumor activity to NSCLC cells were investigated.

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Scheme 1. Schematic illustration of anisamide-functionalized pH-sensitive degradable chimaeric polymersomes (CPs) for efficient loading and targeted delivery of GrB to sigma receptor overexpressing non-small cell lung cancer (NSCLC) cells.

Experimental Section Materials. Acrylic acid (AA, 99%, Alfa Aesar) was purified by distilling under reduced pressure. 2, 2’-Azobisisobutyronitrile (AIBN, 98%, J&K) was re-crystallized twice from methanol. N,N-Dimethylformamide (DMF) was dried by MgSO4 and distilled under reduced pressure. 1,4-dioxane was dried by refluxing over sodium wire under an argon atmosphere and distilled prior to use. α-Amino-ω-hydroxyl poly(ethylene glycol) hydrochloride (HO-PEG-NH2·HCl, Mn = 7.5 kg/mol, ≥95%, Beijing Jenkem Technology Co. Ltd.), triethylamine (99%, Alfa Aesar), dicyclohexyl carbodiimide (DCC, 99%, Alfa Aesar), N-hydroxysuccinimide (NHS, 98%, Alfa Aesar), 4-dimethyl aminopyridine (DMAP, 99%, Alfa Aesar), p-anisic acid (99%, J&K), haloperidol (98%, J&K), fluorescein isothiocyanate (FITC, 95%, Fluka), cytochrome C from equine heart (Sigma), recombinant human granzyme 5


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B (GrB, Biovision), and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)diammonium salt (ABTS, Amresco) were used as received. Annexin V-FITC/propidium iodide (PI, KeyGEN tech.) was used according to the supplier’s instruction. Human large cell lung cancer cells (NCL-H460, H460), human prostate carcinoma cells (PC-3) and human breast adenocarcinoma cell (MCF-7) were obtained from American Type Culture Collection (ATCC). 4-Cyanopentanoic acid dithionaphthalenoate (CPADN) and 2,4,6-trimethoxybenzylidene -1,1,1-tris(hydroxymethyl)ethane methacrylate (TTMA) were synthesized according to the previous reports.39, 54, 55 Characterizations. 1H NMR spectra were recorded on a Unity Inova 400 spectrometer operating at 400 MHz using deuterated chloroform (CDCl3) as a solvent. The molecular weight and polydispersity (PDI) of copolymers were determined with a Waters 1515 gel permeation chromatography (GPC) instrument equipped with three ultra-hydrogel columns following an INLINE precolumn and a differential refractive-index detector. The measurements were performed using DMF containing 1 wt.% LiBr as an eluent at a flow rate of 0.8 mL/min at 30oC and a series of narrow polystyrene standards for the calibration of the columns. The size and size distribution of polymersomes were determined using dynamic light scattering (DLS) at 25oC using a Zetasizer Nano-ZS from Malvern Instruments equipped with a 633 nm He–Ne laser using back-scattering detection. Transmission electron microscopy (TEM) was performed using a Tecnai G220 TEM operated at an accelerating voltage of 120 kV. The samples were prepared by dropping 10 μL of polymersomes dispersion (0.2 mg/mL) on the copper grid followed by staining with phosphotungstic acid. The images of polymersomes and cellular uptake were taken on a confocal laser scanning microscope (CLSM, Leica, TCS-SP5). Synthesis of Anis-PEG-CPADN. Anis-PEG-CPADN macro-RAFT agent was synthesized by reacting HO-PEG-NH2 (Mn 7.5 kg/mol) with N-hydroxysuccinimide (NHS) activated

6


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p-anisic acid (Anis-NHS) and CPADN (Scheme S1). Anis-NHS was obtained with high purity following crystallization from 2-propanol (Figure S1A). Under a nitrogen atmosphere, 2 mL of Anis-NHS (11.0 mg, 0.044 mmol) in DCM was added dropwise to a DCM solution (3 mL) of HO-PEG-NH2·HCl (300 mg, 0.04 mmol) and triethylamine (4.45 mg, 0.044 mmol) at 0oC under constant stirring. After completion of addition, the reaction was allowed to proceed at room temperature (r.t.) for 20 h. The resulting Anis-PEG-OH was recovered by precipitation in cold diethyl ether/ethanol (99/1, v/v) and dried in vacuo for 48 h. Yield: 95%. 1H NMR (400 MHz, CDCl3, Figure S1B): δ 7.77 (ArH-CO-), 6.90 (ArH-OCH3), 3.84 (ArH-OCH3), 3.63 (PEG). The degree of substitution (DS) of anisamide was nearly 100% as calculated from the peak integral ratio of phenyl proton at δ 7.77 to methylene protons at δ 3.63. Under a nitrogen atmosphere, a DCM solution (3 mL) of CPADN (29.6 mg, 0.09 mmol) and DCC (37.2 mg, 0.18 mmol) was stirred over night at r.t.. DMAP (5.5 mg, 0.045 mmol) and Anis-PEG-OH (172.0 mg, 0.022 mmol) in 2 mL DCM was added dropwise. After completion of addition, the reaction was allowed to proceed for another 20 h. The final Anis-PEG-CPADN was recovered by precipitation in cold diethyl ether/ethanol (99/1, v/v) and dried in vacuo. Yield: 80.5%. 1H NMR (400 MHz, CDCl3): δ7.90 and 7.50 (naphthalene), 7.78 (ArH-CO-), 6.90 (ArH-OCH3), 4.27 (-OCH2CH2OCO-), 3.84 (ArH-OCH3), 3.63 (PEG) and 1.99 (-C(CN)(CH3)-S-). Synthesis of Anis-PEG-PTTMA-PAA. Anis-PEG-PTTMA-PAA triblock copolymers were synthesized by RAFT polymerization of TTMA and AA. Under a nitrogen atmosphere, TTMA (0.154 g, 0.42 mmol), Anis-PEG-CPADN (50 mg, 6.7 μmol), AIBN (0.16 mg, 1.0 μmol) and DMF (2 mL) were added into a 10 mL Schlenk flask and the mixture was degassed with nitrogen for 30 min. The flask was then sealed and placed into an oil bath thermostated at 70oC. After 48 h, an aliquot was taken to determine the TTMA conversion and the rest was precipitated in cold diethyl ether, filtrated and dried in vacuo for 48 h obtaining

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Anis-PEG-PTTMA. Under the same conditions, thus obtained Anis-PEG-PTTMA, AA (14.3 mg, 0.20 mmol) and AIBN (0.16 mg, 0.001 mmol) in 2 mL DMF were polymerized at 70oC for 48 h. Yield: 81.2%. 1H NMR (400 MHz, CDCl3): δ 7.78, 6.90 (Anisamide), 6.03 (aromatic protons), 5.83 (Ar-CH-), 4.17 (-COOCH2C-), 3.89 (-OCH2CCH2O-), 3.75 (Ar-OCH3), 3.65 (PEG), 2.23 (-CHCOOH), 1.87 (-CH2CHCOOH,-CH2C-), 0.59-1.26 (CH3CCOO-, CH3C-). Mn (1H NMR) = 27.9 kg/mol. Mn (GPC) = 29.9 kg/mol, PDI (GPC) = 1.8. The synthesis of PEG-PTTMA-PAA was the same as for Anis-PEG-PTTMA-PAA copolymer except that MeO-PEG-CPADN (Mn,PEG = 5 kg/mol) was used as a macro-initiator. Yield: 89.7 %. Mn (1H NMR) = 21.4 kg/mol. Mn (GPC) = 21.7 kg/mol, PDI (GPC) = 1.4. Formation of Chimaeric Polymersomes. 50 μL of DMSO solution (5 mg/mL ) of Anis-PEG-PTTMA-PAA and PEG-PTTMA-PAA at a predetermined weight ratio was added to 950 μL of phosphate buffer (PB, 5 mM, pH 7.4) without stirring. After the slow solvent exchange process, a bluish milky dispersion was obtained. The polymersomes were purified by extensive dialysis (MWCO 7 kDa) against PB. The resulting polymersomes were denoted as

AnisX-CPs,

wherein

X

represents

the

weight

percentage

(wt.%)

of

Anis-PEG-PTTMA-PAA. The size, size distribution and morphology of polymersomes were determined by DLS and TEM, respectively. The critical aggregation concentration (CAC) was determined using pyrene as a fluorescence probe as described in our previous reports.17, 21 Acid-Triggered Hydrolysis of Acetals in the Polymersomes. The acetal hydrolysis was monitored by UV/vis spectroscopy through measuring the absorbance at 292 nm, which is the characteristic absorbance of the hydrolysis product, 2, 4, 6-trimethoxybenzaldehyde.39 In brief, polymersome (1.0 mg/mL, 1 mL) prepared as above was divided into three parts, whose pH was adjusted to 4.0, 5.0 and 7.4, respectively. The solutions were continuously shaken at 37oC. At the desired time intervals, 80 µL aliquot was taken and diluted with 3.5 mL of PB (0.1 M, pH 7.4), and the absorbance at 292 nm was determined. The absorbance at 292 nm of the

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hydrolysis product after incubating with 50 µL concentrated HCl for 2 h was taken as 100% hydrolysis. The change in size and count rate of the polymersomes in response to acetal hydrolysis at pH 5.0 was followed by DLS. Loading and In Vitro Release of Proteins. 50 μL of DMSO solution (15 mg/mL) of Anis-PEG-PTTMA-PAA and PEG-PTTMA-PAA at a predetermined weight ratio was added to 950 μL of HEPES buffer (5 mM, pH varying from 7.4, 8.0 to 8.5) containing FITC-labeled cytochrome C (FITC-CC) without stirring. The theoretical protein loading contents varied from 1 wt.% to 50 wt.%. After 2 h solvent exchange, the dispersion was extensively dialyzed against the same media (MWCO 350 kDa) for 12 h with 5 times change of dialysis media. Thus obtained protein-loaded polymersome dispersion was collected for further use. In order to determine protein loading content (PLC) and protein loading efficiency (PLE), aliquot of protein-loaded polymersome dispersion was taken, lyophilized, weighed, dissolved in DMSO for 3 h under stirring, and measured with UV/vis spectroscopy (U-3900) at 491 nm (FITC). The amount of loaded protein could be estimated based on a calibration curve established with known FITC-CC concentrations. PLC and PLE were calculated according to the following formula: PLC (wt.%) = (weight of loaded protein/ total weight of copolymer and protein) ×100 PLE (%) = (weight of loaded protein/weight of protein in feed) ×100 The in vitro protein release was investigated using a dialysis tube (MWCO 350 kDa) at 37oC with 0.5 mL of FITC–CC-Anis-CPs (0.5 mg/mL) against 25 mL of PBS (pH 7.4, 10 mM, 100 mM NaCl) or acetate buffer (pH 5.0, 10 mM, 100 mM NaCl). At desired time intervals, 5 mL of release media was taken out and replenished with an equal volume of fresh media. The amount of released proteins as well as proteins remaining in the dialysis tube was determined by fluorescence measurements (FLS920, ex. 491 nm, em. 492 to 690 nm). The release

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experiments were conducted in triplicate, and the results presented are the average data with standard deviations. Bioactivity Assay of Released CC. The electron transfer activity of CC released from the polymersomes

was

measured

by

examining

the

catalytic

conversion

of

2,

2’-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS).5 CC-loaded polymersomes were placed into a dialysis tube (MWCO 350 kDa) and incubated in PB (pH 5.0, 10 mM) for 24 h. The released CC was quantified using BCA protein assay (Pierce) and diluted by PB to a final concentration of 4.0 μg/mL. 100 μL of hydrogen peroxide solution (4 mM) and 100 μL of ABTS solution (1.0 mg/mL) in PB were added. The absorbance at 418 nm of the oxidized product was monitored every 20 s for 4 min. Native CC with the same concentration was used as control. MTT Assays. The anti-tumor activity of GrB-Anis-CP in sigma receptor over-expressing H460 and PC-3 cells was evaluated by MTT assays. The cells were seeded in a 96-well plate (5×103 cells per well) in 100 µL of DMEM medium supplemented with 10% FBS, 1% L-glutamine, antibiotics penicillin (100 IU/mL), and streptomycin (100 µg/mL) for 24 h. The medium was aspirated and replaced by 80 µL of fresh medium. 20 µL of GrB-Anis-CP in PB was added to yield final GrB concentrations from 1×10-4 to 0.4 µg/mL. After 4 h incubation, the media were removed and replaced with fresh culture medium, and the cells were further cultured 68 h for H460 cells and 116 h for PC-3 cells. Then, 10 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) solution in PBS (5 mg/mL) was added, and incubated for 4 h. The medium was aspirated, the MTT-formazan generated by live cells was dissolved in 150 µL of DMSO and the absorbance at 570 nm of each well was measured using a microplate reader (Thermo Muitskan FC). The relative cell viability (%) was determined by comparing the absorbance at 570 nm with control wells containing only cell culture medium. Data are presented as average ± SD (n = 4).

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For inhibition experiments, H460 cells were pre-treated with haloperidol (final concentration 30 μM) for 3 h to block the sigma receptors on the cell surface.53 The media were then aspirated and replaced by fresh cell culture media, and GrB-Anis-CP was added. The cytotoxicity of blank polymersomes, Anis20-CP and Anis100-CP to H460, PC-3 or MCF-7 cells was determined in a similar way at polymer concentrations of 0.01, 0.1, 0.2, 0.5 and 1.0 mg/mL, respectively. Confocal Laser Scanning Microscopy (CLSM) Measurements. FICT-CC was used as a model protein to study the cellular uptake and intracellular protein release behaviors. H460 cells were plated on microscope slides in a 24-well plate (2×104 cells per well) under 5% CO2 atmosphere at 37 oC using 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, antibiotics penicillin (100 IU/mL), and streptomycin (100 µg/mL) for 24 h. H460 cells were incubated for 4 h with 50 µL of FITC–CC–Anis30-CP, FITC–CC–CP or free FITC–CC (FITC–CC dosage: 80 µg/mL). Then the medium was aspirated and replaced by fresh medium. After incubation for another 4 h, the cells on microscope plates were washed three times with PBS, and subsequently fixed with 4% paraformaldehyde for 15 min and washed three times with PBS. The cell nuclei were stained with DAPI (5 µg/mL) for 8 min and washed three times with PBS. Images of cells were obtained using a CLSM (Leica, TCS-SP5). For inhibition experiments, H460 cells were pre-treated with haloperidol (final concentration 30 μM) for 3 h to block the sigma receptors.53 The media were then aspirated and replaced by fresh cell culture media, and GrB-Anis30-CP was added. Flow Cytometry Studies. H460 cells were plated in a 24-well plate (5×104 cells/well) under a 5% CO2 atmosphere at 37°C using 1640 medium supplemented with 10% fetal bovine serum, 1% L-glutamine, antibiotics penicillin (100 IU/mL), and streptomycin (100 μg/mL) for 24 h. The cells were treated with a predetermined amount of GrB-Anis-CP or free GrB (GrB

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dosage: 0.4 μg/mL) under a 5% CO2 atmosphere at 37°C for 4 h. Then, the medium was refreshed and the cells were cultured for another 68 h. To quantify the apoptotic cells, an Annexin V-FITC/propidium iodide (PI) kit was used as described by the manufacturer (KenGEN, China). Briefly, cells were digested with EDTA-free trypsin, collected with centrifugation (2000 rpm, 5 min), washed twice with cold PBS, and re-suspended in binding buffer (100 μL) at a concentration of 1×105 cells/mL. The cells were stained with 5 μL of Annexin V-FITC solution and 5 μL of propidium iodide (PI) solution for 15 min at r.t. in the dark. Then, 400 μL of binding buffer was added, and the cells were analyzed immediately using flow cytometry (BD FACSCalibur, Mountain View, CA).

Results and Discussion Synthesis of Anis-PEG-PTTMA-PAA. The aim of this study was set to develop a novel protein delivery vehicle that can efficiently load and protect potent protein drugs, actively target to non-small cell lung carcinoma (NSCLC) cells, and quickly release protein drugs inside the target cells. To this end, anisamide-directed pH-sensitive degradable chimaeric polymersomes (Anis-CPs) were designed and prepared from Anis-PEG-PTTMA-PAA and PEG-PTTMA-PAA triblock copolymers, in which the PEG in Anis-PEG-PTTMA-PAA was devised longer than that in PEG-PTTMA-PAA (7.5 vs. 5.0 kg/mol) to achieve optimal targeting effect of Anis ligands toward the sigma receptors in NSCLC cells and PEG longer than PAA to ensure that PAA is preferentially located in the watery core of the vesicles. Anis-PEG-PTTMA-PAA

was

readily

synthesized

from

sequential

reversible

addition-fragmentation chain transfer (RAFT) polymerization of TTMA and AA using Anis-PEG-CPADN as a macro-RAFT agent (Scheme 2). Anis-PEG-CPADN was obtained by reacting HO-PEG-NH2⋅HCl (Mn = 7.5 kg/mol) with NHS pre-activated anisic acid via amidation reaction followed by esterification reaction with CPADN (Scheme S1, Figure 1S). 12


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CPADN is a versatile RAFT agent, through which we have prepared a series of well-defined di- and tri-block copolymers such as PEG-P(HEMA-co-AC) and PEG-PAA(SH)-PDEA.21, 31 1

H NMR of Anis-PEG-CPADN showed besides resonances of PEG (δ 3.63), signals at δ 7.90,

7.50 and 1.99 attributable to CPADN moieties and δ 6.90 and 7.70 assignable to Anis moieties (Figure S2A). Notably, signals at δ 7.50 and δ 6.90 had an integral ratio close to 2:1, indicating equivalent coupling of CPADN and Anis to PEG. The RAFT polymerization of TTMA and AA was carried out in DMF at 70 oC for 3 and 2 d, respectively. 1H NMR showed besides peaks of PEG at δ 3.63 characteristic signals of PTTMA block at δ 3.75, 5.83 and 6.03 as well as PAA block at δ 1.87-2.23 (Figure S2B). The number average molecular weight (Mn) of PTTMA and PAA blocks in Anis-PEG-PTTMA-PAA were calculated to be 18.4 and 2.0 kg/mol by comparing the integrals of signals at δ 5.83 (acetal methine proton of PTTMA) and δ 1.87-2.23 (PAA) to that of PEG methylene protons at δ 3.63, respectively. It is important to note that resonances attributable to the Anis ligand at δ 6.90 and 7.78 remained unchanged. GPC measurements displayed a unimodal molecular weight distribution and an Mn of 29.9 kg/mol (Table 1), which was close to that determined by

1

H NMR.

Anis-PEG-PTTMA-PAA triblock copolymer was, therefore, acquired with a defined structure and controlled molecular weight. PEG-PTTMA-PAA triblock copolymer, which was used as a non-targeting counterpart, was prepared, as described in our previous report39 with an Mn of 5.0-14.4-2.0 kg/mol as determined by 1H NMR and 21.7 kg/mol by GPC measurement.

13


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O O

O O

O

O O

O

N H

NC

O O

TTMA

S

O

O

S

Anis-PEG-CPADN O OH

O

AA

O

O

N H

m

HO

O

O

O

O

Anis-PEG-PTTMA-PAA

* p O

n O

O

O

Scheme 2. Synthesis of Anis-PEG-PTTMA-PAA. Conditions: (i) DMF, AIBN, 70 oC, 2d; and (ii) DMF, AIBN, 70 oC, 3d.

Table 1. Characterization of PEG-PTTMA-PAA and Anis-PEG-PTTMA-PAA

a

Entry

Polymers

Design

1

PEG-PTTMA-PAA

5.0-16.0-3.0

2

Anis-PEG-PTTMA-PAA

7.5-20.0-3.0

Mn (kg/mol) 1 H NMRa

GPCb

Mw/Mnb

5.0-14.4-2.0

21.7

1.4

7.5-18.4-2.0

29.9

1.8

Determined by comparing the integrals of signals at δ 5.83 (acetal methine proton of PTTMA) and

1.92 (methine proton of PAA), respectively, with that of PEG methylene protons at δ 3.65. b

Using polystyrene standards, DMF as eluent at a flow rate of 0.8 mL/min and 30 °C.

Formation of anis-installed pH-sensitive chimaeric polymersomes (Anis-CPs). Anis-CPs were prepared with different Anis surface densities from co-assembly of Anis-PEG-PTTMA-PAA and PEG-PTTMA-PAA at 20 wt.%, 30 wt.%, 50 wt.%, 70 wt.% and 100 wt.% Anis-PEG-PTTMA-PAA using a solvent displacement method. The resulting polymersomes were denoted as AnisX-CPs, wherein X represents the weight percentage of Anis-PEG-PTTMA-PAA. DLS measurements showed that Anis-CPs had low polydispersities (PDI = 0.04 - 0.11) and small hydrodynamic sizes of 110 - 150 nm, which increased with increasing Anis contents (Figure 1A). TEM micrograph displayed a vesicular structure and an 14


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average size of 100 nm (Figure 1B), close to that determined by DLS. CLSM of large polymersomes simultaneously encapsulated with DOX·HCl (hydrophilic) and nile red (hydrophobic) clearly demonstrated their colocalization, with fluorescence intensity profiles in accordance with location of DOX·HCl in the lumen and nile red in the membrane. Zeta potential measurements revealed that all CPs had a close to neutral surface charge (0.40-0.98 mV), in accordance with selective positioning of PEG and PAA at the outer and inner side of vesicles, respectively. The critical aggregation concentration (CAC) of polymersomes was low (ca. 1.35 mg/L).

30

A

Anis-CPs Anis20-CPs Anis30-CPs Anis50-CPs Anis70-CPs Anis100-CPs

25 Intensity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

20 15

B

10 5 0 100

Size (nm)

1000

Figure 1. Size distribution profiles of Anis-CPs determined by DLS (A) and TEM micrograph of Anis20-CPs (staining with 1 wt.% phosphotungstic acid) (B).

The stability and pH-responsivity of Anis-CPs were studied by DLS. The results showed that Anis20-CPs were sufficiently stable with little size changes in 52 h under physiological conditions (PB, pH 7.4, 37 oC) (Figure 2A), in line with their low CAC. The size and count rate of Anis20-CPs, however, changed rapidly at pH 5.0, in which particle sizes increased to over 1000 nm in 11 h with concomitant decrease of count rates (Figure 2B). After 52 h, Anis-CPs were dissociated into unimers (7-8 nm), indicating complete hydrolysis of acetal bonds. The degradation of Anis20-CPs was monitored by UV at 292 nm. The results showed that the acetal hydrolysis rate of Anis-CPs was highly pH dependent, with a half-life of ca. 4.6

15


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and 13 h at pH 4.0 and 5.0, respectively, at 37 oC (Figure 2C). In contrast, only ca. 7 % acetal bonds were hydrolyzed in 52 h at pH 7.4 under otherwise the same conditions. 50

pH 5.0, 8 h pH 5.0, 12 h pH 5.0, 52 h pH 7.4, 0 h pH 7.4, 52 h

A

Volume (%)

40 30 20 10 0 1

10

100 1000 Size (nm)

10000

B 5000 4000 Size (nm)

200

Size Count Rate

150

3000 100 2000 50

1000 0

5

10 15 20 Time (h)

25

0

30

C

120 Hydrolysis (%)

0

Count Rate (kcps)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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pH 4.0

100

pH 5.0

80 CPs Anis20-CPs Anis100-CPs

60 40 20 0

pH 7.4 0

10

20 30 Time (h)

40

50

16


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Figure 2. Change in size (A) and count rate (B) of Anis20-CPs with time at pH 5.0 (acetate buffer, 60 mM) at 37 oC. (C) pH-Dependent hydrolysis of acetals in Anis70-CPs, Anis20-CPs and CPs at 37 oC.

Encapsulation and pH-Triggered Release of Proteins. We have shown previously that chimaeric polymersomes afford high protein loading contents (PLC) and loading efficiencies.5, 17

Here, using FITC-labeled cytochrome C (FITC–CC) as a model protein, protein-loaded CPs

were prepared by solvent exchange method as described above. Notably, aqueous pH was shown to have an influence on the protein loading efficiency (PLE), in which loading efficiencies of 67.9%, 62.3%, and 57.3% were observed at pH 8.5, 8.0, and 7.4, respectively. This is likely due to a better electrostatic interaction and/or hydrogen bonding between proteins and PAA at higher pH values. In the following, the loading of FITC–CC was investigated at varying protein feed ratios from 1–50 wt.% at pH 8.5. Remarkably, the results showed a quantitative loading of FITC–CC at a protein feed ratio of 1 wt.% and a PLE of 87.6% at 5 wt.% (Table 2). PLE was 40.5% even at a high protein feed ratio of 50 wt.%, which gave a notable PLC of 16.8 wt.%. The sizes of FITC-CC loaded Anis20-CPs increased from 152 to 171 nm with increasing PLC from 0.98 to 16.8 wt.% while PDI remained low (0.06–0.13).

Table 2. Characteristics of FITC-CC loaded Anis20-CPs.

a

Entry

media pH

Feed ratioa (wt.%)

Size b (nm)

PDI b

PLC (wt.%)

PLE (%)

1

7.4

10

153

0.09

5.4

57.3

2

8.0

10

155

0.04

5.9

62.3

3

8.5

1

152

0.07

0.98

~100

4

8.5

5

155

0.06

4.2

87.6

5

8.5

10

158

0.10

6.4

67.9

6

8.5

20

169

0.09

9.0

49.7

7

8.5

50

171

0.13

16.8

40.5

Feed ratio = (weight of CC/weight of polymer)×100%; 17


Biomacromolecules

b

Determined at 25 °C using DLS in PB buffer (pH 7.4, 10 mM).

The in vitro release studies showed that FITC-CC release from Anis-CPs was much faster at pH 5.0 than at 7.4 (Figure 3A). For example, 54.3% and 22.7% of FITC–CC was released from FITC-CC-Anis70-CPs in 24 h at pH 5.0 and pH 7.4, respectively. This is in accordance with triggered degradation of PTTMA membranes. It should further be noted that Anis densities had not much influence on the protein release profile. The release rate of FITC-CC was slower than that of DOX·HCl,39 which is likely due to the fact that protein has a much larger size than small molecule drugs. Importantly, ABTS assay revealed that released CC exhibited almost the same enzymatic activity as native CC in oxidizing ABTS (Figure 3B), indicating that CC released from Anis-CPs has fully maintained its biological activity. A

CPs Anis20-CPs Anis70-CPs

60

0.9

pH 5.0 40 pH 7.4

20 0

B Released CC Native CC

0.8 Abs at 418 nm

80 Cumulative Release (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.7 0.6 0.5 0.4 0.3

0

5

10

15 Time (h)

20

25

0

50

100

150

200

250

300

Time (sec)

Figure 3. Protein release from Anis-CPs. (A) FITC-CC release from FITC-CC-Anis-CPs at pH 5.0 or pH 7.4 at 37 oC; and (B) Oxidation of ABTS catalyzed by CC released from CC-Anis-CPs and native CC in PB (pH 7.4, 5 mM).

Cellular Uptake and Intracellular Protein Release. In the following, confocal laser scanning microscopy (CLSM) was applied to study the internalization and intracellular drug release behaviors of protein-loaded Anis-CPs in sigma receptor over-expressing H460 cells (Figure 4, Figure S3). The results showed that strong FITC fluorescence was observed inside

18


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the H460 cells following 8 h incubation with FITC-CC-Anis30-CPs (Figure 4A). In contrast, weak fluorescence was detected in the H460 cells treated with non-targeting polymersomes (Figure 4B) and little fluorescence in the cells incubated with free FITC-CC (Figure 4C). To further confirm the targetability of Anis-CPs, haloperidol, also specifically recognized by the sigma receptors on the cell surface,52, 53 was used to block the sigma receptors. The results revealed that the cellular level of FITC-CC was significantly reduced upon pre-treating H460 cells with haloperidol (Figure 4D). These observations confirmed that FITC-CC-Anis-CPs were uptaken by H460 cells via a receptor mediated endocytosis mechanism and FITC-CC is quickly released into the cells. A

B

C

D

Figure 4. CLSM images of H460 cells following 8 h incubation with FITC-CC-CPs or free FITC-CC. (A) FITC-CC-Anis30-CPs; (B) non-targeting FITC-CC-CPs; (C) free FITC-CC; and (D) H460 cells were pre-treated with haloperidol (30 μM) for 3 h before adding FITC-CC-Anis30-CPs. FITC-CC concentration was set at 80 μg/mL. For each panel, images from left to right were nuclei stained by DAPI (blue), FITC-CC (green), overlays of two images. The scale bar represents 20 μm. 19


Biomacromolecules

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Anti-Tumor Activity of GrB-Anis-CPs. Here, GrB, a highly potent apoptosis mediator, was employed as a therapeutic protein to study the targetability and protein delivery efficiency of Anis-CPs. It has been demonstrated that exogenous GrB could effectively kill human tumor cells.15, 17 Using the same loading method as for CC, GrB with a pKa of 1056 was encapsulated into Anis-CPs at a protein feed ratio of 0.4 wt.%. The loading of GrB was assumed to be quantitative. The sizes of GrB-Anis-CPs increased from 145 to 179 nm with increasing Anis-PEG-PTTMA-PAA contents from 20 to 100 wt.% (Table S2). Notably, all GrB-Anis-CPs had a low PDI (0.01-0.06). The targetability and anti-tumor activity of GrB-Anis-CP were investigated using MTT assays and flow cytometry in sigma receptor over-expressing H460 cells and human prostate carcinoma cells (PC-3). As shown in Fig. 5, GrB-Anis-CPs caused significantly higher killing effects than free GrB as well as non-targeting GrB-CP counterparts in both H460 and PC-3 cells. The low antitumor activity of free GrB is likely due to its poor cellular internalization. It is interesting to note that GrB-Anis-CP provoked increased antitumor effect to H460 and PC-3 cells with increasing Anis-PEG-PTTMA-PAA contents from 0 to 80 wt.%, but decreased upon further increasing Anis-PEG-PTTMA-PAA content to 100 wt.%. For instance, at a GrB dosage of 0.4 μg/mL and 72 h incubation, GrB-Anis-CPs with 20, 30, 50, 70, 80 and 100 wt.% Anis-PEG-PTTMA-PAA induced ca. 36.3%, 40.4%, 45.8%, 56.1%, 61.6%, and 42.2% of H460 cell death, respectively (Figure 5A). In contrast, much less cell death (21.4 %) was observed for H460 cells treated with non-targeting GrB-CPs. The half-maximal inhibitory concentration (IC50) values of GrB-Anis80-CP and GrB-Anis70-CP were calculated to be ca. 0.12 μg/mL (3.75 nM) and 0.20 μg/mL (6.25 nM), respectively. This high antitumor activity of GrB has also been reported for GrB-loaded galactose-directed reduction-sensitive chimaeric polymersomes to HepG2 cells (ca.2.7 nM),17

20


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and GrB microinjected into MCF-7 cells (0.04 μg/mL GrB led to 40 % apoptosis).15 Notably, GrB-Anis80-CP had a much lower IC50 than tumor targeting antibody-fused GrB which showed an IC50 of 20 nM in human A375-M melanoma cells.16 GrB-Anis80-CP were significantly more potent than free PTX in inhibiting growth of H460 cells (IC50 = 0.12 μM).57 Similar trend was also observed in PC-3 cells (Figure 5B). The IC50 of GrB-Anis70-CP was 0.19 μg/mL (5.94 nM) for PC-3 cells at 120 h incubation. The high antitumor efficacy of GrB-Anis-CPs was due to their much enhanced intracellular GrB concentration, as a result of specific targeting to the sigma receptor over-expressing H460 cells and PC-cells as well as fast protein release inside the tumor cells. It has been reported that GrB concentration is very important for cancer cells to conduct apoptosis. Baginska et. al reported that GrB degradation decreased tumor cell susceptibility to natural killer-mediated lysis.58 Hodge reported that the increase in proteinase inhibitor-9 (PI-9) and/or decrease in GrB in lung cancer cells59 could protect them from apoptosis. Free GrB GrB-CPs GrB-Anis20-CPs GrB-Anis30-CPs

A

GrB-Anis50-CPs GrB-Anis70-CPs GrB-Anis80-CPs GrB-Anis100-CPs

Cell Viability (%)

100

80 *** *** *** ** **

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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60

40 1E-4

1E-3

0.01 [GrB] (μg/mL)

0.1

21


1

Biomacromolecules

B

Cell viability (%)

100

80 *** *** ** ** *

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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60

40 1E-4

1E-3

0.01 [GrB] (μg/mL)

0.1

1

Figure 5. Antitumor activity of GrB-Anis-CPs to H460 cells (A) and PC-3 cells (B). Free GrB was used as a control. GrB dosage varied from 1×10-4 to 0.4 μg/mL. The cells were incubated with GrB-Anis-CP or free GrB for 4 h, media were removed and replenished with fresh culture media, and cells were further cultured for another 68 h for H460 cells and 116 h for PC-3 cells. Data are shown as mean ± SD (n = 4) (Student’s t test, *p< 0.05, **p< 0.01, ***p< 0.001).

The apoptotic activity of GrB-Anis-CPs in H460 cells was also investigated by flow cytometry using annexin V-FITC/PI staining technique. The apoptosis was quantified by the rate of early (lower right) and late apoptotic cells (upper right). The results showed that GrB-Anis-CPs induced progressively higher cell apoptosis with increasing Anis contents, in which GrB-Anis20-CPs, Anis30-CPs, Anis50-CPs, and Anis70-CPs caused ca. 29.3%, 38.0%, 45.3%, and 51.5% cell death, respectively (Figure 6). In contrast, GrB-Anis100-CPs and non-targeting GrB-CPs led to ca. 43.4% and 25.8% cell death, respectively. These results were in accordance with MTT assays.

22


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Figure 6. Contour diagrams of H460 cells stained with annexin V-FITC/PI determined by flow cytometry following 72 h incubation with GrB-CPs (A), GrB-Anis20-CPs (B), GrB-Anis30-CPs (C), GrB-Anis50-CPs (D), GrB-Anis70-CPs (E) and GrB-Anis100-CPs (F). GrB concentration was fixed at 0.4 μg/mL.

To further confirm that Anis-CPs target to sigma receptors in H460 cells, competitive inhibition experiments were performed using haloperidol as an antagonist. The results showed that the anti-tumor activity of GrB-Anis80-CPs was significantly subdued upon pre-treating H460 cells with haloperidol (Figure 7). The cell viability increased from 36.5% to 61.6% at a protein dosage of 0.4 μg/mL. It is evident that GrB-Anis-CPs were internalized by H460 cells via sigma receptor mediated endocytosis. This mechanism of GrB cell entry via active tumor targeting polymersomes presents as an alternative to perforin–mediated translocation or electrostatic binding mediated endocytosis.12 Importantly, MTT assays showed that Anis-CPs were practically non-toxic (cell viabilities > 90 %) to H460, PC-3 and MCF-7 cells up to a tested concentration of 1.0 mg/mL (Figure S4 and S5).

23


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GrB-CPs GrB-Anis80-CPs+HP GrB-Anis80-CPs

Cell Viability (%)

100

80

60

*** **

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 29

40 1E-4

1E-3

0.01 [GrB] (μg/mL)

0.1

1

Figure 7. Antitumor activity of GrB-Anis80-CPs to H460 cells and haloperidol (30 μM, 3 h) pre-treated H460 cells. GrB-CPs was used as a control. GrB dosage varied from 1.0 ×10−4 to 0.4 μg/mL. The cells were incubated with GrB-Anis80-CPs or GrB-CPs for 4 h, media was removed and replenished with fresh culture media, and cells were further cultured for another 68 h. Data are shown as mean ± SD (n = 4) (Student’s t test, **p < 0.01,***p < 0.001).

Conclusions We have demonstrated that anisamide-decorated pH-sensitive degradable chimaeric polymersomes (Anis-CPs) based on anis-PEG-PTTMA-PAA and PEG-PTTMA-PAA could efficiently load, deliver and release apoptotic proteins into sigma receptor-overexpressing non-small cell lung cancer (NSCLC) cells and prostate cancer cells, inducing potent antitumor effects. These multifunctional polymersomes have presented several unique features for protein delivery: (i) they are readily prepared with tunable Anis contents and small sizes of ca. 130−170 nm under mild conditions; (ii) they exhibit active loading of proteins with a loading efficiency of ca. 100 % at a theoretical protein loading content of 1 wt.%; (iii) they show apparent targetability and fast internalization into sigma receptor-overexpressing cells including H460 lung cancer cells and PC-3 prostate cancer cells; and (iv) they are prone to degradation and dissociation under mildly acidic conditions due to hydrolysis of acetals in the 24


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polymersome membrane, thus achieving prompt intracellular protein release and potent tumor cell apoptosis. Our results have shown that granzyme B-loaded Anis-CPs caused superior anti-tumor effects with a particularly low IC50 of 3.75 nM toward H460 cells. These smart polymersomes have appeared as a novel and efficient platform for tumor-targeted protein delivery. In the future, in vivo experiments will be performed to show the potential of these polymersomal protein formulations.

Acknowledgements. This work is financially supported by research grants from the National Natural Science Foundation of China (NSFC 51273139 and 51473111), the National Science Fund for Distinguished Young Scholars (NSFC 51225302), Ph.D. Programs Foundation of Ministry of Education of China (20133201110005), the Major Program of the Natural Science Foundation of Jiangsu Province (14KJA150008) and a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

Supporting Information Synthesis of Anis-NHS; synthesis scheme for Anis-PEG-CPADN maro-RAFT agent; 1H NMR spectra of Anis-NHS, Anis-PEG-OH, Anis-PEG-CPADN and Anis-PEG-PTTMA-PAA; characteristics of GrB-AnisX-CPs; relative green fluorescence intensity in H460 cells; cytotoxicity of CPs and Anis20-CPs toward PC-3, MCF-7 and H460 cells. This material is available free of charge via the Internet at http://pubs.acs.org.

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Anisamide-Decorated pH-Sensitive Degradable Chimaeric Polymersomes Mediate Potent and Targeted Protein Delivery to Lung Cancer Cells Ling Lu, Yan Zou, Weijing Yang, Fenghua Meng*, Chao Deng, Ru Cheng, and Zhiyuan Zhong*

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Anisamide-Decorated pH-Sensitive Degradable ... - ACS Publications

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