Nucleus-Targeted, Echogenic Polymersomes for Delivering a Cancer

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Nucleus-targeted, echogenic polymersomes for delivering a cancer stemness inhibitor to pancreatic cancer cells Fataneh Karandish, Babak Mamnoon, Li Feng, Manas K. Haldar, Lang Xia, Kara N Gange, Seungyong You, Yongki Choi, Kausik Sarkar, and Sanku Mallik Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01133 • Publication Date (Web): 31 Aug 2018 Downloaded from http://pubs.acs.org on August 31, 2018

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Biomacromolecules

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Nucleus-targeted,

echogenic

polymersomes

for

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delivering a cancer stemness inhibitor to pancreatic

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cancer cells

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Fataneh Karandish†, Babak Mamnoon†, Li Feng†, Manas K. Haldar†, Lang Xiaᶩ, Kara N.

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Gangeᶨ, Seungyong You‡, Yongki Choi‡, Kausik Sarkarᶩ, and Sanku Mallik†*

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7



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Washington DC 20052

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ᶨDepartment of Health, Exercise, and Nutrition Sciences, North Dakota State University, Fargo,

Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58108

Department of Mechanical and Aerospace Engineering, The George Washington University,

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ND 58108

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AUTHOR ADDRESS

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Corresponding Author

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*E-mail: [email protected]. Phone: 701-231-7888. Fax: 701-231-7831.

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Present Address

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Department of Pharmaceutical Sciences, North Dakota State University, Fargo, North Dakota

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58108.

Department of Physics, North Dakota State University, Fargo, ND 58108

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KEYWORDS: Nuclear-delivery, targeted drug delivery, dexamethasone, BBI608, napabucasin,

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echogenic, polymersomes.

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ABSTRACT

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Chemotherapeutic agents for treating cancers show considerable side effects, toxicity, and drug

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resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive

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polymeric vesicles (polymersomes) to transport and subsequently release the encapsulated anti-

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cancer drugs within the nuclei of pancreatic cancer cells. We synthesized an alkyne-

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dexamethasone derivative and conjugated it to N3–polyethylene glycol (PEG)–polylactic acid

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(PLA) copolymer employing the Cu2+ catalyzed “Click” reaction. We prepared polymersomes

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from the dexamethasone-PEG-PLA conjugate along with a synthesized stimuli-responsive

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polymer PEG–S–S–PLA. The dexamethasone group dilates the nuclear pore complexes and

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transports the vesicles to the nuclei. We designed the polymersomes to release the encapsulated

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drugs in the presence of a high concentration of reducing agents in the nuclei of pancreatic cancer

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cells. We observed that the nucleus-targeted, stimuli-responsive polymersomes released 70% of

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encapsulated contents in the nucleus-mimicking environment in 80 minutes. We encapsulated the

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cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608 encapsulated

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polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid

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cultures. The polymersomes were prepared following a special protocol so that they scatter

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ultrasound, allowing imaging by a medical ultrasound scanner. Therefore, these echogenic,

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targeted, stimuli-responsive, drug-encapsulated polymersomes have the potential for trackable,

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targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

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Biomacromolecules

INTRODUCTION

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Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of death in the United

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States with the approximate five-year survival rate of less than 10%.1 Pancreatic cancer treatment

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is complicated because of invasiveness, rapid metastasis, and the complex nature of the disease.

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Alteration of different genes, aberrant biochemical pathways, epithelial to mesenchymal transition

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(EMT), hypoxic tumor microenvironment, and the subpopulation of cancer stem cells lead to

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recurrence and drug resistance.2 Considering that pancreatic cancer shows early invasion and

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metastasis, there is an urgent need for developing new treatment of this devastating disease. In the

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solid tumor tissue, EMT leads to a subpopulation of cancer stem cells, which can initiate a tumor,

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self-renew, and increase resistance to chemotherapeutic drugs.3 The small molecule BBI608

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(napabucasin) inhibits signal transducer and activator of transcription 3 (STAT3), reduces the

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expression of cancer stemness markers, inhibits cell proliferation, blocks spherogensis and

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apoptosis in both cancer stem cells, and non-stem cells.2-4 BBI608 is currently in the Phase III

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clinical trials as an adjuvant therapy for a variety of solid tumors, including pancreatic cancer

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(www.clinicaltrials.gov). STAT3 controls many oncogenic pathways and is activated in different

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type of cancers such as breast, ovarian, colorectal, etc.4

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Targeted, stimuli-responsive carriers enhance therapeutic efficacy and reduce toxicity by

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selectively delivering the cytotoxic chemotherapeutic drugs to cancerous tissues.5-7 Polymersomes

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are vesicles prepared from synthetic, amphiphilic block copolymers. Because of higher molecular

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weights, polymersomes have enhanced stability and mechanical robustness compared to

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liposomes, micelles, and polymer micelles.8

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hydrophobic drugs, and the aqueous core incorporates the hydrophilic molecules, enabling

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simultaneous delivery of both drugs.9 Conjugation of antibodies, small molecules or peptides

The bilayer of the polymersomes encapsulate

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enables active targeting of the vesicles to the cancer cells.10, 11 However, due to increased stability,

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the polymer bilayer usually requires a stimulus to rapidly release the encapsulated anticancer

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drugs.12 Polymersomes responsive to pH 13, heat 14, hypoxia 15, and light 16 has been used to deliver

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chemotherapeutic drugs to the cancer cells. Multifunctional polymersomes with drug delivery and

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simultaneous imaging capability are also reported.17, 18 The polymeric nanoparticles accumulate in

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the tumor tissues by the enhanced permeation and retention effect 19; however, for efficient cellular

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internalization, specific ligands are necessary on the vesicle surface.

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Nuclear pore complex is a large multi-protein assembly, which spans the nuclear envelope.

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Approximately 2000 nuclear pore complexes exist in the nuclear envelope. Dexamethasone is a

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synthetic steroid that dilates the nuclear pore complex from 38 nm to 300 nm.20, 21 Glutathione

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(GSH), the abundant cellular reducing agent, is a tripeptide consisting of glutamic acid, cysteine,

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and glycine. GSH is involved in several important processed in the cell nuclei, such as

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transcription, DNA replication, nuclear protein import and export, and chromatin stability.

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High levels of GSH is related to increased cell proliferation. 23 24

22, 23

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Herein, we report nucleus-targeted, echogenic, redox-sensitive polymersomes to deliver the

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cancer stemness inhibitor BBI608 to pancreatic cancer cells. We used dexamethasone as a

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targeting group to dilate the nuclear pore complexes

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nucleus. In the synthesized copolymer, we used polyethylene glycol (PEG) as the hydrophilic

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block and polylactic acid (PLA) as the hydrophobic block. Incorporation polyethylene glycol

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polymer (PEGylation) in the polymersomes composition reduces the interactions of nanoparticle

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with circulating proteins.25 Polylactic acid (PLA) is the hydrophobic part of the polymer and has

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a degradation time of more than one week in aqueous solutions at 37 oC. 26, 27 The two polymer

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blocks were linked by the reduction sensitive disulfide linker.

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and deliver the polymersomes inside the

Several literature reports

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demonstrate the effectiveness of the disulfide bonds to deliver the encapsulated payloads under

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increased cellular reducing agent concentration

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dexamethasone on the surface, internalize in the nuclei of pancreatic cancer cells. The high

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reducing agent concentration in the nuclei

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compromises the polymersome structure, and rapidly releases the encapsulated drug. We observed

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that nucleus-targeted polymersomes encapsulating BBI608 significantly (p < 0.05) decreased the

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viability of the BxPC3 cells compared to control and the non-targeted vesicles. The polymersomes

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were shown to be responsive to ultrasound offering possibility of concurrent ultrasound imaging

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of the cancerous tumors. We have used a preparation protocol incorporating lyophilization in the

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presence of mannitol that has proved previously effective in rendering liposomes

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polymersomes 40 echogenic.

28-31

32, 33

We observed that the vesicles, presenting

cleaves the disulfide bonds of the polymers,

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and

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MATERIALS AND METHODS

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All chemicals and solvents were purchased either from VWR International or TCI America and

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used as received.

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Synthesis of alkyne-dexamethasone. Dexamethasone (100 mg, 0.26 mmol) was dissolved in

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anhydrous pyridine (2 mL) in a round bottom flask and stirred on ice (0 °C) with methane sulfonyl

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chloride (250 µL, 3.2 mmol) under nitrogen for four hours. Then, an additional amount of methane

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sulfonyl chloride (18 µL, 0.23 mmol) was added, and the reaction continued for an additional hour.

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After five hours of stirring under nitrogen, 40 mL of ice water was added to precipitate the product.

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The precipitate was filtered and washed with 40 mL of additional ice water. The crude product

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was purified by recrystallization from tetrahydrofuran (THF) twice to afford the pure product. The

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product (0.026g, 0.06 mmol) and propargylamine (0.10 mL, 1.561 mmol), was stirred in 800 µL

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dimethylformamide (DMF) at 65 °C under nitrogen gas for two hours. After cooling to room

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temperature, the reaction mixture was added dropwise to cold water and centrifuged at 425 g for

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10 minutes. The clear supernatant was decanted, and the precipitate was washed again with water,

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dried under vacuum. The pure product (16 mg, 62%) was characterized by 1H NMR spectroscopy

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(Supporting Information). 1H NMR (400 MHz, chloroform-d) δ ppm: 7.41 (d, 1H), 6.40 (d, 1H),

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6.20 (d, 1H), 4.37 (d, 2H), 4.32 (t, 1H), 3.41 (s, 1H), 2.43 (t, 2H), 2.38 (t, 2H), 1.70 (d, 2H), 1.56

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(s, 3H), 1.06 (s, 3H), 0.95 (s, 3H). HRMS calcd. for C25 H32FNO4: 429.2315. Observed: 429.2306.

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Figure 1. Structure of the synthesized alkyne conjugated dexamethasone

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Synthesis of dexamethasone–PEG1900–PLA8000 polymer conjugate. We synthesized the N3–

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PEG1900–PLA8000 polymer employing a protocol developed in our laboratory (Supporting

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Information). The alkyne-dexamethasone was reacted with the N3–PEG1900–PLA8000 polymer

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using the [2+3]-cycloaddition reaction.41 Briefly, we prepared the copper (II) complex by mixing

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the CuSO4 (71.3 mg in 3 mL water, 0.53 mmol) and pentamethyl diethylenetriamine (440 µL in 3

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mL water, 2 mmol). The N3–PEG1900–PLA8000 polymer (20 mg) and alkyne-dexamethasone (2

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mg) were dissolved in 6 mL tetrahydrofuran (THF), 400 L of the 53 mM copper complex and

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400 L of 53 mM aqueous sodium ascorbate solution were added, and the reaction mixture was

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stirred at room temperature for 24 hours. The reaction mixture was added dropwise to cold ether

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and centrifuged at 425 g for 2 minutes. The clear supernatant was decanted, and the precipitate

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was washed with water three times by vortex and centrifuge, then dried under vacuum. 1H NMR

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(400 MHz, chloroform-d) δ ppm: 6.34 (d, 1H), 6.15 (d, 1H), 5.18 (q, 1 H), 4.37 (d, 2H), 3.66 (t, 4

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H), 2.52 (t, 2H), 2.30 (t, 2H), 1.60 (d, 3 H). From the 1H NMR spectrum, we calculated the

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conversion yield for the dexamethasone coupling reaction to be 39% (Supporting Information).

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Scheme 1. [2+3]-Cycloaddition reaction of N3–PEG1900–PLA8000 polymer and alkyne-

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dexamethasone.

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Preparation of nucleus targeted polymersomes encapsulating BBI608. Polymersomes were

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prepared by the solvent exchange method42 with PEG1900–S–S–PLA6000

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PEG1900–PLA8000 polymer, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-lissamine

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rhodamine B sulfonyl ammonium salt (fluorescent dye, LR, Avanti Polar Lipids) with a molar

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, dexamethasone–

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ratio of 35:60:5, respectively (Figure 2). The polymers were dissolved in THF (9 mg/mL), BBI608

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in THF (3 mg/mL), and LR in chloroform (0.01 mg/mL). First, a rotary evaporator was used to

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evaporate the chloroform from LR lipid to form a thin layer film. The THF solutions of the

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polymers and BBI608 were added to the thin film. The resultant fluorescent THF solution was

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added dropwise to an aqueous HEPES buffer (10 mM, pH 7.4) and stirred for 45 minutes. To

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remove the THF, a gentle stream of air was passed through the mixture for 45 minutes. The

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polymersomes formed were bath sonicated for 60 minutes (Symphony 117 V, 60 Hz, Power level

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9). The polymersomes (1 mg/mL) were passed through a SephadexTM G-100 size exclusion

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column to remove the unencapsulated drug. Drug loading efficacies (DLE) of the polymersomes

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were determined using UV−Vis spectroscopy. After passing through the size-exclusion column,

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the absorption of the polymersomes was recorded at 235 nm to calculate the loading efficiency.

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Figure 2. The structure of synthesized polymers PEG1900–S–S–PLA6000, dexamethasone–

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PEG1900–PLA8000 polymer conjugate, and the commercially available fluorescent lipid 1,2-

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dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-lissamine rhodamine B sulfonyl ammonium

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salt.

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Preparation of control polymersomes. The control polymersomes were prepared following the

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same method as the nucleus-targeted vesicles. PEG1900–S–S–PLA6000, N3–PEG1900–PLA8000, and

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lissamine rhodamine (LR) were used in the molar ratio of 35:60:5, respectively. The PEG1900–S–

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S–PLA6000 and N3–PEG1900–PLA8000 polymers were dissolved in THF. The polymer solution was

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added slowly to the thin film of the LR dye, and then the mixture was added dropwise to a stirred

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10 mM HEPES buffer (pH 7.4). The polymersome solutions were stirred for 45 minutes at room

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temperature, and then air was passed for 45 minutes through the mixture. The polymersomes were

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sonicated for 60 minutes (Symphony 117 V, 60 Hz). Subsequently, the polymersomes were passed

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through a Sephadex G100 (GE Healthcare) size exclusion column to collect lissamine rhodamine

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dye incorporated polymersomes. These polymersomes were used as a control for the cell viability

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assays.

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Polymersomes Size Analysis. The nucleus-targeted polymersomes encapsulating BBI608 and

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control polymersomes were characterized by dynamic light scattering at 90o using a Zeta Sizer

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Nano ZS 90 (Malvern Instrument). Polymersomes were equilibrated for 120 seconds, and five

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measurements were recorded with 10 repeats each.

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Transmission Electron Microscopy (TEM). Samples were prepared on 300 mesh copper grids

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with a formvar-carbon support film. Grids were pretreated with 1% poly-L-lysine and air-dried;

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5 µL of the polymersome suspension was added and allowed to stand 1 min, then wicked off with

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a filter paper. Negative staining was performed using 0.1% phosphotungstic acid for 2 minutes,

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then wicking off and air-dried before observation and imaging in a JEOL JEM-2100 LaB6

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transmission electron microscope.

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Atomic Force Microscopic (AFM) Imaging. The size and morphology of polymersomes were

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characterized using atomic force microscopy. Polymersomes (1 mg/mL) were diluted (20X) in

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HEPES buffer (pH 7.4, 10 mM). Polymersomes were dropped on silica substrates, incubated for a

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minute and extra liquid dried by an air blowgun. The AFM measurements were performed in non-

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contact mode (resonance frequency of 145 kHz and a scanning rate of 1.3 Hz) using an NT-MDT

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INTEGRA instrument (NT-MDT America). The scanning areas were 5 × 5 μm² at the resolution

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of 512 points per line, respectively. The images were flattened to eliminate the background low-

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frequency noise and tilt from the surface using all unmasked portions of scan lines to calculate

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individual least-square fit polynomials for each line.

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Redox-triggered Release studies. Polymersomes encapsulating 20 µM calcein dye were

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prepared to perform the release study. The experiments were conducted by the quenching method

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chloride (10 mM) was used to quench the fluorescence from the unencapsulated calcein outside

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the vesicles. We used 20 μL of calcein-encapsulated polymersomes (total polymer concentration:

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1 mg/mL) in 10 mM HEPES buffer (180 μL, pH =7.4) in a 96-well plate. The release from the

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polymersomes were monitored in the presence of 10 mM glutathione for 40 minutes; subsequently,

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the concentration was increased to 50 mM, and the release was monitored for an additional 40

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minutes using (excitation: 495 nm, emission: 515 nm) employing a fluorescence microplate reader

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(Spectramax M5, Molecular Devices). The amount of calcein release form polymersomes were

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calculated employing this equation:

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Percent Release = [(Emission Intensity after 80 min – Initial Intensity before treatment)/Initial

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Intensity before treatment] ×100.

using cobalt chloride as the quencher and glutathione as the reducing agent. The cobalt (II)

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Preparation and characterization of echogenic polymersomes. Polymersomes were prepared

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by dissolving the PEG1900–S–S–PLA6000 and N3–PEG1900–PLA8000 polymers with the molar ratio

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of 40:60 (total 5 mg in 1 mL of THF), respectively. Then, the polymer mixture was dropwise added

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to 0.32 M mannitol (weak cryoprotectant) prepared in HEPES buffer (10 mM, pH 7.4).

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Subsequently, THF was evaporated for 45 min by passing air through the solution. Then,

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polymersomes were sonicated for 60 minutes (Symphony 117 V, 60 Hz). To make the

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polymersomes echogenic, they were subjected to three freeze (-80 oC, 24 h) and thaw (60 °C)

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cycles. Finally, the polymersomes were lyophilized (Labconco freeze dryer) and reconstituted in

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HEPES buffer (10 mM, pH 7.4) for further experiments.

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Ultrasound Experimental setup to measure scattering in echogenic polymersomes. Two

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spherically focused transducers (each having a central frequency of 2.25 MHz/5 MHz/10 MHz,

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Figure 3) with the same specifications (V310-SU, Olympus NDT) were employed for scattering

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measurements. The transmitting and receiving transducers were placed perpendicularly by two

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separate linear stages (433 series, 360-90, Newport) and immersed in a bigger water tank filled

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with DI water. A 20-mL syringe served as a sample chamber, in which polymersome suspension

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was injected. A function generator (Model AFG 3251; Tektronix) was utilized to generate a 32-

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cycle sinusoidal pulse of 5 MHz frequency at a PRF of 100 Hz. These signals were then amplified

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using a 55dB power amplifier (Model A-300, ENI) and sent to the transmitting transducer. The

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input signals were scattered by the polymersomes inside the focal volume of the transducer. The

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scattered signals were received by the receiving transducer connected to a pulser/receiver

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(DPR300, 475v, JSR) in the through mode with a 27-dB gain. The output signals were then

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transmitted to an oscilloscope (TDS2012, Tektronix) for real-time visualization. The output

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voltage-time RF signals were obtained by the oscilloscope by averaging over every 64 sequences.

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The data from the oscilloscope were finally transmitted and saved onto a desktop computer using

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the software Signal Express Tektronix Edition (version 2.5.1, Labview NI). In time-dependent

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scattering experiments, all the setups and procedures were the same except that the data were

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recorded over a 20-minute period (corresponding to about 344 acquisitions). As for the degassed

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experiment, all the setups and procedures were still the same, only difference being that the PBS

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solution had been degassed using a vacuum pump.

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Figure 3. Experimental setups for the ultrasound scattering measurements from the echogenic

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polymersomes.

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Echogenic polymersomes’ experimental procedure and data reduction. In a scattering

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experiment, the suspension was made by reconstituting the dry powder in phosphate buffered

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saline (PBS) solution to obtain a concentration of 10 μg polymer/mL. We injected 20 mL of the

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resulting suspension into the sample chamber. The measurement was repeated five times to

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guarantee the reliability of the experimental data. The measurement of the control signal, i.e.,

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without polymersomes and the responses due to the polymersomes were acquired by the

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procedures above. The Fourier transform of the signal was performed using a Matlab program to

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get the average scattered power spectra in the frequency domain (50 voltage time acquisitions were

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used for averaging). The scattered response was converted into a dB scale by taking a unit

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reference. Fundamental, second- and sub-harmonic scattered responses were extracted from the

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power spectrum. The final data is reported as an enhancement over the control. We also checked

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the scattered response in the presence of 50 mM of glutathione.

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Ultrasound Imaging. Dried polymersomes were reconstituted in 10 mM HEPES buffer, pH 7.4

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with the concentration of 10 µg/mL. In a 96-well plate, 0.2 mL of polymersomes were dispensed

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into each well; then the plate was covered with parafilm. Subsequently, an ultrasound gel

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(Aquasonic 100, Parker Laboratories) was applied, and a 15 MHz linear ultrasound transducer was

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used for the imaging experiments employing a Terason t3200 instrument (MediCorp LLC).

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Cell Culture. The pancreatic cancer cell line BxPC3 was purchased from the American Tissue

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Culture Consortium (ATCC). The cells were maintained in RPMI 1640 medium (without phenol

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red) supplemented with 1% v/v antibiotics (penicillin and streptomycin) and 10% v/v fetal bovine

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serum. The cell culture flasks were maintained in an incubator at 37 °C in a 5% CO2 atmosphere.

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Nuclear Uptake Studies. The BxPC3 cells (3×10³) were seeded in a 12-well tissue culture plate

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for 24 hours before the experiment. Once the culture is 80-90% confluent, the nucleus-targeted (20

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µL) and non-targeted (20 µL) polymersomes were incubated with the cells for 3 hours.

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Subsequently, the cell culture media and polymersomes were removed, and the cells were washed

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twice with Hanks’ balanced salt solution (HBSS) to remove the non-internalized vesicles. The cell

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nuclei were stained with HOESCHT 33342 dye (Enzo Life Sciences, 1:1000 dilution) and imaged

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using the 20X objective of a Leica fluorescence microscope.

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Cell Viability in Monolayer Cultures. To evaluate the efficacy of the nucleus-targeted

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polymersomes on the human pancreatic cancer cells (BxPC3), the Alamar Blue assay was

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performed. The BxPC3 cells were seeded at a density of 10³/well in a 96-well tissue culture plate

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and were allowed to grow until 80−95% confluent. The plate was divided into four groups: control,

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free drug (BBI608), non-targeted polymersomes, and nucleus-targeted polymersomes

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encapsulating BBI608. The control group did not receive any treatment. Cells treated with BBI608

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received 1, 4, and 8 μM of free and an equivalent amount of encapsulated drug in nucleus-targeted

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polymersomes. The cells were treated for 48 hours at 37 °C, in a 5% CO2 atmosphere.

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Subsequently, the cells were washed with sterile HBSS and replaced with 200 μL of fresh media.

280

Then 20 μL Alamar Blue was added to all the wells and fluorescence were measured after 4 hours.

281

The data presented are normalized to the control.

282 283

Cell Viability in three-dimensional (3D) spheroid cultures. The 24-well 3D petri dishes

284

(Microtissues) were used to prepare BxPC3 spheroids. Briefly, 2% w/v agarose solution was

285

prepared and autoclaved. The BxPC3 cell suspension (104 cells in 60 μL media) was then added

286

to each 3D scaffold. The spheroids were allowed to grow for 7 days. Then scaffolds were divided

287

into four groups: control, non-targeted polymersomes, nucleus-targeted polymersomes

288

encapsulating BBI608, and free drug. Spheroids were treated for 48 hours with the same

289

concentration of drug as used for the monolayer studies. Subsequently, the spheroids were washed

290

with sterile HBSS and then incubated with 100 μL TryPLE (recombinant trypsin, Life

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Technologies) for 10 minutes. The spheroids were removed and subjected to the Alamar Blue

292

assay. The data presented are normalized to the control.

293 294

Results and Discussion

295

Polymer synthesis and polymersome preparation. To prepare nucleus targeted polymersomes,

296

we synthesized an alkyne-dexamethasone conjugate (Figure 1) and linked it to the N3-–PEG1900–

297

PLA8000 polymer (Figure 2 and Supporting Information). The redox-sensitive polymer PEG1900–

298

S–S–PLA6000 (Figure 2) was synthesized as previously reported by our group.

299

dexamethasone–PEG1900–PLA8000 polymer conjugate was synthesized using the Click chemistry

300

[2+3 cycloaddition]. We anticipated that the slightly higher molecular weight of the polymer

301

conjugate would protrude the dexamethasone group from the surface of the vesicles and facilitate

302

the interactions with its receptor. The polymersomes were prepared by the solvent exchange

303

method

304

microscopy (Figure 5), and atomic force microscopy (Figure 6). We encapsulated the stemness

305

gene transcription inhibitor BBI608 (napabucasin) 3 in the polymersomes with an efficiency of 68

306

+ 5%. We observed that the nucleus-targeted polymersomes had a hydrodynamic diameter of 200

307

± 2 nm, with a polydispersity index (PDI) of 0.2 ± 0.02. The average hydrodynamic diameter for

308

the non-targeted polymersomes was 140 ± 3 nm with a PDI of 0.2 ± 0.03. The nucleus-targeted

309

polymersomes were slightly larger than nontargeted polymersomes. We hypothesize that the

310

encapsulation of hydrophobic BBI608 in the polymer bilayer of the vesicles increases the size of

311

vesicles.

312

The dexamethasone–PEG1900–PLA8000 polymer was incorporated into polymersome composition

313

to target the nanoparticles to the cell nucleus. We expected that the dexamethasone on the

42

15, 43, 45

The

and characterized by dynamic light scattering (Figure 4), transmission electron

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21, 46

314

polymersomes would dilate the nuclear pore complex

315

nucleus. The disulfide bond in the redox-sensitive polymer will subsequently be reduced in the

316

nucleus, releasing the encapsulated BBI608.

A

10 8

Intensity%

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

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6 4 2 0

0

100

200

300

Size(nm)

317

400

500

16 14 B 12 10 8 6 4 2 0 0 100

and transport the vesicles into the

200 300 Size (nm)

400

500

318

Figure 4. The hydrodynamic diameters of the (A) non-targeted and (B) nucleus-targeted

319

polymersomes encapsulating the stemness inhibitor BBI608, as determined by dynamic light

320

scattering.

321 322

Figure 5. Transmission electron microscopy images (scale bar: 50 nm) of the non-targeted

323

polymersomes.

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326 327

Figure 6. Atomic force microscopy (AFM) images of the non-targeted polymersomes. (A, B)

328

Polymersomes, (C, D) Polymersomes treated with 10 mM glutathione for 5 minutes, and (E, F)

329

polymersomes treated with 50 mM GSH for 5 minutes.

330 331

Demonstration of reduction-triggered contents release from the polymersomes and

332

structural characterization. We studied the reduction-triggered release of the dye calcein from

333

the polymersomes as a function of time in the presence of different concentrations of added

334

glutathione (GSH). GSH is an important intracellular reducing agent, comprising of glutamic acid,

335

cysteine, and glycine.47 In tumor cells, the concentration of GSH is about 100-1000 time higher

336

than the extracellular fluids.32, 48 It's increased level is correlated with progression and proliferation

337

of various cancers, such as breast 3, colon

338

We observed that the polymersomes released 45% the encapsulated dye in the presence of 10 mM

339

(mimicking the cytosol) GSH. However, the released increased to 70% with 50 mM GSH

49

, lung 49, pancreas

47

, resistance to chemotherapy.47

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(mimicking the nucleus, Figure 7).24 We also observed that both of the releases were rapid. Our

341

atomic force microscopic studies indicated that 10 mM GSH slightly changed the morphology of

342

the polymersomes (Figure 8A); however, 50 mM GSH completely disrupted the vesicle structure

343

(Figure 8B). We checked the size of disruption of polymersomes (with dynamic light scattering)

344

and observed that 50 mM GSH decreased the average diameter of the vesicles from 200 nm (Figure

345

4B) to 100 nm (Figure 8C).

80 [GSH] = 50 mM

Percent 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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70 60 50

[GSH] = 10 mM

40 30 20 10 0 0

346 347

20

40

60

80

Time (min) Figure 7. The glutathione-triggered release of the encapsulated calcein from the polymersomes.

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349

100

200

300

400

500

Size(nm)

350

Figure 8. Structural characterization of the polymersomes after release study employing atomic

351

force microscopy. (A) Polymersomes treated with 10 mM GSH. (B) Polymersomes treated with

352

50 mM GSH. (C) The hydrodynamic diameters of polymersomes after release study, as determined

353

by dynamic light scattering.

354 355

Demonstration of polymersomes’ echogenicity. Polymersomes’ echogenicity was confirmed by

356

a Terason medical ultrasonic imaging system (t3200) using a 15 MHz transducer. We observed

357

that the echogenic polymersomes reflected ultrasound even after a week in the aqueous solution.

358

The ultrasound reflection suggests the presence of air pockets (detailed discussion below) in

359

polymersomes (Figure 9B and C) while control (buffer without any polymersomes) did not show

360

any contrast (Figure 9A).

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362 363

Figure 9. Ultrasound contrast images of the polymersomes at 15 MHz. (A) Control, (B) freshly

364

reconstituted echogenic polymersomes, and (C) echogenic polymersomes after one week in

365

aqueous solution.

366

The scattered power spectra of the polymersomes at three excitation frequencies (2.25, 5,

367

and 10 MHz) are displayed in Figure 10. Power spectra show the responses of the polymersomes

368

as a function of frequencies. Contributions at frequencies other than the excitation frequency

369

indicate nonlinear responses of the polymersomes, which in turn offers the possibility of a

370

nonlinear imaging modality with a potentially better signal to noise ratio 50, 51. Polymersomes are

371

echogenic in aqueous phosphate buffered saline (PBS, pH = 7.4), but not in the degassed solution

372

(Figure 10D). There is a significant nonlinear response, specifically subharmonic, only at 2.25

373

MHz (Figure 10A).

374

Note that although echogenicity of echogenic liposomes (ELIPs), prepared following a

375

particular protocol, has been demonstrated by others and us

376

echogenicity remains unknown. For ELIPs, repeated freeze-thaw cycles followed by lyophilization

377

in the presence of mannitol in the preparation protocol has proved crucial for ensuring echogenicity

378

36, 61

379

that during rehydration generate lipid-coated air-bubbles either inside liposomes or in the bilayer

380

62, 63

36, 52-60

, the exact mechanism of the

. It has been speculated that mannitol, being a weak cryoprotectant, leaves defects in the bilayer

. The bubbles, stabilized against dissolution by the coating64-66, produce strong ultrasound

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echoes. The lack of echogenicity in degassed solution as well as the strong nonlinear response

382

verifies the role of bubbles in the acoustic response of echogenic polymersomes.

-50

-80

2.25 MHz

A

Control Polymersomes

-60

Power Spectrum (dB)

Power Spectrum (dB)

-40

-70 -80 -90 -100

-85

B

5 MHz Control Polymersomes

-90 -95 -100 -105

-110 2

4

6

8

-110

10

5

Frequency (MHz)

383

10

15

20

Frequency (MHz)

-80

-90

C

10 MHz

-95

Power Spectrum (dB)

Power Spectrum (dB)

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Control Polymersomes

-100

-105

-85 -90

5 MHz

D

Control polymersomes in degassed solution

-95 -100 -105

-110

384

10

20

30

40

-110

5

10

15

20

Frequency (MHz)

Frequency (MHz)

385

Figure 10. Scattered responses of echogenic polymersomes in PBS at 500 kPa excitation pressure

386

and excitation frequencies of (A) 2.25 MHz, (B) 5 MHz, and (C) 10 MHz. Control is without

387

polymersomes. (D) Scattered response in degassed solution at 500 kPa and 5 MHz.

388 389

Figure 11(A) shows the enhancements at different excitation frequencies. The

390

enhancement in fundamental response is strongest at 5 MHz excitation, whereas the highest

391

subharmonic enhancement appears at 2.25 MHz. The latter indicates the possibility of

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polymersome aided subharmonic imaging. 36, 67 We also examined the scattered responses over a

393

20-minute period in Figure 11(B) to investigate the long-term stability of their echogenicity crucial

394

for clinical applications. During the 20-minute sonication, the polymersomes produced an almost

395

constant signal indicating their suitability for use in contrast-enhanced imaging. Polymersomes in

396

degassed solution expectedly did not generate any scattered response.

20

-50 Fundamental Subharmonic 2nd-Harmonic

15

10

5

0

2.25 MHz

5 MHz

10 MHz

Fundamental Response (dB)

Enhancment (dB)

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

B -60

Polymersomes Polymersomes in degassed solution

-70 -80 -90 -100 -110

0

5

10

15

20

Time (min)

397 398

Figure 11. (A) Enhancements in fundamental, sub– and second harmonic scattered responses from

399

the echogenic polymersomes at 500 kPa and 2.25, 5, and 10 MHz.

400

fundamental responses of the echogenic polymersomes in normal (blue trace) and degassed PBS

401

(black trace) at 5 MHz and 500 kPa (N = 5).

(B) Time-dependent

402 403

Glutathione cleaves the disulfide bond destabilizing the bilayer of the polymersomes. We

404

repeated the scattering experiments in the presence of 50 mM GSH. As expected, we observed that

405

the fundamental enhancement decreased by 10 dB, and the subharmonic enhancement decreased

406

by 4 dB (Figure 12) due to the loss of structural integrity of the polymersomes.

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30

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25 20 15 10 5 0

Without Glutathione

With Glutathione

407 408

Figure 12. Fundamental (black bars) and sub-harmonic (red bars) enhancements of echogenic

409

polymersomes at 5 MHz and 500 kPa with and without glutathione.

410 411

Nuclear Uptake Studies. To determine the localization within the pancreatic cancer cells, we

412

prepared the polymersomes incorporating 5% of the DSPE–lissamine rhodamine lipid (structure

413

shown in Figure 2) into the bilayer. We anticipated that the dexamethasone would dilate the nuclear

414

pore complexes and transport the polymersomes inside the nucleus.68 We incubated the BxPC3

415

pancreatic cancer cells with nucleus-targeted and non-targeted polymersomes for 3 hours.

416

Subsequently, the cell nuclei were stained with the HOESCHT 33342 dye and imaged employing

417

a fluorescence microscope (Figure 13). We observed localization of the targeted polymersomes in

418

the nuclei of the BxPC3 cells. We also observed similar results employing the breast cancer cells

419

MCF-7 (Supporting Information).

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420 421

Figure 13. Cellular uptake studies with the BxPC3 cells. The non-targeted polymersomes (top

422

panel) did not enter the cell nucleus. The targeted polymersomes (bottom panel) were present in

423

the cell nuclei after 3 hours of incubation (indicated by the overlapping blue and red colors in the

424

Merged panel; scale bar: 50 µm).

425

Viability studies in monolayer cultures of pancreatic cancer cells. After validating efficient

426

nuclear localization, we proceeded to determine the effectiveness of the nucleus-targeted, drug-

427

encapsulated polymersomes in the pancreatic cancer cells. The monolayer culture of the BxPC3

428

cells was treated with the nucleus-targeted polymersomes encapsulating BBI608, the non-targeted

429

vesicles, and the free drug for 48 hours. The cell viability was determined by the Alamar Blue

430

assay (Figure 14). We observed that the control polymersomes without the dexamethasone group

431

decreased the cell viability to 50% at 4 M concentration of encapsulated BBI608 (red columns).

432

At this concentration of encapsulated drug, the targeted polymersomes were more effective,

433

decreasing the viability of the BxPC3 cells to 21% (blue column), similar to the un-encapsulated

434

free BBI608 (pink columns). At lower concentrations of encapsulated BBI608 (1 M and 2 M),

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both encapsulated and free BBI608 were less effective in killing the cancer cells. We determined

436

the IC50 values of the nucleus-targeted polymersomes encapsulating BBI608, the non-targeted

437

vesicles, and the free drug for the BxPC3 cells (48 hours) to be 1.75 + 0.12 M, 3.20 + 0.47 M,

438

and 0.77 + 0.48 M, respectively (Supporting Information).

Control Non-targeted polymersomes encapsulating BBI608 Nucleus-targeted polymersomes encapsulating BBI608 Free BBI608 in DMSO

100

Percent cell viability

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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80 60 40 20 0 1µM

439

2µM

4µM

[BBI608]

440

Figure 14. The viability of the BxPC3 cells in monolayer cultures at three different concentrations

441

of encapsulated BBI608. (A) Cell viability with media only (control, cyan bar), non-targeted

442

polymersomes (red bar), nucleus-targeted polymersomes encapsulating BBI608 (blue bar), and

443

free BBI608 (magenta bar, N = 4).

444

Viability studies in three-dimensional spheroid cultures of pancreatic cancer cells. Compared

445

to the monolayer cultures, the spheroids demonstrate cellular heterogeneity, cell-cell interactions,

446

and better mimic real tumors.45, 69 To evaluate the effectiveness of the polymersome formulations,

447

BxPC3 spheroids were prepared using the 24-well 3D petri dishes (Microtissues). We treated the

448

7-day old spheroids of the BxPC3 cells (Figure 15A) with nucleus-targeted polymersomes

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449

encapsulating BBI608, non-targeted polymersomes encapsulating BBI608, and free drug for 48

450

hours. The cell viability was determined by the Alamar Blue assay. We observed that the nucleus-

451

targeted polymersomes encapsulating BBI608 decreased (p ≤ 0.05) the cell viability to 43% in

452

compared to non-targeted polymersomes (84%) and the control (Figure 15B). We speculate that

453

dexamethasone in the composition of polymersomes opens the nucleus pores, the vesicles enter

454

the nuclei and release the cancer stemness inhibitor, leading to the enhanced toxicity in the BxPC3

455

cells. Control Non-targeted polymersomes encapsulating BBI608 Nucleus-targeted polymersomes encapsulating BBI608

B 100

Free BBI608 in DMSO

*

80 Cell viability%

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

60 40 20 0 8 µM

456

[BBI608]

457

Figure 15. (A) Optical microscopic image of the 7- day old three-dimensional spheroids of the

458

BxPC3 cells (scale bar: 25 µm). (B) The viability of the BxPC3 cells in spheroid cultures. Viability

459

with media only (control, cyan bar), non-targeted polymersomes (red bar), nucleus-targeted

460

polymersomes encapsulating BBI608 (blue bar), and the free BBI608 (magenta bar, N = 4).

461 462 463 464

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465

Conclusions

466

We successfully synthesized alkyne dexamethasone and conjugated it to the N3 – PEG1900–PLA8000

467

polymer. The nucleus-targeted polymer was combined with a redox-sensitive polymer to form

468

stable polymeric vesicles, which encapsulate air bubbles and a stemness inhibitor. Our echogenic

469

nucleus-targeted polymersomes respond to ultrasound and release the encapsulated BBI608 to the

470

nucleus of pancreatic cancer cells. The nucleus-targeted drug-encapsulated polymersomes reduced

471

the viability of pancreatic cancer cells in monolayer and spheroidal cultures to 30% and 43%,

472

respectively. The polymersomes scatter ultrasound and respond to medical ultrasound imager,

473

confirming the echogenicity. They have the potential to image and deliver chemotherapeutic drugs

474

to the tumors tissues simultaneously. This is a non-invasive strategy to monitor targeted drug

475

delivery to improve the therapeutic outcome of chemotherapy. The results of this research will

476

pave the way for other ultrasound reflective nanoparticles for targeted drug delivery and

477

simultaneous imaging capability.

478 479

ASSOCIATED CONTENT

480

Supporting Information. The Supporting Information is available for readers on the ACS

481

Publications website at DOI:

482

The following files are available free of charge.

483

NMR spectra of the alkyne dexamethasone and the dexamethasone–PEG1900–PLA8000 polymer,

484

NMR spectra, and Gel permeation chromatography of the N3-PEG1900–PLA8000 polymer (file

485

type: PDF).

486

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487

FUNDING SOURCE

488

This research was supported by the NSF grant DMR 1306154, and the NIH grant 1 R01GM

489

114080 to SM and KS. SM also acknowledges support from the Grand Challenge Initiative and

490

the Office of the Vice President for Research and Creative Activity, North Dakota State University.

491

We acknowledge Dr. Michael D. Scoot for recording and interpreting the NMR spectrum of the

492

alkyne-dexamethasone conjugate.

493 494 495

REFERENCES

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11. Swain, S.; Sahu, P. K.; Beg, S.; Babu, S. M., Nanoparticles for Cancer Targeting: Current and Future Directions. Curr. Drug Deliv. 2016, 13, (8), 1290-1302. 12. Thambi, T.; Park, J. H.; Lee, D. S., Stimuli-responsive polymersomes for cancer therapy. Biomater. Sci. 2016, 4, (1), 55-69. 13. Chen, W.; Meng, F.; Cheng, R.; Zhong, Z., pH-Sensitive degradable polymersomes for triggered release of anticancer drugs: a comparative study with micelles. J. Controlled Rel. 2010, 142, (1), 40-46. 14. Amstad, E.; Kim, S. H.; Weitz, D. A., Photo‐and thermoresponsive polymersomes for triggered release. Angew. Chem. 2012, 124, (50), 12667-12671. 15. Kulkarni, P.; Haldar, M. K.; You, S.; Choi, Y.; Mallik, S., Hypoxia-Responsive Polymersomes for Drug Delivery to Hypoxic Pancreatic Cancer Cells. Biomacromolecules 2016, 17, (8), 2507-2513. 16. Kamat, N. P.; Robbins, G. P.; Rawson, J.; Therien, M. J.; Dmochowski, I. J.; Hammer, D. A., A generalized system for photoresponsive membrane rupture in polymersomes. Adv. Functional Mater. 2010, 20, (16), 2588-2596. 17. Zhu, D.; Fan, F.; Huang, C.; Zhang, Z.; Qin, Y.; Lu, L.; Wang, H.; Jin, X.; Zhao, H.; Yang, H.; Zhang, C.; Yang, J.; Liu, Z.; Sun, H.; Leng, X.; Kong, D.; Zhang, L., Bubble-generating polymersomes loaded with both indocyanine green and doxorubicin for effective chemotherapy combined with photothermal therapy. Acta Biomater. 2018. 18. Sarkar, B.; Paira, P., Theranostic Aspects: Treatment of Cancer by Nanotechnology. Mini Rev Med Chem 2018, 18, (11), 969-975. 19. Xu, P.; Van Kirk, E. A.; Zhan, Y.; Murdoch, W. J.; Radosz, M.; Shen, Y., Targeted Charge‐ Reversal Nanoparticles for Nuclear Drug Delivery. Angew. Chem. Int. Ed. 2007, 46, (26), 49995002. 20. Shahin, V.; Albermann, L.; Schillers, H.; Kastrup, L.; Schafer, C.; Ludwig, Y.; Stock, C.; Oberleithner, H., Steroids dilate nuclear pores imaged with atomic force microscopy. J. Cell. Physiol. 2005, 202, (2), 591-601. 21. Kastrup, L.; Oberleithner, H.; Ludwig, Y.; Schafer, C.; Shahin, V., Nuclear envelope barrier leak induced by dexamethasone. J. Cell. Physiol 2006, 206, (2), 428-34. 22. Conrad, M.; Moreno, S.; Sinowatz, F.; Ursini, F.; Kölle, S.; Roveri, A.; Brielmeier, M.; Wurst, W.; Maiorino, M.; Bornkamm, G., The nuclear form of phospholipid hydroperoxide glutathione peroxidase is a protein thiol peroxidase contributing to sperm chromatin stability. Mol. Cell. Biol. 2005, 25, (17), 7637-7644. 23. Go, Y.-M.; Jones, D. P., Redox Control Systems in the Nucleus: Mechanisms and Functions. Antioxidants Redox Signaling 2010, 13, (4), 489-509. 24. Anajafi, T.; Scott, M. D.; You, S.; Yang, X.; Choi, Y.; Qian, S. Y.; Mallik, S., Acridine orange conjugated polymersomes for simultaneous nuclear delivery of gemcitabine and doxorubicin to pancreatic cancer cells. Bioconjugate Chem. 2016, 27, (3), 762-771. 25. Molineux, G., Pegylation: engineering improved pharmaceuticals for enhanced therapy. Cancer Treatment Rev. 2002, 28, 13-16. 26. Chen, W. L.; Liu, S. J.; Leng, C. H.; Chen, H. W.; Chong, P.; Huang, M. H., Disintegration and cancer immunotherapy efficacy of a squalane-in-water delivery system emulsified by bioresorbable poly(ethylene glycol)-block-polylactide. Biomaterials 2014, 35, (5), 1686-95. 27. Shah, S. S.; Zhu, K. J.; Pitt, C. G., Poly-DL-lactic acid: polyethylene glycol block copolymers. The influence of polyethylene glycol on the degradation of poly-DL-lactic acid. .J Biomater. Sci. Polym. Ed. 1994, 5, (5), 421-31.

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28. Yang, W.; Zou, Y.; Meng, F.; Zhang, J.; Cheng, R.; Deng, C.; Zhong, Z., Efficient and Targeted Suppression of Human Lung Tumor Xenografts in Mice with Methotrexate Sodium Encapsulated in All-Function-in-One Chimeric Polymersomes. Adv. Mater. 2016, 28, (37), 82348239. 29. Wu, J.; Deng, C.; Meng, F.; Zhang, J.; Sun, H.; Zhong, Z., Hyaluronic acid coated PLGA nanoparticulate docetaxel effectively targets and suppresses orthotopic human lung cancer. J. Controlled Rel. 2017, 259, (Supplement C), 76-82. 30. Fang, Y.; Jiang, Y.; Zou, Y.; Meng, F.; Zhang, J.; Deng, C.; Sun, H.; Zhong, Z., Targeted glioma chemotherapy by cyclic RGD peptide-functionalized reversibly core-crosslinked multifunctional poly(ethylene glycol)-b-poly(ε-caprolactone) micelles. Acta Biomaterialia 2017, 50, (Supplement C), 396-406. 31. Zhu, Y.; Wang, X.; Zhang, J.; Meng, F.; Deng, C.; Cheng, R.; Feijen, J.; Zhong, Z., Exogenous vitamin C boosts the antitumor efficacy of paclitaxel containing reduction-sensitive shell-sheddable micelles in vivo. J. Controlled Rel. 2017, 250, (Supplement C), 9-19. 32. Cheng, R.; Feng, F.; Meng, F.; Deng, C.; Feijen, J.; Zhong, Z., Glutathione-responsive nano-vehicles as a promising platform for targeted intracellular drug and gene delivery. J. Controlled Rel. 2011, 152, (1), 2-12. 33. García-Giménez, J. L.; Markovic, J.; Dasí, F.; Queval, G.; Schnaubelt, D.; Foyer, C. H.; Pallardó, F. V., Nuclear glutathione. Biochim. Biophys. Acta - General Subjects 2013, 1830, (5), 3304-3316. 34. Huang, S.-L., Liposomes in ultrasonic drug and gene delivery. Adv. Drug Deliv. Rev. 2008, 60, (10), 1167-1176. 35. Nahire, R.; Haldar, M. K.; Paul, S.; Mergoum, A.; Ambre, A. H.; Katti, K. S.; Gange, K. N.; Srivastava, D.; Sarkar, K.; Mallik, S., Polymer-coated echogenic lipid nanoparticles with dual release triggers. Biomacromolecules 2013, 14, (3), 841-853. 36. Paul, S.; Russakow, D.; Nahire, R.; Nandy, T.; Ambre, A. H.; Katti, K.; Mallik, S.; Sarkar, K., In vitro measurement of attenuation and nonlinear scattering from echogenic liposomes. Ultrasonics 2012, 52, (7), 962-969. 37. Huang, S.-L., Ultrasound-responsive liposomes. Liposomes: Methods and Protocols, Volume 1: Pharmaceutical Nanocarriers 2010, 113-128. 38. Nahire, R.; Hossain, R.; Patel, R.; Paul, S.; Meghnani, V.; Ambre, A. H.; Gange, K. N.; Katti, K. S.; Leclerc, E.; Srivastava, D., pH-triggered echogenicity and contents release from liposomes. Mol. Pharm. 2014, 11, (11), 4059-4068. 39. Paul, S.; Nahire, R.; Mallik, S.; Sarkar, K., Encapsulated microbubbles and echogenic liposomes for contrast ultrasound imaging and targeted drug delivery. Computational Mech. 2014, 53, (3), 413-435. 40. Nahire, R.; Haldar, M. K.; Paul, S.; Ambre, A. H.; Meghnani, V.; Layek, B.; Katti, K. S.; Gange, K. N.; Singh, J.; Sarkar, K., Multifunctional polymersomes for cytosolic delivery of gemcitabine and doxorubicin to cancer cells. Biomaterials 2014, 35, (24), 6482-6497. 41. Zhang, S.; Zhao, Y., Controlled release from cleavable polymerized liposomes upon redox and pH stimulation. Bioconjugate Chem. 2011, 22, (4), 523-528. 42. Discher, B. M.; Won, Y. Y.; Ege, D. S.; Lee, J. C.; Bates, F. S.; Discher, D. E.; Hammer, D. A., Polymersomes: tough vesicles made from diblock copolymers. Science 1999, 284, (5417), 1143-6.

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TOC graphic:

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TOC Graphic: The polymersomes enter the nuclei of pancreatic cancer cells and deliver a stemness inhibitor.

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