Paclitaxel-Loaded Polymersomes for Enhanced Intraperitoneal Chemotherapy.

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Mol Cancer Ther. Author manuscript; available in PMC 2016 October 01. Published in final edited form as: Mol Cancer Ther. 2016 April ; 15(4): 670–679. doi:10.1158/1535-7163.MCT-15-0713-T.

Paclitaxel-loaded Polymersomes for Enhanced Intraperitoneal Chemotherapy Lorena Simón-Gracia1, Hedi Hunt1, Pablo D. Scodeller1,2, Jens Gaitzsch3,4, Gary B. Braun2, Anne-Mari A. Willmore1, Erkki Ruoslahti2,5, Giuseppe Battaglia3,*, and Tambet Teesalu1,2,*

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1Laboratory

of Cancer Biology, Institute of Biomedicine, Centre of Excellence for Translational Medicine, University of Tartu, Ravila 14b, 50411 Tartu, Estonia 2Cancer Research Center, Sanford-Burnham-Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, 92037 California, USA 3Department of Chemistry, University College London, Gower St. WC1E 6BT London, UK 4Department of Chemistry, University of Basel, Klingelbergstrasse 80, 4056 Basel, Switzerland 5Center for Nanomedicine and Department of Cell, Molecular and Developmental Biology, University of California, Santa Barbara Santa Barbara, 93106 California, USA

Abstract

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Peritoneal carcinomatosis is present in more than 60% of gastric cancer, 40% of ovarian cancer, and 35% of colon cancer patients. It is the second most common cause of cancer mortality, with a median survival of 1–3 months. Cytoreductive surgery combined with intraperitoneal chemotherapy is the current clinical treatment, but achieving curative drug accumulation and penetration in peritoneal carcinomatosis lesions remains an unresolved challenge. Here we employed flexible and pH-sensitive polymersomes for payload delivery to peritoneal gastric (MKN-45P) and colon (CT26) carcinoma in mice. Polymersomes were loaded with Paclitaxel® and in vitro drug release was studied as a function of pH and time. Paclitaxel-loaded polymersomes remained stable in aqueous solution at neutral pH for up to four months. In cell viability assay on cultured cancer cell lines (MKN-45P, SKOV3, CT26), Paclitaxel-loaded polymersomes were more toxic than free drug or albumin-bound Paclitaxel (Abraxane®). Intraperitoneally administered fluorescent polymersomes accumulated in malignant lesions, and immunofluorescence revealed intense signal inside tumors with no detectable signal in control organs. A dual targeting of tumors was observed: direct (circulation independent) penetration, and systemic, blood vessel-associated accumulation. Finally, we evaluated preclinical antitumor efficacy of polymersomes-paclitaxel in treatment of MKN-45P disseminated gastric carcinoma using a total dose of 7 mg/kg. Experimental therapy with polymersome-Paclitaxel improved the therapeutic index of drug over Paclitaxel-Cremophor and Abraxane®, as evaluated by intraperitoneal tumor burden and number of metastatic nodules. Our findings underline the

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CORRESPONDING AUTHORS: Tambet Teesalu. Laboratory of Cancer Biology, Institute of Biomedicine, Centre of Excellence for Translational Medicine, University of Tartu, Ravila 14b, 50411 Tartu, Estonia. Tel: +37253974441, ; Email: [email protected], ; Email: [email protected]. Giuseppe Battaglia. Department of Chemistry, University College London, Gower St. WC1E 6BT London, UK. Tel: +22(0)2076791003, ; Email: [email protected]. Conflicts of interest: E. Ruoslahti and T. Teesalu are shareholders of DrugCendR Inc.

Simón-Gracia et al.

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potential utility of the polymersome platform for delivery of drugs and imaging agents to peritoneal carcinomatosis lesions.

Keywords POLYMERSOMES; INTRAPERITONEAL CHEMOTHERAPY; PACLITAXEL

INTRODUCTION

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Peritoneal carcinomatosis (PC) - dissemination of cancerous tissues in the peritoneal cavity is a common manifestation in digestive-tract and advanced gynecological cancers, such as gastric, colorectal, ovarian, and appendiceal cancer. Up to 60% of patients with gastric cancer show disease progression and die of PC, with a median survival of 1–3 months (1). 40% of colorectal cancers lead to PC (2), and the mortality from ovarian cancer PC is about 35% (3). Systemic chemotherapy is less active against peritoneal metastasis than intraperitoneal chemotherapy (IPC), presumably because early-stage peritoneal tumor nodules are poorly vascularized and drug delivery and penetration into advanced tumors is inefficient. Compared with intravenous (IV) administration, the intraperitoneal (IP) route allows increase in drug concentration within the abdominal cavity; therefore, the local effects of the drug are increased (1–3). Despite proven significant clinical benefit of the IP Paclitaxel® chemotherapy in the optimally debulked epithelial ovarian carcinoma patients (4), the therapy is not widely used, mostly due to complexity of the regimen and drug side effects (5). An ideal IP chemotherapeutic agent should be able to directly target and show high cytotoxic activity towards peritoneal tumors, persist in peritoneal cavity for extended periods, and also result in some degree of systemic exposure (5). When IPC is administered under mild hyperthermia, the effects of chemotherapy are extended by vasodilation and damage of tissue barriers, increasing the penetration of anticancer drugs (1–3). Yet, even in the case of hyperthermic IPC, drug penetration is limited to 3–5 mm and malignant nodules as small as 3 mm need to be excised prior to IPC to achieve adequate therapeutic efficacy (3).

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Paclitaxel® (PTX) is a potent cytotoxic drug used to treat ovarian, breast, lung, and pancreatic cancer, among others. Due to its low solubility in aqueous solutions, PTX is administered with Cremophor EL as solvent, which is responsible for much of the clinical toxicity (6). Abraxane® (ABX) is an alternative formulation, in which PTX is bound to albumin, forming water-soluble nanoparticles of about 130 nm in diameter. ABX is used for treating patients with breast, lung, and pancreatic cancers, and is undergoing clinical evaluation for IP treatment of advanced cancer in the peritoneal cavity. Compared to free anticancer drugs, IP administered nano-formulated drugs have superior antitumor activity in PC (7–11). Nanoparticles can be loaded with poorly soluble hydrophobic anticancer drugs, allowing for higher tumor drug exposure for sustained antitumor activity and enhanced tumor penetration. Polymersomes are nanoscale vesicles formed by self-assembly of amphiphilic blockcopolymers in water. Polymersomes can encapsulate water-insoluble compounds in the

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hydrophobic membrane and water-soluble compounds within the vesicular lumen (12–18). The surface of polymersomes can be functionalized with affinity ligands and dyes for targeted delivery and for tracking (19). The high molecular weight of block copolymers enables the formation of highly entangled membranes (20). This results in increased resilience with elastomer-like mechanical properties, which allows polymersomes to undergo up to 100% surface deformation before rupturing (21–23). These unique mechanical properties enable polymersomes to translocate across pores up to an order of magnitude smaller than their diameter (17).

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Here we use polymersomes assembled from diblock co-polymer poly(oligoethylene glycol methacrylate)-poly(2-(diisopropylamino)ethyl methacrylate) (P[(OEG)10MA]20-PDPA90 or POEGMA-PDPA). POEGMA is a polyethylene oxide-based hydrophilic block that protects the particles from the immune system, extending the in vivo half-life (24), while the PDPA block makes the polymersomes pH-sensitive (Fig. S1). PDPA is not soluble in water at neutral pH, thus allowing the formation of the polymersomes. However under mildly acidic conditions (below pH 6.5) the amine group of the PDPA becomes protonated, resulting in fast disassembly of the polymersomes (25). Upon endocytosis and trafficking within acidic early endosomes, the rapid polymersome destabilization triggers an osmotic shock with consequent endosomal membrane lysis and cytosolic cargo release (14,26,27). PDPA-based polymersomes have been used for intracellular delivery of DNA (14), antibodies (15), antibiotics (28), anticancer drugs (12,13), and peptides (29). Particularly, for cancer cells, an efficient cytoplasmic delivery improves the drug efficacy (12). More recently, it has been demonstrated that polymersome-mediated intracellular delivery enables drugs to act considerably faster than when administered alone (13).

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In the current study, we evaluated IP administration of pH-sensitive polymersomes as a treatment for peritoneal cancer lesions in mice. We demonstrate that polymersomes allow efficient intracellular payload delivery in vitro and in vivo, specific homing to peritoneal tumors, and increase the therapeutic activity of PTX at a very low drug concentration.

MATERIALS AND METHODS Materials CHCl3, MeOH, isopropanol, dimethylformamide (DMF), Rhodamine B octadecyl ester, doxorubicin, paclitaxel, and MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) were purchased from Sigma-Aldrich. Phosphate-buffered saline (PBS) was purchased from Lonza, Belgium.

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MKN45P human gastric cancer cells were isolated from parental MKN45 cells and authenticated as described (30). MKN45P-luc cells were made by infecting MKN-45P cells with lenti-luciferase (Biogenova, Potomac, MD). SKOV-3 and CT-26 cell lines were purchased from ATCC, UK (SKOV-3 ATCC HBT-77, CT.26 ATCC CLR-2638). Cell lines were used without further authentication. The cells were cultivated in DMEM medium (Lonza, Belgium) with 100 I.U/mL of penicillin, streptomycin, and 10% of heat-inactivated fetal bovine serum (FBS, GE Healthcare, UK).



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POEGMA-PDPA co-polymer synthesis

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Athymic nude mice were purchased from Harlan, USA, and Balb/c mice were purchased from Charles River, USA. Animal experimentation procedures were approved by Estonian Ministry of Agriculture, Committee of Animal Experimentation, project #42.

Reaction of Mal-P(OEG10MA)20-PDPA90 with cysteine-terminated Fluorescein (Cys-FAM)

The 4-(2-bromoisobutyryl ethyl)morpholine initiator (MEBr) was prepared according to a previously published procedure (31). The protected maleimide initiator (Mal-Br) was prepared according to a previously published procedure (32). The Rhodamine 6G-based initiator (Rho-Br) was prepared according to a previously published procedure (30). ATRP synthesis and purification of P(OEG10MA)20-PDPA90 from ME-Br, Rho-Br and Mal-Br initiators was carried out as previously described (19). The resulting copolymer composition was determined by 1H NMR in CDCl3 and the polydispersity was determined by size exclusion chromatography in acidic water (0.25 vol% TFA in water). The deprotection of Mal-P(OEG10MA)20-PDPA90 was carried out according to a previously described procedure (19).

The deprotected Mal-P(OEG10MA)20-PDPA90 (20 mg, 0.7 μmol) was dissolved in 1 mL of CHCl3:MeOH 2:1, and 2 eq of Cys-FAM dissolved in 1 mL of nitrogen-purged DMF, were added to the solution. The mixture was stirred at 300 rpm overnight at RT. The CHCl3 and MeOH were evaporated, and the rest of the solution was dialyzed against water using a dialysis cassette (Thermo Scientific, USA) with a molecular weight cut-off of 10 KDa to remove the excess of Cys-FAM. The resulting suspension was freeze-dried and a yellow powder was obtained.

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Polymersomes formation and Paclitaxel, Rhodamine B octadecyl ester, and Doxorubicin (DOX) encapsulation

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For the formation of labeled polymersomes, FAM-P(OEG10MA)20-PDPA90 was mixed with the non-labeled co-polymer in a ratio 2:8. For the labeling of polymersomes with Rhodamine, the Rho-POEGMA-PDPA was mixed with the non-labeled co-polymer at a ratio of 1:9. For the formation of polymersomes and encapsulation of PTX or DOX, 20 mg of co-polymer was dissolved in 4 mL of CHCl3:MeOH 2:1 in a glass vial, and 100 μL of PTX or DOX dissolved in MeOH at a concentration of 5 mM was added to the co-polymer solution (0.25 mM PTX or DOX as a final concentration). For the encapsulation of Rhodamine B octadecyl ester, 20 mg of co-polymer was dissolved in 4 mL of CHCl3:MeOH 2:1 in a glass vial, and 50 μL of Rhodamine B dissolved in CHCl3:MeOH at 1 mg/mL was added. The solution containing the polymer and the dye or drug was dried under vacuum for the formation of the polymer film. The film was hydrated with 2 mL of PBS (pH 7.4) and stirred at 300 rpm for 2 weeks. After that, the suspension was sonicated for 30 min at RT. The polymersome samples were purified by centrifugation. The suspension was spun down at 500 g for 20 minutes at RT, and the resulting supernatant was spun down at 20,000 g for 20 min at RT. The pellet was resuspended in 2 mL of PBS (pH 7.4). Dynamic Light Scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments) was used to assess the polydispersity and average size of polymersome preparations. Z-potential



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measurements were conducted to determine the surface charge of the vesicles, using the Zetasizer Nano ZS, Malvern Instruments. Transmission electron microscopy (TEM) was used to assess the size, surface topology and morphology of assembled vesicles. Polymersomes in PBS were deposited onto copper grids at a concentration of 1 mg/mL, and stained using 0.75% phosphotungstic acid (pH 7). Images were acquired using a transmission electron microscope Tecnai 10, Philips, Netherlands. The encapsulated PTX was quantified by ultra-performance liquid chromatography (UPLC equipment from Waters), using free PTX dissolved in MeOH to make the standard curve. 50 μL of PTX-polymersomes was mixed with 50 μL of MeOH and 2 μL of HCl 1M. 5 uL of this mixture was run using water/ACN as eluent and Acquity Ultraperformance UPLC BEH C18 1.7 uM 2.1×50 mm column.

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Paclitaxel release studies and stability test For PTX release studies, PTX-polymersomes were incubated at 37° C, and after different time points, the sample was centrifuged at 20,000 g. The supernatant was then used for quantification of PTX by UPLC, as described above. For the short-term stability test of polymersomes and PTX-polymersomes, the vesicles were incubated at 37° C over 24 hours in PBS, or in PBS containing 4.5 mg of bovine serum albumin (GE Healthcare, UK), and the stability was studied by monitoring size changes by DLS. For the release test of PTXpolymersomes incubated with cells, 10,000 MKN-45P cells or 5,000 CT26 cells were incubated in 96-well plate with DMEM medium containing FBS for 24 hours. PTXpolymersomes were added to the cells at a final concentration of 10 μM and after 24 hours the culture medium was centrifuged at 20,000 g. The supernatant was mixed with methanol and HCl and analyzed with UPLC for the detection of PTX.

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In vitro cytotoxicity assay

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MKN-45P, CT26, or SKOV-3 cells were plated in 96-well plate (10,000 MKN-45P or SKOV-3 cells for the 6 and 24 hours’ time point; 5,000 MKN-45P cells for the 48 hours’ time point; 5,000 CT26 cells for the 6 and 24 hours’ time point; and 2,500 CT26 cells or the 48 hours’ time point) and incubated for 24 hours at 37° C in 5% CO2 atmosphere. Polymersomes, PTX-polymersomes, ABX, or free PTX samples were then added to the wells at a final concentration of 10, 1, 0.5, or 0.25 μM of drug. Empty polymersomes were used at the same co-polymer concentration as in the PS-PTX sample. For the 6 hours’ time point, the medium was removed from the wells after 6 hours of incubation at 37° C, and fresh medium was added. After 18 hours of incubation at 37° C, the medium was aspirated and 10 μL of MTT reagent was added at a concentration of 5 mg/mL in PBS. After 2 hours of incubation at 37° C, the solution was aspirated and the formed blue crystals were dissolved in 100 μL of isopropanol. The absorbance was measured at 570 nm with a microplate reader (Tecan Austria GmbH). Alternatively, for the 24 and 48 hours’ time point, the cells were incubated for 24 or 48 hours with the samples and the MTT assay was followed as described before.



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Cellular uptake and cargo release experiments

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50,000 MKN-45P, CT26, or SKOV-3 cells were seeded in a 24-well plate with a coverslip. After 24 hours polymersomes loaded with Rho or with DOX were added to the cells at a concentration of 0.8 mg/mL and incubated for 24 more hours. The cells were washed with PBS, fixed with paraformaldehyde, stained with DAPI, and observed under the fluorescence confocal microscopy (Zeizz LSM 510). The images were analyzed with the ZEN lite 2012 image software. In vivo biodistribution studies

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Nude mice were intraperitoneally injected with 106 MKN-45P cells and Balb/c mice received IP injection of 2×106 CT26 cells. Alternatively, nude mice were injected intraperitoneally with 106 MKN-45P cells and subcutaneously with 106 MKN-45P cells in the right flank. The MKN-45P tumors were grown for two weeks and the CT26 tumors for one week. For the ex vivo imaging, FAM-labeled polymersomes were injected IP (0.5 mg in 500 μL of PBS) or IV (0.5 mg in 100 μL of PBS) and after 24 hours the animals were perfused with 10 mL of PBS. The tumors and organs were excised for the fluorescence visualization using Illumatool light source (Lightools Research, US) and fluorescence quantification by Image J software. Tissues were snap-frozen with liquid nitrogen, and kept at −80° C for further analysis. For the in vivo imaging, the same amount of Rho-labeled polymersomes were injected IP into the MKN-45P-bearing mice and the animals were imaged in vivo after 24 hours using the Optix MX3 (Art Advanced Research Technologies Inc.). Immunofluorescence and microscopic imaging

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The excised tumor and organs were cryosectioned, fixed with paraformaldehyde, and immunostained with anti-fluorescein rabbit IgG fragment (Life Technologies, USA) and rat anti-mouse CD31 (BD Biosciences, USA) as primary antibodies, and Alexa fluor 488 goat anti-rabbit IgG and Alexa fluor 647 goat anti-rat IgG (Invitrogen, USA) as secondary antibodies. The nuclei of cells were stained with DAPI. Images of the tissue sections were taken with confocal microscopy (Zeizz LSM 510) and the images were analyzed with the ZEN lite 2012 image software. Experimental tumor therapy

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Athymic nude mice were injected IP with 106 MKN-45P-luciferase (MKN-45P-luc) cells and after 8 days the tumor burden was measured in vivo. Luciferin Firefly (Biosynth, USA) was injected IP at a concentration of 15 mg/mL in PBS and 10 μL/g of body weight, and the bioluminescence was detected after 10 minutes using the IVIS imaging system (Xenogen, Alameda, USA). The animals were distributed in 4 groups with same tumor burden average in each group. The mice were treated every other day with IP injections of 1 mL of PTXCre, ABX, or PTX-polymersomes at the same drug concentration, or PBS. The total dose of PTX was 7 mg/Kg. The tumor burden was monitored every 4 days. After 21 days of tumor induction the animals were perfused with 10 mL of PBS, and the tumors and organs were excised.



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RESULTS Formation and characterization of polymersomes and PTX-loaded polymersomes The formation of POEGMA-PDPA (Fig. S1) polymersomes and loading of PTX were carried out by hydration of a polymer film at neutral pH. Briefly, the co-polymer and the PTX were combined to form a film of polymer/drug, followed by hydration to form vesicles. The loading capacity of the polymersomes was found to be 0.5–0.9 moles PTX/mol polymer, corresponding to 4–7×104 molecules of drug per vesicle (Fig. S2-B).

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TEM was used to characterize the structure of the polymeric vesicles. As shown in Fig. 1, POEGMA-PDPA co-polymer alone or in combination with PTX formed vesicles at pH 7.4, whereas no vesicles were formed at pH 6. The preservation of polymersome structure and size after the loading of the drug was studied by measuring the polymersome size by DLS. We found that the hydrodynamic diameter of PTX-polymersomes (225 nm, PDI 0.2) was very similar to the empty polymersomes (219 nm with a PDI of 0.19), indicating no significant differences in the structure (Fig. 1-A) or size (Fig. 1-B). The surface charge of unloaded POEGMA-PDPA polymersomes and PTX-polymersomes was measured by z-potential and was found to be neutral (Fig. S3). This is an important feature, as it has been demonstrated that a neutral surface charge decreases non-specific cellular interactions of nanoparticles with cells (33–35). Stability of polymersomes and PTX release

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The stability of polymersomes in aqueous solutions was investigated by monitoring the particle size at different time points after vesicle formation. After incubation of polymersomes in PBS (pH 7.4) at 37 °C for 24 h, there were no significant changes in the average hydrodynamic diameter of the vesicles (Fig. 1-B). To evaluate long-term stability of polymersomes, their size was measured after storage at 4 °C for 4 months. There was no aggregation of the polymersome suspension, and the mean diameter of the vesicles remained unchanged over time (Fig. 1-B), indicating that polymersomes in aqueous solutions remain thermodynamically stable.

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The release of drug from polymersomes over time was evaluated by incubating the PTXpolymersomes in PBS (pH 7.4) at 37° C, followed by centrifugation to separate polymersomes from free drug, and quantifying PTX in the supernatant using UPLC. After 72 hours of incubation only 0.7% of drug was released (Fig. S2-B) and no visible aggregation of the particles was observed. After 4 months at 4° C, about 12% of the drug was released from PTX polymersomes (Fig. S2-B). When the empty or PTX-polymersomes were exposed to pH below 6, the colloidal dispersion turned clear, indicating disassembly of the vesicles. The PTX in the solution was quantified by UPLC to estimate the encapsulation efficiency and to confirm the quantitative drug release (Fig. S2-B and S2-C). We also evaluated the stability of polymersomes in cell culture medium in the presence of cultured cells. PTX-polymersomes (10 μM PTX) were incubated with MKN-45P and CT26 cells at 37° C for 24 hours and the medium was analyzed by UPLC for the presence of free and polymersome-encapsulated PTX. No free PTX was detected in the medium for either



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cell line (fig S2-D), demonstrating that PTX is not released from the polymersomes prior to cellular uptake. In vitro activity of PTX-polymersomes in tumor cell lines

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To assess suitability of polymersomes for drug delivery to peritoneal cancer, we first studied in vitro cellular internalization and subcellular distribution of polymersomes loaded with fluorescent cargoes. We used MKN-45P cells, a human gastric cancer cell line with a highpotential for peritoneal dissemination (30), CT26 cells, a mouse colon carcinoma cell line, and SKOV-3 cells, a human ovarian adenocarcinoma. All the cells showed robust uptake of polymersomes loaded with rhodamine B octadecyl ester (Rho) or with Doxorubicin (DOX) as judged by confocal microscopy after 24-hour incubation (Fig. S4). Whereas Rho fluorescence remained limited to the cytosol, DOX fluorescence was also observed in the nuclei of the cells. Nuclear accumulation of DOX suggests that polymersome cargo is released from the endosomes, possibly through endosomal membrane disruption by proton sponge effect (26,27).

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We next investigated in vitro cytotoxicity on a panel of tumor cell lines by the MTT assay (Fig. 2 and S5-A). Treatment of MKN-45P cells with PTX-polymersomes (0.5 μM of PTX, 24 hours of incubation) decreased the cellular viability to about 50% - significantly lower than the viability of cells treated with ABX or with free drug (Fig. 2A and B). Likewise, CT26 cells treated with PTX-polymersomes had significantly lower viability than cells treated with ABX, free PTX, or non-loaded polymersomes. We tested the toxicity of PTX formulations in MKN-45P and CT26 cells over a range of concentrations (0.25–10 μM of PTX) at different time points (6, 24 and 48 hours). We found PTX-polymersomes to be the most toxic formulation across the PTX concentration range at the 6 and 24-hour time points (fig. S5-A). At 48 hour time point, PTX-polymersomes were significantly more toxic than other formulations at 10 μM of PTX, with less pronounced differences at lower doses. To distinguish cellular toxicity from cytostatic effects we stained treated cells with trypan blue vital stain (Fig. S5-B). After 24 hours of treatment, the count of viable MKN-45P and CT26 cells was similar in all groups (Fig. S5-B). However, PTX-polymersomes were significantly more cytostatic (as judged based on total cell count) than ABX or free PTX. The inhibition of cell proliferation observed in our results is in agreement with the results of the MTT assay and the mechanism of action of Paclitaxel, known to target tubulin and interfere with the cell division.

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In addition to studies on MKN-45P and CT26 cells, we tested the effect of the PTX formulations on the viability of the SKOV-3 ovarian carcinoma cells, and observed significant decrease in viability of cells treated with PTX-polymersomes compared with the other PTX formulations (Fig. 2). Homing and penetration of POEGMA-PDPA polymersomes in peritoneal carcinomatosis lesions To determine the potential of the polymersome platform for in vivo targeting of PC lesions, we used MKN-45P peritoneal xenograft and CT26 syngeneic peritoneal tumor models in mice. Animals bearing disseminated peritoneal tumors were IP injected with 0.5 mg of



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polymersomes labeled with fluorescein (FAM-polymersomes). Macroscopic assessment of resected organs revealed accumulation of fluorescent polymersomes in both MKN-45P (Fig. 3-A) and CT26 (Fig. 3-B) tumors, after 24 hours of injection. In the MKN-45P model the fluorescence in the tumor was 3.7-fold higher than in the lungs, 5.5-fold higher than in the kidneys and 12 times higher than in the liver (Fig. 3-C). For the CT26 tumor model the accumulation of polymersomes in the tumor was about 3 times higher than in the lungs and liver, and 4.5 times higher than in kidneys (Fig. 3-C). In tumors and organs resected from non-injected animals bearing MKN-45P and CT26 tumors, no signal over background was observed (Fig. S6).

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In addition, we used in vivo imaging to study accumulation of polymersomes labeled with rhodamine (Rho-polymersomes) to MKN-45P tumors expressing luciferase (Fig. S7). At 24 hours post-injection, the fluorescence of Rho-polymersomes overlapped with the luminescence emitted by the luciferase-expressing tumor. During macroscopic FAM imaging, intrinsic tissue autofluorescence is mapped along with fluorescein signal and intensity of the FAM fluorescence might be affected by the local microenvironment. To confirm polymersome homing using an alternative assay, we immunostained tissue sections of tumors and control organs with anti-fluorescein antibody. Confocal imaging of stained tissue sections confirmed a strong signal in tumors and no detectable signal in the lungs, kidneys or liver (Fig. 3-D and S8).


Confocal fluorescence microscopy of both CT26 and MKN-45P tumors demonstrated selective accumulation and penetration of polymersomes in the peritoneal lesions (Fig. 3-D). Twenty-four hours after IP injection of FAM-polymersomes into CT26 tumor-bearing mice, FAM signal was detected both near the surface of the tumors and also deep inside tumor tissue, indicating effective tumor penetration by polymersomes (Fig. 3-D). In MKN-45P tumors, polymersomes co-localized with tumor vessels (Fig. 3-D, white arrow). In addition, we observed in both MKN-45P and CT26 models polymersomes in areas of the tumors with no detectable CD31-positive blood vessels, in tumor periphery, and deep within the tumor’s parenchyma (Fig. 3-D, pink arrow). This suggests that polymersomes entered into the tumor nodules using dual mode: directly, in a circulation-independent manner, and via a systemic route. To dissect the tumor entry pathways of polymersomes, we injected mice bearing dual peritoneal and subcutaneous MKN-45P tumors with FAM-polymersomes using either IP or IV route. In subcutaneous tumors both administration routes resulted in accumulation of polymersomes in the blood vessels and adjacent areas (Fig. S9). Peritoneal tumors from mice IV injected with polymersomes did not show polymersome accumulation in the periphery and poorly vascularized areas, and showed overall lower accumulation of polymersomes than seen after the IP administration (Fig. S10). The amount of polymersomes trapped in the liver was higher when polymersomes were administrated systemically compared to IP injection (Fig. S8). These observations support that IP polymersomes target peritoneal carcinomatosis using both direct penetration and systemic routes leading to more robust delivery than is possible with IV route.



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Antitumor efficacy of PTX-polymersomes in mice bearing peritoneal MKN-45P gastric tumors Bioluminescence-based imaging of live mice bearing MKN-45P-luc peritoneal tumors was used to measure tumor burden during experimental therapy. Tumor growth was compared in groups of mice that received IP injections of PTX-polymersomes, ABX, or PTX solubilized with Cremophor EL (PTX-Cre). In all treatment groups, the dose of PTX used was 7 mg/Kg. Twelve days after initiation of the treatment, the luminescence detected from the tumor in mice treated with PTX-polymersomes was more than 12 times lower than in the PBS control group (Fig. 4-A), whereas tumor luminescence in the PBS, ABX, and PTX-Cre groups did not differ significantly.

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Analysis of locally disseminated tumor nodules revealed a trend towards a lower number of nodules in the PTX-polymersome treatment group compared to the PTX-Cre and ABX groups (Fig. 4-B). Interestingly, the tumors in the PTX-polymersome group started to shrink after the first day of treatment and also after 10 days of treatment, and continued to do so until day 12, showing that the tumors not only grow more slowly, but also tend to shrink, in contrast to the tumors in other treatment groups.

DISCUSSION


A critical goal in cancer therapy is to increase the tumor selectivity of chemotherapeutics to improve the balance between anticancer activity and side effects of the drug. In the current study, we assessed IP administration of drug-loaded pH-sensitive polymersomes as a treatment for peritoneal cancer. We showed that polymersomes allow efficient payload delivery in tumor cells in vitro and in vivo, using mouse models of PC. Polymersomes are specifically taken up and accumulate in peritoneal tumor lesions, and increase the antitumor activity of a chemotherapeutic drug. Whereas several reports describe applications of polymersomes for anticancer drug delivery to cultured cells, the novelty of this work is the in vivo application of polymersomes for intraperitoneal cancer chemotherapy. We demonstrated here that the pH-sensitive polymersomes loaded with PTX are more effective than ABX, a nanoformulation in clinical use, and PTX-Cremophor, at a very low drug dose in the treatment of MKN-45P gastric tumor. We hypothesized that this efficacy is a result of a drug delivery to tumors by both direct (IP contact of polymersomes with cells) and indirect (via the vasculature) mechanisms, thus the encapsulated drug has effect on both large vascularized tumors and small avascular nodules. To test suitability of polymersomes for payload delivery to IP tumors, we chose to use the IP administration route, as the peritoneal barrier maintains a positive gradient of the drug in the peritoneum, thereby increasing the local effects and reducing the systemic toxicity (1). Moreover, access of systemic drugs to non-vascularized tumor foci, often present in the PC, is limited in the case of systemic therapy (36). IP administered FAM-labeled POEGMA-PDPA polymersomes showed robust accumulation and penetration in malignant lesions in both MKN-45P peritoneal xenografts and CT26 syngeneic peritoneal tumors in mice, and only a background signal in the control organs. In contrast, when polymersomes were administrated systemically, their accumulation was lower in peritoneal tumors and higher in normal organs. Microscopic analysis of the distribution of polymersomes in peritoneal and subcutaneous tumors suggested that



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polymersomes target peritoneal tumors through a combination of direct local penetration and indirect circulation-mediated homing. Such dual targeting is important, as a number of clinical studies indicate that combination of IV and IP chemotherapy for peritoneal carcinomatosis results in better outcome (decreased rate of metastasis, and decreased peritoneal recurrence) than either therapy alone (37). Paclitaxel is very poorly absorbed into the systemic circulation when administered by the IP route, with peak levels in the peritoneal cavity of 1000-fold higher than in the plasma (38), necessitating supplementation of IP paclitaxel with systemic chemotherapies. Our data suggest that formulation of paclitaxel in polymersomes overcomes this limitation.


In a previous reported work, doxorubicin-loaded PMPC[poly(2-(methacryloyloxy)ethyl phosphorylcholine)]-PDPA vesicles showed rapid cellular uptake and cytotoxicity in melanoma cells, but not in normal human dermal fibroblasts (12). In another in vitro study, cultured head and neck cancer spheroids, PMPC-PDPA polymersomes loaded with PTX and/or doxorubicin were found to penetrate into the spheroids and to trigger extensive cell death (13). In latter study, an interaction between the PMPC block displayed on the polymersome surface and the scavenger receptor of tumor cells was described. However, in the present work we used pH-sensitive polymersomes made by the co-polymer POEGMAPDPA, that lack phosphorylcholine groups in the PMPC chains involved in high affinity interaction with class B scavenger receptors. It is possible that tumor tropism of polymersomes may be further improved by active targeting - conjugation of polymersomes to ligands with affinity to malignant cells and cells in tumor stroma. We have previously shown that tumor homing of ABX nanoparticles that possess intrinsic tumor selectivity can be further improved by functionalization with tumor homing and penetrating peptides (39,40). The unique flexibility of polymersomes that allows them to pass through pores much smaller than their size is likely to contribute to enhanced tissue penetration (17). Indeed, the enhanced penetration of polymersomes has been demonstrated in vitro on human mucosa (41), human skin (17), and tumor spheroids (12), and also in vivo (42).

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As a drug for experimental therapy with polymersomes we used PTX, a poorly watersoluble potent anticancer agent known to be rendered more soluble and bioactive by incorporation in nanoparticles (43). Our PTX-polymersomes were more efficient than ABX in inhibiting MKN-45P and CT26 cell growth, suggesting that tumor cell uptake and/or cytoplasmic release of cytotoxic cargo is more efficient for polymersomes compared to albumin-PTX nanoparticles. When PTX-polymersomes were used for experimental therapy of mice bearing IP MKN-45P gastric tumors, they showed potent anti-tumor activity, superior to ABX or PTX-Cre. It has been reported that in the treatment of MKN-45P mice with IP administered NK105 - a PTX-incorporating micellar nanoparticle formulation (8) or a copolymer conjugated with PTX (10), the weight of peritoneal tumor nodules was ~ 5 times lower than in mice treated with PTX-Cre. In another study of IP treatment of ovarian tumors, dendrimer-based nanoparticles loaded with PTX reduced the growth of tumors about 5-fold more than ABX (7). In these studies the total PTX dose used was between 20 and 40 mg/Kg. In our case, tumor burden of the mice treated with PTX-polymersomes was 8 times lower than in PTX-Cre group and 7 times lower compared with ABX. Remarkably, the total dose of PTX we used in our treatment was only 7 mg/kg - the lowest reported PTX dose used in experimental tumor treatment with free PTX or ABX (7,8,10,44–46). The rationale Mol Cancer Ther. Author manuscript; available in PMC 2016 October 01.


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for using the PTX dose below the MTD, with which the PTX-polymersomes are still effective, was to demonstrate that it is possible to reduce the PTX side effects while preserving therapeutic antitumor activity. The high antitumor activity of IP-administered PTX-polymersomes may be due to a combination of factors. On a biodistribution level, intrinsic tumor tropism of polymersomes in combination with dual targeting (direct and circulation-mediated) and ability to pass through narrow pores may allow more extensive tumor accumulation than is possible with other formulations. Loading of a drug in pHsensitive polymersomes may improve its pharmacokinetics, with bioactive drug being released exclusively at endosomal low pH inside the tumor cell.

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In summary, pH-sensitive POEGMA-PDPA polymersomes loaded with PTX internalized and released the drug into cultured malignant cells in a more efficient manner than other PTX formulations. In mouse models of peritoneal carcinomatosis in vivo, IP-administered polymersomes specifically targeted and penetrated into vascularized and non-vascularized peritoneal tumors, and PTX-loaded polymersomes exhibited higher antitumor efficacy than ABX or free PTX. These observations warrant future preclinical and clinical research aimed at the development of polymersome-based drug delivery systems for the treatment of peritoneal carcinomatosis.

Supplementary Material Refer to Web version on PubMed Central for supplementary material.

Acknowledgments FINANCIAL SUPPORT

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This work was supported by the following grants: EMBO Installation grant #2344 (to T. Teesalu), European Research Council Starting Grant (GliomaDDS (to T. Teesalu), MEVIC grant (to G. Battaglia), ERC starting GLIOMADDS from European Regional Development Fund (to T. Teesalu), Wellcome Trust International Fellowship WT095077MA (to T. Teesalu), National Cancer Institute of NIH grant CA152327 (to E. Ruoslahti), and Cancer Center Support Grant CA CA30199 (to E. Ruoslahti). J. Gaitzsch was supported by a German Science Foundation Postdoctoral Fellowship. The authors would like to thank Rein Laiverik (Department of Anatomy, University of Tartu) for the assistance with the TEM equipment in this work.

References

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1. Montori G, Coccolini F, Ceresoli M, Catena F, Colaianni N, Poletti E, et al. The treatment of peritoneal carcinomatosis in advanced gastric cancer: state of the art. Int J Surg Oncol. 2014; 2014:1–7. 2. Klaver YL, Lemmens VE, Nienhuijs SW, Luyer MD, de Hingh IH. Peritoneal carcinomatosis of colorectal origin: incidence, prognosis and treatment options. World J Gastroenterol. 2012; 18:5489–5494. [PubMed: 23112540] 3. Eskander RN, Ansaloni L, Bristow RE, Coccolini F. Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy in epithelial ovarian cancer: state of the art. World J Obstet Gynecol. 2013; 2:94–100. 4. Armstrong DK, Bundy B, Wenzel L, Huang HQ, Baergen R, Lele S, et al. Intraperitoneal cisplatin and Paclitaxel in ovarian cancer. N Engl J Med. 2006; 354:34–43. [PubMed: 16394300] 5. CRC Press, Taylor &Francis group. Intraperitoneal cancer therapy. In: Ceelen, WP.; Levine, EA., editors. Principles and practice. 1. Boca Raton: Broken Sound Parkway; 2016.



Page 13

Author Manuscript Author Manuscript Author Manuscript Author Manuscript

6. Gelderblom H, Verweij J, Nooter K, Sparreboom A. Cremophor EL: the drawbacks and advantages of vehicle selection for drug formulation. Eur J Cancer. 2001; 37:1590–8. [PubMed: 11527683] 7. Xiao K, Luo J, Fowler WL, Li Y, Lee JS, Xing L, et al. A self-assembling nanoparticle for paclitaxel delivery in ovarian cancer. Biomaterials. 2009; 30:6006–16. [PubMed: 19660809] 8. Emoto S, Yamaguchi H, Kishikawa J, Yamashita H, Ishigami H, Kitayama J. Antitumor effect and pharmacokinetics of intraperitoneal NK105, a nanomicellar Paclitaxel formulation for peritoneal dissemination. Cancer Sci. 2012; 103:1304–10. [PubMed: 22429777] 9. Shen YA, Shyu IL, Lu M, He CH, Hsu YM, Liang HF, et al. Bypassing the EPR effect with a nanomedicine harboring a sustained-release function allows better tumor control. Int J Nanomedicine. 2015; 10:2485–502. [PubMed: 25848266] 10. Soma D, Kitayama J, Konno T, Ishihara K, Yamada J, Kamei T, et al. Intraperitoneal administration of Paclitaxel solubilized with poly(2-methacryloxyethyl phosphorylcholine-co nbutyl methacrylate) for peritoneal dissemination of gastric cancer. Cancer Sci. 2009; 100:1979–85. [PubMed: 19604244] 11. Kamei T, Kitayama J, Yamaguchi H, Soma D, Emoto S, Konno T, et al. Spatial distribution of intraperitoneally administrated Paclitaxel nanoparticles solubilized with poly (2-methacryloxyethyl phosphorylcholine-co n-butyl methacrylate) in peritoneal metastatic nodules. Cancer Sci. 2011; 102:200–5. [PubMed: 20942868] 12. Pegoraro C, Cecchin D, Simon-Gracia L, Warren N, Madsen J, Armes SP, et al. Enhanced drug delivery to melanoma cells using PMPC-PDPA polymersomes. Cancer Lett. 2013; 334:328–37. [PubMed: 23402813] 13. Colley HE, Hearnden V, Avila-Olias M, Cecchin D, Canton I, Madsen J, et al. Polymersomemediated delivery of combination anticancer therapy to head and neck cancer cells: 2D and 3D in vitro evaluation. Mol Pharmaceutics. 2014; 11:1176–88. 14. Lomas H, Canton I, MacNeil S, Du J, Armes SP, Ryan AJ, et al. Biomimetic pH-sensitive polymersomes for efficient DNA encapsulation and delivery. Adv Mater. 2007; 19:4238–43. 15. Canton I, Massignani M, Patikarnmonthon N, Chierico L, Robertson J, Renshaw SA, et al. Fully synthetic polymer vesicles for intracellular delivery of antibodies in live cells. FASEB J. 2013; 27:98–108. [PubMed: 23033321] 16. Wang L, Chierico L, Little D, Patikarnmonthon N, Yang Z, Azzouz M, et al. Encapsulation of biomacromolecules within polymersomes by electroporation. Angew Chem Int Ed. 2012; 51:11122–5. 17. Pegoraro C, Cecchin D, Madsen J, Warren N, Armes SP, MacNeil S, et al. Translocation of flexible polymersomes across pores at the nanoscale. Biomater Sci. 2014; 2:680–2. [PubMed: 26828800] 18. Messager L, Gaitzsch J, Chierico L, Battaglia G. Novel aspects of encapsulation and delivery using polymersomes. Curr Opin Pharmacol. 2014; 18:104–11. [PubMed: 25306248] 19. Tian X, Nyberg S, Sharp PS, Madsen J, Daneshpour N, Armes SP, et al. LRP1-mediated intracellular antibody delivery to the central nervous system. Sci Rep. 2015; 5:11990. [PubMed: 26189707] 20. Battaglia G, Ryan AJ. Bilayers and interdigitation in block copolymer vesicles. J Am Chem Soc. 2005; 127:8757–64. [PubMed: 15954782] 21. Discher BM, Won YY, Ege DS, Lee JC, Bates FS, Discher DE, et al. Polymersomes: tough vesicles made from diblock copolymers. Science. 1999; 284:1143–6. [PubMed: 10325219] 22. Bermudez H, Brannan AK, Hammer DA, Bates FS, Discher DE. Molecular weight dependence of polymersome membrane structure, elasticity, and stability. Macromolecules. 2002; 35:8203–8. 23. Battaglia G, LoPresti C, Massignani M, Warren NJ, Madsen J, Forster S, et al. Wet nanoscale imaging and testing of polymersomes. Small. 2011; 7:2010–5. [PubMed: 21695783] 24. Veronese FM, Pasut G. PEGylation, successful approach to drug delivery. Drug Discov Today. 2005; 10:1451–8. [PubMed: 16243265] 25. Du J, Tang Y, Lewis AL, Armes SP. pH-sensitive vesicles based on a biocompatible zwitterionic diblock copolymer. J Am Chem Soc. 2005; 127:17982–3. [PubMed: 16366531] 26. Massignani M, LoPresti C, Blanazs A, Madsen J, Armes SP, Lewis AL, et al. Controlling cellular uptake by surface chemistry, size, and surface topology at the nanoscale. Small. 2009; 5:2424–32. [PubMed: 19634187] Mol Cancer Ther. Author manuscript; available in PMC 2016 October 01.


Page 14

Author Manuscript Author Manuscript Author Manuscript Author Manuscript

27. Massignani M, Canton I, Sun T, Hearnden V, Macneil S, Blanazs A, et al. Enhanced fluorescence imaging of live cells by effective cytosolic delivery of probes. PLoS One. 2010; 5:1–11. 28. Wayakanon K, Thornhill MH, Douglas CW, Lewis AL, Warren NJ, Pinnock A, et al. Polymersome-mediated intracellular delivery of antibiotics to treat porphyromonas gingivalisinfected oral epithelial cells. FASEB J. 2013; 27:4455–65. [PubMed: 23921377] 29. Chierico L, Joseph AS, Lewis AL, Battaglia G. Live cell imaging of membrane/cytoskeleton interactions and membrane topology. Sci Rep. 2014; 4:1–10. 30. Koga A, Aoyagi K, Imaizumi T, Miyagi M, Shirouzo K. Comparison between the gastric cancer cell line MKN-45 and the high-potential peritoneal dissemination gastric cancer cell line MKN-45P. Kurume Me J. 2011; 58:73–9. 31. Weaver JVM, Bannister I, Robinson KL, Bories-Azeau X, Armes SP. Stimulus-responsive watersoluble polymers based on 2-hydroxyethyl methacrylate. Macromolecules. 2004; 37:2395–403. 32. Mantovani G, Lecolley F, Tao L, Haddleton DM, Clerx J, Cornelissen JJ, et al. Design and synthesis of N-maleimido-functionalized hydrophilic polymers via copper-mediated living radical polymerization: a suitable alternative to PEGylation chemistry. J Am Chem Soc. 2005; 127:2966– 73. [PubMed: 15740133] 33. Verma A, Stellacci F. Effect of surface properties on nanoparticle–cell interactions. Small. 2010; 6:12–21. [PubMed: 19844908] 34. Hea C, Hua Y, Yina L, Tanga C, Yin C. Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials. 2010; 31:3657–66. [PubMed: 20138662] 35. Hrkach J, Von Hoff D, Mukkaram AM, Andrianova E, Auer J, Campbell T, et al. Preclinical development and clinical translation of a PSMA-targeted Docetaxel nanoparticle with a differentiated pharmacological profile. Sci Transl Med. 2012; 4:128–39. 36. Sugahara KN, Scodeller P, Braun GB, de Mendoza TH, Yamazaki CM, Kluger MD, et al. A tumorpenetrating peptide enhances circulation-independent targeting of peritoneal carcinomatosis. J Control Release. 2015; 212:59–69. [PubMed: 26071630] 37. Yang S, Feng R, Pan Z-C, Jiang T, Xu Q, Chen Q. A Comparison of Intravenous plus Intraperitoneal Chemotherapy with Intravenous Chemotherapy Alone for the Treatment of Gastric Cancer: A Meta-Analysis. Sci Rep. 2015; 5:12538. [PubMed: 26220081] 38. Markman M, Rowinsky E, Hakes T, Reichman B, Jones W, Lewis JL Jr. Phase I trial of intraperitoneal taxol: a Gynecoloic Oncology Group study. J Clin Oncol. 1992; 10:1485–91. [PubMed: 1355523] 39. Karmali PP, Kotamraju VR, Kastantin M, Black M, Missirlis D, Tirrell M, et al. Targeting Of Albumin-Embedded Paclitaxel Nanoparticles To Tumors. Nanomedicine. 2009; 5:73–82. [PubMed: 18829396] 40. Sugahara KN, Teesalu T, Karmali PP, Kotamraju VR, Agemy L, Girard OM, et al. TissuePenetrating Delivery Of Compounds And Nanoparticles Into Tumors. Cancer Cell. 2009; 16:510– 520. [PubMed: 19962669] 41. Hearnden V, Lomas H, MacNeil S, Thornhill M, Murdoch C, Lewis A, et al. Diffusion studies of nanometer polymersomes across tissue engineered human oral mucosa. Pharm Res. 2009; 26:1718–28. [PubMed: 19387800] 42. Murdoch C, Reeves KJ, Hearnden V, Colley H, Massignani M, Canton I, et al. Internalization and biodistribution of polymersomes into oral squamous cell carcinoma cells in vitro and in vivo. Nanomedicine. 2010; 5:1025–36. [PubMed: 20874018] 43. Ping Ma P, Russell J, Mumper RJ. Paclitaxel nano-delivery systems: a comprehensive review. J Nanomed Nanotechol. 2013; 4:1–16. 44. Yang Y, Niu X, Zhang Q, Hao L, Ding Y, Xu H. The efficacy of Abraxane on osteosarcoma xenografts in nude mice and expression of secreted protein, acidic and rich in cysteine. Am J Med Sci. 2012; 344:199–205. [PubMed: 22222334] 45. Desai N, Trieu V, Soon-Shiong P, Hawkins M. Abraxane (ABI-007) vs Taxotere: a preclinical comparison of toxicity and efficacy. Proc Amer Assoc Cancer Res. 2005; 46:1–5.



Page 15

Author Manuscript

46. Tuan TF, Tsai ML, Yeh KC, Huang HC, Chung CT, Huang CL, et al. Intravenous Paclitaxel against metastasis of human gastric tumors of diffuse type. Cancer Chemother Pharmacol. 2010; 66:773– 83. [PubMed: 20044750]

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Figure 1.

Characterization of empty polymersomes (PS) and PTX-loaded polymersomes (PS-PTX). A: TEM images of freshly prepared empty and PTX-polymersomes. Polymersome samples were deposited on copper grids and stained with phosphotungstic acid at pH 7.4. B: DLS determination of hydrodynamic diameter and polydispersity index (PDI) values of empty and PTX-polymersomes after incubation for indicated time and temperature.

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Figure 2.

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In vitro assessment of cytotoxicity of free and encapsulated PTX to MKN-45P, CT26, and SKOV-3 cells. Cytotoxicity measured by MTT assay of the indicated formulations at 0.5 μM of PTX after 24 hours of incubation. PS, polymersomes; PS-PTX, PTX-loaded polymersomes. Statistical analysis was performed by ANOVA. n=4; error bars indicate +SEM; *** p < 0.001, ** p < 0.01, * p < 0.05.



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Figure 3.

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Intraperitoneally administered fluorescent polymersomes home to peritoneal tumors in vivo. Mice bearing disseminated MKN-45P (A) or CT26 (B) tumors were injected with polymersomes labeled with fluorescein and the mice were perfused after 24 hours. C: Florescence intensity of tumors and different organs from ex vivo imaging quantified with Image J. Results are normalized for tissue area. Panel D: Confocal micrographs of peritoneal tumor sections after 24 hours of FAM-polymersomes injection in MKN-45P and CT26 bearing mice. Blue, DAPI; green, FAM; red, CD31. The white arrow indicates the colocalization of green and red fluorescence and the pink arrow indicates only the green fluorescence. Representative fields from multiple sections of three independent mice are shown. Tu, tumor; Ki, kidney; He, heart; Li, liver; Lu, lung; Sp, spleen. N = 4 for MKN-45 tumor model and n =3 for CT26 tumor model. Error bars indicate + SEM. Scale bars = 20μm, for panel H, 50μm for panels A, C, E, F, G, and 100 μm for panel D.



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Figure 4.

Experimental therapy of MKN-45P tumor-bearing mice. Mice bearing disseminated peritoneal tumors induced with MKN-45P-Luc cells were injected with indicated formulations (1 mg PTX/kg) every other day. A: tumor growth monitored by measurement of luciferase activity. Days after MKN-45P-Luc cells injection are plotted on the x-axis. On the y-axes are the photons recovered from region of interest. The arrows indicate treatment injections. B: Quantification of metastatic nodules after the treatment. n =5 in each group. PS, polymersomes; PS-PTX, PTX-loaded polymersomes. Statistical analyses, ANOVA; error bars, mean + SEM, *** p < 0.001, ** p < 0.01.


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