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Nanomedicine (2010) 5(7), 1025–1036. 1025. Internalization and biodistribution of polymersomes into oral squamous cell carcinoma cells in vitro and in vivo.
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Internalization and biodistribution of polymersomes into oral squamous cell carcinoma cells in vitro and in vivo The prognosis for oral squamous cell carcinoma (OSCC) is not improving despite advances in surgical treatment. As with many cancers, there is a need to deliver therapeutic agents with greater efficiency into OSCC to improve treatment and patient outcome. The development of polymersomes offers a novel way to deliver therapy directly into tumor cells. Here we examined the internalization and biodistribution of two different fluorescently labeled polymersome formulations; polyethylene oxide (PEO)-poly 2‑(diisopropylamino)ethyl methacrylate (PDPA) and poly 2‑(methacryloyloxy)ethyl phosphorylcholine (PMPC)-PDPA, into SCC4 OSCC cells in vitro and in vivo. In vitro SCC4 monolayers internalized PMPC–PDPA and PEO–PDPA at similar rates. However, in vivo PMPC–PDPA polymersomes penetrated deeper and were more widely dispersed in SCC4 tumors than PEO–PDPA polymersomes. In the liver and spleen PMPC–PDPA mainly accumulated in tissue macrophages. However, in tumors PMPC–PDPA was found extensively in the nucleus and cytoplasm of tumor cells as well as in tumor-associated macrophages. Use of PMPC–PDPA polymersomes may enhance polymersome-mediated antitumor therapy. KEYWORDS: biodistribution n cancer n drug delivery n oral squamous cell carcinoma n PMPC n polymer n polymersome n tumor-associated macrophages

Many drugs require intracellular delivery to be effective. Pharmaceutical companies spend much time and effort altering the properties of drugs to allow penetration in the plasma membrane of cells. Often this process is only partially successful, leading to reduced efficacy of the drug. In some instances additional agents are administered with the drug to aid its transfer across the cell membrane. For example, administration of cremophor EL emulsifier with the anticancer drug paclitaxel. However, these agents can often cause additional side effects resulting in unnecessary illness. Moreover, agents such as nucleic acids, therapeutic enzymes, proteins and certain antibiotics cannot cross the cell membrane. Viruses have been used to deliver nucleic acid but there are practical, ethical and safety concerns about their use [1] . Viral vectors also induce an immune response that may result in destruction, reduced circulation times and adverse systemic responses [2] . Recently, liposomes have been used to improve delivery of some therapeutic agents but these have a small load capacity and short circulation times [3] . These problems have led to the development of synthetic block copolymers that self-assemble into membrane-enclosed nanosized vesicles called polymersomes [4–7] . Polymersomes efficiently encapsulate lipophilic molecules within their membranes and hydrophilic molecules within their aqueous core (reviewed in [8,9]).

Polymersomes can carry such diverse agents as anticancer drugs [10,11] , genes [12,13] , proteins [14] , antisense oligonucleotides and siRNA [15] , fluorescent tracking molecules [16,17] and diagnostic probes [18] . Polymersomes possess unique features such as high stability and their adaptable membrane properties permit greater drug loading capacity, better drug retention and longer circulation times than liposomes [18–20] . Their nanometer size allows diffusion through biological fluids/tissues and they are rapidly internalized by cells [16,17] . The first-generation polymer vesicles used polyethylene glycol (PEG) or polyethylene oxide (PEO)-based block co­polymers such as PEG–poly-l-lactic acid, PEG–polycaprolactone or PEO–PBO [21] . We have developed a new generation of polymersomes consisting of the amphiphilic block copolymer poly‑2‑(methacryloyloxy) ethyl phosphorylcholine (PMPC) coupled with a pH-sensitive copolymer, poly2‑(diisopropylamino)ethyl methacrylate (PDPA) [22–24] . PMPC endows polymersomes with similar stealth characteristics to PEG and PEO-based polymersomes [12,25] while PDPA confers pH sensitivity. At pH below 6.4 PDPA is protonated, becomes hydrophilic and the block copolymer unimers dissolve in water. Above pH 6.4, PDPA becomes hydrophobic and the block copolymer molecules self-assemble into membrane-enclosed polymersomes. Thus, any hydrophilic drug in aqueous solution with the copolymer becomes

10.2217/NNM.10.97 © 2010 Future Medicine Ltd

Nanomedicine (2010) 5(7), 1025–1036

Craig Murdoch1, Kim J Reeves2, Vanessa Hearnden1,3,4, Helen Colley1,3, Marzia Massignani3,4, Irene Canton3, Jeppe Madsen3,5, Adam Blanazs5, Steve P Armes5, Andrew L Lewis6, Sheila MacNeil4, Nicola J Brown2, Martin H Thornhill1 & Giuseppe Battaglia†3 School of Clinical Dentistry, Unit of Oral & Maxillofacial Medicine & Surgery, University of Sheffield, Sheffield, UK 2 Department of Surgical Oncology, University of Sheffield, Sheffield, UK 3 Department of Biomedical Science, University of Sheffield, Sheffield, UK 4 Department of Engineering Materials, University of Sheffield, Sheffield, UK 5 Department of Chemistry, University of Sheffield, Sheffield, UK 6 Biocompatibles UK Ltd, Farnham, Surrey, UK † Author for correspondence: Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK Tel.: +44 114 222 2305 Fax: +44 114 276 5413 [email protected] 1

ISSN 1743-5889

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enclosed within the polymersome vesicles as the pH is increased above 6.4, while any hydrophobic drug will be retained in the polymersome hydrophobic bilayer. We have recently shown that PMPC–PDPA polymersomes are taken up by cells via endocytosis  [16,26] . When the PMPC–PDPA polymersomes enter the endosomal compartments of cells they rapidly dissolve into unimers as the pH in these organelles is below 6.4. This rapid transition causes an osmotic shock to the endosome that then ruptures releasing the polymersome contents into the cytosol [13,16] . These properties make PMPC–PDPA polymersomes very efficient at delivering their load into cells. We have recently demonstrated this by using them to deliver plasmid DNA, propidium iodide and rhodamine into several different cell types  [13,16,26] . In the current study, we compare the internalization, retention and biodistribution of PEO–PDPA and PMPC–PDPA polymersomes into human oral squamous cell carcinoma (OSCC) cells in vitro and in vivo.

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under N2 for 20 min, added to the reaction solution and further degassed for 10 min. The mixture was then heated to 50°C and CuCl catalyst (Sigma, Poole, UK) was added under constant N2 flow to allow polymerization for 16 h after which an aliquot was removed for 1H NMR ana­lysis. If the ana­lysis indicated no remaining monomer the reaction was terminated by exposure to air. The copolymer was then dissolved in tetrahydrofuran and the spent catalyst removed by column silica chromatography. To remove unreacted initiator and bpy from the reaction product, the copolymer was dissolved in tetrahydrofuran and dialyzed against water for 7 days followed by dialysis against methanol for 2 days. After evaporation, the copolymer was dried under vacuum and characterized by 1H NMR and GPC.

Methods „„ PMPC25 –PDPA70 copolymer synthesis PMPC25 –PDPA70 copolymer (Figure 1A) was synthesized by atom transfer radical polymerization as reported elsewhere [16,22] . Briefly, a Schlenk flask was charged with CuBr (25.6 mg, 0.178 mmol; Sigma, Poole, UK) and MPC (1.32 g, 4.46 mmol; Biocompatibles Ltd). 2-(4-Morpholino)ethyl 2-bromoisobutyrate (ME-Br) initiator (50.0 mg, 0.178 mmol, prepared as in [27]) and 2,2´-bipyridine (bpy) ligand (55.8 mg, 0.358 mmol; Sigma, Poole, UK) were dissolved in 2  ml methanol and the solution deoxygenated with N2 for 30 min before being injected into the flask. The MPC:ME–Br:CuBr:bpy relative molar ratios were 25:1:1:2, respectively. The polymerization was conducted under a N2 atmosphere at 20°C. After 65 min a mixture of deoxygenated DPA (2.67 g, 12.5 mmol; Scientific Polymer Products, ON, Canada) and methanol (3 ml) was injected into the flask, and after a further 48 h the reaction solution was diluted by adding 200 ml isopropanol and then passed through a silica column (Merk, Darmstadt, Germany) to remove the spent Cu catalyst. The Cu content of the polymer after purification was less than 10 ppm [28] .

„„ Rhodamine–PMPC30 –PDPA60 copolymer synthesis R hodamine–PMPC 30 –PDPA 60 copolymer (Figure 1B) was synthesized by an atom transfer radical polymerization procedure, as previously described [22] . Briefly, a Schlenk flask was charged with MPC (1.20 g, 4.05 mmol). A rhodamine 6G‑based initiator prepared inhouse (83.8 mg, 0.135 mmol) was dissolved in methanol (0.75 ml) and added to the MPC. The solution was deoxygenated by bubbling N2 for 30 min after which a mixture of CuBr (19.37 mg, 0.135  mmol) and bpy ligand (42.17  mg, 0.171 mmol) was added to the reaction mixture. The MPC:Rhodamine:CuBr:bpy relative molar ratios were 30:1:1:2 and the reaction was carried out under a N2 atmosphere at 20°C. After 40 min, a mixture of deoxygenated DPA (1.73 g, 8.10 mmol) and methanol (2 ml) was injected into the flask and 48 h later the reaction solution was diluted with methanol (~70 ml) and opened to the atmosphere. When the suspension turned green, 200 ml chloroform was added to dissolve the copolymer and the solution passed through a silica column to remove the catalyst. After removal of the solvent, the solid was taken up into 3:1 chloroform:methanol and dialyzed for 3 days against this solvent mixture to remove residual bpy ligand. After evaporation, the solid was dispersed in water, freeze-dried and dried in a vacuum oven at 80°C for 48 h.

„„ PEO23 –PDPA15 copolymer synthesis PEO23 –PDPA15 copolymer (Figure 1C) was synthesized as follows: DPA monomer, PEO23 –Br macroinitiator (prepared as in [27]) and bpy were weighed into flask. Methanol was degassed

„„ Production of rhodamine-labeled polymersomes PMPC25 –PDPA70 or PEO23 –PDPA15 were dissolved in a 2:1 chloroform:methanol solution. rho-PMPC30 –PDPA60 (5%; Figure 1C ) was added

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Internalization of polymersomes in vitro & in vivo

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Figure 1. Chemistry, sizing and morphology of the polymersomes. (A) PMPC–PDPA, (B) rhodamine-labeled PMPC–PDPA and (C) PEO–PDPA. The black chain represents PMPC, the blue chain PDPA and the red chain rhodamine. Dynamic light scattering ana­lysis and transmission electron micrographs of (D & F) PMPC–PDPA and (E & G) PEO–PDPA polymersomes, respectively; bar = 50 nm. PDPA: Poly-2-(diisopropylamino)ethyl methacrylate; PEO: Polyethylene oxide; PMPC: Poly-2-(methacryloyloxy)ethyl phosphorylcholine.

to the solution and a copolymer film was formed by evaporating the solvent overnight in a vacuum oven at 50°C. The film was rehydrated using 2 ml of 100 mM phosphate buffered saline (PBS; pH 2) and once the film had dissolved the pH was increased to 7.4. This solution was sonicated for 5  min and then micelles and other small molecular weight impurities were removed from the polymersome fraction by gel permeation future science group

chromatography using a sepharose 4B size exclusion column. The purified polymersomes were subjected to dynamic light scattering ana­lysis and transmission electron microscopy as previously described [16] . Dynamic light scattering and transmission electron microscopy showed both PMPC–PDPA and PEO–PDPA polymersomes to be vesicles with a mean diameter of approximately 130 nm (Figure 1D–G) . www.futuremedicine.com

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„„ Cell culture The human squamous cell carcinoma cell line (SCC4), which is derived from a carcinoma of the tongue [29] , was obtained from the American Type Culture Collection (Rockville, MD, USA). SCC4 cells were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium supplemented with 2 mM l‑glutamine, 400 ng/ml hydrocortisone and 10% fetal bovine serum. Cells were incubated at 37°C in 5% CO2 and were subcultured after brief treatment with trypsin-EDTA. „„ SCC4 cell cytotoxicity SCC4 cells (3 × 105 per well) were plated into 24‑well plates and allowed to adhere overnight. Cells were incubated in the presence of 0.1 or 1  mg/ml rhodamine-labeled PEO–PDPA or PMPC–PDPA polymersomes diluted in medium for 24 and 48 h. Media alone was used as a control. Cell metabolic activity was determined by 3‑(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT, Sigma, Poole, UK) ana­lysis. Briefly, cell monolayers were rinsed with PBS and incubated at 37°C for 40 min with 0.5 mg/ml MTT solution. The solution was then removed and incorporated stain eluted using 400 µl of acidified isopropanol. A total of 150 µl of the eluted dye was transferred into a 96‑well plate and the OD measured spectrophotometrically at 540 nm, with a 630 nm correction reference. „„ Internalization kinetic ana­lysis using flow cytometry SCC4 cells were added to 24‑well plates (3 × 105 per well) and incubated overnight. A total of 500  µl of rhodamine-labeled PEO–PDPA or PMPC–PDPA polymersomes diluted in medium to give a concentration of 1  mg/ml were added to the cell monolayers and incubated at 37°C for increasing lengths of time. At each time point, the media was removed and the cells washed three-times with PBS, trypsinized, resuspended in 2% paraformaldehyde in PBS and stored at 4°C. Fixed cells were analyzed using a FacsArray (BD Biosciences) and the percentage of cells with fluorescence above control cells (cultured in media alone) and median fluorescence of whole cell population calculated. „„ In vivo studies Animals & housing

Male, 6-week-old, immunodeficient CD-1 mice weighing approximately 20–25  g were obtained from Charles River Ltd (Kent, UK) 1028

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and housed in the Field Laboratories at the University of Sheffield (Sheffield, UK) for at least 1  week prior to experimentation. The animals were exposed to a 12:12  h light– dark cycle in a humidity- and temperaturecontrolled environment and allowed access to food and water ad libitum. All procedures were performed under Home Office License approval PPL40/2972 (NJB). Tumors were grown by subcutaneous injection of SCC4 cells (1  ×  10 6 in 100  µl PBS) into the right flank of mice. When the tumors reached more than 200 mm 3 in volume the mice were randomized into treatment (n  =  5) and control (n  =  3) groups and 100  µl of a solution of either rhodamine-labeled PMPC–PDPA or PEO–PDPA polymersomes (10  mg of polymer/ml PBS, 45  mg/kg) or PBS alone (control) were administered by direct injection into the tumor (intratumoral). The fluorescence of tumors was imaged in  situ after 3  h using a macroimaging system (Light-tools, Encinitas, CA). Mice were then sacrificed at 3, 24 and 48 h, tumors excised and the position of fluorescently labeled polymersomes imaged again using the macroimaging system. In addition, tumors, spleen, liver and kidney were removed, snap frozen in OCT compound using liquid nitrogen and stored at -80°C. To quantify the levels of rhodamine-labeled PMPC–PDPA or PEO–PDPA in murine tissues, three frozen sections (5  µM) were cut from each tissue at increasing levels and counterstained for cell nuclei using DAPI (Vector Laboratories, Burlingame, CA, USA). Four random images (×40) were taken per tissue using a Retiga 1300R digital camera (QImaging, Surrey, BC, Canada) attached to a Zeiss Axioplan 2 fluorescence microscope. All images were acquired using the same exposure time with a Cy3 filter to detect rhodamine-derived fluorescence. Image ana­lysis was performed using ImageJ software (NIH, MD, USA). Images were converted to black and white using the threshold setting and the percentage area corresponding to that covered by the fluorescent polymersome calculated per image. To determine the toxicity of polymersomes nontumor-bearing CD-1 mice had 100 µl of 10 mg/ml of rhodamine-labeled PMPC–PDPA or PEO–PDPA polymersomes, or PBS as control (n = 6 per treatment group) administered intravenously. Mice were sacrificed 28  days postinjection and the liver, spleen and kidney were snap frozen and processed for ana­lysis as previously described. future science group

Internalization of polymersomes in vitro & in vivo

Immunocytochemistry

In the in  vitro studies, data are expressed as mean ± standard error of the mean from three independent experiments, and significant differences between groups were examined using the Mann–Whitney U test, with differences considered significant if the p-value was lower than 0.05. For in vivo experiments, all data are means ± standard error of the mean, and differences between groups were analyzed using Kruskal–Wallis one-way ana­lysis of variance on ranks.

Results „„ In vitro studies Monolayers of SCC4 OSCC cells incubated with 1 mg/ml PMPC–PDPA were not affected at 24  h but showed a small but significant (p